Treatment of respiratory infection with TLR agonist

Abstract

To provide methods for the treatment or prevention of rhinovirus-mediated respiratory conditions.SOLUTION: A method of treating or preventing a respiratory condition associated with rhinovirus comprises the step for administering a compound comprising a TLR2 agonist. Preferably, the compound comprising the TLR2 agonist is a pegylated derivative of Pam2Cys, represented by the formula (1).SELECTED DRAWING: None

Description

Technical Field

[0001] [Cross-reference to prior applications] This application claims priority to Australian Provisional Patent Application Nos. 2017901180, 2017905124, 2017905128 and 2018900409, the entire content of each of which is incorporated herein by reference in its entirety.

[0002] [Field of the Invention] The present invention relates to methods, compounds, compositions and kits for the prevention or treatment of respiratory conditions. In particular, the methods, compounds, compositions and kits are particularly useful for, but not limited to, the prevention and / or treatment of rhinovirus infection and the prevention and / or treatment of respiratory exacerbation.

Background Art

[0003] [Background of the Invention] Respiratory infections are one of the most common causes of human disease worldwide and are generally caused by viruses. Rhinovirus (RV) is one of the most common types of viruses that infect humans and is known to cause colds. Unlike sporadic pandemics and seasonal influenza outbreaks, rhinovirus infections occur throughout the year in multiple different serotypes. On average, children experience 5 - 10 colds per year, and more than half of all colds are due to RV infection.

[0004] Viral respiratory infections can exacerbate the severity of respiratory conditions and lead to exacerbations (attacks). Exacerbations can occur in conditions such as asthma and chronic obstructive pulmonary disease (COPD). Asthma and COPD exacerbations are the most clinically and economically important disease forms. Rhinovirus is the most common viral infection associated with asthma exacerbations and thus accounts for the highest burden in terms of morbidity, mortality and healthcare costs.

[0005] In asthma in particular, most exacerbations persist despite the use of the best and most up-to-date available treatments. When an exacerbation occurs, treatment options are limited and have made little progress in recent years. Treatment involves increased doses of inhaled bronchodilators and systemic or oral corticosteroids, which are the same medications that failed to prevent the exacerbation in the first place.

[0006] Therefore, there is a need for novel or improved therapies for the treatment and / or prevention of rhinovirus-borne respiratory conditions. Furthermore, there is a need for novel or improved therapies for the treatment and / or prevention of virus-borne exacerbations.

[0007] No prior art reference in this specification constitutes an endorsement or suggestion that the prior art forms part of the common general knowledge in any jurisdiction, or that the prior art would be understood, considered relevant, and / or naturally expected to be combined with other parts of the prior art by those skilled in the art. [Overview of the project]

[0008] [Overview of the prefecture] The present invention provides a method for treating or preventing a respiratory condition associated with a target rhinovirus, comprising administering a compound containing a TLR2 agonist to thereby treat or prevent the respiratory condition associated with the target rhinovirus.

[0009] Preferably, the method involves administering only compounds containing a TLR2 agonist. In other words, the method does not involve administering agonists of TLRs other than TLR2 homodimers or heterodimers.

[0010] The compound may be administered in a composition. Typically, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient. The composition may be formulated, for example, for inhalation or administration into the nasal airway. The composition does not necessarily have to contain a compound that is an agonist of a TLR other than a TLR2 homodimer or heterodimer. Preferably, the composition consists essentially of or comprises a compound containing a TLR2 agonist and a pharmaceutically acceptable carrier, diluent, or excipient.

[0011] The present invention provides a method for treating or preventing a target rhinovirus infection, comprising administering a compound comprising a TLR2 agonist to thereby treat or prevent the target rhinovirus infection. Preferably, the method further comprises the step of identifying a subject having a rhinovirus infection.

[0012] The present invention provides a method for reducing a target rhinovirus-induced airway inflammation, comprising administering a compound comprising a TLR2 agonist, thereby reducing the rhinovirus-induced airway inflammation.

[0013] The present invention further provides the use of compounds comprising a TLR2 agonist in the preparation of agents for treating or preventing target rhinovirus-associated respiratory conditions. In any embodiment, the present invention also provides the use of compounds comprising a TLR2 agonist for treating or preventing target rhinovirus-associated respiratory conditions.

[0014] The present invention further provides the use of compounds comprising a TLR2 agonist in the preparation of agents for treating or preventing a target rhinovirus infection.

[0015] The present invention also provides the use of compounds containing a TLR2 agonist for the prevention of target rhinovirus infection.

[0016] The present invention provides a method for treating or preventing a virus-mediated exacerbation of a target respiratory condition, comprising administering a compound comprising a TLR2 agonist to the target, thereby treating or preventing a virus-mediated exacerbation of the target respiratory condition. Preferably, the method further comprises the step of identifying a target having a respiratory condition as described herein. For example, the respiratory condition may be chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, or a lung condition associated with lung transplantation or long-term glucocorticosteroid use.

[0017] The present invention also provides a method for improving the ability of a subject to control a respiratory disease during a respiratory viral infection, comprising administering a compound comprising a TLR2 agonist to the subject to thereby improve the subject's ability to control the respiratory disease or respiratory viral infection. Preferably, the infection is a rhinovirus infection.

[0018] The present invention further provides the use of compounds comprising TLR2 agonists in the preparation of agents for the treatment or prevention of virus-mediated exacerbations of target respiratory conditions.

[0019] The present invention further provides the use of compounds comprising a TLR2 agonist for the treatment or prevention of virus-mediated exacerbations of target respiratory conditions.

[0020] In any embodiment of the present invention, the respiratory condition is chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, or a lung condition associated with lung transplantation or long-term glucocorticosteroid use. Preferably, the respiratory condition is asthma or COPD.

[0021] In any embodiment of the present invention, the pathological condition may be caused by a rhinovirus. Furthermore, in any embodiment of the present invention, the virus-mediated exacerbation is rhinovirus-mediated. For example, a virus-mediated exacerbation of asthma is caused by a rhinovirus. The rhinovirus may be any serotype as described herein. Typically, the rhinovirus is rhinovirus serotype 1B (RV1B).

[0022] In any embodiment of the present invention, the TLR2 agonist comprises a lipid, peptidoglycan, lipoprotein, or lipopolysaccharide. Preferably, the TLR2 agonist comprises palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl. The TLR2 agonist may be selected from the group consisting of Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys, and Oct2Cys. In a preferred embodiment, the TLR2 agonist comprises Pam2Cys.

[0023] In any embodiment of the present invention, the compound comprises a soluble TLR2 agonist.

[0024] In any embodiment of the present invention, the TLR2 agonist may be conjugated with other compounds or functional groups. These other compounds or functional groups are any of those described herein. Preferred compounds are selected based on their ability to assist in the dissolution of the TLR2 agonist in a carrier, diluent, excipient, or solvent.

[0025] Depending on the polarity of the solvent, the solubility of the TLR2 agonist can be increased by a solubilizer. Therefore, the compound may contain a TLR2 agonist and a solubilizer. Preferably, the TLR2 agonist and the solubilizer are bonded together. The TLR2 agonist may be PEGylated. Preferably, the solubilizer is any molecule described herein.

[0026] The solubilizer contains, is essentially, or may contain a positively charged or negatively charged group. Preferably, the charged group is a branched or linear peptide. Preferably, the positively charged group contains at least one positively charged amino acid, such as an arginine or lysine residue. Preferably, the negatively charged group contains at least one negatively charged amino acid, such as glutamic acid or aspartic acid. The charged amino acid may be terminal, preferably N-terminal.

[0027] Typically, the solubilizer comprises polyethylene glycol (PEG) or R4. In any embodiment of the present invention, the solubilizer comprises polyethylene glycol (PEG) and R4.

[0028] In any embodiment of the present invention, the compound is PEG 11 It contains Pam2Cys conjugated to it. Preferably, Pam2Cys and PEG 11 The molecule is separated by two serine molecules (PEG 11 -SS-Pam2Cys).

[0029] In any embodiment of the present invention, the TLR2 agonist is not Pam3Cys.

[0030] Compounds comprising a TLR2 agonist that can be considered for use in any aspect of the present invention are any of those described herein.

[0031] In any embodiment of the present invention, the TLR2 agonist is administered once daily, once weekly, or twice weekly.

[0032] In any embodiment of the present invention where prevention or remedy is intended or required, the compound is administered to the subject prior to the onset of clinically or biochemically detectable symptoms of a viral infection, preferably a rhinovirus infection.

[0033] In any embodiment of the present invention, the compound is administered in a composition. Typically, the compound further comprises a pharmaceutically acceptable carrier, diluent, or excipient. The composition does not need to contain a compound that is an agonist of a TLR other than a TLR2 homodimer or heterodimer. Preferably, the composition consists essentially of or comprises a compound containing a TLR2 agonist and a pharmaceutically acceptable carrier, diluent, or excipient.

[0034] In any embodiment of the present invention, the compound or composition is administered into the airway. Typically, the compound or composition is administered into the upper and / or lower airway. For example, the compound or composition may be administered to a subject by inhalation or nasally.

[0035] In any embodiment of the present invention, administration of a TLR2 agonist to a subject reduces the subject's viral load. Preferably, the viral load is reduced in the airways, for example, the upper and / or lower airways. Preferably, the viral load is reduced in the lungs.

[0036] In any aspect of the present invention, administration of a TLR2 agonist to a subject reduces the levels of CXCL1 or TNFα.

[0037] In any aspect of the present invention, treatment or prevention of virus-mediated exacerbations of asthma does not significantly induce interferon expression.

[0038] In any embodiment of the present invention, the subject suffers from mild or moderate asthma. The asthma may develop in childhood or in adulthood. The asthma may have any of the characteristics of the condition outlined in Figure 12a.

[0039] In any aspect of the present invention, the compound or composition may be administered together with a corticosteroid. Specifically, any method or use of the present invention further includes administering a corticosteroid. The compound or composition may be administered simultaneously with or sequentially with a corticosteroid. In one embodiment, the compound or composition may be administered once, twice, or more times over a period of 24 hours or 7 days prior to the administration of the corticosteroid.

[0040] In any embodiment of the present invention, the subject to whom the compound or composition is administered may be receiving or may have received corticosteroids.

[0041] In any aspect of the present invention, the corticosteroid may be a glucocorticoid. Preferably, the glucocorticoid is a glucocorticoid receptor agonist, partial agonist, or allosteric modulator. Preferably, the glucocorticoid is an inhalable glucocorticoid. More preferably, the glucocorticoid is budesonide, cyclocenide, mometasone, beclomethasone, betamethasone, dexamethasone, prednisolone, prednisone, or any other glucocorticoid described herein, such as fluticasone propionate.

[0042] In another embodiment, the present invention also provides compositions comprising, essentially composed of, or consisting of compounds comprising a TLR2 agonist and a corticosteroid.

[0043] Preferably, the compound is any of those described herein, more preferably any one of INNA-001 to INNA-015.

[0044] Preferably, the corticosteroid is a glucocorticoid. Preferably, the glucocorticoid is a glucocorticoid receptor agonist, partial agonist, or allosteric modulator. Preferably, the glucocorticoid is an inhalable glucocorticoid. More preferably, the glucocorticoid is budesonide, cyclocenide, mometasone, beclomethasone, betamethasone, dexamethasone, prednisolone, prednisone, or any other glucocorticoid described herein, such as fluticasone propionate.

[0045] In this embodiment, the composition further comprises a pharmaceutically acceptable diluent, carrier, or excipient. Typically, the diluent, carrier, or excipient is suitable for inhalation or nasal delivery.

[0046] In one embodiment, the only active agent in the composition is a compound comprising a TLR2 agonist and a corticosteroid.

[0047] This composition may be formulated or adapted for administration to the airways, such as the upper or lower airways. Preferably, the composition is formulated or adapted for inhalation or nasal administration. In one embodiment, the composition is an inhalation composition and is formulated as a dry powder suitable for use in a dry powder inhalation device. Alternatively, the composition may be formulated as a spray, mist, or aerosol.

[0048] In preferred embodiments, the composition is formulated as a nasal spray or nasal medication.

[0049] In one embodiment, the present invention relates to the structure: AYB (In the formula, A is, [ka] including or consisting of Here, each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; Y is [ka] And, Here, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; and B contains or consists of polyethylene glycol (PEG). The present invention provides compounds containing a pharmaceutically acceptable salt or prodrug thereof.

[0050] The present invention also provides compounds comprising Pam2Cys and PEG, wherein Pam2Cys and PEG are linked by serine, homoserine, threonine, or phosphoserine residues. Pam2Cys in the compound has the following structure: [ka] It has.

[0051] In one embodiment, the present invention relates to polyethylene glycol (PEG) covalently bonded to [ka] (In the formula, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H.) The present invention provides compounds containing a pharmaceutically acceptable salt or prodrug thereof.

[0052] In one embodiment, the present invention relates to formula (I): [ka] (In the formula, n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. The present invention provides compounds or pharmaceutically acceptable salts or prodrugs thereof.

[0053] In one embodiment, the present invention is represented by formula (II): AY-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(II) (In the formula, A is structure: [ka] Having; Y is [ka] And, Here, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] having where R4 is H; and R5 is an amino acid side chain or a second hydrogen) and providing a compound or a pharmaceutically acceptable salt or prodrug thereof.

[0054] In one embodiment, the compound is of formula (III): Pam2Cys-Y-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(III) (wherein Pam2Cys has the structure:

Chemical formula

Chemical formula

Chemical formula

[0055] In one embodiment, the compound is given by formula (IV): Pam2Cys-Ser-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IV) (In the formula, Pam2Cys-Ser has the following structure: [ka] Having; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. or it has a pharmaceutically acceptable salt or prodrug thereof.

[0056] In one embodiment, the compound is given by formula (V): [ka] (In the formula, n is between 3 and 100; k is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; t is 2, 3, or 4; h is 1, 2, 3, or 4; q is either zero or one; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. or it has a pharmaceutically acceptable salt or prodrug thereof.

[0057] In one preferred embodiment, the compound is compound (1): [ka] It has the structure of or a pharmaceutically acceptable salt or prodrug thereof.

[0058] This compound may also be referred to herein as "Pam2Cys-Ser-PEG" or "INNA-006".

[0059] In other preferred embodiments, the compound is [ka] [ka] JPEG2023081969000020.jpg64149 JPEG2023081969000021.jpg68149 and [ka] It is selected from the group consisting of the following.

[0060] In one particularly preferred embodiment, the compound is [ka] That is the case.

[0061] When used herein, unless the context requires a different interpretation, the term "contains" and its variations, such as "contains," "contains," and "contained," are not intended to exclude any further additives, ingredients, complete products, or steps.

[0062] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs are given as examples and will become apparent from the following description with reference to the accompanying drawings. [Brief explanation of the drawing]

[0063] [Figure 1]Treatment with high-dose TLR-2 agonists reduces viral RNA two days after infection. (a) Schematic diagram showing treatment plans with representative TLR-2 agonists PEG-Pam2Cys-R4 or Pam2Cys-R4. (b) Quantification of viral RNA showing that representative TLR-2 agonists PEG-Pam2Cys-R4 and Pam2Cys-R4 reduce viral RNA at indicated doses in RV-infected mice. Lungs were collected two days after infection, total RNA was extracted, and viral RNA was measured by qPCR. Mean + / - SEM ****p<0.0001, **p<0.01, reduced viral RNA compared to saline-treated RV-infected mice, as evaluated by one-way ANOVA. [Figure 2] Potent antiviral effect of high-dose TLR-2 agonist treatment 7 days prior to RV infection in mice. (a) Schematic diagram showing treatment plans with representative TLR-2 agonists, PEG-Pam2Cys-R4 or Pam2Cys-R4. (b) Agonist treatment at all doses resulted in a very significant reduction in viral load compared to saline-treated RV-infected controls in the presence of representative TLR-2 agonists, PEG-Pam2Cys-R4 or Pam2Cys-R4. Viral RNA in the lungs was measured by qPCR 2 days after infection. **** p<0.0001, reduced viral RNA compared to saline-treated RV-infected mice, as evaluated by one-way ANOVA. [Figure 3] Airway cell inflammation expression induced by high-dose TLR-2 agonist treatment 7 days prior to RV infection in mice. (a-b) Analysis of bronchoalveolar lavage fluid (BAL) cells 2 days post-infection revealed that all treatments significantly increased the total number of immune cells, the majority of which were macrophages. An increase in lymphocyte count was observed at lower agonist treatment doses. Inflammatory cells in BAL were counted and populations were identified by staining 2 days post-infection. Mean + / - SEM **p<0.01, ***p<0.001, ****p<0.0001, increased BAL cells in the treatment group compared to saline-treated RV-infected mice. [Figure 4]Treatment with a high-dose TLR-2 agonist 7 days prior to RV infection suppresses the expression of inflammatory cytokines. Inflammatory cytokines in bronchoalveolar lavage fluid (BAL) were measured by ELISA. (a) Compared to saline-treated RV-infected mice, a significantly reduced production of the neutrophil-mobilizing chemokine CXCL1 was observed in all treatment groups. (b) Compared to saline-treated RV-infected mice, reduced TNFα expression was observed in the higher-dose agonist treatment group and in response to 1 nmol of Pam2Cys-R4. Mean + / - SEM *p<0.05, **p<0.01, reduced protein levels compared to saline-treated RV-infected mice, as evaluated by one-way ANOVA. [Figure 5] Low-dose PEG-Pam2Cys-R4 treatment reduces viral load. (a, b) All doses of PEG-Pam2Cys-R4 significantly inhibited RV replication. Furthermore, the indicated dose of Pam2Cys-R4 also caused a significant decrease in viral RNA compared to untreated saline-treated RV-infected controls. Viral RNA in lung tissue 2 days post-infection was evaluated by qPCR. Mean + / - SEM, *p<0.05, **p<0.01, evaluated by one-way ANOVA. [Figure 6A] Increased BAL macrophages and lymphocytes following low-dose TLR-2 agonist treatment. (a, d) Pam2Cys-R4 caused a significant increase in immune cell count after treatment at the indicated dose compared to untreated saline RV infection controls. (b, e) The increase in BAL cells was primarily due to an increase in macrophage count. (c, f) A significant increase in lymphocyte count was also observed at the indicated dose. Cells were stained and counted 2 days after infection. Mean + / - SEM, *p<0.05, **p<0.01, ***p<0.001, evaluated by one-way ANOVA. [Figure 6B]Increased BAL macrophages and lymphocytes following low-dose TLR-2 agonist treatment. (a, d) Pam2Cys-R4 caused a significant increase in immune cell count after treatment at the indicated dose compared to untreated saline RV infection controls. (b, e) The increase in BAL cells was primarily due to an increase in macrophage count. (c, f) A significant increase in lymphocyte count was also observed at the indicated dose. Cells were stained and counted 2 days after infection. Mean + / - SEM, *p<0.05, **p<0.01, ***p<0.001, evaluated by one-way ANOVA. [Figure 6C] Increased BAL macrophages and lymphocytes following low-dose TLR-2 agonist treatment. (a, d) Pam2Cys-R4 caused a significant increase in immune cell count after treatment at the indicated dose compared to untreated saline RV infection controls. (b, e) The increase in BAL cells was primarily due to an increase in macrophage count. (c, f) A significant increase in lymphocyte count was also observed at the indicated dose. Cells were stained and counted 2 days after infection. Mean + / - SEM, *p<0.05, **p<0.01, ***p<0.001, evaluated by one-way ANOVA. [Figure 7A] Low-dose TLR-2 agonist treatment reduces viral neutrophil inflammation. (a-b) A significant decrease in neutrophils, expressed as a percentage of total BAL cells or total neutrophils, was also observed at the indicated dose. Neutrophils were identified by staining and expressed as a percentage of total BAL cells 2 days after infection. (c) When expressed as the absolute number of total BAL cells, a significant decrease in neutrophil count was observed at the indicated dose compared to saline-treated RV-infected mice. Mean + / - SEM, *=p<0.05. [Figure 7B]Low-dose TLR-2 agonist treatment reduces viral neutrophil inflammation. (a-b) A significant decrease in neutrophils, expressed as a percentage of total BAL cells or total neutrophils, was also observed at the indicated dose. Neutrophils were identified by staining and expressed as a percentage of total BAL cells 2 days after infection. (c) When expressed as the absolute number of total BAL cells, a significant decrease in neutrophil count was observed at the indicated dose compared to saline-treated RV-infected mice. Mean + / - SEM, *=p<0.05. [Figure 8A] Low-dose TLR-2 agonist treatment resulted in a very significant reduction in the neutrophil chemokine CXCL1. (a, b) A very significant reduction in CXCL1 expression was observed in response to all indicated doses of Pam2Cys-R4 and PEG-Pam2Cys-R4 compared to untreated saline-treated RV-infected controls. (c, d) The treatment had no effect on TNFα production. Protein mediators in BAL were measured by ELISA two days post-infection. Evaluated by one-way ANOVA, mean + / - SEM, p<0.0001. Multiple comparisons were evaluated by the Holm-Sidak test. [Figure 8B] Low-dose TLR-2 agonist treatment resulted in a very significant reduction in the neutrophil chemokine CXCL1. (a, b) A very significant reduction in CXCL1 expression was observed in response to all indicated doses of Pam2Cys-R4 and PEG-Pam2Cys-R4 compared to untreated saline-treated RV-infected controls. (c, d) The treatment had no effect on TNFα production. Protein mediators in BAL were measured by ELISA two days post-infection. Evaluated by one-way ANOVA, mean + / - SEM, p<0.0001. Multiple comparisons were evaluated by the Holm-Sidak test. [Figure 9A]Comparison of (i) Peg-SS-Pam2Cys, Peg-S-Pam2Cys, and Pam2CysSK4 treatments and (ii) INNA-011 and Peg-S-Pam2Cys (INNA-006) treatments 7 days prior to infection (dose range: 1 pmol to 10 pmol). (a) TLR2 agonist treatment resulted in a very significant reduction in RV1B copy number in the lungs. Viral RNA in lung tissue on day 2 was evaluated by qPCR. (b) BAL leukocytes were not significantly increased by TLR-agonist treatment. Total BAL leukocytes were assessed 2 days after infection by trypan blue dye exclusion on a hematology plate. Mean + / - SEM *p<0.05, **p<0.01, ***p=0.001, ****p=0.0001. When evaluated by one-way ANOVA, the reduced viral RNA was compared to untreated (saline) RV infection control (vRNA copy number), 10 pmol of Peg-SS-Pam2Cys and Peg-S-Pam2Cys (rhinovirus reduction panel), or 2 pmol of INNA-011. (b) BAL leukocytes were not significantly increased by TLR-agonist treatment. Total BAL leukocytes were assessed 2 days after infection by trypan blue dye exclusion on a hematology plate. Mean + / - SEM and one-way ANOVA. (c) From inflammatory cell analysis, evaluated by one-way ANOVA, Peg-S-Pam2Cys and INNA-011 were found to reduce RV-induced BAL neutrophil inflammation. Cells were stained and counted by light microscopy. *p<0.05, **p<0.01, ****p=0.001, significantly different cell counts compared to saline / RV1B. (d~e) Treatment with TLR-agonists reduced BAL CXCL1 but did not alter TNF-α levels. pi Protein mediators in BAL were measured by ELISA on day 2. Mean + / - SEM *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, reduced CXCL1 compared to the saline RV group by one-way ANOVA. [Figure 9B]Comparison of (i) Peg-SS-Pam2Cys, Peg-S-Pam2Cys, and Pam2CysSK4 treatments and (ii) INNA-011 and Peg-S-Pam2Cys (INNA-006) treatments 7 days prior to infection (dose range: 1 pmol to 10 pmol). (a) TLR2 agonist treatment resulted in a very significant reduction in RV1B copy number in the lungs. Viral RNA in lung tissue on day 2 was evaluated by qPCR. (b) BAL leukocytes were not significantly increased by TLR-agonist treatment. Total BAL leukocytes were assessed 2 days after infection by trypan blue dye exclusion on a hematology plate. Mean + / - SEM *p<0.05, **p<0.01, ***p=0.001, ****p=0.0001. When evaluated by one-way ANOVA, the reduced viral RNA was compared to untreated (saline) RV infection control (vRNA copy number), 10 pmol of Peg-SS-Pam2Cys and Peg-S-Pam2Cys (rhinovirus reduction panel), or 2 pmol of INNA-011. (b) BAL leukocytes were not significantly increased by TLR-agonist treatment. Total BAL leukocytes were assessed 2 days after infection by trypan blue dye exclusion on a hematology plate. Mean + / - SEM and one-way ANOVA. (c) From inflammatory cell analysis, evaluated by one-way ANOVA, Peg-S-Pam2Cys and INNA-011 were found to reduce RV-induced BAL neutrophil inflammation. Cells were stained and counted by light microscopy. *p<0.05, **p<0.01, ****p=0.001, significantly different cell counts compared to saline / RV1B. (d~e) Treatment with TLR-agonists reduced BAL CXCL1 but did not alter TNF-α levels. pi Protein mediators in BAL were measured by ELISA on day 2. Mean + / - SEM *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, reduced CXCL1 compared to the saline RV group by one-way ANOVA. [Figure 9C]Comparison of (i) Peg-SS-Pam2Cys, Peg-S-Pam2Cys, and Pam2CysSK4 treatments and (ii) INNA-011 and Peg-S-Pam2Cys (INNA-006) treatments 7 days prior to infection (dose range: 1 pmol to 10 pmol). (a) TLR2 agonist treatment resulted in a very significant reduction in RV1B copy number in the lungs. Viral RNA in lung tissue on day 2 was evaluated by qPCR. (b) BAL leukocytes were not significantly increased by TLR-agonist treatment. Total BAL leukocytes were assessed 2 days after infection by trypan blue dye exclusion on a hematology plate. Mean + / - SEM *p<0.05, **p<0.01, ***p=0.001, ****p=0.0001. When evaluated by one-way ANOVA, the reduced viral RNA was compared to untreated (saline) RV infection control (vRNA copy number), 10 pmol of Peg-SS-Pam2Cys and Peg-S-Pam2Cys (rhinovirus reduction panel), or 2 pmol of INNA-011. (b) BAL leukocytes were not significantly increased by TLR-agonist treatment. Total BAL leukocytes were assessed 2 days after infection by trypan blue dye exclusion on a hematology plate. Mean + / - SEM and one-way ANOVA. (c) From inflammatory cell analysis, evaluated by one-way ANOVA, Peg-S-Pam2Cys and INNA-011 were found to reduce RV-induced BAL neutrophil inflammation. Cells were stained and counted by light microscopy. *p<0.05, **p<0.01, ****p=0.001, significantly different cell counts compared to saline / RV1B. (d~e) Treatment with TLR-agonists reduced BAL CXCL1 but did not alter TNF-α levels. pi Protein mediators in BAL were measured by ELISA on day 2. Mean + / - SEM *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, reduced CXCL1 compared to the saline RV group by one-way ANOVA. [Figure 9D]Comparison of (i) Peg-SS-Pam2Cys, Peg-S-Pam2Cys, and Pam2CysSK4 treatments and (ii) INNA-011 and Peg-S-Pam2Cys (INNA-006) treatments 7 days prior to infection (dose range: 1 pmol to 10 pmol). (a) TLR2 agonist treatment resulted in a very significant reduction in RV1B copy number in the lungs. Viral RNA in lung tissue on day 2 was evaluated by qPCR. (b) BAL leukocytes were not significantly increased by TLR-agonist treatment. Total BAL leukocytes were assessed 2 days after infection by trypan blue dye exclusion on a hematology plate. Mean + / - SEM *p<0.05, **p<0.01, ***p=0.001, ****p=0.0001. When evaluated by one-way ANOVA, the reduced viral RNA was compared to untreated (saline) RV infection control (vRNA copy number), 10 pmol of Peg-SS-Pam2Cys and Peg-S-Pam2Cys (rhinovirus reduction panel), or 2 pmol of INNA-011. (b) BAL leukocytes were not significantly increased by TLR-agonist treatment. Total BAL leukocytes were assessed 2 days after infection by trypan blue dye exclusion on a hematology plate. Mean + / - SEM and one-way ANOVA. (c) From inflammatory cell analysis, evaluated by one-way ANOVA, Peg-S-Pam2Cys and INNA-011 were found to reduce RV-induced BAL neutrophil inflammation. Cells were stained and counted by light microscopy. *p<0.05, **p<0.01, ****p=0.001, significantly different cell counts compared to saline / RV1B. (d~e) Treatment with TLR-agonists reduced BAL CXCL1 but did not alter TNF-α levels. pi Protein mediators in BAL were measured by ELISA on day 2. Mean + / - SEM *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, reduced CXCL1 compared to the saline RV group by one-way ANOVA. [Figure 10A]Drug combination timing interactions and effects on infection. (a-b) In different time point and administration time combinations, TLR2 agonist treatment resulted in a very significant reduction in RV1B copy number in the lungs. Viral RNA in lung tissue was evaluated by qPCR on day 2. Mean + / - SEM *p<0.05, ****p<0.0001; one-way ANOVA, reduced viral RNA compared to untreated (saline) RV1B infected control (unless otherwise stated). (c-d) BAL neutrophils and lymphocytes were significantly increased by TLR2 agonist treatment. Staining of BAL cells 2 days after infection. Mean + / - SEM #p<0.05, ###p<0.001, ####p<0.0001, compared to saline d-7+d-1 / mock. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA showed significantly different cell counts compared to the saline d-7+d-1 / RV1B group (unless otherwise specified). (e~f) BAL leukocytes are significantly increased by TLR2-agonist treatment. Two days after infection, total BAL leukocytes were assessed by trypan blue dye exclusion on a hemocytometer, and BAL macrophages were assessed by differential cell count. Mean + / - SEM, *p<0.05, **p<0.01, ****p<0.0001, one-way ANOVA compared to saline d-7+d-1 / mock. (g~h) TLR2-agonist treatment on day -1 increases BAL CXCL1 in RV-infected mice, but not with pretreatment on day -7. Protein mediators in BAL were measured by ELISA on day 2 of pi. Mean + / - SEM ****p<0.0001. Unless otherwise specified, values ​​were determined by one-way ANOVA compared to the saline RV group. [Figure 10B]Drug combination timing interactions and effects on infection. (a-b) In different time point and administration time combinations, TLR2 agonist treatment resulted in a very significant reduction in RV1B copy number in the lungs. Viral RNA in lung tissue was evaluated by qPCR on day 2. Mean + / - SEM *p<0.05, ****p<0.0001; one-way ANOVA, reduced viral RNA compared to untreated (saline) RV1B infected control (unless otherwise stated). (c-d) BAL neutrophils and lymphocytes were significantly increased by TLR2 agonist treatment. Staining of BAL cells 2 days after infection. Mean + / - SEM #p<0.05, ###p<0.001, ####p<0.0001, compared to saline d-7+d-1 / mock. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA showed significantly different cell counts compared to the saline d-7+d-1 / RV1B group (unless otherwise specified). (e~f) BAL leukocytes are significantly increased by TLR2-agonist treatment. Two days after infection, total BAL leukocytes were assessed by trypan blue dye exclusion on a hemocytometer, and BAL macrophages were assessed by differential cell count. Mean + / - SEM, *p<0.05, **p<0.01, ****p<0.0001, one-way ANOVA compared to saline d-7+d-1 / mock. (g~h) TLR2-agonist treatment on day -1 increases BAL CXCL1 in RV-infected mice, but not with pretreatment on day -7. Protein mediators in BAL were measured by ELISA on day 2 of pi. Mean + / - SEM ****p<0.0001. Unless otherwise specified, values ​​were determined by one-way ANOVA compared to the saline RV group. [Figure 10C]Drug combination timing interactions and effects on infection. (a-b) In different time point and administration time combinations, TLR2 agonist treatment resulted in a very significant reduction in RV1B copy number in the lungs. Viral RNA in lung tissue was evaluated by qPCR on day 2. Mean + / - SEM *p<0.05, ****p<0.0001; one-way ANOVA, reduced viral RNA compared to untreated (saline) RV1B infected control (unless otherwise stated). (c-d) BAL neutrophils and lymphocytes were significantly increased by TLR2 agonist treatment. Staining of BAL cells 2 days after infection. Mean + / - SEM #p<0.05, ###p<0.001, ####p<0.0001, compared to saline d-7+d-1 / mock. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA showed significantly different cell counts compared to the saline d-7+d-1 / RV1B group (unless otherwise specified). (e~f) BAL leukocytes are significantly increased by TLR2-agonist treatment. Two days after infection, total BAL leukocytes were assessed by trypan blue dye exclusion on a hemocytometer, and BAL macrophages were assessed by differential cell count. Mean + / - SEM, *p<0.05, **p<0.01, ****p<0.0001, one-way ANOVA compared to saline d-7+d-1 / mock. (g~h) TLR2-agonist treatment on day -1 increases BAL CXCL1 in RV-infected mice, but not with pretreatment on day -7. Protein mediators in BAL were measured by ELISA on day 2 of pi. Mean + / - SEM ****p<0.0001. Unless otherwise specified, values ​​were determined by one-way ANOVA compared to the saline RV group. [Figure 11A]Treatment during RV infection in the 1G study. (a) Treatment with Peg-SS-Pam2Cys and Peg-S-Pam2Cys during established infection reduces the RV1B copy number in the lungs. Mice were infected intranasally with RV1B, and the following day, Peg-SS-Pam2Cys and Peg-S-Pam2Cys were administered intranasally. Viral RNA in lung tissue was evaluated by qPCR on day 2. Mean + / - SEM *p<0.05, **p<0.01, reduced viral RNA compared to untreated (saline) RV1B infection. (b~e) Treatment with Peg-SS-Pam2Cys and Peg-S-Pam2Cys during active infection (day 1 post-infection) significantly increases the neutrophil count in BAL. Staining of BAL cells 2 days after infection. Results are graphed as mean + / - SEM. ***p<0.001, ****p<0.0001, one-way ANOVA showed significantly different cell counts compared to the saline RV group. (f~g) Treatment with Peg-SS-Pam2Cys and Peg-S-Pam2Cys on post-infection day 1 induced dose-dependent inflammatory cytokine production. pi Protein mediators in BAL were measured by ELISA on day 2. Results are graphed as mean + / - SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA compared to the saline RV group. [Figure 11B]Treatment during RV infection in the 1G study. (a) Treatment with Peg-SS-Pam2Cys and Peg-S-Pam2Cys during established infection reduces the RV1B copy number in the lungs. Mice were infected intranasally with RV1B, and the following day, Peg-SS-Pam2Cys and Peg-S-Pam2Cys were administered intranasally. Viral RNA in lung tissue was evaluated by qPCR on day 2. Mean + / - SEM *p<0.05, **p<0.01, reduced viral RNA compared to untreated (saline) RV1B infection. (b~e) Treatment with Peg-SS-Pam2Cys and Peg-S-Pam2Cys during active infection (day 1 post-infection) significantly increases the neutrophil count in BAL. Staining of BAL cells 2 days after infection. Results are graphed as mean + / - SEM. ***p<0.001, ****p<0.0001, one-way ANOVA showed significantly different cell counts compared to the saline RV group. (f~g) Treatment with Peg-SS-Pam2Cys and Peg-S-Pam2Cys on post-infection day 1 induced dose-dependent inflammatory cytokine production. pi Protein mediators in BAL were measured by ELISA on day 2. Results are graphed as mean + / - SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA compared to the saline RV group. [Figure 12A]TLR-2 agonist treatment reduces the level of rhinovirus replication in asthma epithelial cells. (a) Patient profiles of subjects with either mild persistent or moderate persistent asthma. Asthma epithelial gas-liquid interface (ALI) cultures were prepared from bronchial epithelial cells from these asthma donors and infected with rhinovirus (RV), but treated with Pam2Cys-R4 either (b) 24 hours before RV infection (pretreatment) (in this case, Pam2Cys-R4 significantly reduced the viral load at 96 hours at 0.02 μM) or (c) 2 hours after RV infection (posttreatment) (in this case, Pam2Cys-R4 significantly reduced the viral load at 96 hours at 0.2 μM). Total cellular RNA was purified 48 and 96 hours after infection, and viral RNA levels were measured by qRT-PCR. When evaluated by a paired t-test with mean + / - SEM *=p<0.05, compared to the RV group. [Figure 12B] TLR-2 agonist treatment reduces the level of rhinovirus replication in asthma epithelial cells. (a) Patient profiles of subjects with either mild persistent or moderate persistent asthma. Asthma epithelial gas-liquid interface (ALI) cultures were prepared from bronchial epithelial cells from these asthma donors and infected with rhinovirus (RV), but treated with Pam2Cys-R4 either (b) 24 hours before RV infection (pretreatment) (in this case, Pam2Cys-R4 significantly reduced the viral load at 96 hours at 0.02 μM) or (c) 2 hours after RV infection (posttreatment) (in this case, Pam2Cys-R4 significantly reduced the viral load at 96 hours at 0.2 μM). Total cellular RNA was purified 48 and 96 hours after infection, and viral RNA levels were measured by qRT-PCR. When evaluated by a paired t-test with mean + / - SEM *=p<0.05, compared to the RV group. [Figure 13A]Reduced viral replication is associated with reduced interferon production. Levels of IFNβ and IFNλ1 / 3 proteins in apical medium were measured by ELISA in rhinovirus (RV)-infected (a, b) n=5 (IFNβ) or (c, d) n=6 (IFNλ) asthma epithelial gas-liquid interface (ALI) cultures, which were treated with Pam2Cys-R4 at the indicated concentration either pre-infection (pre-treatment) or post-infection (post-treatment). Data: Mean + / - SEM. All p-values ​​are compared to RV infection alone at the indicated time point. *p<0.05, **p<0.01, evaluated by Friedman test. [Figure 13B] Reduced viral replication is associated with reduced interferon production. Levels of IFNβ and IFNλ1 / 3 proteins in apical medium were measured by ELISA in rhinovirus (RV)-infected (a, b) n=5 (IFNβ) or (c, d) n=6 (IFNλ) asthma epithelial gas-liquid interface (ALI) cultures, which were treated with Pam2Cys-R4 at the indicated concentration either pre-infection (pre-treatment) or post-infection (post-treatment). Data: Mean + / - SEM. All p-values ​​are compared to RV infection alone at the indicated time point. *p<0.05, **p<0.01, evaluated by Friedman test. [Figure 14A] TLR-2 agonists may increase the expression of pro-inflammatory mediators. Levels of (a, b) IP-10 (CXCL10), (c, d) IL-6, (e, f) IL-8, and (g, h) CCL22 proteins, expressed as mean + / - SEM of n=6 asthma epithelial cultures, were measured by ELISA. *p<0.05, **p<0.01, increased mediator expression in Pam2Cys-R4 treated RV-infected cells compared to untreated RV-infected cells, as assessed by Friedman test; #p<0.05, ##p<0.01, increased mediator expression in Pam2Cys-R4 treated cells compared to untreated cells, as assessed by Friedman test. [Figure 14B]TLR-2 agonists may increase the expression of pro-inflammatory mediators. Levels of (a, b) IP-10 (CXCL10), (c, d) IL-6, (e, f) IL-8, and (g, h) CCL22 proteins, expressed as mean + / - SEM of n=6 asthma epithelial cultures, were measured by ELISA. *p<0.05, **p<0.01, increased mediator expression in Pam2Cys-R4 treated RV-infected cells compared to untreated RV-infected cells, as assessed by Friedman test; #p<0.05, ##p<0.01, increased mediator expression in Pam2Cys-R4 treated cells compared to untreated cells, as assessed by Friedman test. [Figure 14C] TLR-2 agonists may increase the expression of pro-inflammatory mediators. Levels of (a, b) IP-10 (CXCL10), (c, d) IL-6, (e, f) IL-8, and (g, h) CCL22 proteins, expressed as mean + / - SEM of n=6 asthma epithelial cultures, were measured by ELISA. *p<0.05, **p<0.01, increased mediator expression in Pam2Cys-R4 treated RV-infected cells compared to untreated RV-infected cells, as assessed by Friedman test; #p<0.05, ##p<0.01, increased mediator expression in Pam2Cys-R4 treated cells compared to untreated cells, as assessed by Friedman test. [Figure 14D] TLR-2 agonists may increase the expression of pro-inflammatory mediators. Levels of (a, b) IP-10 (CXCL10), (c, d) IL-6, (e, f) IL-8, and (g, h) CCL22 proteins, expressed as mean + / - SEM of n=6 asthma epithelial cultures, were measured by ELISA. *p<0.05, **p<0.01, increased mediator expression in Pam2Cys-R4 treated RV-infected cells compared to untreated RV-infected cells, as assessed by Friedman test; #p<0.05, ##p<0.01, increased mediator expression in Pam2Cys-R4 treated cells compared to untreated cells, as assessed by Friedman test. [Figure 15]Antiviral activity of TLR2 agonists and Pam2CSK4 (a-b). BCi-NS1 cells were cultured in ALI and differentiated. Next, cells were pre-treated with 20 nM-0.2 nM concentrations of Pam2Cys-R4 (INNA-001), Peg-SS-Pam2Cys (INNA-003), Peg-S-Pam2Cys (INNA-006), or Pam2CSK4. 24 hours after treatment, cells were infected with moi0.1 RV1B and harvested 96 hours after infection. Total RNA was extracted and reverse transcribed to cDNA using random hexamer primers. Viral load was evaluated by qPCR and expressed as copy number and percentage of viral RNA in the RV (untreated) well. *p<0.05, **p<0.01, decreased viral RNA compared to the RV (untreated) group. n=2-5 repeat wells. [Figure 16] INNA-006 prevented RV-induced and steroid-resistant neutrophilic inflammation. Cells were stained and counted by light microscopy. Mean + / - SEM *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, increased BAL cells compared to saline Veh PBS (single star), saline Veh RV (below double star), or saline FP RV (above double star). One-way ANOVA. [Figure 17] INNA-006 suppresses RV-induced, steroid-resistant neutrophil chemokine production. CXCL1 protein levels in BAL were measured by ELISA on day 2 of pi. ****p<0.0001, increased mediators compared to saline Veh PBS (black star), saline Veh RV (red star), and saline FP RV (blue star). One-way ANOVA. [Figure 18]Viral lung load increased with FP treatment in vehicle control mice, but the antiviral effect of repeated INNA-006 treatment was enhanced by FP. Lungs were collected on day 2 of pi, and after extraction of total RNA, viral RNA was measured by qPCR. Mean + / - SEM *p<0.05, **p<0.01, ****p<0.0001, increased BAL cells compared to saline Veh RV (single or double star) or saline FP RV (straight star). #p<0.05, increased viral load compared to the saline Veh RV group. One-way ANOVA. [Figure 19] Comparison of the ability of various compounds to stimulate luciferase activity in an NF-κB cell-based reporter system. The columns, from left to right, are as follows: INNA-006 (or compound (1)); INNA-013 (or compound (4)); INNA-014 (or compound (3)); INNA-015 (or compound (2)); INNA-010; INNA-011 (or compound (5)); INNA-012 (or compound (6)); and INNA-009. [Figure 20] Comparison of the ability of INNA-006 or Pam3Cys-Ser-PEG3000 to stimulate luciferase activity in an NF-κB cell-based reporter system. [Figure 21] Representative data demonstrating specific TLR-2 activation by INNA-006. [Modes for carrying out the invention]

[0064] It will be understood that the invention disclosed and defined herein extends to any alternative combination of two or more individual features described or evident from the text or drawings. All of these various combinations constitute various alternative embodiments of the invention.

[0065] Specific embodiments of the present invention are described in detail below. While the present invention is described in conjunction with its embodiments, it will be understood that the present invention is not limited to these embodiments. Conversely, the present invention is intended to encompass all alternative forms, modifications, and equivalents, which may fall within the scope of the present invention as defined by the claims.

[0066] Those skilled in the art will recognize many methods and materials similar to or equivalent to those described herein and usable in carrying out the present invention. The present invention is not limited in any way to the methods and materials described herein. It will be understood that the present invention disclosed and defined herein extends to any alternative combination of two or more individual features described or evident from the text or drawings. All of these various combinations constitute various alternative embodiments of the present invention.

[0067] All patents and publications referenced herein are incorporated herein by reference in their entirety.

[0068] For the purposes of understanding this specification, terms used in the singular form also include their plural forms, and vice versa.

[0069] Viral respiratory infections are the most important trigger for respiratory exacerbations, such as asthma exacerbations. Asthma patients are typically susceptible to more serious effects from viruses that cause the common cold, such as rhinovirus (RV). Viral replication in the airway epithelium leads to the production of inflammation-mediated factors, which can trigger an immune cascade that supports asthma exacerbations. We hypothesized that activation of innate epithelial immunity and / or other intracellular signaling mechanisms by administration of an effective dose of a TLR2 agonist would suppress RV replication and the production of associated inflammatory mediators. We first tested this hypothesis in an in vivo model of RV infection by administering several different doses of a TLR2 agonist before treatment with RV. This was evaluated by measuring parameters including weight loss, viral load, and the expression of inflammatory mediators. In this study, we found that administration of a TLR2 agonist did not induce weight loss but reduced pulmonary viral load and mitigated virus-induced inflammation.

[0070] The inventors further verified the above hypothesis in a therapeutic model of ex vivo gas-liquid interface (ALI) cultures from bronchial epithelium of asthma patients. In this model, TLR2 agonist administration was performed either before or after RV infection of the epithelium. The inventors found that stimulation with TLR2 agonists reduced the viral load in bronchial epithelium affected by asthma.

[0071] One advantage of an aspect of the present invention is the surprising finding that treatment with a TLR2 agonist at the time of established RV infection results in inhibition of RV infection. Therefore, the present invention is particularly applicable to subjects diagnosed with respiratory infections and who have a history of clinical diagnosis of respiratory diseases such as asthma and / or a tendency toward respiratory exacerbations. Another advantage of an aspect of the present invention is the unexpected finding that treatment with lower doses of TLR2 agonists was at least as effective as the higher doses of TLR2 agonists tested. Therefore, the present invention is particularly applicable when a low level of activation of the innate immune system is required or desirable. A further advantage of an aspect of the present invention is the unexpected finding that the TLR2 agonist PEG-Pam2Cys-R4 exhibited excellent antiviral and anti-inflammatory effects in a model of RV-mediated infection. Therefore, agonists with similar functional properties may exhibit similar properties in inhibiting RV-mediated infection and thus preventing and / or treating asthma exacerbations. A further advantage of one aspect of the present invention is the unexpected finding that the antiviral response described herein is independent of the IFN-mediated response. This is important because interferon expression is highly variable, especially in more severe asthma conditions, and therapeutic mechanisms that rely on IFN regulation are uncertain, thus posing problems of either no therapeutic effect or induction of excessive inflammation.

[0072] Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) expressed by diverse cell types that play crucial roles in both innate and adaptive immunity. Cells of the innate immune system respond to TLR activation by producing inflammatory cytokines and chemokines that signal about the clearance of pathogens and damaged self. Upon binding to specific ligands, TLR activation leads to the activation of transcription factors such as nuclear factor κB (NF)-κB, and modulates cytokine expression by activating protein-1 (AP-1) and interferon regulators (IRFs) through several adapter molecules, including the myeloid differentiation primary response gene 88 (MyD88), the Toll-interleukin-1 receptor (TIR) ​​domain-containing adapter protein TIRAP, and the TIR domain-containing adapter-induced interferon βTRIF.

[0073] There are several TLRs belonging to this membrane receptor protein family, including TLR1, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.

[0074] As used herein, the term "TLR2" refers to the Toll-like receptor 2 protein. In humans, TLR2 is encoded by the TLR2 gene. TLR2 is expressed on the surface of several cells and plays a fundamental role in pathogen recognition and activation of innate immunity.

[0075] TLR2 agonists are drugs that bind to Toll-like receptor 2. TLR2 agonists can bind to TLR2 as homodimers or heterodimers and activate it.

[0076] In any embodiment of the present invention, the TLR2 agonist comprises a lipid, peptidoglycan, lipoprotein, or lipopolysaccharide. Preferably, the TLR agonist comprises palmitoyl, myristoyl, stearoyl, lyloyl, octanoyl, or decanoyl. The TLR2 agonist may be selected from the group consisting of Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys, and Oct2Cys. In a preferred embodiment, the TLR2 agonist comprises Pam2Cys.

[0077] An exemplary lipopeptide according to any embodiment of the present invention is the lipopeptide "Pam2Cys". Those skilled in the art will understand that the term "lipopeptide" means any composition comprising one or more conjugated lipid moieties and one or more amino acid sequences. "Pam2Cys" (also known as dipalmitoyl-S-glyceryl-cysteine ​​or S-[2,3-bis(palmitoyloxy)propyl]cysteine) has been synthesized and corresponds to the lipid moiety of MALP-2, i.e., a macrophage-activating lipopeptide isolated from Mycoplasma fermentans. Pam2Cys is known to be a ligand for TLR2.

[0078] Pam2Cys has the following structure: [ka] It has.

[0079] As used herein, the symbol "S" in the chemical structure above defines a sulfur atom.

[0080] Another exemplary lipopeptide is the lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteine, also known as Pam3Cys or Pam3Cys-OH, which is a synthetic version of the N-terminal portion of Braun's lipoprotein that spans the inner and outer membranes of Gram-negative bacteria. Pam3Cys has the following structure: [ka] It has.

[0081] U.S. Patent No. 5,700,910 describes several N-acetyl-S-(2-hydroxyalkyl)cysteines for use as intermediates in the preparation of lipoproteins used as synthetic adjuvants, B lymphocyte stimulating factors, macrophage stimulating factors, or synthetic vaccines. U.S. Patent No. 5,700,910 also teaches the use of these compounds as intermediates in the synthesis of Pam3Cys-OH and lipopeptides containing this lipoamino acid or its analogues at the N-terminus.

[0082] Other lipid moieties that can be used to target cell surface TLRs include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl.

[0083] In addition to Pam2Cys and Pam3Cys, the present invention also considers the use of Ste2Cys, Lau2Cys and Oct2Cys according to the present invention. Those skilled in the art will recognize that Ste2Cys is also known as S-[2,3-bis(stearoyloxy)propyl]cysteine ​​or distearoyl-S-glyceryl-cysteine; Lau2Cys is also known as S-[2,3-bis(lauroyloxy)propyl]cysteine ​​or dilauroyl-S-glyceryl-cysteine; and Oct2Cys is also known as S-[2,3-bis(octanoyloxy)propyl]cysteine ​​or dioctanoyl-S-glyceryl-cysteine.

[0084] Other suitable TLR2 agonists include, but are not limited to, synthetic triacylated and diacylated lipopeptides, FSL-1 (synthetic lipoprotein obtained from Mycoplasma salivarium 1), Pam3Cys (tripalmitoyl-S-glycerylcysteine), and S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine, where "Pam3" is "tripalmitoyl-S-glyceryl". Derivatives of Pam3Cys are also suitable TLR2 agonists, and examples of derivatives, though not limited to them, include S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(Lys)4-hydroxytrihydrochloride;Pam3Cys-Ser-Ser-Asn-Ala;Pam3Cys-Ser-(Lys)4;Pam3Cys-Ala-Gly;Pam3Cys-Ser-Gly;Pam3Cys-Ser;Pam3Cys-OMe;Pam3Cys-OH;PamCAG, palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ala-Gly-OH, etc.

[0085] Other non-limiting examples of suitable TLR2 agonists include Pam2CSK4, Pam2CysSK4 (dipalmitoyl-S-glycerylcysteine-serine-(lysine)4; or Pam2Cys-Ser-(Lys)4), which are synthetic diacylated lipopeptides. Other synthetic TLR agonists are described, for example, in Kellner et al. (1992) Biol. Chem. 373:1:51-5; Seifer et al. (1990) Biochem. J, 26:795-802; and Lee et al. (2003) J. Lipid Res., 44:479-486.

[0086] TLR2 agonists can be conjugated with one or more compounds or functional groups. Specific examples of compounds or functional groups are listed below. One form of compound or functional group may act to increase the solubility of the TLR2 agonist. As will be understood by those skilled in the art, TLR2 agonists are typically nonpolar and therefore soluble in nonpolar solvents, but have low solubility only in polar and aqueous solvents. When the use of TLR2 agonists in polar or aqueous solvents is desired, the TLR2 agonist can be conjugated with a solubilizer.

[0087] The solubilizer may comprise one or more solubilizers, which may be conjugated with a TLR2 agonist to improve the solubility of the TLR2 moiety. The solubilizer generally forms a polar moiety that increases the solubility of the TLR2 moiety in polar or aqueous solvents.

[0088] In any embodiment of the present invention, the solubilizer may be a positively charged group. Examples of positively charged groups of the present invention, but not limited to, include penetratin, HIV Tat 48-60, HIV Rev 34-50, transportan, oligoarginine peptides (linear and branched), oligolysine peptides, pyrrochoricin, α-helix amphiphilic model peptides, polylysine, protamine, FL17, Magnafloc 1697, and polycationic compounds described in U.S. Patent Nos. 6,689,478 and 4,035,558.

[0089] In yet another embodiment of the present invention, the solubilizer comprises, is essentially, or consists of a linear or branched peptide. Typically, the linear or branched peptide contains a positively charged or negatively charged amino acid. The positively charged amino acid may be lysine, arginine, histidine, ornithine, or a combination thereof. The linear or branched peptide may contain at least one lysine or arginine residue. Preferably, the charged amino acid is, for example, terminal, e.g., N-terminus. The branched peptide has the following structure: [ka] It may have one of the following.

[0090] In the above structure, X can independently be either a positively charged or negatively charged residue. Preferably, the positively charged amino acid is lysine, arginine, histidine, or ornithine. Preferably, the negatively charged amino acid is glutamic acid or ascorbic acid.

[0091] As used herein, "PEG" refers to the polymer compound polyethylene glycol. Unless otherwise defined, the designation "PEG" includes polymers of any length of ethylene oxide. The designation PEG also includes substituted PEGs.

[0092] Compounds or functional groups that can act as solubilizers may be one or more of the group consisting of "PEG" (or polyethylene glycol) and polar polypeptides, e.g., "R4", i.e., a highly branched tetraarginine complex; "H4", i.e., a highly branched tetrahistidine complex; "H8", i.e., a linear peptide containing a histidine residue; and "E8", i.e., a linear peptide containing a glutamic acid residue. Other linear and branched lipid solubilizers have also been considered, such as highly branched peptides containing glutamic acid residues (see, for example, "branched E8" below). In yet another embodiment of the present invention, the solubilizer comprises PEG and one or more of the group consisting of R4, H4, H8 and E8 (linear or branched). R4, H4, H8 and E8 are described in PCT / Australian Patent Application Publication No. 2009 / 000469 (International Publication No. 2010 / 115230 brochure) and have the following structures: [ka] [ka] TIFF2023081969000029.tif46149 TIFF2023081969000030.tif109149 [ka] It has.

[0093] The following are schematic diagrams of several examples of branched (structures 1-5) and linear (structures 6-8) immunogenic compositions containing positively charged (arginine, R; lysine, K) or negatively charged (aspartic acid, D; glutamic acid, E) amino acids at their terminal positions, so that their respective charges are presented to the environment. Each immunogenic composition also contains dipalmitoyl-S-glycerylcysteine ​​(Pam2Cys), which is a ligand for Toll-like receptor 2. Two serine residues (Ser) are also incorporated. In the case of construct 2, the peptide structure is assembled in the direction N→C, while all other structures shown in the figure are assembled in the direction C→N. Positive and negative charges are indicated as 2-, 2+, 1-, and 1+ depending on the magnitude of the charge. Ac = In the case of glutamic acid located at the N terminus, this is an acetyl group used to suppress the positive charge of the α-amino acid group. [ka]

[0094] Those skilled in the art will understand that the present invention is not limited to specific exemplary compounds or functional groups that can act as solubilizers, and that other suitable compounds or functional groups, such as carbohydrates, including those known in the art that can act as solubilizers, can be used in accordance with the present invention.

[0095] Methods for conjugating one or more compounds or functional groups (such as solubilizers) to the lipids of the present invention will be well known to those skilled in the art. For example, conjugation via Fmoc chemistry, disulfide or thioether crosslinking, or oxime chemistry can be considered. In a specific embodiment of the present invention, the soluble form of Pam2Cys was prepared by adding O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethylene glycol (Fmoc-PEOn-OH, Merck Ltd) to Pam2Cys. This resulted in Pam2Cys-PEG, the PEGylated form of the lipid. 11 A structure is formed, which is suitable for administration to the target.

[0096] In another embodiment of the present invention, the TLR2 portion includes a conjugate containing a Pam2Cys conjugated to the pendant R4 form. In a preferred embodiment, the pendant Pam2Cys has the following structure: [ka] It is conjugate to R4.

[0097] In a preferred embodiment of any embodiment of the present invention, the TLR2 portion includes a conjugate containing Pam2Cys conjugated to PEG. In a preferred embodiment of any embodiment of the present invention, the TLR2 portion includes PEG 11 or PEG 12 Includes a conjugate containing Pam2Cys conjugated to PEG. Preferably, Pam2Cys and PEG. 11 or PEG 12 The molecule is separated by at least two serine molecules (PEG 11 -SS-Pam2Cys or PEG 12 -SS-Pam2Cys).

[0098] As used herein, the term TLR2 agonist includes its pharmaceutically acceptable salts, solvated compounds, polymorphs, or prodrugs.

[0099] Another compound containing a TLR2 agonist useful in any embodiment of the present invention is described below.

[0100] In any embodiment of the present invention, a compound comprising a TLR2 agonist has the following structure: AYB (In the formula, A is, [ka] including or consisting of Here, each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; Y is [ka] And, Here, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; and B contains or consists of polyethylene glycol (PEG). or including a pharmaceutically acceptable salt or prodrug thereof.

[0101] In any embodiment of the present invention, the compound comprising a TLR2 agonist comprises Pam2Cys and PEG, wherein Pam2Cys and PEG are linked by serine, homoserine, threonine, or phosphoserine residues. Pam2Cys in the compound has the following structure: [ka] It has.

[0102] In one embodiment, the present invention relates to polyethylene glycol (PEG) covalently bonded to [ka] (In the formula, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H.) The present invention provides compounds containing a pharmaceutically acceptable salt or prodrug thereof.

[0103] In any embodiment of the present invention, a compound comprising a TLR2 agonist is defined by formula (I): [ka] (In the formula, n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. It is a compound of the same or a pharmaceutically acceptable salt or prodrug thereof.

[0104] In any embodiment of the present invention, a compound comprising a TLR2 agonist is defined by formula (II): AY-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(II) (In the formula, A is structure: [ka] Having; Y is [ka] And, Here, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. It is a compound of the same or a pharmaceutically acceptable salt or prodrug thereof.

[0105] In any embodiment of the present invention, a compound comprising a TLR2 agonist is defined by formula (III): Pam2Cys-Y-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(III) (In the formula, Pam2Cys has the following structure: [ka] Having; Y is [ka] And, Here, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; When q=1, R3 is H, -NH2, or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. It is a compound of the same or a pharmaceutically acceptable salt or prodrug thereof.

[0106] In any embodiment of the present invention, a compound comprising a TLR2 agonist is defined by formula (IV): Pam2Cys-Ser-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IV) (In the formula, Pam2Cys-Ser has the following structure: [ka] Having; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. It is a compound of the same or a pharmaceutically acceptable salt or prodrug thereof.

[0107] In one embodiment, the compound is given by formula (V): [ka] (In the formula, n is between 3 and 100; k is between 3 and 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17 or 18; p is 2, 3 or 4; t is 2, 3 or 4; h is 1, 2, 3 or 4; q is zero or 1; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 are not both H; when q = 1, R3 is -NH2 or -OH; when q = 0, R3 is H; L is zero or consists of 1 to 10 units, each unit is a natural α - amino acid or is derived from a natural α - amino acid and has the formula:

Chemical formula

[0108] In one preferred embodiment, the compound is the compound (1):

Chemical formula

[0109] This compound may also be referred to herein as "Pam2Cys - Ser - PEG" or "INNA - 006".

[0110] In other preferred embodiments, the compound is

Chemical formula

Chem.

Chem.

[0111] In one particularly preferred embodiment, the compound is

Chem.

[0112] In any aspect of the present invention, the compound comprising a TLR2 agonist is of formula (Ia):

Chem.

[0113] In any embodiment of the present invention, a compound comprising a TLR2 agonist is defined by formula (IIa): AY-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IIa) (In the formula, A is structure: [ka] Having; Y is [ka] And, Here, R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, and R2 and R2' cannot both be H; n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, In the formula, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. It is a compound of the same or a pharmaceutically acceptable salt or prodrug thereof.

[0114] In any embodiment of the present invention, a compound comprising a TLR2 agonist is defined by formula (IIIa): Pam2Cys-Y-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IIIa) (In the formula, Pam2Cys has the following structure: [ka] Having; Y is [ka] And, Here, R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, and R2 and R2' cannot both be H; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. It is a compound of the same or a pharmaceutically acceptable salt or prodrug thereof.

[0115] In any embodiment of the present invention, a compound comprising a TLR2 agonist is defined by formula (IVa): Pam2Cys-Ser-Ser-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IVa) (In the formula, Pam2Cys has the following structure: [ka] Having; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, nor can R2 and R2' both be H; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. It is a compound of the same or a pharmaceutically acceptable salt or prodrug thereof.

[0116] In any embodiment of the present invention, a compound comprising a TLR2 agonist is defined by formula (Va): [ka] (In the formula, n is between 3 and 100; k is between 3 and 100; h is 1, 2, 3, or 4; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; t is 2, 3, or 4; q is either zero or one; R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, nor can R2 and R2' both be H; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of an amino acid. It is a compound of the same or a pharmaceutically acceptable salt or prodrug thereof.

[0117] In one embodiment, the compound has the structure: [ka] It has.

[0118] In a particularly preferred embodiment of the present invention, the compound is compound (1a): [ka] It has the structure of or a pharmaceutically acceptable salt or prodrug thereof.

[0119] In other preferred embodiments, the compound is [ka] [ka] JPEG2023081969000073.jpg63149 JPEG2023081969000074.jpg63149 [ka] and [ka] It is selected from the group consisting of the following.

[0120] Furthermore, the compounds of the present invention also include pharmaceutically acceptable salts or prodrugs of the above compounds (1) to (6) or (1a) to (6a).

[0121] For all of the above structures, if present, one or more of the following features are preferable: n is between 10 and 14, and more preferably, n is 11. n is either 3 or 5. n is between 24 and 30, and more preferably, n is 27. k is between 24 and 30, and more preferably, k is 27. m is 1 to 3, and more preferably, m is 2. h is between 1 and 3, and more preferably, h is 2. g is 10 to 16, more preferably 12 to 14, and most preferably 14. One of R1 and R2 is hydrogen. p is 2. t is 2.

[0122] The term "pharmaceutically acceptable" can be used to describe any pharmaceutically acceptable salt, hydrate, or prodrug or any other compound that, when administered to a subject, can (directly or indirectly) provide the compounds of the present invention described herein or their pharmaceutically acceptable salts, prodrugs, or esters or their active metabolites or residues.

[0123] Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid, or salts of pharmaceutically acceptable organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, malic acid, citric acid, lactic acid, mucoic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0124] Examples of base salts, though not limited to them, include those formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, zinc, and ammonium; alkylammonium salts such as those formed from triethylamine; alkoxyammonium salts such as those formed with ethanolamine; and salts formed from amino acids such as ethylenediamine, choline or arginine, lysine, or histidine. General information regarding the types of pharmaceutically acceptable salts and their formation is well known to those skilled in the art and is described in general texts such as "Handbook of Pharmaceutical Salts" PHStahl, CGWermuth, 1st edition, 2002, Wiley-VCH.

[0125] In the case of solid compounds, it will be understood by those skilled in the art that the compounds, agents, and salts of the present invention may exist in different crystalline forms or polymorphs, all of which are intended to be included within the scope of the present invention and its explicitly stated formulas.

[0126] The term "polymorph" includes all crystalline forms of the compounds of the present invention as described herein, including anhydrous form, hydrated form, solvated compound form, and mixed solvated compound form.

[0127] The compounds of the present invention described herein are intended to encompass solvated and non-solvated forms of the compounds, where applicable. Accordingly, the compounds of the present invention described herein encompass compounds having the indicated structure, including hydrated or solvated forms, as well as non-hydrated and non-solvated forms.

[0128] As used herein, the term “solvated compound” refers to a variable stoichiometric complex formed by a solute (in this invention, a compound of the present invention as described herein or a pharmaceutically acceptable salt, prodrug, or ester thereof) and a solvent. Such solvents for the purposes of this invention should not interfere with the biological activity of the solute. Examples of preferred solvents, but not limited to, include water, methanol, ethanol, and acetic acid. Preferably, the solvent used is a pharmaceutically acceptable solvent. Examples of preferred pharmaceutically acceptable solvents, but not limited to, include water, ethanol, and acetic acid. The most preferred solvent is water.

[0129] Basic nitrogen-containing groups can be quaternized with substances such as chloride, bromide, and halogenated lower alkyls like methyl, ethyl, propyl, and butyl; and dialkyl sulfates like dimethyl sulfate and diethyl sulfate.

[0130] The compounds described herein include isotopic modifications such as the substitution of deuterium with hydrogen.

[0131] The compounds of the present invention may exist in an optically active and racemic form and may be isolated in that form. As will be understood by those skilled in the art, the present invention is intended to encompass all racemic, optically active, or stereoisomeric forms or mixtures thereof of the compounds of formulas (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa) and / or (Va) having the useful properties described herein. Methods for preparing such forms (e.g., by recrystallization of racemic mixtures, synthesis from optically active starting materials, chiral synthesis, or chiral chromatographic separation) are known in the art. In one preferred embodiment, the following* With respect to the carbon shown, the compounds of the present invention are provided in a racemic mixture. In another preferred embodiment, the L-stereoconfiguration or a natural amino acid: [ka] The present invention provides compounds to which are conferred in excess or solely.

[0132] A “prodrug” is a compound that may not fully meet the structural requirements of the compounds provided herein, but which, after administration to a subject or patient, is modified in vivo to produce the compounds of the present invention as described herein. For example, a prodrug may be an acylated derivative of a compound described herein. Prodrugs include compounds in which a hydroxy, carboxy, amine, or sulfhydryl group attached to any group is cleaved upon administration to a mammalian subject to form a free hydroxy, carboxy, amino, or sulfhydryl group, respectively. Examples of prodrugs, but not limited to, include acetic acid, formic acid, phosphoric acid, and benzoic acid derivatives of the alcohol and amine functional groups in the compounds provided herein. Prodrugs of the compounds provided herein can be prepared by modifying the functional groups present in the compound such that the modifier is cleaved in vivo to produce the parent compound.

[0133] Prodrugs include compounds in which a polypeptide chain of amino acid residues or two or more (e.g., 2, 3, or 4) amino acid residues is covalently bonded to the free amino and amide groups of compounds of formulas (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa), and / or (Va). The amino acid residues include 20 naturally occurring amino acids (generally referred to by three-letter abbreviations), and also include 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norbulin, β-alanine, γ-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine, and methionine sulfone. Prodrugs also include compounds in which carbonates, carbamates, amides, and alkyl esters are covalently bonded to the aforementioned substituents of formulas (I), (II), (III), (IV), (V), (Ia), (IIa), (IIIa), (IVa) and / or (Va) or other structures depicted herein.

[0134] The term "respiratory" refers to the process by which oxygen is taken into the body and carbon dioxide is expelled through the bodily system, including the nose, throat, larynx, trachea, bronchi, and lungs.

[0135] As used herein, the airway includes the upper and lower airways. Typically, the upper airway includes the nose and nasal cavity, sinuses, pharynx, and laryngeal portion above the vocal cords. Typically, the lower airway includes the laryngeal portion below the vocal cords, trachea, bronchi, and bronchioles. The lungs may be part of the lower airway or be separate entities, and include respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0136] The term “respiratory disease” or “respiratory condition” refers to any one of several diseases that involve inflammation and affect the components of the respiratory system, including the upper respiratory tract (including the nasal cavity, pharynx, and larynx) and the lower respiratory tract (including the trachea, bronchi, and lungs). Preferably, respiratory diseases are obstructive airway diseases, and such diseases include asthmatic conditions including hay fever, allergen-induced asthma, exercise-induced asthma, pollution-induced asthma, cold-induced asthma, stress-induced asthma and virus-induced asthma; chronic obstructive pulmonary diseases including chronic bronchitis with normal airflow, chronic bronchitis with airway obstruction (chronic obstructive bronchitis), emphysema, asthmatic bronchitis and bullous diseases; and other inflammatory lung diseases including cystic fibrosis, pigeon lover's disease, farmer's lung, acute respiratory distress syndrome, pneumonia, aspiration or inhalation injury, pulmonary fat embolism, pulmonary acidosis inflammation, acute pulmonary edema, acute altitude sickness, post-cardiac surgery, acute pulmonary hypertension, persistent pulmonary hypertension of the newborn, hyalineosis, acute pulmonary thromboembolism, sepsis, persistent asthma and hypoxia. Inflammation of the upper and lower respiratory tracts may be associated with or caused by viral infections or allergens. The anti-inflammatory activity of the compound is expected to be particularly suitable for the treatment of the aforementioned diseases or conditions, either alone or in combination with glucocorticoids.

[0137] Symptoms of respiratory disease may include cough, excessive sputum production, a feeling of shortness of breath with audible wheezing, or chest tightness. Exercise capacity will likely be significantly limited. In asthma, FEV1.0 (forced expiratory volume in one second), as a percentage of what is nomographically predicted based on weight, height, and age, will decrease, as will the maximum expiratory flow rate during forced exhalation. In COPD, FEV1.0 as a ratio of FVC typically decreases to less than 0.7. The impact of each of these conditions can also be measured by lost work / school days, sleep disturbances, need for bronchodilators, and need for glucocorticoids, including oral glucocorticoids.

[0138] The presence, improvement, treatment, or prevention of respiratory disease can be determined by any clinically or biochemically relevant method of the subject or its biopsy. For example, parameters measured may include lung function, the presence or degree of signs and symptoms of obstruction; exercise tolerance; nocturnal awakenings; days lost from school or work; use of bronchodilators; ICS dosage; use of oral gastrointestinal glands; need for other medications; need for medical treatment; and hospitalization.

[0139] As used herein, the term respiratory infection means an infection of any part of the respiratory tract. Examples of respiratory infections include, but are not limited to, the common cold, sinusitis, pharyngeal infection, tonsillitis, laryngitis, bronchitis, pneumonia, or bronchiolitis. Preferably, in any embodiment of the present invention, the respiratory infection is the common cold. Viral testing can identify an individual as having a respiratory infection, in which case the individual may present with symptoms of itchy watery eyes, runny nose, nasal congestion, sneezing, sore throat, cough, headache, fever, discomfort, fatigue, and weakness. In one embodiment, an individual with a respiratory infection may have no other respiratory conditions whatsoever. Detection of the presence or amount of a virus, preferably rhinovirus, can be performed by PCR / sequencing or serology of RNA isolated from clinical samples (nasal lavage fluid, sputum, BAL).

[0140] Rhinovirus-related respiratory conditions can be conditions caused by rhinovirus. Preferably, these conditions are associated with or result from rhinovirus infection. Rhinovirus infection can be determined by the presence of rhinovirus in a sample taken from the respiratory tract of the subject. Individuals with RV infection can be identified by serological viral testing or PCR / sequencing of RNA isolated from clinical samples (nasal lavage, sputum, BAL). Symptoms of RV infection include, but are not limited to, sore throat, runny nose, nasal congestion, sneezing, and cough; sometimes accompanied by muscle pain, fatigue, discomfort, headache, muscle weakness, or loss of appetite.

[0141] In any embodiment of the present invention, the respiratory infection is caused by a rhinovirus (RV). As used herein, the term RV refers to a piconavirus comprising a genomic virus-coding protein in the 5' region and any single-stranded positive-sense RNA having a 3' poly-A tail. It will be understood that the viral particle itself is not encapsulated and has an icosahedral structure. It will also be understood that human rhinovirus is composed of a capsid containing four viral proteins VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 form the majority of the protein capsid. Examples of human rhinoviruses include: HRV-A1, HRV-A2, HRV-A7, HRV-A8, HRV-A9, HRV-A10, HRV-A11, HRV-A12, HRV-A13, HRV-A15, HRV-A16, HRV-A18, HRV-A19, HRV-A20, HRV-A21, HRV-A22, HRV-A23, HRV-A 24, HRV-A25, HRV-A28, HRV-A29, HRV-A30, HRV-A31, HRV-A32, HRV-A33, HRV-A34, HRV-A36, HRV-A38, HR V-A39, HRV-A40, HRV-A41, HRV-A43, HRV-A44, HRV-A45, HRV-A46, HRV-A47, HRV-A49, HRV-A50, HRV-A51 , HRV-A53, HRV-A54, HRV-A55, HRV-A56, HRV-A57, HRV-A58, HRV-A59, HRV-A60, HRV-A61, HRV-A62, HRV- A63, HRV-A64, HRV-A65, HRV-A66, HRV-A67, HRV-A68, HRV-A71, HRV-A73, HRV-A74, HRV-A75, HRV-A76, H RV-A77, HRV-A78, HRV-A80, HRV-A81, HRV-A82, HRV-A85, HRV-A88, HRV-A89, HRV-A90, HRV-A94, HRV-A95, HRV-A96, HRV-A98, HRV-A100, HRV-A101, HRV-A102, and HRV-A103 (collectively known as rhinovirus A viruses);HRV-B3, HRV-B4, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-B26, HRV-B27, HRV-B35, HRV-B37, HRV-B4 2, HRV-B48, HRV-B52, HRV-B69, HRV-B70, HRV-B72, HRV-B79, HRV-B83, HRV-B84, HRV-B86, HRV-B91, H RV-B92, HRV-B93, HRV-B97 and HRV-B99 (collectively known as rhinovirus B viruses); and HRV-C1, HRV-C2, HRV-C3, HRV-C4, HRV-C5, HRV-C6, HRV-C7, HRV-C8, HRV-C9, HRV-C10, HRV-C11, HRV-C12, HRV-C13, HRV-C 14, HRV-C15, HRV-C16, HRV-C17, HRV-C18, HRV-C19, HRV-C20, HRV-C21, HRV-C22, HRV-C23, HRV-C24 , HRV-C25, HRV-C26, HRV-C27, HRV-C28, HRV-C29, HRV-C30, HRV-C31, HRV-C32, HRV-C33, HRV-C34, HR V-C35, HRV-C36, HRV-C37, HRV-C38, HRV-C39, HRV-C40, HRV-C41, HRV-C42, HRV-C43, HRV-C44, HRV-C45, HRV-C46, HRV-C47, HRV-C48, HRV-C49, HRV-C50, and HRV-C51 (collectively known as rhinovirus C viruses).

[0142] In any aspect of the present invention, administration of a TLR2 agonist may enhance the innate immune response.

[0143] In the case of asthma, human rhinovirus is associated with the majority of asthma exacerbations for which current therapies are ineffective. Accordingly, in any embodiment of the present invention, a method for treating or preventing a virus-mediated exacerbation of asthma is provided, comprising administering a TLR2 agonist to the target. Preferably, the virus-mediated exacerbation is caused by a rhinovirus infection.

[0144] As used herein, the term “asthma” refers to a respiratory disorder characterized by incidental dyspnea caused by any one or a combination of three main factors, including 1) bronchospasm (i.e., indefinite and reversible airway obstruction due to airway muscle contraction), 2) inflammation of the airway lining, and 3) bronchial hyperresponsiveness causing excessive mucus production in the airways, which can be triggered by exposure to allergens or combinations of allergens (i.e., dust mites and mold), viral or bacterial infection (i.e., the common cold virus), environmental pollutants (i.e., chemical fumes or smoke), excessive physical exertion (i.e., during exercise), stress, or inhalation of cold air. Individuals may be characterized by, for example, allergen-induced asthma, exercise-induced asthma, pollution-induced asthma, virus-induced asthma, or cold-induced asthma. Asthma will be understood to cause periodic wheezing (a sound produced during breathing), chest tightness, shortness of breath, and coughing.

[0145] As used herein, the term asthma exacerbation refers to an acute or subacute episode of progressively worsening shortness of breath, cough, wheezing, and chest tightness, or a combination thereof, which may also be accompanied by a decrease in expiratory flow. The intensity of the exacerbation is variable. Symptoms may be mild and undetectable to the patient, or they may be very severe and life-threatening. In any embodiment of the present invention, the asthma exacerbation is preferably caused by a rhinovirus infection.

[0146] Individuals with asthma exacerbations can be identified by the degree of airway obstruction, determined by FEV1 or PEF and its impact on gas exchange. FEV1 and PEF are measurements used to assess expiratory flow rate. Depending on the obtained values, an exacerbation is considered mild if the FEV1 or PEF value is equivalent to or greater than 70% of its theoretical value or previous personal best; moderate if the FEV1 or PEF value is between 70% and 50%; and severe if these values ​​are less than 50%. A satisfactory functional response to treatment is considered to be when the FEV1 or PEF value exceeds 45% of the prior measurement and the PEF increases by at least 50 l / min 30 minutes after the start of treatment. The airway obstruction response to initial treatment is an important prognostic factor for assessing aggression. Table 1 (J Investig Allergol Clin Immunol Vol.20, Suppl.1:27-31 (2010)) outlines the diagnostic indicators used to determine whether a person has mild or moderate to severe asthma exacerbations.

[0147] [Table 1]

[0148] In many cases, asthma exacerbations caused by rhinovirus infection can lead to chronic obstructive pulmonary disease (COPD). Therefore, human rhinovirus is associated with COPD, and current therapies for it are inadequate. Accordingly, in any embodiment of the present invention, a method for treating or preventing virus-mediated COPD is provided, comprising administering a TLR2 agonist to the target. Preferably, the virus-mediated COPD is caused by rhinovirus. Preferably, the method is for treating or preventing virus-mediated exacerbations of COPD.

[0149] The terms “chronic obstructive pulmonary disease” and “COPD,” as used interchangeably herein, refer to a chronic disorder or combination of disorders characterized by reduced maximum expiratory flow and slow, forced exhalation, which have not shown significant change over several months and are irreversible or only slightly reversible with conventional bronchodilators. Most commonly, COPD is a combination of chronic bronchitis, namely the presence of cough and sputum for more than three months for approximately two consecutive years, and emphysema, i.e., alveolar damage. However, COPD may include chronic bronchitis with normal airflow, chronic bronchitis with airway obstruction (chronic obstructive bronchitis), emphysema, asthmatic bronchitis and bullous diseases, and combinations thereof. Chronic obstructive pulmonary disease is a condition caused by chronic lung damage, which is usually, but not exclusively, induced by exposure to tobacco smoke. Other non-toxic airborne pollutants, such as indoor cooking exhaust and vehicle exhaust, may also cause or increase the risk of COPD in the long term. Thus, it can be understood that COPD is interchangeable with terms such as "chronic bronchitis" and "emphysema."

[0150] The symptoms of COPD are progressively worsening and include persistent exertional dyspnea, eventually leading to resting dyspnea. The most common symptoms of COPD are shortness of breath (or "need for air"), chronic cough, and sputum (mucus) production. Even everyday activities, such as walking up stairs, and even routine activities, can become extremely difficult as the condition gradually worsens. Patients often experience exacerbations, which are severe episodes of increased shortness of breath, cough, and sputum production that can last from several days to several weeks. These episodes can be so severe that they require emergency medical attention (including hospitalization) and, in some cases, can be fatal.

[0151] Chronic obstructive pulmonary disease (COPD) is usually suspected in people who experience the symptoms described above and can be confirmed by a respiratory test called a spirometry test, which measures how much air a person can forcefully exhale quickly and efficiently.

[0152] Respiratory viruses can also exacerbate cystic fibrosis. For example, viral infection in a subject diagnosed with cystic fibrosis may increase susceptibility to bacterial infection. Accordingly, in any embodiment of the present invention, a method for treating or preventing a virus-mediated exacerbation of cystic fibrosis is provided, comprising administering a TLR2 agonist to the subject. Preferably, the virus-mediated exacerbation is caused by a rhinovirus infection.

[0153] Cystic fibrosis is understood to be a genetic disorder that affects the respiratory, digestive, and reproductive systems, involving the production of an abnormally thick mucous lining in the lungs, and can lead to fatal lung infections. Individuals with cystic fibrosis may present with a variety of symptoms, including very salty skin; a persistent cough, possibly accompanied by sputum, wheezing, or shortness of breath; excessive appetite, but also poor weight gain and large amounts of fatty stools. It is also understood that sweat testing is a standard diagnostic test for cystic fibrosis. This method measures the amount of salt in sweat. High salt levels indicate cystic fibrosis.

[0154] Respiratory viruses can also exacerbate diseases in graft recipient patients. For example, viral infection in lung graft recipients can increase susceptibility to pneumonia, acute rejection, and chronic allograft failure. Accordingly, in any embodiment of the present invention, a method for treating or preventing viral infection in lung graft recipients is provided, comprising administering a TLR2 agonist to the target. Preferably, the viral infection is a rhinovirus infection.

[0155] The present invention also applies to the restoration of antiviral immunity associated with long-term glucocorticosteroid use. Glucocorticoids are understood to be agents that have cortisol-like agonist activity on glucocorticoid receptors, resulting in diverse endocrine and anti-inflammatory effects. The majority of patients with severe asthma and COPD take steroids or glucocorticoids. Steroid use increases during viral exacerbations, which can prolong viral infections and increase susceptibility to secondary bacterial infections.

[0156] Accordingly, in any embodiment of the present invention, a method is provided for treating or preventing a viral infection in a subject to be administered a glucocorticosteroid, comprising administering a TLR2 agonist to the subject. Preferably, the viral infection is a rhinovirus infection. Preferably, the glucocorticosteroid administration is long-term.

[0157] As used herein, “prevent” or “prevention” is intended to mean at least a reduction in the likelihood (or susceptibility) to acquiring a disease or disability (i.e., preventing at least one clinical symptom of the disease from developing in patients who are potentially exposed to or susceptible to the disease but have not experienced or presented symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are well known to physicians. For example, prevention of a viral-induced respiratory infection or a viral-induced exacerbation of asthma may be characterized by a reduction or absence of viral load or suppression of an increase in inflammatory cell mediators or cytokines. In some embodiments, administration of a compound can minimize the occurrence of infection and thus minimize the viral load. Preferably, this reduces the viral load.

[0158] In any preventive or preventive embodiment of the present invention, the subject may not exhibit any detectable symptoms of viral infection, particularly rhinovirus infection, at the time of administration of the compound.

[0159] The terms “treatment” or “to treat” include the application or administration of the compounds of the present invention to a subject (or the application or administration of the compounds of the present invention from a subject to cells or tissues) with the aim of delaying, slowing, stabilizing, curing, restoring, alleviating, reducing, altering, treating, suppressing, mitigating, improving, or acting upon a disease or condition, the symptoms of a disease or condition, or the risk of a disease or condition (or susceptibility to such risk). The term “to treat” means any measure of success in treating or improving an injury, disease, or condition, and includes any objective or subjective parameters such as: mitigation; remission; slower rate of exacerbation; reduced severity of the disease; stabilization, reduction or decrease of symptoms or making the injury, disease, or condition more tolerable to the subject; slower rate of degeneration or debilitation; mitigation of debilitation at the end of degeneration; or improvement of the subject’s physical or mental health.

[0160] The presence, improvement, treatment, or prevention of respiratory infections or exacerbations (e.g., asthma exacerbations) can be determined by clinically or biochemically relevant methods described herein or known to those skilled in the art. Relevant methods may include the measurement of viral load, interferon expression, or inflammatory cell count using bronchoalveolar lavage (BAL), in which a bronchoscope is passed through the mouth or nose to the lungs, the fluid is sprayed onto a small portion of the lung, and then collected for examination. Improvement, treatment, or prevention can also be determined directly from a sample or biopsy of or from the subject. The sample or biopsy may be from the upper or lower respiratory tract. Furthermore, in cases of respiratory infections or asthma exacerbations, a positive response to treatment can also be determined by measuring chemokine and cytokine levels using known assays such as ELISA, as described herein.

[0161] Furthermore, in cases such as respiratory infections or asthma exacerbations, a positive response to treatment is the prevention of further deterioration of lung function as measured by vital capacity measurement, trunk plethysmography, and pulmonary diffusion volume. Particularly with regard to asthma exacerbations, a positive response to treatment is improvement from the initially diagnosed severity (as outlined in Table 1). For example, a subject diagnosed with a moderate exacerbation (FEV1 or PEF measurement of 70%–50%) would show a positive response to treatment if the FEV1 or PEF value exceeds 45% of the prior measurement and the PEF increases by at least 50 l / min 30 minutes after the start of treatment.

[0162] A positive response to treatment may include prevention or reduction of exacerbation of respiratory symptoms following a viral infection of the respiratory tract, such as asthma symptoms. This can be assessed by comparing the mean change in disease scores from baseline to the end of the study period, based on the Juniper Asthma Control Questionnaire (ACQ-6), or by assessing lower respiratory tract symptom scores (LRSS - symptoms of chest compression, wheezing, shortness of breath, and cough) daily after the onset of infection / cold symptoms. Changes from baseline lung function (maximum expiratory flow rate, PEF) can also be assessed, and a positive response to treatment may be a significant reduction in PEF decline. For example, the placebo group may show a significant morning PEF decline of 15% at the peak of exacerbation, while the treatment group may show a non-significant PEF decline of less than 15% from baseline.

[0163] The present invention also provides a method for improving or maintaining a subject's ability to control a respiratory disease during a respiratory viral infection, the method comprising administering a compound comprising a TLR2 agonist to the subject to improve the subject's ability to control the respiratory disease, i.e., a respiratory viral infection. Preferably, the infection is a rhinovirus infection. The improvement or maintenance of the ability to control the respiratory disease may require no additional intervention from the treatments commonly administered for pre-existing respiratory diseases. In other words, the only treatment required for the subject is the treatments normally taken for basic respiratory diseases (i.e., when the subject does not have a viral infection) and the TLR2 agonist-containing compound described herein.

[0164] Typically, therapeutically effective doses are formulated to contain concentrations (by weight) of at least about 0.1% to a maximum of about 50% or more, and any combination and partial combination within that range. Compositions can be formulated to contain one or more compounds or their pharmaceutically acceptable salts, polymorphs or prodrugs at concentrations of about 0.1% to less than about 50%, for example, about 49, 48, 47, 46, 45, 44, 43, 42, 41 or 40%, with concentrations greater than about 0.1%, for example, about 0.2, 0.3, 0.4 or 0.5% to less than about 40%, for example, about 39, 38, 37, 36, 35, 34, 33, 32, 31 or 30%. Exemplary compositions may contain about 0.5% to less than about 30%, for example, about 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20%, with concentrations greater than about 0.5%, for example, about 0.6, 0.7, 0.8, 0.9, or 1% to less than about 20%, for example, about 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10%. Compositions may contain more than about 1%, for example, about 2% to less than about 10%, for example, about 9 or 8%, which includes concentrations greater than about 2%, for example, about 3-4%, and less than about 8%, for example, about 7 or 6%. The active agent may be present at a concentration of, for example, about 5%. In any case, the amount can be adjusted to compensate for differences in the amount of active ingredient actually delivered to the target cells or tissues.

[0165] Although this invention is applicable to humans, it is also useful for therapeutic veterinary purposes. This invention is useful for livestock or farm animals such as cattle, sheep, horses and poultry; pets such as cats and dogs; and zoo animals.

[0166] The compositions of the present invention should be administered in an effective dose. The terms “therapeutic effective dose” or “effective dose” generally refer to the amount of the TLR2 agonist, its pharmaceutically acceptable salt, polymorph, or prodrug of the present invention that (i) treats a particular disease, condition, or disorder; (ii) reduces, improves, or eliminates one or more symptoms of a particular disease, condition, or disorder; or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. Undesirable effects, such as side effects, may sometimes occur along with the desired therapeutic effect; therefore, when determining what constitutes an appropriate “effective dose,” the physician should weigh the potential benefits against the potential risks.

[0167] The exact required dose may vary depending on the subject's species, age, health condition, and administration method. Therefore, it may not be possible to specify a precise "effective dose." However, the appropriate "effective dose" for any given individual case can be determined by a person skilled in the art simply by using standard experiments. In one embodiment, the dose administered to the subject is any dose that reduces the viral load. Preferably, this dose does not significantly increase inflammation, for example, the absolute neutrophil count in the lungs or the percentage of neutrophils in total BAL cells.

[0168] In some embodiments, the effective dose for human subjects is in the range of about 250 nmol / kg body weight / dose to 0.005 nmol / kg body weight / dose. Preferably, this range is in the range of about 250 nmol / kg body weight / dose to 0.05 nmol / kg body weight / dose. In some embodiments, the body weight / dose range is about 250 nmol / kg to 0.1 nmol / kg, about 50 nmol / kg to 0.1 nmol / kg, about 5 nmol / kg to 0.1 nmol / kg, about 2.5 nmol / kg to 0.25 nmol / kg, or about 0.5 nmol / kg to 0.1 nmol / kg body weight / dose. In some embodiments, the dose is 250 nmol, 50 nmol, 5 nmol, 2.5 nmol, 0.5 nmol, 0.25 nmol, 0.1 nmol, or 0.05 nmol / kg body weight / dose or approximately these amounts of the compound. The medication plan can be adjusted to suit the needs of the situation and to provide the optimal therapeutic dose.

[0169] The TLR2 agonists described herein may be compositions formulated as inhalation formulations, including dry powders, sprays, mists, or aerosols. These would be particularly preferred for the treatment of respiratory infections. In the case of inhalation formulations, the compositions or concomitant agents provided herein may be delivered by any inhalation method well known to those skilled in the art. Such inhalation methods and devices include, but are not limited to, metered-dose inhalers containing nebulizers such as CFCs or HFAs, or physiologically and environmentally acceptable nebulizers. Other suitable devices include breath-operated inhalers, multi-dose dry powder inhalers, and aerosol nebulizers. The aerosol formulations used in this method typically contain a nebulizer, a surfactant, and a co-solvent, and may be filled into a conventional aerosol container sealed with a suitable metering valve.

[0170] Inhalation compositions may include liquid or powder compositions containing an active ingredient suitable for spray and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing a fixed amount. Preferred liquid compositions contain the active ingredient in an aqueous, pharmaceutically acceptable inhalation solvent such as isotonic saline or bacteriostatic water. The solution is administered using a pump or a compressed-operated spray dispenser, or by inhaling the required amount of the liquid composition into the patient's lungs, or by any other conventional means that enables lung inhalation. For example, preferred formulations where the carrier is liquid for administration include aqueous or oily solutions of the active ingredient, such as nasal sprays or nasal drops. Alternatively, the composition may be a dry powder and may be administered into the airways as defined herein.

[0171] It will be understood that the specific dose level for any particular patient may vary depending on a variety of factors, including the activity of the specific compound used, age, weight, health status, sex, diet, administration time, route of administration and excretion rate, concomitant drug use (i.e., other drugs used to treat the patient), and the severity of the specific disorder during treatment.

[0172] In another embodiment, a manufacturing kit or product is provided comprising one or more TLR2 agonists described herein, the aforementioned pharmaceutically acceptable salts, diluents or excipients and / or pharmaceutical compositions. The kit may further comprise a corticosteroid described herein. Furthermore, the kit may also comprise instructions for use in any method or application of the present invention described herein.

[0173] In other embodiments, a kit is provided for use in the therapeutic and / or preventive applications described above, and the kit is, - A container for holding a pharmaceutical composition in the form of one or more TLR2 agonists or pharmaceutically acceptable salts, diluents or excipients or pharmaceutical compositions as described herein; - Labels or package inserts containing instructions for use Includes.

[0174] In one embodiment, the kit may contain one or more additional active ingredients or materials for the treatment of respiratory conditions.

[0175] A kit or “product” may include a container and labels or packaging inserts on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold a therapeutic composition effective in treating a condition and may have a sterile access port (for example, the container may be a vial with a stopper that can be punctured by an intravenous infusion bag or a subcutaneous needle). Labels or packaging inserts indicate that the therapeutic composition is used to treat the condition in question. In one embodiment, the label or packaging inserts include instructions for use and indicate that the therapeutic or prophylactic composition can be used to treat the respiratory conditions described herein.

[0176] The kit may comprise (a) a therapeutic or prophylactic composition; and (b) a second container containing a second active ingredient or element. The kit of this embodiment of the present invention may further comprise a package insert indicating that the composition and other active ingredients can be used to treat a disorder resulting from the respiratory conditions described herein or to prevent complications arising therefrom.

[0177] It will be understood that the present invention, as disclosed and defined herein, extends to any alternative combination of two or more individual features described or evident therefrom in the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0178] It will be understood that these embodiments are intended to demonstrate the aforementioned and other aspects of the present invention. It will also be understood that while the embodiments illustrate specific embodiments of the present invention, they do not limit such embodiments to these matters. Various modifications may be made, and equivalents may be used instead, or modified forms may be made, without departing from the aforementioned aspects and / or principles of the present invention. All such modifications, equivalents, and modifications are intended to be included within the claims described herein.

[0179] [Examples] As used herein, including in the following examples, the compounds listed below are described in the table below, and their specific structures are shown elsewhere in this specification.

[0180] [Table 2]

[0181] [Table 3]

[0182] [Example 1] [Inhibition of rhinovirus infection in mouse models] This study was conducted to determine whether activation of the innate immune system by TLR2 agonists reduces viral load and virus-induced inflammation during rhinovirus infection in mice.

[0183] [animal] Female 6-8 week old BALB / c mice were used in all studies. Each group consisted of 5 mice. After the treatment or attack procedure, the mice were monitored daily for weight changes and behavioral or physical changes as specified in the animal experiment ethics approvals for Project A-2016-605. At the time of sample collection, all mice were sacrificed by intraperitoneal administration of pentobarbital sodium. All mice were housed in individually ventilated cages within the HMRI Bioresources facility, with no more than 4 mice per cage. Mice were observed daily from the start of each study, and a health status checklist was maintained.

[0184] [Procedures and treatments for mice] Rhinovirus serotype 1B was first purified from clinical isolates, grown in RD-ICAM cells, and then purified as previously described (Bartlett et al., Nat Med (2008) 14, 199-204; Bartlett et al., Methods Mol Biol (2015) 1221, 181-188). Mice were administered 50 μl of agonist molecules intranasally under mild isoflurane anesthesia in the induction chamber of a Class II biosafety cabinet. At the indicated time point after TLR2 agonist administration, PEG-Pam2Cys-R4 and Pam2Cys-R4 were administered intranasally in the same procedure along with 50 μl containing 5 × 10⁶ TCID50 RV1B. Bronchoalveolar lavage (BAL) was performed on day 2 post-infection to count inflammatory cell infiltration and measure immune mediator protein expression. Lung samples were collected to assess the viral load for total RNA. Mouse RV infection models and related techniques have already been developed (Bartlett et al., Nat Med (2008) 14, 199-204; Bartlett et al., Methods Mol Biol (2015) 1221, 181-188). The experimental groups are shown in Table 2.

[0185] [Table 4]

[0186] [Table 5]

[0187] [Table 6]

[0188] [Table 7]

[0189] [Bronchoalveolar lavage fluid (BAL) cell analysis] After sacrificing mice, a cannula was inserted into the trachea, and the airway was flushed 3-5 times with 1 ml of Hank buffer solution (Hyclone™, GE Life Sciences). BAL cells were pelleted by centrifugation, the supernatant was collected, and stored at -80°C for ELISA. The pelleted cells were lysed erythrocytes, and the remaining cells were counted on a hemocytometer by trypan blue exclusion. The cell suspension was then centrifuged, placed on slides, fixed, and stained with Diff Quick (POCD) solution according to the manufacturer's recommendations. A minimum of 200 cells were counted per slide to determine the number of neutrophils, lymphocytes, and macrophages.

[0190] [RNA extraction and qRT-PCR] Apical lung lobes were collected from each mouse in RNA-later (Ambion). For processing, the lung lobes were transferred to RLT (Qiagen) / 2ME buffer and subjected to tissue dissociation twice at 25 Hz for 2 minutes (with sample rotation) using a TissueLyser II (Qiagen). Cellular debris was pelleted by centrifugation, and RNA, including miRNAs from animal and human cells and tissues, was manually extracted using the miRNeasy kit (Qiagen) according to the supplier's recommended protocol for total RNA extraction. After extraction, RNA concentration was determined by spectrophotometric analysis (Nanodrop), and 200 ng of RNA was used for reverse transcription with random primers and an RNase inhibitor (AB, Applied Biosystems). Subsequently, cDNA was used with a master mix containing ROX (Qiagen) and primers and probes outlined in Table 3 for qPCR analysis on ABI700 using TaqMan and FAM-TAMRA chemistry (Life Technologies). The Ct value of the target gene was determined (starting at 107 copies and performing a 1:10 dilution series, referencing seven standards of known concentrations). The copy numbers of all target genes were normalized relative to the reference gene (18s).

[0191] [Quantification of cytokines using ELISA] The remaining lung lobes, flash-frozen in liquid nitrogen, were homogenized in 600 μl of PBS containing a protease inhibitor (Roche) using a TissueLyser II operating twice at 30 Hz for 4 minutes each. Cellular debris was pelleted by centrifugation, and the sample was diluted 1:2 with PBS and stored at -80°C. Next, the BAL solution was analyzed for KC / IL-8 (CXCL1) and TNF-α formation using a Duoset ELISA (R&D Systems) according to the manufacturer's instructions.

[0192] [Table 8]

[0193] qPCR analysis was performed using TaqMan chemistry on cDNA prepared from RNA extracted from the lung lobe apex of each mouse, with an optimal custom forward / reverse primer ratio, and a total volume of 12.5 µl per reaction.

[0194] [Statistical analysis] One-way ANOVA analysis was performed to compare cohorts of mice treated with saline RV control, Pam2Cys-R4, or PEG-Pam2Cys-R4. A p-value < 0.05 was considered statistical significance.

[0195] [result] As indicated, various doses of PEG-Pam2Cys-R4 and Pam2Cys-R4 (see Table 2) were administered intranasally into the entire airway (50 μl). After treatment, mice were infected with RV1B intranasally. Viral load in the airways was determined by qPCR analysis of viral RNA, and pneumonia was determined by staining of BAL inflammatory cells and measurement of protein immunomediators in BAL fluid.

[0196] [Test 1A: Treatment one day before infection] Mice were treated with the indicated doses of PEG-Pam2Cys-R4 and Pam2Cys-R4 one day prior to intranasal RV infection. Controls that did not receive a TLR agonist were treated with saline (Figure 1a). Lung viral load was evaluated by qPCR. The inventors observed a significant reduction in viral load at all tested doses (Figure 1b).

[0197] [Test 1C: Treatment 7 days before infection] Study 1C was completed simultaneously with Study 1A. Mice were treated with the indicated doses of PEG-Pam2Cys-R4 and Pam2Cys-R4 seven days prior to intranasal RV infection (Figure 2a). Controls not receiving TLR-2 agonists were treated with saline. Agonist treatment at all doses resulted in a very significant reduction in viral load compared to RV-infected controls treated with saline (Figure 2b).

[0198] Analysis of BAL cells on day 2 revealed that all treatments significantly increased the total number of inflammatory cells (the majority of which were macrophages), but an increase in lymphocyte count was observed at lower agonist treatment doses (Figure 3a-b). Inflammatory cytokines in BAL were measured by ELISA. Compared to saline-treated infected mice, significantly lower production of the neutrophil-mobilizing chemokine CXCL1 was observed in all treatment groups (Figure 4a). Reduced TNFα expression was also observed in the higher-dose agonist treatment groups compared to saline-treated infected controls (Figure 4b).

[0199] [Study 1B and 1D: Low-dose treatment 7 days prior to infection] After demonstrating a potent and long-lasting antiviral effect accompanied by a reduction in inflammatory cytokine expression, the design of Study 1C was modified to determine whether the antiviral effect could be maintained at lower doses. Starting with the lowest dose from the previous study (0.1 nmole / mouse), additional groups were treated with 0.05 nmole / mouse and 0.01 nmole / mouse Pam2Cys-R4 or PEG-Pam2Cys-R4 (Study 1B) or 10 pmol / mouse, 5 pmol / mouse, 2 pmol / mouse, or 1 pmol / mouse Pam2Cys-R4 or PEG-Pam2Cys-R4 (Study 1D) 7 days prior to viral treatment. No weight loss was observed by the end of Study 1B or 1D. Lung tissue RV RNA was measured to determine whether the reduced inflammation was associated with a lower viral load (Figure 5a-b). All doses of PEG-Pam2Cys-R4 inhibited RV replication. Pam2Cys-R4 also resulted in a significant reduction in viral RNA at the indicated dose.

[0200] Regarding immune cells, Pam2Cys-R4 and PEG-Pam2Cys-R4 induced a significant increase in cell recruitment after treatment at the indicated dose (Figure 6a, d). The increased BAL cells were primarily due to an increased macrophage count (Figure 6b, e). A significantly increased lymphocyte count was also observed at the indicated dose (Figure 6c, f). As shown in Figure 6c, lymphocytes accounted for approximately 10% of total BAL cells in response to the indicated dose. Neutrophilic inflammation is a typical feature of viral asthma exacerbations and is associated with disease severity. A clinically significant reduction in viral load is expected to be associated with a reduction in viral airway neutrophil inflammation. Compared to saline-treated RV-infected mice, a significant decrease in neutrophils was observed at the indicated dose, expressed as a percentage of total BAL cells or the absolute number of total BAL cells (Figure 7a-c). To provide further evidence of TLR-2 agonist-mediated suppression of virus-induced inflammation, levels of neutrophil-mobilizing chemokine (CXCL1) and the inflammatory cytokine TNFα were measured in both Study 1B and Study 1D. Since viral replication drives CXCL1 expression, this data supports the suppression of viral replication by TLR-2 agonist treatment. A highly significant reduction in CXCL1 expression was observed for all doses of TLR-2 agonist compared to untreated RV-infected controls (Figure 8a, b). Treatment had no effect on TNFα production, confirming that treatment did not induce activation of inflammatory pathways (Figure 8c, d).

[0201] [Study 1E (i) Comparison of treatment with Peg-SS-Pam2Cys, Peg-S-Pam2Cys, and Pam2CysSK4 7 days prior to infection; and (ii) Comparison of treatment with INNA-011 and Peg-S-Pam2Cys (dose range 1 pmol to 10 pmol)] Next, other TLR2 agonists (Peg-SS-Pam2Cys and Peg-S-Pam2Cys) were evaluated. After demonstrating potent and long-lasting antiviral effects accompanied by a reduction in inflammatory cytokine expression using the lowest doses of Pam2Cys-R4 and Peg-Pam2Cys-R4, the inventors attempted to evaluate equivalent doses of Peg-SS-Pam2Cys, Peg-S-Pam2Cys, and INNA-011. Accordingly, the inventors treated mouse groups with 10 pmol / mouse, 5 pmol / mouse, 2 pmol / mouse, and / or 1 pmol / mouse (or 2 pmol / mouse in the case of INNA-011) 7 days prior to infection. Comparisons with commercially available Pam2CysSk4 molecules were also performed using the same doses.

[0202] Next, mouse body weight was evaluated over time. The time-course mouse body weight data showed noticeable clustering among various groups. At baseline (-7 days), there was a significant difference between the saline RV control group and the 1 pmol Peg-SS-Pam2Cys group (p=0.046, one-way ANOVA), and there was a trend toward weight loss within the 1 pmol Peg-S-Pam2Cys group at the time of treatment (p=0.091, one-way ANOVA). Except for the 1 pmol Pam2CysSK4 group, all mouse groups showed a trend toward weight gain or no change in body weight (data not shown). Significant differences were observed between 1 pmol Peg-S-Pam2Cys and saline RV control mice at -4 days and post-infection day 1, but this is likely due to dense clustering of mouse body weight and not due to drug-induced weight loss (data not shown).

[0203] To evaluate the antiviral effects of defined TLR agonists, the number of RV copies in pulmonary lysates from the lung apex of three lobes was quantified by Taqman qPCR. All doses of TLR agonists resulted in a significant reduction in RV infection. Peg-S-Pam2Cys suppression was found to be dose-dependent. Peg-SS-Pam2Cys and Peg-S-Pam2Cys exhibited superior antiviral efficacy compared to Pam2CysSK4 at a 10 pmol dose. Peg-SS-Pam2Cys and Peg-S-Pam2Cys reduced RV infection by approximately 85% (84.82% for 10 pmol of Peg-SS-Pam2Cys and 86.76% for Peg-S-Pam2Cys) compared to only a 58% reduction for the same dose of Pam2CysSK4 (one-way ANOVA, p=0.0246) (Figure 9a). In particular, treatment with INNA-011 reduces RV lung RNA to the same extent as Peg-S-Pam2Cys (INNA-006) (9(a)(ii)).

[0204] Pneumonia assessed by total leukocytes in BAL cells showed no significant difference among all drug treatments compared to saline RV control (Figure 9b). Differential leukocyte counting was impossible in this experiment due to cell loss at either the cytospin preparation stage (cells not adhering to the slide) or the staining stage (cells detach from the slide when fixed or immersed in staining solution). Therefore, the fixation and staining solutions were discarded and replaced. To ensure the success of differential BAL leukocyte counting in future experiments, the cell centrifuge was also replaced and the relative centrifugation force was increased (from 300 rpm to 500 rpm). Evaluation of neutrophils in BAL cells 2 days after infection demonstrated that INNA-011 and Peg-S-Pam2Cys (INNA-006) reduced RV-induced neutrophil inflammation (Figure 9c).

[0205] Treatment with Peg-SS-Pam2Cys, Peg-S-Pam2Cys, or Pam2CysSK4 reduced levels of CXCL1, the major neutrophil chemokine produced in response to RV infection (Figures 9d-e). All doses of Peg-SS-Pam2Cys, as well as 10 pmol and 5 pmol doses of Peg-S-Pam2Cys and Pam2CysSK4 compounds, effectively reduced CXCL1 levels. Furthermore, INNA-011 and Peg-S-Pam2Cys (INNA-006) reduced RV-induced expression of CXCL1. Here, TNF-α was not increased by any of the compounds, which provides evidence that defined TLR agonists do not promote inflammation.

[0206] [Study 1F: Timing interactions and effects of concomitant medications during infection] To determine whether there was a synergistic effect on the antiviral response and inflammation, mice were prophylactically administered either 2 pmol of Peg-SS-Pam2Cys or Peg-S-Pam2Cys either 7 days and / or 1 day before infection. One group was specifically administered 7 days and 1 day before infection. After treatment, mice were intranasally infected with RV1B (or mock-treated), mouse body weight was recorded (measured in grams or as a percentage change from baseline), infection in the BAL was assessed, and viral load in the airways was quantified. Mouse body weight measurements and changes in body weight from baseline (-7 days) did not induce weight loss at any time point, as previously observed (data not shown). More importantly, no weight loss was observed when mice were treated with a second dose of the TLR agonist (6 days after the first dose on the day before infection).

[0207] To test the effect of the timing of administration of these TLR agonists, separate groups of mice were intranasally treated with either 2 pmol of Peg-SS-Pam2Cys or Peg-S-Pam2Cys at either 7 days after infection, 1 day after infection, or a combination of 7 days and 1 day before infection. The mice were then intranasally inoculated with either Mock or RV1B. Pneumonia in the BAL was evaluated 2 days after infection.

[0208] To determine whether the increased pneumonia was due to increased viral load or drug-induced causes, lung RV copy number was evaluated by qPCR. A very significant reduction in viral copy number was observed in each drug-treated group, and the combination of Peg-S-Pam2Cys treatment on day -7 and day -1 enhanced viral clearance compared to mice treated only on day 7 (Figure 10a-b).

[0209] BAL neutrophils were significantly increased only in mice treated with Peg-SS-Pam2Cys one day prior to RV or mock infection. However, lymphocyte counts were induced by Peg-SS-Pam2Cys at all treatment time points (except treatment on day 7 of RV1B infection). A combination of Peg-SS-Pam2Cys treatment seven days prior to infection and one day prior also promoted lymphocyte recruitment in the BAL (Figures 10c-d).

[0210] Total lymphocytes increased in mock control mice administered with a day-1 dose of Peg-SS-Pam2Cys, mock mice administered with Peg-SS-Pam2Cys at both day-7 and day-1, and mice administered with Peg-S-Pam2Cys at day-7, compared to saline mock controls (Figures 10e-f). Interestingly, the same dose scheme in RV1B-infected mice did not induce significantly higher leukocyte recruitment compared to saline RV controls. In contrast to previous experiments, the increase in total BAL leukocytes was not due to macrophage recruitment. Only the day-7 Peg-S-Pam2Cys mock group had a higher number of macrophages compared to its saline mock control group.

[0211] Interestingly, despite significant neutrophil inflammation in the Peg-SS-Pam2Cys d-7 / mock group, CXCL1 production increased only in the Peg-SS-Pam2Cys d-1 / RV1B and Peg-S-Pam2Cys d-1 / RV1B groups. It is also important to note that mice already treated on day -7 were protected from pneumonia induced by administration of either Peg-SS-Pam2Cys or Peg-S-Pam2Cys on day -1 (Figure 10g~h). Consistent with previous experiments, neither Peg-SS-Pam2Cys nor Peg-S-Pam2Cys induced TNF-α in either group.

[0212] [Treatment during RV infection in Experiment 1G] Mice were infected intranasally with RV1B and treated with 10, 5, 2, or 1 pmol doses of Peg-SS-Pam2Cys or Peg-S-Pam2Cys on post-infection day 1 to evaluate the therapeutic antiviral effect and its interaction with the established RV infection during pneumonia. Mouse body weight was recorded after RV infection to determine viral load and inflammation in the airways.

[0213] Administration of TLR agonists during infection significantly reduced the RV copy number in the lungs (Figure 11a). Therefore, studies 1G and 1F (administration of TLR agonists on day 1-1 and day 1 post-infection) accurately represent inflammation from viral load.

[0214] There was no significant difference in total white blood cell count between mice treated with Peg-SS-Pam2Cys or Peg-S-Pam2Cys during active infection compared to infected mice treated with saline (saline RV) or RV and mock controls. However, both Peg-SS-Pam2Cys and Peg-S-Pam2Cys altered the BAL white blood cell profile, significantly reducing macrophage numbers and increasing neutrophil recruitment (Figures 11b-e). Unlike previous studies, there was no change in lymphocyte count.

[0215] Neutrophilic inflammation in BAL is associated with the production of the neutrophil chemokine CXCL1 and the inflammatory cytokine TNF-α, both of which were dose-dependent in response to drug treatment (Figure 11f-g). Importantly, CXCL1 and TNF-α were not increased by the lowest doses of Peg-SS-Pam2Cys or Peg-S-Pam2Cys.

[0216] [Essay] The in vivo program of this trial was conducted in parallel with in vitro experiments in RV-infected primary bronchial epithelial cells. In vitro data provide evidence of the antiviral effects of defined TLR2 agonists. The goal of the mouse trial is to determine whether candidate TLR2 agonists (Pam2Cys-R4, Peg-Pam2Cys-R4, Peg-SS-Pam2Cys, and Peg-S-Pam2Cys) are antiviral against RV in vivo when administered to the lower respiratory tract, and whether the suppression of viral infection provides evidence of clinical benefit by reducing virus-induced airway inflammation.

[0217] In the study, 1A and 1B mice were administered 0.1 nmol, 1.0 nmol, and 5 nmol per mouse. When administered 7 days prior to infection (Study 1B), the lowest dose (0.1 nmol) of Peg-Pam2Cys-R4 did not cause significant weight loss, revealing the potential positive effect of pegylation on the systemic effects of Pam2Cys. Evidence of agonist-induced cell inflammation was present for both administration regimens (-1 and -7 days), which were accompanied by a reduction in viral load in both studies. Treatment 7 days prior to infection achieved a significant reduction in viral load (>90% reduction in viral RNA).

[0218] In Trial 1B, all doses induced immune activation, likely from macrophages involved in neutrophilic infection dissociation. Lower doses (1 and 0.1 nmol) provided evidence of neutropenia and increased lymphocyte recruitment indicative of neutrophilic inflammation dissociation in these groups. Cytokine data supported this. TLR2 agonist treatment 7 days prior to infection reduced levels of the virally induced neutrophilic chemokine CXCL1. Higher doses also reduced TNFα expression. This trial was the first to demonstrate that prophylactic treatment with a TLR2 agonist (7 days prior to infection) can inhibit infection and is associated with a reduction in virally induced inflammatory mediators.

[0219] Study 1A (administration 1 day before infection) and Study 1B (administration 7 days before infection) were conducted in the same mouse group. Based on weight loss and inflammation profiles, it was decided to reduce the dosage range of Study 1C (from 0.1 to 0.01 nmol / mouse) 7 days before rhinovirus infection. No weight loss occurred when 0.01 nmol / mouse was administered with either drug. The data showed that the pegylated form was better tolerated in terms of its effect on weight loss.

[0220] Evaluation of inflammatory cells in Experiment 1C revealed a slight increase of less than twofold total BAL cells, primarily consisting of macrophages. Macrophages are important for the fractionation of neutrophilic inflammation, and this may be mechanistically involved in the agonist-mediated suppression of virus-induced inflammation in this model. Again, Peg-Pam2Cys-R4 treatment at 0.05 nmol / mouse and 0.01 nmol / mouse resulted in low inflammation and no increase in total BAL cells. Significant lymphocyte signaling, albeit at low levels, was evident in all groups except the lowest-dose Peg-Pam2Cys-R4 treatment group. Neutrophils are a key readout for viral inflammation. We observed a near-significant decrease in BAL neutrophils with agonist treatment. Considering the consistency of the neutropenic trend, we are confident that this effect (>50% decrease) will be statistically significant if the dataset size is increased through repeated trials. Since the peak of BAL neutrophils in the mouse RV infection model occurs one day after infection, evaluating it one day earlier may yield clearer signals in future studies focused on suppressing viral neutrophil inflammation with TLR2 agonists.

[0221] Consistent with neutropenia, agonist treatment was highly effective in suppressing CXCL1 expression. No effect was observed on TNFα, confirming that the treatment did not cause significant activation of the inflammatory pathway. Since viral replication drives innate immune activation and CXCL1 expression, these data support the suppression of viral replication and infection-induced inflammation by TLR2 agonist treatment. This was confirmed by viral RNA analysis, with Peg-Pam2Cys-R4 treatment inducing a significant reduction in viral load at all doses. Only the highest dose (0.1 nmol / mouse) of Pam2Cys-R4 resulted in a significant reduction in viral load.

[0222] Having completed the validation of the conceptual study using Pam2Cys-R4 and Peg-Pam2Cys-R4, the inventors then focused on Peg-SS-Pam2Cys, Peg-S-Pam2Cys, and INNA-011. Except for a transient decrease in body weight gain at the highest treatment dose (10 pmol per mouse), Peg-SS-Pam2Cys and Peg-S-Pam2Cys did not affect mouse body weight. The absence of clinically harmful inflammation was consistent with the absence of TNFα induction and significantly reduced neutrophilic inflammation (resulting in a highly significant reduction in viral load (>80%)). The potency of Peg-SS-Pam2Cys and Peg-S-Pam2Cys in terms of antiviral effect was comparable and superior to Pam2CSK4, which reduced pulmonary viral load by 50%. These data confirmed that Peg-SS-Pam2Cys and Peg-S-Pam2Cys, when administered 7 days prior to RV infection, strongly suppressed viral inflammation. Furthermore, INNA-011 also demonstrated a significant inhibitory effect against viral inflammation.

[0223] The inventors subsequently investigated the interaction between multiple doses of Peg-SS-Pam2Cys and Peg-S-Pam2Cys and the proximity of administration to RV infection. Three days after administration, uninfected mice (group d-1) showed a neutrophilic response to pneumonia (only the response to drug treatment was examined) in response to Peg-SS-Pam2Cys (not Peg-S-Pam2Cys). This response was reduced when treatment was administered six days earlier. Thus, this experiment revealed an unexpected effect of multiple doses, where the primary dose resulted in a lower inflammatory response than the secondary dose. One possible explanation for this is that the administration of a second agonist induces inflammatory fractionation pathways, such as anti-inflammatory macrophages that phagocytose apoptotic neutrophils. The same experiment also revealed a difference in the intensity of inflammation between Peg-SS-Pam2Cys and Peg-S-Pam2Cys (Peg-SS-Pam2Cys being more inflammatory). CXCL1 levels were significantly increased in mice treated one day before infection, but this effect was completely eliminated when mice received pretreatment on day -7. Despite the suppression of inflammation after multiple treatments, there was no loss of antiviral immunity. In fact, both treatment on day -7 and day -1 were more effective than a single treatment on day -7 (70% reduction in viral RNA) (approximately 90% reduction in viral RNA).

[0224] In the final trial, the inventors investigated the therapeutic effects of Peg-SS-Pam2Cys and Peg-S-Pam2Cys administered one day after RV1B infection (treatment protocol). Clinical evidence of increased inflammation was observed in relation to weight loss at higher doses. This was represented by dose-dependent expression of inflammatory mediators and neutrophil recruitment. At lower doses (2 pmol and 1 pmol), there was no significant increase in KC or TNFα beyond that induced by untreated RV infection. Significant reduction in pulmonary viral load was observed at doses of 5 pmol or less per mouse. The highest dose (10 pmol per mouse) was less effective and, in the case of Peg-S-Pam2Cys, was not significant. This data is consistent with the viral load data from Trial 1A (Figure 4), which also showed that the highest dose resulted in loss of antiviral effect. This is typical of a bell-shaped response curve, often seen with mixed agonist-antagonists.

[0225] In summary, this study is the first to demonstrate that prophylactic treatment with representative TLR-2 agonists can inhibit virus-borne infections, and that this is associated with a reduction in viral load and virus-induced inflammatory mediators such as the chemokine CXCL1. These studies demonstrate the potent antiviral activity against RV infections of structurally diverse compounds, including TLR2 agonists. Furthermore, antiviral activity against rhinovirus infections can be achieved at agonist doses that do not evoke clinical or immunopathological signals. These data also demonstrate that multiple doses of TLR agonists protect against the acute inflammatory effects of the primary response to agonist treatment without impairing antiviral activity. In addition, post-infection treatment also suppresses viral replication and induces neutrophils at low doses of 1 pmol per mouse, but does not increase inflammatory cytokines at low doses.

[0226] While not bound by any particular theory or mechanism of action, the defense mechanism against infection is thought to involve both non-immune (airway epithelium) and low levels of macrophage and lymphocyte activation.

[0227] [Example 2] [Protective and therapeutic effects of TLR2 agonists against rhinovirus infection in primary asthmatic bronchial epithelial cells] This study was conducted to determine whether TLR2 agonist treatment or prophylaxis reduces viral load and virus-induced immune mediators during rhinovirus infection in human asthmatic bronchial epithelial cells differentiated at the gas-liquid interface (ALI).

[0228] [Differentiation of primary bronchial epithelial cells at the gas-liquid interface in COPD patients] Primary bronchial epithelial cells (Figure 12a) obtained from six patients with mild to moderate persistent asthma were grown in T75 flasks until densely populated (passage 3) and differentiated at the gas-liquid interface (ALI). Briefly, primary cells were grown in complete BEGM (Lonza) with growth factor supplements in liquid monolayer culture, and then these were divided into 2 × 10⁵ cells with 10 ng / ml recombinant human epidermal growth factor (rhEGF), along with 0.1% hydrocortisone, 0.1% bovine insulin, 0.1% epinephrine, 0.1% transferrin, and 0.4% bovine pituitary extract (all in Lonza). Transwells (Corning Cat#3460) in a 12-well plate containing ALI initial medium consisting of 50% BEBM / 50% DMEM containing singlequots (Cat#CC-3171), ethanolamine (final concentration 80 μM), MgCl2 (final concentration 0.3 mM), MgSO4 (final concentration 0.4 mM), bovine serum albumin (final concentration 0.5 mg / ml), aphotericin B (final concentration 250 ug / ml), all-trans retinoic acid (30 ng / ml), and 2% penicillin streptomycin were inoculated until condensation was reached (at least 3 days in the apical and basement membrane compartments). Once condensation was reached, the rhEGF concentration in the ALI phase was changed to 0.5 ng / ml for differentiation in the basement membrane compartment (under the Transwell insert) without apical medium until day 21 after initial inoculation.

[0229] [Transepithelial electrical resistance reading] Transepithelial electrical resistance was measured using a WPI EVOM (an epithelial voltresistor with AC current via an STX2 chopstick electrode set) placed simultaneously in apical and basement membrane media. Starting on day 0 (when inoculated cells reached density), the average of three readings was recorded at each time point, and continued weekly throughout proliferation and differentiation (days 7, 14, and 21), as well as post-differentiation against the time of infection (at -2 hours, 0 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours post-infection). Resistance was expressed in ohms (Ω) / cm².

[0230] [Sample collection from ALI cultures] ALI culture samples were collected at 48 and 96 hours post-infection. At each time point, the apical medium was removed from the culture and stored at -80°C for protein expression analysis. Half of the Transwell membrane was carefully cut from the insert and collected, and introduced into 350 μl of RLT buffer (Qiagen) containing 1% 2-mercaptoethanol (2ME) for downstream molecular analysis by RT-qPCR, while the remaining Transwell membrane was reserved for protein analysis.

[0231] [RNA extraction and qRT-PCR] Cells were isolated from the Transwell membrane in RLT / 2ME buffer by pulse vortexing and lysed. After membrane removal, RNA was extracted on a semi-automated Qiacube platform using the miRNeasy kit (Qiagen) according to the supplier's recommended protocol for extracting total RNA, including miRNAs from animal and human cells and tissues. After extraction, RNA concentration was determined using spectrophotometric method (Nanodrop), and 200 ng of RNA was used for reverse transcription with random primers and an RNase inhibitor (AB, Applied Biosystems). Subsequently, cDNA was used with a master mix containing ROX (Qiagen), primers, and probes outlined in Table 3 for qPCR analysis on ABI700 using TaqMan and FAM-TAMRA chemistry (Life Technologies). The Ct value of the target gene was determined (starting at 107 copies and performed in a 1:10 dilution series, referencing seven known concentrations). The copy number of all target genes was normalized to 18s of the reference gene.

[0232] qPCR analysis was performed using TaqMan chemistry on cDNA prepared from RNA extracted from cell lysates from half of the Transwell membrane at the gas-liquid interface, with an optimal custom forward / reverse primer ratio, at a total volume of 12.5 μl per reaction.

[0233] [Virus Stock] Virus infection after 2 hours of adsorption at MOI 0.1 RV1-B (Stock as of May 2010: 1.55 x 10⁸ TCID / ml) Minimum MOI 1 = 6.45 ul, RV1B + 243.55 ul MOI 0.1 = 1 / 10 of MOI 1 Prepare the minimum MOI of 7W (250µl each) = 175µl RV1B + 1575µl.

[0234] [Time of infection sample collection] At that time: Rereading the TEER (to ensure that virus-treated samples do not mix with each other) Remove the supernatant from the apical side. Collect 500 µl and store at -80°C. Remove the Transwell and transfer it to a collection plate containing 1 ml of PBS. Protein: Extract the protein from half of the membrane in 200 µl of protein lysis buffer according to GLP855 and AR methods (store at -80°C). RNA: According to GLP855, extract RNA from half of the membrane in 350 μl of RLT lysis buffer (store at -80°C).

[0235] [Quantification of cytokines and interferon production] Multiplex cytometry bead arrays (CBA) were used to analyze the production of IL-6 and IP-10 (CXCL10) from the apical supernatant of ALI cultures using the BD CBA Flex set (BD) according to the manufacturer's instructions. Briefly, samples were brought to room temperature, mixed with 50 μl of sample and multiplex beads coated with either anti-human IL-6 or IP-10, and incubated with phycoerythrin (PE)-conjugated detection antibody. Samples were electrophoresed on a 96-well plate format, FACS Canto-II, and IL-6 and IP-10 coated beads were identified based on APC and APC-Cy7 clustering and the PE intensity of unknown substances referencing standard curves of known concentrations using FCAP-Array (version 3) software. ELISA was used according to the manufacturer's instructions for the quantification of IFN-γ, IL-8, and CCL22 (R&D systems Duoset) and IFN-β (PBL Assays).

[0236] [Statistical analysis] An independent nonparametric t-test (Mann-Whitney) was used for all stats comparing the treatment to the saline RV control. A P-value < 0.05 was considered statistically significant. The Friedman test was used to assess interferon expression and inflammatory mediator expression between the saline RV control group and the group treated with the indicated dose of Pam2Cys-R4.

[0237] [result] To determine whether Pam2Cys-R4 can induce an antiviral response in asthma airway epithelial cells, we prepared fully differentiated epithelial cell cultures from five asthma patients. These cultures were treated with two doses of Pam2Cys-R4 (0.2 μM or 0.02 μM) in starvation medium either 24 hours before infection (pre-treatment, prophylactic model) or 2 hours after RV infection (post-treatment; therapeutic model). The medium was added to untreated cells. A consistent trend in the reduction of viral RNA in the treated cultures was observed, reaching statistical significance for the 0.02 μM pre-treatment and 0.2 μM post-treatment groups 96 hours after infection (Figures 12b-e). These data suggest that the kinetics of the antiviral response can be manipulated by dose.

[0238] RV replication generates viral RNA that activates the production of pathogen pattern recognition receptors (PRRs), innate immunity, and type I / III interferons (IFNβ / IFNλ). This process has been shown to be impaired in asthma, particularly in more severe disease forms. To determine whether Pam2Cys-R4 treatment inhibits viral replication, we measured virally induced IFN production. We measured type I (IFNβ) and type III (IFNλ) protein levels in apical medium (Figures 13a-d). Under all treatment conditions, TLR-2 agonist treatment tended to reduce IFN expression, which was consistent with the significant reduction in viral replication in the post-treatment 0.2 μM 96h group shown in Figure 12.

[0239] The agonists did indeed induce the production of inflammatory mediators IP-10, IL-6, IP-8, and CCL22 by both uninfected and infected cells (Figures 14a-h). IL-6 exhibits both inflammatory and anti-inflammatory properties, and its role in asthma is somewhat controversial. Generally, high levels are recognized as being associated with asthma severity. IL-8 is a neutrophil chemokine and another biomarker of severe acute asthma. CCL22 is a chemokine that binds to the CCR4 receptor on the surface of Th2 cells and type 2 innate lymphoid cells and is associated with type 2 inflammation in asthma. These data demonstrate that TLR-2 agonist treatment can reduce the peak and duration of infection without causing a significant increase in inflammation, as evidenced by the measurement of defined inflammatory markers. Clinical trials have revealed that the peak and duration of viral load are associated with disease severity in asthma, supporting the idea that reduced viral replication reduces disease severity.

[0240] The inventors then conducted experiments to evaluate the antiviral activity of TLR2 agonist variants of Pam2Cys-R4, Peg-SS-Pam2Cys, and Peg-S-Pam2Cys compared to commercially available Pam2CSK4. The first experiment was performed using the human bronchial epithelium BCi-NS1 cell line, a minimally immortalized human bronchial epithelium cell line that constitutively expresses human telomamalase reverse transcriptase. This cell line was obtained from airway epithelial swabs of healthy volunteers and retains the characteristics of the original primary cells over more than 40 passages. A key characteristic retained by these cells is their ability to differentiate into ALI. Cells were either untreated or pre-treated with indicated doses of Pam2Cys-R4, Peg-SS-Pam2Cys, and Peg-S-Pam2Cys or Pam2CysSK4 (CSK4). Next, cells were infected with RV1B, and viral RNA levels were measured at 96 hours post-infection (Figure 15a-b). Compared to the control, treatment with Peg-S-Pam2Cys (20nM and 2nM) significantly reduced viral RNA levels by approximately 50% at 96 hours post-infection.

[0241] In summary, this study demonstrates that TLR-2 agonist treatment of fully differentiated asthmatic epithelium isolated from human patients with varying degrees of asthma inhibits viral replication and the production of spontaneously occurring antiviral mediators induced by viral replication. These results are significant as they represent the first demonstration that TLR2 agonists can suppress rhinovirus replication without requiring the initial triggering of INF production and IFN-mediated antiviral responses.

[0242] Epithelial cells for this study were obtained from patients with mild to moderate persistent asthma. Compared to cells from patients with severe disease, these cells were more likely to have a fully functional antiviral response with "normal" interferon expression. Nevertheless, the inventors were able to observe enhanced control of infection by agonist treatment.

[0243] Importantly, the antiviral response is independent of the IFN-mediated response. This is significant because interferon expression is highly variable, especially in more severe asthma phenotypes. Therefore, controlling the therapeutic response to IFN-inducing TLR agonists (e.g., TLR3 or TLR7 agonists) can be problematic, potentially leading to no therapeutic effect or excessive inflammation and associated side effects in clinical trials. Previous clinical trials have observed the variability of the response to recombinant IFN in asthma. Exacerbation reduction has only been observed in clinical trials in the severe subgroup, limiting the applicability of this treatment to asthma. The reduction in viral load in cells from patients with mild and moderate persistent asthma was observed in this experiment, suggesting that targeting of interferon-independent antiviral pathways may be broadly applicable to multiple asthma phenotypes.

[0244] Pam2Cys-R4 induced the production of inflammatory mediators IL-6, IL-8, and CCL22 in both infected and uninfected cells, but not IP-10. Consistent with our results, TLR2 activation has been reported to induce epithelial expression of inflammatory cytokines and chemokines in other systems. IL-6 exhibits both inflammatory and anti-inflammatory properties, and its role in asthma is somewhat controversial. Generally, high levels of IL-6 are recognized as being associated with the severity of asthma. IL-8 is a neutrophil chemokine and another biomarker of severe acute asthma. CCL22 is a chemokine that binds to the CCR4 receptor on the surface of Th2 cells and type 2 innate lymphoid cells and is associated with type 2 inflammation in asthma. The increase in expression of these mediators after treatment with Pam2Cys-R4 was slight (generally less than twofold).

[0245] Alveolar macrophages are the primary commensal immune cells in the airways, and BAL cells from healthy lungs are typically 85% macrophages. To clarify the types and magnitudes of inflammatory cytokines induced, we evaluated the response of BAL macrophages to simultaneous stimulation with RV and Pam2Cys-R4 or Peg-Pam2Cys-R4. We measured IL-6, IL-8, and TNFα. CXCL10 (IP10) was undetectable. For the majority of the experiments, RV attack alone did not induce IL-6, IL-8, and TNFα. This was not surprising, given that macrophages are not tolerant of RV infection.

[0246] Both BECs and macrophages expressed IL-6, IL-8, and TNFα in response to TLR2 activation. CXCL10 was expressed by epithelium in response to RV infection and / or TLR2 stimulation, but not by BAL macrophages. This observation facilitates an understanding of responses that may be expected in human clinical trials (IP10 expression may indicate epithelial activation, while the expression of inflammatory cytokines in the absence of CXCL10 may indicate that the epithelium is not involved and that immune cells (macrophages) are responding).

[0247] After validating proof-of-concept experiments using Pam2Cys-R4 and Peg-Pam2Cys-R4, the inventors proceeded to candidate selection and evaluated the antiviral effects of structural analogs of Pam2Cys-R4 and Peg-Pam2Cys-R4, namely Peg-SS-Pam2Cys and Peg-S-Pam2Cys. Pam2Cys-R4 and the commercially available TLR2 agonist Pam2CSK4 were used as controls.

[0248] The first round of experiments was conducted using the healthy human bronchial epithelial cell line BCi-NS1. These cells behave similarly to primary cells in that they can form pseudostratified epithelium in ALI. The inventors confirmed that structurally related TLR2 agonist compounds exhibit potent antiviral activity. In conclusion, these tests demonstrate the ability of representative TLR-2 agonists to act as antiviral agents against RV infection in asthma epithelial cells.

[0249] [Example 3] [Combination of INNA-003 and INNA-006] [Combination of INNA-003 and INNA-006] Reagents: Solid support: TentaGel S RAM resin (substitution coefficient 0.24 mmol / G; Rapp Polymere, Tuebingen, Germany). Amino acid derivatives: Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Homo-Ser(tBu)-OH, Fmoc-Ser(PO(OBzl)OH)-OH, Fmoc-Thr(tBu)-OH, Fmoc-NH-(PEG)3-COOH, Fmoc-NH-(PEG)5-COOH, Fmoc-NH-(PEG) 11 -COOH, Fmoc-NH-(PEG) 27 -COOH. [ka]

[0250] Note: By using Merck catalog number 851024, the following structures can be obtained as "INNA-003" (sometimes referred to herein as Pam2Cys-SS-PEG) and "INNA-006" (sometimes referred to herein as Pam2Cys-S-PEG).

[0251] [INNA-003:] [ka]

[0252] [INNA-006 or compound (1):] [ka]

[0253] Acylation: A 4x molar excess of Fmoc amino acid, O-benzotriazole-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU), and a 6x molar excess of diisopropylethylamine (DIPEA) are used in all acylation steps. All acylation reactions are carried out for 60 minutes, and completion of the reaction is confirmed by trinitrobenzenesulfonic acid (TNBSA) testing. Removal of the Fmoc protecting group from the α-amino group is achieved by exposing the solid support to 2.5% diazabicyclo[5.4.0]undec-7-ene (DBU; Sigma, Steinheim, Germany) for 2 × 5 minutes. The solid support is washed with dimethylformamide (DMF; Auspep, Melbourne, Australia) during each acylation and deprotection step. Fmoc-NH-(PEG) 11 -COOH coupling (Merck, Bayswater, Australia) is performed in the same way as amino acid coupling.

[0254] Note: First, glycine is placed on the TentaGel S RAM solid support, followed by Fmoc-NH-(PEG) 11 -Couples with COOH

[0255] [Peptide quantification] The peptide-based material was quantified by amino acid analysis performed under vacuum by hydrolysis of the sample in a sealed glass vial at 110°C in the presence of 6N HCl containing 0.1% phenol. Next, amino acid derivatization was performed using Waters AccQTag reagent according to the manufacturer's instructions, and then analysis was performed using an AccQTag Ultra column (2.1 mm × 100 mm; Waters Millipore) on a Waters Acquity UPLC System (Waters Millipore).

[0256] [Preparation of INNA-003 and INNA-006] In the case of INNA-003, after the addition of the PEG portion, two serine residues are sequentially coupled, while in the case of INNA-006, after the addition of the PEG portion, a single serine residue is incorporated.

[0257] [Lipidification (addition of Pam2Cys)] Synthesis of S-(2,3-dihydroxypropyl)cysteine: Triethylamine (6 g, 8.2 ml, 58 mM) is added to an aqueous solution of L-cysteine ​​hydrochloride (3 g, 19 mM) and 3-bromo-propane-1,2-diol (4.2 g, 2.36 ml, 27 mM), and the homogeneous solution is maintained at room temperature for 3 days. The solution is reduced to a white residue under vacuum at 40°C, and then precipitated using acetone (300 ml). The precipitate is isolated by centrifugation. The precipitate is washed twice more with acetone and dried to obtain S-(2,3-dihydroxypropyl)cysteine ​​as a white amorphous powder.

[0258] Synthesis of N-fluorenylmethoxycarbonyl-S-(2,3-dihydroxypropyl)-cysteine ​​(Fmoc-Dhc-OH): S-(2,3-dihydroxypropyl)cysteine ​​(2.45 g, 12.6 mM) is dissolved in 9% sodium carbonate (20 ml). Next, a solution of fluorenylmethoxycarbonyl-N-hydroxysuccinimide (3.45 g, 10.5 mM) in acetonitrile (20 ml) is added, and the mixture is stirred for 2 hours. Dilution with water (240 ml) is followed by extraction with diethyl ether (25 ml x 3). The aqueous phase is acidified to pH 2 with concentrated hydrochloric acid, and then extracted with ethyl acetate (70 ml x 3). The extract is washed with water (50 ml x 2) and saturated sodium chloride solution (50 ml x 2). The extract is dried over anhydrous sodium sulfate and evaporated until dry. The final product is obtained by removing residual solvent under high vacuum.

[0259] Coupling of Fmoc-Dhc-OH and resin-bound peptide: Fmoc-Dhc-OH (100 mg, 0.24 mM) is activated at 0°C for 5 minutes in DCM and DMF (1:1, v / v, 3 mL) containing HOBt (36 mg, 0.24 mM) and DICl (37 μL, 0.24 mM). Next, the mixture is added to a container containing resin-bound peptide (0.04 mM, 0.25 g amino-peptide resin). After shaking for 2 hours, the solution is removed by filtration through a glass sintered funnel (porous 3), and the resin is washed with DCM and DMF (3 × 30 mL each). The reaction is monitored for completion using a TNBSA test. Double coupling is performed if necessary.

[0260] Palmitoylation of the two hydroxyl groups of Fmoc-Dhc-peptide resin: Dissolve palmitic acid (204 mg, 0.8 mM), DIPCDI (154 μL, 1 mM), and DMAP (9.76 mg, 0.08 mM) in 2 mL of DCM and 1 mL of DMF. Suspend the resin-bound Fmoc-Dhc-peptide resin (0.04 mM, 0.25 g) in this solution and shake at room temperature for 16 hours. After removing the solution by filtration, thoroughly wash the resin with DCM and DMF to remove all urea residue. Isolation of the Fmoc group is achieved using 2.5% DBU (2 × 5 min).

[0261] Cleavage of peptides from solid support: Reagent B (93% TFA, 5% water, and 2% triisopropylsilane) over 2 hours. Note: The peptides will not precipitate in cooled ether. Most of the TFA must be removed, and the residue is then dissolved in 50% acetonitrile and immediately purified or lyophilized.

[0262] [Purification and characterization of INNA-003 and INNA-006] After cleavage from the solid support, INNA-003 and INNA-006 were purified by reverse-phase high-performance liquid chromatography using a C4 VYDAC column (10 mm × 250 mm; Alltech, NSW, Australia) mounted on a Waters HPLC system (Waters Millipore, Milford, MA, USA). After determining the identity of the target material by mass spectrometry, the purified material was characterized by analytical HPLC using a VYDAC C8 column (4.6 mm × 250 mm), revealing a purity of over 95%. Mass spectrometry was performed using an Agilent 1100 Series LC / MSD ion trap mass spectrometer (Agilent, Palo Alto, CA, USA).

[0263] Preparation of compound (2) or Pam2Cys-Thr-PEG: After adding the PEG11 portion, a single threonine was incorporated. Pam2Cys was added (lipidization) as described above.

[0264] Preparation of compound (3) or Pam2Cys-homo-Ser-PEG: After adding the PEG11 portion, a single homo-serine was incorporated. Pam2Cys was added (lipidization) as described above.

[0265] Preparation of compound (4) or Pam2Cys-phosphoSer-PEG: After adding the PEG11 portion, a single phosphoserine was incorporated. The addition of Pam2Cys (lipidization) was carried out as described above.

[0266] Preparation of Pam2Cys-Ser-PEG3: After coupling the first amino acid glycine, the PEG3 portion was coupled instead of PEG11. After coupling a single serine residue, Pam2Cys was added (lipidization) as described above.

[0267] Preparation of Pam2Cys-Ser-PEG5: After coupling the first amino acid glycine, PEG5 was coupled in place of the PEG11 portion. After coupling a single serine residue, Pam2Cys was added (lipidization) as described above.

[0268] Preparation of compound (5): After coupling the first amino acid glycine, PEG27 was coupled in place of the PEG11 moiety. After coupling a single serine residue, Pam2Cys was added (lipidization) as described above.

[0269] Preparation of compound (6): After coupling of the first amino acid glycine, the PEG27 portion was coupled twice consecutively. After coupling of a single serine residue, Pam2Cys was added (lipidization) as described above.

[0270] Preparation of compound (2a): After adding the PEG11 moiety, two threonines are incorporated. Pam2Cys is added (lipidization) as described above.

[0271] Preparation of compound (3a): After adding the PEG11 moiety, two homoserines are incorporated. Pam2Cys is added (lipidization) as described above.

[0272] Preparation of compound (4a): After adding the PEG11 moiety, two phosphoserines are incorporated. Pam2Cys is added (lipidization) as described above.

[0273] Preparation of compound (5a): After coupling the first amino acid glycine, PEG27 was coupled in place of the PEG11 moiety. After coupling the two serine residues, Pam2Cys was added (lipidization) as described above.

[0274] Preparation of compound (6a): After coupling of the first amino acid glycine, the PEG27 portion was coupled twice consecutively. After coupling of the two serine residues, Pam2Cys was added (lipidization) as described above.

[0275] [Example 4] The primary objectives of the following studies were to determine whether the antiviral efficacy of compounds containing TLR2 agonists and the subsequent suppression of virus-induced inflammation are maintained by compound treatment in the presence of fission products (FPs), and whether prophylactic treatment of the compounds reverses the suppression of FPs in the innate antiviral defense during rhinovirus infection in mice.

[0276] [Laboratory animals] Female 6-8 week old BALB / c mice were used in all studies. Each group consisted of 8 mice. After the treatment or attack procedure, the mice were monitored daily for weight changes and behavioral or physical changes as specified in the animal experiment ethics approval. At the time of sample collection, all mice were sacrificed by intraperitoneal administration of pentobarbital sodium.

[0277] [Administration of INNA-006 and infection with rhinovirus serotype 1B (RV1B)] Rhinovirus serotype 1B was first purified from clinical isolates, grown in RD-ICAM cells, and then purified as previously described in Nat Med 14,199-204 (2008) and Methods Mol Biol 1221,181-188 (2015). Mice were administered 50 μl of agonist molecule intranasally (in) under mild isoflurane anesthesia in the anesthesia induction chamber of a Class II biosafety cabinet. At the indicated time point after TLR-2 agonist administration, 5 × 10⁶ mice were examined using the same procedure. 6 TCID 50 Mice were infected with 50 μl containing RV1B. Bronchoalveolar lavage (BAL) was performed two days post-infection, inflammatory infiltrating cells were counted, and immune mediator protein expression was measured. Lung samples were collected to assess the viral load for total RNA. The mouse RV infection model and related techniques are described in the publicly available Nat Med 14,199-204 (2008) and Methods Mol Biol 1221,181-188 (2015).

[0278] [Bronchoalveolar lavage fluid (BAL) cell analysis] After sacrificing the mice, a cannula was inserted into the trachea, and the airway was flushed 3-5 times with 1 ml of Hank buffer solution (Hyclone®, GE Life Sciences). BAL cells were pelleted by centrifugation, the supernatant was collected, and stored at -80°C for ELISA. The pelleted cells were lysed erythrocytes, and the remaining cells were counted on a hemocytometer by trypan blue exclusion. The cell suspension was then centrifuged, placed on slides, fixed, and stained with Diff Quick (POCD) solution according to the manufacturer's recommendations. A minimum of 200 cells were counted per slide to determine the number of neutrophils, lymphocytes, and macrophages.

[0279] [RNA extraction and qRT-PCR] Lung lobe apices from each mouse were collected in RNA-later (Ambion). For processing, the lung lobes were transferred to RLT (Qiagen) / 2ME buffer and subjected to tissue dissociation twice at 25 Hz for 2 minutes (with sample rotation) using a TissueLyser II (Qiagen). Cellular debris was pelleted by centrifugation, and RNA, including miRNAs from animal and human cells and tissues, was manually extracted using the miRNeasy kit (Qiagen) according to the supplier's recommended protocol for total RNA extraction. After extraction, RNA concentration was determined using spectrophotometric method (Nanodrop), and 200 ng of RNA was used for reverse transcription with random primers and an RNase inhibitor (AB, Applied Biosystems). Subsequently, cDNA was used for qPCR analysis in Quantstudio 6 using TaqMan and FAM-TAMRA chemistry (Life Technologies), along with a master mix containing ROX (Qiagen) and primers and probes outlined in Table 1. The Ct value of the target gene (referencing 7 standards of known concentrations, 10 7 (Start with a copy and proceed in a 1:10 dilution series). The copy numbers of all target genes were normalized relative to the reference gene (18s).

[0280] [Quantification of cytokines using ELISA] Next, the BAL solution was analyzed using Duoset ELISA (R&D Systems) according to the manufacturer's instructions for the production of KC / IL-8 (CXCL1) and TNF-α.

[0281] [Table 9]

[0282] qPCR analysis was performed using TaqMan chemistry on cDNA prepared from RNA extracted from the lung lobe apex of each mouse, with an optimal custom forward / reverse primer ratio, and a total volume of 12.5 µl per reaction.

[0283] [Test Protocol] Restoration of antiviral immunity after treatment with the corticosteroid (CS) fluticasone propionate (FP). Mice were prophylactically administered with the primary candidate using a pre-determined optimal dosing protocol. Mice were treated with FP (or PBS in the case of controls) one hour before RV1B infection (or mock infection with PBS). Innate antiviral immunity (type I / III IFN proteins in BAL) and lung tissue viral load (viral RNA, qPCR) were evaluated 24 hours after infection.

[0284] [result] Restoration of antiviral immunity after CS (fluticasone propionate FP) treatment. Mice were prophylactically administered 2 pmol of INNA-006 into the whole airway 7 days prior to infection or (individually). Weight loss was monitored daily from the initial treatment. Mice were then treated with FP (PBS for controls) 1 hour prior to RV1B infection (or mock infection with PBS). Lung tissue viral load (viral RNA, qPCR) was assessed 48 hours post-infection, and pneumonia was determined by staining of BAL inflammatory cells and measurement of protein immunomediators in BAL fluid.

[0285] Mice were treated with 2 pmol of INNA-006 (all to the entire airway) on day -7 or a combination of day -7 and day -1 prior to in-in infection by inFP administration and RV. Controls not receiving a TLR agonist were treated with saline. Weight loss from the first treatment day (day -7) was evaluated as a percentage change. No significant weight loss was observed with INNA-006 or FP treatment compared to the relevant controls (data not shown).

[0286] Inflammatory cell analysis revealed that D-7 INNA-006 treatment, when administered with FP, resulted in an increase in macrophages and lymphocytes. The combination of D-7 and D-1 INNA-006 treatment increased macrophages and lymphocytes in the BAL. The increase in macrophages induced by the combined D-7 and D-1 INNA-006 was also observed in FP-treated mice. Surprisingly, INNA-006 treatment completely mitigated the RV-induced and steroid-resistant neutrophil inflammatory response in RV-infected mice (Figure 16).

[0287] The inflammatory mediator CXCL1 in BAL was measured by ELISA (Figure 17). Steroid-resistant RV-induced neutrophilic inflammation was consistent with increased CXCL1 (KC, mouse IL-8) protein production. INNA-006 suppressed CXCL1 production and prevented the steroid-resistant inflammatory response.

[0288] Lung viral load was evaluated by qPCR. FP treatment increased viral lung load only in saline-controlled mice. INNA-006 reduced viral lung load in all groups, but repeated INNA-006 treatment prior to FP actually enhanced the antiviral effect (Figure 18).

[0289] The most striking feature of the BAL data from this study was the complete suppression of RV-induced steroid-resistant neutrophil inflammation by INNA-006 in all treatment protocols. The suppressed neutrophil inflammation coincided with significantly reduced levels of the mouse neutrophil chemokine CXCL1 (KC).

[0290] Repeated treatment with 2 pmol of INNA-006 (days -7 and -1) increased total white blood cell count, which was consistent with macrophage and lymphocyte recruitment. Increased macrophage recruitment was observed in FP-treated mice using either INNA-006 administration protocol, as well as in FP-treated mice. Lymphocyte counts increased in the D-7 INNA-006 FP RV and D-1 & D-1 INNA-006 Veh RV groups by an unknown mechanism. A single dose of 2 pmol of INNA-006 7 days prior to infection resulted in significant TNFα production in the BAL, which was not observed with repeated doses of INNA-006 (D-7 & D-1). FP treatment also reduced INNA-006-stimulated TNFα production.

[0291] Consistent with neutropenia, agonist treatment was highly effective in suppressing CXCL1 expression. Since viral replication drives innate immune activation and CXCL1 expression, this data supports the suppression of viral replication and infection-induced inflammation by TLR2 agonist treatment. Analysis of viral RNA confirmed this, with INNA-006 treatment inducing a significant reduction in viral load in both treatment protocols. The most pronounced suppression of viral lung RNA was indeed observed in mice repeatedly treated with INNA-006 along with FP.

[0292] In conclusion, these studies demonstrate that the antiviral activity of TLR agonists against RV infection is maintained and further enhanced by FP treatment.

[0293] [Example 5 - TLR2 activation by various compounds] We determined a comparison of the ability of various compounds to stimulate luciferase activity in an NF-κB cell-based reporter system. The compounds tested included INNA-006 (or compound (1)); INNA-013 (or compound (4)); INNA-014 (or compound (3)); INNA-015 (or compound (2)); INNA-010; INNA-011 (or compound (5)); INNA-012 (or compound (6)); and INNA-009. HEK293T cells transiently co-transfected with human TLR2 plasmid and luciferase-NF-κB plasmid reporter systems were exposed to various dilutions of each compound. By measuring the luminescence induced by luciferase activity, good reporter binding and subsequent signal transduction events were determined (the results are shown in Figure 19 - for each concentration, the columns from left to right are in the following order: INNA-006 (or compound (1)); INNA-013 (or compound (4)); INNA-014 (or compound (3)); INNA-015 (or compound (2)); INNA-010; INNA-011 (or compound (5)); INNA-012 (or compound (6)); and INNA-009).

[0294] The results revealed that most potent compounds had a single serine, threonine, or homoserine group separating Pam2Cys from PEG, or a 12,28-ethylene oxide monomer, or two groups of 28-ethylene oxide monomer. However, all compounds resulted in good receptor binding followed by signal transduction.

[0295] [Example 6 - Comparison of INNA-006 and Pam3Cys-Ser-PEG3000 using an in vitro luciferase assay] [Comparison of in vitro TLR2 agonist activity of Pam3Cys-Ser-PEG3000 and INNA-006:] HEK293T cells transiently co-transfected with human TLR2 plasmid and luciferase-NF-κB plasmid reporter were exposed to various dilutions of INNA-006 or Pam3Cys-Ser-PEG3000.

[0296] By measuring the luminescence induced by luciferase activity, good reporter binding and subsequent signal transduction events were determined (Figure 20). The results demonstrate that Pam3Cys-Ser-PEG3000 is inferior to INNA-006 in its ability to signal NF-κB within the tested dose range (12.2 pM to 3.125 pM).

[0297] [Example 7 - TLR binding and specificity] INNA-006 was evaluated for its ability to activate various other TLR pattern recognition receptors. These evaluations were performed using both human and mouse TLR panels. These assays detect secreted embryonic alkaline phosphatase (SEAP) reporters under the control of a promoter induced by NF-κB activation in HEK293 cells.

[0298] The secreted embryonic alkaline phosphatase (SEAP) reporter is regulated by a promoter inducible by the transcription factor NF-κB. This reporter gene allows for the monitoring of signal transduction via TLRs based on NF-κB activation. In a 96-well plate (200 μL total volume) containing appropriate cells (50,000–75,000 cells / well), 20 μL of the test substance or positive control ligand was added to each well. The culture medium added to the wells was designed for the detection of NF-κB-induced SEAP expression. After 16–24 hours of incubation, optical density (OD) was read at 650 nm using a Molecular Devices SpectraMax 340PC absorbance detector.

[0299] [Control ligand] hTLR2: 1 × 10⁸ cells / mL of HKLM (heat-sterilized Listeria monocytogenes) hTLR3: 1 μg / mL poly(I:C)HMW hTLR4: 100 ng / mL Escherichia coli (E. coli) K12 LPS hTLR5: 100 ng / mL of Salmonella typhimurium flagellin hTLR7: 1 μg / mL CL307 hTLR8: 1 μg / mL CL075 hTLR9: 1 μg / mL CpG ODN2006

[0300] Under the tested conditions, INNA-006 was confirmed to be able to activate its designated target (TLR-2) and not to activate any other TLRs tested in these assays (Figure 21).

[0301] The present invention may also be in the following embodiments. 1. A method for treating or preventing a respiratory condition associated with a target rhinovirus, comprising administering a compound containing a TLR2 agonist to thereby treat or prevent the respiratory condition associated with the target rhinovirus. 2. The method according to item 1, which does not involve administering an agonist of a TLR other than a TLR2 homodimer or heterodimer. 3. The method according to item 1 or 2, wherein the compound is administered in a composition further comprising a pharmaceutically acceptable carrier, diluent, or excipient. 4. The composition according to item 3, comprising a compound containing a TLR2 agonist and a pharmaceutically acceptable carrier, diluent, or excipient. 5. A method for treating or preventing a target rhinovirus infection, comprising administering a compound containing a TLR2 agonist to thereby treat or prevent the target rhinovirus infection. 6. The method according to item 5, further comprising the step of identifying a subject having a rhinovirus infection. 7. The use of compounds containing TLR2 agonists in the preparation of drugs for the treatment or prevention of respiratory conditions associated with target rhinoviruses. 8. Use of compounds containing TLR2 agonists for the treatment or prevention of respiratory conditions associated with the target rhinovirus. 9. A method for treating or preventing virus-mediated exacerbations of a target respiratory condition, comprising administering a compound containing a TLR2 agonist to the target, thereby treating or preventing virus-mediated exacerbations of the target respiratory condition. 10. The method according to item 9, further comprising the step of identifying a subject having a respiratory condition. 11. The method according to item 9 or 10, wherein the respiratory condition is chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, or a lung condition associated with lung transplantation or long-term glucocorticosteroid use. 12. The use of compounds containing TLR2 agonists in the preparation of drugs for the treatment or prevention of virus-mediated exacerbations of target respiratory conditions. 13. The virus-mediated exacerbation is a rhinovirus-mediated exacerbation, as described or used in any one of items 9 to 12. 14. A method for reducing rhinovirus-induced airway inflammation, comprising administering a compound containing a TLR2 agonist to the target, thereby reducing rhinovirus-induced airway inflammation. 15. The TLR2 agonist comprises a lipid, peptidoglycan, lipoprotein, or lipopolysaccharide, as described or used in any one of items 1 to 14. 16. The TLR2 agonist comprises palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl, as described or used in any one of items 1 to 15. 17. The TLR2 agonist is selected from the group consisting of Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys, and Oct2Cys, and is used or otherwise described in any one of items 1 to 16. 18. The TLR2 agonist, including Pam2Cys, is used or disposed of in the manner described in item 17. 19. The solubility of the TLR2 agonist is increased by a solubilizer, as described or used in any one of items 1 to 18. 20. The compound comprises a TLR2 agonist and a solubilizer, as described or used in any one of items 1 to 19. twenty one. The TLR2 agonist and solubilizer are conjugated, or used, as described in item 19 or 20. twenty two. The solubilizer comprises or consists of a positively charged or negatively charged group, as described or used in any one of items 19 to 21. twenty three. The method or use described in item 22, wherein the charged group is a branched or linear peptide. twenty four. The method or use described in item 22 or 23, wherein the positively charged group comprises at least one positively charged amino acid, preferably an arginine or lysine residue. twenty five. The method or use described in item 22 or 23, wherein the negatively charged group comprises at least one negatively charged amino acid, preferably glutamic acid or aspartic acid. 26. The branched or linear peptide is R4, H4, H8, or E8, as described or used in any one of items 22-25. 27. The aforementioned branched peptide is [ka] The method or use described in any one of items 22-25, including the method described in item 22-25. 27. The solubilizing agent comprises polyethylene glycol (PEG) or R4, as described or used in any one of items 19 to 27. 28. The solubilizing agent comprises polyethylene glycol (PEG) and R4, as described or used in item 27. 29. The aforementioned PEG is PEG 11 or PEG 12 The method or use described in item 28. 30. The compound containing a TLR2 agonist has the following structure: AYB (In the formula, A is, [ka] including or consisting of Here, each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; Y is [ka] And, Here, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; and B contains or consists of polyethylene glycol (PEG). The method or use described in any one of items 1 to 21, or including a pharmaceutically acceptable salt or prodrug thereof. 31. The compound containing the TLR2 agonist comprises Pam2Cys and PEG, wherein Pam2Cys and PEG are linked by serine, homoserine, threonine, or phosphoserine residues. Pam2Cys in the aforementioned compound has the following structure: [ka] The method or use described in any one of items 1 to 21, which includes having the characteristics of any one of items 1 to 21. 32. The aforementioned compound is covalently bonded to polyethylene glycol (PEG). [ka] (In the formula, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H.) The method or use described in any one of items 1 to 21, or including a pharmaceutically acceptable salt or prodrug thereof. 33. The aforementioned compound is given by formula (I): [ka] (In the formula, n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 34. The aforementioned compound is given by formula (II): AY-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(II) (In the formula, A is structure: [ka] Having; Y is [ka] And, Here, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 may not both be H; n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 35. The aforementioned compound is given by formula (III): Pam2Cys-Y-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(III) (In the formula, Pam2Cys has the following structure: [ka] Having; Y is [ka] And, Here, R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; When q=1, R3 is H, -NH2, or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 36. The aforementioned compound is of formula (IV): Pam2Cys-Ser-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IV) (In the formula, Pam2Cys-Ser has the following structure: [ka] Having; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 37. The aforementioned compound is of formula (V): [ka] (In the formula, n is between 3 and 100; k is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; t is 2, 3, or 4; h is 1, 2, 3, or 4; q is either zero or one; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R2 cannot both be H; When q=1, R3 is either -NH2 or -OH; When q=0, R3 is H; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 38. The aforementioned compound is compound (1): [ka] The method or use described in any one of items 1 to 21, having the structure of or a pharmaceutically acceptable salt or prodrug thereof. 39. The aforementioned compound, [ka] [ka] JPEG2023081969000110.jpg63149 JPEG2023081969000111.jpg68149 and [ka] A method or use described in any one of items 1 to 21, selected from the group consisting of the following. 40. The aforementioned compound is given by formula (Ia): [ka] (In the formula, n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, and R2 and R2' cannot both be H; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 41. The aforementioned compound is given by formula (IIa): AY-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IIa) (In the formula, A is structure: [ka] Having; Y is [ka] And, Here, R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, nor can R2 and R2' both be H; n is between 3 and 100; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; q is either zero or one; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 42. The aforementioned compound is given by formula (IIIa): Pam2Cys-Y-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IIIa) (In the formula, Pam2Cys has the following structure: [ka] Having; Y is [ka] And, Here, R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, nor can R2 and R2' both be H; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 43. The aforementioned compound is given by formula (IVa): Pam2Cys-Ser-Ser-NH-(CH2) P -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3(IVa) (In the formula, Pam2Cys has the following structure: [ka] Having; n is between 3 and 100; m is 1, 2, 3, or 4; p is 2, 3, or 4; q is either zero or one; R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, and R2 and R2' cannot both be H; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 44. The aforementioned compound is given by formula (Va): [ka] (In the formula, n is between 3 and 100; k is between 3 and 100; h is 1, 2, 3, or 4; m is 1, 2, 3, or 4; Each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3, or 4; t is 2, 3, or 4; q is either zero or one; R1, R1', R2, and R2' are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, and any one of the alkyl hydrogens may be substituted with a halogen, and R1 and R1' cannot both be H, and R2 and R2' cannot both be H; When q is zero, R3 is H; When q is 1, R3 is either -NH2 or -OH; L is either zero or consists of 1 to 10 units, each unit being a natural α-amino acid or derived from a natural α-amino acid, and the formula is: [ka] It has, Here, R4 is H; and R5 is the side chain or second hydrogen of the aforementioned amino acid. The method or use described in any one of items 1 to 21, of the substance or a pharmaceutically acceptable salt or prodrug thereof. 45. The aforementioned compound has the following structure: [ka] The method or use described in any one of items 1 to 21, which includes having the characteristics of any one of items 1 to 21. 46. The aforementioned compound is compound (1a): [ka] The method or use described in any one of items 1 to 21, having the structure of or a pharmaceutically acceptable salt or prodrug thereof. 47. The aforementioned compound, [ka] [ka] JPEG2023081969000129.jpg65149 JPEG2023081969000130.jpg63149 [ka] and [ka] A method or use described in any one of items 1 to 21, selected from the group consisting of the following. 48. The TLR2 agonist is not Pam3Cys, but is used in any way described in any one of items 1 to 16. 49. The aforementioned compound, [ka] The method or use described in any one of items 1 through 28. 50. The TLR2 agonist is administered once daily, according to the method or use described in any one of items 1 to 49. 51. The TLR2 agonist is administered once weekly, according to the method or use described in any one of items 1 to 49. 52. The compound or composition is administered into the airway, according to or used in any one of items 1 to 51. 53. The compound or composition may be administered to the subject via inhalation or nasally, according to the method or use described in any one of items 1 to 52. 54. The aforementioned asthma is mild asthma, as described in item 11. 55. The method or use described in any one of items 1 to 54, further comprising administering a corticosteroid. 56. The compound or composition described above is administered simultaneously with or sequentially with the corticosteroid, or is used in the manner described in item 55. 57. The compound or composition is administered once, twice, or more times within 24 hours or 7 days prior to the administration of the corticosteroid, as described or used in item 56. 58. The subjects mentioned above are those who are currently receiving or have previously received corticosteroids, and who use or are using any one of the methods described in item 1 to 54. 59. The corticosteroid is a glucocorticoid, as described or used in any one of items 55-58. 60. The glucocorticoid is an agonist, partial agonist, or allosteric modulator of a glucocorticoid receptor, as described or used in item 59. 61. The glucocorticoid is an inhalable glucocorticoid, as described or used in item 60. 62. The glucocorticoid is budesonide, cyclocenide, mometasone, or any other glucocorticoid described herein, such as fluticasone propionate, as described or used in item 61. 63. A compound containing a TLR2 agonist for use in the treatment or prevention of respiratory conditions associated with the target rhinovirus. 64. A pharmaceutical composition comprising a TLR2 agonist for treating or preventing respiratory conditions associated with the target rhinovirus. 65. A compound or pharmaceutical composition as described in item 63 or 64, suitable for administration to the airway. 66. A composition comprising, essentially consisting of, or comprising a compound containing a TLR2 agonist and a corticosteroid. 67. The composition according to item 66, wherein the compound is any one of those described in any one of items 15 to 49. 68. The composition according to item 66 or 67, wherein the corticosteroid is a glucocorticoid. 69. The composition according to item 68, wherein the glucocorticoid is an agonist, partial agonist, or allosteric modulator of a glucocorticoid receptor. 70. The composition according to item 69, wherein the glucocorticoid is an inhalable glucocorticoid. 71. The composition according to item 70, wherein the glucocorticoid is selected from the group consisting of budesonide, cyclocenide, mometasone, beclomethasone, betamethasone, dexamethasone, prednisolone, prednisone, and fluticasone propionate. 72. A composition according to any one of items 66 to 71, further comprising a pharmaceutically acceptable diluent, carrier, or excipient. 73. A composition described in any one of items 66 to 72, formulated or adapted for administration to the airway. 74. The composition described in item 73, formulated or adapted for administration to the upper or lower respiratory tract. 75. The composition described in item 74, formulated or adapted for inhalation or nasal administration. 76. The composition according to item 75, which is an inhalation composition and is formulated as a dry powder suitable for use in a dry powder inhalation device. 77. The composition described in item 75, which is formulated as a nasal spray or nasal medication.

Claims

1. Use of a compound comprising a TLR2 agonist and a solubilizing agent comprising polyethylene glycol (PEG) in the preparation of a medicament for treating or preventing asthma associated with rhinovirus infection, wherein the compound comprising the TLR2 agonist is a lipopeptide comprising a lipid moiety, the lipid moiety being selected from the group consisting of palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl and decanoyl, and the TLR2 agonist and the solubilizing agent are linked together.

2. Use of a compound comprising a TLR2 agonist and a solubilizing agent comprising polyethylene glycol (PEG) in the preparation of a medicament for treating or preventing rhinovirus-mediated exacerbations of asthma, wherein the compound comprising the TLR2 agonist is a lipopeptide comprising a lipid moiety, the lipid moiety being selected from the group consisting of palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl and decanoyl, and the TLR2 agonist and the solubilizing agent are linked together.

3. 3. The use according to claim 1 or 2, wherein the medicament does not comprise an agonist of a TLR other than a TLR2 homodimer or heterodimer.

4. The use according to claim 1 or 2, wherein the medicament further comprises a pharmaceutically acceptable carrier, diluent or excipient.

5. The use according to any one of claims 1 to 4, wherein the lipid moiety comprises palmitoyl.

6. The use according to any one of claims 1 to 5, wherein the TLR2 agonist is selected from the group consisting of Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys and Oct2Cys.

7. The use of claim 6 , wherein the TLR2 agonist comprises Pam2Cys.

8. The use according to any one of claims 1 to 7, wherein the solubilizer comprises a positively or negatively charged group.

9. The compound is 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 and 【Transformation 6】 The use according to any one of claims 1 to 7, wherein the compound is selected from the group consisting of:

10. The use according to any one of claims 1 to 9, wherein the TLR2 agonist is formulated for once-daily or once-weekly administration.

11. The use according to any one of claims 1 to 10, wherein the compound is formulated for administration to the respiratory tract.

12. The use according to any one of claims 1 to 11, wherein the compound is formulated for administration to a subject via inhalation or intranasally.

13. The use according to any one of claims 1 to 12, further comprising administering a corticosteroid.

14. 14. The use according to claim 13, wherein the corticosteroid is a glucocorticoid.

15. 15. The use according to claim 14, wherein the glucocorticoid is an agonist, partial agonist or allosteric modulator of the glucocorticoid receptor.

16. 16. The use according to claim 14 or 15, wherein the glucocorticoid is an inhalable glucocorticoid.

17. 17. The use according to claim 16, wherein the glucocorticoid is budesonide, cyclosenide, mometasone or any other glucocorticoid described herein, such as fluticasone propionate.

18. 1. A pharmaceutical composition comprising a TLR2 agonist and a solubilizing agent comprising polyethylene glycol (PEG) for use in the treatment or prevention of asthma associated with rhinovirus infection, wherein the TLR2 agonist is a lipopeptide comprising a lipid moiety, the lipid moiety being selected from the group consisting of palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and decanoyl, and the TLR2 agonist and the solubilizing agent are linked together.

19. A pharmaceutical composition comprising a TLR2 agonist and a solubilizing agent comprising polyethylene glycol (PEG) for use in the treatment or prevention of rhinovirus-mediated exacerbations of asthma, wherein the TLR2 agonist is a lipopeptide comprising a lipid moiety, the lipid moiety being selected from the group consisting of palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl and decanoyl, and the TLR2 agonist and the solubilizing agent are linked together.

20. 20. The pharmaceutical composition of claim 18 or 19, adapted for administration to the respiratory tract.

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