HK1073109B - Pharmaceutical composition for treatment of male sexual dysfunction - Google Patents
Pharmaceutical composition for treatment of male sexual dysfunction Download PDFInfo
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- HK1073109B HK1073109B HK05105801.3A HK05105801A HK1073109B HK 1073109 B HK1073109 B HK 1073109B HK 05105801 A HK05105801 A HK 05105801A HK 1073109 B HK1073109 B HK 1073109B
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Description
[ field of the invention ]
The present invention relates to compounds and medicaments which may be used for the treatment or prevention of male sexual dysfunction, in particular ejaculatory disorders such as premature ejaculation.
The invention also relates to a method for the prevention and/or treatment of male sexual dysfunction, in particular ejaculatory disorders, such as premature ejaculation.
The invention also relates to an assay for screening compounds useful for the treatment of male sexual dysfunction, in particular ejaculatory disorders, such as premature ejaculation.
[ BACKGROUND OF THE INVENTION ]
Male sexual dysfunction
Sexual Dysfunction (SD) is a significant clinical problem that may affect both men and women. The cause of SD can be either organic or psychiatric, and the organic aspects of SD are typically caused by underlying vascular disease, such as those associated with hypertension or diabetes, by prescribed medicine and/or psychiatric disorders such as depression and the like. Physiological factors include fear, anxiety and opposition among individuals. SD impairs sexual performance, weakens self-esteem, and destroys private relations, thereby inducing personal annoyance. In the clinic, 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 defined as the difficulty or inability of women to find satisfaction in sexual performance, Male Sexual Dysfunction (MSD) is often associated with erectile dysfunction, also known as Male Erectile Dysfunction (MED), and/or with ejaculatory disorders such as premature ejaculation, anorgasmia (inability to achieve orgasm), or sexual desire disorders such as hyposexuality (lack of interest in sex).
Ejaculatory Premature Ejaculation (PE)
PE is a relatively common sexual disorder in men, which has been defined in several different ways, but the most widely accepted is the definition described in the Diagnostic and Statistical Manual of mental disorders IV:
"PE is persistent or recurrent ejaculation for life before, during or after insertion and before the patient's will with minimal sexual stimulation. The clinician must take into account factors that affect the duration of the excitement phase, such as age, freshness of sexual partners or stimuli, and frequency of sexual activity. This obstacle can cause significant annoyance of interpersonal difficulties.
The International Classification of Diseases 10 (International Classification of Diseases 10) is defined as:
"the ejaculation can not be delayed to enjoy love, and any phenomenon is shown as follows: (1) ejaculation occurs shortly before or after the onset of sexual intercourse (if time constraints are required: within 15 seconds before or after the onset of sexual intercourse); (2) ejaculation occurs when there is insufficient erection to enable intercourse to proceed. The problem is not a result of long-term prohibited sexual activity ".
Other definitions that have also been used include classification based on the following criteria:
orgasm associated with partner
Duration between insertion and ejaculation
Number of insertions and ability to consciously control
Mental factors may also be included in PE, with interpersonal problems, anxiety, depression, prior sexual failure, etc. all contributing to a certain degree.
PE prevalence is estimated to be about 22-38% of the male population. Unlike Male Erectile Dysfunction (MED), PE has no clear correlation with age. Estimated as twenty-four million patients in the united states at 30% prevalence (eighty million men aged 18-65 in 1995). Prevalence data in terms of severity are less. It is estimated that the operative definition of PE applies to 5-10% of men, but less than 0.2% are treated. The availability of orally available therapies is highly likely to alter this situation.
Urologists currently constitute a major group of physicians treating PE (59%). GP represents 33% of the physicians treating this condition. Sex therapists, behavior therapists and consultants also treat PE patients. The expert estimates that the diagnosis is due to the impact of the condition on the partner relationship. Stress, difficulty of relationship, and/or impact on quality of life are key causes of disease seeking to treat PE.
Ejaculation is associated with the sympathetic and parasympathetic nervous systems. The efferent impulses to the vas deferens and the epididymis through the sympathetic nervous system produce contraction of smooth muscles moving the semen into the posterior urethra. Similar contractions of the seminal vesicle, prostate and bulbar urethra increase semen volume and fluid content. The expulsion of semen is mediated by the rhythmic contraction of the somatic muscles of the sciatic and urethral lineages and the pelvic floor muscles, with the expulsion impulses originating from the Onf nucleus in the spinal cord passing through the parasympathetic nervous system and promoting the musculus spongiform, sciatic and urethral linea littoralis. Cortical control of ejaculation in humans remains controversial. In rats, the medial anterior retinal region and paraventricular nucleus in the hypothalamus appear to be involved in ejaculation.
There are currently no approved drugs available to treat PE. The most commonly used unofficial approved (off-label) prescription drugs are antidepressants (e.g., clomipramine), and selective 5-hydroxytryptamine reuptake inhibitors (e.g., paroxetine and sertraline). These drugs are often not well accepted by patients because they are all considered antidepressants. They are used as "unofficially approved" prescriptions and are effective when needed (i.e., "prn"), because of their long pharmacokinetic Tmax(the time to reach maximum drug concentration in plasma after oral administration of the drug) so their onset of action may be slow. The side effects common to these drugs can be seen in long-term use. Behavioral therapy is also another control tool, but none are very effective and have high rates of dropped-out and recurrence. Currently, there is a need for new therapies that are more efficient.
Therefore, there is a need to find new methods for the treatment of male sexual dysfunction, in particular for ejaculatory disorders, such as premature ejaculation.
Summary of The Invention
A basic discovery of the present invention is that an increase in ejaculation latency can be achieved following administration of a selective oxytocin antagonist. Thus, it has been shown that treatment of ejaculatory disorders, in particular premature ejaculation, can be achieved using selective oxytocin antagonists. This is achieved by increasing ejaculation latency, preferably to return it to a near normal level.
In particular, the use of selective oxytocin antagonists may lead to ejaculatory disorders, in particular premature ejaculation treatment, while maintaining the mechanisms of erection production, in particular penile erection.
The use of selective oxytocin antagonists in the treatment of ejaculatory disorders, particularly premature ejaculation, may contribute to the treatment of these diseases while maintaining "sexual drive" in the patient. The term "sexual drive" is used herein to refer to sexual desire or desire.
Thus, the compounds of the present invention preferably include the following unexpected advantages: the maintenance of erection mechanisms, particularly penile erection, and/or sexual drive, in comparison to known non-selective oxytocin antagonists.
Role of oxytocin in sexual behavior
Ejaculation comprises two separate phases-ejaculation (emision) and ejaculation. Excretion is the deposition of seminal fluid and sperm from the distal epididymis, vas deferens, seminal vesicles and prostate into the prostatic urethra. This deposition is followed by a forceful expulsion of the semen contents from the urethral meatus. Ejaculation is distinguished from orgasm, which is purely a brain event. These two processes often occur simultaneously.
In mammals, oxytocin pulses in peripheral serum occur with ejaculation. In males, plasma concentrations of oxytocin, but not vasopressin, are significantly elevated during or before ejaculation. Oxytocin does not induce ejaculation per se; this process is 100% under the neural control of sympathetic nerves that pass through the α 1-adrenoreceptor/origin of the lumbar spinal cord. The systemic pulse of oxytocin may have a direct effect in the peripheral ejaculation. For example, it may be used to regulate the contraction of ducts and glandular lobules in the male genital tract, thus affecting the fluid volume of the different ejaculatory components. Oxytocin released centrally into the brain may affect sexual behaviour, subjective perception of sexual arousal (orgasm) and the latency of subsequent ejaculation. The onset of male ejaculation is largely dependent on tactile stimulation of the external genitalia.
It is documented that circulating oxytocin levels increase in sexual stimulation and arousal and reach a maximum in orgasm in both men and women. Murphy et al, (Acta Ant Basel 128: 76-79[1987]) measured plasma oxytocin and Arginine Vasopressin (AVP) concentrations in men during sexual arousal and ejaculation and found a significant increase in plasma AVP but not oxytocin during sexual arousal. However, at ejaculation, mean plasma oxytocin increased approximately 5-fold and returned to baseline concentrations within 30 minutes, whereas AVP had returned to baseline levels at ejaculation and remained stable thereafter.
As detailed in Gimp1 and Fahrenholz (Physiological Reviews Vol.81: No.2.April 2001 pp629-683), oxytocin was found to be one of the most potent substances that can induce penile erection in rats, rabbits and monkeys. In addition, central oxytocin administration is said to shorten the latency to ejaculation and shorten the post-ejaculation interval. Similarly, Meston et al (arch. gen psychotherapy, vol.57, Nov 2000) teach that in male animals, oxytocin can help the penis erect when injected into a specific part of the brain (i.e., the paraventricular nucleus in the hypothalamus) and shorten ejaculation latency and postejaculation intervals when injected centrally or peripherally.
It is well documented in the art that non-contact penile erection can be significantly reduced by the administration of an oxytocin receptor agonist, i.e., 8-seminal oxytocin (see, e.g., Melis et al (Neuroscience Letters 265(1999) 171-. Furthermore, it was also demonstrated in Argiolas et al (European Journal of Pharmacology 149(1988)389-392) that the use of an oxytocin antagonist, 8-vasopressin, by intracerebroventricular Injection (ICV) impairs the sexual performance of experienced males in the presence of recipient females, resulting in the absence of ejaculation, possibly caused by a reduced frequency of insertions. The reduction in frequency of insertion is believed to reflect a reduced ability of the animal to achieve penile erection, as oxytocin antagonists have been found to prevent penile erection.
Although it is suggested in Gimple and Frarenholz (supra) and Meston et al (supra) that oxytocin reduces the latency to ejaculation, other studies demonstrate that oxytocin has no effect on the latency to ejaculation. For example, it was demonstrated in Stoneham et al (J.Endocrinology 107: 97-106, 1985) that intravenous infusion of oxytocin in rats reduces the number of pre-ejaculation insertions in a dose-dependent manner but has no effect on the ejaculation latency. In addition, oxytocin infusion into the third ventricle increased the latency to the first upper body (mount) and insertion and extended the post ejaculation refractory period, but had no effect on injection latency (Stoneham et al supra).
Furthermore, studies have shown that the increase in oxytocin upon elimination of ejaculation is not different with respect to the time required to reach sexual arousal or orgasm. Murphy et al (J.of Clinical Endocrinology and metabolism, Vol.71, No.4(1990) p.1056-1058) demonstrated that the opioid antagonist, naloxone, had no effect on ejaculation in human volunteers, although the serum oxytocin pulse normally observed during ejaculation disappeared. In Ackerman et al (physiol Behav 63: 49-53[1997]), N-methyl-D-aspartate damage that destroys minicell PVN neurons but does not affect macrocellular neurons reduces oxytocin-immunoreactive fibers in the lower lumbar spinal cord (L5-L6). This reduction is accompanied by a significant reduction in ejaculatory semen ejaculatory, but the upper body, insertion and ejaculatory latency are unaffected.
[ summary of the invention ]
In one aspect, the present invention relates to a composition or pharmaceutical composition comprising a selective oxytocin antagonist compound for the treatment and/or prevention of a male ejaculatory disorder, in particular premature ejaculation. In the pharmaceutical composition, the selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient. Here, the composition (as with any other composition set forth herein) can be packaged for subsequent use in treating male ejaculatory disorders, particularly premature ejaculation.
In another aspect, the present invention relates to a composition or pharmaceutical composition comprising a selective oxytocin antagonist compound for the treatment and/or prevention of a male ejaculatory disorder, particularly premature ejaculation, while maintaining an erectile production mechanism, particularly a penile erection, and/or sexual drive; wherein the composition is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
In another aspect, the invention relates to the use of a selective oxytocin antagonist in the manufacture of a medicament (e.g., a pharmaceutical composition) for the treatment of a male ejaculatory disorder, in particular premature ejaculation.
In another aspect, the invention relates to the use of a selective oxytocin antagonist in the manufacture of a medicament (e.g., a pharmaceutical composition) for treating a male ejaculatory disorder, particularly premature ejaculation, while maintaining an erectile production mechanism, particularly a penile erection, and/or sexual drive.
In another aspect, the invention relates to the use of a selective oxytocin antagonist in the manufacture of a medicament (e.g., a pharmaceutical composition) for the treatment of a male ejaculatory disorder, in particular premature ejaculation.
In another aspect, the invention relates to the use of a selective oxytocin antagonist in the manufacture of a medicament (e.g., a pharmaceutical composition) for the treatment of male ejaculatory disorders, particularly premature ejaculation, while maintaining erectile production mechanisms, particularly penile erections, and/or sexual drive.
In one aspect, the invention relates to a method of treating and/or preventing a male ejaculatory disorder, in particular premature ejaculation, in a human or animal, comprising administering to the individual an effective amount of a selective oxytocin antagonist, wherein the selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
In another aspect, the invention relates to a method of treating and/or preventing a male ejaculatory disorder, particularly premature ejaculation, while maintaining an erectile production mechanism, particularly a penile erection, and/or sexual drive in a human or animal, comprising administering to the individual an effective amount of a selective oxytocin antagonist, wherein the selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
Further a pharmaceutical package is given comprising one or more compartments, wherein at least one compartment contains one or more selective oxytocin antagonists.
The invention also provides a process for preparing a pharmaceutical composition of the invention, which process comprises admixing one or more selective oxytocin antagonists with a pharmaceutically acceptable diluent, excipient or carrier.
In another aspect, the invention relates to an assay for identifying a substance useful for treating or preventing a male ejaculatory disorder (hereinafter referred to as a selective oxytocin antagonist), the assay comprising: determining whether the substance to be detected can directly delay the endogenous ejaculation process; wherein the delay is defined as an increase and/or recovery of ejaculation latency (i.e. the time elapsed from the first insertion to ejaculation) in the presence of the test substance as defined herein; such potentiation by a test substance is indicative that the test substance is useful in the treatment and/or prevention of a male ejaculatory disorder, in particular premature ejaculation, and wherein the test substance is a selective oxytocin antagonist. Preferably, the substance has no effect, or no significant effect, on penile erection. That is, preferably, the substance does not adversely affect penile erection; however, the substance enhances endogenous penile erection.
In another aspect, the invention relates to a method comprising the steps of:
(a) carrying out the assay of the invention;
(b) identifying one or more agents capable of increasing and/or restoring ejaculatory latency; and
(c) preparing an amount of the one or more identified substances; wherein the substance is a selective oxytocin antagonist.
In this aspect, the species identified in step (b) may be modified, for example to maximise activity, and step (a) may then be repeated. These steps may be repeated until the desired activity or pharmacokinetic profile is achieved.
Thus, in a further aspect, the present invention relates to a method comprising the steps of: (a1) carrying out the assay of the invention; (b1) identifying one or more agents capable of increasing and/or restoring ejaculatory latency; (b2) modifying one or more of the identified substances; (a2) optionally repeating step (a 1); and (c) preparing an amount of the one or more identified substances (i.e., those modified); wherein the substance is a selective oxytocin antagonist.
In another aspect, the invention relates to a method comprising the steps of:
(i) carrying out the assay of the invention;
(ii) identifying one or more agents capable of increasing and/or restoring ejaculatory latency;
(iii) testing the effect of the identified substance on penile erection in a test animal, e.g., an anesthetized rodent;
(iv) selecting a substance that has no effect, or no significant effect, on penile erection; and
(v) preparing a quantity of the one or more selected substances; wherein the substance is a selective oxytocin antagonist.
In this aspect, the substance identified in step (ii) may be modified, for example to maximise activity, and step (i) may then be repeated. These steps may be repeated until the desired activity or pharmacokinetic profile is achieved.
In another aspect, the invention relates to a diagnostic method comprising isolating one or more samples from a male in the course of sexual stimulation at successive time intervals after the onset of sexual stimulation, i.e. 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes, determining whether the sample contains an entity at those times, wherein the amount of the entity causes ejaculatory disorders in the male, preferably premature ejaculation; and wherein the entity may be modulated by a substance to achieve a beneficial effect, in particular to delay the time required for the entity to appear and/or reach a concentration peak; and wherein the substance is a selective oxytocin antagonist. Preferably, the fruitThe body is oxytocin. The sexual stimulation may be provided by e.g. a penile vibration stimulation device (fertecare,denmark).
In another aspect, the invention relates to a diagnostic composition or kit comprising means for detecting an entity in one or more isolated male samples, wherein the samples are taken at sequential time intervals, i.e. 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes, after the onset of sexual stimulation in the male during sexual stimulation; wherein the means can be used to determine whether the samples contain an entity and the amount thereof at these times will cause a male ejaculatory disorder, preferably premature ejaculation; and wherein the entity may be modulated by a substance to achieve a beneficial effect, in particular to delay the time required for the entity to appear and/or reach a concentration peak; and wherein the substance is a selective oxytocin antagonist. Preferably, the entity is oxytocin. The sexual stimulation may be provided by e.g. a penile vibration stimulation device (fertecare,denmark).
In another aspect, the invention relates to an animal model for identifying an agent capable of treating and/or preventing a male ejaculatory disorder, in particular premature ejaculation, the model comprising a male animal and comprising means for measuring the ejaculatory latency of the animal following introduction of a recipient female; and wherein the substance is a selective oxytocin antagonist.
The animal model may further comprise or be matched with another animal model comprising means for measuring penile erectile change. For example, a suitable additional model may include a anesthetized male animal that includes means to measure its intracavernosal pressure and/or cavernosal blood flow after stimulation of the animal's pelvic nerve; and wherein the substance is a selective oxytocin antagonist.
In another aspect, the invention relates to an assay for identifying an agent that directly enhances endogenous ejaculatory processes to treat or prevent ejaculatory disorders, in particular premature ejaculation, the assay comprising: administering an agent to the animal model of the invention; and measuring ejaculation latency (i.e. the time elapsed from the first insertion to ejaculation) of the animal following introduction of the recipient female; and wherein the substance is a selective oxytocin antagonist.
In another aspect, the invention relates to an assay for identifying a substance that directly enhances endogenous ejaculatory processes without affecting penile erection and/or sexual drive to treat or prevent ejaculatory disorders, in particular premature ejaculation, the assay comprising: administering an agent to the animal model of the invention; and measuring changes in the endogenous ejaculatory process; wherein the change is defined by the ejaculatory latency (i.e., the time elapsed from the first insertion to ejaculation) of the animal following introduction of the recipient female; measuring penile erection and/or sexual drive of the animal model to determine no change or no significant change therein; and wherein the substance is a selective oxytocin antagonist.
For ease of reference, these and further aspects of the invention will be discussed under appropriate sub-headings. However, the teachings under each subheading are not necessarily limited to the particular passage under that subheading.
The terms "selective oxytocin antagonist" and "selective oxytocin receptor antagonist" are used interchangeably and mean an oxytocin receptor antagonist that is selective for the oxytocin receptor relative to a vasopressin receptor, particularly the V1a receptor.
The term "ejaculation latency" is used herein to refer to the time elapsed from the first insertion to ejaculation. The term "recovery of ejaculatory latency" is used herein to refer to the alteration, preferably an increase, in time elapsed from the first insertion to ejaculation. Preferably, the time elapsed from the first insertion to ejaculation is altered (preferably increased) to a near normal level. Typically, a person with premature ejaculation will ejaculate within 30 seconds of the start of intercourse (i.e., from the first insertion) and often within 15 seconds of the start of intercourse (i.e., from the first insertion). In a preferred aspect of the invention, the ejaculation latency is increased to at least more than 30 seconds, preferably to at least more than 60 seconds, more preferably to at least more than 2 minutes, more preferably to at least more than 5 minutes, more preferably to at least more than 10 minutes. Suitably, ejaculation latency can be restored such that the time elapsed from the first insertion to ejaculation is sufficiently extended for the partner to be satisfied.
The term "sexual drive" is used herein to refer to sexual desire or desire.
The term "insertion" is used herein to refer to insertion of the vagina by the penis.
Preferred aspects
In one embodiment, the substance used in the present invention is preferably administered orally.
In another embodiment, the substance used in the present invention is for topical or intranasal application.
Preferably, the substances of the invention are used for the treatment and/or prevention of premature ejaculation.
Preferably, the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular the V1a receptor.
Preferably, the selective oxytocin antagonist is at least 30-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular the V1a receptor.
Preferably, the selective oxytocin antagonist is at least 50-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular the V1a receptor.
Preferably, the selective oxytocin antagonist is at least 100-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular the V1a receptor.
Preferably, the selective oxytocin antagonist is at least 200-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular the V1a receptor.
Preferably, the selective oxytocin antagonist is at least 250-fold selective for an oxytocin receptor as compared with a vasopressin receptor, in particular the V1a receptor.
The present invention also encompasses the administration of the agents of the invention prior to and/or during sexual arousal/stimulation.
Thus, it is highly desirable for certain aspects of the present invention to have a sexual arousal/stimulation step therein.
Here, "sexual arousal/stimulation" may be one or more of the following patterns: visual sexual arousal/stimulation, physical sexual arousal/stimulation, auditory arousal/stimulation or unexpected arousal/stimulation.
Thus, it is preferred that the substances of the invention are administered prior to and/or during sexual arousal/stimulation, particularly when the substances are delivered orally.
Preferred aspects
The present invention proposes the following (numbered) preferred aspects:
1. a composition comprising a selective oxytocin antagonist for the treatment or prevention of a male ejaculatory disorder; wherein the selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
2. The composition according to aspect 1, wherein the male ejaculatory disorder is premature ejaculation.
3. The composition according to aspect 1 or 2, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
4. The composition according to aspect 3, wherein the vasopressin receptor is the V1a receptor.
5. Use of a selective oxytocin antagonist in the manufacture of a medicament for the treatment of a male ejaculatory disorder.
6. The use according to aspect 5, wherein the male ejaculatory disorder is premature ejaculation.
7. The use according to aspect 5 or 6, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
8. The use according to aspect 7, wherein the vasopressin receptor is the V1a receptor.
9. Use according to any of aspects 5 to 8, wherein the selective oxytocin antagonist is administered prior to and/or during sexual arousal.
10. The use according to any of aspects 5-9, wherein the selective oxytocin antagonist is administered orally.
11. A method for treating or preventing a male ejaculatory disorder in a human or animal comprising administering to the individual an effective amount of a selective oxytocin antagonist; wherein the selective oxytocin antagonist is optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient.
12. The method according to aspect 11, wherein the male ejaculatory disorder is premature ejaculation.
13. The method according to aspect 11 or 12, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
14. The method according to aspect 13, wherein the vasopressin receptor is the V1a receptor.
15. The method according to any of aspects 11-14, wherein the selective oxytocin antagonist is administered prior to and/or during sexual arousal.
16. The use according to any of aspects 11-15, wherein the medicament is administered orally.
17. A pharmaceutical package comprising one or more compartments, wherein at least one compartment comprises one or more selective oxytocin antagonists.
18. The pharmaceutical package according to aspect 17, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
19. The pharmaceutical package according to aspect 18, wherein the vasopressin receptor is the V1a receptor.
20. A process for the preparation of a pharmaceutical composition comprising admixing one or more selective oxytocin antagonists with a pharmaceutically acceptable diluent, excipient or carrier.
21. The method according to aspect 20, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
22. The method according to aspect 21, wherein the vasopressin receptor is the V1a receptor.
23. An assay method for identifying an agent useful for treating and/or preventing a male ejaculatory disorder, the assay method comprising: determining whether a substance to be tested can directly enhance the endogenous ejaculation process; wherein the enhancement is defined as an increase and/or a recovery of ejaculation latency in the presence of a test substance as defined herein; such an enhancement of the test substance is indicative that the test substance is useful in the treatment or prevention of a male ejaculatory disorder, and wherein the test substance is a selective oxytocin antagonist.
24. The assay according to aspect 23, wherein the male ejaculatory disorder is premature ejaculation.
25. The assay according to aspect 23 or 24, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
26. The assay according to aspect 25, wherein the vasopressin receptor is the V1a receptor.
27. An agent identified by the assay method of any one of aspects 23 to 26.
28. A substance according to aspect 27 for use in the treatment or prevention of a male ejaculatory disorder.
29. The substance according to aspect 28. Wherein the male ejaculatory disorder is premature ejaculation.
30. A medicament for oral administration to treat a male ejaculatory disorder, wherein the medicament comprises a substance according to aspect 27.
31. The medicament according to aspect 30, wherein the male ejaculatory disorder is premature ejaculation.
32. The medicament according to aspect 30 or 31, wherein the medicament is administered prior to and/or during sexual arousal.
33. The medicament according to any one of aspects 30-32, wherein the medicament is administered orally.
34. A method comprising the steps of: (a) performing the assay method according to any one of aspects 23-26; (b) identifying one or more agents capable of increasing and/or restoring ejaculatory latency; and (c) preparing an amount of the one or more identified substances; wherein the substance is a selective oxytocin antagonist.
35. The method according to aspect 34, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
36. The method according to aspect 35, wherein the vasopressin receptor is the V1a receptor.
37. An animal model for identifying an agent capable of treating or preventing a male ejaculatory disorder, said model comprising a male animal and comprising means for measuring the ejaculatory latency of said animal following introduction into a recipient female; and wherein the substance is a selective oxytocin antagonist.
38. The animal model according to aspect 37, wherein the male ejaculatory disorder is premature ejaculation.
39. The animal model according to aspect 37 or 38, wherein the selective oxytocin antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
40. The animal model according to aspect 39, wherein the vasopressin receptor is the V1a receptor.
41. An assay method for identifying an agent that directly enhances endogenous ejaculatory processes to treat or prevent ejaculatory disorders, the assay method comprising: administering a substance to the animal model according to any one of aspects 37-40; and measuring ejaculation latency of the animal following introduction of the recipient female; and wherein the substance is a selective oxytocin antagonist.
42. Use of a combination of one or more selective oxytocin antagonists and one or more of the following auxiliary active substances for the preparation of a medicament for the treatment and/or prevention of a male ejaculatory disorder:
i) PDE inhibitors, more particularly PDE5 inhibitors, preferably having an IC50 value for the respective enzyme of less than 100 nM;
ii) 5-hydroxytryptamine receptor agonists or modulators, more particularly agonists or modulators of the 5HT2C, 5HT1B and/or 5HT1D receptors, including ampirtoline;
iii) antagonists or modulators of 5-hydroxytryptamine receptors, more particularly antagonists or modulators of 5HT1A, including NAD-299 (robrazol) and WAY-100635, and/or more particularly antagonists or modulators of 5HT3 receptors, including patapride, granisetron, ondansetron, tropisetron (tropistron) and MDL-73147 EF;
iv) antidepressants, in particular i) selective 5-hydroxytryptamine reuptake inhibitors (SSRi) including sertraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, mirtazapine, nefazodone and trazodone; ii) tricyclic antidepressants (TCAs) including clomipramine, desipramine, imipramine, amitriptyline, doxepin (doxepin), amoxapine, maprotiline, nortriptyline, protriptyline, trimipramine and bupropion; and iii) a monoamine oxidase inhibitor;
v) alpha-adrenergic receptor antagonists (also known as alpha-adrenergic blockers, alpha-blockers or alpha-receptor blockers), suitable alpha 1-adrenergic receptor antagonists include: phentolamine, prazosin, phentolamine mesylate (phentolamine mesylate), trazodone, alfuzosin, indoramine, naftopidil, tamsulosin, phenoxybenzamine, rauwolfia alkaloid, Recordatii 15/2739, SNAP 1069, SNAP 5089, RS17053, SL 89.0591, doxazosin, terazosin, and abanoquine; suitable alpha 2-adrenergic receptor antagonists include: tolazoline, tramazoline, efacrine, yohimbine, mizolazine, clonidine and dibenzylamine; suitable non-selective alpha-adrenergic receptor antagonists include dapiprazole; other alpha-adrenergic receptor antagonists are described in the following: WO 99/30697, US4,188,390, US4,026,894, US3,511,836, US4,315,007, US3,527,761, US3,997,666, US2,503,059, US4,703,063, US3,381,009, US4,252,721 and US2,599,000;
vi) rapid onset selective 5-hydroxytryptamine reuptake inhibitors.
43. Use of a combination of one or more selective oxytocin antagonists and one or more PDE inhibitors (PDEi's) for the manufacture of a medicament for the treatment or prevention of a male ejaculatory disorder.
44. The use according to aspect 42 or 43, wherein the male ejaculatory disorder is premature ejaculation.
45. The use according to aspect 43 or 44, wherein the PDEi is a PDE5 inhibitor (PDE5 i).
46. The use according to any of aspects 42-45, wherein the medicament is administered orally.
47. A pharmaceutical composition consisting of one or more selective oxytocin antagonists and one or more PDEi's, optionally in admixture with a pharmaceutically acceptable carrier, diluent or excipient.
48. The pharmaceutical composition according to aspect 47, wherein the PDEi is PDE5 i.
49. The pharmaceutical composition according to aspect 47 or 48, wherein the composition is administered orally.
50. Use of a pharmaceutical composition according to any one of aspects 47-49 in the manufacture of a medicament for the treatment and/or prevention of a male ejaculatory disorder.
Surprising and unexpected discovery
The present invention demonstrates the following surprising and unexpected findings:
(a) administration of selective oxytocin antagonists may increase ejaculation latency. Preferably, administration of the selective oxytocin antagonist may restore ejaculation latency, preferably to a near normal level;
(b) administration of selective oxytocin antagonists may unexpectedly increase ejaculation latency without significantly inhibiting and/or adversely affecting the mechanisms of erection production, particularly penile erection. Preferably, administration of the selective oxytocin antagonist can restore ejaculation latency, preferably to near normal levels, without significantly inhibiting and/or adversely affecting the erection mechanisms, particularly penile erection;
(c) administration of a selective oxytocin antagonist can increase ejaculation latency without significantly inhibiting and/or adversely affecting sexual drive. Preferably, administration of a selective oxytocin antagonist can restore ejaculation latency, preferably to near normal levels, without significantly inhibiting and/or adversely affecting sexual drive.
Advantages of the invention
The present invention is advantageous because:
(a) premature ejaculation can be treated by selective inhibition of oxytocin receptors using selective oxytocin antagonists;
(b) selective inhibition of the oxytocin receptor by use of a selective oxytocin antagonist may unexpectedly result in premature ejaculation without significantly inhibiting and/or adversely affecting the mechanisms of erection production, particularly penile erection;
(c) selective inhibition of oxytocin receptors by the use of selective oxytocin antagonists may unexpectedly lead to premature ejaculation without significant inhibition and/or adverse impact on sexual drive.
Patient group
Patients suffering from ejaculatory disorders, particularly premature ejaculation, should benefit from treatment with a selective oxytocin antagonist.
Early studies indicated that the following group of patients with ejaculatory disorders, in particular premature ejaculation, should benefit from treatment with a selective oxytocin antagonist (or a combination comprising a selective oxytocin antagonist as listed below). These patient groups include those suffering from one or more of the following: neurological disorders, physiological disorders, lack of psychogenic skills, physical illness, physical injury, drug side effects, mental suffering and interpersonal relation suffering.
Oxytocin receptor
As indicated above, the substance may be any suitable substance that is capable of acting as a selective oxytocin antagonist.
Background information relating to the oxytocin receptor has been published by Victor A.McKusick et alhttp: //WWW3.ncbi.nlm.nih.gov/Omim/searchomim.htmIs ready. The following citations relating to the oxytocin receptor were extracted from this source:
the structure and expression of the human oxytocin receptor cDNA isolated by expression cloning was reported by "Kimura et al (1992: Nature 356: 526-529). The encoded receptor is a 388 amino acid polypeptide with 7 transmembrane domains typical of G protein-coupled receptors. Oxytocin receptors expressed in Xenopus oocytes specifically react to oxytocin and induce inward membrane currents. Messenger RNAs for this receptor have two sizes, 3.6kb in the chest and 4.4kb in the ovary, endometrium and myometrium. mRNA levels in the uterine muscle layer are very high during term. Inoue et al (1994 biol. chem.269: 32451-32456) demonstrated the presence of a single copy of the OXTR gene in the human genome by Southern blots. They confirmed that the gene was located at 3p26.2 by fluorescence in situ hybridization. The gene is approximately 17kb in length and contains 3 introns and 4 exons. Exons 1 and 2 correspond to the 5' -non-coding region, and exons 3 and 4 follow to encode the amino acids of the receptor. Intron 3, 12kb, is the largest, which distinguishes the coding regions immediately after the putative 6-transmembrane-spanning domain. The transcription initiation sites were confirmed to be located 618 and 621bp upstream of the methionine start codon by primer extension analysis. Simmons et al (1995 Genomics 26: 623 ═ 625) identified the OXTR gene at 3p25 "by PCR analysis of somatic cell hybrids and fluorescent in situ hybridization.
In Gimple and Fahrenholz (physical reviews Vol.81, No.2, April 2001), detailed reviews are given concerning the receptor architecture. It is stated therein that, in addition to the isolation and identification of cDNAs encoding the human oxytocin receptor (cf. Kimura et al [ supra ]), the oxytocin receptor coding sequences from pigs (Gorbulev et al Eur.J.biochem.215, 1-71993), rabbits (Rozen et al Proc.Natl.Acad.Sci.USA 92: 200-2041995), sheep (Riley et al J.biol.chem.266: 21428-21433, 1991), cattle (Bathgate et al DNA biol.14: 1037 Cell 1048, 1995), mice (Kubota et al mol.cell Endocrinol 124: 25-321996) and macaques (Salvator et al J.Recept Signal Transduct Res.18: 15-24, 1998) have been identified so far.
Oxytocin receptor sequence data
Both the nucleotide and amino acid sequences of the human oxytocin receptor are available in the literature. As an example, only the amino acid sequence of the human oxytocin receptor is given in SEQ ID NO: 1 in (c).
Selective oxytocin antagonists
A detailed description of a suitable assay system for identifying and/or studying oxytocin antagonists is given hereinafter in the section entitled "oxytocin antagonist assay".
An example of a suitable oxytocin antagonist is as follows:
L-368，899
the synthesis of L-368, 899 is described in Williams et al (1994) J.Med.chem.37, 565-571.
L-368, 899 is a selective oxytocin antagonist. L-368, 899 is more than 20-fold selective for the oxytocin receptor than for the vasopressin receptor, particularly the V1a receptor.
Certain known oxytocin antagonists, such as 8-seminal oxytocin, are sometimes referred to as "selective oxytocin antagonists". However, d (CH)2)5Tyr(Me)-Orn88-seminal oxytocin (hereinafter referred to as "8-seminal oxytocin") is only 2-fold to 3-fold selective for the oxytocin receptor over the vasopressin receptor, in particular the V1a receptor. Thus, 8-seminal oxytocin is essentially a non-selective oxytocin/vasopressin antagonist and thus is not within the scope of the term "selective oxytocin antagonist" according to the invention. Preferably, the selective oxytocin antagonist according to the invention is at least 20-fold selective for the oxytocin receptor as compared with the vasopressin receptor, in particular the V1a receptor.
Oxytocin antagonist binding assay and vasopressin V1a receptor binding assay
i) Buffering agent
Cell growth medium Hams F12 Nutrient Mix
10％FCS
2mM L-Glutamine
400 microgram/ml G418
15mM HEPES
Membrane preparation buffer 50mM Tris-HCl, pH7.8
10mM MgCl2
Protease inhibitors
Freezing buffer 50mM Tris-HCl, pH7.8
10mM MgCl2
20% glycerol
Assay Medium 50mM Tris ═ HCl, pH7.8
10mM MgCl2
0.25％BSA
Max. 0.5μM(arg8) -8-fine oxytocin
Prepared in 2.5% DMSO/50mM Tris-HCl,
pH7.8，10mM MgCl2in
Min. 2.5％DMSO/50mM Tris-HCl，pH7.8，
10mM MgCl2
ii) Compound dilution (final concentration in assay 10. mu.M)
a) HTA stock compound, 4mM in 100% DMSO.
b) Dilute the compound in dH2In O, 200. mu.M.
c) Further at 100mM Tris-HCl, pH8，20mM MgCl2Diluted to 100. mu.M. 2.5% DMSO, 50mM Tris-HCl, pH7.8, 10mM MgCl was obtained2To the final concentration of (c).
d) Diluted stock solutions were used in TECAN Genesis 50mM Tris-HCl, pH7.8, 10mM MgCl2Ten 1:2 dilutions were made in 2.5% DMSO.
e) According to the layout requirements of the ECADA assay plate, 10. mu.l of the compound was dispensed in 384-well Optiplates, leaving room for the standard (arg)8) -8-fine oxytocin IC 50. These plates can be stored at 4 ℃.
f) On the day of the assay, 10 microliters of max was added to the + wells, 10 microliters of min was added to the-wells, and duplicate (arg) was added8) 8-addition of Preoxytocin 10 gradients of 1:2 serial dilutions with a maximum concentration of 100nM (20 nM final concentration).
(iii) Maintenance of oxytocin receptor-CHO cells
Cell lines are routinely maintained in continuous culture in 50 ml growth medium in 225 cm flasks
Removal of medium from the cell monolayer, washing with PBS and incubation with trypsin untilUntil dissociation occurred, and the cells were passaged. After knocking the cells off the bottom of the flask, the cells were resuspended in growth medium and grown at 8 × 105The concentration of cells/flask was seeded into a 225 cm square flask.
(iv) Cell growth in roller bottles (roller bottles)
The cells were plated at 6X 106The cells/flask density was seeded into 10 x 850 square centimeter roller bottles and allowed to grow to near confluence.
Cells were removed from the vials using trypsin as described above and seeded into 100x roller bottles (i.e. 1:10 split ratio).
Cells were grown again to near confluence after which the growth medium was removed, 40 ml PBS/flask was added and scraped using CellMate to harvest the cells. The cell suspension was then centrifuged at 2000rpm, washed in PBS, centrifuged again and the cell pellet frozen in portions at-80 ℃.
v) Membrane preparation
The cell pellet was removed from the freezer, thawed on ice and resuspended at a rate of 3 ml membrane preparation buffer per ml clumped cell volume.
The suspension was then homogenized on ice for several 5 seconds using a mechanical homogenizer and centrifuged at 25,000Xg for 30 minutes.
After resuspending the pellet at a ratio of 1 ml of freezing buffer per ml of the original clumped cell volume, the suspension was homogenized slightly to remove small pieces. The protein concentration was then measured and finally the membrane suspension was frozen in portions at a minimum of 5 mg/ml at-80 ℃.
vi) assay
After thawing the membrane on ice, it was diluted to 1 mg/ml in assay buffer. SPA beads were resuspended at 50 mg/ml in assay buffer. At these concentrations, the beads and membrane were pre-coupled by placing 30 micrograms of protein per milligram of bead on a head-to-tail (top-to-tail) shaker for 2 hours at 4 ℃. The beads/membrane were then centrifuged at 2000rpm for 10 minutes and the pellet resuspended at 3 mg/ml.
For125All manipulations of I-OVTA were performed using tips silanized by Sigmacote (tip). All bottles and tubes were also silanized. For every 50. mu. Ci of lyophilized ligand, will125I-OVTA was diluted in 1 ml assay buffer. Then 5 microliters of sample were counted with a liquid scintillation Counter (protocol 61 on Wallac Counter) and repeated once, followed by calculation of the ligand concentration (see examples below). This is to overcome ligand loss due to sticking. Using the measured concentration, the125I-OVTA was diluted to 0.3nM in assay buffer.
Example (b):
if 5. mu.l gives 500000dpm and the specific activity of the ligand is 2200 Ci/mmol:
500000/(2.2 x 2200 x 5) nM
Add 20 μ l of bead/membrane preparation to the prepared Optiplates using Multi-drop. The bead/membrane preparation was kept in suspension using a stirred flask. Then 20 microliters of each well of Optiplates was added using a Multi-drop125I-OVTA. After 4 hours incubation at room temperature, the plates were counted at 30 seconds/well using TopCount NXT.
B. Vasopressin V1a receptor binding assay
i) Material
Cloned human vasopressin Vla Protein/cell
Receptors, in CHO cells, sciences
·HEPES Sigma(H7523)
Magnesium chloride (MgCl)2) Sigma(M2670)
Bovine Serum Albumin (BSA) Sigma (A6003)
Glycerol Sigma (G5150)
Mixed protease inhibitor tablets Roche (1697498)
Pierce BCA protein assay substance Pierce (23225)
8-Arg [ phenylalanyl-3, 4, 5-3H]Vasopressin NEN (NET800)
(3H-AVP)
·d(CH2)5Tyr (Me) AVP [ beta-mercapto-Sigma (V2255)
Beta, beta-Cyclopentamethylenepropionyl, O-Me-Tyr2，Arg8]Blood vessels
Vasopressin (beta MCPVP)
Dimethyl sulfoxide Stores (W34)
96-hole Polypropylene blocks store (D8281)
Polyethyleneimine (PEI) Sigma (P3143)
96-well Unifilter plate GF/C Packard (6005174)
·Topseal A Packard(6005185)
·Microscint-O Packard(6013611)
·SR49059(UK222，633) Compound Control
Equipment: packard Unifilter Unit
Top Counter/NXT Counter
ii) method
Operation solution:
membrane preparation buffer: 25mM HEPES (pH7.4)
5mM MgCl2
Protease inhibitor (1 tablet/50 ml)
Freezing buffer solution: 25mM HEPES (pH7.4)
5mM MgCl2
20% glycerol
Determination of buffer: 25mM HEPES (pH7.4)
5mM MgCl2
0.05％BSA
Washing buffer solution: 25mM HEPES (pH7.4)
5mM MgCl2
35nM solution of H-AVP in assay buffer
(Final assay concentration 0.5nM)
Total 25% DMSO in ddH2In O
NSB in 25% DMSO/ddH210 μ M β MCPVP in O (Final)
The measured concentration was 1. mu.M)
STD SR49059(UK222, 633) diluted in 25% DMSO/ddH2O
In (b), starting from the highest concentration of 1. mu.M (maximum for 100 nM)
Final concentration determination) and continued dilution to 30 with a 0.5Log difference
pM (final assay concentration for 3 pM)
A compound: 50 microliter of compound at a concentration of 4mM in 100% DMSO.
The solution is to be in dH2Diluted 4-fold in O to give 25% DMSO
1mm in the middle. Except for the first dilution (1 mM)
To 300. mu.M) was in 18% DMSO (25% after dilution)
DMSO), compounds were half-logarithmically separated
Further dilutions were made in 25% DMSO. Dilution was with Tecan and
protocol file Kin28IC50dilution2, gem or
The operation is performed manually. Depending on the compound desired, 10pt IC50
The curve is initiated by lower concentrations, but all drugs
The selection was started from 100. mu.M.
0.5% PEI in distillation of H2Preparation of 50% PEI in dH2Diluting in O to
0.5％。
iii) Membrane preparation
Removing the cell pellet from the freezer and gently thawing on ice
3 ml of membrane preparation buffer per ml of primary clumping cell volume was added and the suspension was homogenized on ice using a polytron for several 5 seconds until well dispersed, followed by centrifugation at 1000rpm for 10 minutes.
The supernatant was removed and stored on ice. Another membrane preparation buffer was added to the pellet at a rate of 3 ml of membrane preparation buffer per ml of the original pelleted cell volume, homogenized on ice, and then centrifuged at 1000rpm for 10 minutes.
The supernatant was removed and, after addition to the previously removed supernatant, centrifuged at 25,000Xg for 30 minutes at 4 ℃.
After resuspending the 25,000Xg pellet by homogenization in 1 ml of freezing buffer per 1 ml of primary clumping cell volume, the protein concentration was determined.
iv) protein concentration determination
In dH2BSA was prepared at the following concentrations: 2000, 1000, 500, 250, 12562.5 and 31.25. mu.g/ml.
Add 10. mu.l of the BSA solution to each clear 96-well plate in triplicate (see plate figure in appendix) and add 10. mu.l dH to three blank wells2O。
Add 10. mu.l each of the membrane preparations (triplicate) to the plate, i.e., 1:3(1-in-3), 1:10(1-in-10), 1:30(1-in-30), and 1:100(1-in-100) dilutions of the membrane preparations.
Add 200. mu.l of Pierce protein reaction (50A:1B) to each well and incubate the plate at 37 ℃ for 30 minutes before reading on an Anthos spectrophotometer with absorption at 570 nm.
The protein concentration in the membrane preparation was determined from the BSA standard curve (using membrane preparation dilution falling in the center of the standard curve).
After diluting the membrane preparation to a protein concentration of 5 mg/ml in the freezing buffer, the membrane preparation was frozen in 200 μ l aliquots at-80 ℃.
v) assay protocol
Preparation of assay reagent(s) (ii)3H-AVP, β MCPVP (NSB compound) and test compound-see above working solution). Any peptide solution was kept on ice.
Plate format 10-point IC50-4 compound per plate duplicate panel. The following reagents were added to the appropriate wells of a 96-well polypropylene block and vortexed.
In each total well (T): 25 microliter3H-AVP
(A1, B1, C1, D1, E12, F12, G12& H12) 25 microliter of vehicle
In each NSB well (N): 25 microliter3H-AVP
(A12, B12, C12, D12, E1, F1, G1& H1) 25 microliter of beta MCPVP
At 25. mu.l per assay well3H-AVP
25 microliter of test compound
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | T | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | N |
| B | T | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | N |
| C | T | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | N |
| D | T | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | N |
| E | N | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | T |
| F | N | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | T |
| G | N | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | T |
| H | N | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | T |
C1-concentration 1; c2-concentration 2; c3 concentration 3, and so on.
Row a & B ═ STD
Line C & D ═ compound 1
Line E & F ═ compound 2
Row G & H ═ compound 3
The membrane proteins were gently thawed on ice and diluted to the most appropriate protein concentration for the assay (see appendix for linear protein determination) (about 100. mu.g/ml)
Add 200. mu.l of membrane protein to each well to initiate the reaction, and then incubate the block set at room temperature for 60 minutes with gentle shaking.
The reaction was stopped by filtration through a Unifilter GF/C filter presoaked in 0.5% PEI and washed rapidly with 3x1 ml of ice-chilled wash buffer.
The filter was dried in an oven at 55 ℃ for 2 hours or on a bench overnight (about 16 hours).
The bottom of the filter was sealed and 30. mu.l Microscint-O was added to each well. The filter was then sealed using Topseal A and used on a Packard TopCount (Bld 503/G7A), [2 ]3H]The 96 well unifilter protocol 11 was counted.
C. Data analysis
Data analysis was performed with ECADA.
Specific binding was calculated as follows:
specific binding-average Total cpm-average NSB cpm
For the test compounds, the amount of ligand bound to the receptor is expressed as follows:
% binding ═ sample cpm-mean NSB cpm)/specific binding cpm × 100
Percent inhibition of ligand binding is expressed as% inhibition calculated as follows:
% inhibition of 100%
Oxytocin antagonist function determination method and vasopressin V1a antagonist function determination method
Spontaneous contractions of the myometrium in humans, non-human primates and rodents are sensitive to selective oxytocin receptor antagonists in vitro (see Wilson et al BJOG 2001 Sep; 108 (9): 960-6).
In vitro pharmacology of spontaneous contractions of the myometrium in humans and animals. Human myometrial samples were obtained from caesarean section. Tissue strips were suspended in organ baths to record isometric forces (isomerceforce.) cumulative concentration effect curves for selective oxytocin receptor antagonists and mixed oxytocin/vasopressin V1a receptor antagonists were obtained. In vitro inhibition of spontaneous myometrial contractions was observed.
Combination of
In more detail, the invention further comprises the combination of a compound of the invention with one or more auxiliary active substances (see discussion below regarding suitable examples) for use herein for the treatment of male ejaculatory disorders, in particular premature ejaculation.
The invention further comprises the use of a combination consisting essentially of a selective oxytocin antagonist according to the invention and two auxiliary active substances (see discussion below regarding suitable examples) for the manufacture of a medicament for the treatment and/or prevention of a male ejaculatory disorder, in particular premature ejaculation, as described herein.
The invention further comprises the use of a combination of a selective oxytocin antagonist according to the invention and two auxiliary active substances (see discussion below concerning appropriate examples) for the manufacture of a medicament for the treatment and/or prevention of a male ejaculatory disorder, in particular premature ejaculation, as described herein.
The invention further comprises the use of a combination consisting essentially of a selective oxytocin antagonist according to the invention and an auxiliary active substance (see discussion below regarding suitable examples) for the manufacture of a medicament for the treatment and/or prevention of a male ejaculatory disorder, in particular premature ejaculation, as described herein.
The invention further comprises the use of a combination of a selective oxytocin antagonist according to the invention and an auxiliary active substance (see discussion below concerning appropriate examples) for the manufacture of a medicament for the treatment and/or prevention of a male ejaculatory disorder, in particular premature ejaculation, as described herein.
Thus, the combination aspect of the present invention provides a pharmaceutical combination (for simultaneous, separate or sequential administration) comprising a compound of the present invention together with one or more auxiliary active substances (see discussion below regarding suitable examples).
A further combined aspect of the invention provides a pharmaceutical composition (for simultaneous, separate or sequential administration) consisting essentially of a selective oxytocin antagonist and two auxiliary active substances (see discussion below regarding suitable examples).
A further combined aspect of the invention provides a pharmaceutical composition (for simultaneous, separate or sequential administration) consisting of a selective oxytocin antagonist and two co-active substances (see discussion below regarding suitable examples).
In a further combined aspect the invention provides a pharmaceutical composition (for simultaneous, separate or sequential administration) consisting essentially of a selective oxytocin antagonist and an auxiliary active substance (see discussion below regarding appropriate examples).
In a further combined aspect of the invention there is provided a pharmaceutical composition (for simultaneous, separate or sequential administration) consisting of a selective oxytocin antagonist and an auxiliary active substance (see discussion below regarding appropriate examples).
Auxiliary active substance
Suitable auxiliary actives for use in the compositions of the present invention include:
1) PDE inhibitors, more particularly PDE5 inhibitors (see below), which preferably have an IC50 value of less than 100nM against the corresponding enzyme;
2) 5-hydroxytryptamine receptor agonists or modulators, more specifically agonists or modulators of the 5HT2C, 5HT1B and/or 5HT1D receptors, including ampirtoline;
3) 5-hydroxytryptamine receptor antagonists or modulators, more specifically antagonists or modulators of 5HT1A, including NAD-299 (robalotan) and WAY-100635, and/or more specifically antagonists or modulators of 5HT3 receptors, including batipride, granisetron, ondansetron, tropisetron, and MDL-73147 EF;
4) antidepressants, specifically i) selective 5-hydroxytryptamine reuptake inhibitors (SSRi) including sertraline, fluoxetine, fluvoxamine, paroxetine, citalopram, venlafaxine, mirtazapine, nefazodone and trazodone; ii) tricyclic antidepressants (TCAs) including clomipramine, desipramine, imipramine, amitriptyline, doxepin, amoxapine, maprotiline, nortriptyline, protriptyline, trimipramine and bupropion; and iii) a monoamine oxidase inhibitor;
5) an alpha-adrenergic receptor antagonist (also known as an alpha-adrenergic blocker, an alpha-blocker or an alpha-receptor blocker); suitable alpha 1-adrenergic receptor antagonists include: phentolamine, prazosin, phentolamine mesylate, trazodone, alfuzosin, indoramine, naftopidil, tamsulosin, phenoxybenzamine, rauvolfia alkaloids, Recordati 15/2739, SNAP 1069. SNAP 5089, RS17053, SL 89.0591, doxazosin, terazosin, and abanoquine; suitable alpha 2-adrenergic receptor antagonists include: tolazoline, tramazoline, efaroxan, yohimbine, idazoxan, clonidine and dibenzylamine; suitable non-selective alpha-adrenergic receptor antagonists include dapiprazole; other alpha-adrenergic receptor antagonists are described in the following: WO 99/30697, US4,188,390, US4,026,894, US3,511,836, US4,315,007, US3,527,761, US3,997,666, US2,503,059, US4,703,063, US3,381,009, US4,252,721 and US2,599,000; all incorporated herein by reference;
6) a rapid onset selective 5-hydroxytryptamine reuptake inhibitor (rapid onset SSRI), such as 3- [ (dimethylamino) methyl ] -4- [4- (methylsulfanyl) phenoxy ] benzenesulfonamide (as disclosed in WO 01/72687-example 28).
By cross-reference to a compound contained in a patent or patent application that may be used in the present invention we mean a therapeutically active compound as defined in those claims (especially claim 1) and in the specific examples (all incorporated herein by reference).
If a combination of active substances is administered, they may be administered simultaneously, separately or sequentially.
Auxiliary substances-PDE 5 inhibitors
Suitable cGMP PDE5 inhibitors that may be used in accordance with the present invention include:
pyrazolo [4, 3-d ] pyrimidin-7-ones as disclosed in EP-A-0463756; pyrazolo [4, 3-d ] pyrimidin-7-ones as disclosed in EP-A-0526004; pyrazolo [4, 3-d ] pyrimidin-7-ones as disclosed in published international patent application WO 93/06104; isomeric pyrazolo [3, 4-d ] pyrimidin-4-ones as disclosed in published international patent application WO 93/07149; quinazolin-4-ones disclosed in published international patent application WO 93/12095; pyrido [3, 2-d ] pyrimidin-4-ones as disclosed in published international patent application WO 94/05661; purine-6-ones disclosed in published international patent application WO 94/00453; pyrazolo [4, 3-d ] pyrimidin-7-ones as disclosed in published international patent application WO 98/49166; pyrazolo [4, 3-d ] pyrimidin-7-ones as disclosed in published international patent application WO 99/54333; pyrazolo [4, 3-d ] pyrimidin-4-ones as disclosed in EP-A-0995781; pyrazolo [4, 3-d ] pyrimidin-7-ones as disclosed in published international patent application WO 00/24745, pyrazolo [4, 3-d ] pyrimidin-4-ones as disclosed in EP-A-0995750; the compounds disclosed in published international patent application WO 95/19978; a compound disclosed in published international patent application WO99/24433 and a compound disclosed in published international patent application WO 93/07124.
Pyrazolo [4, 3-d ] pyrimidin-7-ones as disclosed in published international patent application WO 01/27112; pyrazolo [4, 3-d ] pyrimidin-7-ones as disclosed in published international patent application WO 01/27113; compounds disclosed in EP-A-1092718 and compounds disclosed in EP-A-1092719.
Preferred phosphodiesterase type V inhibitors (phosphodiesterase 5(PDE) inhibitors; PDE5 i's) useful in the present invention include:
5- [ 2-ethoxy-5- (4-methyl-1-piperazinylsulfonyl) phenyl ] -1-methyl-3-n-propyl-1, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (sidenafil), also known as 1- [ [3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4, 3-d ] pyrimidin-5-yl) -4-ethoxyphenyl ] sulfonyl ] -4-methylpiperazine (cf. EP-A-0463756);
5- (2-ethoxy-5-morpholinoacetylphenyl) -1-methyl-3-n-propyl-1, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (cf. 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- (2-methoxy-1 (R) -methylethoxy) pyridin-3-yl ] -2-methyl-2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one; also known as 3-ethyl-5- {5- [ 4-ethylpiperazin-1-ylsulfonyl ] -2- ([ (1R) -2-methoxy-1-methylethyl ] oxy) pyridin-3-yl } -2-methyl-2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO 99/54333);
5- [ 2-ethoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl ] -3-ethyl-2- [ 2-methoxy-ethyl ] -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one, also known as 1- { 6-ethoxy-5- [ 3-ethyl-6, 7-dihydro-2- (2-methoxyethyl) -7-oxo-2H-pyrazolo [4, 3-d ] pyrimidin-5-yl ] -3-pyridylsulfonyl } -4-ethylpiperazine (see WO01/27113, example 8);
5- [ 2-isobutoxy-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 (cf. WO01/27113, 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 (cf. WO01/27113, example 66);
5- (5-acetyl-2-propoxy-3-pyridyl) -3-ethyl-2- (1-isopropyl-3-azetidinyl) -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO01/27112, example 124);
5- (5-acetyl-2-butoxy-3-pyridyl) -3-ethyl-2- (1-ethyl-3-azetidinyl) -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO01/27112, example 132);
(6R, 12aR) -2, 3, 6, 7, 12, 12 a-hexahydro-2-methyl-6- (3, 4-methylenedioxyphenyl) -pyrazino [2 ', 1': 6, 1] pyrido [3, 4-b ] indole-1, 4-dione (IC-351), i.e. the compounds of examples 78 and 95 in published International patent application WO 95/19978, and the compounds of examples 1, 3, 7 and 8;
2- [ 2-ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl ] -5-methyl-7-propyl-3H-imidazo [5, 1-f ] [1, 2, 4] triazin-4-one (vardenafil), also known as 1- [ [3- (3, 4-dihydro-5-methyl-4-oxo-7-propylimidazo [5, 1-f ] -as-triazin-2-yl) -4-ethoxyphenyl ] sulfonyl ] -4-ethylpiperazine, i.e. the compounds of examples 20, 19, 337 and 336 in published international patent application WO 99/24433; and
the compound of example 11 (EISA [); and
oteclla DP |, |. Chem., 2000, 43, 1257, compounds 3 and 14.
Other types of cGMP PDE5 inhibitors that may be used with the present invention include: 4-bromo-5- (pyridylmethylamino) -6- [3- (4-chlorophenyl) -propoxy ] -3(2H) pyridazinone; 1- [4- [ (1, 3-benzodioxol-5-ylmethyl) amino ] -6-chloro-2-quinazolinyl ] -4-piperidine-carboxylic acid monosodium salt; (+) -cis-5, 6a, 7, 9, 9, 9 a-hexahydro-2- [4- (trifluoromethyl) -phenylmethyl-5-methyl-cyclopenta [4, 5] -imidazo [2, 1-b ] purin-4 (3H) one; furazlocillin; cis-2-hexyl-5-methyl-3, 4, 5, 6a, 7, 8, 9, 9 a-octahydro-cyclopenta [4, 5] -imidazo [2, 1-b ] purin-4-one; 3-acetyl-1- (2-chlorophenylmethyl) -2-propylindole-6-carboxylate; 3-acetyl-1- (2-chlorophenylmethyl) -2-propylindole-6-carboxylate; 4-bromo-5- (3-pyridylmethylamino) -6- (3- (4-chlorophenyl) propoxy) -3- (2H) pyridazinone; 1-methyl-5- (5-chiorolinoacetyl-2-n-propoxyphenyl) -3-n-propyl-1, 6-dihydro-7H-pyrazolo (4, 3-d) pyrimidin-7-one; 1- [4- [ (1, 3-benzodioxol-5-ylmethyl) amino ] -6-chloro-2-quinazolinyl ] -4-piperidine-carboxylic acid monosodium salt; pharmaprojects No.4516 (GlaxoWellcome); pharmaprojects No.5051 (Bayer); pharmaprojects No.5064(Kyowa Hakko; see WO 96/26940); pharmaprojects No.5069(Schering Plough); GF-196960 (GlaxoWellcome); e-8010 and E-4010 (Eisai); bay-38-3045 & 38-9456(Bayer) and Sch-51866.
The appropriateness of any particular cGMP PDE5 inhibitor can be readily determined by assessing its potency and selectivity using literature methods, followed by assessment of its toxicity, absorption, metabolism, pharmacokinetics, and the like according to standard pharmaceutical practice.
Preferably, the cGMP PDE5 inhibitor has an IC of less than 100nM, more preferably less than 50nM, even more preferably less than 10nM50The value is obtained.
IC50 values for cGMP PDE5 inhibitors can be determined using the PDE5 assay in the test methods section hereinafter.
Preferably the cGMP PDE5 inhibitor used in the pharmaceutical composition of the invention is selective for the PDE5 enzyme. Preferably, they have a selectivity for PDE5 over PDE3 of greater than 100, more preferably greater than 300. More preferably, they have a selectivity for PDE5 over both PDE3 and PDE4 of greater than 100, more preferably greater than 300.
The selectivity ratio can be readily determined by one skilled in the art.
It will be appreciated that the contents of the above-disclosed patent applications, and in particular the compounds of the formulae and implementations described therein, are incorporated herein by reference in their entirety.
Sponge body
As used herein, the term "corpus cavernosum" refers to a mass of tissue in the penis. In this regard, the shaft of the penis is composed of three cylindrical tissue masses, each surrounded by fibrous tissue called tunica albuginea. The paired dorsolateral tissue masses are called sponges (corpus; cavernosum); the smaller, mid-abdominal mass, the corpus cavernosum penis contains the urethral corpus cavernosum and its function is to keep the opening of the urethral corpus cavernosum in ejaculation. All three tissue masses are surrounded by fascia and skin and consist of erectile tissue that is penetrable by the blood sinuses. The corpus cavernosum includes smooth muscle cells.
Ejaculation with ejaculatory effect
Ejaculation involves two separate processes- -ejaculation and ejaculation. Seminiferous is the deposition of semen and spermatozoa from the distal epididymis, vas deferens, seminal vesicles and prostate into the prostatic urethra. This deposition is followed by a forceful expulsion of the semen contents from the urethral meatus. Ejaculation is distinguished from orgasm, which is purely a brain event. These two processes often occur simultaneously.
Erection of penis
As used herein, the term "penile erection" refers to the situation where, following stimulation (which may be visual, tactile, audible, olfactory, or from imagination), the arteries supplying the penis dilate and significant amounts of blood enter the blood sinuses. The expansion of these spaces compresses the veins exiting the penis, resulting in a slowing of the outflow of blood. These vascular changes due to parasympathetic neuroreflex contribute to erection. When the artery constricts and the pressure on the vein is released, the penis returns to its flaccid state.
Smooth muscle
As used herein, the term "smooth muscle" refers to tissue that is specialized to be responsible for contraction, which is composed of smooth muscle fibers (cells), wherein these smooth muscle fibers are located on the wall of a hollow internal organ and are innervated by autonomic motor neurons. The term "smooth muscle" means a muscle that lacks striations, thus giving it a smooth appearance. It is also known as involuntary muscle. Smooth muscle intracellular Ca2An increase in + concentration will initiate contraction as in the striated muscle. However, it is not limited toSmooth muscle lacks sarcoplasmic reticulum (Ca in striated muscle)2+A memory). Calcium ions flow from the extracellular fluid and sarcoplasmic reticulum into the cytosol of smooth muscle cells, although Ca is not present in the smooth muscle because of the absence of a transverse channel2+It takes more time to reach the filaments in the centre of the fibre and trigger the shrinkage process. This phenomenon also explains, in part, the slow onset and prolonged contraction of smooth muscle.
Contraction and relaxation
There are several mechanisms to regulate smooth muscle cell contraction and relaxation. In one mechanism, a regulatory protein called calmodulin binds Ca within the cytosol2+. Calcium ions not only enter the smooth muscle fibers slowly, but as the excitation diminishes, they also move out of the muscle fibers slowly, which delays relaxation. Ca2+Prolonged presence within the cytosol causes smooth muscle tone, a state of sustained partial contraction. Smooth muscles are located on the walls of hollow internal organs, such as blood vessels, airways to the lungs, stomach, intestinal gall bladder, corpus cavernosum of the penis and clitoris.
Treatment of
It is to be understood that the treatment referred to herein includes one or more of curative, palliative and prophylactic treatment.
Sexual stimulation
The invention also encompasses the administration of a selective oxytocin antagonist (if applicable together with an adjuvant) before and/or during sexual stimulation as defined above. The term "sexual stimulation" may be synonymous with the term "sexual arousal" herein. The present invention is advantageous in this respect because it provides systemic (physiological) selectivity.
Therefore, it is advantageous according to the invention to perform the sexual stimulation step at a certain stage. Here, "sexual stimulation" may be one or more of the following: visual stimuli, physical stimuli, auditory stimuli, or ideological stimuli.
Substance(s)
According to the invention, for the treatment of male ejaculatory disorders. In particular the premature ejaculation substance can be any substance which can act as a selective oxytocin antagonist as detailed hereinbefore and, if appropriate, a combination of a selective oxytocin antagonist and an auxiliary substance. As used herein, the term "agent" includes any entity capable of selectively inhibiting the oxytocin receptor.
These substances (i.e., the substances defined above) may be organic compounds or other chemicals. The substance may even be an amino acid sequence or a chemical derivative thereof. The substance may even be a nucleotide sequence, which may be a sense sequence or an antisense sequence. The substance may even be an antibody.
Thus, the term "substance" includes, but is not limited to, compounds that can be obtained or prepared from any suitable source, whether natural or not.
The substance may be designed or derived from a library of compounds, which may include peptides, as well as other compounds, such as small organic molecules, e.g., lead compounds.
For example, the substance can be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or inorganic molecule, a synthetic substance, a semi-synthetic substance, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatized substance, a peptide cleaved from an intact protein, or a synthetically synthesized peptide (e.g., using a peptide synthesizer or using recombinant techniques or combinations thereof), a recombinant substance, an antibody, a natural or non-natural substance, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.
As used herein, the term "substance" can be a single entity or a combination of substances.
If the substance is an organic compound, for some applications, the organic compound may typically include two or more linked hydrocarbyl groups. For some applications, the material preferably includes at least two cyclic groups — optionally, at least one of the cyclic groups is a fused cyclic structure. For some applications, at least one of the cyclic groups is a heterocyclic group. For certain applications, the heterocyclic group preferably includes at least one N in the ring. Examples of such compounds are given herein.
The material may comprise one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene-which may be branched or unbranched.
Substituted by
For the avoidance of doubt, unless otherwise specified, the term "substituted" means substituted by one or more defined groups. While the groups may be selected from a wide variety of alternative groups, the selected groups may be the same or different. For the avoidance of doubt, the term independently means: when more than one substituent is selected from a wide variety of possible substituents, the substituents may be the same or different.
Pharmaceutically acceptable salts
The substance may be in and/or may be administered in the form of a pharmaceutically acceptable salt, such as an acid addition salt or a base salt or a solvate thereof, including a hydrate thereof. 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 with the desired acid or base, if appropriate. The salt may be precipitated from the solution and collected by filtration or may be recovered by evaporation of the solvent.
Suitable acid addition salts may be formed from acids which form non-toxic salts and are exemplified by hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogenphosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, gluconate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.
Suitable base salts are formed from bases which form non-toxic salts and are exemplified by sodium, potassium, aluminum, calcium, magnesium, zinc and diethanolamine salts.
Polymorphous/asymmetric carbons
The substance may exhibit polymorphous forms.
The substance may contain one or more asymmetric carbon atoms and thus have two or more stereoisomeric forms. Cis (E) and trans (Z) isomerism may also occur when a material contains alkenyl or alkenee. The invention includes the individual stereoisomers of the substances and, where appropriate, the individual tautomeric forms thereof, and also mixtures thereof.
Separation of the non-stereoisomers or cis and trans isomers may be achieved by conventional techniques, such as fractional crystallisation of a mixture of non-stereoisomers of the substance or a suitable salt or derivative thereof, chromatography or h.p.l.c.. The individual enantiomers of the substances can also be prepared from the corresponding optically pure intermediates or by resolution of the corresponding racemates using suitable chiral supports, for example h.p.l.c., or, if appropriate, by reaction of the corresponding racemates with suitable optically active acids or bases to form non-stereoisomeric salts and subsequent fractional crystallization.
Isotopic variations
The invention also includes all suitable isotopic variations of the substance or a pharmaceutically acceptable salt thereof. An isotopic variation of a substance of the invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic weight different from the atomic weight usually found in nature. May be incorporated into the substance orExamples of isotopes within pharmaceutically acceptable salts thereof include the following isotopes: hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, e.g. each2H、3H、13C、14C、15N、17O、18O、31P、32P、35S、18F and36and (4) Cl. Certain isotopic variations of the substance or a pharmaceutically acceptable salt thereof, e.g. those incorporating a radioactive isotope such as3H or14C, useful in tissue distribution studies of drugs and/or substrates. Tritiation (i.e., tritiation)3H) And carbon-14 (i.e.14C) Isotopes are particularly preferred for their ease of preparation and detectability. In addition, isotopes such as deuterium (i.e., deuterium) are used2H) Substitution may provide therapeutic advantages due to greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and may be preferred in some circumstances. Isotopic variations of the substance or pharmaceutically acceptable salt thereof can generally be prepared by conventional means using appropriate isotopic variations of the appropriate substance.
Prodrug
Those skilled in the art will appreciate that the substance may also be derived from a prodrug. Examples of prodrugs include entities that have some protected group and may not possess pharmaceutical activity in this state, but in some cases may be metabolized in the body upon administration (e.g., oral or parenteral administration) to form a substance with pharmaceutical activity.
Front body (Pro-moieties)
It will also be appreciated that certain moieties, referred to as "pro-moieties", such as those described in "design precursors" by H.Bundgard, Elsevier, 1985 (the disclosure of which is incorporated herein by reference), may be placed on the appropriate functional groups of the material. These prodrugs are also included within the scope of the present invention.
Inhibitors/antagonists
The term "antagonist" as used herein, in reference to a selective oxytocin antagonist, is considered to be used interchangeably with the term "inhibitor". Likewise, the term "inhibitor" as used herein, in reference to an auxiliary substance such as those listed in the foregoing, is considered to be used interchangeably with the term "antagonist".
As used herein, the term "antagonist" means any substance that can reduce the effect of another substance or target. Antagonism may result from the following: combinations of antagonized substances (chemo-antagonism) either produce opposite effects through different targets (functional antagonism or physiological antagonism) or due to competition for binding sites of intermediates that link target activation and the observed effect (indirect antagonism).
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an agent of the invention and a pharmaceutically acceptable carrier, diluent or excipient, including combinations thereof.
The pharmaceutical compositions may be for human or animal use in human and veterinary medicine and typically include any one or more pharmaceutically acceptable carriers, diluents or excipients. Acceptable carriers or diluents for use in therapy are well known in the Pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Pub rendering co. (a.r. gennaro edit.1985). The choice of pharmaceutical carrier, excipient or diluent can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may additionally comprise any suitable binder, lubricant, suspending agent, coating agent, dissolving agent, or as a carrier, excipient or diluent.
Preservatives, stabilizers, dyes and even flavouring agents may be added to the pharmaceutical compositions. Examples of preservatives include sodium benzoate, sorbic acid and parabens. Antioxidants and suspending agents may also be used.
Different composition/formulation requirements may be present depending on the delivery system. For example, the pharmaceutical compositions of the invention may be formulated for delivery using a micropump or by mucosal route, e.g., as a nasal spray, or inhaled aerosol, or in a digestible form, or for parenteral administration, wherein the composition is formulated in an injectable form for delivery by, e.g., intravenous, intramuscular, or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
In the case where the substance is to be delivered mucosally through the gastrointestinal mucosa, it should be capable of remaining stable during transit through the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acidic pH, and resistant to the detergent effects of bile.
If appropriate, the pharmaceutical composition can be administered by: sucking; administration in the form of a suppository or pessary; topical application in the form of a lotion, solution, cream, ointment or dusting powder; applied as a skin patch; orally in the form of tablets containing excipients such as starch or lactose; administered alone or mixed with excipients in a capsule or ovoid pouch; or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents. Or they may also be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions are preferably in the form of a sterile aqueous solution which may contain other substances, for example, salts or monosaccharides in sufficient amounts to render the solution isotonic with blood. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges (lozenes), which may be prepared by conventional methods.
For certain embodiments, the agents of the present invention may also be used in combination with cyclodextrins. Cyclodextrins are known to form inclusion (inclusion) or non-inclusion complexes with drug molecules. Formation of drug-cyclodextrin complexes can alter the solubility, dissolution rate, bioavailability, and/or stability properties of the drug molecule. Drug-cyclodextrin complexes are generally useful in most dosage forms and routes of administration. In addition to direct complexation with the drug, cyclodextrins may also be used as auxiliary additives, e.g. as carriers, diluents or solubilisers. alphA-, betA-and gammA-cyclodextrins are the most commonly used and suitable examples thereof are described in WO-A-91/11172, WO-A-94/02518 and WO-A-98/55148.
In a preferred embodiment, the agents of the invention are delivered systemically (e.g., orally, buccally, sublingually), more preferably orally.
Thus, the substance is preferably in a form suitable for oral delivery.
Administration of
The term "administering" includes delivery by viral or non-viral techniques. Viral delivery mechanisms include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes viral vectors, retroviral vectors lentiviral vectors, and baculovirus vectors. Non-viral delivery mechanisms include lipid-mediated transfection, liposomes, immunoliposomes, lipofectins, cationic surfactant amphophilic agents (cationic facial amphophiles), and combinations thereof.
The agents of the invention may be administered alone or, generally, in the form of pharmaceutical compositions, for example when the agent is in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
For example, the substance may be administered in the form of a tablet, capsule, ovule, elixir, solution or suspension, which may contain flavoring or coloring agents for immediate, delayed, modified, sustained, pulsed or controlled release applications.
Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dipotassium calcium phosphate and glycine; disintegrants, for example starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders, for example polyvinylpyrrolidone, Hydroxypropylmethylcellulose (HPMC), Hydroxypropylcellulose (HPC), sucrose, gelatin and donkey-hide gelatin (acacia). Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may also be included.
Solid compositions of a similar type may also be employed as fill in gelatin capsules. In this regard, preferred excipients include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the material may be combined with various sweetening or flavouring agents, colouring matter or dyes, emulsifying and/or suspending agents, diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
Routes of administration (delivery) include, but are not limited to, one or more of the following: oral (e.g., in the form of a tablet, capsule, or in the form of an ingestible solution), topical, mucosal (e.g., in the form of a nasal spray or aerosol for inhalation), nasal, parenteral (e.g., in the form of an injection), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, penile, vaginal, epidural, sublingual, and the like.
It will be appreciated that not all substances need be administered by the same route. Likewise, if a composition comprises more than one active ingredient, the ingredients may be administered by different routes.
Where the agent of the invention is administered parenterally, such administration may include one or more of the following: administering the substance intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrathoracic, intracranially, intramuscularly, or subcutaneously; and/or using infusion techniques.
For parenteral administration, the substance is preferably in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solution should be suitably buffered (preferably at a pH of 3 to 9) if necessary. The preparation of suitable parenteral formulations under sterile conditions can be readily accomplished by those skilled in the art using standard pharmaceutical techniques.
As stated, the substances of the invention may be administered intranasally or by inhalation and suitable propellants may be used, for example, in dry powder inhalers or from pressurised containers, pumps, sprayers or nebulisers, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkanes such as 1, 1, 2, 2-tetrafluoroethane (HFA 134A)TM) Or 1, 1, 1, 2, 3,3, 3-heptafluoropropane (HFA 227 EA)TM) Carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by adding a valve to deliver a metered amount. The pressure vessel, pump, spray or nebuliser may contain a solution or suspension of the active compound, for example, in ethanol and a propellant as the solvent, which may also contain a lubricant, for example sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the material and a suitable powdered base such as lactose or starch.
Alternatively, the substances of the present invention may be administered in the form of suppositories or pessaries, or may be administered topically in the form of gels, hydrogels, lotions, solutions, creams, ointments or dusting powders. The substances of the invention can also be administered transdermally or transdermally, for example by using a skin patch. They may also be administered by the pulmonary or rectal route. They may also be administered by the ocular route. For ophthalmic use, the compounds may be formulated as a micronized suspension in isotonic, pH adjusted sterile saline, or preferably as a solution in isotonic, pH adjusted sterile saline, and the like, optionally in combination with a preservative such as benzalkonium chloride. In addition, they can also be formulated in the form of ointments, for example in petrolatum.
For topical application to the skin, the substances of the invention may be formulated as suitable ointments in which the active compound is suspended or dissolved, for example, in a mixture of one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they may be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
The compositions of the present invention may be administered by direct injection.
For certain applications, it is preferred to administer the substance orally.
For certain applications, it is preferred that the substance is administered topically.
Dosage level
Typically, the actual dosage that is most appropriate for an individual patient is determined by a physician. The specific dose level and frequency of dosage for any particular individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The substance and/or pharmaceutical composition of the invention may be administered according to a regimen of 1 to 10 times per day, e.g. once or twice per day.
For oral and parenteral administration to humans, daily dosage levels of the substance may be administered in single or multiple doses.
The substance may be administered in a dose of 0.01 to 30 mg/kg body weight, for example 0.1 to 10 mg/kg, more preferably 0.1 to 1 mg/kg body weight, as required. Of course, the dosages mentioned herein are all exemplary of the general case. There are naturally individual cases where higher or lower doses are required.
Typically, the daily oral dose may be, for example, 20 to 1000 mg, preferably 50 to 300 mg.
Formulation of
The agents of the invention may be formulated into pharmaceutical compositions, for example, by mixing with one or more suitable carriers, diluents or excipients using techniques known in the art.
Some non-limiting examples of formulations are given below.
Formula 1: tablets were prepared using the following ingredients:
weight/mg
The substance 250
Microcrystalline cellulose 400
Fumed silica 10
Stearic acid 5
Total 665
The ingredients were blended and compressed to form tablets weighing 665 mg each.
And (2) formula: an intravenous formulation was prepared as follows:
100 mg of the substance
Isotonic saline 1,000 ml
Individuals
As used herein, the term "individual" refers to a member of a vertebrate, particularly a mammalian species. The term includes, but is not limited to, domestic animals, sport animals, primates and humans.
Bioavailability of the active ingredient
Preferably, the compounds (and compositions) of the present invention are orally available. Oral bioavailability refers to the proportion of a drug administered orally that achieves systemic circulation. Factors that determine the oral bioavailability of a drug are dissolution, membrane penetration rate and metabolic stability. Typically, oral bioavailability is determined using a cascade of screening steps, using first in vitro techniques followed by in vivo techniques.
Dissolution, i.e. dissolution of the drug by the aqueous contents of the gastrointestinal tract (GIT), can be predicted by in vitro dissolution experiments performed at the correct pH to mimic the GIT. Preferably, the compounds of the present invention have a minimum solubility of 50 mg/ml. Solubility can be determined by standard procedures known in the art, for example as described in adv.
Membrane penetration rate refers to the passage of the compound through the GIT cells. Lipophilicity is a key property to predict this, and can be measured in vitro by using organic solvents and buffers7,4The measurements are defined. Preferably, the compounds of the invention have a Log D of from-2 to +4, more preferably from-1 to +27.4The value is obtained. Log D can be determined by standard procedures known in the art, for example, as described in j.pharm.pharmacol.1990, 42: 144, as described in section (a).
Cell monolayer assays such as significant CaCO addition2To predict the favourable membrane penetration rate, the so-called caco-2 flux, in the presence of efflux transporters, e.g. p-glycoproteins. Preferably, the compounds of the present invention have a structure of greater than 2 x 10-6cms1More preferably greater than 5 x 106cms1The Caco-2 flux, Caco flux value, can be determined by standard procedures known in the art, for example as described in j.pharm.sci, 1990, 79, 595-600.
Metabolic stability expresses the ability of GIT or liver to metabolize compounds during absorption: first pass effect (the first pass effect). Metabolic instability can be predicted by assay systems such as microsomes, hepatocytes, etc. Preferably, the compounds of the examples show metabolic stability in the assay system corresponding to a liver extraction rate of less than 0.5. Examples of assay systems and data processing are described in curr. opin. drug disc. devel., 201, 4, 36-44; drug met. disp., 2000, 28, 1518-.
Because of the interplay of the above processes, evidence further supporting that drugs can be orally utilized in humans can be obtained from in vivo experiments in animals. Absolute bioavailability was determined in these studies by oral route, administered separately or in admixture. The intravenous route was also used for absolute value determination (% absorption). Examples of oral bioavailability in animals can be assessed as described in Drug met.disp., 2001, 29, 82-87; med chem, 1997, 40, 827-; drug met.disp., 1999, 27, 221-226.
Chemical synthesis method
Typically, selective oxytocin antagonists (and/or PDEi/PDE5i, if appropriate) suitable for use according to the invention are prepared by chemical synthesis techniques.
The substance or target or variant, homologue, derivative, fragment or mimetic thereof may be produced using chemical methods to synthesise all or part of the substance. For example, peptides can be synthesized by solid phase techniques, cleaved from resins, And purified using preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman And Co., N.Y., NY). The composition of the synthetic peptide can be determined by amino acid analysis or sequencing (e.g., Edman degradation program; Creighton, supra).
Direct synthesis of the substances or variants, homologues, derivatives, fragments or mimetics thereof may be carried out using various solid phase techniques (RobergeJY et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using ABI 431A Peptide Synthesizer (Perkin Elmer) according to the instructions given by the manufacturer. In addition, the amino acid sequence containing the substance or any portion thereof can be altered during direct synthesis and/or chemically combined with sequences from other subunits or any portion thereof to create variant substances or targets, e.g., variant oxytocin receptors.
In another embodiment of the invention, the coding sequence for the target substance or variant, homologue, derivative, fragment or mimetic thereof may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res SympSer 215-23; Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
Simulation object
As used herein, the term "mimetic" refers to any chemical, including but not limited to a peptide, polypeptide, antibody, or other organic chemical, that has the same quantitative activity or effect on a target (e.g., an oxytocin receptor) as a reference substance (e.g., a selective oxytocin antagonist). That is, the mimetic can be a functional equivalent of a known substance.
Chemical derivatives
The term "derivative" or "derivatized" as used herein includes chemical modification of a substance. Examples of such chemical modifications are replacement of hydrogen by halogen, alkyl, acyl or amino groups.
Chemical modification
In one embodiment of the invention, the substance may be a chemically modified substance.
Chemical modification of a substance may enhance or reduce hydrogen bonding interactions, charge interactions, hydrophobic interactions, van der waals interactions, or dipole interactions between the substance and a target.
In one aspect, the identified substance can be used as a model (e.g., template) for the development of other compounds.
Target body
In one aspect of the invention, the oxytocin receptor can be used as a target in a screen to identify agents capable of inhibiting the oxytocin receptor. In this aspect, the target may comprise a target such as seq i DNO: 1 or a variant, homologue, derivative or fragment thereof, which may be prepared by recombinant and/or synthetic means or an expression entity comprising the same.
In another aspect of the invention, oxytocin receptors and vasopressin receptors, preferably the V1a receptor, may be used in the screen as targets to identify agents capable of selectively inhibiting oxytocin receptors. In this aspect, the oxytocin receptor target may comprise an amino acid sequence as set forth in SEQ ID NO: 1 or a variant, homologue, derivative or fragment thereof, prepared by recombinant and/or synthetic means or an expression entity comprising the same, and the vasopressin receptor target may comprise an amino acid sequence as set forth in SEQ ID NO: 2 or a variant, homologue, derivative or fragment thereof, which may be prepared by recombinant and/or synthetic means or an expression entity comprising the same.
Additionally, oxytocin receptors and/or vasopressin receptors (preferably the V1a receptor) may be used as targets to identify agents capable of mediating increased ejaculation latency through selective inhibition of the oxytocin receptor. In this regard, the target may be a suitable tissue extract.
The target may even be a combination of these tissues and/or recombinant targets.
Recombination method
The substances and/or targets of the invention can be prepared by recombinant DNA techniques.
In one embodiment, preferably the substance is a selective oxytocin antagonist. The oxytocin antagonist may be prepared by recombinant DNA techniques.
Amino acid sequence
As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "protein".
The amino acid sequence may be prepared by isolation from an appropriate source, or it may be made synthetically or it may be made by using recombinant DNA techniques.
In one aspect, the invention provides an amino acid sequence that can be used as a target (i.e., oxytocin receptor or vasopressin receptor, preferably V1a receptor) in an assay to identify one or more substances and/or derivatives thereof.
In a second aspect, the present invention provides an amino acid sequence which is a substance capable of selectively inhibiting the oxytocin receptor.
Preferably, the target is an oxytocin receptor.
Preferably, the oxytocin receptor or vasopressin receptor, preferably the V1a receptor, is an isolated receptor and/or purified and/or non-native.
The oxytocin or vasopressin receptors, preferably the V1a receptor, of the invention may be in a substantially isolated form. It will be appreciated that the oxytocin receptor or vasopressin receptor may be mixed with a carrier and/or diluent that does not interfere with the intended purpose of the receptor and/or substance and yet is considered to be substantially separate. The oxytocin or vasopressin receptors of the invention may also be in a substantially pure form, in which case they typically comprise oxytocin or vasopressin receptors in a preparation, wherein more than 90%, e.g. 95%, 98% or 99% of the oxytocin or vasopressin receptors in the preparation have the amino acid sequence of SEQ ID NO: 1 or a variant, homologue, derivative, or fragment thereof, or the amino acid sequence set forth in SEQ ID NO: 2 or a variant, homologue, derivative or fragment thereof.
Nucleotide sequence
As used herein, the term "nucleotide sequence" is synonymous with the term "polynucleotide".
The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin. The nucleotide sequence may be double-stranded or single-stranded, whether representing the sense strand or the antisense strand or a combination thereof.
For some applications, it is preferred that the nucleotide sequence is DNA.
For some applications, it is preferred that the nucleotide sequence is made using recombinant DNA techniques (i.e., recombinant DNA).
For some applications, it is preferred that the nucleotide sequence is a cDNA.
For certain applications, it is preferred in this respect that the nucleotide sequence is identical to the naturally occurring form.
In one aspect, the invention provides a nucleotide sequence encoding a substance capable of acting as a target in an assay to identify one or more substances and/or derivatives thereof.
In one aspect of the invention, the nucleotide sequence encodes an oxytocin receptor.
In another aspect of the invention, the nucleotide sequence encodes a vasopressin receptor, preferably the V1a receptor.
In one aspect of the invention, the nucleotide sequence encodes a substance capable of selectively inhibiting the oxytocin receptor.
It is well understood by those skilled in the art that due to the degeneracy of the genetic code, there may be many different nucleotide sequences encoding the same target (i.e., an oxytocin receptor, such as an oxytocin receptor comprising the amino acid sequence set forth in SEQ ID NO: 1, or a vasopressin receptor, such as a vasopressin receptor comprising the amino acid sequence set forth in SEQ ID NO: 2). Furthermore, those skilled in the art will also appreciate that nucleotide substitutions that do not significantly affect the activity encoded by the nucleotide sequence can be made using conventional techniques to reflect the codon usage of the particular host organism in which the target is to be expressed. Thus, the terms "variant", "homologue" or "derivative" relating to the nucleotide sequence include any substitution, variation, modification, substitution, deletion or addition of one (or more) nucleic acids of the sequence, provided that the resulting nucleotide sequence encodes a functional object of the invention (i.e. e.g. an oxytocin receptor) (or even an agent of the invention if the agent comprises a nucleotide sequence or an amino acid sequence).
Variants/homologues/derivatives
In addition to the specific amino acid sequences mentioned herein, the present invention also encompasses the use of variants, homologues and derivatives thereof. Furthermore, "homology" may be equivalent to "identity".
Herein, homologous sequences include amino acid sequences that are identical to SEQ ID NO: 1 or SEQ ID NO: 2, and preferably at least 95 or 98% identity thereto. In particular, homology is generally considered in terms of regions of the sequence known to be essential for activity. Although homology may also be considered in terms of similarity (i.e., amino acid sequences having similar chemical properties/functions), in this context, it is preferred to express homology in terms of sequence identity.
Homology alignments can be performed visually or, more often, with the aid of readily available sequence alignment programs. These commercially available computer programs can calculate the percent homology between two or more sequences.
Percent homology can be calculated over contiguous sequences, i.e., one sequence is aligned with another sequence and each amino acid in the sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called an "unnotched" alignment. Typically, these ungapped alignments are performed only on a relatively small number of residues.
Although this is a very simple and consistent method, it cannot consider the following: for example, in a non-identical pair of sequences, an insertion or deletion can mis-align subsequent amino acid residues, thus potentially resulting in a substantial reduction in the percentage of homology when performing the overall alignment. Thus, most sequence alignment methods are designed to produce optimal alignments that take into account possible insertions and deletions to produce optimal alignments so as not to unnecessarily reduce overall homology values. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology.
However, these more complex methods assign a "gap penalty" to each gap that occurs in an alignment, such that, for the same number of identical amino acids, an alignment of sequences with as few gaps as possible (reflecting a higher correlation between the sequences of two aligned pairs) will achieve a higher score than an alignment with many gaps. An "affinity gap cost" is typically used, which sets a relatively high cost for the presence of a gap and a small penalty for the subsequent residues of the gap. This is the most commonly used gap scoring system. High gap penalties will of course result in an optimal alignment with fewer gaps. Most alignment programs can adjust gap penalties. However, it is preferable to adopt a default value (default value) when sequence alignment is performed using these software. For example, when using the GCG Wisconsin Bestfit software package (see below), the default gap penalty for a gap in an amino acid sequence is-12 and-4 for each extension.
Thus, the calculation of the maximum percent homology requires first considering the gap penalty to produce an optimal alignment. One suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit software package (University of Wisconsin, U.S. A.; Devereux et al, 1984, Nucleic Acids Research 12: 387). Other examples of software that can perform sequence alignments include, but are not limited to, the BLAST software package (seeSee Ausubel et al, 1999 supra-chapter 18), FASTA (Atschul et al, 1990, j.mol.biol., 403-. Both BLAST and FASTA can be used to perform off-line and on-line searches (see Ausubel et al, 1999, supra, pages 7-58 to 7-60), although the GCG Bestfit program is preferably used. A novel tool, known as BLAST 2 Sequences, can also be used to align protein and nucleotide Sequences (see FEMSMirobiol Lett.1999174 (2): 247-50; FEMS Microbiol Lett 1999177 (1): 187-8 and titiana) ncbi.nlm.nih.gov)。
Although the final percentage of homology can be measured in terms of identity, the alignment program itself is typically not based on all-or-nothing (all-or-nothing) pair comparisons. Instead, a scaled similarity scoring matrix is typically used to assign a score to each pairwise alignment based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix, which is the default matrix of the BLAST package. The GCG Wisconsin program typically uses published default values, or if provided, can use the custom symbol composition table (see further description in the manual of use). Preferably using the public default values of the GCG package or, in case other software is used, using a default matrix, e.g. BLOSUM 62.
Once the software has produced an optimal alignment, the percent homology, preferably the percent sequence identity, can be calculated. Software typically performs such calculations as part of a sequence alignment and produces a numerical result.
The sequences may also have deletions, insertions or substitutions of amino acid residues which result in silent mutations and which result in functionally equivalent substances. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, so long as the substance retains its secondary binding activity. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; while amino acids with uncharged polar head groups and similar hydrophobicity include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example, according to the following table. Amino acids in the same group in the second column and preferably in the same row in the third column may be substituted for each other:
the present invention also encompasses homologous substitutions (both substitution and substitution are used herein to refer to the replacement of an existing amino acid residue by another alternative residue), i.e., reciprocal substitutions (like-for-like) between analogs such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution may also occur, i.e., exchange of one type of residue for another or involving inclusion of unnatural amino acids, such as ornithine (hereinafter Z), diaminobutyric acid ornithine (hereinafter B), norleucine ornithine (hereinafter O), pyridylalanine/thienylalanine, naphthylalanine and phenylglycine.
Substitutions may also be made with unnatural amino acids, including: alpha is alpha*And alpha-disubstituted*Amino acids, N-alkyl*Amino acid, lactic acid*Halogenated derivatives of natural amino acids, e.g. trifluorotyrosine*p-C1-phenylalanine*p-Br-phenylalanine*p-I-phenylalanine*L-allyl-glycine*Beta-alanine*L-alpha-aminobutyric acid*L-gamma-aminobutyric acid*L-alpha-aminoisobutyric acid*L-epsilon-aminocaproic acid#7-amino heptanoic acid*L-methionine sulfone#*L-norleucine*L-norvaline*To, for-nitro-L-phenylalanine*L-hydroxyproline#L-Thioproline*Methyl derivatives of phenylalanine (Phe), e.g. 4-methyl-Phe*pentamethyl-Phe*L-Phe (4-amino)#L-Tyr (methyl)*L-Phe (4-isopropyl)*L-Tic (1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid)*L-diaminopropionic acid#And L-Phe (4-benzyl)*. Symbol*It has been used previously for the purposes discussed above (involving homologous or non-homologous substitution) to indicate the hydrophobic nature of the derivative, where # is used to indicate the hydrophilic nature of the derivative and # refers to the amphiphilic character.
Variant amino acid sequences may include an appropriate spacer (spacer group) which may be inserted between any two amino acid residues in the sequence, including alkyl groups such as methyl, ethyl or propyl and amino acid spacers such as glycine or β -propylamino residues. Another variation includes the presence of one or more amino acid residues in peptoid form (peptoid form), which are familiar to those skilled in the art. For the avoidance of doubt, "peptoid form" is intended to refer to variant amino acid residues in which the alpha-carbon substituent is on the nitrogen atom of the residue and not on the alpha-carbon. Techniques for preparing peptides in peptoid form are known in the art, e.g., Simon RJ et al, RNAS (1992)89(20), 9367-.
Hybridization of
The term "hybridization" as used herein includes: the process by which a nucleic acid strand binds to a complementary strand through a base pairing, and the amplification procedure performed in the Polymerase Chain Reaction (PCR).
Nucleotide sequences capable of selectively hybridizing to a nucleotide sequence encoding an amino acid sequence of the invention or to their complementary strand typically have at least 75%, preferably at least 85 or 90%, and more preferably at least 95% or 98% homology over a region of at least 20, preferably at least 25 or 30, such as at least 40, 60 or 100 or more contiguous nucleotides to the corresponding complementary nucleotide sequence encoding an amino acid sequence set forth herein.
The term "selectively hybridize" refers to a nucleotide sequence that, when used as a probe, is used under conditions that allow the target nucleotide sequence to hybridize to the probe at a level significantly above background. Background hybridization occurs because of the presence of other nucleotide sequences, for example, in the cDNA or genomic DNA library being screened. In this case, the background shows the level of signal generated by the interaction between the probe and the non-specific DNA member in the library, with an intensity 10-fold lower, preferably 100-fold lower, than the specific interaction observed with the target DNA. The intensity of the interaction may be determined by radiolabelling the probe, e.g. with32P is measured.
Hybridization conditions are calculated based on the melting temperature (Tm) of the nucleic acid binding complex, as described in Berger and kimmel (1987, Guide to Molecular Cloning technologies, Methods in Enzymology, Vol.152, Academic Press, San Diego CA), and assigned a definition of "stringency" in the following explanation.
Maximum stringency typically occurs at about Tm-5 ℃ (5 ℃ below the Tm of the probe; high stringency at about 5 ℃ to 10 ℃ below Tm; moderate stringency at about 10 ℃ to 20 ℃ below Tm; whereas low stringency occurs at about 20 ℃ to 25 ℃ below the Tm. As will be appreciated by those skilled in the art, maximum stringency hybridization can be used to identify or detect identical nucleotide sequences, while medium (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
In a preferred aspect, the invention includes a method of treating a subject with a composition that can be under stringent conditions (e.g., 65 ℃ and 0.1XSSC {1XSSC ═ 0.15M NaCl, 0.015M Na3Citrate ph7.0} with a nucleotide sequence encoding an amino acid sequence of the invention. In the case where the nucleotide sequence of the invention is double stranded, both strands of the duplex (either single or a combination thereof) are encompassed by the invention. In the case where the nucleotide sequence is single-stranded, it is to be understood thatThe complementary strand of the nucleotide sequence is also included in the scope of the present invention.
Nucleotide sequences which are not 100% homologous to the nucleotide sequence encoding the amino acid sequence of the present invention but which fall within the scope of the present invention can be obtained in a number of ways. Other variants of the sequences described herein can be obtained, for example, by screening DNA libraries made from a number of sources. In addition, other viral/bacterial, or cellular homologues, particularly cellular homologues in mammalian cells (e.g. rat, mouse, bovine and primate cells) may be obtained and such homologues and fragments thereof will generally be capable of selectively hybridising to the sequences shown in the sequence listing of the present invention. These sequences can be obtained by screening DNA libraries or genomic DNA libraries made from other animal species by probing the libraries with probes comprising all or part of the nucleotide sequences set forth herein under conditions of moderate to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the amino acid sequences and/or nucleotide sequences of the invention.
Variants and strain/species homologues may also be made using degenerate PCR, wherein primers are used which are designed to target sequences within the variants and homologues which are conserved with the amino acid sequence encoding within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning amino acid sequences from several variants/homologues. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used. Primers used in degenerate PCR contain one or more degenerate positions and can be used under less stringent conditions than those used when cloning sequences using single sequence primers against known sequences.
Alternatively, these nucleotide sequences may be obtained by site-directed mutagenesis of the identified sequences, for example, the nucleotide sequences encoding SEQ ID NO: 1 by site-directed mutagenesis. This is useful, for example, when the sequence requires silent codon mutations to optimize codon bias in a particular organism in which the nucleotide sequence is intended to be expressed. Additional sequence changes may also be required to introduce restriction enzyme recognition sites, or to alter the activity of the protein encoded by the nucleotide sequence.
The nucleotide sequence encoding the amino acid sequence of the invention may be used to generate primers, e.g., PCR primers, primers for use in another amplification reaction, probes, e.g., probes that are detectably labeled using a radioactive label or a non-radioactive label by conventional means, or the nucleotide sequence may be cloned into a vector. These primers, probes and other fragments are at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length and are also encompassed by the term "nucleotide sequence of the invention" as used herein.
Nucleotide sequences of the invention, such as DNA polynucleotides and probes, can be made recombinantly, synthetically, or by any means available to those of skill in the art. They can also be cloned using standard techniques.
Generally, primers are generated synthetically, comprising the stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are well known in the art.
Longer nucleotide sequences are usually made using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. This involves making a pair of primers (e.g., about 15 to 30 nucleotides) that flank the region of the target sequence to be cloned; contacting the primer with mRNA or cDNA obtained from an animal or human cell; performing a Polymerase Chain Reaction (PCR) under conditions that facilitate amplification of the desired region; isolating the amplified fragments (e.g., purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers can be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate cloning vector.
Because of the inherent degeneracy of the genetic code, different DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can be used to clone and express a target sequence. As will be appreciated by those skilled in the art, for certain expression systems, non-naturally occurring codons may be used to advantage to make the sequence of interest. Codons preferred by a particular prokaryotic or eukaryotic host may be selected (Murray E et al (1989) Nuc Acids Res 17: 477-508) for example to increase the expression rate of the target or to produce recombinant RNA transcripts with desirable properties, e.g.with a longer half-life than transcripts produced with naturally occurring sequences.
Carrier
In one embodiment of the invention, the substance (i.e., the selective oxytocin antagonist) may be administered directly to the individual.
In another embodiment of the invention, a vector comprising a nucleotide sequence encoding an agent of the invention is administered to an individual.
It is preferred to prepare the recombinant material with a gene vector and/or deliver it to the target site.
As is well known in the art, a carrier is a tool that facilitates the transfer of an entity from one environment to another. According to the present invention, for example, certain vectors used in recombinant DNA techniques may allow for the transfer of entities, such as DNA segments (e.g., heterologous DNA segments, such as heterologous cDNA segments), into hosts and/or target cells for replication of the vector comprising the nucleotide sequence of the present invention and/or expression of the protein of the present invention encoded by the nucleotide sequence of the present invention. 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" means a construct capable of expression in vivo or in vitro/ex vivo.
The term "transformation vector" means a construct capable of being transferred from one species to another.
Naked DNA
Vectors comprising nucleotide sequences encoding the agents of the invention for use in the treatment of ejaculatory disorders, particularly premature ejaculation, may be administered directly in the form of "naked nucleic acid constructs", which preferably further comprise flanking sequences homologous to sequences in the host cell genome.
As used herein, the term "naked DNA" refers to a plasmid comprising a nucleotide sequence encoding an agent of the present invention and a short promoter region controlling its production. This is called "naked" DNA because the plasmid is not carried in any delivery vector, and when this DNA plasmid enters a host cell, such as a eukaryotic cell, the protein encoded by it (e.g., the agent of the invention) is transcribed and translated within the cell.
Non-viral delivery
In addition, vectors comprising nucleotide sequences encoding the amino acids of the invention or the agents of the invention (i.e., selective oxytocin antagonists) or targets of the invention (i.e., oxytocin receptors) may be introduced into suitable host cells using a variety of non-viral techniques known in the art, such as transfection, transformation, electroporation and biolistic transformation.
As used herein, the term "transfection" refers to a procedure that uses a non-viral vector to deliver a gene into a target mammalian cell.
Typical transfection methods include electroporation, DNA biolistic transformation, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofection agents, cation-mediated, cationic surface amphiphiles (CFAs) (Nature Biotechnology 199614; 556), multivalent cations such as spermine, cationic lipids or polylysine, 1, 2-bis (oleoyloxy) 3- (trimethylammonium) propane (DOTAP) -cholesterol complex (Wolf and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
Uptake of naked nucleic acid constructs by mammalian cells can be achieved by several known transfectionsThe techniques are enhanced, including, for example, the use of transfection agents. Examples of such agents include cationic agents (e.g., calcium phosphate and DEAE-dextran) and lipofectant (e.g., lipofectam)TMAnd transfectamTM). Typically, the nucleic acid construct is mixed with a transfection agent to make a composition.
Viral vectors
In addition, vectors comprising the agents or targets of the invention or nucleotide sequences encoding the amino acid sequences of the invention may be introduced into suitable host cells using a variety of viral techniques known in the art, e.g., infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses may be used.
Preferably the vector is a recombinant viral vector. Suitable recombinant viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculoviral vectors, poxviral vectors or parvoviral vectors (see Kestler et al 1999 Human Gene Ther 10 (10): 1619-32). In the case of viral vectors, delivery of the nucleotide sequence encoding the agent of the invention is mediated by viral infection of the target cell.
Targeting vectors
The term "targeting vector" refers to a vector whose ability to infect/transfect/transduce a cell or to be expressed within a host and/or target cell is limited to a particular cell type within the host organism, often cells having a common or similar phenotype.
Replication vector
Thus, in one embodiment of the invention, the invention provides a method of making a subject of the invention comprising introducing a nucleotide sequence of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which cause replication of the vector.
Expression vector
Preferably, the agent of the invention or the nucleotide sequence encoding the amino acid of the invention or the subject of the invention inserted into a vector is operably linked to a control sequence capable of causing a host cell to express the coding sequence, e.g., the coding sequence for the oxytocin receptor of the invention, i.e., the vector is an expression vector. The agent of the invention or the target produced by the recombinant host cell may be secreted or may be contained intracellularly, depending on the sequence and/or vector used. As will be appreciated by those skilled in the art, expression vectors containing coding sequences for the agents or targets of the invention can be designed with signal sequences to direct secretion of the agents or target coding sequences of the invention across the cell membrane of a particular prokaryote or eukaryote.
In vitro expression
The vectors of the invention may be transformed or transfected into appropriate host cells and/or target cells as described below to provide for expression of the agents or targets of the invention. The procedure may comprise culturing a host cell and/or a target transformed with the expression vector under conditions such that the vector expresses a coding sequence encoding the substance or target of the invention, and optionally recovering the expressed substance or target of the invention. The vector may be, for example, a plasmid or viral vector having an origin of replication, optionally a promoter for expression of the polynucleotide and a regulator of the promoter. The vector may comprise one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid, or a neomycin resistance gene for a mammalian vector. The expression of the agents of the invention or targets of the invention may be constitutive, such that they are produced continuously, or inducible, requiring stimulation to initiate expression. In the case of inducible expression, production of the agent or target of the invention may be initiated, if desired, by, for example, addition of an inducer agent to the cell culture medium, such as dexamethasone or IPTG.
Fusion proteins
The oxytocin or vasopressin receptors or substances of the invention (i.e., selective oxytocin antagonists) may be expressed as fusion proteins to aid in extraction and purification and/or delivery of the substances or receptor targets of the invention to individuals and/or to aid in the development of substance screening methods. Examples of fusion protein partners (partners) include glutathione-S-transferase (GST), 6XHis, GAL4(DNA binding and/or transcriptional activation domain) and β -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the target protein sequence to remove the fusion protein sequence. Preferably, the fusion protein does not hinder the activity of the target.
The fusion protein may comprise an antigen or antigenic determinant fused to an agent of the invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein, including substances that may act as adjuvants in providing systemic stimulation of the immune system. The antigen or antigenic determinant may be linked to the amino-terminus or the carboxy-terminus of the substance.
In another embodiment of the invention, the amino acid sequence may be linked to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for substances that affect the activity of the substance, it is useful to encode chimeric substances that express heterologous epitopes that can be recognized by commercially available antibodies.
Host cell
There are a number of host cells which can be used to express nucleotide sequences encoding an agent of the invention (e.g., a selective oxytocin antagonist of the invention) or an oxytocin or vasopressin receptor target of the invention. These cells may be prokaryotic and eukaryotic host cells. Suitable host cells include bacteria such as e.coli (e.coli), yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, such as mouse, CHO, human and monkey cell lines and derivatives thereof.
Examples of suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (Aspergillus) (e.g.as described in EP-A-0184438 and EP-A-0284603) and TrichodermｃA (TrichodermｃA); bacteriｃA such as Bacillus (Bacillus), for example as described in EP-A-0134048 and EP-A-0253455, Streptomyces (Streptomyces) and Pseudomonas (Pseudomonas); and yeasts such as Kluyveromyces (Kluyveromyces) (e.g.as described in EP-A-0096430 and EP-A-0301670) and Saccharomyces (Saccharomyces). For example, a typical expression host may be selected from: aspergillus niger (Aspergillus niger), Aspergillus niger var. tubigenis, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.
The use of appropriate host cells, such as yeast, fungal and plant host cells, may provide post-translational modifications (e.g., myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) to confer optimal biological activity on the recombinant expression products of the invention.
Preferably, the host cell is capable of processing the expression product to produce the 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 substance may be an antibody. Additionally, or alternatively, the target may be an antibody.
Antibodies can be generated by standard techniques, such as immunization with an agent of the invention or use of phage display libraries.
For the purposes of the present invention, unless stated to the contrary, the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chains, Fab fragments, fragments produced by Fab expression libraries, and mimetics thereof. These fragments include fragments of whole antibodies which retain their binding activity to the target substance, FV, F (ab ') and F (ab')2Fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins that include the antigen binding portion of the antibodies. In addition, the antibodies and fragments thereof may be humanized antibodies. Neutralizing antibodies, i.e., antibodies that inhibit the biological activity of a substance polypeptide, are particularly preferred for diagnosis and therapy.
Where polyclonal antibodies are desired, selected mammals (e.g., mice, rabbits, goats, horses, etc.) are immunized with an immunogenic polypeptide that carries an epitope obtainable from the identified agent of the invention. Depending on the host species, various adjuvants may be used to increase the immune response. These adjuvants include, but are not limited to, freund's adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, polyols (pluronic polyols), polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Both BCG (bacillus calmette-Guerin) and Corynebacterium parvum (Corynebacterium parvum) are potentially useful human adjuvants that can be used to administer purified substance polypeptides to immunocompromised individuals to stimulate systemic defenses.
Serum from the immunized animals is collected and processed according to known procedures. If the serum containing polyclonal antibodies against epitopes obtainable from the identified substance of the invention also contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for generating and processing polyclonal antisera are known in the art. To produce such antibodies, the invention also provides polypeptides of the invention or fragments thereof haptenylated to another polypeptide for use as immunogens in animals or humans.
Monoclonal antibodies directed against epitopes obtainable from the identified agents of the invention can also be readily prepared by those skilled in the art. General methods for producing monoclonal antibodies by hybridomas are well known. Immortalized antibody-producing cells can be produced by cell fusion, and other techniques such as direct transformation of B lymphocytes with cancerous DNA, or transfection with Epstein-Barr virus, and the like. The set of monoclonal antibodies made against peripheral (orbit) epitopes can be screened for different properties, i.e., isotype and epitope affinity.
Monoclonal antibodies directed against the substance and/or the identified substance can be prepared using any technique that enables the production of antibody molecules by continuous culture cell lines. These include, but are not limited to, the hybridoma technology originally described by Koehler and Milstein (1975 Nature 256: 495-497), the human B-cell hybridoma technology (kosbor et al (1983) ImmunoToday 4: 72; Cote et al (1983) Proc Natl Acad Sci 80: 2026-2030) and the EBV-hybridoma technology (Cole et al (1985) Monoclonal Antibodies and dCancer Therapy, Alan R LissInc, pp 77-96). In addition, techniques developed for the production of "chimeric antibodies" can be used, i.e., splicing of mouse antibody genes to human antibody genes to yield molecules with the 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). In addition, the techniques used to make single chain antibodies (U.S. Pat. No.4,946,779) can be modified to make such substance-specific single chain antibodies.
Antibodies (both monoclonal and polyclonal) directed against epitopes obtainable from identified substances are particularly useful in diagnostics, whereas neutralizing antibodies can be used in passive immunotherapy. In particular, monoclonal antibodies can be used to generate anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the substance to be protected. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be used in therapy.
Antibodies can also be generated by inducing production in lymphocyte populations in vivo or by screening recombinant immunoglobulin libraries or highly specific binding material sets, as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837) and Winter G and MilsteinC (1991; Nature 349: 293-299).
Antibody fragments containing specific binding sites for the substance may also be produced. For example, such fragments include, but are not limited to, F (ab')2Fragments thereof and fragments obtainable by reacting F (ab')2Fab fragments resulting from reduction of the disulfide bonds of the fragments. In addition, Fab expression libraries can be constructed to quickly and easily identify monoclonal Fab fragments of the desired specificity (Huse WD et al (1989) Science 256: 1275-.
Reporter (Reporters)
A wide variety of reporters are useful in the assay methods (and screens) of the invention, preferably reporters that provide a signal that can be conveniently detected (e.g., by spectroscopy). For example, a reporter gene may encode an enzyme that catalyzes a reaction that alters the light absorbing properties.
Examples of reporter molecules include, but are not limited to, beta-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, beta-glucuronidase, exo-glucanase, and glucoamylase. Alternatively, radiolabeled or fluorescently labeled nucleotides can be incorporated into nascent transcripts, which are then identified upon binding to oligonucleotide probes.
In a preferred embodiment, the production of the reporter molecule is measured by the enzymatic activity of the reporter gene product, e.g., β -galactosidase.
Various protocols for detecting and measuring the expression of a target, such as the use of polyclonal or monoclonal antibodies specific for a protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA) and Fluorescence Activated Cell Sorting (FACS). A dual site monoclonal antibody-based immunoassay utilizing a monoclonal antibody reactive with both non-interfering epitopes on a polypeptide is preferred, although competitive binding assays may also be employed. These and other assays are described, for example, in Hampton R et al (1990, sequential Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 158: 1211).
A wide variety of labels and conjugation methods are known to those skilled in the art and can be used in a variety of nucleic acid and amino acid assays. Means for generating labeled hybridization probes or PCR probes to probe a target polynucleotide sequence include oligomeric labeling (oligomerizing), nick translation (nick translation), end labeling or PCR amplification with labeled nucleotides. Alternatively, the coding sequence, or any portion thereof, can be cloned into a vector to make an mRNA probe. These vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by adding the appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
Several companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, Wis.), and US Biochemical Corp (Cleveland, OH) all supply kits and protocols for these procedures. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent agents, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Patents which teach the use of these markers include 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 may also be produced, as shown in US-A-4816567.
Other Methods of quantifying expression of a particular molecule include radiolabeled nucleotides (Melby PC et al 1993J Immunol Methods 159: 235-44) or biotinylated nucleotides (Duplaa C et al 1993 Anal Biochem 229-36). Co-amplification of control nucleic acids, and a standard curve in which the experimental results are placed. Quantitative analysis of multiple samples can be accelerated by performing an assay in the form of an ELISA, in which the target oligomer is given in multiple dilutions and rapid quantitative analysis can be obtained using spectrophotometric or colorimetric reactions.
Although the presence/absence of expression of the marker gene indicates that the target gene is also present, its presence and expression must still be determined. For example, when a nucleotide sequence is inserted into a marker gene sequence, a recombinant cell containing the same can be identified by the absence of the function of the marker gene. Alternatively, the marker gene may be placed in tandem with the coding sequence of the target, co-located under the control of a single promoter. Expression of a marker gene stressed for induction or selection generally indicates that the target is also expressed.
In addition, host cells that contain the coding sequence of the target and that express the coding region of the target can be identified by a variety of methods known to those skilled in the art. These methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques for detecting and/or quantifying nucleic acids or proteins, including membrane-based, solution-based or chip-based techniques.
Screening
Any one or more appropriate targets-e.g., oxytocin receptors and/or vasopressin, preferably Vla receptors-may be used in a variety of drug screening techniques to identify substances, e.g., selective oxytocin antagonists. The targets used in these assays may be free in solution, immobilized on a solid support, supported on the surface of a cell, or located within a cell. The target may even be located within an animal model, wherein the target may be an exogenous target or an introduced target. The animal model is a non-human animal model. The elimination of target activity or the formation of a binding complex between the target and the test substance can be measured.
The technique used for drug screening can be based on the method described in European patent application 84/03564 to Geysen, published on 9/13 1984. Briefly, a large number of different small peptide test compounds are first synthesized on a solid substrate, such as a plastic needle or some other surface. The peptide test compound is reacted with the appropriate target or fragment thereof and washed. The bound entities are then detected-for example, in a widely accepted manner known in the art. Purified targets can also be coated directly onto plates for use in drug screening techniques. Alternatively, non-neutralizing antibodies are used to capture the peptide and immobilize it on a solid support.
The invention also encompasses the use of competitive drug screening assays in which a neutralizing antibody capable of specifically binding to a target is used to compete with the test compound for binding to the target.
Another screening technique provides High Throughput Screening (HTS) of substances with appropriate binding affinity for the substance and is based on the method detailed in WO 84/03564.
It is anticipated that the assay method of the present invention will be suitable for both small and large scale screening and quantitative assays of test compounds.
In a preferred aspect, the screening of the invention comprises at least the following steps (not necessarily in the same order as below): (a) performing an in vitro screen to determine whether the candidate substance has an associated activity (e.g., modulating an oxytocin receptor); (b) performing one or more selective screens to determine the selectivity of the candidate substance (e.g. determining whether the substance is also a vasopressin, in particular Vla, receptor inhibitor) -for example by using the test protocol given herein; and (c) performing an in vivo screen with the candidate substance (e.g. using a functional animal model comprising determining the selectivity of the substance by determining its effect on a vasopressin, in particular Vla, receptor). Typically, screening (c) is performed if the candidate substance passes through screening (a) and screening (b).
Diagnostic method/composition/kit
The invention also provides a diagnostic method, composition or kit for detecting a pre-disposition of ejaculation. In this aspect, the method, composition or kit comprises means for detecting an entity, preferably oxytocin, in a test sample, preferably a blood sample taken from a sexually aroused male.
To provide a basis for diagnosing premature ejaculation, normal or standard values of the entity must be established. This can be achieved by: body fluids taken from a normal subject (which may be an animal or human) at various times after sexual arousal are combined with antibodies to the entity under conditions well known in the art suitable for complex formation. The amount of standard complex formation can be quantified by comparing it to serial dilutions of a positive control sample in which a known amount of antibody is combined with a known concentration of pure target. Standard values from normal samples can then be compared to values obtained from samples taken from subjects potentially suffering from premature ejaculation. Deviation between the standard value and the subject value indicates the presence of a disease state.
The entity itself, or any portion thereof, can provide the basis for a diagnostic and/or therapeutic compound. For diagnostic purposes, the target polynucleotide sequence may be used to detect and quantify gene expression in conditions, disorders or diseases that may suggest premature ejaculation.
Polynucleotide sequences encoding the target are useful in the diagnosis of premature ejaculation resulting from expression of the target. For example, a polynucleotide sequence encoding an entity can be used to detect expression of the entity in a hybridization assay or PCR assay of tissue obtained from living tissue, or necropsy or biological fluid. Such qualitative or quantitative analysis methods may include southern or northern blotting, dot blotting or other membrane-based techniques, PCR techniques; dip stick (dipstick), needle or chip technology; 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 commercially available diagnostic kits.
These assays can be modified to assess the efficacy of a particular therapeutic regimen and can be used in animal studies, clinical trials, or monitoring treatment of individuals, among others. If the disease is established, an existing therapeutic agent may be administered and a therapeutic profile or value may be generated. Finally, the assay can be repeated on a routine basis to assess whether the value has progressed towards or reverted to a normal or standard pattern. A continuous treatment profile may be used to show the effect of the treatment over a period of days or months.
Measurement method
The assay methods of the invention may employ one or more of the following techniques, including but not limited to: competitive and non-competitive assays, radioimmunoassays, bioluminescent and chemiluminescent assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blot assays, enzyme linked assays, including ELI SA, microtiter plate assays, antibody coated strips or dipsticks for rapid monitoring of urine or blood, immunohistochemistry and immunocytochemistry.
Probe needle
In another aspect, the invention provides nucleic acid hybridization or PCR probes that are capable of detecting (particularly those that are selectively selectable) polynucleotide sequences, including genomic sequences, coding regions of interest, e.g., regions encoding oxytocin receptors, or closely related molecules, e.g., alleles. The specificity of the probe, i.e., whether it is derived from highly conserved, conserved or non-conserved regions or domains, and the stringency of hybridization or amplification (high, medium or low), determines whether the probe will only identify the naturally occurring coding sequence of interest, or related sequences. Probes for probing related nucleic acid sequences are selected from conserved regions or highly conserved nucleotide regions of members of the target family, and these probes can be used in a degenerate probe population. Where the same nucleic acid sequence is to be detected, or where maximum specificity is required, the nucleic acid probe is selected from a non-conserved nucleotide region or a unique region of the target polynucleotide. As used herein, the term "non-conserved nucleotide region" refers to a region of nucleotides that is unique to a target coding sequence disclosed herein and that is not present in a related family member.
PGRs as described in US-A-4683195, US-A-4800195 and US-A-4965188 provide additional uses of oligonucleotides based on target sequences. These oligomers are typically chemically synthesized, although they can also be enzymatically produced or made from recombinant sources. Oligomers usually comprise two nucleotide sequences, one in the sense orientation (5 '→ 3') and the other in the antisense orientation (3 '← 5'), which are applied under optimized conditions to identify a specific gene or disorder. Closely related DNA or RNA sequences can be detected and/or quantified using the same two oligomers, nested oligomers, or even a degenerate population of oligomers, under less stringent conditions.
The nucleic acid sequence of the substance or target may also be used to generate hybridization probes as described above for mapping endogenous genomic sequences. The sequence may be mapped to a particular chromosome or to a particular region of a chromosome using well known techniques. These techniques include in situ hybridization to a chromosomal spreading region (spread) (Verma et al (1988) Human Chromosomes: A Manual of basic techniques, Pergamon Press, New York City), flow sort chromosome preparations, or artificial chromosome constructs such as YACs, Bacterial Artificial Chromosomes (BACs), bacterial PI constructs or single chromosome cDNA libraries.
Both in situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using defined chromosomal markers are important in expanding genetic maps. Examples of genetic maps can be found in Science (1995; 270: 410f and 1994; 265: 1981 f). Gene replacement on a chromosome of another mammalian species can often reveal the linked marker even if the number or arms of a particular human chromosome are unknown. The new sequences can be assigned to the chromosomal arm, or portion thereof, by physical mapping. This provides valuable information to researchers who use positional cloning or other gene discovery techniques to find disease genes. After a disease or syndrome is genetically linked to be roughly localized to a particular genomic region, any sequence localized to that region may represent a related or regulatory gene for further study. The nucleotide sequences of the present invention can also be used to detect differences in chromosomal location between normal, carrier, or diseased individuals due to transposition, inversion, and the like.
Biological organisms
The term "organism" according to the present invention includes any organism containing a target and/or product obtainable therefrom. Examples of organisms may include mammals, fungi, yeast or plants.
The term "transgenic organism" according to the invention includes any organism comprising the target and/or the product obtained therefrom.
Transformation of host cells/host organisms
As indicated earlier, the host organism may be a prokaryote or a eukaryote. Examples of suitable prokaryotic hosts include E.coli and B.subtilis. Descriptions of transformations involving prokaryotic hosts are detailed in the art, and are provided, for example, in Sambrook et al (molecular cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring harbor Laboratory Press) and in Ausubel et al, Current Protocols in molecular Biology (1995), John Wiley & Sons, Inc.
If a prokaryotic host is used, it may be necessary to suitably modify the nucleotide sequence prior to transformation-for example, by removal of introns.
In another embodiment, the transgenic organism is a yeast. In this regard, yeast has also been widely used as a vector for heterologous gene expression. Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Heterologous gene expression in s.cerevisiae has been reviewed, e.g., Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds. pp401-429, Allen and Unwin, London), and King et al (1989, molecular Cell Biology of Yeast, E F Walton and G T Yarronton, eds, pp 107-.
For several reasons, Saccharomyces cerevisiae is well suited for heterologous gene expression. First, they are non-pathogenic to humans and they are unable to produce certain endotoxins. Second, it has a long history of safe use in commercial exploitation for various purposes over the centuries. This makes it widely accepted by the public. Third, the widespread commercial application and research of this organism has resulted in a rich knowledge of the genetics and physiology and large-scale fermentation characteristics of Saccharomyces cerevisiae.
E Hinchcliffe E Kenny (1993, "Yeast as a vehic1E for the expression of heterologous genes", Yeast, Vol.5, Anthony H Rosean and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.) gives a review of the principle of heterologous gene expression and secretion of gene products in s.cerevisiae.
Several yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance; and autonomously replicating plasmid vectors.
To prepare a transgenic yeast, an expression vector is prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast. Several constructs have been developed for heterologous expression. The construct comprises a promoter active in yeast fused to the nucleotide sequence of the present invention, wherein a yeast derived promoter, such as GAL1 promoter, is typically used. Typically, a yeast derived signal sequence is used, such as a sequence encoding the SUC2 signal peptide. A terminator active in yeast terminates the expression system.
For transformation of yeast, several transformation protocols have been developed. For example, the following teachings can be used to prepare transgenic yeasts of the invention: hinnen et al (1978, Proceedings of the national Academy of Sciences of the USA 75, 1929); beggs, J D (1978, Nature, London, 275, 104); and I to, H et al (1983, Jbacteriology 153, 163-.
Transformed yeast cells are selected using a variety of selectable markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, e.g., G418.
Another host organism is a plant. The basic principle of the construction of genetically modified plants is the insertion of genetic information into the plant genome to stably maintain the inserted genetic material. There are several techniques available for inserting genetic information, two main methods being direct introduction of genetic information and introduction of genetic information using vector systems. Reviews relating to the general technique are found in Potrykus (Annu Revplant Physiol Plant Mol Biol [1991] 42: 205-. Further statements concerning plant transformation can be found in EP-A-0449375.
Thus, the invention also provides a method of transforming a host cell with a nucleotide sequence to be a target or to be expressed. Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for expression of the encoded protein and recovery from the cell culture. The protein produced by the recombinant cell may be secreted or contained within the cell according to the sequence and/or vector used. As will be appreciated by those skilled in the art, expression vectors containing a coding sequence can be designed with signal sequences that direct secretion of the coding sequence across the cell membrane of a particular prokaryote or eukaryote. Other recombinant constructs may link the coding sequence to a nucleotide sequence encoding a polypeptide domain that facilitates purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12: 441-53).
PDE inhibitor-assay method
The effect values of the PDEs referred to herein are determined by the following assays:
phosphodiesterase (PDE) inhibition activity
In general, the preferred PDE compounds for use according to the present invention are potent and selective cGMP PDE5 inhibitors. In vitro PDE inhibiting activity against cyclic guanosine 3 ', 5' -monophosphate (cGMP) and cyclic adenosine 3 ', 5' -monophosphate (cAMP) phosphodiesterases can be determined by measuring their IC50Value (concentration of compound required to inhibit 50% of enzyme activity).
The PDE enzymes required can be isolated from a variety of sources, including human cavernosa, human and rabbit platelets, human ventricles, human skeletal muscle, and bovine retina, particularly using the methods of w.j.thompson and m.m.appleman (biochem, 1971,10,311). Specifically, cGMP-specific PDE (PDE5) and cGMP-inhibiting cAMP PDE (PDE3) can be obtained from human cavernous tissue, and cAMP-specific PDE (PDE4) can be obtained from human skeletal muscle. Phosphodiesterase 7-11 can be generated from a full-length human recombinant clone transfected into SF9 cells.
Assays can be performed using w.j.thompson et al. (biochem., 1979, 18, 5228) "batch" (batch) method modification or modification of the protocol described under product No. TRKQ 7090/7100 using Amersham plc employed a scintillation proximity assay that directly probed AMP/GMP. Briefly, the effect of a PDE inhibitor is measured by measuring a fixed amount of the enzyme (in which the substrate cGMP or unlabeled pair of cAMP is present at different inhibitor concentrations and in the presence of a low substrate3H]The ratio of the markers was 3:1 and the concentration was about 1/3Km) Of which IC is studied50≌Ki. With assay buffer [20mM Tris-HCl pH7.4, 5mM MgCl21 mg/ml bovine serum albumin]The final assay volume was adjusted to 100 microliters. Adding an enzyme to initiate the reaction inIncubating at 30 ℃ for 30-60 minutes<30% of the substrate was switched and stopped with 50. mu.l of yttrium silicate SPA beads containing 3mM unlabelled cyclic nucleotides corresponding to PDEs 9 and 11, respectively. The plate was resealed and shaken for 20 minutes, after which the beads were allowed to stand in the dark for 30 minutes and then counted on a TopCount plate reader (Packard, Meriden, CT). Conversion of radioactivity units to% activity of uninhibited control (100%), plotting against inhibitor concentration, and extension using 'Fit Curve' Microsoft Excel to obtain inhibitor IC50The value is obtained.
Functional Activity
This can be assessed in vitro by determining the ability of the compounds of the invention to enhance sodium nitroprusside-induced relaxation of pre-contracted rabbit cavernous tissue strips, as described in s.a. ballard et al (brit.j.pharmacol, 1996, 118(suppl.), abstrate 153P).
The invention will now be further illustrated by way of example with reference to the following figures and sequence listing.
[ attached drawings ]
FIG. 1 shows the effect of the oxytocin receptor antagonist L-368, 899 on the induction of amphetamine (PCA) -induced ejaculation in anesthetized rats;
FIG. 2 shows the effect of a selective oxytocin antagonist (L-368, 899) on seminal vesicle pressure in anesthetized rats.
[ sequence listing ]
SEQ ID NO: 1 shows the amino acid sequence of the human oxytocin receptor.
SEQ ID NO: 2 shows the amino acid sequence of the human vasopressin V1A receptor.
[ embodiment ] A method for producing a semiconductor device
Examples
1.0 method
1.1 animal test methods
1.1.1 penile erection and ejaculation test method of anesthetized rat
To study penile erection and ejaculation, the procedure used was based on the procedure described in Yonezawa et al (2000) Life Sciences 67, 3031-. For ease of reference, the method is cited below:
male Wistar-ST strain rats weighing 350-450 g were used. Animals were housed in groups (2 mice per cage) under a controlled 12 hour light-dark cycle (light started at 07: 00), constant temperature (23 + -1 deg.C) and humidity (55 + -5%) prior to the experiment. They have free access to standard food pellets and water.
Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and placed in a supine position. The penis is extended from its sheath and carefully fixed with a wooden booster placed on the bottom of the penis. Immediately prior to sheath retraction, test compounds were administered intraperitoneally and penile responses were recorded, including penile erection, redness and swelling of the shaft, glans erection, glans and cupular (cup) engorgement and slight flare-up, which was accompanied by a strong opening of the glans. In addition, the time from application of the test compound to the penis to initiate the response and ejaculation was also measured.
The effect of test compounds on PCA-induced ejaculation was also assessed by the amount of ejaculation over 30 minutes. Suitable methods for using conscious rats are described in Renyi (1985) Neuropharmacology, Vol.24, No.8, pp 697-704.
In addition, intracavernosal pressure was also determined in rats anesthetized with pentobarbital (50 mg/kg, i.p.). A small amount of pentobarbital sodium (5 mg) was additionally injected as needed during the experimental period. The penis is extended from its sheath and intracavernosal pressure (ICP) is measured by inserting a stainless steel needle (23-gauge) into one of its sponges. The needle was connected to a teflon tube filled with heparinized saline (10U/ml) and connected to a pressure conducting tube (NEC-San-Ei 7500).
1.1.2 Male sexual behavior model
For all sexual behavior tests, male mice were placed in the observation field (50-60 cm diameter), starting 5 hours from entering the dark cycle and observed under red illumination. After 3 to 4 minutes of placing the male mice in the observation field, a recipient female mouse (ovariectomy, estradiol benzoate/progesterone injected 48 hours before the behavioral study) was introduced into the field and the following parameters were observed:
i) ejaculation latency (EJL; the time elapsed from introduction of the recipient female mouse into the field to ejaculation;
ii) mating efficiency (CE; ejaculation latency/number of times of withdrawal before ejaculation, i.e. the number of seconds between withdrawals);
iii) a decimation frequency (IF; number of insertions before ejaculation);
iv) upper body frequency (MF; number of times of upper body before ejaculation);
v) post ejaculation interval (PEI; the time from ejaculation to the onset of mating behavior).
2.0 Selective oxytocin receptor antagonists
The compounds used in the examples which follow are the following:
the selective oxytocin receptor antagonist L-368, 899. Further details concerning this compound have been provided above. L-368, 899 is more than 20-fold selective for the oxytocin receptor than the Vla receptor [6.3nM OT: 148nM V1a ].
Example 1 in the Presence of a Selective oxytocin receptor antagonist (L-368, 899)
Delaying ejaculation
The oxytocin receptor antagonist L-368, 899 significantly delayed the para-chlorprozamine (PCA) -induced ejaculation in anesthetized rats (0.1-10 mg/kg sc) at oxytocin selective doses. At free plasma concentrations of 5.4 ± 1.5nM (0.9xKi OT, see figure 1), ejaculation was delayed by 140% (near maximal effect) -assuming that any activity at this dose was from antagonism of the oxytocin receptor.
Erectile mechanisms were not substantially affected by oxytocin receptor blockade-penile cupping and unfolding were similar between the control and oxytocin antagonist groups (see table 1 below). Administration of 1 mg/kg-1After sc dose of L-368, 899 (dose which significantly delayed ejaculation), 95% of PCA-induced erections resulted in a cupped penis, while the vehicle control group was 94%, and 61% of PCA-induced erections resulted in a spread of the penis, while the vehicle control group was 63%.
Table 1:
l-368, 899 has very poor CNS penetration and therefore this study shows that oxytocin has a peripheral site of action in PCA-induced ejaculation. PCA is a 5HT releasing agent that activates the non-adrenergic, non-cholinergic nerves that produce penile erection and the sympathetic pathways that control ejaculation. These pro-sexual effects are thought to be mediated by the release of spinal cord 5HT which acts at 5HT1B and 5HT2C receptors. PCT also induces secretion of oxytocin-probably from the posterior pituitary or from the spinal cord center. This increase in oxytocin is involved in the process of ejaculation as in men, since antagonism of the oxytocin receptor has a clear effect on the time taken to reach ejaculation in these studies.
Using a rodent ejaculatory model reflecting human ejaculatory physiology, we demonstrate that the peripheral oxytocin receptor is involved in the mechanism of ejaculation. These effects can be achieved directly or through modulation of sympathetic innervation of the internal reproductive organs. We cannot underestimate the role of the central oxytocin receptor. Furthermore, studies have shown that oxytocin antagonists can treat premature ejaculation by delaying ejaculation.
Example 2 Selective oxytocin antagonist (L-368, 899) in anesthetized rat seminal vesicles
Influence of pressure
L-368, 899 significantly reduced the increase in seminal vesicle pressure stimulated by the splanchnic nerve in anesthetized rats (1-3 mg/kg iv). Seminal vesicle contraction is necessary for ejaculation, and the delivery of semen into the prostatic urethra is thought to trigger ejaculation. Oxytocin has a direct contractile effect on the seminal vesicles of mammals and may additionally have a neuromodulator effect affecting sympathetic innervation in ejaculation. In this study, seminal vesicle contraction was reduced by 41% following injection of a1 mg/kg bolus (see figure 2 below). Preliminary studies have shown that the L-368, 899 concentration in free plasma achieved after 1.0 mg/kg intravenous injection is about 60 nM-based on literature PK and protein plasma binding.
The data show that oxytocin is released upon stimulation of the visceral nerve and that the peptide has a physiological role both in the production of intracapsular pressure and in the ejaculation procedure prior to ejaculation. This study supports the statement that systemic oxytocin affects peripheral ejaculation-these effects can be achieved either directly or through modulation of the sympathetic innervation of the seminal vesicles. Oxytocin regulates the contraction of ducts and glandular lobules throughout the male genital tract and thus affects the fluid volume of the different ejaculatory components. Increasing semen volume is thought to shorten the time to insertion into ejaculation and therefore oxytocin antagonists can be used to treat premature ejaculation by delaying ejaculation.
Example 3 Selective oxytocin antagonists (L-368, 899) mating in rats
Influence of
At doses up to 10 mg/kg sc, L-368, 899 had no effect on the mating behaviour of sexually experienced rats. Rodent mating behavior is characterized by a series of upper body movements, with and without vaginal insertion (50-80% of the upper body causes insertion [ vaginal insertion ]) and ejaculation occurs after 6 to 12 puffs. Each withdrawal lasts for a few seconds-the length of insertion time, i.e. the intravaginal time, cannot be quantified. The effects of L-368, 899 were evaluated in terms of various mating parameters (see above). We focused on intercourse efficiency as a measure to profile vaginal insertion.
At any dose tested (0.05-10 mg/kg sc, see Table 2 below), L-368, 899 had no effect on mating efficiency. Preliminary pharmacokinetic studies showed that free plasma concentrations of 4.5nM and 40nM, respectively, were expected to be achieved 30 minutes after injection at 1 mg/kg sc and 10 mg/kg sc.
Table 2:
| carrier | L-368, 8991 mg/kg sc | L-368, 89910 mg/kg sc | |
| Mating efficiency | 7.0E-2, i.e. 14 s/insertion | 6.2E-2, i.e. 16 s/insertion | 7.0E-2, i.e. 14 s/insertion |
Mean ± sem (n is 14, 8, 5, respectively)
Furthermore, when 50 ng/mouse was administered intracerebroventricularly (icv) with L-368, 899. L-368, 899, there was no significant effect on the mating efficiency of sexually experienced rats.
| Carrier | L-368, 89950 ng/rat icv | |
| Mating efficiency | 3.9E-2, i.e. 25 s/insertion | 3.4E-2, i.e. 29 s/insert |
Mean ± sem (n ═ 4). # p ═ 0.057
Example 4 non-Selective oxytocin antagonist (8-Selenz-vasopression oxytocin) on mating
Effect of behavior
Previous studies explored d (CH)2)5Tyr(Me)Orn8The effect of-8-seminal oxytocin (a peptidergic nonselective oxytocin antagonist) on mating behavior showed no effect on ejaculatory latency at doses up to 25 ng/rat icv. After 5 ng/rat icv injection there was a (significant) reduction in mating efficiency-46 sec/insertion and 25 sec/insertion for vehicle control animals. This effect may be mediated by oxytocin and/or vasopressin receptors. After 50 ng/rat icv administration, mating behavior was completely abolished (Melis et al Neuroscience Letters 205(1999) 171-. In contrast to the results of studies using selective oxytocin receptor antagonists (L-368, 899), the latter data might indicate that antagonism of both the central oxytocin and vasopressin receptors is detrimental to sexual performance including erectile mechanisms and sexual arousal ability.
Abbreviations
Premature ejaculation
Oxytocin (OT ═ oxytocin)
cAMP-3 ', 5' -monophosphate
cGMP-cyclic guanosine 3 ', 5' -monophosphate
PDE ═ phosphodiesterase
PDEcGMPcGMP hydrolysis of PDE
PDEi ═ PDE inhibitors (also known as I: PDE)
PDE5 ═ type 5 phosphodiesterase
PDE5i ═ PDE5 inhibitors
KDa is kilodalton
bp ═ base pair
Kb ═ kilobase pair
Sequence listing(which form part of the specification)
SEQ ID NO：1
MEGALAANWSAEAANASAAPPGAEGNRTAGPPRRNEALARVEVAVLCLILLLALSGNACV
LLALRTTRQKHSRLFFFMKHLSIADLVVAVFQVLPQLLWDITFRFYGPDLLCRLVKYLQV
VGMFASTYLLLLMSLDRCLAICQPLRSLRRRTDRLAVLATWLGCLVASAPQVHIFSLREV
ADGVFDCWAVFIQPWGPKAYITWITLAVYIVPVIVLATCYGLISFKIWQNLRLKTAAAAA
AEAPEGAAAGDGGRVALARVSSVKLISKAKIRTVKMTFIIVLAFIVCWTPFFFVQMWSVW
DANAPKEASAFIIVMLLASLNSCCNPWIYMLFTGHLFHELVQRFLCCSASYLKGRRLGET
SASKKSNSSSFVLSHRSSSQRSCSQPSTA
SEQ ID NO：2
MRLSAGPDAGPSGNSSPWWPLATGAGNTSREAEALGEGNGPPRDVRNEELAKLEIAVLAV
TFAVAVLGNSSVLLALHRTPRKTSRMHLFIRHLSLADLAVAFFQVLPQMCWDITYRFRGP
DWLCRVVKHLQVFGMFASAYMLVVMTADRYIAVCHPLKTLQQPARRSRLMIAAAWVLSFV
LSTPQYFVFSMIEVNNVTKARDCWATFIQPWGSRAYVTWMTGGIFVAPVVILGTCYGFIC
YNIWCNVRGKTASRQSKGAEQAGVAFQKGFLLAPCVSSVKSISRAKIRTVKMTFVIVTAY
IVCWAPFFIIQMWSVWDPMSVWTESENPTITITALLGSLNSCCNPWIYMFFSGHLLQDCV
QSFPCCQNMKEKFNKEDTDSMSRRQTFYSNNRSPTNSTGMWKDSPKSSKSIKFIPVST
Sequence listing
<110>Pfizer Limited(EP(GB)and GB only)
Pfizer Ine，(All other States)
<120> treatment of male sexual dysfunction
<130>PC22047A
<150>GB 02022820
<151>2002-01-31
<160>2
<170>FastSEQ for Windows Version 4.0
<210>1
<211>389
<212>PRT
<213> Intelligent people
<400>1
<210>2
<211>418
<212>PRT
<213> Intelligent people
<400>2
Claims (6)
1. Use of a selective oxytocin receptor antagonist in the manufacture of a medicament for the treatment of premature ejaculation, wherein the selective oxytocin receptor antagonist is at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
2. Use according to claim 1 wherein the selective oxytocin receptor antagonist is at least 30-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
3. Use according to claim 1 or 2, wherein the vasopressin receptor is the V1a receptor.
4. A pharmaceutical package comprising one or more compartments, wherein at least one compartment comprises one or more selective oxytocin receptor antagonists that are at least 20-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
5. A pharmaceutical package according to claim 4 wherein the selective oxytocin receptor antagonist is at least 30-fold selective for an oxytocin receptor as compared with a vasopressin receptor.
6. A pharmaceutical package according to claim 4 or 5 wherein the vasopressin receptor is the V1a receptor.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB0202282.0A GB0202282D0 (en) | 2002-01-31 | 2002-01-31 | Treatment of male sexual dysfunction |
| GB0202282.0 | 2002-01-31 | ||
| PCT/IB2003/000140 WO2003064402A1 (en) | 2002-01-31 | 2003-01-20 | Treatment of male sexual dysfunction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1073109A1 HK1073109A1 (en) | 2005-09-23 |
| HK1073109B true HK1073109B (en) | 2010-04-23 |
Family
ID=
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