WO2018167775A1

WO2018167775A1 - Codrugs of disaccharides and branched-chain amino acids and uses thereof

Info

Publication number: WO2018167775A1
Application number: PCT/IL2018/050283
Authority: WO
Inventors: Dalia Megiddo
Original assignee: Sanonash Pharma Ltd.
Priority date: 2017-03-12
Filing date: 2018-03-12
Publication date: 2018-09-20

Abstract

The present invention relates to codrugs of disaccharides and branched chain amino acids, to conjugates thereof and therapeutic uses thereof.

Description

CODRUGS OF DISACCHARIDES AND BRANCHED-CHAIN AMINO ACIDS

AND USES THEREOF

TECHNOLOGICAL FIELD

The presently disclosed subject matter relates to codrugs of disaccharides and branched chain amino acids and uses thereof.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

[1] WHO Food Additives

(http://www.inchem.ors/documents/iecfa/iecmono/v46je05.htm).

[2] WO 2014/181333.

[3] Jaime L. Schneider and Ana Maria Cuervo, 2014, Liver Autophagy: much more than just taking out the trash, Nat Rev Gastroenterol Hepatol 11(3), 187-200.

[4] Brian J. DeBosch et al., 2016, Trehalose inhibits solute carrier 2 A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis, Science Signaling, Vol 9, Issue 416, pp. ra21 DOI: 10.1126/scisignal.aac5472.

[5] Kazuto Tajiri and Yukihiro Shimizu, 2013, Branched-chain amino acids in liver diseases, World J Gastroenterol, 19(43), 7620-7629.

[6] Daniela Cota et al., 2006, Hypothalamic mTOR Signaling Regulates Food Intake, SCIENCE, vol. 312, 927-930.

[7] Ibrahim A. Aljuffali et al., 2016, The codrug approach for facilitating drug delivery and bioactivity, Expert Opinion on Drug Delivery, vol. 13, No. 9, 1311-1325.

[8] N. Das et al., 2010, Codrug: An efficient approach for drug optimization, European Journal of Pharmaceutical Sciences, 41, 571-588.

[9] Samuele Cazzamalli et al., 2017, Linker stability influences the anti-tumor activity of acetazolamide-drug conjugates for the therapy of renal cell carcinoma. J Control Release, 28; 246: 39-45.

[10] Srinivas R. Chirapu et al., 2014, Undesired vs. Designed Enzymatic Cleavage of Linkers for Liver Targeting, Bioorg Med Chem Lett. 15, 24(4), 1144-1147. [11] http://adcreview.com/news/linkers-for-antibody-drug-conjugates-current-role- and- advancements/.

[12] Ying Wang et al., 2009, Heparin-Paclitaxel Conjugates as Drug Delivery

System: Synthesis, Self-Assembly Property, Drug Release, and Antitumor Activity, Bioconjugate Chem. 20, 2214-2221.

[13] Aikaterini Lalatsa et al., 2012, A Prodrug Nanoparticle Approach for the Oral Delivery of a Hydrophilic Peptide, Leucine5-enkephalin, to the Brain, Mol. Pharmaceutics, 9, 1665-1680.

[14] Prodrug Metabolism (2013)

https://courses.washington.edu/medch570/NEW/pdf/570_5Prodrug_kk2016.pdf.

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

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].

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.

Various therapeutic uses of amino acids are known. In particular, branched chain amino acids (hereafter also "BCAA" or "BCCAs", i.e., leucine, isoleucine and valine) have been shown to affect gene expression, protein metabolism, apoptosis and regeneration of hepatocytes, and insulin resistance. They have also been shown to inhibit the proliferation of liver cancer cells in vitro and are essential for lymphocyte proliferation and dendritic cell maturation [5, 6].

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].

SUMMARY OF THE INVENTION

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).

Thus, in one of its aspects the present invention provides a codrug of Formula I:

D-(x-AA)„

Formula I

wherein,

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 -N¾ 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

n is an integer selected from 1 to 8;

wherein where n>l each of x and AA may be the same or different. In another of its aspects the present invention provides a conjugate (of the aforementioned codrug of Formula I and a targeting moiety) of Formula II:

Formula II

wherein,

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;

wherein where n>l, 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.

In a further one of its aspects, the present invention provides codrugs and/or conjugates according to the invention for use in enhancing autophagy. In yet another one of its aspects, 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.

Yet, in a further one of its aspects the present invention provides 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.

In yet another one of its aspects, the present invention provides the codrugs and/or the targeted conjugates according to the present invention for use in the manufacture of a medicament.

Yet, in a further one of its aspects, 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.

In yet a further one of its aspects, 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.

In a further one of its aspects, 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.

In yet another one of its aspects, 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.

In a further one of its aspects, 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.

In another one of its aspects 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.

In yet another one of its aspects 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.

In yet another one of its aspects, 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.

Yet, in a further one of its aspects, 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.

In yet another one of its aspects, 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.

In a further one of its aspects, 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 codrug.

Yet, in a further one of its aspects, 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.

BRIEF DESCRIPTION OF THE FIGURES

In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

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.

DETAILED DESCRIPTION OF THE 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. 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. Thus, 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. 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. Such codrug may be referred to herein as "targeted codrug" or "targeted codrug conjugate" or "codrug conjugate" .

Accordingly, in one of its aspects the present invention provides a codrug of Formula I:

D-(x-AA)„

Formula I

wherein,

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

n is an integer selected from 1 to 8;

wherein where n>l each of x and AA may be the same or different.

In another of its aspects the present invention provides a conjugate (of the aforementioned codrug of Formula I and a targeting moiety) of Formula II:

[D-(x-AA)J-y-Z

Formula II

wherein, 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);

wherein where n>l, each of x and AA may be the same or different;

It is noted that the functional derivative and/or hydrate and/or salt of the non-reducing disaccharide residue as disclosed herein are pharmaceutically acceptable.

It is noted that in the codrugs and/or the codrugs conjugates according to the present invention each of the non-reducing disaccharide and the BCAA may be further independently substituted. For example, the disaccharide may be further substituted on one or more of its free hydroxyl groups and any one of the BCAA may be further substituted on the free -NH2 or -C(=0)-OH groups thereof. Non limiting examples of 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. Other protecting groups known to those of skill in the art are within the scope of the present disclosure.

The term "residue" 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"). Typically, 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"). For instance, where the drug substance includes a carboxylate functional group, e.g., the BCAA, 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.

As used herein 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). Where 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). Accordingly, the chemical linkage (e.g., a covalent bond) 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). In specific embodiments, the chemical link is such that it is specifically degraded by condition prevailing at or enzymes present at a target site of the codrug.

Some advantages of using codrugs in the treatment of various indications have been detailed in publications [7] to [13], the contents of which are incorporated herein by reference.

The disaccharides according to the present invention are non-reducing disaccharides. As used herein the term "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.

In some embodiments according to the present invention the non-reducing disaccharide may be selected from the group consisting of trehalose α(1→1)α, β,β-trehalose, α,β- 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.

In a particular embodiment the disaccharide is trehalose α(1→1)α (referred to hereinafter as "trehalose"), having the following chemical structure:

Trehalose

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. In specific embodiments, "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'.

It is to be noted that 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.

It is noted that the hydroxyl groups of the disaccharide and the -Nth group and/or the - C(=0)-OH group of the BCAA may be chemically modified and the modified form may be used to bind (directly or via a linker) the disaccharide the BCAA. 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.

Table 2: chemical structure of BCAA

In some embodiments according to the invention BCAA is L-leucine.

In some embodiments according to the invention BCAA is L-Isoleucine.

In some embodiments according to the invention BCAA is L- Valine.

In some embodiments according to the invention "x" is a cleavable bond or linker which may be chemically labile, for example acid labile, and/or enzyme cleavable. Some such linkers are described in [7] and [8], the contents of which are incorporated herein by reference.

In some embodiments "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). By way of example, the carboxylic acid group of the BCAA is esterified with one of the free hydroxyl group of the disaccharide residue.

In some embodiments "x" is a covalent bond between a functional group of D and the carboxyl group of AA.

In some embodiments "x" is a covalent bond between a functional group of D and the - NH2 group of AA. Non-limiting examples of "x" cleavable bonds and/or linkers are -CH2O-, -OCH2O-, - C=0, -C (=S), -C(=0)-(CH₂)-, -C(=0)-NH-, H2PO3, peptide bond, ester (-C(=0)-0-), acyl ester, e.g. gamma-amino butyric ester, carbonate ester, acyloxylalkyl ester, benzyl ester, double ester, O-alkyl carboxylic ester, orthoesters, thioester, phosphate ester, glycine acyloxylalkyl, -NH-, carbamate (-NH- COO-), thiocarbamate, -0-C(=0)-0-, - C(NH2)-COO-, azo (N=N), amide, di-amide, thioamide, thiocarbonate, C4-C5 alkyl chain, glutathione, lipoic acid, succinic acid, nitric oxide (NO), hydrogen sulfide, polyethylene glycol (PEG), disulfide, methylene, acyloxy methylene, glutaric acid, glycolic acid, linear bis-hydroxyl, dicarboxylate, succinate diester, dithiol, cyclic ketal, xanthate, methyl amino carboxymethyl and glycolic acid (-OCH2COO-).

In some embodiments "x" is an ester bond.

In some embodiment "x" is an ester linker.

Table 3 presents exemplary bonds/linkers cleavable by various conditions/enzymes at specific organs/cells [14].

Bond/Linker Cleavable by/at

Ester Liver esterase

Plasma esterase

Liver carboxylesterase

Skin

Carbamate Liver carboxylesterase

Skin

-C(NH₂)-COO- Decarboxylase - blood brain barrier

Azo Gastro intestinal

Nitric oxide Gastro intestinal/Cardio

Hydrogen sulfide Gastro intestinal/Cardio

Amide Gastro intestinal/Skin

PEG Skin 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.

In some embodiments "y" 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).

Various non-limiting examples of "y" bonds and spacers are presented, for example in [9- 12], which are incorporated herein by reference, as follows: thioether spacer N- succinimidyl-4-(N-maleimidomethyl) cylcohexane-l-carboxylate (SMCC); disulfide; acid cleavable/labile [e.g., 4-(4'-acetylphenoxy) butanoic acid, hydrazones and silyl ethers] ; reducible spacer (e.g., glutathione); enzyme cleavable spacers (such as peptide based spacers e.g., valine-citrulline (Val-Cit) dipeptide spacers and phenylalanine-lysine (Phe-Lys) dipeptide spacers), valine-alanine dipeptide spacers and valine-arginine or valine-alanine dipeptide spacers; single amino acid spacers (e.g., valine, leucine, phenylalanine), beta-glucuronide spacer (cleavable by β-glucuromidase); primary and secondary amines; phenols; sulfhydryls; quaternary ammonium spacers (e.g. N-terminal tertiary amine); cathepsin B; cathepsin C, cathepsin K, or 2'-substituted 5-aminovaleric acid carbamate.

In some embodiments "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.

In some embodiments "n" is 1 or 2.

In some embodiments in the codrug/s and/or conjugates according to the invention D is α,α-trehalose, AA is Leucine and 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.

In one embodiment the codrug in accordance with the present disclosure is of the following formula (designated herein as T-Leu):

T-Leu

In the T-Leu codrug 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).

It is noted that the 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.

In one embodiment the codrug in accordance with the present disclosure is of the following formula (designated herein as T-Le¾):

T-Lem

It is noted that the 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.

In some embodiments 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.

Autophagy is the process by which intracellular proteins and organelles are degraded in lysosomes. Early studies in hepatocytes uncovered how nutritional status regulates autophagy and how various circulating hormones modulate the activity of this catabolic process in the liver. The publications by Jaime L. Schneider and Ana Maria Cuervo [3] and Brian J. DeBosch et al. [4], which are incorporated herein by reference, illustrate that trehalose and leucine play a role in autophagy enhancement. Trehalose is known as enhancing autophagy in an mTOR-independent pathway. Leucine is known as enhancing autophagy through mTOR-dependent mechanism. Thus, without being bound by any theory, it can be suggested that by utilizing both a non-reducing disaccharide, for example but not limited to trehalose and BCAA, the codrugs and/or targeted conjugates thereof according to the present disclosure may provide the benefit of enhancing autophagy via this two pronged approach.

Thus, in a further one of its aspects, the present invention provides codrugs and/or conjugates according to the invention for use in enhancing autophagy. In yet another one of its aspects, 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.

In yet another of its aspects, 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. At least in this aspect of the present disclosure, 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. The terms "subject" and "patient" may be used herein interchangingly.

In some embodiments 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.

The compositions according to the present disclosure may optionally further comprise at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.

In yet another one of its aspects, 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). As used herein, the term "autophagy" encompasses one or more of macroautophagy, microautophagy and chaperon-mediated autophagy.

As used herein the term "enhance autophagy " or any lingual variations thereof relates to one or more of inducing autophagy, stimulating autophagy or increasing autophagy.

It is noted that autophagy malfunction may be related to a vast number of disorders. Thus, the ability of the codrugs and/or targeted conjugates of the present invention to enhance autophagy provides them with therapeutic value.

Accordingly, 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. At times, 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. Thus, in a further one of its aspects 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.

In some embodiments according to the present invention 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.

In some embodiments according to the present invention 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.

In some embodiments according to the present invention 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.

In addition, the 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.

In a further one of its aspects 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.

In another one of its aspects 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.

In some embodiments of the present invention 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.

In some embodiments of the present invention 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).

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).

The term "myopathy" as herein defined refers to inherited or acquired or hereditary degenerative disease involving atrophy of the muscle fibers, such as HIBM or ALS; or to muscle dystrophy.

As used herein the term "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.

The term "tauopathy" as herein defined 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).

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). More than 30 different inherited mutations or nucleotide substitutions in the FTDP-17 gene on chromosome 17q21 have been related to neurodegenerative disease manifesting a prototypic intracellular aggregation of tau microfilaments. 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, MPS I Hurler syndrome, MPS I Scheie syndrome, MPS I Hurler-Scheie syndrome, MPS II Hunter syndrome, Sanfilippo syndrome type Maroteau-Lamy (VI), AMPS IIIA, Sanfilippo syndrome type B/MPS IIIB, Morquio type AMPS IVA, Morquio Type B/MPS IVB, MPS IX hyaluronidase deficiency, Niemann-Pick disease (including Type A, Type B, and Type C), neuronal ceroidlipofuscinoses (including CLN6 disease, atypical late infantile, late onset variant, early juvenile Baten-Spielmeyer-Vogt/juvenile NCL/CLN3 disease, Finnish variant late infantile CLN5, Jansky-Bielschowsky disease/late infantile CLN2/TPP1 disease, Kufs/adult-onset NCL/CLN4 disease, northern epilepsy/variant late infantile CLN8, and Santavuori-Haltia/infantile CLNl/PPT disease), beta-mannosidosis, Pompe disease/glycogen storage disease type II, pycnodysostosis, Sandhoff disease/adult onset/GM2 gangliosidosis, Sandhoff disease/GM2 gangliosidosis infantile, Sandhoff disease/GM2 gangliosidosis juvenile, Schindler disease, Salla disease/sialic acid storage disease, Tay-Sachs/GM2 gangliosidosis, Wolman disease, Hermansky-Pudlak disease, Chediak-Higashi, Cistinosis, Salla, Methylmalonic aciduria, Sphingolipid activator protein deficiencies, RNASET2, SCARB2/LJMP-2 deficiency, or Multiple Sulfatase Deficiency.

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 Deficiency; Dejerine-Sottas Disease (DSD); Dermatomyositis (DM); Distal Muscular Dystrophy (DD); Dystrophia Myotonica (Myotonic Muscular Dystrophy); Emery-Dreifuss Muscular Dystrophy (EDMD); Endocrine Myopathies; Eulenberg Disease (Paramyotonia Congenita); Facioscapulohumeral Muscular Dystrophy (FSH or FSHD); Finnish (Tibial) Distal Myopathy; Forbes Disease (Debrancher Enzyme Deficiency); Friedreich's Ataxia (FA); Fukuyama Congenital Muscular Dystrophy; Glycogenosis Type 10; Glycogenosis Type 11; Glycogenosis Type 2; Glycogenosis Type 3; Glycogenosis Type 5; Glycogenosis Type 7; Glycogenosis Type 9; Gowers-Laing Distal Myopathy; Hauptmann-Thanheuser MD (Emery-Dreifuss Muscular Dystrophy); Hereditary Inclusion-Body Myositis; Hereditary Motor and Sensory Neuropathy (Charcot-Marie-Tooth Disease); Hyperthyroid Myopathy; Hypothyroid Myopathy; Inclusion-Body Myositis (IBM); Inherited Myopathies; Integrin-Deficient Congenital Muscular Dystrophy; Kennedy Disease (Spinal-Bulbar Muscular Atrophy); Kugelberg-Welander Disease (Spinal Muscular Atrophy); Lactate Dehydrogenase Deficiency; Lambert-Eaton Myasthenic Syndrome (LEMS); Limb-Girdle Muscular Dystrophy (LGMD); Lou Gehrig's Disease (Amyotrophic Lateral Sclerosis); McArdle Disease (Phosphorylase Deficiency); Merosin-Deficient Congenital Muscular Dystrophy; Metabolic Diseases of Muscle; Mitochondrial Myopathy; Miyoshi Distal Myopathy; Motor Neurone Disease; Muscle- Eye-Brain Disease; Myasthenia Gravis (MG); Myoadenylate Deaminase Deficiency; Myofibrillar Myopathy; Myophosphorylase Deficiency; Myotonia Congenita (MC); Myotonic Muscular Dystrophy (MMD); Myotubular Myopathy (MTM or MM); Nemaline Myopathy; Nonaka Distal Myopathy; Oculopharyngeal Muscular Dystrophy (OPMD); Paramyotonia Congenita; Pearson Syndrome; Periodic Paralysis; Peroneal Muscular Atrophy (Charcot-Marie-Tooth Disease); Phosphofructokinase Deficiency; Phosphoglycerate Kinase Deficiency; Phosphoglycerate Mutase Deficiency; Phosphorylase Deficiency; Phosphorylase Deficiency; Polymyositis (PM); Pompe Disease (Acid Maltase Deficiency); Progressive External Ophthalmoplegia (PEO); Rod Body Disease (Nemaline Myopathy); Spinal Muscular Atrophy (SMA); Spinal-Bulbar Muscular Atrophy (SBMA); Steinert Disease (Myotonic Muscular Dystrophy); Tarui Disease (Phosphofructokinase Deficiency); Thomsen Disease (Myotonia Congenita); Ullrich Congenital Muscular Dystrophy; Walker- Warburg Syndrome (Congenital Muscular Dystrophy); Welander Distal Myopathy; Werdnig-Hoffmann Disease (Spinal Muscular Atrophy); ZASP-Related Myopathy.

In some embodiments 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).

The term "subject in need thereof refers to a subject suffering from a disease as herein defined. In all aspects and embodiments of the present disclosure the subject may be a human subject.

In a specific embodiment 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. A non-limiting example of such disease is cystic fibrosis.

In other specific embodiments, the subject to be treated suffers from any of the above- mentioned disorders.

In some embodiments 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.

In some embodiments 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.

The term "treatment" as used herein 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. Where NAFL is the disease, 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. According to approved safety values, 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. For example, 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 α,α 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. Thus, for example, 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.

Pharmaceutical formulations in accordance with the present disclosure may comprise more than one disaccharide-BCAA codrug as disclosed herein. Depending on the molar ratio of disaccharide:BCAA, 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. The term 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. Alternatively or additionally, 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.

Thus, in some embodiments 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. Specific examples of non-reducing disaccharide-BCAA codrugs are α,α- trehalose-BCAA codrugs.

The 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.

The non-reducing disaccharide-BCAA codrugs according to the present disclosure, for example but not limited to trehalose-BCAA codrugs or pharmaceutical formulations comprising same, 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. Where the codrug is a trehalose-BCAA codrug, 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.

For formulations as herein defined administered as aqueous or other solvent-based dosage forms (e.g., for parenteral administration), 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).

Thus as used herein 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.

For purposes of parenteral administration, 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. Such 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. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

Methods of preparing various pharmaceutical formulations with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art.

In yet another one of its aspects, 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).

Yet, in another one of its aspects, 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. In a further one of its aspects, 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 BCAA (simultaneously or separately) and utilizing one or more means (e.g., chromatographic means, extraction means and the like) to purify the resulted codrug.

In a further one of its aspects, 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 and utilizing chromatographic means to purify the resulted codrug. It is noted that in some embodiments the chromatographic means may be used prior to the de -protection. DETAILED DESCRIPTION OF EMBODIMENTS

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.

Standard molecular biology protocols known in the art not specifically described herein are generally followed essentially as in Sambrook & Russell, 2001.

Standard medicinal chemistry methods known in the art not specifically described herein are generally followed essentially in the series "Comprehensive Medicinal Chemistry" by various authors and editors, published by Pergamon Press.

Thus, the following examples are not in any way intended to limit the scope of the invention as claimed.

Example 1: preparation of trehalose mono-leucine ester and trehalose di-leucine ester codrugs utilizing unprotected disaccharide residue:

The following exemplary co-drugs are prepared:

1. α,α-Trehalose esterified with one residue of leucine ("a trehalose monoleucine ester", referred to as "T-Leu") 2. α,α-Trehalose esterified with two residue of leucine, each on one of the two ot- glucose rings ("a trehalose di-leucine ester", referred to as "T-Leu2")

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.

In general the 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. Alternatively, the Cbz protecting groups are first removed (de -protected) and then the resulting conjugates, i.e., T-Leu and T-Le¾ 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:

It is noted that the 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. 2.1 General methods

Analytical HPLC-MS was performed using an Agilent 1260 series Liquid Chromatograph/Mass Selective Detector (MSD) (Single Quadropole) equipped with an electrospray interface and a UV diode array detector. Analysis were performed by two methods using either an XBridge CI 8 (3.0 x 50 mm) column with a gradient of acetonitrile in ammonium bicarbonate/Ntb (pH=10) over 5 minutes and a flow rate of 1 mL/min, or with a gradient of acetonitrile in 0.1 % aqueous formic acid over 5 minutes and a flow rate of 1 mL/min.

Ή-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. In addition, the commercial names or trivial names were used for the commercial starting materials and reagents.

2.2 Preparation of the trehalose Di-Leucine ester codrug: 1,1 '- ((((2R,2'R,3S,3'S,4S,4'S,5R,5'R)-oxybis(3,4,5-trihydroxytetrahydro-2H- pyran-6,2- diyl) )bis(methylene ) )bis( oxy ) )bis( 4-methyl-l -oxopentan-2-aminium ) dichloride

(compound 4')

The preparation of a hydrochloric salt of di-leucine trehalose ester is illustrated in Fig. 2. In general, 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'. 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').

Provided herein below are further details on the preparation process of the trehalose Di- Leucine ester compound 4' : 2.2.1 Preparation of (3R,3 %4R,4'R,5R,5'R,6R,6'R)-2,2 '-oxybis(4,5-bis((trimethylsilyl)- oxy)-6-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3-ol) (compound 1 '):

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+H₂0]⁺.

2.2.2 Preparation of (3R,3'R,4R,4'R,5R,5'R,6R,6'R)-2,2 '-oxybis(6-(hydroxymethyl)- 4,5- bis((trimethylsilyl)oxy)tetrahydro-2H-pyran-3-ol) (compound 2 '):

The crude mixture of compound 1' above was dissolved in 6 ml of DCM and 20ml

MeOH and 0.12eq of KCO3 was added at 0°C and stirred 15 min at room temperature (r.t). Then glacial acetic acid was added to mixture until pH=7. The residue was filtered, filtrate was diluted and washed with water, brine, MgSO t. Colorless oil in 90% yield. MS (ESI-) m/z 629 [M-H].

2.2.3 Preparation of ((2R,2'R,3S,3'S,4S,4'S,5R,5'R)-oxybis(3,4,5-trihydroxytetrahydro- 2H-pyran-6,2-diyl))bis(methylene)bis(2-((tert-butoxycarbonyl)amino)-4- methylpentanoate) (compound 3')

In a 50ml flask 2-(tert-butoxycarbonylamino)-4-methyl-pentanoic acid (1.25g, 5.41mmol) was dissolved in pyridine (30mL). HATU (2.15g, 5.66mmol) and DIPEA (0.77mL, 5.66mmol) were added and stirred lOmin. Compound 2' (1.55g, 2.46mmol) was dissolved in 5ml pyridine and added to reaction mixture and stirred at r.t. overnight. The solvent was removed, the crude mixture was dissolved in ethyl acetate 50ml and washed with NaCCb aq., extracted from water phase 2*30 ml., then organic layers washed with water, brine and MgSO t. The crude mixture was purified on reverse phase column chromatography in 4% yield. MS (ESI-) m/z 767 [M-H] . 2.2.4 Preparation of l, -((((2R,2'R,3S,3'S,4S,4'S,5R,5'R)-oxybis(3,4,5-tnhydroxy- tetrahydro-2H-pyran-6,2-diyl) )bis( methylene))bis( oxy ) )bis(4-methyl-l-oxopentan-2- aminium) dichloride (compound 4'):

In a 5ml vial compound 3' (80mg, O. lOOOmmol) was dissolved in ethyl acetate (2mL). Then HCl dry 1M in ethyl acetate 1ml were added and stirred over 4 hours (TLC ethyl acetate methanol 10:1 starting material disappeared). The white solid was centrifuged and washed three times with ethyl acetate, then dried on high vacuum to produce 49mg, 80% yield of desired compound 4'. MS (ESI+) m/z 569 [M+H]⁺. The H-NMR spectrum of compound 4' is displayed in Fig. 4.

2.3 Preparation of the trehalose mono-Leucine ester codrug: 4-methyl-l-oxo-l- (((2R,3S,4S,5R)-3,4,5-trihydroxy-6-(((3R,4S,5S,6R)-3,4,5-trihydroxy-6 (hydroxy- methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)pentan-2- a mini 11 in chloride (compound 7')

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. First, trehalose reacted with trityl chloride to produce mono trityl trehalose (compound 5'). Next, esterification of compound 5' was performed with activated BocLeuOH and lead to compound 6' which was purified utilizing column chromatography. Finally, 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').

Provided herein below are further details on the preparation process of the trehalose mono-Leucine ester compound 7':

2.3.1 Preparation of (2R,3S,4S,5R)-2-(hydroxymethyl)-6-(((3R,4S,5S,6R)-

3,4,5-trihydroxy-6-((trityloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran- 3,4,5-triol (compound 5'): To the solution of trehalose leq, 3.75gr in 30ml pyridine leq of trityl chloride was added drop wise under cooling with ice during 30 min and then mixture was stirred for one hour. The solvent was evaporated under vacuum and crude solid contained mixture of mono- and di-tritylated product was dissolved in THF and filtrated from unreacted trehalose. The solvent was evaporated and crude solid was washed with water for 30 min. Mixture was filtrated and water phase contained mono-tritylated product was evaporated and dried under vacuum lead to white solid product in 51 % yield. This product was used immediately in the reaction condensation. MS (ESI+) m/z 583 [M+H]⁺.

2.3.2 Preparation of ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-(((3R,4S,5S,6R)- 3,4,5-trihydroxy-6-((trityloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran- 2- yl)methyl (tert-butoxycarbonyl)leucinate (compound 6'):

2-(tert-butoxycarbonylamino)-4-methyl-pentanoic acid was dissolved in pyridine. HATU 1.3eq, 0.49 gr in 30ml pyridine 1.5eq, 0.26ml DIPEA were added and the mixture was stirred for one hour. Then solution of tritylated trehalose (Compound 5') in 10ml pyridine was added drop wise. Reaction mixture was stirred overnight, pyridine was evaporated in vacuum, crude oil was dissolved in ethyl acetate and washed with NaHCCb solution and with brine. After separation, organic phase was dried over MgSO t, solvent was evaporated and crude solid was purified by column chromatography (reverse phase, acetonitrile-water). After evaporation of solvents 190 mg, 24% yield of desired compound was obtained. MS (ESI ) m/z 796 [M-H] .

2.3.3 Preparation of 4-methyl-l-oxo-l-(((2R,3S,4S,5R)-3,4,5-trihydroxy-6- (((3R,4S,5S,6R)-3,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)- tetrahydro-2H-pyran-2-yl)methoxy)pentan-2-aminium chloride (compound 7')

Solution of compound 6' in HCl dry 1M in ethyl acetate 1ml was stirred for two hour and solvent was evaporated. The crude was washed with ethyl acetate and filtrated. After drying in vacuum 5 mg, of final product was obtained. MS (ESP) m/z 456 [M]⁺. The H-NMR spectrum of compound 7' is displayed in Fig. 5. Example 3: Formulations

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.

Example 4: Preclinical pharmacokinetic studies

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.

All applicable portions of the study conform with the following regulations and guidelines regarding animal care and welfare: 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.

Blood 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

The activity of the above exemplary trehalose-leucine mono- and di-esters (T-Leu and T- Leu2, respectively) (in the following examples "Compound A" and "Compound B") is assessed in various mice models, as follows:

5.1. Con A model:

This model is used for assessing the effect of the codrug on acute hepatic inflammatory response induced by Con A.

24 C57 BL/6 mice 11-12 weeks old are administered with a tested trehalose codrug (up to 1 g/kg) 2 hours before administration of Con A (Con A is given at 500 μπι per mouse at 250 μΐ of saline for 14 hours) by IV, IP or PO administration.

The 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).

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.

5.2. High Fed Diet model:

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%.

Blood samples of the mice are obtained biweekly and are analyzed for serum ALT/ AST, cholesterol, triglycerides and fasting glucose. By 10 weeks of age, 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. In addition a liver biopsy is taken.

5.3. CC Fibrosis model

The effect of the codrug on hepatic fibrosis is assessed in fibrosis model, that imitates the fibrotic element of liver diseases.

8 weeks old C57BL/6 wild type mice are divided into 4 groups each of 6-10 mice.

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.

At the end of the trial the 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.

Claims

CLAIMS:

1. A codrug of the formula:

D-(x-AA)„

Formula I

wherein,

D is a non-reducing disaccharide residue or a functional derivative, a hydrate or a pharmaceutically acceptable salt thereof;

AA is a BCAA residue selected from the group consisting of Leu, He and Val or a pharmaceutically acceptable salt thereof; and

x is a cleavable bond or a linker moiety;

n is an integer selected from 1 to 8;

wherein where n>l each of x and AA may be the same or different.

2. A codrug according to claim 1, wherein x is a cleavable covalent bond between D and AA or a linking moiety selected from the group consisting of-CthO-, -OCH2O-, - C=0, -C (=S), -C(=0)-(CH₂)-, -C(=0)-NH-, H2PO3, peptide bond, ester (-C(=0)-0-), acyl ester, e.g. gamma-amino butyric ester, carbonate ester, acyloxylalkyl ester, benzyl ester, double ester, O-alkyl carboxylic ester, orthoesters, thioester, phosphate ester, glycine acyloxylalkyl, -NH-, carbamate (-NH- COO-), thiocarbamate,-0-C(=0)0-, - C(NH2)-COO-, azo (N=N), amide, di-amide, thioamide, thiocarbonate, C4-C5 alkyl chain, glutathione, lipoic acid, succinic acid, nitric oxide (NO), hydrogen sulfide, polyethylene glycol (PEG), disulfide, methylene, acyloxy methylene, glutaric acid, glycolic acid, linear bis-hydroxyl, dicarboxylate, succinate diester, dithiol, cyclic ketal, xanthate, methyl amino carboxymethyl and glycolic acid (-OCH2COO-).

3. A codrug according to Claim 1 or Claim 2, wherein x is a covalent bond between a functional group of D and the carboxyl group of AA.

4. A codrug according to any one of Claims 1 to 3, wherein said non-reducing disaccharide is α,α-trehalose.

5. A codrug according to any one of Claims 1 to 4, wherein said BCAA is L-leucine.

6. A codrug according to any one of Claims 1 to 5, wherein n is 1 or 2.

7. A codrug according to Claim 5, represented by the following formula T-Leu:

T-Leu

A codrug according to claim 5, represented by the formula the following formula

T-Lem

A codrug conjugate of Formula II:

[D-(x-AA)_n]-y

Formula II wherein,

x is a cleavable bond or a linker moiety, linking D and AA;

AA is a BCAA selected from the group consisting of Leu, He and Val or a pharmaceutically acceptable salt thereof;

n is an integer selected from 1 to 8;

wherein where n>l, each of x and AA may be the same or different;

Z is a targeting moiety that specifically binds to an extracellular ligand on a target cell; and

y is a bond or a spacer linking Z to [D-(x-AA)_n] at any suitable location (as indicated by the dashed line in Formula II.

10. A codrug conjugate according to Claim 9, wherein D is α,α-trehalose, AA is leucine and n is 1 or 2.

11. A codrug conjugate according to Claim 9 or Claim 10, wherein said targeting moiety is an antibody that specifically binds to an extracellular member of a target cell or organ.

12. A pharmaceutical composition comprising at least one codrug of Formula I, wherein the substituents are as defined in any one of Claims 1 to 8, and optionally comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.

13. A pharmaceutical composition according to Claim 12, wherein said codrug is represented by the formula T-Leu.

14. A pharmaceutical composition according to Claim 12, wherein said codrug is represented by the formula T-Leu2.

15. A pharmaceutical composition comprising a codrug conjugate of Formula II, wherein the substituents are as defined in any one of Claims 9 to 11 and optionally comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.

16. 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 codrug as defined in any one of Claims 1 to 8 and/or a targeted conjugate thereof as define in any one of Claims 9 to 11, said composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.

17. A method according to Claim 16, 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 or wherein autophagy has a role in the pathology or treatment of said disease or disorder.

18. 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 in any one of Claims 1 to 8 and/or a targeted conjugate thereof as define in any one of Claims 9 to 11, said composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.

19. A method according to Claim 17 or Claim 18, wherein said at least one disease or disorder is associated with malfunction and/or insufficiency of and/or impaired autophagy mechanisms and processes in said subject.

20. A method according to any one of Claims 17 to 19, wherein said at least one disease or disorder is related to one or more of liver, muscle (including cardiac), kidney and brain and neural disease, disorders or malfunction.

21. A method according to Claim 19 or Claim 20, wherein said impaired or insufficient autophagy associated disease or disorder is 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.

22. 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 in any one of Claims 1 to 8 and/or a targeted conjugate thereof as define in any one of Claims 9 to 11, said composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.

23. 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 in any one of Claims 1 to 8 and/or a targeted conjugate thereof as define in any one of Claims 9 to 11, said composition optionally further comprising at least one of pharmaceutically acceptable additive, carrier, excipient or diluent.

24. A method according to Claim 23, wherein said 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.

25. A method according to Claim 24, wherein said 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.

26. A method according to Claim 21, wherein said protein aggregation disease is any one of poly-alanine aggregation disorder, poly-glutamine aggregation disorder and a tauopathy.

27. A method according to Claim 21, wherein said neurodegenerative disease is any one of Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Landouzy-Dejerine dystrophy (LDMD), Huntington's disease, Creutzfeldt-Jakob disease, spinocerebellar ataxia, Friedrich's ataxia and others.

28. A method according to Claim 21, wherein said lysosomal storage disorder is any one of activator deficiency/GM2 gangliosidosis, alpha-mannosidosis, aspartylglucos- aminuria, 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), GM1 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, MPS I Hurler syndrome, MPS I Scheie syndrome, MPS I Hurler-Scheie syndrome, MPS II Hunter syndrome, Sanfilippo syndrome type Maroteau-Lamy (VI), AMPS IIIA, Sanfilippo syndrome type B/MPS IIIB, Morquio type AMPS IVA, Morquio Type B/MPS IVB, MPS IX hyaluronidase deficiency, Niemann-Pick disease (including Type A, Type B, and Type C), neuronal ceroidlipofuscinoses (including CLN6 disease, atypical late infantile, late onset variant, early juvenile Baten-Spielmeyer-Vogt/juvenile NCL/CLN3 disease, Finnish variant late infantile CLN5, Jansky-Bielschowsky disease/late infantile CLN2/TPP1 disease, Kufs/adult-onset NCL/CLN4 disease, northern epilepsy/variant late infantile CLN8, and Santavuori-Haltia/infantile CLN1/PPT disease), beta-mannosidosis, Pompe disease/glycogen storage disease type II, pycnodysostosis, Sandhoff disease/adult onset/GM2 gangliosidosis, Sandhoff disease/GM2 gangliosidosis infantile, Sandhoff disease/GM2 gangliosidosis juvenile, Schindler disease, Salla disease/sialic acid storage disease, Tay-Sachs/GM2 gangliosidosis, Wolman disease, Hermansky-Pudlak disease, Chediak-Higashi, Cistinosis, Salla, Methylmalonic aciduria, Sphingolipd activator protein deficiencies, RNASET2, SCARB2/LJMP-2 deficiency, or Multiple Sulfatase Deficiency.

29. A method according to Claim 21, wherein said muscular degenerative disease is 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).