CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/379,356, filed August 25, 2016, entitled, "Treatment of Neurodegenerative Disorders and Pain" (Docket No. WRU 0384 MA/40878.522), the contents of which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The present disclosure relates generally to treatment of neurodegenerative disorders and pain, and to pharmaceutical compositions for the treatment of neurodegenerative disorders and pain.

BACKGROUND

[0003] Neurodegenerative disorders, such as, e.g. , Alzheimer' s disease, Parkinson' s disease, and Huntington's disease, generally involve a decrease in neurogenesis. Neurogenesis is decreased by stress and increased by certain growth factors, such as, e.g. , brain-derived neurotrophic factor (hereinafter, "BDNF") and certain sex hormones, such as, e.g., estrogen, prolactin, and testosterone. Further, BDNF increases stem cell proliferation and neurogenesis. Despite ongoing research into neurodegenerative disorders, Alzheimer's disease, Parkinson's disease, and Huntington' s disease remain incurable.

[0004] Thus, ongoing needs exist for effective therapies to treat neurodegenerative disorders.

SUMMARY

[0005] Provided herein are treatments for neurodegenerative disorders and pain. In embodiments, methods for treating neurodegenerative disorders are disclosed. The methods include administering to a subject in need thereof: (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) a therapeutically effective amount of at least one of fluoxetine racemate or a pharmaceutically acceptable salt thereof, enantiopure (S)-fluoxetine or a pharmaceutically acceptable salt thereof, or enantioenriched fluoxetine having an enantiomeric excess of (S) -fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof.

[0006] In embodiments, pharmaceutical compositions for the treatment of neurodegenerative disorders are disclosed. The pharmaceutical compositions include: (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) a therapeutically effective amount of at least one of fluoxetine racemate or a pharmaceutically acceptable salt thereof, enantiopure (S) -fluoxetine or a pharmaceutically acceptable salt thereof, or enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof.

[0007] In embodiments, methods for the treatment or management of pain are disclosed. The methods include administering to a subject in need thereof: (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) therapeutically effective amount of at least one of fluoxetine racemate or a pharmaceutically acceptable salt thereof, enantiopure (S) -fluoxetine or a pharmaceutically acceptable salt thereof, or enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof.

[0008] In embodiments, pharmaceutical compositions for the treatment or management of pain are disclosed. The pharmaceutical compositions include: (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) therapeutically effective amount of at least one of fluoxetine racemate or a pharmaceutically acceptable salt thereof, enantiopure (S) -fluoxetine or a pharmaceutically acceptable salt thereof, or enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof.

[0009] It is to be understood that both the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated into and constitute a part of this specification. The drawings illustrate the various embodiments described herein, and together with the description serve to explain the principles and operations of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is representative images of Ki67 staining in anterior ventricles of 11 month-old female Sprague Dawley rats, wherein Panel (A) shows Ki67 staining in control rats administered vehicle for 30 days, wherein the scale bar shows 100 micrometers; Panel (B) shows Ki67 staining in rats administered simvastatin (1 mg/kg per day), ascorbic acid (20 mg/kg per day), and fluoxetine (5 mg/kg per day) for 30 days; Panel (C) shows the respective mask used to measure the area of Ki67 staining in ImageJ from Panel (A); and Panel (D) shows the respective mask used to measure the area of Ki67 staining in Panel (D);

[0011] FIG. 2 is a graph of Female Sprague Dawley rats administered vehicle (i.e. , Control), simvastatin (i.e., Simvastatin, 1 mg/kg per day), fluoxetine (i.e., Fluoxetine, 5 mg/kg per day), fluoxetine + simvastatin (i.e., Fluoxetine, Statin), or fluoxetine + simvastatin + ascorbic acid (20 mg/kg per day) (i.e., collectively SAAF) for 30 days with respect to Ki67 area (mm ). Each symbol (i.e., circle, square, triangle, shaded circle, and diamond) represents the mean area of Ki67 staining of the subventricular zone of 25 coronal brain sections taken from a single rat. The mean for each group is indicated by the wide horizontal bar. The error bar represents the standard deviation for each group. Statistical test was one way ANOVA with SAAF administration increasing stem cell proliferation over control (P = 0.020);

[0012] FIG. 3 is graph of Male Sprague Dawley rats administered vehicle (i.e., Control), simvastatin (i.e., Statin, 1 mg/kg per day), fluoxetine (i.e., Fluoxetine, 5 mg/kg per day), fluoxetine + simvastatin (i.e., Fluoxetine, Statin), or fluoxetine + simvastatin + ascorbic acid (20 mg/kg per day) (i.e., SAAF) for 30 days with respect to Ki67 area (mm ). Each symbol (i.e., circle, square, triangle, shaded circle, and diamond) represents the mean area of Ki67 staining of the subventricular zone of 30 coronal brain sections (50 μιη thickness) taken from a single rat. The mean for each group is indicated by the wide horizontal bar. The error bar represents the standard deviation for each group. Fluoxetine significantly decreased stem cell proliferation compared to both Control and SAAF (one way ANOVA);

[0013] FIG. 4A is a bar graph of 11-month old Male Sprague Dawley rats and 11-month old Female Sprague Dawley rats administered vehicle (i.e., Control) with respect to Ki67 area (mm ). Each bar represents the mean Ki67 staining area and standard error of the mean (i.e., SEM) for Male versus Female Sprague Dawley rats, wherein the subventricular zone is separated into anterior region (i.e., light gray, speckled bars - left), middle region (i.e., gray bars - middle), and posterior region (i.e., black bars - right). Significant differences were shown by a line over the significantly different group with the P value on top of the line (2-way ANOVA);

[0014] FIG. 4B is a bar graph of Male Sprague Dawley rats and Female Sprague Dawley rats administered 1 mg/kg simvastatin for 30 days with respect to Ki67 area (mm ). Each bar represents the mean Ki67 staining area and SEM for Male versus Female Sprague Dawley rats, wherein the subventricular zone is separated into anterior region (i.e., light gray speckled bars - left), middle region (i.e., gray bars - middle in Male rats, right in Female rats), and posterior region (i.e., black bars - right in Male rats, not present in Female rats). Significant differences were shown by a line over the significantly different group with the P value on top of the line (2- way ANOVA);

[0015] FIG. 4C is a bar graph of Male Sprague Dawley rats and Female Sprague Dawley rats administered 5 mg/kg fluoxetine for 30 days with respect to Ki67 area (mm ). Each bar represents the mean Ki67 staining area and SEM for Male versus Female Sprague Dawley rats, wherein the subventricular zone is separated into anterior region (i.e., light gray speckled bars - left), middle region (i.e., gray bars - middle), and posterior region (i.e., black bars - right). Significant differences were shown by a line over the significantly different group with the P value on top of the line (2-way ANOVA);

[0016] FIG. 4D is a bar graph of Male Sprague Dawley rats and Female Sprague Dawley rats administered 5 mg/kg fluoxetine and 1 mg/kg simvastatin for 30 days with respect to Ki67 area (mm ). Each bar represents the mean Ki67 staining area and SEM for Male versus Female Sprague Dawley rats, wherein the subventricular zone is separated into anterior region (i.e., light gray speckled bars - left), middle region (i.e., gray bars - middle), and posterior region (i.e., black bars - right). Significant differences were shown by a line over the significantly different group with the P value on top of the line (2- way ANOVA);

[0017] FIG. 4E is a bar graph of Male Sprague Dawley rats and Female Sprague Dawley rats administered 5 mg/kg fluoxetine, 1 mg/kg simvastatin, and 20 mg/kg ascorbic acid for 30 days with respect to Ki67 area (mm ). Each bar represents the mean Ki67 staining area and SEM for Male versus Female Sprague Dawley rats, wherein the subventricular zone is separated into anterior region (i.e. , light gray speckled bars - left), middle region (i.e. , gray bars - middle), and posterior region (i.e., black bars - right). Significant differences were shown by a line over the significantly different group with the P value on top of the line (2-way ANOVA);

[0018] FIG. 5 is a graph of female rats administered (S)-Fluoxetine (5 mg/kg), Simvastatin (1 mg/kg) and Ascorbic Acid (20 mg/kg) (i.e., FS-fluoxetine), female rats administered (R)- Fluoxetine (5 mg/kg), Simvastatin (1 mg/kg) and Ascorbic Acid (20 mg/kg) (i.e. , FR- fluoxetine), male rats administered (S)-Fluoxetine (5 mg/kg), Simvastatin (1 mg/kg) and Ascorbic Acid (20 mg/kg) (i.e., MS-fluoxetine), and male rats administered (K)-Fluoxetine (5 mg/kg), Simvastatin (1 mg/kg) and Ascorbic Acid (20 mg/kg) (i.e., MR-fluoxetine) for a period of six days following stroke with respect to Evans Blue dye (i.e., μg/gm) measured in brain tissue surrounding the infarcted region of the brain;

[0019] FIG. 6 is a histological image of the coronal brain sections of male rats administered Fluoxetine (5 mg/kg), Simvastatin (1 mg/kg), and Ascorbic Acid (20 mg/kg) 6-12 hours post- stroke induction (Panel (B)), 20-26 hours post-stroke induction (Panel (C)), or 48-56 hours post- stroke induction (Panel (D)) stained with Hematoxylin and eosin stain. Control rats (Panel (A)) were not administered any drugs. Enclosed dashed lines represent the infarcted area in the right hemisphere of the rats. The stained region within the enclosed dashed lines indicates migration of microglia, macrophages, and/or neutrophils into the cortex surrounding the infarction. The scale bar in Panel (C) is 500 micrometers; and

[0020] FIG. 7 is a graph of male rats administered Fluoxetine (5 mg/kg), Simvastatin (1 mg/kg), and Ascorbic Acid (20 mg/kg) 6- 12 hours post-stroke induction (i.e. , 6- 12 hours), 20-26 hours post-stroke induction (i.e., 20-26 hours), or 48-54 hours post-stroke induction (i.e., 48-54 hours), or administered no drug treatment (i.e. , Control) with respect to volume of infarction (i.e., Volume of Infarcts (mm )). The volume of infarction was measured in coronal brain sections of rats.

DETAILED DESCRIPTION

[0021] While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-described subject matter.

[0022] The terms "treat," "treatment," and "treating," as used herein, refer to delaying acquisition, inhibiting development or progression of, stabilizing, and/or causing regression of a disease, disorder, and/or symptom thereof.

[0023] Depending upon the context of use, the term "subject in need thereof as used herein, refers to a subject at risk for developing a neurodegenerative disorder, a subject exhibiting symptoms associated with a neurodegenerative disorder, a subject having a neurodegenerative disorder, a subject at risk for developing pain, a subject exhibiting symptoms associated with pain, and/or a subject having pain.

[0024] The term "therapeutically effective amount" as used herein, refers to an amount necessary or sufficient to realize a desired biologic effect. The therapeutically effective amount may vary depending on a variety of factors known to those of ordinary skill in the art, including but not limited to, the particular composition being administered, the activity of the composition being administered, the size of the subject, the sex of the subject, the age of the subject, the general health of the subject, the timing and route of administration, the rate of excretion, the administration of additional medications, and/or the severity of the disease or disorder being treated. In some embodiments, the term therapeutically effective amount refers to the amount of the simvastatin, fluoxetine, or ascorbic acid necessary or sufficient to treat a neurodegenerative disorder and/or pain. In some particular embodiments, the term therapeutically effective amount refers to the amount of the simvastatin, fluoxetine, or ascorbic acid necessary or sufficient to increase stem/progenitor cell proliferation and/or neurogenesis in a subject relative to a baseline level. In other specific embodiments, the term therapeutically effective amount refers to the amount of the simvastatin, fluoxetine, or ascorbic acid necessary or sufficient to transform at least one Ml inflammatory type microglial cell to at least one M2 neuroprotective microglial cell. In still other specific embodiments, the term therapeutically effective amount refers to the amount of the simvastatin, fluoxetine, or ascorbic acid necessary or sufficient to restore impermeability of the blood brain barrier. In yet still other specific embodiments, the term therapeutically effective amount refers to the amount of simvastatin, fluoxetine, or ascorbic acid necessary or sufficient to reduce infarction size and/or to reduce migration of macrophages, neutrophils, and/or proliferation of activated microglia within an infarcted region following an ischemic event.

[0025] The term "pharmaceutically acceptable" as used herein, refers to a pharmaceutically active agent and/or other agents/ingredients for use in a pharmaceutical composition which are not deleterious to a subject receiving the pharmaceutical composition and/or which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like commensurate with a reasonable benefit/risk ratio.

[0026] The terms "pharmaceutically acceptable salt" and "pharmaceutically acceptable salts" as used herein, refer to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include: aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium chloride, zinc, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include: salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When a compound is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include: acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, /?-toluenesulfonic acid, and the like. Thus, representative pharmaceutically acceptable salts include but are not limited to acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexyl-resorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, N-methylglucamine, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, trimethylammonium and valerate. It will be understood that, as used herein, the compounds referred to herein are meant to also include the pharmaceutically acceptable salts.

[0027] The term "carrier" as used herein, refers to a solid or liquid filler, diluent or encapsulating substance. These materials are well known to those skilled in the pharmaceutical arts. Some examples of the substances that can serve as pharmaceutical carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols, such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; agar; alginic acid; pyrogen-free water; isotonic saline; and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents and lubricants, such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, tableting agents, and preservatives, can also be present. Formulation of the components into pharmaceutical compositions is done using conventional techniques.

[0028] The term "enantiomeric excess" is used herein according to its commonly understood definition. That is, for two enantiomers A and B that may be present in a mixture in molar amounts MA and MB, respectively, the enantiomeric excess E of the enantiomer present in a higher molar amount in the mixture may be expressed by the relation where E>0%. A "racemic mixture" of the enantiomers A and B, which may be designated by abbreviations such as "rac" and/or "(±)" (or simply lack any reference to enantiomers) has E=0% because MA=M _B . AS a further illustration, a mixture consisting of A and B, in which MA=60% and M _B =40%, has an enantiomeric excess of A equal to 20%. The same mixture may be regarded in the alternative as a mixture consisting of 80% racemic mixture of A and B in combination with 20% enantiopure A, inasmuch as each molecule of B (40% of the mixture) may be paired with a molecule of A in the mixture (40% of the mixture) to leave unpaired an excess of molecules of A (20% of the mixture).

[0029] As used herein, the term "enantiopure" with regard to a molecule having two enantiomers, A and B, refers to a compound or composition containing substantially only one of the enantiomers A or B, but not both A and B . For an "enantiopure" complex, 97%<E<100%.

[0030] As used herein, the term "enantioenriched" refers, in its broadest sense, to a compound or composition containing a molecule having two enantiomers, A and B, such that the compound or composition has an enantiomeric excess of one of the enantiomers, either A or B . Thus, an "enantioenriched mixture of A and B" may refer to a mixture with an enantiomeric excess of A or to a mixture with an enantiomeric excess of B, wherein 0%<E<100% for either A or B . As illustrative examples, the enantiomeric excess of either A or B may be greater than 0.01%, greater than 1%, greater than 10%, greater than 25%, greater than 40%, greater than 50%, greater than 75%, greater than 90%, greater than 98%, greater than 99%, greater than 99.9%, or even equal to 100%.

[0031] Embodiments of the present disclosure relate to methods for treating neurodegenerative disorders or pain, to methods for managing pain, to pharmaceutical compositions for treating neurodegenerative disorders or pain, and to pharmaceutical compositions for managing pain. Embodiments of methods for treating neurodegenerative disorders or pain will now be described in detail. Thereafter, embodiments of the pharmaceutical compositions for managing pain and/or treating neurodegenerative disorders or pain will be described in detail.

A Methods for Managing Pain and/or Treating Neurodegenerative Disorders or Pain

[0032] Methods for managing pain and/or treating neurodegenerative disorders or pain are disclosed. In embodiments, the methods include administering to a subject in need thereof: (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S)- fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof.

[0033] In embodiments, a combination of (A) simvastatin, (B) at least one of fluoxetine racemate, enantiopure (S)-fluoxetine, or enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine, and (C) ascorbic acid is administered to treat neurodegenerative disorders.

[0034] With specific regard to simvastatin, simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase (i.e. , HMG CoA reductase) inhibitor or a statin. Simvastatin is commercially available, such as, e.g., in tablet form, such as Zocor. With specific regard to ascorbic acid, ascorbic acid (i.e. , Vitamin C) is an antioxidant. Ascorbic acid is commercially available.

[0035] With specific regard to fluoxetine, fluoxetine is a selective serotonin reuptake inhibitor (hereinafter, "SSRI"). As used herein, the general term "fluoxetine" may refer to at least one of fluoxetine racemate, enantioenriched fluoxetine having an enantiomeric excess of (K)-fluoxetine, enantiopure (S)-fluoxetine, or enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine. Fluoxetine racemate refers to a racemic mixture of (S) -fluoxetine and (R) -fluoxetine. The structures of (S)-fluoxetine and (K)-fluoxetine are as provided below:

(K)-Fluoxetine

[0036] Fluoxetine is commercially available as fluoxetine hydrochloride. Fluoxetine is available in multiple dosage forms, including, e.g. , capsule form, such as Prozac, and tablet form, such as Sarafem. Commercially available forms of fluoxetine are believed to include either fluoxetine racemate (such as, e.g. , Prozac) or enantioenriched fluoxetine having an enantiomeric excess of (K)-fluoxetine (such as, e.g. , a generic form of fluoxetine). Specifically, with regard to commercially available enantioenriched fluoxetine having an enantiomeric excess of (K)-fluoxetine, it is believed that such commercially available form includes about 60% (R)- fluoxetine and about 40% (S)-fluoxetine.

[0037] (S)-fluoxetine is also commercially available, such as, e.g. , from Sigma Aldrich (St. Louis, MO). In embodiments, (S) -fluoxetine is (S)-(+)-fluoxetine hydrochloride.

[0038] In some embodiments, administering the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid is effective to treat the neurodegenerative disorder by increasing stem cell proliferation in the subject relative to a baseline level. In some particular embodiments, administering the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid is effective to treat the neurodegenerative disorder by increasing neural stem cell proliferation in a subventricular zone (hereinafter, "SVZ") of the subject relative to a baseline level. In some further particular embodiments, administering the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid is effective to treat the neurodegenerative disorder by increasing neural stem cell proliferation in an anterior region, a middle region, and/or a posterior region of a SVZ relative to a baseline level. The SVZ is a paired brain structure situated throughout the lateral walls of the lateral ventricles. The SVZ is a source of neural stem cells.

[0039] In embodiments, the baseline level refers to a level of stem and/or progenitor cell proliferation, such as, e.g., neural stem cell proliferation, in an untreated control. In some embodiments, the untreated control is an untreated control population of subjects having a neurodegenerative disorder and/or an untreated control population of cells known to those of skill in the art for studying neurodegenerative disorders.

[0040] In embodiments, a combination of (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid is administered to treat pain and/or to manage pain. In some embodiments, administering the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid is effective to treat pain and/or to manage pain by transforming at least one Ml inflammatory type microglial cell to at least one M2 neuroprotective microglial cell.

[0041] In embodiments, the methods for managing pain and/or treating a neurodegenerative disorder or pain include administering (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically-acceptable salts thereof, to a subject in need thereof, wherein the subject is a mammal. In one or more particular embodiments, the subject is a mammal chosen from humans, non-human primates, canines, felines, murines, bovines, equines, porcines, and lagomorphs. In some embodiments, the subject is a rat or a human. In some embodiments, the subject is female or male. In some particular embodiments, the subject is female. In other particular embodiments, the subject is a post-menopausal female. In yet other particular embodiments, the subject is male. In yet still other particular embodiments, the subject is a male who is suffering from and/or who has suffered from chronic stress.

[0042] In embodiments, the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, are administered systemically. Systemic administration of the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, may be chosen from sublingual, subcutaneous, intravenous, intramuscular, intranasal, intrathecal, intraperitoneal, percutaneous, intranasal, enteral, oral, or a combination thereof. In one or more embodiments, the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, are administered orally.

[0043] In embodiments, the methods for managing pain and/or treating a neurodegenerative disorder or pain include administering (A) simvastatin, or a pharmaceutically acceptable salt thereof, in a dose of from about 0.5 mg/kg to about 1.0 mg/kg in rats. In embodiments, the methods for managing pain and/or treating a neurodegenerative disorder or pain include administering (B) fluoxetine, or a pharmaceutically acceptable salt thereof, in a dose of from about 5 mg/kg to about 10 mg/kg, or from about 6 mg/kg to about 9 mg/kg, or from about 7 mg/kg to about 8 mg/kg in rats. In embodiments, the methods for managing pain and/or treating a neurodegenerative disorder or pain include administering (C) ascorbic acid, or a pharmaceutically acceptable salt thereof, in a dose of from about 20 mg/kg to about 100 mg/kg, or from about 40 mg/kg to about 80 mg/kg, or from about 50 mg/kg to about 60 mg/kg in rats. In embodiments, such dosages correlate to human dosages of from about 5 mg to about 10 mg of (A) simvastatin, of from about 20 mg to about 40 mg of (B) fluoxetine, and from about 200 mg to about 1000 mg of (C) ascorbic acid. However, translation of rat dosages to human dosages is as described in Reagan-Shaw, S.N., M and Ahmad, N. 2007. Dose translation from animal to human studies revisited. FASEB Journal 22, 659-61; hereinafter, "Reagan-Shaw, 2007".

[0044] In particular embodiments, the methods for managing pain and/or treating a neurodegenerative disorder or pain include administering (A) simvastatin or a pharmaceutically acceptable salt thereof in a dose of about 5 mg, (B) fluoxetine or a pharmaceutically acceptable salt thereof in a dose of about 40 mg, and (C) ascorbic acid or a pharmaceutically acceptable salt thereof in a dose of about 200 mg to a human subject. It is contemplated that such doses serve as non-limiting examples of suitable doses of the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, for a subject in need thereof.

[0045] In embodiments, the dose of the (A) simvastatin, (B) fluoxetine, and/or (C) ascorbic acid, or pharmaceutically acceptable salts thereof, is administered daily. In some embodiments, the (A) simvastatin, (B) fluoxetine, and/or (C) ascorbic acid, or pharmaceutically acceptable salts thereof, is administered at least once a day. In other embodiments, the (A) simvastatin, (B) fluoxetine, and/or (C) ascorbic acid, or pharmaceutically acceptable salts thereof, is administered at least two times a day, at least three times a day, at least four times a day, at least five times a day, and/or at least six times a day. In particular embodiments, the (A) simvastatin, (B) fluoxetine, and/or (C) ascorbic acid, or pharmaceutically acceptable salts thereof, is administered from one time a day.

[0046] In embodiments, the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid are as depicted in Table 1. [0047] In embodiments, the methods for managing pain and/or treating a neurodegenerative disorder or pain further include monitoring disease development and/or progression and repeating administration of the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, one or more times, thereby treating the neurodegenerative disorder. Development and/or progression of the neurodegenerative disorder or pain may be monitored in a variety of ways known to the skilled clinician. In embodiments, the methods for managing pain and/or treating a neurodegenerative disorder or pain further include monitoring disease development and/or progression and repeating administration of the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, one or more times, thereby managing the pain and/or treating the neurodegenerative disorder or the pain. Successive rounds of administering the (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, coupled with monitoring development and/or progression of the pain or the neurodegenerative disorder may be necessary in order to achieve the desired management and/or treatment of the pain or the neurodegenerative disorder.

[0048] In embodiments, the (A) simvastatin, (B) fluoxetine, and/or (C) ascorbic acid, or pharmaceutically acceptable salts thereof, is administered in a pharmaceutical composition including at least one of an excipient, adjuvant, or pharmaceutically acceptable carrier. Examples of suitable excipients include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol®, and vegetable oils. Examples of suitable adjuvants include inorganic compounds (e.g. , aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, and beryllium), mineral oil (e.g. , paraffin oil), bacterial products (e.g. , killed bacteria Bordetelle pertussis, Mycobacterium bovis, and toxoids), nonbacterial organics (e.g. , squalene and thimerosal), delivery systems (e.g. , detergents (Quil A)), cytokines (e.g. , IL- 1, IL-2, and IL- 12), and combinations (e.g., Freund' s complete adjuvant, Freund' s incomplete adjuvant). Examples of pharmaceutically acceptable carriers include a wide range of known diluents (i.e. , solvents), fillers, extending agents, binders, suspending agents, disintegrates, surfactants, lubricants, wetting agents, preservatives, stabilizers, antioxidants, antimicrobials, buffering agents and the like commonly used in this field. Such carriers may be used singly or in combination according to the form of the pharmaceutical preparation. In further embodiments, a preparation resulting from the inclusion of a pharmaceutically acceptable carrier may incorporate, if necessary, one or more solubilizing agents, buffers, preservatives, colorants, perfumes, flavorings and the like, as widely used in the field of pharmaceutical preparation. Examples of suitable preservatives, stabilizers, antioxidants, antimicrobials, and buffering agents include BHA, BHT, citric acid, ascorbic acid, tetracycline, and the like. Cream or ointment bases useful in formulation include lanolin, Silvadene® (Marion), Aquaphor® (Duke Laboratories).

[0049] A pharmaceutical composition for the management of pain and/or treatment of a neurodegenerative disorder or pain may be prepared according to methods known in the pharmaceutical field using a pharmaceutically acceptable carrier. For example, oral forms such as tablets, capsules, granules, pills and the like are prepared according to known methods using excipients such as saccharose, lactose, glucose, starch, mannitol and the like; binders such as syrup, gum arabic, sorbitol, tragacanth, methylcellulose, polyvinylpyrrolidone and the like; disintegrates such as starch, carboxymethylcellulose or the calcium salt thereof, microcrystalline cellulose, polyethylene glycol and the like; lubricants such as talc, magnesium stearate, calcium stearate, silica and the like; and wetting agents such as sodium laurate, glycerol and the like.

[0050] Injections, solutions, emulsions, suspensions, syrups and the like may be prepared according to known methods suitably using solvents for dissolving the simvastatin, fluoxetine, and/or ascorbic acid, or pharmaceutically acceptable salts thereof, such as ethyl alcohol, isopropyl alcohol, propylene glycol, 1,3-butylene glycol, polyethylene glycol, sesame oil and the like; surfactants such as sorbitan fatty acid ester, polyoxyethylenesorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene of hydrogenated castor oil, lecithin and the like; suspending agents such as cellulose derivatives including carboxymethylcellulose sodium, methylcellulose and the like, natural gums including tragacanth, gum arabic and the like; and preservatives such as parahydroxybenzoic acid esters, benzalkonium chloride, sorbic acid salts and the like.

[0051] The pharmaceutical compositions for the management of pain and/or treatment of a neurodegenerative disorder or pain as described herein may be administered to a subject in need thereof in accordance with the methods for managing pain and/or treating a neurodegenerative disorder or pain, as described in an earlier section.

[0052] In embodiments, the methods disclosed herein are useful in the treatment of neurodegenerative disorders including Alzheimer' s disease, Huntington' s disease, Parkinson's disease, amyotrophic lateral sclerosis (hereinafter, "ALS"), and/or damage from brain injury, such as, e.g. , an ischemic event. In particular embodiments the neurodegenerative disorder is Alzheimer's disease.

[0053] In embodiments, the methods disclosed herein are useful in the management or treatment of pain including any pain known in the art, such as, e.g. , pain caused by a disease, disorder, condition, and/or circumstance. In some embodiments, the pain is chosen from nociceptive pain, neuropathic pain, and/or inflammatory pain. In some embodiments, the pain is chosen from chronic pain and/or acute pain.

[0054] In embodiments, (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S)-fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S) -fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof, for use in managing pain and/or treating a neurodegenerative disorder or pain is/are disclosed. In other embodiments, the

(A) therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof,

(B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S)- fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine or a pharmaceutically acceptable salt thereof, and/or (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutical composition for the management of pain and/or treatment of a neurodegenerative disorder or pain and/or are formulated into a dosage form for the management of pain and/or treatment of a neurodegenerative disorder or pain, as previously described. The (A) therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S) -fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S) -fluoxetine or a pharmaceutically acceptable salt thereof, and/or (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof are as previously described. Additionally, the management of pain and/or treatment of a neurodegenerative disorder or pain are also as previously described.

[0055] Embodiments of the methods for managing pain and/or treating a neurodegenerative disorder or pain have been described in detail. Embodiments of pharmaceutical compositions for managing pain and/or treating a neurodegenerative disorder or pain will now be described in detail.

//. Pharmaceutical Compositions for Managing Pain and/or Treating a Neurodegenerative Disorder or Pain

[0056] Pharmaceutical compositions for the management of pain and/or treatment of a neurodegenerative disorder or pain are disclosed. In embodiments, pharmaceutical compositions including: (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S)-fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S) -fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof are disclosed. The (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, are as previously described. The neurodegenerative disorder and pain are also as previously described.

[0057] In embodiments, the pharmaceutical compositions for the management of pain and/or treatment of a neurodegenerative disorder or pain include a combination of (A) simvastatin, (B) fluoxetine, and (C) ascorbic acid, or pharmaceutically acceptable salts thereof, and also include an excipient, an adjuvant, and/or a pharmaceutically acceptable carrier. The excipient, adjuvant, and pharmaceutically acceptable carrier of the pharmaceutical composition are as previously described. Additionally, the pharmaceutical compositions can be prepared as previously described, such as, e.g. , using a pharmaceutically acceptable carrier.

[0058] Embodiments of the pharmaceutical compositions for the management of pain and/or the treatment of a neurodegenerative disorder or pain have been described in detail.

[0059] It should now be understood that various aspects of the present disclosure are described herein and that such aspects may be utilized in conjunction with various other aspects.

[0060] In a first aspect, a method for treating a neurodegenerative disorder is disclosed. The method includes administering to a subject in need thereof: (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S)-fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof.

[0061] In a second aspect, a method according to the first aspect is disclosed, wherein administration of (A), (B), and (C) is effective to treat the neurodegenerative disorder by increasing at least one of stem cell proliferation or progenitor cell proliferation in the subject relative to a baseline level.

[0062] In a third aspect, the method according to any of the first or the second aspects is disclosed, wherein administration of (A), (B), and (C) is effective to treat the neurodegenerative disorder by increasing at least one of neural stem cell proliferation or progenitor cell proliferation in a subventricular zone of the subject relative to a baseline level.

[0063] In a fourth aspect, the method according to any of the first to the third aspects is disclosed, wherein administration of (A), (B), and (C) is effective to treat the neurodegenerative disorder by increasing at least one of neural stem cell proliferation or progenitor cell proliferation in an anterior region, a middle region, and a posterior region of a subventricular zone of the subject relative to a baseline level. [0064] In a fifth aspect, the method according to any of the first to the fourth aspects is disclosed, wherein the subject is a mammal.

[0065] In a sixth aspect, the method according to any of the first to the fifth aspects is disclosed, wherein the subject is a rat or a human.

[0066] In a seventh aspect, the method according to any of the first to the sixth aspects is disclosed, wherein the subject is a female.

[0067] In an eighth aspect, the method according to any of the first to the seventh aspects is disclosed, wherein (A), (B), and (C) are administered systemically.

[0068] In a ninth aspect, the method according to any of the first to the eighth aspects is disclosed, wherein (A) is administered in a daily dose of from about 5 mg to about 10 mg.

[0069] In a tenth aspect, the method according to any of the first to the ninth aspects is disclosed, wherein (B) is administered in a daily dose of about 40 mg.

[0070] In an eleventh aspect, the method according to any of the first to the tenth aspects is disclosed, wherein (C) is administered in a daily dose of from about 200 mg to about 1000 mg.

[0071] In a twelfth aspect, the method according to any of the first to the eleventh aspects is disclosed, wherein at least one of (A), (B), or (C) is administered in a pharmaceutical composition including at least one of an adjuvant, an excipient, or a pharmaceutically acceptable carrier.

[0072] In a thirteenth aspect, the method according to any of the first to the twelfth aspects is disclosed, wherein the neurodegenerative disorder includes at least one of Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), or damage from brain injury.

[0073] In a fourteenth aspect, a pharmaceutical composition for the treatment of a neurodegenerative disorder is disclosed. The pharmaceutical composition includes (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S)-fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S)-fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof.

[0074] In a fifteenth aspect, the pharmaceutical composition according to the fourteenth aspect is disclosed, wherein the neurodegenerative disorder includes at least one of Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), or damage from brain injury.

[0075] In a sixteenth aspect, a method for treating or managing pain is disclosed. The method includes administering to a subject in need thereof: (A) a therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S) -fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S) -fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof.

[0076] In a seventeenth aspect, the method according to the sixteenth aspect is disclosed, wherein at least one of (A), (B), or (C) is administered in a pharmaceutical composition including at least one of an adjuvant, an excipient, or a pharmaceutically acceptable carrier.

[0077] In an eighteenth aspect, a pharmaceutical composition for the treatment or management of pain is disclosed. The pharmaceutical composition includes: (A) a

therapeutically effective amount of simvastatin or a pharmaceutically acceptable salt thereof, (B) at least one of a therapeutically effective amount of fluoxetine racemate or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of enantiopure (S) -fluoxetine or a pharmaceutically acceptable salt thereof, or a therapeutically effective amount of enantioenriched fluoxetine having an enantiomeric excess of (S) -fluoxetine or a pharmaceutically acceptable salt thereof, and (C) a therapeutically effective amount of ascorbic acid or a pharmaceutically acceptable salt thereof. Examples

[0078] The following non-limiting examples illustrate the methods of the present disclosure.

Example 1: Characterization of the Effect of Simvastatin, Fluoxetine, and/or Ascorbic Acid to Increase Stem Cell Proliferation in Rats

[0079] Experimental Protocol. Animals and Animal Husbandry. All animals were purchased as 10-month old Sprague Dawley outbred retired breeders, with an equal number of male and female rats. Animals were weighed once a week, in order to keep the fixed dosage of medication constant for those animals administered drugs and/or drug combinations. Animals were kept on a 12 hour light-dark light cycle and were fed ad lib Harlan rat chow. Animals were administered either vehicle {i.e., 4 grams sugar cookie dough) or drugs encased in 4 grams sugar cookie dough as detailed in Table 2 daily for a period of 30 days. This method of voluntary drug delivery alleviated daily stress associated with other drug delivery methods, such as, e.g., oral gavage and intraperitoneal (hereinafter, "IP") injections. Without being bound by the theory, it is believed that alleviation of daily stress is important to stimulate neurogenesis (See e.g., Corbett, A., McGowin, A., Sieber, S., Flannery, T., Sibbitt, B. 2012. A method for reliable voluntary oral administration of a fixed dosage (mg/kg) of chronic daily medication to rats. Laboratory animals 46(4), 318-24. doi: 10.1258/la.2012.012018; and Corbett, A.M., Sieber, S., Wyatt, N., Lizzi, J., Flannery, T., Sibbit, B., Sanghvi, S. 2015. Increasing neurogenesis with fluoxetine, simvastatin and ascorbic acid leads to functional recovery in ischemic stroke. Recent patents on drug delivery & formulation 9(2), 158-66).

[0080] The rats were euthanized on day 31 after IP injection with 100 mg/kg pentobarbital (i.e., Euthasol) through cardioperfusion with phosphate buffered saline (hereinafter, "PBS") and PBS containing 4% paraformaldehyde. The brain was dissected and prepared for immunocytochemistry. All animal studies in this paper were reviewed and approved by the Institutional Animal Care and Use Committee (i.e., IACUC).

[0081] Immunohistochemistry. The brain was blocked into two coronal pieces. The anterior coronal piece was placed into PBS containing 4% paraformaldehyde (i.e., a fixative) for 24 hours post-fixation. The fixative was poured off the next day and replaced with 30% sucrose for at least a 3 day period to prepare the brain for cryosectioning. The brains sinking into the 30% sucrose (rather than floating on it) is an indication that the sucrose has diffused into the tissue. The coronal section was mounted onto a cryostat post with TissueTek OCT, and frozen using the Peltier device on a model HM 550 cryostat. The brain was cut into 50 micron coronal sections, which were transferred into vials containing phosphate-buffered saline. Brain sections were incubated in blocking solution (i.e., PBS with 0.3% Tween-20 and 3% goat serum) at room temperature for one hour on an orbital shaker. Primary antibody (i.e., AbCam anti-Ki67 (AB 15580)) was added to the blocking solution at a 1: 1000 dilution and incubated overnight at 5 °C on an orbital shaker.

[0082] The next day, the primary antibody solution was discarded and the brain sections were washed twice with PBS containing 0.3% Tween. The brain sections were then placed in blocking solution containing a Vector biotinylated secondary antibody according to manufacturer instructions (i.e., from Vector ABC kit for Rabbit IgG with horseradish peroxidase) and incubated with shaking at room temperature for one hour. This blocking solution was removed from the brain sections and the brain sections were washed twice with PBS containing 0.3% Tween. The ABC reagent (from Vector) was mixed and allowed to sit for at least 30 minutes; then, it was placed onto the brain sections and the brain sections were incubated for one hour. Following incubation, the brain sections were washed twice with PBS containing 0.3% Tween. This solution was then removed from the brain sections. Enzyme substrate (i.e. , 3, 3-diaminobenzidine, hereinafter, "DAB" with nickel enhancement) was placed onto the brain sections for about 10 minutes. At the end of 10 minutes, tap water was squirted into the vials, removed from the brain sections, and the brain sections were resuspended in water. Brain sections were then mounted onto gel-subbed microscope slides, dried, and coverslipped using DPX Mounting Media as a permanent mountant.

[0083] Image Analysis. Digital photos were taken of the SVZ of the lateral ventricles in the coronal sections of the brain using a total 40X magnification (oculars and objective). Multiple images of a single ventricle were montaged into one image using Adobe Photoshop. The montaged image was then opened with ImageJ (i.e. , NTH) and the threshold was adjusted until just the Ki67 staining was highlighted. Apply was pressed and the image was turned into a black and white image with the Ki67 staining in black. The Ki67 staining was enclosed with the free hand tool and the area of particles within the enclosure was measured in mm . For each ventricle, the location of the ventricle (i.e. , anterior, middle or posterior) was determined and the average Ki67 staining area per section for all of the replicate sections was indicated within that region for one particular rat. For each of female rats, an average of 7.5 sections analyzed for the anterior SVZ was employed, an average of 12 sections for the middle SVZ was employed, and an average of 5.8 sections for the posterior SVZ was employed. For each of the male rats, an average of 8.8 sections for the anterior SVZ was employed, an average of 16 sections for the middle SVZ was employed, and an average of 5 sections for the posterior SVZ was employed.

[0084] Graphs and Statistical Analysis. Data was graphed with Graph Pad Prism software and analyzed statistically using the same. In FIGS . 2-3, the average Ki67 staining area from each rat was indicated by a symbol, with each symbol indicating the average of Ki67 staining area from multiple sections in that region (e.g. , the anterior SVZ, the middle SVZ, or the posterior SVZ). The wide horizontal bar indicated mean, and the error bars indicated the standard deviation. The number of rats in each group was given by the number of symbols. A one way ANOVA was used to evaluate statistical differences of the different drugs and/or drug combinations on stem cell proliferation in FIGS. 2-3. In FIGS. 4A-4E, vertical bar charts were used, with each bar indicating the mean and the error bars indicating the standard error of the mean. The same number of animals was used as shown in FIGS. 2 and 3. This was simply a different way to graph them and analyze for 2-way ANOVA, testing for gender differences as well as SVZ location differences.

[0085] Results. Effect of Drugs and/or Drug Combinations on Female Stem/Progenitor Cell Proliferation. Female Sprague Dawley rats were purchased at 10 months of age. The rats were administered various daily doses of drugs and/or drug combinations as set forth in Table 2 for a period of 30 days. All animals were euthanized at 11 months of age using cardio-perfusion to clear the blood and fix the brain with 4% paraformaldehyde. Coronal sections of the brain were incubated with Ki67 antibody overnight, washed, and then incubated with a secondary biotinylated antibody. Because Ki67 marks a protein that only appears during mitosis, the number of stem/progenitor cells in the SVZ undergoing mitosis at the time of the animal's death was determined. This was a measure of stem/progenitor cell proliferation. {See e.g., Cowen, D.S., Takase, L.F., Fornal, C.A., Jacobs, B.L. 2008. Age-dependent decline in hippocampal neurogenesis is not altered by chronic treatment with fluoxetine. Brain research 1228, 14-9. doi: 10.1016/j.brainres.2008.06.059). Vector ABC kits (including horseradish peroxidase) were used to develop the stain using DAB as a substrate. Referencing FIG. 1, the area of Ki67 staining throughout the SVZ in these animals was quantified using the NIH Image J program. In Panel (A) of FIG. 1, a control female rat with Ki67 staining in an anterior ventricle is shown. In Panel (C) of FIG. 1, a mask from ImageJ was used to measure the area of the particles. In Panel (B) of FIG. 1, Ki67 staining of an anterior ventricle from a female rat administered a combination of simvastatin, ascorbic acid, and fluoxetine (collectively, hereinafter, "SAAF") for thirty days with the ImageJ mask was used to measure the area of the particles in Panel (D) is shown.

[0086] In FIG. 2, the area of Ki67 staining from approximately 25 coronal fifty micrometer thick brain sections throughout the SVZ of the lateral ventricles was analyzed, wherein the mean area of Ki67 staining for a section from the SVZ was determined for each animal. The mean area of Ki67 staining for each animal in a group was shown by the individual points and the mean of the group was shown by a broad horizontal line, with error bars representing the standard deviation. As shown in FIG. 2, the mean endogenous female stem/progenitor cell proliferation in the rats administered Vehicle (i.e., Control) in the SVZ was quite low. Without being bound by the theory, it is believed that the low stem/progenitor cell proliferation in the Control rats may be due to the rats being post-menopausal. Additionally, while a daily dose of 1 mg/kg simvastatin (i.e., Simvastatin) in rats increased stem/progenitor cell proliferation, there was wide variation in the data. Further, the Simvastatin rats were not significantly different from the Control rats (P = 0.126, Holm-Sildak post hoc analysis). Rats administered a daily dose of 5 mg/kg fluoxetine (i.e., Fluoxetine) exhibited increased stem/progenitor cell proliferation. However, such increase in the Fluoxetine rats was not significantly different from the Control rats (P = 0.308). Further, rats administered a combination of simvastatin and fluoxetine (i.e., Fluoxetine, Statin) exhibited a surprising effect wherein the increase in stem/progenitor cell proliferation in the Fluoxetine, Statin rats was not additive as compared to the Simvastatin rats or the Fluoxetine rats. Rather, a decrease in stem/progenitor cell proliferation similar to Control rats was observed. Finally, in rats administered a daily combination of 1 mg/kg simvastatin, 20 mg/kg ascorbic acid, and 5 mg/kg fluoxetine (i.e., SAAF), an increase in stem/progenitor cell proliferation was observed. Specifically, approximately a 6.75 fold increase in stem/progenitor cell proliferation was observed in SAAF rats as compared to Control rats and the Fluoxetine, Statin rats. Such increase was significant as determined via ANOVA (P = 0.020).

[0087] Effect of Drugs and Drug Combinations on Male Stem/Progenitor Cell Proliferation. Male Sprague Dawley rats were purchased at 10 months old and subjected to exactly the same drug treatment as the female rats, set forth in Table 2. Euthanasia, cardio- perfusion, fixation, cutting of the brain, and immuno staining were also performed as described previously for the female rats. The average area of Ki67 staining per 50 micrometer coronal brain slice throughout the SVZ was quantified with ImageJ.

[0088] Referencing FIG. 3, each point represented the mean area of Ki67 staining obtained from approximately 30 coronal slices throughout the SVZ of the lateral ventricles for a single male rat. The overall group mean was given by the broad horizontal line in each group, with the error bars represented standard error of the mean. As shown in FIG. 3, endogenous stem/progenitor cell proliferation in rats administered Vehicle (i.e. , Control) was high in male rats. Further, as shown in FIG. 3, rats administered either simvastatin alone (i.e. , Statin) or fluoxetine alone (i.e. , Fluoxetine) exhibited decreased stem/progenitor cell proliferation as compared to Control rats. However, only Fluoxetine rats exhibited a decrease in stem/progenitor cell proliferation that was significant (P = 0.001 ; Holm-Sidak post-hoc test). Also, rats administered a combination of simvastatin and fluoxetine (i.e., Fluoxetine, Statin) exhibited an additive effect, wherein a slight increase in the mean stem/progenitor cell proliferation was observed (but was not statistically different). Finally, rats administered a combination of simvastatin, ascorbic acid, and fluoxetine (i.e. , SAAF) exhibited significantly increased stem/progenitor cell proliferation compared to the Fluoxetine rats (P < 0.001) or the Statin rats (P = 0.006). It was observed that the SAAF rats eliminated the negative effects on stem/progenitor cell proliferation observed in the Statin rats and the Fluoxetine rats, wherein stem/progenitor cell proliferation was brought back to endogenous levels.

[0089] Analysis of Sex and SVZ location differences in Stem/Progenitor Cell Proliferation. The same rats and data of FIGS. 2-3 were re-analyzed with a 2-way ANOVA to detect any differences in stem/progenitor cell proliferation based on either sex or location of the SVZ region (e.g. , anterior, middle, or posterior). For this, each of the coronal brain sections showing the lateral ventricles for each rat was assigned into anterior (i.e. , very small ventricle, signs of Ki67 staining on both sides of the ventricle), middle (i.e., large ventricle with short opening across the top, Ki67 staining primarily on one side), or posterior (i.e. , very large ventricle with broad opening across the top and the bottom of the ventricle beginning to curl toward the midline; Ki67 staining all on one side) regions of the SVZ. Such regional analysis was employed to determine if regional differences existed in the stem/progenitor cell proliferation throughout the SVZ.

[0090] Because the previous coronal section was subdivided into different regions, the Ki67 staining area was averaged over a different number of coronal sections. For female rats, an average of 7.5 coronal sections for the anterior SVZ was employed, an average of 12 sections for the middle SVZ was employed, and an average of 5.8 sections for the posterior SVZ was employed. For male rats, an average of 8.8 coronal sections for the anterior SVZ was employed, an average of 16 sections for the middle SVZ was employed, and an average of 5 sections for the posterior SVZ was employed.

[0091] As shown in FIG. 4A, endogenous stem/progenitor cell proliferation between male and female rats at 11 months of age (i.e. , Control) was studied, with the error bars exhibiting the standard error of the mean. There was a statistical difference in stem/progenitor cell proliferation based on sex of the animals (P < 0.0001, 2-way ANOVA), wherein females exhibited greatly decreased neurogenesis. There was no statistical difference in proliferation as measured by Ki67 staining area between the different regions of the SVZ (P = 0.5512).

[0092] As shown in FIG. 4B, male rats administered a 30 day treatment with 1 mg/kg simvastatin exhibited reduced stem/progenitor cell proliferation as compared to female rats administered a 30 day treatment with 1 mg/kg simvastatin. Administration of 1 mg/kg simvastatin in rats would correlate with approximately an 8 mg daily dose of simvastatin in humans (see e.g. , Reagan-Shaw, S.N., M and Ahmad, N. 2007. Dose translation from animal to human studies revisited. FASEB Journal 22, 659-61 ; hereinafter, "Reagan-Shaw, 2007"). In FIG. 4B, male stem/progenitor cell proliferation was significantly reduced as compared to females (P = 0.03, 2 way ANOVA) with this single drug treatment, but significant differences between the regions of the SVZ were not observed (P = 0.1974).

[0093] As shown in FIG. 4C, male rats administered a 30 day treatment with 5 mg/kg fluoxetine exhibited significantly reduced (P = 0.0017) stem/progenitor cell proliferation as compared to female rats administered a 30 day treatment with 5 mg/kg fluoxetine. Administration of 5 mg/kg fluoxetine in rats would correlate with approximately a 40 mg daily dose of fluoxetine in humans (see e.g. , Reagan-Shaw, 2007). Again, no difference was observed in stem/progenitor cell proliferation between the different regions (i.e. , anterior, middle, or posterior) of the SVZ (P = 0.9665).

[0094] As shown in FIG. 4D, male rats administered a 30 day treatment with a combination of simvastatin and fluoxetine exhibited no difference in stem/progenitor proliferation as compared to female rats administered a 30 day treatment with a combination of simvastatin and fluoxetine (P = 0.2925). Further, no difference was observed in stem/progenitor cell proliferation between the different regions (P = 0.7249). [0095] Finally, as shown in FIG. 4E, female rats administered a 30 day treatment with a combination of simvastatin, fluoxetine, and ascorbic acid (i.e. , SAAF) exhibited a significant increase in stem/progenitor cell proliferation (P = 0.0372) as compared to endogenous male stem/progenitor cell proliferation. Again, no difference was observed in stem/progenitor cell proliferation between the different regions of the SVZ (P = 0.9651).

[0096] Innate Sex Differences in Stem Cell Proliferation in the SVZ of 11-Month Old Rats. 11-month old female rats are generally considered retired breeders. The vast majority of 11-month old rats have very low levels of sex hormones (see e.g. , De Nicola, A.F., Pietranera, L., Beauquis, J., Ferrini, M.G., Saravia, F.E. 2009. Steroid protection in aging and age- associated diseases. Experimental gerontology 44(1-2), 34-40. doi: 10.1016/j.exger.2008.03.005), correlating with the post-menopause stage in humans. However, 11 -month old male rats have elevated levels of testosterone, which could account for their innate high stem cell proliferation level compared to the females (see e.g. , Allen, K.M., Purves-Tyson, T.D., Fung, S.J., Shannon Weickert, C. 2015. The effect of adolescent testosterone on hippocampal BDNF and TrkB mRNA expression: relationship with cell proliferation. BMC neuroscience 16, 4. doi: 10.1186/sl2868-015-0142-x).

[0097] Drug Effects on Subventricular Zone Neurogenesis. As discussed previously, some clear sex based differences were observed in the response of the stem/progenitor cell proliferation in the SVZ with different drugs and/or drug combinations. Fluoxetine, in particular, stimulated stem cell proliferation in post-menopausal female rats but significantly decreased stem cell proliferation in male rats. Without being bound by the theory, it is believed that administration of simvastatin and ascorbic acid with fluoxetine alleviated at least some negative effects observed in rats administered fluoxetine alone.

[0098] In both male and female rats, the highest stem/progenitor cell proliferation was observed in rats administered a combination of simvastatin, ascorbic acid, and fluoxetine. Without being bound by the theory, it is believed that upon administration, simvastatin acts to stimulate endothelial nitric oxide synthase to produce BDNF (see e.g. , Wu, H., Lu, D., Jiang, H., Xiong, Y., Qu, C, Li, B., Mahmood, A., Zhou, D., Chopp, M. 2008. Simvastatin-mediated upregulation of VEGF and BDNF, activation of the PI3K/Akt pathway, and increase of neurogenesis are associated with therapeutic improvement after traumatic brain injury. Journal of neurotrauma 25(2), 130-9. doi: 10.1089/neu.2007.0369), and/or to stimulate production of plasmin, which is capable of cleaving any released pro-BDNF to BDNF (see e.g., Tsai, S.J. 2007. Statins may enhance the proteolytic cleavage of proBDNF: implications for the treatment of depression. Medical hypotheses 68(6), 1296-9. doi: 10.1016/j.mehy.2006.09.043 and Tsai, S.J. 2009. Statins may act through increasing tissue plasminogen activator/plasmin activity to lower risk of Alzheimer's disease. CNS spectrums 14(5), 234-5).

[0099] Without being bound by the theory, it is believed that upon administration, fluoxetine acts on microglial cells in an injury model (see e.g., Corbett, A.M., Sieber, S., Wyatt, N., Lizzi, J., Flannery, T., Sibbit, B., Sanghvi, S. 2015. Increasing neurogenesis with fluoxetine, simvastatin and ascorbic Acid leads to functional recovery in ischemic stroke. Recent patents on drug delivery & formulation 9(2), 158-66), to transform an Ml inflammatory type microglial cell to an M2 neuroprotective microglial cell (see e.g., Su, F., Yi, H., Xu, L., Zhang, Z. 2015. Fluoxetine and S-citalopram inhibit Ml activation and promote M2 activation of microglia in vitro. Neuroscience 294, 60-8. doi: 10.1016/j.neuroscience.2015.02.028). Without being bound by the theory, it is believed that ascorbic acid may act to ensure that substances within the pathways upon which the simvastatin and/or fluoxetine act are not oxidized.

[0100] Additionally, without being bound by the theory, it is specifically contemplated that the conclusions made herein based upon the described observations in rats are also applicable to other mammals, including humans.

Example 2: Characterization of the Effect of Simvastatin, (S)-Fluoxetine, (K)-Fluoxetine, and/or Ascorbic Acid to Increase Blood Brain Barrier Permeability Following Stroke

[0101] Experimental Protocol. Stroke was induced in 10-12 month-old female and male rats with normal endothelin. 20-26 hours post-stroke induction, the rats were administered daily doses of (S)-Fluoxetine (5 mg/kg) or (K)-Fluoxetine (5 mg/kg) (obtained from Sigma Aldrich, St. Louis, MO) along with Simvastatin (1 mg/kg) and Ascorbic Acid (20 mg/kg) for a period of six days. On post-stroke day 7, the rats were euthanized and Evans Blue dye was injected into the heart of the rats about 10 minutes prior to cardioperfusion with phosphate buffered solution (100 mL). [0102] Results. As shown in FIG. 5, the administration of (S)-Fluoxetine in male rats (i.e., MS- fluoxetine) resulted in a significant decrease in blood brain barrier permeability as compared to the administration of (K)-Fluoxetine in male rats (i.e., MR- fluoxetine). Without being bound by the theory, it is believed that Evans Blue dye is normally excluded from the blood brain barrier; however, following a stroke, there tends to be increased permeability in the blood brain barrier. Further, it is believed that increased permeability in the blood brain barrier is a hallmark of reperfusion injury following ischemia. In view of FIG. 5, it is believed that administration of (S)-Fluoxetine resulted in a significant decrease in blood brain barrier permeability to Evans Blue dye measured in μg/gm (P=0.0091; ANOVA). Additionally, a significant decrease in permeability was observed between female rats administered (S)-fluoxetine (i.e., FS -fluoxetine) as compared to male rats administered (R) -fluoxetine.

[0103] Additionally, without being bound by the theory, it is specifically contemplated that the conclusions made herein based upon the described observations in rats are also applicable to other mammals, including humans.

Example 3: Characterization of the Effect of Simvastatin, Fluoxetine, and/or Ascorbic Acid to Increase Migration of Macrophages and/or Neutrophils into Cortex and/or to Proliferate Activated Microglia in Cortex Following Stroke

[0104] Experimental Protocol. Stroke was induced in 10-12 month-old Sprague Daw ley male rats. 6-12 hours post-stroke induction, some rats were administered daily doses of Fluoxetine (5 mg/kg) along with Simvastatin (1 mg/kg) and Ascorbic Acid (20 mg/kg) until day 7 post-stroke. Some rats were administered daily doses of Fluoxetine (5 mg/kg) along with Simvastatin (1 mg/kg) and Ascorbic Acid (20 mg/kg) from 20-26 hours post-stroke induction until day 7 post- stroke. Some rats were administered daily doses of Fluoxetine (5 mg/kg) along with Simvastatin (1 mg/kg) and Ascorbic Acid (20 mg/kg) from 48-56 hours post-stroke induction until day 7 post-stroke. Control rats were not administered drugs. On post-stroke day 7, the rats were euthanized and brain tissue was stained with H&E stain. H&E stains incoming microglia, neutrophils, and macrophages blue. [0105] Results. As shown in panels (A), (B), and (D) of FIG. 6, microglia, macrophage, and neutrophil migration into the cortex surrounding infarction was observed (enclosed in dashed lines). However, where the administration of Fluoxetine, Simvastatin, and Ascorbic Acid occurred 20-26 hours post-stroke induction (see Panel (C) of FIG. 6), a dramatic decrease in the blue stain surrounding infarction injection site was observed (shown as dark grey region within the enclosed dashed lines in FIG. 6). Additionally, as shown in FIG. 7, a significant reduction in the infarction volume in male Sprague Dawley rats was also observed (P=0.0098, one way ANOVA). Without being bound by the theory, it is believed that one hallmark of chronic pain in the spinal cord is the presence of activated microglia following injury.

[0106] Additionally, without being bound by the theory, it is specifically contemplated that the conclusions made herein based upon the described observations in rats are also applicable to other mammals, including humans.

[0107] All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present disclosure.

[0108] It is to be further understood that where descriptions of various embodiments use the term "comprising," and/or "including" those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of or "consisting of."

[0109] While particular embodiments of the present disclosure have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the disclosure. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this disclosure.

[0110] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting. As used in the specification and appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.