WO2011047412A1

WO2011047412A1 - Tyrosine and l-dopa for reducing l-dopa incorporation into proteins

Info

Publication number: WO2011047412A1
Application number: PCT/AU2010/001333
Authority: WO
Inventors: Kenneth Rodgers
Original assignee: The Heart Research Institute Ltd
Priority date: 2009-10-22
Filing date: 2010-10-11
Publication date: 2011-04-28

Abstract

The present invention relates to the field of therapy and prophylaxis of complications and/or conditions associated with dopamine deficiency and/or insufficiency, and compositions for the treatment or prevention of such complications and/or conditions e.g., compositions comprising L-dopa and tyrosine.

Description

TYROSINE AND L-DOPA FOR REDUCING L-DOPA INCORPORATION INTO PROTEINS

Field of the invention

The present invention relates to the field of therapy and prophylaxis of complications 5 and/or conditions associated with dopamine deficiency and/or insufficiency, and compositions for the treatment or prevention of such complications and/or conditions e.g., compositions comprising L-dopa and tyrosine. The compositions may include additional agents that modulate the metabolism of L-dopa or dopamine. 0 Background of the invention

Dopamine is thought to be synthesized by dopaminergic neurons and is thought to play an important role in regulating motor skills and also emotion based behaviour (Chinta, S.J and J.K. Anderson, 2005, Int. J. Biochem. Cell Biol., 37(5): 942-946). Several conditions such as neurodegenerative conditions e.g., Parkinson's disease, Alzheimer's5 disease, Huntington's disease, cerebral aging, age-related cognitive impairment, dementia e.g., senile dementia, and restless leg syndrome, are associated with degeneration of dopaminergic neuron cells in the brain and loss of the neurotransmitter dopamine. Dopamine insufficiency is identified by any one of a number of art- recognized methods e.g., positron emission tomography and/or single photon emission0 tomography. Such conditions may be ameliorated by treatment with L-dopa.

For example, Parkinson's disease (PD) is a progressive, chronic and incurable neurodegenerative disorder affecting over four million people worldwide, with over 1.5 million of these in the US, and over 60,000 of these in Australia alone, causing5 increased disability of movement in afflicted subjects. The main clinical features of Parkinson's disease include resting tremor, rigidity, bradykinesia and postural instability. At the cellular level, Parkinson's disease is characterized by the selective loss of large neuromelanin (NM) pigment-containing dopaminergic neurons in the substantia nigra (SN) region of the brain, and by the presence of Lewy bodies (LB) in0 some surviving neurons. When the first motor symptoms of Parkinson's disease develop, at least 60% of dopaminergic neurons have already degenerated, resulting in severe compromised nigrostrial pathway and a decline in striatal dopamine.

In another example, Alzheimer's disease is a neurodegenerative disorder of the central5 nervous system (CNS) affecting as many as 5.3 million people in the US alone, causing increased disability due to memory loss, mood and personality changes in afflicted subjects. Alzheimer's disease is pathologically characterized by atrophy of the cerebral cortex and by a massive loss of cortical neurons and cholinergic projections of the nuclei basalis towards the cortex. At the cellular level, Alzheimer's disease is characterized by diffuse presence of extracellular and perivascular neuritic plaques which are the result of the aggregation of a beta-amyloid (beta. A) peptide leading to the development of amyloid plaque formation followed by neurodegenerative changes. Although the relationship between protein deposits and neurodegeneration is unclear, the aggregation process is important in neuronal function impairment and it may ultimately lead to neuronal death, and loss of some neurotransmitters including dopamine.

Amongst the various disturbances of neurotransmission accompanying cerebral ageing, dopaminergic insufficiency is undoubtedly the most constant, the earliest and the most severe. Dopaminergic insufficiency affects the three ascending dopaminergic systems and explains many of the motor, emotional, affective and cognitive disorders associated with cerebral ageing e.g., slowness, apathy, learning difficulty, memory loss, reduced problem-solving ability, capacity for abstraction, conceptualization, reasoning,. They probably reflect disafferentation of the frontal lobe, deprived of effective dopaminergic innervation. For example, see Ollat, J Neurol. 239: 1432-1459 (1992).

Although the precise aetiology of primary restless leg syndrome is unknown, it is widely accepted that the condition arises due to a dopamine deficit in the central nervous system (CNS) which develops due to a genetic predisposition e.g., Turjanski et al, Neurol. 52: 932-37 (1999).

The compound L-3,4 dihydroxyphenylalanine (syn. levodopa or L-dopa), is a close structural analogue of tyrosine, has been identified as the precursor of dopamine in catecholaminergic neurons, and has become a widely used therapeutic agent for symptomatic relief of conditions associated with dopamine deficiency, such as Parkinson's disease. Upon administration L-dopa is thought to cross the blood-brain barrier and be taken up by the remaining dopaminergic neurons where it is converted into dopamine by the enzymatic action of aromatic amino acid decarboxylase (AADC) e.g., dopa decarboxylase (DDC). Chronic treatment with L-dopa may be detrimental to subjects being treated with L- dopa (Racol et al., 2003, Ann. Neurol. 53:S3-S15, and Olanow and Mizuno, 2005, Mov. Disord. 20:643-644) with some studies suggesting that L-dopa might even be neurotoxic (Melamed et al., 2000, J. Neurol., 247:11135-139, and Muller et al., 2004, J. Neurol. 251:VI/44-VI46). Initially, oral administration of L-dopa dramatically may improve many of the symptoms associated with Parkinson's disease, however as the disease progresses over several years the behavioural response to L-dopa alters and the subject being treated may develop dyskinesias, motor fluctuation or psychosis. Several in vitro studies have shown that L-dopa is toxic to dopaminergic neurons (Pardo et al., 1995 Brain Res., 682(1-2):133-143; Basma et al., 1995, J. Neurochem., 64(2):825-832; and Clement et al., 2002, J. Neurochem. 81(3):414-421), however no single mechanism of action has been conclusively attributed to this toxicity in dopaminergic neurons. Nevertheless this raises a concern that L-dopa, while initially symptomatically effective in treated subjects such as those with Parkinson's disease, it may in fact accelerate neurodegeneration e.g., following chronic or long term administration. Additionally, only 1-5% of L-dopa enters the dopaminergic neurons. The remaining L-dopa is often metabolised to dopamine elsewhere, causing a wide variety of side effects in other cells and organs. For example, due to feedback inhibition, L-dopa results in a reduction in the endogenous formation of L-dopa, and so eventually becomes counterproductive.

To help prevent the breakdown of L-dopa before it crosses the blood-brain barrier (that is, in the periphery) or to help prevent the metabolism of L-dopa before it reaches the dopaminergic neurons, dopa decarboxylase (DDC) inhibitors e.g., carbidopa and/or benserazide may be co-administered with L-dopa. The addition of carbidopa and/or benserazide increases its half-life, allows lower doses of levodopa to be used, reducing side effects from L-dopa such as nausea and vomiting. Catechol- O-methyl transferase (COMT) is an intracellular enzyme which introduces a methyl group (donated by S- adenosyl methionine /SAM) to catecholamines such as L-dopa. COMT inhibitors thus reduce the metabolism of L-dopa and prolong its in vivo activity. COMT and DDC inhibitors (and any other agents that interfere with L-dopa metabolism) increase the half-life of L-dopa and the availability of L-dopa to the CNS and thus have the potential to increase the toxicity of L-dopa due to its misincorporation into proteins. Recent studies have shown that L-dopa may be used by mammalian cells in vitro as a substrate in protein synthesis, and can become incorporated into proteins by cultured cells (Rodgers et al., 2006, J. Neurochem., 98:1061-1067; Rodgers et al., 2004, Free Radic. Biol. Med., 37(11):1756-1764; and Rodgers et al., 2002, Free Radic. Biol. Med., 32:766-775). Notwithstanding adverse effects of L-dopa e.g., L-dopa associated toxicity, L-dopa remains the most widely used and most effective treatment of conditions associated with dopamine deficiency such as Parkinson's disease, there remains a need in the art for compositions of matter and methods for alleviating, treating or preventing conditions associated with dopamine loss and/or conditions that can be ameliorated by treatment with L-dopa, but which reduce and/or prevent the side effects of L-dopa in a cell and/or tissue and/or subject. Particularly, there is a need for compositions and methods for reducing and/or preventing cellular or tissue toxicity in subjects undergoing treatment e.g., long term or chronic treatment with L-dopa.

Summary of the invention

1. Introduction

In work leading to the present invention, the inventors sought to identify the biochemical mechanisms involved in alleviating and/or reducing and/or preventing the toxicity side effects of L-dopa in a cells and/or tissue associated with L-dopa treatment. The inventors also sought to identify mechanisms that may lead to the toxic effects of L-dopa.

The inventors hypothesized that treatment with L-dopa leads to mis-incorporation of L- dopa into proteins in place of tyrosine during cellular protein synthesis and that such modified proteins have the potential to mis-fold and form covalent cross-links with other amino acids residues in other proteins thereby forming insoluble aggregates in cells. The inventors reasoned that proteins containing mis-incorporated L-dopa accumulate in cells e.g., in the CNS, and contribute to the loss of function and to the toxicity and/or neuronal degeneration that is associated with prolonged L-dopa treatment. Proceeding on this basis, the inventors reasoned that co-administration of L- dopa in combination with tyrosine to a cell, tissue or subject reduces and/or prevents L- dopa incorporation into proteins, thereby reducing adverse side effects such as cellular cytotoxicity and/or neurodegeneration associated with L-dopa treatment. Such advantages are without necessarily impairing the functionality of L-dopa as a substrate for AADC in vivo or decreasing dopamine synthesis.

As used herein, the term "L-dopa" or "levodopa" shall be taken to include L-3,4 dihydroxyphenylalanine or any salt, solvate or hydrate thereof or any functional analogue or derivative thereof or any substantially isolated or purified form thereof capable of being converted to dopamine e.g., in a doperminergic neuron, as formulated in a standard pharmaceutical composition e.g., for the treatment of Parkinson's disease. For example, L-dopa may be formulated as an ester e.g., L-Dopa methyl-ester.

As used herein, the term "tyrosine" shall be taken to mean L-tyrosine, 4- hydroxyphenylalanine or 2-amino-3(4-hydroxyphenyl)-propanoic acid or any salt, solvate or hydrate thereof, racemic mixture, enantiomer or stereoisomer of L-tyrosine capable of being incorporated by cellular translation process(es) into a peptide or protein, and/or converted by cellular process(es) into L-tyrosine, and to any and/or all racemic mixtures comprising such an enantiomer or stereoisomer.

As used herein, the term "dopamine agonist" shall be taken to mean a compound that activates dopamine receptors in such conditions as reduced concentrations or absence of dopamine. Dopamine agonists as described herein refers also to any salt, solvate or hydrate thereof

As used herein, the terms "incorporate", "incorporation" and/or "incorporated" in the context of L-dopa and/or tyrosine residues in a protein, peptide or polysaccharide, shall be taken to include any process by which L-dopa and/or tyrosine are incorporated into proteins in a cell and/or tissue, including any transcription process and/or translation process that leads to the synthesis of a peptide or protein in a cell.

As used herein, the term "administer" or "administration" shall be taken to mean that a composition according to any example hereof is provided or recommended to a cell, tissue or subject such as a cell, tissue or subject in need thereof e.g., in a single or repeated or multiple dosage by any administration route.

As used herein, the term "co-administer" or "co-treat" or similar terms shall be taken to mean a simultaneous and/or sequential administration of a plurality of integers e.g., compositions or formulations, active agents or substances whether or not the plurality of integers are combined or separate. In one example a plurality of integers is combined e.g., into a single formulation, and administered as one or more dosages. In another example, a plurality of integers is administered separately at the same time, or sequentially. As with a combination of the plurality of integers, one or more dosages of each integer of the plurality may be administered separately. As exemplified herein, the inventors have demonstrated using in vitro SH-SY5Y neuronal cell studies that co-treatment with L-dopa and tyrosine prevents and/or reduces L-dopa incorporation into proteins. In particular, the inventors have shown that increasing the tyrosine concentration to above physiological concentrations of tyrosine found in the cerebrospinal fluid (CSF)/brain of Parkinson's disease patients reduced the incorporation of L-dopa into proteins. As exemplified herein, L-dopa can compete with tyrosine for intestinal absorption, uptake into the cells and transport across the blood-brain barrier, but that only L-dopa incorporation into proteins is particularly sensitive to tyrosine concentrations. In one example, such a beneficial effect of co-treatment with L-dopa and tyrosine does not substantially impair the functionality of L-dopa as a precursor of dopamine in vitro. In another example, such a beneficial effect of co-treatment with L-dopa and tyrosine does not substantially impair the functionality of L-dopa as a precursor of dopamine in vivo. The inventors have also demonstrated that even at low tyrosine concentration, where tyrosine competes with L-dopa for uptake by the cells, there is no change in a level of dopamine synthesis, demonstrating that cellular uptake of L-dopa is relatively insensitive to tyrosine concentration. The data herein therefore indicate that tyrosine co-administration with L-dopa is beneficial.

The inventors have also demonstrated that L-dopa at high concentration is cytotoxic to cells, and is a potent inducer of apoptosis. However the inventors did not detect evidence of apoptosis in cells incubated in the presence of both L-dopa and tyrosine, indicating that co-incubation of L-dopa and tyrosine may prevent L-dopa-induced toxicity.

The inventors show that L-dopa incorporated proteins are detected in the brains of L- dopa-treated human subjects, suggesting that L-dopa is generally incorporated into proteins in the brains of subjects treated with L-dopa e.g., subjects suffering from Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age- related cognitive impairment, dementia, restless leg syndrome, or other neurodegenerative conditions characterized by dopamine insufficiency. Similarly, L- dopa is incorporated into proteins of rat brains e.g., 21 days of treatment with L-dopa. These results demonstrate that L-dopa-incorporated proteins accumulate in brain cells and tissue following long term treatment of humans and other mammals with L-dopa. 2. Embodiments

The scope of the invention will be apparent from the claims as filed with the application that follow the examples. The claims as filed with the application are hereby incorporated into the description. The scope of the invention will also be apparent from the following description of specific embodiments and/or detailed description of preferred embodiments.

In one example, the present invention provides a pharmaceutical composition for reducing or preventing L-dopa cytotoxicity, said composition comprises an amount of L-dopa or a salt, solvate or hydrate thereof and an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein.

In another example, the present invention provides a pharmaceutical composition for reducing or preventing incorporation of L-dopa into peptide or protein, wherein said composition comprises an amount of L-dopa or a salt, solvate or hydrate thereof, and an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein. In another example, the present invention provides a pharmaceutical composition for reducing or preventing formation of proteinaceous aggregates e.g., Lewy Bodies, comprising L-dopa in a cell, tissue or subject, wherein said composition comprises an amount of L-dopa or a salt, solvate or hydrate thereof, and an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein, thereby reducing or preventing formation of said proteinaceous aggregates in a cell, tissue or subject.

In yet another example, the present invention provides a pharmaceutical composition for enhancing synthesis of dopamine in a cell, tissue or subject, pharmaceutical composition for enhancing synthesis of dopamine in a cell, tissue or subject, wherein said composition comprises an amount of L-dopa or a salt, solvate or hydrate thereof, and an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein, thereby enhancing dopamine synthesis in a cell, tissue or subject. In one example, a pharmaceutical composition according to any example described hereof, comprises an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce and/or prevent incorporation of L-dopa or a salt, solvate or hydrate thereof into a peptide or protein by incorporation of tyrosine or a salt, solvate or hydrate thereof into said peptide or protein. In one such example, the composition comprises an amount of tyrosine a salt, solvate or hydrate thereof sufficient to compete with L- dopa a salt, solvate or hydrate thereof for incorporation into a peptide or protein by any cellular translation process(es). Preferably, the amount of L-dopa or salt, solvate or hydrate thereof is sufficient to provide a substitution therapy for dopamine when administered to a subject in the presence of the amount tyrosine. For example, the relative amount of L-dopa or salt, solvate or hydrate thereof and tyrosine or salt, solvate or hydrate thereof are such that L-dopa converted to dopamine in a cell, tissue or subject when co-administered with tyrosine sufficient to ameliorate a dopamine deficiency therein.

In one example, a composition as described herein according to any example comprises an amount of L-dopa a salt, solvate or hydrate thereof sufficient for the biosynthesis or formation of dopamine from said L-dopa a salt, solvate or hydrate thereof in a cell, tissue and/or subject e.g., in the presence of tyrosine or a salt, solvate or hydrate thereof. In one example, the composition comprises an amount of L-dopa, a salt, solvate or hydrate thereof sufficient for the formation of dopamine from said L-dopa a salt, solvate or hydrate thereof by an aromatic-L-amino-acid decarboxylase such as DOPA Decarboxylase or DDC present in a cell, tissue and/or subject. In one example, a composition as described herein according to any example is formulated to deliver an amount of L-dopa a salt, solvate or hydrate thereof from 400 to about 1200 mg per day of said L-dopa or a salt, solvate or hydrate thereof to a subject.

In another example, an amount of tyrosine or a salt, solvate or hydrate thereof is sufficient to reduce and/or prevent incorporation of L-dopa or a salt, solvate or hydrate thereof into a peptide or protein without substantially impairing the formation of dopamine from L-dopa or a salt, solvate or hydrate thereof in a cell, tissue and/or subject. In another example, an amount of tyrosine a salt, solvate or hydrate thereof is sufficient to reduce and/or prevent L-dopa cytotoxicity in a cell, tissue and/or subject. In such an example, the composition comprises an amount of tyrosine a salt, solvate or hydrate thereof sufficient to reduce and/or prevent L-dopa cytotoxicity in a cell, tissue and/or subject thereby preventing neuron degeneration, including but not limited to the progressive loss of structure or function of neurons, or death of neurons. For example, the amount of tyrosine a salt, solvate or hydrate thereof may be sufficient to reduce and/or prevent L-dopa cytotoxicity in a cell, tissue and/or subject thereby reducing and/or preventing activation of any cellular apoptosis process(es) that is/are induced by L-dopa and/or by accumulation of peptides or proteins or proteinaceous aggregates comprising L-dopa e.g., Lewy Bodies.

In another example, an amount of tyrosine or a salt, solvate or hydrate thereof is sufficient to reduce and/or prevent incorporation of L-dopa or a salt, solvate or hydrate thereof into a peptide or protein thereby enhancing biosynthesis of dopamine from L- dopa or a salt, solvate or hydrate thereof in a cell tissue and/or subject. For example, the an amount of tyrosine or a salt, solvate or hydrate thereof is sufficient to reduce and/or prevent incorporation of L-dopa or a salt, solvate or hydrate thereof into a peptide or protein thereby increasing the amount of L-dopa or a salt, solvate or hydrate thereof available as substrate for dopamine biosynthesis. In one such example, the composition comprises an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to enhance the formation of dopamine from said L-dopa a salt, solvate or hydrate thereof by an aromatic-L-amino-acid decarboxylase such as DOPA Decarboxylase or DDC present in a cell, tissue and/or subject.

In one example, the pharmaceutical composition according to any example described hereof, further comprises one or more dopamine agonists. A non-limiting example of a dopamine agonist is pergolide, bromocriptine. The one or more dopamine agonists may be administered in conjunction with L-dopa and tyrosine. The administration of a composition that comprises an amount of L-dopa, an amount of tyrosine and an amount of one or more dopamine agonists to a patient may comprise a first step in a delivery method to treat or prevent complications and/or conditions associated with dopamine deficiency and/or insufficiency. The one or more dopamine agonists may be administered in conjunction with L-dopa and tyrosine. The administration of a composition that comprises an amount of L-dopa, an amount of tyrosine and an amount of one or more dopamine agonists to a patient may comprise a therapeutic option in a delivery method to treat or prevent complications and/or conditions associated with dopamine deficiency and/or insufficiency. In one example, the pharmaceutical composition according to any example described hereof, further comprises one or more agents that inhibits or prevents the metabolism of dopamine. An example of an inhibitor of dopamine metabolism is a monoamine oxidase (or MAO) inhibitor such as an inhibitor of MAO-type A and/or M AO-type B. For example, an amount of monoamine oxidase (MAO) inhibitor is sufficient to reduce and/or inhibit oxidative deamination of dopamine upon its formation from L-dopa. In one example, the monoamine oxidase (MAO) inhibitor is but not limited to one or more of L-deprenyl, clorgyline, pargyline, or fluoroallylamine. In one example the monoamine oxidase inhibitor is a fluoroallylamine.. The administration of a composition that comprises an amount of L-dopa, an amount of tyrosine and an amount of one or more MAO inhibitors to a patient may comprise a therapeutic option in a delivery method to treat or prevent complications and/or conditions associated with dopamine deficiency and/or insufficiency. In one example, a pharmaceutical composition that comprises an amount of L-dopa and and an amount of tyrosine may further comprise one or more additional agents. Non- limiting examples of additional agents may be, but not limited to, inhibitors of aromatic-L-amino-acid decarboxylase (DCC) and carboxy-O-methyl transferase (COMT). The presence of an inhibitor of DCC in a composition has the potential to reduce and/or inhibit biosynthesis of L-dopa to dopamine, reduce and/or inhibit the breakdown of L-dopa in the composition during the period immediately after administration of the composition and before it crosses into the brain, and reduce and/or prevent the risk of one or more side effects associated with L-dopa treatment, including but not limited to nausea, vomiting, low blood pressure, arrhythmia, psychosis, gastrointestinal effects including gastrointestinal bleeding, disturbances in breathing function, hair loss, confusion, sleepiness and/or anxiety. In one embodiment, the composition of the invention may comprise an amount of L-dopa, an amount of tyrosine, an amount of a DCC inhibitor and an amount of a COMT inhibitor. The composition may be administered to a patient sequentially or simultaneously with a composition comprising one or more dopamine agonists. Agents administered with L- dopa that are capable of increasing the half-life of L-dopa can also increase exposure of cells in the body to L-dopa potentially increasing the extent/rate at which L-dopa is incorporated into proteins. The addition of tyrosine to such combinations with provide protection against incorporation of L-dopa into proteins. In one example, a composition as described herein according to any example is formulated to deliver an amount of tyrosine a salt, solvate or hydrate thereof that is above physiological concentration of tyrosine a salt, solvate or hydrate thereof present in a cell and/or tissue. In one such example, the composition comprises tyrosine or a salt, solvate or hydrate thereof in an amount sufficient to deliver from about 50 to about 150 mg/ Kg of body weight per day of said tyrosine or a salt, solvate or hydrate thereof to a subject.

As used herein, "L-dopa cytotoxicity" shall be taken to mean a decline in cell function or cell death for example by apoptosis or necrosis or a process resulting ultimately in cell death as a consequence of treatment and/or administration of L-dopa and/or L-dopa accumulation, e.g., a degenerative process such as neurodegeneration ultimately resulting in neuronal death. It is to be understood that a degenerated cell that remains viable may be a consequence of L-dopa cytotoxicity, provided that the degeneration is a consequence of L-dopa administration and/or accumulation. In one example, L-dopa cytotoxicity includes apoptosis or necrosis of a monocytic cell as a consequence of L- dopa administration or accumulation into peptides or proteins. In another example, L- dopa cytotoxicity includes apoptosis or necrosis of a neuronal cell as a consequence of L-dopa administration or accumulation into peptides or proteins. L-dopa cytotoxicity may be diagnosed or determined by any one of a number of means known to the skilled artisan e.g., reduced motor function or loss of motor function, reduced mental capacity, enhanced neuronal degeneration, enhanced autoxidation, enhanced oxidative stress, enhanced membrane destabilization or rupture, enhanced natural killer (NK) response, enhanced lymphokine-activated killer (LAK) response, enhanced cytotoxic T lymphocytes (CTL) response, enhanced complement attack, enhanced DNA fragmentation or activation of a marker of apoptosis e.g., a caspase, annexin. In one example, L-dopa induced cytotoxicity may be determined by measuring annexin-V binding to exposed phosphotidylserine groups on plasma membrane. In another example, L-dopa induced cytotoxicity may be determined by detecting in situ DNA fragmentation using terminal deoxynucelotide transferase (TdT) to transfer biotin- dUTP to strand breaks of fragmented nucleic acids e.g., TUNEL method. In another example, L-dopa induced cytotoxicity may be determined by measuring activity of any mediator of programmed cell death such as measuring caspase activity e.g., caspase-3. Without being bound by any theory or mode of action, the inventors reasoned that L- dopa incorporated proteins accumulate slowly in cells and are a source of oxidative stress to cells which transfer redox damage to other biological molecules in the cells e.g., protein or DNA. For example, L-dopa-containing peptides or proteins may form covalent cross-links with other amino acid residues in proteins, thereby generating protease-resistant aggregates. Post-mitotic cells such as neurons do not normally divide and may not be able to dilute the toxic effect of the accumulation of L-dopa- containing peptides or proteins. Small amounts of misfolded proteins, such as could initially be generated from L-dopa incorporation into protein, can nucleate additional proteins generating larger more toxic aggregates. Post-mitotic cells such as neurons are extremely sensitive to misfolded proteins both in vivo and in vivo and increasing the amount of misfolded proteins results in neurodegenerative changes. The use of tyrosine to prevent incorporation of L-dopa into proteins would protect against the generation of misfolded proteins in all individuals and also individuals with genetic susceptibility to protein misfolding disorders. In one example, a composition as described herein according to any example is formulated for simultaneous or sequential administration of an amount of L-dopa or a salt, solvate or hydrate thereof and an amount of tyrosine or a salt, solvate or hydrate thereof to a cell, tissue or subject in need thereof. Conveniently, the composition is formulated for simultaneous administration of an amount of L-dopa or a salt, solvate or hydrate thereof and an amount of tyrosine or a salt, solvate or hydrate thereof to a cell, tissue or subject in need thereof.

In one example, a composition as described herein according to any example is formulated to be administered to a cell, tissue or subject in need thereof. In one example, the composition is administered by oral means e.g., as a tablet, capsule, liquid formulation. In another example, the composition is administered by inhalation or aspiration e.g., as a powder via the respiratory system of the subject including the nasal passage, buccal cavity, throat or eosophagus or lung. In another example, the composition is administered to the circulatory system of a subject by injection e.g., intramuscularly, subcutaneously, intravenously, intraperitoneally. In another example, the composition is administered rectally. In another example, the composition is administered enterically. For example by duodenal infusion by a pump system such as a portable pump. In one such example, the composition is administered nasoduodenal infusion and optionally followed by intraduodenal infusion e.g., through a transabdominal port. In another example, the composition is administered topically e.g., as a cream for ulcer treatment. In another example, the composition is administered transdermally. In one such example, the composition according to any example hereof is administered by applying a transdermal patch impregnated with the composition in a releasable form to the dermis of the subject. Conveniently the composition is formulated for oral administration e.g., as a tablet or capsule, and/or transdermally by applying a transdermal patch.

The present invention clearly contemplates repeated administration either sequentially or simultaneously of a composition as described according to any example hereof e.g., for reducing or preventing L-dopa cytotoxicity, and/or for reducing or preventing incorporation of L-dopa into peptide or protein, and/or for reducing or preventing formation of proteinaceous aggregates comprising L-dopa without decreasing dopamine concentration and dopamine synthesis in a cell, tissue or subject. For example, repeated oral administration and/or injection and/or inhalation and/or eternal infusion and/or transdermal application of a composition of the present invention may be required to reduce or prevent L-dopa cytotoxicity and/or incorporation of L-dopa into a peptide or protein such as during treatment with L-dopa for a prolonged period of time. Alternatively, or in addition, repeated oral administration and/or injection and/or inhalation and/or eternal infusion and/or transdermal application of a composition of the present invention may be required to ameliorate L-dopa toxicity without decreasing dopamine concentrations or synthesis in dopaminergic neurons for a prolonged period of time. Repeated administration may be timed so as to ensure a sufficiently high concentration of L-dopa and/or tyrosine e.g., in plasma of the subject and/or in the brains at the site of action in the treatment regimen. For example, second and/or subsequent doses may be administered at a time when serum and/or brain concentration of L-dopa and/or tyrosine provided by one or more previous doses fall(s) below a desired level at which it is active or provides sufficient benefit to the cell, tissue or patient. Such repeated and/or booster doses are clearly contemplated in the prophylaxis and/or therapy of one or more complications associated with L-dopa cytotoxicity and/or incorporation of L-dopa into a peptide or protein such as during treatment with L-dopa for a prolonged period of time e.g., in a subject suffering from Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, dementia e.g., senile dementia, or restless leg syndrome, or at risk of suffering from Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, dementia e.g., senile dementia, or restless leg syndrome. Accordingly, in one example, a composition as described according to any example hereof is formulated for daily administration. In one such example, the composition may be administered to a subject in need thereof, once daily. In another example, the composition may be administered to a cell, tissue or subject in need thereof twice daily. In another example, the composition may be administered to a cell, tissue or subject in need thereof three or more times per day. In another example, the composition is formulated for administration every two, three or more days. In another example, the composition may be administered initially once daily, or two, three or more times per day for any given period followed by administration every two, three or more days.

In one example, the pharmaceutical composition according to any example described hereof, is formulated for administration to a neuronal cell. In one example, the cell is a dopaminergic neuron. In one example, the pharmaceutical composition according to any example described hereof, is formulated for administration to brain cell or tissue. In one example, the brain tissue is a region of substantia nigra.

In another example, the pharmaceutical composition according to any example described hereof is formulated for administration to the central nervous system. In another example, the pharmaceutical the pharmaceutical composition according to any example described hereof is formulated for administration to the cerebrospinal fluid (CSF). In one example, a pharmaceutical composition according to any example hereof, further comprises a suitable adjuvant, excipient, carrier or diluent.

As used herein, the term "suitable excipient, carrier or diluent" or "an acceptable excipient, carrier or diluent" shall be taken to mean a compound or mixture thereof that is suitable for administration to a cell, tissue or subject for the treatment or prevention of a condition that may be ameliorated by L-dopa and/or for the treatment or prevention of L-dopa cytotoxicity and/or for reducing or preventing incorporation of L-dopa into a peptide or protein in a cell, tissue or subject, albeit not necessarily limited in use to that context. In one example, the suitable excipient, carrier or diluent is excipient, carrier or diluent that is suitable for absorption of the bio-reactive composition to the gastrointestinal- tract and/or for crossing the blood-brain barrier e.g., to central nervous system and the cerebrospinal fluid (CSF).

An excipient, carrier or diluent useful in a composition described herein according to any example, will generally not inhibit to any significant degree a relevant biological activity of the active component. In one example, the excipient, carrier or diluent will not significantly inhibit the activity of L-dopa a salt, solvate or hydrate thereof with respect of biosynthesis of dopamine from L-dopa. In another example, the excipient, carrier or diluent will not significantly inhibit the activity of tyrosine a salt, solvate or hydrate thereof with respect of reducing and/or preventing incorporation of L-dopa into a peptide or protein. In another example, the excipient, carrier or diluent will not significantly inhibit the activity of tyrosine a salt, solvate or hydrate thereof with respect of reducing and/or preventing biosynthesis of L-dopa from tyrosine.

In another example a excipient, carrier or diluent useful in a composition described herein according to any example, permits the uptake of L-dopa a salt, solvate or hydrate thereof and tyrosine a salt, solvate or hydrate thereof by the cell, and or tissue in a subject.

In one example, a pharmaceutical composition according to any example described hereof, is packaged with written instructions for use of the composition. In one example, the written instructions are for use in the treatment or prevention of a condition ameliorated by L-dopa e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, dementia e.g., senile dementia, or restless leg syndrome.

The present invention also provides a method of treating or preventing a condition ameliorated by L-dopa, wherein said method comprises administering or recommending administration to a subject in need thereof an amount of a pharmaceutical composition according to any example hereof e.g., for a time and under conditions sufficient to produce dopamine without one of more adverse effects associated with prolonged L-dopa therapy e.g., L-dopa cytotoxicity and/or protein aggregate formation and/or apoptosis and/or nausea and/or vomiting and/or low blood pressure and/or arrhythmia and/or psychosis and/or gastrointestinal effects such as gastrointestinal bleeding, disturbances in breathing function and/or hair loss and/or confusion, sleepiness and/or anxiety.

In another example, the invention provides a method of treating or preventing L-dopa cytotoxicity in a cell, tissue or subject comprising administering or recommending administration to said cell, tissue or subject an amount of the composition according to any example described hereof.

In another example, the invention provides a method of reducing or preventing incorporation of L-dopa into a peptide or protein in a cell, tissue or subject, comprising administering or recommending administration to a cell, tissue or subject in need thereof an amount of the pharmaceutical composition according to any example described hereof. In another example, the invention provides a method of reducing or preventing formation of proteinaceous aggregates comprising L-dopa in a cell, tissue or subject, comprising administering or recommending administration to a cell, tissue or subject in need thereof an amount of the pharmaceutical composition according to any example described hereof.

In another example, the invention provides a method of reducing L-dopa toxicity without decreasing dopamine concentrations or synthesis in a cell, tissue or subject, comprising administering or recommending administration to a cell, tissue or subject in need thereof an amount of one or more pharmaceutical compositions in a single or repeated dosage either simultaneously or sequentially according to any example described hereof.

In another example, the invention provides a method of monitoring L-dopa levels in polypeptides and/or proteins in plasma or other tissues. A non-limiting example of a tissue would be tissue contained in a biopsy sample. The advantage of monitoring L- dopa levels is the ability to indicate the extent to which L-dopa has been incorporated into proteins in individual patients. Levels of proteins containing L-dopa in the plasma or other tissues may subsequently be monitored to evaluate the effectiveness of co- administration of L-dopa with tyrosine for each patient as a surrogate marker for levels of L-dopa-containing proteins and/or polypeptides in the brain. In another example, the invention provides a marker to measure the levels of L-dopa- containing proteins and/or polypeptides in the brain of an individual wherein the marker comprises an amount of L-dopa-containing proteins and/or polypeptides in the plasma or other tissues.

As used herein, the term "sequential" or "sequentially" means that a dose of one composition is delivered to a subject or patient after the delivery of another composition regardless of whether the one and another compositions contain the same components, ingredients and/or agents.

As used herein, the term "simultaneously" means that a dose of one composition is delivered to a subject or patient at the same time as the delivery of another composition regardless of whether the one and another compositions contain the same components, ingredients and/or agents.

As used herein, the terms "prevent" and "treat" or similar term shall not be taken to require an absolute i.e., 100% abrogation of a condition ameliorated by L-dopa or L- dopa cytotoxicity or incorporation of L-dopa into a peptide or formation of proteinaceous aggregates comprising L-dopa. The terms "prevent" and "treat" or similar term shall also not be taken to require or an absolute i.e., 100% prevention of the development of one or more complications in a subject having risk factors of developing a condition ameliorated by L-dopa or L-dopa cytotoxicity or incorporation. It is sufficient that there is a significant reduction in the adverse effect(s) using the method of the present invention compared to the absence of prophylaxis or therapy in accordance with the present invention.

As used herein, the terms "ameliorated" or "ameliorate" or "ameliorating" shall not be taken to require abrogation of a condition ameliorated by L-dopa or abrogation L-dopa cytotoxicity or abrogation of L-dopa accumulation and/or incorporation in a cell, tissue or subject that is more than a significant effect compared to the absence of treatment in accordance with the present invention.

Similarly, the terms "inhibit", "repress", "delay", "enhance", "increase", "induce", "activate" and "promote" as used throughout this specification shall not be taken to require any particular quantitative change, merely a modified level and/or activity and/or expression, or modified timing thereof, that is significant compared to the absence of treatment in accordance with the present invention.

As used herein the terms "enhancing synthesis of dopamine" or "increasing synthesis of dopamine" or related terms, shall not be taken to require any particular quantitative change, merely a modified level of formation of dopamine e.g., from L-dopa or a salt, solvate or hydrate thereof that is significant compared to the absence of treatment in accordance with the present invention. Alternatively, or in addition, it is sufficient that the is a significant increase in the formation of dopamine compared to the treatment only with L-dopa or a salt, solvate or hydrate thereof. Alternatively, or in addition, it is sufficient that there is a significant increase in the formation of dopamine compared to the treatment only with tyrosine or a salt, solvate or hydrate thereof.

As used herein the terms "enhancing synthesis of L-dopa" or "increasing synthesis of L-dopa" or related terms, shall not be taken to require any particular quantitative change, merely a modified level of formation of L-dopa e.g., from tyrosine or a salt, solvate or hydrate thereof that is significant compared to the absence of treatment in accordance with the present invention. Alternatively, or in addition, it is sufficient that the is a significant increase in the formation of L-dopa compared to the treatment with a only L-dopa or a salt, solvate or hydrate thereof. Alternatively, or in addition, it is sufficient that there is a significant increase in the formation of L-dopa compared to the treatment only with tyrosine or a salt, solvate or hydrate thereof.

A composition of the invention may be administered to a cell, tissue or subject in need thereof for a time and under conditions sufficient for tyrosine a salt, solvate or hydrate thereof to reduce or prevent incorporation of L-dopa a salt, solvate or hydrate thereof into a peptide or protein in said cell, tissue or subject. Alternatively, or in addition, the composition is administered to a cell tissue or subject in need thereof for a time and under conditions sufficient for formation of dopamine in said cell, tissue or subject from L-dopa a salt, solvate or hydrate thereof in the composition. Alternatively, or in addition, the composition is administered for a time and under conditions sufficient to ameliorate one or more symptoms treatable using L-dopa a salt, solvate or hydrate thereof. Alternatively, or in addition, the composition is administered for a time and under conditions sufficient to ameliorate one or more symptoms caused by or associated with L-dopa cytotoxicity. As used herein, the term "cell or tissue or subject in need thereof shall be taken to mean a cell, tissue or subject that is in need of dopamine therapy or has developed one or more complications of a condition normally ameliorated by treatment with L-dopa and/or has developed one or more complications associated with L-dopa cytotoxicity and/or as a consequence of L-dopa administration and/or accumulation, or is predisposed by virtue of having one or more risk factors to suffering from dopamine deficiency and/or condition ameliorated by treatment with L-dopa and/or associated with L-dopa cytotoxicity and/or as a consequence of L-dopa administration and/or accumulation. A cell, tissue or subject in need thereof may have been adininistered previously with L-dopa or may have never been provided L-dopa before administration of a composition of the invention.

In one example, a condition ameliorated by L-dopa is a neurodegenerative condition. As used herein, the term "neurodegenerative condition" or "neurodegenerative disease" or "neurodegenerative disorder" shall be taken to mean a disease that is characterized by neuronal cell death e.g., as a result from dopamine deficiency and/or administration and/or accumulation of L-dopa. The neuronal cell death observed in a neurodegenerative disease is often preceded by neuronal dysfunction, sometimes by several years. Accordingly, the term "neurodegenerative disease" includes a disease or disorder that is characterized by neuronal dysfunction and eventually neuronal cell death. Neurodegenerative diseases may be characterized by gliosis (e.g., astrocytosis or microgliosis) in a region of neuronal death. Cellular events observed in a neurodegenerative disease often manifest as a behavioural change (e.g., deterioration of thinking and/or memory) and/or a movement change (e.g., tremor, ataxia, postural change and/or rigidity). Examples of neurodegenerative disease include, Parkinson's disease, Alzheimer's disease, frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis, ataxia (e.g., spinocerebellar ataxia or Friedreich's Ataxia), Creutzfeldt- Jakob disease, a polyglutamine disease (e.g., Huntington's disease or spinal bulbar muscular atrophy), Hallervorden-Spatz disease, idiopathic torsion disease, Lewy body disease, multiple system atrophy, neuroanthocytosis syndrome, olivopontocerebellar atrophy, Pelizaeus-Merzbacher disease, Pick's disease, progressive supranuclear palsy, syringomyelia, torticollis, spinal muscular atophy or a trinucleotide repeat disease (e.g., Fragile X Syndrome). Preferably, the neurodegenerative disease is a neurodegenerative disease associated with aberrant dopamine production and/or for which L-dopa administration is beneficial. In one example, the condition ameliorated by L-dopa or the neurodegenerative condition is Parkinson's disease. As used herein, the term "Parkinson's disease" shall be taken to mean a neurological disorder that is characterized by progressive or chronic impairment of motor control, by muscle rigidity, resting tremor slowing of movement (bradykinesia) and, in extreme cases, nearly complete loss of movement (akinesia), cognitive dysfunction, language problems, and depression and loss of mental capacity. Pathologically, a Parkinson's disease is characterized by the selective loss of large neuromelanin (NM) pigment-containing dopaminergic neurons in the substantia nigra (SN) region of the brain, and/or by the presence of proteinaceous cytoplasmic inclusion bodies termed Lewy bodies in some surviving neurons, and/or compromised nigrostrial pathway and/or a decline in striatal dopamine.

In another example, the condition ameliorated by L-dopa or the neurodegenerative condition is dementia. As used herein, the term "dementia" shall be taken to mean a condition that is characterized by chronic loss of mental capacity, particularly progressive deterioration of tliinking and/or memory and/or behaviour and/or personality and/or motor function, and may also be associated with psychological symptoms such as depression and apathy. In this respect, dementia is not caused by, for example, a stroke, an infection or a head trauma. Examples of dementia include, for example, an Alzheimer's disease, vascular dementia, dementia with Lewy bodies and frontotemporal lobar dementia, amongst others.

In another example, the method of the present invention is used to treat or prevent presenile dementia. In this respect, the term "presenile dementia" is understood in the art to mean dementia characterized by the onset of clinically detectable symptoms before a subject is 65 years of age.

In another example, the condition ameliorated by L-dopa or the neurodegenerative condition or the dementia is an Alzheimer's disease or FTLD. By "an Alzheimer's disease" is meant a neurological disorder characterized by progressive impairments in memory, behaviour, language and/or visuo-spatial skills. Pathologically, an Alzheimer's disease is characterized by neuronal loss, gliosis, neurofibrillary tangles, senile plaques, Hirano bodies, granulovacuolar degeneration of neurons, amyloid angiopathy and/or acetylcholine deficiency. The term "an Alzheimer's disease" shall be taken to include early onset Alzheimer's disease (e.g., with an onset of detectable symptoms occurring before a subject is 65 years of age) or a late onset Alzheimer's disease (e.g., with an onset later then, or in, the sixth decade of life). In one example, the Alzheimer's disease is an early onset Alzheimer's disease.

For example, the Alzheimer's disease is a plaque predominant Alzheimer's disease. As used herein, the term "plaque predominant Alzheimer's disease" shall be taken to mean a variant form of Alzheimer's disease characterized by numerous senile plaques in the relative absence of neurofibrillary tangles.

In another example, the neurodegenerative condition is a motor neuron disease. As used herein, the term "motor neuron disease" shall be taken to mean a disease characterized by dysfunction and/or death of motor neurons, e.g., upper motor neurons and/or lower motor neurons. Generally, a motor neuron disease presents as muscle weakness and atrophy, with the weakness often presenting in the limbs and/or as difficulty swallowing. As motor neuron disease progresses an affected subject often develops difficulty walking and lifting objects, and eventually difficulty breathing. Exemplary motor neuron diseases include amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Preferably, the motor neuron disease is ALS.

A cell or tissue or subject to be treated with a composition of the present invention according to any example hereof suffers from dopamine deficiency or a condition known to be ameliorated by treatment with L-dopa e.g., Parkinson's disease or Alzheimer's disease, a neurodegeneration condition or dementia. In another example, the cell or tissue or subject suffers from one or more complications associated with L- dopa cytotoxicity or L-dopa administration and/or accumulation e.g., reduced motor function or loss of motor function, reduced mental capacity, enhanced neuronal degeneration, enhanced cellular autoxidation, enhanced cellular oxidative stress, enhanced cell membrane destabilization or rupture, enhanced natural killer (NK) response, enhanced lymphokine-activated killer (LAK) response, enhanced cytotoxic T lymphocytes (CTL) response, enhanced complement attack, or enhanced cell apoptosis as determined by activation of one or more markers of apoptosis.

In another example, the cell, tissue or subject has not suffered dopamine deficiency and/or has not exhibited a symptom indicative of a complication of a condition ameliorated by treatment with L-dopa. In such a patient class, the subject may exhibit one or more risk factors for dopamine deficiency e.g., one or more risk factors for developing Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, or a dementia. In another example, the cell, tissue or subject has not yet suffered any apparent complication associated with L-dopa cytotoxicity or L-dopa administration and/or accumulation, however has one or more risk factors for developing such complications e.g., receiving or is in need of dopamine therapy and/or L-dopa administration.

In another example, the subject suffers from Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition or a dementia.

A therapeutic or prophylactic method as described according to any example hereof may comprise administering the composition to a cell, e.g., a neuron, such as dopaminergic neuron. Alternatively, or in addition, the composition is administered to a tissue, e.g., brain tissue such as in the substantia nigra (SN), or cerebrospinal fluid (CSF). Such administration may be direct e.g., by parenteral means, or indirect e.g., by oral, transdermal or parenteral means to a site distinct from the stated tissue.

In another example, a method as described herein according to any example, comprises administering or recommending administration of the pharmaceutical composition for a time and under conditions sufficient to deliver an amount of tyrosine or a salt, solvate or hydrate thereof from about 50 to about 150 mg/ Kg of body weight per day to a subject in need thereof, and optionally sufficient to deliver comprising L-dopa or a salt, solvate or hydrate thereof in an amount sufficient to deliver an amount of L-dopa or a salt, solvate or hydrate thereof from 400 to about 1200 mg per day to a subject in need thereof.

In one example, a method as described herein according to any example, comprises administering the pharmaceutical composition orally, parenterally, transdermally, enterically or administration by inhalation to a subject in need thereof.

In one example, a method as described herein according to any example comprises administering the pharmaceutical composition in a tablet or capsule or gel form. In another example a method as described herein according to any example comprises applying transdermal patch impregnated with the composition in a releasable form to the dermis of a subject in need thereof. In another example a method as described herein according to any example comprises administration by duodenal infusion e.g., using a pump system.

In one example, the method of treatment or prophylaxis as described herein according to any example additionally comprises providing or obtaining an amount of a composition described according to any example hereof that reduces or prevents L- dopa cytotoxicity, or reduces or prevents incorporation of L-dopa into a peptide or protein or reduces or prevent formation of L-dopa containing aggregates in a cell, tissue or subject or enhancing synthesis of dopamine in a cell, tissue or subject.

For example, the present invention provides a method of treatment or prophylaxis of a subject in need thereof, said method comprising:

(i) identifying a subject suffering from one or more complications or a condition ameliorated by L-dopa or suffering from one or more complications of L-dopa cytotoxicity or is at risk of developing one or more complications or a condition ameliorated by L-dopa;

(ii) obtaining an amount of a pharmaceutical composition described according to any example hereof that reduces or prevents L-dopa cytotoxicity, or reduces or prevents incorporation of L-dopa into a peptide or protein or reduces or prevent formation of L- dopa containing aggregates without decreasing dopamine concentrations or biosynthesis in a cell, tissue or subject but sufficient to prevent or alleviate one or more complications in the subject associated with L-dopa cytotoxicity in the subject according to any embodiment hereof; and

(iii) administering or recommending said composition to said subject.

In another example, the present invention provides a method of treatment or prophylaxis of a subject in need thereof, said method comprising:

(i) identifying a subject suffering from one or more complications associated with L-dopa cytotoxicity or is at risk of developing one or more complications associated with L-dopa cytotoxicity;

(iii) administering or recommending said composition to said subject. In one example, the present invention also provides a method of preparing the pharmaceutical composition described according to any example hereof, wherein said method comprises admixing an amount of L-dopa or a salt, solvate or hydrate thereof and an amount of tyrosine or a salt, solvate or hydrate thereof. In one example, the method further comprises admixing a suitable adjuvant, excipient a carrier or diluent described according to any example hereof.

In another example, the method further comprises admixing an inhibitor of an aromatic-L-amino-acid decarboxylase. In one such example, the method comprises admixing an amount of an inhibitor of DOPA decarboxylase, such as carbidopa or benserazide. In another example, the method further comprises admixing an amount of an inhibitor of tyrosine hydroxylase such as 3-iodo-L-tyrosine or MPTP or 1-Methyl- 4-phenylpyridinium ion (MPP+). In another example, the method further comprises admixing an inhibitor of a monoamine oxidase (MAO) such as L-deprenyl, clorgyline, pargyline, f N-(2-aminoethyl)-4-chlorobenzamide hydro-chloride, N-(2-aminoethyl)-5- (3-fluorophenyl)-4-thiazolecarboxamide hydrochloride, fluoroallylamine or derivatives thereof.

In yet another example, the method further comprises providing the admixture.

In one example, a method as described in any example hereof additionally comprises providing written instructions for use of the composition. In one example, the method comprises providing written instructions for use in the treatment or prevention of L- dopa cytotoxicity, Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition or a dementia.

The present invention also provides for use of the composition described according to any example hereof in medicine. The present invention also provides for use of the composition described according to any example hereof in the treatment or prevention of a condition ameliorated by L- dopa, e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition or a dementia.

The present invention also provides for use of the composition described according to any example hereof in the treatment or prevention of L-dopa cytotoxicity in a cell, tissue or subject.

The present invention also provides for use of the composition described according to any example hereof in the reduction or prevention of incorporation of L-dopa into a peptide or protein in a cell, tissue or subject. The present invention also provides for use of the composition described according to any example hereof in reducing or preventing formation of proteinaceous aggregates comprising L-dopa in a cell, tissue or subject.

The present invention also provides for use of the composition described according to any example hereof wherein dopamine synthesis in a cell, tissue or subject is not decreased.

The present invention also provides for use of the composition described according to any example hereof in the manufacture of a medicament for the treatment or prevention of a condition a condition ameliorated by L-dopa e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition or a dementia.

The present invention also provides for use of the composition described according to any example hereof in the manufacture of a medicament for the treatment or prevention of L-dopa cytotoxicity in a cell, tissue or subject.

The present invention also provides for use of the composition described according to any example hereof in the manufacture of a medicament for reducing or preventing incorporation of L-dopa into a peptide or protein in a cell, tissue or subject. The present invention also provides for use of the composition described according to any example hereof in the manufacture of a medicament for reducing or preventing formation of proteinaceous aggregates comprising L-dopa in a cell, tissue or subject. The present invention also provides for use of the composition described according to any example hereof in the manufacture of a medicament for enhancing dopamine synthesis in a cell, tissue or subject.

The present invention also provides a kit comprising a i) an amount of L-dopa or a salt, solvate or hydrate thereof; and ii) an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein. In one such example, the kit additionally comprises a suitable adjuvant, excipient, a carrier or diluent and optionally an inhibitor of an aromatic-L-amino-acid decarboxylase such as an inhibitor of DOPA Decarboxylase, an inhibitor of tyrosine hydroxylate and/or an inhibitor of monoamine oxidase such as MAO-type A and/or MAO-type B. .

The kit described according to any example hereof may for the treatment or prevention of a condition ameliorated by L-dopa e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition or a dementia.

A kit described according to any example hereof may also be used for reducing or preventing L-dopa cytotoxicity or for reducing or preventing incorporation of L-dopa into a peptide or protein or for reducing or preventing formation of proteinaceous aggregates comprising L-dopa or for enhancing dopamine biosynthesis.

A kit described according to any example hereof may optionally comprise written instructions for use of the composition and/or the kit. In one example, the kit comprises written instructions use in the treatment or prevention of L-dopa cytotoxicity or a neurodegenerative condition or Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition or a dementia. A subject to which the present invention can be applied is a human or other mammalian subject capable of developing dopamine deficiency and/or impairment dopamine biosynthesis and/or is capable of developing one or more complications associated with L-dopa cytotoxicity and/or incorporation of L-dopa into peptides and/or proteins, including e.g., a domesticated animal such as a domestic pet or commercially-valuable animal. The prophylactic or therapeutic treatment of a dog, cat monkey or horse is clearly encompassed by the present invention.

This invention is useful in the treatment and/or prophylaxis of any condition associated with accumulation of L-dopa in mammalian cells and/or tissues. Such mammalian tissues can be within or outwith the CNS and include for example connective, muscle, epithelial and neural tissues. The cells may be, for example, neurons present in neural tissue of the midbrain or in the striatum. In a non-limiting example, one class of neurons that may be associated with accumulation of L-dopa is midbrain dopaminergic neurons. The midbrain dopaminergic neurons comprise substantia nigra compacta (SNc) neurons and the ventral tegmental area (VTA). It is contemplated herein that the invention is useful in the treatment and/or prophylaxis of any condition associated with accumulation of L-dopa in SNc and VTA neurons. Such conditions include for example neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition, a dementia, atherosclerosis, and cataractogenesis.

In performing a therapeutic method of the invention as described according to any example hereof, it is to be understood that the composition is generally administered for a time and under conditions sufficient to ameliorate or abrogate one or more symptoms or the disease being treated e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition, a dementia, atherosclerosis, or cataractogenesis, without inducing a level of L-Dopa cytotoxicity arising from standard L-Dopa therapy. Subjects treated in accordance with this example will generally have a prior diagnosis of a disease or condition indicating L-Dopa therapy, or alternatively, suffer from one or more symptoms of a disease or condition that is treatable with L- Dopa therapy e.g., resting tremor, rigidity, bradykinesia, postural instability, Lewy Bodies, etc. It will also be apparent from the present disclosure that the invention is also suitable for treatment of such subjects who have been administered L-Dopa previously, or are currently receiving L-Dopa and suffer from L-Dopa-induced cytotoxicity. In this latter patient class, the present therapeutic prevents further neuronal cell death in the patient e.g., by necrotic or apoptotic pathways, and/or will prevent further neuronal cell injury and/or loss of function. Exemplary dosage regimens are described herein. In performing a prophylactic method of the invention as described according to any example hereof, it is to be understood that the composition is generally administered for a time and under conditions sufficient to prevent one or more symptoms of a disease being treated e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition, a dementia, atherosclerosis, or cataractogenesis, without inducing a level of L-Dopa cytotoxicity arising from standard L-Dopa therapy. Subjects treated in accordance with this example will generally have a predisposition to a disease or condition indicating L-Dopa therapy e.g., a genetic predisposition, or have a prior history of a disease or condition indicating L-Dopa therapy e.g., the subject is in remission or presently symptom-free. It will also be apparent from the present disclosure that the invention is also suitable for preventive treatment of such subjects e.g., subjects who are at risk who have been administered L-Dopa previously and suffer from L-Dopa-induced cytotoxicity. In this latter patient class, the present therapeutic prevents further neuronal cell death in the patient e.g., by necrotic or apoptotic pathways, and/or will prevent further neuronal cell injury and/or loss of function. Exemplary dosage regimens are described herein.

3. General

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

Each example described herein is to be applied mutatis mutandis to each and every other example unless specifically stated otherwise.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally- equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, developmental biology, mammalian cell culture, recombinant DNA technology, histochemistry and immunohistochemistry and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference:

1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and HI; 2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;

3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et a/., pp 83-115; and Wu et al. , pp 135-151 ;

4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;

5. Animal Cell Culture: Practical Approach, Third Edition (John R.W. Masters, ed., 2000), ISBN 0199637970, whole of text;

6. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series;

7. Rodgers et al., 2002, Free Radic. Biol. Med., 32:766-775

8. Rodgers et al., 2004, Free Radic. Biol. Med., 37(11):1756-1764

9. Rodgers et al., 2006, J. Neurochem., 98:1061-1067

Brief description of the drawings

Figure 1 is a graphical representation of HPLC results showing levels of dopamine, free dopa and protein incorporated dopa in SH-SY5Y cells after incubation with L- dopa. Human neuronal cells, SH-SY5Y, were incubated in tyrosine-deficient medium containing ΟμΜ, ΙΟΟμΜ, 200μΜ, 500μΜ and 1000 μΜ of L-dopa for 9 hours. Cells were lysed and the free cytosolic fraction and protein fraction separated using trichloroacetic acid (TCA) precipitation. Protein samples were hydrolysed overnight and dopa quantified by HPLC and expressed relative to tyrosine. Dopa and dopamine levels were directly quantified from the cytosolic fraction by HPLC and normalized to protein levels. Cytosolic L-dopa levels are shown in Fig. 1 A, cytosolic dopamine levels are shown in Fig. IB, and levels of L-dopa in hydrolysed cell proteins are shown in Fig. 1C.

Figure 2A is a graphical representation showing liquid scintillation counting (LSC) results of incorporation of [¹⁴C ]-L-dopa into cell proteins by SH-SY5 Y neuronal cells in the presence of tyrosine. SH-SY5Y cells were co-incubated with [¹⁴C]-L-dopa (ΙμΜ) and tyrosine (0 2, 5, 8, 10 and 15 μΜ) for 24 hours. Cell proteins were isolated and the radioactivity (disintegrations per minute/dpm) measured by liquid scintillation counting. L-dopa incorporation into protein (as assessed from DPM mg protein) was significantly reduced when tyrosine was present at 5, 8, 10 and 15μΜ Figure 2B is a graphical representation showing liquid scintillation counting (LSC) results of uptake of [¹⁴C] -L-dopa into SH-SY5Y neuronal cells in the presence of tyrosine. SH-SY5Y were co-incubated with [¹⁴C]-L-DOPA (ΙμΜ) and a range of concentrations of tyrosine (2, 5, 8, 10 and 15 μΜ) for 24 hours. Cells were washed and lysed. The radioactivity (disintegrations per minute, dpm) was measured by liquid scintillation counting and data were normalised to cell protein (cell number). L-dopa uptake into neuronal cells was not significantly reduced when tyrosine was present at 2, 5, 8, and ΙΟμΜ and was reduced by less than 25% at 15μΜ tyrosine.

Figure 2C is a graphical representation showing liquid scintillation counting (LSC) results of incorporation of [¹⁴C ]-L-dopa into cell proteins by SH-S Y5 Y in the presence of cycloheximide (CHX) which blocks protein synthesis by cells. SH-SY5Y cells were co-incubated with [¹⁴C]-L-dopa (ΙμΜ) and tyrosine (8 μΜ) for 24 hours with or without cycloheximide (CHX, 2μg/ml). The presence of cycloheximide prevented incorporation of L-dopa into protein demonstrating that incorporation of L-dopa was protein synthesis-dependent.

Figure 2D is a graphical representation showing dopamine levels in SH-SY5Y cells after incubation with L-dopa (100 μg and 200 μg) with (open bars) or without (black bars) tyrosine (2.5 mM) for 16 hours. Co-administration of L-dopa and tyrosine at concentrations as high as 2.5 mM did not significantly impair biosynthesis of dopamine from L-dopa in SH-SY5Y cells.

In Fig 2 statistical analysis was by one-way ANOVA with Tukey's post-hoc test. * indicates a significance of p<0.05. ** indicates a significance of p<0.01 and *** indicates a significance of pO.001. Figure 3 is a graphical representation showing HPLC analysis of the L-dopa content of hydrolysed proteins from plasma samples obtained from: individuals that did not receive L-dopa (0, n = 11), from patients treated with L-dopa for up to 10 years (1-10, n= 5), 10 to 20 years (10-20, n = 10) and 20 to 30 years (20-30, n= 5). The dopa content of proteins was assessed by HPLC analysis and is shown as a ratio to the L-tyrosine concentration as described previously (Rodgers, 2002, 2004, 2006). L-dopa levels in plasma proteins was significantly increased in individuals that had received L-dopa for 10-20 years and increased significantly after a further decade of treatment. * indicates p < 0.05, ** indicates p < 0.01. Figure 4 is a graphical representation showing levels of L-dopa-containing proteins in brains of L-dopa-treated Parkinson's disease (PD) patients and age-matched controls. Cryopreserved tissue from the anterior cingulated cortex (ACC), motor cortex (MC), occipital cortex (OC) and substantia nigra (SN) of 5 PD patients (4 male and 1 female, average age 78) who were treated with L-dopa for between 5 and 13 years and age matched control individuals (4 male and 1 female, average age 76) was analysed. Tissue was snap frozen in liquid nitrogen and powdered. Proteins were extracted, isolated and L-dopa levels measured by HPLC analysis. L-dopa levels in proteins are expressed as a ratio to tyrosine levels (Rodgers, 2002, 2004, 2006).. L-dopa levels in proteins were significantly increased in the motor cortex (MC), occipital cortex (OC) and substantia nigra (SN) of the L-dopa treated individuals. Statistical analysis was by one-way ANOVA with Tukey's post-hoc test. * indicates a significance of p<0.05.

Figure 5 is a graphical representation of HPLC results showing L-dopa levels in proteins in the substantia nigra (SN) region and the striatum of rat brain following intraperitoneal injection of Sprague-Dawley rats with L-dopa (6.5 mg/kg) and benserazide (1.5mg/kg) twice daily for 21 days (L-DOPA, grey bars) or with vehicle alone (Vehicle, open bars). Incorporation of L-dopa into proteins is significantly elevated in the striatum of rats treated with L-dopa for 21 days.

Figure 6 is a graphical representation showing HPLC results of free dopa concentrations in SH-SY5Y cell lysates after incubation with L-dopa and D-dopa. SH- SY5Y neuronal cells were incubated with 200 μΜ L-dopa or D-dopa. Cells were harvested at lh, 2h, 3h and 24h, and the intracellular levels of dopa measured by HPLC and expressed as a ratio to total protein (DOPA μΜ/mg protein). L-dopa and D-dopa were both efficiently taken up by cells and were detectable in the cell. Levels of D- dopa were significantly higher than those of L-dopa after 3 hours. ** indicates statistically significant differences between L- and D- treated cultures. Statistical analysis was by one-way ANOVA with Tukey's post hoc test.

Figure 7 is a graphical representation of HPLC results showing levels of proteins containing incorporated L-dopa generated in SH-SY5Y cells after incubation with L- dopa. SH-SY5Y cells were incubated in tyrosine-free medium containing L-dopa (L- DOPA), D-dopa (D-DOPA) or medium alone (Control) for 24 hours. The isolated cell proteins were washed, hydrolysed and the dopa and tyrosine content determined by HPLC. The DOPA content of proteins is expressed as a ratio to tyrosine (DOPA μΜο^/ΜοΙε Tyrosine). Only L-Dopa was incorporated into cell proteins. *** indicates statistically significant difference when compared to untreated cells or D- dopa-treated cells (PO.001). Statistical analysis was by one-way ANOVA with Tukey's post-hoc test. Results are from 3 independent experiments.

Figure 8 is a graphical representation showing necrosis of SH-SY5Y neuronal cells as determined by percentage of viable (i.e., non-necrotic) cells in the culture medium which do not release LDH compared to untreated control cells following incubation of SH-SY5Y cells in tyrosine deficient EMEM medium containing either L-dopa or D- dopa (500 μΜ) (grey bars) or in medium containing tyrosine (lOmM) (open bars) for 24 hours. Cell viability (necrosis) was measured using the LDH assay. Tyrosine protected cells from L-dopa induced toxicity. Statistical analysis was by one-way ANOVA with Tukey's post hoc test. *** represents a statistically significant difference between L-dopa and D-dopa toxicity p<0.001

Figure 9 is a graphical representation showing apoptosis in THP1 human monocytic cells after incubation for 24 hours in tyrosine-free medium with L-dopa (L-DOPA, 500μΜ), D-dopa (D-DOPA, 500μΜ), or tyrosine-free medium alone (Untreated/Control) Fig. 9A shows the percentage of cells positive for DNA fragmentation (TUNEL assay), Fig. 9B shows the percentage of cells positive for annexin V binding and Fig. 9C shows the percentage of cells positive for caspase activation. Fig. 9D shows that tyrosine (2.5 mM) protected cells against L-dopa induced apoptotic cell death as indicated by prevention of caspase 3 activation. Statistical analysis was by ANOVA.

Figure 10 is a graphical representation showing activation of apoptosis in SH-SY5Y neuronal cells by L-dopa-containing proteins over time (4, 6, 10 and 16 hours) as determined by percentage of cells positive for caspase activity (Fig 10A) or percentage of cells positive for Annexin V-FITC staining (Fig 10B) compared to untreated control cells at each time point. Cells were incubated in tyrosine-depleted medium containing L- and D-dopa (500 μΜ) or tyrosine-depleted medium alone (control). Statistical analysis was by ANOVA.

Figure 11 is a graphical representation showing activation of apoptosis in SH-SY5Y neuronal cells as determined by percentage of cells showing DNA fragmentation using the COMET assay. Cells were incubated in tyrosine-depleted medium (EMEM), in tyrosine-depleted medium containing 200 μΜ of D-dopa (D-DOPA 200), 200 μΜ of L- dopa (L-DOPA 200) or with L-dopa (200 μΜ) plus 2.5 mM tyrosine (L-DOPA 200+tyr) for 24 hours or with tyrosine alone in culture medium (EMEM+tyr) for 24 hours. Only L-dopa induced a significant level of apoptosis as indicated by DNA fragmentation (% DNA in tail). Tyrosine protected cells against L-dopa induced DNA fragmentation as an art recognized measure of apoptotic cell death.

Figure 12 is a graphical representation showing tyrosine levels in serum of rats injected intraperitoneally with (6.5mg/kg) L-dopa and (1.5mg/kg) benserazide (L-Dopa) or with (6.5mg/kg) L-dopa and (1.5mg/kg) benserazide and (lOOmg/kg) tyrosine (L-Dopa + Tyr). Tyrosine levels in the group of rats that received tyrosine were significantly increased after 0.5 hours and had returned to baseline levels by 2.5 hours. Statistical analysis was by ANOVA.

Figure 13 is a graphical representation showing dopa and dopamine levels in serum of rats injected intraperitoneally with (6.5mg/kg) L-dopa and (1.5mg/kg) benserazide (L- Dopa) or with (6.5mg/kg) L-dopa and (1.5mg/kg) benserazide and (lOOmg/kg) tyrosine (L-Dopa + Tyr). Dopa and dopamine levels in serum did not differ between the two groups of rats at any of the time-points examined. Statistical analysis was by ANOVA.

Figure 14 is a graphical representation of data generated from the HPLC analyses of brains from one group of Sprague-Dawley rats injected intraperitoneally twice daily for 21 days with (6.5mg/kg) L-dopa and (1.5mg/kg) benserazide (Dopa) and a second group of rats injected intraperitoneally twice daily with (6.5mg/kg) L-dopa, (1.5mg/kg) benserazide and (lOOmg/kg) tyrosine (Dopa + Tyr). There were 10 rats in each group and the regions of the brain analysed were the substantia nigra, the motor cortex and the striatum. Tyrosine levels (free) in the group of rats that received tyrosine were significantly increased in the striatum and motor cortex (Fig 14 A, B and C). Levels of dopamine did not differ significantly in the two groups of rats in the three brain regions examined (Fig 14D, E and F). Dopa (free) levels in the striatum were below detection levels and did not differ significantly in the motor cortex (Fig 14G) and the substantia nigra (Fig 14H). Co-administration of tyrosine with L-dopa therefore did not significantly alter levels of L-dopa and dopamine in the brain.

Figure 15 is a graphical representation of data generated from the analyses of proteins extracted from brains of a group of Sprague-Dawley rats injected intraperitoneally twice daily for 21 days with (6.5mg/kg) L-dopa and (1.5mg/kg) benserazide (Dopa) and a group of rats injected intraperitoneally twice daily with (6.5mg/kg) L-dopa, (1.5mg/kg) benserazide and (lOOmg/kg) tyrosine (Dopa + Tyr). There were 10 rats in each group and the regions of the brain analysed were the the motor cortex (Fig 15 A), the substantia nigra (Fig 15B), and the striatum (Fig 15C). Consistent with our previous study (Fig 5) incorporation of L-dopa into proteins was significantly increased in the striatum of the rats treated with L-dopa and benserazide (Fig 15C). Co-administration of tyrosine with L-dopa and benserazide was shown to significantly reduce L-dopa incorporation into proteins such that L-dopa levels in proteins returned to baseline levels (Fig 15C). Detailed description of the preferred embodiments

The present invention contemplates compositions and methods for inhibiting, preventing or reducing L-dopa cytotoxicity in a cell, tissue and/or subject. The present invention also contemplates compositions and methods for inhibiting, preventing or reducing incorporation of L-dopa into peptide or protein in a cell, tissue and/or subject. The present invention further contemplates compositions and methods for inhibiting, preventing or reducing formation of proteinaceous aggregates in a cell, tissue and/or subject. The present invention further contemplates compositions and methods for enhancing the synthesis of dopamine in a cell, tissue and/or subject e.g., in a cell, tissue and/or subject receiving exogenous L-dopa or in need of exogenous L-dopa treatment.

Formulations

1. General

A composition comprising an amount of L-dopa or a salt, solvate or hydrate thereof and/or an amount of tyrosine or a salt, solvate or hydrate thereof is formulated for use with at least one pharmaceutically acceptable carrier or excipient or diluent e.g., suitable for inhalation or oral administration or injection. Formulation of a pharmaceutical composition will vary according to the route of administration selected.

In one example an excipient, carrier or diluent useful in a formulation described herein according to any example, facilitates the storage, administration, and/or the biological activity of an active compound. In one example, the excipient, carrier or diluent will facilitate the biological activity of L-dopa or a salt or solvate or hydrate thereof with respect of biosynthesis of dopamine from L-dopa. In another example, the excipient carrier or diluent will facilitate the biological activity of tyrosine or a salt or solvate or hydrate thereof with respect of reducing and/or preventing incorporation of L-dopa into a peptide or protein. In yet another example, the excipient carrier or diluent will facilitate the biological activity of tyrosine or a salt or solvate or hydrate thereof with respect of biosynthesis of L-dopa from tyrosine.

In one example, an excipient, carrier or diluent useful in a formulation can reduce any undesirable side effects of the active compound. In one such example, the a excipient, carrier or diluent can reduce the reducing side effects from L-dopa such as nausea and vomiting.

In one example, an excipient, carrier or diluent is not capable of reacting with other ingredients in the formulation. In one such example, the excipient, carrier or diluent will not react with L-dopa or a salt or solvate or hydrate thereof and/or with tyrosine or a salt or solvate or hydrate thereof and/or with decarboxylase inhibitor that is optionally present in the formulation. In one example, the excipient, carrier or diluent useful in a formulation described herein according to any example, does not produce significant local or systemic adverse effect in recipients at the dosages and concentrations employed for treatment or prophylaxis using the pharmaceutical compositions of the present invention. In one example a excipient, carrier or diluent useful in a formulation described herein according to any example, permits the uptake of L-dopa a salt, solvate or hydrate thereof and tyrosine a salt, solvate or hydrate thereof by the cell, and or tissue in a subject. In one example, the suitable excipient, carrier or diluent is excipient, carrier or diluent that is suitable for absorption of the bio-reactive composition to the gastrointestinal- tract and/or for crossing the blood-brain barrier e.g., to central nervous system and the cerebrospinal fluid (CSF). Excipients will typically be included in the dosage form e.g., to improve solubility and/or bioadhesion. Suitable excipients include solvents, co-solvents, emulsifiers, plasticizers, surfactants, thickeners, pH modifiers, emollients, antioxidants, and chelating agents, wetting agents, and water absorbing agents. In one example, suitable excipients, and carrier include water, saline, aqueous dextrose, dimethyl sulfoxide (DMSO), and glycols are preferred liquid carriers, particularly (when isotonic) for solutions. In another example, suitable pharmaceutical excipients and carriers include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, ethanol, and the like. In another example, a suitable excipient, carrier or diluent comprises ascorbic acid e.g., 0.1% ascorbic acid. In another example, a suitable excipient, carrier or diluent comprises Tween solution e.g., 0,5% Tween 80 solution. In another example, a suitable excipient, carrier or diluent comprises gum arabic solution e.g., 2% arabic solution. In another example, a suitable excipient, carrier or diluent is a lipid excipient or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include e.g., sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishes and the like (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Pa., 1985).

Formulations may also include one or more additives, for example, dyes, colored pigments, pearlescent agents, deodorizers, and odor maskers.

Diluents or fillers increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.

For inhalation, the composition according to any example hereof can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser). In one example, suitable dispersants include phosphate- buffered saline (PBS), saline, glucose, sodium lauryl sulfate (SLS), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), and hydroxypropylmethylcellulose (HPMC). Formulations may also comprise one or more solubilizing agents to promote dissolution in aqueous media. Suitable solubilizing agents include but not limited to wetting agents such as polysorbates, glycerin, a poloxamer, non-ionic surfactant, ionic surfactant, food acid, food base e.g., sodium bicarbonate, or an alcohol.

Formulations may also comprise one or more stabilizing agents and/or preservatives to inhibit or retard L-dopa or a salt, solvate or hydrate thereof and/or tyrosine a salt, solvate or hydrate thereof decomposition reactions in storage or in vivo. In one example, a salt, solvate or hydrate thereof L-dopa or a salt, solvate or hydrate thereof and/or tyrosine a salt, solvate or hydrate thereof may be susceptible to decomposition by way of example, oxidative reactions, hydrolysis and proteolysis. Stabilizing agents suitable for use in the pharmaceutical composition according to any example hereof include but not limited to protease inhibitors, polysaccharides such as cellulose and cellulose derivatives, and simple alcohols, such as glycerol; bacteriostatic agents such as phenol, m-cresol and methylparaben; isotonic agents, such as sodium chloride, glycerol, and glucose; lecithins, such as example natural lecithins (e.g. egg yolk lecithin or soya bean lecithin) and synthetic or semisynthetic lecithins (e.g. dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoyl- phosphatidylcholine; phosphatidic acids; phosphatidylethanolamines; phosphatidylserines such as distearoyl-phosphatidylserine, dipalmitoylphosphatidylserine and diarachidoylphospahtidylserine; phosphatidylglycerols; phosphatidylinositols; cardiolipins; sphingomyelins. In one example, the stabilizer may be a combination of glycerol, bacteriostatic agents and isotonic agents.

Formulations may also comprise one or more binding agents to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet, bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose ("HPMC"), microcrystalline cellulose ("MCC"), hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone (PVP). Formulations may also comprise one or more lubricants to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

Formulations may also comprise one or more disintegrants to facilitate disintegration of the formulation e.g., a solid formulation, after administration, and include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross-linked PVP.

Formulations may also comprise one or more surfactants, which may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-00 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β- alanine, sodium N-lauryl- β-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

Formulations may also comprise buffer salts for pH control, e.g., sodium phosphate, sodium carbonate or TRIS buffer to maintain the formulation at about physiological pH. Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active agent. In one example, the active agent is L-dopa or a salt, solvate or hydrate thereof. In another example, the active agent is tyrosine or a salt, solvate or hydrate thereof. In another example, the active agent is both L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof. In another example, the active agent may be described as "agent" and may include one or more dopamine agonists and one or more inhibitors as described herein and understood by a person skilled in the art. The concentration of active agent may vary depending upon whether or not the formulation is for prevention or therapy, the route of administration, half-life of the compound following administration by the selected route, and the age, weight and condition of the patient including e.g., the severity of the condition being treated such as Parkinson's disease, Alzheimer's disease, dementia.

In one example, a unit dose may comprise about 1 μg to about 10 μg, or about 0.01 mg to about 2500 mg, or about 2.5 mg to about 10,000 mg, or about 0.1 mg to about 250mg, or about 10 mg to about 25mg of L-dopa or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomer or racemic mixture. In another example, a unit dose may comprise about 1 μg to about 10 μg, or about 0.01 mg to about 2500 mg, or about 2.5 mg to about 10,000 mg, or about 0.1 mg to about 250mg, or about 10 mg to about 25mg of tyrosine or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomer or racemic mixture. In another example, L-dopa and/or tyrosine or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomers or racemic mixtures may be formulated such that the concentration of active agent is at least about 1% (w/w) or at least about 5% (w/w) or at least about 10% (w/w) or at least about 25% (w/w) based on the total weight of the pharmaceutical composition.

As contemplated herein, the ratio of delivered exogenous tyrosine or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomers or racemic mixtures to L-dopa or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomers or racemic mixtures may be about 10:1. The ratio may be lower or higher than 10:1. A ratio that is effective to prevent or obviate accumulation of L-dopa into mammalian tissues and/or cells may depend on, for example, route of delivery of the tyrosine and L-dopa, whether tyrosine is delivered in vitro or in vivo, and/or the types of tissues and/or cells that receive the tyrosine and L-dopa. The cell type, for example, is neurons. In a non-limiting example, one class of neurons that may be associated with accumulation of L-dopa is midbrain dopaminergic neurons, such as substantia nigra compacta (SNc) neurons and the ventral tegmental area (VTA) neurons. The ratio of exogenous tyrosine or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomers or racemic mixtures to L-dopa or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomers or racemic mixtures may may be about 8:1, about 6:1, about 4:1, about 2:1, about 1:1, about 1:2, about 1:4, about 1:6, about 1:8, about 1:10, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90 and about 1:100. Alternatively, the ratio may be about 12:1, about 14:1, about 16:1, about 18:1, about 20:1, about 25:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1 and about 100:1.

As contemplated herein and as described in the examples, the co-administration of tyrosine (whether it be exogenous or endogenous) with L-dopa is beneficial as it (i) stimulates production of homo vanillic acid, a major metabolite of dopamine in, for example, individuals with Parkinson's disease, (ii) obviates L-dopa misincorporation into proteins and (iii) protects against protein misfolding and/or protein aggregation, all without decreasing dopamine concentrations or synthesis.

As used herein "unit dosage" or "unit dosage form" is taken to mean a single or multiple dose form containing a quantity of the active ingredient active material in admixture with or otherwise in association with the an excipient, diluent, carrier and/or adjuvant and quantity being such that one or more predetermined units are normally required for a single therapeutic or prophylactic administration. In the case of multiple dose forms such as liquids or scored tablets, said predetermined unit will be one fraction such as 5 ml (teaspoon) quantity of a liquid or a half or quarter of a scored tablet, of the multiple dose form. In one example, a unit dosage according to any example hereof is further formulated to comprise an amount of an inhibitor of an aromatic-L-amino-acid decarboxylase. In one such example, the a unit dosage is formulated to comprise an amount of inhibitor of DOPA Decarboxylase or DDC such as but not limited to carbidopa or a prodrug thereof and/or benserazide or a prodrug thereof. In another example, the a unit dosage is formulated to comprise an amount of an inhibitor of a monoamine oxidase (MAO) such as but not limited to L-deprenyl, clorgyline, pargyline, N-(2-aminoethyl)-4- chlorobenzamide hydro-chloride, N-(2-aminoethyl)-5-(3-fluorophenyl)-4- thiazolecarboxamide hydrochloride, and derivatives thereof, or a fluoroallylamine and derivatives thereof. In one such example a suitable a fluoroallylamine is selected from the group consisting of 2-isobuty 1-3 -fluoroallylamine, 2-isopropyl-3 -fluoroallylamine, 2-(9-octadecenyl)-3-fluoroallylamine, 2-(3-methyl-3-butenyl)-3 -fluoroallylamine, 2-(4- methoxy-2-butenyl)-3 -fluoroallylamine, 2-isobutylsulfonylmethyl-3 -fluoroallylamine, 2-sec-butyl-3 -fluoroallylamine, 2-butyl-3 -fluoroallylamine, 2-hexyl-3 -fluoroallylamine, 2-heptyl-3 -fluoroallylamine, 2-ethoxymethyl-3 -fluoroallylamine, and 2- thioethoxymethy 1-3 -fluoroallylamine. In another example, the a unit dosage is formulated to comprise an amount of a catechol-O-methyltransferase (COMT) inhibitor such as but not limited to entacapone or tolcapone. In such example, the inhibition of COMT makes an increased amount of L-dopa available for conversion to dopamine by prolonging the plasma-half life and increasing the area under the plasma concentration- time curve of L-dopa.

In one example, a unit dosage of the present invention may further formulated be comprise an effective amount of at least one dehydroepiandrosterone (DHEA) or dehydroepiandrosterone-sulfate (DHEA-S). In one example, the formulations of the L-dopa or salt, solvate or hydrate thereof and the tyrosine or salt, solvate or hydrate thereof are of the same form. In another example, the formulations of the L-dopa or salt, solvate or hydrate thereof and the tyrosine or salt, solvate or hydrate thereof are of different forms. To prepare pharmaceutical formulations, one or more of L-dopa or salt, solvate or hydrate thereof and/or tyrosine or salt, solvate or hydrate thereof and/or the decarboxylase inhibitors and/or the tyrosine hydroxylase inhibitors and/or the MAO inhibitors and or the COMT inhibitors and/or the DHEA and/or the DHEA-S is/are mixed with a pharmaceutically acceptable carrier or excipient for example, by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.). 2. Oral formulations

In one example, an amount of L-dopa or a salt, solvate or hydrate thereof and an amount of tyrosine or a salt, solvate or hydrate thereof can be incorporated into pharmaceutical compositions to be administered orally. Oral administration of the pharmaceutical composition according to any example hereof can result in uptake of the L-dopa and tyrosine throughout the intestine and entry into the systemic circulation for crossing the blood-brain barrier e.g., to central nervous system and the cerebrospinal fluid (CSF).

In one example, of L-dopa or a salt, solvate or hydrate thereof and an amount of tyrosine or a salt, solvate or hydrate thereof are mixed with an excipient, diluted or enclosed within a carrier, which can be in the form of a capsule, sachet, paper or other container. In one example, the excipient serves as a diluent. In one such example, the vehicle or carrier medium for the L-dopa or a salt, solvate or hydrate and/or tyrosine or a salt, solvate or hydrate is a solid, semi-solid, or liquid material.

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules, gel capsules, tablets, pills, powders or granules, lozenges, sachets, cachets, elixirs, suspensions, emulsions, or solutions in aqueous or non-aqueous liquids, or oil-in-water liquid emulsions or water-in-oil liquid emulsions, edible foams or whips, and syrups containing, for example, up to 90% by weight of the active compound using, for example, soft and hard gelatin capsules.

In one example, compositions comprising L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof according to any example hereof are milled to provide an appropriate particle size prior to combining with other ingredients of the formulation. In one example, L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof is/are substantially insoluble. According to this example, L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof is/are are milled to a particle size of less than 200 mesh. In another example, L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof is/are substantially soluble e.g., water soluble. According to this example, L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof is/are are milled to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh. Some examples of suitable excipients for oral formulation of the composition according to any example hereof include but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose.

In one example, the compositions for oral administration are further formulated to comprise lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxy-benzoates, sweetening agents, pH adjusting and buffering agents, toxicity adjusting agents, flavoring agents, and the like.

In one example, the oral formulation comprises an intragranular phase comprising an effective amount of L-dopa or salt, solvate or hydrate thereof and an effective amount of tyrosine or salt, solvate or hydrate thereof according to any example hereof and at least one carbohydrate alcohol and an aqueous binder. In one example, the pharmaceutical formulation is substantially lactose-free. For example, carbohydrate alcohols for such formulations are selected from the group consisting of mannitol, maltitol, sorbitol, lactitol, erythritol and xylitol. In one such example, the carbohydrate alcohol is present at a concentration of about 15% to about 90%. For example, an aqueous binder is selected from the group consisting of hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose sodium, polyvinyl pyrrolidones, starches, gelatins and povidones. In one such example, a binder is generally present in the range of from about 1% to about 15%. In one example, the intragranular phase also comprises one or more diluents, such as a diluent selected from the group consisting of microcrystalline cellulose, powdered cellulose, calcium phosphate-dibasic, calcium sulfate, dextrates, dextrins, alginates and dextrose excipients. In one such example diluents are also present in the range of about 15% to about 90%. In one example, the intragranular phase also comprises one or more disintegrants, for example, a disintegrant selected from the group consisting of a low substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethylcellulose, sodium carboxymethyl cellulose, sodium starch glycollate, crospovidone, croscarmellose sodium, starch, crystalline cellulose, hydroxypropyl starch, and partially pregelatinized starch. In one such example, a disintegrant is generally present in the range of from about 5% to about 20%. In one example, a formulation can comprises one or more lubricants such as a lubricant selected from the group consisting of talc, magnesium stearate, stearic acid, hydrogenated vegetable oils, glyceryl behenate, polyethylene glycols and derivatives thereof, sodium lauryl sulphate and sodium stearyl fumarate. In one such example, a lubricant is generally present in the range of from about 0.5% to about 5%. In one example the formulation comprising intragranular phase are made into a tablet, capsule, or soft gel e.g., by a process comprising mixing an amount of L-dopa or salt, solvate or hydrate thereof and an amount of tyrosine or salt, solvate or hydrate thereof according to any example hereof, and at least one carbohydrate alcohol to form a dry blend. In one such example, the formulation further comprises mixing an inhibitor of an aromatic-L-amino-acid decarboxylase such as carbidopa or benserazide or prodrug thereof and/or an amount of inhibitor of tyrosine hydroxylase (TH) or prodrug thereof. In one example, the formulation further comprises wet granulating the dry blend with an aqueous binder so as to obtain an intragranular phase, and further formulating the resulting intragranular phase so as to provide the formulation.

In one example, tablet or capsules are prepared to contain from about 1 mg to about 10000 mg, such as from about 2.5 mg to about 1000 mg or from about 2.5 mg to about 250 mg or from about lOmg to about lOOmg or from about 15 mg in about 75 mg or from about 17 mg to about 50 mg or about 25 mg of L-dopa or salt, solvate or hydrate thereof per unit dose. In another example, tablet or capsules are prepared to contain from about 1 mg to about 10000 mg, such as from about 2.5 mg to about 1000 mg or from about 2.5 mg to about 250 mg or from about lOmg to about lOOmg or from about 15 mg in about 75 mg or from about 17 mg to about 50 mg or about 25 mg of tyrosine or salt, solvate or hydrate thereof per unit dose.

In one example, tablets or pills comprising L-dopa and/or tyrosine can be coated or otherwise compounded to provide a dosage form affording the advantage of sustained release. For example, a tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over and/or enclosing the former. In one example, the inner dosage and an outer dosage components can be separated by an enteric layer. For example, the enteric layer can serve to resist disintegration in the stomach and/or permit the inner component to pass intact into the duodenum, and/or to delay release. A variety of materials may be used for such enteric layers or coatings. For example, enteric layers or coatings materials include polymeric acids and mixtures of polymeric acids with shellac, cetyl alcohol, or cellulose acetate.

A liquid or semi-solid pharmaceutical formulation for oral administration e.g., a hard gel or soft gel capsule, may be prepared comprising:

(a) a first carrier component comprising from about 10% to about 99.99% by weight of L-dopa or salt, solvate or hydrate thereof and/or from about 10% to about 99.99% by weight of tyrosine or salt, solvate or hydrate thereof according to any example hereof;

(b) an optional second carrier component comprising, when present, up to about 70% by weight of said L-dopa or salt, solvate or hydrate thereof and/or up to about 70% by weight of tyrosine or salt, solvate or hydrate thereof;

(c) an optional emulsifying/solubilizing component comprising, when present, from about 0.01% to about 30% by weight of L-dopa or salt, solvate or hydrate thereof and/or from about 0.01% to about 30% by weight of tyrosine or salt, solvate or hydrate thereof;

(d) an optional anti-cry stallization/solubilizing component comprising, when present, from about 0.01% to about 30% by weight of L-dopa or salt, solvate or hydrate thereof and/or tyrosine or salt, solvate or hydrate thereof; and

(e) an active pharmacological agent comprising from about 0.01% to about 80% of said L-dopa or salt, solvate or hydrate thereof and/or from about 0.01% to about 30% by weight of tyrosine or salt, solvate or hydrate thereof in anhydrous crystal form.

In one example, the first carrier component and optional second carrier component comprise, independently, one or more of lauroyl macrogol glycerides, caprylocaproyl macrogolglycerides, stearoyl macrogol glycerides, linoleoyl macrogol glycerides, oleoyl macrogol glycerides, polyalkylene glycol, polyethylene glycol, polypropylene glycol, polyoxyethylene-polyoxypropylene copolymer, fatty alcohol, polyoxyethylene fatty alcohol ether, fatty acid, polyethoxylated fatty acid ester, propylene glycol fatty acid ester, fatty ester, glycerides of fatty acid, polyoxyethylene-glycerol fatty ester, polyoxypropylene-glycerol fatty ester, polyglycolized glycerides, polyglycerol fatty acid ester, sorbitan ester, polyethoxylated sorbitan ester, polyethoxylated cholesterol, polyethoxylated castor oil, polyethoxylated sterol, lecithin, glycerol, sorbic acid, sorbitol, or polyethoxylated vegetable oil. In one example, the emulsifying/solubilizing component comprises one or more of metallic alkyl sulfate, quaternary ammonium compounds, salts of fatty acids, sulfosuccinates, taurates, amino acids, lauroyl macrogol glycerides, caprylocaproyl macrogolglycerides, stearoyl macrogol glycerides, linoleoyl macrogol glycerides, oleoyl macrogol glycerides, polyalkylene glycol, polyethylene glycol, polypropylene glycol, polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene fatty alcohol ether, fatty acid, polyethoxylated fatty acid ester, propylene glycol fatty acid ester, polyoxyethylene-glycerol fatty ester, polyglycolized glycerides, polyglycerol fatty acid ester, sorbitan ester, polyethoxylated sorbitan ester, polyethoxylated cholesterol, polyethoxylated castor oil, polyethoxylated sterol, lecithin, or polyethoxylated vegetable oil.

In one example, the anti-crystallization/solubilizing component, when present, comprises one or more of metallic alkyl sulfate, polyvinylpyrrolidone, lauroyl macrogol glycerides, caprylocaproyl macrogolglycerides, stearoyl macrogol glycerides, linoleoyl macrogol glycerides, oleoyl macrogol glycerides, polyalkylene glycol, polyethylene glycol, polypropylene glycol, polyoxyethylene-polyoxypropylene copolymer, fatty alcohol, polyoxyethylene fatty alcohol ether, fatty acid, polyethoxylated fatty acid ester, propylene glycol fatty acid ester, fatty ester, glycerides of fatty acid, polyoxyethylene-glycerol fatty ester, polyglycolized glycerides, polyglycerol fatty acid ester, sorbitan ester, polyethoxylated sorbitan ester, polyethoxylated cholesterol, polyethoxylated castor oil, polyethoxylated sterol, lecithin, or polyethoxylated vegetable oil.

In one example, excipients are included in the dosage form e.g., to improve bioadhesion. Suitable excipients include solvents, co-solvents, emulsifiers, plasticizers, surfactants, thickeners, pH modifiers, emollients, antioxidants, and chelating agents, wetting agents, and water absorbing agents. The formulation may also include one or more additives, for example, dyes, colored pigments, pearlescent agents, deodorizers, and odor maskers.

In one example, the composition comprising L-dopa or salt, solvate or hydrate thereof and/or tyrosine or salt, solvate or hydrate thereof are optionally encapsulated or molecularly dispersed in polymers to reduce particle size and increase dissolution. For example, the polymers include polyesters such as but not limited to poly(lactic acid) or P(LA), polycaprylactone, polylactide-coglycolide or P(LGA), poly hydroxybutyrate poly β-malic acid); polyanhydrides such as poly(adipic)anhydride or P(AA), poly(fumaric-co-sebacic)anhydride or P(FA:SA), poly(sebacic)anhydride or P(SA); cellulosic polymers such as ethylcellulose, cellulose acetate, cellulose acetate phthalate, etc; acrylate and methacrylate polymers such as Eudragit RS 100, RL 100, El 00 PO, L100-55, L100, S100 (distributed by Rohm America) or other polymers commonly used for encapsulation for pharmaceutical purposes and known to those skilled in the art. For example other suitable polymers include hydrophobic polymers such as polyimides.

Blending or copolymerization sufficient to provide a certain amount of hydrophilic character can be useful to improve wettability of the materials. For example, about 5% to about 20% of monomers may be hydrophilic monomers. Hydrophilic polymers such as hydroxylpropylcellulose (HPC), hydroxpropylmethylcellulose (HPMC), carboxymethylcellulose (CMC) are commonly used for this purpose.

In one example, formulations for oral delivery of the compositions of the present invention according to any example hereof are relatively lipophilic and readily absorbed by the lining of the stomach and/or the intestine. By appropriate formulation of the compounds in the formulations, the levels of L-dopa or a salt, solvate or hydrate thereof and/or the levels of tyrosine or a salt, solvate or hydrate thereof in body fluids such as blood, plasma and urine may be enhanced, relative to their deposition in adipose tissues. For example, a composition comprising L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof is formulated with a hydrophobic polymer, preferably a bioadhesive polymer and optionally encapsulated in or dispersed throughout a microparticle or nanoparticle. In one example, the bioadhesive polymer improves gastrointestinal retention via adherence of the formulation to the walls of the gastrointestinal tract. For example, suitable bioadhesive polymers include polylactic acid, polystyrene, poly(bis carboxy phenoxy propane-co-sebacic anhydride) (20:80) (poly (CCP:SA)), alginate (freshly prepared); and poly(fumaric anhydride-co-sebacic anhydride (20:80) (poly (FA:SA)), types A (containing sudan red dye) and B (undyed). Other high-adhesion polymers include p(FA:SA) (50:50) and non-water-soluble polyacrylates and polyacrylamides. In one example, bioadhesive polymers are suffciently hydrophobic to be non-water-soluble, but contain a sufficient amount of exposed surface carboxyl groups to promote adhesion e.g., non-water-soluble polyacrylates and polymethacrylates; polymers of hydroxy acids, such as polylactide and polyglycolide; polyanhydrides; polyorthoesters; blends comprising these polymers; and copolymers comprising the monomers of these polymers. In one example, the biopolymers are bioerodable, with preferred molecular weights ranging from 1000 to 15,000 kDa, and most preferably 2000 to 5000 Da. In one example, the bioadhesive polymers are polyanhydrides e.g., polyadipic anhydride ("p(AA)"), polyfumaric anhydride, polysebacic anhydride, polymaleic anhydride, polymalic anhydride, polyphthalic anhydride, polyisophthalic anhydride, polyaspartic anhydride, polyterephthalic anhydride, polyisophthalic anhydride, poly carboxyphenoxypropane anhydride and copolymers with other polyanhydrides at different mole ratios. In one example, blends of hydrophilic polymers and bioadhesive hydrophobic polymers can are employed. In one such example, hydrophilic polymers include e.g., hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, polyvinylalcohols, polyvinylpyrollidones, and polyethylene glycols.

3. Formulations for inhalation

In another example, a formulation comprising L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof is adapted for administration by inhalation. In accordance with this example, the active agent is formulated to produce a fine particle, dust or mist, which may be generated by means of a metered dose inhaler, nebulizer or insufflator. Spray compositions may be formulated as aerosols delivered from pressurized packs, such as a metered dose inhaler, with the use of a suitable liquefied propellant. Capsules and cartridges comprising e.g., gelatine, may be produced for use in an inhaler or insufflator, wherein the L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof comprises a powder contained within the capsule or cartridge. The powder may be produced using a suitable powder base e.g., lactose or starch. Aerosol formulations are preferably arranged so that each metered dose or "puff of aerosol contains about 0.00 ^g to about 2000μg of L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof.

4. Nasal formulations

Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. The overall daily dose and the metered dose delivered by capsules and cartridges in an inhaler or insufflator will generally be double those with aerosol formulations.

5. Parenteral formulations

In another example, a formulation comprising one or more of L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof is adapted for parenteral administration e.g., by subcutaneous or intravenous injection. Such formulations include aqueous and non-aqueous sterile injection solutions which may contain the antioxidants as well as buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

In one example, pharmaceutical composition of L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof used in the method according to any example hereof, is in an intravenous lipid emulsion or a surfactant micelle or polymeric micelle {see., e.g., Jones et al, Eur. J. Pharmaceutics Biopharmaceutics 48, 101-111, 1999; TorchilinJ Clin, release 73, 137-172, 2001).

6. Enteric formulations

The composition of the present invention according to any embodiment thereof may also be formulated for administration enterically.

In one example, the composition is formulated for rectal application. According to this example, the compositions may be formulated as microenemas, suppositories, rectal tablets, rectal devices, sustained-release formulations, and other standard rectal- application forms known in the art. In one example, formulation is a solid suppository comprising a pharmacologically required dose of L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof that is sufficient to provide a therapeutic plasma levels of L-dopa and tyrosine such as to reduce and/ore prevent L- dopa cytotoxicity and/or to prevent incorporation of L-dopa into a peptide or protein, and sufficient suppository base to formulate an acceptable composition. The methods and choice of excipients and suppository bases are well known to those skilled in the art and the composition of said formulations is not limited to solid suppositories by this invention.

In another example, the composition is formulated for infusion to the duodenum to avoid any fluctuations in L-dopa levels in the plasma following oral administration. In one example, duodenal infusion allows delivery of large volumes of the pharmaceutical composition according to any example hereof to the cell, tissue or subject in need thereof. In one such example, an amount of an active agent e.g. an amount of L-dopa or a salt, solvate or hydrate thereof and/or an amount of tyrosine or a salt, solvate or hydrate thereof according to any example hereof are admixed with a suitable carrier, e.g., a gel carrier such as carboxymethyl cellulose. In once such example, using a gel of carboxymethyl cellulose delivers L-dopa and/or tyrosine at concentrations as high as 20 mg/ml each. 7. Topical formulation

The composition of the present invention according to any embodiment thereof may also be formulated for topical administration.

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches impregnated with the composition according to any example hereof. The patches are intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient e.g., L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof may be delivered in a releasable form from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), p318 et seq. (1986).

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For treatments of external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient as L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the as L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof may be formulated in a cream with an oil- in-water cream base or a water-in-oil base. as L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof may also formulated into a cosmetic base for topical application. The pharmaceutical formulation used in the process of the invention according to any example hereof, may also comprise other functional ingredients. It will be apparent to the skilled artisan what functional ingredients are to be included and is dependent upon the symptoms of skin damage that may be associated with L-dopa cytotoxicity that are being treated.

In one example, the formulation further comprises anti-inflammatory agents such as for example, alclometason, amcinonide, benzoyl peroxide, betamethasone, clobetasol, cortisone, hydrocortisone, desonide, desoximetasone, diflorasone, or any agents that treat symptoms associated with dermatitis, including psoriasis, and eczema that may be formulated with an anti-inflammatory agent in a cosmetic base for topical application for local prevention of inflammation and/or tissue damage consequent to inflammation.

A variety of steroidal and non-steroidal anti-inflammatory agents can be combined with the SOD/catalase mimetic. For example, steroidal anti-inflammatory agents, including but not limited to, corticosteroids such as hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fiupreclnisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, eprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof may be used. The preferred steroidal anti-inflammatory for use in the present invention is hydrocortisone.

Specific non-steroidal anti-inflammatory agents useful in the composition of the present invention include, but are not limited to: piroxicam, isoxicam, tenoxicam, sudoxicam, CP- 14,304, aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, fendosal, diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, azemetacin, fentiazac, zomepirac, clidanac, oxepinac, felbinac, mefenamic, meclofenamic, flufenamic, niflumic, tolfenamic acids, ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alinoprofen, tiaprofenic, phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone, among others.

Mixtures of these non-steroidal anti-inflammatory agents may also be employed, as well as the pharmaceutically-acceptable salts and esters of these agents. For example, etofenamate, a flufenamic acid derivative, is particularly useful for topical application. Of the nonsteroidal anti-inflammatory agents, ibuprofen, naproxen, flufenamic acid, mefenamic acid, meclofenamic acid, piroxicam and felbinac are preferred and ibuprofen, naproxen, and flufenamic acid are most preferred. Finally, so-called "natural" anti-inflammatory agents are useful in the present invention. For example, candelilla wax, alpha bisabolol, aloe vera, Manjistha (extracted from plants in the genus Rubia, particularly Rubia Cordifolia) , and Guggul (extracted from plants in the genus Commiphora, particularly Commiphora Mukul) , may be used. In another example, the formulation further comprises a known compound that is useful for wound-healing such as, but not limited to a MMP inhibitor, or any ligand or modulator of the signalling pathways of growth factors belonging to the TGF-β superfamily of ligands. In another example, the formulation further comprises a known compound that is a useful in an anti-ageing formulation, such as, but not limited to a matrix metalloproteinase (MMP) inhibitor, folic acid, creatine, revitol, hydroderm, ceramide c, hyaluronic acid, vitamin A, vitamin C, vitamin E, and glycolic compounds.

In another example, the formulation further comprises sun-block with an SPF rating to block UVB rays and/or compounds such as for example, titanium dioxide, zinc oxide and/or avobenzone, which helps protect against UVA rays.

In another example, the formulation further comprises anti-allergic agents such as for example, any known blocker of histamine release in skin.

In another example, the formulation further comprises anti-acne formulations such as for example, benzoyl peroxide, bacitracin and /or other generic anti-acne compounds.

The pharmaceutical/cosmetic formulations used in the process of the invention according to any embodiment describe herein formulated as solutions typically include a pharmaceutically- or cosmetically-acceptable organic solvent. The terms "pharmaceutically-acceptable organic solvent" and "cosmetically-acceptable organic solvent" refer to an organic solvent which, in addition to being capable of having dispersed or dissolved therein the L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof, and optionally also an anti-inflammatory agent, also possesses acceptable safety (e.g. irritation and sensitization characteristics), as well as good aesthetic properties (e.g., does not feel greasy or tacky). The most typical example of such a solvent is isopropanol. Examples of other suitable organic solvents include: propylene glycol, polyethylene glycol (200-600), polypropylene glycol (425-2025), glycerol, 1,2,4-butanetriol, sorbitol esters, 1 ,2,6-hexanetriol, ethanol, butanediol, water and mixtures thereof. These solutions contain from about 0.0001% to about 20%, preferably from about 0.01% to about 1%, SOD/Catalase mimetic, from about 0.01% to about 5%, preferably from about 0.5% to about 2% of an anti-inflammatory agent, and from about 80% to about 99%, preferably from about 90% to about 98%, of an acceptable organic solvent. As used herein, "emollients" refer to materials used for the prevention or relief of dryness, as well as for the protection of the skin. A wide variety of suitable emollients are known and may be used herein. Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 32-43 (1972), incorporated herein by reference, contains numerous examples of suitable materials. Examples of classes of useful emollients include the following: 1. Hydrocarbon oils and waxes. Examples include mineral oil, petrolatum, paraffin, ceresin, ozokerite, icrocrystalline wax, polyethylene, and perhydrosqualene.

2. Silicone oils, such as dimethyl polysiloxanes, methylphenyl polysiloxanes, water- soluble and alcohol-soluble silicone glycol copolymers.

3. Triglyceride esters, for example vegetable and animal fats and oils. Examples include castor oil, safflower oil, cottonseed oil, corn oil, olive oil, cod liver oil, almond oil, avocado oil, palm oil, sesame oil, and soybean oil.

4. Acetoglyceride esters, such as acetylated monoglycerides.

5. Ethoxylated glycerides, such as ethoxylated glyceryl monostearate.

6. Alkyl esters of fatty acids having 10 to 20 carbon atoms. Methyl, isopropyl, and butyl esters of fatty acids are particularly useful herein. Examples of other useful alkyl esters include hexyl laurate, isohexyl laurate, isohexyl palmitate, isopropyl palmitate, decyl oleate, isodecyl oleate, hexadecyl stearate, decyl stearate, isopropyl isostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyl adipate, diisopropyl sebacate, auryl lactate, myristyl lactate, and cetyl lactate.

7. Alkenyl esters of fatty acids having 10 to 20 carbon atoms. Examples include oleyl myristate, oleyl stearate, and oleyl oleate.

8. Fatty acids having 10 to 20 carbon atoms. Suitable examples include pelargonic, lauric, myristic, palmitic, stearic, isosstearic, hydroxystearic, oleic, linoleic, ricinoleic, arachidic, behenic, and erucic acids.

9. Fatty alcohols having 10 to 20 carbon atoms. Lauryl, myristyl, cetyl, hexadecyl, stearyl, isostearyl, hydroxystearyl, oleyl, ricinoleyl, behenyl, and erucyl alcohols, as well as 2-octyl dodecanol, are examples of satisfactory fatty alcohols.

10. Fatty alcohol ethers. Ethoxylated fatty alcohols of 10 to 20 carbon atoms include the lauryl, cetyl, stearyl, isostearyl, oelyl, and cholesterol alcohols having attached thereto from 1 to 50 ethylene oxide groups or 1 to 50 propylene oxide groups.

11. Ether-esters such as fatty acid esters of ethoxylated fatty alcohols.

12. Lanolin and derivatives. Lanolin, lanolin oil, lanolin wax, lanolin alcohols, lanolin fatty acids, isopropyl lanolate, ethoxylated lanolin, ethoxylated lanolin alcohols, ethoxylated cholesterol, propoxylated lanolin alcohols, acetylated lanolin, acetylated lanolin alcohols, lanolin alcohols linoleate, lanolin alcohols ricinoleate, acetate of lanolin alcohols ricinoleate, acetate of ethoxylated alcohols-esters, hydrogenolysis of lanolin, ethoxylated hydrogenated lanolin, ethoxylated sorbitol lanolin, and liquid and semisolid lanolin absorption bases are illustrative of emollients derived from lanolin. 13. Polyhydric alcohols and polyether derivatives. Propylene glycol, dipropylene glycol, polypropylene glycols 2000 and 4000, polyoxyethylene polyoxypropylene glycols, polyoxypropylene polyoxyethylene glycols, glycerol, sorbitol, ethoxylated sorbitol, hydroxypropyl sorbitol, polyethylene glycols 200-6000, methoxy polyethylene glycols 350, 550, 750, 2000 and 5000, polyethylene oxide] homopolymers (100,000- 5,000,000) , polyalkylene glycols and derivatives, hexylene glycol (2-methyl-2 , 4- pentanediol) , 1,3-butylene glycol, 1,2,6-hexanetriol, ethohexadiol USP (2-ethyl-l, 3- hexanediol) , C15-C18 vicinal glycol, and polyoxypropylene derivatives of trimethylolpropane are examples of this class of materials. 14. Polyhydric alcohol esters. Ethylene glycol mono- and di-fatty acid esters, diethylene glycol mono- and di- fatty acid esters, polyethylene glycol (200-6000) mono- and di-fatty acid esters, propylene glycol mono- and di-fatty acid esters, polypropylene glycol 2000 monooleate, polypropylene glycol 2000 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono- and di-fatty acid esters, polyglycerol poly-fatty acid esters, ethoxylated glyceryl monostearate,

1,3-butylene glycol monostearate, 1,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty acid esters, and polyoxyethylene sorbitan fatty acid esters are satisfactory polyhydric alcohol esters for use herein.

15. Wax esters such as beeswax, spermaceti, myristyl myristate, stearyl stearate.

16. Beeswax derivatives, e.g. polyoxyethylene sorbitol beeswax. These are reaction products of beeswax with ethoxylated sorbitol of varying ethylene oxide content, forming a mixture of ether-esters.

17. Vegetable waxes including carnauba and candelilla waxes.

18. Phospholipids, such as lecithin and derivatives. 19. Sterols. Cholesterol and cholesterol fatty acid esters are examples thereof.

20. Amides such as fatty acid amides, ethoxylated fatty acid amides, solid fatty acid alkanolamides.

Particularly useful emollients which provide skin conditioning are glycerol, hexanetriol, butanetriol, lactic acid and its salts, urea, pyrrolidone carboxylic acid and its salts, amino acids, guanidine, diglycerol and triglycerol. Preferred skin conditioning agents are the propoxylated glycerol derivatives.

6. Immediate and sustained release formulations

In one example, the compositions for administration according to any example hereof are formulated so as to provide immediate release of L-dopa or a salt, solvate or hydrate thereof and/or immediate release of tyrosine or a salt, solvate or hydrate thereof. In one such example the pharmaceutical composition is formulated to release at least 85% (wt/wt) of the L-dopa or a salt, solvate or hydrate thereof and/or at least 85% (wt/wt) of the tyrosine or a salt, solvate or hydrate thereof within 60 minutes in vivo after administration.

In another example, the compositions for administration according to any example hereof are formulated so as to provide a sustained or controlled release of L-dopa or a salt, solvate or hydrate thereof and/or sustained or controlled release of tyrosine or a salt, solvate or hydrate thereof after administration to the subject. In one such example, the pharmaceutical composition is formulated to release L-dopa and/or tyrosine in vivo more slowly than an immediate release formulation. In one such example, it takes longer than 60 minutes to release at least 85% 85% (wt/wt) of the L-dopa or a salt, solvate or hydrate thereof and/or at least 85% (wt/wt) of the tyrosine or a salt, solvate or hydrate thereof in vivo after administration.

In one example, the ratio of L-dopa and/or tyrosine to polymer can be increased. Without being bound by theory or mode of action, increased relative drug concentration is believed to have the effect of increasing the effective compound domain size within the polymer matrix thereby slowing dissolution. In the case of a polymer matrix containing certain types of hydrophobic polymers, the polymer will act for example as a mucoadhesive material and may increase the retention time of the active compound in the gastrointestinal tract. Increased drug dissolution rates combined with the mucoadhesive properties of the polymer matrix increase uptake of the active compound and reduce differences found in the fed and fasted states for the compounds.

In one example, oral compositions formulated to provide sustained release e.g., of L- dopa or a salt, solvate or hydrate thereof and or of tyrosine or a salt, solvate or hydrate thereof comprise beads that on dissolution or diffusion release the L-dopa and/or tyrosine over an extended period of hours, for example of at least 4 hours, or at least 8 hours, or at least 12 hours, or at least 24 hours, or over a period of more than 24 hours. In one example, the L-dopa and/or tyrosine-releasing beads have a central composition or core comprising a of L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof and pharmaceutically acceptable vehicles, and optionally including a lubricant and/or antioxidant and/or buffer. For example, timed- release beads suitable for formulation according to any example hereof are disclosed in Lu, Int. J. Pharm., 1994, 112, 117-124; Pharmaceutical Sciences by Remington, 14th ed, pp. 1626-1628 (1970); Fincher, J. Pharm. Sci., 1968, 57, 1825-1835; and U.S. Pat. No. 4,083,949, incorporated herein by reference in their entirety). For example, suitable time-release tablets are disclosed in Pharmaceutical Sciences by Remington, 17.sup.th Ed, Ch. 90, pp. 1603-1625 (1985) (incorporated herein by reference in its entirety). In one example, oral compositions according to any example hereof are formulated to provide sustained release of the composition using an oral sustained release pump, such as for example as descried in Langer, 1990, Science, 249:1527-1533; Sefton, 1987, CRC Crit. Ref. Biomed. Eng., 14:201; Saudek et al., 1989, N. Engl. J. Med., 321:574. Polymeric materials suitable for oral sustained release delivery of L-dopa and/or tyrosine is described, for example, in "Medical Applications of Controlled Release," Langer and Wise (eds.), CRC Press, Boca Raton, Fla. (1974); "Controlled Drug Bioavailability," Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol Chem., 23:61; Levy et al., 1985, Science, 228: 190; During et al., 1989, Ann. Neurol., 25:351; and Howard et al., 1989, J. Neurosurg., 71 :105.

In one example, enteric-coated oral preparations for sustained release administration of L-dopa and/or tyrosine. In one such embodiment, the coating material includes at least one polymer with a pH-dependent solubility (i.e., pH-controlled release). In another example, the coating material includes at least one polymer with a slow or pH- dependent rate of swelling, dissolution and/or erosion (i.e., time-controlled release). In another example, the coating material includes at least one polymer that can be degraded by enzymes (i.e., enzyme-controlled release). In another example, the coating material includes at least one polymer forming firm layers that can be destroyed by an increase in pressure (i.e., pressure-controlled release).

In one example, drug-releasing lipid matrices or prodrug-releasing waxes can be used for oral sustained release administration.

In one example, a dosage form of the pharmaceutical composition according to any example hereof comprises a L-dopa or a salt, solvate or hydrate thereof and/or tyrosine L-dopa or a salt, solvate or hydrate thereof coated on a erodible, or a nonerodible polymer substrate. Examples of representative biodegradable polymers are described, for example, in Rosoff, Controlled Release of Drugs, Chap. 2, pp. 53-95 (1989); and U.S. Pat. Nos. 3,811,444; 3,962,414; 4,066,747; 4,070,347; 4,079,038; and 4,093,709. In on example, a dosage form of the pharmaceutical composition according to any example hereof comprises L-dopa or a salt, solvate or hydrate thereof and/or tyrosine L-dopa or a salt, solvate or hydrate thereof loaded into a polymer that releases the L- dopa and/or tyrosine by diffusion through a polymer, or by flux through pores or by rupture of a polymer matrix as described, for example, in Coleman et al., Polymers, 1990, 31, 1187-1231; Roerdink et al., Drug Carrier Systems, 1989, 9, 57-100; Leong et al., Adv. Drug Delivery Rev., 1987, 1, 199-233; Roff et al., Handbook of Common Polymers, 1971, CRC Press; and U.S. Pat. No. 3,992,518.

In one example, osmotic delivery systems can also be used for oral sustained release administration as described in Verma et al., Drug Dev. Ind. Pharm., 2000, 26:695-708.

Sustained release injectable formulations are produced e.g., by encapsulating the L- dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof in porous microparticles which comprise a pharmaceutical agent and a matrix material having a volume average diameter between about 1 um and 150 μπι, e.g., between about 5 um and 25 μηι diameter. In example, the porous microparticles have an average porosity between about 5% and 90% by volume. In one example, the porous microparticles further comprise one or more surfactants, such as a phospholipid. The microparticles may be dispersed in a pharmaceutically acceptable aqueous or nonaqueous vehicle for injection. Suitable matrix materials for such formulations comprise a biocompatible synthetic polymer, a lipid, a hydrophobic molecule, or a combination thereof. For example, the synthetic polymer can comprise, for example, a polymer selected from the group consisting of poly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol), polyalkylene oxides such as poly(ethylene oxide), polyalkylene terepthalates such as poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene, polyurethanes and co-polymers thereof, derivativized celluloses such as alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt (jointly referred to herein as "synthetic celluloses"), polymers of acrylic acid, methacrylic acid or copolymers or derivatives thereof including esters, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids"), poly(butyric acid), poly(valeric acid), and poly(lactide-co- caprolactone), copolymers, derivatives and blends thereof. In a preferred embodiment, the synthetic polymer comprises a poly(lactic acid), a poly(glycolic acid), a poly(lactic- co-glycolic acid), or a poly(lactide-co-glycolide).

In one example, a controlled-release system is placed in proximity to the target tissue for L-dopa and/or tyrosine release, thus requiring only a fraction of the systemic dose. In one such example, controlled release system includes a pump and tube system for enteral infusion of L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof into the duodenum as described for example in Nyholm D., Expert Rev Neurother 6:1403-1411 (2006). Other suitable controlled-release system for use in the pharmaceutical formulations according to any example hereof, include for example as described in Goodson, in "Medical Applications of Controlled Release," supra, vol. 2, pp. 115-138 (1984) and/or in Langer, 1990, Science, 249:1527-1533 can also be used. Regardless of the specific form of sustained release formulation used used, L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof can be released from the dosage form, e.g., an orally administered dosage form, over a sufficient period of time to provide prolonged therapeutic concentrations of L-dopa and/or tyrosine in the blood of a patient enabling administration of the dosage form on only a once or twice per day basis. In one example, the formulation can maintain a therapeutic or prophylactic blood concentration of L-dopa and/or tyrosine in the systemic circulation of a patient following administration of L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof over a period of at least 4 hours, or over a period of at least 8 hours, or over a period of at least 12 hours, or over a period of at least 24 hours. Dosages

The pharmaceutical compositions according to any example hereof can be administered for prophylactic and/or therapeutic treatments. Therapeutically effective amounts of L- dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof are amounts sufficient to remedy a disease state or symptoms, or otherwise prevent, hinder, retard, or reverse the progression of disease or any other undesirable symptoms in any way whatsoever. In prophylactic applications, the pharmaceutical compositions are administered to a patient susceptible to or otherwise at risk of a developing a particular disease or condition. Hence, a prophylactically effective amounts of L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof are amounts sufficient to prevent, hinder or retard a disease state or its symptoms.

In one example, L-dopa or a salt, solvate or hydrate thereof is co-administered with tyrosine or a salt, solvate or hydrate thereof in the method according to any example hereof in effective amounts sufficient to reduce or prevent L-dopa incorporation into a peptide or protein. In one such example, the co-administration provides substitution therapy for dopamine. It will be understood, however, that the precise effective amount of any compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, e.g., Parkinson's disease and/or Alzheimer's disease and/or demenia, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. It will be understood that L-dopa or a salt, solvate or hydrate thereof can be coadministered with tyrosine or a salt, solvate or hydrate thereof either substantially at the same time as, or prior to, or after the administration of tyrosine or a salt, solvate or hydrate thereof. When administered prior, L-dopa or a salt, solvate or hydrate thereof can be given up to 12 hours prior, depending on the route of administration and severity of the condition being treated. In one example, L-dopa or a salt, solvate or hydrate thereof is administered up to about 12 hours prior administration of tyrosine or a salt, solvate or hydrate thereof. In another example, L-dopa or a salt, solvate or hydrate thereof is administered up to about 6 hours prior administration of tyrosine or a salt, solvate or hydrate thereof. In another example, L-dopa or a salt, solvate or hydrate thereof is administered up to about 4 hours prior administration of tyrosine or a salt, solvate or hydrate thereof.

In one example L-dopa or a salt, solvate or hydrate thereof can be given up to 12 hours after administration of tyrosine or a salt, solvate or hydrate thereof, depending on the route of administration and severity of the condition being treated. In one example, L- dopa or a salt, solvate or hydrate thereof is administered up to about 12 hours after administration of tyrosine or a salt, solvate or hydrate thereof. In another example, L- dopa or a salt, solvate or hydrate thereof is administered up to about 6 hours after administration of tyrosine or a salt, solvate or hydrate thereof. In another example, L- dopa or a salt, solvate or hydrate thereof and/is administered up to about 4 hours after administration of tyrosine or a salt, solvate or hydrate thereof.

In one example, L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof can be co-administered in a dosage unit, in formulations containing both L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof as active agents. In another example, L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof can be coadministered in a dosage unit, in formulations containing each of L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof as the sole active agent. In either mode of administration, the relative amount of L-dopa or a salt, solvate or hydrate thereof administered as compared to the amount of tyrosine or a salt, solvate or hydrate thereof, will vary depending upon the severity of the condition to be treated, and the compound employed.

In one example, the ratio of the amount of L-dopa or a salt, solvate or hydrate thereof administered as compared to the amount of tyrosine or a salt, solvate or hydrate thereof that is administered is from about 1:1 to about 500:1, or from about 1:1 to about 200:1, or from about 1:1 to about 100:1, or from about 1:1 to about 10:1, or from about 1:1 to 5 : 1 , or from about 1 : 1 to 2: 1. In another example, the ratio of the amount of L-dopa or a salt, solvate or hydrate thereof administered as compared to the amount of tyrosine or a salt, solvate or hydrate thereof that is administered is from about 1:1 to about 1:100, or from about 1:1 to about 1:50, or from about 1:1 to about 1:10, or from about 1 :1 to about 1:5, or from about 1:1 to about 1:2. The appropriate ratio of the amount of L-dopa or a salt, solvate or hydrate thereof administered as compared to the amount of tyrosine or a salt, solvate or hydrate thereof in the pharmaceutical composition according to any example hereof can be determined according to any one of several established protocols. For example, animal studies, such as studies using mice or rats, can be used to determine an appropriate ratio of pharmaceutical compounds in the composition of the invention. The results from animal studies can be extrapolated to determine doses for use in other species, such as for example, humans. In one example, the appropriate ratio may be determined using in vitro studies in dopaminergic SH-SY5Y cell line and/or the rat model described herein. Alternatively, or in addition, a ratio of tyrosine to L-dopa is determined by administering different ratios to another accepted animal model of Parkinson's disease e.g., a reserpine model, a methamphetamine model, a MPTP model, a Paraquat-Maneb model, a rotenone model, a 3-nitrotyrosine model or a transgenic a-synuclein model confirms the effectiveness of this combination therapy in vivo. Alternatively, or in addition, a ratio of tyrosine to L-dopa is determined by administering different ratios to an accepted animal model of Alzheimer's Disease e.g., a murine transgenic amyloid precursor protein model such as the Αβ model described by Lamb, Nature Genet. 9: 4- 6 (1995). The effective amount of L-dopa or a salt, solvate or hydrate thereof administered by the method according to any example hereof typically ranges from about 2.5 mg to about 10 g per day. In one example, an effective amount of L-dopa or a salt, solvate or hydrate thereof administered is from about 10 mg to about 5 g per day. In another example, an effective amount of L-dopa or a salt, solvate or hydrate thereof administered is from about 50 mg to about 2 g per day. In one example, an effective amount of L-dopa or a salt, solvate or hydrate thereof administered is from about 100 mg to 1.5 gram per day. In another example, an effective amount of L-dopa or a salt, solvate or hydrate thereof administered is from about 200 mg to about 1.5 g per day. In another example, an effective amount of L-dopa or a salt, solvate or hydrate thereof administered is from about 300 mg to about 1.5 g per day. In another example, an effective amount of L-dopa or a salt, solvate or hydrate thereof administered is from about 400 mg to about 1.5 g per day. In another example, an effective amount of L- dopa or a salt, solvate or hydrate thereof administered is from 400 mg to about 1.2 g per day. It will be understood that the effective amount of L-dopa or a salt, solvate or hydrate thereof administered according to the method of any example hereof may vary during the course of the treatment or prophylaxis. In one example, L- L-dopa or a salt, solvate or hydrate is administered initially at a dose from about 2.5 mg to about 1 g per day, after which the amount administered is gradually increased for example over a period of 3 to 14 days, or 3 to 7 days, up to a maximum tolerated daily dose of about 10 grams per day.

The effective amount of tyrosine or a salt, solvate or hydrate thereof administered by the method according to any example hereof typically ranges from about 2.5 mg to about 12 g per day. In one example, an effective amount of tyrosine or a salt, solvate or hydrate thereof administered is from about 10 mg to about 2.5 g per day. In another example, an effective amount of tyrosine or a salt, solvate or hydrate thereof administered is from 200 mg to 2.5 g per day. In another example, an effective amount of tyrosine or a salt, solvate or hydrate thereof administered is from about 50 mg to about 1.5 g per day. In one example, an effective amount of tyrosine or a salt, solvate or hydrate thereof administered is from about 100 mg to 1.0 gram per day. In another example, an effective amount of tyrosine or a salt, solvate or hydrate thereof administered is from about 200 mg to about 1.0 g per day. In another example, an effective amount of tyrosine or a salt, solvate or hydrate thereof administered is from about 300 mg to about 1.0 g per day. In another example, an effective amount of L- dopa or a salt, solvate or hydrate thereof administered is from about 400 mg to about 1.0 g per day. In another example, an effective amount of L-dopa or a salt, solvate or hydrate thereof administered is from 500 mg to about 800 mg per day. In one preferred example, the effective amount of L-dopa or a salt, solvate or hydrate thereof administered is from 500 mg to about 720 mg per day.

Alternatively, the effective amount of tyrosine or a salt, solvate or hydrate thereof administered by the method according to any example hereof typically ranges from about 10 to about 200 mg per kg body weight per day. In one example, an effective amount of tyrosine or a salt, solvate or hydrate thereof administered is from about 50 to about 150 mg per kg body weight per day.

It will be understood that the effective amount of tyrosine or a salt, solvate or hydrate thereof administered by the method according to any example hereof may vary during the course of the treatment or prophylaxis. In one example, tyrosine or a salt, solvate or hydrate thereof is administered initially at a dose from about 2.5 mg to about 1 g per day, after which the amount administered is gradually increased for example over a period of 3 to 14 days, or 3 to 7 days, up to a maximum tolerated daily dose of about 12 grams per day.

When an aromatic-L-amino-acid decarboxylase inhibitor such as carbidopa or benserazide is employed in the composition or method according to any example hereof, the amount of decarboxylase inhibitor administered as compared to the amount L-dopa administered will generally vary from about 1:10 to 1:4. In one example, an effective amount of the inhibitor administered typically ranges from about 1 mg to about 1.0 g per day. In one example, an effective amount of the inhibitor administered is about 1 mg to about 800 mg per day, or about 5 mg to about 500 mg per day, or about 10 mg to about 200 mg per day. When an inhibitor of tyrosine hydroxylase (TH) such as 3-iodo-L-tyrosine or MPTP or l-Methyl-4-phenylpyridinium ion (MPP+) is employed is employed in the composition or method according to any example hereof, an effective amount of the inhibitor administered typically ranges from about 1 mg to about 1.0 g per day. In one example, an effective amount of the inhibitor administered is about 1 mg to about 800 mg per day, or about 5 mg to about 500 mg per day, or about 10 mg to about 200 mg per day.

When an inhibitor of monoamine oxidase (i.e., MAO inhibitor) such as L-deprenyl, clorgyline, pargyline, N-(2-aminoethyl)-4-chlorobenzamide hydro-chloride, N-(2- aminoethyl)-5-(3-fluorophenyl)-4-thiazolecarboxamide hydrochloride, fluoroallylamine or derivatives thereofs, is employed in the composition or method according to any example hereof, the dosage of L-dopa may optionally be reduced e.g., 2 to 10 fold, as compared to the dosage of L-dopa that is administered in the absence of an MAO inhibitor. In one example, the amount of MAO inhibitor administered as compared to the amount L-dopa administered will vary from about 1 :20 to 1 :500 depending on the compound employed as the inhibitor. In one example, an effective amount of MAO inhibitor administered is from about 0.1 mg to about 100 mg per day. In another example, the an effective amount of MAO inhibitor administered is from about 1 mg to about 100 mg per day. In another example, the an effective amount of MAO inhibitor administered is from about 5 mg to about 25 mg per day. In another example, the an effective amount of MAO inhibitor administered is from about 10 mg to about 20 mg per day. In one example, fluoroallylamine is used as an inhibitor of MAO. According to this example, an effective amount of the fluoroallylamine that may be administered is about 0.1 mg to about 100 mg per day. At these dosage levels the fluoroallylamine may inhibit both forms of MAO i.e„ type A and MAO-type. In another example, an effective amount of the fluoroallylamine that may be administered is about 0.1 mg to about 0.5 mg per day. According to this example, the fluoroallylamine may selectively inhibit only MAO type B.

It is to be understood that other MAO inhibitors, in addition to fluoroallylamine can have dose-dependent preferential inhibition of MAO-type A or MAO-type B. For example, MAO-A may be preferentially inhibited by clorgyline. In another example, MAO-B may be preferentially inhibited by pargyline and L-deprenyl. The selectivity of an inhibitor for either MAO-A or MAO-B in vivo will be dose-dependent. For example, selectivity being lost as the dosage is increased. According to this example, clorgyline, pargyline, and L-deprenyl are selective inhibitors at lower dosages, but are less selective inhibitors as higher dosages. The literature concerning MAO-A and MAO-B and the selective inhibition thereof is extensive [See, for example, Goodman and Gilman, ibid, pages 204-205; Neff et al., Life Sciences, 14, 2061 (1974); Murphy, Biochemical Pharmacology, 27, 1889 (1978); Knoll, Chapter 10, pages 151-171 and Sandler, Chapter 11, pages 173-181, in Enzyme Inhibitors as Drugs, M. Sandler, Ed., McMillan Press Ltd., London 1980; Lipper et al., Psychopharmacology, 62, 123 (1979); Mann et al., Life Sciences, 26, 877 (1980); and various articles in Monoamines Oxidase: Structure, Function, and Altered Functions, T. Singer et al. Ed., Academic Press. N.Y., 1979].

When a catechol-O-methyltransferase (COMT) inhibitor such as entacapone or tolcapone is employed in the composition or method according to any example hereof, the amount of inhibitor administered typically ranges from about 100 mg to about 2.0 g per day. In one example, an effective amount of the inhibitor administered is about 100 mg to about 1.6 g per day, or about 200 mg to about 1.6 g per day.

Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. In one example, the doses of the L-dopa salt, solvate or hydrate thereof and tyrosine, salt, solvate or hydrate thereof, and optionally the doses of decarboxylase inhibitor and/or inhibitor of tyrosine hydroxylase and/or MAO inhibitor and/or catechol-O- methyltransferase (COMT) inhibitor and/or DHEA and/or DHEA-S are administered sequentially. In another example, the doses of L-dopa salt, solvate or hydrate thereof and tyrosine, salt, solvate or hydrate thereof, and optionally doses of decarboxylase inhibitor and/or inhibitor of tyrosine hydroxylase and/or MAO inhibitor and/or catechol-O-methyltransferase (COMT) inhibitor and/or DHEA and/or DHEA-S are administered simultaneously. In a preferred example, the doses of the L-dopa salt, solvate or hydrate thereof and tyrosine, salt, solvate or hydrate thereof, and optionally the doses of decarboxylase inhibitor and/or inhibitor of tyrosine hydroxylase and/or MAO inhibitor and/or catechol-O-methyltransferase (COMT) inhibitor and/or DHEA and/or DHEA-S are administered to a patient such that the active compounds may be found in the patient's bloodstream at the same time, regardless when the compounds are actually administered, for example, simultaneously or sequentially.

The composition of the invention may be administered according to any standard dosing schedule for L-dopa therapy. The frequency of administration of the composition can vary depending on any of a variety of factors, e.g., severity of the symptoms, etc. For example, the dosages are administered once per day, twice per day, three times per day or at shorter intervals. In another example, the dosage forms are administered once every other day, six times per week, five times per week, four times per week, three times per week, twice per week twice per week, once per week twice per week, or at longer intervals. Higher concentrations of L-dopa and/or greater dosage frequency may be employed relative to standard L-dopa therapy i.e., lacking tyrosine, without the same degree of cytotoxic side effects that would arise from standard L-dopa therapy at such elevated concentrations and/or dosage frequency. The optimum dosage is subject to one or more factors e.g., the severity of the disease or condition to be treated, the overall health of the patient, the method route and dose of administration, and the severity of side affects.

Determination of the appropriate dose may be made by a clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose may commence with an amount somewhat less than the optimum dose and be increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of the disease and/or disorder being treated. Such parameters are described in general e.g., by Maynard, et al, In: A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla. (1996) or Dent In: Good Laboratory and Good Clinical Practice, Urch Publ., London, UK (2001).

Modes of administration

The present invention contemplates any mode of administration of a medicament or formulation as described herein. Combinations of different administration routes are also encompassed.

In one example, a composition of the present invention can be administered to a subject using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. Conventional and pharmaceutically acceptable routes of administration contemplated by the invention include but are not necessarily limited to enteral, parenteral, or inhalational routes. Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, vaginal, rectal, intratracheal, intranasal, intradermal, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrastemal, nasal, topical application, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Enteral routes of administration include, but are not necessarily limited to, oral, rectal (e.g., using a suppository) delivery, and duodenal infusion. Parenteral administration can be carried to effect systemic or local delivery of the composition. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations. The composition can be administered in a single dose or in multiple doses.

In one example, inhalable compositions are administered in an aqueous solution e.g., as a nasal or pulmonary spray. Preferred systems for dispensing liquids as a nasal spray are disclosed in U.S. Pat. No. 4,511,069. Such formulations may be conveniently prepared by dissolving compositions according to the present invention in water to produce an aqueous solution, and rendering the solution sterile. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systems have been described in Transdermal Systemic Medication, Y. W. Chien Ed., Elsevier Publishers, New York, 1985; and in U.S. Pat. No. 4,778,810 (each incorporated herein by reference). Additional aerosol delivery forms may include, e.g., compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, e.g., water, ethanol, or a mixture thereof.

In one example, mucosal formulations are administered as dry powder formulations e.g., comprising L-dopa or a salt, solvate or hydrate thereof and/or tyrosine or a salt, solvate or hydrate thereof in a dry, usually lyophilized, form of an appropriate particle size, or within an appropriate particle size range, for intranasal delivery. Minimum particle size appropriate for deposition within the nasal or pulmonary passages is often about 0.5 micron mass median equivalent aerodynamic diameter (MMEAD), commonly about 1 micron MMEAD, and more typically about 2 micron MMEAD. Maximum particle size appropriate for deposition within the nasal passages is often about 10 micron MMEAD, commonly about 8 micron MMEAD, and more typically about 4 micron MMEAD. Intranasally respirable powders within these size ranges can be produced by a variety of conventional techniques, such as jet milling, spray drying, solvent precipitation, supercritical fluid condensation, and the like. These dry powders of appropriate MMEAD can be administered to a patient via a conventional dry powder inhaler (DPI) which rely on the patient's breath, upon pulmonary or nasal inhalation, to disperse the power into an aerosolized amount. Alternatively, the dry powder may be administered via air assisted devices that use an external power source to disperse the powder into an aerosolized amount, e.g., a piston pump. In another example, the composition of the present invention may be administered by injection. Standard methods are used to administer injectable formulations of the present invention. Preferred means for injection of the compositions of the present invention include intravenous, subcutaneous, percutaneous, intramuscular and intradiscal routes, (e.g., intradiscal injection or intradiscal implant) or injection into the CSF, the only requirement being that of L-dopa or a salt, solvate or hydrate thereof and the tyrosine or a salt, solvate or hydrate thereof integers of the composition are delivered to the region of a subject requiring treatment.

In yet another example, the composition of the present invention are administered enterally by duodenal infusion. In one example the compositions are duodenally infused such as by a portable pump such as CADD-Legacy-Duo-Dopa™ (e.g., Smiths Medicals) which may be attached to a cassette comprising the pharmaceutical composition according to any example hereof. In one example, the composition is administered by intraduodenal infusion e.g., through a transabdominal port and is delivered to the duodenum from the cassette by a percutaneous endoscopic gastrostomy (PEG) tube. For example, the PEG tube may further comprise a smaller bore intestinal tube inside. In one example, the intestinal tube is placed beyond the pylorus to allow for immediate absorption of the composition across the intestinal mucosa in the duodenum or proximal jejunum. The placement of the tube may be carried out by any suitable mean known in the art such as for example surgical means and or by mean of a gastroscope or a metal guide wire under fluoroscopy.

In one example, the composition of the invention according to any embodiment hereof is administered as an adjuvant therapy to a standard L-dopa therapy. In another example, the composition of the present invention is administered in place of standard L-dopa therapy. For example, standard L-dopa therapies include but not limited to oral or intravenous administration of dopamine receptor agonists, or administration of L- dopa in combination with a decarboxylase inhibitor.

When used, other chemotherapeutic agents may be administered before, concurrently, or after administration of a composition of the present invention according to any example hereof. In one example, chemotherapeutic agents additional to the compositions described herein according to any example include

In another example, chemotherapeutic agents additional to the compositions described herein according to any example include non-ergoline dopamine -receptor agonists such as ropinirol or pramipexole. In another example, chemotherapeutic agents additional to the compositions described herein according to any example include ergoline dopamine receptor agonists such as pergolide or cabergoline. In another example, chemotherapeutic agents additional to the compositions described herein according to any example include iso-ergoline dopamine receptor agonists such as lisuride. In another example, chemotherapeutic agents additional to the compositions described herein according to any example include a COMT inhibitor such as entacapone. In another example, chemotherapeutic agents additional to the compositions described herein according to any example include organinc compounds such as 1- aminoadamantane (i.e., amantadine). In another example, chemotherapeutic agents additional to the compositions described herein according to any example include morphine-based dopamine agonist such as apomorphine. In one example chemotherapeutic agents additional to the compositions described herein according to any example include cholinesterase inhibitors typically used in treatment of mild Alzheimer's disease and/or dementia for example, - galantamine, rivastigmine, donepezil, and tacrine. In another example, chemotherapeutic agents additional to the compositions described herein according to any example include those typically used in treatment of advanced Alzheimer's disease and/or dementia for example N-methyl D- aspartate (NMD A) antagonists such as memantine. In another chemotherapeutic agents additional to the compositions described herein according to any example include those typically used in treatment of dementia such as hydergine. In another example, chemotherapeutic agents additional to the compo sitions described herein according to any example include antipsychotic compounds such as haloperidol, cMorpromazine, risperidone or clozapine. In one example, a composition of the present invention is co-administered with one or more other chemotherapeutic agents. By "co-administer" in this context it is meant that the present composition is administered to a subject such that the present composition as well as the co-administered compound may be found in the subject's bloodstream at the same time, regardless when the compounds are actually administered, e.g., simultaneously or sequentially.

Duration of administration

The duration of administration of the composition of the present invention, e.g., the period of time over which the composition is administered, can vary, depending on any of a variety of factors, e.g., subject's response, etc. For example, a composition of the present invention can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.

Kits

The present invention also provides kits for reducing and/or preventing L-dopa cytotoxicity, or for reducing or preventing incorporation of L-dopa into a peptide or protein, or for reducing or preventing formation of proteinaceous aggregates comprising L-dopa, or for enhancing dopamine biosynthesis in a cell tissue and/or subject. For example such kits can comprise i) an amount of L-dopa or a salt, solvate or hydrate thereof; and ii) an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein. In one example, the kits according to any example hereof, further comprise any one or more of the following materials packaging material, and instructions regarding dosage, method or duration of administration for using L-dopa or a salt, solvate or hydrate thereof and tyrosine or a salt, solvate or hydrate thereof to treat or preventing a condition ameliorated by L-dopa or a neurodegenerative condition or Parkinson's disease, or Alzheimer's disease or dementia or atherosclerosis or catatactogenesis.

In one example, the kits contain cells that serve as either positive or negative controls. These control cells can be compared to clinical samples containing similar cells, for instance to determine L-dopa cytotoxicity and/or incorporation of L-dopa into a peptide or protein in the clinical samples.

The present invention is described further in the following non-limiting examples.

EXAMPLE 1

L-DOPA COMPETES WITH THE 'PROTEIN' AMINO ACID L-TYROSINE FOR INCORPORATION INTO PROTEINS.

In this first series of studies the inventors provide evidence that L-dopa, an amino acid, can be mistakenly incorporated into proteins by all cells and in all tissues. The inventors demonstrate that incorporation is such that L-dopa becomes peptide bonded into proteins and becomes part of the polypeptide chain of the protein. In addition they also show that L-dopa directly competes with L-tyrosine for incorporation into proteins.

Example 1A: Evidence that L-dopa is incorporated into cell proteins. This example reports on the relationship between concentrations of L-dopa supplied to dopaminergic neurons in vitro, L-dopa and dopamine levels in the cells, and levels of L-dopa present in (ie. incorporated into) cell proteins. Human dopaminergic neuronal cells, SH-SY5Y, were incubated with a range of L-dopa concentrations of 0 μΜ to 1000 μΜ for 9 hours in L-tyrosine-depleted culture medium. HPLC was used to measure intracellular dopamine levels, intracellular L-dopa levels and, levels of protein-incorporated L-dopa.

Results

There was a linear increase in intracellular levels of L-dopa (Fig 1A) when SH-SY5Y cells were incubated with L-dopa (200 to 1000μΜ). There was a linear increase in intracellular dopamine levels when cells were incubated with 100 μΜ to 500 μΜ L- dopa with no further increase at 750 μΜ and 1000 μΜ of L-dopa (Fig. IB). L-dopa levels in cell proteins increased in a linear fashion with increasing L-dopa concentration in the culture medium (Fig 1C). At the concentration of L-dopa required to maximise dopamine synthesis by dopaminergic neurons in vitro (~500μΜ) there was a significant level of incorporation of L-dopa into cell proteins (Fig. 1C). These studies demonstrate that L-dopa is incorporated into cell proteins in a concentration dependent manner.

Example IB; Evidence that L-dopa competes with L-tyrosine for incorporation into proteins and that incorporation of L-dopa into proteins is dependent on protein synthesis.

This example demonstrates that L-dopa competes with L-tyrosine for incorporation into protein and examines the relationship between the concentrations of L-tyrosine and L- dopa that cells are exposed to and the level of incorporation of L-dopa into protein.

SH-SY5Y neuronal cells were incubated with 1 μΜ [¹⁴C]-L-dopa together with L- tyrosine (0, 2, 5, 8, 10, and 15 μΜ) for 24 hours to reproduce therapeutic levels of L- dopa and physiological levels of L-tyrosine (~5 μΜ) in the brains of L-dopa-treated Parkinson's disease (PD) patients or tyrosine levels that could be achieved by supplying exogenous L-tyrosine to such individuals (Fig 2A and 2B). Levels of [¹⁴C]- L-dopa present in the cell in the free form and present in the protein-bound form were determined by liquid scintillation counting. Results Increasing the concentration of L-tyrosine in the culture medium to 5μΜ (a 5 fold excess over L-dopa) resulted in a significant reduction in the levels of incorporation of L-dopa into cell proteins as assessed from the radioactivity present in isolated cell proteins (Fig. 2A). Incorporation of L-dopa into proteins could be further reduced by increasing the L-tyrosine concentration in the culture medium (Fig. 2A). Intracellular 'free' L-dopa levels decreased by around 25% at 15μΜ L-tyrosine and there was no significant change in L-dopa levels in cells at lower L-tyrosine concentrations (Fig 2B).

Uptake of L-dopa by cells (Fig 2B) was therefore significantly less sensitive to the presence of L-tyrosine that L-dopa incorporation into proteins by cells (Fig 2A). These data demonstrate that preventing/limiting L-dopa incorporation into proteins can be achieved without decreasing L-dopa levels in cells, thus without compromising dopamine synthesis. The cellular uptake of L-dopa, which is believed to occur by a high capacity transporter (large aromatic amino acid transporter), therefore appears to be less sensitive to competition from tyrosine than the direct competition that occurs between tyrosine and L-dopa for incorporation into protein which is believe to occur e.g., via tyrosyl tRNA.

The addition of the protein synthesis inhibitor cycloheximide (CHX) to the culture medium significantly reduced incorporation of L-dopa into protein demonstrating that it was a protein synthesis dependent process (Fig 2C).

In these studies a dopamine-synthesising neuroblastoma cell line SH-SY5Y was selected as a model of neuronal cell culture. The SH-SY5Y cell line has been shown to have a short doubling time and is capable of differentiating into different neuron-like subtypes, in addition to its dopamine synthesising property. SH-SY5Y cells were cultured and incubated with radiolabeled ¹⁴C L-dopa at a concentration reported in the cerebrospinal fluid (CSF) of Parkinson's disease patients in the presence of tyrosine (0- 15μΜ). Cells were then tested for incorporation of ¹⁴C L-dopa into proteins at different concentrations of tyrosine. These in vitro studies were carried out using radio-labelled L-dopa, to provide greater sensitivity, and support the inventor's view that incorporation of L-dopa into proteins could also occur in vivo.

HPLC analysis of dopamine levels was also carried out to on these dopaminergic neurones to determine whether or not co-administration of tyrosine with L-dopa affected levels of dopamine synthesis in cultured SH-SY5Y cells. No significant changes in dopamine synthesis by SH-SY5Y cells was found even in the presence of 2.5mM tyrosine (Fig 2D).

Conclusions from Example 1 - - .

• L-dopa competes with L-tyrosine for incorporation into protein.

• Incorporation of L-dopa into protein is dependent on protein synthesis

• Increasing the concentration of L-tyrosine to which cells are exposed can limit or prevent incorporation of L-dopa into proteins without decreasing intracellular L- dopa concentrations and therefore the ability of cells to synthesise dopamine from L- dopa.

• L-tyrosine can thus prevent 'non-native' or damaged proteins being synthesised from L-dopa. Methods used in Example 1

SH-SY5Y cells were obtained from American Type Tissue Culture Collection, (ATCC, Manassas, VA, USA). Eagle's Minimal Essential Medium (EMEM) deficient in L- tyrosine was custom made by JRH Biosciences (Lenaxa, Kansas, USA). SH-SY5Y cells were seeded at a density of 1 x 10⁶ cells/well in 6-wells plates and allowed to equilibrate overnight. Cells were then incubated in tyrosine-free EMEM for 9 hours in the presence of L-dopa. Cells were then lysed e.g., with 0.1% Triton X-100, and the protein and cytosolic fractions separated by TCA precipitation of proteins and centrifugation (5% final concentration). Protein hydrolysis: Proteins in cell fractions and plasma or brain tissues were precipitated with trichloroacetic acid (TCA; 5%) HPLC grade, washed and de-lipidated by re-suspending the pellet twice in TCA (5%) containing sodium deoxycholate (0.02%) and the reductant, sodium borohydride (25 μg/mL) and then washed twice in ice-cold acetone and once in diethyl ether. The delipidated protein samples were then freeze dried and hydrolysed under anaerobic conditions using a standard gas-phase acid catalysed method (HC1 containing mercaptoacetic acid) (Gieseg S. et al., 1993 Biochemistry 32: 4780-4786). The cell- free serum fraction was diluted 1 in 10 with de-ionized water and analysed by HPLC without hydrolysis. All samples were analysed (blinded to treatment) three to five times on separate occasions, using two different operators, and the average values calculated. HPLC analysis of L-dopa: HPLC analysis of dopamine, L-dopa and L-dopa released from proteins by hydrolysis or proteolysis was performed on an LC-IOA HPLC system (Shimadzu) equipped with a column oven (Waters, 30°C) with methods developed in - our laboratory as described in detail previously (Fu S. et al., 1998, J. Biol. Chem.

273:28 603-28 609). System operation was automated by Class LC-10 software. Chromatography was on a Zorbax ODS column (250 mm x 4.6 mm) with a Pelliguard guard column (LC-18). The mobile phase was a gradient of solvent A (10 mM sodium phosphate buffer pH 2.5 with 100 mM sodium perchlorate) and solvent B (80% v/v methanol) at a flow rate of 1 mL/min. The following gradient was used: isocratic elution with 84% A for 12 min then to 80% A over 8 min; elution at 80% A for 3 min before changing to 50% A in 3 min; isocratic elution at 50% A for a further 3 min, then re-equilibration with 100% A for 10 min. The eluate was monitored by a UV detector (Shimadzu), a fluorescence detector (Hitachi F-1080) and an electrochemical detector, ECD (Intro, Antec Leyden) in series. The fluorescence detector was set at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. The electrode potential in the ECD was set at 1.2 V.

The unmodified p-tyrosine (Analar) was quantified by UV measurement when there was an off-scale response by fluorescent detection. The elution positions of the amino acids and oxidized amino acids were defined on the basis of standards using UV, fluorescent and electrochemical spectra. All samples were analysed in triplicate and the levels of L-dopa in the protein hydrolysates expressed as μπιοΐ L-dopa per mol of tyrosine (parent amino acid). Statistical analysis

Statistical analysis was performed using independent samples by Student's t-test, or the Welch's test if variances were unequal. Significance was inferred if p < 0.05. Otherwise analysis was by ANOVA with an appropriate post-hoc test. Incorporation of radiolabelled dopa into cell proteins measured by liquid scintillation counting (LSC). Cell proteins were isolated by TCA precipitation and centrifugation.

EXAMPLE 2. L-DOPA IS PRESENT IN PROTEINS IN THE BRAIN AND PLASMA AFTER EXPOSURE TO THERAPEUTIC LEVELS OF L-DOPA In this series of studies the inventors examine L-dopa incorporation in to protein in vivo. The inventors demonstrate that L-dopa is present in proteins in living creatures (examples shown are humans and rats) that have been exposed to therapeutic levels of L-dopa. The PD patients examined in the present study all received L-dopa together with a DDC inhibitor which increases the half-life of L-dopa and prevents its peripheral conversion to dopamine and/or a COMT inhibitor. The inventors propose that the use of inhibitors of DDC and COMT increases the likelihood of L-dopa incorporation into protein by maintaining blood/tissue levels of L-dopa and therefore the potential benefit/requirement for co-administration of tyrosine.

Example 2A: L-dopa is present in proteins in plasma of L-dopa treated individuals.

In this example the inventors demonstrate that proteins containing L-dopa are present in the plasma of Parkinson's disease (PD) patients that have been treated with L-dopa. Blood (10 mL) was collected from consenting PD patients (5 females and 15 males, average age of 67) and from consenting partners, relatives or friends at the same time (5 females, 6 males, average age of 68) and processed in an identical manner. Plasma was prepared and analysed for the presence of L-dopa-containing proteins by HPLC.

Results

Significantly higher levels of L-dopa-containing proteins were found in plasma from L- dopa-treated patients compared to non-treated individuals (1,100 vs 2,300 μΜοΙεβ dopa/Mole Tyr respectively, p < 0.005).

Since the L-dopa-treated group included patients that had been treated with L-dopa for less than 3 years as well as patients treated with L-dopa for more than 20 years, we compared the length of time patients had been treated with L-dopa to levels of L-dopa- containing proteins in plasma. A significant increase in levels of L-dopa-containing proteins was found only in patients who had been treated with L-dopa for more than 10 years, and levels increased further after a second decade of treatment (Fig. 3).

Example 2B: L-dopa is present in proteins in the brains of L-dopa treated individuals. The inventors then measured levels of L-dopa-containing proteins in extracts of four brain regions from PD patients treated with L-dopa (and/or COMT and DDC inhibitors) and compared these data to levels of L-dopa-containing proteins from the same brain regions of brains from age-matched individuals that had never received L- dopa.

Results

Levels of L-dopa-containing proteins were significantly increased in the substantia nigra, motor cortex and occipital cortex of L-dopa-treated PD patients relative to individuals not treated with L-dopa (Fig 4).

Example 2C: L-dopa is present in proteins in the brains of rats treated with L- dopa for 21 days. To further examine incorporation of L-dopa into proteins in vivo, we carried out HPLC analysis of extracts of the striatum and substantia nigra from brains of rats that had been treated with L-dopa and rats treated with vehicle alone for 21 days. Male Sprague-Dawley rats were injected intraperitoneally (i.p.) with vehicle (1 ml/kg, sterile water, Braun Medical) or L-DOPA methyl ester (6.5 mg/kg, Sigma) and the peripherally acting DDC inhibitor, benserazide (1.5 mg/kg, Sigma) dissolved in sterile water (Braun Medical), twice daily for 21 days (6 rats in each group).

Results

There was a significant increase in L-dopa-containing protein levels in the striatum in L-dopa/benserazide-treated rats (Fig. 5).

Conclusions from Example 2

• Elevated levels of proteins containing L-dopa were present in plasma proteins and brain proteins of L-dopa (COMT/DDC inhibitor) treated individuals.

• Proteins containing L-dopa are elevated in the brains of rats treated with L-dopa for 21 days.

• These data are consistent with a global incorporation of L-dopa into proteins in living creatures treated with L-dopa with or without compounds that modify its metabolism. Methods used in example 2

Preparation of human plasma samples for HPLC analysis. Blood was collected into Vacuette K3 EDTA tubes (Greiner Bio-One, Solingen, Germany) containing EDTA (1.8 mg/mL). Protein levels in the cell free plasma fraction were quantified using the BCA assay then precipitated (10% TCA), washed and hydrolysed. Hydrolysed samples were reconstituted in water, filtered by centrifugation in 0.2 urn spin column, and 80 μΐ used for HPLC analysis. Brain analysis for L-dopa-containing proteins. Male Sprague- Dawley rats (250-300 g) were maintained under standard housing conditions with constant temperature (22 ± 1°C), humidity (relative, 30%), 12 hour light/dark cycles. Food (Standard pellets, B&K Universal) and water were available ad libitum. Rats were injected intraperitoneally (i.p.) with vehicle (1 ml/kg, sterile water, Braun Medical) or L-DOPA methyl ester (6.5 mg/kg, Sigma) and benserazide (1.5 g/kg, Sigma) dissolved in sterile water (Braun Medical), twice daily for 21 days (6 rats in each group).

The brains were frozen and sections cut (20 um thick). Sections from the striatum and substantia nigra were powdered under liquid nitrogen and proteins solubilized in 2% SDS in 0.1M Tris buffer, pH 6.8 at 37°C for 10 minutes. Proteins were then isolated by TCA precipitation and prepared for HPLC analysis as described previously (Rodgers et al. 2004) . Cryopreserved tissue from the motor cortex, occipital cortex and substantia nigra of 5 PD patients (4 male and 1 female, average age 78) who were treated with L- DOPA for between 5 and 13 years and a control group of patients that did not receive L-DOPA (4 male and 1 female, average age 76) was analysed. Proteins were extracted using a modification of the protocol of Ericsson and colleagues (Ericsson et al. 2007); tissue was snap frozen in liquid nitrogen, and powdered proteins were then extracted into in 0.1M Tris buffer, pH 6.8 containing 2% SDS. Proteins were then isolated by TCA precipitation and prepared for HPLC analysis. HPLC analysis was carried out as described in example 1.

EXAMPLE 3. PROTEINS CONTAINING INCORPORATED L-DOPA ARE TOXIC TO CELLS.

In this series of studies the inventors investigate the possible functional implications of (mis)incorporation of L-dopa into cell proteins. They demonstrated that proteins containing incorporated L-dopa impair cell function and can result in cell death. They also demonstrate that proteins containing incorporated L-dopa are potent inducers of apoptosis in neuronal and non-neuronal cells.

Example 3 A: Both stereoisomers of dopa (L-dopa and D-dopa) are taken up by cells but only L-dopa is incorporated into proteins.

In this series of toxicity studies the inventors compare the toxicity of L-dopa, which can be incorporated into proteins, to that of D-dopa, which cannot be incorporated into proteins but has the same chemistry as L-dopa. For this purpose it was important to determine whether or not D-dopa was taken into SH-SY5Y dopaminergic neurons and was therefore able to exert the same intracellular oxidative stress as L-dopa.

SH-SY5Y cells were incubated in tyrosine-deficient medium containing L-dopa (200 μΜ) or D-dopa (200 μΜ) for 1, 2, 3 and 24 hours. Intracellular dopa concentrations were measured by HPLC as described in Example 1 (and Rodgers, 2002, 2004, 2006) and expressed as a ratio to total cell protein (DOPA μΜοΙεβ/ mg protein). Levels of dopa in proteins was also measured after incubation of SH-SY5Y dopaminergic neurons with tyrosine-deficient medium alone (control), D-dopa (500μΜ) and L-dopa (500μΜ) for 24 hours.

Results

A time-dependent increase in intracellular free dopa was detected in both L-dopa and D-dopa-treated cells (Fig. 6). The data of Fig 6 demonstrate that D-dopa is therefore taken up by SH-SY5Y cells at least as efficiently as L-dopa so is an ideal 'non- incorporatable' control for toxicity studies and allows the toxicity of proteins containing L-dopa to be assessed, as this component of dopa toxicity will be present in L-dopa-treated cells but not in D-dopa-treated cells. HPLC analysis of cell proteins demonstrated that L-dopa was incorporated into proteins but D-dopa was not incorporated into proteins (Fig 7). We previously showed that L-dopa incorporation into protein was blocked by the protein synthesis inhibitor cycloheximide (Fig 2C), we now show that only the L-isomer of dopa is present in proteins (Fig 7). Mammalian cells can only utilise L-amino acids in protein synthesis confirming that L-dopa is incorporated into proteins in a protein synthesis dependent manner. Example 3B: L-dopa is more toxic to cells than D-dopa, demonstrating that proteins containing incorporated L-dopa themselves are cytotoxic. L-tyrosine protects cells against L-dopa toxicity by preventipg limiting the incorporation of L-dopa into proteins.

To examine the toxicity mediated by proteins containing incorporated L-dopa, the inventors examined the viability of cells (SH-SY5Y neurons) after incubation for 24 hours in tyrosine-depleted medium containing either L-dopa (500 μΜ) or D-dopa (500 μΜ) or in medium also containing L-tyrosine (10 mM). Cell viability, as a measure of necrosis, was determined by the release of lactate dehydrogenase (LDH) into the culture medium using LDH viability assay which can be performed with any commercially available kit e.g., Bio Vision Inc., CA.

Results

The cell viability results demonstrate that L-dopa is more neurotoxic than D-dopa (Fig. 8, grey bars). The addition of tyrosine (lOmM) to the culture medium protected neuron cells against toxicity mediated by L-dopa (Fig. 8, open bars). This set of experiments demonstrates that proteins containing L-dopa are toxic to cells and cells (e.g., neurons) can be protected against this toxicity with tyrosine.

Example 3C: Proteins containing incorporated L-dopa impair cell function to such an extent that they can induce apoptotic cell death.

These examples provide further evidence for L-dopa cellular toxicity. The inventors further examine toxicity following incubation of THP-1 human monocytic cells with L- dopa, by measuring caspase 3 activation, annexin V binding and DNA fragmentation (TUNEL assay) as markers of apoptosis (programmed cell death). Caspase 3 activation, annexin V binding and DNA fragmentation demonstrate induction of apoptosis in cells before cell death and accordingly provides more sensitive measures of toxicity/loss of function than LDH viability assay (Fig 8) which measures end-stage necrosis or cell death. Annexin V is a specific phospholipid-binding protein that binds with high affinity to cells undergoing late stage apoptosis. Caspase 3 is an effector caspase that demonstrates the commitment of a cell to apotosis. DNA fragmentation is a late-stage apoptotic event (TUNEL assay or COMET assay).

Results

In these studies, THP-1 human monocytes were incubated with L-dopa, D-dopa or tyrosine-depleted culture medium alone (untreated) for 24 hours. Cells were then analysed for induction of apoptosis by measuring: DNA fragmentation (TUNEL assay, by flow cytometry), annexin-V binding (by flow cytometry) and caspase-3 activation (from enzyme activity). Results are presented as the percentage of positive cells showing the apoptotic change. The results shown in Fig 9 demonstrate that treatment with L-dopa but not with D-dopa, resulted in significant induction of apoptosis of THP- 1 macrophages as measured by TUNEL (Fig 9A, p<0.01), annexin-V binding (Fig 9B, p<0.001) and caspase-3 activation (Fig 9C, pO.001). There was no evidence of increased apoptosis of THP-1 macrophages treated with D-dopa alone (Fig 9). Fig 9D demonstrates that induction of apoptosis from L-dopa incorporation into proteins can be prevented with L-tyrosine (prevention of caspase-3 activation shown as an example). These data demonstrate that induction of apoptosis is due to incorporation of L-dopa into proteins since these monocytic cells are not able to synthesise dopamine from L-dopa (unlike dopaminergic neurones) the increase in apoptosis cannot be due to an increase in dopamine synthesis and is due to the synthesis of L-dopa containing proteins and the consequences of this phenomenon which can include protein misfolding and protein aggregation within the cell.

Having demonstrated that proteins containing incorporated L-dopa impair cell function to such an extent that they induce apoptotic cell death, we examined apoptotic changes in SH-SY5Y neuronal cells over a 16 hour time-course using the annexin V binding assay and caspase 3 activation as measures of apoptosis. SH-SY5Y cells were incubated with D-dopa or L-dopa at 500 μΜ for 4, 6, 10 and 16 hours in tyrosine-free medium, the results shown in Fig. 10 demonstrate that caspase 3 activity was significantly increased at 6, 10 and 16 hours (Fig. 10A). Annexin V binding was significantly higher in L-dopa treated SH-SY5 Y cells than those incubated with D-dopa after 10 hours (Fig. 10B).

SH-S Y5 Y cells were protected from apoptosis induced by incorporation of L-dopa into proteins using tyrosine. This was demonstrated by comparing DNA fragmentation in cells treated with L-dopa, D-dopa and L-dopa with L-tyrosine. SH-SY5Y cells were incubated with 200 μΜ of D-dopa (D-DOPA 200), 200 μΜ of L-dopa (L-DOPA 200) or with 200 μΜ of L-dopa plus 2.5 mM tyrosine (L-DOPA 200+tyr) or with tyrosine alone in culture medium (EMEM+tyr) for 24 hours. DNA fragmentation as a measure of apoptosis was measured using Single Cell Gel Electrophoresis assay also known as comet assay as first described by Singh et al., Experimental Cell Research 175(1): 184- 191 (1988). Results are shown in Fig. 11, and indicate that L-dopa was more toxic than D-dopa. This example demonstrated that tyrosine protected cells such as neurons from apoptotic cell death caused by incorporation of L-dopa into protein as indicated by a reduction in DNA fragmentation in cells co-incubated with L-dopa and tyrosine. These results support that conclusion by the inventors that proteins containing dopa are generated in L-dopa-treated cells and are potent inducers of apoptosis in cells including dopaminergic neurons.

This example demonstrates that in vitro co-administration of L-dopa and tyrosine prevents L-dopa cytotoxicity. Co-incubation of SH-SY5Y cells or THP1 cells with L- dopa and tyrosine confirms the therapeutic benefit of tyrosine for preventing L-dopa induced apoptosis or decline in function in neurons or other cells. The inventors concluded that differences in activation of pro-apoptotic markers observed between L- dopa treated cells and D-dopa or L-dopa and tyrosine treated cells, was due to the incorporation of L-dopa into peptides or proteins in the cells in THP-1 macrophages and SH-S Y5 Y neuronal cells. Co-administration of L-dopa with tyrosine inhibits L- dopa incorporation into peptides or proteins in cells and protects against the pro- apoptotic effect of L-dopa incorporated peptides or proteins. The present invention is described further in the following non-limiting examples: Conclusions from Example 3

• L-dopa impairs cell function and induces apoptosis in cells due to its incorporation into proteins.

• L-tyrosine protects cells against the L-dopa induced decline in cell function and from the induction of apoptosis.

Methods used in example 3

THP-1 cell cells: THP-1 cell cells (a human macrophage cell line ) were obtained from American Tissue Culture Collection (ATCC) and were maintained in 750 cm flasks in DMEM containing 10% FCS.

Annexin-V binding: Annexin-V binding to exposed phosphotidylserine groups on the plasma membrane was measured using an Annexin V-FITC Apoptosis Detection Kit (Becton, BD Biosciences) according to manufacturer's instructions. TUNEL: Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling, or TUNEL, was used to identify apoptotic THP-1 cells following degradation of cellular DNA resulting in strand breaks within the DNA, using the APO-BrdUTMTUNEL Assay Kit (Invitrogen), according to manufacturer's instructions.

Caspase-3 activation: To measure caspase-3 activation cells were lysed in lOOmM HEPES pH 7.2S, 10% sucrose, 0.1% CHAPS, 400 μΐ NP-40 and 2mM DTT was prepared at a final concentration of about 5 μg protein and 1 mM DEVD-AMC was added and changes in fluorescence were read on a CytoFluor fluorescent plate reader.

THP-1 cell culture for L-dopa incorporation: For uptake of dopa into THP-1 cells, the culture medium was replaced with tyrosine-free DMEM (JRH Biosciences) containing 10% fetal calf serum (FCS) supplemented with 500 μΜ L-dopa with or without 1 μΜ tyrosine and incubated as before for 24 hours. Alternatively, culture medium was replaced with tyrosine-free DMEM (JRH Biosciences) containing 10% fetal calf serum (FCS) supplemented with 500 μΜ D-dopa as no dopa-incorporation controls or with PBS as untreated control and incubated for 24 hours. After 24 hours the medium was removed and THP-1 macrophages were washed three times with PBS and recovered by centrifugation. Cells were then analysed for induction of pro-apoptotic markers by annexin-V binding, caspase-3 activation, and TUNEL assays by flow cytometry as described supra at 3.2.1 to 3.2.3. Results were calculated as percentage of positive cells for TUNEL activation, annexin-V and for caspase-3 activity and are shown in Fig. 3 and Fig. 4. Fig 4 demonstrates results of caspase-3 activation following co-incubation of cells with L-dopa and tyrosine. For TUNEL activation (Fig 3A) and annexin-V (Fig 3B) results were calculated as percentage of positive cells of total cells in the culture. For caspase-3 activity (Fig 3C and Fig 4) results were calculated as percentage of positive cells compared to untreated control.

SH-SY5Y cell culture for L-dopa incorporation: For uptake of dopa into SH-SY5Y cells, cells were obtained and cultured as described earlier. For incorporation of L- dopa, the culture medium was replaced with tyrosine-free DMEM (JRH Biosciences) containing 10% fetal calf serum (FCS) and 0, 100, 200 or 500 μΜ L-dopa or D-dopa as no dopa-incorporation controls and incubated for 24 hours. After 24 hours the medium was removed and SH-SY5Y cells were washed three times with PBS and recovered by centrifugation. EXAMPLE 4. L-TYROSINE PREVENTS/LIMITS THE INCORPORATION OF L-DOPA INTO PROTEINS WHEN CO-ADMINISTERED WITH L-TYROSINE.

In this series of studies the inventors demonstrated that co-administration of L-dopa and L-tyrosine to rats can protect against incorporation of L-dopa into brain proteins.

Example 4A: When L-tyrosine is co-administered with L-dopa, tyrosine levels in plasma are elevated but levels of L-dopa, dopamine are not altered. One group of rats was injected intraperitoneally (i.p.) with L-dopa methyl ester (6.5 mg/kg, Sigma) and the DDC inhibitor, benserazide (1.5 g/kg, Sigma) and a second group of rats with L-dopa methyl ester (6.5 mg/kg, Sigma), benserazide (1.5 mg/kg, Sigma) and L-tyrosine (lOOmg/kg). To examine plasma levels of L-dopa, tyrosine and dopamine, blood was collected at different time-points after injection (0.5, 1 and 2 hours: 3 rats in each group) and plasma analysed by HPLC as described previously (section 1 and (Rodgers, 2002, 2004, 2006).).

Results

Tyrosine levels in plasma in the tyrosine-treated rats were approximately 3 fold that in the group of rats that did not receive L-tyrosine after 0.5 hours and returned to basal levels after 2.5 hours (Fig 12). No difference was found in levels of L-dopa (Fig 13A) and L-dopamine (Fig 13B) in plasma at any of the time-points examined when L-dopa was co-administered with L-tyrosine. Example 4B: When L-tyrosine is co-administered with L-dopa and the DDC inhibitor benserazide, levels of tyrosine in the brain are increased, levels of L-dopa and dopamine in the brain are not altered. Incorporation of L-dopa into proteins is significantly reduced. One group of rats was injected intraperitoneally (i.p.) with L-dopa methyl ester (6.5 mg/kg, Sigma) and benserazide (1.5 mg/kg, Sigma) twice daily for 21 days and a second group with L-dopa methyl ester (6.5 mg/kg, Sigma), benserazide (1.5 mg/kg, Sigma) and L-tyrosine (100 mg/kg), twice daily (10 rats in each group). After 21 days, rats were sacrificed, brains collected and analysed as described previously (section 2).

Results L-tyrosine levels in the brain were increased on average around 2 fold in the group of rats that received L-tyrosine (Fig 14 A, B and C). Dopamine levels did not differ significantly between the two groups of rats (Fig 14D, E and F) and L-dopa levels did not differ significantly between the two groups of rats (Fig 14 G and H). L-dopa levels in the substantia nigra were below detection levels. Consistent with the previous study (data presented in Fig 5) levels of protein incorporated L-dopa were shown to be significantly increased in proteins in the striatum of rats treated with L-dopa and a DDC inhibitor for 21 days. In the present study the inventors show that coadministration of tyrosine prevents incorporation of L-dopa into proteins in striatum of rat brains (Fig 15).

Conclusions from Example 4

• Administration of L-tyrosine with L-dopa increases tyrosine levels in plasma and brain

• Administration of L-tyrosine with L-dopa does not decrease levels of dopa and dopamine in the plasma or brain.

• Administration of L-tyrosine and L-dopa prevents/reduces incorporation of L- dopa into proteins

· L-tyrosine can protect against the decline in cell function and induction of apoptosis resulting from the incorporation of L-dopa into proteins by preventing reducing incorporation of L-dopa into proteins in vivo.

Methods used in example 4

Male Sprague-Dawley rats (300-350g) were maintained under standard housing conditions with constant temperature (22 ± 1°C), humidity (relative, 30%), 12 hour light/dark cycles. Food (Standard pellets, B&K Universal) and water were available ad libitum. Rats were injected intraperitoneally (i.p.) with L-dopa methyl ester (6.5 mg/kg, Sigma) and benserazide (1.5 g/kg, Sigma) dissolved in sterile water (Braun Medical), twice daily for 21 days or with L-dopa methyl ester (6.5 mg/kg, Sigma), benserazide (1.5 mg/kg, Sigma) and L-tyrosine (100 mg/kg) dissolved in sterile water (Braun Medical), twice daily for 21 days 10 rats in each group). The brains were removed and tissue from the motor cortex, striatum and substantia nigra powdered under liquid nitrogen and proteins solubilized in 2% SDS in 0.1 M Tris buffer, pH 6.8 at 37°C for 10 minutes. Proteins were then isolated by TCA precipitation and prepared for HPLC analysis as described previously (Rodgers et al. 2004). Free L-dopa, tyrosine and dopamine in the protein-free fraction were analysed by HPLC and normalised to the protein content of the tissue extract which was determined by the BCA procedure. HPLC analysis was carried out as described in example 1.

Summary

As was evident from previous data, short term (eg. 21 day) exposure to L-dopa and a DDC inhibitor leads to a significant incorporation of L-dopa into protein in some regions of the brain (Fig 5). Prolonged exposure to L-dopa leads to a more global increase in L-dopa incorporation into proteins (eg Figs 3 and 4) such as would be experienced by patients treated with L-dopa therapeutically (examples shown are plasma and brain extracts). Using the highly controlled rat study the inventors demonstrate that co-administration of tyrosine with L-dopa and the DDC inhibitor benserazide can prevent incorporation of L-dopa into protein in vivo. Thus the administration of tyrosine with L-dopa (at the same time or at a different time) has a great therapeutic potential by protecting cells against the (mis)incorporation of L-dopa into proteins and from the decline in cell function that could occur leading eventually to apoptosis or cell death.

The results confirm the ability of combination therapy of tyrosine and L-dopa (including other agents that are used to increase the efficacy of L-dopa) to permit the beneficial effects of L-dopa without allowing L-dopa-containing proteins to be generated, and implicate administration of L-dopa and tyrosine in clinical treatment with L-dopa. The relative level of supply of tyrosine and L-dopa that maintains or increased efficacy and limits or abolishes the incorporation of L-dopa into proteins is determined. It is to be understood that efficacy of therapy is also determined by administration of a composition comprising tyrosine and L-dopa to any other accepted animal model of Parkinson's disease, or a combination of different animal models e.g., a reserpine model, a methamphetamine model, a MPTP model, a Paraquat-Maneb model, a rotenone model, a 3-nitrotyrosine model or a transgenic a-synuclein model confirms the effectiveness of this combination therapy in vivo. Accepted animal models of Parkinson's Disease are reviewed e.g., by Betarbet et al, BioEssays 24: 308- 318 (2002). In consideration of the generality of the present invention for therapy and prevention of dopamine insufficiency, it is to be understood that efficacy of therapy may also be determined by administration of a composition comprising tyrosine and L-dopa to an accepted animal model of Alzheimer's Disease e.g., a murine transgenic amyloid precursor protein model such as the Αβ model described by Lamb, Nature Genet. 9: 4- 6 (1995).

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A pharmaceutical composition comprising:

i) an amount of L-dopa or a salt, solvate or hydrate thereof; and

ii) an therapeutic amount of tyrosine or a salt, solvate or hydrate thereof, wherein said amounts in combination are sufficient to treat and/or prevent any condition associated with accumulation of L-dopa in a cell, tissue or subject.

2. A pharmaceutical composition for reducing or preventing L-dopa cytotoxicity comprising:

i) an amount of L-dopa or a salt, solvate or hydrate thereof; and

ii) an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent misincorporation of L-dopa into a peptide or protein.

3. A pharmaceutical composition for reducing or preventing incorporation of L- dopa into peptide or protein, wherein said composition comprises:

i) an amount of L-dopa or a salt, solvate or hydrate thereof; and

ii) an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein.

4. A pharmaceutical composition for reducing or preventing formation of proteinaceous aggregates comprising L-dopa in a cell, tissue or subject, wherein said composition comprises:

i) an amount of L-dopa or a salt, solvate or hydrate thereof; and

ii) an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein, thereby reducing or preventing formation of said proteinaceous aggregates in a cell, tissue or subject.

5. A pharmaceutical composition for reducing or preventing formation of misfolded proteins containing L-dopa in a cell, tissue or subject, wherein said composition comprises:

i) an amount of L-dopa or a salt, solvate or hydrate thereof; and ii) an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein, thereby reducing or preventing formation of said proteinaceous aggregates in a cell, tissue or subject.

6. A pharmaceutical composition for enhancing synthesis of dopamine in a cell, tissue or subject, wherein said composition comprises:

i) an amount of L-dopa or a salt, solvate or hydrate thereof; and

ii) an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein thereby enhancing dopamine synthesis in a cell, tissue or subject.

7. The composition of any one of claims 1, 4 to 6 wherein the tissue comprises neural tissue and the cell is a neuron, wherein the tissue or cell is from a mammal.

8. The composition of claim 7, wherein the neuron is a dopaminergic neuron.

9. The composition of any one of claims 1 to 8 further comprising an amount of one or more dopamine agonists or a salt, solvate or hydrate thereof.

10. The composition of claim 9 wherein said one or more dopamine agonists is selected from the group comprising pergolide and bromocriptine.

11. A method of treating and/or preventing any condition associated with accumulation of L-dopa in a cell, tissue or subject, comprising the administration of a first composition and a second composition to said cell, tissue or subject, wherein said first composition is the composition of any one of claims 1 to 6 and said second composition is a composition comprising one or more dopamine agonists, wherein administration of first and second compositions to said cell, tissue or subject is simultaneous or sequential.

12. Use of at least a first composition and a second composition in treating and/or preventing any condition associated with accumulation of L-dopa in a cell, tissue or subject, wherein said first composition is the composition of any one of claims 1 to 6 and said second composition is a composition comprising one or more dopamine agonists, wherein first and second compositions are administered to said cell, tissue or subject simultaneous or sequential.

13. A method of treating and/or preventing any condition associated with accumulation of L-dopa in a cell, tissue or subject, comprising the administration of one or more dopamine agonists in conjunction with L-dopa and tyrosine to said cell, tissue or subject.

14. Use of one or more dopamine agonists in conjunction with L-dopa and tyrosine to treat and/or prevent any condition associated with accumulation of L-dopa in a cell, tissue or subject, wherein said one or more dopamine agonists in conjunction with L- dopa are administered to said cell, tissue or subject.

15. The method according to claim 11 or 13 wherein administration is to a mammal.

16. Use according to claim 12 or 14 wherein administration is to a mammal.

17. The method according to claim 11 wherein the administration of the first and second compositions comprises a first step delivery method to treat or prevent complications and/or conditions associated with dopamine deficiency and/or insufficiency.

18. A method of treating or preventing complications and/or conditions associated with dopamine deficiency and/or insufficiency comprising therapeutic administration of the composition of claim 9 or claim 10.

19. The composition of any one of claims 1 to 8 further comprising one or more agents that inhibits or prevents the metabolism of dopamine.

20. The composition of claim 19 wherein said agent is a monoamine oxidase (MAO) inhibitor.

21. The composition of claim 20 wherein said inhibitor is selected from the group comprising MAO-type A and MAO-type B inhibitors.

22. The composition of claim 20 wherein said inhibitor is selected from the group comprising L-deprenyl, clorgyline, pargyline, and fluoroallylamine.

23. The composition of claim 23 wherein said inhibitor is fluoroallylamine.

24. A method of reducing and/or inhibiting oxidative deamination of dopamine upon its formation from L-dopa, comprising administration of an amount of the composition of any one of claims 19 to 23 to a subject or patient sufficient to reduce and/or inhibit said oxidative deamination.

25. A method to prevent complications and/or conditions associated with dopamine deficiency and/or insufficiency in a subject or patient comprising therapeutic administration of a composition that comprises an amount of L-dopa, an amount of tyrosine and an amount of one or more MAO inhibitors to said subject or patient.

26. The composition of any one of claims 1 to 8 further comprising one or more inhibitors of aromatic-L-amino-acid decarboxylase (DCC) and/or one or more inhibitors of carboxy-O-methyl transferase (COMT).

27. The composition of claim 26 comprising an amount of L-dopa, an amount of tyrosine, an amount of a DCC inhibitor and an amount of a COMT inhibitor.

28. A method of treating or preventing complications and/or conditions associated with dopamine deficiency and/or insufficiency comprising therapeutic administration of the composition of claim 26 or 27 either sequentially or simultaneously with administration of the composition of claim 9 or 10.

29. The composition of any one of claims 1 to 10, 19 to 23, 26 and 27 further comprising a pharmaceutically acceptable adjuvant, excipient, a carrier or diluent.

30. The composition of any one of claims 1 to 10, 19 to 23, 26, 27 and 29 comprising tyrosine or a salt, solvate or hydrate thereof in an amount sufficient to deliver from about 50 to about 150 mg/ Kg of body weight per day of said tyrosine or a salt, solvate or hydrate thereof to a subject, and optionally comprising L-dopa or a salt, solvate or hydrate thereof in an amount sufficient to deliver from 400 to about 1200 mg per day of said L-dopa or a salt, solvate or hydrate thereof to a subject.

31. The composition of any one of claims 1 to 10, 19 to 23, 27, 29 and 30 wherein the ratio of tyrosine or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomers or racemic mixtures to L-dopa or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomers or racemic mixtures is about 8:1, about 6:1, about 4:1, about 2:1, about 1:1, about 1:2, about 1:4, about 1:6, about 1:8, about 1:10, about 1 :20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 12:1, about 14:1, about 16:1, about 18:1, about 20:1, about 25:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1.

32. The composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 31 comprising about 1 μg to about 10 μg, or about 0.01 mg to about 2500 mg, or about 2.5 mg to about 10,000 mg, or about 0.1 mg to about 250mg, or about 10 mg to about 25mg of L-dopa or a pharmaceutically-acceptable salt, solvate, hydrate thereof, including any isolated stereoisomer or racemic mixture and about 1 μg to about 10 μg, or about 0.01 mg to about 2500 mg, or about 2.5 mg to about 10,000 mg, or about 0.1 mg to about 250mg, or about 10 mg to about 25mg of tyrosine or a pharmaceutically- acceptable salt, solvate, hydrate thereof, including any isolated stereoisomer or racemic mixture.

33. The composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 32 formulated for oral, parenteral, eternal or transdermal administration or administration by inhalation.

34. The composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 32 formulated for administration by duodenal infusion.

35. The composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 34 formulated for daily administration.

36. The composition according to claim 35 formulated to be administrated to a subject one, two, three or more times a day.

37. The composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 36 packaged with instructions for use.

38. The composition according to claim 36 packaged with instructions for use in the treatment or prevention of a condition ameliorated by L-dopa, a neurodegenerative condition or Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition, a dementia, atherosclerosis, or cataractogenesis.

39. A method of treating or preventing a condition ameliorated by L-dopa, comprising administering to a subject an amount of the composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 37.

40. The method according to claim 38, comprising administering the composition for a time and under conditions sufficient for tyrosine or a salt, solvate or hydrate thereof to reduce or prevent incorporation of L-dopa into a peptide or protein in said subject or for formation of dopamine in said subject from L-dopa in the composition or to ameliorate one or more symptoms treatable using L-dopa.

41. The method according to claim 39 or 40, comprising administering the composition to a subject suffering from neurodegeneration or a neurodegenerative disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral aging, age-related cognitive impairment, restless leg syndrome, a neurodegeneration condition, a dementia, atherosclerosis, or cataractogenesis.

42. The method according to any one of claims 39 to 41, comprising administering the composition for a time and under conditions sufficient for tyrosine or a salt, solvate or hydrate thereof to reduce or prevent incorporation of L-dopa into a peptide or protein in said subject or sufficient for formation of dopamine in said subject from L-dopa in the composition or to ameliorate one or more symptoms caused by L-dopa cytotoxicity.

43. A method of reducing or preventing incorporation of L-dopa into a peptide or protein or formation of proteinaceous aggregates comprising L-dopa in a cell, tissue or subject, wherein said method comprises administering to the cell, tissue or subject the composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 37.

44. The method according to any one of claims 39 to 43 comprising administering the composition to a neuron, preferably a dopaminergic neuron.

45. Use of the composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 37 in the treatment or prevention of a condition ameliorated by L-dopa.

46. Use of the composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 37 in the treatment or prevention of L-dopa cytotoxicity in a cell, tissue or subject.

47. Use of the composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 37 in reducing or preventing incorporation of L-dopa into a peptide or protein in a cell, tissue or subject.

48. Use of the composition composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 37 in reducing or preventing formation of proteinaceous aggregates comprising L-dopa in a cell, tissue or subject.

49. A method of monitoring L-dopa levels in polypeptides and/or proteins in plasma or other tissues.

50. The method according to claim 49 wherein said other tissues is tissue from a biopsy sample.

51. A marker to measure the levels of L-dopa-containing proteins and/or polypeptides in the brain of an individual wherein the marker comprises an amount of L-dopa-containing proteins and/or polypeptides in the plasma and/or tissue.

52. The marker of claim 51 wherein the tissue is not brain tissue.

53. A kit comprising a pharmaceutical composition, wherein said composition comprises i) an amount of L-dopa or a salt, solvate or hydrate thereof and ii) an amount of tyrosine or a salt, solvate or hydrate thereof sufficient to reduce or prevent incorporation of L-dopa into a peptide or protein, and optionally instructions for use.

54. The kit according to claim 53 wherein said composition is the composition of any one of claims 1 to 10, 19 to 23, 27 and 29 to 37.