KR100993024B1 - Pharmaceutical formulation for promoting cell proliferation - Google Patents

Abstract

The present invention provides a composition for enhancing cell growth comprising the compound of formula (1), the use of the compound of formula (1) for the preparation of a preparation for enhancing cell growth, cell growth enhancing method and formula comprising treating a compound of formula (1) to a cell Provided is a compound for enhancing cell growth comprising the compound of 1 and having a formulation of lipid nanoparticles, polymer nanoparticles, or polymer gel. Compound of Formula 1 according to the present invention can significantly enhance the growth of existing cells, such as brain cells, neurons, heart cells, stem cells, etc., useful for the treatment of diseases in which transplantation of these cells or an increase in the number of cells is required. It can be used, and by formulating into a formulation such as lipid nanoparticles, polymer nanoparticles can increase the intracellular delivery efficiency. The compound of formula 1 is easy to mass production and quality control, unlike the conventional growth factors, such as growth factors, easy to produce and low production costs, and there is no concern of immune side effects, active ingredients in the composition and preparations for enhancing cell growth This can be usefully used.

Description

Pharmaceutical formulation for promoting cell proliferation

The present invention relates to a composition and preparation for enhancing cell growth.

The apoptosis was introduced by Carl Vogt in 1842, re-examined as the cause of many diseases in 1965, and has been actively studied since the early 1990s. According to the results of the research so far, the cell death mechanism plays an important role in maintaining the development and homeostasis of living organisms, and excessive cell death leads to brain disease, neurological disease, heart disease, circulatory disease, diabetes and the like.

Stem cells can be differentiated into various cells such as blood cells, nerve cells, adipocytes, bone cells, and muscle cells (Pittenger et al., Science 284 (1999) 143-147). Stem cells capable of differentiation into specific cells are currently being studied for the treatment of various clinical diseases. However, when administering stem cells clinically, there is a limitation in that a large amount of cells must be repeatedly administered. This is because the cell viability after administration of stem cells in vivo is less than 10%, and the cell proliferation in vivo is low. If the growth rate of stem cells administered in vivo increases, it may reduce the cost of materials required to cultivate expensive stem cells in large quantities. In addition, the dose and frequency of administration of cells to the patient can be significantly reduced. These dose reductions can lead to lower national medical costs, improve patient compliance, and increase the value added of cellular medicines.

Cell growth rates have been studied in terms of temperature, nutrients, and acidity (pH). Studies on growth mechanisms and enhancers in living organisms have been focused on plant individuals, and studies on cancer cell growth mechanisms have been conducted in animal subjects to promote cancer cell death in the case of cancer cells. Since most animal cells except cancer cells grow slowly or do not grow like nerve cells or brain cells, a method of enhancing cell growth rate is required to cure brain diseases, neurological diseases, heart diseases, and circulatory diseases. .

To date, various growth factors of protein components have been studied to increase cell growth. For example, epidermal growth factor, fibroblast growth factor, transforming growth factor, vascular endothelial growth factor, platelet-derived growth factor (platelet) derived growth factor, neuronal growth factor, insulin-like growth factor, hepatocyte growth factor, placental growth factor, and the like. They are mainly used for cell-level growth and blood vessel production of circulatory diseases and cardiovascular diseases such as leukemia, neutropenia, myelodysplastic syndrome, and bone marrow transplantation and stem cell growth and differentiation. However, these growth factors are expensive to mass production and quality control compared to chemical compounds due to the nature of the protein material. In addition, since these growth factors send cell growth signals by the transporter of the cell membrane, there is a limit to the efficiency of increasing cell growth, as well as the possibility of inducing an immune response in the body if the separation and purification is not good.

The present invention is to provide a composition and preparation for enhancing cell growth, including a compound having excellent economical efficiency, productivity, stability, etc. as an active ingredient that can replace the growth factor that has been used for increasing cell growth.

The present inventors studied a method of enhancing cell growth using the compound as an active ingredient, and found that the compound of Formula 1 can be very useful for enhancing cell growth. Among the compounds of the formula (1), in particular, 4-chloro-2-[[3- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid (NS3694) conventionally inhibits the formation of apoptosome complexes. Although it is known that the present invention has revealed a novel use of the compound of formula 1 to significantly enhance cell growth. In the following examples, unlike z-VAD-fmk or Bax inhibiting peptide P5, which is known to inhibit apoptosis, does not show a significant effect on cell growth enhancement, the compound of formula 1 is an untreated group or apoptosis inhibitor Compared to the treatment group, the effect of enhancing cell growth was found to be remarkable.

Accordingly, the present invention provides a composition for enhancing cell growth comprising the compound of formula (1), the use of the compound of formula (1) for the preparation of a preparation for enhancing cell growth, and cell growth enhancer comprising treating a compound of formula (1) to a cell Provide a method.

[Formula 1]

Where

R ₁ and R ₂ are each independently H, halogen, C _1-4 alkyl, or CF ₃ ;

R ₃ is halogen.

In one embodiment, R ₁ and R ₂ are each independently H, F, Cl, methyl or CF ₃ ; R ₃ may be Cl.

In another embodiment, the compound of formula 1 is 4-chloro-2-[[3- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;

4-chloro-2-[[2- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;

4-chloro-2-[[4- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;

4-chloro-2-[[4-chloro-3- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;

2-[[3,5-bis (trifluoromethyl) phenyl] carbamoylamino] -4-chlorobenzoic acid;

2-[[2-chloro-5- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;

4-chloro-2-[(3-fluorophenyl) carbamoylamino] benzoic acid;

4-chloro-2-[(4-fluorophenyl) carbamoylamino] benzoic acid;

4-chloro-2-[(2-fluorophenyl) carbamoylamino] benzoic acid;

4-chloro-2-[(3-methylphenyl) carbamoylamino] benzoic acid;

4-chloro-2- (phenylcarbamoylamino) benzoic acid;

4-chloro-2-[(3-chlorophenyl) carbamoylamino] benzoic acid;

2-[(3-chlorophenyl) carbamoylamino] -4-fluorobenzoic acid; or

2-[(3-chlorophenyl) carbamoylamino] -5-fluorobenzoic acid.

In addition, when the compound of Formula 1 is formulated and administered, intracellular delivery efficiency is enhanced, thereby significantly increasing cell growth. In addition, it is possible to lower the frequency of drug administration by localizing the drug to specific cells and making a sustained-release polymer gel formulation that can control the rate of drug release in vivo.

Accordingly, the present invention also provides a formulation for enhancing cell growth, which comprises a compound of Formula 1, and has a formulation of lipid nanoparticles, polymer nanoparticles, or polymer gel.

[Formula 1]

Where

R ₁ and R ₂ are each independently H, halogen, C _1-4 alkyl, or CF ₃ ;

R ₃ is halogen.

In one embodiment, the lipid nanoparticles may have a formulation selected from the group consisting of, but not limited to, liposomes, micelles, emulsions, and solid lipid nanoparticles. .

Such lipid nanoparticles include one or more lipids selected from the group consisting of positively charged lipids, neutral lipids and negatively charged lipids. For example, positively charged lipids and neutral lipids may be used to prepare cationic liposomes, neutral lipids may be used to prepare neutral liposomes, and negatively charged lipids and neutral lipids may be used to prepare anionic liposomes.

For example, positively charged lipids include 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoyl glutamide, distea Distearoyl glutamide, dipalmitoyl glutamide, dioleoyl aspartamide, 1,2-dioleoyl-3-dimethylammonium-propane (1,2 -dioleoyl-3-dimethylammonium-propane, DODAP), 3β- [N- (N '(N', N'-dimethylaminoethane) carbamoyl] cholesterol (3β- [N- (N '(N', N'-dimethylaminoethane) carbamoyl] cholesterol DC-Chol), dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3 -ethylphosphocholine, EPC); 1,2-dimyristoyl-3-trimethylammoniumpropane (1,2-dimyristoyl-3-trimethylammonium-propane), 1,2-dipalmitoyl-3-trimethylammoniumpropane (1,2-dipalmitoyl-3- trimethylammonium-propane), 1,2-disteroyl-3-trimethylammoniumpropane (1,2-distearoyl-3-trimethylammonium-propane), 1,2-dioleoyl-3-trimethylammoniumpropane (1,2- dioleoyl-3-trimethylammonium-propane), 1,2-dimyristoyl-3-dimethylammonium propane (1,2-dimyristoyl-3-dimethylammonium-propane), 1,2-dipalmitoyl-3-dimethylammonium Propane (1,2-dipalmitoyl-3-dimethylammonium-propane), 1,2-stearoyl-3-dimethylammonium-propane (1,2-distearoyl-3-dimethylammonium-propane), 1,2-dioleo 1-3-Dimethylammonium propane (1,2-dioleoyl-3-dimethylammonium-propane), dimethyldioctadecylammonium bromide, 1,2-diurauroyl-sn-glycer-3--3-ethylforce Focoline (1,2-dilauroyl-sn-glycero-3-ethylphosphocholine), 1,2-dimyri Toyl-sn-glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (1 2-dipalmitoyl-sn-glycero-3-ethylphosphocholine), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1,2-distearoyl-sn-glycero-3-ethylphosphocholine), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine), 1,2-palmitoyl oleoyl-sn-glycero-3 Ethylphosphocholine (1,2-palmitoyloleoyl-sn-glycero-3-ethylphosphocholine);

Neutral lipids include L-α-phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phophocholine, DSPC), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), cholesterol, isopropyl myristate; 1,2-propionoyl-sn-glycero-3-phosphocholine (1,2-propionoyl-sn-glycero-3-phosphocholine), 1,2-butanoyl-sn-glycero-3-phospho Choline (1,2-butanoyl-sn-glycero-3-phosphocholine), 1,2-pentanoyl-sn-glycero-3-phosphocholine (1,2-pentanoyl-sn-glycero-3-phosphocholine), 1,2-caproyl-sn-glycero-3-phosphocholine (1,2-caproyl-sn-glycero-3-phosphocholine), 1,2-heptanoyl-sn-glycero-3-phosphocholine (1,2-heptanoyl-sn-glycero-3-phosphocholine), 1,2-capriloyl-sn-glycero-3-phosphocholine (1,2-capryloyl-sn-glycero-3-phosphocholine), 1,2-nonanoyl-sn-glycero-3-phosphocholine (1,2-nonanoyl-sn-glycero-3-phosphocholine), 1,2-capryl-sn-glycero-3-phospho Choline (1,2-capryl-sn-glycero-3-phosphocholine), 1,2-undecanoyl-sn-glycero-3-phosphocholine (1,2-undecanoyl-sn-glycero-3-phosphocholine) 1,2-lauroyl-sn-glycero-3-phosphocholine (1,2-lauroyl-sn-glycero-3-phosphocholine), 1,2-tridecanoyl-sn-glycero 3-phosphocholine (1,2-tridecanoyl-sn-glycero-3-phosphocholine), 1,2-myristoyl-sn-glycero-3-phosphocholine (1,2-myristoyl-sn-glycero -3-phosphocholine), 1,2-pentadecanoyl-sn-glycero-3-phosphocholine (1,2-pentadecanoyl-sn-glycero-3-phosphocholine), 1,2-palmitoyl-sn-glycer Rho-3-phosphocholine (1,2-palmitoyl-sn-glycero-3-phosphocholine), 1,2-heptadecanoyl-sn-glycero-3-phosphocholine (1,2-heptadecanoyl-sn- glycero-3-phosphocholine), 1,2-stearoyl-sn-glycero-3-phosphocholine (1,2-stearoyl-sn-glycero-3-phosphocholine), 1,2-nonadecanoyl-sn 1,2-nonadecanoyl-sn-glycero-3-phosphocholine, 1,2-arachidoyl-sn-glycero-3-phosphocholine (1,2-arachidoyl- sn-glycero-3-phosphocholine), 1,2-henicosanoyl-sn-glycero-3-phosphocholine (1,2-heniecosanoyl-sn-glycero-3-phosphocholine), 1,2-behenoyl -sn-glycero-3-phosphocholine (1,2-behenoyl-sn-glycero-3-phosphocholine), 1,2-trussano -sn-glycero-3-phosphocholine (1,2-trucisanoyl-sn-glycero-3-phosphocholine), 1,2-lignoseroyl-sn-glycero-3-phosphocholine (1,2 -lignoceroyl-sn-glycero-3-phosphocholine), 1,2-myristoleoyl-sn-glycero-3-phosphocholine (1,2-myristoleoyl-sn-glycero-3-phosphocholine), 1,2 1,2-myristelaidoyl-sn-glycero-3-phosphocholine, 1,2-palmitoleyl-sn-glycero-3-phospho Choline (1,2-palmitoleoyl-sn-glycero-3-phosphocholine), 1,2-palmitelaidoyl-sn-glycero-3-phosphocholine (1,2-palmitelaidoyl-sn-glycero-3-phosphocholine ), 1,2-petroselinoyl-sn-glycero-3-phosphocholine (1,2-petroselinoyl-sn-glycero-3-phosphocholine), 1,2-oleoyl-sn-glycero-3- Phosphocholine (1,2-oleoyl-sn-glycero-3-phosphocholine), 1,2-Elydoyl-sn-glycero-3-phosphocholine (1,2-elaidoyl-sn-glycero-3- phosphocholine), 1,2-linoleoyl-sn-glycero-3-phosphocholine (1,2-linoleoyl-sn-glyc ero-3-phosphocholine), 1,2-linolenoyl-sn-glycero-3-phosphocholine (1,2-linolenoyl-sn-glycero-3-phosphocholine), 1,2-icosenoyl-sn -Glycero-3-phosphocholine (1,2-eicosenoyl-sn-glycero-3-phosphocholine), 1,2-arachidonoyl-sn-glycero-3-phosphocholine (1,2-arachidonoyl- sn-glycero-3-phosphocholine), 1,2-rukoyl-sn-glycero-3-phosphocholine (1,2-erucoyl-sn-glycero-3-phosphocholine), 1,2-neunoyl- sn-glycero-3-phosphocholine (1,2-nervonoyl-sn-glycero-3-phosphocholine), L-α-phosphatidylethanolamine, 1,2-dicaproyl-sn 1,2-dicaproyl-sn-glycero-3-phosphoethanolamine, 1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine (1,2- dioctanoyl-sn-glycero-3-phosphoethanolamine), 1,2-dicapryl-sn-glycero-3-phosphoethanolamine (1,2-dicapryl-sn-glycero-3-phosphoethanolamine), 1,2-di Lauroyl-sn-glycero-3-phosphoethanolamine (1,2-dilau royl-sn-glycero-3-phosphoethanolamine), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), 1,2 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phospho Ethanolamine (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine), 1,2-diheptadecanoyl-sn 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (1,2 -distearoyl-sn-glycero-3-phosphoethanolamine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 1,2 - Elydoyl-sn-glycero-3-phosphoethanolamine (1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine), 1,2-dilinoyl-sn-glycero-3-phosphoethanol Amine (1,2-dilinoeoyl-sn-glycero-3-phosphoethanolamine), 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine (1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine), 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine), 1,2-docosahexaenoyl-sn -Glycero-3-phosphoethanolamine (1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine), cholesterol, and the like.

In addition, negatively charged lipids include L-α-phosphatidic acid, L-α-phosphatidyl-DL-glycerol, cardiolipin, and L-α. -Phosphatidylinositol (L-α-phosphatidylinositol), L-α-phosphatidylserine, 1,2-dilauroyl-sn-glycero-3- [phospho-rac- (1- Glycerol)] (1,2-dilauroyl-sn-glycero-3- [phospho-rac- (1-glycerol)], DLPG), 1,2-dilauroyl-sn-glycero-3- [phospho -L-serine] (1,2-dilauroyl-sn-glycero-3- [phospho-L-serine], DLPS), 1,2-dilauroyl-sn-glycero-3-phosphate (1,2 -dilauroyl-sn-glycero-3-phosphate, DLPA), 1,2-dimyristoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (1,2-dimyristoyl-sn -glycero-3- [phospho-rac- (1-glycerol)], DMPG), 1,2-dimyristoyl-sn-glycero-3- [phospho-L-serine] (1,2-dimyristoyl -sn-glycero-3- [phospho-L-serine], DMPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (1,2-dimyristoyl-sn-glycero-3-phosphate, DMPA ), 1,2-dioleoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (1,2-dioleoyl-sn-glycero-3- [phospho-rac- (1- glycerol)], DOPG), 1,2-dioleoyl-sn-glycero-3- [phospho-L-serine] (1,2-dioleoyl-sn-glycero-3- [phospho-L-serine] , DOPS), 1,2-dioleoyl-sn-glycero-3-phosphate (1,2-dioleoyl-sn-glycero-3-phosphate, DOPA), 1,2-dipalmitoyl-sn-glycero 3- [phospho-L-serine] (1,2-dipalmitoyl-sn-glycero-3- [phospho-L-serine], DPPS), 1,2-dipalmitoyl-sn-glycero-3- Phosphate (1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPA), 1,2-disteroyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (1,2 -distearoyl-sn-glycero-3- [phospho-rac- (1-glycerol)], DSPG), 1,2-disteroyl-sn-glycero-3- [phospho-L-serine] (1, 2-distearoyl-sn-glycero-3- [phospho-L-serine], DSPS), 1,2-disteroyl-sn-glycero-3-phosphate (1,2-distearoyl-sn-glycero-3- phosphate, DSPA), 1,2-Disteroyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (poly Ethylene glycol 2000] (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (polyethylene glycol) 2000]), 1,2-desteroyl-sn-glycero-3-phosphoethanolamine -N- [maleimide (polyethyleneglycol) 2000] (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) 2000]), 1,2-disteroyl-sn-glycer Rho-3-phosphoethanolamine-N- [PDP (polyethylene glycol) 2000] (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [PDP (polyethylene glycol) 2000]), 1-palmitoyl 2-oleyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (1-palmitoyl-2-oleoyl-sn-glycero-3- [phospho-rac- (1-glycerol) POPG), 1-palmitoyl-2-oleyl-sn-glycero-3- [phospho-L-serine] (1-palmitoyl-2-oleoyl-sn-glycero-3- [phospho-L- serine], POPS), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphate (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, POPA), oleic acid ), L-α-phosphatidylglycerol (L-α-phosphatidylglycerol), 1,2-dicaproyl-sn-letter Vertical-3-phosphoglycerol (1,2-dicaproyl-sn-glycero-3-phosphoglycerol), 1,2-dioctanoyl-sn-glycero-3-phosphoglycerol (1,2-dioctanoyl-sn- glycero-3-phosphoglycerol), 1,2-dicapryl-sn-glycero-3-phosphoglycerol (1,2-dicapryl-sn-glycero-3-phosphoglycerol), 1,2-dilauroyl-sn -Glycero-3-phosphoglycerol (1,2-dilauroyl-sn-glycero-3-phosphoglycerol), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (1,2-dimyristoyl -sn-glycero-3-phosphoglycerol), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol), 1,2-dipita Noyl-sn-glycero-3-phosphoglycerol (1,2-diphytanoyl-sn-glycero-3-phosphoglycerol), 1,2-diheptadecanoyl-sn-glycero-3-phosphoglycerol (1, 2-diheptadecanoyl-sn-glycero-3-phosphoglycerol), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (1,2-distearoyl-sn-glycero-3-phosphoglycerol), 1, 2-dioleoyl-sn-glycero-3-phospho Licerrole (1,2-dioleoyl-sn-glycero-3-phosphoglycerol), 1,2-dielaidoyl-sn-glycero-3-phosphoglycerol (1,2-dielaidoyl-sn-glycero-3- phosphoglycerol), 1,2-dilinoleyl-sn-glycero-3-phosphoglycerol (1,2-dilinoleoyl-sn-glycero-3-phosphoglycerol), 1,2-dilinolenoyl-sn-glycer Rho-3-phosphoglycerol (1,2-dilinolenoyl-sn-glycero-3-phosphoglycerol), 1,2-diarachidonoyl-sn-glycero-3-phosphoglycerol (1,2-diarachidonoyl-sn -glycero-3-phosphoglycerol), 1,2-docosahexaenoyl-sn-glycero-3-phosphoglycerol (1,2-docosahexaenoyl-sn-glycero-3-phosphoglycerol), L-α-phosphatidylinositol (L-α-phosphatidylinositol), 1,2-dioleoyl-sn-glycero-3-phosphoinositol (1,2-dioleoyl-sn-glycero-3-phosphoinositol), δ-erythro-sphingosyl phosphor Inositol (δ-erythro-sphingosyl phosphoinositol), L-α-phosphatidic acid, 1,2-dihexanoyl-sn-glycero-3-phosphate (1,2-d ihexanoyl-sn-glycero-3-phosphate), 1,2-dioctanoyl-sn-glycero-3-phosphate (1,2-dioctanoyl-sn-glycero-3-phosphate), 1,2-detecanoyl -sn-glycero-3-phosphate (1,2-didecanoyl-sn-glycero-3-phosphate), 1,2-didodecanoyl-sn-glycero-3-phosphate (1,2-didodecanoyl-sn- glycero-3-phosphate), 1,2-ditetradecanoyl-sn-glycero-3-phosphate (1,2-ditetradecanoyl-sn-glycero-3-phosphate), 1,2-dihexatecanoyl- sn-glycero-3-phosphate (1,2-dihexadecanoyl-sn-glycero-3-phosphate), 1,2-diphytanoyl-sn-glycero-3-phosphate (1,2-diphytanoyl-sn-glycero -3-phosphate), 1,2-diheptadecanoyl-sn-glycero-3-phosphate (1,2-diheptadecanoyl-sn-glycero-3-phosphate), 1,2-dioctadecanoyl-sn- Glycero-3-phosphate (1,2-dioctadecanoyl-sn-glycero-3-phosphate), 1,2-dioctadecadienoyl-sn-glycero-3-phosphate (1,2-dioctadecadienoyl-sn-glycero -3-phosphate), 1,2-dicosatetraenoyl-sn 1,2-diicosatetraenoyl-sn-glycero-3-phosphate, 1,2-didocosahexaenoyl-sn-glycero-3-phosphate (1,2-didocosahexaenoyl-sn- glycero-3-phosphate), L-α-phosphatidylserine, 1,2-dihexanoyl-sn-glycero-3-phosphoseline (1,2-dihexanoyl-sn-glycero-3 -phosphoserine), 1,2-dioctanoyl-sn-glycero-3-phosphoserine (1,2-dioctanoyl-sn-glycero-3-phosphoserine), 1,2-didecanoyl-sn-glycero- 3-phosphoseline (1,2-didecanoyl-sn-glycero-3-phosphoserine), 1,2-didodecanoyl-sn-glycero-3-phosphoseline (1,2-didodecanoyl-sn-glycero-3- phosphoserine), 1,2-ditetradecanoyl-sn-glycero-3-phosphoserine (1,2-ditetradecanoyl-sn-glycero-3-phosphoserine), 1,2-dihexadecanoyl-sn-glycero 3-phosphoseline (1,2-dihexadecanoyl-sn-glycero-3-phosphoserine), 1,2-diphytanoyl-sn-glycero-3-phosphoseline (1,2-diphytanoyl-sn-glycero-3 -phosphoserine), 1,2-diheptadecano -sn-glycero-3-phosphoseline (1,2-diheptadecanoyl-sn-glycero-3-phosphoserine), 1,2-dioctadecanoyl-sn-glycero-3-phosphoseline (1,2-dioctadecanoyl -sn-glycero-3-phosphoserine), 1,2-dioctadecenoyl-sn-glycero-3-phosphoserine (1,2-dioctadecenoyl-sn-glycero-3-phosphoserine), 1,2-di Octadecadienyl-sn-glycero-3-phosphoseline (1,2-dioctadecadienoyl-sn-glycero-3-phosphoserine), 1,2-diicosatetraenoyl-sn-glycero-3-phosphoseline ( 1,2-dieicosatetraenoyl-sn-glycero-3-phosphoserine), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoserine (1,2-didocosahexaenoyl-sn-glycero-3-phosphoserine), Cholesteryl hemisuccinate; CHEMS), cardiolipin, 1 ', 3'-bis [1,2-ditetradecanoyl-sn-glycero-3-phospho] -sn-glycerol (1', 3'-bis [ 1,2-ditetradecanoyl-sn-glycero-3-phospho] -sn-glycerol), 1 ', 3'-bis [1,2-dioctadecenoyl-sn-glycero-3-phospho] -sn -Glycerol (1 ', 3'-bis [1,2-dioctadecenoyl-sn-glycero-3-phospho] -sn-glycerol) and the like.

In addition to the lipids mentioned above, all lipids that maintain the fat-soluble portion of the lipids, such as galactose lipid derivatives, mannose lipid derivatives, folate lipid derivatives, polystyrene glycol lipid derivatives, biotin lipid derivatives, amino acid lipid derivatives, vitamin lipid derivatives, etc. Available as lipids.

In one embodiment, the lipid may be modified with polyethylene glycol. After the polyethylene glycol is linked to the lipids exemplified by a known method, the preparation of lipid nanoparticles can increase the in vivo working time of the preparation by inhibiting degradation by preventing the recognition of hydrolase. Polyethyleneglycol bound to lipids also facilitates binding of cell targeting ligands. In the following examples modified with polyethylene glycols such as 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -3000 (Avanti Polar Lipid Inc., USA) Lipid derivatives were used, but not limited thereto.

Lipid nanoparticles according to the present invention can be prepared using a general method for preparing liposomes, micelles, emulsions or solid lipid nanoparticles.

For example, in the case of liposomes, an organic solvent in which lipids such as positively charged lipids, neutral lipids, or negatively charged lipids are mixed with an organic solvent in which the compound of Formula 1 is dissolved, and all of the organic solvents are evaporated, the resulting lipid thin film is neutral pH. It can be prepared by hydration with buffer or water. In this case, the buffer solution used may include, for example, TAPS buffer solution, phosphate buffer solution (PBS), HEPES organic buffer solution, Tris buffer solution, Tricine buffer solution, and Tess ( TES) buffer solution, MOPS buffer solution, PIPES buffer solution, Cacodylate buffer solution, and other similar pH solutions in the body can be used alone or in combination with the above buffer solution. Can be. Lipid polymorphic globules in liposomes prepared according to the present invention can also be obtained by known methods, for example, by adding a buffer to the lipid thin film film and then stirring it with a vortex mixer. .

In the case of micelles, an organic solvent in which the compound of Formula 1 and lipids are dissolved can be prepared by dispersing in an aqueous solution, and an amphiphilic lipid is used as the lipid.

Emulsions can also be prepared by known methods, and W / O, O / W and W / O / W emulsions can be prepared, but preferably W / O emulsions in which water is dispersed in oil. This is suitable.

In the case of solid lipid nanoparticles, the drug is added to the lipid and then melted at 60 to 80 ° C. At the same time, an aqueous solution containing a predetermined ratio of a surfactant mixed in a separate container is heated to a similar temperature, followed by After mixing lightly, it can be prepared by maintaining a high temperature and emulsifying for 10 minutes at a speed of 8000rpm or more using a homogenizer and solidifying lipid components by leaving it in cold water at around 10 ° C.

 In one embodiment, the lipid nanoparticles may further comprise a surfactant. For example, surfactants can be used in combination with lipids for the formation of micelles or emulsion formulations.

Such surfactants may use one or more selected from the group consisting of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants.

For example, nonionic surfactants include sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan mono Polysols such as oleate (sorbian monooleate, Span 80), polyoxyethylene sorbitan monolaurate (polysorbate 20, Tween 20), or polyoxyethylene sorbitan monooleate (polysorbate 80, Tween 80) Bait system; Alkylphenol polyethylene oxides such as Triton-X-100; Polyethylene glycol monooleyl ether, ethylene glycol monododecyl ether, diethylene glycol monohexyl ether, triethylene glycol monododecyl ether alkyl (poly) ethylene glycol systems such as ether); Phloxamers; Alkyl polyglucosides such as octyl glucoside or cyclohexylmethyl β-D-maltoside; Alkylamine oxides such as lauryldimethylamine-oxide or dodecyl dimethylamine oxide; Pentaerythrityl palmitate, N-nonanoyl-N-methylglucamine, Steareth-4, Steareth-21, or capryloca Contains prill macrogol glycerides (caprylocaproyl macrogol-8 glyceride, Labrasol).

Cationic surfactants include trimethylhexadecyl ammonium chloride, quaternary ammonium ions, dodecyltrimethyl ammonium bromide, cetyl trimethylammonium bromide, or hexadecyl ammonium bromide hexadecyl trimethyl ammonium bromide).

Zwitterionic surfactants include dodecyl betaine, lecithin, or dodecyl dimethylammonophraphan sulfate (N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate). .

Anionic surfactants are also surfactants or laurosalcosine sodium salts containing sulfates, sulfonates or carboxylate anions such as 3- (N, Ndimethylpalmitylammonio) propane sulfonate (N) salts of fatty acids such as lauroylsarcosine sodium salt).

On the other hand, the polymer nanoparticles or polymer gel which is a form of the cell growth enhancing agent according to the present invention may include a natural polymer or a synthetic polymer.

In one embodiment, the natural polymer is not limited thereto, but may be selected from the group consisting of starch, cellulose, peptide, alginic acid, hyaluronic acid and chitosan.

In another embodiment, the synthetic polymer is polyethylene amine (Polyethylenimine, PEI) polyethylene glycol (Polyethylene glycol), poly vinyl alcohol (Poly vinyl alcohol), poly-N-vinylpyrrolidone (Poly N-vinylpyrrolidone), poly-lactic Poly (lactic-co-glycolic acid), polylactic acid (poly (L-lactic acid)), polyglycolic acid (poly glycolic acid) and beta-tricalcium phosphate It may be selected from the group consisting of.

The target cell receiving the compound of formula 1 through the cell growth enhancing agent of the present invention may be a cell that particularly requires the growth rate of the cell. For example, the proliferation of cells such as neurons, heart cells, pancreatic islets, hematopoietic stem cells, stem cells, skin cells, immune cells, etc., associated with the treatment of brain diseases, neurological diseases, kidney diseases, circulatory diseases, diabetes, leukemia, etc. Cells that are difficult or require massive proliferation of the cells can be target cells.

A composition or formulation comprising a compound of formula 1 according to the invention can be applied to cells on ex vivo or in vivo for enhancing the viability of such cells. For example, neurons, heart cells, pancreatic islets, hematopoietic stem cells, stem cells, etc. are generally transplanted into the body after increasing the number of cells in vitro due to the limited amount of cells. Thus, a composition or formulation comprising a compound of Formula 1 may primarily be used for an increase in the number of cells on ex vivo, or may be additionally used to enhance the growth rate of cells implanted in vivo.

In addition, the formulation comprising the compound of formula 1 according to the present invention may further include a cell targeting ligand for the cells that are subject to growth rate enhancement. The presence of such cell targeting ligands is advantageous because, for example, when the agent according to the present invention is administered in vivo, the compound of formula 1 into the specific cell can increase the delivery efficiency. Receptors present on the surface of nerve cells, hematopoietic stem cells, stem cells, immune cells and the like and ligands that bind specifically to them are well known in the art. Such cell targeting ligands may also be bound to lipids using methods known in the art, and drug delivery agents, such as lipid nanoparticles, may be prepared using lipids bound to cell targeting ligands.

In addition, the formulation comprising the compound of formula 1 according to the present invention may further comprise a second drug. Such a second drug may be a compound medicine, a protein medicine or a nucleic acid medicine. In one embodiment, the nucleic acid used as a nucleic acid medicament may be plasmid small interfering ribonucleic acid (siRNA).

For example, amlodipine, procaine, Bax inhibiting peptide P5, Bad inhibitor peptide, BI-6C9, Pifithrin-α, Pifithrin-μ, Cyclic Pifithrin-μ, Z-DQMD-FMK, AZ10417808, Ivachtin, Z-LEAHD-FMK, Z Known apoptosis inhibitors such as -YVAD-FMK, Z-DEVD-FMK, Z-VAD-FMK, Z-VDAD-FMK, necrostatin-1, MDL 28170, M50054, BBMP, Bongkrekic acid, Fasentin, etc. It can be used as a drug. These apoptosis inhibitors do not show the efficacy of enhancing cell viability, such as the compound of Formula 1 of the present invention, but may assist the cell viability enhancing effect of the compound of Formula 1 by inhibiting cell death. The following example shows the result of enhancing the survival rate of cells by co-administering this second drug together with the compound of Formula 1 according to the present invention.

Compound of Formula 1 according to the present invention can significantly enhance the growth of existing cells, such as brain cells, neurons, heart cells, stem cells, etc., useful for the treatment of diseases in which transplantation of these cells or an increase in the number of cells is required. It can be used, and by formulating into a formulation such as lipid nanoparticles, polymer nanoparticles can increase the intracellular delivery efficiency. The compound of formula 1 is easy to mass production and quality control, unlike the conventional growth factors, such as growth factors, easy to produce and low production costs, and there is no concern of immune side effects, active ingredients in the composition and preparations for enhancing cell growth This can be usefully used.

1 is a graph showing the cell growth promoting effect of the NS3694 containing emulsion on mouse primary microglia.
2 is a graph showing the cell growth promoting effect of NS3694 containing liposomes on rat astrocytes.
Figure 3 is a graph showing the cell growth promoting effect of NS3694 containing liposomes on mouse embryonic stem cells.
4 is a graph showing the cell growth promoting effect of NS3694 containing liposomes on rabbit adipocytes.
5 is a graph showing the cell growth promoting effect of NS3694 containing micelles on primary porcine islet cells.
6 is a graph showing the cell growth promoting effect of the NS3694 containing liposome preparations on stem cells derived from human adipose tissue.
Figure 7 is a graph showing the cell growth promoting effect of NS3694-containing solid lipid nanoparticle preparations on human adipose tissue-derived stem cells.
8 is a graph showing the cell growth promoting effect of the NS3694-containing solid lipid nanoparticle preparations on human bone marrow-derived stem cells.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[Example]

Example 1.Preparation of Negatively Charged PG Liposomes Containing NS3694

5.5 mg of anionic phospholipid phosphatidylglycerol (PG, Avanti Polar Lipid Inc. USA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipid Inc. USA ) 6.8 mg each was dissolved in 1 ml chloroform. 5 mg of NS3694 (sigma, USA) was dissolved in 1 ml of ethanol. Based on the total moles, PG: DOPE: NS3694 is taken at a molar ratio of 5: 5: 10 and mixed in a Pyrex 10 ml glass diaphragm vial, followed by rotary evaporation at low speed until all chloroform has evaporated in a nitrogen environment. It was made into a thin film. Anionic multilayer thin-film liposomes containing NS3694 were prepared by adding 1 ml of HEPES, ACROS organic buffer solution to this thin film and vortexing for 3 minutes. Liposomal suspensions were passed through a particle homogenization maker (extruder, Northern Lipid Inc., Canada) three times equipped with a 0.2 μm polycarbonate membrane to homogenize the size. The resulting NS3694 liposomes were stored at 4 ° C. until use.

Example 2 Preparation of Negatively Charged Cardiolipin Liposomes Containing NS3694

Anionic phospholipid, anionic phospholipid (cardiolipin, Avanti Polar Lipid Inc., USA) 4.06 mg and neutral phospholipid (PC, phosphatidylcholine, Avanti polar lipid Inc, USA) were dissolved in 1 ml of chloroform, respectively. 5 mg of NS3694 (sigma, USA) was dissolved in 1 ml of ethanol. Based on the total moles, cardiolipin: PC: NS3694 is taken at a molar ratio of 5: 5: 5 and mixed in a Pyrex 10 ml glass diaphragm vial, followed by rotary evaporation at low speed until all chloroform has evaporated in a nitrogen environment. Prepared with a lipid thin film. Liposomes containing NS3694 were prepared by adding 1 ml of HEPES, ACROS organic buffer solution to this thin film and vortexing for 3 minutes. Liposomal suspensions were passed through a particle homogenization maker (extruder, Northern Lipid Inc., Canada) three times equipped with a 0.2 μm polycarbonate membrane to homogenize the size. The resulting NS3694 liposomes were stored at 4 ° C. until use.

Example 3. Preparation of NS3694-Containing Positively Charged DOTAP Liposomes

2.8 mg of cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, Avanti Polar Lipid Inc., USA) and cholesterol, a neutral lipid ( 1.93 mg of Avanti Polar Lipid Inc., USA) was dissolved in 1 ml of chloroform, respectively. 5 mg of NS3694 (sigma, USA) was dissolved in 1 ml of ethanol. Based on the total moles, DOTAP: cholesterol: NS3694 is taken at a 5: 5: 10 molar ratio and mixed in a Pyrex 10 ml glass diaphragm vial, followed by rotary evaporation at low speed until all chloroform has evaporated in a nitrogen It was made into a thin film. Liposomes containing NS3694 were prepared by adding 1 ml of an organic buffer solution (HEPES, ACROS) to the thin film and vortexing for 3 minutes. Liposomal suspensions were passed through a particle homogenization maker (extruder, Northern Lipid Inc., Canada) three times equipped with a 0.2 μm polycarbonate membrane to homogenize the size. The resulting NS3694 liposomes were stored at 4 ° C. until use.

Example 4 Preparation of NS3694-Containing Positively Charged EDOPC Liposomes

6.8 mg of cationic phospholipid 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC, Avati Polar Lipid, USA) 6.8 mg of neutral phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipid Inc. USA) was dissolved in 1 ml of chloroform, respectively. 5 mg of NS3694 (sigma, USA) was dissolved in 1 ml of ethanol. Based on the total moles, EDOPC: DOPE: NS3694 is taken at a molar ratio of 5: 5: 5 and mixed in a Pyrex 10 ml glass diaphragm vial, followed by rotary evaporation at low speed until all chloroform has evaporated in a nitrogen environment to provide lipids. It was made into a thin film. Liposome containing NS3694 was prepared by adding 1 ml of an organic buffer solution of HEPES (HEPES, ACROS) to this thin film and vortexing for 3 minutes. Liposomal suspensions were passed through a particle homogenization maker (extruder, Northern Lipid Inc., Canada) three times equipped with a 0.2 μm polycarbonate membrane to homogenize the size. The resulting NS3694 liposomes were stored at 4 ° C. until use.

Example 5 Preparation of Positively Charged DOTMA Liposomes Containing NS3694

Cationic phospholipid N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA, Avati Polar Lipid, USA) 8 mg and neutral phospholipid 1,2-dioleoyl-sn 6.8 mg of -glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipid Inc. USA) was dissolved in 1 ml of chloroform, respectively. 5 mg of NS3694 (sigma, USA) was dissolved in 1 ml of ethanol. Based on the total moles, EPC: DOPE: NS3694 is taken at a molar ratio of 4: 4: 10 and mixed in a Pyrex 10 ml glass diaphragm vial, followed by rotary evaporation at low speed until all chloroform has evaporated in a nitrogen environment. It was made into a thin film. Liposome containing NS3694 was prepared by adding 1 ml of an organic buffer solution of HEPES (HEPES, ACROS) to this thin film and vortexing for 3 minutes. Liposomal suspensions were passed through a particle homogenization maker (extruder, Northern Lipid Inc., Canada) three times equipped with a 0.2 μm polycarbonate membrane to homogenize the size. The resulting NS3694 liposomes were stored at 4 ° C. until use.

Example 6 Preparation of NS3694-Containing Alginic Acid Fine Particles

Alginic acid nanoparticles containing NS3694 were prepared by dissolving NS3694 in a concentration of 0.1 mg / ml in 10 mg / ml of alginic acid solution, and then dropping the solution dropwise into 10 mM CaCl ₂ solution. Alginic acid nanoparticles were stored at 4 ° C. until lyophilization.

Example 7 Preparation of Cationic Solid Lipid Nanoparticles Containing NS3694

7.4 g of cationic phospholipid diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine) (DPhPE, Avanti Polar Lipid, USA) It was melted at 65 ° C., a high temperature, and 10 g of NS3694 was dissolved at 70 ° C. in 10 ml ethanol. A mixture of glycerin, butylene glycol, span20, and span80 in a molar ratio of 4: 3: 8: 5 (20 ml) was added to an organic buffer solution of HEPES, ACROS, to a total of 35 ml, which was 85 degrees. The molten lipid containing drug was added to the aqueous phase by heating until it was added to the aqueous phase, and then emulsified at a speed of 16,000 rpm using a homogenizer while maintaining 85 ° C. Residual organics were removed using a heating mantle. The resulting emulsion was solidified by stirring at 1,000 rpm in a cold water bath at 4 ℃ water. The solid lipid nanoparticles obtained were stored at 4 ° C. until use.

Example 8 Preparation of Neutral Solid Lipid Nanoparticles Containing NS3694

3.4 g of lipid lauric acid (Lauric acid, sigma, USA) was melted at 70 ° C., higher than the melting point of lipids, and 2 g of NS3694 was dissolved at 70 ° C. in 10 ml ethanol. A mixture of Tween 20 and Tween 80 in a 6: 5 molar ratio (20 ml) was added to Hips (HEPES, Acros, USA) in an organic buffer solution to a total of 35 ml, and heated separately until it reached 85 degrees. The molten lipid contained was added to the aqueous phase, and then emulsified at a speed of 14,000 rpm using a homogenizer while maintaining 85 ° C. Residual organics were removed using a heating mantle. The resulting emulsion was solidified by stirring at 1,000 rpm in a cold water bath at 4 ℃ water. The solid lipid nanoparticles obtained were stored at 4 ° C. until use.

Example 9 Emulsion Preparation Containing NS3694

9.2 mg of isopropyl myristate lipid (Fluka, USA) and NS3694 (Sigma, USA), and Tween 80 (USB, USA) were mixed in a 5: 3: 0.5 molar ratio to mix the mixture with phosphate buffer Added to a volume ratio of 1:10 and then homogenized using a homogenizer to prepare a water-in-oil (w / o type) emulsion in which lipids were dispersed in an aqueous solution layer. The resulting emulsion was stored at 4 ° C. until use.

Example 10 Preparation of Mice with NS3694

5: 2: 3 mole of oleic acid (Avanic Polar Lipid, USA) 2.3 mg, surfactant polyethylene glycol monooleyl ether (sigma, USA), NS3694 (sigma, USA) The mixture was taken in a ratio, and then mixed with a phosphate buffer solution in a volume ratio of 1:10, mixed several times with vortexing, and micelles were prepared using an ultrasonic generator for about 1 minute. The micelle obtained was stored at 4 ° C. until use.

Example 11.NS3694 and Preparation of Liposomes Containing Polyethyleneglycol Lipid Derivatives

1,2-Diacyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -3000 (Avanti Polar Lipid Inc., USA), a polyethylene glycol lipid derivative, 4.8 mg, anionic phospholipid 4.06 mg of phosphoric anionic phospholipid (cardiolipin, Avanti Polar Lipid Inc., USA) and 3.81 mg of neutral phospholipid (PC, phosphatidylcholine) were dissolved in 1 ml of chloroform. 5 mg of NS3694 (sigma, USA) was dissolved in 1 ml of ethanol. Anionic liposomes were prepared in the same manner as in Example 2 after the molar ratio of 0.05: 5: 5: 5 based on the total moles. The obtained liposomes were stored at 4 ° C. until use.

Example 12 Preparation of Liposomes Containing NS3694 and Cell Specific Transport Inducing Substances

2 mg of RGD-mPEG-DSPE (Avanti Polar Lipid, USA), a lipid derivative that induces cell-specific transport, 3.08 mg of cardiolipin (Avanti Polar Lipid Inc., USA), an anionic phospholipid, and neutral phospholipid 5.32 mg of phosphorus phosphatidylcholine (PC) was dissolved in 1 ml of chloroform, respectively. 5 mg of NS3694 (sigma, USA) was dissolved in 1 ml of ethanol. Anionic liposomes were prepared in the same manner as in Example 2 after the molar ratio of 0.05: 5: 5: 5 based on the total moles. The micelle obtained was stored at 4 ° C. until use.

Example 13 Preparation of Liposomes Containing NS3694 and siRNA Complex

After preparing the cationic liposome of Example 3, 10 μl of GL2 siRNA was electrostatically coupled to 20 μl liposome solution at room temperature to prepare a liposome containing a combination of NS3694 and siRNA.

Example 14 Preparation of Liposomes Containing NS3694 and Amlodipine in Combination

Anionic liposomes were prepared by the method of Example 2, and then 100 μl of liposomes were positively bound with amlodipine (100 ug / mL) to the liposome surface, followed by PD-10 Columns (GE Healthcare, America). Amlodipine that was not bound to the liposome surface was removed. The resulting NS3694 and amlodipine containing liposomes were stored at 4 ° C. until use.

Comparative Example 1. Preparation of Drug-Free Anionic PG Liposomes

5.5 mg of anionic phospholipid phosphatidylglycerol (PG, Avanti Polar Lipid Inc. USA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipid Inc. USA ) 6.8 mg each was dissolved in 1 ml chloroform. Liposomes were prepared by the method of Example 1 by taking a molar ratio of 5: 5 PG and DOPE based on the total moles. The obtained liposomes were stored at 4 ° C.

Comparative Example 2. Preparation of Anionic PG Liposomes Containing z-VAD-fmk

5.5 mg of anionic phospholipid phosphatidylglycerol (PG, Avanti Polar Lipid Inc. USA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipid Inc. USA ) 6.8 mg each was dissolved in 1 ml chloroform. 5 mg of z-VAD-FMK (Tocris, USA) was dissolved in 1 ml of ethanol. Liposomes were prepared by the method of Example 1 by taking a molar ratio of 5: 5: 10 PG: DOPE: z-VAD-FMK based on the total moles. The obtained liposomes were stored at 4 ° C.

Comparative Example 3. Preparation of Drug-Free Anionic Cardiolipin Liposomes

4.06 mg of anionic phospholipid (cardiolipin, Avanti Polar Lipid Inc., USA) and 3.81 mg of neutral phospholipid (PC, phosphatidylcholine, Avanti Polar Lipid Inc, USA) were dissolved in 1 ml of chloroform. Based on the total number of moles, cardiolipin and PC were taken in a molar ratio of 5: 5, mixed in a Pyrex 10 ml glass diaphragm vial, and then liposomes were prepared by the method of Example 2. The obtained liposomes were stored at 4 ° C.

Comparative Example 4 Preparation of Anionic Cardiolipin Liposomes Containing Bax Inhibiting Peptide P5

4.06 mg of anionic phospholipid (cardiolipin, Avanti Polar Lipid Inc., USA) and 3.81 mg of neutral phospholipid (PC, phosphatidylcholine, Avanti Polar Lipid Inc, USA) were dissolved in 1 ml of chloroform. 4.8 mg of Bax inhibiting peptide P5 (Merck, USA) was dissolved in 1 ml of ethanol. Based on the total number of moles, cardiolipin, PC, Bax inhibiting peptide P5 was taken in a molar ratio of 5: 5: 5, mixed in a Pyrex 10 ml glass diaphragm vial, and liposomes were prepared by the method of Example 2. The obtained liposomes were stored at 4 ° C.

Comparative Example 5. Preparation of Drug-Free Cationic Liposomes

2.8 mg of cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, Avanti Polar Lipid Inc., USA) and cholesterol, a neutral lipid ( 1.93 mg of Avanti Polar Lipid Inc., USA) was dissolved in 1 ml of chloroform, respectively. Liposomes were prepared by the method of Example 3 after DOTAP and cholesterol were taken at a molar ratio of 5: 5 and mixed in a Pyrex 10 ml glass septum vial.

Comparative Example 6. Preparation of Cationic Liposomes Containing z-VAD-fmk

2.8 mg of cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, Avanti Polar Lipid Inc., USA) and cholesterol, a neutral lipid ( 1.93 mg of Avanti Polar Lipid Inc., USA) was dissolved in 1 ml of chloroform, respectively. 6.2 mg of z-VAD-fmk (tocris, USA) was dissolved in 1 ml of ethanol. Based on the total number of moles, DOTAP, cholesterol, z-VAD-fmk was taken in a molar ratio of 5: 5: 10, mixed in a Pyrex 10 ml glass diaphragm vial, and then liposomes were prepared by the method of Example 3.

Comparative Example 7. Preparation of Drug-Free Cationic Solid Lipid Nanoparticles

7.4 g of cationic phospholipid diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine) (DPhPE, Avanti Polar Lipid, USA) Melt at 65 ° C., high temperature. A mixture of glycerin, butylene glycol, span20, and span80 in a molar ratio of 4: 3: 8: 5 (20 ml) was added to an organic buffer solution of HEPES, ACROS, to a total of 35 ml, which was 85 degrees. Separately heated until it was prepared in the same manner as in Example 7. The obtained solid lipid nanoparticles were stored at 4 ° C.

Comparative Example 8. Preparation of Cationic Solid Lipid Nanoparticles Containing Bax Inhibitor Peptide V5

7.4 g of cationic phospholipid diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine) (DPhPE, Avanti Polar Lipid, USA) It was melted at a high temperature of 65 ℃, 1 g Bax inhibitor peptide V5 (tocris, USA) was dissolved in 70 ℃ in 10ml ethanol. A mixture of glycerin, butylene glycol, span20, and span80 in a molar ratio of 4: 3: 8: 5 (20 ml) was added to an organic buffer solution of HEPES, ACROS, to a total of 35 ml, which was 85 degrees. Separately heated until it was prepared in the same manner as in Example 7. The obtained solid lipid nanoparticles were stored at 4 ° C.

Comparative Example 9. Preparation of Cationic Solid Lipid Nanoparticles Containing z-VAD-fmk

7.4 g of cationic phospholipid diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine) (DPhPE, Avanti Polar Lipid, USA) It was melted at 65 ° C. at a high temperature, and 4 g of z-VAD-fmk was dissolved at 70 ° C. in 10 ml ethanol. A mixture of glycerin, butylene glycol, span20, and span80 in a molar ratio of 4: 3: 8: 5 (20 ml) was added to an organic buffer solution of HEPES, ACROS, to a total of 35 ml, which was 85 degrees. Separately heated until it was prepared in the same manner as in Example 7. The obtained solid lipid nanoparticles were stored at 4 ° C.

Comparative Example 10 Preparation of Neutral Solid Lipid Nanoparticles

3.4 g of lauric acid (Sigma, USA) was melted at 70 ° C., above the melting point of the lipid. A mixture of Tween20 and Tween80 in a molar ratio of 6: 5 (20 ml) was added to an organic buffer solution of HEPES, ACROS, to a total of 35 ml, which was heated separately until it reached 85 degrees, and the molten lipid was It was added to the water phase and prepared by the method of Example 8.

Comparative Example 11 Preparation of Neutral Solid Lipid Nanoparticles Containing Necrostatin-1

3.4 g of lipid lauric acid (Sigma, USA) was melted at 70 ° C., higher than the melting point of lipids, and 1.7 g of Necrostatin-1 was dissolved in 70 ml of 10 ml ethanol. A mixture of Tween20 and Tween80 in a molar ratio of 6: 5 (20 ml) was added to an organic buffer solution of HEPES (HEPES, ACROS) to make a total of 35 ml, and the mixture was heated separately until it reached 85 degrees. Prepared.

Comparative Example 12. Drug-Free Emulsion Preparation

9.2 mg of isopropyl myristate lipid (fluka, USA) and Tween 80 (USB, USA) were mixed in a 10: 1 molar ratio, and the mixture was added to the phosphate buffer solution in a volume of 1:10. Prepared by the method of Example 10 below. The obtained emulsion was stored at 4 ° C. until just before use.

Comparative Example 13. Emulsion Preparation Containing z-VAD-fmk

Mix the phosphate buffer by mixing 9.2 mg of isopropyl myristate lipid (fluka, USA) with 5.7 mg of z-VAD-fmk and Tween 80 (USB, USA) in a molar ratio of 5: 3: 0.5. The solution was added in the ratio of 1:10 by volume and then prepared by the method of Example 10. The obtained emulsion was stored at 4 ° C. until just before use.

Comparative Example 14. Preparation of drug-free micelles

Oleic acid (Avanic Polar Lipid, USA) and a surfactant, polyethylene glycol monooleyl ether (sigma, USA), are mixed in a 5: 2 molar ratio, and then mixed solution to phosphate buffer The solution was mixed in a volume ratio of 1:10 and prepared by the method of Example 11. The micelles obtained were stored at 4 ° C. until just before use.

Comparative Example 15 Preparation of Micelle Containing Bax Inhibiting Peptide P5

Oleic acid (Avanic Polar Lipid, USA), surfactant polyethylene glycol monooleyl ether (sigma, USA) and Bax inhibiting peptide P5 is mixed in a molar ratio of 5: 2: 0.1 Then, the mixed solution (20 ml) to the phosphate buffer solution was mixed in a volume ratio of 1:10 and prepared by the method of Example 11. The micelles obtained were stored at 4 ° C. until just before use.

[Experimental Example]

Cell culture

Mouse microglia were cultured for 4-7 days using CMEM-2469 (ATCC, USA) cell line using DMEM (Gibco, USA), followed by extracting only microglia using DMEM (Gibco, USA). And incubated. Rat chondrocytes were extracted from the leg joints of rats, and then the progenase and type II collagenase (Invitrogen USA) were used to separate the chondrocytes that make up the cartilage tissue and to remove DMEM (Gibco, USA). And cultured using. In the case of rat astrocytes, primary astrocytes (Gibco, USA) extracted from a fetus at 19 days were cultured using DMEM (Gibco, USA). The extracted astrocytes were cultured using DMEM (Gibco, USA). Embryonic stem cells were cultured using TK (# 1, ATCC, USA) using DMEM (Gibco, USA). Rabbit adipocytes were extracted from the subcutaneous fat of rabbits and cultured using DMEM (Gibco, USA). Porcine islets were treated with collagenase V by the conventional method, and the cells were sieved and cultured in DMEM (Gibco, USA). Human adipose derived stem cells receive primary adipose cells from donors, extracted using stem cell specific factors, and contain 10% fetal calf serum. Cultured using DMEM ((Dulbecco's modified eagles medium, Gibco, USA). Human bone marrow derived stem cells were used to isolate CD34 ⁺ cells from bone marrow using magnetic beads. Culture was performed using DMEM ((Dulbecco's modified eagles medium, Gibco, USA) containing% calf fetal serum.

Experimental Example 1. Cell proliferation promoting effect of NS3694 containing emulsion on mouse primary microglia

The effect of NS3694-containing emulsion on cell growth was evaluated in mouse primary microglia. Cells were seeded in 24-well plates to be 4 × 10 ⁴ cells per well and incubated for 24 hours, followed by emulsion containing NS3694 prepared in Example 9 and drug-free prepared in Comparative Example 12. The emulsion and 0.2 ml of the emulsion containing z-VAD-fmk of Comparative Example 13 were respectively treated with mouse primary microglia. After 72 hours, the growth rate of the cells was evaluated. MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) was used for cell growth evaluation. For each sample, add MTT solution to 10% of the medium, incubate for another 4 hours, remove supernatant, add 0.04 N isopropanol solution, and absorb its absorbance at 570 nm using an ELISA reader. Was measured. Untreated group cells were used as a control. As shown in FIG. 1, the highest cell growth was observed in the emulsion treatment group containing NS3694.

Experimental Example 2. Cell growth promoting effect of NS3694-containing liposomes on rat astrocytes

The effects of liposomes containing NS3694 on cell growth were evaluated in rat astrocytes. Cells were seeded in 24-well plates to be 4 × 10 ⁴ cells per well and incubated for 24 hours, followed by liposomes containing NS3694 prepared in Example 1 and no drug prepared in Comparative Example 1. 0.1 ml of liposomes containing liposomes and z-VAD-fmk of Comparative Example 2 were treated with rat astrocytes. After 72 hours, the growth rate of the cells was evaluated. MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) was used for cell growth evaluation. For each sample, add MTT solution to 10% of the medium, incubate for another 4 hours, remove supernatant, add 0.04 N isopropanol solution, and absorb its absorbance at 570 nm using an ELISA reader. Was measured. As a control, cells treated with nothing were used. As shown in FIG. 2, the cell growth of rat astrocytes was significantly higher in the liposome treatment group containing NS3694.

Experimental Example 3. Cell growth promoting effect of liposomes containing NS3694 on mouse embryonic stem cells

Mouse embryonic stem cells were evaluated for the effect of NS3694-containing liposomes on cell growth. The cell growth rate of the transplanted cells obtained by adding the NS3694 liposome of Example 11 to the subcutaneous site of the mouse and adding the drug-free liposome of Comparative Example 3 to the obtained mouse embryonic stem cells The change in size was measured and evaluated. As shown in Figure 3, in the case of stem cells administered with the NS3694-containing liposomes, it was observed that the growth of the cells in vivo.

Experimental Example 4. Cell growth promoting effect of liposomes containing NS3694 on rabbit adipocytes

Rabbit fat cells were evaluated for the effect of NS3694-containing liposomes on cell growth. Cells were seeded in 24-well plates to be 5 × 10 ⁴ cells per well and incubated for 24 hours, followed by liposomes containing NS3694 prepared in Example 3 and drug-free prepared in Comparative Example 5. Cells were treated with 0.1 ml of liposomes containing liposomes and z-VAD-fmk of Comparative Example 6, respectively. After 72 hours, the growth rate of the cells was evaluated. MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) was used as a cell growth evaluation method. For each sample, add MTT solution to 10% of the medium, incubate for another 4 hours, remove supernatant, add 0.04 N isopropanol solution, and absorb its absorbance at 570 nm using an ELISA reader. Was measured. As a control, cells treated with nothing were used. As shown in FIG. 4, cell growth of the rabbit adipocytes was significantly higher in the liposome treatment group containing NS3694.

Experimental Example 5. Cell growth promoting effect of NS3694-containing micelles on primary porcine islets

Primary pig pancreatic islet cells were evaluated for the effect of NS3694-containing micelles on cell growth. Cells were seeded in 24-well plates to be 5 × 10 ⁴ cells per well and incubated for 24 hours, followed by micelles containing NS3694 prepared in Example 10 and no drug prepared in Comparative Example 14. 0.1 ml of micelles containing micelles and Bax inhibiting peptide P5 of Comparative Example 15 were treated with cells, and the growth rate of the cells was evaluated after 72 hours. As a cell growth evaluation method, the cell growth evaluation was WST-1 (Roche, Switzerland). WST-1 (Roche) solution was added to 10% of each medium, supernatant medium was removed after 4 hours, and then 0.04 N isopropanol solution was added, followed by using an ELISA reader at 570 nm. Absorbance was measured. As a control, cells treated with nothing were used. As shown in FIG. 5, significantly higher cell growth was observed in the micelle-treated group containing NS3694 compared to the drug-free micelle-treated group or the Bax inhibiting peptide P5 micelle-treated group.

Experimental Example 6. Cell growth promoting effect of NS3694 containing liposome preparations on stem cells derived from human adipose tissue

The cell growth promoting effect of the NS3694-containing liposome preparation was tested on human adipose tissue-derived stem cells in the following manner. Cells were seeded in 24-well plates to be 4 × 10 ⁴ cells per well and incubated for 24 hours, followed by encapsulation of NS3694 prepared in Examples 2, 12, 13 and 14 The liposomes and the drug-free cardiolipin liposomes prepared in Comparative Example 3 and 0.1 ml of cardiolipin liposomes encapsulated with Bax inhibiting peptide P5 of Comparative Example 4 were respectively treated with stem cells derived from human adipose tissue. After 72 hours, the cell growth rate was evaluated by MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) method. For each sample, add MTT solution to 10% of the medium, incubate for another 4 hours, remove supernatant, add 0.04 N isopropanol solution, and absorb its absorbance at 570 nm using an ELISA reader. Was measured. As a control, cells treated with nothing were used. As shown in FIG. 6, in the case of liposomes encapsulated with NS3694, cell growth was significantly higher than cardiolipin liposomes in which nothing was encapsulated, and cardiolipin liposomes containing only Bax inhibiting peptide P5.

Experimental Example 7. Cell growth promoting effect of NS3694-containing solid lipid nanoparticle preparations on human adipose tissue-derived stem cells

The cell growth promoting effect of NS3694-containing solid lipid nanoparticle preparations was tested on human adipose tissue-derived stem cells by the following method. Cells were seeded in 24-well plates to be 4 × 10 ⁴ cells per well and incubated for 24 hours, followed by NS3694-encapsulated solid lipid nanoparticles prepared in Example 7 and prepared in Comparative Example 7. Drug-free solid lipid nanoparticles, solid lipid nanoparticles containing z-VAD-fmk of Comparative Example 8, and 0.3 ml of solid lipid nanoparticles encapsulated with Bax inhibitor peptide V5 of Comparative Example 9 were respectively adjuvant human adipocyte derived stem cells. Was treated. After 72 hours, the cell growth rate was evaluated by MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) method. For each sample, add MTT solution to 10% of the medium, incubate for another 4 hours, remove supernatant, add 0.04 N isopropanol solution, and absorb its absorbance at 570 nm using an ELISA reader. Was measured. As a control, cells treated with nothing were used. As shown in FIG. 7, in the case of solid lipid nanoparticles encapsulated with NS3694, solid lipid nanoparticles containing nothing, solid lipid nanoparticles containing z-VAD-fmk, and solid lipids containing only Bax ch inhibiting peptide V5 Significantly higher cell growth was observed compared to nanoparticles.

Experimental Example 8. Cell growth promoting effect of NS3694-containing solid lipid nanoparticle preparations on human bone marrow-derived stem cells

Cell growth promoting effects of solid lipid nanoparticles encapsulated with NS3694 were tested on human bone marrow-derived stem cells in the following manner. Cells were seeded in 24-well plates to be 4 × 10 ⁴ cells per well and incubated for 24 hours, followed by NS3694-embedded solid lipid nanoparticles prepared in Example 8 and prepared in Comparative Example 10. The drug-free solid lipid nanoparticles and 0.2 ml of the solid lipid nanoparticles containing the necrostatin-1 of Comparative Example 11 were treated with human bone marrow-derived stem cells, respectively. After 72 hours, the cell growth rate was evaluated by MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-di-phenyl tetrazolium bromide) method. For each sample, add MTT solution to 10% of the medium, incubate for another 4 hours, remove supernatant, add 0.04 N isopropanol solution, and absorb its absorbance at 570 nm using an ELISA reader. Was measured. As a control, cells treated with nothing were used. As shown in FIG. 8, in the case of solid lipid nanoparticles encapsulated with NS3694, significantly higher stem cell growth was observed in comparison with solid lipid nanoparticles containing nothing, and solid lipid nanoparticles containing necrostatin-1. It became.

Claims (20)

A composition for enhancing cell growth comprising the compound of Formula 1 below.
[Formula 1]

Where
R ₁ and R ₂ are each independently H, halogen, C _1-4 alkyl, or CF ₃ ;
R ₃ is halogen.
The method of claim 1,
R ₁ and R ₂ are each independently H, F, Cl, methyl or CF ₃ ;
R ₃ is Cl is a composition for enhancing cell growth.
The method of claim 1,
Compound of Formula 1 is
4-chloro-2-[[3- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;
4-chloro-2-[[2- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;
4-chloro-2-[[4- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;
4-chloro-2-[[4-chloro-3- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;
2-[[3,5-bis (trifluoromethyl) phenyl] carbamoylamino] -4-chlorobenzoic acid;
2-[[2-chloro-5- (trifluoromethyl) phenyl] carbamoylamino] benzoic acid;
4-chloro-2-[(3-fluorophenyl) carbamoylamino] benzoic acid;
4-chloro-2-[(4-fluorophenyl) carbamoylamino] benzoic acid;
4-chloro-2-[(2-fluorophenyl) carbamoylamino] benzoic acid;
4-chloro-2-[(3-methylphenyl) carbamoylamino] benzoic acid;
4-chloro-2- (phenylcarbamoylamino) benzoic acid;
4-chloro-2-[(3-chlorophenyl) carbamoylamino] benzoic acid;
2-[(3-chlorophenyl) carbamoylamino] -4-fluorobenzoic acid; or
A composition for enhancing cell growth which is 2-[(3-chlorophenyl) carbamoylamino] -5-fluorobenzoic acid.
A compound for enhancing cell growth, comprising the compound of Formula 1, and having a formulation of lipid nanoparticles, polymer nanoparticles, or polymer gel.
[Formula 1]

Where
R ₁ and R ₂ are each independently H, halogen, C _1-4 alkyl, or CF ₃ ;
R ₃ is halogen.
The method of claim 4, wherein
The lipid nanoparticles of the formulation for cell growth enhancement that has a formulation selected from the group consisting of liposome (liposome), micelles (micelle), emulsion (emulsion) and solid lipid nanoparticles (solid lipid nanoparticles).
The method of claim 4, wherein
The lipid nanoparticles are cells growth enhancing agent comprising one or more lipids selected from the group consisting of positively charged lipids, neutral lipids and negatively charged lipids.
The method of claim 6,
The positively charged lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoyl glutamide, distearoyl glycol. Ruthamide (distearoyl glutamide), dipalmitoyl glutamide, dioleoyl aspartamide, 1,2-dioleoyl-3-dimethylammonium-propane (1,2-dioleoyl- 3-dimethylammonium-propane, DODAP), 3β- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (3β- [N- (N', N'-dimethylaminoethane) carbamoyl] cholesterol; DC- Chol), dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, EPC), 1,2-dimyristoyl-3-trimethylammoniumpropane (1,2-dimyristoyl-3-trimethylammonium-propane), 1,2-dipalmitoyl-3-trimethylammoniumpropane (1,2-dipalmitoyl -3-tr imethylammonium-propane), 1,2-disteroyl-3-trimethylammoniumpropane (1,2-distearoyl-3-trimethylammonium-propane), 1,2-dioleoyl-3-trimethylammoniumpropane (1,2- dioleoyl-3-trimethylammonium-propane), 1,2-dimyristoyl-3-dimethylammonium propane (1,2-dimyristoyl-3-dimethylammonium-propane), 1,2-dipalmitoyl-3-dimethylammonium Propane (1,2-dipalmitoyl-3-dimethylammonium-propane), 1,2-stearoyl-3-dimethylammonium-propane (1,2-distearoyl-3-dimethylammonium-propane), 1,2-dioleo 1-3-Dimethylammonium propane (1,2-dioleoyl-3-dimethylammonium-propane), dimethyldioctadecylammonium bromide, 1,2-diurauroyl-sn-glycer-3--3-ethylforce Pocholine (1,2-dilauroyl-sn-glycero-3-ethylphosphocholine), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3 -ethylphosphocholine), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl- sn-glycero-3-ethylphosphocholine), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1,2-distearoyl-sn-glycero-3-ethylphosphocholine), 1,2-diol Leoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and 1,2-palmitoyloleoyl-sn-glycero-3-ethylphosphocholine (1,2-palmitoyloleoyl-sn-glycero-3-ethylphosphocholine) An agent for enhancing cell growth, which is at least one lipid selected from the group consisting of:
The method of claim 6,
The neutral lipid is L-α-phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phophocholine, DSPC), 1,2-dioleoyl -sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), cholesterol, isopropyl myristate, 1,2-propiono Mono-sn-glycero-3-phosphocholine (1,2-propionoyl-sn-glycero-3-phosphocholine), 1,2-butanoyl-sn-glycero-3-phosphocholine (1,2- butanoyl-sn-glycero-3-phosphocholine), 1,2-pentanoyl-sn-glycero-3-phosphocholine (1,2-pentanoyl-sn-glycero-3-phosphocholine), 1,2-caproyl -sn-glycero-3-phosphocholine (1,2-caproyl-sn-glycero-3-phosphocholine), 1,2-heptanoyl-sn-glycero-3-phosphocholine (1,2-heptanoyl -sn-glycero-3-phosphocholine), 1,2-cap Liloyl-sn-glycero-3-phosphocholine (1,2-capryloyl-sn-glycero-3-phosphocholine), 1,2-nonanoyl-sn-glycero-3-phosphocholine (1, 2-nonanoyl-sn-glycero-3-phosphocholine), 1,2-capryl-sn-glycero-3-phosphocholine (1,2-capryl-sn-glycero-3-phosphocholine), 1,2- Undecanoyl-sn-glycero-3-phosphocholine (1,2-undecanoyl-sn-glycero-3-phosphocholine), 1,2-lauroyl-sn-glycero-3-phosphocholine (1 , 2-lauroyl-sn-glycero-3-phosphocholine), 1,2-tridecanoyl-sn-glycero-3-phosphocholine (1,2-tridecanoyl-sn-glycero-3-phosphocholine), 1, 2-myristoyl-sn-glycero-3-phosphocholine (1,2-myristoyl-sn-glycero-3-phosphocholine), 1,2-pentadecanoyl-sn-glycero-3-phosphocholine (1,2-pentadecanoyl-sn-glycero-3-phosphocholine), 1,2-palmitoyl-sn-glycero-3-phosphocholine (1,2-palmitoyl-sn-glycero-3-phosphocholine), 1 , 2-heptadecanoyl-sn-glycero-3-phosphocholine (1,2-heptadecanoyl-sn-glycero-3-phosphocholine), 1,2-stearoyl-s n-glycero-3-phosphocholine (1,2-stearoyl-sn-glycero-3-phosphocholine), 1,2-nonadecanoyl-sn-glycero-3-phosphocholine (1,2-nonadecanoyl -sn-glycero-3-phosphocholine), 1,2-arachidoyl-sn-glycero-3-phosphocholine (1,2-arachidoyl-sn-glycero-3-phosphocholine), 1,2-henieco Sanoyl-sn-glycero-3-phosphocholine (1,2-heniecosanoyl-sn-glycero-3-phosphocholine), 1,2-behenoyl-sn-glycero-3-phosphocholine (1,2 -behenoyl-sn-glycero-3-phosphocholine), 1,2-trussanoyl-sn-glycero-3-phosphocholine (1,2-trucisanoyl-sn-glycero-3-phosphocholine), 1,2- 1,2-lignoceroyl-sn-glycero-3-phosphocholine, 1,2-myristoleoyl-sn-glycero-3-phosphocholine (1,2-myristoleoyl-sn-glycero-3-phosphocholine), 1,2-myristolaidoyl-sn-glycero-3-phosphocholine (1,2-myristelaidoyl-sn-glycero-3-phosphocholine) 1,2-palmitoleoyl-sn-glycero-3-phosphocholine (1,2-palmitoleoyl-sn-glycero -3-phosphocholine), 1,2-palmitelaidoyl-sn-glycero-3-phosphocholine (1,2-palmitelaidoyl-sn-glycero-3-phosphocholine), 1,2-petroselinoyl-sn -Glycero-3-phosphocholine (1,2-petroselinoyl-sn-glycero-3-phosphocholine), 1,2-oleoyl-sn-glycero-3-phosphocholine (1,2-oleoyl-sn glycero-3-phosphocholine), 1,2-elidoyl-sn-glycero-3-phosphocholine (1,2-elaidoyl-sn-glycero-3-phosphocholine), 1,2-linoleoyl- sn-glycero-3-phosphocholine (1,2-linoleoyl-sn-glycero-3-phosphocholine), 1,2-linolenoyl-sn-glycero-3-phosphocholine (1,2-linolenoyl -sn-glycero-3-phosphocholine), 1,2-icocenoyl-sn-glycero-3-phosphocholine (1,2-eicosenoyl-sn-glycero-3-phosphocholine), 1,2-arachido Noyl-sn-glycero-3-phosphocholine (1,2-arachidonoyl-sn-glycero-3-phosphocholine), 1,2-rukoyl-sn-glycero-3-phosphocholine (1,2- erucoyl-sn-glycero-3-phosphocholine), 1,2-nernoyl-sn-glycero-3-phosphocholine (1,2-nervonoyl-sn- glycero-3-phosphocholine), L-α-phosphatidylethanolamine, 1,2-dicaproyl-sn-glycero-3-phosphoethanolamine (1,2-dicaproyl-sn- glycero-3-phosphoethanolamine), 1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine (1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine), 1,2-dicapryl-sn 1,2-dicapryl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (1,2 -dilauroyl-sn-glycero-3-phosphoethanolamine), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), 1, 2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine), 1,2-dipalmitoyl-sn-glycero-3-force 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn-glycero-3 -p hosphoethanolamine), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine), 1,2-diheptadecanoyl-sn 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (1,2 -distearoyl-sn-glycero-3-phosphoethanolamine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 1,2 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoyl-sn-glycero-3-force 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine (1,2-dilinolenoyl-sn-glycero- 3-phosphoethanolamine), 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine), 1,2-docosahexa One -sn- glycero-3-phospho-ethanolamine (1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine), and cholesterol (cholesterol) one or more lipid formulation for enhancing cell growth is selected from the group consisting of.
The method of claim 6,
The negatively charged lipid is L-α-phosphatidic acid, L-α-phosphatidyl-DL-glycerol, L-α-phosphatidyl-DL-glycerol, cardiolipin, L-α -Phosphatidylinositol (L-α-phosphatidylinositol), L-α-phosphatidylserine, 1,2-dilauroyl-sn-glycero-3- [phospho-rac- (1- Glycerol)] (1,2-dilauroyl-sn-glycero-3- [phospho-rac- (1-glycerol)], DLPG), 1,2-dilauroyl-sn-glycero-3- [phospho -L-serine] (1,2-dilauroyl-sn-glycero-3- [phospho-L-serine], DLPS), 1,2-dilauroyl-sn-glycero-3-phosphate (1,2 -dilauroyl-sn-glycero-3-phosphate, DLPA), 1,2-dimyristoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (1,2-dimyristoyl-sn -glycero-3- [phospho-rac- (1-glycerol)], DMPG), 1,2-dimyristoyl-sn-glycero-3- [phospho-L-serine] (1,2-dimyristoyl -sn-glycero-3- [phospho-L-serine], DMPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (1,2-dimyristoyl-sn-glycero-3-phosphate, DMPA), 1,2-dioleoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (1,2-dioleoyl-sn-glycero-3- [phospho-rac- (1-glycerol) ], DOPG), 1,2-dioleoyl-sn-glycero-3- [phospho-L-serine] (1,2-dioleoyl-sn-glycero-3- [phospho-L-serine], DOPS ), 1,2-dioleoyl-sn-glycero-3-phosphate (1,2-dioleoyl-sn-glycero-3-phosphate, DOPA), 1,2-dipalmitoyl-sn-glycero-3 -[Phospho-L-serine] (1,2-dipalmitoyl-sn-glycero-3- [phospho-L-serine], DPPS), 1,2-dipalmitoyl-sn-glycero-3-phosphate ( 1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPA), 1,2-disteroyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (1,2-distearoyl -sn-glycero-3- [phospho-rac- (1-glycerol)], DSPG), 1,2-disteroyl-sn-glycero-3- [phospho-L-serine] (1,2- distearoyl-sn-glycero-3- [phospho-L-serine] (DPSS), 1,2-disteroyl-sn-glycero-3-phosphate (1,2-distearoyl-sn-glycero-3-phosphate, DSPA), 1,2-Disteroyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (polyethyl Glycol 2000] (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (polyethylene glycol) 2000]), 1,2-desteroyl-sn-glycero-3-phosphoethanolamine- N- [maleimide (polyethylene glycol) 2000] (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) 2000]), 1,2-disteroyl-sn-glycero -3-phosphoethanolamine-N- [PDP (polyethylene glycol) 2000] (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [PDP (polyethylene glycol) 2000]), 1-palmitoyl- 2-oleyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (1-palmitoyl-2-oleoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] , POPG), 1-palmitoyl-2-oleyl-sn-glycero-3- [phospho-L-serine] (1-palmitoyl-2-oleoyl-sn-glycero-3- [phospho-L-serine POPS), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphate (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, POPA), oleic acid , L-α-phosphatidylglycerol, 1,2-dicaproyl-sn-glycer 3-phosphoglycerol (1,2-dicaproyl-sn-glycero-3-phosphoglycerol), 1,2-dioctanoyl-sn-glycero-3-phosphoglycerol (1,2-dioctanoyl-sn-glycero -3-phosphoglycerol), 1,2-dicapryl-sn-glycero-3-phosphoglycerol (1,2-dicapryl-sn-glycero-3-phosphoglycerol), 1,2-dilauroyl-sn- Glycero-3-phosphoglycerol (1,2-dilauroyl-sn-glycero-3-phosphoglycerol), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (1,2-dimyristoyl- sn-glycero-3-phosphoglycerol), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol), 1,2-dipyanoyl -sn-glycero-3-phosphoglycerol (1,2-diphytanoyl-sn-glycero-3-phosphoglycerol), 1,2-diheptadecanoyl-sn-glycero-3-phosphoglycerol (1,2 -diheptadecanoyl-sn-glycero-3-phosphoglycerol), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (1,2-distearoyl-sn-glycero-3-phosphoglycerol), 1,2 -Dioleoyl-sn-glycero-3-phosphogly Roll (1,2-dioleoyl-sn-glycero-3-phosphoglycerol), 1,2-diyladoyl-sn-glycero-3-phosphoglycerol (1,2-dielaidoyl-sn-glycero-3-phosphoglycerol ), 1,2-dilinoleoyl-sn-glycero-3-phosphoglycerol (1,2-dilinoleoyl-sn-glycero-3-phosphoglycerol), 1,2-dilinolenoyl-sn-glycero 3-phosphoglycerol (1,2-dilinolenoyl-sn-glycero-3-phosphoglycerol), 1,2-diarachidonoyl-sn-glycero-3-phosphoglycerol (1,2-diarachidonoyl-sn- glycero-3-phosphoglycerol), 1,2-docosahexaenoyl-sn-glycero-3-phosphoglycerol (1,2-docosahexaenoyl-sn-glycero-3-phosphoglycerol), L-α-phosphatidylinositol ( L-α-phosphatidylinositol), 1,2-dioleoyl-sn-glycero-3-phosphoinositol (1,2-dioleoyl-sn-glycero-3-phosphoinositol), δ-erythro-sphingosyl phosphinositol (δ-erythro-sphingosyl phosphoinositol), L-α-phosphatidic acid, 1,2-dihexanoyl-sn-glycero-3-phosphate (1,2-dihexanoyl -sn-glycero-3-phosphate), 1,2-dioctanoyl-sn-glycero-3-phosphate (1,2-dioctanoyl-sn-glycero-3-phosphate), 1,2-detecanoyl- sn-glycero-3-phosphate (1,2-didecanoyl-sn-glycero-3-phosphate), 1,2-didodecanoyl-sn-glycero-3-phosphate (1,2-didodecanoyl-sn-glycero -3-phosphate), 1,2-ditetradecanoyl-sn-glycero-3-phosphate (1,2-ditetradecanoyl-sn-glycero-3-phosphate), 1,2-dihexatecanoyl-sn 1,2-dihexadecanoyl-sn-glycero-3-phosphate, 1,2-diphytanoyl-sn-glycero-3-phosphate (1,2-diphytanoyl-sn-glycero-) 3-phosphate), 1,2-diheptadecanoyl-sn-glycero-3-phosphate (1,2-diheptadecanoyl-sn-glycero-3-phosphate), 1,2-dioctadecanoyl-sn-glycer Rho-3-phosphate (1,2-dioctadecanoyl-sn-glycero-3-phosphate), 1,2-dioctadecadienoyl-sn-glycero-3-phosphate (1,2-dioctadecadienoyl-sn-glycero- 3-phosphate), 1,2-dicosatetraenoyl-sn-gly Rho-3-phosphate (1,2-dieicosatetraenoyl-sn-glycero-3-phosphate), 1,2-didocosahexaenoyl-sn-glycero-3-phosphate (1,2-didocosahexaenoyl-sn-glycero- 3-phosphate), L-α-phosphatidylserine, 1,2-dihexanoyl-sn-glycero-3-phosphoserine (1,2-dihexanoyl-sn-glycero-3-phosphoserine ), 1,2-dioctanoyl-sn-glycero-3-phosphoserine (1,2-dioctanoyl-sn-glycero-3-phosphoserine), 1,2-didecanoyl-sn-glycero-3- Phosphoserine (1,2-didecanoyl-sn-glycero-3-phosphoserine), 1,2-didodecanoyl-sn-glycero-3-phosphoserine (1,2-didodecanoyl-sn-glycero-3-phosphoserine) 1,2-ditetradecanoyl-sn-glycero-3-phosphoserine, 1,2-dihexadecanoyl-sn-glycero-3 -Phosphoserine (1,2-dihexadecanoyl-sn-glycero-3-phosphoserine), 1,2-diphytanoyl-sn-glycero-3-phosphoserine (1,2-diphytanoyl-sn-glycero-3-phosphoserine ), 1,2-diheptadecanoyl-sn-text Vertical-3-phosphoserine (1,2-diheptadecanoyl-sn-glycero-3-phosphoserine), 1,2-dioctadecanoyl-sn-glycero-3-phosphoserine (1,2-dioctadecanoyl-sn-glycero -3-phosphoserine), 1,2-dioctadecenoyl-sn-glycero-3-phosphoserine (1,2-dioctadecenoyl-sn-glycero-3-phosphoserine), 1,2-diooctadecadienoyl -sn-glycero-3-phosphoseline (1,2-dioctadecadienoyl-sn-glycero-3-phosphoserine), 1,2-dicosatetraenoyl-sn-glycero-3-phosphoseline (1,2- dieicosatetraenoyl-sn-glycero-3-phosphoserine), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoserine (1,2-didocosahexaenoyl-sn-glycero-3-phosphoserine), cholesteryl hexate Immature succinate (cholesteryl hemisuccinate; CHEMS), cardiolipin, 1 ', 3'-bis [1,2-ditetradecanoyl-sn-glycero-3-phospho] -sn-glycerol (1', 3'-bis [ 1,2-ditetradecanoyl-sn-glycero-3-phospho] -sn-glycerol) and 1 ', 3'-bis [1,2-dioctadecenoyl-sn-glycero-3-phospho] -sn Glycerol (1 ', 3'-bis [1,2-dioctadecenoyl-sn-glycero-3-phospho] -sn-glycerol) A formulation for enhancing cell growth, which is at least one lipid selected from the group consisting of:
The method of claim 6,
The lipid is a cell growth enhancing agent modified with polyethylene glycol.
The method of claim 4, wherein
The agent for enhancing cell growth, wherein the lipid nanoparticle further comprises a surfactant.
The method of claim 11,
The surfactant is at least one agent selected from the group consisting of anionic surfactants, cationic surfactants, zwitterionic surfactants and nonionic surfactants for cell growth enhancement.
The method of claim 4, wherein
The polymer nanoparticles or polymer gel is a cell growth enhancing agent comprising a natural polymer or a synthetic polymer.
The method of claim 13,
Wherein the natural polymer is starch, cellulose, peptides, alginic acid, hyaluronic acid and chitosan is selected from the group consisting of cell growth enhancing agent.
The method of claim 13,
The synthetic polymer is a polyethyleneamine (Polyethylenimine, PEI) polyethylene glycol (Polyethylene Glycol), poly vinyl alcohol (Poly vinyl alcohol), poly-N-vinylpyrrolidone (Poly N-vinylpyrrolidone), poly-lactic-co-glyco Selected from the group consisting of polylactic acid (poly (lactic-co-glycolic acid), polylactic acid (poly (L-lactic acid)), polyglycolic acid (poly glycolic acid) and beta-tricalcium phosphate) Formulation for enhancing cell growth.
The method of claim 4, wherein
Said cell is one or more cells selected from the group consisting of neurons, heart cells, pancreatic islet cells, hematopoietic stem cells, stem cells, immune cells and skin cells.
The method of claim 4, wherein
The agent is an agent for enhancing cell growth further comprising a cell targeting ligand.
The method of claim 4, wherein
The agent is a cell growth enhancing agent that further comprises a second drug.
The method of claim 18,
The agent for enhancing cell growth, wherein the second drug is a compound medicine, a protein medicine or a nucleic acid medicine.
The method of claim 19,
The nucleic acid is a plasmid small interference ribonucleic acid (siRNA) formulation for cell growth enhancement.

Images