WO2019000101A1 - Novel formulations useful in treating folate deficiencies - Google Patents

Abstract

Provided herein are novel extended release formulations of L-methylfolate ("5- methyltetrahydrofolate" or "5-MTHF"). The extended release compositions and formulations provided herein may be used to treat a variety of disorders associated with folate deficiencies, including central nervous system (CNS) disorders such as, seizures, ataxia, neuropathy, developmental delay, cognitive dysfunction (as in dementia) and psychiatric disorders as well as cerebral folate deficiency (CFD). Also provided herein are methods of diagnosing and/or treating a disease or disorder in a subject by administering extended release compositions or formulations comprising 5-MTHF.

Description

NOVEL FORMULATIONS USEFUL IN TREATING FOLATE DEFICIENCIES

CROSS-REFERENCE

[0001] This application claims benefit of U.S. Provisional Patent Application No.

62/526,791 filed on June 29, 2017, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Folate and folic acid are forms of B vitamin which perform a number of essential functions within the body. Folate, also commonly referred to as Vitamin B9 or Vitamin M, is naturally occurring in many foods while folic acid is synthetically produced. It is often difficult to consume adequate amounts of folate through diet and hence the need for a synthetic, and also more stable, form of this product. Low levels of folate may be caused by dietary, genetic and/or drug-induced mechanisms. Folate deficiencies are known to cause a number of health issues and thus national programs have been implemented which mandate the fortification of certain foods with folic acid.

[0003] Health issues associated with low folate levels include, for example, memory loss, hearing loss, age-related vision loss, osteoporosis, restless leg syndrome, inflammation, sleep problems, muscle pain and neural tube defects in developing embryos. In addition, folate deficiencies have more recently been linked to a number of central nervous system (CNS) disorders such as, seizures, ataxia, neuropathy, developmental delay, cognitive dysfunction (as in dementia) and psychiatric disorders.

[0004] CNS folate metabolism is tightly linked to the methylation and neurotransmitter cycles within the brain and low CNS folate levels have been associated with hypo-methylation of DNA, proteins and phospholipids; increased homocysteine; impaired neurotransmitter metabolism; and DNA damage. Trafficking of folate into the CNS is largely regulated by folate receptors (particularly folate receptor alpha, FRa), the activities of which may account for folate deficiencies associated with some CNS disorders. Reduced or impaired functioning folate receptors may result from competitive or non-competitive receptor inhibitors, including autoantibodies to the receptors themselves. The presence of such autoantibodies may therefore be indicative of folate treatments for certain CNS disorders. In fact, the diagnostic utility of specific autoantibodies to FRa has recently been investigated for cerebral folate deficiency syndrome (CFD) and, in particular, a focus on its role in schizophrenia and autism spectrum disorders (ASD) has been highlighted (see Sequeira, J. M., et al. (2013) Clin Chem Lab Med 51(3): 545-554; Ramaekers, V. T., et al. (2014) Mol Genet Metab 113(4): 307-314; Frye, R. E. et al. (2013) Molecular Psychiatry 18, 369-381; and Ramaekers, V. T., et al. (2007)

Neuropediatrics 38(6): 276-281).

[0005] The biologically active form of folate or folic acid is the reduced folate, L-5- methyltetrahydrofolate (also known as 5-MTFIF). 5-MTFIF is found in circulation and is known to be transportable across cell membranes as well as the blood-brain barrier and thus is a preferred molecule for CNS treatments. FRoc has a high affinity for 5-MTFIF and other reduced folate derivatives, such as folinic acid. As outlined in Sequeira {supra), 5-MTHF is 8 fold more potent than folic acid in competing with autoantibodies at the FRoc.

[0006] There remains a need for improved 5-MTFIF formulations which prolong delivery of this biologically active folate for sustained periods of time and particularly at a high enough level to normalize functioning of FRoc in those individuals expressing FRoc autoantibodies. It is also important to elevate and maintain 5-MTHF concentrations to levels that provide central exposure in the presence of malfunctioning FRoc (such as in non-competitive or uncompetitive binding of FRoc by FRoc autoantibody, where the antibody complex is not susceptible to disruption by higher levels of 5-MTHF). Through this mechanism, there is more 5-MTHF available and it crosses the blood-brain barrier without the benefit of FRoc through a less efficient process that can be improved with higher 5-MTHF levels on the systemic side.

SUMMARY OF THE INVENTION

[0007] Provided herein are compositions, formulations, medicaments, uses, and methods for enhancing the delivery of L-5-methyltetrahydrofolate ("5-MTHF"). Also provided herein are compositions, formulations, medicaments, uses, and methods for increasing and/or sustaining 5- MTHF plasma and/or CSF levels. In certain embodiments, compositions, formulations, medicaments, uses, and methods described herein provide improved 5-MTHF levels in vivo as compared to conventional dosage forms of 5-MTHF. In some embodiments, provided herein are compositions, formulations, or medicaments comprising 5-MTHF in an extended release dosage form. [0008] The inventors have discovered extended release 5-MTHF oral dosage forms that provide a surprising extended release, biphasic absorption profile upon administration to a subject, as described herein. Without wishing to be bound by theory, this release profile may be explained by the unique and inventive combination of 5-MTHF with (i) a gumming agent (for example guar and/or xanthan gum), which may provide for the maintenance and integrity of the formulation matrix and a degree of resistance to dissolution and degradation and allow passage of the post-burst dosage form as a unit to the small intestine from the stomach, (ii) a

mucoadhesive agent (for example HPMC), which may provide increased adhesion to the surface of the small intestine, (or alternatively, slow transit of the formulation through the proximal segments) which may in turn deliver 5-MTHF in close proximity to the 5-MTHF transporters expressed on the enterocytes of the small intestine, thereby overcoming a possible saturation effect of those transporters.

[0009] In some aspects, the extended release 5-MTHF oral dosage forms of the present invention further comprise an antioxidant, for example ascorbic acid and/or citric acid, which are thought to protect the 5-MTHF against oxidative damage during steps such as filling and storage, thus enhancing shelf life.

[0010] In one aspect, described herein is a 5-methyltetrahydrofolate (5-MTHF) formulation in an extended release oral dosage form comprising 1) a gumming agent selected from guar gum and xanthan gum, or a combination thereof, 2) a mucoadhesion agent wherein the mucoadhesion agent is hydroxypropyl methylcellulose (HPMC), and 3) an antioxidant selected from ascorbic acid and citric acid, or a combination thereof; wherein following administration of the 5-MTHF formulation to a subject, the 5-MTHF formulation exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject; wherein the plasma concentration in the subject increases between about 6-12 hours, between about 6-9 hours, or between about 9-12 hours, following administration of the 5-MTHF formulation to the subject.

[0011] In another aspect, described herein is a 5-methyltetrahydrofolate (5-MTHF) formulation in an extended release oral dosage form comprising 1) a gumming agent, 2) a mucoadhesion agent, and 3) an antioxidant; wherein following administration of the 5-MTHF formulation to a subject, the 5-MTHF formulation exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject; wherein the plasma concentration in the subject increases between about 6-12 hours, between about 6-9 hours, or between about 9-12 hours, following administration of the 5-MTHF formulation to the subject.

[0012] In another aspect, described herein is a 5-methyltetrahydrofolate (5-MTHF) formulation in an extended release oral dosage form wherein following administration of the 5- MTHF formulation to a subject, the 5-MTHF formulation exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject.

[0013] In another aspect, described herein is a 5-methyltetrahydrofolate (5-MTHF) formulation in an extended release dosage form wherein the plasma concentration in a subject increases between about 6-12 hours, between about 6-9 hours, or between about 9-12 hours following administration of the 5-MTHF formulation to the subject.

[0014] In another aspect, described herein is a 5-methyltetrahydrofolate (5-MTHF) formulation in an extended release oral dosage form comprising 1) a gumming agent and 2) a mucoadhesion agent. In some embodiments, the gumming agent is selected from guar gum and xanthan gum, or a combination thereof. In some embodiments, the gumming agent is guar gum. In some embodiments, the gumming agent is xanthan gum. In some embodiments, the gumming agent is a combination of guar gum and xanthan gum. In some embodiments, the mucoadhesion agent is hydroxypropyl methylcellulose (HPMC). In some embodiments, the 5-MTHF formulation further comprising an antioxidant. In some embodiments, the antioxidant is selected from ascorbic acid and citric acid, or a combination thereof. In some embodiments, the antioxidant is ascorbic acid. In some embodiments, the antioxidant is citric acid. In some embodiments, the antioxidant is a combination of ascorbic acid and citric acid. In some embodiments, following administration of the 5-MTHF formulation to a subject, the 5-MTHF formulation exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject. In some embodiments, the plasma concentration in a subject increases between about 6-12 hours, between about 6-9 hours, or between about 9-12 hours following administration of the 5-MTHF formulation to the subject. In some embodiments, the plasma concentration in a subject increases between about 6-12 hours, following administration of the 5-MTHF formulation to the subject. In some embodiments, the plasma concentration in a subject increases between about 6-9 hours, following administration of the 5-MTHF formulation to the subject. In some embodiments, the plasma concentration in a subject increases between about 9-12 hours following administration of the 5-MTHF formulation to the subject.

[0015] In some embodiments of the 5-MTHF formulations described herein, the second increase in the plasma concentration of 5-MTHF (diffusion phase) corresponds with 5-MTHF absorption in the small intestine. In some embodiments, the second increase in the plasma concentration of 5-MTHF (diffusion phase) corresponds with 5-MTHF absorption in the duodenum. In some embodiments, the 5-MTHF absorption in the small intestine is mediated by transporters expressed in the small intestine. In some embodiments, the 5-MTHF absorption in the small intestine is mediated by transporters expressed in the duodenum. In some

embodiments, the trough phase in the subject is about the baseline 5-MTHF plasma

concentration in the subject.

[0016] In some embodiments, the 5-MTHF formulations described herein further comprise a filler. In some embodiments, the filler is selected from microcrystalline cellulose, calcium carbonate, lactose, mannitol, dicalcium phosphate, and starch, or a combination thereof. In some embodiments, the filler is selected from microcrystalline cellulose and calcium carbonate, or a combination thereof.

[0017] In some embodiments, the 5-MTHF formulations described herein further comprise a lubricant. In some embodiments, the lubricant is selected from magnesium stearate, stearic acid, and sodium stearyl fumarate. In some embodiments, the lubricant is magnesium stearate. [0018] In some embodiments of the 5-MTHF formulations described herein, the extended release oral dosage form is a tablet. In some embodiments, the tablet further comprises a coating. In some embodiments, the coating is 2-3% of Opadry II. In some embodiments of the 5-MTHF formulations described herein, the tablet maintains its shape during passage through the stomach.

[0019] Different extended release technologies may be useful in achieving prolonged 5- MTHF delivery. In some embodiments, extended release of 5-MTHF is achieved using methods such as those described in US 2008/0206333, which is incorporated herein by reference in its entirety. In other embodiments, extended release of 5-MTHF is achieved using other methods that a person having ordinary skill in these arts would consider for prolonging the release of an encapsulated agent.

[0020] In some embodiments, 5-MTHF is a crystalline salt of 5-methyl-(6R,S)-, -(6S)- or - (6R)-tetrahydrofolic acid. In some embodiments, 5-MTHF is a crystalline salt of 5-methyl- (6R,S)-, -(6S)- or -(6R)-tetrahydrofolic acid said crystalline salt having a water of crystallization of at least one equivalent per equivalent of 5-methyltetrahydrofolic acid. In some embodiments, 5-MTHF is a crystalline (6S)-tetrahydrofolic acid having a purity by weight of more than 98%.

[0021] In some embodiments, provided herein are compositions which provide steady-state levels of 5-MTHF of 0.1-10,000 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 0.1-5,000 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 0.1-5,000 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 1-5,000 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 1-2,500 nM. In some

embodiments are compositions which provide steady-state levels of 5-MTHF of 1-1,000 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 1-750 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 1-500 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 1- 400 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 1-300 nM. In some embodiments are compositions which provide steady-state levels of 5- MTHF of 1-200 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 1-100 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 3-100 nM. In some embodiments are compositions which provide steady- state levels of 5-MTHF of 5-100 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 5-90 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 5-80 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 5-75 nM. In some embodiments are

compositions which provide steady-state levels of 5-MTHF of 10-100 nM. In some

embodiments are compositions which provide steady-state levels of 5-MTHF of 10-75 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 15-100 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 15-75 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 20-100 nM. In some embodiments are compositions which provide steady-state levels of 5- MTHF of 20-75 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 25-100 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 25-75 nM. In some embodiments are compositions which provide steady- state levels of 5-MTHF of 35-65 nM. In some embodiments are compositions which provide steady-state levels of 5-MTHF of 40-60 nM.

[0022] In some embodiments, provided herein are compositions which provide steady-state levels of 5-MTHF of at least 50 nM. In some embodiments, provided herein are compositions which provide steady-state levels of 5-MTHF of at least 100 nM. In some embodiments, provided herein are compositions which provide steady-state levels of 5-MTHF of at least 125 nM. In some embodiments, provided herein are compositions which provide steady-state levels of 5-MTHF of at least 150 nM.

[0023] In some embodiments, provided herein are compositions which provide plasma 5- MTHF concentrations of at least 50 nM for at least 14 hours. In some embodiments, provided herein are compositions which provide plasma 5-MTHF concentrations of at least 50 nM for at least 16 hours. In some embodiments, provided herein are compositions which provide plasma 5-MTHF concentrations of at least 50 nM for at least 18 hours. In some embodiments, provided herein are compositions which provide plasma 5-MTHF concentrations of at least 50 nM for at least 20 hours. In some embodiments, provided herein are compositions which provide plasma 5-MTHF concentrations of at least 50 nM for at least 22 hours. In some embodiments, provided herein are compositions which provide plasma 5-MTHF concentrations of at least 50 nM for 24 hours or more. In some embodiments, said compositions comprise about 1 to about 100 mg of 5- MTHF. In some embodiments, said compositions comprise about 1 to about 50 mg of 5-MTHF. In some embodiments, said compositions comprise about 10 to about 50 mg of 5-MTHF. In some embodiments, said compositions comprise about 20 mg or 40 mg of 5-MTHF.

[0024] In some embodiments, compositions provided herein comprise an oral dosage form or oral delivery system. In some embodiments, said oral dosage form or oral delivery system is a capsule or non-capsule. In certain specific embodiments, said non-capsule dosage form is a tablet or minitablet. In some embodiments, the composition comprises a transdermal or transmucosal dosage form. In some embodiments, the composition comprises an intramuscular dosage form. In some other embodiments, the composition comprises a dosage form for intravenous administration.

[0025] In some embodiments, the oral dosage form or delivery system are used for administration under fed conditions. In some embodiments, the oral dosage form or delivery system are used for administration under fasted conditions. In some embodiments,

administration of the oral dosage form or delivery system composition under fed conditions reduces retention of said oral dosage form or delivery system in the stomach or a delay in gastric emptying.

[0026] In some embodiments, provided herein are compositions, formulations, or medicaments comprising 5-MTHF and which do not comprise an omega-3 fatty acid.

[0027] In some embodiments, provided herein are compositions, formulations, or medicaments comprising 5-MTHF and which further comprise Vitamin B12. In some embodiments, provided herein are compositions, formulations, or medicaments comprising 5- MTHF and which are administered in conjunction with Vitamin B 12.

[0028] In some embodiments, provided herein are methods of decreasing homocysteine levels in the human body by administering an extended release 5-MTHF formulation. In some embodiments, provided herein are methods of preventing or treating disease associated with increased levels of homocysteine levels in the human body, by administering 5-MTHF in an extended release dosage form.

[0029] In some embodiments, provided herein are methods of increasing tetrahydrobiopterin (BH4) levels in the human body by administering an extended release 5- MTHF formulation. In some embodiments, provided herein are methods of preventing or treating disease associated with decreased levels of tetrahydrobiopterin in the human body comprising administering 5-MTHF in an extended release dosage form.

[0030] In some embodiments, provided herein are methods of increasing s- adenosylmethionine levels in the human body comprising administering an extended release 5- MTHF formulation. In some embodiments, provided herein are methods of preventing or treating disease associated with decreased levels of s-adenosylmethionine in the human body comprising administering 5-MTHF in an extended release dosage form.

[0031] In some embodiments, diseases and/or disorders treatable with 5-MTHF provided herein are selected from a disease and/or disorder associated with and/or caused by folic acid deficiency. In some embodiments, diseases and/or disorders treatable with 5-MTHF provided herein are selected from any disease and/or disorder treatable with folic acid.

[0032] In some embodiments, diseases and/or disorders treatable with 5-MTHF provided herein are selected from the group consisting of, but not limited to, a nervous system or neurodevelopmental disease/disorder (e.g. a central nervous system disease exemplified by Cerebral Folate Deficiency (CFD) or Autism Spectrum Disorder (ASD)), other neurological diseases/disorders (e.g. restless leg syndrome or sleep disorders), conditions associated with injury to or infection of the central nervous system (e.g. viral infection), a mental or psychiatric disorder (e.g. psychotic/mood disorders exemplified by depression), a cancer (e.g. a blood-borne cancer exemplified by acute lymphoblastic leukemia), a joint disease/disorder (e.g. arthritis), an autoimmune disease/disorder (e.g. rheumatoid arthritis), a degenerative disease/disorder (e.g. macular degeneration), a soft-tissue disease/disorder (e.g. fibromyalgia), an inflammatory disease/disorder, a pain disease/disorder, a genetic disorder related to hyper- or hypo- methylation, a gastrointestinal disease/disorder, a cardiovascular disease/disorder, a metabolic disease/disorder (e.g. adult-onset cerebral folate deficiency), anemia, chronic fatigue syndrome, and a disease or disorder induced in whole or in part by aging (exemplified by age-related hearing and/or vision loss).

[0033] In some embodiments, compositions provided herein are used to reduce one or more side-effects caused by the use of one or more other active ingredients. In some embodiments, compositions provided herein are used to reduce one or more side-effects caused by the use of one or more folic acid antagonist or antifolates, including for example, methotrexate. In some embodiments, compositions provided herein are used to reduce one or more side-effects caused by the use of one or more other active ingredient used to treat the diseases and/or disorders selected from the group consisting of, but not limited to, a nervous system or

neurodevelopmental disease/disorder (e.g. a central nervous system disease exemplified by Cerebral Folate Deficiency (CFD) or Autism Spectrum Disorder (ASD)), other neurological diseases/disorders (e.g. restless leg syndrome or sleep disorders), conditions associated with injury to or infection of the central nervous system (e.g. viral infection), a mental or psychiatric disorder (e.g. psychotic/mood disorders exemplified by depression), a cancer (e.g. a blood-borne cancer exemplified by acute lymphoblastic leukemia), a joint disease/disorder (e.g. arthritis), an autoimmune disease/disorder (e.g. rheumatoid arthritis), a degenerative disease/disorder (e.g. macular degeneration), a soft-tissue disease/disorder (e.g. fibromyalgia), an inflammatory disease/disorder, a pain disease/disorder, a genetic disorder related to hyper- or hypo- methylation, a gastrointestinal disease/disorder, a cardiovascular disease/disorder, a metabolic disease/disorder (e.g. adult-onset cerebral folate deficiency), anemia, chronic fatigue syndrome, and a disease or disorder induced in whole or in part by aging (exemplified by age-related hearing and/or vision loss).

[0034] Additional embodiments provided herein relate to combinations of 5-MTHF along with one or more active ingredients that are commonly prescribed or used for treatment of and/or prophylaxis of various diseases or disorders in a subject. "Active ingredient" as used herein is meant to include pharmaceutical, nutritional, dietary or veterinary agents included drugs and/or supplements.

[0035] Also provided herein are methods for treating or preventing and/or prophylaxis in a subject a disease or disorder selected from the group consisting of, but not limited to, a nervous system or neurodevelopmental disease/disorder (e.g. a central nervous system disease exemplified by Cerebral Folate Deficiency (CFD) or Autism Spectrum Disorder (ASD)), other neurological diseases/disorders (e.g. restless leg syndrome or sleep disorders), conditions associated with injury to or infection of the central nervous system (e.g. viral infection), a mental or psychiatric disorder (e.g. psychotic/mood disorders exemplified by depression), a cancer (e.g. a blood-borne cancer exemplified by acute lymphoblastic leukemia), a joint disease/disorder (e.g. arthritis), an autoimmune disease/disorder (e.g. rheumatoid arthritis), a degenerative

disease/disorder (e.g. macular degeneration), a soft-tissue disease/disorder (e.g. fibromyalgia), an inflammatory disease/disorder, a pain disease/disorder, a genetic disorder related to hyper- or hypo-methylation, a gastrointestinal disease/disorder, a cardiovascular disease/disorder, a metabolic disease/disorder (e.g. adult-onset cerebral folate deficiency), anemia, chronic fatigue syndrome, and a disease or disorder induced in whole or in part by aging (exemplified by age- related hearing and/or vision loss), comprising administering an extended release composition comprising 5-MTHF, such that said disease or disorder is treated and/or prevented. In some embodiments, said disease or disorder is a CNS disorder. In certain specific embodiments, said CNS disorder is cerebral folate deficiency (CFD). CFD is typically defined by those having ordinary skill in these arts as any neurological syndrome associated with low cerebrospinal fluid (CSF) 5-MTHF, in the presence of normal folate metabolism outside the nervous system.

[0036] In some embodiments, any of the compositions provided herein is used in the treatment of the diseases and disorders described herein.

[0037] Also provided herein are methods for manufacturing an extended release

composition comprising 5-MTHF. Also provided herein are methods for determining the suitability of using an extended release composition comprising 5-MTHF in a subject, wherein said method comprises measuring the subject' s levels of one or more autoantibodies to a folate receptor. In some embodiments, said folate receptor is folate receptor alpha (FRoc). In specific embodiments, if the level of one or more autoantibodies in said subject is above what is generally accepted by those knowledgeable in the arts as normal levels, then said subject is chosen for treatment with an extended release dosage form comprising 5-MTHF.

[0038] Also provided herein are methods for administering an extended release composition comprising 5-MTHF wherein said method comprises administering said composition to a patient or selected subject group that have fasted prior to administration of said composition. "Fasted" typically is meant to be an overnight fast such that patients (or subjects) are administered the composition at least one hour prior to their first meal of the day (i.e. typically breakfast). In some embodiments, "fasted" conditions are such that subjects begin fasting at least 10 or 12 hours before drug administration and fasting continues for 1 or 4 hours following drug administration. Also provided herein are methods for administering an extended release composition comprising 5-MTHF wherein said method comprises administering said

composition to a patient or selected subject group under fed conditions. "Fed" conditions are typically such that the patients/subjects ingest a meal approximately 1-2 hours before being administered a composition disclosed herein. In certain embodiments, under "fed" conditions, subjects start fasting at least 12 hours before morning breakfast and then receive a meal approximately 30 minutes before drug administration.

[0039] The details of one or more embodiments are set forth in the description below.

Other features, objects and advantages will be apparent from the description, the drawings, and the claims. In addition, the disclosures of all patents and patent applications referenced herein are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

[0040] FIG. 1 is a theoretical plot showing the steady-state delivery of 5-MTHF using an extended release (ER) technology disclosed herein in comparison to delivery of 5-MTHF using an immediate release (IR) formulation.

[0041] FIG. 2 is a graph of the average 5-MTHF plasma concentration with the standard error of the mean from animal subjects who were administered a single 10 mg dose of Prototype-

I, a representative 5-MTHF extended release formulation.

[0042] FIG. 3 is a graph of the average 5-MTHF plasma concentration with the standard error of the mean from animal subjects who were administered a single 10 mg dose of Prototype-

II, a representative 5-MTHF extended release formulation.

[0043] FIG. 4 is a graph of the dissolution profiles of various 5-MTHF formulations.

[0044] FIG. 5 is a graph of the average 5-MTHF plasma concentration with the standard error of the mean from animal subjects who were administered a single 20 mg dose of a 5-MTHF extended release formulation of Example 4. 5-MTHF plasma concentration of animals receiving Deplin 15, an immediate release 20 mg 5-MTHF formulation, in a separate study is shown for comparison purposes.

DETAILED DESCRIPTION OF THE INVENTION Certain Terminology

[0045] The singular forms "a," "an," and, "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a drug" includes reference to one or more of such drugs, and reference to "an excipient" includes reference to one or more of such excipients. When ranges are used herein, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range varies between 1% and 15% of the stated number or numerical range.

[0046] The terms "formulation" and "composition," as used herein, are used

interchangeably and refer to a mixture of two or more compounds, elements, or molecules. In some aspects the terms "formulation" and "composition" may be used to refer to a mixture of one or more active agents with a carrier or other excipients.

[0047] As used herein, "5-MTHF" refers to the compound 5-methyltetrahydrofolate, including salt forms, which is also known as levomefolic acid, L-5-methyltetrahydrofolate (L-5- MTHF), L-5-methyltetrahydrofolic acid, (6S)-5-methyltetrahydrofolic acid, (6S)-5-MTHF, (6S)- 5-methyl-5,6,7,8-tetrahydropteroyl-L-glutamic acid, 5-methyltetrahydropteroylglutamate, L- methylfolate, and LMF. In some embodiments, pharmaceutically acceptable salts, prodrugs, polymorphs, solvates, isomers, and derivatives of 5-MTHF are used in the compositions and methods disclosed herein. Examples of salts, prodrugs, polymorphs, solvates, isomers, and derivatives of 5-MTHF that are used in the compositions and methods disclosed herein include, but are not limited to, calcium and/or magnesium salts of 5-MTHF including calcium salt [(L-5- MTHF-Ca)], calcium salt [(6S)-5-MTHF-Ca]; it is also meant to includes the CAS Registry Numbers 129025-21-4 (Calcium salt with an unspecified ratio of L-5-MTHF/Ca2+) and 151533- 22-1 (Calcium salt with specified 1 : 1 ratio of L-5-MTHF/Ca2+). In some embodiments, the salt form of the 5-MTHF is a glucosamine salt.

[0048] L-5-MTHF-Ca has two chiral carbon atoms: the C-atom in position 6 of the pteroyl moiety and the a-C atom in the L-glutamic acid moiety. Consequently there exists the possibility of four stereoisomers: (6S,aS), (6S,aR), (6R,aS), (6R,aR). The naturally occurring isomers of tetrahydrofolic acid and its 5-substituted derivatives are the (6S,aS) and (6R,aS)

diastereoisomers, all of which are included for use in this invention according to the instant disclosure.

Extended Release 5-MTHF Formulations

[0049] Provided herein are extended release oral dosage formulations comprising 5- methyltetrahydrofolate (5-MTHF). In some embodiments, the extended release 5-MTHF oral dosage formulations of the present invention exhibit a biphasic absorption profile upon administration to a subject. In some embodiments, the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject.

[0050] In some embodiments, the release profile exhibits an increase in plasma

concentration between about 6 and 9 hours after administration to the subject, an increase between 6 and 12 hours after administration to the subject, and/or an increase between about 9 and 12 hours after administration to the subject.

[0051] In some embodiments, the extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulations of the present invention comprise a 5-MTHF and one or more of a gumming agent, a mucoadhesion agent, and an antioxidant. In some preferred embodiments, the extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulations of the present invention comprise 5-MTHF, guar gum and/or xanthan gum, hydroxypropyl methylcellulose (HMPC), and ascorbic acid and/or citric acid.

[0052] In a preferred embodiment, the extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulations of the present invention exhibit a biphasic absorption profile upon administration to a subject. In some embodiments, the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject, wherein the plasma concentration in the subject increases between about 6 and 9 hours, between about 6 and 12 hours, or between about 9 and 12 hours after administration to the subject, further wherein the extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulations comprise: (i) 5- MTHF; (ii) one or both of xanthan gum or guar gum; (iii) HPMC; and (iv) ascorbic acid.

[0053] The inventors of the presently disclosed extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulations have made the surprising discovery that these formulations demonstrate a biphasic absorption profile upon administration to a subject, as depicted for example at FIG. 5. Without wishing to be bound by theory, the observed biphasic release profile may be explained by an initial burst phase caused by rapid dissolution and ionization in the acidic stomach of the subject, and corresponding absorption across the stomach wall of 5-MTHF ions, or absorption of dissolved 5-MTHF through transit to the small intestine.

[0054] As demonstrated in FIG. 4, the inventors have demonstrated that inclusion of gumming agents in 5-MTHF formulation provides a decreased dissolution rate in vitro. Again without wishing to be bound by theory, the presence of gumming agents, for example guar and/or xanthan gum, in the 5-MTHF oral dosage formulations of the present inventions, may provide resistance to further dissolution of the oral dosage formulations, which may explain the decrease in absorption rate after the burst phase of the biphasic absorption profile, and may allow a substantial portion of the formulation to then pass to the small intestine as an intact unit, more specifically to the ileum and/or duodenum, of the subject.

[0055] Upon delivery of the oral dosage formulations to the ileum and/or the duodenum, where the pH is substantially higher than in the stomach, dissolution, and absorption of 5-MTHF is expected to slow, corresponding with the drop in plasma levels following the burst phase. Alternatively, the intermediate diminishment of 5-MTHF systemic levels may be due to exhaustion of the immediate release, burst pay-load of the dosage form, followed by a delay in transit of the remaining intact unit from the stomach to the small intestine. Still without any wish to be bound by theory, further absorption of 5-MTHF may then be mediated by transporters expressed on the surface of the ileum and duodenum. This may correspond with the presence of a mucoadhesion agent, for example HPMC, in the oral dosage formulations. The inclusion of a mucoadhesion agent may aid in binding to the surface of the ileum and duodenum, and in this way providing proximity of 5-MTHF to the transporters discussed above. Over time, the mucoadhesive properties of the oral dosage formulations provided herein may overcome a saturation effect, allowing for an extended delivery (and extended increase or steady state plasma levels) of 5-MTHF over time, when compared with formulations lacking a mucoadhesive agent. [0056] In some embodiments, the extended release 5-MTHF oral dosage forms of the present invention further comprise an antioxidant, for example ascorbic acid and/or citric acid, which are thought to protect the 5-MTHF against oxidative damage during steps such as filling and storage, thus enhancing shelf life.

[0057] In sum, the inventors have discovered extended 5-MTHF oral dosage forms that provide a surprising extended release, biphasic release profile upon administration to a subject, as described herein. Without wishing to be bound by theory, this release profile may be explained by the unique and inventive combination of 5-MTHF with (i) a gumming agent (for example guar and/or xanthan gum), which may provide resistance to rapid dissolution and degradation and allows passage of the dosage form to the small intestine from the stomach, (ii) a mucoadhesive agent (for example HPMC), which may provide increased adhesion to the surface of the small intenstine, delivering 5-MTHF in close proximity to the transporters expressed on the surface of the small intestine, thereby overcoming a possible saturation effect; and (iii) an antioxidant (for example ascorbic acid), which may provide protection of the 5-MTHF against oxidative degradation during the formulations' passage through the upper GI tract of the subject.

[0058] In some embodiments, compositions disclosed herein are administered twice a day (b.i.d.). In some embodiments, compositions disclosed herein are administered once a day (q.d.). A variety of methods have been used to prepare extended release compositions of various drugs; and it is contemplated that at least one of these methodologies can be used to prepare extended release 5-MTHF compositions according to the instant disclosure. For example, U.S. Pat. No. 6,759,395 (incorporated herein by reference in its entirety) provides gelatin capsules capable of being adapted to provide extended release of 5-MTHF, e.g. by including within the gelatin capsules granules of 5-MTHF coated with a controlled-release coating, optionally including a pore former, such as sodium alginate and/or a fatty acid, such as stearic acid, or another water- soluble pore former. In some embodiments, extended release 5-MTHF dosage forms disclosed herein include for example osmotic dosage forms, extended release matrices, gastric retentive or mucoadhesive formulations, pulsatile release formulations and extended release formulations coated with one or more enteric coatings, though any technology known to prolong release of an active agent may be used. In some embodiments, an extended release matrix (monolithic core) containing 5-MTHF may be coated with an extended release coating, which may optionally include a pore former (such as sodium alginate, stearic acid or both). Thus, in some embodiments, an extended release formulation of 5-MTHF includes any formulation that has, as a substantial part of that formulation, an extended release component comprising 5-MTHF - that is a component that releases 5-MTHF over a period of more than about 6 hours, particularly about 6 to 24, 12 to 24, 14 to 24 or 18 to 24 hours.

[0059] In some embodiments the 5-MTHF pharmaceutical formulations described herein comprise one or more of the following components in the following amounts:

guar gum 30-70 10.5% - 24.6% xanthan gum 5-15 1.8% - 5.3%

Excipients

[0061] Suitable optional excipients for use in the 5-MTHF pharmaceutical formulations described herein include any commonly used excipients in pharmaceutics and are selected on the basis of compatibility with the active pharmaceutical agent and the release profile properties of the desired dosage form. Excipients include, but are not limited to, binders, fillers, flow aids, disintegrants, lubricants, glidants, gelling agents, plasticizers, stabilizers, surfactants, and the like. A summary of excipients described herein, may be found, for example in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa. : Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999), herein incorporated by reference in their entirety.

[0062] Binders impart cohesiveness to solid oral dosage form formulations: for powder filled capsule formulation, they aid in plug formation that can be filled into soft or hard shell capsules and for tablet formulation, they ensure the tablet remaining intact after compression and help assure blend uniformity prior to a compression or fill step. Materials suitable for use as binders in the solid dosage forms described herein include, but are not limited to,

carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropyl methylcellulose (FIPMC), hydroxypropylmethylcellulose acetate stearate (Aqoate HS-LF and HS),

hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Prosolv 90M® and Avicel®), microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acids, bentonites, gelatin,

polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, a sugar, such as sucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, starch, polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone® XL- 10, and Povidone® K-12), larch arabogalactan,

Veegum®, polyethylene glycol, waxes, sodium alginate, and the like. In some embodiments, the binder is HPMC, hydroxypropyl cellulose, or ethyl cellulose. In some embodiments, the binder is HPMC. In some embodiments, the binder is hydroxypropyl cellulose. In some embodiments, the binder is ethyl cellulose.

[0063] Fillers or diluents increase bulk in the pharmaceutical formulation. Such compounds include e.g., lactose; starch; mannitol; sorbitol; dextrose; microcrystalline cellulose such as Avicel®; dibasic calcium phosphate; dicalcium phosphate dihydrate; tricalcium phosphate;

calcium phosphate; anhydrous lactose; spray-dried lactose; pregelatinzed starch; compressible sugar, such as Di-Pac® (Amstar); hydroxypropylmethylcellulose; sucrose-based diluents;

confectioner's sugar; monobasic calcium sulfate monohydrate; calcium sulfate dihydrate;

calcium lactate trihydrate; dextrates; hydrolyzed cereal solids; amylose; powdered cellulose; calcium carbonate; glycine; kaolin; sodium chloride; inositol; bentonite; and the like. In some embodiments of the pharmaceutical formulations described herein, the filler is lactose, mannitol, or microcrystalline cellulose. In some embodiments, the filler is lactose. In some embodiments, the filler is mannitol. In some embodiments, the filler is microcrystalline cellulose.

[0064] Glidants improve the flow characteristics of a powder mixtures. Such compounds include, e.g., colloidal silicon dioxide such as Cab-o-sil®; tribasic calcium phosphate, talc, corn starch, DL-leucine, sodium lauryl sulfate, magnesium stearate, calcium stearate, sodium stearate, kaolin, and micronized amorphous silicon dioxide (Syloid®)and the like. In some embodiments of the pharmaceutical formulations described herein, the glidant is colloidal silicon dioxide or talc. In some embodiments, the glidant is talc. In some embodiments, the glidant is colloidal silicon dioxide.

[0065] Lubricants are compounds which prevent, reduce or inhibit adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid; calcium hydroxide, talc; a

hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), Lubritab®, Cutina®; higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, magnesium stearate, glycerol, talc, waxes, Stearowet®, boric acid, sodium acetate, leucine, a polyethylene glycol or a methoxypolyethylene glycol such as Carbowax™, sodium oleate, glyceryl behenate (Compitrol 888®), glyceryl palmitostearate (Precirol®), colloidal silica such as Syloid™, Carb-O-Sil®, a starch such as corn starch, silicone oil, a surfactant, and the like. Hydrophilic lubricants include, e.g., sodium stearyl fumerate (currently marketed under the trade name PRUV®), polyethylene glycol (PEG), magnesium lauryl sulfate, sodium lauryl sulfate (SLS), sodium benzoate, sodium chloride, and the like. In some embodiments of the

pharmaceutical formulations described herein, the lubricant is magnesium stearate, stearic acid, or sodium stearyl fumarate. In some embodiments, the lubricant is stearic acid. In some embodiments, the lubricant is sodium stearyl fumarate. In some embodiments, the lubricant is magnesium stearate.

[0066] Disintegrants facilitate breakup or disintegration of the pharmaceutical formulation after administration. Examples of disintegrants include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®; a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® PI 00, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose; a cross- linked starch such as sodium starch glycolate; a cross-linked polymer such as crospovidone; a cross-linked polyvinylpyrrolidone; alginate such as alginic acid or a salt of alginic acid such as sodium alginate; a clay such as Veegum® HV (magnesium aluminum silicate); a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth; sodium starch glycolate; bentonite; a natural sponge; a resin such as a cation-exchange resin; citrus pulp; sodium lauryl sulfate;

sodium lauryl sulfate in combination starch; and the like. In some embodiments of the pharmaceutical formulations described herein, the disintegrant is povidone, crospovidone, HPMC, hydroxypropyl cellulose, or polyvinyl alcohol. In some embodiments, the disintegrant is polyvinyl alcohol. In some embodiments, the disintegrant is hydroxypropyl cellulose. In some embodiments, the disintegrant is HPMC. In some embodiments, the disintegrant is povidone. In some embodiments, the disintegrant is crospovidone.

[0067] Gelling agents include compounds such as polyvinylpyrrolidone, e.g.,

polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30; polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400; polysorbate-80; sodium alginate; gums, such as, e.g., gum tragacanth, locust bean gum, gum acacia, carrageenan gum, guar gum; xanthans, including xanthan gum; sugars;

cellulosics, such as, e.g., sodium carboxymethylcellulose, ethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose; polysorbate-80; sodium alginate; polyethoxylated sorbitan monolaurate; polyethoxylated sorbitan monolaurate; povidone and the like.

[0068] Stabilizers include compounds such as any anti-oxidation agents, e.g., butylated hydroxytoluene (BHT), sodium ascorbate, and tocopherol; buffers, acids, and the like.

[0069] Surfactants include compounds such as sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl

monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF); and the like.

[0070] The aforementioned excipients are given as examples only and are not meant to include all possible choices. Other suitable excipient classes include coloring agents, granulating agents, preservatives, anti-foaming agents, plasticizers and the like. Additionally, many excipients can have more than one role or function, or can be classified in more than one group; the classifications are descriptive only, and are not intended to limit any use of a particular excipient.

Folate Receptor Autoantibodies to Define Drug Levels

[0071] As mentioned above, the presence of autoantibodies to FRoc have recently been investigated for their use in predicting treatment outcomes with reduced folates for certain CNS diseases associated with low CSF folate levels. In particular, a recent study by Frey et al, shows that children having autism spectrum disorder (ASD) who tested positively for folate receptor autoantibodies had significantly higher improvement ratings after folinic acid treatment over a mean period of 4 months compared to those ASD children not expressing such autoantibodies (Frye, R. E. et al. (2013) Molecular Psychiatry; 18, 369-381.) Though not wishing to be bound by any particular theory, the present investigators suggest that treatment with reduced folates, e.g. 5-MTHF, must produce adequate serum and/or plasma folate levels over prolonged periods of time so as to displace the autoantibodies from the folate receptor and thus help to normalize CSF folate levels. Furthermore, the binding affinity of a particular folate receptor autoantibody is used to determine the minimum serum and/or plasma folate concentration needed to displace that autoantibody. As demonstrated in the Examples, an extended release dosage form is a means to prolong delivery of 5-MTHF. Extended release dosage forms may thus allow for bi-daily or, once-a-day dosing.

[0072] The present investigators have therefore determined that the presence of one or more autoantibody to a folate receptor, e.g. FRa, can be used to predict a need for treatment with extended release 5-MTFIF formulations. More specifically, the present investigators have determined that the autoantibody binding affinity to a specific folate receptor can be used to define the duration and/or the 5-MTFIF plasma level needed to out-compete binding at said folate receptor. Extended release 5-MTHF formulations are customized to release at least a defined amount of 5-MTHF over a specific period of time such that adequate 5-MTHF is available to compete with one or more folate receptor autoantibody. CSF folate levels are then normalized using extended release 5-MTHF formulations disclosed herein. Thus, provided herein are methods for predicting the suitability of using an extended release composition comprising 5- MTHF in a subject, wherein said method comprises measuring the subject' s levels of one or more autoantibodies to a folate receptor. In some embodiments, said folate receptor is folate receptor alpha (FRa). In specific embodiments, if the level of one or more autoantibodies in said subject is above what is generally accepted by those knowledgeable in the arts as a normal level, then said subject is chosen for treatment with an extended release dosage form of 5-MTHF. In other embodiments, the binding affinity of a specific folate receptor autoantibody is used to determine the minimum 5-MTHF plasma and/or CSF levels to be achieved by an extended release formulation disclosed herein.

Dosing with Multiple Dosage Units

[0073] In some embodiments, the instant disclosure provides methods for the treatment of one or more diseases or disorders selected from the group consisting of a nervous system or neurodevelopmental disease/disorder (e.g. a central nervous system disease exemplified by Cerebral Folate Deficiency (CFD) or Autism Spectrum Disorder (ASD)), other neurological diseases/disorders (e.g. restless leg syndrome or sleep disorders), conditions associated with injury to or infection of the central nervous system (e.g. viral infection), a mental or psychiatric disorder (e.g. psychotic/mood disorders exemplified by depression), a cancer (e.g. a blood-borne cancer exemplified by acute lymphoblastic leukemia), a joint disease/disorder (e.g. arthritis), an autoimmune disease/disorder (e.g. rheumatoid arthritis), a degenerative disease/disorder (e.g. macular degeneration), a soft-tissue disease/disorder (e.g. fibromyalgia), an inflammatory disease/disorder, a pain disease/disorder, a genetic disorder related to hyper- or hypo- methylation, a gastrointestinal disease/disorder, a cardiovascular disease/disorder, a metabolic disease/disorder (e.g. adult-onset cerebral folate deficiency), anemia, chronic fatigue syndrome, and a disease or disorder induced in whole or in part by aging (exemplified by age-related hearing and/or vision loss) in a patient, comprising administering to the patient an extended release dosage comprising a therapeutically effective amount of 5-MTHF, or a pharmaceutically acceptable salt thereof. In some particular embodiments, the therapeutically effective dose is administered on a once-a-day basis. In some embodiments, the once-a-day dose may be administered in a single dosage unit~e.g. a single tablet, capsule, caplet, etc. In other

embodiments, the dose may be administered as multiple tablets, capsules or caplets. In some embodiments, for instance, a dosage of 1 to 1000 mg of 5-MTHF per day may be divided into two, three, four or more tablets, capsules or caplets of 10 to 100 mg of 5-MTHF per unit dose. In some embodiments, the daily dose is two, three or four tablets, capsules or caplets of 10 to 50 mg of 5-MTHF per dose.

Fed vs. Fasted Dosing

[0074] In some embodiments of the methods disclosed herein, it is advantageous to ensure that the patient is either fed or fasted (e.g. overnight for at least about 6, e.g. about 8, hours). It is considered that food or a carbonated beverage administered at the same time, immediately (i.e. less than about 30, or less than about 15 minutes) before or soon after (e.g. less than about 10 minutes) an extended release 5-MTHF formulation is administered to the patient may increase the rate of gastric emptying, thus increasing the rate of uptake of 5-MTHF from the extended release formulation. Thus, in some embodiments, an extended release 5-MTHF formulation is administered with food or a carbonated beverage. In some embodiments, administration of an extended release 5-MTHF formulation with food or a carbonated beverage hastens the onset of action of 5-MTHF without significantly affecting the long-acting characteristics of the extended release 5-MTHF formulation. In some embodiments of the methods, administration of the 5- MTHF extended release formulation with food impacts the steady-state levels achievable upon multi-dosing.

Combinations of MTHF with Other Active Ingredients

[0075] Some embodiments of the present disclosure relate to combinations of 5-MTHF with one or more active ingredients that are commonly prescribed or used for treatment of and/or prophylaxis of various diseases or disorders listed herein. In some other embodiments, 5-MTHF extended release formulations provided herein are used to reduce one or more side-effects caused by the use of one or more other active ingredient used to treat the diseases and/or disorders selected from the group consisting of, but not limited to, a nervous system or

neurodevelopmental disease/disorder (e.g. a central nervous system disease exemplified by Cerebral Folate Deficiency (CFD) or Autism Spectrum Disorder (ASD)), other neurological diseases/disorders (e.g. restless leg syndrome or sleep disorders), conditions associated with injury to or infection of the central nervous system (e.g. viral infection), a mental or psychiatric disorder (e.g. psychotic/mood disorders exemplified by depression), a cancer (e.g. a blood-borne cancer exemplified by acute lymphoblastic leukemia), a joint disease/disorder (e.g. arthritis), an autoimmune disease/disorder (e.g. rheumatoid arthritis), a degenerative disease/disorder (e.g. macular degeneration), a soft-tissue disease/disorder (e.g. fibromyalgia), an inflammatory disease/disorder, a pain disease/disorder, a genetic disorder related to hyper- or hypo- methylation, a gastrointestinal disease/disorder, a cardiovascular disease/disorder, a metabolic disease/disorder (e.g. adult-onset cerebral folate deficiency), anemia, chronic fatigue syndrome, and a disease or disorder induced in whole or in part by aging (exemplified by age-related hearing and/or vision loss). "Active ingredient" as used herein is meant to include

pharmaceutical, nutritional, dietary or veterinary agents included drugs and/or supplements.

[0076] The present embodiments are further described by the following examples. These examples, while illustrating certain specific aspects of the present embodiments, should not be considered to limit or circumscribe the scope of the disclosed embodiments.

EXAMPLES

[0077] The examples below describe some embodiments of the methods and compositions described herein. Methods and materials that are not specifically described in the following examples are within the scope of the instant disclosure and will be apparent to those skilled in the art with reference to the disclosure herein.

EXAMPLE 1

Steady State 5-MTHF Concentrations for Displacing FRa Autoantibodies

[0078] Folate receptor alpha (FRa) is a member of the folate receptor (FOLR) family and has high affinity for 5-MTFIF and other reduced folic acid derivatives such as folinic acid. FRa transports reduced folates into cells and its absence, through mutation or knock-out, has been associated with a number of neurological and behavioral conditions.

[0079] Function of FRa has also been found to be reduced by the presence of autoantibodies (AuAb) against FRa where antibodies that recognize this self-antigen bind to the receptor and reduce its function through recruitment of immune mediators or the AuAb are able to block the binding of reduced folates to the receptor and inhibit the uptake of folates. The presence of AuAb has also been associated with several disorders including autism, cerebral folate deficiency (CFD) and depression.

[0080] The binding affinity of AuAb to FRa is determined in vitro by incubating increasing amounts of purified, unbound FRa with a constant amount of serum containing AuAb. The amount of FRa that is blocked by AuAb is measured by adding 5-MTHF and then subtracting the amount of bound 5-MTHF from the total 5-MTHF -FRa binding capacity. The ratio of blocked FRa to unbound FRa is used to determine the binding affinity.

[0081] Through a similar methodology, but with fixed ratios of FRa and AuAb, it is found that concentrations of 50 nM or greater of 5-MTHF can displace the FRa AuAb.

[0082] A single 40 mg dose of extended release 5-MTHF keeps the plasma concentration above 50 ng/mL for the duration of treatment and normalizes the CSF folate levels. The theoretical plot in Fig. 1 shows that for an extended release formulation of 40 mg (for the same total daily dose of 2x20 mg) a steady state concentration of 5-MTHF is achieved over the entire 24 hour period.

EXAMPLE 2 Preparation of Mucoadhesive Extended Release 5-MTHF Formulations (Prototype I and II)

[0083] The powder mixture of the extended release (ER) layer was weighed (approximately 502-505 mg) and transferred to the die. The powder was slightly pressed with 500PSI to form the shape. Another 502-505 mg of the powder for the immediate release (IR) layer was weighed and transferred to the die. The final bilayer tablet was formed by pressing with the top punch with 3000PSI pressure.

[0084] The core tablets were coated with Opadry II from Colorcon bilayer modified release (MR) tablets), with about 4% gains in weight.

Prototype I: Composition of 10 mg 5-MTHF Bilayer Tablets:

Prototype I: Composition of 5 mg 5-MTHF IR Layer

Prototype I: Composition of 5 mg 5-MTHF ER Layer

Chitosan 75.00 15.00%

Xanthan Gum 150.00 30.00%

HPMC 25.00 5.00%

Citric Acid/ Antioxidant 15.00 3.00%

Calcium Carbonate 30.00 6.00%

Magnesium Stearate (NF) 35.00 7.00%

500.00 100.00%

Prototype II: Composition of 10 mg 5-MTF1F SR Layer of Bilayer Tablets: Prototype II: Composition of 5 mg 5-MTHF IR Layer

Prototype II: Composition of 5 mg 5-MTHF ER Layer

Percentage (%) Based on

API Excipients Quantity/Tablet (mg)

Core

5-L-MTHF-Ca 6.48 1.30%

Prosolv SMCC 90M (q.s.) 153.52 30.70% Chitosan 85.00 17.00%

Xanthan Gum 150.00 30.00%

HPMC 25.00 5.00%

Calcium Carbonate 30.00 6.00%

Citric Acid/ Antioxidant 15.00 3.00%

Magnesium Stearate (NF) 35.00 7.00%

500.00 100.00%

EXAMPLE 3

Beagle Plasma 5-MTFiF Levels Using Extended Release (ER) 5-MTFiF Formulations (Prototype I and II)

[0085] In vivo experiments were conducted using two different 5-MTHF extended release prototypes.

[0086] Bi-layer extended release tablets comprising 10 mg of 5-MTHF (5 mg immediate release (IR) layer and 5 mg extended release (ER) layer) were formed using various compression techniques. In addition to 5-MTHF, the tablets also contained excipients including an antioxidant, lubricant and thickening agent.

[0087] Beagle dogs were administered a single, orally delivered 10 mg dose of either a representative extended release 5-MTHF composition or a 5-MTHF control (immediate release) composition. Plasma samples were drawn at regular intervals and 5-MTHF levels were recorded for each formulation.

[0088] The graph in Fig. 2 shows the average plasma 5-MTHF concentration from two dogs that were administered a 10 mg dose of prototype-I, a representative bilayer extended release 5- MTHF tablet. As shown in Table 1 below, the average area under the curve (AUC) is 1560 ng.h/mL with an approximate oral bioavailability of 30%.

[0089] The graph in Fig. 3 shows the average plasma 5-MTHF concentration from three dogs that were administered a 10 mg dose of prototype-II. Prototype-II is a 5-MTHF bilayer tablet that has a different extended release layer in comparison to prototype-I. The average AUC using prototype-II is 2223 ng h/mL with an approximate oral bioavailability of 43% (see Table 1).

Table 1 : In Vivo Extended Release 5-MTHF Pharmacokinetics

'AUC has not been corrected for endogenous 5MTHF levels; t=0-8 hours for "Control" and t=0-12 hours for Prototype-I and II

EXAMPLE 4

Preparation of Mucoadhesive Extended Release 5-MTHF Formulations

[0090] The four extended release tablet formulations shown below were prepared according to the following process.

Preparation of Blend for Tableting

[0091] The Prosolv 90M, Calcium Carbonate, Citric Acid, Magnesium Stearate, Ascorbic Acid, HPMC (hydroxypropyl methyl cellulose), guar gum, and xanthan gum were mixed in a mixer for 10 minutes (no ascorbic acid in MR Tablet Formulation #1). 5-L-MTHF-Ca was added to the above mixture and mixed for 5 minutes. The blend was discharged in a container lined with double polyethylene bag liners and labeled as "Final Blend for Core Preparation".

Compression of tablets

[0092] The blend was compressed using single tablet press. The diameter of the tablets was 0.375 inch. The force used for making tablet was 3000psi.

[0093] During compression, the following controls were performed periodically: tablet weight, tablet hardness, and tablet thickness. In addition, dissolution tests were conducted to verify the performance of the tablets.

Coating

[0094] The obtained core tablets were coated with 2-3% of Opadry II (with or without color).

Calcium Carbonate 10 3.5

Citric Acid 30 10.5

Magnesium Stearate 10 3.5

Ascorbic Acid 40 14.0

HPMC 30 10.5

Guar Gum 50 17.5

Xanthan Gum 10 3.5

Prototype V-290 mg %

5-L-MTHF-Ca 26 9.0

Prosolv 90M 79 27.2

Calcium Carbonate 10 3.4

Citric Acid 30 10.3

Magnesium Stearate 10 3.4

Ascorbic Acid 40 13.8

HPMC 35 12.1

Guar Gum 50 17.2

Xanthan Gum 10 3.4

EXAMPLE 5

Dissolution Testing of Mucoadhesive Extended Release 5-MTHF Formulations

[0095] SR8 PLUS Hanson dissolution apparatus was equipped with baskets rotating clockwise at lOORPM during the studies until the last 15 minutes, which 200RPM was used.

Water bath temperature and rotation speed were calibrated internally and externally with thermometers and tachometer. The apparatus temperature control was also within specification of U.S. Pharmacopeia (USP) and National Formulary (NF) (37.0 ± 0.5°C).

[0096] Each 500 mL of 0. IN HC1 media was transferred to the individual vessel.

Approximately 250 mg of ascorbic acid was added into each vessel. The water bath was heated and maintained at 37°C throughout the studies, while maintaining rotation at lOORPM until the last 15 minutes.

[0097] Once the system stabilized, one tablet from the test article was placed into the 0. IN HC1. 1 mL of solution from each vessel was removed from given time interval. The sampled solution was mixed with 3.0 mL of 0.0 IN HC1 solution. The mixed solution was vortexed and filtered through a 25mm syringe filter with 0.2μπι Nylon membrane and polypropylene housing (VWR Cat. No. 28145-487). The obtained samples were stored at 5°C before analysis with HPLC.

[0098] The amount of 5-MTHF release was determined using the assay method developed in house. Fig. 4 shows the dissolution profiles of various 5-MTHF formulations (Deplin 15 is a commercially available immediate release 5-MTHF formulation; Prototype-II and Prototype-V are extended release 5-MTHF formulations prepared in Examples 2 and 4).

EXAMPLE 6

Beagle Plasma 5-MTHF Levels Using Extended Release (ER) 5-MTHF Formulations of Example 5

[0099] In a similar manner as described in Example 3, in vivo experiments were conducted using a 5-MTHF extended release formulation of Example 4 (Prototype-V).

[00100] The data for this study (Fig. 5) is a graph of the average (N = 6) 5-MTHF plasma concentration with the standard error of the mean from animal subjects administered a single 20 mg dose of a 5-MTHF extended release formulation of Example 4. 5-MTHF plasma

concentration of animals receiving Deplin 15, an immediate release 20 mg 5-MTHF formulation (N = 6), in a separate study is shown for comparison purposes. As shown in Fig. 5, an extended release formulation of Example 4 (Prototype-V) exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase), then a decrease in the plasma concentration of 5-MTHF to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) from 6-12 hours and a prolonged 5- MTHF exposure out to 15 hours. In contrast, the Deplin 15 immediate release 5-MTHF formulation shows an initial spike in plasma concentration followed by a return to near baseline levels at 5 hours.

[00101] The graph in Fig. 5 shows the average plasma 5-MTHF concentration from six dogs that were administered a 20 mg dose of prototype-V. Prototype-V is a 5-MTHF single layer tablet that has an extended release (ER) profile in comparison to Deplin 15. The advantage of the ER formulation, in comparison to the IR formulation of Deplin 15 is that the extended absorption that is clearly evident in the second peak provides systemic, therapeutic L-MTHF without the need for additional dosing. Single dosing is an advantage of multi-dosing for a number of reasons. A once-a-day formulation is more convenient and this translates to a much higher likelihood of compliance on the part of patients. In some patient groups, the advantage extends beyond convenience and better compliance, and into the area where the ER formulation makes the difference between therapeutic success, or not. There can be, with some conditions, a strong, difficult to overcome, aversion to solid oral dosage forms. In autism, for example, aversion to ingestion of pills is prevalent. With once-a-day dosing, the timing of the dose can be such as to be in a window of time optimal for the preferences of the patient.

[00102] Additionally, the ER profile lends itself to accumulation of systemic levels from one day to the next. It's anticipated that a multi-day dosing will give rise to a pharmacokinetic steady state wherein the accumulation and elimination rates are balanced, and a constant, narrow range of system 5-MTHF is achieved. This has a therapeutic advantage over an IR formulation in that the variance of the therapeutic, systemic level of 5-MTHF is more narrow. In the case of a pathology where consistent therapeutic drug levels would be an advantage, (such as Cerebral Folate Deficiency) an ER formulation, providing a steady state narrow range of active is significantly superior to an IR product taken multiple times / day, (with the compliance challenges described above) with, even in the ideal situation, widely varying drug levels (from endogenous baseline to Cmax, and then back down again).

[00103] Another advantage of the ER formulation pertains to dose proportionality. 5-MTHF is actively transported in the small intestine, and the transporters are proximally localized. IR formulations therefore are expected to reach a saturation point in dose vs. exposure, beyond which, higher dosing does not give rise to higher absorption. ER formulations displaying the pharmacokinetic profile shown in figure 5, with a second peak separated in time from the first, have the potential to maintain dose proportionality at higher dosing levels. The time separation between peaks allows for renewed transport after the initial burst phase has elapsed. This increases the therapeutic potential of an ER form over IR.

Table 2: In Vivo Extended Release 5-MTHF Pharmacokinetics

[00104] The various embodiments described above can be combined to provide further embodiments. All of the U. S. patents, U. S. patent application publications, U. S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further

embodiments.

[00105] These and other changes can be made to the embodiments in light of the above- detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMS What is claimed is:

1. An extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulation comprising 1) a gumming agent selected from guar gum and xanthan gum, or a combination thereof, 2) a mucoadhesion agent wherein the mucoadhesion agent is hydroxypropyl

methylcellulose (HPMC), and 3) an antioxidant selected from ascorbic acid and citric acid, or a combination thereof; wherein following administration of the 5-MTHF formulation to a subject, the 5-MTHF formulation exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject; wherein the plasma concentration in the subject increases between about 6-12 hours, between about 6-9 hours, or between about 9-12 hours, following administration of the 5-MTHF formulation to the subject.

2. An extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulation comprising 1) a gumming agent, 2) a mucoadhesion agent, and 3) an antioxidant; wherein following administration to a subject, the formulation exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject; wherein the plasma concentration in the subject increases between about 6- 12 hours, between about 6-9 hours, or between about 9-12 hours, following administration of the 5-MTHF formulation to the subject.

3. An extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulation wherein following administration of the formulation to a subject, the formulation exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma

concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject.

4. An extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulation wherein the plasma concentration in a subject increases between about 6-12 hours, between about 6-9 hours, or between about 9-12 hours following administration of the formulation to the subject.

5. An extended release 5-methyltetrahydrofolate (5-MTHF) oral dosage formulation comprising 1) a gumming agent and 2) a mucoadhesion agent.

6. The formulation of claim 5, wherein the gumming agent is selected from guar gum and xanthan gum, or a combination thereof.

7. The formulation of claim 6, wherein the gumming agent is guar gum.

8. The formulation of claim 6, wherein the gumming agent is xanthan gum.

9. The formulation of claim 6, wherein the gumming agent is a combination of guar gum and xanthan gum.

10. The formulation of any one of claims 5-9, wherein the mucoadhesion agent is hydroxypropyl methylcellulose (HPMC).

11. The formulation of any one of claims 5-10, further comprising an antioxidant.

12. The formulation of claim 11, wherein the antioxidant is selected from ascorbic acid and citric acid, or a combination thereof.

13. The formulation of claim 12, wherein the antioxidant is ascorbic acid.

14. The formulation of claim 13, wherein the antioxidant is citric acid.

15. The formulation of claim 14, wherein the antioxidant is a combination of ascorbic acid and citric acid.

16. The formulation of any one of claims 5-15, wherein following administration of the formulation to a subject, the formulation exhibits a biphasic absorption profile, wherein the profile consists of a first increase in the plasma concentration of 5-MTHF (burst phase) in the subject, then a decrease in the plasma concentration of 5-MTHF in the subject to a trough phase, and then a second increase in the plasma concentration of 5-MTHF (diffusion phase) in the subject.

17. The formulation of any one of claims 5-16, wherein the plasma concentration in a subject increases between about 6-12 hours, between about 6-9 hours, or between about 9-12 hours following administration of the formulation to the subject.

18. The formulation of claim 17, wherein the plasma concentration in a subject increases between about 6-12 hours following administration of the formulation to the subject.

19. The formulation of claim 17, wherein the plasma concentration in a subject increases between about 6-9 hours following administration of the formulation to the subject.

20. The formulation of claim 17, wherein the plasma concentration in a subject increases between about 9-12 hours following administration of the formulation to the subject.

21. The formulation of any one of claims 1-4 and 16-20, wherein the second increase in the plasma concentration of 5-MTHF (diffusion phase) corresponds with 5-MTHF absorption in the small intestine.

22. The formulation of claim 21, wherein the second increase in the plasma concentration of 5-MTHF (diffusion phase) corresponds with 5-MTHF absorption in the duodenum.

23. The formulation of claim 21, wherein the 5-MTHF absorption in the small intestine is mediated by transporters expressed in the small intestine.

24. The formulation of claim 23, wherein the 5-MTHF absorption in the small intestine is mediated by transporters expressed in the duodenum.

25. The formulation of any one of claims 1-4 and 16-24, wherein the trough phase in the subject is about the baseline 5-MTHF plasma concentration in the subject.

26. The formulation of any one of claims 1-25, wherein the formulation further comprises a filler.

27. The formulation of claim 26, wherein the filler is selected from microcrystalline cellulose, calcium carbonate, lactose, mannitol, dicalcium phosphate, and starch, or a

combination thereof.

28. The formulation of claim 27, wherein the filler is selected from microcrystalline cellulose and calcium carbonate, or a combination thereof.

29. The formulation of any one of claims 1-28, wherein the formulation further comprises a lubricant.

30. The formulation of claim 29, wherein the lubricant is selected from magnesium stearate, stearic acid, and sodium stearyl fumarate.

31. The formulation of claim 30, wherein the lubricant is magnesium stearate.

32. The formulation of any one of claims 1-31, wherein the extended release oral dosage form is a tablet.

33. The formulation of claim 32, wherein the tablet further comprises a coating.

34. The formulation of claim 33, wherein the coating is 2-3% of Opadry II.

35. The formulation of any one of claims 32-34, wherein the tablet maintains its shape during passage through the stomach.

36. An extended release dosage form comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof.

37. An extended release dosage form comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof, which does not comprise an omega-3 fatty acid.

38. An extended release dosage form comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof, which further comprises vitamin B12 and/or s-adenosylmethionine.

39. A pharmaceutical formulation comprising an extended release dosage form comprising 5- MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof.

40. A method of treating a disease or disorder comprising administering an extended release dosage form of any one of claims 36-38, or a pharmaceutical formulation of claim 39 to a patient in need thereof.

41. A method of treating a disease or disorder in a subject who expresses one or more folate receptor autoantibodies, comprising administering an extended release dosage form of any one of claims 36-38, or a pharmaceutical formulation of claim 39 to the subject.

42. An extended release dosage form of any one of claims 36-38, or a pharmaceutical formulation of claim 39 which provides steady-state levels of 5-MTHF of at least 50 nM.

43. An extended release dosage form of any one of claims 36-38, or a pharmaceutical formulation of claim 39 which provides plasma 5-MTHF concentrations of at least 50 nM for at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours or at least 24 hours or more.

44. An extended release dosage form of any one of claims 36-38, or a pharmaceutical formulation of claim 39 which comprises about 1 to about 100 mg of 5-MTHF.

45. A method of decreasing homocysteine levels in the human body by administering an extended release dosage form comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof.

46. A method of preventing or treating a disease associated with increased levels of homocysteine levels in the human body, comprising administering an extended release dosage form comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof.

47. A method of increasing tetrahydrobiopterin levels in the human body comprising administering an extended release formulation comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof.

48. A method of preventing or treating a disease associated with decreased levels of tetrahydrobiopterin levels in the human body, comprising administering an extended release dosage form comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof.

49. A method of increasing s-adenosylmethionine levels in the human body comprising administering an extended release formulation comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof.

50. The formulation of any one of claims 1-35, comprising a calcium and/or magnesium salt of 5-MTHF.

51. The formulation of claim 50, wherein the salt is [(L-5-MTHF-Ca)], calcium salt [(6S)-5- MTHF-Ca]; CAS Registry Number 129025-21-4 (Calcium salt with an unspecified ratio of L-5- MTHF/Ca2+), or CAS Registry Number 151533-22-1 (Calcium salt with specified 1 : 1 ratio of L-5-MTHF/Ca2+).

52. A method of preventing or treating disease associated with decreased levels of s- adenosylmethionine levels in the human body, comprising administering an extended release dosage form comprising 5-MTHF, or a pharmaceutically acceptable salt, prodrug, polymorph, solvate, isomer, or derivative thereof.