JP4318985B2 - Somatostatin analog derivatives and uses thereof - Google Patents

Description

  The present invention relates to a somatostatin analog derivative, a complex of the derivative and a metal, and a pharmaceutical composition containing the same, which are useful for diagnostic imaging and treatment using a metal.

（式中、Ａ’はPheまたはTyrを表す）
で示されるソマトスタチンアナログのＮ末端に1,4,7,10-テトラ−アザシクロドデカン−１,4,7,10−4酢酸（1,4,7,10-テトラ−アザシクロドデカン−１,4,7,10-tetraacetic acid、以下、「ＤＯＴＡ」と略称）と放射性核種との錯体を含有するソマトスタチン受容体陽性癌および転移癌の治療のための医薬組成物が開示されている。上記の式でＡ’がPheであるオクトレオチド（Octreotide)とジエチレントリアミン５酢酸（Diethylenetriamine pentaaceticAcid、以下「ＤＴＰＡ」と略称）との複合体が¹¹¹Inに配位している、¹¹¹In-DTPA-Phe¹-octreotideは、既に欧米で臨床使用されている（非特許文献１、非特許文献２）。
また、特許文献５には、式：

で示される化合物のＣ末端またはＮ末端にキレート剤（ＤＯＴＡまたはＴＥＴＡ）が結合してなる化合物と金属との錯体の、腫瘍組織の破壊、診断における使用について開示されている。 Somatostatin is a peptide consisting of 14 amino acids synthesized in the hypothalamus and pancreas of mammals including humans, and is expressed on the cell surface constituting a wide range of tissues such as the central nervous system, spinal cord, pancreas, and gastrointestinal tract. It binds to a specific receptor and exerts its physiological activity. Since most endocrine tumors in these tissues have a high affinity somatostatin binding site, somatostatin receptor positive tumors can be diagnosed and treated based on the high expression of somatostatin receptor. Conventionally, peptides (somatostatin analogs) that bind to various somatostatin receptors have been reported (for example, Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4) and Patent Document 5). A therapeutic / diagnostic drug in which a compound of such a peptide and a chelator is labeled with a metal is also known.

(In the formula, A ′ represents Phe or Tyr)
1,4,7,10-tetra-azacyclododecane-1,4,7,10-4 acetic acid (1,4,7,10-tetra-azacyclododecane-1, A pharmaceutical composition for the treatment of somatostatin receptor positive cancer and metastatic cancer containing a complex of 4,7,10-tetraacetic acid (hereinafter abbreviated as “DOTA”) and a radionuclide is disclosed. Complex of the above octreotide A 'is Phe by formula (octreotide) and diethylenetriaminepentaacetic acid (Diethylenetriamine pentaaceticAcid, hereinafter abbreviated as "DTPA") is coordinated to ^{111 In, 111 In-DTPA-} Phe 1 -octreotide has already been clinically used in the West (Non-patent Document 1, Non-patent Document 2).
Patent Document 5 discloses a formula:

And the use of a complex of a compound obtained by binding a chelating agent (DOTA or TETA) to the C-terminus or N-terminus of a compound represented by formula (II) with a metal in tumor tissue destruction or diagnosis.

  Thus, compounds belonging to various somatostatin analogs can be used as a complex with a chelator, such as tumor cells and endocrine cells that express therapeutic and / or diagnostic metals such as radionuclides with high density of somatostatin receptors. Since it can reach the system selectively, it is useful as a metal carrier, and its development and function have been studied.

Conventional therapeutic and diagnostic substances containing somatostatin analogs cross-react with not only somatostatin-binding receptors on the surface of target cells such as tumor cells, but also receptors expressed on normal cells. Problems with the above safety have been pointed out. For example, the above-mentioned tumor scintigraphy using ¹¹¹ In-DTPA-Phe ¹ -octreotide shows specific tumors (gastrinoma, carcinoid, insulinoma, etc.) positively with high sensitivity and non-invasive whole body search It is said that the immunogenicity is low, the blood clearance and the tissue transferability are excellent, and an early diagnosis after administration is made possible to reduce the exposure of the subject (Non-patent Document 3). . However, it is known to show non-specific accumulation and retention of radioactivity in the kidney, and there are problems such as reduced diagnostic accuracy and side effects in treatment. Means for solving such problems have been studied, and there are reports that a method using lysine or arginine in combination (Patent Document 2) and a method using lysine in combination (Non-Patent Document 4) are useful. In addition, there is a report that if the N-terminal amino acid is substituted with an amino acid having low fat solubility, radioactivity accumulation in the kidney can be reduced (Non-patent Document 5). However, these documents do not disclose carrier materials for therapeutic and / or therapeutic metals, including somatostatin analogs with low renal accumulation that can actually be applied clinically. Under such circumstances, development of clinically useful substances containing somatostatin analogs that can be used as highly accurate diagnostic agents and highly safe therapeutic agents has been demanded.

British Patent Application No. 2225579 Japanese Patent No. 3054346 Japanese Patent No. 3117218 Japanese Patent No. 3288055 Special table 2001-515494 Krenning, E.P., et al., Eur.J.Nucl.Med., 20, 716-731 (1993) Kwekkeboom, D. et al., J. Nucl. Med., 41, 1704-1713 (2000) Krenning, E.P., et al., J. Nucl. Med., 33, 652-658 (1992) Bert F. et al., J. Nucl. Med., 38, 1929-1933 (1997)

The present invention specifically delivers a metal for diagnosis or treatment of a tumor or the like expressing a somatostatin-binding receptor at a high density to the target site while excretion with minimal accumulation in the kidney. It is an object to provide a carrier material that can be allowed to flow.
Another object of the present invention is to provide a pharmaceutical composition for diagnosis and treatment of the above carrier substance and metal complex, and a somatostatin receptor positive tumor containing the same.
In particular, an object of the present invention is to improve renal accumulation of a substance containing a somatostatin analog moiety useful for diagnosis and / or treatment of a somatostatin positive tumor such as octreotide.

（式中、Ｒはキレーター残基、Ｘは１つ以上の遊離のカルボキシル基またはアミノ基を有するフェニル基またはナフチル基、ＹはThr-OH、Thr‐NH₂及びTrp‐NH₂から選択される基、ＡはPheまたはTyr、ＢはThrまたはValを表す）
で示される、診断及び／または治療用の金属をソマトスタチン結合性受容体を発現している標的細胞に送達させるためのキャリア物質を提供するものである。 The present inventors have found that renal accumulation is significantly reduced when the N-terminal phenylalanine of ¹¹¹ In-DTPA-Phe ¹ -octreotide is modified, and the present invention has been completed.
That is, the present invention relates to the formula (I):

Wherein R is a chelator residue, X is a phenyl or naphthyl group having one or more free carboxyl groups or amino groups, and Y is selected from Thr-OH, Thr-NH ₂ and Trp-NH ₂ Group, A represents Phe or Tyr, B represents Thr or Val)
A carrier substance for delivering a diagnostic and / or therapeutic metal represented by the above to a target cell expressing a somatostatin-binding receptor is provided.

  In the context of the present invention, “target cell expressing a somatostatin-binding receptor” means a cell expressing somatostatin receptor at a high density in a different manner from normal cells, specifically, somatostatin Receptor positive tumor cells and cells expressing somatostatin receptors in high density in connection with endocrine disease are included.

  In the formula (I), examples of the chelator residue represented by R include the aforementioned DTPA, DOTA and 1,4,7,10-tetraazacyclododecane-1-aminoethylcarbamoylmethyl-4,7,10-tris [ (R, S) methylacetic acid] (1,4,7,10-Tetraazacyclododecane-1-aminoethylcarbamoylmethyl-4,7,10-tris [(R, S) -methylacetic acid, hereinafter abbreviated as “DO3MA”) A group derived from a ligand is preferably used. Particularly preferred ligands are DTPA and DOTA.

The part other than the group R in the formula (I) is a somatostatin receptor-binding derivative of a somatostatin analog, and cyclo [Cys-A-] is present on the C-terminal side of N-terminal carboxylated or aminated phenylalanine or naphthylalanine. A cyclic peptide represented by (D-Trp) -Lys-B-Cys] -Y is bound. Preferred combinations of amino acids constituting this cyclic peptide moiety are as follows.
(1) A is Phe, B is Thr, Y is Thr (ol)
(2) A is Tyr, B is Thr, Y is Thr (ol)
(3) A is Tyr, B is Val, Y is Trp-NH ₂
(4) A is Tyr, B is Val, Y is Thr-NH ₂
(5) A is Tyr, B is Val, Y is Thr-OH
The combination of the N-terminal amino acid (carboxylated / aminated Phe ¹ or Na ¹ ) with the cyclic peptide described above gives derivatives of known somatostatin analogues.
That X is a carboxy or amino phenyl, C is the case terminal peptide of (1) octreotide (octreotide), in the case of (2) Tyr ³ - Octreotide (Tyr ³ -octreotide), in the case of (3) corresponding to a derivative of octreotate (Tyr ³ -octreotate) - vapreotide (vapreotide), in the case of (5) Tyr ^3. When X is carboxy or aminonaphthyl and the C-terminal peptide is (4), it corresponds to a lanreotide derivative. Therefore, when X is phenyl having a carboxy or amino substituent, A, B, Y are any combination of (1), (2), (3) and (5), and X is carboxy or amino substituted In the case of a naphthyl having a group, A, B and Y are preferably a combination of (4), in particular X is phenyl having a carboxy or amino substituent, and A, B and Y are those of (1) A combination is preferred.
In the present specification, unless otherwise specified, the amino acid may be either D-form or L-form.

As described in Examples below, the present inventors have the formula (I) wherein R is DTPA, X is carboxyphenyl or aminophenyl, A is Phe, B is Thr, and Y is Thr (ol). A compound of the present invention (amino form, carboxy form) and a compound (methyl form) that differs only in that X is methylphenyl were synthesized, and these were labeled with ¹¹¹ In to produce the following three types of complexes. The pharmacokinetics was examined. As a control, a compound in which X is Phe (Phe body) was used.

The test substance was intravenously administered to mice, and the pharmacokinetics was examined by measuring the radioactivity (count) in blood, tissue, and urine after a certain period of time. In all cases, the radioactivity in blood was administered 3 Almost disappeared by time, and a rapid clearance from blood was observed.
Further, in all cases, higher radioactivity accumulation was observed in the kidney than in other organs, but the accumulation amount and its mode were considerably different in the amino and carboxy forms, and in the methyl and Phe forms. In particular, in the case of the amino form, it was almost the same as the Phe form 10 minutes after administration, but the renal radioactivity accumulation was significantly reduced 30 minutes after administration. In the case of the carboxy form, the kidney radioactivity accumulation was lower than that of the Phe form over the entire period tested. In particular, it was significantly lower from 30 minutes to 6 hours after administration. On the other hand, in the case of the methyl body, improvement in renal accumulation was not observed as compared with the Phe body. In the image diagnosis using metal, diagnosis is generally performed within several tens of minutes to 3 days after administration, and the above results indicate that the carboxy form or amino form can improve the accuracy of diagnosis. This result also indicates that these substances are highly safe therapeutics. These compounds correspond to octreotide and its derivatives, but it is known that the pharmacokinetics of octreotide D-Phe and L-Phe are almost unchanged ("Renal Metabolism of ¹¹¹ In-DTPA-D-Phe ^1- octreotide in vivo. "Biocojugate Chem. 9 (6), 662-670, 1998).

  The carrier substance of the present invention having the above characteristics has a function of delivering a metal for treatment or diagnosis to a somatostatin receptor-positive tumor cell or the like expressing somatostatin receptor at a high density, and a metal kidney. It can be excreted while suppressing accumulation and retention. Therefore, the complex of the carrier substance and metal of the present invention is useful as an active ingredient of a pharmaceutical composition for diagnosis and / or treatment. The present invention also provides such a pharmaceutical composition.

  The metal can be arbitrarily selected from known paramagnetic and radioactive metals coordinated to the chelator in the above formula (I) and used for diagnosis and / or treatment. Examples of radioactive metals include Tc-99m, In-111, Cu-62, Ga-67, Ga-68, Re-186, Re-188, Y-90, Cu-64, Sn-117m and Sm-153. Examples of paramagnetic metals include Gd, Dy, Fe and Mn.

  The complex can be appropriately prepared according to a conventional method according to the metal used and the type of chelator residue of the carrier substance. Usually, a complex is formed by mixing a solution of the carrier substance of the present invention and a solution of a compound containing metal ions. Examples of the compound containing a metal ion include, but are not limited to, a metal chloride and a metal bromide. Moreover, as a solvent, although polar solvents, such as water, an acid, a base, and DMF, or a mixture thereof can be used, it is not limited to these. However, there is a preferred method in each case.For example, in the case of technetium-99m (Tc-99m), a reducing agent such as stannous chloride is added to the carrier material and mixed with a sodium pertechnetate solution. It can be suitably prepared. In the case of In-111, it is preferable to mix a carrier substance solution and an acidic aqueous solution containing In-111 ions. If necessary, unreacted metal ions may be removed by HPLC or the like.

  The pharmaceutical composition of the present invention specifically accumulates and stays in somatostatin receptor-positive tumor cells expressing somatostatin receptor at high density and cells suffering from endocrine diseases, while being rapidly excreted from the kidney. Therefore, the accuracy in diagnosis is extremely high. In addition, since the nephrotoxicity of metals can be reduced, it is extremely safe during treatment.

When the pharmaceutical composition of the present invention is a diagnostic agent for image diagnosis of a tumor site in a living body by a nuclear medicine technique, the metals include Tc-99m, In-111, Cu-62, Ga-67 and Ga-68. A radioactive metal selected from is preferred.
Further, when the pharmaceutical composition of the present invention is used for radiotherapy for destroying a somatostatin receptor positive tumor, Re-186, Re-188, Y-90, Cu-64, Sn-117m and Sm are used as metals. A radioactive metal selected from -153 is preferred.
Furthermore, when the pharmaceutical composition of the present invention is a diagnostic agent for diagnosing an MRI image of a tumor site in a living body, a paramagnetic metal selected from Gd, Dy, Fe and Mn is preferable.

  The pharmaceutical composition can be prepared according to a conventional method from the metal complex of the present invention and a pharmaceutically acceptable carrier or diluent. If necessary, other additives such as pH adjusters such as pharmaceutically acceptable aqueous buffers, excipients such as D-mannitol and citric acid, tartaric acid which help to improve the radiochemical purity , Malonic acid and the like may be contained. The pharmaceutical composition of the present invention may be in the form of a composition for preparation at the time of use (kit) in which the metal ion solution and the carrier substance are housed in separate containers. The kit may contain usual additives such as pharmaceutically acceptable salts, buffers, preservatives and the like for adjusting osmotic pressure. The components of the kit can be provided in liquid or lyophilized form.

When the pharmaceutical composition of the present invention is a radiodiagnostic agent for image diagnosis by a nuclear medicine technique, such a radiodiagnostic agent is effective immediately after parenteral administration such as intravenous injection by bolus administration. To target sites such as tumors, achieve rapid distribution of radioactive material, provide a target site / background ratio sufficient for imaging approximately 1 hour after administration, and suitable for imaging imaging It has a favorable characteristic that it accumulates at the target site for a long time and is then rapidly excreted from the kidney into the urine. The diagnosis can be performed using a normal radiation imaging apparatus.
The radiodiagnostic agent of the present invention can be administered by generally used parenteral means such as intravenous injection by bolus administration, and the dosage is based on the patient's weight, age and various conditions such as an appropriate radiation imaging apparatus, The amount of radioactivity that can be considered to be imaged is determined. When targeting humans, a range of 37 to 1,110 MBq is usually preferable.

  When the pharmaceutical composition of the present invention is a radiotherapeutic agent for destroying somatostatin receptor positive tumor (cancer), the therapeutic agent is parenterally, particularly intravenously, for example in an injectable solution or suspension. It can be administered in the form. Depending on the site to be treated, such as a tumor, it may be administered as close as possible, for example by a catheter. Administration can be continuous or repeated. The dosage is determined by a doctor in consideration of various conditions such as the patient's weight, age, and symptoms, but in the case of human subjects, it is usually in the range of 37 to 11,100 MBq.

The therapeutic agent of the present invention can specifically accumulate in somatostatin receptor-positive cancer without accumulating and staying in the kidney, destroying cancer cells, and exhibiting a therapeutic effect. Therefore, it can be applied to patients with renal disorders that have been difficult to treat due to the fear of nephrotoxicity, and can be administered in a larger amount depending on the case. it can.
Examples of cancers that can be treated with the therapeutic agents of the present invention include pituitary, gastrointestinal pancreatic cancer, central nervous system, breast, prostate, ovarian or colon cancer, small cell lung cancer, paraganglioma, kidney cancer, skin cancer, neuroblastoma Pheochromocytoma, medullary thyroid cancer, myeloma, lymphoma, Hodgkin and non-Hodgkin's disease, bone cancer and their metastatic cancer.
The radiotherapeutic agents of the present invention can also be used to treat autoimmune or inflammatory diseases that express somatostatin receptors, such as rheumatoid arthritis.

When the pharmaceutical composition of the present invention is a diagnostic agent for MRI imaging of a tumor site in a living body, the metal in the metal complex of the present invention can be obtained by, for example, literature (Weissleder, R., “Scaling Down Imaging: Molecular Mapping of Cancer in Mice ", Nature Reviews Cancer 2002; 2: 11-18) and various paramagnetic or ferromagnetic metals such as Gd, Mn, Dy, iron oxide by methods known to those skilled in the art. Replace with etc. The MRI diagnostic agent thus prepared can be administered by a commonly used means such as intravenous administration or local administration, and the dosage depends on the type of metal used, the degree of relaxation of the MRI diagnostic agent, and other physicochemicals. In consideration of various conditions such as properties, patient weight, configuration of the MRI imaging apparatus, and MRI imaging method, it is determined within a range where imaging is possible. For example, when intravenously administered to humans and the metal species used is Gd, the range of 0.01 to 0.3 mmol / kg per body weight is usually preferred.
The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention.

1. In the following examples and experimental examples, octreotide derivatives are synthesized by introducing substituents (amino group, carboxy group, methyl group) having almost the same molecular size and different charges into the N-terminal amino acid Phe of octreotide, and DTPA as a chelator A metal complex with In-111, ie ¹¹¹ In-DTPA-p-amino-Phe ¹ -octreotide (amino), ¹¹¹ In-DTPA-p-carboxy-Phe ¹ -octreotide (carboxy), or ¹¹¹ In-DTPA-p-methyl-Phe ¹ -octreotide (methyl) was prepared, and the radioactivity dynamics in the body was examined.

2. Experimental Method 2-1: Reagents Reagents used in the following examples are as follows.
Diethylenetriamine: First grade Tokyo Chemical Industry Co., Ltd. Ethyl trifluoroacetate: First grade Tokyo Chemical Industry Co., Ltd.
N, N-Diisopropylethylamine (DIPEA): First grade Tokyo Chemical Industry Co., Ltd. Silica Gel 60 H: MERCK Wakogel C-200: Wako Pure Chemical Industries, Ltd. Bromoacetic acid tert-Butyl: First grade Tokyo Chemical Industry Co., Ltd. Sodium hydride: Special grade Ishizu Pharmaceutical Co., Ltd. Hydrazine anhydride: First grade Tokyo Chemical Industry Co., Ltd. Benzyl bromoacetate: First grade Tokyo Chemical Industry Co., Ltd. Palladium / carbon (Pd / C): First grade Nakarai Tesque Co., Ltd. 9-Fluoromethoxy carbonyl -Ot- butyl -L- threonine ^{(Fmoc-Thr (Bu t)} -OH): Corporation peptide Institute, ethylene glycol dimethyl ether: special grade Tokyo Chemical industry Co., Ltd.
N-methylmorpholine: Special grade Nakarai Tesque Co., Ltd. Isobutyl chloroformate: Special grade Nacalai Tesque Co., Ltd.
2-chlorotrityl chloride resin: NOVA biochem company Dehydrated pyridine: Kanto Chemical Co., Ltd. Piperidine: Special grade Nakarai Tesque Co., Ltd.
1,3-diisopropylcarbodiimide (DIPCDI): Grade 1 Sigma Aldrich Chem. Co.
1-hydroxybenzotriazole (HOBt ・ H ₂ O): 1st grade Tokyo Chemical Industry Co., Ltd.
9-Fluorenylmethoxycarbonyl-S-acetamidomethyl-L-cysteine
(Fmoc-Cys (Acm) -OH): Peptide Institute, Inc.
N ^α -9-fluorenylmethoxycarbonyl-N ^ε -tert-butyloxycarbonyl-L-lysine
(Fmoc-Lys (Boc) -OH): Peptide Institute, Inc.
9-Fluorenylmethoxycarbonyl-D-tryptophan
(Fmoc-D-Trp-OH): Peptide Institute, Inc.
9-Fluorenylmethoxycarbonyl-L-phenylalanine
(Fmoc-Phe-OH): Peptide Institute, Inc.
Fmoc-p-carboxy-Phe (O ^t Bu) -OH: BACHEM
Fmoc-p-amino-Phe (Boc) -OH: BACHEM
Fmoc-p-methyl-Phe-OH: BACHEM Thioanisol: First grade Tokyo Chemical Industry Co., Ltd. Trifluoroacetic acid (TFA): First grade Tokyo Chemical Industry Co., Ltd. Guanidine hydrochloride: Special grade Nakarai Tesque Iodine (I ₂ ): Special grade Ishizu Pharmaceutical Co., Ltd.
L-(+)-Ascorbic acid: Special grade Ishizu Pharmaceutical Co., Ltd. Indium chloride-111 (129.5 MBq / ml in 0.02 N HCl) ( ¹¹¹ InCl ₃ ): Nippon Mediphysics Corporation Human serum albumin: Sigma Aldrich Chem. Co.
n-Octanol: Special grade Wako Pure Chemical Industries, Ltd.
HPLC eluent: distilled water obtained with HPLC grade organic solvent and distilled water production equipment (MILLI-Q-Labo, MILLIPORE) All other reagents are reagent grade.

2-2: Apparatus The apparatus used in the examples and the method of using the apparatus are as follows.
(1) Fast atom bombardment mass spectra (FAB-MS)
A 70-SE type mass spectrometer (manufactured by VG) was used for measurement of the FAB-MS spectrum.

(2) reversed phase high-pressure liquid chromatography (RP-HPLC)
(2) -1 RP-HPLC experiment of non-radioactive compound Two LC-9A (Shimadzu Corporation) were used for the liquid feed pump, and the high pressure gradient method was used. In addition, an ultraviolet-visible absorbance meter SPD-6AV (Shimadzu Corporation) was used as a detector, and a C-R6A CHROMATOPAC (Shimadzu Corporation) was used as a recorder.
COSMOSIL 5C18-ARII column (size: 20 × 250 mm, Nacalai Tesque, Inc.) is used for fractionation, the mobile phase is 0.1% trifluoroacetic acid (TFA) / H ₂ O and acetonitrile, and the flow rate is 5 ml / At min, acetonitrile was increased with a linear gradient in 60 minutes.
(DTPA-p-amino-Phe ¹ -octreotide: 15 → 40%, DTPA-p-carboxy-Phe ¹ -octreotide: 20 → 40%, DTPA-p-methyl-Phe ¹ -octreotide: 20 → 40%)
For analysis, use a COSMOSIL 5C18-ARII column (size: 4.6 x 150 mm, Nacalai Tesque, Inc.) connected to a COSMOSIL 5C18-ARII column (size: 4.6 x 10 mm, Nacalai Tesque, Inc.) as a guard column. The phase was 0.1% trifluoroacetic acid (TFA) / H ₂ O and acetonitrile, and acetonitrile was increased in a linear gradient over 30 minutes at a flow rate of 0.9 ml / min. (DTPA-p-amino-Phe ¹ -octreotide: 15 → 40%, DTPA-p-carboxy-Phe ¹ -octreotide: 20 → 40%, DTPA-p-methyl-Phe ¹ -octreotide: 20 → 40%).

(2) -2 RP-HPLC experiment of radioactive compound Two LC-10ATs (Shimadzu Corporation) were used for the liquid feed pump, and the high pressure gradient method was used.
Use a COSMOSIL 5C18-MS column (size: 4.6 × 150 mm, Nacalai Tesque, Inc.) as the column, 0.05 M acetate buffer (pH 5.5) and methanol as the mobile phase at a flow rate of 1 ml / min. Gradient elution. In the case of the amino form, methanol was kept at 0% in the first 10 minutes, methanol was increased from 0% to 50% in the following 15 minutes, and methanol was kept at 50% for the last 20 minutes. In the case of the carboxy form, methanol was kept at 0% in the first 10 minutes, methanol was increased from 0% to 45% in the following 15 minutes, and methanol was kept at 45% for the last 20 minutes. In the case of the methyl form, methanol was kept at 5% for the first 10 minutes, methanol was increased from 5% to 65% in the following 15 minutes with a linear gradient, and methanol was kept at 65% for the last 20 minutes.
Moreover, the eluate was fractionated with a fraction collector (RediFrac, Pharmacia LKB), and the radioactivity (count) of each fraction was measured with a scintillation counter.

(3) Cellulose acetate electrophoresis (CAE)
Use Separax-SP (Jokoh Co. Ltd., Tokyo) for the cellulose acetate membrane, PS 1510 (Jokoh Co. Ltd., Tokyo) for the power supply, EC-100 (Adbantec) for the electrophoresis tank, and CAE. went.

(4) Amino acid analysis Amino acid analysis was performed by acid hydrolysis with 6 N hydrochloric acid at 110 ° C for 24 hours, and then analysis with a Hitachi L 8500 amino acid analyzer (Hitachi).

(5) Scintillation counter For measurement of radioactivity, Aloka ARC 2000, Tokyo was used.

(6) Metabolic cage room A metabolic cage (Metabolica, MM type; former Sugiyama Medical Instrument Co., Ltd.) was used to collect mouse urine and feces.

a：CF₃COOC₂H₅；b：BrCH₂COOBu^t, DIPEA；c：BrCH₂COOBu^t, NaH；d：NH₂NH₂, Bu^tOH；e：BrCH₂COOCH₂C₆H₅, DIPEA；f：Pd/C, H₂
Bu^t:tert-ブチル
Bz:ベンジル Example 1 Synthesis of DTPA-p-amino-Phe ¹ -octreotide, DTPA-p-carboxy-Phe ¹ -octreotide, and DTPA-p-methyl-Phe ¹ -octreotide
1． Synthesis of monoreactive DTPA (mDTPA)
mDTPA was synthesized according to the method of Arano et al. (Arano, Y., Uezono, T., Akizawa, H. et al., J. Med. Chem., 39, 3451-3460 (1996)).

_{a: CF 3 COOC 2 H 5} ; b: BrCH 2 COOBu t, DIPEA; c: BrCH 2 COOBu t, NaH; d: NH 2 NH 2, Bu t OH; e: BrCH 2 COOCH 2 C 6 H 5, DIPEA F: Pd / C, H ₂
Bu ^t: tert- butyl
Bz: benzyl

Compound (2)
Synthesis of di-tert-butyl 3-((tert-butoxycarbonyl) methyl) -6- (2-((trifluoroacetyl) amino) ethyl) -3,6-diazaoctanedioic acid Diethylenetriamine (1) (3.18 g, 3.36 ml, 30.9 mmol) of anhydrous acetonitrile solution (24 ml) was kept below 0 ° C, and thereto was added dropwise an anhydrous acetonitrile solution (36 ml) of ethyl trifluoroacetate (4.38 g, 3.69 ml, 30.9 mmol). After stirring for an hour, the mixture was further stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure and the residue was dissolved in anhydrous acetonitrile solution (120 ml), cooled to 0 ° C or lower, and then in the presence of N, N-diisopropylethylamine (13.95 g, 18.81 ml, 108 mmol), bromoacetic acid. tert-Butyl (21.06 g, 17.43 ml, 108 mmol) was added dropwise, protected from light, and stirred at room temperature for 24 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate (300 ml), washed twice with saturated sodium hydrogen carbonate (300 ml), the organic layer was dried over anhydrous calcium sulfate (CaSO ₄ ), and the solvent was removed under reduced pressure. Distilled off. The residue was subjected to silica gel chromatography using ethyl acetate: hexane (1: 8) as a developing solvent to obtain the desired product as a pale yellow oily substance (yield: 10.25 g, yield: 61.4%).

Compound (3)
Synthesis of di-tert-butyl 3,6-bis ((tert-butoxycarbonyl) methyl) -9- (trifluoro-acetyl-3,6,9-triazaundecanedioic acid
Sodium hydride (0.908 g, 22.71 mmol) suspended in N, N-dimethylformamide (82 ml) was kept at -15 ° C or lower, and to this was added N, N of compound (2) (10.25 g, 18.93 mmol). -A dimethylformamide solution (14 ml) was added dropwise, and the mixture was stirred at room temperature for 1 hour. After cooling to 0 ° C. or lower, tert-butyl bromoacetate (5.54 g, 4.59 ml, 28.4 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 hours. Ethyl acetate (400 ml) was added to the reaction solution, washed twice with saturated sodium bicarbonate (400 ml), the organic layer was dried over CaSO ₄ , and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel chromatography using ethyl acetate: hexane (1: 8) as a developing solvent to obtain the desired product as a pale yellow oil (yield: 7.51 g, yield: 60.5%).
FAB-MS (C ₃₀ H ₅₃ N ₃ O ₉ F ₃ ) (MH ⁺ ): m / z calculated, 656; found, 656.

Compound (4)
Synthesis of 1-benzyl tert-butyl 3,6,9-tris ((tert-butoxycarbonyl) methyl) -3,6,9-triazaundecanedioic acid Compound (3) (7.4 g, 11.3 mmol) tert- A butyl alcohol solution (74 ml) was kept at 0 ° C. or lower, and anhydrous hydrazine (35.4 ml, 1.13 mol) was added thereto, followed by stirring for 3 hours. Chloroform (400 ml) was added to the reaction solution, washed twice with saturated sodium bicarbonate (400 ml), the organic layer was dried over CaSO ₄ , and the solvent was distilled off under reduced pressure. In the presence of N, N-diisopropylethylamine (2.19 g, 2.95 ml, 16.9 mmol), benzyl bromoacetate (3.89 g, 2.68 ml, 9.6 mmol) was added dropwise to the residue, and the mixture was stirred at room temperature for 24 hours. Ethyl acetate (350 ml) was added to the reaction solution, washed twice with saturated sodium hydrogen carbonate (350 ml), the organic layer was dried over CaSO ₄ , and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel chromatography using ethyl acetate: hexane (1: 3) as a developing solvent to obtain the desired product as a pale yellow oily substance (yield: 3.4 g, yield: 42.5%).
FAB-MS (C ₃₇ H ₆₂ N ₃ O ₁₀ (MH ⁺ ): m / z calculated, 708; found, 708.

Compound (5)
Synthesis of tert-butyl 3,6,9-tris ((tert-butoxycarbonyl) methyl) -3,6,9-triazaundecanedioic acid (mDTPA) Compound (4) (1.55 g, 2.19 mmol) in ethyl acetate (5.6 ml), hydrogen substitution was performed in the presence of 10% palladium / carbon (0.1 g), and the mixture was stirred at room temperature for 4.5 hours. After removing palladium / carbon by filtration, the solvent was distilled off under reduced pressure to obtain mDTPA as a pale yellow oily substance (yield: 1.26 g, yield: 93.1%).
FAB-MS (C ₃₀ H ₅₆ N ₃ O ₁₀ ) (MH ⁺ ): m / z calculated, 618; found, 618.

Fmoc-Thr(Bu^t)-OH Fmoc-Thr(Bu^t)-ol
a：N-メチルモルホリン, クロロギ酸イソプロピル；b：NaBH₄
Fmoc: 9-フルオレニルメトキシカルボニル
Bu^t: tert-ブチル
Fmoc-Thr(Bu^t)-ol は、ロドリゲッツら（Rodrigues, M., Lilnares, M., Doulut, S. et al., Tetrahedron Lett., 32, 923-926 (1991)）の方法に従って合成した。
Fmoc-Thr(Bu^t)-OH (1.99 g, 5 mmol)のN,N-ジメチルホルムアミド溶液(5 ml) を -15 ℃に保ち、これにN-メチルモルホリン (0.65 ml, 5 mmol)と クロロギ酸イソプロピル(0.65 ml, 5 mmol)を加え、1 分間撹拌した。速やかに沈殿物を取り除き、0 ℃以下に冷却した後、水(2.5 ml)に懸濁させたホウ水素化ナトリウム(0.57 g, 15 mmol)を加え、30 分間撹拌した。水(125 ml)を加えて反応を終了させ、酢酸エチル (50 ml)により 3 回抽出した。有機層を合わせ、5 % 炭酸水素ナトリウム溶液(150 ml)で 2 回洗浄し、有機層をCaSO₄ で乾燥させ、溶媒を減圧留去した。残さをクロロホルム:メタノール(19 : 1)を展開溶媒とするシリカゲルクロマトグラフィーに付し、目的物を白色油状物質として得た(収量: 1.21 g, 収率: 36.2 %)。
FAB-MS（C₂₃H₂₉NO₄）(MH⁺): m/z 計算値, 384; 実測値, 384 2. Synthesis of DTPA-p-amino-Phe ¹ -octreotide, DTPA-p-carboxy-Phe ¹ -octreotide, and DTPA-p-methyl-Phe ¹ -octreotide
(1) Synthesis of Fmoc-Thr (Bu ^t ) -ol

Fmoc-Thr (Bu ^t ) -OH Fmoc-Thr (Bu ^t ) -ol
a: N-methylmorpholine, isopropyl chloroformate; b: NaBH ₄
Fmoc: 9-fluorenylmethoxycarbonyl
Bu ^t: tert- butyl
Fmoc-Thr (Bu ^t ) -ol was synthesized according to the method of Rodriguez et al. (Rodrigues, M., Lilnares, M., Doulut, S. et al., Tetrahedron Lett., 32, 923-926 (1991)). .
A solution of Fmoc-Thr (Bu ^t ) -OH (1.99 g, 5 mmol) in N, N-dimethylformamide (5 ml) was kept at -15 ° C, to which N-methylmorpholine (0.65 ml, 5 mmol) and chloroform Isopropyl acid (0.65 ml, 5 mmol) was added and stirred for 1 minute. After quickly removing the precipitate and cooling to 0 ° C. or lower, sodium borohydride (0.57 g, 15 mmol) suspended in water (2.5 ml) was added and stirred for 30 minutes. Water (125 ml) was added to terminate the reaction, and the mixture was extracted 3 times with ethyl acetate (50 ml). The organic layers were combined, washed twice with 5% sodium bicarbonate solution (150 ml), the organic layer was dried over CaSO ₄ , and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel chromatography using chloroform: methanol (19: 1) as a developing solvent to obtain the desired product as a white oil (yield: 1.21 g, yield: 36.2%).
FAB-MS (C ₂₃ H ₂₉ NO ₄ ) (MH ⁺ ): m / z calculated, 384; measured, 384

2-クロロトリチルクロリドレジン Fmoc-Thr(Bu^t)-O-レジン
a：Fmoc-Thr(Bu^t)-ol, 脱水ピリジン
ウエンシュらの方法（Wenschuh, H., Beyerman, M., Haber, H. et al., J.Org.Chem., 60, 405-410 (1995)）に従って、Fmoc-Thr(Bu^t)-olを2-クロロトリチル クロリドレジンに導入した。
2-クロロトリチルクロリドレジン(1.26 mmol/g; 200 mg, 0.252 mmol)と、Fmoc-Thr(Bu^t)-ol (289 mg, 0.76 mmol)を脱水ジクロロメタン (1.0 ml)と脱水N,N-ジメチルホルムアミド(1.0 ml)の混合溶媒に溶解し、脱水ピリジン(0.12 ml)を加え、窒素置換を行い、室温で 24 時間撹拌した。その後、N,N-ジメチルホルムアミド で洗浄し、メタノール(2.5 ml)を加え、30 分間撹拌することにより、未反応のトリチル基をエンドキャップした。次いで、N,N-ジメチルホルムアミドとジクロロメタンで洗浄し、減圧下で乾燥させた。 (2) Introduction of Fmoc-Thr (Bu ^t ) -ol into the resin

2-chlorotrityl chloride resin Fmoc-Thr (Bu ^t ) -O-resin
a:.. Fmoc-Thr ( Bu t) -ol, dehydrated pyridine Uenshu et al. method (Wenschuh, H., Beyerman, M. , Haber, H. et al, J.Org.Chem, 60, 405-410 ( according 1995)), was introduced Fmoc-Thr (Bu ^t) -ol to 2-chlorotrityl Kuroridorejin.
2-chlorotrityl chloride resin (1.26 mmol / g; 200 mg, 0.252 mmol) and Fmoc-Thr (Bu ^t ) -ol (289 mg, 0.76 mmol) were mixed with dehydrated dichloromethane (1.0 ml) and dehydrated N, N-dimethyl. The product was dissolved in a mixed solvent of formamide (1.0 ml), dehydrated pyridine (0.12 ml) was added, nitrogen substitution was performed, and the mixture was stirred at room temperature for 24 hours. Then, it wash | cleaned by N, N- dimethylformamide, methanol (2.5 ml) was added, and the unreacted trityl group was endcapped by stirring for 30 minutes. Then, it was washed with N, N-dimethylformamide and dichloromethane and dried under reduced pressure.

(3) Resin on Fmoc-Thr (Bu ^t) Quantitative Main hot fur et al method -ol (Meienhofer, J., Waki, M., Heimer, EP et al., Int.J.Pept.Protein Res., 13, according to 35-42 (1979)), was quantified Fmoc-Thr (Bu ^t) -ol bound to the resin.
Resin sample was dried under vacuum to constant weight, accurately weighed 1-2 × 10 ^-3 g (X), piperidine (0.4 ml) and dichloromethane (0.4 ml) were added and reacted for 30 minutes . Methanol (1.6 ml) was added to terminate the reaction, dichloromethane was added to make the total volume exactly 10 ml, and the solution was mixed. The absorbance (A ₃₀₁ ) of the sample solution with respect to the control solution was measured at 301 nm, and the substitution rate was determined by the following formula, and found to be 0.243 mmol / g.
Substitution rate (amino acid mmol / resin 1 g) = (A301 / 7800) × (10 ml / X)

(4) Synthesis of protected DTPA-p-amino-Phe ¹ -octreotide (6), protected DTPA-p-carboxy-Phe ¹ -octreotide (7), and protected DTPA-p-methyl-Phe ¹ -octreotide (8)
20% piperidine / dimethylformamide (2 ml) was added and stirred for 20 minutes to remove the Fmoc group. Next, after washing with dimethylformamide, 2.5 equivalents of side chain protected F-moc-amino acid derivative and the condensing agent DIPCDI and HOBt · H ₂ O were added, and the condensation reaction was carried out in dimethylformamide (1 ml) for 1.5 hours. It was. This condensation reaction was repeated until it became negative by the Kaiser Test (Kaiser, E., Colescot, RL, Bossinger, CD et al., Anal. Biochem., 34, 595-598 (1970)). This removal of the F-moc group and condensation of the F-moc-amino acid derivative are repeated to extend the protected peptide chain to the respective N-terminal amino acid p-amino-Phe, p-carboxy-Phe, or p-methyl-Phe. did. The F-moc- amino acid, Fmoc-Cys (Acm) -OH , Fmoc-Thr (Bu t) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp-OH, Fmoc-Phe-OH, Fmoc-p-amino-Phe (Boc) -OH, Fmoc-p-carboxy-Phe (OtBu) -OH, Fmoc-p-methyl-Phe-OH were used. Then add 2.5 equivalents of mDTPA and DIPCDI and HOBt.H ₂ O and conduct a condensation reaction in dimethylformamide (1 ml) for 1.5 hours to obtain protected DTPA-p-amino-Phe ¹ -octreotide (6), protected DTPA. -p-Carboxy-Phe ¹ -octreotide (7) and protected DTPA-p-methyl-Phe ¹ -octreotide (8) were obtained.

(5) [Cys (Acm) 2,7] -DTPA-p-amino-Phe ¹ -octreotide (9), [Cys (Acm) 2,7] -DTPA-p-carboxy-Phe ¹ -octreotide (10) And [Cys (Acm) 2,7] -DTPA-p-methyl-Phe ¹ -octreotide (11) Protected DTPA-p-amino-Phe ¹ -octreotide (6) (100 mg), protected DTPA-p -Carboxy-Phe ¹ -octreotide (7) (100 mg) or protected DTPA-p-methyl-Phe ¹ -octreotide (8) (100 mg) was kept below 0 ° C and thioanisole (400 μl) In the presence, trifluoroacetic acid (TFA) (4 ml) was added and stirred at room temperature for 2 hours. After cooling to below 0 ° C, ether (5 ml) was added and crude [Cys (Acm) 2,7] -DTPA-p-amino-Phe ¹ -octreotide, crude [Cys (Acm) 2,7] -DTPA -p-Carboxy-Phe ¹ -octreotide or crude [Cys (Acm) 2,7] -DTPA-p-methyl-Phe ¹ -octreotide (11) was precipitated. The precipitate was extracted with 6 M guanidine hydrochloride (5 ml), and the resin (resin) was removed using a glass filter. After pretreatment using an aqueous filter (DISMIC-13HP, Tokyo Filter Paper Co., Ltd.), purification was performed by RP-HPLC. Monitor at 230 nm, collect fractions containing the desired product, lyophilize, [Cys (Acm) 2,7] -DTPA-p-amino-Phe ¹ -octreotide (9) (Yield: 8.09 mg, Yield: 15.4%), [Cys (Acm) 2,7] -DTPA-p-carboxy-Phe ¹ -octreotide (10) (Yield: 6.05 mg, Yield: 14.27%), or [Cys (Acm) 2,7] -DTPA-p-methyl-Phe ¹ -octreotide (11) (yield: 5.56 mg, yield: 10.57%) was obtained as a white powder.

In addition, the obtained [Cys (Acm) 2,7] -DTPA-p-amino-Phe ¹ -octreotide, [Cys (Acm) 2,7] -DTPA-p-carboxy-Phe ¹ -octreotide, and [Cys The white powder of (Acm) 2,7] -DTPA-p-methyl-Phe ¹ -octreotide was analyzed by RP-HPLC. Major peaks were observed at retention times of 16.70 min, 16.04 min, and 20.82 min, respectively. It was.
[Cys (Acm) 2,7] -DTPA-p-amino-Phe ¹ -octreotide:
FAB-MS (C ₆₉ H ₁₀₁ N ₁₆ O ₂₁ S ₂ ) (MH ⁺ ): m / z calculated, 1554; measured, 1554.
[Cys (Acm) 2,7] -DTPA-p-carboxy-Phe ¹ -octreotide:
FAB-MS (C ₇₀ H ₁₀₀ N ₁₅ O ₂₃ S ₂ ) (MH ⁺ ): m / z calculated, 1582; measured, 1582.
[Cys (Acm) 2,7] -DTPA-p-methyl-Phe ¹ -octreotide
FAB-MS (C ₇₀ H ₁₀₁ N ₁₅ O ₂₁ S ₂ ) (MH ⁺ ): m / z calculated, 1552; measured, 1552.

(6) Synthesis of DTPA-p-amino-Phe ¹ -octreotide (12), DTPA-p-carboxy-Phe ¹ -octreotide (13) and DTPA-p-methyl-Phe ¹ -octreotide (14)
[Cys (Acm) 2,7] -DTPA-p-amino-Phe ¹ -octreotide (9) (1 mg, 0.64 μmol), [Cys (Acm) 2,7] -DTPA-p-carboxy-Phe ^1- Octreotide (10) (1 mg, 0.63 μmol) or [Cys (Acm) 2,7] -DTPA-p-methyl-Phe ¹ -octreotide (11) (1 mg, 0.64 μmol) in 80% acetic acid (324 μl 20% I2 / methanol (16.6 μl, 13.02 μmol) was added, and the mixture was stirred at room temperature for 60 minutes. A 1 M aqueous ascorbic acid solution was added to reduce the excess amount of iodine, and after pretreatment using an aqueous filter (DISMIC-13HP, Tokyo Filter Paper Co., Ltd.), purification was performed by RP-HPLC. Monitor at 230 nm, collect fractions containing the desired product, lyophilize, DTPA-p-amino-Phe ¹ -octreotide (9) (Yield: 292 μg, Yield: 32.19%), DTPA-p-carboxy- Phe ¹ -octreotide (10) (yield: 163 μg, yield: 17.92%) or DTPA-p-methyl-Phe ¹ -octreotide (11) (yield: 306 μg, yield: 33.71%) as a white powder Got as.
The resulting white powders of DTPA-p-amino-Phe ¹ -octreotide, DTPA-p-carboxy-Phe ¹ -octreotide, and DTPA-p-methyl-Phe ¹ -octreotide were analyzed by RP-HPLC. However, major peaks were observed at retention times of 17.13 minutes, 17.65 minutes, and 22.52 minutes, respectively.
DTPA-p-amino-Phe ¹ -octreotide
FAB-MS (C ₆₃ H ₈₉ N ₁₄ O ₁₉ S ₂ ) (MH ⁺ ): m / z calculated, 1410; measured, 1410
Amino acid composition ratio after acid degradation with 6 N hydrochloric acid:
Thr 1.00 (1) Phe 1.08 (1) Lys 0.99 (1) [Theoretical composition ratios in parentheses]
DTPA-p-carboxy-Phe ¹ -octreotide
FAB-MS (C ₆₄ H ₈₈ N ₁₃ O ₂₁ S ₂ ) (MH ⁺ ): m / z calculated, 1439; measured, 1439
Amino acid composition ratio after acid degradation with 6 N hydrochloric acid:
Thr 1.00 (1) Phe 1.63 (1) Lys 1.07 (1) [Theoretical composition ratios in parentheses]
DTPA-p-methyl-Phe ¹ -octreotide
FAB-MS (C ₆₄ H ₉₀ N ₁₃ O ₁₉ S ₂ ) (MH ⁺ ): m / z calculated, 1409; measured, 1409
Amino acid composition ratio after acid degradation with 6 N hydrochloric acid:
Thr 1.00 (1) Phe 0.88 (1) Lys 1.02 (1) [Theoretical composition ratios in parentheses]

Example 2 ¹¹¹ In-DTPA-p-amino-Phe ¹ -octreotide (amino form), ¹¹¹ In-DTPA-p-carboxy-Phe ¹ -octreotide (carboxy form), and ¹¹¹ In-DTPA-p-methyl-Phe Synthesis of ¹ -octreotide (methyl)
After dissolving 10 μg DTPA-p-amino-Phe ¹ -octreotide, DTPA-p-carboxy-Phe ¹ -octreotide, or DTPA-p-methyl-Phe ¹ -octreotide in 0.1% acetic acid (100 μg), ¹¹¹ InCl ₃ in 0.02 N HCl (7.62-12.5 μl) was added and left at room temperature for 1 hour. The samples obtained here were used for comparison of electrical properties and fat solubility. Moreover, the sample obtained by the following operation was used for the in-vivo radioactivity distribution. To the above sample, 10 mM HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) buffer (pH 7.6) containing 0.1 HSA (human serum albumin) was further added. A solution of μg / 100 μl was prepared.
When amino, carboxy and methyl were analyzed by RP-HPLC, major peaks were observed at retention times of 39 min, 38 min and 34 min, respectively.
The prepared amino form, carboxy form, and methyl form were confirmed to have a radiochemical purity of 95% or more by analysis by RP-HPLC and used in the subsequent experiments.

Experimental Example 1 ¹¹¹ In-DTPA-p-amino-Phe ¹ -octreotide (amino form), ¹¹¹ In-DTPA-p-carboxy-Phe ¹ -octreotide (carboxy form), and ¹¹¹ In-DTPA-p-methyl-Phe Electrical properties of ¹ -octreotide (methyl)
¹¹¹ In-DTPA-p-amino-Phe ¹ -octreotide (amino form), ¹¹¹ In-DTPA-p-carboxy-Phe ¹ -octreotide (carboxy form), and ¹¹¹ In-DTPA-p-methyl-Phe ¹ -octreotide Cellulose acetate electrophoresis (CAE) was performed in order to compare the electrical properties of (methyl). Separax-SP (Jokoh Co. Ltd., Tokyo) is used for the cellulose acetate membrane, in pH 5.0, 6.0, 7.0 corresponding to urine pH, and pH 7.4 corresponding to plasma pH in 20 mM phosphate buffer. For 60 minutes. The results are shown in FIG. In the figure, A represents pH 5.0, B represents pH 6.0, C represents pH 7.0, and D represents pH 7.4. As shown in FIG. 1, at pH 5.0, 6.0, 7.0 corresponding to the pH of urine and pH 7.4 corresponding to the pH of plasma, the amino isomer moves to the minus side compared to the Phe isomer, and the carboxy isomer is positive. Moved to the side. Moreover, the methyl body moved to the same position as the Phe body. These results show that in urine and plasma, amino is positively charged and carboxy is negatively charged compared to Phe and methyl, reflecting the side chain charge of Phe ¹ residue. Show.

Experimental Example 2 ¹¹¹ In-DTPA-p-amino-Phe ¹ -octreotide (amino form), ¹¹¹ In-DTPA-p-carboxy-Phe ¹ -octreotide (carboxy form), and ¹¹¹ In-DTPA-p-methyl-Phe Fat solubility of ¹ -octreotide (methyl) In order to compare the fat solubility of amino, carboxy and methyl, the n-octanol / buffer partition ratio was determined as follows.
n-Octanol and phosphate buffer were mixed and allowed to saturate overnight. 3 ml each of the organic layer and the aqueous layer were taken from a plastic tube coated with silicon (Siliconize L-25, Fuji System Corporation), and an amino, carboxy, or methyl solution (5 μl) was added. [1 minute vortex-left for 1 minute] was repeated 3 times and then left for 20 minutes. This operation was repeated 3 times and centrifuged. 1 ml each of the upper layer (n-octanol layer) and the lower layer (buffer layer) was taken, the radioactivity (count) was measured with a scintillation counter, and the distribution ratio (PC) was calculated by the following formula.
PC = [Radioactivity of n-octanol layer] / [Radioactivity of buffer layer]
The buffer used was a 0.1 M phosphate buffer with pH 5.0, 6.0, 7.0 corresponding to urine pH and pH 7.4 corresponding to plasma pH. The results are shown in Table 1. As apparent from Table 1, both the amino and carboxy forms showed lower fat solubility than the Phe form, but at pH 5.0 and 6.0, the carboxy form showed higher fat solubility than the amino form, and pH 7.0 and 7.4. Then, the amino form showed higher fat solubility than the carboxy form. In addition, the methyl body showed higher fat solubility than the Phe body at any pH. However, both fat solubility was very low.

Table 1 Fat solubility of amino, carboxy, and methyl forms

Experimental Example 3 ¹¹¹ In-DTPA-p-amino-Phe ¹ -octreotide (amino form), ¹¹¹ In-DTPA-p-carboxy-Phe ¹ -octreotide (carboxy form), and ¹¹¹ In-DTPA-p-methyl-Phe Plasma stability of ¹ -octreotide (methyl) Under ether anesthesia, blood was collected from 6-week-old ddY male mice, and mouse plasma was prepared by centrifugation at 900 g for 20 minutes at 4 ° C. Amino form (5 μl, 0.5 μg, 126.4 kBq), carboxy form (5 μl, 0.5 μg, 126.4 kBq), or methyl form (5 μl, 0.5 μg, 126.4 kBq) were prepared from fresh mouse plasma (450 μl) and incubated at 37 ° C. After 1 and 3 hours, the sample solution was taken out, filtered through an ultrafiltration membrane (Microcon-10, Amicon Inc., Beverly, MA) with a molecular weight cut off of 10,000, and then analyzed by RP-HPLC. The results are shown in FIG. The vertical axis represents the ratio (%) of unchanged substance when the ratio of unchanged substance at 0 hour is 100%.
In the amino, carboxy and methyl forms, 98, 97 and 97% existed unchanged after 1 hour of incubation and 98, 97 and 96% remained unchanged after 3 hours, respectively. These results indicate that the amino, carboxy, and methyl isomers exist stably in the blood like the Phe isomers.

Experimental Example 4 ¹¹¹ In-DTPA-p-amino-Phe ¹ -octreotide (amino form), ¹¹¹ In-DTPA-p-carboxy-Phe ¹ -octreotide (carboxy form), and ¹¹¹ In-DTPA-p-methyl-Phe ¹ -Octreotide (methyl) in vivo radioactivity distribution 6 groups of 6-week-old male ddY mice (body weight 28-30 g) consisting of 5-6 mice (amino acids (0.1 ng, 100 μl) from the tail vein) ), Carboxy (0.1 ng, 100 μl), or methyl (0.1 ng, 100 μl), and after 10 and 30 minutes, 1, 3, 6, and 24 hours, decapitation, dissection, and organ of interest (Liver, kidney, spleen, pancreas and lung) were removed and the weight and radioactivity of blood and organs of interest were measured. In addition, in order to investigate the in vitro excretion route and excretion of radioactivity, mice were kept in a metabolic cage for 24 hours, and urine and feces excreted from immediately after administration to 24 hours later were collected and their radioactivity was measured. did. As a control, ¹¹¹ In-DTPA-Phe ¹ -octreotide (Phe body) was used for the test. The results are shown in Tables 2-5. Moreover, the time-dependent change of the radioactive activity in the kidney of an amino body, a carboxy body, a methyl body, and Phe body is shown in FIG.

Table 2 ¹¹¹ In-DTPA-p- amino -Phe in mice ¹ - body radioactivity distribution ^a post octreotide intravenous administration

Table ¹¹¹ in 3 mice In-DTPA-p- carboxy -Phe ¹ - body radioactivity distribution ^a post octreotide intravenous administration

Table ¹¹¹ in 4 mice In-DTPA-p- methyl -Phe ¹ - body radioactivity distribution ^a post octreotide intravenous administration

Table ^{5 111 In-DTPA-p-} Phe in mice ¹ - body radioactivity distribution ^a post octreotide intravenous administration

From Tables 2 to 5, it can be seen that the radioactivity in the blood almost disappeared by 3 hours after administration and disappeared from the blood promptly. In all cases, the kidneys showed higher radioactivity accumulation than other organs, but the accumulation amount was greatly different in amino, carboxy and methyl forms. That is, in the case of the amino form, compared with the Phe form, it was the same at 10 minutes after administration, but was significantly lower at 30 minutes after administration, and higher at 24 hours after administration. The carboxy form showed a lower accumulation at all time points examined than the Phe form, and was significantly lower from 30 minutes to 6 hours after administration. In the case of the methyl form, it was significantly higher than that of the Phe form at 1 hour and 6 hours after administration, but in other cases, the behavior was almost the same (FIG. 3).
In other organs, there was a significant difference, but the amount of radioactivity accumulated was very small.
In addition, the radioactivity excreted in urine and feces by 24 hours after administration was 82.03% and 5.80% in the amino form, and 86.30% and 5.72% in the carboxy form, respectively. In the case of methyl, they were 66.67% and 11.74%, respectively. On the other hand, it was 57.54% and 22.81% for Phe bodies, respectively. From this result, it was found that metal was excreted mainly in urine.

Experimental Example 5 Analysis of Amino, Carboxy and Methyl in Urine Amino (100 μl, 0.1 ng, 9.25 kBq), Carboxy (100 μl, 0.1 ng, 9.25 kBq), or Methyl (100 μl, 0.1 ng, 9.25 kBq) was administered to 6-week-old male ddY mice (body weight 28-30 g), the mice were kept in a metabolic cage for 24 hours, and urine excreted immediately after administration until 24 hours was collected. The collected urine was filtered through an ultrafiltration membrane (Microcon-10, Amicon Inc., Beverly, MA) having a fractional molecular weight of 10,000, and then analyzed by RP-HPLC. The results are shown in FIGS. 4, 5 and 6, respectively. In the case of the amino form, the retention time was 37 minutes (FIG. 4), in the case of the carboxy form, the retention time was 41 minutes (FIG. 5), and in the case of the methyl form, the retention time was 33 minutes (FIG. 6). We observed a major peak that seems to be of the body.
Further, based on the results (FIGS. 4-6), the proportion of the radioactivity excreted in the urine by the unchanged form was calculated (FIG. 7). The amino form was 76.00% and the carboxy form was 86.69. In the methyl form, 71.14% was unchanged.

From the experimental results described in the above experimental examples, the following points became clear.
(1) Amino, carboxy, methyl, and Phe forms are all stable in blood and promptly show clearance from blood, and thus are considered to be transferred to an organ without change.
(2) In any embodiment, high radioactivity accumulation was observed in the kidney among the tested organs, and the radioactivity was mainly excreted in the urine, and more than 70% conformed to ¹¹¹ In-DTPA-Phe ¹ -octreotide It is an unchanged body (FIG. 7). This result shows that the amino, carboxy, methyl, and Phe isomers are mostly unchanged, glomerularly filtered and excreted in the urine, and partly taken up by kidney cells. It shows that it shows almost the same radioactivity dynamics. That is, it is suggested that the difference in fat solubility and electrical properties does not affect the rate of uptake into kidney cells.
(3) Regarding the accumulation of radioactivity in the kidney, as is apparent from FIG. 3, in the case of the amino form of the present invention, it was lower than the Phe form 30 minutes to 3 hours after administration, and in the case of the carboxy form, At the time, it was suppressed lower than that of Phe body, and showed rapid clearance after uptake into kidney cells.
Both the carboxy form and amino form of the present invention are promising as diagnostic agents because they exhibit significantly lower renal accumulation throughout the period required for post-administration image diagnosis compared to Phe form and methyl form. Moreover, the property of being rapidly excreted after uptake into kidney cells is extremely important in avoiding the expression of cytotoxicity to kidney cells, and is extremely promising as a therapeutic agent.

Industrial utility

  The carrier substance of the present invention maintains a therapeutic and diagnostic metal stably in the blood, can be rapidly delivered to cells of target somatostatin receptor positive tumors and endocrine diseases, and the metal kidney. It has an excellent property of keeping the accumulation low. By coordinating an appropriate radioactive or paramagnetic metal to this carrier substance, a diagnostic / therapeutic drug with high accuracy and low side effects can be obtained. According to the present invention, it is possible to perform a more accurate and safe diagnosis and treatment by greatly reducing the exposure and burden on the patient, and at the same time, it is difficult or impossible to apply the kidney or a disease or tumor around the kidney. It is possible to carry out nuclear medicine diagnosis and treatment, MRI diagnosis, and the like.

¹¹¹ In-DTPA-p-amino-Phe ¹ -octreotide (amino form), ¹¹¹ In-DTPA-p-carboxy-Phe ¹ -octreotide (carboxy form), and ¹¹¹ In-DTPA-p-methyl-Phe ¹ -octreotide It is a figure which shows the radioactivity (count) profile in the cellulose acetate electrophoresis of (methyl body). It is a figure which shows the stability of the amino body, carboxy body, and methyl body in mouse | mouth plasma measured by RP-HPLC. It is a graph which shows the time-dependent change of the radioactive activity in a kidney after administering an amino body, a carboxy body, a methyl body, and a Phe body to a mouse | mouth. It is a figure which shows the result of having analyzed the state of the radioactive clearance in the mouse | mouth urine of an amino body by HPLC. It is a figure which shows the result of having analyzed the state of the radioactive clearance in the mouse | mouth urine of a carboxy body by HPLC. It is a figure which shows the result of having analyzed the state of the radioactive clearance to the mouse | mouth urine of a methyl body by HPLC. It is a graph which shows the ratio for which an unchanged body accounts among the radioactivity excreted in the urine created based on the analysis result of FIGS. 4-6.

Claims (8)

Formula (I):

Wherein R is DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10-tetra-azacyclododecane-1,4,7,10-4 acetic acid) or 1,4,7,10- A chelator residue selected from tetraazacyclododecane -1-aminoethylcarbamoylmethyl-4,7,10-tris [(R, S) methylacetic acid] ;
X is carboxyphenyl or aminophenyl ,
Y is Thr-OH ,
A is Phe ,
B is a Thr)
A carrier substance for delivering a diagnostic and / or therapeutic metal represented by a target cell expressing a somatostatin-binding receptor. R is, carrier substances 請 Motomeko 1 is a DTP A. A tumor-accumulating complex of a diagnostic and / or therapeutic metal and the carrier substance according to claim 1 or 2. Radioactive metal consisting of Tc-99m, In-111, Cu-62, Ga-67, Ga-68, Re-186, Re-188, Y-90, Cu-64, Sn-117m and Sm-153 And a tumor-accumulating complex according to claim 3, selected from paramagnetic metals consisting of Gd, Dy, Fe and Mn. A pharmaceutical composition for diagnosis or treatment of a tumor containing the complex according to claim 4. 6. The diagnostic imaging method according to claim 5, wherein the metal is a radioactive metal selected from Tc-99m, In-111, Cu-62, Ga-67 and Ga-68. Pharmaceutical composition. 6. The pharmaceutical composition according to claim 5, for radiotherapy, wherein the metal is a radiometal selected from Re-186, Re-188, Y-90, Cu-64, Sn-117m and Sm-153. The pharmaceutical composition according to claim 5, for MRI imaging of a tumor site in a living body, wherein the metal is a paramagnetic metal selected from Gd, Dy, Fe and Mn.

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