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WO2018167775A1 - Co-médicaments de disaccharides et d'acides aminés à chaîne ramifiée et leurs utilisations - Google Patents

Co-médicaments de disaccharides et d'acides aminés à chaîne ramifiée et leurs utilisations Download PDF

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Publication number
WO2018167775A1
WO2018167775A1 PCT/IL2018/050283 IL2018050283W WO2018167775A1 WO 2018167775 A1 WO2018167775 A1 WO 2018167775A1 IL 2018050283 W IL2018050283 W IL 2018050283W WO 2018167775 A1 WO2018167775 A1 WO 2018167775A1
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Prior art keywords
disease
codrug
disorder
ester
autophagy
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PCT/IL2018/050283
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English (en)
Inventor
Dalia Megiddo
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Sanonash Pharma Ltd.
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Publication of WO2018167775A1 publication Critical patent/WO2018167775A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines

Definitions

  • the presently disclosed subject matter relates to codrugs of disaccharides and branched chain amino acids and uses thereof.
  • Trehalose a non-reducing disaccharide of glucose (a-G-glucopyranosyl a-D- glucopyranoside), is found in many plants, fungi, bacteria, insects and other invertebrates, where it serves as a natural sugar and energy source. Due to its unique physical and biochemical properties demonstrated in its ability to sustain and preserve a wide array of biological molecules, trehalose has found its use in several food and cosmetic products and most notably in as an excipient in pharmaceutical products. Trehalose is an approved ingredient in all major markets designated as Generally Regarded as Safe (GRAS) food ingredient by the Food and Drug Administration (FDA) [1].
  • GRAS Generally Regarded as Safe
  • FDA Food and Drug Administration
  • Proposed therapeutic uses of trehalose are, among others, treatment of myopathies, neurodegenerative disorders or tauopathies associated abnormal protein aggregation [2].
  • Trehalose is also known for its ability to induce cellular autophagy, in particular liver autophagy [3]. Recently it has been reported that trehalose triggers beneficial cellular autophagy by inhibiting glucose transport [4].
  • Amino acids are biologically important organic compounds containing amine and carboxyl functional groups, along with a side-chain specific to each amino acid. Because of their biological significance, amino acids are important in nutrition and are commonly used in nutritional supplements and food technology.
  • BCAA branched chain amino acids
  • BCCAs i.e., leucine, isoleucine and valine
  • Codrug or mutual prodrug is a drug design approach to chemically bind two or more drugs to improve therapeutic efficiency or decrease adverse effects.
  • the codrug can be cleaved in the body to generate the free parent active agents.
  • the codrug itself can be inactive, less active, or more active than the parent agents. It has been demonstrated that codrugs possess some benefits over conventional drugs, including enhanced solubility, increased permeation for passing across biomembranes, prolonged half-life for extending the therapeutically effective period, and reduced toxicity. Codrugs are predominantly used to treat some conditions such as neurodegenerative, cardiovascular, cancerous, infectious, and inflammatory disorders. Many codrugs have been developed to increase lipophilicity for better transport into/across biomembranes, especially the skin and cornea. A targeted delivery of codrugs to specific tissues or organs thus can be achieved to promote bioavailability [7, 8].
  • the targeting of the codrug to specific organs may also be achieved by specific selection of the linkers which connect the different drugs in the codrug entity [9, 10].
  • Various linkers for antibody-drug conjugates are known. Linker research is aiming to produce linkers that suit the particular antibody and drug being used, provide stability before entering the cell, and provide efficient payload release once inside the target cell [11].
  • Therapeutic benefits of codrugs may be improved by using nanocarriers.
  • the nanoparticles exhibit specific physicochemical properties for improving the delivery of drugs, including codrugs [7, 12, 13].
  • Sugars like trehalose contain abundant reactive -OH groups that can be utilized to attach bioactive molecules such as amino acids and/or peptides through a reaction such as esterification. Conjugation of compounds such as amino acids and/or peptides with a carbohydrate molecule may provide a convenient matrix for one or more of stabilization, targeting and controlled release of the parent compound (e.g., amino acid and/or peptide).
  • the present invention provides a codrug of Formula I:
  • D is a non-reducing disaccharide residue or a functional derivative (e.g., a derivative thereof possessing similar activities), a hydrate (e.g., dihydrate) or any pharmaceutically acceptable salt thereof (e.g., acid derived salts such as HC1);
  • a functional derivative e.g., a derivative thereof possessing similar activities
  • a hydrate e.g., dihydrate
  • any pharmaceutically acceptable salt thereof e.g., acid derived salts such as HC1;
  • AA is a BCAA residue selected from the group consisting of Leu, He and Val or any pharmaceutically acceptable salt thereof (e.g., acid derived salts such as HC1);
  • x is a cleavable bond (e.g., a covalent bond directly between a functional group of the D residue and the carboxyl group and/or the -N3 ⁇ 4 group of the AA residue, for example between a hydroxyl group of D and the carboxyl group of AA to form an ester bond) or a linker moiety, which in specific embodiments (e.g., in vivo) is degradable by one or more specific factors (e.g., specific substances such as enzymes and/or specific organ or cellular physico-chemical conditions such as pH ) at a target site, to thereby release both the D and AA active moieties (i.e., in their original form) at the target site; and
  • a linker moiety which in specific embodiments (e.g., in vivo) is degradable by one or more specific factors (e.g., specific substances such as enzymes and/or specific organ or cellular physico-chemical conditions such as pH ) at a
  • n is an integer selected from 1 to 8;
  • each of x and AA may be the same or different.
  • the present invention provides a conjugate (of the aforementioned codrug of Formula I and a targeting moiety) of Formula II:
  • D is a non-reducing disaccharide residue or a functional derivative (e.g., a derivative thereof possessing similar activities), a hydrate (e.g., dihydrate) or any pharmaceutically acceptable salt thereof (e.g., acid derived salts such as HC1);
  • a functional derivative e.g., a derivative thereof possessing similar activities
  • a hydrate e.g., dihydrate
  • any pharmaceutically acceptable salt thereof e.g., acid derived salts such as HC1;
  • AA is a BCAA residue selected from the group consisting of Leu, He and Val or any pharmaceutically acceptable salt thereof (e.g., acid derived salts such as HC1);
  • x is a cleavable bond (e.g., a covalent bond directly between a functional group of the D residue and the carboxyl group and/or the -NH2 group of the AA residue, for example between a hydroxyl group of D and the carboxyl group of AA to form and etser bond) or a linker moiety, linking D and AA, which cleavable direct bond or bonded linking moiety are in specific embodiments degradable (e.g., in vivo) by one or more specific factors (e.g., specific substances such as enzymes and/or specific organ or cellular physico-chemical conditions such as pH) at a target site, to thereby release both the D and AA active moieties (i.e., in their original form) at the target site; n is an integer selected from 1 to 8;
  • each of x and AA may be the same or different;
  • Z is a targeting moiety (e.g., an antibody or any pharmaceutically acceptable substance that specifically bind to an extracellular member on a target cell, e.g. a receptor,); and y is a bond or a spacer linking Z to [D-(x-AA) n ] at any suitable location thereof (as indicated by a dashed line in Formula II), wherein said bond or spacer is chemically degradable (e.g., acid labile) and/or enzymatically degradable (e.g., via endosomal and/or lysosomal activity receptor ) and degradation thereof releases the codrug moiety D-(x-AA) n at the target cell.
  • a targeting moiety e.g., an antibody or any pharmaceutically acceptable substance that specifically bind to an extracellular member on a target cell, e.g. a receptor
  • y is a bond or a spacer linking Z to [D-(x-AA) n ] at any suitable location thereof (a
  • the present invention provides codrugs and/or conjugates according to the invention for use in enhancing autophagy.
  • the present invention provides a pharmaceutical composition comprising at least one codrug of Formula I according to the invention and optionally comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a codrug conjugate of Formula II according to the invention and optionally comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides the codrugs and/or the targeted conjugates according to the present invention for use in the manufacture of a medicament.
  • the present invention provides the use of the codrugs and/or the targeted conjugates according to the present invention for the manufacture of a medicament.
  • the present invention provides the codrugs and/or the targeted conjugates according to the present invention for the treatment of one or more of the disorders/diseases detailed herein.
  • the present invention provides a method for enhancing autophagy in a human subject in need thereof, the method comprising administration to the subject of a composition comprising at least one said codrug as defined herein and/or said targeted conjugate thereof as defined herein, the composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a method for treating a human subject in need thereof, wherein said subject suffers from at least one disease or disorder in which autophagy has a role in the disease or disorder pathology or treatment, the method comprising administration to the subject of a composition comprising at least one said codrug as defined herein and/or said targeted conjugate thereof as defined herein, wherein the composition optionally further comprises at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a method of treating a human subject in need, wherein said human subject suffers from at least one disease or disorder in which autophagy has a role in the disease or disorder pathology or treatment and/or in the pathology or treatment of which autophagy has a role, the method comprising administration to the subject of a composition comprising at least one codrug as defined herein and/or a targeted conjugate thereof as define herein, said composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a method of treating a disease in a human subject in need thereof, wherein the disease exhibits at any stage thereof liver steatosis, inflammation or fibrosis, the method comprising administration to the subject of a composition comprising at least one codrug according to the present invention and/or a targeted conjugate thereof, the composition optionally may further comprise at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a method of treating a hepatic disease in a human subject in need thereof, wherein the hepatic disease exhibits at any stage thereof liver steatosis, inflammation or fibrosis, the method comprising administration to the subject of a composition comprising at least one codrug according to the invention and/or a targeted conjugate thereof, the composition optionally may further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a method of treating a subject in need thereof, by administration to the subject of a composition comprising at least one codrug according to the invention and/or a targeted conjugate thereof, the composition optionally may further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent, wherein said subject suffers from a disease or disorder related to impaired or insufficient autophagy, which may be any one of liver (hepatic) disease, cancer, neurodegenerative disease, neurodevelopmental disease, myopathic muscular dystrophy, infectious condition, metabolic disorder, severe neuronal and muscular degenerative disease, protein aggregation disease, lysosomal storage disorder (LSD) or genetic disease associated with deficient or mutated autophagy related genes or RNA.
  • a disease or disorder related to impaired or insufficient autophagy which may be any one of liver (hepatic) disease, cancer, neurodegenerative disease, neurodevelopmental disease, myopathic muscular dystrophy, infectious condition, metabolic disorder, severe neuronal and muscular degenerative disease, protein aggregati
  • the present invention provides a process for the preparation of the codrugs according to the present invention, the process comprises providing a disaccharide, reacting the disaccharide with protected BCAA under conditions allowing the coupling of the disaccharide with the protected BCAA to thereby provide a mixture of disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA, utilizing chromatographic means to separate between said conjugates (i.e., disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA) and subjecting the separated conjugates to conditions allowing the removal of the protecting group/s.
  • the present invention provides a process for the preparation of the codrugs according to the present invention, the process comprises providing a disaccharide, reacting the disaccharide with protected BCAA under conditions allowing the coupling of the disaccharide with the protected BCAA to thereby provide a mixture of disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA, subjecting the conjugates (i.e., the disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA) to conditions allowing the removal of the protecting group/s and utilizing chromatographic means to separate between the resulted co-drugs.
  • the conjugates i.e., the disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA
  • the present invention provides a process for the preparation of the codrugs according to the present invention, the process comprises providing a disaccharide, protecting the disaccharide with a protecting group under conditions allowing the reaction of all hydroxyl groups in the disaccharide with the protecting group, subjecting the resulted protected disaccharide to basic conditions allowing selective de- protection of the primary hydroxyls of the disaccharide, reacting the resulted disaccharide (i.e., a disaccharide with the secondary hydroxyl groups thereof protected) with protected BCAA under conditions allowing the coupling of the primary hydroxyl group of said disaccharide with the protected BCAA, subjecting the resulted conjugate (of the protected disaccharide and the protected BCAA) to conditions allowing the removal of the protecting group/s of the saccharide and the BCAA (simultaneously or separately) and utilizing one or more means (e.g., chromatographic means, extraction means and the like) to purify the resulted
  • one or more means e
  • the present invention provides a process for the preparation of the codrugs according to the present invention, the process comprises providing a disaccharide, protecting the disaccharide with a protecting group under conditions allowing the reaction of one of the primary hydroxyl groups in the disaccharide with the protecting group, reacting the resulted disaccharide (i.e., a disaccharide with the one primary hydroxyl group thereof protected) with protected BCAA under conditions allowing the coupling of the other (non- protected) primary hydroxyl group of the disaccharide with the protected BCAA, subjecting the resulted conjugate (i.e., disaccharide in which one primary hydroxyl is protected with a protecting group and the other primary saccharide is couples to a protected BCAA) to acidic conditions allowing the removal of the protecting group/s of both the saccharide and the BCAA and utilizing chromatographic means to purify the resulted codrug.
  • a disaccharide protecting the disaccharide with a protecting group under conditions allowing the reaction of one
  • FIG. 1 demonstrates a schematic illustration of the preparation of two exemplary codrugs according to some embodiments of the present invention.
  • FIG. 2 demonstrates a schematic illustration of a synthetic method of an exemplary di- leucine trehalose ester codrug according to some embodiments of the present invention.
  • FIG. 3 demonstrates a schematic illustration of a synthetic method of an exemplary mono-leucine trehalose ester codrug according to some embodiments of the present invention.
  • FIG. 4 demonstrates the H-NMR spectrum of an exemplary di-leucine trehalose ester codrug according to some embodiments of the present invention.
  • FIG. 5 demonstrates the H-NMR spectrum of an exemplary mono-leucine trehalose ester codrug according to some embodiments of the present invention.
  • the present disclosure makes use of the chemically available hydroxylic groups of non- reducing disaccharides such as, but not limited to trehalose, for linking with either the amine or the carboxyl functional groups of one or more of the branched-chain amino acids leucine (Leu), isoleucine (He) and valine (Val), at times via a linker moiety, to produce conjugates of disaccharides and branched chain amino acids, which may be used as codrugs for specific therapeutic uses.
  • non- reducing disaccharides such as, but not limited to trehalose
  • the chemical bond which connects the two drugs, i.e., the disaccharide and the BCAA, either directly or via a linker moiety may be chemically cleavable under specific organ or cellular conditions (e.g., the bond may be acid labile and susceptible to pH conditions such as low pH) and/or cleavable by enzymes, e.g., proteases, esterases (such as for example hydrolases, phosphatases, etc.) specific to the target cell/organ, or different substances in the target.
  • the bond may undergo degradation at a specific target cell or organ, to thereby release the parent drugs, i.e., the disaccharide and BCAA, at a desired site.
  • the codrugs of disaccharide and at least one BCAA, as disclosed herein, may be further conjugated to a targeting moiety (e.g., a macromolecule), for example an antibody, via a bond or a spacer.
  • a targeting moiety e.g., a macromolecule
  • the linkage between the codrug and the targeting moiety may be at any suitable location on the codrug (i.e., at any location on the disaccharide and/or on the BCAA).
  • Such a conjugation may further assist in specific targeting of the codrug to the target cell/organ.
  • codrug may be referred to herein as “targeted codrug” or “targeted codrug conjugate” or “codrug conjugate” .
  • the present invention provides a codrug of Formula I:
  • D is a non-reducing disaccharide residue or a functional derivative (e.g., a derivative thereof possessing similar activities), a hydrate (e.g., dihydrate) or any pharmaceutically acceptable salt thereof (e.g., acid derived salts such as HC1);
  • a functional derivative e.g., a derivative thereof possessing similar activities
  • a hydrate e.g., dihydrate
  • any pharmaceutically acceptable salt thereof e.g., acid derived salts such as HC1;
  • AA is a BCAA residue selected from the group consisting of Leu, He and Val or any pharmaceutically acceptable salt thereof (e.g., acid derived salts such as HC1);
  • x is a cleavable bond (e.g., a covalent bond directly between a functional group of the D residue and the carboxyl group and/or the -NH2 group of the AA residue, for example between a hydroxyl group of D and the carboxyl group of AA to form an ester bond) or a linker moiety, which in specific embodiments (e.g., in vivo) is degradable by one or more specific factors (e.g., specific substances such as enzymes and/or specific organ or cellular physico-chemical conditions such as pH ) at a target site, to thereby release both the D and AA active moieties (i.e., in their original form) at the target site; and
  • a linker moiety which in specific embodiments (e.g., in vivo) is degradable by one or more specific factors (e.g., specific substances such as enzymes and/or specific organ or cellular physico-chemical conditions such as pH ) at a target site
  • n is an integer selected from 1 to 8;
  • each of x and AA may be the same or different.
  • the present invention provides a conjugate (of the aforementioned codrug of Formula I and a targeting moiety) of Formula II:
  • D is a non-reducing disaccharide residue or a functional derivative (e.g., a derivative thereof possessing similar activities), a hydrate (e.g., dihydrate) or any pharmaceutically acceptable salt thereof (e.g., acid derived salts such as HC1);
  • AA is a BCAA residue selected from the group consisting of Leu, He and Val or any pharmaceutically acceptable salt thereof (e.g., acid derived salts such as HC1);
  • x is a cleavable bond (e.g., a covalent bond directly between a functional group of the D residue and the carboxyl group and/or the -NH2 group of the AA residue, for example between a hydroxyl group of D and the carboxyl group of AA to form and etser bond) or a linker moiety, linking D and AA, which cleavable direct bond or bonded linking moiety are in specific embodiments degradable (e.g., in vivo) by one or more specific factors (e.g., specific substances such as enzymes and/or specific organ or cellular physico-chemical conditions such as pH) at a target site, to thereby release both the D and AA active moieties (i.e., in their original form) at the target site; n is an integer selected from 1 to 8;
  • each of x and AA may be the same or different;
  • Z is a targeting moiety (e.g., an antibody or any pharmaceutically acceptable substance that specifically bind to an extracellular member on a target cell, e.g. a receptor,); and y is a bond or a spacer linking Z to [D-(x-AA) n ] at any suitable location thereof (as indicated by a dashed line in Formula II), wherein said bond or spacer is chemically degradable (e.g., acid labile) and/or enzymatically degradable (e.g., via endosomal and/or lysosomal activity receptor ) and degradation thereof releases the codrug moiety D-(x-AA) n at the target cell.
  • a targeting moiety e.g., an antibody or any pharmaceutically acceptable substance that specifically bind to an extracellular member on a target cell, e.g. a receptor
  • y is a bond or a spacer linking Z to [D-(x-AA) n ] at any suitable location thereof (a
  • each of the non-reducing disaccharide and the BCAA may be further independently substituted.
  • substituted BCAA are BCAA groups substituted by protecting groups, said protecting groups form protective derivatives of BCAA such as but not limited to (tert-butyloxycarbonyl) BOC substituted BCAA e.g., BOC-Leu, carboxybenzyl (Cbz) substituted BCAA e.g., Cbz-Leu etc.
  • Other protecting groups known to those of skill in the art are within the scope of the present disclosure.
  • Non limiting examples of substituted non-reducing disaccharides are non-reducing disaccharides substituted by one or more protecting groups of trimethyl silyl ether (TMS), one or more protecting trityl groups etc.
  • TMS trimethyl silyl ether
  • Other protecting groups known to those of skill in the art are within the scope of the present disclosure.
  • reduct when applied to non-reducing disaccharide and/or BCAA as used herein means a part of the disaccharide and/or BCAA that is substantially identical to the disaccharide and/or BCAA, respectively, from which it is derived, with minor differences arising by virtue of having one or more atoms removed to provide points of attachment for the linker "x" (and when applicable also for the spacer "y").
  • At least one functional group of the residue will be altered (relative to the parent active agent i.e., disaccharide and/or BCAA) to covalently bind directly to the other active member when "x" is a covalent bond, or to the linker "x" when "x” is a linking group (and when applicable to covalently bind to the spacer "y”).
  • This will typically involve removal of an exchangeable hydrogen and/or a functional group such as but not limited to -OH, leaving a free valence for attachment of either the second active residue or the linker "x" (and at times the spacer "y”).
  • the residue of the BCAA formed by removal of a hydroxyl group from the carboxylate may form an ester bond with a hydroxyl group on the disaccharide residue, which itself is formed by removal of a hydrogen atom from a hydroxyl group from the disaccharide.
  • the term "codrug” is envisaged as an entity in which two or more different drug molecules, the "parent active drugs", specifically a non-reducing disaccharide and a branched-chain amino acid, are chemically linked (directly or via a linker moiety).
  • the codrug comprises more than two drug molecules, such as for example one molecule of a disaccharide and two or more branched-chain amino acids, at least one of them is different from the others, i.e., where more than one amino acid is present, the amino acids may be independently identical or different.
  • the codrug is cleaved in the body (i.e., in vivo) to generate the parent active drugs.
  • the constituent drugs may be indicated for the same disease, but may exert different therapeutic effects via disparate mechanisms of action.
  • the codrug itself may be pharmacologically inactive, but releases the constituent drugs upon breakage (e.g., biochemical breakage) of the chemical linkage, preferably at the target tissue (where their therapeutic effects are needed).
  • the chemical linkage e.g., a covalent bond
  • the chemical link may be biodegradable, for example by hydrolysis, by an enzymatic or non-enzymatic mechanism (e.g., at specific conditions such as pH, e.g. where the link is acid labile).
  • the chemical link is such that it is specifically degraded by condition prevailing at or enzymes present at a target site of the codrug.
  • the disaccharides according to the present invention are non-reducing disaccharides.
  • non-reducing disaccharide or any lingual variations thereof refers to a disaccharide with two monosaccharide units bond through an acetal linkage between their anomeric centers and neither monosaccharide moiety has a free hemiacetal.
  • Pharmaceutically acceptable functional derivatives of the non-reducing disaccharides are also contemplated within the scope of the present disclosure.
  • the non-reducing disaccharide may be selected from the group consisting of trehalose ⁇ (1 ⁇ 1) ⁇ , ⁇ , ⁇ -trehalose, ⁇ , ⁇ - trehalose, cellobiose (the hydrolysis product of the polysaccharide cellulose), isomaltulose, lactulose, melibiose, sucrose, lactose, maltose (the hydrolysis product of the polysaccharide starch), chitobiose (the hydrolysis product of the polysaccharide chitin), kojibiose, nigerose, isomaltose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiulose, rutinose, rutinulose and xylobiose.
  • cellobiose the hydrolysis product of the polysaccharide cellulose
  • the disaccharide is trehalose ⁇ (1 ⁇ 1) ⁇ (referred to hereinafter as “trehalose”), having the following chemical structure:
  • Trehalose has eight available sites for conjugation with BCAA.
  • Table 1 below provides non limiting exemplary list of possible conjugates of trehalose and BCAAs according to the invention.
  • Table 1 also identifies the integer "n" of the entity [D-(x-AA) n ] according to the present invention, which designates the number of BCAA residues linked to the disaccharide residue, which, when n>l, can be identical or different.
  • "n" can be determined in accordance with maximum tolerated dose of each of the disaccharide and the BCAA, as detailed herein.
  • the long bonds in the depicted structures indicate that the bond between the heterocyclic ring and the bonded group X-AA may be via any one of the hydroxyl positions in the heterocyclic ring i.e., any one of positions 2, 3 and 4 and their corresponding positions 2', 3' and 4'.
  • the number of BCAA residues linked to the disaccharide, specifically to trehalose, as well as the specific position on the disaccharide molecule, specifically trehalose, to which they are linked may be designed in consideration of steric hindrance, mainly in consideration of the size of the BCAA and the length of the linker "x".
  • the structure of the disaccharide and the space around its core may also affect the degree of substitution.
  • Amino acids bear both amine and carbonyl functional groups which may be used to conjugate the amino acids.
  • the BCAA of the codrugs according to the invention may be connected to the disaccharide (and/or to the targeting moiety, when applicable), directly or via a linker moiety, either via the amine functional group or via the carboxyl functional group.
  • the modified groups may also be used for binding (directly or via a spacer) to a targeting moiety.
  • BCAAs employed in the codrugs according to the present disclosure may be in their Inform, D-form, racemic or any other mixture thereof.
  • Table 2 depicts the chemical structure of exemplary three BCAA with the specific functional groups.
  • BCAA is L-leucine
  • BCAA is L-Isoleucine.
  • BCAA is L- Valine.
  • x is a cleavable bond or linker which may be chemically labile, for example acid labile, and/or enzyme cleavable.
  • x may be attached to any suitable heteroatom present in the non- reducing disaccharide and BCAA that carries an exchangeable hydrogen (such as -OH, NH2, and COOH groups).
  • an exchangeable hydrogen such as -OH, NH2, and COOH groups.
  • the carboxylic acid group of the BCAA is esterified with one of the free hydroxyl group of the disaccharide residue.
  • x is a covalent bond between a functional group of D and the carboxyl group of AA.
  • x is a covalent bond between a functional group of D and the - NH2 group of AA.
  • x is an ester bond
  • x is an ester linker
  • Table 3 presents exemplary bonds/linkers cleavable by various conditions/enzymes at specific organs/cells [14].
  • the targeted conjugates of the codrugs according to the present invention include a member designated "y".
  • "y” is a chemical bond or a spacer linking the member Z to the D-(x-AA) n entity (the codrug) of the codrug targeted conjugates of the present disclosure, at any suitable location thereof, "y” is a degradable member, capable of being degraded (either chemically or enzymatically for example via endosomal and/or lysosomal activity receptor), thereby release the entity D-(x-AA) n at a target cell.
  • y may be attached to any suitable heteroatom present in the non- reducing disaccharide and BCAA that carries an exchangeable hydrogen (such as-OH, NH 2 , and COOH groups).
  • n is 1. In some embodiments “n” is 2. In some embodiments “n” is 3. In some embodiments “n” is 4. In some embodiments “n” is 5. In some embodiments “n” is 6. In some embodiments “n” is 7. In some embodiments “n” is 8.
  • n is 1 or 2.
  • D is ⁇ , ⁇ -trehalose
  • AA is Leucine
  • n is 1 or 2.
  • Exemplary specific codrugs in accordance with the present disclosure may be trehalose to which one or two of leucine molecules are attached by an ester bond at various positions thereof.
  • codrug in accordance with the present disclosure is of the following formula (designated herein as T-Leu):
  • an ester bond is formed directly between the trehalose and the leucine (i.e., the "x" group in the codrug according to the invention is an ester bond).
  • T-Leu codrug may be conjugated (to a targeting moiety as herein described) at any further position to form targeted conjugate/s according to the present disclosure.
  • the codrug in accordance with the present disclosure is of the following formula (designated herein as T-Le3 ⁇ 4):
  • T-Leu2 codrug may be conjugated (to a targeting moiety as herein described) at any further position to form targeted conjugate/s according to the present disclosure.
  • the codrugs, and particularly the codrug targeted conjugates according to the present invention are nanoparticulate, with e.g., nano-scale size such as 50 nm, 100 nm, 150 nm, 200 nm.
  • the codrugs and/or targeted conjugates thereof according to the present disclosure may provide the benefit of enhancing autophagy via this two pronged approach.
  • the present invention provides codrugs and/or conjugates according to the invention for use in enhancing autophagy.
  • the present invention provides a pharmaceutical composition comprising at least one codrug of Formula I according to the invention and optionally comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a codrug conjugate of Formula II according to the invention and optionally comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a method for enhancing autophagy in a subject, specifically a human subject in need thereof, the method comprising administration to the subject of a composition comprising a codrug and/or targeted conjugate thereof according to the present invention.
  • the subject in need of treatment may be a subject, specifically a human subject, suffering from a disease in which autophagy has a role in the disease pathology or treatment.
  • subject and patient may be used herein interchangingly.
  • autophagy has a role in the disease or disorder pathology or treatment. In some embodiments autophagy has a role in the pathology or treatment of the disease or disorder. In some embodiments the subject suffers from at least one disease or disorder in which autophagy has a role, or wherein autophagy has a role in the pathology or treatment of said disease or disorder.
  • compositions according to the present disclosure may optionally further comprise at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides the codrugs and/or the targeted conjugates according to the present invention for use in the manufacture of a medicament (e.g., for enhancing autophagy).
  • autophagy encompasses one or more of macroautophagy, microautophagy and chaperon-mediated autophagy.
  • the term "enhance autophagy" or any lingual variations thereof relates to one or more of inducing autophagy, stimulating autophagy or increasing autophagy.
  • autophagy malfunction may be related to a vast number of disorders.
  • the ability of the codrugs and/or targeted conjugates of the present invention to enhance autophagy provides them with therapeutic value.
  • the codrugs and/or the conjugates of the present invention may be used for the treatment of one or more disorders which may be related to malfunction and/or insufficiency of and/or impaired autophagy mechanisms and processes in a subject in need, specifically a human subject.
  • the disorders may be related to one or more of liver, muscle (including cardiac), kidney and brain and neural diseases, disorders or malfunction.
  • Non-limiting examples of such impaired or insufficient autophagy associated disorders/diseases are: liver (hepatic) diseases, cancer, neurodegenerative diseases, neurodevelopmental diseases, myopathic muscular dystrophies, infectious conditions, metabolic disorders, severe neuronal and muscular degenerative diseases, protein aggregation diseases, lysosomal storage disorder (LSD), and genetic diseases associated with deficient or mutated autophagy related genes or RNA, etc.
  • Hepatic diseases as referred to in the present disclosure include diseases that exhibit at any stage thereof liver steatosis, inflammation or fibrosis.
  • Non-limiting examples of hepatic diseases are Alpha 1 antitrypsin deficiency, fatty liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, liver cancer, hepatic encephalopathy and others.
  • these hepatic diseases may be accompanied by at least one of metabolic symptoms such as obesity, diabetes mellitus, hyperlipidemia, hypercholesterolemia hypertriglyceridemia, and other symptoms of the metabolic syndrome.
  • the present invention provides a method of treating a human subject in need, wherein said human subject suffers from at least one disease or disorder in which autophagy has a role in the disease or disorder pathology or treatment and/or in the pathology or treatment of which autophagy has a role, the method comprising administration to the subject of a composition comprising at least one codrug as defined herein and/or a targeted conjugate thereof as define herein, said composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the at least one disease or disorder may be associated with malfunction and/or insufficiency of and/or impaired autophagy mechanisms and processes in the subject.
  • the at least one disease or disorder may be related to one or more of liver, muscle (including cardiac), kidney and brain and neural disease, disorders or malfunction.
  • the impaired or insufficient autophagy associated disease or disorder may be any one of liver (hepatic) disease, cancer, neurodegenerative disease, neurodevelopmental disease, myopathic muscular dystrophy, infectious condition, metabolic disorder, severe neuronal and muscular degenerative disease, protein aggregation disease, lysosomal storage disorder (LSD) or genetic disease associated with deficient or mutated autophagy related genes or RNA.
  • codrugs and/or the codrug conjugates of the present invention may be used for the treatment of any disease that exhibit at any stage thereof liver steatosis, inflammation or fibrosis.
  • the present invention provides a method of treating a disease in a human subject in need thereof, wherein said disease exhibits at any stage thereof liver steatosis, inflammation or fibrosis, the method comprising administration to the subject of a composition comprising at least one codrug as defined herein and/or a targeted conjugate thereof as define herein, said composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a method of treating a hepatic disease in a human subject in need thereof, wherein said hepatic disease exhibits at any stage thereof liver steatosis, inflammation or fibrosis, the method comprising administration to the subject of a composition comprising at least one codrug as defined herein and/or a targeted conjugate thereof as define herein, said composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the hepatic disease may be any one of Alpha 1 antitrypsin deficiency, fatty liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, liver cancer and hepatic encephalopathy.
  • the hepatic disease may be accompanied by at least one of metabolic symptoms such as obesity, diabetes mellitus, hyperlipidemia, hypercholesterolemia hypertriglyceridemia or other symptoms of the metabolic syndrome.
  • Non- limiting examples of protein aggregation diseases as disclosed herein are: poly- alanine aggregation disorder, poly-glutamine aggregation disorder and a tauopathy, for example any one of oculopharyngeal muscular dystrophy (OPMD), spinocerebellar ataxias (SCA), Friedreich's ataxia, spinal and bulbar muscular atrophy (SBMA), Huntington's disease, Parkinson's disease, Alzheimer's disease and Amyotrophic Lateral Sclerosis (ALS), Dentatorubral-Pallidoluysian Atrophy (DRPLA), Pick's disease, Corticobasal degeneration (CBD), Progressive Supranuclear Palsy (PSP) and Frontotemporal Dementia and Parkinsonism linked to chromosome 17 (FTDP-17).
  • OPMD oculopharyngeal muscular dystrophy
  • SCA spinocerebellar ataxias
  • Friedreich's ataxia spinal and bulbar muscular atrophy
  • poly-glutamine As used herein the terms “poly-glutamine” , “poly -alanine aggregation disorder” or “protein codon reiteration disorder” as herein defined, refer to disorders associated with formation of intracellular polyglutamine or polyalanine aggregates, preferably referring to Oculopharyngeal Muscular Dystrophy (OPMD), Huntington's disease (HD), Spinal and Bulbar Muscular Atrophy (SBMA), Dentatorubral-Pallidoluysian Atrophy (DRPLA) and Spino-Cerebellar' Ataxia (SCA).
  • Oculopharyngeal Muscular Dystrophy OPMD
  • HD Huntington's disease
  • SBMA Spinal and Bulbar Muscular Atrophy
  • DPLA Dentatorubral-Pallidoluysian Atrophy
  • SCA Spino-Cerebellar' Ataxia
  • myopathy refers to inherited or acquired or hereditary degenerative disease involving atrophy of the muscle fibers, such as HIBM or ALS; or to muscle dystrophy.
  • neurodegenerative disorder or any lingual variation thereof refers to hereditary or sporadic conditions characterized by progressive nervous system dysfunction.
  • Non limiting examples of such disorders are Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Landouzy-Dejerine Dystrophy (LDMD), Huntington's disease, Creutzfeldt-Jakob disease, Spino-Cerebellar Ataxia, Friedrich's Ataxia and others.
  • tauopathy refers to neurodegenerative diseases associated with tau-pathology, prototypic intracellular aggregation of tau microfilaments, in the context of present disclosure particularly referring to Pick's disease, CorticoBasal Degeneration (CBD), Progressive Supranuclear Palsy (PSP) and Frontotemporal Dementia and Parkinsonism linked to chromosome 17 (FTDP-17).
  • CBD CorticoBasal Degeneration
  • PSP Progressive Supranuclear Palsy
  • FTDP-17 Frontotemporal Dementia and Parkinsonism linked to chromosome 17
  • Tauopathies are known as diseases caused by mutations leading to misfolding of Tau microtubule-associated protein that binds and stabilizes microtubules in neuronal cells.
  • Tau pathology is a prominent feature of the sporadic Alzheimer's disease (AD), but is also seen in a variety of other related neurodegenerative diseases, such as Pick's disease, Corticobasal degeneration (CBD), Progressive supranuclear palsy (PSP) and Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).
  • AD sporadic Alzheimer's disease
  • CBD Corticobasal degeneration
  • PSP Progressive supranuclear palsy
  • FTDP-17 Frontotemporal dementia and parkinsonism linked to chromosome 17
  • Tau mutation and by analogy tau dysfunction in inherited and in sporadic diseases may be pathogenic through mechanisms involving both loss of function (decreased microtubules stabilization) and toxic gain of function (increased fibril formation).
  • Non- limiting examples of lysosomal storage disorders are: activator deficiency/GM2 gangliosidosis, alpha-mannosidosis, aspartylglucosaminuria, cholesteryl ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Anderson- Fabry disease, Farber disease, fucosidosis, sialidosis, galactosialidosis, Kanzaki, Gaucher disease (including Type I, Type II, and Type III), GMl gangliosidosis (including infantile, late infantile/juvenile, adult/chronic), I-cell disease/mucolipidosis II, infantile free sialic acid storage disease/ISSD, juvenile hexosaminidase A deficiency, Krabbe disease (including infantile onset, late onset), metachromatic leukodystrophy, pseudo- Hurler polydystrohpy/mucolipidosis IIIA,
  • Non limiting examples of muscle diseases are: Autophagic Vacuolar Myopathies and X- linked myopathy with excessive autophagy; Douchenne Muscular Dystrophy (DMD), Friedrich's ataxias; Acid Maltase Deficiency (AMD); Amyotrophic Lateral Sclerosis (ALS); Andersen-Tawil Syndrome; Becker Muscular Dystrophy (BMD); Becker Myotonia Congenita; Bethlem Myopathy; Bulbospinal Muscular Atrophy (Spinal-Bulbar Muscular Atrophy); Carnitine Deficiency; Carnitine Palmityl Transferase Deficiency (CPT Deficiency); Central Core Disease (CCD); Centronuclear Myopathy; Charcot- Marie-Tooth Disease (CMT); Congenital Muscular Dystrophy (CMD); Congenital Myasthenic Syndromes (CMS); Congenital Myotonic Dystrophy; Cori Disease (Debrancher Enzyme Deficiency); Debrancher Enzyme
  • the muscular degenerative disease may be any one of Autophagic Vacuolar Myopathies and X-linked myopathy with excessive autophagy; Friedrich's ataxias; Amyotrophic Lateral Sclerosis (ALS); Congenital Muscular Dystrophy (CMD); Congenital Myasthenic Syndromes (CMS); Congenital Myotonic Dystrophy; Duchenne Muscular Dystrophy (DMD); or Lou Gehrig's Disease (Amyotrophic Lateral Sclerosis).
  • ALS Amyotrophic Lateral Sclerosis
  • CMD Congenital Muscular Dystrophy
  • CMS Congenital Myasthenic Syndromes
  • DMD Duchenne Muscular Dystrophy
  • Lou Gehrig's Disease Amyotrophic Lateral Sclerosis
  • subject in need thereof refers to a subject suffering from a disease as herein defined.
  • the subject may be a human subject.
  • the subject in need of treatment with a codrugs or codrug targeted conjugate in accordance with the present disclosure is a subject suffering from NAFLD at any stage thereof, including NAFL, NASH, liver fibrosis and liver cirrhosis and other hepatic disorders/diseases, as well as other diseases exhibiting at any stage thereof hepatic steatotis, hepatic inflammation or hepatic fibrosis.
  • NAFLD any stage thereof
  • NASH hepatic steatotis
  • hepatic inflammation or hepatic fibrosis a non-limiting example of such disease is cystic fibrosis.
  • the subject to be treated suffers from any of the above- mentioned disorders.
  • the present invention provides a method of treating a disease in a human subject in need thereof, wherein the disease exhibits at any stage thereof liver steatosis, inflammation or fibrosis, the method comprising administration to the subject of a composition comprising at least one codrug according to the present invention and/or a targeted conjugate thereof, the composition optionally may further comprise at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the present invention provides a method of treating a hepatic disease in a human subject in need thereof, wherein the hepatic disease exhibits at any stage thereof liver steatosis, inflammation or fibrosis, the method comprising administration to the subject of a composition comprising at least one codrug according to the invention and/or a targeted conjugate thereof, the composition optionally may further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.
  • the hepatic disease is any one of Alpha 1 antitrypsin deficiency, fatty liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, liver cancer and hepatic encephalopathy.
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • liver fibrosis liver fibrosis
  • cirrhosis liver cancer and hepatic encephalopathy.
  • the hepatic disease is accompanied by at least one of metabolic symptoms such as obesity, diabetes mellitus, hyperlipidemia, hypercholesterolemia hypertriglyceridemia or other symptoms of the metabolic syndrome.
  • treatment is to be taken to mean achieving a therapeutic effect, ameliorating, relieving or reducing the severity and/or frequency of at least one sign or symptom associated with diseases as herein defined, elimination of signs or symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause (e.g., prophylactic therapy), improvement or remediation of damage and eliminating or reducing the severity of the disease to be treated.
  • treatment at least reduce fat accumulation in the liver, specifically in hepatocytes.
  • the therapeutically amounts of a codrug in accordance with the present disclosure are based on the respective effective amounts of their parent active non-reducing disaccharide and BCAA, for example trehalose and leucine, in consideration of the maximum tolerated dose of each of these parent drugs constituting the codrug.
  • the amount of leucine for administration is from about 25 mg and up to 500 mg/Kg body weight/day, and the amount of trehalose from about 250 mg and up to 1000 mg/Kg body weight/day.
  • the structure of the codrug can be designed in accordance with recommended amounts of each parent active member, in consideration of its molecular weight.
  • the molecular weight of each of leucine and isoleucine is 131.18 gr/mole and that of valine is 131.16;
  • the molecular weight of ⁇ , ⁇ trehalose is 342.296 gr/mole (anhydrous) and 378.33 gr/mole (dihydrate).
  • the molar ratio of non-reducing disaccharide (e.g. trehalose) to BCAA (e.g. leucine) in the codrug can be from 1 : 1 to 1 :8. Specific ratios are 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1:7 or 1 :8.
  • a codrug of trehalose and leucine may comprise from 1 to 8 leucine residues for each molecule of trehalose. Specific examples are one or two molecules of leaucine to one molecule of trehalose.
  • compositions in accordance with the present disclosure may comprise more than one disaccharide-BCAA codrug as disclosed herein.
  • disaccharide-BCAA codrug for example, but not limited to trehalose:leucine
  • formulations of several codrugs may allow, for example, for flexibility in dosaging, considering recommended doses vis-a-vis molar ratios between the two parent active members of the codrug.
  • Optimal dosing schedules may be calculated from measurements of drug accumulation in the body of the patient. Optimal doss can be determined by dosing methodologies and repetition rates. In general, dosage is calculated according to body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every few years. Persons of ordinary skill in the art can readily estimate repetition rates for dosing based on measured residence times and concentrations of the combined composition of the invention in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the combined composition of the invention is administered in maintenance doses, once or more daily.
  • Formulations comprising at least one codrug according to the invention may be designed for administration by any suitable route, for example, but not limited to oral administration, parenteral administration, intrarectal administration, intranasal administration, ocular administration or topical administration.
  • parenteral administration as herein defined refers to a route of administration where the desired effect is systemic and the active agent (for example a trehalose codrug, such as a trehalose-leucine codrug), administered via a route other than the digestive tract, for example by intravenous, intramuscular, intraperitoneal or subcutaneous administration.
  • Administration can be based on a daily regime as a single dose or multiple doses by a single parenteral administration or as multiple doses by multiple parenteral administrations, respectively.
  • administration can be periodic, for example every other day, three times weekly, twice weekly, once weekly, or once monthly, and frequency of administration can be varied according to the patient condition.
  • the pharmaceutical formulation as herein defined comprises the non-reducing disaccharide-BCAA codrug as sole active ingredient, and optionally further comprises at least one pharmaceutically acceptable additive, carrier, excipient or diluent.
  • non-reducing disaccharide-BCAA codrugs are ⁇ , ⁇ - trehalose-BCAA codrugs.
  • formulations in accordance with the present disclosure may be designed for any one of immediate, controlled, sustained, delayed or extended release of the codrug/s or codrug targeted conjugate/s comprised therein.
  • non-reducing disaccharide-BCAA codrugs according to the present disclosure may be used in combination with other therapies or treatments for treating or alleviating any of the diseases described above.
  • the formulations described herein comprise at least one non-reducing disaccharide- BCAA codrug, for example a trehalose-BCAA codrug as active ingredient, and may optionally further comprise an additional active ingredient such as an anti-inflammatory agent, and at least one pharmaceutically acceptable additive, carrier, excipient or diluents as well known in the art.
  • these formulations may further comprise a trehalase (a glycoside hydrolase enzyme catalyzing the conversion of trehalose to glucose found in the intestine, kidney and liver) inhibitor, i.e., competitive or other inhibitor of the trehalase enzyme.
  • a variety of liquid carriers may be used, in particular water or saline.
  • Aqueous solutions may include salts, buffers, and the like.
  • the carrier can be solvent or dispersion medium suitable for parenterally-administrable compositions containing, for example, water, saline, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Water is an essential additive (or carrier).
  • the term "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. It is contemplated that the active agent can be delivered by any acceptable parenteral route and in any pharmaceutically acceptable dosage form.
  • formulations in suitable oil such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts.
  • aqueous formulations may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • these aqueous formulations are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.
  • the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
  • the present invention provides a process for the preparation of the codrugs according to the present invention, the process comprises providing a disaccharide (e.g., trehalose), reacting said disaccharide with protected BCAA (e.g., Cbz-Leu) under conditions allowing the coupling of said disaccharide with the protected BCAA to thereby provide a mixture of disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA, utilizing chromatographic means to separate between said conjugates (i.e., disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA) and subjecting the separated conjugates to conditions allowing the removal of the protecting group/s (de -protection).
  • a disaccharide e.g., trehalose
  • protected BCAA e.g., Cbz-Leu
  • the present invention provides a process for the preparation of the codrugs according to the present invention, the process comprises providing a disaccharide (e.g., trehalose), reacting said disaccharide with protected BCAA (e.g., Cbz-Leu) under conditions allowing the coupling of said disaccharide with the protected BCAA to thereby provide a mixture of disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA, subjecting said conjugates (i.e., the disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA) to conditions allowing the removal of the protecting group/s (de -protection) and utilizing chromatographic means to separate between the resulted co-drugs.
  • a disaccharide e.g., trehalose
  • protected BCAA e.g., Cbz-Leu
  • said conjugates i.e., the disaccharides conjugated via an ester bond to one or more molecules of the protected BCAA
  • the present invention provides a process for the preparation of the codrugs according to the present invention, the process comprises providing a disaccharide (e.g., trehalose), protecting the disaccharide with a protecting group (e.g., trimethyl silyl ether) under conditions allowing the reaction of all hydroxyl groups in said disaccharide with the protecting group, subjecting the resulted protected disaccharide to basic conditions allowing selective de -protection of the primary hydroxyls of the disaccharide, reacting the resulted disaccharide (i.e., a disaccharide with the secondary hydroxyl groups thereof protected) with protected BCAA (e.g., BoC-Leu) under conditions allowing the coupling of the primary hydroxyl group of said disaccharide with the protected BCAA, subjecting the resulted conjugate (of the protected disaccharide and the protected BCAA) to conditions allowing the removal of the protecting group/s (de- protection) of the saccharide and the process comprises providing a disacchari
  • the present invention provides a process for the preparation of the codrugs according to the present invention, the process comprises providing a disaccharide (e.g., trehalose), protecting the disaccharide with a protecting group (e.g., trityl group) under conditions allowing the reaction of one of the primary hydroxyl groups in said disaccharide with the protecting group, reacting the resulted disaccharide (i.e., a disaccharide with the one primary hydroxyl group thereof protected) with protected BCAA (e.g., BoC-Leu) under conditions allowing the coupling of the other (nonprotected) primary hydroxyl group of the disaccharide with the protected BCAA, subjecting the resulted conjugate (disaccharide in which one primary hydroxyl is protected with a protecting group and the other primary saccharide is couples to a protected BCAA) to acidic conditions allowing the removal of the protecting group/s (de- protection) of both the saccharide and the BCAA
  • Example 1 preparation of trehalose mono-leucine ester and trehalose di-leucine ester codrugs utilizing unprotected disaccharide residue:
  • T-Leu trehalose monoleucine ester
  • T-Leu2 trehalose di-leucine ester
  • FIG. 1 A schematic illustration of the preparation of two above two exemplary codrugs according to the present invention, T-Leu and T-Leu2, is presented in Fig. 1.
  • codrugs are produced by utilizing carboxybenzyl (Cbz) protected amino acid without the necessity of protecting the hydroxyl groups of the disaccharide residue.
  • Cbz protected leucine is reacted with trehalose under specific coupling conditions (e.g., EDC-HOBT coupling reaction known in the art) which provide a mixture of conjugates of trehalose conjugated via an ester bond to one molecule of Cbz protected leucine (designated in Fig. 1 as Trehalose-Cbz Leu) and trehalose conjugated to two molecules of Cbz protected leucine [designated in Fig. 1 as Trehalose-(Cbz Leu)2].
  • the conjugates may be separated (e.g., by chromatographic means) followed by removal of the Cbz protecting group/s (de -protection) under conditions known in the art.
  • the Cbz protecting groups are first removed (de -protected) and then the resulting conjugates, i.e., T-Leu and T-Le3 ⁇ 4 are separated. It is noted that by utilizing the Cbz chemistry only the hydroxyl groups on positions 6 and 6' of the trehalose molecule (i.e., -CH2-OH) interact with the protected amino acid to form an ester bond with the carboxylic group of the amino acid.
  • All prepared codrugs according to the present disclosure are characterized e.g., by MP, NMR, Mass, DLS, GPC, FT-IR etc.
  • Example 2 preparation of trehalose mono-leucine ester and trehalose di-leucine ester codrugs utilizing protected disaccharide residue:
  • codrugs of the present invention may also be produced by utilizing protected amino acid in combination with one or more protected hydroxyl groups of the disaccharide residue, as will be demonstrated herein below.
  • MSD Liquid Chromatograph/Mass Selective Detector
  • ⁇ -NMR spectra were recorded on a Bruker 400MHz instrument at 25 °C in CD3OD.
  • the compounds were named using the software ChemDraw Professional 16.0.
  • the commercial names or trivial names were used for the commercial starting materials and reagents.
  • a hydrochloric salt of di-leucine trehalose ester is illustrated in Fig. 2.
  • the preparation started by protection of trehalose with trimethyl silyl ether (TMS) to produce compound 1' following selective de -protection of primary hydroxyls in basic conditions to produce compound 2'.
  • TMS trimethyl silyl ether
  • the appropriate protected trehalose (compound 2') reacted with activated BocLeuOH to produce compound 3' followed by removal of the Boc group to produce the hydrochloric salt of the di-leucine ester of trehalose (compound 4').
  • the trimethyl silyl ether protecting groups were used on six hydroxyl groups of trehalose.
  • Preparation of di-leucine ester of trehalose was investigated and the best conditions which gave 90% conversion were obtained with using of 0.2 eq. of trimethylsilyl chloride (TMSCl) and 6 eq. of hexamethyldisilazane (HMDS) in DMF, followed by evaporation to dryness. Then crude mixture was dissolved in 50ml DCM and 10ml water, was extracted with two portions of DCM, washed with brine and dried over MgSO/t. MS (ESI+) m/z 792 [M+H 2 0] + .
  • the mono-leucine ester of trehalose was prepared using selective protecting group strategy.
  • the preparation of a hydrochloric salt of mono-leucine trehalose ester is illustrated in Fig. 3.
  • trehalose reacted with trityl chloride to produce mono trityl trehalose (compound 5').
  • esterification of compound 5' was performed with activated BocLeuOH and lead to compound 6' which was purified utilizing column chromatography.
  • de-protection of trityl and tert-butyloxycarbonyl of compound 6' was completed in acidic conditions to produce the hydrochloric salt of the mono- leucine trehalose ester (compound 7').
  • Formulations comprising the above exemplary trehalose monoleucine ester and/or trehalose di-leucine ester are prepared e.g., by dissolving the ester in a suitable aqueous medium, for example saline.
  • the plasma and/or muscle and/or liver and/or brain and/or kidney concentrations of trehalose, BCAA e.g., leucine, the codrugs of same and glucose (which is the metabolite of trehalose) are measured in male Sprague-Dawley (SD) rats and are determined after intravenous bolus (IV) and oral gavage (PO) administration.
  • BCAA e.g., leucine
  • the codrugs of same and glucose which is the metabolite of trehalose
  • AAALAC International and NIH guidelines as reported in the "Guide for the Care and Use of Laboratory Animals," National Research Council ILAR, Revised 1996.
  • the study includes SD rats (male, 250 to 350 grams in weight, the Shanghai SLAC Laboratory Animal Co. Ltd.). Animals are administered with the codrugs (and/or conjugates) of the invention intravenously or orally.
  • Plasma samples are collected after each dose administration and processed for plasma. Samples e.g., muscle samples (hind leg muscle) are collected and homogenized. The concentrations of trehalose, leucine, the codrugs of same and glucose in plasma and in samples, e.g., muscle homogenate samples, are analyzed by qualified bioanalytical LC/MS/MS methods.
  • Example 5 Activity
  • This model is used for assessing the effect of the codrug on acute hepatic inflammatory response induced by Con A.
  • mice are divided into 4 groups, each of 6 animals, 1 group for treatment with Compound A (trehalose-leucine monoester), 1 group for treatment with Compound B (trehalose-leucine diester) and 2 non-treated control groups (one for each treated group).
  • Compound A trehalose-leucine monoester
  • Compound B trehalose-leucine diester
  • 2 non-treated control groups one for each treated group.
  • mice are sacrificed on the next day and measured for serum ALS and AST, Interferon gamma (ELISA). In addition liver biopsy is taken for pathology.
  • ELISA Interferon gamma
  • High fed diet model creates a fatty liver disease in mice including steatosis and inflammation. The effect of the drug on these and other specific parameters is assessed in this model.
  • C57BL mice in 4 groups of 6-10 are fed a high fat diet ad libitum (about 2.5g/mouse/day). Fat content of the diet at least 40%.
  • mice Blood samples of the mice are obtained biweekly and are analyzed for serum ALT/ AST, cholesterol, triglycerides and fasting glucose.
  • treatment is started either orally or IP (up to lg/kg/day - 2 days/3 days etc. It is noted that on the first 10 weeks mice are only on high fed diet.
  • One group is treated with Compound A (trehalose-leucine monoester), 1 group is treated with Compound B (trehalose-leucine diester) and 2 control groups (one for each treated group) are non-treated.
  • mice are sacrificed on week 16 and measured for serum ALT/AST, cholesterol, glucose, insulin, and in addition, liver fat fraction is assessed by MRI, and level of triglycerides in the liver is measured.
  • the levels of inflammatory cells such as CD4, CD8 as well as a battery of inflammatory cytokines including interleukins, interferons, TNF-ot, etc. are also measured.
  • a liver biopsy is taken.
  • the effect of the codrug on hepatic fibrosis is assessed in fibrosis model, that imitates the fibrotic element of liver diseases.
  • Hepatic fibrosis is induced by intraperitoneal injection of carbon tetrachloride (5 ⁇ litexlg body weight) for 7 weeks. Therapy is started in different groups at different times, starting from week 12 (3 weeks before the end of the trial).
  • One group is treated with Compound A (trehalose-leucine monoester), 1 group is treated with Compound B (trehalose-leucine diester) and 2 groups serve as non-treated controls.
  • mice are sacrificed and serum is measured for ALT/AST and bilirubin. Liver is assessed histologically for injury and for relative fibrosis quantification. In addition inflammatory level is assessed through levels of inflammatory cytokines and T cells.

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Abstract

La présente invention concerne des co-médicaments de disaccharides et d'acides aminés à chaîne ramifiée, leurs conjugués et leurs utilisations thérapeutiques.
PCT/IL2018/050283 2017-03-12 2018-03-12 Co-médicaments de disaccharides et d'acides aminés à chaîne ramifiée et leurs utilisations WO2018167775A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
RU2810089C2 (ru) * 2021-12-24 2023-12-21 федеральное государственное автономное образовательное учреждение высшего образования "Санкт-Петербургский политехнический университет Петра Великого" (ФГАОУ ВО "СПбПУ") Способ использования бинарной системы, состоящей из агентов различной природы, для разработки нового класса антибактериальных препаратов

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2810089C2 (ru) * 2021-12-24 2023-12-21 федеральное государственное автономное образовательное учреждение высшего образования "Санкт-Петербургский политехнический университет Петра Великого" (ФГАОУ ВО "СПбПУ") Способ использования бинарной системы, состоящей из агентов различной природы, для разработки нового класса антибактериальных препаратов

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