JP2010209075A - Treatment of male sexual dysfunction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for use in the treatment and/or prevention of a male ejaculatory disorder. <P>SOLUTION: The composition comprises a selective oxytocin antagonist for use in the treatment and/or prevention of a male ejaculatory disorder. The selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to compounds and medicaments useful for the treatment and / or prevention of male sexual dysfunction, particularly ejaculation disorders such as premature ejaculation.

  The present invention also relates to a method for preventing and / or treating male sexual dysfunction, particularly ejaculation disorders such as premature ejaculation.

  The present invention also relates to a method of screening for compounds useful in the treatment of male sexual dysfunction, particularly ejaculation disorders such as premature ejaculation.

Male Sexual Dysfunction Sexual dysfunction (SD) is an important clinical problem that can affect both males and females. The cause of SD can be both organic as well as psychological. The organic aspects of SD are typically caused by the underlying vascular disease, such as that associated with hypertension or diabetes, by prescription medication, and / or by mental illness such as depression . Physiological factors include fear, performance anxiety and interpersonal conflict. SD disrupts interpersonal relationships by harming sexual performance, damaging self-esteem, and causing personal distress. In the clinic, 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 is best defined as the difficulty or inability of a woman to find satisfaction in sexual expression. Male sexual dysfunction (MSD) is usually erectile dysfunction, also known as male erectile dysfunction (MED) and / or ejaculation disorders such as premature ejaculation, anorgasmia (cannot obtain orgasm) Or related to either sexual desire disorder (lack of interest in sexual intercourse).

Early firing (PE)
PE is a relatively common sexual dysfunction in men. It has been defined in several different ways, but the most widely accepted is the Diagnostic and Statistical Manual of Mental Disorder and Statistical Manual IV
Disorders IV)
“PE is lifelong or recurrent, ejaculation with minimal sexual stimulation before, at or immediately after penetration and before the patient wants it. Clinicians are new to age, sexual partner or stimulation Factors that affect the duration of the arousal phase, such as sexuality and frequency of sexual activity, must be taken into account, which can cause significant distress of interpersonal relationships. ”

The International Classification of Diseases 10
definition) is
“Following: (1) Ejaculation occurs before or very soon after sexual intercourse (before time of sexual intercourse or within 15 seconds if time restrictions are required); (2) Enough to allow sexual intercourse “The problem is not the result of a long-term ban on sexual activity.”

Other definitions that have been used include classification on the following criteria:
● related to the orgasm of the opponent ● duration between penetration and ejaculation ● ability to control by number of push-in and free will

  Psychological factors may be involved in PE, and relationship problems, anxiety, depression, and previous sexual failure all play a role.

  The estimated prevalence of PE is approximately 22-38% of the male population. Unlike male erectile dysfunction (MED), PE has no clear correlation with age. With an average prevalence of 30%, there are an estimated 24 million sufferers in the United States (male age 18-65 was 80 million in 1995). There are few data on the prevalence by severity. The operational definition of PE may apply to 5-10% of men, but it is estimated that less than 0.2% requires treatment. The effectiveness of orally effective treatment is likely to change this situation.

  Urologists currently form the majority (59%) of physicians treating PE. General practitioners form 33% of physicians treating symptoms. Sex therapists, behavioral therapists and counselors also treat PE patients. Experts estimate that 50% of filers do so because of the impact the symptoms have on the relationship with the other. Stress, relationship difficulties and / or effects on quality of life are important triggers for sufferers seeking PE treatment.

  Ejaculation depends on the sympathetic and parasympathetic nervous systems. Centrifugal impulses into the vas deferens and epididymis through the sympathetic nervous system result in smooth muscle contraction and move sperm into the dorsal urethra. Similar contractions of seminal vesicles, prostate and urethral glands increase semen volume and fluid contents. Semen ejection is mediated by efferent impulses originating from the intraneuronal nucleus of the spinal cord that cause rhythmic contraction of the bulbous, sciatic, and pelvic floor muscles through the parasympathetic nervous system. The cortical control of ejaculation is still controversial in humans. In rats, the medial preoptic area and paraventricular nucleus in the hypothalamus are thought to be involved in ejaculation.

Currently there are no approved drugs available to treat PE. The most common off-label prescription drugs are antidepressants (eg, clomipramine) and selective cetronin reuptake inhibitors (eg, paroxetine and sertraline). These drugs are often not well accepted by patients because they are considered antidepressants. They are used 'off-label' and are effective when used (ie 'adaptively'), but long pharmacokinetics T _max (after oral administration of the drug, maximum in plasma The action seems to start gradually due to the time to drug concentration). When used regularly, side effects common to this class of drugs may be seen. Behavioral therapy has been another means of control but is not very effective and has a high withdrawal and recurrence rate. New and more effective therapies are needed.

  Accordingly, it is desirable to find new ways to treat male sexual dysfunction, particularly ejaculation disorders such as premature ejaculation.

  A promising discovery of the present invention is that an increase in latency to ejaculation can be obtained by administering a selective oxytocin antagonist. Thus, it has been shown that the use of selective oxytocin antagonists can achieve treatment of ejaculatory disorders, particularly early firing. This can be achieved by increasing the ejaculation latency, preferably by restoring the ejaculation latency to near normal levels.

  In particular, the use of selective oxytocin antagonists is attributed to the treatment of ejaculatory disorders, particularly premature ejaculation, while maintaining erection formation mechanisms, particularly penile erection.

  Treatment of ejaculatory disorders, particularly premature ejaculation, with selective oxytocin antagonists allows the treatment while maintaining the patient's sexual impulse. The term “sexual drive” as used herein means libido or sexual desire.

  Thus, the compounds according to the invention preferably comprise the unexpected advantage of maintaining an erection formation mechanism, in particular penile erection and / or sexual impulse, compared to known non-selective oxytocin antagonists.

The role of oxytocin in sexual behavior Ejaculation involves two separate components-injection and ejaculation. Ejection is the deposition of semen and sperm from the distal epididymis, vas deferens, seminal vesicles and prostate to the urethral prostate. Following this deposit is a powerful ejection of semen content from the ureteral orifice. Ejaculation is different from orgasm, which is purely a brain event. Often the two processes coincide.

  Pulses of oxytocin in peripheral serum are associated with ejaculation in mammals. In humans, oxytocin plasma concentrations, but not vasopressin, are significantly elevated during or near ejaculation. Oxytocin does not itself induce ejaculation, and this process is 100% under neural control through the α1-adrenergic receptor / sympathetic nerve originating from the lumbar portion of the spinal cord. Systemic pulses of oxytocin may have a direct role in the peripheral ejaculatory response. It alters the contraction of ducts and glandular lobules in the male genital tract and can thus serve to influence the fluid volume of different semen components, for example. Oxytocin released primarily into the brain can affect sexual behavior, subjective perception of arousal (orgasm) and the latency to subsequent ejaculation. The occurrence of ejaculation in males is critically dependent on tactile stimulation of the external genitals.

  It is well documented that circulating oxytocin levels increase during sexual stimulation and arousal and peak during orgasm in both men and women. Murphy et al. (Acta. Anat. Basel 128: 76-79 [1987]) measured plasma oxytocin and arginine vasopressin (AVP) concentrations in men during sexual arousal and ejaculation, and plasma AVP, but not oxytocin. It was found that during sexual arousal, it increases markedly. However, mean plasma oxytocin increased approximately 5-fold upon ejaculation and returned to basal concentration within 30 minutes, however AVP had already returned to basal levels at the time of ejaculation and remained stable thereafter.

Gimpl and Fahrenholz (Physiological
Reviews 81: 2 April 2001 pages 629-683), oxytocin is one of the most potent substances inducing penile erection in rats, rabbits and monkeys. Has been discovered. In addition, central administration of oxytocin is claimed to reduce the latency to achieve ejaculation and shorten post-ejaculation intervals. Similarly, Meston et al. (Arch. Gen Psychiatry, 57, November 2000) in male animals injected into specific areas of the brain (ie, the periventricular nucleus of the hypothalamus). Oxytocin states that it facilitates penile erection and shortens the ejaculation latency and post-ejaculation intervals when injected either centrally or peripherally.

It has been well documented in the art that administration of an oxytocin receptor agonist, vasotocin, significantly reduces non-contact penile erections (see, eg, Melis et al. (Neuroscience Letters 265 (1999) 171-174). In addition, intracerebroventricular (ICV) injections of the oxytocin antagonist vasotocin impair the sexual performance of experienced male rats in the presence of receiving females and eliminate ejaculation (probably caused by reduced insertion frequency) ) Argiolas, etc. (European Journal of Pharmacology)
Journal of Pharmacology) 149 (1988) 389-392). Since oxytocin antagonists have been shown to prevent penile erections, the reduced insertion frequency was thought to reflect the animals' reduced ability to achieve penile erections.

Although Gimpur and Farenholtz (above) and Meston et al. (Above) suggested that oxytocin reduces the latency to achieve ejaculation, another study has shown that oxytocin has no effect on ejaculation latency. Show. For example, Stoneham et al. (J. Endocrinology 107: 97-106,
(1985) show that intravenous infusion of oxytocin in rats subordinately reduces the number of insertions made prior to ejaculation but has no effect on ejaculation latency. In addition, the injection of oxytocin into the third ventricle increased the latency to the first back and insertion, prolonging the post-ejaculation refractory period, but had no effect on the ejaculation latency (Stoneham, etc.) .

  In addition, studies have shown that eliminating the increase in oxytocin during ejaculation does not make any difference in the time it takes to achieve arousal or orgasm. In Murphy et al. (J. of Clinical Endocrinology and Metabolism, 71, 4 (1990) 1056-1058), the opioid antagonist naloxone is It was shown that it has no effect on ejaculation in volunteers. In Ackerman et al. (Physiol Behav 63: 49-53 [1997]), N-methyl-D-aspartate injury, which leaves large cell neurons intact but destroys small cell PVN neurons, is inferior to the lower lumbar spinal cord (L5 -L6) reduced oxytocin-immunoreactive fibers. This decrease is associated with a significant decrease in semen ejection at the time of ejaculation, but backrest, insertion and ejaculation latency were not affected.

  In one embodiment, the present invention relates to a composition or pharmaceutical composition comprising a selective oxytocin antagonist compound for use in the treatment and / or prevention of male ejaculation disorders, particularly premature ejaculation. In the pharmaceutical composition, the selective oxytocin antagonist may optionally be mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive. Here, the composition (as well as the other compositions described herein) can be packaged for subsequent use in the treatment of male ejaculation disorders, particularly premature ejaculation.

  In another aspect, the present invention provides selective oxytocin for use in the treatment and / or prevention of male ejaculation disorders, particularly premature ejaculation, while maintaining erection formation mechanisms, particularly penile erections, and / or sexual impulses. With respect to a composition or pharmaceutical composition comprising an antagonist compound, the composition may optionally be mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive.

  In another aspect, the invention relates to the use of a selective oxytocin antagonist in the manufacture of a medicament (eg, a pharmaceutical composition) for use in the treatment of male ejaculation disorders, particularly premature ejaculation.

  In another aspect, the present invention provides a medicament (eg, pharmaceutical composition) for use in the treatment of male ejaculation disorders, particularly premature ejaculation, while maintaining erection formation mechanisms, particularly penile erections, and / or sexual impulses. ) For the use of selective oxytocin antagonists.

  In another aspect, the present invention relates to the use of a selective oxytocin antagonist in the preparation of a medicament (eg, pharmaceutical composition) for use in the treatment of male ejaculation disorders, particularly premature ejaculation.

  In another aspect, the present invention provides a medicament (eg, pharmaceutical composition) for use in the treatment of male ejaculation disorders, particularly premature ejaculation, while maintaining erection formation mechanisms, particularly penile erections, and / or sexual impulses. ) In the preparation of a selective oxytocin antagonist.

  In one embodiment, the present invention provides a method of treating or preventing male ejaculation disorders, particularly premature ejaculation, in humans or animals, wherein the selective oxytocin antagonist is a pharmaceutically acceptable carrier, diluent or It relates to a method comprising administering to an individual an effective amount of a selective oxytocin antagonist, optionally mixed with a pharmaceutical additive.

  In one embodiment, the present invention is a method for treating or preventing male ejaculation disorders, particularly premature ejaculation, while maintaining erection formation mechanisms, particularly penile erections, and / or sexual impulses in humans or animals. Administering to the individual an effective amount of the selective oxytocin antagonist, optionally mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive. Regarding the method.

  Further provided is a pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises one or more selective oxytocin antagonists.

  The present invention further provides a method for preparing a pharmaceutical composition according to the present invention, wherein the method mixes one or more selective oxytocin antagonists with a pharmaceutically acceptable diluent, pharmaceutical additive or carrier. Including that.

  In a further aspect, the present invention is a measurement method for identifying substances (hereinafter referred to as selective oxytocin antagonists) that can be used to treat or prevent male ejaculation disorders, particularly premature ejaculation. Relates to a measurement method comprising measuring whether a test substance can directly delay the endogenous ejaculation process, wherein this delay is the ejaculation latency in the presence of the test substance as defined herein. Defined as an increase in time (ie, the time taken from the first insertion to ejaculation) and / or recovery; such a potentiation effect by the test substance is useful for treating or preventing male ejaculation disorders, particularly early ejaculation Where the test substance is a selective oxytocin antagonist. Preferably, the substance has no or substantially no effect on penile erection. In other words, preferably the substance does not adversely affect penile erection, however, the substance can enhance endogenous penile erection.

In a further aspect, the present invention provides:
(A) carry out the measuring method according to the invention;
(B) identifying one or more substances capable of increasing and / or restoring ejaculation latency; and (c) preparing a quantity of one or more of these identified substances (where The substance is a selective oxytocin antagonist)
It relates to a method comprising steps. For this embodiment, the substance identified in step (b) can be modified, for example, to maximize activity, and then step (a) can be repeated. These steps can be repeated until the desired activity or pharmacokinetic profile is achieved.

Accordingly, in a further aspect, the present invention provides:
(A1) carry out the measurement method according to the invention; (b1) identify one or more substances that can directly increase and / or restore ejaculation latency; (b2) one or more of these identified (A2) step (a1) may optionally be repeated; and (c) preparing an amount of one or more of these identified substances (ie modified) (here) The substance is a selective oxytocin antagonist).

In a further aspect, the present invention provides:
(I) carrying out the measuring method according to the invention;
(Ii) identifying one or more substances capable of increasing and / or restoring ejaculation latency;
(Iii) testing the identified substances for their effects on penile erection in test animals such as anesthetized rodents;
(Iv) selecting a substance that has no or substantially no effect on penile erection; and (v) preparing an amount of one or more of the selected substances (where the substance is selective Oxytocin antagonist)
It relates to a method comprising steps. With respect to this embodiment, the substance identified in step (ii) can be modified, for example, to maximize activity, and then step (i) can be repeated. These steps can be repeated until the desired activity or pharmacokinetic profile is achieved.

  In a further aspect, the present invention is a diagnostic method comprising the steps of sexual stimulation at successive time intervals after initiation of sexual stimulation, i.e. 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes. One or more samples obtained from males during mechanical stimulation and determine whether the samples contain substances present in such amounts at times that cause male ejaculation disorders, preferably premature ejaculation Where the factor can specifically adjust the time it takes for the factor to appear and / or delay the concentration peak so as to achieve a beneficial effect through the use of the substance Where the substance is a selective oxytocin antagonist. Preferably, the factor is oxytocin. Sexual stimulation can be generated, for example, by a penile vibration stimulator (FertiCare, Horsholm, Denmark).

  In a further embodiment, the present invention provides that the male sample is collected at a continuous time interval after initiation of sexual stimulation, i.e. 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes. Relates to a diagnostic composition or kit comprising means for detecting an agent in one or more isolated samples taken during sexual stimulation; wherein the means comprises that the sample is a male ejaculatory disorder, preferably premature ejaculation Can be used to determine whether or not it contains a factor in such an amount at a time such that it causes a beneficial effect from the use of the substance, especially if the factor is The time it takes to appear can be adjusted and / or the concentration peak can be delayed; here the substance is a selective oxytocin antagonist. Preferably, the factor is oxytocin. Sexual stimulation can be caused, for example, by a penile vibration stimulator (Feltycare, Horsholm, Denmark).

  In a further aspect, the present invention relates to an animal model used to identify substances capable of treating and / or preventing male ejaculation disorders, particularly premature ejaculation, which model after introduction of the receiving female. Includes male animals that include means for measuring the animal's ejaculation latency (ie, the time taken from initial insertion to ejaculation), where the substance is a selective oxytocin antagonist.

  The animal model can further include or be used in conjunction with additional animal models that include means for measuring changes in penile erections. For example, a suitable additional model may include an anesthetized male animal that includes means for measuring changes in the intracavernous pressure and / or cavernous blood flow of the animal after stimulation of the pelvic nerve. Where the substance is a selective oxytocin antagonist.

  In a further aspect, the present invention relates to a measurement method for identifying substances capable of directly enhancing the endogenous ejaculation process in order to treat or prevent ejaculation disorders, in particular premature ejaculation, the measurement method comprising: Measuring the ejaculation latency of the animal after introduction of the receiving female (ie, the time taken from initial insertion to ejaculation); wherein the substance is selected Oxytocin antagonist.

  In a further aspect, the present invention provides a substance capable of directly enhancing the endogenous ejaculation process without affecting penile erection and / or sexual impulses to treat or prevent ejaculation disorders, particularly premature ejaculation. With regard to the measurement method for identification, the measurement method comprises administering a substance to the animal model of the invention; and measuring a change in the endogenous ejaculation process; wherein the change is after introduction of the receiving female Defined as the ejaculation latency of this animal (ie, the time it takes from first insertion to ejaculation); measuring penis erections and / or sexual impulses in animal models with no or substantially no change in that respect Here, this substance is a selective oxytocin antagonist.

  For ease of discussion, these and further aspects of the present invention are now discussed under the appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.

  The terms “selective oxytocin antagonist” and “selective oxytocin receptor antagonist” have the same meaning and mean an oxyton receptor antagonist that is selective for the oxytocin receptor compared to the vasopressin receptor, particularly the V1a receptor. To do.

  As used herein, the term “ejaculation latency” means the time required from initial insertion to ejaculation. As used herein, the term “recovery of ejaculation latency” means that the time required from initial insertion to ejaculation varies, preferably increases. Preferably, the time required from insertion to ejaculation varies (preferably increases) to near normal levels. Typically, a person suffering from premature ejaculation ejaculates within 30 seconds of sexual initiation (ie, from the first insertion) and often within 15 seconds of sexual initiation (ie, from the first insertion). In a preferred embodiment of the present invention, the ejaculation latency is increased to at least 30 seconds, preferably at least 60 seconds, more preferably at least 2 minutes, more preferably at least 5 minutes, more preferably at least 10 minutes. . Suitably, ejaculation latency can be recovered so that the time required from insertion to ejaculation is sufficiently delayed to satisfy the opponent.

  The term “sexual drive” as used herein means libido or sexual desire. The term “insertion” as used herein means vaginal penetration by the penis.

Preferred Embodiments In one embodiment, preferably the substance for use according to the invention is for oral administration.

  In another embodiment, the substance for use according to the invention is for topical or intranasal administration.

  Preferably, the substance according to the invention is for use in the treatment and / or prevention of premature ejaculation.

  Preferably, the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor, particularly the V1a receptor.

  Preferably, the selective oxytocin antagonist is at least 30-fold selective for the oxytocin receptor relative to the vasopressin receptor, particularly the V1a receptor.

  Preferably, the selective oxytocin antagonist is at least 50-fold selective for the oxytocin receptor relative to the vasopressin receptor, particularly the V1a receptor.

  Preferably, the selective oxytocin antagonist is at least 100-fold selective for the oxytocin receptor relative to the vasopressin receptor, particularly the V1a receptor.

  Preferably, the selective oxytocin antagonist is at least 200-fold selective for the oxytocin receptor relative to the vasopressin receptor, particularly the V1a receptor.

  Preferably, the selective oxytocin antagonist is at least 250-fold selective for the oxytocin receptor relative to the vasopressin receptor, particularly the V1a receptor.

  The present invention also encompasses administration of a substance of the present invention prior to and / or during sexual arousal / stimulation.

  Thus, in some aspects of the invention, it is highly desirable to have a sexual arousal / stimulation stage.

  Here, “sexual arousal / stimulation” may be one or more visual arousal / stimulation, physical awakening / stimulation, auditory arousal / stimulation or thought arousal / stimulation.

  Thus, preferably the substances of the present invention are delivered before or during sexual arousal / stimulation, especially when those substances are for oral delivery.

Preferred Embodiments The present invention provides the following (numbered) preferred embodiments:
1. A composition comprising a selective oxytocin antagonist for use in the treatment or prevention of male ejaculation disorders, wherein the selective oxytocin antagonist may optionally be mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive. object.
2. The composition of embodiment 1, wherein the male ejaculation disorder is premature ejaculation.
3. The composition of embodiment 1 or embodiment 2, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor compared to the vasopressin receptor.
4). The composition according to embodiment 3, wherein the vasopressin receptor is a V1a receptor.
5). Use of a selective oxytocin antagonist in the manufacture of a medicament for use in the treatment of male ejaculation disorders.
6). Use according to aspect 5, wherein the male ejaculation 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 the oxytocin receptor when compared to the vasopressin receptor.
8). Use according to aspect 7, wherein the vasopressin receptor is a V1a receptor.
9. The use according to any one of aspects 5-8, wherein the selective oxytocin antagonist is administered before and / or during sexual arousal.
10. The use according to any one of aspects 5-9, wherein the selective oxytocin antagonist is administered orally.
11. An effective method of treating or preventing male ejaculation disorders in humans or animals, wherein the selective oxytocin antagonist may optionally be mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive Administering an amount of a selective oxytocin antagonist to the individual.
12 12. The method according to aspect 11, wherein the male ejaculation disorder is premature ejaculation.
13. Embodiment 13. The method of embodiment 11 or embodiment 12, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor.
14 14. The method according to aspect 13, wherein the vasopressin receptor is a V1a receptor.
15. 15. The method according to any one of aspects 11-14, wherein the selective oxytocin antagonist is administered before and / or during sexual arousal.
16. The method according to any one of aspects 11-15, wherein the medicament is administered by the oral cavity.
17. A pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises one or more selective oxytocin antagonists.
18. The pharmaceutical pack according to embodiment 17, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor.
19. The pharmaceutical pack according to aspect 18, wherein the vasopressin receptor is a V1a receptor.
20. A method for preparing a pharmaceutical composition comprising mixing one or more selective oxytocin antagonists with a pharmaceutically acceptable diluent, pharmaceutical additive or carrier.
21. 21. The method of embodiment 20, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor.
22. The method according to embodiment 21, wherein the vasopressin receptor is a V1a receptor.
23. A method for identifying substances that can be used to treat and / or prevent male ejaculation disorders, comprising determining whether a test substance can directly enhance the endogenous ejaculation process Where the enhancement is defined as an increase and / or recovery in ejaculation latency in the presence of the test substance as defined herein; It has been shown that the substance can be useful in the treatment or prevention of male ejaculation disorders, wherein the test substance is a selective oxytocin antagonist.
24. 24. The measurement method according to aspect 23, wherein the male ejaculation disorder is premature ejaculation.
25. 25. The assay according to embodiment 23 or embodiment 24, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor.
26. The measurement method according to embodiment 25, wherein the vasopressin receptor is a V1a receptor.
27. A substance identified by the measurement method according to any one of aspects 23 to 26.
28. 28. A substance according to aspect 27 for use in treating or preventing male ejaculation disorders.
29. 29. A substance according to aspect 28, wherein the male ejaculation disorder is premature ejaculation.
30. A medicament for oral administration for treating a male ejaculation disorder, wherein the medicament comprises the substance according to aspect 27.
31. The medicament according to aspect 30, wherein the male ejaculation disorder is premature ejaculation.
32. 32. The medicament according to aspect 30 or aspect 31, wherein the medicament is administered before and / or during sexual arousal.
33. The medicament according to any one of aspects 30-32, wherein the medicament is administered by the oral cavity.
34. (A) performing any one of the methods of Embodiments 23-26; (b) identifying one or more substances capable of increasing and / or restoring ejaculation latency; and (c) a certain amount of A method comprising the step of preparing one or more of the identified substances; wherein the substance is a selective oxytocin antagonist.
35. 35. The method of embodiment 34, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor.
36. 36. The method of embodiment 35, wherein the vasopressin receptor is a V1a receptor.
37. An animal model for identifying a substance capable of treating or preventing male ejaculation disorders, said model comprising a male animal comprising means for measuring the ejaculation latency of this animal after introduction of a receiving female; This substance is a selective oxytocin antagonist.
38. The animal model according to aspect 37, wherein the male ejaculation disorder is premature ejaculation.
39. 39. The animal model of embodiment 37 or embodiment 38, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor compared to the vasopressin receptor.
40. 40. The animal model according to aspect 39, wherein the vasopressin receptor is a V1a receptor.
41. A method for identifying a substance capable of directly enhancing an endogenous ejaculation process to treat or prevent ejaculation disorders, wherein the substance is administered to an animal model of any one of embodiments 37 to 40; and A method comprising measuring the ejaculation latency of the animal after introduction of the receiving female; wherein the substance is a selective oxytocin antagonist.
42. Use of a multi-drug combination consisting of one or more selective oxytocin antagonists and one or more of the following auxiliary active substances in the manufacture / preparation of a medicament for the treatment and / or prevention of male ejaculation disorders:
i) PDE inhibitors, more particularly PDE5 inhibitors, which inhibitors preferably have an IC50 of less than 100 nM for the respective enzyme;
ii) Serotonin receptor agonists or modulators, more particularly agonists or modulators of 5HT2C, 5HT1B and / or 5HT1D receptors, including ampitrine;
iii) Serotonin receptor antagonists or modulators, more particularly 5HT1A antagonists or modulators, including NAD-299 (robalzotan) and WAY-100635, and / or more particularly batanopirde, granisetron, ondansetron Antagonists or modulators of 5HT3 receptors, including tropistron and MDL-73147EF;
iv) antidepressants, in particular i) selective serotonin reuptake inhibitors (SSRI) including sertraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, mirtazapine, nefazodone and trazodone; ii) clomipramine, desapramine Tricyclic antidepressants (TCAs), including imipramine, amitriptyline, doxepin, amoxapine, maprotiline, nortriptyline, protriptyline, trimipramine and buproprion; and iii) monoamine oxidase;
v) α-adrenergic receptor antagonists (also known as α-adrenergic blockers, α-blockers or α-receptor blockers); suitable α1-adrenergic receptor antagonists include phentolamine, prazosin , Phentolamine mesylate, trazodone, alfuzosin, indolamine, naphthopidyl, tamsulosin, phenoxybenzamine, laurophia total alkaloid, recorda 15/2739, SNAP1069, SNAP5089, RS17053, SL89.0591, doxazosin, terazosin Suitable α2-adrenergic receptor antagonists include dibenarnine, tolazoline, trimazosin, efaloxane, yohimbine, idazo Suitable non-selective α-adrenergic receptor antagonists include dapiprazole; additional α-adrenergic receptor antagonists include WO99 / 30697, US 4,188,390, US 4, 026,894, US 3,511,836, US 4,315,007, US 3,527,761, US 3,997,666, US 2,503,059, US 4,703,063, US 3,381,009, US 4,252 721 and US 2,599,000;
vi) Selective serotonin reuptake inhibitors with rapid efficacy.
43. Use of a multi-drug combination consisting of one or more selective oxytocin antagonists and one or more PDE inhibitors (PDEi's) in the manufacture / preparation of a medicament for the treatment or prevention of male ejaculation disorders.
44. 45. Use according to aspect 42 or aspect 43, wherein the male ejaculation disorder is premature ejaculation.
45. 45. Use according to aspect 43 or aspect 44, wherein the PDEi is a PDE5 inhibitor (PDE5i).
46. The use according to any one of aspects 42-45, wherein the medicament is administered by the oral cavity.
47. A pharmaceutical composition comprising one or more selective oxytocin antagonists and one or more PDEi's, optionally mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive.
48. 48. The pharmaceutical composition according to aspect 47, wherein the PDEi is PDE5i.
49. 49. The pharmaceutical composition according to any one of aspects 47 or 48, wherein the composition is administered by the oral cavity.
50. Use of the pharmaceutical composition according to any one of aspects 47-49 in the preparation of a medicament for the treatment and / or prevention of ejaculation disorders.

Surprising and unexpected discovery The present invention
(A) Administration of a selective oxytocin antagonist increases ejaculation latency. Preferably, administration of a selective oxytocin antagonist restores ejaculation latency, preferably to near normal levels;
(B) Administration of a selective oxytocin antagonist unexpectedly increases ejaculation latency without substantially disturbing and / or adversely affecting erection formation mechanisms, particularly penile erections. Preferably, administration of a selective oxytocin antagonist restores ejaculation latency, preferably to near normal levels, without substantially disturbing and / or adversely affecting erection formation mechanisms, particularly penile erections;
(C) administration of a selective oxytocin antagonist increases ejaculation latency without substantially preventing and / or adversely affecting sexual impulses. Surprising and unexpected discovery that administration of a selective oxytocin antagonist preferably restores ejaculation latency, preferably to near normal levels, without substantially impeding and / or adversely affecting sexual impulses To demonstrate.

Advantages The present invention provides:
(A) selectively inhibiting the oxytocin receptor through the use of selective oxytocin antagonists results in the treatment of premature ejaculation;
(B) Selective inhibition of oxytocin receptors through the use of selective oxytocin antagonists results in unexpectedly early firing without substantially disturbing and / or adversely affecting erection formation mechanisms, particularly penile erections Attributed to the treatment of sperm;
(C) Selectively inhibiting oxytocin receptors through the use of selective oxytocin antagonists can be used for the treatment of unexpectedly early-launched sperm without substantially preventing and / or adversely affecting sexual impulses. It is advantageous to return.

Patient group ejaculation disorders, especially those with early firing, should benefit from treatment with selective oxytocin antagonists.

  Initial investigations will elucidate ejaculatory disorders described below, particularly those with early-fired ejaculation, to benefit from treatment with selective oxytocin antagonists (or multidrugs that contain selective oxytocin antagonists as described below). Suggest a deaf. These patient groups include one or more of the following: neurological disorders, physiological disorders, lack of psychological sexual skills, physical illness, physical damage, pharmacological side effects, psychological distress and urgency of relationships People who suffer from.

Oxytocin Receptor As indicated above, the substance may be any suitable substance that can act as a selective oxytocin antagonist.

Background teachings on oxytocin receptors can be found at http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm It has been prepared by Victor A. McKusick and others. The following text on oxytocin receptors has been extracted from its source:
Kimura et al. (1992:
Nature 356: 526-529) reported the structure and expression of a human oxytocin receptor cDNA isolated by expression cloning. The encoded receptor was a 388 amino acid polypeptide with a seven transmembrane domain typical of G protein coupled receptors. The oxytocin receptor expressed in Xenopus oocytes specifically responded to oxytocin and induced an inward membrane current. The messenger RNAs for this receptor were of two sizes, 3.6 kb in the breast and 4.4 kb in the ovary, endometrium and myometrium. MRNA levels in the myometrium were very high as of the expected date of delivery. Inoue et al. (1994 Biol. Chem. 269: 32451-32456) showed by Southern blotting that one copy of the OXTR gene was present in the human genome. fluorescence
By in situ hybridization, they showed that the gene is located on 3p26.2. The gene spans about 17 kb and has 3 introns and 4 exons. Exons 1 and 2 correspond to the 5-prime non-coding region, followed by exons 3 and 4 encoding the receptor amino acids. The largest intron 3 at the 12 kb position disrupts the coding region immediately following the putative 6-transmembrane domain. The transcription start sites shown by primer extension analysis are 618 and 621 bp upstream of the methionine start codon. PCR analysis and fluorescence of somatic cell hybrids
By in situ hybridization, Simmons et al. (1995 Genomics 26: 623-625) assigned the OXTR gene to 3p25. '

A detailed review of the receptor structure is provided in Gimpur and Fahrenholtz (Physiological Reviews 81: 2 April 2001). Among them, to date, in addition to the isolation and identification of cDNA encoding the human oxytocin receptor (see Kimura et al. Above), pigs (Gorbulev et al. Eur. J. Biochem. 215, 1-7 1993), rat (Rozen et al. Proc. Natl. Acad. Sci. USA
92: 200-204 1995), sheep (Riley et al. J. Biol. Chem. 266: 21428-21433, 1991), cattle (Bathgate et al. DNA Cell Biol. 14: 1037-1048,
1995), mice (Kubota et al. Mol. Cell Endocrinol 124: 25-32 1996) and rhesus monkeys (Salvatore et al. J. Recept Signal Transduct Res.
18: 15-24, 1998), the sequence encoding the oxytocin receptor was also identified.

Oxytocin receptor sequence data The nucleotide and amino acid sequences of the human oxytocin receptor are available in the literature. By way of example, only the amino acid sequence of the human oxytocin receptor is provided in SEQ ID NO: 1.

Details of the preferred measurement system to identify and / or studying the selective oxytocin antagonist oxytocin antagonist is provided in the section entitled subsequent Bu oxytocin antagonist measurement゛To.

An example of a suitable oxytocin antagonist is shown below:

The synthesis of L-368,899 is described by Williams et al. (1994) J. Med. Chem. 37, 565-571.
Is taught.

  L-368,899 is a selective oxytocin antagonist. L-368,899 is more than 20-fold selective for the oxytocin receptor over the vasopressin receptor, particularly the V1a receptor. Preferably the selectivity is both binding and functional selectivity.

Certain known oxytocin antagonists, such as vasotocin, are sometimes referred to as “selective oxytocin antagonists”. _{However, d (CH 2) 5 Tyr} (Me) -Orn 8 - vasotocin (hereinafter, referred to Bu vasotocin゛To) are only selective 2-3 times the oxytocin receptors than vasopressin receptor particular V1a receptor . As such, vasotocin is a substantially non-selective oxytocin / vasopressin antagonist and does not fall within the scope of the term “selective oxytocin antagonist” according to the present invention. Preferably, the selective oxytocin antagonist according to the invention is at least 20-fold selective for the oxytocin receptor over the vasopressin receptor, in particular the V1a receptor.

Ｃ１＝濃度１、Ｃ２＝濃度２、Ｃ３＝濃度３、等。
列Ａ＆Ｂ＝ＳＴＤ
列Ｃ＆Ｄ＝化合物１
列Ｅ＆Ｆ＝化合物２
列Ｇ＆Ｈ＝化合物３
_● 膜蛋白質を、氷上で穏やかに解凍し、測定に最適な蛋白質濃度に希釈する（蛋白質線形性測定用付録参照）（およそ１００μｇ／ｍｌ）
_● ２００μｌの膜蛋白質を各ウェルに加えて反応を開始し、ブロックを、次いで、ＲＴで６０分間穏やかに振とうしながらインキュベートする。
_● 反応は、０．５％ＰＥＩに予め浸しておいたユニフィルターＧＦ／Ｃフィルターを通じた濾過および３ｘ１ｍｌの氷冷洗浄バッファーでの素早い洗浄により終了する。
_● フィルターを、５５℃の乾燥器で２時間乾燥するか、またはベンチ上で一晩（〜１６時間）放置する。
_● フィルターを底に封じ、３０μｌのマイクロシンチ−Ｏを各ウェルに入れる。フィルターを、次いで、トップシールＡで封じ、［^３Ｈ］９６ウェルユニフィルタープロトコール１１を用いてパッカードトップカウンター（Ｂｌｄ５０３／Ｇ７Ａ）によりカウントする。
Ｃ．データ分析
データ分析をＥＣＡＤＡにより行う
特異的結合を次の通りに算定する：
特異的結合＝平均合計ｃｐｍ−平均ＮＳＢｃｐｍ
試験化合物では、受容体に結合したリガンドの量を、次の通りに表す：
％結合＝（試料ｃｐｍ−平均ＮＳＢｃｐｍ）／特異的結合ｃｐｍｘ１００
リガンド結合のパーセンテージ阻害を、次の通りに算定される％阻害で報告する：
％阻害＝１００−％結合 Oxytocin antagonist binding assay and vasopressin V1a binding assay
A. Oxytocin receptor ligand binding IC50 measurement i) Buffer cell growth medium Hams F12 nutrient mix
10% FCS
2 mM L-glutamine
400 μg / ml G418
15 mM HEPES
Membrane preparation buffer 50 mM Tris-HCl, pH 7.8
10 mM MgCl ₂
Protease inhibitor frozen buffer 50 mM Tris-HCl, pH 7.8
10 mM MgCl ₂
20% glycerol measurement medium 50 mM Tris-HCl, pH 7.8
10 mM MgCl ₂
0.25% BSA
Max. 2.5% DMSO / 50 mM Tris-HCl,
0.5 μM (arg ⁸ ) -baso prepared at pH 7.8
Tocin, 10 mM MgCl ₂
Min. 2.5% DMSO / 50 mM Tris-HCl,
pH 7.8, 10 mM MgCl ₂
ii) Compound dilution (final concentration of 10 μM in the measurement)
a) 4 mM HTA stock compound in 100% DMSO b) Compound is diluted to 200 μM in dH ₂ O.
c) The compound is further diluted to 100 μM in 100 mM Tris-HCl, pH 7.8, 20 mM MgCl ₂ . This results in a final concentration of 2.5% DMSO, 50 mM Tris-HCl, pH 7.8, 10 mM MgCl ₂ .
d) Using diluted stock, prepare 1: 2 dilution over 10 points in 50 mM Tris-HCl, pH 7.8, 10 mM MgCl ₂ , 2.5% DMSO containing TECAN Genesis .
e) Dispense 10 μl of compound into 384 well Optiplate according to the plate layout required for analysis by ECADA leaving space for standard (arg ⁸ ) -vasotocin IC50. These plates can be stored at 4 ° C.
f) On the day of measurement, 10 μl Max. In + wells and 10 μl of Min. To the wells and do a 1: 2 serial dilution over 10 points with (arg ⁸ ) -vasotocin at the highest concentration of 100 nM (final 20 nM).
iii) maintenance of oxytocin receptor-CHO cells
_• Regularly maintain the cell line as a continuous culture in 50 ml growth medium in a 225 cm ² flask.
_• Cells are passaged by removing the medium from the monolayer, washing with PBS, and incubating with trypsin until the cells show signs of dissociation. After smashing to drop the cells from the bottom of the flask, the cells are resuspended in growth medium and seeded in a 225 cm ² flask at a concentration of 8 × 10 ⁵ cells / flask.
iv) Cell growth in roller bottles
_● The cells were seeded at a concentration of 6x10 ⁶ cells / bottle in roller bottles 10X850cm ^2, to reach the confluent nearby.
_● Cells were removed from the bottle using trypsin as described above, cells are seeded in 100x roller bottles (i.e. 1:10 split ratio of).
_● the growth medium was removed, added PBS / bottle 40 ml, and before collecting by scraping with Serumeito (CellMate), the cells again, to reach near confluence. The cell suspension is then centrifuged at 2000 rpm, washed in PBS, centrifuged again, and the pellet is aliquoted and frozen at −80 ° C.
v) Membrane preparation
_● removed cell pellets from the freezer, thawed on ice, resuspended in membrane preparation buffer of packed cells volume ml per 3 ml.
_• Before centrifuging at 25,000 xg for 30 minutes, the suspension is then homogenized with several bursts of 5 seconds on ice using a mechanical homogenizer.
_● After resuspending the pellet in 1 ml of freezing buffer per ml of the initially packed cell volume, homogenize the suspension briefly to remove fine clumps. The protein concentration is then measured and the membrane suspension is finally frozen at −80 ° C. in portions with a minimum of 5 mg / ml.
vi) Measurement
_• Thaw the membrane on ice before diluting to 1 mg / ml in measurement buffer. SPA beads are resuspended in measurement buffer at 50 mg / ml. From these concentrates, the beads are pre-bound to the membrane by incubating 30 μg of protein per mg of bead on a top-to-tail shaker at 4 ° C. for 2 hours. The beads / membrane are then centrifuged at 2000 rpm for 10 minutes and the pellet is resuspended at 3 mg / ml.
_• All operations of ¹²⁵ I-OVTA are performed using chips that have been silanized using SigmaCote. All bottles and tubes are also silane treated. ¹²⁵ I-OVTA is diluted in 1 ml of measurement buffer per 50 μCi of lyophilized ligand. A 5 μl sample is then counted in duplicate using liquid scintillation counting (protocol 61 with Wallac Counter) and the concentration of the ligand is calculated (see example below). This will overcome the loss of ligand due to stickiness. Using the measured concentration, ¹²⁵ I-OVTA is diluted to 0.3 nM in the measurement buffer.
Example:
If 5 μl becomes 500,000 dpm and the specific activity of the ligand is 2200 Ci / mmol:
Concentration = 500,000 / (2.2 × 2200 × 5) nM
_• Add 20 μl of bead / membrane preparation to the prepared Optiplate using Multi-drop. The bead / membrane preparation is maintained in suspension using a stirred flask. 20 μl of ¹²⁵ I-OVTA is then added to each well of the Optiplate using a multi-drop. After 4 hours incubation at room temperature, the plates are counted using a TopCount NXT for 30 s / well.
B. Vasopressin V1A receptor binding assay i) Substance
_● Human clone protein / cell science vasopressin V1a receptor in CHO cells (Protein / cell sciences)
_● HEPES Sigma (H7523)
_● Magnesium chloride (MgCl ₂ ) Sigma (M2670)
_● Bovine serum albumin (BSA) Sigma (A6003)
_● Glycerol Sigma (G5150)
_● Protease inhibitor cocktail tablets Roche (1697498)
_● Pierce BCA protein assay reagent Pierce (23225)
_● 8-Arg [Phenylalanyl-3,4,5
^-3 H] -vasopressin ( ³ H-AVP) NEN (NET 800)
_{● d (CH2) 5Tyr (Me} ) AVP [β- mercapto-beta, beta-cyclopenta methylene ^{propionyl, O-Me-Tyr 2,} Arg 8] - vasopressin (βMCPVP) Sigma (V2255)
_● Dimethyl sulfoxide stores (W34)
_● 96-well polypropylene blocks stores (D8281)
_● Polyethyleneimine (PEI) Sigma (P3143)
_● 96-well Unifilter Packard Plate GF / C (Packard) (60000514)
_● Topseal A Packard (60000515)
_● Microscint-O Packard (6013611)
_● SR49059 (UK222,633) control compounds device: Packard Unifilter unit Top Counter / NXT Counter ii) how working solution:
Membrane preparation buffer: 25 mM HEPES (pH 7.4)
5 mM MgCl ₂
Protease inhibitor (1 tablet per 50 ml)
Freezing buffer: 25 mM HEPES (pH 7.4)
5 mM MgCl ₂
20% glycerol measurement buffer: 25 mM HEPES (pH 7.4)
5 mM MgCl ₂
0.05% BSA
Washing buffer: 25 mM HEPES (pH 7.4)
5 mM MgCl ₂
5 nM solution in ³ H-AVP measurement buffer (final measured concentration of 0.5 nM)
Total 25% DMSO in ddH ₂ O
10 μM βMCPVP (1 μM final measured concentration) in NSB 25% DMSO / ddH ₂ O
STD SR49059 (UK222,633) was started at the highest concentration of 1 μM (100 nM final measured concentration) and continued to 25 pM with 30% (3 pM final measured concentration) in 25% DMSO / ddH ₂ O Dilute in.
Compound: 50 μl of 4 mM compound in 100% DMSO. This is diluted 4-fold in dH ₂ O to obtain the highest concentration of 1 mM in 25% DMSO. Compounds are further diluted in a semi-log step (25% DMSO after dilution) in 25% DMSO except for the first dilution (1 mM to 300 μM) that will be in 18% DMSO. Dilution is performed manually or by Tecan and protocol file Kin28IC50 dilution2. Perform using gem. The 10 pt IC50 curve starts at a lower concentration as required by the compound, but all drugs are initially screened starting at 100 μM.
0.5% PEI Prepare 50% PEI in distilled H ₂ O and dilute to 0.5% in dH ₂ O.
iii) Membrane preparation
_● Remove the frozen cell pellet from the freezer, gently thawed on ice.
_● Add 3 ml of membrane preparation buffer per ml of packed cell initially and centrifuge at 1000 rpm for 10 minutes before homogenizing the suspension with polytron using several bursts of 5 seconds on ice. To do.
_● Remove the supernatant and store on ice. An additional 3 ml of membrane preparation buffer per ml of initially packed cell volume is added to the pellet, homogenized on ice, and then centrifuged at 1000 rpm for 10 minutes.
_● removed supernatant was added to the supernatant volume taken out previously, then centrifuged for 30 minutes at 25,000xg and 4 ° C..
_• Resuspend the 25,000 xg pellet by homogenization in 1 ml of freezing buffer per ml of initially packed cell volume and determine the protein concentration.
iv) Measurement of protein concentration
_● The BSA, the following concentrations: at 2000,1000,500,250,125,62.5 and 31.255μg / ml is prepared in distilled _{H 2} O.
_● Each BSA solution 10 [mu] l, duplicate addition 3 times the plate clear 96-well (see accompanying plate map) is added distilled of _{H 2} O 10 [mu] l into three blank wells.
_● The 10μl of membrane preparations, as is 1 / 3,1 / 10,1 / 30 and 1/100 dilution of membrane preparation is added to triplicate to the plate.
_• 200 μl of Pierce protein reagent (50A: 1B) is added to each well and the plate is incubated at 37 ° C. for 30 minutes and then read on an Anthos spectrophotometer at an absorption setting of 570 nm.
_{• From the} BSA standard curve, determine the concentration of protein in the membrane preparation (using the dilution of the membrane preparation located in the center of the standard curve).
_• Dilute the membrane preparation to a protein concentration of 5 mg / ml in freezing buffer before freezing 200 μl at -80 ° C.
v) Measurement protocol
_● preparing a measurement reagent ^{(3 H-AVP, βMCPVP (} NSB compounds) and test compound - see above working solution). All peptide solutions are kept on ice.
_The plate format is 10 points of IC50-4 compound per separate row of plate overlap experiments. The following reagents are added to the appropriate wells of a 96 well polypropylene block and stirred.
(T) in each and every well: 25 μl ³ H-AVP
(A1, B1, C1, D1, E12, 25 μl excipient F12, G12 & H12)
(N) in each NSB well: 25 μl ³ H-AVP
(A12, B12, C12, D12, 25 μl βMCPVP
E1, F1, G1 & H1)
In each measurement well: 25 μl ³ H-AVP
25 μl test compound

C1 = concentration 1, C2 = concentration 2, C3 = concentration 3, etc.
Row A & B = STD
Row C & D = Compound 1
Row E & F = Compound 2
Row G & H = Compound 3
_● Thaw membrane protein gently on ice and dilute to the optimal protein concentration for measurement (see appendix for protein linearity measurement) (approximately 100 μg / ml)
_• 200 μl of membrane protein is added to each well to initiate the reaction, and the block is then incubated for 60 minutes at RT with gentle shaking.
_The reaction is terminated by filtration through a unifilter GF / C filter presoaked in 0.5% PEI and a quick wash with 3 × 1 ml ice-cold wash buffer.
_● a filter, and dried for 2 hours in an oven of 55 ° C., or bench on overnight (16 h) to stand.
_● sealed the filter to the bottom, put the micro scintigraphy -O of 30μl to each well. The filter is then sealed with Top Seal A and counted with a Packard Top Counter (Bld503 / G7A) using [ ³ H] 96 well Unifilter protocol 11.
C. Data analysis Specific analysis performed by ECADA for data analysis is calculated as follows:
Specific binding = average total cpm-average NSBcpm
For test compounds, the amount of ligand bound to the receptor is expressed as follows:
% Binding = (sample cpm−average NSBcpm) / specific binding cpmx100
Percentage inhibition of ligand binding is reported as% inhibition calculated as follows:
% Inhibition = 100-% binding

Oxytocin antagonist function measurement and vasopressin V1a antagonist function measurement Spontaneous myometrial contraction from humans, non-human primates and rodents is sensitive to selective oxytocin receptor antagonists in vitro (Wilson) Et al. BJOG September 2001; 108 (9): 960-6).

  In vitro pharmacology of spontaneous contraction of myometrium from humans and animals. Human myometrial samples are obtained by caesarean section. The tissue strip is suspended in an organ bath for isometric force recording. Cumulative concentration effect curves for selective oxytocin receptor antagonists and mixed oxytocin / vasopressin V1a receptor antagonists can be obtained. Inhibition of spontaneous myometrial contraction is observed in vitro.

More particularly multiple drug combination drug, the present invention is male ejaculatory disorders as outlined herein, a particularly premature compounds of the present invention for the treatment of ejaculation and one or more auxiliary active agent (suitably Examples further include multidrug combinations (see discussion below).

  The present invention further includes a selective oxytocin antagonist according to the present invention and two types of supplementation in the manufacture of a medicament for the treatment and / or prevention of male ejaculation disorders, particularly premature ejaculation, as outlined herein. Including the use of multi-drug combinations consisting of active substances (see discussion below for suitable examples).

  The present invention further relates to a selective oxytocin antagonist and two auxiliary active substances according to the invention in the manufacture of a medicament for the treatment and / or prevention of male ejaculation disorders, in particular premature ejaculation, as outlined herein (suitable Examples include the use of multi-drug combinations consisting of a later discussion).

  The present invention further relates to a selective oxytocin antagonist according to the present invention and one kind in the manufacture or preparation of a medicament for the treatment and / or prevention of male ejaculation disorders, in particular premature ejaculation, as outlined herein. Including the use of multi-drug combinations consisting of a supplementary active substance (see discussion below for suitable examples).

  The invention further relates to a selective oxytocin antagonist and one auxiliary active substance according to the invention in the manufacture or preparation of a medicament for the treatment and / or prevention of male ejaculation disorders, in particular premature ejaculation, as outlined herein (A suitable example includes the use of a combination drug consisting of a discussion below).

  Accordingly, further multi-drug embodiments of the present invention include multi-drug combinations (simultaneous, separate or sequential) comprising a compound of the present invention and one or more auxiliary active substances (see discussion below for suitable examples). For administration).

  A still further multi-drug embodiment of the present invention is a pharmaceutical composition (simultaneously, separately or separately) consisting essentially of a selective oxytocin antagonist and two auxiliary active substances (see discussion below for suitable examples). For continuous administration).

  A still further multi-drug embodiment of the present invention is a pharmaceutical composition (for simultaneous, separate or sequential administration) comprising a selective oxytocin antagonist and two auxiliary active substances (see discussion below for suitable examples). )I will provide a.

  A still further multi-drug embodiment of the present invention is a pharmaceutical composition (simultaneous, separate or concise) consisting essentially of a selective oxytocin antagonist and one auxiliary active substance (see discussion below for suitable examples). For continuous administration).

  A still further multi-drug embodiment of the present invention is a pharmaceutical composition (for simultaneous, separate or sequential administration) comprising a selective oxytocin antagonist and one auxiliary active substance (see discussion below for suitable examples). )I will provide a.

Supplementary Active Substances Suitable auxiliary active substances for use in the combination drug of the present invention include the following:
1) PDE inhibitors, more particularly PDE5 inhibitors (see below), which inhibitors preferably have an IC50 of less than 100 nM for the respective enzyme;
2) Serotonin receptor agonists or modulators, more specifically 5HT2C, 5HT1B and / or 5HT1D receptor agonists or modulators, including ampitrine;
3) Serotonin receptor antagonists or modulators, more specifically 5HT1A antagonists or modulators, including NAD-299 (Rovarzotan) and WAY-100635, and / or more particularly Batanopiled, Granisetron, Ondansetron, Tropistron and MDL- Antagonists or modulators of the 5HT3 receptor, including 73147EF;
4) antidepressants, in particular i) selective serotonin reuptake inhibitors (SSRi) including sertraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, mirtazapine, nefazodone and trazodone; ii) clomipramine, desapramine, imipramine, Tricyclic antidepressants (TCAs) including amitriptyline, doxepin, amoxapine, maprotiline, nortriptyline, protriptyline, trimipramine and buproprion; and iii) monoamine oxidase;
5) α-adrenergic receptor antagonists (also known as α-adrenergic blockers, α-blockers or α-receptor blockers); suitable α1-adrenergic receptor antagonists include phentolamine, prazosin , Phentolamine mesylate, trazodone, alfuzosin, indolamine, naphthopidyl, tamsulosin, phenoxybenzamine, laurophia total alkaloids, Recordati 15/2739, SNAP1069, SNAP5089, RS17053, SL89.0591, doxazosin, terazosin and abanokill; Suitable α2-adrenergic receptor antagonists include dibenalnin, trazoline, trimazosin, efaloxane, yohimbine, idazoxan, clonidine and dibenal. Suitable non-selective α-adrenergic receptor antagonists include dapiprazole; additional α-adrenergic receptor antagonists, each of which is incorporated herein by reference, WO 99 / 30697, US 4,188,390, US 4,026,894, US 3,511,836, US 4,315,007, US 3,527,761, US 3,997,666, US 2,503,059, US 4,703,063, Described in US 3,381,009, US 4,252,721 and US 2,599,000;
6) Rapid action, such as 3-[(dimethylamino) methyl] -4- [4- (methylsulfanyl) phenoxy] benzenesulfonamide (as published in WO01 / 72687-Example 28) Selective serotonin reuptake inhibitor (fast-acting SSRI).

  Regarding cross-references herein to compounds contained in patents and patent applications that can be used according to the invention, the inventors claim (especially claim 1) and specific examples ( All of which are meant to be therapeutically active compounds as defined herein).

  When administering a combination of active agents, they can be administered simultaneously, separately or sequentially.

Auxiliary Substance- PDE5 Inhibitors Suitable cGMP PDE5 inhibitors for use according to the present invention include:
Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in EP-A-0463756; Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in EP-A-0526004; Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in published international patent application WO 93/06104; isomeric pyrazolo [4,3-d] disclosed in published international patent application WO 93/07149 Pyrimidin-4-ones; quinazolin-4-ones disclosed in published international patent application WO 93/12095; pyrido [3,2-d] pyrimidines disclosed in published international patent application WO 94/05661 4-ones; purine-6-ones disclosed in published international patent application WO 94/00453; published international patent application WO 98/49166 The indicated pyrazolo [4,3-d] pyrimidin-7-ones; the pyrazolo [4,3-d] pyrimidin-7-ones disclosed in published international patent application WO 99/54333; EP-A- Pyrazolo [4,3-d] pyrimidin-4-ones disclosed in 0995751; pyrazolo [4,3-d] pyrimidin-7-ones disclosed in published international patent application WO 00/24745; EP- Pyrazolo [4,3-d] pyrimidin-4-ones disclosed in A-099750; compounds disclosed in published international application WO 95/19978; compounds disclosed in published international application WO 99/24433 and And compounds disclosed in published international application WO 93/07124.

  Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in published international application WO01 / 27112; pyrazolo [4,3-d] pyrimidine-7 disclosed in published international application WO01 / 27113 -Ones: compounds disclosed in EP-A-1092718 and compounds disclosed in EP-A-1092719.

Preferred type V phosphodiesterase inhibitors (= phosphodiesterase 5 (PDE) inhibitors; PDE5i's) for use according to the present invention include the following:
1-[[3- (6,7-Dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d] pyrimidin-5-yl) -4-ethoxyphenyl] sulfonyl]- 5- [2-Ethoxy-5- (4-methyl-1-piperazinylsulfonyl) phenyl] -1-methyl-3-n-propyl-1,6-dihydro-, also known as 4-methylpiperazine 7H-pyrazolo [4,3-d] pyrimidin-7-one (sildenafil) (see EP-A-0463756);
5- (2-Ethoxy-5-morpholinoacetylphenyl) -1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-one (EP-A- 052604));
3-ethyl-5- [5- (4-ethylpiperazin-1-ylsulfonyl) -2-n-propoxyphenyl] -2- (pyridin-2-yl) methyl-2,6-dihydro-7H-pyrazolo [ 4,3-d] pyrimidin-7-one (see WO 98/49166);
3-ethyl-5- [5- (4-ethylpiperazin-1-ylsulfonyl) -2- (2-methoxyethoxy) pyridin-3-yl] -2- (pyridin-2-yl) methyl-2,6 Dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-one (see WO 99/54333);
3-ethyl-5- {5- [4-ethylpiperazin-1-ylsulfonyl] -2-([(1R) -2-methoxy-1-methylethyl] oxy) pyridin-3-yl} -2-methyl (+)-3-ethyl-5- [5- (4-ethylpiperazin-1-yl), also known as -2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-one Sulfonyl) -2- (2-methoxy-1 (R) -methylethoxy) pyridin-3-yl] -2-methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-one (See WO99 / 54333);
1- {6-Ethoxy-5- [3-ethyl-6,7-dihydro-2- (2-methoxyethyl) -7-oxo-2H-pyrazolo [4,3-d] pyrimidin-5-yl]- 5- [2-Ethoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl] -3-ethyl-2- [also known as 3-pyridylsulfonyl} -4-ethylpiperazine 2-methoxyethyl] -2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-one (see WO 01/27113, Example 8);
5- [2-Iso-butoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl] -3-ethyl-2- (1-methylpiperidin-4-yl) -2,6- Dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-one (see WO 01/27113, Example 15);
5- [2-Ethoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl] -3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo [4,3-d ] Pyrimidin-7-one (see WO01 / 27113, Example 66);
5- (5-acetyl-2-propoxy-3-pyridinyl) -3-ethyl-2- (1-isopropyl-3-azetidinyl) -2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidine- 7-one (see WO01 / 27112, Example 124);
5- (5-acetyl-2-butoxy-3-pyridinyl) -3-ethyl-2- (1-ethyl-3-azetidinyl) -2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidine- 7-one (see WO01 / 27112, Example 132);
(6R, 12aR) -2,3,6,7,12,12a-Hexahydro-2-methyl-6- (3,4-methylenedioxyphenyl) pyrazino [2 ′, 1 ′: 6,1] pyrido [ 4,3-b] indole-1,4-dione (IC-351), ie the compounds of Examples 78 and 95 of published international application WO 95/19978 and the compounds of Examples 1, 3, 7 and 8;
1-[[3- (3,4-Dihydro-5-methyl-4-oxo-7-propylimidazo [5,1-f] -as-triazin-2-yl) -4-ethoxyphenyl] sulfonyl]- 2- [2-Ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl] -5-methyl-7-propyl-3H-imidazo, also known as 4-ethylpiperazine [ 5,1-f] [1,2,4] triazin-4-one (vardenafil), ie the compounds of Examples 20, 19, 337 and 336 of published international application WO99 / 24433; and published International application WO 93/07124, Example 11 compound (EISAI); and Rotella DP (Rotella
DP), J. Med. Chem., 2000, 43, 1257.

Yet another type of cGMP PDE5 inhibitor useful for linking with the present invention includes: 4-bromo-5- (pyridylmethylamino) -6- [3- (4-chlorophenyl) -propoxy] -3 (2H ) Pyridazinone; 1- [4- (1,3-benzodioxol-5-ylmethyl) amino] -6-chloro-2-quinazolinyl] -4-piperidine-carboxylic acid, monosodium salt; (+)-cis -5,6a, 7,9,9,9a-hexahydro-2- [4- (trifluoromethyl) -phenylmethyl-5-methyl-cyclopent-4,5] imidazo [2,1-b] purine-4 ( 3H) ON; furazlocillin; cis-2-hexyl-5-methyl-3,4,5,6a, 7,8,9,9a-octahydrocyclopent [4,5] -imidazo [2,1 -B] Pudding -4-one; 3-acetyl-1- (2-chlorobenzyl) -2-propylindole-6-carboxylic acid ester; 3-acetyl-1- (2-chlorobenzyl) -2-propylindole-6-carboxylic acid Acid ester; 4-bromo-5- (3-pyridylmethylamino) -6- (3- (4-chlorophenyl) propoxy) -3- (2H) pyridazinone; 1-methyl-5 (5-morpholinoacetyl-2- n-propoxyphenyl) -3-n-propyl-1,6-dihydro-7H-pyrazolo (4,3-d) pyrimidin-7-one; 1- [4-[(1,3-benzodioxole- 5-ylmethyl) amino] -6-chloro-2-quinazolinyl] -4-piperidinecarboxylic acid, monosodium salt; Pharmaprojetcts no. 4516 (Glaxo Welcome
Wellcome); Pharma Projects No. 5051 (Bayer; Pharma Projects No. 5064 (Kyowa Hakko; see WO96 / 26940); Pharma Projects No. 5069 (Schering Plow); GF-196960 (Glaxowelcome); E-8010 and E-4010 (Eisai); Bay-38-3045 & 38-9456 (Bayer) as well as Sch-51866.

  The suitability of any particular cGMP PDE5 inhibitor is readily determined by assessment of its potency and selectivity using literature methods followed by assessment of its toxicity, absorption, metabolism, pharmacokinetics, etc. according to standard pharmaceutical practice be able to.

Preferably, the cGMP PDE5 inhibitor has an IC ₅₀ of less than 100 nanomolar, more preferably less than 50 nanomolar, still more preferably less than 10 nanomolar.

  IC50 values of cGMP PDE5 inhibitors can be measured using the PDE5 measurement in the Test Methods section that follows.

  Preferably, the cGMP PDE5 inhibitor used in the combination drug according to the present invention is selective for the PDE5 enzyme. Preferably they have a selectivity over PDE5 over PDE3, more preferably over 300. More preferably, the PDE5 has a selectivity of more than 100, more preferably more than 300, over both PDE3 and PDE4.

  The selectivity ratio can be easily measured by a skilled person.

  The contents of the above published patent applications, in particular the general formulas therein and the exemplified compounds, can be understood to be incorporated herein in their entirety by reference thereto.

Penile cavernous body As used herein, the term "penile cavernous body" refers to a mass of tissue, particularly in the penis. In this respect, the penis body is composed of three cylindrical masses each surrounded by a fibrous tissue called white membrane. The paired dorsal mass is called the penile cavernous body (body = main body; cavernous = hole); the smaller mid-bodied mass, the urethral cavernous body, has a cavernous urethra, It functions to keep the spongy urethra open during ejaculation. All three masses are composed of erectile tissue that is encased by fascia and skin and filled by the vascular sinus. The penile corpus cavernosum contains smooth muscle cells.

Ejaculation ejaculation involves two separate components-injection and ejaculation. Ejection is the deposition of semen and sperm from the distal epididymis, vas deferens, seminal vesicles and prostate to the urethral prostate. Following this deposit is a powerful ejection of semen content from the ureteral orifice. Ejaculation is different from orgasm, which is purely a brain event. Often the two processes coincide.

Penile erection As used herein, the term “penile erection” refers to the arterial supply to the penis due to stimuli that may be visual, tactile, auditory, olfactory, or imaginary, causing large amounts of blood to It refers to the situation that enters the sinus. The expansion of these spaces tightens the veins that allow blood to flow out of the penis, so the blood flow is slow. These vascular changes due to parasympathetic reflexes result in erections. When the artery contracts and the pressure on the vein is removed, the penis returns to its messy state.

Smooth muscle As used herein, the term “smooth muscle” refers to a tissue specialized in contraction that consists of smooth muscle fibers (cells) located in the walls of hollow internal organs and innervated by autonomic motor neurons. . The term “smooth muscle” means a muscle that is free of striated and thus gives it a smooth appearance. It is also called involuntary muscle. Increasing Ca ²⁺ concentration in the smooth muscle cytosol initiates contraction just as in striated muscle. However, the sarcoplasmic reticulum (Ca ²⁺ reservoir in striated muscle) is scarce in smooth muscle. Calcium ions enter the smooth muscle cytosol from both extracellular fluid and sarcoplasmic reticulum, but smooth muscle fibers do not have transverse tubules, so Ca ²⁺ reaches the filaments in the center of the fibers and triggers the contraction process. It takes longer to draw. This in part causes a slow onset and prolonged smooth muscle contraction.

Contraction and relaxation Several mechanisms control smooth muscle cell contraction and relaxation. For one, a regulatory protein called calmodulin binds to cytosolic Ca ²⁺ . Not only do calcium ions gradually enter the smooth muscle fibers, they slowly exit the muscle fibers when the excitement subsides and delay relaxation. The prolonged presence of Ca ^{2+ in} the cytosol provides for smooth muscle tone and continued partial contraction. Smooth muscle tissue resides in hollow internal walls such as blood vessels, lung airways, stomach, intestinal gallbladder, urinary bladder, penile corpus cavernosum and clitoris.

Treatment It should be understood that all references to treatment herein include one or more curative, palliative and prophylactic treatments.

Sexual Stimulation The present invention also encompasses the use as defined above via administration of a selective oxytocin antagonist (and auxiliary substance if applicable) prior to and / or during sexual stimulation. Here, the term “sexual stimulation” may be synonymous with the term “sexual arousal”. This aspect of the invention is advantageous because it provides systemic (physiological) selectivity.

  Thus, according to the present invention, it is highly desirable to have a sexual stimulation stage in some situations. Here, “sexual stimulation” may be one or more visual stimuli, physical stimuli, auditory stimuli, or thought stimuli.

Substances for use in the treatment of male ejaculation disorders, in particular premature ejaculation according to the invention, can act as selective oxytocin antagonists and are selective oxytocin antagonists and multiple agents of auxiliary substances as described in detail above Any suitable substance for use in combination may be used. As used herein, the term “substance” includes any agent that can selectively inhibit the oxytocin receptor.

  Such a substance (ie, a substance as defined above) may be an organic compound or other chemical substance. This object may even be an amino acid sequence or a chemical derivative thereof. The substance may even be a nucleotide sequence which may be a sense sequence or an anti-sense sequence. The substance may even be an antibody.

  Thus, the term “substance” includes, but is not limited to, compounds that can be obtained from or produced by any suitable source, whether natural or not.

  Substances can be designed or derived from libraries of compounds that may include peptides, as well as other compounds, such as small organic molecules such as lead compounds.

  By way of example, substances can be natural objects, biological macromolecules, or extracts made from biological materials such as bacteria, fungi or animal (especially mammalian) cells or tissues, organic or inorganic molecules, synthetic substances, Semi-synthetic materials, structural or functional mimetics, peptides, peptidomimetics, derived materials, peptides degraded from whole proteins, or synthetically synthesized peptides (for example, using a peptide synthesizer, Or by recombinant techniques or any combination thereof), recombinant materials, antibodies, natural or non-natural materials, fusion proteins or equivalents and mutants, derivatives or combinations thereof.

  As used herein, the term “substance” may be a single factor or a combination of substances.

  If the material is an organic compound, in some applications, the organic compound can typically include two or more linked hydrocarbyl groups. For some applications, preferably the material comprises at least two cyclic groups, optionally one of the cyclic groups may be a fused cyclic ring structure. For some applications, at least one cyclic group is a heterocyclic group. For some applications, preferably the heterocyclic group contains at least one N in the ring. An example of such a compound is provided herein.

  The material may have one or more alkyl, alkoxy, alkenyl, alkylene and alkenylene groups, which may be unbranched or branched.

For the avoidance of substitution doubt, unless otherwise indicated, the term substituted means substituted by one or more defined groups. If the group can be selected from a number of alternative groups, the selected group may be the same or different. For the avoidance of doubt, the term independently means that when more than one substituent is selected from a number of possible substituents, the substituents may be the same or different.

The pharmaceutically acceptable salt substance may be in the form of a pharmaceutically acceptable salt, such as an acid addition salt or a base salt, or a solvate thereof including a hydrate thereof, And / or can be administered as such. A review on suitable salts can be found in Berge et al., J. Pharm. Sci., 1977, 66 ,
See 1-19.

  Typically, a pharmaceutically acceptable salt can be readily prepared by using the desired acid or base, if 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 that form non-toxic salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, phosphorus Oxyhydrogen salt, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharide, benzoate, methanesulfonate, ethanesulfonate Benzene sulfonate, p-toluene sulfonate and pamoate.

  Suitable base salts are formed from bases that form non-toxic salts, examples being sodium, potassium, aluminum, calcium, magnesium, zinc and diethanolamine salts.

Polymorphic / asymmetric carbon materials can exist in polymorphic forms.

  A substance can have one or more asymmetric carbons and therefore exists in two or more stereoisomeric forms. If the material has an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur. The present invention includes the individual stereoisomers of the material as well as its individual tautomeric forms, if appropriate, with mixtures thereof.

  Separation of diastereoisomers or cis and trans isomers can be accomplished by conventional techniques of stereoisomeric mixtures of substances or their appropriate salts or derivatives, eg, fractional crystallization, chromatography or H.P. P. L. C. Can be achieved. Individual enantiomers of a substance can also be obtained from the corresponding optically pure intermediate or by resolution, for example, the corresponding racemic H.P. using a suitable chiral support. P. L. C. Or, where appropriate, by fractional crystallization of diastereoisomeric salts formed by reaction of the corresponding racemate with the appropriate optically active acid or base.

Isotope variants The invention also includes all suitable isotopic variants of the substance or a pharmaceutically acceptable salt thereof. Isotopic variations of the substances of the invention or pharmaceutically acceptable salts thereof are those in which at least one atom has the same atomic number but an atomic mass that differs from the atomic mass normally found in nature Defined as being replaced by. Examples of isotopes that can be incorporated into the substance and its pharmaceutically acceptable salts include ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³¹ P, respectively. , ³² P, ³⁵ S, ¹⁸ F, and ³⁶ Cl, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine. Certain isotopic variations of substances and their pharmaceutically acceptable salts, for example those incorporating radioisotopes such as ³ H or ¹⁴ C, are useful for drug and / or substrate tissue distribution studies It is. Tritiated, ie ³ H, and carbon-14, ie ¹⁴ C, isotopes are particularly preferred due to their ease of preparation and detectability. Furthermore, deuterium, i.e., substitution with isotopes such as ^{2 H,} the advantages of the particular treatment resulting from greater metabolic stability, for example, be obtained in vivo half-life and increased or reduced dosage requirements Yes, and therefore may be preferable in certain situations. Isotopic variants of the substance and its pharmaceutically acceptable salts can be prepared by conventional techniques, usually using the appropriate isotope variants of the appropriate reagents.

It should be understood by those skilled in the art that prodrug substances can be derived from prodrugs. Examples of prodrugs have certain protected groups and cannot themselves have pharmacological activity, but are administered in certain cases (eg, orally or parenterally) Factors that are subsequently metabolized in the body to form pharmacologically active substances are included.

Pro-parts such as H.I. Known as “pro-part” as described in “Prodrug Design” by H. Bundgaard, Elsevier, 1985, the disclosure of which is hereby incorporated by reference. It should be further understood that certain moieties can be placed on appropriate functional groups of the material. Such prodrugs are also included in the scope of the present invention.

Inhibitor / Antagonist The term antagonist as used herein in connection with a selective oxytocin antagonist can be considered to have the same meaning as the term inhibitor. Similarly, the term inhibitor as used herein in connection with the ancillary materials provided above (such as PDEi or PDE5i compounds where applicable) can be considered to have the same meaning as the term antagonist.

  As used herein, the term “antagonist” means any substance that reduces the action of another substance or target. Antagonizing effects are the result of counteracting objects (chemical alliances) or opposite effects through different targets (functional or physiological alliances) or intermediate binding sites that link target activation to the observed effects. It may be due to a combination as a result of competition (indirect conflict).

Pharmaceutical Compositions The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a substance of the present invention and a pharmaceutically acceptable carrier, diluent or pharmaceutical additive (including combinations thereof). To do.

The pharmaceutical composition may be for human or animal use in human and veterinary medicine, typically any one or more pharmaceutically acceptable diluents, carriers, or pharmaceutical additives. Including things. Carriers or diluents that can be tolerated for therapeutic applications are well known in the pharmaceutical industry, eg, Remington's Pharmaceutical Science, Mack Publishing Company.
Publishing Co.) (AR Gennaro ed. 1985). The choice of pharmaceutical carrier, pharmaceutical additive or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can include any suitable binder, lubricant, suspending agent, coating agent, solubilizer, as or in addition to the carrier, pharmaceutical additive or diluent.

  Even preservatives, stabilizers, dyes and flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-oxybenzoic acid. Antioxidants and suspending agents may be also used.

  There may be different composition / formulation requirements depending on the different delivery systems. By way of example, the pharmaceutical composition of the present invention can be obtained using a mini-pump or by a mucosal route, for example as an intranasal spray or inhalable aerosol or ingestible solution, or the composition can be, for example, by intravenous, intramuscular or subcutaneous routes. It can be formulated to be delivered parenterally which is formulated in an injectable form for delivery. Alternatively, the formulation may be designed to be delivered by both routes.

  If the substance is to be delivered by the mucosa through the gastrointestinal mucosa, it should still be able to remain stable during passage through the gastrointestinal tract; for example, resistant to protein degradation, stable at acid pH, and Should withstand the cleaning action of bile.

  Where appropriate, the pharmaceutical composition can be obtained by inhalation, in the form of suppositories or pessaries, topically in the form of lotions, solutions, creams, ointments or dusts, by the use of skin patches, Contains any capsule or vaginal suppository, or flavoring or coloring agent, either orally in the form of a tablet containing a pharmaceutical additive such as lactose, or alone or mixed with a pharmaceutical additive They can be administered in the form of elixirs, solutions or suspensions, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the composition can best be 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. . For buccal or sublingual administration, the compositions can be administered in the form of tablets or lozenges that can be formulated in conventional manner.

  In some embodiments, the materials of the invention can also be used in combination with cyclodextrins. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of a drug-cyclodextrin complex can modify the solubility, dissolution rate, bioavailability and / or stability properties of the drug molecule. Drug-cyclodextrin complexes are usually useful for most dosage forms and administration routes. As an alternative to direct complexation reactions with drugs, cyclodextrins can be used as auxiliary additives, for example as carriers, diluents or solubilizers. 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. Yes.

  In preferred embodiments, the substances of the invention are delivered systemically (eg, oral, buccal, sublingual), more preferably orally.

  Hence, preferably the substance is in a form that is suitable for oral delivery.

The administration term “administration” includes delivery by viral or non-viral techniques. Viral delivery mechanisms include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic surface amphiphiles (CFAs) and combinations thereof.

  The substances of the invention can be administered alone, but typically the substance is mixed with, for example, appropriate pharmaceutical pharmaceutical additives, diluents or carriers selected with regard to the intended route of administration and standard pharmaceutical practice. In some cases, it is administered as a pharmaceutical composition.

  For example, the substance may contain tablets, capsules, vaginal suppositories, elixirs that may contain flavoring or coloring agents for immediate-, delayed-, modified-, sustained-, pulsatile- or controlled-release applications. It can be administered in the form of an agent, solution or suspension (eg, orally or topically).

  Tablets include microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine excipients, starch (preferably corn, potato or tapioca starch), starch starch glycolate, croscarmellose Contain disintegrants such as sodium and certain complex silicates and granulated binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and gum arabic it can. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

  Similar types of solid compositions can also be used as excipients in gelatin capsules. Preferred pharmaceutical additives in this respect include lactose, starch, cellulose, lactose or high molecular weight polyethylene glycols. Aqueous suspensions and / or elixirs contain substances such as various sweetening or flavoring agents, coloring materials or dyes, emulsifying agents and / or suspending agents, and water, ethanol, propylene glycol and glycerin and It can be combined with a diluent such as the combination.

  Administration (delivery) routes include, but are not limited to, oral (eg, as tablets, capsules, or as ingestible solutions), topical, mucosal (eg, nasal spray or aerosol for inhalation). Intranasal, parenteral (eg, injectable form), gastrointestinal, intrathecal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intraventricular , Intracerebral, subcutaneous, ocular (including intravitreal or intraocular), transdermal, rectal, oral, penis, vagina, epidural, sublingual.

  It should be understood that not all substances need to be administered by the same route. Similarly, if a composition contains more than one active ingredient, those ingredients may be administered by different routes.

  When the substance of the invention is administered parenterally, examples of such administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular or subcutaneous. Administration of substances by one or more of the following; and / or using infusion techniques.

  For parenteral administration, the substance 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 solution should be appropriately buffered if necessary (preferably a pH of 3 to 9). Preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

As indicated, the substances of the invention can be administered intranasally or by inhalation and are suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkanes such as 1, Pressurization using 1,1,2-tetrafluoroethane (HFA 134A ^™ ) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA ^™ ), carbon dioxide or other suitable gas Conveniently delivered in the form of a dry powder inhaler or aerosol spray delivery from a sealed container, pump, spray or nebulizer. In the case of a pressurized aerosol, the dosage unit can be measured by providing a valve to deliver a metered amount. Pressurized containers, pumps, sprays or nebulizers may additionally contain a lubricant, for example sorbitan trioleate, for example a solution or suspension of the active compound using a mixture of ethanol and propellant as solvent Can be entered. Capsules and cartridges (eg, made of gelatin) for inhaler or insufflator applications can be formulated to contain a powder mix of the material and a suitable powder base such as lactose or starch. .

  Alternatively, the substances of the invention can be administered in the form of suppositories or pessaries, or topically applied in the form of a gel, hydrogel, lotion, solution, cream, ointment or spray. be able to. The substances according to the invention can also be administered dermally or transdermally, for example by the use of skin patches. They can also be administered by the pulmonary or rectal route. They can also be administered by the ocular route. For ophthalmic applications, the compound may optionally be combined with a preservative such as benzylalkonium chloride, as a micronized suspension in isotonic pH-adjusted sterile saline, or preferably It can be formulated as a solution in sterile saline adjusted to pH. Alternatively, they can be formulated in an ointment such as petrolatum.

  For topical application to the skin, the substances according to the invention can be used, for example, as a mixture with one or more of the following: mineral oil, liquid petrolatum, white pet, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water It can be formulated as a suitable ointment containing the active compound suspended or dissolved therein. Alternatively, it may be, for example, in a mixture with one or more of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Can be formulated as a suitable lotion or cream suspended or dissolved in

  The composition of the present invention can be administered by direct injection.

  For some applications, preferably the substance is administered orally.

  For some applications, preferably the substance is administered topically.

Dosage levels Typically, a physician will determine the actual dosage that is most appropriate for an individual subject. The specific dose level and frequency for a particular individual can vary and the activity of the particular compound used, the metabolic stability and length of action of that compound, age, weight, general health, sex, diet, administration It depends on a variety of factors, including the mode and time of the excretion, the rate of excretion, the combination of drugs, the severity of the particular condition, and the treatment the individual receives. The substances and / or pharmaceutical compositions of the invention can be administered according to a regimen of 1 to 10 times per day, such as once or twice per day.

  For oral and parenteral administration to humans, the daily dose level of the substance may be a single or divided amount.

  Depending on requirements, the substance may be administered at a dose of 0.01 to 30 mg / kg body weight, for example 0.1 to 10 mg / kg body weight, more preferably 0.1 to 1 mg / kg body weight. Of course, the doses described herein are typical of the average case. Of course, there can be individual cases where higher or lower capacity ranges deserve.

  Typically, the daily oral dose may be for example 20-1000 mg, preferably for example 50-300 mg.

Formulation The substance of the invention may be formulated into a pharmaceutical composition by using techniques known in the art, for example by mixing with one or more suitable carriers, diluents or pharmaceutical additives. Can do.

  The following provides non-limiting examples of formulations.

Formulation 1: Tablets are prepared using the following ingredients:
Weight / mg
Substance 250
Cellulose, microcrystal 400
Silicon dioxide, fumes 10
Stearic acid 5
Total 665
The ingredients are mixed and compressed to form tablets each weighing 665 mg.

Formulation 2: Intravenous formulations can be adjusted as follows:
Substance 100mg
1,000ml isotonic saline

Individual As used herein, the term “individual” refers to a member of a vertebrate, particularly a mammalian species. The term includes, but is not limited to, domestic animals, sport animals, primates and humans.

Bioavailability Preferably, the compounds of the present invention (and combination drugs) are orally bioavailable. Oral bioavailability refers to the percentage of orally administered drug that reaches the systemic circulation. Factors that determine the oral bioavailability of a drug are solubility, membrane permeability and metabolic stability. Typically, oral bioavailability is determined using a sorting cascade of in vitro techniques first, then in vitro.

  Solubilization, solubilization of the drug by the aqueous contents of the gastrointestinal tract (GIT) can be predicted from in vitro solubility experiments performed at an appropriate pH that mimics GIT. Preferably, the compounds of the invention have a minimum solubility of 50 mg / ml. Solubility can be measured by standard techniques known in the art as described in Adv. Drug Deliv. Rev. 23, 3-25, 1997.

Membrane permeability refers to the passage of compounds through the cells of GIT. Lipophilicity is an important property to anticipate this and is defined by in vitro Log D _7.4 measurements using organic solvents and buffers. Preferably, the compounds of the invention have a Log D _7.4 of -2 to +4, more preferably -1 to +2. Log D can be measured by standard techniques known in the art as described in J. Pharm. Pharmacol. 1990, 42: 144.

In the presence of an efflux carrier such as p-glycoprotein, a cell monolayer measurement such as CaCO ₂ , the so-called caco-2 flux, is substantially added to the prediction of advantageous membrane permeability. Preferably, the compounds of the present invention ^is greater than 2x10 -6 ^{cms -1,} more preferably from ^5x10 -6 ^{cms -1} greater than caco-2 flux. The caco flux value can be measured by standard techniques known in the art as described in J. Pharm. Sci., 1990, 79, 595-600.

Metabolic stability addresses the ability of the GIT or liver to metabolize compounds during the absorption process: the first pass effect. Measurement systems such as microsomes, hepatocytes, etc. are predictive of ease of metabolism. Preferably, the compounds of this example exhibit metabolic stability in a measurement system that balances liver extracts below 0.5. Examples of measurement systems and data handling are Curr. Opin. Drug Disc. Devel., 201, 4, 36-44, Drug Met. Disp., 2000,
28, 1518-1523.

Due to the interaction of the above processes, further support that the drug is orally bioavailable in humans can be obtained by in vivo experiments in animals. Absolute bioavailability in these studies is measured by administering the compounds separately or mixed by the oral route. The venous route is also used for absolute measurements (% absorbed). Examples of evaluation of oral bioavailability in animals are Drug Met. Disp., 2001, 29, 82-87; J. Med. Chem, 1997, 40, 827-829,
Drug Met. Disp., 1999, 27, 221-226.

Chemical Synthesis Methods Typically, selective oxytocin antagonists (and / or PDEi / PDE5i where applicable) suitable for use in accordance with the present invention are prepared by chemical synthesis techniques.

A substance or target or variant, homologue, derivative, fragment or mimetic thereof can be produced using chemical methods that synthesize the substance in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (eg, Creighton (1983) Proteins Structures and Molecular Principles, WH
Freeman and Co, New York NY). The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing methods (eg, Edman degradation techniques; Kraton, above).

  Direct synthesis of substances 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 ), Automated synthesis can be achieved, for example, using an ABI 43 1 A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer. Furthermore, the amino acid sequence comprising the substance or any part thereof may be altered directly during synthesis and / or combined with a sequence obtained from other subunits or any part thereof using chemical methods, For example, targets such as variant oxytocin receptors can be obtained.

In another aspect of the invention, the coding sequence of a substance, target or variant, homologue, derivative, fragment or mimetic thereof is synthesized in whole or in part using chemical methods well known in the art. (Calthers MH) (1980) Nuc Acids Res Symp
Ser 215-23; Horn T etc.
(1980) Nuc Acids Res Symp Ser 225-232).

As used mimetics herein, the term Bu mimetic゛Ha relates to any chemical, to, Ronkyu substances i.e. such as a selective oxytocin antagonist, the same qualitative activity or effect as a target i.e. for example oxytocin receptor Including, but not limited to, peptides, polypeptides, antibodies or other organic chemicals. That is, the mimetic may be functionally equivalent to a known substance.

Chemical Derivative As used herein, the term “derivative” or “derived” includes chemical modification of a substance. Specific examples of such chemical modification would be the replacement of hydrogen with a halo group, alkyl group, acyl group or amino group.

Chemical Modification In one embodiment of the present invention, the substance may be a chemically modified substance.

  Chemical modification of a substance either enhances or reduces hydrogen bond interactions, charge interactions, hydrophobic interactions, van der Waals interactions or dipole interactions between the substance and target, and Also good.

  In one aspect, the identified substance can serve as a model for developing other compounds (eg, a template).

Target In one aspect of the invention, the oxytocin receptor can be used as a target in a screen to identify substances that can inhibit the oxytocin receptor. In this regard, the target can include the amino acid sequence shown as SEQ ID NO: 1 prepared by recombinant and / or synthetic methods, or variants, homologues, derivatives or fragments thereof or expression factors comprising them.

  In a further aspect of the invention, both the oxytocin receptor and the vasopressin receptor, in particular the V1a receptor, can be used as targets in screening to identify substances that can selectively inhibit the oxytocin receptor. In this regard, the oxytocin receptor target may comprise the amino acid sequence shown as SEQ ID NO: 1 prepared by recombinant and / or synthetic methods, or variants, homologues, derivatives or fragments thereof or expression factors comprising them. And the vasopressin receptor target can comprise the amino acid sequence shown as SEQ ID NO: 2 or a variant, homologue, derivative or fragment thereof or an expression factor comprising these prepared by recombinant and / or synthetic methods .

  Alternatively, oxytocin receptor and / or vasopressin receptor (preferably V1a receptor) is used as a target to identify substances that can mediate increased ejaculation latency through selective inhibition of oxytocin receptor Can do. In this regard, the target may be a suitable tissue extract.

  The target may even be a combination of such tissues and / or recombinant targets.

Recombinant Methods The materials and / or targets of the present invention can be prepared by recombinant DNA techniques.

  In one aspect, preferably the substance is a selective oxytocin antagonist. Oxytocin antagonists can be prepared by recombinant DNA techniques.

Amino Acid Sequence As used herein, the term “amino acid sequence” has the same meaning as the term “polypeptide” and / or the term “protein”. In some cases, the term “amino acid sequence” has the same meaning as the term “peptide”. In some cases, the term “amino acid sequence” has the same meaning as the term “protein”.

  The amino acid sequence can be isolated and prepared from a suitable source, can be made synthetically, or can be prepared by use of recombinant DNA techniques.

  In one aspect, the invention provides an amino acid sequence that can serve as a target (ie, an oxytocin receptor or vasopressin receptor, preferably a V1a receptor) in a measurement for identification of one or more substances and / or derivatives thereof. I will provide a.

  In a second aspect, the present invention provides an amino acid sequence that allows a substance to selectively inhibit an oxytocin receptor.

  Preferably the target is an oxytocin receptor.

  Preferably the oxytocin receptor and / or vasopressin receptor, preferably the V1a receptor, is an isolated receptor and / or purified and / or not native.

  The oxytocin receptor and / or vasopressin receptor, preferably the V1a receptor of the present invention may be in a substantially isolated form. The oxytocin receptor or vasopressin receptor can be mixed with a carrier or diluent that does not interfere with the intended purpose of the receptor and / or substance, and is still considered substantially isolated. The oxytocin receptor or vasopressin receptor of the present invention may also be present in substantially pure form, in which case it is usually greater than 90% in the formulation, eg 95%, 98% or 99 % Oxytocin receptor or vasopressin receptor, each having the amino acid sequence represented by SEQ ID NO: 1, or a variant, homologue, derivative or fragment thereof, or peptide having the amino acid sequence represented by SEQ ID NO: 2, or Including oxytocin receptor or vasopressin receptor in the formulation, which are variants, homologues, derivatives or fragments thereof.

Nucleotide Sequence As used herein, the term “nucleotide sequence” has the same meaning as the term “polynucleotide”.

  The nucleotide sequence may be genomic or synthetic or recombinant DNA or RNA. The nucleotide sequence may be double-stranded or single-stranded, whether representing the sense or antisense strand, or a combination thereof.

  For some applications, preferably the nucleotide sequence is DNA.

  For some applications, preferably the nucleotide sequence is prepared by use of recombinant DNA techniques (eg, recombinant DNA).

  For some applications, preferably the nucleotide sequence is cDNA.

  For some applications, preferably the nucleotide sequence may be the same as the naturally occurring form for this embodiment.

  In one aspect, the invention provides a nucleotide sequence that encodes an object that can serve as a target for measurement for identification of one or more substances and / or derivatives thereof.

  In one aspect of the invention, the nucleotide sequence encodes an oxytocin receptor.

  In a further embodiment of the invention, the nucleotide sequence encodes a vasopressin receptor, preferably a V1a receptor.

  In one embodiment of the invention, the nucleotide sequence encodes a substance that can selectively inhibit the oxytocin receptor.

  A number of different nucleotide sequences can result in the same target as a result of the degeneracy of the genetic code (ie, an oxytocin receptor, eg, an oxytocin receptor comprising the amino acid sequence shown in SEQ ID NO: 1, or a vasopressin receptor, eg, SEQ ID NO: 2). It should be understood by those skilled in the art that a vasopressin receptor comprising the amino acid sequence shown in FIG. In addition, skilled artisans can use conventional techniques to make nucleotide substitutions that do not substantially affect the activity encoded by the nucleotide sequence that reflects the codon convention of the particular host organism in which the target is to be expressed. It should be considered possible. Thus, the terms “variant”, “homolog” or “derivative” in relation to a nucleotide sequence include that the resulting nucleotide sequence is a functional target according to the present invention (ie, for example, an oxytocin receptor) (or its substance is a nucleotide Any substitution, modification, modification, exchange, deletion or deletion of one (or more) nucleic acid from or to the sequence, provided that it encodes a sequence or even an amino acid sequence (including an amino acid sequence) Includes appends.

Variants / Homologues / Derivatives In addition to the specific amino acid sequences described herein, the present invention also encompasses the use of variants, homologues and derivatives thereof. Here, the term “homology” may be equated with “identical”.

  In this context, a homologous sequence comprises an amino acid sequence that may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. It is understood. In particular, homology should typically be considered with respect to regions of the sequence known to be essential for activity. Homology can also be considered from the standpoint of similarity (ie, amino acid residues having similar chemical properties / functions), but in the context of the present invention, it is preferred to represent homology from the same standpoint of sequence.

  Homologous comparisons can be made by eye or more commonly by readily available sequence comparison programs. These commercially available computer programs can calculate% homology between two or more sequences.

  % Homology can be calculated over adjacent sequences, ie, one sequence is aligned with the other sequence, and each amino acid in one sequence is residueed one residue at a time with the corresponding amino acid in the other sequence. Compare directly. This is called “ungapped alignment”. Typically, such ungapped door alignments are performed only on those with relatively few residues.

  Although this is a very simple and consistent method, for example, in one otherwise identical pair of sequences, one insertion or deletion will cause the next amino acid residue to be taken out of alignment. Therefore, it does not take into account that if the overall alignment is performed, it is likely to result in a large reduction in% homology. Thus, most sequence comparison methods are designed to obtain an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to maximize local homology.

  However, these more complex methods, with the same number of identical amino acids, reflect a higher correlation between the two compared sequences, and sequence alignments with as few gaps as possible have many gaps Assign a “gap penalty” to each gap present in the alignment so that a higher score is obtained. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap evaluation system. High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

The calculation of maximum% homology therefore first requires the production of an optimal alignment taking into account the gap penalty. A suitable computer program for performing such an alignment is the GCG Wisconsin Best Fit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acid Research)
12: 387). Examples of other software that can perform sequence comparisons include the BLAST package (see Ausubel et al., 1999 ibid, Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol. 403- 410) and a set of comparison tools for GENEWORKS, but are not limited to these. Both BLAST and FASTA are available for offline and online searching (see Ausberg et al., 1999 ibid, pages 7-58 to 7-60). However, it is preferred to use the GCG best fit program. A new tool called BLAST2 Sequences is also available to compare protein and nucleotide sequences (FEMS Microbiol Lett 1999 174 (2): 247-50; FEMS Microbiol Lett 1999
177 (1): 187-8 and tatiana@ncbi.nlm.nih.gov).

  Although the final% homology can be measured from the same point of view, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a proportional similarity score matrix is typically used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix, which is the BLAST suite of default programming matrices. GCG Wisconsin programs typically use either official default values or custom symbol comparison tables (see user manual for further details) when supplied. It is preferred to use a public default value for the GCG package, or in the case of other software, a default matrix such as BLOSUM62.

  Once the software has produced an optimal alignment, it is possible to calculate% homology, preferably% sequence identity. The software typically does this as part of the sequence comparison and produces a numerical result.

  The sequence may also have deletions, insertions or substitutions of amino acid residues that cause silent changes and that result in a functionally equivalent object. Intentional amino acid substitutions can be made based on residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic similarity so long as the secondary binding activity of the object is retained . For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and uncharged polar head groups with similar hydrophilicity values Examples of amino acids having leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

また、相同置換（本明細書で、置換および交換を両方とも用いて、現存するアミノ酸残基の代替残基での交換を意味する）、即ち塩基性には塩基性、酸性には酸性、極性には極性等のような似たもの同士の置換も包含する本発明もあってもよい。また、非相同置換、即ち、一方のクラスの残基から他方へ又は代わりにオルニチン（以後、Ｚと称する）、ジアミノ酪酸オルニチン（以後、Ｂと称する）、ノルロイシンオルニチン（以後、Ｏと称する）、ピリルアラニン、チエニルアラニン、ナフチルアラニンおよびフェニルグリシンのような非天然のアミノ酸の含有を含むことが起こってもよい。 Conservative substitution can be made, for example, according to the following table. Amino acids in the same block in the second column, preferably in the same line of the third column, can be substituted for each other:

Also, homologous substitution (in this specification, both substitution and exchange are used to mean replacement of an existing amino acid residue with an alternative residue), ie, basic for basic, acidic for acidic, polar The present invention may also include substitution of similar ones such as polarity. Non-homologous substitutions, ie, ornithine (hereinafter referred to as Z), ornithine diaminobutyrate (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O) from one class of residues to the other or instead. Inclusion of unnatural amino acids such as, pyrylalanine, thienylalanine, naphthylalanine and phenylglycine may occur.

The exchange also includes alpha ^* and alpha-disubstituted ^* amino acids, N-alkyl amino acids ^* , lactic acid ^* , halide derivatives of natural amino acids such as trifluorotyrosine ^* , p-Cl-phenylalanine ^* , p-Br-phenylalanine ^*. , P-I-phenylalanine ^* , L-allyl-glycine ^* , beta-alanine ^* , L-α-aminobutyric acid ^* , L-γ-aminobutyric acid ^* , L-α-aminoisobutyric acid ^* , L-εaminocaproic acid ^# , 7-aminoheptanoic acid ^* , L-methionine sulfone ^{# *} , L-norleucine ^* , L-norvaline ^* , p-nitro-L-phenylalanine ^* , L-hydroxyproline ^# , L-thioproline ^* , phenylalanine (Phe) methyl derivatives such as 4-methyl -Phe ^*, pentamethyl -Phe ^*, L-P he (4-amino) ^# , L-Tyr (methyl) ^* , L-Phe (4-isopropyl) ^* , L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) ^* , L- It can also be performed with non-natural amino acids including diaminopropionic acid ^# and L-Phe (4-benzyl) ^* . Note ^* is used for the purposes of the above discussion (related to homologous or non-homologous substitutions) to indicate the hydrophobicity of the derivative, while ^# is used to indicate the hydrophilicity of the derivative, and ^** is amphiphilic. Show the characteristics.

  Variant amino acid sequences can be inserted between any two amino acid residues of a sequence containing an alkyl group such as a methyl, ethyl or propyl group in addition to an amino acid spacer such as a glycine or β-alanine residue. Any suitable spacer group can be included. Additional forms of variation involving the presence of one or more amino acid residues within the peptoid form should be well understood by those skilled in the art. For the avoidance of doubt, “peptoid form” is used to mean a variant amino acid residue in which the α-carbon substituent is on the nitrogen atom rather than the α-carbon of the residue. Methods for preparing peptoid forms of peptides are known in the art, for example, Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. 1995) 13 (4), 132-134.

Hybridization As used herein, the term “hybridization” includes “a method in which a single-stranded nucleic acid binds to a complementary strand through base pairing” and an amplification method as performed in polymerase chain reaction (PCR) technology. included.

  The nucleotide sequence that can selectively hybridize to the nucleotide sequence encoding the amino acid sequence of the present invention or a complement thereof has at least 20 regions, preferably at least 25 or 30, such as at least 40, 60 or 100 or more. Is usually at least 75%, preferably at least 85 or 90%, more preferably at least 95% or 98% homologous to the corresponding complementary nucleotide sequence encoding the amino acid sequence set forth herein with respect to .

The term “can selectively hybridize” means that when a nucleotide sequence is used as a probe, it is used under conditions that find the target nucleotide sequence and hybridize to the probe well above background. To do. Background hybridization can occur, for example, due to the selected cDNA or other nucleotide sequences present in a genomic DNA library. In this case, the background arises from interactions between the probe and the non-specific DNA members of the library that are less than 10 times, preferably less than 100 times the specific interaction observed with the target DNA. It means the level of signal. The intensity of interaction may, for example, can be measured by radiolabelling the probe for example, ^{32 P.}

  Hybridization conditions are described by Berger and Kimmel (1987, Molecular Cloning Techniques, Methods in Enzymology, Volume 152, Academic Press, San Diego). Is based on the melting temperature (Tm) of the nucleic acid binding complex as taught in CA) and gives a defined stringency as described below.

  Maximum stringency is typically about Tm-5 ° C (5 ° C below the Tm of the probe); exact stringency is Tm minus about 5 ° C to 10 ° C; intermediate stringency is Tm minus about 10 ° C And moderate stringency occurs at Tm minus about 20 ° C to 25 ° C. As will be appreciated by those skilled in the art, maximal stringency hybridization can be used to identify or detect identical nucleotide sequences, whereas intermediate (or gradual) stringency hybridization is similar or related. Can be used to identify or detect the polynucleotide sequence of.

In a preferred embodiment, the present invention, under stringent conditions (e.g., 65 ° C. and 0.1 × SSC {1 × SSC = 0.15 M of NaCl, citric acid _Na 3 pH 7.0 in 0.015 M) the amino acid sequence of the present invention It includes nucleotide sequences that can hybridize to the encoding nucleotide sequence. Where the nucleotide sequence of the present invention is double stranded, both duplexes are encompassed by the present invention either individually or in combination. Where the nucleotide sequence is single stranded, complementary sequences of that nucleotide sequence are also considered to be within the scope of the invention.

  Nucleotide sequences that are not 100% homologous to the nucleotide sequence encoding the amino acid sequence of the invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein can be obtained, for example, by probing DNA libraries made from a range of sources. In addition, homologues of other viruses / bacteria or cells can be obtained, in particular cell homologues found in mammalian cells (eg rat, mouse, bovine and primate cells), such homologues and fragments thereof Can normally selectively hybridize to the sequences shown in the sequence listing of the present specification. Such sequences include genomic DNA libraries derived from other animal species or cDNA libraries generated therefrom, and nucleotide sequences shown herein under moderate to strict stringency conditions. Can be obtained by probing with a probe including all or part of the above. 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 are also obtained using degenerate PCR with primers designed to target variants encoding conserved amino acid sequences within sequences of the invention and sequences within homologues. be able to. Conserved sequences can be predicted, for example, by aligning amino acid sequences obtained from several variants / homologs. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used. Primers used for degenerate PCR have one or more degenerate positions and are used under milder stringency conditions than those used for cloning sequences using single sequence primers for a plurality of known sequences.

  Alternatively, such a nucleotide sequence can be obtained by site-directed mutagenesis of a confirmed sequence, such as the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1. This is useful, for example, when silent codon changes require the sequence to optimize the codon preference of the particular host cell in which the nucleotide sequence is expressed. Other sequence changes may be desired to introduce restriction enzyme recognition sites or to alter the activity of the protein encoded by the nucleotide sequence.

  Using the nucleotide sequence encoding the amino acid sequence of the present invention, it is possible to produce a probe labeled with a primer, eg, a PCR primer, another amplification reaction primer, eg, a label revealed by a conventional method using a radioactive or non-radioactive label. The nucleotide sequence can be cloned into a vector. Such primers, probes and other fragments are at least 15, preferably at least 20, such as at least 25, 30 or 40 nucleotides in chain length and, as used herein, are also the terms of the present invention. Of the nucleotide sequence.

  Nucleotide sequences such as DNA polynucleotides and probes according to the present invention can be produced by recombination, synthesis or any method available to those skilled in the art. They can also be cloned by standard techniques.

  Usually, primers are made by synthetic methods that involve the stepwise production of one nucleotide at a time of the desired nucleic acid sequence. Techniques for accomplishing this using automated techniques are readily available in the art.

  Longer nucleotide sequences are usually produced using recombinant methods, for example using PCR (polymerase chain reaction) cloning techniques. This creates a pair of primers (eg, about 15 to 30 nucleotides) adjacent to the region of the target sequence that it is desired to clone and contacts the primer with mRNA or cDNA obtained from animal or human cells. Perform a polymerase chain reaction (PCR) under conditions that result in amplification of the desired region, isolate the amplified fragment (eg, by purifying the reaction mixture on an agarose gel), and recover the amplified DNA Including doing. The primers can be designed with appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate cloning vector.

  Due to the inherent degeneracy of the genetic code, target sequences can be cloned and expressed using a variety of DNA sequences that encode substantially the same or functionally equivalent amino acid sequences. As will be appreciated by those skilled in the art, it may be advantageous to create a target sequence having a non-naturally occurring codon for a particular expression system. The codons preferred by a particular prokaryotic or eukaryotic host (Murray E etc. Nuc Acid Res 17-477-508) can be selected, for example, to increase target expression or to obtain from naturally occurring sequences. Recombinant RNA transcripts having desirable properties such as a longer half-life than the resulting transcript can be obtained.

In one aspect of the invention, the substance (ie, a selective oxytocin antagonist) can be administered directly to an individual.

  In another aspect of the invention, a vector comprising a nucleotide sequence encoding a substance of the invention is administered to an individual.

  Preferably, the recombinant material is prepared and / or delivered to the target site using a genetic vector.

  As is well known in the art, a vector is a tool that allows or facilitates the transfer of factors from one environment to another. According to the present invention, by way of example, some vectors used in recombinant DNA technology replicate vectors containing the nucleotide sequence of the invention and / or express the protein of the invention encoded by the nucleotide sequence of the invention. For this purpose, factors such as segments of DNA (eg, heterologous DNA segments such as heterologous cDNA segments) can be introduced into the host and / or target cells. Examples of vectors used in recombinant DNA technology include, but are not limited to, plasmids, chromosomes, artificial chromosomes or viruses.

  The term “vector” includes expression vectors and / or transformation vectors.

  The term “expression vector” means a construct capable of in vivo or in vitro / ex vivo expression.

  The term “transformation vector” means a construct that can be introduced from one species into another.

Naked DNA
A vector comprising a nucleotide sequence encoding a substance of the invention for use in the treatment of ejaculatory disorders, in particular early ejaculation, is preferably administered directly as a “naked nucleic acid construct” further comprising a flanking sequence homologous to the host cell genome. Can do.

  As used herein, the term “naked DNA” refers to a plasmid that contains a nucleotide sequence encoding a substance of the invention together with a short promoter region that controls its production. It is called “naked” DNA because the plasmid is not carried by any delivery vehicle. When such a DNA plasmid enters a host cell such as a eukaryotic cell, the protein it encodes (eg, a substance of the present invention) is transcribed and translated within the cell.

Non-viral delivery or a vector comprising a nucleotide sequence encoding an amino acid of the invention or a substance of the invention (ie a selective oxytocin antagonist) or a target of the invention (ie an oxytocin receptor) may be used for transfection, A variety of non-viral techniques known in the art such as poration and biolistic transformation can be used to introduce into a suitable host cell.

  As used herein, the term “transfection” refers to a method that uses a non-viral vector to deliver a gene to a target mammalian cell.

Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compressed DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic cations Amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), polyvalent cations such as spermine, cationic lipids or polylysine, 1,2-bis (oleoyloxy) -3- (tothymethylammonio) propane (DOTAP) -cholesterol complex (Wolff and Trubetskoy 1998 Nature
Biotechnology 16: 421) and combinations thereof.

The uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques, including those involving the use of transfection agents. Examples of these substances include cationic substances (eg, calcium phosphate and DEAE-dextran) and lipofectants (eg, lipofectam ^™ and transfectam ^™ ). Typically, a nucleic acid construct is mixed with a transfection agent to produce a composition.

Viral vectors or vectors comprising a nucleotide sequence encoding a substance or target of the invention or an amino acid sequence of the invention are known in the art, such as infection using recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses. A variety of known viral techniques can be used to introduce into a suitable host cell.

Preferably the vector is a recombinant viral vector. Suitable recombinant viral vectors include adenovirus vectors, adeno-associated virus (AAV) vectors, herpes-virus vectors, retrovirus vectors, lentivirus vectors, baculovirus vectors, poxvirus vectors or parvovirus vectors (Kestler et al. 1999) Human Gene Ther 10 (10):
1619-32), but is not limited to these. In the case of a viral vector, delivery of the nucleotide sequence encoding the substance of the invention is mediated by viral infection of the target cell.

The target vector term “target vector” refers to the expression of a particular cell type in a host organism, usually a common or similar, that is capable of infecting / transfecting / transducing cells or being expressed in a host and / or target cell. A vector that is restricted to cells having a type.

Replicate Vectors Nucleotide sequences encoding substances of the invention (ie selective oxytocin antagonists or PDEi or PDE5i where applicable) or targets (eg oxytocin receptor) can be incorporated into recombinant replicable vectors. Vectors can be used to replicate nucleotide sequences in compatible host cells. Thus, in one aspect of the invention, the invention introduces a nucleotide sequence of the invention into a replicable vector, introduces the vector into a compatible host cell, and grows the host cell under conditions that result in replication of the vector. Provides a method for producing the target of the present invention. The vector can be recovered from the host cell.

A nucleotide sequence encoding a substance of the invention or an amino acid of the invention or a target of the invention inserted into an expression vector vector provides expression of a coding sequence, such as the coding sequence of an oxytocin receptor of the invention, by a host cell. The vector is an expression vector that is operably linked to a control sequence that is capable of. The substance or target of the invention produced by the host recombinant cell can be secreted or contained within the cell depending on the sequence and / or vector used. As understood by those skilled in the art, an expression vector having a sequence encoding a substance or target of the present invention directs secretion of the substance or target of the present invention encoded by the sequence through a specific prokaryotic or eukaryotic cell membrane. Can be designed with a signal sequence.

In Vitro Expression The vectors of the present invention can be transformed or transfected with appropriate host cells and / or target cells as described below to prepare for expression of the substances or targets of the present invention. This method comprises culturing a host cell and / or target cell transformed with an expression vector under conditions ready for expression by a vector of a coding sequence encoding the substance or target of the invention and expressing the expressed substance or target of the invention May optionally be recovered. The vector may be, for example, a plasmid or viral vector that provides an origin of replication, optionally a promoter for expression of the polynucleotide, and optionally a regulator of the promoter. The vector can have one or more selectable marker genes, such as an ampicillin resistance gene in the case of bacterial plasmids, or a neomycin resistance gene in mammalian vectors. Expression of the substances of the invention or the targets of the invention may be constitutive such that they are produced continuously or may be inducible requiring a stimulus to initiate expression. In the case of inducible expression, production of a substance or target of the invention can be initiated when required, for example, by addition of an inducer such as dexamethasone or IPTG to the culture medium.

Fusion protein oxytocin receptor or vasopressin receptor or substance of the invention (ie selective oxytocin antagonist) aids in the extraction and purification of the substance or receptor target of the invention and / or delivery to an individual and / or substance It may also be expressed as a fusion protein that facilitates the development of the selection. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation domain) and β-galactosidase. It may also be advantageous to include a proteolytic site between the fusion protein partner and the protein sequence of interest to allow removal of the fusion protein sequence. Preferably, the fusion protein does not interfere with the target activity.

  The fusion protein may comprise an antigen or antigenic determinant fused to the object of the invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein comprising an object that can act as an adjuvant in the sense of providing a generalized stimulus of the immune system. The antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the object.

  In another embodiment of the invention, the amino acid sequence can be ligated to a heterologous sequence encoding a fusion protein. For example, for the selection of peptide libraries of substances that can affect the activity of an object, it may be useful to encode a chimeric object that expresses a heterologous epitope recognized by a commercially available antibody. .

Host cells A variety of host cells can be used to express nucleotide sequences encoding substances such as selective oxytocin antagonists of the invention or oxytocin or vasopressin receptor targets of the 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 mouse, CHO, human and monkey cell lines and derivatives thereof.

  Examples of suitable expression hosts within the scope of the present invention are Aspergillus species (eg those described in EP-A-0184438 and EP-A-0284603) and fungi such as Trichoderma species; Bacillus species (eg EP -As described in A-0134048 and EP-A-0253455), bacteria such as Streptomyces sp. And Pseudomonas sp .; and kluyveromyces sp. (E.g. EP-A-0096430 and EP- A. 0301670) and yeasts such as Saccharomyces species. By way of example, representative expression hosts are Aspergillus niger, Aspergillus niger var. Choose from Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae be able to.

  The use of appropriate host cells, such as yeast, fungal and plant host cells, may require post-translational modifications (e.g., for imparting optimal biological activity to the recombinant expression product of the invention (e.g., , Myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation).

  Preferred host cells can process the expressed product to produce the appropriate mature polypeptide. Examples of processing include, but are not limited to, glycosylation, ubiquitination, disulfide bond formation and general post-translational modifications.

Antibody In one embodiment of the present invention, the substance may be an antibody. In addition or alternatively, the target may be an antibody.

  Antibodies can be obtained by standard techniques such as by immunization with an object of the invention, or by using phage display libraries.

For the purposes of the present invention, unless specified to the contrary, the term “antibody” includes polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments obtained from a Fab expression library, and mimetics thereof. However, it is not limited to these. Such fragments include fragments of whole antibodies that retain their binding activity to the target object, Fv, F (ab ′) and F (ab ′) ₂ fragments, and single chains comprising the antigen binding site of the antibody. Antibodies (scFv), fusion proteins and other synthetic proteins are included. Furthermore, the antibodies and fragments thereof may be humanized antibodies. Neutralizing antibodies, ie those that inhibit the biological activity of the object polypeptide, are particularly preferred for diagnostic and therapeutic methods.

  If polyclonal antibodies are desired, selected mammals (eg, mice, rabbits, goats, horses, etc.) can be obtained with an immunogenic polypeptide having an epitope that can be obtained from the identified substances and / or objects of the present invention. Immunize. Depending on the host species, various adjuvants can be used to enhance the immunological response. Such adjuvants include Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. However, it is not limited to these. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are used if the purified object polypeptide is administered individually, immunologically compromised for the purpose of stimulating systemic defenses. A potentially useful human adjuvant that can.

  Serum obtained from the immunized animal is collected and processed according to known procedures. If sera containing polyclonal antibodies to epitopes obtainable from the identified substances and / or objects of the invention contain 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 to produce such antibodies, the present invention also provides a polypeptide of the present invention or a fragment thereof haptenized against another polypeptide for use as an immunogen in animals or humans.

  Monoclonal antibodies directed against epitopes obtainable from the identified substances and / or objects of the present invention can also be readily produced by those skilled in the art. The general methodology for producing monoclonal antibodies with hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion and other techniques such as direct transformation of B lymphocytes with oncogene DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against orbital epitopes can be screened for various properties, ie isotype and epitope affinity.

Monoclonal antibodies against objects and / or identified substances can be prepared using any technique that provides for the production of antibody molecules by continuous cell line culture. These include Koehler and Milstein (1975 Nature
256: 495-497), the hybridoma technique first described by 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 EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies and
Cancer Therapy, Alan R Liss Inc., pp 77-96), but is not limited. In addition, techniques developed for the production of “chimeric antibodies”, using splicing of mouse antibody genes to human antibody genes to obtain molecules with appropriate antigen specificity and biological activity Can (Morrison et al. (1984) Proc Natl Acad Sci 81:
6851-6855; Neuberger, etc.
Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454). Alternatively, the techniques described for the production of single chain antibodies (US Pat. No. 4,946,779) can be adapted to produce object specific single chain antibodies.

  Both monoclonal and polyclonal antibodies directed against epitopes obtainable from identified substances and / or objects are particularly useful in diagnosis, and those that are neutral are useful in passive immunotherapy. Monoclonal antibodies can in particular be used to grow anti-idiotype antibodies. Anti-idiotypic antibodies are immunoglobulins that have an “internal image” of the object and / or substance for which protection is desired. Techniques for growing anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may again be useful for therapy.

Antibodies can also be induced by in vivo production of lymphocyte populations, or by recombinant immunoglobulin libraries or Orlandi et al. (1989, Proc Natl Acad Sci 86:
3833-3837), and by screening a panel of very specific binding reagents as disclosed in Winter G and Milstein C (1991; Nature 349: 293-299) Can do.

Antibody fragments having specific binding sites for the object can also be obtained. For example, such fragments include F (ab ′) ₂ fragments that can be obtained by pepsin digestion of antibody molecules and Fab fragments that can be obtained by reducing disulfide bridges of F (ab ′) ₂ fragments. However, it is not limited. Alternatively, Fab expression libraries are constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al. (1989) Science 256: 1275-1281).

A variety of receptor reporters can be used in the assay methods (also screened) of the invention, with preferred reporters providing signals that can be conveniently detected (eg, spectroscopy). As an example, the reporter gene can encode an enzyme that catalyzes a reaction that alters light absorption properties.

  Examples of reporter molecules include, but are not limited to, β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, β-glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabeled or fluorescent tag labeled nucleotides can then be incorporated into nascent transcripts that are identified when bound to oligonucleotide probes.

  In one preferred embodiment, the production of the reporter molecule is measured by the enzymatic activity of a reporter gene product such as β-galactosidase.

Various protocols are known in the art to detect and measure target expression, such as by using either polyclonal or monoclonal antibodies specific for the protein. Examples include solid phase enzyme immunoassay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell sorter (FACS). A two-site monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on the polypeptide is preferred, but a competitive binding assay may be used. These and other measurement methods include, among others, Hampton R et al. (1990, Serological Methods, A
Laboratory Manual, APS Press (APS Press), St. Paul MN, Maddox DE, etc. (1983, J Exp Med 15 8: 121 1).

  Various labels and linking techniques are known by those skilled in the art and can be used for various nucleic acid and amino acid measurements. Methods for producing labeled hybridization or PCR probes for target polynucleotide sequence detection include oligolabelling, nick translation, end-labelling or PCR amplification using labeled nucleotides. Alternatively, the coding sequence, or any part of it, can be cloned into an mRNA probe production vector. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by the addition of appropriate RNA polymerases such as T7, T3 or SP6 and labeled nucleotides. Can do.

  Many, such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI) and US Biochemical Corp (Cleveland, OH) Companies supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic materials and substrates, cofactors, inhibitors, magnetic particles, and the like. Patents teaching the use of such labels include: US-A-3178737; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241 is mentioned. Recombinant immunoglobulins can also be made as shown in US-A-4816567.

Additional methods for quantifying the expression of specific molecules include radiolabels (Melby PC et al. 1993 J Immunol Methods
159: 235-44) or biotinylated (Duplaa C et al. 1993 Anal Biochem 229-36) nucleotides, auxiliary amplification of control nucleic acids, and standard curves with interpolated experimental results. The quantification of a large number of samples can be expedited by taking measurements in an ELISA format in which the oligomer in question is provided at various dilutions and the spectrophotometric or calorimetric response results in rapid quantification.

  The presence / absence of marker gene expression suggests that the gene in question is still present, but its presence and expression should be confirmed. For example, if a nucleotide sequence is inserted into a marker gene sequence, recombinant cells containing it can be identified by the absence of marker gene function. Alternatively, the marker gene can be aligned with the target coding sequence under the control of a single promoter. Expression of a marker gene in response to induction or selection usually indicates target expression as well.

  Alternatively, a host cell having a target coding sequence and expressing a target coding region can be identified by various techniques known to those skilled in the art. These techniques include DNA-DNA or DNA-RNA hybridization and protein biological quantification methods, including membrane-based, solution-based, or chip-based technologies for the detection and / or quantification of nucleic acids or proteins. Or an immunoassay technique is mentioned, However It is not necessarily limited.

Screening Any one or more suitable targets, such as oxytocin receptors and / or vasopressin receptors, preferably V1a receptors, are used to identify substances such as selective oxytocin receptor antagonists in various drug screening techniques be able to. The target used in such a test may be free in solution, fixed to a solid support, placed on the cell surface, or located intracellularly. The target may even be in an animal model, where the target may be a foreign target or an introduced target. The animal model is a non-human animal model. Loss of target activity or formation of binding complexes between the target and the substance being tested can be measured.

  The drug sorting technique may be based on the method described in Geysen, European Patent Application 84/03564, published September 13, 1984. In summary, a number of different small peptide test compounds are synthesized on a solid substrate such as a plastic pin or another surface. The peptide test compound is reacted with an appropriate target or fragment thereof and washed. The bound agent is then detected, for example by appropriate adaptation of methods well known in the art. The purified target can again be coated directly onto a plate for use in drug sorting techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.

  The present invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies that can specifically bind to the target compete with the test compound for binding to the target.

  Another sorting technique provides for high throughput sorting (HTS) of substances with appropriate binding affinity for objects and is based on the method described in detail in WO 84/03564.

  The assay of the present invention is expected to be suitable for both small and large scale screening of test compounds and quantitative assays.

  In a preferred embodiment, the screening of the present invention comprises at least the following steps (which need not be in this same sequential order): (a) the candidate substance has the appropriate activity (such as an oxytocin receptor change) Perform in vitro sorting to determine whether; (b) see, for example, whether this substance is also a vasopressin receptor, particularly a V1a receptor inhibitor, for example by using the measurement protocol set forth herein Performing one or more selective screens that measure the selectivity of the candidate material; and (c) performing an in vivo screen using the candidate material (eg, a material for a vasopressin receptor, particularly a V1a receptor). Using functional animal models, including measuring the selectivity of a substance by measuring its action). Typically, if this candidate substance has passed through sorting (a) and sorting (b), then sorting (c) is performed.

Diagnostic Methods / Compositions / Kits The present invention also provides diagnostic methods, compositions or kits for the detection of previous predisposition to premature ejaculation. In this regard, the method, composition or kit comprises a means for detecting an agent, preferably oxytocin, in a test sample, preferably a blood sample taken from a sexually aroused male.

  In order to provide diagnostic criteria for early firing, normal or standard values for the factors should be established. It is suitable for complex formation, as well as antibodies to body fluids and factors collected from normal subjects, either animals or humans, collected at various times after sexual arousal, well known in the art. Can be achieved by combining under conditions. The amount of standard complex formation can be quantified by comparing it to a series of dilutions of a positive control that combines a known amount of antibody with a known concentration of purified target. The standard value obtained from a normal sample can then be compared to the value obtained from a sample obtained from a subject potentially affected by early firing. Deviation between standard and subject values establishes the presence of a disease state.

  The factor itself, or part thereof, can provide the basis for diagnostic and / or therapeutic compounds. For diagnostic purposes, the target polynucleotide sequence can be used to detect and quantify gene expression in a condition, disorder or disease that can be associated with early firing.

  The polynucleotide sequence encoding the target can be used for diagnosis of premature ejaculation resulting from target expression. For example, a polynucleotide sequence encoding an agent can be used in a tissue or biological fluid hybridization or PCR assay obtained from a biopsy or autopsy to detect expression of the agent. Such qualitative or quantitative forms include Southern or Northern analysis, dot blot or other membrane based technology; PCR technology; dip stick, pin or chip technology; and ELISA or other sample formats Technology may be included. All these techniques are well known in the art and in fact are the basis of many commercially available diagnostic kits.

  Such measures can be tailored to assess the efficacy of a particular therapy treatment plan and can be used for animal studies, clinical trials, or monitoring of individual treatment. Once the disease has been established, the existing therapeutic agent can be administered to obtain a treatment profile or value. Finally, the measurement can be repeated periodically to assess whether the value proceeds or returns towards a normal or standard pattern. A continuous treatment profile can be used to show the efficacy of treatment over a period of days or months.

Assays The assays according to the present invention include, but are not limited to, one or more of the following techniques: competitive and non-competitive assays, radioimmunoassays, bioluminescence and chemiluminescence assays, fluorimetry Methods, 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 immune cells Can be used.

Nucleic Acids Another aspect of the probe present invention, capable of detecting polynucleotide sequences, including genomic sequences encoding the target coding regions, such as regions encoding closely related molecules, such as oxytocin receptor or allele The provision of hybridization or PCR probes (especially those that can be selectively selected). The specificity of the probe, ie whether it is derived from a highly conserved, conserved or non-conserved region or domain, and the stringency of hybridization or amplification (strict, intermediate or gradual) Determine whether to identify only naturally occurring target coding sequences 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 can be used in a pool of degenerate probes. For the detection of identical nucleic acid sequences, or where maximum specificity is desired, the nucleic acid probe is selected from a non-conserved nucleotide region or unique region of the target polynucleotide. As used herein, the term “non-conserved nucleotide region” refers to a nucleotide region that is unique to the target coding sequence disclosed herein and is not present in related family members.

  PCR as described in US-A-4683195, US-A-4800195 and US-A-4965188 provides further use of oligonucleotides based on target sequences. Such oligomers are usually chemically synthesized, but they can be produced enzymatically or made from recombinant sources. Oligomers are typically two nucleotide sequences used under conditions optimized for the identification of a particular gene or condition, those with sense orientation (5 '-> 3') and those with antisense (3 '< −5 ′). The same two oligomers, nested sets of oligomers, or even a condensed pool of oligomers, under milder stringent conditions for the detection and / or quantification of closely related DNA or RNA sequences Can be used.

The substance or target nucleic acid sequence can also be used to create a hybridization probe as previously described for mapping of endogenous genomic sequences. Sequences can be mapped to specific chromosomes or specific regions of chromosomes using well-known techniques. These include in situ hybridization 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 constructs such as YACs, bacterial artificial chromosomes (BACs), bacterial PI constructs or single chromosomal cDNA libraries are included.

  Physical mapping techniques such as in situ hybridization of chromosomal preparations and linkage analysis using established chromosomal markers are invaluable for the expansion of genetic maps. Examples of genetic maps can be found in Science (1995; 270: 410f and 1994; 265: 1981f). Often, even if the number or arm of a particular human chromosome is unknown, the placement of the gene on the chromosome of another mammalian species may reveal an associated marker. New sequences can be assigned to chromosome arms, or parts thereof, by physical mapping. This provides valuable information to seekers who investigate disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome has been localized as is by genetic linkage to a particular genomic region, sequence mapping for that region can show related or regulatory genes for further exploration. The nucleotide sequences of the present invention can also be used to detect chromosomal location differences due to translocations, inversions, etc. between normal individuals, carriers or affected individuals.

Organism The term “organism” in relation to the present invention includes any organism that can contain a target and / or a product derived therefrom. Examples of organisms may include mammals, fungi, yeasts or plants.

  The term “transgenic organism” in relation to the present invention includes any organism that can contain a target and / or a product derived therefrom.

As indicated before transformation of the host cell / host organism , the host organism may be prokaryotic or eukaryotic. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings regarding transformation of prokaryotic hosts are well documented in the art, such as Sambrook et al. (Molecular Cloning: Experimental Manual)
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.

  If a prokaryotic host is used, the nucleotide sequence may need to be appropriately modified prior to transformation, for example by removal of introns.

In another embodiment, the transgenic organism may be a yeast. In this respect, yeast has been widely used as a vehicle for expressing heterologous genes. The species Saccharomyces cerevisiae has a long history of industrial uses, including its use for heterologous gene expression. Heterologous gene expression in Saccharomyces cerevisiae has been described by Goodey et al. (1987, Yeast Biotechnology, DR Berry et al., Pp 401-429, Allen and Unwin (Allen and Unwin)
Unwin), London) and King et al. (1989,
Have been reviewed by Molecular and Cell Biology of Yeasts, edited by EF Walton and GT Yarronton, pp 107-133, Blackie, Glasgow.

  For several reasons, Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and cannot produce certain endotoxins. Second, it has a long history of safe use as a result of centuries of commercial use for various purposes. This provides wide public tolerance. Thirdly, the widespread commercial use and research directed at this organism has resulted in a wealth of knowledge about the genetics and physiology of Saccharomyces cerevisiae and large-scale fermentation characteristics.

  An overview of the principles of heterologous gene expression and gene product secretion in Saccharomyces cerevisiae can be found in E Hinchcliffe E Kenny (1993, Yeast as a vehicle for the expression of heterologous genes), Yeasts, Volume 5, Anthony H Rose and J Stuart Harrison, 2nd edition, Academic Publishers.

  Several types of yeast vectors are available, including integrative vectors that require recombination using the host genome to maintain them and plasmid vectors that replicate automatically.

  To prepare transgenic Saccharomyces, an expression construct is prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The construct has promoter activity in yeast fused to the nucleotide sequence of the present invention, and usually a yeast-derived promoter such as the GAL1 promoter is used. Usually, a signal sequence derived from yeast such as a sequence encoding the SUC2 signal peptide is used. Terminator activity in yeast terminates the expression system.

Several transformation protocols have been developed for transformation of yeast. For example, the transgenic Saccharomyces according to the present invention is described by Hinnnen et al. (1978, Proceedings of the National Academy of Science of the USA 75,
1929); Beggs, JD (1978, Nature, London, 275, 104); and Ito, H et al. (1983, J Bacteriology)
153, 163-168).

  Transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and major antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.

  Another host organism is a plant. The basic principle in the construction of genetically modified plants is to insert genetic information into the plant genome so as to obtain a stable maintenance of the inserted genetic material. There are several techniques for inserting genetic information and the two main principles are the direct introduction of genetic information and the introduction of genetic information through the use of vector systems. General techniques are reviewed 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). Can be found in the paper by. Further teachings regarding plant transformation can be found in EP-A-0449375.

  Thus, the present invention also provides a method of transforming a host cell with a nucleotide sequence that should be the target or should express the target. Host cells transformed with the nucleotide sequence can be cultured under conditions suitable for expression and recovery of the encoded protein from the cell culture. Proteins produced by recombinant cells can be secreted or can be intracellular, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, expression vectors containing a coding sequence can be designed to have a signal sequence that directs secretion of the coding sequence through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructs can link the coding sequence to a nucleotide sequence encoding a polypeptide domain that facilitates purification of soluble proteins (Kroll DJ et al. (1993) DNA Cell Biol 12: 441-53 ).

PDE Inhibitor-Test Method The PDE potency value discussed herein is determined by the following assay:

Phosphodiesterase (PDE) inhibitory activity Preferred PDE compounds for use according to the present invention are potent selective cGMP PDE5 inhibitors. In vitro PDE inhibitory activity against cyclic guanosine 3 ′, 5′-monophosphate (cGMP) and cyclic adenosine 3 ′, 5′-monophosphate (cAMP) phosphodiesterase is determined by their IC ₅₀ values (50% of enzyme activity). Can be determined by measuring the concentration of the compound required for inhibition)

W. J. et al. Thompson and M.M. M.M. Appleman (MM
PDE enzymes substantially required by the method of Appleman) (Biochem., 1971, 10 , 311) are human penile corpus cavernosum, human and rabbit platelets, human ventricle, human skeletal muscle and bovine retina. Can be isolated from a variety of sources including: In particular, cGMP-specific PDE (PDE5) and cAMP PDE that inhibits cGMP (PDE3) can be obtained from human penile cavernous tissue, and cAMP-specific PDE (PDE4) can be obtained from human skeletal muscle. . Phosphodiesterase 7-11 can be obtained from a full-length human recombinant clone transfected into SF9 cells.

Measurements are taken from W.W. J. et al. Thompson, etc. (Biochem., 1979, 18, 5228) Bu batch or use of variants of Bu method, or product code TRKQ7090 / 7100 Amersham (Amersham) direct AMP / GMP using a modification of the protocol are described by plc of Either a scintillation proximal measurement method for visual detection can be performed. In short, the effect of the PDE inhibitor is that the inhibitor and the low substrate (concentration ˜1 / 3 Km, unlabeled 3: 1 to [ ³ H] -label, so that the IC ₅₀ is approximately equal to Ki. Investigate by measuring a fixed amount of enzyme in the presence of a proportion of cGMP or cAMP). The final measurement volume is brought to 100 μl with measurement buffer [20 mM Tris-HCl pH 7.4, 5 mM MgCl ₂ , 1 mg / ml bovine serum albumin]. The reaction was initiated with enzyme, incubated at 30 ° C. for 30-60 minutes to <30% substrate turnover, and 50 μl of yttrium silicate SPA beads (3 mM unlabeled cyclic nucleotides for PDEs 9 and 11, respectively). Contain). After resealing the plate and shaking for 20 minutes, the beads are allowed to settle for 30 minutes in the dark and then counted on a top count plate reader (Packard, Meriden, CT). Radioactivity units are converted to% activity of uninhibited control (100%), plotted against inhibitor concentration, and inhibitor IC50 values are expressed as 'Fit Curve' Microsoft Excel. Extension (Microsoft
Excel extension).

Functional activity A. SA Ballard et al. (Brit. J. Pharmacol., 1996,
118 (suppl.), Summary 153P) to assess in vitro by measuring the ability of the compounds of the present invention to enhance sodium nitroprusside-induced relaxation of pre-contracted rabbit penile cavernous tissue strips. Can do.

1.0 Method 1.1. Animal test method 1.1.1. Penile Erection and Ejaculation Test Method in Anesthetized Rats The method used to study penile erection and ejaculation is based on the methodology taught in Yonezawa et al. (2000) Life Sciences 67, 3031-3039. For ease of discussion, this methodology is described below.

  Male Wistar-ST strain rats weighing 350-450 g are used. Prior to the experiment, the animals were divided into groups (2 animals per cage) under a controlled 12 hour light / dark cycle (lights on 07:00), constant temperature (23 ± 1 ° C.) and humidity (55 ± 5%). Rats are raised. They have free access to standard food pellets and water.

  Rats are anesthetized with sodium pentobarbital (50 mg / kg, ip) and placed in a supine position. Push the penis out of its sheath and gently support it with a wooden applicator at the base of the penis. Recorded penile responses including sheath contraction and penis erection, flushing and swelling of penis body, glans erection, glans and cup engorgement and slight flaring, glans erection with glans strong overhang Just prior to being administered, the test compound is administered intraperitoneally. The latency from test compound administration to initial penile response and ejaculation is also measured.

  The effect of the test compound on PCA-induced ejaculation is also assessed by weighing the semen accumulated over 30 minutes. A suitable method using conscious rats is described in Renyi (1985) Neuropharmacology, 24, 8, 697-704.

  Intracavernous pressure is again measured in rats anesthetized with sodium pentobarbital (50 mg / kg, ip). In addition, a small additional amount (5 mg) may be injected throughout the experimental period as needed. The penis is pushed out of its sheath and the intracavernous pressure (ICP) is measured by inserting a stainless steel needle (23 gauge) into one penile cavernous body. The needle is attached to a fluororesin (Teflon®) tube filled with heparinized physiological saline (10 U / ml) and connected to a pressure transducer (NEC-San-Ei7500).

1.1.2 Male Sexual Behavior Model:
In all sexual behavior tests, male rats are placed on the observation stage (diameter 50-60 cm), starting at 5 o'clock and going to the dark cycle and observing under red illumination. Three to four minutes after placing the male on stage, an accepting female (ovariectomized, estradiol benzoate / progesterone injection 48 hours prior to behavioral study) is introduced to the stage and the following parameters are noted:
i) Ejaculation latency (EJL; the time it takes to ejaculate after introducing the receiving female to the stage);
ii) intercourse efficiency (CE; ejaculation latency / number of insertions until ejaculation, ie, number of seconds between insertions);
iii) insertion frequency (IF; number of insertions until ejaculation);
iv) Backrest frequency (MF; number of backrests until ejaculation);
v) Interval after ejaculation (PEI; time taken from ejaculation to start of sexual behavior).

2.0 Selective Oxytocin Receptor Antagonists The compounds used in the following examples are as follows:
Selective oxytocin receptor antagonist L-368,899. Further details regarding this compound are provided above. L-368,899 is more than 20-fold selective for the oxytocin receptor compared to the V1a receptor [6.3 nM OT: 148 nM V1a].

Example 1. Delay ejaculation oxytocin receptor antagonist L-368,899 in the presence of selective oxytocin receptor antagonists (L-368,899) are anesthetized rats (0.1-10mgkg ^-1, subcutaneous (sc)) in p- Chloroamphetamine (PCA) induced ejaculation was significantly delayed by oxytocin selective doses. Ejaculation was delayed 140% (near maximum effect) at a free plasma concentration of 5.4 ± 1.5 nM (0.9 × Ki OT, see FIG. 1) —at this dose, some activity is produced by antagonism of the oxytocin receptor it is conceivable that.

Ｌ−３６８，８９９は、ＣＮＳ侵入がほとんどなく、それだけで、この研究は、オキシトシンが、ＰＣＡが誘導する射精において末梢部位の作用を有することを示す。ＰＣＡは、陰茎勃起をもたらす非アドレナリン作動性非コリン作動性神経および射精を制御する交感神経経路を活性化する５ＨＴ遊離薬である。これらのプロセクシュアル(prosexual)効果は、５ＨＴ１Ｂおよび５ＨＴ２Ｃ受容体に対し作用する脊髄の５ＨＴの放出を経て仲介されると考えられる。ＰＣＡは、また、おそらく下垂体後葉または脊髄中心から、オキシトシンの分泌も誘導する。男性におけるようなオキシトシンのこの増加は、これらの研究におけるオキシトシン受容体の拮抗作用が、射精を達成するのにかかる時間に対して著しい効果があることから、射精過程に関与する。 The erection formation mechanism was not significantly affected by oxytocin receptor blockade-the number of penis cups and overhangs were similar in control and oxytocin antagonist studies (see Table 1 below). After a ¹ mg kg ⁻¹ sc dose of L-368,899 (dose that significantly delays ejaculation), 95% of PCA-induced erections were attributed to the penis cup and increased compared to 94% in the vehicle control group. 61% of PCA-induced erections were attributed to penile overhang compared to 63% in the form control group.

L-368,899 has little CNS invasion and as such, this study shows that oxytocin has a peripheral site effect in PCA-induced ejaculation. PCA is a 5HT releaser that activates non-adrenergic non-cholinergic nerves that lead to penile erections and sympathetic pathways that control ejaculation. These prosexual effects are thought to be mediated through the release of 5HT in the spinal cord acting on 5HT1B and 5HT2C receptors. PCA also induces oxytocin secretion, possibly from the posterior pituitary gland or the spinal cord center. This increase in oxytocin as in men is involved in the ejaculation process, since the oxytocin receptor antagonism in these studies has a significant effect on the time it takes to achieve ejaculation.

  Using a rodent model of ejaculation that reflects human ejaculation physiology, the inventors have shown that peripheral oxytocin receptors are involved in the mechanism of ejaculation. These effects may be direct or may undergo modulation of internal genital sympathetic innervation. The inventors cannot discount the role of the central oxytocin receptor. Furthermore, studies indicate that oxytocin antagonists are useful for the treatment of premature ejaculation by slowing ejaculation.

Example 2 Effect of selective oxytocin antagonist (L-368,899) on seminal vesicle pressure in anesthetized rats L-368,899 stimulates visceral nerves in seminal vesicle pressure in anesthetized rats (1-3 mgkg ⁻¹ , vein) The increase was significantly reduced. Seminal vesicle contraction is essential for ejection, and semen supplied to the prostate urethra is thought to trigger ejaculation. Oxytocin has a direct contractile effect on mammalian seminal vesicles and may also have a neuromodulator role that affects sympathetic innervation during ejaculation. In this study, seminal vesicle contraction was reduced 41% after a bolus injection of ¹ mg kg ⁻¹ (see FIG. 2 below). Preliminary studies suggest that the free plasma concentration of L-368,899 obtained after 1.0 mg kg ⁻¹ intravenous injection is approximately 60 nM based on literature PK and protein plasma binding.

  The data suggests that oxytocin is released during visceral nerve stimulation and that this peptide plays a physiological role in the process of intracapsular pressure generation and ejection before ejaculation. This study supports the role of systemic oxytocin in affecting the peripheral ejaculatory response-these effects may be direct or may be through the regulation of seminal sympathetic innervation. Oxytocin alters the contraction of ducts and glandular lobules in the male genital tract, thus affecting the fluid volume of different semen components. An increase in semen volume is believed to shorten the time from insertion to ejaculation, and thus oxytocin antagonists can be useful in the treatment of premature ejaculation by delaying semen ejection.

Example 3 Effect of selective oxytocin antagonist (L-368,899) on sexual behavior in rats L-368,899 has no effect on sexual behavior in sexually experienced rats at doses up to 10 mg kg ⁻¹ sc. Rodent sexual behavior is characterized by a series of backrests with and without vaginal insertion (50-80% of backrests are attributed to insertion [vaginal penetration]) and ejaculation is 6 to 12 times Get up after insertion. Each insertion lasts approximately a few seconds-it is not possible to quantify the length of the insertion, i.e. the intravaginal latency. The effect of L-368,899 was evaluated with respect to a number of sexual parameters (see above). We focused on sexual efficiency as a measure to summarize vaginal penetration.

Ｌ−３６８，８９９は、また、脳室内（ｉｃｖ）により投与された。Ｌ−３６８，８９９は、５０ｎｇ／ラットｉｃｖ投与された場合、性的に経験を積んだラットにおける性交効率に対し何ら有意の影響がない。

There was no effect of L-368,899 on intercourse efficiency at any dose tested (0.05-10 mg kg ⁻¹ sc, see Table 2 below). Preliminary pharmacokinetic studies suggest that free plasma concentrations of 4.5 nM and 40 nM would be expected 30 minutes after injection of ¹ mgkg ⁻¹ sc and 10 mgkg ⁻¹ sc, respectively.

L-368,899 was also administered intraventricularly (icv). L-368,899 has no significant effect on intercourse efficiency in sexually experienced rats when administered at 50 ng / rat icv.

Example 4 Non-selective oxytocin antagonist for intercourse action _{d (CH} 2) against the effects intercourse action ^{(vasopressin) 5 Tyr (Me) -Orn 8} - vasopressin, namely the study prior to study the effects of peptidic non-selective oxytocin antagonist, It was shown that there was no effect on ejaculation latency at doses up to 25 ng / rat icv. There was a (significant) decrease in intercourse efficiency after 5 ng / rat icv injection-46 seconds / insertion compared to 25 seconds / insertion in vehicle control animals. This effect may be mediated by oxytocin and / or vasopressin receptors. Sexual behavior disappeared after administration of 50 ng / rat icv (Melis et al. Neuroscience Letters 265 (1999)
171-174) showed that 100 ng / rat icv reduced the number of contactless erections, and 100 ng / rat icv completely eliminated drug-induced erections. This latter data shows that, in contrast to studies with selective oxytocin receptor antagonists (L-368,899), antagonism of both the central oxytocin and vasopressin receptors impairs erection mechanisms and arousal abilities. It can be shown that it may be disadvantageous for sexual behavior involving.

Abbreviations PE = early fired sperm OT = oxytocin cAMP = cyclic adenosine-3 ', 5'-monophosphate cGMP = cyclic guanosine-3', 5'-monophosphate PDE = phosphodiesterase PDE _cGMP = cGMP PDE
PDEi = inhibitor of PDE (I: also known as PDE)
PDE5 = phosphodiesterase type 5 PDE5i = inhibitor of PDE5 kDa = kilodalton bp = base pair kb = kilobase pair

Sequence listing (forms part of the description)

  The invention will now be further described with reference to the following figures and sequence listings only by way of example.

FIG. 1 shows a graph depicting the effect of the oxytocin receptor antagonist L-368,899 on ejaculation induced by p-chloroamphetamine (PCA) in anesthetized rats; FIG. 2 shows a graph depicting the effect of a selective oxytocin antagonist (L-368,899) on seminal vesicle pressure in anesthetized rats.

Sequence Listing SEQ ID NO: 1 shows the amino acid sequence of the human oxytocin receptor; and SEQ ID NO: 2 shows the amino acid sequence of the human vasopressin V1A receptor.

Claims (30)

A selective oxytocin antagonist comprises a selective oxytocin antagonist for use in the treatment or prevention of male ejaculation disorders, optionally mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive Composition. The composition of claim 1, wherein the male ejaculation disorder is premature ejaculation. 3. The composition of claim 1 or claim 2, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor. Use of a selective oxytocin antagonist in the manufacture of a medicament for use in the treatment of male ejaculation disorders. Use according to claim 4, wherein the male ejaculation disorder is premature ejaculation. 6. Use according to claim 4 or claim 5, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor. An effective method for treating or preventing male ejaculation disorders in humans or animals, wherein the selective oxytocin antagonist may optionally be mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive Administering an amount of a selective oxytocin antagonist to the individual. 8. The method of claim 7, wherein the male ejaculation disorder is premature ejaculation. 9. The method of claim 7 or claim 8, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared to a vasopressin receptor. A pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises one or more selective oxytocin antagonists. 11. The pharmaceutical pack according to claim 10, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor compared to a vasopressin receptor. A method for preparing a pharmaceutical composition comprising mixing one or more selective oxytocin antagonists with a pharmaceutically acceptable diluent, pharmaceutical additive or carrier. 13. The method of claim 12, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor. A method for identifying substances that can be used to treat and / or prevent male ejaculation disorders, comprising determining whether a test substance can directly enhance the endogenous ejaculation process A measurement method comprising (wherein said enhancement is defined as an increase and / or recovery of ejaculation latency in the presence of a test substance as defined herein; It shows that the substance can be useful in the treatment or prevention of male ejaculation disorders, where the test substance is a selective oxytocin antagonist). The measurement method according to claim 14, wherein the male ejaculation disorder is premature ejaculation. 16. A method according to claim 14 or claim 15, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor. The substance identified by the measuring method of any one of Claim 14 to 16. An orally administered medicament for treating male ejaculation disorders, wherein the medicament comprises the substance of claim 17. The medicament according to claim 18, wherein the male ejaculation disorder is premature ejaculation. (A) performing the measurement method of any one of claims 14 to 16; (b) identifying one or more substances capable of increasing and / or restoring ejaculation latency; and (c) constant Preparing a quantity of those one or more identified substances, wherein the substance is a selective oxytocin antagonist. 21. The method of claim 20, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor. An animal model for identifying a substance capable of treating or preventing male ejaculation disorders, wherein said model comprises a male animal comprising means for measuring ejaculation latency of said animal after introduction of a receiving female The substance is a selective oxytocin antagonist). 23. The animal model according to claim 22, wherein the male ejaculation disorder is premature ejaculation. 24. The animal model of claim 22 or claim 23, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor relative to the vasopressin receptor. 25. A measurement method for identifying a substance capable of directly enhancing an endogenous ejaculation process in order to treat or prevent ejaculation disorders, comprising administering the substance to the animal model according to any one of claims 22 to 24. And a measurement method comprising measuring the ejaculation latency of the animal after introduction of the receiving female (wherein the substance is a selective oxytocin antagonist). Use of a multi-drug combination consisting of one or more selective oxytocin antagonists and one or more of the following auxiliary active substances in the manufacture / preparation of a medicament for the treatment and / or prevention of male ejaculation disorders:
i) PDE inhibitors, more particularly PDE5 inhibitors, which inhibitors preferably have an IC50 of less than 100 nM for the respective enzyme;
ii) Serotonin receptor agonists or modulators, more particularly agonists or modulators of 5HT2C, 5HT1B and / or 5HT1D receptors, including ampitrine;
iii) Serotonin receptor antagonists or modulators, more particularly 5HT1A antagonists or modulators, including NAD-299 (robalzotan) and WAY-100635, and / or more particularly batanopirde, granisetron, ondansetron Antagonists or modulators of 5HT3 receptors, including tropistron and MDL-73147EF;
iv) antidepressants, in particular i) selective serotonin reuptake inhibitors (SSRI) including sertraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, mirtazapine, nefazodone and trazodone; ii) clomipramine, desapramine Tricyclic antidepressants (TCAs), including imipramine, amitriptyline, doxepin, amoxapine, maprotiline, nortriptyline, protriptyline, trimipramine and buproprion; and iii) monoamine oxidase;
v) α-adrenergic receptor antagonists (also known as α-adrenergic blockers, α-blockers or α-receptor blockers); suitable α1-adrenergic receptor antagonists include phentolamine, prazosin , Phentolamine mesylate, trazodone, alfuzosin, indolamine, naphthopidyl, tamsulosin, phenoxybenzamine, laurophia total alkaloid, recorda 15/2739, SNAP1069, SNAP5089, RS17053, SL89.0591, doxazosin, terazosin Suitable α2-adrenergic receptor antagonists include dibenarnine, tolazoline, trimazosin, efaloxane, yohimbine, idazo Suitable non-selective α-adrenergic receptor antagonists include dapiprazole; additional α-adrenergic receptor antagonists include WO99 / 30697, US 4,188,390, US 4, 026,894, US 3,511,836, US 4,315,007, US 3,527,761, US 3,997,666, US 2,503,059, US 4,703,063, US 3,381,009, US 4,252 721 and US 2,599,000;
vi) Selective serotonin reuptake inhibitors with rapid efficacy. 27. Use according to claim 26, wherein the male ejaculation disorder is premature ejaculation. A pharmaceutical composition comprising one or more selective oxytocin antagonists and one or more PDEi's optionally mixed with a pharmaceutically acceptable carrier, diluent or pharmaceutical additive. 30. The pharmaceutical composition according to claim 28, wherein the PDEi is PDE5i. 30. Use of a pharmaceutical composition according to claim 28 or claim 29 in the preparation of a medicament for the treatment and / or prevention of ejaculation disorders.

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