TREATMENT OF INFLAMMATION AND IMMUNE-RELATED DISORDERS

FIELD OF THE INVENTION

The present invention relates to the use of metadoxine and derivatives thereof in the treatment of inflammatory and immune-related disorders. Particularly, the present invention provides the use of metadoxine and derivatives thereof, as an antiinflammatory agents and/or as an immune-modulators, when comprised in sustained-, controlled-or delayed-release formulations, which may be combined with immediate- release treatment.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.

Metadoxine (pyridoxol L-2-pyrrolidone-5-carboxylate) is an ion-pair salt of pyridoxine (vitamin B6) and pyrrolidone carboxylate (PCA). It has been used in the treatment of acute alcohol intoxication and in the treatment of alcohol withdrawal syndrome. Over 40 years of clinical use have shown metadoxine to be safe and effective as a blood alcohol reducing agent, and in the treatment of alcoholic steatosis [see e.g., Sung Hwan Ki et al. (2007) Chem-Biol. Interac. 169:80-90; WO 08/066353; Calabrese et al. (1995) Int. J. Tiss. Reac. XVII(3): 101-108; Arosio et al. (1993) Pharmacol. Toxicol. 73: 301304; Annoni et al. (1992) Pharmacol. Res. 25(l):87-93].

Several studies have reported the use of metadoxine in the treatment of liver diseases, such as Non-alcoholic Steatohepatitis (NASH) [see, e.g., Feher and Lengyel (2003) J. Intern. Med. Res. 31 :537-551; Kadayifci et al. (2007) Clin. Liver Dis. 11 :119- 140]; or chronic hepatitis [see e.g., Cacciatore et al. (1988) Clin. Trials. J. 25(3):220- 226].

These publications describe human studies, or animal studies, either standard animals [e.g. WO 08/0663531, or, for example, the CCI4 model [Arosio et al., id ibid.]. Studies in established "inflammatory" models, have not been described, and thus the effect of metadoxine in the diseases described in the prior art cannot be attributed to prevention of inflammation, but may be due to reducing, e.g., necrosis by changing toxic conditions, or other effects, which would not necessarily be relevant to other inflammatory or immune-related conditions.

Concanavalin A (Con A), a plant lectin and T cell mitogen, rapidly induces severe immune-mediated hepatitis in mice, which is associated with increased TNFa, IFNy, IL-12, IL-18, and IL-4 expression, and in which NKT lymphocytes, CD4+ T cells and Kupffer cells have a contributory role. It is manifested by a robust increase of cytokines and liver damage. Together with TNFa, IFNy is considered to have a key role in the pathogenesis of the immune-mediated damage in this model. In addition, the IFNy-inducing cytokines, such as IL-12 and IL-18, and macrophage inflammatory protein-2 (MIP-2) contribute to the liver injury in this model, whereas IL-6, IL-11 and IL-10 may have a protective role [Gantner F. et al., Exp Cell Res (1999) 229:137-46; Knolle PA et al., Hepatology (1996) 24:824-9; Koerber K et al., Hepatology (2002) 36:1061-9; Kunstle G. et al., Hepatology (1999) 30:1241-51; Sass G et al., Cytokine (2002) 19:115-20; Tiegs G., Acta Gastroenterol Belg (1997) 60:176-9; Tiegs G. et al., Exp Toxicol Pathol (1996) 48:471-6; Tiegs G. et al., J. Clin. Invest. (1992) 90:196-203; Trautwein C. et al., J. Clin. Invest. (1995) 101 :1960-9].

Both CD4 and CD8 lymphocytes can be typed as either Thl cells that produce IL-2 and IFNy, or Th2 cells that produce IL-4, and IL-10. The way the immune system responds to foreign and self antigens is the result of a balance between the two subtypes of responses [Weiner, H.L., et al., Immunol. Today 18: 335-343 (1997); Adorini, L., et al., Immunol. Today 18:209-211 (1997)]. A Thl type response is involved in the pathogenesis of several autoimmune and chronic inflammatory disorders [Adorini, L., et al., (1997) ibid.; Mizoguchi, A., et al., J. Exp. Med. 183:847-856, (1996)]. Thus certain inflammatory disorders in humans can be perceived as an imbalance between proinflammatory Thl -type and anti-inflammatory Th2-type cytokines. It has been recently shown, in both animals and humans, that anti-inflammatory cytokines such as IL10 can down regulate the pro-inflammatory effects of Thl -mediated cytokines, thereby alleviating immune-mediated disorders [Mizoguchi, A., et al., (1996) ibid.; Madsen, K.L., et al., Gastroenterology 113:151-159 (1997); Van Deventer Sander, J., et al., Gastroenterology 113:383-389 (1997)].

The Con A treated mouse can serve as a murine model for immunemediated, T cell-or cytokine-mediated disorders, and has been previously described, e.g., in WO 02/051986. The present inventors employed the Con A mouse model to study the antiinflammatory and immune modulating effect of metadoxine, particularly when contained in special formulations and dosage forms. SUMMARY OF THE INVENTION

In a first aspect, the present invention provides the use of metadoxine in the preparation of a pharmaceutical composition for the treatment of an immune-related disease, wherein said composition is formulated in a sustained-, delayed- or controlled- release dosage form.

In another aspect the invention provides a use of a salt adduct comprising:

a positively charged moi

wherein

Ri is straight or branched CpC alkyl;

R ₂ is selected from -OH, straight or branched Ci-C ₆ alkoxy, and straight or branched Ci-C ₆ alkoxy carbonyl;

R.3 and R4 are each independently selected from formyl, straight or branched Cj-Q alkyl optionally substituted by at least one halogen, amine, hydroxyl, Cj-C ₆ alkoxy, thiol, C]-C alkoxycarbonyl; and

- a negatively charged carboxylated lactam ring selected from the group consisting of:

(III) and

wherein

R is selected from H, straight or branched C C ₆ alkyl optionally substituted by at least one halogen, straight or branched C ₂ -C ₆ alkenyl, straight or branched C ₂ -C alkynyl, cycloalkyl, aryl and heteroaryl optionally substituted by a C!-C ₆ alkyl;

R ₇ , R ₈ , R ₉ , R ₁₀ Rn, Ri2, Rn and R ₁₄ are each independently selected from H, straight or branched C[-C alkyl, C ₂ -C ₆ alkenyl, C ₂ -C alkynyl, cycloalkyl, aryl and heteroaryl optionally substituted by at least one group selected from C _\ -C alkyl, halogen, amino, cyano, nitro, thiol, Cj- C ₆ alkoxy, aminocarbonyl, C C alkoxycarbonyl, Ci-C ₆ carboxyalkyl, Q-Q alkoxycarbonylalkyl and amidino;

for the preparation of a pharmaceutical composition for the treatment of an immune-related disease, condition or disorder.

In some embodiments, said carboxylated lactam ring is a compound of formula

(II):

(II) and

said positively charged moiety is compound (2): In am ring is compound (1):

said positively charged moiety is a compound of formula (I):

In further embodiments R _\ is a C C ₆ alkyl and R ₂ , R ₃ and R4 are as defined above. In other embodiments, R ₂ is selected from-OH and C _\ -C alkoxy; and R _l5 R ₃ and R4 are as defined hereinabove. In yet further embodiments, R ₃ is -CH ₂ R ₁₅ , wherein R ₁₅ is selected from -C{-Ce alkoxy, -OH and -NH ₃ ⁺ ; and R _l5 R ₂ and R4 are as defined hereinabove. In further embodiments, R4 is selected from formyl and -CH ₂ R ₁₆ , wherein R ₁₆ is selected from -Ci-Ce alkoxy and -OH; and Ri, R ₂ and R ₃ are as defined hereinabove.

In some embodiments of the invention, said carboxylated lactam ring is a compound of formula (II):

In some embodiments wherein said carboxylated lactam ring is a compound of formula (II), Re is Cj-C ₆ alkyl. In further said embodiments, R ₉ is CrC ₆ alkyl.

In other embodiments of the invention, said carboxylated lactam ring is a compound of formula (III):

III) and

said p ompound of formula (I):

In other embodiments of the invention, said carboxylated lactam ring is a compound of formula (IV):

said positively charged moiety is a compound of formula (I):

I).

In some embodiments wherein said carboxylated lactam ring is a compound of formula (III) or (IV), said positively charged moiety is compound (2):

.

In some embodiments of the invention, said salt adduct is selected from following group:

1)

In one specific embodiment, said composition is formulated in a sustained-, delayed- or controlled-release dosage form combined with an immediate or burst effect dosage form.

In particular, said composition is formulated to deliver metadoxine or salt adduct defined herein above, up to 0.1-1000 mg/kg body weight/day, preferably 1-400 mg/kg body weight, in a single dose administration or portion thereof, wherein said composition is formulated for enteral or parenteral administration. Said enteral administration may be oral or rectal administration. Alternatively, said parenteral administration may be by intramuscular, intraperitoneal, intratechal or intravenous, or sublingual, buccal, nasal, transdermal, transmucosal, or subcutaneous mode.

In another specific embodiment, said pharmaceutical composition may further comprise at least one additional pharmaceutically active agent.

In a further specific embodiment, said immune-related disease is an autoimmune disease, graft rejection pathology, inflammatory disease, non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, or Metabolic Syndrome or any of the conditions comprising the same.

In another embodiment, said immune-related disease is an autoimmune disease, graft rejection pathology, inflammatory disease, hyperlipidemia, atherosclerosis, or Metabolic Syndrome or any of the conditions comprising the same. In another aspect, the present invention provides a method of treating an immune-related disease in a subject in need thereof, said method comprising the step of administering a therapeutically effective dosage of metadoxine, or a composition comprising thereof, to said subject.

In a further aspect the invention provides a method of treating an immune- related disease in a subject in need, said method comprising administering a therapeutically effective dosage of a salt adduct or a composition comprising thereof, to said subject, wherein said salt adduct comprises: a positively charged moiety of formula (I), as defined herein above; and a negatively charged carboxylated lactam ring selected from the group consisting of: compounds of formulae (II), (III) and (IV), as defined herein above.

In one embodiment of the method of the invention, said metadoxine or salt adduct defined herein above,, or composition comprising the same, is formulated in a sustained-, delayed- or controlled-release dosage form, which may optionally be combined with an immediate or burst effect dosage form.

In another particular embodiment of said method, said metadoxine or salt adduct defined herein above,, or composition comprising the same, is formulated in a dosage form to deliver 0.1-1000 mg/kg body weight/day, preferably 1-400 mg/kg body weight metadoxine or salt adduct defined herein above,, in a single dose administration or portion thereof.

In certain embodiments, said composition is formulated for enteral or parenteral administration, wherein said enteral administration is oral or rectal administration, whereas said parenteral administration is by intramuscular, intraperitoneal, intratechal or intravenous, or sublingual, buccal, nasal, transdermal, transmucosal, or subcutaneous mode.

In an optional embodiment, said pharmaceutical composition further comprises at least one additional pharmaceutically active agent.

In a particular embodiment of the method of the invention, said immune-related disease is selected from the group consisting of an autoimmune disease, graft rejection pathology, inflammatory disease, non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, Metabolic Syndrome and any conditions, syndromes, disorders or diseases comprising the same.

In a particular embodiment of the method of the invention, said immune-related disease is selected from the group consisting of an autoimmune disease, graft rejection pathology, inflammatory disease, hyperlipidemia, atherosclerosis, Metabolic Syndrome and any conditions, syndromes, disorders or diseases comprising the same.

The present invention further provides a kit for use in treating an immune- related disease in a subject in need thereof, said kit comprising: (a) metadoxine or a composition comprising the same; (b) means for administering said metadoxine or composition comprising the same; and (c) instructions for use.

The invention further encompasses a kit for treating an immune-related disease in a subject in need, said kit comprising: (a) salt adduct or a composition comprising the same; (b) means for administering said salt adduct or composition comprising the same; and (c) instructions for use; wherein said salt adduct comprises: a positively charged moiety of formula (I), as defined herein above; and a negatively charged carboxylated lactam ring selected from the group consisting of: compounds of formulae (II), (III) and (IV), as defined herein above.

In one embodiment of said kit, said metadoxine or salt adduct defined herein above, or composition comprising thereof is provided in a sustained-, delayed- or controlled-release dosage form.

In a particular embodiment, said composition is formulated in a sustained-, delayed or controlled-release dosage form combined with an immediate or burst effect dosage form.

In another particular embodiment of said kit, said immune-related disease is an autoimmune disease, graft rejection pathology, inflammatory disease, non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, Metabolic Syndrome or any conditions, syndromes, disorders or diseases comprising the same.

In another particular embodiment of said kit, said immune-related disease is an autoimmune disease, graft rejection pathology, inflammatory disease, hyperlipidemia, atherosclerosis, Metabolic Syndrome or any conditions, syndromes, disorders or diseases comprising the same.

In another aspect, the present invention provides a use of metadoxine in the preparation of a pharmaceutical composition for the treatment of a disorder or condition associated with or related to an imbalance in Thl and Th2 cells response. In certain embodiments, the treatment shifts the balance to favor Th2 cells response.

In yet a further aspect the invention provides a use of a salt adduct comprising: a positively charged moiety of formula (I), as defined herein above; and a negatively charged carboxylated lactam ring selected from the group consisting of: compounds of formulae (II), (III) and (IV), as defined herein above; in the preparation of a pharmaceutical composition for the treatment of an imbalance in Thl and Th2 cells

In another aspect, the present invention provides a method of treating a disorder or condition associated with or related to an imbalance in Thl and Th2 cells response in a subject in need thereof, said method comprising administering a therapeutically effective dosage of metadoxine, or a composition comprising thereof, to said subject. In certain embodiments, the treatment shifts the balance to favor Th2 cells response.

In a further aspect, the invention provides a method of treating an immune disorder associated with an imbalance in Thl and Th2 cells response in a subject in need, said method comprising administering a therapeutically effective dosage of a salt adduct, or a composition comprising thereof, to said subject, wherein said salt adduct comprises a positively charged moiety of formula (I), as defined herein above; and a negatively charged carboxylated lactam ring selected from the group consisting of: compounds of formulae (II), (III) and (IV), as defined herein above.

As used herein the term "salt adduct" is meant to encompass a salt product of a direct addition of two or more distinct ions, wherein the overall charge of the salt adduct is zero. In certain embodiments, the salt adduct comprises one positively charged moiety having a single positive charge functional group (i.e., the positively charged moiety is charged with +1 net charge) and one negatively charged moiety having a single negative charge functional group (i.e., the negatively charged moiety is charged with -1 net charge). In certain embodiments, the salt adduct comprises one positively charged moiety having two positively charged functional groups, which may be the same or different (i.e., the positively charged moiety is charged with +2 net charge) and two negatively charged moieties, which may be the same or different, and each having a single negative charged functional group (i.e., each negatively charged moiety is charged with -1 net charge). In certain embodiments, the salt adduct comprises two positively charged moieties, which may be the same or different, having each one positively charged functional group (i.e., each positively charged moiety is charged with +1 net charge) and one negatively charged moiety, having two negatively charged functional groups, being the same or different (i.e., the negatively charged moiety is charged with -2 net charge). In certain embodiments, the salt adduct comprises a positively charged moiety charged with +n net charge (originating from one or more positively charged functional groups, which may be the same or different), and a negatively charge moiety having -n (originating from one or more negatively charged functional groups, which may be the same or different) net charge, wherein n is an integer which may be equal to 1, 2, 3, 4, 5 or 6.

As used herein, a "positively charged moiety of a salt adduct" of the invention is the corresponding acid of pyridoxine, or any derivative thereof. In certain embodiments, the positive charge of the positively charged moiety stems from the protonated basic nitrogen atom of pyridoxine (as for example in compound (2)) or any derivative thereof (such as for example compounds of formula (I)). In certain embodiments, the positively charged pyridoxine derivative is substituted with a positively charged functional group such as for example -NH3+, -CH2NH3+, -NH2R+, -NHR2+ (wherein each R is independently a C1-C6 alkyl), which may, in some embodiments, be present in addition to the positively charged protonated basic aromatic nitrogen atom in the pyridine ring.

As used herein, a "carboxylated 5- to 7-membered lactam ring" of a salt adduct of the invention, is meant to encompass a γ-lactam, δ-lactam or ε-lactam rings, having a negatively charged carboxylate group (-COO-) substituted thereto. In certain embodiments, said carboxylate group is substituted at the lactam ring carbon atom adjacent to the amide nitrogen. In another embodiment said carboxylated lactam ring is a L-2-pyrrolidone-5-carboxylate (compound (1)). In other embodiments said carboxylate group is substituted on any position of the lactam ring. In certain embodiments, said carboxylated 5- to 7-membered lactam ring may be substituted by another substituent at any position on the lactam ring.

The term "halogen" as used herein means F, CI, Br or I.

The term "C1-C6 alkyl" as used herein represents a saturated, branched or straight hydrocarbon chain having 1, 2, 3, 4, 5 or 6 carbon atoms. Typical Cl-6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl and the like.

The term "C2-C6 alkenyl" as used herein represents a branched or straight hydrocarbon chain having 2, 3, 4, 5 or 6 carbon atoms and at least one double bond positioned between any two carbons of the chain. Examples of such groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1,3-butadienyl, 1- butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 1-hexenyl, 2-hexenyl and the like. The term "C2-C6 alkynyl" as used herein represents a branched or straight hydrocarbon chain having 2, 3, 4, 5 or 6 carbon atoms and at least one triple bond positioned between any two carbons of the chain. Examples of such groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2- pentynyl, 1-hexynyl, 2-hexynyl and the like.

The term "CI- C6 alkoxy" as used herein refers to the radical -O-Cl-6 alkyl, wherein CI -6 alkyl is as defined above. Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.

The term "CI- C6 alkylthio" as used herein refers to the radical -S-Cl-6 alkyl, wherein CI -6 alkyl is as defined above. Representative examples are methylthio, ethylthio, isopropylthio, n-propylthio, butylthio, pentylthio and the like.

The term "cycloalkyl" as used herein represents a monocyclic, carbocyclic group having 3, 4, 5, 6, 7 or 8 carbon atoms, but may also include heteroatoms such as N, O and/or S. Representative examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

The term "aryl" as used herein is intended to include carbocyclic aromatic ring systems such as phenyl, biphenylyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, indenyl, pentalenyl, azulenyl and the like. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 1,2,3,4-tetrahydronaphthyl, 1,4- dihydronaphthyl and the like.

The term "heteroaryl" as used herein refers to ring systems in which at least one ring is an aromatic ring in which at least one atom is a non-carbon atom, either substituted or non-substituted, where the non-carbon atom may be, for example, a nitrogen, sulfur or oxygen atom.

The term "C1-C6 alkoxycarbonyl" as used herein refers to the radical -C(0)0- Cl-6 alkyl, wherein CI -6 alkyl is as defined above.

The terms "C1-C6 alkoxy" and "C1-C6 alkylthio" as used herein refers to the radicals CI -6-0- and C1-6-S-, respectively, wherein CI -6 alkyl is as defined above. The term "hydroxyl" as used herein refers to the radical -OH. As used herein the term "thiol" refers to the radical -SH. As used herein the term "formyl" refers to the radical -COH. As used herein the term "cyano" refers to the radical -CN. As used herein the term "nitro" refers to the radical -N02.

The term "amine" as used herein refers to -NH3 or any primary (-NH2R), secondary (-NHR2), tertiary (-NR3) or quarternary amines (-NR4+), wherein each R may be the same or different and independently selected from H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl as defined herein above.

The term "C1-C6 carboxyalkyl" as used herein refers to the radical -CO-(Cl-C6 alkyl), wherein CI -6 alkyl is as defined above.

The term "aminocarbonyl" as used herein refers to the radical -CONR2, wherein each of groups R is independently selected from H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl as defined herein above.

The term "alkoxycarbonylalkyl" as used herein refers to the radical -OCO-(Cl- C6alkyl), wherein CI -6 alkyl is as defined above.

The term "amidino" as used herein refers to the radical -C(=NH)-NH2.

The term "optionally substituted" as used herein means that the moieties referred to are either unsubstituted or substituted with one or more of the substituents specified. When the moieties referred to are substituted with more than one substituent the substituents may be the same or different.

Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, an alkylthio, an acyloxy, a phosphoryl, a phosphate, a phosphonate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aryl or heteroaryl moiety.

It should be understood that moieties of a salt adduct of the invention may contain each at least one chiral center, and thus may exist in, and be isolated as, any stereoisomer thereof including, enantiomers, diastereomers or any mixtures thereod including, but not limited to racemic mixtures. The present invention includes any possible stereoisomer (e.g. enantiomers, diastereomers), any mixtures thereof including, but not limited to, racemic mixtures, of any of the individual moieties of a salt adduct of the invention. Where the herein-described processes for the preparation of each of the moieties of a salt adduct of the invention give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques, such as preparative chromatography. The moieties of a salt adduct of the invention may be each prepared in any mixture of possible stereoisomers thereof, including but not limited to racemic mixtures thereof, or individual stereoisomers (e.g. enantiomers, diastereomers) may be prepared either by enantiospecific synthesis or by chiral chromatographic separation of a racemate. Whenever referring to amino acids, the invention should be understood to encompass natural and non-natural amino acids or any derivative thereof.

The term "non-natural amino acid" as used herein refers herein to amino acids, or any derivative thereof, that are not among the amino acids which are the building blocks of proteins having L as well as D-configurations, while "natural amino acids", refer to amino acids or any derivative thereof, which are the building blocks of proteins, having L as well as D-configurations.

Carboxylated 5- to 7-membered lactam rings, which may be optionally substituted as defined hereinabove, may be synthetically prepared by any method known to a person skilled in the art or by methods described herein. In certain embodiments, said carboxylated lactam may be prepared from the corresponding amino acids, such as by methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1: Histogram showing the level of liver enzymes (AST and ALT) in ConA-treated mice following the administration of metadoxine versus control.

Figure 2: Histogram showing the level of IFNy in the serum of ConA-treated mice following the administration of metadoxine versus control. Figure 3: Histogram showing NO supernatant concentration (μΜ) after LPS induction with or w.o. pyridoxal (Mean±SE, n=3), treatment of 0.3 mg/ml and 0.03 mg/ml pyridoxal were statistically significant.

Figure 4: Histogram showing IL-l-beta supernatant concentration (pg/ml) after LPS induction with or w.o. pyridoxal (Mean±SE, n=3), treatment of 0.3 mg/ml pyridoxal was statistically significant.

DETAILED DESCRIPTION OF EMBODIMENTS

The present inventors found, as shown in the following examples, that treatment of Con A model mice with metadoxine induced a decrease in liver enzymes such as AST and ALT, as well as a decrease in the levels of IFNy in the serum, strongly suggesting that metadoxine may be used as a therapeutic agent in the treatment of immune-related disorders, and as a modulator of the Thl-Th2 cell balance.

The effect of metadoxine on IFNy serum levels suggests that metadoxine may be used as a modulator of Thl/Th2 balance. High IFNy levels induce the differentiation of naive CD4 cells into Thl cells, and inhibit the proliferation of Th2 cells. The inhibition of IFNy by metadoxine may thus shift the Thl-Th2 balance in the opposite direction, favoring Th2 cells. In specific conditions, it may be desirable to shift the balance towards Thl cells.

Accordingly, the invention provides a method for treating an imbalance between pro-inflammatory Thl -type and anti-inflammatory Th2type cytokines in a subject in need thereof.

In a first aspect, the invention relates to a process for the modulation of the Thl/Th2 cell balance, toward anti-inflammatory or pro-inflammatory cytokine producing cells, in a subject suffering from an inflammatory or immune-related disorder, said modulation being effected by administering to said subject an effective amount of metadoxine or a salt adduct defined herein above,, optionally in combination with another pharmaceutically active agent.

In an alternative embodiment, the invention relates to a method for the treatment of immune-related disorders in a mammalian subject, this method involving manipulation of NK T cell population by ex vivo education of the said NK T cells, such that the educated NK T cells have the capability to modulate the ThllTh2 balance toward anti-inflammatory or pro-inflammatory cytokine producing cells. This method comprises the steps of: (a) obtaining NK T cells from said subject; (b) ex vivo educating the NK T cells obtained in step (a) such that the resulting educated NK T cells have the capability of modulating the ThllTh2 cell balance toward anti-inflammatory or proinflammatory cytokine producing cells; and (c) re-introducing to the subject the educated NK T cells that were obtained in step (b).

Most notably, step (b) is effected in culture, bringing the NK T cells into contact with metadoxine or a salt adduct defined herein above,, for any suitable time effective to confer unto said cells the capability of modulating the Thl-Th2 cell balance toward anti-inflammatory or pro-inflammatory cytokine producing cells. As used in the present application, once conferred, at least to some extent, with said capability to modulate the Thl-Th2 cell balance toward anti-inflammatory or pro-inflammatory cytokine producing cells, the cells are considered "educated". The "education" toward antiinflammatory or pro-inflammatory cytokine producing cells will depend on the point of origin of the (to be) treated cells. Naturally, this is dependent on the immune system of the subject from whom the cells were obtained.

Modulation of the Thl-Th2 cell balance toward anti-inflammatory cytokine producing cells results in an increase of the quantitative ratio between at least one of IL- 4 and IL-10 to IFNy. In counterpart, modulation of the ThllTh2 cell balance toward proinflammatory cytokine producing cells, results in a decrease of the quantitative ratio between at least one of IL-4 and IL-10 to IFNy (i.e., an increase in the pro-inflammatory cytokines IL-2 and IFNy).

The above-described method may optionally further comprise the step of eliciting in the subject up or down regulation of the immune response to the immune- related disorder. In certain embodiments, said regulation of the immune response is by oral tolerization.

The ex vivo educated NK T cells according to the method of the invention are re-introduced by adoptive transfer to the treated subject.

Thus, the present invention also provides a therapeutic composition for the treatment of an immune-related disorder in a mammalian subject, which composition comprises as an effective ingredient ex vivo educated autologous NK T cells capable of modulating the Thl-Th2 cell balance toward anti-or pro-inflammatory cytokine producing cells. Most importantly, such ex vivo educated autologous NK T cells capable of modulating the Thl-Th2 cell balance toward anti- or pro-inflammatory cytokine producing cells are produced by being in contact with metadoxine or salt adduct defined herein above, or a composition comprising same.

Said composition may optionally further comprise one or more pharmaceutically acceptable carrier, diluent, excipient or additive.

In a further aspect, the invention relates to the use of metadoxine or salt adduct defined herein above, in the treatment of inflammatory and immune-related disorders. Of particular interest is such use of metadoxine or salt adduct defined herein above, when comprised in sustained-, controlled- or delayed-release compositions, as detailed below.

The term "metadoxine" as used herein refers to the currently known and available form of metadoxine, which is an ion-pair between pyrrolidone carboxylate (PCA) and pyridoxine (vitamin B6) with the two compounds linked in a single product by salification. The term "metadoxine" as used herein is also intended to refer to and encompass any other active form of pyrrolidone carboxylate (PCA) in stable association with pyridoxine (vitamin B6). Stable association between the PCA and pyridoxine may be non-covalent, such as through a salt link, or other hydrostatic or electrostatic forces. Stable association, in other cases, may be accomplished by covalent bonding, e.g., by means of a linker or other chemical bond between the peA and pyridoxine in which metadoxine activity is retained. The skilled artisan will be able to test the biological activity of any such complexes in standard metadoxine assays.

The term "acceptable derivative" with respect to metadoxine or a salt adduct as defined herein above, refers to any salt, conjugate, ester, complex or other chemical derivative of metadoxine or a salt adduct as defined herein above, or any of the moieties comprising the same, which, upon administration to a subject, is capable of providing (directly or indirectly) metadoxine or a salt adduct as defined herein above, or a metabolite or functional residue thereof, or measurable metadoxine activity. The term "physiologically compatible metadoxine derivative" may be used interchangeably herein with the term "acceptable derivative" and refers to a functional, active, pharmaceutically acceptable derivative of metadoxine or a salt adduct as defined herein above. The term "excipient" refers to an inactive substance used as a carrier for the active ingredient in a formulation.

In one aspect, the present invention provides for the use of metadoxine or a salt adduct as defined herein above, in the preparation of a pharmaceutical composition for the treatment of an inflammatory or an immune-related disease.

The preparation of pharmaceutical compositions is well known in the art and has been described in many articles and textbooks, see e.g., Remington's Pharmaceutical Sciences, Gennaro A. R. ed., Mack Publishing Company, Easton, Pennsylvania, 1990, and especially pages 1521-1712 therein.

In certain embodiments, said composition is formulated III a sustained- or controlled-release dosage form, or such form combined with immediate release form.

The term "controlled release" refers to any formulation which delivers an agent at a controlled rate for an extended time and is designed to achieve a desired agent level profile.

The term "sustained release" is used in its conventional sense to refer to a formulation that provides for gradual release of an active material over an extended period of time, which in certain embodiments may also further result in substantially constant blood levels over an extended time period, i.e., controlled release.

The term "immediate release" is used in its conventional sense to refer to a formulation that provides for non delayed or controlled release of an active material upon administration.

The treatment regimen selected may be chronic or non-chronic. Optimal dosing schedules may be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. In general, dosage is calculated according to body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the combined composition of the invention in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the composition of the invention is administered in maintenance doses, once or more daily. Administration of metadoxine or a salt adduct as defined herein above, may be via enteral (via the digestive tract) and/or parenteral (other than digestive tract) routes, depending on the delivery systems of choice, which will also define the selection of the carrier, e.g. appropriate surfactants/co-surfactants composition or micro/nano particles (such as liposomes or nano-liposomes) entrapping the active ingredients, or other additives or excipients, for the delivery system of interest.

The enteral delivery systems may be designed for oral administration (tablets, sachets, lozenges, capsules, gelcaps, drops, or other palatable form, drop solutions, syrups) or rectal administration (suppository or (mini) enema form).

The systems for parenteral administration include subcutaneous, transdermal (diffusion through the intact skin), transmucosal (diffusion through a mucous membrane), sublingual, buccal (absorbed through cheek near gumline) administration, or administration by inhalation. In certain embodiments, the compositions of the invention are not administered by invasive modes of treatment (i.e., are non-invasive). In certain embodiments, the metadoxine or salt adduct as defined herein above, or compositions comprising thereof may be administered by intravenous, intraperitoneal or intramuscular injection.

In certain embodiments, metadoxine or a salt adduct as defined herein above, or the composition comprising thereof is delivered as a microcrystalline powder or a solution suitable for nebulization; for intravaginal or intrarectal administration, pessaries, suppositories, creams or foams. The composition may be formulated for oral administration. In another embodiment, the composition may be formulated for topical administration. In another embodiment, the composition may be formulated for transmucosal administration, sublingual, buccal (absorbed through cheek near gumline) administration, administration by inhalation or ocular administration, e.g., in eye drops. In another embodiment, the composition may be formulated for injection.

Administration of metadoxine for medical uses requires safe and efficient delivery systems. The present invention can provide delivery systems for safe delivery of the active ingredient metadoxine or a salt adduct as defined herein above, due to their special physico-chemical features, particularly direct absorption, by non-invasive means, and consequent avoidance of side effects. The delivery systems significantly enhance efficiency and quality of metadoxine or a salt adduct as defined herein above absorption based on their unique physicochemical features, which enable lower concentrations or amounts of the active substance to be delivered to a subject in a biologically active form. The delivery systems of the invention provide for the direct access of the active substance to the tissues and thus provide immediate or near- immediate effects of metadoxine or a salt adduct as defined herein above to the subject in need thereof.

In certain embodiments, the drug delivery systems comprising the metadoxine or a salt adduct as defined herein above, may be designed for oral, nasal, ocular, rectal, subcutaneous, transdermal, transmucosal, sublingual, buccal or inhalation administration. The drug delivery systems may provide the active substance in a controlled release mode. In certain embodiments, the drug delivery systems of the invention may further comprise at least one additional pharmaceutically active agent.

The delivery systems used in the invention may generally comprise a buffering agent, an agent which adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and/or additives as known in the art. Supplementary pharmaceutically acceptable active ingredients can also be incorporated into the compositions. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, preservatives and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except when any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. It is contemplated that the active agent can be delivered by any pharmaceutically acceptable route and in any pharmaceutically acceptable dosage form.

Oral forms include, but are not limited to, tablets, capsules, pills, sachets, lozenges, drops, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Also included are oral rapid-release, time controlled-release, and delayed- release pharmaceutical dosage forms. The active drug components can be administered in a mixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier"), materials suitably selected with respect to the intended form of administration.

Where the delivery system is for oral administration and is in the form of a tablet or capsule or the like, the active drug components can be combined with a non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, modified sugars, modified starches, methylcellulose and its derivatives, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, and other reducing and non-reducing sugars, magnesium stearate, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate and the like. For oral administration in liquid form, the active drug components can be combined with non-toxic pharmaceutically acceptable inert carriers such as ethanol, glycerol, water and the like. When desired or required, suitable binders, lubricants, disintegrating agents and coloring and flavoring agents can also be incorporated into the mixture. Stabilizing agents such as antioxidants, propyl gallate, sodium ascorbate, citric acid, calcium metabisulphite, hydroquinone, and 7- hydroxycoumarin can also be added to stabilize the dosage forms. Other suitable compounds can include gelatin, sweeteners, natural and synthetic gums such as acacia, tragacanth, or alginates, carboxymethylcellulose, polyethylene, glycol, waxes and the like.

Additional suitable pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat. In some embodiments, the pharmaceutically acceptable carrier is magnesium stearate. Additional pharmaceutical excipients commonly accepted and used are found in, for example, Remington's Pharmaceutical Sciences (Gennaro, A., ed., Mack Pub., 1990).

For purposes of parenteral administration, solutions in suitable oil such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art. Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art.

The half-life of metadoxine in human serum is very short. Lu Yuan et al. (Chin. Med. J. 2007 120(2) 160-168) shows a mean half life of about 0.8 hour. One way of prolonging serum levels of active moiety is by administering the material in a sustained- release formulation. Because metadoxine is freely soluble in water and in various biological fluids, it is difficult to sustain its release and prolong its absorption time. Therefore, a controlled release dosage form of metadoxine, which may be based on a predetermined gradual release of the active ingredient in the biological fluids, resulting in a sustained action with small fluctuations of the plasma level over a prolonged period of time, may be significantly advantageous in the treatment of diseases.

In certain embodiments, the method of administration will be determined by the attending physician or other person skilled in the art after an evaluation of the subject's condition and requirements. An embodiment of the method of the present invention is to administer the therapeutic compound described herein in a sustained release form. Any controlled or sustained release method known to those of ordinary skill in the art may be used with the compositions and methods of the invention such as those described in Langer, Science 249(4976): 1527-33 (1990). Such method comprises administering a sustained-release composition, a suppository, or a coated implantable medical device so that a therapeutically effective dose of the composition of the invention is continuously delivered to a subject of such a method. Sustained release may also be achieved using a patch designed and formulated for the purpose. The composition of the invention may be delivered via a capsule which allows sustained-release of the agent over a period of time. Controlled or sustained-release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Sustained release formulae or devices, or any topical formulations, may additionally contain compositions to stabilize the composition or permeate physiological barrier such as skin or mucous membrane. Exemplary additional components may include any physiologically acceptable detergent, or solvent such as, for example, dimethylsulfoxide (DMSO).

The advantages of sustained release formulations include:

(a) Sustained blood levels: sustained release formulations slower the rate of absorption of drug and allow less blood concentrations fluctuations within a dosing interval. For drugs with short half-lives, like metadoxine, the use of extended-release formulation may maintain therapeutic concentrations over prolonged periods of time.

(b) Attenuation of adverse effects: absorption of conventional dosage forms results in high peak blood concentrations which may be reached soon after administration with possible adverse effects related to the transiently high concentration. The use of metadoxine sustained release product avoids the initial high blood concentrations which may cause a sudden side effect, like diarrhea.

(c) Improved patient compliance: drugs with short half-lives often need to be given at frequent intervals to maintain blood concentrations within the therapeutic range. There is an inverse correlation between the frequency of dosing and patient compliance. A reduction in the number of daily doses offered by extended-release products has the potential to improve compliance.

(d) Improved economical value: improvement of adherence to treatment can provide both clinical and economic value by improving treatment outcomes and reduction of use of medical services (for example: fewer doctor visits).

(e) Improved compliance in special populations: reduction in the number of daily doses offered by extended-release products are especially important in children and elderly who are often sensitive to adverse effects or must adhere to complex medication regiments. For example: sustained release formulation avoids the need of children to take medication during school activity.

In certain embodiments, the metadoxine or a salt adduct as defined herein above, in compositions to be used in the invention may be in immediate, fast or burst release form.

In certain embodiments, the metadoxine or a salt adduct as defined herein above, in compositions to be used in the invention may be in a combination of sustained or slow release and immediate or fast release forms. In certain embodiments, the relative proportion of sustained or slow release metadoxine to immediate or fast release metadoxine or a salt adduct as defined herein above, is, e.g., 1 to 99, 5 to 95, 10 to 90, 15 to 85, 20 to 80, 25 to 75, 30 to 70, 35 to 65, 40 to 60, 45 to 55, 50 to 50, 55 to 45, 60 to 40, 65 to 35, 70 to 30, 75 to 25, 80 to 20, 85 to 15, 90 to 10, 95 to 5, or 99 to 1.

In certain embodiments, a polymeric material is used to sustain or control release of metadoxine or a salt adduct as defined herein above. In certain embodiments, the type of polymeric material and the amount of which is used, have a strong influence on the rate of release of metadoxine or a salt adduct as defined herein above, from the product of the present invention. Examples of polymers include both hydrophobic and hydrophilic polymers. Examples of hydrophobic polymers include, but are not limited to, ethyl cellulose and other cellulose derivatives, fats such as glycerol palmito-stereate, beeswax, glycowax, castorwax, carnaubawax, glycerol monostereate or stearyl alcohol, hydrophobic polyacrylamide derivatives and hydrophobic methacrylic acid derivatives, as well as mixtures of these polymers. Hydrophilic polymers include, but are not limited to, hydrophilic cellulose derivatives such as methyl cellulose, hydroxypropylmethyl cellulose, hydroxyethylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, sodium carboxymethylcellulose and hydroxyethyl methylcellulose polyvinyl alcohol, polyethylene, polypropylene, polystyrene, polyacrylamide, ethylene vinyl acetate copolymer, polyacrylate, polyurethane, polyvinylpyrrolidone, polymethylmethacrylate, polyvinyl acetate, polyhydroxyethyl methacrylate, as well as mixtures of these polymers. Furthermore, any mixture of one or more hydrophobic polymer and one or more hydrophilic polymer could optionally be used.

In certain embodiments, a polymeric material to be used in compositions of the invention is e.g., but not limited to, microcrystalline cellulose such as "Avicel PH 101" manufactured by FMC BioPolymer's, Hydroxypropyl Methylcellulose such as "Metholose" produced by Shin-Etsu Chemical Co., Ethyl cellulose such as "Ethocel™" manufactured by The Dow Chemical Company, an acrylic polymer such as "Eudragit RSTM" produced by Rohm GmbH, a colloidal silicone dioxide such as "Aerosil™" manufactured by Degussa, a Poly (Vinyl Acetate) such as "Kollicoat SR" manufactured by BASF, or an Ethyl Acetate and Vinyl Acetate solution such as "Duro-Tak" manufactured by Delasco Dermatologic Lab & Supply, Inc.

In certain embodiments, delivery systems of the invention comprise delivery devices. In certain embodiments, the compositions of the invention are delivered by an osmotic process at a controlled rate such as by an osmotic pump. The system may be constructed by coating an osmotically active agent with a rate controlling semipermeable membrane. This membrane may contain an orifice of critical size through which agent is delivered. The dosage form after coming into contact with aqueous fluids, imbibes water at a rate determined by the fluid permeability of the membrane and osmotic pressure of the core formulation. This osmotic imbibitions of water result in formation of a saturated solution of active material within the core, which is dispensed at controlled rate from the delivery orifice in the membrane.

In certain embodiments, the compositions of the invention are delivered using biodegradable microparticles. In certain embodiment, the system to prepare microparticles consists of an organic phase comprised of a volatile solvent with dissolved polymer and the material to be encapsulated, emulsified in an aqueous phase. In certain embodiments, the biodegradable polymers that can be used for the microparticle matrix, comprises polylactic acid (PLA) or the copolymer of lactic and glycolic acid (PLAGA). The PLAGA polymer degrades hydrolytic ally over time to its monomeric components, which are easily removed from the body through natural life processes.

The preparation may also contain an absorption enhancer and other optional components. Examples of absorption enhancers include, but are not limited to, cyclodextrins, phospholipids, chitosan, DMSO, Tween, Brij, glycocholate, saponin, fusidate and energy based enhancing absorption equipment.

Optional components present in the dosage forms include, but are not limited to, diluents, binders, lubricants, surfactants, coloring agents, flavors, buffering agents, preservatives, stabilizing agents and the like.

Diluents, also termed "fillers" include, for example, dicalcium phosphate dihydrate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, hydrolyzed starches, silicon dioxide, colloidal silica, titanium oxide, alumina, talc, microcrystalline cellulose, and powdered sugar. For administration in liquid form, the diluents include, for example, ethanol, sorbitol, glycerol, water and the like.

Binders are used to impart cohesive qualities to the formulation. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinzed starch), gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums, e.g., acacia, tragacanth, sodium alginate, celluloses, and Veegum, and synthetic polymers such as polymethacrylates and polyvinylpyrrolidone .

Lubricants are used to facilitate manufacture; examples of suitable lubricants include, for example, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, and polyethylene glycol.

Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. In certain embodiments, the surfactants are anionic. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions, associated with cations such as sodium, potassium and ammonium ions. In certain embodiments, the surfactants include, but are not limited to: long alkyl chain sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylhexyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.

Stabilizing agents such as antioxidants, include, but are not limited to, propyl gallate, sodium ascorbate, citric acid, calcium metabisulphite, hydroquinone, and 7- hydroxycoumarin.

If desired, the present compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, preservatives, and the like.

Any of the compositions of the invention may be used alone or in combination with one or more additional therapeutic agents, for the treatment of inflammatory conditions and/or immune-related disorders.

Optional additional therapeutic agents of the composition may be selected from the group consisting of analgesics, antibiotics, anti-inflammatory agents, anti-neoplastic agents, glucocorticoids, hematopoietic agents, glycolipids, and immunosuppressants, but are not limited thereto.

The amount of both metadoxine or a salt adduct as defined herein above, and the additional therapeutic agent that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. In certain embodiments, the compositions of this invention may be formulated so that a dosage of between 0.1-1000 mg/kg body weight/day can be administered. In certain embodiments, the compositions of this invention may be formulated so that a dosage of between 1-400 mg/kg body weight can be administered. The dose of the compound depends on the condition and the illness of the patient, and the desired daily dose. In human therapy, the oral daily dose may be 50-3000 mg. These doses are administered in unit dosage forms, which may be divided into 2-3 smaller doses for each day in certain cases, especially in oral treatment.

In certain embodiments, the compositions of the present invention may act synergistically in combination with each other and may further act synergistically in the presence of an additional therapeutic agent. Therefore, the amount of compound(s) and additional therapeutic agent(s) in such compositions will be less than that required in a mono-therapy utilizing only that therapeutic agent. In such compositions a dosage of between 1-400 mg/kg bodyweight/day of the additional therapeutic agent can be administered.

The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance or substances. The term thus means any substance intended for use in diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions In an animal or human. The term "therapeutically effective amount" means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. In certain embodiments, a therapeutically-effective amount of a compound or composition will depend on its therapeutic index, solubility, and the like. For example, certain metadoxine formulations of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to a selected treatment, as may be determined by the skilled artisan.

Thus, the present invention provides a method of treating an immune-related disease in a subject in need, said method consisting of administering a therapeutically effective dosage of metadoxine or a salt adduct as defined herein above, or a composition comprising thereof, to said subject.

According to another embodiment, dosage unit forms used by the methods of the invention may be either for a single or for repeated administration. Administration of said dosage unit form may be repeated, for example, everyone to five, ten or twenty- four hours, for a therapeutically sufficient period of time. According to an alternative embodiment, the dosage unit form may be a sustained-released dosage unit form which provides continued pH-independent drug release for a considerable period of time after administration.

The term "effective amount" refers to the amount of a therapeutic reagent that when administered to a subject in an appropriate dose and regimen produces at least one desired result.

A "subject" or "patient" to be treated by a method of the invention may mean either a human, or non-human animal, preferably a mammal.

The term "treating" or "treatment" refers to mitigating or alleviating at least one symptom of a condition, disease or disorder in a mammal, such as a human, or the improvement of an ascertainable measurement associated with a condition, disease or disorder.

As shown in the examples below, the data suggest that metadoxine can serve for the treatment of any inflammatory disorder in which the immune system plays a role and which is either cytokine-mediated or cell mediated. These include but not limited: any Thl- or Th2-mediated disorder, and T cell or B cell, or dendritic cells, or any other cell of the immune system-mediated disease. Any disease in which the immune system may be a direct or indirect role in the pathogenesis of.

Modulation of the Thl/Th2 balance towards an anti-inflammatory Th2 response may be particularly applicable in immune related disorders having an undesired unbalanced pro-inflammatory Thl reaction. For example, such immune-related disorders may be an autoimmune disease, graft rejection pathology, inflammatory disease, non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, Metabolic Syndrome or any of the conditions comprising the same.

This metadoxine may be administered to a subject to treat or prevent disorders associated with an abnormal or unwanted immune response associated with cell, tissue or organ transplantation, e.g., renal, hepatic, and cardiac transplantation, e.g., graft versus host disease (GVHD), or to prevent allograft rejection.

More generally, immune-related disorders to be treated with metadoxine in accordance with the invention may be autoimmune disease, graft rejection pathology, inflammatory disease, non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, Metabolic Syndrome or any of the conditions comprising the same.

Examples of autoimmune disorders to be treated with metadoxine in accordance with the invention include, but are not limited to, Alopecia Areata, Lupus Erythematosus, Ankylosing Spondylitis, Meniere's Disease, Antiphospholipid Syndrome, Mixed Connective Tissue Disease, Autoimmune Addison's Disease, Multiple Sclerosis, Autoimmune Hemolytic Anemia, Myasthenia Gravis, Autoimmune Hepatitis, Pemphigus Vulgaris, Behcet's Disease, Pernicious Anemia, Bullous Pemphigoid, Polyarteritis Nodosa, Cardiomyopathy, Polychondritis, Celiac Sprue- Dermatitis, Polyglandular Syndromes, Chronic Fatigue Syndrome (CFIDS), Polymyalgia Rheumatica, Chronic Inflammatory Demyelinating, Polymyositis and Dermatomyositis, Chronic Inflammatory Polyneuropathy, Primary Agammaglobulinemia, Churg-Strauss Syndrome, Primary Biliary Cirrhosis, Cicatricial Pemphigoid, Psoriasis, CREST Syndrome, Raynaud's Phenomenon, Cold Agglutinin Disease, Reiter's Syndrome, Crohn's Disease, Rheumatic Fever, Discoid Lupus, Rheumatoid Arthritis, Essential Mixed, Cryoglobulinemia Sarcoidosis, Fibromyalgia, Scleroderma, Grave's Disease, Sjogren's Syndrome, GuillainBarre Syndrome, Stiff-man Syndrome, Hashimoto's Thyroiditis, Takayasu Arteritis, Idiopathic Pulmonary Fibrosis, Temporal Arteritis/Giant Cell Arteritis, Idiopathic Thrombocytopenia Purpura (ITP), Ulcerative Colitis, IgA Nephropathy, Uveitis, Insulin Dependent Diabetes (Type I), Vasculitis, Lichen Planus, and Vitiligo

Finally, the present invention provides a kit for treating an immunerelated disease III a subject in need thereof, said kit compnsmg: (a) metadoxine or a composition comprising metadoxine; (b) means for administering said metadoxine or composition; and (c) instructions for use.

The present invention is defined by the claims, the contents of which are to be read as included within the disclosure of the specification.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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

Examples

Example 1:

Methods

A number of immunological techniques are not in each instance described herein in detail, like for example ELISA, as they are well known to the person of skill in the art, and these are described in detail in e.g., Harlow and Lane (1988) Antibodies: a laboratory manual, Cold Spring Harbour Laboratory.

Animal model

Ten weeks old, male C57B1/6 mice were obtained from Jackson Harlan Laboratories, Jerusalem, Israel (http://www.harlanisrael.com) and maintained in the Animal Core of the Hadassah-Hebrew University Medical School. Mice were administered standard laboratory chow and water ad libitum, and kept in 12-hour light/dark cycles. Animal experiments were carried out according to the guidelines of the Hebrew University-Hadassah Institutional Committee for Care and Use of Laboratory Animals, and with the committee's approval.

Induction of Con A Hepatitis:

Concanavalin A (MP Biomedicals, USA) was dissolved in 50mM Tris pH 7, 150mM NaCl, 4mM CaCl ₂ and injected into the tail vein at a dose of 500 μg/mouse (20mg/kg).

Drug administration Metadoxine was dissolved in double-distilled water (DDW) and administered two hours prior to induction of disease (22 hours before sacrifice), IP 400mg/kg = 10 mg/mouse.

Experimental groups:

Two groups of mice were tested, n=5 per group. The first group (A) was treated with PBS served as controls and the second (B) with Metadoxine

Follow up parameters:

Animals were followed at 20 hours following induction of disease for liver enzymes (AST, ALT), and for serum IFN-γ levels.

Liver enzymes:

Sera from individual mice were obtained. Serum AST and ALT levels were measured by an automatic analyzer.

Cytokine measurement:

Serum IFN-γ levels were measured in each animal by "sandwich" ELISA, using commercial kits (Quantikine, R&D Systems, MN, USA).

Results:

Administration of Metadoxine decreases both ALT and AST liver enzymes Administration of Metadoxine decreases both ALT and AST liver enzymes, as shown in Tables la and lb below, showing the results from control and experimental groups, respectively :

Table la Table lb

Group A: DDW Group B: Metadoxine

# AST ALT # AST ALT

1 3480 4530 1 1210 1050

2 4700 599 2 1320 920

3 3120 304 3 1230 790

4 2290 2890 4 990 380 5 4060 6340 5 580 480

Average 3530 2932.6 Average 1066 724

Stdev 916.2423 2574.835 Stdev 297.5399 285.8846

b. Administration of Metadoxine decreases IFNy serum levels:

Reduction of IFNy serum levels by metadoxine:

Summary of the Results:

Administration of Metadoxine had a profound anti-inflammatory effect in the immune-mediated Con A hepatitis model.

Example 2: Effect of Pyridoxal L-2-pyrrolidone-5-carboxylate on Nitric Oxide and cytokine IL-l-beta secretion following LPS induction in mouse macrophage cells

The study was aimed in finding and characterizing anti-inflammatory effects of pyridoxal L-2-pyrrolidone-5-carboxylate (referred to as "pyridoxal") in a macrophage murine cellular system (RAW 264.7) before stimulation with Lipopolysaccharide (LPS), an endotoxin which induces a strong response from normal animal immune systems.

The inflammatory effect was demonstrated, among others, by secretion of ILlbeta in the culture media as well as the production of Nitric oxide (NO). IL-lbeta is a pro-inflammatory cytokine and is expressed by many cells including macrophage, NK cells, monocytes, and neutrophils. Inflammatory hypersensitivity has been found to be the result of IL-Ιβ activation of cyclooxygenase-2 (PTGS2/COX2). IL-lbeta has also been associated with septic shock, and wound healing. IL-l-beta levels were assessed using a commercial ELISA kit.

Nitric oxide is generated by phagocytes (monocytes, macrophages, and neutrophils) as part of the human immune response. Phagocytes are armed with inducible nitric oxide synthase (iNOS), which is activated by IFN-γ as a single signal or by tumor necrosis factor along with a second signal. NO production was measured by Griess reagent which detects the nitrite (N02) production. The minimum dose (0.003 mg/ml) of pyridoxal was calculated according to the following calculation:

Human dose/amount of fluid in average human body = amount of compound per ml of fluid. The average human body contains 6 liters, or 6000 ml of fluid. The dose given to humans in clinical trials (for treatment of alcohol intoxication) was 20 mg/kg, the therapeutic dose per ml is 0.003 mg/ml. Since this was an exploratory study, this dose was determined as the minimum concentration, with higher concentrations being 10 and 100 fold this minimum dose.

The anti-inflammatory effect of Pyridoxal was demonstrated by a significant dose dependent reduction in NO production (Greiss reaction, Figure 3) and in ILlbeta secretion at the highest dose of pyridoxal (Figure 4).

Example 3: In vivo Inflammation Effect

Metadoxine and pyridoxal L-2-pyrrolidone-5-carboxylate (referred to as "pyridoxal") are analyzed for their ability to reduce Experimental Autoimmune Encephalomyelitis (EAE) burden in a mouse model. The EAE model is an established animal model of brain inflammation, or more specifically - multiple sclerosis (MS, despite known differences between the human and murine model of the disease), and is achieved in mice by injection of central nervous system proteins in order to produce an inflammatory response and the demyelination characteristic of MS.

The rodent species is the CB57/BLJ female mouse. Healthy adult animals (10 weeks old) are employed, with a total of 36 mice, divided into 3 groups. All three groups are immunized with an emulsion containing 300 μg of Myelin Oligodendrocyte Glycoprotein and an equal amount of Freund's adjuvant, with one group being treated with Metadoxine, the second one is treated with Pyridoxal and the third one is treated with the vehicle (the control group).

The mice are treated with their respective compound from day 1 (in conjunction with the CNS protein injection) until the study is terminated due to morbidity or until the last day of the study (day 42). The study endpoints are mortality and mobility observations as well as measurements of neurological scores (tail tonicity, hind leg weakness/paralysis).

The dose administered in the study is based on the Body Surface Area (BSA) dose translation: Human Equivalent Dose (mg/kg) = Animal Dose (mg/kg) multiplied by (Mouse Km/Human Km); 20 mg/kg = Animal Dose (mg/kg) multiplied by (3/37) = 246.67 mg/kg.

The above dose in mice would be the equivalent of the dose given in humans to treat alcohol intoxication, however, since this is the first in vivo experiment carried out for treatment of inflammation with these compounds, a twofold dose of 500 mg/kg/day per mouse will be used in order to obtain an effect if one is indeed present. Higher doses are not recommended in order to avoid potential toxicity issues which may affect study outcome.