WO2023277628A1 - Nouveau peptide de pénétration cellulaire et son utilisation - Google Patents

Nouveau peptide de pénétration cellulaire et son utilisation Download PDF

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WO2023277628A1
WO2023277628A1 PCT/KR2022/009463 KR2022009463W WO2023277628A1 WO 2023277628 A1 WO2023277628 A1 WO 2023277628A1 KR 2022009463 W KR2022009463 W KR 2022009463W WO 2023277628 A1 WO2023277628 A1 WO 2023277628A1
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cell
peptide
lys
general formula
amino acids
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English (en)
Korean (ko)
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김한주
김용호
김남형
이은아
안용일
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주식회사 아임뉴런
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Publication of WO2023277628A1 publication Critical patent/WO2023277628A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • Liposome is an artificially created phospholipid carrier that can encapsulate both lipophilic and hydrophilic drugs, and is non-toxic because it is a biocompatible material and protects the drug from the external environment.
  • absorption is delayed, distribution is limited, metabolic rate is low, and it is quickly removed from the blood by being captured by cells of the liver or spleen.
  • Micelles have characteristics that can increase the solubility and bioavailability of drugs, but much research is still needed on the effect of the movement of substances into cells and their basic medical and clinical applicability. Because of these limitations, there is a need for new agents that can effectively deliver biomaterials into the living body, have no cytotoxicity, and do not enter through endocytosis in particular.
  • the cell-penetrating peptide is a kind of signal peptide, which is a peptide in which a specific amino acid sequence is used for the purpose of delivering high molecular substances such as proteins, DNA, RNA, etc. into cells.
  • signal peptide which is a peptide in which a specific amino acid sequence is used for the purpose of delivering high molecular substances such as proteins, DNA, RNA, etc. into cells.
  • Antennapedia (Penetratin) derived from Drosophila protein, VP22 derived from HSV-1 virus, and Pep-1 derived from Simian Virus 40 large antigen T are typical first-generation cells It is considered a penetrating peptide and has been widely used together with TAT.
  • peptides such as poly Arginine and poly Lysine, in which several cationic amino acids such as arginine and lysine are repeatedly linked, have been reported to have excellent cell permeability (Korean Patent Publication No. 10-2020- 0104524), which is applied to various mass transfers.
  • the inventors of the present invention designed and synthesized cell-permeable peptides whose amino acid sequences were variously modified based on the existing reference peptides, and their excellent cell-permeability As confirmed, this completed the present invention.
  • one aspect is to provide novel cell penetrating peptides.
  • Another aspect is to provide a complex comprising the cell-penetrating peptide and a biologically active substance.
  • Another aspect is to provide a composition for mass delivery comprising the complex, and a mass delivery method comprising bringing the composition into contact with cells.
  • Another aspect is to provide a polynucleotide encoding the cell penetrating peptide.
  • One aspect provides a cell-permeable peptide consisting of an amino acid sequence represented by the following [General Formula I].
  • X a is SHH, GHH, GHA, AHA, or GQH,
  • X b is Arg(R) or Ala(A);
  • X c is SD, SE, SN, SH, LD, or AD;
  • X d is Glu(E), Thr(T), Gln(Q), Lys(K), His(H), or Arg(R);
  • X e is Ser(S), Asp(D), Glu(E), Lys(K), Asn(N), or Thr(T), and
  • X f can be Thr(T), Glu(E), Lys(K), Asn(N), Gln(Q), or Arg(R).
  • One aspect provides a cell-permeable peptide consisting of an amino acid sequence represented by the following [General Formula II].
  • X h is absent, GH, SH, or H
  • X i is His(H) or Ala(A);
  • X j is Arg(R) or Ala(A);
  • X k is KS, KL, KA, KQ, or RS;
  • X l is Glu(E), Lys(K), or Thr(T);
  • X m is WS, IS, or WD;
  • X n is Thr(T), Arg(R), Asn(N), Lys(K), or Glu(E);
  • X o is SG, QG, SA, or SV
  • X 1 and X 2 is absent or present at least one, wherein X 1 is L, AL, KWSAL, or ESKAVKWSAL, and X 2 can be G, GL, GLIES, or GLIESESAET.
  • One aspect relates to a cell-permeable peptide that can be usefully utilized in the field of basic research, diagnosis and treatment of various diseases, etc., a basic platform peptide structure consisting of 16 amino acids that can be extended to an unlimited number of designs and other not limited It relates to a basic platform peptide structure composed of various amino acids that can be extended to an unlimited number of designs.
  • amino acid herein includes the 22 standard amino acids that are naturally incorporated into peptides, as well as D-isomers and modified amino acids. Accordingly, the peptide may be a peptide containing D-amino acids. Meanwhile, the peptides of the present invention may include post-translational modified non-standard amino acids.
  • post-translational modifications are phosphorylation, glycosylation, acylation (including eg acetylation, myristoylation and palmitoylation), alkylation ), carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-elimination deimidation) , deamidation) and structural changes (eg formation of disulfide bridges).
  • changes in amino acids caused by chemical reactions occurring in the course of bonding with crosslinkers to form a peptide conjugate include changes in amino acids, such as changes in an amino group, a carboxy group, or a side chain.
  • the amino acid sequences used in the present specification are abbreviated according to the IUPAC-IUB nomenclature.
  • cell permeability refers to the ability or property of a peptide to permeate a cell (membrane) and penetrate into the cell.
  • peptide is a polymer of amino acids, and a form in which a small number of amino acids are linked is usually called a peptide, and a form in which many amino acids are linked is called a protein. Connections between amino acids in these peptide and protein structures consist of amide bonds or peptide bonds.
  • a peptide bond is a bond between a carboxyl group (-COOH) and an amino group (-NH 2 ) in which water (H 2 O) escapes to form -CO-NH-.
  • the peptide may be isolated, artificially synthesized, or non-naturally occurring or engineered, and “non-naturally occurring or engineered” refers to a natural state. It means a state created by applying artificial deformation, not a state as it exists in .
  • the artificial modification is to improve cell permeability, and may include substitution, deletion, or addition of an amino acid sequence.
  • cell penetrating peptide refers to a peptide that can move a cargo into a cell in vitro and / or in vivo .
  • carrier includes all substances that can bind to cell-permeable peptides and move into cells, for example, all substances that want to increase cell penetration efficiency, for example, through a general route It may mean a biologically active substance that is not easily moved into cells.
  • the peptide may consist of an amino acid sequence represented by the following [General Formula IA].
  • X aa is Asp (D), Glu (E), Asn (N), or His (H)
  • X ba is Glu (E), Thr (T), Gln (Q), Lys (K), His(H), or Arg(R)
  • X ca is Ser(S), Asp(D), Glu(E), Lys(K), Asn(N), or Thr(T)
  • X da can be Thr(T), Glu(E), Lys(K), Asn(N), or Gln(Q).
  • the peptide may consist of an amino acid sequence represented by the following [General Formula IB].
  • X ab is Arg (R) or Ala (A)
  • X bb is Asp (D), Asn (N), Glu (E), or His (H)
  • X cb is Glu ( E), Thr(T), Gln(Q), Lys(K), His(H), or Arg(R)
  • X db is Ser(S), Asp(D), Glu(E), Lys( K), Asn(N), or Thr(T)
  • X eb can be Thr(T), Glu(E), Lys(K), Asn(N), or Gln(Q).
  • the peptide may consist of an amino acid sequence represented by the following [general formula IC].
  • X ac is GHA, AHA, or GQH
  • X bc is Arg (R) or Ala (A)
  • X cc is Ser (S), Leu (L), or Ala (A)
  • X dc can be Thr(T), Arg(R), or Lys(K).
  • the peptide may consist of an amino acid sequence represented by the following [general formula IIA].
  • X ha is GH or SH
  • X ia is His (H) or Ala (A)
  • X ja is Glu (E), Lys (K), or Thr (T)
  • X ka is WS, IS, or WD
  • X la is Thr(T), Arg(R), Lys(K), or Glu(E)
  • X ma is SG or QG
  • X la is absent or L
  • X 2a can be absent or G, GL, GLIES, or GLIESESAET.
  • the peptide may consist of an amino acid sequence represented by the following [General Formula IIB].
  • X hb is absent or His (H)
  • X ib is His (H) or Ala (A)
  • X jb is Arg (R) or Ala (A)
  • X kb is Lys (K) or Arg(R)
  • X lb is Ser(S), Leu(L), Ala(A), or Gln(Q)
  • X mb is Trp(W) or Ile(I)
  • X nb is Thr(T), Asn(N), or Lys(K)
  • X ob can be SG, QG, SA, or SV.
  • the novel peptides of Formulas I and II may be representatively any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 126, but are not limited thereto.
  • the cell-penetrating peptide is 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 91%, 92%, or 93% of the amino acid sequences represented by SEQ ID NOs: 1 to 126, respectively.
  • %, 94%, 95%, 96%, 97%, 98% may include an amino acid sequence having a sequence homology of 99% or more.
  • novel peptide of Formula I may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 102, and the novel peptide of Formula II typically consists of SEQ ID NOs: 103 to 126 It may be any one amino acid sequence selected from the group.
  • the novel peptide of Formula IA may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 47, and the novel peptide of Formula IB is typically SEQ ID NO: 48 to SEQ ID NO: 48 It may be any one amino acid sequence selected from the group consisting of 96, and the new peptide of general formula IC may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 97 to SEQ ID NO: 102, and the new peptide of general formula IIA
  • the peptide may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 103 to SEQ ID NO: 114, and the novel peptide of formula IIB is any one amino acid sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 126 can
  • the amino acid of the peptide may be changed, and such a change refers to changing the physicochemical properties of the peptide.
  • Amino acid changes can be made, for example, to improve the thermostability of the peptide, to alter the substrate specificity, to change the pH optimum, and the like.
  • Various amino acids capable of enhancing the effect as the cell penetrating peptide may be additionally added or deleted to the N-terminus and C-terminus of the cell penetrating peptide represented by Formula I or II.
  • the peptide can be synthesized by N- or C of the peptide to obtain chemical stability, enhanced pharmacological properties (half-life, uptake, potency, potency, etc.), altered specificity (eg, broad biological activity spectrum), and reduced antigenicity.
  • -A protecting group may be selectively bonded to the terminal.
  • the N-terminus of the peptide is an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group group), a stearyl group, a butoxycarbonyl group, an aryloxycarbonyl group, and polyethylene glycol (PEG).
  • the C-terminus of the peptide is bound to any one protecting group selected from the group consisting of an amino group (amino group, -NH 2 ), a tertiary alkyl group, and an azide (-NHNH 2 )
  • the peptide may optionally further include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or stability of the peptide.
  • the term “stability” may refer to storage stability (eg, storage stability at room temperature) as well as in vivo stability that protects the peptide from attack by proteolytic enzymes in vivo.
  • the cell penetrating peptide is permeable to the type of cell, but is not limited thereto, for example, brain endothelial cells, cancer cells, blood cells, lymphocytes ), immune cells, stem cells, induced pluripotent stem cells (iPSC), neural stem cells (NSC), T cells, B cells, natural killer cells (NK cells), It may be any one selected from the group consisting of macrophages, neurons, glial cells, microglia, astrocytes, and muscle cells.
  • iPSC induced pluripotent stem cells
  • NSC neural stem cells
  • B cells natural killer cells
  • NK cells natural killer cells
  • the peptides of the present invention can be produced so that the purity of each peptide is 90% or more through conventional peptide synthesis methods or production methods known to those skilled in the art, and can be directly synthesized or purchased after requesting production from a peptide manufacturer. .
  • the peptides are prepared in the form of D-form or L-form, peptides consisting only of D-form or L-form, or racemic forms thereof through conventional peptide synthesis methods or preparation methods known to those skilled in the art. can In addition, other conventional modifications known in the art are possible to increase the stability of the peptide.
  • the peptide was preferably synthesized using a solid state peptide synthesis method, but as described above, the method and conditions for synthesizing the peptide are not limited thereto.
  • the present inventors discovered cell-permeable peptides with excellent cell-penetrating ability, and in order to additionally discover peptides with excellent cell-penetrating ability, based on the sequence and structure of the discovered peptides, modified amino acids at specific positions based on 16 amino acids Alternatively, various peptide candidates were designed and synthesized by modifying their length, and by analyzing their cell permeability, various cell penetrating peptides having excellent cell penetrating ability according to the present invention were discovered.
  • the amino acids at the 1st to 3rd positions from the N-terminus in the basic framework are maintained as SHH, but the amino acids at the remaining positions are Modified into a peptide containing at least one or more substitutions, or ii) amino acids at the 1st to 3rd positions from the N-terminus in the basic framework are maintained as GHH, but amino acids at the remaining positions are peptides containing at least one substitution or iii) amino acids at the 1st to 3rd positions from the N-terminus in the basic framework are GHA, AHA, or GQH, and the amino acids at the remaining positions do not contain substitutions or contain at least one substitution.
  • the 4th, 6th, 9th, and 13th in the 16-mer reference peptide Using a peptide with amino acids as a basic skeleton, i) the amino acids at the 1st and 2nd positions from the N-terminus in the basic skeleton are maintained as GH or SH, but at least any of the C-terminus and the N-terminus of the peptide Modification to a peptide containing an additional amino acid sequence in one region, or ii) deletion of at least one amino acid among amino acids at the 1st and 2nd positions from the N-terminus in the basic framework, and at least 1 amino acid at the remaining position
  • Cell-penetrating peptide candidates were designed and synthesized by modifying the peptides containing the above substitutions (peptides other than 16-mer).
  • the first and second amino acids from the N-terminus are GH or SH, and at least one of the C-terminal and N-terminal regions of the peptide contains amino acids
  • amino acid regions at positions 4 to 9, 13, and 16 from the N-terminus are conserved
  • at least one amino acid among amino acids at positions 1 and 2 from the N-terminus is deleted. In this case, it was found that preservation of amino acid regions at positions 4, 6, 9, 10, 12, and 13 from the N-terminus is important for maintaining cell permeability.
  • Another aspect provides a complex comprising the cell-penetrating peptide and a biologically active substance.
  • the complex includes both those formed by simply mixing a peptide and a substance, those formed by mixing a peptide and a substance, and those formed by linking or conjugating these by a bond.
  • the term "complex" may be used interchangeably with conjugate or conjugate.
  • the complex may be a complex formed by expressing the peptide and the biologically active material in a fused state.
  • a gene expressing the peptide and a biologically active substance is inserted into one vector, and then an organism is transformed with the vector to express the gene inserted into the vector, the peptide and the biologically active substance form a fusion protein (fusion protein).
  • fusion protein When expressed as a fusion protein, an optional linker may be incorporated between the peptide and the biologically active substance.
  • the biologically active material is a protein
  • specific examples of binding to the complex that is, the fusion protein, are as follows: When preparing a primer to produce the fusion protein, the biologically active material is expressed.
  • a nucleotide encoding a carrier cell penetrating peptide is added in front of the nucleotide, and then the obtained nucleotide is inserted into a vector (eg, pET vector) using a restriction enzyme, transformed into an appropriate host cell, and expressed.
  • a vector eg, pET vector
  • the fusion protein can be effectively expressed by treating with an expression inducer such as IPTG (isopropyl-1-thio- ⁇ -D-galactopyranoside).
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
  • the expressed fusion protein may be purified by a method such as His tag purification, dialyzed using PBS, and then concentrated by centrifugation.
  • the cell-permeable peptide may include a single or a plurality of combined forms in order to efficiently deliver the biologically active substance into the cell, and the cell-permeable peptide may be combined according to the biologically active substance to be delivered
  • the number can be easily selected or adjusted by a person skilled in the art.
  • the biologically active substance capable of forming a complex by being bound to a cell-penetrating peptide preferably means a 'substance having biological or pharmaceutical activity', which is penetrated into cells (cytoplasm or nucleus) to regulate physiological activity. It refers to substances that can be involved or express pharmacological effects, or substances that have biological activity in various parts of the body, such as cells, tissues, interstitial cells, and blood, which are transported and acted upon.
  • the compound is a broad concept including chemical substances that can function as drugs, including natural or synthetic chemical substances.
  • the nucleic acids include, for example, DNA, RNA, ASO (Antisense oligonucleotide), microRNA (miRNA), small interfering RNA (siRNA), circular RNA, long noncoding RNA lncRNA), small activating RNA (saRNA), messenger RNA (MRNA), aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino ), and may additionally include decoy DNA, plasmid, shRNA, antisense RNA, oligoribonucleotides, or transfer RNA, but is not limited thereto.
  • the nucleases include, for example, CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS ⁇ , CAS variants, Cfp1 (CxxC-finger protein-1), ZEN (zinc-finger nucleases) and TALEN (Transcription activator- like effector nuclease), but is not limited thereto.
  • the drug is a chemical drug, a bio drug, a nucleic acid drug, a peptide drug, a protein drug, a natural product drug, It may be selected from the group consisting of hormones, contrast agents, and antibodies, but is not limited thereto.
  • contrast agent is a broad concept including all materials used for imaging structures or fluids in a living body in medical imaging.
  • Suitable contrast agents include, but are not limited to, radiopaque contrast agents, paramagnetic contrast agents, superparamagnetic contrast agents, computed tomography (CT) contrast agents, and other contrast agents.
  • radiopaque contrast agents for X-ray imaging
  • inorganic iodine compounds and organic iodine compounds e.g. diatrizoat
  • radiopaque metals and their salts e.g. silver, gold, platinum, etc.
  • other radiopaque compounds eg calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide.
  • Suitable paramagnetic contrast agents include gadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and its derivatives, and other gadolinium, manganese, iron, dysprosium, copper, europium (europium), erbium, chromium, nickel and cobalt complexes such as 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid ( DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,-N',N"-triacetic acid (D03A), 1,4,7-triazacyclononane -N,N',N"-triacetic acid (NOTA), 1,4,8,10-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA), hydroxybenzyl ethylene-d
  • Suitable superparamagnetic contrast agents include magnetite, super-paramagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO) and monocrystaline iron oxide.
  • SPIO super-paramagnetic iron oxide
  • USPIO ultrasmall superparamagnetic iron oxide
  • Other suitable contrast agents are iodinated and non-iodinated, ionic and nonionic CT contrast agents, and contrast agents such as spin-label, or other diagnostically effective agents.
  • biodrug refers to various biopharmaceuticals such as (original) biologics, biogenerics, biobetters, and biosuperiors.
  • the biodrug refers to any drug manufactured, secreted, or semi-synthesized from a biological origin, and includes, but is not limited to, vaccines, blood products, antigens, cell products, gene therapy products, stem cells, and the like.
  • the nanoparticles may be selected from the group consisting of iron oxide, gold, carbon nanotubes, and magnetic beads, but are not limited thereto.
  • compositions for mass delivery comprising the complex as an active ingredient.
  • the substance delivery composition may be used to deliver a biologically active substance into living tissue or blood or promote cell permeation.
  • the composition may be delivered through cells constituting biological tissues or cell-to-cell junctions, but the delivery method is not limited.
  • the biological tissue refers to one or more epithelial tissue, muscle tissue, nerve tissue, and connective tissue, and each organ may consist of one or more tissues, such as mucous membrane, skin, brain, lung, liver, kidney, spleen, lung, heart, and stomach. , large intestine, digestive tract, bladder, ureter, urethra, ovary, testis, genital organ, muscle, blood, blood vessel, lymphatic vessel, lymph node, thymus, pancreas, adrenal gland, thyroid, parathyroid gland, larynx, tonsils, bronchi, and alveoli. It may, but is not limited thereto.
  • the biologically active substance is an extracellular portion protein of a ligand capable of selectively binding to a receptor specifically expressed in a specific cell, tissue or organ, or A complex may be formed by combining with a monoclonal antibody (mAb) capable of specifically binding to these receptors or ligands and a modified form.
  • mAb monoclonal antibody
  • the binding of the peptide and the biologically active substance is by indirect linkage by a cloning technique using an expression vector at the nucleotide level, or by direct linkage by chemical or physical covalent or non-covalent bonds between the peptide and the biologically active substance. can do.
  • the composition including the complex when used as a pharmaceutical composition, the composition may include about 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition.
  • composition may contain at least one active ingredient exhibiting the same or similar function in addition to the active ingredient.
  • composition may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • a pharmaceutically acceptable carrier may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and a mixture of one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents and other conventional additives may be added.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injections such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets, and may act specifically on target organs.
  • a target organ-specific antibody or other ligand may be used in combination with the carrier.
  • it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's literature.
  • compositions containing the complex as an active ingredient are intravenous, intraperitoneal, intramuscular, intrathecal, intracerebroventricular, subcutaneous, and intradermal. , It can be delivered into the body by injection through routes such as nasal, mucosal, inhalation, and oral.
  • the dosage varies depending on the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.
  • Formulations for oral administration may be tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions, but are not limited thereto.
  • Formulations for parenteral administration may be injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays, but are not limited thereto.
  • Another aspect provides a method for transferring a substance into a cell comprising the step of bringing the composition for substance delivery into contact with the cell.
  • the cells include brain endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells (iPSC), neural stem cells ( neural stem cell (NSC), T cell, B cell, natural killer cell (NK cell), macrophage, neuron, glial cell, microglia , It may be selected from the group consisting of astrocytes and muscle cells. In addition, the cells may be cells of humans and/or other mammals.
  • the cell-penetrating peptide having a substance transfer function according to the present invention is a very small peptide, biological interference with the active substance that may occur can be minimized.
  • Another aspect provides a polynucleotide encoding the peptide.
  • the polynucleotide may be in the form of RNA or DNA, including cDNA and synthetic DNA.
  • DNA can be single-stranded or double-stranded. If single-stranded, it may be the coding strand or the non-coding (antisense) strand, and the coding sequence encodes the same polypeptide as a result of degeneracy or redundancy of the genetic code.
  • the polynucleotide may also include a variant of a polynucleotide described herein, which variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • An allelic variant is an alternating form of a polynucleotide sequence that may have a substitution, deletion, or addition of one or more nucleotides that does not substantially alter the function of the polynucleotide being encoded (encoded). It is well known in the art that a single amino acid can be encoded by more than one nucleotide codon and that the polynucleotide can be readily modified to produce alternating polynucleotides encoding the same peptide.
  • the novel cell-penetrating peptide according to one aspect has excellent cell-penetrating ability, it can effectively deliver substances having biological activity into cells, tissues, etc. in vivo, and can be usefully used in the field of basic research, diagnosis and treatment of various diseases, etc. there is.
  • 1 is a peptide candidate group (#1 to #7, #10) in the basic skeleton of a reference peptide, wherein amino acids at positions 1 to 3 from the N-terminus are SHH, and amino acids at other positions include at least one substitution. to #47; 4 ⁇ M) showing the results of cell permeability analysis.
  • Figure 2 is a peptide candidate group (#1 to #47; 8 ⁇ M) in which amino acids at the 1st to 3rd positions from the N-terminus are SHH in the basic skeleton of the reference peptide, and amino acids at the remaining positions contain at least one substitution. This is the result showing the cell permeability analysis results for.
  • Figure 3 is a peptide candidate group (#48 to #94; 4 ⁇ M) in which amino acids at the 1st to 3rd positions from the N-terminus are GHH in the basic skeleton of the reference peptide, and the amino acids at the remaining positions contain at least one substitution. This is the result showing the cell permeability analysis results for.
  • peptide candidate group (#48 to #94; 8 ⁇ M) in which amino acids at positions 1 to 3 from the N-terminus are GHH in the basic skeleton of a reference peptide, and amino acids at other positions include at least one substitution. This is the result showing the cell permeability analysis results for.
  • Figure 5 is a peptide candidate group (#95 and #96; 4 ⁇ M) in which amino acids at positions 1 to 3 from the N-terminus are GHH in the basic skeleton of the reference peptide, and amino acids at other positions include at least one substitution. This is the result showing the cell permeability analysis results for.
  • Figure 6 shows that in the basic skeleton of the reference peptide, the amino acids at the 1st to 3rd positions from the N-terminus are GHA, AHA, or GQH, Amino acids at the remaining positions are results of cell permeability analysis for peptide candidates (#97 to #102; 4 ⁇ M) containing at least one substitution.
  • peptide candidate group comprising an additional amino acid sequence in at least one of the C-terminal and N-terminal regions of the reference peptide, wherein the first and second positions of amino acids are GH or SH in the basic skeleton of the reference peptide ( These are the results of cell permeability analysis for #103 to #114; 4 ⁇ M).
  • peptide candidate group in which at least one or more amino acids among the amino acids at the first and second positions from the N-terminus are deleted in the basic skeleton of the reference peptide, and the amino acids at the remaining positions include at least one substitution (#115 to #126; 4 ⁇ M) shows the result of cell permeability analysis.
  • cell-permeable peptides having variously modified sequences were designed and synthesized.
  • the amino acid at the 4th position (E), the amino acid at the 6th position (L), the amino acid at the 7th position (K), the amino acid at the 11th position (W), the 13th position A cell-permeable peptide candidate group was designed by substituting amino acids at the remaining positions without modifying the amino acid (V), the amino acid (S) at the 15th position, and the amino acid (G) at the 16th position.
  • the present inventors used a solid state peptide synthesis (SPPS) method to synthesize each of the peptides described in Example 1-1.
  • SPPS solid state peptide synthesis
  • This method is an organic synthesis method in which the C-terminus of an amino acid whose N-terminus is protected by F-moc is coupled to the N-terminus of the resin one by one.
  • N , N -dimethylformamide (DMF) was used as the solvent for all reactions, and amino acid coupling conditions were 0.5M DIC ( N,N- Diisopropylcarbodiimide, DIC) 1 ml, 1 M Ethyl cyanohydroxyiminoacetate (Oxyma) was mixed with 0.5 ml and reacted in a microwave synthesizer.
  • amino acids were prepared by varying the reaction time, temperature, or microwave voltage for each amino acid sequence.
  • the F-moc of the previous amino acid must be removed.
  • the F-moc protecting group was deprotected twice for 2 minutes at 80 °C using 80% DMF and 20% piperidine solution ( deprotecting). Between all coupling processes and deprotection processes, a process of washing alternately three times using DMF and methylene chloride (dichloromethane, DCM) was performed.
  • a fluorescent substance containing a carboxyl group may be linked to the N-terminus of the peptides by a chemical bonding method in order to observe and quantify cell permeability later.
  • the fluorescent materials that can be used at this time include 5-carboxyfluorescein (5-FAM), Fluorescein-5-isothiocyanate (FITC), Cyanine 3 carboxylic acid, Cyanine 5 carboxylic acid, Cyanine 7 carboxylic acid ( Cyanine 7 carboxylic acid) and the like, and one or more of the fluorescent materials may be used.
  • the present inventors first synthesized the last amino acid of the synthesized peptide in a solid phase resin, mixed 5-FAM: DIC: Oxyma: resin in a ratio of 2: 2.5: 4: 1, and then reacted in the resin. It proceeded for 2 hours at room temperature, and a magnetic stirrer was used. Next, when the resin color changed from yellow to deep yellow or orange during the synthesis process, a process of washing DMF and methylene chloride alternately three times was performed.
  • the lyophilized peptide was dissolved in distilled water or acetonitrile (ACN), and separated and purified using reverse phase high-performance liquid chromatography.
  • Solvent A distilled water 99.9%, TFA 0.1%)
  • Solvent B distilled water 9.9%, acetonitrile 90%, TFA 0.1%) were used as the mobile phase solvents of the HPLC.
  • the HPLC mobile phase was separated by starting with 90% of Solvent A and 10% of Solvent B and increasing Solvent B at a gradient of 1%/min. Thereafter, the separated peptide solution was lyophilized to remove the solvent, and then dissolved in a desired solvent to conduct the experiment.
  • Example 2 In vitro cell permeability analysis of 16-mer cell penetrating peptide candidates
  • Example 1 In order to evaluate the cell permeability of the peptide candidates synthesized in Example 1, the present inventors performed an in vitro permeability assay for blood-brain barrier cells.
  • the hCMEC/D3 cells were cultured at 37° C. under 5% CO 2 conditions using a synthetic culture medium of Endothelial Cell Basal Medium 2 (EBM2) containing growth factors. Thereafter, when the cell confluency reached 80%, the cells were separated, and 4 ⁇ 10 3 cells were added to 40 ⁇ L of the culture medium, which was dispensed into a 384-well plate, centrifuged for 10 seconds, and 37°C, 5% CO 2 Cells were adhered to the plate by culturing for 18 hours or longer under the conditions.
  • EBM2 Endothelial Cell Basal Medium 2
  • the amino acids at the remaining positions are based on a peptide conserving the 4th, 6th, 7th, 11th, 13th, 15th, and 16th amino acids in the reference peptide as a basic backbone Peptide candidates with various modifications were designed. Specifically, based on the amino acid position of the reference peptide, amino acids at positions 1 to 3 from the N-terminus were maintained as SHH, but amino acids at the remaining positions contained at least one substitution. However, the position of the amino acid sequence that did not appear to be modified among the modified peptides was preserved in the sequence of the basic skeleton, and specific sequence information is shown in Tables 1 and 2 below. In Tables 1 and 2, portions marked in bold represent regions where amino acids are substituted.
  • the amino acids at the 1st to 3rd positions from the N-terminus are SHH, and the 4th to 8th and 11th positions from the N-terminus , 13th, 15th, and 16th positions along with conservation of amino acid regions, it was found that even when amino acids at other positions were substituted, the unique structure of the peptide exhibiting excellent cell permeability was maintained.
  • the amino acids at the remaining positions are based on a peptide conserving the 4th, 6th, 7th, 11th, 13th, 15th, and 16th amino acids in the reference peptide as a basic backbone Peptide candidates with various modifications were designed. Specifically, based on the amino acid position of the reference peptide, amino acids at positions 1 to 3 from the N-terminus were maintained as GHH, but amino acids at other positions were designed to contain at least one substitution. However, among the modified peptides, the position of the amino acid sequence that did not appear to be modified was preserved in the sequence of the basic skeleton, and specific sequence information is shown in Tables 3 and 4 below. In Tables 3 and 4, portions marked in bold represent regions where amino acids are substituted.
  • the amino acids at the 1st to 3rd positions from the N-terminus are GHH, and the 4th, 6th to 8th amino acids from the N-terminus , 11th, 13th, 15th, and 16th positions along with conservation of amino acid regions, even when amino acids at other positions are substituted, it was found that the unique structure of the peptide exhibiting excellent cell permeability was maintained.
  • the amino acids at the remaining positions are based on a peptide conserving the 4th, 6th, 7th, 11th, 13th, 15th, and 16th amino acids in the reference peptide as a basic backbone Peptide candidates with various modifications were designed.
  • the amino acids at the 1st to 3rd positions from the N-terminus based on the amino acid position of the reference peptide are GHA, AHA, or GQH
  • Amino acids at the remaining positions were designed as peptide candidates containing no substitution or at least one substitution.
  • the position of the amino acid sequence that did not appear to be modified was preserved in the sequence of the basic skeleton, and specific sequence information is shown in Table 5 below. In Table 5, portions marked in bold represent regions where amino acids are substituted.
  • the amino acids at the 1st to 3rd positions from the N-terminus are GHA, AHA, or GQH, and the 4th from the N-terminus, With conservation of amino acid regions at positions 6, 7, 9 to 13, 15, and 16, even when amino acids at other positions are substituted, the unique structure of the peptide exhibiting excellent cell permeability is maintained And it was found.
  • cell-permeable peptides having variously modified sequences and lengths were designed and synthesized using Korean Patent Application No. 2020-0049621 peptide (GHHERLKSDEWSVTSG, hereinafter referred to as reference peptide) as a basic framework.
  • reference peptide Korean Patent Application No. 2020-0049621 peptide
  • the amino acid at position 4 (E), the amino acid at position 6 (L), the amino acid at position 9 (D), and the amino acid at position 13 (V) are not modified.
  • a cell-penetrating peptide candidate group was designed by substituting or deleting amino acids at the remaining positions and adding amino acid sequences to their N- and/or C-terminus.
  • the first and second amino acids in the reference peptide are GH or SH, and the first peptide candidate group comprising an additional amino acid sequence in at least one of the C-terminal and N-terminal regions of the peptide, the first and a second peptide candidate group in which at least one or more of the amino acids at position 2 is deleted, and a cell-permeable peptide candidate group was designed and synthesized.
  • the present inventors used a solid state peptide synthesis (SPPS) method to synthesize each of the peptides described in Example 3-1, and synthesized and separated and purified the peptides in the same manner as in Example 1-2 .
  • SPPS solid state peptide synthesis
  • Example 4 In vitro cell permeability analysis of cell permeable peptide candidates other than 16-mer
  • Example 3 In order to evaluate the cell permeability of the peptide candidates other than the 16-mers synthesized in Example 3, the present inventors performed in vitro permeability analysis for blood-brain barrier cells in the same manner as in Example 2-1.
  • the amino acids at the 1st and 2nd positions from the N-terminus are GH or SH, and the 4th to 9th from the N-terminus, With the conservation of the amino acid region at the 13th and 16th positions, even when amino acids are added to at least one of the C-terminal and N-terminal regions of the peptide, the unique structure of the peptide exhibiting excellent cell permeability it was found to be maintained.
  • Example 3-1 using a peptide with the 4th, 6th, 9th, and 13th amino acids conserved in the reference peptide as a basic skeleton, designing a peptide candidate in which amino acids at the remaining positions are variously modified did Specifically, based on the amino acid position of the reference peptide, at least one or more amino acids among amino acids at the first and second positions from the N-terminus are deleted, and amino acids at the remaining positions include at least one substitution. A peptide candidate group was designed. . However, among the modified peptides, the position of the amino acid sequence that did not appear to be modified was preserved in the sequence of the basic skeleton, and specific sequence information is shown in Table 7 below. In Table 7, portions marked in bold indicate regions where amino acids are substituted.
  • the peptide candidate group based on the amino acid position of the reference peptide, conservation of the amino acid region at the 4th, 6th, 9th, 10th, 12th, and 13th positions from the N-terminus. In addition, it was found that the unique structure of the peptide exhibiting excellent cell permeability is maintained even when at least one or more amino acids of the first and second positions from the N-terminus are deleted.

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Abstract

La présente invention concerne un nouveau peptide de pénétration cellulaire et son utilisation ; l'invention concerne un peptide de pénétration cellulaire comprenant un acide aminé représenté par la formule générale I ou la formule générale II, un polynucléotide codant pour le peptide de pénétration cellulaire, un complexe comprenant le peptide de pénétration cellulaire et une substance biologiquement active, ainsi qu'une composition pour administrer une substance, comprenant le complexe.
PCT/KR2022/009463 2021-07-02 2022-06-30 Nouveau peptide de pénétration cellulaire et son utilisation WO2023277628A1 (fr)

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US20190046652A1 (en) * 2012-10-04 2019-02-14 Aadigen, Llc Cell penetrating peptides for intracellular delivery of molecules
KR20200104524A (ko) * 2019-02-27 2020-09-04 주식회사 아임뉴런바이오사이언스 신규의 세포 투과성 펩타이드 및 이의 용도
KR102274876B1 (ko) * 2020-12-24 2021-07-08 주식회사 아임뉴런바이오사이언스 신규한 세포 투과성 펩타이드 및 이의 용도
KR102386478B1 (ko) * 2021-07-01 2022-04-15 주식회사 아임뉴런바이오사이언스 신규한 세포 투과성 펩타이드 및 이의 용도
KR102386477B1 (ko) * 2021-07-01 2022-04-15 주식회사 아임뉴런바이오사이언스 신규한 세포 투과성 펩타이드 및 이의 용도

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190046652A1 (en) * 2012-10-04 2019-02-14 Aadigen, Llc Cell penetrating peptides for intracellular delivery of molecules
KR20150145132A (ko) * 2014-06-18 2015-12-29 한국과학기술연구원 신규 세포투과성 펩타이드 및 이의 용도
KR20170015852A (ko) * 2015-07-31 2017-02-09 이화여자대학교 산학협력단 신규한 세포투과성 펩타이드
CN106916228A (zh) * 2017-01-13 2017-07-04 华南理工大学 可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用
KR20200104524A (ko) * 2019-02-27 2020-09-04 주식회사 아임뉴런바이오사이언스 신규의 세포 투과성 펩타이드 및 이의 용도
KR102274876B1 (ko) * 2020-12-24 2021-07-08 주식회사 아임뉴런바이오사이언스 신규한 세포 투과성 펩타이드 및 이의 용도
KR102386478B1 (ko) * 2021-07-01 2022-04-15 주식회사 아임뉴런바이오사이언스 신규한 세포 투과성 펩타이드 및 이의 용도
KR102386477B1 (ko) * 2021-07-01 2022-04-15 주식회사 아임뉴런바이오사이언스 신규한 세포 투과성 펩타이드 및 이의 용도

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