Liquid composition containing a mixture of DNA fragments at a high concentration and having fluidity, and method for producing same
阅读说明：本技术 以高浓度含有dna片段混合物且具有流动性的液剂组合物及其制备方法 (Liquid composition containing a mixture of DNA fragments at a high concentration and having fluidity, and method for producing same ) 是由 金益洙 李修渊 于 2019-03-07 设计创作，主要内容包括：本发明涉及一种以高浓度含有DNA片段混合物的液剂组合物及其制备方法,制备了含有具有流动性的高浓度的DNA片段混合物的液剂组合物,并确认到这种含有具有流动性的高浓度的DNA片段混合物的液剂组合物能够维持药剂活性且即使长时间保存也具有高稳定性,并且能够给有效地注射至人体内,从而可期待利用本发明的具有流动性的高浓度的DNA片段混合物适用于开发多种剂型,如注射剂、液态制剂、霜剂等。(The present invention relates to a liquid composition containing a DNA fragment mixture at a high concentration and a method for preparing the same, and it is confirmed that the liquid composition containing a DNA fragment mixture at a high concentration having fluidity can maintain the activity of a medicament and has high stability even when stored for a long time, and can be effectively injected into the human body, and thus it is expected that the DNA fragment mixture at a high concentration having fluidity of the present invention is suitable for developing various dosage forms such as injections, liquid preparations, creams, and the like.)
1. A liquid composition having fluidity, characterized by containing a DNA fragment mixture at a high concentration and having a viscosity of 1 to 1,000 mPas at 20 ℃.
2. The liquid composition having fluidity according to claim 1,
the liquor composition comprises a cationic additive.
3. The liquid composition having fluidity according to claim 2,
the liquid composition is prepared by mixing a DNA fragment mixture and a cationic additive in a weight ratio of 1: 1-5.
4. The liquid composition having fluidity according to claim 1,
the liquid composition comprises 2-20 wt% of a DNA fragment mixture based on the total weight of the liquid composition.
5. The liquid composition having fluidity according to claim 1,
the molecular weight of the DNA fragment mixture is 50-10,000 kDa.
6. The liquid composition having fluidity according to claim 1,
the DNA fragment mixture is one or more selected from the group consisting of a polydeoxyribonucleotide and a polynucleotide.
7. The liquid composition having fluidity according to claim 1,
the DNA fragment mixture is isolated from the testis or semen of the fish.
8. The liquid composition having fluidity according to claim 7,
the fish is Salmonidae.
9. The liquid composition having fluidity according to claim 2,
the cationic additive is one or more selected from the group consisting of cationic peptides, cationic lipids, and cationic polymers.
10. The liquid composition having fluidity according to claim 9,
the cationic peptide is one or more selected from the group consisting of polylysine, protamine, and KALA peptide.
11. The liquid composition having fluidity according to claim 9,
the cationic lipid is prepared from N- [1- (2,3-dioleoyloxy) propyl ] -N, n-trimethyl ammonium chloride, 1,2-dioleoyloxy-3- (trimethyl ammonium) propane, 1,2-dioleoyl-3- (4' -trimethyl ammonium) butyryl-sn-glycerol, 1,2-diacyl-3-dimethyl ammonium-propane, 1,2-diacyl-3-trimethyl ammonium-propane, 1,2-diacyl-sn-glycerol-3-ethylphosphocholine, 3 β - [ N- (N ', N ' -dimethylaminoethane) -carbamoyl ] cholesterol, dimethyl octacosyl ammonium bromide and copolymers thereof.
12. The liquid composition having fluidity according to claim 9,
the cationic polymer is one or more selected from the group consisting of chitosan, spermine, putrescine, cadaverine, and spermidine.
13. The liquid formulation composition having fluidity according to any one of claims 1 to 12,
the liquid composition is added with and mixed with at least one selected from the group consisting of hyaluronic acid, carboxymethyl cellulose, collagen, alginate, poloxamer, antibody, protein, peptide, miRNA, siRNA, aptamer, synthetic drug and bone graft material.
14. The liquid formulation composition having fluidity according to any one of claims 1 to 12,
the liquid composition is used for injection, liquid preparation, cream, ointment, gel, emulsion, internal liquid, external liquid and transdermal absorbent.
15. The liquid composition having fluidity according to claim 1 or 2,
the liquid formulation composition is prepared by a process consisting of:
a first step of adding a DNA fragment mixture to a buffer solution and dissolving at a high temperature of 80 ℃ or higher using a thermal stirrer to prepare a DNA fragment mixture storage solution;
a second step of adding the cationic additive to the buffer solution to be dissolved, to prepare a cationic additive storage solution; and
and a third step of mixing and stirring the DNA fragment mixture storage solution of the first step and the cationic additive storage solution of the second step in a thermal stirrer at 80 ℃ or higher while cooling the temperature to room temperature.
Technical Field
The present invention relates to a liquid composition containing a DNA fragment mixture at a high concentration and having fluidity and a method for producing the same, and more particularly, to a liquid composition containing a DNA fragment mixture at a high concentration in a stabilized state while maintaining fluidity at room temperature.
Background
Nucleic acids (nucleotides acid), which are DNA and RNA present in the nucleus, are polymers of nucleotides (nucleotides) consisting of purine (purine) bases (adenine, a; guanine, G) or pyrimidine (pyrimidine) bases (cytosine, C; thymine, T; uracil, U), five-carbon sugars and phosphates. Such nucleic acids have been considered as substances for storing and transmitting genetic information, but recently, it is well known that nucleic acids are decomposed into small pieces outside cells and exert a function of selectively binding to receptors on the cell surface to activate signals intracellularly and small pieces exerting nucleic acids move into cells to rapidly induce DNA synthesis with little energy, and thus new functions and technologies for adopting the functions are being developed.
It is known that polydeoxyribonucleotides (polydeoxyribonucleotides) are a mixture of DNA fragments extracted from animals or plants and having a specific size, stimulate adenosine A2receptors (adenosines A2receptors), and have anti-inflammatory (anti-inflammation), cell proliferation (cell proliferation), and tissue regeneration (tissue regeneration) effects (Kim, y.h., et al., 2010). In Europe and Korea, a mixture of DNA fragments extracted from the semen of a fish of the Salmonidae family such as salmon or trout is namedAnd is used as a raw material for pharmaceuticals for cell proliferation and tissue regeneration (Yu)n，J.K.，et al.，2015)。
Polynucleotides (hereinafter referred to as PN) andsimilarly, a DNA fragment mixture obtained by extracting the seminal fluid of a fish belonging to the family Salmonidae such as salmon or trout but having a chain length ratio of nucleic acidLong and of greater molecular weight. These PN are used as a physical carrier as a raw material for medical devices by exhibiting cell adhesion and lubrication and buffering action.
In the development of drugs and medical devices, the concentration, solubility, physical properties and shape of a raw material substance affect the method of application of the drugs and medical devices and the absorption of the raw material substance in the human body. In addition, when a plurality of additional components such as excipients and carriers are added to the raw material substance, it is important to uniformly mix the raw material substance and the additional components, and in this case, the solubility of the raw material substance and the additional components plays an important role.
To improve the quality of
Here, the present inventors prepared a liquid composition containing a DNA fragment mixture at a high concentration having fluidity in the course of studying the liquid composition containing the DNA fragment mixture, and confirmed that such a liquid composition can maintain the pharmaceutical activity and can be effectively injected into a human body. Further, the present inventors have completed the present invention by confirming that the fluidity of the prepared compound can be continuously maintained without any state change even if it is stored for a long time to have high stability.
In addition, when a large amount of DNA fragment mixture is injected, unlike the case where a plurality of injections are repeated, the injection frequency can be reduced by the liquid composition of the present invention containing a DNA fragment mixture at a high concentration, and other effective components can be mixed in a high concentration state, thereby improving the industrial availability.
As the prior art, korean registered patent No. 1459185 discloses a nucleic acid delivery complex in which nucleic acid is bound to spermine copolymer, but does not disclose nor suggest at all the effect of dissolving a high concentration of DNA fragment mixture using the cationic additive of the present invention. Further, korean laid-open patent No. 2011-0129969 discloses a preparation comprising a polyamine derivative and a nucleic acid, but does not disclose nor suggest an effect of dissolving the fragment mixture of the present invention at a high concentration at all.
As a non-patent document, Sung, B.K. (2012) discloses DNA condensation using spermine, but does not disclose gelation of the high concentration DNA fragment mixture of the present invention and the effect of preventing gelation to dissolve the high concentration DNA fragment mixture.
Disclosure of Invention
Problems to be solved by the invention
The purpose of the present invention is to provide a liquid composition having fluidity and containing a DNA fragment mixture at a high concentration, which is capable of preparing a liquid composition containing a DNA fragment mixture at a high concentration that is also flowable and stable at room temperature, and a method for preparing the same.
Means for solving the problems
The present invention relates to a liquid composition having fluidity and containing a mixture of DNA fragments at a high concentration, the viscosity of which at 20 ℃ is 1 to 1,000 mPas.
The liquor composition may comprise a cationic additive.
The liquid composition may be a mixture of DNA fragments and a cationic additive mixed at a weight ratio of 1:1 to 5.
The liquid formulation composition may include 2 to 20 wt% of the DNA fragment mixture based on the total weight of the liquid formulation composition.
The molecular weight of the DNA fragment mixture can be 50-10,000 kDa.
The mixture of DNA fragments may be one or more selected from the group consisting of polydeoxyribonucleotides and polynucleotides.
The DNA fragment mixture can be isolated from the testis or semen of fish.
The fish may be salmonidae.
The cationic additive may be one or more selected from the group consisting of cationic peptides, cationic lipids, and cationic polymers.
The cationic peptide may be one or more selected from the group consisting of polylysine (polylysine), protamine (protamine) and KALA peptide (cationic lipophilic peptide).
The cationic lipid may be a lipid prepared from N- [1- (2,3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (N- [1- (2,3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), 1,2-dioleoyloxy-3- (trimethylammonium) propane (1,2-dioleoyloxy-3- (trimethylammonium) propane), 1,2-dioleoyl-3- (4 '-trimethylammonium) butyryl-sn-glycerol (1,2-dioleoyl-3- (4' -trimethylammonium) butyryl-sn-glycerol), 1, 2-dioleoyl-3-dimethylammonium-propane (1, 2-dioleoyl-3-dimonium-propane), 1,2-diacyl-3-trimethylammonium-propane (1, 2-diacyl-3-trimethylammoniummonio-propane), 1,2-diacyl-sn-glycerol-3-ethylphosphocholine (1,2-diacyl-sn-glycerol-3-ethylphosphocholine), 3 β - [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol (3 β - [ N- (N ', N' -dimethyllaminoethane) -carbamoyl ] cholestol), dimethyldioctadecylammonium bromide (dimethyldioctadecylammonium bromide) and copolymers thereof.
The cationic polymer may be one or more selected from the group consisting of chitosan (chitosan), spermine (speramine), putrescine (putrescine), cadaverine (cadeverine), and spermidine (speramine).
The liquid composition may further contain one or more selected from the group consisting of hyaluronic acid (hyaluronic acid), carboxymethylcellulose (carboxymethylcellulose), collagen (collagen), alginate (alginate), poloxamer (poloxamer), antibodies (antibody), proteins (protein), peptides (peptide), mirna (microrna), sirna (small interfering rna), aptamers (aptamer), synthetic drugs (chemical drug), and bone graft materials (bone graft materials).
The liquid composition can be used as injection, liquid preparation, cream, ointment, gel, emulsion, internal liquid, external liquid and transdermal absorbent.
The liquid formulation composition may be prepared by a process consisting of: a first step of adding a DNA fragment mixture to a buffer solution and dissolving at a high temperature of 80 ℃ or higher using a thermal stirrer to prepare a DNA fragment mixture storage solution; a second step of adding the cationic additive to the buffer solution to be dissolved, to prepare a cationic additive storage solution; and a third step of mixing and stirring the DNA fragment mixture storage solution of the first step and the cationic additive storage solution of the second step in a thermal stirrer at 80 ℃ or higher while cooling the temperature to room temperature.
The present invention will be described in detail below.
The present invention relates to a liquid composition having fluidity and containing a mixture of DNA fragments at a high concentration, the viscosity of which at 20 ℃ is 1 to 1,000 mPas.
The fluidity refers to the property of flowing and moving like a liquid, is related to viscosity, and may affect the uniformity and content of a formulation when the formulation is developed. In addition, in the case of injectable formulations, it is described as an important property in determining whether or not an injection is possible. Generally, it is known that the viscosity of water is about 1mPa · s at 20 ℃ and the viscosity of honey is about 10,000mPa · s, and the viscosity similar to water is very effective not only for injection preparations but also for other preparations.
The liquid composition having fluidity of the present invention may exhibit a viscosity of 1 to 1,000 mPas at 20 ℃. Preferably, the viscosity is 1 to 100 mPas, which can be adjusted according to the choice of the formulation to be mixed, and is preferably capable of maintaining fluidity.
The liquid composition having the above-mentioned fluidity can maintain the fluidity and has high stability without changing physical properties even when stored at room temperature for a long time.
The DNA fragment mixture is in a form of extracting DNA corresponding to a biopolymer consisting of phosphoric acid, four bases, and deoxyribose (deoxyribose), and is mixed in a form of DNA fragments having various molecular weights.
The DNA fragment mixture may be gelled (gelation) during dissolution at a high concentration, resulting in precipitation of a DNA fragment mixture having low fluidity or not dissolved, thereby making it difficult to use industrially. Therefore, to prepare a liquid composition having fluidity containing a mixture of DNA fragments at a high concentration, a cationic additive may be added.
When the DNA fragment mixture accounts for 2% by weight or more of the total weight of the liquid composition containing the DNA fragment mixture alone, gelation of the DNA fragment mixture may occur.
The liquid composition having fluidity to which the cationic additive is added may be a mixture of DNA fragments and the cationic additive in a weight ratio of 1:1 to 10. Preferably 1:1 to 5 weight ratio, more preferably 1:3 to 5 weight ratio.
The liquid composition containing the mixture of DNA fragments at a high concentration may contain the mixture of DNA fragments in an amount of 2% by weight or more based on the total weight of the liquid composition having fluidity. Preferably 2 to 20% by weight, more preferably 2 to 10% by weight.
The liquid composition containing the DNA fragment mixture at a high concentration can reduce the number of injections when a large amount of the DNA fragment mixture is injected, and can be mixed with other active ingredients at a high concentration, thereby improving the industrial availability.
The molecular weight of the DNA fragment mixture can be 50-10,000 kDa. When the molecular weight of the DNA fragment mixture is less than 50kDa, the DNA fragment mixture can be dissolved at a sufficiently high concentration without using a cationic additive and can maintain fluidity, and when it exceeds 10,000kDa, the solubility in water is low and the fluidity is lowered, so that it is difficult to industrially use the DNA fragment mixture.
The mixture of DNA fragments may be one or more selected from the group consisting of polydeoxyribonucleotides and polynucleotides.
The DNA fragment mixture can be isolated from the testis or semen of fish.
The fish may be of the salmonidae family, preferably salmon or trout, most preferably salmon.
The cationic additive may be one or more selected from the group consisting of cationic peptides, cationic lipids, and cationic polymers.
The cationic peptide may be one or more selected from the group consisting of polylysine (polylysine), protamine (protamine) and KALA peptide (cationic lipophilic peptide). However, it is not limited thereto.
The cationic lipid may be a lipid prepared from N- [1- (2,3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (N- [1- (2,3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), 1,2-dioleoyloxy-3- (trimethylammonium) propane (1,2-dioleoyloxy-3- (trimethylammonium) propane), 1,2-dioleoyl-3- (4 '-trimethylammonium) butyryl-sn-glycerol (1,2-dioleoyl-3- (4' -trimethylammonium) butyryl-sn-glycerol), 1, 2-dioleoyl-3-dimethylammonium-propane (1, 2-dioleoyl-3-dimonium-propane), 1,2-diacyl-3-trimethylammonium-propane (1, 2-diacyl-3-trimethylammoniummonio-propane), 1,2-diacyl-sn-glycerol-3-ethylphosphocholine (1,2-diacyl-sn-glycerol-3-ethylphosphocholine), 3 β - [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol (3 β - [ N- (N ', N' -dimethyllaminoethane) -carbamoyl ] cholestol), dimethyldioctadecylammonium bromide (dimethyldioctadecylammonium bromide) and copolymers thereof. However, it is not limited thereto.
The cationic polymer may be one or more selected from the group consisting of chitosan (chitosan), spermine (speramine), putrescine (putrescine), cadaverine (cadeverine), and spermidine (speramine). Preferably, the one or more compounds selected from the group consisting of spermine, spermidine and putrescine. Spermine is most preferred. However, it is not limited thereto.
The liquid composition may further contain a physiologically active substance or a material for medical equipment.
The physiologically active substance may be an antibody (antibody), a protein (protein), a peptide (peptide), mirna (microrna), sirna (small interfering rna), an aptamer (aptamer), a synthetic drug (chemicaldrug), or the like, but is not limited thereto.
The medical device material may be hyaluronic acid (hyaluronic acid), carboxymethyl cellulose (carboxymethyl cellulose), collagen (collagen), alginate (alginate), poloxamer (poloxamer), bone graft materials (bone graft materials), etc., but is not limited thereto.
The liquid composition can be used as an injection, a liquid preparation, a cream, an ointment, a gel, an emulsion, an internal liquid, an external liquid, a transdermal absorbent, and the like, and is not limited as long as the liquid composition can be used.
The liquid formulation composition may be prepared by a process consisting of: a first step of adding a DNA fragment mixture to a buffer solution and dissolving at a high temperature of 80 ℃ or higher using a thermal stirrer to prepare a DNA fragment mixture storage solution; a second step of adding the cationic additive to the buffer solution to be dissolved, to prepare a cationic additive storage solution; and a third step of mixing and stirring the DNA fragment mixture storage solution of the first step and the cationic additive storage solution of the second step in a thermal stirrer at 80 ℃ or higher while cooling the temperature to room temperature.
The buffer solution used for preparing the DNA fragment mixture stock solution of the first step may use disodium hydrogen phosphate dodecahydrate (sodium phosphate), sodium chloride (sodium chloride), magnesium chloride (magnesium chloride), potassium chloride (potassium chloride), phosphate buffer solution (phosphate buffer), or HEPES (N- (2-hydroxyethoxy) -piperazine-N' -2-ethylhanesulfonic acid) buffer solution, etc., but is not limited thereto.
The buffer solution used for preparing the cationic additive storage solution of the second step may use disodium hydrogen phosphate dodecahydrate, sodium chloride, magnesium chloride, potassium chloride, phosphate buffer solution, HEPES buffer solution, acetic acid (acetic acid), hydrochloric acid (hydrochloric acid), ascorbic acid (ascorbyl acid), lactic acid (lactic acid), nitric acid (nitrile), and the like, but is not limited thereto.
In addition, the present invention provides a liquid formulation having fluidity containing a mixture of DNA fragments at a high concentration and a pharmaceutical composition comprising a pharmaceutical excipient.
The pharmaceutical composition can be used as an injection, a liquid preparation, a cream, an ointment, a gel, an emulsion, an internal liquid, an external liquid, a transdermal absorbent and the like according to a conventional method. Examples of the carrier, excipient, and diluent that can be contained in the pharmaceutical composition include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When the agent is formulated, it is produced by using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant, which is generally used.
The dosage of the pharmaceutical composition of the present invention varies depending on the age, sex, body weight, pathological state of the disease, severity of the pathological state, administration route, and judgment of the prescribing physician of the subject to be treated.
The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock and humans by various routes. All modes of administration are envisioned, e.g., oral, rectal or intravenous, intramuscular, subcutaneous, dermal, epidural or intracerebral intravascular injection, topical application.
Effects of the invention
The present invention relates to a liquid composition containing a mixture of DNA fragments at a high concentration and a method for preparing the same, and more particularly, to a liquid composition containing a mixture of DNA fragments at a high concentration, which has fluidity and stability at room temperature (1 to 30 ℃), and which is confirmed to be capable of maintaining pharmaceutical activity and effectively injecting into a human body, and to be capable of continuously maintaining fluidity and having high stability without changing the physical properties of the composition even when stored at room temperature for a long period of time.
Thus, the DNA fragment mixture containing a high concentration and having fluidity of the present invention is expected to be suitable for the development of various dosage forms such as injections, liquid preparations, creams and the like, and by promoting the further mixing of various polymers and active factors in the high concentration DNA fragment mixture in a liquid state, it is expected to contribute to the development of a composition with enhanced activity by combining with each of the various substances.
Drawings
Fig. 1 shows the results of analyzing changes in storage modulus (G') and loss modulus (G ″) and complex viscosity (η ″) according to the magnitude of vibrational strain (a) and temperature change (B) of a liquid formulation composition containing a mixture of DNA fragments at a high concentration according to the present invention.
FIG. 2 shows the results of visual observation of the change in physical properties of the liquid composition of the present invention containing a mixture of DNA fragments at a high concentration.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein, and may be embodied in other forms. The following examples are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.
<Example 1 preparation of a liquid composition containing a mixture of DNA fragments at high concentrations>
The mixture of DNA fragments of the present invention utilizes polydeoxyribonucleotides (product name:pharmaceutical research products, ltd, korea), and the cationic additive utilizes spermine (speramine).
As a mixture of DNA fragmentsAdding to 0.4% sodium chloride (NaCl) buffer solution, and dissolving at 80 deg.C for 20 min with a thermal stirrer to prepare
As a cationic additive, spermine was added to a 0.4% sodium chloride (NaCl) buffer solution and dissolved at normal temperature for more than one hour to prepare a spermine storage solution.
Prepared by the aboveThe stock solutions and the spermine stock solution were mixed to the concentration conditions shown in table 1 below to prepare the compositions of examples 1-1 to 1-12. At this time, the mixture was stirred at 80 ℃While storing the solution, the spermine storage solution was slowly added and stirred for 1 hour or more and mixed, and then the temperature was lowered to room temperature while continuously stirring in a stirrer to prepare the liquid formulation composition of the present invention.
[ TABLE 1 ]
<Comparative example 1 preparation of liquid composition containing high concentration of DNA fragment mixture to be compared>
For comparison with the high concentration DNA fragment mixture of the present invention, liquid agent compositions of comparative examples 1-1 to 1-10 shown in Table 2 below were prepared in the same manner as the preparation method of the example 1.
[ TABLE 2 ]
<Example 2 confirmation of physical Properties of liquid composition containing DNA fragment mixture at high concentration>
The stability and physical properties of the liquid composition have a great influence on the development of a preparation using the liquid composition and a treatment method depending on a treatment site. Therefore, it was confirmed whether or not the fluidity of the liquid composition containing a mixture of DNA fragments at a high concentration of the present invention is stably maintained by analyzing whether or not the physical properties of the liquid composition vary depending on external force or temperature.
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