KR20210102157A - Composition comprising doxorubicin as effective ingredient and method for preparing the same - Google Patents

Abstract

Disclosed are a nano-cancer agent composition for inducing immunogenic cell death and uses thereof. A composition according to the present invention can exhibit a synergistic effect in terms of anti-cancer efficacy when combined with an immuno-cancer agent by inducing immunogenic cell death. According to one aspect of the present invention, the present invention contains doxorubicin, an amphiphilic block copolymer, and polylactic acid salts as active components.

Description

Composition comprising doxorubicin as an effective ingredient and method for preparing the same

The present invention relates to a composition for controlling the release of doxorubicin, and more particularly, to a nano-anticancer composition having improved anticancer effect and immunogenic apoptosis inducing ability of doxorubicin, and to a use thereof.

It is known that some anticancer drugs not only directly kill cancer cells through chemotoxicity, but also cause immune death, that is, immunogenic cell death (ICD), against cancer cells by activating adaptive immunity after cancer cell death. Cancer cells killed by anticancer drugs known to induce ICD release damage-associated molecular patterns (DAMPs) such as Carleticulin (CRT), high mobility group box1 (HMGB1), and adenosine triphosphate (ATP) to the outside of the cell membrane. known to be exposed. These DAMPs play a role as an alarm to inform surrounding tissues or cells of danger, and are recognized by antigen-presenting cells such as dendritic cells to induce cancer cell-specific anti-tumor immunity. As such, anticancer agents that induce ICD include Doxorubicin, Idarubicin, Mitoxantrone, Oxaliplatin, Cyclosphosphamide, Maposfamide, Bortezomib. (Bortezomib) is known.

Because nano-cancer drugs can accumulate in the tumor due to the enhanced permeability and retention (EPR) effect, which penetrates between the loose blood vessels of neovascularization around the tumor and accumulates inside the tumor tissue, it is a suitable material for the delivery of ICD-induced anticancer drugs. . However, since nano-cancer drugs can affect the release and stability of active substances, it is still a challenge to develop nano-cancer drugs that can exert anti-cancer effects or induce ICD by actually exceeding the function of active substances. .

The global anti-cancer drug market is growing rapidly, centering on immuno-oncology drugs. As the proportion of immuno-oncology drugs in the anti-cancer drug market is increasing, the possibility that the immuno-oncology/chemo-cancer drug combination therapy will be adopted as a standard treatment for various cancers has increased. Therefore, it is necessary to develop an anticancer agent that induces ICD that contributes to immune activity as an anticancer agent that can create synergy in terms of anticancer efficiency when combined with immunotherapy.

An object of the present invention is to provide a nanocomposite composition comprising doxorubicin as an active ingredient.

Another object of the present invention is to provide a nanocomposite composition for treating cancer in combination with an immunotherapy.

Another object of the present invention is to provide a method for preparing the nanocomposite composition.

According to one aspect of the present invention, doxorubicin as an active ingredient; amphiphilic block copolymers; and polylactic acid salt; the amphiphilic block copolymer is an AB-type double block copolymer composed of a hydrophilic A block and a hydrophobic B block, wherein the hydrophilic A block is polyalkylene glycol, polyvinyl alcohol, polyvinyl. It is at least one selected from the group consisting of pyrrolidone, polyacrylamide, and derivatives thereof, and the hydrophobic B block is selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester, and polyphosphazine. It is one or more selected, wherein the doxorubicin is characterized in that it is encapsulated in the nanoparticle structure formed by the amphiphilic block copolymer and the polylactate, the nanocomposite composition is provided.

In one embodiment of the present invention, the molar ratio of the sum of the amphiphilic block copolymer and the polylactate to doxorubicin is 8 to 12: 1, and the molar ratio of the polylactate to doxorubicin may be 1 to 6: 1.

In another embodiment of the present invention, the molar ratio of the sum of the amphiphilic block copolymer and the polylactate to doxorubicin is 18 to 22:1, and the molar ratio of the polylactate to doxorubicin may be 1 to 15:1.

According to another aspect of the present invention, there is provided a method comprising the steps of: (a) dissolving doxorubicin in an aqueous solvent; (b) adding and dissolving polylactic acid salt to the solution obtained in step (a); (c) adding and dissolving the amphiphilic block copolymer to the solution obtained in step (b); and (d) drying the mixture obtained in step (c), wherein the amphiphilic block copolymer is an AB-type diblock copolymer composed of a hydrophilic A block and a hydrophobic B block, wherein the hydrophilic A block is At least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, and derivatives thereof, and the hydrophobic B block is polyester, polyanhydride, polyamino acid, polyol At least one selected from the group consisting of a bovine ester, and polyphosphazine, wherein the doxorubicin is encapsulated in the nanoparticle structure formed by the amphiphilic block copolymer and the polylactate, Preparation of a nanocomposite composition A method is provided.

According to another aspect of the present invention, there is provided a composition for treating cancer, comprising the nanocomposite composition.

According to another aspect of the present invention, there is provided a composition for treating cancer in combination with an immuno-cancer agent, including the nanocomposite composition.

According to another aspect of the present invention, there is provided a method for preparing a composition for treating cancer in combination with an immuno-oncology agent, comprising using the nanocomposite composition.

According to still another aspect of the present invention, there is provided a composition for treating cancer, comprising the nanocomposite composition and an immunotherapy agent.

The composition according to the present invention may exhibit a synergistic effect in terms of anti-cancer efficacy when combined with an immuno-cancer agent by inducing immunogenic cell death.

1 is a graph showing the ratio of tumor free mice at the end of the Vaccination assay experiment performed in the Example of the present invention.
2 is a graph showing changes in ^{CD8 +} T cells and MDSC upon administration of Doxorubicin-PNP in the immune cell recruitment assay performed in the Example of the present invention.

In the present specification, 'immunogenic apoptosis or ICD-induced anticancer agent' refers to the death of cancer cells by anticancer agents while releasing damage-associated molecular patterns (DAMPs) such as CRT, HMGB1, and ATP from the inside of the cells to the surrounding tissues. It refers to a drug that plays a role as an alarm to inform cells of danger and induces cancer cell-specific anti-tumor immunity by recognizing DAMP as antigen-presenting cells such as dendritic cells. Examples of ICD-induced anticancer drugs include Doxorubicin, Idarubicin, Mitoxantrone, Oxaliplatin, Cyclosphosphamide, Mafosfamide, Bortezomib. , paclitaxel, docetaxel.

As used herein, the term 'nano-cancer agent' is a concept including polymer micelles (PM) and polymer nanoparticles (PNP).

The present applicant has developed a formulation using a block copolymer having a hydrophilic portion and a hydrophobic portion for solubilization of a poorly water-soluble drug. Polymeric micelles (PM) can increase the solubility of poorly soluble drugs with a solubility of several μg/mL to several mg/mL. This block copolymer has a low critical micelle concentration of 2 μg/mL, and the polymer micelles have a uniform particle distribution of 20 nm on average. It is suitable for the injection production process because it can be sterilized by filtration. Genexol-PM, a polymer micelle of Paclitaxel, and Nanoxel M, a polymer micelle of Docetaxel, have been commercialized.

Polymeric Nano Particles (PNP) uses water-soluble polylactic acid (PLA) in a block copolymer having a hydrophobic portion and a hydrophilic portion. Polymeric nanoparticles are more stable in aqueous phase than polymer micelles because they are prepared through ionic crosslinking by adding calcium ions, which are divalent cations. Polymeric nanoparticles can increase the solubility of poorly soluble drugs with a solubility of several μg/mL to several mg/mL. Polymer nanoparticles have a uniform particle distribution with an average size of 20 nm and can be sterilized by filtration, making them suitable for injection production processes. Polymer nanoparticles, which are stable nanoparticles, increase the blood retention of the drug and exhibit excellent tumor accumulation effect by EPR effect. It has been formulated by applying polymer nanoparticle technology to various drugs such as paclitaxel-PNP, docetaxel-PNP, and doxorubicin-PNP.

Hereinafter, the present invention will be described in more detail.

doxorubicin

Doxorubicin used in the nanocomposite composition of the present invention is a drug that induces immune death of cancer cells, that is, immunogenic cell death (ICD), and is an active ingredient of the finally prepared composition.

In one embodiment, the doxorubicin may be included in an amount of 0.01 to 10% by weight, more specifically, 0.1 to 5% by weight, based on the dry weight of the final composition, but is not limited thereto.

In one embodiment of the present invention, the molar ratio of the sum of the amphiphilic block copolymer and the polylactate to doxorubicin may be 8 to 12:1, and the molar ratio of polylactate to doxorubicin may be 1 to 6:1.

More specifically, the molar ratio of the sum of the amphiphilic block copolymer and the polylactate to doxorubicin may be 9 to 11: 1, and the molar ratio of polylactate to doxorubicin may be 3 to 6:1.

In another embodiment of the present invention, the molar ratio of the sum of the amphiphilic block copolymer and the polylactate to doxorubicin may be 18 to 22:1, and the molar ratio of polylactate to doxorubicin may be 1 to 15:1.

More specifically, the molar ratio of the sum of the amphiphilic block copolymer and the polylactate to doxorubicin may be 19 to 21:1, and the molar ratio of polylactate to doxorubicin may be 9 to 15:1.

amphiphilic block copolymer

The amphiphilic block copolymer used in the nanocomposite composition of the present invention is an AB-type diblock copolymer including a hydrophilic A block and a hydrophobic B block, and nanoparticles formed by the amphiphilic block copolymer and polylactate Doxorubicin is encapsulated inside the structure.

The AB-type diblock copolymer is a core-shell type in which the hydrophobic B block forms the core (inner wall) and the hydrophilic A block forms the shell (outer wall) in the aqueous phase, and controls the body distribution of the polymer carrier or the carrier is It is possible to increase the efficiency of delivery into cells.

The hydrophilic A block is at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, and derivatives thereof. More specifically, the hydrophilic A block may be at least one selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, a copolymer of polyethylene and propylene glycol, and polyvinylpyrrolidone. In one embodiment, the hydrophilic A block may have a number average molecular weight of 200 to 50,000 Daltons, more specifically 1,000 to 20,000 Daltons, and even more specifically 1,000 to 5,000 Daltons.

The hydrophobic B block, as a biocompatible biodegradable polymer, is at least one selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazine. More specifically, the hydrophobic B block is polylactide, polyglycolide, polycaprolactone, polydioxan-2-one, a copolymer of polylactide and glycolide, polylactide and polydioxan-2-one It may be at least one selected from the group consisting of a copolymer of, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone. In one embodiment, the hydrophobic B block may have a number average molecular weight of 50 to 50,000 Daltons, more specifically 200 to 20,000 Daltons, and even more specifically 1,000 to 5,000 Daltons. In addition, in one embodiment, in order to improve the stability of the nanoparticles by increasing the hydrophobicity of the hydrophobic B block, the terminal hydroxyl group of the hydrophobic B block is one selected from the group consisting of tocopherol, cholesterol, and fatty acids having 10 to 24 carbon atoms. It may be modified above.

In the amphiphilic block copolymer, the composition ratio of the hydrophilic block (A) and the hydrophobic block (B), based on the weight of the copolymer, the hydrophilic block (A) is 30 to 80 wt%, specifically 40 to 70 wt% % range. If the proportion of the hydrophilic block (A) is less than 30% by weight, the polymer has a low solubility in water and thus it is difficult to form nanoparticles, so that the copolymer has sufficient solubility in water to form nanoparticles. It is good that the ratio of A) is 30% by weight or more, while if it exceeds 80% by weight, the hydrophilicity is too high, and the stability of the polymer nanoparticles is lowered, so it is difficult to use it as a solubilization composition of the composite. ) is preferably 80% by weight or less.

In one embodiment, the amphiphilic block copolymer may be included in an amount of 1 to 99.99% by weight, and more specifically, in an amount of 5 to 99% by weight, based on the dry weight of the total composition to be finally prepared. not limited

polylactate

The polylactic acid salt (eg PLA-COONa) used in the nanocomposite composition of the present invention is distributed in the core (inner wall) of the nanoparticles to strengthen the hydrophobicity of the core to stabilize the nanoparticles and at the same time to stabilize the reticuloendothelial system in the body ( RES) is effectively avoided. That is, the carboxylate anion of polylactic acid binds to the virus more effectively than polylactic acid and reduces the surface potential of polymer nanoparticles. It has the advantage of being less captured by , and thus excellent in delivery efficiency to a target site (eg, cancer cells, inflammatory cells, etc.).

The nanocomposite of the present invention may be for controlling the release of doxorubicin. For example, the nanocomposite according to one embodiment has an emission of 20% or less (eg, greater than 0% and 20% or less), preferably 15% or less, more preferably 7% or less, based on 24 hours. It may have speed. The release rate can be generally measured using a well-known method, for example, it can be measured after 24 hours by shaking and stirring at 150 to 350 rpm in a PBS solution of 35 to 38 ℃, pH 6.5 to 8.0.

As a separate component from the amphiphilic block copolymer, the polylactate contained as a component of the inner wall of the nanoparticles preferably has a number average molecular weight of 500 to 50,000 Daltons, specifically, 1,000 to 10,000 Daltons. If the molecular weight is less than 500 Daltons, the hydrophobicity is too low to be difficult to exist in the core (inner wall) of the nanoparticles, and if the molecular weight exceeds 50,000 Daltons, there is a problem in that the particles of the polymer nanoparticles become large.

The polylactic acid salt may be used in an amount of 1 to 500 parts by weight, specifically 20 to 400 parts by weight, and more specifically 40 to 300 parts by weight based on 100 parts by weight of the amphiphilic block copolymer. When the content of polylactate exceeds 500 parts by weight based on 100 parts by weight of the amphiphilic block copolymer, the size of the nanoparticles increases, making filtration using a sterile membrane difficult, and when the content is less than 1 part by weight, the desired effect cannot be sufficiently obtained.

In one embodiment, the terminal of the polylactic acid salt opposite to the carboxylic acid-metal (eg, sodium) is hydroxy, acetoxy, benzoyloxy, decanoyloxy, palmitoyloxy and alkoxy having 1 to 2 carbon atoms. may be substituted with one selected from the group consisting of

In one embodiment, the polylactic acid salt may be at least one selected from the group consisting of compounds of Formulas 1 to 6 below.

[Formula 1]

RO-CHZ-[A] _n -[B] _m -COOM

In Formula 1, A is -COO-CHZ-; B is -COO-CHY-, -COOCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - or -COO-CH ₂ CH ₂ OCH ₂ -; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y are each a hydrogen atom, or a methyl or phenyl group; M is Na, K, or Li; n is an integer from 1 to 30; m is an integer from 0 to 20.

[Formula 2]

RO-CHZ-[COO-CHX] _p -[COO-CHY'] _q -COO-CHZ-COOM

In Formula 2, X is a methyl group; Y' is a hydrogen atom or a phenyl group; p is an integer from 0 to 25, q is an integer from 0 to 25, with the proviso that p+q is an integer from 5 to 25; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is Na, K, or Li; Z is a hydrogen atom, methyl or phenyl group.

[Formula 3]

RO-PAD-COO-W-M'

또는

이고; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체 및 D,L-락트산과 1,4-디옥산-2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이며; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이고; M은 독립적으로 Na, K, 또는 Li이다.In Equation 3, W-M' is

or

ego; PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, D,L-lactic acid and copolymers of caprolactone and copolymers of D,L-lactic acid and 1,4-dioxan-2-one; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is independently Na, K, or Li.

[Formula 4]

S-O-PAD-COO-Q

이고; L은 -NR₁- 또는 -O-이며, 여기서 R₁은 수소원자 또는 C_1-10 알킬이고; Q는 -CH₃, -CH₂CH₃, -CH₂CH₂CH₃, -CH₂CH₂CH₂CH₃, 또는 -CH₂C₆H₅이고; a는 0 내지 4의 정수이며; b는 1 내지 10의 정수이고; M은 Na, K, 또는 Li이며; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체, 및 D,L-락트산과 1,4-디옥산-2-온의 공중합체로 이루어진 군에서 선택되는 하나 이상이다.In Equation 4, S is

ego; L is -NR ₁ - or -O-, wherein R ₁ is a hydrogen atom or C _1-10 alkyl; Q is —CH ₃ , —CH ₂ CH ₃ , —CH ₂ CH ₂ CH ₃ , —CH ₂ CH ₂ CH ₂ CH ₃ , or —CH ₂ C ₆ H ₅ ; a is an integer from 0 to 4; b is an integer from 1 to 10; M is Na, K, or Li; PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, D,L-lactic acid and at least one selected from the group consisting of a copolymer of caprolactone, and a copolymer of D,L-lactic acid and 1,4-dioxan-2-one.

[Formula 5]

또는

or

In Formula 5, R' is -PAD-OC(O)-CH ₂ CH ₂ -C(O)-OM, wherein PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, D ,L-lactic acid and glycolic acid copolymer, D,L-lactic acid and mandelic acid copolymer, D,L-lactic acid and caprolactone copolymer, D,L-lactic acid and 1,4-dioxane-2 -one is selected from the group consisting of copolymers, M is Na, K, or Li; a is an integer from 1 to 4.

[Formula 6]

YO-[-C(O)-(CHX) _a -O-] _m -C(O)-R-C(O)-[-O-(CHX') _b -C(O)-] _n -OZ

In Formula 6, X and X' are independently hydrogen, alkyl having 1 to 10 carbons, or aryl having 6 to 20 carbons; Y and Z are independently Na, K, or Li; m and n are independently integers from 0 to 95, provided that 5 < m + n <100; a and b are independently integers from 1 to 6; R is -(CH ₂ ) _k -, divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbon atoms, or a combination thereof, where k is 0 to 10 is the integer of

In one embodiment, the polylactate may be a compound of Formula 1 or Formula 2.

divalent or trivalent metal ions

The nanocomposite composition of the present invention may further include a divalent or trivalent metal ion.

The divalent or trivalent metal ion is preferably calcium (Ca ²⁺ ), magnesium (Mg ²⁺ ), barium (Ba ²⁺ ), chromium (Cr ³⁺ ), iron (Fe ³⁺ ), manganese ( Mn ²⁺ ), nickel (Ni ²⁺ ), copper (Cu ²⁺ ), zinc (Zn ²⁺ ), or aluminum (Al ³⁺ ) may be selected.

The metal ion may be added to the polymer nanoparticle composition in the form of sulfate, hydrochloride, carbonate, phosphate and hydroxide, preferably calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ ), zinc chloride (ZnCl ₂ ), chloride Aluminum (AlCl ₃ ), iron chloride (FeCl ₃ ), calcium carbonate (CaCO ₃ ), magnesium carbonate (MgCO ₃ ), calcium phosphate (Ca ₃ (PO ₄ ) ₂ ), magnesium phosphate (Mg ₃ (PO ₄ ) ₂ ), Aluminum phosphate (AlPO ₄ ), magnesium sulfate (MgSO ₄ ), calcium hydroxide (Ca(OH) ₂ ), magnesium hydroxide (Mg(OH) ₂ ), aluminum hydroxide (Al(OH) ₃ ), zinc hydroxide (Zn(OH) ₂ ) or mixtures thereof may be added.

The divalent or trivalent metal ion can control the release rate of the drug encapsulated in the polymer nanoparticles by controlling the equivalent weight. Specifically, when 1 equivalent or less of the divalent or trivalent metal ion is included in the polymer nanoparticle composition with respect to the equivalent of the carboxyl group of the polylactate, the number of bonds with the carboxy terminal group of the polylactate is small, and the release rate of the drug is increased, When 1 equivalent or more is included, the release rate of the drug is delayed because the number of bonds with the carboxy terminal group of the polylactate is large. Accordingly, a small amount of metal ions may be used to increase the release rate of the drug in the blood, and a large amount of metal ions may be used to delay the release rate of the drug.

In addition, the divalent or trivalent metal ion may be included in an amount of 0.01 to 10 equivalents, 0.1 to 5 equivalents, or 0.2 to 2 equivalents based on the equivalent of the carboxy terminal group of the polylactic acid salt.

immunotherapy

The nanocomposite composition of the present invention may be used to treat cancer, and more specifically, may be used to treat cancer in combination with an immuno-oncology agent.

In one embodiment, the immuno-cancer agent that can be used in combination with the nanocomposite composition of the present invention may be an anti-PD-1 drug, and the anti-PD-1 drug is, for example, nivolumab or pembrolizumab. , Atezolizumab, Avelumab, Durvalumab, Semiplimab, and combinations thereof may be selected from the group consisting of.

How to prepare the composition

The nanocomposite composition of the present invention comprises the steps of: (a) dissolving doxorubicin in an aqueous solvent; (b) adding and dissolving polylactic acid salt to the solution obtained in step (a); (c) adding and dissolving the amphiphilic block copolymer to the solution obtained in step (b); and (d) drying the mixture obtained in step (c).

The aqueous solvent may be an aqueous solution of a lower alcohol (methanol, ethanol, propanol, etc.), distilled water, water for injection, or a buffer, and a preferred buffer may be phosphate buffered saline.

In one embodiment, the method for preparing the composition of the present invention may further include (e) adding a divalent or trivalent metal ion after step (d).

In one embodiment, the method for preparing the composition of the present invention, (f) after the step (e), may further include the step of freeze-drying. At this time, in step (e) or (f), a freeze-drying aid may be added to the resultant of step (e).

The freeze-drying aid used in the present invention allows the freeze-dried composition to maintain a cake shape, or after freeze-drying compositions such as amphiphilic block copolymer and polylactate, uniformly within a short time in the process of reconstitution. It is added to help dissolve, and specifically, it may be one or more selected from the group consisting of lactose, mannitol, sorbitol and sucrose. The content of the freeze-drying aid is, based on the total dry weight of the freeze-dried composition, 1 to 90% by weight, more specifically 4 to 20% by weight.

In addition, in the composition prepared according to an aspect of the present invention, since doxorubicin remains encapsulated in the nanoparticle structure formed by the amphiphilic block copolymer and polylactate, safety and stability in blood or body fluid This is improved.

In a preferred embodiment, the particle size of the nanoparticles in the composition is 10 to 300 nm, more specifically, preferably 10 to 150 nm.

The composition according to the present invention may be administered through an administration route such as blood vessel, muscle, subcutaneous, oral, bone, transdermal or topical tissue, and may be formulated into various oral or parenteral administration formulations suitable for such administration route. can The oral administration formulation may include various formulations such as tablets, capsules, powder formulations, liquid formulations, and the like, and parenteral formulations may include various formulations such as eye drops and injections. In a preferred embodiment, the composition is an injection formulation, more preferably It may be a formulation for intravenous injection. For example, when the composition according to the present invention is freeze-dried, it can be reconstructed with distilled water for injection, 0.9% physiological saline, and 5% dextrose aqueous solution to prepare a formulation for injection.

One aspect of the present invention provides a method of treating cancer in an individual, comprising the step of encapsulating doxorubicin in a nanoparticle structure formed by an amphiphilic block copolymer and a polylactate and administering the same to an individual in need thereof do.

In one embodiment, the nanocomposite composition of the present invention may have a dissolution rate of 1% to 20% by shaking and stirring at a temperature of 150 to 250 rpm for 20 to 24 hours in an aqueous solution having a pH of 7.0 to 7.5 at 35 ° C. to 38 ° C.

Hereinafter, the present invention will be described in more detail based on the following examples, but these are only for illustrating the present invention and the scope of the present invention is not limited in any way by these.

[Example]

1. Preparation and evaluation of nano-cancer drugs

1.1. Preparation of Doxorubicin-PNP

Doxorubicin-PNP was prepared according to the following method with the composition of Tables 1 and 2 below:

(1) After dissolving doxorubicin hydrochloride (Doxorubicin HCl) in an aqueous ethanol solution (ethanol:water = 9:1, v/v) in a round flask, polylactic acid sodium salt (PLA-COONa) was added as a polylactic acid salt. Complete dissolution at 50°C for 30 minutes.

(2) mPEG-PLA (monomethoxy polyethylene glycol-polylactide block copolymer) as an amphiphilic block copolymer was added to the resultant of (1), and all of them were dissolved.

(3) The resultant of (2) was first dried at 50° C. with an aspirator, and then dried secondarily with a vacuum pump.

(4) The dried product of (3) was placed in a round flask, and dissolved by adding physiological saline.

_{(5) CaCl 2} ·2H ₂ O and mannitol (Mannitol) were added to the resultant of (4), and then filtered with a 0.22㎛ syringe filter.

(6) The resultant of (5) was put into a glass vial in an amount corresponding to 10 mg of doxorubicin and lyophilized to obtain doxorubicin-polymer nanoparticles (Doxorubicin-PNP).

1.2. Assessment of Doxorubicin-PNP

1.2.1. Release test of Doxorubicin-PNP

1) Test method

① Reconstruct each example with physiological saline at a concentration of 1 mg/mL

② Put 18 mL of pH 7.4 PBS in a 37℃ water bath and add 2 mL of each sample (0.2mg/mL)

③ After shaking and stirring at 200rpm in a 37℃ water bath, after 24 hours, take 500uL of membrane NMWL 30kDa centrifugal filter and filter at 14000rpm for 15 minutes.

④ Analysis by HPLC content analysis method.

2) Analysis of the content of Doxorubicin-PNP

- Preparation of standard solution

① Precisely weigh 10 mg of doxorubicin, put it in a 10 mL volumetric flask, and adjust to the mark (1 mg/mL) with water for injection

② Prepare standard solution by concentration by diluting the solution with water for injection

- HPLC analysis conditions

① Column: Kromasil 100-10, C18, 4.6 x 250 mm

② Flow rate: 0.8 mL/min

③ Mobile phase: 0.01M sodium acetate: Acetic acid: MeOH=50:2.1:50

④ Injection volume: 50μL

⑤ Wavelength and column temperature: 480nm

⑥ Column temperature: 25℃

- Sample solution preparation and analysis method

① Add water for injection to each example and completely dissolve it to prepare 1 mg/mL

② Take 1 mL of the above solution, put it in a 20 mL volumetric flask, mark it with water for injection, and prepare it at a concentration of 50 μg/mL

③ Analyze 50 μl of the solution by HPLC content analysis

1.2.2. Encapsulation rate of Doxorubicin-PNP

- Sample solution preparation and analysis method

① Add water for injection to each example and completely dissolve it to prepare 1 mg/mL

② Take 500 μl of the solution and filter it with a 100 kDa centrifugal filter

③ Analyze 50 μl of the solution by HPLC content analysis

1.2.3. Particle size of Doxorubicin-PNP

- Sample solution preparation and analysis method

① Add water for injection to each example and completely dissolve it to prepare 1 mg/mL

② Take 1 mL of the above solution and analyze the particle size by DLS

1.3. Experiment result

1) Doxorubicin-PNP release test

As can be seen from Tables 3 and 4 above, it can be seen that in particular Examples 1, 5, and 6 were successfully reconstructed and the release rate could be controlled.

2) Doxorubicin-PNP content and encapsulation rate

The content and encapsulation rate of nanoparticles were repeated three times for Example 6 and the average was obtained.

As can be seen from Tables 5 and 6, Example 6 showed excellent results in the reconstruction rate and the encapsulation rate.

3) Particle size of Doxorubicin-PNP

Example 6 was repeated three times to obtain the average particle diameter of the nanoparticles.

As can be seen from Table 7, Example 6 has a preferred nanoparticle particle diameter of 50 nm or less.

2. In vivo vaccination assay

2.1. Experimental purpose

Vaccination assay was performed to confirm that Doxorubicin-PNP is an ICD inducer. For each concentration, the concentration determined by checking _{IC 10} , IC ₅₀ , and IC ₉₀ in the in vitro cytotoxicity test was used.

2.2. Experimental method

In this experiment, as shown in Table 8 below, CT26 cancer cells (murine colon carcinoma cells) were used, and for vaccination, cancer cells were treated with _{an IC 90 concentration of the test drug.} The dying cancer cells 3 x 10 ⁶ were administered subcutaneously to the left side of female BALB/c mice (8w, n=10/group). One week later, 5 x 10 ⁵ living cancer cells were administered subcutaneously to the right, and the tumor size of the left and right mice was measured. The end of the experiment was measured until the size of the tumor became 2000mm ³ or 45 days after the start of the experiment. Example 6 was used as Doxorubicin-PNP.

If the Doxorubicin-PNP to be verified has the ability to induce ICD, the secondary transplanted cells will not grow. Vaccination assay results were expressed as a percentage (Tumor free mice Proportion (%)) of the number of mice that did not grow cancer cells in each group.

2.3. Experiment result

As can be seen from Figure 1, the nano-anticancer drug Doxorubicin-PNP showed increased ICD induction and increased vaccination effect compared to simple API. In particular, Doxorubicin-PNP showed more improved effects than paclitaxel and docetaxel, which are other anticancer drugs tested.

3. Immune cell recruitment assay

3.1. Experimental purpose

In this experiment, the expression and decreased immune cells were identified by ICD induction, and the secretion of cytokines by the immune cells was confirmed.

3.2. Experimental method

In this experiment, the CT26 murine colon carcinoma BALB/c model was used, and the test material was administered to mice transplanted with cancer cells 7 days later. Among the test substances, 5 mg/kg of Mitoxantrone and Doxorubicin were used as a positive control group. And 5 mg/kg of Doxorubicin-PNP was administered as a nano-cancer agent. After administration of the test substance, tumors were excised on days 2 and 8, and the expression of immune cells in the tumor was confirmed by FACS and IHC. If anticancer immunity is increased by ICD, CD8 ⁺ T cells increase and myeloid-derived suppressor cells (MDSCs) decrease.

3.3 Experimental results

As can be seen from FIG. 2 , compared with simple AIP, Doxorubicin-PNP, which is a nano-cancer drug, ^{showed a tendency of overall CD8 +} T cells to be expressed more than equal to and also to decrease MDSC.

Claims (6)

Doxorubicin as an active ingredient;
amphiphilic block copolymers;
polylactate;
mannitol; and
Calcium divalent ion (Ca ²⁺ );
The amphiphilic block copolymer is an AB-type diblock copolymer composed of a hydrophilic A block and a hydrophobic B block, wherein the hydrophilic A block is monomethoxypolyethylene glycol, and the hydrophobic B block is polylactide,
the polylactic acid salt is polylactic acid sodium salt;
The doxorubicin is encapsulated in the nanoparticle structure formed by the amphiphilic block copolymer and the polylactate,
The molar ratio of the sum of the amphiphilic block copolymer and the polylactate to doxorubicin is 19 to 21:1, and the molar ratio of the polylactate to doxorubicin is 9 to 15:1,
The number average molecular weight of the hydrophilic A block is 1,000 to 5,000 daltons, the number average molecular weight of the hydrophobic B block is 1,000 to 5,000 daltons,
The content of the hydrophilic A block in the amphiphilic block copolymer is, based on the weight of the copolymer, 40 to 70% by weight,
Nanocomposite composition. The nanocomposite composition according to claim 1, which is used to treat cancer. The nanocomposite composition according to claim 2, which is used to treat cancer in combination with an immuno-oncology agent. The nanocomposite composition according to claim 3, wherein the immuno-oncology agent is an anti-PD-1 drug. 5. The method of claim 4, wherein the anti-PD-1 drug is nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, semiplumab (Cemiplimab) and combinations thereof, which is selected from the group consisting of, the nanocomposite composition. (a) dissolving doxorubicin in an aqueous solvent;
(b) adding and dissolving polylactic acid salt to the solution obtained in step (a);
(c) adding and dissolving the amphiphilic block copolymer to the solution obtained in step (b);
(d) drying the mixture obtained in step (c);
(e) adding calcium divalent ions (Ca ²⁺ ) and mannitol to the mixture obtained in step (d); and
(f) drying the mixture obtained in step (e);
The amphiphilic block copolymer is an AB-type diblock copolymer composed of a hydrophilic A block and a hydrophobic B block, wherein the hydrophilic A block is monomethoxypolyethylene glycol, and the hydrophobic B block is polylactide,
the polylactic acid salt is polylactic acid sodium salt;
The doxorubicin is encapsulated in the nanoparticle structure formed by the amphiphilic block copolymer and the polylactate,
The molar ratio of the sum of the amphiphilic block copolymer and the polylactate to doxorubicin is 19 to 21:1, and the molar ratio of the polylactate to doxorubicin is 9 to 15:1,
The number average molecular weight of the hydrophilic A block is 1,000 to 5,000 daltons, the number average molecular weight of the hydrophobic B block is 1,000 to 5,000 daltons,
The content of the hydrophilic A block in the amphiphilic block copolymer is, based on the weight of the copolymer, 40 to 70% by weight,
A method for preparing a nanocomposite composition.

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