WO2024050300A2 - Tensioactifs synthétiques pour inhiber une infection à coronavirus - Google Patents

Tensioactifs synthétiques pour inhiber une infection à coronavirus Download PDF

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Publication number
WO2024050300A2
WO2024050300A2 PCT/US2023/072984 US2023072984W WO2024050300A2 WO 2024050300 A2 WO2024050300 A2 WO 2024050300A2 US 2023072984 W US2023072984 W US 2023072984W WO 2024050300 A2 WO2024050300 A2 WO 2024050300A2
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amino acid
seq
acid sequence
peptide
fragment
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PCT/US2023/072984
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English (en)
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Frans J. Walther
Alan J. Waring
Larry M. Gordon
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Lundquist Institute For Biomedical Innovation At Harbor-Ucla Medical Center
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Publication of WO2024050300A2 publication Critical patent/WO2024050300A2/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • COVID-19 pandemic has resulted in hundreds of millions of infected patients, with disease severity ranging from mild flu-like symptoms to severe acute respiratory distress syndrome (ARDS) and death.
  • ARDS severe acute respiratory distress syndrome
  • infection with the SARS-CoV-2 virus can lead to an acute viral infection with a mean incubation time of around 5 days.
  • Clinical symptoms include fever, cough, fatigue, muscle pain, and dyspnea.
  • a subsequent rapid worsening of respiratory problems may require invasive mechanical ventilation with oxygen supplementation and an administration of antivirals, monoclonal antibody therapy, and anti-coagulants.
  • membrane-bound protein angiotensin-converting Enzyme 2 is expressed in the epithelium of the lungs, small intestines, heart, liver, and kidneys.
  • ACE2 is mainly present on the membranes of alveolar epithelial Type 2 cells. Binding of the receptor- binding domain (RBD) of the SARS-CoV-2 Spike 1 (S1) protein to ACE2 plays an essential role in viral entry and results in damage to the alveoli. Following infection, SARS-CoV-2 replicates in the cells of the respiratory and intestinal epithelium, leading to tissue damage and associated clinical symptoms.
  • RBD receptor- binding domain
  • S1 SARS-CoV-2 Spike 1
  • the peptide includes an N-terminal helix, connected optionally through a turn, to a C-terminal helix of the ⁇ -helix of surfactant protein (SP)-B.
  • the N-terminal or C-terminal helix can be modified, as compared to the natural SP-B peptide, with one or more substitutions of the cysteine and/or methionine residues or adding ion pair residue substitutions.
  • the turn is a natural or designer loop peptide sequence that facilitates formation of a helix-turn-helix structure. Example sequences are provided below.
  • a first aspect of the present invention relates to surface active peptides that bind with high affinity to the SARS-CoV-2 Receptor Binding Domain and the ACE2 receptor in a manner that block the virus from entering the host cell.
  • the surfactant peptide comprises (i) a first fragment comprising the amino acid sequence of XWLXRALIKRIQAZI (SEQ ID NO: 1) or a first amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 and (ii) a second fragment comprising the amino acid sequence of RZLPQLVXRLVLRXS (SEQ ID NO: 2) or a second amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein X and Z each can be any amino acid.
  • X is any amino acid but at least one amino acid at the X positions is not cysteine.
  • Z is any amino acid but at least one amino acid at the Z positions is not methionine.
  • the peptide further comprises (iii) a turn between the first fragment and the second fragment.
  • the turn comprises PKGG (SEQ ID NO:3).
  • the turn can form a salt bridge between amino acids within the turn or between the turn and the first or second fragment.
  • the turn comprises DATK (SEQ ID NO:4).
  • the first fragment is at the N-terminal end of the second fragment.
  • the peptide further comprises an insertion sequence at the N-terminal end of the first fragment.
  • the insertion sequence comprises FPIPLPY (SEQ ID NO:5).
  • the peptide is 100 amino acids in length or shorter. In some aspects, the peptide is 80 amino acids in length or shorter.
  • at least one amino acid at the X positions is not cysteine. In some aspects, each amino acid at the X positions is not cysteine. In some aspects, the amino acid at each X position is selected from the group consisting of Y, L, A, and F. [0015] In some aspects, at least one amino acid at the Z positions is not methionine.
  • each amino acid at the Z position is not methionine. In some aspects, the amino acid at each X position is leucine. [0016] In some aspects, the first fragment comprises any amino acid sequence of SEQ ID NO:11-18, an amino acid sequence having at least 90% sequence identity to any amino acid sequence of SEQ ID NO:11-18, or an amino acid sequence derived from any amino acid sequence of SEQ ID NO: 11-18 with one, two or three amino acid addition, deletion and/or substitution.
  • the second fragment comprises any amino acid sequence of SEQ ID NO:19-26, an amino acid sequence having at least 90% sequence identity to any amino acid sequence of SEQ ID NO:19-26, or an amino acid sequence derived from any amino acid sequence of SEQ ID NO: 19-26 with one, two or three amino acid addition, deletion and/or substitution.
  • the peptide comprises any amino acid sequence of SEQ ID NO: 27-58, an amino acid sequence having at least 90% sequence identity to any amino acid sequence of SEQ ID NO:27-58, or an amino acid sequence derived from any amino acid sequence of SEQ ID NO: 27-58 with one, two or three amino acid addition, deletion and/or substitution.
  • a composition comprising a peptide of the present disclosure and one or more phospholipid.
  • the one or more phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylglycerol (PG), palmitoyloleoylphosphatidylglycerol (POPG), cholesterol (Chol), 1,2-Dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1- palmitoyl-2-oleoylsn-glycero phosphocholine (POPS), 1,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-Dipalmitoyl-s
  • DPPC dipalmitoylphosphati
  • the one or more phospholipid comprises DPPC, POPC and POPG.
  • the DPPC, POPC and POPG are at ratio of about (4-6):(2-4):(1-3).
  • a method of inhibiting infection by a virus in a subject in need thereof comprising administration to the patient a composition of the present disclosure.
  • the virus is a coronavirus.
  • the coronavirus is SARS-CoV-2, SARS-CoV, Middle East respiratory syndrome–related coronavirus (MERS-CoV).
  • the virus is an influenza virus.
  • influenza virus is the H5N1 influenza A virus or the H7N9 influenza A virus.
  • FIG. 1 Molecular illustration of SARS-CoV-2 bound to synthetic surfactant B-YL peptide. B-YL backbone and aromatic side chains are highlighted in red. SARS-CoV-2 protein backbone is green and the RBD domain with interactive aromatic amino acid side chains is highlighted in blue. Tyr-8 of B-YL paired with Tyr-473 of the RBD, Tyr-11 of B-YL paired with Tyr-489 RBD, Tyr-34 of B-YL interacted with Tyr-495 and Phe-497 of the RBD, and Tyr-40 of B-YL interacted with Tyr-490 RBD.
  • FIG. 2 Molecular illustration of B-YL bound to the ACE2 lung cell receptor.
  • the interactive B-YL backbone with aromatic and ionic residue side chains is shown in red.
  • the ACE2 protein backbone is highlighted in green, while the interactive domain residues with aromatic and ionic side chains is shown in blue.
  • Ion pairs are shown between ACE2 Glu-30 and B-YL Arg-12, while hydrophobic ring interactions are labeled between ACE2 Tyr-83 and B-YL Phe-1.
  • FIG. 3 Surface plasmon resonance (SPR) sensorgrams of rbCovid-19-RBD protein construct and human ACE2 receptor binding to immobilized B-YL peptide.
  • SPR Surface plasmon resonance
  • the B-YL peptide was biotinylated to facilitate attachment to the Biacore chip as described in Methodology. Solutions of 1 ⁇ M recombinant protein in HBS-EP buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) were then flowed over the respective chip-linked peptides. The concentration dependent binding of the viral RBD construct (A) and the human ACE-2 receptor (B) to the B-YL lung surfactant peptide are shown with the SPR response indicated in relative response units (RU) on the graph’s Y-axis. [0025] FIG. 4.
  • SPR Surface plasmon resonance
  • compositions and methods when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) claimed. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. [0030] The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or ( ⁇ ) 10%, 5% or 1%.
  • sequence identity refers to a level of amino acid residue or nucleotide identity between two peptides or between two nucleic acid molecules. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position.
  • a peptide (or a polypeptide or peptide region) has a certain percentage (for example, at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% or at least about 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • sequences having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% or at least about 99% sequence identity to the reference sequence are also within the disclosure.
  • the present disclosure also includes sequences that have one, two, three, four, or five substitution, deletion or addition of amino acid residues or nucleotides as compared to the reference sequences.
  • analogs of a peptide comprising any amino acid sequence described herein are also provided, which have at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity to any of reference amino acid sequences.
  • the analogs include one, two, three, four, or five substitution, deletion or addition of amino acid residues as compared to the reference sequences.
  • the substitution is a conservative substitution.
  • a “conservative substitution” of an amino acid or a “conservative substitution variant” of a peptide refers to an amino acid substitution which maintains: 1) the secondary structure of the peptide; 2) the charge or hydrophobicity of the amino acid; and 3) the bulkiness of the side chain or any one or more of these characteristics.
  • hydrophilic residues relate to serine or threonine.
  • Hydrodrophobic residues refer to leucine, isoleucine, phenylalanine, valine or alanine, or the like.
  • “Positively charged residues” relate to lysine, arginine, ornithine, or histidine. “Negatively charged residues” refer to aspartic acid or glutamic acid. Residues having “bulky side chains” refer to phenylalanine, tryptophan or tyrosine, or the like. A list of illustrative conservative amino acid substitutions is given in Table A. Table A For Amino Acid Replace With [0035] as used herein, the term “composition” refers to a preparation suitable for administration to an intended patient for therapeutic purposes that contains at least one pharmaceutically active ingredient, including any solid form thereof.
  • the composition may include at least one pharmaceutically acceptable component to provide an improved formulation of the peptide, such as a suitable carrier.
  • the composition is formulated as a film, gel, patch, or liquid solution.
  • pharmaceutically acceptable indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile.
  • the term “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body, or to deliver an agent to the internal surface of the lung.
  • pharmaceutically acceptable carrier refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body, or to deliver an agent to the internal surface of the lung.
  • Surfactant Peptides For Treating or Preventing Viral Infections [0038] COVID-19 pneumonia is initiated by the binding of the viral receptor-binding domain (RBD) of SARS-CoV-2 to the cellular receptor angio
  • the instant inventors studied the binding of two synthetic surfactant protein B (SP-B) peptide mimics, Super Mini-B (SMB, SEQ ID NO:7) and B-YL (SEQ ID NO:27), to a recombinant human ACE2 receptor protein construct using molecular docking and surface plasmon resonance (SPR) to evaluate their potential as antiviral drugs.
  • SP-B synthetic surfactant protein B
  • SMB Super Mini-B
  • B-YL SEQ ID NO:27
  • one embodiment of the present disclosure provides a method for preventing, inhibiting or treating the infection of a virus in a subject in need thereof, or treating the infection or an associated symptom in the subject.
  • the method entails administering to the subject a surfactant peptide, such as any one of the SP-B peptide mimics as disclosed herein.
  • the surfactant peptide includes an N-terminal helix, connected optionally through a turn, to a C-terminal helix of the alpha helix of surfactant protein (SP)-B.
  • the N-terminal or C-terminal helix can be modified, as compared to the natural SP-B peptide, with one or more substitutions at the cysteine and/or methionine residues.
  • the turn is a natural or designer loop peptide sequence that facilitates formation of a helix-turn- helix structure.
  • Mini-B further includes a “PKGG” turn (SEQ ID NO:3).
  • Super Mini-B then further includes the “insertion sequence” (SEQ ID NO:5) from the natural SP-B peptide.
  • the Mini-B and Super Mini-B peptides can be modified by replacing the PKGG turn with another turn, such as DATK (SEQ ID NO: 4) which is discovered to be able to increase molecular stability and improve the ease of synthesis, folding and purification of the peptides.
  • Example analogs in this respect include SMB-DATK (SEQ ID NO:8) and MB-DATK (SEQ ID NO:10).
  • any of these amino acid sequences can further be modified within either or both the helix regions.
  • at least one, two, three, or four, or all of the cysteines in the helix is substituted with another amino acid.
  • at least one cysteine in each helix is substituted wither another amino acid.
  • at least one of the helices has no cysteine residue.
  • the entire peptide includes no cysteine.
  • the substitution is with Y, L, A, or F.
  • the peptide can still form a desired helix-turn-helix structure and is more stable and effective.
  • the cysteines when the cysteines are substituted with one or more tyrosine residues, the hydrophobic core formed by the tyrosine residues can further help stabilize the peptide.
  • at least one of the methionine residues is substituted with another amino acid. In one embodiment, both of the methionine residues are substituted. In some embodiments, the substitution is with leucine.
  • the surfactant peptide includes (i) a first fragment comprising the amino acid sequence of XWLXRALIKRIQAZI (SEQ ID NO: 1) or a first amino acid sequence having at least 90% (or at least 80%, 85% or 95%) sequence identity to SEQ ID NO: 1 and (ii) a second fragment comprising the amino acid sequence of RZLPQLVXRLVLRXS (SEQ ID NO: 2) or a second amino acid sequence having at least 90% (or at least 80%, 85% or 95%) sequence identity to SEQ ID NO: 2, wherein X is any amino acid and Z is any amino acid.
  • X is any amino acid but at least one amino acid at the X positions is not cysteine.
  • Z is any amino acid but at least one amino acid at the Z positions is not methionine.
  • Non-limiting examples of SEQ ID NO:1 include SEQ ID NO:11-18.
  • Non-limiting examples of SEQ ID NO:2 include SEQ ID NO:19-26.
  • the peptide further includes a turn between the first fragment and the second fragment.
  • a “turn” as used herein, refers to a relatively short (e.g., less than 50 amino acids in length) amino acid fragment that forms a secondary structure in a polypeptide chain where the polypeptide chain reverses its overall direction.
  • turns include, without limitation, ⁇ -turns, ⁇ -turns, ⁇ -turns, ⁇ -turns, ⁇ -turns, loops, multiple turns and hairpins.
  • the turn is typically from one amino acid to about 50 amino acids (or to about 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6 or 5 amino acids) in length.
  • the turn does not include cysteine.
  • the turn does not include methionine.
  • the turn includes an amino acid that forms a salt bridge with either of the helices.
  • the turn includes amino acids to form a salt bridge within.
  • Non-limiting examples of turns include PKGG (SEQ ID NO:3), DATK (SEQ ID NO:4) and amino acids 23-63 of SEQ ID NO:6 or a portion or combination of portions thereof.
  • the helices can be orientated either way.
  • SEQ ID NO:1 (or the first fragment) can be at the N-terminal direction of SEQ ID NO:2 (or the second fragment).
  • SEQ ID NO:1 (or the first fragment) can be at the C- terminal direction of SEQ ID NO:2 (or the second fragment).
  • the surfactant peptide further includes an insertion sequence at the N-terminal end of the peptide.
  • the peptide further includes an insertion sequence at the N-terminal direction of the first fragment or the N-terminal direction of the second fragment.
  • the insertion sequence in some embodiments, includes at least one proline. In another embodiment, the insertion sequence includes at least a leucine or isoleucine.
  • a non- limiting example of the insertion sequence is FPIPLPY (SEQ ID NO:5).
  • the total length of the surfactant peptide varies from 20 amino acids to about 100 amino acids. In one embodiment, the peptide is not longer than about 100, or 90, 80, 70, 60 or 50 amino acids long.
  • Non-limiting examples of the surfactant peptide include SEQ ID NO: 7-10 and 59 or an amino acid sequence having at least 90% (or at least 80%, 85% or 95%) sequence identity to any amino acid sequence of SEQ ID NO: 7-10 and 59, or an amino acid sequence derived from any amino acid sequence of SEQ ID NO: 7-10 and 59 with one, two or three amino acid addition, deletion and/or substitution.
  • Non-limiting examples of the surfactant peptide include SEQ ID NO: 27-58 or an amino acid sequence having at least 90% (or at least 80%, 85% or 95%) sequence identity to any amino acid sequence of SEQ ID NO:27-58, or an amino acid sequence derived from any amino acid sequence of SEQ ID NO: 27-58 with one, two or three amino acid addition, deletion and/or substitution.
  • This surfactant peptides are shown to have dual clinical roles in alleviating surfactant insufficiency and inhibition of binding of the SARS-CoV-2 Receptor Binding Domain to the ACE2 receptor on the host cells.
  • Table B lists the amino acid sequences, SEQ ID NOs and, in some cases, short names for various peptides disclosed in the present application.
  • Table B Peptide Sequences and Names SEQ ID NO:1 XWLXRALIKRIQAZI SEQ ID NO:2 RZLPQLVXRLVLRXS SEQ ID NO:3 PKGG SEQ ID NO:4 DATK SEQ ID NO:5 FPIPLPY SEQ ID NO:11 YWLYRALIKRIQALI SEQ ID NO:12 LWLYRALIKRIQALI SEQ ID NO:13 AWLYRALIKRIQALI SEQ ID NO:14 FWLYRALIKRIQALI SEQ ID NO:15 YWLFRALIKRIQALI SEQ ID NO:16 LWLFRALIKRIQALI SEQ ID NO:17 AWLFRALIKRIQALI SEQ ID NO:18 FWLFRALIKRIQALI SEQ ID NO:19 RLLPQLVYRLVLRYS SEQ ID NO:20
  • the peptides described herein can be ordered from a commercial source or partially or fully synthesized using methods well known in the art (e.g., chemical and/or biotechnological methods).
  • the peptides are synthesized according to solid phase peptide synthesis protocols that are well known in the art.
  • the peptide is synthesized on a solid support according to the well-known Fmoc protocol, cleaved from the support with trifluoroacetic acid and purified by chromatography according to methods known to persons skilled in the art.
  • the peptide is synthesized utilizing the methods of biotechnology that are well known to persons skilled in the art.
  • a DNA sequence that encodes the amino acid sequence information for the desired peptide is ligated by recombinant DNA techniques known to persons skilled in the art into an expression plasmid (for example, a plasmid that incorporates an affinity tag for affinity purification of the peptide), the plasmid is transfected into a host organism for expression, and the peptide is then isolated from the host organism or the growth medium, e.g., by affinity purification.
  • Recombinant DNA technology methods are described in Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), incorporated herein by reference, and are well-known to the skilled biochemist.
  • the peptides can be also prepared by using recombinant expression systems. Generally, this involves inserting the nucleic acid molecule into an expression system to which the molecule is heterologous (i.e., not normally present). One or more desired nucleic acid molecules encoding a peptide of the disclosure may be inserted into the vector. When multiple nucleic acid molecules are inserted, the multiple nucleic acid molecules may encode the same or different peptides.
  • the heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′ ⁇ 3′) orientation relative to the promoter and any other 5′ regulatory molecules, and correct reading frame.
  • the nucleic acid molecules can be derived from the known SP-B nucleotides.
  • nucleic acid constructs can be carried out using methods well known in the art.
  • U.S. Pat. No. 4,237,224 to Cohen and Boyer which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture. Other vectors are also suitable.
  • a suitable expression vector Once a suitable expression vector is selected, the desired nucleic acid sequences are cloned into the vector using standard cloning procedures in the art. The vector is then introduced to a suitable host.
  • Purified peptides may be obtained by several methods. The peptide is preferably produced in purified form (preferably at least about 80% or 85% pure, more preferably at least about 90% or 95% pure) by conventional techniques. Depending on whether the recombinant host cell is made to secrete the peptide into growth medium (see U.S. Pat. No.
  • the peptide can be isolated and purified by centrifugation (to separate cellular components from supernatant containing the secreted peptide) followed by sequential ammonium sulfate precipitation of the supernatant.
  • the fraction containing the peptide is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the peptides from other proteins. If necessary, the peptide fraction may be further purified by HPLC.
  • the peptide of interest is not secreted, it can be isolated from the recombinant cells using standard isolation and purification schemes.
  • the peptide of interest may also contain a purification tag (such as poly-histidine, a glutathione-5-transferase, or maltose-binding protein (MBP-)), which assists in the purification but can later be removed, i.e., cleaved from the peptide following recovery.
  • a purification tag such as poly-histidine, a glutathione-5-transferase, or maltose-binding protein (MBP-)
  • MBP- maltose-binding protein
  • Protease-specific cleavage sites can be introduced between the purification tag and the desired peptide.
  • compositions and Formulations Surfactants and compositions that include any one or more of the peptides as disclosed herein are also provided. In one embodiment, the composition includes any one or more of the peptides and one or more phospholipid. [0068] There are an abundance of kinds of phospholipids suitable for use in surfactants.
  • Non- limiting examples include dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylglycerol (PG), palmitoyloleoylphosphatidylglycerol (POPG), cholesterol (Chol), glycerophospholipids such as 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1-palmitoyl-2-oleoylsn-glycero phosphocholine (POPS), 1,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoglyce
  • the phospholipids can be mixed at suitable ratios, in some embodiments.
  • DPPC:POPC:POPG can be used a ratio of about 5:3:2, DPPC:POPG at a ratio of about 7:3, DEPN-8:PG-1 at about 9:1 or 8:2.
  • the phospholipids include DPPC, POPC and POPG.
  • the DPPC, POPC and POPG are at ratio of about (4-6):(2-4):(1- 3).
  • the peptides described herein can be modified by the inclusion of one or more conservative amino acid substitutions.
  • the surfactant compositions can further include any one or more of a non-phospho surfactant.
  • a non-phospho surfactant refers to surface active compounds that do not possess a phospho group (e.g., phosphate, phosphonate, etc.).
  • Exemplary non- phospho surfactants include, without limitation, a free fatty acid, hexadecanol, or cholesterol.
  • Preferred free fatty acids include saturated and monounsaturated C10 to C24 hydrocarbons, more preferably C 12 -C 20 hydrocarbons, most preferably C 14 -C 18 hydrocarbons. Of these, saturated hydrocarbons are preferred.
  • the peptides or compositions of the present disclosure can be used for delivering pharmaceutical agents to a subject in need thereof.
  • the composition (or formulation) includes a peptide or composition of the earlier disclosure and a therapeutic agent.
  • the therapeutic agent can be any agent that is shown, tested, or proposed to have therapeutic effects.
  • the method entails administration to the patient a composition of the present disclosure.
  • the virus is a coronavirus.
  • the coronavirus is SARS-CoV-2, SARS-CoV, Middle East respiratory syndrome–related coronavirus (MERS-CoV).
  • the virus is an influenza virus.
  • the influenza virus is the H5N1 influenza A virus or the H7N9 influenza A virus.
  • Treatment is an approach for obtaining beneficial or desired results including clinical results.
  • beneficial or desired clinical results may include one or more of the following: a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and/or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and/or preventing or delaying the spread (e.g., metastasis) of the disease or condition); and/or c) relieving the disease, that is, causing the regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or total remission of the disease or condition, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.
  • a) inhibiting the disease or condition e.g., decreasing one or more symptoms resulting from the disease or condition
  • prevention or “preventing” means any treatment of a disease or condition that causes the clinical symptoms of the disease or condition not to develop.
  • Peptides may, in some embodiments, be administered to a subject (including a human) who is at risk or has a family history of the disease or condition.
  • Subject refers to an animal, such as a mammal (including a human), that has been or will be the object of treatment, observation or experiment. The methods described herein may be useful in human therapy and/or veterinary applications.
  • the subject is a mammal.
  • the subject is a human.
  • terapéuticaally effective amount or “effective amount” of a compound described herein or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analog thereof means an amount sufficient to effect treatment when administered to a subject, to provide a therapeutic benefit such as amelioration of symptoms or slowing of disease progression.
  • a therapeutically effective amount may be an amount sufficient to decrease a symptom of a disease or condition.
  • the therapeutically effective amount may vary depending on the subject, and disease or condition being treated, the weight and age of the subject, the severity of the disease or condition, and the manner of administering, which can readily be determined by one or ordinary skill in the art.
  • the methods described herein may be applied to cell populations in vivo or ex vivo.
  • “In vivo” means within a living individual, as within an animal or human. In this context, the methods described herein may be used therapeutically in an individual.
  • “Ex vivo” means outside of a living individual. Examples of ex vivo cell populations include in vitro cell cultures and biological samples including fluid or tissue samples obtained from individuals. Such samples may be obtained by methods well known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, and saliva. In this context, the compounds and compositions described herein may be used for a variety of purposes, including therapeutic and experimental purposes.
  • the compounds and compositions described herein may be used ex vivo to determine the optimal schedule and/or dosing of administration of a compound of the present disclosure for a given indication, cell type, individual, and other parameters. Information gleaned from such use may be used for experimental purposes or in the clinic to set protocols for in vivo treatment. Other ex vivo uses for which the compounds and compositions described herein may be suited are described below or will become apparent to those skilled in the art.
  • the selected compounds may be further characterized to examine the safety or tolerance dosage in human or non-human subjects. Such properties may be examined using commonly known methods to those skilled in the art.
  • the peptides disclosed herein may be used in combination with one or more additional therapeutic agent that are being used and/or developed to treat viral infections, such as nirmatrelvir/ritonavir (marketed as Paxlovid) for treating COVID-19.
  • the one or more additional therapeutic agent may be remdesivir.
  • the one or more additional therapeutic agent may be favipiravir, fingolimod, and/or methylprednisolone.
  • the one or more additional therapeutic agent may be bevacizumab.
  • the one or more additional therapeutic agent may be chloroquine phosphate, chloroquine, or hydroxychloroquine sulfate.
  • Surfactant peptides provided herein are usually administered in the form of pharmaceutical compositions.
  • suitable pharmaceutically acceptable vehicles may include, for example, inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.
  • Such compositions are prepared in a manner well known in the pharmaceutical art. See, e.g., Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed.
  • Aerosol delivery of the compositions can be accomplished with a vibrating mesh nebulizer. However, both liquid and dry-powder nebulizers can also be used. The type of nebulizer can vary and the drug combination can be nebulized as a wet or dry preparation. [0084] Nebulizers and pressurized metered-dose inhalers can be used to deliver aerosolized drugs.
  • Nebulizers transform liquid formulations and suspensions into medical aerosol and are understood to include jet nebulizers, ultrasonic nebulizers and mesh nebulizers.
  • Mesh nebulizers use lower-frequency waves and eliminate the heating issues that can denature proteins during aerosolization.
  • Mesh nebulizers typically force liquid medications through multiple apertures in a mesh or aperture plate to generate aerosol.
  • Mesh nebulizers are advantageous because they provide consistent aerosol generation and a predominately fine-particle fraction reaching into the peripheral lung, low residual volume, and the ability to nebulize in low drug volumes.
  • mesh nebulizers can be classified as active mesh nebulizers and passive mesh nebulizers. Active mesh nebulizers use a piezo element that contracts and expands on application of an electric current and vibrates a precisely drilled mesh in contact with the medication to generate aerosol.
  • One suitable mesh nebulizer for the compositions described herein is the Aeroneb (Aerogen, Galway, Ireland) nebulizers which can be used for both spontaneously breathing and ventilator-dependent patients.
  • the aerosolized composition can be administered using either a mouthpiece or a face mask, with a face mask being suitable for infants, small children, and patients with cognitive problems.
  • the aerosolized composition can have a median mass aerodynamic diameter (MMAD) of 0.5 ⁇ m, 1 ⁇ m, 1.5 ⁇ m, 2 ⁇ m, 2.5 ⁇ m, 3 ⁇ m, 3.5 ⁇ m, 4 ⁇ m, 4.5 ⁇ m, 5 ⁇ m, 5.5 ⁇ m, 6 ⁇ m, 6.5 ⁇ m, 7 ⁇ m, 7.5 ⁇ m, 8 ⁇ m, 8.5 ⁇ m, 9 ⁇ m, 9.5 ⁇ m, or 10 ⁇ m.
  • MMAD median mass aerodynamic diameter
  • the MMAD may also be within a range between any two of the foregoing values.
  • the specific dose level of a peptide of the present application for any particular subject will depend upon a variety of factors including the activity of the specific peptide employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the subject undergoing therapy.
  • a dosage may be expressed as a number of milligrams of a peptide described herein per kilogram of the subject’s body weight (mg/kg). Dosages of between about 0.1 and 150 mg/kg may be appropriate. In some embodiments, about 0.1 and 100 mg/kg may be appropriate.
  • a dosage of between 0.5 and 60 mg/kg may be appropriate. Normalizing according to the subject’s body weight is particularly useful when adjusting dosages between subjects of widely disparate size, such as occurs when using the drug in both children and adult humans or when converting an effective dosage in a non-human subject such as dog to a dosage suitable for a human subject.
  • the peptides of the present application or the compositions thereof may be administered once, twice, three, or four times daily, using any suitable mode described above. Also, administration or treatment with the peptides may be continued for a number of days; for example, commonly treatment would continue for at least 7 days, 14 days, or 28 days, for one cycle of treatment.
  • B-YL – Surfactant is a synthetic lung surfactant that has been developed to treat mammalian lungs that either lack native lung surfactant, such as in premature infants with respiratory distress syndrome (RDS), or have less efficacy because of damaged endogenous lung surfactant, such as acute respiratory distress syndrome (ARDS).
  • This surfactant formulation is composed of a single peptide, B-YL (SEQ ID NO:27), and emulates the lipid transport function of native Surfactant Protein B (SP-B) that facilitates rapid transport of lipids to and from the monolayer of phospholipids critical in maintaining lung functionality.
  • SP-B Surfactant Protein B
  • the peptide-lipid formulation also contains several synthetic lipids that represent major phospholipid molecular species found in native lung surfactant, thereby optimizing emulation of native lung surfactant for replacement therapy.
  • Some of the important advantages of this synthetic lung surfactant preparation are that it can be produced in large amounts very economically, has a very good shelf-life compared to preparations derived from animal sources, and can be administered to the lungs in various ways.
  • These treatment approaches include an aqueous dispersion of the peptide- lipid material, as an aerosol spray, or as a dry powder.
  • the B-YL family of peptides also has potential as antiviral therapy.
  • This technique allows the direct measurement of the binding of a peptide or protein by attaching one protein to a sensor chip surface and flowing a solution containing a second protein over the surface.
  • the binding affinities derived from such measurements are very similar to those observed in vivo.
  • the resulting sensorgram shows the time-course of association of the mobile phase protein with that of the sensor chip attached protein and directly measures the off and on rates of protein-protein interactions that can then be used to accurately determine protein binding affinities.
  • the binding affinity of the B-YL lung surfactant peptide to the coronavirus recombinant spike protein construct of SARS-CoV-2 (viral spike protein amino acid sequence) that included the Receptor Binding Domain (RBD) was measured with SPR spectroscopy using a Biacore T200 system (GE Healthcare Bio-Sciences Corp, Piscataway, NJ 08855). Because of limited aqueous solution solubility, the B-YL peptide was biotinylated to increase the peptide’s specific interaction with series S Senor Chip SA (GE, BR100530).
  • Binding measurements were then made by flowing a running buffer solution of the test in HBS-EP buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) over the chip-associated peptide at a flow rate of 30 ⁇ l/min to determine the binding affinity at 37 o C.
  • HBS-EP buffer 10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20
  • B-YL peptide binding to the recombinant SARS-CoV-2 RBD domain dissolved in a series of seven concentrations (1,000, 500, 250, 125, 62.5, 31.25 and 0 nM) in the analyte and was determined from the sensorgrams, in which arbitrary response units (RUs) are recorded as a function of time.
  • RUs arbitrary response units
  • SPR Surface Plasmon Resonance
  • the B- YL peptide was biotinylated so to increase the peptides specific interaction with series S Senor Chip SA (GE, BR100530). Binding measurements were then made by flowing a running buffer solution of the test in HBS-EP buffer (i.e., 10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) over the chip-associated peptide at a flow rate of 30 ⁇ l/min to determine the binding affinity at 37 o C.
  • HBS-EP buffer i.e., 10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20
  • SPR surface plasmon resonance
  • SPR Surface Plasmon Resonance

Abstract

L'invention concerne également des compositions et des méthodes qui sont utiles pour traiter et prévenir des infections virales, telles que celles par le SARS-CoV-2. On a découvert que des tensioactifs synthétiques tels que SMB (SEQ ID NO : 7) et B-YL (SEQ ID NO : 27) peuvent se lier au domaine de liaison au récepteur (RBD) du SARS-CoV-2 et au récepteur ACE2, ce qui permet d'inhiber l'interaction entre le virus et ses cellules cibles dans le poumon, réduisant à un minimum l'infectivité du virus.
PCT/US2023/072984 2022-08-29 2023-08-28 Tensioactifs synthétiques pour inhiber une infection à coronavirus WO2024050300A2 (fr)

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