FIELD OF THE INVENTION:

The present invention relates to levodopa beta-glycosyl derivatives, their stereoisomers, their pharmaceutically acceptable salts, pharmaceutically acceptable solvates, pharmaceutical compositions containing them, methods of their preparation and use for the treatment of pathological central and peripheral nervous system dysfunctions, neuromotor conditions and cardiovascular diseases and associated clinical symptoms.

BACKGROUND OF THE INVENTION:

Parkinson's disease is a progressive, neurodegenerative disorder of the extrapyramidal nervous system affecting the mobility and control of the skeletal muscular system, whose major symptoms are stiffness of the muscles, difficulty in moving, and tremors, rigidity, and bradykinetic movements. Diseases related by clinical symptomology, and progressive clinical symptomology in Parkinson's patients, include post-encephalitic syndromes, Wilson's disease, Parkinsonism secondary to cerebrovascular trauma and strole, dementia, Alzheimer's disease, Lou Gehrig's disease, psychomotor retardation, certain schizophreniform behaviour, anxiety and depression.

In biochemical terms, Parkinson's disease is characterised by a reduced concentration of the neurotransmitter, dopamine in the brain and treatment is directed towards ensuring an adequate supply of dopamine. A possible therapy based on the use of dopamine itself is hampered by the fact that this substance is unable to cross the blood brain barrier (BBB) because of its hydrophilicity and the absence of specific mechanisms of transport. Levodopa

(or L-DOPA), a dopamine precursor, has been and is one of the most commonly prescribed drugs for patients diagnosed with Parkinson's despite new therapies entering the market. When levodopa is administered orally, it is rapidly decarboxylated to dopamine in extracerebral tissues so that only a small portion of a given L-DOPA dose is transported unchanged to the central nervous system. One of the main problems in using levodopa is that it is unstable and metabolised in the body, and therefore it is not possible to get a sufficiently stable high concentration in the blood to drive the transport over the Blood Brain Barrier to reach a stable therapeutic concentration in the brain (Chukwuermeka S. Okereke in J Pharm Pharmaceutical Sci 5(2), (2002):146-161 ).

Oral delivery of CNS-drugs requires intestinal penetration, blood borne delivery, blood brain barrier penetrability and maintenance of functional utility. Various dopamine and L-DOPA derivatives have been synthesized in an attempt to address at least some of these needs and include certain glycoside derivatives described in more detail hereinbelow.

Glycoside derivatives are attractive potential candidates as many cellular mechanisms exist for transporting glycosides. For instance, intestinal intracellular transport vesicles containing Na+/glucose co-transporters (SGLTs) are reported to drive active transport of glucose and galactose across the intestinal brush border by harnessing Na+ gradients across the membrane. Unlike, intestinal transport, neural glycosides transport at the blood brain barrier appears to be mediated by endothelial cells and a sodium- independent transporter (GLUT1 , 2, 3, 4 and 5), although the possible involvement of the SGLT transporter family cannot be completely excuded (A. Tsuji, NeuroRx (2005) 2:54-62.

For example, Fernandez, C. et al. (Carbohydrate Research 327 (2000) 353- 365; Org. Biomol. Chem. 1 (2003) 767-771 ) describe the synthesis of certain

glycosyl dopamine derivatives as potential antiparkinsonian agents, some of which were more stable in buffer or in plasma than dopamine, lntraperitonal administration of some of these derivatives to reserpinized mice did not result in any significant antiparkinsonian properties, however. The glucoside dopamine derivatives were considered unsuitable prodrug candidates due to their lack of affinity for the glucose transporter GLUT-1.

US 6.548.484 relates to N-glycosylated dopamine, where only dopamine receptor binding and dopamine transporter binding activity (2 compounds) but not stability, intestinal transport or blood-brain barrier pass data is demonstrated.

Bonina, F. et al, J. Drug Targeting 11/2 (2003) 25-36, describes 3-O-[N-( _L - 3,4-dihydroxyphenylalanine)-succinamyl]-α-β- _D -glucopyranose (III) and 6-O- [N-( _L -3,4-dihydroxyphenylalanine)-succinamyl]-α-β- _D -galactopyranose (IV), (both (III) and (IV) may be considered as Levodopa N-glycosides). Their chemical and enzymatic stability data are similar to dopamine, see Table 1.

Table 1. t 1/2 Ih)

Compound pH 7.4 buffer Rat Plasma

III** 16.5 2.8

IV** 22.1 5.1

* Dopamine 27 2

* Data from Fernandez, C, Org. Biomol. Chem., 1 (2003) 767-771

** Data from Table 1 of the Bonina, F. et al article.

There therefore remains a need for new therapeutics for the treatment, or alleviation of the symptoms, of Parkinsons disease and its related diseases and this invention meets that need.

SUMMARY OF THE INVENTION:

The problem to be solved by the present invention is to provide a levodopa derivative with improved bioavailability and stability, improved intestinal transport and/or blood-brain barrier passage. The solution is based on that the finding by the present inventors that levodopa beta-glycosyl derivatives, as compared to levodopa aglycon, have improved hydrophilicity, prolonged bioavailability and stability, with improved intestinal transport and blood-brain barrier pass.

Accordingly, a first aspect of the invention relates to a levodopa beta-glycosyl derivative compound according to formula I, their stereoisomers, pharmaceutically acceptable salts and pharmaceutically acceptable solvates

wherein: R2 is not H and is preferably -COOH, an alkyl ester (eg methyl ester) or -

COO-beta-saccharide; R ₅ is selected from the group consisting of H and (C ₁ -

C ₄ )-alkyl; and at least one of R ₁ , R ₁ -, R ₃ or R ₄ is the beta anomer of a radical of a saccharide or R ₂ is -COO-beta-saccharide, with the proviso that when R ₁ , R ₃ , R ₄ and R ₅ are H, and R ₂ is -COOH, then R _r is not 3-O-succinamyl-α-β-D-glucopyranose or 6-O-succinamyl-α-β-D-galactopyranose.

A second aspect of the invention relates to a pharmaceutical composition comprising an effective amount of levodopa beta-glycoside or derivative thereof, their stereoisomers, salts, solvates or mixtures in combination with pharmaceutically acceptable carriers. Accordingly, the present invention

relates to a pharmaceutical composition as defined above for use in a method for metabolic replacement therapy or prevention of Parkinson and Parkinson's related diseases or associated clinical symptoms in a human person.

A third aspect of the invention relates to use of a levodopa beta-glycosyl derivative, their stereoisomers, salts or solvates as described herein, for the manufacture of a medicament for the treatment of pathological central and peripheral nervous system dysfunctions, neuromotor conditions and cardiovascular diseases and associated clinical symptoms in a human person, including Parkinsons disease and a Parkinson's related disease.

This third aspect may alternatively be formulated as a method for treatment of the diseases mentioned above in a human comprising administering to a human in need thereof an effective amount of pharmaceutical product as described herein.

A fourth aspect of the invention relates to a method for enzymatically making a levodopa beta-glycoside of formula I, their stereoisomers, pharmaceutically acceptable salts and pharmaceutically acceptable solvates

wherein:

R2 is not H and is preferably -COOH, an alkyl ester (eg methyl ester) or - COO-beta-saccharide; R ₅ is selected from the group consisting of H and (Ci- C ₄ )-alkyl; and

at least one of R ₁ , R ₁ -, R ₃ or R ₄ is the beta anomer of a radical of a saccharide or R ₂ is -COO-beta-saccharide, comprising the steps:

(i) contacting, under suitable conditions, a levodopa aglycon or a levodopa glycoside with a glycosyltransferase or a glycosidase capable of glycosylating the aglycon or capable of adding one or more saccharides to the saccharide of the levodopa glycoside or to glycosylate one of the other positions of the levodopa glycoside; and (ii) recovering the levodopa glycoside.

DEFINITIONS:

Prior to describing the more detailed embodiments of the invention, definitions of specific terms relating to the main aspects of the invention are provided below.

The term "aglycon" denotes the non-carbohydrate part of the corresponding glycosylated form of the aglycon. It may also be defined as an acceptor compound capable of being conjugated to a sugar. In a number of relevant examples, the aglycon is an alcohol with a hydroxy group suitable for being glycosylated. An aglycon may also be glycosylated at groups other than a hydroxy group, in particular at other nucleophilic groups such as a carboxylic acid group or an amine group. An example of this is levodopa which has two hydroxy groups, one carboxylic group and one amino group that can be conjugated (glycosylated) to provide Levodopa glycoside derivatives (the corresponding glycosylated form of the aglycon Levodopa). For example, where the sugar is glucose the aglycon may be termed aglucone. Further, when the sugar is glucose the term glucosylated may be used instead of glycosylated.

The term "glycoside" denotes a compound which on hydrolysis may give a saccharide and a non-saccharide (aglycon) residue or a glycoside with at least one glycosyl unit less, e.g. glucosides give glucose, galactosides give galactose, disaccharide derivatives give monosaccharide derivatives. To illustrate, when glycosylation has been made at an -OH position of levodopa, the glycoside is an O-glycoside, and when glycosylation has been made at a NH ₂ position of levodopa, the glycoside is an N-glycoside.

The term "glycosyltransferase" denotes a glycosyltransferase capable of conjugating a saccharide to an aglycon or to a glycoside as described herein. When the substrate of the glycosyltransferase is a glycoside, one can recover two, three or poly-saccharides conjugated to the aglycon, the aglycon conjugated to a di-, tri- or oligo-saccharide or mixtures of them or a glycoside glycosylated in more than one position.

The term "glycosidase" denotes an enzyme capable of deglycosylating (via hydrolysis) a glycoside to give a saccharide and a non-saccharide (aglycon) residue, i.e. are glycosyltransferases that use water as an acceptor molecule, and as such, are typically glycoside-hydrolytic enzymes. When the saccharide is glucose the enzyme is termed glucosidase. An example of a glycosidase is beta-glycosidase.

The term "recovering" in relation to "recovering the levodopa glycoside" of step ii) of the fourth aspect of the invention should be understood broadly in the sense that the compound is recovered in order to provide the compound in a higher state of purity relative to before the recovery step. The recovery step may include additional purification steps. Preferably the compound after the recovery step is present in a composition where the composition comprises at least 4% (w/w) of the compound, more preferably at least 10% (w/w) of the compound, even more preferably at least 20% (w/w) of the compound and most preferably at least 50% (w/w) of the compound. The

skilled person is aware of suitable purification protocols (e.g. by using solubility in different solvents, adequate purification columns, TLC or HPLC) to obtain the desired purity.

Preferably after the recovering step(s), there is at least 1 mg compound, more preferably at least 10 mg compound, even more preferably at least 1 g compound, and most preferably at least 10 g compound is recovered.

If the method of the fourth aspect is e.g. an in vivo based enzymatic method (see below) the compound may be recovered from the cell or from e.g. the supernatant of the medium whereby the cell for instance is fermented in order to obtain the compound in a more pure form relative to before the recovery step.

The term "glycoside transporter" is intended to mean a cellular membrane protein capable of binding a saccharide and transporting that saccharide from one location to another of the cell. Representative examples of glycoside transporters include glucose transporters (e.g. GLUT 1 , 2, 3, 4 and 5), galactose transporters, mannose transporters, hexose transporters (SGLT1 ) and the like. Those skilled in the art are aware of methods by which one may test compounds according to the invention in order to test their capability of binding to a glycoside transporter.

The term "brain penetration index", abbreviated BPI, is intended to mean the mathematical ratio calculated by the amount of one or more of the compounds in brain tissue per gram of brain tissue, divided by the amount of the compound (or compounds) in liver tissue per gram of liver tissue. The liver is chosen as a reference organ because of its intimate contact with blood and relative lack of barriers. The mathematical ratio is commonly expressed as a percentage.

In the definitions of the groups in formula (I), the alkyl group and the alkyl moiety of the alkanoyl group are a straight-chain, branched or cyclic alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and hexyl. An "alkanoyl" group is exemplified by a straight, branched or cyclic (C ₂ -C ₇ )- alkanoyl, and specifically, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, hexanoyl, etc. An "alkoxy" group is exemplified by a straight or branched chain (CrC ₆ ) alkoxy. Alkoxy groups are alkyl groups terminating with an oxygen. Examples of alkoxyl groups are methoxy, ethoxyl, propoxyl, isopropoxyl, butoxyl, isobutoxyl, pentoxyl, hexoxyl. Alkoxy groups also include polyethers, such as methoxyethoxy.

Amino acids

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, v- carboxyglutamate, and O-phosphoserine. "Amino acid analogue" refers to entities that have the same basic chemical structure as a naturally occurring amino acid, e.g., a carbon that is linked to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogues have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetic" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

Natural amino acids include glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, lysine, glutamic acid, glutamine, arginine, histidine, phenylalanine, cysteine, tryptophan, tyrosine, methionine, and proline. Others include lanthionine, cystathionine, and homoserine. Some unusual or modified amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-amino-caproic acid, 2-amino-heptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, 6-N- methyllysine, N-methylvaline, norvaline, norleucine, and ornithine.

Non-natural amino acids include 1-aminosuberic acid, 3-benzothienylalanine, 4,4'-biphenylalanine, 4-bromophenylalanine, 2-chlorophenylalanine, 3- chlorophenylalanine, 4-chlorophenylalanine, 3-cyanophenylalanine, 4- cyanophenylalanine, 3,4-dichlorophenylalanine, 3,4-difluorophenylalanine, 3,5-difluorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4- fluorophenylalanine, 3,3-diphenylalanine, homophenylalanine, 2- indanylglycine, 4-iodophenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4- nitrophenylalanine, pentafluorophenylalanine, 2-pyridylalanine, 3- pyridylalanine, 4-pyridylalanine, tetrahydroisoquinoline-3-COOH, 4- thiazolalanine, 2-thienylalanine, 3-trifluoroethylphenylalanine, 4- trifluoromethylphenylalanine, and 3,4,5-trifluoromethylphenylalanine.

Acyl or amino group of amino acids include the -CO or -N terminal group of natural and non-natural amino acids, which may be protected or unprotected, or otherwise modified.

The mono-, di-, tri- tetra-, or pentapeptide residue derived from the group consisting of natural and non-natural aminoacids may be linked through the

acyl or amino group of one of the amino acids.

Embodiments of the present invention are described below, by way of example only.

DETAILED DESCRIPTION OF THE INVENTION:

Levodopa qlvcosyl derivatives

The levodopa beta-glycosyl derivatives as described herein may be made synthetically or enzymatically. Preferred enzymatic processes are described below.

According to an embodiment of the invention, it relates to a levodopa glycoside derivative of formula I, wherein Ri and Rr are each independently selected from the group consisting of H, (CrC ₆ )-alkyl, NH ₂ , (C ₂ -C ₇ )-alkanoyl, beta-saccharide and an acyl group of a mono-, di-, tri-, tetra- or pentapeptide residue derived from the group consisting of natural and non-natural aminoacids; R ₅ is selected from the group consisting of H and (Ci-C ₄ )-alkyl; R ₂ , is selected from the group consisting of (d-C ₆ )-alkyl, NH ₂ , OH, (CrC ₆ )- alkoxy and -COOR ₆ ; wherein R ₆ is selected from the group consisting of H, (Ci-C ₆ )-alkyl, beta-saccharide and an amino group of a mono-, di-, tri-, tetra- or pentapeptide residue derived from the group consisting of natural and non- natural amino acids; R ₃ and R ₄ are each independently selected from the group consisting of H, (Ci-C ₄ )-alkyl and beta-saccharide; and at least one of Ri, Rr, R ₃ , R ₄ or R ₆ is not H.

Preferably, peptide residues are derived from glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, lysine, glutamic acid, glutamine, arginine, histidine, phenylalanine, cysteine, tryptophan, tyrosine, methionine, and proline. Similarly, preferably amino acid

residues are natural amino acid residues.

In a more preferred embodiment, the levodopa glycosyl derivative is that wherein R ₅ is H; R ₂ is -COOR ₆ ; Ri, Rr, R3, R ₄ and R ₆ are each independently selected from H and beta-saccharide; and at least one of Ri, Rr, R ₃ , R ₄ or R ₆ is different of H. In particular R ₆ is an alkyl, such as a methyl group.

Glycosylation of levodopa derivatives according to the present invention may be at any suitable position, according to formula I, but is preferably at OH in position 3 or 4 (i.e., R ₃ and/or R ₄ is preferably a saccahride or glycoside). The glycoside can comprise more than one glycosidic unit, in particular 2, 3 or 4 glycosidic units. Preferred, however, are those having 1 glycosidic unit linked at OH in position 3 or 4 (i.e., R ₃ and/or R ₄ is preferably a saccahride or glycoside). In general, any form of glycosylation, according to formula I may be employed, provided that it is substantially non-toxic to the patient. Glycosylation of levodopa glycosyl derivatives is also possible at any suitable position of the levodopa glycoside: the OH (R ₃ or R ₄ ), NH ₂ (Ri or R ₁ -) or COOH (R ₂ ) position of the levodopa moiety or at any suitable position of the saccharide moiety, thereby allowing formation of a di- tri- or poly-saccharide conjugated to the levodopa or levodopa glycosylated at more than one position. The levodopa glycosyl derivative can comprise more than one glycosidic unit, in particular 1 , 2, 3 or 4 glycosidic units, each of these glycosidic units being mono-, di-, tri- or oligo-saccharides.

According to the fourth aspect of the invention, the levodopa glycosides as described herein are made enzymatically, as is described below in more detail.

The compound of the general formula (I) may be converted into its pharmaceutically acceptable salts, or its pharmaceutically acceptable

solvates by conventional methods. For example, such salts may be prepared by treating one or more of the instant compounds with an aqueous solution of the desired pharmaceutically acceptable metallic hydroxide or other metallic base and evaporating the resulting solution to dryness, preferably under reduced pressure in a nitrogen atmosphere. Alternatively, a solution of the levodopa glycosyl derivative may be mixed with an alkoxide to the desired metal, and the solution subsequently evaporated to dryness. The pharmaceutically acceptable hydroxides, bases, and alkoxides include cations, such as, without limitation potassium, sodium, ammonium, calcium, and magnesium. Other representative pharmaceutically acceptable salts include hydrochloride, hydrobromide, sulphate, bisulphate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate and the like. Salts may include acid addition salts which are sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides, acetates, tartrates, maleates, citrates, succinates, palmoates, methanesulfonates, benzoates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, glycerophosphates, ketoglutarates and the like.

Pharmaceutically acceptable solvates may be hydrates or comprising other solvents of crystallization such as alcohols.

Saccharide residues:

By "saccharide" are meant mono-, di-, tri- or oligosaccharide made up of n sugar subunits linked to each other by glycosidic bonds, which subunits, when n is grater that 1 , may be the same or different in respect to the type of constituent sugar residues (e.g. homo- or heteropolymeric), the localization of axial and equatorial ring substituents, the number of carbon atoms and the ring carbon locations and orientations of hydroxyl groups.

Saccharides exist as stereoisomers, optical isomers, anomers, and epimers.

The meaning of "saccharide" as used herein encompasses any isomers such as stereoisomers, optical isomers, and epimers, limited to the beta anomer. For example, a hexose can be either an aldose or a ketose, and can be of D- or L-configuration, can assume either an alpha or beta conformation, and can be a dextro- or levo-rotatory with respect to plane-polarized light. Thus, all stereoisomers, optical isomers, and epimers are suitable saccharides to perform the glycosylation of levodopa or levodopa glycosyl derivatives according to the present invention, but only the beta anomer is used. Accordingly, the term saccharide as used herein encompasses any stereoisomer, optical isomer and epimer of the beta conformer.

It has been established that in the chair conformations, the molecules have a lower energy if the larger substituents on the carbons are roughly in the plane of the ring itself. This positions are called "equatorial" to distinguish them from the other positions, roughly perpendicular to the ring, called "axial".

Glycosides with the substituent in the anomeric carbon in equatorial position are named beta-glycosides, and those with axial position are named alpha- glycosides. A compound which can have all of its substituents in an equatorial position is more stable than one which cannot.

Further, a list of suitable sugars can be seen in US 2003/0130205A1 paragraphs [0029] to [0036]. Exemplary monosaccharide residues include residues of a pentose, hexose, or a heptose. Non-limiting examples of pentoses include arabinose, ribose, ribulose, xylose, lyxose, and xylulose. Non-limiting examples of hexoses include glucose, galactose, fructose, fucose, mannose, allose, altrose, talose, idose, psicose, sorbose, and tagatose. Non-limiting examples of heptoses include mannoheptulose and sedoheptulose.

Thus according to the foregoing disclosure, in a preferred embodiment of the invention the saccharides are beta-glycosyl saccharides.

Some saccharide residues may have a hydroxyl group at the C2 position in an axial or equatorial conformation. For example, glucosyl residue has a hydroxyl group in equatorial conformation at its C2 position, whereas a mannosyl residue, for example, has a hydroxyl group in axial conformation at its C2 position. In some cases, the saccharide residue may possess a group other than a hydrogen or hydroxyl at its C2 position.

The saccharides of the present invention may also be substituted with various groups. Such substitutions may include (C1-C6)-alkyl, (C1-C6)-alkoxy, (C1- C6)-acyl, acylamine, amine, (C1-C6)-alkylamine, (C1-C6)-alkylamide, halo, thio, nitro, keto, and phosphatyl groups, wherein the substitution may be at one or more positions on the saccharide, except for the anomeric carbons which form the glycosidic bond. Moreover, the saccharides may also be present as a deoxy saccharide. Examples of such substituted saccharides include: D-glucosamine and D-galactosamine, which are 2-amino-2-deoxy glucose and 2-amino-2-deoxy galactose, respectively. Examples of carboxy- containing saccharides include aldonic, aldaric, and uronic acids. Examples of carboxy-containing amino sugars include N-acetylmuramic acid and N- acetylneuraminic acid, wherein each is a six-carbon amino sugar linked to a three-carbon sugar acid.

Preparation of substituted sugars with the above-listed substituents is well within the ordinary skill in the art.

According to one embodiment of the present invention, the linkage of the saccharide moiety to the hydroxy, carboxy or amine group of levodopa occurs through a single bond formed between the subject reacting group of the levodopa (or levodopa glycosyl derivative) and either of the hydroxy groups or suitable substituents thereof of the subject saccharide. The linkage of the saccharide moiety to the hydroxy, carboxy or amine group of levodopa

derivative occurs through the beta- conformation of the hydroxy groups or suitable substituents thereof of the subject saccharide moiety. Accordingly, the levodopa glycosyl derivative of the present invention is a levodopa beta- glycosyl derivative.

Accordingly, an embodiment of the invention representative examples of monosaccharide residues include the following: namely, polyhydroxy aldehydes (e.g. aldoses and ketoaldoses); polyols resulting from e.g., reduction of the aldehyde carbonyl to a hydroxyl (e.g., alditols and ketoses); polyhdyroxy acids resulting e.g., from oxidation of the aldehyde and/or the chain terminal hydroxyl (e.g., aldonic, ketoaldonic, aldaric and ketoaldaric); amino-sugars resulting from replacement of any hydroxyl in the chain with an amino (e.g., aldosamines and ketosamines); aldehydo-acids resulting e.g. from oxidation of only the chain terminal hydroxyl in an aldehydo-sugar (e.g., uronic acids and ketouronic acids); and their various lactones, i.e., cyclic esters of hydroxy carboxylic acids containing one 1-oxacycloalkan-2-one structure.

Preferably, the subject sugars may be straight chains and/or cyclic 3-, 4-, 5-, 6-, 7-, 8- and 9-membered sugar residues (e.g., hemiacetals and acetals) optionally substituted and linked with the hydroxyl, carboxyl or amine group of levodopa. Representative triosyl residues (i.e. 3-membered residues) include the aldoses D- and L-glyceraldehyde and derivatives thereof e.g., glyceraldehyde and glyceric acid phosphates; the keto-sugars D- and L- dihydroxyacetone and derivatives thereof. Representative tetraosyl residues include the aldoses D- and L-erythrose, threose, streptose and apiose; the keto-sugars D- and L-erythrulose; and derivatives thereof. Representative pentosyl residues (i.e. 4-membered residues) include the D- and L-aldoses ribose, arabinose, xylose and lyxose; the D- and L-ketoses ribulose and xylulose; and, derivatives thereof. Representative hexosyl residues (i.e. 6- membered residues) include aldosyl, furanosyl and pyranosyl sugars, e.g.,

cyclic and acyclic D- and L-aldoses such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, fructose, glucono-1 ,4-lactone, glucaro-1 ,4:6,3-dilactone, gluconofuranono-6,3-lactone; the ketoses ribo- hexulose, arabino-hexulolose, xylo-hexulose and lyxo-hexulose; and derivatives thereof. Representative heptosyl residues (i.e., 7-membered residues) include e.g., sedoheptulose and derivatives thereof; and, representative nonosyl residues (i.e., 9-membered residues) include N- acetylneuraminic acid and derivatives thereof. Also representative are, 2- deoxy-ribose, 6-deoxyglucose and 2-deoxyglucose, xyloascorbyllactone, digitoxose (2-deoxyaltromethylose), fucose (6-deoxy-galactose), gluconolactone, galaconolactone, rhamnose (6-deoxy-mannose), fructose (2- keto-arabohexose), aldaric acids, alditols, aldonic acids, ketoaldonic acids, and amino sugars. Representative alditols include e.g., erythritol, threitol, ribitol, arabinitol, xylitol, lyxitol, glucitol, allositol, altrositol, mannositol, gulositol, idositol, galactositol, talositol and their derivatives. Representative aldonic acids include erythronic acid, threonic acid, ribonic acid, arabinonic acid, xylonic acid, lyxonic acid, gluconic acid, allonic acid, altronic acid, mannonic acid, gulonic acid, idonic acid, galactonic acid, tolonic acid and their derivatives. Representative ketoaldonic acids include erythro- tetraulosonic acid, threo-tetraulosonic acid, ribo-pentulosonic acid, arabino- pentulosonic acid, xylo-pentulosonic acid, lyxo-pentulosonic acid, gluco- hexulosonic acid, allo-hexulosonic acid, altro-hexulosonic acid, manno- hexulosonic acid, gulo-hexulosonic acid, ido-hexulosonic acid, galacto- hexulosonic acid, talo-hexulosonic acid and their derivatives. Representative aldaric acids include erythraric acid, threaric acid, ribaric acid, arabinaric acid, xylaric acid, lyxaric acid, allaric acid, altraric acid, glucaric acid, mannaric acid, gularic acid, idaric acid, galactaric acid, talaric acid and their derivatives. Representative of amino sugar include erhtyrosamine, threosamine, ribosamine, arabinosamine, xylosamine, lyxosamine, allosamine, altrosamine, glucosamine, N-acetylglucosamine, N- methlglucosamine mannosamine, gulosamine, idosamine, galactosamine,

talosamine and their derivatives. Representative uronic acids include erythrosuronic acid, threosuronic acid, ribosuronic acid, arabinosuronic acid, xylosuronic acid, lyxosuronic acid, allosuronic acid, altrosuronic acid, glucuronic acid, mannosuronic acid, gulosuronic acid, idosuronic acid, galactosuronic acid, talosuronic acid and their derivatives. Representative keto-uronic acids include keto-erythrosuronic acid, keto-threosuronic acid, keto-ribosuronic acid, keto-arabinosuronic acid, keto-xylosuronic acid, keto- lyxosuronic acid, keto-allosuronic acid, keto-altrosuronic acid, keto-glucuronic acid, keto-mannosuronic acid, keto-gulosuronic acid, keto-idosuronic acid, keto-galactosuronic acid, keto-talosuronic acid and their derivatives.

Representative lactones include erythrolactone, threolactone, ribolactone, arabinolactone, xyloslactone, lyxoslactone, allolactone, altrolacone, glucolactone, mannolactone, gulolactone, idolactone, galactolactone, talolactone and their stereoisomers, optical isomers, epimers and derivatives.

Preferably, the subject sugar comprises an aldose or ketose pentose or hexose sugar selected from the group consisting of D- and L-enantiomers of ribose, glucose, galactose, mannose, arabinose, allose, altrose, gulose, idose, talose and their substituted derivatives. Most preferably, the subject sugar comprises an aldose pentosyl or hexosyl sugar selected from ribose, glucose, galactose, glucosamine, galactosamine, N-acetylglucosamine, N- acetylgalactosamine, N-acetyl ribosamine, xylose, mannose and arabinose, their stereoisomers, optical isomers, epimers and derivatives.

The term "Di-saccharide", when used in regard to the subject Rr, R ₁ , R ₃ , R ₄ and R ₆ , is intended to mean a polymeric assemblage of 2 sugar residues. Preferable examples of disaccharides include homo-polymeric (e.g., maltose and cellobiose) and hetero-polymeric (e.g., lactose and sucrose) assemblages of sugars as set forth supra.

The term "Tri-saccharide", when used in regard to the subject R ₁ -, R ₁ , R ₃ , R ₄

and R ₆ , is intended to mean a polymeric assemblage of 3 sugar residues, e.g., as set forth supra. Preferable examples of trisaccharides include homo- polymeric and hetero-polymeric assembled sugars, each of the subject constituent sugars is linked one-to-another in a serial array through a series of glycosyl bonds.

"Oligosaccharide", when used in relation to the subject Rr, R ₁ , R ₃ , R ₄ and R ₆ , is intended to mean a polymeric assembly of preferably about 4 to about 10 constituent homomonosaccharide sugars (i.e., all the same constituent) or hetero-monosaccharide (i.e., different constituent) sugars. Each of the subject constituent sugars is linked one-to-another in a serial array through a series of glycosyl bonds. Preferred oligosaccharide of this invention may comprise of 2 to 6 monosaccharide units, preferably, 2 to 4 monosaccharide units, and more preferably, 2 to 3 monosaccharide units.

Non-limiting examples of oligosaccharides include lactose, maltose, cellobiose, gentiobiose, melibiose, isomaltose, mannobiose and xylobiose. The preparation of several oligosaccharides is well-known in the art. They can be obtained by partial hydrolysis of polysaccharides or synthesized from the desired monosaccharides. In addition, several oligosaccharides can be purchased from commercial sources or can be custom made using ordinary skill in the art.

Oligosaccharides also exist in many stereoisomeric, isomeric, epimeric and anomeric forms and the oligosaccharides described herein include such stereoisomeric, isomeric, epimeric and anomeric forms, linked to the hydroxy, carboxy or amine group of the levodopa derivative occurs through the beta- conformation of the hydroxy groups or suitable substituents thereof of the subject saccharide moiety.

Preferably, the subject di-, tri- and oligosaccharide saccharides are

metabolizable and/or acid hydrolyzable to mono-, di- and tri-saccharides and transportable by saccharide transporters in mammals; most preferably, when present as an oligosaccharide the subject Rr, R ₁ , R ₃ , R ₄ and R ₆ -moiety comprises a residue selected from the group of metabolizable di-and tri- saccharides consisting of: (i) homopolymers such as an erythran, a threan, a riban, an arabinan, a xylan, a lyxan, an allan, an altran, a glucan (e.g. maltose, isomaltose, cellobiose), a mannan, a gulan, an idan, a galactan, a talan and their substituted derivatives; (ii) heteropolymers such as erythrosides, threosides, ribosides, arabinosides, xylosides, lyxosides, allosides, altrosides, glucosides (e.g., sucrose; (Glc-.beta.1 ,4-Frc), galactosides (e.g., lactose; Gal-.beta.1 ,4-Glc), mannosides, gulosides, idosides, talosides and their substituted derivatives. Other representative oligosaccharides include the following: namely, sucrose glycogen, fucosidolactose, lactulose, lactobionic acid, amylose, fructose, fructofuranose, scillabiose, panose, raffinose, amylopectin, hyaluronic acid, chondroitin sulfate, heparin, laminarin, lichenin and inulin. Preferably, the subject R- moiety, when present as an oligosaccharide, is selected from the group consisting of glucosyl and galactosyl homo- and heteropolymers, e.g., glucans, galactans, glucosides and galactosides.

Thus according to the foregoing disclosure, embodiments of the invention provide a variety of compounds which are within the spirit of the present invention.

Accordingly, an embodiment, of the invention relates to O-linked and/or N- linked transportable and metabolizable levodopa beta-glycosyl derivatives according to formula I. Accordingly, in a more preferred embodiment of the invention relates to O-linked levodopa beta-glycosyl derivatives, and most preferred is O-linked at OH in position 3 or 4 of levodopa.

In an embodiment of the invention, the saccharide is selected from the group

consisting of the beta conformer of glucose, mannose, galactose, fructose, xylose, fucose, rhamnose, apiose, glucuronic acid, mannuronic acid, galacturonic acid and their stereoisomers, optical isomers, epimers and derivatives thereof.

In a still more preferred embodiment, the saccharide is β-glucose.

Contacting the levodopa aglycon or the levodopa glycoside with a glvcosyltransferase or a glvcosidase - Enzymatically synthesized glycosides

The levodopa aglycon or the levodopa glycoside is contacted with a glycosyltransferase or a glycosidase under suitable conditions to ensure proper functioning of the enzymatic process. This may be done in different manners.

For example, this can be done in vitro where the levodopa aglycon or the levodopa glycoside and the glycosyltransferase or the glycosidase are present in a receptacle, e.g. a tube. Suitable conditions refer to adequate buffer solutions, temperature, time, etc. as is apparent to the artisan. In light of the teachings provided herein, the skilled person may routinely identify such suitable conditions.

Accordingly in one embodiment of the fourth aspect of the invention, the contacting of step (i) is carried out in vitro under suitable conditions.

In a preferred embodiment, in the contacting step (i), a glycosyltransferase is used rather than a glycosidase.

The in vitro method is performed in an adequate reaction mixture comprising a suitable glycosyltransferase and the aglycon of interest, which is incubated under suitable conditions. The enzymatically made glycoside derivative may

be qualitatively analysed by thin layer chromatography (TLC) and quantitatively analyzed by LC-MS, as described herein below in more detail.

Altematviely, glycosylation may be achieved in vivo, for example through fermentation of a suitable microorganism cell (e.g., yeast). Accordingly to this embodiment of the fourth aspect of the invention, the contacting of step (i) is done in a microorganism cell fermented in a suitable medium, where the microorganism is capable of growing. In a preferred embodiment, the cell comprises a glycosyltransferase gene encoding a glycosyltransferase capable of glycosylating the levodopa aglycon or the levodopa glycoside, or a glycosidase capable of transglycosylation.

The in vivo procedure may be implemented by adding the aglycon or glycoside of interest to the fermentation media comprising the cells, the cells comprising a glycosyltransferase e.g. through expression of a recombinant heterologous glycosyltransferase gene. The aglycon or glycoside enters the cell and is glycosylated in vivo. The resulting glycoside may then be released from the cells into the surrounding fermentation medium form which it may be recovered. (See for example, WO04/111254).

If considered advantageous, one may also construct a cell that besides the glycosyltransferase gene also comprises a gene encoding a product involved in the biosynthesis pathway leading to the aglycon compound of interest. If the cell comprises e.g. all relevant biosynthesis pathway genes leading to in vivo biosynthesis of the aglycon compound of interest from e.g. a suitable amino acid then such a cell could advantageously be used to make the aglycon of interest. The aglycon of particular interest herein is Levodopa or derivatives thereof.

A preferred microorganism cell suitable to be used in a method as described herein is a microorganism cell selected from the group consisting of a yeast

cell and a prokaryotic cell.

A preferred yeast cell is a yeast cell selected from the group consisting of Ascomycetes, Basidiomycetes and fungi imperfecti. Preferably a yeast cell selected from the group consisting of Ascomycetes. Preferred Ascomycetes yeast cell selected from the group consisting of Ashbya, Botryoascus, Debaryomyces, Hansenula, Kluveromyces, Lipomyces, Saccharomyces spp e.g. Saccharomyces cerevisiae, Pichia spp., Schizosaccharomyces, spp. e.g. Schizosaccharomyces pombe. A preferred yeast cell is a yeast cell selected from the group consisting of Saccharomyces spp e.g. Saccharomyces cerevisiae, and Pichia spp. and Schizosaccharomyces pombe.

In a method as described herein a very preferred cell is a prokaryotic cell. A preferred prokaryotic cell is selected from the group consisting of Bacillus, Streptomyces, Corynebacterium, Pseudomonas, lactic acid bacteria and in particular an E. coli cell. A preferred Bacillus cell is B. subtilis, B. amyloliquefaciens or B. licheniformis. A preferred Streptomyces cell is S. setonii. A preferred Corynebacterium cell is C. glutamicum. A preferred Pseudomonas cell is P. putida or P. fluorescens.

A preferred cell is a cell without active or with reduced levels of beta- glycosidase, for example a cell without at least some of the genes encoding a beta-glycosidase that deglycosylates the glycosylated form of the aglycon compound of interest to be produced as described herein. Further, in some embodiments, the cell may comprise a permease or other transport protein enabling the cell to release or secrete the glycoside to the medium or to an internal compartment other than the one where it is glycosylated.

Transformation of suitable DNA containing vectors into the cells described above is routine work for the skilled person. Suitable vectors can be constructed, containing appropriate regulatory sequences, including promoter

sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990).

Preferably, the microorganism cell is a cell wherein the cell expresses a heterologous glycosyltransferase gene as described herein. In this connection, the term "heterologous glycosyltransferase gene" should be understood according to the art as a glycosyltransferase gene that has been introduced into a cell that, before the introduction, did not express the glycosyltransferase gene. Alternatively, the glycosyltransferase gene may be an endogenous glycosyltransferase gene naturally produced by the cell.

Method for identification of glvcosyltransferases to be used

A method for identifying a suitable production method for glycosylation of a drug substance comprises the steps of: i) providing a library containing samples of the same or different biological materials with glycosylation activity, ii) treating the drug substance by use of one of the following steps a) and b) depending on the water solubility of the drug substance a) if the drug substance is soluble in water, the drug substance is dissolved in water b) if the drug substance is not readily soluble in water, the drug substance is subjected to one of the following: b1 ) dissolving the drug substance in an organic solvent and diluting it with water optionally comprising a surfactant, and b2) dissolving the drug substance in a solvent optionally

comprising a surfactant, followed by evaporation of the solvent to obtain the drug substance on amorphous form, which subsequently is dissolved in water or in an aqueous medium as defined in step b1 ), iii) assaying the library for glycosylation ability towards the drug substance by c) incubating the drug substance treated as defined in step ii) with the samples of biological material in the presence of one or more sugar donors, and d) detecting sample(s) of biological material contained in the library that is/are capable of glycosylating the drug substance, iv) identifying one or more biological materials suitable for producing the glycosylated drug substance, and v) optionally producing the glycosylated drug substance.

Preferably, the library of step i) contains more than one sample, preferably 5, 10, 15, 20 or more samples of the same or different biological materials with glycosylation activity.This method for identifying a suitable production method for glycosylation of a drug substance is described in more detail in European patent application No. EP04106058.3, the content of which is hereby incorporated by reference into this present application.

Glvcosyltransferase:

A very large number of glycosyltransferases are known in the art. The choice of glycosyltransferase to be used in the present invention is not limiting, provided that it can utilize a modified sugar as a sugar donor and link it to the aglycon (or further glycosylate a glycosyl derivative). Examples of such enzymes include Leloir pathway glycosyltransferases, such as galactosyltransferase, N-acetylglucosaminyltransferase, N- acetylgalactosaminyltransferase, fucosyltransferase, sialyltransferase,

mannosyltransferase, xylosyltransferase, glucurononyltransferase and the like.

For enzymatic saccharide syntheses that involve glycosyltransferase reactions, glycosyltransferases can be cloned, or isolated from any source. Many cloned glycosyltransferases are known, as are their polynucleotide sequences. Glycosyltransferases that can be employed in the methods of the invention include, but are not limited to, galactosyltransferases, fucosyltransferases, glucosyltransferases, N- acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases, glucuronyltransferases, sialyltransferases, mannosyltransferases, glucuronic acid transferases, galacturonic acid transferases, and oligosaccharyltransferases.

Suitable glycosyltransferases include those obtained from eukaryotes (eg, plants), as well as from prokaryotes. DNA encoding glycosyltransferases may be obtained by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by combinations of these procedures. Screening of mRNA or genomic DNA may be carried out using specific oligonucleotide probes (ie selective for a glycosyltransferase) generated from the glycosyltransferases nucleic acid sequence. Probes may be labelled with a detectable label, such as, but not limited to, a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with known procedures and used in conventional hybridization assays. In the alternative, glycosyltransferases nucleic acid sequences may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the glycosyltransferases nucleic acid sequence. Preferred glycosyl transferases are described in WO0140491 , which is hereby incorporated by reference in its entirety.

A glycosyltransferases enzyme may be synthesized in a host cell transformed with a vector containing DNA encoding the glycosyltransferase. A vector is a replicable DNA construct. Vectors are used either to amplify DNA encoding the glycosyltransferases enzyme and/or to express DNA which encodes the glycosyltransferases enzyme. An expression vector is a replicable DNA construct in which a DNA sequence encoding the glycosyltransferases enzyme is operably linked to suitable control sequences capable of effecting the expression of the glycosyltransferases enzyme in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.

The art describes a number of glycosyltransferases that can glycosylate a low molecular weight organic aglycon or a levodopa glycoside compound of interest. Based on DNA sequence homology of the sequenced genome of the plant Arabidopsis thaliana it is believed to contain around 100 different glycosyltransferases. These and numerous others have been analyzed in Paquette, S. et al, Phytochemistry 62 (2003) 399-413.

Glycosyltransferase amino acid sequences and nucleotide sequences encoding glycosyltransferases from which the amino acid sequences can be deduced are also found in various public available databases, including GenBank, Swiss-Prot, EMBL, CAZY and others.

On the filing date on the present invention the web link for this CAZY database was http://afmb.cnrs-mrs.fr/CAZY/index.html

In the CAZY database, there can be found suitable glycosyltransferase sequences from virtually all species including, animal, insects, plant, microorganisms. For instance, there can be found glycosyltransferase sequences able of form disaccharides, e.g. EC 2.4.1.11 , EC 2.4.1.11 , EC 2.4.1.22 and EC 2.4.1.69.

In a preferred embodiment, the glycosyltransferase uses nucleotide diphospho-sugar or nucleotide monophospho-sugars or sugar phosphate as sugar donors. See below for more details for description for sugar donors.

In a more preferred embodiment, the glycosyltransferase is a NDP- glycosyltransferase. Such glycosyltransferases have been identified in plants, animals, fungi, bacteria and viruses. These glycosyltransferases are characterized by utilization of NDP-activated sugar moieties as the donor molecule and contain a conserved UGT-defining sequence motif near the C- terminus.

In an even more preferred embodiment, the glycosyltransferase is a UDPG- glycosyltransferase (UGT). UGTs have been identified in plants, animals, fungi, bacteria and viruses. These glycosyltransferases are characterized by utilization of UDP-activated sugar moieties as the donor molecule and contain a conserved UGT-defining sequence motif near the C-terminus.

Preferably, the glycosyltransferase is a glycosyltransferase that conjugates a monosaccharide to an aglycon or to the glycosidic unit of a glycoside as described herein or to the levodopa glycoside in a different position. Most preferably glycosyltransferase is a glycosyltransferase that conjugates a monosaccharide sugar to an aglycon as described herein.

In a preferred embodiment, the glycosyltransferase is a glucosyltransferase.

Herein most preferred glycosyltransferase is a glycosyltransferase selected from the group consisting of the glucosyltransferase from Sorghum bicolour termed UGT85B1 ; UGT85A2, and UGT89B1.

A glycosyltransferase gene as described herein may be introduced into a cell in order to make a cell express a heterologous glycosyltransferase gene as described herein, in particular a microorganism cell or a filamentous fungi cell that expresses a heterologous glycosyltransferase gene as described herein. Alternatively, the glycosyltransferase gene may be an endogenous glycosyltransferase gene naturally produced by the cell.

In addition genes giving raise to increased expression of the glycosyltransferase or increased yield of the glycoside may be introduced. Such genes may encode regulatory proteins, protease inhibitors, repressors of protease gene, genes increasing the level or precursors, especially the relevant NDP-sugars, genes involved in NDP-metabolism, permeases and other transporters, genes reducing the metabolism of the aglycone etc.

Glvcosidase

Glycosidases normally catalyze the hydrolysis of a glycosidic bond. However, under appropriate conditions, they can be used for the formation of glycosidic bonds in vitro by controlling the thermodynamics or kinetics of the reaction mixture.

A glycosidase can function by retaining the stereochemistry at the bond being broken during hydrolysis or by inverting the stereochemistry at the bond being broken during hydrolysis, classifying the glycosidase as either a "retaining" glycosidase or an "inverting" glycosidase, respectively. Retaining glycosidases have two critical carboxylic acid moieties present in the active

site, with one carboxylate acting as an acid/base catalyst and the other as a nucleophile, whereas with the inverting glycosidases, one carboxylic acid functions as an acid and the other functions as a base.

Most glycosidases used for carbohydrate synthesis are exoglycosidases; the glycosyl transfer occurs at the non-reducing terminus of the substrate. The glycosidase binds a glycosyl donor in a glycosyl-enzyme intermediate that is either intercepted by water to yield the hydrolysis product, or by an acceptor, to generate a new glycoside or oligosaccharide. An exemplary pathway using an exoglycosidase is the synthesis of the core trisaccharide of all N-linked glycopeptides, including the β-mannoside linkage, which is formed by the action of β-mannosidase (Singh et al., Chem. Commun. 993-994 (1996)).

Although their use is less common than that of the exoglycosidases, endoglycosidases are also utilized to prepare carbohydrates. Methods based on the use of endoglycosidases have the advantage that an oligosaccharide, rather than a monosaccharide, is transferred. Oligosaccharide fragments have been added to substrates using endo-β-N-acetylglucosamines such as endo-F, endo-M (Wang et al., Tetrahedron Lett. 37: 1975-1978); and Haneda et al., Carbohydr. Res. 292: 61-70 (1996)). Glycosidases useful in the invention include, but are not limited to, sialidase, galactosidase, endoglycanase, mannosidase, xylosidase, fucosidase, Agrobacterium sp. β- glucosidase, Cellulomonasfimi mannosidase 2A, Humicola insolens glycosidase, Sulfolobus solfataricus glycosidase and Bacillus licheniformis glycosidase.

Using glycosidase there are two possible reaction pathways:

1. Transglycosylation where the glycosidase uses a poly-saccharide such a di or tri-saccharide as substrate. This known enzymatic reaction may be described as:

(i) Levadopa aglycon + poly(n)-saccharide => Levadopa glycoside + poly(n-1 )-saccharide; or

(ii) Levadopa (x) glycoside + poly(n)-saccharide => Levadopa (x+1 ) glycoside + poly(n-1 )-saccharide.

2. Direct glycosylation where the glycosidase uses a mono-saccharide as substrate: This known enzymatic reaction may be described as:

(i) Levadopa aglycon + mono-saccharide => Levadopa glycoside

(ii) Levadopa (x) glycoside + mono-saccharide => Levadopa (x+1 ) glycoside.

In light of the present disclosure, the skilled person is able of identify a suitable glycosidase with respect to a particular reaction of interest. A preferred glycosidase is the almond β-glucosidase (E. C. 3.2.1.21 ) that can be obtained from Sigma.

Sugar donors

A preferred sugar donor is a nucleotide diphospho-sugar, a nucleotide monophospho-sugar or a sugar phosphate. Normally, the nucleoside is selected from adenosine, cytidine, guanidine, thymidine and uridine. In a preferred aspect, the nucleoside is uridine.

The chosen sugar donor leads to the formation of a selected type of glycoside. For example the choice of UDP glucose (i.e. uridine-diphosphate glucose) will give glucosides, UDP galactose will give galactosides and UDP xylose will give xylosides. All of these can have different practical applications as side groups of drug candidates since they differ in stability and in transport properties. Therefore different sugars can be chosen in connection with the assaying and in connection with the synthesis of glycoside derivatives of the compounds of formula I.

Preferably, the sugar donor comprises a monosaccharide selected from the group consisting of aldoses or ketoses. More specifically, the monosaccharide is a pentose, a hexose or a heptose. Normally, the monosaccharide is selected from the group consisting of glucose, xylose, arabinose, mannose, fucose, rhamnose, galactose, and apiose. In one embodiment, the monosaccharide is a sugar carboxylic acid selected from the group glucuronic acid, mannuronic acid, galacturonic acid. In another embodiment the monosaccharide is an amino sugar. More specifically the amino sugar is derived from a pentose , hexose or heptose. Normally, the amino sugar is selected from the group consisting of glucosamine, galactosamine, N-acetyl-D-glucosamine, mannosamine, mycaminose, 2,6- diamino-D-glucose.

As mentioned above, more than one sugar donor may be employed as described herein.

The sugar donor may be labeled in order to facilitate detection of the glycosylated substance. The label may be a radiolabel, or a fluorescent or phosphorescent label.

Improved bioavailability

Methods for assessing improved bioavailability of glycosides are known in the art. For example, that a compound according to the present invention has good bioavailability may be determined e.g., using a method based on the method described in WO 04/52841. Briefly, rats are pre-cannulated in the both the ascending colon and the jugular vein. Animals are conscious at the time of the experiment. All animals are fasted overnight and until around 4 hours post-dosing of levodopa (or a derivative thereof). Levodopa beta- glycoside is administered as a suitable solution (e.g. in water or citrate buffer)

either orally, or intraperitoneally or intracolonically at a suitable dose that corresponds to a clinical relevant dose for a specific levodopa (or derivative) of interest. Levodopa aglycon is administered in a comparative similar way. Blood samples are obtained from the jugular cannula at suitable intervals. Blood is then quenched with methanol/perchloric acid to prevent hydrolysis of the prodrug. Blood samples are analyzed by LC/MS/MS.

Intestinal Transport:

Methods for assessing intestinal transport of glycosides are known. For example, that a compound according to the present invention is transportable by an intestinal saccharide transporter may be determined e.g., using the Caco-2 cell culture model or a perfused rat intestinal model, based on the method described in Liu, Y. et al Drug Metab. Disp. 30/4 (2002) 370-377. The Caco-2 model is excellent for studying the mechanism of transepithelial transport but often lacks or poorly expresses phase I and phase Il enzymes. On the other hand, the rat perfusion model, an in situ model with intact circulation, is very suited to study regional absorption and metabolism but not secretory transport.

The transport experiment in the Caco-2 cell culture model is performed using a method based on that described in Hu, M. et al, J Drug Targeting 2 (1994) 79-89 and Hu, M. et al, Pharm Res 11 (1994) 1405-1413. Briefly, the cell monolayers are suitably washed. The transepithelial electrical resistance values are measured, and those with transepithelial electrical resistance values less than a selected value are discarded. The monolayers are loaded with a solution containing the compound of interest and the amount of compound transported transepithelially is followed as a function of time by HPLC.

Transport experiments in the perfused rat intestinal model is performed as

described in Hu, M. et al, J Theor Biol 131 (1988) 107-114 and Hu, M. et al, Pharm Res 12 (1995) 1120-1125. Briefly, after a suitable intestinal segment is cannulated, it is suitable washed. Samples are collected at different times. In general, steady-state transport is usually achieved within 30 min after the perfusion of a solution containing the compound of interest and e.g. PEG4000 (as a water flux marker) begin, and it is maintained throughout the experimental period. After perfusion, the length of the intestine is measured. The outlet concentrations of test compounds in the luminal perfusate (or perfusate) are determined by HPLC, and the radioactivity of labelled PEG4000 in the perfusate is determined by liquid scintillation spectrophotometry.

Liu, Y. et al Drug Metab. Disp. 30/4 (2002) 370-377 describes the intestinal absorption of genistein and its glycoside, (genistin or genistein-7-Oglucoside) in Caco-2 cell culture model and in a perfused rat intestinal model, where permeabilities of aglycones (e.g., genistein) were comparable to well absorbed compounds, such as testosterone and propranolol. In the Caco-2 model, permeabilities of aglycones were at least 5 times higher (p < 0.05) than their corresponding glycosides (e.g., genistin), and the vectorial transport of aglycones was similar (p > 0.05). Surprisingly, while other levodopa beta-glycosyl derivatives were shown not to have affinity for theGLUH receptor, glycosyl derivatives according to the present invention are shown in the Examples below to bind to the SGLT1 transporter.

Blood-brain barrier pass:

Assays for determining that a glycoside is capable of passing the blood-brain barrier are also known. For example, a test compound according to the invention may be injected intravenously into test mice and passage of the blood-brain-barrier may be evaluated by measuring a brain penetration index (BPI), wherein the amount of test compound measured per gram of brain

tissue is divided by the amount of test compound measured per gram of liver tissue. For comparison, the BPI value for gamma amino butyric acid (GABA) is about 1.0%.

Ex-Vivo assays for determining blood brain barrier transport are also known, e.g., Duport et al., Proc. Natl. Acad. Sci. 95/4 (1998) 1840-1845, using in vitro organ culture to measure the likelihood that a test compound will passage the blood brain barrier. Briefly, in the latter assay slices of selected brain regions are overlaid onto an endothelial cell monolayer in vitro and allowed to form tight junctions over the course of about 10 days of culture. Test compounds are then perfused into the endothelial side of culture and blood brain barrier penetration is detected by measuring the levels of the test compound which enter into the organ slice.

Stability of levodopa glycosides

Methods for assessing stability of glycosides are known and such methods may be applied to the levodopa derivatives of the present invention e.g., the derivatives can be tested using the method described in Fernandez et al. (Carbohydrate Research 327 (2000) 353-365, in particular p363).

Briefly, all incubations are carried at a suitable temperature, typically using a shaker or agitator. The levodopa beta-glycoside derivatives (eg, those described in example 2) are tested for stability by incubating them in the presence of specific enzymes or plasma. For example, bovine liver esterase (e.g. EC 3.1.1.1 ), alpha-chymotrypsin from bovine pancreas (e.g. EC 3.4.21.1 ) or β-glucosidase from almonds (e.g. EC 3.2.1.21 ) are commercially available (eg, Sigma) and can be used for the stability assays.

Enzyme stability studies

Esterase.

To a solution of a levodopa beta-glycosyl derivative in suitable buffer preincubated at suitable temperature, suitable amounts of buffer or an esterase solution in buffer, are added. Aliquots are removed and immediately analysed by HPLC.

Protease.

To a solution of a levodopa beta-glycosyl derivative in a suitable buffer, are added suitable amounts of buffer or an enzyme solution in buffer. Aliquots are removed and analysed by HPLC.

Glucosidase.

A levodopa beta-glycosyl derivative is dissolved in a suitable buffer at a suitable concentration. To this solution, suitable amounts of buffer or of a glucosidase solution in buffer are added. The samples are incubated and analysed by HPLC.

Plasma stability

A levodopa beta-glycosyl derivative can be tested for stability in plasma by incubation of the derivative with a plasma preparation from rat or human blood. The progress of the incubations are monitored by high-performance liquid chromatography (HPLC) using a reverse-phase column of aliquots removed from incubation at various time intervals.

Stability of the levodopa beta-glycosides of the present invention are more stable under physiological conditions comparable to the conditions in blood, than the levodopa aglycon. In blood, the levodopa aglycon is rapidly

decarboxylated to dopamine resulting in undesired side effects. The levodopa glycosides as described herein are more stable and do not exhibit dopamine- like side effects because of the glycoside groups. In addition, decarboxylation is significantly reduced when the glycoside protects the levodopa carboxyl group, i.e. a glycoside is at the R ₂ position. Some deglycosylation at this site may occur but the pharmaceutically active component is significantly stabilized. Dopamine is known to have some unwanted side-effects outside the brain such as in the blood. Accordingly, it is an advantage that the levodopa beta-glycosides as described herein are more stable in e.g. the blood.

Bioavailability to exert physiological effect

Although not wishing to be bound by theory, delivery of the Levodopa glycoside derivative across the blood brain barrier may be expected to be via glucose transporters (given the affinity of the glycoside derivatives of the present invention for glucose transporters, such as SGLT1 ) and/or via amino acid transporters (eg, phenylalanine transporter, which is thought to be a transporter of levodopa). Once in the brain, the glycosyl levodopa derivative is activated by a brain glycosidase, in order to release levodopa into the brain and to allow levodopa to exert its physiological effects.

To test the ability of brain tissue to deglycosylate the levodopa glycoside derivatives, the derivatives are therefore incubated with brain extract and deglycosylation monitored.The test is carried out essentially as follows (see also Fernandez et al., Carbohydrate Research 327 (2000) 353-365, in particular p363, second column). Briefly, a rat brain is removed after incision of the skull, and is homogenated under suitable conditions. Samples of the brain extract are incubated with the levodopa glycosyl derivative and at various time intervals, aliquots are removed and treated under suitable conditions. See Example 6 below.

Dopaminergic activity:

Methods for determining that a glycoside derivative according to the present invention is dopaminergic, e.g., the compound is activated by a glycosidase in order to release levodopa and then the levodopa is transformed to dopamine which is capable of binding a dopamine receptor, may be determined according to methods known in the art, e.g. using a method essentially as described in Fernandez et al. (Carbohydrate Research 327 (2000) 353-365).

Briefly, mice are housed in groups of e.g. 12 (mice) under regulated conditions in standard e.g. Makrolon cages (215 x 465 x 145 mm). The animals receive standard laboratory chow and tap water ad lib until the beginning of the experiments. Post-synaptic D2 receptor antagonist activity is confirmed by increased locomotor activity in reserpine-treated mice. Experimental animals are pretreated with suitable amounts of reserpine intraperitoneally, e.g. 18 h prior to experiments. Locomotive activity, rearing and velocity, are measured always at the same time of day, to avoid variation due to circadian rhythms, with a video computerised animal observation system. Activity (total distance travelled in cm), rearing (changes upper 15% in corporal surface) and velocity, are recorded in four square test arenas (50 x 50 x 30 cm) via a video camera fixed on the ceiling above the arenas. Animals are tested for e.g. 1 h and the recorded information is relayed to a monitor and a video tracking motion analysis.

Derivatives:

The levodopa glycosides may be converted into derivatives thereof, which, within a pharmaceutical composition as described herein, are all covered by the present invention. The derivatives can be prepared by standard procedures known to one skilled in the art.

Generally, a derivative of levodopa denotes herein a derivative that at least is capable of triggering a response in vivo that in essence corresponds to the response in vivo of levodopa. Levodopa is a dopamine precursor, dopamine being a neurotransmitter that binds to dopamine receptors in the brain. A derivative of levodopa denotes herein a derivative that is capable of releasing levodopa and/or dopamine in the brain. The skilled person is perfectly capable of making e.g. minor structural modifications of the levodopa moiety of the levodopa glycoside derivative to produce a derivative that fulfils the criteria above.

Pharmaceutical product:

The pharmaceutical product may be provided as: (i) a container comprising a composition comprising an effective amount of levodopa beta-glycoside derivative or derivative thereof and a pharmaceutically acceptable carrier;

(ii) instructions for administration of the compositions for use in a method for metabolic replacement therapy or prevention of Parkinson's disease or Parkinson's related diseases or associated clinical symptoms in a human person.

"Metabolic replacement therapy", as used herein, is intended to mean that the levodopa beta-glycoside, when administered in the pharmaceutical composition, is effective, following transport into a neural cell, to satisfy one or more metabolic requirements of catecholamine synthesis in the neural cell of a subject having a nigrostriatal dopamine insufficiency.

The term "container" should be understood broadly as some kind of physical item. It could e.g. be a tablet, an injector, a capsule, an inhaler etc.

Accordingly to an embodiment of the second aspect of the invention, the pharmaceutical composition also comprises at least one beta-glycosidase inhibitor. The amount of the glycosidase inhibitor should be an amount that is sufficient to decrease hydrolysing of the levodopa beta-glycoside in the intestine. Non-limiting examples of suitable examples of beta-glycosidase inhibitors are N-(n-butyl)-deoxygalactonojirimycin ("Absorption of quercetin-3- glucoside and quercetin-4-glucoside in the rat small intestine: the role of lactase phlorizin hydrolase and the sodium-dependent glucose transporter."; Andrea J. Day et al. Biochemical Pharmacolcogy 65 (2003) 1199-1206.); or d- gluconolactone (commercially available).

In some embodiments, the pharmaceutical composition also comprises at least one glycosidase. Non-limiting examples of suitable glycosidases are described in the section "glycosidase" herein (see above).

In a further embodiment, the pharmaceutical composition may comprise an inhibitor of enzymes that metabolize levodopa, such as an inhibitor of a dopa-decarboxylase (eg carbidopa or benserazide). Alternatively, stability of the levodopa can be achieved chemically as described above and derivatives such as alkyl esters.e.g., methyl esters of the levodopa carboxyl group.

The invention provides pharmaceutical compositions containing one or more of the levodopa beta-glycoside or derivative thereof (with or without a glycosidase inhibitor or with or without a glycosidase), their stereoisomers, pharmaceutically acceptable salts or pharmaceutically acceptable solvates, in combination with optional stabilizers, carriers, binders, buffers, excipients, emollients, disintegrants, lubricating agents, antimicrobial agents and the like. For oral administration, the instant pharmaceutical compositions may be liquid, solid or encapsulated. For parenteral administration, the instant pharmaceutical compositions may be sterile liquids or solids may be provided in a form suitable for reconstitution, e.g., powdered or granulated.

Preferably, the composition comprises from 50 mg to 1000 mg of levodopa glycoside or derivative thereof, more preferably 100 mg to 400 mg, depending on the levodopa glycoside or derivative thereof used.

By "pharmaceutically acceptable carrier" as used herein is meant one or more compatible solid or liquid filler diluents, or encapsulating substances. By "compatible" as used herein is meant that the components of the composition are capable of being commingled without interacting in a manner which would substantially decrease the pharmaceutical efficacy of the total composition under ordinary use situations.

The levodopa glycosides may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers may include inert solid diluents or fillers, sterile aqueous solutions, and various nontoxic organic solvents. The pharmaceutical compositions formed by combining the instant compound with the pharmaceutically acceptable carrier may then be readily administered in a variety of dosage forms such as tablets, lozenges, syrups, injectable solutions, inhalants and the like. These pharmaceutical carriers can, if desired, contain additional ingredients such as flavourings, binders, excipients, and the like.

Thus, for purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate, and calcium phosphate may be employed along with various disintegrants such as starch, and preferably potato or tapioca starch, alginic acid, and certain complex silicates, together with binding agents such as polyvinylpyrolidone, sucrose, gelatin, and acacia. Additionally, lubricating agents, such as magnesium stearate, sodium lauryl sulphate, and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed as fillers in salt and

hard-filled gelatin capsules. Preferred materials for this purpose include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions of elixirs are desired for oral administration, the instant compound therein may be combined with various sweetening or flavoring agents, colored matter or dyes, and if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof. For parenteral administration, solutions of the instant compound in sesame or peanut oil or in aqueous polypropylene glycol may be employed, as well as sterile aqueous saline solutions of the corresponding water-soluble pharmaceutically acceptable metal salts previously described. Such an aqueous solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal injection. The sterile aqueous media employed are all readily obtainable by standard techniques well known to those skilled in the art.

It may prove desirable to stabilize the instant compounds e.g. to increase their shelf life and/or pharmacokinetic half-life. Shelf-life stability may be improved by adding excipients such as: a) hydrophobic agents (e.g., glycerol); b) non-linked sugars (e.g., sucrose, mannose, sorbitol, rhamnose, xylose); c) non-linked complex carbohydrates (e.g., lactose); and/or d) bacteriostatic agents. Pharmacokinetic half-life of the instant compounds varies depending upon the pyranosyl or furanosyl moiety selected, whether the saccharide units therein are multimeric, whether the multimer constitutes an oligosaccharide, and whether the multimers or oligosaccharide are derivatized, i.e., chemically modified by methylation, sulphation, nitration and the like. Pharmacokinetic half-life and pharmacodynamics may also be modified by: a) encapsulation (e.g., in liposomes); b) controlling the degree of hydration (e.g., by controlling the extent and type of saccharide units); and, c) controlling the electrostatic charge and hydrophobicity of the saccharide

units.

Wetting agents, fillers and lubricants such as sodium lauryl sulphate, as well as colouring agents, flavoring agents, lubricants, excipients, tabletting agents, stabilizers, anti-oxidants and preservatives, can also be present.

This list is not meant to be exclusive, but instead merely representative of the classes of excipients and the particular excipients that may be used in preferred dosage forms of the present invention.

A pharmaceutical product as described herein can be administered orally, transdermally, parenterally, intramuscularly, intravenously, subcutaneously, by inhalation or by other modes of administration. Preferably, the pharmaceutical product can be administered orally.

A pharmaceutical product, as described herein, may include other pharmaceutically active substances. It can be prepared by mixing the active compounds with one or more pharmacologically tolerated auxiliaries and/or excipients such as, for example, fillers, emulsifiers, lubricants, masking flavours, colorants, or buffer substances, and converting the mixture into a suitable pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules, powders, emulsions, suspensions, or solutions.

Examples of auxiliaries and/or excipients which may be mentioned are tragacanth, lactose, talc, agar, polyglycols, ethanol, and water. It is also possible to administer the active substances as such, without vehicles or diluents, in a suitable form, for example, in capsules.

Preferably, the pharmaceutical product, as described herein, is provided together with suitable pharmaceutically relevant instructions. The instructions preferably explain pharmaceutically relevant information such as e.g.

qualitative and quantitative of composition, pharmaceutical form, therapeutic indications and method of administration (including recommend doses).

Use for the treatment of clinical symptoms:

Accordingly to the third aspect of the invention, the levodopa glycosides or derivatives thereof according to the invention, may advantageously be used to treat a variety of pathological central and peripheral nervous system dysfunctions, neuromotor conditions and cardiovascular diseases and associated clinical symptoms in subjects in need of treatment.

In a preferred embodiment, the subject conditions include, but are not limited to, i) toxic dystrophy, (e.g., chemical or drug-induced secondary dystrophy in the nervous system), ii) vascular impairment e.g. resulting in damage to nervous tissues, iii) central nervous system degeneration or peripheral nerve degeneration, iv) nervous system lesions induced by physical trauma, v) nervous system complications of illnesses and infections (e.g., viral or bacterial); and vi) hereditary nervous system impairment. Representative illness, diseases, and conditions having neurologic dysfunction have been classified and codified ("International Classification of Diseases, Washington D.C., 1989).

In line of above, a more preferred embodiment, the subject conditions are Parkinson's disease, a Parkinson's related disease or associated clinical symptoms.

Representative examples of subjects in need of treatment may include humans and domestic animals having e.g., a condition of hyper- or hypo- dopaminergic activity, such as may be evident in a patient with schizoprenia, Parkinson's disease, epilepsy, locomotor deficiency, hyperprolactinemia, Tourette's syndrome, Huntington's disease, psychosis, chronic psychiatric

illness with amotivation, apathy, asociality, psychomotor adverse effects of drugs of abuse (e.g., cocaine, amphetamine, neuroleptics), subolivopontocerebellar atrophy (sOPCA), multiple system atrophy (MSA), bipolar disorder, chronic alcoholism, cocaine abuse, mood disorders, attention deficit disorder, physiologic stress, pesticide exposure (e.g., organochlorine insecticides), juvenile neuronal ceroid lipofuscinosis (JNCL), detached personality syndromes (as e.g. determined using the Karolinska Scales of Personality questionnaire) and the like. Representative examples of conditions exhibiting hyper-dopaminergic activity include schizophrenia, chronic psychiatric illness with hallucinations and delusions. Also representative are, patients with coronary hypertension, angina, ischemic myocardium and the like. In addition, prophylactic methods are envisaged for lowering aortic and pulmonary artery pressure during and after coronary bypass surgery and liver, kidney and heart transplant surgery. Vasodilation mediated by the instant compounds is without impairment of oxygen delivery or impairment of intrinsic neural or hormonal control systems.

The pharmaceutical product can be administered in multiple doses per day, if desired, to achieve the total desired daily dose. Typically, the human will be treated over the course of several months or years, or even life-long to ameliorate the signs and symptoms. In a typical therapeutic regimen, the pharmaceutical product is administered to the human at least once daily (e.g., orally, subcutaneously, or delivered by transdermal, nasal or depot methods) for at least days, determined by those skilled in the art.

"Parkinson's related disease", as used herein, is intended to mean a disease characterized by one or more symptoms which are also evidenced clinically in a patient with Parkinson's disease. Representative examples of symptoms evidenced in patients with Parkinsonism include seizure, loss of neuromotor control of muscle movements, tarditive dyskinesia, Alzheimer's disease, Wilson's disease, post-encephalitic syndromes, Parkinsonism secondary to

trauma and stroke, dementia, Lou Gehrig's disease, psychomoter retardation, schizophreniform behavior, anxiety and depression. Clinical features of Parkinson's related diseases are disclosed in Hurtig, H. I., Expert. Neurol. 144 (1997) 10-16.

Diseases related by clinical symptomology, and progressive clinical symptomology in Parkinson's patients, include post-encephalitic syndromes, Wilson's disease, Parkinsonism secondary to cerebrovascular trauma and stroke, dementia, Alzheimer's disease, Lou Gehrig's disease, psychomotor retardation, certain schizophreniform behavior, anxiety and depression.

In order that this invention may be better understood, the following examples are set forth to illustrate various aspects of the present invention. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention or the scope of the claims in any manner.

All publications referred to herein are incorporated by reference in their entirety, as if each publication were referred to individually.

EXAMPLES:

Example 1 : In vitro method for enzymatic synthesis of levodopa glycosides

Enzymatic glycosylatioin of levadopa is essentially carried out as described by Hansen et al., Phytochemistry 64 (2003) 143-151. Briefly, Levodopa or its methylester (for example, when glycosylation at a site other than the carboxyl group is desired) is incubated with a suitable glycosyltransferase (eg UGT89B1 ). The resulting glycoside may be qualitatively analyzed by thin layer chromatography (TLC) and quantitatively analyzed by LC-MS.

Recombinant protein and enzyme assays

Heterologous expression and subsequent isolation of glycosyltransferase from Sorghum bicolour termed UGT89B1 and enzyme assays are performed in a suitable reaction volume (depending on the amount of desired product) essentially based on the method described by Jones et al., J. Biol. Chem. 249, 5826-5830 (1999). Glycerol can be added to the isolated pure enzyme, as this results in improved enzyme activity compared to when glycerol is absent. Due to their lability, the aglycon and enzyme are added last to the reaction mixture.

TLC

Reaction mixtures from qualitative and quantitative assays can be applied to Silica Gel 60 F254 plates (Merck), dried and developed in a suitable solvent system. Plates are dried and expose to phosphoimaging screens (Molecular Dynamics).

Substrates

Levodopa and levodopa methylester are commercially available (eg Sigma).

LC-MS

Assays are performed essentially as outlined for qualitative assays, except that crude E. coli extracts expressing either UGT89B1 or transformed with control vector lacking the UGT89B1 coding sequence are optionally employed instead of isolated pure protein. Likewise, unlabelled UDP-glucose is included in each assay. Finally, the enzymatic reaction is stopped and the supernatant obtained after centrifugation is subjected to LC-MS.

Results:

The following levodopa beta-glycoside derivates are made and isolated (Formula I is as described herein):

(A): R ₃ is β-glucose and R ₁ , Rr, R ₄ and R ₅ are H, and R ₂ is -COOH. (B): R ₄ is β-glucose and R ₁ , Rr, R ₃ and R ₅ are H, and R ₂ is -COOH.

Example 2: Chemical synthesis of glycoside derivatives of levodopa or levodopa methylester

This example describes the chemical synthesis of mono- and diglycosylated L-DOPA derivatives (numbers refer to scheme 1 shown below). Mono- and di- glucosylated L-DOPA derivatives 11 , 12 and 13 were prepared from N-benzyloxycarbonyl-L-DOPA benzyl ester (1 , prepared from commercially available L-DOPA as described in T.R. Burke Jr. et al, Tetrahedron 54 (1998) 9981-9994, and commercially available 1 ,2,3,4,6- penta-O-acetyl-D-beta-glucopyranoside (2, "glucose pentaacetate"). The following steps were carried out:

N-benzyloxycarbonyl-L-DOPA benzyl ester (1) and 1 ,2,3,4,6-penta-O- acetyl-beta-D-glucopyranoside (2) were reacted at room temperature in dry toluene using boron trifluoride etherate as promotor. A mixture of meta- glucosylated derivative 3, para-glucosylated derivative 4 and di-glucosylated derivative 5 was obtained in a total yield of about 60% and a 45:45:10 ratio. This mixture proved difficult to separate into its pure components, therefore the crude mixture was O-acetylated with pyridine-acetic anhydride to yield a mixture of 5, 6, and 7. From this mixture, it was possible to obtain some pure para-glucosylated derivative 7 by crystallization. The mother liquor from the crystallization was evaporated and purified by repeated silica gel chromatography to give more 7 and the two other desired products, 5 and 6.

Catalytic hydrogenation of 5, 6 and 7 with palladium on charcoal in acetic acid followed by treatment with sodium methoxide in methanol gave the final products 11 , 12 and 13 in 80-95 % yield, isolated as off-white powders by precipitation from hot isopropanol. Scheme 1 :

As is apparent to those of skill in the art the above procedure may be modified to produce the desired product, for example by varying reaction times or conditions (eg, by varying the glycoside used).

Example 3: Intestinal Transport of levodopa glycoside derivates

The principal objective of this study is to assessing intestinal transport of levodopa glycosides.

The levodopa beta-glycoside derivates made and isolated as described in example 2 are tested in a Caco-2 cell culture model similar to that described in the section entitled "Intestinal Transport" hereinabove. The control is a glycoside known to cross the Caco-2 cell epithelium.

Briefly, the monolayers are placed in an experimental setup with an "apical chamber" and a "basolateral chamber". To asses transepithelial transport, the "apical chamber" is loaded with 100 μM or 4.5 mM α-methyl-D-glucose (MDG), and the amount of α-MDG transported is determined by sampling from the "basolateral chamber at time intervals followed by appropriate HPLC analysis. To address the affinity of the α-MDG transporter for L-DOPA- glucosides, each is added in combination with α-MDG in equimolar amounts, and inhibition of α-MDG is taken as evidence of affinity of the transport molecule for the added glucoside in question.

Both L-DOPA-4-O glucoside and L-DOPA-3-O glucoside have a significant inhibition of α-MDG transport. In comparison to the control methylglucose having 100 arbitrarily assigned affinity units for the transporter, L-DOPA-3-O glucoside had about 75 affinity units and L-DOPA-4-O glucoside about 25 affinity units.

The tests show that levodopa beta-glycoside derivates have an unexpected affinity for the α-MDG transporter (SGLT1 ), indicating the potential of trans epithelial transport, such as in the intestine.

The levodopa glycoside derivates made and isolated as described in example 2 are also tested in perfused rat intestinal model as described in section "Intestinal Transport" hereinabove. The control is levodopa aglycon.

The levodopa beta-glycoside derivates have an improved Intestinal Transport as compared to the levodopa aglycon. When the saccharide is a β-glycosyl saccharide, the resulting levodopa β-glycosyl derivatives are transported more efficiently than α-glycosyl derivatives.

Example 4: Stability of levodopa glycosides

A levodopa beta-glycosyl derivative is tested for stability in plasma by incubation of the derivative with a plasma preparation from rat or human blood. The progress of the incubations are monitored by high-performance liquid chromatography (HPLC) using a reverse-phase column of aliquots removed from incubation at various time intervals.

The tests show that under physiological conditions comparable to the conditions in blood the levodopa beta-glycosides are more stable than the levodopa aglycon. Stability of the levodopa glycoside derivatives are important either to increase the circulating amount of aglycon to thereby drive transport across the blood brain barrier or to allow more delivery of the prodrug across the blood brain barrier.

Example 5: Blood-brain barrier test of levodopa glycoside derivatives

The principal objective of this study is to establish that levodopa glycosides as described herein are capable of passing through the blood-brain barrier. The levodopa glycoside derivates made and isolated as described in example 2 are tested in a blood-brain barrier test as described in the section entitled "Blood-brain barrier test" hereinabove. The control is levodopa aglycon. The tests show that all of the levodopa beta-glycoside derivates are capable of passing over the blood-brain barrier.

Example 6: Improved bioavailability

The principal objective of this example is to establish that the levodopa glycosides as described herein provide improved bioavailability of levodopa in the brain.

The levodopa beta-glycoside derivates made and isolated as described in example 2 are tested by incubating with a sample of brain homogenate. Briefly, a brain homogenate with β-glycosidase activity is obtained as described by Withers and co-workers (Biochem. J., 301 (1994) 343-348). β- Glucosidase activity of the homogenate is measured and expressed as nmol of p-nitrophenyl β-D-glucopyranoside hydrolysed per h and per mg of tissue. Tissue homogenates are frozen and stored at a suitable temperature, typically -70C or in liquid nitrogen. 300 μl of a solution of levodopa beta- glycoside is added to the brain homogenate and incubated at 37 ⁰ C. At various time intervals, 50 μl aliquots are removed and combined with one volume of methanol, so as to precipitate macromolecules. The samples are centrifuged for 5 min at 16.1xg. Supematants are then analyzed by LC-MS to separate the glycosylated L-DOPA from L-DOPA. The results indicate that L- DOPA-3-OH-glucoside and L-DOPA-4-OH-glucoside are slowly de- glycosylated by beta-glucosidase present in the rat brain. This is a highly desirable effect to allow sustained release of levodopa in the brain and resulting in a more constant physiological effect.

Example 7: In Vivo Effect of levodopa glycosyl derivatives

The principal objective of this study is to determine an in vivo effect of levodopa beta-glycosides in a Parkinsons disease model.

Briefly, female NMRI mice (BOM, DK), weight 22-25g, house grouped, with free access to food and water were used. The mice were habituated to the animal facility for one week with a light/dark cycle of 7:00AM/7:00PM.

Mouse Locomotor Activity was assessed using the ActiMot system (TSE systems, DE) with standard home cages. The standard frame features a grid of 2 x 6 infra-red sensor pairs whose interruptions are used to monitor locomotor activity inside the cage.

Central stimulant effects, such as sniffing, stimulated activity and stereotypy are recorded by visual observation.

3-gly-O-levodopa and 4-gly-O-levodopa were tested in the mice. After ip administration of 100, 300 or 1000 mg/kg of either glycoside derivative, no change in normal locomotor behaviour was induced. After iv administration of 3-gly-O-levodopa, some enhanced vigilance (increase in rearings and locomotor activity) was observed from 30-240 minutes after administration (300 and 1000 mg/kg iv). However, the mice were grooming normally, did not show any sign of stereotypy and were sleeping normally together from 4-6 hours after iv administration under the conditions used. Twenty four hours after iv administration the mice were climbing, eating and showing normal behaviour.

The mice can be euthanized by cervical dislocation and brain tissue removed for analytical chemical determination of L-DOPA concentration in whole brain

as well as of any remaining glycosylated derivatives.

Table 2 Summary of Mouse activity after iv admin., 3-gly-O-levodopa.

(Mot- motility; Red mot- reduced motility)