CN1802177A

CN1802177A - Inhibitors of phosphodiesterases in infertility

Info

Publication number: CN1802177A
Application number: CNA2004800145039A
Authority: CN
Inventors: S·S·帕尔马; S·D·麦肯纳; S·J·阿金斯托; A·艾斯克尔; M·C·麦克纳米
Original assignee: Applied Research Systems ARS Holding NV
Priority date: 2003-04-01
Filing date: 2004-04-01
Publication date: 2006-07-12
Also published as: ZA200507228B

Abstract

The present invention is directed to methods of increasing oocyte production in a mammal. More specifically, the specification describes methods and compositions for inducing follicular maturation using a PDE inhibitor. The inhibitor may be used alone at high doses. Alternatively, the follicular maturation is achieved by combining a low dose of FSH with the PDE inhibitor treatment.

Description

The phosphodiesterase inhibitor that is used for infertility

The application advocates the U.S. Provisional Patent Application No.60/458 that on April 1st, 2003 submitted to, 955, the U.S. Provisional Patent Application No.60/470 that submitted on May 15th, 2003,434, the U.S. Provisional Patent Application No.60/540 that submitted on January 28th, 2004, the U.S. Provisional Patent Application No.60/544 that on February 12nd, 301 and 2004 submitted to, 003 priority, more than each patent application in to fit into this paper for referencial use.

Technical field

The present invention relates generally to biology of reproduction and auxiliary procreation technology (ART), as super irritate (COH) of controlled ovary of external fertilization (IVF).More specifically say, the present invention relates to single one or more phosphodiesterase inhibitors of using, or with one or more promoting sexual gland hormone couplings, induce the method and composition of follicle maturity in the body, comprise irritating follicle maturity, for example ovulation induction (OI).In addition, the present invention relates to single one or more phosphodiesterase inhibitors of using, or with one or more chorionic-gonadotropin hormone couplings, the method and composition of external evoked oocyte maturation.

Background technology

Women's reproductive cycle depends on multiple promoting sexual gland hormone.Mainly hypophysis cerebri follicle stimulating hormone (FSH) and lutropin (LH) in these hormones.The period of development of ovum, i.e. the process of female sex cell ovum generation occurs in the follicle.In the ovary that contains oocyte (ovum), follicle is the set of cell.Finally cause the follicle maturity of ovulating, depend on the stimulation of FSH.

The every wheel in the menstrual cycle, the maturation of oocyte will be replenished many follicles.When about 28 days menstrual cycle began, follicle existed with the original hase form, and it is an oocyte round a confluent monolayer cells.Growth and maturation along with FSH activation follicle form the multilamellar granulosa cell round initial cell monolayer, and this process lasts till midcycle.These granulosa cell are responsible for nutrition and generation being provided and discharging estrogen to oocyte.The FSH that hypophysis cerebri produces can induce aromatase activity in this granulosa cell, thereby improves estrogen production.Therefore, early stage follicle maturity and the estrogen production increase at 28 days menstrual cycle takes place simultaneously.Follicle also contains the receptor of second kind of pituitary gonadotropic hormone LH.Along with follicle continued growth maturation to mid-term (about 14 days), the inboard space (hole) that produces of granulosa cell group.LH produces and increases sharply during mid-term, acts on the LH receptor and causes follicular rupture and discharge oocyte, is transported to fertilization then in the fallopian tube.Each menstrual cycle, the women of normal ovulation will replenish about 300 jejune oocytes.In the normal menstrual cycle, all follicles will be degenerated (locking) except that a follicle, and a dominant follicle will occur up to discharging an oocyte.

Ovulation induction (OI) and complementary reproductive technology (ART) scheme are the hormone schemes, comprise two main period: irritate the phase and the onset of ovulation.In the time of suitably, inhibitory stage can be prior to the phase of irritating.Inhibitory stage comprises and gives GnRH agonist, is used to suppress LH or estradiol level.The phase of irritating starts from having the preparation (as FSH) of short follicular activity, reaches 6-10 days usually.Patient generally passes through or induces three days of when beginning of passing through spontaneous, and every day, the FSH with about 150IU treated.The FSH administration continues up to a follicle average diameter being arranged more than or equal to 16-18mm (available ultrasonic investigation follicular development).At this moment, has an active preparation of LH with heavy dose of (5000-10000IU), as human chorionic gonadotropin (hCG) induced ovulation, make the peak or the surge (occurring in mid-term) of follicular rupture and the natural lutropin of simulation (LH), be discharged in the fallopian tube to trigger an oocyte.Give to inform patient's sexual intercourse in 24-38 hour behind heavy dose of hCG.Irritate in the phase, give follicle stimulating hormone (FSH), or have the preparation that irritates follicular activity, maybe can irritate the preparation that endogenous FSH discharges, irritate ovarian follicular growth.Irritating does not need to continue to give FSH or has the active preparation of FSH sample in the phase, maybe can irritate the preparation that endogenous FSH discharges, and does not need to last till that the phase that irritates finishes.Perhaps need other preparation that can act on the phase of irritating or the onset of ovulation, particularly oral formulations.The IV type phosphodiesterase inhibitor that United States Patent (USP) 2002/0103106A1 (Palmer etc.) discloses the oral and subcutaneous use of using the onset of ovulation triggers ovulation.United States Patent (USP) 2003/0018037 (Westbrook Lempriere etc.) discloses and has adopted the ODE5 inhibitor to improve embryo survival, improve birth weight, increase uterine blood flow and improve the Progesterone serum levels in the ovulation non-complementary cycle of back.

Ovulation induction (OI) is the treatment to anovulation and menolipsis women, carries out internal fertilization or in utero insemination (IUI) in the fallopian tube to cause that single oocyte is discharged into.The purpose of OI scheme is the release that causes single oocyte, thereby avoids multiple gestation.In the conventional scheme of OI, the first phase of treatment is called stimulation period.

Among the patient of some experience OI, may wish with the inhibitory stage begin treatment.In this inhibitory stage, begin to give gonadotropin-releasing hormone (GnGH) agonist and suppress the hypophysis gonadotropin with before the FSH treatment.Generate in the phase (usually menstrual cycle about 20 days) at the corpus luteum of menstrual cycle and to begin to give the GnRH agonist.Usually suppress ovarian function with the GnRH agonist and take 8-21 days, can monitor (LH＜5IU/L, E2＜50pg/ml is general, and expression fully suppresses) by monitoring LH or estradiol (E2) level.Give FSH then and begin the phase of irritating.Adopt the GnRH agonist to suppress to trigger natural LH peak or the surge that immature oocyte discharges.This is able to regularly discharge oocyte better, has sexual intercourse then.The patient who suffers from polycystic ovary syndrome (PCOS) also needs to adopt the GnRH agonist, because these patients often have unsuitable endogenous LH high level, also should suppress LH in the phase whole irritating, and making can have better reaction to FSH.

The preparation that can excite endogenous FSH to discharge also commonly used carries out OI as citric acid clomiphene or aromatase inhibitor and irritates the phase.Give citric acid clomiphene (3-7 of menstrual cycle days or 5-9 days, common dose 50-100mg) in the phase of irritating and to cause that endogenous FSH secretion raises, and causes ovarian follicular growth.Aromatase inhibitor, for example letrozole (Letrozole), my bent azoles (Anastrozole), YM-511 can give when 3-7 days of menstrual cycle or 5-9 days, also can excite to discharge endogenous FSH, see WO/083239, include this paper list of references in.

Opposite with OI, when in complementary reproductive technology (ART) scheme, needing an ovulation follicle and an oocyte, need in one-period, collect oocyte as much as possible.Adopt ART, for example sperm injection (ICSI), the interior transfer method (GIFT) of gamete fallopian tube and interior transfer method (ZIFT) treatment of zygote fallopian tube are sterile in external fertilization (IVF), the endochylema, need controlled ovary super irritate (COH) improve the female gamete number (Healy etc., Lancet.343:1539-1544,1994).For example, external fertilization (IVF) now has been usually used in treating women and the low person of male fertility, and this is a kind of based on catching the ART technology that people's mature oocyte is fertilized the female knot of sophisticated ovum born of the same parents with sperm then.Under the IVF therapeutic scheme, adopt long-term hormone therapy scheme, as raising people's mature oocyte over 30 days.The FSH and the LH that adopt gonadotropin-releasing hormone GnRH or its congener to suppress patient self start this scheme, and injection of exogenous promoting sexual gland hormone then is as FSH and/or LH, to guarantee the growth of a plurality of preovulatory follicles.In the suitable stage of ovarian follicular growth, a plurality of oocytes of ovulation eve collected at suction.The oocyte of external fertilization suction and cultivation then at the 4-8 cell stage, transferred to the embryo who produces in the uterus after general three days.

Say that more specifically the COH standard scheme of ART is included in inhibitory stage, is also referred to as in the downward modulation phase, give the GnRH agonist and suppress endogenous LH that the corpus luteum of beginning menstrual cycle generates the phase (usually menstrual cycle about 20 days).Usually need 8-21 days with GnRH agonist inhibition ovarian function, can monitor (LH＜5IU/L, fully inhibition of the general expression of E2＜50pg/ml) by monitoring LH or estradiol level.Irritating in the phase after downward modulation gives follicle stimulating hormone (FSH) every day, uses always and induces follicular development over about 75-600IU/ days.

Perhaps, when beginning to irritate follicle with FSH, 1,2 or 3 day in spontaneous menstruation or after inducing menstruation can adopt the GnRH antagonist to replace the GnRH agonist usually, and the back began to give the generation that the GnRH antagonist suppresses endogenous LH in about 6 days passing through.GnRH antagonist and FSH administration will continue to trigger the regulation of dosage hCG up to meeting following ovulating.

In the phase of irritating, use the ultrasound investigation ovary, understand and the measurement follicular development.Because a plurality of follicular developments are purposes of the COH scheme of ART, when showing that ovary contains at least 3 average diameters greater than the follicle of 16mm (preferably one of them is 18mm), injection hCG (5000-10000IU) is to cause ovulation.36-38 hour recovery oocyte behind the injection hCG.Suction method commonly used reclaims the oocyte in the preovulatory follicle.

Although it is for many years clinical that such scheme has been used for, these schemes are not have some significant disadvantage.The FSH half-life is limit quite short, irritates in the phase, and OI or ART adopt the FSH treatment to relate to the heavy dose of FSH (75-600IU FSH/ days) of injection every day.Every day, injection can cause that patient does not accommodate inconvenience, and expense is higher.It is bigger that so a large amount of injection generation every day ovaries surpass the risk that irritates syndrome (OHSS), may life-threatening when serious.Other side effect of gonadotropin goods comprises weight increase, edema, feels sick, vomiting, the time of observation process and unknown permanent back risk of cancer.When normally finishing women's (its sterile problem is and the difference correlation of male partner sperm quality) of these programs when IVF is given, these hormone therapy schemes often become and more are a problem.Therefore, the current therapeutic scheme existing problems that produce oocyte with IVF.Still need to produce the method that the greater number oocyte satisfies ovulation.

Because the gonadotropin administration relates to risk, has proposed various alternative methods.Reducing a kind of method that secondary controlled ovary irritates risk, side effect and the uneconomical shortcoming of scheme relates to and catches immature oocyte then at maturation in vitro.In the method, do not irritate parent or only accept small irritating, make the Oocyte in Vitro of catching accept HORMONE TREATMENT.This maturation in vitro (IVM) scheme significantly reduces/has eliminated above-mentioned side effect, and secondly benefit economically is the hormone amount that has reduced treatment usefulness.Yet though the external maturation of zoopery (IVM) becomes the effective ways that IVF produces oocyte, the success rate that records of people IVM is very low clinically.

Need another kind of method, replace FSH, avoid the OHSS risk with the other medicines that can help ovarian follicular growth.And, provide goods to cause that ovarian follicular growth may also be sought after with FSH effect, because it can improve the low-level of endogenous FSH, cause those because the low and patient's of not ovulating the ovarian follicular growth of endogenous FSH level, maybe can improve the effect of exogenous FSH, improve ART Low Response person's reaction.Or identical reaction is arranged when making low dosage ART, and/or reduce the FSH frequency injection, avoid the OHSS risk simultaneously.

Summary of the invention

In some aspects, the invention provides a kind of method that increases follicle maturity, this method comprises can effectively irritate phosphodiesterase (PDE) the inhibitor for treating mammal of ovarian follicular growth and sophisticated amount.On the other hand, the invention provides the ovarian follicular growth that the employing phosphodiesterase inhibitor irritates patient's ovary.Also have on the one hand, the present invention provides the method that irritates the folliculus ovarii growth to the patient of needs treatment, comprises the phosphodiesterase inhibitor that gives patient's effective dose.A further aspect of the present invention is the method that increases follicle maturity, comprises that wherein, PDE inhibitor and promoting sexual gland hormone provide with the effective dose of associating, to irritate ovarian follicular growth and maturation with PDE inhibitor and promoting sexual gland hormone treatment mammal.

On the other hand, the invention provides the purposes of PDE inhibitor in preparing the medicine that can irritate the growth of patient's folliculus ovarii.Preferably align patient, prepare the medicine that can irritate its folliculus ovarii growth with the PDE inhibitor induced ovulation.More specifically say, irritate patient, adopt the PDE inhibitor to prepare and to irritate the medicine that its folliculus ovarii is grown adopting the controlled ovary of complementary reproductive technology to surpass.Before irritating phase ovulation, begin to give the PDE inhibitor, should have the active preparation of LH before the onset of ovulation or the PDE inhibitor of stopping using the same day of beginning giving heavy dose (as 5000-10000hCG).More preferably before giving hCG, stopped the administration of PDE inhibitor in 2,1 or 0 days.Most preferably giving the hCG same day inactive PDE inhibitor.

Described medicine can with FSH, or have the active preparation of FSH, the preparation that maybe can cause endogenous FSH to discharge, simultaneously, give respectively or successively.Preferred described medicine and FSH are simultaneously, give respectively or successively.Perhaps, described medicine can with have the active preparation of FSH, the preparation that maybe can cause endogenous FSH to discharge, simultaneously, give respectively or successively.Described medicine should be when menstruation begins, or administration in 2-3 days after the menstruation.When administration every day gets the hCG (dosage is preferably 5000-10000IU) that enough main points trigger ovulation up to ovarian follicular growth.When described medicine gives with FSH, can reduce the dosage of FSH, the amount of required FSH is low during without the PDE inhibitor than this patient, and to promoting ovarian follicular growth to obtain identical result.

In the present invention's preferred embodiment in this respect, the PDE inhibitor is to be selected from least a inhibitor that is selected from the type of 1,5 and 6 types.This PDE inhibitor is selected from following chemical compound: 5-[2-ethyoxyl-5-(4-methyl isophthalic acid-piperazinyl sulfonyl) phenyl]-1-methyl-3-just-propyl group-1,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones (sildenafil, sldenafil); Zaprinast (Zaprinast); Persantin (dipyridamole); 5-(2-ethyoxyl-5-morpholino acetylphenyl)-1-methyl-3-just-propyl group-1,6-dihydro-7H-20 pyrazolo [4,3-d] pyrimidin-7-ones; 3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-just-propoxy phenyl]-2-(pyridine-2-yl) methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-(2-methoxy ethoxy) pyridin-3-yl]-2-(pyridine-2 base) methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; (+)-3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-(2-methoxyl group-1 (R)-methyl ethoxy) pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-ethyoxyl-5-(4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-[2-methoxy ethyl]-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-is different-butoxy-5-(4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-1-methyl piperidine-4-base-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-ethyoxyl-5-(4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-(5-acetyl group-2-propoxyl group-3-pyridine radicals)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-(5-acetyl group-2-butoxy-3-pyridine radicals)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; (6R, 12aR)-2,3,6,7,12,12a-six hydrogen-2-methyl-6-(3, the 4-methylenedioxyphenyl) pyrazolos [2 ', 1 ': 6,1] pyridos [3,4-b] indole-1,4-diketone (Tadalafil; IC-351); The embodiment 78 of disclosed International Patent Application WO 95/19978 and 95 chemical compound, and embodiment 1 wherein, 3,7 and 8 chemical compound: 2-[2-ethyoxyl-5 (4-ethyl-piperazine-1-base-1-sulfonyl)-phenyl]-5-methyl-7-propyl group-3H-imidazo-[5,1f] [1,2,4] triazine-4-ketone (Vardenafil); Disclosed embodiment 11 chemical compounds of International Patent Application WO 93/07124 (EISAI); Rotella, DP., J Med Chem., 2000, the chemical compound 3 and 14 of 43:1257; 4-bromo-5-(pyridine radicals methylamino)-6-[3-(4-chlorphenyl)-propoxyl group]-3 (2H) 2H-Pyridazin-3-one; 1-[4-[(1, dioxy between the 3-benzo-5-ylmethyl) amino]-6-chloro-2-quinazolyl]-4-piperidyl-carboxylic acid one sodium salt; (+)-suitable-5,6a, 7,9,9,9a-six hydrogen-2-[4-(trifluoromethyl)-benzyl-5-methyl-cyclopenta-[4,5] imidazo [2,1-b] purine-4 (3H) ketone; Furaziocillin; Suitable-2-hexyl-5-methyl-3,4,5,6a, 7,8,9,9a-octahydro cyclopenta [4,5]-imidazo [2-1 ,-b] purine-4-ketone; 3-acetyl group-1-(2-benzyl chloride base)-2-propyl indole-6-carboxylate; 3-acetyl group-1-(2-benzyl chloride base)-2-propyl indole-6-carboxylate; 4-bromo-5-(3-pyridine radicals methylamino)-6-(3-(4-chlorphenyl) propoxyl group)-3-(2H) 2H-Pyridazin-3-one; 1-methyl-5 (5-morpholino acetyl group-2-just-propoxy phenyl)-3-just-propyl group-1,6-dihydro-7H-pyrazoles (4,3-d) pyrimidin-7-ones; 1-[4-[(1,3-benzo dioxole-5-ylmethyl) amino]-6-chloro-2-quinazolyl]-4-piperidine carboxylic acid one sodium salt; Pharmaprojects No.4516 (Glaxo Wellcome); Pharmaprojects No.5051 (Bayer); Pharmaprojects No.5064 (Kyowa Hakko; See WO 96/26940); PharmaprojectsNo.5069 (Schering Plough); GF-196960 (Glaxo Wellcome); E-4010 (Eisai); Bay-38-3145 ﹠amp; Bay-38-9456 (Bayer), vinpocetine (Vinpocetine) (Richter Gideon); SCH-51866 (Schering-Plough), SCH-59498; (6aR, 9aS)-2-(xenyl methyl)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-3 (benzyl) Pentamethylene. is imidazo [2,1-b] purine-4 (3H)-ketone also-[4,5]; 5 '-methyl-2 ' (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1,7 ' (8 ' H)-[3H] imidazoles [2,1-b] purine]-4 (5 ' H)-ketone; (6aR, 9aS)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-2-(benzyl)-3-(phenylacetylene base) Pentamethylene. is imidazo [2,1-b] purine-4 (3H)-ketone also-[4,5]; Persantin; AWD-12-171 and AWD-12-217 (ASTS Medica); BMS-341400 (Bristol Meyers Squibb); UK-343,664 (Pfizer); 5E-3623,5E-3569,5E-3657, E4021 (Eisai); KS-505a (KyowaHakko Kogyo); YC-1 (Yung Shin Pharmaceutical Industries); IDDB reference number 323951 (Bayer); WIN-61691 (Sanofi Winthrop); FR226807 (Fujisawa); IDDB reference number 461317,462503,461321,461324,466146 (Johnson ﹠amp; Johnson); Pyridin-4-yl methyl-3-(1,3-benzodioxole-5-yl)-9-oxo-1,3,4,9 tetrahydrochysenes-2H-pyrrolo-[3,4-b] quinolyl-2-carboxylate:

Jiang etc., J Med Chem, the chemical compound that the table 1 of 46:441-444 (2003) is listed, particularly chemical compound 20b, 20e, 20f, 20l, 20o, 20p, (-)-20q, 20t, 20u, 20v, 20w and 26a.

The PDE inhibitor can be PDE1 or PDE5, or the selective depressant of PDE1 and PDE5.The PDE inhibitor can be selected from: sldenafil and zaprinast, persantin and (6aR, 9aS)-2-(xenyl methyl)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-3-(benzyl) cyclopentadienyl group-[4,5] imidazo [2,1-b] purine-4 (3H)-ketone; And 5 '-methyl-2 ' (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1,7 ' (8 ' H)-[3H] imidazo [2,1-b] purine]-4 (5 ' H)-ketone (are respectively people such as Ahn, J Med Chem, " the chemical compound no.31 " described in the 40:2196-2210 (1997) and " chemical compound no.33 ".

On the other hand, the invention provides the method for external oocyte maturation, comprise that wherein PDE and promoting sexual gland hormone provide with the amount of associating of enough guiding oocyte maturations with the PDE inhibitor or with PDE inhibitor and promoting sexual gland hormone extracorporeal treatment oocyte.Should improve the number of mature oocyte, 0-1 oocyte when not handling by adopting methods described herein to make brought up to the oocyte more than 4.

Of the present invention one concrete aspect, the method that increases the animal follicle maturity is provided, comprise giving the compositions that this animal contains at least a PDE inhibitor and promoting sexual gland hormone, wherein PDE and promoting sexual gland hormone provide with the amount of associating that can effectively improve the oocyte number that the human chorionic gonadotropin is reacted.In particularly preferred embodiments, the PDE inhibitor is the PDE4 inhibitor.Adoptable exemplary PDE inhibitor comprises but is not limited to be selected from following these: Piclamilast, Roflumilast, Ariflo, Filaminast, Mesopram, D4418, Arofylline and CL1044, also can adopt other PDE4 inhibitor.Many will illustrating following, but should be understood that and also can adopt the PDE4 that has of these chemical compounds to suppress active congener and derivant.Consider that the inventive method can only adopt a kind of PDE inhibitor.Perhaps, can adopt the mixture of multiple PDE inhibitor.This mixture can comprise one or more PDE4 inhibitor and one or more other PDE inhibitor.The described compositions of special consideration can contain at least a PDE4 inhibitor and at least a other PDE inhibitor, is selected from PDE1 inhibitor, PDE5 inhibitor, PDE6 inhibitor, PDE7 inhibitor, PDE9 inhibitor, PDE10 inhibitor, PDE11 inhibitor.In specific embodiments, method of the present invention adopts the compositions that contains two kinds or multiple PDE4 inhibitor.

In the method for the present invention, be used to irritate or the promoting sexual gland hormone that increases follicle maturity is selected from: follicle stimulating hormone (FSH), lutropin (LH) and chorionic gona dotropin (CG) and their combination.Preferred promoting sexual gland hormone is FSH.In other embodiment, this method comprises promoting sexual gland hormone such as the LH that gives FSH and give non--FSH.Perhaps, this method comprises stimulant, agonist or the co-adjuvant that gives FSH and add FSH.Perhaps, this method comprises that the stimulant, agonist or the co-adjuvant that give FSH add the PDE4 inhibitor.Irritating the active exemplary medicament of FSH comprises: aromatase inhibitor such as Letrozole, Auastrozole and Vorozole (see PCT/EP01/14730 and U.S. Patent application No, 20020103106, include this paper list of references in).

In specific embodiments, PDE inhibitor and promoting sexual gland hormone treatment can give simultaneously.Utilizing " promoting sexual gland hormone treatment " of the present invention, is single with FSH treatment, the non--FSH promoting sexual gland hormone of FSH treatment associating; Single with the active stimulant of FSH or other agonist treatment or with FSH and/or with the therapeutic alliance of other non--FSH promoting sexual gland hormone.In certain embodiments, give the PDE inhibitor earlier and give promoting sexual gland hormone again.In other embodiments, give promoting sexual gland hormone earlier and give the PDE inhibitor again.The consumption of FSH can be any routine dose of usefulness clinically.For example, the FSH dosage range that can give is about 5-450IU/ days, more preferably gives dosage range 5-75IU/ days of FSH.In specific embodiments, consider to give PDE4 or other PDE inhibitor to reduce the FSH consumption of common individuality.Be this kind application, the FSH of " inferior suitable dosage (suboptimal) " is the FSH of any this reduction dosage, and promptly this FSH consumption is lower than the dosage that can produce the sophisticated follicle effect of best increase.This FSH can be Recombinant FSH (r-FSH), the people FSH (hFSH) of preferred reorganization.In other embodiment, this FSH purification derives from urine.

The present invention also provides the method for follicular development preovulatory phase of increasing mammal in the body, and this method comprises and gives at least a PDE4 inhibitor of this mammal and external source FSH hormone.The method suppresses the generation of this mammal endogenous FSH and LH before also can being included in and giving PDE4 inhibitor and FSH hormone.Preferred this exogenous FSH hormone is the Recombinant FSH hormone.In other embodiment, this exogenous FSH hormone is a urine FSH hormone.

In specific embodiments, the dosage that the PDE inhibitor gives is about 0.05-5mg/ days.Consider the dosage of this inhibitor should be according to the concrete inhibitor and the patient's that treats concrete condition difference.Consider that this dosage can be 0.05mg/ days, 0.075mg/ my god, 0.10mg/ my god, 0.125mg/ my god, 0.150mg/ my god, 0.175mg/ my god, 0.20mg/ my god, 0.225mg/ my god, 0.250mg/ my god, 0.275mg/ my god, 0.30mg/ my god, 0.325mg/ my god, 0.350mg/ my god, 0.375mg/ my god, 0.40mg/ my god, 0.425mg/ my god, 0.450mg/ my god, 0.475mg/ my god, 0.50mg/ my god, 0.525mg/ my god, 0.550mg/ my god, 0.575mg/ my god, 0.60mg/ my god, 0.625mg/ my god, 0.650mg/ my god, 0.675mg/ my god, 0.70mg/ my god, 0.725mg/ my god, 0.750mg/ my god, 0.775mg/ my god, 0.80mg/ my god, 0.825mg/ my god, 0.850mg/ my god, 0.875mg/ my god, 0.90mg/ my god, 0.925mg/ my god, 0.950mg/ my god, 0.975mg/ my god, 1.0mg/ my god, 1.25mg/ my god, 1.5mg/ my god, 1.75mg/ my god, 2.0mg/ my god, 2.25mg/ my god, 2.50mg/ my god, 2.75mg/ my god, 3.0mg/ my god, 3.25mg/ my god, 3.5mg/ my god, 3.75mg/ my god, 4.0mg/ my god, 4.25mg/ my god, 4.5mg/ my god, 4.75mg/ my god, 5.0mg/ it or more.In preferred embodiments, the about 10--200mg/ of dosage days of consideration PDE4 inhibitor.Those skilled in the art will know that these are exemplary dosage and scope.Should understand the consumption of any individuality of concrete consideration and quote between the dosage range above, the consumption of each individuality between these dosage all belongs in the scope of the present invention.Each numerical value of simple abreviation does not belong to scope of the present invention because it is not listed between above-mentioned concrete dosage.Other non-PDE inhibitor combination to this paper administration is like this too.

In other embodiments, consider that FSH gives with 5IU FSH/ days to 75IU FSH/ days dosage range.But the dosage that FSH can 150IU FSH/ days gives.Preferred FSH gives with single agent.Other embodiments are considered to give FSH with multi-agent.But FSH intramuscular, subcutaneous or by any method afford easily.Preferred FSH gives between the 2nd day to the 14th day of mammiferous menstrual cycle.In specific embodiments, FSH gave between the 7th to 12 day continuously.

In specific embodiments, suppress endogenous FSH and LH and comprise to mammal and give gonadotropin-releasing hormone (GnRH) or its congener.More specifically say, gave mammalian GnRH in preceding 30 days giving PDE4 inhibitor and FSH hormone.The dosage of GnRH can be any dosage that routine is used to suppress the endogenous promoting sexual gland hormone.These embodiments generally can adopt GnRH or its antagonist, and dosage is about 0.25-3mg GnRH every day.

In the method for the present invention, give the mammal of PDE4 inhibitor and FSH, compare, can produce the oocyte that more hCG can be ovulated with the animal that does not give PDE4 inhibitor and FSH.For example, the object of shortage PDE4 treatment can produce the oocyte that 0-1 hCG can be ovulated; And through the oocyte that the hCG more than 4 can be ovulated that produces of PDE treatment.Therefore adopt the inventive method can produce 4,5,6,7,8,9,10 or more a plurality of oocyte of gathering in the crops.The inventive method can be after giving PDE4 inhibitor and FSH hormone at first 12 days results oocytes.This method also comprises the oocyte of external fertilization results, and the fertilized oocyte of cultivating this results is to the 4-8 cell stage.And then the fertilized oocyte of this 4-8 cell stage transferred in the mammal uterus.This animal can be same the animal of collecting oocyte, maybe can be and collection oocyte animal inequality.

The present invention also provides the test kit of treatment infertility, first compositions that is included at least a PDE4 inhibitor in the pharmaceutically acceptable preparation is housed in this test kit and is included in second compositions that contains FSH in the pharmaceutically acceptable preparation.This test kit can be equipped with urine FSH or Recombinant FSH.The all preferred people FSH of FSH under these two kinds of situations.FSH in the test kit should provide with the about 5-75IU FSH of unit dose.This test kit also is equipped with LH the 3rd compositions that is included in the pharmaceutically acceptable preparation.The LH typical flat dosage that is adopted in the test kit is between 75IU LH to 150IULH.

Will be appreciated that other features and advantages of the present invention from following detailed description.Yet should understand, though these are described in detail and specific embodiment has just illustrated preferred implementation of the present invention with the elaboration mode, those skilled in the art are clear can to make various variations and modification all belongs to design of the present invention and scope from this detailed description.

Description of drawings

The following drawings constitutes the part of this description, further specifies the present invention.Can understand the present invention with reference to these accompanying drawings better in conjunction with specific embodiment provided herein.

Fig. 1 is the FSH (" low FSH ": 151.5ng * 4 every Mus) treatment after of immature female rats with suboptimal dose, with add persantin (Dipyridamole) (1,5, the every Mus in 25mg/kg * 4) treatment with the FSH of suboptimal dose (" low FSH ") after, every oocyte number that rat is discharged.

Fig. 2 is the FSH (" low FSH ": 151.5ng * 4 every Mus) treatment after of immature female rats with suboptimal dose, with add assorted Pu Site ((zaprinast) (1,5, the every Mus in 25mg/kg * 4) treatment with the FSH of suboptimal dose (" low FSH ") after, every oocyte number that rat is discharged.

Fig. 3 is the FSH (" low FSH ": 151.5ng * 4 every Mus) treatment after of immature female rats with suboptimal dose, with add sldenafil (Sildenafil) (1,5, the every Mus in 25mg/kg * 4) treatment with the FSH of suboptimal dose (" low FSH ") after, every oocyte number that rat is discharged.

Fig. 4 for low dosage FSH (" low FSH ": the every Mus in 151.5ng * 3), high dose (" high FSH ": the every Mus in 606.2ng * 3) and low dosage FSH add the secondary follicle number of the rat that sldenafil (the every Mus in 25mg/kg * 3) treats.

Fig. 5 for low dosage FSH (" low FSH ": the every Mus in 151.5ng * 3), high dose (" high FSH ": the every Mus in 606.2ng * 3) and low dosage FSH add the hole follicle number of the rat that the 75mg/kg sldenafil treats.

Fig. 6 for immature female rats give the low dosage FSH that buffer saline liquid joins (" low FSH ": the every Mus in 151.5ng * 3), high dose (" high FSH ": the every Mus in 606.2ng * 3) and low dosage FSH (the every Mus in 151.5ng * 4) add the oocyte number of sldenafil (1,5, the every Mus in 25mg/kg * 4) treatment back rat discharge.

Fig. 7 do not give FSH (" no FSH ") for immature female rats, give the suitable dose F SH in Asia that buffering liquid joins (" low FSH ": the every Mus in 151.5ng * 3), after inferior suitable dose F SH (the every Mus in 151.5ng * 4) adds tadanafil (Tadalafil) (the every Mus in 25mg/kg * 4) and high dose (the every Mus in " high FSH " 606.2ng * 3) treatment, the oocyte number of every rat discharge.

The every Mus in 151.5ng * 3), inferior suitable dose F SH (" low FSH ": the every Mus in 151.5ng * 4) add chemical compound no.31 (6aR Fig. 8 do not give FSH (" no FSH ") for immature female rats, give the suitable dose F SH in Asia that buffer saline liquid joins (" low FSH ":, 9aS)-2 (xenyl methyl)-5.6a, 7,8,9,9a-six hydrogen-5-methyl-3 (benzyl) cyclopenta [4.5] imidazoles-[2,1-b] purine-4 (3H)-ketone) after the treatment of (the every Mus in 25mg/kg * 4) and high dose (the every Mus in 606.2ng * 3), every oocyte number that rat is discharged.

The every Mus in 151.5ng * 3), inferior suitable dose F SH (" low FSH ": the every Mus in 151.5ng * 4) add chemical compound no.33 (5 '-methyl-2 ' (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1 Fig. 9 do not give FSH (" no FSH ") for immature female rats, give the suitable dose F SH in Asia that buffer saline liquid joins (" low FSH ":, 7 ' (8 ' H)-[3H] imidazo [2,1-b] purine radicals]-4 (5 ' H)-ketone) after (the every Mus in 25mg/kg * 4) and high dose FSH (the every Mus in the 606.2ng * 3) treatment, every oocyte number that rat is discharged.

Figure 10 A-10C is that the cAMP that the pig granulosa cell (JC410/FSHR) of rat granulosa cell (rat ovary dispersate) and/or expressing human fsh receptor was induced or increased to the PDE4 inhibitor is measured in vitro study.

Figure 11 gives low dosage FSH associating variable concentrations (0.08mg/kg for proof in the body; 0.4mg/kg; 2mg/kg) Piclami last can induce follicle maturity.

Figure 12 gives low dosage FSH associating variable concentrations (0.08mg/kg for proof in the body; 0.4mg/kg; 2mg/kg) roflumilast (Roflumilast) can be induced follicle maturity.

Figure 13 is for showing inducing follicle maturity in the body.Competitive list is with hanging down and high dose FSH; Variable concentrations (0.08mg/kg; 0.4mg/kg; 2mg/kg) RPR 73401 (Piclamilast) irritates and with low dosage FSH and variable concentrations (0.08mg/kg; 0.4mg/kg; When 2mg/kg) Piclamilast irritates, the generation of oocyte.The result who shows the secondary independent experiment.

Figure 14 is for showing that cumulative data shows, with low dosage FSH and variable concentrations (0.08mg/kg to the inducing of follicle maturity in the body; 0.4mg/kg; 2mg/kg) Piclamilast unites and irritates the back oocyte and produce to increase.The result who shows the secondary independent studies.

Figure 15 is for showing in the body the inducing of follicle maturity, and cumulative data shows, with low dosage FSH and variable concentrations (0.0032,0.016,0.08mg/kg; 0.4mg/kg; 2mg/kg) Mesopram unites and irritates the back oocyte and produce to increase.The result who shows the secondary independent studies.

The specific embodiment

Now have recognized that, adopt promoting sexual gland hormone to irritate the meaningful therapeutic scheme that ovary can be used for having inferior suitable level spouse's fertility.The IVF program depends on FSH and other promoting sexual gland hormone that adopts remarkable dosage, is used for fertilization to irritate the generation oocyte.Yet this ovary irritates and often causes tangible harmful side effect.Therefore, need other oocyte that can promote to ovulate to produce but just do not depend on the method that irritates ovary with promoting sexual gland hormone.

The present invention relates to adopt phosphodiesterase (PDE) inhibitor to induce follicle maturity.Found when giving immature P of Rats DE inhibitor, can cause the hCG-oocyte number purpose of can ovulating to increase.Say that more specifically the PDE4 inhibitor has been induced the increase greatly of this oocyte number when the FSH with the suitable dosage in Asia gives.The inventor also is surprised to find, and belongs to phosphodiesterase (PDE) inhibitor molecules in the scope of the invention, is preferably selected from 1,5,6,7,9,10,11 types of PDE, and more preferably 1 of PDE and 5 types can help ovarian follicular growth when having the FSH of suboptimal dose.

In specific embodiments, discovery gives PDE4 inhibitor Piclamilast and Roflumilast has promoted the inductive follicle maturity of FSH.The most significant effect sees low dosage FSH and the PDE4 inhibitor is united when giving.Be less than the ovary that irritates with high dose FSH but give low dosage FSH (seeing Figure 11 and the 12) oocyte that every rat ovary is discharged from the data notice of invitation of Figure 12-14, common every the rat of high dose FSH can produce 15-18 ovum.But the problem of employing high dose FSH as mentioned above.The present invention proves that the oocyte number that can discharge with the rat of low dosage FSH substantially exceeds radix when with the PDE4 inhibitor for treating.Really, as Figure 11 and 12 findings, the Piclamilast of low dosage FSH and 0.4mg/kg or Roflumilas administering drug combinations, even the single FSH with high dose of ratio has produced the oocyte that more can discharge (Figure 11 and 12).In addition, from Figure 14 as seen, notice that single Piclamilast (being 2mg/kg) inadequacy FSH with high dose has also increased the oocyte number that hCG can be ovulated.

In view of above-mentioned experimental data, consider that PDE inhibitor, particularly PDE4 inhibitor can be used for the oocyte that generation can be discharged in the body.This is particularly useful in the treatment female infertility, but even normal women also can benefit from this treatment, if when particularly wishing to improve pregnancy rate.In a more particular embodiment, consider that the PDE4 inhibitor is particularly useful in promotion or increases follicle maturity, the oocyte number of ovulating that produces in mammalian body increases than the ovulated oocyte number that is produced without the PDE4 inhibitor.

In view of Figure 14 finding, consideration gives the PDE4 inhibitor separately and does not irritate with low dosage FSH simultaneously, can enough increase the oocyte of can ovulating.Therefore, some method of the present invention does not need to irritate ovary with exogenous FSH fully.Perhaps, Figure 11-14 finds that method of the present invention also comprises and gives low dosage FSH combined PD E4 inhibitor, is enough to produce the remarkable increase of oocyte number of can ovulating.No matter the PDE4 inhibitor whether give separately or as with the part of low dosage FSH therapeutic alliance, method of the present invention produces the oocyte number of can ovulating by remarkable reduction effectively increases required FSH quantity of stimulus, and obvious improvement is provided.This remarkable reduction of FSH consumption benefits, because it has reduced or eliminated and the relevant many problems of fertility treatment promoting sexual gland hormone administration.Particularly, the attenuating of FSH consumption will reduce the probability that causes fertility treatment women that OHSS takes place.

Though this paper has been used as many discussion with regard to making of PDE4 inhibitor, but the use of other PDE inhibitor is supported in discovery of the present invention, singly use and the coupling of PDE4 inhibitor, with low dosage FSH coupling, or with PDE4 inhibitor and low dosage FSH coupling, for example other inhibitor can be ovulated in the method for oocyte number for the PDE5 inhibitor can be used for increasing mammal.The method can singly be used the PDE5 inhibitor; The method with the PDE5 inhibitor or with the coupling of PDE4 inhibitor, or with low dosage FSH coupling; But but the method coupling PDE5 inhibitor and PDE4 inhibitor add low dosage FSH.In preferred embodiments, described inhibitor is selected from phosphodiesterase 1 type and/or 5 types (" selectivity " inhibitor of PDE1 type and/or 5 types for example refers to, shows the IC of PDE1 type and/or 5 types ₅₀Molecule, promptly than the IC of other PDE type molecule ₅₀Low about 10 times, preferably low 100 times, more preferably hang down 1000 times molecule).The IC of PDE enzyme molecule ₅₀Available in vitro tests is measured, and for example sees Thompson etc., Adv Cyclic NucleotideRes.10:69-92 (1979).Also preferred PDE inhibitor molecules amount is below 500Da.In another preferred embodiment, the PDE inhibitor is a PDE1 type inhibitor, and optimum option is the selective depressant of PDE1 type.The PDE inhibitor should be non-peptide class PDE inhibitor.Another preferred alternative method is that PDE4 inhibitor and PDE5 inhibitor add low dosage FSH method for combined use.Consideration in the method, when giving the PDE inhibitor consumption of FSH less than or needed about 75% when equaling same patient without the PDE inhibitor, and can obtain the follicle maturity of par.More preferably, the consumption of FSH less than or needed about 50% when equaling same patient without the PDE inhibitor, even more preferably, the consumption of FSH less than or when equaling same patient needed about 30% without the PDE inhibitor.To more elaborate the method for PDE inhibitor and the coupling of FSH compositions below this paper.

FSH and other gonadotropin compositions

FSH is a kind of hypophysis cerebri glycoprotein hormones of being made up of two subunits.α-subunit is that FSH and other glycoprotein LH, HCG and TSH are common, and β-subunit is given FSH specificity.Because the progress of sterility treatment, existing many FSH goods are commercialized supply, can be used in the method for the present invention.These class commercialization goods comprise FSH compositions and the Recombinant FSH compositions that derives from urine.These compositionss comprise as Pergnol ^TM, Fertinexl ^TM, Repronex ^TM, Bravelle ^TM, Humegon ^TM, Gonal-F, Follistim ^TMThese are exemplary commercialization FSH goods.Those skilled in the art will know that might produce other this class FSH goods is used for method and composition of the present invention.Above-mentioned composition provides preparation and the dosage guide about available FSH herein, and they will be described in further detail following.Yet, be to be understood that, be not difficult to revise these preparations and dosage and still can be used for the present invention, as long as the ovulated oocyte number that produces in vivo when this FSH dosage and preparation and one or more PDE inhibitor couplings, the oocyte number increase that produces than time spent not gets final product.

Humegon ^TM(Organon, West Orange NJ) are the active gonadotropin purification of a kind of FSH of having and LH goods.Adopt character, guide and the program of these gonadotropin goods to see Physician ' sDesk Reference (PDR for details ^TM, 52 editions, 1998, the 1949-1951 page or leaf is included this paper list of references in).In brief, every bottle is equipped with 75IU or 150IU FSH, 75IU or 150IU LHo Humegon ^TMWith hCG mode administration in turn, cause the infertility women's of not ovulating ovulation and gestation, it is functional that these women are not ovulated, and is not because the ovary decline.Show participating in the ovulation patient of external fertilization program, Humegon ^TMCan irritate a plurality of follicles similarly with hCG and produce (promptly irritating follicular development).Usually need adjust the Humegon that is used to increase sophisticated follicle to each patient ^TMRequired dosage and individuation.Yet the dosage of general first recommendation is 75IU FSH/LH/ days, intramuscular administration 7-12 days, in the end potion Humegon ^TMOne day after, give potion hCG (5000U-10000U).Common Humegon ^TMTreating a course of treatment should be continuously above 12 day.For administration, with bottled Humegon ^TM(containing 75IU FSH/LH or 150FSH/LH) is dissolved in the 2ml sterile saline, intramuscular injection immediately.

Pergnol ^TM(Serono Laboratoyies Inc., Randolph MA), see Physician ' s DeskReference (PDR ^TM, 52 editions, 1998,2773-2775 page or leaf), be the another kind of commercialization purification gonadotropin goods of women's urine preparation after the menopause, can provide the inventive method used FSH compositions.But the said composition intramuscular injection of preparation.In addition, this is the pharmaceutical compositions of a kind of 75IU of containing FSH/LH or 150IUFSH/LH dosage unit.This pharmaceutical preparation is the known compositions that is used to not have ovarian failure women administration promotion ovarian follicular growth.In order to act on ovary is not follicle maturity, gives Pergnol ^TM7-12 days, give the injection of hCG medicine group then.

Repronex ^TM(Ferring Pharmaceutical Inc., Tarrytown, NJ), wind Physician ' sDesk Reference (PDR ^TM, 57 editions, 2003,1325-1327 page or leaf), be another kind of after the menopause women's urine separate the exemplary purification gonadotropin goods that obtain.Can obtain the said composition of 75IU or 150IU FSH/LH unit dose, administration 7-12 days to promote the folliculus ovarii growth.In case follicle is enough ripe, gives hCG induced ovulation.Can be with Repronex ^TMSubcutaneous or intramuscular ovulate sterile patient seldom or the patient who is just experiencing complementary reproductive therapy.In the previous case, what patient accepted is the treatment intervention that suppresses the endogenous gonadotropin, Repronex ^TMPreceding 5 days preliminary dosage of drug treatment are 150IU/ days.According to clinical monitoring,, can adjust subsequent dose up and down according to patient's individual reaction situation as serum estradiol level and the monitoring of transvaginal sonography ripple.Should not make dose titration every other day, each adjustment changes should not surpass 75-150IU.Repronex ^TMThe maximum daily dose of treatment should not surpass 450IU, treats natural law continuously and should not surpass 12 days, and this dose titration guide can be used for other FSH goods described herein.If patient presents the follicle maturity signal, at Repronex ^TMTreatment finishes to give one day after hCG.

Adopt Repronex in complementary reproduction therapy ^TMAmong the patient of treatment, patient accepts gonadotropin suppression therapy (suppressing as GnRH or antagonist hypophysis) earlier, and typical preliminary dosage can be the Repronex of 225IU ^TM, adjust according to the individual patient response situation then.In case the evidence that follicle reaches full growth occurs, give hCG (5000-10000USP unit) and induce follicle finally ripe, carry out oocyte and catch.When because Repronex ^TMAnd when ovary taking place increasing unusually, do not give hCG, to reduce the probability that OHSS takes place.As mentioned above, the subcutaneous said composition that gives.For subcutaneous administration, with Repronex ^TMMix the administration of hypogastric region subcutaneous injection with 2ml saline.

Fertinex ^TM(Serono Laboratoyies Inc., Randolph MA), see Physician ' s DeskReference (PDR ^TM, 52 editions, 1998, the 2771-2773 page or leaf), be high-purity FSH that purification obtains women's urine after menopause.Adopt mouse anti FSH monoclonal antibody by immunoaffinity chromatography purification FSH compositions, the FSH specific activity that obtains is about 8500-13500IU FSH/mg albumen.Be not difficult to adopt this kind purification process to originate as obtaining FSH women's urine after menopause from non-commercial.Fertinex ^TMSubcutaneous injection, the bottle that provides contain 75IU FSH or 150IU FSH.The common dose scope that gives this FSH is different between 75IU-300IU/ days.

Bravelle ^TM(Ferring Pharmaceutical Inc., Tarrytown NJ), see Physician ' s DeskReference (PDR ^TM, 57 editions, 2003, the 1325-1327 page or leaf), be that another kind can be used for the exemplary high purification FSH goods in the inventive method.Bravelle ^TMBut can subcutaneous or intramuscular administration, available unit dose is 75IU and 150IU.

Gonal F ^TMBe a kind of hypodermic Recombinant FSH goods that are fit to.Gonal F ^TMPerformance and feature see Physician ' s Desk Reference (PDR ^TM, 57 editions, 2003, the 3124-3128 page or leaf) described in.

Except these commercial obtainable compositionss, those skilled in the art can adopt technology well known in the art, select from natural origin, obtain FSH (seeing for example U.S. Patent No. 5,767,067) as the urine purification of women after menopause.

Perhaps, those skilled in the art can adopt technology well known in the art, select the production Recombinant FSH.Special consideration can be adopted long-acting FSH agonist in the methods of the invention.For example, the half-life of known hCG is longer than FSH.These two kinds of gonadotropin have common α-subunit, and its activity specific is by β-subunit decision.Before prove that a kind of α-subunit of gonadotropin can share with the β-subunit of another kind of gonadotropin, still produce the mosaic gonadotropin of physiologically active.And the biological activity that has proved hCG because of the carboxyl terminal peptide of its β-subunit than LH height (Matzu etc., Endocrinology.126:376-383,1990).Can produce the long-acting agonist of FSH, it extends at the carboxylic end peptide of FSH β-subunit.(LaPolt etc., Endocrinology, 131:6,2514-2520,1992).With wild type FSH relatively, this mosaic molecule shown circulating half-life with remarkable increase and activity intensity (Fares etc., Proc Natl AcadSci., 89:4304-4308,1992).

No matter the source of FSH, this paper proves and adopts the FSH of inferior suitable dosage can realize significantly inducing follicle maturity.As mentioned above, the FSH typical doses scope that the fertility treatment scheme gives is 75IU FSH/ days---450IU FSH/ days, and about 7--12 days.In the inventive method, the FSH dosage that irritates follicle maturity of consideration and PDE4 or other PDE inhibitor coupling, can be identical with the used dosage of present treatment oligo-ovulation and/or complementary reproductive technology (promptly changing between FSH/ days) at 75IU FSH-450IU.Yet, in view of the present invention says,, when with PDE4 inhibitor administering drug combinations, also might reach follicle maturity even adopt the FSH of inferior suitable dosage, therefore preferably adopt the amount lower than these typical doses.

The inventive method can consider to adopt the amount that is low to moderate 5IU FSH/ days.Therefore any given therapeutic scheme can adopt 5IU FSH/ days, and 10IU FSH/ days, 15IU FSH/ days, 20IU FSH/ days, 25IU FSH/ days, 30IU FSH/ days, 35IU FSH/ days, 40IU FSH/ days, 45IU FSH/ days, 50IU FSH/ days, 55IU FSH/ days, 60IU FSH/ days, 65IU FSH/ days, 70IU FSH/ days, 75IU FSH/ days, 150IU FSH/ days, or the FSH/ of more units days.Certainly, should understand these is exemplary daily dose, and other integer dosage also can be used for Therapeutic Method of the present invention between the above-mentioned particular value.In addition, should be understood that during the FSH administration and can raise or reduce described dosage.Can give FSH by any approach that is usually used in the promoting sexual gland hormone administration.Most preferably by intramuscular or subcutaneous injection administration.In the whole therapeutic process, monitoring patient's untoward reaction signal comprises the OHSS signal.

Except that FSH, other promoting sexual gland hormone that the inventive method adopts can be packaged in the mentioned reagent box.These hormones comprise hCG.The hCG such as the Novarel that can buy by commercial sources ^TM(FerringPharmaceutical Inc., Tarrytown NJ), see Physician ' s Desk Reference (PDR ^TM, 57 editions, 2003, the 1324-1325 page or leaf), be the gonadotropin that people's Placenta Hominis produces, can be available from gravid woman's urine.Another commercialization goods of hCG are Pregnyl ^TM(Organon West Orange, NJ) performance of this hormone, pointer and operational version describe in detail and see Physician ' s Desk Reference (PDR ^TM, 57 editions, 2003,2401 pages).These two kinds of goods all are intramuscular injection.Usually the dosage range that gives this hormone comes induced ovulation between about 5000-10000 unit.

Another hormone that this paper can adopt and pack is GnRH.This hormone has many commercializations source.Can purchase having of the GnRH that sells for and its analog: Cetrotide ^TM(Serono PDR ^TM, 57 editions, 2003, the 3119-3121 page or leaf); Eligard ^TM(Sanofi-Synthelabo PDR ^TM, 57 editions, 2003,2994 pages); Lupron ^TM(PDR ^TM, 57 editions, 2003,3185 pages); And Zoladex ^TM(AstraZeneca PDR ^TM, 57 editions, 2003,695 pages).The LH/FSH that adopts these preparations to suppress the women produces, thereby can be used for delaying ovulation.The typical doses of these preparations is 0.25mg-3mg.Irritating treatment with FSH as ovary generally began at the 2nd or 3 day of menstrual cycle.In early days to mid-term (" irritating the phase "), give GnRH or its congener (low dosage such as 0.25mg) once a day at follicle, or potion (as 3mg).Give GnRH till that day that gives hCG.When ultrasound wave announcement follicle was enough big, it was ripe at last to give hCG induced ovulation and oocyte.

Phosphodiesterase inhibitor

Phosphodiesterase (PDE) is to be responsible for second message,second messenger's cyclisation AMP (cAMP) and the metabolic enzyme family of cyclisation GMP (cGMP) in the born of the same parents.PDE4 is the cAMP specific PDE, and it is the main metabolic enzyme that sees the cAMP in inflammatory cell and the immunocyte, if not the only, the cyclisation AMP metabolism in the smooth muscle is had remarkable effect.PDE4 be subjected to antidepressant Rolipram (Rolipram) (4-[3-(cyclopentyloxy)-4-methoxyl group-phenyl]-2-Pyrrolidone; AG Scientific, Inc .San Diego CA) suppresses.Rolipram is the first generation PDE4 inhibitor (seeing Cont, Biology ofReproduction.67:1653-1661,2002) of exploitation.Identify other this class inhibitor subsequently, include but not limited to Piclamilast, Roflumilast, Ariflo, Filaminast, Mesopram, D4418, Arofylline, C11044.In addition, other PDE inhibitor such as Sildenafil, AS701948/1 and AS701947/1 also can be used for the present invention.Therefore being used for preferred especially PDE4 inhibitor of the present invention comprises: lirimilast (Bayer AG); CDP-840 (Cetttech Group PLC), NCS-613 (Centre Nationalde la Recherche Scientifique (CNRS), E-402 (Eisai Co Ltd), GRC-3785 (GlenmarkPharmaceuticals Ltd), IC-485 (ICOS Corp); IPL-455903 (InflazymePharmaceuticals Ltd), ONO-6126 (Ono Pharmaceutical Co Ltd), Tofimilast (PfizerInc.), Piclamilast (Rhone-Poulenc SA (Aventis SA)), Cilomilast (SmithklineBeecham PLC), Filaminast (Wyeth-Ayerst Pharmaceuticals Inc), WAY-126120 (Wyeth-Ayerst Pharmaceuticals Inc0.), Msopram (Schering) and Roflumilast (Altana).

Above-mentioned and PDE4 inhibitor is that these those skilled in the art know, and describes to some extent in following patent: U.S. Pat No.6649633, US No.6624181, US No.6127363; DE1545687, DE2028869, DE2123328, DE2315801, DE2402908, DE2413935, DE3900233, EP0103497, EP0139464, EP0158380, EP0163965, EP0335386, EP0389282, EP0428302, EP0435811, EP0459505, EP0470805, EP0490823, EP0506194, EP0511865, EP0527117, EP0393500, EP0510562, EP0553174, EP0557016, EP0626939, EP0664289, EP0671389, EP0685474, EP0685479, EP0736532, EP0738715, EP0748805, EP0763534, EP0816357, EP0819688, EP0819689, EP0832886, EP0834508, EP0848000, JP92234389, JP94329652, JP95010875, JP98072415, JP98147585, USNo.5,703,098; 5,739,144;

WO9117991, WO9200968, WO9212961, WO9307146, WO9315044, WO9315045, WO0318024, WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517, W09402465, WO0412461, WO9420455, WO9422852, WO9427947, WO9501338, WO9501980, WO9503794, WO9504045, WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836, WO9514667, WO9514680, WO9514681, WO9517392, WO9517399, WO9519362, WO9520578, WO9522520, WO9524381, WO9527692, WO9535281, WO9535283, WO9535284, WO9600218, WO9601825, WO9606843, WO9611690, WO9611917, WO9612720, WO9631486, WO9631487, WO9635683, WO9636595, WO9636596, WO9636611, WO9636625, WO9636638, WO9638150, WO9639408, WO9640636, WO9703967, WO9704779, WO9705105, WO9708143, WO9709345, WO9712895, WO9718208, WO9719078, WO9720833, WO9722585, WO9722586, WO9723457, WO9723460, WO9723461, WO9724117, WO9724355, WO0725312, WO0728131, WO9730999, WO9731000, WO9732853, WO9735854, WO9736905, WO9743288, WO9744036, WO9744322, WO9747604, WO9746697, WO9804534, WO9805327, WO9806692, WO9806704, WO9807715, WO9808828, WO9808830, WO9808841, WO9808844, WO9809946, WO9809961, WO9811113, WO9814448, WO9818796, WO9821208, WO9822453, WO9845268, WO9855481, WO9856756, WO9905111, WO9905112, WO9505113, WO9906404, WO9918095, WO9501338, WO9603399, WO9636625, WO9636626, WO9735854, WO9821208, WO9831674, WO9840392, WO9855481, WO9905111, WO9905112, WO9905113, WO9931071 and WO9931090.Can adopt in these materials any as PDE4 inhibitor combination of the present invention.The special material that preferably has good oral utilization rate.

These PDE4 inhibitor can be by any administration that is usually used in the administration of PDE inhibitor.Usually these chemicals can be made into peroral dosage form.The tablet of preparation can contain 50 μ g, 100 μ g, 200 μ g, 250 μ g, 300 μ g, 400 μ g, 450 μ g, 500 μ g.Developing roflumilast at present and treating as pulmonary disease, its oral dose is about 400 μ g/ days.Consider that this dosage also can be used for the present invention.The PDE4 inhibitor daily dose that gives patient is 200 μ g/ days, 300 μ g/ days, 400 μ g/ days, 500 μ g/ days or even nearly 1-5mg/ days.Patient accepts few to 100 μ g/ days during the general treatment.Will of course be appreciated that according to the individuation requirement, patient can accept PDE4 inhibitor more or less.Generally should avoid being higher than 100mg/ days dosage.Can single agent carry the PDE4 inhibitor, perhaps can be divided into multi-agent and in preset time, give.Can be once a day, secondary, three times or repeatedly contain the oral tablets of this inhibitor.Though it is preferred should also be understood that oral administration, can give similar dosage by other conventional route of administration.

Sldenafil is that preparation and route of administration that it can be the PDE4 inhibitor provide extra guide (to see PDR through the medicinal PDE5 inhibitor of approval ^TM, 57 editions, 2003, the 2653-2656 page or leaf).For therapeutic scheme, those of ordinary skills can utilize this used guide of this pharmacy PDE inhibitor.Other PDE5 and PDE1 inhibitor can be used for the present invention.This type of inhibitor is seen U.S. Patent application No.60/470,434, exercise question is " the PDE enzyme inhibitor in sterile " and U.S. Patent application No.10/014,812 (United States Patent (USP) publication No.20020103106, it is for referencial use to fit into this paper in it), be for described in " method of induced ovulation ", this two application has been described the various PDE3/4 inhibitor that can be used for triggering in the follicle maturity method of the present invention ovulation.The method that should note the present invention relates to increasing the method for follicle maturity and induced ovulation is different.Other compositions is seen described in the PCT/EP01/14730.

The exemplary inhibitor that can be used for the above-mentioned therapeutic alliance of this paper comprises but is not limited to: 5-[2-ethyoxyl-5-(4-methyl isophthalic acid-piperazine sulfonyl) phenyl]-1-methyl-3-just-propyl group-1,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones (sildenafil); Zaprinast (Zaprinast); Persantin (dipyridamole); 5-(2-ethyoxyl-5-morpholino acetylphenyl)-1-methyl-2-just-propyl group-1,6-dihydro-7H-20 pyrazolo [4,3-d] pyrimidin-7-ones; 3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-just-propoxy phenyl]-2-(pyridine-2-yl) methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidine radicals-7-ketone; 3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-(2-methoxy ethoxy) pyridine-just-the 3-yl]-2-(pyridine-2 base) methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidine radicals-7-ketone; (+)-3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-(2-methoxyl group-1 (R)-methyl ethoxy) pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-ethyoxyl-5 (4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-[2-methoxy ethyl]-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-is different-butoxy-5-(4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-1-methyl piperidine-4-base-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-ethyoxyl-5-(4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-(5-acetyl group-2-propoxyl group-3-pyridine radicals)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-isobutoxy-5-(4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-(1-methyl piperidine-4-yl)-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-ethyoxyl-5-(4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-(5-acetyl group-2-butoxy-3-pyridine radicals)-3-ethyl-2-(1-base-3-azetidinyl)-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; (6R, 12aR)-2,3,6,7,12,12a-six hydrogen-2-methyl-6-(3,4-weapon base dioxy phenyl) pyrazolo [2 ', 1 ': 6,1] pyrido [3,4-b] indole-1,4-diketone (Tadalafil; IC-351); The embodiment 78 of disclosed International Patent Application WO 95/19978 and 95 chemical compound, and embodiment 1 wherein, 3,7 and 8 chemical compound: 2-[2-ethyoxyl-5 (4-ethyl-piperazine-1-base-1-sulfonyl)-phenyl]-5-methyl-7-propyl group-3H-imidazo [5,1f] [1,2,4] triazine-4-ketone (Vardenafil); Disclosed embodiment 11 chemical compounds of International Patent Application WO 93/07124 (EISAI); Rotella, DP., J Med Chem., 2000, the chemical compound 3 and 14 of 43:1257; 4-bromo-5-(pyridine radicals methylamino)-6-[3-(4-chlorphenyl)-propoxyl group]-3 (2H) 2H-Pyridazin-3-one; 1-[4-[(1,3-benzodioxole base-5 ylmethyl) amino]-6-chloro-2-quinazolyl]-4-piperidines-carboxylic acid one sodium salt; (+)-suitable-5,6a, 7,9,9,9a-six hydrogen-2-[4-(trifluoromethyl)-benzyl-5-methyl-Pentamethylene. is imidazo [2,1-b] purine-4 (3H) ketone also-[4,5]; Furaziocillin; Suitable-2-hexyl-5-methyl-3,4,5,6a, 7,8,9a-ten octahydro cyclopenta [4,5]-imidazo [2-1 ,-b] purine-4-ketone; 3-acetyl group-1-(2-benzyl chloride base)-2-propyl indole-6-carboxylate; 3-ethyl-1-(2-benzyl chloride base)-2-propyl indole-6-carboxylate; 4-bromo-5-(3-pyridine radicals methylamino)-6-(3-(4-chlorphenyl) propoxyl group)-3-(2H) 2H-Pyridazin-3-one; 1-methyl-5 (5-morpholino acetyl group-2-just-propoxyl group phenyl)-3-just-propyl group-1,6-dihydro-7H-pyrazolo (4,3-d) pyrimidin-7-ones; 1-[4-[(1,3-benzodioxole-5-ylmethyl) amino]-6-chloro-2-quinazolyl]-4-piperidine carboxylic acid one sodium salt; Pharmaprojects No.4516 (Glaxo Wellcome); PharmaprojectsNo.5051 (Bayer); Pharmaprojects No.5064 (Kyowa Hakko; See WO 96/26940); Pharmaprojects No.5069 (Schering Plough); GF-196960 (Glaxo Wellcome); E8010 and E-4010 (Eisai); Bay-38-3145 ﹠amp; Bay-38-9456 (Bayer), vinpocetine (Vinpocetine) (Richter Gideon); SCH-51866 (Schering-Plough), SCH-59498; (6aR, 9aS)-2-(xenyl methyl) 5,6a, 7,8,9,9a-six hydrogen-5-methyl-3 (benzyl) cyclopenta-[4,5] imidazo [2,1-b] purine-4 (3H)-ketone; 5 '-methyl-2 ' (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1,7 ' (8 ' H)-[3H] imidazo [2,1-b] purine]-4 (5 ' H)-ketone; (6aR, 9aS)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-2-(benzyl)-3-(benzyl) Pentamethylene. is [4,5] imidazo [2,1-b] purine-4 (3H)-ketone also; Persantin; AWD-12-171 and AWD-12-217 (ASTS Medica); BMS-341400 (Bristol Meyers Squibb); UK-343,664 (Pfizer); 5E-3623,5E-3569,5E-3657, E4021 (Eisai); KS-505a (Kyowa Hakko Kogyo); YC-1 (Yung ShinPharmaceutical Industries); IDDB reference number 323951 (Bayer); WIN-61691 (SanofiWinthrop); FR226807 (Fujisawa); IDDB reference number 461317,461321,461324,466146 (Johnson ﹠amp; Johnson); Pyridin-4-yl methyl-3-(1,3-benzodioxole-5-yl)-9-oxo-1,3,4,9 tetrahydrochysenes-2H-pyrrolo-[3,4-b] quinolyl-2-carboxylate:

Particularly preferred PDE inhibitor is: and sldenafil and zaprinast, persantin and chemical compound no.31 and no.33 (6aR, 9aS)-2-(xenyl methyl)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-3-(benzyl) Pentamethylene. is [4,5] imidazo [2,1-b] purine-4 (3H)-ketone also; And 5 '-methyl-2 ' (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1,7 ' (8 ' H)-[3H] imidazo [2,1-b] purine]-4 (5 ' H)-ketone.

Can be used for the pharmaceutical compositions that contains the PDE inhibitor in the inventive method, comprise that the PDE inhibitor dosage that wherein contains can effectively reach all compositionss of therapeutic purposes.In addition, this pharmaceutical composition can contain suitable pharmaceutically acceptable carrier, includes to be beneficial to reactive compound is processed into excipient and the co-adjuvant that can be used as the pharmacy goods.Suitable pharmaceutically acceptable carrier is well known in the art, for example see canonical reference book Gennaro Remington ' the s PharmaceuticalSciences of this field Gennaro etc., the 8th part, the 20th edition, Merck PublishingCompany, Easton is described in the Pennsylvania (2000).Can be according to administering mode and the dissolubility and the pharmaceutically acceptable carrier of stability selection of PDE inhibitor.For example, the dosage form of intravenously administrable can comprise sterile water solution, also can contain buffer agent, diluent and other suitable additive.Biomaterial that adopt to carry medicine and other polymer and different technology and pattern verify disclosed AD HOC in the document (Luo etc., Exp Opin Ther Patents, 11:1395-1410 (2001); Cleland etc., Curr OpinBiotechnol., 12:212-9 (2001).

Can any method afford PDE inhibitor, as long as reach therapeutic purposes.For example administration can have many different approach, includes but not limited in subcutaneous, intravenous, Intradermal, intramuscular, intraperitoneal, the brain, in the trachea, in intranasal, oral, rectum, transdermal, intranasal or mouthful cheek.Preferred oral gives PDE inhibitor.

Can inject or progressively pour into for a long time and carry out the intestines and stomach external administration by medicine group.Should know that dosage depends on receiver's age, health and body weight, current treatment, if any, the character of therapeutic frequency and required effect.Each treatment can divide multi-agent or potion to give required accumulated dose.

The goods of intestines and stomach external administration comprise aquesterilisa or non-aqueous solution, and emulsion, can contain co-adjuvant known in the art or excipient.The intestines and stomach external administration preparation that is fit to comprises the aqueous solution of reactive compound water-soluble form, for example water soluble salt.In addition, can give the suspension such as the oiliness injectable formulation of this reactive compound.

Depend on the route of administration of wanting, can be mixed with injectable the PDE inhibitor or Orally administered composition.Liquid preparations for oral administration can be taked bulk liquid solution or suspensions, or the batch powder.Yet more normal said composition is provided with unit dosage form and help accurate quantification.Term " unit dosage form " refers to be suitable for the unit that physically separates of patient or other animal dose, and per unit contains calculates the good scheduled volume active substance that can produce required therapeutic effect, and suitable pharmaceutical excipient.Typical unit doses comprises the prefill of fluid composition, the ampoule or the needle tubing of scheduled volume, or pill of solid composite, tablet, capsule etc.In this based composition, the PDE inhibitor is the less component (account for about 0.1-50% of weight, preferably account for 1-40%) of content normally, and all the other are various carriers and the material that helps to form required dosage form.

Be fit to oral liquid dosage form and can comprise suitable aqueous or non-aqueous carrier and buffer agent, suspending agent and dispersant, aromatic etc.Solid dosage forms can comprise, but the chemical compound of for example any following composition similarity: binding agent such as microcrystalline Cellulose, Tragacanth or gelatin; Excipient such as starch or lactose; Disintegrating agent such as alginic acid, virgin rubber (Primogel) corn starch; Lubricant such as magnesium stearate; Lubricant such as colloidal silicon dioxide; Sweeting agent such as sucrose or glucide; Or aromatic such as Oleum menthae, methyl salicylate or orange flavor.

Injectable composition is generally based on the known injectable sterile saline in this field or phosphate-buffered saline or other injectable carrier.

Above-mentioned oral administration or injectable compositions fraction are representational.Other material and process technology etc. are (Gennaro etc., 2000) that the technical staff knows.

The PDE inhibitor also can slow release formulation or is administered systemically from the slow-releasing medicament transport.These those skilled in the art know that also representational slow-release material describes (volume such as Karsa: capsule and controlled release (Encapsulation andControlled Release), Springer Verlag, 1993; Yacobi etc., oral sustained release dosage form: design and evaluation (Oral Sustained Release Formulations:Design and Evaluation), PergamonPress, 1998.

When " effective dose " referred to add or do not add exogenous FSH or FSH substitute, the consumption of PDE inhibitor enough reached the concentration that can promote ovarian follicular growth.These those skilled in the art can conventionally determine this concentration.Actual administered compound amount is looked relevant situation by the doctor usually, comprises that age, body weight, the reactivity of disease to be treated, selected route of administration, the particular compound that gives, individual patient, patient's endogenous FSH level etc. determine.

Term " pharmaceutically acceptable " refers to comprise not can interferon activity component biological action, to the avirulent any carrier of the host of administration.For example, above-mentioned active component can be formulated in the unit dosage forms with carrier such as saline, dextran solution, serum albumin and ringer's solution parenteral.

Except pharmaceutically acceptable carrier, the compositions that contains the PDE inhibitor also can contain trace mineral supplement, as stabilizing agent, excipient, buffer agent and antiseptic.

Those skilled in the art can be with reference to U.S. Provisional Patent Application No.60/470, and 434, exercise question is " the PDE enzyme inhibitor in sterile ".In described description, described and adopted PDE1 and/or 5 inhibitor induced ovulations and the super method and composition that irritates external fertilization of control ovary.The whole this paper that includes in of document content is as the reference of its described inhibitor and dosage regimen.Its disclosed any inhibitor can be used among the present invention program.

The especially preferably PDE4 inhibitor that can be used for this paper comprises but is not limited to: (method for preparing it is seen J MedChem. to Piclamilast to Roflumilast (the visible WO9501338 of method and composition for preparing this chemical compound), 37:1696-1703, described in 1994), (method for preparing it is seen J MedChem. to Ariflo/Cilomilast, 41:821-835, described in 1998), Mesopram (method for preparing it is seen WO97/15561), Filaminast (method for preparing it is seen described in the EP0470805B1).

Unite and carry FSH and phosphodiesterase inhibitor to induce follicle maturity

Some aspect, the compositions that the inventive method consideration coupling contains PDE4 inhibitor and FSH increases follicle maturity.Yet based on the treatment of carrying the FSH/PDE4 associating, method of the present invention is also considered and the third compositions therapeutic alliance that can strengthen therapy follicle maturity of the present invention except only.This third compositions can be second kind of PDE4 inhibitor, or second kind of PDE inhibitor beyond the PDE4 inhibitor.For example, method of the present invention can be considered also to comprise and gives PDE1 and/or PDE5 inhibitor.In addition, method of the present invention also provides LH and/or hCG.Should understand and give the PDE4 inhibitor separately and can reach effective treatment.

In order to obtain good therapeutic outcome in the therapeutic alliance that this paper considers, promptly obtaining the mammal to be treated oocyte number of can ovulating increases, and generally will give patient FSH and PDE4 inhibitor combination.The effective dose that these compositionss can be united provides to produce required therapeutic effect.Usually as implied above, the FSH treatment is that administration every day continues 7-12 days.The administration of PDE4 inhibitor can give simultaneously with the FSH treatment.At this moment, PDE4 inhibitor and FSH can be contained in the same bottle as mixture.Perhaps, can before or after the FSH treatment, take the PDE4 inhibitor combination.And, though the FSH treatment should be considered to give the PDE4 inhibitor continuously in the whole cycle, as long as described administration can not cause harmful side effect menstrual cycle given period administration 7-12 days.Perhaps, the administration number of times of PDE4 inhibitor can be less than the FSH treatment.

In other embodiment of the third medicine, this third medicine can with FSH and PDE4 inhibitor for treating compositions one or both of administration simultaneously, or can before or after the FSH/PDE4 inhibitor for treating, give.

In giving all embodiments of two kinds or multiple therapeutic composition respectively, should guarantee usually can be not expired between each administration, and for example PDE4 inhibitor and FSH administration and/or the third medicine still can be brought into play effective synergy by pair cell.At this moment, consideration can give all three kinds of compositionss in 12-24 hour, more preferably in 6-12 hour, and about 12 hours of best slack time.Under the certain situation, may need significant prolongation treatment time, yet can be several days the blanking time of administration respectively (2,3,4,5,6 or 7 days).Equally, when repeating whole proposal, may in whole consecutive periods, repeat this scheme, or the doctor need between the secondary therapeutic scheme of the present invention can determine

interval

1,2,3,4 or more a plurality of cycle.

Patient selection and monitoring

The patient who accepts the present invention treatment is the aeg, and preferred age is between 20-45 year.The selected patient of the inventive method uses the treatment of above-mentioned FSH for the basis, can adopt PDE ^TMThe identical parameters of scheme.For example, before the treatment, patient accepts whole gynecologial examinations and endocrine evaluation, comprises the pelvic anatomy assessment.Should get rid of the decline of constitutional ovarian function by measuring basic serum gonadotropin level, should guarantee that patient does not have pregnancy.

In whole therapeutic scheme of the present invention, should be before treatment, treatment neutralization back evaluating patient monitoring OHSS signal.The symptom of OHSS includes but not limited to: abdominal pain, abdominal distention; That gastrointestinal symptom comprises is nauseating, diarrhoea, serious ovary increase, weight increase, dyspnea and oliguria.Show as clinically that hypovolemia, blood concentrate, electrolyte loss of equilibrium, ascites, intraperitoneal hemorrhage, pleural effusion, the poverty-stricken and thromboembolism of the impatient property of hydrothorax lung.When giving when FSH treatment or any other can irritate the preparation of follicle maturity the OHSS symptom to take place, should stop administration and patient is carried out medical monitoring, determining whether needs to be in hospital or other intervention.Other symptom that can be used for monitoring the FSH treatment comprises that colpocytology changes, the mucous wire drawing and the Ferning phenomenon of vaginal mucosa outward appearance and volume, cervical mucosa.These symptoms of back are indexs that this treatment estrogen works, and should monitor because the FHS administration will irritate estrogen production.Preferably should monitor the growth of these estrogenic effects and the more direct mensuration follicle of combination, as measuring serum estradiol and ultrasonic image.

Can obtain the clinical ovulation performance except that gestation by the generation of directly or indirectly measuring progesterone.This class sign comprises: basal body temperature raises, serum progesterone increases, body temperature changes the back menstruation takes place.In conjunction with the sign that above-mentioned progesterone produces, available ovary ultrasound observation helps to determine whether can ovulation.This class morphology monitoring can comprise the existence of estimating liquid, ovarian follicular stigma and atrophic follicle in the ovary fornix.Ultrasonic mensuration helps also to determine whether ovary increases among the OHSS.

Utilize the induced ovulation of PDE inhibitor

Should be at the about 2-10 of menstrual cycle days, preferably about 3-8 days, more preferably from about irritating in the phase of beginning in 2 or 3 days gave PDE inhibitor.For OI, should continue medication up to a follicle average diameter being arranged more than or equal to about 16-18mm (with having less than two follicle average diameters) greater than 14mm.Should give hCG to begin the onset of ovulation same day at inactive PDE inhibitor.

For induced ovulation, can give PDE inhibitor (working) separately with endogenous FSH one, or with FSH or have the FSH activity and maybe can irritate preparation that endogenous FSH discharges and unite and give.Give PDE inhibitor and another follicle stimulant and can be simultaneously, give respectively or successively.Have the active preparation of FSH and comprise reorganization or urine FSH, and FSH mixes mutually with not commensurability LH such as hMG.Have the active preparation of FSH and also comprise the congener of FSH, Recombinant FSH as the long-acting modification of CTP-FSH, form by wild type α-subunit and hybridization β-subunit, wherein, the carboxyl terminal peptide of hCG is blended in the C-end of FSH β-subunit, sees LaPolt etc., Endocrinology, 131:2514-2520,1992; Human Reprod.18:50-56 such as Klein, 203].Also comprise strand FSH-CTP, a kind of single chain molecule of forming by following sequence (holding the C-end) from N-;

βFSH

βhCG-CTP(113-145)

αFSH

Wherein β-FSH represents β-subunit of FSH, the carboxyl terminal peptide of β-hCG CTP (113-145) expression hCG, and α-FSH represents α-subunit of FSH, sees Klein etc., Fertility ﹠amp; Sterility, 77:1248-1255, (2002).The preparation that can excite or cause endogenous FSH to produce or discharge comprises citric acid clomifene and aromatase inhibitor, as letrozole (Letrozole), YM-511, my bent azoles (Anastrozole), Arensm (Fadrozole).The purposes of these molecules in OI seen for example WO02/083241 and WO 02/083239.

In the preferred version of OI, give patient P DE inhibitor, preferred PDE1 and/or 5 inhibitor, more be preferably optionally PDE1 and/or 5 inhibitor, more preferably the PDE1 inhibitor particularly preferably is selectivity PDE1 inhibitor most, should be after menstruation immediately or began in three days to use.This indicates that the phase of irritating begins.Giving the PDE inhibitor should continue once a day, but but also every day secondary or every other day once, or even give single dose.The developmental process of follicle can be used monitoring ultrasonic.When the average diameter of a follicle more than or equal to 16-18mm, the average diameter of preferred two following follicles is during greater than 16mm, judge that growth is enough, with having the active preparation of LH-, as hCG (5000-10000IU commonly used), LH (25000-70000IU) or PDE 1V inhibitor (seeing laid-open U.S. Patents application No2002/010106A1) provoked ovulation (indicating that the phase that irritates finishes the onset of ovulation and begins).Inform patient's sexual intercourse in 24-36 hour after the provoked ovulation administration.Perhaps send into the sperm fertilization by intrauterine.

The FSH serum levels is irritated and do not reduced (free period) and is lower than patient's (measuring immediately or in two days after the menstruation) that about 2IU/ rises, should or have the FSH activity with PDE inhibitor associating FSH, the preparation that maybe can irritate endogenous FSH release carries out ovulation induction.The FSH dosage that needs is lower than the required dosage without PDE inhibitor patient, with regard to about 16mm of average diameter or the bigger follicle number, can reach identical or better reaction when irritating 8 days.

When adopting FSH in OI, or have follicle and irritate activity, in the time of maybe irritating the preparation that endogenous FSH discharges, can give to give FSH before or after the PDE inhibitor, perhaps these two kinds of preparations can begin to give simultaneously.Preferred FSH administration and the administration of PDE inhibitor begin simultaneously, or begin the previous day in the administration of PDE inhibitor.The phase that irritates is from the administration for the first time of FSH or PDE inhibitor, if whichever first administration or only adopt the PDE inhibitor then begins the phase of irritating in its administration that day.

Adopt super the irritating of PDE inhibitor control ovary

Super irritate (COH) of ovary when controlling ART, can utilize PDE inhibitor of the present invention and need not exogenous FSH (or have follicle irritate active preparation), improve endogenous FSH level and cause, and produce a plurality of follicles of ovulating the super control that irritates of ovary.If patient FSH serum levels is irritated and downward modulation (free period) and be lower than about 5IU/ liter (after the menstruation immediately or in two days mensuration), should adopt aforesaid PDE inhibitor associating FSH, or have follicle and irritate activity, maybe can irritate the preparation that endogenous FSH discharges and carry out COH.When in COH, adopting FSH, or have follicle and irritate activity, in the time of maybe irritating the preparation that endogenous FSH discharges, can before or after giving the PDE inhibitor, begin to give FSH, or these two kinds of preparations begin simultaneously to give.Preferred FSH administration and the administration of PDE inhibitor begin simultaneously, or begin the previous day in the administration of PDE inhibitor.The phase that irritates is from the administration for the first time of FSH or PDE inhibitor, if whichever first administration or only adopt the PDE inhibitor then begins the phase of irritating in its administration that day.

In the preferred version of COH, when begin the luteal phase of patient's menstrual cycle (often for menstrual cycle about 20 days), give gonadotropin-releasing hormone (GnRH), the downward modulation patient is to suppress its endogenous lutropin (LH) earlier.Be everlasting 8-21 days with GnRH agonist inhibition ovarian function, monitor by monitoring LH or estradiol level (LH＜5IU/L, E2＜50pg/ml generally show and suppress fully).The GnRH agonist for example comprises: buserelin (Buserselin), goserelin (Goserelin), leuprorelin (Leuprorelin), triptorelin (Triptorelin) and naphthalene method Rayleigh (Nafarelin).Adopt the PDE inhibitor, preferred PDE1 and/or 5 inhibitor, more preferably optionally PDE1 and/or 5 inhibitor, more preferably PDE1 inhibitor, PDE1 inhibitor optionally most preferably irritates the follicular development of phase, downward modulation then.Can singly use the PDE inhibitor, or unite and give follicle and irritate element (FSH) or have follicle and irritate activity, maybe can irritate the preparation that endogenous FSH discharges.With that day that PDE inhibitor or FSH begin to irritate, whicheverly begin earlier, all be defined as and irritate the time limit and begin.If single FSH that use during COH adopts the PDE inhibitor can allow same patient coupling low dosage FSH (or equivalent), and when irritating 8 days with regard to about 16mm of average diameter or bigger follicle number, can obtain identical or better reaction.When giving the PDE inhibitor, when the consumption of FSH should be less than or equal to same patient without the PDE inhibitor about 75% of required FSH dosage, to obtain identical follicle reaction, when more preferably the consumption of FSH is less than or equal to same patient without the PDE inhibitor about 50% of required FSH dosage, when most preferably the consumption of FSH is less than or equal to same patient without the PDE inhibitor about 30% of required FSH dosage.

When giving the PDE inhibitor, the consumption of FSH is preferably about 25-600IU FSH/ days, more preferably from about 50-450IU/ days.Most preferably 50-300IU/ days.When lacking or have exogenous FSH (or equivalent), irritate and to last till and judge that ovarian follicular growth enough greatly the time, promptly has three follicle average diameters at least greater than about 16mm (should have one greater than 18mm), and continues to give the GnRH antagonist with the PDE inhibitor.Then as described in the OI, what give provoked ovulation dosage has an active preparation of LH (as the hCG of 5000-10000IU).36-38 hour time behind the provoked ovulation for the recovery oocyte.Usually from the follicle that is about to ovulate, reclaim oocyte with suction method.Collected the back about 72-96 hour, to the oocyte graduation, external fertilization selects the embryo to transport in the uterus.

In another preferred version of COH, adopt the GnRH antagonist.Do not give the GnRH agonist, be everlasting spontaneous or inductive menstruation after 1, the beginning follicle irritated in 2 or 3 days, adopted the PDE inhibitor, preferred PDE1 and/or 5 inhibitor, more be preferably optionally PDE1 and/or 5 inhibitor, more preferably PDE1 inhibitor, most preferably selectivity PDE1 inhibitor irritates element (FSH) in conjunction with giving follicle, or have follicle and irritate activity, maybe can irritate the preparation that endogenous FSH discharges.Menstruation began to give the GnRH antagonist in 6 days.The GnRH antagonist comprises, for example: cetrorelix (Cetrorelix), Nal-Glu, An Taite (Antide), ganirelix (Ganirelix), Antazoline B (Azaline B) and Antarelix (Antarelix).Continue to irritate up to having three follicle average diameters at least with PDE inhibitor and FSH (or equivalent) greater than about 16mm (should have one) greater than 18mm.Then as described in the OI, what give provoked ovulation dosage has an active preparation of LH.Give the GnRH antagonist up to that day that ovulation takes place.

In another preferred COH scheme, should be after menstruation gave aromatase inhibitor in 3-6 days, in conjunction with the PDE inhibitor, preferred PDE1 and/or 5 inhibitor, more be preferably optionally PDE1 and/or 5 inhibitor, more preferably PDE1 inhibitor, most preferably selectivity PDE1 inhibitor begins follicle and irritates.Gave GnRH antagonist at about the 6th day, aromatase inhibitor and start injection FSH stop using.Continue to give PDE inhibitor, FSH and GnRH antagonist up to the hCG that gives provoked ovulation dosage.

The fertilization of employing Oocyte in Vitro

Adopt the inventive method to produce the oocyte that hCG can be ovulated.PDE4 inhibitor/FSH therapeutic alliance can be short-term or long-term treatment, adds or do not add hypophysis downward modulation, or with and without the GnRH antagonist, and usefulness or without hCG.Give PDE4 inhibitor/FSH therapeutic alliance up to follicle fully ripe in the follicle of size (10-25mm size, preferred 16-20mm follicle) fully by the time.If the oocyte of this class follicle endogenous gonadotropin in vivo is not suppressed, can increases sharply by the hCG of patient self menstrual cycle, or ovulate to the exogenous hCG of patient injection (as 5000-10000IU).

Perhaps, under instructing, ultrasound wave waits until in the suction results that the follicle of size (10-25mm size, preferred 16-20mm follicle) is used for external fertilization fully.Seek the ovarian cumulus oocyte complex (COC) in the aspirated liquid, stereoscopic microscope (with or without embryo's filter) in a single day identify down, COC is placed cultivation.These those skilled in the art know various oocyte culture medium or nutrient media components.This class culture medium may needn't contain human serum albumin (HAS).

Behind the In vitro culture or when cultivating,, or can cause the conventional fertilization method of germ cell, make this oocyte fertilization with any other with conventional IVF or the interior sperm injection (ICSI) of kytoplasm.Can should shift the embryo who grows at 2-3 days at after fertilization 1-6 days, or shift or the transfer of a plurality of ovum as single ovum.

Before the program jump ovum that is individual design and afterwards, patient can accept progesterone and/or estrogen to start and to keep suitable acceptance endometrium pedigree.

Method of the present invention provides more ovulated oocyte number for above-mentioned IVF method.Yet the inventive method also can be used for the maturation in vitro of immature oocyte.In this class embodiment, save oocyte from the ovary hole follicle that contacts the commendable increase of midcycle gonadotropin, it is characterized in that immature or incomplete sophisticated oocyte.Think that people's oocyte and the oocyte of other animal have seldom or do not have mound shape expansion (a kind of genitality vesicle, nonpolarity corpusculum), this is that IVF treatment those skilled in the art are not indiscernible.A kind of potential use of therapy of the present invention is in the IVF scheme, replaced C C or FSH, and this treatment this moment can improve natural fertilization but not complementary reproductive technology is gathered in the crops the maturation of the single or dominant follicle of usefulness.

Though this paper has narrated the maturation of many human follicles, consider that the inventive method is available as other mammal, produce oocyte as house pet (cat, Canis familiaris L. or Cavia porcellus) or zoo animal (as primates).In other preferred embodiment, described mammal is the animal industry, the part of preferred farming animals garden such as calf, horse, pig, mink, goat or sheep industry.In most preferred embodiment, described mammal is human.For maturation in vitro, use the PDE inhibitor, or PDE inhibitor and promoting sexual gland hormone extracorporeal treatment immature oocyte, to produce fertilizable mature oocyte.

Pharmaceutical composition and test kit

The pharmaceutical composition that is used for administration of the present invention can contain at least a promoting sexual gland hormone, and FSH of the present invention is preferably with pharmaceutically acceptable form, and is randomly combined with pharmaceutically acceptable carrier.These compositionss can anyly can reach the method administration of therapeutic purposes.Adopt the inventive method to give individuation dosage and the scheme that the FSH compositions irritates follicle maturity, the guide that those skilled in the art are not difficult to utilize Physician ' sDesk Reference to provide is determined, adopts this based composition treatment ovulation to stop the complementary reproductive technology that is used for of disease.As mentioned above, those skilled in the art can adopt medical science teach in the book at present the FSH dosage and the scheme of usefulness earlier.For reaching this effect, those skilled in the art refer in particular among above-mentioned Physician ' the s Desk Reference each chapters and sections and in the middle of their instruction give above-mentioned Fertimex ^TM, Gonal ^TM, bravelle ^TMChapters and sections Deng the method and composition of preparation fit into this paper as a reference in it.Each chapters and sections among Physician ' the s Desk Reference provide preparation type, route of administration and the therapeutic scheme about can be used for the FSH administration.Any scheme as herein described, preparation, route of administration etc. are used for the present invention after all being not difficult to revise.

Belong to the compositions in the scope of the invention, comprise the compositions that contains at least a PDE4 inhibitor of the present invention, its consumption can effectively reach and irritate, induces or increase can the ovulate purpose of oocyte number of animal, though individually dosed, or preferably unite low dosage FSH administration.Be used for the PDE4 inhibitor of the inventive method and/or any method afford that other active ingredient can adopt conventional administration.The technical staff can be specifically with reference to U.S. Patent application No.60/470,434 exercise questions " the PDE enzyme inhibitor in sterile ", United States Patent (USP) publication No.20020103106 and PCT/EP01/14730, their each self-described in the relevant application of fertilization the dosage and the approach of PDE inhibitor.The compositions used of orally give the present invention most preferably.

Though individual need difference, the optimum range of determining each chemical compound effective dose are the things within the art technology scope.The typical doses of FSH comprises about 5IU FSH/ days-75IU FSH/ days, and 7-12 days altogether.The typical doses of PDE4 inhibitor is from about 5mg/ days-100mg/ days or higher and different.Though want give PDE4 continuous every day, may when stopping the FSH administration, stop the PDE4 administration.Certainly FSH treats administration 7-12 days traditionally, but can give once among the present invention, maybe may need the FSH of low dosage several times, produces the useful result of therapeutic who increases sophisticated follicle.When the OHSS symptom, should stop treatment.

The suitable dose that should know the present composition depends on receptor's age, health status and body weight, the kind (if any) of treatment at present, the frequency of treatment and the character of required effect.Yet, confirmable such with need not too much testing as is known to the person skilled in the art, can determine its optimal dosage for individual subject.This is usually directed to the adjustment of standard dose, should reduce dosage as patient body's heavy and light.

As mentioned above, can required accumulated dose be divided into multi-agent or potion gives with treat at every turn.Can give this compositions separately, or other therapeutic agent of sick therewith or this sick other symptom is united and is given.

From disclosure that this paper provided as can be known, the clinical practice of conjoint therapy is considered to have in the extensive aspect that the present invention uses, and comprises first kind of compositions that contains the PDE inhibitor and the second kind of compositions that contains FSH.Therefore, these compositionss should be mixed with suitable pharmaceutical composition, promptly use the appropriate format of this therapeutic alliance in the body.Usually this comprises that preparation does not have pyrogen and other compositions to human or animal's possibility objectionable impurities basically.The identical preparation of FSH goods as herein described that FSH can be mixed with and can buy at present.The PDE4 inhibitor can be mixed with and be similar to Viagra ^TMThe preparation of (the PDE5 inhibitor of promptly knowing).

Usually needs adopt suitable salt and buffer agent that said composition is stablized and said composition are ingested at target site.Generally provide hormonal composition of the present invention, before administration, rebuild, the PDE4 inhibitor combination can be mixed with tablet form with lyophilized form.Buffer and the solution of rebuilding hormone can provide with this pharmaceutical preparation, supply with medicine to produce water-soluble compositions of the present invention.This water-soluble compositions contains each medicine that is dissolved in or is scattered in the effective dose in pharmaceutically acceptable carrier or the water-soluble medium.This compositions is also referred to as inoculum.Phrase " pharmaceutically or acceptable on the pharmacology " refers to can not have side effects when giving the animal or human, the molecule and the compositions of irritated or other untoward reaction.This paper " pharmaceutically acceptable carrier " comprise any He all solvents, disperse medium, coating materials, antibacterium and antifungal drug, etc. blend absorption delay agent etc.Being used for pharmaceutically, this class medium and the preparation of active substance are that this field is known.Except known to so far with inconsistent any conventional media of this therapeutic combination or preparation, all consider to can be used in this therapeutic combination.Also the active component that adds can be mixed in the said composition.

Active compound of the present invention comprises described herein and the FSH pharmaceutical preparations known classics of these those skilled in the art.The PDE4 inhibitor also is that these those skilled in the art are known.Target tissue can give these compositionss of the present invention through any approach commonly used, as long as can obtain this compositions by this approach.The most normal these compositionss are mixed with oral administration.Yet, other conventional route of administration, as in subcutaneous, intravenous, Intradermal, intramuscular, the mammary gland, in the intraperitoneal, trachea, behind the ophthalmic, eyeball, in the lung under (being mature release), aerosol, the cheek, nose, anus, rectum, vagina or transdermal delivery, or operation implants specific part, also can adopt when problem is arranged when oral.Multi-agent treatment in available potion or regular period.

This reactive compound can be prepared into the aqueous solution of free alkali or pharmaceutically-acceptable salts, suitably mix the back administration with surfactant such as hydroxypropyl cellulose.Also available glycerol, liquid macrogol and their mixture and oil are prepared into dispersion liquid.Under common storage and service condition, these goods should contain antiseptic and grow with prophylaxis of microbial.

Be fit to the pharmaceutical dosage form of injection, comprise sterile water solution or dispersion liquid and sterile powder, the sterilization Injectable solution or the dispersion liquid of interim preparation.Under all situations, this dosage form must be aseptic, must be that liquid easily is contained in the syringe.Must be stable under manufacturing and storage requirement, must be able to prevent the pollution of microorganism such as antibacterial and fungus.Described carrier can be solvent or disperse medium, for example comprises water, ethanol, polyhydric alcohol (as glycerol, Polyethylene Glycol and liquid macrogol etc.), their suitable mixture and vegetable oil.Can by size and the employing surfactant of keeping the required particle of dispersion liquid, keep suitable flowability by adopting coating such as lecithin.Available various antibacterium and antifungal drug are as the effect of prophylaxis of microbial such as p-Hydroxybenzoate (parabens), methaform, phenol, sorbic acid, thimerosal.Preferably comprise isotonic agent amount such as sugar and sodium chloride under many situations.The absorption that prolongs Injectable composition can be adopted the preparation of delayed absorption in said composition.As aluminum monostearate and gelatin.

This reactive compound is mixed in the appropriate solvent with aequum, prepare the Injectable solution of sterilization with above-mentioned various other compositions, but as needs filtration sterilization then.Usually, various sterilizing activity compositions are joined in the sterilization carrier that contains basic dispersion medium and above-mentioned required other composition the preparation dispersion liquid.With sterile powder preparation sterilization Injectable solution, preferred manufacturing procedure is vacuum drying and freeze drying technology, can produce to contain this active component and the powder of other any required composition of filtration sterilization solution in advance.

As used herein, " pharmaceutically acceptable carrier " comprise any He all solvents, disperse medium, coating materials, antibacterium and antifungal drug, etc. blend absorption delay agent etc.Being used for pharmaceutically, this class medium and the preparation of active substance are that this field is known.Except known to so far with inconsistent any conventional media of this therapeutic combination or preparation, all consider to can be used in this therapeutic combination.Also the active component that adds can be mixed in the said composition.

Compositions of the present invention can be mixed with neutrality or salt form.Pharmaceutically acceptable salt comprises acid-addition salts (forming with proteinic free amine group), available mineral acid example hydrochloric acid or phosphoric acid, or organic acid such as acetic acid, oxalic acid, tartaric acid, mandelic acid etc. form this kind salt.The salt that forms with free carboxy also can be derived from inorganic base, as sodium hydroxide, potassium, ammonium, calcium or ferrum and organic base such as 2-aminopropane., Trimethylamine, histamine, procaine etc.

After the preparation, can compatible form with dosage form, give solution with the treatment effective dose.Be not difficult to different dosage form, give these preparations as Injectable solution, drug release capsules etc.For example, the gastrointestinal tract external administration of aqueous solution if desired, should suitably cushion this solution, earlier with enough saline or glucose this liquid diluting liquid etc. is oozed.These graininess aqueous solutions are particularly suitable for intravenous, subcutaneous and intraperitoneal administration.

" unit dose " refers to be dispersed in the uniqueness amount of the therapeutic composition in the suitable carrier.The example of FSH and PDE inhibitor preferred dose as mentioned above.A kind of gastrointestinal tract external administration of or two kinds of therapeutic combinations, can be first the injection of medication group, continuous infusion is to keep the therapeutic blood circulation level of drug products then.Those of ordinary skills are not difficult by the clinical condition of good medical code of practice and individual patient, optimize effective dose and dosage regimen.

Administration frequency depends on the pharmacokinetic parameter and the route of administration of said preparation.Those skilled in the art can determine optimum pharmaceutical preparation according to route of administration and required dosage.For example see, Remington ' sPharmaceutical Sciences, 18 editions, Mark Easton, PA (1990), it is for referencial use to fit into this paper in it.This type of preparation may have influence on the interior rate of release of physical state, stability, body, the homoiostasis speed of the medicine of giving.According to route of administration, can calculate proper dosage by body weight, body surface area or organ size.Determine the further renewal of the required value of calculation of appropriate therapeutic dosage, can need not too much test by this field those of ordinary skill routinely, specifically rely on dosage information disclosed herein and test, and in the clinical trial of animal or human's body observed pharmacokinetic data, formulate.

Can utilize the test of the mensuration medicine blood levels of having set up, the dose response data relevant with combination are determined proper dosage.Last dosage is by being responsible for the factor that the doctor considers to influence pharmaceutically active, as the specific activity of medicine, the order of severity of damage, patient's reactivity, patient's age, state, body weight, sex and diet, the seriousness of infection, time of administration and other clinical factor and determine.Along with the carrying out of research, will occur about the suitable dose level of specified disease and disease and the further information of treatment persistent period.

Pharmaceutical composition of the present invention and Therapeutic Method be can understand and physianthropy and veterinary applications can be used for.Therefore, the object of treatment can be a mammal, preferred people and other animal.For the veterinary, object comprises that for example farming animals comprises milch cow, sheep, pig, horse and goat; That companion animals such as Canis familiaris L. and cat, foreign country introduce and/or zoo animal, laboratory animal comprise mice, rat, rabbit, Cavia porcellus and hamster; With poultry such as chicken, turkey, duck and goose.

The present invention also comprises the test kit that is used for the treatment of infertility.This test kit is equipped with at least the first kind of compositions and second kind of compositions that contains at least a PDE4 inhibitor in pharmaceutically acceptable carrier of containing FSH in pharmaceutically acceptable carrier.This test kit also can be equipped with the solution or the buffer that can influence first and second delivery of composition.This test kit also is equipped with and contains other PDE inhibitor, as other PDE4 inhibitor or other PDE1 or 5 inhibitor, and/or other compositions of other hormone such as hCG, LH etc.This test kit also can be equipped with conduit, syringe or other conveyer device, is used to one or more compositionss of carrying the inventive method used.This test kit also can be equipped with the description that comprises the therapeutic scheme treatment sequence.

Embodiment

The embodiment that below comprises has shown preferred implementation of the present invention.The technology disclosed in these embodiment that it will be understood by those skilled in the art that is disclosed by the invention, implements the good representative art of work of the present invention, therefore can think to have constituted and implement preference pattern of the present invention.Yet, one skilled in the art will appreciate that to rely on content disclosed herein, can make many modifications to the disclosed specific embodiment and still can obtain same or analogous result, but these modifications all belong within design of the present invention and the scope.

Material and method

Animal: adopt jejune Sprague Dawley CD (SD) BR female rats, body weight 36-39 gram when accepting.Animal feeding is under following standard environment condition: temperature 22 ± 2, relative humidity 55% ± 10, exchange of air 15-20 time (filtering 99.99% through HEPA) per hour, artificial lighting's circulation (7h00-19h00) in 12 hours.

Rat remains in the rearging cage that (the random feeding standard particle of device food (4RF21 that is produced by Charles River Italia ' s licensee Mucedola s.r.l.) and water are raised with rustless steel in cm.40.5 * 38.5 * 18h) during the whole research.

Chemical substance: people's Puregon (r-FSH) and people recombinate the short gonad (r-hCG) of chorion by Laboratoires Serono Aubonne (LSA, Aubonne, Switzerland) supply.Test compounds or synthetic according to the synthetic method of the chemical compound of delivering, or available from commercial source.Specifically, the technical staff can be with reference to the method for preparing Roflumilast described in the WO9501338; J Med Chem.1994,37:1696-1703 describes the method for preparing Piclamilast in detail; J Med Chem.1998,41:821-835 has described the method for preparing Ariflo/Cilomilast; WO 97/15561 has described the method for preparing Mesopram; EP0407805 B1 has described the method for preparing Filaminast. and these methods can modifiedly be produced other PDE inhibitor.Other test compounds (persantin, zaprinast, sldenafil) or synthetic according to the synthetic method of the chemical compound of delivering, or available from commercial source.In addition, can adopt and include this paper U.S. Patent application No.60/470 for referencial use in, 434 exercise questions " the PDE enzyme inhibitor in sterile ", other related compound that United States Patent (USP) publication No.20020103106 and PCT/EP01/14730 are mentioned.

Rat follicle maturity test in the body: transport to jejune female rats the Friday before the experiment, and the 18-19 age in days is with the female Mus of lactication (10 young Mus of every female Mus of lactication).All rats are randomized into enforcement group (6-8/group) in wean next Monday (21-22 age in days).

Give rat skin lower injection (cervical region) r-hFSH or carrier (PBS) every day 2 times, each 250 microlitres, two days (8.30-9.00,15.30-16.00 for the second time for the first time).FSH injected dose or suboptimal dose (606ng/ Mus, total amount are divided into 4 injections, are labeled as " low FSH " among the figure) or a large amount (2424.8ng/ Mus, total amount are divided into 4 injections, labelling among the figure " high FSH ") are as positive control.

Except that above injection, with the FSH injection while, rat is the totally two days every day 2 times of test compounds carrier of dosage shown in the subcutaneous injection (mg/kg/ per injection) also.These chemical compounds or with NP3S (n-N-methyl-2-2-pyrrolidone N-5%, PEG400 30%, polypropylene glycol 20025%, propylene glycol 20%, saline 20%) or dilute with water.Therefore, total frequency injection of the promotion ovarian follicular growth that every Mus is accepted is 8 times, comprises the carrier of 4 injection r-hFSH or r-hFSH, adds the carrier of 4 ejection testing chemical compounds or test compounds.

The last injection of this experiment and r-hFSH second day, the subcutaneous again r-hCG of rat (the every Mus of 1430ng/) injection for curing, induce all or great majority mature follicle ovulate.

10 of mornings are used CO behind the injection hCG ₂Suffocate and put to death rat.Animal is lain on the back, and downside ethanol spraying disinfection keeps the not sagging influence of Mus hair to dissect.Under the shears tweezers help, begin to cut skin and muscle from pubic symphysis along abdomen mouth line to breastbone, Dirty in exposing takes out small intestinal and puts aside.Take out ovary, cornua uteri and body of uterus folder degrease and connective tissue.Whole reproductive tract are inserted (1 animal/hole) in the hole that contains PBS in 24 orifice plates.

After putting to death all animals collection ovaries, take out fallopian tube from ovary gently, immerse among the PBS, place on the microscope slide.Take out ovary, cleaning then, and place PBS weighing (abandoning the uterus).

A pair of fallopian tube is placed on the microscope slide, place the first microscope slide top to guarantee that the hair end of microscope slide sticks together microscope slide with an adhesive tape then.Fallopian tube is placed the microscope slide bottom, be stacked on the microscope slide of top, tack non-burr with stick, fallopian tube pressurizes between two microscope slides.Check fallopian tube with optical microscope under anti-phase condition (minimum 40 times of amplifications), if having ovum in two ampulla of uterine tubees, counting.The result draws with the oocyte average that every rat is discharged.The error rod reflects the standard deviation of each cell mean.

Embodiment 1: with various PDE inhibitor associating low dosage FSH induced ovulation

Experimentize by rat follicle maturity test in the above-mentioned body.In an experiment, in the end second day intermediate collection ovary of treatment made histologic analysis before the last injection.These rats have just been accepted 3 doses of FSH and test compounds earlier before putting to death and collecting organ.Counting secondary follicle sum comprises little follicle (ripe mid-term have multilamellar granulosa and one-level diffusive cavity but do not have the hole chamber) and hole follicle (have hole and expand, about 500 microns or higher of external diameter is with or without GCL and thickens), the evaluation secondary follicle.Also count respectively the hole follicle (〉=500mcm).

Data analysis

Calculate the ratio of ovulation animal from the various values of each experimental group, the ovum average and the ovary average weight that exist in every rat ampulla of uterine tube are drawn relevant figure.All figure are expressed as mean+SD.Among the figure, the rod on the standard deviation is represented the number that this group has the rat of one or more discharge ovums to compare with this group rat sum, and for example X/Y refers to that X rat in the total number Y has the ovum of one or more discharges.

The chemical compound of test

In above scheme, tested following chemical compound:

(i) zaprinast (PDE1,5,6,7,9,10,11 inhibitor)

(ii) sldenafil (PDE1,5 and 6 inhibitor)

(iii) persantin (PDE5,6,7,8,10,11 inhibitor)

(iv) tadanafil (PDE5 and 6 inhibitor)

(v) chemical compound no.31[(6aR, 9aS)-2-(xenyl methyl)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-3-(benzyl) encircles penta dioxane-[4,5] imidazo [2,1-b] purine-4 (3H)-ketone] (inhibitor of PDE1)

(Vi) chemical compound no.33[5 '-methyl-2 ' (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1,7 ' (8 ' H)-[3H] imidazo [2,1-b] purine]-4 (5 ' H)-ketone] (inhibitor of PDE1)

Proved that these molecules show following selectivity to various PDE enzymes:

Molecule	IC ₅₀PDE1	IC ₅₀PDE5	IC ₅₀PDE6[ ^**]	IC ₅₀PDE3[ ^*]
Molecule	IC ₅₀PDE1	IC ₅₀PDE5	IC ₅₀PDE6[ ^**]	IC ₅₀PDE3[ ^*]	Zaprinast	9400nM ^ref1	2000nM ^ref1		＞100000nM ^ref1
Sldenafil	260nM ^ref1	3.0-3.6nM ^ref1	Selectivity ratios PDE6 to PDE5 is high more than 10 times ^ref2	65000nM ^ref1	Zaprinast	9400nM ^ref1	2000nM ^ref1		＞100000nM ^ref1
Sldenafil	260nM ^ref1	3.0-3.6nM ^ref1		65000nM ^ref1	Tadanafil	＞30000nM ^ref3	6.7nM ^ref3	To the high 187-193 of selectivity ratios PDE6 of PDE5 doubly ^ref3	＞100,000
Chemical compound no.31	0.07nM ^ref4	305nM ^ref4		3500	Tadanafil	＞30000nM ^ref3	6.7nM ^ref3		＞100,000
Chemical compound no.31	0.07nM ^ref4	305nM ^ref4		3500	Chemical compound no.33	0.6nM ^ref4	200nM ^ref4		7000

People such as ref 1:Terreft: Biooganic and Medicinal Chemistry Letters, 6 (15): 1819-1824,1996.

Ref 2:Physicians Desk Reference, 57 ^Th, version 2003.

People such as ref 3:Corbin: International Journal of Clinical Practice, 56 (6): 453-459.2002.

Ref4:Ahn. wait the people: J Med Chem., 40:2196-2210,1997.

^*PDE3 expresses in oocyte, and preferred PDE inhibitor is selective to PDE3, because the oocyte that PDE3 suppresses can cause not being optimum is grown.People such as Wiersma, J ClinInvst., 102 (3): 532-537,1998.

^*PDE6 expresses in amphiblestroid bar and awl; The PDE6 inhibitor can cause slight ocular disorders; Preferred PDE inhibitor is to PDE6 selective (the energy force rate inhibition PDE6 that promptly preferably suppresses PDE1 or 5 is high at least about 100 times).

The result:

The persantin that every rat gives to join with NP3S (PDE5,6,7,8,10,11 inhibitor),

dosage

1,5 and 25mg/kg * 4 injection (subcutaneous) and low dosage FSH, add the contrast of NP3S carrier with low dosage FSH and compare, the oocyte number that causes every rat to be discharged increases.Only only collect an average oocyte with every rat of FSH of low dosage, 10 Mus have only 4 ovulations.On the contrary, when low dosage FSH coupling persantin (1,5 or 25mg/kg), every Mus oocyte average is respectively 7.5,6.8 and 6.2,10

Mus

7,6 and 10 ovulations respectively.Fig. 1 has shown these results.

The zaprinast that every rat gives to join with NP3S (PDE1,5,6,7,9,10,11 inhibitor),

dosage

1,5 and 25mg/kg * 4 injection (subcutaneous) and low dosage FSH, add the contrast of NP3S carrier with low dosage FSH and compare, the oocyte number that causes every rat to be discharged is dose dependent to be increased.Only only collect average 2.4 oocytes with every rat of FSH of low dosage, 10 Mus have only 5 ovulations (experiment 1).On the contrary, when low dosage FSH coupling zaprinast (1,5 or 25mg/kg), every Mus oocyte average is respectively 3.25,6.4 and 9.5,10

Mus

6,9 and 10 ovulations respectively.Fig. 2 has shown these results.

The sldenafil that every rat gives to join with NP3S (PDE1,5 and 6 inhibitor),

dosage

1,5 and 25mg/kg * 4 injection (subcutaneous) and low dosage FSH, add the contrast of NP3S carrier with low dosage FSH and compare, the oocyte number that causes every rat to be discharged is dose dependent to be increased.Only only collect an average oocyte with every rat of FSH of low dosage, 10 Mus have only 3 ovulations.On the contrary, when low dosage FSH coupling sldenafil (1,5 or 25mg/kg), every Mus oocyte average is respectively 3.5,5.5 and 6.9,8

Mus

6,5 and 7 ovulations (experiment 1) respectively.Fig. 3 has shown these results.

Fig. 4 shows secondary follicle (two ovaries) sum with every Mus in the rat of carrier, low FSH, high FSH or the treatment of low FSH+75mg/kg sldenafil.The secondary follicle sum of sldenafil+FSH group is higher than any other group [carrier=69, low FSH=64, low FSH+ sldenafil=84, high FSH=64].

Fig. 5 shows hole follicle (two ovaries) sum with the every Mus of rat of carrier, low FSH, high FSH or the treatment of low FSH+75mg/kg sldenafil.The hole follicle sum of sldenafil+FSH treatment rat is higher than the hole follicle sum [carrier=3.2, low FSH=4.1, low FSH+ sldenafil=7.4, high FSH=9.5] with low FSH treatment rat.

Every rat gives

sldenafil

1,5 and 25mg/kg * 4 injection (subcutaneous) and the low dosage FSH that the use carrier is joined, and only adds the contrast of aqueous carrier with low dosage FSH and compares, and causes the oocyte number of every rat discharge to be the dose dependent increase.Only only collect average 1 oocyte with every rat of FSH of low dosage, 8 Mus have only 2 ovulations.On the contrary, when low dosage FSH coupling sldenafil (5mg/kg), every Mus oocyte average is respectively 20,8 Mus 7 ovulations.Fig. 6 has shown these results [note: the difference of Fig. 6 and Fig. 3 is that carrier is water rather than organic NP3S-SM].

Every rat gives the Tadalafil that water joins (PDE5 and 6 inhibitor) dosage 25mg/kg * 4 injection (subcutaneous) and low dosage FSH, add the contrast of NP3S carrier with low dosage FSH and compare, the oocyte number that causes every rat to be discharged is dose dependent to be increased.Every rat is collected less than 1 oocyte during without FSH, and these the results are shown in Figure 7.When not giving FSH, every Mus is on average collected 1 oocyte.(" low FSH ": the every Mus in 151.5ng * 4) average every Mus is collected 5 oocytes with suboptimal dose FSH.When the average every Mus of this suboptimal dose FSH associating Tadalafil (25mg/kg * 4 time every Mus, " low FSH adds Tadalafil ") is collected 16 oocytes.(" high FSH ": the every Mus in 606.2ng * 3) every Mus is on average collected 21 oocytes to high dose FSH.

Ahn. wait the people: J Med Chem., 40:2196-2210,1997 chemical compound no.31[(6aR, 9aS)-2-(xenyl methyl)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-3-(benzyl) Pentamethylene. also [4,5] imidazo [2,1-b] purine-4 (3H)-ketone] (inhibitor of PDE1) show below:

Every rat gives the chemical compound of joining with NP3S no.31, and dosage 25mg/kg * 4 injection (subcutaneous) and low dosage FSH adds the contrast of NP3S carrier with low dosage FSH and compares, and the oocyte number that causes every rat to be discharged is dose dependent to be increased.These the results are shown in Figure 8.When not giving FSH, every Mus is on average collected 1 oocyte.(" low FSH ": the every Mus in 151.5ng * 4) average every Mus is collected 5 oocytes to give suboptimal dose FSH.When the average every Mus of this suboptimal dose FSH amalgamation compound no.31 (25mg/kg * 4 time every Mus) is collected 18 oocytes.(" high FSH ": the every Mus in 606.2ng * 3) every Mus is on average collected 21 oocytes to high dose FSH.

Ahn. wait the people: J Med Chem., 40:2196-2210, chemical compound no.33[5 '-methyl-2 ' of 1997 (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1,7 ' (8 ' H)-[3H] imidazole radicals [2,1-b] purine radicals]-4 (5 ' H)-ketone] (inhibitor of PDE1) show below:

Every rat gives the chemical compound of joining with NP3S no.33, and dosage 25mg/kg * 4 injection (subcutaneous) and low dosage FSH adds the contrast of NP3S carrier with low dosage FSH and compares, and the oocyte number that causes every rat to be discharged is dose dependent to be increased.These the results are shown in Figure 9.When not giving FSH, every Mus is on average collected 1 oocyte.(" low FSH ": the every Mus in 151.5ng * 4) average every Mus is collected 5 oocytes to give suboptimal dose FSH.When the average every Mus of this suboptimal dose FSH amalgamation compound no.33 (25mg/kg * 4 time every Mus) is collected 27 oocytes.(" high FSH ": the every Mus in 606.2ng * 3) every Mus is on average collected 20 oocytes to high dose FSH.

Give 0.08,0.4 and 2mg/kg/ days Mesopram and low dosage FSH.Only in the group of accepting low FSH, see the remarkable increase of follicle maturity.During higher dosage, it seems that follicle maturity is counter reduces.Give then than low dosage 0.032,0.016,0.08mg/kg/ days Mesopram and low dosage FSH show that follicle maturity is dose dependent and increases, and reach the most remarkable during 0.08mg/kg.Figure 15 has shown these results.

Test other PDE inhibitor with above-mentioned pattern, the results are shown in table 1.

The various PDE inhibitor of table 1 are to the effect of ovarian follicular growth

Chemical compound	The PDE selectivity	With the facilitation of FSH to ovarian follicular growth
Chemical compound	The PDE selectivity	With the facilitation of FSH to ovarian follicular growth	Papavarne	Non-selectivity	Do not have
Sldenafil	PDE	1、5、6	Papavarne	Non-selectivity	Do not have	Have
Sldenafil	PDE	1、5、6	Ariflo	PDE		Have
4	Suppress ovarian follicular growth		Ariflo	PDE
4	Suppress ovarian follicular growth	Persantin	PDE
5、6、7、8、10、 11	Have	Persantin	PDE
5、6、7、8、10、 11	Have	Zaprinast	PDE
1、5、6、7、9、10、 11	Have	Zaprinast	PDE
1、5、6、7、9、10、 11	Have	CDP840	PDE	4	Suppress ovarian follicular growth
Tadanafil	PDE	CDP840	PDE	4	Suppress ovarian follicular growth
Tadanafil	PDE	5、6	Have
Chemical compound no.31	PDE 1	5、6	Have	Have
Chemical compound no.31	PDE 1	Chemical compound no.33	PDE 1	Have	Have

Embodiment 2: cAMP is induced in various PDE inhibitor and low dosage FSH coupling

Except estimating the granulosa cell that produces, as mentioned above for the cAMP that measures in the cell line has adopted JC-410 pig granulosa cell.These cells are available from Jorge doctor Chedrese (Sakatchewan university).Cell maintains among the DNEN/F12 that is added with 5% new-born calf serum (Gibco) and 5 μ g/ml insulins (Gibco).By adopting the standard rotaring dyeing technology and selecting, the cDNA commentaries on classics beam of people LH and fsh receptor has been set up stable cell line in cell with 300 μ g/ml Geneticins (Gibco).Cell is maintained in the Geneticin of same concentrations after the selection.Measure for cAMP, cell is seeded in 96 orifice plates with the density in 25,000 cell/every holes measures after one day.Next day,, exist as shown or irritated cell 1 hour when not having 1nM FSH with the inhibitor molecules of incremental change.(DMEM/F12 0.1%BSA-Sigma) dilutes all chemical compounds with the test buffer that contains 4%DMSO (ultimate density of test is 0.5%).After irritating 1 hour, cell lysis is measured cAMP with Tropix cAMP-screening test (Applied Biosystems) by manufacturers protocol.

Following monitoring ovary of former generation disperses the cAMP in the culture.Collect the ovary of Sprague-Dawley rat (22 age in days) and under anatomic microscope, slough outer coatings with 3-5 milliliter Digestive system.This Digestive system is formed by testing culture fluid (being added with the McCoy 5A culture fluid of 1mg/ml BSA (Sigma), 5 μ g/ml gentamycins and 3 μ g/ml amphotericin Bs (Gibco)) and 8mg/ml collagenase (Sigma) and 0.05%DNA enzyme (Invitrogen).After sloughing coating, ovary is slit into small pieces with No. 27 syringe needles decomposition, transfers in the 15ml test tube, 37 shake digestion 45 minutes.The tissue of digestion filters by 70 μ m Nalgene filters, 1000rpm centrifugal filtrate 5 minutes.Precipitation is washed recentrifuge with the test culture fluid.The gained precipitation is suspended in the 2ml grown cultures liquid (being added with the McCoy 5A culture fluid of 5%FBS, 5 μ g/ml gentamycins and 3 μ g/ml amphotericin Bs (Gibco)) counting cells.With grown cultures liquid diluting cells, 25-30,000 cell/every hole is inoculated, and cultivates to irritate then in 48 hours.Irritate the cAMP of raji cell assay Raji cell line as mentioned above.

The result

Testing in vitro one group of PDE4 inhibitor rat granulosa cell of handling or do not handle at FSH and/or the pig granulosa cell of expressing human fsh receptor be among the JC410 (JC410FSHR), the ability of inducing cAMP.In this model, some of PDE4 inhibitor induce cAMP active high, and some activity are medium, the very little or non-activity of some activity.Notice that the PDE4 inhibitor seldom maybe can not be induced cAMP (seeing Figure 10 A-10C) when lacking FSH.The source of granulosa cell no matter, the intensity of all inhibitor is identical, promptly cell and former generation granulosa cell the two is similar.

In the body, two kinds of exemplary PDE4 inhibitor: Piclamilast and Roflumilast have promoted the inductive follicle maturity of FSH.The Piclamilast of low dosage (0.08 and 0.4mg/kg) is not enough to induce follicle maturity (Figure 13) when lacking low-level endogenous FSH.Yet Piclamilast has induced follicle maturity (Figure 13) really during higher dosage 2mg/kg.When having low dosage FSH, as can discharge oocyte number increase prove, give Piclamilast and induced follicle maturity very significantly.

Above result shows, when giving mammal with suboptimal dose FSH, the PDE4 inhibitor can cause increasing the hCG-oocyte number of can ovulating.Narrated more than this paper and explored the used method and composition of this discovery.

Rely on content disclosed herein, need not too much test and to prepare and to carry out disclosed herein and all compositionss and/or method claim.Though by preferred embodiment having narrated the compositions and methods of the invention, but those skilled in the art will know that, can be to all compositionss of this paper and/or method, and the order of all steps of method or all steps applies various changes but does not break away from notion of the present invention, thinking and scope.Say that more specifically some reagent that obvious available physicochemical property is relevant substitutes reagent as herein described still can obtain same or analogous result.Those skilled in the art will know that similar the substituting and modify of all these classes all belongs within the defined thinking of appended claims book of the present invention, scope and the notion.

The degree of the list of references that this paper quoted, patent and patent publications makes them provide exemplary program or other additional in detail to content described herein, and all these are quoted thing and all specifically include in this paper list of references.

Claims

1.PDE the purposes of enzyme inhibitor in preparation stimulation women folliculus ovarii growth medicine.

2. purposes as claimed in claim 1 is characterized in that described patient will experience ovulation induction.

3. purposes as claimed in claim 1 or 2 is characterized in that, described patient will experience the super stimulation of in check ovary.

4. as the described purposes of claim 1,2 or 3, it is characterized in that, described medicine and FSH or have the active preparation of FSH, or the preparation that can stimulation of endogenous FSH discharges, simultaneously, give respectively or successively.

5. as claim 1,2 or 3 described purposes, it is characterized in that described medicine and FSH are simultaneously, give respectively or successively.

6. as claim 1,2 or 3 described purposes, it is characterized in that, described medicine with have an active preparation of FSH, or the preparation that can stimulation of endogenous FSH discharges, simultaneously, give respectively or successively.

7. any one described purposes of claim as described above is characterized in that, described medicine began to give after menstruation in about 2-3 days.

8. any one described purposes of claim as described above is characterized in that, give every day described medicine up to ovarian follicular growth to enough, trigger the hCG of ovulation dosage this moment.

9. purposes as claimed in claim 8 is characterized in that, the triggering ovulation dosage of described hCG is 5000-10000IU.

10. any one described purposes of claim as described above is characterized in that described medicine gives with FSH, and wherein, the dosage of FSH is lower than same patient required dosage when lacking the PDE inhibitor, so that can obtain the identical result of stimulation ovarian follicular growth.

11. any one described purposes of claim is characterized in that as described above, described PDE inhibitor is the inhibitor of at least a PDE1 of being selected from, 5 and 6 types.

12. any one described purposes of claim is characterized in that as described above, described PDE inhibitor is selected from:

5-[2-ethyoxyl-5-(4-methyl isophthalic acid-piperazinyl sulfonyl) phenyl]-1-methyl-3-just-propyl group-1,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones (sildenafil, sldenafil); Zaprinast (Zaprinast); Persantin (dipyridamole); 5-(2-ethyoxyl-5-morpholino acetylphenyl)-1-methyl-2-just-propyl group-1,6-dihydro-7H-20 pyrazolo [4,3-d] pyrimidin-7-ones; 3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-just-the propoxyl group phenyl]-2-(pyridine-2-yl) methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-(2-methoxy ethoxy) pyridine-just-the 3-yl]-2-(pyridine-2-yl) methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-4-one; (+)-3-ethyl-5-[5-(4-ethyl piperazidine-1-base sulfonyl)-2-(2-methoxy-1 (R)-methyl ethoxy) pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-ethyoxyl-5 (4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-[2-methoxy ethyl]-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-is different-butoxy-5-(4-ethyl piperazidine-1-base sulfonyl) pyridin-3-yl]-3-ethyl-2-(methyl piperidine-4-yl)-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-[2-ethyoxyl-5-4-ethyl piperazidine-1-base sulfonyl] pyridin-3-yl]-3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-(5-acetyl group-2-propoxyl group-3-pyridine radicals)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; 5-(5-acetyl group-2-butoxy-3-pyridine radicals)-3-ethyl-2-(1-base-3-azetidinyl)-2,6-dihydro-7H-pyrazolo [4,3-d] pyrimidin-7-ones; (6R, 12aR)-2,3,6,7,12,12a-six hydrogen-2-methyl-6-(3, the 4-methylenedioxyphenyl) pyrazolos [2 ', 1 ': 6,1] pyridos [3,4-b] indole-1,4-diketone (tadanafil (Tadalafil); IC-351); 2-[2-ethyoxyl-5 (4-ethyl-piperazine-1-base-1-sulfonyl)-phenyl]-5-methyl-7-propyl group-3H-imidazo [5,1f] [1,2,4] triazine-4-ketone (Vardenafil); 4-bromo-5-(pyridine radicals methylamino)-6-[3-(4-chlorphenyl)-propoxyl group]-3 (2H) 2H-Pyridazin-3-one; 1-[4-[(1,3-benzodioxole-5-ylmethyl) amino]-6-chloro-2-quinazolyl]-4-piperidyl-carboxylic acid one sodium salt; (+)-suitable-5,6a, 1,7,9,9,9a-six hydrogen-2-[4-(trifluoromethyl)-benzyl-5-methyl-cyclopentano-[4,5] imidazo [2,1-b] purine-4 (3H) ketone; Furaziocillin; Suitable-2-hexyl-5-methyl-3,4,5,6a, 7,8,9a-octahydro cyclopentano [4,5]-imidazo [2-1 ,-b] purine-4-ketone; 3-acetyl group-1-(2-benzyl chloride base)-2-propyl indole-6-carboxylate; 3-acetyl group-1-(2-benzyl chloride base)-2-propyl indole-6-carboxylate; 4-bromo-5-(3-pyridine radicals methylamino)-6-(3-(4-chlorphenyl) propoxyl group)-3-(2H) 2H-Pyridazin-3-one; 1-methyl-5 (5-morpholino acetyl group-2-just-propoxy phenyl)-3-just-propyl group-1,6-dihydro-7H-pyrazolo (4,3-d) pyrimidin-7-ones; 1-[4-[(1,3-benzodioxole-5-ylmethyl) amino]-6-chloro-2-quinazolyl]-4-piperidine carboxylic acid one sodium salt; Pharmaprojects No.4516; PharmaprojectsNo.5051; Pharmaprojects No.5064; Pharmaprojects No.5069; GF-196960; E-8010 and E-4010; Bay-38-3145 ﹠amp; Bay-38-9456; Vinpocetine (Vinpocetine); SCH-51866; SCH-59498; (6aR, 9aS)-2-(xenyl methyl)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-3-(benzyl) cyclopentano-[4,5] imidazo [2,1-b] purine-4 (3H)-ketone; 5 '-methyl-2 ' (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1,7 ' (8 ' H)-[3H] imidazo [2,1-b] purine]-4 (5 ' H)-ketone; (6aR, 9aS)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-2-(phenylacetylene base)-3-(benzyl) cyclopentano-[4,5] imidazo [2,1-b] purine-4 (3H)-ketone; Persantin; AWD-12-171 and AWD-12-217; BMS-341400; UK-343,664; 5E-3623,5E-3569,5E-3657, E4021; KS-505a; YC-1; IDDB reference number 323951; WIN-61691; FR226807; IDDB reference number 461317,461321,461324,466146; Pyridin-4-yl methyl-3-(1,3-benzodioxole-5-yl)-9-oxo-1,3,4,9 tetrahydrochysenes-2H-pyrrolo-[3,4-b] quinaldic acid's salt:

13. any one described purposes of claim is characterized in that as described above, described PDE inhibitor is selected from:

Sldenafil, zaprinast, persantin, (6aR, 9aS)-2-(xenyl methyl)-5,6a, 7,8,9,9a-six hydrogen-5-methyl-3-(benzyl) cyclopentano-[4,5] imidazo [2,1-b] purine-4 (3H)-ketone; And 5 '-methyl-2 ' (xenyl methyl)-3 '-(benzyl) spiral shell [Pentamethylene .-1,7 ' (8 ' H)-[3H] imidazo [2,1-b] purine]-4 (5 ' H)-ketone.

14., it is characterized in that described PDE inhibitor is a zaprinast as any one described purposes of claim 1-11.

15., it is characterized in that described PDE inhibitor is a sldenafil as any one described purposes of claim 1-11.

16., it is characterized in that described PDE inhibitor is a tadanafil as any one described purposes of claim 1-11.

17., it is characterized in that described PDE inhibitor is the selective depressant of PDE1 and PDE5 as any one described purposes of claim 1-11.

18., it is characterized in that described PDE inhibitor is the selective depressant of PDE1 as any one described purposes of claim 1-11.

19., it is characterized in that described PDE inhibitor is the selective depressant of PDE5 as any one described purposes of claim 1-11.

20. a method that increases follicle maturity, this method comprise with the combination treatment women who contains phosphodiesterase (PDE) inhibitor that stimulates the follicle maturity effective dose.

21. a method that increases oocyte maturation, this method comprise with the compositions extracorporeal treatment oocyte that contains the PDE inhibitor that causes the oocyte maturation effective dose.

22., it is characterized in that described compositions contains at least a PDE4 inhibitor as claim 20 or 21 described methods.

23. as claim 20 or 21 described methods, it is characterized in that described compositions contains at least a Piclamilast of being selected from, Roflumilast, Ariflo, Filaminast, Mesopram, D4418, the PDE4 inhibitor of Arofylline and CL1044.

24. as claim 20 or 21 described methods, it is characterized in that, described compositions contains at least a PDE4 inhibitor and a kind of other PDE inhibitor, and described other PDE inhibitor is selected from PDE1 inhibitor, PDE7 inhibitor, PDE9 inhibitor, PDE10 inhibitor and PDE11 inhibitor.

25., it is characterized in that this method also comprises the promoting sexual gland hormone treatment with at least a FSH of being selected from, lutropin and chorionic gona dotropin as claim 20 or 21 described methods.

26. method as claimed in claim 22 is characterized in that, this method also comprises the promoting sexual gland hormone treatment with at least a FSH of being selected from, lutropin and chorionic gona dotropin.

27. method as claimed in claim 23 is characterized in that, this method also comprises the promoting sexual gland hormone treatment with at least a FSH of being selected from, lutropin and chorionic gona dotropin.

28. method as claimed in claim 24 is characterized in that, this method also comprises the promoting sexual gland hormone treatment with at least a FSH of being selected from, lutropin and chorionic gona dotropin.

29., it is characterized in that this method also comprises with FSH treats as claim 20 or 21 described methods.

30. as claim 20 or 21 described methods, it is characterized in that, this method also comprise give FSH and at least a non--promoting sexual gland hormone of FSH.

31. method as claimed in claim 30 is characterized in that, described non--the FSH promoting sexual gland hormone is a lutropin.

32. method as claimed in claim 30 is characterized in that, described non--the FSH promoting sexual gland hormone is a chorionic gona dotropin.

33., it is characterized in that this method comprises stimulant, agonist or the co-adjuvant that gives FSH separately as claim 20 or 21 described methods, or combined PD E4 inhibitor.

34. method as claimed in claim 33 is characterized in that, the stimulant of described FSH is selected from: letrozole, my bent azoles and R 83842.

35. method as claimed in claim 25 is characterized in that, the administration simultaneously of described PDE inhibitor and promoting sexual gland hormone.

36. method as claimed in claim 25 is characterized in that, described PDE inhibitor and FSH are contained in the bottle as mixture.

37. phial that the single dosage of PDE4 inhibitor and FSH mixture is housed.

38. method as claimed in claim 25 is characterized in that, described PDE inhibitor gave before the promoting sexual gland hormone treatment.

39. method as claimed in claim 25 is characterized in that, described PDE inhibitor gives after the promoting sexual gland hormone treatment.

40. method as claimed in claim 25 is characterized in that, the dosage range of described FSH administration is about 5-450IU/ days.

41. method as claimed in claim 25 is characterized in that, the dosage scope of described FSH is about 5-75IU/ days.

42. method as claimed in claim 20 is characterized in that, this method comprises and gives the compositions that the women contains at least a PDE4 inhibitor and a kind of exogenous FSH hormone.

43. method as claimed in claim 42 is characterized in that, described exogenous FSH hormone is the Recombinant FSH hormone.

44. method as claimed in claim 42 is characterized in that, described exogenous FSH hormone is a urine FSH hormone.

45. method as claimed in claim 42 is characterized in that, the dosage of described PDE4 inhibitor is about 0.05-5mg/ days.

46. method as claimed in claim 42 is characterized in that, the dosage of described PDE4 inhibitor is about 10-200mg/ days.

47. method as claimed in claim 42 is characterized in that, the dosage scope of described FSH is 5-75 IU FSH/ days.

48. method as claimed in claim 42 is characterized in that, the dosage of described FSH is IU FSH every days 150.

49. method as claimed in claim 42 is characterized in that, described FSH gives with a dosage.

50. method as claimed in claim 42 is characterized in that, described FSH gives with a plurality of dosage.

51. method as claimed in claim 42 is characterized in that, described FSH is through intramuscular or subcutaneous giving.

52. method as claimed in claim 42 is characterized in that, described FSH gives between 2-14 days of women's menstrual cycle.

53. method as claimed in claim 42 is characterized in that, described FSH administration continues 7-12 days.

54. method as claimed in claim 42 is characterized in that, this method also is included in and gives before PDE4 inhibitor and the FSH hormone, and the endogenous FSH and the LH that suppress the women produce.

55. method as claimed in claim 54 is characterized in that, women GnRH or its congener suppress described endogenous FSH and LH produces by giving.

56. method as claimed in claim 54 is characterized in that, before giving at least a PDE4 inhibitor and exogenous FSH hormone, and when giving the described GnRH of women or its congener and being 30 days.

57. method as claimed in claim 55 is characterized in that, the dosage range of the described GnRH of giving or its congener is about 0.25-3mg GnRH every day.

58. method as claimed in claim 42 is characterized in that, described women can produce 4 or more a plurality of oocyte of gathering in the crops.

59. method as claimed in claim 58 is characterized in that, this method also is included in and gives behind PDE4 inhibitor and the FSH 12 days first, the step of results oocyte.

60. method as claimed in claim 59, it is characterized in that, this method also is included in the oocyte that external fertilization is gathered in the crops, and cultivates the fertilized oocyte of being gathered in the crops and transfers to the intrauterine step of women to the 4-8 cell stage with this 4-8 cell stage fertilized oocyte.

61. a test kit for the treatment of infertility, this test kit is equipped with:

In pharmaceutically acceptable preparation, contain first compositions of at least a PDE4 inhibitor and in pharmaceutically acceptable preparation, contain second compositions of FSH.

62. test kit as claimed in claim 61 is characterized in that, this test kit is equipped with urine FSH or Recombinant FSH.

63. test kit as claimed in claim 62 is characterized in that, this test kit is equipped with people FSH.

64. test kit as claimed in claim 61 is characterized in that, the FSH unit dose that this test kit provides is between about 5-75 IU FSH.

65. test kit as claimed in claim 61 is characterized in that, this test kit also is equipped with the 3rd compositions that contains LH in pharmaceutically acceptable preparation.

66., it is characterized in that the LH unit dose that this test kit provides is between about 75-150 IU LH as the described test kit of claim 65.