WO2012142666A1 - Method of modulating amine oxidase activity and agents useful for same - Google Patents

Method of modulating amine oxidase activity and agents useful for same Download PDF

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Publication number
WO2012142666A1
WO2012142666A1 PCT/AU2012/000407 AU2012000407W WO2012142666A1 WO 2012142666 A1 WO2012142666 A1 WO 2012142666A1 AU 2012000407 W AU2012000407 W AU 2012000407W WO 2012142666 A1 WO2012142666 A1 WO 2012142666A1
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app
oxidase activity
amine oxidase
disorder
agent
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PCT/AU2012/000407
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French (fr)
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James Alexander DUCE
Ashley Ian Bush
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The Mental Health Research Institute Of Victoria
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Priority claimed from AU2011901468A external-priority patent/AU2011901468A0/en
Application filed by The Mental Health Research Institute Of Victoria filed Critical The Mental Health Research Institute Of Victoria
Publication of WO2012142666A1 publication Critical patent/WO2012142666A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/315—Zinc compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates generally to a method of treating conditions characterised by aberrant amine oxidase activity and agents useful for same. More particularly, the present invention relates to a method of treating conditions characterised by aberrant amine oxidase activity by modulating the activity of amyloid precursor protein.
  • the method of the present invention is useful, inter alia, in the treatment and/or prophylaxis of conditions including, but not limited to, clinical depression.
  • catecholamines mediate alertness and stress responses, increasing heart rate, blood pressure, gluconeogenesis, as well as the release of free fatty acids and the secretion of various hormones (Kvetnansky R. et al., 2009). Sustained elevation of catecholamines has been shown to suppress the proliferation and survival of lymphocytes directly (Bergquist J. et al., 1998) and by lowering prolactin levels (Clevenger CV. et al., 1991).
  • epinephrine adrenaline
  • norepinephrine norepinephrine
  • dopamine dopamine
  • catecholamines dopamine (DA) and norepinephrine (NE) are neurotransmitters, while NE and epinephrine (E) are released into blood by the adrenal medulla (Kobayashi S and Coupland RE., 1993). Endogenous catecholamines are also produced by peripheral organs such as the heart (Diarra A. et al., 1989) and immunocompetent cells (Bergquist J. et al., 1998).
  • Catecholaminergic inactivation occurs by diffusion, transporter uptake and enzymatic breakdown (Galli A. et al., 1998; Ford CP et al., 2010).
  • catecholamines are transported into vesicles or degraded by multiple intracellular pathways including methylation (e.g. catechol-O-methyltransferase) and amine oxidation (Kvetnansky R. et al., 2009).
  • Perisynaptic catecholamine concentrations of 30-400 ⁇ are estimated (Galli A. et al., 1998; Esler M. et al., 1990) but drop to nM levels in plasma (Esler M. et al., 1990) due to dilution and possible extracellular degradation either by methylation through catechol-O- methyltransferases (COMT) or by deamination through amine oxidases.
  • COMP catechol-O- methyltransferases
  • High catecholamine levels in blood is typically associated with stress, which can be induced from psychological reactions or environmental stressors such as elevated sound levels, intense light, or low blood sugar levels.
  • Extremely high levels of catecholamines also known as catecholamine toxicity
  • Extremely high levels of catecholamine can also be caused by
  • Catecholamines also cause general physiological changes, including increased heart rate, blood pressure, alertness and blood glucose levels, opening of airways and a general increase in sympathetic nervous system activity.
  • Catecholamine levels also tend to peak early in the day, which may contribute to the increased risk of heart attacks and strokes during the morning hours.
  • catecholamine levels For example, one of the first effective antidepressant drugs were the amine oxidase inhibitors (e.g., Nardil®, Parnate®) and tricyclic antidepressants (e.g., Elavil®, Tofranil® and Pamelor®).
  • amine oxidase inhibitors e.g., Nardil®, Parnate®
  • tricyclic antidepressants e.g., Elavil®, Tofranil® and Pamelor®.
  • the therapeutic effect of these drugs appears to be based largely on their ability to increase catecholamine levels in the brain by blocking the normal metabolic degradation or reuptake of catecholamine molecules that are released into the synapse.
  • Vitamins B6, B 12 and folic acid have also been proposed as useful compounds for the treatment of clinical depression. Vitamin B6 levels have been found to be low in patients diagnosed with clinical depression. Moreover, studies show that supplementing vitamin B6 in people with affective disorders is associated with increased levels of NE and serotonin and relief of depression. Folic acid and vitamin B 12 are necessary for the synthesis of a substance called S-adenosylmethionine, which is vital for neurotransmitter metabolism. A deficiency of either folic acid or vitamin B 12 may cause similar neurologic and psychiatric disturbances, including depression, dementia, and demyelinating myopathy.
  • a folate deficiency may specifically affect the metabolism of catecholamines and indoleamines and aggravate depressive disorders.
  • Folic acid and vitamins B6 and B12 are also required for the conversion of homocysteine to methionine. This prevents the accumulation of homocysteine, which has been linked to the formation of atherosclerotic plaque in arteries as well as to neurotoxic effects that can produce neurologic and psychiatric disturbances.
  • serotonin In addition to catecholamines, serotonin (5-HT), a monoamine neurotransmitter, also plays a very important role in the regulation of aggression, mood, body temperature, sleep, vomiting, sexual drive and appetite.
  • the role of serotonin in these clinical settings may be regulated, at least in part, by agonistic or antagonistic action on serotonin receptors.
  • the concentration of synaptic serotonin can also be controlled directly by its reuptake into the presynaptic terminal and, thus, drugs blocking serotonin transport have been used with some success for the treatment of depression.
  • cardiovascular modulating effects both vasoconstrictor and vasodilator
  • potent pro- thrombotic activity potent pro- thrombotic activity
  • endothelial mitogenic action as well as immune modulating effects.
  • One method for modulating peripheral serotonin levels is the chronic use of serotonin-selective reuptake inhibitor drugs (SSRIs) such as alaproclate, dapoxetine, etoperidone, citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline and zimelidine, which are routinely used for treating CNS conditions such as clinical depression, obsessive-compulsive disorder, and additional mood disorders and are hence known to belong to the class of antidepressant drugs.
  • SSRIs serotonin-selective reuptake inhibitor drugs
  • Serotonin-selective reuptake inhibitors such as fluoxetine (ProzacTM)
  • TCAs tricyclic antidepressants
  • SSRIs have a more favourable adverse reaction profile in comparison to the TCAs and are typically much easier to tolerate.
  • SSRIs exert their therapeutic effect by blocking the reuptake of serotonin into the presynaptic nerve terminal, thus increasing the synaptic concentration of serotonin.
  • SSRIs may also increase the efficacy of the serotonin neurons by desensitizing 5-HT autoreceptors located on the presynaptic serotonin nerve terminals.
  • the ability of the 5-HT autoreceptors to inhibit the release of serotonin decreases after long-term treatment with SSRIs, with the net effect being that a greater amount of serotonin is released per impulse. As a consequence, the release of serotonin from these neurons becomes more efficient.
  • Research in the treatment of depression remains focused on serotonin because a good number of selective serotonin reuptake blockers tested in the clinical setting have been effective in treating major depression.
  • the general mechanism of action is hypothesized as enhanced serotonin neurotransmission due to the increased availability of serotonin in the synapse of these neurons.
  • SSRIs generally have a 50- to 100-fold or greater selectivity for the inhibition of serotonin uptake in vitro when compared to their ability to inhibit NE or DA uptake.
  • amyloid precursor protein is a functional amine oxidase which simultaneously oxidizes amines and catecholamines catalytically. This activity is inhibited by Zn + . Since reduced levels of catecholamines and amines such as serotonin are linked to conditions such as clinical depression, the determination that Zn 2+ inhibits APP amine oxidase activity has now provided the basis for the development of a new treatment approach. These findings provide the basis for the development of a new treatment approach for conditions characterised by aberrant amine oxidase activity, such as, but not limited to, clinical depression.
  • the term "derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of "a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
  • the subject specification contains amino acid sequence information prepared using the programme Patentin Version 3.5, presented herein after the bibliography.
  • Each amino acid sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (e.g. ⁇ 210>1, ⁇ 210>2, etc).
  • the length, type of sequence (protein, etc) and soufce organism for each sequence is indicated by information provided in the numeric indicator fields ⁇ 21 1>, ⁇ 212> and ⁇ 213>, respectively.
  • Amino acid sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (e.g. SEQ ID NO: l , SEQ ID NO:2, etc.).
  • sequence identifier referred to in the Specification correlates to the information provided in numeric indicator field ⁇ 400> in the sequence listing, which is followed by the sequence identifier (e.g. ⁇ 400>1, ⁇ 400>2, etc). That is SEQ ID NO.l as detailed in the specification correlates to the sequence indicated as ⁇ 400>1 in the sequence listing.
  • One aspect of the present invention is directed to a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate the functional interactivity of Zn 2+ with said APP wherein antagonising the interaction of Zn 2+ with said APP increases APP amine oxidase activity and facilitating the interaction of Zn 2+ with said APP decreases APP amine oxidase activity.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate GFD potentiation of APP amine oxidase activity wherein facilitating the interaction of GFD with APP increases APP amine oxidase activity and antagonising the GFD interaction with APP decreases APP amine oxidase activity.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to antagonise the interaction of Zn 2+ with said APP.
  • said antagonist is a zinc chelator, ionophore or metal protein attenuating compound.
  • the Zn chelator is a moderate affinity chelator which is hydrophobic. Examples include the 8-hydroxy quinolines, such as clioquinol, PBT2, M30, VK28 or related molecules, pyrithione, diethyl pyrocarbamate, l,2-bis-(2-(amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid and derivatives, the bicyclam analogue JKL169 (1 ,1 '-xylyl bis- 1 ,4,8,1 1 tetraaza cyclotetradecane), DP 109 and related compounds.
  • APP-mediated amine oxidase activity can be reduced by administering Zn 2+ to said subject.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof for a time and under conditions sufficient to potentiate APP-mediated amine oxidase activity.
  • SEQ ID NO:2 represents the amino acid sequence of the growth factor domain (GFD) of APP.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent which antagonises the interaction of GFD with APP.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to inhibit APP-mediated amine oxidase activity.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to inhibit APP-mediated amine oxidase activity.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject which condition is characterised by insufficient catecholamine levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
  • Conditions characterised by insufficient catecholamine levels include, but are not limited to. Major depression. Minor depression. Atypical depression. Dysthvmia, Melancholia, Anergic depression. Treatment-resistant depression, Headache, Extrapyramidal disorders. Generalised anxiety disorder. Lichen simplex chronicus. Insomnia, Panic disorder, Stress disorder, Posttraumatic stress disorder (PTSD), Attention deficit disorder, Hyperactivity, Conduct disorder, Narcolepsy, Social phobia and anxiety, Obsessive-compulsive disorder.
  • Eating disorder Bulimia, Drug withdrawal syndromes and drug dependence disorders, Atypical facial pain, Chronic pain syndrome, Parkinson's disease, Hypertension, Irritable bowel syndrome (IBS), Jet lag (desynchronosis) and Premature ejaculation.
  • IBS Irritable bowel syndrome
  • Jet lag desynchronosis
  • Conditions characterised by insufficient serotonin levels include, but are not limited to, increased aggressive and angry behaviours, clinical depression, obsessive-compulsive disorder (OCD), migraine, irritable bowel syndrome (IBS), tinnitus, fibromyalgia, bipolar disorder, anxiety disorders, intense religious experiences, depression, anxiety disorders and other affective disorders, eating disorders such as bulimia, anorexia and obesity, phobias, dysthymia, premenstrual syndrome, cognitive disorders, impulse control disorders, attention deficit hyperactivity disorder and drug abuse.
  • the anxiety disorders may include, but are not limited to, general anxiety disorders, panic anxiety, obsessive compulsive disorder, acute stress disorder, post trauma stress disorder or social anxiety disorder.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
  • a condition characterised by excessive serotonin levels includes, but is not limited to, Serotonin Syndrome.
  • said agent is a Zn 2+ chelator, ionophore or metal protein attenuating compound as hereinbefore described.
  • said agent is SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof.
  • the present invention provides use of an agent which decreases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient catecholamine levels.
  • the present invention provides use of an agent which increases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted catecholamine levels.
  • the present invention provides use of an agent which increases the level of Zn 2+ in the manufacture of a medicament for the treatment of a condition characterised by unwanted APP-mediated amine oxidase activity.
  • the present invention provides use of an agent which antagonises the interaction of APP with Zn 2+ in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP-mediated amine oxidase activity.
  • the present invention provides use of a compound of SEQ ID NO: 1
  • the present invention provides use of an agent which inhibits GFD-mediated potentiation of APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient catecholamine levels.
  • the present invention provides use of an agent which decreases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient serotonin levels.
  • the present invention provides use of an agent which increases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted serotonin levels.
  • the present invention provides use of a compound of S EQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof which increases APP- mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted serotonin levels.
  • the present invention provides use of an agent which inhibits GFD-mediated potentiation of APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient serotonin levels.
  • modulatory agents as herein described, when used in the methods of the present invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an agent as hereinbefore described and one or more pharmaceutically acceptable carriers and/or diluents for use in the methods of the present invention, as herein described.
  • Said pharmaceutical composition may additionally comprise molecules with which it is to be co-administered.
  • a method of screening for a compound that modulates the APP-mediated amine oxidase activity comprising exposing said compound to APP and an APP substrate under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said compound is indicative that said compound modulates APP-mediated amine oxidase activity.
  • the level of oxidised APP substrate increases as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound increases APP- mediated amine oxidase activity.
  • the level of oxidised APP substrate is decreased as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound decreases APP-mediated amine oxidase activity.
  • a method of screening a biological sample for APP-mediated amine oxidase activity comprising exposing a biological sample that putatively comprises APP or a functional derivative thereof to an APP substrate under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said sample is indicative that said sample comprises APP or a functional deri vative thereof.
  • a functional derivative of APP includes, but is not limited to, soluble APP (sAPP).
  • APP substrates for use in the screening method of the present invention
  • substrates for use in the screening method of the present invention include, but are not limited to:
  • FIG. 1 Cp-Iike amine oxidase activity of APP695a. Oxidation rates catalysed by Cp compared to APP695a at optimal pH, as a product of initial o-dianisidine (a) and p- phenylenediamine (b) concentration. Proteins (500 nM) were incubated in (a) 75 mM Na acetate, pH 5.0, or 50 mM HEPES, pH 7.2, or (b) 75 mM Na acetate, pH 5.5 at 37 °C. (c) Kinetic values of Cp and APP695a as calculated using Michaelis-Menten equations, are comparable with each substrate.
  • Bovine albumin was inactive in this assay (not shown), (d) Schematic of the positions of the ferroxidase domain (FD1 ) 5 within the E2 -domain of APP (APP-E2), and the growth factor domain (GFD) within the El -domain of APP (APP-E1 ) in relation to other recognized domains of N-terminal APP including the copper binding domain (CuBD) and the zinc binding domain (ZnBD).
  • APP 770 isoform is shown, APP 75 i lacks the OX-2 domain, and APP 6 95 lacks both OX-2 and Kunitz protease inhibitor (KPI) domains,
  • KPI Kunitz protease inhibitor
  • FDl (E415N) -APP695a is APP695a with a mutation at the second glutamic acid in the REXXE motif ofFDl .
  • APP deficient mice have increased catecholamines and concomitant physiological changes.
  • HPLC-ECD revealed increased NE (a), E (b) and DA (c), here represented as a percentage relative to wild-type age matched controls, in tissues as shown. Specific catecholamine concentrations within each tissue are shown in Table 2.
  • Cardiovascular alterations consistent with chronic catecholamine increase included elevated basal heart rate (d) and systolic blood pressure (e) in 6-month APP-/- mice compared to age-matched wild- type controls,
  • Plasma prolactin is decreased in APP-/- mice, consistent with elevated brain stem DA.
  • the percentage of lymphocytes as a proportion of white cell count is reduced in APP-/- mice. Total white cell count was not significantly altered.
  • APLP2 has no oxidase activity and zinc specific inhibition of oxidase activities differentiates APP.
  • Cp was inhibited by azide (Topham RW. and Frieden E. 1970) while APP695a was inhibited by zinc (Duce, J.A. et ai, 2010). Neither was inhibited by MAO inhibitors.
  • APLP2 which lacks ferroxidase activity (Duce JA. et al, 2010), similarly had no amine oxidase activity.
  • Wild-type mouse plasma contains oxidase activity that can be inhibited by zinc and azide.
  • O-dianisidine oxidase activity of wild-type mouse plasma inhibited by azide (lOmM) leaves a residual activity similar to total activity in APP-/- mice (Fig. 2c).
  • the present invention is predicated, in part, on the determination that amyloid precursor protein (APP) comprises amine oxidase activity and that this activity can be inhibited by Zn 2+ . Still further, the inventors have determined that the amine oxidase activity is ascribed, at least in part, to the E2 domain of APP.
  • one aspect of the present invention is directed to a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate the functional interactivity of Zn 2+ with said APP wherein antagonising the interaction of Zn 2+ with said APP increases APP amine oxidase activity and facilitating the interaction of Zn 2+ with said APP decreases APP amine oxidase activity.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate GFD potentiation of APP amine oxidase activity wherein facilitating the interaction of GFD with APP increases APP amine oxidase activity and antagonising the GFD interaction with APP decreases APP amine oxidase activity.
  • APP amyloid precursor protein
  • sAPP soluble forms of APP
  • any isoforms which arise from alternative splicing of APP mRNA allelic variants, polymorphic variants or various post translational forms of APP which undergo modification at, for example, the level of glycosylation, phosphorylation, tyrosine sulfation and proteolytic processing.
  • APP is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation and neural plasticity.
  • APP is best known and most commonly studied as the precursor molecule whose proteolysis generates beta amyloid ( ⁇ ), a 39- to 42-amino acid peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients. APP also undergoes proteolytic cleavage to produce a soluble form of the protein (sAPP) that is active in plasma.
  • ⁇ beta amyloid
  • sAPP soluble form of the protein
  • the gene for APP is located on chromosome 21 and contains at least 18 exons in 240 kilobases.
  • Several alternative splicing isoforms of APP have been observed in humans, ranging in length from 365 to 770 amino acids, with certain isoforms preferentially expressed in neurons. Changes in the neuronal ratio of these isoforms have been associated with Alzheimer's disease.
  • Homologous proteins have been identified in other organisms such as Drosophila (fruit flies), C. elegans (roundworms), and all mammals.
  • a number of distinct, largely independently-folding structural domains have been identified in the APP sequence.
  • the extracellular region much larger than the intracellular region, is divided into the E I and E2 domains, linked by an acidic domain (AcD).
  • El contains two subdomains including a growth factor-like domain (GFLD) and a copper-binding domain (CuBD) interacting tightly together.
  • GFLD growth factor-like domain
  • CuBD copper-binding domain
  • APP can be referred to by different names including, but not limited to:
  • amine oxidase activity should be understood as a reference the oxidation of a wide range of biogenic amines including many neurotransmitters, histamine and xenobiotic amines.
  • Amine oxidases also referred to herein as monoamine oxidases (MAO; e.g., EC 1.4.3.4), are a family of enzymes that catalyze the oxidation of monoamines. They are found bound to the outer membrane of mitochondria in most cell types in the body. In humans there are two types of MAO: MAO-A and MAO-B. Both are found in neurons and astroglia. Outside the central nervous system, MAO-A is also found in the liver, gastrointestinal tract, and placenta, whereas MAO-B is mostly found in blood platelets.
  • monoamine oxidases catalyze the oxidative deamination of monoamines.
  • Oxygen is used to remove an amine group from a molecule, resulting in the corresponding aldehyde and ammonia.
  • M AO-A is particularly important in the catabolism of monoamines ingested in food.
  • Both MAOs are also vital to the inactivation of monoaminergic neurotransmitters, for which they display different specificities. For example, serotonin, melatonin, norepinephrine, and epinephrine are mainly broken down by MAO-A. Phenethylamine is mainly broken down by MAO-B. Both forms break down dopamine equally. Specific reactions catalyzed by MAO include:
  • MAO dysfunction (too much or too little MAO activity) is thought to be responsible for a number of neurological disorders.
  • unusually high or low levels of MAOs in the body have been associated with depression, schizophrenia, substance abuse, attention deficit disorder, migraines, and irregular sexual maturation.
  • Monoamine oxidase inhibitors are one of the major classes of drug prescribed for the treatment of depression, although they are last-line treatment due to risk of the drug's interaction with diet or other drugs. Excessive levels of catecholamines (for example, epinephrine, norepinephrine, and dopamine) may lead to a hypertensive crisis. PET research has shown that MAO is also heavily depleted by use of tobacco cigarettes.
  • APP has been determined to possess amine oxidase activity.
  • reference to "APP-mediated amine oxidase activity” should be understood as a reference to the amine oxidase activity of the amyloid precursor protein itself. It has been found that the amine oxidase activity of APP resides in the E2 domain of APP.
  • Reference to "aberrant" APP-mediated amine oxidase activity should be understood as a reference to a level of APP-mediated amine oxidase activity which is problematic or otherwise not appropriate. This may be either inadequate amine oxidase activity (for example, potentially leading to localised areas of increased catecholamine and/or serotonin levels in tissue) or too much (i.e., unwanted) amine oxidase activity (for example, leading to reduced catecholamine and/or serotonin levels in tissue). It should be appreciated that, in some situations, the level of A P-mediated amine oxidase activity may be physiologically normal.
  • the amine oxidase activity is "aberrant" within the context of this invention since it is an unwanted level of activity when considered in light of the individual's overall physiological state.
  • the method of the present invention provides a means of modulating APP-mediated amine oxidase activity in order to improve the individual's physiological state. In terms of inadequate amine oxidase activity, this may take the form of either a reduction in the level of amine oxidase activity relative to normal levels or a complete ablation of amine oxidase activity.
  • GFD potentiates APP amine oxidase activity.
  • a further treatment method has been developed based on modulating GFD potentiation functionality.
  • antagonising GFD interaction with APP provides a means of reducing amine oxidase activity while facilitating the interaction of GFD with APP, such as via the use of GFD mimetics, provides a means for increasing APP amine oxidase activity.
  • This increase in APP activity will typically be finite, as the ratio of GFD mimetic with APP will generally not exceed 1 : 1 (see Figure I f).
  • APP-mediated amine oxidase activity is entirely abrogated, it would be appreciated by the skilled person that even reducing the extent or degree of APP-mediated amine oxidase activity can nevertheless produce a valuable therapeutic outcome, such as increasing catecholamine levels and/or serotonin levels in a subject. Conversely, in terms of increasing APP-mediated amine oxidase activity, any level of increase (even if not restoration of normal level of activity) may nevertheless be desirable if it at least partially restores APP-mediated amine oxidase activity levels and produce a valuable therapeutic outcome, such as decreasing catecholamine levels and/or serotonin levels in a subject.
  • agent should be understood as a reference to any proteinaceous or non-proteinaceous molecule which modulates the interaction of Zn 2+ or GFD with APP.
  • the subject agent may be linked, bound or otherwise associated with any proteinaceous or non- proteinaceous molecule.
  • it may be associated with a molecule which permits targeting to a specific tissue, such as the brain.
  • Said proteinaceous molecule may be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening.
  • Said non-proteinaceous molecule may be derived from natural sources, such as for example natural product screening or may be chemically synthesised.
  • the agent is either an antagonist which interacts with Zn 2+ to prevent its interaction with APP or is Zn 2+ , itself, or a molecule which results in the formation or release of Zn 2+ , thereby reducing APP amine oxidase functionality.
  • the present invention contemplates Zn" chelators which are exemplified later in this document.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to antagonise the interaction of Zn 2+ with said APP.
  • said antagonist is a zinc chelator, ionophore or metal protein attenuating compound.
  • Zn 2+ chelators provide one method to prevent Zn 2+ from inhibiting APP amine oxidase activity.
  • a Zn 2+ chelator, as described herein, is any compound that binds Zn 2+ (whether or not it is a true chelator). Accordingly, any molecule that has the ability to ligand or chelate to a Zn 2+ molecule can be used.
  • the Zn 2+ chelator is any ligand that is able to form two or more coordination bonds with a zinc ion.
  • the zinc chelator is hydrophobic and is able to pass through the blood brain barrier, and optionally binds to zinc with moderate affinity.
  • chelators that bind to zinc with high affinity may also be effective.
  • the zinc chelator may include a cyclic group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc or a cyclic group which includes at least one heteroafom such as nitrogen, oxygen or sulfur and in which the cyclic group is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc.
  • the cyclic group is a heteroaryl group that is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc.
  • the heteroaryl group is especially selected from quinazolinyl, quinoxalinyl, naphthyridinyl, pyrimidopyrimidinyl, cinnolinyl, phenazinyl, acridinyl, phenanthrolinyl, pyridopyrimidinyl, pyridopyrazinyl, pyranopyridinyl, dibenzoquinolizinyl, quinolinyl, isoquinolinyl, pyfidinyl and pyrimidinyl groups, especially pyridinyl and quinolyl groups.
  • Suitable zinc chelators that include a pyridyl group include pyrithione, deferiprone and ⁇ , ⁇ , ⁇ ', ⁇ '-tetrakis (2-pyridyImethyl) ethylenediamine (TPEN), especially pyrithione.
  • Suitable quinolines may include a hydroxy substituent especially in the 8-position.
  • Suitable quinolines may include clioquinol, iodoquinol, PBT2, M30 and related molecules such as those discussed in US 7,619,091. Further compounds that include a heteroaryl group substituted with a functional group that is able to carry a negative charge are discussed in US 7,692,01 1 and US 6,855,71 1. '
  • the cyclic group is an aryl group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc.
  • Suitable aryl groups include phenyl and naphthyl groups.
  • Exemplary compounds that include an aryl group that is substituted with a functional group that is able to donate electrons to a coordination bond with zinc are discussed in US 6,855,71 1.
  • Functional groups able to donate electrons to a coordination bond with zinc include atoms with lone pairs of electrons.
  • such groups include heteroatoms and functional groups that are able to bear a negative charge.
  • Suitable heteroatoms include nitrogen, oxygen and sulfur.
  • Suitable functional groups that are able to carry a negative charge include hydroxy, mercapto. ester, carboxylate, oxtme, aldehyde and ketone groups, especially hydroxy and mercapto groups.
  • Another group of zinc chelators useful in the present invention include a heterocyclyl macrocyclic group, such as a cyclam or a bicyclam.
  • Cyclams are compounds comprising a 1 ,4,8,1 1 -tetraazacyclotetradecane ring, which may be optionally substituted. Suitable substituents include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl and aryl groups.
  • Bicyclams comprise two cyclam rings linked by an aromatic or aliphatic linker.
  • a suitable bicyclam is ⁇ , ⁇ -xylyl bis- 1 , 4,8, 1 1 -tetraazacyclotetradecane (J L 169).
  • the zinc chelator may also be a polycarboxylic acid, such as ethylene diamine tetraacetic acid (EDTA), nitrilotriacetic acid, nitrilotripropionic acid, diethylenetriamine pentaacetic acid, 2-hydroxyethyl-ethylenediamine-triacetic acid, 1 ,6-diamino-hexamethylene- tetraacetic acid, 1 ,2-diamino-cycIohexane tetraacetic acid, 0,0'-bis(2-aminoethyl)- ethyleneglycol-tetraacetic acid, 1 ,3-diaminopropane-tetraacetic acid, N,N-bis(2- hydroxybenzyl)ethylenediamine-N,N-diacetic acid, ethylenediamine-N,N'-diacetic acid, ' ethylenediamine-N,N'-dipropionic acid, tri
  • the polycarboxylic acid is l ,2-bis-(2-amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid or ethylenediamine tetraacetic acid (EDTA), especially l,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid.
  • the zinc chelator may also be an ester of these polycarboxylic acids.
  • Diesters of (HOOC- CH2-)2N-A-N -CH 2 COOH)2 (where A is a saturated or unsaturated, aliphatic, aromatic or heterocyclic divalent linking radical containing, in a direct chain link between the two depicted nitrogen atoms, 2-8 carbon atoms in a continuous chain which may be interrupted by 2-4 oxygen atoms, provided that the chain members directly connected to the two depicted nitrogen atoms are not oxygen atoms) are discussed, for example, in US 6,458,837.
  • the ester of the polycarboxylic acid is an alkyl ester.
  • the zinc chelator is BAPTA-AM (l,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), DP-b99 (l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, N,N'-di(octyloxyethyl ester), ⁇ , ⁇ '-disodium salt) or DP-109, especially DP-109.
  • BAPTA-AM l,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester
  • DP-b99 l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, N,N'-di(octyloxyethyl ester), ⁇ , ⁇ '
  • the zinc chelator includes two carbamate groups linked by an aromatic or aliphatic linker or a heteroatom such as oxygen, nitrogen or sulfur, such as in diethylpyrocarbamate.
  • the zinc chelator is an amino carboxylic acid that includes a functional group that is able to donate electrons to a coordination bond with zinc. Suitable amino carboxylic acids include penicillamine, cysteine, aspartic acid and glutamic acid, and also esters of these amino carboxylic acids.
  • the amino carboxylic acid is d-penicillamine.
  • the ester of the amino carboxylic acid is an alkyl ester.
  • the zinc chelator includes a hydroxamide group, such as desferrioxamine.
  • the zinc chelator may also be a substituted transition metal including two or more functional groups that are able to carry a negative charge.
  • Suitable transition metals include molybdenum, and a suitable zinc chelator is tetrathiolmolybdenate.
  • aromatic or aliphatic linker refers to a divalent group that connects two or more groups that are able to chelate zinc.
  • Suitable aromatic or aliphatic linkers include optionally substituted arylene, alkylene, alkenylene, cycloalkylene and cycloalkenylene groups, especially optionally substituted arylene, more especially optionally substituted phenylene, most especially divalent xylene.
  • the alkylene or alkenylene groups may have one or more non-consecutive carbon atoms replaced by a heteroatom such as nitrogen, oxygen or sulfur.
  • alkyl refers to a straight chain or branched saturated hydrocarbon group having 1 to 12 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, which includes alkyl groups having 1 , 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, /j-propyl, /-propyl, w-butyl, /-butyl, t- butyl, M-pentyl, heptyl, octyl, nonyl and dodecyl.
  • alkylene refers to a divalent alkyl group.
  • alkenyl refers to a straight-chain or branched
  • alkenyl group having one or more double bonds between carbon atoms and having 2 to 12 carbon atoms.
  • the alkenyl group may have a specified number of carbon atoms.
  • C 2 -C6 as in "Cj-Cealken l” includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement.
  • suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl. hexadienyl, heptenyl, octenyl. nonenyl, decenyl, undecenyl and dodecenyl.
  • alkenylene refers to a divalent alkylene group.
  • alkynyl refers to a straight-chain or branched
  • C 2 -C6 as in "C 2 -C 6 alkynyl” includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement.
  • suitable alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, octynyl, nonynyl, decynyl, undecynyl and dodecynyl.
  • cycloalkyl refers to a saturated cyclic hydrocarbon.
  • the cycloalkyl ring may include a specified number of carbon atoms.
  • a 3 to 8 membered cycloalkyl group includes 3, 4, 5, 6, 7 or 8 carbon atoms.
  • suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentanyl, cyclohexanyl and cycloheptanyl.
  • cycloalkylene refers to a divalent cycloalkyl group.
  • cycioaikenyl refers to a cyclic hydrocarbon having at least one double bond, which is not aromatic.
  • the cycioaikenyl ring may include a specified number of carbon atoms.
  • a 4 to 8 membered cycioaikenyl group contains at least one double bond and 4, 5, 6, 7 or 8 carbon atoms.
  • cycioaikenyl groups include, but are not limited to cyclopentenyl, cyclopenta-l,3-dienyl, cyclohexenyl, cyclohexen- 1 ,3-dienyl and cyclohexen-l ,4-dienyl.
  • cycloalkenylene refers to a divalent cycloalkenyl group.
  • aryl is intended to mean any stable, monocyclic, bicyclic or tricyclic carbon ring of up to 7 atoms in each ring, -wherein at least one ring is aromatic. When more than one ring is present, the rings may be fused to one another.
  • aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indartyl, biphenyi, binaphthyl, anthracenyl, phenanthrenyl, phenalenyl and fluorenyl.
  • arylene refers to a divalent aryl group.
  • heterocyclyl refers to a cycloalkyl or cycloalkenyl group in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. For example, between 1 and 4 carbon atoms in each ring may be replaced by heteroatoms independently selected from N, S and O. If the heterocyclyl group includes more than one ring in a ring system, at least one ring is heterocyclic.
  • heterocyclyl groups examples include tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolinyl, dithiolyl, 1 ,3-dioxolanyl, pyrazolinyl, imidazolinyl, imidazolidonyl, dioxanyl, dioxinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyranyl, dithianyl, and
  • heteroaryl represents a stable monocyclic, bicyclic or tricyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • heteroaryl groups examples include quinazolinyl, quinoxalinyl, naphthyridinyl, pyrimidopyrimidinyl, cinnolinyl, phenazinyl, acridinyl, phenanthrolinyl, pyridopyrimidinyl, pyridopyrazinyl, pyranopyridinyl, dibenzoquinolizinyl, quinolinyl, isoquinolinyl, pyridinyl and pyrimidinyl.
  • the Zn 2+ chelator is a moderate affinity chelator which is hydrophobic
  • Examples include the 8-hydroxy quinolines, such as clioquinol, PBT2, M30, VK28 or related molecules, pyrithione, diethyl pyrocarbamate. 1 ,2-bis-(2-(amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid and derivatives, the bicyclam analogue JKL169 (1,1 '-xylyl bis-1 ,4,8,1 1 tetraaza cyclotetradecane), DP109 and related compounds.
  • APP-mediated amine oxidase activity can be induced by administering Zn 2+ to said subject.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof for a time and under conditions sufficient to potentiate APP-mediated amine oxidase activity.
  • said condition is characterised by unwanted catecholamine levels and/or unwanted serotonin levels, examples of which are herein described.
  • SEQ ID NO:2 represents the amino acid sequence of the growth factor domain (GFD) of APP.
  • fragments include parts and portions, mutants, variants and mimetics from natural, synthetic or recombinant sources including fusion proteins.
  • Parts or fragments include, for example, active regions of GFD.
  • Mimetics may be derived from insertion, deletion or substitution of amino acids.
  • Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • An example of substitutional amino acid variants are conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • Additions to amino acid sequences include fusions with other peptides; polypeptides or proteins or cyclising the peptide, for example to yield a pharmacologically active form.
  • a "homologue” refers to a sequence in another animal or organism which has at least about 70% identity, preferably 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identify to the human GFD molecule.
  • Analogues include chemical and functional equivalents of GFD. These should be understood as molecules exhibiting any one or more of the functional activities of GFD and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
  • the fragments may have the active sites of GFD fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
  • Analogues contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBF ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (T BS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH. ⁇ .
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent dertvatisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuri- benzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4- nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethyipyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acids contemplated, herein is shown in Table 2.
  • Analogues may also include competitors for the active site of APP (e.g., APP-E2).
  • Analogues may also include peptoids (poly-N-substituted glycines), which are a class of peptide mimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the a-carbons. Peptoids are typically resistant to proteolysis and, therefore, are advantageous for therapeutic applications where proteolysis is of concern.
  • peptoids poly-N-substituted glycines
  • Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala ⁇ -amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutarnic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methyl isol leucine Nmile
  • D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
  • agents which are administered to a subject in accordance with the present invention may also be linked to a targeting means, such as a monoclonal antibody, which provides specific delivery of these molecules to target tissue regions.
  • a targeting means such as a monoclonal antibody, which provides specific delivery of these molecules to target tissue regions.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent which antagonises the interaction of GFD with APP.
  • One aspect of the present invention is directed to treating a condition characterised by aberrant APP amine oxidase activity.
  • This should be understood as a reference to any disease or condition in respect of which aberrant APP-mediated amine oxidase activity is a cause, symptom or side effect.
  • This includes, for example, conditions which occur as a side effect of a treatment regime for an unrelated disease condition.
  • the subject condition may be congenital or acquired and may be in an acute or chronic phase.
  • Conditions characterised by aberrant APP-mediated amine oxidase activity will include conditions characterised by aberrant catecholamine levels within the peripheral circulation ⁇ e.g., plasma) or biological tissue of said subject, including, but not limited to, the central nervous system. Said condition may be characterised by excessive catecholamine levels or inadequate catecholamine levels.
  • said condition is characterised by insufficient catecholamine levels, attributed at least in part to excessive APP-mediated amine oxidase activity.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to inhibit APP-mediated amine oxidase activity.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
  • Conditions characterised by insufficient catecholamine levels include, but are not limited to, Major depression, Minor depression. Atypical depression, Dysthymia, Melancholia, Anergic depression, Treatment-resistant depression. Headache, Extrapyramidal disorders, Generalised anxiety disorder, Lichen simplex chronicus, Insomnia, Panic disorder, Stress disorder, Posttraumatic stress disorder (PTSD), Attention deficit disorder, Hyperactivity, Conduct disorder, Narcolepsy, Social phobia and anxiety.
  • Obsessive-compulsive disorder Eating disorder, Bulimia, Drug withdrawal syndromes and drug dependence disorders, Atypical facial pain, Chronic pain syndrome, Parkinson's disease, Hypertension, Irritable bowel syndrome (IBS), Jet lag (desynchronosis), and premature ejaculation.
  • IBS Irritable bowel syndrome
  • Jet lag desynchronosis
  • said agent is Zn 2+ or an antibody or other interactive molecule directed to GFD (e.g., a peptoid).
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
  • said agent is a Zn 2+ chelator, ionophore or metal protein attenuating compound as hereinbefore described.
  • said agent is SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof.
  • Conditions characterised by excessive catecholamine levels include, but are not limited to, stress, which can be induced from psychological reactions or environmental stressors such as elevated sound levels, intense light, or low blood sugar levels, catecholamine toxicity as result of central nervous system trauma (e.g., due to stimulation and/or damage of nuclei in the brainstem, in particular those nuclei affecting the sympathetic nervous system), neuroendocrine tumors of the adrenal medulla (e.g., pheochromocytoma), tachycardia, hypertension, conditions associated with increased sympathetic nervous system activity, Schizophrenia, psychoses, gambling addiction and Obsessive Compulsive Disorder (OCD).
  • stress which can be induced from psychological reactions or environmental stressors such as elevated sound levels, intense light, or low blood sugar levels
  • catecholamine toxicity as result of central nervous system trauma (e.g., due to stimulation and/or damage of nuclei in the brainstem, in particular those nuclei affecting the sympathetic nervous system), neuroendocrine tumors
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effecti ve amount of an agent for a time and under conditions sufficient for said agent to inhibit APP-mediated amine oxidase activity.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
  • Conditions characterised by insufficient serotonin levels include, but are not limited to, increased aggressive and angry behaviours, clinical depression, obsessive-compulsive disorder (OCD), migraine, irritable bowel syndrome (IBS), tinnitus, fibromyalgia, bipolar disorder, anxiety disorders, intense religious experiences, depression, anxiety disorders and other affective disorders, eating disorders such as bulimia, anorexia and obesity, phobias, dysthymia, premenstrual syndrome, cognitive disorders, impulse control disorders, attention deficit hyperactivity disorder and drug abuse.
  • the anxiety disorders may include, but are not limited to, general anxiety disorders, panic anxiety, obsessive compulsive disorder, acute stress disorder, post trauma stress disorder or social anxiety disorder.
  • a method for the therapeutic or prophylactic treatment of a condition in a subject comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
  • a conditions characterised by excessive serotonin levels includes, but is not limited to, Serotonin Syndrome.
  • the present invention provides use of an agent which decreases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient serotonin levels.
  • the present invention provides use of an agent which increases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted serotonin levels.
  • the present invention provides use of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof which increases APP- mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted serotonin levels.
  • the present invention provides use of an agent which inhibits GFD-mediated potentiation of APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient serotonin levels.
  • subject as used herein includes humans, primates, livestock animals (e.g., sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), companion animals (e.g., dogs, cats), captive wild animals (e.g., foxes, kangaroos, deer), aves or reptiles.
  • livestock animals e.g., sheep, pigs, cattle, horses, donkeys
  • laboratory test animals e.g., mice, rabbits, rats, guinea pigs
  • companion animals e.g., dogs, cats
  • captive wild animals e.g., foxes, kangaroos, deer
  • aves or reptiles e.g., the mammal is human or a laboratory test animal. Even more preferably, the mammal is a human.
  • an "effective amount” typically means an amount necessary to at least partly attain the desired response; for example, to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition.
  • the amount varies depending upon the health and physical condition of the subject to be treated, the taxonomic group of the subject to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors known to those skilled in the art.
  • the method of the present invention preferably facilitates said condition being reduced, retarded or otherwise inhibited.
  • Reference to "reduced, retarded or otherwise inhibited” should be understood as a reference to inducing or facilitating the partial or complete inhibition of any one or more causes or symptoms of said condition.
  • conditions such as clinical depression are extremely complex comprising numerous physiological events which often occur simultaneously.
  • the present invention contemplates both relieving any one or more symptoms of said condition (for example, improving one or more cognitive function) or facilitating retardation or cessation of the cause of said condition.
  • Administration of an agent of the present invention in the form of a pharmaceutical composition may be performed by any convenient means.
  • the agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the form of modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • the agent may be administered in a convenient manner by any suitable route.
  • Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeal ⁇ , intravenously, intraperitoneally, subcutaneously, intracranial ly, intradermally, intramuscularly, intraoccularly, intrathecal ly, intracereberally, intranasally, infusion,- orally, rectally, via IV drip patch and implant (e.g.. using slow release molecules).
  • the subject undergoing treatment or prophylaxis may be any human or animal in need of therapeutic or prophylactic treatment.
  • treatment and prophylaxis are to be considered in its broadest context.
  • the term “treatment” does not necessarily imply that a mammal is treated to total recovery.
  • prophylaxis does not necessarily mean that the subject will not eventually contract said condition.
  • treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • the term “prophylaxis” may be considered as reducing the severity of the onset of a particular condition. “Treatment” may also reduce the severity of an existing condition.
  • said APP is central nervous system APP.
  • the present invention provides use of an agent which decreases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient catecholamine levels.
  • the present invention provides use of an agent which increases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted catecholamine levels.
  • the present invention provides use of an agent which increases the level of Zn 2+ in the manufacture of a medicament for the treatment of a condition characterised by unwanted APP-mediated amine oxidase activity.
  • the present invention provides use of an agent which antagonises the interaction of APP with Zn 2+ in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP-mediated amine oxidase activity.
  • the present invention provides use of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof which increases APP- mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted catecholamine levels.
  • the present invention provides use of an agent which inhibits GFD-mediated potentiation of APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient catecholamine levels.
  • modulatory agents as herein described, when used in the methods of the present invention.
  • the agent defined in accordance with the present invention may be co-administered with one or more other compounds or molecules.
  • coadministered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours or days between administration of the two types of molecules. These molecules may be administered in any order.
  • the method of the present invention may also be combined with currently known methods for treatment and/or prophylaxis of said conditions.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an agent as hereinbefore described and one or more pharmaceutically acceptable carriers and/or diluents for use in the methods of the present invention, as herein described.
  • Said pharmaceutical composition may additionally comprise molecules with which it is to be co-administered. These agents are typically referred to as the active ingredients.
  • the methods of the present invention are preferably achieved via the intravenous or oral administration of the subject agent, it should be understood that the present invention is not limited to this method of administration and may encompass any other suitable method of administration.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which- contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.
  • the active ingredients When the active ingredients are suitably protected they may be orally administered,, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least I % by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 g and 3000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavouring agent such as peppermint, oil of wintergreen, or
  • any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amounts employed.
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • a method of Screening for a compound that modulates the APP-mediated amine oxidase activity comprising exposing said compound to APP and an APP substrate under conditions that allow for APP- mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said compound is indicative that said compound modulates A PP-mediated amine oxidase acti vity.
  • oxidised APP substrate increases as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound increases APP-mediated amine oxidase activity.
  • level of oxidised APP substrate is decreased as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound decreases APP-mediated amine oxidase activity.
  • a method of screening for a compound that modulates the APP-mediated amine oxidase activity comprising exposing said compound to APP and an APP substrate under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said compound is indicative that said compound modulates APP-mediated amine oxidase activity.
  • the level of oxidised APP substrate increases as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound increases APP- mediated amine oxidase activity.
  • the level of oxidised APP substrate is decreased as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound decreases APP-mediated amine oxidase activity.
  • a compound is screened for its ability to modulate APP- mediated amine oxidase activity whilst having a negligible effect on ceruloplasmin amine oxidase activity. Ceruloplasmin activity is typically dependent on copper and inhibited by NaN 3 , whereas neither NaN 3 nor Cu 2+ alter APP-mediated amine oxidase activity.
  • a compound may be screened for ceruloplasmin amine oxidase activity by exposing said compound to APP, an APP substrate and NaN 3 under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of NaN 3 is indicative that said compound modulates ceruloplasmin amine oxidase activity.
  • catecholamine and/or serotonin levels in vivo This could provide a number of advantages. For example, where it is desirable to increase catecholamine and/or serotonin levels in vivo, a compound that is capable of selectively inhibiting APP-mediated amine oxidase activity is likely to provide tighter control and thus avoid an excessive increase in said catecholamine and/or serotonin levels that would otherwise lead to undesirable outcomes.
  • APP substrates for use in the screening methods of the present in vention, as herein described (also referred to herein as “APP substrates”) include, but are not limited to:
  • Test agents can be administered to the reaction system at a single concentration or, alternatively, at a range of concentrations from about 1 nM to 1 tnM.
  • the AAP substrate is o-diamsidine dihydrochloride.
  • Diversity libraries such as random combinatorial peptide or nonpeptide libraries can be screened.
  • Many publicalty or commercially available libraries can be used such as chemically synthesized libraries, recombinant (e.g., phage display libraries) and in vitro translation-based libraries.
  • phage display libraries are described by Scott and Smith., (1990); Devlin et al, (1990); Christian R.B et al, (1992); Lenstra,, (1992); Kay et al, ( 1993) and .
  • test compound can be a macromolecule, such as biological polymer, including polypeptides or polysaccharides.
  • Compounds useful as potential therapeutic agents can be generated by methods well known to those skilled in the art, for example, well known methods for producing pluralities of compounds, including chemical or biological molecules such as simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, antibodies, and the like, are well known in the art and are described, for example, in Huse, U.S. Patent No. 5,264,563; Francis et ah, Curr. Opin. Chem. Biol., 2:422-428 ( 1998); Tietze et al, Curr. Biol , 2:363-381 (1 98); Sofia, Molecule.
  • test compound can be preselected based on a variety of criteria. For example, suitable test compounds having known modulating activity on a pathway suspected to be involved in APP amine oxidase activity can be selected for testing in the screening methods. Alternatively, the test compounds can be selected randomly and tested by the screening methods of the present invention. Test compounds can be administered to the reaction system at a single concentration or, alternatively, at a range of concentrations from about 1 nM to 1 mM.
  • test compounds examined using the methods of the invention will depend on the application of the method. It is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay.
  • the methods can be performed in a single or multiple sample format. Large numbers of compounds can be processed in a high-throughput format which can be automated or semi-automated.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (see, e.g., Gallop et al. ( 1994) 37(9): 1233- 1250).
  • Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art, see, e.g., U.S. Patent No.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175; Furka (1991 ) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991 ) Nature, 354: 84-88).
  • chemistries for generating chemical diversity libraries include, but are not limited to: peptoids (see, e.g., WO 91/1 735), encoded peptides (see, e.g., WO 93/20242), random bio-oligomers (see, e.g., WO 92/00091 ), benzodiazepines (see, e.g., U.S. Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (see, e.g., Hobbs (1993) Proc. Nat. Acad. Set.
  • peptoids see, e.g., WO 91/1 735
  • encoded peptides see, e.g., WO 93/20242
  • random bio-oligomers see, e.g., WO 92/00091
  • benzodiazepines see, e.g., U.S. Patent No. 5,
  • polypeptides of the invention such as GFD and the E2 domain of APP
  • potential modulators e.g., inhibitors or activators
  • Such peptides and polypeptides may be bound to a solid support.
  • Solid supports can include, e.g., membranes (e.g., nitrocellulose or nylon), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dip stick (e.g., glass, PVC, polypropylene, polystyrene, latex and the like), a microfuge tube, or a glass, silica, plastic, metallic or polymer bead or other substrate such as paper.
  • a metal e.g., cobalt or nickel
  • Adhesion of peptides to a solid support can be direct (i.e., the protein contacts the solid support) or indirect (a particular compound or compounds are bound to the support and the target protein binds to this compound rather than the solid support).
  • Peptides can be immobilized either covalently (e.g., utilizing single reactive thiol groups of cysteine residues (see, e.g., Colliuod (1993) Bioconj gate Chem. 4:528-536) or non-covalently but specifically (e.g., via immobilized antibodies (see, e.g., Schuhmann (1991) Adv. Mater. 3 :388-391 ; Lu (1995) Anal. Chem.
  • Indirect binding can be achieved using a variety of linkers which are commercially available.
  • the reactive ends can be any of a variety of functionalities including, but not limited to: amino reacting ends such as N-hydroxysuccinimide (NHS) active esters, imidoesters, aldehydes, epoxides, sulfonyl halides, isocyanate, isothiocyanate, and nitroaryl halides; and thiol reacting ends such as pyridyl disulfides, maleimides, thiophthalimides, and active halogens.
  • NHS N-hydroxysuccinimide
  • thiol reacting ends such as pyridyl disulfides, maleimides, thiophthalimides, and active halogens.
  • the heterobifunctional crosslinking reagents have two different reactive ends, e.g., an amino-reactive end and a thiol-reactive end, while homobitunctional reagents have two similar reactive ends, e.g., bismaleimidohexane (BMH) which permits the cross-linking of sulfhydryl-containing compounds.
  • the spacer can be of varying length and be aliphatic or aromatic.
  • Examples of commercially available homobifunctional cross-linking reagents include, but are not limited to, the imidoesters such as dimethyl adipimidate dihydrochloride (DMA); dimethyl pimelimidate dihydrochloride (DMP); and dimethyl suberimidate dihydrochloride (DMS).
  • DMA dimethyl adipimidate dihydrochloride
  • DMP dimethyl pimelimidate dihydrochloride
  • DMS dimethyl suberimidate dihydrochloride
  • Heterobifunctional reagents include commercially available active halogen-NHS active esters coupling agents such as N-succinimidyl bromoacetate and N- succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) and the sulfosuccinimidyl derivatives such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) (Pierce).
  • active halogen-NHS active esters coupling agents such as N-succinimidyl bromoacetate and N- succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) and the sulfosuccinimidyl derivatives such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) (Pierce).
  • Another group of coupling agents is the heterobifunctional and thiol cleavable agents such as N-succinimidyl 3- (2-pyridyidithio)propionate (SPDP) (Pierce Chemicals, Rockford, IL).
  • SPDP N-succinimidyl 3- (2-pyridyidithio)propionate
  • Antibodies can be used for binding polypeptides and peptides to a solid support. This can be done directly by binding peptide-specific antibodies to the column or it can be done by creating fusion protein chimeras comprising motif-containing peptides linked to, e.g., a known epitope (e.g., a tag (e.g., FLAG, myc) or an appropriate immunoglobulin constant domain sequence (an "immunoadhesin,” see, e.g., Capon (1989) Nature 337:525-531 (1989).
  • a known epitope e.g., a tag (e.g., FLAG, myc)
  • an appropriate immunoglobulin constant domain sequence an "immunoadhesin," see, e.g., Capon (1989) Nature 337:525-531 (1989).
  • the present invention is directed to the use of antibodies to GFD to antagonise its activity.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies or may be specifically raised.
  • antibody includes a peptide or polypeptide derived from, modelled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush ( 1992) J. Biochem. Biophys. Methods 25:85-97.
  • the term antibody includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including
  • Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • a F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;
  • a Fd fragment consisting of the VH and CH I domains;
  • a Fv fragment consisting of the VL and V H domains of a single arm of an antibody,
  • a dAb fragment Ward et al, (1989) Nature 341 :544-546, which consists of a VH domain; and
  • CDR complementarity determining region
  • Antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies or may be specifically raised to these polypeptide and gene products.
  • the present invention extends to recombinant and synthetic antibodies and to antibody hybrids.
  • a “synthetic antibody” is considered herein to include fragments and hybrids of antibodies.
  • the agent may be an antibody that specifically binds to Zn 2+ and thereby inhibits the interaction of Zn 2+ with APP.
  • the agent is an antibody that specifically binds to the GFD of APP-E1 , (as herein described) to facilitate inhibition of APP-mediated amine oxidase activity.
  • said method comprises exposing said compound to APP and an APP substrate in the presence of Zn 2+ .
  • said method comprises exposing said compound to APP and an APP substrate in the presence of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof.
  • a method of screening a biological sample for APP-mediated amine oxidase activity comprising exposing a biological sample that putatively comprises APP or a functional derivative thereof to an APP substrate ⁇ e.g., as herein described) under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said sample is indicative that said sample comprises APP or a functional derivative thereof.
  • a biological sample in accordance with this aspect of the present invention may include, but is not limited to, whole blood, plasma, urine and biological tissue.
  • the biological sample is plasma.
  • E2-APP E2 domain of APP
  • a three-dimensional (3D) prediction of the APP-E2 domain is provided in Figure 8 and it is within the skill of the person in the art to design, for example, in silico, agents which would appropriately interact.
  • the crystal structure of the E2 domain (containing the amine oxidase site but also other domains) has also been described elsewhere (see, e.g., Wang et al. (2004) Molecular Cell, Vol. 15:343-353).
  • mice All mouse studies were performed with the approval of the IACUC and in accordance with statutory regulations.
  • APP knockout mice Zheng, H. et ai, 1995
  • background controls C57BL6/SV129
  • Plasma was separated immediately from red blood cells when required by centrifugation at 4,000 x g for l Omin. Tissues was then taken and either used immediately or stored at -80°C until required.
  • the synthetic peptide FD1 was synthesized using solid-state Fmoc chemistry, in a microwave synthesizer, using Fmoc -PAL-PEG-PS as resin from Applied Biosystems. All the amino acids were coupled to the resin using a 3-fold excess where 0.5 M 2-(lH-Benzotriazole- l -yl)-l , l ,3,3-tetramethyluronium hexafluoro-phosphate and 0.5 of N,N- diisopropylethylamine were used as activators.
  • DMF dimethylformamide
  • the acetylation step was performance for 30 min at room temperature. Resin was then washed with DMF and methanol, and freeze-dried.
  • the peptide was cleaved from the resin by stirring in a solution of 1% water, 0.5%
  • dihydrochloride substrate was added (1.576 mM final concentration), mixed and then incubated for 5 and 60 minutes. 9M sulfuric acid was used to stop the reaction at these times. Oxidized product was monitored by absorbance at 540nm from where the velocity was plotted. When not specified, 500nM of pure protein or 5 ⁇ 1 plasma was used to measure oxidase activity. Sodium azide (10 mM) abolished the activity of pure ceruloplasmin and zinc (10 ⁇ ) abolished the activity of pure APP695a in the assay.
  • Tissue or plasma was homogenized by pulse sonication in 0.4M perchloric acid, 0.15% Sodium metabisulfite and 0.05% ethylenediaminetetraacetic acid disodium salt (EDTA) before centrifugation at 13,000 rpm at 4°C for 10 minutes.
  • NE, E and DA were measured in supernatants using an HPLC system (ESA Biosciences; model 584) coupled to an HPLC system (ESA Biosciences; model 584) coupled to an
  • electrochemical detector ESA Biosciences; Coulochem III detector
  • El El :-l 50mV
  • a MD-150 reverse phase C18 column ESA Biosciences
  • elution was performed at a flow rate of 0.6ml/min in mobile phase (75mM sodium dihydrogen phosphate, 1.7mM 1-octanesulfonic Acid Sodium Salt, l OOmL/L triethylamine, 25mM EDTA, 10% acetonitrile, pH 3). Peaks were identified by retention times set to known standards. Data were normalized for total wet weight of sample.
  • Systolic blood pressure and pulse rate were measured using tail cuff plethysmography and a photoelectric sensor, respecti vely. Briefly, the tail-cuff method required mild restraint of the mouse in a dark chamber on a heated platform with their tails threaded through a tail cuff. Following a period of training (3 days), readings of blood pressure from the tail were then taken by brief cuff inflation (-5-10 seconds). This procedure was repeated 2-3 times for each mouse.
  • a 96-well microplate was coated with ⁇ of capture antibody and incubated overnight at room temperature. Plates were washed and incubated with blocking buffer for I h before washing again and then adding 1 ⁇ of detection antibody for a further 1 h. Plates were again washed before ⁇ ⁇ of streptavidin-HRP was added for 20 minutes. After a final wash, detection of prolactin from standards and plasma (50 ⁇ 1 of each in triplicate) was carried out by adding samples to wells and incubating for 20 minutes. Stop reaction was stopped with 50 ⁇ of stop solution. Optical density of each well was measured at 450nm.
  • NE, E and DA in tissue or plasma homogenized supernatants were injected onto a MD-150 reverse phase CI 8 column (ESA Biosciences) using a HPLC system (ESA Biosciences; model 584) coupled to an electrochemical detector (ESA Biosciences;
  • Coulochem I I detector (El :- 150mV, E2:+220mV, and guard cell: +250mV). Peaks were identified by retention times set to known standards in mobile phase (75mM Sodium dihydrogen phosphate, l .7mM 1 -octanesulfonic Acid Sodium Salt, lOOmL/L triethylamine, 25mM EDTA, 10% acetonitrile, pH 3) at a flow rate of 0.6ml/min.
  • stolic blood pressure and heart rate were determined non-invasively by tail-cuff plethysmography (Hatteras Instruments, MC4000 Blood Pressure Analysis System).
  • APP695a exhibits near-maximal o-dianisidine oxidase activity through a pH range of 4.5 - 7.5, with maxima at pH 5.0 and 7.0 (Fig. 4), and is distinctly different from its profile for ferroxidase activity (Duce, J.A. et al., 2010), indicating that the amine oxidase active site on APP may not be identical to the ferroxidase domain.
  • ferroxidase domain peptide alone, which retains ferroxidase activity (Duce JA. et al., 2010), did not oxidize o-dianisidine (FD l , Fig. l e).
  • ferroxidase and amine oxidase activities of CP are also overlapping but not identical (Bielli P. and Calabrese L., 2002).
  • As-isolated soluble APPa isoforms 695, 751 and 770 had similar oxidase activity (Fig. 5), however, the APP-E2 domain had 60% the activity of APP695ot (Fig. le), proportional to its ferroxidase activity (Duce JA. et ai, 2010). While purified APP-El domain (Fig. I d & e) possessed no oxidase activity, equimolar concentrations of APP-El doubled APP-E2 activity (Fig. I f) to that of APP695a (Fig. le). We mapped this potentiating effect to the growth factor domain (GFD) (Fig. Id & e) within APP-El which has a similar potentiating effect on the ferroxidase activity of APP-E2 (Duce JA. et al., 2010).
  • GFD growth factor domain
  • amine oxidase activity of APP-E2 was diminished compared to APP695a, it similarly was inhibited by Zn 2+ (Fig. 2a) but not by azide or physiological concentrations of other divalent metal ions; e.g. 20 ⁇ Cu 2+ (data not shown).
  • APLP2 did not possess amine oxidase activity (Fig. 6).
  • the selective inhibitors to monoamine oxidases A (clorgyline) and B (selegiline) had no effect on either APP695a or CP (Fig. 6).
  • O-dianisidine oxidation is used in clinical pathology as an assay for plasma CP activity (Lehmann HP. et al., 1974). We mapped the components that oxidize this amine substrate by assaying plasmas from CP knockout (CP-/-), APP knockout (APP-/-) as well as age- and gender-matched background control mice. Both azide and genetic ablation of CP induced a -60% drop in plasma amine oxidase activity (Fig. 2b and Fig. 7) and the remaining oxidase activity in the CP-/- plasma could not be inhibited by azide (Fig. 2b).
  • NE, E and DA were found to competitively inhibit the oxidation of o-dianisidine by APP in a dose dependent manner (Fig. 2d). Each catecholamine completely inhibited o- dianisidine oxidation at 1 : 1 stoichiometry (Fig. 2d). APP catalytically oxidized NE and DA as shown by a fluorometric assay of exposed primary amine groups that precluded the assay of epinephrine (Fig. 2e). Both catecholamines were oxidized by APP695q with similar K m values and comparable to previous reports with CP (Richards DA., 1983).
  • APP-/- mouse tissues relevant to the sympathetic nervous system had relatively marked and significantly higher concentrations of NE (Fig. 3a), E (Fig. 3b) and DA (Fig. 3c) than age-matched background controls (concentrations are reported in Table 3).
  • APP-/- mice exhibited increased heart rate (Fig. 3d) and systolic blood pressure (Fig. 3e) consistent with the increased NE and E (Kvetnansky R. et al., 2009) present in the heart and plasma (Fig. 3a & b). Similar effects have been observed with the inhibition (Anderson MC. et al., 1993) or genetic ablation (Desir GV. 2009) of other catecholamine oxidases. [0215J We also tested for the impact of elevated brain DA levels on the tuberoinfundibular- dopaminergic system by assaying prolactin, a pituitary hormone whose release is suppressed by the presence of DA.
  • prolactin levels were significantly decreased in APP-/- mice (Fig. 3 ' f), consistent with increased brain stem DA in these mice (Fig. 3c).
  • Prolactin has multiple effects upon growth, metabolism and immunomodulation, as well as reproduction, for which it is best characterized. Decreased prolactin could explain the 15-20% reduction in adult body weight seen in APP -/- mice (Zheng H. et al., 1995), since prolactin suppression results in decreased food intake and body weight (Gerardo-Gettens T., et al. 1989). Plasma glucose is also decreased in APP-/- mice ( eedham BE. et al , 2008).
  • prolactin suppression also elevates adipokines ⁇ e.g. adiponectin) causing decreased plasma glucose (Shibli-Rahhal A. and Schlechte J., 2009).
  • Prolactin, acting as a cytokine also promotes the proliferation and survival of lymphocytes (Clevenger CV. et al,, 1991), while elevated catecholamines impair lymphocyte survival (Kvetnansky R. et al.. 2009). Accordingly, the lymphocyte count of APP- /- mice was significantly decreased compared to wild-type (Fig. 3g). ⁇
  • APP Within tissue, the active site of full-length APP is on the extracellular surface of the plasma membrane (Fig. Id). Unlike CP (Duce JA. et al., 2010), APP is expressed in all neurons, at synaptic junctions (Priller C. et al, 2006; Storey E. et al, 1996), which may make it available for controlling catecholamine concentrations in the vicinity, modulated by extracellular Zn 2+ concentrations transferring from nearby synapses (Sensi SL. et al, 2009). The K m of APP is suitable for the spillover of concentrations of catecholamines that are not cleared by uptake transporters.
  • APP may be a significant factor in the clearance of catecholamine. Indeed, the degree of
  • catecholaminergic accumulation in APP-/- mice is similar to amine oxidase deficient mice (e.g. monoamine oxidase-A; see Cases O. et al., 1995).
  • the modulation of catecholamine levels in the brain and sympathetic target organs is an important end-effect for several major classes of psychotropic and cardiovascular drugs.
  • the discovery that APP has an adjustable activity that influences catecholamine levels at these sites has important implications for clinical pharmacology.
  • Hephaestin is a ferroxidase that maintains partial activity in sex-linked anemia mice. Blood 103 (10), 3933-3939 (2004).
  • An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol 32 (2), 415-438 ( 1967).
  • Spurck, T., Pickett-Heaps, J., Beyreuther, K., & Masters, C.L. The amyloid precursor protein of Alzheimer's disease is found on the surface of static but not activity motile portions of neurites. Brain Res 735 (1), 59-66 (1996).
  • Chromaffin cells express Alzheimer amyloid precursor protein in the same manner as brain cells. Neurosci Lett 168 ( 1 -2), 57-60 ( 1994).

Abstract

The present invention relates generally to a method of treating or preventing conditions characterised by aberrant amine oxidase activity and agents useful for same. More particularly, the present invention relates to a method of treating or preventing conditions characterised by aberrant amine oxidase activity by modulating the activity of amyloid precursor protein. The method of the present invention is useful, inter alia, in the treatment and/or prophylaxis of conditions including, but not limited to, clinical depression.

Description

METHOD OF MODULATING AMINE OXIDASE ACTIVITY AND AGENTS
USEFUL FOR SAME
FIELD OF THE INVENTION
[0001] The present invention relates generally to a method of treating conditions characterised by aberrant amine oxidase activity and agents useful for same. More particularly, the present invention relates to a method of treating conditions characterised by aberrant amine oxidase activity by modulating the activity of amyloid precursor protein. The method of the present invention is useful, inter alia, in the treatment and/or prophylaxis of conditions including, but not limited to, clinical depression.
BACKGROUND OF THE INVENTION
[0002] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
[0003] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an
acknowledgment or admission or any form of suggestion that that prior publication (or . information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
[0004] As neurotransmitters and hormones, catecholamines mediate alertness and stress responses, increasing heart rate, blood pressure, gluconeogenesis, as well as the release of free fatty acids and the secretion of various hormones (Kvetnansky R. et al., 2009). Sustained elevation of catecholamines has been shown to suppress the proliferation and survival of lymphocytes directly (Bergquist J. et al., 1998) and by lowering prolactin levels (Clevenger CV. et al., 1991). In the human body, the most abundant catecholamines are epinephrine (adrenaline), norepinephrine (noradrenaline) and dopamine, all of which are produced from phenylalanine and tyrosine.
[0005] Through vesicular release, the catecholamines dopamine (DA) and norepinephrine (NE) are neurotransmitters, while NE and epinephrine (E) are released into blood by the adrenal medulla (Kobayashi S and Coupland RE., 1993). Endogenous catecholamines are also produced by peripheral organs such as the heart (Diarra A. et al., 1989) and immunocompetent cells (Bergquist J. et al., 1998). [0006] Catecholaminergic inactivation occurs by diffusion, transporter uptake and enzymatic breakdown (Galli A. et al., 1998; Ford CP et al., 2010). Following uptake, catecholamines are transported into vesicles or degraded by multiple intracellular pathways including methylation (e.g. catechol-O-methyltransferase) and amine oxidation (Kvetnansky R. et al., 2009).
Mechanisms around the synapse clear 80-90% of the neurotransmitter, but spillover of excess catecholamines may act on targets at a distance and enter plasma (Esler M. et al., 1990).
Perisynaptic catecholamine concentrations of 30-400 μΜ are estimated (Galli A. et al., 1998; Esler M. et al., 1990) but drop to nM levels in plasma (Esler M. et al., 1990) due to dilution and possible extracellular degradation either by methylation through catechol-O- methyltransferases (COMT) or by deamination through amine oxidases.
[0007] High catecholamine levels in blood is typically associated with stress, which can be induced from psychological reactions or environmental stressors such as elevated sound levels, intense light, or low blood sugar levels. Extremely high levels of catecholamines (also known as catecholamine toxicity) can also occur in central nervous system trauma due to stimulation and/or damage of nuclei in the brainstem, in particular those nuclei affecting the sympathetic nervous system. Extremely high levels of catecholamine can also be caused by
neuroendocrine tumors in the adrenal medulla, a treatable condition known as
pheochromocytoma. Catecholamines also cause general physiological changes, including increased heart rate, blood pressure, alertness and blood glucose levels, opening of airways and a general increase in sympathetic nervous system activity.
[0008] Catecholamine levels also tend to peak early in the day, which may contribute to the increased risk of heart attacks and strokes during the morning hours.
[0009] Reduced levels of catecholamines are typically associated with clinical depression. Hence, a major effort of the last decade in the treatment of depression (and other disorders associated with reduced levels of catecholamines) has been to modulate aberrant
catecholamine levels. For example, one of the first effective antidepressant drugs were the amine oxidase inhibitors (e.g., Nardil®, Parnate®) and tricyclic antidepressants (e.g., Elavil®, Tofranil® and Pamelor®). The therapeutic effect of these drugs appears to be based largely on their ability to increase catecholamine levels in the brain by blocking the normal metabolic degradation or reuptake of catecholamine molecules that are released into the synapse.
[0010] Other regimes for the treatment of clinical depression have included taking supplements containing 1-phenylalanine and/or 1-tyrosine, the metabolic precursors for norepinephrine and dopamine. Examples include the natural supplement Hypericum perforatum, better known as St. John's wort, which has been used as an antidepressant nutrient in the treatment of depression, anxiety, apathy and insomnia.
[0011] Vitamins B6, B 12 and folic acid have also been proposed as useful compounds for the treatment of clinical depression. Vitamin B6 levels have been found to be low in patients diagnosed with clinical depression. Moreover, studies show that supplementing vitamin B6 in people with affective disorders is associated with increased levels of NE and serotonin and relief of depression. Folic acid and vitamin B 12 are necessary for the synthesis of a substance called S-adenosylmethionine, which is vital for neurotransmitter metabolism. A deficiency of either folic acid or vitamin B 12 may cause similar neurologic and psychiatric disturbances, including depression, dementia, and demyelinating myopathy. A folate deficiency, in particular, may specifically affect the metabolism of catecholamines and indoleamines and aggravate depressive disorders. Folic acid and vitamins B6 and B12 are also required for the conversion of homocysteine to methionine. This prevents the accumulation of homocysteine, which has been linked to the formation of atherosclerotic plaque in arteries as well as to neurotoxic effects that can produce neurologic and psychiatric disturbances.
[0012] In addition to catecholamines, serotonin (5-HT), a monoamine neurotransmitter, also plays a very important role in the regulation of aggression, mood, body temperature, sleep, vomiting, sexual drive and appetite. The role of serotonin in these clinical settings may be regulated, at least in part, by agonistic or antagonistic action on serotonin receptors. The concentration of synaptic serotonin can also be controlled directly by its reuptake into the presynaptic terminal and, thus, drugs blocking serotonin transport have been used with some success for the treatment of depression.
[0013] Low levels or low bioavailability of serotonin are associated with several disorders such as increased aggressive and angry behaviours, clinical depression, obsessive-compulsive disorder (OCD), migraine, irritable bowel syndrome (IBS), tinnitus, fibromyalgia (FM or FMS), bipolar disorder, anxiety disorders and intense religious experiences. In addition, abnormal serotonergic neurons have been associated with the risk of sudden infant death syndrome (SIDS). When taken orally, serotonin does not pass into the serotonergic pathways of the central nervous system (CNS) because it cannot cross the blood-brain barrier. [0014] In addition to its CNS activities, serotonin is involved in several peripheral activities; including cardiovascular modulating effects (both vasoconstrictor and vasodilator), potent pro- thrombotic activity, endothelial mitogenic action, as well as immune modulating effects.
[0015] One method for modulating peripheral serotonin levels is the chronic use of serotonin-selective reuptake inhibitor drugs (SSRIs) such as alaproclate, dapoxetine, etoperidone, citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline and zimelidine, which are routinely used for treating CNS conditions such as clinical depression, obsessive-compulsive disorder, and additional mood disorders and are hence known to belong to the class of antidepressant drugs.
[0016] Serotonin-selective reuptake inhibitors (SSRIs), such as fluoxetine (Prozac™), have traditionally been the mainstay of treatment for clinical depression, replacing the more toxic tricyclic antidepressants (TCAs). SSRIs have a more favourable adverse reaction profile in comparison to the TCAs and are typically much easier to tolerate. SSRIs exert their therapeutic effect by blocking the reuptake of serotonin into the presynaptic nerve terminal, thus increasing the synaptic concentration of serotonin. SSRIs may also increase the efficacy of the serotonin neurons by desensitizing 5-HT autoreceptors located on the presynaptic serotonin nerve terminals. The ability of the 5-HT autoreceptors to inhibit the release of serotonin decreases after long-term treatment with SSRIs, with the net effect being that a greater amount of serotonin is released per impulse. As a consequence, the release of serotonin from these neurons becomes more efficient. Research in the treatment of depression remains focused on serotonin because a good number of selective serotonin reuptake blockers tested in the clinical setting have been effective in treating major depression. The general mechanism of action is hypothesized as enhanced serotonin neurotransmission due to the increased availability of serotonin in the synapse of these neurons. Moreover, SSRIs generally have a 50- to 100-fold or greater selectivity for the inhibition of serotonin uptake in vitro when compared to their ability to inhibit NE or DA uptake.
[0017] Unfortunately, however, SSRIs are not innocuous drugs and in spite of the progress in the treatment of depression with the advent of the SSRIs, many adverse side effects still remain; most notably, sexual dysfunction.
[0018] Despite existing regimes for the treatment of clinical depression and other conditions associated with aberrant catecholamine and/or serotonin levels, there is a need for the development of new therapeutic and prophylactic approaches to such conditions. Current treatments are, at best, marginally effective and with the ever increasing incidence of clinical depression, the need to develop better methods of treatment and prophylaxis is urgent.
[0019] In work leading up to the present invention, it has been determined that amyloid precursor protein (APP) is a functional amine oxidase which simultaneously oxidizes amines and catecholamines catalytically. This activity is inhibited by Zn +. Since reduced levels of catecholamines and amines such as serotonin are linked to conditions such as clinical depression, the determination that Zn2+ inhibits APP amine oxidase activity has now provided the basis for the development of a new treatment approach. These findings provide the basis for the development of a new treatment approach for conditions characterised by aberrant amine oxidase activity, such as, but not limited to, clinical depression.
SUMMARY OF THE INVENTION
[0020] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and
"comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
[0021] As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of "a", "and" and "the" include plural referents unless the context clearly dictates otherwise.
[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0023] The subject specification contains amino acid sequence information prepared using the programme Patentin Version 3.5, presented herein after the bibliography. Each amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, etc). The length, type of sequence (protein, etc) and soufce organism for each sequence is indicated by information provided in the numeric indicator fields <21 1>, <212> and <213>, respectively. Amino acid sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (e.g. SEQ ID NO: l , SEQ ID NO:2, etc.). The sequence identifier referred to in the Specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (e.g. <400>1, <400>2, etc). That is SEQ ID NO.l as detailed in the specification correlates to the sequence indicated as <400>1 in the sequence listing.
[0024] A summary of the sequence identifiers disclosed in the subject specification is provided in Table 1.
TABLE 1
Figure imgf000007_0001
[0025] One aspect of the present invention is directed to a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate the functional interactivity of Zn2+ with said APP wherein antagonising the interaction of Zn2+ with said APP increases APP amine oxidase activity and facilitating the interaction of Zn2+ with said APP decreases APP amine oxidase activity.
[0026] In another aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate GFD potentiation of APP amine oxidase activity wherein facilitating the interaction of GFD with APP increases APP amine oxidase activity and antagonising the GFD interaction with APP decreases APP amine oxidase activity.
[0027] In yet another aspect there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to antagonise the interaction of Zn2+ with said APP.
[0028] In one embodiment, said antagonist is a zinc chelator, ionophore or metal protein attenuating compound. [0029] In another embodiment, the Zn chelator is a moderate affinity chelator which is hydrophobic. Examples include the 8-hydroxy quinolines, such as clioquinol, PBT2, M30, VK28 or related molecules, pyrithione, diethyl pyrocarbamate, l,2-bis-(2-(amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid and derivatives, the bicyclam analogue JKL169 (1 ,1 '-xylyl bis- 1 ,4,8,1 1 tetraaza cyclotetradecane), DP 109 and related compounds.
[0030] In still another embodiment, APP-mediated amine oxidase activity can be reduced by administering Zn2+ to said subject.
[0031] In a further aspect of the present invention, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient APP-mediated amine oxidase activity, said method comprising administering to said subject an effective amount of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof for a time and under conditions sufficient to potentiate APP-mediated amine oxidase activity.
[0032] SEQ ID NO:2 represents the amino acid sequence of the growth factor domain (GFD) of APP. By administering a composition comprising this sequence, or a functional fragment, mimetic, analogue or homologue thereof, it has been found that APP-mediated amine oxidase can be potentiated.
[0033] In still another aspect there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by unwanted APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent which antagonises the interaction of GFD with APP.
[0034] In another aspect of the present invention, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient catecholamine levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to inhibit APP-mediated amine oxidase activity.
[0035] In another aspect of the present invention, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient serotonin levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to inhibit APP-mediated amine oxidase activity. [0036] In a further aspect there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient catecholamine levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
(i) increase the level of Zn2+; or
(ii) antagonise the interaction of GFD with APP.
[0037] In a further aspect there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient serotonin levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
(i) increase the level of Zn2+; or
(ii) antagonise the interaction of GFD with APP.
[0038] Conditions characterised by insufficient catecholamine levels include, but are not limited to. Major depression. Minor depression. Atypical depression. Dysthvmia, Melancholia, Anergic depression. Treatment-resistant depression, Headache, Extrapyramidal disorders. Generalised anxiety disorder. Lichen simplex chronicus. Insomnia, Panic disorder, Stress disorder, Posttraumatic stress disorder (PTSD), Attention deficit disorder, Hyperactivity, Conduct disorder, Narcolepsy, Social phobia and anxiety, Obsessive-compulsive disorder. Eating disorder, Bulimia, Drug withdrawal syndromes and drug dependence disorders, Atypical facial pain, Chronic pain syndrome, Parkinson's disease, Hypertension, Irritable bowel syndrome (IBS), Jet lag (desynchronosis) and Premature ejaculation.
[0039] Conditions characterised by insufficient serotonin levels include, but are not limited to, increased aggressive and angry behaviours, clinical depression, obsessive-compulsive disorder (OCD), migraine, irritable bowel syndrome (IBS), tinnitus, fibromyalgia, bipolar disorder, anxiety disorders, intense religious experiences, depression, anxiety disorders and other affective disorders, eating disorders such as bulimia, anorexia and obesity, phobias, dysthymia, premenstrual syndrome, cognitive disorders, impulse control disorders, attention deficit hyperactivity disorder and drug abuse. The anxiety disorders may include, but are not limited to, general anxiety disorders, panic anxiety, obsessive compulsive disorder, acute stress disorder, post trauma stress disorder or social anxiety disorder.
[0040] In yet another aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by excessive catecholamine levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
(i) antagonise the interaction of Zn2+ with APP; or
(ii) facilitate the interaction of GFD with APP. ;
[0041] In yet another aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by excessive serotonin levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
(i) antagonise the interaction of Zn2+ with APP; or
(ii) facilitate the interaction of GFD with APP.
[0042] A condition characterised by excessive serotonin levels includes, but is not limited to, Serotonin Syndrome.
[0043] In one embodiment, said agent is a Zn2+ chelator, ionophore or metal protein attenuating compound as hereinbefore described.
[0044] In yet another embodiment, said agent is SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof.
[0045] In still yet another aspect, the present invention provides use of an agent which decreases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient catecholamine levels.
[0046] In yet still another aspect, the present invention provides use of an agent which increases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted catecholamine levels.
[0047] In still yet another aspect, the present invention provides use of an agent which increases the level of Zn2+ in the manufacture of a medicament for the treatment of a condition characterised by unwanted APP-mediated amine oxidase activity.
[0048] In yet another aspect, the present invention provides use of an agent which antagonises the interaction of APP with Zn2+ in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP-mediated amine oxidase activity.
[0049] In a further aspect, the present invention provides use of a compound of SEQ ID
NO:2 or functional fragment, mimetic, analogue or homologue thereof which increases APP- mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted catecholamine levels. [0050] In yet another aspect, the present invention provides use of an agent which inhibits GFD-mediated potentiation of APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient catecholamine levels.
[0051] In still yet another aspect, the present invention provides use of an agent which decreases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient serotonin levels.
[0052] In yet still another aspect, the present invention provides use of an agent which increases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted serotonin levels.
[0053] In a further aspect, the present invention provides use of a compound of S EQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof which increases APP- mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted serotonin levels.
[0054] In yet another aspect, the present invention provides use of an agent which inhibits GFD-mediated potentiation of APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient serotonin levels.
[0055] In still yet another aspect of the present invention, there is provided modulatory agents, as herein described, when used in the methods of the present invention.
[0056] In a further aspect, the present invention provides a pharmaceutical composition comprising an agent as hereinbefore described and one or more pharmaceutically acceptable carriers and/or diluents for use in the methods of the present invention, as herein described. Said pharmaceutical composition may additionally comprise molecules with which it is to be co-administered.
[0057] In still another aspect of the present invention, there is provided a method of screening for a compound that modulates the APP-mediated amine oxidase activity, said method comprising exposing said compound to APP and an APP substrate under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said compound is indicative that said compound modulates APP-mediated amine oxidase activity. For example, where the level of oxidised APP substrate increases as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound increases APP- mediated amine oxidase activity. Conversely, where the level of oxidised APP substrate is decreased as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound decreases APP-mediated amine oxidase activity.
[0058] In still another aspect of the present invention, there is provided a method of screening a biological sample for APP-mediated amine oxidase activity, said method comprising exposing a biological sample that putatively comprises APP or a functional derivative thereof to an APP substrate under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said sample is indicative that said sample comprises APP or a functional deri vative thereof. For example, where the level of oxidised APP substrate increases as compared to the level of oxidized APP substrate in the absence of said sample, this would be indicative that said sample comprises APP or a functional derivative thereof. A functional derivative of APP includes, but is not limited to, soluble APP (sAPP).
[0059J Appropriate substrates for use in the screening method of the present invention (also referred to herein as "APP substrates") include, but are not limited to:
• Benzidine
• N,N-dimethyl-p-phenylenediamine
• p-phenylenediamine (PPD)
• o-dianisidine dihydrochloride
• Histamine
• Tyramine
• Tryptamine
• Phenethylamine (PEA)
• Serotonin
• Norepinephrine (NE; noradrenaline)
• Epinephrine (E, adrenaline) and
• Dopamine (D).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Cp-Iike amine oxidase activity of APP695a. Oxidation rates catalysed by Cp compared to APP695a at optimal pH, as a product of initial o-dianisidine (a) and p- phenylenediamine (b) concentration. Proteins (500 nM) were incubated in (a) 75 mM Na acetate, pH 5.0, or 50 mM HEPES, pH 7.2, or (b) 75 mM Na acetate, pH 5.5 at 37 °C. (c) Kinetic values of Cp and APP695a as calculated using Michaelis-Menten equations, are comparable with each substrate. Bovine albumin was inactive in this assay (not shown), (d) Schematic of the positions of the ferroxidase domain (FD1 )5 within the E2 -domain of APP (APP-E2), and the growth factor domain (GFD) within the El -domain of APP (APP-E1 ) in relation to other recognized domains of N-terminal APP including the copper binding domain (CuBD) and the zinc binding domain (ZnBD). The APP770 isoform is shown, APP75i lacks the OX-2 domain, and APP695 lacks both OX-2 and Kunitz protease inhibitor (KPI) domains, (e) Activities of the APP-E2 ± GFD compared to APP695ct GFD, FD1 and FDl (E415N)-APP695a alone using o-dianisidine as the substrate (pH 5). FDl (E415N)-APP695a is APP695a with a mutation at the second glutamic acid in the REXXE motif ofFDl . (0 Oxidation of o- dianisidine by the APP-E2 (500 nM) is potentiated by the APP-El in a concentration- dependent manner up to a 1 : 1 stoichiometry. Data are means ± SEM. n= 3.
Figure 2. APP amine oxidase activity is selectively inhibited by zinc and oxidizes catecholamines, (a) Inhibition curves of o-dianisidine oxidase activities of CP (by azide), APP695a and APP-E2 (by Zn2+). Inhibition is saturable with similar K, values between APP695a and APP-E2. (b) O-dianisidine oxidase activity of plasma from Cp-/- mice is markedly decreased compared to background control mice, but is not inhibited by azide (consistent with APP azide resistance), (c) O-dianisidine oxidase activity of APP-/- plasma is markedly decreased compared to background control mice, and is completely inhibited by azide (consistent with the residual activity present in APP-/- mice being CP), (d) APP- mediated oxidation of o-dianisidine is competitively inhibited by catecholamines NE, E and DA, suggesting that catecholamines bind to the active site (e) Catalytic oxidation of NE and DA by APP695q (50nM) using the fluorescamine amine assay. All assays were performed at pH 5.0. Data are means ±SEM, (a-d) n= 3 & (e) n= 6.
Figure 3. APP deficient mice have increased catecholamines and concomitant physiological changes. HPLC-ECD revealed increased NE (a), E (b) and DA (c), here represented as a percentage relative to wild-type age matched controls, in tissues as shown. Specific catecholamine concentrations within each tissue are shown in Table 2. Cardiovascular alterations consistent with chronic catecholamine increase included elevated basal heart rate (d) and systolic blood pressure (e) in 6-month APP-/- mice compared to age-matched wild- type controls, (f) Plasma prolactin is decreased in APP-/- mice, consistent with elevated brain stem DA. (g) The percentage of lymphocytes as a proportion of white cell count is reduced in APP-/- mice. Total white cell count was not significantly altered. Data are means ± SEM, (a-c & f ) n=5 & (d-e & g) n=6. * = p<0.05, ** = p<0.0 l, *** = p<0.001 analyzed by 2-tailed t- tests.
Figure 4. pH optima for o-dianisidine oxidation by APP695a. APP (500 nM) was incubated with 500 μΜ o-dianisidine in various pH buffers, 37 °C. Oxidized product was measure at 540nm. For these studies buffers (50 mM) were: pH 4.0 - 6.0 Na Acetate, pH 6.5 - 7.5 HEPES, pH 7.5 - 9.0 Tris. Optimal pH was found to be close to biologically relevant pH (cerebral spinal fluid is pH.7.2 and plasma is pH 7.4). Data are means ±SEM, n= 3.
Figure 5. Similar oxidase activity present in all isoforms of APP. Oxidase activity, as measured with o-dianisidine as the substrate, has no significant difference between all 3 as- isolated soluble PPa isoforms (all confirmed by ICP-MS to have no Zn2+). As shown in Fig. Id, APP770 isoform contains both the Kunitz protease inhibitor ( Pl) and OX-2 domains, APP751 lacks the OX-2 domain, and APP695 lacks both OX-2 and PI domains. Values are means ± SEM, n= 3.
Figure 6. APLP2 has no oxidase activity and zinc specific inhibition of oxidase activities differentiates APP. The effects of azide (lOmM), Zn2+ (10μΜ) and the monoamine oxidase inhibitors clorgyline (MAO-A specific) and selegiline (MAO-B specific) upon o-dianisidine oxidation by 500nM Cp, APP695a and APLP2, are shown. As previously reported, Cp was inhibited by azide (Topham RW. and Frieden E. 1970) while APP695a was inhibited by zinc (Duce, J.A. et ai, 2010). Neither was inhibited by MAO inhibitors. APLP2, which lacks ferroxidase activity (Duce JA. et al, 2010), similarly had no amine oxidase activity.
Figure 7. Wild-type mouse plasma contains oxidase activity that can be inhibited by zinc and azide. O-dianisidine oxidase activity of wild-type mouse plasma inhibited by azide (lOmM) leaves a residual activity similar to total activity in APP-/- mice (Fig. 2c).
Alternatively, plasma after zinc (Ι ΟμΜ) inhibition is -50% of the total activity and is proposed to represent total multi-copper oxidase activity. Addition of oxidase activities after azide or zinc inhibition is observed to have combined activity as the total. All o-dianisidine assays were performed in 75 mM Na acetate, pH 5.0, 37 °C. Values are means ± SEM, n= 3. Table 2. Increased catecholamine concentrations within APP deficient mice. Biological sample from APP-/- mice compared to 6-month age-matched wild-type controls were measured for catecholamines and values calculated using a standard curve for each catecholamine. Data are means ± SEM of n=5.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The present invention is predicated, in part, on the determination that amyloid precursor protein (APP) comprises amine oxidase activity and that this activity can be inhibited by Zn2+. Still further, the inventors have determined that the amine oxidase activity is ascribed, at least in part, to the E2 domain of APP. These findings have now permitted the rational design of therapeutic and prophylactic treatments for conditions characterised by aberrant amine oxidase activity. Such conditions include, but are not limited to, those characterised by aberrant catecholamine and/or serotonin levels, as well as those characterised by normal catecholamine and/or serotonin levels but which would otherwise be responsive to changes {i.e., an increase or decrease) in those levels.
[0061] Accordingly, one aspect of the present invention is directed to a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate the functional interactivity of Zn2+ with said APP wherein antagonising the interaction of Zn2+ with said APP increases APP amine oxidase activity and facilitating the interaction of Zn2+ with said APP decreases APP amine oxidase activity.
[0062] In a related aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate GFD potentiation of APP amine oxidase activity wherein facilitating the interaction of GFD with APP increases APP amine oxidase activity and antagonising the GFD interaction with APP decreases APP amine oxidase activity.
[0063] Reference to "amyloid precursor protein" ("APP") should be understood as a reference to all forms of APP including, for example, soluble forms of APP (sAPP), any isoforms which arise from alternative splicing of APP mRNA, allelic variants, polymorphic variants or various post translational forms of APP which undergo modification at, for example, the level of glycosylation, phosphorylation, tyrosine sulfation and proteolytic processing. Without limiting the present invention to any one theory or mode of action, APP is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation and neural plasticity. APP is best known and most commonly studied as the precursor molecule whose proteolysis generates beta amyloid (Αβ), a 39- to 42-amino acid peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients. APP also undergoes proteolytic cleavage to produce a soluble form of the protein (sAPP) that is active in plasma.
[0064] In humans, the gene for APP is located on chromosome 21 and contains at least 18 exons in 240 kilobases. Several alternative splicing isoforms of APP have been observed in humans, ranging in length from 365 to 770 amino acids, with certain isoforms preferentially expressed in neurons. Changes in the neuronal ratio of these isoforms have been associated with Alzheimer's disease. Homologous proteins have been identified in other organisms such as Drosophila (fruit flies), C. elegans (roundworms), and all mammals.
[0065] A number of distinct, largely independently-folding structural domains have been identified in the APP sequence. The extracellular region, much larger than the intracellular region, is divided into the E I and E2 domains, linked by an acidic domain (AcD). El contains two subdomains including a growth factor-like domain (GFLD) and a copper-binding domain (CuBD) interacting tightly together. A serine protease inhibitor domain, absent from the isoform differentially expressed in the brain, is found between the acidic region and E2 domain.
[0066] It should also be understood that APP can be referred to by different names including, but not limited to:
• amyloid beta (A4) precursor protein
• ABPP
• ADl
• amyloid A4 protein precursor
• peptidase nexin-II, Alzheimer disease
• amyloid precursor protein
• APP1 • PN-II
• PreA4
• protease nexin 2 *
• protease nexin-II
[0067] Reference to "amine oxidase activity" should be understood as a reference the oxidation of a wide range of biogenic amines including many neurotransmitters, histamine and xenobiotic amines. Amine oxidases, also referred to herein as monoamine oxidases (MAO; e.g., EC 1.4.3.4), are a family of enzymes that catalyze the oxidation of monoamines. They are found bound to the outer membrane of mitochondria in most cell types in the body. In humans there are two types of MAO: MAO-A and MAO-B. Both are found in neurons and astroglia. Outside the central nervous system, MAO-A is also found in the liver, gastrointestinal tract, and placenta, whereas MAO-B is mostly found in blood platelets.
[0068] Without limiting the present invention to any one theory or mode of action, monoamine oxidases catalyze the oxidative deamination of monoamines. Oxygen is used to remove an amine group from a molecule, resulting in the corresponding aldehyde and ammonia. M AO-A is particularly important in the catabolism of monoamines ingested in food. Both MAOs are also vital to the inactivation of monoaminergic neurotransmitters, for which they display different specificities. For example, serotonin, melatonin, norepinephrine, and epinephrine are mainly broken down by MAO-A. Phenethylamine is mainly broken down by MAO-B. Both forms break down dopamine equally. Specific reactions catalyzed by MAO include:
• Epinephrine or norepinephrine to 3,4-Dihydroxymandelic acid
• Metanephrine or normetanephrine to vanillylmandelic acid (VMA)
• Dopamine to dihydroxyphenylacetic acid
• 3-Methoxytyramine to homovanillic acid
[0069] Because of the vital role that MAOs play in the inactivation of neurotransmitters, MAO dysfunction (too much or too little MAO activity) is thought to be responsible for a number of neurological disorders. For example, unusually high or low levels of MAOs in the body have been associated with depression, schizophrenia, substance abuse, attention deficit disorder, migraines, and irregular sexual maturation. Monoamine oxidase inhibitors are one of the major classes of drug prescribed for the treatment of depression, although they are last-line treatment due to risk of the drug's interaction with diet or other drugs. Excessive levels of catecholamines (for example, epinephrine, norepinephrine, and dopamine) may lead to a hypertensive crisis. PET research has shown that MAO is also heavily depleted by use of tobacco cigarettes.
[0070] APP has been determined to possess amine oxidase activity. Hence, reference to "APP-mediated amine oxidase activity" should be understood as a reference to the amine oxidase activity of the amyloid precursor protein itself. It has been found that the amine oxidase activity of APP resides in the E2 domain of APP.
[0071] Reference to "aberrant" APP-mediated amine oxidase activity should be understood as a reference to a level of APP-mediated amine oxidase activity which is problematic or otherwise not appropriate. This may be either inadequate amine oxidase activity (for example, potentially leading to localised areas of increased catecholamine and/or serotonin levels in tissue) or too much (i.e., unwanted) amine oxidase activity (for example, leading to reduced catecholamine and/or serotonin levels in tissue). It should be appreciated that, in some situations, the level of A P-mediated amine oxidase activity may be physiologically normal. However, there may be other factors which are acting to cause problematic catecholamine and/or serotonin levels and in respect of which modulation of APP-mediated amine oxidase activity would nevertheless assist in addressing the problematic catecholamine levels. In this case, the amine oxidase activity is "aberrant" within the context of this invention since it is an unwanted level of activity when considered in light of the individual's overall physiological state. The method of the present invention provides a means of modulating APP-mediated amine oxidase activity in order to improve the individual's physiological state. In terms of inadequate amine oxidase activity, this may take the form of either a reduction in the level of amine oxidase activity relative to normal levels or a complete ablation of amine oxidase activity.
[0072] It has also been determined that APP amine oxidase activity is inhibited by Zn2+. Thus, a method has been developed based on modulating the functional interactivity of Zn2+ with APP. To this end, reference to modulating the "interaction" of Zri2+ with APP should be understood as a reference to either antagonising the subject interaction, such that Zn2+ inhibition of APP-mediated amine oxidase activity is either minimised or entirely abrogated, or else facilitating the interaction of Zn2+ with APP such that Zn2+ inhibition of APP-mediated amine oxidase activity is induced. [0073] In a related aspect, it has also been determined that GFD potentiates APP amine oxidase activity. Accordingly, a further treatment method has been developed based on modulating GFD potentiation functionality. For example, antagonising GFD interaction with APP provides a means of reducing amine oxidase activity while facilitating the interaction of GFD with APP, such as via the use of GFD mimetics, provides a means for increasing APP amine oxidase activity. This increase in APP activity will typically be finite, as the ratio of GFD mimetic with APP will generally not exceed 1 : 1 (see Figure I f).
[0074] It should be understood that reference to antagonising "interactivity" is a reference to either entirely preventing the interaction of Zn2+ with APP or else to sufficiently disrupting this process such that the functional outcome of inhibiting APP amine oxidase activity is either abrogated or at least reduces.
[0075] Although it is preferable that, in some embodiments, APP-mediated amine oxidase activity is entirely abrogated, it would be appreciated by the skilled person that even reducing the extent or degree of APP-mediated amine oxidase activity can nevertheless produce a valuable therapeutic outcome, such as increasing catecholamine levels and/or serotonin levels in a subject. Conversely, in terms of increasing APP-mediated amine oxidase activity, any level of increase (even if not restoration of normal level of activity) may nevertheless be desirable if it at least partially restores APP-mediated amine oxidase activity levels and produce a valuable therapeutic outcome, such as decreasing catecholamine levels and/or serotonin levels in a subject.
[0076] Reference to "agent" should be understood as a reference to any proteinaceous or non-proteinaceous molecule which modulates the interaction of Zn2+ or GFD with APP. The subject agent may be linked, bound or otherwise associated with any proteinaceous or non- proteinaceous molecule. For example, it may be associated with a molecule which permits targeting to a specific tissue, such as the brain.
[0077] Said proteinaceous molecule may be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening. Said non-proteinaceous molecule may be derived from natural sources, such as for example natural product screening or may be chemically synthesised. In one embodiment, the agent is either an antagonist which interacts with Zn2+ to prevent its interaction with APP or is Zn2+, itself, or a molecule which results in the formation or release of Zn2+, thereby reducing APP amine oxidase functionality. For example, the present invention contemplates Zn" chelators which are exemplified later in this document.
[0078] Accordingly, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to antagonise the interaction of Zn2+ with said APP.
[0079] In one embodiment, said antagonist is a zinc chelator, ionophore or metal protein attenuating compound.
[0080] Zn2+ chelators provide one method to prevent Zn2+ from inhibiting APP amine oxidase activity. A Zn2+ chelator, as described herein, is any compound that binds Zn2+ (whether or not it is a true chelator). Accordingly, any molecule that has the ability to ligand or chelate to a Zn2+ molecule can be used.
[0081] In one embodiment, the Zn2+ chelator is any ligand that is able to form two or more coordination bonds with a zinc ion. In particular embodiments, the zinc chelator is hydrophobic and is able to pass through the blood brain barrier, and optionally binds to zinc with moderate affinity. However, chelators that bind to zinc with high affinity may also be effective.
[0082] The zinc chelator may include a cyclic group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc or a cyclic group which includes at least one heteroafom such as nitrogen, oxygen or sulfur and in which the cyclic group is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc.
[0083] In one embodiment, the cyclic group is a heteroaryl group that is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc. The heteroaryl group is especially selected from quinazolinyl, quinoxalinyl, naphthyridinyl, pyrimidopyrimidinyl, cinnolinyl, phenazinyl, acridinyl, phenanthrolinyl, pyridopyrimidinyl, pyridopyrazinyl, pyranopyridinyl, dibenzoquinolizinyl, quinolinyl, isoquinolinyl, pyfidinyl and pyrimidinyl groups, especially pyridinyl and quinolyl groups. Suitable zinc chelators that include a pyridyl group include pyrithione, deferiprone and Ν,Ν,Ν',Ν'-tetrakis (2-pyridyImethyl) ethylenediamine (TPEN), especially pyrithione. Suitable quinolines may include a hydroxy substituent especially in the 8-position. Suitable quinolines may include clioquinol, iodoquinol, PBT2, M30 and related molecules such as those discussed in US 7,619,091. Further compounds that include a heteroaryl group substituted with a functional group that is able to carry a negative charge are discussed in US 7,692,01 1 and US 6,855,71 1. '
[0084] In another embodiment, the cyclic group is an aryl group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc. Suitable aryl groups include phenyl and naphthyl groups. Exemplary compounds that include an aryl group that is substituted with a functional group that is able to donate electrons to a coordination bond with zinc are discussed in US 6,855,71 1.
[0085] Functional groups able to donate electrons to a coordination bond with zinc include atoms with lone pairs of electrons. For example, such groups include heteroatoms and functional groups that are able to bear a negative charge. Suitable heteroatoms include nitrogen, oxygen and sulfur. Suitable functional groups that are able to carry a negative charge include hydroxy, mercapto. ester, carboxylate, oxtme, aldehyde and ketone groups, especially hydroxy and mercapto groups.
[0086] Another group of zinc chelators useful in the present invention include a heterocyclyl macrocyclic group, such as a cyclam or a bicyclam. Cyclams are compounds comprising a 1 ,4,8,1 1 -tetraazacyclotetradecane ring, which may be optionally substituted. Suitable substituents include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl and aryl groups. Bicyclams comprise two cyclam rings linked by an aromatic or aliphatic linker. A suitable bicyclam is Ι , Γ-xylyl bis- 1 , 4,8, 1 1 -tetraazacyclotetradecane (J L 169).
[0087] The zinc chelator may also be a polycarboxylic acid, such as ethylene diamine tetraacetic acid (EDTA), nitrilotriacetic acid, nitrilotripropionic acid, diethylenetriamine pentaacetic acid, 2-hydroxyethyl-ethylenediamine-triacetic acid, 1 ,6-diamino-hexamethylene- tetraacetic acid, 1 ,2-diamino-cycIohexane tetraacetic acid, 0,0'-bis(2-aminoethyl)- ethyleneglycol-tetraacetic acid, 1 ,3-diaminopropane-tetraacetic acid, N,N-bis(2- hydroxybenzyl)ethylenediamine-N,N-diacetic acid, ethylenediamine-N,N'-diacetic acid, ' ethylenediamine-N,N'-dipropionic acid, triethylenetetraamine hexaacetic acid, iminodiacetic acid, l ,3-diamino-2-hydroxypropane-tetraacetic acid, 1 ,2-diaminopropane-tetraacetic acid, triethylenetetramine-hexaacetic acid and l ,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'- tetraacetic acid. In one embodiment, the polycarboxylic acid is l ,2-bis-(2-amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid or ethylenediamine tetraacetic acid (EDTA), especially l,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid. The zinc chelator may also be an ester of these polycarboxylic acids. Diesters of (HOOC- CH2-)2N-A-N -CH2COOH)2 (where A is a saturated or unsaturated, aliphatic, aromatic or heterocyclic divalent linking radical containing, in a direct chain link between the two depicted nitrogen atoms, 2-8 carbon atoms in a continuous chain which may be interrupted by 2-4 oxygen atoms, provided that the chain members directly connected to the two depicted nitrogen atoms are not oxygen atoms) are discussed, for example, in US 6,458,837. In one embodiment, the ester of the polycarboxylic acid is an alkyl ester. In another embodiment, the zinc chelator is BAPTA-AM (l,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), DP-b99 (l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, N,N'-di(octyloxyethyl ester), Ν,Ν'-disodium salt) or DP-109, especially DP-109.
[0088] In another embodiment, the zinc chelator includes two carbamate groups linked by an aromatic or aliphatic linker or a heteroatom such as oxygen, nitrogen or sulfur, such as in diethylpyrocarbamate. In a further embodiment, the zinc chelator is an amino carboxylic acid that includes a functional group that is able to donate electrons to a coordination bond with zinc. Suitable amino carboxylic acids include penicillamine, cysteine, aspartic acid and glutamic acid, and also esters of these amino carboxylic acids. In one embodiment, the amino carboxylic acid is d-penicillamine. In one embodiment, the ester of the amino carboxylic acid is an alkyl ester.
[0089] In another embodiment, the zinc chelator includes a hydroxamide group, such as desferrioxamine.
[0090] The zinc chelator may also be a substituted transition metal including two or more functional groups that are able to carry a negative charge. Suitable transition metals include molybdenum, and a suitable zinc chelator is tetrathiolmolybdenate.
[0091] The term "aromatic or aliphatic linker" refers to a divalent group that connects two or more groups that are able to chelate zinc. Suitable aromatic or aliphatic linkers include optionally substituted arylene, alkylene, alkenylene, cycloalkylene and cycloalkenylene groups, especially optionally substituted arylene, more especially optionally substituted phenylene, most especially divalent xylene. In some embodiments, the alkylene or alkenylene groups may have one or more non-consecutive carbon atoms replaced by a heteroatom such as nitrogen, oxygen or sulfur. [0092] As used herein, the term "alkyl" refers to a straight chain or branched saturated hydrocarbon group having 1 to 12 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example,
Figure imgf000023_0001
which includes alkyl groups having 1 , 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, /j-propyl, /-propyl, w-butyl, /-butyl, t- butyl, M-pentyl, heptyl, octyl, nonyl and dodecyl. The term "alkylene" refers to a divalent alkyl group.
[0093] As used herein, the term "alkenyl" refers to a straight-chain or branched
hydrocarbon group having one or more double bonds between carbon atoms and having 2 to 12 carbon atoms. Where appropriate, the alkenyl group may have a specified number of carbon atoms. For example, C2-C6 as in "Cj-Cealken l" includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl. hexadienyl, heptenyl, octenyl. nonenyl, decenyl, undecenyl and dodecenyl. The term "alkenylene" refers to a divalent alkylene group.
[0094] As used herein, the term "alkynyl" refers to a straight-chain or branched
hydrocarbon group having one or more triple bonds between carbon atoms and having 2 to 12 carbon atoms. Where appropriate, the alkynyl group may have a specified number of carbon atoms. For example, C2-C6 as in "C2-C6alkynyl" includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, octynyl, nonynyl, decynyl, undecynyl and dodecynyl.
[0095] As used herein, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon. The cycloalkyl ring may include a specified number of carbon atoms. For example, a 3 to 8 membered cycloalkyl group includes 3, 4, 5, 6, 7 or 8 carbon atoms. Examples of suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentanyl, cyclohexanyl and cycloheptanyl. The term "cycloalkylene" refers to a divalent cycloalkyl group.
[0096] As used herein, the term "cycioaikenyl" refers to a cyclic hydrocarbon having at least one double bond, which is not aromatic. The cycioaikenyl ring may include a specified number of carbon atoms. For example, a 4 to 8 membered cycioaikenyl group contains at least one double bond and 4, 5, 6, 7 or 8 carbon atoms. Examples of suitable cycioaikenyl groups include, but are not limited to cyclopentenyl, cyclopenta-l,3-dienyl, cyclohexenyl, cyclohexen- 1 ,3-dienyl and cyclohexen-l ,4-dienyl. The term "cycloalkenylene" refers to a divalent cycloalkenyl group.
[0097] As used herein, the term "aryl" is intended to mean any stable, monocyclic, bicyclic or tricyclic carbon ring of up to 7 atoms in each ring, -wherein at least one ring is aromatic. When more than one ring is present, the rings may be fused to one another. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indartyl, biphenyi, binaphthyl, anthracenyl, phenanthrenyl, phenalenyl and fluorenyl. The term "arylene" refers to a divalent aryl group.
[0098] The term "heterocyclyl" as used herein, refers to a cycloalkyl or cycloalkenyl group in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. For example, between 1 and 4 carbon atoms in each ring may be replaced by heteroatoms independently selected from N, S and O. If the heterocyclyl group includes more than one ring in a ring system, at least one ring is heterocyclic. Examples of suitable heterocyclyl groups include tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolinyl, dithiolyl, 1 ,3-dioxolanyl, pyrazolinyl, imidazolinyl, imidazolidonyl, dioxanyl, dioxinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyranyl, dithianyl, and
tetrahydropyranyl.
[0099] The term "heteroaryl" as used herein, represents a stable monocyclic, bicyclic or tricyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Examples of suitable heteroaryl groups include quinazolinyl, quinoxalinyl, naphthyridinyl, pyrimidopyrimidinyl, cinnolinyl, phenazinyl, acridinyl, phenanthrolinyl, pyridopyrimidinyl, pyridopyrazinyl, pyranopyridinyl, dibenzoquinolizinyl, quinolinyl, isoquinolinyl, pyridinyl and pyrimidinyl.
[0100] The alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl and heteroaryl groups may be optionally substituted, for example with one or more optional substituents selected from R, R-0-(CH2)m-, R-S-(CH2)m-, HO-(CH2)m-, HS-(CH2)m-, R-C(=0)-0-(CH2)m-, R-O- C(=0)-(CH2)m-, R-C(=0)-(CH2)m-, R2N-C(=0)-(CH2)m-, RS(0)n-(CH2)m-, R2N-(CH2)m-, cyano, nitro and halo, wherein each R is independently selected from H, alkyl, alkenyl, alkynyl, -(CH2)p-aryl, -(CH2)p-heteroaryl, -(CH2)p-cycloalkyl, -(CH2)p-cycloalkenyl or -(CH2)P- heterocyclyl; m and p are 0 or an integer from 1 to 6, and n is 0 or an integer of 1 or 2. [0101] As used herein, the term "halo" represents fluoro, chloro, bromo or iodo.
[0102] In one particular embodiment, the Zn2+ chelator is a moderate affinity chelator which is hydrophobic, Examples include the 8-hydroxy quinolines, such as clioquinol, PBT2, M30, VK28 or related molecules, pyrithione, diethyl pyrocarbamate. 1 ,2-bis-(2-(amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid and derivatives, the bicyclam analogue JKL169 (1,1 '-xylyl bis-1 ,4,8,1 1 tetraaza cyclotetradecane), DP109 and related compounds.
[0103] In another embodiment, APP-mediated amine oxidase activity can be induced by administering Zn2+ to said subject.
[0104] It has also been found that purified APP-E l (the E l domain of APP) increases the amine oxidase activity of APP. This potentiating effect was mapped to the growth factor domain (GFD) within APP-El .
[0105] Thus, in a further aspect of the present invention, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient APP-mediated amine oxidase activity, said method comprising administering to said subject an effective amount of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof for a time and under conditions sufficient to potentiate APP-mediated amine oxidase activity. In some embodiments, said condition is characterised by unwanted catecholamine levels and/or unwanted serotonin levels, examples of which are herein described.
[0106] SEQ ID NO:2 represents the amino acid sequence of the growth factor domain (GFD) of APP. By administering a composition comprising this sequence, or a functional fragment, mimetic, analogue or homologue thereof, it has been found that APP-mediated amine oxidase can be potentiated.
[0107] As used herein, "fragments" include parts and portions, mutants, variants and mimetics from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of GFD. Mimetics may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides; polypeptides or proteins or cyclising the peptide, for example to yield a pharmacologically active form.
[0108] A "homologue" refers to a sequence in another animal or organism which has at least about 70% identity, preferably 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identify to the human GFD molecule.
[0109] Analogues include chemical and functional equivalents of GFD. These should be understood as molecules exhibiting any one or more of the functional activities of GFD and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
[0110] The fragments may have the active sites of GFD fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
[0111] Analogues contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.
[0112] Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBF ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (T BS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH.^.
[0113] The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. [0114] The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent dertvatisation, for example, to a corresponding amide.
[0115] Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuri- benzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4- nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
[0116] Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
[0117] Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethyipyrocarbonate.
[0118] Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated, herein is shown in Table 2.
[0119] Analogues may also include competitors for the active site of APP (e.g., APP-E2).
[0120] Analogues may also include peptoids (poly-N-substituted glycines), which are a class of peptide mimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the a-carbons. Peptoids are typically resistant to proteolysis and, therefore, are advantageous for therapeutic applications where proteolysis is of concern.
TABLE 2
Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutarnic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methyl isol leucine Nmile
D-alanine Dai L-N-methyl leucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys
D-aspartic acid Dasp L-N-methylmethionine Nmmet
D-cysteine Dcys L-N-methylnorleucine Nmnle
D-glutamine Dgln L-N-methylnorvaline Nmnva
D-glutamic acid Dglu L-N-methylornithine Nmo n
D-histidine Dhis L-N-methylphenylalanine Nmphe
D-isoleucine Dile L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser
D-lysine Dlys L-N-methylthreonine Nmthr
D-methionine Dmet L-N-fnethyltryptophan Nmt
D-ornithine Dorn L-N-methyltyrosine Nmtyr
D-phenylalanine Dphe L-N-methyl valine Nmval
D-proline Dpro L-N-methylethylglycine Nmetg
D-serine Dser L-N-methyl-t-butylglycine Nmtbug
D-threonine Dthr L-norleucine Nle
D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr a-methyl-aminoisobutyrate Maib
D- valine Dval a-methyl- -aminobutyrate Mgabu
D-a-methylalanine Dmala a-methylcyclohexylalanine Mchexa
D-a-methylarginine Dmarg a-methylcylcopentylalanine Mcpen
D-a-methylasparagine Dmasn a-methyl-a-napthylalanine Manap
D-a-methylaspartate Dmasp a-methylpenicillamine Mpen
D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-a-methylisoleucine Dmile N-amino-a-methylbutyrate Nmaabu
D-a-methyl leucine Dmleu a-napthylalanine Anap
D-a-methyllysirte Dmlys N-benzylgJycine Nphe
D-a-methylmethionine Dmmet N-(2-carbamy lethy l)glyc ine Ngln
D-a-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-a-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-a-methylproIine Dmpro N-(carboxymethyl)glycine Nasp
D-a-methylserine Dmser N-cyclobutylglycine Ncbut
D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-a-methyltryptophan Dmtrp N-cycIohexylglycine Nchex
D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-a-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine bhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N -(hydroxyethy l))glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methy 1th reon ine Dnmthr
D-N-methyltryptophan Dnmtrp N-( 1 -methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyI)glycine Nhtyr
L-i-butylglycine Tbug N-(thiomethyl)glycine Ncys
L-ethylglycine Etg penicillamine Pen
L-homophenylalanine Hphe L-a-methylalanine Mala
L-a-methylarginine Marg L-a-methylasparagine Masn
L-a-methylaspartate Masp L-a-methyl-t-butylglycine Mtbug
L-a-methylcysteine Mcys L-methylethylglycine Metg
L-a-methylglutamine Mgln L-a-methylglutamate Mglu
L-a-methylhistidine his L-a-methylhomophenylalanineMhphe
L-a-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet
L-a-methylleucine Mleu L-a-methyllysine Mlys
L-a-methylmethionine Mmet L-a-methylnorleucine Mnle
L-a-methy lnorval i ne Mnva L-a-methylornithine Morn
L- -methylphenylalanine Mphe L-a-methylproline pro
L-a-methylserine Mser L-a-methylthreonine Mthr
L-a-methyltryptophan Mtrp L-a-methyltyrosine Mtyr
L-a-methylvaline Mval L-N-methylhomophenylalanine
N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamy Imethy l)gly c i ne carbamylmethyl)glycine
l -carboxy-l-(2,2-diphenyl-Nmbc ethylamino)cyclopropane
[0121] Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunetional imido esters having (CH2)n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.
[0122] The agents which are administered to a subject in accordance with the present invention may also be linked to a targeting means, such as a monoclonal antibody, which provides specific delivery of these molecules to target tissue regions.
[0123] In still another aspect there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by unwanted APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent which antagonises the interaction of GFD with APP.
[0124] One aspect of the present invention is directed to treating a condition characterised by aberrant APP amine oxidase activity. This should be understood as a reference to any disease or condition in respect of which aberrant APP-mediated amine oxidase activity is a cause, symptom or side effect. This includes, for example, conditions which occur as a side effect of a treatment regime for an unrelated disease condition. The subject condition may be congenital or acquired and may be in an acute or chronic phase. Conditions characterised by aberrant APP-mediated amine oxidase activity will include conditions characterised by aberrant catecholamine levels within the peripheral circulation {e.g., plasma) or biological tissue of said subject, including, but not limited to, the central nervous system. Said condition may be characterised by excessive catecholamine levels or inadequate catecholamine levels. Some examples of conditions that could be treated or prevented in accordance with the methods of the present invention are described herein.
[0125] In one embodiment, said condition is characterised by insufficient catecholamine levels, attributed at least in part to excessive APP-mediated amine oxidase activity.
[0126] Thus, in another aspect of the present invention, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which cond ition is characterised by insufficient catecholamine levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to inhibit APP-mediated amine oxidase activity.
[0127] There is therefore provided, in one embodiment, a method for the therapeutic or prophylactic treatment of a condition in a subject, which condit ion is characterised by insufficient catecholamine levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
(i) increase the level of Zn2+; or
(ii) antagonise the interaction of GFD with APP.
[0128] Conditions characterised by insufficient catecholamine levels include, but are not limited to, Major depression, Minor depression. Atypical depression, Dysthymia, Melancholia, Anergic depression, Treatment-resistant depression. Headache, Extrapyramidal disorders, Generalised anxiety disorder, Lichen simplex chronicus, Insomnia, Panic disorder, Stress disorder, Posttraumatic stress disorder (PTSD), Attention deficit disorder, Hyperactivity, Conduct disorder, Narcolepsy, Social phobia and anxiety. Obsessive-compulsive disorder, Eating disorder, Bulimia, Drug withdrawal syndromes and drug dependence disorders, Atypical facial pain, Chronic pain syndrome, Parkinson's disease, Hypertension, Irritable bowel syndrome (IBS), Jet lag (desynchronosis), and premature ejaculation.
[0129] In one embodiment, said agent is Zn2+ or an antibody or other interactive molecule directed to GFD (e.g., a peptoid).
[0130] In yet another embodiment, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by excessive catecholamine levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
(i) antagonise the interaction of Zn2+ with APP; or
(ii) facilitate the interaction of GFD with APP.
[0131] In one embodiment, said agent is a Zn2+ chelator, ionophore or metal protein attenuating compound as hereinbefore described.
[0132] In yet another embodiment, said agent is SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof.
[0133] Conditions characterised by excessive catecholamine levels include, but are not limited to, stress, which can be induced from psychological reactions or environmental stressors such as elevated sound levels, intense light, or low blood sugar levels, catecholamine toxicity as result of central nervous system trauma (e.g., due to stimulation and/or damage of nuclei in the brainstem, in particular those nuclei affecting the sympathetic nervous system), neuroendocrine tumors of the adrenal medulla (e.g., pheochromocytoma), tachycardia, hypertension, conditions associated with increased sympathetic nervous system activity, Schizophrenia, psychoses, gambling addiction and Obsessive Compulsive Disorder (OCD).
[0134] In another aspect of the present invention, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient serotonin levels, said method comprising administering to said subject an effecti ve amount of an agent for a time and under conditions sufficient for said agent to inhibit APP-mediated amine oxidase activity.
[0135] In a further aspect there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient serotonin levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
(i) increase the level of Zn2+; or
(ii) antagonise the interaction of G FD with APP.
[0136] Conditions characterised by insufficient serotonin levels include, but are not limited to, increased aggressive and angry behaviours, clinical depression, obsessive-compulsive disorder (OCD), migraine, irritable bowel syndrome (IBS), tinnitus, fibromyalgia, bipolar disorder, anxiety disorders, intense religious experiences, depression, anxiety disorders and other affective disorders, eating disorders such as bulimia, anorexia and obesity, phobias, dysthymia, premenstrual syndrome, cognitive disorders, impulse control disorders, attention deficit hyperactivity disorder and drug abuse. The anxiety disorders may include, but are not limited to, general anxiety disorders, panic anxiety, obsessive compulsive disorder, acute stress disorder, post trauma stress disorder or social anxiety disorder.
[0137] In yet another aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by excessive serotonin levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:
(i) antagonise the interaction of Zn2+ with APP; or
(ii) facilitate the interaction of GFD with APP.
[0138] A conditions characterised by excessive serotonin levels includes, but is not limited to, Serotonin Syndrome.
[0139] In still yet another aspect, the present invention provides use of an agent which decreases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient serotonin levels.
[0140] In yet still another aspect, the present invention provides use of an agent which increases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted serotonin levels.
[0141] In a further aspect, the present invention provides use of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof which increases APP- mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted serotonin levels.
[0142] In yet another aspect, the present invention provides use of an agent which inhibits GFD-mediated potentiation of APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient serotonin levels.
[0143] The term "subject" as used herein includes humans, primates, livestock animals (e.g., sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), companion animals (e.g., dogs, cats), captive wild animals (e.g., foxes, kangaroos, deer), aves or reptiles. Preferably, the mammal is human or a laboratory test animal. Even more preferably, the mammal is a human.
[0144] An "effective amount" typically means an amount necessary to at least partly attain the desired response; for example, to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition. The amount varies depending upon the health and physical condition of the subject to be treated, the taxonomic group of the subject to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors known to those skilled in the art.
[0145] The method of the present invention preferably facilitates said condition being reduced, retarded or otherwise inhibited. Reference to "reduced, retarded or otherwise inhibited" should be understood as a reference to inducing or facilitating the partial or complete inhibition of any one or more causes or symptoms of said condition. In this regard, it should be understood that conditions such as clinical depression are extremely complex comprising numerous physiological events which often occur simultaneously. In terms of the aspect of the subject method of treatment and/or prophylaxis, it should be understood that the present invention contemplates both relieving any one or more symptoms of said condition (for example, improving one or more cognitive function) or facilitating retardation or cessation of the cause of said condition.
[0146] Administration of an agent of the present invention in the form of a pharmaceutical composition, may be performed by any convenient means. The agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the form of modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The agent may be administered in a convenient manner by any suitable route. Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeal^, intravenously, intraperitoneally, subcutaneously, intracranial ly, intradermally, intramuscularly, intraoccularly, intrathecal ly, intracereberally, intranasally, infusion,- orally, rectally, via IV drip patch and implant (e.g.. using slow release molecules).
[0147] In a related aspect of the present invention, the subject undergoing treatment or prophylaxis may be any human or animal in need of therapeutic or prophylactic treatment. In this regard, reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated to total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract said condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity of the onset of a particular condition. "Treatment" may also reduce the severity of an existing condition.
[0148] In some embodiments, said APP is central nervous system APP.
[0149] In yet another aspect, the present invention provides use of an agent which decreases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient catecholamine levels.
[0150] In yet another aspect, the present invention provides use of an agent which increases APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted catecholamine levels.
[0151] In still yet another aspect, the present invention provides use of an agent which increases the level of Zn2+ in the manufacture of a medicament for the treatment of a condition characterised by unwanted APP-mediated amine oxidase activity.
[0152] In yet another aspect, the present invention provides use of an agent which antagonises the interaction of APP with Zn2+ in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP-mediated amine oxidase activity.
[0153] In yet another aspect, the present invention provides use of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof which increases APP- mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by unwanted catecholamine levels. [0154] In yet another aspect, the present invention provides use of an agent which inhibits GFD-mediated potentiation of APP-mediated amine oxidase activity in the manufacture of a medicament for the treatment of a condition characterised by insufficient catecholamine levels.
[0155] In yet another aspect of the present invention, there is provided modulatory agents, as herein described, when used in the methods of the present invention.
[0156] In accordance with these methods, the agent defined in accordance with the present invention may be co-administered with one or more other compounds or molecules. By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between administration of the two types of molecules. These molecules may be administered in any order.
[0157] The method of the present invention may also be combined with currently known methods for treatment and/or prophylaxis of said conditions.
[0158] In yet another aspect, the present invention provides a pharmaceutical composition comprising an agent as hereinbefore described and one or more pharmaceutically acceptable carriers and/or diluents for use in the methods of the present invention, as herein described. Said pharmaceutical composition may additionally comprise molecules with which it is to be co-administered. These agents are typically referred to as the active ingredients.
[0159] Although the methods of the present invention are preferably achieved via the intravenous or oral administration of the subject agent, it should be understood that the present invention is not limited to this method of administration and may encompass any other suitable method of administration. In this regard, the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0160] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which- contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.
[0161] When the active ingredients are suitably protected they may be orally administered,, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least I % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 g and 3000 mg of active compound.
[0162] The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.
[0163] In another aspect of the present invention, there is provided a method of Screening for a compound that modulates the APP-mediated amine oxidase activity, said method comprising exposing said compound to APP and an APP substrate under conditions that allow for APP- mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said compound is indicative that said compound modulates A PP-mediated amine oxidase acti vity. For example, Where the level of oxidised APP substrate increases as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound increases APP-mediated amine oxidase activity. Conversely, where the level of oxidised APP substrate is decreased as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound decreases APP-mediated amine oxidase activity.
[0164] In still another aspect of the present invention, there is provided a method of screening for a compound that modulates the APP-mediated amine oxidase activity, said method comprising exposing said compound to APP and an APP substrate under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said compound is indicative that said compound modulates APP-mediated amine oxidase activity. For example, where the level of oxidised APP substrate increases as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound increases APP- mediated amine oxidase activity. Conversely, where the level of oxidised APP substrate is decreased as compared to the level of oxidized APP substrate in the absence of said compound, this would be indicative that said compound decreases APP-mediated amine oxidase activity.
[0165] In some embodiments, a compound is screened for its ability to modulate APP- mediated amine oxidase activity whilst having a negligible effect on ceruloplasmin amine oxidase activity. Ceruloplasmin activity is typically dependent on copper and inhibited by NaN3, whereas neither NaN3 nor Cu2+ alter APP-mediated amine oxidase activity. Thus, in some embodiments, a compound may be screened for ceruloplasmin amine oxidase activity by exposing said compound to APP, an APP substrate and NaN3 under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of NaN3 is indicative that said compound modulates ceruloplasmin amine oxidase activity. Conversely, where there is no or negligible change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of NaN3 is indicative that said compound selectively modulates APP-mediated amine oxidase activity. Without being bound by theory, compounds that selectively modulate APP- mediated amine oxidase activity (i.e., having negligible effect on ceruloplasmin amine oxidase activity) are likely to provide greater specificity and tighter control in modulating
catecholamine and/or serotonin levels in vivo. This could provide a number of advantages. For example, where it is desirable to increase catecholamine and/or serotonin levels in vivo, a compound that is capable of selectively inhibiting APP-mediated amine oxidase activity is likely to provide tighter control and thus avoid an excessive increase in said catecholamine and/or serotonin levels that would otherwise lead to undesirable outcomes.
[0166] Appropriate substrates for use in the screening methods of the present in vention, as herein described (also referred to herein as "APP substrates") include, but are not limited to:
• Benzidine
• N,N-dimethyl-p-phenylenediamine
• p-phenylenediamine (PPD)
• o-dianisidine dihydrochloride
• Histamine
• Tyramine • Tryptamine
• Phenethylamine (PEA)
• Serotonin
• Norepinephrine (NE; noradrenaline)
• Epinephrine (E, adrenaline) and
• Dopamine (D).
[0167] Test agents can be administered to the reaction system at a single concentration or, alternatively, at a range of concentrations from about 1 nM to 1 tnM.
[0168] In some embodiments, the AAP substrate is o-diamsidine dihydrochloride.
[0169] Methods of screening for compounds or agents which antagonise or agonise the interaction of GFD with E2 domain of APP or the interaction of Zn2+ with APP would also be well known to those of skill in the art.
[0170] Diversity libraries, such as random combinatorial peptide or nonpeptide libraries can be screened. Many publicalty or commercially available libraries can be used such as chemically synthesized libraries, recombinant (e.g., phage display libraries) and in vitro translation-based libraries.
[0171] Examples of chemically synthesized libraries are described in Fodor et al, (1991); Houghten et ai, (1991 ); Lam et al, (1991 ); Medynski., ( 1994); Gallop et al., (1994);
Ohlmeyer et al, (1 93); Erb et al, (1994); Houghten et al, (1992); Jayawickreme et al, (1994); Salmon et al., (1993); International Patent Publication No. WO 93/20242; and Brenner and Lerner., (1992).
[0172] Examples of phage display libraries are described by Scott and Smith., (1990); Devlin et al, (1990); Christian R.B et al, (1992); Lenstra,, (1992); Kay et al, ( 1993) and .
International Patent Publication No. WO 94/18318.
[0173] In vitro translation-based libraries include but are not limited to those described in Mattheakis et al, (\ 994).
[0174] Without limiting the present invention in any way a test compound can be a macromolecule, such as biological polymer, including polypeptides or polysaccharides.
Compounds useful as potential therapeutic agents can be generated by methods well known to those skilled in the art, for example, well known methods for producing pluralities of compounds, including chemical or biological molecules such as simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, antibodies, and the like, are well known in the art and are described, for example, in Huse, U.S. Patent No. 5,264,563; Francis et ah, Curr. Opin. Chem. Biol., 2:422-428 ( 1998); Tietze et al, Curr. Biol , 2:363-381 (1 98); Sofia, Molecule. Divers., 3:75-94 (1998); Eichler et ah, Med. Res. Rev. 15:481 -496 (1995); and the like. Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources. Combinatorial libraries of molecules can be prepared using well known combinatorial chemistry methods (Gordon et l., J. Med. Chem. 37: 1233-1251 (1994); Gordon et al., J. Med. Chem. 37: 1385-1401 (1994); Gordon et ah, Acc. Chem. Res. 29: 144-154 (1996); Wilson and Czarnik, eds., Combinatorial Chemistry: Synthesis and Application, John Wiley & Sons, New York (1997).
[0175] Additionally, a test compound can be preselected based on a variety of criteria. For example, suitable test compounds having known modulating activity on a pathway suspected to be involved in APP amine oxidase activity can be selected for testing in the screening methods. Alternatively, the test compounds can be selected randomly and tested by the screening methods of the present invention. Test compounds can be administered to the reaction system at a single concentration or, alternatively, at a range of concentrations from about 1 nM to 1 mM.
[0176] The number of different test compounds examined using the methods of the invention will depend on the application of the method. It is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. The methods can be performed in a single or multiple sample format. Large numbers of compounds can be processed in a high-throughput format which can be automated or semi-automated.
[0177] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents.' For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (see, e.g., Gallop et al. ( 1994) 37(9): 1233- 1250). Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art, see, e.g., U.S. Patent No.
6,004,617; 5,985,356. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175; Furka (1991 ) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991 ) Nature, 354: 84-88). Other chemistries for generating chemical diversity libraries include, but are not limited to: peptoids (see, e.g., WO 91/1 735), encoded peptides (see, e.g., WO 93/20242), random bio-oligomers (see, e.g., WO 92/00091 ), benzodiazepines (see, e.g., U.S. Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (see, e.g., Hobbs (1993) Proc. Nat. Acad. Set. USA 90: 6909- 6913), vinylogous polypeptides (see, e.g., Hagihara (1992) J Amer. Chem. Soc. 1 14: 6568), non-peptidal peptidomimetics with a Beta-D-Glucose scaffolding (see, e.g., Hirschmann (1992) J. Amer. Chem. Soc. 1 14: 9217-9218), analogous organic syntheses of small compound libraries (see, e.g., Chen (1994) J. Amer. Chem. Soc. 1 6: 2661 ), oligocarbamates (see, e.g., Cho ( 1993) Science 261 : 1303), and/or peptidyl phosphonates (see, e.g., Campbell ( 1994) J. Org. Chem. 59: 658). See, e.g., U.S. Patent No. 5,539,083; for antibody libraries, see, e.g., Vaughn (1996) Nature Biotechnology 14:309-314; for carbohydrate libraries, see, e.g., Liang et al. (1996) Science 274: 1520-1522, U;S. Patent No. 5,593,853; for small organic molecule libraries, see, e.g., for isoprenoids U.S. Patent 5,569,588; for thiazolidinones and
metathiazanones, U.S. Patent No. 5,549,974; for pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; for morpholino compounds, U.S. Patent No. 5,506,337; for benzodiazepines U.S. Patent No. 5,288,514.
[0178] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., U.S. Patent No. 6,045,755; 5,792,431 ; 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Y, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). A number of robotic systems have also been developed for solution phase chemistries. These systems include automated workstations, e.g., like the automated synthesis apparatus developed by Takeda Chemical Industries, L TD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).
[0179J In practicing this aspect of the method of the invention, a variety of apparatus and methodologies can be used to in conjunction with the polypeptides of the invention (such as GFD and the E2 domain of APP), e.g., to screen compounds as potential modulators (e.g., inhibitors or activators). Such peptides and polypeptides may be bound to a solid support. Solid supports can include, e.g., membranes (e.g., nitrocellulose or nylon), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dip stick (e.g., glass, PVC, polypropylene, polystyrene, latex and the like), a microfuge tube, or a glass, silica, plastic, metallic or polymer bead or other substrate such as paper. One solid support uses a metal (e.g., cobalt or nickel)-comprising column which binds with specificity to a histidine tag engineered onto a peptide.
(0180] Adhesion of peptides to a solid support can be direct (i.e., the protein contacts the solid support) or indirect (a particular compound or compounds are bound to the support and the target protein binds to this compound rather than the solid support). Peptides can be immobilized either covalently (e.g., utilizing single reactive thiol groups of cysteine residues (see, e.g., Colliuod (1993) Bioconj gate Chem. 4:528-536) or non-covalently but specifically (e.g., via immobilized antibodies (see, e.g., Schuhmann (1991) Adv. Mater. 3 :388-391 ; Lu (1995) Anal. Chem. 67:83-87; the biotin/strepavidin system (see, e.g., Iwane ( 1997) Biophys. Biochem. Res. Comm. 230:76-80); metal chelating, e.g., Langmuir-Blodgett films (see, e.g., Ng (1995) Langmuir 1 1 :4048-55); metal-chelating self-assembled monolayers (see, e.g., Sigal (1.996) Anal. Chem. 68:490-497) for binding of polyhistidine fusions.
[0181] Indirect binding can be achieved using a variety of linkers which are commercially available. The reactive ends can be any of a variety of functionalities including, but not limited to: amino reacting ends such as N-hydroxysuccinimide (NHS) active esters, imidoesters, aldehydes, epoxides, sulfonyl halides, isocyanate, isothiocyanate, and nitroaryl halides; and thiol reacting ends such as pyridyl disulfides, maleimides, thiophthalimides, and active halogens. The heterobifunctional crosslinking reagents have two different reactive ends, e.g., an amino-reactive end and a thiol-reactive end, while homobitunctional reagents have two similar reactive ends, e.g., bismaleimidohexane (BMH) which permits the cross-linking of sulfhydryl-containing compounds. The spacer can be of varying length and be aliphatic or aromatic. Examples of commercially available homobifunctional cross-linking reagents include, but are not limited to, the imidoesters such as dimethyl adipimidate dihydrochloride (DMA); dimethyl pimelimidate dihydrochloride (DMP); and dimethyl suberimidate dihydrochloride (DMS). Heterobifunctional reagents include commercially available active halogen-NHS active esters coupling agents such as N-succinimidyl bromoacetate and N- succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) and the sulfosuccinimidyl derivatives such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) (Pierce). Another group of coupling agents is the heterobifunctional and thiol cleavable agents such as N-succinimidyl 3- (2-pyridyidithio)propionate (SPDP) (Pierce Chemicals, Rockford, IL).
[0182] Antibodies can be used for binding polypeptides and peptides to a solid support. This can be done directly by binding peptide-specific antibodies to the column or it can be done by creating fusion protein chimeras comprising motif-containing peptides linked to, e.g., a known epitope (e.g., a tag (e.g., FLAG, myc) or an appropriate immunoglobulin constant domain sequence (an "immunoadhesin," see, e.g., Capon (1989) Nature 337:525-531 (1989).
[0183] In some embodiments, the present invention is directed to the use of antibodies to GFD to antagonise its activity. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies or may be specifically raised. The term
"antibody" includes a peptide or polypeptide derived from, modelled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush ( 1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including
(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains;
(ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH I domains; (iv) a Fv fragment consisting of the VL and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody." [0184] Antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies or may be specifically raised to these polypeptide and gene products. The present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies.
[0185] In some embodiments, the agent may be an antibody that specifically binds to Zn2+ and thereby inhibits the interaction of Zn2+ with APP. In other embodiments, the agent is an antibody that specifically binds to the GFD of APP-E1 , (as herein described) to facilitate inhibition of APP-mediated amine oxidase activity.
[0186] In practicing this aspect of the method of the present invention, a variety of apparatus and methodologies (described hereinbefore in detail) can be used to in conjunction with the polypeptides of the invention (such as GFD) to screen compounds as potential agents (e.g., agonists or antagonists). Thus, in some embodiments, said method comprises exposing said compound to APP and an APP substrate in the presence of Zn2+. In some embodiments, said method comprises exposing said compound to APP and an APP substrate in the presence of a compound of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof.
[0187] In still another aspect of the present invention, there is provided a method of screening a biological sample for APP-mediated amine oxidase activity, said method comprising exposing a biological sample that putatively comprises APP or a functional derivative thereof to an APP substrate {e.g., as herein described) under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said sample is indicative that said sample comprises APP or a functional derivative thereof. For example, where the level of oxidised APP substrate increases as compared to the level of oxidized APP substrate in the absence of said sample, this would be indicative that said sample comprises APP or a functional derivative thereof. In some embodiments, the functional derivative of APP is soluble APP (sAPP). A biological sample in accordance with this aspect of the present invention may include, but is not limited to, whole blood, plasma, urine and biological tissue. In one embodiment, the biological sample is plasma.
[0188] In other embodiments, one may design and test compounds which can modulate APP- mediated amine oxidase activity, for example, by modulating the interaction of Zn2+ with the E2 domain of APP (E2-APP), as herein described and/or by modulating the interaction of the GFD of APP-El . A three-dimensional (3D) prediction of the APP-E2 domain is provided in Figure 8 and it is within the skill of the person in the art to design, for example, in silico, agents which would appropriately interact. The crystal structure of the E2 domain (containing the amine oxidase site but also other domains) has also been described elsewhere (see, e.g., Wang et al. (2004) Molecular Cell, Vol. 15:343-353).
[0189] The present invention is further described by reference to the following non-limiting examples.
EXAMPLE 1
Experimental Procedures
Reagents
[0190] Reagents were all analytical grade and were purchased from Sigma, Australia, unless otherwise specified. Purified human ceruloplasmin was purchased from Vital Products (USA).
Mice
[0191] All mouse studies were performed with the approval of the IACUC and in accordance with statutory regulations. APP knockout mice (Zheng, H. et ai, 1995) and background controls (C57BL6/SV129) were thoracotomized under deep anesthesia, and heparinized blood obtained from the left atrium. Plasma was separated immediately from red blood cells when required by centrifugation at 4,000 x g for l Omin. Tissues was then taken and either used immediately or stored at -80°C until required.
Recombinant Protein Production and Purification
[0192] The recombinant fragments of the human soluble APP695a, human APLP2 ectodomain and APP-E1 (amino acids 28- 189) and APP-GFD (amino acids 28-123) were expressed in the methylotrophic yeast Pichia pastoris strain GS 1 15 and purified as previously described (Henry A. et al., 1997; Cappai R. et al., 1999). APP-E2 (amino acids 290-495) was amplified in the E. coli BL21 (DE3). Following induction by IPTG the bacterial cells were collected by centrifugation for subsequent protein purification.
[0193] All fragments required a two-step procedure performed using an AK.TA FPLC (GE Healthcare). APP695a and APP-E l was purified from culture media as previously described (Cappai R. et al, 1999) using anion exchange on a Q-Sepharose column (1.6 x 25 cm column, GE Healthcare) followed by hydrophobic exchange with phenyl-Sepharose (0.5 x 5cm column, GE Healthcare). APP-E2 and APP-GFD purification was achieved by heparin chromatography using heparin-Sepharose (1.6 x 12cm; GE Healthcare) and anion exchange on Q-Sepharose (1.6 x 25 cm column, GE Healthcare; see Andersen OM. et al, 2006).
Synthetic peptide synthesis
[0194] The synthetic peptide FD1 was synthesized using solid-state Fmoc chemistry, in a microwave synthesizer, using Fmoc -PAL-PEG-PS as resin from Applied Biosystems. All the amino acids were coupled to the resin using a 3-fold excess where 0.5 M 2-(lH-Benzotriazole- l -yl)-l , l ,3,3-tetramethyluronium hexafluoro-phosphate and 0.5 of N,N- diisopropylethylamine were used as activators. Once synthesis was complete, the resin was swelled in dimethylformamide (DMF) and the acetylation step was performance for 30 min at room temperature. Resin was then washed with DMF and methanol, and freeze-dried. The peptide was cleaved from the resin by stirring in a solution of 1% water, 0.5%
triisopropylsilane in trifluoroacetic acid (TFA) for 3 h. HPLC of peptide was performed using a preparative C8 Vydac Column, Buffer A = 0.1 % TFA in water and Buffer B = 0.1 % TFA, 50% isopropanol in acetonitrile. The gradient used was 1 5-70% B for 30rnin. The peptides were collected, lyophilized and re-analyzed by analytical HPLC and MALDI-TOF MS.
o-Dianisidine Oxidase Activity Assay
[0195] Samples were incubated at 37°C after being mixed with either 75 mM sodium acetate buffer pH 5.0 or 50 mM HEPES, pH 7.2 in 150 mM NaCl. 7.88 mM o-dianisidine
dihydrochloride substrate was added (1.576 mM final concentration), mixed and then incubated for 5 and 60 minutes. 9M sulfuric acid was used to stop the reaction at these times. Oxidized product was monitored by absorbance at 540nm from where the velocity was plotted. When not specified, 500nM of pure protein or 5μ1 plasma was used to measure oxidase activity. Sodium azide (10 mM) abolished the activity of pure ceruloplasmin and zinc (10 μΜ) abolished the activity of pure APP695a in the assay.
p-Phenylenediamine Oxidase Activity Assay
[0196] Method was based upon the published procedures of Chen et al., 2004). Samples were incubated with 0.01% pPD substrate in acetate buffer, pH 5.45, for 30 minutes at 37°C in the dark. Color development was monitored as absorbance at 570 nm.
Fluorometric detection of catecholamines
[0197] In a 96-well format varying concentrations of NE and DA were added to 50nM APP695a in acetate buffer, pH5.5. Each sample was made to a total volume of 200μ1 with buffer and shaken at 220rpm for 1 h at 37°C in the dark. Free amines in 90μ1 of sample were measured by adding ΙΟμΙ of fluorescamine (50mM dissolved in acetone) and detecting at 390nm excitation and 465nm emission with a 455nm cut-off. Oxidised substrate was calculated by subtracting the residual unmodified amines detected in this assay from the total added as a substrate. To control for auto-oxidation by NE and DA, samples incubated with APP were subtracted from the average absorbance of control samples that contained substrate oxidation without APP. APP oxidized NE and DA was converted to the μΜ equivalent by plotting against a standard curve.
HPLC with electrochemical detector
[0198] Tissue or plasma was homogenized by pulse sonication in 0.4M perchloric acid, 0.15% Sodium metabisulfite and 0.05% ethylenediaminetetraacetic acid disodium salt (EDTA) before centrifugation at 13,000 rpm at 4°C for 10 minutes. NE, E and DA were measured in supernatants using an HPLC system (ESA Biosciences; model 584) coupled to an
electrochemical detector (ESA Biosciences; Coulochem III detector) (El :-l 50mV,
E2:+220mV, and guard cell: +250m V). For catecholamine detection within each sample, 50μί, supernatant was injected onto a MD-150 reverse phase C18 column (ESA Biosciences) and elution was performed at a flow rate of 0.6ml/min in mobile phase (75mM sodium dihydrogen phosphate, 1.7mM 1-octanesulfonic Acid Sodium Salt, l OOmL/L triethylamine, 25mM EDTA, 10% acetonitrile, pH 3). Peaks were identified by retention times set to known standards. Data were normalized for total wet weight of sample.
Physiological testing
[0199] Systolic blood pressure and pulse rate (heart beats per minute) were measured using tail cuff plethysmography and a photoelectric sensor, respecti vely. Briefly, the tail-cuff method required mild restraint of the mouse in a dark chamber on a heated platform with their tails threaded through a tail cuff. Following a period of training (3 days), readings of blood pressure from the tail were then taken by brief cuff inflation (-5-10 seconds). This procedure was repeated 2-3 times for each mouse.
Prolactin assay
[0200] A 96-well microplate was coated with ΙΟΟμΙ of capture antibody and incubated overnight at room temperature. Plates were washed and incubated with blocking buffer for I h before washing again and then adding 1 ΟΟμΙ of detection antibody for a further 1 h. Plates were again washed before Ι ΟΟμΙ of streptavidin-HRP was added for 20 minutes. After a final wash, detection of prolactin from standards and plasma (50μ1 of each in triplicate) was carried out by adding samples to wells and incubating for 20 minutes. Stop reaction was stopped with 50μΙ of stop solution. Optical density of each well was measured at 450nm.
Measuring lymphocyte count in blood
[0201] All analytes were measured from complete blood counts and % lymphocytes were automatically calculated through the manufactures software using the formula; ([100 x Lymphocyte count] / PHA cells) - %BASO.
o-Dianisidine and p-Phenylenediamine Oxidase Activity Assays
[0202] The assays were based upon published procedures and standardized to international enzyme units (U/L) as described (Lehmann HP., Schosinsky KH. and Beeler MF., 1974; Rice EW., 1962).
Fluorometric detection of catecholamines
[0203] Varying concentrations of N E and DA were added to 50nM APP695a in acetate buffer, pH5.5. In total volume of 200μ1 samples were shaken (220 rpm) for 1 h at 37°C in the dark. Free amines were detected with fluorescamine (5mM) at 390 nm excitation and 465 nm emission with a 455 nm cut-off.
HPLC with electrochemical detection (ECD) of catecholamines
[0204] NE, E and DA in tissue or plasma homogenized supernatants (50μί) were injected onto a MD-150 reverse phase CI 8 column (ESA Biosciences) using a HPLC system (ESA Biosciences; model 584) coupled to an electrochemical detector (ESA Biosciences;
Coulochem I I detector) (El :- 150mV, E2:+220mV, and guard cell: +250mV). Peaks were identified by retention times set to known standards in mobile phase (75mM Sodium dihydrogen phosphate, l .7mM 1 -octanesulfonic Acid Sodium Salt, lOOmL/L triethylamine, 25mM EDTA, 10% acetonitrile, pH 3) at a flow rate of 0.6ml/min.
Physiological testing
[0205] Systolic blood pressure and heart rate were determined non-invasively by tail-cuff plethysmography (Hatteras Instruments, MC4000 Blood Pressure Analysis System).
Prolactin assay
[0206] Prolactin from plasma (50μ1 of each in triplicate) was assayed using a commercially available kit (R&D systems) ahd following the manufacturers recommended procedure. Lymphocyte count in blood
[0207] Blood was collected in lithium heparin tubes. Complete blood counts were analyzed within lh of collection using the Advia 120 hematology analyzer (Bayer Diagnostics) Results
[0208] The aromatic amine oxidase activity of CP has been extensively characterized with the synthetic substrates o-dianisidine dihydrochloride and p-phenylenediamine (Lehmann HP. et al, 1974; Rice EW., 1962). We found that APP695a possesses oxidase activity similar to CP with these substrates, with m values for o-dianisidine (0.55 mM at pH 7.2 and 0.21 at pH 5) and PPD (0.24 mM at pH 5.5) very similar to those of CP (Fig. 1 a-c), which matched previous results (Lehmann HP. et ai, 1974). APP695a exhibits near-maximal o-dianisidine oxidase activity through a pH range of 4.5 - 7.5, with maxima at pH 5.0 and 7.0 (Fig. 4), and is distinctly different from its profile for ferroxidase activity (Duce, J.A. et al., 2010), indicating that the amine oxidase active site on APP may not be identical to the ferroxidase domain. We mapped the amine oxidase site of APP to within the APP-E2 domain (Fig. Id & e) and dependent on the intact ferroxidase domain since a mutation within the consensus REXXE ferroxidase motif abolished activity (FD1 E415N-APP, Fig. le). However, the ferroxidase domain peptide alone, which retains ferroxidase activity (Duce JA. et al., 2010), did not oxidize o-dianisidine (FD l , Fig. l e). By analogy, the ferroxidase and amine oxidase activities of CP are also overlapping but not identical (Bielli P. and Calabrese L., 2002).
[0209] As-isolated soluble APPa isoforms 695, 751 and 770 had similar oxidase activity (Fig. 5), however, the APP-E2 domain had =60% the activity of APP695ot (Fig. le), proportional to its ferroxidase activity (Duce JA. et ai, 2010). While purified APP-El domain (Fig. I d & e) possessed no oxidase activity, equimolar concentrations of APP-El doubled APP-E2 activity (Fig. I f) to that of APP695a (Fig. le). We mapped this potentiating effect to the growth factor domain (GFD) (Fig. Id & e) within APP-El which has a similar potentiating effect on the ferroxidase activity of APP-E2 (Duce JA. et al., 2010).
[0210] The oxidase activity of CP was inhibited by azide (Fig. 2a; Fig. 6) as expected (Zaitsev VN. et al, 1999), but, as with APP695a ferroxidase activity (Duce JA. et al., 2010), azide did not inhibit amine oxidase activity (Fig. 6). However, Zn2+ inhibits APP695a ferroxidase activity (Duce JA. et al., 2010) and also satiirably inhibited APP695a amine oxidase activity (Ki= 759 μΜ, Fig. 2a; Fig. 6). Although the amine oxidase activity of APP-E2 was diminished compared to APP695a, it similarly was inhibited by Zn2+ (Fig. 2a) but not by azide or physiological concentrations of other divalent metal ions; e.g. 20 μΜ Cu2+ (data not shown). As with ferroxidase activity (Duce JA. et al^ 2010), APLP2 did not possess amine oxidase activity (Fig. 6). The selective inhibitors to monoamine oxidases A (clorgyline) and B (selegiline) had no effect on either APP695a or CP (Fig. 6).
[0211] O-dianisidine oxidation is used in clinical pathology as an assay for plasma CP activity (Lehmann HP. et al., 1974). We mapped the components that oxidize this amine substrate by assaying plasmas from CP knockout (CP-/-), APP knockout (APP-/-) as well as age- and gender-matched background control mice. Both azide and genetic ablation of CP induced a -60% drop in plasma amine oxidase activity (Fig. 2b and Fig. 7) and the remaining oxidase activity in the CP-/- plasma could not be inhibited by azide (Fig. 2b). By comparison, genetic ablation of APP caused a =40% decrease in plasma oxidase activity with the residual activity completely inhibited by azide (Fig. 2c), consistent with it being due to CP. Zn2+ inhibited the same proportion of amine oxidase activity in wild-type plasma as the activity lost in APP-/- plasma (Fig. 7). The loss of either oxidase activity in each type of knockout mouse was not associated with increased expression of an alternative amine oxidase, indicating that the activities are not redundant (Fig. 2b & c).
[0212] NE, E and DA were found to competitively inhibit the oxidation of o-dianisidine by APP in a dose dependent manner (Fig. 2d). Each catecholamine completely inhibited o- dianisidine oxidation at 1 : 1 stoichiometry (Fig. 2d). APP catalytically oxidized NE and DA as shown by a fluorometric assay of exposed primary amine groups that precluded the assay of epinephrine (Fig. 2e). Both catecholamines were oxidized by APP695q with similar Km values and comparable to previous reports with CP (Richards DA., 1983).
[0213] APP-/- mouse tissues relevant to the sympathetic nervous system (adrenal, heart, brain stem, plasma) had relatively marked and significantly higher concentrations of NE (Fig. 3a), E (Fig. 3b) and DA (Fig. 3c) than age-matched background controls (concentrations are reported in Table 3). These findings are consistent with previous reports describing high APP expression in brain stem neurons (Tanaka S. et al., 1989), the medullae chromaffin cells of the adrenal gland (Takeda M. et al., 1994), the heart (Duce JA. et al., 2010), and the presence of soluble APP within plasma (Bush Al. et al. , 1990).
[0214] APP-/- mice exhibited increased heart rate (Fig. 3d) and systolic blood pressure (Fig. 3e) consistent with the increased NE and E (Kvetnansky R. et al., 2009) present in the heart and plasma (Fig. 3a & b). Similar effects have been observed with the inhibition (Anderson MC. et al., 1993) or genetic ablation (Desir GV. 2009) of other catecholamine oxidases. [0215J We also tested for the impact of elevated brain DA levels on the tuberoinfundibular- dopaminergic system by assaying prolactin, a pituitary hormone whose release is suppressed by the presence of DA. Indeed plasma prolactin levels were significantly decreased in APP-/- mice (Fig. 3'f), consistent with increased brain stem DA in these mice (Fig. 3c). Prolactin has multiple effects upon growth, metabolism and immunomodulation, as well as reproduction, for which it is best characterized. Decreased prolactin could explain the 15-20% reduction in adult body weight seen in APP -/- mice (Zheng H. et al., 1995), since prolactin suppression results in decreased food intake and body weight (Gerardo-Gettens T., et al. 1989). Plasma glucose is also decreased in APP-/- mice ( eedham BE. et al , 2008). While this may be due to increased metabolism resulting from chronically elevated catecholamines, prolactin suppression also elevates adipokines {e.g. adiponectin) causing decreased plasma glucose (Shibli-Rahhal A. and Schlechte J., 2009). Prolactin, acting as a cytokine, also promotes the proliferation and survival of lymphocytes (Clevenger CV. et al,, 1991), while elevated catecholamines impair lymphocyte survival (Kvetnansky R. et al.. 2009). Accordingly, the lymphocyte count of APP- /- mice was significantly decreased compared to wild-type (Fig. 3g). ^
[0216] Within tissue, the active site of full-length APP is on the extracellular surface of the plasma membrane (Fig. Id). Unlike CP (Duce JA. et al., 2010), APP is expressed in all neurons, at synaptic junctions (Priller C. et al, 2006; Storey E. et al, 1996), which may make it available for controlling catecholamine concentrations in the vicinity, modulated by extracellular Zn2+ concentrations transferring from nearby synapses (Sensi SL. et al, 2009). The Km of APP is suitable for the spillover of concentrations of catecholamines that are not cleared by uptake transporters. In addition, full-length APP has been identified on the mitochondrial membrane (Anandatheerthavarada HK. et al, 2003), where monoamine oxidase A and B are also predominantly expressed (Sottocasa GL. et al., 1967). Therefore, APP may be a significant factor in the clearance of catecholamine. Indeed, the degree of
catecholaminergic accumulation in APP-/- mice is similar to amine oxidase deficient mice (e.g. monoamine oxidase-A; see Cases O. et al., 1995). The modulation of catecholamine levels in the brain and sympathetic target organs is an important end-effect for several major classes of psychotropic and cardiovascular drugs. The discovery that APP has an adjustable activity that influences catecholamine levels at these sites has important implications for clinical pharmacology. [0217] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in · this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
TABLE 3
Figure imgf000053_0001
Table 3. Increased catecholamine concentrations within APP deficient mice.
Biological sample from APP-/- mice compared to 6-month age-matched wild-type controls were measured for catecholamines and values calculated using a standard curve for each catecholamine. Data are means ± SEM of n=5.
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Claims

1. A method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate the functional interactivity of Zn2+ with said APP, wherein antagonising the interaction of Zn2+ with said APP increases APP amine oxidase activity and facilitating the interaction of Zn2+ with said APP decreases APP amine oxidase activity.
2. A method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP amine oxidase activity, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate GFD potentiation of APP amine oxidase activity, wherein facilitating the interaction of GFD with APP increases APP amine oxidase activity and antagonising the interaction of GFD with APP decreases APP amine oxidase activity.
3. The method of claim 1, wherein the condition is characterised by insufficient APP amine oxidase activity and said method comprises administering to said subject an effecti ve amount of an agent for a time and under conditions sufficient for said agent to antagonise the interaction of Zn2+ with said APP.
4. The method of claim 3, wherein said agent is a zinc (Zn2+) chelator, an ionophore or a metal protein attenuating compound.
5. The method of claim 4, wherein said Zn2+ chelator can form two or more coordination bonds with a zinc ion.
6. The method of claim 5, wherein the Zn2+ chelator is a hydrophobic Zn2+ chelator.
7. The method of claim 6, wherein said Zn2+ chelator is of moderate affinity.
8. The method of claim 4, wherein said Zn2+ chelator includes a cyclic group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc.
9. The method of claim 4, wherein said Zn chelator includes a cyclic group thatincludes at least one heteroatom amd wherein the cyclic group is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc.
10. The method of claim 6, wherein the Zn2+ chelator is selected from the group consisting of 8-hydroxy quinoline, pyrithione, diethyl pyrocarbamate, 1 ,2-bis-(2-(amino- phenoxy)ethane-N,N,N\N'-tetraacetic acid, Ι,Γ-xylyl bis-1 ,4,8,1 1 tetraaza
cyclotetradecane, DP 109. and functional derivatives thereof.
1 1. The method of claim 10, wherein the 8-hydroxy quinoline is selected from the group consisting of clioquinol, PBT2, M30 and VK28.
12. The method of claim 2, wherein the condition is characterised by insufficient APP- mediated amine oxidase activity and said method comprises administering to said subject an effective amount of a peptide comprising SEQ ID NO:2 or a functional fragment, mimetic, analogue or homologue thereof, for a time and under conditions sufficient to potentiate APP-mediated amine oxidase activity in said subject.
13. The method of claim 1, wherein the condition is characterised by unwanted APP amine oxidase activity and said method comprises administering to said subject an effective amount of an agent that increases the level of Zn2+ in said subject, for a time and under conditions sufficient to facilitate the interaction of Zn2+ with said APP.
14. The method of claim 13, wherein said agent is Zn2+.
15. The method of claim 2, wherein the condition is characterised by unwanted APP amine oxidase activity and said method comprises administering to said subject an effective amount of an agent which antagonises the interaction of GFD with APP.
16. The method of any one of claims 13 to 15, wherein the condition is characterised by insufficient catecholamine levels and/or insufficient serotonin levels in said subject.
17. The method of claim 16, wherein the condition characterised by insufficient catecholamine levels is selected from the group consisting of Major depression, Minor depression, Atypical depression, Dysthymia, Melancholia, Anergic depression, Treatment- resistant depression, Headache, Extrapyramidal disorders, Generalised anxiety disorder, Lichen simplex chronieus, Insomnia, Panic disorder, Stress disorder, Posttraumatic stress disorder (PTSD), Attention deficit disorder, Hyperactivity, Conduct disorder, Narcolepsy, Social phobia and anxiety, Obsessive-compulsive disorder, Eating disorder, Bulimia, Drug withdrawal syndromes and drug dependence disorders, Atypical facial pain, Chronic pain syndrome, Parkinson's disease, Hypertension, Irritable bowel syndrome (IBS), Jet lag (desynchronosis) and Premature ejaculation.
18. The method of claim 16, wherein the condition characterised by insufficient serotonin levels is selected from the group consisting of increased aggressive and angry behaviours, clinical depression, obsessive-compulsive disorder (OCD), migraine, irritable bowel syndrome (IBS), tinnitus, fibromyalgia, bipolar disorder, anxiety disorders, intense religious experiences, depression, anxiety disorders and other affective disorders, eating disorders such as bulimia, anorexia and obesity, phobias, dysthymia, premenstrual syndrome, cognitive disorders, impulse control disorders, attention deficit hyperactivity disorder and drug abuse.
19. The method of claim 18, wherein the anxiety disorder is selected from the group consisting of general anxiety disorders, panic anxiety, obsessive compulsive disorder, acute stress disorder, post trauma stress disorder and social anxiety disorder.
20. The method of any one of claims 3 to 12, wherein the condition is characterised by excessive catecholamine levels and/or excessive serotonin levels in the subject.
21. The method of claim 20, wherein the condition characterised by excessive catecholamine levels is selected from the group consisting of stress, including stress induced from psychological reactions or environmental stressors including elevated sound levels, intense light, or low blood sugar levels, catecholamine toxicity as result of central nervous system trauma, including catecholamine toxicity as a result of stimulation and/or damage of nuclei in the brainstem, neuroendocrine tumors of the adrenal medulla, including pheochromocytoma, tachycardia, hypertension, conditions associated with increased sympathetic nervous system activity, Schizophrenia, psychoses, gambling addiction and Obsessive Compulsive Disorder (OCD).
22. The method of claim 20, wherein the condition characterised by excessive serotonin levels is Serotonin Syndrome.
23. Use of an agent that increases the level of Zn2+ in a subject in need thereof, in the manufacture of a medicament for the treatment of a condition characterised by unwanted APP-mediated amine oxidase activity in a subject
24. Use of claim 23, wherein the agent is Zn2+.
25. Use of an agent that antagonises the interaction of APP with Zn2+ in a subject in need thereof, in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP-mediated amine oxidase activity in a subject.
26. Use of an agent that antagonises the interaction of GFD with APP in the manufacture of a medicament for the treatment of a condition characterised by unwanted APP-mediated amine oxidase activity in a subject.
27. Use of claim 25, wherein said agent is a zinc (Zn2+) chelator, an ionophore or a metal protein attenuating compound.
28. Use of claim 27, wherein the Zn chelator can form two or more coordination bonds with a zinc ion.
29. Use of claim 28, wherein the Zn + chelator is a hydrophobic Zn2+ chelator.
30. Use of claim 29, wherein said Zn2+ chelator is of moderate affinity.
31. Use of claim 27, wherein said Zn2+ chelator includes a cyclic group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc.
32. Use of claim 27, wherein said Zn2+ chelator includes a cyclic group thatincludes at least one heteroatom amd wherein the cyclic group is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc.
33. Use of claim 29, wherein the Zn + chelator is selected from the group consisting of 8-hydroxy quinoline, pyrithione, diethyl pyrocarbamate, l,2-bis-(2-(amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid, Ι,Γ-xyiyl bis-1,4,8,11 tetraaza
cyclotetradecane, DP 109, and functional derivatives thereof.
34. Use of claim 33, wherein the 8-hydroxy quinoline is selected from the group consisting of clioquinol, PBT2, M30 and VK28.
35. Use of a peptide comprising SEQ ID NO:2, or functional fragment, mimetic, analogue or homologue thereof, in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP-mediated amine oxidase activity in a subject.
36. Use of claim 23 or claim 24, wherein the condition is characterised by insufficient serotonin levels and/or insufficient catecholamine levels in the subject.
37. Use of claim 36, wherein the condition characterised by insufficient catecholamine levels is selected from the group consisting of Major depression, Minor depression, Atypical depression, Dysthymia, Melancholia, Anergic depression, Treatment-resistant depression, Headache, Extrapyramidal disorders, Generalised anxiety disorder, Lichen simplex chronicus, Insomnia, Panic disorder, Stress disorder, Posttraumatic stress disorder (PTSD), Attention deficit disorder, Hyperactivity, Conduct disorder, Narcolepsy, Social phobia and anxiety, Obsessive-compulsive disorder, Eating disorder, Bulimia, Drug withdrawal syndromes and drug dependence disorders. Atypical facial pain, Chronic pain syndrome, Parkinson's disease, Hypertension, irritable bowel syndrome (IBS), Jet lag (desynchronosis) and Premature ejaculation.
38. Use of claim 36, wherein the condition characterised by insufficient serotonin levels is selected from the group consisting of increased aggressive and angry behaviours, clinical depression, obsessive-compulsive disorder (OCD), migraine, irritable bowel syndrome (IBS), tinnitus, fibromyalgia, bipolar disorder, anxiety disorders, intense religious experiences, depression, anxiety disorders and other affective disorders, eating disorders such as bulimia, anorexia and obesity, phobias, dysthymia, premenstrual syndrome, cognitive disorders, impulse control disorders, attention deficit hyperactivity disorder and drug abuse.
39. Use of claim 38, wherein the anxiety disorder is selected from the group consisting of general anxiety disorders, panic anxiety, obsessive compulsive disorder, acute stress disorder, post trauma stress disorder and social anxiety disorder.
40. Use of any one of claims 25 to 35, wherein the condition is characterised by excessive serotonin levels and/or excessived catecholamine levels in the subject.
41. Use of claim 40, wherein the condition characterised by excessive catecholamine levels is selected from the group consisting of stress, including stress induced from psychological reactions or environmental stressors including elevated sound levels, intense light, or low blood sugar levels, catecholamine toxicity as result of central nervous system trauma, including catecholamine toxicity as a result of stimulation and/or damage of nuclei in the brainstem, neuroendocrine tumors of the adrenal medulla, including
pheochromocytoma, tachycardia, hypertension, conditions associated with increased sympathetic nervous system activity, Schizophrenia, psychoses, gambling addiction and Obsessive Compulsive. Disorder (OCD).
42. Use of claim 40, wherein the condition characterised by excessive serotonin levels is Serotonin Syndrome.
43. A pharmaceutical composition comprising Zn2+ or an agent that increases the level of Zn2+ in a subject in need thereof and one or more pharmaceutically acceptable carriers and/or diluents for use in the method of claim 12.
44. The pharmaceutical composition of claim 43, wherein the agent is Zn2+.
45. A pharmaceutical composition comprising an agent that antagonises the interaction of GFD with APP and one or more pharmaceutically acceptable carriers and/or diluents for use in the method of claim 15.
46. A pharmaceutical composition comprising an agent that antagonises the interaction of APP with Zn2+ in a subject in need thereof and one or more pharmaceutically acceptable carriers and/or diluents for use in the methods of any one of claims 3 to 11.
47. The pharmaceutical composition of claim 46, wherein said agent is a zinc (Zn ) chelator, an ionophore or a metal protein attenuating compound.
48. The pharmaceutical composition of claim 47, wherein said agent is a zinc (Zn2+) chelator, an ionophore or a metal protein attenuating compound.
49. The pharmaceutical composition of claim 48, wherein the Zn2+ chelator can form two or more coordination bonds with a zinc ion.
50. The pharmaceutical composition of claim 49, wherein the Zn2+ chelator is a hydrophobic Zn + chelator.
51. The pharmaceutical composition of claim 50, wherein said Zn chelator is of moderate, affinity.
52. The pharmaceutical composition of claim 47, wherein said Zn2+ chelator includes a cyclic group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc.
53. The pharmaceutical composition of claim 47, wherein said Zn2+ chelator includes a cyclic group thatincludes at least one heteroatom amd wherein the cyclic group is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc.
54. The pharmaceutical composition of claim 50, wherein the Zn chelator is selected from the group consisting of 8-hydroxy quinoline, pyrithione, diethyl pyrocarbamate, 1,2- bis-(2-(amino-phenoxy)ethane-N.N,N',N'-tetraacetic acid, Ι,Γ-xylyl bis-1 ,4,8,1 1 tetraaza cyclotetradecane, DP 109, and functional derivatives thereof.
55. The pharmaceutical composition of claim 54, wherein the 8-hydroxy quinoline is selected from the group consisting of clioquinol, PBT2, M30 and V 28.
56. A pharmaceutical composition comprising a peptide comprising SEQ ID NO:2, or functional fragment, mimetic, analogue or homologue thereof and one or more
pharmaceutically acceptable carriers and/or diluents for use in the methods of claim 12.
57. A method of screening for a compound that modulates the APP-mediated amine oxidase activity, said method comprising exposing said compound to APP and an APP substrate under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the leyel of oxidized APP substrate in the absence of said compound is indicative that said compound modulates APP-mediated amine oxidase activity.
58. The method of claim 57, wherein an increase in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said compound is indicative that said compound increases APP-mediated amine oxidase activity.
59. The method of claim 57, wherein a decrease in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said compound is indicative that said compound decreases APP-mediated amine oxidase activity.
60. A method of screening a biological sample for APP-mediated amine oxidase activity, said method comprising exposing a biological sample that putatively comprises APP or a functional derivative thereof to an APP substrate under conditions that allow for APP-mediated oxidation of the APP substrate, and measuring the level of oxidised APP substrate, wherein a change in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said biological sample is indicative that said biological sample comprises APP or a functional derivative thereof.
61. The method of claim 60, wherein an increase in the level of oxidised APP substrate as compared to the level of oxidized APP substrate in the absence of said biological sample is indicative that said biological sample comprises APP or a functional derivative thereof.
62. The method of claim 60 or claim 61 , wherein the APP substrate is selected from the group consisting of Benzidine, N,N-dimethyl-p-phenylenediamine, p-phenylenediamine (PPD), o-dianisidine dihydrochloride, Histamine, T ramine, Tryptamine, Phenethylamine (PEA), Serotonin, Norepinephrine (NE; noradrenaline), Epinephrine (E, adrenaline) and Dopamine (D).
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