HK1247112B - Method of treating neurological conditions with extract of nerium species or thevetia species - Google Patents

Description

Methods of treating neurological disorders with extracts of Nerium species or Nerium oleander species

The present application is a divisional application of Chinese patent application No. 201180065127.6, filed on November 3, 2011, entitled “Methods for treating neurological disorders using extracts of Nerium species or Nerium spp.”

Technical Field

The present invention relates to methods for treating neurological conditions using extracts of Nerium species or Thevetia species, or preparations (compositions, formulations) containing them. In particular, the present invention relates to methods for treating neurological diseases or disorders by administering the extracts to a subject in need thereof. The present invention also includes pharmaceutical compositions containing the extract fractions or subfractions, as well as methods for their use and preparation.

Background Art

Neurological diseases and disorders affect brain function. Attempts have been made to cure or improve therapies for these diseases and disorders; however, while many drug treatments have proven effective for many different diseases and disorders, no widespread or universal cure has yet been established.

Huntington's disease (HD) is a genetic disease of the brain that affects the nervous system. It is caused by a defective gene that is passed from parent to child. The HD gene interferes with the production of a specific protein called "huntingtin", which appears to be critical for proper brain development. Typical signs of HD include emotional, cognitive and movement disorders. Huntington's disease is characterized by rapid involuntary movements (chorea), but sometimes causes stiffness without abnormal movements, changes in the use of limbs (apraxia), loss of control of body functions and dementia, including progressive deterioration of memory, thinking speed, judgment and lack of problem awareness and planning awareness. There is no known cure for Huntington's disease. Although many drugs help control the symptoms associated with HD, such as emotional and movement problems, there is no treatment to stop or reverse the progression of the disease. Huntington's disease has been recognized as a disease with universal membrane abnormalities. Significantly elevated levels and activity (10-fold increase) of Na,K-ATPase have been observed in the membranes of erythrocytes and basal ganglia of Huntington's disease patients (Schroeder F, Goetz IE, Roberts E, Membrane anomalies in Huntington's disease fibroblasts. J. Neurochem. 43:526-539, 1984) compared to normal (Butterfield DA, Oeswein JQ, Prunty ME, Hisle KC, Markesbery WR., Increased sodium potassium adenosine triphosphatase activity in erythrocyte membrane in Huntington's disease, Ann Neurology, 4:60-62, 1978) fibroblasts obtained from the skin of Huntington's disease patients.

Alzheimer's disease is a form of dementia—a neurodegenerative disorder that impairs intellectual function (memory, orientation, calculation, etc.) while typically sparing motor function. In Alzheimer's disease, mental abilities gradually deteriorate, causing memory loss, confusion, disorientation, impaired judgment, and other problems that can affect a person's ability to perform normal daily activities. The type, severity, sequence, and progression of mental changes vary widely. There is no known cure for Alzheimer's disease, nor are there known ways to slow its progression. For some people in the early or middle stages of the disease, medications such as tacrine can relieve some cognitive symptoms. Aricept (donepezil) and Exelon (rivastigmine) are reversible acetylcholinesterase inhibitors indicated for the treatment of mild to moderate dementia of the Alzheimer's type. These medications (called cholinesterase inhibitors) work by increasing brain levels of the neurotransmitter acetylcholine, helping to restore connections between brain cells. Some medications can help control behavioral symptoms such as insomnia, agitation, wandering, anxiety, and depression. The goal of these treatments is to make the patient more comfortable. Although there is no known cure for Alzheimer's disease, cholinesterase inhibitors may improve performance in daily activities or alleviate behavioral problems. Drugs currently being tested for the treatment of Alzheimer's disease include estrogen, nonsteroidal anti-inflammatory drugs, vitamin E, selegiline (Carbex, midopyril), and the botanical product Ginkgo biloba.

Nerium oleander is an ornamental plant found widely in subtropical Asia, the southwestern United States, and the Mediterranean. Its medicinal and toxicological properties have long been recognized. It has been used, for example, to treat hemorrhoids, ulcers, leprosy, snake bites, and even to induce abortion. Oleandrin, an important but not exclusive component of oleander extract, is a cardiac glycoside.

Extraction of glycosides from plants of the genus Nerium has provided pharmacologically/therapeutically active ingredients from oleander. These include oleandrin, nerifolin B, and other cardiac glycoside compounds. Oleandrin extract, obtained by hot water extraction of oleander (sold under the trademark ANVIRZEL ^™ ), contains oleander hot water extract in concentrated or powdered form. Phase I trials of hot water oleander extract (i.e., Anvirzel ^™ ) have been completed (Mekhail et al., Am. Soc. Clin. Oncol., vol. 20, p. 82b, 2001). It is hypothesized that the oleander extract could provide approximately 57 μg of oleandrin/day, which could be safely administered at doses up to 1.2 ml/ ^m² /day. No dose-related toxicity has been observed.

Summary of the Invention

The present invention provides a method for treating a neurological disorder, comprising administering to a subject in need thereof an effective amount of a composition comprising an extract of an Nerium species or an Nerium species to treat the neurological disorder. The present invention provides embodiments in which a fraction or subfraction of a fraction of an extract is used in place of an unfractionated extract, and wherein the fraction or subfraction of the extract does not contain oleandrin and oleandrin-2-hydroxybutyrate.

In one aspect, the present invention provides a method of treating a neurological disease or disorder in a subject in need thereof with a composition comprising an extract of Nerium sp. or Nerium spp., the method comprising:

Identification of a subject with a neurological disease or disorder; and

Administration of a composition comprising an extract of Nerium sp. or Nerium spp. to a subject is indicated.

Certain embodiments of the present invention include those wherein 1) a therapeutically relevant dose of the composition is prescribed and administered to a subject; 2) the subject takes the composition according to the prescribed dosing regimen; 3) the extract comprises one or more therapeutically effective agents extracted from Nerium species or Nerium species; 4) the composition further comprises one or more other therapeutically effective agents; 5) the extract is obtained by extracting Nerium species or Nerium species using hot water, cold water, a supercritical fluid, an organic solvent, or a combination thereof; 6) the extract does not comprise cardiac glycosides; 7) the extract does not comprise a therapeutically effective amount of cardiac glycosides; 8) the extract does not comprise oleandrin; 9) the composition comprises a component of an extract of Nerium species or Nerium species; 10) the composition comprises a component of an extract of Nerium species or Nerium species, wherein the component has been prepared by liquid chromatography fractionation of the extract; 11) the Nerium species is Oleander oleander and the Nerium species is Nerium oleander. neriifolia); 12) an extract that does not contain neriside B; 13) a composition comprising a subfraction of a fraction of an extract of an Nerium sp. or an Nerium flavescentis sp., wherein the subfraction has been prepared by liquid chromatographic fractionation of a fraction of the extract, and the subfraction does not contain oleandrin and neriside B; 14) an extract of an Nerium sp. or an Nerium flavescentis sp., if it contains cardiac glycosides, that, when administered in a dosage form to a subject suffering from a neurological disease or disorder, provides an improved clinical response or clinical effect compared to the pure cardiac glycoside administered to a subject at the same dose of the cardiac glycoside in an otherwise similar dosage form; or 15) a combination thereof.

The present invention also provides a method of treating a neurological disorder in a subject in need thereof, the method comprising:

determining whether the subject's neurological condition is Alzheimer's disease, Huntington's disease, stroke, or another neurological condition;

Indication for administering an extract of Nerium species or Nerium spp.;

administering an initial dose of the extract to the subject for a period of time according to the prescribed initial dosing regimen;

periodically determining the subject's clinical response to treatment with the extract and/or the adequacy of the therapeutic response; and

If the subject's clinical response and/or therapeutic response is adequate, continue treatment with the extract as needed until the desired clinical endpoint is achieved; or

If the subject's clinical and/or therapeutic response is inadequate at an initial dose and initial dosing regimen, the dose is titrated upward or downward until the desired clinical and/or therapeutic response is achieved in the subject.

The present invention also provides a method for preventing or reducing the incidence of a neurological disorder in a population of subjects at risk for a neurological disorder, the method comprising:

An effective dose of an extract of Nerium sp. or Nerium spp. is administered on a regular basis and for an extended period of time to one or more subjects in a subject population at risk for a neurological disorder, such as Alzheimer's disease, Huntington's disease, stroke, or other neurological disorder, thereby preventing or reducing the incidence of the neurological disorder in the population.

The present invention includes embodiments wherein: a) the method further comprises directing administration of the extract to one or more subjects; b) the method further comprises administering an effective dose of the extract to the subject for a period of time according to a prescribed dosing regimen; c) the method further comprises periodically determining the adequacy of the clinical response and/or therapeutic response of the one or more subjects to treatment with the extract; d) if the subject's clinical response and/or therapeutic response is adequate, then the method further comprises continuing treatment with the extract as needed until a desired clinical endpoint is achieved; e) if the subject's clinical response and/or therapeutic response is inadequate at the initial dose and initial dosing regimen, then the method further comprises gradually increasing or decreasing the dose until the desired clinical response and/or therapeutic response in the subject is achieved; f) the extract is administered to a plurality of subjects in a population; g) the regular basis is daily, every other day, every two days, every three days, every four days, every five days, every six days, weekly, every other week, every two weeks, every three weeks, monthly, bimonthly, semi-monthly, every other month, every h) the extended period is one or more weeks, one or more months, one or more quarters and/or one or more years; i) the effective dose is administered one or more times a day; j) the method further comprises identifying a population of subjects at risk for a neurological disorder, such as Alzheimer's disease, Huntington's disease, stroke or other neurological disorder; k) the population of subjects at risk is characterized by the subjects' increasing age, family history of a neurological disorder, a genetic susceptibility to developing a neurological disorder, the presence and expression of the ApoE4 gene in the subject, female sex (women are twice as likely as men to develop Alzheimer's disease), cardiovascular disease (e.g., high blood pressure and high cholesterol levels), diabetes (particularly type 2 or adult-onset diabetes), Down syndrome, head injury, low level of formal education, smoking, excessive alcohol consumption and/or drug abuse; l) the extract does not contain a therapeutically effective amount of cardiac glycosides; m) the extract does not contain cardiac glycosides; or n) a combination thereof.

The present invention also provides a time-delayed method for treating a stroke in a subject, comprising:

administering an initial dose of an extract of Nerium sp. or Nerium spp. according to an initial dosing schedule during a delayed period after the subject has suffered a stroke;

Determining the subject's clinical response and/or the adequacy of the therapeutic response to treatment with the extract; and

If the subject's clinical response and/or therapeutic response is adequate, then treatment with the extract is continued as needed until the desired clinical endpoint is reached; or

If the subject's clinical and/or therapeutic response is inadequate at an initial dose and initial dosing regimen, the dose is titrated upward or downward until the desired clinical and/or therapeutic response is achieved in the subject.

Certain embodiments of the invention include those wherein: 1) the delay period is 10 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, or 10 minutes or less; 2) determining the adequacy of the subject's clinical and/or therapeutic response is accomplished by assessing any weakness on one side of the body, an arm, and/or leg, paralysis on one side of the body, an arm, and/or leg, inability to understand spoken language, inability to speak or speak clearly, inability to write, dizziness and/or unbalanced gait, double vision, and unusually severe headache; or 3) a combination thereof.

The present invention also provides use of an extract of an Nerium species or a Nerium genus in preparing a medicament for treating a neurological condition in a subject. In certain embodiments, the preparation of the medicament comprises: providing an extract of an Nerium species or a Nerium genus; including a dosage of the extract of an Nerium species or a Nerium genus ... The preparation may also include one or more additional steps such as: delivering the packaged dosage form to a supplier (retailer, wholesaler, and/or distributor); selling or otherwise providing the packaged dosage form to a subject suffering from a neurological disorder; including a label and package insert within the drug that provides instructions for use, dosing regimen, administration, content, and toxicological properties of the dosage form. In certain embodiments, the treatment of a neurological disorder comprises: determining that the subject suffers from a neurological disorder or condition; instructing the subject to administer the extract or a component thereof according to a dosing regimen; administering to the subject one or more pharmaceutical dosage forms containing the extract, wherein the one or more pharmaceutical dosage forms are administered according to the dosing regimen.

The present invention also provides an extract or composition of Nerium species or Nerium yellow flower species, i.e., a pharmaceutical formulation or dosage form comprising an extract of Nerium species or Nerium yellow flower species for treating a neurological disorder. In certain embodiments, the extract can be obtained from Nerium species or Nerium yellow flower species as described herein or in U.S. Patent No. 7,402,325, PCT International Patent Application PCT/US06/29061, U.S. Patent Application No. 12/019435, or Newman et al. (Mol. Interven. (2008), 8, 36-49), the entire disclosures of which are incorporated herein by reference.

The present invention provides a method for preparing an extract fraction of an Nerium species or a Nerium species, comprising: extracting a substance containing the Nerium species or the Nerium species to form an unfractionated extract thereof, wherein the extract contains one or more pharmacologically active ingredients for treating neurological disorders; and fractionating the unfractionated extract to form two or more fractions thereof, wherein at least one fraction contains one or more pharmacologically active ingredients other than cardiac glycosides. In certain embodiments, a) at least one fraction does not contain a cardiac glycoside; b) at least one fraction contains at least a cardiac glycoside; c) extraction is performed using a supercritical liquid, water, an organic solvent, or a combination thereof; d) fractionation is performed using liquid chromatography or solvent extraction; e) the at least one fraction does not contain oleandrin and oleandrin; or f) a combination thereof.

The present invention also provides a method for fractionating an extract of an Nerium species or a yellow flower Nerium species to provide one or more therapeutically effective components thereof. The method comprises: a) providing an extract of an Nerium species or a yellow flower Nerium species; b) fractionating the extract to provide two or more components of the extract, a first extract component comprising one or more pharmacologically active agents (which are not cardiac glycosides) and not comprising cardiac glycosides (oleandrin and oleandrin B), and a second extract component comprising one or more cardiac glycosides and one or more pharmacologically active agents (which are not cardiac glycosides). Fractionation can also be performed as described herein. In certain embodiments, the first extract component and the second extract component are further fractionated to provide two or more different subcomponents, wherein the first subcomponent comprises one or more steroids and the second subcomponent comprises one or more triterpenes. In certain embodiments, fractionation is performed by liquid chromatography having a stationary phase and a mobile phase.

The present invention also provides a composition comprising an extract fraction obtained from Nerium species or Nerium spp., whereby the fraction has been obtained by fractionating an extract obtained from Nerium species or Nerium spp. In certain embodiments, the extract fraction comprises one or more steroids and one or more triterpenes, and optionally does not comprise cardiac glycosides (oleandrin and oleandrin-B).

The present invention also provides a composition comprising a subfraction of an extract component obtained from an Nerium species or a Nerium species, whereby the subfraction has been obtained from an extract component obtained from an Nerium species or a Nerium species by further fractionation. In certain embodiments, the subfraction of the extract component comprises one or more steroids, cardiac glycosides, a related aglycone of a cardiac glycoside, such as oleandrin, cardiosteroids, or triterpenoids, and one or more triterpenes. In certain embodiments, the subfraction of the extract component comprises one or more triterpenes and does not comprise a steroid. Each subfraction independently and optionally does not comprise a cardiac glycoside (oleandrin and cardioside B).

In certain embodiments: a) the extract further comprises at least two pharmacologically active agents obtained (extracted) from a species of the genus Nerium or a species of the genus Oleander; b) the at least two pharmacologically active agents additively or synergistically enhance the therapeutic efficacy of the extract when the extract is administered to a subject; c) neither of the at least two pharmacologically active agents is a cardiac glycoside; and/or d) the at least two pharmacologically active agents are selected from the group of cardiac glycosides, related aglycones of cardiac glycosides, such as oleandrin, cardiosteroids, and triterpenoids.

In certain embodiments: 1) the cardiac glycoside is selected from the group consisting of oleandrin, odoroside, oleandrin, G-strophanthin, bufalin, digitoxin, cinobufatalin, cinobufagin, and resibufogenin; 2) the extract is present in the pharmaceutical formulation or composition; 3) the extract has been obtained from oleander plant material or neriifolia plant material; 4) the plant material comprises a species of the genus Nerium, such as Nerium oleander; or a species of the genus Nerium, such as Thevetia neriifolia or Thevetia neriifolia. peruviana (also known as yellow oleander); 5) the extract is prepared by supercritical fluid (SCF) extraction, optionally in the presence of a modifier; 6) the cardiac glycoside is oleandrin; 7) the extract is prepared by hot water extraction, cold water extraction, organic solvent extraction, or aqueous organic solvent extraction.

In certain embodiments, the extract (or its components or sub-components) comprises less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01% polysaccharide by weight. In certain embodiments, the extract (or its components or sub-components) comprises betulin (urs-12-ene-3β,28-diol); 28-norurs-12-ene-3β-ol; urs-12-ene-3β-ol; 3β,3β-hydroxy-12-oleanone-28-oic acid; 3β,20α-dihydroxyurs-21-en-38-oic acid; 3β,27-dihydroxy-12-ursen-38-oic acid. acid); 3β,13β-dihydroxyursolic-11-ene-28-oic acid; 3β,12α-dihydroxyoleanane-28,13β-lactone and 3β,27-dihydroxy-12-oleanan-28-oic acid. In certain embodiments, the extract (or its components or subcomponents) comprises one or more cardiac glycoside precursors selected from the glycosyl components of cardiac glycosides. In certain embodiments, the glycosyl is selected from the group consisting of glucosides, fructosides and glucuronides. In certain embodiments, the extract (or its components or subcomponents) comprises oleandrin, ursolic acid, betulinic acid, odoroside, oleandrin, oleanolic acid and one or more triterpenes and less than 0.5% by weight of polysaccharides.

In certain embodiments, a subject suffering from a neurological disorder, i.e., a subject in need thereof, is part of such a population of subjects. The present invention provides methods for improving the clinical status of a statistically significant number of subjects in a population of subjects suffering from a neurological disorder, comprising: administering an extract of Nerium spp. or Nerium spp., or a composition comprising an extract of Nerium spp. or Nerium spp.; and determining the clinical status of the subjects. In certain embodiments, the statistically significant number is at least 5% of the population.

In certain embodiments, the neurological disorder is Alzheimer's disease, Huntington's disease, stroke, tauopathies, or other neurological disorders, such as those described herein. A medicament can be prepared by including the extract in a pharmaceutical dosage form containing one or more pharmaceutically acceptable excipients.

Treatment of the subject with the extract or composition containing the extract is continued as needed. The dosage or administration regimen may be adjusted as needed until the patient achieves the desired clinical endpoint, such as a reduction or alleviation of a specific neurological symptom associated with the disease. Determination of the clinical response and/or adequacy of the therapeutic response may be performed by a clinician familiar with the neurological condition being treated.

In certain embodiments, the neurological condition is selected from the group consisting of neurological disease, neurological disorder, tauopathy and stroke. In certain embodiments, the neurological disease is a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is selected from the group consisting of Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, bovine spongiform encephalopathy, multiple sclerosis, diabetic neuropathy, autism and juvenile neuronal ceroid lipofuscinosis. In certain embodiments, the stroke is an ischemic injury caused by a stroke. In certain embodiments, the neurological condition is tauopathy, which is a neurodegenerative disease with an etiology related to an imbalance in the Tau3R/Tau4R ratio in the subject. Tauopathy is a class of neurodegenerative diseases caused by pathological aggregation of tau protein (tau) in the human brain. In certain embodiments, tauopathy is Down syndrome, Pick's disease, basal ganglia degeneration, certain variants of prion disease, Alzheimer's disease, progressive supranuclear palsy or frontotemporal dementia. The individual steps of the process according to the invention can be carried out in separate facilities or in the same facility.

In certain embodiments, the neuron is in vitro, ex vivo, or in vivo. In certain embodiments, the neuron is a CA-1 neuron.

In certain embodiments, the present invention provides extracts of Nerium species or Neriifolia species, or components thereof, or subfractions thereof, having a ¹ H NMR spectrum as described herein. In certain embodiments, the present invention provides extracts of Nerium species or Neriifolia species, or components thereof, or subfractions thereof, that, when administered to a subject, exhibit therapeutic activity as described herein. In certain embodiments, the present invention provides extracts of Nerium species or Neriifolia species, or components thereof, or subfractions thereof, that have an HPLC chromatogram as described herein. In certain embodiments, the methods of the present invention utilize extracts, components thereof, or subfractions thereof as described herein. In certain embodiments, compositions of the present invention comprise extracts, components thereof, or subfractions thereof as described herein.

The present invention includes all combinations of aspects, embodiments and sub-embodiments of the invention described herein. Unless otherwise indicated herein, the term "extract" may refer to an unfractionated extract or a fractionated extract (i.e., a component of an extract) or a subfractionated extract (i.e., a sub-component of a component of an extract).

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings constitute a part of this specification and depict exemplary embodiments of the claimed invention. Based on these drawings and the description herein, one skilled in the art will be able to implement the invention without undue experimentation.

FIG1A depicts concentration-response data obtained from a comparative evaluation of oleandrin compared to oxygen or glucose deprivation (OGD) controls in a neuroprotective brain slice-based "stroke" assay (Example 8), wherein the number of healthy cortical neurons was determined 5-6 minutes after oxygen and glucose deprivation (OGD = stroke) in the presence or absence of oleandrin.

Figure IB depicts the results of a concentration-response assay for an unfractionated SCF extract of Nerium oleander in a neuroprotective brain slice-based "stroke" assay as described herein (Example 8), using either anaerobic or glucose deprivation as controls.

Figures 2A-2C depict the results of a comparative evaluation of oleandrin compared to Nerium oleander unfractionated SCF extract in a neuroprotective brain slice-based "Alzheimer" assay (Example 9), wherein the number of healthy cortical neurons was determined following APP/Aβ-induced degeneration in the absence or presence of varying amounts of those preparations.

Figures 3A-3D depict results from replicate experiments comparatively evaluating oleandrin (Figures 3A-3B) (Figures 3C-3D) in a "Huntington's disease" neuroprotection assay based on corticostriatal co-cultured neurons (Example 10), wherein the percent rescue of cortical neurons relative to controls compared to striatal neurons transfected with a mutant form of huntingtin (htt) protein was determined in the absence or presence of varying amounts of oleandrin.

Figures 4A-4E depict the results of a neuroprotective brain slice-based "stroke" assay as described herein, wherein an SCF extract containing oleandrin has been fractionated by liquid chromatography (Example 13) and five different fractions (described below) were subjected to the assay (Example 15): Fraction O-H (Figure 4A), Fraction O-2 (Figure 4B), Fraction O-3 (Figure 4C), Fraction O-4 (Figure 4D), and Fraction O-5 (Figure 4E).

5 depicts the results of a concentration-response brain slice-based "stroke" assay (Example 15) for Fraction 0-4 of Nerium oleander SCF extract compared to total (parent) unfractionated Nerium oleander SCF extract (PBI or PBI-05204).

Figure 6 depicts the results of a comparative evaluation of the fractions of Nerium oleander SCF extract (O-4 or O-4A) compared to untreated (cells not transfected with APP/Aβ) in the APP-based "Alzheimer" assay (Example 11), wherein the number of healthy cortical neurons was determined after APP/Aβ-induced degeneration in the absence or presence of varying amounts of those agents.

Figure 7 depicts the results of a comparative evaluation of a fraction (O-4) of an Nerium oleander SCF extract compared to untreated (cells not transfected with APP/Aβ) in a Tau4R-based "Alzheimer's" assay (Example 12), wherein the number of healthy and damaged cortical neurons was determined following Tau4R in the absence or presence of varying amounts of those agents.

8A-8D depict chromatograms obtained by HPLC analysis of fractions prepared according to Example 13.

Figures 9A-9I depict HNMR spectra for various components present in the fraction O-4 Nerium oleander SCF extract. Figure 9A depicts the HNMR spectrum of the O-4 fraction prior to subfractionation according to Example 17. Figures 9B-9I depict the HNMR spectra of various subfractions obtained by silica gel flash chromatography of the O-4 fraction according to Example 17.

DETAILED DESCRIPTION

The present invention provides a method for treating a neurological condition by administering an effective dose of an extract of Nerium species or Nerium spp. to a subject in need thereof. The extract is administered according to a dosing regimen most suitable for the subject, the dosage and dosing regimen being clinically determined based on conventional clinical practice and clinical therapeutic endpoints for the neurological condition being treated.

In certain embodiments, the neurodegenerative disease or neurological condition being treated has an etiology associated with overexpression of tau protein and/or an imbalance in the Tau3R/Tau4R ratio in the subject. Such conditions are called tauopathy. Exemplary tauopathy conditions include Down syndrome, Pick's disease, certain variants of prion disease, Alzheimer's disease, progressive supranuclear palsy or frontotemporal dementia, basal ganglia degeneration, Parkinson's disease syndrome, argyrophilic grain dementia, Niemann-Pick disease type C, and dementia pugilistica.

In certain embodiments, the neurodegenerative disease or neurological condition being treated has an etiology associated with abnormal or atypical proteolysis of β-amyloid precursor protein, accumulation of β-amyloid protein in neuronal synapses, formation of amyloid fibrils in neuronal synapses, or formation of amyloid plaques in neuronal synapses. An example of such a disease or condition is Alzheimer's disease. A subject treated according to the present invention will exhibit a therapeutic response. "Therapeutic response" means that a subject suffering from a disease or condition will experience at least one of the following clinical benefits as a result of treatment with the extract: improvement of the disease or condition, reduction in the occurrence of symptoms associated with the disease or condition, partial relief of the disease or condition, complete relief of the disease or condition, or prolonged progression. In other words, the therapeutic response can be a complete or partial therapeutic response.

A therapeutic response can also be described as an improvement in the quality of life of a patient affected by a neurodegenerative disease. Improved quality of life can occur, for example, by a decrease in the occurrence, frequency, or severity of symptoms associated with the disease (e.g., tremors, involuntary muscle movements, loss or partial loss of neuromuscular coordination, memory retention, etc.).

"Preventing the development of a neurological disorder in a population of subjects at risk" means that in a demographically predetermined population at risk for developing a neurological disorder, the neurological disorder will not develop within a predetermined time period. The prevention within the predetermined time period is due to the fact that the subjects in said population have taken the extract according to the present invention. As an example, when the extract or a composition containing the extract is administered to subjects in a population at risk for developing a stroke for a predetermined time period, no stroke will develop in those subjects within the predetermined time period. In particular, the composition containing the extract is administered chronically over a period of one year to a population of subjects at risk for developing Alzheimer's disease or any tauopathology-related disease, and the subjects in said population do not exhibit symptoms associated with Alzheimer's disease within the one-year period.

"Reducing the incidence of a neurological disorder in a population of at-risk subjects" is related in meaning to "preventing the occurrence," except that "reducing the incidence" allows for the occurrence of a neurological disorder in a demographically predetermined population of subjects, but at a reduced incidence or severity level compared to an otherwise similar demographically predetermined population of at-risk subjects who are not taking the extract-containing composition according to the present invention.

As used herein, "progression time" is the period of time, length of time, or duration after a disease is diagnosed (or treated) before the disease begins to worsen. This is the period of time during which the disease level remains, without further progression of the disease, and ends when the disease begins to progress again. Disease progression is determined by "staging" a subject with a neurological disorder before or at the start of treatment. For example, the neurological health of the subject is determined before or at the start of treatment. The subject is then treated with the extract, and the neurological health is monitored regularly. At some later time point, the symptoms of the neurological disorder may worsen, thereby marking the progression of the disease and the end of the "progression time." The period of time during which the disease does not progress or during which the disease level or severity does not worsen is the "progression time."

The dosage regimen includes the treatment-related dose (or effective dose) of the extract used according to the dosing plan.Therefore, the treatment-related dose is the therapeutic dose at which the therapeutic response of the disease or condition treated by the extract is observed, and is the therapeutic dose at which the subject can take the extract without excessive undesirable or harmful side effects. The treatment-related dose is not lethal for the subject, although it may cause certain side effects in the patient. It is the dose at which the level of clinical benefit exceeds the level of harmful side effects experienced by the subject due to taking the extract for the subject taking the extract. According to a variety of known pharmacological, pharmacodynamic and pharmacokinetic principles, the treatment-related dose is different between subjects. However, the treatment-related dose is generally in the range of 0.1 to 100 micrograms of extract/day, and the extract is solid, liquid or semi-solid form. It is known in the art that the actual amount of the pharmacologically active agent providing the target treatment result in the subject can be different between subjects according to pharmaceutical basic principles.

According to any dosage regimen commonly used in neurological diseases or disorders or neurodegenerative diseases or disorders, a treatment-related dose can be administered. Treatment-related doses can be administered once, twice, three times or more daily dosing arrangements. Treatment-related doses can be administered every other day, every three days, every four days, every five days, every half week, every week, every two weeks, every three weeks, every four weeks, every month, every two months, every half month, every three months, every four months, every six months, every year, or according to any of the above combinations to achieve suitable dosing arrangements. For example, a treatment-related dose can be administered once daily for one or more weeks.

The following examples include evidence of the extract's effectiveness in neurological conditions such as neurological diseases, neurological disorders, or stroke. Example 3 details a method of treating Alzheimer's disease with an extract of Nerium species, or a combination thereof, an extract of Nerium species, or a combination thereof, or a combination thereof, in combination with one or more other therapeutic agents. Example 4 details a method of treating Huntington's disease with an extract or a combination of extracts in combination with one or more other therapeutic agents. Example 5 details a method of treating ischemic brain damage caused by stroke or non-stroke ischemic brain damage with an extract or a combination of extracts in combination with one or more other therapeutic agents.

In general, the following treatment is used to treat a subject suffering from a neurological disorder. The subject showing a neurological disorder is assessed to determine whether the neurological disorder is Alzheimer's disease, Huntington's disease, stroke or other neurological disorder. If the subject receives a positive diagnosis, the extract or a composition containing the extract is administered. An initial dose of the extract or composition is administered to the subject for a period of time according to the prescribed dosage regimen. The clinical response and therapeutic response level of the subject are regularly determined. If the level of therapeutic response is too low at one dose, the dose is gradually increased according to a pre-determined dose escalation schedule until the desired therapeutic response level is reached in the subject. If the subject exhibits undesirable side effects or unacceptable levels of side effects, the dose is gradually reduced until the desired balance of therapeutic response level compared to the side effect profile is reached in the subject. As needed, the subject is continued to be treated with the extract or composition. The dosage or dosage regimen can be adjusted as needed until the patient achieves a desired clinical endpoint, such as cessation of the disease itself, a reduction in disease-related symptoms, and/or a decline in the progression of the disease.

Extracts, particularly unfractionated extracts, contain one or more pharmacologically active compounds. Some of those compounds have not yet been identified, and some may be oleandrin or other cardiac glycosides, oleaside, oleandrin, oleandrin, oleandrin (Wang X, Plomley JB, Newman RAand Cisneros A.LC/MS/MSanalyses of an oleander extract for cancer treatment, Analytical Chem.72:3547-3552,2000) and other plant materials. Unfractionated SCF extracts from supercritical fluid processes generally contain oleandrin in the theoretical range of 0.9% to 2.5% by weight. SCF extracts containing different amounts of oleandrin have been obtained. In one embodiment, the SCF extract contains about 2% oleandrin by weight.

The extractable, unidentified components of the Nerium species or Nerium spp. extract may include at least one (non-cardiac glycoside) pharmacologically active ingredient that contributes to the efficacy of the SCF extract or its components. Two or more pharmacologically active extractable components may contribute additively or synergistically to the observed efficacy. In other words, the Nerium species or Nerium spp. extract of the present invention contains one or more pharmacologically active ingredients that are not cardiac glycosides, although one or more cardiac glycosides may be additionally included in the extract. The extract can be fractionated into a variety of different components, some of which contain cardiac glycosides, one or more pharmacologically active ingredients that are not cardiac glycosides, or a combination thereof. In addition, each extract component can be further fractionated into two or more different subcomponents.

Evidence for the presence of one or more pharmacologically active ingredients in the SCF extract other than oleandrin comes from comparing the concentration-response curves of solutions containing pure oleandrin with those containing the SCF extract. FIG1A depicts the results of a concentration-response experiment on a solution containing pure oleandrin in a neuroprotective brain slice-based "stroke" assay as described in Example 8. The oleandrin concentration in the solution varied from 0.0069 to 230 μg/ml. FIG1B depicts the results of a concentration-response experiment on an extract of the SCF Nerium species containing oleandrin in a neuroprotective brain slice-based "stroke" assay as described herein (Example 8). The data indicate that the extract is more effective than pure oleandrin, suggesting that the extract contains one or more pharmacologically active agents that provide neuroprotection.

Example 8 details an in vitro assay used to evaluate the efficacy of the extract, or a combination thereof, for treating ischemic neurological damage caused by stroke. The assay is a brain slice-based assay for oxygenated glucose deprivation (OGD), which is designed to induce a loss of ≥50% of healthy cortical neurons within 24 hours. A total unfractionated SCF extract of an Nerium species, such as Nerium oleander, is used as a positive control. This total extract is then fractionated according to Example 13 to provide the components of the Nerium species extract. The components are analyzed according to Examples 6, 14, and 17.

The HNMR characteristics of triterpenes are 7 methyl signals upfield, olefin protons at approximately 5.3 ppm, and an oxidized methine signal at approximately 3.4 ppm, as well as numerous methylene and methine proton signals upfield (approximately 1.0 to 2.5 ppm). The HNMR spectrum (Figures 9B-9I) indicates that the major components are steroids and triterpenes. No significant glycoside signals were observed. No signals were observed for α,β-unsaturated γ- or δ-lactones, which are characteristic of cardiac glycosides, indicating that cardiac glycosides or their aglycones are not present in the Fr-O-4 component. The HNMR spectrum in Figure 9C corresponds to a subfraction containing at least one steroid and at least one or at least two different triterpenes. The HNMR spectrum in Figure 9B corresponds to a subfraction containing a mixture of at least two different triterpenes, such as two ursolanes, and does not contain a steroid.

Accordingly, component O-4 contains at least one triterpene and at least one steroid. In certain embodiments, component O-4 contains at least two different triterpenes and at least two different steroids, or the component contains multiple different triterpenes and multiple different steroids. The O-4 component evaluated in this example does not contain a therapeutically effective amount of a cardiac glycoside. In certain embodiments, component O-4 does not contain a cardiac glycoside. In certain embodiments, the first subcomponent of component O-4 contains at least one steroid and at least one triterpene or at least two different triterpenes, and the second subcomponent contains at least two different triterpenes and does not contain a steroid. In certain embodiments, each of the first and second subcomponents does not contain a cardiac glycoside.

Fraction O-4 was tested in brain slices treated with OGD (a stroke model) and brain slices treated with non-OGD (i.e., a control) (a non-stroke model). The data showed that when a solution of the extract O-4 fraction was used at a concentration range of 100 ng/mL to 1 μg/mL, the extract O-4 fraction provided substantial neuroprotection, and when a solution of the extract O-4 fraction was used at a concentration range of 1 μg/mL to 1 mg/mL, even higher neuroprotection was provided. Therefore, a liquid dosage form containing 100 ng/mL to 1 mg/mL of the extract fraction per mL of liquid dosage form will provide neuroprotection in subjects to whom it is administered.

Although direct measurements have not been performed in the human brain following systemic dosing of the extract, it is hypothesized that one or more of the physiologically active components of the extract will cross the blood-brain barrier when administered to a subject. Oleandrin, either as a pure compound or contained in an SCF extract designated PBI-05204, has been shown to effectively cross the blood-brain barrier and enter the brain in a rodent (mouse) model. It is reasonable to expect that oleandrin will do the same for the human blood-brain barrier.

Accordingly, the present invention provides a method for protecting neurons from loss of activity caused by oxygen deprivation or oxygen-glucose deprivation by exposing oxygen-deprived and/or glucose-deprived neurons to an effective amount of an extract of an Nerium species or a Nerium species to minimize the loss of activity, reduce the rate of loss of activity, stop the loss of activity, slow the occurrence of loss of activity, and/or protect the function of neurons caused by exposure to conditions of oxygen deprivation and/or glucose deprivation. In certain embodiments, the method employs an effective amount of a component or subcomponent of an Nerium species extract or a Nerium species extract. In certain embodiments, the component or subcomponent has been prepared by liquid chromatography fractionation of the extract. In certain embodiments, the component does not comprise cardiac glycosides, and in other embodiments, the component or subcomponent comprises one or more cardiac glycosides, particularly those described herein.

Example 9 details an in vitro assay for evaluating the extract's efficacy in treating Alzheimer's disease. This assay is a brain slice-based assay targeting APP/Aβ (APP: β-amyloid precursor protein)-induced degeneration of cortical pyramidal neurons. After cleavage by secretases, APP is broken down into Aβ peptides, which are believed to be the causative factor in the formation of β-amyloid plaques. Aβ protein is associated with the formation of β-amyloid plaques and is believed to be a hallmark (if not the cause) of Alzheimer's disease. Biolistic transfection is used to introduce necessary markers, such as YFP (yellow fluorescent protein marker) and disease gene constructs, into the same neuronal population in brain slices. Co-transfection of YFP with APP isoforms results in progressive degeneration of cortical pyramidal neurons within three to four days after brain slice preparation and transfection. The data (Figures 2A-2C) demonstrate that the Nerium species SCF extract provides concentration-dependent neuroprotection in APP-transfected brain slices, resulting in rescue levels nearly equivalent to those provided by BACE inhibitor drugs, i.e., β-secretase inhibitors. β-Secretase cleaves the APP precursor protein into the toxic Aβ protein. SCF extracts containing oleandrin were shown to provide greater neuroprotection than oleandrin alone. The data in Figures 2A-2C are significant in that few compounds or therapeutic strategies in the literature have demonstrated any significant neuronal protection in this in vitro assay representative of Alzheimer's disease.

The APP-WT brain slice-based Alzheimer's disease test (Example 11) was repeated using components of the European oleander SCF extract. After APP/Aβ-induced degradation, the number of healthy cortical neurons was determined in the presence of different amounts of the SCF extract O-4A component (0.01 to 100 μg/mL). Exposure to oxygen and glucose deprivation was used as an internal positive control to produce neuronal damage similar to that caused by a stroke. The negative control was simply relatively healthy brain slice neurons, without OGD treatment or exposure to treatment. The data are depicted in Figure 6, where lighter colored columns indicate significant differences relative to the APP-WT condition, based on ANOVA and subsequent Dunnett's post hoc comparison test at a confidence level of 0.05. The data indicate that the O-4A component provides neuroprotection in this test, even though it does not contain cardiac glycosides.

Fraction O-4 (O-4A) of the Nerium oleander SCF extract was evaluated in a brain slice-based Alzheimer's disease assay utilizing the tau4R (Example 12). The number of healthy cortical neurons was measured. Efficacy in this assay was defined as or based on the following: the relative total number of healthy compared to unhealthy and the percentage of degraded neurons in the presence of varying amounts of the SCF extract O-4A fraction (0.3-100 μg/ml, concentrations determined by weight of the extract). Negative controls in these experiments consisted of brain slices not exposed to OGD, while brain slices exposed to OGD but not treated with the fraction derived from the unfractionated Nerium oleander extract served as internal positive controls. The data are depicted in Figure 7, where lighter bars indicate significant differences relative to damaged neurons, as determined by ANOVA followed by Dunnett's post hoc comparisons at a 0.05 confidence level. The data suggest that fraction O-4A provides neuroprotection in this assay, even though it does not contain cardiac glycosides.

Accordingly, the present invention provides a method for protecting neurons from loss of activity caused by Alzheimer's disease, the method comprising: exposing neurons exhibiting Alzheimer's disease characteristics to an effective amount of an extract of an Nerium species or a Nerium species to minimize the loss of activity, reduce the rate of loss of activity, stop the loss of activity, slow the occurrence of loss of activity, and/or the key functions of neurons caused by Alzheimer's disease. In certain embodiments, the method employs an effective amount of a component of an Nerium species extract or a Nerium species extract. In certain embodiments, the component has been prepared by liquid chromatography fractionation of the extract. In certain embodiments, the component does not comprise cardiac glycosides, and in other embodiments, the component comprises one or more cardiac glycosides, particularly those described herein.

Example 10 details an experiment conducted to evaluate the efficacy of an extract for treating Huntington's disease. Mutant htt protein was introduced into a high-density, mixed co-culture of cortical neurons, striatal neurons, and glia by electroporation. Striatal and cortical neurons were transfected with different colored fluorescent proteins, facilitating the separate identification of different neuronal types within the co-culture. Color fluorescent proteins are fluorescent and "emit" color upon excitation with a light source of appropriate wavelength. The data (Figures 3A-3D) demonstrate that oleandrin and the SCF extract of Nerium oleander were more effective than KW6002 (an adenosine 2a receptor antagonist) in increasing the number of viable neurons. The data also demonstrate that the SCF extract was more effective than oleandrin alone, suggesting that the extract, further comprising one or more therapeutically effective agents in addition to oleandrin, could be used to treat Huntington's disease. Such other agents could be used in conjunction with or without oleandrin or other cardiac glycosides. Accordingly, the present invention provides a method for protecting neurons from loss of activity caused by Huntington's disease, the method comprising: exposing neurons exhibiting Huntington's disease characteristics to an effective amount of oleandrin or an extract containing oleandrin to minimize the loss of activity, reduce the rate of loss of activity, stop the loss of activity, slow the occurrence of loss of activity, and/or the normal function of the neurons caused by Huntington's disease.

Example 16 details an exemplary brain slice assay that can be used to evaluate the efficacy of the extract in treating stroke in subjects after a delayed period following a stroke. A brain slice assay utilizing oxygen glucose deprivation was performed as described herein; however, rather than treating the brain slices prophylactically with the extract, they were treated with the extract after delayed periods of 0, 1, 2, 4, and 6 hours. The data demonstrate that inclusion of the extract is effective in providing significant neuroprotection for delayed periods of up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, and up to approximately 6 hours following a stroke.

Accordingly, the present invention provides a method for delaying the treatment of stroke in a subject by administering to the subject a dose of an extract of an Nerium species or a Nerium species after the subject has suffered a stroke. An initial dose of the extract is administered according to an initial dosing regimen within an acceptable delay period after the subject has suffered a stroke. Thereafter, the appropriateness of the subject's clinical response and/or therapeutic response to the extract treatment is determined. If the subject's clinical response and/or therapeutic response is appropriate, treatment with the extract is continued as needed until the desired clinical endpoint is reached. Alternatively, if the subject's clinical response and/or therapeutic response is inappropriate at the initial dose and initial dosing regimen, the dose is gradually increased or gradually decreased until the desired clinical response and/or therapeutic response is achieved in the subject. The dose can be gradually increased or decreased in combination with a change in the dosing regimen, such as a change in the frequency of administration or the total period of dosing.

Certain brain slice experiments herein were conducted under conditions in which brain tissue was treated with the extract prior to OGD. Under those conditions, the data demonstrate the utility of the extract in prophylactically providing neuroprotection against stroke-induced damage.

If a clinician seeks to treat a subject with a neurological condition with a combination of an extract or a composition thereof and one or more other therapeutic agents, and it is known that the subject has a particular neurological condition that is at least partially responsive to treatment with the one or more other therapeutic agents, then the present method invention comprises administering to a subject in need thereof a therapeutically relevant dose of the extract (or a component or subcomponent thereof) and a therapeutically relevant dose of the one or more other therapeutic agents, wherein the extract (or a component or subcomponent thereof) is administered according to a first dosing regimen and the one or more other therapeutic agents are administered according to a second dosing regimen. In certain embodiments, the first and second dosing regimens are the same. In certain embodiments, the first and second dosing regimens are different.

If the neurological condition being treated is Alzheimer's disease, the one or more additional therapeutic agents may be selected from the group consisting of a BACE inhibitor or an acetylcholinesterase inhibitor. In certain embodiments, the one or more additional therapeutic agents may be selected from the group consisting of Namenda ^{™ (} memantine HCl), Aricept ^™ (donepezil ^{), Razadyne™ (} galantamine), Exelon ^™ (rivastigmine), and Cognex ^™ (tacrine).

If the neurological disorder being treated is Huntington's disease, the one or more other therapeutic agents may be selected from the group consisting of natural products, anticonvulsants, NMDA (n-methyl d-aspartate) receptor antagonists, and sodium channel blockers. Exemplary therapeutic agents include vitamin E, baclofen (a CoQ10 derivative), lamotrigine (an anticonvulsant), remaramide (an anesthetic that is a low-affinity NMDA antagonist), and riluzole (a Na channel antagonist). The efficacy of each of these agents is considered low on its own (Mestre T. et al, Chochrane Database Systematic Reviews July 8, 2009; 8(3): CD006455); however, it is expected that administration of a dosage form containing the extract to a subject receiving one or more of these other agents will provide an improved clinical effect for a subject suffering from a neurological disorder compared to administration of these agents without the extract.

If the neurological disorder being treated is ischemic brain damage caused by stroke (ischemic stroke), then in addition to the extract, the therapeutic treatment disclosed in the literature (Gutierrez M. et al. "Cerebral protection, brain repair, plasticity and cell therapy in ischemic stroke" Cerebrovasc. Dis. 2009; 27 Suppl 1: 177-186) such as intravenous thrombolysis can be used. In certain embodiments, the one or more therapeutic agents can be selected from the group consisting of drugs such as alteplase (thrombolytic agent).

The invention relates to the invention of the present invention and the invention relates to a kind of therapeutic agent that can be used for the treatment of the present invention.Can dosage and be recognized as the effective dosage regimen for treatment according to clinician, or be recognized as the effective dosage of sub-treatment with clinician.The clinical benefit and/or the therapeutic effect provided by the combination of extract and one or more other therapeutic agents can be additive or synergistic, and the level of this benefit or effect is by determining relatively using the use of the combination with the use of independent extract and one or more other therapeutic agents.Can dosage and according to the dosage regimen such as U.S. Food and Drug Administration (U.S.F.D.A.), World Health Organization (W.H.O), European Medicines Agency (E.M.E.A.), Drug Administration (TGA, Australia), Pan American Health Organization (PAHO), Drug and Medical Device Safety Agency (Medsafe, New Zealand) or multiple health ministries in the world suggest or illustrate use one or more other therapeutic agents.

If a cardiac glycoside is used according to the present invention, it can be any cardiac glycoside known to have Na,K-ATPase binding activity. The cardiac glycoside should be able to cross the blood-brain barrier and remain in brain tissue for an extended period after administration. In this regard, the cardiac glycoside should remain in the brain for at least 8 hours after administration due to tissue binding and the resulting low clearance rate.

If present, the cardiac glycoside may be present in pure form or in admixture with one or more other compounds. The cardiac glycoside may be present as an extract.

The extract can be prepared by supercritical fluid (SCF) carbon dioxide (CO ₂ ) extraction or a chemically modified form of such an extract (e.g., an extract comprising ethanol or prepared using SCF CO ₂ and ethanol; Example 1). The extract can be obtained by extracting the plant material with an organic solvent, such as ethanol, methanol, propanol, or other such solvents. The extract can be obtained from the plant material. The plant material can be, for example, plant material obtained from a species of the genus Nerium, such as Nerium oleander, or plant material from a species of the genus Nerium, such as Nerium candidum (also known as yellow oleander). The extraction process can be performed on dried powder of European oleander leaves, which is prepared according to the process described in the following documents: currently pending U.S. provisional application 60/653,210 filed on February 15, 2005 in the name of Addington, U.S. patent application 11/340,016 filed on January 26, 2006 in the name of Addington, U.S. patent application 11/191,650 filed on July 28, 2006 in the name of Addington (now U.S. Patent 7,402,325 issued on July 22, 2008), or PCT International Patent Application PCT/US06/29061 filed on July 26, 2006, or Newman et al. (Mol. Interven. (2008), 8, 36-49), the entire disclosures of which are incorporated herein by reference, or prepared according to the process described herein. These methods can also be used to prepare unfractionated extracts of Nerium species or Nerium spp. Unless otherwise indicated, the term "extract" as used herein can refer to an "unfractionated extract" or a fraction of an extract or a subfraction of a fraction of an extract. The term "unfractionated extract" generally means an extract obtained by extracting plant material, wherein the extract has not been fractionated, for example, by chromatography or solvent extraction, or separated into individual components or groups of components after the initial preparation of the extract.

As used herein, the term "oleandrin" means all known forms of oleandrin, unless otherwise indicated. Oleandrin can exist in racemic form, optically pure form, or optically enriched form. Nerium oleander plant material can be obtained, for example, from commercial plant supplier Aldridge Nursery (Atlas, Texas).

Unfractionated extracts can be obtained by modified (e.g., ethanol) or unmodified supercritical fluid extraction of cardiac glycoside-containing plant material, such as plant material containing Nerium species or Nerium spp. The supercritical fluid extract may contain one or more pharmacologically active agents extracted from the plant material that, when administered to a subject, contribute to the therapeutic efficacy of the extract. When two or more such agents are present, they contribute additively or synergistically to the therapeutic efficacy of the extract.

Unfractionated extracts can be prepared by a variety of different processes. Extracts can be prepared as above or according to a process developed by Dr. Huseyin Ziya Ozel (U.S. Patent No. 5,135,745), which describes a hot water extraction procedure for preparing plant extracts in water. The aqueous extract is reported to contain several polysaccharides, oleandrin, oleandrin, oleandrin, and oleandrin, with molecular weights varying from 2 kD to 30 kD. Polysaccharides are reported to include acidic homopolygalacturonans or arabinogalaturonans. U.S. Patent No. 5,869,060, issued to Selvaraj et al., discloses hot water extracts of Nerium species and methods for their production, such as Example 2. The resulting extract can then be lyophilized to produce a powder. U.S. Patent No. 6,565,897 (U.S. Pre-Grant Publication No. 20020114852 to Selvaraj and PCT International Publication No. WO 2000/016793) discloses a hot water extraction process for preparing a substantially sterile extract. Erdemoglu et al. (J. Ethnopharmacol. (2003) Nov. 89(1), 123-129) disclose results comparing aqueous and ethanol extracts of plants, including oleander, based on their antinociceptive and anti-inflammatory activities. Adome et al. (Afr. Health Sci. (2003) Aug. 3(2), 77-86; ethanol extract), el-Shazly et al. (J. Egypt Soc. Parasitol. (1996) Aug. 26(2), 461-473; ethanol extract), Begum et al. (Phytochemistry (1999) Feb. 50(3), 435-438; methanol extract), Zia et al. (J. Ethnolpharmacol. (1995) Nov. 49(1), 33-39; methanol extract), and Vlasenko et al. (Farmatsiia. (1972) Sept.-Oct. 21(5), 46-47; alcohol extract) disclose organic solvent extracts of oleander. U.S. Pre-granted Patent Application Publication No. 20040247660 by Singh et al. discloses a protein-stabilized liposomal formulation for the preparation of oleandrin for the treatment of cancer. US Pregrant Patent Application Publication No. 20050026849 to Singh et al. discloses a water-soluble formulation of oleandrin containing cyclodextrin. US Pregrant Patent Application Publication No. 20040082521 to Singh et al. discloses a protein-stabilized nanoparticle formulation of oleandrin prepared from a hot water extract.

SCF extraction can be carried out in the presence of a modifier, for example an alcohol such as methanol, in a supercritical fluid to enhance the extraction of the desired compound from the plant material (PCT/US06/29061, filed July 26, 2005; US 7,402,325 and USSN 12/019435, filed January 4, 2008, or Newman et al. (Mol. Interven. (2008), 8, 36-49), the entire disclosures of which are incorporated herein by reference). The modifier typically has a volatility between that of the supercritical fluid and the compound being extracted, and they must be miscible with the supercritical fluid. In certain embodiments, the modifier is a liquid at ambient conditions. By way of example and not limitation, the modifier may be selected from the group consisting of ethanol, methanol, propanol, acetone, ethyl acetate, dichloromethane, and the like.

It is possible that the extracts also differ in their relative properties, as determined by efficacy in the assays included herein. Even so, if one or more pharmacologically active agents are present in the extract in sufficiently high amounts or concentrations to enable preparation of a therapeutically relevant dose, then the extract is considered part of the present invention.

Example 13 describes a chromatographic method for fractionating an SCF extract into five different fractions: O-H, O-2, O-3, O-4, and O-5. The fractions were prepared by loading the unfractionated extract onto an ODS-silica gel column equilibrated with water, then eluting the different fractions of the extract by sequentially passing multiple portions of an aqueous mobile phase with varying methanol contents (30%, 55%, 80%, and 100%) through the column, collecting the respective effluents (fractions), and concentrating the effluents by removing the solvent by evaporation under reduced pressure, thereby providing fractions O-1 (or O-H), O-2, O-3, O-4, and O-5. The fractions were analyzed according to Example 14, and their composition in terms of cardiac glycosides and other components was determined by thin-layer chromatography using sensitive dye indicators attached to (and therefore useful for detecting) cardiac glycosides. In addition, these fractions were analyzed for the presence or absence of cardiac glycosides using liquid chromatography/tandem mass spectrometry or DAD-UV detection.

The components or subcomponents of the extract can be analyzed by liquid chromatography using a stationary phase other than ODS-silica gel and/or by using a mobile phase other than water. Exemplary suitable stationary phases are further described herein.

Figures 8A-8D depict chromatograms obtained after HPLC analysis of fractions Fr-O-1, Fr-O-2, Fr-O3, and Fr-O-4 of Example 13. Based on comparison of retention times obtained using corresponding external reference samples, fractions (Fr-O-2 and Fr-O-3) were determined to contain oleandrin derivatives (cardiac glycosides), oleandrin (Rt = 8.3 min), and other unidentified components. The majority of oleandrin found in the original unfractionated SCF extract was primarily in the Fr-O-3 fraction. Fr-O-4 did not contain any quantifiable amounts of cardiac glycosides. Accordingly, the composition of the fractions varied depending on the oleandrin content, cardiac glycosides, and other unidentified components.

Components Oleandrin (Y/N) Other cardiac glycosides (Y/N) Neuroprotection (Y/N) O-H N N Y O-2 N Y N O-3 Y Y Y O-4(O-4A) N N Y O-5 N N N

These components were then subjected to the neuroprotective brain slice-based experiments detailed in Example 15 to determine the level of neuroprotection provided by each. The data are depicted in Figures 4A-4E, where the neuroprotective activity of an aqueous solution containing SCF extract (23 μg/mL) was compared with other solutions containing 0.03, 0.3, or 3 μg/mL of other ingredients. All components were weighed and compared on an equal weight basis. It was determined that the components containing oleandrin or cardiac glycosides (described herein) and certain components that did not contain oleandrin or cardiac glycosides could provide neuroprotection.

The performance of the O-4 fraction of the SCF extract in a brain slice-based stroke assay was compared to that of an unfractionated SCF extract (PBI-05204). The performance of the O-4 fraction at varying doses (0.03 to 300 μg/mL) was compared to that of a fixed dose of the extract (23 μg oleandrin mL). The data ( FIG5 ) clearly demonstrate that the O-4 fraction of the Nerium oleander SCF extract retains its efficacy despite not containing oleandrin or any other cardiac glycosides in detectable amounts. The light-colored bars in FIG5 represent significant differences relative to stroke symptoms (set to 0) based on an ANOVA followed by a Dunnett's post hoc comparison test at a 0.05 confidence level.

Accordingly, the present invention provides multiple therapeutic fractions of an extract of Nerium sp. or Nerium spp. selected from the group consisting of: a) a fraction comprising one or more pharmacologically active agents but not oleandrin and other cardiac glycosides, wherein the fraction provides neuroprotection; b) a fraction comprising one or more pharmacologically active agents, oleandrin, and one or more other cardiac glycosides, wherein the fraction provides neuroprotection; and c) a different fraction comprising one or more pharmacologically active agents (other than those in a) above) but not oleandrin and other cardiac glycosides, wherein the fraction provides neuroprotection.

The present invention also provides a method for fractionating an extract of an Nerium species or a Nerium species to provide one or more therapeutically active components thereof. The method comprises: a) providing an extract of an Nerium species or a Nerium species; b) fractionating the extract to provide two or more components of the extract, a first extract comprising one or more pharmacologically active agents (which are not cardiac glycosides) and not containing cardiac glycosides, and a second extract comprising one or more pharmacologically active agents (which are not cardiac glycosides) and one or more cardiac glycosides. In certain embodiments, fractionation is accomplished by liquid chromatography having a stationary phase and a mobile phase. In certain embodiments, the stationary phase comprises a medium selected from the group consisting of a "reversed phase" resin, an inert non-polar substance used for chromatography that achieves sufficient packing, such as a short carbon chain (C8 to C18) bound to silica gel, cyano-bound silica gel or phenyl-bound silica gel, an ion exchange resin (cationic or anionic), a "normal phase" resin, such as silica gel or an organic moiety having cyano and amino functional groups. In certain embodiments, the mobile phase comprises a solvent selected from the group consisting of water, methanol, ethanol, acetonitrile, tetrahydrofuran, an aqueous buffer solution, or a mixture thereof. In certain embodiments, the mobile phase comprises aqueous methanol, wherein the methanol content increases sequentially from about 30% to 100%, and the stationary phase is ODS-silica gel. Chromatography can be performed using a gradient elution mobile phase, a step elution mobile phase, or a stationary composition mobile phase.

The extract components can be sub-fractionated to provide two or more different sub-fractions of the extract components. Sub-fractionation can be completed by liquid chromatography of the components. Suitable stationary phases for liquid chromatography can include silica gel or other resins such as ion exchange media, aluminum oxide or non-bound C18 materials, and suitable mobile phases for liquid chromatography can include a combination of two or more organic solvents with different polarities: an organic solvent with less polarity and an organic solvent with greater polarity. Suitable polar organic solvents can be tetrahydrofuran, dichloromethane, ethyl acetate, acetone, dimethylformamide, acetonitrile, n-butanol, isopropyl alcohol, n-propanol, ethanol, methanol, acetic acid and water. Suitable non-polar organic solvents can be ethyl acetate pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane, chloroform or ether.

Buffers for use in buffered solutions include those known in the art of liquid chromatography. Exemplary buffers include those containing phosphate, acetate, citrate, formates, phosphates, trifluoroacetic acid, chloroacetate, sulfonates, alkylamines, TAE, TBE, ammonia, BuffAR, carbonates, HEPES, MES, thiocyanates, CAPS, CHES, guanidine, MOPS, PIPES, TRIS, sulfates, hydroxides, alkali metal halides, trimethylglycine or amino acid ions or combinations thereof. One or more ion pairing agents and/or one or more organic modifiers may also be included in the mobile phase.

The other types of chromatography that can be used for fractionation extract comprise size exclusion chromatography, normal phase chromatography, ion exchange chromatography, hydrophobic interaction chromatography or their combination.Also may use the combined form of different types of chromatograms.Stationary phase can be comprised as the medium of the combination of two or more different media, and described two or more different media are used for reversed-phase chromatography, size exclusion chromatography, ion exchange chromatography or hydrophobic interaction chromatography, for example the combination of reversed-phase stationary phase and size exclusion stationary phase, the combination of reversed-phase stationary phase and ion exchange stationary phase or other such combinations or two, three or four different stationary phase media.Stationary phase medium can be porous, non-porous, surface porous, dispersive porous or fully porous.

The present invention provides a method for fractionating an extract, comprising: a) providing an extract obtained from a species of the genus Nerium or a species of the genus Oleander; b) fractionating the extract by column chromatography using ODS-silica gel as a stationary phase and aqueous methanol as a mobile phase to provide at least two different components: a first component comprising at least one cardiac glycoside and at least one pharmacologically active agent other than a cardiac glycoside, and another component comprising no cardiac glycoside and at least one pharmacologically active agent other than a cardiac glycoside; c1) subfractionating another component of b) by column chromatography using silica gel as a stationary phase and a mixture of at least two organic solvents having different polarities as a mobile phase to provide at least two different subfractions: a subfraction comprising one or more steroids and one or more triterpenes, and another subfraction comprising two or more different triterpenes and no steroids, wherein the subfractions do not comprise a cardiac glycoside.

In certain embodiments, the method further comprises: c2) subfractionating the first component of b) by column chromatography using silica gel as the stationary phase and a mixture of at least two organic solvents of different polarity as the mobile phase to provide at least two different subfractions: a first subfraction comprising one or more steroids and one or more triterpenes, and another subfraction comprising two or more different triterpenes and no steroids, wherein either or both of the subfractions further comprise a cardiac glycoside.

The extract or its components or subcomponents can be formulated into any suitable pharmaceutically acceptable dosage form. Parenteral dosage forms, ear dosage forms, eye dosage forms, nasal dosage forms, inhalable dosage forms, oral dosage forms, sublingual dosage forms, enteral dosage forms, topical dosage forms, oral dosage forms, oral dosage forms and injectable dosage forms are particularly useful. Specific dosage forms include fixed or liquid dosage forms. Exemplary suitable dosage forms include tablets, capsules, pills, caplets, lozenges, sachet (sache), solution, suspension, dispersion, vial (vial), bag, bottle, injection liquid, i.v. (intravenous), i.m. (intramuscular) or i.p. (intraperitoneal) administrable liquid and other such dosage forms known to those skilled in the art of pharmacy.

The amount of the extract or its components or subcomponents contained in the medicament of the present invention will be at least one or more dosage forms and selected according to known pharmaceutical principles. In particular, an effective amount or a therapeutically relevant amount of the therapeutic compound is expected. By the term "effective amount", it is understood that a pharmaceutically effective amount is expected relative to, for example, a drug. A pharmaceutically effective amount is an amount or mass of active ingredient sufficient for the required or desired therapeutic response, in other words, an amount sufficient to cause a perceptible biological response when administered to a patient. This perceptible biological response can occur due to the administration of a single or multiple doses of the active substance. The dose may comprise one or more dosage forms. It will be understood that for any patient, a specific dosage level will depend on a variety of factors, including the indication being treated, the severity of the indication, the patient's health status, age, sex, weight, diet, pharmacological response, the specific dosage form administered, and other such factors.

For oral administration, the desired dosage is up to 5 dosage forms, although as few as one dosage form and as many as ten dosage forms can be taken as a single dose. Exemplary dosage forms contain 0.1 to 5 mg of SCF extract per dosage form, suitable for a total of 0.1 to 500 mg per dose (1 to 10 dose levels). The dosage will be administered according to a dosing regimen that is predetermined and/or adjusted to achieve a specific therapeutic response or clinical benefit in the subject.

For the application of treatment mammals, extract or its component or subcomponent can be included in dosage form.Some embodiment of dosage form is not enteric, and releases their extract load in 0.5 to 1 hour or shorter time.Some embodiment of dosage form is enteric, and for example releases their cardiotonic load from jejunum, ileum, small intestine and/or large intestine (colon) in the downstream of stomach.Enteric coating formulation will release extract into systemic circulation in 1-10 hour after oral administration.

It should be noted that the compounds herein may have one or more functions in the formulations of the present invention. For example, a compound may act as both a surfactant and a water-miscible solvent, or as both a surfactant and a water-insoluble solvent.

The liquid composition may comprise one or more pharmaceutically acceptable liquid carriers. The liquid carrier may be aqueous, non-aqueous, polar, non-polar and/or organic. By way of example and not limitation, the liquid carrier includes water-miscible solvents, water-immiscible solvents, water, buffers and combinations thereof.

As used herein, the interchangeable terms "water-soluble solvent" or "water-miscible solvent" refer to an organic liquid that does not form a two-phase mixture with water, or an organic liquid that is sufficiently water-soluble to provide an aqueous solvent mixture containing at least five percent solvent without liquid phase separation. The solvent is suitable for administration to humans or animals. By way of example and not limitation, exemplary water-soluble solvents include PEG (polyethylene glycol), PEG 400 (polyethylene glycol with an approximate molecular weight of about 400), ethanol, acetone, alkanones, alcohols, ethers, propylene glycol, glycerol, triacetin, polypropylene glycol, PVP (polyvinyl pyrrolidone), dimethyl sulfoxide, N,N-dimethylformamide, formamide, N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

As used herein, the terms "water-insoluble solvent" or "water-immiscible solvent," which are used interchangeably, refer to an organic liquid that forms a biphasic mixture with water or undergoes phase separation when the concentration of the solvent in water exceeds five percent. The solvent is suitable for administration to humans or animals. By way of example and not limitation, exemplary water-insoluble solvents include medium/long chain triglycerides, oils, castor oil, corn oil, vitamin E, vitamin E derivatives, oleic acid, fatty acids, olive oil, softisan 645 (Diglyceryl Caprylate/Caprate/Stearate/Hydroxystearate Adipate), migliol, captex (Captex 350: Tricaprylate/Caprate/Laurate triglyceride, Captex 355: Glyceryl Tricaprylate/Caprate triglyceride, Captex 355EP/NF: Glyceryl Tricaprylate/Caprate medium chain triglyceride).

Suitable solvents are listed in the "International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Guidance for Industry Q3C Impurities: Residual Solvents" (1997), which provides recommendations on what amounts of residual solvents are considered safe in pharmaceuticals. Exemplary solvents are listed as Class 2 or Class 3 solvents. Class 3 solvents include, for example, acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, methyl tert-butyl ether, cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, or propyl acetate.

Other materials that can be used as water-immiscible solvents in the present invention include: Captex 100: Propylene Glycol Dicaprylate; Captex 200: Propylene Glycol Dicaprylate/Dicaprate; Captex 200P: Propylene Glycol Dicaprylate/Dicaprate; Propylene Glycol Dicaprylocaprate; Captex 300: Glyceryl Tricaprylate/Caprate; Captex 300EP/NF: Caprylic/Capric Medium Chain Triglycerides; Captex 350: Caprylic/Capric/Lauric Triglycerides; Captex 355: Caprylic/Capric Triglycerides; Captex 355EP/NF: Caprylic/Capric Medium Chain Triglycerides; Captex 500: Glyceryl Triacetate; Captex 500P: Glyceryl Triacetate (Pharmaceutical Grade); Captex 800: Propylene Glycol Bis(2-Hexyl Acetate); Captex Capmul 810D: Caprylic/Capric/Linoleic Triglyceride; Captex 1000: Tricaprin; Captex CA: Medium Chain Triglycerides; Captex MCT-170: Medium Chain Triglycerides; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50EP/NF: Glyceryl Monooleate; Capmul MCM: Medium Chain Mono &Diglycerides; Capmul MCM C8: Glyceryl Monocaprylate; Capmul MCM C10: Glyceryl Monocaprate; Capmul PG-8: Propylene Glycol Monocaprylate; Capmul PG-12: Propylene Glycol Monolaurate; Caprol 10G10O: Decaglyceryl Decaoleate; Caprol 3GO: Triglyceryl Monooleate; Caprol ET: Polyglyceryl Esters of Mixed Fatty Acids; Caprol MPGO: Hexaglyceryl Dioleate; Caprol PGE 860: Decaglyceryl Monooleate, Decaglyceryl Dioleate.

As used herein, "surfactant" refers to a compound that contains a polar or charged hydrophilic portion and a nonpolar hydrophobic (lipophilic) portion; that is, the surfactant is amphiphilic. The term surfactant can refer to a single compound or a mixture of compounds. A surfactant can be a solubilizer, an emulsifier, or a dispersant. A surfactant can be hydrophilic or hydrophobic.

The hydrophilic surfactant can be any hydrophilic surfactant suitable for use in pharmaceutical compositions. Such surfactants can be anionic, cationic, zwitterionic or nonionic, although nonionic hydrophilic surfactants are currently preferred. As mentioned above, these nonionic hydrophilic surfactants will typically have an HLB value greater than about 10. Mixtures of hydrophilic surfactants are also within the scope of the present invention.

Similarly, the hydrophobic surfactant can be any hydrophobic surfactant suitable for use in pharmaceutical compositions. Typically, suitable hydrophobic surfactants have an HLB value of less than about 10. Mixtures of hydrophobic surfactants are also within the scope of the present invention.

Examples of other suitable solubilizers include: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediol and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, reducing glycols, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycol having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (tetrahydrofuran glycol ether, commercially available from BASF under the trade name Tetraglycol) or methoxy PEG (Union Carbide Corporation); Carbide); amides such as 2-pyrrolidone, 2-piperidone, caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esters such as ethyl propionate, tributyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, triethyl citrate, ethyl oleate, ethyl octanoate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, caprolactone and its isomers, valerolactone and its isomers, butyrolactone and its isomers; and other solubilizers known in the art, such as dimethylacetamide, isosorbide dimethyl ether (Arlasolve DMI (ICI)), N-methylpyrrolidone (Pharmasolve (ISP)), monooctanoin, diethylene glycol monoethyl ether (available from Gattefosse under the trade name Transcutol) and water. Mixtures of solubilizing agents are also within the scope of the present invention.

Unless otherwise indicated, the compounds referred to herein are readily available from standard commercial sources.

A transparent liquid composition will be visually clear to the unaided eye in that it will contain less than 5%, less than 3%, or less than 1% by weight of suspended solids based on the total weight of the composition.

Although not necessary, the compositions or kits of the present invention may contain chelating agents, preservatives, antioxidants, adsorbents, acidifiers, alkalizers, antifoaming agents, buffers, colorants, electrolytes, salts, stabilizers, osmotic modifiers, diluents, other pharmaceutical excipients, or combinations thereof.

As used herein, the term "antioxidant" means an agent that inhibits oxidation and is therefore used to prevent the degradation of an article by oxidative processes. By way of example and not limitation, such compounds include ascorbic acid, ascorbyl palmitate, vitamin E, vitamin E derivatives, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metalbisulfite, and other such materials known to those skilled in the art.

As used herein, the term chelating agent refers to a compound that chelates metal ions in solution. Exemplary chelating agents include EDTA (tetrasodium ethylenediaminetetraacetic acid), DTPA (pentasodium diethylenetriaminepentaacetic acid), HEDTA (trisodium salt of N-(hydroxyethyl)-ethylene-diaminetriacetic acid), NTA (trisodium nitrilotriacetic acid), disodium ethanoldiglycinate ( _Na2EDG ), sodium diethanolglycinate (DEGNa), citric acid, and other compounds known to those skilled in the art.

As used herein, the term "adsorbent" means an agent capable of holding other molecules on its surface by physical or chemical (chemisorption) means. By way of example and not limitation, such compounds include powdered activated carbon and other materials known to those skilled in the art.

As used herein, the term "alkalizing agent" means a compound used to provide an alkaline medium. By way of example and not limitation, such compounds include ammonia, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and other compounds known to those skilled in the art.

As used herein, the term "acidifying agent" means a compound used to provide an acidic medium. By way of example and not limitation, such compounds include acetic acid, amino acids, citric acid, fumaric acid and other α-hydroxy acids, hydrochloric acid, ascorbic acid, and other compounds known to those skilled in the art.

As used herein, the term "defoaming agent" means a compound that prevents or reduces the amount of foam formed on the surface of a filling composition. By way of example and not limitation, suitable defoaming agents include dimethicone, simethicone, octylphenol polyether, and other defoaming agents known to those skilled in the art.

As used herein, the term "buffer" means a compound used to counteract changes in pH upon dilution or the addition of an acid or base. By way of example and not limitation, such compounds include potassium metaphosphate, potassium phosphate, monobasic sodium acetate, and anhydrous and dehydrated sodium citrate, and other such materials known to those skilled in the art.

As used herein, the term "diluent" or "filler" means an inert substance that acts as a filler to create the desired bulk, flowability, and compression characteristics in the preparation of tablets and capsules. By way of example and not limitation, such compounds include dibasic calcium phosphate, kaolin, lactose, sucrose, mannitol, microcrystalline cellulose, powdered cellulose, precipitated calcium carbonate, sorbitol, and starch, and other materials known to those skilled in the art.

As used herein, the term "preservative" means a compound used to prevent the growth of microorganisms. By way of example and not limitation, such compounds include benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, phenylmercuric acetate, thimerosal, m-cresol, meperidinium chloride, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, sorbic acid, thymol, and methyl, ethyl, propyl, or butyl benzoates, and other compounds known to those skilled in the art.

As used herein, the term "colorant" means a compound used to impart color to a pharmaceutical product. By way of example and not limitation, such compounds include FD&C Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C Blue No. 2, FD&C Green No. 5, FD&C Orange No. 5, FD&C Red No. 8, caramel, and iron oxides (black, red, and yellow), other FD&C dyes, and natural colorants such as grape skin extract, beet red powder, beta-carotene, tartrate, carmine, turmeric, paprika, combinations thereof, and other such materials known to those skilled in the art.

As used herein, the term "stabilizer" means a compound used to stabilize an active agent against physical, chemical, or biochemical processes that would otherwise reduce the therapeutic activity of the active agent. By way of example and not limitation, suitable stabilizers include albumin, sialic acid, creatinine, glycine and other amino acids, nicotinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycol, sodium caprylate, and sodium saccharin, and other stabilizers known to those skilled in the art.

As used herein, the term "osmotic modifier" means a compound used to adjust the osmotic pressure of a liquid formulation. Suitable osmotic modifiers include glycerol, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose, and other osmotic modifiers known to those skilled in the art.

The compositions of the present invention may also include oils such as fixed oils, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil; fatty acids such as oleic acid, stearic acid, and isostearic acid; and fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. The compositions may also include alcohols such as ethanol, isopropyl alcohol, cetyl alcohol, glycerol, and propylene glycol; glycerol ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol; ethers such as polyethylene glycol 450; petroleum hydrocarbons such as mineral oil and petrolatum; water; a pharmaceutically suitable surfactant, suspending agent, or emulsifier; or mixtures thereof.

It should be understood that compounds used in the field of pharmaceutical formulations often provide multiple functions or purposes. Therefore, if a compound specified herein is only mentioned once or is used to define more than one term herein, its purpose or function should not be construed as being limited to the specified purpose or function.

One or more ingredients in the formula can exist in the form of its free radical or pharmaceutically or analytically acceptable salt. As used herein, "pharmaceutically or analytically acceptable salt" refers to a compound that has been modified by reacting the compound with the required acid to form an ion-bound pair. Examples of acceptable salts include conventional non-toxic salts formed, such as those formed from non-toxic inorganic or organic acids. Suitable non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, sulfamic acid, phosphoric acid, nitric acid, and other inorganic acids known to those skilled in the art. Salts prepared from organic acids such as amino acids, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetylbenzoic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and other organic acids known to those skilled in the art. Lists of other suitable salts are found in Remington's Pharmaceutical Sciences, ^17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the relevant disclosure of which is incorporated herein by reference.

The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and/or dosage forms which are suitable, within sufficient medical determination, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or any other problem or complication commensurate with a reasonable benefit/risk ratio.

The dosage forms can be prepared by any conventional method known in the pharmaceutical industry. A liquid dosage form can be prepared by providing at least one liquid carrier and oleandrin or an oleandrin-containing extract to a container. One or more other excipients can be included in the liquid dosage form. A solid dosage form can be prepared by providing at least one solid carrier and oleandrin or an oleandrin-containing extract. One or more other excipients can be included in the solid dosage form.

The dosage form can be packaged using conventional packaging equipment and materials. It can be contained in a bag, bottle, vial, bag, syringe, envelope, packet, blister, box, ampoule or other such container.

The present invention includes a method for improving the clinical status of a statistically significant number of subjects in a population with a neurological condition, the method comprising: administering an extract of an Nerium species or a Nerium spp., or a combination thereof, to the population of subjects; and determining the clinical status of the subjects to establish an improved clinical status. In certain embodiments, the statistically significant number is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 9% of the population. In certain embodiments, the extract comprises one or more other pharmacologically active compounds. In other embodiments, the extract comprises one or more other pharmacologically active compounds, including those that cooperate with oleandrin or other cardiac glycosides to improve the clinical status of the subject.

Based on the above description and the following examples, those skilled in the art will be able to practice the present invention without undue experimentation. The foregoing will be better understood with reference to the following examples, which detail specific processes for preparing/making embodiments of the present invention. All references to these examples are for illustrative purposes only. The following examples should not be considered exhaustive, but rather illustrate only a few of the many embodiments contemplated by the present invention.

Oleandrin can be purchased from Sigma Chemical (St. Louis, MO).

Example 1: Supercritical Fluid Extraction of Powdered Oleander Leaves

Method A uses carbon dioxide

Powdered oleander leaf was prepared by harvesting, washing and drying oleander leaf material, followed by passing the oleander leaf material through comminution and dewatering equipment such as those described in US Patents 5,236,132, 5,598,979, 6,517,015 and 6,715,705. The weight of the starting material used was 3.94 kg.

In an extractor unit, the starting material is combined with pure _CO₂ at a pressure of 300 bar (30 MPa, 4351 psi) and a temperature of 50°C (122°F). A total of 197 kg _{of CO₂} is used, resulting in a solvent to raw material ratio of 50:1. The mixture of _CO₂ and raw material is then passed through a separator unit, which changes the pressure and temperature of the mixture and separates the extract from the carbon dioxide.

The extract (65 g) was obtained as a brown, viscous material with a pleasant aroma. The color is likely due to chlorophyll. To determine the exact yield, the test tube and separator were rinsed with acetone, and the acetone was evaporated to obtain an additional 9 g of extract. The total amount of extract was 74 g. Based on the weight of the starting material, the yield of the extract was 1.88%. The content of oleandrin in the extract was calculated by high pressure liquid chromatography and mass spectrometry to be 560.1 mg, or a yield of 0.76%.

Method B uses a mixture of carbon dioxide and ethanol

Powdered oleander leaf was prepared by harvesting, washing and drying oleander leaf material, followed by passing the oleander leaf material through comminution and dewatering equipment such as those described in US Patents 5,236,132, 5,598,979, 6,517,015 and 6,715,705. The weight of the starting material used was 3.85 kg.

In an extractor unit, the starting material is combined with pure _CO₂ and 5% ethanol at a pressure of 280 bar (28 MPa, 4061 psi) and a temperature of 50°C (122°F). A total of 160 kg _CO₂ and 8 kg ethanol are used, resulting in a solvent-to-raw material ratio of 43.6 to 1. The mixture of _CO₂ , ethanol, and raw material is then passed through a separator unit that changes the pressure and temperature of the mixture and separates the extract from the carbon dioxide.

After removing the ethanol, an extract (207 g) was obtained as a dark green, viscous substance which clearly contained some chlorophyll. The yield of the extract was 5.38% based on the weight of the starting material. The content of oleandrin in the extract was calculated by high pressure liquid chromatography and mass spectrometry to be 1.89 g, or a yield of 2.1%.

Example 2: Hot water extraction of powdered oleander leaves

Hot water extraction is typically used to extract oleandrin and other active ingredients from oleander leaves. Examples of hot water extraction methods are found in U.S. Patents 5,135,745 and 5,869,060.

Hot water extraction was performed using 5g of powdered oleander leaves. Ten volumes of boiling water (based on the weight of the oleander starting material) were added to the powdered oleander leaves and the mixture was stirred for 6 hours. The mixture was then filtered, and the leaf residue was collected and extracted again under the same conditions. The filtrates were combined and freeze-dried. The extract was brown. The dried extract weighed approximately 1.44g. 34.21mg of the extract material was dissolved in water and analyzed for oleandrin content using high pressure liquid chromatography and mass spectrometry. The amount of oleandrin was determined to be 3.68mg. Based on the amount of extract, the oleandrin yield was calculated to be 0.26%. The following table shows a comparison between the oleandrin yields of the two supercritical carbon dioxide extractions and the hot water extraction for Example 1.

Yield comparison

Extraction medium Oleandrin yield based on the total weight of the extract Supercritical Carbon Dioxide: Example 1, Method A 0.76% Supercritical Carbon Dioxide: Example 1, Method B 2.1% Hot water extraction: Example 2 0.26%

Example 3: Treatment of Neurological Disorders Including but Not Limited to Alzheimer's Disease

Method A Extract Therapy

A prescription for a cardiac glycoside is prescribed for a subject exhibiting Alzheimer's disease, and a therapeutically relevant dose is administered to the subject for a period of time according to the prescribed dosage regimen. The level of the subject's therapeutic response is regularly measured. If the level of therapeutic response is too low at one dose, the dose is gradually increased according to a predetermined dose escalation schedule until the desired level of therapeutic response is achieved in the subject. Treatment of the subject with the extract, or its components or subcomponents, is continued as needed, and the dosage or dosage regimen may be adjusted as needed until the patient reaches the desired clinical endpoint.

Method B Combination therapy: extract and another therapeutic agent

Following Method A above, in addition to prescribing and administering one or more other therapeutic agents to the subject for treating Alzheimer's disease or its symptoms, the one or more other therapeutic agents may be administered before, after, or concurrently with the extract. The dosage of the one or more other therapeutic agents may also be gradually increased (or decreased). Suitable one or more other therapeutic agents include memantine ^HCl , Aricept ^™ (donepezil), galantamine ^™ , Exelon ^™ (rivastigmine), Cognex ^™ (tacrine), and amantadine.

Example 4: Treatment of Neurological Disorders Including but Not Limited to Huntington's Disease

Method A Extract Therapy

The extract is prescribed to a subject exhibiting Huntington's disease, and a therapeutically relevant dose is administered to the subject according to the prescribed dosing regimen for a period of time. The subject's level of therapeutic response is periodically measured. If the level of therapeutic response is too low at one dose, the dose is gradually increased according to a predetermined dose escalation schedule until the desired level of therapeutic response is achieved in the subject. Treatment of the subject with the extract continues as needed, and the dose or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint. The doses administered may be similar to those of Example 3, or as otherwise described herein.

Method B Combination therapy: extract and another therapeutic agent

Following Method A above, in addition to prescribing and administering to the subject one or more other therapeutic agents for treating Huntington's disease or its symptoms. The one or more other therapeutic agents may be administered before, after, or simultaneously with the cardiac glycoside. The dose of the one or more other therapeutic agents may also be gradually increased (or gradually decreased). Suitable one or more other therapeutic agents include vitamin E, baclofen (a derivative of CoQ10), lamotrigine (an anticonvulsant), remaramide (an anesthetic that is a low-affinity NMDA antagonist), and riluzole (a Na ion channel antagonist).

Example 5: Treatment of Neurological Disorders Including but Not Limited to Ischemic Stroke

Method A Extract Therapy

The extract is prescribed for a subject experiencing ischemic stroke, and a therapeutically relevant dose is administered to the subject for a period of time according to the prescribed dosage regimen. The level of the subject's therapeutic response is regularly measured. If the level of therapeutic response is too low at one dose, the dose is gradually increased according to a predetermined dose escalation schedule until the desired level of therapeutic response is achieved in the subject. Treatment of the subject with the extract is continued as needed, and the dose or dosage regimen may be adjusted as needed until the patient reaches the desired clinical endpoint. The dosage administered may be similar to that of Example 3, or as otherwise described herein.

Method B Combination therapy: extract and another therapeutic agent

Following Method A above, in addition to prescribing and administering to the subject one or more other therapeutic agents for treating ischemic stroke or its symptoms. The one or more other therapeutic agents may be administered before, after, or simultaneously with the extract. The dosage of the one or more other therapeutic agents may also be gradually increased (or gradually decreased).

Example 6: HPLC analysis of a solution containing oleandrin

Samples (oleandrin standard, SCF extract, and hot water extract) were analyzed on an HPLC (Waters) using the following conditions: a Symmetry C18 column (5.0 μm, 150 × 4.6 mm ID; Waters); a mobile phase of methanol:water (54:46 v/v) and a flow rate of 1.0 ml/min. The detection wavelength was set at 217 nm. Samples were prepared by dissolving the compound or extract in a fixed amount of HPLC solvent to achieve the approximate target concentration of oleandrin.

Example 7: Determination of α3 and α1 Expression in Normal Nervous Tissue

The procedure set forth in PCT International Application No. PCT/US08/82641, filed on November 6, 2008, in the name of Phoenix Biotechnology, Inc., the entire disclosure of which is incorporated herein by reference, may be followed.

Example 8: In vitro tests on cardiac glycosides and the extracts of the present invention in stroke and non-stroke

Assessment

Method A Stroke: Preparation of Cortical Brain Slices and OGD

Neocortical brain slices were prepared from PND 7 Sprague-Dawley newborn rats. The cerebral cortex was dissected, cut into 400 μ thick slices, and transferred to a container containing cold artificial cerebrospinal fluid containing 1 μM MK-801 before patching; MK-801 was not included in any subsequent procedures. To simulate ischemic injury using transient oxygen-glucose deprivation (OGD), slices from each cerebral hemisphere were exposed to glucose-free, _N2 -bubbled artificial cerebrospinal fluid in a low _O2 (0.5%) environment for 7.5 min. OGD slices were then slid side by side with control slices from the contralateral hemisphere onto nitrocellulose or Millicell (Millipore) permeable membranes, which were prepared identically except for the absence of OGD. Thirty minutes after slitting, paired brain slices were transfected, transferred to 24-well plates, and incubated in a humidified chamber at 37°C, 5% _CO2 . In each experiment, 5-6 minutes of oxygen-glucose deprivation (OGD) was used to cause a loss of >50% of healthy cortical cells within 24 hours. A set concentration (3 μM) of oleandrin (cardiac glycoside) was used as an internal positive control. For oleandrin (cardiac glycoside), all three concentrations from 0.3 to 3 μM were shown to provide neuroprotection in the first two rounds of experiments, so the oleandrin concentration tested was reduced in the third round, suggesting that the threshold concentration for neuroprotection is between 0.1 and 0.3 μM. Unfractionated extracts, such as unfractionated extracts of Nerium species, or components thereof, may also be used as described for oleandrin.

Method B Non-stroke: Brain slice test

Oleandrin and PBI-05204 (an unfractionated SCF extract of Nerium oleander) were tested on "non-stroke" brain slices; that is, slices that had been sliced and transfected with YFP but had not undergone the additional trauma of OGD. See the experimental procedures outlined above. We have observed that many neuroprotective compounds, including scutellarin, can provide modest levels of neuroprotection to these brain slices, presumably by protecting against the trauma caused by the slicing process and culture itself. The data demonstrate that oleandrin and the extract were shown to provide similar levels of neuroprotection to scutellarin in such "non-OGD" brain slices, suggesting that cardiac glycosides mediate neuroprotection even in the absence of oxygen and glucose deprivation.

Example 9: Evaluation of cardiac glycosides and extracts in an in vitro assay for Alzheimer's disease

In a rat brain slice model targeting APP/Aβ-induced cortical pyramidal neuron degeneration, biolistic transfection of essential markers, such as YFP, along with disease gene constructs, was used to introduce the same neuronal population into the brain slice. Thus, the APP/Aβ brain slice model co-transfected with YFP and APP isoforms resulted in progressive degeneration of cortical pyramidal neurons within three to four days following brain slice preparation and transfection. The data demonstrated that both oleandrin and PBI-05204, an unfractionated SCF extract of Nerium oleander, were able to provide concentration-dependent neuroprotection in APP-transfected brain slices, resulting in rescue levels approaching those provided by BACE inhibitor drugs.

Example 10: Evaluation of Cardiac Glycosides and Extracts in an In Vitro Corticostriatal Co-culture Assay for Huntington's Disease

In this experiment, instead of using intact brain slices, mutant htt proteins were introduced into high-density, mixed co-cultures of cortical neurons, striatal neurons, and glia arranged in 96-well plates by electroporation. The goal of this experimental platform is to combine the biological/clinical relevance of complex primary culture systems that recapitulate the key aspects of the interconnectedness of disease-related neuronal populations in vivo with the ability to perform large-scale, fully automated screening. In this experiment, the transfected mutant htt construct caused progressive degeneration of both striatal neurons and pyramidal neurons over a period of 1-2 weeks in vitro, after which the neurons were quantified on the Cellomics Arrayscan VTI platform using automated image acquisition and object detection algorithms. Each data point was obtained from 6 wells, each with 16 images captured, processed, and analyzed on the Cellomics Arrayscan using a program developed during large-scale screening in conjunction with the Cure Huntington's Disease Initiative. In a complete round, approximately 25,000 images were collected and analyzed per cycle, with 4 cycles per week.

Cortex-striatum co-culture experimental platform

Pure glial cultures are prepared before neuron plating to establish 96-well plates with confluent glial beds. Cortical and striatal tissues are then individually isolated and "nucleofected" with appropriate DNA constructs, which can then be distinguished by the expression of different fluorescent proteins such as YFP, CFP, and mCherry. These individually transfected cortical and striatal neurons are then thoroughly mixed and plated into 96-well plates containing previously seeded glial monolayers.

Both oleandrin and PBI-05204 (a supercritical _CO₂ extract of Nerium oleander) were tested in this cortex-striatum co-culture platform. Preliminarily, these compounds appear to be the strongest targets we have observed to date among the >400 late-stage drug molecules evaluated in this assay system. For comparison, a dose-response graph for KW6002 (an adenosine 2a receptor antagonist) is included, along with compounds that we routinely include as positive controls in this co-culture assay. Oleandrin's efficacy was comparable to that of KW6002, however, its potency appeared approximately 100-fold higher (Figures 3A-3D).

Example 11: Evaluation of components of the SCF extract of Nerium oleander in an in vitro APP assay for Alzheimer's disease

The components were prepared according to Example 13. The experiment was performed similarly to Example 9. The data in Figure 6 demonstrates a concentration-dependent effect of component O-4A in preventing neurodegeneration associated with the introduction of the APP construct. In particular, the data demonstrate neuroprotection in the concentration range of 3 to 30 μg/mL.

Example 12: Evaluation of components of Nerium oleander SCF extract in an in vitro tau4R assay for Alzheimer's disease

The components were prepared according to Example 13. The data in Figure 7 demonstrate a concentration-dependent effect of component O-4A in preventing neurodegeneration associated with the introduction of a Tau construct. Specifically, the data demonstrate neuroprotection across a concentration range of 3 to 30 μg/mL. Significant differences were observed between cells treated with the Tau construct and cells exposed to a solution of component O-4A.

Example 13: Chromatographic fractionation of SCF extract

A supercritical extract of oleander leaves (5 g) (obtained as described herein by extracting plant matter with a mixture of supercritical CO ₂ and ethanol added as a cosolvent/modifier) was suspended in water (150 mL) and partitioned three times with hexane (150 mL each). The aqueous layer was fractionated on an ODS C-18 (octadecyl-functionalized silica gel, labeled 20-22%, 200-400 mesh) open column (400 mm (L) x 38 mm (ID)) by loading the aqueous layer directly onto a bed of water-equilibrated ODS resin. The column was treated sequentially with a mixture of water and methanol (30% methanol in water, 1000 mL; 55% methanol in water, 1000 mL; 80% methanol in water, 1000 mL; 100% methanol in water, 1000 mL) and an acetone:methanol mixture (2 volume: 1 volume; 1000 mL). The effluent from each mixture (1000 mL) was collected. The solvent was removed from each fraction by evaporation to yield five fractions, namely Fr-O-1, Fr-O-2, Fr-O-3, Fr-O-4 and Fr-O-5. The fractions were then analyzed by HPLC chromatography as in Example 14.

Example 14: HPLC Analysis of SCF Extract Components

The purpose of this experiment was to identify the extract fractions (from the above) that contained cardiac glycosides. Samples of each fraction obtained according to Example 13 were analyzed as follows. 1-3 mg of each fraction was dissolved in 1-5 mL of aqueous methanol (80% methanol in water). The diluted samples (10-25 μL) were analyzed using an Agilent Zorbax SB-C18 column using 80% methanol in water as the mobile phase at a flow rate of 0.7 mL/min and DAD-UV effluent monitoring at the following wavelengths: 203, 210, 217, 230, 254, 280, 310, and 300 nm.

Example 15: Brain slice assay for determining neuroprotection provided by extract components

The experiment was performed according to Example 8. The data demonstrated that PBI-05204 provided significant levels of protection compared to untreated stroke (OGD)-induced neuronal damage in brain slices. Similar levels of protection were provided by component O-4A (FIGS. 4A and 5), as well as component O-3 (FIG. 4C) and component O-1 (FIG. 4D). In contrast, components O-2 (FIG. 4B) and O-5 (FIG. 4E) demonstrated no neuroprotective effect in the OGD model of ischemic brain injury caused by stroke.

Example 16: Time-Delayed Brain Slice Assay for Determining Neuroprotection

The study was conducted according to Example 8, except for the following changes. A specific duration was allowed between OGD and the introduction of the proposed neuroprotectant. The ability of PBI-05201 to provide neuroprotection to brain slices was determined if treatment was delayed relative to the time of OGD treatment. The data showed that a 2-hour delay in the administration of oleander extract was well tolerated, showing a similar level of neuroprotection to that obtained by administering PBI-05204 immediately after OGD treatment. The neuroprotective benefit decreased when PBI-05204 was administered with a 4- to 6-hour delay, but was still at a significant and physiologically relevant level of neuroprotection.

Example 17: Identification of compounds in the fractions of the Nerium oleander SCF extract obtained according to Example 13

The water and methanol in the Fr-O-4 fraction were removed by evaporation under reduced pressure. The residue from the Fr-O-4 fraction of Example 13 was subjected to silica gel chromatography (below) to provide subfractions, which were then analyzed by thin layer chromatography (TLC). Fractions with similar TLC characteristics were combined and their solvents were removed by evaporation under reduced pressure. The remaining residue was analyzed by HNMR.

Thin layer chromatography

TLC was performed on conventional analytical grade TLC plates using a mixture of hexane:ethyl acetate (7:3 _v :v). Compounds were visualized using _H2SO4 , with the steroid appearing blue and the triterpenes appearing purple.

Before further fractionation by flash chromatography, TLC analysis of the Fr-O-4 fraction showed the presence of one major spot and more than five minor spots. The color reaction showed that the major spot contained a mixture of steroids and triterpenes, and most of the minor spots contained steroids.

Silica gel flash chromatography

Silica gel (Biotage; 10-15 g) was packed into a column and equilibrated with a mixture of ethyl acetate (3%) and hexane (97%). The residue from the Fr-O-4 fraction was placed in a mixture of ethyl acetate (3%) and hexane (97%) (0.2-0.5 mL) and loaded onto the column. Flash chromatography was performed using a solvent gradient of ethyl acetate (3%-30%) in hexane (97%-70%, respectively) followed by 100% methanol. The subfractions collected from the column were analyzed by TLC (above), and those fractions with similar TLC color characteristics were combined and concentrated to remove the solvent.

HNMR chromatography

A sample of each concentrated subfraction from the flash chromatography was analyzed by HNRM using conventional methods to determine the structural type of the major component.

Example 18: Determination of the SCF of the Oleander obtained according to Example 1 (Method B) in unfractionated form

Compounds in the extract

The SCF extracts were analyzed by MS-DART TOF analysis as follows: A JEOL AccuTOF-DART mass spectrometer (Jeol USA, Peobody, MA, USA) was used.

A JEOL AccuTOF-DART mass spectrometer (Jeol USA, Peabody, MA, USA) was used. Analyses were performed in positive ion mode (DART+), generating masses corresponding to the M+H+ ions generated by DART-MS. A range of instrument settings were used to determine optimal conditions for the analysis of oleander. Typical DART+ settings included: needle voltage 3500 V; orifice 1-2–20 V; ring lens 2–5 V; orifice 2-2–5 V; and peak voltage 1000 V. Calibration was performed internally with each sample using a 10% solution of PEG 600, which provided mass markers throughout the required mass range of 100–1000 mass units. Other analyses were performed in DATR mode, which consisted of the following: needle voltage 3500 V; heating element 250°C; electrode 1–150 V; electrode 2–250 V; and He gas flow rate 3.79 LPM. Mass spectrometer settings: MCP 2600 V; aperture 1-15 V; ring lens -5 V, aperture 2-5 V; and peak voltage 1000 V. Calibration was performed internally for each sample using a perfluorinated carboxylic acid solution that provided markers over the required mass range of 100–1000 mass units. The oleander samples were introduced into the DART helium plasma using the closed end of a borosilicate glass melting point tube. The capillary was held in the He plasma for approximately 3–5 seconds per analysis. Molecular formulas were determined using the elemental composition and isotope matching program provided by the JEOLAccuTOF D-MS instrument. A searchable database of oleander constituents developed by Herbal Science (Naples, FL, USA) was used.

The SCF extract was found to contain at least the following components present in the indicated relative abundance (%).

Element Relative abundance (%) Oleandrin 2.99 Oleandrin 3.31 Ursolic acid/betulinic acid 15.29 Odonoside 0.80 Oleanolic acid 0.60 Urso-12-ene-3β,28-diol/Buttiol 5.44 3β,3β-Hydroxy-12-oleanone-28-oic acid 14.26 28-norurso-12-en-3β-ol 4.94 Urso-12-en-3β-ol 4.76

As used herein, unless otherwise indicated, the term "about" or "approximately" means ±10%, ±5%, ±2.5% or ±1% of a particular value. As used herein, unless otherwise indicated, the term "substantially" means "to a large extent," "at least a majority," greater than 70%, greater than 85%, greater than 90%, greater than 95%, greater than 98% or greater than 99%.

The above is a detailed description of specific embodiments of the present invention. It will be understood that although specific embodiments of the present invention have been described herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the present invention is not limited except by the appended claims. All embodiments disclosed and claimed herein can be made and implemented without undue experimentation in light of the present disclosure.

Claims (8)

1. A pharmaceutical composition for treating neurological disorders selected from Alzheimer's disease and stroke, wherein the pharmaceutical composition is prepared by loading an extract obtained from the leaves of *Nerium oleander* via supercritical carbon dioxide fluid extraction onto a water-equilibrium ODS-silica column, followed by eluting different components of the extract by sequentially passing a multipart aqueous mobile phase with methanol contents of 30%, 55%, 80%, and 100%, collecting the fraction eluted with 100% methanol, and removing the solvent from the fraction eluted with 100% methanol; and The pharmaceutical composition contains oleanolic acid, ursolic acid and betulinic acid, and the composition does not contain cardiac glycosides obtained from European oleander, and the composition contains less than 0.5% by weight of polysaccharides. 2. The composition of claim 1, wherein the composition further comprises a steroid. 3. The composition of claim 2, wherein the steroid is oleandrin. 4. The composition of claim 1, wherein the composition further comprises one or more therapeutically effective components selected from cardiac glycoside aglycones, triterpenoids, betulin, 28-nor-ursode-12-en-3β-ol, ursode-12-en-3β-ol, 3β,3β-hydroxy-12-oleaneno-28-acid, 3β,20α-dihydroxyursode-21-en-38-acid, 3β,27-dihydroxy-12-ursode-38-acid, 3β,13β-dihydroxyursode-11-en-28-acid, 3β,12α-dihydroxyoleanane-28,13β-lactone, or 3β,27-dihydroxy-12-oleaneno-28-acid. 5. The composition of claim 4, wherein the aglycone of the cardiac glycoside is a cardiac steroid. 6. The composition of claim 1, wherein the extract obtained by supercritical carbon dioxide fluid extraction is prepared by supercritical fluid extraction of leaves in the presence of a modifier. 7. The composition of claim 6, wherein the modifier is selected from the group consisting of ethanol, methanol, propanol, acetone, ethyl acetate and dichloromethane. 8. Use of the pharmaceutical composition of any one of claims 1-7 in the preparation of a medicament for treating neurological disorders selected from Alzheimer's disease and stroke.