Composition for fertilization in vitro

FIELD: medicine.

SUBSTANCE: the present innovation deals with composition for fertilization in vitro and the system for its delivering (device). The composition suggested contains steroid at the quantity of below 5% (weight/weight), that is: 4.4-dimethyl-5α-cholesta-8.14.24-trien-3β-ol, hemisuccinate of 4.4-dimethyl-5α-cholest-8.14.24-trien-3β-ol; 5α-cholest-8.14-dien-3β-ol; hemisuccinate of 5α-cholest-8.14-dien-3β-ol; (20S)-cholest-5-en-3, 20-diol; N-(methionine)amide of 3β-hydroxy-4.4-dimethyl-5α-chol-8.14-dien-24-oic acid or cholest-5-en-16β-ol, and, also, additive (water-soluble protein or phosphoglyceride). Delivering system has got either one foramen or one cavity that contains the composition mentioned as a solid product or solution. The composition of sterols contains no constituents negatively affecting oocytes and could be dissolved in aqueous medium without physical impact (that is, heating, mixing or ultrasound treatment).

EFFECT: higher efficiency of fertilization in vitro.

8 cl, 5 ex, 3 tbl

 

The present invention relates to a solid product that can be used in connection with in vitro fertilization.

Prerequisites to the creation of inventions

There are several substances that trigger meiosis (in this text, these substances are identified as MAS). When MAS are in the medium containing the oocytes, they become more capable of fertilization. However, the main problem associated with the use of MAS, is that they have very low solubility.

A brief description of the invention

One of the objectives of this invention to provide compositions containing the MAS or their derivatives, which can dissolve in water.

Another objective of the invention to provide compositions containing the MAS or their derivatives, which can be dissolved in water without any physical effects, such as heating, stirring or sonication.

Detailed description of the present invention

The preferred solubility MAS, i.e. FF-MAS, in water is very low, i.e. approximately 20 PG/ml (respectively 2×10-5µg/ml), in ethanol solubility is much higher, i.e. approximately 4 mg/ml According to preliminary studies, the highest solubility of FF-MAS in a mixture of ethanol and water (1:2,5) is arr is siteline 0.4 mg/ml Several other MAS have such a low solubility in water.

Unexpectedly it was found that the solid composition containing the MAS and an additive, soluble in water. Additives are components, adding them to the MAS leads to such compositions that can be used to prepare an aqueous solution containing MAS.

Examples of additives are water-soluble proteins, such as serum albumin, such as serum albumin human (denoted in the text as HSA), not necessarily in recombinant form, the enzymes and phosphoglyceride, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol.

Preferably, when the composition according to the present invention have a water content below 10%, preferably below 5%, more preferably below 1% (wt./wt.).

Preferably, when the composition according to the present invention have a content of organic solvent is less than 10%, preferably below 5%, more preferably below 1% (wt./wt.).

Preferably, when the composition according to the present invention have a content of MAS below 1%, preferably below 0.1%, more preferably below 0.05% (wt./wt.).

Preferably, when the composition according to the present invention have a content of the additive is higher than 99%, more is preferably higher than 99,9%.

Preferred compositions according to the present invention are those which can be processed in an aqueous medium containing no organic solvent or containing only low concentrations, which leads to the solution containing the MAS. Preferably, when this water contains less than 1%, preferably less than 0.5%, more preferably less than 0.1% of an organic solvent (wt./wt.).

Taken earlier attempts to prepare compositions that meet the specified requirement, failed.

In this text the term "substances which activate meiosis" (MAS), means compounds that indirectly cause meiosis of oocytes. More precisely, MAS are the compounds in the test which in the example 1 described below, the collapse of the germinal vesicle (herein designated as GVB), expressed in percent, was significantly higher decay in control. Preferred MAS are those substances which contribute GVB, constituting at least 50%, preferably at least 80%. Examples MAS preferred are 4,4-dimethyl-5α-cholesta-8,14,24-triene-3β-ol (herein denoted as FF-MAS); hemisuccinate 4,4-dimethyl-5α-cholesterol-8,14,24-triene-3β-ol; 5α-cholesterol-8,14-Dien-3β-ol; hemisuccinate 5α-cholesterol-8,14-Dien-3β -ol; (20S)-cholesterol-5-ene-3β,20-diol; N-(methionine) amide 3β-hydroxy-4,4-dimethyl-5αhole-8,14-Dien-24-OIC acid and cholesterol-5-ene-16β-ol. The following examples MAS mentioned in WO 96/00235, 96/27658, 97/00884, 98/28323, 98/54965 and 98/55498, more precisely, in paragraph 1 of the claims.

One method of preparation of compositions according to this invention is that MAS mix a solution in an organic solvent, such as ethanol, with an aqueous solution of the additive and then leave the mixture as long as the solvent does not evaporate. The evaporation can be accelerated by application of a constant airflow over the product by vacuum or any other possible ways to remove the solvent. The product can be a delivery system having one recess or a cavity or more. These in-depth cavity are mutually designated by the cavities. At least one of these cavities contains a composition according to the present invention. A suitable way to conclude a solid composition in the cavity includes first making a solution containing MAS and additive in the cavity and then evaporation of the solution. In this way the residue after evaporation, i.e. the composition according to the present invention, is located directly in the cavity of the specified device (delivery system).

Since the composition according to the SNO present invention is to be used for processing of oocytes an important factor is that the composition contains no parts which may adversely affect the oocyte.

One of the ways of application of the compositions according to the present invention consists in the dissolution of the composition in aqueous medium, such as water, and then, if desirable, the addition of component parts, which can have a beneficial effect on oocyte maturation.

Another method of application of the composition is dissolved in an environment that is typically used for maturation in vitro.

The present invention is illustrated by the following examples, which, however, should not be construed as limiting the scope of protection of the invention. Properties disclosed in the description and in the examples, in any combination, can be used for carrying out the invention in its various types.

Example 1

A method of determining whether a substance MAS or not

Oocytes were obtained from immature female mice (C 57 BL/6J × DBA/2J Fl, Bomholtgaard, Denmark) weighing 13-16 grams, which were kept at controlled temperature (20-22° (C)lighting (light from 06.00 to 18.00) and relative humidity (50-70%). Mice were injected intraperitoneally with 0.2 ml of gonadotropins (Gonal-F, Serono)containing 20 international units (IU) of follicle stimulating hormone (FSH), and after 48 hours the animals were killed by way of the shift of the cervical vertebrae. The ovaries were removed and oocytes were isolated in the NC environment (see below) under a stereo microscope by manual rupture of the follicle, using a pair of needle size 27. Spherical oocytes detecting intact germinal vesicle (in the text denoted as GV), were divided into oocytes surrounded ovarian tubercle (in this text labeled as SEO), and naked oocytes (in this text are marked as NO) and placed in α-minimum essential support environment (α-MEM without ribonucleosides, Gibco BRL, Cat. No. 22561), supplemented with 3 mg/ml serum albumin bovine (BSA, Sigma Cat. No. A-7030), 5 mg/ml serum albumin human (HSA, State Serum Institute, Denmark), 0.23 mmol pyruvate (Sigma, Cat. No. S-8636), 2 mmol glutamine (Flow Cat. No. 16-801), 100 IU/ml penicillin and 100 µg/ml streptomycin (Flow, Cat No. 16-700). This medium was supplemented with 3 mmol gipoksantina (Sigma, Cat. No. H-9377) and was designated as NC-environment.

Oocytes were washed three times in NC environment and uniform size of the oocytes were divided into groups of CEOS and NO. SEO and NO cultured in 4-hole plates (Nunclon, Denmark)in which each well contained 0.4 ml NC-environment and the test compound at a concentration of 10 Microm. One control well (i.e. 35-45 oocytes were cultured in the same medium without addition of the test compound) is always contained in the culture conditions simultaneously with 3 test holes (35-45 oocytes per well with relax the authorized test connection).

Oocytes were cultured in a humidified atmosphere of air containing 5% CO2during 24 hours at 37°C. At the end of the cultivation period, the number of oocytes with GV, GVB and polar cells (in the text is given as PB), respectively, were counted using a stereomicroscope (Wildt, Leica MZ 12). The percentage of GVB, indicated as a percentage of oocytes that survived GVB, the total number of oocytes in this hole, was calculated as follows:

%GVB=((number of GVB + number RV)/total number of oocytes) × 100.

Example 2

The method of determining whether to use the connection as an additive in the compositions according to the present invention or not

Additives for compositions containing FF-MAS, characterized by the fact that:

improve the solubility of FF-MAS in a mixture of ethanol/water (1:2.5 to about./vol.);

contribute to the fact that the solution of FF-MAS remains transparent after recreating the song α-MEM medium.

The resulting research level GVB amounted to at least 50%, preferably 80%, the test compound on the oocytes obtained from immature female mice.

Prepared saturated solution of FF-MAS in ethanol. Mixed with the aqueous solution of the additive in the ratio of 1:2,5. By visual inspection was controlled so that the solution contained an excessive amount of FF-MAS. They mixed the solution for 24 hours at room temperature the re. Filtered the solution through a filter with pore size 0.22 μm, determined the content of FF-MAS by HPLC and calculated solubility. Bore 350 μl of a solution in a 4-well plate and left to evaporate to dryness at room temperature. Added 500 μl of alpha-MEM medium (Gibcobal). If within half an hour had formed a clear solution, the composition was tested on oocytes obtained from nedorazvitia female mice. The resulting research level GVB was at least 50%, preferably 80%, see example 1.

Example 3

A composition comprising a serum albumin human (HSA)

In this example were obtained three products. As can be seen from the table below, the basic solution of FF-MAS was used for product 1, 2 and 3, containing 50, 500 and 3330 μg/ml, respectively. For each product the basic HSA solution contained 20% HSA. The number of these used the basic solutions are presented in the table. For example, for product 1 the General solution of FF-MAS in the amount of 400 μl was mixed with 1000 ál of the basic solution of HSA. After mixing these basic solutions the solutions were transparent and were not observed precipitation. After mixing, the number of solutions indicated in the table, was transferred to a 4-hole plates (Nuclon, Denmark). For example, for product 1 mixture in an amount of 350 µl of the bore is in the tablet. Finally, the solution evaporated to dryness at room temperature. After evaporation of some of the products formed opalescense transparencies in tablets, other items were for the human eye. The highest concentration of FF-MAS dissolved in this example, was 0.95 mg/ml

Before using 500 μl of alpha-MEM medium (Gibcobal) was added and obtained clear solution of FF-MAS and HSA within half an hour at room temperature.

4-well plate No. 14-well plate No. 24-well plate No. 3
A solution of FF-MAS in ethanol, 50 mg/ml400 ál--
A solution of FF-MAS in ethanol, 500 mg/ml-400 ál-
A solution of FF-MAS in ethanol, to 3.33 mg/ml--450 ál
The HSA solution in water, 20%1000 ál1000 ál1125 μl
The amount transferred to tablet350 ál350 ál525 ál
The ratio of FF-MAS and HSA1:100001:10001:150
The appearance of the solution prior to evaporationprozrachnye colorless solution without precipitate

Example 4

Compositions containing serum albumin human (HSA)

As described in the previous example, the way the solutions of FF-MAS in a mixture of water/ethanol containing HSA were obtained in the following concentrations by simple mixing at room temperature. After preparation, the solutions were transparent and the precipitate was not observed. The solutions were transferred into 4-well plates (Nuclon, Denmark). Finally, the solution evaporated to dryness at room temperature.

Before using 500 μl of alpha-MEM medium (Gibcobal) was added and within half an hour at room temperature was obtained a clear solution of FF-MAS and HSA.

The compositions were tested on oocytes obtained from immature female mice. Levels GVB, expressed in percent, for the respective compositions indicated in the table below.

4-well plate No. 14-well plate No. 24-well plate No. 3
A solution of FF-MAS in ethanol, 5,22 mg/ml100 µl--
A solution of FF-MAS in ethanol, and 26.1 mg/ml-100 µl-
A solution of FF-MAS in ethanol, 261 mg/ml--100 µl
The HSA solution in water,20% 250 ál250 ál250 ál
The ratio of FF-MAS and HSA1:100001:20001:200
theoretical amount of FF - MAS on well0.5 mg2, 5 mcg25 mcg
% GVB729391

Example 5

Compositions containing serum albumin human (HSA)

As described in the previous example, the way the solutions of FF-MAS in a mixture of water/ethanol containing HSA were obtained in the following concentrations by simple mixing at room temperature. After preparation, the solutions were transparent and the precipitate was not observed. The solutions were transferred into 4-well plates (Nuclon, Denmark). Finally, the solution evaporated to dryness at room temperature.

Before using 500 μl of alpha-MEM medium (Gibcoba-l) was added and within half an hour at room temperature was obtained a clear solution of FF-MAS and HSA.

The concentration of FF-MAS after reconstruction was determined by HPLC, the results indicated in the table below.

1. Composition, activating the meiosis of oocytes for fertilization in vitro, contains a steroid and an additive, characterized in that the activating meiosis steroids are contained in the number menee% (wt./wt.) and represent 4,4-dimethyl-5α -cholesta-8,14,24-triene-3β-ol, hemisuccinate 4,4-dimethyl-5α-cholesterol-8,14,24-triene-3β-ol; 5α-cholesterol-8,14-Dien-3β-ol; hemisuccinate 5α-cholesterol-8,14-Dien-3βOla; (2OS)-cholesterol-5-ene-3β,20-diol; N-(methionine)amide 3β-hydroxy-4,4-dimethyl-5αhole-8,14-Dien-24-OIC acid or cholesterol-5-ene-16β-ol, and the additive is a water-soluble protein or phosphoglyceride.

2. The composition according to claim 1, wherein the steroid is 4,4-dimethyl-5A-cholesta-8,14,24-triene-3β-ol.

3. The composition according to claim 1 or 2, characterized in that it can be used to prepare aqueous solution.

4. The composition according to claim 1, 2 or 3, characterized in that it is an aqueous solution in which the content of the steroid is at least about 0.001 μg/ml, preferably at least 0,01 μg/ml, more preferably at least 0.1 mg/ml, most preferably at least 0.5 μg/ml

5. Composition according to claims 1, 2, 3 or 4, characterized in that the content of the steroid in aqueous solution is not more than 0.1 g/ml, preferably not more than 0.01 g/ml.

6. Composition according to any one of the preceding paragraphs, characterized in that it further contains an organic solvent in a quantity of less than 0.1%, preferably less than 0.05%, most preferred less than 0.01%.

7. Composition according to any one of the preceding pun is tov, characterized in that the additive is serum albumin human, not necessarily in recombinant form.

8. The device, which is a delivery system having one recess or one cavity containing composition in the form of solid or solution according to any one of the preceding paragraphs.



 

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