A method of obtaining a composite product containing nucleic acid

 

The way to obtain a product containing a complex of a cationic carrier with a nucleic acid polymer, is that two single-stranded polymer of nucleic acids, capable of at least partially, to form a double-stranded structure, added separately, each in a separate circuit, or cationic carrier or to the material from which to form a cationic carrier, and all these ingredients are subjected to dispersion processing. Effect: method provides a homogeneous composite of the preparation of good quality, containing nucleic acid, which can be sterilized by filtration, and is distinguished by the absence of large particles with a size of 7 μm or more, which are considered unsafe for humans. 3 S. and 7 C.p. f-crystals, 1 tab., 2 Il.

Field of invention the Present invention relates to complex preparations containing cationic complex media and polymers of nucleic acids. Complex sometimes referred to as lipoplexes.

As used herein, the term "cationic carrier" refers to drug carriers having positive charges in the water, which awsumest. Recently, cationic carriers have been widely studied as drug delivery vehicles for transferring genes into cells and RNA such as poly CR>

The expression "plastics nucleic acid" refers to natural or synthetic or synthetically derived from substances of natural origin, polynucleotides (DNA, RNA) and natural or synthetic or synthetically derived from substances of natural origin oligonucleotides.

The prior art Contains nucleic acid complex drugs, which include cationic complex media and polyanionic double chain polymer of nucleic acids having a double helical structure can be obtained only by mixing the cationic carrier and a polymer of nucleic acids with a double chain.

However, in obtained in this way complex preparations containing nucleic acid particles of the drugs are typically large, with a diameter of several micrometers to several hundred micrometers and heterogeneous. Containing nucleic acid complex drugs with such large and heterogeneous particles are uncomfortable in relation to the lack of homogeneous data is the definition of the problem, on large particle sizes are difficulties in the implementation of sterilization on an industrial scale and that the injection needle and the capillaries in the case of intravenous administration may cause embolization and the like, despite the fact that pharmaceutical products derived under the assumption that they are safe when administered to humans. Such problems difficult to be solved not only by using methods, which are complex drugs only by mixing, as described above, but also using the ways to obtain that use the dispersion process using suitable emulsifying dispersion devices.

Aggregation of particles is also a problem that occurs when the freeze-drying process undertaken to stabilize the complex preparations.

It is desirable that the polymers of nucleic acids in complex products had a high concentration to reduce the dosage and reduce stress for patients and health workers in the implementation process, and to achieve productive efficiency of the complex products. However, conventional methods of obtaining, when the total amount of polymer Amu in terms of the method of obtaining the product and produced huge sediments or suspended solids, easily visible to the naked eye. They may not be dispersed sufficiently by using a method of dispersing.

It is generally accepted that double-stranded RNA having a double helical structure, is commonly used as gene and RNA such as poly I:C, from the point of view of their physiological characteristics and stability for various nucleases. For example, it is known that sufficient pharmacological efficacy is not achieved by the separate introduction of poly I and poly C instead of poly I:C, which has a physiological activity, such as a strong induction of the activity of interferon and immunopotency action (Archives of Virology, 51, 199-215 (1976)). Thus, I believe that the double chain having a double helical structure, are essential for gene and RNA such as poly CR>

For containing nucleic acid complex products, which include cationic complex media and polymers of nucleic acids, the necessity of a double helical structures were not discussed, and usually in ways of producing complex products use double-stranded DNA and double-stranded RNA having a double helical structure.

The present applicants filed sakurada drugs in cancer cells, because of the complex preparations containing nucleic acid consisting of cationic carriers and double chains of RNA, such as poly I: C, activate nucleases in cancer cells, which is effective in the treatment of cancer, and they filed a patent application relating to complex preparations containing nucleic acids as pharmaceutical agents for HCV, as these drugs for an extended period of time induce an effective amount of interferon that are specific to the liver and spleen (PCT/JP98/04695, PCT/JP99/01438).

Description of the invention an Object of the present invention is, in particular, how to obtain a homogeneous complex products of good quality, containing a nucleic acid, characterized in that these preparations it is possible to obtain the so-called sterilization by filtration, and they do not contain coarse particles with a size of 7 μm or more, which are considered unsafe for humans.

The authors present invention first found that the above problems can be solved by a single achievement, without affecting the pharmacological activity of drugs by obtaining, using polymers and nucleic without double chain structure, without the use of the double DNA chain or double chain RNA, usually having a double helical structure, in the production method of containing maleinovyi acid complex products that contain cationic carrier and polymers of nucleic acids, and thus have accomplished the present invention.

Therefore, the present invention includes a method of obtaining containing nucleic acid complex preparations, characterized in that the two polymer single-stranded nucleic acid, which can, at least partially, to form double chains, added separately in the form of a single chain to a cationic carrier or to the source material before the formation of the cationic carrier, and two polymer-stranded nucleic acid is subjected to a dispersion treatment in the production method of the specified complex preparation containing nucleic acid which comprises a cationic carrier and polymers of nucleic acids (hereinafter referred to as "containing nucleic acid complex drugs").

Hereinafter the invention will be described in detail.

"Cationic carriers" that can be used in the present invention may include native medicinal EE as compound A), represented by the following structural formula [I], and drug delivery vehicles, formed by phospholipids as mandatory components, and drug delivery vehicles, such as polylysine, in addition to commercially available lipofectin (trade name), lipofectamine (trade name), lipofectin (trade name) and DMRIE-C (trade name).

"Two polymer single-stranded nucleic acids", suitable for use in the present invention, may include, but are not limited to specific them, if they represent two single-stranded polymer of nucleic acids, which can at least partly form double chains, for example, two single-stranded DNA and RNA, which are natural genes or artificially modified genes (e.g., plasmid) and two single-stranded RNA such as poly I and poly C, poly I and poly C12U, poly I with partial chemical modification (for example, poly (7-easeinsine acid) and poly C, poly I and poly C With partial chemical modification (for example, poly(branitelja acid), poly (theoritically acid)). In the present invention it is possible to use two separate chains Rinoa inducing action of interferons. Used in this description, the terms "poly I, poly C, poly A, poly U and poly C12U mean polyinosinic acid, polycytidylic acid, polyadenylic acid, prioritylevel acid and the copolymer of cititravel acid and pridelava acid, where one pridelava acid replaces approximately every 12 Cotidianul acid, respectively.

The expression "can, at least partially, to form double chain" refers to the fact that the complementary bases exist in two-stranded polymers of nucleic acids, are such an extent that they can form double chains under physiological conditions, and the degree varies depending on the types of polymers-stranded nucleic acids and the length of each polymer of nucleic acids, and specific commonality lies in the fact that the number of complementary bases is 20 or more.

An appropriate number of reasons, which contains each polymer single-stranded nucleic acid and which is not specifically limited, is 10,000 or less, preferably 2000 or less. The number of bases may be selected appropriately depending on the nature, which has each a polymer of nucleic acid to the number of bases. Each nucleic acid polymer usually has a certain distribution, composed of different numbers of bases, but each number of bases refers to the number of grounds for maximum distribution and is referred to in this description as "the average number of grounds."

Further, for example, the average number of bases of poly I and poly C in the present invention can be determined on the basis of the balance between efficiency and security. In particular, a suitable range is from 30 to 3000 bases, preferably in the range from 60 to 2000 bases, and more preferably range from 100 to 500 bases.

Phospholipids in drug delivery vehicles (cationic carriers) formed by the above-mentioned compound a and phospholipids as mandatory components that are not restricted if they are pharmaceutically acceptable. For example, they include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin and lecithin. It is also possible to use hydrogenated phospholipids. Preferred phospholipids can include phosphatidylcholine from egg yolk, egg yolk lecithin, soybean lecithin and phosphated egg yolk. You can use two or more types of postupniy with phosphatidylethanolamine, usually used in cationic carriers.

The ratio of the components of phospholipids to the connection And varies depending on the form of phospholipids and of the two polymers used single-stranded nucleic acids, however, the number of phospholipids, respectively, ranges from 0.1 to 10 weight parts, preferably in the range of from 0.5 to 5 weight parts and more preferably in the range from 1 to 2 weight parts per 1 weight part of the connection A. This ratio is the same when the phospholipid replace lecithin.

The mixing ratio of the polymers of nucleic acids to cationic carrier varies depending on the source of the cationic carrier and the polymer of nucleic acids, however, the total number of polymers of nucleic acids, respectively, is in the range from 0.05 to 10 weight parts, preferably in the range from 0.1 to 4 weight parts and more preferably in the range from 0.5 to 2 weight parts to 10 weight parts of a cationic carrier. Similarly, when the complex is formed of poly I and poly With together with cationic carrier obtained from the compounds a and phospholipids as a prerequisite, provided that two polymer one is respectively in the range from 0.05 to 10 weight parts, preferably in the range from 0.1 to 4 weight parts and more preferably in the range from 0.5 to 2 weight parts to 10 weight parts of a cationic carrier.

Containing nucleic acid complex preparations of the present invention (hereinafter called "the preparations of the present invention") can be obtained, for example, a dispersion method using conventional methods using suitable emulsifying dispersing device, after adding the two polymers, single-stranded nucleic acid sequence or simultaneously to the aqueous solution, which is dispersed commercially available cationic carrier or aqueous solution, in which the cationic carrier is produced by dispersion method using a conventional method of processing an aqueous solution in which is dispersed raw materials before receiving cationic carrier using a suitable emulsifying dispersing device, or to aqueous solution, in which dispersed the source material before the formation of the cationic carrier. The preparations of the present invention can also be obtained by methods in which two separate chains of polymers of nucleic acids is added to solid is tamago emulsifying dispersing device. The sequence is added, the added amounts of added concentration and the concentration of cationic carriers and their source materials in the solutions chosen arbitrarily, and they are not specifically limited in the present invention.

More specifically, when using a cationic carrier obtained from the compounds a and phospholipids as a mandatory component, the drug of the present invention may be emulsifying dispersion treatment of the mixture formed by gradually adding dropwise the aqueous solutions of poly I and poly With separately in aqueous solution, in which the dispersed cationic carrier. Also the drug of the present invention can be obtained by a method in which the compound a, a phospholipid, poly I and poly With weighed and loaded into a chemical glass and roughly dispersed after adding water by using a homogenizer, and then dispersed under pressure by means of the emulsifying dispersion device.

You can use two polymer single-stranded nucleic acid obtained by the division of polymer double-stranded nucleic acid using conventional procedures. In particular, the manipulation may include non-enzymatic processing, such as Narnia media can be used without processing or using an appropriate treatment.

The above aqueous solutions can include water for injection, distilled water for injection and electrolyte solutions, such as saline, and glucose.

The above-mentioned emulsifying dispersing device may include, for example, homemaker, a homogenizer, an ultrasonic dispersing device, an ultrasonic homogenizer, emulsifying dispersion device high-pressure Microfluidizer (trade name), Nanomizer (trade name), Ultimizer (trade name), DeBEE2000 (trade name) homogenizer high pressure Manton-Gaulin type, but enough are the ones that are suitably used for medical purposes. The machining conditions, the timing and temperature of treatment is chosen appropriately.

The preparations of the present invention may contain appropriate amounts of pharmaceutically acceptable additives such as emulsifying dispersant excipient, stabilizer, isotonic agent, lipoprotein and pH regulator. In particular, they may include fatty acids containing from 6 to 22 carbon atoms (for example, Caprylic acid, capric acid, lauric acid, myristic sour docosahexaenoic acid), their pharmaceutically acceptable salts (e.g. sodium salt, potassium salt and calcium salt), emulsifying, dispersing AIDS, such as albumin and dextran, stabilizers, such as cholesterin and phosphatidic acid, isotonic agents, such as sodium chloride, glucose, maltose, lactose and sucrose, neoprotestant and pH regulators, such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide and triethanolamine.

The above optional pharmaceutically acceptable excipients can be added by suitable means before or after dispersion.

After dispersion, the drugs may not necessarily be filtered through a 0.2 μm sterilizing filter membrane, and then Packed in ampoules and vials. Particle diameter of almost all drugs of the present invention is 200 nm or less. Therefore, almost 100% of the preparations according to the present invention can pass through 0.2 μm sterilizing membrane.

Through the above-mentioned methods of obtaining, in accordance with the present invention, can be obtained containing nucleic acids can be obtained containing nucleic acid complex preparations, containing polymers of nucleic acids in solution at a concentration of 0.1 mg/ml or more.

Therefore, obtained by the above methods of preparation, containing nucleic acid complex drugs and obtained by the above methods of obtaining containing nucleic acid complex drugs, including polymers of nucleic acids in solution at a concentration in the range from 0.1 to 10 mg/ml, in the range from 0.5 to 10 mg/ml, in the range from 1 to 10 mg/ml or in the range from 2 to 10 mg/ml, may also be included in the scope of the present invention. However, the present invention does not exclude the polymers of nucleic acids with concentrations in solution 10 mg/ml or more.

In addition, if the preparations of the present invention, obtained by dispersion, as described above, is subjected to freeze-drying, they can be a liofilizovannye preparations of the present invention. Therefore, liofilizovannye drugs may also be included as one of the drugs of the present invention. Freeze-drying implement common ways.

Liofilizovannye preparations of the present invention, for example, is subjected to sublimally distribution of bubbles, followed by primary drying at 0-10oWith under reduced pressure, followed by secondary drying at 15-25oWith under reduced pressure. Usually the bubbles fill the inside with gaseous nitrogen and clog bubbles, getting liofilizovannye preparations of the present invention. In the case when the drugs were subjected to freeze-drying, it is preferable to use iprotectyou, which form liofilizovannye tortillas. Especially suitable are the saccharides and disaccharides, in particular, among the other most preferred is maltose.

Restore moisture liofilizovannyh preparations according to the invention can generally be carried out by adding an optional suitable solution (replenishing solution) with subsequent use. These restorative solutions can include water for injection, glucose, electrolyte solutions, such as saline solution and other infusion solutions. Fluid volume data recovery solutions may vary depending on the application and is not specifically limited, but are suitable amounts of from 0.5 to two times the amount of liquid before drying or 500 ml or modified means for injection or drip, or in the form liofilizovannyh drugs.

The preparations of the present invention can type animals, including humans, different routes of administration, such as, for example, intravenously, intraarterially, subcutaneously, intramuscularly, by inhalation, nasal, ophthalmologist, oral and rectal. Preparative dosage form and the dosage can be selected appropriately in accordance with the wishes. In addition, drugs may also be used as a variety of reagents and drugs for cultivated cells of animals, plants, fungi and bacteria.

The best way of carrying out the invention Hereinafter, the invention will be described in more detail using examples, comparative examples and case studies.

As initial products in examples 1-5 using the single-stranded RNA poly I and poly C, each of which has an average number of grounds of approximately 200. In addition, after dispersion in each example can be carried out sterilization by filtration without clogging of the filter during the process of sterilization by filtration using a 0.2 μm membrane filter, and the outputs of the leachate are in the range of 98 to 1 for 100%.

Example 1 Forty grams of maltose dissolved in 100 ml of water for injection was added to 2.0 g of compound a and 2.0 g of purified egg yolk lecithin, mixed by stirring, and was dispersively for 5 minutes using a homogenizer, obtaining the crude dispersion solution of cationic carrier. Crude dispersion solution further was dispersively during the hours using small laboratory emulsifying dispersion device, and then brought water for injection solution volume of 250 ml, obtaining a dispersion solution of cationic carrier. To 250 ml of this dispersion solution under stirring was added seventy-five ml of an aqueous solution containing 250 mg poly I, then added with stirring, 75 ml of an aqueous solution containing 250 ml poly C, and the mixture was additionally dispersible during the hours using small laboratory emulsifying dispersion device, followed by sterilization by filtration through a 0.2 μm membrane filter, receiving the drug of the present invention. The average diameter of the complex particles for the preparation of the present invention was 138 nm, when measured using a device to measure the dia is a mini light scattering. In addition, there were no particles with a diameter of 1 μm or more.

Subsequently, 1 ml of this drug according to the present invention were distributed in each vial and subjected to processing in liofilizovannye the drug in accordance with conventional ways. Restoring moisture content obtained liofilizirovannogo of the drug was carried out by adding 0.9 ml of water for injection. The average diameter of the complex particles in the recovered drug of the present invention was 140 nm when measured by using a device for measuring particle diameter (DLS-700, production Otsuka Electronics Inc. , then used the same instrument), using the method of dynamic light scattering. In addition, there were no particles with a diameter of 1 μm or more.

Example 2 Four kilograms of sucrose dissolved in 10 l of water for injection was added to 50 g of compound a and 30 g of purified phosphatide egg yolk, mixed by stirring, and was dispersively the mixture for 10 minutes by using a homogenizer (Manton-Gaulin high pressure, and then bringing the solution volume up to 25 l of water for injection, obtaining a dispersion solution of cationic carrier. Six liters of an aqueous solution containing 50 g of poly C was added to 20 l Yes is holding 50 g of poly I. the pH of this dispersion solution was brought to 5.5 with hydrochloric acid and the dispersion solution was additionally dispersible within 30 minutes by using a homogenizer (Manton-Gaulin high pressure, and then sterilized by filtration through 0.2 μm membrane filter, receiving the drug of the present invention. The measured mean diameter of the complex particles of the drug of the present invention was 150 nm. In addition, there were no particles with a diameter of 1 μm or more.

Subsequently, 20 ml of this drug according to the present invention were distributed in each vial and subjected to processing in liofilizovannye the drug in accordance with conventional ways. Restoring moisture content obtained liofilizirovannogo of the drug was carried out by adding a commercially available 5% infusion glucose solution (500 ml). The average diameter of the complex particles in the recovered drug of the present invention was 151 nm when measured by using a device for measuring the diameter of the particles using the method of dynamic light scattering. In addition, there were no particles with a diameter of 1 μm or more.

Example 3 Two grams of compound a, 2 g soy lecithin 25 mg poly I and 25 mg floor the contents of the beaker were mixed by stirring and dispersively for 5 minutes using a homogenizer. Crude dispersion solution was dispersively during the hours using small laboratory emulsifying dispersion device high pressure (800 kg/cm2and brought water for injection solution volume to 400 ml, and then sterilized by filtration through 0.2 μm membrane filter, receiving the drug of the present invention. The average diameter of the complex particles of this drug according to the present invention was 121 nm, when measured using a device for measuring the diameter of the particles using the method of dynamic light scattering. In addition, there were no particles with a diameter of 1 μm or more.

Example 4 Forty grams of maltose dissolved in 100 ml of water for injection was added to 1.2 g of compound a and 2.0 g of purified egg yolk lecithin, mixed by stirring, and was dispersively the mixture for 30 minutes using a small laboratory emulsifying dispersion device, high pressure, and then brought water for injection solution volume of 250 ml, obtaining a dispersion solution of cationic carrier. To 250 ml of this dispersion under stirring at the same time was gradually added dropwise seventy-five ml of an aqueous solution of codereaders using small laboratory emulsifying dispersion device with pressure (1,100 kg/cm2), followed by sterilization by filtration through a 0.2 μm membrane filter, receiving the drug of the present invention. The average diameter of the complex particles of the drug of the present invention was 124 nm, when measured using a device for measuring the diameter of the particles using the method of dynamic light scattering. In addition, there were no particles with a diameter of 1 μm or more.

In addition, when the size distribution of particle diameter for a given drug of the present invention was measured using the device for measuring the diameter of the particles (LA-910, riba Ltd., then used it), which uses the method of laser diffraction scattering were obtained the results shown in Fig.1. In accordance with these results, the peak of the size distribution of the particle diameter was located at 139 nm and larger particles were not detected.

Example 5
Forty grams of maltose dissolved in 100 ml of water for injection was added to 4.8 g of compound a and 8.0 g of purified egg yolk lecithin, mixed by stirring, was dispersively the mixture for 30 minutes using a small laboratory emulsifying dispersion device, high pressure, and then the argument of the second dispersion under stirring at the same time was gradually added dropwise seventy-five ml of an aqueous solution, containing 400 mg of poly I and 75 ml of an aqueous solution containing 400 ml poly With, and additionally they dispersible for 2 hours using a small laboratory emulsifying dispersion device high pressure (1,100 kg/cm2), followed by sterilization by filtration through a 0.2 μm membrane filter, receiving the drug of the present invention. The average diameter of the complex particles of the drug of the present invention was 138 nm, when measured using a device for measuring the diameter of the particles using the method of dynamic light scattering. In addition, there were no particles with a diameter of 1 μm or more.

Example 6
One ml of an aqueous solution containing 100 μg of a commercially available DNA plasmid vector (pMC1neo), was heated on a water bath at 70oWith stirring for 3 hours. To this solution under stirring was added to two ml of the dispersion solution containing 2 mg of commercially available lipofectin (trade name), similarly heated to 70oC, and the mixture was dispersively for 10 minutes at 70oWith using an ultrasonic dispersing device probe type, followed by sterilization by filtration is TIC in this preparation of the present invention was 145 nm, when measured with the use of the device for measuring the diameter of the particles using the method of dynamic light scattering. In addition, there were no particles with a diameter of 1 μm or more.

Example 7
As the source materials used single chain RNA poly I and poly C, the average number of bases in each of which was approximately 1500.

Forty grams of maltose dissolved in 100 ml of water for injection was added to 1.2 g of compound a and 2.0 g of purified egg yolk lecithin, mixed by stirring, and was dispersively the mixture for 30 minutes using a small laboratory emulsifying dispersion device, high pressure, and then brought water for injection solution volume of 250 ml, obtaining a dispersion solution of cationic carrier. To 250 ml of this dispersion under stirring gradually were simultaneously added dropwise seventy-five ml of an aqueous solution containing 100 mg of poly I and 75 ml of an aqueous solution containing 100 ml of poly With, and additionally they dispersible for 2 hours using a small laboratory emulsifying dispersion device with pressure (1,100 kg/cm2), followed by sterilization by filtration through 0.2 of the rata according to the present invention was 134 nm, when measured with the use of the device for measuring the diameter of the particles using the method of dynamic light scattering. In addition, there were no particles with a diameter of 1 μm or more.

Example 8
The drug of the present invention with an average diameter of particles constituting 130 nm, obtained by the method similar to that described in example 7, at a pressure dispersion 800 kg/cm2using 200 mg poly I with the average number of grounds about 350 and 200 mg poly With an average of approximately 350 bases.

Example 9
The drug of the present invention with an average diameter of particles constituting 150 nm, obtained by the method similar to that described in example 7, at a pressure dispersion 800 kg/cm2using 200 mg poly I with the average number of bases around 1450 and 200 mg poly With an average number of grounds around 1450.

Example 10
The drug of the present invention with an average diameter of particles constituting 135 nm, obtained by the method similar to that described in example 7, at a pressure dispersion 800 kg/cm2using 400 mg of poly I with the average number of grounds about 80 and 400 mg poly With an average number of grounds about 80.

Comparative example 1 (getting the usual way is,2 g of compound a and 2.0 g of purified egg yolk lecithin, mixed by stirring, and was dispersively the mixture for 30 minutes using a small laboratory emulsifying dispersion device, high pressure, and then brought water for injection solution volume of 250 ml, obtaining a dispersion solution of cationic carrier. 150 ml of an aqueous solution containing 200 mg of double-chain poly I:C, which has about 200 base pairs, was gradually added dropwise to 250 ml of the specified dispersion solution with stirring, and the mixture was additionally dispersible for 2 hours using a small laboratory emulsifying dispersion device high pressure (1,100 kg/cm2), receiving a comparative drug. The average diameter of the complex particles of this comparative drug was $ 182 nm, when measured using a device for measuring the diameter of the particles using the method of dynamic light scattering.

In addition, when the distribution of the size of the diameter of the particles in the preparation of the present invention was measured using the device for measuring the diameter of the particles, which used the method of laser diffraction scattering, in the same way as in example 4, results were obtained that show the 243 nm, however, 20% were found in the form of large particles with diameters of 3 to 20 μm, with a peak in the distribution at 8000 nm, indicating a bimodal distribution of particles.

In addition, when you try to filter this comparative drug through a 0.2 μm membrane filter, only 50 ml of the drug passed through the filter, resulting in clogging of the filter, and sterilization by filtration could not be realized.

Comparative example 2 (getting the usual manner consistent with example 5).

Forty grams of maltose dissolved in 100 ml of water for injection was added to 4.8 g of compound a and 8.0 g of purified egg yolk lecithin, mixed by stirring, was dispersively for 30 minutes using a small laboratory emulsifying dispersion device, high pressure, and then brought water for injection solution volume of 250 ml, obtaining a dispersion solution of cationic carrier. One hundred fifty ml of an aqueous solution containing 800 mg of double-chain poly I:C, which has about 200 base pairs, was gradually added dropwise to 250 ml of the specified dispersion solution with stirring, and additional dispersible for 2 hours using a small laboratory emulgel uravnitelny the product was a white, prone to selection in sediment suspension solution, and it was deposited with the aggregation within 5 minutes after collection. He was like a suspension solution of the final sludge. The diameters of the particles could not be measured because their size exceeded the measurement range of the particle diameter measuring device, using the method of dynamic light scattering. Filter sterilized through a 0.2 μm membrane filter was impossible.

Test example 1
The biological activity of the drug of the present invention obtained in example 4 and the comparative activity of the preparation of comparative example 1 were evaluated for their suppressive (inhibitory) effect on the proliferation of cancer cells of the cervix (Hela S3).

In this experiment, cells Hela S3 were sown in 96-well tablets at a concentration of 104cells/well, which was confirmed adhesion to the wells after cultivation for 5 hours or more, followed by the addition of each drug to the culture, then the cultivation was continued and after 3 days after addition of drugs by the MTT method counted the number of viable cells. Relationship of inhibition was obtained from the following formula and calculated znaczenia group cells/absorption values in the group of cells, treated with saline)X100%
The values of the IC50presented as the concentration of the total amount of the polymers of nucleic acids, obtained by combining poly I and poly C.

As shown in the table, the differences in biological activity between the drug of the present invention according to example 4 and comparative preparation according to comparative example 1 is not available.

The results of the invention
The present invention, for example, has the following results.

(1) Can be obtained homogeneous containing nucleic acid complex drugs of high quality, do not contain large complex particles.

(2) Can be obtained homogeneous containing nucleic acid complex drugs of high quality, which essentially does not contain large complex particles. This result is more remarkable at higher concentrations of polymers of nucleic acids.

(3) When restoring moisture liofilizovannyh preparations containing nucleic acid complex products obtained according to the present invention, can be restored complex drugs, equivalent to those that existed prior to sublimage to go through a 0.2 μm sterilizing filter with nearly 100% efficiency.

Brief description of drawings
In Fig.1 shows the distribution of the diameters of the complex particles in the preparation of the present invention according to example 4. The horizontal axis, the left vertical axis and the right vertical axis indicates the particle diameter (µm) frequency (%) and the integral frequency, respectively.

In Fig. 2 shows the distribution of the diameters of the complex particles in comparative preparation according to comparative example 1. The horizontal axis, the left vertical axis and the right vertical axis indicates the particle diameter (µm) frequency (%) and the integral frequency, respectively.


Claims

1. The method of obtaining containing nucleic acid complex of the drug, wherein the two polymer single-stranded nucleic acids, which can, at least partially, to form double chains, are mixed separately in the form of a separate circuit with a cationic carrier or the raw material for cationic carrier before the formation of the cationic carrier, and then the mixture is dispersed in an aqueous solution at the time of receipt containing the nucleic acid complex of the drug.

3. The method according to p. 1, in which two single-stranded polymer of nucleic acids, which can, at least partially, to form double chains represent poly I and poly C.

4. The method according to p. 1, in which the cationic carrier is a carrier of drugs containing 2-o-(2-diethylaminoethyl)carbarnoyl-1,3-o-dioleoylglycerol and phospholipids as mandatory components.

5. The method according to p. 4, in which the phospholipid is a lecithin.

6. Containing nucleic acid complex preparation obtained by mixing the two polymers, single-stranded nucleic acids, which can, at least partially, to form double chains, in the form of a separate circuit, with a cationic carrier or the raw material for cationic carrier before the formation of the cationic carrier in an aqueous solution, and the mixture is dispersed in an aqueous solution at the time of receipt containing the nucleic acid complex of the drug, where the complex contains cationic carrier and two polymer-stranded nucleic acids, which can, at least partially, to form double chains, and where h is iofilizirovanny form.

7. Containing nucleic acid complex drug under item 6 or lyophilized form, where the particle of the complex preparation in the middle has a diameter of less than about 150 nm, being in a state of solution.

8. Complex drug under item 7 or in its dried form, where the particles of the integrated product have a diameter of less than about 200 nm, being in a state of solution.

9. Containing nucleic acid complex preparation obtained by mixing the two polymers, single-stranded nucleic acids, which can, at least partially, to form double chains, in the form of a separate circuit, with a cationic carrier or the raw material for cationic carrier before the formation of the cationic carrier in an aqueous solution, and the mixture is dispersed in an aqueous solution at the time of receipt containing the nucleic acid complex of the drug, where the complex contains cationic carrier and two polymer-stranded nucleic acids, which can, at least partially, to form double chains, and where the complex particles of the drug can pass through the sterilizing filtration membrane with a size of 0.2 μm, being in a state of solution or lyophilized form.

10.you is 0.1-10 mg/ml in a state of solution.

 

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The invention relates to new optically active proizvodnim of benzopyran formula

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where R and R are independently selected from the group consisting of hydroxyl and a moiety that can be converted in vivo in hydroxyl, such as acyloxy, -OR4, -OC(O)R7or-OC(O)OR4(where R4represents alkyl, alkenyl, quinil or aryl; and R7represents amino, alkylamino, aminoalkyl and alkylsulfonyl); and R3represents-CH2- or-CH2CH2-; or its pharmaceutically acceptable salt, where the specified compound or salt is optically active because they contain more than 50% (by weight relative to all stereoisomers) 2S stereoisomers

The invention relates to medicine, namely to preventive Oncology

The invention relates to purified and dedicated not naturally occurring RNA ligands to vascular endothelial growth factor (VEGF) (indicated oligonucleotide sequence)

The invention relates to purified and dedicated not naturally occurring RNA ligands to vascular endothelial growth factor (VEGF) (indicated oligonucleotide sequence)

The invention relates to medicine and relates to the application of sodium nukleospermata as a drug for the treatment of HIV infection

The invention relates to new methods for the production of medicines and new ways to treat infections of hepatitis b virus

The invention relates to medicine and for the creation of antiviral immunotropic drugs

The invention relates to medicine, in particular to cancer, and can be used for the treatment of generalized tumors

The invention relates to the field of biotechnology and relates to new compounds mobilarena, its pharmaceutically acceptable salts or complex and simple esters

The invention relates to veterinary medicine and can be used to combat diseases of bees: ascosphaerosis and European grilcom, and pest honeycomb wax moth

The invention relates to veterinary
The invention relates to veterinary medicine, namely to medicines used to treat parvovirus enteritis in dogs, and to methods of using these medicines
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