Methods and Compositions for Treating COVID Infection

Abstract

The present invention relates to a peptide linker having the formula: n wherein each peptide is independently an HRC peptide or a targeting peptide, with the proviso that at least one peptide is an HRC peptide, each linker is independently a bivalent linking moiety, and B is a multivalent moiety comprising a cysteine, X, and optionally Y, and / or optionally Z, where X, Y, and Z are defined herein and n is an integer selected from 1, 2, 3 or more, and methods therewith for treating or preventing a viral infection in a subject in need thereof.

Description

[Technical field]

[0001] This application is a continuation-in-part of U.S. Patent Application No. 17 / 870,158 and U.S. Patent Application No. 17 / 870,174, both filed on July 21, 2022. This application also claims priority to U.S. Provisional Application No. 63 / 307,371, filed on February 7, 2022. The entire teachings of the above applications are incorporated herein by reference. [Background technology]

[0002] A variety of viruses are known to cause respiratory infections in humans, resulting in diseases that are typically classified according to their clinical presentation, such as the common cold, influenza, bronchiolitis, croup, or pneumonia. Although such infections are generally self-limited, they can lead to more severe and potentially life-threatening diseases, including pneumonia, in some patients, especially the elderly, infants, and people with compromised immune systems. Most drug treatments prescribed for these diseases only provide symptomatic relief, and few drugs are available to modify the course of any of these diseases. Furthermore, new respiratory diseases caused by zoonotic viruses have emerged in recent years, such as the severe acute respiratory syndrome coronavirus (SARS-CoV), identified in 2002, the Middle East respiratory syndrome coronavirus (MERS-CoV), identified in 2012, and most recently, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a virus first described in December 2019 that causes the disease called COVID-19. The SARS-CoV-2 virus remains a global threat. SARS-CoV and MERS-CoV are not currently a threat to the human population, but they can exist in animal reservoirs and may pose a threat to humans in the future.

[0003] Moreover, coronaviruses are mutating rapidly, raising concerns that new strains of the virus could emerge that can evade existing treatments, necessitating the rush to develop new drugs.

[0004] There remains a need in the art for methods of treating or preventing viral respiratory infections. Summary of the Invention

[0005] The present invention is based on the discovery that new antiviral conjugates can be synthesized on demand by conjugating coronavirus spike proteins and other competing ligands to lipid molecules, such as cholesterol or tocopherol, using click chemistry. The method provides a programmable rapid response platform for the on-demand synthesis of potent antiviral materials. The platform includes four modules: a viral fusion peptide inhibitor, a membrane anchoring unit (such as a lipid molecule), an ACE2 targeting peptide, and a spike-binding peptide. Covalent combination with at least one module includes a fusion peptide inhibitor that is conjugated to another module to provide an effective anticoronavirus material. The modules can be connected via a covalent framework consisting of at least one sulfur or nitrogen aryl linkage. The other modules consist of a connector consisting of a triazole, amide, or sulfur-sp3 carbon bond. These connectors can be rapidly created using click chemistry.

[0006] The invention also provides compounds, compositions and methods of their use, such as compounds identified by the platform, for treating or preventing infectious diseases.

[0007] In one embodiment, the present invention provides compounds comprising one, two, three or more HRC sequences of a viral spike peptide (also called fusion peptide) conjugated to a membrane anchoring moiety (such as a hydrophobic moiety) via an optional linker. The hydrophobic moiety can be a membrane-integrating ligand, such as cholesterol, sphingolipid, glycolipid, glycerophospholipid, etc. The viral spike peptide is preferably a coronavirus spike protein. The peptides of the present invention inhibit viral fusion.

[0008] The present invention includes compositions for delivery, e.g., pulmonary or nasal delivery, of the compounds of the present invention. The present invention provides a method for treating or preventing a viral infection, e.g., SARS-CoV-2 (COVID-19) infection, in a subject in need thereof, comprising administering an effective amount of a compound of the present invention. The present invention further provides a method for making the compositions of the present invention.

[0009] In embodiments, antiviral compounds useful for treating coronaviruses include compounds of the invention having the formula shown in FIG. 1G, FIG. 1H, or FIG. 1I.

[0010] The present invention also contemplates providing an efficient generation of molecular libraries, the platform and libraries that can be generated allow for the identification of linkers, spacers, sequences and targeting moieties with improved antiviral activity against SARS-CoV2 and its mutations, including but not limited to alpha, beta, gamma, delta and omicron mutants, by examining the mutants.

[0011] This patent file or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0012] The foregoing and other objects, features and advantages of the present invention will become apparent from the following more detailed description of preferred embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. [Brief description of the drawings]

[0013] [Figure 1A] FIG. 1 depicts exemplary compounds of the present invention. [Figure 1B] FIG. 1 depicts exemplary compounds of the present invention. [Figure 1C] FIG. 1 depicts exemplary compounds of the present invention. [Figure 1D] FIG. 1 depicts exemplary compounds of the present invention. [Figure 1E] FIG. 1 depicts exemplary compounds of the present invention. [Figure 1F] FIG. 1 depicts exemplary compounds of the present invention. [Figure 1G] FIG. 1 depicts exemplary compounds of the present invention. [Figure 1H] FIG. 1 depicts exemplary compounds of the present invention. [Diagram 2] FIG. 1 depicts an outline of the pseudotyped virus neutralization assay. [Diagram 3] A diagram showing the different SARS-CoV2 variants and the regions where these mutations can be found. [Figure 4] This is a model of the coronavirus-derived HRC peptide described in Duquerroy et al, Virology (2005) 335:276-285. [Diagram 5] FIG. 2 is a cross-sectional view of the S protein trimer. [Figure 6] FIG. 1 illustrates the atomic order of various atoms in an amino acid side chain. [Figure 7] FIG. 1 is a schematic diagram of the study design for Example 2. [Figure 8]FIG. 1 depicts mean percentage body weight change (y-axis) and x-axis corresponds to time period from -1 and -2 (baseline) to day 7. [Figure 9] Figure 1. Percentage body weight change (y-axis) whereas x-axis corresponds to treatment group. Ordinary one-way ANOVA with multiple comparisons against control group 1. Significant differences are indicated by asterisks when present. P>0.05 * P≦0.05 ** P≦0.01 *** P≦0.001 **** P≦0.0001. [Figure 10] Figure 14. Estimation of oral swab viral load by genome copy number by RT-qPCR (y-axis normalized to Log10) from days 1 to 7 (x-axis). G1 from days 1 to 2 had n=6 due to accidental death. Groups G2 to G5 all had n=8 during this period. From days 3 to 7, G1 had n=3, while G2-G5 had n=4. [Figure 11] Figure 1. Estimation of viral load by genome copy number in oropharyngeal swabs by RT-qPCR on a daily basis (y-axis is Log10 normalized) and x-axis corresponds to treatment group. Ordinary one-way ANOVA with multiple comparisons against control group 1. Significant differences are indicated with asterisks when present. ns P>0.05 * P≤0.05 ** P≤0.01 *** P≤0.001 **** P≤0.0001. [Figure 12] Figure 1. Estimation of viral load by genome copy number by RT-qPCR (y-axis is Log10 normalized) in lungs collected on day 2 (n=4) and day 7 (n=4), where the x-axis corresponds to treatment group. Ordinary one-way ANOVA with multiple comparisons versus control group 1. Significant differences are indicated with asterisks when present. ns P>0.05, * P≤0.05, ** P≤0.01, *** P≤0.001, or **** P≤0.0001. [Figure 13]Figure 1. Live SARS-CoV-2 titration assay in lungs on study days 2 and 7. TCID50 / mg lung tissue on the y-axis was normalized using Log10. Graph shows lung titers and averages for all individual animals by group (x-axis). Groups (2, 3, 4 and 5) had significantly reduced viral loads on day 2 when compared to control group 1 by TCID50. (**, P<0.01 or not shown if not significant). [Figure 14] Figure 1. Live SARS-CoV-2 titration assay in nasal turbinates on days 2 and 7. TCID50 / mg nasal turbinate tissue is shown on the y-axis. The graph shows the lung titers and the averages for all individual animals by group (x-axis). Groups (2, 3, 4 and 5) had significantly reduced viral loads on day 2 when compared to control group 1 by TCID50 (**, P<0.01 or not shown if not significant). [Figure 15] FIG. 1 is a schematic diagram of the study design for Example 3. [Figure 16] FIG. 1 depicts the mean percentage body weight change (y-axis) while the x-axis corresponds to days −1 and −2 (baseline) to day 7 of the study. [Figure 17] Figure 1. Percentage body weight change (y-axis) whereas x-axis corresponds to treatment group. Ordinary one-way ANOVA with multiple comparisons against control group 1. Significant differences are indicated by asterisks when present. P>0.05 * P≦0.05 ** P≦0.01 *** P≦0.001 **** P≦0.0001. [Figure 18] Figure 1. Estimation of oral swab viral load by genome copy number by RT-qPCR from days 1 to 7 (x-axis) (y-axis is Log10 normalized). [Figure 19]Figure 1. Estimation of viral load by genome copy number in oropharyngeal swabs by RT-qPCR on a daily basis (y-axis is Log10 normalized) and x-axis corresponds to treatment group. Ordinary one-way ANOVA with multiple comparisons against control group 1. Significant differences are indicated with asterisks when present. ns P>0.05 * P≤0.05 ** P≤0.01 *** P≤0.001 **** P≤0.0001. [Figure 20] Figure 1. Estimation of viral load by genome copy number by RT-qPCR (y-axis is Log10 normalized) in lungs collected on day 2 (n=6) and day 7 (n=6), where the x-axis corresponds to treatment group. Ordinary one-way ANOVA with multiple comparisons versus control group 1. Significant differences are indicated with asterisks when present. ns P>0.05, * P≤0.05, ** P≤0.01, *** P≤0.001, or **** P≤0.0001. [Figure 21] Figure 1. Estimation of viral load by TCID50 (y-axis is Log10 normalized) in lungs collected on days 2 (n=6) and 7 (n=6), where the x-axis corresponds to treatment group. Ordinary one-way ANOVA with multiple comparisons against control group 1. Significant differences are indicated with asterisks when present. ns is not significant. P>0.05, *P≦0.05, **P≦0.01, ***P≦0.001, or ****P≦0.0001. [Figure 22] FIG. 1 shows the efficacy of antiviral therapeutic compounds of the invention against the WA1 strain. [Figure 23] FIG. 1 shows the efficacy of antiviral therapeutic compounds of the invention against Delta strains. [Figure 24] FIG. 1 shows the efficacy of antiviral therapeutic compounds of the invention against Omicron strains. [Diagram 25] FIG. 1 shows the efficacy of antiviral therapeutic compounds of the invention against the P1 strain. [Figure 26] FIG. 1 shows the efficacy of antiviral therapeutic compounds of the invention against SARS-CoV-1 strains. [Figure 27]FIG. 1 shows the efficacy of antiviral therapeutic compounds of the invention against MERS-CoV strains. [Figure 28] FIG. 1 shows the efficacy of antiviral therapeutic compounds of the invention against the OC43 strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Coronaviruses target human cells through the spike protein binding domain (RBD) that attaches to the human angiotensin-converting enzyme 2 (hACE2) receptor on the host cell. The coronavirus spike (S) glycoprotein is a class I viral fusion protein on the outer membrane of the virion and plays a crucial role in viral infection by recognizing host cell receptors and mediating the fusion of the viral and cellular membranes. Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein, which forms a homotrimer protruding from the viral surface. S contains two functional subunits responsible for binding to the host cell receptor (S1 subunit) and fusion of the viral membrane with the cellular membrane (S2 subunit).

[0015] S1 plays a role in receptor binding and contains an N-terminal signal peptide (SP), an N-terminal domain (NTD) and a receptor binding domain (RBD).S2 functions in membrane fusion to facilitate cell entry and contains a fusion peptide (FP) domain, an internal fusion peptide (IFP), two heptad repeat domains (HR1 and HR2), a transmembrane domain and a C-terminal domain.

[0016] After binding, the spike protein is activated by the host cell's transmembrane protease / serine subfamily member 2 (TMPRSS2), which results in the virus undergoing fusion with the endosomal membrane for cell entry. As the membrane fusion domain of the spike protein in coronaviruses is highly conserved, targeting membrane fusion may provide durable and long-lasting therapeutics.

[0017] The present invention provides a programmable rapid response platform for on-demand synthesis of potent anti-coronavirus materials. The platform preferably comprises a coronavirus fusion peptide inhibitor, a membrane anchoring moiety, and a multimeric core (B) that covalently links the peptide to the membrane anchoring moiety. The platform may further comprise a spike-binding peptide. The platform may further comprise a targeting peptide, such as an ACE2 targeting peptide or a receptor binding domain targeting peptide.

[0018] The compounds of the present invention have the general formula: (Peptide-Linker) n -B - Hydrophobic part wherein the linker is optional and B is a multimeric core covalently linking each peptide moiety to the hydrophobic moiety, comprising a cysteine, X, and optionally Y, and / or optionally Z, where X, Y and Z are defined herein and n is an integer selected from 1, 2, 3 or more. In a preferred embodiment, the compound is prepared by chemically conjugating each module as a separate building block. Click chemistry is preferably used for the conjugation.

[0019] Each peptide is independently an HRC peptide and / or a targeting peptide from a coronavirus spike protein, provided that at least one HRC peptide from a coronavirus spike protein is present. A preferred embodiment of the invention utilizes natural or wild-type peptides, but non-natural peptides can be used as well. Amino acids found in one or more mutations (e.g., omicron mutations) can be combined with natural sequences from other viruses (e.g., delta viruses). The so-called HRC peptides or HRC regions of coronavirus spike proteins are preferred. HRC peptides inhibit viral fusion, a critical early step in the infection process. Wild-type HRC peptides are conserved regions of the spike protein, i.e., the S protein, across coronaviruses. The conserved nature of the fusion region (HRC / HRN) and fusion mechanism of class I enveloped viruses makes them ideal targets for developing pan-coronavirus inhibitors.

[0020] A preferred wild-type HRC peptide is Ac n -DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO: 1) [where n is 0 or 1] and binding fragments thereof.

[0021] For the HRC peptide in SEQ ID NO:1, conventional amino acid numbering starts at 1150, D. 1151I, 1154I and 1158V are in the prefusion hydrophobic interface, and 1159V is exposed. These amino acids stabilize the helix. Upon conformational change (e.g., release of FP by protease clipping), 1158V becomes exposed and 1159V is in the hydrophobic surface that interacts with the HRN trimer. 1155N and 1176N are involved in N-linked glycosylation, which is conserved in coronaviruses. The first seven amino acids are involved in HRN binding. The "N-cap" region spans 1159V and 1171V. 1171V is a hydrophobic residue conserved in coronaviruses that stabilizes the HRC hydro-core and participates in HRN interaction.

[0022] The hydrophobic core spans 1161I and 1175L and is a helix in both pre-fusion and post-fusion. These isoleucines, leucines, and alanines are important for the folding and stability of the coiled coil. The C-cap region spans 1176N and 1185L. 1179L, 1182L, and 1185L are in the hydrophobic interface of the pre-fusion and 1180I is exposed. These amino acids stabilize the helix. 1185Y may be involved in the hydrophobic packing between the three polypeptide chains in the trimeric coiled coil. Upon conformational change (e.g., release of FP by protease clipping), 1179L becomes exposed and 1180I is in the hydrophobic surface that interacts with the HRN trimer. 1164E and 1184E form salt bridges between HRC and HRN. In addition, 1159V, 1160N, 1171V and 1180I have been shown to interact with HRN in the crystal structure. Figure 5 shows a cross-sectional view of the S protein trimer.

[0023] In certain embodiments, the peptide is Ac n -DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO: 1) [where n is 0 or 1] and binding fragments thereof.

[0024] In the context of protein mutants, the term "mutant" is defined as a peptide having at least one amino acid deletion, addition or substitution compared to the wild-type sequence, e.g., SEQ ID NO: 1 or other naturally occurring sequences described herein. Mutants preferably bind to the cognate ligand of the wild-type sequence. For example, peptides in which one, two, three, four or five amino acids of SEQ ID NO: 1 are substituted can be used. Such substituted amino acids can preferably be selected from one or more corresponding amino acids identified in different coronavirus strains by sequence alignment as shown above. For example, as described in the alignment above, one or both of the underlined isoleucines can be substituted with leucine and / or methionine. The underlined alanine can be substituted with valine, leucine or isoleucine. One or both of the underlined leucines can be independently substituted with isoleucine, tyrosine, alanine or valine. Other conservative or non-conservative substitutions (lysine for glutamine or aspartic acid for glutamic acid) can be similarly selected, e.g., as shown in the sequence alignment above. In embodiments, amino acids that are conserved among two, three, four, five or more coronaviruses (e.g., coronaviruses isolated from bats or mutant or variant strains of SARS-CoV2) remain conserved in the non-natural HRC peptide.

[0025] For example, the wild-type HRC sequence can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional amino acids at the N-terminus and / or C-terminus that are natural to the S protein. For example, a glycine can be added to the N-terminus. Furthermore, the wild-type HRC peptide fragment can inhibit infection even if 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted from the N-terminus and / or C-terminus. Typically, a total of 10 or fewer amino acids are deleted overall. For example, it can be expected that inhibitory activity will be retained even if those 10 amino acids are deleted from the C-terminus.

[0026] In certain embodiments, modifications to the wild-type sequence are desirable. For example, one or more D-amino acids may improve the pharmacokinetics and half-life of the peptide. Thus, in certain embodiments, the invention encompasses peptides characterized by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more D-amino acids. The D-amino acids can preferably be the corresponding L-amino acids of the wild-type sequence. In certain embodiments, the D-amino acids are amino acids located at or near (e.g., within 1, 2 or 3 amino acids) the protease cleavage site. In certain embodiments, the D-amino acids are hydrophobic amino acids that participate in binding to the HRN peptide, preferably with higher affinity than the corresponding wild-type sequence. Alternatively or additionally, the D-amino acids are hydrophilic amino acids, such as lysine, aspartic acid, glutamic acid or arginine. Alternatively or additionally, the D-amino acids can be selected from the seven amino acids at the N-terminus of SEQ ID NO:1. Peptides improved by incorporating D-amino acids are described in U.S. Patent Application No. 63 / 140,387, filed January 22, 2021, which is incorporated by reference in its entirety into this specification.

[0027] However, replacing one or more D-amino acids with the corresponding L-amino acids may alter the topology of the peptide, affecting its function. Thus, a preferred non-natural HRC peptide is a retro-inversion HRC peptide, or "RI HRC peptide." Retro-inversion HRC peptides are preferably characterized by a binding affinity to their cognate ligand that is at least about 50% of that of wild-type HRC peptide in standard binding assays, and reduced susceptibility to mammalian protease degradation. Retro-inversion is defined as inverting the D-peptide sequence of a helical peptide, i.e., "flipping" both ends, thereby restoring the presentation of the side chain to a binding ligand or target. See Kim et al, Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PBS, PNAS, February 13, 2018, 115(7)1505-1510, which is incorporated herein by reference in its entirety. Thus, a non-naturally occurring peptide of the invention can include a peptide having the sequence of SEQ ID NO:1, where the amino acids, e.g., amino acids in a region, are D-amino acids inverted so that the N-terminus becomes the C-terminus. For example, the N-terminus can be retro-inverted as shown in SEQ ID NO:2, where each D amino acid is preceded by a "d": dIdGdSdIdD NASVVN I QKE I DRLNEV A KN L NES L IDLQEL (sequence number 2).

[0028] This example gives a single RI region of 5 amino acids. However, it is also possible to select only two amino acids (e.g. the two N-terminal amino acids). For example, the RI region can span the hydrophobic core, 1160N-1176N, or the C-cap region or a portion thereof. Alternatively, the entire peptide can be an RI peptide. Furthermore, it is also possible to include two, three or more RI regions. For example, both the N-terminal region and the C-cap region can be RI regions while retaining the hydrophobic core of L-amino acids.

[0029] For example, when using mirror image phage display to screen HRC mutants, the first D-peptide can be synthesized from the HRN coronavirus peptide or the first L-peptide. The first L-peptide can be a naturally occurring L-peptide or a peptide chimera. The method can further include screening the HRC peptide or the second L-peptide that specifically binds to the first D-peptide, and then synthesizing a second D-peptide that is a mirror image of the second L-peptide. In one embodiment of the D-peptide screening method described herein, the N-trimer target can be first synthesized with D-amino acids to create a mirror image of the natural LN-trimer target. The DN-trimer target can be used in standard peptide-based screening, such as phage display, ribosome display, and / or CIS display, to identify L-peptides that bind to the DN-trimer. The identified L-peptide can then be synthesized with D-amino acids. By the rules of symmetry, the resulting D-peptides will bind to the natural LN-trimer and thus target the N-trimer region of the coronavirus HRN intermediate, thereby inhibiting infection. This screening method is also described in Schumacher, et al., Identification of D-peptide ligands through mirror-image phage display, Science, 1996 Mar 29;271(5257):1854-7, which is incorporated herein by reference in its entirety.

[0030] Hotspot residues of HRC peptides can be identified by the crystal structure or NMR solution structure of HRC peptides. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids selected from 1150D, 1151I, 1154I, 1155N, 1158V, 1159V, 1161I, 1164E, 1171V, 1175L, 1176L, 1179L, 1180I, 1182L, 1184E and / or 1185Y, such as one or more amino acids selected from 1159V, 1160N, 1163E, 1171V, 1180I, 1184E and / or 1185L of SEQ ID NO: 1, can be designated as hotspot residues.

[0031] In some embodiments, the peptide comprises a cell targeting peptide. In some embodiments, the targeting peptide is selected from, but not limited to, an ACE2 targeting peptide or a receptor binding domain targeting peptide. In some embodiments, the targeting peptide is an ACE2 targeting peptide. In some embodiments, the targeting peptide is a receptor binding domain targeting peptide.

[0032] In embodiments where the compound comprises only one peptide, the peptide is an HRC peptide derived from a coronavirus spike protein. In embodiments where the compound comprises two or more peptides, each peptide can be an HRC peptide derived from a coronavirus spike protein. In embodiments where the compound comprises two or more peptides, the peptides can be selected from HRC peptides derived from a coronavirus spike protein and / or targeting peptides, provided that there is at least one HRC peptide derived from a coronavirus spike protein. Preferably, only one of the peptides is a targeting peptide, and the other peptide or peptides are HRC peptides derived from a coronavirus spike protein.

[0033] In certain embodiments, the optional "linker" is defined as a divalent moiety or group that is covalently attached to the peptide (preferably at its C-terminus or N-terminus) and also covalently attached to B. In embodiments, the linker can have 1, 2, 3, 4, 5 or more subunits or segments. In embodiments, the linker comprises one or more amino acid subunits. The amino acids may be naturally occurring or synthetic. Thus, the linker may be (Gly) n+1 , (GlySerGly) n or (Gly-Pro) n (wherein n is 1 or more, for example 1 to 12, 1 to 6 or 1 to 4). GlySerGly is an example of a sequence of amino acids that may form a linker or part of a linker.

[0034] In certain embodiments, the linker may include a non-amino acid subunit. In some embodiments, an example of a non-amino acid subunit of the linker is -(OCH2CH2). m -, where m is 1 to 15, for example 2 to 10, 2 to 6, or 4. The introduction of a (poly)ethylene glycol group aids in solubility in aqueous media. In some embodiments, examples of non-amino acid moieties of the linker are -CH2C(O)- and -CH2C(O)NHCH2CH2(OCH2CH2)4C(O)-.

[0035] For example, it may be advantageous to use a linker having three subunits. The first optional subunit is a flexible peptide, e.g. -(G) m -or-(GS) mG-, where m is an integer of 1, 2, 3, 4, 5 or more, e.g., 2. The second subunit can be a residue of a chemical reaction (such as a click chemistry reaction), e.g., a peptide bond, ester, ether, or thioether, involving the N-terminus, C-terminus, or side chain of the first subunit. The residue can be non-cleavable, such as those formed with carbodiimides or sulfhydrylmaleimides. The third optional subunit can be a hydrophilic spacer, e.g., polyethylene glycol, polyethyleneamine, polyacetal polymer, poly(1-hydroxymethylethylenehydroxymethyl-formal) (PHF), or carbohydrate. The hydrophilic spacer is generally a polymer and can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. Polyethylene glycol (PEG4) with four monomers is sufficient. The length of the hydrophilic spacer can correspond to the full length of the S protein gap, to facilitate orientation of the peptide for binding to the HRN domain. PEG can also reduce aggregation and improve solubility. The other subunit can be the residue of a chemical reaction (such as a click chemistry reaction) between B and a linker or its subunit.

[0036] B can be a multimeric core that provides a framework that covalently links one or more peptide-linker moieties to hydrophobic moieties. B can include one or more cysteines, one or more X, and optionally Y, and / or optionally Z, where X, Y, and Z are defined herein. In some embodiments, the one or more cysteines connect one or more peptide-linker moieties to other components of B.

[0037] There is a need to be able to covalently link peptides to B using tunable and simple chemical reactions that are non-toxic to cells and can occur at fast rates under biological conditions. The present invention addresses this need. In a broad aspect, the present disclosure provides click chemistry compatible functional groups, also called "click chemistry handles", that react via click chemistry.

[0038] As used herein, "click chemistry compatible" structures, functional groups, monomers, oligomers, etc. should be understood to refer to compounds, materials, etc. that are structurally characterized by including one or more chemical moieties suitable for participating in a click chemistry reaction.

[0039] A click chemistry handle is a chemical moiety that provides a reactive group that can participate in a click chemistry reaction. Click chemistry reactions and suitable chemical groups for click chemistry reactions are well known to those skilled in the art, and include, but are not limited to, terminal alkynes, azides, strained alkynes, dienes, dienophiles, alkoxyamines, carbonyls, phosphines, hydrazides, thiols, and alkenes. For example, in some embodiments, azides and alkynes are used in click chemistry reactions.

[0040] Some embodiments of the invention provide modified peptides, such as proteins, that contain a C-terminal or N-terminal click chemistry handle, in which case such peptides can be covalently conjugated to B, which contains a moiety that can react with the click chemistry handle of the protein.

[0041] In embodiments in which copper-catalyzed azide-alkyne cycloaddition (CuAAC) is the click chemistry used to functionalize the materials disclosed herein, the "click chemistry-compatible" compounds contain terminal alkyne and / or terminal azide functional groups.

[0042] An exemplary click chemistry is CuAAC, although one of skill in the art will appreciate that other click chemistry compatible reactions that would be understood to be equivalent to CuAAC may be used without departing from the scope of the inventive concepts described herein. For example, in various embodiments, click chemistry compatible reactions may include strained alkene reactions such as CuAAC, strain-promoted azide-alkyne cycloaddition (SPAAC), strain-promoted alkyne-nitrone cycloaddition (SPANC), alkene-azide cycloaddition, and the like. As will be understood by one of skill in the art upon reading this disclosure, click chemistry compatible reactions may also be considered to include alkene-tetrazine reverse solicited Diels-Alder reactions, alkene-tetrazole photoclick reactions, Michael addition of thiols, nucleophilic substitution of thiols with amines, as well as certain Diels-Alder reactions and their equivalents as disclosed by Becer, et al. "Click chemistry beyond metal-catalyzed cycloaddition." Angew. Chem. Int. Ed. 2009, 48: p. 4900-4908.

[0043] Thus, in various embodiments, click chemistry compatible groups, compounds, etc. should be understood to include one or more suitable chemical moieties that convey the ability to participate in any combination of the exemplary click chemistries set forth above.

[0044] B is a multimeric core that provides a framework that covalently links one or more peptide-linker moieties to hydrophobic moieties. B comprises one or more cysteines, one or more X, and optionally Y, and / or optionally Z, where X, Y, and Z are defined herein.

[0045] The one or more cysteine ​​residues, one or more X, optional Y and optional Z can be in any order, where the first listed component of B is attached to the peptide-linker and the last listed component is attached to the hydrophobic moiety. For example, if B comprises, in order, one or more cysteine ​​residues, one or more X, and Z, the peptide-linker is attached to the one or more cysteines and Z is attached to the hydrophobic moiety. In some embodiments, B comprises, in order, one or more cysteines and one or more X. In some embodiments, B comprises a cysteine ​​and X.

[0046] In embodiments, B comprises one or more cysteines, one or more X, and Z. In embodiments, B comprises Z, one or more cysteines, and one or more X.

[0047] In embodiments, B comprises Y, one or more cysteines, and one or more X. In embodiments, B comprises Y, one or more cysteines, one or more X, and Z.

[0048] In embodiments, the one or more cysteines link the one or more peptide-linker moieties to the other components of B. In embodiments, the one or more cysteines are attached to the one or more X via a thioether bond. In embodiments, the one or more cysteines have the following structure: [ka] [In the formula, R4 is OH or NH2] has.

[0049] In embodiments, X comprises one or more sulfur aryl linkages, nitrogen aryl linkages, or other linkages, such as triazole, amide, sulfur-sp3 carbon bond, or hydrophilic linker. In embodiments, the hydrophilic linker is selected from, for example, polyethylene glycol (PEG), polyethyleneimine, polyacetal polymer, poly(1-hydroxymethylethylenehydroxymethyl-formal) (PHF), or carbohydrate. In embodiments, the hydrophilic linker is a polymer and can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. In embodiments, the hydrophilic linker is polyethylene glycol (PEG). In embodiments, the hydrophilic linker is polyethylene glycol (PEG4) with four monomers. In one embodiment, X comprises one or more sulfur aryl linkages. In one embodiment, X comprises one or more nitrogen aryl linkages. In one embodiment, X comprises one or more sulfur-sp3 carbon bonds.

[0050] In embodiments, the one or more X's are attached directly to the hydrophobic moiety by a thioether bond. In embodiments, the one or more X's are attached to Z, if present, which is then attached to the hydrophobic moiety.

[0051] In certain embodiments, B further comprises Y. In embodiments, when Y is present, Y is interposed between the one or more peptide-linker moieties and the one or more cysteines, linking the one or more peptide-linker moieties to the one or more cysteines which in turn are linked to other components of B.

[0052] In embodiments, Y comprises one or more amino acids. The amino acids may be naturally occurring or synthetic. Y may comprise one or more, e.g., 1-12, 1-6, or 1-4 amino acids. The one or more amino acids may be added to the linker in a stepwise manner. For example, a first amino acid is added to a cysteine ​​of B, then a peptide-linker is attached to the first amino acid prior to the addition of the second amino acid. After the peptide-linker is attached to the first amino acid, the next amino acid is attached to the previous amino acid to allow for the attachment of a further peptide-linker, etc. In some embodiments, the amino acid of the linker is one or more lysines.

[0053] In embodiments, when Z is present, it is interposed between X and the hydrophobic moiety and links B to the hydrophobic moiety.

[0054] In embodiments, when Z is present, it is selected from the group consisting of formula (I): [ka] and a moiety having the structure In the formula, each of R1 and R2 is (i) R3 (ii) Formula (II): [ka] The structure of and (iii) Formula (III): [ka] Structure of are independently selected from the group consisting of During the ceremony, In each case, W is -HC(O)-O-, -OC(O)-H-, -C(O)-O-, -OC(O)-, -(CH2) mand, most preferably, W is -C(O)-H-; V is in each case -(CH2) m -, -(CH2) m -C(X)-H-, -HC(X)-(CH2) m -, -(CH2) m -HC(O)-O-, -OC(O)-H-(CH2) m -, -(CH2) m -C(O)-O-, -OC(O)-(CH2) m and, most preferably, V is -CH2CH2-C(O)-H-; D is in each case either O, S or H, A at each occurrence is independently selected from -C(O)CH-, -CHC(O)-, -HCH-, -CHH-, -HC(O)-, -C(O)H-, -H-, -CH-, -CHC(O)H- and -HC(O)CH-, most preferably Y is -HCH-; Q, at each occurrence, is independently selected from -CH2-, -H-, -O-, -CH2O-, -HCH2-, and -OCH2-, most preferably Z is -O-; R3, in each case, is independently selected from any of the above polypeptides, and they may be the same or different; m in each occurrence is independently selected from an integer from 0 to 5, i.e. 0, 1, 2, 3, 4, or 5, preferably 0 to 3, preferably m is the same in each occurrence; n in each case is independently selected from an integer from 0 to 40, i.e. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, preferably from 3 to 10, and preferably n is the same in each case; o in each occurrence is independently selected from an integer from 0 to 5, i.e., 0, 1, 2, 3, 4, or 5, preferably 2, and preferably o is the same in each occurrence; p in each occurrence is independently selected from an integer from 0 to 5, i.e. 0, 1, 2, 3, 4, or 5, preferably 0 to 3, and preferably p is the same in each occurrence; q is in each case independently selected from an integer from 0 to 5, i.e. 0, 1, 2, 3, 4 or 5, preferably 0 to 3, preferably q is the same in each case, and / or preferably q <pであり、 M is the hydrophobic moiety; In the formula, * represents the position where structures (II-III) are linked to structure (I).

[0055] In embodiments, when Z is present, it is Formula (I): [ka] It does not contain a portion having the structure:

[0056] In embodiments, when Z is present, it is selected from the group consisting of formula (IV): [ka] and a moiety having the structure wherein R5 is selected from a hydrophilic linker selected from polyethylene glycol (PEG), polyethyleneimine, polyacetal polymer, poly(1-hydroxymethylethylenehydroxymethyl-formal) (PHF) or a carbohydrate, and the like; W, in each instance, is independently selected from a direct bond or a hydrophilic linker selected from polyethylene glycol (PEG), polyethyleneimine, polyacetal polymer, poly(1-hydroxymethylethylenehydroxymethyl-formal) (PHF) or a carbohydrate, and the like.

[0057] In some embodiments, the hydrophilic linker of R5 is a polymer and can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. In some embodiments, the hydrophilic linker of R5 is polyethylene glycol (PEG). In some embodiments, the hydrophilic linker of R5 is polyethylene glycol (PEG4) with four monomers.

[0058] In embodiments, the hydrophilic linker of W, in each instance, can be independently polymeric and can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. In embodiments, the hydrophilic linker is polyethylene glycol (PEG). In embodiments, the hydrophilic linker of W, in each instance, can be independently polyethylene glycol (PEG4) with four monomers.

[0059] The hydrophobic moiety can be a membrane-integrating lipid, such as cholesterol, sphingolipid, sphingomyelin, glycolipid, glycerophospholipid (such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine), ergosterol, 7-dihydrocholesterol and stigmasterol.Preferably, it is cholesterol.Typically, B is linked directly or indirectly to the hydroxyl group of cholesterol, such as 3-OH.The hydrophobic moiety can facilitate the insertion of the compound of the present invention into cell membrane and inhibit the entry of viruses.

[0060] B may be attached at any convenient position on the hydrophobic moiety. In some embodiments, attachment may be via a hydroxy group on the hydrophobic moiety. For example, if the hydrophobic moiety is cholesterol, B may be attached via the group -C(O)- or -C 1~4 It may be attached to cholesterol by an alkylene C(O)-, such as -CH2C(O)-.

[0061] Phospholipids include lipids such as egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and phosphatidic acid (EPA), those lipids derived from soybean, soybean phosphatidylcholine (SPC), SPG, SPS, SPI, SPE, and SPA, those lipids derived from hydrogenated egg and soybean (e.g., HEPC, HSPC), other phospholipids composed of ester bonds of fatty acids containing chains of 12 to 26 carbon atoms at the 2- and 3-positions of glycerol and various head groups including choline, glycerol, inositol, serine, ethanolamine at the 1-position of glycerol, and the corresponding phosphatidic acids. The chains on these fatty acids can be saturated or unsaturated, and phospholipids can be composed of fatty acids of various chain lengths and various degrees of unsaturation. In particular, the compositions of the present formulations may include dipalmitoylphosphatidylcholine (DPPC), the main component of naturally occurring pulmonary surfactant. Other examples include mixed phospholipids such as dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatideholine (DPPQ) and dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPQ) and distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolamine (DOPE), and palmitoylstearoylphosphatidyl-choline (PSPC) and palmitoylstearolphosphatidylglycerol (PSPG), and monoacylated phospholipids such as monooleoylphosphatidylethanolamine (MOPE).

[0062] Examples of cholesterols include cholesterol, cholesterol esters such as cholesterol hemisuccinate, cholesterol salts such as cholesterol hydrogen sulfate, ergosterol, ergosterol esters such as ergosterol hemisuccinate, ergosterol salts such as ergosterol hydrogen sulfate and ergosterol sulfate, lanosterol, lanosterol esters such as lanosterol hemisuccinate, lanosterol salts such as lanosterol hydrogen sulfate and lanosterol sulfate. Examples of tocopherols include tocopherol, tocopherol esters such as tocopherol hemisuccinate, tocopherol salts such as tocopherol hydrogen sulfate and tocopherol sulfate.

[0063] In embodiments, the compound of the invention has the structure shown in FIG. 1H (also referred to as "Decoy 101" or "DCOY101").

[0064] In embodiments, the compound of the invention has the structure shown in FIG. 1I (also referred to as "Decoy 102" or "DCOY102").

[0065] In embodiments, the compound of the invention has the structure shown in FIG. 1G (also referred to as "Decoy 103" or "DCOY103").

[0066] The linker of DCOY102 and DCOY103 is a new linker, which gives improved stability over DCOY101. Furthermore, DCOY102 and DCOY103 show improved solubility in PBS buffer as follows: Decoy101: about 0.04 mg / mL = 0.004 mM; Decoy102: about 0.35 mg / mL = 0.035 mM, Decoy103: about 0.19 mg / mL = 0.018 mM.

[0067] The composition of the present invention comprises a compound described herein and a pharma- ceutically acceptable carrier. For example, the composition can be administered systemically or locally. The composition can be administered, for example, for oral, intravenous, intramuscular, rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, inhalation, or vaginal delivery. Thus, the composition can take the form of, for example, a tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel, including hydrogel, paste, ointment, cream, plaster, drench, osmotic delivery device, suppository, enema, injection, implant, spray, or aerosol. The composition can be formulated according to conventional pharmaceutical practice (see, for example, Remington: The Science and Practice of Pharmacy, 22). nd edition,2013,ed.LVAllen,Pharmaceutical Press,Philadelphia and Encyclopedia of Pharmaceutical Technology,4 th Edition, ed. J. Swarbrick, 2013, CRC Press, New York).

[0068] The compounds can be formulated in a variety of ways known in the art. For example, one or more compounds of the present invention and any additional biologically active agent, if present, as defined herein, can be formulated together or separately.

[0069] Each compound of the present invention, alone or in combination with one or more active agents described herein, can be formulated for controlled release (e.g., sustained or metered) administration, as described in U.S. Patent Application Publication Nos. 2003 / 0152637 and 2005 / 0025765, which are incorporated herein by reference.For example, the compound of the present invention, alone or in combination with one or more biologically active agents described herein, can be incorporated into a capsule or tablet that is administered to a patient.

[0070] Controlled release formulations known in the art include specially coated pellets, polymer preparations or polymer matrices for surgical insertion or as sustained release microparticles, such as microspheres or microcapsules for implantation, insertion, injection or injection, where the sustained release of the active pharmaceutical agent is brought about by sustained or controlled diffusion from the matrix and / or selective degradation of the preparation's coating or selective degradation of the polymer matrix. Other formulations or vehicles for controlled, sustained or immediate delivery of the agent to a preferred localized site in the patient include, for example, suspensions, emulsions, gels, liposomes, and any other suitable delivery vehicle or formulation known in the art that is acceptable for subcutaneous or intramuscular administration.

[0071] Any suitable biocompatible polymer can be utilized as the controlled release material. The polymeric material may include a biocompatible biodegradable polymer, preferably a copolymer of lactic acid and glycolic acid in certain preferred embodiments. Preferred controlled release materials useful in the formulations of the present invention include polyanhydrides, polyesters, copolymers of lactic acid and glycolic acid (where the weight ratio of lactic acid to glycolic acid is preferably 4:1 or less, i.e., 80% or less lactic acid to 20% or more glycolic acid by weight), and polyorthoesters containing a catalyst or degradation-promoting compound, e.g., containing at least 1% by weight of anhydride catalyst, e.g., maleic anhydride. Examples of polyesters include polylactic acid, polyglycolic acid, and polylactic acid-polyglycolic acid copolymers. Other useful polymers include protein polymers, such as collagen, gelatin, fibrin, and fibrinogen, and polysaccharides, such as hyaluronic acid.

[0072] In further embodiments, the controlled release material, which in effect acts as a carrier for the compounds of the invention, may further comprise a bioadhesive polymer, such as pectin (polygalacturonic acid), mucopolysaccharides (hyaluronic acid, mucin) or non-toxic lectins, or the polymer itself may be a bioadhesive, such as a polyanhydride or a polysaccharide, such as chitosan. In embodiments in which the biodegradable polymer comprises a gel, one such useful polymer is a thermogelling polymer, such as a polyethylene oxide, polypropylene oxide (PEO-PPO) block copolymer, such as PLURONIC F127 from BASF Wyandotte.

[0073] Formulations for oral use include tablets containing one or more active ingredients in a mixture with non-toxic pharma- ceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches such as potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives such as microcrystalline cellulose, starches such as potato starch, croscarmellose sodium, alginates, or alginic acid), binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol), and lubricants, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other examples of pharma- ceutically acceptable excipients include colorants, flavoring agents, plasticizers, humectants, buffers, and flavoring agents (eg, hydroxypropylmethylcellulose, hydroxypropylcellulose).

[0074] One or more compounds of the present invention may be mixed together or dispensed in a tablet, capsule or other vehicle. In one example, a compound of the present invention is contained on the inside of the tablet and the biologically active agent is on the outside of the tablet, such that a substantial portion of the biologically active agent is released prior to the release of the compound of the present invention.

[0075] Formulations for oral use may be provided as chewable tablets or as hard gelatin capsules in which one or more active ingredients are mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredients are mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil. Powders, granules, and pellets may be prepared in a conventional manner, for example using mixers, fluidized bed equipment, or spray drying equipment, using the ingredients mentioned above in the tablet and capsule sections. Oral preparations may also be provided as mouthwashes, mouth sprays, mouthwashes, or mouth ointments, or mouth gels.

[0076] Dissolution or diffusion controlled release can be achieved by suitable coating of tablet, capsule, pellet or granule formulations of the compound or by incorporating the compound in a suitable matrix. The controlled release coating can include one or more of the above mentioned coating materials and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitosterate, ethyl cellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3-butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In controlled release matrix formulations, the matrix material may also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons.

[0077] Liquid forms into which the compounds and compositions of the present invention can be incorporated for oral administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions using edible oils, such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0078] Suitable formulations for parenteral administration (e.g., by injection) include aqueous or non-aqueous isotonic pyrogen-free sterile liquids (e.g., solutions, suspensions) in which the compound is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may further contain other pharma- ceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the compound in the liquid is about 1 ng / ml to about 10 μg / ml, e.g., about 10 ng / ml to about 1 μg / ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0079] The composition of the present invention can comprise a liquid medium suitable for nasal administration.The medium is preferably an aqueous solution.More preferably, the medium is an aqueous solution that contains a thickening agent and optionally one or more additional excipients, such as those that improve the stability and / or comfort of administration of the formulation.

[0080] A variety of thickening agents are known in the art. Thickening agents include hydrophilic polymers, such as polysaccharides, polysaccharide derivatives, proteins and synthetic polymers. Examples include, but are not limited to, gum arabic, gum tragacanth, alginic acid, carrageenan, locust bean gum, guar gum, gelatin, hyaluronic acid, polyacrylates, polyacrylate / alkyl acrylate copolymers, polyvinyl alcohol, polyvinylpyrrolidone, starch, polypropylene glycol alginate, maltodextrin, and cellulose ether derivatives, such as methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, and carboxymethylcellulose. Where possible, the salt form of any of the above substances is preferred. Preferred thickening agents include hyaluronic acid, such as sodium hyaluronate, carboxymethylcellulose, such as sodium carboxymethylcellulose and calcium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and hydroxypropylcellulose.

[0081] The composition optionally includes one or more additional excipients, such as those that enhance ease of administration, comfort of the subject, or stability of the composition. Suitable additional excipients include, but are not limited to, tonicity adjusting agents such as sodium chloride and dextrose, antioxidants such as butylated hydroxyanisole, buffers such as sodium bicarbonate, sodium citrate and sodium phosphate, preservatives such as benzalkonium chloride, ethanol, propylene glycol, benzoyl alcohol, phenethyl alcohol, chlorobutanol or methylparaben, pH adjusting agents such as hydrochloric acid, sulfuric acid, sodium hydroxide, surfactants such as polysorbate 80, polysorbate 20 and polyoxyl 400 stearate, chelating agents such as disodium EDTA, antioxidants, cosolvents such as ethanol, PEG400 and propylene glycol, penetration enhancers such as oleic acid, and humectants such as glycerin (S. Thorat, Sch. J. App. Med. Sci. 2016,4(8D):2976-2985; D. Marx et al. al., IntechOpen, DOI:10.5772 / 59468, available at intechopen.com / books / drug-discovery-and-development-from-molecules-to-medicine / intranasal-drug-administration-an-attractive-delivery-route-for-some-drugs.

[0082] In one embodiment, the vehicle consists of sodium hyaluronate, aloe vera, allantoin, sodium chloride, sodium bicarbonate, glycerin, propylene glycol, benzalkonium chloride, and USP grade purified water. A suitable vehicle is sold by NEILMED™ under the trademark NASOGEL™.

[0083] The amount of active agent in the composition can vary, for example, from about 0.5% to about 25% by weight.

[0084] The pH of the formulation is acceptable within the nasal cavity, and is preferably at least about 8.0. Buffers that can be used in the composition include, but are not limited to, phosphate, Tris, [tris(hydroxymethyl)methylamino]propanesulfonic acid, 2-(bis(2-hydroxyethyl)amino)acetic acid, and N-[tris(hydroxymethyl)methyl]glycine, and Alkaline Buffer (Seachem).

[0085] A pharmaceutical composition suitable for nasal or pulmonary administration, comprising a water-soluble solvent selected from the group consisting of propylene glycol, glycerin, polyethylene glycol, and combinations thereof. The composition may further comprise one or more of a polysaccharide gum, a non-ionic surfactant, and a preservative. An exemplary polysaccharide gum is sclerotium gum. An exemplary surfactant is a poloxamer, such as, but not limited to, poloxamer 188. The preservative may be, for example, benzalkonium chloride.

[0086] The compositions can be either a dry powder, suspended in a propellant and delivered by a dry powder inhaler, or as an aqueous suspension or solution, delivered by a nebulizer.

[0087] For example, a solution or suspension of an active agent and a pulmonary excipient, such as lactose, can be spray-dried to form particles having a fine particle fraction sufficient for delivery to the lungs or upper respiratory system. Alternatively, an aqueous solution or suspension can be sonicated, thereby aerosolizing the solution / suspension into droplet sizes that can be inhaled, such as by a nebulizer.

[0088] Excipients include carbohydrates, such as monosaccharides, disaccharides and polysaccharides. For example, monosaccharides such as dextrose (anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol, sorbose, disaccharides such as lactose, maltose, sucrose, trehalose, trisaccharides such as raffinose, other carbohydrates such as starch (hydroxyethyl starch), cyclodextrins and maltodextrins. Other excipients suitable for use in the present invention are known in the art, including amino acids, such as those disclosed in WO95 / 31479, WO96 / 32096 and WO96 / 32149. Furthermore, mixtures of carbohydrates and amino acids are also within the scope of the present invention. The inclusion of both inorganic (e.g. sodium chloride, etc.), organic acids and their salts (e.g. carboxylic acids and their salts, e.g. sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, etc.) and buffers is also envisioned.

[0089] The composition can be used in the form of dry powder or in the form of a stabilized dispersion containing a non-aqueous phase.Thus, the dispersion or powder of the present invention can be used together with metered dose inhaler (MDI), dry powder inhaler (DPI), sprayer, nebulizer or liquid dose instillation (LDI) technology to provide for effective drug delivery.With respect to inhalation therapy, those skilled in the art will understand that the hollow and porous microparticles of the present invention are particularly useful in DPI.Traditional DPI includes powder formulations and devices in which a predetermined dose of medicament is delivered for inhalation as a dry powder aerosol, alone or combined with lactose carrier particles.

[0090] The medicament is formulated so that it disperses readily into discrete particles, the mass median aerodynamic diameter of the powder typically being in the range of about 0.5 to 10, preferably about 0.5 to 5.0 microns MMAD.

[0091] As discussed above, the stabilized dispersions disclosed herein may also be administered to a patient's nasal or pulmonary airways by aerosolization, for example, using a metered dose inhaler. MDIs are well known in the art and may be readily used to administer the present dispersions without undue experimentation. Breath-actuated MDIs, including other types of improvements already developed or to be developed, are also contemplated as being compatible with the stabilized dispersions and the present invention and are therefore within the scope. However, it should be emphasized that in preferred embodiments, the stabilized dispersions may be administered by MDIs using a number of different routes, including, but not limited to, topical, nasal, pulmonary, or oral. Those skilled in the art will appreciate that such routes are well known and may readily devise dosages and administration procedures for the stabilized dispersions of the present invention.

[0092] In conjunction with the above-described embodiments, the stabilized dispersions of the present invention may be used in conjunction with a nebulizer as disclosed in PCT WO99 / 16420, the disclosure of which is incorporated herein by reference in its entirety, to provide an aerosolized medicament that can be administered to the pulmonary airways of a patient in need thereof. Nebulizers are well known in the art and could be readily used to administer the dispersions of the present application without undue experimentation. Breath-actuated nebulizers, including other types of improvements already developed or to be developed in the future, are also contemplated as being compatible with and within the scope of the stabilized dispersions and the present invention.

[0093] It will be understood that, in addition to DPIs, MDIs and nebulizers, the stabilized dispersion of the present invention can be used with liquid dose instillation techniques, i.e. LDI techniques, as disclosed, for example, in WO99 / 16421, the entirety of which is incorporated herein by reference. In liquid dose instillation, the stabilized dispersion is administered directly to the lung. In this regard, direct pulmonary administration of bioactive compounds is particularly effective, especially in the treatment of disorders in which poor vascular circulation in diseased parts of the lung reduces the effectiveness of intravenous drug delivery. With respect to LDI, the stabilized dispersion is preferably used in conjunction with partial or total liquid ventilation. Furthermore, the present invention can further comprise introducing a therapeutically beneficial amount of a physiologically acceptable gas, such as nitric oxide or oxygen, into the pharmaceutical microdispersion before, during or after administration.

[0094] How to use The present invention also includes a method of using the composition of the present invention to treat or prevent an infectious disease in a subject in need of such treatment or prevention. The method includes administering an effective amount of the composition to the subject. The infectious disease can be an infection of the gastrointestinal tract or the upper or lower respiratory tract, such as the common cold, influenza, respiratory syncytial virus infection, severe acute respiratory syndrome, Middle East respiratory syndrome, COVID-19, or a disease caused by another emerging zoonotic virus, such as a zoonotic coronavirus. In certain embodiments, the method of the present invention treats a viral respiratory infection, such as a SARS-CoV-2 (COVID-19) respiratory infection.

[0095] In one embodiment, the present invention provides a method for treating a respiratory infection caused by a mutant SAR-Cov-2 strain in a subject in need of such treatment. In some embodiments, the method comprises administering to a subject an effective amount of a peptide of the formula: (peptide-linker) nadministering to a subject a compound having a B-hydrophobic moiety, wherein each peptide is independently an HRC peptide or a targeting peptide, with the proviso that at least one peptide is an HRC peptide, each linker is independently a bivalent linking moiety, and B is a multivalent moiety comprising a cysteine, X, and optionally Y, and / or optionally Z, where X, Y and Z are defined herein and n is an integer selected from 1, 2, 3 or more, wherein said mutant comprises at least five mutations, and said at least five mutations are independently in the spike protein S1 subunit or S2 subunit or a combination thereof.

[0096] In some embodiments, the compound comprises a targeting peptide and one or more HRC peptides. In some embodiments, the targeting peptide is an ACE2 targeting peptide or a receptor binding domain peptide. In some embodiments, the hydrophobic moiety is cholesterol.

[0097] In some embodiments, the at least five mutations are independently in the N-terminal domain (NTD), the receptor binding domain (RBD), the fusion peptide (FP) domain, the heptad repeat 1 (HR1) domain, or a combination thereof. In some embodiments, the at least five mutations are independently selected from the mutations listed in FIG.

[0098] In some embodiments, the at least five mutations are independently selected from at least five mutations from a SAR-Cov-2 alpha variant, at least five mutations from a SAR-Cov-2 beta variant, at least five mutations from a SAR-Cov-2 delta variant, or at least five mutations from a SAR-Cov-2 omicron variant. In some embodiments, the at least five mutations are independently selected from at least five mutations from a SAR-Cov-2 alpha variant. In some embodiments, the at least five mutations are independently selected from at least five mutations from a SAR-Cov-2 beta variant. In some embodiments, the at least five mutations are independently selected from at least five mutations from a SAR-Cov-2 delta variant. In some embodiments, the at least five mutations are independently selected from at least five mutations from a SAR-Cov-2 omicron variant.

[0099] In some embodiments, the mutant strain comprises at least 10 mutations. In some embodiments, the mutant strain comprises at least 15 mutations. In some embodiments, the mutant strain comprises at least 20 mutations.

[0100] In embodiments, the SARS-CoV-2 variants include at least one variant selected from B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), B.1.429 / B.1.427 (epsilon), B.1.617.1 (kappa), B.1.525 (eta), B.1.526 (iota), P.3 (theta), P.2 (zeta), and B.1.1.529 (omicron).

[0101] In several embodiments, the SARS-CoV-2 variants include at least one variant selected from A.1-A.6, B.3-B.7, B.9, B.10, B.13-B.16, B.2, B.1 lineage, P.1, P.2, P.3, and R.1.

[0102] In some embodiments, the B.1 lineage may be any of a variety of strains, including, but not limited to, B.1, B.1.1, B.1.1.7, B.1.1.7 with E484K, B.1.2, B.1.5 to B.1.72, B.1.9, B.1.13, B.1.22, B.1.26, B.1.37, B.1.3 to B.1.66, B.1.177, B.1.24 3, and at least one of B.1.313, B.1.351, B.1.427, B.1.429, B.1.525, B.1.526, B.1.526.1, B.1.526.2, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, B.1.619, B.1.620, and B.1.621.

[0103] In some embodiments, administration is accomplished using an intranasal spray, inhaler, or nebulizer.

[0104] In embodiments, the compound is administered in combination with at least one other antiviral active agent or treatment.

[0105] The subject, preferably a human, can be an individual diagnosed with an infectious disease, either symptomatic, pre-symptomatic, or asymptomatic, or at risk of developing an infectious disease. For example, the subject can be at risk of developing a viral respiratory infection due to direct or indirect exposure or potential exposure to the virus (SARS-CoV-2 or its variants), for example, by exposure to an infected individual or a virally contaminated fomites. The subject can be a resident or visitor in an area where a viral respiratory infection has been identified, for example, the subject can be a family member of an infected person, or the subject can be a person working in a medical setting who provides medical care to an infected person. In certain embodiments, the subject at risk of infection is asymptomatic and has tested negative for the presence of the virus before the start of treatment. In a specific embodiment, the subject can be at risk of developing COVID-19 due to exposure to the SARS-CoV-2 virus, for example, from respiratory droplets or aerosols of an infected person and / or by contact with a contaminated fomites. In further embodiments, the subject has COVID-19, including subjects with mild, moderate or severe COVID-19.

[0106] In certain embodiments of the methods of the invention, the subject suffers from another disease or condition, such as chronic obstructive pulmonary disease (COPD) or ulcerative colitis, that may be exacerbated by infection.

[0107] The composition is preferably administered to the subject before the subject becomes symptomatic (e.g., pre-symptomatic) or at the onset of symptoms. The composition can be administered in various dosing schedules. For example, the composition can be administered once or multiple times over one or several days. In certain embodiments, the composition is administered once or multiple times per day for 1-10 days. In certain embodiments, the composition is administered once or multiple times per day until the subject becomes asymptomatic and / or the virus tests negative.

[0108] The composition can be administered to the nasal passages using routine methods and devices (see D. Marx et al., IntechOpen, DOI:10.5772 / 59468, available at https: / / www.intechopen.com / books / drug-discovery-and-development-from-molecules-to-medicine / intranasal-drug-administration-an-attractive-delivery-route-for-some-drugs). For example, the composition can be administered to the nasal passages as droplets or as an aerosol spray, such as with an aerosol bottle or multi-dose spray pump that can provide a uniform metered dose. The volume per dose varies, but is typically about 50 to about 150 μl. The desired volume will depend on the desired dose of the active agent and the concentration of the active agent in the composition.

[0109] When delivery to the pulmonary system or lungs is desired, it may be effective to aerosolize a low concentration solution of the active agent for an extended period of time, for example overnight.

[0110] Combination treatment The compounds or compositions described herein can be co-administered with other active agents and treatments.

[0111] In some embodiments, the other active agent includes, but is not limited to, an antibody against SARS-CoV-2. Suitable antibodies are described, for example, in US2022 / 0017604, US2022 / 0017614, US2021 / 0403550, US2021 / 0395345, US2021 / 0403537, US2021 / 0388066, US2021 / 0388065, US2021 / 0347859, or US2021 / 0309733, which are incorporated herein by reference. In some embodiments, the antibody is a monoclonal antibody, such as casirivimab, imdevimab, bamlanivimab, or etesevimab. In embodiments, the antibody is a monoclonal antibody therapy, such as casirivimab + imdevimab, bamlanivimab, or bamlanivimab + etesevimab.

[0112] The active agents and compositions of the invention are also intended for use in conjunction with general medical care provided to patients with viral infections, such as parenteral fluids (dextrose saline and lactated Ringer's solution) and nutrition, antibiotics (including metronidazole and cephalosporin antibiotics, such as ceftriaxone and cefuroxime) and / or antiviral prophylaxis, antipyretics (e.g., acetaminophen) and analgesics, antiemetics (such as metoclopramide) and / or antidiarrheals, vitamin and mineral supplements (e.g., vitamin K and zinc sulfate), anti-inflammatories (such as ibuprofen), analgesics, and medications for other common illnesses in the patient population, such as artemether, artesunate-lumefantrine combination therapy, quinolone antibiotics, such as ciprofloxacin, macrolide antibiotics, such as azithromycin, cephalosporin antibiotics, such as ceftriaxone, or aminopenicillins, such as ampicillin, or medications for shigellosis.

[0113] The combination treatment may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

[0114] Co-administration of a compound of the invention with one or more other active therapeutic agents generally refers to the simultaneous or sequential administration of a compound of the invention with one or more other active therapeutic agents such that a therapeutically effective amount of both the compound of the invention and the one or more other active therapeutic agents are present in the patient's body.

[0115] Co-administration includes administration of a unit dose of a compound of the invention before or after administration of a unit dose of one or more other active therapeutic agents, e.g., administration of a compound of the invention within seconds, minutes or hours of administration of one or more other active therapeutic agents, and / or administration of a unit dose of a compound of the invention as part of the same treatment regimen. For example, a unit dose of a compound of the invention can be administered first, followed within seconds or minutes or days by administration of a unit dose of one or more other active therapeutic agents. Alternatively, a unit dose of one or more other therapeutic agents can be administered first, followed within seconds or minutes or days by administration of a unit dose of a compound of the invention. In some instances, it may be desirable to administer a unit dose of a compound of the invention first, followed by a unit dose of one or more other active therapeutic agents several hours (e.g., 1-12 hours) later. In other instances, it may be desirable to administer a unit dose of one or more other active therapeutic agents first, followed by a unit dose of a compound of the invention several hours (e.g., 1-12 hours) later.

[0116] Combination treatments may provide "synergy" or "synergistic effects", i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects obtained when the compounds are used separately.

[0117] As used herein, the words "a" and "an" are intended to include one or more, unless otherwise specified. For example, the term "an agent" includes a single agent and a combination of two or more agents.

[0118] The term "treat" or "treatment" as used herein refers to the treatment of a disease or condition of interest (e.g., respiratory infection) in a mammal, preferably a human, having said disease or condition, and includes, for example, preventing or delaying the onset of said disease or condition from occurring in a mammal, particularly when such a mammal is at risk of developing said disease but is not yet symptomatic and / or has not yet been diagnosed as having said disease; inhibiting said disease or condition, i.e., arresting its onset; relieving said disease or condition, i.e., causing regression of said disease or condition; and / or stabilizing said disease or condition. Treatment includes improving or alleviating the severity of symptoms of a disease or condition, and / or inhibiting further progression or worsening of those symptoms. Treatment also includes shortening the time course and / or severity of a disease or condition, as compared with the expected or past time course and / or severity of the disease.

[0119] As used herein, the term "prevent" means not developing clinical symptoms of a disease or condition, and includes inhibiting the development of a viral infection in a subject who may be exposed to or susceptible to a viral infection, but who has not yet experienced or exhibited symptoms of the infection.

[0120] An "effective amount" or "therapeutically effective amount" of a compound or composition described herein refers to an amount of compound sufficient to achieve a particular effect or result and / or to prevent or treat a disease or condition and / or symptom, such that, for example, the symptoms associated with the disorder or condition are alleviated in whole or in part, or the further progression or worsening of those symptoms are halted or slowed, or the disorder or condition is prevented or prevented. An "effective amount" and a "therapeutically effective amount" specifically include an antiviral amount of a compound of the invention or a composition described herein (alone or in combination with other active agents).

[0121] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0122] The patents and scientific literature referred to herein reveal the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and foreign patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. The relevant teachings of all patents, published applications and references cited herein are incorporated herein by reference in their entirety.

[0123] Example 1 Compounds were tested using an optimized SARS-CoV-2 pseudotyped virus neutralization assay (PNA), which is used to quantify neutralizing antibody titers or half-maximal inhibitory concentrations (IC50) of molecules against SARS-CoV-2 (Figure 2). Pseudotyped virus particles are generated using a genetically modified vesicular stomatitis virus with deleted glycoprotein G (VSVΔG). VSVΔG virus is transduced into HEK293T cells previously transfected with spike glycoproteins from various SARS-CoV-2 coronavirus strains (WT (Wuhan), alpha (B.1.1.7, UK), beta (B.1.351, South Africa) or gamma (P.1, Brazil) strains) with the last 19 amino acids of their cytoplasmic tails deleted (ΔCT). The resulting pseudoparticles (VSVΔG-spikeΔCT) contain a luciferase reporter that can be quantified in relative luminescence units (RLU).

[0124] Neutralizing activity against SARS-CoV-2 pseudoviruses was measured using a single-round infection assay in 293T / ACE2 target cells. Pseudotyped virus particles were produced in 293T / 17 cells (ATCC) by co-transfection of a plasmid encoding the codon-optimized SARS-CoV-2 full-length spike (containing G at position 614), the packaging plasmid pCMVΔR8.2, and the luciferase reporter plasmid pHR'CMV-Luc. Wild-type and B.1.1.529 (Omicron) mutant spike plasmids, packaging plasmids, and luciferase plasmids were kindly provided by Dr. Barney Graham (Vaccine Research Center, NIH). Spike plasmids for the B.1.351 (beta) and B.1.617.2 (delta) variants were kindly provided by Dr. Bing Chen (Children's Hospital, Boston, MA). The 293T cell line stably overexpressing the human ACE2 cell surface receptor protein was kindly provided by Dr. Michael Farzan and Dr. Huihui Ma (Scripps Research Institute). For neutralization assays, serial dilutions of peptides were performed in duplicate and then pseudovirus was added. Pooled serum samples from convalescent COVID-19 patients or PBS / 10% DMSO dilution buffer were used as positive and negative controls, respectively. Plates were incubated at 37 °C for 1 h before inoculation with 293 / ACE2 target cells (1 × 10 4 / well) was added. Wells containing cells + pseudovirus (no sample) or cells alone served as positive and negative infection controls, respectively. Assays were harvested on day 3 using Promega BrightGlo luciferase reagent and luminescence was detected using a Promega GloMax luminometer. Titers were calculated as the concentration of peptide that inhibited 50% or 80% of viral infection (IC50 Titer and IC 80 All neutralization experiments were repeated twice with similar results.

[0125] A pseudotyped virus neutralization assay was used to evaluate the inhibitory potential of the compounds of the present invention against four SARS-CoV-2 strains (WT, alpha, beta and gamma). Compounds were tested at a starting concentration of 5 μM and serially diluted in 96-well plates according to three different serial dilution schemes, namely 10-fold, 5-fold and 3-fold dilutions. Since the compounds were initially resuspended in DMSO, to address the diluent effect of the compounds on the assay, 3-fold serial dilutions of the DMSO solution (starting concentration 0.052% DMSO) were also tested in PNA with the same strains. A predetermined amount of each pseudotyped virus (corresponding to approximately 1,200,000 RLU / well) was applied to the plate and incubated with the diluted compounds to allow binding of the compounds to the pseudotyped viruses. A positive control for the assay was also included on each plate and tested with all four spike-pseudotyped virus strains. This control consisted of human serum containing neutralizing antibodies against the SARS-CoV-2 spike protein, which is routinely used in PNA testing.

[0126] After incubation of the compound / serum-pseudotyped virus complexes, the medium was removed from the 96-well plate of Vero E6 cells, and the compound-pseudotyped virus complexes were transferred onto the cells. The test plate was incubated overnight at 37°C and 5% CO2. After incubation, luciferase substrate was added to the Vero E6 cells, and the plate was read using a plate reader that detects luminescence. The intensity of the light emitted is inversely proportional to the level of neutralizing ability of the compound. Compound IC50s were then calculated using a standard four-parameter logistic model for all dilutions of the Wuhan strain and spike mutant strains tested. The calculated half-maximal inhibitory concentrations (IC50s) and corresponding R-squared values ​​for the 10-fold serial dilutions are shown in Table 1. The IC50s of the EK1 positive control for WT, omicron, delta, and beta were 0.622 μM, 0.241 μM, 0.244 μM, and 0.527 μM, respectively (data not shown). [Table 1] [Table 2-1] [Table 2-2]

[0127] Example 2 - Efficacy of potential antiviral therapeutics against SARS-CoV-2 in hamsters The objective of this study was to test the in vivo efficacy of DCOY101 as a potential antiviral therapeutic against SARS-CoV-2, the causative pathogen of COVID-19, in a hamster model. The test article (TA), i.e., DCOY101, solution, and dosing are defined in Table 3. Bulk test article was stored at approximately -20 ± 5°C and protected from light until use. Dosing formulations of test article were prepared at the start of dosing, aliquoted, and stored at -20 ± 5°C. [Table 3]

[0128] Hamsters were administered vehicle or test article dosing formulations intranasally once daily (Qd) for 9 days (study days -2 through -6) according to the experimental design (Figure 7) and detailed in Table 2. On study day 0, SARS-CoV-2 B.1.617.2 was administered at 5 x 10 4 TCID 50 Hamsters were intranasally administered the drug at a target dose of 100 mg / animal and treated with TA 4±2 hours later. [Table 4]

[0129] Experimental endpoints were observation of moribundity / death, body weight and body weight change, determination of clinical signs, gross necropsy, viral shedding in oral swabs by RT-qPCR, and TCID 50 This consisted of viral load in lung tissue and nasal turbinates by viral titer and viral genome copy number in lungs by RT-qPCR, and lung histopathological analysis.

[0130] None of the deaths were related to SARS-CoV-2 disease, but two animals in the control group died accidentally during treatment.

[0131] While animals in vehicle-designated control group 1 exhibited an average weight loss of -10.5%, all treatment groups even gained weight during the study, with a maximum of -1.1% in group 3 and +3.4% in group 2 (Figure 8). Specifically, no differences were observed between any of the groups on day -2. On day -1, group 2 with the high dose of 10 mg / kg exhibits a significant (P < 0.0187, *) weight loss compared to vehicle control group 1 (Figure 9). On day 0, group 3 with the second highest dose of DCOY, 5 mg / kg, exhibits a significant (P < 0.0315, *) weight loss compared to vehicle control group 1 (Figure 9). On day 1, no significant differences were observed in any of the groups in response to administration of TA at the high dose. During the period from day 2 to day 4, slight differences (*) were consistently observed in the low dose groups, G4-DCOY-2.5 mg / kg and G5-DCOY-1 mg / kg. Strong significant differences in unconventional drug response concentrations were observed during study days 5-7. Compared to vehicle control group 1 (mean weight change was -10.5%), the high dose DCOY 10 mg / kg group 2 (mean weight change was +3.4%) and the low dose group DCOY 1 mg / kg (mean weight change was +0.9%) had the largest weight gains (***, **) (Figure 9).

[0132] Viral shedding in oral swabs was estimated as viral genome copy number analyzed by RT-qPCR from day 1 to day 7. A summary of the shedding profiles is shown in Figure 10. There is no statistical difference between any of the groups on days 1, 4, 5, 6 or 7. However, there is a significant reduction on days 2 and 3. When compared to the control group 1, group 2 showed a reduction of *** P ≤ 0.001, group 3 **** P ≤ 0.0001, group 4 *** P ≤ 0.001 and group 5 **** P ≤ 0.0001 on day 2. On day 3, the reduction was not significant in groups 3, 4 and 5, while group 2 remained at *** P ≤ 0.001. (Figure 11).

[0133] RT-qPCR titers from lung samples showed broadly consistent results within each group and followed an inverse correlation with body weight change. Viral copy numbers were estimated by one-step RT-qPCR using RNA isolated from left lung tissue samples and oral swabs. Briefly, RNA was extracted from lung tissue samples or oral swab supernatants using the Quick-RNA Virus Kit (Zymo Research) and stored in RNA / DNA Shield. RT-qPCR analysis was performed using the BlazeTaq Probe One-Step RT-qPCR Kit (GeneCopoeia, Rockville, MD) with isolated RNA as template according to the manufacturer's protocol. The following RT-qPCR cycle conditions were used: 50°C for 15 min (RT), 95°C for 2 min (denaturation), then 40 cycles of 95°C for 10 s followed by 62°C for 45 s (extension). The following primers and probes were used for detection of SARS-CoV-2: [Table 5]

[0134] To estimate viral copy number, samples were compared to a standard curve with synthetic RNA. There is more variability in viral load on day 2 than on day 7. However, as shown in Figure 12, in lung tissue harvested, there is a significant reduction in groups 2, 3, 4, and 5 compared to the vehicle control group 1 on both days 2 and 7 (ns P>0.05, *P≦0.05, **P≦0.01, ***P≦0.001).

[0135] On days 2 and 7, TCID 50 The assay estimated the viral load in the lungs. On day 2, animals in groups 2, 3, 4 and 5 showed a significant reduction in the accumulation of viral load in the lungs when compared to group 1 (vehicle control) (**P≦0.01), but no clear dose response was observed. These results are consistent with the information read out by RT-qPCR (Figure 13). On day 7, TCID 50Although the assay no longer detected infectious virus, small amounts of residual viral genome were still detected by RT-qPCR.

[0136] Viral load in the nasal turbinates of the upper respiratory tract is TCID 50 The results of this analysis showed that on day 2, titers were higher in vehicle control Group 1 than in Groups 2, 3, 4, or 5, but this difference was not statistically significant. On day 7, titers were higher than TCID 50 No infectious virus was detected in the assay (Figure 14).

[0137] Based on the reduced weight loss consistent with a significant reduction in viral load in the lungs of animals treated with DC101 regardless of the dose utilized, the data from this study suggests that DCOY101 reduces SARS-CoV-2 associated phenotypes, reduces viral titers in the lungs at the peak of disease, and reduces shed viral load in a hamster model under the conditions tested.

[0138] Example 3 - Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PeP) efficacy of DCOY101 against SARS-CoV-2 in a Syrian hamster model The objective of this study was to test the efficacy of DCOY101 against SARS-CoV-2, the causative agent of COVID-19, in a hamster model when treatment was initiated pre- or post-infection at various time points. The test article (TA), i.e., DCOY101, solution, and dosing are defined in Table 6. Bulk test article was supplied by the sponsor and stored at approximately -20±5°C and protected from light until use. Test article dosing formulations were prepared at the start of dosing, aliquoted, and stored at -20±5°C. [Table 6]

[0139] Hamsters were administered vehicle or test article dosing formulations intranasally once daily (Qd) for 9 days, as detailed in Table 5, according to the experimental design (Figure 15). On study day 0, SARS-CoV-2 B.1.617.2 was administered at 5 x 10 4 TCID 50 Hamsters were intranasally administered the drug at a target dose of 100 mg / animal and treated with TA 4±2 hours later. [Table 7]

[0140] Experimental endpoints were observation of moribundity / death, body weight and body weight change, determination of clinical signs, gross necropsy, viral shedding in oral swabs by RT-qPCR, and TCID 50 This consisted of viral load in lung tissue by viral titer and viral genome copy number in lung by RT-qPCR, and lung histopathological analysis.

[0141] None of the deaths were related to SARS-CoV-2 disease.

[0142] Animals in vehicle-designated control Group 1 exhibited an average weight loss of -10.84%, while all treatment groups had a BWL of -1.39%, with the greatest observed in Group 6. Groups 2 (+3.31%), 3 (+2.09%), and 4 (+1.02%) all gained weight over the course of the study (Figure 16). Days -2 and -1 showed no differences between any of the groups. On day 1, Groups 2 (P < 0.0272, *), 3 (P < 0.0038, **), 4 (P < 0.0076, **), and 6 (P < 0.0015, **) all exhibited significant weight loss compared to vehicle control Group 1 (Figure 17). On days 2 through 4, Group 2, which received 5 mg / kg DCOY101 as intranasal antiviral prophylaxis starting 48 hours prior to challenge through Day 6, consistently demonstrated significant differences (P≦0.0029, **; P≦0.0003, ***; P<0.0001 ****). Strong significant differences were observed in the post-exposure groups (Groups 3-6) during study days 5-7, consistent with normative time-response curves. Briefly, Group 2 (pre-treatment -48 hours) had a mean weight gain of +3.31%, while Group 6 (post-treatment +36 hours) had a mean weight change of -1.39%.

[0143] Viral shedding in oral swabs was estimated as viral genome copy numbers analyzed by RT-qPCR from days 1 to 6. Data from day 7 were excluded from the analysis due to lack of reliability. A summary of the shedding profile is shown in Figure 18. No statistical differences were found for any group on days 1, 2, 3, 4 or 6, although there were some numerical differences for control group 1, especially on day 3, with the exception of day 5, where group 4 (pre-exposure +36 hours) showed a significant increase when compared to control group 1 (**P≦0.0030). Detailed information on oral swabs by RT-qPCR per day is shown in Figure 19.

[0144] Briefly, RT-qPCR (using primers in Table 4 above) titers from lung samples showed some numerical differences, especially for pre-exposure treatment (PrEP), but again, no significant differences were found. However, as shown in Figure 20, at day 7, there is a significant decrease in lung tissue collected in groups 2 (PrEP), 3 (PEP+2) and 5 (PEP+24) compared to the vehicle control group 1 (P≦0.0028,**; P≦0.0017,** and P≦0.0019,**).

[0145] On days 2 and 7, TCID 50 The assay estimated the viral load in the lungs. On day 2, there were some numerical differences, with the majority of animals (n=5 / 6) in group 2 (PrEP) showing titers below the detection limit, but one animal with an unusual distance from the other values, resulting in an overall non-significant reduction in the accumulation of viral load in the lungs when compared to group 1 (vehicle control) (Figure 21). The same phenomenon was observed in groups 4, 5 and 6, with 2 out of 6 animals exhibiting low titers consistent with antiviral efficacy, although not all animals followed the same consistency. These results are consistent with the information read out by RT-qPCR. On day 7, TCID 50 Although the assay did not detect infectious virus (Figure 21), small amounts of residual RNA viral genome or fragments were still detected by RT-qPCR (Figure 20).

[0146] Based on a significant reduction in body weight loss, data from this study demonstrated that administration of DCOY101 at 5 mg / kg intranasally in 50 μl once daily for 6 days to hamsters either pre-exposure or after exposure up to 36 hours post-challenge reduced TCID 50 These results suggest that vaccination conferred partial protection against the B.1.617.2 variant of SARS-CoV-2, as indicated by reduced lung viral load and reduced viral genome copy numbers in the lungs on day 7, and, to a lesser extent, by low viral copy numbers in oral swabs on day 3.

[0147] DCOY101 appears to reduce SARS-CoV-2 disease pathology but does not appear to affect viral load in the lungs or reduce viral shedding in a hamster model under the conditions tested.

[0148] Example 4 - In Vitro Efficacy of Candidate Antiviral Compounds Against Betacoronavirus The aim of this study is to test the in vitro efficacy of DCOY101 as a potential antiviral therapeutic against the beta-CoVs SARS-CoV-1, MERS-CoV, WA1, and the delta and omicron variants of SARS-CoV-2.

[0149] The test articles (TA) are identified in Table 7. [Table 8]

[0150] Test articles were prepared as lyophilized powders and diluted to a stock concentration of 10 mg / ml (or 967.49 μM) in DMSO according to the scheme in Table 8. After dilution, test articles were placed in 1× media (1×DEME with 2% FBS). [Table 9]

[0151] Dose-dependent antiviral effect at 0.005TCID 50 Viruses were grown and titered in African green monkey kidney (Vero E6) cells, human epithelial cells (Calu-3), human fibroblasts (MRC-5), Macaca mulatta rhesus monkey kidney cells (LLC-MK2), or human colorectal epithelial adenocarcinoma cells (Caco-2) as listed in Table 9. Cells were maintained in Dulbecco's minimum essential medium with 10% fetal bovine serum and antibiotics. For potency assays, FBS was reduced to 2%. [Table 10]

[0152] Antiviral activity was determined for each test article using a pre- and post-treatment regimen. TA was mixed with virus and incubated for 60±10 minutes before being added in triplicate to confluent Vero E6 cell monolayers. TA:virus was then added to the cell monolayer and the virus was allowed to adsorb for 60-90 minutes. Following adsorption, cells were washed with 1× PBS or medium and 1× medium containing TA was replaced on top of the cells. Remdesivir free base was evaluated in parallel as a positive control compound. Cells with DMSO added were evaluated in parallel as a negative control. The results are shown in Figures 22-28.

[0153] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0154] The patents and scientific literature referred to herein reveal the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and foreign patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. The relevant teachings of all patents, published applications and references cited herein are incorporated herein by reference in their entirety.

Claims

1. formula: (Peptide-Linker) ｎ -B-hydrophobic part wherein each peptide is independently an HRC peptide or a targeting peptide, with the proviso that at least one peptide is an HRC peptide, each linker is independently a bivalent linking moiety, and B is a multivalent moiety comprising a cysteine, X, and optionally Y, and / or optionally Z, where X, Y, and Z are defined herein, and n is an integer selected from 1, 2, 3, or more. A compound having the formula:

2. The compound of claim 1 , comprising a targeting peptide and one or more HRC peptides.

3. The compound of claim 1 or 2, wherein the targeting peptide is an ACE2 targeting peptide or a receptor binding domain peptide.

4. 3. The compound of claim 1 or 2, wherein the hydrophobic moiety is cholesterol.

5. wherein Z is formula (I): 【Chemistry 1】 The compound according to claim 1 or 2, which does not contain the structure:

6. The following structure 【Chemistry 2】 wherein the peptide-linker is DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL-GSGSG-.

3. The compound of claim 1 or 2, having the formula:

7. The following structure 【Transformation 3】 wherein the peptide-linker is DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL-GSGSG-.

3. The compound of claim 1 or 2, having the formula:

8. A pharmaceutical composition comprising a compound of claim 1 or 2 and a pharmaceutically acceptable carrier.

9. 1. A pharmaceutical composition for treating a respiratory infection associated with a SAR-Cov-2 variant in a subject in need thereof, comprising an effective amount of a compound of the formula: (Peptide-Linker) ｎ -B-hydrophobic part wherein each peptide is independently an HRC peptide or a targeting peptide, with the proviso that at least one peptide is an HRC peptide, each linker is independently a bivalent linking moiety, and B is a multivalent moiety comprising cysteine, and X, and optionally Y, and / or optionally Z, where X, Y, and Z are defined herein, and n is an integer selected from 1, 2, 3, or more. and a compound having the formula: wherein the mutant strain comprises at least five mutations, and the at least five mutations are independently in the spike protein S1 subunit or S2 subunit, or a combination thereof. Pharmaceutical compositions.

10. 10. The pharmaceutical composition of claim 9, wherein the compound comprises a targeting peptide and one or more HRC peptides.

11. The pharmaceutical composition of claim 9 or 10, wherein the targeting peptide is an ACE2 targeting peptide or a receptor binding domain peptide.

12. 11. The pharmaceutical composition of claim 9 or 10, wherein the hydrophobic moiety is cholesterol.

13. 11. The pharmaceutical composition of claim 9 or 10, wherein the at least five mutations are independently in the N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP) domain, heptad repeat 1 (HR1) domain, or a combination thereof.

14. 11. The pharmaceutical composition of claim 9 or 10, wherein the at least five mutations are independently selected from the mutations listed in Figure 4.

15. The at least five mutations are (i) at least five mutations from the SAR-Cov-2 alpha variant; (ii) at least five mutations from the SAR-Cov-2 beta variant; (iii) at least five mutations from the SAR-Cov-2 delta variant, and (iv) at least five mutations from the SAR-Cov-2 Omicron variant 15. The pharmaceutical composition of claim 14, wherein the compound is independently selected from the group consisting of:

16. 14. The pharmaceutical composition of claim 13, wherein the mutant strain comprises at least 10 mutations.

17. 14. The pharmaceutical composition of claim 13, wherein the mutant strain comprises at least 15 mutations.

18. 14. The pharmaceutical composition of claim 13, wherein the mutant strain comprises at least 20 mutations.

19. 10. The pharmaceutical composition of claim 9, wherein the SARS-CoV-2 comprises at least one variant selected from B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), B.1.429 / B.1.427 (epsilon), B.1.617.1 (kappa), B.1.525 (eta), B.1.526 (iota), P.3 (theta), P.2 (zeta), and B.1.1.529 (omicron).

20. 20. The pharmaceutical composition of claim 19, wherein the SARS-CoV-2 comprises at least one variant selected from A.1 to A.6, B.3 to B.7, B.9, B.10, B.13 to B.16, B.2, B.1 lineage, P.1, P.2, P.3, and R.

1.

21. The B.1 lineage includes, but is not limited to, B.1, B.1.1, B.1.1.7, B.1.1.7 with E484K, B.1.2, B.1.5 to B.1.72, B.1.9, B.1.13, B.1.22, B.1.26, B.1.37, B.1.3 to B.1.66, B.1.177, B.1.243, B.1.313, B.1.351, B.1.427, B.1.429, B.1.525, B.1.526, B.1.526.1, B.1.526.2, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, B.1.617.4, B.1.617.5, B.1.617.6, B.1.617.7, B.1.617.8, B.1.617.9, B.1.617.1 ...1, B.1.617.1, B.1.617.2, B.1.617.3, B.1.617.4, B.1.617.5, B.1.617.6, B.1.617.7, B.1.617.8, B.1.617 21. The pharmaceutical composition of claim 20, comprising at least one of B. 1.617.2, B. 1.617.3, B. 1.619, B. 1.620, and B. 1.

621.

22. 11. The pharmaceutical composition of claim 9 or 10, wherein the pharmaceutical composition is administered using an intranasal spray, an inhaler, or a nebulizer.

23. 11. The pharmaceutical composition according to claim 9 or 10, wherein said pharmaceutical composition is administered in combination with at least one other antiviral active agent or treatment.

24. The compound has the following structure: 【Chemistry 4】 wherein the peptide-linker is DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL-GSGSG-.

11. The pharmaceutical composition of claim 9 or 10, having the formula:

25. The compound has the following structure: 【Transformation 5】 wherein the peptide-linker is DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL-GSGSG-.

11. The pharmaceutical composition of claim 9 or 10, having the formula:

26. The compound has the following structure: 【Transformation 6】 wherein the peptide-linker is DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL-GSGSG-.

11. The pharmaceutical composition of claim 9 or 10, having the formula:

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