CA2333320A1 - Treatment of infertility with camp-increasing compounds alone or in combination with at least one meiosis-stimulating compound - Google Patents
Treatment of infertility with camp-increasing compounds alone or in combination with at least one meiosis-stimulating compound Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
- A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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Abstract
The present invention relates to a pharmaceutical composition comprising c-AMP-increasing compounds in low dose and at least one meiosis-stimulating compound for the treatment of infertility and to the use of low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound to increase the rate of fertilization in a mammal and for the preparation of medicaments. The preferred c-AMP-increasing compound is Hypoxanthine. The meiosis-stimulating compound is FF-MAS (4,4-dimethyl-5alpha-cholest-8,14,24-trien-3beta-ol).
Description
Treatment of infertility with cAMP-increasing Compounds alone or in combination with at feast one Meiosis-stimulating Compound The present invention relates to a pharmaceutical composition and its use to treat infertility.
Meiosis is the unique and ultimate event of germ cells on which sexual reproduction is based. Meiosis comprises two meiotic divisions. During the first division, exchange between maternal and paternal genes take place before the pairs of chromosomes are separated into the two daughter cells. These contain only half the number (1 n) of chromosomes and 2c DNA. The second meiotic division proceeds without a DNA
synthesis. This division therefore results in the formation of the haploid germ cells with only 1 c DNA.
The meiotic events are similar in the male and female germ cells, but the time schedule and the differentiation processes which lead to ova and to spermatozoa differ profoundly. All female germ cells enter the prophase of the first meiotic division early in life, often before birth, but all are arrested as oocytes later in the prophase (dictyate state) until ovulation after puberty. Thus, from early life the female has a stock of oocytes which is drawn upon until the stock is exhausted. Meiosis in females is not completed until after fertilization, and results in only one ovum and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells enter meiosis from puberty and leave a stem population of germ cells throughout Life. Once initiated, meiosis in the male cell proceeds without significant delay and produces 4 spem~atozoa.
Only little is known about the mechanisms which control meiosis in the male and in the female. In the oocyte, recent studies indicate that follicular purines, such as hypoxanthine, and adenosine, could be responsible for meiotic arrest (Downs, S.M.
et al. (1985), Dev. Biol. 82: 454-458, Eppig, J.J. et al. (1986) Dev. Biol.
119: 313-321, Downs, S.M. et al. (1993), Mol. Reprod. Dev. 35: 82-94). These purine bases were found in follicular fluid in millimofar concentrations (Eppig, J.J. et al.
(1985) 8iol. Reprod.
33: 1041-1049). However, the purine bases induced arrest was reversible. This was provided by experiments in which mice and human oocvtes were maintained in meiotic arrest for 24 hours with hypoxanthine followed by a 16-30 hour culture in inhibitor-tree medium (Downs. S.M. et al. (1986) Gamet Res. 15: 305-316, Cha.
K.Y. et al.
(1992) Reprod. i=ertil. Dev. 4: 695-701 ). Nearly 100% of the arrested mice oocytes resumed maturation and, furthermore, the mature oocytes were successfully fertilized and demonstrated complete pre-and post-implantation development. These data collectively support the idea that purines such as hypoxanthine and adenosine are physiologically important in the mechanisms controlling meiotic arrest in vivo.
Cyclic adenosine 5'-monophosphate (CAMP) plays a pivotal role as a second messenger in the signal transduction pathway during meiosis in the oocyte.
cAMP is generated by the action of adenylate cycfase (AC). CAMP is degraded by the family of phosphodiesterase enzymes (PDE), which produces inactive second messenger products. Hypoxanthine is an inhibitor of CAMP PDE (Eppig, J.J. et al. (1985) Biol.
Reprcd. 33: 10.1-1049). As such, it can prevent the hydrolysis of oocyte cANIP
and thereby maintain elevated levels of cAMP in the oocyte. in addition to hypoxanthine, agents acting upstream or downstream of CAMP are able to increase cAMP levels.
By this way activation of AC with forskolin, inhibition of PDE with the nonselective 3-isobutyl-1-methyixanthine (IBMX) or inhibition of the oocyte=specifiic isoform with a specific PDE3-inhibitor, e.g. milrinone, leads to meiotic arrest by maintaining elevated levels of c-AMP within the oocytes (Downs SM and Hunzicker-Dunn M
(1995) Dev Biol 172: 72-85; Tsafri A et al. (1996) Dev Biol 178: 393-402).
A PDE3 specific inhibitor has been described as a contraceptive agent (W098/107fi5).
The presence of a diffusible meiosis regulating substance was ftrst described (Byskov, A.G. et al. (1976) Dev. Biol. 52: 193-200) in the fetal mouse gonades. A meiosis activating substance (MAS) was secreted in the fetal mouse ovary in which meiosis was ongoing, and a meiosis preventing substance (MPS) was released from the morphologically differentiated testis with resting, non-meiotic germ cells. It was therefore suggested that the relative concentrations of MAS and MPS regulated the beginning, arrest and resumption of meiosis in the male and in the female gems cells (Byskov, A.G. and Hsyer P.E. (1994), The Physiology of Reproduction, Knobil, E.
and NeiII,J.D. (eds.), Raven Press, New York, pp 487-540). A recent article (Byskov, A.G. et al. (1995) Nature 374: 559-562) describes the isolation of certain sterols from preovulatory ovarian follicular fluid, defined as FF-MAS, and bull testicular testis, defined as T-MAS. that activate oocyte meiosis. This was confirmed by Grandahl et al. (1998, Biol. Reprod. in press) showing that de novo synthesized FF-MAS is capable of mediating resumption of meiosis in mice oocytes.
Although the use of MAS improves the rate of fertility compared to the currently applied methods, it is still desirable to increase this rate even further.
This is achieved by the present invention which provides a pharmaceutical composition comprising c-AMP-increasing compounds in low dose and at least one ~ 5 meiosis-stimulating compound for the treatment of infertility in mammals, particulary in hump~s, more particulary in females. The increase of fertility by low dose c-AMP-increasing compounds is surprising since the teaching of the prior art is that c-AMP-increasing compounds are responsible for meiotic arrest and a PDE3 specific inhibitor has even been used as a contraceptive agent. The combination of the low dose CAMP-increasing compounds with at least one meiosis-stimulating compound shows a significantly higher stimulation rate than the meiosis-stimulating compounds) atone.
In a further embodiment the invention relates to the use of low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound as active substances for the production of a pharmaceutical composition for the treatment of infertiiity.in mammals, particular in humans, more particularly in females. Low dose c-AMP-increasing compounds alone increase the fertilization rate compared to the control where no compounds were added and the combination of c-AMP-increasing compounds in a low dose with a meiosis-stimulating compound shows an even higher rate of fertilization than the meiosis-stimulating compound alone or the low dose c-AMP-increasing compound alone.
In a still further embodiment the invention relates to the use of low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound to increase the rate of fertilization in a mammal, particularly in humans, more particularly in females. This use may be to regulate the fertilization rate in a fertilization culture media.
In another aspect, the invention includes the use of a low dose c-AMP-increasing compounds alone or in combination with at feast one meiosis-stimulating compound for the administration to a germ cell. The germ cell may be an ooc a Yt or a spermatozoon.
In another embodiment the invention relates to a method of stimulating meiosis in a mammalian germ cell comprising administering ex vivo or in vivo or in vitro to a germ cell in need of such a stimulation an effective amount of low dose c-AMP-increasin compounds alone or in combination with at least one meiosis-stimulating coin ound.
P
The germ cell may be an oocyte or a spermatozoon.
In a further embodiment the invention relates to a pharmaceutical kit comprising a dosage unit for a ,c-AMP-increasing compound in low dose and a dosage unit of at least one meiosis-stimulating compound. The c-AMP-increasing coin ound an P d the meiosis-stimulating compound may be provided in the same application form or in different application forms. Application forms means e.g. tablets, liquid compositions for injections, paste and others well known in the art.
Meiosis-stimulating compounds according to the present invention are a!I
compounds that can activate meiosis. Compounds being known to stimulate meiosis and their synthesis are described, i.e., in WO 96127658, W097100884, W096/00235, WO98/28323 and W098/52965. In preferred embodiments of al! modes of the invention the meiosis-stimulating compound is FF-MAS (4,4-dimethyl-5a-cholesta 8,14,24-trien-3f3-o!).
Under a significantly higher stimulation rate of fertilizatibn according to the present invention it is meant that the stimulation rate is at (east 40 - 50 %, referred P
50 - 75%, and more preferred 75 -100 %.
Low dose CAMP-increasing compounds according to the present invention is a dose of CAMP-increasing compounds that lead to meiotic maturation without inducin meiotic arrest. In preferred embodiments of the present invention the cAMP-increasing compounds are applied in a dose below 3mM, more prefen-ed in a dose of 0.003 -1 mM, and especially preferred in a dose of 0.1-0.5mM.
In specially preferred embodiments of all modes of the invention the cAMP
increasing compounds are purines, unspecific PDE-inhibitors, specific PDE
mhibitors or synthetic membrane permeable cAMP. A purine is e.g. hypoxanthine or adenosine. Unspecific PDE-inhibitors are nonselective inhibitors which inhibit all types of PDEs. e.g. IBMX, theophylline or SQ20.006 (1-ethyl-4-hydrazino-14-pyrazolo-(3,4-b)-pyridine-5-carboxyiicacidethylester). Examples of specific inhibitors are miirinone, ciiostamide, amrinone, enoximone, lixazinone, indolidan and other inhibitors as listed in W098/10765. This reference also gives the methods of preparation of these compounds. An example for a synthetic membrane permeable CAMP is dibutyryi-c-AMP (dbcAMP).
In a further specially prefer-ed embodiments of ail modes of the invention the meiosis-stimulating compound is FF-MAS and the cAMP-increasing compound is a purine, preferably hypoxanthine.
As described above the present invention relates in further embodiments to pharmaceutical compositions. The compositions may comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disintegrating agents and other ingredients which are conventionally 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 waxes and cacao butter.
Liquid compositions include sterile solutions, suspensions and emulsions. Such liquid compositions may be suitable for injection or for use in connection with ex vivo, in vivo ,and in vitro fertilization. The liquid compositions may contain other ingredients which are conventionally used in the art, some of which are mentioned in the list above. Further, a composition for transdermal administration of a compound of this invention may be provided in the form of a patch, a composition for nasal administration may be provided in the form of a nasal spray in liquid or powder form and a composition for intro-vaginal administration may be provided in the form of a tampon or other intro-vaginal devices.
The dosage to be administered depends to a large extent on the condition and the size of the subject being treated as well as the frequency of treatment and the route of administration.
In general, the compositions of the invention are prepared by intimately bringing into association the active compound or compounds with the liquid or solid auxiliary ingredients and then, if necessary, shaping the product into the desired formulation.
Meiosis is the unique and ultimate event of germ cells on which sexual reproduction is based. Meiosis comprises two meiotic divisions. During the first division, exchange between maternal and paternal genes take place before the pairs of chromosomes are separated into the two daughter cells. These contain only half the number (1 n) of chromosomes and 2c DNA. The second meiotic division proceeds without a DNA
synthesis. This division therefore results in the formation of the haploid germ cells with only 1 c DNA.
The meiotic events are similar in the male and female germ cells, but the time schedule and the differentiation processes which lead to ova and to spermatozoa differ profoundly. All female germ cells enter the prophase of the first meiotic division early in life, often before birth, but all are arrested as oocytes later in the prophase (dictyate state) until ovulation after puberty. Thus, from early life the female has a stock of oocytes which is drawn upon until the stock is exhausted. Meiosis in females is not completed until after fertilization, and results in only one ovum and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells enter meiosis from puberty and leave a stem population of germ cells throughout Life. Once initiated, meiosis in the male cell proceeds without significant delay and produces 4 spem~atozoa.
Only little is known about the mechanisms which control meiosis in the male and in the female. In the oocyte, recent studies indicate that follicular purines, such as hypoxanthine, and adenosine, could be responsible for meiotic arrest (Downs, S.M.
et al. (1985), Dev. Biol. 82: 454-458, Eppig, J.J. et al. (1986) Dev. Biol.
119: 313-321, Downs, S.M. et al. (1993), Mol. Reprod. Dev. 35: 82-94). These purine bases were found in follicular fluid in millimofar concentrations (Eppig, J.J. et al.
(1985) 8iol. Reprod.
33: 1041-1049). However, the purine bases induced arrest was reversible. This was provided by experiments in which mice and human oocvtes were maintained in meiotic arrest for 24 hours with hypoxanthine followed by a 16-30 hour culture in inhibitor-tree medium (Downs. S.M. et al. (1986) Gamet Res. 15: 305-316, Cha.
K.Y. et al.
(1992) Reprod. i=ertil. Dev. 4: 695-701 ). Nearly 100% of the arrested mice oocytes resumed maturation and, furthermore, the mature oocytes were successfully fertilized and demonstrated complete pre-and post-implantation development. These data collectively support the idea that purines such as hypoxanthine and adenosine are physiologically important in the mechanisms controlling meiotic arrest in vivo.
Cyclic adenosine 5'-monophosphate (CAMP) plays a pivotal role as a second messenger in the signal transduction pathway during meiosis in the oocyte.
cAMP is generated by the action of adenylate cycfase (AC). CAMP is degraded by the family of phosphodiesterase enzymes (PDE), which produces inactive second messenger products. Hypoxanthine is an inhibitor of CAMP PDE (Eppig, J.J. et al. (1985) Biol.
Reprcd. 33: 10.1-1049). As such, it can prevent the hydrolysis of oocyte cANIP
and thereby maintain elevated levels of cAMP in the oocyte. in addition to hypoxanthine, agents acting upstream or downstream of CAMP are able to increase cAMP levels.
By this way activation of AC with forskolin, inhibition of PDE with the nonselective 3-isobutyl-1-methyixanthine (IBMX) or inhibition of the oocyte=specifiic isoform with a specific PDE3-inhibitor, e.g. milrinone, leads to meiotic arrest by maintaining elevated levels of c-AMP within the oocytes (Downs SM and Hunzicker-Dunn M
(1995) Dev Biol 172: 72-85; Tsafri A et al. (1996) Dev Biol 178: 393-402).
A PDE3 specific inhibitor has been described as a contraceptive agent (W098/107fi5).
The presence of a diffusible meiosis regulating substance was ftrst described (Byskov, A.G. et al. (1976) Dev. Biol. 52: 193-200) in the fetal mouse gonades. A meiosis activating substance (MAS) was secreted in the fetal mouse ovary in which meiosis was ongoing, and a meiosis preventing substance (MPS) was released from the morphologically differentiated testis with resting, non-meiotic germ cells. It was therefore suggested that the relative concentrations of MAS and MPS regulated the beginning, arrest and resumption of meiosis in the male and in the female gems cells (Byskov, A.G. and Hsyer P.E. (1994), The Physiology of Reproduction, Knobil, E.
and NeiII,J.D. (eds.), Raven Press, New York, pp 487-540). A recent article (Byskov, A.G. et al. (1995) Nature 374: 559-562) describes the isolation of certain sterols from preovulatory ovarian follicular fluid, defined as FF-MAS, and bull testicular testis, defined as T-MAS. that activate oocyte meiosis. This was confirmed by Grandahl et al. (1998, Biol. Reprod. in press) showing that de novo synthesized FF-MAS is capable of mediating resumption of meiosis in mice oocytes.
Although the use of MAS improves the rate of fertility compared to the currently applied methods, it is still desirable to increase this rate even further.
This is achieved by the present invention which provides a pharmaceutical composition comprising c-AMP-increasing compounds in low dose and at least one ~ 5 meiosis-stimulating compound for the treatment of infertility in mammals, particulary in hump~s, more particulary in females. The increase of fertility by low dose c-AMP-increasing compounds is surprising since the teaching of the prior art is that c-AMP-increasing compounds are responsible for meiotic arrest and a PDE3 specific inhibitor has even been used as a contraceptive agent. The combination of the low dose CAMP-increasing compounds with at least one meiosis-stimulating compound shows a significantly higher stimulation rate than the meiosis-stimulating compounds) atone.
In a further embodiment the invention relates to the use of low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound as active substances for the production of a pharmaceutical composition for the treatment of infertiiity.in mammals, particular in humans, more particularly in females. Low dose c-AMP-increasing compounds alone increase the fertilization rate compared to the control where no compounds were added and the combination of c-AMP-increasing compounds in a low dose with a meiosis-stimulating compound shows an even higher rate of fertilization than the meiosis-stimulating compound alone or the low dose c-AMP-increasing compound alone.
In a still further embodiment the invention relates to the use of low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound to increase the rate of fertilization in a mammal, particularly in humans, more particularly in females. This use may be to regulate the fertilization rate in a fertilization culture media.
In another aspect, the invention includes the use of a low dose c-AMP-increasing compounds alone or in combination with at feast one meiosis-stimulating compound for the administration to a germ cell. The germ cell may be an ooc a Yt or a spermatozoon.
In another embodiment the invention relates to a method of stimulating meiosis in a mammalian germ cell comprising administering ex vivo or in vivo or in vitro to a germ cell in need of such a stimulation an effective amount of low dose c-AMP-increasin compounds alone or in combination with at least one meiosis-stimulating coin ound.
P
The germ cell may be an oocyte or a spermatozoon.
In a further embodiment the invention relates to a pharmaceutical kit comprising a dosage unit for a ,c-AMP-increasing compound in low dose and a dosage unit of at least one meiosis-stimulating compound. The c-AMP-increasing coin ound an P d the meiosis-stimulating compound may be provided in the same application form or in different application forms. Application forms means e.g. tablets, liquid compositions for injections, paste and others well known in the art.
Meiosis-stimulating compounds according to the present invention are a!I
compounds that can activate meiosis. Compounds being known to stimulate meiosis and their synthesis are described, i.e., in WO 96127658, W097100884, W096/00235, WO98/28323 and W098/52965. In preferred embodiments of al! modes of the invention the meiosis-stimulating compound is FF-MAS (4,4-dimethyl-5a-cholesta 8,14,24-trien-3f3-o!).
Under a significantly higher stimulation rate of fertilizatibn according to the present invention it is meant that the stimulation rate is at (east 40 - 50 %, referred P
50 - 75%, and more preferred 75 -100 %.
Low dose CAMP-increasing compounds according to the present invention is a dose of CAMP-increasing compounds that lead to meiotic maturation without inducin meiotic arrest. In preferred embodiments of the present invention the cAMP-increasing compounds are applied in a dose below 3mM, more prefen-ed in a dose of 0.003 -1 mM, and especially preferred in a dose of 0.1-0.5mM.
In specially preferred embodiments of all modes of the invention the cAMP
increasing compounds are purines, unspecific PDE-inhibitors, specific PDE
mhibitors or synthetic membrane permeable cAMP. A purine is e.g. hypoxanthine or adenosine. Unspecific PDE-inhibitors are nonselective inhibitors which inhibit all types of PDEs. e.g. IBMX, theophylline or SQ20.006 (1-ethyl-4-hydrazino-14-pyrazolo-(3,4-b)-pyridine-5-carboxyiicacidethylester). Examples of specific inhibitors are miirinone, ciiostamide, amrinone, enoximone, lixazinone, indolidan and other inhibitors as listed in W098/10765. This reference also gives the methods of preparation of these compounds. An example for a synthetic membrane permeable CAMP is dibutyryi-c-AMP (dbcAMP).
In a further specially prefer-ed embodiments of ail modes of the invention the meiosis-stimulating compound is FF-MAS and the cAMP-increasing compound is a purine, preferably hypoxanthine.
As described above the present invention relates in further embodiments to pharmaceutical compositions. The compositions may comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disintegrating agents and other ingredients which are conventionally 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 waxes and cacao butter.
Liquid compositions include sterile solutions, suspensions and emulsions. Such liquid compositions may be suitable for injection or for use in connection with ex vivo, in vivo ,and in vitro fertilization. The liquid compositions may contain other ingredients which are conventionally used in the art, some of which are mentioned in the list above. Further, a composition for transdermal administration of a compound of this invention may be provided in the form of a patch, a composition for nasal administration may be provided in the form of a nasal spray in liquid or powder form and a composition for intro-vaginal administration may be provided in the form of a tampon or other intro-vaginal devices.
The dosage to be administered depends to a large extent on the condition and the size of the subject being treated as well as the frequency of treatment and the route of administration.
In general, the compositions of the invention are prepared by intimately bringing into association the active compound or compounds with the liquid or solid auxiliary ingredients and then, if necessary, shaping the product into the desired formulation.
The entire disclosure of ail applications, patents and publications, cited above and below are hereby incorporated by reference.
The present invention will be illustrated in detail in the following examples.
These examples are included for illustrative purposes and should not be considered to Limit the present invention.
Examples ~ 5 Example 1: Test of hypoxanthine, FF-MAS and the combination of FF-MAS and hypoxanthine in the oocyte assay Material and Methods:
Naked oocytes (NO) and cumulus enclosed oocytes (CEO) were isolated from follicles from immature (C57B1l6J x DBAI2J)Fi mice (age 21-24 days), that had received 10 LU. PMSG i.p. 48h prior to collection. The oocytes were cultured in 4-well multidishes in a modified a-MEM medium containing 1 mg fetuinlmi culture medium. Each well contained 0.4 ml of the oocyte culture medium and 35-45 oocytes. The control and test cultures were made with different concentrations of the compounds to be tested as indicated in the tables.
The cultures were kept at 37°C and 100% humidity with 5% C02 in the air for 18 hours.
Oocytes arrested in meiosis are characterised by an intact nucleus with a prominent nucleolus, known as germinal vesicle (GV). Upon reinitiation of meiosis the nucleolus and the nuclear envelope disappear and this is characterised by a breakdown of the GV, which than is called germinal vesicle breakdown (GVB).
The present invention will be illustrated in detail in the following examples.
These examples are included for illustrative purposes and should not be considered to Limit the present invention.
Examples ~ 5 Example 1: Test of hypoxanthine, FF-MAS and the combination of FF-MAS and hypoxanthine in the oocyte assay Material and Methods:
Naked oocytes (NO) and cumulus enclosed oocytes (CEO) were isolated from follicles from immature (C57B1l6J x DBAI2J)Fi mice (age 21-24 days), that had received 10 LU. PMSG i.p. 48h prior to collection. The oocytes were cultured in 4-well multidishes in a modified a-MEM medium containing 1 mg fetuinlmi culture medium. Each well contained 0.4 ml of the oocyte culture medium and 35-45 oocytes. The control and test cultures were made with different concentrations of the compounds to be tested as indicated in the tables.
The cultures were kept at 37°C and 100% humidity with 5% C02 in the air for 18 hours.
Oocytes arrested in meiosis are characterised by an intact nucleus with a prominent nucleolus, known as germinal vesicle (GV). Upon reinitiation of meiosis the nucleolus and the nuclear envelope disappear and this is characterised by a breakdown of the GV, which than is called germinal vesicle breakdown (GVB).
Results:
Table 1: Activation of meiosis in oocytes using FF-MAS, low dose hypoxanthine and the combination of FF-MAS + hypoxanthine Compounds Oocyte n Oocytes GVB (%) type Control (no compounds NO 187 g5,1 added) 0.4mM Hx NO 19C 95.8 a' 3mM Hx NO 170 12.4 10NM FF-MAS + 0.4mM Hx NO 172 98.3 10NM FF-MAS + 3mM Hx NO 223 92.3 Hx = Hypoxanthine NO = naked oocytes GVB = germinal vesicle breakdown n = number of The hypoxanthine used was obtained from Sigma, Deisenhofen, Deutschland.
Similar to the control, low dose hypoxanthine (0.4mM) is able to lead to meiotic maturation in most oocytes. However, 3mM hypoxanthine nearly completely prevents meiotic maturation. It is evident that FF-MAS not only when given together with a meiosis - inhibiting dose of hypoxanthine (3mM), but also when given together with low dose hypoxanthine (0.4mM) is able to lead to meiotic maturation in nearly all oocytes.
Example 2: Test of hypoxanthine, FF-MAS and the combination of FF-MAS and hypoxanthine in the in-vitro fertilization (IVF) assay Material and Methods:
NO and CEO from immature mice (C57B1/6J x DBAJ/2)F, were isolated and cultured under the same conditions as described for the oocyte assay. After 18 hours oocytes that exhibited germinal vesicle breakdown (GVB) were shortly washed in hypoxanthine-free. medium and transferred to the insemination dishes prepared in advance, which consisted of a motile sperm preparation from the caudal epididymis of male mice. The dishes were then incubated under defined gas conditions (5%
C021 at 37°C in a modified a-MEM IVF-medium. Neither the insemination ~-medium nor the IVF-medium contained hypoxanthine. Examination of the oocytes were carried out 20-22 hours after insemination, in order to check fertilization and to record the number of 2-cell embryos. The percentage fertilization (= fertilization rate) was determined from counts of oocytes that had cleaved into two-cell embryos.
s Results:
Table 2: In vitro fertilization rate (IVF-rate) of oocytes cultured with FF-MAS, low dose hypoxanthine and the combination of FF-MAS + hypoxanthine Compounds Oocyte n / GVB oocytes Two-cell embryos type used after in (%) vitro culture Control (no compound NO 177 34.1 added) u.5 ~ i-ix NO 182 522 3mM Hx NO 21 0 10NM FF-MAS + 0.4mM Hx NO 167 64.7 10NM FF-MAS + 3mM Hx NO 102 51.0 Hx = hypoxanthine NO = naked oocytes n = number of GVB = germinal vesicle breakdown The hypoxanthine used was obtained from Sigma, Deisenhofen, Deutschland.
Oocytes cultured in the presence of 3mM hypoxanthine were unable to get fertilized.
0.4mM hypoxanthine and 10NM FF-MAS + 3 mM hypoxanthine increased the fertilization rate of the control group by about 50%. However, the combination of 10uM FF-MAS + 0.4 mM hypoxarlthine could nearly double the control IVF-rate.
Table 1: Activation of meiosis in oocytes using FF-MAS, low dose hypoxanthine and the combination of FF-MAS + hypoxanthine Compounds Oocyte n Oocytes GVB (%) type Control (no compounds NO 187 g5,1 added) 0.4mM Hx NO 19C 95.8 a' 3mM Hx NO 170 12.4 10NM FF-MAS + 0.4mM Hx NO 172 98.3 10NM FF-MAS + 3mM Hx NO 223 92.3 Hx = Hypoxanthine NO = naked oocytes GVB = germinal vesicle breakdown n = number of The hypoxanthine used was obtained from Sigma, Deisenhofen, Deutschland.
Similar to the control, low dose hypoxanthine (0.4mM) is able to lead to meiotic maturation in most oocytes. However, 3mM hypoxanthine nearly completely prevents meiotic maturation. It is evident that FF-MAS not only when given together with a meiosis - inhibiting dose of hypoxanthine (3mM), but also when given together with low dose hypoxanthine (0.4mM) is able to lead to meiotic maturation in nearly all oocytes.
Example 2: Test of hypoxanthine, FF-MAS and the combination of FF-MAS and hypoxanthine in the in-vitro fertilization (IVF) assay Material and Methods:
NO and CEO from immature mice (C57B1/6J x DBAJ/2)F, were isolated and cultured under the same conditions as described for the oocyte assay. After 18 hours oocytes that exhibited germinal vesicle breakdown (GVB) were shortly washed in hypoxanthine-free. medium and transferred to the insemination dishes prepared in advance, which consisted of a motile sperm preparation from the caudal epididymis of male mice. The dishes were then incubated under defined gas conditions (5%
C021 at 37°C in a modified a-MEM IVF-medium. Neither the insemination ~-medium nor the IVF-medium contained hypoxanthine. Examination of the oocytes were carried out 20-22 hours after insemination, in order to check fertilization and to record the number of 2-cell embryos. The percentage fertilization (= fertilization rate) was determined from counts of oocytes that had cleaved into two-cell embryos.
s Results:
Table 2: In vitro fertilization rate (IVF-rate) of oocytes cultured with FF-MAS, low dose hypoxanthine and the combination of FF-MAS + hypoxanthine Compounds Oocyte n / GVB oocytes Two-cell embryos type used after in (%) vitro culture Control (no compound NO 177 34.1 added) u.5 ~ i-ix NO 182 522 3mM Hx NO 21 0 10NM FF-MAS + 0.4mM Hx NO 167 64.7 10NM FF-MAS + 3mM Hx NO 102 51.0 Hx = hypoxanthine NO = naked oocytes n = number of GVB = germinal vesicle breakdown The hypoxanthine used was obtained from Sigma, Deisenhofen, Deutschland.
Oocytes cultured in the presence of 3mM hypoxanthine were unable to get fertilized.
0.4mM hypoxanthine and 10NM FF-MAS + 3 mM hypoxanthine increased the fertilization rate of the control group by about 50%. However, the combination of 10uM FF-MAS + 0.4 mM hypoxarlthine could nearly double the control IVF-rate.
Claims (33)
1. A pharmaceutical composition comprising c-AMP-increasing compounds in low dose and at least one meiosis-stimulating compound for the treatment of infertility in mammals.
2. Pharmaceutical composition according to claim 1 wherein the mammal is a human.
3. Pharmaceutical composition according to one of the claims 1 or 2 wherein c-AMP-increasing compounds are purines, unspecific PDE-inhibitors, specific PDE3-inhibitors and synthetic membrane permeable cAMP.
4. Pharmaceutical composition according to one of the claims 1, 2 or 3 wherein the meiosis-stimulating compound is FF-MAS.
5. Pharmaceutical composition according to one of the claims 1-4 wherein the c-AMP-increasing compound is hypoxanthine.
6. Use of low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound as active substances for the production of a pharmaceutical composition for the treatment of infertility in mammals.
7. Use according to claim 6, wherein the mammal is a human.
8. Use according to one of the claims 6 or 7, wherein the c-AMP-increasing compounds are used alone.
9. Use according to one of the claims 6, 7 or 8, wherein the c-AMP-increasing compound is selected from the group consisting of purines, unspecific PDE-inhibitors, specific PDE3-inhibitor and synthetic membrane permeable cAMP.
10. Use according to one of the claims 6-9, wherein the c-AMP-increasing compound is hypoxanthine.
11. Use according to one of the claims 6, 7, 9 or 10 , wherein the meiosis-stimulating compound is FF-MAS.
12. Use of low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound to increase the rate of fertilization in a mammal.
13. Use according to claim 12, wherein the mammal is a human.
14. Use according to one of the claims 12 or 13 to regulate the fertilization rate in fertilization culture media.
15. Use according to one of the claims 12-14, wherein the c-AMP-increasing compounds are used alone.
16. Use according to one of the claims 12-15, wherein the c-AMP-increasing compound is selected from the group consisting of purines, unspecific PDE-inhibitors, specific PDE3-inhibitors and synthetic membrane permeable cAMP.
17. Use according to one of the claims 12-16, wherein the c-AMP-increasing compound is hypoxanthine.
18. Use according to one of the claims 12-14 or 16-17, wherein the meiosis-stimulating compound is FF-MAS.
19. Use of a low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound for the administration to a germ cell.
20. Use according to claim 19, wherein the germ cell is an oocyte.
21. Use according to claim 19, wherein the germ cell is a spermatozoon.
22. Use according to one of the claims 19, 20 or 21 , wherein c-AMP-increasing compounds are used alone.
23. Use according to one of the claims 19-22, wherein the c-AMP-increasing compound is selected from the group consisting of purines, unspecific PDE-inhibitors, specific PDE3-inhibitors and synthetic membrane permeable cAMP.
24. Use according to one of the claims 19-23, wherein the c-AMP-increasing compound is hypoxanthine.
25. Use according to one of the claims 19-21 or 23-24 wherein the meiosis-stimulating compound is FF-MAS.
26. A method of stimulating meiosis in a mammalian germ cell comprising administering ex vivo or in vivo or in vitro to a gems cell in need of such a stimulation an effective amount of low dose c-AMP-increasing compounds alone or in combination with at least one meiosis-stimulating compound.
27. A method according to claim 26, wherein the germ cell is an oocyte
28. A method according to claim 26, wherein the germ cell is a spermatozoon.
29. A method according to one of the claims 26-28, wherein c-AMP-increasing compounds are used atone.
30. A method according to one of the claims 28-31, wherein c-AMP-increasing compound is selected from the group consisting of purines, unspecific PDE-inhibitors, specific PDE3-inhibitors and synthetic membrane permeable CAMP.
31. A method according to one of the claims 26-30 wherein the c-AMP-increasing compound is hypoxanthine.
32. A method according to one of the claims 26-28, 30 or 31, wherein the meiosis-stimulating compound is FF-MAS.
33. A pharmaceutical kit comprising a dosage unit for a c-AMP-increasing compound in low dose and a dosage unit of at least one meiosis-stimulating compound.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP98250177 | 1998-05-26 | ||
| EP98250177.7 | 1998-05-26 | ||
| PCT/EP1999/003138 WO1999061010A2 (en) | 1998-05-26 | 1999-05-07 | Treatment of infertility with cam-increasing compounds alone or in combination with at least one meiosis-stimulating compound |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2333320A1 true CA2333320A1 (en) | 1999-12-02 |
Family
ID=8234594
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002333320A Abandoned CA2333320A1 (en) | 1998-05-26 | 1999-05-07 | Treatment of infertility with camp-increasing compounds alone or in combination with at least one meiosis-stimulating compound |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1098652A2 (en) |
| JP (1) | JP2002516272A (en) |
| KR (1) | KR20010043828A (en) |
| AU (1) | AU5408999A (en) |
| CA (1) | CA2333320A1 (en) |
| NO (1) | NO20005954L (en) |
| WO (1) | WO1999061010A2 (en) |
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|---|---|---|---|---|
| JP2003509365A (en) | 1999-09-16 | 2003-03-11 | ノボ ノルディスク アクティーゼルスカブ | Composition for IVF |
| ES2284542T3 (en) * | 1999-11-25 | 2007-11-16 | Novo Nordisk A/S | TREATMENT OF HUMAN INFERTILITY. |
| US6544166B1 (en) | 1999-11-25 | 2003-04-08 | Groendahl Christian | Treatment of human infertility |
| WO2001062260A2 (en) * | 2000-02-25 | 2001-08-30 | Schering Aktiengesellschaft | Improvement of implantation rate |
| WO2001062258A2 (en) * | 2000-02-25 | 2001-08-30 | Schering Aktiengesellschaft | Improvement of implantation rate using ff-mas |
| AU2001248282A1 (en) * | 2000-04-06 | 2001-10-23 | Novo-Nordisk A/S | Synchronization of the cytoplasmatic and the nuclear maturation of oocytes in vitro |
| EP1147774A1 (en) * | 2000-04-20 | 2001-10-24 | Stichting Dienst Landbouwkundig Onderzoek | Method for improving the quality of sperm for artificial insemination of animals |
| AU2001263924A1 (en) * | 2000-05-18 | 2001-11-26 | Novo-Nordisk A/S | Fertilization of aged oocytes |
| EP1245572A1 (en) | 2001-03-26 | 2002-10-02 | Schering Aktiengesellschaft | Aminosterol compounds, their use in the preparation of meiosis-regulating medicaments and method for their preparation |
| WO2003051344A1 (en) * | 2001-12-14 | 2003-06-26 | Applied Research Systems Ars Holding N.V. | Methods of inducing ovulation_using a non-polypeptide camp level modulator |
| AU2003203145A1 (en) * | 2002-02-22 | 2003-09-09 | Novo Nordisk A/S | A transducer of mas signalling |
| US9624278B2 (en) | 2011-08-30 | 2017-04-18 | Centre National De La Recherche Scientifique | Protein complex comprising MCM8 and MCM9 proteins and their use |
| GB201117453D0 (en) * | 2011-10-10 | 2011-11-23 | Univ Dundee | Improved sperm function/activity |
| CN108531447B (en) * | 2018-04-13 | 2021-11-23 | 上海市生物医药技术研究院 | Compound for regulating sperm motility and assisted reproduction and application thereof |
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|---|---|---|---|---|
| EP0196530A3 (en) * | 1985-03-26 | 1989-03-22 | CTA Finanz AG | Agent and process for increasing growth, for optimizing fertility and for stimulating the immune system in humans and animals |
| US5716777A (en) * | 1994-06-23 | 1998-02-10 | Novo Nordisk A/S | Regulation of meiosis using sterols |
| WO1996027658A1 (en) * | 1995-03-06 | 1996-09-12 | Novo Nordisk A/S | Stimulation of meiosis |
| US5800625A (en) * | 1996-07-26 | 1998-09-01 | Cauldron Limited Partnership | Removal of material by radiation applied at an oblique angle |
| CA2199663C (en) * | 1997-03-11 | 2004-08-10 | Ruth Miriam Moses | In vitro maturation and fertilization of mammalian oocytes |
-
1999
- 1999-05-07 JP JP2000550470A patent/JP2002516272A/en active Pending
- 1999-05-07 WO PCT/EP1999/003138 patent/WO1999061010A2/en not_active Application Discontinuation
- 1999-05-07 KR KR1020007013278A patent/KR20010043828A/en not_active Application Discontinuation
- 1999-05-07 CA CA002333320A patent/CA2333320A1/en not_active Abandoned
- 1999-05-07 AU AU54089/99A patent/AU5408999A/en not_active Abandoned
- 1999-05-07 EP EP99939972A patent/EP1098652A2/en not_active Withdrawn
-
2000
- 2000-11-24 NO NO20005954A patent/NO20005954L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU5408999A (en) | 1999-12-13 |
| NO20005954D0 (en) | 2000-11-24 |
| KR20010043828A (en) | 2001-05-25 |
| EP1098652A2 (en) | 2001-05-16 |
| WO1999061010A3 (en) | 2000-04-27 |
| NO20005954L (en) | 2000-11-24 |
| WO1999061010A2 (en) | 1999-12-02 |
| JP2002516272A (en) | 2002-06-04 |
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