Cross Reference

This application claims priority to U.S Provisional Patent Application Serial Number 62/682055 filed June 7, 2018, incorporated by reference herein in its entirety.

Bac!tgroimo

Despite the potential beneficial effects of polyphenol derivatives, such as catechins, there are many bioavailability problems associated with the oral delivery of catechins especially into the brain. Catechins do not easily penetrate tire human digestive tract and are subject to intestine-based metabolism and rejection, and thus very _' limited amounts of orally administered catechins enter the plasma. Furthermore, the small amount of catechin that enters the bloodstream is rapidly metabolized by the liver and in plasma. Therefore, only very small amounts of orally administered catechins enter the blood and in the brain tissue.

Summary

In one aspect, the disclosure provides intranasal pharmaceutical formulations comprising a polyphenoiic compound and a pharmaceutically acceptable diluent or carrier.

In one embodiment, the polyphenoiic compound comprises a compound selected from the group consisting of flavonoids, curcumin, resveratrol, or pharmaceutically acceptable salts thereof in another embodiment, the phenolic derivative comprises a catechin or a pharmaceutically acceptable salt thereof. In a further embodiment, the catechin comprises one or more of (2S,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro -2H-l- benzopyran-3-yl 3,4,5-trihydroxybenzoate [i.e. (-)GCG], (2R,3S)-5,7-dihydroxy-2-(3,4,5- trihydroxyphenyl)-3, 4-dihydro- 2H- !-benzopyran-3-yl 3,4,5-trihydroxybenzoate [ e

(+)GCG], (2S,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4-dihydro-2H -l -benzopyran-3- yl 3,4,5-trihydroxybenzoate [i.e. (-)CGj, (2R,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy- 3,4-dihydro-2H- l-benzopyran-3-yl 3,4,5-trihydroxybenzoate [i.e. (+)CG], (2R,3R)-2-(3,4- dihydroxyphenyl)-5,7-dihydroxy-3,4-dihydro-2H- 1 -benzopyran-3 -yl 3,4,5- trihydroxybenzoate [i .e. (-)-ECG], (2S,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4- dihydro-2H- l-benzopyran-3-yl 3,4,5-trihydroxybenzoate [i .e. (+)-ECG], (2R,3R)5,7- dihydroxy-2-(3 ,4,5-trihydroxyphenyl)-3, 4-dihydro- 2H-l-benzopyran-3-yl 3,4,5- Trihydroxybenzoate [i .e. (-)EGCG], (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4- dihydro-2H-l-benzopyran-3-yl 3,4-dihydroxy-5- methoxybenzoate [i.e. EGCG-3”-OMe j); (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro -2H- l -benzopyran-3-yl 3,4- Dihydroxybenzoate; (2R, 3R)-5,7-dihydroxy-2-(3, 4, 5-trihydroxyphenyl)-3, 4-dihydro- 2H- 1- benzopyran-3-yl 4-hydroxybenzoate, or pharmaceutically acceptable salts thereof. In another embodiment, die catechin is present in the formulation at between about 1% to about 40% w/w, between about 1% to about 30% w/w, between about 1% to about 25% w/w, between about 4% to about 30% w/w, between about 5% to about 30% w/w, between about 6% to about 30% w/w, between about 8% to about 30% w/w, between about 10% to about 25% w/w, between about 1 1% to about 25% w/w, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,

9%, 10%, 11%, 12% w/w, 24%, 25%, or 30%

In one embodiment, the formulation may further comprise one or more of:

(a) 0 1 -20% w/w'% of a permeation enhancer;

(b) 0.1 _“ 20% w/w% of a chelator/anti-oxidant;

(c) 1 -30% w/w% of a humectant; and/or

(d) 0.03%-2% preservative;

wherein die formulation has a pH of between 4.0 and 6.5

In another embodiment, the permeation enhancer is present at 1.0% to about 20%, 1- 18%, 2-18%, 3-17%, 4-16%, 5-15%, 6-14%, 7-13%, 8-12%, 9-11%, 2.3-10%, 0.1% to 2% or about 10% w/w %. In a further embodiment, the permeation enhancer comprises one or more compounds selected from the group consisting of cyclodextrin or analogs thereof, glycerin, PEG 400, sucrose monolaurate, chitosan, transmucosal delivery enhancement agents including but not limited to alkylsaccharide transmucosal deliver} _' enhancement agents (including but not limited to tetradecyl maltoside (TDM)), or pharmaceutically acceptable salts diereof. In one embodiment, the permeation enhancer comprises (2~HydiOxypropyi)~fl- cyclodextrin (HR-b-cyclodextrin); also referred to as HR-b-CD, or Hydroxypropyl betadex), randomly methylated cyclodextrin (also referred to as IIM-b-€E>), sulfobutylether-b- cyciodextrin (also referred to as 8BE-b-€E>), sucrose monolaurate, pharmaceutically acceptable salts thereof, or combinations thereof.

In another embodiment, the anti-oxidant/chelators is present at 0.05-15%, 0.8-15%, 0.1-15%, 0.1-10%, 0.1-9%, 0.1-6%, or about 0.2%, 1%. 2%, 3%, 4%, 5% w/w%. In a further embodiment, the anti -oxidant comprises one or more compounds selected from the group consisting of ascorbic acid, sodium metabisulfite, sodium bisulfite, tocopherol, or pharmaceutically acceptable salts thereof. In one embodiment, the anti-oxidant comprises ascorbic acid or a pharmaceutically acceptable salt thereof.

In another embodiment, the humectant is present at 1-25%, 1-20%, 1-15%, 1-10%, 1- 9%, 2-8%, 3-7%, 4-6%, or about 5% w/w%. In a further embodiment, the humectant comprises a compound selected from the group consisting of glycerin, PEG (including but not limited to PEG 300, PEG400, and PEG 600), or a pharmaceutically acceptable salt thereof.

In another embodiment, the formulation comprises a pH modifier present at 0.1-2%, 0.5-1.5% or about 1% w/w%. In a further embodiment, the pH modifier comprises a compound selected from the group consisting of sodium hydroxide or a pharmaceutically acceptable salt thereof.

In another embodiment, the formulation comprises a liquid, a powder, a spray, a nose drop, a gel, an ointment, or a combination thereof.

In another aspect, tire disclosure provides methods for treating a central nervous system disorder, a congenital hyperimsulinemia (CHI) disorder, a tumor, diabetes, obesity, or a systemic disorder, comprising administering to a subject in need thereof an amount effective of the intranasal pharmaceutical formulation of any embodiment or combination of embodiments of the disclosure to treat the disorder in the subject.

In another aspect, the disclosure provides methods for treating a central nervous system disorder, a congenital hyperinsulinemia (CHI) disorder, a tumor, diabetes, obesity, or a systemic disorder, comprising intranasal administration to a subject need thereof of an amount effective of a catechin or a pharmaceutically acceptable salt thereof to treat the disorder in the subject. In one embodiment, the catechin comprises one or more of (2S,3R)- 5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-!-be nzopyran-3-yi 3 ,4,5- trihydroxy benzoate [i.e. (-)GCGJ, (2R,3S)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4- dihydro-2H- l-benzopyran-3-yl 3,4,5-trihydroxybenzoate [i.e. (+)GCG], (2S,3R)-2-(3,4- dihydroxyphenyi)~5 ,7~dihydroxy-3 ,4-dihydro-2H~ 1 -benzopyran -3 -yl 3,4,5- trihydroxybenzoate [i .e, (-)CGJ, (2R,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4- dihydro-2H-l-benzopyran-3-yl 3,4,5-trihydroxybenzoate [i.e. (+)CG], (2R,3R)-2-(3,4- dihydroxyphenyl)-5 ,7-dihydroxy-3 ,4-dihydro-2H- 1 -benzopyran-3 -yl 3,4,5- trihydroxybenzoate [i.e. (-)-ECG], (2S,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4- dihydro-2H- l-benzopyran-3-yl 3,4,5-trihydroxybenzoate [i.e. (+)-ECG], (2R,3R}5,7- dihydroxy-2-(3,4,5-trihydroxyphenyi)-3,4-dihydro-2H-l-benzop yram-3-yl 3,4,5- Trihydroxybenzoate [i.e. (-)EGCG], (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4- dihydro-2H-l-benzopyran-3-yl 3,4-dihydroxy-5- methoxybenzoate [i.e. EGCG-3”-OMej); (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro -2H-l-benzopyran-3-yl 3,4- Dihydroxybenzoate; (2R, 3R)-5,7-dihydroxy-2-(3, 4, 5-trihydroxyphenyl)-3, 4-dihydro- 2H-1- benzopyran-3-yl 4-hydroxybenzoate, or pharmaceutically acceptable salts thereof.

In other embodiments of the methods of tire disclosure, the intranasal adminis tration comprises administration to an olfactory mucosa in the upper third of the nasal cavity, or comprises administering to nasal respirator} _' mucosa. In various further embodiments, the disorder comprises a neurodegenerative disease (including but not limited to Alzheimer's Disease, Down Syndrome, multiple sclerosis, Fragile X syndrome, and Parkinson disorders), systemic amyloidosis, cancer (including but not limited to glioblastoma, and a congenital hyperinsulinemia (CHI) disorder (including but not limited to Hyperinsulinemia- Hyperammonemia Syndrome (HHS or HI/HA) and short-chain 3-hydroxyacyl-CoA dehydrogenase (SCAD) deficiency).

In another aspect, the di sclosure provides methods for treating a congenital hyperinsulinemia (CHI) disorder, comprising administration to a subject in need thereof of an amount effective of (-)EGCG, (-)GCG, (+)-GCG, (-)CG, or a pharmaceutically acceptable salt thereof to treat the disorder in the subject. In one embodiment, the CHI disorder comprises Hyperinsulinemia-Hyperammonemia Syndrome (HHS or HI/HA) and short-chain 3-hydroxyacyl-CoA dehydrogenase (SCAD) deficiency. In another embodiment, the administration comprises oral, intravenous, intranasal, intrapulmonary, intrabronchial, subcutaneous, buccal, sublingual, cutaneous, intradermal, intramuscular administration.

Detailed Description

All references cited herein are incorporated by reference m their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure.

Indeed, the present disclosure is in no way limited to the methods and materials described.

As used in the description herein and throughout the claims that follow, the meaning of“a,”“an,” and“the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of“in” includes“in” and“on” unless the context clearly dictates otherwise.

All embodiments disclosed herein can be combined unless the context clearly precludes such combination.

As used herein, the term '‘about ^” means +/- 5% of the recited parameter.

In a first aspect the disclosure provides intranasal pharmaceutical formulations comprising a polyphenolic compound and a pharmaceutically acceptable diluent or carrier.

As used herein, a“polyphenolic compound” is a compound characterized by the presence of 2 or more (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) phenol structural units.

The formulations and methods disclosed herein improve overall bioavailability of polyphenolic compounds by administering via the nasal route in order to deliver them through the nasal mucosa and to reduce the dose required for a beneficial effect.

It was surprisingly discovered herein that polyphenolic compounds exemplified by catechins can be successfully administered by the intranasal route and that the drug is surprisingly more rapidly absorbed following intranasal administration, including in the brain, compared to the corresponding oral dose, leading to a more rapid onset of action and efficacy at lower doses.

in one embodiment, the polyphenolic compound comprises a compound selected from the group consisting of fiavonoids, curcumin, resveratrol, or pharmaceutically acceptable salts thereof. Curcumin is (lA,6/i)-l,7-Bis(4-hydroxy-3-methoxyphenyl)hepta-l,6-diene- 3,5-dione (CAS 458-37-7). Resveratrol is 3,5,4'-trihydroxy-/rara-stilbene (CAS 501-36-0). Fiavonoids have a general structure of a 15-carbon skeleton, which consists of two phenyl rings (A and B) and heterocyclic ring (C). This carbon structure can be abbreviated C6-C3- C6. They can be classified into:

Flavones derived from 2-phenylchromen-4-one

Flavono!s derived from 3-hydroxy-2-phenylchromen-4-one

_* Flavanones derived from 2,3-dihydro-2-phenylchromen-4-one

isoflavenoids, derived from 3-phenylchromen-4-one (3-phenyl- 1,4- benzopyrone) structure

_* neof!avenoids, derived from 4-phenylcoumarine (4-phenyl- 1,2- benzopyrone) structure

_* Flavans which include Flavan-3-ols derived from 2-(3,4- dihydroxyphenyl)-3,4-dihydro-2H-l-benzopyran-3,5,7-triol

Anthocyamdins derived from 2-phenylchromenylium Theaflavins which contain the 3,4,5-trihydroxy- i,8-bis(3, 5,7- trihydroxy-3,4-diliydro-2H- l-benzopyran-2-yl)-6H-benzo[7]annulen-

6-one

In another embodiment, the phenolic derivative comprises a catechin or a pharmaceutically acceptable salt thereof. As used herein, a catechin is a flavonoid compound of formula or its derivatives. Green tea and its major constituent polyphenols are also known as green tea catechins (GTCs); Epigallocatechin-3-gallate (EGCG), the major component of GTCs, accounts for approximately 59% of the total polyphenols in dry green tea leaves. Several other components of GTCs are epicatechin ga!iate (ECG), epigallocatechin (EGC), epicatechin (EC), catechin (C), gallocatechin galiate (GCG), catechin galiate (GC), and gallocatechin (GCj. EGCG and analogs thereof bearing a gallic group in position 3’ can be used for: protection against degenerative diseases;

antiproliferative activity; hypolipidemic activity; prevention of hepatotoxicity;

antitumorigenic effects; prevention of oxidative stress and thus anti-inflammatory actions; reducing blood glucose levels and body weight, thus reducing the risk of stroke and coronar - heart disease. EGCG and analogs thereof bearing a gallic group in position 3 ^’ can also be used for the treatment of numerous central and peripheral di sorders including Down

Syndrome (DS), Alzheimer’s disorders (AD), Multiple sclerosis (MS), Parkinson disorders (PD), Fragile X syndrome, systemic amyloidosis, cancer, and hyperinsulinemia

hyperammonemia syndrome (HHS).

The poor pharmacokinetic properties of EGCG (including limited/no access to the brain, poor absorption, high metabolism in the gastrointestinal tract and liver) and related analogs has precluded its therapeutic use other than at very high concentrations that lead to toxic side effects. The intranasal formulations described herein surprisingly provide significantly enhanced access of the active agent to the systemic circulation and to the brain. This permits use of significantly lower doses thereby causing lesser side effect. The formulations can be used for intranasal deli very of all polyphenoiic compounds.

In various embodiments, the catechin comprises one or more of (2S,3R)-5,7- dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H- Tbenzopyran-3-yl 3,4,5- trihydroxybenzoate j i.e. (-)GCG], (2R,3S)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4- dihydro-2H- l-benzopyran-3-yl 3,4,5 -trihydroxybenzoate [i.e. (+)GCG|, (2S,3R)-2-(3,4- dihydroxyphenyi)-5 ,7-dihydroxy-3 ,4-dihydro-2H- 1 -benzopyran-3 -yl 3,4,5- trihydroxybenzoate [i.e. (-)CG], (2R,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4- dihydro-2H-l-benzopyran-3-yl 3,4,5-trihydroxybenzoate [i.e. (+)CG], (2R,3R)-2-(3,4- dihydroxyphenyl)-5,7-dihydroxy-3,4-dihydro-2H- 1 -benzopyran-3 -yl 3,4,5- trihydroxybenzoate [i.e. (-)-ECG], (2S,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4- dihydro-2H~l -benzopyran-3 -yl 3,4,5-trihydroxybenzoate [i.e. (+)-ECG], (2R,3R)5,7- dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-l-benzop yran-3-yl 3,4,5- Trihydroxybenzoate [i.e. (-)EGCG], (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4- dihydro-2H-l -benzopyran-3 -yl 3,4-dihydroxy-5- methoxy benzoate [i.e. EGCG-3”-OMe]); (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro -2H-l-benzopyran-3-yl 3,4- Dihydroxybenzoate; (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro -2H- 1- benzopyran-3-yl 4-hydroxybenzoate, or pharmaceutically acceptable salts thereof.

In another embodiment, the catechin is present in the formulation at between about 1% to about 40% w/w, between about 1% to about 30% w/w, between about 1% to about 25% w/w, between about 4% to about 30% w/w, between about 5% to about 30% w/w, between about 6% to about 30% w/w, between about 8% to about 30% w/w, between about 10% to about 25% w/w, between about 11% to about 25% w7w, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% w/w, 24%, 25%, or 30%.

In a further embodiment, the formulation may further comprise one or more of:

(a) 0.1-20% w/w% of a permeation enhancer;

(b) 0.1-20% w/w% of a chelator/anti-oxidant;

(e) 1-30% w/w% of a humectant; and/or

(d) 0.03%-2% preservative;

wherein the formulation has a pH of between 4.0 and 6.5

A pemieation enhancer increases permeability of the poiyphenohc compound through title nasal membrane. A humectant helps increase solubility. An anti-oxidant/chelator helps stabilize the poiyphenohc compound from auto-oxidation. A pH modifier helps make the pH physiological and non-irritating (pH 5.0 - 6.5 for nasal mucosa). These components also help in obtaining ideal osmolarity (i.e. 300 - 700 mOsmol/kg) and increasing the viscosity of the solution as such increasing the residence time and improving absorption.

In one embodiment, permeation enhancer is present at about 1.0% to about 20%, 1- 18%, 2-18%, 3-17%, 4-16%, 5-15%, 6-14%, 7-13%, 8-12%, 9-1 1%, 2.3-10%, 0.1 % to 2% or about 10% w/w%. By using a permeation enhancer, it is possible to further improve the aqueous solubility of the polyphenolic compounds such as catechin. Examples of suitable permeation enhancers include HR-b-CD, glycerin, and chitosan, transmucosal delivery enhancement agents including but not limited to alkyisaccharide transmucosal delivery enhancement agents (including but not limited to tetradecyl maltoside (TDM)), or combinations thereof. In various embodiments the permeation enhancer comprises HR-b-CD (such as at a concentration of from about 1.0% to about 20% w/w or any of the alternative embodiments listed for permeation enhancers in general), chitosan (such as at a concentration of from about 0.1% to about 2% w/w or any of the relevant alternative embodiments listed for permeation enhancers in general), glycerin (such as at a concentration of from about 1 % to about 10% w/w or any of tire relevant alternative embodiments listed for permeation enhancers in general), PEG 300 (such as at a concentration of from about 1% to about 20% w/w or any of the relevant alternative embodiments listed for penneation enhancers in general), PEG 400 (such as at a concentration of from about 1% to about 20% w/w or any of the relevant alternative embodiments listed for permeation enhancers in general), PEG 600 (such as at a concentration of from about 1% to about 20% w/w or any of the relevant alternative embodiments listed for permeation enhancers in general), and/or transmucosal delivery- enhancement agents including but not limited to alkyisaccharide, including but not limited to tetradecyl maltoside (TDM) (such as at a concentration of from about 0.1% to about 2% w/w or any of the relevant alternative embodiments listed for permeation enhancers in general).

Using such penneation enhancers we can increase the solubility of polyphenol derivatives such as catechms in water to more than 10% w/w. This allows a more concentrated solution to be administered facilitating a rapid onset of action and reducing irritancy.

One particular formulation comprises catechin, such as GCG, between about 1% and about 30% w/w% or about 10% and about 25% w/w%, or about 10% and about 25% w/w%, or about 1% and about 12% w/w%, m a buffered aqueous solution. The pH of the solution may be adjusted to pH 3-5, or to about pH 4.2 by the addition of a base such as sodium hydroxide. Other particular formulations are exemplified in the examples that follow.

In one embodiment, the permeation enhancer comprises one or more compounds selected from the group consisting of cyclodextrin or analogs thereof, glycerin, PEG 400, sucrose inonolaurate, chitosan, transmucosal delivery- enhancement agents including but not limited to alkyisaccharide transmucosal delivery enhancement agents (including but not limited to tetradecyl maltoside (TDM)), or pharmaceutically acceptable salts thereof. In another embodiment, the permeation enhancer comprises one or more compounds selected from the group consisting of (2-Hydroxypropyl)-p-cyciodextrin (HR-b-cyclodextrin); also referred to as HR-b-CD, or Hydroxypropyl betadex), randomly methylated cyclodextrin (also referred to as KM-b-CD), sulfobutylether-P-cyclodextrin (also referred to as SBE-p-CD), sucrose monolaurate, pharmaceutically acceptable salts thereof, or combinations thereof.

In another embodiment, the anti-oxidant/chelators is present at 0.05-15%, 0.8-15%, 0.1-15%, 0.1-10%, 0.1-9%, 0.1-6%, or about 0.2%, 1%. 2%, 3%, 4%, 5% w/w%.

Additional stabilizers may be used to improve chemical stability of tire formulations; i.e. anti-oxidants such as ascorbic acid, sodium metabisulfite, sodium bisulfite or tocopherol, or metal chelators such as ethyienedaminetetraacetic acid (EDTA).

In one embodiment, the anti-oxidant comprises one or more compounds selected from the group consisting of ascorbic acid, sodium metabisulfite, sodium bisulfite, tocopherol, or pharmaceutically acceptable salts thereof. In another embodiment, the anti-oxidant comprises ascorbic acid or a pharmaceutically acceptable salt thereof.

In a further embodiment, humectants may also be added to improve the patient acceptability of the formulations. In one embodiment, the humeetant may be present at 1- 25%, 1-20%, 1-15%, 1-10%, 1-9%, 2-8%, 3-7%, 4-6%, or about 5% w/w%. In another embodiment, the humeetant comprises a compound selected from the group consisting of glycerin, PEG (including but not limited to PEG 300, PEG400, and PEG 600), or a pharmaceutically acceptable salt thereof

In another embodiment, to avoid nasal irritation the formulations may be buffered to pH 3-8, more preferably 4 to 7 using buffer systems, such as citrate, lactate, sodium hydroxide, or phosphate buffers to control the pH. In addition, osmo!arity may be adjusted so that the formulation is isotonic using standard osmogens (e.g. sodium chloride, mannitol or glucose). In one embodiment, the formulation comprises a pH modifier present at 0.1-2%, 0.5-1.5% or about 1% w/w%. In another embodiment, the pH modifier comprises a compound selected from the group consisting of sodium hydroxide or a pharmaceutically acceptable salt thereof.

The intranasal pharmaceutical formulation may comprise any suitable form for intranasal administration ln various embodiments, the formulation may comprise a liquid, a powder, a spray, a nose drop, a gel, an ointment, or a combination thereof. The

pharmaceutical composition can be formulated, for example, as a nasal emulsion, ointment, gel, (which offer advantages for local application because of their viscosity) or can be, for example powder formulations or nasal sprays. Such sprays typically comprise a solution of the active drug in physiological saline or other pharmaceutically suitable carrier liquids. Various nasal spray compression pumps can be used and calibrated to deliver a

predetermined dose of the active poiyphenolie compound

For example, the nasal formulations may be capable of delivering a poiyphenolie compound dose (such as catechins) between about 1 mg to about 100 mg, or between about 5 mg to 20 mgs per shot (i.e.: per pump of a nasal spray) which can be given as one or more shots per nostril.

For solution formulations typical volumes used to deliver between about 1 rng to about 100 mg, or between about 5 mg to 20 mgs man are 25 to 200 pL, or 75 to 150 pL per dose in each nostril. The intranasal solution formulations can be administered as drops from a nasal dropper bottle or as aerosols after being applied from squeeze bottles, single unit dose or metered-dose pump sprays.

Idle dose of poiyphenolie compound such as a catechin can be combined with a mucoadhesive to enhance its contact with the olfactory mucosa. In some embodiments, the mucoadhesive is selected from the group consisting of a hydrophilic polymer, a hydrogel and a thermoplastic polymer. Preferred hydrophilic polymers include cellulose-based polymers (such as methylce!lulose, hydroxyethyl cellulose, hydroxy propyl methyl cellulose, sodium carboxy methyl cellulose), a carbomer chitosan and plant gum. In some embodiments, the mucoadhesive is selected from the group consisting of poly(lactic acid) ("PLA") and po!y(glycolic acid) ("PGA"), and copolymers thereof. In some embodiments, the mucoadhesive formulation includes a penetration enhancer such as sodium g!yeocholate, sodium taurocholate, L-lysophosphotidy! choline, DMSO and a protease inhibitor. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier, a lipophilic micelle, a liposome, or a combination thereof. For example, the lipophilic micelle or liposome may comprise a ganglioside, a phosphatidylcholine, a

phosphatidylserine, or a combination thereof.

According to some embodiments of intranasal delivery, it can be desirable to prolong the residence time of the pharmaceutical composition in the nasal cavity (e.g , in the olfactory region and/or in the sinus region), for example, to enhance absorption. Thus, the

pharmaceutical composition can optionally be formulated with a bioadhesive polymer, a gum (e.g., xanthan gum), chitosan (e.g., highly purified cationic polysaccharide), pectin (or any carbohydrate that thickens like a gel or emulsifies when applied to nasal mucosa), a microsphere (e.g., starch, albumin, dextran, cyclodextrin), gelatin, a liposome, carbamer, polyvinyl alcohol, alginate, acacia, chitosans and/or cellulose (e.g., methyl or propyl: hydroxyl or carboxy; carboxymethyl or hydroxylpropyl), which are agents that enhance residence time in the nasal cavity. As a further approach, increasing the viscosity of the dosage formulation can also provide a means of prolonging contact of agent with olfactory epithelium.

The pharmaceutical formulation can also optionally include an absorption enhancer, such as an agent that inhibits enzyme activity, reduces mucous viscosity or elasticity, decreases mucociliary clearance effects, opens tight junctions, and/or solubilizes the active compound. Chemical enhancers are known in the art and include chelating agents (e.g., EDTA), fatty acids, bile acid salts, surfactants, and/or preservatives. Enhancers for penetration can he particularly useful when formulating compounds that exhibit poor membrane permeability, lack of lipophilicity, and/or are degraded by aminopeptidases. The concentration of the absorption enhancer in the pharmaceutical composition will vary depending upon the agent selected and the formulation.

To extend shelf-life, preservatives can optionally be added to the pharmaceutical formulation. Suitable preservatives include but are not limited to benzyl alcohol, parabens, thimerosal, chlorobutanol and benzalkoniurn chloride, and combinations of tire foregoing.

The concentration of the preservative will van _' depending upon the preservative used, the compound being formulated, the formulation, and the like. In some representative embodiments, the preservative is present in an amount of 2%, 1%, 0.5%, 0.2%, 0.1%, 0.03%, by weight or less (or not present at all).

The pharmaceutical formulation can optionally contain an odorant to provide a sensation of odor, to aid in inhalation of the composition so as to promote delivery _' to the olfactory epithelium and/or to trigger transport by the olfactory neurons. The pharmaceutical formulations may also optionally include a thickener, which may be present in an amount of 1 %, 0.5%, 0.2%, 0.1 % by weight or less (or not present at all).

Single unit-dose spray can be prepared aseptically or terminally sterilized to produce a sterile final product.

In the formulations, effecti ve concentrations of one or more compounds or pharmaceutically acceptable derivatives is (are) mixed with a suitable pharmaceutical carrier or vehicle. The compounds may be derivatized as the corresponding salts, esters, enol ethers or esters, acids, bases, solvates, hydrates or prodrugs prior to formulation. Any suitable carrier or di luent may be used, including but not limited to a sol vent of di spersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. In another aspect, the disclosure provides method for treating a central nervous system disorder, a congenital hyperinsulinemia (CHI) disorder, a tumor, diabetes, obesity, or a systemic disorder, comprising intranasal administration to a subject in need thereof of an amount effective of a polyphenolic compound (such as a catechin) or a pharmaceutically acceptable salt thereof to treat the disorder in the subject, or by administering an intranasal pharmaceutical formulation of any embodiment of the disclosure, to treat the disorder in the subject. As discussed above, it was surprisingly discovered herein that polyphenolic compounds exemplified by catechins can be successfully administered by the intranasal route and that the drug is surprisingly more rapidly absorbed following intranasal administration, including in the brain, compared to the corresponding oral dose, leading to a more rapid onset of action and efficacy at lower doses. Thus, the methods disclosed herein provide significant benefits over previous methods for using catechins to treat such disorders. Despite the beneficial effects of polyphenol derivati ves, such as catechins, the present inventor noted that there are many bioavailability problems associated with the oral delivery of catechins especially into the brain. The formulations disclosed herein significantly increase exposure in the brain and allow the use of significantly lower therapeutic doses as such increasing the overall safety and significantly reducing toxicity- . Increasing bioavailability of the drag using the formulations disclosed herein will also be beneficial for indications outside the CNS.

In various embodiments, the polyphenolic compound may- comprise one or more of (2S,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro -2H- l-benzopyran-3-yl 3,4,5- trihydroxybenzoate [i.e. (-)GCG], (2R,3S)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4- dihydro-2H-l-benzopyran-3-yl 3,4,5-trihydroxybenzoate [i.e. (+)GCG], (2S,3R)-2-(3,4- dihy droxyphenyl)-5 ,7 -dihydroxy-3 ,4-dihydro-2H- 1 -benzopyran-3 -yl 3 ,4,5 - trihydroxybenzoate [i.e. (-)CGj, (2R,3S)-2-(3 ,4-dihydroxyphenyl)-5, 7-dihydrox -3,4- dihydro-2H-l -benzopyran-3 -yd 3,4,5-trihydroxybenzoate [i.e. (+)GG], (2R„3R)~2-(3,4- dihydroxyphenyi)~5 ,7-dihydroxy-3 ,4-dihydro~2H~ 1 -benzopyran-3 -yl 3,4,5- trihydroxybenzoate [i.e. (-)-ECG], (2S,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4- dihydro-2H-l -benzopyran-3 -yl 3,4,5-trihydroxybenzoate [i.e. (+}~ECG], (2R,3R)5,7- dihydroxy-2-(3, 4, 5-trihydroxyphenyl)-3, 4-dihydro- 2H-l-benzopyran-3-yl 3,4,5- Trihydroxybenzoate [i.e. (-)EGCGj, (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4- dihydro-2H- l-benzopyran-3-yl 3,4-dihydroxy-5- methoxybenzoate [i.e. EGCG-3”-OMe]); (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro -2H-l-benzopyran-3-yl 3,4- Dihydroxy benzoate; (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro -2H-l- benzopyran-3-yl 4-hydroxybenzoate, or pharmaceutically acceptable salts thereof.

In some embodiments, the method of the present disclosure includes applying the pharmaceutical composition to an olfactory area in the upper third of the nasal cavity, such as the olfactory mucosa. In some embodiments, the method of the present invention includes applying the pharmaceutical composition to the nasal respiratory mucosa in order to deliver drug systemically.

In one embodiment, the disorder comprises a neurodegenerative disease, including but not limited to Alzheimer's Disease, Down Syndrome, multiple sclerosis, and Parkinson disorders. In another embodiment, the disorder is selected from the group consisting of systemic amyloidosis, Fragile X, and cancer. In one such embodiment, the cancer comprises glioblastoma.

In another embodiment, the disorder comprises a congenital hyperinsulinemia (CHI) disorder. Congenital hyperinsulinemia is a medical temi referring to a variety of congenital disorders in which hypoglycemia is caused by excessive insulin secretion. Congenital forms of hyperinsulinemia hypoglycemia can be transient or persistent, mild or severe. HHS is a rare autosomal dominant disease manifested by hypoglycemic symptoms triggered by fasting or high-protein meals, and by elevated serum ammonia. It is the second most common cause of hyperinsulinemic hypoglycemia m infancy. It is a rare genetic disease (prevalence is estimated at 1 in 200,000 new bom/yr) caused by activating mutations in GLIJD1, a gene located on chromosome 10q23.3., composed of 13 exons that encode the mitochondrial enzyme glutamate dehydrogenase (GDH) {Hsu, B.Y., A. Kelly, P.S. Thornton, C.R.

Greenberg, L.A. Billing, and C.A. Stanley. J Pedia.tr. 2001, 138(3), 383-389 ]. HHS has profound effects on several major organs. In the pancreas, dysregulated GDH causes an exaggerated insulin response to amino acid consumption, leading to hypoglycemia. In the liver and/or kidneys, increased glutamate catabolism decreases urea synthesis by

carbamoylphosphate synthetase (CPS) because of reduced formation of its activator, N- acetyl-glutamate. In the CNS, there is a high correlation between HHS and childhood-onset epilepsy, learning disabilities, and seizures j Bahi-Buisson, LZ, E. Roze, C. Dionisi, F.

Escande, V. Valayannopoulos, F. Fei!let, and C. Heinrichs. Dev. Med. & Child Neurol. 2008. 50; 945-949] . The over activity of GDH in the brain leads to chronic depletion of brain pools of glutamate and other amino acids that feed into the glutamate pool, especially glutamine. This depletion of glutamate pools could lead to disequilibrium between glutamate and GABA. Altogether, this disequilibrium could contribute to the occurrence of neurological disorders, which could result in epilepsy or developmental delay. Inhibitors of GDH can also be very useful for the treatment of another congenital hyperinsulinemia disorder associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD). It is caused by a mutation in the HADH gene (4q22-q26) encoding the SCHAD protein that has a dual function both as an enzyme and an inhibitor of GDH. It is an ultra-rare genetic disease whose prevalence is estimated at <1 in 1,000,000 new born/yr).

The loss of GTP inhibition of Glutamate Dehydrogenase (GDH) leads to excess insulin secretion. GDH is found in all living organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P) as a coenzyme. GTP is a potent inhibitor of the reaction and acts by increasing the binding affinity for the product, thereby decreasing enzymatic turnover. ADP is an activator of GDH that acts in an opposite manner to GTP by facilitating product release. Leucine is a poor substrate for GDH and an allosteric activator for the enzyme. Its activation is akin to ADP but acts at a site distinct from ADP. Children with HHS have increased b-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. This is likely due to uncontrolled amino acid catabobsm, yielding high ATP levels that stimulate insulin secretion as well as high serum ammonium levels. During glucose-stimulated insulin secretion in normal individuals, it has been proposed that the generation of high energy phosphates (mainly GTP) inhibit GDH and promotes conversion of glutamate to glutamine, which, alone or combined, might amplify the release of insulin. Subsequently, it was postulated that glutamine could also play a secondary messenger role, via a yet unidentified mechanism, and that GDH plays a role in its regulation. Work from Smith, Stanley, and Li has shown that the loss of GTP inhibition of GDH and consequent increase in a-ketoglutarate, cascades to (1) increased mitochondrial oxidation (2) increased cellular phosphate potential (3) elevated ATP/ADP ratio (4) closing of the plasma membrane KATP channels (5) depolarization of the membrane potential (6) opening of voltage gated Ca ²⁺ channels and finally (7) exocytosis of insulin granules [Stanley, C.A., Y.K Lieu, B. Y. Hsu, A.B. Burlina, C.R. Greenberg, NJ Hopwood, K. Perlman, B.H. Rich, E.

Zamrnarchi, and M. Poncz, New England Journal of Medicine 1998, 338, 1352-1357] (-) Epigallocatechin gallate (EGCG), a potent inhibitor of GDH, has been shown to restore GDH function to alleviate HHS-related pathologies [Li C, Allen A, Kwagh J, Doliba N M, Qin W, Najafi H, Collins H Matschinsky F, Stanley CA, and Smith TJ. JBiol. Chern. 2006, 281(15), 10214 10221] As noted earlier, the need to reach the brain is very important to overcome the neurological disorders associated with HHS. Earlier study about EGCG’s brain exposure showed that this molecule is not able to reach the brain in a significant way. Despite some reports which showed some brain exposure, a more in-depth search together with our experimental data showed that EGCG is not able to readily cross the BBB. This data agrees with known algorithms like the CNS MPO model [Wager, TT, Hou, X, Verhoest, PR, and Villalobos, A. ACS Ghent. Neurosci. 2010. 1(6), 435-449] which predict a poor tendency for EGCG (MPO=3.5) to cross the BBB mostly due to high TPS (total polar surface) and HBD (hydrogen bond donors). The methods and formulations of the present disclosure overcome these problems.

In one embodiment, the CHI disorder comprises Hyperins ulinemia-Hyperammonemia Syndrome (HHS or HI/HA) and short-chain 3 -hydroxy acyl-CoA dehydrogenase (SCAD) deficiency.

In another aspect, the disclosure provides methods for treating a congenital hyperinsuiinemia (CHI) disorder, comprising administration to a subject in need thereof of an amount effective of (-)EGCG, (-)GCG, (+)-GCG, (-)CG, or a pharmaceutically acceptable salt thereof to treat the disorder in the subject. The inventor show's in the examples that follow below that (-)GCG, (+)-GCG, (-)CG are significantly better GDH inhibitors that

EGCG, which would be unexpected to those of skill in the art. In one embodiment, the CHI disorder comprises Hyperinsulinemia-Hyperamnionemia Syndrome (HHS or HI/HA) and short-chain 3-hydroxyacyl-CoA dehydrogenase (SCAD) deficiency. In various

embodiments, the administration comprises oral, intravenous, intranasal, intrapulrnonary, intrabronchial, subcutaneous, buccal, sublingual, cutaneous, intradermal, intramuscular administration.

In all embodiments of the methods disclosed herein, the subject may be any suitable subject that might benefit from treatment, including any mammal that might benefit. In one embodiment, the subject is a human subject.

In all embodiments of the methods disclosed herein, "treat" or "treating" means accomplishing one or more of tire following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorders) being treated; (c) inhibiting worsening of symptoms characteristic of the disorders) being heated; (d) limiting or preventing recurrence of the disorders) in patients that have previously had the disorders); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder) s).

The catechins and formulations can be administered as tire sole active pharmaceutical agent, or they can he used in combination with one or more other compounds useful for carrying out the methods of the invention. When administered as a combination, tire therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

compounds for use in the methods described herein are administered to an individual in need thereof.

In therapeutic applications, compositions are administered in an amount sufficient to ca ' out the methods of the disclosure. Amounts effective for these uses depend on factors including, but not limited to, the nature of the compound (specific activity, etc.), the route of administration, the stage and severity of the disorder, the weight and general state of health of the subject, and the judgment of the prescribing physician. The active compounds are effective over a wide dosage range, such as those disclosed herein. However, it will be understood that the amount of the compound actually administered will be determined by a physician, in the light of the above relevant circumstances. Therefore, the above dosage ranges are not intended to limit the scope of the invention in any way.

For administration to non-human mammals, the composition may also be added to the animal teed or drinking water. It may be convenient to formulate these animal feed and drinking water compositions so that the animal ingests an appropriate quantity of the composition during a meal or throughout the course of the day. It may also be convenient to present the composition as a premix for addition to the feed or drinking water.

The following examples illustrate preparation of the formulations of the disclosure.

EXAMPLE 1

An mtranasal solution fonnulation for (-)EGCG was prepared of the following

composition :

Formulation composition follows table above for total test article amount of 100 am. to be performed at room temperature (RT) Adjustments to total preparation amount may be scaled appropriately meeting composition stated in the Table above

Step 1. Add 60 gm of Purified Water, USP to a 250 inL glass beaker containing a magnetic stir bar. Place the glass beaker on a stirring plate and stir the Purified Water, USP at approximately 250 revolutions per minute (RPM).

Step 2. To Step 1 preparation, add 5 gm of Ascorbic Acid, USP in small increments and allow to dissolve.

Step 3. To Step 2 preparation, add 1 gm of Sodium Hydroxide, NF, USP in small increments and allow to dissolve.

Step 4. To Step 3 preparation, add 10 gm of HP-p-cyclodextrin in small increments and allow to hydrate and dissolve. This step may take a few' minutes since it consists in the dissolution of the polymer.

Step 5. To Step 4 preparation, add 5 gm of Glycerin, USP and mix properly to ensure a solution preparation.

Step 6. To Step 5 preparation, add (-)-EGCG in small increments and allow to dissolve.

Step 7. To Step 6 preparation, Q.S. (dilute to weight) to total formulation amount of 100 gm. Using a pH meter (previously calibrated at 4.0 and 7.0 buffer solution) or pH indicator strips for 4.0 - 7.0, verify that final pH of the formulation in Step 6 is approximately 5.5 (range 5.0 - 6.0). Allow solution to continuously mix before vivo dosing.

EXAMPLE 2

Intranasal solution formulation for (-)-GCG was prepared of the following composition;

Formulation composition follows table above for total test article amount of 100 gm. to be performed at room temperature (RT). Adjustments to total preparation amount may be scaled appropriately meeting composition stated in the Table above. Step 1. Add 60 gm of Purified Water, USP to a 250 rnL glass beaker containing a magnetic stir bar. Place the glass beaker on a stirring plate and stir the Purified Water, USP at approximately 250 revolutions per minute (RPM).

Step 2. To Step 1 preparation, add 5 gm of Ascorbic Acid, USP in small increments and allow to dissolve.

Step 3. To Step 2 preparation, add 1 gm of Sodium Hydroxide, NF, USP in small increments and allow to dissolve.

Step 4. To Step 3 preparation, add 10 gm of HR-b-cyclodextrin in small increments and allow to hydrate and dissolve. This step may take a few' minutes since it consists in the dissolution of the polymer.

Step 5. To Step 4 preparation, add 5 gm of Glycerin, USP and mix properly to ensure a solution preparation.

Step 6. To Step 5 preparation, add (-)-GCG in small increments and allow to dissolve.

Step 7. To Step 6 preparation, Q.S. (dilute to weight) to total formulation amount of 100 gm. Using a pH meter (previously calibrated at 4.0 and 7.0 buffer solution) or pH indicator strips for 4.0 - 7.0, verify that final pH of tire formulation in Step 6 is approximately 5.5 (range 5.0 - 6.0). Allow solution to continuously mix before in vivo dosing.

EXAMPLE 3

Intranasal solution formulation for (-)-CG was prepared of the following composition:

Formulation composition follows table above for total test article amount of 100 gm. to be performed at room temperature (RT). Adjustments to total preparation amount may be scaled appropriately meeting composition stated in the Table above Step 1 Add 60 gm of Purified Water, USP to a 250 inL glass beaker containing a magnetic stir bar. Place the glass beaker on a stirring plate and stir the Purified Water, USP at approximately 250 revolutions per minute (RPM).

Step 2. To Step 1 preparation, add 5 gm of Ascorbic Acid, USP in small increments and allow to dissolve.

Step 3. To Step 2 preparation, add 1 gm of Sodium Hydroxide, NF, USP in small increments and allow to dissolve.

Step 4. To Step 3 preparation, add 10 gm of HR-b-cyclodextrin in small increments and allow to hydrate and dissolve. This step may take a few' minutes since it consists in the dissolution of the polymer.

Step 5. To Step 4 preparation, add 5 gm of Glycerin, USP and mix properly to ensure a solution preparation.

Step 6. To Step 5 preparation, add (-)-CG in small increments and allow' to dissolve.

Step 7. To Step 6 preparation, Q.S. (dilute to weight) to total formulation amount of 100 gm. Using a pH meter (previously calibrated at 4.0 and 7.0 buffer solution) or pH indicator strips for 4.0 - 7.0, verify that final pH of tire formulation in Step 6 is approximately 5.5 (range 5.0 - 6.0). Allow _' solution to continuously mix before in vivo dosing.

EXAMPLE 4

Intranasal solution formulation for (-)-GCG was prepared of the following

composition :

Formulation composition follows table above for total test article amount of 100 gm. to be performed at room temperature (RT). Adjustments to total preparation amount may be scaled appropriately meeting composition stated in the Table above.

Step 1. Add 60 gm of Purified Water, USP to a 250 mL glass beaker containing a magnetic stir bar. Place the glass beaker on a stirring plate and stir the Purified Water, USP at approximately 250 revolutions per minute (RPM).

Step 2. To Step 1 preparation, add 10 gm of PEG400 in small increments and allow to dissolve.

Step 3. To Step 2 preparation, add 0.2 gm of EDTA in small increments and allow to dissolve.

Step 4. To Step 3 preparation, add 0.03gm or benzyl alcohol

Step 5. To Step 4 preparation, add 10 gm of HR-b-cyclodextrin in small increments and allow to hydrate and dissolve. This step may take a few minutes since it consists in the dissolution of the polymer.

Step 6. To Step 5 preparation, add 10 gm of (-)-GCG in small increments and allow to dissolve.

Step 7. To Step 6 preparation, Q.S. (dilute to weight) to total formulation amount of 100 gm.

EXAMPLE 5

Intranasal solution formulation for (-)-GCG was prepared of the following

composition :

Formulation composition follows table above for total test article amount of 100 gm. to be performed at room temperature (RT). Adjustments to total preparation amount may be scaled appropriately meeting composition stated in the Table above.

Step 1. Add 60 gm of Purified Water, USP to a 250 mL glass beaker containing a magnetic stir bar. Place the glass beaker on a stirring plate and stir the Purified Water, USP at approximately 250 revolutions per minute (RPM).

Step 2. To Step 1 preparation, add 11 gm of PEG400

Step 3. To Step 2 preparation, add 0.08 gm of EDTA and allow to dissolve.

Step 4. To Step 3 preparation, add 0.1 gm of sucrose monolaurate

Step 5. To Step 4 preparation, add 2.2 gm of HR-b-cyclodextrin

Step 6. To Step 5 preparation, add 11 gm of (-)-GCG in small increments and allow to dissolve.

Step 7. To Step 6 preparation, Q.S. (dilute to weight) to total formulation amount of

100 gm.

EXAMPLE 6

Intranasal solution formulation for (-)-GCG was prepared of the following composition;

Formulation composition follows table above for total test article amount of 100 gm. to be performed at room temperature (RT). Adjustments to total preparation amount may be scaled appropriately meeting composition stated in the Table above.

Step 1. Add 60 gm of Purified Water, USP to a 250 mL glass beaker containing a magnetic stir bar. Place the glass beaker on a stirring plate and stir the Purified Water, USP at approximately 250 revolutions per minute (RPM).

Step 2. To Step 1 preparation, add 10 gm of PEG400

Step 3. To Step 2 preparation, add 0.2 gm of EDTA and allow to dissolve.

Step 4. To Step 3 preparation, add Q.G3g of benzyl alcohol

Step 5. To Step 4 preparation, add dlO gm of Methyi-p-cyclodextrin

Step 6. To Step 5 preparation, add 1 gm of (-)-GCG in small increments and allow to dissolve.

Step 7. To Step 6 preparation, Q.S. (dilute to weight) to total formulation amount of 100 gm.

EXAMPLE 7

Intranasal solution formulation for (-)-GCG was prepared of the following composition:

Formulation composition follows table above for total test article amount of 100 gm. to be performed at room temperature (RT). Adjustments to total preparation amount may be scaled appropriately meeting composition stated in the Table above.

Step 1. Add 60 gm of Purified Water, USP to a 250 mL glass beaker containing a magnetic stir bar. Place the glass beaker on a stirring plate and stir the Purified Water, USP at approximately 250 re volutions per minute (RPM).

Step 2. To Step 1 preparation, add 10 gm of PEG400

Step 3. To Step 2 preparation, add 0.2 gm of EDTA and allow to dissolve.

Step 4. To Step 3 preparation, add 0.03g of benzyl alcohol

Step 5. To Step 4 preparation, add dlO gm of HR-b-cyclodextrin

Step 6: to Step 5 add 0.1 g of Hydroxypropyl methyl cellulose (HPMC)

Step 7. To Step 6 preparation, add 1 gm of (-)-GCG in small increments and allow to dissolve.

Step 8. To Step 7 preparation, Q.S. (dilute to weight) to total formulation amount of

100 gm.

To measure the efficacy of intranasal drug delivery system applied to catechm we have tested the above formulations in mice and measured the concentration of drag both in blood and brain. The table below shows the exposure level using tranasal route and the comparison of exposure level to other route of administrations (i.e. oral and subcutaneous).

ND = Not Detected; N/A = Not Available

Using the formulation in the intranasal route we achieved several fold improvements in systemic exposure (blood) compared to oral administration and many folds improvement in brain exposure was achieved for both catechins tested. The remarkable improvement in bioavailability will allow dosing with much lower level of the drug to achieve therapeutic effects. Since the drag is mostly metabolized in the gastrointestinal tract and liver, the fonnulations can achieve much higher drag level in blood. Furthermore, because liver toxicity at high doses are seen for some catechins (i.e. EGCG), the use of the present formulations which use less drug and bypass the liver (first-pass metabolism) should help in reducing important side-effects that precluded the use of it in a clinical setting.

Example 8. GDH ICS O’ s GDH Inhibition by Catechins

Example 9. GDH in vitro assay

The results in the above table resulted from performing the following assay:

The bovine GDH (bGDH) used for these assays was obtained from Sigma Aldrich Chemical Co as a concentrated stock solution preserved with 50% glycerol. GDH was diluted to a final concentration of 0.01 mg/mL in 0.1 M sodium phosphate buffer, pH 8.0. The substrate mix consisted of 2.0 mM NAD and 200 mM sodium glutamate in 0.1 M sodium phosphate buffer, pH 8.0. The stop solution for this reaction was 0.8% sodium dodecyl sulfate in water. Test compound is dissolved in DMSO and added to the cuvete. The enzyme solution was added followed by the addition of the substrate solution. Hie resulting solution is quickly mixed and NADH production is monitored at 340 nm wavelength.