MXPA01008218A - Shortened-chain polynucleotides and process for the preparation thereof - Google Patents

Abstract

Shortened-chain polynucleotides useful as drugs and medicinal compositions containing the same. Specifically, shortened-chain polynucleotides or salts thereof, characterized by the content of 2'-5'phosphoric diester linkage of 3%or below based on all the phosphoric diester linkages;and medicinal compositions containing both.

Description

TRIMMED CHAIN POLYUCLEOTIDE AND METHOD FOR THE PREPARATION OF THE SAME. Technical Field The present invention discloses a truncated chain polynucleotide particularly useful as a medicine, and a method for preparing the same. More specifically, the present invention • Describes a synthetic truncated chain polynucleotide or salts thereof, wherein the percentage of a 2'-5'-phosphodiester bond is up to 3% based on the total phosphodiester bonds, i.e. the percentage of the groups of phosphate transposed from the 3 'to 2' positions in the total phosphate groups of the phosphodiester linkages (the percentage of phosphate rearrangement) is up to 3%, and a method for preparing the same. TECHNICAL BACKGROUND Polynucleotides typified by polyinosine nico-id polyclide, ie poly (I) poly (C), are compounds well known in the art, and the potentiality as a medicine for the treatment of hepatitis or cancer has been investigated in view of its action of induction of interferon, Ref: 131855 action of immune activation, and the like. The pharmacological action of these nucleotides has a high correlation with the length of the chain, and the longer the length of the chain the stronger the action of induction of interferon and the like. On the other hand, the longer the length of the chain, the stronger the toxicity manifested. Recently, there has been an approach to reduce toxicity by keeping the pharmacological action of a polynucleotide useful, by means of a method wherein a synthetic polynucleotide having a relatively short chain prepared by the hydrolysis of a polynucleotide is included in a carrier such as an effective cationic liposome for introducing a medicament into a cell (e.g., PCT O99 / 20283, PCT 099/48531). It is known that when a polynucleotide is hydrolyzed to shorten the long chain as described above, some phosphate groups cause an intramolecular rearrangement of the 3 'position to the 2' position through a mechanism called pseudo rotation simultaneously with the shortening of the chain (see for example, "Protein Nucleic acid Enzyme", Vol 40, No. 10 pp. 1233 'to 1332 (1995)). As a result, a portion of 3'-5 'phosphodiester linkages in the cut-chain polynucleotide molecule is replaced by 2'-5'-phosphodiester linkages. In any case it has never been known that a phenomenon of the transposition of the phosphate affects the pharmacological action. DESCRIPTION OF THE INVENTION It is an object of the present invention to provide, firstly, a cut-chain polynucleotide or a salt thereof and a trimmed chain polynucleotide. double strand or a salt thereof, which are safe and effective as a medicine. The present inventors have studied intensively and have found that the problems described above can be solved by means of a cut-chain polynucleotide which contains a 2'-5'-phosphodiester linkage produced mainly in a chain-cut reaction only in a proportion particular or minor, or a salt thereof, and carrying out the present invention. One aspect of the present invention is a truncated chain polynucleotide that contains a 2'-5'-phosphodiester linkage in a ratio of 3% or less, preferably 2% or less, based on the total phosphodiester linkages, or a come out of them. The present invention also includes as an embodiment a double stranded cut-chain polynucleotide or a salt thereof, which is formed of two cut-chain polynucleotides or salts thereof capable of forming a double strand, while included in the polynucleotide. chain cut described above containing a 2'-5'-phos fodies ter link in a percentage of 3% or less, preferably 2% or less, based on the total phosphodiester linkages. further, the present invention also includes a composition comprising a complex formed of an effective carrier for introducing a medicament into a cell and the cut-out strand polynucleotide described above or a salt thereof wherein the percentage of a 2'-5 'linkage -fosfodies ter is 3% or less based on the entire phosphodiester linkages, or the polynucleotide chain double-stranded or a salt thereof formed of two cut-chain polynucleotides or salts thereof capable of forming a double strand as an essential ingredient. The polynucleotide used in the present invention is a compound comprising at least about 20 nucleotides, which is formed by the polymerization linearly through a phosphodiester bond and includes natural and synthetic compounds. Specific examples include polinosinic acid [called, poly (I)] or an analogue thereof, idyllic polycyclic acid [called, poly (C)] or an analog thereof, polyadenylic acid [called, poly (A) ] or an analogue thereof, and the polyuridyl acid [called, poly (U)] or an analogue thereof. The polinosinic acid analogue is a homopolymer wherein all or part of the inosinic acid is chemically modified or a copolymer of the inosinic acid with another nucleotide, for example, poly (7 -deazainosinic acid) and poly (2'-azidoinosinic acid). The analog of polycyclic acid is a homopolymer in which all or part of the cytidyl acid is chemically modified or a copolymer of the cididic acid with another nucleotide, for example, poly (5-bromocit idol acid), poly (2-thiocytidyl acid) ), poly (cycididine-5-thiophosphoric acid), poly (cycidyl acid, uridilic acid), poly (cycidyl acid, 1-t-iuridic acid), poly (1-vinyl iditic acid). The analogue of polyadenyl acid and the analogue of polyuridyl acid are also defined. Among these, polyinosinic acid and polycyclic acid are suitable in the present invention. The average chain length of the cut chain polynucleotide of the present invention is suitable from 0.1 K bases to 1 K base. The term "base" means the base number and "1 k base" indicates a base number of 1000, and later, "base (s)" is abbreviated to "b". The average length of the chain is preferably from 200 b to 800 b, and more preferably from 300 b to 60 LC b. The average chain length can be easily determined, for example, by gel permeation chromatography (formerly referred to as "GPC") as described above in Experiment 5. In the trimmed chain polynucleotide of the present invention , the percentage of the phosphate rearrangement is 3% or less, preferably 2% or less or between 0.1% and 2%, and more preferably 1% or less or between 0.1% and 0.1%. 1 9- The rearrangement of the phosphate group from the 3 'position to the 2' position in the polynucleotide can be easily confirmed, for example, by a method as described in Experiment 6. That is, a polynucleotide is degraded with a nuclease Pi, which specifically hydrolyzes the linkage 3'-5 'phosphodiester levels as a nucleoside, nucleotide and oligonucleotide, and then treated with alkaline phosphatase, which specifically hydrolyzes the terminal phosphate group, to convert the total nucleotides in nucleosides. On the other hand an oligonucleotide having a 2'-5'-phosphodiester bond, which is not hydrolyzed by the Pi nuclease is not degraded to the nucleoside even by means of the treatment with an alkaline phosphatase because a 2'-5 'linkage intramolecular phosphodiester is not hydrolyzed. The percentage of transposition of the phosphate can be calculated by means of the determination of nucleosides and oligonucleotides (most of them are dimers) by liquid chromatography, or the like. Loe examples of a pair of polynucleotides trimmed string capable of forming a double strand with respect to the present invention include polyinosinic acid and polycytidylic acid, polyadenylic acid and polyuridylic acid analogue of polyinosinic acid and polycytidylic acid, polyinosinic acid and analog polycytidylic acid analogue of polyinosinic acid and analog polycytidylic acid analogue of polyadenylic acid and polyuridylic acid, polyadenylic acid and analog polyuridylic acid, and the analogue of polyadenylic acid analogue of polyuridilic acid. Thus examples of polynucleotides chain cut double-stranded formed between two polynucleotide chains cut capable of forming a double strand include polyinosinic acid - polycytidylic, polyadenylic acid - polyuridylic, polyinosinic analogue - polycytidylic acid analogue polyinosinic acid - polycytidylic, polyinosinic analogue - polycytidylic acid analogue, polyadenylic analogue - polyuridylic acid, polyadenylic acid analogue • - polyuridylic, and polyadenylic analogue - polyuridylic acid analogue. For the purposes of the present invention, the polyinosinic-polycytidylic acid can be a double-stranded cut-chain polynucleotide. The average chain length of the double-strand truncated chain polynucleotide is reasonably contemplated as that corresponding to the average chain length of the total trimmed chain polynucleotides. Accordingly, the latter can be used to show the average of the length of the apparent strand of the double strand truncated chain polynucleotide in terms of the number of base pairs (bp). Therefore, the average chain length of the double-stranded cropped chain polynucleotide is 0.1 k bp to 1 k bp. The term "bp" means the number of base pairs and "1 k bp" corresponds to the base even number of 1000. The average chain length of the double-stranded polynucleotide is preferably from 200 bp to 800 bp, and more preferably from 300 bp to 600 bp. The salt of a truncated chain polynucleotide and that of a double-stranded stranded chain polynucleotide of the present invention are not particularly restricted whereby they are pharmaceutically acceptable, and examples therinclude a sodium salt and a potassium salt . As an effective carrier for introducing a drug into a cell, those having a positive charge are exemplified, and specific examples include cationic polymers such as poly-L-lysine, cationic liposomes such as Lipofectin, Lipofectamina®, Lipofectase®, DMRIE-C®, etc., and carriers considered to be the same species which is described in PCT W094 / 19314. Carriers for carrying a medicament are formed, for example, 2-0-. { 2-diethylaminoet il} carbamoyl-1, 3-0- dioleoyl glycerol of the formula [I] And a phospholipid (for example, phosphatidyl choline, phosphatidyl ethanolamine, yolk lecithin, soy lecithin, hydrogenated phospholipid thereof) as essential structural components. It is believed that the cationic liposome described above is positively charged and forms an electrostatic complex with a polynucleotide or a negatively charged oligonucleotide. When the resulting complex fuses with the cell membrane, the polynucleotide or oligonucleotide is commonly introduced into the cell. Just like the complex is sometimes called "lipoplex". A method for preparing the trimmed chain polynucleotide of the present invention will be described in detail. The trimmed chain polynucleotide of the present invention can be prepared, for example, by hydrolyzing the starting nucleotide in solution in a suitable pH range while heating to a suitable temperature range. The suitable pH of the aqueous solution in this process is basic, that is, the pH of 7 or more, preferably 7 to 10. Considering the reaction ratio of the chain shortening reaction and the stability of the base radical, more preferably pH of the solution should be between 8 and 9. The reaction temperature is suitable within a range of 20 to 110 ° C, preferably 40 to 100 ° C, from the standpoint of the stability of the base. However, considering the sufficient hydrolysis ratio and the stability of the base radical, the most preferable reaction temperature will be between 50 and 90 ° C. More specifically, for example, a polynucleotide is dissolved in water such as an injectable water, and the pH of the solution is adjusted from 8 to 9 with a buffer or a pH regulator. When hydrolysis is conducted by heating the solution in the reaction temperature range of 50 to 90 ° C for 0.5 to 60 hours while monitoring the average chain length and percentage of phosphate rearrangement, a chain polynucleotide Cropped that contains fewer transposed phosphate groups and that has an average of the chain length of between 0.1 kb and 1 kb may be prducted. Pharmaceutically acceptable additives such as a buffer or a pH regulator can be used to adjust the pH.

Specific examples include buffers and pH regulators such as aminoacetic acid (synonym: glycerin), tr is (hydroxymethyl) aminomethane (synonym: Tris), sodium carbonate, sodium hydrogen carbonate (synonym: sodium bicarbonate), sodium hydroxide, diethanolamine, triethanolamine and the like. There are no limitations with respect to the group, the combination, the concentration and the similar of these additives. Unnecessary monomers and salts, impurities, side products produced during the chain shortening reaction and the like can be removed from the system by treating the reaction solution by dialysis or activated carbon. The trimmed chain polynucleotide of the present invention can also be prepared by treatment with the starting polynucleotide in solution with a phosphodiesterase in a suitable pH range while heating in a suitable temperature range. The suitable pH of the aqueous solution in this process is between 4 and 9, preferably between 5 and 8. Considering the transposition of the phosphate during the chain shortening reaction, more preferably the pH of the solution should be between 6 and 7. The reaction temperature is suitable within a range of 20 to 60 ° C, preferably 25 to 50 ° C, from the point of view of the characteristics of the enzyme. However, the most preferable reaction temperature should be between 30 and 40 ° C taking the ratio of sufficient hydrolysis, preventing the influence of nonenzymatic hydrolysis such as hydrolysis with heating, and preventing the transposition of the phosphate group taken into consideration. More specifically, for example, a polynucleotide is dissolved in water such as injectable water, distilled water of injection or physiological saline with agitation. The pH of the solution is optionally adjusted by the addition of a buffer or a pH regulator, if necessary. To the solution is added phosphodiesterase such as a Px nuclease and the resulting mixture is subjected to the chain shortening reaction at a temperature of between 30 to 40 ° C while monitoring the average of the chain length and the percentage of the phosphate rearrangement to obtain a trimmed chain polynucleotide containing fewer transposed phosphate groups and having an average chain length between 0.1 kb and 1 kb. There are no limitations regarding the concentration of the enzyme or the reaction conditions. Unnecessary monomers and salts, impurities, side products produced during the chain shortening reaction and the like can be removed from the system by treating the reaction solution by ethanol precipitation, dialysis or with carbon activated The trimmed chain polynucleotide can be purified by an appropriate separation method with a membrane. For example, the ultrafiltration membrane is suitable for the purpose of fractionating polynucleotides of average chain length between 0.1 kb and 1 kb of the present invention. There are no limitations with respect to the quality of the material or the pore size of the membrane. As a starting material, any of the polynucleotides can be used without regard to the origin such as natural or synthetic, type of salt or chain length. Examples of the natural polynucleotides include tRNA and polyadenic acid. On the other hand, a synthetic polynucleotide can be produced from an RNA synthetase such as the polynucleotide phosphorylase or the immobilized enzymes thereof. In addition, sodium polynite, sodium polyidiidylate, etc., which are commercially available as induction reagents of the inferred, are also used as a starting material. The double-stranded truncated chain polynucleotide of the present invention can be prepared by mixing, in a suitable solution (e.g., containing 0.15 M NaCl Tris buffer-10 mM hydrochloric acid (Tris-HCl buffer, pH 7)), two polynucleotides chain cut capable of forming a double strand between the trimmed chain polynucleotides containing fewer transposed phosphate groups as prepared above, or alternatively, by allowing them to be strengthened in a conventional manner. As the annealing method, there exists, for example, a method in which a solution containing two truncated chain polynucleotides capable of forming a double strand is heated to 70 to 80 ° C, and then cooled gradually. A truncated chain polynucleotide with fewer transposed phosphate groups or a double-stranded cut-chain polynucleotide with fewer transposed phosphate groups obtained as described above can be treated by means of lyophilization to provide a lyophilized product that can be stored for a long period of time. The lyophilization treatment can be conducted in a conventional manner. For example, a lyophilized product can be obtained as follows: a solution of a truncated chain polynucleotide obtained under the above conditions is sterilized by filtration, the filtrate is then poured into a metal bath previously treated by sterilization in dry heating, it conducts a pre-freeze at a storage temperature of -40 to 20 ° C for about 1 to 4 hours, and the primary drying is conducted before the secondary drying which is affected under reduced pressure at a storage temperature of 15 ° C. at 30 ° C (for about 10 to 15 hours). In general, said lyophilized product can be used after reconstitution (re-constitution) by the addition of an appropriate solution such as injectable water, distilled water by injection, physiological saline, maltose solution, glucose solution, or similar. The composition of the present invention can be prepared in a manner similar to those generally used for the preparation of the liposome, while the composition comprises a complex (hereinafter referred to as the present complex) formed with an effective carrier for introducing a medicament into a cell and two cut-chain polynucleotides containing 2'-5'-phosphodiester bonds in a 3% percentage. or less based on the total phosphodiester bonds and which are capable of forming a double strand, or a double strand truncated chain polynucleotide formed between said two strand chain polynucleotides capable of forming a double strand as an essential ingredient. Specifically, a composition for injection of the present invention can be prepared by the following steps comprising the addition of water (injectable water, distilled water by injection, physiological saline and the like) to an effective carrier to introduce a medicament into a cell, for example, a cationic liposome or raw material thereof (for example, glycerol derivative such as 2-0- (2-diethylaminoethyl) carbamoyl -1, 3-0-dioleoyl glycerol and the like and the phospholipid); the mixture is stirred; the mixture is treated with a device suitable for dispersion, for example, a homomixer, a homogenizer, an ultrasonic dispersion device, an ultrasonic homogenizer, a high-pressure emulsifying dispersant device, a microfluidizer (trade name), a nanomizer (trade name), De Bee 2000 (trade name), Ultimizador (commercial name) or high pressure homogenizer type Mant on-Gaulin; a truncated chain polynucleotide or a double-stranded crop chain polynucleotide of the present invention is added to the resulting lipid dispersion; the re-dispersion of the mixture by treatment with a suitable dispersion device; and then subjecting the resulting composition to sterilization by filtration or the like. Any other additives can be added at an appropriate stage during the preparation without any particular limitation. Alternatively, a composition of the present invention containing the present complex can be prepared as follows. Thus, a mixture of an effective carrier for introducing a medicament into a cell, for example, a cationic liposome or a raw material thereof (eg, glycerol derivative such as 2-0- [2-diethylaminoet il) carbamoi 1 1, 3-0-dioleoyl glycerol and the like and phospholipid) and a trimmed chain polynucleotide or a double-stranded stranded chain polynucleotide of the present invention are prepared first. Water is added to the previously prepared mixture while conducting the dispersion treatment at the same time. In addition, a composition of the present invention can be prepared via a suitable crude dispersing step during the methods described above. The composition resulting from the present invention can be lyophilized, thereby obtaining a lyophilized preparation of the composition of the present invention stored for a long period. The lyophilization can be conducted in a conventional manner. For example, a proportionate amount of a composition of the present invention, which has been obtained by the aforementioned dispersion and sterilization by filtration, is dispersed within a flask. The lyophilization is carried out by subjecting the bottle to a pre-freezing at a temperature between about -40 and -20 ° C for about 2 to 3 hours; primary drying at a temperature between about 0 to 10 ° C under reduced pressure, and secondary drying at a temperature between about 15 to 25 ° C under reduced pressure. In general, after replacing the internal space with an inert gas such as nitrogen gas, the bottle is covered to provide a lyophilized preparation of a composition of the present invention. The lyophilized preparation of a composition of the present invention can be used after reconstitution by the addition of an appropriate solution before use. Examples of such solutions for reconstitution include injectable water, distilled water for injection, physiological saline, maltose solution, glucose solution and other general infusion solutions and the like. The compositions of the present invention and the lyophilized preparation thereof can be used in the form of a pharmaceutical preparation as a medicine. The composition of the present invention and the lyophilized preparation as a medicine exhibits a pharmacological activity due to the active polynucleotide. Specific examples of the medicine include interferon-inducing agents, immune activating agents, cellular nuclease activation agents, cancer preventive or treatment agents, and hepatitis preventive or treatment agents. BEST MODE FOR CARRYING OUT THE PRESENT INVENTION The following examples and experiments further illustrate the present invention in more detail. The present invention is not restricted by these examples and experiments in any way. REFERENCE EXAMPLE 1 500 mL of a sodium hydroxide buffer - 0.1 M glycerin is added to 8 g of inos ina-5 '- trisodium diphosphate and 1 g of magnesium chloride, and the mixture is stirred for dissolution. After adjusting the pH to 9.3 by the addition of 6N sodium hydroxide, the mixture is left to stand for 1 hour at a temperature of 38 ° C. 1 mL of a polynucleotide phosphorylase solution is added to the mixture, and the reaction is conducted at 38 ° C for 18 hours. The reaction is then quenched by the addition of 25 mL of ethylacetamine ethylacetamine 0.2 M (EDTA), 10 mL of saturated saline and 500 mL of pure ethanol are added to precipitate the polyinosinic acid (1973b).

REFERENCE EXAMPLE 2 Sodium hydroxide - 0.2 M sodium hydrogen carbonate buffer is added to 10 g of cytidine-5'-trisodium di-phosphate and 3 g of magnesium chloride, and the mixture is stirred by dissolution . After adjusting the pH to 9.8 by the addition of 6N sodium hydroxide, the mixture is allowed to remain for about 1 hour at 3 ° C. 2 mL of a purified polynucleotide phosphorylase solution is added to the mixture, and the reaction is conducted at 36 ° C for 24 hours. The reaction is quenched by the addition of 50 mL of 0.2 M EDTA. Then 20 mL of saturated saline and 1 L of pure ethanol are added to the mixture to precipitate the polycytidylic acid (3300 b). EXAMPLE 1 The polyosinic acid obtained in reference to Example 1 is separated by centrifugation. The precipitate is re-dissolved in 500 mL of injectable water and subjected to dialysis. The internal solution is treated after dialysis with an activated carbon and filtered to remove the activated carbon. 6N sodium hydroxide is added to the filtrate to adjust the pH to 8.5. The hydrolysis is conducted by heating at 70 ° C for 8 hours to cut the polyinosinic acid chain. The resulting solution of the chain polynucleotide cut with activated carbon is treated, filtered to remove the activated carbon, and dialyzed. The interior solution is sterilized after dialysis by filtration. The filtrate is subjected to lyophilization in a conventional manner to obtain 1.9 g of a freeze-dried product of the trimmed chain polynucleotide (poly i inosine, sodium salt) of the present invention with fewer transposed phosphate groups (percentage of phosphate rearrangement). : 0.2%, average of the length of the chain: 360 b). EXAMPLE 2 The polycyclic acid obtained from reference example 2 is separated by centrifugation. The precipitate is re-dissolved in 500 mL of injectable water and subjected to dialysis. The internal solution after dialysis is treated with activated carbon and filtered to remove the activated carbon. 6N sodium hydroxide is added to the filtrate to adjust the pH to 9.0. hydrolysis is conducted by heating at a temperature of 80 ° C for 4 hours to cut the polycyclic acid chain. The resulting truncated chain polynucleotide solution is treated with activated charcoal, filtered to remove the activated charcoal and dialyzed. The interior solution after dialysis is sterilized by filtration. The filtrate is lyophilized in a conventional manner to obtain 2.7 g of lyophilized chain-cut polynucleotide product (polycyclic idilate, sodium salt) of the present invention with fewer transposed phosphate groups (percentage of phosphate rearrangement: 0.1%, Average length of the chain: 3.18 b). EXAMPLE 3 200 mL of 0.1 M tris-HCl buffer is added (pH 8.9) to 1 g of brass or sodium polyadeni (S ° 2o, w (sedimentation constant): 7.2 Seikagaku Corporation), and the mixture is stirred for dissolution. The hydrolysis is conducted by heating at 60 ° C for 48 hours to shorten the polyadenyl acid chain while monitoring the average chain length according to the method described in Experiment 5. The solution of the cut chain polynucleotide is subjected to the dialysis. The internal solution after dialysis is subjected to lyophilization in a conventional manner to obtain 0.3 g of a lyophilized product of trimmed chain polynucleotide (polynucleotide, sodium salt) of the present invention with fewer transposed phosphate groups (percentage of transposition of phosphate: 1.9%, average chain length: 154 b). EXAMPLE 4 200 mL of 0.2 M NaOH glycerin buffer (pH 9.0) is added to 1 g of po 1 iur id 1 at or sodium (S ° 2o, w (settling constant): 6.5 Seikagaku Corporation), and the mixture for dissolution. The hydrolysis is conducted by heating at 60 ° C for 25 hours to cut the polyadenylic acid chain while monitoring the average of the chain length according to the method described in Experiment 5. The resulting solution of the cut chain polynucleotide is subject to dialysis. The internal solution of the dialysis is subjected to lyophilization in a conventional manner to obtain 0.2 g of a lyophilized product of trimmed polynucleotide chain (polyuridyl, sodium salt) of the present invention with fewer transposed phosphate groups (percentage of phosphate rearrangement: 1.2%, average chain length: 108 b). 50 mL of 0.1 M tris-HCl buffer (pH 8.0) is added to 250 mg of pol i inosianat or sodium (S ° 2o, w (sedimentation constant): 8.0 Yamasa Corporation), and the mixture is stirred for dissolution. The solution is heated to a temperature of 50 to 120 ° C suitable for shortening the chain. The polyinosinic acid having an arbitrary chain length is sampled while monitoring the average molecular length by the method described in Example 5. The results are shown in Table 1. The resulting sampled solutions are dialyzed, and lyophilized in a manner conventional to obtain lyophilized products.

Table 1 EXAMPLE 6 50 mL of 0.1 M tris-HCl buffer (pH 9.0) is added to 250 mg of sodium tidylatil or poly (S ° 2o, w (sedimentation constant): 8.6 Yamasa Corporation), and the mixture is stirred for dissolution. The solution is heated to a temperature of 55 to 120 ° C suitable for cutting the chain. The polycyclic acids having an arbitrary chain length are sampled while monitoring the average molecular length by the method described in Experiment 5. The resulting sampled solutions are dialyzed, and lyophilized in a conventional manner to obtain lyophilized products. Table 2 This is apparent from the results obtained in Examples 5 and 6 that, in the process of chain shortening of the commercially available polyunsatinate or sodium or sodium polycydolate, sufficient hydrolysis may not be carried out when chain shortening it is carried out at 55 ° C or less. As a result. The average chain length exceeds 1 kb equal after about 50 hours of the chain shortening reaction. Otherwise, in the sampling where the chain shortening is carried out at a temperature of 120 ° C or 90 ° C, the control of chain shortening is made difficult due to the rapid rate of hydrolysis. Particularly, in the sample where the shortening of the chain is carried out at 120 ° C, the chain is shortened to such a degree around the level of the oligonucleotide approximately even after 1 to 1.5 hours of the shortening of the chain. In this sample, the phosphate transposition ratio is high and degradation of the base radical is also recognized. EXAMPLE 7 Dissolve in 100 mL of buffer 0.1 M Tris-HC1 (pH 7.0), 100 mg of sodium polyadenylate (S ° 2GJ, W (sedimentation constant): 7.2, Seikagaku Corporation). P nuclease is added to the solution (derived from Penicillum Citrinum, Seikagaku Corporation). The mixture is incubated at a temperature of 25 ° C for 3 hours while monitoring the average of the length of the chain according to the method described in Experiment 5 to shorten the chain of piloadenylic acid (percentage of phosphate rearrangement: 0.1% , average of the length of the chain: 287b). EXAMPLE 8 100 mL of sodium hydroxide-0.2M sodium hydrogen carbonate buffer is added to 1 g of uridine-5'-trisodium di-phosphate and 0.3 g of magnesium chloride, and the mixture is stirred for dissolution. After adjusting the pH to 9.5 with 1 N sodium hydroxide, 0.2 mL of polynucleotide phosphorylase is added. The mixture is then allowed to react for 10 hours at a temperature of 25 ° C while monitoring the average of the length of the chain according to the method described in Experiment 5. The reaction is then quenched by adding 5 mL of 0.2 M EDTA , add 2 mL of a saturated saline solution and 100 mL of ethanol to precipitate the polyuridilic acid (549 b). The polyuridilic acid is separated by means of centrifugation. The precipitate is redissolved in 50 mL of injectable water and dialyzed. The inner solution of the dialysis is adjusted to pH 8.5 with 1 N sodium hydroxide and hydrolyzed at a temperature of 80 ° C for 30 minutes to regulate the length of the polyuridylic acid chain. The resulting solution of the trimmed chain polynucleotide is subjected to a membrane separation by means of ultrafiltration to adjust the chain length distribution, as well as to remove unnecessary salt, side products during the chain shortening reaction or the similar (percentage of phosphate transposition: 0.1%, average of the length of the chain: 485 b). EXAMPLE 9 40 g of maltose dissolved in 100 mL of water for injection are added to 1 g of 2-0- (2-diethylaminoet yl) -carbamoyl-1,3-O-dioleoyl glycerol and 2 grams of purified yolk lecithin, the mixture is stirred and dispersed for 5 minutes with a homogenizer to obtain a crude dispersion of a cationic liposome (carrier) The crude dispersion is further dispersed for 1 hour with a small laboratory type emulsified dispersion device to obtain a cationic liposome solution. About 50 mL of an aqueous solution containing 200 mg of each of the cut chain sodium polyososinate and sodium poly iditrate each containing less transposed phosphate groups are added gradually to a solution of cationic liposome, which is obtained in Examples 2 and 1, with agitation. The mixture is then treated with a small laboratory-type emulsified dispersion device for another 2 hours, and finally a volume of 400 mL is adjusted with injectable water to obtain a composition containing the present complex. The composition containing the present complex is sterilized by means of filtration, distributed in 1 mL aliquots in a flask, and converted into a lyophilized preparation in a conventional manner. When the lyophilized preparation is reconstituted by the addition of injectable water to produce the volume of 1 mL, the average particle diameter of the present complex is 133 nm when evaluated by means of the photon correlation method. EXAMPLE 10 1 kg of sucrose dissolved in 3 1 of water for injection is added to 50 g of 2-0- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoyl glycerol and 25 g of soy lecithin. The mixture is stirred and dispersed for 30 minutes with a Manton-Gaulin high pressure homogenizer, and the volume is adjusted to 5 liters with injectable water to obtain a dispersion of a cationic liposome (carrier). About 2 liters of an aqueous solution containing one gram of each of the cut chain sodium polyinosinate and sodium polycytidylate each containing less rearranged phosphate groups are added to the dispersion of the carrier, which are obtained in examples 1 and 2, with agitation. The dispersion is adjusted to pH 5.5 with IN hydrochloric acid and dispersed with a Manton-Gaulin high pressure homogenizer for another hour, and finally the volume is adjusted to 10 L with injectable water to obtain a composition containing the present complex. . The composition containing the present complex is distributed in 20 mL aliquots into a bottle and converted into a lyophilized preparation in a conventional manner. When the lyophilized preparation is reconstituted by the addition of injectable water to produce the volume of 20 mL, the average particle diameter of the present complex is 150 nM when evaluated by means of photon correlation.

EXAMPLE 11 40 g of glucose dissolved in 10 ml of water for injection are added to 1 g of 2-0- (2-diethylaminoet i 1) -ca bamoyl-1,3-O-dioleoyl glycerol and 2 g of the yolk phosphatide. The mixture is stirred and dispersed for 5 minutes with a homogenizer, and the volume is adjusted to 500 mL to obtain a crude dispersion of a cationic (carrier) liposome. The raw dispersion is further dispersed for 1 hour with a small laboratory type emulsified dispersion device to obtain a cationic liposome solution. The solution is distributed in 10 mL aliquots in a bottle and lyophilized in a conventional manner. 10 mL of an aqueous solution containing 5 mg of each of the cut chain sodium polyinosinate, polycyclic idilat or sodium each containing less phosphate groups rearranged to the lyophilized product, which are obtained in Examples 1, 2 or 5, 6, and 5 mg of each of the commercially available sodium polycyanate (S ° 2rj 'w (settling constant): 8.8, Yamasa Corporation) and sodium polycytidylate (S ° 2c w (settling constant): 8.6, Yamasa Corporation). The mixture is subjected to a dispersion treatment for 10 minutes with a probe-type ultrasonic dispersion device to obtain a composition containing the present complex. EXAMPLE 12 An aqueous solution (1 L) containing 100 μg of the trimethylated sodium polyadenylate and the sodium polyuridylate each containing less rearranged phosphate groups is mixed with stirring., which is obtained in Examples 3 and 4, and 2 mL of an aqueous solution containing 2 mg of a commercially available Lipofectin (tradename). The mixture is dispersed for 15 minutes with a probe-type ultrasonic dispersion device to obtain a composition having the complex present. EXAMPLE 13 The 0.2 M sodium acetate acetic acid buffer (pH 5.2) is added to about 250 mg of the sodium polyinosinate (S ° 20 'w (settling constant): 8.8, Yamasa Corporation), and the mixture is stirred for dissolution. The mixture is heated to a temperature of 80 ° C and the polyinosinic acids having a transposition percentage of the arbitrary phosphate are sampled while monitoring the percentage of phosphate rearrangement by the method described in the experiment. The polyinosinic acid of the transposition percentage of each phosphate is heated to a temperature of 60 ° C in a borate buffer (pH 8.5) to cut the length of the chain until the average length of the chain is between 200 ± 50 b while the length of the chain is monitored according to the method described in experiment 5. The results are shown in table 3. The respective cut-chain polyinosinic acid solutions thus obtained are dialyzed and lyophilized in a conventional manner to obtain a lyophilized product.

Table 3 EXAMPLE 14 The buffer 0.2 M sodium acetate acetic acid (pH 5.2) is added to about 250 mg of polycitidyl or sodium (S ° 20 'w (settling constant): 8.6, Yamasa Corporation), and the mixture is stirred for its dissolution. The mixture is heated to a temperature of 80 ° C and id polycyclic acids having a transposition percentage of the arbitrary phosphate are sampled while monitoring the percentage of phosphate rearrangement by the method described in Example 6. The polycyclic acid of the transposition percentage of each phosphate is heated to a temperature of 70 ° C in a borate buffer (pH 8.5) to cut the length of the chain until the average length of the chain is between 200 ± 50 b while monitoring the length of the chain according to the method described in experiment 5. The results are shown in table 4. The solutions of the respective cut-chain polyosinic acid thus obtained are dialysed and lyophilized in a conventional manner to obtain a lyophilized product . Table 4 EXAMPLE 15. Double-stranded chain-cut polynucleotide. The double-stranded cut-chain polynucleotides (double-stranded RNAs) of the following combinations are prepared: polyinosinic acid (percentage of phosphate rearrangement: 0.7%) and polycytidyl acid (percentage of phosphate rearrangement: 1.2%); polyinosinic acid (percentage of transposition of phosphate 2.0%) and polycyclic acid (percentage of transposition of phosphate 1.9%); polyinosinic acid (percentage of transposition of phosphate 2.8%) and polycytidyl acid (percentage of transposition of phosphate 2.7%), which have been obtained according to the method of example 1,2,11 or 12. Each of the polynucleotides of double-stranded cut chain are prepared by dissolving the sodium polyinosinate and the sodium polycytidylate in a Tri-HCl buffer (pH 7, containing 0.15 M NaCl) by heating the solution to a temperature of 80 ° C for 15 minutes, and gradually cooling the solution. COMPARATIVE EXAMPLE 1 This comparative example corresponds to Example 1 above, wherein the preparation is carried out by means of a conventional method (preparation 1). 10 mL of injectable water is added to 5 mg of sodium polyinocinate (S ° 20-w (settling constant): 8.8, Yamasa Corporation), and the mixture is stirred for dissolution. 10 mL of formamide is added to the solution, and the mixture is heated at a temperature of 80 ° C for 5 hours (percentage of phosphate rearrangement: 8.9%, average of the average chain length: 628 b). COMPARATIVE EXAMPLE 2 This comparative example corresponds to Example 2 above, wherein the preparation is carried out by means of a conventional method (preparation 1). 10 mL of injectable water are added to 5 mg of sodium polycytidylate (S ° 2o 'w (sedimentation constant): 8.6, Yamasa Corporation), and the mixture is stirred for dissolution. 10 mL of formamide is added to the solution, and the mixture is heated at a temperature of 80 ° C for 5 hours (percentage of phosphate rearrangement: 4.2%, average of the average chain length: 751 b). COMPARATIVE EXAMPLE 3 This comparative example corresponds to Example 1 above, wherein the preparation is carried out by means of a conventional method (preparation 2). 10 mL of injectable water is added to 5 mg of sodium polyinosinate (S ° 2o'W (settling constant): 8.8, Yamasa Corporation), and the mixture is stirred for dissolution. The solution is heated at 90 ° C for 8 hours (percentage of phosphate rearrangement: 7.1%, average chain length: 213 b). COMPARATIVE EXAMPLE 4 This comparative example corresponds to Example 2 above, wherein the preparation is carried out by means of a conventional method (preparation 2). 10 mL of injectable water are added to 5 mg of sodium polycytidylate (S ° 2o 'w (sedimentation constant): 8.6, Yamasa Corporation), and the mixture is stirred for dissolution. This solution is heated to a temperature of 90 ° C for 12 hours (percentage of phosphate rearrangement: 4.2%, average chain length: 289 b). COMPARATIVE EXAMPLE 5 The double-stranded cut-chain polynucleotide (double-stranded RNAs) is prepared by combining the polyinosinic acid (percentage of phosphate rearrangement: 4.2%) with the polycytidyl acid (percentage of phosphate rearrangement: 3.8%), the which has been prepared in examples 11 and 12 respectively. Said double-stranded chain-cut polynucleotide is prepared by dissolving the sodium polyinosinate and the sodium polycytidylate in a Tris-HCl buffer (pH 7, containing 0.15 M NaCl) by heating the solution at a temperature of 80 ° C for 5 minutes. , and gradually cooling the solution. EXPERIMENT 1. Influence of the Average of the Length of the Chain on the Pharmacological Activity. The pharmacological activity of the composition of Example 9 is evaluated in vitro according to the inhibitory action of growth on the HeLa S 3 cancer cells. Inoculated HeLa S3 cancer cells are inoculated into 96-well plates at a density of 10 4 cells / well and are cultured for 24 hours or more when sufficient adhesion of the cell to the plate is confirmed. A composition of the present invention is added to the plate, and cultivation is continued. After three days of cultivation in a C02 incubator the number of viable cells is counted by means of an MTT method. The percentage of inhibition of cell growth is calculated according to the following formula (I). The results are shown in table 5.

Percentage of inhibition of cell growth (%) = Absorbance of the complex - treated group X 100 (I) Absorbance of the control group Table 5 poly (I) = polyinosinic acid poly (I), Na = polyinosinate, sodium salt **: poly (C) = polycyclic acid poly (C), Na = polycydoidylate, sodium salt (a): polyinosinate, sodium salt (Yamasa Corporation) (b): polycytidylate, sodium salt (Yamasa Corporation) It is apparent from Table 5 that the action of inhibiting the growth of the cells of each composition of HeLa S3, a strain of the cancer cell, is highly correlated with the average of the length of the chain. Table 5 reveals that polyinosinic-polycytidylic acids have long chain lengths of greater than 1000 b, which are generally used as interferon induction agents, show stronger inhibition of growth action. The cut chain polyinosinico-polycytidylic acids of the present invention have an average chain length of between 0.1 kb to 1 kb and contains fewer transposed phosphate groups that still have sufficiently high growth inhibitory activity, although it is slightly lower. Such inhibitory action of growth decreases steeply in a polynucleotide having the average length of the chain below 100 b, and those having the average length of the chain similar to those of the oligonucleotides, shows a small action. EXPERIMENT 2. Influence on the cells of the marrow. The evaluation of toxicity is carried out on the basis of bone marrow toxicity. Each of the following samples is administered by intravenous injection to male ddY mice (males, 8 weeks old): commercially available sodium polyinosinate (S ° 2o '(settling constant): 8.8, Yamasa Corporation) and polysitidylate sodium (S ° 20 'w (sedimentation constant): 8.6, Yamasa Corporation), and polyinosinic acid and polysididylic acid containing less transposed phosphate groups, which have the average chain length of 982 by 907 b respectively, and are prepared in Examples 5 and 6. On the next day the bone marrow cells are collected from the thigh bone, stained with New Methylene Blue and Giemsa, and observed to count the number of reticulum and erythrocytes. mature. The results are shown in Table 6, where the bone marrow toxicity is expressed as a percentage of the number of reticulocytes with the total number of mature erythrocytes as defined by the following formula. The mark * means that there is a significant difference based on Dunnett's method of comparison at a significant level of p < 0.01 among the test groups was the control group that received intravenous administration of a polynucleotide without a vehicle. Bone marrow toxicity Reticulocite number Reticulocite number + Mature erythrocyte number Table 6 1) poly (I) = polyinosinic acid 2): poly (C) = polycytidylic acid (a): polyinosinate, sodium salt (Yamasa Corporation) (b): polycytidylate, sodium salt (Yamasa Corporation) It is apparent from Table 6 that the change in bone marrow toxicity based on that of control achieved as high as 39% at a dose of 1 mg / kg in cases where the polyinosinic-polycytidyl acid has a long chain length above 1000 by and is generally used as an interferon induction agent, while there is no significant difference compared to the control in the case of polycyclic cut chain polyclinic acid having an average chain length of between 0.1 kb and 1 kb and containing fewer phosphate groups transposed even in doses of 25 times. The above results reveal that the toxicity of polyinosinic acid and polycyclic acid is highly correlated with the average chain length, similarly to the pharmacological activity thereof. It was unexpectedly and surprisingly found that the trimmed chain polynucleotide of the present invention can significantly improve such toxicity. EXPERIMENT 3. Inhibitory Action of Cell Growth (In vitro) in cancer cells A431 (average chain length: 200 ± 50 b A composition is prepared in a manner similar to that described in Example 9 using polyinosinic acid (percentage of phosphate rearrangement: 0.7 - 4.2%) and polycytidyl acid (percentage of phosphate rearrangement: 1.2 - 3.8%) obtained in examples 11 and 12, and the trimethylated polyinosinic acid and polycyclic acid obtained in examples 1 and 2. The A431 cancer cells are inoculated into a 96-well plate at a density of 10 4 cells / well and cultured for 5 hours or more, when sufficient adhesion of the cell to the plate is confirmed. of the compositions and cultures After three days of culture in a C02 incubator, the number of viable cells is counted by means of an MTT method.The percentage of inhibition of the growth of the The formulas are calculated according to the formula (I), and the total concentration of the sodium polyinosinate and the sodium polycytidylate corresponds to 50% of the percentage of reduction of the growth of the cell.

(IC50) is calculated. The results are shown in Table 7 Table 7 As apparent from Table 7, the inhibitory action of cell growth of the composition in the cancer cell A431 is highly correlated with the percentage of phosphate rearrangement. That is, as the increase of the phosphate groups transposed from the 3 'position to the 2' position, the inhibitory action of growth tends to be very weak, without consideration of polyinosinic acid or polycytidyl acid. It is notable that the inhibitory action of growth is remarkably strong in a combination of chain cut polyinosinic acid with polysit idyllic chain-cut acid each containing less transposed phosphate groups (percentage of phosphate rearrangement is 3% or less, particularly 2). % or less) of the present invention. On the other hand, in a combination of the polyinosinic acid with the acid each having a percentage of transposition of the phosphate of more than 2%, particularly 3% more, the action tends to be synergistically weaker. For example, in Table 7, a combination of polyinosinic acid (percentage of phosphate rearrangement: 2.0%) with polysit idic acid (percentage of phosphate rearrangement: 1.9%) show the IC50 percentage improved by 12 times or 47 times compared to combinations of polyinosinic acid (percentage of phosphate rearrangement: 2.8%) with polycyclic acid (percentage of phosphate rearrangement: 2.7 %), and polyinosinic acid (percentage of phosphate transposition: 4.2%) with idyllic poicitic acid (percentage of phosphate transposition: 3.8%), respectively. It is extremely unexpected that the pharmacological activity which increases in this manner steeply as the percentage of transposition of the phosphate group from the 3 'position to the 2' position decreases to 3%, particularly 2% as the limit. EXPERIMENT 4. Influence of Phosphate Transposition on Fusion Temperature (Tm) and Pharmacological Activity. The double-stranded RNA is dissociated into two single-stranded RNAs when the temperature rises to a given degree. The temperature varies depending on the type of bases that make up an RNA and is specific to that. In accordance, said temperature is defined as the melting temperature of a double-stranded RNA and is generally referred to as a "Tm value". There are several methods to measure the Tm value. In the present experiment, the Tm values of the double-stranded RNAs shown in Example 13 and Comparative Example 5 are measured by the most common method, ie, absorptometry. The results are shown in table 8. The IC50 values shown in Table 8 are obtained in experiment 3.

Table 8 As a result of measuring the Tm value in the polyinosinic-polycytidylic acid having the phosphate transposition percentage in the range of about 0 to 4%, no noticeable difference was found in each combination. (for the polyinosinic-polycytidyl acid having a long chain length generally used as the interferon induction agent, the Tm value is 61 ° C). It is revealed that polycydoidic polyinosinic acid having a phosphate transposition percentage between about 0 and 4% can form a double-spiral structure characteristic of double-stranded RNA. However, it is also revealed that the action of immune activation and the carcinoestic action, which are the main medicinal effects polyinosinic-polycytidylic acid are affected not only by the double-spiral structure of a double-stranded RNA but also by the percentage of transposition of the phosphate group, because the pharmacological activity varies as much as by four times to 50 times or more when the phosphate transposition percentage changes from 2-3% as a limit, as shown in experiment 3. In addition, unexpectedly and frequently the pharmacological activity increases steeply when the percentage of transposition of the phosphate group from the 3 'position to the 2' position changes from 2-3% as a limit. EXPERIMENT 5. Measurement of the Average Chain Length of the Truncated Chain Polynucleotide (GCP method). The length of the average chain is measured using an aqueous solution of 1 mg / mL of the polynucleotide by means of gel impregnation chromatography (GPC) under the following conditions: GCP Opposition Conditions : Detection: UV at 260 nm Column: Tosoh gel TSK G5000PWXL 7.8 irarif x 300 mm Mobile phase: 50 mM Tris-HCl buffer (pH 7.5) containing 7 M urea 0.5 ml / min. . Flow rate: 0.5 ml / min. Size indicator: RNA ladder (1770 b, 1520 b, 1280b, 780 b, 530 b, 400 b, 280 b, 155 b) (Gibco BRL). EXPERIMENT 6. Measurement of the percentage of phosphate transposition. 3.2 mL of an aqueous solution of 500 U / mL nuclease Pl (derived from Penicillium Citrinum, Seikagaku Corporation) is added to 1 mL of an aqueous solution of 1 mg / mL of the polynucleotide, and diluted with water to reach the volume of 5. mL. The aqueous solution reacts for one hour in a water bath at a temperature of 37 ° C. Water is added to the reaction mixture to obtain a volume of 10 mL and a 3.2 mL portion is separated from it. To the separated solution is added 0.8 mL of an aqueous solution of 0.1 U / mL of alkaline phosphatase (derived from bovine intestine, Seikagaku Corporation) and allowed to react in a water bath at a temperature of 37 ° C for 30 minutes. The solution is appropriately diluted and examined by liquid chromatography under the following conditions to determine a dimer having a phosphodiester bond 2'-5 'and the percentage of phosphate rearrangement. Operating conditions of liquid chromatography: Detection: UV at 260 nm Shiseido Capcell column pack CX8 UG120 5 μm 4.6 mmf x 250 mm Mobile phase: mixed solution (95: 5) of 50 mM phosphate amphotiguther (pH 8) containing 5 mM of tetrabutylammonium hydrogen sulfate and methanol Flow rate: 1 ml / min. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A cut-chain polynucleotide or a salt thereof characterized in that the percentage of a 2'-5'-phosphodiester bond is in the range of 0.1 % to 3% based on total phosphodiester bonds.
2. 2. The polynucleotide or a salt thereof according to claim 1, characterized in that the average of the length of the chain is between 0.1 kb and 1 kb.
3. 3. A method for preparing a polynucleotide or a salt thereof wherein the percentage of the 2'-5'-phosphodiester bond is up to 3% based on the total phosphodiester bonds and the average of the chain length is between 0.1 k bases and 1 k bases, characterized in that a percentage of phosphate transposition is measured in the course of the preparation.
4. 4. A method for preparing a cut-chain polynucleotide or a salt thereof wherein the percentage of a 2'-5'-phosphodiester linkage is up to 3% based on the entire phosphodiester linkages, characterized in that a polynucleotide or a salt thereof is reacted in a solution at a pH of 7 to 10 at a temperature between 20 and 11 ° C to shorten the chain with the measurement of a percentage of transposition of the phosphate.
5. 5. A method for preparing a cut-chain polynucleotide or a salt thereof wherein the percentage of the 2'-5'-phosphodiester bond is up to 3% based on the total phosphodiester bonds, characterized in that a polynucleotide or a salt thereof is treated with a phosphodiesterase to shorten the chain by measuring a percentage of phosphate rearrangement.
6. 6. A polynucleotide or a salt thereof wherein the average of the length of the chain is between 0.1 k bases and 1 k bases, characterized in that the percentage of the 2'-5'-phosphodiester bond is in the range of 0.1 to 3% based on the total phosphodiester bonds.
7. 7. The polynucleotide or a salt thereof according to any one of claims 1, 3 and 14, characterized in that the polynucleotide is the polyinosinic acid or an analogue thereof, the polycytidylic acid or an analogue thereof, the polyadenylic acid or an analog thereof, the polyuridyl acid or an analogue thereof.
8. 8. A polynucleotide or a salt thereof according to any one of claims 1, 3, 14 and 15, characterized in that it is in the form of a double-stranded polynucleotide or a salt thereof formed of two polynucleotides or salts thereof. which are capable of forming a double strand.
9. 9. The double-stranded polynucleotide or a salt thereof according to claim 16, characterized in that two of the polynucleotides which are capable of forming a double strand are selected from the group consisting of a combination of the polyinosinic acid and the polycyclic acid , polyadenylic acid and polyuridylic acid, polyinosinic acid analogue and polycyclic acid, polyinosinic acid and polycyclic acid analog, polyinosinic acid analogue and polycyclic acid analogue, polyadenylic acid analog and polyuridylic acid , of the polyadenyl acid and the analogue of the polyuridyl acid, and of the polyadenyl acid analogue and of the polyuridyl acid analogue.
10. A composition characterized in that it comprises a complex formed of an effective carrier for introducing a medicament into a cell and a polynucleotide or a salt thereof according to any one of claims 1, 3, 14 and 15 or a double polynucleotide. or a salt thereof according to claim 16 or 17 as an essential ingredient.
11. 11. The composition according to claim 18, characterized in that the effective carrier for introducing a medicament into a cell is a positively charged carrier.
12. 12. The composition according to claim 19, characterized in that -, the positively charged carrier is a cationic liposome.
13. The composition according to claim 18, characterized in that the effective carrier for introducing a medicament into a cell is a carrier formed from 2-0- (2-diethylaminoethyl) carbamoi 1-1, 3-0-dioleoylglycerol and a phospholipid fos as essential constituent components.
14. 14. The composition according to any one of claims 18 to 21, characterized in that it is in the form of a pharmaceutical preparation.
15. 15. The composition according to claim 22, characterized in that the pharmaceutical preparation is an induction agent of interferon, an immune activating agent, an intracellular nuclease activation agent, an agent for the treatment of cancer or preventive agent, or a hepatitis treatment agent or a preventive agent.