JP2012131743A - Tumor accumulation type anticancer agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new means of utilizing membrane-permeating peptide; and to provide a new anticancer agent and a new method for treating a cancer utilizing the new means of utilizing membrane-permeating peptide.SOLUTION: The carrier is used for accumulating a cytotoxicity substance, comprising oligo-arginine peptide represented by (Arg)n [n denotes an integer of 4 to 12], on a tumor.

Description

  The present invention relates to a cell-integrated anticancer agent using a membrane-permeable peptide and a method for treating cancer.

  HIV-1 Tat (48-60) and oligoarginine (Rn: n = 6 to 12) are known to have the ability to move into cells while being a short-chain peptide of about 10 residues, It is called a membrane penetrating peptide (hereinafter referred to as “CPP” as appropriate) (Non-patent Documents 1 and 2). Several studies on CPP have been reported so far.

  Non-Patent Documents 1 and 2 report examples in which CPP is applied as an intracellular delivery vector for substances including proteins and nucleic acids that cannot be transferred into cells alone.

  Non-Patent Document 3 reports that protein transduction domain (PTD) from Tat protein was combined with β-galactosidase and injected intraperitoneally into mice, resulting in the delivery of the fusion protein to all tissues of mice. is doing.

  In Non-Patent Document 4, the distribution of a conjugate of HIV-1 REV peptide and Fab fragment after intravenous administration to rats is examined using autoradiography, and the REV peptide is bound. It is described that significantly higher radioactivity was detected in the adrenal gland, spleen and liver compared to the non-fabricated Fab fragment.

  However, the kinetics of CPP in the body is not fully understood, and information on its distribution and metabolism is limited.

Futaki, S .; (Ed.) Adv. Drug Delivery Rev. (2008) 60, 447-614. Futaki, S .; Biopolymers (2006) 84, 241-249. Schwarze, S .; R. Ho, A .; , Vocero-Akbani, A .; , Dowdy, S .; F. Science (1999) 285, 1569-1572. Kameyama, S .; Okada, R .; , Kikuchi, T .; Omura, T .; Nakase, I .; , Takeuchi, T .; Sugiura, Y .; , Futaki S. Mol. Pharm. (2006) 3, 174-180.

  An object of the present invention is to provide a new means for using a membrane-permeable peptide, an anticancer agent using the peptide, and a method for treating cancer.

As a result of intensive studies to solve the above-mentioned problems, the present inventor said that, among the oligopeptides conventionally known to have intracellular translocation ability, oligoarginine peptides accumulate in tumors in tumor-bearing model mice. It was found to have properties. Based on this finding, the present inventors have further researched, and by binding a substance having cytotoxic activity to the oligoarginine peptide and administering it to a cancer-bearing mouse statically, the enlargement of the tumor is significantly increased. It was confirmed that it was possible to suppress. In addition, the present inventors have significantly increased non-specific cytotoxicity by administering the substance having cytotoxic activity bound to the oligoarginine peptide as compared with the case where the cytotoxic active substance is administered alone. It was found to be reduced. Furthermore, the present inventors have found that oligoarginine composed of D-arginine has superior cell migration ability and strong tumor accumulation property than conventional oligoarginine composed of L-arginine. As a result of further improvements based on these findings, the present inventors have completed the present invention.
Below, typical this invention is shown.

I. Item 1. Carrier comprising oligoarginine (Arg) A carrier for accumulating a cytotoxic substance in a tumor, comprising an oligoarginine peptide represented by n [n represents an integer of 4 to 12].
Item 2. Item 2. The carrier according to Item 1, wherein Arg is D-arginine.
Item 3. Item 3. The carrier according to Item 1 or 2, wherein n is 6 to 10.
Item 4. Item 4. The carrier according to any one of Items 1 to 3, wherein the cytotoxic substance is an anticancer agent.

II. Item 5. Tumor-accumulating anticancer agent Item 4. A tumor accumulation type anticancer agent, wherein the carrier according to any one of Items 1 to 3 and a cytotoxic substance are bound directly or indirectly.
Item 6. Item 6. The tumor-accumulated anticancer agent according to Item 5, wherein the carrier and the cytotoxic substance are bound via a linker.
Item 7. Item 7. The tumor-accumulating anticancer agent according to Item 5 or 6, wherein the cytotoxic substance is an anticancer agent.

III. Method for treating cancer Item 8. A method comprising administering to a patient in need of cancer treatment an oligoarginine peptide represented by (Arg) n [n represents an integer of 4 to 12] conjugated with a cytotoxic substance.
Item 9. Item 9. The method according to Item 8, further comprising accumulating the conjugate in a tumor.

The carrier of the present invention has a property of accumulating in a tumor in an animal body. Therefore, by combining the carrier of the present invention with another substance, the other substance can be efficiently delivered to the tumor. For example, by adopting a cytotoxic substance as another substance, the cytotoxic substance can be efficiently caused to act on the tumor to treat the tumor.
In addition, the use of the carrier of the present invention can reduce the non-specific action of a substance having cytotoxic activity. For this reason, the toxicity by the substance (for example, anticancer agent) which has cytotoxic activity can be reduced by utilizing the carrier of this invention.

  Since the tumor-accumulating anticancer agent of the present invention has a tumor accumulating ability, it can efficiently treat cancer with a smaller dose of active ingredient. In addition, the tumor-accumulating anticancer agent of the present invention has reduced toxicity because of its non-specific action due to its ability to accumulate tumors. Therefore, the tumor accumulation type anticancer agent of the present invention is useful for the treatment of cancer.

FIG. 1 shows the dynamics when a fluorescently labeled oligopeptide is administered to a tumor-bearing mouse. FIG. 2 shows the results of measuring the ability of oligoarginine peptides to move into cells. FIG. 3 shows the tumor accumulation property of oligoarginine administered to cancer-bearing mice. FIG. 4 shows the tumor accumulation properties of oligoarginine administered to cancer-bearing mice. The influence on the tumor size by the tumor accumulation type | mold anticancer agent which combined doxorubicin with the oligo arginine peptide is shown. The influence on the body weight of a mouse | mouth by the cell accumulation type | mold anticancer agent which combined doxorubicin with the oligo arginine peptide is shown.

Hereinafter, the present invention will be described in detail.
I. Carrier comprising oligoarginine peptide The present invention relates to a method for accumulating a cytotoxic substance in a tumor, comprising an oligoarginine peptide represented by (Arg) n [n represents an integer of 4 to 12]. A carrier (hereinafter referred to as “the carrier of the present invention” as appropriate) is provided.

Oligoarginine peptide The oligoarginine peptide contained in the carrier of the present invention means an oligoarginine peptide represented by (Arg) n [n represents an integer of 4 to 12] unless otherwise indicated. . Arg means arginine.

  An oligoarginine peptide is a linear oligomer in which 4 to 12 arginines are linked to each other by peptide bonds. The oligoarginine peptide is preferably composed of 5 to 10 arginines, more preferably composed of 6 to 10 arginines, more preferably composed of 7 to 9 amino acids, and even more. Preferably, it is composed of 8 arginines. In other words, a preferable value of n is 5 to 10, more preferably 6 to 10, still more preferably 7 to 9, and still more preferably 8. Such an oligoarginine peptide is hereinafter referred to as “oligoarginine” as appropriate.

  The arginine constituting the oligoarginine may be L-arginine or D-arginine. Oligoarginine may be composed of a combination of L-arginine and D-arginine as long as it retains the ability to penetrate cells. The arginine constituting the oligoarginine is preferably D-form arginine. In the oligoarginine, preferably all constituent arginines are D-arginine.

  The oligoarginine may have a structure in which the arginine molecule constituting the oligoarginine is modified as long as it has the ability to penetrate cells. Examples of the modified oligoarginine include a structure in which an acetyl group or other acyl group is bonded to the N-terminal arginine, and an amide or ester is bonded to the C-terminal arginine. The modified oligoarginine may have a structure in which a methyl group or an ethyl group is bonded to the nitrogen atom of any arginine (one or more) constituting the modified oligoarginine.

  Oligoarginine can be produced using any known method. Oligoarginine can be synthesized by organic chemical synthesis or genetic engineering techniques. As an organic chemical synthesis method, for example, a solid phase peptide synthesis method (for example, Fmoc synthesis method, Boc synthesis, etc.) can be suitably used. As a genetic engineering technique, for example, by preparing a DNA fragment encoding oligoarginine, inserting it into an appropriate vector, transforming an appropriate host using it, and expressing the DNA, Oligoarginine peptides can be obtained. In order to easily purify the produced oligoarginine peptide, it is also possible to design the oligoarginine peptide to be provided with a tag. Such methods are known in the art.

  Oligoarginine has the ability to permeate the cell membrane and migrate into the cell. Without being bound by theory, cell penetrating peptides containing oligoarginine accumulate on the cell surface by recognizing sulfated polysaccharides such as heparan sulfate and chondroitin sulfate on the cell membrane surface, and as a result, It is thought that tosis is induced and moves into cells.

  Oligoarginine has the property of accumulating not only in the above-mentioned cell membrane permeability but also in tumors present in the animal body. Preferably, oligoarginine can be accumulated in tumors existing in the body by being introduced into blood vessels. Therefore, by using oligoarginine as a carrier, other substances (for example, substances having cytotoxic activity) can be accumulated in the tumor and allowed to act.

  The carrier of the present invention contains oligoarginine, and its structure is not particularly limited as long as it is possible to accumulate a cytotoxic active substance in a tumor, and may contain additional components. For example, the carrier of the present invention may be a combination of oligoarginine and another carrier. The carrier of the present invention preferably consists essentially of oligoarginine (that is, a substance or functional group that does not interfere with the function of the carrier is not bound).

Cytotoxic substance A cytotoxic substance is a substance having cytotoxic activity. The cytotoxic substance can be used without particular limitation as long as it is transported to the tumor by the carrier of the present invention and can act on the tumor to kill and / or suppress the growth of tumor cells. As long as such actions and effects are exhibited, the type and origin of the cytotoxic substance is not limited. Therefore, any substance that can kill and / or suppress the growth of cells can be selected and used in accordance with the type of target tumor and the symptoms of the patient. For example, compounds having cytotoxic activity (for example, low molecular weight compounds), peptides, nucleic acids and the like can be used. Preferably, substances known as anticancer agents and substances that will be developed as anticancer agents in the future can be used as cytotoxic substances.

  Typical known anticancer agents that can be used as cytotoxic substances in the present invention include the following: acivicin; aclarubicin; acodazole hydrochloride; abiraterone; aclarubicin; acylfulvene; adecipenor; adzelesin; aldesleukin; Antagonists; altrethamine; ambastin; amidostin; amifostine; aminolevulinic acid; amrubicin; amsulin, anagrelide; anastrozole; andrographolide; acronin; adzelesin; aldesleukin; actinomycin; Amsacrine; anastrozole; anthramycin; 1-β-D-arabinofuranosylcytosine; Asperine; Azacitidine; Azetepa; Azotomycin; Battimastat; Benzodepa; Bicalutamide; Bisantren hydrochloride; Bisnafide dimesylate; Caremycin; carsterone; caracemide; capecitabine; carbetimer; carboplatin; oxaliplatin; carmustine; carubicin hydrochloride; carzelesin; camptothecin; cedephingol; chlorambucil; cilolemycin; cisplatin; gradribin; Doxyfluridine; dactinomycin; daunorubicin hydrochloride; temozolo Decitabine; Dexormaplatin; Dexamethasone; Dezaguanine; Dezaguanine mesylate; Dinostatin stimaramer; Diaziquan; Docetaxel; Doxorubicin; Doxorubicin hydrochloride; Droloxifene; Droloxifene citrate; Edatrexate; eflonitin hydrochloride; elsamitrucin; exemestane; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbrozole; esorubicin hydrochloride; estramustine; Fazalabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; Foscidon; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosin; interleukin II, interferon α-2a; interferon α-2b; interferon α-n1; interferon α-n3; Interferon β-Ia; interferon γ-Ib; iproplatin; irinotecan hydrochloride hydrate; lanreotide acetate; letrozole; leuprolide acetate; riarosol hydrochloride; lometrexol sodium; lomustine; ranimustine; rosoxantrone hydrochloride; masoprocol; ; Megestrol acetate; melengestrol acetate; melphalan; menogalyl; mercaptopurine; Methotrexate sodium; methoprin; metredepa; mitindamide; mitocalysin; mitocrine; mitodiline; mitmarcin; mitomycin; Oxythran; paclitaxel; pegaspargase; peromycin; pentamuthine; pepromycin sulfate; pemetrexed sodium hydrate; perphosphamide; N-morphonoacetyl-L-aspartate; pipbloman; prednisolone; pupelomachine; Pricamycin; promestan; porfimer sodium; porphyromycin Prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; levofolinate calcium; ribopurine; logretimide; saphingol; safingol hydrochloride; semustine; simtrazene; sodium spalfosate; sparsomycin; Thromdomide; spiroplatin; streptonigrin; streptozocin; slofenal; tagafur; thalisomycin; tecogalane sodium; Tilapazamine; topotecan; toremifene citrate; trestron acetate; triphosphate Livin; Trimetrexate; Trimetrexate glucuronide; Triptorelin; Tubrosol hydrochloride; Uracil mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine sulfate; Vindesine sulfate; Vinepidine sulfate; Vinoresin sulfate tartrate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; borosol; xeniplatin; dinostatin; zorubicin hydrochloride.

  From the viewpoint of better membrane permeability using oligoarginine, a low molecular weight anticancer agent is preferable, for example, a molecular weight of 1500 or less, preferably 1200 or less, more preferably 1000 or less, and still more preferably 800 or less. In addition, examples of preferable anticancer agents from another viewpoint include anthracycline anticancer agents such as doxorubicin, daunorubicin, pirarubicin, epirubicin, idarubicin, aclarubicin, and amrubicin. Any of the cytotoxic substances listed above are commercially available.

Carrier The carrier in the present invention means a carrier for carrying a cytotoxic substance introduced into an animal body to a tumor and accumulating it in the tumor. The carrier of the present invention contains the above oligoarginine. As described above, arginine has a property of accumulating in a tumor in the body. As long as the cell accumulation ability by arginine is not inhibited, the carrier of the present invention may contain other components. In a preferred embodiment, oligoarginine itself is the carrier of the present invention.

  Delivery of the cytotoxic substance to the tumor by the carrier can be performed by binding the cytotoxic substance and the carrier directly or indirectly and introducing it into the blood vessel of the animal having the tumor. A method for introducing a carrier to which a cytotoxic substance is bound into a blood vessel of an animal is not particularly limited, but is generally introduced directly into a blood vessel (preferably a vein) by injection. Intravenous introduced carriers reach the tumor through the blood vessels and accumulate. The binding between the cytotoxic substance and the carrier will be described later.

Tumors Tumors on which the carrier of the present invention accumulates generally include those recognized as tumors and related diseases. Specific examples include the following tumors and related diseases: bone sarcoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, malignant giant cell tumor, bone fibrosarcoma, chordoma, periosteal osteosarcoma , Bone tissue and connective tissue such as, soft tissue sarcoma, angiosarcoma, hemangiosarcoma, fibrosarcoma, Kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma Sarcoma; glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroma, nonglial tumor, vestibular schwannoma, craniopharyngioma, medulloblastoma, medulla Membranous, pineal tumor, pineoblastoma, primary brain lymphoma brain tumor; adenocarcinoma, lobular (small cell) cancer, intraductal cancer, medullary breast cancer, thoracic mucinous cancer, thoracic tubular adenocarcinoma, Breast nipple cancer, pa Breast cancer such as jet disease and inflammatory breast cancer; adrenal cancer such as pheochromocytoma and adrenocortical carcinoma; thyroid cancer such as papillary or follicular thyroid cancer, medullary thyroid cancer and undifferentiated thyroid cancer; insulinoma; Pancreatic cancer such as gastrinoma, glucagonoma, bipoma, somatostatin secreting tumor, and carcinoid or Langerhans islet cell tumor; pituitary cancer such as Cushing's disease, prolactin secreting tumor, acromegaly, and diabetes insipidus; Ocular cancers such as ocular melanoma (eg, iris melanoma, choroidal melanoma and ciliary body melanoma) and retinoblastoma; vaginal cancer, eg squamous cell carcinoma, Adenocarcinoma and melanoma; vulvar cancer, eg squamous cell carcinoma, melanoma, adenocarcinoma, base Cell carcinoma, sarcoma, and Paget's disease; squamous cell carcinoma, and cervical cancer such as adenocarcinoma; uterine cancer such as endometrial carcinoma and uterine sarcoma; ovarian epithelial carcinoma, borderline tumor Ovarian cancer, such as germ cell tumor, and stromal tumor; squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, myxoid epidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma , Esophageal cancers such as wart carcinoma, and oat cell (small cell) carcinoma; adenocarcinoma, fungating (polypoid), ulcerous, superficial spread, diffuse spread, malignant lymphoma, fat Gastric cancer such as sarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer such as hepatocellular carcinoma and germ cell tumor, gallbladder cancer (eg adenocarcinoma); papillary, nodular Cholangiocarcinoma; lung cancer, eg non-small cell lung cancer Squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; embryonic tumor, seminoma, undifferentiated, spermatogenic, nonseminoma, embryo Testicular cancer such as stage carcinoma, teratocarcinoma, choriocarcinoma (yolk sac tumor), prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; squamous cell carcinoma Oral cancer such as tumor; basal cancer; salivary gland cancer such as adenocarcinoma, mucinous epidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer such as squamous cell carcinoma and warts; Skin cancer such as carcinoma, squamous cell carcinoma and melanoma, superficial enlarged melanoma, nodular melanoma, malignant melanoma, terminal melanoma; renal cell carcinoma, adenocarcinoma, adrenal gland Tumor, fibrosarcoma, translocation Wilms'tumor; kidney cancer of epithelial cell cancers such as transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, bladder cancer, such as cancer sarcoma

II. Tumor accumulation type anticancer agent The present invention relates to the above-mentioned I. Provided is a tumor-accumulating anticancer agent in which the carrier of the present invention described above and a cytotoxic substance are bound directly or indirectly. In other words, the tumor-accumulating anticancer agent is obtained by binding one or more carriers of the present invention to a cytotoxic substance.

  Here, the carrier and the cytotoxic substance of the present invention are described in I. above. As explained in That is, the carrier of the present invention is a carrier containing oligoarginine and having a function of carrying a cytotoxic substance to a tumor. On the other hand, a cytotoxic substance is a substance having a property capable of suppressing the growth and / or expansion of a tumor and reducing the size of the tumor by killing and / or inhibiting the growth of cells. .

Binding between carrier and cytotoxic substance The binding between carrier and cytotoxic substance is optional unless the accumulation of the carrier on the tumor is inhibited by the binding and the cytotoxic activity of the cytotoxic substance is not inhibited. It is possible to implement by this method. In a preferred embodiment, the carrier and the cytotoxic substance are bound by directly or indirectly coupling (preferably covalently coupling) the oligoarginine contained in the carrier and the cytotoxic substance. The

  As long as oligoarginine binds to cytotoxic substances, as long as it does not inhibit the tumor accumulation of oligoarginine and the cytotoxic activity of cytotoxic substances, any known binding method may be used in consideration of the structure of the cytotoxic substance. You can choose to do it. The direct bond is a bond not via a linker or the like, and the indirect bond is a bond via a linker or the like. The cytotoxic substance may be bound to any arginine of oligoarginine. For example, it may be bonded through a free carboxyl group or amino group of the terminal amino acid, or may be bonded through a guanidino group of any arginine. From the viewpoint of facilitating the coupling, it is preferable to bind directly or indirectly to the C-terminal or N-terminal arginine.

  When the oligoarginine and the cytotoxic substance are directly bound, for example, the guanidino group and amino group of the oligoarginine are protected with an appropriate protecting group, and then the free carboxyl group and the cytotoxic substance are present. A group capable of reacting with a carboxyl group (for example, a primary hydroxyl group) or an amine can be reacted to form an ester bond or an amide for bonding. Similarly, a group capable of reacting with the amino group or guanidino group present in the cytotoxic substance using the guanidino group possessed by any N-terminal amino group of oligoarginine or any arginine constituting oligoarginine It is also possible to combine directly with.

In a preferred embodiment, the oligopeptide and the cytotoxic agent are indirectly bound. Various techniques for indirectly binding an oligopeptide and a cytotoxic substance via a linker are known. For example, cysteine can be used as a linker, and the C-terminus or N-terminus of oligoarginine can be modified with a cysteine (Cys) residue. For example, oligo-arginine is at its C-terminus, can be modified to include -Gly-Cys-NH _2. When the peptide contains a Cys residue, a disulfide (—S—S—) bond can be formed between the —SH group of the Cys group and the —SH group present in the cytotoxic substance. When the cytotoxic substance does not inherently have an —SH group, it can be modified by adding an —SH group as long as the cytotoxic activity is not inhibited. Such a technique is known in the art. It is.

  Another linker includes a cross-linking agent having at least two functional groups capable of binding oligoarginine and a cytotoxic substance. Examples of such a cross-linking agent include 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, 3,3-dithiobis (succinimidylpropionate), bis-N-maleimide-1, Bifunctional cross-linking reagents such as 8-octane, heterobiology such as maleimidobenzoyl-N-hydroxysuccinimide (MBS), N- (6-maleimidocaproyloxy) succinimide, N- (4-maleimidobutyloxy) succinimide A functional crosslinking reagent etc. can be mentioned.

  In addition to these, it is also possible to use a photocleavable linker or a linker that can be cleaved by an enzyme in a living body. Indirect binding between the cytotoxic substance and oligoarginine includes binding using liposomes or polymer micelles.

  As described above, the tumor-integrated anticancer agent of the present invention can be produced by binding oligoarginine and a cytotoxic substance.

III. Method for treating cancer According to the present invention, a cytotoxic substance (for example, a known substance) is administered by administering an oligoarginine to which a cytotoxic substance is bound (that is, a tumor-accumulating anticancer agent) to a patient in need of treatment for the tumor. Anticancer agents) can be effectively acted on tumors, so that such patients can be treated. The administration method is not particularly limited as long as the administered oligoarginine can accumulate in the tumor in the body, and can be appropriately selected according to the type and site of the tumor to be treated. The preferred administration method is parenteral administration, more preferably intravascular administration by injection.

  The dose of the tumor-accumulating anticancer agent is not particularly limited as long as the effect of suppressing the growth or expansion of the tumor is obtained, and is appropriately determined according to the type of tumor, the site, the age and weight of the patient, the type of cytotoxic substance, etc. You can choose. In general, the dosage can be about 0.01 μg to 10 mg / kg / day for a patient.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these.

Example 1: Synthesis of oligoarginine peptide and labeling thereof Peptides having 11 amino acid sequences shown in Table 1 below were prepared using the Fmoc solid phase method. The preparation was performed according to the method described in Biochemistry 46: 492-501 (2007). After deprotecting the resin with trifluoroacetic acid and 1,2-ethanedithiol (95: 5), the resulting peptide was purified by HPLC. The obtained peptide was identified by confirming the molecular weight with MALDI-TOFMS. The peptides thus prepared are shown below.

^* R and (Arg) represent L-arginine.
^** r and _D (Arg) represent D-arginine.

Via the thiol group of the cysteine residue side chain in which the prepared peptide is arranged at the C-terminus, Alexa Fluor _{660 C 2 -maleimide (Alexa660) (} Molecular Probes, Invitrogen Inc.) were labeled with or _{Alexa Fluor 488 C 5 -maleimide (Alexa488} ) (Molecular Probes, Invitrogen Inc.). Specifically, 1.1 equivalent of a fluorescent label was mixed with 1 equivalent of peptide in a DMF / MeOH (1: 1) solvent, and the mixture was stirred (light-shielded) at room temperature for 3 hours for fluorescent labeling. The fluorescently labeled peptide was purified by HPLC, and the molecular weight was confirmed by MALDI-TOFMS.

Example 2: Behavior of cell-penetrating peptide in cancer-bearing mice HeLa-bearing mice: After transplanting HeLa cells subcutaneously to the right shoulder of nude mice at 5.0 × 10 ⁶ cells, the tumor size becomes 150 mm ³ or more Was raised until. This was used as a HeLa-bearing mouse in the test. Tumor volume was calculated by (major axis) × (minor ^diameter) 2/2.

  Next, Alexa660 fluorescently labeled oligopeptides (Tat, Penetratin, and R8) prepared in Example 1 and GC without a cell-penetrating peptide were dissolved in PBS, and 3 nmol of each tail vein was added to the HeLa-bearing mice prepared above. Administered. Fluorescence images up to 24 hours after administration were acquired over time (excitation wavelength 640 nm, detection wavelength 700 nm). Imaging was performed using IVIS Spectrum (manufactured by Xenogen) after anesthetizing the mouse with isoflurane (2.5%). The result is shown in FIG.

  In FIG. 1, the arrow indicates the location of the carried tumor. In mice administered with fluorescently labeled R8, the fluorescence intensity at the tumor location is shown to be significantly higher, indicating that the administered R8 has accumulated in the tumor. In contrast, in mice administered with fluorescently labeled Tat and Penet, some fluorescence is observed at the tumor location, but the fluorescence intensity in the kidney is relatively stronger, and it is considered that there is no accumulation in the tumor. It is done. Little fluorescence was observed in mice administered with fluorescently labeled GC. This is thought to be because GC is not a cell-penetrating peptide, but circulates in the body in the bloodstream but is not taken into the cells. From the test results, it was revealed that among the cell-permeable peptides, the oligoarginine peptide has a high tumor accumulation property. Alexa660 used as a fluorescent label has a molecular weight of 902 and is also a model low molecular weight compound. Therefore, this test result shows that it is possible to carry an oligoarginine peptide to a tumor by using it as a carrier for a low molecular weight compound.

Example 3: Intracellular transfer ability of oligoarginine The intracellular transfer ability of oligoarginine was measured by the following procedure. HeLa cells derived from human cervical cancer were cultured under conditions of 37 ° C. and 5% CO ₂ using a DMEM medium containing 10% fetal bovine serum (FBS). Cells were cultured in 100-mm or 150-mm dishes and passaged every 2-3 days.

HeLa cells were subcultured in 24-well microplate (Iwaki) at a rate of 1.0 × 10 ⁵ cells / well for 24 hours under conditions of 37 ° C. and 5% CO ₂ , and then 1 μM prepared in Example 1 was used. Alexa488 labeled oligonucleotide arginine (R2, r6, r8, R8 , r10, r12, R12 and R16) culture solution containing the (10% FBS-containing DMEM) was added in 200iL to each well, 37 ° C., of 5% CO ₂ Cells were cultured for 1 hour under conditions. In addition, fluorescently labeled GC was also used as a comparative control. Thereafter, the cells were washed three times with phosphate buffered saline (PBS) containing heparin (0.5 mg / ml), treated with 0.01% trypsin (10 minutes, 37 ° C.), and then the cells were collected from the microplate. The collected cells were centrifuged (3 minutes, 800 g, 4 ° C.) and washed with PBS three times, and the fluorescence intensity of the cells was measured with a flow cytometer (FACS). The results are shown in FIG.

  FIG. 2 shows the intracellular translocation ability of each oligoarginine by relatively showing the fluorescence intensity of each oligoarginine when the fluorescence intensity by GC having no intracellular translocation ability is 100. From this result, when the number of arginine which comprises a peptide was below a fixed value (12), the tendency for intracellular translocation ability to become high according to the increase in the number of arginine was shown. Moreover, it was shown that the peptide comprised by D-arginine has a higher intracellular translocation ability than the peptide comprised by L-arginine. This is considered to be due to the fact that a peptide composed of D-arginine is more difficult to be degraded by a peptide degrading enzyme than a peptide composed of L-arginine.

Example 4: Tumor accumulation of oligoarginine 1
The tumor accumulation in vivo of oligoarginine was examined. 3 nmol each of the fluorescently labeled oligoarginine (R2, r8, R8, r12, R12 and R16) prepared in Example 1 was administered to the HeLa-bearing mice prepared in the same manner as in Example 2 in the tail vein. As in Example 2, fluorescence images were obtained 24 hours after administration. The result is shown in FIG.

  In FIG. 3, the position of the tumor in the carrier mouse is indicated by an arrow. In all the images shown in FIG. 3, since fluorescence is seen at the position of the tumor, it can be seen that all oligoarginines have tumor accumulation properties. Surprisingly, it was shown that oligoarginine consisting of 8 D-arginines has far stronger tumor accumulation than r12, which has the highest intracellular translocation ability in Example 3. Thus, it was shown that the tumor accumulation property by oligoarginine is not a property depending solely on the ability of oligoarginine to translocate into the cell as shown in Example 3.

Example 5: Tumor accumulation of oligoarginine 2
After subcutaneously transplanting 4.0 × 10 ⁶ cells of CHO-K1 cells into the left shoulder of a nude mouse, the mice were reared until the size of CHO-K1 reached 512 mm ³ . To the CHO-K1 tumor-bearing mouse prepared as described above, 3 nmol tail vein was administered with oligoarginine (r8) composed of the fluorescently labeled D-arginine prepared in Example 1. And the fluorescence image of the mouse | mouth was acquired 24 hours after administration. The result is shown in FIG.

  In FIG. 4, the arrows indicate the positions of tumors formed by carrying CHO-K1 cells. From this result, it was confirmed that oligoarginine clearly shows the accumulation property to the tumor formed with CHO-K1 cells.

Example 6: Preparation of cell-integrated anticancer agent Doxorubicin was used as a representative example of a cytotoxic substance. In order to bind doxorubicin to a peptide, doxorubicin-maleimide was prepared according to the method described in Journal of Controlled Release 110: 362-369. Specifically, 23 mg of Doxorubicin hydrochloride (40 nmol, 1 equivalent) and 24 mg of N- (β-maleidopropionic acid) hydride (80 nmol, 2 equivalents) were dissolved in 10 mL of methanol (dehydrated), and then trifluoroacetic acid was added. Two drops were added and gently stirred at room temperature for 4 hours (light-shielding). After the reaction, the solvent was removed by an evaporator, and precipitation purification was performed three times with ethyl acetate (7 mL). Then, it light-shielded and air-dried and obtained doxorubicin-maleimide. ^The target product was confirmed by ¹ H-NMR and FAB-MS measurement (FAB-MS measurement value: 709, theoretical value [M + H] ⁺ : 709.2).

Next, conjugates (conjugates) of three types of oligoarginine (r6, r8 and r10) composed of D-arginine and doxorubicin were prepared. After each oligoarginine (4.7 nmol, 1 equivalent) and doxorubicin-maleimide (4.7 nmol, 1 equivalent) prepared in Example 1 were dissolved in 400 μL of methanol, 0.5 μL of N-methylmorpholine was added, The mixture was stirred at room temperature for 3 hours (shielded from light). After purification by HPLC, the product was freeze-dried and the target product was confirmed by MALDI-TOFMS measurement (yield about 50%) (r6-doxorubicin: MALDI-TOFMS measurement: 1824.8, theoretical value [M + H] ⁺ : 1823.6, r8-doxorubicin: MALDI-TOFMS measured value: 2136.0, theoretical value [M + H] ⁺ : 2136.0, r10-doxorubicin: MALDI-TOFMS measured value: 2448.8, theoretical value [M + H] ⁺ : 2448.3).

Example 7: Treatment of tumor with tumor-accumulating anticancer agent The effect of each conjugate of oligoarginine and doxorubicin prepared in Example 6 on the tumor was examined. Each conjugate was dissolved in PBS and administered to the HeLa-bearing mouse prepared in the same manner as in Example 2 via the tail vein (once daily for 3 consecutive days). As a comparative control, doxorubicin alone (4 or 6 mg / kg) was also administered into the tail vein. Each conjugate was administered so that the dose of doxorubicin was 4 mg / kg. The tumor size and body weight of each mouse were measured 24 hours before the administration and every 48 hours or 72 hours after the continuous administration for 3 days. The results regarding tumor size are shown in FIG. The measurement results of the body weight of the mouse are shown in FIG. In each test, the n number is 5-6. For control, phosphate buffered saline (PBS) was used.

  As shown in FIG. 5, when doxorubicin (4 mg / kg) was administered or when the control was performed, the size of the tumor at the end of the measurement increased about four times as compared with the start of the measurement. In contrast, when a conjugate of each oligoarginine (r6, r8 or r10) and a cytotoxic substance is administered to a tumor-bearing mouse, the size of the tumor at the end of measurement is about It was 2 to 2.5 times. This shows that a cytotoxic substance can be effectively made to act on a tumor by administering a cytotoxic substance in association with oligoarginine. In addition, the results shown in FIG. 6 show that the weight of the mouse administered with the cytotoxic substance bound to oligoarginine is not significantly different from that of the control not administered with the cytotoxic substance. It was. On the other hand, when doxorubicin was administered in a large amount of 6 mg / kg, the body weight of the mice was significantly reduced. From these results, it is shown that by administering a cytotoxic substance bound to oligoarginine, it can act on the tumor specifically, and as a result, an effect of reducing toxicity due to the cytotoxic substance can be obtained. It was.

  When the above results are examined together with the results of the previous examples (that is, oligoarginine has a tumor accumulation property, a substance bound thereto (Alexa660) is transported specifically to the tumor), a cytotoxic substance is obtained. Is administered in combination with oligoarginine to efficiently accumulate cytotoxic substances in the tumor due to tumor accumulation by oligoarginine, and the cytotoxic substances are more effective due to the ability of oligoarginine to penetrate the cell membrane. It was strongly suggested that it is possible to act on the cells constituting the tumor.

Claims (7)

(Arg) A carrier for accumulating a cytotoxic substance in a tumor, comprising an oligoarginine peptide represented by n [n represents an integer of 4 to 12]. The carrier according to claim 1, wherein Arg is D-arginine. The carrier according to claim 1 or 2, wherein n is an integer represented by 6 to 10. The carrier according to any one of claims 1 to 3, wherein the cytotoxic substance is an anticancer agent. A tumor accumulation type anticancer agent, wherein the carrier according to any one of claims 1 to 3 and a cytotoxic substance are bound directly or indirectly. The tumor-accumulating anticancer agent according to claim 5, wherein the carrier and the cytotoxic substance are bound via a linker. The tumor-accumulating anticancer agent according to claim 5 or 6, wherein the cytotoxic substance is an anticancer agent.

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