WO2006116808A1 - Treating peripheral neuropathies - Google Patents

Abstract

The invention provides a method of treating or preventing a peripheral neuropathy or treating nerves by administering to a patient an effective amount of an antagonist of a neuronal nicotinic acetyl choline receptor (nAChR), for example an antibody or antisense molecule or an α conotoxins.

Description

TREATING PERIPHERAL NEUROPATHIES

The invention relates to methods for treating peripheral neuropathies.

BACKGROUND

A peripheral neuropathy is a disorder that affects the peripheral nerves, most often manifested as one or a combination of motor, sensory, sensorimotor, or autonomic neural dysfunction.

The peripheral nerves make up an intricate network that connects the brain and spinal cord to the muscles, skin, and internal organs. Peripheral nerves come out of the spinal cord and are arranged along lines in the body called dermatomes. Typically, damage to a nerve will affect one or more dermatomes, which can be tracked to specific areas of the body.

As a group, peripheral neuropathies are common, especially among people over the age of 55. The conditions collectively affect 3% to 4% of people in this group.

The wide variety of morphologies exhibited by peripheral neuropathies can each be uniquely attributed to an equally wide variety of causes. It is often difficult to discern the cause and in up to one in three cases, the cause cannot be determined. Diabetes is responsible for another third of cases . Other known causes include several rare inherited diseases, alcoholism, toxins and certain environmental agents, some medications, poor nutrition or vitamin deficiency, trauma due to compression, herniated discs in the back, certain kinds of cancer, conditions where nerves are mistakenly attacked by the body's own immune system or damaged by an overaggressive response to injury, particular medications, kidney disease, thyroid disease, and infections such as Lyme disease, shingles, HIV or AIDS.

Hereditary neuropathies are diseases of the peripheral nerves that are genetically passed from parent to child. The most common of these is Charcot-Marie-Tooth disease type 1, which is characterized by weakness in the legs and, to a lesser degree, the arms. Caused by degeneration of the myelin that normally surrounds the nerves and helps them conduct the electrical impulses needed for them to trigger muscle movement, symptoms usually appear between mid-childhood and age 30.

Neuropathies are typically classified either according to the problems they cause or according to the root of the damage. They may also be described in terms that express how extensively the nerves have been damaged. Damage to a single peripheral nerve is called a mononeuropathy. Physical injury or trauma such as from an accident is the most common cause of mononeuropathy. Prolonged pressure on a nerve, caused by extended periods in sedentary positions such as in a wheelchair or bed, or continuous, repetitive motions can trigger mononeuropathy. If the fibrous, shock-absorbing discs that lie between the bones in the back are damaged, they could press on a nerve and cause this type of neuropathy.

Carpal tunnel syndrome is another common type of mononeuropathy. It is called an overuse strain injury and occurs when the nerve that extends through the wrist is compressed, damaging the nerve. In time, carpal tunnel injuries can weaken the muscles in the hand.

Neuropathy can affect a variety of nerves, those that affect muscle movement and those that detect sensations such as coldness or pain, in some cases, it can affect internal organs, such as the heart, blood vessels, or the bladder and intestines. Neuropathy that affects internal organs is called an autonomic neuropathy. Polyneuropathy accounts for the greatest number of peripheral neuropathy cases. It is caused when many peripheral nerves throughout the body malfunction at the same time. Polyneuropathy can have a wide variety of causes, including exposure to certain toxins, such as when undergoing chemotherapy or drug abuse, poor nutrition (particularly vitamin B deficiency) , as a result of diseases such as diabetes, HIV, AIDS and auto-immune diseases, and complications from diseases such as cancer, liver or kidney failure. The most common symptoms of polyneuropathy are tingling, numbness, burning pain and loss of sensation in the arms and legs. Because people with chronic polyneuropathy often lose their ability to sense temperature and pain, they can burn themselves and develop open sores as the result of injury or prolonged pressure. If the nerves serving the organs are involved, diarrhea or constipation may result, as well as loss of bowel or bladder control. Sexual dysfunction and abnormally low blood pressure also can occur. Joints are particularly vulnerable to stress in people with polyneuropathy because they are often insensitive to pain.

One of the most common forms of chronic polyneuropathy is diabetic neuropathy, a condition that occurs in diabetics as a result of poorly controlled blood sugar levels. Though less common, diabetes can also cause mononeuropathy, often characterized by weakness of ocular or thigh muscles . Estimates of the prevalence of neuropathy in diabetes vary widely, from a low of 5% to a high of 80%, largely due to the numerous definitions and clinical descriptions of neuropathy. Nevertheless, the additive effects of neuropathy in the suffering diabetic patient are well known and documented. The effect of the neuropathy is complex. The loss of sensory information from the foot is related to abnormal and prolonged pressure on the areas of the foot (sensory neuropathy) . Motor neuropathy leads to deformity, further increasing pressure loading on the foot . In autonomic neuropathy, loss of innervation of the sweat glands results in dry skin that cracks creating an environment amenable to infection. Autonomic dysfunction contributes further by altering the distribution of micro-circulatory blood flow, directing the blood flow through shunts and away from the nutritive skin capillaries. These factors as a whole, in conjunction with foot trauma, result in skin breakdown and ulcers . Scientists have not yet determined the mechanism that leads to nerve damage in diabetic subjects, but it is believed to be multifactorial. These factors include genetic predisposition, metabolic and vascular abnormalities, and lack of perturbation of growth factors. The response of the peripheral nervous system to the metabolic effects of diabetes does not appear to differ between type 1 and type 2 diabetes, which suggests a likelihood of similar clinical response to therapies in the two primary forms of the disease. There seem to be a number of susceptibility factors, as yet unknown, for the development of neuropathy, which operate in the presence of hyperglycemia (high blood sugar) . Currently, there are a limited number of drugs available for the treatment of peripheral neuropathies . Most treatments composition and methods are directed towards relief of pain. For example, current methods to treat neuropathic pain include administration of local anesthetic blocks targeted to trigger points, peripheral nerves, plexi, dorsal roots, and to the sympathetic nervous system. However, these treatments have only shortlived antinociceptive effects and do not treat the cause of the neuropathic pain.

There are currently no drugs on the market for the treatment of diabetic neuropathy. There are some drugs in trials or awaiting trials, including alond (zopolrestat ; Pfizer), zenarestat (Fujisawa), timcodar dimesylate (Vertex) , the NMDA antagonist memantine, neurulin (Cortec) , and an IGF-II product (Aurogen) .

Other approaches are being tried or being considered, including aldose reductase inhibitors, which are thought to inhibit the increased flux through the polyol pathway caused by high blood glucose, mimicking the effect of improved glycemic control, nerve growth factor, alpha-lipoic acid, gamma-linolenic acid as a food supplement, insulin-like growth factor hormones, immunoglobulin, myo-inositol , or aminoguanidine . However, there is still a substantial need for an improved treatment for diabetic neuropathy, particularly a treatment that can actually slow or reverse the degeneration of the nerves involved.

There is also a need for treatments for other peripheral neuropathies. There is a particular need for methods that protect against nerve damage, or can stimulate nerve growth or regeneration, particularly without inducing hyperalgesia.

It is an aim of a preferred embodiment of the invention to provide a method of treating a peripheral neuropathy, particularly a peripheral neuropathy associated with diabetes.

SUMMARY

Accordingly, a first aspect of the invention provides a method of treating or preventing a peripheral neuropathy in a patient, said method comprising administering to the patient an effective amount of a neuronal nicotinic acetyl choline receptor (nAChR) antagonist .

In a second aspect the present invention provides the use of an antagonist of a neuronal nAChR in the manufacture of a medicament for treating or preventing a peripheral neuropathy.

Also provided is a pharmaceutical or veterinary agent for treating or preventing a peripheral neuropathy, which agent comprises an antagonist of a neuronal nAChR.

Further provided is a pharmaceutical or veterinary composition for the treatment or prevention of a peripheral neuropathy, the composition comprising an antagonist of a neuronal nAChR, together with a pharmaceutically-acceptable carrier.

In a preferred embodiment of each aspect of the invention, the peripheral neuropathy is a diabetic neuropathy. It will be clearly understood that the diabetic neuropathy may be associated with Type 1 (insulin-dependent) diabetes, Type 2 (non-insulin- dependent) diabetes, or both.

In other preferred embodiments, the neuropathy is a non-diabetic neuropathy. Such a non-diabetic neuropathy may be genetically acquired, such as Charcot- Marie-Tooth syndrome. In other embodiments the peripheral neuropathy can result from a systemic or infectious disease such as HIV, or an infectious disease condition such as AIDS. In further embodiments, the peripheral neuropathy is post herpetic neuropathy. In another embodiment the peripheral neuropathy manifests as a post surgical complication. In other embodiments the peripheral neuropathy is induced by a toxic agent. For example, the peripheral neuropathy can be caused by a chemotherapeutic agent such as taxol, vincristine, cisplatin, an agent used for the treatment of infectious diseases such as streptomycin, didanosine or zalcitabine, or any other chemically toxic agent . In another embodiment peripheral neuropathies induced by infectious diseases, conditions such as post- polio syndrome or AIDS-associated neuropathy, are specifically contemplated. Other peripheral neuropathies contemplated for treatment or prevention in accordance with the present invention include HIV associated neuropathy; vitamin B_i2- deficiency associated neuropathy; cranial nerve palsies; drug-induced neuropathy, including drug abuse related neuropathy; industrial neuropathy; lymphomatous neuropathy; myelomatous neuropathy; multi-focal motor neuropathy; chronic idiopathic sensory neuropathy; carcinomatous neuropathy; acute pan autonomic neuropathy; alcoholic neuropathy; compressive neuropathy; vasculitic/ischaemic neuropathy; and mono- and polyneuropathies . BRIEF DESCRIPTION QF FIGURES

Figure 1 provides a graph showing the antiallodynic effect of ACVl, a neuronal nAChR antagonist, in diabetic rats. Figure 2 provides graphs showing indicators of tissue injury. Figure 2a shows lipid hydroperoxide levels in nerve biopsy samples from animals treated with ACVl versus control levels. Figure 2b shows nitrotyrosine levels in systemic blood samples from animals treated with ACVl versus control levels. * indicates significantly different (P value < 0.05) from undiabetic vehicle, # indicates significantly different (P value < 0.05) from diabetic vehicle, and $ P = 0.0058 when compared to diabetic vehicle Figure 3 provides a graph showing in vitro measurement of the neuroprotective effect of ACVl in cerebellar microexplants against a toxic agent 3NP/glutamate.

Figure 4 provides a graph showing in vitro measurement of the neuroprotective effect of ACVl in rat DRG (peripheral) neurons against the toxicity causes by chemotherapeutic agent cisplatin. DETAILED DESCRIPTION

Nicotinic acetyl choline receptors (nAChRs) are ligand-gated ion channels which consist of five subunits arranged around a cation-conducting pore.

There are two main classes of nAChRs, the neuronal type; and the muscle type.

Distinct receptor subtypes that comprise the nicotinic acetylcholine receptor (nAChR) family are found on skeletal muscle at the neuromuscular junction, within the brain and spinal cord, on sensory nerves and some peripheral nerve terminals. These receptors function as ligand-gated ion channels. Upon binding ligands which are agonists, nAChRs are transiently converted to an open channel state (active conformation) which allows cation influx and subsequent depolarization of the cell.

Examples of ligands which function as agonists are the natural neurotransmitter, acetylcholine, its nonhydrolyzable analog, carbamylcholine, DMPP, epibatidine and anatoxin-ce. Antagonist ligands include d-tubocurarine, and the snake venom α-neurotoxins, such as α-bungarotoxin.

Muscle type receptors are composed of α, β, γ and ε (or δ) subunits . In the peripheral and central nervous systems, it is recognized that there is a molecular diversity of neuronal nAChRs subtypes composed of pentameric oligomers from a multi-gene family containing at least 13 members (cei-ce_9; βz-βs) ■ Molecular and biochemical approaches have allowed neuronal nAChR subunits to be classified as either subunits involved in binding of acetylcholine (α-subunits) or structural subunits (termed either as non-α or as β) . The acetylcholine binding subunits have been defined on the basis of adjacent cysteine residues (Cys 192 and 193) in the primary sequences that are known to be part of the agonist binding site and by reactivity with acetylcholine affinity alkylating agents.

There are at least nine neuronal α-subunits (a_±- (X₉) which can be divided into two classes on the basis of their ability to bind α-bungarotoxin (subunits Ot₇ and oϋ₈) and at least four neuronal β subunits (_/62-_/6₅) • A variety of functional neuronal nAChR subtypes have been constructed in heterologous expression studies. Pairwise coexpression of either a₂, 01₃, or α₄ with β₂ or /3₄ subunits has produced active acetylcholine-gated ion channels. These expressed receptor subtypes differ in their pharmacological profiles with respect to both agonist and antagonist sensitivities, as well as blockade by κ-bungarotoxin and are thereby pharmacologically distinguishable. In contrast to other nAChR subunits, a₇ has been shown to form homo oligomer receptors when expressed in Xenopus oocytes, and these active channels are characterized by high Ca²⁺ conductance and rapid desensitization.

Clearly, there is a multitude of possible neuronal nAChR subtype variations based upon combinations of five receptor subunits. Some of the pharmacological profiles for the expressed receptor subunit combinations are correlated with properties of endogenously expressed receptors found in ganglia, the CNS and in cell lines. nAChRs comprised of α₄ and β₂ subunits (nicotine binding sites) and oι₇ (which bind α-bungarotoxin) represent the predominant subtypes in the mammalian brain. Non- α₄ β₂ nAChRs have a more limited localization within the CNS.

Receptor subtypes containing α₃ subunits are characteristic of human ganglionic nAChRs and are found in IMR-32 cells.

Evidence indicates neuronal nicotinic cholinergic channel agonists can function as potent analgesic agents by acting through neuronal nAChRs.

Recently, discovery of the potent antinociceptive actions of epibatidine have led to the identification and development of novel neuronal nAChR subtype-selective nAChR ligands with therapeutic potential as analgesic drugs. Substantial preclinical and clinical data suggest that compounds that selectively activate neuronal nAChR subtypes will have therapeutic utility for the treatment of moderate and severe pain across a wide range of conditions that include: acute, persistent inflammatory and neuropathic pain states. The specificity inherent in drugs targeted at neuronal receptor subtypes allows for a defined mechanism of action with reduced side effect liabilities associated with interactions with nAChRs at the neuromuscular junction.

Other known agents that act through neuronal nAChRs include certain conotoxins . These are nAChR antagonists. Conotoxins are also proposed in the art to be useful in the treatment of any disorder regulated at nAChR, including stroke, pain, epilepsy, nicotine addiction, schizophrenia, Parkinson's disease, small cell lung carcinoma and Alzheimer's disease. International patent application, published as

WO 02/0279236 and which is incorporated herein by reference, describes novel alpha conotoxins with analgesic properties. These conotoxins are proposed to act through neuronal nAChRs. The specification proposes the use of the novel conotoxins in the treatment of pain and conditions associated with neurogenic pain.

Despite knowledge that agents that modulate neuronal nAChRs have utility in treating neuropathic pain, the neuronal nAChR has not previously been proposed as a target in the treatment of peripheral neuropathies.

Surprisingly, the inventors have found that markers of tissue damage through oxidative stress are reduced as well as long term relief provided from tactile allodynia and mechanical hyperalgesia in rats with streptozotocin-induced diabetes, to whom the a conontoxin ACVl, a known neuronal nAChR antagonist, has been administered. These superoxides are known to cause with metabolic and vascular imbalances which bring about the neuropathy, and achieving a reduction in these markers, most often by good glycemic control, is known to prevent development of the neuropathy in humans. Accordingly, the inventors propose that neuronal nAChR antagonists can treat and prevent diabetic neuropathy and other peripheral neuropathies .

Whilst not wishing to be bound by theory, the inventors propose that neuronal nAChR antagonists decrease oxidative stress and thus prevent or decrease nerve damage, or may reduce the effect of oxidative stress on nerve damage. The modulator may act directly on the C fibre or other nerve fibres or alternatively stimulate the glial cells to release neuroprotective factors, to prevent or reduce damage to the nerves or surrounding tissues. Antagonism of neuronal nAChRs may also treat peripheral neuropathies by increasing microcirculation, nitric oxide stabilization, and facilitating healing of skin ulcers and lesions, inhibiting protein kinase C, providing an anti-inflammatory effect, protecting against radiation damage (particularly ultraviolet radiation) , blocking the formation of leukotrienes, stabilizing cell membranes, and/or promoting the synthesis of nerve growth factor. It is further proposed that antagonism of the neuronal nAChR may promote healing of the nerve, for example by encouraging release of neuropeptides that bring about nerve regeneration.

A neuropathy is a disease or disorder involving damage to nerves causing nerve dysfunction. The function of the nerve or nerves that is disrupted may involve the rate of flow of the electrical current through the nerve or may involve ectopic firing (firing in the absence of stimulus) of the nerve, or may involve inappropriate or inadequate firing of the nerve in response to a stimulus. Peripheral neuropathy as used herein is defined as a disorder resulting from damage to peripheral nerves. It may be acquired, caused by diseases of the nerves or as the result of systemic illness.

Neuropathies included within the scope of the term peripheral neuropathy/neuropathies include HIV associated neuropathy; vitamin Bχ₂-deficiency associated neuropathy; cranial nerve palsies; drug-induced neuropathy; industrial neuropathy; lymphomatous neuropathy; myelomatous neuropathy; multi-focal motor neuropathy; chronic idiopathic sensory neuropathy; carcinomatous neuropathy; acute pan autonomic neuropathy; alcoholic neuropathy; compressive neuropathy; vasculitic/ischaemic neuropathy; mono- and polyneuropathies .

Also included within the scope of the term neuropathy/neuropathies are neuropathies associated with systemic diseases such as: uremia; childhood cholestatic liver disease; chronic respiratory insufficiency; alcoholic polyneuropathy; multiple organ failure; sepsis; hypo-albuminemia; eosinophilia-myalgia syndrome; hepatitis; porphyria; hypo-glycemia; vitamin deficiency; chronic liver disease; primary biliary cirrhosis; hyperlipidemia; leprosy; Lyme disease; herpes zoster; Guillain-Barre syndrome; chronic inflammatory demyelinating polyradiculoneuropathy; sensory perineuritis; acquired immunodeficiency syndrome (AIDS)-- associated neuropathy; Sjogren's syndrome; primary vasculitis (such as polyarteritis nodosa) ; allergic granulomatous angiitis; hypersensitivity angiitis; Wegener's granulomatosis; rheumatoid arthritis; systemic lupus erythematosis; mixed connective tissue disease; scleroderma; sarcoidosis; vasculitis; systemic vasculitides; acute tunnel syndrome; pandysautonomia; primary, secondary, localized or familial systemic amyloidosis; hypothyroidism; chronic obstructive pulmonary disease; acromegaly; malabsorption (sprue, celiac disease) ; carcinomas (sensory, sensorimotor, late and demyelinating) ; lymphoma (including Hodgkin's), polycythemia vera; multiple myeloma (lytic type, osteosclerotic, or solitary plasmacytoma) ; benign monoclonal gammopathy; macroglobulinemia; cryoglobulinemia; tropical myeloneuropathies; herpes simplex infection; cytomegalovirus infection; and diabetes .

Also included within the scope of the term neuropathy/neuropathies are genetically acquired neuropathies, including peroneal muscular atrophy (Charcot-Marie-Tooth Disease) hereditary amyloid neuropathies, hereditary sensory neuropathy (type I and type II) , porphyric neuropathy, hereditary liability to pressure palsy, Fabry's Disease, adrenomyeloneuropathy, Riley-Day Syndrome, Dejerine-Sottas neuropathy (hereditary motor-sensory neuropathy-III), Refsum's disease, ataxia- telangiectasia, hereditary tyrosinemia, anaphalipoproteinemia, abetalipoproteinemia, giant axonal neuropathy, metachromatic leukodystrophy, globoid cell leukodystrophy, and Friedri'ch's ataxia. As used herein a "neuronal nAChR" refers to a member of a heterogeneous family of ligand-gated ion channels that are differentially expressed in many regions of the central nervous system (CNS) and peripheral nervous system, for example in the ganglia of the autonomic nervous system and in the central nervous system, in postsynaptic locations, and in pre- and extra-synaptic locations. Neuronal nAChRs as defined as such herein by their ability to respond to nicotine in a similar way as they respond to their natural ligand, the neurotransmitter acetyl choline.

As used herein, an "antagonist of a neuronal nAChR" refers to a compound or signal that alters the activity or numbers of neuronal nAChR so that cellular activity through the nAChR receptors is reduced in the presence of the compound or signal as compared to that in the absence of the compound or signal . The term "antagonist" refers to a substance that interferes with receptor function or reduces receptor number .

Typically, the effect of an antagonist is observed as a blocking of activation by an agonist . The antagonist may bind to the agonist and neutralise its effect at the receptor. Antagonists include competitive and non-competitive antagonists. A competitive antagonist (or competitive blocker) interacts with or near the site on the receptor specific for the agonist (e.g., ligand or neurotransmitter) for the same or closely situated site. A non-competitive antagonist or blocker inactivates the functioning of the receptor by interacting with a site on the receptor other than the site that interacts with the agonist. Some agonists, termed super agonists, have a desensitising effect and are effective antagonists as they fatigue the receptor and therefore reduce or block its function. Suitable antagonists for use in the present invention include antibodies to the neuronal nAChR or antisense molecules able to prevent expression of the neuronal nAChR . Antisense or siRNA may alternatively be used to reduce expression of the receptor protein and thereby reduce the total nAChR-induced activity in the cells. Suitable antagonists for use in the present invention include dihydro-/3-Erythroidine, Erysodine, Mecamylamine, Neosurugatoxin (NSTX) , which blocks ACh- elicited currents in oocytes containing α?₂|6₂, OJ₄₁S₂, and Qf₃₁S₂ nAChR subtypes, neuronal bungarotoxin (n-BgT) which completely blocks ACh-induced currents in oocytes injected with oι₃β₂ and partially blocks the α₄/3₂ subtypes. Other suitable antagonists include known a conotoxin peptides such as :

Mil (SEQ ID NO: 1) GCCSNPVCHLEHSNLC from Conus magus, acting through neuronal 0!_3/S₂ receptors;

PnIA (SEQ ID NO : 2) GCCSLPPCAANNPDYC from C. pennaceus, acting through neuronal ce₃/3₂ receptors;

PnIB (SEQ ID NO: 3) GCCSLPPCALSNPDYC from C. pennaceus, acting through neuronal OJ₇ receptors;

EpI (SEQ ID NO: 4) GCCSDPRCNMNNPDYC from C. episcopatus, acting through neuronal α₃/3₂/α!₃/3₄ receptors; AuIA (SEQ ID NO : 5) GCCSYPPCFATNSDYC from C. aulicus, acting through neuronal o2₃/3₄ receptors;

AuIC (SEQ ID NO: 6) GCCSYPPCFATNSGYC from C. aulicus, acting through neuronal ce₃/3₄ receptors;

AuIB (SEQ ID NO: 7) GCCSYPPCFATNPDYC from C. aulicus, acting through neuronal α_3lβ₄ receptors; and

ImI (SEQ ID NO: 8) GCCSDPRCAWR from C. imperialis, acting through neuronal α₇ receptors . Other suitable neuronal nAChR antagonists, as described in WO 02/079236, are α-conotoxin-like peptides having the sequence of amino acids

XaaiCCSXaa₂Xaa3Xaa₄CXaa5Xaa6Xaa7Xaa8Xaa₉XaaioXaaiiC-NH₂ (SEQ ID NO: 9) in which Xaai is G or D; Xaa₃ is proline, hydroxyproline or glutamine; each of Xaa₂ to Xaa₈ and Xaa_u is independently any amino acid; Xaa₉ is proline, hydroxyproline or glutamine; Xaa₁₀ is aspartate, glutamate or γ-carboxyglutamate; Xaau is optionally absent; and the C-terminus is optionally amidated, with the proviso that the peptide is not α-conotoxin EpI, α-conotoxin ImI, α- conotoxin PnIA or α-conotoxin PnIB.

Preferred peptides are those in which Xaa₂ is D, H or N; Xaa₄ is A, P or R; Xaa₅ is N, A or Y; Xaa₆ is A, Y, H, M or V; Xaa₇ is N or D; Xaa₈ is H or N; and Xaau is I or Y.

More preferably the peptide comprises the sequence XaaiC C S Xaa₂ Xaa₃Xaa₄ C Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉Xaai₀XaaiiC-NH₂ (SEQ ID NO: 10) in which Xaai is G or D; Xaa₂ is D₇H or N;

Xaa₃ is P or 4-Hyp, Xaa₄ is A, P or R; Xaa₅ is N,A or Y; Xaa₆ is A, Y, H, M or V; Xaa₇ is N or D; Xaa₈ is H or N; Xaa₉ is proline, hydroxyproline or glutamine; Xaa_i0 is aspartate, glutamate or γ-carboxyglutamate; Xaau is I or Y; and the C-terminus is amidated.

Preferred peptides are those in which Xaai is G or D, Xaa₄ is R, Xaa₅ is N, Xaa₆ is Y, Xaa₇ is D, Xaa₈ is H, and Xaau is I •

More preferably, the peptide comprises the sequence GCCSDXaa₁RCNYDHXaa₂Xaa₃IC (SEQ ID NO: 11) , in which Xaai is proline, hydroxyproline or glutamine; Xaa₂ is proline or hydroxyproline; Xaa₃ is aspartate, glutamate or γ -carboxyglutamate; and the C-terminus is optionally amidated.

Most preferred neuronal nAChR antagonists are GCCSDPRCNYDHPEIC-NH₂ (SEQ ID NO: 12) ;

GCCSD4-HypRCNYDHPgammacarboxy-GluIC-NH₂ (SEQ ID NO: 13) ;

DCCSNPPCAHNNPDC-NH₂ (SEQ ID NO: 14) ; and GCCSHPACYANNQDYC-NH₂ (SEQ ID NO: 15) in each of which the C-terminal cysteine is amidated.

The most preferred neuronal nAChR antagonist for use in the present invention is GCCSDPRCNYDHPEIC (SEQ ID NO: 16), termed ACVl. Other neuronal nAChR antagonists that may be used in the present invention may be identified by those of skill in the art using appropriate assay methods. Assay methods for identifying compounds that antagonise human neuronal nicotinic AChR activity (antagonists) generally require comparison to a control. One type of a "control" cell or "control" culture is a cell or culture that is treated substantially the same as the cell or culture exposed to the test compound, except the control culture is not exposed to test compound. For example, in methods that use voltage clamp electrophysiological procedures, the same cell can be tested in the presence and absence of test compound, by merely changing the external solution bathing the cell. Another type of "control" cell or "control" culture may be a cell or a culture of cells which are identical to the transfected cells, except the cells employed for the control culture do not express functional human neuronal nicotinic AChRs. In this situation, the response of test cell to test compound is compared to the response (or lack of response) of receptor-negative (control) cell to test compound, when cells or cultures of each type of cell are exposed to substantially the same reaction conditions in the presence of compound being assayed.

Preferably the antagonist for use in accordance with the present invention is specific for the neuronal nAChR. Specific as referred to herein means that the antagonist has a higher affinity for the neuronal nAChR than for any other receptor. The antagonist may have at least 2x greater affinity for the neuronal nAChR than for any other receptor. The antagonist may have at least 5x greater affinity for the neuronal nAChR than for any other receptor. The antagonist may have at least 10x greater affinity for the neuronal nAChR than for any other receptor. The antagonist may have at least 2Ox greater affinity for the neuronal nAChR than for any other receptor. The antagonist may have at least 5Ox greater affinity for the neuronal nAChR than for any other receptor.

If the antagonist is an antibody, it is preferred that the antibody is specific (as defined herein) for the neuronal nAChR. Preferred antagonists for use in accordance with the present invention are those that act via a neuronal nAChR comprising an ce₃ subunit . Preferably the neuronal nAChR is comprises the subunits 0!_3/S₄Ce₅, Ce₃CX₇₁S₄ or Ce₃₁S₄. Preferably the antagonist does not affect muscle-type nAChRs .

Preferably the antagonist of a neuronal nAChR is effective in treating peripheral neuropathies due to its ability to reduce oxidative stress or nerve damage caused by oxidative stress.

"Oxidative stress" as used herein relates to the attack of reactive oxygen species (ROS) on biological molecules.

Reactive oxygen species are another term for free radicals. ROS are derived from the metabolism of oxygen and exist inherently in all aerobic organisms. There are many different routes by which reactive oxygen species may be generated. Most reactive oxygen species are the result of normal metabolic function, although exposure to cigarette smoke and environmental pollutants and bacterial, viral and fungal infections are all risk factors for producing ROS. Oxidative stress caused by ROS is associated with apoptosis and necrotic cell death. Certain cells, particularly neurons, are highly sensitive to cell death cause by ROS. Oxidative stress can damage nerves to such an extent that nerve function is reduced and neurons die. Oxidative stress occurs when there is an excess of ROS, a decrease in antioxidant levels, or both.

By the term "effective amount" of a compound as provided herein is meant at least a sufficient amount of the compound to provide the desired effect . A "therapeutically effective amount" of a compound herein, used to treat a mammalian individual suffering from a condition, disorder or disease that is responsive to administration of a neuronal nAChR antagonist, is an amount that is non-toxic but sufficient to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular neuronal nAChR antagonist and mode of administration, and the like. Thus, it is not possible to specify an exact "therapeutically effective amount." However, an appropriate "therapeutically effective" amount in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.

Dosage levels of compound may be of the order of about lOμg to about 20 mg per kilogram body weight, with a preferred dosage range between about 0.5 mg to about 10 mg per kilogram body weight per day (from about 0.5 mg to about 3 g per patient per day) . The amount of compound that may be combined with the carrier materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 0.5 mg to Ig of a compound with an appropriate and convenient amount of carrier material which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 0.5 mg to 500 mg of a compound.

Optionally, the compound may be administered in a divided dose schedule, such that there are at least two administrations in total in the schedule. Administrations are given preferably at least every two hours for up to four hours or longer; for example a compound may be administered every hour or every half hour. In one preferred embodiment, the divided-dose regimen comprises a second administration of a compound after an interval from the first administration sufficiently long that the level of compound in the blood has decreased to approximately from 5-30% of the maximum plasma level reached after the first administration, so as to maintain an effective content of compound in the blood. Optionally one or more subsequent administrations may be given at a corresponding interval from each preceding administration, preferably when the plasma level has decreased to approximately from 10-50% of the immediately-preceding maximum.

The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms (prophylaxis) and/or their underlying cause, and improvement or remediation of damage. Thus, for example, the present method of "treating" a disorder that is responsive to a neuronal nAChR antagonist, as the term "treating" is used herein, encompasses both prevention of the disorder in a predisposed mammalian individual and treatment of the disorder in a clinically symptomatic individual.

"Treating" as used herein covers any treatment of, or prevention of a condition in a vertebrate, a mammal, particularly a human, and includes: inhibiting the condition, i.e., arresting its development; or relieving or ameliorating the effects of the condition, i.e., cause regression of the effects of the condition. "Prophylaxis" or "prophylactic" or "preventative" therapy as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it . The term "subject" as used herein refers to any animal having a disease or condition which requires treatment with a pharmaceutically-active agent. The subject may be a mammal, preferably a human, or may be a non-human primate or non-primates such as used in animal model testing. While it is particularly contemplated that the agents according to the invention are suitable for use in medical treatment of humans, it is also applicable to veterinary treatment , including treatment of companion animals such as dogs and cats, and domestic animals such as Galliformes, Anseriformes, horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates .

Suitable mammals include members of the Orders Primates, Rodentia, Lagomorpha, Cetacea, Carnivora, Perissodactyla and Artiodactyla. Members of the Orders Perissodactyla and Artiodactyla are particularly preferred because of their similar biology and economic importance.

For example, the Order Artiodactyla comprises approximately 150 living species distributed through nine families: pigs (Suidae) , peccaries (Tayassuidae) , hippopotamuses (Hippopotamidae) , camels (Camelidae) , chevrotains (Tragulidae) , giraffes and okapi (Giraffidae) , deer (Cervidae) , pronghorn (Antilocapridae) , and cattle, sheep, goats and antelope (Bovidae) . Many of these animals are used as feed animals in various countries. More importantly, many of the economically important animals such as goats, sheep, cattle and pigs have very similar biology and share high degrees of genomic homology.

The Order Perissodactyla comprises horses and donkeys, which are both economically important and closely related.

Pharmaceutical or veterinary compositions of the present invention or usable in the methods of the present invention comprise at least one antagonist of a neuronal nAChR, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic agents. Each carrier, diluent, adjuvant and/or excipient must be pharmaceutically "acceptable" .

By "pharmaceutically acceptable carrier" is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected active agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Similarly, a "pharmaceutically acceptable" salt or ester of a novel compound as provided herein is a salt or ester which is not biologically or otherwise undesirable .

Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, ocular or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.

The compositions may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association a compound with the carrier which constitutes one or more accessory ingredients .

In general, the compositions are prepared by uniformly and intimately bringing into association the compound with liquid carriers, diluents, adjuvants and/or excipients or finely divided solid carriers or both, and then if necessary shaping the product.

The compound may additionally be combined with other medicaments to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically-active agents, as long as the combination does not eliminate the activity of the modulator. It will be appreciated that the compound and the other medicament may be administered separately, sequentially or simultaneously. As used herein, a "pharmaceutical carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the agent to the subject. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Each carrier must be pharmaceutically "acceptable" in the sense of being not biologically or otherwise undesirable i.e. the carrier may be administered to a subject along with the agent without causing any or a substantial adverse reaction. The compound or composition may be administered orally, topically, or parenterally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes subcutaneous injections, aerosol for administration to lungs or nasal cavity, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques .

The compound or composition may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs. The composition for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable preservatives include sodium benzoate, vitamin E, alphatocopherol , ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. The tablets may contain the agent in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid; (3) binding agents, such as starch, gelatin or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Coating may also be performed using techniques described in the U.S. Pat. Nos. 4,256,108; 4 ,160 ,452 ; and 4,265,874 to form osmotic therapeutic tablets for control release .

If the compound is not generally orally available, there are methods by which oral availability may be improved. An example of such an agent is provided in Australian provisional application no. 2005904150, the contents of which are incorporated herein by reference. This application describes methods for improving the oral or sublingual bioavailability of a molecule by linking the molecule to a peptide comprising a basic amino acid and 2 to about 5 hydrophobic amino acids.

As well as oral administration, antagonists or compositions useful in the method of the invention, such ACVl, can be administered, for in vivo application, parenterally by injection or by gradual perfusion over time independently or together. Administration may be intravenously, intraarterial, intraperitoneally, intramuscularly, subcutaneously, subconjunctivalIy, intracavity, transdermally or infusion by, for example, osmotic pump. For in vitro studies the compounds or compositions may be added or dissolved in an appropriate biologically acceptable buffer and added to a cell or tissue. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-microbials, anti -oxidants, chelating agents, growth factors and inert gases and the like.

The antagonist may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art . Examples of such veterinary compositions include those adapted for:

(a) oral administration, external application, for example drenches (e.g. aqueous or nonaqueous solutions or suspensions); tablets or boluses ; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced in the udder via the teat;

(c) topical applications, e.g. as a cream, ointment or spray applied to the skin; or

(d) intravaginally, e.g. as a pessary, cream or foam.

In the present specification, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It must be noted that, as used in the present specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a diuretic" includes a single diuretic, as well as two or more diuretics; and so forth. All references, including any patents or patent application, cited in this specification are hereby incorporated by reference to enable full understanding of the invention. Nevertheless, such references are not to be read as constituting an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

EXAMPLES

The invention will now be described in detail by way of reference only to the following non- limiting example.

Example 1

The compound under test was ACVl , the neuronal nicotinic acetylcholine receptor antagonist described in WO 02/079236 Al. ACVl is a peptide having amino acid sequence GCCSDPRCNYDHPEIC (SEQ ID NO: 16) with a first disulphide bond between the first and third cysteine residues and a second disulphide bond between the second and fourth cysteine residues. It may be prepared synthetically.

Animals

Outbred male Sprague-Dawley rats were obtained from Monash University Animal Services, Clayton. Rats were non SPF.

Induction and assessment of diabetes

Outbred male Sprague-Dawley rats weighing 130- 17Og (6-7 weeks old) were injected with streptozotocin (75mg/kg body weight dissolved in 0. IM sodium citrate buffer pH4) after an overnight fast. They were given 5% sucrose solution for 48h and then placed on standard food (Barrostock GR2) and water at libitum. 2-3 days later, the presence of diabetes was determined originally by urine glucose assay (Diastix, Bayer Australia Ltd) . Rats were assessed for the level of hyperglycemia via testing a drop of blood taken from tail artery or vein for blood glucose assay (Reflolux S, Boehringer Mannheim Australia Pty Ltd) . Rats with blood glucose values >27mmol/l were included in this study and they were also monitored for changes in body weight during the experimental period. Rats were sacrificed if they lost 10% of their body mass.

The development of neuropathy in the rats was confirmed by lowered allodynia and mechanical hyperalgesia thresholds .

Method of assessment of allodynia

Von Frey filaments applied to the plantar surface of the hindpaw were used to determine the lowest mechanical threshold required for paw withdrawal . The filaments used produced a weight of 2 , 4, 6, 8, 10, 12, 15 and 20 g. Absence of a response after 5 seconds prompted use of the next filament of increasing weight. A score of 2Og was given to animals that did not respond to any of the von Frey filaments. Note: control rats usually withdraw at a force of 2Og. Animals were tested twice at each time point using the filaments, with 10 minute resting interval in between, and an average of the two readings was taken. All assessments were done only on the right hind foot .

Treatment Protocol

Six weeks after the STZ injection, animals were given daily subcutaneous injection into the back of the neck of ACVl (3ug/kg, 30ug/kg or 300ug/kg) or vehicle (n=6 per group) over a period of 4 weeks, weekends excluded. In the first and fourth weeks of treatment animals were tested for allodynia at 0 , 1, 3 and 6 hours post ACVl or vehicle injection. In the second and third weeks animals were only tested for allodynia immediately prior to injection. Only the allodynia measurements taken immediately before the daily injection (t=0) are showed here for simplicity. Seven to ten days post ACVl treatment period, the animals were measured again for allodynia levels. Blood samples and nerve tissue samples from the high dose group (300ug/kg) and vehicle dose group were also taken at 7-10 days post ACVl treatment period. Blood samples were then measured for levels of nitro-tyrosine and lipid hydroperoxidase, indicators of tissue injury.

Tissue Injury Markers Previous data had suggested that ACVl may have anti-oxidant properties. To test this further 2 oxidative stress markers were assessed in samples taken from the animals. One indication of oxidative stress is an increase in nerve lipid hydroperoxide, which was measured in this study. Another marker for tissue injury, nitro-tyrosine, was also measured. Briefly, at 7-10 days post treatment animals from the ACVl 300μg/kg group and diabetic control group had nerve biopsies and blood samples taken from them. The two biomarkers were then quantified following procedures contained in Appendix 1 & 2.

Nitro-tyrosine :

Nitrotyrosine has been identified as a marker for cell and tissue damage, infection and inflammation. Nitrotyrosine has been found to be a sensitive marker for neurodegenerative processes involving oxidative stress . In this study levels of nitro-tyrosine in systemic blood samples were assessed according to the method in Appendix

1.

Lipid hydroperoxidases :

Lipid peroxidation is used as a basic marker to assess the role of oxidative injury in pathophysiological disorders such as diabetes. In this study, levels of lipid hydroperoxidases were measured in nerve biopsy samples according to Appendix 2.

Results :

As shown in Figure 1, during the four week treatment period, baseline paw withdrawal thresholds continually increased from their neuropathy-sensitized state in the 30 and 300μg/kg groups. The post treatment levels of allodynia were measured at 7-10 days post ACVl treatment period. The results show that even after treatment ceased, the anti-allodynia effect continued, indicating repair. Figure 2 represents indicators of tissue injury.

In Figure 2, lipid hydroperoxide levels from a nerve biopsy (a) and nitrotyrosine levels from systemic blood (b) were measured as indicators of tissue injury. Samples were taken at 7-10 days post treatment with ACVl. Undiabetic vehicle data is historical, and not part of the data set performed during this study.

Chronic effect: The anti-allodynic effect of ACVl is cumulative, with daily baseline thresholds increasing from 5g to 12.4g for the 300μg/kg treated group during the course of treatment. Historical data (not shown) indicates that normal rats have a threshold of 15- 2Og. If 15g is used as the standard then at the end of the study, the diabetic rats treated with 300μg/kg ACVl were about 70% normalised in their pain levels. A similar cumulative or chronic effect was seen in the 30μg/kg group, though no such effect was observed in the 3μg/kg group. In contrast, the saline treated diabetic rats showed a gradual reduction in threshold over the period of testing.

Post treatment effect : The results show that the anti-allodynic effect of ACVl was partially maintained up to 10 days following cessation of treatment.

Anti-oxidative effect: Graph 2 shows that the untreated diabetic animals of this study had higher levels of both oxidative stress markers than historical data from normal animals. This is consistent with a higher level of tissue damage occurring in diabetic animals.

Diabetic animals treated with ACVl had lower levels of both oxidative stress markers compared with untreated diabetic animals, and for the nitro-tyrosine assay the difference was highly significant (P=O.00004) . In addition, ACVl treated diabetic animals even had a lower nitro-tyrosine recording than the historical normal animal data. This indicates that ACVl normalised the level of oxidative stress (or tissue injury) occurring in diabetic animals. A similar trend of reduced oxidative stress in the same diabetic animals treated with ACVl was also seen in the lipid hydroperoxide assay, though it was not quite significant (P = 0.058) when compared to untreated diabetic animals.

This study shows that ACVl has cumulative analgesic effects in diabetic animals, to the point of normalising their levels of painful neuropathy, in addition to long term maintenance of that effect after cessation of treatment.

Pharmacokinetic studies have shown the elimination half life of ACVl after subcutaneous injection to be approximately 40 minutes so long term maintenance is not the result of ACVl persisting in the animal .

The long term maintenance and increasing chronic effect of ACVl seen here is caused by ACVl promoting neuronal recovery. Oxidative stress and oxidative damage to tissues is a well known feature of diabetes and likely to be the cause of the neuropathy. Since ACVl is shown from this study to be likely to be able to promote nerve repair, then it could be used to treat not only the pain associated with diabetic neuropathy, but also to treat or prevent the underlying neuropathy itself. The relationship between the pain, vascular, immune and endocrine systems over the course of trauma and healing remains has been widely acknowledged. Tissue damage resulting from oxidative stress liberates a variety of pro-inflammatory agents, including acetylcholine, that cause pain as well as promoting the healing of injured tissue thereafter.

Conclusion:

ACVl administered at 30 and 300μg/kg subcutaneously has an anti-allodynic effect in diabetic rats with peripheral neuropathy which increased over the 4 weeks of treatment and is maintained to a large degree for 7-10 days following cessation of injection.

A surprising feature of ACVl found in this study is its ability to reduce levels of oxidative stress parameters in both nerve tissue and blood plasma.

Example 2

Cerebellar microexplants - Injury paradigm and survival analysis after NNZ-4921 treatment

Laminated cerebellar cortex is extracted from P4 , P7/8 wistar rat pups and triturated through a 125 μ.m gauze to obtain uniformly sized microexplants. After centrifugation for 3 minutes at 60xg, the pellet was resuspended in StartV medium (Biochrom-Germany) and equally amounts of cell suspension is seeded on poly-D-lysine (lOOμg/ml) coated glass coverslips in 6-well plates and incubated for 3 hrs to allow adherence, before 1 ml StartV per well was added. The irreversible succinate dehydrogenase inhibitor 3-nitropropionic acid (3 -NP prepared as 50μM stock, pH 7) and L-glutamate (50μM stock) are added at 0.5mM final concentration. Cerebellar cells receive simultaneously the drug and the above described injury paradigm. The explants are cultivated at 34⁰C at 5% CO₂ and 100% humidity for 48 hrs. For analysis microexplants are fixed in increasing amounts of paraformaldehyde (0.4/ 1.2 /3 and 4%) in PBS. For quantitative survival analysis, 4 microscopic fields (phase-contrast) displaying the highest cell density are selected for each well. Cells are counted as viable that show neurite outgrowth. Statistical significance is analysed by ANOVA.

Referring now to the result in Figure 3, it can be seen that ACVl is neuroprotective over a range of concentrations, with an optimum concentration of 10 nM.

Example 3

In this example, conducted in the laboratories of Neurofit, France, an evaluation of the in-vitro neuroprotective effect of ACVl compound against neuronal death induced by cisplatin was performed.

Materials and Methods

Pregnant female rats of 15 days gestation were killed by cervical dislocation (Rats Wistar; Janvier, Le Genest-St- Isle, France) and the foetuses were removed from the uterus. DRG were collected, placed in ice-cold medium of Leibovitz (L15, Gibco, Invitrogen, Cergy-Pontoise, France) and dissociated by trypsinization (trypsin, 0.05%; Gibco) for 30 min at 37°C in the presence of DNAase I (Roche Meylan) . The reaction was stopped by addition of DMEM containing 10% of foetal bovine serum (FBS) . The suspension was triturated with a 10 ml pipette and cells were then mechanically dissociated by several passages through the 21 gauge needle of a syringe. Cells were then centrifuged at 337 x g for 10 min at room temperature. The pellet of dissociated cells was resuspended in a mixture of culture media (Neurobasal and B27: 97% and 2% in proportion, respectively) supplemented with glutamine (200 ttiM) and 3°ng/ml NGF. 3 hr later, DRG cells suspension was recovered, counted in a Neubauer cytometer using the trypan blue exclusion test (Sigma) and seeded on the basis of 20 000 cells per well in 96 well-plates (Nunclon) precoated with poly-L-lysine-laminin. Test compound (ACVl) or reference compound (NGF) prepared in DMEM was added to the culture preparation and plates were then maintained at 37°C in a humidified incubator, in an atmosphere of air (95 %.) -CO₂ (5%) .

After 24 hr, cisplatin prepared in DMEM were added to the culture to yield a final cisplatin concentration of 3 μg/ml or 0.5 μg/ml .

Acid phosphatase activity

After 48h, neuronal survival was assayed by measuring acid phosphatase activity. Briefly, after removal of the culture medium, cells were rinsed twice with 100 μl of PBS (Phosphatase Buffer Saline) , and 100 μl of buffer containing 0. IM sodium acetate (pH 5.5), 0.1% Triton XlOO and 1 mg/ml p-nitrophenyl phosphate (Sigma) is added. Reaction was stopped by addition of 10 μl of IN NaOH. Enzyme activity was measured at 405 nm in a multiplate reader (Labsystems Multiskan Bichromatic) . Statistical analysis

Anova was used to assess the difference between experimental conditions. Multiple comparisons were performed using Fisher's Protected Least Significant Difference (PLSD) . p value less or equal to 0.05 was deemed significant.

#, p < 0.05, significantly different as compared to the control condition

*, p < 0.05, significantly different as compared to the cisplatin-intoxicated condition

Referring now to Figure 4, the results show that ACVl is effective in lessening the damage caused by cisplatin, with the most effective concentration about 10 nM. ACV is more effective than the positive control, Nerve Growth

Factor (NGF) when high cisplatin concentrations are used, and slightly less effective when low cisplatin concentrations are used.

In conclusion, ACVl has potential as a protection from or treamtent for chemical induced neuropathy. The direct neuroprotective effect further strengthens the interpretation of the results in example 1 of fundamental repair of the neuropathy provided by ACVl rather than sustained analgesia.

Appendix 1 - Nitro-tyrosine assay

ELISA for NITRO-TYROSINE (NT-E28 & others) Plate : Costar

Antigen: NT-BSA from Cayman Cat No: 89542, stored at

250μg/mL=5μg/20μL in -80C/9/2

NT-BSA coating buffer: Carbonate buffer (0.06M), pH 9.6.

PBS (0.01M) , pH 7.2-7.4 Blocking soln: 0.1%GEL in PBS

Antibody: Rabbit anti-NT-BSA from Cayman Cat No: 189540 in

-80C/9/1, 25μg/ml, 20μl vols

Antibody diluent= PBS-O . l%TX100. It may be good to filter the solutions through a 0.2μ filter.

DAYl:

NT-BSA: Coat plate with NT-BSA iμg/mL (100ng/well) diluted in carbonate buffer.

Place 100 μL/well and incubate overnight at room-temp. (A 20μl, 5μg aliquot makes 5.0 mL)

DAY2:

Rabbit anti-NT-BSA: Dilute in PBS-O . l%TX100 to make 0.1 μg/ml (lOOng/mL) . See Below

Standards : Dilute the standards in medium if the samples are in medium or :

70mg/mL HSA in PBS+0. l%TX100 if the samples are human plasma 55mg/mL BSAor HSA in PBS+0. l%TX100 for rat plasma and

39mg/mL HSA in PBS+0. l%TX100 for human peritoneal fluid.

There can be two standards (see below) :

1. NT-BSA and/or

2. Nitrotyrosine

To ImL PP tubes add an aliquot of standard and samples (eg 60μl-100μL for single test or 110-200 μL for duplicates. The bigger the volume the better) To all the tubes add an equal volume of 0. lμg/mL rabbit anti-NT-BSA diluted in PBS+0. l%TX100. (The final concentration of antibody is now .05μg/mL=50ng/mL) .

Vortex each tube individually and incubate tubes with standards and samples at 37°C for 180 mins . After about lhr, wash the plate 5x with PBS-O .1%TX1OO and Block wells with 200μl of 0.1%GEL in PBS during the incubation of stds/samples with Ab. Incubate at 37°C for approximately 2 hrs

Wash the plate 5x with PBS-O . l%TX100 , flick off wash solution and blot 6X.

Dispense lOOμL or 2xl00μL (duplicate) standards and samples to the plate. Incubate at 37°C for 60 mins

Wash the plate 5x with PBS-O . l%TX100

Sheep anti-rabbit Igs-HRP (Silenus) Cat No:

Dilute 1/1500 in PBS+0. l%TX100+0.05%HSA and addlOOμl/well . (Adding 0.5mg/mL of HSA or BSA may help stop non-specific binding. In NT-E40 I had this and it was a good run) Incubate at 37°C for 90 mins. (Use this antibody at 1/2000 when new) . TMB substrate (from KPL labs)

Remove equal amounts of part A and B and leave at RT for -15 mins

Wash plate 5x with PBS-TXlOO Mix A and B Wash plate 5x with water

Place 100 μl/well of TMB substrate

Incubate at RT for about 5 mins .

Stop with lOOμl 2M H2SO4 and read at 450-620nm. NT-BSA: (1-11-04) 200μg is supplied at a cone of

400μg/mL in 2OmM TRIS pH 8.

Therefore we have 200μg in 500μL. To this add 300μL fresh

2OmM TRIS= 200μg in 800μL =250ug / mL. Freeze 20 and 40 μL aliquots at -80C/9/2.

Rabbit anti-NT-BSA dilution:

This is supplied as 500μg/mL in TRIS, 15OmM NaCl, 0.5mg/mL

BSA, 50% glycerol, 0.02% NaN3 and is stored at -20⁰C in

"GG" freezer. Working dilution: take lOμL and add 90μL of TRIS (2OmM) ,

15OmM NaCl, 0.5mg/mL BSA, 50% glycerol, 0.02%NaN3. This

1/10 = 50μg/mL. Store at -20C (will not freeze) .

Dilution of standards (based on the results of NT-E28 and

NT-E40) NT-BSA is stored at 250μg/ml in 20μl aliquots i.e. 5 μg/20μl [Nitrotyrosine-BSA] μg/mL ng/mL

1 20μl 250μg/mL + 480μL PBS-TXlOO 10 10000 Total vol 500μl

2 200μl lOug/ml + 200μl PBS-TXlOO 5 5000

3 200μl 5μg/ml + 200μl PBS-TXlOO 2.5 2500

4 200μl 2.5μg/ml + 200μl PBS-TXlOO 1.25 1250

5 200μl 1.25μg/ml + 200μl PBS-TXlOO 0.625 625

6 200μl 0.625μg/ml + 200μl PBS-TXlOO 0.3125 312.5

7 200μl 0.3125μg/ml + 200μl PBS-TXlOO ) 0.156 156

8 200μl 0.156μg/ml + 200μl PBS-TXlOO 0.078 78

9 200μl 0.078μg/ml + 200μl PBS-TXlOO 0.039 39

10 200μl 0.039μg/ml + 200μl PBS-TXlOO ) 0.0195 19.5

11 200μl 0.02μg/ml + 200μl PBS-TXlOO 0.01 10

12 200μl of PBS-TXlOO 0

[Nitrotyrosine-BSA] μg/mL ng/ml

1 19.71μl 250μg/mL + 365.3μL PBS-TXlOO 12.8 12800 Total vol 385μl

2 200μl 12.8μg/ml + 200μl PBS-TXlOO 6.4 6400

3 200μl 6.4μg/ml + 200μl PBS-TXlOO 3.2 3200

4 200μl 3.2μg/ml + 200μl PBS-TXlOO 1.6 1600

5 200μl 1.6μg/ml + 200μl PBS-TXlOO 0.8 800

6 200μl 0.8μg/ml + 200μl PBS-TXlOO 0.4 400

7 200μl 0.4μg/ml + 200μl PBS-TXlOO 0.2 200

8 200μl 0.2μg/ml + 200μl PBS-TXlOO 0.1 100

9 200μl O.lμg/ml + 200μl PBS-TXlOO 0.05 50

10 200μl 0.05μg/ml + 200μl PBS-TXlOO 0.025 25

11 200μl 0.025μg/ml + 200μl PBS-TXlOO 0.0125 12.5

12 200μl PBS-TXlOO 0 0

This will allow 150μl of standard + 150 μl of rabbit anti- NT antibody to be mixed together.

NOTE: diluent can vary, eg medium or PBS+TX100 +HSA or BSA. [Nitrotyrosine-BSA] μg/mL ng/ml

1 20μl 250ug/mL + 380μL PBS-TXlOO 12.5 12500 Total vol 400μl

2 200μl 12.5μg/ml + 200μl PBS-TXlOO 6.25 6250

3 200μl 6.25μg/ml + 200μl PBS-TXlOO 3.12 3125 4 200μl 3.125μg/ml + 200μl PBS-TXlOO 1.5625 1563

5 200μl 1.5625μg/ml + 200μl PBS-TXlOO 0.78125 781

6 200μl 0.78125μg/ml + 200μl PBS-TXlOO 0.3906 390

7 200μl 0.3906μg/ml + 200μl PBS-TXlOO 0.1953 195

8 200μl 0.1953μg/ml + 200μl PBS-TXlOO 0.097656 97.6 9 200μl 0.0976μg/ml + 200μl PBS-TXlOO 0.0488 48.8

10 200μl 0.0488μg/ml + 200μl PBS-TXlOO 0.0244 24.4

11 200μl 0.0244μg/ml + 200μl PBS-TXlOO 0.0122 12.2 12 200μl PBS-TXlOO 0

Nitrotyrosine : 1000 and 250μg/mL stocks

Make up fresh NT at lmg/mL (lOOOμg/mL) in PBS. Dilute by Vi eg 250μL lOOOμg/mL + 750μL PBS. Microfuge for 30 sec, then take 8μL or 16 μL for dilution (see below) .

1 8μl 1000μg/τnl +1992μl dil =4000ng/ml (4 μg/ml)

1 8ul 500μg/ral + 992μl dil =4000ng/ml

1 8μl 250μg/ml + 492μl dil =4000ng/ml

1 16μl 250μg/ml + 984μl dil =4000ng/ml**

2 200μl of 4000 + 200μl dil = 2000ng/ml

3 200μl of 2000 + 200μl dil = lOOOng/mL

4 200μl of 1000 + 200μl dil = 500ng/mL

5 200μl of 500 + 200μl dil = 250ng/mL

6 200μl of 250 + 200μl dil = 125ng/mL

7 200μl of 125 + 200μl dil = 62.5ng/mL

8 200μl of 62.5 + 200μl dil = 31.25ng/mL

9 200μl of 31.25 + 200μl dil = 15.63ng/mL

10 200μl of 15.63 + 200μl dil = 7.82ng/mL

11 200μl of 7.82 + 200μl dil = 3.9ng/mL

12 200μl of dil = Ong/mL SET UP TWO OR MORE ZERO TUBES

Therefore, stds 1-10 and 2 or more zero tubes.

NOTES :

*It is preferable to make the set of standards in a larger volume for accuracy (eg 500 μL) , then transfer to a new set of tubes the required volume. Eg 120μL-150μL

* Dilute the standards in medium if the samples are in medium.

* If nitrotyrosine is 500μg/mL then 8μL of 500μg/mL + 992μL of diluent =4μg/mL=4000ng/mL

If nitrotyrosine is 250μg/mL then 8μL of 250μg/mL + 492μL of diluent =4μg/mL=4000ng/mL

* A 10 point standard curve is enough. i.e. 1-10 and zero. It's worth having two 'zero' tubes.

* In the last standard, remove the carry-over volume eg 200μl to leave the same in each tube; ONLY if adding antibody to the same tube. When transfering a fixed volume to a new set of tubes, which is the preferable method, this is not necessary) . * The sheep anti-rabbit Ig-HRP is used at 1/2000 when new. Closer to expiry date I use it at 1/1500. The OLD method

Dilution of standards

NT-BSA is stored at 250μg/ml in 20μl aliquots i.e. 5 μg/20μl Thaw a 20μl aliquot (5μg) + .230μl PBS-TW-O .1%GEL = 5μg in 250μl = 20μg/ml (STOCK)

For duplicate standards

Tube 1: Add HOμl of 20μg/ml (lOμg/ml) Tube 2: Add HOμl of 20μg/ml to HOμl of PBS-TW-0.1% GEL = lOμg/ml (5)

Tube 3 : take HOμl of lOμg/ml + HOμl of PBS-TW- 0 . 1% GEL =

5 . 0μg/ml (2 . 5 )

Tube 4 : take HOμl of 5 . 0μg/ml + HOμl of PBS-TW- 0 . 1% GEL = 2 . 5μg/ml (1 . 25 )

Tube 5 : take HOμl of 2 . 5μg/ml + HOμl of PBS-TW- 0 . 1% GEL

= 1 . 25μg/ml ( . 625 )

Tube 6: take HOμl of 1.25μg/ml + HOμl of PBS-TW-0.1% GEL

= 0.625μg/ml (.3125) Tube 7: take HOμl of 0.625μg/ml + HOμl of PBS-TW-0.1%

GEL = 0.3125μg/ml (.156)

Tube 8: take HOμl of 0.3125μg/ml + HOμl of PBS-TW-0.1%

GEL = 0.156μg/ml (.078)

Tube 9: take HOμl of 0.156μg/ml + HOμl of PBS-TW-0.1% GEL = 0.078μg/ml (.039)

Tube 10: take HOμl of 0.078μg/ml + HOμl of PBS-TW-0.1%

GEL = 0.039μg/ml (0.0195)

Tube 11: take HOμl of 0.039μg/ml + HOμl of PBS-TW-0.1%

GEL = 0.0195μg/ml (0.01)* Tube 12: Add HOμl of PBS-TW-O.1% GEL = O.Olμg/ml (0)

• Remove HOμl of sample from tube 11 to leave llOμl.

FINAL CONCENTRATIONS OF NT-BSA AND ANTIBODY IN THE MIXTURE:

NT-BSA Rabbit anti-NT-BSA

Tubel: 10 μg/ml 0.05 μg/ml

Tube2 5.0 μg/ml 0.05 μg/ml Tube3 2.5 μg/ml 0.05 μg/ml Tube4 1.25 μg/ml 0.05 μg/ml Tube5 0.625 μg/ml 0.05 μg/ml Tube6 0.3125 μg/ml 0.05 μg/ml Tube7 : 0.156 μg/ml 0.05 μg/ml Tube8 : 0.078 μg/ml 0.05 μg/ml Tube9 : 0.039 μg/ml 0.05 μg/ml Tube10 : 0.0195 μg/ml 0.05 μg/ml Tubell: 0.01 μg/ml 0.05 μg/ml Tube12 : 0 0.05 μg/ml

NITROTYROSINE.

FW= 226. 2 IM = 226.2 gram/litre =226. .2mg/mL ImM = 226.2mg/L =226..2μg/mL IuM = 226.2μg/L =226..2ng/mL InM = 226.2ng/L =226,.2pg/mL

If limit of detection is IOng/mL = lOμg/L lμM = 226.2μg/L xuM = lOμg/L x=10/226.2 = 0.0442μM =442nM

BSA FW=66_/000

Sprague Dawley rat plasma total protein=5.5+-0. lg/dL ie

55mg/mL

50 days old, 223.9 +- 2.3g

Rudy M Ortiz 22/2/2000. Water balance in rats exposed to centrifugation.

J Applied Physiol 84, 50-60, 2000.

Appendix 2 : Lipid hydroperoxide assay

Lipid hydroperoxide assay Lipid hydroperoxide standard: Cat number 705014 Caymen chemicals .

50μM 13-HpODE in methanol. Store on ice during experiment and store at -80C. Chromogen Reagent 1: 4.5mM ferrous sulphate in 0.2M HCl. Stable for 1 year at 4C. I make up every 6 months.

Chromogen Reagent 2: 3% solution of ammonium thiocyanate in methanol . Stable for 1 year at 4C. I make up every 6 months.

"Extract R": Metaphosphoric acid (HPO3)n. Store at 4C in container with dessicant.

Cat No: 23927-5 Sigma. Stable for 2yr at 4C. A fresh solution is made on the day of the test. Weigh out lOOmg and add to glass tube containing 15mL deoxygenated methanol . Vortex the tube thoroughly for 2 mins .

The methanol becomes cloudy and most of the solid remains undissolved.

Use within two hours .

Human or rat plasma: Use 300μl plasma and add an equal volume of ER

Nerve tissue: Weigh, Freeze dry and store nerve tissue at -80C until required. Try freeze same lenghts/quantity (about lcm=20mg) . Solvents

Deoxygenate approximately 50 mL of chloroform and methanol in 10OmL glass bottles by bubbling nitrogen gas for at least 30 mins. (I do this on ice) .

Cold chloroform is required for sample extraction. Solvent mixture: Mix 2 volumes of chloroform with 1 volume of methanol and keep on ice. (Approx ImL of mixture is required for each sample) .

Sample treatment : 1) Plasma:

300μL of human or rat plasma is aliquoted into a glass test tube (12x75mm) . Add 300μL of extract R to each tube and vortex and sit on ice for 5 mins

Add ImL of cold chloroform to each tube and thoroughly vortex for 6 sees. Sit on ice for 5 mins

Centrifuge the mixture at 1500rpm (2800rpm) for 5mins at

OC.

Remove the top layer by sucking off with pasteur pipette attached to water vacuum pump. Also remove part of the protein layer on the side of the tube.

Using a glass Pasteur pipette attached to "gasfirn pump" pierce the protein layer on the side of the tube and take up the bottom sample layer (chloroform layer) and transfer to clean glass tubes on ice. (The method recommends that the samples be tested in triplicate, i.e. 3 extractions per sample) .

2) Nerve tissue:

Thaw nerve and keep on ice . Cut section to be tested and weigh. Aim for 25-30 mg

(~lcm)

Add 500μL Milliq water to glass test tube (12x75mm) and keep on ice .

Add the nerve to the tube. Using a glass rod "homogenise the nerve for 2 minutes.

Add 500μL of extract R and vortex thoroughly for 5 seconds

Sit on ice for 5 minutes

Add ImL of degassed cold chloroform and vortex thoroughly for 5 sees. Sit on ice for 6 mins

Centrifuge the mixture at 150Og (2800rpm) for 5mins at OC.

(I spin for 10 mins)

Remove the top layer by sucking off with pasteur pipette attached to water vacuum pump. Using a glass Pasteur pipette attached to "gasfirn pump" take up the bottom sample layer (chloroform layer) and transfer to clean glass tubes on ice. (volume recovered is usually 700-800μL)

(The method recommends that the samples be tested in triplicate, i.e. 3 extractions per sample).

Test samples immediately or store at -80C.

ASSAY MIXTURE:

1) STANDARD curve: The method recommends to do each cone in triplicate (I do single), in glass tubes. (I use 4ml glass screw capped tubes) Also, I only set up standards A, B, D, F and H Tube HP Std(μL) Chlor:Meth 2:1 (μL) Final [HP] nmol To do

A 0 950 0 #

Blank

B 10 940 0.5

C 20 930 1.0 #

D 30 920 1.5 #

E 40 910 2.0

F 60 890 3.0 #

G 80 870 4.0

H 100 850 5.0 #

2) SAMPLES:

Place 450μL of assay diluent in each tube (screw capped) Place 500μL of each sample into tubes Vortex

Make up chromogen by mixing equal parts of solution 1 and solution 2- (USE NEW TIP) . Add 50μL of chromogen to each tube Vortex

Place 3x300μL of standards and samples into the glass plate .

Allow to sit at RT for a few minutes.

(If some wells appear cloudy/opaque then incubate plate at 37C for a few minutes) .

Switch on the ELISA plate reader about 15 mins before use. Read the plate at 490 nm (490-510nm is OK preferably at 500nm)

NOTE: with nerve extraction, usually over 500μL of sample is recovered. In this case over 500μL of sample may be used in the assay. Eg 600μL sample (instead of 500μL) 350μL of assay diluent (2:1 Chlor:Meth) 50 μL chromogen lOOOμL total volume.

DATA analysis:

Average the absorbance of standards and samples (if done in duplicate or triplicate) .

Subtract from the absorbance values the standard A (zero) absobance value from itself and all other standards and samples

Plot these corrected absorbance values against lipid hydroperoxide (nmol) to generate a standard curve which should be a straight line.

A data reduction program may be used. The concentration of hydroperoxide (nmol) in the samples is read off the standard curve.

To calculate the amount of lipid hydroperoxide in the original sample:

HPST nmol=hydroperoxide in the sample tubes-read off the Standard curve.

VE (mL) =volume of extract used for the assay (0.5mL for plasma, and 0.6mL for nerve) SV (mL) =volume of the original sample used for the extraction. (0.3mL plasma & 0.5mL for nerve)

Cone of hydroperoxide in SAMPLE in uM= HPST X ImL

VE SV

For nerve/tissue samples divide the hydroperoxide value (μM) by the amount of tissue used. Eg, 0.3mL of plasma. Hydroperoxide value off std curve = O.lΞnmol Volume of extracted sample used in the assay=0.5mL

Cone of hydroperoxide in SAMPLE in μM= 0.15 X ImL

0.5 0.3

Eg; nerve extracted in 0.5mL water,

Hydroperoxide value read off std curve = 0.22nmol Volume of extracted sample used in the assay=0.6mL (instead of 0.5mL) Cone of hydroperoxide in SAMPLE in μM= 0.22 X ImL

0.6 0.5 then divide this value by the mg of tissue to give μM hydroperoxide/mg of tissue.

Tissue treatment/extraction for Lipid Hydroperoxide

Remove tissue from animal and freeze dry Store at -80C

Remove samples from -80oC for testing

Keep samples on ice

Try remove any suture

Weigh (aim for 20-25 mg of tissue, about 10mm) Transfer to glass tube with 500μL ice cold milliQ water

(Turn on centrifuge to cool down to 4oC)

Using glass rod "homogenize" each sample for two minutes. Keep tubes on ice .

To each sample, add 500μL of phosphoric acid in degassed methanol (100mg/15mL methanol) .

Vortex for 5 sees. Keep on ice for 5 mins .

Add ImL of degassed chloroform.

Vortex for 5 sees .

Keep on ice for 5 mins .

Centrifuge for 10 mins at 4oC at 3000rpm. Aspirate off the top layer.

Carefully collect the sample/bottom (chloroform) layer.

Normally over 600μL is collected (700-800μL) .

Place sample in glass tubes and cap tightly.

Keep samples on ice until testing or store samples at - 8OC.

Standard assay using 500μL If over 500μL is of sample: collected:

500μL sample 600μL sample 450μL diluent (2:1 Ch:Me) 350μL diluent (2:lCh:Me)

50μL chromogen 50μL chromogen

Total vol lOOOμL lOOOμL

When samples are placed in the glass 96 well plate they often go cloudy. This will give an incorrect reading. Place the plate at 37oC for a few minutes until the samples look clear, then read the plate.

The plate is read at 490nm (500nm is recommended but 490- 510nm is suitable) .

Claims

Claims :

1. A method of treating or preventing a peripheral neuropathy in a patient, said method comprising administering to the patient an effective amount of an antagonist of a neuronal nicotinic acetyl choline receptor (nAChR) .

2. A method according to claim 1, in which the peripheral neuropathy involves causing nerve dysfunction involving abnormal rate of flow of the electrical current through the nerve, abnormal ectopic firing of the nerve, or inappropriate or inadequate firing of the nerve in response to a stimulus.

3. A method according to claim 1 or claim 2, in which the peripheral neuropathy results from damage to peripheral nerves that is genetically acquired, caused by diseases of the nerves, is associated with exposure to a toxic agent or is the result of systemic illness.

4. A method according to claim 3, in which the neuropathy is associated with exposure to a toxic agent.

5. A method according to claim 4, in which the toxic agent is a chemotherapeutic agent including taxol, vincristine, cisplatin, an agent used for the treatment of infectious diseases including streptomycin, didanosine or zalcitabine, or any other chemically toxic agent, including alcohol .

6. A method according to claim 3, in which the neuropathy is associated with nerve injury due to trauma.

7. A method according to claim 3, in which the neuropathy is associated with a systemic disease.

8. A method according to claim 7, in which the systemic disease is uremia, childhood cholestatic liver disease, chronic respiratory insufficiency, alcoholic polyneuropathy, multiple organ failure, sepsis, hypo- albuminemia, eosinophilia-myalgia syndrome, hepatitis, porphyria, hypo-glycemia, vitamin deficiency, chronic liver disease, primary biliary cirrhosis, hyperlipidemia, leprosy, Lyme disease, herpes zoster, Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, sensory perineuritis, acquired immunodeficiency syndrome (AIDS) --associated neuropathy, Sjogren's syndrome, primary vasculitis (such as polyarteritis nodosa), allergic granulomatous angiitis, hypersensitivity angiitis, Wegener's granulomatosis, rheumatoid arthritis, systemic lupus erythematosis, mixed connective tissue disease, scleroderma, sarcoidosis, vasculitis, systemic vasculitides, acute tunnel syndrome, pandysautonomia, primary, secondary, localized or familial systemic amyloidosis, hypothyroidism, chronic obstructive pulmonary disease, acromegaly, malabsorption (sprue, celiac disease) , carcinomas (sensory, sensorimotor, late and demyelinating), lymphoma (including Hodgkin's), polycythemia vera, multiple myeloma (lytic type, osteosclerotic, or solitary plasmacytoma), benign monoclonal gammopathy, macroglobulinemia, cryoglobulinemia, tropical myeloneuropathies, herpes simplex infection, cytomegalovirus infection or diabetes.

9. A method according to claim 3, in which the neuropathy is genetically acquired.

10. A method according to claim 9, in which the neuropathy is peroneal muscular atrophy (Charcot-Marie- Tooth Disease) , hereditary amyloid neuropathy, hereditary sensory neuropathy (type I and type II) , porphyric neuropathy, hereditary liability to pressure palsy, Fabry's Disease, adrenomyeloneuropathy, Riley-Day Syndrome, Dejerine-Sottas neuropathy (hereditary motor- sensory neuropathy-III), Refsum's disease, ataxia- telangiectasia, hereditary tyrosinemia, anaphalipoproteinemia, abetalipoproteinemia, giant axonal neuropathy, metachromatic leukodystrophy, globoid cell leukodystrophy or Friedrich's ataxia.

11. A method according to claim 1, in which the nephropathy is diabetic neuropathy.

12. A method according to claim 1, in which the antagonist is a competitive antagonist.

13. A method according to claim 1, in which the antagonist is a non-competitive antagonist.

14. A method according to claim 1, in which the antagonist is a super agonist.

15. A method according to claim 1, in which the antagonist is an antibody specific for the neuronal nAChR or an antisense molecule specific for neuronal nAChR and able to prevent expression of a functional neuronal nAChR.

16. A method according to claim 1, in which the antagonist is an a conotoxins selected from:

Mil (SEQ ID NO: 1) GCCSNPVCHLEHSNLC from Conus magus, acting through neuronal Qf₃(S₂ receptors;

PnIA (SEQ ID NO : 2) GCCSLPPCAANNPDYC from C. pennaceus, acting through neuronal α₃/3₂ receptors;

PnIB (SEQ ID NO: 3) GCCSLPPCALSNPDYC from C. pennaceus, acting through neuronal oι₇ receptors;

EpI (SEQ ID NO: 4) GCCSDPRCNMNNPDYC from C. episcopatus, acting through neuronal α^^/α^^ receptors; AuIA (SEQ ID NO: 5) GCCSYPPCFATNSDYC from C. aulicus, acting through neuronal «₃(6₄ receptors;

AuIC (SEQ ID NO: 6) GCCSYPPCFATNSGYC from C. aulicus, acting through neuronal α₃β₄ receptors;

AuIB (SEQ ID NO: 7) GCCSYPPCFATNPDYC from C. aulicus, acting through neuronal Of₃₁S₄ receptors; and

ImI (SEQ ID NO: 8) GCCSDPRCAWR from C. imperialis, acting through neuronal α_η receptors.

17. A method according to claim 1, in which the antagonist is an α-conotoxin-like peptide having the sequence of amino acids XaaiCCSXaa₂Xaa3Xaa4CXaa₅Xaa6Xaa₇Xaa8Xaa₉XaaioXaa₁iC-NH2 (SEQ ID NO: 9) in which Xaai is G or D; Xaa₃ is proline, hydroxyproline or glutamine; each of Xaa₂ to Xaa₈ and Xaau is independently any amino acid; Xaa₉ is proline, hydroxyproline or glutamine; Xaaχ₀ is aspartate, glutamate or γ-carboxyglutamate; Xaau is optionally absent; and the C-terminus is optionally amidated, with the proviso that the peptide is not α-conotoxin EpI, α-conotoxin ImI, α- conotoxin PnIA or α-conotoxin PnIB.

18. A method according to claim 17, in which Xaa₂ is D, H or N; Xaa₄ is A, P or R; Xaa₅ is N,A or Y; Xaa₆ is A, Y, H, M or V; Xaa₇ is N or D; Xaa₈ is H or N; and Xaau is I or Y.

19. A method according to claim 17, in which the peptide comprises the sequence Xaa_xC C S Xaa₂ Xaa₃Xaa₄ C Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉Xaa₁₀Xaa₁₁C-NH₂ (SEQ ID NO: 10) in which Xaai is G or D; Xaa₂ is D, H or N; Xaa₃ is P or 4 -Hyp, Xaa₄ is A, P or R; Xaa₅ is N,A or Y; Xaa_s is A,Y,H,M or V; Xaa₇ is N or D; Xaa₈ is H or N; Xaa₉ is proline, hydroxyproline or glutamine; Xaaio is aspartate, glutamate or γ-carboxyglutamate; Xaau is I or Y; and the C-terminus is amidated.

20. A method according to claim 17, in which Xaa_x is G or D, Xaa₄ is R, Xaa₅ is N, Xaa₆ is Y, Xaa₇ is D, Xaa₈ is H, and Xaan is I .

21. A method according to claim 17, in which the peptide comprises the sequence GCCSDXaaiRCNYDHXaa₂Xaa₃IC (SEQ ID NO: 11), in which Xaai is proline, hydroxyproline or glutamine; Xaa₂ is proline or hydroxyproline; Xaa₃ is aspartate, glutamate or γ -carboxyglutamate; and the C- terminus is optionally amidated.

22. A method according to claim 17, in which the peptide is selected from

GCCSDPRCNYDHPEIC-NH₂ (SEQ ID NO: 12) GCCSD4-HypRCNYDHPgammacarboxy-GluIC-NH₂ (SEQ ID NO: 13)

DCCSNPPCAHNNPDC-NH₂ (SEQ ID NO: 14) and GCCSHPACYANNQDYC-NH₂ (SEQ ID NO: 15) in each of which the C-terminal cysteine is amidated.

23. A method according to claim 17, in which the peptide comprises GCCSDPRCNYDHPEIC (SEQ ID NO: 16) .

24. A method according to claim 1, in which the antagonist acts via a neuronal nAChR comprising an oι₃ subunit .

25. A method according to claim 24, in which the neuronal nAChR is comprises the subunits QJ_3JS₄Of₅, α;₃α₇/3₄ or α₃/3₄.

26. A method according to any preceding claim, in which the antagonist of a neuronal nAChR is capable of reducing oxidative stress or nerve damage caused by oxidative stress .

27. A method of treating or preventing nerve damage comprising administering an effective amount of an antagonist of a neuronal nAChR.

28. Use of an antagonist of a neuronal nAChR in the manufacture of a medicament for treating or preventing a peripheral neuropathy.

29. A pharmaceutical or veterinary agent for treating or preventing a peripheral neuropathy, which agent comprises an antagonist of a neuronal nAChR and a pharmaceutically- acceptable carrier.