JP2023164109A - Polymer targeting L-type amino acid transporter 1 - Google Patents

Abstract

To provide a conjugate for cancer-targeted therapy, with extended residual time in the body, and a drug delivery system.SOLUTION: A conjugate has a drug bound to an L-type amino acid transporter 1 (LAT1) binding site, where the LAT1 binding site is a compound having the following structure or a pharmaceutically acceptable salt thereof, where R1 is a methionine-like structure or a tyrosine-like structure.SELECTED DRAWING: None

Description

The present invention relates to methionine/tyrosine modified polymers and conjugates targeting L-type amino acid transporter 1 (LAT1) for targeted cancer therapy.

It is well known that cancer cells overexpress glucose transporters, but recent studies have revealed that they also overexpress amino acid transporters (Non-Patent Documents 1 and 2). In particular, L-type amino acid transporter 1 (LAT1), also called SLC7A5, has attracted attention. LAT1 is classified as a Na ⁺ -independent neutral amino acid transporter that transports neutral amino acids with bulky hydrophobic side chains such as phenylalanine and methionine (Non-patent Documents 3 and 4).

Previous studies have shown that overexpression of LAT1 can be observed in various types of cancer cells (Non-Patent Documents 5-7), but it is extremely limited in normal organs such as the blood-brain barrier, placenta, and neurovascular areas. It is expressed only in certain areas (Non-Patent Document 4). Therefore, LAT1 has been considered a promising target for cancer diagnosis and treatment. For example, ¹¹ C-methionine-(3- ¹⁸ F)-α-methyltyrosine is taken into cancer cells via LAT1 and is used for cancer imaging using PET (Non-patent Document 8). . p-boronophenylalanine (BPA) used in boron neutron capture therapy is also a therapeutic agent that is taken up via LAT1 (Non-Patent Documents 9 and 10).

All of the above-mentioned drugs against LAT1 are low molecules, and their application to bulky drugs or polymer drugs that cannot pass through transporters is still limited. Several studies have reported polymers and nanoparticles decorated with phenylalanine derivatives, showing efficient cellular uptake through LAT1 recognition, while successful in vivo drug delivery via LAT1 targeting has been reported. There are only a few studies that have focused on this, Non-Patent Documents 11 and 12). In particular, there have been no reports of successful in-vivo photodynamic therapy using polymers targeting LAT1. This is thought to be because it is difficult to develop a drug delivery system that maintains selectivity for LAT1 even in the complex environment of in vivo.

E.L. Lieu, T. Nguyen, S. Rhyne, J. Kim, Amino acids in cancer, Exp. Mol. Med. 52 (2020) 15-30. https://doi.org/10.1038/s12276-020-0375- 3 Y.D. Bhutia, E. Babu, S. Ramachandran, V. Ganapathy, Amino acid transporters in cancer and their relevance to "glutamine addiction": Novel targets for the design of a new class of anticancer drugs, Cancer Res. 75 (2015) 1782 -1788. https://doi.org/10.1158/0008-5472.CAN-14-3745 E.M. del Amo, A. Urtti, M. Yliperttula, Pharmacokinetic role of l-type amino acid transporters lat1 and lat2, Eur. J. Pharm. Sci. 35 (2008) 161-174. S.E. Jin, H.E. Jin, S.S. Hong, Targeting l-type amino acid transporter 1 for anticancer therapy: Clinical impact from diagnostics to therapeutics, Expert. Opin. Ther. Targets 19 (2015) 1319-1337. H. Betsunoh, T. Fukuda, N. Anzai, D. Nishihara, T. Mizuno, H. Yuki, A. Masuda, Y. Yamaguchi, H. Abe, M. Yashi, Y. Fukabori, K. Yoshida, T. Kamai, Increased expression of system large amino acid transporter (lat)-1 mRNA is associated with invasive potential and unfavorable prognosis of human clear cell renal cell carcinoma, BMC Cancer 13 (2013) 509. https://doi.org/10.1186/ 1471-2407-13-509 Z. Liang, H.T. Cho, L. Williams, A. Zhu, K. Liang, K. Huang, H. Wu, C. Jiang, S. Hong, R. Crowe, M.M. Goodman, H. Shim, Potential biomarkers of l -type amino acid transporter 1 in breast cancer progression, Nucl. Med. Mol. Imaging 45 (2011) 93-102. https://doi.org/10.1007/s13139-010-0068-2 O. Yanagida, Y. Kanai, A. Chairoungdua, D.K. Kim, H. Segawa, T. Nii, S.H. Cha, H. Matsuo, J. Fukushima, Y. Fukasawa, Y. Tani, Y. Taketani, H. Uchino, J.Y. Kim, J. Inatomi, I. Okayasu, K. Miyamoto, E. Takeda, T. Goya, H. Endou, Human l-type amino acid transporter 1 (lat1): Characterization of function and expression in tumor cell lines, Biochim Biophys. Acta 1514 (2001) 291-302. https://doi.org/10.1016/s0005-2736(01)00384-4 K. Kubota, T. Matsuzawa, M. Ito, K. Ito, T. Fujiwara, Y. Abe, S. Yoshioka, H. Fukuda, J. Hatazawa, R. Iwata, S. Watanuki, T. Ido, Lung- Tumor imaging by positron emission tomography using c-11 l-methionine, J. Nucl. Med. 26 (1985) 37-42. P. Wongthai, K. Hagiwara, Y. Miyoshi, P. Wiriyasermkul, L. Wei, R. Ohgaki, I. Kato, K. Hamase, S. Nagamori, Y. Kanai, Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by atb0,+, lat1 and lat2, Cancer Sci. 106 (2015) 279-286. https://doi.org/10.1111/cas.12602 A. Wittig, W. Sauerwein, J. Coderre, J. Coderre, Mechanisms of transport of p-borono-phenylalanine through the cell membrane in vitro, Radiat. Res. 153 (2000) 173-180 E. Puris, M. Gynther, S. Auriola, K.M. Huttunen, L-type amino acid transporter 1 as a target for drug delivery, Pharm. Res. 37 (2020) 1-17. https://doi.org/Artn 8810.1007/S11095-020-02826-8 S. Takano, K. Sakurai, S. Fujii, Internalization into cancer cells of zwitterionic amino acid polymers via amino acid transporter recognition, Polym. Chem. 12 (2021) 6083-6087. https://doi.org/10.1039/d1py01010g

An object of the present invention is to provide a conjugate for targeted cancer therapy, which remains in the body for a long time, and a drug delivery system.

The present inventors have demonstrated that by introducing a methionine or tyrosine structure, which serves as a substrate for LAT1, into the side chain, drugs can selectively accumulate in tumors targeting LAT1, thereby providing efficient anticancer therapy. We have discovered that this can be done, and have completed the present invention.

When using a polymer in which cysteine, which has a structure similar to methionine, is introduced into the side chain, there is no selectivity for LAT1 compared to a polymer in which methionine is introduced, and tumor accumulation is relatively low. It was done. From this, it has also been found that selectivity to LAT1 can be increased by finely adjusting the side chain structure.

That is, the present invention is as follows.
[1] A conjugate in which a drug is bound to the L-type amino acid transporter 1 (LAT1) binding site,
The LAT1 binding site has the following structure:

[During the ceremony,
R ₁ represents a methionine-like structure or a tyrosine-like structure, where the methionine-like structure is

and the tyrosine-like structure is

And;
R ₂ is a hydrogen atom, a hydroxy group, a carboxy group, an optionally substituted amino group, an optionally substituted C _1-10 alkyl group, an optionally substituted C _1-10 alkoxy group, a thiol group , a cyano group, an azide group, or a drug;
R ₃ and R ₄ are independently a hydrogen atom or -L ₃ -drug;
L ₁ , L ₂ , and L ₃ are independently linkers or absent;
x is an integer from 0 to 1000;
y is an integer from 2 to 1000; and the bonding order of each repeating unit is arbitrary.]
or a pharmaceutically acceptable salt thereof.
[2] The conjugate according to [1], wherein R ₁ is a methionine-like structure.
[3] The conjugate according to [1], wherein R ₁ is a tyrosine-like structure.
[4] L ₁ , L ₂ , and L ₃ are each independently a C _1-40 alkylene group, and the methylene group in the C _1-40 alkylene group has 1 to 10 oxo groups, NH , or the conjugate according to any one of [1] to [3], which may be substituted with a polyoxyalkylene having a degree of polymerization of 2 to 2,000.
[5] The conjugate according to any one of [1] to [4], wherein x is an integer of 30 to 100 and y is an integer of 10 to 70.
[6] The conjugate according to any one of [1] to [5], wherein the value of y/(x+y) is 0.5 or less.
[7] The conjugate according to any one of [1] to [6], wherein the drug is selected from the group consisting of low-molecular compounds, middle-molecular compounds, and antibodies.
[8] The drug is selected from the group consisting of anticancer drugs, photosensitizers, nucleic acid drugs, cytotoxic proteins, and in-vivo diagnostic agents, in any one of [1] to [6] Conjugates as described.
[9] The conjugate according to [8], wherein the drug is an anticancer drug or a photosensitizer.
[10] Brain cancer, head and neck cancer, esophageal cancer, thyroid cancer, small cell cancer, non-small cell cancer, breast cancer, stomach cancer, gallbladder/bile duct cancer, lung cancer, liver cancer, and hepatocyte cancer. Cancer, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, chorioepithelial cancer, endometrial cancer, cervical cancer, renal pelvic/ureteral cancer, bladder cancer, prostate cancer, penis cancer. For the treatment of cancer selected from the group consisting of testicular cancer, embryonal cancer, Wilms cancer, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor, and soft tissue sarcoma. The conjugate according to [8] or [9], which is used.
[11] A pharmaceutical composition comprising the conjugate according to any one of [1] to [10] and a pharmaceutically acceptable carrier.
[12] Brain cancer, head and neck cancer, esophageal cancer, thyroid cancer, small cell cancer, non-small cell cancer, breast cancer, stomach cancer, gallbladder/bile duct cancer, lung cancer, liver cancer, and hepatocyte cancer. Cancer, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, chorioepithelial cancer, endometrial cancer, cervical cancer, renal pelvic/ureteral cancer, bladder cancer, prostate cancer, penis cancer. For the treatment of cancer selected from the group consisting of testicular cancer, embryonal cancer, Wilms cancer, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor, and soft tissue sarcoma. The pharmaceutical composition according to [11], which is used.

By using the methionine/cysteine-modified polymer and conjugate of the present invention, drugs can be selectively delivered to cancer.

¹ H NMR spectrum of PEG-PBLA ₉₇ (2 mg/ml in DMSO-d6, room temperature) is shown. ¹ H NMR spectrum of PEG-PBLA ₂₇ (2 mg/ml in DMSO-d6, room temperature) is shown. ¹ H NMR spectrum of PBLA ₁₀₀ (2 mg/ml, in DMSO-d6, room temperature) is shown. ¹ H NMR spectrum of PBLA ₅₇ (2 mg/ml, in DMSO-d6, room temperature) is shown. Figure 3 shows a ¹ H NMR spectrum of PAsp ₁₁₀ (10 mg/ml in _D2O , room temperature). Figure 3 shows the ¹ H NMR spectrum of PAsp ₅₇ (10 mg/ml in _D2O , room temperature). ¹ H NMR spectrum of PEG-PAsp ₉₇ (10 mg/ml, in D ₂ O, room temperature) is shown. ¹ H NMR spectrum of PEG-PAsp ₂₇ (10 mg/ml in D ₂ O, room temperature) is shown. The ¹ H NMR spectrum of PEG-P[Asp ₆₀ /Asp(All) ₃₇ ] (10 mg/ml in D ₂ O, room temperature) is shown. The ¹ H NMR spectrum of PEG-P[Asp ₁₂ /Asp(All) ₁₅ ] (10 mg/ml in D ₂ O, room temperature) is shown. The ¹ H NMR spectrum of P[Asp ₅₉ /Asp(All) ₅₁ ] (10 mg/ml in D ₂ O, room temperature) is shown. The ¹ H NMR spectrum of P[Asp ₁₉ /Asp(All) ₃₈ ] (10 mg/ml in D ₂ O, room temperature) is shown. ^1H NMR spectrum of P[Asp ₇₀ /Asp(Met) ₄₀ ] (10 mg/ml in D ₂ O, room temperature) is shown. ^1H NMR spectrum of P[Asp ₃₃ /Asp(Met) ₂₄ ] (10 mg/ml in D ₂ O, room temperature) is shown. ¹ H NMR spectrum of PEG-P[Asp ₆₀ /Asp(Met) ₃₇ ] (10 mg/ml in D ₂ O, room temperature) is shown. ¹ H NMR spectrum of PEG-P[Asp ₁₄ /Asp(Met) ₁₃ ] (10 mg/ml in D ₂ O, room temperature) is shown. ^1H NMR spectrum of P[Asp ₇₄ /Asp(Cys) ₃₆ ] (10 mg/ml in D ₂ O, room temperature) is shown. Figure 3 shows the ¹ H NMR spectrum of PEG-P[Asp ₆₀ /Asp(Cys) ₃₇ ] (10 mg/ml in D ₂ O at room temperature). Figure 3 shows evaluation of cellular uptake of Cy5-labeled Met/Cys polymer by FCM. (a) SKOV3-Luc, (b) CT26, (c) BxPC3 and NIH3T3. Results are expressed as mean±SD (n=5). Statistical analysis was performed using one-way analysis of variance according to the Tukey method ( ^*** p<0.0001). Figure 3 shows evaluation of cellular uptake of Cy5-labeled Met polymer by FCM. (a) SKOV3-Luc, (b) CT26, (c) BxPC3. Results are expressed as mean±SD (n=5). Statistical analysis was performed using one-way analysis of variance according to the Tukey method ( ^*** p<0.0001). FIG. 3 is a diagram showing quantitative analysis of cellular uptake of Cy5-labeled polymers. (a) SKOV3-Luc, (b) CT26, (c) BxPC3. Results are expressed as mean±SD (n=6). Statistical analysis was performed using two-way analysis of variance with multiple comparison method ( ^*** p<0.0001). FIG. 3 is a diagram showing the intracellular distribution of polymers. (a) Fluorescence images of the intracellular distribution of the Met-functionalized polymer after 30 min, 1 h, 2 h, and 3 h of incubation with 2 μM Cy5 concentration in SKOV3-Luc and BxPC3 cells. (b) Fluorescence image of the intracellular distribution of Cys-functionalized polymer after 3 hours of incubation with 2 μM Cy5 concentration in SKOV3-Luc and BxPC3 cell lines. (c) Fluorescence image of the intracellular distribution of Met/Cys functionalized polymer after 3 hours of incubation with 2 μM Cy5 concentration in CT26 cell line. Scale bar indicates 10 μm. FIG. 3 shows inhibition studies analyzed by flow cytometry. SKOV3-Luc was incubated with BCH (system L inhibitor), BzlSer (ASCT2 inhibitor) or MeAIB (system A inhibitor). Results are expressed as mean±SD (n=5). Statistical analysis was performed using Student's t-test ( ^* p<0.05, ^** p<0.01, ^*** p<0.001, ^*** p<0.0001). FIG. 3 shows inhibition studies analyzed by flow cytometry. CT26 was incubated with BCH (system L inhibitor), BzlSer (ASCT2 inhibitor) or MeAIB (system A inhibitor). Results are expressed as mean±SD (n=5). Statistical analysis was performed using Student's t-test ( ^* p<0.05, ^** p<0.01, ^*** p<0.001, ^*** p<0.0001). FIG. 3 shows inhibition studies analyzed by flow cytometry. BxPC3 was incubated with BCH (system L inhibitor), BzlSer (ASCT2 inhibitor) or MeAIB (system A inhibitor). Results are expressed as mean±SD (n=5). Statistical analysis was performed using Student's t-test ( ^* p<0.05, ^** p<0.01, ^*** p<0.001, ^*** p<0.0001). In vitro PDT of Ppa-P[Asp ₉₂ /Asp(Met) ₃₅ ] is shown. (a) Phototoxicity, (b) Dark toxicity. Cells were incubated with Ppa-P [Asp ₉₂ /Asp(Met) ₃₅ ] for 3 h, and for phototoxicity, cells were washed twice with PBS and then treated with halogen at 3.0 mW/cm ² for 1 h in fresh medium. Lamp light irradiation was performed. For dark toxicity, cells were washed three times with PBS and incubated in fresh medium without light irradiation. Results are expressed as mean±SEM (n=6). Statistical analysis was performed using two-way analysis of variance with multiple comparison method ( ^*** p<0.05). LAT1 immunohistochemistry is shown. The intraperitoneal dissemination model was established by intraperitoneal injection of cancer cells. Tissues were harvested from mice and fixed in 4% PFA for 2 hours before sectioning at 5 μm. Samples subjected to hematoxylin staining are shown at 20x magnification. The scale bar indicates 50 μm. Figure 2 shows biodistribution evaluation of polymers in a peritoneal dissemination model. (a) Biodistribution in the SKOV3-Luc peritoneal dissemination model. Tissues were removed after 1 hour of intraperitoneal injection of the indicated polymers. Tissue bioluminescence/fluorescence (ex/em=630nm/690nm) images and Cy5 distribution were captured by IVIS. (b) Quantitative analysis of polymer biodistribution in the SKOV3-Luc tumor model. Polymer radiative efficiency measurements were automatically assigned by the LiveImage software used for image processing. The average radiant efficiency of the tumor was expressed as the total fluorescence of the bioluminescent area. Results are shown as mean±SD (n=3). Statistical analysis was performed using two-way analysis of variance with Tukey's multiple comparison method ( ^** p<0.01). (c) Representative fluorescence (ex/em=610nm/690nm) images of tissue polymer accumulation after 1 hour of polymer injection using IVIS in the BxPC3 tumor model. (d) Biodistribution of the polymer in the BxPC3 tumor model, quantified by fluorescence intensity (ex/em = 630 nm/680 nm) and expressed as percentage of injected dose/g tissue. (e) Tumor/blood and tumor liver ratio. Results are expressed as mean±SD (n=3). Statistical analysis was performed using Student's t-test ( ^* p<0.05, ^*** p<0.001). Biodistribution in the CT26 peritoneal dissemination model is shown. (a) Tissue fluorescence (ex/em=630nm/690nm) images and Cy5 distribution were captured by IVIS. (b) Quantitative analysis of polymer biodistribution in the CT26 tumor model. Tissues were removed 1 hour after intraperitoneal injection of polymer. Images of the tumor and distribution of Cy5 were taken by IVIS. (c) Biodistribution in normal organs after 1 hour of intraperitoneal injection of the polymer. Figure 3 shows anti-tumor potential in the SKOV3-Luc peritoneal dissemination model. Bioluminescent image of SKOV3-Luc tumor after PDT. The dose of Ppa modified polymer is 0.1 mg/mouse. Luminescence images were collected at 0, 5, 10, 15, and 20 days after light irradiation. Average tumor growth curves are shown. Tumor growth was expressed as total bioluminescence in the mouse abdomen. Results are expressed as mean±SEM (n=5). Statistical analysis was performed using two-way analysis of variance with Tukey's multiple comparison method ( ^* p<0.05, **p<0.01, ^*** p<0.001). Bioluminescent images of SKOV3-Luc tumor without light irradiation after polymer injection are shown. The dose of Ppa modified polymer is 0.1 mg/mouse. Luminescence images were collected on days 0, 5, 10, 15, and 20. Ex vivo images of liver and pancreatic tumor growth in the SKOV-Luc peritoneal dissemination model at the end of the anti-tumor study (photostimulation group) are shown. After intraperitoneal injection of luciferin for 5 minutes, tissues were removed. FIG. 4 shows relative mouse weight curves with and without PDT. The Met-modified polymer showed tumor-inhibiting ability upon photoirradiation, but did not induce side effects with or without photoirradiation. These results suggest that Met-modified polymers have great potential in PDT. ¹ H NMR spectrum of N ₃ -PEG _9k -PBLA ₉₅ is shown. ¹ H NMR spectrum of N ₃ -PEG _9k -PAsp ₉₅ is shown. The ¹ H NMR spectrum of N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ] is shown. The ¹ H NMR spectrum of N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ] is shown. ¹ H NMR spectrum of N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] is shown. The ¹ H NMR spectrum of N ₃ -PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] is shown. Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-[Asp ₇₄ /Asp(TEG -Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]. After treating BxPC3 cells with 10 mM BCH solution, 2.0 μM of each of Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ / Asp(Tyr) ₃₀ ], Cy5-PEG-[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] in 10 mM BCH containing medium solution. Time-treated and flow cytometry analysis was performed. Results are shown as mean±SD (n=3). Statistical significance was assessed using Student's t-test ( ^* p<0.05, ^** p<0.01, ^*** p<0.005). Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-P[Asp ₇₄ /Asp( Cellular uptake of TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] is shown. After BxPC3 cells were treated with 10 mM BCH solution, 2.0 μM of Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], and Cy5-PEG-P [Asp ₆₅ /Asp (Tyr) ₃₀ ], Cy5-PEG-[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] in 10 mM BCH containing medium solution for 3 hours. processed and subjected to flow cytometry analysis. Results are shown as mean±SD (n=3). Statistical significance was assessed using Student's t-test ( ^* p<0.05, ^** p<0.01). Shows the fluorescence observed in normal organs (spleen, kidney, liver, lung, heart, tumor) by IVIS (ex/em=640/700 nm) in a BxPC3 xenograft tumor model (n=3). Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], in BxPC3 xenograft tumor model (n = 3). Tumor accumulation of Cy5-PEG-[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] is shown. Results are shown as mean±SD (n=3). Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey's multiple comparison method ( ^* p<0.05, ^** p<0.01). Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P[ in normal organs (spleen, kidney, liver, lung, heart) of BxPC3 xenograft tumor model. The biodistribution of Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] is shown. . Results are shown as mean±SD (n=3). Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-[ in BxPC3 xenograft tumor model. Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] blood retention. Results are shown as mean±SD (n=3). Phototoxicity of Ppa-conjugated polymers in BxPC3 cells is shown. Results are shown as mean±SD (n=6). Figure 3 shows the dark toxicity of PPa-conjugated polymers in BxPC3 cells. For dark toxicity, cells were washed three times with PBS and incubated in fresh medium without light irradiation. Results are shown as mean±SD (n=6). The results of observing changes in tumor size over time after intraperitoneal administration of various polymers in Balb/c nude mice subcutaneously seeded with BxPC3 cells are shown. The results of observing changes in body weight over time after intraperitoneal administration of various polymers in Balb/c nude mice subcutaneously seeded with BxPC3 cells are shown.

The L-type amino acid transporter 1 (hereinafter sometimes simply referred to as "LAT1") binding site and conjugate according to the present invention will be explained below. LAT1 is an amino acid transporter expressed in various cancer cells and is considered a promising target molecule in tumor targeting.

According to the present invention, a drug delivery system targeting LAT1 can be provided by binding one or more methionine/tyrosine structures to the side chain of a hydrophilic polymer. As described below, a methionine (Met)-modified LAT1-binding site or a tyrosine (Tyr)-modified LAT1-binding site is efficiently taken up into cancer cells by LAT1-mediated endocytosis and exhibits excellent tumor accumulation. Furthermore, binding an anti-cancer agent (or anti-tumor agent) can bring about a significant anti-tumor effect.

1. Amino Acid Transporter Amino acid transporters exist in very limited normal tissues (eg, cerebral vascular endothelial cells, placenta) in living organisms, but their expression is known to be significant in cancer cells. Furthermore, some amino acid transporters are expressed specifically in cancer cells. LAT1, which is cancer-specific, is attracting attention as a cancer diagnostic marker and a molecular target for therapy.

It is well known that there are 21 types of natural amino acids that constitute proteins, and that the properties of each amino acid vary greatly depending on the side chains of these amino acids. In living organisms, there are various amino acid transporters that use amino acid groups with similar properties as substrates to transport such diverse amino acids to appropriate cells. Amino acid transporters are classified into a vesicular membrane type existing in intracellular organelles and a different cell membrane type, and the latter was classified as an amino acid transport system based on its functional characteristics before molecular cloning. It was classified as L, system B ^0,+, etc.

Some of the information shown in Table 1 below has been obtained regarding amino acid transporters whose expression is increased in cancer cells (Nagamori et al., Biochemistry, Vol. 85, Vol. 3). No., pp. 338-344 (2014)).

2. Methionine/tyrosine modified LAT1 binding site The methionine/tyrosine modified LAT1 binding site of the present invention has the following general formula (I):

It is a polymer containing polyaspartic acid having a structure represented by: Hereinafter, in order to explain the above-mentioned polymer of the present invention in more detail, in this specification, the right-hand part separated by "/ (slash)" in the above-mentioned formula (I) will be referred to as "A segment", and the left-hand side segment will be referred to as "A segment". The portion is sometimes referred to as a "B segment." In order to better understand the polymer of the present invention, for convenience, the above formula (I) is described in the order of B segment - A segment, but the structure and order of the repeating units specified by x and y are arbitrary. For example, it may be in the form of blocks or it may be in the form of random. As will be described later, the B segment can have x=0, so it may be in the form of a homopolymer in which the A segment is repeated alone.

(1) A segment The LAT1 binding site of the present invention is taken into cells (especially cancer cells) by LAT1-mediated endocytosis. _R1 in the A segment represents a transport substrate for LAT1, and examples include methionine, tyrosine, methionine derivatives, and tyrosine derivatives. As the transport substrate, methionine and tyrosine are particularly preferred. In order to clarify the bonding positions of methionine, tyrosine, methionine derivatives, and tyrosine derivatives, the terms ``methionine-like structure'' is used for methionine and methionine derivatives, and ``tyrosine-like structure'' is used for tyrosine and tyrosine derivatives. , will be explained in detail. Note that these amino acids are preferably L-type.

"Methionine-like structure" is

(wherein R ₃ is a hydrogen atom or -L ₃ -drug)

However, according to the present invention, an A segment having a methionine derivative can be used if it is possible to obtain the same effect as an A segment having a natural methionine (i.e., _R3 is a hydrogen atom) in its structure. It's okay. Methionine derivatives include stereoisomers and enantiomers and may exist in different isomeric forms. Methionine derivatives include, but are not limited to, esters of methionine, methionine amides, esters of methionine amide, hydroxy analogs of methionine (i.e., 2-hydroxy-4-(methylthio)butanoic acid), and hydroxy analogs of methionine. Examples include esters and salts thereof. The esters include methyl ester, ethyl ester, n-propyl ester, isopropyl ester, butyl ester (i.e., n-butyl ester, s-butyl ester, isobutyl ester and t-butyl ester), pentyl ester (n-pentyl ester), esters, isopentyl esters) and hexyl esters (particularly n-hexyl esters and isohexyl esters).

"Cysteine-like structure" is

(In the formula, R ₅ is a hydrogen atom or -L ₃ -drug)

has. The cysteine-like structure is similar to the above-mentioned methionine-like structure, but although it does not show selectivity for LAT1 compared to the use of a LAT1-binding site that has a methionine-like structure in its side chain, it has a low tumor accumulation potential. . The cysteine-like structure can also be used as a control for the LAT1 binding site that has a methionine-like structure. Cysteine derivatives include stereoisomers and enantiomers, and can exist in different isomeric forms.

"Tyrosine-like structure" is

(wherein R ₄ is a hydrogen atom or -L ₃ -drug)

However, according to the present invention, an A segment having a tyrosine derivative can be used if it is possible to obtain the same effect as an A segment having a natural tyrosine (that is, _R4 is a hydrogen atom) in its structure. It's okay. Tyrosine derivatives include stereoisomers and enantiomers and may be capable of existing in different isomeric forms. Examples of tyrosine derivatives include, but are not limited to, methyl (2R)-2-amino-3-(2-chloro-4hydroxyphenyl)propanoate, D-tyrosine ethyl ester hydrochloride, methyl (2R)-2-amino- 3-(2,6-dichloro-3,4-dimethoxyphenyl)propanoate HD-tyrosine (tBu)-allyl ester hydrochloride, methyl (2R)-2-amino-3-(3-chloro-4,5 -dimethoxyphenyl)propanoate, methyl(2R)-2-amino-3-(2-chloro-3-hydroxy-4-methoxyphenyl)propanoate, methyl(2R)-2-amino-3-(4-[(2 -chloro-6-fluorophenyl)methoxy]phenyl)propanoate, methyl (2R)-2-amino-3-(2-chloro-3,4-dimethoxyphenyl)propanoate, methyl (2R)-2-amino-3- (3-chloro-5-fluoro-4-hydroxyphenyl)propanoate, diethyl 2-(acetylamino)-2-(4-[(2-chloro-6-fluorobenzyl)oxy]benzylmalonate, methyl (2R) -2-amino-3-(3-chloro-4-methoxyphenyl)propanoate, methyl (2R) -2-amino-3-(3-chloro-4-hydroxy-5-methoxyphenyl)propanoate, methyl (2R) -2-amino-3-(2,6-dichloro-3-hydroxy-4-methoxyphenyl)propanoate, methyl (2R)-2-amino-3-(3-chloro-4-hydroxyphenyl)propanoate, H- DL-tyrosine methyl ester hydrochloride, H-3,5-diiodo-tyrosine-methyl ester hydrochloride, HD-3,5-diiodo-tyrosine-methyl ester hydrochloride, HD-tyrosine-methyl ester hydrochloride , D-tyrosine methyl ester hydrochloride, D-tyrosine methyl ester hydrochloride, methyl D-tyrosinate hydrochloride, HD-tyrosine methyl ester hydrochloride, D-tyrosine methyl ester hydrochloride, HD-tyrosine methyl Ester hydrochloride, (2R)-2-amino-3-(4-hydroxyphenyl)propionic acid, (2R)-2-amino-3-(4-hydroxyphenyl)methyl ester hydrochloride, methyl (2R)- 2-amino-3-(4-hydroxyphenyl)propanoate hydrochloride, methyl (2R)-2-azanyl-3-(4-hydroxyphenyl)propanoate hydrochloride, 3-chloro-L-tyrosine, 3-nitro-L -Tyrosine, 3-nitro-L-tyrosine ethyl ester hydrochloride, DL-m-tyrosine, DL-o-tyrosine, Boc-tyrosine (3,5-I2)-OSu, Fmoc-tyrosine (3-NO2)-OH , α-methyl-L-tyrosine, α-methyl-D-tyrosine, and α-methyl-DL-tyrosine.

In one embodiment, the integer "y" for specifying the number of A segments is 0-1000, such as 0-50, 0-100, 0-200, 0-500, etc.

In one embodiment, the content of A segment is 30-60% by weight relative to the LAT1 binding site with methionine-like structure of the present invention. Preferably it is 50% by weight.

In one embodiment, the average molecular weight of the A segment is at least 5000. Preferably, it is 10,700 or more, 12,000 or more, or in the range of 5,000 to 11,000, or 5,000 to 13,000.

In another aspect, the content of A segment is 25-70% by weight relative to the LAT1 binding site with tyrosine-like structure of the invention. Preferably it is 61% by weight.

In one embodiment, the average molecular weight of the A segment is at least 5000. Preferably, it is 10,000 or more, 11,000 or more, or in the range of 5,000 to 11,000, or 10,000 to 23,000.

"L ₁ " in the A segment is a linker or is absent, and a methionine-like structure or a tyrosine-like structure is bonded to the (poly)aspartic acid of the main skeleton through or without the linker. I can do it. Furthermore, “L ₃ ” in R ₃ and R ₄ above can represent a hydrogen atom or a linker. The type, structure, and length of the linker are not limited as long as the transport substrate (such as methionine) is recognized by the amino acid transporter and the action of the bound drug is not inhibited.

When L ₁ and L ₃ are linkers, for example, but not limited to, C1-40 alkylene groups. Here, the methylene group in the C1-40 alkylene group may be substituted with 1 to 10 oxo groups, or adjacent methylene groups may be connected to each other with 1 to 10 unsaturated bonds. , Also, among the methylene groups in the alkylene group, 1 to 20 methylene groups are NH, N (C1-10 alkyl), O, S, C6-14 arylene, 5- to 10-membered heteroarylene, or polymerization degree may be replaced with 2 to 2,000 polyoxyalkylene. Additionally, the linker has the following structure:

[wherein X and Y are independently C1-5 alkylene groups or absent; m is 1 to 2,000]
or

[wherein X and Y are independently C1-5 alkylene groups or absent; m is 1 to 2,000]
It may be.

(2) B segment The terminal portion of the B segment can take, for example, an R ₂ -NH- structure depending on the case, and has a certain length, so that the LAT1 binding site of the present invention can be - Helps control transfer to the liver, etc. Moreover, by having a certain length, it is expected that even if a bulky structure is introduced at the end, the interaction between the polymer and LAT1 will not be inhibited. More specifically, R ₂ is -CH ₂ -CH ₂ -R ₅ (where R ₅ is hydrogen, methyl, azide, or methoxy), -CH ₂ -CH ₂ -(O-CH ₂ -CH ₂ ) _m -O-R ₆ (wherein R ₆ is hydrogen, methyl, azide, or methoxy; m is an integer from 0 to 1500, and may be an integer from 10 to 1000 , may be an integer from 100 to 500), or -CH ₂ -CH ₂ -(O-CH ₂ -CH ₂ ) _n -R ₅ (where R ₅ is hydrogen, methyl, azide, or methoxy n is an integer of 0 to 1500, may be an integer of 10 to 1000, or may be an integer of 100 to 500).

In one embodiment, the content of B segment within the LAT1 binding site of the invention is 30-60% by weight. Preferably it is 38% by weight.

In another aspect, the average molecular weight of the B segment is at least 1500. It is preferably 7,000 or more, 8,000 or more, or in the range of 1,500 to 7,500, or 1,500 to 8,500.

In one embodiment, the content of B segment is 15-40% by weight. Preferably it is 16% by weight.

In another aspect, the average molecular weight of the B segment is at least 5000. Preferably, it is 7,000 or more, 8,000 or more, or in the range of 7,000 to 9,000, or 5,000 to 9,000.

L ₂ is a linker or is absent, and the R ₂ group can be bonded to the backbone (poly)aspartic acid through or without the linker. The type, structure, and length of the linker are not limited as long as the transport substrate (such as methionine) bound via the linker can be recognized by the amino acid transporter and the action of the bound drug is not inhibited.

When L ₂ is a linker, it is, for example, but not limited to, a C1-40 alkylene group. Here, the methylene group in the C1-40 alkylene group may be substituted with 1 to 10 oxo groups, or adjacent methylene groups may be connected to each other with 1 to 10 unsaturated bonds. , Also, among the methylene groups in the alkylene group, 1 to 20 methylene groups are NH, N (C1-10 alkyl), O, S, C6-14 arylene, 5- to 10-membered heteroarylene, or polymerization degree may be replaced with 2 to 2,000 polyoxyalkylene. Additionally, the linker has the following structure:

[wherein X and Y are independently C1-5 alkylene groups or absent; m is 1 to 2,000]
or

[wherein X and Y are independently C1-5 alkylene groups or absent; m is 1 to 2,000]
It may be.

(3) LAT1 binding site The LAT1 binding site of the present invention consisting of A segment and B segment can be produced using commercially available raw materials. Moreover, when it is not commercially available, the raw material or intermediate can be manufactured by a method well known to those skilled in the art. In the Examples described below, LAT1 binding sites were synthesized with various polymerization ratios of A segment and B segment, and it was confirmed that the desired products were obtained according to conventional methods. For example, methods using NMR (nuclear magnetic resonance) and gas chromatography are common.

It is possible to obtain a product in which the ratio of A segment and B segment constituting the LAT1 binding site is changed as appropriate. The fraction of A segment or B segment is preferably 20 to 50 mol%. The term "mol %" as used herein refers to the number of moles of a component divided by the sum of the moles of each component in a multicomponent system. For example, if the composition of the LAT1 binding site is expressed as the number of monomer units of the A segment and the B segment using "y" and "x", for example, the fraction of the A segment is expressed by the formula: y x 100/ It can be determined by (x+y).

Additionally, according to the present invention, the average molecular weight (e.g., weight average molecular weight (Mw) and number average molecular weight (Mn)) of the LAT1 binding site can be determined by methods well known to those skilled in the art, such as chromatography (GPC) methods, viscosity It can be measured by method, end group quantitative method, osmotic pressure method, light scattering method, and sedimentation velocity method. As used herein, the term "weight average molecular weight ( _Mw )" means the molecular weight of a polydisperse polymeric compound. Specifically, the weight average molecular weight in a polydisperse system with n _i molecules (i=1, 2, . . . ) having a molecular weight M _i is calculated by the following formula:

It is expressed as The weight average molecular weight (M _w ) of the LAT1 binding site having a methionine-like structure of the present invention is preferably 50,000. More preferably, it is 40,000. On the other hand, the weight average molecular weight (M _w ) of the LAT1 binding site having a tyrosine-like structure of the present invention is preferably 50,000. More preferably, it is 76,000.

On the other hand, the term "number average molecular weight (M _n )" is the average molecular weight when a molecule is composed of two or more atoms, and if the i-th atomic weight is M _i and the number is N _i , then the following formula :

It is expressed as The number average molecular weight (M _n ) of the LAT1 binding site having a methionine-like structure of the present invention is preferably 25,000. More preferably, it is 20,000. On the other hand, the number average molecular weight (M _n ) of the LAT1 binding site having a tyrosine-like structure of the present invention is preferably 25,000. More preferably, it is 38,000.

As measured in Examples described later, the molecular weight of the LAT1 binding site is M _w =42,000 and M _n =21,000, so it can be said that it has a very large molecular weight.

The molecular weight distribution of the LAT1 binding site of the present invention can be expressed by M _w /M _n (dispersion ratio). The dispersion ratio is preferably 1.1 or more, more preferably 1.1 or more and 3 or less.

Since the LAT1 binding site of the present invention has a large molecular weight, it is characterized in that it takes a long time to be degraded when administered to a living body. Therefore, a conjugate in which a drug is bound to the LAT1 binding site described below is not rapidly metabolized in vivo and is retained for a long time, so that the amount of drug added can be kept significantly low. For example, when aiming at clinical effects such as cancer treatment, typically the LAT1 binding site of the present invention is preferably one listed below.

2. Conjugate As mentioned above, the LAT1-binding site disclosed herein is taken up by LAT1-targeted endocytosis, so by utilizing such an action mechanism, it can be conjugated to the LAT1-binding site. Therapeutic agents can be specifically delivered to cancer cells. Examples of therapeutic agents that can be used in the present invention include, but are not limited to, adriamycin, paclitaxel, docetaxel, cisplatin, mitomycin-C, daunomycin, 5-FU (fluorouracil), gemcitabine hydrochloride, emtansine, calicheamycin, toxins such as , diphtheria toxin, Pseudomonas aeruginosa exotoxin, etc.), boron-containing drugs (e.g., bortezomib, or drugs such as p-boronophenylalanine that can be used in boron neutron capture therapy (BNCT)), nucleic acid drugs (e.g., Decoy nucleic acids, antisense nucleic acids, siRNA, miRNA, ribozymes, aptamers), photosensitizers that can be used in photodynamic therapy (PDT) (eg, porphyrin derivatives), and the like.

As used herein, "anti-cancer agent" or "anti-tumor agent" refers to a drug that produces a targeted cytotoxic effect on cancer cells (or tumor cells). Examples of anticancer agents include, but are not limited to, bortezomib, imatinib, seliciclib, afatinib, alectinib, axitinib, belinostat, bortezomib, bosutinib, cabozantinib, carfilzomib, ceritinib, cobimetinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, fitinib, ibrutinib, Idelalisib, imatinib, ixazomib, lapatinib, lenvatinib, nilotinib, olaparib, osimertinib, palbociclib, panobinostat, pazopanib, ponatinib, regorafenib, ruxolitinib, cipleucel-T, sonidegib, sorafenib, temsirolimus, tofacitinib, trametinib vandetanib, vemurafenib, vismodegib, and vorinostat can be mentioned.

Substances that can be conjugated to the LAT1 binding site of the present invention include various compounds in addition to therapeutic drugs, including toxins, contrast agents, photosensitizers, nucleic acid drugs, boron-containing drugs, etc. It will be done. A method of conjugating such a substance to the LAT1 binding site of the present invention includes, for example, conjugating such a substance to the carboxy group or amino group of the methionine-like structure or tyrosine-like structure corresponding to _R1 of the A segment, directly or via a linker. Alternatively, this can be achieved by covalently bonding the functional group of the above substance to the structure corresponding to R ₂ of the B segment, either directly or via a linker, and can be carried out by a method well known to those skilled in the art.

As used herein, a "photosensitizer" is a compound that can be promoted to an excited state upon absorption of light, and is sometimes referred to as a "photosensitive molecule." Photosensitizers activated (photoexcited) by light irradiation can become toxic or produce toxic molecules, and thus become cytotoxic to the irradiated cancer cells. Examples of such photosensitizers include, but are not limited to, fluorescent dyes, infrared absorbing dyes, near infrared absorbing dyes, porphyrin molecules, chlorophyll molecules, and the like.

When conjugating the therapeutic agent of the present invention, it is preferable to appropriately adjust the amount of the therapeutic agent bound and administer the conjugate in an appropriate therapeutically effective amount for the target disease to be treated. As used herein, the term "therapeutically effective amount" refers to the effect of the conjugate of the present invention on preventing or ameliorating the pathology of a disease or the progression of a disease when administered to cells, tissues, or organs of a subject. refers to the amount of a therapeutic drug.

3. Pharmaceutical Composition According to the present invention, the polymer disclosed herein, or a salt, derivative, or hydrate thereof, and other pharmaceutically acceptable ingredients (carrier, excipient, disintegrant, Pharmaceutical compositions for treating cancer are provided, including buffering agents, emulsifying agents, suspending agents, soothing agents, stabilizers, preservatives, preservatives, saline, etc.).

4. Cancer Treatment The conjugate of the present invention specifically accumulates in cancer, and can treat cancer with the bound drug. The cancer to be treated is not particularly limited, but is preferably a solid cancer. Solid cancers include colon cancer, stomach cancer, breast cancer, colorectal cancer, gallbladder cancer, lung cancer (specifically adenocarcinoma), colorectal cancer (CRC) such as metastatic CRC, head and neck cancer, Selected from various forms of cancer, including liver cancer, pancreatic cancer, osteosarcoma, etc. A person skilled in the art, such as a doctor, will be able to determine the dosage, concentration, number of doses, and frequency of the LAT1 binding site of the present invention as an active ingredient for treating cancer, depending on the type and malignancy of cancer, and the subject ( Appropriate selection can be made taking into consideration the gender, age, weight, condition of the affected area, etc. of the subject (subject).

According to the present invention, the LAT1 binding site, conjugate, and pharmaceutical composition of the present invention can be administered intravenously or locally to a subject using a syringe or the like. The subject to be treated is preferably a mammal, including but not limited to humans, primates such as monkeys, rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, Examples include sheep, pigs, cows, horses, donkeys, goats, and ferrets.

The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples in any way.

Representative reagents and measuring instruments used in this example are listed below. Furthermore, all animal experiments were conducted with approval from the Institutional Animal Care and Use Committee of Tokyo Institute of Technology.

(1) Reagent/α-methoxy-ω-amino-poly(ethylene glycol) (MeO-PEG-NHOA): Nofu Co., Ltd. (Tokyo, Japan)
・Propylamine: Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・β-Benzyl L-aspartic acid N-carboxy anhydride (BLA-NCA): Chuo Kasei ・N,N-dimethylformamide (DMF): Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・Dichloromethane (DCM): Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・Ethyl acetate (EtOAc): Kanto Kagaku Co., Ltd. (Tokyo)
・Diethyl ether: Kanto Kagaku Co., Ltd. (Tokyo)
・Hexane: Kanto Kagaku Co., Ltd. (Tokyo)
・Hydrochloric acid (HCl): Nacalai Tesque (Kyoto, Japan)
・Sodium hydroxide (NaOH): Nacalai Tesque (Kyoto, Japan)
・N-Methyl-2-pyrrolidone: Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・Allylamine: Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM): Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・Triethylamine (TEA): Nacalai Tesque (Kyoto, Japan)
・DL-Homocysteine: Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan)
・Lcysteine: Tokyo Chemical Industry (Tokyo, Japan)
・2,2-dimethoxy-2-phenylacetophenone (DMPA): Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan)
・Acetonitrile (CH3CN): Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・Sodium chloride (NaCl): Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・Sodium dihydrogen phosphate (NaH2PO4): Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka Prefecture)
・Cy5 NHS ester: Lumiprobe (Maryland, USA)
・Pheophorbide a (Ppa): Funakoshi Co., Ltd. (Tokyo, Japan)
・Azide-poly(ethylene glycol)-amine (N3-PEG9k-NH2) [Mw: 9K]
・Benzene: Nacalai Tesque (Kyoto, Japan)
・β-Benzyl L-aspartic acid N-carboxy anhydride (BLA-NCA): Chuo Kasei Co., Ltd. ・N-Methylpyrrolidone (NMP): Wako Pure Chemical Industries, Ltd.
・Dimethyl sulfoxide (DMSO): Nacalai Tesque (Kyoto, Japan)
・NH ₂ -Tyr (synthesized in the laboratory)
・NH ₂ -TEG-Tyr (provided by Daiichi Sankyo)
・1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC・HCl): TCI chemical substance ・N-hydroxysuccinimide (NHS): Wako Pure Chemical Industries, Ltd. ・Trifluoroacetic acid (TFA): TCI Chemical substance

(2) Measurement equipment/NMR (nuclear magnetic resonance): BRUKER AVANCE III 400 (400MHz, BRUKER BioSpin) (Billerica, USA)
- GPC (gel permeation chromatography): Jasco International Co. , Ltd. (Tokyo Japan)
·column:
TSK-gel superAW3000, superAW4000 column: Tosoh Corporation (Tokyo)
Superdex 200 Increase 10/300 GL (GE Healthcare (Tokyo))
GL sciences WP300 C18 5μm 4.6 x 250mm (Tokyo, Japan)
·Detector:
Refractive index-2031 (RI-2031): Jasco International Co. , Ltd. (Tokyo Japan)
Ultraviolet-visible spectroscopy-2070 (UV-2070): Jasco International Co. , Ltd. (Tokyo Japan)
・Disposable PD-10 desalination column: GE Healthcare (Tokyo)
- Nanodrop microvolume spectrophotometer and fluorometer: Thermo Fisher Scientific, Inc. (Waltham, MA)
・Microplate reader: Spark, Tecan Japan Co. , Ltd. (Kawasaki City, Japan)

For the abbreviations of compounds used herein, please refer to the corresponding official names below:
・PEG: Poly(ethylene glycol)
・PAsp: Poly(aspartic acid)
・PEG-PAsp: Poly(ethylene glycol)-block-poly(aspartic acid)
・P[Asp _m /Asp(Met) _n ]: Poly[aspartic acid-ran-(3-((3-amino-3-carboxypropyl)thio)propyl)aspartamide] (Note: m and n are respectively (The same applies hereafter.)
・PEG-P[Asp _m /Asp(Met) _n ]: Poly(ethylene glycol)-block-poly[aspartic acid-ran-(3-((3-amino-3-carboxypropyl)thio)propyl)aspart Amide]
・P[Asp _m /Asp(Cys) _n ]: poly[aspartic acid-ran-(3-((2-amino-2-carboxyethyl)thio)propyl)aspartamide]
・PEG-P[Asp _m /Asp(Cys) _n ]: Poly(ethylene glycol)-block-poly[aspartic acid-ran-(3-((2-amino-2-carboxyethyl)thio)propyl)aspart Amide]
・P[Asp _m /Asp(Met/Cys) _n ]: poly[aspartic acid-ran-(3-((3-amino-3-carboxypropyl)thio)propyl)aspartamide]; and poly[aspartic acid -ran-(3-((2-amino-2-carboxyethyl)thio)propyl)aspartamide]
・PEG-P[Asp _m /Asp(Met/Cys) _n ]: Poly(ethylene glycol)-block-poly[aspartic acid-ran-(3-((3-amino-3-carboxypropyl)thio)propyl) aspartamide]; and poly(ethylene glycol)-block-poly[aspartic acid-ran-(3-((2-amino-2-carboxyethyl)thio)propyl)aspartamide]
・P[Asp _m /Asp(All) _n ]: Poly(aspartic acid-ran-allylaspartamide)
・PEG-P[Asp _m /Asp(All) _n ]: Poly(ethylene glycol)-block-poly[aspartic acid-ran-allylaspartamide]
・P[Asp _m /Asp(Tyr) _n ]: Poly[aspartic acid-ran-(3-(4-(2-amino-2-carboxyethyl)phenoxy)propyl)aspartamide]
・P[Asp _m /Asp(TEG-Tyr) _n ]: Poly[aspartic acid-ran-(16-(4-(2-amino-2-carboxyethyl)phenoxy)-3,6,9,12-tetra oxahexadecanyl) aspartamide]
・PEG-P[Asp _m /Asp(Tyr) _n ]: Poly(ethylene glycol)-block-poly[aspartic acid-ran-(3-(4-(2-amino-2-carboxyethyl)phenoxy)propyl) Aspartic acid]
・PEG-P[Asp _m /Asp(TEG-Tyr) _n ]: Poly(ethylene glycol)-block-poly[aspartic acid-ran-(16-(4-(2-amino-2-carboxyethyl)phenoxy) -3,6,9,12-tetraoxahexadecanyl)aspartamide]

Example 1: Synthesis of methionine- or cysteine-modified polymer (1) Overview Water-soluble polymer poly(aspartic acid _m /aspartic acid (methionine) _n ) (hereinafter referred to as P[Asp _m /Asp(Met) _n ]), Polyethylene glycol-poly(aspartic acid _m /aspartic acid (methionine) _n ) (hereinafter PEG-P [Asp _n /Asp(Met) _m ]), and poly(aspartic acid _m / aspartic acid (cysteine) _n ) (hereinafter referred to as PEG-P [Asp n /Asp(Met) m ]) , P[Asp _m /Asp(Cys) _n ]), polyethylene glycol-poly(aspartic acid _m / aspartic acid (cysteine) _n ) (hereinafter referred to as PEG-P[Asp _m /Asp(Cys) _n ]) were synthesized. Describe the process. PBLA _n (n indicates the degree of polymerization) was synthesized by N-carboxy anhydride (NCA) polymerization using propylamine as an initiator and BLA-NCA as a monomer. PEG-PBLA _n was synthesized by NCA polymerization using α-methoxy-2-amino-poly(ethylene glycol) as an initiator and BLA-NCA as a monomer. The side chain carboxy group was deprotected under basic conditions to obtain PAsp _n and PEG-PAsp _n (n indicates the degree of polymerization). Thereafter, the amino group of allylamine was bonded to the carboxy group of PAsp _n and PEG-PAsp _n to obtain poly[Asp _m /Asp(allylamine) _n ] (hereinafter, (P[Asp _m /Asp(All) _n ]). P[Asp _m /Asp(Met) _n ] and P[Asp _m /Asp(Cys) _n ] are converted into DL-homocysteine and L- _cysteine by photochemical radical thiol-ene click reaction. Asp(All) _n ] was synthesized by introducing it into the side chain carbon double bond.

(2) Synthesis method and analysis (2-1) Synthesis of PEG-PAsp _n (n = 27 and 97) and PAsp _n (n = 57 and 110) Poly(aspartic acid) (PAsp) and polyethylene glycol-poly(asparagine) The synthesis scheme of (PEG-PAsp) is shown below.

PEG-PAsp _n and PAsp _n (n indicates the degree of polymerization of PAsp) were synthesized by ring-opening polymerization of BLA-NCA and subsequent deprotection (hydrolysis). For the synthesis of PEG-b-poly(β-benzyl-L-aspartate) (PEG-PBLA ₉₇ ), MeO-PEG10k-NH2 (500 mg, 0.05 mmol) was lyophilized in benzene. Dissolve BLA-NCA (1.195 g, 4.8 mmol) in a mixed solvent of DCM (16.2 ml) and DMF (1.8 ml), and dissolve in a mixed solvent of DCM (6.3 ml) and DMF (0.7 ml). Polymerization was initiated by mixing with the prepared MeO-PEG _10k -NH ₂ at room temperature. After stirring for 2 days under an argon atmosphere, the mixture was added dropwise to a mixed solvent of hexane and ethyl acetate (v/v=3/2) to perform reprecipitation. The precipitate was collected by vacuum filtration and vacuum drying to obtain 1.23 g.

For the synthesis of poly(β-benzyl-L-aspartate) (PBLA ₉₇ ), BLA-NCA (3.05 g, 12.7 mmol) was dissolved in a mixed solvent of DCM (20 mL) and DMF (2 mL), and propyl Polymerization was carried out by mixing with amine (9 μL, 0.12 mmol) and stirring at room temperature for 2 days under an argon atmosphere. Thereafter, it was added dropwise to 150 mL of diethyl ether to obtain a precipitate. The precipitate was collected by vacuum filtration and vacuum drying to obtain 2.4 g. Other PEG-PBLA and PBLA were polymerized in the same manner.

PEG-PBLA
^{The 1} H NMR spectrum shows a peak derived from PEG [3.20-3.63 ppm (-CH ₂ -CH ₂ -O-)] and a peak derived from PBLA [2.53-2.93 ppm (-CH ₂ -CO-O-)]. -), 4.86-5.18 (-O-CH ₂ -Ph), 7.13-7.43 ppm (Ph)], the degree of polymerization of PBLA DP = 27 (n = 27) and DP=97 (n=97), number average molecular weight Mn=15,600 (n=27), and Mn=29,900 (n=97). In addition, from the GPC curve, the molecular weight dispersity of the obtained polymer was found to be PDI = 1.13 (n = 27) and PDI = 1.13 (n = 97), confirming that it has a narrow molecular weight distribution ( (data not shown).

PEG-PBLA
¹ H NMR (400 MHz, DMSO-d6): δ8.27-8.06 (br, -CO-CH-NH ₂ ), 7.43-7.13 (br, =CH-CH=), 5.18 -4.86 (br, -O-CH ₂ -), 4.71-4.48 (br, -CO-CH-NH-), 3.71-3.65 (br, -O-CH ₂ - CH ₂ ), 3.63-3.20 (br, -CH ₂ -CH ₂ -O-), 3.28-3.21 (br, -CH ₂ -CH ₂ -CH ₂ -), 2.93 -2.53 (br, -CH ₂ -CO-), 1.25-1.22 (br, CH ₂ -CH ₂ -NH-)

^{The 1} H NMR spectra of PEG-PBLA ₉₇ and PEG-PBLA ₂₇ (both 2 mg/ml in DMSO-d6 at room temperature) are shown in Figures 1A and 1B.

PBLA
In ^{the 1} H NMR spectrum, a peak derived from the initiator [0.71-0.78 ppm (-CH ₃ --CH ₂ --)] and a peak derived from PBLA [2.54-2.94 ppm (-CH ₂ --CO-O- ), 4.90-5.14 (-O-CH ₂ -Ph), 7.13-7.43 ppm (Ph)], the degree of polymerization of PBLA DP = 57 (n = 57) and DP = 110 (n = 110), number average molecular weight Mn = 11,700 (n = 57), and Mn = 22,600 (n = 110). In addition, from the GPC curve, the molecular weight dispersity of the obtained polymer was found to be PDI = 1.07 (n = 57) and PDI = 1.08 (n = 110), confirming that it has a narrow molecular weight distribution (data ¹ H NMR spectra of PBLA ₁₁₀ and PBLA ₅₇ (both 2 mg/ml in DMSO-d6 at room temperature) are shown in Figures 2A and 2B.
¹ H NMR (400 MHz, DMSO-d6): δ8.29-8.03 (br, -CO-CH-NH ₂ ), 7.43-7.13 (br, =CH-CH=), 5.14 -4.90 (br, -O-CH ₂ -), 4.71-4.88 (br, -CO-CH-NH-), 3.70-3.55 (br, -CH ₂ -NH) , 2.94-2.54 (br, -CH ₂ -CO-), 1.83-1.70 (br, -CH ₂ -CH ₂ -), 1.37-1.29 (br, -CH ₂ -CH ₂ -NH-), 0.78-0.71 (br, CH ₃ -CH ₂ -).

Deprotection was carried out by placing 1.0 g of PEG-PBLA or PBLA in 20 ml of 0.5 M NaOH aqueous solution and stirring at room temperature for 12 hours. The solution was dialyzed against a 0.01 M HCl aqueous solution and pure water at room temperature using a dialysis membrane (MWCO, 3,500 Da), and then lyophilized and collected. (Yield: PEG-PAsp: 95%, PAsp: 94%). The physical properties of the polymer were confirmed by ¹ H NMR and size exclusion chromatography.

PEG-PAsp
The integrated value of the PEG-derived peak [3.48-3.90 ppm (-CH ₂ -CH ₂ -O-)] and the α-hydrogen peak of aspartic acid (2.68-3.02 ppm) in ^{the 1} H NMR spectrum. From the ratio, the degree of polymerization of Asp was determined to be DP = 97 (n = 97) and DP = 27 (n = 27), and the number average molecular weight Mn = 21,200 (n = 110) and Mn = 13,200 (n = 27). It was done. Furthermore, it was confirmed from the GPC curve that the obtained polymer had a narrow monomodal molecular weight distribution (data not shown).

PAsp
From the ratio of the integral value of the peak derived from the initiator [0.79-0.97 ppm (CH ₃ - CH ₂ -)] in the ¹ H NMR spectrum and the peak of α hydrogen of aspartic acid (2.87-3.05 ppm) The degree of polymerization of Asp was determined to be DP = 110 (n = 110) and DP = 57 (n = 57), and the number average molecular weight Mn = 12,700 (n = 110) and Mn = 6,600 (n = 57). . Furthermore, it was confirmed from the GPC curve that the obtained polymer had a narrow monomodal molecular weight distribution (data not shown). The ¹ H NMR spectra of PAsp ₁₁₀ and PAsp ₅₇ (both 10 mg/ml in D ₂ O at room temperature) are shown in Figures 3A and 3B.
¹ H NMR (400 MHz, D ₂ O): δ 4.76-4.67 (br, -CO-CH-NH-), 3.15-3.07 (br, -CH ₂ -CH ₂ -NH-) , 3.05-2.87 (br, -CH ₂ -CO-) 1.52-1.40 (br, -CH ₂ -CH ₂ -), 0.91-0.79 (br, CH ₃ - _CH2- ).

PEG-PAsp
^{The 1} H NMR spectra of PEG-PAsp ₉₇ and PEG-PAsp ₂₇ (both 10 mg/ml in D ₂ O at room temperature) are shown in Figures 4A and 4B.
¹ H NMR (400 MHz, D ₂ O): δ 4.77-4.66 (br, -CO-CH-NH-), 3.90-3.48 (br, -CH ₂ -CH ₂ -O-) , 3.21-3.28 (br, -CH ₂ -CH ₂ -), 3.02-2.68 (br, -CH ₂ -CO-), 1.83-1.72 (br, -CH ₂ - _CH2 -NH-).

(2-2) Synthesis of PEG-P[Asp _m /Asp(All) _n ] and P[Asp _m /Asp(All) _n ] The synthesis scheme of allylamine-modified PAsp and PEG-PAsp is shown below.

PEG-P[Asp _m /Asp(All) _n ] and P[Asp _m /Asp(All) _n ] were synthesized by a condensation reaction between a polymer and allylamine. For the synthesis of PEG-P [Asp ₆₀ /Asp(All) ₃₇ ], PEG-PAsp ₉₇ (200 mg) was dissolved in DMF (15 ml), DMT-MM (0.5 equivalent to Asp unit, 58 mg), TEA (3 drops), allylamine (0.5 equivalents per Asp unit, 12 mg) were added to the solution. The reaction was allowed to proceed overnight at room temperature. After the reaction, it was dialyzed in an aqueous NaHCO ₃ (10 mM) solution and deionized water (MWCO: 3,500 Da), and recovered by freeze-drying (yield = 93.5%).

For the synthesis of P[Asp ₅₉ /Asp(All) ₅₁ ], PAsp (100 mg) was dissolved in DMF (20 mL), DMT-MM (0.8 equivalents to Asp units, 382.35 mg), TEA ( 3 drops) and allylamine (2 equivalents per Asp unit, 442.34 mg) were added, and the mixture was stirred at 40° C. overnight to react. Thereafter, it was collected by dialysis and freeze-drying in the same manner as above (yield = 94.7%). The physical properties of all polymers were evaluated by ¹ H NMR spectroscopy (solvent: D ₂ O) and GPC.

PEG-P[Asp _m /Asp(All) _n ]
The peak derived from the initiator in the ¹ H NMR spectrum [0.79-0.97 ppm (CH ₂ -CH ₂ -O-)] and the allylamine peak [(3.78-3.81 ppm (-NH-CH ₂ -) , 5.05-5.26 ppm (-CH=CH ₂ ), 5.73-5.94 ppm (-CH ₂ -CH=)], the number of units into which allylamine was introduced was 37 (m= 60, n=37) and 15 (m=12, n=15), number average molecular weight Mn=22,700 (m=60, n=37) and Mn=13,800 (m=12, n=15) Further, it was confirmed from the GPC curve that the obtained polymer had a narrow monomodal molecular weight distribution (data not shown).

PEG-P [Asp _n /Asp(All) _m ]
^1H NMR spectra of PEG-P[Asp ₆₀ /Asp(All) ₃₇ ] and PEG-P[Asp ₁₂ /Asp(All) ₁₅ ] (both 10 mg/ml in D ₂ O, room temperature) are shown in Figures 5A and 5B. It was shown to.
¹ H NMR (400 MHz, D ₂ O): δ5.94-5.73 (br, -CH ₂ -CH=), 5.26-5.05 (br, -CH=CH ₂ ), 4.75- 4.36 (br, -CO-CH-NH-), 3.91-3.78 (br, -NH-CH ₂ -), 3.79-3.62 (br, -CH2-CH ₂ -O -), 3.31-3.17 (br, -CH ₂ -CH ₂ -CH ₂ -), 2.96-2.45 (br, -CH ₂ -CO-), 1.35-1.25 (br, -CH ₂ -CH ₂ -NH-).

P[Asp _m /Asp(All) _n ]
The peak derived from the initiator [0.81-0.94 ppm (CH ₃ -CH ₂ -)] and the peak of allylamine [ ⁽ 3.69-3.90 ppm (-NH-CH ₂ -), 5 From the ratio of the integral values of .07-5.29 ppm (-CH=CH ₂ ), 5.74-5.95 ppm (-CH ₂ -CH=)], the number of units into which allylamine was introduced was 51 (m = 59, n = 51) and 38 (m = 19, n = 38), number average molecular weight Mn = 14,700 (m = 59, n = 51) and Mn = 8,100 (m = 19, n = 38). In addition, it _was confirmed from the GPC curve that the obtained polymer had a monomodal narrow molecular weight distribution (data _not shown). ₁₉ /Asp(All) ₃₈ ] ( ^both 10 mg/ml in D ₂ O, room temperature) are shown in Figures 6A and 6B.
¹ H NMR (400 MHz, D ₂ O): δ 5.95-5.74 (br, -CH ₂ -CH=), 5.29-5.07 (br, =CH ₂ ), 4.73-4. 59 (br, -CO-CH-CH ₂ -CO-), 4.60-4.39 (br, -CO-CH-CH ₂ -COOH), 3.90-3.69 (br, -CONH- CH ₂ -), 3.17-3.10 (br, -CH ₂ -CH ₂ -CH ₂ -), 3.02-2.44 (br, -CH ₂ -CO-), 1.55-1 .44 (br, -CH ₂ -CH ₂ -NH-), 0.94-0.81 (br, CH ₃ -CH ₂ -).

(2-3) Synthesis of PEG-P[Asp _m /(Asp-Met/Cys) _n ] and P[Asp _m /(Asp-Met/Cys) _n ] Methionine (Met)/cysteine (Cys) functionalized PAsp The synthesis scheme of PEG-PAsp and PEG-PAsp is shown below.

Met and Cys were introduced into P[Asp _m /Asp(All) _n ] and PEG-P [Asp _m /Asp(All) _n ] by photochemical radical thiol-ene click reaction. In the synthesis of PEG-P[Asp _m /Asp(Met) _n ] and P[Asp _m /Asp(Met) _n ], PEG-P[Asp _m /Asp(All) _n ] or P[Asp _m /Asp( Weigh out 30 _mg of DL-homocysteine, dissolve 150-450 equivalents of DL-homocysteine in water (7 mL) based on Asp(All) units, and dissolve 30-100 equivalents of DMPA(Asp(All)) units in water (7 mL). An acetonitrile solution (3 mL) was added. After stirring for 30 minutes, the reaction mixture was stirred at room temperature for 1.5 to 2 hours while irradiated with light using a handy UV lamp (365 nm). After dialysis in acetone and water (MWCO: 3,500), lyophilization gave PEG-P[Asp _m /Asp(Met) _n ] or P[Asp _m /Asp(Met) _n ] (yield >90%).

PEG-P[Asp _m /Asp(Cys) _n ] and P[Asp _m /Asp(Cys) _n ] were synthesized by using L-cysteine instead of DL-homocysteine. All obtained polymers were analyzed by ¹ H NMR (solvent: D ₂ O) and GPC [Column: Superdex 200 increase 10/300GL (GE Healthcare Ltd); Eluent: 10 mM phosphate buffer (pH 7.4) (150 mM Flow rate: 0.6 ml/min; Temperature: room temperature].

PEG-P[Asp _m /Asp(Met) _n ]
In ^{the 1} H NMR spectrum, a peak derived from PEG [3.61-3.81 ppm (CH ₂ -CH ₂ -O-)] and a peak of methionine [(1.70-1.89 ppm (-CH ₂ -CH ₂ -S) -), 2.30-2.02ppm (-S-CH ₂ -CH ₂ -), 2.51-2.71ppm (-CH ₂ -S-CH ₂ -), 2.72-2.99ppm (- Ratio of integral values of CH-CH ₂ -CO-), 3.20-3.41ppm (-NH-CH ₂ -CH ₂ -), 3.81-3.96ppm (-CO-CH-NH ₂ )] The number of units into which methionine was introduced was 37 (m = 60, n = 37) and 13 (m = 14, n = 13), and the number average molecular weight Mn = 28,300 (m = 60, n = 37) and Mn = 16,000 (m = 14, n = 13). Also, from the GPC curve, it was confirmed that the obtained polymer had a narrow monomodal molecular weight distribution (data not shown).

P[Asp _m /Asp(Met) _n ]
The peak derived from the initiator [0.80-0.92ppm (CH ₃ -CH ₂ -)] and the peak of methionine [(1.70-1.86ppm-CH ₂ -CH ₂ -S-) in the ¹ H NMR spectrum , 2.30-2.01ppm (-S-CH ₂ -CH ₂ -), 2.49-2.71ppm (-CH ₂ -S-CH ₂ -), 2.74-2.99ppm (-CH- _{Methionine _ _ _} The number of units introduced is 40 (m = 70, n = 40) and 24 (m = 33, n = 24), number average molecular weight Mn = 21,100 (m = 70, n = 40) and Mn = 19 , 700 (m=33, n=24). Furthermore, it was confirmed from the GPC curve that the obtained polymer had a narrow monomodal molecular weight distribution (data not shown).

P[Asp _m /Asp(Met) _n ]
^{The 1} H NMR spectra of P[Asp ₇₀ /Asp(Met) ₄₀ ] and P[Asp ₃₃ /Asp(Met) ₂₄ ] (both 10 mg/ml in D ₂ O at room temperature) are shown in Figures 7A and 7B.
¹ H NMR (400 MHz, D ₂ O): δ 4.70-4.50 (br, -CO-CH-NH-), 3.96-3.78 (br, -CO-CH-NH ₂ ), 3 .43-3.19 (br, -NH-CH ₂ -CH ₂ -), 3.17-3.07 (br, CH ₃ -CH ₂ -CH ₂ -), 2.99-2.74 (br , -CH-CH ₂ -CO-), 2.71-2.49 (br, -CH ₂ -S-CH ₂ -), 2.30-2.01 (br, -S-CH ₂ -CH ₂ -), 1.86-1.70 (br, -CH ₂ -CH ₂ -S-), 1.29-1.18 (br, -CH ₂ -CH ₂ -NH), 0.92-0. 80 (br, CH ₃ -CH ₂ -).

PEG-P[Asp _m /Asp(Met) _n ]
^1H NMR spectra of PEG-P[Asp ₆₀ /Asp(Met) ₃₇ ] and PEG-P[Asp ₁₄ /Asp(Met) ₁₃ ] (both 10 mg/ml in D ₂ O, room temperature) are shown in Figures 8A and 8B. It was shown to.
¹ H NMR (400 MHz, D ₂ O): δ 4.73-4.51 (br, -CO-CH-NH-), 3.96-3.81 (br, -CO-CH-NH ₂ ), 3 .81-3.61 (br, -CH ₂ -CH ₂ -O-), 3.54-3.50 (br, CH ₃ -O-CH ₂ -), 3.41-3.20 (br, -CH ₂ -CH ₂ -CH ₂ -), 2.99-2.72 (br, -CH-CH ₂ -CO-), 2.71-2.51 (br, -CH ₂ -S-CH ₂ -), 2.30-2.02 (br, -S-CH ₂ -CH ₂ -CH), 1.89-1.70 (br, NH-CH ₂ -CH ₂ -CH ₂ -S-), 1.27-1.20 (br, -NH-CH ₂ -CH ₂ -).

PEG-P[Asp _m /Asp(Cys) _n ]
In the ¹ H NMR spectrum, a peak derived from PEG [3.58-3.78 ppm (CH ₂ -CH ₂ -O-)] and a cysteine peak [(1.68-1.91 ppm (-CH ₂ -CH ₂ -CH _2- ), 2.51-2.67ppm (-CH ₂ -CH ₂ -S-), 2.96-3.18ppm (-S-CH ₂ -CH-), 3.21-3.42ppm (- NH-CH ₂ -CH ₂ -), 3.91-3.99 ppm (-CO-CH-NH2)], the number of units into which cysteine was introduced was 40 (m = 57, n = 40) , number average molecular weight Mn = 27, 200 (m = 57, n = 40). Also, from the GPC curve, it was confirmed that the obtained polymer had a narrow monomodal molecular weight distribution (data (not shown).

P[Asp _m /Asp(Cys) _n ]
The peak derived from the initiator in the ¹ H NMR spectrum [0.82-0.91 ppm (CH ₃ -CH ₂ -)] and the peak of cysteine [(1.69-1.89 ppm (-CH ₂ -CH ₂ -CH ₂ -), 2.52-2.67ppm (-CH ₂ -S-CH ₂ -), 2.97-3.20ppm (-S-CH ₂ -CH-), 3.21-3.41ppm (-NH -CH ₂ -), 3.87-4.02 ppm (-CO-CH-NH ₂ )], the number of units into which cysteine was introduced was 36 (m = 74, n = 36), and the number average molecular weight It was found that Mn=19,700 (m=74, n=36). Furthermore, it was confirmed from the GPC curve that the obtained polymer had a narrow monomodal molecular weight distribution.

P[Asp _m /Asp(Cys) _n ]
The ¹ H NMR spectrum of P[Asp ₇₄ /Asp(Cys) ₃₆ ] (10 mg/ml in D ₂ O, room temperature) is shown in FIG.
¹ H NMR (400 MHz, D ₂ O): δ 4.72-4.49 (br, -CO-CH-NH-), 4.02-3.87 (br, -CO-CH-NH ₂ ), 3 .41-3.21 (br, -NH-CH ₂ -), 3.20-2.97 (br, -S-CH ₂ -CH-, -CH ₂ -CH ₂ -CH ₂ -), 2. 98-2.67 (br, -CH-CH ₂ -CO-), 2.67-2.52 (br, -CH ₂ -S-CH ₂ -), 1.89-1.69 (br, - CH ₂ -CH ₂ -NH-), 0.91-0.82 (br, CH ₃ -CH ₂ -).

PEG-P[Asp _m /Asp(Cys) _n ]
^{The 1} H NMR spectrum of PEG-P[Asp ₅₇ /Asp(Cys) ₄₀ ] (10 mg/ml in D ₂ O, room temperature) is shown in FIG.
¹ H NMR (400 MHz, D ₂ O): δ 4.75-4.50 (br, -CO-CH-NH-), 3.99-3.91 (br, -CO-CH-NH ₂ ), 3 .90-3.80 (br, -O-CH ₂ -CH ₂ -) 3.78-3.58 (br, -CH ₂ -CH ₂ -O-), 3.55-3.50 (br, CH ₃ -O-CH ₂ -), 3.42-3.21 (br, -NH-CH ₂ -CH ₂ -), 3.18-2.96 (br, -S-CH ₂ -CH-, -CH ₂ -CH ₂ -CH ₂ -), 2.97-2.67 (br, -CH-CH ₂ -CO-), 2.67-2.51 (br, -CH ₂ -CH ₂ -S -), 1.91-1.68 (br, -CH ₂ -CH ₂ -NH-).

(2-4) Fluorescent labeling of polymer Met/Cys-modified polymer (10 mg) was dissolved in water, and a DMSO solution of Cy5-NHS (1.0 equivalent to the polymer) was added and reacted. . After stirring overnight, the solution was dialyzed in deionized water (MWCO: 3,500), passed through a PD-10 desalting column (GE Healthcare) to remove unreacted dye, and recovered by lyophilization. (Yield: Cy5-PEG-P[Asp _m /Asp(Met) _n ] = 70%, Cy5-P[Asp _m /Asp(Met) _n ] = 88%). GPC for the product [Column: Superdex 200 Increase 10/300GL (GE Healthcare Ltd); Eluent: 10 mM phosphate buffer (pH 7.4) (containing 150 mM NaCl); Flow rate: 0.6 mL/min; Temperature: Room temperature ] and confirmed that unreacted Cy5 was removed. Cy5 was similarly introduced into PEG20k-NH ₂ .

The fluorescence of the Cy5-modified polymer in PBS was measured using a microplate reader (Spark, Tecan Japan Co., Ltd. Kawasaki, Japan).

A synthetic scheme for conjugation (fluorescent labeling) of Met/Cys functionalized polymer and Cy5 is shown below.

(2-5) Binding of photosensitizer (pheophorbide a:Ppa) to PEG-P[Asp _m /Asp(Met/Cys) _n ] and P[Asp _m /Asp(Met/Cys) _n ] Met functionality A synthetic scheme for the conjugation of conjugated PAsp and Ppa is shown below.

For photosensitizer binding, Ppa, DCC, and NHS (1:1.2:1.4, 30 mg: 12 mg: 8.22 mg) were dissolved in DMF (5 ml) and stirred in the dark for 2 h. Thereafter, it was purified using a silica gel column (solvent: dichloromethane/methanol, 10/1 volume ratio), and the solvent was removed by evaporation to recover Ppa NHS ester (yield = 75%). The purified Ppa NHS ester and the Met-modified polymer were dissolved in a carbonate buffer at a mixing ratio of 2:1 (0.42 mg:10 mg) with a catalytic amount of TEA (pH = 7-8), and then In the evening, the reaction was carried out at room temperature in the dark. Dialyzed in deionized water (MWCO: 3,500), passed through a PD-10 desalting column to remove free Ppa, and lyophilized to recover the Ppa-bound polymer (yield: Ppa-P[Asp _m /Asp(Met) _n ] = 30%). GPC for the product [Column: Superdex 200 Increase 10/300GL (GE Healthcare Ltd); Eluent: 10 mM phosphate buffer (pH 7.4) (containing 150 mM NaCl); Flow rate: 0.6 mL/min; Temperature: Room temperature ] and confirmed that unreacted Ppa was removed (data not shown).

Example 2: Evaluation of cultured cells (1) Overview The amount of cellular uptake of methionine/cysteine (Met/Cys) modified polymers labeled with a fluorescent dye (Cy5) was compared. Thereafter, the intracellular distribution of the Met/Cys-modified polymer was observed using a confocal microscope. Subsequently, specificity to LAT1 was examined using an amino acid transporter inhibitor. Finally, the cancer cell phototoxicity of the Met/Cys-modified polymer labeled with a photosensitizer (Ppa) was evaluated by CCK-8 assay.

(2) Evaluation of the amount of cellular uptake of Met/Cys functionalized polymer (2-1) Measurement of cellular uptake using a flow cytometer (FCM) Cells were placed in a 24-well plate at a density of 1.0 x 10 ⁵ cells/well. McCoy's 5A (containing 10% fetal bovine serum/penicillin) for SKOV3-Luc cells, RPMI (containing 10% fetal bovine serum/penicillin) for BxPC3 and CT26 cells, and DMEM (10% fetal bovine serum) for NIH3T3 cells. / penicillin) and precultured for 24 hours at 37°C and 5% _CO2 . After replacing the medium, 500 μL of the following sample solution was added and incubated for 3 hours.
・Cy5-Met/Cys modified polymer (Cy5: 2 μM)
・PEG20k-Cy5 (Cy5: 2μM)

After washing twice with 0.5 mL of D-PBS, 150 μL of trypsin-EDTA solution was added and incubated for 3 minutes for SKOV3-Luc cells and CT26 cells, 10 minutes for BxPC3 cells, and 5 minutes for NIH3T3 cells. After confirming that the cells were detached using a light microscope, use McCoy's 5A (containing 10% fetal bovine serum/penicillin) for SKOV3-Luc cells, and RPMI (containing 10% fetal bovine serum/penicillin) for BxPC3 and CT26 cells. For NIH3T3 cells, 350 μL of DMEM (containing 10% fetal bovine serum/penicillin) was added and suspended, and then cell uptake was measured by FCM. The obtained results are shown in FIGS. 11 and 12.

(2-2) Measurement of cell uptake by fluorometric determination of lysate Cells were seeded in a 96-well plate at 1.0×10 ⁴ cells/well and precultured at 37° C. and 5% CO ₂ for 24 hours. 100 μL of the following sample solution was added and incubated for 3 hours.
・Cy5-Met/Cys modified polymer (Cy5: 2 μM)
・PEG20k-Cy5 (Cy5: 2μM)

After washing twice with 0.1 mL of D-PBS(+), 0.1 mL Lysis buffer solution was added and incubated for 30 minutes. After transferring 50 μL of the solution to a black plate, the fluorescence intensity was measured using a microplate reader (ex/em=635/690 nm). The amount of uptake calculated using the calibration curve is shown in FIG.

In cancer cells overexpressing LAT1, the Met-modified polymer showed higher cellular uptake than the Cys-modified polymer and PEG20k (FIG. 11). On the other hand, the Met-modified polymer showed an extremely small amount of uptake into NIH3T3 cells, which are normal cells. Similar results were obtained in the fluorescence assay of cell lysate (FIG. 12). Furthermore, the amount of cellular uptake was dependent on the degree of polymerization, and P[Asp ₇₀ /Asp(Met) ₄₀ ] showed a higher uptake than P[Asp ₃₃ /Asp(Met)24] (FIG. 13).

Example 3: Observation of intracellular distribution using a confocal microscope (1) Observation using a confocal microscope Cells were seeded in a 35 mm ² glass dish at a density of 5.0 x 10 ⁴ cells/dish, and incubated at 37°C at 5% Preincubation was performed for 24 hours under _CO2 . 1 mL of 2.0 μM Cy5-Met/Cys modified polymer solution was added to each dish and incubated for 3 hours. After washing with 1 mL of D-PBS(-), 1 mL of 100 nM LysoTracker (registered trademark) red DND-99/(D-PBS:RPMI=1:9) solution was added and incubated for 30 minutes. After washing with 1 mL of D-PBS, add 1 mL of 5.0 μg/ml Hoechst/D-PBS solution, and after washing twice with 1 mL of D-PBS, add McCoy's 5A for SKOV3-Luc cells, and 2 mL of RPMI for BxPC3 and CT26 cells. was added and observed by CLSM. The obtained results are shown in FIG. 14.

The Met/Cys-modified polymer was localized in endosomes/lysosomes, suggesting that it is taken up into cells by endocytosis.

(2) Specificity Evaluation Cells were seeded in a 24-well plate at 1.0×10 ⁵ cells/well and precultured at 37° C. and 5% CO ₂ for 24 hours. 500 μL of the following sample solution was added and incubated for 30 minutes.
・BCH (System L inhibitor): 10mM
・BzlSer (ASCT2 inhibitor): 10mM
・MeAIB (System A inhibitor): 10mM

Thereafter, 500 μL of the following sample solution was added and incubated for 3 hours.
・Cy5-Met modified polymer (Cy5: 2μM), BCH 10mM
・Cy5-Met modified polymer (Cy5: 2μM), BzlSer 10mM
・Cy5-Met modified polymer (Cy5: 2μM), MeAIB 10mM
・Cy5-Cys modified polymer (Cy5: 2μM), BCH 10mM
・Cy5-Cys modified polymer (Cy5: 2μM), BzlSer 10mM
・Cy5-Cys modified polymer (Cy5: 2μM), MeAIB 10mM

After washing twice with 0.5 mL of D-PBS, 150 μL of trypsin-EDTA solution was added and incubated for 3 minutes for SKOV3-Luc cells and CT26 cells and 10 minutes for BxPC3 cells. After confirming that the cells were detached using an optical microscope, 350 μL of McCoy's 5A was added for SKOV3-Luc cells, and 2 mL of RPMI was added for BxPC3 and CT26 cells, and after suspension, cell uptake was measured using FCM. Specificity evaluation of the Cys-modified polymer was performed in the same manner as above. The obtained results are shown in FIG. 15.

LAT1 inhibitor could significantly inhibit the cellular uptake of Met-modified PAsp in SKOV3-Luc, CT26 and BxPC3, whereas ASCT2 and system A inhibitors did not inhibit the cellular uptake of Met-modified PAsp. It was suggested that endocytosis was mediated by LAT1. On the other hand, the cellular uptake of Met-modified PEG-PAsp Cys-modified polymer was not reduced under the treatment of inhibition. This suggested that the chemical structure of the side chain contributes to LAT1 targeting.

Example 4: Evaluation of cell phototoxicity The cancer cell proliferation inhibitory effect of the Ppa-Met modified polymer was evaluated by CCK-8 assay.
(1) Cytotoxicity measurement Cells were seeded in a 96-well plate at 1.0×10 ⁴ cells/well, and precultured at 37° C. and 5% CO ₂ for 24 hours. 100 μL of the following sample solution was added and incubated for 3 hours.
・Ppa-Met modified polymer (Ppa: 2 μM)

After washing twice with 0.1 mL of D-PBS(-), use McCoy's 5A (containing 10% fetal bovine serum/penicillin) for SKOV3-Luc cells, RPMI (containing 10% fetal bovine serum/penicillin) for BxPC3 cells, For NIH3T3 cells, 100 μL of DMEM (containing 10% fetal bovine serum/penicillin) was added and irradiated with light using a 680 nm halogen lamp for 1 hour. After 48 hours of incubation, 100 μL of 10% CCK-8 solution was added to each well, and after 1.5 hours of incubation, absorbance at 450 nm was measured. The obtained results are shown in FIG. 16.

P[Asp ₉₂ /Asp(Met) ₃₅ ]-Ppa showed extremely high phototoxicity to cancer cells, but low phototoxicity to NIH3T3 cells (FIG. 16(a)). This result was consistent with the amount of cellular uptake (FIG. 16(b)). On the other hand, without light irradiation, P[Asp ₉₂ /Asp(Met) ₃₅ ]-Ppa cytotoxicity was not induced. In other words, it was suggested that the polymer-photosensitizer itself has no significant toxicity.

Example 5: Effect on peritoneal dissemination tumor mouse model (LAT1 expression level in tumor, tumor accumulation, and antitumor effect)
(1) Overview Met/Cys-modified polymers labeled with a fluorescent dye (Cy5) were intraperitoneally injected into peritoneal disseminated tumor mouse models (SKOV3-Luc, BxPC3, and CT26), and their pharmacokinetics were evaluated. Furthermore, a Met/Cys-modified polymer labeled with a photosensitizer (Ppa) was intraperitoneally injected into a SKOV3-Luc (human ovarian cancer cell line) peritoneally disseminated tumor mouse model to evaluate the antitumor effect.

(2) Evaluation of tumor LAT1 expression level by immunohistochemistry (2-1) Preparation of peritoneal dissemination tumor mouse model CT26: Cell suspension (3.0 × 10 ⁶ cells/ml) was applied to Balb/c mice. 200 μl was injected intraperitoneally.
SKOV3-Luc・BxPC3: 200 μl of the cell suspension (5.0×10 ⁶ cells/ml) was intraperitoneally injected into Balb/c nude mice.

(2-2) Immunohistochemistry After paraffin-embedded tissue sections with a thickness of 5 μm were deparaffinized and dehydrated, microwave treatment was performed in citrate buffer for 10 minutes for antigen retrieval. Treatment with 3% H ₂ O ₂ for 20 minutes was performed to block endogenous peroxedase activity. After blocking with 3% BSA PBST solution for 30 minutes, rabbit polyclonal anti-LAT1 antibody (5 μg/ml, Massachusetts, USA) was placed on the tissue sections, incubated for 2 hours at room temperature, and washed three times with PBST. Histofine® Simple StainTM Rabbit MAX PO (Nichirei biosciences Inc. Tokyo, Japan) was placed on the tissue section and allowed to react at room temperature for 15 minutes. Finally, nuclear staining was performed with hematoxylin using 3-3' diaminobenzine (DAB; Agilent Technologies Japan, Ltd.) as a reaction dye, and then observation was made with an optical microscope. The obtained results are shown in FIG. 17.

Strong expression of LAT1 was observed in all of the SKOV3-Luc, BxPC3, and CT26 peritoneal dissemination tumor mouse models.

Example 6: Kinetics of methionine/cysteine functionalized polymer (1) Preparation of peritoneal dissemination tumor mouse model CT26: 200 μl of cell suspension (3.0×10 ⁶ cells/ml) was added to Balb/c mice. Injected intraperitoneally.
SKOV3-Luc・BxPC3: 200 μl of the cell suspension (5.0×10 ⁶ cells/ml) was intraperitoneally injected into Balb/c nude mice.

(2) Evaluation of pharmacokinetics Two weeks after the peritoneal dissemination of cancer cells, 250 μL of the following sample D-PBS(-) solution was intraperitoneally administered (Cy5 10 μM, 250 μL/mouse).
・Cy5-Met modified polymer ・Cy5-Cys modified polymer ・PEG20k-Cy5

Mice were euthanized 1 hour after sample injection. For the SKOV3 tumor model, 200 μL of luciferin (30 mg/ml) was administered intraperitoneally 5 minutes before euthanasia. After the tumor and organs were removed, they were carefully washed with PBS, excess water was wiped off with tissue paper, and Cy5 fluorescence was measured using an in vivo imaging system (ex/em = 630/690 nm; IVIS, Perkin Elmer, Waltham, MA). ). For the SKOV3 model, luciferin luminescence was also measured.

For quantification, a tumor near the pancreas was excised and homogenized by adding Passive Lysis Buffer. Various organs were also homogenized in the same way. Homogenization was performed using a multibead shocker. The homogenized solution was transferred to a black 96-well plate (Thermo Fisher Scientific Inc. USA), and fluorescence was measured using a microplate reader (SPARK TKS01, TEACN, Switzerland) (ex/em=630 nm/680 nm). In addition, a calibration curve was created using an organ homogenization solution, and the amount of Cy5 contained in each sample was calculated, thereby converting it into dosage amount %/g tissue. The results are shown in FIGS. 18 and 19.

Met/Cys-modified polymers rapidly accumulated in tumors. On the other hand, it did not accumulate much in normal tissues.

Example 7: Antitumor effect Two weeks after peritoneal dissemination of SKOV3-Luc cells into Balb/c nude mice, 250 μL of the following sample D-PBS(-) solution was intraperitoneally administered (Ppa 10 μM, 250 μL/mouse e). .
・Ppa-P[Asp ₉₂ /Asp(Met) ₃₅ ]
・Ppa-P [Asp ₈₉ /Asp(Cys) ₃₈ ]
・Ppa-PEG20k

Regarding the light irradiation group, one hour after the sample administration, the abdomen of the mouse was irradiated with a 200 mW/cm ² 680 nm laser for 600 seconds. The day of irradiation was defined as the first day, and tumor growth was measured every 5 days using IVIS, and body weight was measured using an electronic balance for a total of 20 days.

The evaluated samples are summarized as follows (n=5).
・Ppa-P [Asp ₉₂ /Asp(Met) ₃₅ ] (Light)
・Ppa-P [Asp ₈₉ /Asp(Cys) ₃₈ ] (Light)
・Ppa-P[Asp ₉₂ /Asp(Met) ₃₅ ] (Dark)
・Ppa-P [Asp ₈₉ /Asp(Cys) ₃₈ ] (Dark)
・Ppa-PEG20k (Light)
・No treatment

The results are shown in FIGS. 20 to 24.

Example 8: Synthesis of tyrosine-modified polymers (1) N ₃ -PEG _9k -poly[aspartic acid _m /aspartic acid (tyrosine) _n ] and N ₃ -PEG _9k -poly[aspartic acid _m /aspartic acid (triethylene) Synthesis of the present tyrosine ligand-modified polymers N ₃ -PEG _9k -poly[aspartic acid _m /aspartic acid (tyrosine) _n ] and N ₃ -PEG _9k -poly[aspartic acid _m / aspartic acid (tyrosine) n _] ethylene glycol-tyrosine) _n ] (hereinafter referred to as N ₃ -PEG-P[Asp _m /Asp(Tyr) _n ] and N ₃ -PEG-P [Asp _m /Asp(TEG-Tyr) _n ] (m = Asp The synthesis method of the number of units (n=number of units of Asp(Tyr) or Asp(TEG-Tyr)) is described.

The synthesis scheme of N ₃ -PEG-P [Asp _m /Asp(Tyr) _n ] is shown below.

The synthesis scheme of N ₃ -PEG-P [Asp _m /Asp(TEG-Tyr) _n ] is shown below.

N ₃ -PEG _9k -PBLA _n was synthesized by N-carboxy anhydride (NCA) polymerization using N ₃ -PEG _9k -NH ₂ as an initiator and BLA-NCA as a monomer. The side chain carboxy group was deprotected under basic conditions to obtain N ₃ -PEG _9k -PAsp _n . Thereafter, the amino groups of the tyrosine structure ligands NH ₂ -Tyr and NH ₂ -TEG-Tyr having a propyl spacer and a triethylene glycol spacer are respectively bonded to the carboxyl group of N ₃ -PEG _9k -PAspn, and N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and N ₃ - PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] was obtained.

(2) Synthesis method (2-1) Synthesis of N ₃ -PEG _9k -PBLA ₉₅ Weigh out 0.40 g (0.044 mmol) of N ₃ -PEG _9k -NH ₂ into a 300 mL two-necked eggplant flask, and add 8.0 mL of benzene. After dissolving it in the solution, it was freeze-dried. 1.26 g (5.07 mmol) (n=95) of BLA-NCA was weighed into a 100 mL two-necked eggplant flask under an argon atmosphere. 1.36 mL of DMF purified by distillation and 13.6 mL of DCM were added to N ₃ -PEG9k-NH ₂ . Additionally, 3.55 mL of DMF and 35.5 mL of DCM were added to BLA-NCA and dissolved. The BLA-NCA solution was added to the N ₃ -PEG _9k -NH ₂ solution and stirred at room temperature under an argon atmosphere for 48 hours. Each reaction solution was added dropwise to 800 mL of a mixed solvent of hexane and ethyl acetate (hexane: ethyl acetate = 3:2), and purified by reprecipitation. Thereafter, it was dried under reduced pressure to obtain 1.20 g of white solid N ₃ -PEG _9k -PBLA ₉₅ in a yield of 96%. ^{The 1} H NMR spectrum is shown in FIG. 25. ^1H NMR (400MHz, DMSO-d6): δ8.41-8.02 (br, -NH-C=O-), 7.48-7.06 (br, =CH-CH=), 5.30 -4.83 (br, -O-CH ₂ -C-), 4.82-4.32 (br, -CO-CH-NH-), 3.73-3.64 (br, -CH ₂ - CH ₂ -NH-), 3.57-3.43 (br, -CH ₂ -CH ₂ -O-), 2.94-2.72, 2.70-2.55 (br, -CH-CH ₂ -C=O-), 2.36-2.29 (br, -N ₃ -CH ₂ -CH ₂ -).

(2-2) Deprotection of N ₃ -PEG _9k -PBLA _n (n=95) Weigh out 1.05 _g (0.037 mmol) of PEG _9k -PBLA 95 into a 50 mL eggplant flask, add 15 mL of 0.1 M NaOHaq, and 15 mL of NMP. was added and stirred at room temperature overnight. The reaction solution was put into a dialysis membrane (MWCO=3.5 kDa) and dialyzed three times against 2 L of pure water. The solution was lyophilized to yield 787 mg of white solid N ₃ -PEG _9k -PAsp ₉₅ with a yield of 93%. ^{The 1} H NMR spectrum is shown in FIG. ^1H NMR (400MHz, D ₂ O): δ7.34-7.12, 7.11-6.83 (br, -CH=CH-), 4.75-4.35 (br, -C=O -CH-NH), 4.17-3.99 (br, -CH ₂ -CH ₂ -O-), 3.98-3.85 (br, -CH ₂ -CH-C=O-), 3 .86-3.60 (br, -CH ₂ -CH ₂ -O-), 3.47-3.27 (br, -CH ₂ -CH ₂ -), 3.26-3.13, 3.12 -2.93 (br, -CH-CH ₂ -CH-), 2.94-2.51 (br, -CH-CH ₂ -C=O-), 2.08-1.78 (br, - _CH2 - _CH2 - _CH2- ).

(2-3) N ₃ -PEG-P [Asp ₇₅ /Asp (Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp (Tyr) ₃₀ ], N ₃ -PEG-P [Asp ₇₄ /Asp (TEG-Tyr) ₂₁ ] and N ₃ -PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] Weigh out 150 _mg (7.5 μmol) of N ₃ -PEG _9k -PAsp 95 into a 20 mL screw tube bottle. , was dissolved in 10 mL of a mixed solvent of H ₂ O and DMF (H ₂ O:DMF=2:8). There, 84 mg (214 μmol) of NH ₂ -Tyr (N ₃ -PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ]) and 211 mg (534 μmol) (N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]), NH ₂ -TEG-Tyr 125 mg (214 μmol) (N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and 313 mg (534 μmol) (N ₃ -PEG-P[Asp ₅₂ / Asp(TEG-Tyr) ₄₃ ]), TEA 298 μL (2.138 mmol), EDC/HCl 55 mg (285 μmol) (N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ]), 137 mg (713 μmol) (N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ]), 55 mg (285 μmol) (N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and 137 mg (713 μmol) (N ₃ - PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]), NHS 33 mg (285 μmol) (N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ]), 82 mg (713 μmol) (N ₃ -PEG -P[Asp ₆₅ /Asp(Tyr) ₃₀ ]), 33 mg (285 μmol) (N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and 82 mg (713 μmol) (N ₃ -PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]) were respectively added and stirred at room temperature overnight. The reaction solution was put into a dialysis membrane (MWCO=3.5 kDa), and each membrane was dialyzed twice with 2 L of 0.01 M NaOH, and then four times with 2 L of pure water. The obtained solution was freeze-dried to form white solids N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG- Yield 177 mg of P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] (N ₃ -PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ]), 219 mg (N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]), 213 mg (N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and 302 mg (N ₃ -PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]), yield 86% (N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ]), 93% (N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ]), 89% (N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and 92% (N ₃ -PEG-P[Asp ₅₂ /Asp( TEG-Tyr) ₄₃ ]).

(2-4) N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P [Asp ₇₄ /Asp (TEG-Tyr) ₂₁ ] and N ₃ -PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] ₁₅₀ mg each in a 20 mL screw tube bottle _{. 20} ] (5.5 μmol), N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ] (4.8 μmol), N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] (4 .7 μmol) and N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] (3.4 μmol) were weighed out, dissolved in 5 mL of 95% TFA, and stirred overnight at room temperature. The reaction solution was put into a dialysis membrane (MWCO=3.5 kDa) and dialyzed twice with 2 L of 0.01M NaOH each time, and then four times with 2 L of pure water. The solution was lyophilized to form white solids N ₃ -PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] in a yield of 95 mg (3.9 μmol) (N ₃ -PEG-P[Asp ₇₅ /Asp( Tyr) ₂₀ ]), 91 mg (3.4 μmol) (N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]), 107 mg (3.7 μmol) (N ₃ -PEG-P [Asp ₇₄ /Asp( TEG-Tyr) ₂₁ ]) and 124 mg (3.3 μmol) (N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]), yield 71% (N ₃ -PEG-P[Asp ₇₅ / Asp(Tyr) ₂₀ ]), 71% (N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]), 79% (N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ) and 97% (N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]). ¹ H NMR spectra are shown in Figure 27 (N ₃ -PEG-P [Asp ₇₅ /Asp (Tyr) ₂₀ ]), Figure 28 (N ₃ -PEG-P [Asp ₆₅ /Asp (Tyr) ₃₀ ]), Figure 29 ( N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and FIG. 30 (N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]).

_N3- PEG-P[ _Aspm /Asp(Tyr) _n ] (n=20 and 30): ^1H NMR (400MHz, _D2O ): δ7.34-7.12, 7.11-6.83 (br, -CH=CH-), 4.75-4.35 (br, -C=O-CH-NH), 4.17-3.99 (br, -CH ₂ -CH ₂ -O-) , 3.98-3.85 (br, -CH ₂ -CH-C=O-), 3.86-3.60 (br, -CH ₂ -CH ₂ -O-), 3.47-3. 27 (br, -CH ₂ -CH ₂ -), 3.26-3.13, 3.12-2.93 (br, -CH-CH ₂ -CH-), 2.94-2.51 (br , -CH-CH ₂ -C=O-), 2.08-1.78 (br, -CH ₂ -CH ₂ -CH ₂ -).

_N3- PEG-P[ _Aspm /Asp(TEG-Tyr) _n ] (n=21 and 43): ^1H NMR (400MHz, _D2O ): δ7.35-7.16, 7.10-6 .91 (br, -CH=CH-), 4.74-4.38 (br, -C=O-CH-NH), 4.19-4.03 (br, -CH ₂ -CH ₂ -O -), 4.02-3.85 (br, -CH ₂ -CH ₂ -O-), 3.82-3.63 (br, -CH ₂ -CH ₂ -O-), 3.47-3 .32 (br, -C-CH ₂ -CH-), 3.31-3.16 (br, -O-CH ₂ -CH ₂ -), 3.17-2.97 (br, -CH ₂ - CH ₂ -O-), 2.96-2.50 (br, -CH-CH ₂ -C=O-), 2.48-2.29 (br, -CH ₂ -CH-C=O-) , 1.96-1.80 (br, -CH ₂ -CH ₂ -CH ₂ -), 1.79-1.64 (br, -CH ₂ -CH ₂ -CH ₂ -).

(2-5) tert-butyl O-(16-amino-5,8,11,14-tetraoxahexadecan-1-yl)-N-(tert-butoxycarbonyl)-L-tyrosinate (“NH ₂ -TEG -Tyr'' (intermediate) NH ₂ -TEG-Tyr was synthesized according to the following synthesis scheme.

Synthesis of Scheme 1.4. Reagents and conditions: (a) trans-1,4-dibromo-2-butene, tetra-n-butylammonium hydrogen sulfate, NaOH, CH ₂ Cl ₂ , H ₂ O, 58%. (b) Benzyl (2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}ethyl)carbamate, tetra-n-butylammonium hydrogen sulfate, NaOH, CH ₂ Cl ₂ , H ₂ O, 30% (c) H ₂ (1 atm), Pd/C, AcOEt, MeOH, 51%.

(2-5-1) tert-butyl O-[(2E)-4-bromobut-2-en-1-yl]-N-(tert-butoxycarbonyl)-L-tyrosinate (2)

A dichloromethane solution of tert-butyl N-(tert-butoxycarbonyl) L-tyrosinate (0.20 g, 0.59 mmol) and trans-1,4-dibromo-2-butene (0.25 g, 1.2 mmol) ( 1.8 mL), an aqueous solution (0.95 mL) of tetra-n-butylammonium hydrogen sulfate (0.20 g, 0.59 mmol) and sodium hydroxide (0.047 g, 1.2 mmol) were added, and the mixture was heated to room temperature. The mixture was stirred for 45 minutes. Dichloromethane and water were added to the reaction solution to separate the layers, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane/ethyl acetate) to obtain the title compound (0.16 g, yield: 58%) as a colorless solid.
^1H NMR ( _CDCl3 ) δ: 7.08 (2H, d, J = 8.5Hz), 6.82 (2H, d, J = 8.5Hz), 6.11-5.95 (2H, m ), 4.97 (1H, d, J = 7.9Hz), 4.53 (2H, dd, J = 4.8, 1.2Hz), 4.40 (1H, q, J = 6.7Hz) , 3.99 (2H, d, J=7.3Hz), 2.99 (2H, d, J=3.6Hz), 1.42 (9H, s), 1.41 (9H, s). HRMS (ESI ⁺ ₎ m/z: 470.1555 ₍ M+H) ⁺ (calc.: _C22H33BrNO5 :470.1537).

(2-5-2) tert-butyl N-(tert-butoxycarbonyl)-O-[(18E)-3-oxo-1-phenyl-2,7,10,13,16-pentaoxa-4-azaicos- 18-en-20-yl]-L-tyrosinate (3)

tert-Butyl O-[(2E)-4-bromobut-2-en-1-yl]-N-(tert-butoxycarbonyl)-L-tyrosinate (1.0 g, 2.1 mmol) and benzyl (2 -{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}ethyl)carbamate (0.84 g, 2.6 mmol) in dichloromethane solution (1.8 mL) was added with tetra-n-butylammonium hydrogen sulfate (0 .72 g, 2.1 mmol) and an aqueous solution (0.95 mL) of sodium hydroxide (0.17 g, 4.3 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Dichloromethane and water were added to the reaction solution to separate the layers, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane/ethyl acetate) to obtain the title compound (0.45 g, yield: 30%) as a colorless liquid.
^1H NMR ( _CDCl3 ) δ: 7.35 (4H, d, J = 5.1Hz), 7.32-7.30 (1H, m), 7.07 (2H, d, J = 8.5Hz) ), 6.81 (2H, d, J=8.5Hz), 5.92 (2H, s), 5.49 (1H, s), 5.10 (2H, brs), 4.98 (1H, d, J = 8.5Hz), 4.49 (2H, brs), 4.40 (1H, q, J = 6.7Hz), 4.03 (2H, s), 3.64-3.55 ( 14H, m), 3.39 (2H, q, J = 5.2Hz), 2.99 (2H, d, J = 5.4Hz), 1.42 (9H, s), 1.41 (9H, s). HRMS ( _ESI ⁺ ) m/z: _717.4009 (M+H) ⁺ (calcd: _C38H57N2O11 :717.3957) _.

(2-5-3) tert-butyl O-(16-amino-5,8,11,14-tetraoxahexadecan-1-yl)-N-(tert-butoxycarbonyl)-L-tyrosinate (4)

tert-Butyl N-(tert-butoxycarbonyl)-O-[(18E)-3-oxo-1-phenyl-2,7,10,13,16-pentaoxa-4-azaicos-18-en-20-yl ] - To a solution of L-tyrosinate (0.46 mg, 0.63 mmol) in ethyl acetate (3 mL) and methanol (3 mL), 10% palladium-activated carbon (87 mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 4 hours. did. After filtering through Celite, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate/methanol) to obtain a crude product (0.19 g, yield: 51%) as a colorless liquid.
^1H NMR ( _CDCl3 ) δ: 7.06 (2H, d, J = 9.1 Hz), 6.80 (2H, d, J = 8.5 Hz), 4.98 (1H, d, J = 8 .5Hz), 4.40 (1H, q, J = 6.9Hz), 3.95 (2H, t, J = 6.0Hz), 3.68-3.59 (12H, m), 3.54 -3.49 (4H, m), 2.99 (2H, d, J = 5.4Hz), 2.86 (2H, t, J = 5.1Hz), 1.88-1.72 (4H, m), 1.42 (9H, s), 1.41 (9H, s). HRMS (ESI ⁺ ) m/z: 585.3761 _{( M} +H) ⁺ ( _calcd : _C30H53N2O9 :585.3746).

Example 9: Analysis (1) N ₃ -PEG _9k -PBLA ₉₅
^{The 1} H NMR spectrum shows a peak derived from the initiator N ₃ -PEG _9k -NH ₂ [3.57-3.43 (br, -CH ₂ -CH ₂ -O-)] and a peak derived from BLA-NCA [8. 41-8.02 (br, -NH-C=O-), 5.30-4.83 (br, -O-CH ₂ -C-), 2.94-2.72, 2.70-2 .55 (br, -CH-CH ₂ -C=O-)], it was determined that the degree of polymerization DP of PBLA was 95 and the number average molecular weight Mn was 28,500. Furthermore, from the GPC curve, the molecular weight dispersity of the obtained polymer was found to be PDI=1.11, confirming that it had a narrow molecular weight distribution.

(2) N ₃ -PEG _9k -PAsp ₉₅
^{The 1} H NMR spectrum has a peak derived from the initiator N ₃ -PEG _9k -NH2 [3.57-3.43 (br, -CH ₂ -CH ₂ -O-)] and a peak of α hydrogen of aspartic acid (3.57-3.43 (br, -CH 2 -CH 2 -O-)). From the ratio of the integral values of 01-2.40 (br, -CH-CH ₂ -C=O-), it was determined that the degree of polymerization DP of Asp was 95 (n = 95) and the number average molecular weight Mn was 20,000. Furthermore, it was confirmed from the GPC curve that the obtained polymer had a narrow monomodal molecular weight distribution (data not shown).

(3) N ₃ -PEG-P [Asp ₇₅ /Asp (Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp (Tyr) ₃₀ ], N ₃ -PEG-P [Asp ₇₄ /Asp (TEG -Tyr) ₂₁ ] and N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]
The α-hydrogen peak of aspartic acid in the ¹ H NMR spectrum (3.01-2.40 (br, -CH-CH ₂ -C=O-) and the hydrogen peak on the benzene ring of NH ₂ -Tyr [7. 34-7.12, 7.11-6.83 (br, -CH=CH-)], the number of NH ₂ -Tyr introduced is 20 (N ₃ -PEG-P [Asp ₇₅ /Asp (Tyr) ₂₀ ]) and 30(N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]), the number average molecular weight is Mn=24,300 (N ₃ -PEG-P[Asp ₇₅ /Asp(Tyr) ) ₂₀ ]) and Mn=26,500 (N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]).Similarly, the α hydrogen peak of aspartic acid in the ¹ H NMR spectrum (3. 01-2.40 (br, -CH-CH ₂ -C=O-) and hydrogen peaks on the benzene ring of NH ₂ -TEG-Tyr [7.34-7.12, 7.11-6.83 (br, -CH=CH-)], the number of introduced NH ₂ -TEG-Tyr is 21 (N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and 43 ( N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]), number average molecular weight Mn=28,500 (N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and It was determined that Mn = 37,200 (N ₃ -PEG-P [Asp ₅₂ /Asp (TEG-Tyr) ₄₃ ]). Also, from the GPC curve, the obtained polymer had a narrow monomodal molecular weight distribution. This was confirmed (data not shown).

Example 10: Evaluation of cultured cells (1) Overview N ₃ -PEG-PAsp _n (Cy5-PEG-PAsp _n ), N ₃ -PEG-P [Asp ₇₅ /Asp (Tyr) labeled with fluorescent dye (Cy5) ₂₀ ](Cy5-PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ]), N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ](Cy5-PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]), N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] (Cy5-PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] (Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]) was compared. Next, the amount of uptake in co-culture with BCH (LAT1 inhibitor) was measured and LAT1 specificity was compared.

(2) N ₃ -PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P[Asp ₇₄ /Asp(TEG -Tyr) ₂₁ ] and N ₃ -PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] Fluorescent dye (Cy5) was injected into N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ], and N ₃ -PEG-P [Asp ₅₂ /Asp (TEG-Tyr) ₄₃ ] and added to the cells, and the intracellular Cy5 fluorescence intensity was measured by flow cytometry.

(3) Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] By click chemistry, N ₃ -PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and N ₃ -PEG-P [Asp ₅₂ /Asp(TEG- Cy5-DBCO was bonded to the terminal azide group of Tyr) ₄₃ ]. 50.0 mg (2.50 μmol) of N ₃ -PEG-PAsp ₉₅ , 70.0 mg (2.88 μmol) of N ₃ -PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], and 70.0 mg (2.88 μmol) of N ₃ -PEG-P [ Asp ₆₅ /Asp(Tyr) ₃₀ ], 70.0 mg (2.64 μmol), N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ], 80.0 mg (2.81 μmol), and N ₃ - 80.0 mg (2.15 μmol) of PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] was weighed into a 10 mL vial and dissolved in 5.00 mL of pure water. Next, 202 μL (N 3 -PEG-PAsp 95 ) and 233 μL (N ₃ -PEG-PAsp ₉₅ ) and 233 μL (N ₃ -PEG-PAsp 95 ) of Cy5-DBCO (12.5 mg/ml Cy5-DBCO/DMSO solution) were added to each polymer solution in an equivalent amount. [Asp ₇₅ /Asp (Tyr) ₂₀ ]), 213 μL (N ₃ -PEG-P [Asp ₆₅ /Asp (Tyr) ₃₀ ]), 227 μL (N ₃ -PEG-P [Asp ₇₄ /Asp (TEG-Tyr) ₂₁ ]) and 174 μL (N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]) were added to the polymer solution and stirred overnight at room temperature. The reaction solution was put into a dialysis membrane (MWCO=3.5 kDa) and dialyzed four times with 2 L of pure water each time. Thereafter, further purification was performed using a PD-10 column, and the polymer solution was freeze-dried to give a blue solid of 44.3 mg (N ₃ -PEG-PAsp ₉₅ ), 64.8 mg (N ₃ -PEG-P[Asp ₇₅ / Asp(Tyr) ₂₀ ]), 61.2 mg (N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]), 62.2 mg (N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ]) and 58.9 mg (N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]).

(4) Measurement of intracellular Cy5 fluorescence intensity BxPC3 cells were seeded in a 24-well plate at a density of 1.0×10 ⁵ cells/well, and precultured at 37° C. and 5% CO ₂ for 24 hours. Each of the above polymer solutions was prepared in a medium to a concentration of 2.0 μM, and 500 μL each was added to the wells and incubated for 3 hours. After washing twice with 500 μL of D-PBS, 150 μL of trypsin-EDTA solution was added and incubated for 7 minutes. After confirming that the cells were detached using an optical microscope, 350 μL of RPMI medium was added, suspended, and the amount taken into the cells was measured by flow cytometry (Ex/Em = 650/670 nm). The obtained results are shown in FIG. 31.

All of the tyrosine-modified polymers showed a significant increase in the amount of uptake compared to the non-tyrosine-modified control, Cy5-PEG-PAsp ₉₅ . Cy5-PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] are Cy5-PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ] and Cy5-PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ], and both showed an increase in uptake as the number of tyrosine ligands increased.

(5) N ₃ -PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P[Asp ₇₄ /Asp(TEG -Tyr) ₂₁ ] and N ₃ -PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] by BCH. N ₃ -PEG-P [Asp ₇₅ /Asp introduced with fluorescent dye (Cy5) (Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ], and N ₃ -PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ], the cells were co-cultured with BCH, a LAT1 inhibitor, and the intracellular Cy5 fluorescence intensity was measured by flow cytometry.

BxPC3 cells were seeded in a 24-well plate at 1.0×10 ⁵ cells/well and precultured at 37° C. and 5% CO ₂ for 24 hours. BCH was prepared in a medium to a concentration of 10.0 mM, and 500 μL of the solution was added to each well and incubated for 30 minutes. After washing twice with 500 μL of D-PBS, each of the above polymer solutions and BCH were prepared in a medium at 2.0 μM and 10.0 mM, respectively, and 500 μL each was added to the wells and incubated for 3 hours. After washing twice with 500 μL of D-PBS, 150 μL of trypsin-EDTA solution was added and incubated for 7 minutes. After confirming that the cells were detached using an optical microscope, 350 μL of RPMI medium was added, suspended, and the amount taken into the cells was measured by flow cytometry (Ex/Em = 650/670 nm). The obtained results are shown in FIG. 32.

Under co-culture with BCH (LAT1 inhibitor), no change was observed in the amount of Cy5-PEG-PAsp ₉₅ and Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ] taken into cells. On the other hand, tyrosine-modified polymers Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] was confirmed. From the above results, Cy5-PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P[Asp ₅₂ /Asp( It was suggested that TEG-Tyr) ₄₃ ] was efficiently taken into cells by endocytosis specifically through LAT1.

Example 11: Effect on subcutaneous tumor mouse model (blood retention and tumor accumulation)
(1) Overview Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ], and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] were intravenously injected into the body. The dynamics were evaluated.

(2) N ₃ -PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ], N ₃ -PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], N ₃ -PEG-P[Asp ₇₄ /Asp(TEG -Tyr) ₂₁ ] and N _{3 -PEG} -P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] in the BxPC3 subcutaneous tumor mouse model _. /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] was injected intravenously, and blood retention and tumor accumulation were evaluated.

(3) Preparation of BxPC3 subcutaneous tumor mouse model 100 μl of BxPC3 cell suspension (5.0×10 ⁷ cells/ml) was subcutaneously injected into Balb/c mice.

(4) Evaluation of body dynamics 100 μL of the above 50.0 μM polymer preparation solution was administered through the tail vein to a model mouse whose tumor size had reached approximately 200 mm ³ . The dosage was 0.10 mg Cy5-PEG-PAsp ₉₅ , 0.12 mg Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], and 0.13 mg Cy5-PEG-P [Asp ₆₅ /Asp] per mouse. (Tyr) ₃₀ ], 0.14 mg Cy5-PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and 0.19 mg Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]. The animals were dissected 1, 3, 6, and 24 hours after sample administration, and blood and various organs were collected into multibead shocker crushing tubes. Thereafter, various organs were washed with PBS, and the fluorescence intensity was observed using IVIS (Ex/Em=640/700 nm), and the results are shown in FIG. 33. 800 μL of Passive Lysis Buffer was added to 100 mg of the collected organ, homogenized with a multi-bead shocker, and then transferred to a 96-well plate, and the fluorescence intensity was quantitatively measured using TECAN (Ex/Em = 640 nm/700 nm). The results are shown in Figures 34-36.

Administered Cy5-PEG-PAsp ₉₅ showed accumulation in the liver, while Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ] , Cy5-PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] showed accumulation in the kidney even after 24 hours. Moreover, compared to Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-P[ Tumor tissue treated with Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] showed relatively high fluorescence (FIG. 33).

The tumor uptake of the administered Cy5-PEG-PAsp ₉₅ was maintained at approximately 1.0% dose/g tissue over 24 hours. On the other hand, Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ] and Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ] showed a slight increase in tumor accumulation 6 hours after administration; No significant difference was observed. On the other hand, in agreement with the results in FIG. 34, Cy5-PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] are Cy5- It showed a significant increase in tumor accumulation compared to PEG-PAsp ₉₅ , showing approximately 1.5% dosage/g tissue and 2.0% dosage/g tissue, respectively, 6 hours after administration. This tyrosine-modified polymer was shown to achieve tumor accumulation through tyrosine modification.

Cy5-PEG-PAsp ₉₅ , Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ], Cy5-PEG-P at 6 hours after administration. The blood concentrations of [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] were below 5.0% of the dose/ml, 24 hours after administration. The post-blood concentration was below 1.0% dosage/ml, indicating rapid disappearance from the blood (Figure 35).

Administered Cy5-PEG-PAsp ₉₅ showed accumulation in the liver, while Cy5-PEG-P [Asp ₇₅ /Asp(Tyr) ₂₀ ], Cy5-PEG-P [Asp ₆₅ /Asp(Tyr) ₃₀ ] , Cy5-PEG-P [Asp ₇₄ /Asp(TEG-Tyr) ₂₁ ] and Cy5-PEG-P [Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ] showed accumulation in the kidney. Furthermore, from FIG. 36, it was suggested that the tyrosine-modified polymer was rapidly excreted from the body through the kidneys after administration.

Example 12: Evaluation of cell phototoxicity The cancer cell proliferation inhibitory effect of the Ppa-Tyr modified polymer was evaluated by CCK-8 assay.
(1) Cytotoxicity measurement Cells were seeded in a 96-well plate at 1.0×10 ⁴ cells/well, and precultured at 37° C. and 5% CO ₂ for 24 hours. 100 μL of the following sample solution was added and incubated for 3 hours.
・Ppa-Tyr modified polymer (Ppa: 1 μM)

After washing twice with 0.1 mL of D-PBS(-), 100 μL of RPMI (containing 10% fetal bovine serum/penicillin) was added to the BxPC3 cells, and the cells were irradiated with light using a 680 nm halogen lamp for 1 hour. After 48 hours of incubation, 100 μL of 10% CCK-8 solution was added to each well, and after 1.5 hours of incubation, absorbance at 450 nm was measured. The obtained results are shown in FIGS. 37 and 38.

None of the Ppa-Tyr-modified polymers showed phototoxicity to cancer cells unless irradiated with light (FIG. 38). On the other hand, in the case of light irradiation, N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]-Ppa, N ₃ -PEG-PAsp ₉₅ -Ppa, N ₃ - PEG-P[Asp ₇₅ /Asp(Tyr) ₂₀ ]-Ppa, N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]-Ppa and N ₃ -PEG-P[Asp ₇₄ /Asp(TEG-Tyr) ) ₂₁ ]-Ppa showed higher phototoxicity. (Figure 37). That is, it was shown that the present tyrosine-modified polymer achieves increased phototoxicity than tyrosine modification.

According to the present invention, it is possible to treat cancer by using a conjugate in which an anticancer drug is bound to a methionine- or tyrosine-modified LAT1-binding site targeting the amino acid transporter (LAT1).

All publications and patent documents cited herein are incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for illustrative purposes, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Example 13: Antitumor Effect Two weeks after BxPC3 cells were subcutaneously inoculated into Balb/c nude mice, 100 μL of the following sample D-PBS(-) solution was intraperitoneally administered (Ppa 50 μM, 100 μL/mouse e).
・N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]-Ppa
・N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]-Ppa
・N ₃ -PEG-PAsp ₉₅ -Ppa

Six hours after the sample administration, the mice were subcutaneously irradiated with a 200 mW/cm ² 680 nm laser for 600 seconds. The day of irradiation was defined as day 1, and tumor growth was measured every 3 days using a vernier scale, and body weight was measured using an electronic balance for a total of 28 days. The results are shown in FIGS. 39 and 40.

In the case of light irradiation, N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]-Ppa and N ₃ -PEG-P[Asp ₆₅ /Asp(Tyr) ₃₀ ]-Ppa are N ₃ -PEG-PAsp ₉₅ showed higher antitumor effect than -Ppa. Furthermore, compared to the control group to which no Ppa-Tyr modified polymer was administered, N ₃ -PEG-P[Asp ₅₂ /Asp(TEG-Tyr) ₄₃ ]-Ppa showed a significant difference in higher antitumor efficacy. (Figure 39). Even after light irradiation of any of the Ppa-Tyr modified polymers, the body weight of Balb/c nude mice did not change significantly (FIG. 40). That is, the present tyrosine-modified polymer was shown to achieve increased anti-tumor effects on the BxPC3 subcutaneous tumor model through tyrosine modification.

Claims (12)

A conjugate in which a drug is bound to the L-type amino acid transporter 1 (LAT1) binding site,
The LAT1 binding site has the following structure:
[During the ceremony,
R ₁ represents a methionine-like structure or a tyrosine-like structure, where the methionine-like structure is
and the tyrosine-like structure is
And;
R ₂ is a hydrogen atom, a hydroxy group, a carboxy group, an optionally substituted amino group, an optionally substituted C _1-10 alkyl group, an optionally substituted C _1-10 alkoxy group, a thiol group , a cyano group, an azide group, or a drug;
R ₃ and R ₄ are independently a hydrogen atom or -L ₃ -drug;
L ₁ , L ₂ , and L ₃ are independently linkers or absent;
x is an integer from 0 to 1000;
y is an integer from 2 to 1000; and the bonding order of each repeating unit is arbitrary.]
or a pharmaceutically acceptable salt thereof. 2. The conjugate of claim 1, wherein R ₁ is a methionine-like structure. 2. The conjugate of claim 1, wherein R ₁ is a tyrosine-like structure. L ₁ , L ₂ , and L ₃ are each independently a C _1-40 alkylene group, and the methylene group in the C _1-40 alkylene group has 1 to 10 oxo groups, NH, or polymerized The conjugate according to any one of claims 1 to 3, optionally substituted with polyoxyalkylene having a degree of 2 to 2,000. The conjugate according to any one of claims 1 to 4, wherein x is an integer from 30 to 100 and y is an integer from 10 to 70. The conjugate according to any one of claims 1 to 5, wherein the value of y/(x+y) is 0.5 or less. The conjugate according to any one of claims 1 to 6, wherein the drug is selected from the group consisting of low molecular weight compounds, medium molecular weight compounds, and antibodies. The conjugate according to any one of claims 1 to 6, wherein the drug is selected from the group consisting of an anticancer drug, a photosensitizer, a nucleic acid drug, a cytotoxic protein, and an in-vivo diagnostic agent. 9. The conjugate according to claim 8, wherein the drug is an anticancer drug or a photosensitizer. Brain cancer, head and neck cancer, esophageal cancer, thyroid cancer, small cell cancer, non-small cell cancer, breast cancer, stomach cancer, gallbladder/bile duct cancer, lung cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer Cancer, colon cancer, rectal cancer, ovarian cancer, chorioepithelial cancer, endometrial cancer, cervical cancer, renal pelvis/ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicle used for the treatment of cancer selected from the group consisting of cancer, embryonal cancer, Wilms' cancer, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor and soft tissue sarcoma; The conjugate according to claim 8 or 9. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier. Brain cancer, head and neck cancer, esophageal cancer, thyroid cancer, small cell cancer, non-small cell cancer, breast cancer, stomach cancer, gallbladder/bile duct cancer, lung cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer Cancer, colon cancer, rectal cancer, ovarian cancer, chorioepithelial cancer, endometrial cancer, cervical cancer, renal pelvis/ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicle used for the treatment of cancer selected from the group consisting of cancer, embryonal cancer, Wilms' cancer, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor and soft tissue sarcoma; The pharmaceutical composition according to claim 11.

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