PHARMACEUTICAL COMPOSITION CONTAINING 211AT-LABELED AMINO ACID DERIVATIVE, AND METHOD FOR PRODUCING SAID PHARMACEUTICAL COMPOSITION

Abstract

To develop a compound which is highly accumulated in cancer cells and capable of emitting an α-ray and provide a pharmaceutical composition for cancer treatment including the compound.

A pharmaceutical composition for cancer treatment including a compound having a structure in which .sup.211At is introduced as a substituent into an amino acid derivative having an affinity with an amino acid transporter LAT1 or a pharmaceutically acceptable salt of the compound.

Claims

1. A method of treating a cancer associated with expression of LAT1 in a subject, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising a compound having a structure in which .sup.211At is introduced as a substituent into an amino acid derivative having an affinity with an amino acid transporter LAT1 or a pharmaceutically acceptable salt of the compound, wherein the compound is one or more compounds having a structure represented by a following formula (I), ##STR00013## wherein: L is a direct bond or an optionally substituted C.sub.1-C.sub.5 alkylene group; M is a hydrogen atom or a halogen atom; R.sup.1 is a substituted C.sub.1-C.sub.5 alkyl group; R.sup.2 is a hydrogen atom, a C.sub.1-C.sub.5 alkyl group, a C.sub.6-C.sub.14 aryl group, or a C.sub.7-C.sub.16 arylalkyl group; and R.sup.3 is a hydrogen atom or a C.sub.1-C.sub.3 alkyl group.

2. The method according to claim 1, wherein: L is a direct bond; M is a hydrogen atom or a halogen atom; R.sup.1is a substituted C.sub.1-C.sub.5 alkyl group; R.sup.2 is a hydrogen atom or a C.sub.1-C.sub.5 alkyl group; and R.sup.3 is a hydrogen atom.

3. The method according to claim 1, wherein the compound is any of compounds having following structures or a combination of these compounds, ##STR00014## wherein M represents I or Br.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0041] FIG. 1 is an image illustrating stability of a .sup.211At-labeled amino acid derivative compound (.sup.211At-AAMT) in the presence and absence of ascorbic acid.

[0042] FIG. 2 is an image illustrating stability of radioactivity in blood and urine of normal rats (two animals) one hour after intravenous injection of .sup.211At-AAMT (with ascorbic acid).

[0043] FIG. 3 is a graph showing biodistribution of .sup.211At-AAMT in major organs when .sup.211At-AAMT is intravenously injected in normal mice with or without ascorbic acid.

[0044] FIG. 4 is a graph showing accumulation of .sup.211At-AAMT in a human pancreatic cancer cell line (PANC-1).

[0045] FIG. 5 is a graph comparing accumulation of .sup.211At-AAMT in cancer cells in the presence and absence of ascorbic acid.

[0046] FIG. 6 is a graph showing a cell-killing effect of .sup.211At-AAMT on the human pancreatic cancer cell line (PANC-1).

[0047] FIG. 7 is a graph comparing the cell-killing effect of .sup.211At-AAMT in the presence and absence of ascorbic acid.

[0048] FIG. 8 is graphs showing a change in a tumor size (FIG. 8(A)) and a change in body weight (FIG. 8(B)) of a cancer-bearing mouse to which .sup.211At-AAMT is administered.

[0049] FIG. 9 is SPECT images of .sup.211At-AAMT (with ascorbic acid, at a dose of 1 MBq each by intravenous injection) in pancreatic cancer (PANC-1) transplanted mice (two animals).

[0050] FIG. 10 is SPECT images (20 min to 1 hr) of .sup.211At-AAMT (without ascorbic acid, at a dose of 1 MBq by intravenous injection) in normal rats.

[0051] FIG. 11 is a graph showing the number of nodules in melanoma cell (B16) transplanted mice to which .sup.211At-AAMT is administered.

[0052] FIG. 12 is a graph showing a change in body weight of the melanoma cell (B16) transplanted mice to which .sup.211At-AAMT is administered.

DESCRIPTION OF EMBODIMENTS

[0053] Embodiments of the present invention are described below. The embodiments described below do not restrict the scope of the present invention, and changes other than according to the cited examples below may also be implemented as appropriate in a range not compromising the intent of the present invention.

[0054] 1. Definitions

[0055] In the present specification, an “alkyl or alkyl group” may be any aliphatic hydrocarbon group including a straight chain, a branched chain, a ring, or any combination thereof. The carbon number of the alkyl group is not particularly limited, and may be, e.g., 1 to 20 (C.sub.1-.sub.20), 1 to 15 (C.sub.1-15), and 1 to 10 (C.sub.1-10). In the present specification, alkyl groups may have one or more of any substituent. For example, C.sub.1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like. Alkoxy groups, halogen atoms (the term “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), amino groups, mono- or di-substituted amino groups, substituted silyl groups, or acyl groups and the like can be cited as examples of substituents, but the possible substituents are not thus limited. When an alkyl group has two or more substituents, the substituents may be the same or different. The same applies for the alkyl portions of other substituents which include an alkyl portion (e.g., alkoxy groups, arylalkyl groups, and the like).

[0056] In the present specification, an “alkylene” is a divalent group including a straight-chain or branched saturated hydrocarbon, and examples thereof include methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1-diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyltrimethylene, and the like.

[0057] In the present specification, “aryl or aryl group” may refer to any monocyclic or condensed polycyclic aromatic hydrocarbon group, and may include one or more hetero atoms (e.g., oxygen atoms, nitrogen atoms, sulfur atoms, or the like) as annular atoms. In this case, the group is sometimes referred to as a “heteroaryl” group or a “heteroaromatic” group. Bonding may occur at all possible positions whether the aryl has a monocyclic or a condensed ring structure. In the present specification, an aryl group may have one or more of any substituent on the ring thereof. Alkoxy groups, halogen atoms, amino groups, mono—or di-substituted amino groups, substituted silyl groups, or acyl and the like can be cited as examples of substituents, but the possible substituents are not thus limited. When an aryl group has two or more substituents, the substituents may be the same or different. The same applies for the aryl portions of other substituents which include an aryl portion (e.g., aryloxy groups, arylalkyl groups, and the like).

[0058] In the present specification, the term “arylalkyl” signifies an alkyl substituted with an abovementioned aryl. The arylalkyl may have one or more of any substituent. Alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, or acyl groups and the like can be cited as examples of substituents, but the possible substituents are not thus limited. When an acyl group has two or more substituents, the substituents may be the same or different. Representatively, arylalkylbenzyl, p-methoxybenzyl, and the like can be mentioned.

[0059] In the present specification, when a certain functional group is defined as being “optionally substituted”, a type of substituent, a substitution position, and the number of substituents are not particularly limited. When there are two or more substituents, they may be the same or different. Examples of the substituent can include an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group, without being limited thereto. Further substituents may be present in these substituents. Examples of such a case can include a halogenated alkyl group without being limited thereto.

[0060] The term “amide” used in the present specification includes both RNR′CO—(Alkylaminocarbonyl- when R is an alkyl) and RCONR′—(alkylcarbonylamino- when R is an alkyl).

[0061] The term “ester” used in the present specification includes both ROCO—(alkoxycarbonyl- when R is an alkyl) and RCOO—(alkylcarbonyloxy- when R is an alkyl).

[0062] In the present specification, a certain substituent may form a ring structure with another substituent, and in such cases where substituents are bonded, a person skilled in the art can understand the specific substitution, e.g., the formation of a bond to hydrogen. Consequently, when specific substituents are described as together forming a ring structure, a person skilled in the art can understand that the ring structure can be formed by a normal chemical reaction and easily be generated. Such ring structures as well as the processes for forming the same are within the cognizance of a person skilled in the art. Further, the heterocyclic structure may include any substituent on the ring.

[0063] 2. .sup.211at-Labeled Amino Acid Derivative

[0064] A pharmaceutical composition of the present invention is characterized by including a compound having a structure in which .sup.211At is introduced as a substituent into an amino acid derivative having an affinity with an amino acid transporter LAT1 (in the present specification, also referred to as a “.sup.211At-labeled amino acid derivative compound”, or a pharmaceutically acceptable salt thereof.

[0065] It has been known that LAT1 is one of amino acid transporters which are membrane proteins required for cellular uptake of neutral branched chain amino acids and aromatic amino acids, and is specifically expressed in cancer cells. LAT1 has a role in supplying amino acids as nutrients in cancer tissues. In the present specification, the term “amino acid derivative having affinity with LAT1” refers to an amino acid derivative that can be taken up by LAT1, more preferably, an amino acid derivative having properties of being taken up more easily by LAT1 than amino acid transporters (e.g., LAT2) in normal cells. As the amino acid derivative having an affinity with LAT1, an aromatic amino acid derivative is preferably mentioned, and as a typical example thereof, a tyrosine derivative such as α-methyltyrosine can be mentioned.

[0066] On the other hand, .sup.211At is a radioactive isotope of astatine (At), which is an element belonging to halogen. .sup.211At decays to stable lead (.sup.207Pb) with the emission of a high-energy α-ray having cell-killing activity. A half-life of .sup.211At is 7.2 hours. That is, when .sup.211At, which has a short half-life and high cell-killing activity, is used as a substituent for labeling and brought to an affected site, it becomes possible to efficiently destroy the cancer tissue. Further, when .sup.211At having such a feature is used as a substituent for labeling a LAT1-affinity compound, it becomes possible to provide a therapeutic effect in which α-ray irradiation can be applied using .sup.211At that is specifically accumulated in the cancer cells expressing LAT1 with almost no risk of having side effects as LAT1 itself is rarely expressed in normal cells.

[0067] In a preferred embodiment of the present invention, the above “compound having a structure in which .sup.211At is introduced as a substituent into an amino acid derivative having an affinity with an amino acid transporter LAT1” has a structure represented by the following formula (I). The compound represented by the formula (I) has tyrosine as a basic structure. Note that one type of the compound represented by the formula (I) may be solely used, or, in a preferred embodiment, two or more types of the compounds represented by the formula (I) can also be used in combination.

##STR00006##

[0068] L represents a direct bond or an optionally substituted C.sub.1-C.sub.5 alkylene group; M represents a hydrogen atom or a halogen atom; R.sup.1 represents a hydrogen atom or an optionally substituted C.sub.1-C.sub.5 alkyl group; R.sup.2 represents a hydrogen atom, a C.sub.1-C.sub.5 alkyl group, a C.sub.6-C.sub.14 aryl group, or a C.sub.7-C.sub.16 arylalkyl group; and R.sup.3 represents a hydrogen atom or a C.sub.1-C.sub.3 alkyl group.

[0069] Preferably, L represents a direct bond; R.sup.1 represents an optionally substituted C.sub.1-C.sub.5 alkyl group; R.sup.2 represents a hydrogen atom or a C.sub.1-C.sub.5 alkyl group; and R.sup.3 represents a hydrogen atom.

[0070] A halogen atom in M is preferably iodine (I) or bromide (Br), more preferably, I. In a preferred embodiment, M is a hydrogen atom or I.

[0071] .sup.211At-L, which can exist at any position on the benzene ring, preferably exists in an ortho position with respect to the OH group. More preferably, .sup.211At-L can exist in an ortho position with respect to the OH group and in a meta position with respect to the side chain (a linking group to a moiety having R.sup.1, COOR.sup.2, and NHR.sup.3) on the benzene ring. Further, M, which can exist at any position on the benzene ring (any position other than a position where .sup.211At-L or the OH group exists), preferably can exist in an ortho position with respect to the OH group and in a meta position with respect to .sup.211At-L (in this case, M is in a meta position with respect to the side chain).

[0072] Non-limiting specific examples of the amino acid derivative compound represented by the formula (I) include the following compound (also referred to as “.sup.211At-AAMT” in the present specification). In this compound, .sup.211At is directly introduced on the benzene ring of α-methyltyrosine.

##STR00007##

[0073] Further, in another preferred embodiment, non-limiting specific examples of the amino acid derivative compound represented by the formula (I) can include the following compound (also referred to as “.sup.211At-AIAMT” in the present specification). In this compound, similarly to the above .sup.211At-AAMT, .sup.211 At is directly introduced on the benzene ring of α-methyltyrosine. Additionally, it has a structure in which a halogen atom M is introduced in an ortho position with respect to the OH group and in a meta position with respect to .sup.211At-L. In the formula, M is I or Br.

##STR00008##

[0074] As described above, .sup.211At-AAMT and .sup.211At-AIAMT each can be solely used, or these two compounds can be used in combination.

[0075] The .sup.211At-labeled amino acid derivative compound used in the pharmaceutical composition of the present invention can be provided as a form of a pharmaceutically acceptable salt. Such salt is not particularly limited, and base addition salts, acid addition salts, amino acid salts, and the like can be cited as examples thereof. Sodium salts, potassium salts, calcium salts, magnesium salts, and other alkaline earth metal salts; ammonium salts; or triethylamine salts, piperidine salts, morpholine salts, and other organic amine salts can be cited as examples of base addition salts; and hydrochlorides, hydrobromates, sulfates, nitrates, phosphates, and other mineral acid salts; and salts of methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, salicylic acid, and other organic acids can be cited as examples of acid addition salts. Glycinates, asparaginates, glutamates, and the like can be cited as examples of amino acid salts. The salt may also be an aluminum salt or other metal salt.

[0076] The .sup.211At-labeled amino acid derivative compound used in the pharmaceutical composition of the present invention can be in a D form or an L form. However, it is preferably in an L form. Further, the .sup.211At-labeled amino acid derivative compound may have one or two or more asymmetric carbons depending on the types of substituents, and it may exist as a stereoisomer such as an optical isomer or a diastereomer. A stereoisomer in a pure form, any mixture of stereoisomers, racemate, and the like are all encompassed within the scope of the present invention.

[0077] The .sup.211At-labeled amino acid derivative compound or a pharmaceutically acceptable salt thereof used in the pharmaceutical composition of the present invention may also exist as a hydrate or a solvate. Any of these substances are encompassed within the scope of the present invention. The type of solvent for forming a solvate is not particularly limited, but ethanol, acetone, isopropanol, and other solvents can be cited as examples thereof.

[0078] 3. Pharmaceutical Composition

[0079] The pharmaceutical composition of the present invention includes the .sup.211At-labeled amino acid derivative compound or a pharmaceutically acceptable salt thereof. Here, the term “composition” subsumes not only products including active components and inactive components (pharmaceutically acceptable excipients) for constituting a carrier, but also any product directly or indirectly generated as a result of association, complexing, or aggregation of any two or more components, as a result of dissociation of one or more components, or as a result of another type of reaction or interaction of one or more components. The pharmaceutical composition of the present invention can include a combination of two or more kinds of compounds corresponding to the “.sup.211At-labeled amino acid derivative compound”.

[0080] In a preferred embodiment of the present invention, the pharmaceutical composition is used for cancer treatment. Cancer targeted by the pharmaceutical composition of the present invention is not particularly limited as long as LAT1 is expressed in cancer cells and includes any malignant tumor. Example of the cancer include pancreatic cancer, leukemia (including both myeloid leukemia and lymphocytic leukemia), melanoma, colorectal cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, kidney cancer, laryngeal cancer, esophageal cancer, liver cancer, lymphoma, myeloma, head and neck cancer, ovarian cancer, bladder cancer, childhood cancer, childhood leukemia, or a brain tumor. The pharmaceutical composition can be preferably used for intractable cancer or advanced cancer that is difficult to be treated by a surgical treatment.

[0081] Further, the pharmaceutical composition of the present invention may further include a stabilizer for the purpose of reducing degradation of the compound by stabilizing the bond between .sup.211At and a carbon atom and enhancing stability of the compound in the living body in addition to the .sup.211At-labeled amino acid derivative compound as an active ingredient. As such a stabilizer, a compound having reducing activity can be typically mentioned. Examples of the stabilizer can include ascorbic acid, an alkali metal salt of ascorbic acid, an alkaline earth metal salt of ascorbic acid, cysteine, glutathione, gentisic acid, and glucose. Of these, ascorbic acid, an alkali metal salt of ascorbic acid, or an alkaline earth metal salt of ascorbic acid is preferable, and ascorbic acid or sodium ascorbate is more preferable. As shown in the following Examples, in the present invention, it has been surprisingly found that using the .sup.211At-labeled amino acid derivative compound and the reducing agent such as ascorbic acid in combination can not only stabilize the compound for a long period of time as described above but also increase stability of the compound in the living body even after the compound is taken up in the living body, thereby making it possible to improve accumulation of the compound in the cancer cells. These are unconventional characteristics not found in the related art.

[0082] The pharmaceutical composition of the present invention can further include a probe compound capable of detecting a cancer tissue. Such a probe compound is preferably a probe compound for positron emission tomography (PET). As a probe compound for PET (PET agent), a publicly known probe compound for PET in the related art can be used. For example, a PET agent labeled with fluorine (.sup.18F) can be used. Using both the .sup.211At-labeled amino acid derivative compound capable of performing α-ray treatment and the probe compound capable of detecting the cancer tissue makes it possible to monitor delivery of the agent to the affected site, the condition of the affected site after administration, and the like during the treatment.

[0083] The type of pharmaceutical composition is not particularly limited, and can be prepared as a pharmaceutical composition for oral administration in a form such as granules, fine granules, a powder, a hard capsule, a soft capsule, a syrup, an emulsion, a suspension, or a liquid, or as a pharmaceutical composition for parenteral administration in the form of an injection such as for intravenous injection, intramuscular injection, or subcutaneous injection, or in a form such as drops, a percutaneous absorption preparation, a transmucosal absorption preparation, nasal drops, an inhalant, or a suppository. These preparations are prepared in accordance with the usual methods. A preferable dosage form is a liquid preparation for oral administration or injection.

[0084] Such a liquid preparation is formulated by dissolving the .sup.211At-labeled amino acid derivative compound of the present invention in water. However, if necessary, it may be dissolved in a physiological saline solution or a glucose solution, or a buffer or a preservative may be added to it. Further, as described above, the reducing agent such as ascorbic acid can be further included. Particularly, to manufacture an injection, the active ingredient may be dissolved in distilled water for injection together with hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium hydrogen phosphate, sodium dihydrogen phosphate, or another pH adjuster, and sodium chloride, glucose, or another isotonizing agent as needed, and sterile-filtered and filled into an ampoule, or mannitol, dextrin, cyclodextrin, gelatin, or the like may be further added and the product vacuum freeze-dried to obtain an injection to be dissolved at the time of use thereof. Lecithin, polysorbate 80, polyoxyethylene hardened castor oil, or the like may also be added to the active ingredient and emulsified in water to obtain an emulsion for injection.

[0085] The types of additives for preparation used in manufacturing the pharmaceutical composition, the ratios of additives for preparation with respect to the active ingredient, or the method for producing the pharmaceutical composition can appropriately be selected by a person skilled in the art in accordance with the form of the composition. Inorganic or organic substances, or solid or liquid substances may be used as additives for preparation, and additives for preparation may generally be blended in the range of 1 wt % to 90 wt % with respect to the weight of the active ingredient.

[0086] The dosage and number of doses of the pharmaceutical composition of the present invention are not particularly limited, and can appropriately be selected by the judgment of a physician in accordance with the purpose for preventing and/or treating the worsening or progress of the disease to be treated, the type of disease, the body weight or age of the patient, the degree of serious ness of the disease, and other conditions.

[0087] In some cases, the pharmaceutical composition can be provided in a form of a kit including the .sup.211At-labeled amino acid derivative compound of the present invention and the reducing agent such as ascorbic acid or an ascorbic acid salt. In such a case, the pharmaceutical composition is used as an aqueous solution in which the compound and the reducing agent such as ascorbic acid are mixed at the time of use.

[0088] 4. Production Method of .sup.211At-Labeled Amino Acid Derivative

[0089] In another embodiment, the present invention also relates to a method for producing the .sup.211At-labeled amino acid derivative. More specifically, the production method of the present invention is a method for producing the compound represented by the above formula (I) and characterized by including the following steps A) to C).

[0090] Step A):

[0091] A step for adding a mercury compound to a compound represented by the following formula (Ia) to obtain a precursor compound represented by the following formula (Ib-1) or formula (Ib-2).

##STR00009##

[0092] in which L, R.sup.1, R.sup.2, and R.sup.3 have the same definition as in the above formula (I);

##STR00010##

[0093] in which L, R.sup.1, R.sup.2, and R.sup.3 have the same definition as in the above formula (I).

[0094] The step A) is a step in which a mercury compound is added to an amino acid derivative of the formula (Ia) serving as a starting substance not including .sup.211At to obtain a precursor in which an aromatic hydrocarbon-mercury bond is formed. In this step, the mercury compound is not particularly limited as long as including a mercury ion (Hg.sup.2+). For example, HgSO.sub.4 or Hg(OAc).sub.2 can be used. The step A) is preferably performed under an acidic condition by adding sulfuric acid or the like. Further, the precursor compound represented by the formula (Ib-1) or the formula (Ib-2) can be selectively produced by changing the amount of the mercury compound used in the step A). More specifically, the precursor compound of the formula (Ib-2) in which mercury ions (Hg) are introduced in two positions on the benzene ring can be produced by increasing the mole ratio of the mercury compound with respect to the compound represented by the formula (Ia) serving as a raw substance, typically by performing a reaction under the condition in which the mole ratio [mercury compound/compound represented by formula (Ia)] is 1 or greater. Note that both the precursor compounds of the formula (Ib-1) and the formula (Ib-2) can be produced under some condition. However, such a step is also permitted as long as the compound corresponding to the formula (I) is produced via the following steps B) and C).

[0095] Step B):

[0096] The step B) is a step for adding .sup.211At and a halide constituted by a halogen element other than astatine to the precursor compound represented by the formula (Ib-1) or the formula (Ib-2) obtained in the step A). This reaction is performed to introduce .sup.211At in replacement of Hg.sup.+ in the formula (Ib) by using a halide ion as a carrier.

[0097] .sup.211At used in the step B) can be obtained from bismuth (Bi) by using an accelerator (cyclotron). More specifically, .sup.211At is produced by a nuclear reaction of .sup.209Bi(α, 2n) .sup.211At in which bismuth is irradiated with a helium particle accelerated by 28 MeV or more using cyclotron. .sup.211At is captured and dissolved in water or an organic solvent to obtain a raw liquid of .sup.211At, which can be used for the reaction in the step B).

[0098] The halide used in the step B) is preferably a triiodide. An alkali metal triiodide salt such as KI.sub.3 can be more preferably used. Further, after addition of the triiodide, the corresponding monoiodide can be further added. For example, .sup.211At and KI.sub.3 are added in the same time and then KI can be further added after .sup.211At and KI.sub.3 are stirred for a predetermined time.

[0099] Step C):

[0100] The step C) is a step for purifying the reaction product obtained in the above step B) to obtain the compound represented by the above formula (I). This step is for removing a byproduct other than the desired compound represented by the formula (I) from the product in the step B).

[0101] Purification in the step C) is preferably performed by column chromatography using an ion exchange resin. For example, the desired product can be isolated and purified by performing cation exchange column chromatography and then performing anion exchange column chromatography. Note that, as the ion exchange resin, an ion exchange resin conventionally used in the related art can be used

[0102] Further, in a preferred embodiment, a step for adding ascorbic acid or an ascorbic acid salt can be further included after the purification in the step C). This reduces degradation of the purified compound of the formula (I), making it possible to store the compound for a long period of time.

[0103] Regarding reaction conditions such as a solvent and a reaction temperature in each step of the production method of the present invention described above, a representative example thereof will be described in detail in the following Examples. However, the reaction conditions are not necessarily limited thereto, and a person skilled in the art could appropriately select the reaction conditions on the basis of knowledge common in the field of organic synthesis.

EXAMPLES

[0104] Below, the present invention will be described in further detail using Examples. However, the present invention is not limited to these Examples.

Example 1

[0105] 1-1. Synthesis of .sup.211at-Labeled Amino Acid Derivative compound (.sup.211At-AAMT) [0106] (1) Preparation of .sup.211at Aqueous Solution

[0107] A target substance Bismuth was irradiated with a helium particle (28 MeV, 1 to 2 μA) accelerated by cyclotron to cause a nuclear reaction of .sup.209Bi(α, 2n).sup.211At, thereby producing .sup.211At in the target substance. The target substance including .sup.211At was heated to 850° C. in an electric heater to be melted. The transpired .sup.211At was dissolved in a small amount of water (0.1 to 2 mL) to prepare a .sup.211At aqueous solution having a radioactivity concentration of about 5 MBq/ml.

[0108] (2).sup.211at Labeling Reaction

[0109] α-methyl-L-tyrosine (22 μmol) and HgSO.sub.4 (20 μmol) were added to 0.5 mL of a H.sub.2SO.sub.4 aqueous solution (0.2 M), and the resulting mixture was stirred at room temperature for 2 hours. NaCl (45 μmol) was added to the reaction solution and the reaction solution was stirred for 5 minutes. To the reaction mixture, 100 μL of the .sup.211At aqueous solution (about 5 MBq/ml) obtained in the above (1) and 5 μL of 1M KI.sub.3 were added and the reaction solution was stirred for 30 minutes. Subsequently, an appropriate amount of KI was added to the reaction mixture until the suspension solution became transparent, thereby terminating the reaction. The reaction solution thus obtained was passed through a cation column (Dowex™ 50 W×8, 100-200 mesh, 0.2 M NH.sub.3 aqueous solution) and then an anion column (Dowex™ 1×8, 50-100 mesh, 2% AcOH aqueous solution) to remove salts, thereby obtaining the following compound (.sup.211At-AAMT). The yield was from 60 to 80%.

##STR00011##

[0110] 1-2. Synthesis of .sup.211at-Labeled Amino Acid Derivative compound (.sup.211At-AIAMT)

[0111] (1) Preparation of .sup.211at Aqueous Solution

[0112] The .sup.211At aqueous solution having a radioactivity concentration of about 5 MBq/ml was prepared by the same procedure as described above.

[0113] (2).sup.211at Labeling Reaction

[0114] α-methyl-L-tyrosine (22 μmol) and HgSO.sub.4 (100 μmol) were added to 0.5 mL of a H.sub.2SO.sub.4 aqueous solution (0.2 M), and the resulting mixture was stirred at room temperature for 2 hours. NaCl (45 μmol) was added to the reaction solution and the reaction solution was stirred for 5 minutes. To the reaction mixture, 100 μL of the .sup.211At aqueous solution (about 5 MBq/ml) obtained in the above (1) and 5 μL of 1M KI.sub.3 were added and the reaction solution was stirred for 30 minutes. Subsequently, an appropriate amount of KI was added to the reaction mixture until the suspension solution became transparent, thereby terminating the reaction. The reaction solution thus obtained was passed through a cation column (Dowex™ 50 W×8, 100-200 mesh, 0.2 M NH.sub.3 aqueous solution) and then an anion column (Dowex™ 1×8, 50-100 mesh, 2% AcOH aqueous solution) to remove salts, thereby obtaining the following compound (.sup.211At-AIAMT). The yield was from 24.5 to 41.5%.

##STR00012##

Example 2

[0115] 2. Stability of .sup.211at-Labeled Amino Acid Derivative compound (.sup.211At-AAmT) [0116] (1) In Vitro Stability

[0117] The stability of .sup.211At-AAMT synthesized in Example 1 was compared in the presence and absence of sodium ascorbate using thin-layer chromatography (TLC). The result is shown in FIG. 1.

[0118] As a result, it was found that, in the absence of sodium ascorbate, almost all of .sup.211At-AAMT was degraded in about 40 minutes, while, in the presence of sodium ascorbate, .sup.211At-AAMT could stably exist even after the lapse of seven or more hours. This shows that the presence of ascorbic acid, which is a reducing agent, can stabilize .sup.211At-AAMT by reducing dissociation of aromatic carbon-211At.

[0119] (2) In Vivo (Inside of Mouse Living Body) Stability

[0120] .sup.211At-AAMT to which ascorbic acid was added at a final concentration of 1% was intravenously injected in normal rats, and, after one hour, blood and urine were collected and analyzed by thin-layer chromatography (TLC). As a thin layer plate, silica gel G60F254 (Merck KGaA) was used, and, as a development solvent, a mixture of butanol: water: acetic acid (4:1:1) was used (Nacalai Tesque, Inc.). The thin layer plate after development was analyzed by using a bioimaging analysis apparatus (Typhoon 7000, GE Healthcare) (FIG. 2). As a result, only .sup.211At-AAMT was detected in both the blood and the urine, but free .sup.211At was not observed. That is, it was shown that .sup.211At-AAMT to which ascorbic acid was added could stably exist in the living body without being metabolized or degraded.

[0121] (3) Effect of Addition of Ascorbic Acid on Biodistribution of .sup.211at-AAMT

[0122] Next, an effect of ascorbic acid on biodistribution of .sup.211At-AAMT in the mouse body was compared. ddY mice (male, 5 weeks old) purchased from Japan SLC, Inc. were used after being acclimated for one week (6 weeks old at the time of experiment). .sup.211At-AAMT with 1% ascorbic acid or without ascorbic acid was intravenously injected in normal mice (N=3), and the mice were dissected after one hour. The organs were all put in polyethylene bags with zipper (A-4, 0.04 mm, SEISANNIPPONSHA Ltd.), and radioactivity of the organs was measured using Wizard.sup.2 gamma counter (PerkinElmer). The result of .sup.211At distribution in each major organ is shown in FIG. 3. As a result, it was confirmed that accumulation in the stomach and the kidney was significantly reduced in the 1% ascorbic acid addition group as compared with the ascorbic acid free group. This result shows that adding ascorbic acid has an effect of causing .sup.211At-AAMT that has been administered to the living body to stably exist in the body.

Example 3

[0123] 3. Specific Accumulation of .sup.211At-AAMT in Cancer cells (in vitro experiment)

[0124] A human pancreatic cancer cell line (PANC-1) was treated with .sup.211At-AAMT and cultured for a predetermined time. Then, the cells were washed to remove unincorporated .sup.211At-AAMT. The cellular uptake of .sup.211At-AAMT was measured from the radiation dose remaining in the cells. The result thereof is shown in FIG. 4.

[0125] As a result, .sup.211At-AAMT was taken up immediately after addition of .sup.211At-AAMT, and the uptake of .sup.211At-AAMT reached the maximum value in about 10 minutes. Further, the uptake of .sup.211At-AAMT was reduced by competition with a LAT1 inhibitor, BCH (2-aminobicyclo[2.2.1]heptane-2-carboxylic acid) or methyltyrosine performed in Comparative example. From this result, it was found that .sup.211At-AAMT was specifically accumulated in the cancer cells via LAT1.

[0126] Further, an effect of ascorbic acid on the cellular accumulation of .sup.211At-AAMT was also examined. The measurement result of the radiation dose in the human pancreatic cancer cell line in the absence and presence of ascorbic acid is shown in FIG. 5. As a result, it was found that accumulation of .sup.211At-AAMT in the pancreatic cancer cell line (PANC-1) was significantly increased by addition of ascorbic acid (0.1%).

[0127] Considering this result and the result of Example 2(3) together, addition of ascorbic acid increased accumulation of .sup.211At-AAMT in the tumor cells, while addition of ascorbic acid rather reduced accumulation of .sup.211At-AAMT in normal cells having no tumor, thus it was found that addition of ascorbic acid caused an effect of facilitating clearance of .sup.211At-AAMT in organs other than the target organ.

Example 4

[0128] 4. Examination of Cell-Killing Effect of .sup.211at-AAMT

[0129] A cell survival rate of the human pancreatic cancer cell line (PANC-1) administered with .sup.211At-AAMT under the same conditions as in Example 3 is shown in FIG. 6. As a result, it was confirmed that nearly all of the cancer cells could be killed by .sup.211At-AAMT. On the other hand, the cellular uptake of .sup.211At-AAMT was reduced in the presence of the inhibitor BCH or methyltyrosine. As a result, a significant reduction in the cell survival rate was not observed.

[0130] Similarly, an effect of ascorbic acid on the cell-killing effect was compared, and the result thereof is shown in FIG. 7. As a result, it was found that the cancer cell-killing activity of .sup.211At-AAMT was increased by addition of ascorbic acid. This is because the cellular uptake of .sup.211At-AAMT in the cancer cells increased about four times by addition of ascorbic acid.

Example 5

[0131] 5. Administration of .sup.211at-AAMT to Cancer-Bearing mouse

[0132] A cancer-bearing mouse created by using the human pancreatic cancer cell line (PANC-1) was intravenously administered with .sup.211At-AAMT at a dose of 0.6 MBq per mouse. A change in the tumor size caused by the administration is shown in FIG. 8(A), while a change in the body weight of the cancer-bearing mouse is shown in FIG. 8(B).

[0133] As shown in FIG. 8(A), a regression effect of the tumor was obtained 7 days after the administration. On the other hand, as shown in FIG. 8(B), a substantial reduction of the body weight was not observed 7 days after the administration. This showed that .sup.211At-AAMT could provide the tumor regression effect without causing significant side effects.

[0134] Note that, as a result of performing image analysis of in vivo localization of .sup.211At-AAMT administered to the normal rat using a gamma camera, accumulation of the radioactivity was observed in the kidney and its surrounding organs, confirming quick excretion of .sup.211At-AAMT by the kidney.

Example 6

[0135] 6. Administration of .sup.211at-AAMT to Pancreatic Cancer mouse

[0136] Balb/c-nu/nu mice (female, 5 weeks old) purchased from Japan SLC, Inc. were acclimated in a breeding space for one week. A human pancreas cell line PANC-1 (RIKEN cell bank), which was maintained in an RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics (100×, FUJIFILM Wako Pure Chemical Corp.), was adjusted to a concentration of 5×10.sup.6 cells/100 μL. Subsequently, the cells were mixed with Matrigel (Growth Factor Reduced, BD) in a 1:1 ratio, and the mixture was transplanted to a subcutaneous region of the hinder back of a mouse using a syringe (FN syringe 27G, Terumo Corp.). Subsequently, after one-month follow-up observation, the mouse having a sufficiently large tumor size was used in the experiment. These pancreatic cancer transplanted mice (animal 1:25.5 g, animal 2:30.2 g) were intravenously injected with .sup.211At-AAMT with 1% ascorbic acid, at a dose of 1 MBq each), and SPECT imaging was performed after 10 minutes. Imaging was performed using a gamma camera E-Cam (Siemens) (imaging time: 10 minutes, collimator: low energy all purpose, pixel size: 1.2 mm×1.2 mm, matrix: 256×256, zoom: 2-fold, energy window: 79±20%). The obtained result is shown in FIG. 9.

[0137] As a result, the tumors were clearly visualized in both cases. On the other hand, the thyroid was not visualized, showing that was unlikely that free .sup.211At was produced in the living body under the present conditions. That is, it was found that .sup.211At-AAMT added with ascorbic acid was highly accumulated in the pancreatic cancer and stable in the living body.

Example 7

[0138] 7. SPECT Imaging in Normal Rat

[0139] Normal rats (Wister, 3 months old, male, 3 animals, body weight of 289.8±5.5 g) were intravenously injected with .sup.211At-AAMT (without ascorbic acid, 1 MBq), and SPECT imaging was performed after from 20 minutes to 1 hour. Imaging was performed using a gamma camera E-Cam (Siemens) (imaging time: 20 minutes, collimator: low energy all purpose, pixel size: 2.4 mm×2.4 mm×2.4 mm (SPECT), matrix: 128×128). The obtained result is shown in FIG. 10.

[0140] As a result, accumulation of the radioactivity was observed in the thyroid and the stomach in all animals, suggesting that, in the absence of ascorbic acid, .sup.211At-AAMT was metabolized in the living body to produce free .sup.211At, which was then accumulated in the thyroid and the stomach.

Example 8

[0141] 8. Examination of Treatment Effect of .sup.211at-AAMT on Melanoma Metastasis to Lung

[0142] C57BL/6 mice (female, 5 weeks old) purchased from Japan SLC, Inc. were used in the experiment after being acclimated in a breeding space for one week. Melanoma cells B16F10 (a high lung-metastatic cell line cloned from B16, R.sup.1KEN cell bank) were maintained in a D-MEM medium (high glucose, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics (100×, FUJIFILM Wako Pure Chemical Corp.). These cells in an amount of 2×10.sup.5 cells were intravenously transplanted in each mouse. On the following day, the mice (N=5) was intravenously injected with .sup.211At-AAMT (with 1% ascorbic acid) at a dose of 0.0 MBq (control group), 0.1 MBq, or 1 MBq. Fourteen days later, the mice were dissected, and the number of nodules spread to the lung was visually counted. The results of the number of the nodules and a change in the body weight thus obtained were shown in FIG. 11 and FIG. 12, respectively.

[0143] As a result, it was found that the number of the melanoma nodules in the lung decreased more in the group with more doses of .sup.211At (FIG. 11). Further, during this period, there was no difference in the body weight change between the .sup.211At-AAMT group and the control group (FIG. 12). These results show that .sup.211At-AAMT (with 1% ascorbic acid) is effective in the melanoma metastasis treatment in a dose dependent manner.