Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/22—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with hetero atoms directly attached to ring nitrogen atoms
  • C07D295/26—Sulfur atoms
• • 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
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
• • 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
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/4965—Non-condensed pyrazines
• • 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
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to a compound and a pharmaceutical that is useful for the treatment and/or prevention of male sexual dysfunction, in particular ejaculatory disorders, such as premature ejaculation.
• SD sexual dysfunction
• the causes of SD may be both organic as well as psychological.
• Organic aspects of SD are typically caused by underlying vascular diseases, such as those associated with hypertension or diabetes mellitus, by prescription medication and/or by psychiatric disease such as depression.
• Physiological factors include fear, performance anxiety and interpersonal conflict. SD impairs sexual performance, diminishes self-esteem and disrupts personal relationships thereby inducing personal distress.
• SD disorders have been divided into female sexual dysfunction (FSD) disorders and male sexual dysfunction (MSD) disorders (Melman et al 1999 J. Urology 161 5-11).
• FSD female sexual dysfunction
• MSD male sexual dysfunction
• MSD Male sexual dysfunction
• erectile dysfunction also known as male erectile dysfunction (MED)
• MED male erectile dysfunction
• ejaculatory disorders such as premature ejaculation, anorgasmia (unable to achieve orgasm) or desire disorders such as hypoactive sexual desire disorder (lack of interest in sex).
• PE is a lifelong persistent or recurrent ejaculation with minimal sexual stimulation before, upon or shortly after penetration and before the patient wishes it.
• the clinician must take into account factors that affect duration of the excitement phase, such as age, novelty of the sexual partner or stimulation, and frequency of sexual activity.
• the disturbance causes marked distress of interpersonal difficulty.
• PE erectile dysfunction
• Urologists currently form the bulk (59%) of physicians treating PE. GP's form 33% of doctors treating the condition. Sex therapists, behavioural therapists and counsellors also treat patients with PE. Experts estimate that 50% of presenters do so because of the impact the condition has on the relationship with the partner. Stress, relationship difficulties and/or effect on quality of life are the key triggers for sufferers to seek treatment for PE.
• Ejaculation is dependent on the sympathetic and parasympathetic nervous systems. Efferent impulses via the sympathetic nervous system to the vas deferens and the epididymis produce smooth muscle contraction, moving sperm into the posterior urethra. Similar contractions of the seminal vesicles, prostatic glands and the bulbouretheral glands increase the volume and fluid content of semen. Expulsion of semen is mediated by efferent impulses originating from the nucleus of Onuf in the spinal cord, which pass via the parasympathetic nervous system and cause rhythmic contractions of the bulbocavemous, ischiocavernous and pelvic floor muscles. Cortical control of ejaculation is still under debate in humans.
• the medial pre-optic area and the paraventricular nucleus of the hypothalamus seem to be involved in ejaculation.
• the most commonly off-label prescribed medications are the anti-depressants (for example clomipramine) and the selective serotonin re-uptake inhibitors (for example
• a selective oxytocin antagonist results in the treatment of ejaculatory disorders, in particular premature ejaculation, whilst maintaining erectogenic mechanisms, in particular penile erection.
• ejaculatory disorders in particular premature ejaculation
• a selective oxytocin antagonist allows the treatment thereof whilst maintaining the patient's sexual drive.
• sexual drive means libido or sexual desire.
• compounds according to the present invention preferably comprise the unexpected advantage of maintaining erectogenic mechanisms, in particular penile erection, and/or sexual drive, as compared with known non-selective oxytocin antagonists.
• vasotocin significantly reduces non-contact penile erections (see, for example, Melis ef al (Neuroscience Letters 265 (1999) 171-174).
• intracerebroventricular (ICV) injection of the oxytocin antagonist vasotocin was shown in Argiolas et al (European Journal of Pharmacology 149 (1988) 389-392) to impair sexual performance in experienced male rats in the presence of a receptive female, with the abolishment of ejaculation (probably caused by a decreased intromission frequency).
• the decrease in intromission frequency was thought to reflect a decreased capacity of the animals to achieve penile erection, as the oxytocin antagonist was found to prevent penile erection.
• the present invention relates to the use of a selective oxytocin antagonist in the manufacture of a medicament (such as a pharmaceutical composition) for use in the treatment of a male ejaculatory disorder, in particular premature ejaculation.
• a medicament such as a pharmaceutical composition
• the present invention relates to the use of a selective oxytocin antagonist in the preparation of a medicament (such as a pharmaceutical composition) for use in the treatment of a male ejaculatory disorder, in particular premature ejaculation.
• a medicament such as a pharmaceutical composition
• the present invention relates to a method of treating and/or preventing a male ejaculatory disorder, in particular premature ejaculation, whilst maintaining erectogenic mechanisms, in particular penile erection, and/or sexual drive, in a human or animal which method comprises administering to an individual an effective amount of a selective oxytocin antagonist, wherein said selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
• 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 selective oxytocin antagonists with a pharmaceutically acceptable diluent, excipient or carrier.
• the present invention relates to an assay method for identifying an agent (hereinafter referred to as a selective oxytocin antagonist) that can be used to treat or prevent a male ejaculatory disorder, in particular premature ejaculation, the assay comprising: determining whether a test agent can directly delay the endogenous ejaculatory process; wherein said delay is defined as an increase in and/or restoration of ejaculatory latency (i.e.
• the agent has no, or substantially no, effect on penile erection. That is to say, preferably, the agent does not adversely affect penile erection; however, the agent may enhance endogenous penile erection.
• the present invention relates to a process comprising the steps of:
• 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 increase and/or restore ejaculatory latency; (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 a selective oxytocin antagonist.
• the present invention relates to a process comprising the steps of:
• step (iv) selecting agents with no, or substantially no, effect on penile erection; and (v) preparing a quantity of those one or more selected agents; and wherein said agent is a selective oxytocin antagonist.
• the agent identified in step (ii) may be modified so as to maximise, for example, activity and then step (i) may be repeated. These steps may be repeated until the desired activity or pharmacokinetic profile has been achieved.
• the present invention relates to a diagnostic method, the method comprising isolating one or more samples from a male during sexual stimulation at successive time intervals, i.e. 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes following the commencement of sexual stimulation, determining whether the sample(s) contains an entity present at such a time and in such an amount as to cause a male ejaculatory disorder, preferably premature ejaculation; and wherein said entity can be modulated, in particular the time taken for the entity to appear and/or the peak in concentration can be delayed, to achieve a beneficial effect by use of an agent; and wherein said agent is a selective oxytocin antagonist.
• the entity is oxytocin.
• the sexual stimulation may be caused by a penile vibratory stimulation device (FertiCare, H ⁇ rsholm, Denmark), for example.
• the present invention relates to a diagnostic composition or kit comprising means for detecting an entity in one or more isolated male samples which sample(s) is taken at successive time intervals, i.e. 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes following the commencement of sexual stimulation, during sexual stimulation of said male; wherein the means can be used to determine whether the sample(s) contains the entity at such a time and in such an amount as to cause a male ejaculatory disorder, preferably premature ejaculation; and wherein said entity can be modulated, in particular the time taken for the entity to appear and/or the peak in concentration can be delayed, to achieve a beneficial effect by use of an agent; and wherein said agent is a selective oxytocin antagonist.
• the entity is oxytocin.
• the sexual stimulation may be caused by a penile vibratory stimulation device (FertiCare, H ⁇ rsholm, Denmark), for example.
• the present invention relates to an animal model used to identify agents capable of treating and/or preventing a male ejaculatory disorder, in particular premature ejaculation, said model comprising a male animal including means to measure ejaculation latency (i.e. time taken from first intromission to ejaculation) of said animal following introduction of a receptive female; and wherein said agent is a selective oxytocin antagonist.
• the animal model may further comprise or be used in conjunction with an additional animal model comprising means to measure changes in penile erection.
• a suitable additional model may be one 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 a selective oxytocin antagonist.
• the present invention relates to an assay method for identifying an agent that can directly enhance the endogenous ejaculatory processes in order to treat or prevent ejaculatory disorders, in particular premature ejaculation, the assay method comprising: administering an agent to the animal model of the present invention; and measuring ejaculation latency (i.e. time taken from first intromission to ejaculation) of said animal following introduction of a receptive female; and wherein said agent is a selective oxytocin antagonist.
• the present invention relates to an assay method for identifying an agent that can directly enhance the endogenous ejaculatory process without affecting penile erection and/or sexual drive in order to treat or prevent ejaculatory disorders, in particular premature ejaculation, the assay method comprising: administering an agent to the animal model of the present invention; and measuring the change in the endogenous ejaculatory process; wherein said change is defined as ejaculation latency (i.e.
• selective oxytocin antagonist and “selective oxytocin receptor antagonist” are interchangeable and mean an oxytocin receptor antagonist which is selective towards oxytocin receptors as compared with vasopressin, in particular Via, receptors.
• ejaculatory latency means the time taken from first intromission to ejaculation.
• restoration of ejaculatory latency means that the time take from first intromission to ejaculation is modified, preferably increased.
• the time taken from intromission to ejaculation is modified (preferably increased) to near normal levels.
• a person suffering from premature ejaculation ejaculates within 30 seconds of the beginning of intercourse (i.e. from first intromission) and often within 15 seconds of the beginning of intercourse (i.e. from first intromission).
• ejaculatory latency is increased to at least above 30 seconds, preferably to at least above 60 seconds, more preferably to at least above 2 minutes, more preferably to at least above 5 minutes, more preferably to at least above 10 minutes.
• ejaculatory latency may be restored such that the time taken from intromission to ejaculation is sufficiently delayed to allow for partner satisfaction.
• the agent for use according to the present invention is for oral administration.
• the agent for use according to the present invention may be for topical administration or intranasal administration.
• the agent according to the present invention is for use in the treatment and/or prevention of premature ejaculation.
• the selective oxytocin antagonist is at least 20-fold selective for an L5 oxytocin receptor as compared with a vasopressin receptor, in particular a Via receptor.
• the selective oxytocin antagonist is at least 30-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular a Via 20 receptor.
• the selective oxytocin antagonist is at least 50-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular a Via receptor.
• the selective oxytocin antagonist is at least 100-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular a Via receptor.
• the selective oxytocin antagonist is at least 200-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular a Via receptor.
• the selective oxytocin antagonist is at least 250-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular a Vi a receptor.
• the present invention also encompasses administration of the agent of the present invention before and/or during sexual arousal/stimulation.
• 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 the following (numbered) preferred aspects:
• a composition comprising a selective oxytocin antagonist for use in the treatment or prevention of a male ejaculatory disorder; which selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
• a composition according to aspect 1 wherein the male ejaculatory disorder is a premature ejaculation.
• the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
• the vasopressin receptor is a Via receptor.
• a selective oxytocin antagonist in the manufacture of a medicament for use in the treatment of a male ejaculatory disorder. 6. The use according to aspect 5 wherein the male ejaculatory disorder is premature ejaculation. 7. The use according to aspect 5 or aspect 6 wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor, 8. The use according to aspect 7 wherein the vasopressin receptor is a Vi a receptor.
• a method of treating or preventing a male ejaculatory disorder in a human or animal comprises administering to an individual an effective amount of a selective oxytocin antagonist; wherein said selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
• vasopressin receptor is a Via receptor.
• a pharmaceutical pack comprising one or more compartments wherein at least one compartment comprises one or more of a selective oxytocin antagonist.
• vasopressin receptor is a Via receptor.
• a process of preparation of a pharmaceutical composition comprising admixing one or more selective oxytocin antagonists with a pharmaceutically acceptable diluent, excipient or carrier.
• An assay method for identifying an agent that can be used to treat and/or prevent a male ejaculatory disorder comprising: determining whether a test agent can directly enhance the endogenous ejaculatory
• said enhancement is defined as an increase in and/or restoration of ejaculatory latency 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 treatment or prevention of a male ejaculatory disorder, and wherein said test agent is a selective oxytocin antagonist.
• a medicament for oral administration to treat a male ejaculatory disorder wherein the medicament comprises the agent according to aspect 27.
• a process comprising the steps of: (a) performing the assay method of any one of aspects 23-26; (b) identifying one or more agents capable of increasing and/or restoring ejaculatory latency; and (c) preparing a quantity of those one or more identified agents; and wherein said agent is a selective oxytocin antagonist.
• the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
• the vasopressin receptor is a Via receptor. 37.
• An animal model for identifying an agent capable of treating or preventing a male ejaculatory disorder comprising a male animal including means to measure ejaculation latency of said animal following introduction of a receptive female; and wherein said agent is a selective oxytocin antagonist.
• said agent is a selective oxytocin antagonist.
• vasopressin receptor is a Via receptor.
• An assay method for identifying an agent that can directly enhance the endogenous ejaculatory processes in order to treat or prevent ejaculatory disorders comprising: administering an agent to the animal model of any one of aspects 37 to 40; and measuring ejaculation latency of said animal following introduction of a receptive female; and wherein said agent is a selective oxytocin antagonist.
• a PDE inhibitor more particularly a PDE 5 inhibitor, said inhibitors preferably having an IC50 against the respective enzyme of less than 100nM
• a serotonin receptor agonist or modulator more particularly agonists or modulators for 5HT2C, 5HT1B and/or 5HT1D receptors, including anpirtoline
• a serotonin receptor antagonist or modulator more particularly antagonists or modulators for 5HT1A, including
• An antidepressant in particular i) a selective serotonin re- uptake inhibitor (SSRi), including sertraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, mirtazapine, nefazodone and trazodone;
• SSRi selective serotonin re- uptake inhibitor
• TCA tricyclic antidepressant
• clomipramine desapramine, imipramine, amitriptyline, doxepine, amoxapine, maprotiline, nortriptyline, protriptyline, trimipramine and buproprion
• monoamine oxidase v) An ⁇ -adrene
• a pharmaceutical composition consisting of one or more selective oxytocin antagonists and one or more PDEi's, optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
• the present invention demonstrates the surprising and unexpected findings that:
• administration of a selective oxytocin antagonist increases ejaculatory latency.
• administration of a selective oxytocin antagonist restores ejaculatory latency, preferably to near normal levels;
• administration of a selective oxytocin antagonist unexpectedly increases ejaculatory latency without substantially inhibiting and/or adversely affecting erectogenic mechanisms, in particular penile erection.
• administration of a selective oxytocin antagonist restores ejaculatory latency, preferably to near normal levels, without substantially inhibiting and/or adversely affecting erectogenic mechanisms, in particular penile erection;
• administration of a selective oxytocin antagonist increases ejaculatory latency without substantially inhibiting and/or adversely affecting sexual drive.
• administration of a selective oxytocin antagonist restores ejaculatory latency, preferably to near normal levels, without substantially inhibiting and/or adversely affecting sexual drive.
• patient groups should benefit from treatment with a selective oxytocin antagonist (or a combination comprising a selective oxytocin antagonist as set out hereinafter).
• a selective oxytocin antagonist or a combination comprising a selective oxytocin antagonist as set out hereinafter.
• patient groups include those suffering from one or more of the following: a neurological disorder, a physiological disorder, psychosexual skills deficit, a physical illness, a physical injury, pharmacological side effects, physchological distress and relationship distress.
• the agent may be any suitable agent that can act as a selective oxytocin antagonist.
• Background teachings on oxytocin receptors have been prepared by Victor A. McKusick et al on http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm. The following text concerning oxytocin receptors has been extracted from that source:
• L-368,899 is a selective oxytocin antagonist.
• L-368,899 is over 20-fold selective for oxytocin receptors over vasopressin, in particular Via, receptors.
• the selectivity is both binding and functional selectivity.
• vasotocin Certain known oxytocin antagonists, such as vasotocin for instance are sometimes referred to a being “selective oxytocin antagonists".
• vasotocin d(CH 2 ) 5 Tyr(Me)-Orn 8 -vasotocin (hereinafter referred to as "vasotocin") in only 2- to 3-fold selective for oxytocin receptors over vasopressin, in particular Via, receptors.
• vasotocin in a substantially non-selective oxytocin/vasopressin antagonist does not fall within the scope of the term "selective oxytocin antagonist” according to the present invention.
• the selective oxytocin antagonist according to the present invention is at least 20-fold selective for oxytocin receptors over vasopressin, in particular Via, receptors.
• the suspension is then homogenised using a mechanical homogeniser for several bursts of 5 sees on ice before centrifuging at 25,000 x g for 30 mins. • After resuspending the pellet in 1 ml of freezing buffer per 1 ml of the original packed cell volume the suspension is briefly homogenised to remove small lumps. Protein concentrations are then measured and the membrane suspension is finally frozen in aliquots at a minimum of 5 mg/ml at -80°C.
• Membranes are thawed on ice before diluting to 1 mg/ml in assay buffer. SPA beads are resuspended at 50 mg/ml in assay buffer. From these concentrations, beads are pre-coupled with membranes by incubating 30 ⁇ g of protein per mg of bead on a top-to-tail shaker for 2 hours at 4 °C. The bead/membranes are then centrifuged at 2000 rpm for 10 mins and the pellet is resuspended at 3 mg/ml.
• the 125 l-OVTA is diluted in 1 ml assay buffer per 50 ⁇ Ci of lyophilised ligand. A 5 ⁇ l sample is then counted in duplicate using liquid scintillation counting (protocol 61 on Wallac Counter) and the concentration of the ligand is calculated (see example below). This is to overcome any loss of ligand due to stickiness. Using the measured concentration, the 125 l-OVTA is diluted to 0.3 nM in assay buffer.
• STD SR49059 (UK222.633) will be diluted in 25% DMSO/ddH 2 0 starting with a top concentration of 1 ⁇ M (for final assay concentration of
• BSA is prepared in distilled H 2 0 at the following concentrations: 2000, 1000, 500, 250, 125, 62.5 and 31.25 5 yg/ml
• the concentration of protein in the membrane prep is determined (the dilution of the membrane prep which lies on the centre of the standard curve is used).
• the membrane prep is diluted to a protein concentration of 5mg/ml in freezing buffer before being frozen in 200 ⁇ l aliquots at -80°C
• Membrane protein is thawed gently on ice and diluted to the optimum protein concentration for the assay (see appendix for protein linearity determination)
• % bound (sample cpm- mean NSB cpm)/ specific binding cpm x 100
• the present invention further comprises the combination of a compound of the invention for the treatment of a male ejaculatory disorder, in particular premature ejaculation, as outlined herein with one or more of auxiliary active agents (see later discussion for suitable examples).
• the present invention further comprises the use of a combination consisting essentially of a selective oxytocin antagonist 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 and/or prevention of a male ejaculatory disorder, in particular premature ejaculation, as outlined herein.
• the present invention further comprises the use of a combination consisting of a selective oxytocin antagonist 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 and/or prevention of a male ejaculatory disorder, in particular premature ejaculation, as outlined herein.
• the present invention further comprises the use of a combination consisting of a selective oxytocin antagonist 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 and/or prevention of a male ejaculatory disorder, in particular premature ejaculation, as outlined herein.
• 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 a selective oxytocin antagonist 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 a selective oxytocin antagonist 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: 1) A PDE inhibitor, more particularly a PDE 5 inhibitor (see hereinafter), said inhibitors preferably having an IC50 against the respective enzyme of less than 100nM;
• a serotonin receptor agonist or modulator more particularly agonists or modulators for 5HT2C, 5HT1 B and/or 5HT1 D receptors, including anpirtoline;
• a serotonin receptor antagonist or modulator more particularly antagonists or modulators for 5HT1A, including NAD-299 (robalzotan) and WAY- 100635, and/or more particularly antagonists or modulators for 5HT3 receptors, including batanopirde, granisetron, ondansetron, tropistron and
• An antidepressant in particular i) a selective serotonin re-uptake inhibitor (SSRi), including sertraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, mirtazapine, nefazodone and trazodone; ii) a tricyclic antidepressant (TCA), including clomipramine, desapramine, imipramine, amitriptyline, doxepine, amoxapine, maprotiline, nortriptyline, protriptyline, trimipramine and buproprion; and iii) monoamine oxidase;
• SSRi selective serotonin re-uptake inhibitor
• TCA tricyclic antidepressant
• An ⁇ -adrenergic receptor antagonist also known as ⁇ -adrenergic blockers, ⁇ -blockers or ⁇ -receptor blockers
• suitable ⁇ 1 -adrenergic receptor antagonists include: phentolamine, prazosin, phentolamine mesylate, trazodone, alfuzosin, indoramin, naftopidil, tamsulosin, phenoxybenzamine, rauwolfa alkaloids, Recordati 15/2739, SNAP 1069, SNAP 5089, RS17053, SL 89.0591 , doxazosin, terazosin and abanoquil;
• suitable ⁇ 2-adrenergic receptor antagonists include dibenarnine, tolazoline, trimazosin, efaroxan, yohimbine, idazoxan clonidine and dibenarnine; suitable non-selective ⁇ -
• a combination of active agents may be administered simultaneously, separately or sequentially.
• Suitable cGMP PDE5 inhibitors for the use according to the present invention include:
• PDE phosphodiesterase 5 (PDE) inhibitors
• PDE ⁇ i's PDE inhibitors
• Still other type cGMP PDE5 inhibitors useful in conjunction with the present invention include:4-bromo-5-(pyridylmethylamino)-6-[3-(4-chlorophenyl)-propoxy]- 3(2H)pyridazinone; 1-[4-[(1 ,3-benzodioxol-5- ylmethyl)amiono]-6-chloro-2- quinozolinyl]-4-piperidine-carboxylic acid, monosodium salt; (+)-cis-5,6a,7,9,9,9a- hexahydro-2-[4-(trifluoromethyl)-phenylmethyl-5-methyl-cyclopent-
• 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.
• the cGMP PDE5 inhibitors have an IC 50 at less than 100 nanomolar, more preferably, at less than 50 nanomolar, more preferably still at less than 10 nanomolar.
• IC50 values for the cGMP PDE5 inhibitors may be determined using the PDE5 assay in the Test Methods Section hereinafter.
• the cGMP PDE5 inhibitors used in the pharmaceutical combinations according to the present invention are selective for the PDE5 enzyme.
• they have a selectivity of PDE5 over PDE3 of greater than 100 more preferably greater than 300.
• the PDE5 has a selectivity over both PDE3 and PDE4 of greater than 100, more preferably greater than 300.
• the term "corpus cavemosum” 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 cavemosum comprises smooth muscle cells.
• Ejaculation comprises two separate components - emission and ejaculation.
• Emission is the deposition of seminal fluid and sperm from the distal epididymis, vas deferens, seminal vesicles and prostrate into the prostatic urethra. Subsequent to this deposition is the forcible expulsion of the seminal contents from the urethral meatus.
• Ejaculation is distinct from orgasm, which is purely a cerebral event. Often the two processes are coincidental.
• the term "penile erection” refers to the situation whereby, upon stimulation, which may be visual, tactile, auditory, olfactory or from the imagination, the arteries supplying the penis dilate and large quantities of blood enter the blood sinuses. Expansion of these spaces compresses the veins draining the penis, so blood outflow is slowed. These vascular changes, due to a parasympathetic reflex, result in an erection. The penis returns to its flaccid state when the arteries constrict and pressure on the veins is relieved.
• smooth muscle refers to a tissue specialised for contraction composed of smooth muscle fibres (cells) which are located in the walls of hollow internal organs and innervated by autonomic motor neurons.
• smooth muscle means muscle lacking striations, hence giving it a smooth appearance. It is also called involuntary muscle.
• An increase in the concentration of Ca 2+ in smooth muscle cytosol initiates contraction, just as in striated muscle.
• sacroplasmic reticulum (the reservoir for Ca 2+ in striated muscle) is scanty in smooth muscle.
• calmodulin binds to Ca 2+ in the cytosol. Not only do calcium ions enter smooth muscle fibres slowly, but they also move slowly out of the muscle fibre when excitation declines, which delays relaxation.
• the prolonged presence of Ca 2+ in the cytosol provides for smooth muscle tone, a state of continued partial contraction.
• Smooth muscle tissue is located in the walls of hollow internal organs such as blood vessels, airways to the lungs, the stomach, intestinal gall bladder, urinary bladder, the corpus cavernosa of the penis and the clitoris.
• references herein to treatment include one or more of curative, palliative and prophylactic treatment.
• the present invention also encompasses use as defined hereinbefore via administration of a selective oxytocin antagonist (and an auxiliary agent where applicable) before and/or during sexual stimulation.
• a selective oxytocin antagonist and an auxiliary agent 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.
• 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 a male ejaculatory disorder, in particular premature ejaculation, according to the present invention may be any suitable agent that can act as a selective oxytocin antagonist and, where appropriate a combination of a selective oxytocin antagonist and an auxiliary agent as detailed hereinbefore.
• agent includes any entity capable of selectively inhibiting oxytocin receptors.
• agents can be an organic compound or other chemical.
• the substance may even be an amino acid sequence or a chemical derivative thereof.
• 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.
• 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.
• the term "agent” may be a single entity or it may be a combination of agents.
• 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. An example of such a compound is presented herein.
• the agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.
• substituted means substituted by one or more defined groups.
• groups may be selected from a number of alternative groups, the selected groups may be the same or different.
• the term independently means that where more than one substituent is selected from a number of possible substituents, those substituents may be the same or different.
• 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 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.
• antagonist as used herein in relation to the selective oxytocin antagonist is to be regarded as being interchangeable with the term inhibitor.
• inhibitor as used herein, in relation to the auxiliary agents hereinbefore presented for example (such as where applicable PDEi or PDE5i compounds), 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 from 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. 1985).
• 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.
• Alpha-, beta- and gamma-cyclodextrins are most commonly used and suitable examples are described in WO-A- 91/11172, WO-A-94/02518 and WO-A-98/55148.
• 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 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
• composition comprises more than one active component, then those components may be administered by different routes.
• agents of the present invention are administered parenterally, then 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.
• the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
• the preparation of 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.
• 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 daily dosage level of the agent may be in single or divided doses.
• the daily oral dose may be, for instance, between 20-1000 mg, preferably 50-300 mg for example.
• 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 bioavailablity 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 bioavailablity.
• the solubilisation of the drug by the aqueous contents of the gastrointestinal tract can be predicted from in vitro solubility experiments conducted at appropriate pH to mimic the GIT.
• the compounds of the invention have a minimum solubility of 50 mg/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 4 measurements using organic solvents and buffer.
• the compounds of the invention have a Log D 74 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, 595-600
• 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.
• the selective oxytocin antagonist (and/or PDEi/PDE5i 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 (1995) 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 oxytocin receptor.
• 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, i.e. to a selective oxytocin antagonist for example, to a target, i.e. to a oxytocin receptor for example. 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 oxytocin receptor may be used as a target in screens to identify agents capable of inhibiting oxytocin receptors.
• the target may comprise an amino acid sequence shown as SEQ ID NO: 1 or a variant, homologue, derivative or fragment thereof which is prepared by recombinant and/or synthetic means or an expression entity comprising same.
• both an oxytocin receptor and a vasopressin, particularly Via, receptor may be used as targets in screens to identify agents capable of selectively inhibiting oxytocin receptors.
• the oxytocin receptor target may comprise an amino acid sequence shown as SEQ ID NO: 1 or a variant, homologue, derivative or fragment thereof which is prepared by recombinant and/or synthetic means or an expression entity comprising same and the vasopressin receptor target may comprise an amino acid sequence shown as SEQ ID NO: 2 or a variant, homologue, derivative or fragment thereof which is prepared by recombinant and/or synthetic means or an expression entity comprising same.
• an oxytocin receptor and/or a vasopressin receptor may be used as a target to identify agents capable of mediating an increase in ejaculatory latency through the selective inhibition of the oxytocin receptor.
• the target may be suitable tissue extract.
• the target may even be a combination of such tissue and/or recombinant targets.
• agent and/or target of the present invention may be prepared by recombinant DNA techniques.
• the agent is a selective oxytocin antagonist.
• the oxytocin antagonist 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 (i.e. an oxytocin receptor or a vasopressin, preferably a Via, receptor) in an assay for the identification of one or more agents and/or derivatives thereof.
• a target i.e. an oxytocin receptor or a vasopressin, preferably a Via, receptor
• the present invention provides an amino acid sequence that 5 is an agent is capable of selectively inhibiting an oxytocin receptor.
• the target is an oxytocin receptor.
• the oxytocin receptor and/or vasopressin, preferably Via, receptor is o an isolated receptor and/or is purified and/or is non-native.
• the oxytocin receptor or the vasopressin, preferably Via, receptor of the present invention may be in a substantially isolated form. It will be understood that the oxytocin receptor or the vasopressin receptor may be mixed with carriers or
• the oxytocin receptor or the vasopressin receptor of the present invention may also be in a substantially pure form, in which case it will generally comprise the oxytocin receptor or vasopressin receptor in a preparation in which more than 90%, e.g.
• the oxytocin receptor or vasopressin receptor in the preparation is a peptide having the amino acid sequence shown in SEQ ID NO: 1 or variants, homologues, derivatives or fragments thereof or SEQ ID NO: 2 or variants, homologues, derivatives or fragments thereof, respectively.
• 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 oxytocin receptor.
• nucleotide sequence encodes a vasopressin receptor, preferably a Via receptor.
• the nucleotide sequence encodes an agent capable of selectively inhibiting oxytocin receptors.
• nucleotide sequences can encode the same target (i.e. oxytocin receptor, such as an oxytocin receptor comprising the amino acid sequence shown in SEQ ID NO: 1 , or a vasopressin receptor, such a vasopressin receptor comprising the amino acid sequence shown in SEQ ID NO: 2) as a result of the degeneracy of the genetic code.
• oxytocin receptor such as an oxytocin receptor comprising the amino acid sequence shown in SEQ ID NO: 1
• vasopressin receptor such as a vasopressin receptor comprising the amino acid sequence shown in SEQ ID NO: 2
• skilled persons may, using routine techniques, make nucleotide substitutions that do not substantially affect the activity encoded by a nucleotide sequence to reflect the codon usage of any particular host organism in which the target is to be expressed.
• the terms “variant”, “homologue” or “derivative” in relation to the nucleotide sequence 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 (i.e. oxytocin receptor for example) 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).
• 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, 85 or 90% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, 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 et al., 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): 247-50; 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 omithine
• 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*, methyl
• 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
• 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.
• 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 capable of selectively hybridising to nucleotide sequences encoding the amino acid sequences of the present invention, 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 encoding the amino acid 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.
• 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.
• 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 nucleotide sequences encoding the amino acid 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 sequences encoding the amino acid sequences shown in SEQ ID NO: 1 for example. 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.
• Nucleotide sequences encoding the amino acid 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. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the nucleotide sequences may be cloned into vectors.
• a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the nucleotide sequences may be cloned into vectors.
• Such 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 stepwise 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.
• 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.
• an agent i.e. a selective oxytocin antagonist
• a selective oxytocin antagonist 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.
• vectors comprising nucleotide sequences encoding an agent of the present invention for use in treating ejaculatory disorders, in particular premature ejaculation 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. NON-VIRAL DELIVERY
• the vectors comprising nucleotide sequences encoding the amino acids of the present invention or an agent of the present invention (i.e. a selective oxytocin antagonist) or a target of the present invention (i.e. an oxytocin receptor) 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.
• 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 encoding amino acid 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.
• Nucleotide sequences encoding an agent (i.e. a selective oxytocin antagonist or, where applicable, a PDEi or PDE5i) of the present invention or a target (such as an oxytocin receptor) 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 encoding an amino acid of the 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 oxytocin receptor 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 oxytocin receptor 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 oxytocin receptor or vasopressin receptor or an agent (i.e. a selective oxytocin antagonist) 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 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 a selective oxytocin antagonist of the present invention - or an oxytocin or vasopressin 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.
• 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-0253455), 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 posttranslational 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 and Corynebacterium 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 o 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 5 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 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 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 include, but are not limited to, the F(ab') 2 fragments which can
• 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-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 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 159:235-44) 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.
• 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 a 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.
• an appropriate target - such as an oxytocin receptor and/or a vasopressin, preferably a Via, receptor - may be used for identifying an agent, e.g. a selective oxytocin receptor antagonist, 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 an oxytocin receptor); (b) conducting one or more selectivity screens to determine the selectivity of said candidate agent (e.g. to see if said agent is also a vasopressin, particularly Via, receptor inhibitor) - such as by using the assay protocol presented herein; 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 vasopressin, particularly Via, receptors).
• screen (c) is performed.
• the present invention also provides a diagnostic method, composition or kit for the detection of a pre-disposition for premature ejaculation.
• the method, composition or kit will comprise means for detecting an entity, preferably oxytocin, in a test sample, preferably a blood sample taken from a sexually aroused male.
• an entity preferably oxytocin
• a test sample preferably a blood sample taken from a sexually aroused male.
• normal or standard values for an entity should be established. This may be accomplished by combining body fluids taken from normal subjects, either animal or human, taken at various time periods following sexual arousal, with an antibody to the entity 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 premature ejaculation. Deviation between standard and subject values establishes the presence of the disease state.
• target polynucleotide sequences may be used to detect and quantify gene expression in conditions, disorders or diseases in which premature ejaculation may be implicated.
• the target encoding polynucleotide sequence may be used for the diagnosis of premature ejaculation resulting from expression of the target.
• polynucleotide sequences encoding an entity may be used in hybridisation or PCR assays of tissues from biopsies or autopsies or biological fluids, to detect expression of the entity.
• 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. 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 assay methods according to the present invention may use one or more of 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.
• 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 a region encoding an oxytocin 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.
• oligonucleotides based upon target sequences.
• Such 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 io with antisense (3' ⁇ -5') 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 20 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 (1995), 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.
• constructs used for heterologous expression have been developed.
• 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
• a terminator active in yeast ends the expression system.
• For the transformation of yeast several transformation protocols have been developed.
• a transgenic Saccharomyces according to the present invention 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] 42:205-225) 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 a/ (1993) DNA Cell Biol 12:441-53).
• 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 PDE5 inhibitors.
• In vitro PDE inhibitory activities against cyclic guanosine 3',5'-monophosphate (cGMP) and cyclic adenosine 3',5'-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 cavemosum, 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 cavemosum tissue, and the cAMP-specific PDE (PDE4) from human skeletal muscle.
• 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 al. (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-HCI 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).
• Figure 1 shows a graph which depicts the effect of an oxytocin receptor antagonist L-368,899 on p-chloroamphetamine (PCA)-induced ejaculation in anaesthetised rats; and
• Figure 2 shows a graph depicting the effect of a selective oxytocin antagonist (L-368,899) on seminal vesicle pressure in anaesthetised rats.
• SEQ ID NO: 1 shows an amino sequence for human oxytocin receptor
• SEQ ID NO: 2 shows an amino acid sequence for human vasopressin V1A receptor.
• mice Male Wistar-ST strain rats, weighing 350-450 g, are used. Prior to the experiments the animals are housed in groups (2 rats per cage) under controlled 12 h light-dark cycle (lights on at 07:00), constant temperature (23 ⁇ 1°C) and humidity (55 ⁇ 5%). They have free access to standard food pellets and water.
• Rats are anesthestised with sodium pentobarbital (50mg/kg, i.p.) and are placed in the supine position.
• the penis is extruded from its sheath and gently held by a wooden applicator positioned at the base of the penis.
• the test compounds are administered i.p. immediately before the sheath retraction and the penile responses, including penile erection, redding and expansion of the penile body, glans erection, engorgement and slight flaring of the glans and cup, glans erection with intense flaring of the glans, are recorded. Latencies from test compound administration to the initial penile response and ejaculation were also measured.
• Intracavernosal pressure is also determined in rats anesthetised with sodium pentobarbitol (50 mg/kg, i.p.). Further small additional amounds (5mg) may be injected throughout the experimental period as required.
• the penis is extruded from its sheath and the intracavernosal pressuer (ICP) was measured by inserting a stainless steel needle (23-guauge) into one corpus cavemosum. The needle is attached to a heparinized saline (10U/ml)-filled teflon tube and connected to a pressure transducer (NEC-San-Ei 7500).
• the male rats were placed in an observation arena (50-60 cm diameter), starting 5 hours into the dark cycle and observed under red illuminationion. Three to four minuted after placing the male in the arena, a receptive female (ovariectomised, oestradiol benzoate / progesterone injection
• EJL ejaculatory latency
• CE copulatory efficiency
• IF intromission frequency
• MF mount frequency
• PEI post ejaculatory interval
• Example 1 Delaying ejaculation in the presence of a selective oxytocin receptor antagonist (L-368,899)
• PCA p-chloroamphetamine
• L-368,899 has very poor CNS penetration and as such this study shows that oxytocin has a peripheral site of action in PCA-induced ejaculation.
• PCA is a 5HT releaser which activates non-adrenergic non-cholinergic nerves that produce penile erection and the sympathetic pathways that control ejaculation. These prosexual effects are thought to be mediated via release of spinal 5HT acting on 5HT1B and 5HT2C receptors.
• PCA also induces the secretion of oxytocin - possibly from the posterior pituitary or from spinal centres. This increase in oxytocin, like in man, is involved in the ejaculatory process since antagonism of oxytocin receptors in these studies has significant effects on the time taken to achieve ejaculation.
• Oxytocin may be modulating the contraction of ducts and glandular lobules throughout the male genital tract thus influencing the fluid volume of different ejaculate components. Increasing ejaculate volume is thought to shorten the time from intromission to ejaculation and hence an oxytocin antagonist could be useful in the treatment of premature ejaculation by delayed seminal emission.
• L-368,899 has no effect on copulatory behaviour in sexually-experienced rats at doses upto 10mgkg '1 sc.
• Rodent copulatory behaviour is characterised by a series of mounts, with and without vaginal insertion (50-80% of mounts result in intromission [vaginal penetration]) and ejaculation occurs after 6 to 12 intromissions. Each intromission lasts a matter of seconds - it is not possible to quantify intromission length i.e. intravaginal latency.
• the effect of L-368,899 was assessed on a number of copulatory parameters (see above). We have focused copulatory efficiency as a measure that summarises vaginal penetration.
• L-368,899 was also administered intracerebroventricularly (icv). L-368,899 has no significant effects on copulatory efficiency in sexually-experienced rats when dosed 50ng/rat icv.
• PE Premature ejaculation
• cAMP cyclic adenosine-3',5'-monophosphate
• cGMP cyclic guanosine-3',5'-monophosphate
• PDE phosphodiesterase
• PDEi inhibitor of a PDE (also known as l:PDE)
• PDE5 phosphodiesterase type 5
• PDE5i inhibitor of PDE5

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