KR20010043828A - Treatment of Infertility with cAMP-Increasing Compounds Alone or in Combination with at Least One Meiosis-Stimulating Compound - Google Patents

Abstract

The present invention is directed to pharmaceutical compositions for the treatment of infertility and to low doses of c-AMP-enhancing compounds alone to increase the rate of fertilization of mammals, comprising low doses of c-AMP-enhancing compounds and one or more mitosis-stimulating compounds. Or to combinations with one or more meiosis-stimulating compounds and for the manufacture of a medicament.

Description

Treatment of infertility by combination of one or more cAMP-enhancing compounds alone or in combination with one or more meiosis-stimulating compounds {Treatment of Infertility with cAMP-Increasing Compounds Alone or in Combination with at Least One Meiosis-Stimulating Compound}

The present invention relates to pharmaceutical compositions and their use for treating infertility.

Meiosis is a unique and eventual germ cell event that underlies sexual reproduction. Meiosis involves two stages of meiosis. During the first division, the exchange between maternal and paternal genes occurs before the chromosome pairs separate into two daughter cells. They contain only half (1n) and 2c DNA of the chromosome. The second sensitive division proceeds without DNA synthesis. Therefore, this division forms half the germ cells with only 1c DNA.

The meiosis event is similar in male and female germ cells, but the predetermined time and differentiation process leading to egg and sperm are completely different. All female germ cells enter the early stages of life, often before the first susceptible division, but until ovulation after puberty, they all cease as oocytes later in the electricity (net). Therefore, from the beginning of life, women accumulate oocytes, which are used until the accumulation is exhausted. In females, meiosis is completed after fertilization, resulting in only one egg and two dysfunctional polar bodies per germ cell. In contrast, only a few of the male germ cells enter meiosis from puberty and most of the germ cells remain throughout life. Mitosis in male cells, once initiated, proceeds without significant delay and produces four sperm.

Little is known about the mechanisms by which meiosis is regulated in men and women. In oocytes, recent studies have shown that purines of vesicles such as hypoxanthine and adenosine can be responsible for meiosis arrest (Downs, SM et al. (1985), Dev. Biol. 82: 454-458, Eppig, JJ et al. (1986) Dev. Biol. 119: 313-321, Downs, SM et al. (1993), Mol. Reprod. Dev. 35: 82-94). These purine bases were found in millimolar concentrations in the vesicles (Eppig, J. J. et al. (1985) Biol. Reprod. 33: 1041-1049). However, the stop induced by purine base was reversible. This was provided by experiments in which mouse and human oocytes were meiogenicly stopped with hypoxanthine for 24 hours and then incubated for 16-30 hours in medium without inhibitor (Downs, SM et al. (1986) Gamet Res 15: 305-316, Cha. KY et al. (1992) Reprod. Fertil. Dev. 4: 695-701). Almost 100% of quiescent mouse oocytes resumed maturation, and furthermore mature oocytes were successfully fertilized and full pre- and post-implantation development was demonstrated. These data collectively support the hypothesis that purines such as hypoxanthine and adenosine are physiologically important in the mechanisms that regulate meiosis arrest in the body.

Cyclic adenosine 5'-monophosphate (cAMP) plays a pivotal role as a second messenger in the meiotic signal transduction pathway in oocytes. cAMP is produced by the action of adenylate cyclase (AC). cAMP is degraded by a group of phosphodiesterase enzymes (PDEs) that produce an inactive second messenger product. Hypoxanthin is an inhibitor of cAMP PDE (Eppig, J. J. et al. (1985) Biol. Reprod. 33: 1041-1049). As such, it is possible to prevent hydrolysis of oocyte cAMP and thus to maintain elevated levels of cAMP in oocytes. In addition to hypoxanthine, agents acting upstream or downstream of cAMP can raise cAMP levels. Thus activating AC with forskolin, inhibiting PDE with non-selective 3-isobutyl-1-methylxanthine (IBMX) or oocytes with specific PDE3-inhibitors such as milrinone Inhibiting specific isoform PDE3 to stop meiosis by maintaining elevated levels of c-AMP in oocytes (Downs SM and Hunzicker-Dunn M (1995) Dev Biol 172: 72-85; Tsafiri A et. al. (1996) Dev Biol 178: 393-402).

PDE3 specific inhibitors have been described as contraceptives (WO 98/10765).

The presence of diffuse mitogenic regulators in mouse fetal gonads is described by Byskov, A. G. et al. (1976) Dev. Biol. 52: 193-200. Mitosis activating substance (MAS) was secreted from meiotic mouse fetal ovaries, and anti-mitotic substance (MPS) was secreted from morphologically differentiated testes with stationary non-meiotic germ cells. Therefore, it was suggested that the relative concentrations of MAS and MPS regulate the onset, stop, and resumption of meiosis in male and female germ cells (Byskov, AG and Hoyer. PE (1994), The Physiology of Reproduction, Knobil, E and Neill, JD (eds.), Raven Press, New York, pp 487-540]. Recent papers [Byskov, A. G. et al. (1995) Nature 374: 559-562, which is isolated from preovulatory ovarian vesicles, which activate oocyte meiosis, is separated from certain sterols and bull testes defined as FF-MAS, and defined as T-MAS. Sterols are described. This was confirmed by Grondahl et al. 1988, Biol. Reprod. In press, which revealed that the newly synthesized FF-MAS can mediate the resumption of meiosis in mouse oocytes.

Although the use of MAS improves the modification rate compared to currently applied methods, it is still desirable to raise this rate further.

This is achieved by the present invention which provides a pharmaceutical composition comprising a low dose of c-AMP-enhancing compound and one or more meiosis-stimulating compounds for the treatment of infertility in mammals, in particular humans, more particularly women. Conventional teachings suggest that c-AMP-enhancing compounds are responsible for mitosis cessation and that PDE3 specific inhibitors have even been used as contraceptives, so an increase in fertility by small doses of c-AMP-enhancing compounds was unexpected. Combinations of low dose cAMP-enhancing compounds and one or more meiosis-stimulating compounds showed significantly higher stimulation rates than meiosis-stimulating compound (s) alone.

In another embodiment the invention provides a low dose c-AMP-enhancing compound alone or a meiosis-stimulating compound as an active substance for the manufacture of a pharmaceutical composition for the treatment of infertility in a mammal, in particular in humans, more particularly in women. It relates to one or more uses in combination. Small doses of c-AMP-enhancing compounds alone increase the fertilization rate compared to the control to which no compounds are added, and a combination of low doses of c-AMP-enhancing compounds and meiosis-stimulating compounds results in meiosis-stimulation. It shows a higher rate of fertilization than compound alone or a small dose of c-AMP-enhancing compound alone.

In another embodiment the invention relates to the use of a low dose c-AMP-enhancing compound alone or in combination with one or more meiotic-stimulating compounds for raising the fertilization rate in mammals, in particular in humans, more particularly in women. will be. This use may be for controlling the fertilization rate in fertilized culture medium.

In another aspect, the invention relates to the use of a low dose of c-AMP-enhancing compound alone or in combination with one or more meiotic-stimulating compounds for administration to germ cells. Germ cells may be oocytes or sperm.

In another embodiment, the present invention comprises administering an effective amount to a germ cell in need of meiosis stimulation, either alone or in combination with one or more meiosis-stimulating compounds, in an ex vivo or in vitro or ex vivo manner. And methods for stimulating meiosis in mammalian germ cells. Germ cells may be oocytes or sperm.

In another embodiment, the invention is directed to a pharmaceutical kit comprising a dosage unit of a low dose of c-AMP-enhancing compound and one or more meiosis-stimulating compound. The c-AMP-enhancing compound and the meiosis-stimulating compound may be provided in the same application form or in different application forms. Application form means, for example, tablets, liquid compositions for injection, ointments and others well known in the art.

Fission-stimulating compounds according to the invention are all compounds capable of activating meiosis. Compounds known to stimulate meiosis and their synthesis are described in WO 96/27658, WO 97/00884, WO 96/00235, WO 98/28323 and WO 98/52965. It is. In a preferred embodiment of the present invention in all forms, the fission-stimulating compound is FF-MAS (4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol).

A significantly higher fertility stimulation rate according to the present invention means that the stimulation rate is at least 40-50%, preferably 50-75% and more preferably 75-100%.

The low dose cAMP-enhancing compound according to the invention is the dose of cAMP-enhancing compound that leads to maturation of meiosis without inducing cessation of meiosis. In a preferred embodiment of the invention the cAMP-enhancing compound is applied at a dose of up to 3 mM, more preferably at a dose of 0.003 to 1 mM and particularly preferably at a dose of 0.1 to 0.5 mM.

In a particularly preferred embodiment of the present invention of all types, the cAMP-enhancing compound is a purine, non-specific PDE-inhibitor, specific PDE ₃ -inhibitor, or synthetic membrane permeable cAMP. Furin is for example hypoxanthine or adenosine. Non-specific PDE-inhibitors include all forms of PDE, such as IBMX, theophylline or SQ20,006 (1-ethyl-4-hydrazino-14-pyrazolo- (3,4-b) -pyridine -5-carboxylic acid ethyl ester). Examples of specific PDE3 inhibitors are milnonone, cilostamide, amlinone, enoximon, lyxazinone, indolidan and other inhibitors such as those listed in WO 98/10765. The document also provides a process for the preparation of these compounds. An example of a synthetic membrane permeable cAMP is dibutyryl-c-AMP (dbcAMP).

In another particularly preferred embodiment of the present invention in all forms of the meiotic-stimulating compound is FF-MAS and the cAMP-enhancing compound is purine, preferably hypoxanthine.

As described above, the present invention relates to pharmaceutical compositions in another embodiment. The composition may comprise pharmaceutically acceptable excipients well known in the art such as carriers, diluents, absorption enhancers, preservatives, buffers, osmotic pressure modifiers, tablet disintegrants and other ingredients commonly used in the art. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, low melting wax and cacao butter.

Liquid compositions include sterile solutions, suspensions, and emulsions. Such liquid compositions may be suitable for infusion or for use associated with disassembly, in vivo and in vitro fertilization. Liquid compositions may contain other ingredients conventionally used in the art, some of which are mentioned in the list above. In addition, a composition for transdermal administration of a compound of the present invention may be provided in the form of a patch, the composition for intranasal administration may be provided in the form of a nasal spray in liquid or powder form and the composition for intravaginal administration may be a tampon or It may be provided in the form of other vaginal devices.

Dosage depends a lot on the condition and size of the patient to be treated as well as on the frequency and route of administration.

In general, the compositions of the present invention are prepared by tightly combining the active compound or compounds with liquid or solid auxiliary ingredients and, if necessary, in the form of preparations which require the product.

What is disclosed in all the applications, patents, and publications cited above and below are hereby incorporated by reference.

The invention is illustrated in detail in the following examples. These examples are included for illustrative purposes and should not be considered as limiting the invention.

<Example 1>: Test of hypoxanthine, FF-MAS, and the combination of FF-MAS and hypoxanthine in oocyte analysis

Substances and Methods:

Oocytes (NO) and enclosed oocytes exposed from vesicles of immature (C57B1 / 6J x DBA / 2J) F ₁ mice (ages 21-24 days) to which 10 IU PMSG were administered intraperitoneally 48 hours prior to collection (CEO) was separated. Oocytes were cultured in modified α-MEM medium containing 1 mg fetuin / ml culture medium in 4-well multidish. Each well contained 0.4 ml of oocyte culture medium and 35 to 45 oocytes. Control and test cultures were prepared with different concentrations of compounds to be tested as indicated in the table.

Cultures were stored for 18 hours at 37 ° C. and 5% CO _{2 in} air and 100% humidity.

Oocytes stopped during meiosis are characterized by an intact nucleus with raised glomeruli, known as germ vesicles (GV). When meiosis resumes, glomeruli and nuclei disappear, characterized by the destruction of GV called reproductive vesicle destruction (GVB).

result:

Activation of meiosis in oocytes using FF-MAS, low dose hypoxanthine and FF-MAS + hypoxanthine combination compound Oocyte Morphology O of oocyte GVB (%) Control Standard (No Compound) NO 187 95.1 0.4 mM Hx NO 190 95.8 3 mM Hx NO 170 12.4 10 μM FF-MAS + 0.4 mM Hx NO 172 98.3 10 μM FF-MAS + 3 mM Hx NO 223 92.3 Hx = hypoxanthine NO = exposed oocytes GVB = destruction of reproductive vesicles n = number

Hypoxanthine used was obtained from Sigma, Deisenhofen, Deutchland. Similar to the control, low dose hypoxanthine (0.4 mM) was able to reach meiotic maturation in most oocytes. However, 3 mM hypoxanthine almost completely prevented meiosis maturation. Mitosis maturation is reached in almost all oocytes not only when FF-MAS is given with a mitotic-inhibitory dose of hypoxanthine (3 mM) but also with a low dose of hypoxanthine (0.4 mM). It is obvious that it can be done.

<Example 2>: Test of hypoxanthine, FF-MAS and the combination of FF-MAS and hypoxanthine in in vitro fertilization (IVF) assay

Substances and Methods:

NO and CEO were isolated from immature mice (C57B1 / 6J × DBA / 2J) F ₁ and cultured under the same conditions as described in the oocyte assay. After 18 hours, oocytes showing germ vesicle destruction (GVB) were immediately washed in hypoxanthine-free medium and transferred to a prepared fertilization dish consisting of a motile sperm blend obtained from the tail epididymis of the male mouse. The dish was incubated in α-MEM IVF-medium modified at 37 ° C. under defined gas conditions (5% CO ₂ ). Both the modified medium and the IVF-medium did not contain hypoxanthine. Examination of oocytes was performed 20-22 hours after fertilization to confirm fertilization and record the number of 2-cell embryos. The fertilization rate (= fertilization rate) was determined from the number of oocytes cut into 2-cell embryos.

result:

In vitro fertilization rate (IVF-ratio) of oocytes cultured with a combination of FF-MAS, low dose hypoxanthine and FF-MAS + hypoxanthine compound Oocyte Morphology N / GVB of oocytes used after in vitro culture 2-cell embryo (%) Control (no compound added) NO 177 34.1 0.4 mM Hx NO 182 52.2 3 mM Hx NO 21 0 10 μM FF-MAS + 0.4 mM Hx NO 167 64.7 10 μM FF MAS + 3 mM Hx NO 102 51.0 Hx = hypoxanthine N0 = exposed oocytes n = number GVB = destruction of reproductive vesicles

The hypoxanthine used was obtained from Sigma. Oocytes cultured in the presence of 3 mM hypoxanthine could not be fertilized. 0.4 mM hypoxanthine and 10 μM FF-MAS + 3 mM hypoxanthine raised the fertilization rate of the control by about 50%. However, the combination of 10 μM FF-MAS + 0.4 mM hypoxanthine could nearly double the control IVF-rate.

Claims (33)

A pharmaceutical composition for treating infertility in a mammal comprising a low dose of c-AMP-enhancing compound and one or more mitosis-stimulating compounds. The pharmaceutical composition of claim 1, wherein the mammal is a human. The pharmaceutical composition according to claim 1 or 2, wherein the c-AMP-enhancing compound is purine, non-specific PDE-inhibitor, specific PDE ₃ -inhibitor, and synthetic membrane permeable cAMP. The pharmaceutical composition according to any one of claims 1 to 3, wherein the fission-stimulating compound is FF-MAS. The pharmaceutical composition according to any one of claims 1 to 4, wherein the c-AMP-enhancing compound is hypoxanthine. Use of a low dose of c-AMP-enhancing compound alone or in combination with one or more meiosis-stimulating compounds as an active substance for the manufacture of a pharmaceutical composition for treating infertility in a mammal. 7. Use according to claim 6, wherein the mammal is a human. 8. Use according to claim 6 or 7, wherein the c-AMP-enhancing compound is used alone. The use according to any one of claims 6 to 8, wherein the c-AMP-enhancing compound is selected from the group consisting of purine, non-specific PDE-inhibitors, specific PDE ₃ -inhibitors, and synthetic membrane-permeable cAMP. The use according to any one of claims 6 to 9, wherein the c-AMP-enhancing compound is hypoxanthine. Use according to any of claims 6, 7, 9 or 10, wherein the fission-stimulating compound is FF-MAS. Use of a low dose of c-AMP-enhancing compound alone or in combination with one or more meiosis-stimulating compounds to raise the rate of fertilization of a mammal. 13. Use according to claim 12, wherein the mammal is a human. Use according to claim 12 or 13 for controlling the rate of fertilization in fertilization culture medium. The use according to any one of claims 12 to 14, wherein the c-AMP-enhancing compound is used alone. The use according to any one of claims 12 to 15, wherein the c-AMP-enhancing compound is selected from the group consisting of purine, nonspecific PDE-inhibitors, specific PDE ₃ -inhibitors, and synthetic membrane permeable cAMP. The use according to any one of claims 12 to 16, wherein the c-AMP-enhancing compound is hypoxanthine. 18. The use according to any one of claims 12-14 or 16 or 17, wherein the meiotic-stimulating compound is FF-MAS. Use of a low dose of c-AMP-enhancing compound alone or in combination with one or more meiosis-stimulating compounds for administration to germ cells. 20. The use of claim 19, wherein the germ cells are oocytes. 20. The use of claim 19, wherein the germ cells are sperm. 22. The use of any one of claims 19, 20 or 21, wherein the c-AMP-enhancing compound is used alone. 23. The use of any one of claims 19-22, wherein the c-AMP-enhancing compound is selected from the group consisting of purine, nonspecific PDE-inhibitors, specific PDE ₃ -inhibitors, and synthetic membrane permeable cAMP. Use according to any of claims 19 to 23, wherein the c-AMP-enhancing compound is hypoxanthine. The use according to any of claims 19 to 21 or 23 or 24, wherein the meiotic-stimulating compound is FF-MAS. Meiosis stimulation of mammalian germ cells, including ex vivo or in vitro or ex vivo administration of low doses of c-AMP-enhancing compounds alone or in combination with one or more meiosis-stimulating compounds to germ cells in need of meiosis stimulation Way. The method of claim 26, wherein the germ cells are oocytes. The method of claim 26, wherein the germ cells are sperm. 29. The method of any one of claims 26-28, wherein the c-AMP-enhancing compound is used alone. 32. The method of any one of claims 28-31, wherein the c-AMP-enhancing compound is selected from the group consisting of purine, nonspecific PDE-inhibitors, specific PDE ₃ -inhibitors, and synthetic membrane permeable cAMP. 31. The method of any one of claims 26-30, wherein the c-AMP-enhancing compound is hypoxanthine. 32. The method of any one of claims 26-28, 30 or 31, wherein the meiotic-stimulating compound is FF-MAS. A pharmaceutical kit comprising a dosage unit of a low dose of c-AMP-enhancing compound and one or more meiosis-stimulating compound.