KR20230082178A - Composition for photodynamic therapy for HER2-expressing cancer and use thereof - Google Patents

Abstract

The present invention relates to a composition for photodynamic treatment of HER2-expressing cancer and its use. Specifically, the composition for photodynamic therapy according to the present invention maximizes the accumulation of radioactive isotopes in cancer cells using a cancer-targeting antibody and induces the accumulation of a photosensitizer in cancer cells by including a photosensitizer precursor rather than the photosensitizer itself. By doing so, it is possible to minimize damage to normal tissue and maximize the therapeutic effect of photodynamic therapy without separate light irradiation.

Description

Composition for photodynamic therapy of HER2-expressing cancer and use thereof {Composition for photodynamic therapy for HER2-expressing cancer and use thereof}

The present invention relates to a composition for photodynamic treatment of HER2-expressing cancer and its use.

In photodynamic therapy (PDT), a photosensitizer excited by light of a specific wavelength reacts with oxygen to generate active oxygen such as singlet oxygen ( ¹ O ₂ ) to kill surrounding cells. As a non-invasive and highly efficient treatment method, it is used for the treatment of acne, fungal infection, and cancer. However, in the case of cancer treatment, due to limitations in light transmission, only superficial cancer and endoscopic light source accessible cancer are selectively used.

Recently, in order to overcome these limitations, studies using Cherenkov luminescence, which is generated when a radioactive isotope reacts with a surrounding medium, as an excitation source for a photosensitizer are being conducted.

Cherenkov luminescence is a phenomenon that occurs when charged particles move faster than light in a specific dielectric medium, and beta rays and positrons from radioactive isotopes can generate Cherenkov luminescence. Cherenkov emission has the greatest intensity at about 400 nm, and photosensitizers such as porphyrin and chlorin have high absorbance at about 400 nm, so they can be excited by Cherenkov emission. .

As such, radioactive isotopes such as ¹⁸ F, ⁶⁴ Cu, ⁸⁹ Zr, ⁹⁰ Y, ¹⁷⁷ Lu, and ¹³¹ I can generate Cherenkov luminescence, and photodynamic therapy without external light irradiation by grafting with a photosensitizer. , PDT) is being researched. Until now, studies evaluating the PDT effect through Cherenkov radiation energy transfer by loading radioactive isotopes such as ⁶⁴ Cu, ⁸⁹ Zr, and ¹⁸ F and photosensitizers on one platform have been preceded ( Nat Nanotechnol , 2015, 10(4) , 370-379; ACS Nano , 2020, 14(10), 13004-13015; ACS Appl Mater Interfaces , 2016, 8(40), 26630-26637).

However, these conventional techniques do not use a targeting moiety when delivering a radioisotope to a cancer, so that the radioisotope accumulation in the cancer is not efficient, and the radioisotope and photosensitizer are together on one platform. Since it is loaded, there is a problem that normal tissues can be damaged by active oxygen generated before cancer reaches and accumulates.

Accordingly, the inventors of the present invention minimized damage to normal tissue and maximized the therapeutic effect without separate light irradiation by injecting a radioisotope-labeled compound and a photosensitizer (or a precursor of the photosensitizer) into an antibody targeting cancer cells, respectively. Focusing on the possibility of realizing photodynamic therapy that can be performed, the present invention has been completed.

Republic of Korea Patent Registration No. 10-1918205

An embodiment of the present invention for solving the above problems is a compound of Formula 1 or a pharmaceutically acceptable salt thereof; And to provide a composition for photodynamic therapy comprising a photosensitizer or a precursor of the photosensitizer:

[Formula 1]

R-L-Ab

In Formula 1,

R is a radioactive isotope,

L is a chelator or a direct bond;

Ab is an antibody.

Another embodiment of the present invention to solve the above problems is to provide a pharmaceutical composition for preventing or treating cancer comprising the composition for photodynamic therapy as an active ingredient.

Another embodiment of the present invention to solve the above problems is to provide a food composition for preventing or improving cancer containing the composition for photodynamic therapy as an active ingredient.

Another embodiment of the present invention to solve the above problems, (a) comprising the step of administering the composition for photodynamic therapy to a subject other than a human or in vitro ( in vitro ), It is to provide a photodynamic treatment method that is performed without irradiation of.

On the other hand, the technical problems to be achieved in the present invention are not limited to the above-mentioned technical problems, and other technical problems that are not mentioned will become clear to those skilled in the art from the description below. You will be able to understand.

Photodynamic therapy composition

According to one embodiment of the present invention, in order to solve the above-described technical problem, the compound of Formula 1 or a pharmaceutically acceptable salt thereof; And a composition for photodynamic therapy comprising a photosensitizer or a precursor of the photosensitizer is provided:

[Formula 1]

R-L-Ab

In Formula 1,

R is a radioactive isotope,

L is a chelator or a direct bond;

Ab is an antibody.

Conventionally, research to implement photodynamic therapy (PDT) without external light irradiation by using Cherenkov Luminescence, which is generated by the reaction of radioactive isotopes with surrounding media, as an excitation source for photosensitizers. has progressed In this regard, up to now, studies evaluating the PDT effect through Cherenkov radiation energy transfer by loading radioactive isotopes such as ⁶⁴ Cu, ⁸⁹ Zr, and ¹⁸ F and a photosensitizer on one platform have been preceded.

However, these conventional techniques do not use a targeting moiety when delivering a radioisotope to a cancer, so that the radioisotope accumulation in the cancer is not efficient, and the radioisotope and photosensitizer are combined in one platform. Since it is mounted, there is a problem that normal tissues can be damaged by active oxygen generated before reaching and accumulating cancer.

In order to solve the above problems, in the present invention, using the composition for photodynamic therapy, minimizing damage to normal tissue and implementing photodynamic therapy capable of maximizing the therapeutic effect without separate light irradiation, It is intended to provide a photodynamic treatment method.

Hereinafter, the mechanism of photodynamic therapy (PDT) according to the present invention will be described in detail with reference to FIG. 1 .

In photodynamic therapy using conventional light, a photosensitizer (PS) excited by light of a specific wavelength reacts with oxygen to form active oxygen (ROS) such as singlet oxygen ( ¹ O ₂ ). can cause the death of surrounding cells. At this time, when the surrounding cells are cancer cells, the generated active oxygen can kill the cancer cells. Although PDT has been used for acne, fungal infection, and cancer treatment, in the case of cancer treatment, it is selectively used only for superficial cancer and endoscopic light source accessible cancer due to limitations in light transmission. A problem existed.

Referring to FIG. 1, the photodynamic therapy according to the present invention preferentially induces the accumulation of a photosensitizer through the absorption of a photosensitizer, specifically a precursor of the photosensitizer, into cancer cells, and at the same time, a radioisotope-antibody platform (e.g. ⁶⁴ Cu-Trastuzumab) can maximize the accumulation of radioactive isotopes in cancer cells through targeting with antibodies.

Then, the radioactive isotope accumulated in the cancer cells reacts with the surrounding medium to generate Cherenkov emission, and uses the generated Cherenkov emission as an excitation source of the photosensitizer to perform photodynamic therapy without external light irradiation. therapy, PDT) can be implemented.

More specifically, the photosensitizer accumulated in the cancer cells is excited by Cherenkov light emission and then reacts with oxygen to generate reactive oxygen species (ROS) such as singlet oxygen ( ¹ O ₂ ) to kill cancer cells. .

As such, the composition for photodynamic therapy according to the present invention, through the above mechanism, can be used without light irradiation, in contrast to the prior art, which has been selectively used only for superficial cancer and cancer accessible to an endoscope light source due to limitations in light transmission. Photodynamic therapy can be applied even to cancer that exists deep in the body, maximizing the therapeutic effect, and using two platforms (radioisotope-antibody and photosensitizer or photosensitizer precursor) before cancer reaches and accumulates. It has the effect of minimizing damage to normal tissues because there is no generation of active oxygen.

The radioisotope-antibody platform may be coupled by a chelator that binds the radioisotope and the antibody. Also, depending on the type of radioisotope labeled on the antibody, the chelator may not be attached (bound) to the radioisotope.

The compounds of Formula 1 of the present invention may contain one or more asymmetric carbons and thus may exist as racemates, racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers.

These isomers can be separated by conventional techniques, for example, the compound of Formula 1 is separated by column chromatography or HPLC. Alternatively, each stereoisomer of the compound represented by Formula 1 can be stereospecifically synthesized using optically pure starting materials and/or reagents of known arrangement.

In the present invention, the pharmaceutically acceptable salt refers to a salt commonly used in the pharmaceutical industry, for example, an inorganic ion salt prepared from calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, inorganic acid salts prepared from iodic acid, perchloric acid and sulfuric acid; Acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid organic acid salts prepared from acids, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like; sulfonic acid salts prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid; amino acid salts made of glycine, arginine, lysine, and the like; and amine salts prepared with trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the types of salts meant in the present invention are not limited by these listed salts.

In Formula 1, R may be a radioactive isotope. As a specific example, the radioisotope is a group consisting of Cu-64, Cu-67, Ga-68, Zr-89, Lu-177, Y-90, In-111, I-123, I-124 and I-131. It may be one or more selected from, but is not limited thereto, and an appropriate radioisotope may be selected according to the use of the composition for photodynamic therapy. As a more specific example, the radioactive isotope may be Cu-64 or I-124, and in this case, an excellent cancer cell killing effect may be exhibited compared to cases containing other types of radioactive isotopes. In addition, the radioisotope may be coordinately bonded to the chelator.

In Formula 1, L may be a chelator or a direct bond. As a specific example, the chelator is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTA-NCS, DOTA-NHS ester, DOTA-Bz-NCS, tris (tbu) DOTA, HBED-CC-TFP ester, DTPA (diethylene triamine penta aeetic acid), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid), NOTA (1,4,7-triazacyclononane- 1,4,7-triacetic acid), NODAGA (1,4,7-Triazacyclononane, 1-glutaric acid-4,7-acetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N', N'',N'''-tetraacetic acid), TE3A (1,4,8,11-tetraazacyclotetradecane-1,4,8-triacetic acid), TE2A (1,4,8,11-Tetraazabicyclohexadecane-4,11 -diacetic acid), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1,11,13-triene-3,6,9,-triacetic acid), and DFO (Deferrioxamine). It may be one or more selected from, but is not limited thereto, and an appropriate chelator may be selected according to the use of the composition for photodynamic therapy. As a more specific example, the chelator is DOTA (CAS NO.: 60239-18-1), specifically DOTA-NHS ester (CAS No.: 1823122-52-6) or p-SCN-Bn-DOTA (CAS No. : 127985-74-4), and in this case, the accumulation effect of radioactive isotopes in cancer cells can be improved compared to the case of including other types of chelators.

The chelator refers to a molecule forming a complex with a metal atom, wherein the complex is stable under physiological conditions. Specifically, the chelator may be synthesized with a radioactive isotope and be physiologically stable.

In Formula 1, Ab is an antibody. As a specific example, the antibody is from the group consisting of trastuzumab, pertuzumab, cetuximab, rituximab, brentuximab and panitumumab. It may be one or more selected, but is not limited thereto, and an appropriate antibody may be selected according to the purpose of the composition for photodynamic therapy. As a more specific example, the antibody may be trasuzumab, and in this case, the antibody has excellent targeting effect in HER2-expressing cancer cells. The HER2-expressing cancer may be breast cancer or gastric cancer, but is not limited thereto.

According to one embodiment of the present invention, the photosensitizer or the precursor of the photosensitizer is 5-aminolevulinic acid (5-ALA), protoporphyrin IX (PPIX), chlorine e6 (Ce6) ), methyl-aminolevulinate (MAL), hexyl-aminolevulinic acid (hexylaminolevulinate, HAL), and one or more selected from the group consisting of derivatives thereof, but is not limited thereto, and photodynamic therapeutic composition An appropriate photosensitizer or a precursor of the photosensitizer may be selected according to the use of the photosensitizer. As a specific example, the composition for photodynamic therapy according to the present invention may include a precursor of a photosensitizer, and the precursor of the photosensitizer may be 5-ALA.

The photosensitizer or precursor of the photosensitizer may be selectively accumulated in cancer tissues through receptors overexpressed in cancer cells.

5-ALA and its derivatives are precursors of photosensitizers, and compared to general photosensitizers, which are insoluble in water and do not accumulate in cells by themselves, they dissolve well in water and are easily applied in vivo, and are absorbed into cancer cells after being absorbed into cancer cells. It can be converted into protoporphyrin IX (PPIX) photosensitizer in the mitochondria. Specifically, the PPIX molecule can be chelated with iron by the enzyme ferrochelatase (FECH) to produce heme. In cancer cells, the activity of the enzyme (FECH) is absent or reduced, so PPIX can accumulate in cancer cells (FIG. 2). reference).

As described above, compared to general photosensitizers that are not easily accumulated in cancer cells, 5-ALA and its derivatives are converted into photosensitizers (PPIX) in cancer cells and then accumulated, thereby releasing active oxygen before reaching and accumulating in cancer cells. It has the effect of reducing damage to normal tissues by minimizing the occurrence of

According to one embodiment of the present invention, the composition for photodynamic therapy may be used for photodynamic therapy of cancer without light irradiation, and the cancer is breast cancer, stomach cancer, ovarian cancer, brain cancer, and uterine cancer. , It may be selected from the group consisting of endometrial cancer, pancreatic cancer, kidney cancer, peritoneal cancer and lung cancer. Specifically, the cancer may be breast cancer or gastric cancer. More specifically, the cancer may be breast cancer.

Use of composition for photodynamic therapy

The present invention provides a use of the composition for photodynamic therapy.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the composition for photodynamic therapy as an active ingredient.

The cancer may be selected from the group consisting of breast cancer, stomach cancer, ovarian cancer, brain cancer, uterine cancer, endometrial cancer, pancreatic cancer, kidney cancer, peritoneal cancer, and lung cancer. Specifically, the cancer may be breast cancer or gastric cancer. More specifically, the cancer may be breast cancer.

For administration, the pharmaceutical composition of the present invention may further include at least one pharmaceutically acceptable carrier in addition to the composition for photodynamic therapy. A pharmaceutically acceptable carrier may be a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and, if necessary, antioxidants and buffers. , bacteriostatic agents and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Accordingly, the pharmaceutical composition of the present invention may be a patch, liquid, pill, capsule, granule, tablet, suppository or the like. These formulations may be prepared by a conventional method used for formulation in the art or a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA, and formulated into various formulations depending on each disease or component It can be.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) depending on the desired method, and the dosage is determined by the patient's body weight, age, sex, The range varies according to health status, diet, administration time, administration method, excretion rate, and type and severity of disease. The daily dose of the pharmaceutical composition of the present invention is about 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day.

The pharmaceutical composition of the present invention may further contain at least one active ingredient exhibiting the same or similar medicinal effect in addition to the composition for photodynamic therapy.

The present invention provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of the composition for photodynamic therapy to a mammal, including a human.

The term "therapeutically effective amount" used in the present invention refers to an amount of the composition for photodynamic therapy effective for preventing or treating cancer.

The treatment method of the present invention includes not only addressing the disease itself prior to the onset of symptoms, but also inhibiting or avoiding its symptoms, by administering the photodynamic therapeutic composition. In the management of disease, the prophylactic or therapeutic dose of a particular active ingredient will vary depending on the nature and severity of the disease or condition and the route by which the active ingredient is administered. Dosage and frequency of dosing will vary according to the age, weight and response of the individual patient. A suitable dosage regimen can be readily selected by those skilled in the art who take these factors into account. In addition, the treatment method of the present invention may further include the administration of a therapeutically effective amount of an additional active agent helpful for disease treatment together with the photodynamic treatment composition, and the additional active agent is the photodynamic treatment composition Together with can exhibit synergistic or auxiliary effects.

In addition, the present invention provides a food composition for preventing or improving cancer comprising the composition for photodynamic therapy as an active ingredient.

The cancer may be selected from the group consisting of breast cancer, stomach cancer, ovarian cancer, brain cancer, uterine cancer, endometrial cancer, pancreatic cancer, kidney cancer, peritoneal cancer, and lung cancer. Specifically, the cancer may be breast cancer or gastric cancer. More specifically, the cancer may be breast cancer.

The food composition of the present invention can be used as a health functional food. The term “health functional food” refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Health Functional Food Act No. 6727, and “functional” refers to the structure of the human body. And it refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients for functions or physiological functions.

The food composition of the present invention may contain conventional food additives, and the suitability as the "food additive" is determined in accordance with the General Rules of the Code of Food Additives and General Test Methods approved by the Korea Food and Drug Administration, unless otherwise specified. It is judged according to the standards and standards for

For the purpose of preventing and/or improving cancer, the food composition of the present invention contains the radioisotope-labeled antibody and photosensitizer (or photosensitizer precursor), or the photodynamic therapeutic composition in an amount of 0.01 to 95% based on the total weight of the composition. %, preferably from 1 to 80% by weight percentage. In addition, for the purpose of preventing and/or improving cancer-related diseases, it can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, beverages, and the like.

The terms “prevention,” “treatment,” “pharmaceutical composition,” and the like are all defined the same unless specifically defined.

In addition, the present invention provides a photodynamic treatment method comprising the step of (a) administering the composition for photodynamic treatment according to the present invention to a subject other than a human or in vitro . The photodynamic treatment method is characterized in that it is performed without irradiation of light.

According to one embodiment of the present invention, after step (a), (b) accumulating the composition in target cells of the subject may be further included.

At this time, the concentration of the photosensitizer (or precursor of the photosensitizer) is 10 to 20 mM, 10 to 18 mM, or 10 to 15 mM, and the step (b) is performed for 1 to 24 hours, 2 to 10 hours, or 3 to 5 hours. It may be performed, and within the concentration range of the photosensitizer (or the precursor of the photosensitizer) and the execution time range of step (b), there is an effect of improving the accumulation of the photosensitizer. Specifically, when 5-ALA is used as a precursor of the photosensitizer, the accumulation of the photosensitizer is further improved, thereby maximizing the therapeutic effect.

The target cells may be cancer cells, specifically breast cancer or gastric cancer cells, but are not limited thereto.

Matters mentioned in the composition, use, and treatment method of the present invention are equally applied unless contradictory to each other.

The composition for photodynamic therapy according to the present invention maximizes the accumulation of radioactive isotopes in cancer cells using cancer-targeting antibodies and induces the accumulation of photosensitizers in cancer cells by including photosensitizer precursors instead of the photosensitizer itself, thereby inducing the accumulation of photosensitizers in normal tissues. It is possible to maximize the therapeutic effect of photodynamic therapy without separate light irradiation while minimizing damage to the skin.

On the other hand, the effects obtainable in the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below. You will be able to.

1 is an image showing the mechanism of a photodynamic treatment method using a photodynamic treatment composition according to the present invention.
FIG. 2 is an image showing the mechanism by which protoporphyrin IX (PPIX) and heme are produced intracellularly upon external injection of 5-ALA.
3 is (a) a graph of trastuzumab and (b) a MALDI-TOF measurement result graph of DOTA-Trastuzumab synthesized from Example 1 of the present invention, and (c) a result table.
4 is a graph showing the Kd value of ⁶⁴ Cu-DOTA-Trastuzumab synthesized in Example 1 of the present invention for a breast cancer cell line (BT-474) (a) the total amount of ⁶⁴ Cu-DOTA-Trastuzumab and cells treated in the cell line A graph comparing the amount of ⁶⁴ Cu-DOTA-Trastuzumab combined with (b) its Scatchard plot, and to obtain the immunoreactive fraction (c) cell-bound ⁶⁴ Cu-DOTA-Trastuzumab according to the number of cells A graph showing the amount change, (d) a double inverse plot thereof, and (e) a schematic diagram showing the chemical structure of the synthesized ⁶⁴ Cu-DOTA-Trastuzumab.
Figure 5 shows (a-d) PPIX fluorescence images according to 5-ALA concentration and (e) PPIX fluorescence spectrum over time [5-ALA (a) 0 mM, (b) 3 mM, (c) 6 mM, (d) 12 mM, (e) 12 mM].
6 is a graph showing (a-d) PPIX fluorescence images and (e) average light radiance intensity over time when 5-ALA was injected intravenously into a gastric cancer (NCI-N87) mouse model. [(a) 1 hour, (b) 3 hours, (c) 6 hours, (d) 9 hours].
7 is a graph showing (a-d) PPIX fluorescence images and (e) average light radiance intensity over time upon intraperitoneal injection of 5-ALA into a gastric cancer (NCI-N87) mouse model. [(a) 1 hour, (b) 4 hours, (c) 5 hours, (d) 9 hours].
8 is (a) an image and (b) a graph showing the intensity of Cherenkov luminescence according to the intensity of radioactivity of ⁶⁴ Cu, and shows the change in ⁶⁴ Cu-based Cherenkov luminescence intensity according to PPIX concentration (c) image and (d) graph.
Figure 9 shows negative control (Ctrl), 5-ALA (12mM) (Treatment 1), ⁶⁴ Cu (500μCi) (Treatment 2), 5-ALA (12mM) and ⁶⁴ Cu-DOTA-trastuzumab (500μCi) treated in sequence. It is a graph showing relative breast cancer cell viability for the group (Treatment 3) and the group treated with a mixture of 5-ALA (12 mM) and ⁶⁴ Cu-DOTA-trastuzumab (500 μCi) (Treatment 4).
Figure 10 is a negative control (Control), 5-ALA alone, Ce6 photosensitizer alone, ⁶⁴ Cu alone, ⁶⁴ Cu-DOTA-Trastuzumab alone, 5-ALA and ⁶⁴ Cu-DOTA-Trastuzumab mixed, Ce6 and ⁶⁴ Cu-DOTA-Trastuzumab It is a graph showing breast cancer cell viability according to the treatment of combination, ¹²⁴ I alone, and 5-ALA and ¹²⁴ I-Trastuzumab combination.
11 is a graph showing the growth of cancer cells over time through intraperitoneal injection of 5-ALA and intravenous injection of ⁶⁴ Cu-DOTA-trastuzumab into a gastric cancer (NCI-N87) mouse model.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Embodiments of the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples. This embodiment is provided to more completely explain the present invention to those skilled in the art. Accordingly, the shapes of elements in the figures are exaggerated to emphasize clearer description.

<Example>

ingredient

5-aminolevulinic acid (5-ALA, Cat# A7793) was purchased from Sigma-Aldrich, p-SCN-Bn-DOTA (Cat# B-205) was purchased from Macrocyclics, and trastuzumab (Herceptin ^® ) was purchased from Roche. ⁶⁴ Cu and ¹²⁴ I were produced using a 50 MeV accelerator at Korea Institute of Atomic Energy and Medicine.

Example 1. Synthesis of radioisotope-labeled antibody (radioisotope-antibody)

(1) Synthesis of DOTA-Trastuzumab

After dissolving trastuzumab (19.8 mg, 0.137 mmol) in HEPES buffer (HEPES 50 mM / NaCl 150 mM, pH 8.6), add p-SCN-Bn-DOTA (0.942 mg, 1.37 mmol) and react at 25 °C for 16 hours. did After the reaction, unconjugated p-SCN-Bn-DOTA was removed with an Amicon Ultra centrifugal device (30K, Milipore), and DOTA-Trastuzumab was separated from the filter using PBS (pH 7.4). The amount of conjugated DOTA was obtained through mass comparison using MALDI-Mass (TOF/TOF™ 5800 System, Ab Sciex).

Referring to FIG. 3 , it can be confirmed that 1.3 mol of DOTA was conjugated to 1 mol of trastuzumab.

(2) DOTA-Trastuzumab ⁶⁴ Cu cover

Add DOTA-Trastuzumab (166.7 mg, 4.6 mL PBS, pH 7.4) and ⁶⁴ Cu (500 mCi, 2.8 mL 0.01 M HCl solution) sequentially to 0.25 M NH ₄ OAc, pH 5.0 buffer (12.6 mL) at 37 °C. After reacting for 30 minutes, the radiochemical purity of ⁶⁴ Cu-DOTA-Trastuzumab was confirmed using radio-TLC (B-AR2000-1, Eckert & Ziegler).

(3) Trastuzumab ¹²⁴ I cover

PBS (pH 7.4, 98.2 mL), trastuzumab (275 mg, 13.4 mL PBS, pH 7.4), and ¹²⁴ I (550 mCi, 38.4 mL 0.1 M NaOH solution) were placed in an iodination tube (Pierce™ Iodination tubes, Thermo Scientific) in that order. It was put as it was and reacted at 37 °C for 30 minutes, and the radiochemical purity of ¹²⁴ I-trastuzumab was confirmed using radio-TLC (B-AR2000-1, Eckert & Ziegler).

(4) ⁶⁴ Evaluation of Kd value and immunoreactive fraction of Cu-DOTA-Trastuzumab

For Kd values, BT-474 cells (2 × 10 ⁶ cells, PBS containing 1% BSA) were incubated with various concentrations of ⁶⁴ Cu-DOTA-Trastuzumab (0.27 ~ 68.96 nM) at 4 °C for 3 hours. After that, ⁶⁴ Cu-DOTA-Trastuzumab not bound to cells was washed away with PBS, and a graph comparing the total amount of ⁶⁴ Cu-DOTA-Trastuzumab and the amount of ⁶⁴ Cu-DOTA-Trastuzumab bound to cells and its Scatchard plot (Fig. 4a , b) was used to derive the Kd value (Kd = 17.03 nM).

For the immunoreactive fraction, ⁶⁴ Cu-DOTA-Trastuzumab (20 nM) was incubated with various numbers of BT-474 cells (0.059 ~ 15.0 × 10 ⁶ cells, PBS containing 1% BSA) at 4 °C for 3 hours. After that, the cell-bound ⁶⁴ Cu-DOTA-Trastuzumab was washed away with PBS, and the change in the amount of cell-bound ⁶⁴ Cu-DOTA-Trastuzumab according to the number of cells was graphed, and a double inverse plot thereof (FIG. 4c,d Reference) was used to derive the immunoreactive fraction (immunoreactive fraction = 91.5%). It can be seen that 91.5% of the synthesized ⁶⁴ Cu-DOTA-Trastuzumab maintained well the immunoreactivity of Trastuzumab.

Experimental Example 1. PPIX accumulation experiment through 5-ALA treatment

(1) in breast cancer cell line (BT474) In vitro Experiment

For cell fluorescence images, BT-474 cells (5 × 10 ⁶ cells per well) were seeded on a 4-well glass-bottom slide (Nunc ^® Lab-Tek ^® II Chamber Slide™ system, Lab-Tek) and after 24 hours After treatment with various concentrations of 5-ALA (0 to 12 mM) and incubation for 24 hours, fluorescence images of PPIX in cells were acquired (DMI3000 B, Leica).

Referring to FIGS. 5A to 5D , it can be confirmed that when the breast cancer cell line (BT-474) is treated with 5-ALA, PPIX accumulated in proportion to the concentration of 5-ALA increases.

In the case of the PPIX fluorescence spectrum, BT-474 cells (2 × 10 ⁴ cells per well) were seeded in a 96-well plate, and after 24 hours, a constant concentration of 5-ALA (12 mM) was incubated for various times, and the intensity of PPIX fluorescence was measured (SpectraMax ^® i3x, Molecular Devices).

Referring to FIG. 5e, it can be confirmed that the highest PPIX fluorescence intensity appears when 4 hours have elapsed after 5-ALA treatment.

(2) 5-ALA intravenous injection (i.v. injection) into a mouse model In vivo Experiment

5-ALA (500 mg/kg, 100 mL PBS pH 7.4) was injected intravenously into a mouse model transplanted with HER2 overexpressing gastric cancer cell line (NCI-N87), and PPIX accumulation over time was measured by in vivo fluorescence. It was confirmed using a measuring device (IVIS Spectrum, Perkin Elmer).

6a to 6e, the accumulation of PPIX increased after intravenous injection, showing the highest accumulation at 3 to 6 hours.

(3) 5-ALA intraperitoneal injection (i.p. injection) into a mouse model In vitro Experiment

5-ALA (500 mg/kg, 100 mL PBS pH 7.4) was injected intraperitoneally into a mouse model transplanted with HER2 overexpressing gastric cancer cell line (NCI-N87), and PPIX accumulation over time was evaluated in vivo. It was confirmed using a fluorescence measurement equipment (IVIS Spectrum, Perkin Elmer).

Referring to Figures 7a to 7e, the accumulation of PPIX increased after 3 hours after intraperitoneal injection, showing the highest accumulation at 4 to 9 hours.

Experimental example 2. ⁶⁴ Confirmation of Cherenkov radiation and energy transfer by Cu

In the case of experiments to confirm Cherenkov emission by ⁶⁴ Cu, ⁶⁴ Cu of various radioactivity (50 ~ 500 mCi) was put into a 96-well black plate and Cherenkov emission was measured using a fluorescence measurement equipment (IVIS Spectrum, Perkin Elmer). did

Referring to FIGS. 8A and 8B , it can be seen that Cherenkov emission caused by ⁶⁴ Cu increases in proportion to the radioactivity of ⁶⁴ Cu.

In the case of experiments confirming the transfer of ⁶⁴ Cu-based Cherenkov emission to PPIX, ⁶⁴ Cu with constant radioactivity (100 mCi) was put into a 96-well black plate, and various concentrations of PPIX (0 ~ 1.0 mM) were added to detect Cherenkov emission. It was measured using a fluorescence measurement equipment (IVIS Spectrum, Perkin Elmer).

Referring to FIGS. 8C to 8D , Cherenkov emission by ⁶⁴ Cu was reduced by PPIX, confirming that Cherenkov radiation energy was transferred to PPIX.

Experimental Example 3. For breast cancer cell lines In vitro Cytotoxicity test

(1) 5-ALA (12 mM) and ⁶⁴ Confirmation of cytotoxicity by Cu-DOTA-trastuzumab (500 μCi)

BT-474 cells (2 × 10 ⁴ cells per well) were seeded in a 96-well plate and 24 hours later, 5-ALA (12 mM) alone, ⁶⁴ Cu-DOTA-trastuzumab (500 mCi) alone, 5-ALA (12 mM) and ⁶⁴ Cu-DOTA-trastuzumab (500 mCi) sequentially or by mixing 5-ALA (12 mM) and ⁶⁴ Cu-DOTA-trastuzumab (500 mCi), incubated for 48 hours, and then used for MTT assay to evaluate cell viability.

Referring to FIG. 9, when only 5-ALA (12 mM) or ⁶⁴ Cu (500 μCi) was treated alone (Treatment 1 and Treatment 2), there was no cytotoxicity, and 5-ALA (12 mM) and ⁶⁴ Cu-DOTA- By showing that cytotoxicity exists only when trastuzumab (500 μCi) is present at the same time, it is confirmed that PPIX is excited by Cherenkov emission generated by ⁶⁴ Cu and cells are killed by reactive oxygen species (ROS) generated thereby. can

(2) Comparison of cytotoxicity according to types of radioactive isotopes and photosensitizers

BT-474 cells (2 × 10 ⁴ cells per well) were seeded in a 96-well plate and 24 hours later, 5-ALA (12 mM) alone, Ce6 (30 mM) alone, ⁶⁴ Cu (500 mCi) alone, ⁶⁴ Cu -DOTA-trastuzumab (500 mCi) alone, 5-ALA (12 mM) and ⁶⁴ Cu-DOTA-trastuzumab (500 mCi) mixed, Ce6 (30 mM) and ⁶⁴ Cu-DOTA-trastuzumab (500 mCi) mixed, After treatment with ¹²⁴ I (200 mCi) or a mixture of 5-ALA (12 mM) and ¹²⁴ I-trastuzumab (200 mCi) and incubation for 48 hours, cell viability was evaluated using MTT assay.

Referring to Figure 10, when 5-ALA (12mM), ⁶⁴ Cu (500μCi) or ¹²⁴ I (200μCi) was treated alone, there was no cytotoxicity, and when the two components were present at the same time, significant cytotoxicity was observed when compared to the control. representation can be confirmed. In addition, it was confirmed that there was significant cytotoxicity even when the photosensitizer Ce6 and ⁶⁴ Cu-DOTA-Trastuzumab, which are commonly used in PDT studies, were mixed and treated, thereby confirming the universal applicability of the present technology.

In particular, it can be confirmed that the treatment with a mixture of 5-ALA and ⁶⁴ Cu-DOTA-Trastuzumab exhibits more improved cytotoxicity. It is believed that this is because 5-ALA, a precursor of a photosensitizer, dissolves in water and is absorbed into cancer cells, and then is easily accumulated in cancer cells compared to a general precursor through a process in which 5-ALA is converted into a PPIX photosensitizer.

Experimental Example 4. Gastric cancer mouse model In vivo cancer treatment experiment

5-ALA (500 mg/kg, ip injection) was injected into a mouse model transplanted with HER2 overexpressing gastric cancer cell line (NCI-N87), and ⁶⁴ Cu-DOTA-trastuzumab (1 mCi, iv injection) was injected 2 hours later. . ⁶⁴ 5-ALA (500 mg/kg, ip injcetion) was additionally injected 2 hours and 6 hours after the injection of Cu-DOTA-trastuzumab, respectively, and the treatment effect was evaluated.

Referring to FIG. 11, when comparing the experimental group (5-ALA + ⁶⁴ Cu-DOTA-trastuzumab) and the control group (Saline, 5-ALA alone, or ⁶⁴ Cu-DOTA-trastuzumab alone injection), the growth of the tumors in the experimental group was significantly delayed. can confirm that

The above detailed description is illustrative of the present invention. In addition, the foregoing is intended to illustrate and describe preferred embodiments of the present invention, and the present invention can be used in various other combinations, modifications, and environments. That is, changes or modifications are possible within the scope of the concept of the invention disclosed in this specification, within the scope equivalent to the written disclosure and / or within the scope of skill or knowledge in the art. The written embodiment describes the best state for implementing the technical idea of the present invention, and various changes required in the specific application field and use of the present invention are also possible. Therefore, the above detailed description of the invention is not intended to limit the invention to the disclosed embodiments. Also, the appended claims should be construed to cover other embodiments as well.

Claims (13)

A compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof; And a composition for photodynamic therapy comprising a photosensitizer or a precursor of the photosensitizer:
[Formula 1]
RL-Ab
In Formula 1,
R is a radioactive isotope,
L is a chelator or a direct bond;
Ab is an antibody. According to claim 1,
The radioisotope is selected from the group consisting of Cu-64, Cu-67, Ga-68, Zr-89, Lu-177, Y-90, In-111, I-123, I-124 and I-131 One or more, and a composition for photodynamic therapy that is coordinately bonded to the chelator. According to claim 1,
The chelator is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTA-NCS, DOTA-NHS ester, DOTA-Bz-NCS, tris(tbu)DOTA, HBED -CC-TFP ester, DTPA (diethylene triamine penta aeetic acid), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid), NOTA (1,4,7-triazacyclononane-1,4 ,7-triacetic acid), NODAGA (1,4,7-Triazacyclononane,1-glutaric acid-4,7-acetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), TE3A (1,4,8,11-tetraazacyclotetradecane-1,4,8-triacetic acid), TE2A (1,4,8,11-Tetraazabicyclohexadecane-4,11-diacetic acid) ), PCTA (3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1,11,13-triene-3,6,9, -triacetic acid), and DFO (Deferrioxamine) selected from the group consisting of At least one composition for photodynamic therapy. According to claim 1,
The antibody is one selected from the group consisting of trastuzumab, pertuzumab, cetuximab, rituximab, brentuximab, and panitumumab. More than, a composition for photodynamic therapy. According to claim 1,
The photosensitizer or the precursor of the photosensitizer is 5-aminolevulinic acid (5-ALA), protoporphyrin IX (PPIX), chlorin e6 (Ce6), methyl-aminolevulinic acid ( At least one selected from the group consisting of methylaminolevulinate (MAL), hexyl-aminolevulinic acid (hexylaminolevulinate, HAL), and derivatives thereof, a composition for photodynamic therapy. According to claim 1,
The composition is for use in photodynamic therapy of cancer without irradiation of light, a composition for photodynamic therapy. According to claim 6,
Wherein the cancer is selected from the group consisting of breast cancer, stomach cancer, ovarian cancer, brain cancer, uterine cancer, endometrial cancer, pancreatic cancer, kidney cancer, peritoneal cancer and lung cancer, photodynamic therapy composition. A pharmaceutical composition for preventing or treating cancer comprising the composition for photodynamic therapy according to any one of claims 1 to 7 as an active ingredient. A food composition for preventing or improving cancer comprising the composition for photodynamic therapy according to any one of claims 1 to 7 as an active ingredient. (a) administering the composition for photodynamic therapy according to any one of claims 1 to 7 to a subject other than a human or in vitro ,
Photodynamic therapy, which is performed without light irradiation. According to claim 10,
After step (a), (b) further comprising accumulating the composition in target cells of the subject,
The concentration of the photosensitizer is 10 to 20 mM,
Wherein step (b) is performed for 1 to 24 hours, a photodynamic treatment method. According to claim 11,
The concentration of the photosensitizer is 10 to 15 mM,
Wherein step (b) is performed for 3 to 5 hours, a photodynamic treatment method. According to claim 11,
The photosensitizer is 5-aminolevulinic acid photodynamic therapy method.

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