Pain-relieving Compositions And Uses Therefor

TITLE OF THE INVENTION

"PAIN-RELIEVING COMPOSITIONS AND USES THEREFOR"

FIELD OF THE INVENTION

[0001] This invention relates generally to compositions and methods for inducing, promoting or otherwise facilitating pain relief. More particularly, the present invention relates to the use of sub-normovasodilatory doses of nitric oxide donors in the therapeutic management of vertebrate animals including humans, for the prevention or alleviation of pain, especially neuropathic pain. According to some embodiments of the present invention, nitric oxide donors are administered by any suitable route so as to provide concentrations of NO that are about 1A to 10"15 of those known to induce vasodilation in normal circulations.

BACKGROUND OF THE INVENTION

[0002] Painful diabetic neuropathy (PDN) is a common and debilitating peripheral nerve complication of diabetes mellitus. By 1 year after the initial diagnosis of diabetes, 7% of patients report symptoms (e.g. pain, abnormal sensations) with the prevalence rising to 50% by 25 years of diabetes diagnosis (Sima and Sugimoto, Diabetologia 42: 773-788 1999; Cameron et al. Diabetologia 44: 1973-88 2001). Patients may present with one or more symptoms including burning sensations, lancinating and deep aching pains, depending upon the extent of nerve injury {Boulton, Diabetes Metab Res Rev 19: S16-21 2003). Numbness, tingling, and a sensation of tightness in the extremity are also commonly associated with PDN (Boulton, Diabetes Metab Res Rev 19: S 16-21 2003). In addition, unpleasant abnormal sensations (dysaethesia), enhanced sensitivity to stimulation (hyperaesthesia), a heightened response to painful stimuli (hyperalgesia) and a distorted sense of touch producing allodynia (innocuous stimuli such as light brushstrokes of the skin produce pain) are all commonly reported by patients with diabetic neuropathy (Boulton, Diabetes Metab Res Rev 19: S 16-21 2003).

[0003] There are no preventative treatments for PDN (Sima et al. Diabetologica 42: 773-788 1999), hence the therapeutic management of the condition is primarily palliative. This palliative management also represents a significant therapeutic obstacle, as the most efficient analgesic pharmaceuticals available, the //-opioid receptor agonists such as morphine, are reportedly ineffective for the relief of PDN (Attaϊ, Clin J Pain 16: Sl 18-30 2006). The mechanism underpinning the development of this opioid agonist hyposensitivity is unclear, but investigations have shown that poor glycaemic control can reduce pain tolerance and pain threshold and thus reduce the effectiveness of analgesics such as morphine (Morley et al. Am JMedll{\): 79-83 1984). In addition, there may be diabetes-associated alterations in morphine pharmacokinetics (Courteix et al. J Pharmacol Exp Ther 285(1): 63-70 1998) and/or changes in opioid receptor function {Chen et al. Anesthesiology 97: 1602-1608 2002).

[0004] Although PDN is attributed primarily to poor glycaemic control over a prolonged period, the exact pathogenesis is poorly understood (Sima and Sugimoto, Diabetologia 42: 773-788 1999; Feldman et al. Curr Opin Neurol 12: 553-63 1999) Presently, there are two broad theories regarding the development of PDN: the vascular dysfunction theory and the metabolic dysfunction theory.

[0005] The vascular dysfunction theory proposes that changes in the blood supply to the nerves (the neurovasculature or vasa nervorum) occur secondary to haemodynamic abnormalities (such as accelerated platelet aggregation and increased blood viscosity) (Fusman et al. Acta Diabetol 38(3): 129-34 2001). In addition, pathological changes in the small blood vessels of the neurovasculature may occur (such as reduction of the production of nitric oxide from the endothelial cells of blood vessels and acceleration of the reactivity on vasoconstrictive substances) (McAuley et al. Clin Sci (Lond) 99(3): 175-9 2000). These haemodynamic and vascular changes, acting independently or synergistically, are capable of causing the perineurial ischemia and subsequent endoneurial hypoxia observed in human patients and animal models of diabetes (Cameron et al. Diabetologia 44(11): 1973-88 2001). The end result of these abnormalities is nerve damage capable of causing the symptoms and signs of PDN.

[0006] On the other hand, in the metabolic dysfunction theory, the causes of nerve damage are mediated through the activation of the polyol metabolic pathway and through non-enzymatic protein glycation. These pathways induce mitochondrial and cytosolic NAD+/NADH redox imbalances and energy deficiencies in the nerves which can culminate in damage to neural and neurovascular tissues (Obrosova et al FASEB J 16(1): 123-5 2002). In addition, these metabolic changes are thought to activate protein kinase C (PKC) which is capable of heightening pain responses (Kamei et al. Expert Opin Investig Drugs 10(9): 1653-64 2001) and also of producing μ-opioid agonist hyposensitivity (Wang et al. Brain Res 723(1-2): 61-9 1996). Furthermore, heightened PKC activity is thought to reduce the binding affinity of //-opioid receptors for ligands (Ohsawa et al. Brain Res 764 244-8 1998). The consequences of these metabolic abnormalities are nerve damage and the development of μ-opioid agonist hyposensitivity, as seen in patients with PDN.

[0007] It is likely that neither theory is mutually exclusive and proponents of both theories converge in the belief that, downstream of vascular dysfunction or metabolic abnormalities, there is an imbalance in the production of vaso-active compounds in the vasa nervorum which leads to hypoxic ischemia of diabetic nerves.

[0008] Of all the endogenous vasodilators, nitric oxide is the most potent and hence is a likely candidate for reduced synthesis and consequent diabetes-induced constrictions in vascular tone. As well as relaxing vascular smooth muscle, it also inhibits the processes of platelet aggregation, mitogenesis and proliferation of cultured vascular smooth muscle, and leucocyte adherence (Wroblewski et al. Prev Cardiol

3(4): 172-177 2000). Nitric oxide is produced by the vascular endothelium by a group of enzymes called nitric oxide synthases. There are three isoforms of nitric oxide synthase (NOS) named according to their activity or the tissue type in which they were first described. These enzymes all convert the endogenous substrate, L-arginine, into L- citrulline, producing NO in the process.

[0009] Recent studies by the present inventors revealed unexpectedly that nitric oxide donors such as L-arginine can broadly prevent, attenuate and/or reverse the development of reduced analgesic sensitivity to an opioid receptor agonist such as morphine in neuropathic conditions, including peripheral neuropathic conditions such as PDN (see International Publication No. WO 2003/078437). This finding that nitric oxide donors can restore the analgesic sensitivity of opioid analgesics such as morphine in subjects with neuropathic conditions was significant because it allowed the use of these analgesics for treating or preventing pain in conditions, for which they were previously considered ineffective. SUMMARY OF THE INVENTION

[0010] The present inventors have surprisingly discovered that nitric oxide (NO) donors which directly or indirectly generate NO at concentrations that are smaller than those known to induce vasodilation in normal circulations (also referred to herein as sub-normovasodilatory (SNV) concentrations), are effective in producing analgesia in subjects with a neuropathic condition without the need for co-administering opioid analgesics. Based on this discovery, the present inventors consider that specific embodiments of SNV concentrations can broadly range from about 1A to about 10"15 of those known to induce vasodilation in normal circulations. [0011] Accordingly, one aspect of the present invention provides methods for the treatment or prophylaxis of a neuropathic condition in a subject. In some embodiments, the neuropathic condition is treated or prevented by administering to the subject at least one NO donor at a level that enhances NO and that does not alter normal systemic vascular tone in the subject. The NO donor is suitably administered without co-administration of an opioid analgesic. Thus, in these embodiments, the methods of treating or preventing the neuropathic condition consist essentially of administering the NO donor(s). Suitably, the level of NO is a sub-normovasodilatory (SNV) concentration that ranges from about Vi to about 10"15 of a reference concentration required to induce vasodilation in an anatomical site of a reference subject lacking a vascular condition, which suitably but not exclusively associates with the neuropathic condition to be treated or prevented. Illustrative anatomical sites include kidney, skin, skeletal muscle, arm, leg, tail and gastro-intestinal tract.

[0012] The NO donor is suitably administered in the form of a composition comprising a pharmaceutically acceptable carrier and/or diluent. The composition may be administered by injection, by topical application, or by the buccal, sublingual, rectal or oral routes, including sustained-release modes of administration, over a period of time and in amounts which are effective for delivering a SNV concentration of NO as broadly described above. In some embodiments, the NO donor is provided in a sustained release formulation {e.g., transdermal patch), which delivers a SNV concentration of NO as broadly described above. In some embodiments, the NO donor is a slow-release NO donor that delivers an SNV concentration of NO as broadly described above. [0013] In accordance with the present invention, SNV concentrations of NO have been shown to prevent or attenuate the pain associated with a neuropathic condition. Thus, in another aspect, the invention provides methods for preventing or attenuating neuropathic pain, especially peripheral neuropathic pain, in a subject. In some embodiments, neuropathic pain is prevented or attenuated by administering to the subject at least one NO donor at a level that enhances NO and that does not alter normal systemic vascular tone in the subject, wherein the NO donor is suitably in the form of a composition comprising a pharmaceutically acceptable carrier and/or diluent.

[0014] In a further aspect, the present invention contemplates the use of a composition that consists essentially of at least one NO donor for producing analgesia in a subject, especially in a subject having a neuropathic condition, which is suitably a peripheral neuropathic condition such as PDN or a related condition, wherein the composition comprises at least one NO donor at a level that enhances NO and that does not alter normal systemic vascular tone in the subject. Suitably, the composition excludes an opioid analgesic or is used to produce analgesia in the absence of coadministering an opioid analgesic.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a graphical representation showing that tactile (mechanical) allodynia is folly developed by -9-10 wks post-STZ administration.

[0016] Figure 2 is a graphical representation showing that single s.c. bolus doses (8-800 fmol/kg) of the NO donor, PRGl 00, produces dose-dependent relief of tactile allodynia, the defining symptom of painful diabetic neuropathy (PDN), in STZ- diabetic rats at 10 wks post-STZ administration

[0017] Figure 3 is a graphical representation showing that single s.c. bolus doses (8-800 pmol/kg) of the NO donor, PRGlOO, produces dose-dependent relief of tactile allodynia, the defining symptom of painful diabetic neuropathy (PDN), in STZ- diabetic rats at 14 and 24 wks post-STZ administration.

[0018] Figure 4 is a graphical representation showing that neither single s.c. bolus doses of vehicle (DMSO:water, 90:10) nor the NO donor PRGlOO at 80 fmol/kg- 8 μmol/kg, significantly altered mean (± SEM) systolic blood pressure of adult male normotensive non-diabetic Wistar rats for up to 1 h post-dosing. By contrast, single s.c. bolus doses of PRGlOO at 80 μmol/kg to similar animals, significantly reduced systolic blood pressure at 30 min post-dosing relative to pre-dosing baseline measurements of systolic blood pressure.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0020] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0021] As used herein, the term "about" refers to a quantity, level, concentration, value, dimension, size, or amount that varies by as much as 30%, 20%, or 10% or even as much as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, concentration, value, dimension, size, or amount.

[0022] The term "allodynia" as used herein refers to pain that results from a non-noxious stimulus i.e., a stimulus that does not normally provoke pain. Examples of allodynia include, but are not limited to, cold allodynia, tactile allodynia (pain due to light pressure or touch), and the like. [0023] The term "analgesia" is used herein to describe states of reduced pain perception, including absence from pain sensations as well as states of reduced or absent sensitivity to noxious stimuli. Such states of reduced or absent pain perception are induced by the administration of a pain-controlling agent or agents and occur without loss of consciousness, as is commonly understood in the art. The term analgesia encompasses the term "antinociception", which is used in the art as a quantitative measure of analgesia or reduced pain sensitivity in animal models.

[0024] The term "causalgia" as used herein refers to the burning pain, allodynia and hyperpathia after a traumatic nerve lesion, often combined with vasomotor and sudomotor dysfunction and later trophic changes. [0025] By "complex regional pain syndromes" is meant the pain that includes, but is not limited to, reflex sympathetic dystrophy, causalgia, sympathetically maintained pain, and the like.

[0026] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. The phrases "consisting essentially of," "consists essentially of and the like refer to the components which are essential in order to obtain the advantages of the present invention and any other components present would not significantly change the properties related to the inventive concept. Put another way, these phrases refer to the inclusion of any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrases "consisting essentially of," "consists essentially of and the like indicate that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0027] By "effective amount", in the context of treating or preventing a condition (e.g. a neuropathic condition) is meant the administration of that amount of active to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. [0028] By "hyperalgesia" is meant an increased response to a stimulus that is normally painful.

[0029] By "neuropathic pain" is meant any pain syndrome initiated or caused by a primary lesion or dysfunction in the peripheral or central nervous system. Examples of neuropathic pain include, but are not limited to, thermal or mechanical hyperalgesia, thermal or mechanical allodynia, painful diabetic neuropathy, postherpetic neuralgia, pantom limb pain, sciatica, chemotherapy-induced neuropathy, HIV- AIDS-associated neuropathy, nerve entrapment pain, and the like.

[0030] By "nitric oxide donor," "NO donor" and the like is meant any substance that is converted into, degraded or metabolised into, or provides a source of in vivo nitric oxide or NO and includes any and all forms of NO which exist under physiological conditions. The, the term "NO donor" includes and encompasses any compound which mimics the effects of NO, generates or releases NO through biotransformation, any compound which generates NO spontaneously, any compound which spontaneously releases NO, or any compound which in any other manner generates NO or a NO-like moiety when administered to a subject.

[0031] "Nociceptive pain" refers to the normal, acute pain sensation evoked by activation of nociceptors located in non-damaged skin, viscera and other organs in the absence of sensitization. [0032] The term "pain" as used herein is given its broadest sense and includes an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage and includes the more or less localised sensation of discomfort, distress, or agony, resulting from the stimulation of specialised nerve endings. There are many types of pain, including, but not limited to, lightning pains, phantom pains, shooting pains, acute pain, inflammatory pain, neuropathic pain, complex regional pain, neuralgia, neuropathy, and the like (Dorland's Illustrated Medical Dictionary, 28th Edition, W. B. Saunders Company, Philadelphia, Pa.). The goal of treatment of pain is to reduce the severity of pain perceived by a treatment subject. [0033] By "pharmaceutically acceptable carrier" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in topical, local or systemic administration. [0034] The term "pharmaceutically compatible salt" as used herein refers to a salt which is toxicologically safe for human and animal administration. This salt may be selected from a group including hydrochlorides, hydrobromides, hydroiodides, sulphates, bisulphates, nitrates, citrates, tartrates, bitartrates, phosphates, malates, maleates, napsylates, fumarates, succinates, acetates, terephthalates, pamoates and pectinates.

[0035] The term "prodrug" is used in its broadest sense and encompasses those compounds that are converted in vivo to a NO donor according to the invention. Such compounds would readily occur to those of skill in the art, and include, for example, compounds where a free hydroxy group is converted into an ester derivative. Prodrug forms of compounds may be utilised, for example, to improve bioavailability, mask unpleasant characteristics such as bitter taste, alter solubility for intravenous use, or to provide site-specific delivery of the compound.

[0036] By "slow-release nitric oxide donor" or "slow-release NO donor" is meant any substance that is converted or degraded or metabolised into, or provides a source of in vivo nitric oxide or NO over an extended period of time, thereby delivering a low concentration of nitric oxide into the blood stream. Suitably the slow-release nitric oxide donor is administered in an amount such that nitric oxide is delivered at a rate of 0.000001 nmol/kg/hour to 2.0 nmol/kg/hour. [0037] The terms "subject" or "individual" or "patient", used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, primates, avians, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes). A preferred subject is a human in need of treatment or prophylaxis for a peripheral neuropathic condition, especially PDN. However, it will be understood that the aforementioned terms do not imply that symptoms are present. [0038] The term "sub-normovasodilatory concentration" or "SNC concentration" as used herein refers to a level of NO donor, which enhances NO and that does not alter normal systemic vascular tone in the subject. [0039] The term "sustained-release formulation of nitric oxide donor" as used herein refers to a formulation of an NO donor that is adapted to release nitric oxide at a rate of 0.000001 nmol/kg/hour to 2.0 nmol/kg/hour or a range selected from 0.00001 nmol/kg/hour to 2.0 nmol/kg/hour, 0.0002 nmol/kg/hour to 1.0 nmol/kg/hour, 0.0005 nmol/kg/hour to 1.0 nmol/kg/hour, 0.0001 nmol/kg/hour to 0.5 nmol/kg/hour, 0.0002 nmol/kg/hour to 0.2 nmol/kg/hour, 0.0005 nmol/kg/hour to 0.1 nmol/kg/hour or 0.001 nmol/kg/hour to 0.05 nmol/kg/hour, 0.005 nmol/kg/hour to 0.01 nmol/kg/hour. The sustained release formulation may be any formulation capable of releasing NO at this rate. Illustrative sustained release formulations are transdermal patches adapted to deliver 0.1 nmol to 500 nmol per 24 hours, especially 10 nmol to 100 nmol per 24 hours, more especially 20 nmol to 60 nmol per 24 hours, most especially about 50 nmol over 6, 9, 12, 18, 24 or 30 hours.

[0040] By "does not alter normal systemic vascular tone" is meant not affecting mean arterial pressure so as to produce inappropriate systemic vasodilation with effects such as hypotension, headache, flushing in a normal subject or in a subject lacking a vascular condition (e.g., a neuropathic condition such as PDN).

2. Methods for the production of analgesia

[0041] The present invention provides methods for producing analgesia in a subject having a neuropathic condition. These methods generally comprise administering to the subject at least one NO donor at a level that enhances NO and that does not alter normal systemic vascular tone in the subject. Suitably, this level equates to one that does not induce vasodilation, or not appreciably, in "healthy" or non-NO deficient circulations. Suitably, the level of NO is a sub-normovasodilatory (SNV) concentration that ranges from about Vz to about 10"ls of those currently used in clinical applications.

[0042] The method of the present invention has particular utility in the prevention and/or alleviation of the painful symptoms associated with neuropathic conditions. There are many possible causes of neuropathic conditions and it will be understood that the present invention contemplates the treatment and/or prevention of pain associated with any neuropathic condition regardless of the cause. In one embodiment, the neuropathic conditions are a result of diseases of the nerves (primary neuropathy) and neuropathy that is caused by systemic disease (secondary neuropathy), such, as but not limited to diabetic neuropathy, Herpes Zoster (shingles)-related neuropathy, phantom limb pain, uraemia-associated neuropathy, amyloidosis neuropathy, HIV sensory neuropathies, hereditary motor and sensory neuropathies (HMSN), hereditary sensory neuropathies (HSNs), hereditary sensory and autonomic neuropathies, hereditary neuropathies with ulcero-mutilation, nitrofurantoin neuropathy, tomaculous neuropathy, neuropathy caused by nutritional deficiency and neuropathy caused by kidney failure. Other causes include repetitive activities such as typing or working on an assembly line, medications known to cause peripheral neuropathy such as several AIDS drugs (DDC and DDI), antibiotics (metronidazole, an antibiotic used for Crohn's disease, isoniazid used for tuberculosis), gold compounds (used for rheumatoid arthritis), some chemotherapy drugs (such as vincristine and others) and many others. Chemical compounds are also known to cause peripheral neuropathy including alcohol, lead, arsenic, mercury and organophosphate pesticides. Some peripheral neuropathies are associated with infectious processes (such as Guillian-Barre syndrome). In another embodiment, the neuropathic condition is a peripheral neuropathic condition such as PDN or related condition.

[0043] The neuropathic condition may be acute or chronic and, in this connection, it will be understood by persons of skill in the art that the time course of a neuropathy will vary, based on its underlying cause. With trauma, the onset of symptoms may be acute, or sudden, with the most severe symptoms being present at the onset or developing subsequently. Inflammatory and some metabolic neuropathies have a subacute course extending over days to weeks. A chronic course over weeks to months usually indicates a toxic or metabolic neuropathy. A chronic, slowly progressive neuropathy over many years occurs with most hereditary neuropathies or with a condition termed chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Neuropathic conditions with symptoms that relapse and remit include the Guillian-Barre syndrome.

[0044] The NO donor includes and encompasses any substance that is converted into, or degraded or metabolised into, or provides a source of, in vivo NO, inclusive of NO in its various redox forms. The presence of nitric oxide (NO) in biological systems is usually inferred based on its physiological effect. However, several different redox forms of NO such as the NO free radical (NO.), the nitrosonium cation (NO+), the nitroxyl anion (NO-) or other oxides of nitrogen (NOx) are known to exist under physiological conditions and there is no clear evidence to suggest that one form is favored over another (Butler et al. 1995, Trends Pharmacol. Sci. 16:18-22; Stamler et al. 1992, Science 258:1898-1902). NO is also know to react with thiols to form S-nitrosothiols (RS-NO) and may represent a long-term storage form for NO. This category includes compounds having differing structural features. For example, NO donors include, but are not limited to: metabolic precursors of NO such as L-arginine and L-citrulline; so-called "organonitrates" such as nitroglycerin (GTN), glyceryl trinitrate, isosorbide 5-mononitrate (ISMN), isosorbide dinitrate (ISDN), pentaerythritol tetranitrate (PETN), erythrityl tetranitrate (ETN), amino acid derivatives such as N- hydroxy-L-arginine (NOHA), N6-( 1 -iminoethyl)lysine) (L-NIL)5 L-N5-( 1 - iminoethyl)ornithine (LN-NIO), N-methyl-L-arginine (L-NMMA), and S- nitrosoglutathione (SNOG); other compounds which generate or release NO under physiologic conditions such as S,S-dinitrosodithiol (SSDD), [N-[2-(nitroxyethyl)]-3-p- yridinecarboxamide (nicorandil), sodium nitroprusside (SNP), S-nitroso-N- acetylpenicillamine (SNAP), 3-morpholmo-sydnonimine (SIN-I), molsidomine, DEA- NONOate (2-(N,N-diethylamino)-diazenolate-2-oxide), spermine NONOate (N-[4-[l- (3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]bu- tyl-l,3-propanediamine), and NO gas, or a functional equivalent thereof. The organic nitrates GTN, ISMN, ISDN, ETN, and PETN, as well as nicorandil are commercially available in pharmaceutical dosage forms. SIN-I, SNAP, S-thioglutathione, L-NMMA5 L-NIL, L-NIO5 spermine

NONOate, and DEA-NONOate are commercially available from Biotium, Inc. 183 Shoreline Court, Richmond, Calif., USA. In other embodiments, the NO donor is suitably selected from [3-(lH-Imidazol-4-yl)propyl]guanidines-containing furoxan moieties, as for example, described in Bertinaria et al. (2003, Bioorganic & Medicinal Chemistry 11: 1197-1205), NO-donor phenols as described, for example, in Boschi et al. (2006, J Med. Chem. 49: 2886-2897), pseudojujubogenin glycosides such as dammarane-type triterpenoid saponins (e.g., bacopasaponins) as well as their derivatives or analogues. In specific embodiments, the NO donor is PRGlOO, as disclosed in Example 1. [0045] In accordance with the present invention, an NO donor is administered at a level that enhances NO and that does not alter normal systemic vascular tone in the subject. Suitably, the level of NO is a sub-normovasodilatory (SNV) concentration that ranges from about 1A to about 10"15 of a reference concentration required to induce vasodilation in an anatomical site of a reference subject lacking a vascular condition, which is suitably the neuropathic condition. Vasodilation may be measured using any suitable technique for defining SNV concentrations. Illustrative methods for measuring vasodilation include, but are not limited to, measuring systolic blood pressure (e.g., in a limb or tail), by measuring blood flow in ears or using the vasodilation assay described in Pharmacol Res. 39(3): 217-20 (1999). In a specific embodiment, systolic blood pressure is measured in normotensive experimental animals (e.g., rats) that are lightly sedated via intraperitoneal injection of Zoletil (tiletamine 15 mg/kg, zolazepam 15 mg/kg), using an inflatable tail-cuff. Representative SNV concentration ranges include from about V2 to about 1/20, V2 to about 1/50, 1A to about 10"1, 10'1 to about 10"15, 10"2 to about 10"15, 10'3 to about 10'15, 10"4 to about 10"15, lO'5 to about 10"15, 10"6 to about 10"15, 10'2 to about 10-'3, 10'2 to about 10'12, 10"2 to about 10"11, 10'2 to about 10"10, 10"2 to about 10"9, 10"2 to about 10"8, 10"2 to about 10"7, or 10"2 to about 10"6 of the reference concentration. In some embodiments in which the NO donor is in slow-release form, the amount of NO donor that is administered as a bolus is in the range of 0.000001 nmol/kg to 2 nmol/kg, 0.00001 nmol/kg to 2 nmol/kg, 0.0001 nmol/kg to 2 nmol/kg, 0.001 nmol/kg to 2 nmol/kg, 0.001 nmol/kg to 1 nmol/kg, 0.001 nmol/kg to 0.6 nmol/kg, 0.004 nmol/kg to 0.4 nmol/kg, preferably in a range selected from 0.005 nmol/kg to 0.3 nmol/kg, 0.006 nmol/kg to 0.2 nmol/kg, 0.007 nmol/kg to 0.1 nmol/kg, 0.008 nmol/kg to 0.09 nmol/kg, 0.009 nmol/kg to 0.08 nmol/kg, 0.01 nmol/kg to 0.07 nmol/kg, 0.02 nmol/kg to 0.06 nmol/kg, and especially 0.03 nmol/kg to 0.05 nmol/kg. In other embodiments in which the NO donor is formulated in a sustained release formulation, the NO donor is adapted to release nitric oxide at a rate of 0.00001 nmol/kg/hour to 2.0 nmol/kg/hour, 0.0001 nmol/kg/hour to 2.0 nmol/kg/hour, 0.0002 nmol/kg/hour to 2.0 nmol/kg/hour or in a range selected from 0.001 nmol/kg/hour to 1.0 nmol/kg/hour, 0.005 nmol/kg/hour to 1.0 nmol/kg/hour, 0.001 nmol/kg/hour to 0.5 nmol/kg/hour, 0.002 nmol/kg/hour to 0.2 nmol/kg/hour, 0.005 nmol/kg/hour to 0.1 nmol/kg/hour, or 0.01 nmol/kg/hour to 0.05 nmol/kg/hour. In illustrative examples of this type, the NO donor is a transdermal patch adapted to release 0.5 nmol to 500 nmol, especially 10 nmol to 100 nmol, more especially 20 nmol to 60 nmol and even more especially about 50 nmol over 6, 9, 12, 18, 24 or 30 hours.

[0046] Suitably, the level of NO donor administered is effective for treating or preventing a neuropathic condition, including a peripheral neuropathic condition such as PDN or a related condition, and especially for the treatment or prevention of pain in neuropathic conditions, including the prevention of incurring pain, holding pain in check, and/or treating existing pain. Whether pain has been treated is determined by measuring one or more diagnostic parameters which is indicative of pain (e.g., subjective pain scores, tail-flick tests and tactile allodynia) compared to a suitable control. In the case of an animal experiment, a "suitable control" is an animal not treated with the nitric oxide donor, or treated with the pharmaceutical composition without nitric oxide donor. In the case of a human subject, a "suitable control" may be the individual before treatment, or may be a human (e.g., an age-matched or similar control) treated with a placebo. In accordance with the present invention, the treatment of pain includes and encompasses without limitation: (i) preventing pain experienced by a subject which may be predisposed to the condition but has not yet been diagnosed with the condition and, accordingly, the treatment constitutes prophylactic treatment for the pathologic condition; (ii) inhibiting pain initiation or a painful condition, i.e., arresting its development; (iii) relieving pain, i.e., causing regression of pain initiation or a painful condition; or (iv) relieving symptoms resulting from a disease or condition believed to cause pain, e.g., relieving the sensation of pain without addressing the underlying disease or condition.

3. NO donor-containing compositions [0047] Another aspect of the present invention provides compositions for producing analgesia and especially for treating, preventing and/or alleviating the painful symptoms of a neuropathic condition. These analgesic compositions generally comprise at least one NO donor at a level that enhances NO and that does not alter normal systemic vascular tone in the subject, as broadly described above. The effect of the compositions of the present invention may be examined by using one or more of the published models of pain/nociception or of neuropathy, especially peripheral neuropathy, and more especially PDN, known in the art. This may be demonstrated, for example using an animal model which assesses the onset and development of tactile allodynia, the defining symptom of PDN, as for example described herein. The analgesic activity of the compounds of this invention can be evaluated by any method known in the art. Examples of such methods are the Tail-flick test (D 'Amour et al. 1941, J Pharmacol. Exp. and Ther. 72: 74-79); the Rat Tail Immersion Model, the Carrageenan-induced Paw Hyperalgesia Model, the Formalin Behavioral Response Model (Dubuisson et al. 1977, Pain 4: 161-174), the Von Frey Filament Test (Kim et al. 1992, Pain 50: 355-363), the Chronic Constriction Injury, the Radiant Heat Model, and the Cold Allodynia Model (Gogas et al. 1997, Analgesia 3: 111-118), the paw pressure test of mechanical hyperalgesia (Randall and Selitto, 1997, Arch Int

Pharmacodyn 111: 409-414; Hargreaves et al. 1998, Pain, 32: 77-88). An in vivo assay for measuring the effect of test compounds on the tactile allodynia response in a rat model of painful diabetic neuropathy is described in Example 1. Compositions which test positive in such assays are particularly useful for the treatment or prevention of neuropathic pain.

[0048] The NO donors may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, maleic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

[0049] The dose of NO donor administered to a patient should be sufficient to achieve a beneficial response in the patient over time such as a reduction in, or relief from, pain, especially neuropathic pain. The quantity of the compound(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. This quantity, however, will be one that enhances NO and that does not alter normal systemic vascular tone in the subject. In this regard, precise amounts of the NO donor(s) for administration will depend on the judgement of the practitioner. In determining the effective amount of the NO donor(s) to be administered in the production of analgesia, the physician may evaluate severity of the pain symptoms associated with nociceptive or inflammatory pain conditions or numbness, weakness, pain, loss of reflexes and tactile allodynia associated with neuropathic conditions, especially peripheral neuropathic conditions such as PDN. In any event, those of skill in the art may readily determine suitable dosages of the nitric oxide donors of the invention without undue experimentation. [0050] In some embodiments, and dependent on the intended mode of administration, the NO donor-containing compositions will generally contain about 0.001% to 90%, about 0.1% to 50%, or about 1% to about 25%, by weight of NO donor, the remainder being suitable pharmaceutical carriers and/or diluents etc. The dosage of the nitric oxide donor can depend on a variety of factors, such as the individual nitric oxide donor, mode of administration, the species of the affected subject, age and/or individual condition. [0051] Depending on the specific neuropathic condition being treated, the NO donor(s) may be formulated and administered systemically, topically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include buccal, sublingual, oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the therapeutic agents of the invention may be formulated in aqueous solutions, suitably 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.

[0052] Alternatively, the compositions of the invention can be formulated for local or topical administration. In this instance, the subject compositions may be formulated in any suitable manner, including, but not limited to, creams, gels, oils, ointments, solutions and suppositories. Such topical compositions may include a penetration enhancer such as benzalkonium chloride, digitonin, dihydrocytochalasin B, capric acid, increasing pH from 7.0 to 8.0. Penetration enhancers which are directed to enhancing penetration of the active compounds through the epidermis are advantageous in this regard. Alternatively, the topical compositions may include liposomes in which the active compounds of the invention are encapsulated.

[0053] The compositions of this invention may be formulated for administration in the form of liquids, containing acceptable diluents (such as saline and sterile water), or may be in the form of lotions, creams or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity and appearance. Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, emulsifying agents such as non- ionic organic and inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives, and hydrophilic beeswax derivatives.

[0054] Alternatively, the NO donor(s) can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration, which is also preferred for the practice of the present invention. Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for buccal or sublingual administration or oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water. [0055] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be 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 that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilisers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0056] Pharmaceutical preparations for buccal, sublingual or oral use can be obtained by combining the active compounds with solid excipients, 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 are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as., for example, maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilising processes.

[0057] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, 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 characterise different combinations of active compound doses. [0058] For buccal or sublingual administration, the formulations of the invention can be provided in the form of a tablet, patch, troche, or in free form, such as a gel, ointment, cream, or gum. Examples of suitable buccal or sublingual formulations and devices are disclosed, for example, in U.S. Pat. Nos. 5,863,555, 5,849,322, 5,766,620, 5,516,523, 5,346,701, 4,983,395, and 4,849,224. Such formulations and devices can use a suitable adhesive to maintain the device in contact with the buccal mucosa. Examples of suitable adhesives are found, for example, in U.S. Pat. Nos. 3,972,995, 4,259,314, 4,680,323; 4,740,365, 4,573,996, 4,292,299, 4,715,369, 4,876,092, 4,855,142, 4,250,163, 4,226,848, and 4,948,580. Typically, the adhesive comprises a matrix of a hydrophilic, e.g., water soluble or swellable, polymer or mixture of polymers that can adhere to a wet, mucous surface. These adhesives can be formulated as ointments, thin films, tablets, troches, and other forms. Other non-limiting buccal or sublingual formulations are disclosed in U.S. Pat. Nos. 7,067,116; 7,025,983; 6,923,981; 6,596,298; 6,726,928; 6,709,669; 6,509,040; 6,413,549; 5,976,577; 5,827,541; 5,738,875; 5,648,093; 5,631,023; 5,188,825; 4,020,558; 4,229,447; 3,972,995; 3,870,790; 3,444,858; 2,698,822; 3,632,743, and U.S. Published Application Nos. 20070059361; 20040247648; 20040131661; and 20040028730. In some embodiments, the dosage forms are prepared using pharmaceutically acceptable excipients. Illustrative excipients that are commonly formulated into buccal and sublingual dosage forms include maltodextrin, colloidal silicon dioxide, starch, starch syrup, sugar and α-lactose.

[0059] Pharmaceuticals which can be used orally include push-fit capsules made of gelatine, as well as soft, sealed capsules made of gelatine and a plasticiser, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilisers. 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, stabilisers may be added. [0060] Dosage forms of the NO donors may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an active compound of the invention may be achieved by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, controlled release may be achieved by using other polymer matrices, liposomes and/or microspheres.

[0061] The NO donors may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the neuropathic condition being treated, whether a recurrence of the condition is considered likely, etc. The administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, e.g., active compounds may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable. [0062] The compositions of the present invention may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebuliser, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients. In such a case, the particles of the formulation may advantageously have diameters of less than 50 micrometers, suitably less than 10 micrometers. [0063] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

EXAMPLE 1

ANTI-ALLODYNIC EFFICACY AND POTENCY OF THE NO DONOR PRGlOO [0064] This study was designed as a dose identification study such that each rat received 3-4 single bolus doses of the NO donor, PRGlOO, with each dose separated by an approximately 3 -day washout period. Single bolus doses of PRGlOO were administered by subcutaneous (s.c.) injection into the back of the neck of STZ-diabetic rats at 10, 14 and 24 wks post-STZ administration. Von Frey filaments were used to quantify paw withdrawal thresholds in the rat hindpaws. [0065] Tactile (mechanical) allodynia was fully developed in the hindpaws of

STZ-diabetic Wistar rats by ~ 9-10 wks post-STZ administration (Figure 1). Following administration of single s.c. bolus doses of the NO donor, PRGlOO, to STZ-diabetic rats at 10 wks post-STZ administration, dose-dependent relief of tactile allodynia was produced (Figure 2) such that at doses of 80 and 800 fmol/kg, tactile allodynia was completely alleviated. At 14 and 24 wks post-STZ administration in the same STZ- diabetic rats, ~ 1000-fold larger doses of PRGlOO were required to produce dose- dependent relief of tactile allodynia (Figure 3). At PRGlOO doses of 80 and 800 pmol/kg, tactile allodynia was essentially fully relieved.

EXPERIMENTAL

PREPARATION OF PRGlOO

[0066] PRGlOO is a [3-(lH-Imidazol-4-yl)propyl]guanidines-containing furoxan, which was prepared according to the method described in Bertinaria et al. (2003 , Bioorganic & Medicinal Chemistry 11: 1197-1205). EXPERIMENTAL ANIMALS

[0067] Adult male Wistar rats (225 g - 250 g) were purchased from the Central Animal Breeding House, The University of Queensland (Brisbane, Australia) and were housed in a temperature-controlled environment (21 ± 20C) with a 12 h/12 h dark/light cycle; food and water were available ad libitum. Rats were given at least a 24 h acclimatisation period prior to experimentation. Ethics approval for these experiments was obtained from the Animal Experimentation Ethics Committee of The University of Queensland.

DRUGS AND MATERIALS [0068] PRG 100 was synthesized by Dr Craig Williams, Department of

Chemistry, The University of Queensland. Streptozotocin, citric acid, and trisodium citrate were purchased from Sigma-Aldrich Pty Ltd (Sydney, Australia). Xylazine (Ilium Xylazil ™), Zoletil 100® solution (containing: tiletamine HCl 2.5 mg/ml and zolazepam HCL 2.5 mg/ml), sodium benzylpenicillin (Benpen™) vials containing 600 mg of powder and topical antibiotic powder (containing: neomycin sulphate 2.5 mg, sulfacetamide sodium 100 mg, nitrofurazone 2 mg, phenylmercuric nitrate 0.05 mg and benzocaine 5 mg in 50 g soluble powder) were purchased from Provet Queensland Pty Ltd (Brisbane, Australia). Blood glucose meters (Precision QID, Accucheck advantage 2) and glucose testing electrodes (Precision Plus™ and Accucheck Advantage™) were purchased from the Campus Pharmacy at The University of Queensland (Brisbane,

Australia). Medical grade CO2 and O2 were purchased from BOC gases Ltd (Brisbane, Australia) and isoflurane (Isoflo™) was purchased from Abbott Australasia Pty Ltd (Sydney, Australia).

DIABETES INDUCTION [0069] Whilst anaesthetized with a mixture of Zoletil 100® (tiletamine HCl

0.625 mg and zolazepam 0.625 mg) and xyalazine HCl (5 mg) administered by intraperitoneal injection to induce deep and stable anaesthesia, adult male Wistar rats were administered single intravenous bolus doses of streptozotocin (STZ, 75 mg/kg) via a cannula surgically inserted into the superior aspect of the bifurcation of the internal and external jugular veins. Following this, the jugular cannula was withdrawn, the vein tied off, and the incision sutured. Following surgery, rats received antibiotic prophylaxis in the form of topical dusting powder over the sutured incisions, as well as a subcutaneous injection of benzylpenicillin (60 mg). Rats were placed in individual cages, kept warm and monitored closely during the surgical recovery period. Rats were classified as diabetic if on day 10 post-STZ administration, their daily water intake was > 100 mL and their corresponding blood glucose levels (BGLs) were > 15 mM.

DEVELOPMENT OF TACTILE (MECHANICAL) ALLODYNIA

[0070] Calibrated von Frey filaments were used to document the time course for the development and maintenance of tactile allodynia (defining symptom of PDN). Tactile allodynia was considered to be fully developed when von Frey paw withdrawal thresholds in the hindpaws of STZ-diabetic rats were < 6g compared with ~12 g in the same animals prior to the induction of diabetes with STZ..

[0071] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0072] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [0073] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

EXAMPLE 2

EFFECTS OF THE NO DONOR PRGlOO ON SYSTOLIC BLOOD PRESSURE IN

NORMOTENSIVE WlSTAR RATS [0074] This study was designed to assess the effects of single subcutaneous

(s.c.) bolus doses of the NO donor, PRGlOO, relative to vehicle (DMS O: water, 90:10) on systolic blood pressure in normotensive, non-diabetic, adult male Wistar rats. Single bolus doses of PRGlOO were administered by s.c. injection into the back of the neck of adult male normotensive, non-diabetic Wistar rats in the dose range, 80 fmol/kg to 80 μmol/kg. Specifically, the doses of PRGlOO tested were 80 fmol/kg (n=3), 800 pmol/kg (n=8), 8 nmol/kg (n=6), 800 nmol/kg (n=4), 8 μmol/kg (n=4), 80 μmol/kg (n=3).

[0075] Following administration of single s.c. bolus doses of the NO donor, PRGlOO, to normotensive non-diabetic adult male Wistar rats, there were no significant effects (p > 0.05) on systolic blood pressure for doses in the range, 80 fmol/kg to 8 μmol/kg (Figure 4). However, at the highest dose tested (80 μmol/kg, n=3), mean (± SEM) systolic blood pressure decreased significantly (p < 0.05) from 111.3 (±1.5) mm Hg just prior to administration of PRGlOO (80 μmol/kg) to 79.8 (±0.5) mm Hg at the time of peak effect at 30 min post-dosing (Figure 4). By 1 h post-dosing, mean systolic blood pressure in the same animals (112.6 ± 0.4 mm Hg) did not differ significantly (p > 0.05) from baseline systolic blood pressure determined just prior to dose administration (Figure 4).

EXPERIMENTAL

EXPERIMENTAL ANIMALS [0076] Adult male Wistar rats (300 g - 350 g) were purchased from UQBR

(University of Queensland Biological Resources), The University of Queensland (Brisbane, Australia) and were housed in a temperature-controlled environment (21 ± 20C) with a 12 h/12 h dark/light cycle; food and water were available ad libitum. Rats were given at least a 24 h acclimatisation period prior to experimentation. Ethics approval for these experiments was obtained from the Animal Experimentation Ethics Committee of The University of Queensland.

DRUGS AND MATERIALS

[0077] PRGl 00 was synthesized by Dr Craig Williams, Department of Chemistry, The University of Queensland. Zoletil 100® solution (containing: tiletamine HCl 2.5 mg/ml and zolazepam HCL 2.5 mg/ml), was purchased from Provet Queensland Pty Ltd (Brisbane, Australia).

BLOOD PRESSURE MEASUREMENT

[0078] Sedation was induced by the intraperitoneal administration of Zoletil® (tiletamine 15 mg kg'1 and zolazepam 15 mg kg"1). Using a tail pulse transducer (MLTlOlO) and an inflatable tail cuff with a Capto SP844 physiological pressure transducer (MLT844/D), systolic blood pressure was recorded via a PowerLab data acquisition unit (ADInstruments, Sydney, Australia). Tails were warmed with lukewarm water prior to each blood pressure reading to increase peripheral circulation so as to facilitate blood pressure recordings using the tail cuff method.

WHAT IS CLAIMED IS:

1. A method for treating or preventing a neuropathic condition in a subject, the method consisting essentially of administering to the subject at least one NO donor at a level that enhances NO and that does not alter normal systemic vascular tone in the subject.

2. A method according to claim 1 , wherein the at least one NO donor is administered without co-administration of an opioid analgesic.

3. A method according to claim I5 wherein the level of NO is a sub- normovasodilatory (SNV) concentration that ranges from about 1A to about 10"15 of a reference concentration required to induce vasodilation in an anatomical site of a reference subject lacking a vascular condition.

4. A method according to claim 3, wherein the anatomical site is selected from kidney, skin, skeletal muscle, arm, leg, tail and gastro-intestinal tract.

5. A method according to claim 3, wherein the vascular condition associates with the neuropathic condition.

6. A method according to claim 5, wherein the neuropathic condition is PDN.

7. A method according to claim 1, wherein the at least one NO donor is administered in the form of a composition comprising a pharmaceutically acceptable carrier and/or diluent. 8. A method according to claim 7, wherein the composition is administered by injection, by topical application or by sublingual, rectal or oral routes over a period of time and in an amount which is effective for delivering the SNV concentration of NO to the subject.

9. A method according to claim I5 wherein the at least one NO donor is provided in a sustained release formulation.

10. A method according to claim I5 wherein an individual NO donor is a slow- release NO donor that delivers the SNV concentration of NO to the subject.

11. A method according to claim 10, wherein the NO donor is administered as a bolus in the range of 0.000001 nmol/kg to 2 nmol/kg. 12. A method according to claim 10, wherein the NO donor is a transdermal patch adapted to release 0.5 nmol to 500 nmol of NO over at least 6 hours.

13. A method according to claim 1, wherein the at least one NO donor is selected from: L-arginine, L-citrulline, nitroglycerin (GTN)5 glyceryl trinitrate, isosorbide 5 -mononitrate (ISMN), isosorbide dinitrate (ISDN), pentaerythritol tetranitrate (PETN), erythrityl tetranitrate (ETN), N-hydroxy-L-arginine (NOHA), N6- (l-iminoethyl)lysine) (L-NIL), L-N5-(l-iminoethyl)ornithine (LN-NIO), N-methyl-L- arginine (L-NMMA), S-nitrosoglutathione (SNOG); S,S-dinitrosodithiol (SSDD), [N- [2-(nitroxyethyl)]-3-p- yridinecarboxamide (nicorandil), sodium nitroprasside (SNP), S- nitroso-N-acetylpenicillamine (SNAP), 3-morpholino-sydnonimine (SIN-I), molsidomine, DEA-NONOate (2-(N,N-diethylamino)-diazenolate-2-oxide), spermine NONOate (N-[4-[l -(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]bu- tyl-1 ,3- propanediamine), NO gas, [3-(lH-Imidazol-4-yl)propyl]guanidines-containing furoxan moieties, NO-donor phenols and pseudojujubogenin glycosides.

14. A method according to claim 1, wherein the at least one NO donor is a [3- ( 1 H-Imidazol-4-yl)propyl] guanidine-containing furoxan

15. A method according to claim 14, wherein the [3-(lH-Imidazol-4- yl)propyl] guanidine-containing furoxan is PRGlOO. 16. A method for preventing or attenuating neuropathic pain in a subject, the method consisting essentially of administering to the subject at least one NO donor at a level that enhances NO and that does not alter normal systemic vascular tone in the subject.

17. A method according to claim 16, wherein the at least one NO donor is administered without co-administration of an opioid analgesic.

18. Use of a composition that consists essentially of at least one NO donor for producing analgesia in a subject having a neuropathic condition, wherein the at least one NO donor is provided at a level that enhances NO and that does not alter normal systemic vascular tone in the subject. 19. A use according to claim 18, wherein the composition excludes an opioid analgesic.

20. Use of at least one NO donor in the manufacture of a medicament for producing analgesia in a subject having a neuropathic condition and in the absence of co-administering an opioid analgesic to the subject, wherein the at least one NO donor is formulated in an amount that enhances NO and that does not alter normal systemic vascular tone in the subject.

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