KR100829262B1 - Treatment of male sexual dysfunction - Google Patents

Abstract

A composition for use in treating and / or preventing ejaculation disorders in a male, comprising an optional oxytocin antagonist optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient.

Description

How to treat sexual dysfunction in males {TREATMENT OF MALE SEXUAL DYSFUNCTION}             

The present invention relates to compounds and pharmaceutical compositions useful for treating and / or preventing sexual dysfunction in males, especially ejaculation disorders such as premature ejaculation.

The invention also relates to methods of preventing and / or treating male dysfunction, in particular ejaculation disorders such as premature ejaculation.

The invention also relates to assays for selecting compounds useful for treating male dysfunction, particularly ejaculation disorders such as premature ejaculation.

Male dysfunction

Sexual dysfunction (SD) is a serious clinical problem that can affect both men and women. The cause of SD may be biological or psychological. The biological aspect of SD is typically caused by underlying vascular diseases such as those involving high blood pressure or diabetes mellitus, by prescribed medications and / or by mental illnesses such as depression. Psychological factors include fear, performance anxiety, and interpersonal conflict. SD causes personal distress by impairing sexual function, reducing self-esteem and disrupting relationships. In hospitals, SD disorders are 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 a condition in which women are less or less satisfied with sexual expression. Male sexual dysfunction (MSD) is a sexual disorder such as erectile dysfunction and / or premature ejaculation, also commonly known as male erectile dysfunction (MED), sexual desire such as insensitivity (unreaching orgasm) or impaired libido (lack of interest in sex) It is associated with disability.

Premature Ejaculation (PE)

PE is a relatively common sexual dysfunction in men. This has been defined in several different ways, but the most widely accepted is that "PE lasts for a lifetime, with minimal sexual stimulation, before, at or after insertion, and before the patient desires ejaculation. The doctor should consider factors that affect the persistence of the excitement, such as age, whether the sexual partner or stimulus is new, and the frequency of sexual activity. Cause, "the Diagnostic and Statistical Manual of Mental Disorders IV.

International Disease Classification Criteria 10 defines:

"It is not possible to delay ejaculation sufficiently to enjoy sexual intercourse and manifests as: (1) ejaculation occurs before or immediately after commencement of insertion (if a time limit is required, before commencement or within 15 seconds of insertion). (2) ejaculation occurs without sufficient erection to allow insertion. This problem does not arise as a result of prolonged sexual intercourse. "

Other definitions used include classifications based on the following criteria:

Relationship to the orgasm of the other party

Duration between insertion and ejaculation

Number of piston movements and voluntary control

Psychological factors may be involved in PE, with pre-sexual disability relational disorders, anxiety, and depression all playing a role.

The estimated PE prevalence is about 22 to 38% of the male population. Unlike male erectile dysfunction (MED), PE has no clear correlation with age. At an average prevalence of 30%, approximately 24 million people suffer in the United States (80 million men aged 18 to 65 in 1995). There is very little prevalence data based on severity. Applicable definitions of PE may apply to 5-10% of men, but less than 0.2% are estimated to be receiving treatment. If effective oral therapies are available, this situation can be changed.

Urologists currently make up the vast majority (59%) of doctors who treat PE. GP makes up 33% of physicians who treat the disease. Sex therapists, behavioral therapists and counselors also treat PE patients. Experts estimate that 50% of participants do so because of the impact of the disease on their relationship with the other. Stress, difficulties in relationships and / or effects on quality of life are major causes for patients to treat PE.

Ejaculation depends on the sympathetic and parasympathetic nervous system. The impulse nerve impulses through the sympathetic nervous system and into the lens tube and epididymis cause smooth muscle contraction and move sperm to the posterior dorsal urethra. Similar contractions of the seminal vesicles, prostate gland and the urethral canal lead to an increase in the volume and fluid content of semen. The discharge of semen is mediated by the impulse nerve impulses, starting from the nucleus of Onuf of the spinal cord, passing through the parasympathetic nervous system and causing periodic contractions of the mandibular cavernous, sciatic and pelvic floor muscles. Cortical control of ejaculation in humans is still under debate. In rats, the anterior medial region and the hypothalamus nucleus appear to be involved in ejaculation.

There is currently no approved medicament that can be used to treat PE. The most common off-label prescribed drugs are antidepressants (eg clomipramine) and selective serotonin reuptake inhibitors (eg paroxetine and sertraline). Because patients think of these drugs as antidepressants, they are often not well received. These drugs are used 'off-label' and are effective when needed (ie 'prn'), but long pharmacokinetic T _max (the time it takes to reach maximum drug concentration in plasma after oral administration of a drug) May cause the action to begin slowly. Long-term use can cause side effects common to this class of medications. Behavioral therapy is another means of treatment but is not very effective and has a high dropout rate and relapse rate. More efficient new therapies are needed.

Therefore, it is desirable to find new methods of treating male dysfunction, especially ejaculation disorders such as premature ejaculation.

Summary of the Invention

A promising finding of the present invention is that it is possible to increase ejaculation potential by administering selective oxytocin antagonists. Thus, it has been found that the use of selective oxytocin antagonists can treat ejaculation disorders, particularly premature ejaculation. By increasing the ejaculation potential, preferably by recovering the ejaculation potential to near normal levels, this can be achieved.

Specifically, selective oxytocin antagonists can be used to treat ejaculation disorders, especially premature ejaculation, while maintaining an erection mechanism, particularly penile erection.

Treatment of ejaculatory disorders, especially premature ejaculation, with selective oxytocin antagonists, allows for treatment while maintaining the patient's sexual drive. As used herein, the term "sexual desire" means libido or libido.

Thus, the compounds according to the invention preferably have the unexpected advantage of maintaining an erection mechanism (especially penile erection) and / or sexual desire when compared to known non-selective oxytocin antagonists.

Role of Oxytocin in Sexual Behavior

The assessment includes two separate components: emission and injection. A spermatozoa is the semen and sperm from the distal epididymis, the lens tube, the seminal vesicles and the prostate into the prostate urethra. After being driven in this way, the contents of the semen from the urethral cavity are strongly pressed and released. Ejaculation is different from orgasm, a purely cerebral event. Often these two processes occur at the same time.

Oxytocin beats in peripheral blood occur simultaneously with ejaculation in mammals. In men, the concentration of oxytocin in plasma increases significantly at ejaculation or at the time of ejaculation, but not for vasopressin. Oxytocin does not induce ejaculation itself. This process is under neural control via the α1-adrenergic receptor / sympathetic nerve starting in the lumbar region of the 100% spinal cord. Systemic rhythms of oxytocin can play a direct role in peripheral ejaculation reactions. This may serve to regulate the contraction of the duct and gland lobules throughout the male reproductive tract, such as affecting the fluid volume of different ejaculation components. Oxytocin, released into the central nervous system towards the brain, can affect sexual behavior, subjective perception of excitement (orgasm), and latent potential for subsequent ejaculation. Ejaculation in men is strictly dependent on tactile stimulation of the external reproductive organs.

It is well reported that levels of circulating oxytocin increase during sexual stimulation and excitement 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) levels in males during sexual arousal and ejaculation, resulting in a significant increase in plasma AVP during sexual arousal. Oxytocin, on the other hand, was found not. At ejaculation, however, the mean plasma oxytocin increased about five-fold and returned to baseline concentration within 30 minutes, whereas AVP had already returned to baseline at assessment and remained stable thereafter.

As described in detail in Gimpl and Fahrenholz (Physiological Reviews Vol. 81: No. 2. April 2001 pp. 629-683), oxytocin is the penis in rats, rabbits and monkeys. It has been found to be one of the most potent drugs that induce erection. In addition, central administration of oxytocin is claimed to reduce the late phase to ejaculation and shorten the intermediate phase after ejaculation. Similarly, Meston et al. (Arch. Gen Psychiatry, Vol. 57, November 2000) reported that penile oxytocin is injected into certain regions of the brain (ie, the periventricular nucleus of the hypothalamus) in animal males. It is described as facilitating an erection and shortening the late phase of ejaculation and the middle phase of ejaculation in the case of central or peripheral injection.

It is well documented in the art that administration of vasotocin, an oxytocin receptor agonist, significantly reduces contactless penile erection (see, for example, Melis et al., Neuroscience Letters 265 (1999) 171-174). . In addition, Argiolas et al. (European Journal of Pharmacology 149 (1988) 389-392) reported that intravenous (ICV) injection of vasotosin, an oxytocin antagonist, resulted in the presence of receptive females. Male rats have been shown to have impaired isosexual function, possibly by ejaculation (probably due to reduced insertion frequency). The decrease in the frequency of insertion was thought to reflect a decrease in the animal's ability to erect penis, as oxytocin antagonists were found to prevent penile erection.

The literature by Gimple and Farenholz, and Meston et al. Suggested that oxytocin reduces latent ejaculation, but other studies have shown that oxytocin has no effect on ejaculatory latent. For example, Stoneham et al. (J. Endocrinology 107: 97-106, 1985) show that intravenous injection of oxytocin into rats significantly reduces the number of insertions made before ejaculation but has no effect on ejaculation potential. Showed. In addition, injecting oxytocin into the third ventricle increases the latent period up to the first insertion and insertion and prolongs refractory, but has no effect on the latent phase of ejaculation (Stoneham et al., Supra).

In addition, several studies have shown no difference in the time to reach excitement or orgasm unless oxytocin is elevated during ejaculation. In Murphy et al., J. of Clinical Endocrinology and Metabolism, Vol. 71, No. 4 (1990) p 1056-1058, naloxone, an opioid antagonist, is a human in the absence of serum oxytocin rhythms typically observed at ejaculation. It showed no effect on the applicants' circumstances. In Ackerman et al. (Physiol Behav 63: 49-53 [1997]), N-methyl-D-aspartic acid disorders that destroy parvo cell PVN neurons, while leaving large cell neurons intact, result in lumbar spine (L5). -L6) decreased oxytocin-immunorefied fibers. This reduction was associated with a significant reduction in spermatozoa during ejaculation, but the ride, insertion and ejaculation potentials were not affected.

Detailed aspect

In one aspect, the invention relates to a composition or pharmaceutical composition comprising an optional oxytocin antagonist compound for use in treating and / or preventing ejaculation disorders, particularly premature ejaculation, in men. In pharmaceutical compositions, the optional oxytocin antagonist is optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient. At this time, the composition (as with any other composition mentioned herein) may later be packaged for use in treating male ejaculation disorders, in particular premature ejaculation.

In another aspect, the present invention includes an optional oxytocin antagonist compound for use in treating and / or preventing ejaculation disorders, especially premature ejaculation, in men while maintaining an erection mechanism (especially penile erection) and / or sexual desire. To a pharmaceutically acceptable acceptable carrier, diluent or excipient.

In another aspect, the invention relates to the use of selective oxytocin antagonists in the manufacture of a medicament (eg, pharmaceutical composition) for use in treating ejaculation disorders, in particular premature ejaculation, in men.

In another aspect, the invention provides selective oxytocin in the manufacture of a medicament (eg, pharmaceutical composition) for use in treating ejaculation disorders, particularly premature ejaculation, in men while maintaining an erection mechanism (especially penile erection) and / or sexual desire. It relates to the use of antagonists.

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

In another aspect, the present invention is directed to a method for preparing a medicament (eg, pharmaceutical composition) for use in treating ejaculation disorders, especially premature ejaculation, in men while maintaining an erection mechanism (especially penile erection) and / or sexual desire. It relates to the use of oxytocin antagonists.

In another embodiment, the present invention comprises administering to a subject an effective amount of a selective oxytocin antagonist, which is optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient, in a human or animal ejaculation disorder. And, in particular, methods of treating and / or preventing premature ejaculation.

In another aspect, the invention provides an erectile mechanism (especially in humans or animals) comprising administering to an individual an effective amount of a selective oxytocin antagonist, which is optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient. , Penile erection) and / or sexual desire while maintaining and / or preventing ejaculation disorders, in particular premature ejaculation, of males.

Also provided are pharmaceutical packs comprising one or more compartments, wherein the one or more compartments comprise one or more optional oxytocin antagonists.

The invention also provides a method of preparing a pharmaceutical composition according to the invention, comprising mixing at least one optional oxytocin antagonist with a pharmaceutically acceptable diluent, excipient or carrier.

In another aspect, the invention provides a method that can be used to treat or prevent ejaculation disorders, particularly premature ejaculation, in males (hereinafter, selective oxytocin), comprising determining whether the test drug can directly delay the endogenous ejaculation process. Analysis method for identifying an antagonist, wherein the delay is in the presence of a test drug defined herein as an increase in ejaculation potential (ie, time from initial insertion to ejaculation) and / or recovery of ejaculation potential As defined, this increase in potency by the test drug is an indication of whether the test drug may be useful for treating and / or preventing ejaculation disorders, especially premature ejaculation, in males, wherein the test drug is a selective oxytocin antagonist. Preferably, the drug has no effect or substantially no effect on penile erection. That is, preferably, the drug can enhance endogenous penile erection without adversely affecting penile erection.

In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of (a) performing an analysis method according to the present invention; (b) identifying one or more drugs that can increase and / or recover ejaculation potential; And (c) preparing a large amount of one or more identified drugs, wherein the drug is a selective oxytocin antagonist. In this embodiment, step (a) may be repeated after modifying the drug identified in step (b), for example to maximize activity. These steps can be repeated until the desired activity or pharmacokinetic profile is obtained.

Thus, in another aspect, the present invention provides a method for producing a pharmaceutical composition comprising (a1) performing an analysis according to the present invention; (b1) identifying one or more drugs that can directly increase and / or recover ejaculation potential; (b2) modifying at least one drug identified above; (a2) optionally repeating step (a1); And (c) preparing a large amount of one or more identified drugs (ie, modified drugs).

In another embodiment, the present invention provides a method for producing a pharmaceutical composition comprising the steps of (i) performing an analysis method according to the present invention; (ii) identifying one or more drugs that can increase and / or recover ejaculation potential; (iii) in a test animal, such as anesthetized rodent, testing for penile erection of the identified drug; (iv) selecting a drug that has no or substantially no effect on penile erection; And (v) preparing a large amount of one or more selected drugs, wherein the drug is a selective oxytocin antagonist. In this embodiment, the drug identified in step (ii) can be modified such as 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 another embodiment, the invention isolates one or more samples from a male during sexual stimulation at successive time intervals of 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, and 5 minutes after initiating sexual stimulation. Doing; And determining whether the sample (s) contains a particular substance present in such an amount at a time that causes ejaculation disorders, preferably premature ejaculation, of the male, wherein the particular substance is In order to achieve a beneficial effect by the use of the drug, it may be specifically modified to delay the time it takes for a particular substance to appear and / or reach a maximum concentration, the drug being a selective oxytocin antagonist. Preferably, the specific substance is oxytocin. For example, sexual stimulation was triggered by a penis vibratory stimulator (FertiCare, Holsham, Denmark).

In another embodiment, the invention provides one or more isolated isolates taken at consecutive time intervals of 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes after initiating sexual stimulation during male sexual stimulation. A diagnostic composition or kit comprising means for detecting a particular substance in a male sample, wherein said means is such amount at said time that said sample (s) causes ejaculation disorders, preferably premature ejaculation of the male. It may be used to determine whether it contains a particular substance, and the particular substance may be delayed to achieve a beneficial effect by using the drug, specifically the time it takes for the specific substance to appear and / or reach a maximum concentration. And the drug is a selective oxytocin antagonist. Preferably, the specific substance is oxytocin. Sexual stimuli can be triggered, for example, by penile vibration stimulators (Puttycare, Holmö, Denmark).

In another aspect, the present invention treats ejaculatory disorders, in particular premature ejaculation, of a male, including an animal male having a means for measuring ejaculation potential (ie, time from initial insertion to ejaculation) of a male after introducing a receptive female. And / or to an animal model used to identify drugs that can be prevented, the drugs being selective oxytocin antagonists.

Animal models can include or be used with additional animal models with means for measuring changes in penile erection. For example, a suitable further model may be an anesthetized animal male having means for measuring changes in the cavernous pressure and / or cavernous blood flow in the animal after stimulation of the penis nerve, wherein the drug is an optional oxytocin antagonist. to be.

In another aspect, the present invention provides a method of treating a drug, comprising administering a drug to an animal model of the present invention; And measuring the ejaculation potential of the animal (ie, the time from initial insertion to ejaculation) after introducing a receptive female, directly improving the endogenous ejaculation process to treat or prevent ejaculation disorders, particularly premature ejaculation. An assay method for identifying a drug that can be used, wherein the drug is a selective oxytocin antagonist.

In another embodiment, the present invention provides a method of administering a drug comprising administering a drug to an animal model of the present invention; Measuring a change in the endogenous ejaculation process; And measuring penile erection and / or sexual desire in the animal model to confirm that there is no change or substantially no change in them, in order to treat or prevent ejaculation disorders, in particular premature ejaculation, The present invention relates to an analytical method for identifying a drug that can directly improve the endogenous ejaculation process without affecting sexual desire, wherein the change in the endogenous ejaculation process is the assessment of the animal after introduction of a receptive female. Defined as latent stage (ie, time from initial insertion to ejaculation), the drug is a selective oxytocin antagonist.

For ease of reference, these and other aspects of the invention are discussed under appropriate paragraph headings. However, the teachings of each paragraph are not necessarily limited to each particular paragraph.

The terms "selective oxytocin antagonist" and "selective oxytocin receptor antagonist" are interchangeable and refer to oxytocin receptor antagonists that are selective for oxytocin receptors as compared to vasopressin, in particular V1a receptors.

As used herein, the term "cumshot latent" means the time from initial insertion to ejaculation. As used herein, the term "recovery of ejaculation potential" means that the time from initial insertion to ejaculation changes (preferably increased). Preferably, the time from insertion to ejaculation is altered (preferably increased) to near normal levels. Typically, premature ejaculation patients ejaculate within 30 seconds after initiation of coitus (ie, first insertion) and often within 15 seconds after initiation of coitus (ie, first insertion). In a preferred embodiment of the present invention, ejaculation potential is increased to at least 30 seconds, preferably at least 60 seconds, more preferably at least 2 minutes, even more preferably at least 5 minutes, even more preferably at least 10 minutes. Suitably, the ejaculation potential can be restored such that the time from insertion to ejaculation is delayed sufficiently to satisfy the other party.

As used herein, the term "sexual desire" means libido or libido.

The term "insertion" as used herein refers to vaginal penetration by the penis.

Preferred Embodiment

In one embodiment, the drug for use according to the invention is preferably for oral administration.

In another embodiment, the drug for use according to the invention may be for topical or intranasal administration.

Preferably, the drug according to the invention is for use in treating and / or preventing premature ejaculation.

Preferably, the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor, in particular the Vla receptor.

Preferably, the selective oxytocin antagonist is at least 30-fold selective for the oxytocin receptor as compared to the vasopressin receptor, in particular the Vla receptor.

Preferably, the selective oxytocin antagonist is at least 50-fold selective for the oxytocin receptor as compared to the vasopressin receptor, in particular the Vla receptor.

Preferably, the selective oxytocin antagonist is at least 100-fold selective for the oxytocin receptor as compared to the vasopressin receptor, in particular the Vla receptor.

Preferably, the selective oxytocin antagonist is at least 200-fold selective for the oxytocin receptor as compared to the vasopressin receptor, in particular the Vla receptor.

Preferably, the selective oxytocin antagonist is at least 250-fold selective for the oxytocin receptor as compared to the vasopressin receptor, in particular the Vla receptor.

The invention also includes administering a drug of the invention prior to and / or during sexual arousal / stimulation.

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

Here, "sexual excitation / stimulation" may be one or more of visual excitation / stimulation, physical excitation / stimulation, auditory excitation / stimulation, or imaginary excitation / stimulation.

Thus, preferably, when the drugs of the present invention are particularly for oral delivery, these drugs are delivered before or during sexual arousal / stimulation.

A chosen embodiment

The present invention provides the following (numbered) selected embodiments:

1. A composition for use in treating or preventing ejaculation disorders in a male, comprising an optional oxytocin antagonist optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient.

2. The composition of aspect 1 wherein the male ejaculatory disorder is premature ejaculation.

3. The composition of aspect 1 or 2, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor.

4. The composition of aspect 3, wherein the vasopressin receptor is a Vla receptor.

5. Use of an optional oxytocin antagonist in the manufacture of a medicament for use in treating male ejaculation disorders.

6. Use according to the fifth aspect, wherein the male ejaculatory disorder is premature ejaculation.

7. Use according to the fifth or sixth aspect, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor.

8. Use according to the seventh aspect, wherein the vasopressin receptor is a Vla receptor.

9. Use according to any one of the fifth to eighth embodiments, wherein the optional oxytocin antagonist is administered before and / or during sexual arousal.

10. The use of any one of the fifth to ninth embodiments, wherein the optional oxytocin antagonist is administered by mouth.

11. A method of treating or preventing a male ejaculation disorder or an animal male ejaculation disorder comprising administering to a subject an effective amount of a selective oxytocin antagonist optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient.

12. The method of aspect 11, wherein the male ejaculation disorder is premature ejaculation.

13. The method of aspect 11 or 12, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor.

14. The method of embodiment 13, wherein the vasopressin receptor is a Vla receptor.

15. The method of any one of the eleventh through fourteenth aspects, wherein the optional oxytocin antagonist is administered prior to and / or during sexual arousal.

16. The method of any one of the eleventh to fifteenth aspects, wherein the medicament is administered through the mouth.

17. A pharmaceutical pack comprising one or more compartments, wherein the one or more compartments comprise one or more optional oxytocin antagonists.

18. The pharmaceutical pack of embodiment 17, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor.

19. The pharmaceutical pack of embodiment 18, wherein the vasopressin receptor is a V1a receptor.

20. A method of making a pharmaceutical composition comprising mixing at least one optional oxytocin antagonist with a pharmaceutically acceptable diluent, excipient or carrier.

21. The method of embodiment 20, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor.

22. The method of aspect 21, wherein the vasopressin receptor is a Vla receptor.

23. An analytical method for identifying a drug that can be used to treat and / or prevent an ejaculation disorder in a male, comprising determining whether the test drug can directly improve the endogenous ejaculation process, wherein Enhancement is defined as an increase and / or recovery of ejaculation potential in the presence of a test drug as defined herein, wherein an increase in potency by the test drug may be useful for treating or preventing ejaculation disorders in males. An indicator, wherein said test drug is a selective oxytocin antagonist.

24. The analysis method according to aspect 23, wherein the male ejaculation disorder is premature ejaculation.

25. The assay method of aspect 23 or 24, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor.

26. The assay of aspect 25, wherein the vasopressin receptor is a Vla receptor.

27. A drug identified by the method of analysis according to any one of items 23 to 26.

28. The drug of aspect 27, for use in treating or preventing ejaculation disorders in males.

29. The method of aspect 28, wherein

A drug in which the male ejaculation disorder is premature ejaculation.

30. A medicament for oral administration for the treatment of male ejaculation disorders comprising the medicament according to aspect 27.

31. The agent according to aspect 30, wherein the male ejaculatory disorder is premature ejaculation.

32. An agent according to aspect 30 or 31, which is administered prior to and / or during sexual arousal.

33. The agent according to any one of aspects 30 to 32, which is administered through the mouth.

34. (a) carrying out the method of analysis according to any one of the twenty-third to twenty-sixth aspects; (b) identifying one or more drugs that can increase and / or recover ejaculation potential; And (c) preparing a large amount of one or more of these identified drugs, wherein the drug is a selective oxytocin antagonist.

35. The method of embodiment 34, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor.

36. The method of embodiment 35, wherein the vasopressin receptor is a Vla receptor.

37. A male animal having a means for measuring the ejaculation potential of an animal male after introduction of a receptive female, wherein the drug is a selective oxytocin antagonist identifying a drug capable of treating or preventing ejaculation disorders in the male Animal model for

38. The animal model of aspect 37, wherein the male ejaculatory disorder is premature ejaculation.

39. The animal model of aspect 37 or 38, wherein the selective oxytocin antagonist is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor.

40. The animal model of aspect 39, wherein the vasopressin receptor is a Vla receptor.

41. administering the drug to an animal model of any one of aspects 37-40; And measuring the ejaculation potential of the animal after introducing a receptive female, wherein the drug is a selective oxytocin antagonist, the drug capable of directly enhancing endogenous ejaculation processes to treat or prevent ejaculation disorders. Analytical method to confirm.

42. Use of a combination of one or more optional oxytocin antagonists and one or more of the following auxiliary active agents in the manufacture / preparation of a medicament for treating and / or preventing male ejaculation disorders:

i) PDE inhibitors, more specifically PDE 5 inhibitors, having an IC50 of less than 100 nM for individual enzymes;

ii) serotonin receptor agonists or modulators, more specifically agents or modulators for 5HT2C, 5HT1B and / or 5HT1D receptors (including ampitolins);

iii) a serotonin receptor antagonist or modulator, more specifically an antagonist or modulator for 5HT1A (including NAD-299 (Rovalzotan) and WAY-100635), and / or more specifically an antagonist or modulator for 5HT3 receptor (batano Fidr, granistron, ondansetron, trophytron and MDL-73147EF);

iv) antidepressants, specifically i) selective serotonin reuptake inhibitors (SSRi) including serraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, myrzazapine, nefazodone and trazodone; ii) tricyclics, including clomipramine, desipramine, imipramine, amitriptyline, doxepin, amoxapine, maprotilin, nortriptyline, protriptiline, trimipramine and buproprion Antidepressant (TCA); And iii) monoamine oxidase inhibitors;

v) α-adrenergic receptor antagonists (also known as α-adrenergic blockers, α-blockers or α-receptor blockers); Suitable α1-adrenergic receptor antagonists are phentolamine, prazosin, phentolamine mesylate, trazodone, alfuzosin, indoramine, naphthopidyl, tamsulosin, phenoxybenzamine, lauwalpa alkaloids, Recordati 15/2739, including SNAP 1069, SNAP 5089, RS 17053, SL 89.0591, doxazosin, terrazosin and abanoquil; Suitable α2-adrenergic receptor antagonists include dibenamine, tolazoline, trimazosine, eparoxane, yohimbine, idazoic acid clonidine and dibenamine; Suitable non-selective α-adrenergic receptor antagonists include dapiprazole; Other α-adrenergic receptor antagonists 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, US 3,381,009, US 3,381,009 4,252,721 and US 2,599,000;

vi) rapid onset selective serotonin reuptake inhibitor.

43. Use of a combination of one or more optional oxytocin antagonists and one or more PDE inhibitors (PDEi) in the manufacture / preparation of a medicament for treating or preventing male ejaculation disorders.

44. Use according to aspect 42 or 43, wherein the male ejaculation disorder is premature ejaculation.

45. Use according to aspect 43 or 44, wherein the PDEi is a PDE 5 inhibitor (PDE5i).

46. The use according to any one of aspects 42 to 45, wherein the medicament is administered by mouth.

47. A pharmaceutical composition consisting of one or more optional oxytocin antagonists and one or more PDEi, optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient.

48. The pharmaceutical composition of embodiment 47, wherein the PDEi is PDE5i.

49. The pharmaceutical composition according to aspect 47 or 48, which is administered through the mouth.

50. Use of a pharmaceutical composition according to any one of embodiments 47-49 in the manufacture of a medicament for treating and / or preventing ejaculation disorders.

Amazing and unexpected discovery

The present invention illustrates the following surprising and unexpected findings:

(a) Administration of selective oxytocin antagonists increases ejaculation potential. Preferably, administration of the selective oxytocin antagonist restores the ejaculation potential, preferably to near normal levels.

(b) Administration of selective oxytocin antagonists unexpectedly increases ejaculation potential without substantially inhibiting an erection mechanism, particularly penile erection, and / or without adversely affecting penile erection. Preferably, administration of the selective oxytocin antagonist restores ejaculation potential, preferably to nearly normal levels, without substantially inhibiting the erection mechanism, especially penile erection, and / or without adversely affecting penile erection.

(c) Administration of selective oxytocin antagonists increases ejaculation potential without substantially inhibiting sexual desire and / or without adversely affecting sexual desire. Preferably, administration of the selective oxytocin antagonist restores the ejaculation potential, preferably to nearly normal levels, without substantially inhibiting sexual desire and / or without adversely affecting sexual desire.

advantage

The present invention is advantageous for the following reasons:

(a) treating premature ejaculation by selectively inhibiting the oxytocin receptor with a selective oxytocin antagonist;

(b) selectively inhibiting the oxytocin receptor with selective oxytocin antagonists to unexpectedly treat premature ejaculation without substantially inhibiting an erection mechanism, particularly penile erection and / or without adversely affecting penile erection;

(c) Treatment of premature ejaculation with selective oxytocin antagonists to selectively inhibit oxytocin receptors unexpectedly without substantially inhibiting sexual desire and / or without adversely affecting sexual desire.

Patient group

Ejaculation disorders, especially premature ejaculation patients, should benefit from treatment with selective oxytocin antagonists.

Initial studies suggest that the ejaculatory disorders mentioned below, in particular the premature ejaculation patient group, should be benefited by treating with an optional oxytocin antagonist (or a combination comprising the optional oxytocin antagonist described later). These groups of patients include patients suffering from one or more of the following diseases: nervous system disorders, physiological disorders, mental sexual skills deficiency, physical illness, physical injury, pharmacological side effects, mental grievances and relationship difficulties.

Oxytocin receptor

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

The background for oxytocin receptors has been taught by Victor A. McKusick et al. ( Http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm ). From the above, the following is drawn about the oxytocin receptor:

"Kimura et al. (1992: Nature 356: 526-529) reported the structure and expression of human oxytocin receptor cDNA isolated by expression cloning. The encoded receptor is seven membranes typical for G protein-coupled receptors. It was a polypeptide of 388 amino acids with a transverse domain.Oxytocin receptors expressed in Xenopus oocytes responded specifically to oxytocin and induced inward membrane currents. , 3.6 kb in the chest and 4.4 kb in the ovary, endometrium and uterine muscle layer, and mRNA levels in the uterine muscle layer were very high at the border.Inoue et al. (1994, Biol. Chem. 269: 32451-32456) Showed that the OXTR gene was present in the human genome as a single copy, and by in situ fluorescent hybridization, they demonstrated that the gene was located at 3p26.2. Reaches about 17 kb, and contains 3 introns and 4 exons, exons 1 and 2 correspond to the 5-prime noncoding region, followed by exons 3 and 4 encoding the amino acids of the receptor. Large intron 3 separates the coding region immediately after the domain, which is estimated to span six times across the membrane The transcription start site, as evidenced by primer extension analysis, lies 618 and 621 bp upstream of the methionine initiation codon PCR analysis of somatic hybrids and By in situ fluorescent hybridization, Simons et al. (1995, Genomics 26: 623-625) gave the OXTR gene at 3p25. "

Gimple and Farenholz's (Physiological reviews Vol. 81, No. 2, April 2001) provides a detailed overview of the receptor structure. To this document, to date, in addition to the isolation and identification of cDNA encoding human oxytocin receptors (see above, Kimura et al.), Swine (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) (Salvatore) et al., J. Receipt Signal Transduct Res. 18: 15-24, 1998).

Oxytocin Receptor Sequence Data

Nucleotide and amino acid sequences for human oxytocin receptors can be obtained from the literature. By way of example, the amino acid sequence of a human oxytocin receptor is provided in SEQ ID NO: 1.

Selective Oxytocin Antagonists

Details of suitable assay systems for identifying and / or studying oxytocin antagonists are provided in the paragraph titled "Oxytocin antagonist assays".

Examples of suitable oxytocin antagonists are indicated below:

L-368 899

Synthesis of L-368,899 is taught in Williams et al. (1994, J. Med. Chem. 37, 565-571).

L-368,899 is a selective oxytocin antagonist. L-368,899 is 20 times more selective for oxytocin receptors than vasopressin, especially Vla receptors. Preferably, the selectivity is both binding selectivity and function selectivity.

Certain known oxytocin antagonists such as, for example, vasotocin, are often referred to as "selective oxytocin antagonists." However, d (CH ₂ ) ₅ Tyr (Me) -Orn ⁸ -Vasatocin (hereinafter referred to as "Vasatocin") is only two to three times more selective for oxytocin receptors than vasopressin, especially Vla receptors. 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 antagonists according to the invention are at least 20 times more selective for the oxytocin receptor than for the vasopressin, in particular the Vla receptor.

Oxytocin Antagonist Binding Assay and Vasopressin V1a Binding Assay

A. Oxytocin Receptor Ligand Binding IC50 Assay

i) buffer

ii) compound dilution (final concentration of 10 μM in the assay)

a) 4 mM HTA stock compound in 100% DMSO

b) dilute the compound to 200 μM in dH ₂ O

c) Further diluting the compound to 100 μM in 100 mM Tris-HCl, pH 7.8, 20 mM MgCl ₂ . This produces a final concentration of 2.5% DMSO, 50 mM Tris-HCl, pH 7.8, 10 mM MgCl ₂ .

d) Using diluted raw material, prepare 1: 2 dilution in 50 mM Tris-HCl, pH 7.8, 10 mM MgCl ₂ , 2.5% DMSO at 10 or more points using TECAN Genesis.

e) Dispense 10 μl of compound into 384 well Optiplate according to the plate layout required for analysis by ECADA leaving room for standard (arg ⁸ ) -Vasatocin IC50. These plates can be stored at 4 ° C.

f) On day of analysis, Max. Add 10 μl-Min. 10 μl is added and serial dilution 1: 2 in duplicate (arg ⁸ ) -basothosine to a maximum concentration of 100 nM (final 20 nM) above 10 points.

iii) maintenance of oxytocin receptor-CHO cells

Cell lines are routinely maintained in 50 ml of growth medium in 225 cm ² flasks as continuous culture.

Cells are passaged by removing medium from the molecular membrane, washing with PBS and incubating with trypsin until the cells show signs of dissociation. After knocking 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 x 10 ⁵ cells / flask.

iv) growth of cells in roller bottles

Cells are inoculated into 10 850 cm ² roller bottles at a density of 6 × 10 ⁶ cells / bottle and covered with almost a single cell.

Trypsin is used to remove cells from the bottle as described above and the cells are inoculated into 100 roller bottles (ie 1:10 split ratio).

Allow to cover again with almost one cell, then harvest by removing the growth medium, adding 40 ml PBS / bottle and scraping off with CellMate. The cell suspension is then centrifuged at 2000 rpm, washed in PBS and then centrifuged again and the pellet frozen in aliquots at -80 ° C.

v) membrane preparation

Remove the cell pellet from the freezer and thaw on ice and resuspend in 3 ml of membrane preparation buffer per ml of packaged cell volume.

• Homogenize the suspension several times on ice for 5 seconds using a mechanical homogenizer and then centrifuge for 30 minutes at 25,000 × g.

Resuspend the pellet in 1 ml of freezing buffer per 1 ml of original packaged cell volume, then simply homogenize the suspension to remove small clumps. Protein concentration is measured and the membrane suspension is finally frozen at an aliquot of at least 5 mg / ml at -80 ° C.

vi) method

Thaw the membrane on ice and dilute to 1 mg / ml in assay buffer. SPA beads are resuspended at 50 mg / ml in assay buffer. From these concentrates, the beads are prebound to the membrane by incubating 30 μg of protein per mg of beads in the top-to-bottom shaker at 4 ° C. for 2 hours. Beads / membrane are centrifuged at 2000 rpm for 10 minutes and the pellet is resuspended at 3 mg / ml.

Perform all manipulations of ¹²⁵ I-OVTA using tips silanized with SigmaCote. Both bottles and tubes are also silanized. Dilute ¹²⁵ I-OVTA in 1 ml of assay buffer per 50 μCi of lyophilized ligand. 5 μL samples are counted in duplicate using the liquid scintillation count (protocol 61 of the Wallac Counter) and the concentration of ligand is calculated (see example below). This is to overcome the loss of ligand due to stickiness. Using the measured concentration, ¹²⁵ I-OVTA is diluted to 0.3 nM in assay buffer.

Yes:

If 5 μl represents 500000 dpm and the specific activity of the ligand is 2200 Ci / mmolol:

Density = 500000 / (2.2 × 2200 × 5) nM

Using Multi-drop, add 20 [mu] l of bead / membrane formulation to the prepared Optiplate. The stirred flask is used to hold the bead / membrane formulation in suspension. 20 μl of ¹²⁵ I-OVTA is then added to each well of the opticplate using multi-drop. After 4 hours of incubation at room temperature, the plates are counted for 30 seconds per well using TopCount NXT.

B. Vasopressin V1A Receptor Binding Assay

i) substance

Human vasopressin V1a receptor cloned in CHO cells: Protein / cell science

HEPES: Sigma (H7523)

Magnesium Chloride (MgCl ₂ ): Sigma (M2670)

Bovine serum albumin (BSA): Sigma (A6003)

Glycerol: Sigma (G5150)

Protease Inhibitor Cocktail Table: Roche (1697498)

Pierce BCA Protein Assay Reagent: Pierce (23225)

· 8-Arg [phenyl alanyl -3,4,5- ³ H] - vasopressin ⁽³ H-AVP): norbornene (NEN) (NET800)

D (CH2) 5Tyr (Me) AVP [β-mercapto-β, β-cyclopentamethylene propionyl, O-Me-Tyr ² , Arg ⁸ ] -vasopressin (βMCPVP): sigma (V2255)

Dimethyl sulfoxide: Stores (W34)

Polypropylene block with 96 wells: Stores (D8281)

Polyetherinimine (PEI): Sigma (P3143)

Unifilter plate GF / C with 96 wells: Packard (6005174)

Topseal A: Packard (6005185)

Microscint-O: Packard (6013611)

SR49059 (UK222,633): Compound Control

Instrument: Packard Unifilter Unit, Top Counter / NXT Counter

ii) method

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

Assay Buffer: 25 mM HEPES (pH 7.4), 5 mM MgCl ₂ , 0.05% BSA

Wash buffer: 25 mM HEPES (pH 7.4), 5 mM MgCl ₂

³ H-AVP: 5 nM solution in assay buffer (final assay concentration 0.5 nM).

Total: 25% DMSO in ddH ₂ O.

NSB: 10 μΜ βMCPVP (final assay concentration 1 μΜ) in 25% DMSO / ddH ₂ O.

STD: Dilute SR49059 (UK222,633) in 25% DMSO / ddH ₂ O by starting with a maximum concentration of 1 μM (final assay concentration of 100 nM) and continuing decreasing in 30 log steps to 30 pM (final assay concentration of 3 pM). .

Compound: 50 μL of 4 mM compound in 100% DMSO. This is diluted 4-fold in dH ₂ O to create a maximum concentration of 1 mM in 25% DMSO. The compound is further diluted in 0.5 log steps in 25% DMSO except for the initial dilution (1 mM to 300 μM) in 18% DMSO (25% DMSO after dilution). Dilute by hand or using the Tecan and protocol file Kin28IC50dilution2.gem. Depending on the compound, the 10pt IC50 curve is started at lower concentrations if necessary, but all drugs are selected and initially started at 100 μM.

0.5% PEI: 50% PEI prepared in distilled H ₂ O is diluted to 0.5% in dH ₂ O.

iii) membrane preparation

Remove frozen cell pellets from the freezer and slowly thaw on ice.

Add 3 ml of membrane preparation buffer per 1 ml of the original packed cell volume, homogenize the suspension with polytron while triggering several times on ice for 5 seconds until well dispersed, then centrifuge at 1000 rpm for 10 minutes.

Remove supernatant and store on ice. An additional 3 ml of membrane preparation buffer per 1 ml of original packaged cell volume is added to the pellet, homogenized on ice and centrifuged for 10 minutes at 1000 rpm.

Remove the supernatant and add it to the previously removed volume of supernatant, then centrifuge for 30 min at 25,000 x g and 4 ° C.

Re-suspend 25,000 × g pellets by homogenizing in 1 ml of freezing buffer per 1 ml of original packaged cell volume and determine protein concentration.

iv) determination of protein concentration

Prepare BSA in distilled H ₂ O at the following concentrations: 2000, 1000, 500, 250, 125, 62.5 and 31.25 μg / ml.

Add 10 μl of each BSA solution in triplicate to a clear 96 well plate (see plate map in the attached document) and add 10 μl of distilled H ₂ O to three blank wells.

10 μl of membrane preparation is added in triplicate to the plate as 1/3, 1/10, 1/30 and 1/100 dilutions of the membrane preparation.

200 μl of Pierce Protein Reagent (50 A: 1 B) is added to each well, the plates are incubated at 37 ° C. for 30 minutes and then read on an Anthos spectrometer at an absorbance setting of 570 nm.

From the BSA standard curve, determine the concentration of protein in the membrane formulation (use dilution of the membrane formulation centered in the standard curve).

The membrane preparation is diluted to a protein concentration of 5 mg / ml in freezing buffer and then frozen in 200 μl aliquots at −80 ° C.

v) analysis protocol

Prepare analytical reagents ( ³ H-AVP, βMCPVP (NSB compound) and test compound—see working solution above). Any peptide solution is kept on ice.

Plate format is 10 pt IC50-4 compound for separate double rows of plates. The following reagents are added to the appropriate wells of a 96 well polypropylene block and vortexed.

To each total well (T) (A1, B1, C1, D1, E12, F12, G12 & H12): 25 μl ³ H-AVP, 25 μl vehicle

To each NSB well (N) (A12, B12, C12, D12, E1, F1, G1 & H1): 25 μl ³ H-AVP, 25 μl βMCPVP

In each assay well: 25 μl ³ H-AVP, 25 μl test compound

A & B column = STD

C & D Heat = Compound 1

E & F Heat = Compound 2

G & H Heat = Compound 3

Thaw membrane proteins slowly on ice and dilute to optimal protein concentration for analysis (see attached document for protein first order determination) (about 100 μg / ml).

200 μl of membrane protein is added to each well to initiate the reaction, and the blocks are incubated with gentle shaking for 60 minutes at room temperature.

The reaction is terminated by filtration through a unifilter GF / C filter pre-soaked in 0.5% PEI and washing three times with 1 ml of ice cold wash buffer.

The filter is dried in an oven at 55 ° C. for 2 hours or placed on a bench overnight (-16 hours).

Seal the filter to the bottom and add 30 μL of MicroSint-O to each well. The filter is then sealed in TopSill A and counted on Packard TopCount (Bld 503 / G7A) using Unifilter Protocol 11 of [ ³ H] 96 wells.

C. Data Analysis

Data analysis performed by ECADA

Calculate the nonbonding as follows:

Unbound = Average Total cpm-Average NSB cpm

For test compounds, the amount of ligand bound to the receptor is expressed as follows:

% Binding = (sample cpm-average NSB cpm) / unbound cpm × 100

Inhibition of ligand binding is reported as percent inhibition calculated as follows:

% Inhibition = 100-% binding

Oxytocin Antagonist Function Assay and Vasopressin V1a Antagonist Function Assay

Spontaneous contraction of the uterine muscle layer from humans, non-human primates and rodents is sensitive to selective oxytocin receptor antagonism in vitro (Wilson et al., BJOG September 2001; 108 (9): 960-6) .

In vitro pharmacology of spontaneous contraction of uterine muscular layer from humans and animals. Samples of the human uterine muscle layer were obtained at the caesarean section. Tissue strips were suspended in tracheal baths for isometric force recording. Cumulative concentration effect curves for selective oxytocin receptor antagonists and mixed oxytocin / vasopressin Vla receptor antagonists can be obtained. Inhibition of spontaneous contraction of the uterine myometrium is observed in vitro.

Combination

More specifically, the present invention further comprises a combination of one or more adjuvant active agents (see later discussion of suitable examples) and a compound of the present invention for treating ejaculation disorders, in particular premature ejaculation, of males as outlined herein. Include.

The present invention is essentially an optional oxytocin antagonist and two adjuvant active agents according to the present invention in the manufacture of a medicament for treating and / or preventing ejaculation disorders, in particular premature ejaculation, of males as outlined herein (suitable examples). The use of the combination).

The present invention provides an optional oxytocin antagonist and two adjuvant active agents according to the present invention in the manufacture of a medicament for treating and / or preventing ejaculation disorders, in particular premature ejaculation, of males, as outlined herein. And the use of combinations).

The invention is essentially an optional oxytocin antagonist and one adjuvant active agent according to the invention in the manufacture of a medicament for treating and / or preventing ejaculation disorders, in particular premature ejaculation, of males as outlined herein (a suitable example). The use of the combination).

The present invention provides a selective oxytocin antagonist and one adjuvant active agent according to the present invention in the manufacture of a medicament for treating and / or preventing ejaculation disorders, in particular premature ejaculation, of males, as outlined herein. Further discussion).

Accordingly, other combination embodiments of the present invention provide pharmaceutical combinations (simultaneously, separately or continuously administered) comprising a compound of the present invention and one or more auxiliary active agents (see later discussion of suitable examples).

Another combination aspect of the present invention provides a pharmaceutical composition (administered simultaneously, separately or continuously) consisting essentially of an optional oxytocin antagonist and two auxiliary active agents (see later discussion of suitable examples).

Another combination aspect of the present invention provides a pharmaceutical composition (simultaneously administered separately or continuously) consisting of an optional oxytocin antagonist and two auxiliary active agents (see later discussion of suitable examples).

Another combination aspect of the present invention provides a pharmaceutical composition (administered simultaneously, separately or continuously) consisting essentially of the optional oxytocin antagonist and one adjuvant active agent (see later discussion for suitable examples).

Another combination aspect of the present invention provides a pharmaceutical composition (simultaneously administered separately or continuously) consisting of an optional oxytocin antagonist and one adjuvant active agent (see later discussion of suitable examples).

Co-active agent

Supplementary active agents suitable for use in the combination of the present invention include the following:

1) PDE inhibitors, more specifically PDE 5 inhibitors (see below), which inhibitors preferably have an IC50 of less than 100 nM for individual enzymes;

2) serotonin receptor agonists or modulators, more specifically agents or modulators for 5HT2C, 5HT1B and / or 5HT1D receptors (including ampitoline);

3) serotonin receptor antagonists or modulators, more specifically antagonists or modulators for 5HT1A (including NAD-299 (Rovalzotan) and WAY-100635) and / or more specifically antagonists or modulators for 5HT3 receptor (batanopyr) Deg, granistron, ondansetron, trophytron and MDL-73147EF);

4) antidepressants, specifically i) selective serotonin reuptake inhibitors (SSRi), including sertraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, mirtazapine, nefazodone and trazodone; ii) tricyclics, including clomipramine, decipramine, imipramine, amitriptyline, doxepin, amoxapine, maprotilin, nortriptyline, protriptiline, trimipramine and buproprion Antidepressant (TCA); And iii) monoamine oxidase inhibitors;

5) α-adrenergic receptor antagonists (also known as α-adrenergic blockers, α-blockers or α-receptor blockers); Suitable α1-adrenergic receptor antagonists are phentolamine, prazosin, fentolamine mesylate, trazodone, alfuzosin, indoramine, naphthopidyl, tamsulosin, phenoxybenzamine, lauwalpa alkaloids, recordoda 15/2739 , SNAP 1069, SNAP 5089, RS 17053, SL 89.0591, doxazosin, terrazosin and abanoquil; Suitable α2-adrenergic receptor antagonists include dibenamine, tolazoline, trimazosine, eparoxane, yohimbine, idazoic acid clonidine and dibenamine; Suitable non-selective α-adrenergic receptor antagonists include dapiprazole; Other α-adrenergic receptor antagonists include 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 2,053,059, US 4,703,063, US 3,381,009 4,252,721 and US 2,599,000, each of which is incorporated herein by reference;

6) rapid onset selective serotonin such as, for example, 3-[(dimethylamino) methyl] -4- [4- (methylsulfanyl) phenoxy] benzenesulfonamide (disclosed in Example 28 of WO 01/72687) Reuptake inhibitors (fast onset SSRIs).

By cross-reference herein to compounds contained in the above patents and patent applications that may be used in accordance with the present invention, the inventors have indicated that the therapeutically active compounds and specific examples as defined in the claims (specifically claim 1) (All of which are incorporated herein by reference).

If a combination of active agents is administered, they may be administered simultaneously, separately or sequentially.

Co-Activator-PDE 5 Inhibitor

Suitable cGMP PDE5 inhibitors for use in accordance with the present invention include the following compounds:

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 WO 93/06104; The isomer pyrazolo [3,4-d] pyrimidin-4-ones disclosed in WO 93/07149; Quinazolin-4-ones disclosed in WO 93/12095; Pyrido [3,2-d] pyrimidin-4-ones disclosed in WO 94/05661; Purin-6-ones disclosed in WO 94/00453; Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in WO 98/49166; Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in WO 99/54333; Pyrazolo [4,3-d] pyrimidin-4-ones disclosed in EP-A-0995751; Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in WO 00/24745; Pyrazolo [4,3-d] pyrimidin-4-ones disclosed in EP-A-0995750; Compounds disclosed in WO 95/19978; Compounds disclosed in WO 99/24433; And compounds disclosed in WO 93/07124.

Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in WO 01/27112; Pyrazolo [4,3-d] pyrimidin-7-ones disclosed in WO 01/27113; The compounds disclosed in EP-A-1092718; And compounds disclosed in EP-A-1092719.

Preferred Type V phosphodiesterase inhibitors (= phosphodiesterase 5 (PDE) inhibitors; PDE5i) for use according to the invention include the following compounds:

1-[[3- (6,7-Dihydro-1-methyl-7-oxo-3-propyl-1 H-pyrazolo [4,3-d] pyrimidin-5-yl) -4-ethoxyphenyl 5- [2-ethoxy-5- (4-methyl-1-piperazinylsulfonyl) phenyl] -1-methyl-3-n-propyl-1, also known as] sulfonyl] -4-methylpiperazine , 6-dihydro-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] pyrimidine-7- On (see EP-A-0526004);

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 (+)-3-ethyl-5- [5- (4-ethylpiperazine, also known as -methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-one -1-ylsulfonyl) -2- (2-methoxy-1 (R) -methylethoxy) pyridin-3-yl] -2-methyl-2,6-dihydro-7H-pyrazolo [4,3 -d] pyrimidin-7-one (see WO 99/54333);

1- {6-Ethoxy-5- [3-ethyl-6,7-dihydro-2- (2-methoxyethyl) -7-oxo-2H-pyrazolo [4,3-d] pyrimidine- 5- [2-ethoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl]-also known as 5-yl] -3-pyridylsulfonyl} -4-ethylpiperazine. 3-ethyl-2- [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 WO 01/27113, Example 66);

5- (5-acetyl-2-propoxy-3-pyridinyl) -3-ethyl-2- (1-isopropyl-3-azetidinyl) -2,6-dihydro-7H-pyrazolo [4 , 3-d] pyrimidin-7-one (see WO 01/27112, Example 124);

5- (5-acetyl-2-butoxy-3-pyridinyl) -3-ethyl-2- (1-ethyl-3-azetidinyl) -2,6-dihydro-7H-pyrazolo [4, 3-d] pyrimidin-7-one (see WO 01/27112, Example 132);

(6R, 12aR) -2,3,6,7,12,12a-hexahydro-2-methyl-6- (3,4-methylenedioxyphenyl) -pyrazino [2 ', 1': 6,1 ] Pyrido [3,4-b] indole-1,4-dione (IC-351), ie the compounds of Examples 78 and 95 of WO 95/19978, and Examples 1, 3, 7 and Compound of 8;

1-[[3- (3,4-Dihydro-5-methyl-4-oxo-7-propylimidazo [5,1-f] -as-triazin-2-yl) -4-ethoxy 2- [2-ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl] -5-methyl-, also known as phenyl] sulfonyl] -4-ethylpiperazine 7-propyl-3H-imidazo [5,1-f] [1,2,4] triazin-4-one (vardenafil), ie Examples 20, 19, 337 and 336 of WO 99/24433 Compound of; And

The compound of Example 11 (EISAI) in International Patent Publication WO 93/07124; And

Rotella D P, J. Med. Chem., 2000, 43, 1257; and 3 and 14.

Other types of cGMP PDE5 inhibitors that may be used with the present invention include the following compounds: 4-bromo-5- (pyridylmethylamino) -6- [3- (4-chlorophenyl) -propoxy ] -3 (2H) pyridazinone; 1- [4-[(1,3-benzodioxol-5-ylmethyl) amino] -6-chloro-2-quinozolinyl] -4-piperidine-carboxylic acid, monosodium salt; (+)-Cis-5,6a, 7,9,9,9a-hexahydro-2- [4- (trifluoromethyl) -phenylmethyl-5-methyl-cyclopent-4,5] imidazo [ 2,1-b] purin-4 (3H) one; Furazlocillin; Cis-2-hexyl-5-methyl-3,4,5,6a, 7,8,9,9a-octahydrocyclopent [4,5] -imidazo [2,1-b] purin-4-one ; 3-acetyl-1- (2-chlorobenzyl) -2-propylindole-6-carboxylate; 3-acetyl-1- (2-chlorobenzyl) -2-propylindole-6-carboxylate; 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) pyrimidine- 7-one; 1- [4-[(1,3-benzodioxol-5-ylmethyl) amino] -6-chloro-2-quinazolinyl] -4-piperidinecarboxylic acid, monosodium salt; Pharmaprojects No. 4516 (Glaxo Wellcome); Pharmaprojects No. 5051 (Bayer); Pharmaprojects No. 5064 (Kyowa Hakko; see WO 96/26940); Pharmaprojects No. 5069 (Schering Plough); GF-196960 (Glaxo Welcome); E-8010 and E-4010 (Eisai); Bay-38-3045 & 38-9456 (Bayer) and Sch-51866.

Easily determine the suitability of the cGMP PDE5 inhibitor by assessing the efficacy and selectivity of any particular cGMP PDE5 inhibitor using the methods described in the literature and then evaluating its toxicity, absorption, metabolism, pharmacokinetics, etc. according to standard pharmaceutical practice. Can be.

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

IC50 values of cGMP PDE5 inhibitors can be determined using the PDE5 assay in the test method paragraph below.

Preferably, the cGMP PDE5 inhibitor used in the pharmaceutical combination according to the invention is selective for the PDE5 enzyme. Preferably, they have a selectivity of PDE5 that is at least 100, more preferably at least 300, greater than PDE3. More preferably, PDE5 has a selectivity of at least 100, more preferably at least 300, greater than PDE3 and PDE4.

Selectivity ratios can be readily determined by one skilled in the art.

It is to be understood that the contents of the published patent applications, in particular the general formulas and compounds exemplified therein, are incorporated herein by reference.

Spongy

As used herein, the term "cavernous body" refers to a mass of tissue that is found in the penis itself. In this regard, the penis body consists of three cylindrical tissue masses, each of which is surrounded by fibrous tissue called the white membrane. Paired posterior peripheral masses are called corpus cavernosum (corpora = major body; cavernosa = hollow); The smaller central mass, the urethral cavernous penis, contains the cavernous urethra and functions to keep the cavernous urethra open during ejaculation. All three clumps are surrounded by fascia and skin and consist of erectile tissue penetrated by blood cavities. The cavernous body contains smooth muscle cells.

circumstances

The assessment includes two separate components, namely, tack and injection. A spermatozoa is the semen and sperm from the distal epididymis, the lens tube, the seminal vesicles and the prostate into the prostate urethra. After this drive, the contents of semen from the urethral cavity are strongly pressed and released. Ejaculation is different from orgasm, a purely cerebral event. Often these two processes occur simultaneously.

Penis erection

As used herein, the term "penis erection" refers to a condition in which an artery supplied to the penis is expanded and a large amount of blood enters the blood cavity when stimulated, which may be visual, tactile, auditory, olfactory or imaginary Refers to. Expansion of these spaces compresses the veins exiting the penis, thus slowing blood outflow. These vascular changes due to parasympathetic reflexes cause an erection. When the arteries are constricted and the blood pressure in the veins is reduced, the penis returns to its relaxed state.

Smooth muscle

As used herein, the term "smooth muscle" refers to a tissue specific for contraction, consisting of smooth muscle fibers (cells) located on the walls of hollow internal organs and governed by autonomic motor neurons. The term "smooth muscle" means muscles that have no pattern and thus have a smooth appearance. It is also called involuntary muscle. When the Ca ²⁺ concentration in the smooth muscle cytoplasm increases, contraction begins, as in rhabdomyolysis. However, smooth muscle lacks the cytoplasmic reticulum (a storage container for Ca ²⁺ in rhabdomyo). Calcium ions enter the smooth muscle cytoplasm from extracellular fluid and myoplasmic reticulum, but since smooth muscle fibers lack transverse tubules, Ca ²⁺ takes longer to reach the fiber-centric filaments and trigger the contraction process. This is partly the reason for the contraction of smooth muscle, which starts slowly and lasts long.

Contraction and relaxation

Several mechanisms regulate the contraction and relaxation of smooth muscle cells. In one mechanism, a regulatory protein called calmodulin binds to cellular Ca ²⁺ . Calcium ions not only slowly enter smooth muscle fibers but also slowly exit muscle fibers when excitement is reduced, thus delaying relaxation. Prolonged presence of Ca ²⁺ in the cytoplasm provides smooth muscle tension, a state of partial partial contraction. Smooth muscle tissue is located in the walls of hollow internal organs such as blood vessels, airways to the lungs, stomach, gallbladder, bladder, cavernous body of the penis and clitoris.

cure

Reference to treatment herein should be understood to include one or more treatment, alleviation and prophylactic treatments.

Sexual stimulation

The invention also encompasses the use as defined above, via the administration of selective oxytocin antagonists (and co-activators, if applicable) prior to and / or during sexual stimulation. Here, the term "sexual stimulation" may have the same meaning as "sexual excitement." This aspect of the invention is advantageous because it provides systemic (physiological) selectivity.

Thus, according to the invention, it is highly desirable that there is a sexual stimulation step in some steps. Here, the "sexual stimulus" may be one or more of a visual stimulus, a physical stimulus, an audio stimulus, or an imaginary stimulus.

drug

Drugs for use in treating male ejaculation disorders, in particular premature ejaculation, in accordance with the present invention may be any suitable drug that can act as a combination of a selective oxytocin antagonist and, where appropriate, a selective oxytocin antagonist and a co-active agent as previously described in detail. Can be. As used herein, the term "drug" includes any substance that can selectively inhibit the oxytocin receptor.

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

Thus, the term "drug" includes, but is not limited to, a compound that can be obtained or produced from any suitable source, whether natural or not.

The drug can be designed or obtained from a library of compounds that can include peptides and other compounds such as small organic molecules such as lead compounds.

By way of example, a drug may be a natural substance, biological macromolecule, or extract, organic or inorganic molecule, synthetic drug, semisynthetic drug, structural or functional analogue, peptide prepared from organisms such as bacteria, fungi or animal (especially mammalian) cells or tissues. , Peptide analogs, derived drugs, peptides synthesized from whole proteins or peptides synthesized by synthetic methods (eg, using peptide synthesizers or by recombinant techniques, or by a combination thereof), recombinant drugs, antibodies, natural Or non-natural drugs, fusion proteins or equivalents thereof, mutants, derivatives or combinations thereof.

The term "drug" as used herein may be a single substance or may be a combination of drugs.

If the drug is an organic compound, in some applications the organic compound may typically comprise two or more linked hydrocarbyl groups. In some applications, preferably the drug comprises two or more cyclic groups (optionally one may be a cyclic ring structure in which one of these cyclic groups is fused). In some applications, at least one of the cyclic groups is a heterocyclic group. In some applications, the heterocyclic group preferably contains one or more N in the ring. Examples of such compounds are described herein.

The drug may contain one or more alkyl, alkoxy, alkenyl, alkylene and alkenylene groups which may be non-branched or branched.

substitution

For the avoidance of doubt, unless otherwise indicated, the term substituted means substituted with one or more defined groups. If a group can be selected from a number of other groups, the selected groups can 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, these substituents may be the same or different.

Pharmaceutically acceptable salts

The drug may be in the form of a solvate, including pharmaceutically acceptable salts such as acid addition salts or base salts or hydrates thereof, and / or may be administered in such form. For a review of suitable salts, see Berge et al., J. Pharm. Sci., 1977, 66 , 1-19.

Typically, pharmaceutically acceptable salts can be readily prepared by using the desired acid or base, if appropriate. The salt may precipitate out of solution and be collected by filtration or may be recovered by evaporation of the solvent.

Suitable acid addition salts are prepared from acids which form non-toxic salts, for example hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, Lactate, tartrate, citrate, gluconate, succinate, saccharide, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p -toluenesulfonate and pamoate salts.

Suitable base salts are formed from bases which produce non-toxic salts, for example sodium, potassium, aluminum, calcium, magnesium, zinc and diethanolamine salts.

Polymorph (s) / Asymmetric Carbon (s)

Drugs can exist in polymorphs.

The drug may contain one or more asymmetric carbon atoms and therefore exist in two or more stereoisomeric forms. If the drug contains alkenyl or alkenylene groups, the cis (E) and trans (Z) isomers may occur. The present invention includes the individual stereoisomers of the drugs, and, if appropriate, their individual tautomeric forms, with mixtures thereof.

By conventional techniques, diastereomers or cis isomers and trans isomers can be separated, for example, by fractional crystallization, chromatography or H.P.L.C. of stereoisomer mixtures of the drug or a suitable salt or derivative thereof. The individual enantiomers of the drug can be separated from the corresponding optically pure intermediates, or by cleavage such as by HPLC of the corresponding racemates using a suitable chiral support, or the reaction of the corresponding racemates with a suitable optically active acid or base. It can be prepared by fractional crystallization of diastereomeric salts formed by

Isotope change

The invention also encompasses all suitable isotopic variants of the drug or pharmaceutically acceptable salts thereof. Isotope variants of the drugs of the present invention or pharmaceutically acceptable salts thereof are defined as being replaced with atoms having one or more atoms having the same atomic number but having an atomic mass different from the atomic mass commonly found in nature. Examples of isotopes that may be incorporated into the drug and its pharmaceutically acceptable salts include ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, Isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine such as ¹⁸ F and ³⁶ Cl. The incorporation of certain isotope variants of the drug and its pharmaceutically acceptable salts, such as radioactive isotopes such as ³ H or ¹⁴ C, is useful for pharmaceutical and / or matrix tissue distribution studies. Tritium (ie ³ H) and carbon-14 (ie ¹⁴ C) isotopes are particularly preferred because of their ease of preparation and detection. In addition, substitution with an isotope such as deuterium (ie ² H) may provide certain therapeutic benefits due to greater metabolic stability, eg, increased in vivo half-life or reduced dosage requirements, and therefore in some situations It may be desirable. Isotope variants of the drug and its pharmaceutically acceptable salts can be conventionally prepared by conventional procedures using appropriate isotopic variants of suitable reagents.

Prodrug

Those skilled in the art will appreciate that the drug may be derived from prodrugs. Examples of prodrugs have specific protected group (s) and do not have pharmacological activity on their own but in certain circumstances will be metabolized in the body after being administered (eg oral or parenteral) to form a pharmacologically active drug. It can contain substances that can be.

Pro-moiety

Certain residues, known as “progenitor-residues” as described, for example, in Bundgaard (“Design of Prodrugs”, Elsevier, 1985), the contents of which are hereby incorporated by reference, may be used as appropriate functional groups of the drug. You will also find that it can be located at. Such prodrugs are also included within the scope of the present invention.

Inhibitors / antagonists

The term antagonist as used herein in connection with an optional oxytocin antagonist should be considered to be compatible with the term inhibitor. Likewise, the term inhibitor used herein in connection with, for example, a previously described auxiliary active agent (such as a PDEi or PDE5i compound, where applicable) should be considered to be compatible with the term antagonist.

As used herein, the term “antagonist” means any drug that reduces the action of another drug or target. Antagonism is competition (indirect antagonism) of binding sites associated with the binding of antagonistic components (chemical antagonism) or the generation of opposite effects through different targets (functional antagonism or physiological antagonism) or target activation to the observed effect. Action).

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the drug of the invention and a pharmaceutically acceptable carrier, diluent or excipient, including combinations thereof.

The pharmaceutical composition may be for human or animal use in human and veterinary medicaments and typically comprises one or more pharmaceutically acceptable diluents, carriers or excipients. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (edited by A. R. Gennaro, 1985). The choice of pharmaceutical carrier, excipient, or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition comprises any suitable binder (s), lubricant (s), suspending agent (s), coating agent (s), solubilizer (s) as a carrier, excipient or diluent, or in addition to the carrier, excipient or diluent. can do.

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

There may be different composition / mixing conditions depending on different delivery systems. By way of example, the pharmaceutical compositions of the present invention may be prepared using a mini-pump or by, for example, nasal sprays or aerosols or ingestible solutions for inhalation, by the mucosal route, or for example in the intravenous, intramuscular or subcutaneous route. Formulated for delivery by a parenteral route that is formulated in an injectable form for delivery by. Alternatively, the formulation may be designed to be delivered by both routes.

If the drug is delivered through the gastrointestinal mucosa, it must be able to remain stable during transport through the gastrointestinal tract. For example, the drug must be resistant to degradation due to proteolysis, stable at acidic pH, and resistant to the detergent effect of bile.

Where appropriate, the pharmaceutical composition contains excipients such as starch or lactose, by inhalation, in the form of suppositories or pessaries, locally in the form of lotions, solutions, creams, ointments or spray powders, using skin patches. Or capsules or ovules in the form of tablets or actives alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents, or they are parenteral, such as intravenous, muscle It can be injected intravenously or subcutaneously. For parenteral administration, the composition may best be used in the form of a sterile aqueous solution which may contain sufficient salt or monosaccharides to make other components, for example, the solution isotonic with blood. For lingual or sublingual administration, the compositions may be administered in the form of tablets or lozenges which may be formulated in a conventional manner.

In some embodiments, the drugs of the present invention can also be used with cyclodextrins. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of the drug-cyclodextrin complex can alter the solubility, dissolution rate, bioavailability and / or stability of the drug molecule. Drug-cyclodextrin complexes are usually useful for most dosage forms and administration routes. As an alternative to direct complexation with the medicament, the cyclodextrin may be used as an auxiliary additive such as a carrier, diluent or solubilizer. 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. .

In a preferred embodiment, the drug of the invention is delivered systemically (eg oral, buccal, sublingual), more preferably orally.

Thus, preferably the drug is in a form suitable for oral delivery.

administration

The term "administration" includes delivery by viral or non-viral techniques. Virus delivery mechanisms include, but are not limited to, adenovirus vectors, adenovirus-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentiviral vectors, and baculovirus vectors. Non-viral delivery mechanisms include lipid mediated transfections, liposomes, immune liposomes, lipofectin, cationic facial amphiphilic compounds (CFAs), and combinations thereof.

The drug of the present invention may be administered alone, but is generally administered as a pharmaceutical composition when the drug is mixed with a suitable pharmaceutical excipient, diluent or carrier, for example, selected in connection with the intended route of administration and standard pharmaceutical practice.

For example, the drug may contain tablets, capsules, orbs, elixirs, which may contain flavoring or coloring agents for immediate-release, delayed-release, modified-release, sustained-release, pulse-release or controlled-release applications. It may be administered in the form of a solution or suspension (eg oral or topical).

Tablets may include excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; Disintegrants such as starch (preferably corn starch, potato starch or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates; And granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and gum arabic. Lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may also be included.

Solid compositions of a similar type may also be used as fillers for gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactose or high molecular weight polyethylene glycols. In the case of aqueous suspensions and / or elixirs, the drug may be mixed with various sweetening or flavoring agents, colorants or dyes, emulsifiers, suspending agents and diluents such as water, ethanol, propylene glycol and glycerine, and combinations thereof. Can be.

The route of administration (delivery) can be oral (eg, as a tablet, capsule or ingestible solution), topical, mucosal (eg, as a nasal spray or inhalation aerosol), nasal, parenteral (eg, by injectable form). , Gastrointestinal tract, spinal cord, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intraventricular, intracranial, subcutaneous, eye (intravitreal or anterior), transdermal, rectal , But not limited to, buccal, penis, vagina, epidural, sublingual.

Note that not all drugs need to be administered by the same route. Likewise, if the composition comprises more than one active ingredient, these ingredients can be administered by different routes.

When the drug of the present invention is administered parenterally, examples of such administration include administering the drug intravenously, intraarterally, intraperitoneally, intradural, intraventricular, urethra, intestinal, intracranial, intramuscular or subcutaneously. One or more of using an injection technique.

For parenteral administration, the drug is best used in the form of a sterile aqueous solution which may contain sufficient salt or glucose to make other components, for example, the solution isotonic with blood. The aqueous solution should be suitably buffered (preferably up to pH 3-9) if necessary. Suitable parenteral formulations are readily prepared under sterile conditions by standard pharmaceutical techniques well known to those skilled in the art.

As indicated, the drugs of the present invention may be administered intranasally or by inhalation, and may be administered with a dry powder inhaler, or with a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro Lutein, hydrofluoroalcaine (e.g., 1,1,1,2-tetrafluoroethane (HFA 134A®) or 1,1,1,2,3,3,3-heptafluoropropane ( HFA 227EA®), conveniently delivered in the form of aerosol sprays from pressurized containers, pumps, sprays or nebulizers using carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a weighed amount. Pressurized vessels, pumps, sprays or nebulizers may contain a solution or suspension of the active compound, for example using a mixture of ethanol and propellant as solvent, which may further contain a lubricant, for example sorbitan trioleate. Can be. Capsules and cartridges (such as made of gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mixture of the drug and a suitable powder base (eg lactose or starch).

Alternatively, the drugs of the present invention may be administered in the form of suppositories or pessaries, or they may be administered locally in the form of gels, hydrogels, lotions, solutions, creams, ointments or spray powders. Drugs of the invention can also be administered dermal or transdermal, such as by using skin patches. They may also be administered by the pulmonary or rectal route. They may also be administered by the eye route. For ophthalmic use, the compounds may optionally be formulated with a preservative such as benzylalkonium chloride as a finely divided suspension in isotonic pH adjusted sterile saline, or preferably as a solution in isotonic pH adjusted sterile saline. Alternatively, they may be formulated in an ointment such as petrolatum.

When topically administered to the skin, the drugs of the present invention are suspended or dissolved in, for example, mineral oils, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes and mixtures with one or more of water. It can be formulated as a suitable ointment containing the compound. Alternatively, it may be, for example, one or more of mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water It can be formulated as a suitable lotion or cream suspended or dissolved in the mixture.

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

In some uses, the drug is preferably administered orally.

In some applications, the drug is preferably administered locally.

Dose level

Typically, the physician will determine the actual dosage that is best suited for the individual patient. Dosage levels and frequency of administration specific for any particular subject may vary and include the activity, metabolic stability, and time of action, age, weight, general health, sex, diet, mode of administration of the particular compound employed, It depends on a variety of factors including the time of administration, the rate of secretion, the combination of the drugs, the severity of the particular disease, and the therapy that proceeds individually. Drugs and / or pharmaceutical compositions of the invention may be administered according to a dosing schedule from 1 to 10 times per day (eg, once or twice per day).

For oral and parenteral administration to humans, the daily dose level of the drug may be single or divided doses.

If desired, the drug may be administered at a dosage of 0.01 to 30 mg / kg body weight, such as 0.1 to 10 mg / kg body weight, more preferably 0.1 to 1 mg / kg body weight. Naturally, the dosages mentioned herein are examples of average cases. Of course, there may be individual cases where higher or lower dosages are advantageous.

Typically, the daily oral dosage can be, for example, 20 to 1000 mg, preferably 50 to 300 mg.

Formulation

Drugs of the present invention may be formulated into pharmaceutical compositions by mixing with one or more of one or more suitable carriers, diluents or excipients using techniques known in the art.

Below are some non-limiting examples of formulations.

Formulation 1: A tablet is prepared using the following ingredients.

The ingredients are blended and compressed to form tablets weighing 665 mg each.

Formulation 2: Intravenous formulations can be prepared as follows:

individual

The term "individual" as used herein refers to a member of a vertebrate, in particular a mammal. The term includes, but is not limited to, livestock, race animals, primates, and humans.

Bioavailability

Preferably, the compounds (and combinations) of the invention are administered orally for use in vivo. Bioavailability after oral administration refers to the proportion of orally administered agents that are systemically circulated. Factors that determine bioavailability after oral administration of a medicament are solubility, membrane permeability and metabolic stability. Typically, bioavailability after oral administration is first determined using a series of screening methods using in vitro techniques followed by in vivo techniques.

Dissolution, solubilization of the medicament by the aqueous contents of the gastrointestinal tract (GIT) can be predicted from in vitro solubility experiments performed at an appropriate pH to mimic GIT. Preferably, the compound of the present invention has a minimum solubility of 50 mg / ml. Solubility can be determined by standard procedures known in the art as described in Adv. Drug Deliv. Rev. 23, 3-25, 1997.

Membrane permeability refers to the passage of a compound through the cells of GIT. Lipophilic is a key characteristic in predicting this and is defined as an in vitro Log D _7.4 measurement with organic solvents and buffers. Preferably, the compounds of the present invention have a Log D _7.4 of -2 to +4, more preferably -1 to +2. Log D can be determined by standard procedures known in the art, such as those described in J. Pharm. Pharmacol. 1990, 42: 144.

Cellular membrane assays such as CaCO ₂ substantially help to predict the desired membrane permeability, so called caco-2 flux, in the presence of efflux transporters such as p-glycoproteins. Preferably, the compounds of the present invention have caco-2 fluxes greater than 2 × 10 ⁻⁶ cms ⁻¹ , more preferably greater than 5 × 10 ⁻⁶ cms ⁻¹ . The caco flux value can be determined by standard procedures known in the art, such as those described in J. Pharm. Sci. 1990, 79, 595-600.

Metabolic stability deals with GIT or liver's ability to metabolize compounds during the absorption process: first pass effect of the drug. Analytical systems, such as microsomal bodies and hepatocytes, predict metabolic charges. Preferably, the compounds of the examples exhibit metabolic stability in an assay system in which liver extracts of less than 0.5 are suitable. Examples of assay systems and data manipulations are described in Curr. Opin. Drug Disc. Devel., 201, 4, 36-44, Drug Met. Disp., 2000, 28, 1518-1523.

Because of the interaction of these processes, in vivo experiments in animals can provide additional evidence that the medicament is efficient in vivo after oral administration to humans. Absolute bioavailability is determined in these studies by administering the compounds by oral route either separately or in combination. Intravenous routes are also used to obtain absolute determinations (% absorption). Examples of methods for assessing bioavailability after oral administration in animals are described in Drug Met. Disp., 2001, 29, 82-87; J. Med Chem., 1997, 40, 827-829; Drug Met.Disp. , 1999, 27, 221-226).

Chemical synthesis method

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

Agents or targets, or variants, homologues, derivatives, segments or analogs thereof, may be prepared in whole or in part using chemical methods of synthesizing drugs. For example, proteins can be synthesized by solid phase techniques, cleaved from resins, 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 synthetic peptides can be confirmed by amino acid analysis or sequencing (eg, Edman degradation procedure; Creighton, supra).

Various solid phase techniques (Roberge JY et al., (1995) Science 269: 202-204) can be used to directly synthesize a drug, or a variant, homologue, derivative, segment or analog thereof, such as provided by a manufacturer. Automated synthesis can be accomplished using an ABI 43 1 A peptide synthesizer (Perkin Elmer) according to the instructions. In addition, amino acid sequences comprising a drug or any portion thereof can be changed during direct synthesis and / or combined with sequences from other subunits or any portion thereof using chemical methods, thereby modifying the drug or target, eg For example, modified oxytocin receptors.

In another embodiment of the invention, chemical methods well known in the art (Caruthers MH et al., (1980), Nuc Acids Res Symp Ser 214-23; Horn T et al., (1980), Nuc Acids Res Symp Ser 225-232) can be used to fully or partially synthesize coding sequences of drug targets or variants, homologues, derivatives, segments or analogs thereof.

Analogue

As used herein, the term “analogue” includes, but is not limited to, peptides, polypeptides, antibodies or other organic compounds that have the same activity or effect as related drugs (ie, selective oxytocin antagonists) to the target (ie oxytocin receptor). It relates to any chemical that is not limited. In other words, the analog may be functionally identical to a known drug.

Chemical derivatives

As used herein, the term "derivative" or "derived" includes chemical modification of the drug. Examples of such chemical modifications may be the replacement of hydrogen with a halo, alkyl, acyl or amino group.

Chemical modification

In one embodiment of the invention, the drug may be a chemically modified drug.

Chemical modification of the drug may enhance or reduce hydrogen bond interactions, charge interactions, hydrophobic interactions, Van Der Waals interactions or dipole interactions between the drug and the target.

In one embodiment, the identified drugs can act as models (eg, templates) in developing other compounds.

Target

In one embodiment of the invention, the oxytocin receptor can be used as a target in screening for identifying drugs that can inhibit the oxytocin receptor. In this regard, the target may comprise the amino acid sequence shown in SEQ ID NO: 1, or variants, homologs, derivatives or fragments thereof produced by recombinant and / or synthetic means, or an expression comprising them.

In another embodiment of the present invention, both oxytocin receptors and vasopressin (particularly V1a) receptors can be used as targets in screening to identify drugs that can selectively inhibit oxytocin receptors. In this regard, the oxytocin receptor target may comprise the amino acid sequence shown in SEQ ID NO: 1, or variants, homologs, derivatives or fragments thereof, prepared by recombinant and / or synthetic means, or an expression comprising them The vasopressin receptor target may comprise the amino acid sequence shown in SEQ ID NO: 2, or variants, homologs, derivatives or fragments thereof produced by recombinant and / or synthetic means, or an expression comprising them.

Alternatively, oxytocin receptors and / or vasopressin receptors (preferably V1a receptors) can be used as targets for identifying drugs that can mediate ejaculation potential increase through selective inhibition of oxytocin receptors. In this regard, the target may be a suitable tissue extract.

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

Recombination Method

Drugs and / or targets of the invention can be prepared by recombinant DNA techniques.

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

Amino acid sequence

The term "amino acid sequence" as used herein is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term “amino acid sequence” is synonymous with the term “peptide”. In some cases, the term “amino acid sequence” has the same meaning as “protein”.

Amino acid sequences can be isolated and prepared from a suitable source, or they can be synthesized, or they can be prepared using recombinant DNA techniques.

In one aspect, the invention provides amino acid sequences that can act as targets (ie, oxytocin receptors or vasopressin (preferably V1a) receptors) in assays to identify one or more drugs and / or derivatives thereof.

In a second aspect, the present invention provides an amino acid sequence which is a drug capable of selectively inhibiting an oxytocin receptor.

Preferably, the target is an oxytocin receptor.

Preferably, the oxytocin receptor and / or vasopressin (preferably Vla) receptor are isolated receptors and / or are not purified and / or naturally occurring.

The oxytocin receptor or vasopressin (preferably Vla) receptor of the invention may be in substantially isolated form. It will be appreciated that the oxytocin receptor or vasopressin receptor can be mixed with a carrier or diluent that is likewise considered substantially isolated without interfering with the intended purpose of the receptor and / or drug. The oxytocin receptor or vasopressin receptor of the present invention may be in substantially pure form, in which case generally at least 90%, such as 95%, 98% or 99%, of the oxytocin receptor or vasopressin receptor in the formulation is in SEQ ID NO: 1, respectively. Oxytocin receptors or vasopressin receptors in a formulation which is a peptide having the amino acid sequence shown or variants, homologs, derivatives or fragments thereof, or the amino acid sequence shown in SEQ ID NO: 2 or variants, homologs, derivatives or fragments thereof.

Nucleotide sequence

The term "nucleotide sequence" as used herein, has the same meaning as the term "polynucleotide".

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

In some applications, preferably the nucleotide sequence is DNA.

In some applications, preferably, nucleotide sequences are prepared by using recombinant DNA techniques (eg, recombinant DNA).

In some applications, preferably the nucleotide sequence is cDNA.

In some applications, preferably the nucleotide sequence may be identical to that naturally occurring.

In one aspect, the present invention provides nucleotide sequences encoding components that can serve as targets in assays for identifying one or more drugs and / or derivatives thereof.

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

In another embodiment of the invention, the nucleotide sequence encodes a vasopressin receptor, preferably the Vla receptor.

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

One of ordinary skill in the art will appreciate that many different nucleotide sequences may have the same target due to the degeneracy of the genetic code (i.e., oxytocin receptor, e.g., oxytocin receptor, or vasopressin receptor, such as SEQ ID NO: SEQ ID NO: 1). It will be appreciated that the vasopressin receptor comprising the amino acid sequence shown in Fig. 2) can be encoded. In addition, those skilled in the art, using routine techniques, produce nucleotide substituents that do not substantially affect the activity encoded by the nucleotide sequence, reflecting the codon usage of any particular host organism to which the target should be expressed. You will know that you can. Thus, the term “variant”, “homolog” or “derivative” in relation to a nucleotide sequence is a nucleotide sequence produced by the optional substitution, modification, alteration, replacement, deletion or addition of one or more nucleic acids from or into a sequence. To encode a functional target according to the invention (ie oxytocin receptor) (or even a drug according to the invention, if the drug comprises a nucleotide sequence or an amino acid sequence).

Variant / homolog / derivative

In addition to the specific amino acid sequences mentioned herein, the invention also encompasses the use of variants, homologs and derivatives thereof. Here, the term “homology” can be equated with “identity”.

In this regard, the cognate sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90%, preferably at least 95 or 98% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In particular, homology should typically be considered in terms of regions of sequence known to be essential for activity. Homology should be considered in terms of similarity (ie, amino acid residues with similar chemical properties / functions), but in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be performed with the naked eye, or more commonly, homology comparisons can be performed using readily available sequence comparison programs. These commercial computer programs can calculate% homology between two or more sequences.

% Homology can be calculated on consecutive sequences. That is, after aligning one sequence with another sequence, each amino acid of one sequence is directly compared with the corresponding amino acid of the other sequence (one residue at a time). This is called an "ungapped" alignment. Typically, such gapless alignment is performed only on relatively few residues.

This is a very simple and consistent method, but in other words, for example, in the same sequence pair, the subsequent amino acid residues are not aligned by one insertion or deletion, resulting in a significant reduction in percent homology when aligned as a whole. It does not consider the possibility. As a result, most sequence comparison methods are designed to optimally align in view of possible insertions and deletions without excessively damaging the overall homology scores. This is accomplished by putting "gaps" in the sequence alignment to optimize local homology.

However, these more complex methods impart a “gap penalty” to each gap that occurs during alignment, so that, for the same number of identical amino acids, sequence alignment with as few gaps as possible (more than between two sequences being compared) Reflecting high relevance) achieves higher grades than having many gaps. “Related gap loss” is typically used, which bears a relatively high loss for the presence of a gap and erases a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. The high gap penalty will of course be optimally aligned to have fewer gaps. Most alignment programs allow you to adjust the gap penalty. However, it is desirable to use default values when using such software for sequence comparison. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalty for the amino acid sequence is -12 for the gap and -4 for each extension.

Therefore, the calculation of the maximum homology% needs to be optimally aligned first taking into account the gap penalty. A suitable computer program for this sorting is the GCG Wisconsin Best Fit Package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include the BLAST package (Ausubel et al., 1999, see chapter 18 of the same document), Pasta (Atschul et al., 1990, J. Mol) Biol., 403-410) and GENEWORKS suites of comparison means. Blasts and pasta are available for offline and online searches (see Osbel et al., 1999, same document, pages 7-58 to 7-60). However, it is desirable to use the GCG Best Fit program. A new means called Blast 2 Sequences can also be used to compare protein and nutotide sequences (FEMS Microbiol Lett 1999 174 (2): 247-50; FEMS Microbiol Lett 1999 177 (1): 187-). 8 and tatiana@ncbi.nlm.nih.gov).

The final percent homology can be measured in terms of identity, but the alignment process itself is typically not based on all-or-nothing pair comparisons. Instead, a graded similarity score matrix is generally used, which scores each pair comparison based on chemical similarity or evolution distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix-the default matrix for the blast suite of programs. GCG Wisconsin programs typically use public default values or custom code comparison tables, if supplied (see the user manual for further details). It is advisable to use public default values for the GCG package, or default matrix such as BLOSUM62 for other software.

Once the software has generated the optimal alignment,% homology, preferably% sequence identity, can be calculated. Software typically does this as part of sequence comparisons and produces numerical results.

The sequence may have deletions, insertions or substitutions of amino acids that result in silent changes and functionally produce the same substance. Careful amino acid substitutions can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residue, as long as the incidental binding activity of the substance is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid; Positively charged amino acids include lysine and arginine; Amino acids having uncharged polar head groups with similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

For example, conservative substitutions can be made according to the table below. Amino acids in the same block of the second column and preferably in the same row of the third column may be substituted for one another:

The present invention also encompasses cognate substitutions where similar ones are substituted such as basic to basic, acid to acid, polar to polar, etc. (substitution and substitution herein means that the present amino acid residues are interchanged with other residues). do. Residues of one class are substituted with residues of another class or ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O) Non-cognate substitutions may also be made, including non-natural amino acids such as pyridylalanine, thienylalanine, naphthylalanine and phenylglycine.

Halide derivatives of natural amino acids such as alpha ^* and alpha-disubstituted ^* amino acids, N-alkyl amino acids ^* , lactic acid ^* , trifluorotyrosine ^* , p-Cl-phenylalanine ^* , p-Br-phenylalanine ^* , pI-phenylalanine ^* , L-allyl-glycine ^* , β-alanine ^* , L-α-aminobutyric acid ^* , L-γ-amino butyric acid ^* , L-α-amino isobutyric acid ^* , L-ε-amino caproic acid ^# , 7 -Amino heptanoic acid ^* , L-methionine sulfone ^{# *} , L-norleucine ^* , L-novalin ^* , p-nitro-L-phenylalanine ^* , L-hydroxyproline ^# , L-thioproline ^* , 4- Methyl derivatives of phenylalanine (Phe), such as methyl-Phe ^* , pentamethyl-Phe ^* , L-Phe (4-amino) ^# , L-Tyr (methyl) ^* , L-Phe (4-isopropyl) ^* , L- May be replaced by non-natural amino acids including Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) ^* , L-diaminopropionic acid ^# and L-Phe (4-benzyl) ^* . The * mark was used in the above discussion (for cognate or non-cognate substitutions) to indicate the hydrophobicity of the derivative, while # was used to indicate the hydrophilicity of the derivative and # * indicates amphotericity.

Modified amino acid sequences may include suitable spacer groups (including alkyl groups such as methyl, ethyl or propyl groups) that can be inserted between any two amino acid residues of the sequence in addition to amino acid spacers (eg glycine or β-alanine residues). . Other types of modifications include the presence of one or more amino acid residues in peptoid form and are well known to those skilled in the art. To avoid uncertainty, “peptoid form” is used to refer to modified amino acid residues in which the α-carbon substituent is on the nitrogen atom of the residue rather than the α-carbon. Methods for preparing peptoid-type peptides are known in the art (eg, Simon RJ et al., PNAS (1992) 89 (20), 9367-9371; and Harwell DC, Trends Biotechnol. (1995) 13 (4), 132-134.

Hybrid formation

As used herein, the term "hybrid formation" includes "a process in which a nucleic acid strand is joined to a complementary strand through base pairing" and an amplification process performed by a polymerase chain reaction (PCR) technique.

Nucleotide sequences encoding amino acid sequences of the present invention, or nucleotide sequences capable of selectively hybridizing with their complementary sequences, are usually at least 20, preferably at least 25 or at least 30, such as at least 40, 60 At least 75%, preferably at least 85% or at least 90%, more preferably at least 95% relative to the corresponding complementary nucleotide sequence encoding an amino acid sequence set forth herein over at least or over a region of at least 100 consecutive nucleotides Or 98% or more homologous.

The term "optionally hybridizable" means that the nucleotide sequence used as the probe is used under conditions that the target nucleotide sequence has been found to hybridize with the probe to a level significantly higher than the background level. Background Hybridization can occur due to, for example, other nucleotide sequences present in the cDNA or genomic DNA libraries being searched for. In this case, the background represents the signal level generated by the interaction between the probe and the non-specific DNA member of the library, which is 10 times, preferably 100 times less potent than the specific interactions observed in the target DNA. The intensity of the interaction can be measured, for example, by radiolabeling the probe at ³² P, for example.

As taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego, Calif.), Hybridization conditions are described in terms of nucleic acid binding complexes. Based on the melting point (Tm), it gives the stringency described below.

Maximum stringency typically occurs at about Tm-5 ° C (5 ° C lower than the Tm of the probe); High stringency at about 5 ° C. to 10 ° C. below the Tm; Medium stringency at about 10 ° C. to 20 ° C. lower than Tm; Low stringency occurs at temperatures about 20 ° C. to 25 ° C. lower than Tm. As will be appreciated by those skilled in the art, maximal stringency hybridization can be used to identify or detect identical nucleotide sequences, while hybrid stringent to medium (or low) stringency to identify or detect similar or related polynucleotide sequences. Formation can be used.

In a preferred embodiment, the present invention provides a nucleotide encoding the amino acid sequence of the present invention under stringent conditions (eg, 65 ° C. and 0.1 × SSC {1 × SSC = 0.15} M NaCl, 0.015M Na ₃ citrate, pH 7.0). Nucleotide sequences capable of hybridizing with the sequence. Where the nucleotide sequence of the invention is double-stranded, each strand of the double strand is included in the invention both individually or together. If the nucleotide sequence is single-stranded, the complementary sequence of the nucleotide sequence is also included within the scope of the present invention.

Nucleotide sequences that are not 100% homologous to the nucleotide sequences encoding amino acid sequences of the invention but fall within the scope of the invention can be obtained in a number of ways. For example, rigorous exploration of DNA libraries made from multiple sources can yield other variants of the sequences described herein. It is also possible to obtain other virus / bacterial or cell analogs, in particular cell homologs found in mammalian cells (eg, rat, mouse, bovine and primate cells), and these analogs and their segments are generally described herein. It may optionally hybridize with the sequences shown in the sequence listing. These sequences can be obtained by strictly exploring cDNA libraries or genomic DNA libraries prepared from other animal species, and by exploring the libraries with probes comprising all or a portion of the nucleotide sequences described herein under conditions of medium to high stringency. Can be. Similar considerations apply to obtaining homologue and allelic variants of the amino acid and / or nucleotide sequences of the invention.

Variants and strains / homologs can also be obtained using degenerate PCR using primers designed to target sequences within homologs and variants encoding amino acid sequences that are conserved within the sequences of the invention. Conserved sequences can be predicted, for example, by aligning amino acid sequences from several variants / homologs. Sequences can be aligned using computer software known in the art. For example, the GCG Wisconsin FileUp program is widely used. Primers used for degenerate PCR contain one or more degenerate positions and are used at lower stringency conditions than those used to clone sequences into single sequence primers against known sequences.

Alternatively, such nucleotide sequences may be obtained by site induced mutation of a sequence in which features such as, for example, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 are determined. This may be useful if, for example, a silent codon change is required in the sequence to optimize codon priority for the particular host cell in which the nucleotide sequence is expressed. Other sequence changes may be necessary to introduce restriction enzyme recognition sites or to change the activity of the protein encoded by the nucleotide sequence.

Nucleotide sequences encoding amino acid sequences of the present invention are used to generate probes labeled with exposure labels by conventional means using primers such as PCR primers, primers for other amplification reactions, such as radioactive or non-radioactive labels. Or nucleotide sequences can be cloned into a vector. Such primers, probes, and other segments are at least 15, preferably at least 20, such as at least 25, at least 30, or at least 40 nucleotides in length and are included in the term nucleotide sequence of the present invention as used herein.

Nucleotide sequences, such as DNA polynucleotides and probes according to the present invention, can be prepared by recombinant methods, by synthesis, or by any means available to those skilled in the art. They may also be cloned by standard techniques.

In general, primers are prepared by synthetic means, including stepwise preparation of the desired nucleic acid sequence (one nucleotide at a time). Techniques for achieving this using automated techniques are readily available in the art.

Generally, recombinant means is used to generate longer nucleotide sequences, for example using PCR (polymerase chain reaction) cloning techniques. This produces a pair of primers (eg, about 15 to 30 nucleotides) that contiguous the region of the targeting sequence to be cloned, contacts the primers with mRNA or cDNA obtained from animal cells or human cells, and then the desired region. Performing polymerase chain reaction (PCR) under conditions that amplify, isolating the amplified segments (eg, by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. Primers can be designed that contain a suitable restriction enzyme recognition site so that the amplified DNA can be cloned into a suitable cloning vector.

Due to the inherent degeneracy of the genetic code, various DNA sequences that encode substantially identical or functionally identical amino acid sequences can be used to clone and express target sequences. As will be appreciated by those skilled in the art, for certain expression systems, it may be beneficial to generate target sequences with non-naturally occurring codons. Codons preferred by particular prokaryotic hosts or eukaryotic hosts (Murray E et al., (1989) Nuc Acids Res 17: 477-508) are selected to, for example, increase target expression rates or generate from naturally occurring sequences. It is possible to produce recombinant RNA transcripts with desirable properties such as longer half-lives than the resulting transcripts.

vector

In one embodiment of the invention, drugs (ie, selective oxytocin antagonists) may be administered directly to the subject.

In another embodiment of the invention, a vector comprising a nucleotide sequence encoding a drug of the invention is administered to a subject.

Preferably, the genetic vector is used to prepare the recombinant drug and / or deliver it to the target site.

As is well known in the art, vectors are tools that enable or facilitate the transfer of one substance from one environment to another. In accordance with the present invention, and also by way of example, some vectors used in preparative DNA techniques are capable of copying and / or presenting a material, such as a segment of DNA (heterologous DNA segment, such as a heterologous cDNA segment), to a vector comprising the nucleotide sequence of the present invention. In order to express a protein of the invention encoded by the nucleotide sequence of the invention, it can be delivered into a host and / or target cell. Examples of vectors used in recombinant DNA techniques 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” refers to a construct capable of expression in vivo or in vitro / in vitro.

The term "transformation vector" refers to a construct that can be transferred from one species to another.

Naked DNA

Direct administration of a vector comprising a nucleotide sequence encoding a drug of the invention for use in treating ejaculation disorders, in particular premature ejaculation, as an "exposed nucleic acid construct" further comprising a contiguous sequence homologous to the host cell genome. can do.

As used herein, the term “exposed DNA” refers to a plasmid comprising a nucleotide sequence encoding a drug of the invention with a short promoter region for controlling the manufacture of the drug of the invention. Since the plasmid is not carried in any transport vehicle, it is called "exposed" DNA. When this DNA plasmid enters a host cell, such as a eukaryotic cell, the protein it encodes (eg, the drug of the invention) is transcribed and translated in the cell.

Non-Virus Transport

Alternatively, a vector comprising a nucleotide sequence encoding an amino acid of the invention or a drug of the invention (ie, a selective oxytocin antagonist) or a target of the invention (ie, an oxytocin receptor) can be transfected, transformed, electrophoresed. And various non-viral techniques known in the art, such as biolistic transformation, can be introduced into suitable host cells.

As used herein, the term “transfection” refers to a method of using non-viral vectors to deliver genes to target mammalian cells.

Typical transfection methods include electrophoresis, DNA biolistics, lipid-mediated transfection, compressed DNA-mediated transfection, liposomes, immune liposomes, lipofectin, cationic drug-mediated, cationic facial amphiphiles ( Nature Biotechnology 1996 14; 556), polyvalent cations such as spermine, cationic lipids or polylysines, 1,2-bis (oleoyloxy) -3- (trimethylammonio) propane (DOTAP) Cholesterol complexes (Wolff and Trubetskoy, 1998, Nature Biotechnology, 16: 421) and combinations thereof.

Uptake of exposed nucleic acid constructs by mammalian cells is enhanced by some known transfection techniques, such as the use of transfection agents. Examples of such transfection agents include cationic agents (eg, calcium phosphate and DEAE-dextran) and lipofectants (eg, lipofectam® and transfectam®). Typically, the nucleic acid construct is mixed with a transfection agent to prepare the composition.

Virus vector

Alternatively, such as by infecting a drug or target of the invention or a vector comprising a nucleotide sequence encoding an amino acid sequence of the invention with a recombinant viral vector such as a retrovirus, herpes simplex virus and adenovirus. Various viral techniques known in the art can be used to introduce into suitable host cells.

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

Targeted vector

The term “targeted vector” refers to a vector whose ability to infect / transfect / transform a cell or to be expressed in a host and / or target cell is limited to a particular cell type in the host organism, typically a cell having a common or similar phenotype. Say.

Clone vector

Nucleotide sequences encoding the drugs of the invention (ie, selective oxytocin antagonists, or PDEi or PDE5i, if applicable) or targets (eg, oxytocin receptors) can be incorporated into replicable recombinant vectors. Vectors can be used to replicate nucleotide sequences in compatible host cells. Thus, in one embodiment of the invention, the invention provides a method of introducing a nucleotide sequence of the invention into a replicable vector, introducing the vector into a suitable host cell, and growing the host cell under conditions such that the vector is replicated. Provided are methods of making the target of the invention. The vector can be recovered from homework cells.

Expression vector

Preferably, a nucleotide sequence encoding a drug of the invention or an amino acid of the invention or a target of the invention inserted into a vector is capable of expressing by a host cell a coding sequence such as the coding sequence of the oxytocin receptor of the invention. Operatively link to the sequence. That is, the vector is an expression vector. The drug or target of the invention produced by the host recombinant cell may be secreted or contained within the cell, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, expression vectors containing a drug or target coding sequence of the present invention may be used to select a signal sequence that induces secretion of the drug or target coding sequence of the present invention through a specific prokaryotic or eukaryotic cell membrane. Can be designed to have.

In vitro expression

Vectors of the invention can be transformed or transfected into suitable host cells and / or target cells described below to allow expression of the drugs or targets of the invention. The method cultures host cells and / or target cells transformed with the expression vector under conditions such that the coding sequence encoding the drug or target of the invention can be expressed by the vector, and optionally the expressed drug of the invention. Or recovering the target. The vector can be, for example, a plasmid or viral vector provided with a source of replication, optionally a promoter for expression of said polynucleotide and optionally a modulator of the promoter. The vector may contain one or more selectable marker genes, for example ampicillin resistance genes for bacterial plasmids, or neomycin resistance genes for mammalian vectors. Expression of a drug of the invention or a target 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, the production of a drug or target of the invention can be initiated, for example, when an inducer component is required by addition to the medium (eg dexamethasone or IPTG).

Fusion protein

The oxytocin receptor or vasopressin receptor or drug (i.e., selective oxytocin antagonist) of the present invention may facilitate extraction and purification of the drug or receptor target of the present invention and / or delivery to the subject and / or to facilitate the selection of the drug. Can be expressed as a fusion protein. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation domains) and β-galactosidase. It may be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest so that the fusion protein sequence is removed. Preferably, the fusion protein does not interfere with the activity of the target.

Fusion proteins may include antigens or antigenic determinants fused to components of the invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein comprising a component that can act as an adjuvant in that it provides a generalized stimulus to the immune system. The antigen or antigenic determinant may bind to either the amino or carboxy terminus of the component.

In other embodiments of the invention, the amino acid sequence may be linked to a heterologous sequence to encode a fusion protein. For example, in the search of peptide libraries for drugs that may affect the activity of the component, it may be useful to encode a chimeric component that expresses a heterologous antigenic determinant recognized by a commercially available antigen.

Host cell

A wide range of host cells can be used to express a drug of the invention (eg, an optional oxytocin antagonist of the invention) or a nucleotide sequence encoding the oxytocin or vasopressin receptor target of the invention. These cells can be prokaryotic host cells and eukaryotic host cells. Suitable host cells are E. coli. Bacteria such as E. coli , yeast, fibrous fungi, insect cells, typically permanent mammalian cells such as mouse, CHO, human and monkey cell lines and derivatives thereof.

Examples of suitable expression hosts within the scope of the present invention include fungi such as Aspergillus species (eg, those described in EP-A-0184438 and EP-A-0284603) and Trichoderma species; Bacillus (Bacillus) species (e. G., EP-A-0134048 will be described in the arc and arc EP-A-0253455), Streptomyces (Streptomyces) and bacteria such as Pseudomonas species (Pseudomonas) species; And yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. For example, typical expression hosts include Aspergillus niger , Aspergillus niger var. Tubigenis , Aspergillus niger var. Awamori, Aspergillus niger, Aspergillus niger var. Aspergillus aculeatis , Aspergillus nidulans , Aspergillus orvzae , Trichoderma reesei , Bacillus subtilis , Bacillus Bacillus licheniformis , Bacillus amyloliquefaciens , Kluyveromyces lactis and Saccharomyces cerevisiae .

Using suitable host cells (eg yeast, fungal and plant host cells), post-translational modifications (eg myristoylation, glycosylation, cleavage, etc.) may be necessary to confer optimal biological activity to the recombinant expression products of the invention. , Lapidation and tyrosine, serine or threonine phosphorylation).

Preferred host cells can be treated so that the expression product produces an appropriate mature polypeptide. Examples of treatments include, but are not limited to, glycosylation, ubiquitination, disulfide bond formation, and general post-translational modifications.

Antibodies

In one embodiment of the invention, the drug may be an antibody. In another embodiment, the target may be an antibody.

Antibodies can be prepared by standard techniques, such as by immunization with a component of the invention, or by using phage display libraries.

In the present invention, the term "antibody" includes, but is not limited to, polyclonal, monoclonal, chimeric, short chain, Fab fragments, segments produced by Fab expression libraries, and analogs thereof. Such fragments are fragments of whole antibodies that retain binding activity for target components, ie Fv, F (ab ') and F (ab') ₂ segments, and antigen-binding sites of single-chain antibodies (scFv), fusion proteins and antibodies. It includes other synthetic proteins, including. In addition, the antibody and its fragments may be human antibodies. Neutralizing antibodies, ie antibodies which inhibit the biological activity of the component polypeptides, are particularly preferred for diagnosis and treatment.

If polyclonal antibodies are desired, immunogenic polypeptides having the identified drugs and / or antigenic determinant (s) obtainable from the components of the present invention may be selected from mammals (eg, mice, rabbits, goats, Horses, etc.). Depending on the species of the host, various adjuvants may be used to increase the immune response. Such adjuvants include, but are not limited to, Freund's mineral gels such as aluminum hydroxide, and surface active agents such as lysocithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limbal hemocyanin and dynitrophenols. It is not limited to. BCG ( Bacilli Calmette-Guerin and Corynebacterium parvum ) can be used when purified component polypeptides are administered to immunized individuals to stimulate systemic defense. Which can be used in humans.

Sera from immunized animals are collected and treated according to known procedures. If the serum containing the polyclonal antibody against the antigenic determinant obtainable from the identified drugs and / or components of the invention contains an antibody against another antigen, the polyclonal antibody can be purified by immunoaffinity chromatography. Can be. Techniques for producing and processing polyclonal antisera are known in the art. To prepare such antibodies, the present invention also provides a polypeptide of the invention or a fragment thereof attached to another polypeptide for use as an immunogen in an animal or human.

Monoclonal antibodies directed against antigenic determinants obtainable from the identified drugs and / or components of the invention can also be readily prepared by those skilled in the art. General methods for preparing monoclonal antibodies by hybridomas are well known. Cell fusion can also produce permanent antibody-producing cell lines, such as by direct transformation of B lymphocytes with carcinogenic DNA or by transfection with Epstein-Barr virus. A set of monoclonal antibodies generated against orbit epitopes can be screened for a variety of properties, namely isotypes and epitope affinity.

Any technique of generating antibody molecules by continuous cell lines in culture can be used to prepare monoclonal antibodies against components and / or identified drugs. These are the hybridoma technique first described by Koehler and Milstein (1975, Nature 256: 495-497), the human B-cell hydridoma 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 to these. In addition, techniques developed for the generation of “chimeric antibodies” may be used, ie, techniques for splicing mouse antibody genes into human antibody genes to obtain molecules with appropriate antigen specificity and biological activity (Morrison et al. (1984) Proc Natl Acad Sci 81: 6851-6855; Neuberger et al. (1984) Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454. Alternatively, component specific single chain antibodies can be prepared by employing the techniques described for preparing single chain antibodies (US Pat. No. 4,946,779).

Monoclonal and polyclonal antibodies directed against antigenic determinants that can be obtained from the identified drugs and / or components are particularly useful for diagnosis, and neutralizing monoclonal and polyclonal antibodies are useful for passive immunotherapy. In particular, monoclonal antibodies can be used to generate anti-idiotypic antibodies. Anti-idiotypic antibodies are immunoglobulins that carry "internal images" of components and / or drugs in need of protection. Techniques for generating anti-idiotypic antibodies are known in the art. These anti-idiotypic antibodies may also be useful for treatment.

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 Winter G and Milstein (1991); Antibodies can also be prepared by searching a group of highly specific binding reagents as disclosed in Nature 349: 293-299).

Antibody fragments may also be generated that contain binding sites specific for the components. For example, such segments include Fab segments that can be produced by reducing disulfide bridges of F (ab ') ₂ segments and F (ab') ₂ segments, which can be produced by pepsin digestion of antibody molecules, However, it is not limited to these. Alternatively, Fab expression libraries can be constructed to quickly and easily identify monoclonal Fab segments with the desired specificity (Huse WD, et al. (1989) Science 256: 1275-1281).

Reporter

Various reporters can be used in the analytical methods (and screening methods) of the present invention, and preferred reporters provide signals that can be conveniently detected (eg, by spectroscopy). As an example, the reporter gene may encode an enzyme that promotes a reaction that changes the absorbance.

Examples of reporter molecules include β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, β-glucuronidase, exo-glucanase and glucoamylase It is not limited to these. Alternatively, incorporation of radiolabeled or fluorescent tag-labeled nucleotides into neoplastic transcripts can be identified when bound to oligonucleotide probes.

In one preferred embodiment, the production of reporter molecules is determined by the enzymatic activity of the reporter gene product (eg β-galactosidase).

Various protocols are known in the art for detecting and measuring expression of a target, such as using either polyclonal or monoclonal antibodies specific for a protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell sorting. Two-site monoclonal immunoassays using monoclonal antibodies reactive with two non-interfering antigenic determinants on a polypeptide are preferred, but competitive binding assays may also be used. These and other assays are described in particular in Hampton R et al. (1990, Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn.) And Madox DE et al. (1983, J Exp Med 15 8). 121 1).

A wide range of labeling and conjugate techniques are known to those skilled in the art and can be used for a variety of nucleic acid and amino acid assays. Means for preparing labeled hybridization or PCR probes to detect target polynucleotide sequences include oligo labeling, gap translation, end-labeling or PCR amplification using labeled nucleotides. Alternatively, the coding sequence, or any portion thereof, can be cloned into a vector to prepare an mRNA probe. Such vectors are known in the art and are commercially available and can be used to synthesize RNA probes in vitro by adding appropriate RNA polymerases and labeled nucleotides such as T7, T3 or SP6.

Many companies include Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, Ohio). Commercial kits and protocols are provided for this procedure. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent agents, chemiluminescent or colorants and substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels are described in US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149; And US-A-4366241. In addition, recombinant immunoglobulins can be prepared as shown in US-A-4816567.

Other methods of quantifying expression of specific molecules include radiolabeling of nucleotides (Melby PC et al., 1993, J Immunol Methods 159: 235-44) or biotinylation (Duplaa C) et al., 1993, Anal Biochem 229-36), simultaneous amplification of regulatory nucleic acids and standard curves in which experimental results are entered. Quantification of several samples can be accelerated by performing the assay in ELISA format (where oligomers of interest are provided in various dilutions and quantified rapidly by spectroscopic or thermometric reactions).

Although the presence / absence of marker gene expression suggests that there is also the gene of interest, its presence and expression should be confirmed. For example, when 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 located in series with the target coding sequence under the control of a single promoter. Expression of a marker gene corresponding to induction or selection usually also indicates expression of the target.

Alternatively, various procedures known to those skilled in the art can identify host cells that contain a coding sequence for a target and express a target coding region. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybrid formation and protein bioassay or immunoassay techniques (including techniques based on membranes, solutions, or chips for detection and / or quantification of nucleic acids or proteins). no.

Selection

Any one or more suitable targets (eg, oxytocin receptors and / or vasopressin, preferably V1a receptors) can be used to identify drugs, eg, selective oxytocin receptor antagonists, in any of a variety of drug selection techniques. Targets used for such testing can be free in solution, fixed to a solid support, produced on the cell surface or located within the cell. The target may even be in an animal model, where the target may be an exogenous target or an introduced target. Animal models are non-human animal models. Removal of target activity or production of binding complexes between the target and the drug being tested can be measured.

Drug selection techniques are based on the method described in Geysen, European Patent Publication No. 84/03564 (published Sep. 13, 1984). In summary, many different small peptide test compounds are synthesized on a solid substrate (eg, plastic pins or some other surface). Peptide test compounds are reacted with a suitable target or segment thereof and washed. The bound material is detected by, for example, a suitably adopted method which is well known in the art. For use in drug selection techniques, the purified target may also be coated directly onto the plate. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

Also contemplated herein are the use of competitive drug screening assays in which neutralizing antibodies capable of binding a target specifically compete with a test compound to bind the target.

Another screening technique provides high throughput screening (HTS) of drugs with suitable binding affinity for the component, which is based on the method described in detail in WO 84/03564.

The analytical method of the present invention is expected to be suitable for small and large scale screening and quantitative analysis of test compounds.

In a preferred embodiment, the selection of the present invention comprises at least the following steps (not necessarily in the same consecutive order): (a) In vitro selection is carried out such that the candidate drug is associated with relevant activity (eg, modulation of oxytocin receptors). Determining whether or not); (b) performing one or more selectivity selections, such as by using an assay protocol described herein to determine the selectivity of the candidate drug (e.g. to determine whether the drug is a vasopressin, in particular a V1a receptor inhibitor) for); And c) performing in vivo selection using said candidate drug (e.g., using a functional animal model to determine the drug's selectivity by determining the effect of the drug on vasopressin, in particular V1a receptors). ). Typically, if the candidate drug passes through selection (a) and selection (b), then selection (c) is performed.

Diagnostic method / composition / kit

The invention also provides diagnostic methods, compositions or kits for detecting propensity to premature ejaculation. In this regard, the method, composition or kit will comprise means for detecting a substance, preferably oxytocin, in a test sample, preferably a blood sample taken from a sexually excited male.

To provide a basis for premature ejaculation diagnosis, a normal or standard value of the substance should be established. This can be achieved by mixing body fluids taken from normal individuals, whether animals or humans, after various times of sexual excitement, with antigens for the substance, under conditions suitable for complex formation well known in the art. The amount of standard complex formation can be quantified by comparing it with a series of dilutions of the positive control in which a known amount of antibody is mixed with a known concentration of purified target. The standard values obtained from normal samples can then be compared with the values obtained from samples of individuals likely to premature ejaculation. Deviations between standard and individual values demonstrate the presence of the disease state.

The substance itself, or any portion thereof, may provide the basis for a diagnostic and / or therapeutic compound. For diagnosis, the target nucleotide sequence can be used to detect and quantify gene expression in a disease, disorder or condition in which premature ejaculation may be involved.

In the diagnosis of premature ejaculation resulting from expression of a target, a polynucleotide sequence encoding the target can be used. For example, polynucleotide sequences encoding a substance can be used for hybridization or PCR analysis of body fluids of tissues or organisms from a biopsy or autopsy to detect expression of the substance. Forms of such qualitative or quantitative methods may include Southern or Northern analysis, point blots or other techniques based on membranes; PCR technique; Dipstick, pin or chip technique; And ELISA or other multiple sample format techniques. All of these techniques are well known in the art and are in fact the basis of many commercial diagnostic kits.

Such assays can be tailored to assess the effectiveness of a particular therapeutic regimen and can be used for animal studies, clinical trials, or individual treatment monitoring. Once the disease is demonstrated, the therapeutic agent present can be administered and a therapeutic profile or therapeutic value can be obtained. Finally, by repeating the assay on an accredited basis, one can assess whether the value proceeds toward or returns to a normal or standard pattern. Continuous treatment profiles can be used to indicate the effectiveness of treatment over days or months.

Analytical Method

Analytical methods according to the present invention may employ one or more of the following techniques, including but not limited to: competitive or non-competitive assays, radioimmunoassays, bioluminescence and chemiluminescence assays, fluorescence assays , Sandwich assays, immunoradiometric assays, dot blots, enzyme linkage assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, immunohistochemistry and immune cell chemistry.

Probe

Another aspect of the invention is a nucleic acid hybrid formation or PCR brob that is capable of detecting polynucleotide sequences, including genomic sequences encoding target coding regions, such as regions encoding oxytocin receptors or closely related molecules such as alleles. In particular, it is possible to provide selectively). The specificity of the probe, i.e. whether it is derived from a highly conserved region or domain, a conserved region or domain or an unconserved region or domain, and the stringency of the hybrid formation or amplification (high, medium or low) is that the probe is natural Determine whether only the developing target coding sequence is identified or related sequences. Probes for detecting related nucleic acid sequences are selected from conserved or highly conserved nucleotide regions of target family members, which can be used in a pool of degenerate probes. For detection of the same nucleic acid sequence, or where maximum specificity is required, nucleic acid probes are selected from unconserved nucleotide regions or specific regions of the target polynucleotide. As used herein, the term “unconserved nucleotide region” refers to a nucleotide region that is specific for the target coding sequence disclosed herein and does not occur in a family member involved.

PCR described in US-A-4683195, US-A-4800195 and US-A-4965188 provides additional uses for oligonucleotides based on target sequences. Such oligomers are usually synthesized chemically, but they may be produced by enzymes or may be produced from recombinant sources. Oligomers typically comprise two nucleotide sequences, one with a sense orientation (5 '→ 3') and the other with an antisense orientation (3 '← 5'), which are optimized to identify specific genes or conditions Used under conditions. For detection and / or quantification of closely related DNA or RNA sequences, one can use the same two oligomers, a paired set of oligomers or a degenerate pool of oligomers under less stringent conditions.

Using the nucleic acid sequence of the drug or target. The hybridization probes described previously for mapping endogenous genomic sequences can be prepared. Well known techniques can be used to map sequences to specific chromosomes or to specific regions of chromosomes. These include in situ hybridization with chromosomal spreads, flow-classified chromosomal preparations or artificial chromosome constructs (eg, YACs), bacterial artificial chromosomes (BACs), bacterial PI constructs, or single chromosomal cDNA libraries (Verma (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City).

In situ hybridization and physical mapping techniques of chromosomal preparations (eg, binding assays using proven chromosomal markers) are very important for extending genetic maps. Examples of genetic maps can be found in Science 1995; 270: 41 and 1994; 265: 1981f. Often, placing a gene on a chromosome of another mammalian species can reveal related markers even if the number of arms of a particular human chromosome is unknown. By physical mapping, new sequences can be assigned to chromosomal cancers or portions thereof. This provides important information for researchers searching for disease genes using location cloning or other gene discovery techniques. Once gene binding has revealed a particular genomic region from which the disease or syndrome originated, any sequence mapping to this region may represent a relevant or regulatory gene for further study. The nucleotide sequence of the present invention can be used to detect differences in chromosomal position due to translocation or inversion, etc. between normal individuals, carriers or individuals with disease.

Creature

The term "organism" in connection with the present invention includes any organism that may include a target and / or a product obtained therefrom. Examples of organisms can include mammals, fungi, yeasts or plants.

The term "gene pre-organism" in the context of the present invention includes any organism, including targets and / or products obtained therefrom.

Transformation of Host Cells / Host Organisms

As indicated above, the host organism may be a eukaryotic or prokaryotic organism. Examples of suitable prokaryotic hosts are E. coli. E. coli and Bacillus subtilis. Teachings on the prokaryotic host transformation can be found in the art, such as in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, 1989, Cold Spring Harbor Laboratory Press) and Osbel et al. It is well described in Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.

If a prokaryotic host is used, it may be necessary to modify the nucleotide sequence appropriately (eg, removal of introns) prior to transformation.

In other embodiments, the gene transfer organism can be yeast. In this regard, yeast has been widely used as a vehicle for heterologous gene expression.

Saccharomyces cerevisiae has been used industrially for a long time, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been described by Goodey et al. (1987, Yeast Biotechnology, DR Berry et al., Pp 401-429, Allen and Unwin, London) and King. (1989, Molecular and Cell Biology of Yeasts, edited by EF Walton and GT Yarronton, pp 107-133, Blackie, Glasgow).

For several reasons, Saccharomyces cerevisiae is suitable 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, with centuries of commercial development for various purposes. This was widely accepted by the public. Third, extensive commercial use and research on living organisms has enriched knowledge of the genetics and physiology of Saccharomyces cerevisiae , and the large-scale fermentation characteristics.

A review of the principle of heterologous gene expression in Saccharomyces cerevisiae is described by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol. 5, Anthony H Rose and J Stuart Harrison, 2nd edition, Academic Press Ltd.

Several types of yeast vectors are available, including integration vectors that require recombination with the host genome for maintenance and plasmid vectors that autonomously replicate.

To prepare gene transfer Saccharomyces , expression constructs are prepared by inserting the nucleotide sequences of the invention into constructs designed to express in yeast. Several types of constructs have been developed for use in heterologous expression. The construct contains a promoter active to yeast fused to the nucleotide sequence of the present invention and typically uses a promoter generated from yeast (eg, a GAL1 promoter). Typically, signal sequences generated from yeast such as sequences encoding SUC 2 signal peptides are used. Terminators active in yeast terminate the expression system.

For transformation of yeast, several transformation protocols have been developed. For example, Hinnen et al. (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, JD (1978, Nature, London, 275, 104); And by following the teachings of Ito (Ito, H), etc. (1983, J Bacteriology 153, 163-168 ), it can be prepared my process (Saccharomyces) a transgenic Saccharomyces according to the present invention.

A variety of selective markers are used to select transformed yeast cells. Among the markers used for transformation are many, including ureotropic markers such as LEU2, HIS4 and TRP1, and aminoglycoside antibiotic markers such as potent antibiotic resistance markers such as G418.

Other host organisms are plants. The basic principle of the construction of genetically modified plants is to insert genetic information into the plant genome to keep the inserted genetic material stable. There are several techniques for embedding genetic information, two main principles being the introduction of genetic information by direct introduction and the use of vector systems. An overview of the general technique can be found in Potrykus' paper (Annu Rev Plant Physiol Plant Mol Biol [1991] 42: 205-225) and Christou's paper (Agro-Food-Industry Hi-Tech 1994 3). February / April 17-27). Further teaching on plant transformation can be found in EP-A-0449375.

Accordingly, the present invention also provides a method of transforming a host cell with a nucleotide sequence that is targeted or expresses a target. Host cells transformed with the nucleotide sequence can be cultured under conditions suitable for expressing and recovering the encoded protein from the cell culture. The protein produced by the recombinant cell may be secreted or contained within the cell, depending on the sequence and / or the vector used. As will be appreciated by those skilled in the art, expression vectors containing coding sequences can be designed to have signal sequences that direct direct secretion of the coding sequence through a particular eukaryotic membrane or prokaryotic membrane. Other recombinant constructs may bind coding sequences to nucleotide sequences encoding polypeptide domains that facilitate purification of soluble proteins (Kroll DJ et al. (1993) DNA Cell Biol 12: 441-53).

PDE Inhibitors-Test Methods

PDE action potency values referred to herein are determined by the following assay:

Phosphodiesterase (PDE) inhibitory activity

Preferred PDE compounds suitable for use according to the invention are potent and selective cGMP PDE5 inhibitors. In vitro PDE inhibitory activity against cyclic guanosine 3 ', 5'-monophosphate (cGMP) and cyclic adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase was shown to have an IC ₅₀ value (50% enzyme activity). Can be determined by measuring the concentration of compound required to inhibit).

The PDE enzymes required are essentially human cavernous bodies, human and rabbit platelets, human ventricles, human skeletal muscles by the methods of WJ Thompson and MM Appleman (Biochem., 1971, 10 , 311). And bovine retinas. Specifically, cGMP-specific PDE (PDE5) and cGMP-inhibited cAMP PDE (PDE3) can be obtained from human cavernous tissue, and cAMP-specific PDE (PDE4) can be obtained from human skeletal muscle. Phosphodiesterases 7-11 can be prepared from full length human recombinant clones that are transfected into SF9 cells.

AMP / GMP using a variation of the "batch" method of Thompson et al. (Biochem., 1979, 18 , 5228) or using a modification of the protocol described under the trade name TRKQ7090 / 7100 by Amersham plc. The analysis can be performed using a flash proximity assay for direct detection of. Briefly, in the presence of inhibitor concentrations and low substrate concentrations (unlabeled at concentrations ˜1 / 3 km _m to [ ³ H] -labeled ratio of 3: 1 cGMP or cAMP) such that IC ₅₀ is similar to K _i . The effect of PDE inhibitors was investigated by analyzing a fixed amount of enzyme. Final assay volumes were made up to 100 μl with assay buffer [20 mM Tris-HCl pH 7.4, 5 mM MgCl ₂ , 1 mg / ml bovine serum albumin]. Enzyme was used to initiate the reaction and incubated at 30 ° C. for 30-60 minutes to provide substrate conversion of less than 30%, and 50 μl yttrium silicate SPA beads (each unlabeled cyclic nucleotide for PDE 9 and PDE 11). 3 mM). The plates were resealed and shaken for 20 minutes, after which the beads were allowed to settle in the dark for 30 minutes and counted in a top count plate reader (Packard, Meriden, Connecticut). Convert radioactivity units to% for uninhibited control (100%) activity, plot for inhibitor concentrations, and then inhibit IC ₅₀ values using a 'Fit Curve' Microsoft Excel extension. Obtained.

Function active

Sodium nitroproside-induced cavernous tissue strips of preconstricted rabbits, as described in SA Ballard et al. (Brit. J. Pharmacol., 1996, 118 (supplementary version), abstract 153P). By determining the ability of the compounds of the present invention to enhance relaxed relaxation, this can be assessed in vitro.

The invention is described in more detail by the following examples with reference to the figures and the sequence listing.

1 is a graph depicting the effect of oxytocin receptor antagonist L-368,899 on p-chloroamphetamine (PCA) -induced ejaculation in anesthetized rats.

2 is a graph depicting the effect of selective oxytocin antagonists (L-368,899) on seminal vesicle pressure in anesthetized rats.

Sequence list

SEQ ID NO: 1 shows the amino acid sequence of a human oxytocin receptor.

SEQ ID NO: 2 shows the amino acid sequence of human vasopressin V1A receptor.

1.0 way

1.1. Animal testing methods

1.1.1. Penile Erection and Ejaculation Test Methods in Anesthetized Rats

The method used to study penile erection and ejaculation was based on the method taught in Yonezawa et al. (2000) Life Sciences 67, 3031-3039. To facilitate reference, this method is quoted below:

Wistar-ST male rats weighing 350-450 g are used. Before experimenting, group animals (12 rats per cage) for 12 hours lighting-dark cycle (lights up at 07:00), constant temperature (23 ± 1 ° C) and humidity (55 ± 5%) Lock it up. They have free access to standard food pellets and water.

Rats are anesthetized with sodium pentobarbital (50 mg / kg intraperitoneal) and placed in a flat lying position. The penis protrudes from the epidermis and is gently gripped by a wood applicator placed at the bottom of the penis. Immediately before the epidermis is pulled back, the test compound is administered intraperitoneally and penis including penile erection, redness and enlargement of the penis body, glans of the glans, glans of the glans and cups, and slight redness, strong redness of the glans erection Record the reaction. Latency from the administration of the test compound to the initial penile response and ejaculation was also measured.                 

The effect of the test compound on PCA induced ejaculation is assessed by weighing the ejaculate collected for 30 minutes. Suitable methods of using conscious rats are described in Renyi (1985) Neuropharmacology, Vol. 24, No. 8, pp 697-704.

Intracavernosal pressure is also measured in rats anesthetized with sodium pentobarbitol (50 mg / kg, intraperitoneal). If desired, additional small doses (5 mg) may be injected throughout the duration of the experiment. The intracavernosal pressure (ICP) was measured by protruding the penis from the epidermis and inserting a stainless steel needle (23-gauge) into the cavernous body. Attach a needle to a Teflon tube filled with heparinized saline (10 U / ml) and connect to a pressure transducer (NEC-San-Ei 7500).

1.1.2 Male Sexual Behavior Model:

In all sexual behavior tests, male rats were placed in the observation field (50-60 cm in diameter) and observed under red illumination, starting with a 5 hour dark cycle. Three to four minutes after the males were placed on the field, a receptive female (ovariectomized, injected with estradiol benzoate / progesterone 48 hours before initiation of the behavioral study) was introduced into the field and the following variables were noted: do:

i) ejaculation latent (EJL; time to ejaculate after receiving a receptive female into the field);

ii) mating efficiency (CE; ejaculation latent / inserted ejaculation, ie second interval between insertions);

iii) frequency of insertion (IF; number of insertions assessed);                 

iv) frequency of rides (MF; number of rides assessed);

v) Intermediate post-assessment (PEI; time from assessment to initiation of mating behavior).

2.0 Selective Oxytocin Receptor Antagonists

The compounds used in the following examples were as follows:

Selective oxytocin receptor antagonist L-368,899. Further details regarding this compound are provided above. L-368,899 is at least 20 times more selective for oxytocin receptors compared to V1a receptors [6.3 nM OT: 148 nM V1a].

Example 1 Delay of Ejaculation in the Presence of Selective Oxytocin Receptor Antagonists (L-368,899)

Oxytocin receptor antagonist L-368,899 significantly delayed p-chloroamphetamine (PCA) -induced ejaculation at selective oxytocin doses (0.1-10 mg / kg, subcutaneous) in anesthetized rats. At a free plasma concentration of 5.4 ± 1.5 nM (0.9 × Ki OT, see FIG. 1), ejaculation was delayed by 140% (almost maximal effect), and at this dose it was estimated that any activity arises from antagonism of oxytocin receptors. .

The mechanism causing the erection was not significantly affected by oxytocin receptor occlusion. That is, the number of penis depressions and enlargements was similar in the control and oxytocin antagonist studies (see Table 1 below). After 1 mg / kg subcutaneous administration of L-368,899 (substantially delayed dose), 95% of PCA-induced erection in the penis depression was comparable to 94% of the vehicle control group and occurred in the penis enlargement. 61% of PCA-induced erections are comparable to 63% of the vehicle control group.                 

L-368,899 has very poor CNS penetration, and by itself this study shows that oxytocin has a peripheral nerve action site in PCA-induced ejaculation. PCA is a 5HT releaser that activates non-adrenergic non-cholinergic nerves that produce sympathetic pathways that cause penile erection and control ejaculation. These pre-sex effects are thought to be mediated through the release of spinal cord 5HT acting on 5HT1B and 5HT2C receptors. PCA also possibly induces the release of oxytocin from the anterior pituitary or spinal cord centers. It increases oxytocin and is involved in the ejaculation process as in men (because in these studies antagonists of oxytocin receptors have a significant effect on the time it takes to reach ejaculation).

Using a rodent model of ejaculation that reflects human ejaculation physiological function, the inventors have found that peripheral nerve oxytocin receptors are involved in ejaculation mechanisms. This effect may be direct or may be by adjustment of the sympathetic distribution of the internal reproductive organs. We have to consider the role of the central nervous system for oxytocin receptors. In addition, this study shows that oxytocin antagonists are useful for treating premature ejaculation by delaying ejaculation.                 

Example 2 Effect of Selective Oxytocin Antagonist (L-368,899) on Sperm Pressure in Anesthetized Rats

L-368,899 significantly reduced visceral neuro-stimulated increase in seminal vesicle pressure in anesthetized rats (1-3 mg / kg, intravenously). The seminal vesicle contraction is essential for spermatozoa, and semen carried into the prostate urethra is thought to trigger ejaculation. Oxytocin has a direct contractile effect on the seminal vesicles in mammals and may additionally act as a neuromodulator that affects sympathetic distribution during ejaculation. In this study, the seminal vesicle shrinkage was reduced by 41% after a 1 mg / kg pill injection (see Figure 2 below). Preliminary studies show that the free plasma concentration of L-368,899 achieved after 1.0 mg / kg intravenous injection is about 60 nM based on the literature PK and protein plasma binding.

The data indicate that oxytocin is released during visceral nerve stimulation, and the peptides play a physiological function in the development of intravesicular pressure and in spermatologic processes before ejaculation. This study supports the role of systemic oxytocin in influencing peripheral neuronal ejaculation responses, which may be either direct or by adjusting the sympathetic distribution of the seminal vesicles. Oxytocin can regulate the contraction of the tube and gland lobules throughout the male reproductive tract, affecting the fluid volume of different ejaculate components. Increasing ejaculation volume is believed to shorten the time from insertion to ejaculation, and therefore oxytocin antagonists may be useful for treating premature ejaculation with delayed lacrimal puncture.

Example 3 Effect of Selective Oxytocin Antagonists (L-368,899) on Mating Behavior in Rats

L-368,899 has no effect on mating behavior in sexually experienced rats at doses below 10 mg / kg (subcutaneous). The mating behavior of rodents is characterized by a series of climbs that are inserted or not inserted into the vagina (insertion in 50 to 80% of the climb) (vaginal penetration), and ejaculation occurs after 6 to 12 insertions. Each insertion lasts in seconds, making it impossible to quantify the length of insertion (ie, vaginal latent). The effect of L-368,899 was evaluated on a number of mating parameters (see above). The inventors focused on mating efficiency as a measure to outline vaginal penetration.

At any dose tested (0.05-10 mg / kg, subcutaneous, see Table 2 below) L-368,899 had no effect on mating efficiency. Preliminary pharmacokinetic studies indicate that free plasma concentrations of 4.5 nM and 40 nM are expected 30 minutes after injection of 1 mg / kg (subcutaneous) and 10 mg / kg (subcutaneous), respectively.

L-368,899 was also administered intraventricular (icv). L-368,899 has no significant effect on the mating efficiency of sexually experienced rats when administered at 50 ng / rat (ventricular).

Example 4 Effect of Non-Selective Oxytocin Antagonist (Basotocin) on Mating Behavior

Previous studies investigating the effect on the mating behavior of d (CH ₂ ) ₅ Tyr (Me) -Orn ⁸ -Vasatosine, a peptidic non-selective oxytocin antagonist, at doses below 25 ng / rat (ventricular) As a result, it showed no effect on ejaculation potential. After 5 ng / rat intraventricular administration, there was a (significant) decrease in mating efficiency (45 sec / insertion versus 25 sec / insertion of vehicle control animals). This effect is probably mediated by oxytocin and / or vasopressin receptors. After 50 ng / rat intraventricular administration, mating behavior disappeared (Melis et al., Neuroscience Letters 265 (1999) 171-174). This showed that the number of non-contact erections was reduced at 100 ng / rat ventricular administration and that the drug-induced erection was completely eliminated at 100 ng / rat ventricular administration. These data may indicate that, in contrast to studies with selective oxytocin receptor antagonists (L-368,899), antagonism of central oxytocin and vasopressin receptors may be detrimental to sexual behavior, including erectile mechanisms and excitability.

Abbreviation

PE: premature ejaculation

OT: Oxytocin

cAMP: cyclic adenosine-3 ', 5'-monophosphate

cGMP: cyclic guanosine-3 ', 5'-monophosphate

PDE: phosphodiesterase                 

PDE _cGMP : cGMP hydrolysis PDE

PDEi: Inhibitor of PDE (also known as I: PDE)

PDE5: phosphodiesterase type 5

PDE5i: Inhibitors of PDE5

kDa: kilodaltons

bp: base pair

kb: kilobase pair

List of sequences (part of detailed description)

SEQ ID NO: 1

SEQ ID NO: 2

Attach sequence list electronic file  

Claims (30)

delete delete delete delete delete delete A non-peptidic selective oxytocin receptor antagonist of at least 20-fold selective and orally bioavailable to the oxytocin receptor as compared to the vasopressin receptor, mixed with or not mixed with a pharmaceutically acceptable carrier, diluent or excipient. Comprising a block containing L-368,899, Pharmaceutical packs for treating or preventing premature ejaculation of humans or animals:

delete delete An analytical method for identifying a drug that can be used to treat or prevent premature ejaculation, comprising determining whether a test drug can directly improve the endogenous ejaculation process, Wherein the test drug is an orally bioavailable non-peptide selective oxytocin receptor antagonist that is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor, Wherein said improvement is defined as an increase in ejaculation potential or recovery of ejaculation late in the presence of the test drug and is an indication of whether the test drug may be useful for treating or preventing premature ejaculation. The method of claim 10, An assay method wherein the selective oxytocin receptor antagonist is at least 30-fold selective for the oxytocin receptor as compared to the vasopressin receptor. The method of claim 10 or 11, An assay method wherein the vasopressin receptor is the V1a receptor. (a) performing an analysis method according to claim 10; (b) identifying a drug capable of increasing or restoring ejaculation potential; And (c) preparing a large amount of the identified drug, Wherein said drug is an orally bioavailable non-peptide selective oxytocin receptor antagonist that is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor. The method of claim 13, Wherein the selective oxytocin receptor antagonist is at least 30-fold selective for the oxytocin receptor as compared to the vasopressin receptor. The method according to claim 13 or 14, The vasopressin receptor is a V1a receptor. An animal model for identifying drugs that can treat or prevent premature ejaculation, Wherein the drug is an orally bioavailable non-peptide selective oxytocin receptor antagonist that is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor, An animal model comprising an animal male having a means for measuring ejaculation potential of a male after introducing a receptive female. The method of claim 16, Animal model wherein the selective oxytocin receptor antagonist is at least 30-fold selective for the oxytocin receptor as compared to the vasopressin receptor. The method according to claim 16 or 17, Animal model wherein the vasopressin receptor is the V1a receptor. Administering the drug to an animal model according to claim 16; Measuring the ejaculation potential of the animal after introducing a receptive female, As an analytical method to identify drugs that can directly improve the endogenous ejaculation process to treat or prevent premature ejaculation, Wherein the drug is an orally bioavailable non-peptide selective oxytocin receptor antagonist that is at least 20-fold selective for the oxytocin receptor as compared to the vasopressin receptor. The method of claim 19, An assay method wherein the selective oxytocin receptor antagonist is at least 30-fold selective for the oxytocin receptor as compared to the vasopressin receptor. The method of claim 19 or 20, An assay method wherein the vasopressin receptor is the V1a receptor. delete delete delete A non-peptidic selective oxytocin receptor antagonist of at least 20-fold selective and orally bioavailable to the oxytocin receptor as compared to the vasopressin receptor, mixed with or not mixed with a pharmaceutically acceptable carrier, diluent or excipient. L-368,899; And phosphodiesterase inhibitors (PDEi), Pharmaceutical compositions for treating or preventing ejaculation disorders:

delete delete The method of claim 25, Pharmaceutical composition wherein the phosphodiesterase inhibitor is a phosphodiesterase 5 inhibitor (PDE5i). delete delete

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