WO2009152483A2 - Nitric oxide induced adaptive resistance as a therapy for central nervous system diseases and trauma - Google Patents

Abstract

Disclosed herein are methods for treating Central Nervous System (CNS) diseases and CNS injuries. The methods provide to cells an amount of nitric oxide that induces in the cells an adaptive resistance to high levels of nitric oxide which are destructive and which are associated with diseases of the Central Nervous System, inter alia, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease and diabetic neuropathy. Also disclosed are methods for determining a therapeutically effective amount of nitric oxide. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present disclosure.

Description

NITRIC OXIDE INDUCED ADAPTIVE RESISTANCE AS A THERAPY FOR CENTRAL NERVOUS SYSTEM DISEASES AND TRAUMA

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 61/061,696 filed June 12, 2008, which is incorporated herein by reference in its entirety.

FIELD

Disclosed herein are methods for treating Central Nervous System (CNS) diseases and CNS injuries. The methods provide to cells an amount of nitric oxide that induces in the cells an adaptive resistance to high levels of nitric oxide which are destructive and which are associated with diseases of the Central Nervous System, inter alia, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and multiple sclerosis (MS).

BACKGROUND

Nitric oxide (NO) is a free radical gas that has a Janus nature. In the cell, NO can either function as a beneficial physiological agent utilized for essential functions such as differentiation or neurotransmission, or as a pathological agent that causes or exacerbates central nervous system disease and injury. Whether NO is helpful or harmful depends on a variety of factors, such as the cellular environment in which NO is released, the rate of NO flux, as determined by which NOS isozyme is activated, and what array of second messenger cascades are available for utilization by NO for beneficial or toxic cell signaling. Understanding the mechanisms by which NO is beneficial in one set of circumstances and toxic in another is critical and offers therapeutic targets for the mitigation of NO-mediated damage seen during CNS disease and injury.

Affecting how the actions of NO are transduced in the cell leads to more targeted application of therapies that utilize induced adaptive resistance (IAR) as a method for treating diseases related to the central nervous system. As such, there is a need for methods of treating CNS diseases and CNS-related injuries, as well as tumors that can self-induce resistance to NO.

BRIEF DESCRIPTION OF THE FIGURES

Figure Ia is a micrograph of NSC34 cells that were untreated and which did not receive a subsequent challenge with a high dose of nitric oxide.

Figure Ib is a micrograph of NSC34 cells that were pre-treated with a low dose of nitric oxide, but which were not subsequently challenged with a high dose of nitric oxide. Figure Ic is a micrograph of NSC34 cells not pre-treated with a low dose of nitric oxide, but which were subsequently challenged with a high dose of nitric oxide.

Figure Id is a micrograph showing the induced adaptive resistance of NSC34 cells that were pre-treated with a low dose of nitric oxide and which were subsequently challenged with a high dose of nitric oxide.

Figure 2 is a graph of the results depicted in Figures 1-4.

Figure 3 is a micrograph of a Western blot of the NSC34 cells depicted in Figures 1-4 indicating the presence of nitrotyrosine.

Figure 4 is a graph showing the relative amounts of nitrotyrosine present in the NSC34 cells depicted in Figures 1-4 correlates with the cytotoxicity of NO to cells.

Figure 5 is a graph depicting the amount of nitrotyrosine present in the non- pretreated but nitric oxide challenged NSC34 cells of Example 2.

Figure 6 is a graph depicting the amount of nitrotyrosine present in the pre-treated and nitric oxide challenged NSC34 cells of Example 2 showing that induced adaptive response is correlated to the absence of increase nitrotyrosine formation.

Figure 7a is a micrograph depicting the induced adaptive resistance of NSC34 cells when pre-treated with a low dose of nitric oxide and subsequently challenged with a high dose of nitric oxide.

Figure 7b is a micrograph depicting NSC34 cells not receiving pre-treatment with a low dose of nitric oxide, but which were subsequently challenged with a high dose of nitric oxide

Figure 7c is a micrograph depicting NSC34 cells not receiving pre-treatment with a low dose of nitric oxide that contained the peroxynitrite scavenger uric acid prior, but were not challenged with a high dose of nitric oxide. Figure 7d is a micrograph depiction NSC34 cells not receiving pre-treatment with a low does of nitric oxide that contained the peroxynitrite scavenger uric acid prior to being challenged with a high dose of nitric oxide.

Figure 7e is a micrograph depicting NSC34 cells not receiving pre-treatment with a low dose of nitric oxide that contained the nitric oxide scavenger 2-phenyl-4,4,5,5,- tetramethylimidazoline-1-oxyl 3-oxide (PTIO) prior, but were not challenged with a high dose of nitric oxide.

Figure 7f is a micrograph depiction NSC34 cells not receiving pre-treatment with a low does of nitric oxide that contained the nitric oxide scavenger 2-phenyl-4,4,5,5,- tetramethylimidazoline-1-oxyl 3-oxide (PTIO) prior to being challenged with a high dose of nitric oxide.

Figure 8 is a graph depicting the percentage of cell survival for samples 7a and 7b. Figure 9 is a graph depicting the percentage of cell survival for samples 7c and 7d. Figure 10 is a graph depicting the percentage of cell survival for samples 7e and 7f.

Figure 11 depicts the plots of the amount of in vivo total plasma nitric oxide found for study group Group 1 (□) and Group 2 (■) over time.

DETAILED DESCRIPTION

Before the present compounds, composites, compositions, and/or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which need to be independently confirmed.

Throughout the description and claims of this specification the term "comprise" and other forms of the term, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other elements, additives, components, integers, or steps. Thus, such terms are inclusive or open-ended transitional terms and do not exclude additional, unrecited elements, additives, components, integers, or steps, hi one aspect, these terms are synonymous with "having," "including," "containing," or "characterized by."

As used herein, the terms "consisting essentially of or "consists essentially of are generally open-ended transitional terms, but limit the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

As used in the description and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nanoparticle" includes mixtures of two or more such nanoparticles. "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed, then "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed, then "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout the application data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. "Nitric oxide" as used herein refers to the free radical having the formula NO. The nitric oxide disclosed herein can have any source, for example, a gas, solution, as a stabilized combination with one or more ingredients, or the nitric oxide can be generated in situ from one or more nitric oxide generators.

"Pre-treatment" as used herein refers to exposing cells for a period of time to a flux of nitric oxide sufficient to induce adaptive resistance to the exposed cells.

"Adaptive resistance" or "adaptive resistant cells" as used herein refers to cells that when challenged by a flux of nitric oxide of at least about 110 pmol/sec will have at least about 20% more cells that survive than like cells that are exposed to the same challenging flux of nitric oxide but that have not been pre-treated in a manner that the adaptive resistant cells have been pre-treated.

"Nitric oxide challenge" as used herein refers to exposing or contacting cells with a nitric oxide flux of at least about 110 pmol/sec. It has been found that found that an adaptive response can be induced into cells which protect the cells from the high nitric oxide flux that can occur during injury, during the onset or during certain neurological diseases. As such, the present disclosure provides a number of medical and diagnostic advantages, non-limiting examples of which include:

1) A method for treating a disease that affects neuronal cells and glial (oligodendrocytes) cells ;

2) A method for preventing a disease that affects neuronal cells;

3) A method for determining if a disease is resistant to nitric oxide mediation treatment; and

4) A method for determining the therapeutic amount of nitric oxide pre- treatment necessary to either prevent or to mediate a disease that affects neuronal cells.

Nitric oxide is a free radical that under normal conditions and is released and utilized by the central nervous system for neurotransmission and differentiation. Nitric oxide, however, is toxic to cells if released where it is not utilized, released in excessive amounts, or at a high flux rate. Nitric oxide can be released after an injury, inter alia, spinal cord trauma, or during neurodegenerative diseases, inter alia, multiple sclerosis (MS), Alzheimer's, and amyotrophic lateral sclerosis (ALS).

The concentration of nitric oxide present in a mixture or a sample can be determined using a chemiluminescent reaction involving ozone (Fontijn, A. et al, (1970). "Homogeneous chemiluminescent measurement of nitric oxide with ozone." Analytical Chemistry 42(6): 575-579). A sample containing nitric oxide is mixed with a large quantity of ozone. The nitric oxide reacts with the ozone to produce oxygen, nitrogen dioxide, and light which is measured with a photodetector. The amount of light produced is proportional to the amount of nitric oxide in the sample.

Other methods of testing include electroanalysis (amperometric approach), where NO reacts with an electrode to induce a current or voltage change. The detection of NO radicals in biological tissues can be difficult due to the short lifetime and concentration of NO radicals in tissues. One method for determing the amount of nitric oxide in tissue is spin trapping the nitric oxide with iron-dithiocarbamate complexes and subsequent detection of the mono-nitrosyl-iron complex using Electron Paramagnetic Resonance (EPR) (Vanin A. F. et al; Methods in Enzymology vol 359 (2002) 27 - 42 and Nagano T. et al; "Bioimaging of nitric oxide," Chemical Reviews vol 102 (2002) 1235 - 1269). A further method relates to fluorescent dye indicators available in an acetylated form for intracellular measurements. One example is 4,5-diaminofluorescein (DAF-2) (Culotta E. et al, "NO news is good news. (nitric oxide; includes information about other significant advances & discoveries of 1992) (Molecule of the Year)." Science 258 (5090): 1862-1864).

It has been discovered that pre-treating neuronal cells in vivo, in vitro, or ex vivo with from about 0.01 picomoles of nitric oxide per sec (pmol/sec) to about 100 pmol/sec can induce adaptive resistance to cells. In one embodiment, the amount of nitric oxide is from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide. In another embodiment, the amount of nitric oxide is from about 0.1 pmol/sec to about 20 pmol/sec of nitric oxide. In a further embodiment, the amount of nitric oxide is from about 0.01 pmol/sec to about 10 pmol/sec of nitric oxide, hi a yet further embodiment, the amount of nitric oxide is from about 0.1 pmol/sec to about 10 pmol/sec of nitric oxide. In a still further embodiment, the amount of nitric oxide is from about 0.5 pmol/sec to about 8 pmol/sec of nitric oxide. In a yet another embodiment, the amount of nitric oxide is from about 0.5 pmol/sec to about 5 pmol/sec of nitric oxide. In a still yet further embodiment, the amount of nitric oxide is from about 1 pmol/sec to about 5 pmol/sec of nitric oxide. However, the amount of nitric oxide that comprises the pre-treatment flux can have any value, for example, 1 pmol/sec, 2 pmol/sec, 3 pmol/sec, 4 pmol/sec, 5 pmol/sec, 6 pmol/sec, 7 pmol/sec, 8 pmol/sec, 9 pmol/sec, and 10 pmol/sec, or the amount can be any fractional amount, for example, 1.5 pmol/sec, 2.9 pmol/sec, and the like.

When a neuronal cell is pre-treated as disclosed herein with nitric oxide then exposed to a nitric oxide flux of at least about 110 pmol/sec, the pre-treated cells will have at least about 30 % more cells living cells after about 24 hours than neuronal cells exposed to at least about 110 pmol/se of nitric oxide flux but not pre-treated with nitric oxide. In another embodiment, the pre-treated cells will have at least about 40% more living cells after about 24 hours. In further embodiment, the pre-treated cells will have at least about 50% more living cells after about 24 hours. In a yet further embodiment, the pre-treated cells will have at least about 60% more living cells after about 24 hours. In a still further embodiment, the pre-treated cells will have at least about 70% more living cells after about 24 hours. In a yet still further embodiment, the pre-treated cells will have at least about 80% more living cells after about 24 hours. In a yet another embodiment, the pre-treated cells will have at least about 90% more living cells after about 24 hours. However, the measurement of living cells can be made at any time after exposure to the challenge flux of nitric oxide of at least about 110 pmol/sec, for example, 1 hour, 2 hours, 3 hours, 4, hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hour, 10 hours, 11 hours, 12, hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hour, 18 hours, 19 hours, 20, hours, 21 hours, 22 hours, 23 hours, 31 hours, 48 hours, and 72 hours. The time can also be any fractional amount of an hour, for example, 30 minutes, 6.7 hours, and the like.

PROCEDURES Treatment of CNS Cell Lines or Cells with Nitric Oxide Donor

Motor neuron cell lines (human or mouse) are pretreated with varying flux levels of nitric oxide gas. Alternatively, subdermal implants of a nitric oxide donor at varying concentrations can be used which provide a pretreatment flux of about 2 pmol/sec.

Group I: After pretreatment, cells are challenged with a nitric oxide flux yielding from about 100 pmol/sec to about 1 μmol/sec.

Group II: A control group is established wherein the cells receive from about 100 pmol/sec to about 1 μmol/sec nitric oxide flux challenge but no pre-treatment.

Group III: This group receives the nitric oxide pre-treatment, but does not receive the from about 100 pmol/sec to about 1 μmol/sec nitric oxide flux challenge.

Group IV: This group receives no pre-treatment or nitric oxide challenge. This protocol is then tested at varying levels of pre-treatment doses, i.e., from about 0.01 pmol/sec to about 50 pmol/sec over a range to time courses. The data can be used to establish the effective dose of nitric oxide gas or nitric oxide donor suitable to treat the infected cells. Nitric Oxide Treated CNS Cell Assay

After the cells have been treated under the above protocol for 1 day, 2 days, 3 days, and 4 days, the cells or cell lines (rats or human) are analyzed for cell death by the Trypan blue cell stain method and TUNEL assay as described herein. The percent difference in cell death in CNS cells which have received a pre-treatment prior to toxic nitric oxide challenge versus the percent cell death for CNS cells not receiving the pre-treatment but which receive the toxic nitric oxide challenge are plotted and the data analyzed for effective pre-treatment levels. In addition, the amount of nitric oxide mediated nitrotyrosine formation is evaluated for each sample group. The nitrotyrosine level has been established as a marker for multiple sclerosis, amyotrophic lateral sclerosis, and spinal injury. Whole Animal Procedure Rats are pretreated with a variety of fluxes of nitric oxide gas or injections of a selected nitric oxide donor. Dosing is such that the pretreatment levels can range from about 0.01 pmole/sec to about 50 pmol/sec. After dosing, the pretreated rats are given a nitric oxide challenge with a nitric oxide flux of from about 100 pmol/sec to about 1 mmol/sec of nitric oxide. Other animals are treated according to the procedure of Groups III and IV as further described herein. After 1 day, 2 days, 3 days, 4 days the rats are sacrificed and the brain slices and spinal cords are isolated by the above described methods. They are analyzed for cell death by the Trypan blue cell stain method and TUNEL assay described in methods. The percent difference in cell death in CNS tissue of rats who have received pretreatment before toxic nitric oxide challenge versus rats who received toxic nitric oxide challenge without nitric oxide pretreatment is quantified. Also quantified is whether nitric oxide mediated nitrotyrosine (3NY) formation is mitigated or not. 3NY is a quantifiable marker for NO mediated damage. Intact Animal Assay The rats are assayed for any neurological toxicity by the hanging and gripping assay where the rats are hung by the tail and their grasping and reaching is videotaped and quantified. Death and sickness in the animals are assayed by outward physiological signs such as uneven gait, irritability, sluggishness and death and these parameters are analyzed in animals who have received the pretreatment dose before toxic nitric oxide challenge, animals who received the toxic nitric oxide challenge alone, well as control animals treated with the vehicle alone. Methods: Tissue Culture: Growth and maintenance of NSC34 cells

The protocols for growth and maintenance of the NSC 34 cells according to Bishop A. et al. , "Adaptive resistance to nitric oxide in motor neurons." Free Radical Biology & Medicine 26(7/8) 978-986 (1999) and Bishop A. et al, "Decreased resistance to nitric oxide in motor neurons of HO-I null mice." BBRC 325:3-9 (2004), both of which are incorporated by reference in their entirety, are used. NSC34 cells are made from a fusion of primary spinal cord motor neurons with spinal neuroblastoma cells. These cells provide a homogenous and partially immortalized line of cells that have all the characteristics of motor neurons for which the these assays are relevant. The motor neuron characteristics that are exhibited by these cells are an expression of key neurofilament proteins, the generation of characteristic action potentials, production of acetylcholine, induction of acetylcholine receptors when cultured in the presence of myotubules, as well as innervation and twitching of co-cultured myotubules. These cells exhibit none of the characteristics of the neuroblastoma except for one beneficial characteristic for experimental purposes-the ability to divide and therefore can be manipulated for biochemical and molecular studies. These cells spontaneously differentiate in a controlled and predictable manner and can be terminally differentiated by the application of retinoic acid. The cells are grown in a humidified 5% CO₂ environment in plastic T25 flasks in Dulbecco's modified Eagle's medium (Mediatech; Logan, UT) without sodium pyruvate and supplemented with 10% heat-inactivated, fetal bovine serum. Vertebrate animals (mice and rats) The mice (Mus musculus) wildtype were derived from a 129 SV/BalbC strain.

Females are mated and monitored for pregnancy. On day El 3 (13^th day of pregnancy) the pregnant females are euthanized. The animals are anesthetized by flooding an airtight container with carbon dioxide gas. Death or unconsciousness is assessed by unresponsiveness to tail pinch or loud noise. The mouse is sterilized by ethanol wash and the aorta immediately severed to terminate mice that were merely unconscious. The uteri are extracted and the embryos harvested for cells isolated from the spinal cord. Or the brain and spinal cord is extracted from the pregnant mother or non pregnant mother after treatment.

The following procedure is utilized for confirmatory studies, for all the aims where primary cells are used. Wild-type mice are mated then after 13.5-14 days of pregnancy, the dams are sacrificed and the embryos harvested (Stage E13.5-14). The spinal cord is isolated from each embryo and the meninges layers are dissected away from the isolated spinal cord to decrease the amount of fibroblasts in the culture. The dorsal roots are removed to eliminate the cell bodes of the sensory neurons thereby decreasing the sensory neuron population. The ventral spinal cord with ventral roots are utilized to increase the amount of motor neurons. The spinal cord is minced and the cells from the minced spinal cord explants are separated by buoyancy into fractions that are enriched for motor neurons. This is repeated several times to "enrich" further for motor neuronal cell types in one fraction. The enriched cells are plated and maintained without the glial feeder layer and with motor neurons media.

Primary Motor Neurons from Treated Animals

The following procedure is identical to the procedure disclosed herein above except that the non pregnant mice or rats are nitric oxide treated first and then the spinal cords are isolated from the treated rats or mice. This procedure is utilized for confirmatory studies. Wild-type mice are treated with nitric oxide then the upper spinal cord is isolated from each adult mouse. The meninges layers are dissected away from the isolated spinal cord to decrease the amount of fibroblasts in the culture. The dorsal roots are removed to eliminate the cell bodes of the sensory neurons thereby decreasing the sensory neuron population. The ventral spinal cord with ventral roots are utilized to increase the amount of motor neurons. The spinal cord is minced and the cells from minced spinal cord explants are separated by buoyancy into fractions that are enriched for motor neurons. This procedure is repeated several times to "enrich" further for motor neuronal cell types in one fraction. The enriched cells are plated and maintained without the glial feeder layer and with motor neurons media.

Motor neurons are identified morphologically as cells with a cell body and a single, robust axon. With Hoechst staining the motor neuronal nuclei are sharp, small and dense while any small amount of glial cells (astrocytes), have larger more diffuse nuclei. Propidium iodide staining is used to differentiate the motor neurons which have long axons and significantly more discrete cell bodies as compared to glial cells (astrocytes). In addition, cells are stained with antibodies specific to the different cell types (neurons, oligodendrocytes, astrocytes).

For immunocytochemistry studies the medium is removed, and the cells (NSC34 or primary cells from spinal cord explants) are fixed in and labeled with a variety of Ab by standard procedures. T he monoclonal neurofilament antibody, SMI-32 (specific motor neuron), can be obtained from Affiniti Research Products Ltd. and this antibody is used to label all motor neurons-(NSC34 cells or primary neurons from spinal cord explants) that are undifferentiated or differentiated. The antibodies to neurofilaments or NGFR detect neurofilaments, or NGFR or other epitopes are displayed by only differentiated neurons. Primary motor neurons are identified by additional antibodies to detect an active NOS system indicative of fully differentiated motor neurons. These antibodies are the polyclonal antibodies NMDARl, NMDAR2A/B,GluRl, GluR2/3, GluR4 which are obtained from Chemicon; or Upstate Biotechnology. Visualization of the cells that bind the primary Ab of choice can be obtained by using a variety of secondary antibodies depending on which primary Ab is used. One secondary antibody is anti-rabbit IgG (Molecular Probes), conjugated to the Oregon Green (a color suitable for our filter sets). These can to be visualized by fluorescence microscopy. For example, visualization can be accomplished by using either FITC or rhodamine-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) or peroxidase-conjugated secondary antibodies (Jackson Laboratory, West Grove, PA) with antibody positive cells detected by diaminobenzidine (DAB) staining for a non fluorescent assay. Nuclei are stained with 1 mg Hoechst 33342/mL (Molecular Probes, Eugene, OR) in PBS for 5 minutes at room temperature. Nitric Oxide Treatment The following general procedure is used to determine the level of induced adaptive response. Media containing spent nitric oxide donor is added as a control for all the following experiments. This procedure is use to induce adaptive response, to quantify adaptive response and to evaluate the level which cell are able to withstand the nitric oxide challenge flux. Diazenium-diolate NO-donors and nitroglycerin (glyceryl trinitrate) are used as the source of nitric oxide. Low nitric oxide pre-treatment doses can be effectively provided using (Z)- 1 -[2-aminoethyl)-iV-(2-ammonioethyl)amino]diazen- 1 -ium- 1 ,2-diolate] (DETA- NONOate). DETA has a relatively slow decay rate (half-life ~16 h) and releases slower and steadier nitric oxide fluxes thus allowing a nitric oxide flux that mimics physiological conditions. Spermine-NONOate, with a half-life of -40 min, can be used for cytotoxic nitric oxide challenges. The amount of spermine NONOate can be adjusted such that the nitric oxide is release in a burst that effectively mimics the toxic fluxes of nitric oxide release by macrophages and microglia. The nitric oxide donor is added to the slightly basic culture medium and the nitric oxide release commences once the pH of the medium is adjusted to about 7.4. Amounts of spermine nonoate for the nitric oxide challenge can range from 0.2 μmol NO to 0.3 μmol NO per gram of body weight. Nitric Oxide Pre-treatment and Challenge

The NSC34 cells were pre-treated with DETA-NONOate™. DETA-NONOate™ has a decay rate, T_1Z2, of approximately 16 hours and releases a nitric oxide flux that simulates a physiological exposure level.

N-(2-Aminoethyl)-N-(2-hydroxyl-2-nitrosohydrazino)-l,2-ethylenediamine (spermine-NONOate™; #567703, Calbiochem.), has a half-life, T_1/2, of approximately 40 minutes. Spermine-NONOate™ was used for the cytotoxic nitric oxide challenges. Spermine-NONOate™ releases nitric oxide at a high rate in a burst mimicking the pathological burst a cell experiences during cytotoxic nitric oxide release by macrophages and microglia. One advantage of using NONOate nitric oxide donors is the ease of application. In addition, both DETA-NONOate™ and Spermine-NONOate™ release nitric oxide at a predictable flux that can be adjusted by changing the donor concentration, pH, temperature, as well as the duration of exposure to the media. DETA-NONOate™ and Spermine-NONOate™ were each added separately to the culture medium and nitric oxide release commenced once the medium was adjusted to pH 7.4. Standard curves can be easily established indicating how much nitric oxide is released at pH7.4, 37⁰C over various time periods. One method for identifying the release of nitric oxide is the use of a colorimetric nitric oxide assay kit that is based on the Greiss Reaction. Determination of Nitric Oxide Concentration

The amount of nitric oxide released by the reagents can be calculated by measuring the extinction coefficient and absorbance exhibited by the sample at various concentrations and time. Standardized curves can be obtained using a commercially available kit, for example, a kit based upon the Greiss Reaction sold by R&D Systems Inc. Minneapolis, MN. Addition of Test Agents

Addition of lOμM 2-phenyl-4,4,5,5,-tetramethylimidazoline-l-oxyl 3-oxide (PTIO), 20μM zinc protoporphyrin IX (ZnPPIX) or lOμM uric acid to cells is always compared to a control for that agent. Controls are established for each sample wherein the test reagent is incubated alone with the cells in order to establish whether any toxicity is due to the PTIO, ZnPPIX or uric acid. Cell Survival: Trypan Blue Assay

The percent cell survival was assayed by cell number, loss of neurites, and loss of trypan blue exclusion which indicated cell death. After cells are tested for induced adaptive resistance of nitric oxide toxicity the cells were stained with 0.2% trypan blue and then fixed with 2% glutaraldehyde or methanol vapor and those that no longer exclude Trypan Blue were counted as dead. In addition, any cells that detached from the side of the plate was assayed for trypan exclusion. In general, all of cells that detached did so because they are dead and thus no longer exclude trypan blue.

EXAMPLES

Example 1 demonstrates the induction of adaptive resistance into NSC34 cells by the disclosed method.

EXAMPLE 1 NSC34 cells are harvested and equally divided into four samples.

1. Control. Sample 1 was not pre-treated by the disclosed methods nor was this sample subsequently challenged as further described herein.

2. Pre-treated/Unchallenged. Sample 2 was pretreated with (Z)-l-[2-aminoethyl)-N-(2- ammonioethyl)-amino]diazen-l-ium-l,2-diolate] DETA-NONOate™ which released nitric oxide at a flux rate of about 4 nM (4 pmol/sec) with a total amount of 14 nmoles of nitric oxide released.

3. Not pre-treated/Challenged. Sample 3 was not pre-treated but was instead given a cytotoxic challenge with spermine-NONOate™ as follows. N-(2-aminoethyl)-N-(2- hydroxyl-2-nitrosohydrazino)-l ,2-ethylenediamine (spermine-NONOate™;

#567703, Calbiochem.) which decomposed (Tv₂ = 39 minutes) to yield 2 M nitric oxide per 1 M adduct, was dissolved in TBS (pH 10). The solution was kept cold on ice until used. Spermine-NONOate™ was used for this sample at a concentration that provided a flux of nitric oxide at a rate of approximately 110 pmol/sec for a total amount of nitric oxide of 400 nmoles.

4. Pre-treated/Challenged. Sample 4 was pre-treated with DETA as in sample 2, which affords induced adaptive resistance after which this sample is treated with a cytotoxic challenge using Spermine-NONOate™ as described for sample 3. Samples 2 and 4 were pre-treated with DETA-NONOate™ and incubated for 2 hours. After 2 hours, Samples 3 and 4 were challenged by treatment of the cells with Spermine-NONOate™ as described above. Data were collected over the next 24 hours.

Figures Ia- Id are micrographs depicting the results of Example 1. Figure 2 graphically depicts the results depicted in Figures Ia- Id. As seen in Figure 2 the cells from sample Ic have a reduced viable population while the majority of cells from sample Id are viable, especially when compared to the number of surviving cells in sample Ib, which were only treated with DETA-NONOate™_.

Peroxynitrite is formed in cells from nitric oxide. Nitrotyrosine is formed in cells damaged by peroxynitrite which is present due to nitric oxide and thus the presence of this nitrated amino acid is used as a marker in studying the presence of and the degree to which nitric oxide has damaged cells. As such, the degree to which nitrotyrosine has formed when cells are challenged with a cytotoxic dose of nitric oxide can be another measure of the degree to which induced adaptive resistance occurs in cells.

EXAMPLE 2

NSC34 cells were treated under the same protocol as the samples described in Example 1. After each of the nitric oxide regimens the cells were incubated at 37⁰C and at 0, 2, or 24 hours, protein lysates were taken and analyzed by Western blot (Figure 3). Figure 4 indicates that the relative amounts of nitrotyrosine present in the NSC34 cells from Example 1 correlates with the cytotoxicity of nitric oxide toward cells. Figures 5 and 6 depict the results of the tests performed in Example 2. Figure 5 indicates that there was an increase in the percentage of nitrotyrosine present in the sample that was challenged but not pretreated. Whereas Figure 6 shows that the same pre-treated by the disclosed method prior to nitric oxide challenge had the same or less nitrotyrosine present at 2 and 24 hours as in the initial sample.

EXAMPLE 3

As seen in Examples 1 and 2, the high nitric oxide flux challenge leads to cell death, as well as nitrotyrosine formation. In this example, cells are treated with the nitric oxide scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-l-oxyl 3-oxide (PTIO). Because nitric oxide is scavenged by PTIO, no peroxynitrite can form. Thus, any induced adaptive resistance to cell death when cells are challenged by high levels of nitric oxide, would only be presumed to be due to heme-mediated nitration (Pfeiffer S. et al, "Interference of carboxy-PTIO with nitric oxide- and peroxynitrite-mediated reactions." Free Radio Biol Med; 22(5): p. 787-94 (1997)). Further in this example, cells are treated with the peroxynitrite scavenger uric acid.

Uric acid does not affect nitric oxide. Thus, any induced adaptive resistance to cell death or inhibition of nitrotyrosine formation would be presumed to be due to peroxynitrite mediated protein nitration (Espey M. G. et al, "Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms." PNAS 99; p. 3481-3486 (2002)). a) NSC34 cells were treated with a 2 pmol/sec pre-treatment of nitric oxide and subsequently challenged with a 110 pmol/sec dose of nitric oxide. Figure 7a is a micrograph depicting the surviving cells. b) NSC34 cells were not pre-treated, but were challenged with a 110 pmol/sec dose of nitric oxide. Figure 7b is a micrograph depicting the surviving cells. c) NSC34 cells containing uric acid were not pre-treated, but and were not challenged with a 110 pmol/sec dose of nitric oxide. Figure 7c is a micrograph depicting the surviving cells. d) NSC34 cells containing uric acid were not treated with a 2 pmol/sec pre- treatment of nitric oxide and subsequently challenged with a 110 pmol/sec dose of nitric oxide. Figure 7d is a micrograph depicting the surviving cells. e) NSC34 cells containing 2-phenyl-4,4,5,5,-tetramethylimidazoline-l-oxyl 3- oxide (PTIO) that were not pre-treated and were not challenged with a 110 pmol/sec dose of nitric oxide. Figure 7e is a micrograph depicting the surviving cells. f) NSC34 cells containing 2-phenyl-4,4,5,5,-tetramethylimidazoline-l-oxyl 3- oxide (PTIO) were not treated with a 2 pmol/sec pre-treatment of nitric oxide and were challenged with a 110 pmol/sec dose of nitric oxide. Figure 7f is a micrograph depicting the surviving cells. Figure 8 is a graph of the percent survival of cells from Examples 3a and 3b as depicted in the micrographs Figures 7a and 7b. Figure 9 is a graph of the percent survival of cells from Examples 3c and 3d as depicted in the micrographs Figures 7c and 7d. Figure 10 is a graph of the percent survival of cells from Examples 3e and 3f as depicted in the micrographs Figures 7e and 7f. The percent survival of cells pre-treated with 2 pmol/sec nitric oxide exceeds the percent survival of cells treated with a peroxynitrite scavenger (uric acid) or a nitric oxide scavenger (PTIO).

EXAMPLE 4

An in vivo study utilizing three hundred 8-week old male C57BL6/J mice was conducted to demonstrate the induced adaptive resistance from low dose nitric oxide using nitroglycerin and a toxic nitric oxide challenge using spermine nonoate as a model for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Alzheimer's Disease (AD), diabetic neuropathy, and Parkinson's disease. Nitric Oxide Pharmacokinetic Studies

A total of 60 C57BL6/J mice were divided into two separate groups comprising 30 mice each. One group was dosed with a sample formulation comprising 0.002% of nitroglycerin (Group 1) and one group was dosed with a sample comprising 2% nitroglycerin (Group 2). Tables I and II herein below provide a summary of the results for these two groups. The samples were delivered topically to the scruff of the neck and subsequently three animals were sacrificed at the following times: 0, 5, 15, and 30 minutes, 1, 2, 6, 12, 24, and 48 hours.

A series of standardized curves were used to determine the amount of total nitric oxide present in each plasma sample. Assay Designs Total Nitric Oxide Assay Kit™ (cat. # 917-020) was used to measure the total amount of nitrates and nitrites present in each sample and each sample was run in duplicate. Dilution standards were made up to final concentration of 0 μM (blank), 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

The plasma samples were diluted 1 : 1 with distilled water after which the protein was removed by centrifuging at 400 x g for 1 hour using 10,000 MWCO dialysis units available from Pierce (cat # 69572). The filtrate was then directly assayed, and the concentration determined by measuring the absorbance at 540 nm using a VERS Amax™ spectrophotometer. The following standardized curves were used to determine the plasma levels of nitric oxide as indicated in Tables I and II herein below

Standard Curve A

Standard Curve B

Standard Curve C

Standard Curve D

TABLEI

TABLE II

Figure 11 depicts the plots of the amount of total plasma nitric oxide found for Group 1 (D) and Group 2 (■) over time. For Group 1 the level of nitric oxide remained approximately the same throughout the course of this experiment thereby providing a more level nitric oxide flux. The flux of nitric oxide for Group 2 leveled off at approximately 6 hours and converged with the levels found for animals dosed with 0.002% nitroglycerin (Group 1). Nitric Oxide Challenge

Ten wild type mice were injected with approximately 0.07 mg/g of spermine NONOate. This level of spermine NONOate produces a mitochondrial challenge that can potentially induce death and was chosen because mice given this amount of this nitric oxide flux producing compound had an approximately 25% survival rate. After receiving a single IV injection of spermine NONOate, the animals' response to several tests were monitored. One key test was the ability of the animal to right itself from its back 30 seconds after injection. Mice given this amount of spermine NONOate was used in the adaptive resistance challenge test. Adaptive Resistance Challenge Test

Similar to the in vitro tests described herein above, an adaptive resistance challenge test was conducted to determine if mice pretreated with a therapeutic amount of nitroglycerin would survive a nitric oxide flux challenge. Forty mice were divided into cohorts often mice each and treated as described in Table III.

TABLE III

Each animal in Group I was given the nitroglycerin treatment followed by an IV injection of spemine NONOate. For a 20 g animal approximately 1.4 jf|g of spermine NONOate is administered. The survival rate of the animals was then monitored to determine if a greater than 25% survival rate is achieved. Adaptive Response in ALS Mouse Model Predicated on the level of the nitric oxide flux providing compound found to be efficacious in Protocols 1 and 3 various levels of a nitric oxide flux providing compound can be tested in an ALS mouse model. SOD1*G93A C57BL/6 mice available from Jackson Laboratories, West Sacramento, CA 95605 can be used for this protocol.

For example, cohorts of 15 male mice each can be treated with varying amounts of a nitric oxide flux providing compound and the overall increase in life span for each group is monitored. In one embodiment, the same criteria used in protocol 2 can be used to assess the animals treated in this protocol. For example, the ability to no longer right itself after an animal is placed on its back.

In addition, further end points or test observations can be included in this protocol, for example, amount of a nitric oxide flux providing compound necessary to extend life for a finite period, i.e., 25% more time. In addition, rotarod and grip strength tests can be used as a clinical observation.

METHODS

Disclosed herein are methods for treating a neurodegenerative disease, comprising administering to a human or mammal having a neurodegenerative disease with from about 0.01 pmol/sec to about 500 pmol/sec of nitric oxide. The disclosed methods include providing a nitric oxide flux by any means of administration. In addition, disclosed herein are methods for treating a human or a mammal diagnosed with a neurodegenerative disease, comprising administering to a human or mammal diagnosed as having a neurodegenerative disease with from about 0.01 pmol/sec to about 500 pmol/sec of nitric oxide. In one embodiment, the amount of nitric oxide is from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide, hi another embodiment, the amount of nitric oxide is from about 0.1 pmol/sec to about 20 pmol/sec of nitric oxide, hi a further embodiment, the amount of nitric oxide is from about 0.01 pmol/sec to about 10 pmol/sec of nitric oxide, hi a yet further embodiment, the amount of nitric oxide is from about 0.1 pmol/sec to about 10 pmol/sec of nitric oxide. In a still further embodiment, the amount of nitric oxide is from about 0.5 pmol/sec to about 8 pmol/sec of nitric oxide. In a yet another embodiment, the amount of nitric oxide is from about 0.5 pmol/sec to about 5 pmol/sec of nitric oxide, hi a still yet further embodiment, the amount of nitric oxide is from about 1 pmol/sec to about 5 pmol/sec of nitric oxide.

The present methods include a treatment of a neurodegenerative disease, comprising administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.001 mg to about 0.3 mg of nitric oxide in plasma per kg of body weight on a continuous basis, hi one embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.01 mg to about 0.3 mg of nitric oxide in plasma per kg of body weight on a continuous basis, hi another embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.001 mg to about 0.01 mg of nitric oxide in plasma per kg of body weight on a continuous basis, hi a further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.005 mg to about 0.01 mg of nitric oxide in plasma per kg of body weight on a continuous basis, hi a still further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.01 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis, hi a yet still further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.001 mg to about 0.05 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

The present methods further include a treatment of a neurodegenerative disease, comprising administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.001 mg to about 0.1 mg of nitric oxide per hour per kg of body weight, hi one embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.001 mg to about 0.1 mg of nitric oxide per hour per kg of body weight. In another embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.001 mg to about 0.01 mg of nitric oxide per hour per kg of body weight. In a further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.001 mg to about 0.05 mg of nitric oxide per hour per kg of body weight, hi a still further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.01 mg to about 0.1 mg of nitric oxide per hour per kg of body weight. In a yet still further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator that provides from about 0.05 mg to about 0.1 mg of nitric oxide per hour per kg of body weight.

The present methods yet further include a treatment of a neurodegenerative disease, comprising administering to a human or mammal a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 1 μM to about 500 μM. hi another embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 1 μM to about 300 μM. hi a further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 10 μM to about 100 μM. In a yet further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 25 μM to about 50 μM. In a still further embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 100 μM to about 500 μM. hi a yet another embodiment, the method comprises administering to a human or mammal a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 250 μM to about 500 μM. The method for delivering nitric oxide on a continuous basis can be in any manner convenient to the user. For example, a compound that provides a source of nitric oxide to the body can be introduced by various methods known in the art. These methods include intravenous delivery, transdermal patch, suppository, sublingually (including but not limited to a sublingual spray), or by a topical formulation, for example, a cream or lotion.

The plasma nitric oxide level can be determined by any method chosen by the formulator, for example, using a kit or device designed to measure the total nitrates and nitrites in plasma. The Total Nitric Oxide Assay Kit™ (cat. # 917-020) available from Assay Designs is one kit that can be used to measure the nitric oxide level or nitric oxide flux value.

Further disclosed herein are methods for determining the effective amount of nitric oxide or nitric oxide generator necessary to achieve induced adaptive resistance. The disclosed method comprises: a) pre-treating neuronal cells by contacting the cells with from about 0.01 pmol/sec to about 50 pmol/sec of nitric oxide; b) challenging the cells with at least about 110 pmol/sec; and c) comparing the number of surviving cells from Step (b) with cells that have been challenged with at least about 110 pmol/sec of nitric oxide but have not been pre- treated with from about 0.01 pmol/sec to about 50 pmol/sec of nitric oxide.

The method can be used with cells in vivo, in vitro, or ex vivo. Incubation of the cells can take place for from about 0.1 hour to about 48 hours prior to Step (b). In another embodiment, incubation of the cells can take place for from about 0.1 hour to about 24 hours prior to Step (b). In a further embodiment, incubation of the cells can take place for from about 01 hour to about 10 hours prior to Step (b). In a yet further embodiment, incubation of the cells can take place for from about 1 hour to about 4 hours prior to Step (b). In a still further embodiment, incubation of the cells can take place for about 2 hours prior to Step (b). The nitric oxide can be provided by a nitric oxide generator. Non-limiting examples of nitric oxide generators include sodium nitroprusside, (Z)-l-[2-aminoethyl)-N-(2- ammonioethyl)-amino]diazen-l-ium-l,2-diolate], N-(2-Aminoethyl)-N-(2-hydroxyl-2- nitrosohydrazino)-! ,2-ethylenediamine, or 3-morpholino-sydnonimine. Disclosed herein are methods for treating a human or a mammal having a neurodegenerative disease. The method can be used to prevent the onset of a neurodegenerative disease, for example, in a human or an animal having the propensity for developing a neurodegenerative disease base upon DNA testing. As such, the method relates to the prophylactic use of the methods for treating a human or a mammal.

The methods can also be used to treat a human or a mammal suffering from a neurodegenerative disease. The methods can be used to titer the level of nitric oxide treatment to meet the development of the disease or to establish an effective level of treatment. The disclosed methods comprise administering to a human or a mammal an effective amount of nitric oxide or a nitric oxide generator.

The disclosed methods comprise providing adaptive resistance to the healthy cells of a human or a mammal against the effects of nitric oxide or peroxynitrite released by unhealthy cells due to a neurodegenerative disease. The disclosed methods comprise providing adaptive resistance to a cell against cell death due to a high flux of nitric oxide or peroxynitrite. The source of high nitric oxide or peroxynitrite flux can be due to trauma or a neurodegenerative disease. As such, the disclosed methods comprise a method of controlling the effects of a high nitric oxide or peroxynitrite flux experienced by healthy cells by inducing adaptive resistance to cell death due to fluxes of nitric oxide or peroxynitrite.

FORMULATIONS

Adaptive resistance can be induced by providing a therapeutic amount of nitric oxide to a human or a mammal. The term "effective amount" as used herein means "an amount of nitric oxide effective at dosages and for periods of time necessary to achieve the desired or therapeutic result." An effective amount may vary according to factors known in the art, such as the disease state, age, sex, and weight of the human or animal being treated. Although particular dosage regimes may be described in examples herein, a person skilled in the art would appreciated that the dosage regime may be altered to provide optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. In addition, the compositions of the present disclosure can be administered as frequently as necessary to achieve a therapeutic amount.

The present disclosure also relates to compositions or formulations which comprise nitric oxide. In general, the compositions of the present disclosure comprise: a) an effective amount nitric oxide according to the present disclosure can be used for treating trauma or a neurodegenerative disease, non-limiting examples of which include amyotrophic lateral sclerosis (ALS), Alzheimer's, and multiple sclerosis (MS); and b) one or more excipients. The formulator will understand that excipients are used primarily to serve in delivering a safe, stable, and functional pharmaceutical, serving not only as part of the overall vehicle for delivery but also as a means for achieving effective absorption by the recipient of the active ingredient. An excipient may fill a role as simple and direct as being an inert filler, or an excipient as used herein may be part of a pH stabilizing system or coating to insure delivery of the ingredients safely to the stomach. The formulator can also take advantage of the fact the compounds of the present disclosure have improved cellular potency, pharmacokinetic properties, as well as improved oral bioavailability.

Non-limiting examples of compositions according to the present disclosure include: a) an amount of nitric oxide sufficient to provide from about 0.1 pmol/sec to about 50 pmol/sec of a flux of nitric oxide for at least 24; and b) one or more excipients.

Another example according to the present disclosure relates to a cylinder containing gaseous nitric oxide, the composition comprising: a) an amount of nitric oxide sufficient to provide from about 0.1 pmol/sec to about 50 pmol/sec of a flux of nitric oxide for at least 24; and b) one or more carrier gasses.

A further example according to the present disclosure relates to the following compositions: a) an amount of nitric oxide sufficient to provide from about 0.1 pmol/sec to about 50 pmol/sec of a flux of nitric oxide for at least 24; and b) oxygen gas or a source of oxygen gas.

As described herein above, the formulations of the present disclosure include pharmaceutical compositions comprising nitric oxide or nitric oxide generator that can initiate adaptive resistance to cells and therefore is suitable for use in treating amyotrophic lateral sclerosis (ALS), Alzheimer's, and multiple sclerosis (MS) (or a pharmaceutically- acceptable salt thereof) and a pharmaceutically-acceptable carrier, vehicle, or diluent. Those skilled in the art based upon the present description and the nature of any given inhibitor identified by the assays of the present invention will understand how to determine a therapeutically effective dose thereof. The pharmaceutical compositions may be manufactured using any suitable means, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in a conventional manner using one or more physiologically or pharmaceutically acceptable carriers (vehicles, or diluents) comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any suitable method of administering a pharmaceutical composition to a patient may be used in the methods of treatment of the present invention, including injection, transmucosal, oral, inhalation, ocular, rectal, long acting implantation, liposomes, emulsion, or sustained release means.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For ocular administration, suspensions in an appropriate saline solution are used as is well known in the art. For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols, hi addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. A pharmaceutical aerosol spray containing 0.001% to 0.1% nitroglycerin is another buccal administration route.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. hi the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds maybe prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen- free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

One type of pharmaceutical carrier for hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.

The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD: 5 W) consists of VPD diluted 1 :1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.

Furthermore, the identity of the co-solvent components maybe varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. , polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed. Additionally, the compounds may be delivered using any suitable sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a prolonged period of time. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Many of the agents of the invention may be provided as salts with pharmaceutically acceptable counterions. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. Other aspects of the present invention include methods of treating a condition or a disease in a mammal comprising administering to said mammal a pharmaceutical composition of the present invention.

The administration of nitric oxide can be carried out by its introduction into the patient as a gas along with other normal inhalation gases given to breathe the patient. In one embodiment, a cylinder under pressure is provided and that can be at pressures of from about 1000 psi to about 2000 psi. The cylinder comprises an admixture of nitric oxide and nitrogen with a concentration of nitric oxide of from about 800 ppm to about 2000 ppm. A pressure reduction apparatus is used in conjunction with the disclosed cylinder, such that the amount of nitric oxide being administered to the human or mammal is from about 0.01 pmol/sec to about 100 pmol/sec. Regulators capable of metering out the proper amount of nitric oxide are commercially available, for example by MKS Instruments, Inc. of Andover, MA. One such device for delivery of nitric oxide to a patient is disclosed in U.S. 5,558,083 include herein by reference in its entirety.

While particular embodiments of the present disclosure have been illustrated and described, it would be obvious to those 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.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a neurodegenerative disease, comprising administering to a human or mammal having a neurodegenerative disease with a sufficient amount of a nitric oxide generator to provide a nitric oxide flux of from about 0.01 pmol/sec to about 500 pmol/sec of nitric oxide.

2. A method for treating a neurodegenerative disease, comprising administering to a human or mammal diagnosed as having a neurodegenerative disease with a sufficient amount of a nitric oxide generator to provide a nitric oxide flux of from about 0.01 pmol/sec to about 500 pmol/sec of nitric oxide.

3. The method according to either Claim 1 or 2, wherein the amount of nitric oxide is from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide.

4. The method according to either Claim 1 or 2, wherein the amount of nitric oxide is from about 0.1 pmol/sec to about 20 pmol/sec of nitric oxide.

5. The method according to either Claim 1 or 2, wherein the amount of nitric oxide is from about 0.01 pmol/sec to about 10 pmol/sec of nitric oxide.

6. The method according to either Claim 1 or 2, wherein the amount of nitric oxide is from about 0.1 pmol/sec to about 10 pmol/sec of nitric oxide.

7. The method according to either Claim 1 or 2, wherein the amount of nitric oxide is from about 0.5 pmol/sec to about 8 pmol/sec of nitric oxide.

8. The method according to either Claim 1 or 2, wherein the amount of nitric oxide is from about 0.5 pmol/sec to about 5 pmol/sec of nitric oxide.

9. The method according to either Claim 1 or 2, wherein the amount of nitric oxide is from about 1 pmol/sec to about 5 pmol/sec of nitric oxide.

10. A method for treating a neurodegenerative disease, comprising administering to a human or mammal having a neurodegenerative disease with a sufficient amount of a nitric oxide generator to provide from about 0.001 mg to about 0.3 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

11. A method for treating a neurodegenerative disease, comprising administering to a human or mammal diagnosed as having a neurodegenerative disease with a sufficient amount of a nitric oxide generator to provide from about 0.001 mg to about 0.3 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

12. The method according to either Claim 10 or 11, wherein the amount of nitric oxide in plasma is from about 0.01 mg to about 0.3 mg of nitric oxide per kg of body weight.

13. The method according to either Claim 10 or 11, wherein the amount of nitric oxide in plasma is from about 0.001 mg to about 0.01 mg of nitric oxide per kg of body weight.

14. The method according to either Claim 10 or 11, wherein the amount of nitric oxide in plasma is from about 0.005 mg to about 0.01 mg of nitric oxide per kg of body weight.

15. The method according to either Claim 10 or 11, wherein the amount of nitric oxide in plasma is from about 0.001 mg to about 0.05 mg of nitric oxide per kg of body weight.

16. A method for treating a neurodegenerative disease, comprising administering to a human or mammal having a neurodegenerative disease with a sufficient amount of a nitric oxide generator to provide from about 0.001 mg to about 0.3 mg of nitric oxide per hour per kg of body weight.

17. A method for treating a neurodegenerative disease, comprising administering to a human or mammal diagnosed as having a neurodegenerative disease with a sufficient amount of a nitric oxide generator to provide from about 0.001 mg to about 0.3 mg of nitric oxide per hour per kg of body weight.

18. The method according to either Claim 16 or 17, wherein the amount of nitric oxide in plasma is from about 0.001 mg to about 0.01 mg of nitric oxide per hour per kg of body weight.

19. The method according to either Claim 16 or 17, wherein the amount of nitric oxide in plasma is from about 0.001 mg to about 0.05 mg of nitric oxide per hour per kg of body weight.

20. The method according to either Claim 16 or 17, wherein the amount of nitric oxide in plasma is from about 0.01 mg to about 0.3 mg of nitric oxide per hour per kg of body weight.

21. The method according to either Claim 16 or 17, wherein the amount of nitric oxide in plasma is from about 0.05 mg to about 0.3 mg of nitric oxide per hour per kg of body weight.

22. A method for treating a neurodegenerative disease, comprising administering to a human or mammal having a neurodegenerative disease with a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 1 μM to about 500 μM.

23. A method for treating a neurodegenerative disease, comprising administering to a human or mammal diagnosed as having a neurodegenerative disease with a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 1 μM to about 500 μM.

24. The method according to either Claim 22 or 23, wherein the concentration of nitric oxide as measured in cells or in plasma is from about from about 10 μM to about 100 μM.

25. The method according to either Claim 22 or 23, wherein the concentration of nitric oxide as measured in cells or in plasma is from about 25 μM to about 50 μM.

26. The method according to either Claim 22 or 23, wherein the concentration of nitric oxide as measured in cells or in plasma is from about 100 μM to about 500 μM.

27. The method according to either Claim 22 or 23, wherein the concentration of nitric oxide as measured in cells or in plasma is from about 250 μM to about 500 μM.

28. The method according to any of Claims 1 to 27, wherein the human or mammal is administered nitric oxide gas.

29. The method according to any of Claims 1 to 27, wherein the human or mammal is administered a nitric oxide generator.

30. The method according to any of Claims 1 to 27, wherein the human or mammal is administered a nitric oxide generator chosen from sodium nitroprusside, (Z)- 1 -[2- aminoethyl)-N-(2-ammonioethyl)-amino]diazen-l-ium-l,2-diolate], Λ/-(2- Aminoethyl)-N-(2-hydroxyl-2-nitrosohydrazino)- 1 ,2-ethylenediamine, or 3- morpholino-sydnonimine.

31. The method according to any of Claims 1 to 30, wherein the neurodegenerative disease is chosen from amyotrophic lateral sclerosis, Alzheimer's disease, or multiple sclerosis.

32. A method for determining the effective amount of nitric oxide pre-treatment for neuronal cells, comprising: a) pre-treating neuronal cells by contacting the cells with from about 0.01 pmol/sec to about 50 pmol/sec of nitric oxide; b) challenging the cells with at least about 110 pmol/sec; and c) comparing the number of surviving cells from step (b) with cells that have been challenged with at least about 110 pmol/sec of nitric oxide but have not been pre-treated with from about 0.01 pmol/sec to about 50 pmol/sec of nitric oxide.

33. The method according to Claim 32, wherein the cells are treated in vivo.

34. The method according to Claim 32, wherein the cells are treated in vitro.

35. The method according to Claim 32, wherein the cells are treated ex vivo.

36. The method according to any of Claims 32 to 35, wherein the cells are incubated for from about 0.1 hour to about 24 hours prior to step (b).

37. The method according to any of Claims 32 to 35, wherein the cells are incubated for from about 1 hour to about 10 hours prior to step (b).

38. The method according to any of Claims 32 to 35, wherein the cells are incubated for from about 1 hour to about 4 hours prior to step (b).

39. The method according to any of Claims 32 to 35, wherein the cells are incubated for from about 2 hours prior to step (b).

40. A method for treating a human or a mammal suffering from a neurodegenerative disease, comprising administering to a human or a mammal an effective amount of nitric oxide to initiate adaptive resistance to cell death due to the release of nitric oxide by unhealthy cells.

41. A method for treating a human or a mammal suffering from trauma, comprising administering to a human or a mammal an effective amount of nitric oxide to initiate adaptive resistance to cell death due to the release of nitric oxide by unhealthy cells.

42. A method for treating trauma, comprising administering to a human or mammal a sufficient amount of a nitric oxide generator to provide a nitric oxide plasma flux of from about 0.01 pmol/sec to about 500 pmol/sec of nitric oxide.

43. A method for treating trauma, comprising administering to a human or mammal a sufficient amount of a nitric oxide generator to provide from about 0.001 mg to about 0.3 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

44. A method for treating trauma, comprising administering to a human or mammal a sufficient amount of a nitric oxide generator to provide from about 0.001 mg to about 0.3 mg of nitric oxide per hour per kg of body weight.

45. A method for treating trauma, comprising administering to a human or mammal a sufficient amount of a nitric oxide generator such that the concentration of nitric oxide as measured in cells or in plasma is from about 1 μM to about 500 μM.