WO2024129497A1 - Conjugués peptidiques de thyclotide ayant une perméabilité cellulaire et activité inhibitrice - Google Patents

Conjugués peptidiques de thyclotide ayant une perméabilité cellulaire et activité inhibitrice Download PDF

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WO2024129497A1
WO2024129497A1 PCT/US2023/082871 US2023082871W WO2024129497A1 WO 2024129497 A1 WO2024129497 A1 WO 2024129497A1 US 2023082871 W US2023082871 W US 2023082871W WO 2024129497 A1 WO2024129497 A1 WO 2024129497A1
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cancer
thyclotide
salt
peptide
peptide conjugate
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PCT/US2023/082871
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English (en)
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Daniel H. Appella
Hongchao Zheng
Victor CLAUSSE
Harsha C. AMARASEKARA
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2024129497A1 publication Critical patent/WO2024129497A1/fr

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    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • peptides typically are poor drugs as they do not penetrate cell membranes and therefore have low bioactivity in vivo. This limitation often prevents peptides from being developed into a therapeutic drug.
  • many strategies have been examined to improve the cellular uptake of peptides, such as cyclization of a linear peptide into a macrocyclic ring, methylation of selected amide bonds within the peptide sequence, and introduction of a hydrocarbon staple that links sidechains within the peptide. None of these approaches guarantees cellular uptake of a peptide, but there are selected cases where cellular uptake may be improved when using one or more of these strategies (Methods Mol.
  • CPPs cell penetrating peptides
  • the present invention provides a thyclotide-peptide conjugate of the formula (I): Tn-L-P (I), wherein T is a thyclotide unit of the formula: , wherein the configuration of the tetrahydrofuranyl ring the two nitrogen atoms can be (R,R) and/or (S,S), and wherein the thyclotide units are optionally intercepted by segments of polypeptide which can be the same as or different from P, n is about 5 to about 25, L is a linker, P is a polypeptide, and B is a nucleobase or a heterocyclyl moiety, or a pharmaceutically acceptable salt thereof.
  • the present invention further provides pharmaceutical compositions containing one or more of the thyclotides, as well as method of treating diseases and disorders in a human or animal by the administration of one or more of these conjugates.
  • Leydig 769584 HHS E-033-2023-0-PC-01 3 [0010]
  • the thyclotide peptide of the present invention show reduced or no aggregation and advantageously penetrate the cell membranes, and therefore have advantageous bioavailability, which results in efficacious treatment of diseases many of which have been considered intractable heretofore.
  • FIG.1 illustrates the structures of (S,S) and (R,R) thyclotides in accordance with an aspect of the invention.
  • FIG.2A-2C display FACS (fluorescence activated cell sorting analysis) data of three replicate experiments, showing that the thyclotide-peptide conjugate molecule 3 was able to enter cells while the control peptide (molecule 1) and aegPNA-peptide conjugate (molecule 2) could not.
  • FIG.2D displays the statistical analysis of the mean FACS cell uptake data.
  • FIG.3A-3C display fluorescence microscopy data showing that the thyclotide- peptide conjugate 3 is able to enter the nuclei of cells.
  • FIG.4 displays a series of Western blots that use an antibody to detect a methylated form of the protein p53 (p53K382Me) showing that the thyclotide-peptide conjugate 6 was able to inhibit the SETD8 enzyme in cells while the peptide along (molecule 4) had no activity.
  • FIG.5 displays the change in cell proliferation as a function of concentration of thyclotide-peptide conjugate 6.
  • FIG.6 depicts the HPLC chromatogram of peptide 1.
  • FIG.7 depicts the mass spectrum of peptide 1.
  • FIG.8 depicts the HPLC chromatogram of thyclotide-peptide conjugate 2.
  • FIG.9 depicts the mass spectrum of thyclotide-peptide conjugate 2.
  • FIG.10 depicts the HPLC chromatogram of thyclotide-peptide conjugate 3.
  • FIG.11 depicts the mass spectrum of thyclotide-peptide conjugate 3.
  • FIG.12 depicts the HPLC chromatogram of peptide 4.
  • FIG.13 depicts the mass spectrum of peptide 4.
  • FIG.14 depicts the HPLC chromatogram of thyclotide-peptide conjugate 5.
  • FIG.15 depicts the mass spectrum of thyclotide-peptide conjugate 5.
  • FIG.16 depicts the HPLC chromatogram of thyclotide-peptide conjugate 6.
  • Leydig 769584 HHS E-033-2023-0-PC-01 4 depicts the mass of thyclotide-peptide conjugate 6.
  • FIG.18 depicts the HPLC chromatogram of peptide 7.
  • FIG.19 depicts the mass spectrum of peptide 7.
  • FIG.20 depicts the structures of peptide 1.
  • FIG.21 depicts the structure of thyclotide-peptide 2.
  • FIG.22 depicts the structure of thyclotide-peptide 3.
  • the molecule is shown split into two parts at the vertical dashed line for the sake of illustration.
  • FIG.23 depicts the structure of peptide 4.
  • FIG.24 depicts the structure of thyclotide-peptide 5.
  • the molecule is shown split into two parts at the vertical dashed line for the sake of illustration.
  • FIG.25 depicts the structure of thyclotide-peptide 6.
  • FIG.26 depicts the structure of peptide 7.
  • FIG.27 depicts the structures of peptide 8. The molecule is shown split into two parts at the vertical dashed line for the sake of illustration.
  • FIG.28 depicts the structures of peptide 9. The molecule is shown split into two parts at the vertical dashed line for the sake of illustration.
  • FIG.29 depicts the FACS data for peptide 8.
  • FIG.30 depicts the Western blot data for peptide 8, showing that the peptide suppresses the activity of the target enzyme, Setd8, in SK-N-AS cells which are derived from neuroblastoma.
  • the present invention provides thyclotide-peptide conjugates represented by the formula (I): T n -L-P (I), wherein T is a thyclotide unit of the formula: , wherein the configuration of the tetrahydrofuranyl ring carbon nitrogen atoms can be (R,R) and/or (S,S), and wherein the thyclotide units are optionally intercepted by segments of polypeptide which can be the same as or different from P, Leydig 769584 HHS E-033-2023-0-PC-01 5 n is about 5 to about 25, L is a linker, P is a polypeptide, and B is a nucleobase or a heterocyclyl moiety, or a pharmaceutically acceptable salt thereof.
  • T is a thyclotide unit of the formula: , wherein the configuration of the tetrahydrofuranyl ring carbon nitrogen atoms can be (R,R) and/or (S,S), and wherein the
  • n of the thyclotide-peptide conjugate or salt can be from about 8 to about 20, for example, about 10 to about 15.
  • the nucleobase B of the thyclotide-peptide conjugate or salt can be any of adenosine, guanosine, cytosine, thymine, and uracil. Numerous nucleobases of non-natural nucleotides are known in the art.
  • nucleobases of non-natural nucleotides include, for example, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, and pseudouridine.
  • a readily available reference for modified RNAs and nucleobases and nucleotides contained therein is available. See Cantara, W.A. et al., Nucleic Acids Research, Vol.39, Database issue D195– D201 (2011) and references described therein.
  • heterocyclyl “heterocycle”, or “heterocyclic” as used herein, means a monocyclic heterocyclic or a bicyclic heterocyclic group.
  • the monocyclic heterocyclic is a three-, four-, five-, six- or seven-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, N(H) and S.
  • the three- or four- membered ring can contain zero or one double bond and a heteroatom selected from the group consisting of O, N, N(H) and S.
  • the five-membered ring can contain zero or one double bond, and one, two or three heteroatoms selected from the group consisting of O, N, N(H) and S.
  • the six-membered ring can contain zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N, N(H) and S.
  • the seven- membered ring can contain zero, one, two, or three double bonds and one, two or three heteroatoms selected from the group consisting of O, N, N(H) and S.
  • the monocyclic heterocycle can be unsubstituted or substituted and is connected to the parent molecular moiety through any substitutable carbon atom or any substitutable nitrogen atom contained within the monocyclic heterocycle.
  • heterocyclyl groups include pyridyl, piperidinyl, piperazinyl, pyrazinyl, pyrolyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolidinyl, furanyl, Leydig 769584 HHS E-033-2023-0-PC-01 6 tetrahydrofuranyl, thiophenyl, purinyl, pyrimidinyl, thiazolyl, thiazolidinyl, thiazolinyl, oxazolyl, triazolyl, tetrazolyl, tetrazinyl, benzoxazolyl, morpholinyl, thiomorpholinyl, quinolinyl, and isoquinolinyl, preferably tetrazolyl.
  • any of the heterocyclyl group can be optionally substituted with one or more substituents, which can be electron withdrawing and/or electron donating or hydrophilic and/or hydrophilic.
  • substituents on the heterocyclyl groups include halo, hydroxy, carboxy, phosphoryl, phosphonyl, phosphono C1-C6 alkyl, carboxy C1-C6 alkyl, dicarboxy C1-C6 alkyl, dicarboxy halo C1-C6 alkyl, sulfonyl, cyano, nitro, alkoxy, alkylthio, acyl, acyloxy, thioacyl, acylthio, aryloxy, amino, aminoalkyl, alkylamino, dialkylamino, trialkylamino, guanidino, aldehydo, ureido, aminocarbonyl, a cationic group, and an anionic group.
  • the linker L of the thyclotide-peptide conjugate or salt can be a linear or cyclic linker.
  • the linker is a linear linker.
  • linear linkers include linkers comprising an alkylenedioxy group, an amido group, or both alkylenedioxy and amido groups.
  • cyclic linkers include linkers comprising a cyclic aliphatic linker, a cyclic aromatic linker, and/or a cyclic alicyclic linker.
  • the thyclotide unit has two chiral carbon atoms at positions 3 and 4 of the tetrahydrofuran ring: B O .
  • the tetrahydrofuran the two chiral centers can form four diastereomeric pairs: (3R,4R), (3R,4S), (3S,4R), and (3S,4S).
  • the monomeric subunit has the (3R,4R) configuration.
  • the monomer When the monomer has the (3R,4R) configuration, the monomer induces a right-handed helix into the PNA, which matches the right-handed helicity of DNA and RNA, thereby allowing for complex formation (by Watson-Crick binding) of the conjugate and DNA or RNA.
  • P of the thyclotide-peptide conjugate or salt is of the formula: Leydig 769584 HHS E-033-2023-0-PC-01 7 , wherein R is a side the peptide can be anywhere from 1 amino acid to up to about 30 amino acids, preferably from about 3 to about 25 amino acids, more preferably from about 5 to about 20 amino acids, and particularly preferably from about 8 to about 18 amino acids.
  • R is a side the peptide can be anywhere from 1 amino acid to up to about 30 amino acids, preferably from about 3 to about 25 amino acids, more preferably from about 5 to about 20 amino acids, and particularly preferably from about 8 to about 18 amino acids.
  • An example of a thyclotide-peptide conjugate is 6, which is of the formula: .
  • Further examples of the thyclotide-peptide conjugate according to formula (I) include compounds 8 and 9 having the following structures:
  • the present invention further provides a pharmaceutical composition comprising the thyclotide-peptide conjugate or salt as described above and a pharmaceutically acceptable carrier.
  • anticancer peptides are known in the art, for example, Mellitin (active against human and leukemic cells), Polybia-MP1 (active against human prostate cancer cells and bladder cancer cells), NRC-03 and NRC-07 (active against human breast Leydig 769584 HHS E-033-2023-0-PC-01 9 cancer cells and murine mammary carcinoma cells), D-peptides A, B, C, and D (active against human lung, cervix, glioma cancer cell, and mouse myeloma cells), Magainin 2 (active against human bladder cancer cells), Gomesin (active against human colon, breast, and cervix adenocarcinoma cells), SVS-1 (active against human epidermis, leukemia, and various carcinomas), Lactoferrici B (active against human fibrosarcoma), LL-37 (active against human squamous cell carcinoma and leukemic cells), LTX-315 (active against human osteosarcoma), and Pard
  • the thyclotide-peptide conjugates of the present invention can be made by any suitable methods known to those skilled in the art, for example, by the use of “click chemistry” based “click reactions.”
  • click reactions include cycloadditions such as 1,3-dipolar cycloadditions and hetero-Diels-Alder cycloadditions; nucleophilic ring opening reactions which involve openings of strained heterocyclic electrophiles such as aziridines, epoxides, cyclic sulfates, aziridinium ions, and episulfonium ions; and carbonyl chemistry of the non-aldol type such as the formation of ureas, thioureas, hydrazones, oxime ethers, and aromatic heterocycles; and additions to carbon-carbon multiple click reactions.
  • Cycloadditions include Cu1 catalyzed Huisgen 1,3- dipolar cycloadditions of azides and alkynes as illustrated herein: . to include non- toxic salts synthesized from the parent conjugate which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these conjugates with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
  • non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Suitable bases for forming salts include inorganic bases such as alkali and alkaline earth metal bases, such as those containing metallic cations such as sodium, potassium, magnesium, calcium and the like.
  • suitable bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate.
  • Suitable acids for forming salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p- toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, maleic acid, tartaric acid, fatty acids, long chain fatty acids, and the like.
  • Preferred pharmaceutically acceptable salts of inventive conjugates having an acidic moiety include sodium and potassium salts.
  • Preferred pharmaceutically acceptable salts of inventive conjugates having a basic moiety include hydrochloride and hydrobromide salts.
  • the conjugates of the present invention containing an acidic or basic moiety are useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof.
  • the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
  • conjugates and salts may form solvates, or exist in a substantially uncomplexed form, such as the anhydrous form.
  • solvate refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice. When the solvent incorporated in the solvate is water, the molecular complex is called a hydrate.
  • Pharmaceutically acceptable solvates include hydrates, alcoholates such as ethanolates, acetonitrilates and the like. These conjugates can also exist in polymorphic forms.
  • the conjugates and salts thereof of the present invention can be prepared by any suitable method, in particular as disclosed herein.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the thyclotide-peptide conjugate or salt and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier be one that is chemically inert to the active conjugates and one that has no detrimental side effects or toxicity under the conditions of use.
  • Leydig 769584 HHS E-033-2023-0-PC-01 11 [0064] The choice of carrier will be determined in part by the particular conjugate or salt of the present invention chosen, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.
  • compositions for parenteral administration that comprise a solution or suspension of the inventive conjugate or salt dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous isotonic sterile injection solutions.
  • Such solutions can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the conjugate or salt of the present invention may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxy
  • Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary
  • the parenteral formulations can contain preservatives and buffers.
  • such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17.
  • HLB hydrophile-lipophile balance
  • the quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight.
  • Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Topical formulations including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the invention for application to skin. Topically applied compositions are generally in the form of liquids, creams, pastes, lotions and gels.
  • the liquid vehicle can include other materials, such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain Leydig 769584 HHS E-033-2023-0-PC-01 13 the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved in water or a biocompatible solvent prior to use to form a solution that is substantially neutral or that has been rendered substantially neutral and that can then be applied to the target site.
  • other materials such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain Leydig 769584 HHS E-033-2023-0-PC-01 13 the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as a therapeutically effective amount of the inventive conjugate or salt thereof dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules, (c) powders, (d) suspensions in an appropriate liquid, and (e) suitable emulsions.
  • liquid solutions such as a therapeutically effective amount of the inventive conjugate or salt thereof dissolved in diluents, such as water, saline, or orange juice
  • capsules, sachets, tablets, lozenges, and troches each containing a predetermined amount of the active ingredient, as solids or granules
  • powders such as powders, (d) suspensions in an appropriate liquid, and (e) suitable emulsions.
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • the conjugate or salt of the present invention alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation.
  • the conjugates or salts are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of active conjugate are 0.01%-20% by weight, preferably 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such surfactants are the esters or partial esters of fatty acids Leydig 769584 HHS E-033-2023-0-PC-01 14 containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25%-5%.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included as desired, such as lecithin for intranasal delivery.
  • These aerosol formulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations may be used to spray mucosa.
  • the conjugate or salt of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • the conjugate or salt of the present invention may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes serve to target the conjugates to a particular tissue, such as lymphoid tissue or cancerous hepatic cells. Liposomes can also be used to increase the half-life of the inventive conjugate.
  • Liposomes useful in the present invention include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the active agent to be delivered is incorporated as part of a liposome, alone or in conjunction with a suitable chemotherapeutic agent.
  • liposomes filled with a desired inventive conjugate or salt thereof can be directed to the site of a specific tissue type, hepatic cells, for example, where the liposomes then deliver the selected compositions.
  • Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • lipids are generally guided by consideration of, for example, liposome size and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980), and U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • a ligand to be incorporated into the liposome can include, for example, antibodies or fragments thereof Leydig 769584 HHS E-033-2023-0-PC-01 15 specific for cell surface determinants of the tissue type.
  • a liposome suspension containing a conjugate or salt of the present invention may be administered intravenously, locally, topically, etc. in a dose that varies according to the mode of administration, the agent being delivered, and the stage of disease being treated.
  • Encapsulation formulations including liposome formulations are well suited for use in oral administration of the inventive oligomer, optionally further comprising permeation enhancers such as sodium caprate (see, e.g., van Putten, M. et al, Mol Ther Nucleic Acids, Nov; 3(11): e211 (2014).
  • the conjugates or salts thereof can be used in any suitable dose. Suitable doses and dosage regimens can be determined by conventional range finding techniques.
  • the dosages range from about 0.001 to about 1000 mg/kg body weight of the animal being treated/day.
  • the conjugates or salts may be administered from about 100 mg/kg to about 300 mg/kg, from about 120 mg/kg to about 280 mg/kg, from about 140 mg/kg to about 260 mg/kg, from about 150 mg/kg to about 250 mg/kg, from about 160 mg/kg to about 240 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
  • the present invention further provides a method of treating an animal afflicted with a disease or disorder comprising administering to the animal an effective amount of a thyclotide-peptide conjugate or salt or a pharmaceutical composition as described herein.
  • the thyclotide-peptide conjugate or salt thereof is administered intravenously or subcutaneously.
  • the disease or disorder is cancer. Any suitable can be treated.
  • the cancer is selected from the group consisting of leukemia, adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, brain cancer, basal cancer of the skin, bile duct cancer, Ewing Sarcoma, bladder cancer, lung cancer, breast cancer, gastrointestinal cancer, central nervous system cancer, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasm, colorectal cancer, T-cell lymphoma, uterine cancer, esophageal cancer, head and neck cancer, Leydig 769584 HHS E-033-2023-0-PC-01 16 eye cancer
  • EXAMPLE 1 This example illustrates methods of preparation of peptides, thyclotides, peptide- PNA conjugates and thyclotide-peptide conjugates in accordance with aspects of the invention.
  • Reagents and Materials All 9-fluorenylmethoxycarbonyl (Fmoc)-PNA monomers were purchased from PolyOrg, Inc. (Leominster, MA, USA).
  • Fmoc-Val-OH and Fmoc-Leu-OH were purchased from Advanced ChemTech (Louisville, KY, USA).
  • Fmoc-his(trityl)-OH, Fmoc-Nle-OH, and Fmoc- Lys(Boc)-OH were purchased from Sigma-Aldrich (St Louis, MO, USA).
  • High purity water (18 M ⁇ ) was generated from a Millipore (Billerica, MA, USA) Milli-Q water system.
  • Rink Amide ProTide resin (LL, 100–200 mesh, 0.19 mmol/g) was purchased from CEM corporation (Matthews, NC, USA).
  • the resin was resubmitted to fresh cleavage cocktail and cleaved for 1 hour, and was drained into the first cleavage fraction.
  • the volatiles were removed by flowing dry N2 over the solution to produce a yellow-brown oil.
  • Approximately 10 mL of Et2O was added to the cleavage oil to create a suspended white precipitate.
  • the suspension was partitioned into five 2 ml microcentrifuge tubes and chilled over dry ice for 10 minutes. The tubes were centrifuged at 12,000 r.p.m. for 40 seconds to produce a white pellet. Et 2 O was carefully decanted, leaving the white crude peptide or peptide-thyclotide conjugate solid.
  • Solvent A was 0.05% TFA in water and Solvent B consisted of 90% acetonitrile in water.
  • Peptide and thyclotide-peptide conjugate HPLC isolates were characterized using electrospray ionization-mass spectrometry on a Waters/Micromass LCT Premier time-of-flight mass spectrometer. The instrument was operated in W-mode at a nominal resolution of 10,000.
  • the electrospray capillary voltage was 2 kV and the sample cone voltage was 60 V.
  • the desolvation temperature was 275 °C and the desolvation gas was N 2 with a flow rate of 300 L h ⁇ 1 . Accurate masses were obtained using the internal reference standard method.
  • EXAMPLE 2 This example illustrates the molecules prepared and tested and the results obtained in accordance with the present invention. Illustrative molecules are presented in Table 1. Molecules 1, 2, and 3 are designed to test for cell uptake. Each molecule had a fluorescent group (fluorescein (FI)) attached to the end. Molecules 4, 5, and 6 were used to test for biological activity. For the thyclotide and aegPNA portions of molecules 2, 3, 5, 6, and 7, thymine (T) nucleobase was used. Leydig 769584 HHS E-033-2023-0-PC-01 19 Table 1.
  • Obserd thyclotide conjugates 1 1 0 0 7 FI H] 2+ , [M+H+NH 4 ] 2+ or triply charged ion [M+3H] 3+ .
  • the Petros peptide was used as a negative control in molecules 1 and 4.
  • the aegPNA-peptide conjugates are also negative controls, shown as molecules 2 and 5.
  • the (S,S) THF-thyclotide with ten T residues is shown as molecule 7.
  • FIG.2A-2D depicts that the SETD8 inhibitor peptide conjugated to a T 10 - thyclotide showed a significantly better cell uptake than the inhibitor alone in SH-SY5Y neuroblastoma cells.
  • the SETD8 inhibitor peptide conjugated to a T10- thyclotide can enter the nuclei of SH-SY5Y neuroblastoma cells.3D volume reconstruction of the nuclei of SH-SY5Y cells treated with 5 ⁇ M of fluorescein-labelled SETD8 peptide inhibitor-thyclotide conjugate (molecule 3) for 16 hours at 37°C. Cells were fixed with 4% PFA then stained with 2 ⁇ g/mL Hoechst 33342 solution in PBS, and mounted on glass slides with Prolong glass mounting medium.
  • FIG.3A-3C display fluorescence microscopy data showing that the thyclotide-peptide conjugate molecule 3 was able to enter the nuclei of cells. This is important because the SETD8 enzyme is in the nucleus of cells, and therefore any molecule that will inhibit the SETD8 must be able to reach the nucleus.
  • the data in FIG.4 represent a series of Western blots that used an antibody to detect a methylated form of the protein p53 (p53K382Me).
  • the SETD8 enzyme will methylate the p53 protein at lysine 382. If the SETD8 enzyme is inhibited, then the amount of methylated p53 at lysine 382 will decrease. Treatment of the cells with molecule 6 resulted in a decrease in p53K382Me, while treatment with the peptide alone (molecule 4) showed no decrease in p53K382Me.
  • Leydig 769584 HHS E-033-2023-0-PC-01 21 [0092] FIG.5 showed the following: The SETD8 inhibitor peptide conjugated to a T 10 - thyclotide (molecule 3) can efficiently decrease SETD8-dependent p53 methylation on lysine 382.
  • SH-SY5Y neuroblastoma cells were either untreated or treated with 5 ⁇ M of the SETD8 peptide inhibitor only (molecule 4), or 5 ⁇ M of the SETD8 inhibitor peptide conjugated to a T 10 -thyclotide (molecule 6), for 24, 48, or 72 hours. There is no significant difference between untreated cells and cells treated with the peptide inhibitor (molecule 4) only. However, cells treated with the inhibitor conjugated to the thyclotide (molecule 6) showed significant decrease in p53 methylation in lysine 382, detected with a monoclonal antibody targeting the specific methylation on this amino acid residue.
  • the thyclotide conjugated to the SETD8 inhibitor (molecule 6) can deliver the peptide inside the nucleus of the cell to allow inhibition of SETD8, preventing the SETD8-dependent methylation of p53.
  • the data shown in FIG.5 reveal that the thyclotide-peptide conjugate molecule 6 was able to inhibit the SETD8 enzyme in cells while the peptide along (molecule 4) had no activity.
  • FIG.5 depicts the following.
  • the SETD8 inhibitor peptide conjugated to a T 10 - thyclotide prevents the proliferation of SH-SY5Y neuroblastoma cells.50,000 SH-SY5Y neuroblastoma cells were seeded in each wells of 24 well-plates and were either untreated or treated with 10 or 20 ⁇ M of the SETD8 inhibitor alone (molecule 4) or the inhibitor conjugated to the T10-thyclotide (molecule 6).3 days after the treatment, a CyQUANT proliferation assay was performed, showing that the conjugate (molecule 6) efficiently prevents the proliferation of the neuroblastoma cells, whereas the inhibitor (molecule 4) alone doesn’t show any significant difference compared with untreated cells.
  • FIG.29 depicts the FACS data for peptide 8. Peak 1 depicts the untreated cell count, peak 2 depicts the cell count when treated with the peptide alone, peak 3 correspond to the cell count when treated with peptide 8, and peak 4 corresponds to the cell count when treated with a THF polynucleotide (6).
  • FIG.30 depicts the Western blot data for peptide 8, showing that the peptide suppresses the activity of the target enzyme, Setd8, in SK-N-AS cells which are derived from neuroblastoma.
  • the target of methylation of Setd8 is p53, and the lowering of methylation in the + lane compared to the NT (not treated) lane shows that the peptide works for the intended purpose.

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Abstract

La présente divulgation concerne des conjugués de thyclotide-peptide de formule (I) : Tn-L-P (I), dans laquelle T est une unité de thyclotide de la formule, les unités de thyclotide étant éventuellement interceptées par des segments de polypeptide qui peuvent être identiques ou différents de P, n étant d'environ 5 à environ 25, L étant un lieur, P étant un polypeptide, et B étant une nucléobase ou une fraction hétérocyclyle. La présente divulgation concerne également des compositions pharmaceutiques et des méthodes de traitement de maladies telles que des cancers en administrant une quantité efficace d'un conjugué thyclotide-peptide.
PCT/US2023/082871 2022-12-13 2023-12-07 Conjugués peptidiques de thyclotide ayant une perméabilité cellulaire et activité inhibitrice WO2024129497A1 (fr)

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Citations (5)

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