COMPOSITION AND METHOD FOR FREEZE-DRYING PHARMACEUTICAL COMPOSITION CONTAINING ANIONIC DRUG

Abstract

Disclosed are a composition and a method for freeze-drying which allow excellent stability, safety, and efficacy to be exhibited at the time of freeze-drying and reconstituting a composition for anionic drug delivery.

Claims

1. A composition for freeze-drying of a composition for delivering anionic drug, which comprises: a composition for delivering anionic drug comprising an anionic drug as an active ingredient, a cationic compound, an amphiphilic block copolymer and one or more salts of polylactic acid selected from the group consisting of the compounds of the following Formulas 1 to 6, wherein the anionic drug forms a complex with the cationic compound by electrostatic interaction, the complex is entrapped in a nanoparticle structure formed by the amphiphilic block copolymer and the salt of polylactic acid; and sorbitol as a cryoprotectant:
ROCHZ-[A].sub.n-[B].sub.mCOOM[Formula 1] wherein A is COOCHZ; B is COOCHY, COOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2 or COOCH.sub.2CH.sub.2OCH.sub.2; R is a hydrogen atom, or acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl; each of Z and Y is a hydrogen atom, or methyl or phenyl; M is Na, K or Li; n is an integer of from 1 to 30; and m is an integer of from 0 to 20;
ROCHZ[COOCHX].sub.p[COOCHY].sub.gCOOCHZCOOM[Formula 2] wherein X is methyl; Y is a hydrogen atom or phenyl; p is an integer of from 0 to 25, q is an integer of from 0 to 25, with the proviso that p+q is an integer of from 5 to 25; R is a hydrogen atom, or acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl; M is Na, K or Li; and Z is a hydrogen atom, methyl or phenyl;
RO-PAD-COOW-M[Formula 3] wherein W-M is ##STR00005## PAD is selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, copolymer of D,L-lactic acid and glycolic acid, copolymer of D,L-lactic acid and mandelic acid, copolymer of D,L-lactic acid and caprolactone, and copolymer of D,L-lactic acid and 1,4-dioxane-2-one; R is a hydrogen atom, or acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl; and M is independently Na, K or Li;
SO-PAD-COO-Q[Formula 4] wherein S is ##STR00006## L is NR.sub.1 or O, wherein R.sub.1 is a hydrogen atom or C.sub.1-10 alkyl; Q is CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.2CH.sub.3, or CH.sub.2C.sub.6H.sub.5; a is an integer of from 0 to 4; b is an integer of from 1 to 10; M is Na, K or Li; and PAD is one or more selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, copolymer of D,L-lactic acid and glycolic acid, copolymer of D,L-lactic acid and mandelic acid, copolymer of D,L-lactic acid and caprolactone, and copolymer of D,L-lactic acid and 1,4-dioxane-2-one; ##STR00007## wherein R is -PAD-OC(O)CH.sub.2CH.sub.2C(O)OM, wherein PAD is selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, copolymer of D,L-lactic acid and glycolic acid, copolymer of D,L-lactic acid and mandelic acid, copolymer of D,L-lactic acid and caprolactone, and copolymer of D,L-lactic acid and 1,4-dioxane-2-one, M is Na, K or Li; and a is an integer of from 1 to 4;
YO[C(O)(CHX).sub.aO-].sub.mC(O)RC(O)[O(CHX).sub.bC(O)].sub.nOZ[Formula 6] wherein X and X are independently hydrogen, C.sub.1-10 alkyl or C.sub.6-20 aryl; Y and Z are independently Na, K or Li; m and n are independently an integer of from 0 to 95, with the proviso that 5<m+n<100; a and b are independently an integer of from 1 to 6; and R is (CH.sub.2).sub.k, C.sub.2-10 divalent alkenyl, C.sub.6-20 divalent aryl or a combination thereof, wherein k is an integer of from 0 to 10.

2. The composition for freeze-drying of a composition for delivering anionic drug of claim 1, which comprises the sorbitol in an amount of 1 to 5,000 parts by weight, based on 1 part by weight of the anionic drug.

3. The composition for freeze-drying of a composition for delivering anionic drug of claim 1, wherein the anionic drug is nucleic acid.

4. The composition for freeze-drying of a composition for delivering anionic drug of claim 1, wherein the cationic compound is one or more selected from the group consisting of cationic lipids and cationic polymers.

5. The composition for freeze-drying of a composition for delivering anionic drug of claim 4, wherein the cationic lipid is one or more selected from the group consisting of N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammoniumbromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammoniumchloride (DOTAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,2-diacyl-3-dimethylammonium-propane (DAP), 3-[N(N,N,N-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3[N(N,N-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3-[N(N-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3-[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), cholesteryloxypropane-1-amine (COPA), N(N-aminoethane)carbamoylpropanoic tocopherol (AC-tocopherol) and N(N-methylaminoethane)carbamoylpropanoic tocopherol (MC-tocopherol).

6. The composition for freeze-drying of a composition for delivering anionic drug of claim 4, wherein the cationic lipid is a cationic lipid represented by the following Formula 7: ##STR00008## wherein each of n and m is 0 to 12 with the proviso that 2n+m12, each of a and b is 1 to 6, and each of R.sub.1 and R.sub.2 is independently selected from the group consisting of saturated and unsaturated C.sub.11-25 hydrocarbons.

7. The composition for freeze-drying of a composition for delivering anionic drug of claim 6, wherein each of R.sub.1 and R.sub.2 is independently selected from the group consisting of lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, cerotyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl and cerotyl.

8. The composition for freeze-drying of a composition for delivering anionic drug of claim 1, wherein the amphiphilic block copolymer is an A-B type di-block copolymer comprising a hydrophilic A block and a hydrophobic B block, wherein the hydrophilic A block is one or more selected from the group consisting of polyalkyleneglycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide and derivatives thereof, and the hydrophobic B block is one or more selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazine.

9. The composition for freeze-drying of a composition for delivering anionic drug of claim 8, wherein a hydroxyl group at the end of the hydrophobic B block is modified by one or more selected from the group consisting of cholesterol, tocopherol and C.sub.10-24 fatty acid.

10. The composition for freeze-drying of a composition for delivering anionic drug of claim 1, further comprising a fusogenic lipid.

11. The composition for freeze-drying of a composition for delivering anionic drug of claim 10, wherein the fusogenic lipid is one or more selected from the group consisting of dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dilinoleoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1,2-diphytanoyl-3-sn-phosphatidylethanolamine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1,2-diphytanoyl-3-sn-phosphatidylcholine, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, dioleoyl phosphatidic acid, dilinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatidic acid, 1,2-diphytanoyl-3-sn-phosphatidic acid, cholesterol and tocopherol.

12. A method for freeze-drying of a composition for delivering anionic drug, comprising conducting the freeze-drying by using a composition according to claim 1.

13. A freeze-dried product of a composition for delivering anionic drug freeze-dried by the method according to claim 12.

Description

BRIEF EXPLANATION OF THE DRAWINGS

[0064] FIG. 1 is a schematic structure of the polymer nanoparticle delivery system according to an embodiment of the present invention, in which a complex of the anionic drug and the cationic compound is entrapped.

BEST MODES FOR CARRYING OUT THE INVENTION

[0065] The present invention will be explained below in more detail with reference to the following Examples. However, the Examples are only to illustrate the invention, and the scope of the present invention is not limited thereby in any manner.

[Preparation Example] Preparation of Polymeric Nanoparticles Containing KRAS siRNA/1,6-dioleoyl triethylenetetramide (dioTETA)/mPEG-PLA-tocopherol/PLANa/DOPE

[0066] 5 g of KRAS siRNA was dissolved in 94.52 l of distilled water, and 94.52 g of dioTETA was dissolved in 94.52 l of 20 mM acetate buffer (pH 4.6), and the solutions were mixed dropwise in sonicated state. The resulting mixture was freeze-dried to a powdery state, and the powder was dissolved in with 10 l of ethyl acetate. To this, a solution of 300 g of PLANa dissolved in 15 l of ethyl acetate, a solution of 104.2 g of DOPE dissolved in 5.2 l of ethyl acetate, and a solution of 1000 g of mPEG-PLA-tocopherol dissolved in 20 of ethyl acetate were added in this order, and mixed. While adding the resulting mixture dropwise to 100 l of distilled water, a complex emulsion was prepared by using a sonicator. The prepared complex emulsion was put into a 1-necked round flask and distilled under reduced pressure in a rotary evaporator for selective removal of ethyl acetate, to prepare polymeric nanoparticles containing siRNA/1,6-dioleoyl triethylenetetramide (dioTETA)/mPEG-PLA-tocopherol/PLANa/DOPE.

TABLE-US-00001 TABLE 1 Cationic Polymer Polymer Helper Composition Ratio siRNA lipid 1 2 lipid Preparation siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g Example 1 tocopherol/PLANa/DOPE 0.3-1

[Examples 1 to 4] Preparation of Freeze-Dried Formulation of Polymeric Nanoparticles Containing KRAS siRNA/1,6-Dioleoyl Triethylenetetramide (dioTETA)/mPEG-PLA-Tocopherol/PLANa/DOPE/Sorbitol

[0067] The concentration of the polymeric nanoparticle prepared in Preparation Example was fixed to 100 ng/l of siRNA and sorbitol was added thereto, and the mixture was frozen in an ultradeep freezer, and then the freeze-drying was conducted. The freeze-drying conditions were the same as provided in the following Table 2.

TABLE-US-00002 TABLE 2 Temperature condition Vacuum Process Set temper- Mode for degree time Step ature ( C.) change (mTorr) (min.) Initial freezing 40 Lowering Atmospheric 90 Freezing 40 Maintaining Atmospheric 240 Cold Trap 50 Lowering Atmospheric 20 1 Step 40 Maintaining 200 60 2 Step 10 Elevating 200 240 3 Step 10 Maintaining 200 360 4 Step +5 Elevating 200 120 5 Step +5 Maintaining 150 1,200 6 Step +25 Elevating 150 120 7 Step +25 Maintaining 150 240 Total 2,690

[0068] To the freeze-dried powder, sterilized distilled water at room temperature was added and the mixture was shaken to dissolve the powder, to prepare a composition containing siRNA/dio-TETA/mPEG-PLA-tocopherol/PLANa/DOPE/sorbitol.

TABLE-US-00003 TABLE 3 Cationic Polymer Polymer Helper Composition Ratio siRNA lipid 1 2 lipid Sorbitol Example siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 0.25 mg 1 tocopherol/PLANa/DOPE/sorbitol 0.3-1 Example siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 2.5 mg 2 tocopherol/PLANa/DOPE/sorbitol 0.3-1 Example siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 5 mg 3 tocopherol/PLANa/DOPE/sorbitol 0.3-1 Example siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 10 mg 4 tocopherol/PLANa/DOPE/sorbitol 0.3-1

[Comparative Examples 1 to 3] Preparation of Freeze-Dried Formulation of Polymeric Nanoparticles Containing KRAS siRNA/1,6-Dioleoyl Triethylenetetramide (dioTETA)/mPEG-PLA-Tocopherol/PLANa/DOPE/Trehalose

[0069] By using trehalose as a cryoprotectant, polymeric nanoparticles containing siRNA/dio-TETA/mPEG-PLA-tocopherol/PLANa/DOPE/trehalose were prepared in the same manner as described in Example 1.

TABLE-US-00004 TABLE 4 Cationic Polymer Polymer Helper Composition Ratio siRNA lipid 1 2 lipid Trehalose Comp. siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 2.5 mg Example tocopherol/PLANa/DOPE/trehalose 0.3-1 1 Comp. siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 5 mg Example tocopherol/PLANa/DOPE/trehalose 0.3-1 2 Comp. siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 10 mg Example tocopherol/PLANa/DOPE/trehalose 0.3-1 3

[Comparative Example 4] Preparation of Freeze-Dried Formulation of Polymeric Nanoparticles Containing KRAS siRNA/1,6-Dioleoyl Triethylenetetramide (dioTETA)/mPEG-PLA-Tocopherol/PLANa/DOPE/Mannitol

[0070] By using mannitol as a cryoprotectant, polymeric nanoparticles containing siRNA/dio-TETA/mPEG-PLA-tocopherol/PLANa/DOPE/mannitol were prepared in the same manner as described in Example 1.

TABLE-US-00005 TABLE 5 Cationic Polymer Polymer Helper Composition Ratio siRNA lipid 1 2 lipid Mannitol Comp. siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 2.5 mg Example tocopherol/PLANa/DOPE/mannitol 0.3-1 4

[Comparative Example 5] Preparation of Freeze-Dried Formulation of Polymeric Nanoparticles Containing KRAS siRNA/1,6-Dioleoyl Triethylenetetramide (dioTETA)/mPEG-PLA-Tocopherol/PLANa/DOPE/Sucrose

[0071] By using sucrose as a cryoprotectant, polymeric nanoparticles containing siRNA/dio-TETA/mPEG-PLA-tocopherol/PLANa/DOPE/sucrose were prepared in the same manner as described in Example 1.

TABLE-US-00006 TABLE 6 Cationic Polymer Polymer Helper Composition Ratio siRNA lipid 1 2 lipid Sucrose Comp. siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 2.5 mg Example tocopherol/PLANa/DOPE/sucrose 0.3-1 5

[Comparative Example 6] Preparation of Freeze-Dried Formulation of Polymeric Nanoparticles Containing KRAS siRNA/1,6-Dioleoyl Triethylenetetramide (dioTETA)/mPEG-PLA-Tocopherol/PLANa/DOPE/Glucose

[0072] By using glucose as a cryoprotectant, polymeric nanoparticles containing siRNA/dio-TETA/mPEG-PLA-tocopherol/PLANa/DOPE/glucose were prepared in the same manner as described in Example 1.

TABLE-US-00007 TABLE 7 Cationic Polymer Polymer Helper Composition Ratio siRNA lipid 1 2 lipid Glucose Comp. siRNA/dioTETA/mPEG-PLA- 5-18-1- 5 g 94.5 g 1000 g 300 g 104.2 g 2.5 mg Example tocopherol/PLANa/DOPE/glucose 0.3-1 6

[Experimental Example 1] Size Measurement and Stability Comparison (Heparin Competition Analysis) for Polymeric Nanoparticles Containing siRNA/dioTETA/mPEG-PLA-Tocopherol/PLANa/DOPE/Cryoprotectant

[0073] Size measurement was conducted for the polymeric nanoparticles prepared with different cryoprotectants. The sizes of the particles were measured by using Dynamic Light Scattering (DLS) method. Specifically, a HeNe laser was used as a light source, and Zetasizer Nano ZS90 (MALVERN) device was operated according to the manual.

[0074] In addition, heparin competition analysis was conducted to evaluate in vivo stability of the polymeric nanoparticles according to the kind of cryoprotectant. 10 l of each formulation (siRNA 300 ng) was treated with 40 g of heparin and reacted for 10 minutes at room temperature, and then the amount of disintegrated siRNA was measured through electrophoresis. The lower disintegration degree of siRNA indicates the better stability of the formulation. In addition, the formulation itself alone was subjected to electrophoresis to measure the amount of unentrapped siRNA in the formulation.

[0075] The measured results of the unentrapped siRNA, particle size, and disintegration degree of siRNA for the formulations of Example 2 and Comparative Examples 1 and 4 to 6 prepared with different cryoprotectants are shown in the following Table 8.

TABLE-US-00008 TABLE 8 Unentrapped Particle Disintegration Composition siRNA size degree of siRNA (%) Preparation Aqueous solution formulation of 0% 38 nm 5% Example siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE Example Freeze-dried formulation of 0% 46 nm 13% 2 siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE/ sorbitol 2.5 mg Comp. Freeze-dried formulation of 5% 23 nm 8% Example siRNA/dioTETA/mPEG-PLA- 1 tocopherol/PLANa/DOPE/ trehalose 2.5 mg Comp. Freeze-dried formulation of 18% 25 nm 14% Example siRNA/dioTETA/mPEG-PLA- 4 tocopherol/PLANa/DOPE/ mannitol 2.5 mg Comp. Freeze-dried formulation of 0% 154 nm 37% Example siRNA/dioTETA/mPEG-PLA- 5 tocopherol/PLANa/DOPE/ sucrose 2.5 mg Comp. Freeze-dried formulation of 0% 34 nm 15% Example siRNA/dioTETA/mPEG-PLA- 6 tocopherol/PLANa/DOPE/ glucose 2.5 mg

[0076] As can be know from Table 8, in case of using trehalose or mannitol, the amount of unentrapped siRNA increased, and in case of using sucrose, the disintegration degree of siRNA was high. Thus, it could be confirmed that trehalose, mannitol and sucrose are not suitable cryoprotectants.

[0077] The analysis results for the formulations of Examples 1 to 4 and Comparative Examples 1 to 3 prepared with different amounts of sorbitol or trehalose among the cryoprotectants are shown in the following Table 9.

TABLE-US-00009 TABLE 9 Unentrapped Particle Disintegration Composition siRNA size degree of siRNA (%) Preparation Aqueous solution formulation of 0% 38 nm 5% Example siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE Example Freeze-dried formulation of 0% 42 nm 16% 1 siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE/ sorbitol 0.25 mg Example Freeze-dried formulation of 0% 46 nm 13% 2 siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE/ sorbitol 2.5 mg Example Freeze-dried formulation of 0% 48 nm 8% 3 siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE/ sorbitol 5 mg Example Freeze-dried formulation of 0% 34 nm 13% 4 siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE/ sorbitol 10 mg Comp. Freeze-dried formulation of 5% 23 nm 8% Example siRNA/dioTETA/mPEG-PLA- 1 tocopherol/PLANa/DOPE/ trehalose 2.5 mg Comp. Freeze-dried formulation of 25% 43 nm 28% Example siRNA/dioTETA/mPEG-PLA- 2 tocopherol/PLANa/DOPE/ trehalose 5 mg Comp. Freeze-dried formulation of 14% 52 nm 26% Example siRNA/dioTETA/mPEG-PLA- 3 tocopherol/PLANa/DOPE/ trehalose 10 mg

[0078] As can be known from Table 9, in case of using trehalose, the amount of unentrapped siRNA increased, and the disintegration degree of siRNA became higher as the amount of trehalose increased. Thus, it could be confirmed that trehalose is not a suitable cryoprotectant.

[Experimental Example 2] Cell Efficacy and Toxicity Comparison of Polymeric Nanoparticles Containing siRNA/dioTETA/mPEG-PLA-Tocopherol/PLANa/DOPE/Cryoprotectant

[0079] For the polymeric nanoparticles containing siRNA/dioTETA/mPEG-PLA-tocopherol/PLANa/DOPE/cryoprotectant prepared in Preparation Example, Examples 2 to 4 and Comparative Examples 1 and 6, the efficacy of delivering siRNA to A549 lung cancer cell line was evaluated in mRNA level. The cells were seeded to a 96-well cell culture plate at 5000 cells/well concentration. After 24 hours, it was confirmed that about 50 to 60% of cells in each well were grown uniformly. Then, the medium in the well was removed, and 90 l of fresh medium containing serum at 10% of final volume was added. To the cell culture medium, each of the compositions of Preparation Example, Examples 2 to 4 and Comparative Examples 1 and 6 was added so that siRNA might be contained at 400 nM, 200 nM, 100 nM, 50 nM, 5 nM, 0.5 nM, 0.05 nM concentration. The cells were cultured in an incubator at 37 C. with 5% CO.sub.2 for 48 hours and the medium was removed, and then 100 l of cell lysis mixture was added and reacted at 50 C. for 18 hours. Thereafter, in order to evaluate mRNA expression, Branched DNA assay (bDNA, Quantigene 2.0 Assay kit, Panomics, QS0009) was used. According to the protocol, 2.0 substrate was added and reacted at room temperature for 5 minutes, and then microplate fluorescence reader (Bio-Tek, Synergy HT) was used to measure the fluorescence expression amount. Furthermore, in order to analyze intracellular toxicity, Cell Titer-Glo luminescent cell viability assay (Promega, G7571) was used. According to the protocol, to the cell plate at room temperature, the sample for analysis (100 l) and a cell titer assay reagent (100 l) were added and reacted for 30 minutes, and then microplate fluorescence reader (Bio-Tek, Synergy HT) was used to measure the value. The results of evaluating efficacy and toxicity for the cell line are shown in the following Table 10.

TABLE-US-00010 Composition LC.sub.50 IC.sub.50 Preparation Aqueous solution formulation of 138 32.3 Example siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE Example Freeze-dried formulation of 262 22.1 2 siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE/ sorbitol 2.5 mg Example Freeze-dried formulation of 290.7 11.2 3 siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE/ sorbitol 5 mg Example Freeze-dried formulation of 238.8 9.0 4 siRNA/dioTETA/mPEG-PLA- tocopherol/PLANa/DOPE/ sorbitol 10 mg Comp. Freeze-dried formulation of 293.6 4.6 Example siRNA/dioTETA/mPEG-PLA- 1 tocopherol/PLANa/DOPE/ trehalose 2.5 mg Comp. Freeze-dried formulation of 179.9 6.3 Example siRNA/dioTETA/mPEG-PLA- 2 tocopherol/PLANa/DOPE/ glucose 2.5 mg

[0080] As clearly described in Table 10, the results showed the tendency that the freeze-drying reduced the toxicity and improved the efficacy since after the freeze-drying, LC.sub.50 was increased and IC.sub.50 was decreased, as compared with those before the freeze-drying. As a result of calculating LC.sub.50/IC.sub.50, the samples showed the efficacy in the order of sorbitol 2.5 mg>sorbitol 5 mg>sorbitol 10 mg>glucose 2.5 mg>trehalose 2.5 mg.