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WO2002047670A1 - Traitement du dysfonctionnement sexuel male - Google Patents

Traitement du dysfonctionnement sexuel male Download PDF

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Publication number
WO2002047670A1
WO2002047670A1 PCT/IB2001/002399 IB0102399W WO0247670A1 WO 2002047670 A1 WO2002047670 A1 WO 2002047670A1 IB 0102399 W IB0102399 W IB 0102399W WO 0247670 A1 WO0247670 A1 WO 0247670A1
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WO
WIPO (PCT)
Prior art keywords
npy
agent
med
inhibitor
npyi
Prior art date
Application number
PCT/IB2001/002399
Other languages
English (en)
Inventor
Alasdair Mark Naylor
Pieter Hadewijn Van Der Graaf
Christopher Peter Wayman
Original Assignee
Pfizer Limited
Pfizer Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0030647A external-priority patent/GB0030647D0/en
Priority claimed from GB0108730A external-priority patent/GB0108730D0/en
Priority claimed from GB0109910A external-priority patent/GB0109910D0/en
Priority claimed from GB0111037A external-priority patent/GB0111037D0/en
Priority claimed from US09/895,367 external-priority patent/US20020028799A1/en
Priority claimed from US09/905,846 external-priority patent/US6878529B2/en
Priority claimed from GB0120679A external-priority patent/GB0120679D0/en
Priority to HU0400528A priority Critical patent/HUP0400528A2/hu
Priority to CA002431747A priority patent/CA2431747A1/fr
Priority to IL15577501A priority patent/IL155775A0/xx
Priority to AU2002220977A priority patent/AU2002220977A1/en
Priority to JP2002549244A priority patent/JP2004522720A/ja
Application filed by Pfizer Limited, Pfizer Inc. filed Critical Pfizer Limited
Priority to NZ526925A priority patent/NZ526925A/en
Priority to EP01270206A priority patent/EP1347750A1/fr
Priority to KR10-2003-7007946A priority patent/KR20030061441A/ko
Publication of WO2002047670A1 publication Critical patent/WO2002047670A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • 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
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • 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
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/344—Disorders of the penis and the scrotum and erectile dysfuncrion

Definitions

  • the present invention relates to a compound and a pharmaceutical that is useful for the treatment and/or prevention of male sexual dysfunction (MSD), in particular male erectile dysfunction (MED).
  • MSD male sexual dysfunction
  • MED male erectile dysfunction
  • the present invention also relates to a method of prevention and/or treatment of MSD, in particular MED.
  • the present invention also relates to assays to screen for the compounds useful in the treatment of MSD, in particular MED.
  • SD sexual dysfunction
  • MSD male sexual dysfunction
  • MED male erectile dysfunction
  • Penile erection is a haemodynamic event which is dependent upon the balance of contraction and relaxation of the corpus cavernosal smooth muscle and vasculature of the penis (Lerner et al 1993).
  • Corpus cavernosal smooth muscle is also referred to herein as corporal smooth muscle or in the plural sense corpus cavernosa. Relaxation of the corpus cavernosal smooth muscle leads to an increased blood flow into the trabecular spaces of the corpus cavernosa, causing them to expand against the surrounding tunica and compress the draining veins. This produces a vast elevation in blood pressure which results in an erection (Naylor, 1998).
  • NANC neurotransmitters found in the penis, other than NO, such as calcitonin gene related peptide (CGRP) and vasoactive intestinal peptide (VIP).
  • CGRP calcitonin gene related peptide
  • VIP vasoactive intestinal peptide
  • NO nitric oxide
  • NOS nitric oxide synthase
  • sGC soluble guanylate cyclase
  • cGMP intracellular cyclic guanosine 3',5'-monophosphate
  • Sildenafil citrate also known as ViagraTM
  • Pfizer As the first oral drug treatment for MED.
  • Sildenafil acts by inhibiting cGMP breakdown in the corpus cavernosa by selectively inhibiting phosphodiesterase 5 (PDE5), thereby limiting the hydrolysis of cGMP to 5'GMP (Boolel et al., 1996; Jeremy et al., 1997) and thereby increasing the intracellular concentrations of cGMP and facilitating corpus cavernosal smooth muscle relaxation.
  • PDE5 phosphodiesterase 5
  • MED therapies on the market such as treatment with prostaglandin based compounds i.e. alprostadil which can be administered intra-urethrally (available from Vivus Inc., as MuseTM) or via small needle injection (available from Pharamcia & Upjohn, as CaverjectTM), are either inconvenient and/or invasive.
  • Other treatments include vacuum constriction devices, vasoactive drug injection or penile prostheses implantation (Montague et al., 1996).
  • injectable vasoactive drugs show high efficacy, side effects such as penile pain, fibrosis and priapism are common, and injection therapy is not as convenient as oral therapy therefore sildenafil currently represents the most preferred therapy on the market.
  • a seminal finding of the present invention is the ability to selectively treat a male suffering from sexual dysfunction, in particular MED, with use of a neuropeptide Y inhibitor (NPYi), preferably a NPY Y1 receptor inhibitor (NPY Y1i), without peripheral side effects.
  • NPYi neuropeptide Y inhibitor
  • NPY Y1i NPY Y1 receptor inhibitor
  • the term "without peripheral side effects" as used herein means that the NPYi, preferably NPY Y1i, is devoid, or substantially devoid, of any activity on the cardiovascular system.
  • the NPYi preferably NPY Y1i
  • the NPYi is inactive in the cardiovascular system, thus reducing or eliminating the prospect of cardiovascular events, such as a drop in blood pressure when the NPYi, preferably NPY Y1i, is administered systemically (i.e. by mouth).
  • Peripheral side effects are those resulting from the inhibition of NPY or NPY Y1 receptors other than those in the genitalia and/or central nervous system.
  • the NPYi, preferably NPY Y1i, according to the present invention may, in addition to acting on NPY, preferably NPY Y1 receptors, in the genitalia, act centrally on the central nervous system to effectively treat for example abnormal drink and food intake disorders, such as obesity, anorexia, bulimia and metabolic disorders.
  • the NPYi, preferably NPY Y1i, according to the present invention when in use preferably has no, or substantially no, peripheral activity, i.e. on the cardiovascular system and/or the gastrointestinal system, other than that in respect of the genitalia.
  • peripheral activity i.e. on the cardiovascular system and/or the gastrointestinal system
  • NPY neuropeptide Y
  • NPY Y1 receptor an inhibitor of a neuropeptide Y
  • MED MED
  • NPY neuropeptide Y
  • an inhibitor of a neuropeptide Y preferably an inhibitor of a NPY Y1 receptor, which when in use is selective, or highly selective, for NPY or NPY Y1 receptors associated with male genitalia for enhancing nerve-stimulated erectile process.
  • the NPY/NPY Y1 inhibitors for use in the treatment of male sexual dysfunction, in particular MED according to the present invention have an IC50 of less than 100 nanomolar (nM), preferably of less than 75 nM, more preferably of less than 50 nM.
  • NPY inhibitors according to the present invention have greater than about 100-fold, more preferably greater than about 300-fold selectivity for NPY, in particular NPY Y1 receptors, in male genitalia, preferably in the corpus cavernosum, over NPY Y2 or NPY Y5 receptors.
  • the NPYi or NPY Y1 i has no, or substantially no, activity towards endopeptidase NEP EC 3.4.24.11 and/or angiotensin converting enzyme (ACE).
  • ACE angiotensin converting enzyme
  • the NPY or NPY Y1 inhibitors according to the present invention have no activity towards endothelin converting enzyme (ECE).
  • the NPY or NPY Y1 inhibitors according to the present invention have greater than 300-fold, more preferably greater than 500-fold, more preferably greater than 1000-fold selectivity for NPY/NPY Y1 over NEP and/or ACE.
  • the NPYi also has a greater than 1000-fold selectivity over ECE. This reduces the prospect of cardiovascular events (e.g. drop in blood pressure) when the NPYi or NPY Y1i is administered systemically (e.g. by mouth).
  • cardiovascular events e.g. drop in blood pressure
  • the term "highly selective” as used herein means the NPY/NPY Y1 inhibitors according to the present invention have greater than about 400-fold selectivity, preferably at least about 500-fold selectivity, preferably at least about 600-fold selectivity, preferably at least about 700-fold selectivity, preferably at least about 800-fold activity, preferably at least about 900-fold activity, preferably at least about 1000-fold selectivity for NPY, in particular NPY Y1 receptors, in male genitalia (particularly in the corpus cavernosum) over NPY Y2 or NPY Y5 receptors.
  • the NPYi or NPY Y1 i has no, or substantially no, activity towards NEP and/or ACE and/or ECE.
  • the term "highly selectively” should be construed accordingly.
  • the NPY inhibitors for use in the treatment of male sexual dysfunction, in particular MED, according to the present invention when in use are highly selective for the reproductive tract.
  • an NPY inhibitor preferably an NPY Y1i
  • an NPYi preferably an NPY Y1 i
  • an NPYi preferably an NPY Y1i
  • the NPYi or the NPY Y1i may be used in, for example, a manufacturing step and/or an identification preparative step and/or a modification preparative step of an agent according to the present invention.
  • NPY acts as a vasoconstrictor and causes restriction of penile veins, in particular those which regulate the flow of blood from the penis.
  • NPY was thought to aid the maintenance of an erection by causing constriction of the penile veins, therefore, preventing or reducing the flow of blood from the corpus cavernosum in the penis.
  • inhibition of NPY would have been expected to result in the relaxation of the penile veins regulating the flow of blood from the corpus cavernosum and, thus, the administration of an NPYi or an NPY Y1i would have been expected to result in the detumescence of the penis. In other words, inhibition of NPY, prior to the present invention, would have been expected to maintain the penis in a flaccid state.
  • NPY inhibitors and in particular use of antagonists of NPY Y1 receptors, results in an increase in the intracavernosal pressure of the penis and, thus, facilitates and/or causes penile erection.
  • an inhibitor which when in use is highly selective for NPY, preferably NPY Y1 receptors, associated with sexual responses, preferably in the genitalia.
  • the present invention further provides an NPYi, preferably an NPY Y1i, which when in use is highly selective for NPY receptors, preferably NPY Y1 receptors, associated with an increase the intracavernosal pressure in the corpus cavernosum.
  • the present invention further provides the use of an NPYi, preferably an NPY Y1i, in the manufacture of a medicament for selectively increasing the intracavernosal (i.e.) pressure during sexual arousal.
  • the NPYi preferably NPY Y1i, advantageously enhances the sexual arousally mediated increase in i.e. pressure, suitably the NPY/NPY Y1 inhibitors of the present invention selectively enhance the sexually arousally mediated increase in i.e. pressure.
  • the NPYi and/or NPY Y1i may be used to treat MED by increasing i.e. pressure, for example by influencing genital blood flow.
  • the present invention provides an NPYi, preferably an NPY Y1i, compounds for use in the selective treatment and/or selective prevention of MED.
  • an inhibitor of a NPY Y2 receptor may be used.
  • the term NPYi used herein includes a NPY Y2 inhibitor.
  • the present invention is advantageous as it provides a means for restoring a normal sexual arousal response - namely increased penile blood flow leading to erection of the penis.
  • the present invention provides a means to restore, or potentiate, the normal sexual arousal response.
  • NPYi neuropeptides
  • NPY Y1i neuropeptides
  • compounds were tested as agents and were found to be useful for enhancing the endogenous erectile process, and thereby being useful in the treatment of MED.
  • the present invention relates to NPYi (preferably NPY Y1i) compounds and pharmaceutical compositions comprising an NPYi, preferably an NPY Y1i, and pharmaceutical combinations consisting of an NPYi, preferably an NPY Y1i, and a PDEi, preferably a PDE5i, for use (or when in use) in the selective treatment and/or selective prevention of male sexual dysfunction, in particular MED.
  • NPYi or NPY Y1i and PDEi or PDE5i, if present
  • the composition (like any of the other compositions mentioned herein) may be packaged for subsequent use in the treatment of male sexual dysfunction, in particular MED.
  • the present invention relates to the use of an NPYi, preferably am NPY Y1 i, in the manufacture of a medicament (such as a pharmaceutical composition) for use in the selective, or highly selective, treatment of male sexual dysfunction, in particular MED.
  • a medicament such as a pharmaceutical composition
  • the present invention relates to the use of an NPYi, preferably an NPY Y1i, in the preparation of a medicament (such as a pharmaceutical composition) for use in the selective, or highly selective, treatment of male sexual dysfunction, in particular MED.
  • a medicament comprising an NPYi, preferably an NPY Y1i, which inhibitor when in use is selective for NPY, preferably NPY Y1 receptors, in genitalia.
  • the present invention relates to a method of selectively, or highly selectively, treating or preventing MED in a human or animal which method comprises administering to an individual an effective amount of an NPYi, preferably an NPY Y1i, wherein the NPYi, preferably the NPY Y1i, is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention relates to a method of treating a male suffering from male sexual dysfunction, in particular MED; the method comprising delivering to the male an NPYi, preferably an NPY Y1i, that is capable of selectively increasing the intracavernosal pressure during sexual arousal, without peripheral side effects.
  • a pharmaceutical pack comprising one or more compartments wherein at least one compartment comprises one or more of an NPYi, preferably an NPY Y1 i.
  • the present invention further provides a process of preparation of a pharmaceutical composition according to the present invention, said process comprising admixing one or more NPYi, preferably an NPY Y1i, with a pharmaceutically acceptable diluent, excipient or carrier.
  • the present invention further provides the use of an NPYi, preferably NPY Y1i, in accordance with the present invention in the manufacture or preparation of a medicament for both the selective treatment and/or selective prevention of male sexual dysfunction, particularly MED, and the treatment and/or prevention of abnormal drink and food intake disorders, in particular, obesity, anorexia, bulimia and metabolic disorders.
  • the present invention relates to an assay method for identifying an agent (hereinafter referred to as an NPYi or an NPY Y1i) that can be used to selectively treat or prevent male sexual dysfunction, in particular MED, the assay comprising: determining whether a test agent can directly enhance the endogenous erectile process; wherein said enhancement is defined as a potentiation of intracavernosal (i.e.) pressure (and/or cavernosal blood flow) in the presence of a test agent as defined herein; such potentiation by a test agent is indicative that the test agent may be useful in the selective treatment or prevention of male sexual dysfunction, in particular MED and wherein said test agent is an NPYi, preferably an NPY Y1i.
  • the agent inhibits NPY, preferably NPY Y1 receptors, associated with the genitalia, particularly in association with the corpus cavernosum.
  • the agent has no, or substantially no, effect
  • the present invention relates to an assay method for identifying an agent that can directly enhance the endogenous erectile process in order to treat or prevent male sexual dysfunction, in particular MED, the assay method comprising: contacting a test agent which has a moiety capable of inhibiting the metabolic breakdown of a peptide (preferably a fluorescent labelled peptide), said peptide being normally metabolised by NPY or NPY Y1; and measuring the activity and/or levels of peptide remaining after a fixed time (for example via fluorometric analysis); wherein the change in the level of the peptide measured by fluorescence is indicative of the potency (IC 5 o) of the test agent and is indicative that the test agent may be useful in the treatment or prevention of male sexual dysfunction, in particular MED; and wherein said agent is an NPYi or NPY Y1i.
  • a test agent which has a moiety capable of inhibiting the metabolic breakdown of a peptide (preferably a fluorescent labelled peptide), said peptide being
  • the present invention relates to a process comprising the steps of: (a) performing the assay method according to the present invention; (b) identifying one or more agents capable of inhibiting NPY, preferably NPY Y1 ; and
  • step (c) preparing a quantity of those one or more identified agents; and wherein said agent is a NPYi or an NPY Y1i.
  • the agent identified in step (b) may be modified so as to maximise, for example, activity and then step (a) may be repeated. These steps may be repeated until the desired activity or pharmacokinetic profile has been achieved.
  • the present invention relates to a process comprising the steps of: (a1) performing the assay according to the present invention; (b1) identifying one or more agents that can directly enhance the endogenous erectile process ; (b2) modifying one or more of said identified agents; (a2) optionally repeating step (a1); and (c) preparing a quantity of those one or more identified agents (i.e. those that have been modified); and wherein said agent is an NPYi or an NPY YIi.
  • the present invention relates to a process comprising the steps of: (i) performing the assay method according to the present invention;
  • NPY Y1 (iii) testing identified agents for their effect on arterial blood pressure in test animials, such as anaesthetised rabbits; (iv) selecting agents with no, or substantially no, effect on arterial blood pressure; and (v) preparing a quantity of those one or more selected agents; and wherein said agent is a NPYi or an NPY Y1i.
  • the agent identified in step (b) may be modified so as to maximise, for example, activity and then step (a) may be repeated. These steps may be repeated until the desired activity or pharmacokinetic profile has been achieved.
  • the present invention relates to a method of treating or preventing male sexual dysfunction, in particular MED, by potentiating the nerve stimulated endogenous erectile process in vivo (e.g. in rabbit) by measuring the intracavernosal pressure or cavernosal blood flow with an agent; wherein the agent is capable of directly inhibiting the metabolic breakdown of a fluorescent peptide (as detailed hereinbefore) in an in vitro assay method; wherein the in vitro assay method is the assay method according to the present invention; and wherein said agent is an NPYi or an NPY Y1i.
  • the present invention relates to the use of an agent in the preparation of a pharmaceutical composition for the selective treatment or selective prevention of male sexual dysfunction, in particular MED, wherein the agent is capable of directly inhibiting the metabolic breakdown of a fluorescent peptide when assayed in vitro by the assay method according to the present invention; and wherein said agent is an NPYi or an NPY Y1i.
  • the present invention relates to the use of an agent in the manufacture of a pharmaceutical composition for the selective treatment or selective prevention of male sexual dysfunction, in particular MED, wherein the agent is capable of directly inhibiting the metabolic breakdown of a fluorescent peptide when assayed in vitro by the assay method according to the present invention; and wherein said agent is an NPYi or an NPY Y1i.
  • the present invention relates to an animal model used to identify agents capable of treating or preventing male sexual dysfunction (in particular MED), said model comprising an anaesthetised male animal including means to measure changes in intracavernosal pressure and/or cavernosal blood flow of said animal following stimulation of the pelvic nerve thereof; and wherein said agent is an NPYi or an NPY Y1i.
  • the animal model may further comprise means to measure the arterial blood pressure of said animal.
  • the present invention relates to an assay method for identifying an agent that can directly enhance the endogenous erectile process in order to selectively treat or selectively prevent MED, the assay method comprising: administering an agent to the animal model of the present invention; and measuring the change in the endogenous erectile process; wherein said change is defined as a potentiation of intracavernosal pressure (and/or cavernosal blood flow) in the animal model in the presence of a test agent as defined; and wherein said agent is an NPYi or an NPY Y1i.
  • the present invention relates to an assay method for identifying an agent that can directly enhance the endogenous erectile process without effect on arterial blood pressure in order to selectively treat or selectively prevent MED, the assay method comprising: administering an agent to the animal model of the present invention; and measuring the change in the endogenous erectile process; wherein said change is defined as a potentiation of intracavernosal pressure (and/or cavernosal blood flow) in the animal model in the presence of a test agent as defined; measuring the arterial blood pressure in the animal model to ensure no or substantially no change in blood pressure; and wherein said agent is an NPYi or an NPY Y1i.
  • the present invention relates to a diagnostic method, the method comprising isolating a sample from a male; determining whether the sample contains an entity present in such an amount as to cause male sexual dysfunction, preferably MED; wherein the entity has a direct effect on the endogenous erectile process in the corpus cavernosum of the male; and wherein said entity can be modulated to achieve a beneficial effect by use of an agent; and wherein said agent is an NPYi or an NPY Y1i.
  • the present invention relates to a diagnostic composition or kit comprising means for detecting an entity in an isolated male sample; wherein the means can be used to determine whether the sample contains the entity and in such an amount to cause male sexual dysfunction, preferably MED, or is in an amount so as to cause male sexual dysfunction, preferably MED; wherein the entity has a direct effect on the endogenous erectile process and wherein said entity can be modulated to achieve a beneficial effect by use of an agent; and wherein said agent is an NPYi or an NPY Y1 i.
  • NPY and NPY Y1 receptors are present throughout the body, it is very unexpected that NPYi and/or NPY Y1i can be administered systemically and achieve a therapeutic response in the male genitalia without provoking intolerable (adverse) side effects, in particular intolerable (adverse) peripheral side effects.
  • NPY Y1i alone (particularly having a selectivity as above) and NP PDE5 combination when administered systemically increased genital blood flow, upon sexual arousal (mimicked by pelvic nerve stimulation) without adversely affecting cardiovascular parameters, such as causing a significant hypotensive or hypertensive effect.
  • an NPYi preferably NPY Y1i
  • systemic administration preferably by mouth e.g. swallowable tablet or capsule, or a sublingual or buccal formulation
  • a combination comprising one or more NPYi's, preferably NPY Y1i's, and one or more PDEi's, preferably PDE ⁇ i's, in the manufacture/preparation of a medicament for the selective treatment or selective prevention of male sexual dysfunction, in particular MED.
  • the present invention provides the use of a combination consisting of one or more NPYi's, preferably NPY Y1i's, and one or more PDEi's, preferably PDE ⁇ i's, in the manufacture/preparation of a medicament for the treatment or prevention of male sexual dysfunction, in particular MED.
  • NPYi's particularly NPY Y1i's
  • PDEi's particularly PDE ⁇ i's
  • said combined treatment comprises a combination of one or more NPYi's, preferably NPY Y1i's, with one or more PDEi's, preferably PDE ⁇ i's. More preferably such a combination provides for the concomitant administration of one or more NPYi's, particularly NPY Y1i's with one or more PDEi's, particularly PDE ⁇ i's, for the treatment of MED.
  • the present invention provides the use of a pharmaceutical composition
  • a pharmaceutical composition comprising one or more NPYi's, preferably NPY Y1 i's, with one or more PDEi's, preferably PDE ⁇ i's, (without the presence of further active components/ingredients, for example other inhibitors, such as neutral endopeptidase (NEP) inhibitors for example) for the treatment or prevention of MED.
  • the pharmaceutical composition consists solely of one or more NPYi's, particularly NPY Y1i's, with one or more PDEi's, particularly PDE ⁇ i's, as the active components/ingredients for the treatment of MED.
  • the present invention provides the use of a pharmaceutical composition
  • a pharmaceutical composition comprising one or more NPYi's, preferably NPY Y1i's, one or more PDEi's, preferably PDE ⁇ i's, and one further auxiliary active agent as disclosed hereinbelow.
  • the present invention provides a pharmaceutical composition consisting of one or more NPYi's, preferably NPY Y1i's, and one or more PDEi's, preferably PDE ⁇ i's, optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
  • a potent and selective NPYi's preferably NPY Y1i's
  • a potent and selective PDEi preferably PDE ⁇ i
  • a pharmaceutical composition consisting of one or more NPYi's, preferably NPY Y1i's, and one or more PDEi's, preferably PDE ⁇ i's, for the selective treatment of MED.
  • said combined administration of a NPYi preferably a NPY Y1i, and a PDEi, preferably a PDE ⁇ i
  • Concomitant administration as defined herein encompasses simultaneous (separate) administration, simultaneous combined administration, separate administration, combined administration, sequential administration and co- formulated combined administration of a PDEi (particularly PDE ⁇ i) and a NPYi (particularly NPY Y1 i).
  • the present invention further proposes that, concomitant administration of a PDEi (particularly PDE ⁇ i) and NPYi (particularly NPY Y1i) can effect an increase in the efficacy as compared with that obtainable by PDE, particularly PDE ⁇ -alone associated MED therapy.
  • an NPYi preferably an NPY Y1i
  • a PDEi preferably a PDE ⁇ i
  • the present invention additionally provides the use of a fast-acting composition for the treatment of MED.
  • a fast acting MED composition as defined herein means that following i.v.
  • composition consisting of a NPYi, preferably NPY Y1i, and a PDEi, preferably PDE ⁇ i
  • the time to maximal effect on intracavernosal pressure is reduced versus the equivalent time obtained for the same dose of the PDEi or PDE ⁇ i alone.
  • a further aspect of the invention provides a fast acting pharmaceutical compositions comprising an NPYi, preferably an NPY Y1i, and a PDEi, preferably a PDE ⁇ i, for use in the selective treatment of MED.
  • a further aspect of the invention provides a fast acting pharmaceutical compositions consisting of an NPYi, preferably an NPY Y1i, and a PDEi, preferably a PDE ⁇ i, for use in the selective treatment of MED.
  • NPY Y1i/PDE ⁇ i combination may enhance the efficacy of the PDE ⁇ i thereby enabling a reduction in the dose of PDE ⁇ inhibitor required for a specific efficacy.
  • a formulation comprising a NPY Y1i and a reduced amount of a PDE ⁇ i as defined herein means that a reduced amount of a given PDE ⁇ i is required to effect a particular response when combined with an effective amount of a NPY Y1i according to the present invention than the required amount of PDE ⁇ i alone.
  • Such reduced dose compositions for the treatment of MED reduce the potential nitrate interactions of PDE ⁇ .
  • it may be desirable for particular individuals such as for example men with mild MED. This may be particularly advantageous to individuals who respond poorly to a PDE ⁇ inhibitor alone (e.g. sildenafil).
  • a pharmaceutical combination adapted for administering by mouth in the preparation of a medicament for the selective treatment of male sexual dysfunction, said combination comprising an inhibitor of NPY or NPY Y1 having an IC 50 against NPY or NPY Y1 , respectively, of less than 100nM and a selectivity for NPY or NPY Y1 receptors in the genitalia over angiotensin converting enzyme of greater than 1000, and an inhibitor of phosphodiesterase type 5 enzyme (PDE5) having an IC 50 against PDE ⁇ of less than 100nM and a selectivity for PDE ⁇ over PDE3 of greater than 100.
  • an inhibitor of NPY or NPY Y1 having an IC 50 against NPY or NPY Y1 , respectively, of less than 100nM and a selectivity for NPY or NPY Y1 receptors in the genitalia over angiotensin converting enzyme of greater than 1000
  • PDE5 phosphodiesterase type 5 enzyme
  • the PDE ⁇ i used herein is sildenafil, preferably sildenafil citrate.
  • the present invention relates to the use of a composition consisting essentially of an NPYi, preferably an NPY Y1i, as the sole active ingredient in the manufacture of a medicament (such as a pharmaceutical composition) for use in the treatment of male sexual dysfunction, in particular MED.
  • a composition consisting essentially of an NPYi, preferably an NPY Y1i, as the sole active ingredient in the manufacture of a medicament (such as a pharmaceutical composition) for use in the treatment of male sexual dysfunction, in particular MED.
  • the present invention relates to the use of a composition consisting of an NPYi, preferably an NPY Y1 i, as the sole active ingredient in the manufacture of a medicament (such as a pharmaceutical composition) for use in the treatment of male sexual dysfunction, in particular MED.
  • a composition consisting of an NPYi, preferably an NPY Y1 i, as the sole active ingredient in the manufacture of a medicament (such as a pharmaceutical composition) for use in the treatment of male sexual dysfunction, in particular MED.
  • active ingredient means an ingredient which is active in the treatment of male sexual dysfunction, in particular MED.
  • active ingredient means an ingredient which is active in the treatment of male sexual dysfunction, in particular MED.
  • the agents for use in the selective treatment or selective prevention of MED according to the present invention are preferably NPY inhibitors and/or NPY Y1 inhibitors.
  • the agents for use in the selective treatment or selective prevention of MED according to the present invention are preferably highly selective when in use for NPY and/or NPY Y1 receptors in genitalia compared with NPY and/or NPY Y1 receptors at other peripheral locations in the body, for example in the cardiovascular system and/or the gastrointestinal system.
  • agents for use in the selective treatment or prevention of MED according to the present invention in addition to being when in use highly selective for NPY and/or NPY Y1 receptors in genitalia are active against NPY and/or NPY Y1 receptors in the central nervous system and thus may be used in the treatment or prevention of both MED and food and drink intake disorders.
  • the agent for the use according to the present invention is for oral administration.
  • the agent for the use according to the present invention may be for topical administration or intracavernosal administration.
  • the present invention also encompasses administration of the agent of the present invention before and/or during sexual arousal/stimulation. This is advantageous because it may provide systemic selectivity activity, such that the NPYi or NPY Y1i is active at the genitalia but not active at the cardiovascular system for example. This selectivity may be due a physiological effect rather than a pharmacological effect.
  • this step can provide systemic selectivity.
  • sexual arousal/stimulation may be one or more of a visual arousal/stimulation, a physical arousal/stimulation, an auditory arousal/stimulation or a thought arousal/stimulation.
  • the agents of the present invention are delivered before or during sexual arousal/stimulation, particularly when those agents are for oral delivery.
  • the present invention provides for the use of an agent in the manufacture of a medicament for the selective treatment or selective prevention of male sexual dysfunction, in particular MED; wherein said agent is capable of inhibiting NPYi, preferably NPY Y1i, in the genitalia of an individual without peripheral side effects; and wherein said individual is sexually aroused/stimulated before or during administration of said medicament.
  • the medicament is delivered orally to said individual.
  • the present invention provides for a method of treating an individual; the method comprising delivering to the individual an agent that is capable of inhibiting NPYi, preferably NPY Y1i, in the genitalia without peripheral side effects; wherein the agent is in an amount to selectively treat or selectively prevent MED; wherein the agent is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient; and wherein said individual is sexually aroused/stimulated before or during administration of said agent.
  • an agent that is capable of inhibiting NPYi, preferably NPY Y1i, in the genitalia without peripheral side effects
  • the agent is in an amount to selectively treat or selectively prevent MED
  • the agent is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient
  • said individual is sexually aroused/stimulated before or during administration of said agent.
  • said agent is delivered orally to said individual.
  • the present invention demonstrates the surprising and unexpected findings that:
  • An agent which inhibits NPY or NPY Y1 may be useful in enhancing the erectile response and may help to overcome an erectile dysfunction such as MED without peripheral side effects;
  • NPYi preferably an NPY Y1 i
  • systemically results in selective treatment of MED without peripheral side effects, in particular without any adverse cardiovascular events, such as a drop in blood pressure.
  • an NPYi preferably an NPY Y1 i
  • systemically may be useful in the selective treatment or selective prevention of both MED and food or drink intake disorders, because in addition to acting peripherally on the genitalia (although not elsewhere peripherally) the agent may also act on the central nervous system.
  • the present invention is advantageous because: (i) Agents which inhibit NPY, particularly NPY Y1 receptors, in genitalia can provide a means for selectively preventing and/or selectively treating and/or selectively restoring a normal sexual response, such as a male erectile response, by inducing an increased intracavernosal pressure and/or blood flow into the corpus cavernosum without the risk of adverse side effects, such as adverse cardiovascular events.
  • the present invention provides a means to restore, or mimic, the normal erectile response.
  • vasoactive drugs which are currently used to treat MED.
  • Patients with mild to moderate MED should benefit from treatment with a NPYi or NPY Y1i, and patients with severe MED may also respond.
  • early investigations suggest that the responder rate of patients with mild, moderate and severe MED will be greater with a NPY or NPY Y1 inhibitor/PDE ⁇ inhibitor combination.
  • Mild, moderate and severe MED will be terms known to the man skilled in the art, but guidance can be found in : The Journal of Urology, vol. 151, 54-61 (Jan 1994).
  • Early investigations suggest the below mentioned MED patient groups should benefit from treatment with a NPYi/NPY Y1 i and a PDE ⁇ i (or other combination set out hereinafter). These patient groups, which are described in more detail in Clinical Andrology vol.
  • the agent may be any suitable agent that can act as an inhibitor of NPY (sometimes referred to as an NPY antagonist).
  • Neuropeptide Y is an abundant and widespread peptide in the mammalian nervous system. It shows sequence homology to peptide YY and over 60% homology in amino acid and nucleotide sequence to pancreatic polypeptide (PNP; 167780). NPY is a 36-amino acid peptide. Minth et al. (1984) cloned the NPY gene starting from mRNA of a pheochromocytoma. Takeuchi et al. (198 ⁇ , 1986) isolated cDNA clones of the NPY and PNP genes from a pheochromocytoma and a pancreatic endocrine tumour, respectively.
  • NPY is one of the most highly conserved peptides known, with, for example, only 3 amino acid differences between human and shark.
  • Neuropeptide Y is a neuromodulator implicated in the control of energy balance and is overproduced in the hypothalamus of ob/ob mice.
  • NPY is a central effector of leptin deficiency.
  • Genetic linkage analysis of rats that were selectively bred for alcohol preference identified a chromosomal region that included the NPY gene (Carr et al., 1998). Alcohol- preferring rats had lower levels of NPY in several brain regions compared with alcohol-nonpreferring rats. Thiele et al. (1998) therefore studied alcohol consumption by mice that completely lacked NPY as a result of targeted gene disruption (Erickson et al., 1996).
  • NPY-deficient mice showed increased consumption, compared with wildtype mice, of solutions containing 6%, 10%, and 20% (by volume) ethanol. NPY-deficient mice were also less sensitive to the sedative/hypnotic effects of ethanol, as shown by more rapid recovery from ethanol-induced sleep, even though plasma ethanol concentrations did not differ significantly from those of controls. In contrast, transgenic mice that overexpressed a labelled NPY gene in neurons that usually express it had a lower preference for ethanol and were more sensitive to the sedative/hypnotic effects of ethanol than controls. These data provided direct evidence that alcohol consumption and resistance are inversely related to NPY levels in the brain. As part of an on-going study of the genetic basis of obesity, Karvonen et al.
  • pro7 NPY As the impact of pro7 NPY on serum cholesterol levels could not be found in normal-weight Dutchmen, it can be assumed that obese persons may be more susceptible to the effect of the gene variant. It was calculated that the probability of having the pro7 in NPY could be as high as ⁇ O to 60% in obese subjects with a total serum cholesterol equal to or higher than 8 mmol/L. At least among Finns, the pro7 form of NPY is one of the strongest genetic factors affecting serum cholesterol levels. See also Allen and Bloom (1986); Dockray (1986); Maccarrone; and Minth et al. (1986)."
  • Neuropeptide Y is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiologic activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system.
  • NPY neuropeptide Y1 and Y2
  • Y1 and Y2 Two major subtypes of NPY (Y1 and Y2) have been defined by pharmacologic criteria.
  • the NPY Y1 receptors have been identified in a variety of tissues, including brain, spleen, small intestine, kidney, testis, placenta, and aortic smooth muscle.
  • the Y2 receptor is found mainly in the central nervous system.
  • Herzog et al. (1992) reported cloning of a cDNA encoding a human NPY receptor which they confirmed to be a member of the G protein-coupled receptor superfamily. When expressed in Chinese hamster ovary (CHO) or human embryonic kidney cells, the receptor exhibited characteristic ligand specificity.
  • the receptor was coupled to a pertussis toxin- sensitive G protein that mediated the inhibition of cyclic AMP accumulation.
  • the receptor was coupled not to inhibition of adenylate cyclase but rather to the elevation of intracellular calcium.
  • the second messenger coupling of the NPY receptor was cell type specific, depending on the specific repertoire of G proteins and effector systems present in the cell type.
  • Larhammar et al. (1992) independently cloned and characterised the neuropeptide Y receptor.
  • Herzog et al. (1993) determined the molecular organisation and regulation of the human NPY Y1 receptor gene.
  • NPY Y1 receptor gene In contrast to the contiguous structure of most G protein-coupled receptor genes, they found that the NPY Y1 receptor gene has 3 exons. They also identified a common Pstl polymorphism in the first intron of the gene. By high-resolution fluorescence in situ hybridisation, they localised the gene to 4q31.3-q32. Herzog et al. (1997) found that the NPY1 R and NPY ⁇ R (602001) genes are colocalized on chromosome
  • the 2 genes are transcribed in opposite directions from a common promoter region.
  • One of the alternately spliced ⁇ -prime exons of the Y1 receptor gene is a part of the coding sequence of the Y ⁇ receptor.
  • This unusual arrangement suggested to Herzog et al. (1997) that the 2 genes arose by a gene duplication event and that they may be coordinately expressed.
  • Lutz et al. (1997) mapped the Npyl r and Npy2r genes to conserved linkage groups on mouse chromosomes 8 and 3, respectively, which correspond to the distal region of human chromosome 4q.”
  • NPY and it associated receptors As indicated background teachings on NPY and it associated receptors has been prepared by Victor A. McKusick et al (ibid). The following text concerning NPY Y2 has been extracted from that source.
  • Neuropeptide Y signals through a family of G protein-coupled receptors present in the brain and sympathetic neurons. At least 3 types of neuropeptide Y receptor have been defined on the basis of pharmacologic criteria, tissue distribution, and structure of the encoding gene; see 162641 and 162643. Rose et al. (199 ⁇ ) reported the expression cloning in COS cells of a cDNA for the human type 2 receptor, NPY2R. Transfected cells showed high affinity for NPY (162640), peptide YY (PYY; 600781), and a fragment of NPY including amino acids 13 to 36.
  • the predicted 381 -amino acid protein has 7 transmembrane domains characteristic of G protein-coupled receptors and is only 31 % identical to the human Y1 receptor (NPY1 R; 162641).
  • a 4-kb mRNA was detected on Northern blots of tissue samples from several regions of the nervous system.
  • Gerald et al. (1996) cloned the cDNA corresponding to the human Y2 receptor from a human hippocampal cDNA expression library using a radiolabeled PYY-binding assay. They expressed the Y2 gene in COS-7 cells and performed a hormone-binding assay which showed that the Y2 receptor binds (from highest to lowest affinity) PYY, NPY, and pancreatic polypeptide (PP;
  • Ammar et al. (1996) cloned and characterised the human gene encoding the type 2 NPY receptor. The transcript spans 9 kb of genomic sequence and is encoded in 2 exons. As in the type 1 NPY receptor gene, the ⁇ -prime untranslated region of NPY2R is interrupted by a 4.5-kb intervening sequence.
  • Ammar et al. (1996) demonstrated by Southern analysis of rodent-human cell hybrids followed by fluorescence in situ hybridisation (FISH) that the NPY2R gene maps to 4q31 , the same region containing the NPY1 R gene, suggesting that these subtypes may have arisen by gene duplication despite their structural differences.
  • FISH fluorescence in situ hybridisation
  • Nucleotide sequences and amino acid sequences for NPY and its receptors are available in the literature. Some sequences are presented as Figures 4-6.
  • NPY inhibitors or NPY Y1 inhibitors are disclosed and discussed in the following review articles:
  • Buttle LA Anti-obesity drugs: on target for huge market sales.
  • SR 120819A Two potent and selective, orally-effective antagonists for NPY Y1 receptors Soc Neurosci Abstr 1994 20 Pt 1 907 -Abs 376.14
  • NPY inhibitors and/or NPY Y1 inhibitors are selected from the following structures:
  • the cell line SK-N-MC was employed. This cell line was available from Sloane-Kettering Memorial Hospital, New York.
  • DMEM Dulbecco's Minimal Essential Media
  • the pellets were resuspended using a glass homogeniser in 25 mM HEPES (pH7.4) buffer containing 2.5 mM calcium chloride, 1 mM magnesium chloride and 2 g/L bacitracin. Incubations were performed in a final volume of 200 ⁇ l containing 0.1 nM 125 I-peptide YY (2200 Ci/mmol) and 0.2-0.4 mg protein for about two hours at room temperature. Nonspecific binding was defined as the amount of radioactivity remaining bound to the tissue after incubating in the presence of 1 ⁇ M neuropeptide Y. In some experiments various concentrations of compounds were included in the incubation mixture.
  • the present invention further comprises the combination of a compound of the invention for the treatment of male sexual dysfunction as outlined herein (more particularly male erectile dysfunction) with one or more of auxiliary active agents (see later discussion for suitable examples).
  • the combination provides a treatment for erectile dysfunctions of organic, vascular, neurogenic, drug induced and/or psychogenic origin.
  • the present invention further comprises the use of a combination consisting essentially of an NPYi or an NPY Y1i according to the present invention and two auxiliary active agents (see later discussion for suitable examples) in the manufacture of a medicament for the treatment or prevention of male sexual dysfunction as outlined herein (more particularly male erectile dysfunction).
  • the combination provides a treatment for erectile dysfunctions of organic, vascular, neurogenic, drug induced and/or psychogenic origin.
  • the present invention further comprises the use of a combination consisting of an NPYi or an NPY Y1i according to the present invention and two auxiliary active agents (see later discussion for suitable examples) in the manufacture of a medicament for the treatment or prevention of male sexual dysfunction as outlined herein (more particularly male erectile dysfunction).
  • the combination provides a treatment for erectile dysfunctions of organic, vascular, neurogenic, drug induced and/or psychogenic origin.
  • the present invention further comprises the use of a combination consisting essentially of an NPYi or an NPY Y1 i according to the present invention and one auxiliary active agent (see later discussion for suitable examples) in the manufacture or preparation of a medicament for the treatment or prevention of male sexual dysfunction as outlined herein (more particularly male erectile dysfunction).
  • the combination provides a treatment for erectile dysfunctions of organic, vascular, neurogenic, drug induced and/or psychogenic origin.
  • the present invention further comprises the use of a combination consisting of an NPYi or an NPY Y1i according to the present invention and one auxiliary active agent (see later discussion for suitable examples) in the manufacture or preparation of a medicament for the treatment or prevention of male sexual dysfunction as outlined herein (more particularly male erectile dysfunction).
  • the combination provides a treatment for erectile dysfunctions of organic, vascular, neurogenic, drug induced and/or psychogenic origin.
  • a further combination aspect of the invention provides a pharmaceutical combination (for simultaneous, separate or sequential administration) comprising a compound of the invention and one or more auxiliary active agents (see later discussion for suitable examples).
  • a yet further combination aspect of the invention provides a pharmaceutical composition (for simultaneous, separate or sequential administration) consisting essentially of an NPYi or an NPY Y1i and two auxiliary active agents (see later discussion for suitable examples).
  • a yet further combination aspect of the invention provides a pharmaceutical composition (for simultaneous, separate or sequential administration) consisting of an NPYi or an NPY Y1i and two auxiliary active agents (see later discussion for suitable examples).
  • a yet further combination aspect of the invention provides a pharmaceutical composition (for simultaneous, separate or sequential administration) consisting essentially of an NPYi or an NPY Y1i and one auxiliary active agent (see later discussion for suitable examples).
  • a yet further combination aspect of the invention provides a pharmaceutical composition (for simultaneous, separate or sequential administration) consisting of an NPYi or an NPY Y1i and one auxiliary active agent (see later discussion for suitable examples).
  • Suitable auxiliary active agents for use in the combinations of the present invention include:
  • prostaglandins for use herein include compounds such as alprostadil, prostaglandin E-i .prostaglandin E 0 , 13, 14 - dihydroprosta glandin E ⁇ , prostaglandin E 2 ⁇ eprostinol, natural synthetic and semi-synthetic prostaglandins and derivatives thereof including those described in WO- 00033825 and/or US 6,037,346 issued on 14th March 2000 all incorporated herein by reference, PGE 0 , PGEL PGAL PGBI , PGFI ⁇ ,
  • ⁇ - adrenergic receptor antagonist compounds also known as ⁇ - adrenoceptors or ⁇ -receptors or ⁇ -blockers.
  • Suitable compounds for use herein include: the ⁇ -adrenergic receptor blockerss as described in
  • ⁇ i-adrenoceptor blockers include: phentolamine, phentolamine mesylate, trazodone, alfuzosin, indoramin, naftopidil, tamsulosin, dapiprazole, phenoxybenzamine, idazoxan, efaraxan, yohimbine, rauwolfa alkaloids, Recordati 15/2739, SNAP 1069, SNAP 5089, RS17053, SL 89.0591 , doxazosin, terazosin, abanoquil and prazosin; ⁇
  • NO-donor compounds for use herein include organic nitrates, such as mono- di or tri-nitrates or organic nitrate esters including glyceryl trinitrate (also known as nitroglycerin), isosorbide 5-mononitrate, isosorbide dinitrate, pentaerythritol tetranitrate, erythrityl tetranitrate, sodium nitroprusside (SNP), 3-morpholinosydnonimine molsidomine, S-nitroso- N-acetyl penicilliamine (SNAP) S-nitroso-N-glutathione (SNO-GLU), N-hydroxy -N-N-glutathione (SNAP) S-nitroso-N-glutathione (SNO-GLU), N-hydroxy -
  • potassium channel openers or modulators include nicorandil, cromokalim, levcromakalim, lemakalim, pinacidil, cliazoxide, minoxidil, charybdotoxin, glyburide, 4-amini pyridine, BaCI 2 ;
  • Dopaminergic agents preferably apomorphine or a selective D2, D3 or
  • D2/D 3 agonist such as, pramipexole and ropirinol (as claimed in WO- 0023066),PNU95666 (as claimed in WO-0040226);
  • Vasodilator agents include nimodepine, pinacidil, cyclandelate, isoxsuprine, chloroprumazine, halo peridol, Rec 16/2739, trazodone;
  • 6,037,346 issued on 14th March 2000 and include acetergamine, brazergoline, bromerguride, cianergoline, delorgotrile, disulergine, ergonovine maleate, ergotamine tartrate, etisulergine, lergotrile, lysergide, mesulergine, metergoline, metergotamine, nicergoline, pergolide, propisergide, proterguride and terguride;
  • Atrial naturetic factor also known as atriai naturetic peptide
  • B type and C type naturetic factors such as inhibitors or neutral endopeptidase
  • 11)Angiotensin receptor antagonists such as losartan
  • Substrates for NO-synthase such as L-arginine
  • Calcium channel blockers such as amlodipine
  • Antagonists of endothelin receptors and inhibitors or endothelin-converting enzyme are antagonists of endothelin receptors and inhibitors or endothelin-converting enzyme
  • Cholesterol lowering agents such as statins (e.g. atorvastatin/ Lipitor- trade mark) and fibrates;
  • Antiplatelet and antithrombotic agents e.g. tPA, uPA, warfarin, hirudin and other thrombin inhibitors, heparin, thromboplastin activating factor inhibitors;
  • Insulin sensitising agents such as rezulin and hypoglycaemic agents such as glipizide;
  • Acetylcholinesterase inhibitors such as donezipil
  • Estrogen receptor modulators and/or estrogen agonists and/or estrogen antagonists preferably raloxifene or lasofoxifene, (-)-cis-6-phenyl-5-[4- (2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol and pharmaceutically acceptable salts thereof the preparation of which is detailed in WO 96/21656; 22) A PDE inhibitor, more particularly a PDE 2, 3, 4, 5, 7 or 8 inhibitor, preferably PDE2 or PDE ⁇ inhibitor and most preferably a PDE5 inhibitor (see hereinafter), said inhibitors preferably having an IC ⁇ O against the respective enzyme of less than 100nM (with the proviso that PDE3 and 4 inhibitors are only administered topically or by injection to the penis;
  • a NEP inhibitor preferably having an IC50 for NEP of less than 300nM, more preferably less than 100nM;
  • Vasoactive intestinal protein VIP
  • VIP mimetic a compound having a wide range of properties of interest
  • VIP analogue a compound having a wide range of properties of interest
  • VPAC1 NPAC or PACAP pituitory adenylate cyclase activating peptide
  • VIP receptor agonist or a VIP analogue eg Ro-125-1563
  • VIP fragment eg. Invicorp, Aviptadil
  • Invicorp Aviptadil
  • a melanocortin receptor agonist or modulator or melanocortin enhance such as melanotan II, PT-14, PT-141 or compounds claimed in WO- 09964002, WO-00074679, WO-09956679, WO-00106401 , WO- 00068361 , WO-00114879, WO-00113112, WO-09954358;
  • a serotonin receptor agonist, antagonist or modulator more particularly agonists, antagonists or modulators for 5HT1 A (including VML 670), 6HT2A, 5HT2C, 5HT3 and/or 5HT6 receptors, including those described in WO-09902169, WO-00002650 and/or WO-00028993;
  • a testosterone replacement agent including dehydroandrostendione), testosternone (Tostrelle), dihydrotestosterone or a testosterone implant;
  • Estrogen, estrogen and medroxyprogesterone or medroxyprogesterone acetate i.e. as a combination
  • estrogen and methyl testosterone hormone replacement therapy agent e.g. HRT especially Premarin, Cenestin, Oestrofeminal, Equin, Estrace, Estrofem, Elleste Solo, Estring, Eastraderm TTS, Eastraderm Matrix, Dermestril, Premphase, Preempro, Prempak, Premique, Estratest, Estratest HS, Tibolone);
  • a purinergic receptor agonist and/or modulator A purinergic receptor agonist and/or modulator
  • a neurokinin (NK) receptor antagonist including those described in WO- 09964008;
  • An opioid receptor agonist, antagonist or modulator preferably agonists for the ORL-1 receptor
  • An agonist or modulator for oxytocin/vasopressin receptors preferably a selective oxytocin agonist or modulator
  • a bombesin receptor antagonist more particularly a bombesin BBi, BB 2 , or BB 3 receptor antagonist, preferably a bombesin BBi inhibitor (see hereinafter), said inhibitors preferably having an IC50 against the respective enzyme of less than 100nM;
  • a SEP inhibitor for instance a SEPi having an IC 50 at less than 100 nanomolar, more preferably, at less than 50 nanomolar.
  • the SEP inhibitors according to the present invention have greater than 30-fold, more preferably greater than 50-fold selectivity for
  • ACE angiotensin converting enzyme
  • SEPi also has a greater than 100-fold selectivity over endothelin converting enzyme (ECE);
  • IKc a intermediate conductance calcium-activated potassium
  • a combination of active agents may be administered simultaneously, separately or sequentially.
  • cGMP PDE5 inhibitor The suitability of any particular cGMP PDE5 inhibitor can be readily determined by evaluation of its potency and selectivity using literature methods followed by evaluation of its toxicity, absorption, metabolism, pharmacokinetics, etc in accordance with standard pharmaceutical practice.
  • IC50 values for the cGMP PDE ⁇ inhibitors may be determined using the PDE5 assay (see hereinbelow).
  • the cGMP PDE ⁇ inhibitors used in the pharmaceutical combinations according to the present invention are selective for the PDE5 enzyme.
  • they are selective over PDE3, more preferably over PDE3 and PDE4.
  • the cGMP PDE5 inhibitors of the invention have a selectivity ratio greater than 100 more preferably greater than 300, over PDE3 and more preferably over PDE3 and PDE4. Selectivity ratios may readily be determined by the skilled person.
  • IC50 values for the PDE3 and PDE4 enzyme may be determined using established literature methodology, see S A Ballard et al, Journal of Urology, 1998, vol. 159, pages 2164-2171 and as detailed herein after.
  • Suitable cGMP PDE5 inhibitors for the use according to the present invention include:
  • PDE5 inhibitors for the use according to the present invention include:
  • EP-A-0463756 5-(2-ethoxy-5-morpholinoacetylphenyl)-1-methyl-3-n- propyl-1 ,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see EP-A- 0526004); 3-ethyl-5-[5-(4-ethylpiperazin-1 -ylsulphonyl)-2-n- propoxyphenyl]-2-(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3- d]pyrimidin-7-one (see WO98/49166); 3-ethyl-5-[5-(4-ethylpiperazin-1- ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3-yl]-2-(pyridin-2-yl)methyl-2,6- dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (
  • PDE5 inhibitors include:
  • the required PDE enzymes were isolated from a variety of sources, including human corpus cavernosum, human and rabbit platelets, human cardiac ventricle, human skeletal muscle and human and canine retina, essentially by the method of W.J. Thompson and M.M. Appleman (Biochem., 1971, 10, 311).
  • the cGMP-specific PDE (PDE5) and the cGMP-inhibited cAMP PDE (PDE3) were obtained from human corpus cavernosum or human platelets; the cGMP-stimulated PDE (PDE2) was obtained from human corpus cavernosum and human platelets; the calcium/calmodulin (Ca/CAM)- dependent PDE (PDE1) from human cardiac ventricle; the cAMP-specific PDE (PDE4) from human skeletal muscle and human recombinant, expressed in SF9 cells; and the photoreceptor PDE (PDE6) from human or canine retina.
  • Phosphodiesterases 7-11 were generated from full length human recombinant clones transfected into SF9 cells.
  • Assays can be performed either using a modification of the "batch" method of W.J. Thompson et al. (Biochem., 1979, 18, 6228) or using a scintillation proximity assay for the direct detection of AMP/GMP using a modification of the protocol described by Amersham pic under product code TRKQ7090/7100.
  • PDE inhibitors were investigated by assaying a fixed amount of enzyme in the presence of varying inhibitor concentrations and low substrate, (cGMP or cAMP in a 3:1 ratio unlabelled to [ 3 H]-labelled at a cone ⁇ 1/3 K m ) such that IC 50 ⁇ K,.
  • the final assay volume was made up to 100 ⁇ l with assay buffer [20 mM Tris-HCl pH 7.4, 5 mM MgCI 2 , 1 mg/ml bovine serum albumin]. Reactions were initiated with enzyme, incubated for 30-60 min at 30°C to give ⁇ 30% substrate turnover and terminated with 60 ⁇ l yttrium silicate SPA beads (containing 3 mM of the respective unlabelled cyclic nucleotide for PDEs 9 and 11).
  • Functional activity can be assessed in vitro by determining the capacity of a compound of the invention to enhance sodium nitroprusside or electrical field stimulation-induced relaxation of pre-contracted rabbit corpus cavernosum tissue strips, using methods based on that described by S.A. Ballard et al. (Brit. J. Pharmacol., 1996, 118 (suppl.), abstract 153P) or S.A. Ballard et a]. (J. Urology, 1998, 159, 2164-2171).
  • Compounds can be screened in vivo in test animals, such as anaesthetised rabbits, to determine their capacity, after i.v. administration, to enhance the pressure rises in the corpora cavernosa of the penis induced by intracavernosal injection of sodium nitroprusside, using a method based on that described by Trigo-Rocha et a[. (Neurourol. and Urodyn., 1994, 13, 71).
  • potent and selective PDE5 inhibitors are potent and selective PDE5 inhibitors.
  • NEP EC3.4.24.11 (FEBS Lett. 229(1), 206-210 (1988)), also known as enkephalinase or neprilysin, is a zinc-dependent neutral endopeptidase. This enzyme is involved in the breakdown of several bioactive oligopeptides, cleaving peptide bonds on the amino side of hydrophobic amino acid residues (Reviewed in Turner et al., 1997).
  • NEP neuronally released bioactive agents or neuropeptides metabolised by NEP
  • natriuretic peptides such as atrial natriuretic peptides (ANP) as well as brain natriuretic peptide and C- type natriuretic peptide, bombesin, bradykinin, calcitonin gene-related peptide, endothelins, enkephalins, neurotensin, substance P and vasoactive intestinal peptide.
  • Some of these peptides have potent vasodilatory and neurohormone functions, diuretic and natriuretic activity or mediate behaviour effects. Background teachings on NEP have been presented by Victor A. McKusick et al on http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm.
  • the l:NEP have a selectivity over ACE of greater than 300.
  • IC60 values and selectivity ratios for ACE may be determined by methods described in EP1097719A1.
  • NEP inhibitors are disclosed and discussed in the following review articles: Pathol. Biol., 46(3), 1998, 191; Current Pharm. Design, 2(5), 1996, 443; Biochem. Soc. Trans., 21(3), 1993, 678; Handbook Exp. Pharmacol., 104/1 , 1993, 547; TiPS, 11 , 1990, 245; Pharmacol. Rev., 46(1), 1993, 87; Curr. Opin. Inves. Drugs, 2(11), 1993, 1175; Antihypertens. Drugs, (1997), 113; Chemtracts, (1997), 10(11), 804; Zinc Metalloproteases Health Dis. (1996), 105; Cardiovasc.
  • NEP inhibitors are disclosed in the following documents: EP-509442A; US-192435; US-4929641 ; EP-599444B; US-884664; EP- 544620A; US-798684; J. Med. Chem. 1993, 3821; Circulation 1993, 88(4), 1; EP-136883; JP-85136654; US-4722810; Curr. Pharm. Design, 1996, 2, 443; EP-640594; J. Med. Chem.
  • NEP inhibitors are disclosed in EP1097719-A1 , in particular compounds FXII to FXIII therein.
  • Preferred NEP inhibitors are compounds FV to FXI and F57 to F65 of EP1097719-A1.
  • bombesin receptor antagonists are useful in the treatment of male sexual dysfunction, especially drug-induced male sexual dysfunction and psychogenic male sexual dysfunction associated with generalised unresponsiveness and ageing-related decline in sexual arousability.
  • proceptive behaviour in the rat includes "hopping and darting" movement, with rapid vibration of the ears.
  • Tests to assess the eagerness to seek sexual contact (sexual motivation) have been reported as the most appropriate way to measure proceptivity (Meyerson B.J, Lindstrom L.H., Acta Physiol. Scand., 1973; 389 (Suppl.): 1-80).
  • Receptivity, in the rat is demonstrated when the female assumes a lordotic position. This occurs when, on mounting, the male exerts pressure with his forepaws on the flanks of the receptive female.
  • VNN ventromedial nucleus
  • MCG midbrain central grey area
  • Bombesin is a 14-amino acid peptide originally isolated from the skin of the European frog Bombina bombina (Anastasi A. et al., Experientia, 1971 ; 27: 166). It belongs to a class of peptides which share structural homology in their C-terminal decapeptide region (Dutta A.S., Small Peptides; Chemistry, Biology, and Clinical Studies, Chapter 2, pp 66-82). At present, two mammalian bombesin-like peptides have been identified, the decapeptide neuromedin B (NMB) and a 23-residue amino acid, gastrin-releasing peptide (GRP).
  • NMB decapeptide neuromedin B
  • GFP 23-residue amino acid, gastrin-releasing peptide
  • BBi receptor binds neuromedin B (NMB) with higher affinity than gastrin-related peptide (GRP) and neuromedin C (NMC) and BB receptors bind GRP and NMC with greater affinity than NMB. More recently evidence has emerged of two more receptor subtypes denoted BB 3 and BB 4 but due to limited pharmacology, little is known of their function at present. BBi and BB 2 receptors have a heterogeneous distribution within the central nervous system indicating that the endogenous ligands for these receptors may differentially modulate neurotransmission. Among other areas, BBi receptors are present in the ventromedial hypothalamus (Ladenheim E.E et al, Brain Res., 1990; 537: 233-240).
  • One preferred genus of bombesin receptor antagonists disclosed in WO 98/07718 comprises compounds of the formula (I)
  • n 0, 1 or 2;
  • Ar is phenyl, pyridyl or pyrimidyl, each unsubstituted or substituted by from 1 to 3 substituents selected from alkyl, halogen, alkoxy, acetyl, nitro, amino, -CH 2 NR10R11, cyano, -CF 3 , -NHCONH 2 , and -C0 2 R 12 ;
  • R 1 is hydrogen or straight, branched, or cyclic alkyl of from 1 to 7 carbon atoms
  • R 8 is hydrogen or forms a ring with R 1 of from 3 to 7 carbon atoms;
  • R 2 is hydrogen or straight, branched, or cyclic alkyl of from 1 to 8 carbon atoms which can also contain 1 to 2 oxygen or nitrogen atoms;
  • R 9 is hydrogen or forms with R 2 a ring of from 3 to 7 carbon atoms which can contain an oxygen or nitrogen atom; or R 2 and R 9 can together be a carbonyl;
  • Ar 1 can be independently selected from Ar and can also include pyridyl-N-oxide, indolyl, imidazolyl, and pyridyl;
  • R 4 , R 5 , R 6 , and R 7 are each independently selected from hydrogen and lower alkyl; R 4 can also form with R 5 a covalent link of 2 to 3 atoms which may include an oxygen or a nitrogen atom;
  • R 3 can be independently selected from Ar or is hydrogen, hydroxy, -
  • R 10 , R 11 , R 12 , R 13 and R 14 are each independently selected from hydrogen or straight, branched, or cyclic alkyl of from 1 to 7 carbon atoms.
  • a particularly preferred compound within the above genus is (S) 3-(1H- lndol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[3-(4- nitro-phenyl)-ureido]-propionamide and its pharmaceutically acceptable salts.
  • BB-j and BB2 binding were as follows. CHO-K1 cells stably expressing cloned human NMB (for (BBi assay) and GRP receptors (for BB2 assay) were routinely grown in
  • Ham's F12 culture medium supplemented with 10% foetal calf serum and 2 mM glutamine.
  • cells were harvested by trypsinization, and stored frozen at -70°C in Ham's F12 culture medium containing ⁇ % DMSO until required. On the day of use, cells were thawed rapidly, diluted with an excess of culture medium, and centrifuged for 5 minutes at 2000 g.
  • a SEPi is a compound which inhibits or selectively inhibits a polypeptide having SEP activity.
  • SEP is a soluble secreted endopeptidase.
  • Endopeptidases including serine proteases, cysteine proteases and metalloendopeptidases, cleave at a sequence within an peptide.
  • Zinc metalloproteases An important group of endopeptidases known as zinc metalloproteases are characterised by having a requirement for the binding of a zinc ion in their catalytic site.
  • Zinc metalloproteases can be subdivided into classes (for review see FEBS Letters 364 (1994) pp. 1-6), with one such class being the neprilysin (NEP)-like zinc metalloproteases (FASEB Journal, Vol 11 , 1997 pp. 356-384).
  • NEP neprilysin
  • the NEP class includes at least 7 enzymes that are structurally related to each other (see later). They are typically membrane-bound, with a large carboxy- terminal extracellular domain, a short membrane-spanning region, and a short intracellular domain at the amino terminus.
  • neprilysin also called NEP, CD10, CALLA, enkephalinase or EC 3.4.24.11
  • ECE-1 and ECE-2 endothelin-converting enzymes
  • PEX PEX
  • KELL PEX
  • KELL PEX
  • XCE/DINE X-converting enzyme/damage induced neural endopeptidase
  • SEP/NEPII soluble secreted endopeptidase/neprilysin II
  • NEPII is likely to be a rat equivalent of SEP, which is a mouse enzyme, as they share 91 % amino acid identity. They are the members of this class closest to NEP in their amino acid sequence, both being 54% identical to human NEP.
  • the mRNA of both is highly abundant in the testis and can also be detected at low levels in a broad range of other tissues.
  • rat NEPII the mRNA has also been found at comparatively high levels in the brain and pituitary. When produced recombinantly in mammalian cells, both mouse SEP and rat NEPII can be found in the growth media.
  • Mouse SEP and rat NEPII like some other members of this class such as ECE-1 , exhibit splice variation. In the case of mouse SEP and rat NEPII, this splice variation results in isoforms with alterations in sequences involved in membrane localisation and secretion. The physiological significance of this is unclear but it is likely there could be membrane-bound, circulating, and intracellular forms of these enzymes.
  • Mouse SEP has been shown to be able to cleave a range of important biological peptides including enkephalin, endothelin, big-endothelin, Bradykinin and substance P. Like NEP, therefore, it has a fairly broad substrate specificity and may have several physiological functions in different tissues.
  • Enzymes in this NEP class like other metalloprotease enzymes, have been shown to be amenable to inhibition by small drug-like molecules (for example, thiorphan and phosphoramidon). This, together with the emerging nature of the physiological function of some members of the NEP-like enzymes in modulating peptidergic signalling, makes them attractive targets for pharmaceutical intervention.
  • SEP sequences for SEP are presented in WO99/53077, EP 1069188 and WO00/47750 and also in Figures 7-9 (SEQ ID No.s 4-6).
  • SEP sequences mentioned herein for, for example, assays include references to any one or more of the sequences presented in WO99/53077, EP 1069188 or WO00/47760 or presented as SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 or variants, fragments, homologues, analogues or derivatives thereof.
  • SEQ ID No. 4 and SEQ ID No. ⁇ each disclose a nucleotide sequence (cDNA) coding for human SEP.
  • SEQ ID No. ⁇ includes 5' and 3' partial vector sequences.
  • SEQ ID No. 6 shows a human SEP protein.
  • any particular SEPi can be determined by evaluation of its potency and selectivity using, for example, the following assays followed by evaulation of its toxicity, absorption, metabolism, pharmacokinetics, etc. in accordance with standard pharmaceutical practice.
  • SEP assay that may be used to detect candidate inhibitors of SEP is a fluorescence resonance energy transfer (FRET) assay.
  • FRET fluorescence resonance energy transfer
  • said labelled substrate peptide is Rhodamine green-Gly-Gly-c/Phe-Leu-Arg-Arg-Val- Cys(QSYTM-7)- ⁇ Ala-NH 2 .
  • the SEP FRET assay is based on an assay developed by Carvalho et al. for use with NEP (Carvalho et al., Annal. Biochem. 237, pp. 167-173 (1996)).
  • the SEP FRET assay utilises a similar intramolecularly quenched fluorogenic peptide substrate, but with a novel combination of fluorogenic donor/acceptor dyes, specifically Rhodamine green (Molecular Probes, Inc., Eugene, OR, USA) and QSYTM-7 (abbreviated hereafter as "QSY-7" or "QSY7"; Molecular Probes, Inc.).
  • the endopeptidase activity of SEP is measured by monitoring its ability to proteolyse the synthetic peptide substrate Rhodamine green-Gly-Gly-dPhe- Leu-Arg-Arg-Val-Cys(QSY7)- ⁇ Ala-NH 2 .
  • the two fluorophores (fluorogenic dyes) chosen for this assay have overlapping emission and absorption spectra and hence are suitable for energy transfer.
  • the Rhodamine green acts as a donor and when excited at 485 nm gives out an emission (fluorescence) at 536 nm which in turn excites the QSY7 (FRET is occurring).
  • the QSY7 is fluorescently silent and so gives off no emission above 535 nm hence no signal is observed (the Rhodamine green emission is quenched).
  • Rhodamine green and QSY7 moieties move apart and so upon excitation at 485 nm, energy transfer can no longer take place. As a result, an increase in fluoresence is observed at 535 nm for the Rhodamine green.
  • DIEA diisopropylethyl amine
  • MALDI-MS Matrix Assisted Laser Desorption lonization mass spectrometry
  • CP4 is the desired synthetic peptide substrate Rhodamine green- Gly-Gly-dPhe-Leu-Arg-Arg-Val-Cys(QSY7)- ⁇ Ala-NH 2 .
  • FMOC-Arg(Pbf)-OH was purchased from AnaSpec, Inc., CA, USA;
  • 2,2,2-Trifiuoroethanol was purchased from Aldrich, WI, USA. Sodium Carbonate was purchased from Fisher, PA, USA.
  • MALDI-MS were obtained on a Perseptive Biosystems Voyager-DE linear mass spectrometer using alpha cyano 4-hydroxy cinnamic acid matrix (Hewlett Packard, CA, USA) and reported masses based on external calibration.
  • CP4 synthetic peptide substrate Rhodamine green-Gly-Gly- ⁇ Phe-Leu-Arg- Arg-Val-Cys(QSYTM-7)- ⁇ Ala-NH 2 ) is synthesised by incorporating the key intermediate CP3 in a solid phase peptide synthesis scheme.
  • FMOC-PAL-PEG Resin is elaborated using Solid Phase Peptide Synthesis protocols optimised for efficiency of yield and time. These cycles (full details supra) incorporate 2 FMOC deprotections, washes, a single coupling of HBTU activated amino acid, washes, capping and finally, washing first with NMP then with 1 :1 trifluoroethanol / dichloromethane. These washes help to relax resin secondary structure allowing for thorough deprotection and efficient coupling of the next incoming amino acid during the next cycle.
  • Reagents for the assay are first prepared as follows:
  • a substrate solution is made up by resuspending the substrate Rhodamine green-Gly-Gly- Phe-Leu-Arg-Arg-Val-Cys(QSY7)- ⁇ Ala-NH 2 in 50mM HEPES buffer pH7.4 (Sigma, UK) at a concentration of 2 ⁇ M, then adding 1 EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics, UK) per 2 ⁇ ml.
  • a 4% DMSO solution comprised of 4ml DMSO plus 96ml 50mM HEPES pH7.4 is prepared.
  • a product solution is prepared by adding ⁇ OO ⁇ l of substrate solution to 250 ⁇ l enzyme solution plus 2 ⁇ 0 ⁇ l of 4% DMSO solution, and incubating at 37°C for 16 hours.
  • a black 96 well microtitre plate 100 ⁇ l of substrate solution is added to ⁇ O ⁇ l of 4% DMSO solution.
  • a similar non-specific background blank is also set up in which the ⁇ O ⁇ l of 4% DMSO solution additionally contains 40 ⁇ M phosphoramidon.
  • ⁇ O ⁇ l of enzyme solution is added to the assay and blank, and the 96 well plate placed in a BMG galaxy fluorescence reader, operating with the Biolise software package (BMG Lab technologies, Offenberg, Germany).
  • the plate is incubated in the fluorescence reader for 1 hour at 37°C and a fluorescence measurement taken every 3 minutes (Excitation (Ex) 486 nm / Emission (Em) 536 nm).
  • the proteolytic activity of SEP corresponds to the rate of increase in fluorescence of the sample - rate of increase in fluorescence units of the non-specific background blank.
  • the maximum velocity measurement (MaxV) calculated by the software over four successive readings is used for this calculation.
  • a fluorescence measurement taken from 200 ⁇ l of product in a well on an identical microtitre plate is taken. If required this value is used, together with the measured fluorescence units from the 60 min timepoint of the SEP assay, to calculate the percentage (%) of the substrate proteolysed during the 1 hour incubation period or to convert the measured rates of fluorescence increase into other useful units such as ng substrate proteolysed/min/ml enzyme.
  • the assay is used to calculate enzyme kinetic parameters such as Vmax and Km following standard principles described in Fundamentals of Enzyme Kinetics by Athel Cornish Bowden, 1979, published by Butterworths.
  • SEP inhibitors for example phosphoramidon
  • multiple SEP assays are performed as described above with a range of test concentrations of inhibitor included in the 50 ⁇ l of DMSO solution (made by appropriate dilution of a 10mM 100% DMSO stock of inhibitor with 4% DMSO/50mM HEPES pH7.4.).
  • a sigmoidal dose response curve is fitted to a plot of log inhibitor concentration versus MaxV (or % inhibition or % activity).
  • the IC 50 is calculated as the inhibitor concentration causing 50 % maximal inhibition.
  • a dose range of at least 10 inhibitor concentrations differing in half log unit increments is used.
  • the SEP assay is used to determine the Ki and mode of inhibition (i.e. whether the inhibition is competitive, mixed, non-competitive, etc.) following standard enzymology principles as described, for example, in Fundamentals of Enzyme Kinetics by Athel Cornish Bowden, 1979, published by Butterworths.
  • calcium-activated potassium channels includes large conductance calcium activated (BKc a ) channels (also referred to as Maxi K+ channels), small conductance calcium activated (SKca) channels and intermediate conductance calcium activated (IK Ca ) channels which are sometimes referred to as an hSK 4 channels or IK channels or hlKi channels.
  • BKc a large conductance calcium activated
  • SKca small conductance calcium activated
  • IK Ca intermediate conductance calcium activated
  • SKc a small conductance calcium activated
  • BK channels are gated by the concerted actions of internal calcium ions and membrane potential and have a unit conductance of 100 to 220 picoSiemens (pS); whereas Intermediate conductance (IK) and small conductance (SK) channels are gated solely by internal calcium ions.
  • IKca and SK Ca channels have a unit conductance of 20 to 85 pS and 2 to 20 pS, respectively, and are more sensitive to calcium than are BK channels. Each type of channel shows a distinct pharmacology (Ishii et al 1997).
  • intermediate conductance calcium activated (IKca) channel refers to a subtype of the calcium activated potassium channels which is characterised by the degree of ionic conductance that passes through the channel pore during a single opening (Fan et al 1995).
  • BK large conductance
  • IK intermediate conductance
  • modulating IKca channel activity means any one or more of: improving, increasing, enhancing, agonising, depolarising or upregulating IKca channel activity or that the Ca 2+ sensitivity of the IKca channel is increased - that is, the calcium concentration required to elicit IKca channel activity/opening is lowered.
  • the increase in the Ca 2+ sensitivity of the IKca channel may be increased/enhanced by a direct or indirect opening of the IKc a channels.
  • This increase in the Ca 2+ sensitivity of the IK Ca channel may result in a modification of the IK Ca channel characteristics such that the IK Ca channel opening is affected in such a way that the IKca channel opens earlier and/or at lower intracellular calcium concentrations and/or for longer periods of time and/or with an increased open time probability.
  • modulating IK Ca channel activity also includes the upregulation of IKca channel expression in corpus cavernosum smooth muscle tissue such as, for example, by an agent that increases the expression of the IK Ca channel and/or by the action of an agent on a substance that would otherwise impair and/or antagonise the modulation of IK Ca channel activity and/or the expression of the IKc a channel.
  • the modulator may have the structure of formula (I):
  • R1 is a H or a suitable substituent, such as an alkyl group which may be optionally substituted;
  • R2 is a H or a suitable substituent, preferably H
  • R3 represents one or more suitable optional substituents.
  • the modulator may have the structure of formula (1) :
  • X is selected from NR, O or S wherein R is H or alkyl (preferably lower alkyl, more preferably C1-6 alkyl) R1 is alkyl (preferably lower alkyl, more preferably C1-6 alkyl)
  • R2 is selected from H, halide, alkyl (preferably lower alkyl, more preferably C1- 6 alkyl), alkoxy (preferably lower alkoxy, more preferably C1-6 alkoxy)
  • R3 is selected from H, halide, alkyl (preferably lower alkyl, more preferably C1- 6 alkyl), alkoxy (preferably lower alkoxy, more preferably C1-6 alkoxy)
  • R4 is selected from H, halide, alkyl (preferably lower alkyl, more preferably C1- 6 alkyl), alkoxy (preferably lower alkoxy, more preferably C1-6 alkoxy)
  • R5 is selected from H, halide, alkyl (preferably lower alkyl, more preferably C1- 6 alkyl), alkoxy (preferably lower alkoxy, more preferably C1-6 alkoxy).
  • Aminophenols and aminothiophenols are usually prepared from the respective corresponding nitrophenols (4a) or nitrothiophenols (4b) by reduction. Many substituted nitrophenols (4a) and nitrothiophenols (4b) are commercially available.
  • the modulator is EBIO (1-ethyl-2-benzimidazolinone) or a mimetic thereof or a pharmaceutically acceptable salt of any thereof.
  • EBIO 1-ethyl-2-benzimidazolinone
  • the structure of EBIO is:
  • the agent has an IC 5 o value of less than 300nM, 250nM, 200nM, 1 ⁇ 0nM, preferably less than about 100 nM, preferably less than about 75 nM, preferably less than about 50 nM, preferably less than about 25 nM, preferably less than about 20 nM, preferably less than about 15 nM, preferably less than about 10 nM, preferably less than about 5 nM.
  • the agent has at least about a 26, 50, 75, 100 fold selectivity to the desired target, preferably at least about a 150 fold selectivity to the desired target, preferably at least about a 200 fold selectivity to the desired target, preferably at least about a 260 fold selectivity to the desired target, preferably at least about a 300 fold selectivity to the desired target, preferably at least about a 360 fold selectivity to the desired target.
  • the term "corpus cavernosum” refers inter alia to a mass of tissue found in the penis.
  • the body of the penis is composed of three cylindrical masses of tissue, each surrounded by fibrous tissue called the tunica albuginea.
  • the corpus cavernosum comprises smooth muscle cells.
  • the present invention also encompasses use as defined hereinbefore via administration of an NPYi, preferably an NPY Y1 i (and a PDEi, preferably a PDE ⁇ i, where applicable) before and/or during sexual stimulation.
  • NPYi preferably an NPY Y1 i
  • PDEi preferably a PDE ⁇ i, where applicable
  • sexual stimulation may be synonymous with the term "sexual arousal”.
  • This aspect of the present invention is advantageous because it provides systemic (physiological) selectivity. The natural cascade only occurs at the genitalia and not in other locations - e.g. in the heart etc. Hence, it is possible to achieve a selective effect on the genitalia via the MED treatment according to the present invention.
  • sexual stimulation may be one or more of a visual stimulation, a physical stimulation, an auditory stimulation, or a thought stimulation.
  • Agents for use in the treatment of male sexual dysfunction, in particular MED, according to the present invention may be any suitable agent that can act as an NPYi, preferably an NPY Y1i, and, where appropriate a combination of an NPYi, preferably an NPY Y1i, and a PDEi, preferably a PDE ⁇ i.
  • the term "agent” includes any entity capable of inhibiting NPY and/or NPY Y1 receptors.
  • agents can be an amino acid sequence or a chemical derivative thereof.
  • the substance may even be an organic compound or other chemical.
  • the agent may even be a nucleotide sequence - which may be a sense sequence or an anti-sense sequence.
  • the agent may even be an antibody.
  • the term "agent” includes, but is not limited to, a compound which may be obtainable from or produced by any suitable source, whether natural or not.
  • the agent may be designed or obtained from a library of compounds which may comprise peptides, as well as other compounds, such as small organic molecules, such as lead compounds.
  • the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised agent, a peptide cleaved from a whole protein, or a peptide synthesised synthetically (such as, by way of example, either using a peptide synthesiser or by recombinant techniques or combinations thereof, a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.
  • ACE assay An ACE assay is presented hereinbelow.
  • agents i.e. those that also display ACE inhibitory action
  • the NPY or NPY Y1 inhibitors according to the present invention has no, or substantially no, activity towards ACE.
  • agent may be a single entity or it may be a combination of agents.
  • the agent is an organic compound then for some applications - such as if the agent is an NPYi or an NPY Y1i - that organic compound may typically comprise two or more linked hydrocarbyl groups.
  • the agent comprises at least two cyclic groups - optionally wherein one of which cyclic groups may be a fused cyclic ring structure.
  • at least one of the cyclic groups is a heterocyclic group.
  • the heterocyclic group comprises at least one N in the ring. Examples of such compounds are presented herein.
  • the agent is an organic compound then for some applications - such as if the agent is an PDE ⁇ i - that organic compound may typically comprise two or more linked hydrocarbyl groups.
  • the agent comprises at least two cyclic groups - wherein one of which cyclic groups may be a fused cyclic ring structure.
  • at least one of the cyclic groups is a heterocyclic group.
  • the heterocyclic group comprises at least one N in the ring. Examples of such compounds are presented in the PDE ⁇ section herein.
  • the agent may contain halo groups.
  • halo means fluoro, chloro, bromo or iodo.
  • the agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.
  • the agent may be in the form of - and/or may be administered as - a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof.
  • a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof.
  • a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate.
  • the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
  • Suitable acid addition salts are formed from acids which form non-toxic salts and examples are the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p_- toluenesulphonate and pamoate salts.
  • Suitable base salts are formed from bases which form non-toxic salts and examples are the sodium, potassium, aluminium, calcium, magnesium, zinc and diethanolamine salts.
  • the agent may exist in polymorphic form.
  • the agent may contain one or more asymmetric carbon atoms and therefore exists in two or more stereoisomeric forms. Where an agent contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur.
  • the present invention includes the individual stereoisomers of the agent and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
  • Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of the agent or a suitable salt or derivative thereof.
  • An individual enantiomer of the agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
  • the present invention also includes all suitable isotopic variations of the agent or a pharmaceutically acceptable salt thereof.
  • An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
  • isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 0, 18 0, 31 P, 32 P, 35 S, 18 F and 36 CI, respectively.
  • isotopic variations of the agent and pharmaceutically acceptable salts thereof are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent and pharmaceutically acceptable salts thereof can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
  • the agent may be derived from a prodrug.
  • prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form the agent which are pharmacologically active.
  • pro-moieties for example as described in "Design of Prodrugs” by H. Bundgaard, Elsevier, 1985 (the disclosure of which is hereby incorporated by reference), may be placed on appropriate functionalities of the agents. Such prodrugs are also included within the scope of the invention.
  • inhibitor as used herein in relation to the NPYi or NPY Y1i (and where applicable PDEi or PDE ⁇ i compounds and other auxiliary active agents) is to be regarded as being interchangeable with the term antagonist.
  • the term "antagonist” means any agent that reduces the action of another agent or target.
  • the antagonistic action may result form a combination of the substance being antagonised (chemical antagonism) or the production of an opposite effect through a different target (functional antagonism or physiological antagonism) or as a consequence of competition for the binding site of an intermediate that links target activation to the effect observed (indirect antagonism).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the agent of the present invention and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1986).
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Preservatives may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be delivered by both routes.
  • the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
  • compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the agents of the present invention may also be used in combination with a cyclodextrin.
  • Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of a drug- cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes.
  • the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser.
  • the agents of the present invention are delivered systemically (such as orally, buccally, sublingually), more preferably orally.
  • the agent is in a form that is suitable for oral delivery.
  • the agent - when in use - in addition to acting peripherally on the genitalia the agent also acts on the central nervous system.
  • the agent - when in use - is not peripherally acting other than in respect of receptors located in the genitalia and preferably those associated with the corpus cavernosum.
  • the term "administered” includes delivery by viral or non-viral techniques.
  • Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno- associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.
  • Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • agents of the present invention may be administered alone but will generally be administered as a pharmaceutical composition - e.g. when the agent is in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the agent can be administered (e.g. orally or topically) in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
  • the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the routes for administration include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, penile, vaginal, epidural, sublingual.
  • oral e.g. as a tablet, capsule, or as an ingestable solution
  • mucosal e.g. as a nasal spray or aerosol for inhalation
  • nasal parenteral (e.g. by an injectable form)
  • gastrointestinal intraspinal, intra
  • agents need be administered by the same route.
  • the composition comprises more than one active component, then those components may be administered by different routes.
  • examples of such administration include one or more of: intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.
  • the agent is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • the agent of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrof luoroalkane such as 1 , 1 , 1 ,2-tetrafluoroethane (HFA 134ATM) or 1 ,1 ,1,2,3,3,3-heptafluoropropane (HFA 227EATM), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrof luoroalkane such as 1 , 1 , 1
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent and a suitable powder base such as lactose or starch.
  • the agent of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the agent of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route.
  • the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
  • a preservative such as a benzylalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • the agent of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • compositions of the present invention may be administered by direct injection.
  • the agent is administered orally.
  • the agent is administered topically.
  • a physician will determine the actual dosage which will be most suitable for an individual subject.
  • the specific dose level and frequency of dosage for any particular individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the agent and/or the pharmaceutical composition of the present invention may be administered in accordance with a regimen of from 1 to 10 times per day, such as once or twice per day.
  • the daily dosage level of the agent may be in single or divided doses.
  • the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • the dosages mentioned herein are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
  • the daily oral dose may be, for instance, between 20-1000 mg, preferably 60-300 mg for example.
  • the agents of the present invention may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.
  • Formulation 1 A tablet is prepared using the following ingredients:
  • the components are blended and compressed to form tablets each weighing 665mg.
  • Formulation 2 An intravenous formulation may be prepared as follows:
  • the term "individual” refers to vertebrates, particularly members of the mammalian species. The term includes but is not limited to domestic animals, sports animals, primates and humans.
  • the compounds of the invention are orally bioavailable.
  • Oral bioavailabiity refers to the proportion of an orally administered drug that reaches the systemic circulation.
  • the factors that determine oral bioavailability of a drug are dissolution, membrane permeability and metabolic stability.
  • a screening cascade of firstly in vitro and then in vivo techniques is used to determine oral bioavailabiity.
  • Dissolution the solubilisation of the drug by the aqueous contents of the gastro-intestinal tract (GIT)
  • GIT gastro-intestinal tract
  • the compounds of the invention have a minimum solubility of 50 mcg/ml. Solubility can be determined by standard procedures known in the art such as described in Adv. Drug Deliv. Rev. 23, 3-25, 1997.
  • Membrane permeability refers to the passage of the compound through the cells of the GIT. Lipophilicity is a key property in predicting this and is defined by in vitro Log D 7 . 4 measurements using organic solvents and buffer.
  • the compounds of the invention have a Log D7.4 of -2 to +4, more preferably -1 to +2.
  • the log D can be determined by standard procedures known in the art such as described in J. Pharm. Pharmacol. 1990, 42:144.
  • caco-2 flux Cell monolayer assays such as CaC0 2 add substantially to prediction of favourable membrane permeability in the presence of efflux transporters such as p-glycoprotein, so-called caco-2 flux.
  • compounds of the invention have a caco-2 flux of greater than 2x10 "6 cms "1 , more preferably greater than 5x10 "6 cms "1 .
  • the caco flux value can be determined by standard procedures known in the art such as described in J. Pharm. Sci, 1990, 79, 695-600
  • Metabolic stability addresses the ability of the GIT or the liver to metabolise compounds during the absorption process: the first pass effect.
  • Assay systems such as microsomes, hepatocytes etc are predictive of metabolic liability.
  • the compounds of the Examples show metabolic stability in the assay system that is commensurate with an hepatic extraction of less then 0.5. Examples of assay systems and data manipulation are described in Curr. Opin. Drug Disc. Devel., 201 , 4, 36-44, Drug Met. Disp.,2000, 28, 1518-1523.
  • NPYi/NPY Y1 i (and/or PDEi/PDE ⁇ i where applicable) suitable for the use according to the present invention will be prepared by chemical synthesis techniques.
  • agent or target or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesise the agent in whole or in part.
  • peptides can be synthesised by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • Direct synthesis of the agent or variants, homologues, derivatives, fragments or mimetics thereof can be performed using various solid-phase techniques (Roberge JY et al (1996) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising the agent or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant agent or target, such as, for example, a variant NPY or NPY Y1.
  • the coding sequence of the agent target or variants, homologues, derivatives, fragments or mimetics thereof may be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215- 23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
  • mimetic relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a reference agent to a target. That is a mimetic may be a functional equivalent to a known agent.
  • derivative or "derivatised” as used herein includes chemical modification of an agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
  • the agent may be a chemically modified agent.
  • the chemical modification of an agent may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the agent and the target.
  • the identified agent may act as a model (for example, a template) for the development of other compounds.
  • an NPY or NPY Y1 receptor may be used as a target in screens to identify agents capable of inhibiting NPY or NPY Y1.
  • the target may comprise an amino acid sequence encoded by the nucleotide sequences shown as SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or a variant, homologue, derivative or fragment thereof which is prepared by recombinant and/or synthetic means or an expression entity comprising same.
  • an NPY or NPY Y1 receptor may be used to as a target to identify agents capable of mediating an increase in intracavernosal pressure through the inhibition of NPY or NPY Y1.
  • the target may be suitable tissue extract.
  • the target may even be a combination of such tissue and/or recombinant targets.
  • the agent of the present invention may be prepared by recombinant DNA techniques.
  • the agent is an NPYi or an NPY Y1i.
  • the NPYi or the NPY Y1 i may be prepared by recombinant DNA techniques.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “protein”.
  • amino acid sequence may be prepared isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
  • the present invention provides an amino acid sequence that is capable of acting as a target in an assay for the identification of one or more agents and/or derivatives thereof.
  • the target is an NPY or NPY Y1 receptor.
  • the NPY or NPY Y1 receptor is an isolated NPY or NPY Y1 receptor and/or is purified and/or is non-native.
  • the NPY or NPY Y1 receptor of the present invention may be in a substantially isolated form. It will be understood that the NPY or NPY Y1 receptor may be mixed with carriers or diluents which will not interfere with the intended purpose of the receptor and which will still be regarded as substantially isolated.
  • the NPY or NPY Y1 receptor of the present invention may also be in a substantially pure form, in which case it will generally comprise the NPY or NPY Y1 receptor in a preparation in which more than 90%, e.g. 9 ⁇ %, 98% or 99% of the NPY or NPY Y1 receptor in the preparation is a peptide obtainable from the expression of SEQ ID No. 1, 2 or 3 or variants, homologues, derivative or fragments thereof.
  • nucleotide sequence is synonymous with the term “polynucleotide”.
  • the nucleotide sequence may be DNA or RNA of genomic or synthetic or of recombinant origin.
  • the nucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
  • the nucleotide sequence is DNA.
  • the nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA).
  • the nucleotide sequence is cDNA.
  • the nucleotide sequence may be the same as the naturally occurring form for this aspect.
  • the present invention provides a nucleotide sequence encoding a substance capable of acting as a target in an assay for the identification of one ore more agents and/or derivative thereof.
  • the nucleotide sequence encodes an NPY or an NPY Y1 receptor.
  • nucleotide sequences can encode the same target as a result of the degeneracy of the genetic code.
  • skilled persons may, using routine techniques, make nucleotide substitutions that do not substantially affect the activity encoded by the nucleotide sequence of the present invention to reflect the codon usage of any particular host organism in which the target is to be expressed.
  • variant in relation to the nucleotide sequence set out in the attached sequence listings include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence encodes a functional target according the present invention (or even an agent according to the present invention if said agent comprises a nucleotide sequence or an amino acid sequence).
  • sequence homology preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the NPY sequence cross referenced to herein. More preferably there is at least 95%, more preferably at least 98%, homology.
  • Nucleotide homology comparisons may be conducted as described above.
  • a preferred sequence comparison program is the GCG Wisconsin Bestfit program described above.
  • the default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch.
  • the default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.
  • the present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length. These sequences could be used a probes, such as in a diagnostic kit.
  • the present invention also encompasses the use of variants, homologue and derivatives thereof.
  • the term "homology" can be equated with "identity”.
  • an homologous sequence is taken to include an amino acid sequence which may be at least 75, 8 ⁇ or 90% identical, preferably at least 95 or 98% identical.
  • homology should typically be considered with respect to those regions of the sequence known to be essential for an activity.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • BLAST and FASTA are available for offline and online searching (see Ausubel etal., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
  • a new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-60; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • Non- homologous substitution may also occur i.e.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine thienylalanine
  • naphthylalanine phenylglycine
  • Replacements may also be made by unnatural amino acids include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine * , p-CI- phenylalanine*, p-Br-phenylalanine*, p-l-phenylalanine*, L-allyl-glycine*, ⁇ - alanine*, L- ⁇ -amino butyric acid*, L- ⁇ -amino butyric acid*, L- ⁇ -amino isobutyric acid*, L- ⁇ -amino caproic acid*, 7-amino heptanoic acid * , L-methionine sulfone* * , L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine * , L- hydroxyproline* L-thioproline*,
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ -alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ -alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the - carbon substituent group is on the residue's nitrogen atom rather than the ⁇ - carbon.
  • Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.
  • hybridisation shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • Nucleotide sequences of the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the corresponding complementary nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
  • the term "selectively hybridizable" means that the nucleotide sequence, when used as a probe, is used under conditions where a target nucleotide sequence is found to hybridise to the probe at a level significantly above background.
  • the background hybridisation may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened.
  • background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
  • Hybridisation conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • a maximum stringency hybridisation can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences.
  • both strands of the duplex either individually or in combination, are encompassed by the present invention.
  • the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
  • Nucleotide sequences which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of sources. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • mammalian cells e.g. rat, mouse, bovine and primate cells
  • sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the nucleotide sequence set out in herein under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the amino acid and/or nucleotide sequences of the present invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences, such as the nucleotide sequence set out in SEQ ID Nos 1 or 2 of the sequence listings of the present invention. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the protein encoded by the nucleotide sequences.
  • the nucleotide sequences of the present invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g.
  • nucleotide sequences may be cloned into vectors.
  • primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term nucleotide sequence of the invention as used herein.
  • nucleotide sequences such as a DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • PCR polymerase chain reaction
  • This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • PCR polymerase chain reaction
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used to clone and express the target sequences. As will be understood by those of skill in the art, for certain expression systems, it may be advantageous to produce the target sequences with non-naturally occurring codons.
  • Codons preferred by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of the target expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
  • an agent i.e. an NPYi or an NPY Y1i
  • an agent may be administered directly to an individual.
  • a vector comprising a nucleotide sequence encoding an agent of the present invention is administered to an individual.
  • the recombinant agent is prepared and/or delivered to a target site using a genetic vector.
  • a vector is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a host and/or a target cell for the purpose of replicating the vectors comprising the nucleotide sequences of the present invention and/or expressing the proteins of the invention encoded by the nucleotide sequences of the present invention.
  • vectors used in recombinant DNA techniques include but are not limited to plasmids, chromosomes, artificial chromosomes or viruses.
  • vector includes expression vectors and/or transformation vectors.
  • expression vector means a construct capable of in vivo or in vitro/ex vivo expression.
  • transformation vector means a construct capable of being transferred from one species to another.
  • the vectors comprising nucleotide sequences encoding an agent of the present invention for use in treating MSDs such as MED may be administered directly as "a naked nucleic acid construct", preferably further comprising flanking sequences homologous to the host cell genome.
  • naked DNA refers to a plasmid comprising a nucleotide sequences encoding an agent of the present invention together with a short promoter region to control its production. It is called “naked” DNA because the plasmids are not carried in any delivery vehicle.
  • a DNA plasmid enters a host cell, such as a eukaryotic cell, the proteins it encodes (such as an agent of the present invention) are transcribed and translated within the cell.
  • the vectors comprising nucleotide sequences of the present invention or an agent of the present invention (i.e. NPYi or NPY Y1 i) or a target of the present invention (i.e. NPY or NPY Y1) may be introduced into suitable host cells using a variety of non-viral techniques known in the art, such as transfection, transformation, electroporation and biolistic transformation.
  • transfection refers to a process using a non-viral vector to deliver a gene to a target mammalian cell.
  • Typical transfection methods include electroporation, DNA biolistics, lipid- mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic lipids or polylysine, 1 , 2,-bis (oleoyloxy)-3- (trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
  • CFAs cationic facial amphiphiles
  • Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
  • transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM).
  • cationic agents for example calcium phosphate and DEAE-dextran
  • lipofectants for example lipofectamTM and transfectamTM.
  • nucleic acid constructs are mixed with the transfection agent to produce a composition.
  • the vectors comprising an agent or target of the present invention or nucleotide sequences of the present invention may be introduced into suitable host cells using a variety of viral techniques which are known in the art, such as for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses.
  • the vector is a recombinant viral vectors.
  • Suitable recombinant viral vectors include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, a retroviral vector, lentiviral vectors, baculoviral vectors, pox viral vectors or parvovirus vectors (see Kestler et al 1999 Human Gene Ther 10(10): 1619-32).
  • AAV adeno-associated viral vectors
  • herpes-virus vectors a retroviral vector
  • lentiviral vectors lentiviral vectors
  • baculoviral vectors pox viral vectors or parvovirus vectors
  • target vector refers to a vector whose ability to infect/transfect/transduce a cell or to be expressed in a host and/or target cell is restricted to certain cell types within the host organism, usually cells having a common or similar phenotype.
  • the nucleotide sequences encoding an agent (i.e. NPYi or NPY Y1 i or PDEi or PDE ⁇ i) of the present invention or a target (such as NPY or NPY Y1) may be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleotide sequence in a compatible host cell.
  • the invention provides a method of making a target of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • an agent of the present invention or a nucleotide sequence of present invention or a target of the present invention which is inserted into a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence, such as the coding sequence of the NPY or NPY Y1 of the present invention by the host cell, i.e. the vector is an expression vector.
  • a control sequence that is capable of providing for the expression of the coding sequence, such as the coding sequence of the NPY or NPY Y1 of the present invention by the host cell, i.e. the vector is an expression vector.
  • An agent of the present invention or a target produced by a host recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing an agent or target of the present invention coding sequences can be designed with signal sequences which direct secretion of the agent or target of the present invention coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • the vectors of the present invention may be transformed or transfected into a suitable host cell and/or a target cell as described below to provide for expression of an agent or a target of the present invention.
  • This process may comprise culturing a host cell and/or target cell transformed with an expression vector under conditions to provide for expression by the vector of a coding sequence encoding an agent or a target of the present invention and optionally recovering the expressed agent or target of the present invention.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampiciUin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • the expression of an agent of the present invention or target of the present invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
  • inducible expression production of an agent of the present invention or a target can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
  • the NPY or NPY Y1 or an agent (i.e. NPYi or NPY Y1i) of the present invention may be expressed as a fusion protein to aid extraction and purification and/or delivery of the agent of the present invention or the NPY/NPY Y1 receptor target to an individual and/or to facilitate the development of a screen for agents.
  • fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target.
  • the fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention.
  • the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system.
  • the antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
  • the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence for example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.
  • host cells can be employed for expression of the nucleotide sequences encoding the agent - such as an agent of the present invention - or a NPY/NPY Y1 receptor target of the present invention. These cells may be both prokaryotic and eukaryotic host cells. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof.
  • bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof.
  • suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A- 0253465), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species.
  • typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var.
  • suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
  • post-translational modifications e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • Preferred host cells are able to process the expression products to produce an appropriate mature polypeptide.
  • processing includes but is not limited to glycosylation, ubiquitination, disulfide bond formation and general post-translational modification.
  • the agent may be an antibody.
  • the target may be an antibody.
  • Antibodies may be produced by standard techniques, such as by immunisation with the substance of the invention or by using a phage display library.
  • the term "antibody”, unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library, as well as mimetics thereof.
  • Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
  • the antibodies and fragments thereof may be humanised antibodies. Neutralising antibodies, i.e., those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • an immunogenic polypeptide bearing a epitope(s) obtainable from an identified agent and/or substance of the present invention.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebactehum parvum are potentially useful human adjuvants which may be employed if purified the substance polypeptide is administered to immunologically compromised individuals for the purpose of stimulating systemic defence.
  • Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from an identified agent and/or substance of the present invention contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
  • Monoclonal antibodies directed against epitopes obtainable from an identified agent and/or substance of the present invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
  • Monoclonal antibodies to the substance and/or identified agent may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96).
  • Antibodies both monoclonal and polyclonal, which are directed against epitopes obtainable from an identified agent and/or substance are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
  • Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the substance and/or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for the substance may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1).
  • reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
  • reporter molecules include but are not limited to ⁇ -galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, ⁇ -glucuronidase, exo-glucanase and glucoamylase.
  • radiolabelled or fluorescent tag-labelled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes.
  • the production of the reporter molecule is measured by the enzymatic activity of the reporter gene product, such as ⁇ - galactosidase.
  • a variety of protocols for detecting and measuring the expression of the target such as by using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non-interfering epitopes on polypeptides is preferred, but a competitive binding assay may be employed.
  • Means for producing labelled hybridisation or PCR probes for detecting the target polynucleotide sequences include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled nucleotide.
  • the coding sequence, or any portion of it may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labelled nucleotides.
  • reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
  • Patents teaching the use of such labels include US-A-3817837; US-A-3860762; US-A-3939360; US-A-3996345; US-A-4277437; US-A-4276149 and US-A-4366241.
  • recombinant immunoglobulins may be produced as shown in US-A-4816567.
  • Additional methods to quantify the expression of a particular molecule include radiolabeling (Melby PC et al 1993 J Immunol Methods 169:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantification of multiple samples may be speeded up by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or calorimetric response gives rapid quantification.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed.
  • the nucleotide sequence is inserted within a marker gene sequence, recombinant cells containing the same may be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a target coding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the target as well.
  • host cells which contain the coding sequence for the target and express the target coding regions may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridisation and protein bioassay or immunoassay techniques which include membrane-based, solution-based, or chip-based technologies for the detection and/or quantification of the nucleic acid or protein.
  • any one or more of appropriate targets - such as an amino acid sequence and/or nucleotide sequence - may be used for identifying an agent, e.g. NPYi or an NPY Y1i, in any of a variety of drug screening techniques.
  • the target employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the target may even be within an animal model, wherein said target may be an exogenous target or an introduced target.
  • the animal model will be a non-human animal model. The abolition of target activity or the formation of binding complexes between the target and the agent being tested may be measured.
  • Techniques for drug screening may be based on the method described in Geysen, European Patent Application 84/03564, published on September 13, 1984.
  • large numbers of different small peptide test compounds are synthesised on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known in the art.
  • a purified target can also be coated directly onto plates for use in a drug screening techniques.
  • non-neutralising antibodies can be used to capture the peptide and immobilise it on a solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a target specifically compete with a test compound for binding to a target.
  • HTS high throughput screening
  • the screen of the present invention comprises at least the following steps (which need not be in this same consecutive order): (a) conducting an in vitro screen to determine whether a candidate agent has the relevant activity (such as modulation of NPY, in particular NPY Y1, such as NPY or NPY Y1 from dog kidney); (b) conducting one or more selectivity screens to determine the selectivity of said candidate agent (e.g. to see if said agent is also an ACE inhibitor - such as by using the assay protocol presented herein and/or see if said agent is active against NPY Y2 and/or NPY Y5); and (c) conducting an in vivo screen with said candidate agent (e.g. using a functional animal model, including determining the selectivity of the agent by determining the effect of the agent on arterial blood pressure).
  • screen (c) is performed.
  • the present invention also provides a diagnostic composition or kit for the detection of a pre-disposition for MED.
  • the composition or kit will comprise an entity that is capable of indicating the presence of one or more - or even the absence of one or more - of the targets in a test sample.
  • the test sample is obtained from the penis.
  • the diagnostic composition may comprise any one of the nucleotide sequences mentioned herein or a variant, homologue, fragment or derivative thereof, or a sequence capable of hybridising to all or part of any one of the nucleotide sequence.
  • normal or standard values from a target should be established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with an antibody to a target under conditions suitable for complex formation which are well known in the art.
  • the amount of standard complex formation may be quantified by comparing it to a dilution series of positive controls where a known amount of antibody is combined with known concentrations of a purified target. Then, standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by MED. Deviation between standard and subject values establishes the presence of the disease state.
  • a target itself, or any part thereof, may provide the basis for a diagnostic and/or a therapeutic compound.
  • target polynucleotide sequences may be used to detect and quantify gene expression in conditions, disorders or diseases in which MED may be implicated.
  • the target encoding polynucleotide sequence may be used for the diagnosis of MED resulting from expression of the target.
  • polynucleotide sequences encoding a target may be used in hybridisation or PCR assays of tissues from biopsies or autopsies or biological fluids, to detect abnormalities in target expression.
  • the form of such qualitative or quantitative methods may include Southern or northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin or chip technologies; and ELISA or other multiple sample formal technologies. All of these techniques are well known in the art and are in fact the basis of many commercially available diagnostic kits.
  • Such assays may be tailored to evaluate the efficacy of a particular therapeutic treatment regime and may be used in animal studies, in clinical trials, or in monitoring the treatment of an individual.
  • a normal or standard profile for target expression should be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with the target or a portion thereof, under conditions suitable for hybridisation or amplification. Standard hybridisation may be quantified by comparing the values obtained for normal subjects with a dilution series of positive controls run in the same experiment where a known amount of purified target is used. Standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to expression of the target coding sequence.
  • Deviation between standard and subject values establishes the presence of the disease state. If disease is established, an existing therapeutic agent is administered, and treatment profile or values may be generated. Finally, the assay may be repeated on a regular basis to evaluate whether the values progress toward or return to the normal or standard pattern. Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.
  • the present invention relates to the use of a target polypeptide, or variant, homologue, fragment or derivative thereof, to produce anti-target antibodies which can, for example, be used diagnostically to detect and quantify target levels in MED.
  • the present invention further provides diagnostic assays and kits for the detection of a target in cells and tissues comprising a purified target which may be used as a positive control, and anti-target antibodies.
  • Such antibodies may be used in solution-based, membrane-based, or tissue-based technologies to detect any disease state or condition related to the expression of target protein or expression of deletions or a variant, homologue, fragment or derivative thereof.
  • the present invention also includes a diagnostic composition or diagnostic methods or kits for (i) detection and measurement of NPY and NPY Y1 activity in biological fluids and tissue; and/or (ii) localisation of a NPY and NPY Y1 activity in erectile tissues; and/or for (iii) the detection of a predisposition to a male sexual dysfunction, such as MED.
  • the composition or kit will comprise an entity that is capable of indicating the presence of one or more - or even the absence of one or more - targets, such as NPY or NPY Y1 activity in a test sample.
  • the test sample is obtained from male sexual genitalia or a secretion thereof or therefrom.
  • the diagnostic composition may comprise any one of the nucleotide sequences mentioned herein or a variant, homologue, fragment or derivative thereof, or a sequence capable of hybridising to all or part of any one of the nucleotide sequence.
  • normal or standard values from a target should be established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with, for example, an antibody to a target under conditions suitable for complex formation which are well known in the art.
  • the amount of standard complex formation may be quantified by comparing it to a dilution series of positive controls where a known amount of antibody is combined with known concentrations of a purified target.
  • standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a male sexual dysfunction (such as MED). Deviation between standard and subject values establishes the presence of the disease state.
  • a target itself, or any part thereof, may provide the basis for a diagnostic and/or a prophylactic and/or therapeutic compound.
  • target polynucleotide sequences may be used to detect and quantify gene expression in conditions, disorders or diseases in which male sexual dysfunction, particularly MED, may be implicated.
  • the target encoding polynucleotide sequence may be used for the diagnosis of SD resulting from expression of the target.
  • polynucleotide sequences encoding a target may be used in hybridisation or PCR assays of tissues from biopsies or autopsies or biological fluids, to detect abnormalities in target expression.
  • the form of such qualitative or quantitative methods may include Southern or northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin or chip technologies; and ELISA or other multiple sample formal technologies. All of these techniques are well known in the art and are in fact the basis of many commercially available diagnostic kits.
  • Such assays may be tailored to evaluate the efficacy of a particular therapeutic treatment regime and may be used in animal studies, in clinical trials, or in monitoring the treatment of an individual.
  • a normal or standard profile for target expression should be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with the target or a portion thereof, under conditions suitable for hybridisation or amplification. Standard hybridisation may be quantified by comparing the values obtained for normal subjects with a dilution series of positive controls run in the same experiment where a known amount of purified target is used. Standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to expression of the target coding sequence.
  • Deviation between standard and subject values establishes the presence of the disease state. If disease is established, an existing therapeutic agent is administered, and treatment profile or values may be generated. Finally, the assay may be repeated on a regular basis to evaluate whether the values progress toward or return to the normal or standard pattern. Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.
  • the present invention relates to the use of a target polypeptide, or variant, homologue, fragment or derivative thereof, to produce anti-target antibodies which can, for example, be used diagnostically to detect and quantify target levels in male sexual dysfunctioning states.
  • the present invention further provides diagnostic assays and kits for the detection of a target in cells and tissues comprising a purified target which may be used as a positive control, and anti-target antibodies.
  • a purified target which may be used as a positive control, and anti-target antibodies.
  • Such antibodies may be used in solution-based, membrane-based, or tissue-based technologies to detect any disease state or condition related to the expression of target protein or expression of deletions or a variant, homologue, fragment or derivative thereof.
  • compositions and/or kits comprising these entities may be used for a rapid, reliable, sensitive, and specific measurement and localisation of NPY or NPY Y1 activity in erectile tissue extracts.
  • the kit may indicate the existence of male sexual dysfunction, such as MED.
  • the diagnostic compositions and/or methods and/or kits may be used in the following techniques which include but are not limited to; competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, immunohistochemistry and immunocytochemistry.
  • an immunohistochemistry kit may also be used for localisation of NPY or NPY Y1 activity in genital tissue.
  • This immunohistochemistry kit permits localisation of NPY or NPY Y1 in tissue sections and cultured cells using both light and electron microscopy which may be used for both research and clinical purposes. Such information may be useful for diagnostic and possibly therapeutic purposes in the detection and/or prevention and/or treatment of MED.
  • the range, sensitivity, precision, reliability, specificity and reproducibility of the assay are established. Intraassay and interassay variation is established at 20%, 60% and 80% points on the standard curves of displacement or activity.
  • nucleic acid hybridisation or PCR probes which are capable of detecting (especially those that are capable of selectively selecting) polynucleotide sequences, including genomic sequences, encoding a target coding region, such as an NPY or an NPY Y1 receptor, or closely related molecules, such as alleles.
  • the specificity of the probe i.e., whether it is derived from a highly conserved, conserved or non-conserved region or domain, and the stringency of the hybridisation or amplification (high, intermediate or low) will determine whether the probe identifies only naturally occurring target coding sequence, or related sequences.
  • Probes for the detection of related nucleic acid sequences are selected from conserved or highly conserved nucleotide regions of target family members and such probes may be used in a pool of degenerate probes.
  • nucleic acid probes are selected from the non-conserved nucleotide regions or unique regions of the target polynucleotides.
  • non-conserved nucleotide region refers to a nucleotide region that is unique to a target coding sequence disclosed herein and does not occur in related family members.
  • PCR as described in US-A-4683196, US-A-4800195 and US-A-4966188 provides additional uses for oligonucleotides based upon target sequences.
  • oligomers are generally chemically synthesised, but they may be generated enzymatically or produced from a recombinant source.
  • Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'- >3') and one with antisense (3' ⁇ - ⁇ ') employed under optimised conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantification of closely related DNA or RNA sequences.
  • the nucleic acid sequence for an agent or a target can also be used to generate hybridisation probes as previously described, for mapping the endogenous genomic sequence.
  • the sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in situ hybridisation to chromosomal spreads (Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City), flow-sorted chromosomal preparations, or artificial chromosome constructions such as YACs, bacterial artificial chromosomes (BACs), bacterial PI constructions or single chromosome cDNA libraries.
  • nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. between normal, carrier or affected individuals.
  • organism in relation to the present invention includes any organism that could comprise the target and/or products obtained therefrom.
  • organisms may include a mammal, a fungus, yeast or a plant.
  • transgenic organism in relation to the present invention includes any organism that comprises the target and/or products obtained therefrom.
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1996), John Wiley & Sons, Inc.
  • nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • the transgenic organism can be a yeast.
  • yeast have also been widely used as a vehicle for heterologous gene expression.
  • the species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression.
  • Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
  • Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
  • yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
  • expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast.
  • the constructs contain a promoter active in yeast fused to the nucleotide sequence of the present invention, usually a promoter of yeast origin, such as the GAL1 promoter, is used.
  • a promoter of yeast origin such as the GAL1 promoter
  • a signal sequence of yeast origin such as the sequence encoding the SUC2 signal peptide, is used.
  • a terminator active in yeast ends the expression system.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
  • the transformed yeast cells are selected using various selective markers.
  • markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, e.g. G418.
  • Another host organism is a plant.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • Several techniques exist for inserting the genetic information the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system.
  • a review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-226) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
  • the present invention also provides a method of transforming a host cell with a nucleotide sequence that is to be the target or is to express the target.
  • Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).
  • Potency values for ACE or selectivity values for inhibitors of NPY or NPY Y1 over ACE are determined by the following assay.
  • Soluble ACE activity is obtained from the kidney cortex and assayed by measuring the rate of cleavage of the ACE substrate Abz-Gly-p-nitro-Phe-Pro- OH to generate its fluorescent product, Abz-Gly.
  • Tris (Fisher T/P630/60) is diluted in 1 litre of water and the pH adjusted to 7.1 using 6M HCl at room temperature. To this 18.22g Mannitol (Sigma M- 9546) is added.
  • ACE substrate is stored as a powder at -20°C.
  • a 2mM stock is made by gently re-suspending the substrate in ACE buffer, this must not be vortexed or sonicated. 400 ⁇ l aliquots of the 2mM stock are stored at -20°C for up to one month. 1.7 Total product
  • Samples corresponding to 100% substrate to product conversion are included on the plate to enable the % substrate turnover to be determined (see calculations).
  • the total product is generated by incubating 1ml of 2mM substrate with 20 ⁇ l of enzyme stock for 24 hours at 37°C. 1.8 Stop solution.
  • EDTA Promega CAS[6081/92/6]
  • DMSO Dimethyl sulphoxide
  • 3.1 Human ACE is obtained from the kidney cortex using a method adapted from Booth, A.G. & Kenny, A.J. (1974) Biochem. J. 142, 676-581.
  • 3.3 Frozen kidneys are allowed to thaw at room temperature and the cortex is dissected away from the medulla.
  • the cortex is finely chopped and homogenised in approximately 10 volumes of homogenisation buffer-1 (1.4) using a Braun miniprimer (2.2).
  • the homogenate is centrifuged at 1 ,500g (3,820rpm) for 12 minutes in a Beckman centrifuge (2.3) before removing the supernatant to a fresh centrifuge tube and discarding the pellet.
  • the activity of the previously aliquoted ACE is measured by its ability to cleave the ACE specific peptide substrate.
  • Porcine ACE (1.2) is defrosted and resuspended in ACE buffer (1.6) at 0.004U/ ⁇ l, this is frozen down in 50 ⁇ l aliquots.
  • the 2mM substrate stock is diluted 1 :100 to make a 20 ⁇ M solution. 100 ⁇ l of 20 ⁇ M substrate is added to each well (final concentration in the assay 10 ⁇ M). 4.5 50 ⁇ l of a range of enzyme dilutions is added to initiate the reaction (usually 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200 are used). 50 ⁇ l of ACE buffer is added to blank wells.
  • Steps 5.2 and 5.3 can be carried out either by hand or using the Packard multiprobe robots
  • the 2mM substrate stock is diluted 1:100 in ACE buffer to make a 20 ⁇ M solution (10 ⁇ M final concentration in the assay) (110 ⁇ l of 2mM substrate added to 10.89ml buffer is enough for 1 plate).
  • the 0.5mM EDTA stock is diluted 1:250 to make a 2mM stock (44 ⁇ l EDTA to 10.96ml ACE buffer).
  • Table 1 Reagents added to 96 well plate.
  • the reaction is initiated by the addition of the ACE enzyme before incubating at 37°C for 1 hour in a shaking incubator.
  • reaction is stopped by the addition of 100 ⁇ l 2mM EDTA and incubated at 37°C for 20 minutes in a shaking incubator, before being read on the BMG Fluostar Galaxy (ex320/em420).
  • the activity of the ACE enzyme is determined in the presence and absence of compound and expressed as a percentage.
  • Mean FU of controls - Mean FU of blanks X 100 Mean FU of totals - Mean FU of blanks
  • a sigmoidal dose-response curve is fitted to the % activities (% of control) vs compound concentration and IC 50 values calculated using LabStats fit-curve in Excel.
  • PDE action potency values referred to herein are determined by the following assays:
  • Phosphodiesterase (PDE) inhibitory activity Phosphodiesterase (PDE) inhibitory activity
  • Preferred PDE compounds suitable for use in accordance with the present invention are potent and selective cGMP PDE ⁇ inhibitors.
  • In vitro PDE inhibitory activities against cyclic guanosine 3', ⁇ '-monophosphate (cGMP) and cyclic adenosine 3', ⁇ '-monophosphate (cAMP) phosphodiesterases can be determined by measurement of their IC 50 values (the concentration of compound required for 50% inhibition of enzyme activity).
  • the required PDE enzymes can be isolated from a variety of sources, including human corpus cavernosum, human and rabbit platelets, human cardiac ventricle, human skeletal muscle and bovine retina, essentially by the method of W.J. Thompson and M.M. Appleman (Biochem., 1971, 10, 311).
  • the cGMP-specific PDE (PDE5) and the cGMP-inhibited cAMP PDE (PDE3) can be obtained from human corpus cavernosum tissue, human platelets or rabbit platelets; the cGMP-stimulated PDE (PDE2) was obtained from human corpus cavernosum; the calcium/calmodulin (Ca/CAM)-dependent PDE (PDE1) from human cardiac ventricle; the cAMP-specific PDE (PDE4) from human skeletal muscle; and the photoreceptor PDE (PDE6) from bovine retina.
  • Phosphodiesterases 7-11 can be generated from full length human recombinant clones transfected into SF9 cells.
  • Assays can be performed either using a modification of the "batch" method of W.J. Thompson et a[. (Biochem., 1979, 18, 5228) or using a scintillation proximity assay for the direct detection of AMP/GMP using a modification of the protocol described by Amersham pic under product code TRKQ7090/7100.
  • the final assay volume was made up to 100 ⁇ l with assay buffer [20 mM Tris-HCl pH 7.4, 5 mM MgCI 2 , 1 mg/ml bovine serum albumin]. Reactions were initiated with enzyme, incubated for 30-60 min at 30°C to give ⁇ 30% substrate turnover and terminated with 50 ⁇ l yttrium silicate SPA beads (containing 3 mM of the respective unlabelled cyclic nucleotide for PDEs 9 and 11).
  • FIG. 2 which shows a graph
  • Figure 3 which shows a graph
  • Figure 8 which shows a nucleotide sequence
  • Figure 1 shows the effect of an NPY Y1 antagonist BIBP3226 (1-100 ⁇ g/kg iv) on intracavernosal pressure (ICP) at stimulation in an anaesthetised rabbit. (*P ⁇ 0.05, Students T-test);
  • Figure 2 shows the effect of a PDE ⁇ inhibitor on intracavernosal pressure (ICP) at stimulation in the anaesthetised rabbit. Data is expressed in percentage increase in ICP over control increases. (*P ⁇ 0.01, Students T-test unpaired compared with control increases;
  • Figure 4 shows a human neuropeptide Y (NPY) nucleotide sequence (SEQ ID No. 1);
  • Figure ⁇ shows a human NPY Y1 receptor nucleotide sequence (SEQ ID No. 2);
  • Figure 6 shows a human NPY Y2 receptor nucleotide sequence (SEQ ID No. 3);
  • Figures 7 and 8 show nucleotide sequences (cDNAs) coding for human SEP (SEQ ID No. 4 and SEQ ID No. ⁇ respectively).
  • SEQ ID No. ⁇ includes ⁇ ' and 3' partial vector sequences;
  • Figure 9 shows an amino acid sequence of a human SEP protein (SEQ ID No. 6).
  • Figure 10 shows the effect of an NPY Y1 receptor antagonist, a PDE ⁇ inhibitor, and a combination of an NPY Y1 receptor antagonist and PDE ⁇ inhibitor on intracavernosal pressure (ICP) at stimulation in the anaesthetised rabbit. Data is expressed in percentage increase in ICP over control increases.
  • mice Male New Zealand rabbits ( ⁇ 2. ⁇ kg) were pre-medicated with a combination of Medetomidine (Domitor®) 0.5ml/kg i.m., and Ketamine (Vetalar®) 0.2 ⁇ ml/kg i.m. whilst maintaining oxygen intake via a face mask.
  • the rabbits were tracheotomised using a PortexTM uncuffed endotracheal tube 3 ID., connected to ventilator and maintained at a ventilation rate of 30-40 breaths per minute, with an approximate tidal volume of 18-20 ml, and a maximum airway pressure of 10 cm H 2 0. Anaesthesia was then switched to Isoflurane and ventilation continued with 0 2 at 21/min.
  • the right marginal ear vein was cannulated using a 23G or 24G catheter, and Lactated Ringer solution perfused at O. ⁇ ml/min.
  • the rabbit was maintained at 3% Isoflurane during invasive surgery, dropping to 2% for maintenance anaesthesia.
  • the left jugular vein was exposed, isolated and then cannulated with a PVC catheter (17G) for the infusion of drugs and compounds.
  • the left groin area of the rabbit was shaved and a vertical incision was made approximately 5cm in length along the thigh.
  • the femoral vein and artery were exposed, isolated and then cannulated with a PVC catheter (17G) for the infusion of drugs and compounds. Cannulation was repeated for the femoral artery, inserting the catheter to a depth of 10cm to ensure that the catheter reached the abdominal aorta.
  • This arterial catheter was linked to a Gould system to record blood pressure. Samples for blood gas analysis were also taken via the arterial catheter. Systolic and diastolic pressures were measured, and the mean arterial pressure calculated using the formula
  • Heart rate was measured via the pulse oxymeter and Po-ne-mah data acquisition software system (Ponemah Physiology Platform, Gould Instrument Systems Inc).
  • a ventral midline incision was made into the abdominal cavity.
  • the incision was about 5cm in length just above the pubis.
  • the fat and muscle was bluntly dissected away to reveal the hypogastric nerve which runs down the body cavity. It was essential to keep close to the side curve of the pubis wall in order to avoid damaging the femoral vein and artery which lie above the pubis.
  • the sciatic and pelvic nerves lie deeper and were located after further dissection on the dorsal side of the rabbit. Once the sciatic nerve is identified, the pelvic nerve was easily located.
  • the term pelvic nerve is loosely applied; anatomy books on the subject fail to identify the nerves in sufficient detail.
  • the pelvic nerve was freed away from surrounding tissue and a Harvard bipolar stimulating electrode was placed around the nerve. The nerve was slightly lifted to give some tension, then the electrode was secured in position. Approximately 1ml of light paraffin oil was placed around the nerve and electrode. This acts as a protective lubricant to the nerve and prevents blood contamination of the electrode. The electrode was connected to a Grass S88 Stimulator. The pelvic nerve was stimulated using the following parameters:- 5V, pulse width O. ⁇ ms, duration of stimulus 20 seconds with a frequency of 16Hz. Reproducible responses were obtained when the nerve was stimulated every 15-20 minutes.
  • the compound(s) to be tested were infused, via the jugular vein, using a Harvard 22 infusion pump allowing a continuous 15 minute stimulation cycle.
  • the skin and connective tissue around the penis was removed to expose the penis.
  • a catheter set (Insyte-W, Becton-Dickinson 20 Gauge 1.1 x 48mm) was inserted through the tunica albica into the left corpus cavernosal space and the needle removed, leaving a flexible catheter.
  • This catheter was linked via a pressure transducer (Ohmeda 5299-04) to a Gould system to record intracavernosal pressure.
  • the NPY inhibitor was made up in saline + 10% 1M NaOH, the phosphodiesterase type 5 (PDE5) inhibitor was made up in saline + 5% 1M HCl.
  • the inhibitors and vehicle controls were infused at a rate of 0.1 ml/second.
  • NPY inhibitors and PDE C A MP inhibitors were left for 15 minutes prior to pelvic nerve stimulation.
  • NPY Y1 receptor antagonists and PDE5 or combinations of the two have no significant effect on un-stimulated ICP i.e. they do not directly induce an increase in ICP in the absence of sexual drive/arousal.
  • This is highly advantageous as the only other marketed therapy for MED which requires sexual stimulation to work is sildenafil thus the present invention provides a viable alternative oral therapy to sildenafil and all other PDE ⁇ alone based drugs.
  • NPY receptor antagonist BIBP 3226
  • PDE ⁇ i 3-ethyl-5- ⁇ 5-[4-ethylpiperzino)sulphonyl-2-propoxyphenyl ⁇ -2-(2- pyridylmethyI)-6,7-dihydro-2H-pyrazolo[4,3-cflpyrimidin-7-one also known as 3- ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-n-propoxyphrenyl]-2-(pyridin-2- yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see W098/491066).
  • IC ⁇ O against human native PDE5 1.1nM
  • selectivity for PDE5 over PDE3 is greater- than 90,000 and selectivity over PDE4 is 18,545.
  • Example 1 Inhibition of NPY Y1 receptors dose-dependentlv potentiates nerve stimulated increases in intracavernosal pressure in anaesthetised rabbit model of erection.
  • ICP intracavernosal pressure
  • Example 2 PDE5 inhibition significantly increases the efficacy of PDE ⁇ inhibitor to enhance penile erection in an anaesthetised rabbit model of erection.
  • Intravenous administration of BIBP3226 (a selective NPY Y1 antagonist had no substantial effect the mean arterial blood pressure in the anaesthetised rabbit model of penile erection.
  • the graph shown in Figure 3 demonstrates that BIBP3226 has no significant effect on mean arterial pressure in the anaesthetised rabbit at doses that enhanced pelvic nerve stimulated increases in intracavernosal pressure.
  • Light grey bars represent the basal MAP prior to drug administration and dark grey bars represent the MAP following intravenous application of BIBP3226.
  • White bars represent vehicle controls. * P ⁇ 0.06, Students t-test unpaired compared with control increases.
  • NPY 1 receptor antagonists significantly increases the efficacy of PDE 5 inhibitor to enhance penile erection in an anaesthetised rabbit model of erection.
  • a selective PDE ⁇ inhibitor (1 mg/kg) further enhanced nerve-stimulated increases in ICP to a maximum increase of 360% (see Figure 10).
  • Example ⁇ NPY Y1 receptor antagonists potentiate the erectile effects of PDE ⁇ inhibitors and speeds up the onset of action of PDE 5 inhibitors in anaesthetised rabbit model of erection.
  • NPY Y1 receptor antagonists beneficially potentiates the efficacy of a PDE ⁇ inhibitor and speeds up the onset of PDE ⁇ inhibitor action in the anaesthetised rabbit model.
  • PDEcGMP cGMP hydrolysing PDE
  • PDEi inhibitor of a PDE (also known as l:PDE)
  • PDE5 phosphodiesterase type
  • NPY neuropeptide Y
  • NPYi inhibitor of NPY
  • NPYY1 neuropeptide Y Y1 receptor
  • NPY YIi __ inhibitor of NPY Y1

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Abstract

L'invention porte sur l'utilisation d'un inhibiteur d'un neuropeptide Y (NPY), de préférence d'un récepteur de NPY Y1, cet inhibiteur étant sélectif pour un récepteur de NPY ou NPY Y1 associé aux organes génitaux mâles, dans la préparation/fabrication d'un médicament destiné à être utilisé dans le traitement ou la prévention de la dysérection.
PCT/IB2001/002399 2000-12-15 2001-12-10 Traitement du dysfonctionnement sexuel male WO2002047670A1 (fr)

Priority Applications (8)

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KR10-2003-7007946A KR20030061441A (ko) 2000-12-15 2001-12-10 남성 성기능 장애의 치료
EP01270206A EP1347750A1 (fr) 2000-12-15 2001-12-10 Traitement du dysfonctionnement sexuel male
NZ526925A NZ526925A (en) 2000-12-15 2001-12-10 Treatment of male sexual dysfunction
HU0400528A HUP0400528A2 (hu) 2000-12-15 2001-12-10 Neuropeptidáz Y inhibitor alkalmazása férfiak szexuális funkciózavarának kezelésére alkalmas gyógyszerkészítmény előállítására
JP2002549244A JP2004522720A (ja) 2000-12-15 2001-12-10 雄性性的機能障害の処置
AU2002220977A AU2002220977A1 (en) 2000-12-15 2001-12-10 Treatment of male sexual dysfunction
IL15577501A IL155775A0 (en) 2000-12-15 2001-12-10 Treatment of male sexual dysfunction
CA002431747A CA2431747A1 (fr) 2000-12-15 2001-12-10 Traitement du dysfonctionnement sexuel male

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GB0030647A GB0030647D0 (en) 2000-12-15 2000-12-15 Treatment of male sexual dysfunction
GB0030647.2 2000-12-15
GB0108730A GB0108730D0 (en) 2001-04-06 2001-04-06 Treatment of male sexual dysfunction
GB0108730.3 2001-04-06
GB0109910.0 2001-04-23
GB0109910A GB0109910D0 (en) 2001-04-23 2001-04-23 Treatment of sexual dysfunction
GB0111037A GB0111037D0 (en) 2001-05-04 2001-05-04 Treatment of sexual dysfunction
GB0111037.8 2001-05-04
US09/895,367 2001-06-29
US09/895,367 US20020028799A1 (en) 2000-07-06 2001-06-29 Treatment of male sexual dysfunction
US09/905,846 US6878529B2 (en) 2000-07-14 2001-07-13 Compounds for the treatment of sexual dysfunction
US09/905,846 2001-07-13
GB0120679A GB0120679D0 (en) 2001-08-24 2001-08-24 Treatment of male sexual dysfunction
GB0120679.6 2001-08-24

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EP1635813A2 (fr) * 2003-06-06 2006-03-22 Merck &amp; Co. Inc. Polytherapie permettant de traiter la dyslipidemie
US7189755B2 (en) 2001-08-10 2007-03-13 Palatin Technologies, Inc. Pyrrolidine melanocortin-specific compounds
US7354923B2 (en) 2001-08-10 2008-04-08 Palatin Technologies, Inc. Piperazine melanocortin-specific compounds
US7456184B2 (en) 2003-05-01 2008-11-25 Palatin Technologies Inc. Melanocortin receptor-specific compounds
US8101371B2 (en) 2007-10-18 2012-01-24 Musc Foundation For Research Development Methods for the diagnosis of genitourinary cancer
CN111840508A (zh) * 2020-07-17 2020-10-30 北京清大赛尔生物科技有限公司 一种预防及治疗男性性功能障碍的外用制剂及其应用

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EP1925307A1 (fr) * 2006-11-03 2008-05-28 Emotional Brain B.V. Utilisation de 3-alpha-androstanediol pour le traitement des dysfonctions sexuelles
DE102009018668A1 (de) 2009-04-23 2010-11-25 Igor Lier Faltbare Duschkabine
SMT201800145T1 (it) * 2010-11-15 2018-05-02 Neuroderm Ltd Somministrazione continua di l-dopa, di inibitori della dopa decarbossilasi, di inibitori della catecol-o-metil trasferasi e composizioni per essa

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IL155775A0 (en) 2003-12-23
CN1496254A (zh) 2004-05-12
EP1347750A1 (fr) 2003-10-01
CA2431747A1 (fr) 2002-06-20

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