KR101577274B1 - Nucleotide having endosomolysis activity and uses thereof - Google Patents

Abstract

The present invention relates to a non-viral drug delivery system comprising a nucleotide and a non-viral drug delivery system, said composition having proton buffering activity or hemolysis activity, wherein the endosomolysis ) To induce endosomal escape of the delivered drug. The non-viral drug delivery compositions of the present invention are for effective drug delivery vehicles capable of escaping drugs delivered by hydrogen ion buffering activity or hemolytic activity of nucleotides from endosomes to other organelles in the cell.

Description

Nucleotides having endosomal resolution and uses thereof Nucleotide having endosomolysis activity and uses thereof < RTI ID = 0.0 >

The present invention relates to nucleotides having endosome resolution and their uses.

(Eg, high molecular weight peptides, proteins and genetic material as well as low molecular weight chemical drugs and contrast agents) to specific organs, tissues, cells, cytoplasm, mitochondria, perinuclear regions and nucleus regions There is a growing interest in developing an effective system to do so. Unlike traditional formulations, position-specific nanotherapeutics have been designed to maximize the bioavailability of therapeutic agents delivered to the target site and can be used to treat the disease with low side effects and to increase therapeutic efficacy. This drug delivery technology is a high value added technology and it is increasingly used as a method to improve the compliance of patients and the convenience of taking drugs.

Endosomal sequestration of the biological agent delivered into the cell is an important hurdle that can be overcome through endosome-membrane destabilization or destruction induced by polymers or monomers with amines, sulfonamides, or carboxylic acids . These polymers / monomers give improved results in nonviral gene delivery and in the cytoplasmic delivery of chemical drugs. At endosomal pH, proton buffering and conformational transition properties are known to induce lipid membrane destabilization or endosomolysis.

Korean Patent Application No. 10-2009-0134481 discloses a composition for delivering an anionic drug comprising polylactic acid and a method for preparing the same, but the present invention is not limited to the use of the nucleotide described in the present invention, There is no.

Accordingly, the present inventors have found that a nucleotide has a hydrogen ion buffering ability, destabilizes or destroys the endosomal membrane by causing a hemolytic action, and has biofunctional ability to escape the delivered drug from endosomes to other organelles in the cell And developed a drug delivery system using the same, thereby completing the present invention.

It is an object of the present invention to provide a non-viral drug delivery system comprising a nucleotide and a non-viral drug delivery system. The nucleotides provide a role for destabilizing or destroying endosomes in order to enhance the efficacy of the drug in non-viral drug delivery vehicles, including drugs of varying properties.

In order to accomplish the above object, the present invention provides a non-viral drug delivery composition comprising a nucleotide and a non-viral drug delivery system.

Specifically, the nucleotide is selected from the group consisting of adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine monophosphate Guanosine diphosphate (GDP), guanosine triphosphate (GTP), Cytidine monophosphate (CMP), Cytidine diphosphate (CDP), Cytidine triphosphate (CTP) A thymidine monophosphate (TMP), a thymidine diphosphate (TDP), or a thymidine triphosphate (TTP).

In addition, the composition has hydrogen ion proton buffering activity or hemolysis activity, thereby inducing endosomal escape of the drug through endosomolysis.

Specifically, the non-viral drug delivery system includes any one or more drugs selected from the group consisting of hydrophilic and hydrophobic chemical drugs, protein drugs, peptide drugs, gene drugs and diagnostic drugs, and includes polyethyleneimine (bPEI), polylysine (PLA), poly (arginine), polyhistidine, poly (amido amine), poly (beta amino ester), poly Chitosan, protamine, histone, poly (tertiary amine methacrylate), poly (2- dimethylamino) ethyl methacrylate (poly (2- (dimethylamino) ) ethyl methacrylate), poly (N- [N- (2-aminoethyl) -2-aminoethyl] aspartamide) Phospholipids, polymer-derived nanostructures, metal-derived nanostructures, carbon-derived nanostructures, calcium phosphate nano- That includes one or more delivery agent is selected from the group consisting of a crude product, porous silica nanostructure, and a complex thereof, characterized, but is not limited to.

The compositions according to the present invention may comprise a pharmaceutically effective amount of the drug alone or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount as used herein refers to an amount sufficient for a drug to be administered to an animal or a human to exhibit a desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the age, body weight, health condition, sex, administration route and treatment period of the subject to be administered.

The term "pharmaceutically acceptable" as used herein means physiologically acceptable and does not generally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

The composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, and the dose of the drug may be varied depending on various factors such as route of administration, age, sex, Can be appropriately selected. In addition, the composition of the present invention may be administered in combination with a known compound capable of raising the desired effect of the drug.

The " drug " used in the present invention is a substance capable of inducing a desired biological or pharmacological effect by promoting or inhibiting a physiological function in the body of an animal or human, and is a chemical or biological substance (1) prevent undesired biological effects such as infection prevention and have a preventive effect on the organism, (2) alleviate the condition caused by the disease, for example, the pain or infection resulting from the disease And (3) mitigate, reduce or completely eliminate disease from organic matter.

The present invention relates to a nucleotide having an ability to degrade endosomes, and a use thereof, wherein a drug transferred from a non-viral drug delivery vehicle containing the nucleotide due to hydrogen ion buffering activity or hemolysis of the nucleotide is transferred from endosomes to other sub- Effective < / RTI > bioactive material that can escape.

Figure 1 shows the results of proton buffering capacity evaluation of nucleotides.
FIG. 2 shows the results of evaluation of hemolysis phenomenon of ATP and GTP under pH environment.
Figure 3 shows the particle size of bPEI _25kDa / ATP-pDNA complexes and PLL / ATP-pDNA complexes (Fig. 3a) and surface charge (Fig. 3b).
Fig. 4 shows gene expression efficiency of bPEI _{25 kDa} / ATP-pDNA and bPEI _{25 kDa} / GTP-pDNA complex in HepG2 cells.
FIG. 5 shows gene expression efficiency of bPEI _{25 kDa} / ATP-pDNA and bPEI _{25 kDa} / GTP-pDNA complex in HepG2 cells in the presence of Chloroquine or Bafilomycin A1.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Nucleotide Hydrogen ion  Buffer capacity ( proton buffering  capacity evaluation

The nucleotides were dissolved in a 150 mM aqueous solution of sodium chloride at a concentration of 1 mg / mL, and a small amount of 1 M sodium hydroxide aqueous solution was added thereto to prepare an aqueous solution having a pH of about 11. The pH change was measured while 0.1 M hydrogen chloride was added to the aqueous solution.

As shown in FIG. 1, it was confirmed that the sodium chloride aqueous solution had no hydrogen ion buffering ability at pH 4 to 7, which is the endosomal pH region, and adenosine triphosphate (ATP) has hydrogen ion buffering ability. In addition, guanosine triphosphate (GTP), cytidine triphosphate (CTP), and thymidine triphosphate (TTP) also had hydrogen ion buffering capacity. The hydrogen ion buffering capacity of these nucleotides in the endosomal pH environment can destabilize or destroy the endosomal membrane due to the impact of osmotic pressure differences, such as the well known "proton sponge effect".

< Example  2> Nucleotide  Hemolysis Hemolysis ) evaluation

To confirm that the nucleotides destroy endosomes through interaction with endosomal membranes, the ability of the nucleoside to destroy endosomes was evaluated using erythrocytes with a structure similar to that of the endosomal membrane. The red blood cells are separated from the blood collected from the rat (Rat). Prepare erythrocytes 1 x 10 ⁸ cells are mixed in DPBS solution (pH 4-7.5) containing 1 mg / mL of ATP and GTP with different pH and mixed well. This solution is left in a thermostat at 37 ° C for 1 hour. After centrifuging again, transfer the supernatant to a 96-well plate and measure the absorbance at 450 nm to calculate the hemolysis. In FIG. 2, when ATP was added, hemolysis was observed at a pH of 6 or less. In the case of GTP, hemolysis was not observed.

From the results of Example 1 and Example 2, it can be seen that ATP escapes the endosomes through interaction with the endosomal membrane as well as the hydrogen ion buffering ability. GTP, I could see that the cotton was escaping.

< Example  3> Nucleotide  Gene Expression Efficiency of Polymeric Gene Drug Delivery Systems Included

In order to encapsulate the nucleotides in a well-known polymer / gene complex, a solution of positively charged polymeric polyethyleneimine (bPEI _{25 kDa} , molecular weight 25 kDa) and a solution of pDNA and a negative charge containing nucleotides were prepared. The two solutions were mixed to form bPEI _{25 kDa} / pDNA complexes. N / P ratio 5, except that the phosphate of the nucleotide was not taken into consideration, but was calculated only with the amine of pPEI _{25 kDa} and the phosphate of pDNA.

The prepared bPEI _{25 kDa} / nucleotide-pDNA complex was compared with the bPEI _{25 kDa} / DNA complex without nucleotide to evaluate the ability of the encapsulated nucleotide to improve the gene expression efficiency. 5 × 10 ⁵ cells per well of a 6-well plate were added and cultured for 24 hours. One hour before transfection, cells were transfected with serum-free transfection medium, and the prepared bPEI _{25 kDa} / nucleotide-pDNA complex or bPEI _{25 kDa} / pDNA complex solution was transfected into the cells. After culturing for 4 hours, the cells were changed with serum-containing cell culture medium, and then the gene expression efficiency was evaluated after further incubation for 44 hours.

As shown in FIG. 3, the particle size of bPEI _{25 kDa} / ATP-pDNA complex was about 200 nm up to 0.5, 4, 8 nmol of ATP per 1 microgram of pDNA, and positive charge up to 2 nmol of surface charge and negative charge Respectively. The particle size of the PLL / ATP-pDNA complex was about 200 nm up to about 9 nmol of ATP per microgram of pDNA and the surface charge was positive up to about 18 nmol.

When the concentration of ATP and GTP encapsulated in FIG. 4 was 0, the control complex, bPEI _{25 kDa} / pDNA complex, was formed and set to 1 for relatively comparative gene expression efficiency in HepG2 cells of this complex. bPEI _25kDa / ATP-pDNA complexes is about 80 times the gene expression efficiency at an ATP concentration of _{4nmol / μg pDNA, bPEI 25kDa /} GTP-pDNA complexes is increased about 35 times the gene expression efficiency when the GTP concentration of 2nmol / μg pDNA Respectively.

< Example  4> Bafilymycin  A1 or Chloroquine In this case Nucleotide  Gene Expression Efficiency of Polymeric Gene Drug Delivery Systems Included

In general, when a polymer gene drug transporter enters the cell through endocytosis, escape from the endosome is one of several important factors that determine gene expression efficiency. In order to evaluate whether the polymer gene drug transporter was introduced into cells using endocytosis, a reagent (chloroquine; 25 μM) or an endosomal breakdown inhibitor (Bafilomycin A1; 20 nM) was used in the transfection of bPEI _{25 kDa} / ATP- ) Was added to the medium and then the gene expression efficiency was evaluated.

When the polymer gene drug delivery system is introduced into cells via endocytosis, the polymer drug delivery system rapidly escapes from the endosome when chloroquine is added, as compared with the polymer gene carrier in the control experiment in which neither chloroquine nor Bafilomycin A1 is added. As the expression efficiency increases, the efficiency of gene expression is lowered or not affected because the polymer drug delivery system is difficult to escape into the endosome when Bafilomycin A1 is added. The same result was shown in the gene expression efficiency using the bPEI _{25 kDa} / ATP-pDNA complex as shown in Fig. That is, a polymer gene complex containing a nucleotide can enter the cell through endocytosis, and the nucleotide can destroy the endosomal membrane by hydrogen ion buffering.

Claims (4)

Adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP) (GTP), Cytidine monophosphate (CMP), Cytidine diphosphate (CDP), Cytidine triphosphate (CTP), Thymidine monophosphate (TMP) , Thymidine diphosphate (TDP), and thymidine triphosphate (TTP), wherein the nucleotide destabilizes or destroys the endosomal membrane, resulting in endosome excretion of the delivery drug (Endosomal escape). &Lt; / RTI &gt; delete Adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP) (GTP), Cytidine monophosphate (CMP), Cytidine diphosphate (CDP), Cytidine triphosphate (CTP), Thymidine monophosphate (TMP) (TTP), thymidine triphosphate (TTP), and the like. The charged nucleotides are proton-buffered, Has activity or hemolysis activity and is characterized by inducing endosomal escape of the drug through endosomolysis. Non-viral drug delivery composition.
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