WO2009152479A1 - Compositions comprising nitric oxide or nitric oxide donors for the treatment of neurodegenerative diseases of trauma - Google Patents

Abstract

Disclosed herein are compositions for treating Central Nervous System (CNS) diseases and CNS injuries. The compositions 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). 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

COMPOSITIONS COMPRISING NITRIC OXIDE OR NITRIC OXIDE DONORS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES OF TRAUMA

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 61/060,971 filed June 12, 2008, which is incorporated herein by reference in its entirety.

FIELD

Disclosed herein are compositions that can be used for treating Central Nervous System (CNS) diseases and CNS injuries. The compositions can induce adaptive resistance to cells which provides cells with protection against the high nitric oxide flux released by unhealthy cells that are associated with diseases of the Central Nervous System, inter alia, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, multiple sclerosis (MS), and trauma to the Central Nervous System.

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. Unhealthy cells associated with neurodegenerative diseases or trauma release a high flux of nitric oxide which leads to cell death. Providing protection against this high flux of nitric oxide provides a method for treating neurodegenerative diseases or for mitigating the effects of trauma. One method is to induce into cells a tolerance of or adaptive resistance to the high concentrations of nitric oxide released by unhealthy cells. As such, there is a need for compositions useful for treating CNS diseases and CNS-related injuries, as well as tumors that can self-induce resistance to nitric oxide.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts the plots of the amount of in vivo total plasma nitric oxide found for study group Group 1 (G) 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. In 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.

"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.

"Nitric oxide generator" or "nitric oxide donor" as used herein refers to a compound that is capable of releasing nitric oxide, for example, nitroglycerin (glyceryl trinitrate) is capable of releasing 3 equivalents of nitric oxide. It has been found that an adaptive response can be induced into cells, which protects the cells from the high nitric oxide flux that can occur during injury, during the onset of or during the course of certain neurological diseases. As such, the present disclosure provides a number of medical and diagnostic advantages, non-limiting examples of which include: 1) Compositions for treating a disease that affects neuronal cells and glial

(oligondendrocytes) cells;

2) Compositions for preventing a disease that affects neuronal cells;

3) Compositions for treating neurological trauma;

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

5) 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).

It has been discovered that compositions that can release a nitric oxide flux of from about 0.01 picomoles of nitric oxide per sec (pmol/sec) to about 100 pmol/sec can induce cells with an adaptive resistance to the nitric oxide flux caused by the unhealthy cells associated with neurodegenerative diseases or trauma. As such, the disclosed compositions can be used to provide a human or a mammal with an effective amount of nitric oxide useful for providing cells with an adaptive resistance to high nitric oxide fluxes caused by damage or diseased cells and thus provide a method for treating a neurodegenerative disease or trauma.

The compositions can be used for pre-treating neuronal cells in vivo, in vitro, or ex vivo with from about 0.1 pmol/sec to about 100 pmol/sec of nitric oxide. In one embodiment, the amount of nitric oxide provided by the composition is from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide. In another embodiment, the amount of nitric oxide provided by the composition is from about 0.1 pmol/sec to about 20 pmol/sec of nitric oxide. In a further embodiment, the amount of nitric oxide provided by the composition is from about 0.01 pmol/sec to about 10 pmol/sec of nitric oxide. In a yet further embodiment, the amount of nitric oxide provided by the composition 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 provided by the composition 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 provided by the composition 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 provided by the composition is from about 1 pmol/sec to about 5 pmol/sec of nitric oxide. However, the amount of nitric oxide provided by the composition 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.

The disclosed compositions comprise: a) a source of nitric oxide in an amount sufficient to provide a flux of from about 0.01 pmol/sec to about 100 pmol/sec of nitric oxide; and b) one or more pharmaceutically acceptable ingredients. In one embodiment, the compositions comprise: a) nitric oxide that is provided in an amount sufficient to provide a flux of from about 0.01 pmol/sec to about 100 pmol/sec of nitric oxide; and b) one or more pharmaceutically acceptable carrier gases. In another embodiment, the compositions comprise: a) a nitric oxide donor in an amount sufficient to provide a flux of from about

0.01 pmol/sec to about 100 pmol/sec of nitric oxide; and b) one or more pharmaceutically acceptable ingredients. In another aspect, the disclosed compositions comprise: a) a source of nitric oxide in an amount sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis; and b) one or more pharmaceutically acceptable ingredients. In one embodiment, the compositions comprise: a) nitric oxide that is provided in an amount sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis; and b) one or more pharmaceutically acceptable carrier gases. In another embodiment, the compositions comprise: a) a nitric oxide donor in an amount sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis; and b) one or more pharmaceutically acceptable ingredients. In a further aspect, the disclosed compositions comprise: a) a source of nitric oxide in an amount sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide per hour per kg of body weight; and b) one or more pharmaceutically acceptable ingredients. In one embodiment, the compositions comprise: a) nitric oxide that is provided in an amount sufficient to provide from about

0.001 mg to about 0.1 mg of nitric oxide per hour per kg of body weight; and b) one or more pharmaceutically acceptable carrier gases. In another embodiment, the compositions comprise: a) a nitric oxide donor in an amount sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide per hour per kg of body weight; and b) one or more pharmaceutically acceptable ingredients. In a yet further aspect, the disclosed compositions comprise: a) a source of nitric oxide in an amount sufficient to provide a concentration of nitric oxide as measured in cells or in plasma from about 1 μM to about 500 μM; and b) one or more pharmaceutically acceptable ingredients. In one embodiment, the compositions comprise: a) nitric oxide that is provided in an amount sufficient to provide a concentration of nitric oxide as measured in cells or in plasma from about 1 μM to about 500 μM; and b) one or more pharmaceutically acceptable carrier gases. In another embodiment, the compositions comprise: a) a nitric oxide donor in an amount sufficient to provide a concentration of nitric oxide as measured in cells or in plasma from about 1 μM to about 500 μM; and b) one or more pharmaceutically acceptable ingredients.

The nitric oxide can be provided by nitric oxide gas or by a nitric oxide donor or generator. As used herein the terms "donor" and "provider" and "generator" are use interchangeably throughout the specification and claims and mean compounds that can result in increased levels of nitric oxide in a cell, plasma, or any other part of a subject. One aspect of the disclosed compositions relates to nitric oxide provided by nitric oxide gas. In another aspect, the nitric oxide can be provided by a nitric oxide donor. In one embodiment, the nitric oxide donor is a compound chosen from nitroglycerin (glyceryl trinitrate); amyl nitrate; octyl nitrate; propatyl nitrate (2-ethyl-2-(hydroxymethyl)-l,3- propanediol trinitrate); isosorbide mononitrate; (8-nitrooxy-2,6-dioxabicyclo[3.3.0]octan-4- ol); trolnitrate (2,2',2"- nitrilotrisethanol, trinitrate (ester)); ethylene glycol dinitrate, glyceryl l,2dinitrate, glyceryl 1,3-dinitrate, glyceryl 1 -mononitrate; butane 1,2,3-triol nitrate, pentaerythrityl trinitrate, isosorbide mononitrate, isosorbide mononitrate, or erythrityl tetranitrate.

In another embodiment, the nitric oxide donor is a compound chosen from sodium nitroprusside, (Z)-l-[2-aminoethyl)-N-(2-ammonioethyl)-amino]diazen-l-ium-l,2-diolate], N-(2-Aminoethyl)-N-(2-hydroxyl-2-nitrosohydrazino)-l ,2-ethylenediamine, or 3- morpholino-sydnonimine. In a further embodiment, the nitric oxide donor is a compound chosen from S- nitroso-N-acetylpenicillamine, S-nitrosoglutathione, S-nitroso-N-acetylcysteine, S-nitroso- cysteine, S-nitroso-homocystein, S-nitrosopantathoein, or S-nitroso-captopril.

Compositions comprising a nitric oxide donor can deliver a nitric oxide flux from about 0.01 picomole per second (pmol/sec) to about 100 pmol/sec of nitric oxide. In one embodiment, the compositions can deliver a nitric oxide flux from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide. In another embodiment, the compositions can deliver a nitric oxide flux from about 0.1 pmol/sec to about 20 pmol/sec of nitric oxide. In a further embodiment, the compositions can deliver a nitric oxide flux from about 0.5 pmol/sec to about 20 pmol/sec of nitric oxide, hi a still further embodiment, the compositions can deliver a nitric oxide flux from about 0.5 pmol/sec to about 10 pmol/sec of nitric oxide. Li a yet still further embodiment, the compositions can deliver a nitric oxide flux from about 0.1 pmol/sec to about 8 pmol/sec of nitric oxide.

One aspect of the disclosed compositions relates to compositions comprising: a) nitroglycerin in an amount sufficient to provide a flux of from about 0.01 pmol/sec to about 100 pmol/sec of nitric oxide; and b) one or more pharmaceutically acceptable ingredients. Nitroglycerin (glyceryl trinitrate) provide there equivalents of nitric oxide per equivalent of nitroglycerin. As such, the disclosed compositions can comprise from about 1 x 10^" mg nitroglycerin per gram of composition (0.00001%) to about 0.1 mg per gram of composition (0.01%). In one embodiment, the disclosed compositions comprise from about 5 x 10^"4 mg nitroglycerin per gram of composition (0.00005%) to about 0.05 mg per gram of composition (0.005%). In another embodiment, the disclosed compositions comprise from about 5 x 10^"4 mg nitroglycerin per gram of composition (0.00005%) to about 1 x 10^"2 mg per gram of composition (0.001%). In a further embodiment, the disclosed compositions comprise from about 1 x 10^"3 mg nitroglycerin per gram of composition (0.0001%) to about 1 x 10^"2 mg per gram of composition (0.001%). In a yet further embodiment, the disclosed compositions comprise from about 5 X lO^"4 mg nitroglycerin per gram of composition (0.00005%) to about 5 x 10^"3 mg per gram of composition (0.0005%). In a still further embodiment, the disclosed compositions comprise from about 1 x 10^"3 mg nitroglycerin per gram of composition (0.0001%) to about 5 x 10^"2 mg per gram of composition (0.001%).

Compositions comprising nitroglycerin can deliver a nitric oxide flux from about 0.01 picomole per second (pmol/sec) to about 100 pmol/sec of nitric oxide. In one embodiment, the compositions can deliver a nitric oxide flux from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide, hi another embodiment, the compositions can deliver a nitric oxide flux from about 0.1 pmol/sec to about 20 pmol/sec of nitric oxide, hi a further embodiment, the compositions can deliver a nitric oxide flux from about 0.5 pmol/sec to about 20 pmol/sec of nitric oxide, hi a still further embodiment, the compositions can deliver a nitric oxide flux from about 0.5 pmol/sec to about 10 pmol/sec of nitric oxide, hi a yet still further embodiment, the compositions can deliver a nitric oxide flux from about 0.1 pmol/sec to about 8 pmol/sec of nitric oxide. hi another embodiment, the nitric oxide generators include nitroglycerin, sodium nitroprusside, (Z)-l-[2-aminoethyl)-N-(2-ammonioethyl)-amino]diazen-l-ium-l,2-diolate], N-(2-Aminoethyl)-N-(2-hydroxyl-2-nitrosohydrazino)- 1 ,2-ethylenediamine, or 3- morpholino-sydnonimine. One embodiment comprises nitroglycerin as the nitric oxide generator.

COMPOSITIONS The disclosed compositions can be formulated to release a continuous flux of nitric oxide for about 0.01 pmol/sec to about 100 pmol/sec. The nitric oxide flux necessary to provide induced adaptive resistance in cells is well below the level of nitric oxide necessary to provide vasodilatation. For embodiments which utilize nitroglycerin as the source of nitric oxide, 3 moles of nitric oxide are released per mole of nitroglycerin. The formulator can use the following example calculation to increase or decrease the amount of nitroglycerin that comprises a formulation based upon the need of the patient or the use thereof.

The following example compares a prior art formulation that releases a vasodilating flux of nitric oxide. As shown below, the prior art formulations are too concentrated for a patient to apply a limited amount of the prior art formulation in order to treat a neurodegenerative disease or trauma. For example, one commonly prescribed prior art formulation that is a cream applied by the user to a small area of skin in order to induce vasodilation, comprises 2% by weight of nitroglycerin. This amount corresponds to a formulation that comprises 2 g (2000 mg) of nitroglycerin per 100 g of formulation. The number of millimoles of nitroglycerin delivered per gram of formula is therefore:

(2000 _mg NG/100 g form.) _{= 0}.088 mmol NG/g form. 227 mg NG/mmol

Since 3 moles of nitric oxide are released per mole of nitroglycerin, 2 grams of nitroglycerin provides:

0.264 mmol NO/g form. Expressing this amount in picomoles of nitric oxide:

0.264 mmol NO/g form. = 2.6 x 10⁸ pmol/g form.

The amount of this prior art formula needed to provide, for example, a flux of 2 pmol/sec, which is an amount within the range of the nitric oxide flux levels provided by the disclosed compositions, can be determined by the formula:

[2 pmol NO/sec] [86400 sec/day] = 0.00066 g form./day [2.6 x 10⁸ pmol/g form.]

This commercially available prior art formulation, which comprises 2% nitroglycerin, is directed by package insert instructions to be applied twice daily. The normal amount of this formula that is applied to achieve vasodilatation is approximately 1 g of formula. One gram of this prior art formulation delivers a bolus of approximately 15 mg of nitroglycerin. Therefore, prior art formulations that provide an amount of nitroglycerin necessary to achieve vasodilatation are highly concentrated when compared to the disclosed compositions. As such, it would impractical, or otherwise ineffective, for a patient to apply as little as 0.00066 g of the prior art 2% by weight nitroglycerin formula, use the highly concentrated prior art levels of nitroglycerin as compositions useful for treating neurodegenerative diseases or trauma.

EXAMPLE A For example, a 0.0004% nitroglycerin formulation comprises 0.4 mg of nitroglycerin per 100 grams of formulation.

(0.4 mgNG/100 g form.) _{n ^nn Λ C}, , _^ , _c - — - = 0.0000l8 mmol NG/g form.

227 mg NG/mmol ^&

Since 3 moles of nitric oxide are released per mole of nitroglycerin, 0.4 mg of nitroglycerin provides:

0.000054 mmol NO/g form. Expressing this amount in picomoles of nitric oxide:

0.000054 mmol NO/g form. = 5.4 x I0⁴ pmol/g form.

The amount of formula needed to provide, for example, a flux of 2 pmol/sec can be determined by the formula:

[2 pmol NO/sec] [86400 sec/day] = 3.2 g form./day

[5.4 x l0⁴ pmol/g form.]

As such, the user would need to apply approximately 1.6 g of a 0.0004% nitroglycerin comprising formula twice a day to achieve a 2 pmol/sec flux of nitric oxide. Ointments Dosage forms for the topical administration of the disclosed nitric oxide releasing compositions include ointments, pastes, salves, creams, lotions, and gels. The nitric oxide releasing compounds can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any petrolatum, lanolin, methylcellulose, sodium carboxymethylcellulose, hydroxpropylcellulose, sodium alginate, carbomers, glycerin, glycols, oils, glycerol, benzoates, parabens, surfactants, preservatives, or buffers. The disclosed topical preparations can be prepared by combining the nitric oxide releasing compounds with conventional pharmaceutical diluents and carriers commonly used in topical liquid and cream formulations. Ointment and creams can, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Such bases can include water and/or petroleum based oil, inter alia, liquid paraffin or a vegetable oil, inter alia, peanut oil or castor oil. Thickening agents which can be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and the like. Lotions can be formulated with an aqueous or oily base and, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like. The disclosed ointments, pastes, creams and gels also can contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

The disclosed ointments, pastes, salves, creams, lotions, and gels comprise: a) an amount of a nitric oxide releasing compound sufficient to release from about 0.01 pmol/sec to about 100 pmol/sec of nitric oxide; and b) one or more pharmaceutically acceptable carrier or adjunct ingredient. One embodiment relates to ointments that can release from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide over a period of from about 8 hours to about 24 hours. Li a further embodiment, the compositions can release from about 0.5 pmol/sec to about 10 pmol/sec of nitric oxide over a period of from about 8 hours to about 24 hours. In another embodiment, the compositions can release from about 0.5 pmol/sec to about 5 pmol/sec of nitric oxide over a period of from about 8 hours to about 24 hours. In a yet further embodiment, the compositions can release from about 0.1 pmol/sec to about 4 pmol/sec of nitric oxide over a period of from about 8 hours to about 24 hours. In a still further embodiment, the compositions can release from about 0.5 pmol/sec to about 10 pmol/sec of nitric oxide over a period of from about 8 hours to about 12 hours. In a still yet further embodiment, the compositions can release from about 1.5 pmol/sec to about 2.5 pmol/sec of nitric oxide over a period of from about 8 hours to about 12 hours. In addition to a nitric oxide releasing compound and carriers or adjunct ingredients, the compositions can further comprise one or more active ingredients. For example, a composition comprising: a) an amount of a nitric oxide donor sufficient to release from about 0.01 pmol/sec to about 20 pmol/sec of nitric oxide; b) an effective amount of one or more physiologically active ingredients; and c) one or more pharmaceutically acceptable carrier or adjunct ingredient. The one or more physiologically active ingredients can be a pharmaceutical agent that protects the skin during long exposure to the carriers or adjunct ingredients, or to exposure, for example, sunlight or extreme cold. The active ingredient can also be an agent used to treat a symptom or disease associated with the illness, disease, syndrome, or condition being treated by the nitric oxide. For example, a local anti-inflammatory agent, inter alia, naproxen, pramoxicam, a corticosteroid, inter alia, cortisone, hydrocortisone, an anti-itch agent, inter alia, a growth-promoting and/or wound healing-promoting agent known to promote re-epithelialization, inter alia, platelet-derived growth factor PDGF, interleukin-11 (IL-Il), or an anti-microbial agent, inter alia, neosporin, polymyxin B sulfate, bacitracin zinc.

In addition, the disclosed ointments or creams can comprise one or more preservatives or bacteriostatic agents, inter alia, methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, and benzalkonium chlorides. Further, the disclosed compositions can comprise other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.

The following Tables I-IX provide non-limiting examples of ointments, creams, salves, and the like that can deliver a nitric oxide flux of from about 0.5 pmol/sec to about 27.5 pmol/sec. All values are expressed in weight percent.

TABLE I

TABLE VIII

Transdermal Patch Disclosed herein are transdermal nitroglycerin comprising patches that can release a sufficient amount of nitroglycerin to provide a nitric oxide flux of from about 0.01 pmol/sec to about 100 pmol/sec. As used herein, the term "transdermal" is used in its conventional sense, and means the introduction and delivery of a pharmacological or medicinal compound through the skin of a patient in need of such treatment to elicit a systemic effect. Hence, transdermal delivery of a drug is effective for treating illnesses, conditions or disorders beyond mere topical applications of a medicinal agent. The transdermal system described herein is useful for the delivery of nitroglycerin to elicit an induced adaptive response to a human or an animal.

The patches can further comprise one of more carriers, skin-penetrating enhancers, and the like. The patches comprise a nitroglycerin comprising composition deposed upon a pressure sensitive adhesive patch. The patch comprises an impermeable backing material. One embodiment of a patch, comprises two regions, a center region containing the nitroglycerin composition and an outer region comprising a pharmaceutically acceptable adhesive. Carriers can be pharmaceutically acceptable solvents, non-limiting examples of which include dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), glycerin, propylene glycol, glyme, diglyme, xylitol, and the like, hi one embodiment, dimethyl sulfoxide is the solvent. In a further embodiment, N-methyl-2-pyrrolidone is the solvent, hi another embodiment, glycerin is the solvent. In a yet further embodiment, a mixture of glycerin and N-methyl-2-pyrrolidone is the solvent. The disclosed patches can further comprise one or more skin penetration enhancers. Non-limiting examples of skin penetration enhancers include oleic acid, linoleic acid, isopropyl linoleate (e.g., Ceraphyl™. IPL, from Van Dyk Division of Mallinckrodt, Inc., Belleville, NJ.), oleyl alcohol, l-dodecyl-azacycloheptan^-one and butanediol. In one embodiment, oleic acid is the skin penetration enhancer.

The transdermal patch described herein is any conventional patch from, e.g., adhesive matrix, polymeric matrix or reservoir patch, and is generally comprised of one or more backing layers, adhesive, nitroglycerin, one or more penetration enhancers, an optional rate controlling membrane and a release liner which is removed to expose the adhesive prior to application. Polymeric matrix patches also comprise a polymeric-matrix forming material. The backing layer used herein may be any conventional transdermal backing material which does not adversely react with the nitroglycerin or the other components in the patch. Examples are foam, metal foil, polyester, low density polyethylene, copolymers of vinyl chloride and polyvinylidine chloride and laminates thereof. One embodiment relates to a patch comprising water resistant polyethylene or vinyl.

The adhesive used in the patch described herein can be any pharmaceutically acceptable pressure sensitive polymeric adhesive, such as an acrylic, vinyl acetate, silicone or synthetic or natural rubber adhesive. For example, acrylic adhesives such as RA 2484, RA 2333, RA 2397, R 363 and R 362 from Monsanto Co. are appropriate. Other suitable acrylic adhesives, such as Durotak™, manufactured by Morton Thiokol, Inc., and Neocryl™ XA5210 by Polyvinyl Chemicals, Ltd. maybe utilized. Numerous silicone based adhesives may be used, such as Q72929, Q27406, X72920 and 355, each manufactured by Dow-Corning. Vinyl acetate adhesives include Flexcryl 1614, 1617, 1618 and 1625 from Air

Products. Natural and synthetic rubber adhesives include polyisobutylenes, neoprenes, polybutadienes and polyisoprenes. The adhesives may be used singly or combined in the patch.

The adhesive material may also be modified through the use of diluents or thickeners, if necessary. The preferred diluents are organic or inorganic solvents such as ethanol or water. The preferred thickeners include acrylic polymer thickeners such as Union Amsco RES 6038 by Unocal. Thickeners are used to adjust viscosity of the adhesive mixtures to about 6,000-10,000 cps for coating on the backing material. A crosslinking agent such as Aerotex Resin 3730 (American Cyanamid) may be added to facilitate curing. Non-limiting examples of polymeric matrix materials are polyvinyl alcohols, polyvinyl pyrrolidones, gelatin and partially hydrolyzed polyvinyl alcohols. Other agents may be incorporated into the matrix material, such as gelling agents, e.g., Klucel™, povidone or gelatin, or hygroscopic agents, e.g., glycerin, sorbitol or glycols. Such agents make the matrix material easier to handle and affect the rate of nitroglycerin delivery. Materials suitable for rate-controlling membranes include ethylene- vinyl acetate (EVA) copolymer membranes (e.g. 1-20% vinyl acetate), polyvinylalcohol (PVA) gels and silicone films.

Protective release liners used to prevent dirt from sticking to the patch during shipment and storage are made from such materials as polyethylene and polyethylene coated paper, polystyrene and polycarbonates, preferably silicon-coated to facilitate removal.

Adhesive matrix transdermal devices are preferred and methods for preparing them are known in the art. A preferred method for preparing adhesive matrix transdermal devices of the present invention comprises casting a thin layer of the polymer blend (i.e., the mixture of adhesive, active, skin penetration enhancer and adhesive diluents or thickeners) onto the material to be used as the release liner, curing the polymer blend to form the polymer adhesive (including drying in an oven), and laminating the backing material to the resultant adhesive layer. Suitably sized patches may then be punched out automatically, and the patches are preferably sealed into protective pouches.

The layer of polymer blend cast on the release liner according to the preferred method is preferably about 0.127 millimeters to about 0.254 millimeters thick. The cast layer is preferably dried at a temperature of about 80 ⁰C for a period of about 20 minutes. A specific example of a formulation is shown below. Polymeric matrix transdermal patches are also prepared by known methods. When a polymeric matrix is present, the adhesive can be used to coat the backing layer and to adhere the polymeric matrix to the backing-layer, leaving an adhesive margin around the polymeric matrix in order to affix the patch to the skin while allowing the drug to transfer directly from the matrix to the skin. Alternatively, the polymeric matrix can be glued to the backing and adhesive may be coated on the backing around the matrix.

Reservoir-type patches may also be made by known procedures. For example, a layer of adhesive may be applied to the release liner, the rate-controlling membrane may be laminated to the adhesive side, a portion of a solution comprising nitroglycerin and one or more penetration enhancer (e.g., the polymer blend) may be placed on the membrane, and the backing material may then be heat-sealed to the rate-controlling membrane around the edges of the patch.

Representative examples of formulations which generate unexpectedly superior flux for nitroglycerin transdermal patches are described in the following examples in Tables X- XIII. All values are in weight percent.

TABLE X

Combine the adhesive, the penetration enhancers, the thickener and the nitroglycerin and mix until smooth. Check the viscosity, and if necessary, add thickener to increase the viscosity of the adhesive blend to the required level. Cast a 0.127 10 0.254 millimeter (5-10 mil) layer of polymer blend onto the release liner. Dry the layer at 80 ⁰C for 20 minutes. Laminate the backing material to the dry polymer film using conventional equipment. Using an automatic punch machine, punch out the desired size patches. Using a pouch machine, enclose the patches in pouches and heat-seal closed. Tablet/Capsules

One aspect of this category of nitric oxide flux releasing compositions relates to delayed and sustained-release delivery of orally delivered nitric oxide generators. As used herein, "delayed release" means that the nitric oxide generators are slowly released from the pharmaceutical matrix, typically post-gastric. In one example, the sustained release of the nitric oxide generator is over a 2 hour to 12 hour period, hi another example, the sustained release of the nitric oxide generator is over a 4 hour to 6 hour period. hi one embodiment, a composition is provided as an enteric coated capsule containing a nitric oxide generator in the form of a powder (microgranules) that is coated with a one or more coating materials, for example, methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, an acrylic resin comprising one or more acrylic or methacrylic acid esters comprising a low content of quaternary ammonium groups. For example, a suitable coating includes EUDRAGIT™ L, EUDRAGIT™ S, EUDRAGIT™ RL, and EUDRAGIT™ RS. Once coated the microgranules can be compressed into tablets or packed into hard gelatin capsules suitable for oral administration. hi a further embodiment, the nitric oxide generator can be combined with a saturated polyglycolized glyceride excipient, wherein the excipient comprises a monoglyceride, a diglyceride, or triglyceride or mono- or di- fatty acid ester of polyethylene glycol hydrophobic matrix. The polyglycolized glyceride can be adapted to have any hydrophobic-lipophobic balance (HLB) desired by the formulator. For example, for a more lipophilic nitric oxide generator the melting point/HLB can be 42/12, 44/14, or 50/13. hi another example, for more hydrophilic nitric oxide generators, inter alia, water soluble salts, the melting point/HLB can be 46/07, 48/09, or 53/10.

In one example wherein nitroglycerin is the nitric oxide donor, the melting point/HLB can be 42/12 or 50/13. A non-limiting example of a suitable polyglycolized excipient that can be used with nitroglycerin is GELUCIRE™. A non-limiting example of a polyacrylic acid derivative that can be combined with nitroglycerin is a carbomer, for example CARBOPOL™ available from B. F. Goodrich Co. Such polymers are commercially available from B.F. Goodrich under the designation CARBOPOL™ 420, CARBOPOL™ 430, CARBOPOL™ 475, CARBOPOL™ 488, CARBOPOL™ 493, CARBOPOL™ 910, CARBOPOL™ 934, CARBOPOL™ 934P and the like. Carbopols are versatile controlled-release polymers, as described by Brock (Pharmacotherapy, 14:430- 7 (1994)) and Durrani (Pharmaceutical Res. (Supp.) 8:S-135 (1991)), both of which are included herein by reference, and belong to a family of carbomers which are synthetic, high molecular weight, non- linear polymers of acrylic acid, crosslinked with polyalkenyl polyether. In one embodiment the carbomer is CARBOPOL™ 974P NF.

Representative examples of table or capsule formulations that can provide a nitric oxide flux sufficient to induce adaptive resistance to cells are described in the following examples in Tables XIV-XVII. All values are in weight percent.

TABLE XIV

Ingredients 66 67 68 69 70

Active ingredients

Nitroglycerin 0.035 0.04 0.045 0.05 0.055

Glycerin 99.965 99.96 99.955 99.95 99.945

Tablet composition

Active ingredients 1.0 1.0 1.0 1.0 1.0 iV-methyl-2-pyrrolidone 1.0 1.0 1.0 1.0 1.0 β-lactose 9.0 9.0 9.0 9.0 9.0

Lactose balance balance balance balance balance

TABLE XV

Ingredients 71 72 73 74 75

Active ingredients

Nitroglycerin 0.06 0.065 0.07 0.075 0.08

Glycerin 99.94 99.935 99.93 99.925 99.92

Tablet composition

Active ingredients 1.0 1.0 1.0 1.0 1.0

N-methyl-2-pyrrolidone 1.0 1.0 1.0 1.0 1.0 β-lactose 9.0 9.0 9.0 9.0 9.0 lactose balance balance balance balance balance

TABLE XVI

TABLE XVII

A syrup of active ingredients is formulated by combining the nitroglycerin and glycerin in one liter of purified water and 1.5 liters of ethanol (SD 3A) and stirring. Polyvinyl pyrrolidinone is added to the alcohol-water solution and thoroughly dispersed. The syrup is then added to the β-lactose/lactose admixture in a tumbling mixer and blended to form tablets on a conventional Colton tablet molding machine employing a tablet molding plate having cavities designed to make tablets from the above described composition weighing approximately 1 mg, 1.5 mg, 2 mg, and 5 mg. each. The molded tablets are spread on drying trays and the moisture and alcohol removed therefrom by drying overnight at ambient conditions. The resulting dry nitroglycerin tablets are combined in glass jars which held about 5 lbs. of the dry finished tablets. Samples of the nitroglycerin tablets are removed from representative jars and the tablets analyzed individually for their nitroglycerin content. Coefficient of variation percentages were computed from the individual analyses.

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-diaminofiuorescein (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). Microparticles

The compositions disclosed herein can also be delivered by microparticles. The term "microparticle" is used herein to include nanoparticles, microspheres, nanospheres, microcapsules, nanocapsules, and particles, in general. As such, the term microparticle refers to particles having a variety of internal structure and organizations including homogeneous matrices such as microspheres (and nanospheres) or heterogeneous core-shell matrices (such as microcapsules and nanocapsules), porous particles, multi-layer particles, among others. The term "microparticle" refers generally to particles that have sizes in the range of about 10 nanometers (run) to about 2 mm (millimeters). The disclosed microparticles comprise: a) one or more homopolymers or copolymers in an amount sufficient to form a wall forming matrix; and b) an amount of a nitric oxide donor sufficient to provide a nitric oxide flux of from about 0.01 pmol/sec to about 100 pmol/sec.

The following are non-limiting examples of polymers that can comprise the disclosed microparticles. Block co-polymers comprising a hydrophilic block and a hydrophobic block, for example, polymers wherein: a) the hydrophilic block comprises one or more of the following: i) polyalkylene glycols, for example, polyethylene glycol, polypropylene glycol, and the like; ii) polyvinyl pyrrolidone and derivatives thereof; iii) naturally occurring, synthetic, or modified polysacharrides; iv) peptides and/or proteins; and v) other hydrophilic units, oligomers, homopolymers, or copolymers; and b) the hydrophobic block comprises one or more of the following: i) lactide, glycolide, caprolactone, and mixtures thereof; ii) polyester, polyhydroxy acids, polyanhydrides, polyorthoesters, polyetheresters, polyesteramides, polyphosphazines, polyphosphoesters, polyphosphates, polyphosphonates, polycarbonates, polyorthocarbonates, polyamides, or copolymers thereof.

The fibers can comprise one or more homopolymers or copolymers chosen from: i) poly(lactide)-co-(polyalkylene oxide); ii) poly(lactide-co-glycolide)-co-(polyalkylene oxide); iii) poly(lactide-co-caprolactone)-b-(polyalkylene oxide); iv) poly(lactide-co-glycolide-co-caprolactone)-b-(polyalkylene oxide); v) poly(lactide)-co-(polyvinyl pyrrolidone); vi) poly(lactide-co-glycolide)-co-(polyvinyl pyrrolidone); vii) poly(lactide-co-caprolactone)-b-(polyvinyl pyrrolidone); viii) poly(lactide-co-glycolide-co-caprolactone)-b-(polyvinyl pyrrolidone); ix) poly(lactide); x) poly(lactide-co-glycolide); xi) poly(lactide-co-caprolactone) ; xii) poly(lactide-co-glycolide-co-caprolactone); xiii) poly(glycolide-co-caprolactone); xiv) poly(caprolactone); xv) polyesters; for example, polyethylene, polypropylene; xvi) polyurethanes; xvii) polyhydroxy acids; xviii) polyanhydrides; xix) polyorthoesters, xx) polyetheresters, xxi) polyesteramides, xxii) polyphosphazines, xxiii) polyphosphoesters, xxiv) polyphosphates, xxv) polyphosphonates, xxvi) polycarbonates, xxvii) polyorthocarbonates, xxviii) polyamides. In one embodiment, the microparticles comprise a polymer chosen from poly(ethylene terephthalate), nylon, polyethyleneimine, poly(vinylidene fluoride), polyethylene, polysiloxane, polystyrene, polyethylene glycol, or a mixture thereof. In another embodiment, the microparticles comprise a polymer chosen from polystyrene-polyvinyl pyridine, polystyrene-polybutadiene, polystyrene-hydrogenated polybutadiene, polystyrene-polyisoprene, polystyrene-hydrogenated polyisoprene, polystyrene-poly(methyl methacrylate), polystyrene-polyalkenyl aromatics, polyisoprene- poly(ethylene oxide), polystyrene-poly(ethylene propylene), poly(ethylene oxide)- polycaprolactones, polybutadiene-poly(ethylene oxide), polyisoprene-poly(ethylene oxide), polystyrene-poly(t-butyl methacrylate), poly(methyl methacrylate)-poly(t-butyl methacrylate), poly(ethylene oxide)-poly(propylene oxide), polystyrene-poly(t- butylacrylate), and polystyrene-poly(tetrahydrofuran).

The molecular weight of the polymers that comprise the disclosed microparticles can be from about 500 daltons to about 2,000,000 daltons. In one embodiment, the molecular weight of the polymer can be from about 2,000 daltons to about 200,000 daltons. In another embodiment, the molecular weight of the polymer can be from about 500 daltons to about 5,000 daltons. Wherein a further aspect of this embodiment comprises copolymers wherein the polymer has an average molecular weight of from 500 daltons to 1,500 daltons. In a yet further embodiment, the molecular weight of the polymer can be from about 1,000 daltons to about 200,000 daltons. m another further embodiment, the molecular weight of the polymer can be from about 4,000 daltons to about 150,000 daltons. And in a yet further embodiment, the molecular weight of the polymer can be from about 4,000 daltons to about 100,000 daltons. The molecular weight of the polymer of the copolymers of the present disclosure can be from about 100 daltons to about 100,000 daltons. In another embodiment, the molecular weight of the polymer can be from about 100 daltons to about 40,000 daltons. In yet another embodiment, the molecular weight of the polymer can be from about 100 daltons to about 8,000 daltons. A further embodiment comprises a polymer having a molecular weight of from about 1,000 daltons to about 8,000 daltons. A yet another further embodiment comprises a polymer having a molecular weight of from about 1,000 daltons to about 6,000 daltons. hi a still yet another embodiment comprises a polymer having a molecular weight of from about 10,000 daltons to about 100,000 daltons. hi a still yet further embodiment comprises a polymer having a molecular weight of from about 5,000 daltons to about 50,000 daltons. Another further embodiment comprises a polymer having a molecular weight of from about 3,000 daltons to about 12,000 daltons. A still further embodiment comprises a polymer having a molecular weight of from about 400 daltons to about 4,000 daltons.

The polymer average molecular weights can be obtained be Gel Permeation Chromatography (GPC), for example, as described by L. H. Sperling of the Center for Polymer Science and Engineering & Polymer Interfaces Center, Materials Research Center, Department of Chemical Engineering and Materials Science and Engineering Department, Lehigh University, 5 E. Packer Ave., Bethlehem, PA 18015-3194, as first described in: ACS Division of Polymeric Materials: Science and Engineering (PMSE), 81, 569 (1999). Alternatively the molecular weights can be described by their measured Inherent Viscosity (IV) as determined by capillary viscometry. Molecular weights of the polymers described herein can be about 0.05 dL/g to about 2.0 dL/g wherein dL is deciliter. In another embodiment the inherent viscosity can be from about 0.05 dL/g to about 1.2 dL/g. In a further embodiment the inherent viscosity can be form about 0.1 dL/g to about 1.0 dL/g. A yet further embodiment of the polymers and copolymers of the present disclosure can have an inherent viscosity of from about 0.1 dL/g to about 0.8 dL/g. And yet another embodiment of the polymers of the present disclosure can have an inherent viscosity of from about 0.05 dL/g to about 0.5 dL/g. Alternatively, the formulator can express the inherent viscosity in cm³/g if convenient. Preparation of Nitroglycerin Containing Microspheres EXAMPLE 86

A 1 weight percent polymer solution is prepared by dissolving 2 g of 50:50 poly(DL-lactide-co-glycolide) ("DL-PLG") in 198 g of dichloromethane (The DL-PLG had an inherent viscosity of 1.27 dL/g.). Two grams of a glycerin/nitroglycerin solution comprising 0.001% by weight of nitroglycerin is suspended in the polymer solution by homogenization. The glycerin/nitroglycerin suspension is then poured into 300 ml resin kettle and stirred at 3500 rpm with a 1.5 inch Teflon impeller. Silicone oil (350 cs) is pumped into the resin kettle at a rate of about 2 mL per min. After approximately 50 mL of oil is added, the contents of the resin kettle are poured into 3.5 L of heptane. The heptane is stirred at 900 rpm with a 2.5 inch stainless steel impeller. After 0.5 hour of stirring, the glycerin/nitroglycerin microsphere suspension is poured through a stainless steel sieve with 45 μm openings to remove microspheres larger than 45 μm in diameter. Microspheres less than 45 μm in diameter are collected on a fritted glass filter funnel and dried at room temperature in a vacuum oven for about 48 hours. The glycerin/nitroglycerin microspheres are then collected in tared glass scintillation vials and stored under desiccant at 40 ⁰C. The glycerin/nitroglycerin admixture is encapsulated in two types of copolymer excipients made in accordance with Example A. One copoylmer has a 50:50 mole ratio of lactide to glycolide and the other copolymer has a 65:35 mole ratio. In view of the higher lactide content of the 65:35 copolymer, this copolymer will take longer to biodegrade than the 50:50 copolymer. Thus, the delivery time of the 65:35 copolymer can be longer than the delivery time of the 50:50 copolymer. Additional variations of the actual proportions of lactide and glycolide in the copolymer and the copolymer morphology can be manufactured to more or less custom adjust the rate and amount of nitroglycerin molecules being released into the central nervous system. As such the delivery of nitric oxide via nitroglycerin at a flux rate of from about 0.01 pmol/sec to about 100 pmol/sec can be accomplished by varying the size and the composition of the microparticle.

The final microspheres are free- flowing powders consisting of spherical particles approximately 1 to 45 μm in diameter. These microspheres can easily be suspended in aqueous vehicles and injected through conventional hypodermic needles. Although the amount of nitroglycerin contained in each microsphere may vary, the microspheres manufactured and used in the following example consisted of from about 40% (by weight) glycerin/nitroglycerin and of about 60% (by weight) of the poly(DL-lactide-co-glycolide). The formulator can, however, change the ratio of glycerin/nitroglycerin to polymer ratio. The standard release tests can be used to determine the rat of glycerin/nitroglycerin admixture release and thus the nitric oxide flux.

EXAMPLE 87

A 20 weight percent polymer solution Is prepared by dissolving 1 g of poly- caprolactone in 4 g of dichloromethane. (The polycaprolactone has an inherent viscosity of 1.0 dl/g.) A dispersion is formed by suspending 1 g of glycerin/nitroglycerin admixture comprising about 0.0005% by weight nitroglycerin in the polymer solution. An emulsion is formed when the glycerin/nitroglycerin /polymer dispersion is transferred into a 300 mL resin kettle containing 188 g of process medium stirring at 1200 rpm with a 1.5 in. Teflon impeller. The process medium consists of 5 wt % polyvinyl alcohol) and 16 wt % calcium chloride saturated with 4.4 g of dichloromethane. After 1 min of stirring, the dopamine microspheres are hardened by extracting the dichloromethane from the microspheres. This extraction is done by adding the content of the resin kettle to a bath containing 1022 g of a 32 weight percent calcium chloride solution stirring at 200 rpm. At 10 and 20 minutes, this post addition, 500 mL of water are slowly added to the extraction bath. (Total extraction time is 30 min.) The contents of the extraction bath are next centrifuged at 1800 x G for 45 minutes. After centrifugation, the microspheres are collected on a fritted glass funnel and dried at room temperature in a vacuum oven for 48 hours. The microspheres are processed through a stainless steel sieve with 45 μm openings to remove microspheres larger than 45 μm in diameter. The glycerin/nitroglycerin microspheres are then collected in tared glass scintillation vials and stored under desiccant at 40 ⁰C.

EXAMPLE 88

A 15 weight percent polymer solution is prepared by dissolving 0.75 g of polyhydroxybutyrate/polyhydroxyvalerate copolymer (PHBV) in 4.3 g of dichloromethane. A dispersion is formed by suspending 1 g of glycerin/nitroglycerin admixture comprising about 0.0005% by weight nitroglycerin in the polymer solution. An emulsion is formed when the glycerin/nitroglycerin/polymer dispersion is transferred into a 300-mL resin kettle containing 179 g of process medium stirring at 1400 rpm with a 1.5-in. Teflon impeller. The process medium consisted of 5 weight percent poly(vinyl alcohol) saturated with 4.3 g of dichloromethane. After 1 minute of stirring, the glycerin/nitroglycerin microspheres are hardened by extracting the dichloromethane from the microspheres. This extraction is done by adding the contents of the resin kettle to a bath containing 1021 g of water stirring at 740 rpm. After 30 minutes of stirring, the microspheres are centrifuged at 1800 x G for 45 minutes. Then, the microspheres are collected on a fritted glass funnel and dried at room temperature in a vacuum oven for 48 hours. The microspheres are processed through a stainless steel sieve with 45 μm openings to remove microspheres larger than 45 μm in diameter. The glycerin/nitroglycerin microspheres are then collected in tared glass scintillation vials and stored under desiccant at 4 ⁰C.

In one embodiment the disclosed microparticles comprise: a) one or more homopolymers or copolymers in an amount sufficient to form a wall forming matrix; b) an amount of a nitric oxide donor sufficient to provide a nitric oxide flux of from about 0.01 pmol/sec to about 100 pmol/sec; and c) a component chosen from a rapidly biodegradable component, a rapidly dissolving component, a rapidly swelling component, and a component that causes osmotic rupture of the encapsulated composition.

The optional components of the microparticles, which is referred to herein as component (c), provides for the rapid release of the nitric oxide donor. Component (c) can be but is not limited to 1) a rapidly biodegradable component, 2) a rapidly dissolving component, 3) a component that causes osmotic rupture of the encapsulated polymeric composition or 4) a component that causes the encapsulated nitric oxide donor comprising composition to swell and form a gel-like structure. Component (c) can be a single component that has one or any combination of more than one property described above (i.e. rapidly biodegradable, rapidly dissolving, rapidly swelling or osmotic rupture property). Component (c) can be, for example, a rapidly biodegradable component, but also can have any of the other three properties, depending upon the composition of component (c). Thus, component (c) can have any single or combination of the four features described above. Component (c) can also comprise a mixture of two or more different type components described above. The terms "rapidly biodegradable" and "rapidly dissolving" when describing component (c) of the present invention are defined herein such that component (c) will, at a minimum, biodegrade or dissolve at a higher rate than the polymer matrix that encapsulates the nitric oxide donor comprising composition. The microparticles can biodegrade by a number of methods, which include but are not limited to hydrolysis or enzymatic degradation. The rate at which the nitric oxide donor composition and component (c) biodegrades or dissolves relative to the matrix forming polymer depends upon the materials selected for the polymers and component (c). More specifically, the term "rapidly" indicates that component (c) biodegrades or dissolves or swells at a rate so as to enable the release of the nitric oxide donor within a period of time for producing a nitric oxide flux of from about 0.01 pmol/sec to about 100 pmol/sec. Such a release can occur within any time period determined by the formulator.

Materials that are useful for component (c) include but are not limited to an acidic salt, a basic salt, a neutral salt, a carbohydrate, a starch, a polyelectrolyte, biocompatible hydrophilic materials, swellable materials, a gelatin, an amine, a surfactant, an inorganic acid or base, an organic acid or base, an amino acid, a monomer, an oligomer, a polymer or a mixture thereof, hi one iteration of this embodiment, component (c) can include but is not limited to sodium chloride, sodium phosphate, bile salts, ammonium sulfate, ammonium chloride, sodium carbonate or potassium carbonate, polyethylene glycol, polyoxoethylene alkyl ethers, trehalose, mannitol, sorbitol, dextrose, dextrin, sucrose, lactose, saccharides, polysaccharides, oligosaccharides, saccharin, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose or sodium starch glycolate, citric acid, lactic acid, glycolic acid, acetic acid, ascorbic acid, tartaric acid, malic acid, maleic acid, benzoic acid, arginine, glycine, threonine, choline, ethanolamine, protamine, sodium alginate, heparin, docusate sodium, glycerin, glycofurol, propylene glycol, polysorbate, povidone, and albumin. EXAMPLE 89

A polymer solution is prepared by dissolving 1.05 g of 60:40 DL-PLG (Birmingham Polymers Inc.) with an inherent viscosity of 0.49 dL/g in chloroform and 0.45 g of DL- lactide (Rl 04, Boehringer Ingelheim) with a molecular weight of 2000 in 5.5 g of ethyl acetate. Next, approximately 12.5 mg of an admixture of glycerin and nitroglycerin, wherein nitroglycerin comprises about 0.001% by weight of the admixture, is dissolved in 0.5 mL of sterile water. The glycerin/nitroglycerin admixture is added to the polymer solution while it is being homogenized with a Brinkmann Polytron (Model 10, PTA-10S probe, Brinkmann Instrument Co., Westbury, NY). In a separate container, 280 g of an aqueous solution consisting of 1.0 wt % poly( vinyl alcohol)(PVA) and 0.7 wt % carboxymethyl cellulose (CMC), which is saturated with ethyl acetate, is equilibrated to 12 ⁰C. The standard mixing head of a Silverson Laboratory Mixer (Silverson Machines Inc., East Longmeadow, MA) is positioned beneath the surface of the P V A/CMC solution. With the Silverson mixer operating at a stir rate of about 3850 rpm, the glycerin/nitroglycerin/polymer emulsion is immediately transferred to the PVA/CMC solution. The resulting water-in-oil-in-water emulsion is stirred for 45 seconds, after which the emulsion is transferred to a 5.2 L of sterile water being stirred with a stainless steel impeller. The resulting microspheres are stirred in the water for approximately 15 minutes. The sterile water and microspheres are then transferred to 6, 1-L centrifuge bottles.

The bottles are centrifuged at 4200 rpm using a Beckman J6M centrifuge (Beckman Instruments, Inc. Fullerton, CA) for 45 minutes. After centrifuging the suspension, the supernatant is removed. The microspheres are resuspended in additional sterile water. The contents of each centrifuge bottle are diluted to a total volume of 800 mL. The centrifuge bottles are again centrifuged at 4200 rpm for 55 minutes. This washing process is repeated one additional time, after which the microspheres are suspended in a total volume of approximately 400 mL of sterile water, a total of 300 mg of mannitol is added to the suspension, which is then transferred to a 1-L freeze-drying flask. The microsphere suspension is then frozen and lyophilized (FTS Systems, Stone Ridge, NY). As described herein above, the formulations of the present disclosure include pharmaceutical compositions comprising nitric oxide or nitric oxide donor 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. 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. In 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. 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. In 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:5W) 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 may be 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.

PROCEDURES The following procedures can be used to monitor cell vitality following treatment of a human or mammal with a disclosed composition, hi addition, the following procedures can be used to determine the effective amount of nitric oxide necessary to effectively treat a particular individual human or mammal. Also the following procedures can be used to determine the suitability of one nitric oxide generator over another for a particular patient or for a particular condition, for example, a type of neurodegenerative trauma. Growth and maintenance of NSC34 cells

The motor neuron cell line, NSC34, was used to demonstrate and test the level of effectiveness of the disclosed compositions. The procedure for growth and maintenance of the NSC34 cells was performed as previously described (Bishop A. et al., "Adaptive resistance to nitric oxide in motor neurons." Free Radical Biology & Medicine (1999) 26(7- 8): 978-986 and Bishop A. et al., (2004) "Decreased resistance to nitric oxide in motor neurons of HO-I null mice." BBRC 325:3-9). NSC34 cells were made from a fusion of primary mouse spinal cord motor neurons with spinal neuroblastoma cells. These cells provide a homogenous and partially immortalized line of cells that have many of the characteristics of motor neurons assayed for thus far and are an accepted model of motor neurons. They spontaneously differentiate in a controlled and predictable manner and can be terminally differentiated by the application of retinoic acid. The cells were 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. 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 the 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 rV: This group receives no pre-treatment of 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 an 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 (Bishop et al, 1999, 2004, ms in prep). 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), m 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 Affmiti 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-coηjugated 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. In Vivo Testing

An in vivo study utilizing three hundred 8-week old male C57BL6/J mice was conducted to determine the pharmacokinetics of nitric oxide induced adaptive resistance to amyotrophic lateral sclerosis (ALS) and efficacy of a compound that provides an in vivo source of nitric oxide. 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 VERSAmax™ 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

Analyte Calculated

Absorbance %

Concentration Concentration at 540 nm Difference

(μM) (μM)

0 0.011 -3.49 NA

6.25 0.068 5.96 -4.6

12.5 0.116 14.3 14.4

25 0.203 28.8 15.2

50 0.321 48.7 -2.6

100 0.622 99.5 -0.5

TABLE I

TABLE II

Figure 1 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/kg 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 rv 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.

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 μg 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 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).

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, hi addition, the compositions of the present disclosure can be administered as frequently as necessary to achieve a therapeutic amount. The disclosed compositions can be used for treating one or more neurodegenerative diseases or a neurodegenerative state caused by trauma. The disclosed compositions provide from about 0.01 pmol/sec to about 100 pmol/sec of nitric oxide when administered to a subject having a neurodegenerative disease or diagnosed as having a neurodegenerative disease. In one embodiment, the disclosed compositions can provide from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide. In another embodiment, the disclosed compositions can provide from about 0.1 pmol/sec to about 20 pmol/sec of nitric oxide, hi a further embodiment, the disclosed compositions can provide from about 0.01 pmol/sec to about 10 pmol/sec of nitric oxide, hi a yet further embodiment, the disclosed compositions can provide from about 0.1 pmol/sec to about 10 pmol/sec of nitric oxide, hi a still further embodiment, the disclosed compositions can provide from about 0.5 pmol/sec to about 8 pmol/sec of nitric oxide, hi a yet another embodiment, the disclosed compositions can provide from about 0.5 pmol/sec to about 5 pmol/sec of nitric oxide. In a still yet further embodiment, the disclosed compositions can provide from about 1 pmol/sec to about 5 pmol/sec of nitric oxide. The disclosed compositions provide a sufficient amount of a nitric oxide generator that provides from about 0.001 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis. In one embodiment, the disclosed compositions provide 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 another embodiment, the disclosed compositions provide 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 disclosed compositions provide 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. In a still further embodiment, the disclosed compositions provide 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. In a yet still further embodiment, the disclosed compositions provide 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 compositions further provide 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 one embodiment, the disclosed compositions provide 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 disclosed compositions provide 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 disclosed compositions provide 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. In a still further embodiment, the disclosed compositions provide 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 disclosed compositions provide 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 compositions yet further provide 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. In another embodiment, the disclosed compositions provide 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. In a further embodiment, the disclosed compositions provide 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 disclosed compositions provide 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 disclosed compositions provide 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. In a yet another embodiment, the disclosed compositions provide 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, 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.

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 composition comprising, nitric oxide or a nitric oxide donor or provider, wherein the composition delivers a flux of from about 0.01 pmol/sec to about 100 pmol/sec of nitric oxide.

2. The composition according to Claim 1, wherein the flux is from about 0.1 pmol/sec to about 50 pmol/sec of nitric oxide.

3. The composition according to Claim 1, wherein the flux is from about 0.1 pmol/sec to about 20 pmol/sec of nitric oxide.

4. The composition according to Claim 1, wherein the flux is from about 0.01 pmol/sec to about 10 pmol/sec of nitric oxide.

5. The composition according to Claim 1, wherein the flux is from about 0.1 pmol/sec to about 10 pmol/sec of nitric oxide.

6. The composition according to Claim 1, wherein the flux is from about 0.5 pmol/sec to about 8 pmol/sec of nitric oxide.

7. The composition according to Claim 1, wherein the flux is from about 0.5 pmol/sec to about 5 pmol/sec of nitric oxide.

8. The composition according to Claim 1, wherein the flux is from about 1 pmol/sec to about 5 pmol/sec of nitric oxide.

9. A composition comprising, nitric oxide or a nitric oxide donor or provider, wherein the composition provides from about 0.001 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

10. The composition according to Claim 9, wherein the composition 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.

11. The composition according to Claim 9, wherein the composition from about 0.001 mg to about 0.01 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

12. The composition according to Claim 9, wherein the composition 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.

13. The composition according to Claim 9, wherein the composition from about 0.01 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

14. The composition according to Claim 9, wherein the composition from about 0.001 mg to about 0.05 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

15. A composition comprising, nitric oxide or a nitric oxide donor or provider, wherein the composition provides from about 0.001 mg to about 0.1 mg of nitric oxide per hour per kg of body weight.

16. The composition according to Claim 15, wherein the composition provides from about 0.001 mg to about 0.01 mg of nitric oxide per hour per kg of body weight.

17. The composition according to Claim 15, wherein the composition provides from about 0.001 mg to about 0.05 mg of nitric oxide per hour per kg of body weight.

18. The composition according to Claim 15, wherein the composition provides from about 0.01 mg to about 0.1 mg of nitric oxide per hour per kg of body weight.

19. The composition according to Claim 15, wherein the composition provides from about 0.05 mg to about 0.1 mg of nitric oxide per hour per kg of body weight.

20. A composition comprising, nitric oxide or a nitric oxide donor or provider, wherein the composition provides a concentration of nitric oxide as measured in cells or in plasma from about 1 μM to about 500 μM.

21. The composition according to Claim 20, wherein the composition provides a concentration of nitric oxide as measured in cells or in plasma from about 1 μM to about 300 μM.

22. The composition according to Claim 20, wherein the composition provides a concentration of nitric oxide as measured in cells or in plasma from about 10 μM to about 100 μM.

23. The composition according to Claim 20, wherein the composition provides a concentration of nitric oxide as measured in cells or in plasma from about 25 μM to about 50 μM.

24. The composition according to Claim 20, wherein the composition provides a concentration of nitric oxide as measured in cells or in plasma from about 100 μM to about 500 μM.

25. The composition according to Claim 20, wherein the composition provides a concentration of nitric oxide as measured in cells or in plasma from about 250 μM to about 500 μM.

26. The composition according to any of Claims 1 to 25, wherein the nitric oxide donor or provider is chosen from amyl nitrate; octyl nitrate; propatyl nitrate (2-ethyl-2- (hydroxymethyl)- 1,3 -propanediol trinitrate); isosorbide mononitrate; (8-nitrooxy- 2,6-dioxabicyclo[3.3.0]octan-4-ol); trolnitrate (2,2',2"- nitrilotrisethanol, trinitrate (ester)); ethylene glycol dinitrate, glyceryl 1 ,2dinitrate, glyceryl 1,3-dinitrate, glyceryl 1 -mononitrate; butane 1,2,3-triol nitrate, pentaerythrityl trinitrate, isosorbide mononitrate, isosorbide mononitrate, or erythrityl tetranitrate.

27. The composition according to any of Claims 1 to 25, wherein the nitric oxide donor or provider is chosen from sodium nitroprusside, (Z)-l-[2-aminoethyl)-N-(2- ammonioethyl)-amino] diazen- 1 -ium- 1 ,2-diolate] , N-(2- Aminoethyl)-N-(2-hydroxyl- 2-nitrosohydrazino)- 1 ,2-ethylenediamine, or 3 -morpholino-sydnonimine.

28. The composition according to any of Claims 1 to 25, wherein the nitric oxide donor or provider is chosen from S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, S- nitroso-N-acetylcysteine, S-nitroso-cysteine, S-nitroso-homocystein, S- nitrosopantathoein, or S-nitroso-captopril

29. The composition according to any of Claims 1 to 25, wherein the nitric oxide donor or provider is nitroglycerin.

30. The composition according to any of Claims 1 to 29, wherein the composition is in the form of an ointment, paste, salve, cream, lotion, gel, transdermal patch, pill, or capsule.

31. A composition comprising: a) nitroglycerin in an amount sufficient to provide a flux of from about 0.01 pmol/sec to about 100 pmol/sec of nitric oxide; and b) one or more pharmaceutically acceptable ingredients.

32. A composition comprising: c) nitroglycerin in an amount sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis; and d) one or more pharmaceutically acceptable ingredients.

33. A composition comprising: e) nitroglycerin in an amount sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide per hour per kg of body weight; and f) one or more pharmaceutically acceptable ingredients.

34. A composition comprising: g) nitroglycerin in an amount sufficient to provide a concentration of nitric oxide as measured in cells or in plasma from about 1 μM to about 500 μM; and h) one or more pharmaceutically acceptable ingredients.

35. The composition according to any one of Claims 31 to 34, comprising from about 1 x 10^"4 mg to about 0.1 mg of nitroglycerin per gram of composition.

36. The composition according to any one of Claims 31 to 34, comprising from about 5 x 10^"4 mg to about 0.05 mg of nitroglycerin per gram of composition.

37. The composition according to any one of Claims 31 to 34, comprising from about 5 x 10^"4 mg to about 1 x 10^'2 mg of nitroglycerin per gram of composition.

38. The composition according to any one of Claims 31 to 34, comprising from about 7.5 x 10^"7 gram to about 2 x 10^"5 gram of nitroglycerin per gram of composition.

39. The composition according to any one of Claims 31 to 34, comprising from about 1 x 10^" mg to about 1 x 10^" mg of nitroglycerin per gram of composition.

40. The composition according to any one of Claims 31 to 34, comprising from about 5 x 10^"4 mg to about 5 x 10^"3 mg of nitroglycerin per gram of composition.

41. The composition according to any one of Claims 31 to 34, comprising from about 1 x 10^" mg to about 5 x 10^"2 mg of nitroglycerin per gram of composition.

42. The composition according to any one of Claims 31 to 34, wherein the composition is in the form of an ointment, paste, salve, cream, lotion, gel, transdermal patch, pill, or capsule.

43. A composition comprising: a) nitric oxide; and b) one or more carrier gases.

44. The composition according to Claim 43, wherein the carrier gas is chosen from nitrogen, helium, argon, oxygen, or a mixture thereof.

45. A microparticle comprising: a) one or more homopolymers or copolymers in an amount sufficient to form a wall forming matrix; and b) an amount of a nitric oxide donor sufficient to provide a nitric oxide flux of from about 0.01 pmol/sec to about 100 pmol/sec.

46. A microparticle comprising: a) one or more homopolymers or copolymers in an amount sufficient to form a wall forming matrix; and b) an amount of a nitric oxide donor sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide in plasma per kg of body weight on a continuous basis.

47. A microparticle comprising: a) one or more homopolymers or copolymers in an amount sufficient to form a wall forming matrix; and b) an amount of a nitric oxide donor sufficient to provide from about 0.001 mg to about 0.1 mg of nitric oxide per hour per kg of body weight.

48. A microparticle comprising: a) one or more homopolymers or copolymers in an amount sufficient to form a wall forming matrix; and b) an amount of a nitric oxide donor sufficient to provide a concentration of nitric oxide as measured in cells or in plasma from about 1 μM to about 500 μM.

49. The microparticle according to any of Claim 45 to 48, wherein the homopolymers or copolymers are chosen from: i) poly(lactide)-co-(polyalkylene oxide); ii) poly(lactide-co-glycolide)-co-(polyalkylene oxide); iii) poly(lactide-co-caprolactone)-b-(polyalkylene oxide); iv) poly(lactide-co-glycolide-co-caprolactone)-b-(polyalkylene oxide); v) poly(lactide)-co-(polyvinyl pyrrolidone); vi) poly(lactide-co-glycolide)-co-(polyvinyl pyrrolidone); vii) poly(lactide-co-caprolactone)-b-(polyvinyl pyrrolidone); viii) poly(lactide-co-glycolide-co-caprolactone)-b-(polyvinyl pyrrolidone); ix) poly(lactide); x) poly^actide-co-glycolide); xi) poly(lactide-co-caprolactone) ; xii) poly(lactide-co-glycolide-co-caprolactone); xiii) poly(glycolide-co-caprolactone) ; xiv) poly(caprolactone); xv) polyesters; for example, polyethylene, polypropylene; xvi) polyurethanes; xvii) polyhydroxy acids; xviii) polyanhydrides; xix) polyorthoesters, xx) polyetheresters, xxi) polyesteramides, xxii) polyphosphazines, xxiii) polyphosphoesters, xxiv) polyphosphates, xxv) polyphosphonates, xxvi) polycarbonates, xxvii) polyorthocarbonates, xx viii) polyamides.

50. The microparticle according to any of Claim 45 to 48, wherein the homopolymer or copolymer is chosen from: i) poly(lactide)-co-(polyalkylene oxide); ii) poly(lactide-co-glycolide)-co-(polyalkylene oxide); iii) poly(lactide-co-caprolactone)-b-(polyalkylene oxide); iv) poly(lactide-co-glycolide-co-caprolactone)-b-(polyalkylene oxide); v) poly(lactide)-co-(polyvinyl pyrrolidone); vi) poly(lactide-co-glycolide)-co-(polyvinyl pyrrolidone); vii) poly(lactide-co-caprolactone)-b-(polyvinyl pyrrolidone); viii) poly(lactide-co-glycolide-co-caprolactone)-b-(polyvinyl pyrrolidone); ix) poly(lactide); x) poly(lactide-co-glycolide); xi) poly(lactide-co-caprolactone); xii) poly(lactide-co-glycolide-co-caprolactone); xiii) poly(glycolide-co-caprolactone); and xiv) poly(caprolactone).

51. The microparticle according to any of Claim 45 to 48, wherein the homopolymer or copolymer is chosen from: i) poly(lactide); ii) poly(lactide-co-glycolide); iii) poly(lactide-co-caprolactone); iv) poly(lactide-co-glycolide-co-caprolactone); and v) poly(glycolide-co-caprolactone).

52. The microparticle according to any of Claim 45 to 48, further comprising sodium chloride, sodium phosphate, bile salts, ammonium sulfate, ammonium chloride, sodium carbonate or potassium carbonate, polyethylene glycol, polyoxoethylene alkyl ethers, trehalose, mannitol, sorbitol, dextrose, dextrin, sucrose, lactose, saccharides, polysaccharides, oligosaccharides, saccharin, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose or sodium starch glycolate, citric acid, lactic acid, glycolic acid, acetic acid, ascorbic acid, tartaric acid, malic acid, maleic acid, benzoic acid, arginine, glycine, threonine, choline, ethanolamine, protamine, sodium alginate, heparin, docusate sodium, glycerin, glycofurol, propylene glycol, polysorbate, povidone, and albumin.