WO2013086582A1 - Genetic polymorphism and responsiveness to vitamin therapy of migraine - Google Patents

Abstract

A method and kit for determining the responsiveness of a human to vitamin treatment of migraine is provided, wherein an A66G methionine synthase reductase (MTRR) genotype indicates the responsiveness of a human to B complex vitamin treatment of migraine. AG heterozygotes and AA homozygotes have a relatively increased responsiveness to vitamin treatment of migraine. A GG genotype is indicative of a relatively reduced or lower responsiveness to vitamin treatment compared to an A allele carrier. The method and kit may detect an isolated nucleic acid or an encoded protein, due to a methionine/isoleucine polymorphism encoded by the A66G polymorphism. A method of treatment of migraine using vitamins is also provided, wherein the responsiveness of the patient to vitamin treatment has been assessed by determining the patient's A66G genotype. The vitamins are typically vitamin B6, vitamin B12 and folic acid.

Description

TITLE

GENETIC POLYMORPHISM AND RESPONSIVENESS TO VITAMIN THERAPY OF MIGRAINE FIELD THIS INVENTION relates to treatment of migraine. More particularly, this invention relates to a genetic polymorphism which indicates the responsiveness of an individual to vitamin and folate therapy of migraine.

BACKGROUND

Homocysteine related dysfunction of the vascular endothelium may potentially influence migraine susceptibility (Chen, 2001; Parsons, 2003; Storer, 1997). Hyperhomocysteinemia related endothelial injury may activate trigeminal fibres leading to an inflammatory reaction occurring in the meninges, along with dilation of the large cerebral vessels. It is this reaction that is thought to participate in the characteristic head pain common to both migraine with aura (MA) and migraine without aura (MO) (Lea, 2009; Lea, 2004; Parsons, 2003). Various factors determine the levels of circulating plasma homocysteine, in particular dietary deficiencies in the co-factors such as folic acid, vitamin !½ and Ββ essential for metabolising homocysteine and mutations in the genes of key enzymes participating in homocysteine metabolism such as cystathionine β-synthase (CBS), methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and methionine synthase reductase (MTRR) (Silaste, 2001).

The MTRR gene reduces inactive cobalamin II to active cobalamin I and methylates it to methylcobalamin using S-adenosylmethionine as the methyl donor (Elmore, 2007). MTRR therefore plays a pertinent role in maintaining adequate supply of active cobalamin I and may also be a critical determinant of homocysteine concentrations (Gaughan, 2001). A common variant, A66G in the MTRR gene results in the replacement of a methionine (Met 22) with an isoleucine in the enzyme. The MTRR A66G polymorphism is also associated with an increase in plasma homocysteine, with the GG genotype having a greater effect than an AG genotype (Gaughan, 2001). However, Vaughn, 2004 found no distinction between AG and GG genotypes in relation to plasma homocysteine and suggested that the MTRR A66G polymorphism is uninformative with regard to plasma homocysteine levels. SUMMARY

Surprisingly and notwithstanding the conclusion of the prior art that the MTRR A66G polymorphism is uninformative with regard to plasma homocysteine levels, the present inventors have identified that the MTRR A66G polymorphism does influence plasma homocysteine levels. Furthermore, the present inventors have found that A allele carriers of the MTRR A66G polymorphism are more responsive to vitamin and folic acid treatment of migraine.

In a first aspect, the invention provides a method for determining the responsiveness of a human to vitamin and/or folate treatment of migraine, said method including the step of determining the methionine synthase reductase (MTRR) genotype of the human to thereby determine the responsiveness of a human to vitamin treatment of migraine.

In a second aspect, the invention provides a method for preventing or treating migraine in a human, said method including the step of administering to the human a therapeutically effective amount of one or more vitamins, wherein the human is, or has been, identified or selected as having a methionine synthase reductase (MTRR) genotype that determines the responsiveness of the human to vitamin and/or folic acid treatment of migraine.

In a third aspect, the invention provides a kit for determining the responsiveness of a human to vitamin treatment of migraine, said kit comprising one or more reagents for determining the methionine synthase reductase (MTRR) genotype of the human to thereby determine the responsiveness of the human to vitamin treatment of migraine.

In certain embodiments, the one or more reagents of the kit may comprise one or more primers, probes and/or antibodies.

Suitably, the MTRR genotype is in relation to an A66G polymorphism. Preferably, an A allele is indicative of a relatively increased responsiveness to vitamin and/or folic acid treatment of migraine.

In one embodiment of the aforementioned aspects, the MTRR genotype may be determined by detection of an A66G polymorphism in an isolated nucleic acid.

In another embodiment of the aforementioned aspects, the MTRR genotype may be determined by detection of an isolated protein comprising an amino acid encoded by an A66G polymorphism. In a particular embodiment, the vitamin or vitamins are of the vitamin B complex.

Preferably, the vitamin or vitamins include vitamin B_¾, vitamin B12 and/or folic acid.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Patient flow chart for the trial

Figure 2: Change in (a) homocysteine, (b) folate, (c) and (d) B12 levels after trial in placebo and vitamin treated groups.

Figure 3: Change in frequency of high-level of migraine disability as measured by the Migraine Disability Score (MIDAS) instrument (MIDAS> 11) over the treatment period in vitamin and placebo groups.

Figure 4: Change in average pain score over treatment period for placebo and vitamin groups. Values are medians. Quartiles are not shown but reductions are statistically significant (P<0.05).

Figure 5: Change in homocysteine levels over the treatment period in vitamin treated group stratified by the MTRRA66G variant. Values are represented as mean ± SEM.

DETAILED DESCRIPTION

The present invention relates to migraine, particularly to a genetic basis underlying the responsiveness of migraineurs to a nutritional supplement that includes a therapeutically effective amount of a mixture of vitamins for preventing, reducing the frequency of or reducing the severity of migraine. More particularly, the present invention has arisen from the finding that the methionine synthase reductase (MTRR) gene polymorphism A66G is a unique, MTRR SNP that is at leas partly indicative of the responsiveness of a migraine sufferer to treatment with vitamins such as B complex vitamins Be, B12 and/or folic acid.

The method may be used independently of clinical diagnosis or may be used in conjunction therewith to confirm or assist clinical diagnosis and vitamin and/or folic acid treatment of migraine, inclusive of migraine with aura and migraine without aura.

Furthermore, the method of the invention may be used in combination with methods that identify other genetic polymorphisms associated with responsiveness to treatment with B complex vitamins such as vitamin B₆, B12 and/or folic acid.

The term ''f' olic acid or "folate" refers to a water-soluble vitamin of the vitamin B complex. Folic acid is also known as vitamin B9.

The term "vitamin Bi? describes cobalamin or cyanocobalamin, a member of the vitamin B complex that contains cobalt.

The term "vitamin B_<s" encompasses any of a group of water-soluble members of the itamin B complex, including pyridoxine, pyridoxal and pyridoxamine.

By "migraine" is meant a neurological disorder with symptoms that include head pain that can vary in intensity, duration (4-72 hours) and frequency. The head pain is usually accompanied by symptoms such as nausea, vomiting, photophobia, phonophobia, variations of the visual field and other temporary, reversible neurological symptoms. The most common forms of migraine have been classified as migraine with aura and migraine without aura and are diagnosed according to the International Classification of Headache disorders 2^nd Edition as described by the International Headache Society.

With regard to the MTRR gene polymorphism A66G, an A at position 66 (i.e an A allele) is associated with a relatively increased or higher responsiveness to treatment with vitamins such as vitamins Ββ, B 12 and/or folic acid.

A homozygote individual having an A at position 66 in both MTRR alleles (i.e an AA genotype) has a relatively increased or higher responsiveness to treatment with B complex vitamins such as vitamin B and Bi₂ and/or folic acid.

A heterozygote individual having an A at position 66 in one MTRR allele and a G in another allele (i.e an AG genotype) has a relatively increased or higher responsiveness to treatment with B complex vitamins such as vitamin Be, B12 and/or folic acid.

Typically, a homozygote individual having a guanine at position 66 in both

MTRR alleles (i.e a GG genotype) will display relatively reduced or lower responsiveness to treatment with B complex vitamins such as vitamin B₆, Bi₂ and/or folic acid compared to an A allele carrier. Accordingly, one particular aspect of the invention includes a method of determining the MTRR genotype of a human to thereby determine or predict the responsiveness of the human to treatment with B complex vitamins such as vitamin B₆, B 12 and/or folic acid.

In one embodiment, the method includes the step of isolating a nucleic acid from the human, wherein the nucleic acid comprises a nucleotide sequence of at least a fragment of an MTRR gene, which nucleotide sequence includes nucleotide 66 of the MTRR gene.

For the purposes of this invention, by "isolated' is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native or recombinant form.

The term "nucleic acid^'' as used herein designates single-or double-stranded mRNA, RNA, cRNA and DNA inclusive of cDNA and genomic DNA and DNA- RNA hybrids.

A "polynucleotide'^'' is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide" has less than eighty (80) contiguous nucleotides.

A "probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

A "primer" is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™. Non-limiting examples of primers useful according to the invention comprise respective nucleotide sequences set forth in SEQ ID NO:l and SEQ ID NO:2. However, it will be readily appreciated by persons skilled in the art that the human MTRR gene sequence may be used as the basis for designing alternative primers that allow amplification of a fragment of an MTRR gene comprising nucleotide 66. The terms "anneaF, "hybridize" and "hybridization" are used herein in relation to the formation of bimolecular complexes by base-pairing between complementary or partly-complementary nucleic acids in the sense commonly understood in the art. It should also be understood that these terms encompass base- pairing between modified purines and pyrimidines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (for example thiouridine and methylcytosine) as well as between A,G,C,T and U purines and pyrimidines. Factors that influence hybridization such as temperature, ionic strength, duration and denaturing agents are well understood in the art, although a useful operational discussion of hybridization is provided in to Chapter 2 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 2000), particularly at sections 2.9 and 2.10.

The term "gene" is used herein as a discrete nucleic acid unit or region that may comprise one or more of introns, exons, open reading frames, splice sites and regulatory sequences such as promoters and polyadenylation sequences.

The term "single nucleotide polymorphism (SNP)" is used herein to indicate any nucleotide sequence variation in an allelic form of a gene that occurs in a human population. This term encompasses mutation, insertion, deletion and other like terms that indicate specific types of SNPs.

In the context of the present invention by "corresponds to" and

"corresponding to" is meant that an isolated nucleic acid comprises a nucleotide sequence that is, or is complementary to, a nucleotide sequence of at least a fragment of an MTRR gene comprising nucleotide 66. The MTRR gene numbering used herein is that used by the Genbank Accession number AH008763, SNP number rsl 801394, Gaughan et al., 2001 and/or Wilson et al., 1999.

Nucleic acid-based methods may include the step of obtaining at least a fragment of said isolated MTRR nucleic acid from said individual, wherein the fragment comprises nucleotide 66 of the MTRR gene.

Said at least a fragment of the isolated nucleic acid may be in the form of genomic DNA, RNA or cDNA reverse-transcribed from isolated RNA.

Typically, in certain embodiments fragments may have at least 9, 15, 20, 50 or up to 80 contiguous, nucleotides (such as oligonucleotide primers and probes). In other embodiments, fragments may have 80, 100, 150, 200, 300, 500 or more contiguous nucleotides (such as PCR amplification products).

In a particular embodiment of the invention, a fragment may be a product of nucleic acid sequence amplification.

A non-limiting example of such fragments is a 118 bp fragment comprising nucleotide 66 of a human MTRR gene. In one embodiment,, said fragment is capable of being amplified by a nucleic acid sequence amplification technique using primers that comprise respective nucleotide sequences according to 5'- GCA A AG GCC ATC GCA GAA GAC AT -3' (SEQ ID NO:l) and 5'-AAA CGG TAA AAT CCA CTG TAA CGG C-3' (SEQ ID NO:2).

In this regard, it will be appreciated that preferred diagnostic methods employ a nucleic acid sequence amplification technique.

Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et al. supra; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et al, 1996, J. Am. Chem. Soc. 118 1587 and International application WO 92/01813, and Lizardi et al, (International Application WO 97/19193); nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et al, 1994, Biotechniques 17 1077; ligase chain reaction (LCR) as for example described in International Application WO89/09385; Q-β replicase amplification as for example described by Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93 5395; and helicase- dependent amplification as for example described in International Publication WO 2004/02025.

As used herein, an "amplification product" is a nucleic acid produced by a nucleic acid sequence amplification technique.

A preferred nucleic acid sequence amplification technique is PCR. It will be appreciated that PCR includes any of a number of techniques including "RT-PCR", "quantitative", "semi-quantitative", "competitive" and "real time" PCR. Another particular example of a PCR method that may also be useful is Bi-PASA (Bidirectional PCR Amplification of Specific Alleles), as for example described in Liu et al. 1997, Genome Res. 7 389-399. Non-limiting examples of primers suitable for PCR amplification comprise respective nucleotide sequences 5'- GCA AAG GCC ATC GCA GAA GAC AT -3' (SEQ ID NO:l) and 5'-AAA CGG TAA AAT CCA CTG TAA CGG C-3' (SEQ ID NO:2).

However, it will be readily appreciated by persons skilled in the art that the human MTRR gene may be used as the basis for designing alternative primers that allow amplification of a fragment of a MTRR gene comprising nucleotide 66.

It will also be well understood by the skilled person that identification of the MTRR gene SNP may include any of a variety of techniques (alternatively or in addition to PCR) such as melting analysis, SSCP analysis, denaturing gradient gel electrophoresis (DGGE), probe hybridization, restriction endonuclease digestion or direct sequencing of amplification products.

A preferred method includes melting analysis of PCR amplification products. Melting analysis includes "high resolution melting (HRM)" and "melt curve" analysis. Preferably, fluorescent DNA-intercalating dyes are incorporated during PCR (i.e in "real time") and the PCR amplicons are then denatured post amplification to determine the presence of differences in nucleotide make-up between amplicons of a known sequence. These differences alter the melting temperature (T_m) of the amplicons that can be measured by fluorescence based on the release of the fluorescent DNA-intercalating dyes. The shift in T_m indicates the presence of a variant allele and a genotype can be deduced. Non-limiting examples of fluorescent DNA-intercalating dyes include SYBR Green I, LC Green, LC Green Plus, ResoLight, EvaGreen, Chromofy and SYTO 9.

In alternative embodiments, melting analysis can be performed using fluorochrome-labeled allele-specific probes which form base-pair mismatches when annealing to DNA strands. The base-pair mismatches are revealed by virtue of their lower melting temperature (T_m) compared to fully complementary annealing between the probe and one or the other amplified DNA strands.

Another potentially useful method is allele-specification oligonucleotide hybridization, as for example described in Aitken et al., 1999, J Natl Cancer Inst 91 446-452.

DGGE also exploits T_m differences, but uses differential electrophoretic migration through gradient gels as a means of distinguishing subtle nucleotide sequence differences between alleles. Examples of DGGE methods can be found in Fodde & Losekoot, 1994, Hum. Mutat. 3 83-9 and United States Patents 5,045,450 and 5,190,856.

The MTRR gene SNP used according to the invention may be identified by direct sequencing of a PCR amplification product, for example. An example of nucleic acid sequencing technology is provided in Chapter 7 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et at. (John Wiley & Sons NY USA 1995-2001).

In yet another embodiment, mass spectroscopy (such as MALDI-TOF) may be used to identify nucleic acid polymorphisms according to mass. In a preferred form, such methods employ mass spectroscopic analysis of primer extension products, such as using the MassARRAY™ technology of Sequenom.

In a further embodiment, said MTRR gene SNP may be identified by a microarray method of the invention.

Microarray technology has become well known in the art and examples of methods applicable to microarray technology are provided in Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al (John Wiley & Sons NY USA.1995-2001).

With respect to the present invention, a preferred microarray format comprises a substrate such as a glass slide or chip having an immobilized, ordered grid of a plurality of nucleic acid molecules, such as cDNA molecules, although without limitation thereto.

A microarray would typically comprise a nucleic acid having said MTRR gene polymorphism together with control MTRR nucleic acids.

Such a microarray could also include a plurality of other nucleic acids indicative of other diseases that have an underlying genetic basis and be useful in large scale genetic screening, for example.

In a preferred embodiment, the method includes the steps of:

(i) obtaining a nucleic acid sample from a human;

(ii) subjecting the nucleic acid samples to PCR amplification using primers to produce an amplification product that comprises a nucleotide sequence of a fragment of an MTRR gene comprising nucleotide 66; and

(iii) determining whether nucleotide 66 is an A or a G by high resolution melting analysis. Non-limiting examples of primers in (ii) comprise respective nucleotide sequences 5'- GCA AAG GCC ATC GCA GAA GAC AT -3' (SEQ ID NO:l) and ^' 5'-AAA CGG TAA AAT CCA CTG TAA CGG C-3' (SEQ ID NO:2).

It will also be appreciated from the foregoing that the invention contemplates a kit for determining the responsiveness of a human to vitamin treatment of migraine, said kit comprising one or more reagents for determining the methionine synthase reductase (MTRR) genotype of the human to thereby determine the responsiveness of a human to vitamin treatment of migraine.

In one particular embodiment, the kit comprises primers for nucleic acid sequence amplification of at least a fragment of a MTRR gene comprising nucleotide 66.

Non-limiting examples of primers comprise respective nucleotide sequences 5'- GCA AAG GCC ATC GCA GAA GAC AT -3' (SEQ ID NO:l) and S'-AAA CGG TAA AAT CCA CTG TAA CGG C-3' (SEQ ID NO:2).

One or more other reagents are contemplated such as. probes for hybridization-based methods and detection reagents useful in enzymatic, colorimetric and/or radionuclide-based detection of nucleic acids, although without limitation thereto.

In another embodiment, the method includes the step of isolating an MTRR protein from the human and determining whether the protein comprises either a methionine or an isoleucine encoded by a codon comprising nucleotide 66 of an MTRR gene.

The methionine/isoleucine polymorphism is with respect to the methionine at residue 22 of the MTRR protein, underlined in the following amino acid sequence ¹⁸IAEEMCEQAV²⁷ (SEQ ID NO:3).

Typically, a methionine at residue 22 is associated with a relatively increased or higher responsiveness to treatment with vitamins (such as vitamin B and Bi₂) and/or folic acid.

Typically, an isoleucine at residue 22 is associated with a relatively reduced or lower responsiveness to treatment with vitamins (such as vitamin Be and B₁₂) and/or folic acid compared to a methionine.

By "protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art. A "peptide" is a protein having less than fifty (50) amino acids.

A "polypeptide" is a protein having fifty (50) or more amino acids.

Such MTRR protein detection methods are well known in the art and include western blotting, ELISA, two dimensional protein profiling, protein arrays, immunoprecipitation, radioimmunoassays and radioligand binding and MTRR enzyme activity assays, although without limitation thereto.

In one advantageous embodiment, the method uses an antibody specific for a methionine or isoleucine at residue 22 of an MTRR protein to determine whether the isolated MTRR protein comprises said methionine or isoleucine.

This embodiment would be particularly suited to an ELISA-based method of detection.

It is also known that an MTRR protein having methionine at residue 22 has greater affinity for methionine synthase substrate than does an MTRR protein having an isoleucine at residue 22. Accordingly, the invention contemplates measurement of the affinity of an MTRR protein for its methionine synthase substrate to thereby determine whether the MTRR protein has a methionine or an isoleucine at residue 22.

It will also be appreciated from the foregoing that the invention contemplates a kit for determining the responsiveness of a human to vitamin and/or folate treatment of migraine, said kit comprising one or more reagents for determining the methionine synthase reductase (MTRR) genotype of the human by detection of an isolated protein comprising an amino acid encoded by an A66G polymorphism.

In one particular embodiment, the kit comprises an antibody for detecting a methionine and/or antibody for detecting an isoleucine encoded by the A66G polymorphism.

One or more other reagents are contemplated such as a secondary antibody and or detection reagents useful in enzymatic, colorimetric and or radionuclide-based detection of the protein, although without limitation thereto. The antibody may be directly labeled, such as with an enzyme (e.g horseradish peroxidase or alkaline phosphatase), a fluorochrome (e.g. FITC, Texas Red, R^hywerythrin), digoxogenin, biotin or a radionuclide, or the secondary antibody may be labeled.

According to the aforementioned aspects and embodiments, MTRR nucleic acids and proteins are suitably isolated from a biological sample obtained from the human. Non-limiting examples of biological samples include blood, serum, plasma, saliva, urine, skin, and blood cells.

Another aspect of the invention relates to a method for preventing or treating migraine in a human, said method including the step of administering to the human a therapeutically effective amount of a vitamin and/or folic acid, wherein the human is, or has been, identified or selected as having a methionine synthase reductase (MTRR) genotype that determines the responsiveness of the human to vitamin and/or folic acid treatment of migraine.

Suitably, the determination of the MTRR genotype of the human is performed according to any of the methods hereinbefore described.

As used herein "treatment or "treating" migraine in a human means a therapeutic course of action which alleviates, ameliorates or reduces existing symptoms of migraine in the human. It is to be understood that such treatment need not be absolute to be beneficial to a subject.

As used herein, "preventing", "prevent" or "prevention", refers to a prophylactic course of action initiated prior to the onset of a migraine symptom so as to alleviate, ameliorate or reduce the migraine symptom. It is to be understood that such treatment need not be absolute to be beneficial to a subject.

Preferably, treatment of prevention of migraine comprises at least one of: reducing the severity of migraine symptoms in the human; reducing the duration of migraine symptoms in the human; and reducing the frequency of migraine symptoms in the human.

By "reducing", as in "reducing the severity of migraine", or "reducing the frequency of migraine" or "reducing the duration of migraine", is meant a lessening or shortening of a symptom of or associated with migraine, or of the number of migraines a human experiences, or of the length of time a human experiences a symptom of or associated with migraine. It is to be understood that such reducing need not be absolute to be beneficial to the human.The term "therapeutically effective amount" describes a quantity of a therapeutic agent(s) (e.g. vitamins B6, Bj₂ and/or folic acid) sufficient to achieve a desired effect in a subject being treated with the agent(s). For example, this may be the amount of folic acid, vitamin Bi₂ and vitamin B necessary to prevent or treat migraine, or reduce the severity of, the frequency of or the duration of migraine. Ideally, a therapeutically effective amount of an agent is an amount sufficient to effect the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for preventing or treating migraine, or reducing the severity of, the frequency of or the duration of migraine will be dependent on the subject being treated, the severity of the migraine, and the manner of administration of the therapeuticagents.

A therapeutically effective amount of vitamin agents such as folic acid, vitamin B₁₂ and vitamin B₆ may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the frequency of administration is dependent on the preparation applied, the subject being treated, the severity and type of migraine, and the manner of administration of thetherapeutic agent.

The forms of the vitamins such as vitamin B₆, Bj₂ and folic acid described herein may be varied as known to one of ordinary skill in the art. In one embodiment, the pyridoxine may be present, at least partially, as pyridoxine HCL or its coenzyme form pyridoxal 5-phosphate. Likewise, cobalamin may be present, at least partially, as methyl cobalamin or hydroxycobalamin. Furthermore, one or more of the components may also be present in a chelated form. In one embodiment, amino acid chelates are used. The chelated forms facilitate a more effective absorption and increased biological activities as well as increased shelf life.

Furthermore, the present invention includes as substitutes for the vitamins described herein, those compounds that are converted into any of the forgoing through the physiological processes of a subject.

The vitamins of the present disclosure (e.g vitamin B₆, Bj₂ and/or folic acid), can be administered by any conventional method available, preferably in the form of a pharmaceutical composition. The pharmaceutical compositions of the present disclosure may be administered alone or may be administered with additional active agents if desired and/or may comprise a pharmaceutically acceptable carrier, diluent and/or excipient, as is well understood in the art.

The pharmaceutical compositions described can be used in the form of a medicinal preparation, for example, in aerosol, solid, semi-solid, or liquid form which contains the vitamins disclosed herein (e.g vitamin B₆, Bi₂ and or folic acid), as active ingredients. In addition, the compositions may be used in an admixture with an appropriate pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers include, but are not limited to, organic or inorganic carriers, excipients or diluents suitable for pharmaceutical applications. The active ingredients may be compounded, for example, with the usual non-toxic pharmaceutically acceptable carriers, excipients or diluents for tablets, pellets, capsules, inhalants, suppositories, solutions, emulsions, suspensions, aerosols, and any other form suitable for use. _r

Pharmaceutically acceptable carriers for use in pharmaceutical compositions are well known in the art, and are described, for example, in Remington: The Science and Practice of Pharmacy Pharmaceutical Sciences, Lippincott Williams and Wilkins (A. R. Gennaro editor, 20^th edition). Such materials are nontoxic to the recipients at the dosages and concentrations employed and include, but are not limited to, water, talc, gum acacia, gelatin, magnesium trisilicate, keratin, colloidal silica, urea, buffers such as phosphate, citrate, acetate, and other organic acid salts, antioxidants such as ascorbic acid, peptides, low molecular weight (less than about ten residues) peptides such as, but not limited to, polyargjnine, proteins, such as, but not limited to, serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as, but not limited to, polyvinylpyrrolidinone, amino acids such as, but not limited to, glycine, glutamic acid, aspartic acid, or arginine, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, lactose, mannitol, glucose, mannose, dextrins, potato or corn starch or starch paste, chelating agents such as, but not limited to, EDTA, sugar alcohols such as mannitol or sorbitol, counterions such as, but not limited to, sodium, and nonionic surfactants such as, but not limited to, the Tweens, Pluronics or polyethyleneglycol. In addition, the compositions may comprise auxiliary agents, such as, but not limited to, taste^ enhancing agents, stabilizing agents, thickening agents, coloring agents and the like.

The pharmaceutical compositions may be prepared for storage or administration by mixing the active ingredients, each having a desired degree of purity, with physiologically acceptable carriers, excipients, stabilizers, auxiliary agents, and the like, as is known in the art. Such compositions may be provided in sustained release or timed release formulations.

The pharmaceutical compositions containing the active ingredients may be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. Furthermore the compositions containing the active ingredients may be administered parenterally, in sterile liquid dosage forms, by transmucosal delivery via solid, liquid or aerosol forms or transdermally via a patch mechanism or ointment. Various types of transmucosal administration include respiratory tract mucosal administration, nasal mucosal administration, oral transmucosal (such as sublingual and buccal) administration, and rectal transmucosal administration.

For preparing solid compositions such as, but not limited to, tablets or capsules, the vitamins described herein may be mixed with appropriate pharmaceutically acceptable carriers, such as conventional tableting ingredients (e.g., lactose, sucrose, mannitol, com starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, gums, colloidal silicon dioxide, croscarmellose sodium, talc, sorbitol, stearic acid magnesium stearate, calcium stearate, zinc stearate, stearic acid, and dicalcium phosphate), other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers, as well as diluents (e.g., water, saline or buffering solutions) to form a substantially homogenous composition. The substantially homogenous composition means the components are dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

The solid compositions described may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, tablets or pills can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

The pharmaceutical compositions may be in rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides. The solid compositions may also comprise a capsule, such as hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.

For intranasal administration, intrapulmonary administration or administration by other modes of inhalation, the pharmaceutical compositions may be delivered in the form of a solution or suspension from a pump spray container or as an aerosol spray presentation from a pressurized container or nebulizer, with the use of a suitable propellant {e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, nitrogen, propane, carbon dioxide, or other suitable gas) or as a.dry powder. In the case of an aerosol or dry powder format, the amount (dose) of the composition delivered may be determined by providing a valve to deliver a metered amount.

Liquid forms may be administered orally, parenterally or via transmucosal administration. Suitable forms for liquid administration include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic natural gums, such as tragacanth, acacia, alginate, dextran, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone, and gelatin. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol), preservatives (e.g., methyl or propyl p- hydroxybenzoates or sorbic acid), and artificial or natural colors and/or sweeteners.

Liquid formulations may include diluents, such as water and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, and the polyethylene alcohols), either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.

For buccal or sublingual administration, the pharmaceutical composition may take the form of tablets or lozenges formulated in conventional manners. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.

The pharmaceutical compositions may be formulated for parenteral administration. Parenteral administration includes, but is not limited to, intravenous administration, subcutaneous administration, intramuscular administration, intradermal administration, intrathecal administration, intraarticular administration, intracardiac administration, retrobulbar administration, and administration via implants, such as sustained release implants.

The pharmaceutical compositions may be presented in unit-dose or multi- dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. The requirements for effective pharmaceutically acceptable carriers for injectable compositions are well known in the art.

Therapeutic treatments can include a therapeutically effective amount of vitamins (e.g. folic acid, vitamin Bi₂ and vitamin B₆)necessary to prevent or treat migraine, or reduce the severity of, the frequency of or the duration of migraine. Ideally, a therapeutically effective amount of an agent is an amount sufficient to effect the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for preventing or treating migraine, or reducing the severity of, the frequency of or the duration of migraine will be dependent on the subject being treated, the severity of the migraine, and the manner of administration of the therapeutic composition. Effective amounts can be determined by standard clinical techniques.

For example, when administering a pharmaceutical composition comprising folic acid, vitamin Bj₂ and vitamin Be, the precise dose to be employed in the formulation will depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each subject's circumstances. The concentration of an active ingredient (such as folic acid, vitamin Bi₂ or vitamin B₆) in a topical composition (such as an ointment, cream, gel, or lotion) is typically from about 0.2% to about 1% (by weight relative to the total weight of the topical composition); for example, from about 0.3% to about 0.9%, from about 0.4% to about 0.8%, and from about 0.5% to about 0.7%. Within the ranges, higher concentrations allow a suitable dosage to be achieved while applying the lotion, ointment, gel, or cream in a lesser amount or with less frequency.

In other embodiments, a dosage range for non-topical administration (such as oral administration, or intravenous or intraperitoneal injection) of a pharmaceutical composition containing folic acid, vitamin B|₂ and vitamin B_¾ is from about 0.1 to about 200 mg kg body weight for each vitamin in single or divided doses; for example from about 1 to about 100 mg/kg, from about 2 to about 50 mg/kg, from about 3 to about 25 mg/kg, or from about 5 to about 10 mg/kg.

Acceptable daily dosages of the active ingredients (i.e., folic acid, vitamin Bj₂and vitamin B₆) of the pharmaceutical compositions of the present invention include between 0.5 and 5.0 mg of folic acid (e.g., 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, and 5.0 mg), between 0.02 and 1.0 mg of vitamin B]₂ (e.g., 0.02 mg, 0.04 mg, 0.06 mg, 0.08 mg, 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, and 1.0 mg) and between 2.0 and 50.0 mg of vitamin B₆ (e.g., 2.0 mg, 5.0 mg, 10.0 mg, 15.0 mg, 20.0 mg, 25.0 mg, 30.0 mg, 35.0 mg, 40.0 mg, 45.0 mg, and 50.0 mg).

In one non-limiting exemplary embodiment, 2 mg folic acid, 25 mg vitamin Βό and 0.4mg vitamin Bj₂ may be administered daily according to the method disclosed herein.

In one non-limiting exemplary embodiment, a phannaceutical composition may comprise, in combination, 2 mg folic acid, 25 mg vitamin Be and 0.4 mg vitamin B₁₂. According to this embodiment, the pharmaceutical composition is preferably suitable for once-daily administration.

The vitamins (eg. vitamin B6, Bn and/or folic acid) of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (for example, in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the migraine, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term treatment with a disclosed pharmaceutical composition is contemplated.

So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples. EXAMPLES

Introduction

Migraine is a chronic disabling condition that may in part be caused by endothelial and cerebrovascular disruption induced by hyperhomocysteinemia. Migraine is a polygenic multifactoral disorder. With multiple genes reported to contribute to migraine susceptibility, it is plausible that different genotypes in these genes may cause varying disease manifestations and also varying response to medication. The current study examined the genotypic effects of an MTR variant on vitamin supplementation treatment response in homocysteine-lowering and migraine disability including frequency and severity.

This was a randomised, double blinded placebo controlled trial of daily vitamin supplementation for 6 months in 206 female patients diagnosed with migraine with aura.

Vitamin supplementation significantly reduced homocysteine levels

(P<0.001), migraine head pain severity (P=0.017) and high migraine disability (P=0.022) in the studied population of migraineurs compared to the placebo effect (P>0.1). When the vitamin treatment group analysis was stratified by genotype, the A allele carriers of the MTRRA66G variants showed the highest level of reduction in homocysteine levels (PO.OOl), migraine pain severity (P=0.002) and percentage of high migraine disability (P=0.006). Interestingly, a combined analysis of the MTRRA66G and the known MTHFRC677T variant showed that carriers of both the A and C allele respectively had the most significant reduction in migraine head pain severity (P=0.002) and high migraine disability (P=0.015), however the effect of the combined analysis was not greater than the independent effect of the two variants on treatment response in migraineurs.

This study has provided evidence that the MTHFR and MTRR variants may be acting independently from one another in influencing vitamin treatment response in migraineurs

Methods

Study design and participant group

This study analysed the genotypic effect of the MTRRA66G polymorphisms on daily folic acid and E&6 and B₎₂ vitamin treatment for lowering homocysteine, migraine disability, frequency and pain severity by conducting a randomized, double blinded placebo controlled clinical trial over a 6 month period. The trial guidelines were designed using the guidelines for controlled trials of drugs in migraine (Tfelt- Hansen, 2000). The study recruited female Caucasian adult between the ages of 18 and 60, of European ancestry from all over Australia. All participants were interviewed and completed a detailed questionnaire that was administered through Griffith University's Genomics Research Centre (GRC). As migraine is more prevalent in females and the possibility that there may be a difference in migraine susceptibility and response in relation to treatment, the current study only focussed on one gender. Females between the ages of 18 and 60 were recruited and participants were included if they had suffered migraine for over 20 years and had a current diagnosis of MA (>90% of their migraine attacks were associated with aura), and a 1-year history of severe, long lasting attacks (at least 4 attacks lasting more than 48h), and had a family history of migraine. Confirmation of migraine diagnosis was carried out using the IHS criteria. Participants who were currently taking vitamin supplementation, pregnant, or had been diagnosed with a clinically recognised co-morbid disease such as vascular disease, depression or epilepsy were excluded from the trial to reduce clinical and pathological heterogeneity. Participants that had taken part in another clinical trial or had received any experimental therapy within the last one month were also excluded from the-trial. The patient group was not selected on the basis of pre existing folic acid, B12 or B6 deficiency.

Treatment

245 female patients meeting the inclusion criteria were randomly assigned into either the placebo or the treatment group. A blocked random allocation sequence was generated using Microsoft Excel (Microsoft, USA). Participants and everyone involved in this trial were blinded to randomisation and group allocation. Participants received either vitamin tablets containing 2mg of folic acid, 25mg of vitamin B₆ and 400 μg of vitamin Bi₂ or the placebo tablet. Participants were instructed to take one tablet daily for 6 months. Both the vitamin and placebo tablets were produced by Blackmores and were identical in appearance.

Baseline and Follow-up assessment

Before commencing the trial all participants were assessed for migraine disability using the Migraine Disability Assessment Score (MIDAS) instrument, which provides a measure of productive days lost to migraine headache in previous 3 months (i.e. migraine disability), headache frequency and pain severity (Lea, 2009; Stewart, 1999). Studies have shown that the MIDAS instrument is a valid and clinically useful instrument for assessing health-related quality of life in migraineurs. Based on the 5-question MIDAS rating, participants were arbitrarily categorized into a 'low' disability group if they had a MIDAS rating of 0-10 and into a 'high' disability group if they had a MIDAS rating greater than 1 l(Lea, 2009 #21 ; Stewart, 1999; Stewart, 1999). Questions 6 and 7 of the MIDAS instrument were on migraine frequency and head pain severity respectively. These were measured as number of days with headache (over a 3 month period) and a pain score (based on a scale of 1- 10), respectively (Lea, 2009; Stewart, 1999; Stewart, 1999).

Participants were asked to complete a daily diary during the trial period to record the details of their migraine symptoms (duration, frequency and severity) and treatment compliance. Participants were also instructed to take their usual migraine treatment for acute attacks. A blood sample was collected for baseline measurement of plasma homocysteine (μιηοΙ/L), folate (nmol/L), vitamin Be and Bi₂ (pmol/L) concentration. 2mL of venous blood was collected for Genomic DNA extraction and genotyping purposes. Participants were contacted after 3 months of starting the trial for headache diary and compliance checking. At the end of the 6 months trial the patients were reassessed at the GRC clinic. They were questioned about their migraine history in the last 6 months since the start of the trial. A second collection of blood samples was done for measurement of homocysteine, folate, B_¾ and Bj₂ concentrations. The Plasma homocysteine, folate, B₆ and B₁₂ levels were measured in an accredited pathology laboratory.

Genotyping

High resolution melt analysis was used to genotype A66G polymorphisms. Primers used for genotyping the MTRR A66G polymorphism are "Forward 5': GCA AAG GCC ATC GCA GAA GAC AT 3' (SEQ ID NO:l) and "Reverse 5': AAA CGG TAA AAT CCA CTG TAA CGG C 3' (SEQ ID NO:2). The reaction mixture used HotStarTaq (Qiagen, Hilden Germany) and consisted of 40 ng of genomic DNA, 10x PCR buffer, 25 mM MgCl₂, 5uM of each primer, 2.5mM of dNTPs, 50 μΜ of SYTO 9 (Invitrogen, Carlsbad, USA), 0.5 U of HotStarTaq polymerase and PCR grade water in a volume of 25 μL· All PCR reactions were performed in duplicate. PCR cycling and HRM analysis was performed on the Rotor-Gene™ 6000 (Corbett Research, Mortlake, New South Wales, Australia). The PCR cycling conditions for the MTRR A66G were as follows; one cycle of 95°C for 5 minutes; 45 cycles of 95°C for 5 seconds, 45 cycles of 60°C for 10 seconds, 72°C for 20 seconds; one cycle of 95°C for 1 second, 72°C for 90 seconds and a HRM step from 75 to 85°C rising at 0.1 °C per second.

Dietary consumption

Variation in the participant's diet is a potential confounding factor for this study. Participants were thus required to keep a daily diary of food type, amount and frequency. Each participant was given 2 diary packs, each pack consisting of 7 days of daily diet intake to be recorded. The diet dairy was designed to estimate the usual dietary intake of nutrients such as B₆, Bj₂ and dietary folate over a typical week. Participants were asked to complete their diet dairy once a fortnight, on only one day until each day of the week has been recorded. The nutrient intake of participants was analysed using the NUTTAB version 2010 database, which is based on the Australian New Zealand food standard code.

Statistical analysis

The analysis for the current trial was conducted on a modified intention-to- treat (ITT) principle. The modified ITT population was composed of all randomised participants who started the trial and consumed study supplements on at least one occasion, excluding those who withdrew from the trial after the randomisation process had taken place but before the commencement of study supplement consumption.

At baseline, the unpaired samples Mests were used to test the group means. The median were compared using the Mann Whitney U tests and proportions were compared using the χ² test of independence. The primary hypothesis of this trial, that vitamin supplementation reduced migraine disability was tested by comparing proportions of high disability migraineurs before and after the 6 month trial in both the vitamin and placebo groups. The χ² test of independence was used to compare the proportion changes. Mean changes were compared before and after treatment using paired samples t-tests and median changes were compared using nonparametric Wilcoxin signed rank tests for related samples. The relationships among the baseline biochemical variables were assessed using the Pearson's correlation tests. Post treatment means for treatment and placebo groups were compared at 6 months using the unpaired samples /-tests. The significance threshold was set at a level of 0.05. Linear regression analysis was performed using the "successive steps" method, to determine the independent predictors of the difference in homocysteine levels before and after the trial, allowing the introduction of a new variable if the P value of the new model was less than 0.05, and excluding those yielding a P value higher than 0.10 in each step. The independent variables were age, genotype, treatment group (placebo vs vitamin), and dietary intake of B₆, Bi₂ and folate. All analyses were performed using the Statistical Package for Social Sciences (SPSS version 18.0).

Results

Figure 1 illustrates the patient flow through the trial from January 2009 to January 2010. Six hundred and twenty nine migraine patients were assessed for eligibility prior to enrolment into trial. 384 migraine patients were excluded from enrolment due to reasons such as not meeting inclusion criteria, refusal to participate in placebo controlled trial and other reasons. 245 participants were initially enrolled in the trial and were randomly assigned to either the placebo group or the vitamin treated group but 3 participants dropped out before the commencement of the trial and the remaining 242 participants received baseline assessment and commenced the trial. Hundred and nineteen participants were on the vitamin treated group and the remaining 123 participants were in the placebo treated group. Forty four participants were lost to follow up due to lack of compliance and 162 participants completed the trial (76 vitamin: 86 placebo).

Baseline analysis

Table 1 shows the baseline clinical characteristics of the participant group. For the total migraine group (n= 206), mean folate concentration was 30.1 nmol/L, which is above the average for a general Caucasian population replete for folate (13.7nmol/L). The mean plasma homocysteine concentration for the migraine group was 11.5 μιηοΙ/L, which is also above the average for a general Caucasian population (8.9 μπιοΙ/L). The mean levels of Ββ and Bi₂ at baseline fell within the normal range for this patient group. For the total group, plasma homocysteine concentration was negatively correlated with plasma folate (Pearson's r = - 0.057, P = 0.438). Vitamin B₆ (Pearson's r = -0.212, P = 0.05), vitamin Bi₂ (Pearson's r = - 0.279, P= 0.000). The percentage of participants with high migraine disability did not differ significantly between the placebo and the vitamin treated group (P=0.18). Similarly the migraine attack frequency (P=0.41) and migraine pain severity (P= 0.38) did not differ significantly between the placebo and the vitamin treated group. There were no statistically significant differences between the vitamin and placebo groups for the test variables at baseline.

Six-month follow-up analysis

A total of 162 participants completed the trial. 86 of them were on placebo and 76 of them were on vitamin supplementation. Figure 2 shows the post treatment change in plasma folate, B$, Bj₂ and homocysteine concentration in vitamin and placebo group. After 6 months of treatment, the vitamin treated group had marked increases in folate, B^, Bj₂ concentration compared with baseline and the placebo group (P< 0.001). In the placebo group, the mean folate levels increased by 11.8% after 6 months (28.7 - 32.1 nmol/I, P= 0.852). The B₁₂ levels decreased by 6.5% (328.6-307.1 pmol/I, P= 0.059) and the B₆ levels decreased by 7.8% (80.8- 74.5 pmol/I, P= 0.292). In the vitamin treated group, the median homocysteine levels reduced by 20% after 6 months (11.5-9.2 pmol/I, PO.001) compared with 4.8% reduction observed for the placebo group (P=0.121). The effect of treatment on the reduction of homocysteine levels remained significant after correction for confounding factors such as age, genotype and dietary consumption of vitamin e, B_t2 and dietary folate (^=0.042; P = 0.019) (Table 2). The dietary consumption of B₆, B]₂ and dietary folate between the vitamin and placebo groups were not significantly different at the 6 month follow-up analysis (B₆, P=0.721 ; B₁₂, P= 0.891; Dietary folate, P= 0.373).

In the vitamin treated group the frequency of high migraine disability decreased after 6 months of supplementation from 74% to 56.9% (P=0.022). The reduction in the placebo group was not statistically significant (65.3% to 53.2% (P=0.098) (Figure 3). Headache frequency did not decrease from a median of 2 for both the vitamin (P=0.46) and placebo (P=0.147) groups after 6 months. The vitamin treated' group reported a decrease in pain severity from a median score of 7 to 6 (P= 0.017), whereas the placebo group reported no change (P>0.1) (Figure 4).

Treatment response and MTRRA66G genotype

When the vitamin- treated group was stratified by MTRRA66G genotype, the mean homocysteine reduction was 27% for the AA carriers (P<0.001), 16% for the AG carriers (P= 0.001) compared to 13% for the GG carriers (P=0.03) (Figure 5). For further analysis the AA and the AG genotypes of the A66G variant, which showed the largest decrease in homocysteine levels after vitamin supplementation were grouped and their effect on migraine frequency, pain severity and disability were analysed. Again there was no significant change in migraine frequency between the two groups. The A allele carriers of the MTRRA66G variant showed a significant reduction in migraine pain severity with the pain scores decreasing from 7.1 to 6.2 after the trial (P=0.002). In the GG genotype group however, the pain scores increased from 6.3 to 7 (P=0.05). When the percentage of high migraine disability was analysed in the two genotype groups, the percentage of highly disabled migraineurs decreased from 75.3% to 53.8% in the A allele carriers ( P= 0.006), while it only decreased from 68.8% to 66.7% in the GG genotype group (P=0.45).

Discussion

Migraine is one of the most burdensome headache disorders in the world. This chronic and often disabling condition has an enormous impact on both the individual sufferer and on society at large (Leonardi, 2005). There are several acute care therapies and medications currently available that have been successful in either treating migraine symptoms or decreasing migraine attacks and several other medications and therapies in various stages of development (Rapoport, 2011). However current medications and therapies work with differing efficacy in migraineurs and are often associated with adverse effects (Lea, 2009). Thus the search for effective, safe and inexpensive migraine therapies to combine with or replace current therapies still continues (Lea, 2009). One of the most significant findings in recent times is the involvement of genetics in migraine aetiology, which has added further complexity to understanding the pathophysiology underlying migraine. This has made migraine diagnosis and treatment options varied and continuously improved.

Mild hyperhomocysteinemia has been reported to increase the risk of artherosclerotic vascular disease (Wald, 2002). MA sufferers have been linked to increased risk of vascular brain lesions and ischaemic stroke (Kurth, 2007; Merikangas, 1997; Tzourio, 2001). It is still not clear if homocysteine levels are raised in migraineurs; however there is evidence to suggest that the CSD phenomenon observed in MA can also occur during a stroke episode (Scher, 2006). Based on the potential role homocysteine may play on the cerebrovascular system and the co morbidity of migraine and stroke, it is plausible that homocysteine levels may be involved in the underlying pathophysiology of both MA and stroke (Silberstein, 2001). The reasons for hyperhomocysteinemia may be varied: mutations in the genes for MTHFR, MTRR and CBS or possible nutritional deficiencies in cofactors in the homocysteine metabolism (Selhub, 1993). Alternatively, migraineurs may be hypersensitive to folate at normal levels.

The current study examined the genotypic effects of the MTRR gene on folate and vitamin B treatment response in migraineurs. Vitamin supplementation in the current trial was well tolerated by the study grou with no reports of adverse reactions. At baseline, the homocysteine levels were mildly elevated in the migraine group compared to the general Caucasian population with some but not all studies of homocysteine in migraine (Di Rosa, 2007; Lea, 2009; Scher, 2006). Results of this trial showed the vitamin treatment compared with the placebo significantly decreases the homocysteine levels at six months by an average of 2.2 μτηόΛ, an effect size, in Australian Caucasian female MA sufferers. This decrease in homocysteine levels remained significant after correcting for dietary consumption of folate, B6 and B12.

The 2.2 μπιοΐ ΐ reduction in the homocysteine levels at 6 months in the current trial approximates the 2.0 μιηοΐ/ΐ reduction observed in the "Vitamin Intervention for Stroke Prevention (VISP) trial, which randomised 3680 stroke survivors of primarily white origin from North America to receive high-dose (folic acid 2.5mg; 25mg: Bi₂ 0.4 mg) or low dose folic acid 20 μg; Ββ 200ug; Bi₂ 0.6 μg) (Toole, 2004). However the 2.2 μιηοΐ/ΐ reduction in homocysteine levels observed in the current trial is lower compared to the 4.0 μπκ>1/1 reduction observed in a study by Lea et al in 2009 that investigated the effect of vitamin supplementation and MTHFR (C677T) genotype on homocysteine-lowering and migraine disability in 52 Australian Caucasians (Lea, 2009). The smaller than expected treatment effect of the prescribed dosage of vitamins seen in the current trial is most likely attributable to the implementation on September 2009, of fortification of wheat flour for bread making with folic acid in Australia, which coincided with the conduct of the trial. This may also be the reason for the slight increase in folate levels observed in the placebo group at 6 months follow up assessment. There was significant reduction in migraine pain severity and disability according to the MIDAS instrument scores in the vitamin treated group. This decrease was greater overall compared with the placebo effect, which was not statistically significant (Figure 3). However the frequency of migraine headache did not decrease from a median of 2 in the vitamin treated group, which is inconsistent to that observed in the pilot study by Lea et al that reported a decrease from a median of 4 to 1 in migraine headache frequency in the vitamin treated group. The absence of significant reduction in migraine headache frequency observed in this trial may be attributable to the fact that the trial population only had a median headache frequency of 2 at baseline. A population of median headache frequency of 4 and more at baseline may have yielded a significant reduction in headache frequency after the prescribed vitamin treatment.

When the effects of the MTRRA66G variant were studied, the A allele carriers showed the largest reduction in homocysteine levels and migraine pain severity and high migraine disability under vitamin supplementation compared to the mutant homozygote GG genotype carriers.

The MTHFR product, 5-methyl-THF, donates a methyl group for the remethylation of homocysteine to methionine, which is catalysed by MTR in a vitamin Bi₂ dependent reaction. MTR may become inactive due to oxidation of its vitamin Bj₂ cofactor and restoration of MTR activity is dependent on reductive remethylation of vitamin I½ by MTRR (Leclerc, 1996). The functional effects of the MTRR A66G variant have not been fully understood, however in vivo experiments suggest that the A66G variant MTRR enzyme restores MTR activity less efficiently than wild-type (Olteanu, 2002), and has also been shown to increase plasma homocysteine levels in humans(Leclerc, 1998; Wilson, 1999).

Genes involved in the homocysteine pathway play a crucial role in the amount of homocysteine in the extracellular media such as plasma (Woodside, 1998). The allele groups of MTRR that showed the largest reduction in homocysteine levels showed the most significant reduction in migraine pain severity and disability under vitamin treatment. There is an undeniable relationship between homocysteine levels and migraine disability. The homozygote mutant allele carriers of the MTRR variant may need a higher dose of vitamin supplementation to experience the same effect as the wild type allele carriers of the variants, in migraine pain severity and disability reduction. Further clinical trials of higher doses of vitamin supplementation are required to make an evidence based argument of this idea. The effects of hyperhomocysteinemia may be a partial determinant for the neuro and/or vascular pathologies underlying MA and stroke (Lea, 2009).

Conclusion

The current study has produced the first evidence that homocysteine reduction through vitamin supplementation may reduce migraine disability in a subgroup of patients and that the MTRR A66G genotype contributes an effect on migraine treatment response.

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference.

Table 1:

P-

Variable Total Vitamin Placebo value^*

No.patients 206 103 103

Age in years, mean(SD)^b 44(13) 42(13) 45(13) 0.65

Female 206

Folate, mean (SD)^b 30.1(12.5) 31.4(12.8) 28.7(12.2) 0.56

Vitamin B₁₂, mean (SD)^b 322.7(148.5) 315.7(114.9) 328.6(175.7) 0.15

Vitamin B₆, mean (SD)^b 78.8(12.6) 77(11.9) 80.8(13.5) 0.82

Homocysteine, mean (SD)^b 11.5(3.4) 11.6(3.6) 11.5(3.3) 0.7^"

B12ug consumption, mean (SD)^b 1.01(0.5) 1.1(0.5) 0.9(0.5) 0.9

B6mg consumption, mean (SD)^b 1.7(0.6) 1.7(0.7) 1.7(0.6) 0.13

Folate ug consumption, median (range)⁸ 428.7(225.6) 433(239) 424(212) 0.74

High migraine disability (%)^a 69.7 74% 65.30% 0.18

Attack frequency median (range)³ 2 (1-6) 2(1-6) 2(1-4) 0.41

Head pain score median (range)³ 7(3.5-10) 7(1-10) 7(3.5-10) 0.38

MTHFR C677T (TT) genotype % 15.3 18.3 12.4 0.46 TRR A66G (GG) genotype % 25.4 19.6 30.9 0.15 ,

MTHFR C677T, methlenetetrahydrofolate gene

MTRR A66G Methionine synthase reductase gene

⁸ Based on a 3-month history.

Average daily consumption.

* All P values are two-tailed.

Table 2: Regression analysis model predicting homocysteine reduction after trial

Regression ANOVA

Model Variable R² F P

1 Treatment 0.042 5.67 0.019

Model 1 : The correlation for difference in homocysteine levels after trial was corrected_, for age, genotype, and dietary consumption of B6, B12 and dietary folate

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Claims

1. A method for determining the responsiveness of a human to vitamin and/or folate treatment of migraine, said method including the step of determining the methionine synthase reductase (MTRR) genotype of the human to thereby determine the responsiveness of a human to vitamin treatment of migraine.

2. The method of Claim 1, wherein the MTRR genotype is in relation to an A66G polymorphism.

3. The method of Claim 2, wherein an A allele is indicative of a relatively increased responsiveness to vitamin treatment of migraine.

4. The method of Claims 2 or Claim 3, wherein, an AA or an AG genotype is indicative of a relatively increased responsiveness to vitamin treatment of migraine.

5. The method of Claim 2, wherein a GG genotype is indicative of a relatively reduced or lower responsiveness to vitamin treatment of migraine compared to an A allele carrier.

6. The method of any preceding claim, wherein the MTRR genotype is determined by detection of an A66G polymorphism in an isolated nucleic acid.

7. The method of Claim 6, wherein the MTRR genotype is determined by PCR amplification of a nucleic acid comprising the A66G polymorphism.

8. The method of Claim 7, wherein the MTRR genotype is determined by PCR amplification using at least one of primers 5'- GCA AAG GCC ATC GCA GAA GAC AT -3' (SEQ ID NO:l) and.5'-AAA CGG TAA AAT CCA CTG TAA CGG C-3' (SEQ ID NO:2).

9. The method of Claim 7 or Claim 8, wherein the A66G polymorphism is detected by high resolution melting.

10. The method of any one of Claims 1-5, wherein the MTRR genotype is determined by detection of an isolated protein comprising an amino acid encoded by an A66G polymorphism.

11. The method of Claim 10, wherein a methionine at residue 22 of an MTRR protein is associated with a relatively increased or higher responsiveness to vitamin treatment of migraine.

12. The method of Claim 10, wherein an isoleucine at residue 22 is associated with a relatively reduced or lower responsiveness to vitamin treatment of migraine compared to a methionine.

13. The method of any preceding claim, wherein the vitamin is of the vitamin B complex.

14. The method of Claim 13, wherein the B complex vitamin is vitamin Be vitamin Bi₂ and/or folic acid.

15. A method for preventing or treating migraine in a human, said method including the step of administering to the human a therapeutically effective amount of one or more vitamins, wherein the human has been determined to have a methionine synthase reductase (MTRR) genotype according to the method of any one of Claims 1-14.

16. The method of Claim 15, wherein a therapeutically effective amount of one or more B complex vitamins is administered to the human.

17. The method of Claim 16, wherein a therapeutically effective amount of vitamin Β_δ, vitamin B₁₂ and/or folic acid is administered to the human.

18. A kit for determining the responsiveness of a human to vitamin and/or folate treatment of migraine according to the method of any one of Claims 1-14, said kit comprising one or more reagents for determining the methionine synthase reductase (MTRR) genotype of the human to thereby determine the responsiveness of a human to vitamin treatment of migraine.

19. The kit of Claim 18, wherein the one or more reagents of the kit may comprise one or more primers, probes and/or antibodies for determining the MTRR genotype.