Title:

Compounds, compositions and methods for treating neuropsychiatric disorders

Document Type and Number:

United States Patent 7407778

Abstract:

Acetyl-L-carnitine (ALCAR) and L-carntine (LCAR) are nutraceuticals with indications in treating a variety of mental health disorders. A metabolomics-guided bioprocess method is presented to enhance ALCAR and LCAR formation in germinating plant seeds. Metabolic fluxes are manipulated by germination in bioreactors to increase oxygen availability as well as provide an aseptic environment to alter carbohydrate consumption and feedback repress gluconeogenesis. Large shifts in sunflower seed fatty acid, phospholipid and high-energy metabolism change the germination environment and these metabolic changes lead to an approximate 1000-fold increase in natural LCAR and ALCAR production by the seeds. The resulting LCAR and ALCAR products from the seeds are used for treating mental health disorders including Alzheimer's disease, geriatric depression, non-geriatric depression and schizophrenia.

Inventors:

Pettegrew, Jay W. (630 S. Linden Ave., Pittsburgh, PA, US)
Gershon, Samuel (1981 Squaw Run Rd., Fox Chapel, PA, US)
Panchalingam, Kanagasabai (1214 Holy Cross Dr., Monroeville, PA, US)

Plaque It!

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a Continuation-In-Part (CIP) of U.S. application Ser. No. 11/117,126, filed Apr. 27, 2005, which is a CIP of Ser. No. 10/359,560, filed Feb. 7, 2003, which claims priority to U.S. Provisional application No. 60/354,323, filed Feb. 7, 2002, contents of all of which are incorporated by reference.

Claims:

We claim:

1. A method for producing a pharmaceutical composition comprising: i) a manipulated germination process wherein a sucrose controlled and oxygen controlled environment are applied to a plurality of germinating sunflower seeds to enhance the generation of a metabolite selected from L-camitine (LCAR), acetyl-L-camitine (ALCAR) and combinations thereof wherein the oxygen controlled environment includes at least 40% O₂; ii) isolating said metabolite therefrom by solvent extraction; and iii) mixing said metabolite with one or more pharmaceutically acceptable carriers or excipients.

2. A method for producing L-camitine (LCAR) and acetyl-L-camitine (ALCAR), comprising: i) enhancing fatty acid β-oxidation with sucrose suppression of a plurality of germinating sunflower seeds; and ii) stimulating β-oxidation of the fatty acids in the plurality of sunflower seeds with an oxygen enhanced environment for a pre-determined time period, wherein the oxygen enhanced environment comprises at least 40% O₂, thereby enhancing LCAR and/or ALCAR synthesis in the plurality of germinating sunflower seeds.

3. The method of claim 2 further comprising: extracting LCAR and ALCAR from the plurality of germinating sunflower seeds by contacting said seeds with an organic solvent.

4. The method of claim 3 wherein the step of extracting LCAR and ALCAR from the plurality of germinating sunflower seeds comprises contacting said seeds with an organic solvent or solvents selected from the group consisting of hexane, methylene chloride and methyl alcohol.

5. The method of claim 4 further comprising contacting said extracted LCAR and ALCAR with a pharmaceutically acceptable carrier(s) or excipient(s) to generate a pharmaceutically acceptable composition suitable for human consumption.

6. A method for producing L-camitine (LCAR) and/or acetyl-L-camitine (ALCAR), comprising: i) enhancing fatty acid β-oxidation with sucrose suppression of a plurality of glycoxylate cycles in a plurality of germinating sunflower seeds over a pre-determined time period; and ii) stimulating β-oxidation of the fatty acids in the plurality of sunflower seeds with O₂for a pre-determined time period, wherein the O₂enhanced environment comprises at least 40% O₂, thereby enhancing LCAR and/or ALCAR synthesis in the plurality of germinating sunflower seeds.

Description:

FIELD OF THE INVENTION

The present invention relates to compounds, compositions and methods for synthesis and use adapted for treating neuropsychiatric disorders, generally, and of Alzheimer's disease, geriatric depression, non-geriatric depression and schizophrenia specifically.

BACKGROUND OF THE INVENTION

The clinical response to antidepressant treatment in later life follows a variable temporal response, with a median time to remission of 12 weeks. Newer antidepressants still demonstrate a disturbing side-effect profile in this fragile patient population. Thus, there is a need for the development of newer antidepressants. One such candidate is acetyl-L-carnitine (ALCAR), a molecule that is naturally present in human brain demonstrating only few side effects.

Seven parallel, double-blind, placebo-controlled studies have examined ALCAR efficacy in various forms of geriatric depression. Phosphorus magnetic resonance spectroscopy (³¹P MRS) directly provides information on membrane phospholipid and high-energy phosphate metabolism in defined, localized brain regions. Although in vivo ³¹P MRS studies in major depression are limited, there is evidence of altered high-energy phosphate and membrane phospholipid metabolism in the prefrontal and basal ganglia regions. Increased levels of precursors of membrane phospholipids [i.e., increased phosphomonoesters (PME) levels] in the frontal lobe of major depressed subjects compared to controls was reported. Other researchers also observed higher PME levels in bipolar subjects in their depressive phase compared with the euthymic state. In terms of high-energy phosphates, reduced levels of adenosine triphosphate (ATP) have been observed in both the frontal and basal ganglia of major depressed subjects. The level of the high-energy phosphate buffer, phosphocreatine (PCr), was lower in severely depressed subjects compared with mildly depressed subjects. Accordingly, the relationship between membrane phospholipid and high-energy phosphate metabolism as assessments of beneficial results in the treatment of depression are recognized.

Epidemiology of Depressive Disorders

Depressive disorders (i.e., major depression, dysthymia, bipolar disorder) are among the most common and disabling medical conditions throughout the world. For example, about 9.5% of the US adult population will suffer from a form of depression during any given year which is approximately 18.8 million people. In addition, 16-18% of women and 10% of men (3-4 million) will experience some form of depression. The lifetime risk for depression is approximately 15-20% regardless of gender.

When one episode of depression is experienced, there is a 50% likelihood of recurrent episodes. When a second episode of depression occurs, there is a 80-90% likelihood of recurrent episodes and 75% of depressive disorders are recurrent.

It is estimated 20% of depressed individuals will attempt suicide and 6% will be successful. 75% of those committing suicide have a depressive disorder. The rate of successful suicide is four times greater in men.

About 10% of people with depression also will experience episodes of mania. Bipolar depressive episodes usually last longer, have a greater likelihood of psychotic features, and convey a greater risk of suicide. Bipolar disorder may be misdiagnosed as depression resulting in inappropriate treatment that may worsen the disease progression and outcome.

Depression is a acotraveler with a number of other medical and psychiatric conditions and numerous medications can cause depressive symptoms.

The prevailing dogma concerning the pathophysiology of depressive disorders (major depression, dysthymia, bipolar disorder) is that of an altered neurotransmitter receptor and many studies have been conducted to find such an alteration. To date, there has been no demonstration of an alteration in the binding site for any of the targeted neurotransmitters. Another problem with the altered neurotransmitters receptor dogma is that although the tricyclic antidepressants and selective neurotransmitter reuptake inhibitor drugs quickly enter brain and bind to their targeted sites, the clinical therapeutic effect does not occur for 4-6 weeks even though the onset of side effects is immediate.

Studies by Samuel Gershon over the years, since early 1950, have questioned the concepts of the established modes of action of antidepressants and those of the etiology of affective disorders.

In the early 50's a number of papers appeared suggesting that lithium not only had anti-manic properties but that it also exhibited anti-depressant and prophylactic activity in depression. These observations were confirmed by the controlled studies carried out by Schou et al. in Denmark and Prien et al. in Australia. This tended to indicate that perhaps a single neurotransmitter and a single receptor site would not qualify as the full explanation of their effects. In 1961, Gershon published a report in the Lancet on the psychiatric sequelae of organo-phosphorus insecticides in an exposed human population. Thus a role for acetylcholine in contributing to the production of major depressive disorder (MDD) was presented. This added to the complexity of current theories. In the 1970's an antidepressant Ludiomil was marketed with the effect of being a specific norepinephrine (NE) uptake inhibitor and thus exerting its effect by this route. This was an effective agent and was taken off the market because of other adverse effects (AE). In 1970 Gershon and colleagues carried out a number of experiments with synthesis inhibitors in patients undergoing treatment with different antidepressants and showed that only the inhibition of serotonin synthesis and not NE synthesis interfered with antidepressant outcomes.

These experiments demonstrated that a single transmitter or a single receptor could not account for therapeutic activity and clearly suggested other mechanisms are involved relating to membrane effects and second messenger systems. Antidepressant use has now clearly been associated with treatment emergent mania and the induction of rapid cycling in affective disorder patients (Tamada et al., 2004).

In addition to the concerns that have been established with the more classic bipolar I (BPI) type, much controversy surrounds the use of antidepressants in bipolar II (BPII) depression, a growing population.

Antidepressant induced cycle acceleration has been reported to be more likely in BPII patients than in BPI (Altshuler et al., 1995; Joffe et al., 2002; Benazzi, 1997; Henry et al., 2001; Ramasubbu, 2001).

The data has increasingly shown the need for the use of effective antidepressants but at the same time has produced data indicating the need for caution with the agents available. These effective antidepressants cause both the risk of switch into mania and the even more serious effect of rapid cycling of the affective disorder and an alteration of the frequency and severity of episodes.

A different conceptual approach has been the subject of almost 3 decades of research by Jay W. Pettegrew. This concept is that there is nothing structurally wrong with neurotransmitter receptors, but the receptors are in a membrane environment that has altered molecular structure and dynamics. It is these membrane alterations that alter the functional dynamics of neurotransmitter receptors which in turn alters their physiological function. Dr. Pettegrew was one of the first to demonstrate alterations in membrane molecular dynamics in living cells obtained from patients with neuropsychiatric disorders. Alterations were similarly demonstrated in cells obtained from patients with depression (Pettegrew et al., 1979c; Pettegrew et al., 1980a; Pettegrew et al., 1981a; Pettegrew et al., 198; Pettegrew et al., 1979b; Pettegrew et al., 1980b; Pettegrew et al., 1981b; Pettegrew et al., 1982b; Pettegrew et al., 1987b; Pettegrew et al., 1993b; Pettegrew et al., 1990c; Pettegrew et al., 1993a; Pettegrew et al., 1990b). Lithium was shown to correct the membrane dynamic alterations observed in depressive patients.

Given the rather striking changes in membrane molecular dynamics, Dr. Pettegrew turned to investigate alterations in membrane metabolism (Pettegrew et al., 1978; Pettegrew and Minshew, 1981; Pettegrew et al., 1982a; Glonek et al., 1982a) (Pettegrew et al., 1979a; Glonek et al., 1982b; Cohen et al., 1984; Pettegrew et al., 1986; Pettegrew et al., 1987a; Pettegrew et al., 1988a; Pettegrew et al., 1988b; Pettegrew et al., 1990a; Pettegrew et al., 1991; Keshavan et al., 1991; Kanfer et al., 1993; Pettegrew et al., 1994; Singh et al., 1994; Pettegrew et al., 1995; Klunk et al., 1996; Geddes et al., 1997; Klunk et al., 1998; Pettegrew et al., 2001; Keshavan et al., 2003; Sweet et al., 2002) and again significant alterations were observed in several neuro-psychiatric disorders including major depressive disorder (Pettegrew et al., 2002). Again, lithium was shown to correct the alteration in membrane metabolism observed in patients with depression.

Concerns About Current Classes of Antidepressants in Depressive Disorders

Concerns have been accumulating on the widespread use of all the current classes of antidepressants. This is reflected in the recently published North American based treatment guidelines (Grunze et al., 2002; Hirschfeld et al., 2002); including those of the APA (Sachs et al., 2000). These recommendations have voiced considerable limitations and a conservative attitude to their use, recommending use be restricted to severe bipolar depressions (Goodwin & Jamison, 1990; Murray & Lopez, 1996; Bostwick & Pankratz, 2000). The recommendations go on to suggest that if antidepressants are used they should be withdrawn as early as possible; thus we are now seeing a shift away from both the use of the current classes of antidepressants and recommendations for their long term use since they are associated with the following problems.

1. The risk for induced mania. There is now established a considerable risk of antidepressant induced manic switching and/or rapid cycling. This is seen in both short term and long term exposures. For example with selective reuptake inhibitors (SRIs) clinical samples demonstrate length of switch that are not minimal, that is 15 to 27%. The authors of a number of review articles on this topic suggest that the real rates are around 40% for tricyclic antidepressants (TCAs) and 20% with new SRI antidepressants. Substance abuse has been shown to be a major predictor of antidepressant-induced mania.

2. The risk of suicide in bipolar depressed patients. This risk is in and of itself a significant issue of concern. An analysis of SRIs and other novel antidepressants submitted to the FDA totaling nearly 20 thousand cases showed that there was no significant difference in completed or attempted suicides between patients on antidepressants and placebo treated groups. Simply stated, it appears that antidepressants as a group have not been shown to adequately reduce suicide rates. However, the data on lithium is in contrast to this with a very well established finding of its prophylactic effects against suicidality in a variety of diagnostic categories.

3. Antidepressant efficiency in treating bipolar depression. Prophylactic studies with antidepressants are not robust in the treatment of depressive episodes in bipolar disorders. Again, in contrast, the evidence of efficiency in treating bipolar depression with mood stabilizers is much higher (e.g., lithium and lamotrogine).

4. The potential value of other antidepressant classes. Based on this extensive new information as to the cautions that need to be employed in the use of the standard and SRI antidepressant classes, there is an urgent need for new classes of antidepressant thymoleptics. One such agent, ALCAR has a body of literature that supports the possibility of its therapeutic value in a number of depressive categories.

In view of its unique biochemical effects on the nervous system and its stabilizing effects on membrane functions, ALCAR's antidepressant activity may indeed provide a unique opportunity to address the above-described concerns. Since ALCAR is a natural substance and has been shown to have antidepressant properties without significant side effects and without the potential to induce mania, it is a logical new therapeutic approach.

ALCAR has been shown to have beneficial effects on age-related neurodegenernation and brain energetic stress providing a rationale for its use in Major Depressive Disorder (MDD). In European clinical trials to date, ALCAR has demonstrated antidepressant activity in MDD subjects without significant side effects (Villardita et al., 1983; Tempesta et al., 1987; Nasca et al., 1989; Bella et al., 1990; Fulgente et al., 1990; Garzya et al., 1990).

Overview of Biological Findings in Major Depressive Disorder

MDD has been shown to be associated with changes in: (1) neurotransmitter systems such as serotonin, acetylcholine, and noradrenergic; (2) membranes (e.g., composition, metabolism, biophysical parameters, signal transduction, and ion transport); (3) brain energy metabolism; and (4) brain structure. Computed tomography (CT) and magnetic resonance imaging (MRI) studies in subjects with non-demented, geriatric, major depressive disorder suggest neurodegenerative changes are associated with vascular risk factors (Krishnan, 1993). Beyond brain structural changes, there is evidence from functional neuroimaging studies for molecular, metabolic, and physiologic brain changes suggestive of energetic stress in subjects with MDD. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies show a reduced fluorodeoxyglucose metabolic rate (rCMRg) (Buchsbaum et al., 1986) and reduced regional cerebral blood flow (rCBF) (Schlegel et al., 1989) in the basal ganglia and a decrease in rCMRg and rCBF in the frontal lobes of subjects with MDD (Mayberg et al., 1994). Of the neuroimaging methods, ³¹P and ¹H magnetic resonance spectroscopic imaging (³¹P-¹H MRSI) studies provide direct information on membrane phospholipid and high-energy phosphate metabolism (³¹P MRSI) as well as a marker for neuronal structural and metabolic integrity (¹H MRSI). ³¹P and ¹H MRS studies of subjects with MDD indicate alterations in high-energy phosphate and membrane phospholipid metabolism in basal ganglia and prefrontal cortex (Moore et al., 1997a; Charles et al., 1994; Pettegrew et al. 2002).

Neuromorphometric Changes in MDD

Neuroimaging studies have enhanced our understanding of the pathophysiology of MDD. MRI studies provide neuromorphometric correlates of MDD (reviewed by Botteron & Figiel, 1997). MRI studies of third ventricle size in major depression give mixed results; Coffey et al. (1993) report no difference in ventricle size and Rabins et al. (1991) report increased third ventricle size in subjects with MDD compared with controls. Brain MRI subcortical white matter hyperintensities have been reported in the basal ganglia, periventricular region, and frontal lobe of elderly depressed (Coffey et al., 1988; Figiel et al., 1989; Rabins et al., 1991). There have been reports of decreased volumes of the basal ganglia in MDD; Husain et al. (1991) found reduced volume in the putamen, Krishnan et al. (1993) found reduced volume in the caudate, and Dupont et al. (1995) found reduced volume in the caudate, lenticular nucleus, and thalamus. Coffey et al. (1993) report an approximately 7% reduction in bilateral frontal lobe volume in subjects with MDD.

These studies reveal neurodegenerative change in MDD. Other as yet unknown molecular and metabolic factors could predispose to both depression and the neuromorphometric changes associated with it.

Magnetic Resonance Spectroscopy Studies of Major Depressive Disorder (MDD)

While there are several MRS studies in bipolar disorder (reviewed by Moore & Renshaw, 1997b), there are only two ³¹P MRS studies (Kato et al., 1992; Moore et al., 1997a) and one ¹H MRS analysis of MDD (Charles et al., 1994). Kato et al. (1992), using a coronal slice DRESS ³¹P MRS protocol, examined the frontal cortex of 12 subjects (age 35.3∀12.1 years) with MDD, 10 subjects (age 42∀8.6 years) with bipolar disorder and 22 control subjects (age 36.1∀11.5 years). Although the pH and PME levels were significantly higher in euthymic MDD subjects compared with euthymic bipolar subjects, no significant differences were found for ³¹P MRS parameters of MDD subjects compared with control subjects. A study by Moore et al. (1997a) using a ³¹P MRS ISIS protocol, measured ³¹P metabolites in a 45 cm³voxel containing the bilateral basal ganglia in 35 unmedicated subjects (age 37.2∀lain 8.5 years) with MDD and 18 control subjects (age 38.2∀9.9 years). There was a 16% reduction in ATP (β-ATP peak) in the MDD subjects. The PCr/Pi ratio of MDD subjects compared with control subjects did not change. This study indicates that an abnormality in basal ganglia high-energy phosphate metabolism is associated with MDD. A ¹H MRS study by Charles et al. (1994), using a combination of the STEAM technique for spatial lipid suppression and 1D CSI for additional spatial localization of the basal ganglia and thalamus, examined seven subjects with MDD (age range 63-76, mean=71.4 years ) compared with ten control subjects (age range 65-75, mean=68.9 years). The subjects with MDD were medication free for two (1 subject) or three (6 subjects) weeks. The authors report an increase in the TMA MRS peak in the basal ganglia of MDD subjects and subsequent drop in the trimethlyamine (TMA) peak in four subjects after treatment. We have recently observed an increase in PME and a decrease in PCr in two subjects with MDD (Pettegrew et al., unpublished results).

Molecular and Metabolic Effects of ALCAR

There is neuroimaging evidence for neurodegeneration and a reduction in energy metabolite levels and rCBF in MDD. These findings provide a rationale for the use of ALCAR in MDD as there is a considerable body of research that indicates that ALCAR has a positive modulating influence on membrane structure, function and metabolism, energy metabolism, and the physiology and metabolism of neurotrophic factors. There also is clinical evidence that ALCAR is beneficial in the treatment of neurodegenerative disorders as well as normal aging-related processes and the treatment of geriatric depression. A thorough review of the possible CNS actions of ALCAR has appeared (Calvani & Carta, 1991; Pettegrew et al., 2000). What follows is a brief review of the metabolic, physiologic, behavioral, and clinical roles for ALCAR.

ALCAR'S Effect on Energy Metabolism

ALCAR has been shown to exert a beneficial effect on brain metabolism after energetic stresses. In a canine model of complete, global cerebral ischemia and reperfusion, ALCAR treated animals exhibited significantly lower neurological deficit scores (p=0.0037) and more normal cerebral cortex lactate/pyruvate ratios than did vehicle-treated control animals (Rosenthal et al., 1992). In a rat cyanide model of acute hypoxia, increased rate of phosphatidic acid formation, possibly reflecting increased phospholipase C activity was observed and spatial navigation performance in a Morris task was impaired. Chronic treatment with ALCAR attenuated the cyanide-induced behavioral deficit but had no effect on energy-dependent phosphoinositide metabolism suggesting ALCAR affected free fatty acid metabolism by increasing the reservoir of activated acyl groups involved in the reacylation of membrane phospholipids (Blokland et al., 1993). In a canine model employing 10 minutes of cardiac arrest followed by restoration of spontaneous circulation for up to 24 hours, ALCAR eliminated the reperfusion elevation of brain protein carbonyl groups which reflect free radical-induced protein oxidation (Liu et al., 1993). In a rat streptozotocin-induced model of brain hypoglycemia, ALCAR attenuated both the streptozotocin-induced impairment in spatial discrimination learning and decrease in hippocampal choline acetyltransferase activity (Prickaerts et al., 1995). A deficiency in ALCAR has been shown to be a cause for altered nerve myo-inositol content, Na⁺—K⁺-ATPase activity, and motor conduction velocity in the streptozotocin-diabetic rat (Stevens et al., 1996). Finally, sparse-fur mice have a deficiency of hepatic ornithine transcarbamylase resulting in congenital hyperammonemic with elevated cerebral anmonia and glutamine and reduced cerebral cytochrome oxidase activity and a reduction in cerebral high-energy phosphate levels. ALCAR treatment increased cytochrome oxidase subunit I mRNA, and restored both cytochrome oxidase activity and the levels of high-energy phosphates (Rao et al., 1997). Our studies of hypoxia in Fischer 344 rats demonstrate ALCAR's beneficial effect on brain membrane phospholipid and high-energy phosphate metabolism (Pettegrew et al., unpublished results.

ALCAR'S Effect on Membrain Composition, Structure, and Dynamics

ALCAR has been shown to effect membrane structure and function in a number of different systems. ALCAR administration affects the inner mitochondrial membrane protein composition in rat cerebellum (Villa et al., 1988), increases human erythrocyte membrane stability possibly by interacting with cytoskeletal proteins (Arduini et al., 1990), increases human erythrocyte cytoskeletal protein-protein interactions (Butterfield & Rangachari, 1993), and alters the membrane dynamics of human erythrocytes in the region of the glycerol backbone of membrane phospholipid bilayers (Arduini et al., 1993).

ALCAR∝S Enhancement of Nerve Growth Factor Activity

A number of studies have demonstrated that ALCAR enhances the neurotrophic activity of nerve growth factor (NGF). ALCAR increases NGF binding in aged rat hippocampus and basal forebrain (Angelucci et al., 1988), increases NGF receptor expression in rat striatum, and increases choline acetyltranferase activity in the same area (De Simone R. et al., 1991), enhances PC12 cells response to NGF (Taglialatela et al., 1991), increases the level of NGF receptor (P75NGFR) mRNA (Taglialatela et al., 1992), increases choline acetyltransferase activity and NGF levels in adult rats following total fimbria-fomix transection (Piovesan et al., 1994; 1995), and enhances motomeuron survival in rat facial nucleus after facial nerve transection (Piovesan et al., 1995).

Influence of ALCAR on Cholinergic and Serotonergic Neurotransmitter Systems

ALCAR has some cholinergic activity (Fritz, 1963; Tempesta et al., 1985), possibly because it shares conformational properties with acetylcholine (Sass & Werness, 1973). This is interesting as acetylcholine may play an important role in the chronobiological organization of the human body (Morley & Murrin, 1989; Wee & Turek, 1989), mediating also some effects of light on the circadian clock (Wee & Turek, 1989). Acetylcholine is implicated in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis (Mueller et al., 1977; Risch et al., 1981) and cholinomimetics are effective on the HPA axis (Janowsky et al., 1981; Risch et al., 1981). ALCAR also seems to interfere with the serotonergic system (Tempesta et al., 1982; 1985). There is ample evidence supporting a reduction in serotonergic activity in depression (Ashcroft et al., 1966; Asberg et al., 1976; Cochran et al., 1976; Traskman et al., 1981; Stanley & Mann, 1983); although these results have not always been confirmed (Bowers, 1974; Murphy et al., 1978). The efficacy of 5-HTP also has been reported in involutional depression (Aussilloux et al., 1975). Moreover the selective serotonin reuptake inhibitors (SSRI) antidepressants increase serotonergic transmission and are currently widely used in treating MDD (Aberg-Wistedt et al., 1982; Stark & Hardison, 1985). Serotonin plays an important role in the regulation of circadian rhythms (Kordon et al., 1981; Leibiwitz et al., 1989) and there is consistent evidence that it affects cortisol secretion (Imura et al., 1973; Krieger, 1978; Meltzer et al., 1982).

ALCAR'S Effect on Aging-Relating Metabolic Changes

ALCAR has been demonstrated to reverse aging-related changes in brain ultrastructure, neurotransmitter systems, membrane receptors, mitochondrial proteins, membrane structure and metabolism, memory, and behavior. ALCAR restores the number of axosomatic and giant bouton vesicles in aged rat hippocampus (Badiali et al., 1987), reduces aging-related lipofuscin accumulation in prefrontal pyramidal neurons and hippocampal CA3 neurons in rats (Kohjimoto et al., 1988; Amenta et al., 1989), and reduces aging-related changes in the rat hippocampal mossy fiber system (Ricci et al., 1989). ALCAR reduces the age-dependent loss of glucocorticoid receptors in rat hippocampus (Ricci et al., 1989), attenuates the age-dependent decrease in NMDA receptors in rat hippocampus (Fiore & Rampello, 1989; Castorina et al., 1993; 1994; Piovesan et al., 1994; and reviewed by Castorina & Ferraris, 1988), and reduces age-related changes in the dopaminergic system of aging mouse brain (Sershen et al., 1991). Age-related changes in mitochondria also are reduced by ALCAR. ALCAR increases cytochrome oxidase activity in rat cerebral cortex, hippocampus, and striatum (Curti et al., 1989), restores to normal reduced transcripts of mitochondrial DNA in rat brain and heart but not liver (Gadaleta et al., 1990), increases cytochrome oxidase activity of synaptic and non-synaptic mitochondria (Villa & Gorini, 1991), reverses age-related reduction in the phosphate carrier and cardiolipin levels in heart mitochondria (Paradies et al., 1992), reverses age-related reduction in cytochrome oxidase and adenine nucleotide transferase activity in rat heart by modifying age-related changes in mitochondrial cardiolipin levels (Paradies et al., 1994; 1995), and reverses age-related alteration in the protein composition of the inner mitochondrial membrane (Villa et al., 1988). ALCAR also increases synaptosomal high-affinity choline uptake in the cerebral cortex of aging rats (Curti et al., 1989; Piovesan et al., 1994), increases choline acetyltransferase activity in aged rat striatum (De Simone R. et al., 1991; Taglialatela et al., 1994), modulates age-related reduction in melatonin synthesis (Esposti et al., 1994), reverses the age-related elevation in free and esterified cholesterol and arachidonic acid (20:4) in rat plasma (Ruggiero et al., 1990), and increases PCr and reduces lactate/pyruvate and sugar phosphate levels in adult and aged rat brain (Aureli et al., 1990). Age-related changes in NGF are reduced by ALCAR: ALCAR increases NGF receptor expression in rat striatum (De Simone R. et al., 1991) and in PC12 cells (Castorina et al., 1993); enhances the effect of NGF in aged dorsal root ganglia neurons (Manfridi et al., 1992); exerts a neurotrophic effect in three month old rats after total fimbria transection (Piovesan et al., 1994); and increases NGF levels in aged rat brain (Taglialatela et al., 1994). ALCAR has been shown in aged rats to modulate synaptic structural dynamics (Bertoni-Freddari et al., 1994) and improve measures of behavior (Angelucci, 1988; Kohjimoto et al., 1988) as well as memory (Barnes et al., 1990; Caprioli et al., 1990; 1995). ALCAR has been reported to normalize the pituitary-adrenocortical hyperactivity in pathological brain aging (Nappi et al., 1988; Ghirardi et al., 1994). We have reported that ALCAR improves standardized clinical measures and measures of membrane phospholipid and high-energy phosphate metabolism in subjects with Alzheimer's disease (AD) measured by in vivo ³¹P MRS (Pettegrew et al., 1995). We now have data in a rat hypoxia model which demonstrate that ALCAR has more beneficial effects on aged rats (30 months) than on adolescent (1 month) or adult (12 months) animals (Pettegrew et al., unpublished results).

Antidepressant Effects of ALCAR

In European clinical trials, ALCAR has been shown to have significant antidepressant activity in geriatric depressed subjects with minimal or no side effects (Villardita et al., 1983; Tempesta et al., 1987; Nasca et al., 1989; Bella et al., 1990; Fulgente et al., 1990; Garzya et al., 1990; Gecele et al., 1991). Villardita et al. (1983) reported a double-blind ALCAR/placebo study of 28 subjects (18 males, 10 females; 72.3∀7.3 years). Sixteen subjects were treated with ALCAR (1.5 gm/day; baseline HDRS=26.3∀3.3) and 12 Patients were treated with placebo (baseline HDRS=26.6∀3.2) for 40 days. By day 40, the ALCAR treated subjects showed significant improvement (p<0.001) in the Hamilton Depressive Rating Scale (HDRS) but the placebo treated subjects did not. There were no side effects to ALCAR. Tempesta et al. (1987) in an open label, cross over study of 24 subjects over the age of 70 years, all of whom were nursing home residents, reported ALCAR (2 gm/day) to be highly effective in reducing HDRS scores, especially in subjects with more severe clinical symptoms. Again there were no reported ALCAR side effects. In a simple blind ALCAR/placebo study of 20 subjects (10 ALCAR treated subjects; 62.5∀5.7 years, 8 males, 2 females, baseline HDRS=44.9∀3.1 and 10 placebo treated subjects; 62.5∀5.3 years, 8 males, 2 females, baseline HDRS=43.9∀2.8), Nasca et al. (1989) demonstrated a significant improvement in the HDRS scores of ALCAR treated subjects at day 40 of treatment (p<0.001). There was no improvement in the placebo treated group. Similar significant beneficial effects of ALCAR on the HDRS were observed in randomized, double-blind, ALCAR/placebo studies of Garzya et al. (1990) (28 subjects; ages 70-80 years; ALCAR 1.5 gm/day), Fulgente et al. (1990) [60 subjects; 70-80 years; ALCAR 3.0 gm/day; baseline HDRS (ALCAR=25; placebo=23); day 60 HDRS (ALCAR=12; placebo=22); p # 0.0001], and Bella et al. (1990) [60 subjects, 60-80 years, ALCAR 3.0 gm/day; baseline HDRS (ALCAR=22; placebo=21); day 60 HDRS (ALCAR=11; placebo=20); p # 0.0001]. ALCAR was well tolerated in these studies even at the higher dosages. A double-blind, ALCAR/placebo study by Gecele et al. (1991) (30 subjects, 66-79 years, ALCAR 2 gm/day) not only showed a significant improvement in the HDRS of ALCAR treated subjects (p<0.001) but a significant reduction in both mean cortisol levels (p<0.001) as well as 12 am (p<0.001) and 4 pm (p<0.01) cortisol levels.

Since acetyl-1-carnitine (ALCAR) is a natural substance and has been shown to have antidepressant properties without significant side effects and without the potential to induce mania, it is a logical new therapeutic approach.

LCAR is important in the β-oxidation of fatty acids and ALCARc ontains carnitine and acetyl moieties, both of which have neurobiological properties. LCAR is important in the β-oxidation of fatty acids and the acetyl moiety of ALCAR can be used to maintain acetyl-CoA levels. Other reported neurobiological effects of ALCAR include modulation of: 1) brain energy and phospholipid metabolism; 2) cellular macromolecules including neurotrophic factors and neurohormones; 3) synaptic morphology; and 4) synaptic transmission of multiple neurotransmitters. Potential molecular mechanisms of ALCAR include: 1) acetylation of —NH2 and —OH functional groups in amino acids and N-terminal amino acids in peptides and proteins resulting in modification of their structure, dynamics, function and turnover; and 2) acting as a molecular chaperone to a larger molecule.

There is data demonstrating that ALCAR can acetylate lysine 28 in the Aβ(1-40) peptide derived from the ubiquitous protein, amyloid precursor protein. ALCAR is reported to have neuroprotective effects (Pettegrew et al., 2000) and could become recognized as a dietary component important to mental health—particularly in older populations. ALCAR has therapeutic indications for Alzheimer's disease (Pettegrew et al., 1995); geriatric depression (Pettegrew et al., 2002); schizophrenia (Masterson and Wood, 2000).

However, LCAR and ALCAR are difficult to produce. There is a need for producing a low-cost nutraceutical to treat major mental illnesses, especially in poor countries such as LCAR and ALCAR.

Thus, it is desirable to provide compositions, compounds and methods that can be used to produce LCAR and ALCAR.

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Most of the studies have been directed toward geriatric subjects. However, it is also desirable to use acetyl-L-carnitine (ALCAR) for non-geriatric human subjects as well as for adolescent human subjects.

SUMMARY OF THE INVENTION

In accordance with preferred embodiments of the present invention, some of the problems presently associated with producing LCAR and ALCAR are overcome. Compounds, compositions and methods for treating neuropsychiatric disorders are presented.

A metabolomics-guided bioprocess method is presented to enhance LCAR and ALCAR formation in germinating plant seeds. Metabolic fluxes are manipulated by germination in bioreactors to increase oxygen availability as well as provide an aseptic environment to alter carbohydrate consumption and feedback repress gluconeogenesis. Large shifts in germinating seed fatty acid, phospholipid and high-energy metabolism change the germination environment and these metabolic changes lead to an approximate 1000-fold increase in natural LCAR and ALCAR production by the germinating seeds.

The foregoing and other features and advantages of preferred embodiments of the present invention will be more readily apparent from the following detailed description. The detailed description proceeds with references to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention are described with reference to the following drawings, wherein:

FIG. 1B is a graph showing the correlation of PME(s-τ_c) levels from the prefrontal region with HDRS scores for both depressed patients (● subject #1; ♦ subject #2);

FIG. 1A is a graph showing the correlation of PCr levels from the prefrontal region with HDRS scores for both depressed patients (● subject #1; ♦ subject #2);

FIG. 2A is a graph showing PME(s-τ_c) and FIG. 2B is a graph showing PCr levels in the a) prefrontal region of the two depressed patients (● subject #1; ♦ subject #2) and normal controls (O, n=6) at baseline and at 6 and 12 weeks follow up. The control values include meanplusSD;

FIG. 2C is a graph showing PME(s-τ_c) FIG. 2D is a graph showing and PCr levels in the basal ganglia region of the two depressed patients (● subject #1; ♦ subject #2) and normal controls (O, n=6) at baseline and at 6 and 12 weeks follow up. The control values include meanplusSD;

FIG. 3A is a phosphorous magnetic resonance spectroscopic image showing the Z-scores of the two depressed subjects compared with controls at entry and 12 weeks for PME(s-τ_c) metabolite levels for those regions with significant differences. The intensity of the color is scaled to the z-score (mean difference/SD) given on the scale below the image. Z-scores for PME(s-τ_c) and PCr levels in the frontal region exceed 3.0 and 2.0, respectively;

FIG. 3B is a phosphorous magnetic resonance spectroscopic image showing the Z-scores of the two depressed subjects compared with controls at entry and 12 weeks for PCr metabolite levels for those regions with significant differences. The intensity of the color is scaled to the z-score (mean difference/SD) given on the scale below the image. Z-scores for PME(s-τ_c) and PCr levels in the frontal region exceed 2.0 and 2.0, respectively;

FIG. 4 is a block diagram illustrating an effect of ALCAR on in vitro ³¹P MRS α-GP and PCr levels under hypoxic (30 seconds) and normoxic conditions in Fischer 344 rats;

FIG. 5 is a block diagram illustrating an effect of ALCAR on in vitro ³¹P MRS phospholipid levels under hypoxic and normoxic conditions in Fischer 344 rats;

FIG. 6 is a block diagram illustrating a percent change of in vivo

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5668117	Methods of treating neurological diseases and etiologically related symptomology using carbonyl trapping agents in combination with previously known medicaments	September, 1997	Shapiro
5773024	Container with multi-phase composition for use in diagnostic and therapeutic applications	June, 1998	Unger
5837219	Labelled acyl-L-carnitine and diagnostic agent	November, 1998	Watanabe
5846517	Methods for diagnostic imaging using a renal contrast agent and a vasodilator	December, 1998	Unger
5853985	Promoter of the gene for the human precursor of the alzheimer's disease and its use	December, 1998	Salbaum
5866427	In vitro proton MRS detection of frequency and amount of alcohol self-administration	February, 1999	Mendelson
5879884	Diagnosis of depression by linkage of a polymorphic marker to a segment of chromosome 19P13 bordered by D19S247 and D19S394	March, 1999	Peroutka	435/6
5889055	L-carnitine and acetyl-L-carnitine combined for prevention and treatment of syndromes related to diseases of energy metabolism	March, 1999	Howard
5973004	L-carnitine, acetyl-L-carnitine, and pantothenic acid or ubiquinone, combined for prevention and treatment of syndromes related to ineffective energy metabolism	October, 1999	Howard
5976816	Cell tests for alzheimer's disease	November, 1999	Alkon
6037373	Use of L-acetylcarnitine, L-isovalerylcarnitine, L-propionylcarnitine for increasing the levels of IGF-1	March, 2000	De Simone
6066664	Method for decreasing the appetite in bulimic, overweight individuals	May, 2000	Cavazza
6071494	Methods for diagnostic imaging using a contrast agent and a renal vasodilator	June, 2000	Unger
6080582	Cell tests for Alzheimer's disease	June, 2000	Alkon
6107050	Diagnostic test for alzheimers disease	August, 2000	Alkon
6114175	Compound for the antemortem diagnosis of Alzheimer's Disease and in vivo imaging and prevention of amyloid deposition	September, 2000	Kiunk
6133259	Alkyl, alkenyl and alkynyl chrysamine G derivatives for inhibition of cell degeneration and toxicity associated with amyloid deposition	October, 2000	Kiunk
6139819	Targeted contrast agents for diagnostic and therapeutic use	October, 2000	Unger
6166077	Use of L-acetylcarnitine, L-isovalerylcarnitine, L-propionylcarnitine for increasing the levels of IGF-1	December, 2000	De Simone
6168776	Alkyl, alkenyl and alkynyl Chrysamine G derivatives for the antemortem diagnosis of Alzheimer's disease and in vivo imaging and prevention of amyloid deposition	January, 2001	Klunk
6175061	Production of polyhydroxyalkanoate in plants	January, 2001	Bright
6183777	Controlled release tacrine dosage form	February, 2001	Chen
6231834	Methods for ultrasound imaging involving the use of a contrast agent and multiple images and processing of same	May, 2001	Unger
6255057	Detection of cellular exposure to ethanol	July, 2001	Gordon
6300085	Diagnostic method for Alzheimer's disease	October, 2001	Alkon
6313159	Metabotropic glutamate receptor ligand derivatives as naaladase inhibitors	November, 2001	Jackson
6335021	Composition for controlling mood disorders in healthy individuals	January, 2002	Cavazza
6337197	Coenzymes useful for the synthesis of L-carnitine	January, 2002	Elssner
6346282	Neuroprotective composition for the prevention and/or treatment of nervous and behavioural alterations due to anxiety states or depression, comprising acetyl-L-carnitine and hypericin	February, 2002	Cavazza
6348464	Pyrrolecarbonylimino derivatives as naaladase inhibitors	February, 2002	Jackson
6379936	Coenzymes useful for the synthesis of L-carnitine	April, 2002	Elssner
6380244	Nutritional and therapeutic uses of 3-hydroxyalkanoate oligomers	April, 2002	Martin
6380252	Use of L-acetylcarnitine, L-isovalerylcarnitine, L-propionylcarnitine for increasing the levels of IGF-1	April, 2002	De Simone
6403056	Method for delivering bioactive agents using cochleates	June, 2002	Unger
6416740	Acoustically active drug delivery systems	July, 2002	Unger
6417178	Amyloid binding nitrogen-linked compounds for the antemortem diagnosis of alzheimer's disease, in vivo imaging and prevention of amyloid deposits	July, 2002	Klunk
6420599	Chemical process for the stereoselective synthesis of R-(-)-carnitine	July, 2002	Marzi
6479034	Method of preparing gas and gaseous precursor-filled microspheres	November, 2002	Unger
6521211	Methods of imaging and treatment with targeted compositions	February, 2003	Unger
6548047	Thermal preactivation of gaseous precursor filled compositions	April, 2003	Unger
6551576	Container with multi-phase composition for use in diagnostic and therapeutic applications	April, 2003	Unger
6576220	Non-invasive methods for surgery in the vasculature	June, 2003	Unger
6589547	Sustained-release preparation for AII antagonist, production and use thereof	July, 2003	Igari
6589948	Cyclic salen-metal compounds: reactive oxygen species scavengers useful as antioxidants in the treatment and prevention of diseases	July, 2003	Malfroy-Camine
6653112	Method for producing L-carnitine from crotonobetaine using a two stage continuous cell-recycle reactor	November, 2003	Kleber
6708053	Biochemical markers of brain function	March, 2004	Brooks
6716412	Methods of ultrasound treatment using gas or gaseous precursor-filled compositions	April, 2004	Unger
6808720	Charged lipids and uses for the same	October, 2004	Unger

WO/1994/009371	April, 1994	ANTEMORTEM DIAGNOSTIC TEST FOR ALZHEIMER'S DISEASE
WO/1999/066914	December, 1999	NEUROPROTECTIVE COMPOSITION FOR THE PREVENTION AND/OR TREATMENT OF NERVOUS AND BEHAVIOURAL ALTERATIONS DUE TO ANXIETY STATES OR DEPRESSION, COMPRISING ACETYL-L-CARNITINE AND HYPERICIN
WO/2003/066041	August, 2003	CARNITINE IN THE TREATMENT OF GERIATRIC DEPRESSION
WO/2004/054567	July, 2004	USE OF CARNITINES FOR THE PREVENTION AND/OR TREATMENT OF DISORDERS CAUSED BY THE ANDROPAUSE