JPH1095737A - Peptide derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a support bindable to a somatostatin receptor, to be used for preparing detectable element-bearing medicinal agents, by binding a chelate group to a somatostatin peptide. SOLUTION: This derivative, a somatostantin derivative, is obtained by chemically binding a chelate group to the terminal amino group of a somatostatin peptide. This derivative is to be used as a support for detectable elements. The support is bindable to a somatostaitn receptor; therefore, after supporting a radioactive element thereon, can be used as e.g. a medicine for treating somatostaitn receptor-positive tumor, or as an imaging reagent for in vivo diagnoses. The somatostatin analog mentioned above is e.g. a derivative of the formula (A is a 7-10C phenylakyl, etc.; A' is H, an alkyl, etc.; Y1 and Y2 are each a direct bond, H, etc.; B is Phe or Tyr; C is a (substituted)(L)-Trp, etc.; D is Lys, etc.; E is Thr, Ser, etc.; G is COOR7 , etc.; R7 is H, etc.).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to polypeptides and methods for producing them, drugs containing them, and use as, for example, a medicament for treating somatostatin receptor-positive tumors or as an imaging reagent for in vivo diagnosis. .

[0002]

BACKGROUND OF THE INVENTION During the past few years, somatostatin receptors have been used in various human tumors, such as pituitary tumors,
It is increasingly found in central nervous system tumors, breast tumors, gastrointestinal / pancreatic tumors and their metastases. Some of them are small and slow growing tumors, which make it difficult to accurately locate them using conventional diagnostic methods.

[0003] The somatostatin receptor is a radioiodinated somatostatin or somatostatin analog in vitro, such as [ ¹²⁵ I-Tyr ¹¹ ] somatostatin-1.
4 [Taylor, JE, et al., Life Sciences (Life
Science (1983) 43 : 421] or [ ¹²⁵ I-Tyr.
³ ] SMS 201-995 (also referred to as 204-090)
[Lyubi, JC Brain Research (Brain Res.)
(1987) 406 : 891; Ryubi, JC et al., Journal of Clinical Endocrinology and Metabolism (J. Clin. Endocr. Metab.) (198).
7) 65 : 1127; Ryubi, JC, et al., Cancer.
Research (Cancer Res.) (1987) 47 : 551; Ryubi, JC, et al., Cancer Research.
s.) (1987) 47 : 5758] and the like.

[0004]

DESCRIPTION OF THE INVENTION A new somatostatin peptide has been discovered according to the present invention which is therapeutically beneficial and which can be labeled for in vivo diagnostic and therapeutic administration. The present invention provides a somatostatin peptide having at least one chelating group for a detectable element (eg, a detectable element). This chelating group is linked to the amino group of the peptide, and this amino group does not show significant binding affinity for the somatostatin receptor. These compounds are hereinafter referred to as "ligands of the invention" or "ligands of the invention". These have a detectable element, such as a radionuclide, a radiopaque (X-ray visible) element, or one chelating (forming) group capable of reacting with a paramagnetic ion to form a complex; Furthermore, it can bind to a somatostatin receptor that is expressed or overexpressed by, for example, a tumor or metastasis. The chelating group is covalently linked to the amino group of the peptide. Preferably, the chelating group is attached to the N-terminal amino group of the somatostatin peptide.

According to the present invention, the chelating group can be linked to the somatostatin peptide directly or indirectly, ie via a spacer group. One group of "ligands" include those in which a chelating group is directly attached to the amino group of the somatostatin peptide. There is also a group of “ligands” that have a chelating group that is indirectly linked to the amino group of the somatostatin peptide by a bridge or an intervening group. Preferably, the chelating group is attached to the peptide via an amide bond. The term somatostatin peptide also includes natural somatostatin (tetradecapeptide) and analogs or derivatives thereof.

Derivatives and analogs as used herein are any linear or cyclic polypeptides derived from the natural tetradecapeptide somatostatin, in which one or more amino acid units have been removed. And / or substituted with one or more other amino acid groups, and / or one or more functional groups are substituted with one or more other functional groups, and / or One or more groups are
It is substituted with one or several other isosteric groups. In general, the term encompasses all modified derivatives that are biologically active, and the derivatives exhibit qualitatively similar effects to the unmodified somatostatin peptide. For example, it binds to the somatostatin receptor and suppresses hormone secretion.

[0007] Cyclic, bridged cyclic and linear somatostatin analogs are known compounds. Regarding those compounds and the production method, for example, European Patent Specification EP-A-
1295; 29579; 215171; 203030;
214872; 298732; 277419. Preferred "ligands of the invention" are those derived from the somatostatin analogs shown below.

A. Analogs of formula I

Embedded image In the formula, A 1 is a C _1-12 alkyl, a C _7-10 phenylalkyl or a group represented by the formula RCO—. Where i) R is hydrogen, C _1-11 alkyl, phenyl or C
_7-10 phenylalkyl or ii) RCO- is a) optionally F, Cl,, Br, NO 2, NH 2, OH, C
_An L- or D-phenylalanine residue which may be ring-substituted by _1-3 alkyl and / or C _1-3 alkoxy; b) a natural or synthetic α-amino acid residue other than those defined in a) above. Group, or corresponding D-amino acid residue,
Or c) the individual amino acid residues are the same or different,
The α-amino group of the dipeptide amino acid residues a) and b) selected from those defined in a) and / or b) above, and the N-terminal amino group of the dipeptide residue c) are optionally mono- Or di C _1-12 alkylated or C _1-8
It may be substituted by alkanoyl.

A ′ is hydrogen, C _1-12 alkyl or C
_7-10 phenylalkyl, Y ₁ and Y ₂ together form a direct bond, and Y ₁ and Y _{2 are} each independently hydrogen or a group of formulas (1) to (5).

Embedded image Wherein Ra is methyl or ethyl, Rb is hydrogen, methyl or ethyl, m is an integer from 1 to 4, n is an integer from 1 to 5, Rc is (C _1-6 ) alkyl, Rd
Is a substituent (including hydrogen) bonded to the α-carbon atom of a natural or synthetic α-amino acid; Re is (C _1-5 ) alkyl;
a 'and Rb' are independently hydrogen, methyl, or ethyl;
R ₈ and R ₉ are independently hydrogen, halogen, (C _1-3 ) alkyl, or (C _1-3 ) alkoxy, p is 0 or 1, q is 0 or 1, r is 0,1, Or 2,

B is halogen, NO ₂ , NH
₂ , -Phe- (including pentafluoroalanine) or β-naphthyl-Ala optionally substituted with OH, C _1-3 alkyl and / or C _1-3 alkoxy. C
May be optionally α-N-methylated, and the benzene ring is optionally halogen, NO ₂ , NH ₂ , O
(L) -Trp- or (D) -Trp-, D optionally substituted with H, C _1-3 alkyl and / or C _1-3 alkoxy
Is Lys, L whose side chain contains 0 or S at the β-position.
ys, γF-Lys or δF-Lys, which may be optionally α-N-methylated, or a 4-aminocyclohexyl Ala or 4-aminocyclohexyl Gly residue, E is Thr, Ser, Val , Phe, Ile, or aminoisobutyric acid or aminobutyric acid residue,

G is -COOR ₇ , -CH ₂ OR ₁₀ ,

Embedded image Wherein R ₇ is hydrogen or C _1-3
Alkyl, R ₁₀ is hydrogen, or physiologically acceptable,
The residue of a physiologically hydrolysable ester, R ₁₁ is hydrogen, C _1-3 alkyl, phenyl, or C _7-10 phenylalkyl; R ₁₂ is hydrogen, C _1-3 alkyl, or —C
H (R ₁₃₎ groups represented by -X _1, R ₁₃ is CH ₂ OH, -
_{(CH 2) 2 -OH, -} (CH 2) -OH or -CH (CH,
₃ ) a substituent (including hydrogen) that is OH or attached to the α-carbon atom of a natural or synthetic α-amino acid;

X ₁ is —COOR ₇ , —CH ₂ OR ₁₀ , or

Embedded image Wherein R ₇ and R ₁₀ have the meanings indicated above,
R ₁₄ is hydrogen or C _1-3 alkyl, R ₁₅ is hydrogen,
C _1-3 alkyl, phenyl, or C _7-10 phenylalkyl, R ₁₆ represents hydrogen or hydroxy. Provided that when R ₁₂ is —CH (R ₁₃ ) —X ₁ , R ₁₁ is hydrogen or methyl, B, D and E residues are L-forms, and residues at positions 2 and 7 And Y ₁ (4) and Y ₂
Any of the residues in (4) are independently in the (L) or (D) configuration. It is preferred that the definitions of A and A 'in Formula I be selected such that the compound has a terminal -NH- group capable of binding to a chelating agent. For the compounds of formula I, the following definitions are each preferred, either independently or in combination or as a sub-combination.

1. A is C _7-10 phenylalkyl, especially phenethyl, or a group represented by the formula RCO.
A is preferably a group represented by the formula of RCO. 1.1. R is preferably C _1-11 alkyl, or C _7-10 phenylalkyl, especially C _7-10 phenylalkyl, and more preferably phenethyl. Also, RCO has a), b)
Or the meaning of c) is preferred. 1.2. When RCO has the meaning of a), b) or c), the α-amino group of amino acid residues a) and b) or a dipeptide residue
the N-terminal amino group of c) is not alkylated,
Preference is given to C _{1 -12} monoalkylation, especially C _{1 -8} alkylation, furthermore methylation. Most preferably, the N-terminus is not alkylated. 1.3. When RCO means a), this may optionally be N-C _{1-12 -monoalkylated} ,
-Or D-phenylalanine, or -tyrosine residue a '). More preferably, a ') is an L- or D-phenylalanine residue. 1.4. When RCO has the meanings of b) and c), the defined residues are preferably lipophilic. Preferred residues b)
Is an α-amino acid having b ′) a hydrocarbon side chain.
For example alkyl having 3, preferably 4 or more carbon atoms, for example up to 7 carbon atoms, naphthyl-methyl or heteroaryl, for example 3-
(2- or 1-naphthyl) -alanine, pyridyl-methyl, or tryptophan residues, and the above-mentioned residues include L-form and D-form. Preferred residues c) are dipeptide residues, the individual amino acid residues of which are the same or different, which are selected from those defined in a ′) and b ′) above. By way of example, residue c) is, for example, a 3- (2-naphthyl) -alanine residue. 1.5. Most preferably, RCO means a), especially a ').

2. B is B ', B' is Phe, or T
yr. 3. C is C 'and C' is (D) Trp. 4. D is D ', D' is Lys, MeLys, or Ly
s (ε-Me), especially Lys. 5. E is E ', E' is Val or Thr, especially Thr. 6. G is G ', G' is

Embedded image A group represented by the formula

Embedded image (In this case, R ₁₁ = H or C
H ₃ ). In the latter case, the —CH (R ₁₃ ) —X ₁ moiety is preferably in the L form.

6.1. Preferably, R ₁₁ is hydrogen. 6.2. Natural amino acid _{(i.e., H 2 N-CH (R} 13) -
R ₁₃ is --CH ₂ OH, --CH (CH ₃ ), such as a substituent attached to the α-carbon atom of
Preferably it is -OH, isobutyl or butyl.
Or R ₁₃ is — (CH ₂ ) ₂ —OH or — (CH ₂ ) ₃ —O
H. Especially, -CH ₂ OH or -CH (CH ₃₎
-OH is preferred. 6.3. X ₁ is

Embedded image Or a group represented by the formula -CH ₂ -OR _10, especially -
It is preferably a group represented by the formula CH ₂ —OR ₁₀ .
R ₁₀ is preferably hydrogen, but means that indicated by 7 below. Most preferably, R ₁₀ is hydrogen.

The following individual compounds are illustrative of compounds of formula I:

Embedded image It is also known as octreotide.

Embedded image

B. Analogues of formula II

Embedded image [Veil et al., Metabolism 27, Addendum 1, 139 (1978)]

Embedded image (See EP-A-200188) The entire contents of the above publications, including the specific compounds, are hereby incorporated by reference.

Particularly preferred "ligands" are derived from:

Embedded image

Suitable chelating groups are those that are physiologically acceptable and capable of forming a complex with a detectable element. The chelate group preferably has sufficient hydrophilicity. Examples of chelating groups include, for example, those derived from iminodicarboxyl groups, polyaminopolycarboxyl groups, and acyclic ligands, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylene glycol-O, O '-Bis (2-aminoethyl) -N, N, N', N '
-Tetraacetic acid (EGTA), N, N'-bis (hydroxybenzyl) ethylenediamine-N, N'-diacetic acid (HBE
D) and triethylenetetraamine hexaacetic acid (TTH
A), those derived from substituted EDTA or -DTPA, such as p-isothiocyanate benzyl-ED
TA, -DTPA, those derived from macrocyclic ligands, such as 1,4,7,10-tetraazacycloododecane-N, N ', N ", N"'-tetraacetic acid (DOTA), and 1, 4,8,11-tetraazacyclotetradecane-N,
Those derived from N-substituted or C-substituted macrocyclic amines including N ', N ", N"'-tetraacetic acid (TETA) cyclams, for example, EP-304780A1 and WO 89 / 01476-A Formula IV or V published in
A group represented by the formula,

Embedded image Wherein R ₁ , R ₂ , R ₃ are independently C _1-6 alkyl C _6-8 aryl, or C _7-9 arylalkyl, each optionally being OH, C _1-4 alkoxy, COOH, Or may be substituted with SO ₃ H, and n ′ is 1 or 2, i
Is an integer from 2 to 6, TT is independently an α or β amino acid (attached to each other via an amide bond), a group derived from a bisaminothiol derivative, for example, a compound represented by formula VI,

Embedded image In the formula, each of R ₂₀ , R ₂₁ , R ₂₂ and R ₂₃ is independently hydrogen or C _1-4 alkyl, a group capable of reacting with the N-amino group of the X ₂ peptide, m ′ is 2 or 3, Groups derived from thiacemicarbazone derivatives, such as compounds of formula VII

Embedded image Wherein X ₂ is as defined above, a group derived from a propyleneamine oxime derivative, for example a compound of the formula VIII

Embedded image In the formula, R ₂₄ , R ₂₅ , R ₂₆ , R ₂₇ , R ₂₈ , and R ₂₉ are each independently hydrogen or C _1-4 alkyl, X ₂ and m ′ have the meanings defined above, and diamide dimercaptide Groups derived from the class, for example compounds of formula IX:

Embedded image In the formula, X ₃ is a divalent group, which may be optionally substituted, and has a group capable of reacting with the N-amino group of the peptide, for example, a C _1-4 alkylene having an X ₂ group, or phenylene , Y ₅ is hydrogen or CO ₂ R ₃₀ ;
R ₃₀ is a C _1-4 alkyl or a group derived from a porphyrin, for example, N-benzyl-5,10,1.
5,20-tetrakis- (4-carboxyphenyl) porphine, or a TPP having an X ₂ group as defined above
and so on. Aryl is preferably phenyl. Arylalkyl is preferably benzyl.

Examples of X ₂ include-(X ₄ ) n "-X _{5 wherein} X ₄ is C _1-6 alkylene or, optionally, a carbon atom to which an oxygen atom or -NH- is bonded. An optionally substituted C _1-6 alkylene, etc. n ″ is 0 or 1,
X ₅ is —NCS, a carboxy group, or a functional derivative thereof, such as an acid halide, anhydride, or hydrazide. X ₂ is-[CH
₂ ] m'-, or = CH-CH = is believed to be attached to one of the carbon atoms.

[0021] The chelating group is the N of the somatostatin peptide.
-Can be bound directly or indirectly to an amino group. When bonded indirectly, it is preferable to bond via a crosslink or an intervening group. For example, it is a group of the formula (α ₁ ). ZR ₃₅ -CO- (α ₁ ) R ₃₅ is C _1-11 alkylene, C _2-11 alkenylene, or —CH (R ′) —, wherein R ′ is a natural or synthetic α.
-A residue linked to the α-position of an amino acid. For example, hydrogen, C _1-11 alkyl, benzyl, optionally substituted benzyl, naphthyl-methyl, pyridyl-
And methyl. Z is a functional group capable of reacting and covalently binding with a chelating reagent. Z is, for example, a group that can be combined with a chelating group to form an ether, ester, or amide. Chelating groups, carboxy, when containing a -SO ₃ H and / or amino groups,
It can be present in free or salt form. Preferred chelating groups are those derived from polyamino-polycarboxyl groups. For example, EDTA, DTPA, DOTA,
Such as those derived from TETA, or a substitution of EDTA or DTPA. Most preferably, the chelating group is derived from DTPA.

In the “ligand of the present invention”, a chelating group having a plurality of functional groups binds to one somatostatin peptide molecule, or to one or more somatostatin peptide molecules, for example, two somatostatin peptide molecules. Can be. "Ligands of the invention" can exist, for example, in free or salt form. Salts include acid addition salts, such as salts formed with organic, polymeric, or inorganic acids, such as hydrochlorides, acetates, and salts obtained with carboxylic and sulfonic acids present in the chelating group, such as sodium and potassium. Such an alkali metal or a substituted or unsubstituted ammonium salt.

The present invention also includes a method for producing the “ligand of the present invention”. These can be manufactured in a manner analogous to known methods. "Ligands of the invention" can be prepared, for example, as follows: a) Remove at least one protecting group present on a somatostatin peptide having a chelating group. Or b) linking the two peptide fragments with an amide bond. Each of the peptide fragments has at least one amino acid or amino alcohol, in protected or unprotected form, one of which contains a chelating group. Among them, the amide bond or the bond is formed in such a manner that a desired amino acid sequence can be obtained. Then, if desired, step a) of the reaction is performed. Or c) Bind the desired somatostatin peptide, in protected or unprotected form, with the chelating reagent such that the chelating group is on the desired N-amino group of the peptide. Then, if desired, the step a) is performed. Or d) removing an unprotected or protected functional group of the peptide having a chelating group, or converting it to another functional group to provide another unprotected having a chelating group, Alternatively, a protected form of the peptide is obtained. In the latter case, step a) of the reaction is performed. Or e) oxidize the somatostatin peptide modified with the chelating group. In that peptide, the mercapto group of the Cys group is present in a free form to create an analog in which the two Cys groups are SS cross-linked. The "ligand" thus obtained is regenerated in free or salt form.

The above-mentioned reaction is carried out, for example, by a method similar to the known method described in the Examples, in particular, the methods a) and c) described above. If the chelating group is amide linked, this can be done in a manner similar to that used for amide formation. In these reactions, a protecting group suitable for use of a peptide or a target chelating group can be used for a functional group which does not participate in the reaction, if desired. The term protecting group may also include polymer resins having functional groups.

It is desired that a chelating group be attached to the N-terminal amino group of the peptide or peptide fragment used as a starting material, and if the peptide contains one or more side-chain amino groups, these groups Chain amino groups are conveniently protected with protecting groups, such as those used in peptide chemistry.

It is desired that a chelating group be attached to the side chain amino group of the peptide or peptide fragment used as starting material, and if the peptide contains a free N-terminal amino group, the latter is protected by a protecting group. Preferably.

[0027] The peptide fragment having a chelating group and used in step b) may be obtained by reacting a peptide fragment containing at least one amino acid or amino alcohol in a protected or unprotected form with a chelating reagent. Can be prepared. The reaction is a process
This can be done in a manner similar to c). The chelating group of formula IV or V can be attached to the peptide by reacting a chelating reagent of formula IV 'or V'.

Embedded image Wherein X is an activating group capable of forming an amide bond with the N-amino group of the peptide. The reaction is EP2478
66A1.

The chelating reagent used in step c) of the reaction is known, and can be prepared by a method similar to a known method. Preferably, the chelating reagent is one that is capable of introducing the desired chelating group or somatostatin peptide. For example, the disclosed polyaminopolycarboxylic acids, their salts or anhydrides.

In the above reaction, when the amino acid, peptide or peptide fragment used as a starting material is bonded to the peptide via a chelating group or a peptide via a bridge or an intervening group, for example, via a group of the above formula (α ₁ ) When bonded together, such amino acids, peptide fragments or peptides include the corresponding amino acids or peptides without cross-linking or intervening groups, and the corresponding cross-linking or interposing group-forming compounds, for example, cross-linking groups or interposing groups, It can be prepared by reacting an acid or a reactive acid derivative, for example, an acid of the formula ZR ₃₅ -COOH or its reactive acid derivative such as an active ester. By way of example, active ester groups or carboxy activating groups such as 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, succinimidyl, or 1-
And hydroxy-benzotriazolyl.

Alternatively, the chelating reagent can first be reacted with a compound that forms a bridging or intervening group to provide a bridging or intervening group, and then reacted with a peptide, peptide fragment or amino acid in a conventional manner.
The “ligand of the present invention” can be purified by a conventional method, for example, by chromatography. Preferably, the "ligand of the invention" has less than 5% by weight of the content of the peptide that does not have a chelating group attached. According to another aspect, the "ligand of the invention" is capable of forming a complex with a detectable element.

Accordingly, the present invention relates to a “ligand of the invention” (hereinafter referred to as a “ligand of the invention” complexed with a detectable element in free or salt form (hereinafter referred to as the chelate of the invention) as defined above. , Referred to as chelates of the present invention), and methods for their manufacture, as well as uses for in vivo diagnostic and therapeutic treatments. Detectable elements include any element that can be suitably detected in a therapeutic or in vivo diagnostic technique, preferably a metal ion. For example, a metal ion that emits detectable radiation, or N
Metal ions that affect the MR relaxation characteristics.

Suitable detectable metal ions include, by way of example, heavy metal ions, or rare earth ions such as those used in CAT scans (computer axial tomography), paramagnetic ions such as Gd ³⁺ , Fluorescent metal ions such as Fe ³⁺ , Mn ²⁺ , Cr ²⁺ ,
For example, Eu ³⁺ , and radionuclides such as γ-emitting radionuclides, β-ray emitting radionuclides, α-ray emitting radionuclides, positron emitting radionuclides, such as ⁶⁸ Ga.

Suitable gamma-emitting radionuclides include those useful in diagnostic techniques. The gamma-emitting radionuclide advantageously has a half-life of 1 hour to 40 days, preferably 5 hours to 4 days, more preferably 12 hours to 3 days. For example, radionuclides derived from gallium, indium, technetium, ytterbium, rhenium and thallium, for example, ⁶⁷ Ga, ¹¹¹ In, ^99m T
C, ¹⁶⁹ Yb and ¹⁸⁶ Re. It is preferred to select a gamma radionuclide in accordance with the metabolism of the "ligand of the invention" or the somatostatin peptide used. More preferably, the "ligand of the invention" chelates gamma-emitting nuclides with a half-life longer than that of the somatostatin peptide on the tumor. Suitable radionuclides for use in imaging are positron emitting radionuclides, such as those described above.

Suitable beta-emitting radionuclides include those useful for therapeutic administration. For example, ⁹⁰ Y, ⁶⁷ -Cu,
¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁶⁹ Er, ¹²¹ Sn, ¹²⁷ Te, ¹⁴³ Pr,
¹⁹⁸ Au, ¹⁰⁹ Pd, ¹⁶⁵ Dy, ³² P, ¹⁴² Pr and the like. β
The radiation-emitting nuclide advantageously has a half-life of 2.3 hours to 14.3 days, with 2.3-100 hours being preferred.
Preferably, the beta-emitting radionuclide is selected to have a longer half-life on the tumor than the somatostatin peptide. Suitable alpha-emitting radionuclides are those used in therapeutic treatments, such as ²¹¹ At, ²¹² Bi.

The "chelates of the present invention" can be prepared by reacting a "ligand" with a corresponding compound for forming a detectable element, for example, a metal salt, preferably a water-soluble salt.
The reaction may be performed by a known method, for example, perine, an organic ligand chemical data series (Organic Ligand, Ch.).
emical Data Series 22. New York Pergamon Press (1982), Grejukarito and Tucker, Biophysical and Biochemical Research Communications (Biophys. Biohem. Res. Com.) 7 .
7 : 581 (1977) and Wagner and Werch, Journal of Nuclear Medicine (J. Nucl.
Med.) 20 428 (1979). Preferably, the "ligand" complex is formed at a pH at which the "ligand of the invention" exhibits a physiologically stable state.

[0036] Alternatively, the detectable element can also be added to the solution as a complex with a mediating chelating reagent.
Mediating chelating agents include, for example, the physiological
pH, a chelate reagent that forms a chelate complex that solubilizes the element, but is more thermodynamically unstable than the chelate. Examples of such mediating chelating agents include 4,5
-Dihydroxy-1,3-benzene-disulfonic acid (Tiron). In such reactions, the detectable element replaces the ligand.

A "chelate of the invention" also binds a chelating reagent complexed with a detectable element and a protected or unprotected form of a somatostatin peptide, and if present, at least It can be made by removing one protecting group. The same reaction occurs with a chelating reagent that forms a complex with an undetectable metal ion, and in the resulting peptide complex, the metal ion can be replaced with the desired detectable element.

A "chelate of the invention" also binds a chelating reagent complexed with a detectable element and a protected or unprotected form of a somatostatin peptide fragment containing at least one amino acid,
Peptide synthesis can then be continued in a stepwise manner until the final peptide sequence is obtained, if desired, by removing at least one protecting group present. Instead of a detectable element, the chelating reagent can form a complex with the undetectable metal, and the resulting somatostatin peptide can then replace this metal with the detectable element.

When the chelating group is linked to the somatostatin peptide via a bridging or intervening group, for example via a group of formula (α ₁ ) as defined above,
The somatostatin peptide and either the peptide fragment or the chelating agent can have the above-mentioned cross-linking or intervening groups.

The reactions described above can be carried out in analogy to known methods. Depending on the chelating groups present, the labeling efficiency will be close to 100% and may not require purification. For example, Technetium-9
Radionuclides such as 9 m are oxidized, for example Tc-99
Used in a form such as m-pertechnetate, which can complex under reduced conditions.

The above-described reaction is preferably carried out in a state in which trace amounts of metal impurities are removed. Deionized distilled water, ultrapure reagents, chelation-grade radioactivity, etc. are used to reduce the effects of trace metals.

The "chelate of the present invention" can exist, for example, as a free form or as a salt. Salts include, for example, acid addition salts with organic, polymeric, or inorganic acids, such as hydrochloric acid, acetic acid, and the like, and salts obtained with carboxylic acids present in molecules that do not participate in chelation, such as sodium Examples include alkali metal salts such as potassium, or substituted or unsubstituted ammonium salts.

The "chelates of the invention" and pharmaceutically acceptable salts exhibit pharmacological activity. Therefore, it can be complexed with, for example, paramagnetic ions, gamma-emitting metal ions or positron-emitting radionuclides to visualize somatostatin receptor-positive tumors or metastases, or complexed with α or β radionuclides. Are useful as radiopharmaceuticals for treating somatostatin receptor positive tumors or metastases in vivo. These can be proven by standard tests. In particular, the "chelates of the invention" have an affinity for the somatostatin receptor expressed or overexpressed by tumors and metastases, as shown by standard binding assays in vitro.

A somatostatin receptor-positive tumor derived from the human gastrointestinal tract is removed from a patient and immediately placed on ice,
Freeze at -80 ° C for up to 30 minutes. For further autoradiography, the frozen material was cut into 10 μm pieces in a cryostat (Rights 1720), mounted on washed microscope slides and at least 3 ° C. at −20 ° C.
Store for days to increase tissue adsorption to slides. The fragment was pre-incubated for 10 minutes at room temperature in Tris-HCl buffer (50 mM, pH 7.4) containing CaCl ₂ (2 mM) and KCl (5 mM), and then incubated in the same buffer without salt. Wash twice for minutes. This fragment was then obtained from bovine serum albumin (10 g / l), bacitracin (4
0 mg / l) and MgCl ₂ (5 mM) in Tris-HCl buffer (170 mM, pH 7.4) at room temperature for 2 hours with the “chelates of the invention”
Inhibits endogenous proteases. Non-specific binding is

Embedded image At a concentration of 1 μM and quantified. The incubated fragments are washed twice for 5 minutes with cold incubation buffer containing 0.25 g / l BSA. Immersed short while in distilled water to remove excess salts, fragments rapidly dried, put into ^[3 H] -LKB film. After exposure to an X-ray cassette for about one week, the "chelates of the invention", eg, the "chelates" of radionuclides, have a concentration of 10 ^-10 to 10 ^-3 M
When added at a concentration of, very good results are obtained in labeling the tumor tissue without labeling the surrounding healthy tissue. This binding of the in vitro assay revealed that the compound of Example 5 highly specifically labeled somatostatin receptor positive tumor tissue with low non-specific background.

The affinity of the "chelates of the invention" for the somatostatin receptor can also be demonstrated in in vivo tests. Rats with transplantable exocrine pancreatic somatostatin receptor positive tumors were treated with an intravenous injection of the "chelates of the invention". The injection site is the blood vessel of the penis. Immediately after dosing, animals were placed on a gamma camera collimator and the distribution of radioactivity was monitored at various time intervals. The biodistribution of radioactivity can also be determined by serially necropsying a number of rats so treated and assaying the organ for radioactivity. The "chelates of the invention", e.g. "chelates" of radionuclides, e.g.
"Ligand" labeled with ~ 2 mCi of radionuclide
After administration at a dose of 5 μg / kg, the tumor site becomes detectable along with the organs that are actually secreted. The compound of Example 5 is administered intravenously to animals at a dose corresponding to 4 μg / kg body weight of the labeled compound of Example 1 at 0.5 mCi ¹¹¹ In. Radioactivity was 3 minutes, 10 minutes, 15 minutes after injection.
Minutes, 20 minutes, 20 hours, 48 hours. 3 after injection
In minutes, radioactivity was detected in kidney, bladder, and tumor sites. Twenty hours after injection, the radioactivity increases and is very highly localized at the tumor site.

Thus, in a series of specific or alternative embodiments, the present invention also provides: 1. An in vivo method for detecting a somatostatin receptor-positive tumor or metastasis in a subject, comprising: a) administering the "chelate of the invention" to the subject; and b) recording the location of the receptor targeted by the "chelate". "Chelates of the Invention" for Use in the In Vivo Detection Methods of the Invention
Is a gamma-emitting radionuclide, a positron-emitting radionuclide or a paramagnetic metal ion, eg, a "chelate" complexed with those described above.

The "chelate of the present invention" used as an image forming agent in the method (1) may be parenterally, preferably intravenously,
It can be administered, for example, in the form of injectable solutions or suspensions, preferably in a single injection. Suitable doses will, of course, vary with, for example, the "ligand" and the type of detection element used, for example, the radionuclide. Suitable injection doses are those that can be imaged by photographic scanning methods known in the art. Radiolabeled "chelates of the invention"
When used, the radioactivity is 0.1 to 50 mCi, preferably 0.1 to 30 mCi, more preferably 0.1 to 20 mCi.
It may be advantageous to administer a dose having Ci.

In animals, the indicated dose is 0.1-2 mCi.
The "ligand" labeled with a gamma-emitting radionuclide, for example ¹¹¹ In, can range from 0.1 to 10 μg / kg. For large animals, for example humans, the indicated dose is 0.1-50 mC.
i, preferably 0.1-30 mCi, for example 3-15 mCi, of a "ligand" 1-2 labeled with a gamma-emitting radionuclide, the amount of which depends on the gamma-emitting radionuclide used.
It can be in the range of 00 μg. For example, in the case of In, it is preferable to use a low range of radioactivity, and in the case of Tc, it is preferable to use a high range of radioactivity.

After enrichment of the tumor (cancer) forming site by "chelate", the corresponding imaging technique, such as a nuclear medicine imaging device such as a scanner, gamma camera, rotary gamma
Techniques using a line camera (preferably each computerized), a PET scanner (positron emission tomography), an MRI device or a CAT scanning device may be used. Most of the "chelates of the invention", e.g., gamma-emitting "chelates" are substantially excreted from the kidneys and do not significantly accumulate in the liver.

2. An in vivo method of treating somatostatin receptor positive tumors and metastases in a subject in need of treatment comprising administering to the subject a therapeutically effective amount of a "chelate of the invention". A "chelate of the invention" for use in the in vivo treatment method of the invention is a "chelate" complexed with an alpha or beta emitting radionuclide as described above. The dose used in practicing the therapy of this invention will, of course, depend, for example, on the particular condition to be treated, such as the volume of the tumor, the particular “chelate” used, eg, the half-life of the “chelate” in the tumor, and Depends on treatment. Generally, doses are calculated based on the radioactivity distribution for each organ and the observed target uptake. For example, 0.1-3 mCi
/ Kg body weight, for example 1-3 mCi, preferably 1-1.5 m
"Chelates" can be administered in a daily dose range having a radioactivity of Ci / Kg.

For animals, the indicated dose is 0.1-3 mCi.
Α- or β-emitting radionuclides, eg ⁹⁰ Y labeled “ligands” may range from 0.1-5 μg / kg. In the case of large animals, for example humans, the indicated daily dose is 0.1-3 m.
"Ligand" 1-labeled with an alpha or beta emitting radionuclide at Ci / kg body weight, for example 0.1-1.5 mCi / kg body weight.
It may be in the range of 200 μg, which is conveniently administered in divided doses no more than four times a day.

The α- or β-emitting radionuclides of the invention can be administered by any conventional route, in particular by the parenteral route, for example in the form of injectable solutions or suspensions. They may also be advantageously administered by infusion, eg, 30-60 minutes. Depending on the site of the tumor, they are as close as possible to the site of the tumor and can be administered, for example, by a catheter. The administration method can be selected according to the dissociation rate and excretion rate of the “chelate” used. The "chelates of this invention" can be administered in free form or in pharmaceutically acceptable salt form. Such salts can be prepared by conventional methods and exhibit the same order of activity as the free compounds.

The "chelate of the present invention" used in the method of the present invention is produced immediately before administration to a subject, that is, radiolabeled with a target metal ion, particularly a target α, β or γ radionuclide. Is administered slightly prior to administration. “Chelates of the invention” include tumors of the pituitary gland, gastrointestinal pancreas, central nervous system, breast (breast), prostate, ovary or colon, small cell lung cancer, paragangliomas,
Suitable for imaging or treating tumors such as neuroblastoma, pheochromocytoma, myeloid thyroid carcinoma, myeloma and their metastases.

According to another aspect of the invention, gamma-emitting "chelates of the invention" can also be used as imaging agents for evaluation of renal function. A group of 5 mice was used. Each mouse was intravenously injected via the tail vein with 0.1 ml of a solution containing 1 mCi of the "chelate of the invention". Mice were placed in a metabolic cage for urine collection. Injection 1
The urethra was ligated 0 or 120 minutes later, and the mice were anesthetized with chloroform. The urine-producing system was imaged by conventional imaging techniques. In this study, imaging of renal excretion was demonstrated when the gamma-emitting "chelates of the invention" were administered at a dose of 0.1-30 mCi. Accordingly, the invention also provides an in vivo method for assessing renal function in a subject, comprising administering to the subject an effective amount of a gamma-emitting "chelate" and recording renal function.

According to another aspect of the present invention, there is provided: i) A pharmaceutical composition comprising a free form of the "ligand of the invention" or a pharmaceutically acceptable salt form thereof together with one or more pharmaceutically acceptable carriers or diluents. ii) A pharmaceutical composition comprising the free form of the "chelate" of the invention or a pharmaceutically acceptable salt form thereof together with one or more pharmaceutically acceptable carriers or diluents.

Such a composition can be produced by a conventional method. The composition of the invention can be supplied as a separate package with instructions for mixing the "ligand" with the metal ion and administering the resulting "chelate". It can also be supplied in a single package, ie, a twin pack, containing separate unit doses of the "ligand" and the detectable metal ion, along with instructions for mixing and administering the "chelate". The carrier or diluent can be in unit dosage form. In the following examples, all temperatures are in ° C.
And uncorrected together with the [α] ²⁰ _D value. Use the following abbreviations: Boc: t-butoxycarbonyl TFA: trifluoroacetic acid DTPA: diethylenetriaminepentaacetic acid

Embodiment 1

Embedded image Of the free base form

Embedded image 1.1 g (1 mM) was dissolved in 5 l of dioxane / water 1/1 (v / v) and reacted with 5 g of NaHCO ₃ . 520 mg of DTPA dianhydride were slowly added with stirring. The reaction mixture was stirred for a further 30 minutes and lyophilized. The residue was dissolved in 250 ml of water and the pH was adjusted to 2.5 with concentrated HCl. The precipitated product was filtered, washed and dried with phosphorus pentachloride. After silica gel column chromatography, the following product was isolated.

Embedded image 230 mg and the corresponding dimer

Embedded image 500 mg. 3 ml of TFA

Embedded image 200 mg were mixed. After 5 minutes at room temperature, the mixture was precipitated with diisopropyl ether, filtered and dried. The residue was desalted and lyophilized with Duolite to give 150 mg of the title compound. [α] ²⁰ _D = −26.6 ° (c = 1, 95% acetic acid)

The starting materials can be prepared as follows.

Embedded image Di-tert-butyl-pyrocarbonate dissolved in 30 ml of DMF 2.
25 g in 100 ml DMF

Embedded image The solution was slowly added dropwise to the 10 g solution at room temperature. After 2 hours at room temperature, the solvent was removed by suction under vacuum and 200 ml of diisopropyl ether were added to the residue. The precipitate formed was filtered, washed with diisopropyl ether and dried. Silica gel chromatography of the crude product (eluent: CH
₍ Cl ₂ / MeOH 9/1), after which a white amorphous powder was isolated. [α] ²⁰ _D = 29.8 ゜ (c in DMF = 1.28)

Embodiment 2

Embedded image Intermediate product obtained in Example 1

Embedded image Was treated as described above for the corresponding monomeric form to remove the Boc protecting group to give the title compound. [α] ²⁰ _D = −24.5 ° (c = 0.55, 95% acetic acid)

Embodiment 3

Embedded image

Embedded image 0.56 g, 0.5 mmol of Fmoc-ε-aminocaproic acid and 115 mg of hydroxybenzotriazole in DMF
Dissolved in 10 ml and cooled to -30 ° C. Add DM to this solution
A solution of 115 mg of dicyclohexylcarbodiimide in 5 ml of F (cooled to -10 DEG C.) was added. During the reaction for 24 hours, the mixture was returned to room temperature, and then the resulting dicyclohexylurea was filtered, and the filtrate was diluted with 10 times the amount of water. The precipitated reaction product was filtered, washed and dried over phosphorus pentachloride. The crude product was used for the next step without further purification.

B) Fmoc cleavage 0.5 g of the crude product from the coupling reaction (a) was added to DMF /
The mixture was treated with piperidine 4/1 v / v (clear liquid) at room temperature for 10 minutes, and then mixed with 100 ml of diisopropyl ether. The reaction product precipitate was filtered, washed and dried. The crude product was used for the next step without further purification.

C) BOC cleavage 300 mg of the crude product obtained in (1.b)
(Complete dissolution) 5 ml was treated with 5 ml at room temperature, and then mixed with 50 ml of diisopropyl ether. After adding 2 ml of HCl / diethyl ether, the resulting precipitate was filtered, washed and dried under high vacuum. The final product is purified by silica gel chromatography (CHCl ₃ / MeOH / AcOH 7/3 /
Purification by 0.5 / 0.5) and desalted with Duolite followed _{(gradient: H 2 O / AcOH 95/5 ---} H 2 O /
Dioxane / AcOH 45/50/5). The title mixture was obtained as the acetate (white lyophilizate). [α] ²⁰ _D = −32 ° (c = 0.5, 95% acetic acid) The reaction of DTPA according to the steps of Examples 1 and 2 was used for the resulting compound.

Example 4 The following ligand was prepared according to the method described in Examples 3 and 1.

Embedded image [α] ²⁰ _D = 14.8 ° (c = 0.5, 95% acetic acid)

Embodiment 5

Embedded image

Embedded image 1 mg was dissolved in 5 ml of 0.01M acetic acid. The resulting solution was passed through a 0.22 μMirex-GV filter and 0.1 ml
And stored at -20 ° C. ^This was carried out by pre-diluting ¹¹¹ InCl ₃ (Amersham, 1 mCi / 100 μl) with the same volume of 0.5 M sodium acetate, and mixing and gentle homogenization of the InCl ₃ solution and ligand at room temperature. HEPES buffer and pH 7.4 were added to make a 10 ^-6 M solution.

Example 6 ⁹⁰ Y-labelled

Embedded image The ⁹⁰ Y were obtained from ^{90 Sr-90} Y radionuclide generator. The instrument configuration, elution and conversion of [ ⁹⁰ Y] EDTA to acetate complex is described in Journal of Nuclear Medicine.
(J. Nucl. Med.) 28: 1465-1470 (19
1987). Tinol and D.I. J. Performed by the method described by Natwich. Dissolved in 5 ml of 0.01 M acetic acid

Embedded image 1 g is brought to room temperature and ⁹⁰ Y 1.0 in 50 μl of sterile 0.5 M acetic acid.
mCi was added. The mixture was then left gently for 30 minutes to 1 hour to maximize chelation.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Stephen V. J. Lamberz 3055 Gäu J. Rotterdam, Molenlan 172 (72) Inventor Janos Press 4054 Basel, Switzerland, Cruise Strasse 24

Claims (8)

[Claims] The present invention relates to a somatostatin receptor consisting of a somatostatin peptide and a chelate group chemically bonded to a terminal amino group of the peptide via a bridging group or an intervening group or not. A carrier for preparing a detectable element-containing drug. 2. The carrier according to claim 1, wherein the detectable element is a radioactive element and the drug is a radiopharmaceutical. 3. The carrier according to claim 1, wherein the medicament is for treating a disease. 4. The carrier according to claim 1, wherein the drug is for diagnosis of a disease. 5. A carrier comprising a somatostatin peptide and a chelating group chemically bound to a terminal amino group of the peptide via a bridging group or an intervening group with or without a bridging group, carrying a detectable element. An agent having the property of binding to the somatostatin receptor. 6. The drug according to claim 5, wherein the detectable element is a radioactive element and the drug is a radiopharmaceutical. 7. The drug according to claim 5, wherein the drug is for treating a disease. 8. The drug according to claim 5, wherein the drug is for diagnosing a disease.