FI102540B - Somatostatin peptide derivative with a chelating group, for diagnostic use - Google Patents

Description

102540

FOR THE DIAGNOSTIC USE OF SOMATOSTATIN PEPTIDE DERIVATIVES CONTAINING A CHELATING GROUP

The invention relates to polypeptide derivatives, to a process for their preparation, to their use as diagnostic agents and to diagnostic kits containing them.

Over the last couple of years, a large number of somatostatin receptors have been shown to be present in a large number of human tumors, e.g., pituitary tumors, central nervous system tumors, breast tumors, gastrointestinal pancreatic tumors, and their metastases. Some of them are small or slow-growing tumors that are difficult to pinpoint accurately by conventional diagnostic methods.

In vitro visualization of somatostatin receptors has been performed by autoradiography of tumor tissue using radioiodinated somatostatin or somatostatin analogs, e.g., [125 I-Tyru] somatostatin-14 (Taylor, JE et al., Life Science (1988) 43: 20 421). or [125I-Tyr3] SMS 201-995 is also said to be [125I] 204-090 (Reubi, JC et al., Brain Res. (1987) 406: 891; Reubi, JC et al., J. Clin. Endocr. Metab.

(1987) 65: 1127; Reubi, J.C. you do not. ai., Cancer Res.

(1987) 47: 551; Reubi, J.C. you do not. ai., Cancer Res.

25 (1987) 47: 5758).

GB 2 109 407, EP patent applications 35765, 38546, 103049, 174853, 233619, 238196, FI patent publication 81263 and U.S. patent publication 4 287 362 disclose conjugates for therapeutic or diagnostic use in which the active ingredient a chelating group is attached to the substance, which in turn is accompanied by a suitable detectable element, and the active ingredient discloses a protein, e.g., human serum albumin, an antibody such as an antibody to carcinoembryo antigen (CEA), or an antibody fragment, an antibiotic, hormone 2 102540 ni, saccharide, fatty acid, etc. EP 248506 describes a method for attaching a metal ion to a protein or polypeptide, the method comprising attaching the metal ion to the active ingredient via a chelating group 5. Examples are antibodies, with only a general mention of polypeptides. .

Ko. common to the publications is that the proteins, antibodies, enzymes, etc. disclosed therein are large molecules with a high molecular weight and contain a number of reactive side chains that can be used as a chelating group attachment site, e.g., amino, carboxy, SH- groups, etc. These groups are randomly distributed throughout the structure of the protein or the like. For example, the reaction between a protein and a chelating agent generally produces a randomly substituted protein. In this case, it is unclear to which chelating groups are attached and how many chelating groups are attached to one molecule. Due to random substitution, labeled proteins and antibodies have many disadvantages: poor homogeneity and reproducibility cause problems, purification is problematic, they lack sensitivity to small tumors, the ability of the antibody to bind antigen is reduced, and so on.

Novel somatostatin peptides which are: useful as diagnostic agents and which can be labeled for in vivo diagnostics have now been invented.

According to the invention, there is provided a somatos-30 tatine peptide having at least one detectable. chelating group, this chelating group is attached to the amino group of said peptide, and this amino group has no significant binding affinity for somatostatin receptors.

These compounds are hereinafter referred to as ligands of the invention. They have one chelating group which can react with a detectable 3 102540 element, e.g. a radionuclide, an X-ray opaque element or a paramagnetic ion, to form a complex and can further bind to somatostatin receptors, e.g.

5 expressed or overexpressed by tumors or metastases.

The chelating group is covalently attached to the amino group of the peptide.

The chelating group is preferably attached to the terminal N-amino group of the soma-10 tostatin peptide.

According to the invention, the chelating group may be attached either directly or indirectly, e.g. by means of a spacer group, to the amino group of the somatostatin peptide.

One group of ligands is one in which the chelating group is attached directly to the amino group of the somatostatin peptide.

The second ligand moiety is one in which the chelating moiety is indirectly attached to the amino group of the somatostatin peptide by a bridging bond or a spacer group.

Preferably, the chelating group is attached to the peptide by an amide bond.

The term somatostatin peptide means naturally occurring somatostatin (tetradecapeptide) and its analogs or derivatives.

By derivatives or analogs, as the terms are used herein, is meant any straight-chain or cyclic polypeptide derived from a naturally occurring tetradecapeptide somatostatin in which one or more amino acid units have been removed and / or replaced with one or more other amino acid residues. wherein one or more functional groups have been replaced by one or more functional groups and / or one or more groups have been replaced by one or more other isosteric groups. Usually, the term encompasses all modified derivatives of 4,102,540 biologically active peptides that show qualitatively similar potency to the unmodified somatostatin peptide, e.g., they bind to somatostatin receptors and reduce hormone secretion.

Cyclic, crosslinked cyclic and straight chain somatostatin analogs are known compounds. Such compounds and their preparation are described, for example, in EP-A-1295; 10 29,579; 215.171; 203.031; 214.872; 298.732; 277.419.

Preferred ligands of the invention are those derived from the following somatostatin analogs: A. Analogs of formula I CH2-S-Yi Y2-S-CH2 I (I)

RCO-NH-CH-CO-B-C-Lys-E-NH-CH-G

Wherein RCO- is a) an L- or D-phenylalanine residue optionally substituted with F, Cl, Br, NO 2, NH 2, OH, C 1-3 alkyl and / or C 1-3 alkoxy, • - - or D-Trp or 3- ( 2-naphthyl) -D-Ala

Yj. and Y 2 are together a direct bond or Y 1 and Y 2 are independently hydrogen B is -Phe- optionally ring substituted with halogen, NO 2, NH 2, OH, C 1-3 alkyl and / or C 1-3 alkoxy (including pentafluoroananine) or B -naphthyl-Ala, 35 C is (L) -Trp- or (D) -Trp-, 5 102540 E is Thr, Ser, Val, Phe or Ile G is a group of formula 5 -CON <^ R12 where 10 R 11 is hydrogen or C 1-6 alkyl, R 12 is hydrogen, C 1-3 alkyl or a group of the formula - CH (R 13) - X 1, R 13 is CH 2 OH, - (CH 2) 2 -OH, - (CH 2) 3 -OH, -CH ( CH3) OH, isobutyl or butyl or is a substituent attached to the α-carbon atom of Trp or β-15 Nairn, X1 is a group of the formula -CH2OR10 or - CONR14R35, wherein R10 is hydrogen or a residue of a physiologically acceptable physiologically hydrolyzable ester r14 is hydrogen or C 1-3 alkyl and r 15 is hydrogen or C 1-3 alkyl, provided that when R 12 is -CH (R 13) -X 1, then R 11 is hydrogen or methyl, wherein residues B and E are in the L-configuration and 2 and 7 are the residues in position are each different independently in the (L) or (D) configuration.

In the compounds of the formula I, the following meanings are preferred, either individually or in combination or subcombination: 1.1. RCO is preferably a '), L- or D-phenylalanine 3 or a tyrosine residue, optionally mono-N-C 1-12 alkylated. Most preferably a ') is an L- or D-phenylalanine residue.

β 102540 1.2. RCO is an α-amino acid residue having a hydrocarbon side chain, e.g. alkyl having 3, more preferably 4, or more C atoms, e.g. 7 C atoms, naphthylmethyl or heteroaryl, e.g. 3- (2- or 1-naphthyl) alanine, a pyridylmethyl or tryptophan residue and said residues are in the L or D configuration.

1.3. Most preferably RCO is a) especially a ') · 2. B is B', where B 'is Phe or Tyr.

3. C is C, wherein C is (D) Trp.

10 4. E is E ', where E' is Val or Thr, in particular

Thr.

5. G is G ', wherein G' is a group of the formula -CO-N 2, in particular a group of the formula 15 R 12 -CO-N <^ ^ CH (R 13) -X! (where Ru = H or CH3). In the latter case, the group -CH (R13) -X1 is suitably in the L-configuration.

5.1. RX1 is suitably hydrogen.

5.2. Since the substituent attached to the α-carbon atom of a natural amino acid (i.e. the formula H2N-CH (R13) -COOH), R13 is suitably -CH2OH, -CH (CH3) -OH, isobutyl or butyl or R13 is - (CH2) 2 -OH or - (CH 2) 3 -OH. In particular, it is -CH2OH or -CH (CH3) OH.

5.3. X 3 is, in particular, a group of the formula -CO-N 2 O 3. or -CH 2 -OR 10, in particular the formula -CH 2 -OR 10, and R 10 is suitably hydrogen or has the same meaning as in 7 below. Most preferably R10 is hydrogen.

The following individual compounds describe compounds of formula I: H- (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-Thr-ol 7 102540 is also known as octreotide

I I

(D) Phe-Cys-Thr- (D) Trp-Lys-Val-Cys-ThrNH2 I-1 5 (D) Phe-Cys-Tyr- (D) Trp-Lys-Val-Cys-TrpNH2 I-1 ( D) Trp-Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 I-1 10 (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 I-1 3- (2-Naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val-Cys-ThrNH2 I-1 15 3- (2-Naphthyl) - (D) Ala-Cys-Tyr- (D) ) Trp-Lys-Val-Cys-β-

Nal-NH2 I-1 3- (2-Naphthyl) - (D) Ala-Cys-fi-Nal- (D) Trp-Lys-Val-Cys-ThrNH2 20, -1 (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-B-Nal-NH 2 B. Analogs of formula II I-1

25 H-Cys-Phe-Phe- (D) -Trp-Lys-Thr-Phe-Cys-ol II

[See. Vale et. ai., Metabolism, 27, Supp, 1, 139, (1978)] I-1H-Cys-His-His-Phe-Phe- (D) Trp-Lys-Thr-Phe-Thr-Ser-Cys-

30 OH III

(see EP-A-200,188)

Including the contents of all of the above publications, the defined compounds are incorporated herein by reference.

Particularly preferred are ligands derived from 8 102540

I I

H- (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-Thr-ol.

Suitable chelating groups include physiologically acceptable chelating groups that can form a complex with a detectable element. Preferably, the chelating group has a substantially hydrophilic nature. Examples of chelating groups include, e.g., iminodicarboxylic groups, polyaminopolycarboxylic groups, e.g., those derived from non-cyclic ligands, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-pentaacetic acid (DTPA), ethylene-DTPA), ethyl (2-aminoethyl) -N, N, N ', N'-tetraacetic acid (EGTA), N, N'-bis (hydroxybenzyl) ethylenediamine-N, N'-diacetic acid (HBED) and triethylenetetraamine hexacetic acid ( TTHA), those derived from substituted EDTA or DTPA, e.g. p-isothiocyanato-benzyl-EDTA or -DTPA derived from macrocyclic ligands, e.g. 1,4,7,10-tetra- 20 azacyclododedecane-Ν, Ν ', N' ', N' '' - tetraacetic acid (DOTA) and 1,4,8,11-tetra-azacyclotetradecane-Ν, Ν ', Ν' ', N' '' - tetraacetic acid ( TETA), and those derived from N-substituted or C-substituted macrocyclic amines, including cyclames, e.g. as in EP 304,780 A1 and WO 89/01476-A described, groups of formula IV or V O 0 0.

Il H II

30 Ri-C-S- (CH2) n.-C- (TT) i-C- (IV) 35 9 102540

O O

Il II

R2-C-S- (CH2) n, -C-NH-CH2O O

II 5 (V) R 3 -C 5 - (CH 2) n, -C-Nff ilo 10 wherein R 3, R 2 and R 3 are each independently C 1-8 alkyl, C 6-8 aryl or C 7-9 arylalkyl, each optionally substituted with OH, C 1-4 alkoxy, COOH or SO 3 H, n 'is 1 or 2, i is an integer from 2 to 6, and TT is independently α or β amino acids linked together by amide bonds, groups derived from bis-aminothiol derivatives, e.g. compounds of formula VI ÄH <VI> 25 * 2Γ <n / SH HS s23 20 wherein each R20 'R21 / R22 and R23 independently of one another is hydrogen or C1-4alkyl, -. X 2 is a group capable of reacting with the N-amino group of the peptide, and m 'is 2 to 3.35 groups derived from dithiasemicarbazone derivatives, e.g. compounds of formula VII 102540 f * / τ \ 5 Α ° ΗΝ ΝΗ 1 Λ; (νιι) ΗΝ ^ SH HS ΝΗ where 10 Χ2 means the same as above, groups derived from propyleneamine oxime derivatives, e.g. compounds of formula VIII 15 A ch23? ^

ΗΝ NH

20 R24 | ^

OH OH

wherein each of R24, R25, R26, R27, R2e 3a R29 is independently hydrogen or C1-4alkyl and X2 and m 'are as defined above, groups derived from diamide mercaptides, e.g., compounds of formula IX

Y

"5 I I

35 H5CgCO COCgH- (IX) 11 102540 wherein X3 is a divalent radical which is optionally substituted and has a group which can react with the N-amino group of the peptide, e.g. C1-4alkylene or phenylene having a group X2 and Y5 is hydrogen or CO 2 R 30 where R 30 is C 1-4 alkyl or groups derived from porphyrins, e.g. N-benzyl-5,10,15,20-tetrakis- (4-carboxy-phenyl) porphine or TPP having a group X 2 which is defined as above.

Aryl is preferably phenyl. Aralkyl is preferably benzyl.

Examples of X2 groups contain radicals of the formula - (X4) n ..- X5, wherein X4 is C1-4alkylene; or C 1-6 alkylene optionally bonded to a carbon atom by an oxygen atom or -NH-, n '' is O or 1 and X 5 is -NCS, a carboxy group or a functional derivative thereof, e.g. an acid halide, anhydride or hydrazide. It is clear that X2 is attached to one carbon atom - [CH2] m.- or = CH-CH = by replacing one hydrogen atom.

The chelating group may be attached either directly or indirectly to the N-amino group of the somatostatin peptide. When indirectly bonded, it is suitably bonded via a bridging bond or via a space-consuming group, for example, a group of formula (ai).

Z-r35-CO- (ai) R35 is C1-alkenylene, C2-11alkenylene or CH (R ') -, wherein R' is a residue attached to a natural or synthetic α-amino acid of α-carbon, e.g. hydrogen , C 1-6 alkyl, benzyl, optionally substituted benzyl, naphthylmethyl, pyridylmethyl, Z is a functional group that can react covalently with a chelating agent.

12 102540 Z may, for example, be a group which may form an ether, ester or amide bond with the chelating group. Z is preferably amino.

The chelating group, when it contains carboxy, 5-SO 3 H and / or amino groups, may be in free or salt form.

Preferred chelating groups are those derived from polyamino-polycarboxyl groups, e.g., those derived from EDTA, DTPA, DOTA, TETA, or substituted EDTA or DTPA. The most preferred are chelating groups derived from DTPA.

In the ligands of the invention, the chelating group, when polyfunctional, may be attached to either one somatostatin peptide molecule or to more than one somatostatin peptide molecule, e.g., two somatostatin peptide molecules.

The ligands of the invention may be in free or salt form. Salts mean acid addition salts, e.g. with organic acids, polymeric acids or inorganic acids, e.g. hydrochlorides and acetates, as well as salt forms obtained with carboxylic or sulphonic acid groups present in the chelating group, e.g. alkali metal salts such as sodium. - or potassium, or substituted or unsubstituted ammonium salts.

The present invention also relates to a process for the preparation of the ligands according to the invention. They can be prepared analogously according to known methods.

The ligands of the invention can be prepared, e.g., by: a) removing at least one protecting group present in a somatostatin peptide having a chelating group, or b) linking two peptide fragments each containing at least one amino acid or amino alcohol in a protected or amide bond. in an unprotected form and one of which contains a chelating group in which the amide bond is such that the desired amino acid sequence is obtained, and optionally followed by step a), or c) linking the chelating agent and the desired soma-5 tostatin peptide in protected or unprotected form with in such a way that the chelating group is attached to the desired N-amino group of the peptide, and optionally then carrying out step a), or d) removing the functional group from the unprotected or protected peptide having a chelating group or converting it to another functional group n to obtain a second unprotected or protected peptide having a chelating group, and in the latter case carrying out step a), or e) oxidizing a somatostatin peptide modified with a chelating group in which the mercapto groups in the Cys radicals are in free form to form an analog , in which the two Cys radicals are linked via an SS bridge, and by regenerating the ligand thus obtained in free form or in salt form.

The above reactions can be carried out analogously to known methods, e.g. as described in the following examples, in particular methods a) and c). When the chelating group is attached by an amide dis bond, this can be done analogously to the methods used to prepare the amide. If desired, protecting groups suitable for use with the peptides or in the desired chelating groups as functional groups which do not participate in the reaction may be used in these reactions. The term protection; The va group also comprises a polymeric resin having functional groups.

If it is desired to link the chelating group to the N-amino group or peptide fragment of the peptide used as a starting material and containing one or more amino groups in the side chain, these side chain amino groups are conventionally protected with a protecting group, e.g. as used in peptide chemistry.

If it is desired to link the chelating group to an amino group or peptide fragment of the peptide side chain used as a starting material, and the peptide contains a free terminal N-amino group, the latter usually being protected by a protecting group.

The peptide fragment having a chelating group used in step b) can be prepared by reacting a peptide fragment containing at least one amino acid or amino alcohol in protected or unprotected form with a chelating agent. The reaction can be carried out analogously to step c).

Chelating groups of formulas IV or V can be attached to the peptide by introducing chelating agents of formulas IV or V, O 0 0

Il II II

20 R1-C-S- (CH2) n.-C- (TT) i-C-X (IV) o o

Il H

R2-C-S- (CH2) n.-C-NH-CH2 O

25 \ «(V)

R3-C-S- (CH2) n. -C-NTT

Il I

o o 30 wherein X is an activating group that can form an amide bond to react with the N-amino group of the peptide. The reaction can be carried out as described in EP 247,866 A1.

The chelating agent used in step c) may be known in the art or may be prepared according to methods known in the art. The compound used is such as to allow the introduction of the desired chelating group 10254040 into the somatostatin peptide, e.g. a polyaminopolycarboxylic acid as described, a salt or anhydride thereof.

In the above method, if the amino acid-5, peptide fragments or peptides used as starting materials have a chelating group attached to the peptide via a bridging bond or a spacer group, e.g. a radical of formula (ai) as defined above, such amino acids, peptide fragments 10 or the peptides can be prepared by reacting the corresponding amino acids or peptides without a bridging or bulking group in a conventional manner with a corresponding bridging or bulking compound, for example an acid or a reactive acid derivative containing a bridging or bulking group, e.g. an acid of formula Z-R35-COOH or a reactive acid derivative thereof such as an active ester. Examples of active esters or carboxy activating groups are, for example, 4-20 nitrophenyl, pentachlorophenyl, pentafluorophenyl, succinimidyl or 1-hydroxybenzotriazolyl.

Alternatively, the chelating agent may first be reacted with a bridging or bulking group agent to form a bridging or bulking group, and then reacted with the peptide, peptide fragment or amino acid in a conventional manner.

The ligands of the invention can be purified in a conventional manner, e.g. by chromatography. Preferably, the ligands of the invention contain less than 5% by weight of peptides without chelating groups.

The ligands according to the invention in free form or in the form of physiologically acceptable salts are valuable compounds. According to another embodiment, the ligands of the invention may form a complex with a detectable element.

Thus, the present invention also provides ligands of the invention, as defined above, which form complexes with a detectable element (hereinafter referred to as chelates of the invention), in free form or in salt form, their preparation and their use in vivo for diagnostic purposes. purposes.

By detectable element is meant any element, preferably a metal ion, which exhibits a property detectable in in vivo diagnostic methods, e.g., a metal ion which imparts detectable radiation, or a metal ion capable of affecting NMR relaxation properties. .

Suitable detectable metal ions are, for example, heavy elements or rare earth metal ions, e.g. as used in CAT (Computer axial tomography) scanning, paramagnetic ions, e.g. Gd3 +, Fe3 +, Mn2 + and Cr2 +, fluorescent metal ions. ions, e.g. Eu3 +, and radionuclides, e.g. γ- radiating radionuclides, positron emitting radionuclides e.g. 68Ga.

Radionuclides suitable for diagnostic methods include γ-emitting radionuclides. The γ-25 radiating radionuclides preferably have a half-life of 1 h to 40 days, preferably 5 h to 4 days, most preferably 12 h to 3 days. Examples are radionuclides obtained from gallium, indium, technetium, ytterbium, rhenium and thallium, e.g. 67Ga, luIn, 99mTc, 169Yb and 186Re. Preferably, the γ-radionuclide is selected depending on which metabolism or somatostatin peptide of the ligand of the invention is used. Most preferably, the ligand of the invention is chelated with a γ-radionuclide having a longer half-life than the somatostatin peptide in the tumor.

17 102540

Other radionuclides suitable for use in imaging include radionuclides emitting positrons, e.g., as described above.

The chelates of the invention can be prepared by reacting the ligand to the appropriate level. with an elemental compound, e.g. a metal salt, suitably a water-soluble salt. The reaction can be carried out analogously to known methods, e.g. as described in: Perrin, Organic Ligang, Chemical Data Series 22. NY Pergamon Press (1982); Krejcarit and Tucker, Biophys. Biochem. Res. Corn. 77: 581 (1977) and Wagner and Welch, J. Nucl. Med. 20: 428 (1979).

Most preferably, the ligand complex is formed at a pH at which the ligand of the invention is physiologically stable.

Alternatively, the detectable element may be introduced into solution complexed with another co-chelating agent, e.g., a chelating agent that forms a chelating complex that solubilizes the element at the physiological pH of the ligand but is thermodynamically less stable than the chelate. An example of such an auxiliary chelating agent is 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). In such a method, the detectable element changes the ligand.

The chelates of the invention may also be prepared by combining a chelating agent, complexed with a detectable element, and somatostat in protected or unprotected form. peptide, and if desired by removing at least one protecting group present. The same reaction can be performed when the chelating agent forms a complex with an undetectable metal ion, and in the resulting complex peptide, the metal ion can be replaced with the desired detectable element.

18 102540

The chelates of the invention may also be prepared by combining a chelating agent, complexed with a detectable element, and a somatostatin peptide fragment containing at least one 5 amino acid in protected or unprotected form, and then continuing peptide synthesis step by step until the final peptide sequence is obtained, and if desired. removing at least one protecting group present. Instead of a detectable element, the coil-10 agent may form a complex with a non-detectable element, and this metal may be replaced with a detectable element in the final complex in the somatostatin peptide.

When the chelating group is attached to the somatos-15 tatine peptide via a bridging bond or a spacer group, e.g., via a radical of formula (ai) as described above, the bridging bond or spacer group may be in either the somatostatin peptide or peptide fragment or chelate. in a desirable substance.

The above reactions can be carried out analogously according to known methods. Depending on the chelating group present, the labeling efficiency may be close to 100% so that no purification is required. Radionuclides such as technetium-99m can be used in oxidized form, e.g., Tc-99m pertechnetate, which can be complexed under reducing conditions.

The above reactions are conventionally carried out under conditions which avoid trace metal contamination. Preferably, distilled deionized water, ultrapure reagents, chelating asteroid activity, etc. are used to reduce the effects of the trace metal.

The chelates of the invention may, for example, be in free or salt form. Salts include acid addition salts with e.g. organic acids, polymeric acids or inorganic acids, e.g. hydrochlorides and acetates, as well as salt forms obtained from carboxylic acid groups present in the molecule which do not participate in chelation, e.g. alkali metal salts such as sodium or potassium, or substituted or unsubstituted ammonium salts.

The chelates of the invention and their physiologically acceptable salts are useful either as an imaging agent, e.g., in the visualization of somatostatin receptor-positive tumors and metastases to form complexes with a paramagnetic, γ-emitting metal ion or a positron emitting radionuclide.

In particular, the chelates of the invention show affinity for somatostatin receptors, which are expressions or overexpressions of tumors and metastases, as demonstrated in in vitro binding experiments.

A somatostatin receptor-positive tumor from the human gastrointestinal tract is removed from the patient and immediately placed on ice and cooled within a maximum delay of 30 minutes at -80 ° C. In addition, for autoradiography, this frozen material is cut into 10 μπι slices with a cryostat (Leitz 1720), placed on pre-cleaned microscope glass, and stored at -20 ° C for at least 3 days to improve tissue adhesion to the glass. The slices are preincubated in Tris-HCl buffer (50 mM, pH 7.4), containing CaCl 2 (2 mM) and KCl (5 mM), for 10 min at room temperature. and then washed twice for 2 min with the same ‘buffer without the addition of salts. The slices are then incubated with the chelate of the invention for 2 h at room temperature in Tris-HCl buffer (170 mM, pH 7.4) containing bovine serum albumin (10 g / l), bacitracin (40 mg / l) and MgCl 2 (5 mM ) to inhibit endogenous protease. Non-specific binding is determined by adding the corresponding unlabeled, unmodified somatostatin peptide at a concentration of 1 μ 1. The incubated slices are washed twice for 5 min with cold medium containing 0.25 g / l BSA. After a short immersion in distilled water, the slices are quickly dried and checked for [3H] -LKB film to remove excess salts. After about a week of exposure in X-ray cassettes, it is found that the chelates of the invention, e.g.

The affinity of the chelates of the invention for somatostatin receptors can also be demonstrated in vivo.

Rats with transplantable exocrine pancreatic somatostatin receptor-positive tumors are given an intravenous injection of a chelate of the invention. The injection site is the penile vein. Immediately after dosing, the animals are placed on a gamma camera collimator, and the distribution of radioactivity in the animals is monitored at various time intervals.

The biodistribution of radioactivity can also be determined by killing a series of rats treated in this manner and determining the radioactivity of the organs. After administration of a chelate according to the invention, e.g. a radionuclide chelate, e.g. γ-radiating chelate, a dose of 1-5 pg / kg of ligand labeled with 0.1 to 2 30 mCi of radionuclide, the location of the tumor should be detected; together with the organs in which the secretion essentially takes place.

Thus, in specific or alternative embodiments, the invention also provides methods: 351025 1. A method for detecting somatostatin receptor-positive tumors or metastases in a subject in vivo, wherein a) a chelate of the invention is administered to a subject and b) receptors for targeting said chelate are targeted. is recorded.

The chelates of the invention used in the in vivo method of the invention are those that form complexes with a γ-emitting radionuclide, a positron emitting radionuclide 10, or a paramagnetic metal ion, e.g., as indicated above.

The chelates of the invention used as imaging agents in method (1) can be administered parenterally, preferably directly intravenously, e.g., as an injectable solution or suspension, preferably as a single injection. The appropriate dose will, of course, vary with, for example, the li-Gand and the detectable element, e.g. the radionuclide, used. A suitable injectable dose is in the range of 20 which allows the use of photoscanning methods known in the art of imaging. When the radiolabeled chelate of the invention is used, it may preferably be administered at a dose of 0.1 to 50 mCi, suitably 0.1 to 30 mCi, more suitably 0.1 to 20 mCi. As indicated, the dose amount: · may be 1 to 200 μg of ligand-labeled 0.1 to 50 mCi, suitably 0.1 to 30 mCi, e.g. 3 to 15 mCi, of γ-radiating radionuclide, depending on which γ-radiating ra dionuclide is used. For example, when using In, 30, it is preferable to use lower radioactivity, while when using Te, it is preferable to use radioactivity that is at the upper limit.

Chelation enrichment at tumor sites can be monitored by similar imaging techniques, e.g., using nuclear drug imaging equipment, e.g., scanner, γ-camera, rotating γ-camera, preferably 'computer assisted'; PET scanner 22 102540 (Positron emission tomography); MRI device or CAT scanning device.

The chelates of the invention, e.g. most of the γ-radiating chelates, are excreted substantially by the kidneys and do not accumulate in the liver.

The chelates according to the invention can be administered in free form or in the form of a physiologically acceptable salt. Such salts can be prepared by conventional methods and show an activity of the same order of magnitude as the free compounds.

The chelates of the invention used in the method of the present invention may conveniently be prepared just prior to administration to a subject, i.e. radiolabeling with the desired detectable metal ion, in particular the desired γ-radionuclide, may be performed immediately prior to dosing.

The chelates of the invention may be suitable for imaging tumors, such as pituitary, gastrointestinal, central nervous, breast, prostate, ovarian or colon tumors, small cell lung cancer, paragangio tissue tumors, neuroblastoma, peochromocytoma only (peochromocytoma) medullary thyroid carcinoma, myeloma, etc. 25 as well as their metastases.

The γ-radiating chelates of the invention according to a further embodiment of the invention can also be used as imaging agents in the evaluation of renal function.

Groups of five mice were used. Each mouse is injected directly into a vein, one tail vein, with 0.1 ml containing 1 mCi of the chelate of the invention. Mice are then placed in metabolic cages to collect excreted urine. 10 and 35 After 120 min of injection, the ureters are ligated and the mice were anesthetized with chloroform. The urinary system is imaged using standard imaging techniques. In this experiment, the γ-radiating chelates of the invention improve the imaging of renal secretion when administered at doses of 0.1 to 30 mCi.

Thus, the present invention can be used to evaluate renal function in vivo in a subject, wherein an effective amount of γ-radiating chelate is administered to the subject and renal function is recorded.

According to another embodiment of the invention, there is provided: i. A diagnostic composition comprising a ligand of the invention in free or physiologically acceptable salt form together with one or more pharmaceutically acceptable carriers or diluents therefor; 15 ii. a diagnostic composition comprising a chelate of the invention in free form or in the form of a pharmaceutically acceptable salt together with one or more pharmaceutically acceptable carriers or diluents thereof.

Such compositions may be prepared by conventional methods.

The composition of the invention may also be placed in separate packages together with instructions describing how the ligand is mixed with the metal ion and how the resulting chelate is dosed. It can also be prepared in the form of a double package,. i.e. as a single package containing the ligand and the detectable metal ion in separate unit doses together with instructions 30 for mixing and chelating the dosage. The diluent or carrier may be present in unit dosage form.

In the following examples, all temperatures are ° C and [a] D values are uncorrected. The following 35 abbreviations have been used:

Boc tert-butoxycarbonyl TFA trifluoroacetic acid 24 102540 DTPA diethylenetriaminepentaacetic acid

I I

EXAMPLE 1: DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol 5 I-1 1.1 g DPhe-Cys-Phe-DTrp-Lys (ε-Boc) -Thr-Cys-Thr -ol in free base (ImM) is dissolved in 5 l of dioxane / H 2 O 1/1 (v / v) and reacted with 5 g of NaHCO 3.

520 mg of DTPA dianhydride are added slowly with stirring. The reaction mixture is stirred for a further 30 minutes and freeze-frozen. The residue is dissolved in 250 ml of water and the pH is adjusted to pH 2.5 with concentrated HCl. The precipitated product is filtered off, washed and dried over phosphorus pentoxide. After silica gel column chromatography, the following products are isolated:

I I

230 mg DTPA-DPhe-Cys-Phe-DTrp-Lys (ε-Boc) -Thr-Cys-Thr- ol 25 102540 I-1 10 g H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr -ol acetate and 100 ml DMF. After 2 h at room temperature, the solution is removed in vacuo and 200 ml of diisopropyl ether are added to the residue. The precipitate that forms is filtered off, washed with diisopropyl ether and dried. The crude product is purified by chromatography on silica gel (eluent: CH2Cl2 / MeOH 9/1) and then isolated as a white amorphous powder.

10 [α] D 20 = 29.8 (c = 1.28 in DMF).

I-1 EXAMPLE 2: DTPA- (DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol) 2 I-15 Fraction containing the intermediate DTPA-DPhe-Cys-Phe 1 DTrp-Lys (ε- Boc) -Thr-Cys-Thr-ol) 2, as obtained in Example 1, is treated as described above for the corresponding monomeric product, the Boc protecting groups are removed to give the title compound: [<x] D20 = -24.5 ° (c - 0.55 95% AcOH).

EXAMPLE 3: H2N- (CH2) 5-CO-DPhe-Cys-Phe-DTrp-Lys-Thr-I

25 Cys-Thr-ol

I I

a. 0.56 g of H-DPhe-Cys-Phe-DTrp-Lys (BOC) -Thr-Cys-Thr-ol, 0.5 mM Fmoc-e-aminocaproic acid and 115 mg of hydroxybenzotriazole are dissolved in 10 ml of DMF and cooled 30-30 ° C. A solution of 115 mg of di- is added to the solution. . . cyclohexylcarbodiimide and 5 ml DMF (cooled .. -10 ° C). After a reaction time of 24 h, during which the mixture is warmed to room temperature, the resulting dicyclohexylurea is filtered off and the filtrate is diluted with water to a volume of 10 to 35 times. The precipitated reaction product is filtered off, washed and dried over phosphorus pentoxide. The crude product is used for the next step without further purification.

b. Fmoc cleavage 0.5 g of the crude product obtained from the coupling reaction 5 (a) is treated with 5 ml of DMF / piperidine 4/1 v / v (clear solution) for 10 min at room temperature and then mixed with 100 ml of diisopropyl ether. The reaction product which precipitates is filtered off, washed and dried. The crude product is used in the next step without further purification.

c. Cleavage of BOC 300 mg of the crude product obtained (l.b) are treated for 5 min at room temperature with 5 ml of 100% TFA (completely dissolved) and then mixed with 50 ml of diisopropyl ether. The precipitate obtained after addition of 2 ml of HCl / diethyl ether is filtered off, washed and dried under high vacuum. The final product is purified by chromatography on silica gel (CHCl 3 / MeOH / H 2 O / AcOH, 7/3 / 0.5 / 0.5), the salt is then removed with Duolite (gradient: H 2 O / AcOH 95/5) --- H 2 O. / dioxane / AcOH, 45/50/5).

The title compound is obtained as acetate (white lyophilisate).

[α] D 20 = - 32 0 (c = 0.5 95% AcOH).

The obtained compound can be used in the reaction with DTPA according to the methods of Examples 1 and 2.

EXAMPLE 4:

The following ligand can be prepared following the method described in Examples 1 and 3:

I I

.. DTPA-FiAla-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol.

[α] D 20 = - 14.8 ° (c = 0.55% AcOH).

I- 35 EXAMPLE 5: InIn Labeled DTPA-DPhe-Cys-Phe-DTrp-Lys - 1

Thr-Cys-Thr-ol 27 102540

I I

1 mg of DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol is dissolved in 5 ml of 0.01 M acetic acid. The resulting solution is passed through a 0.22 μ Millex-GV filter and divided into 0.1 5 ml portions and stored at -20 ° C. inInCl3 (Amersham, 1 mCi / 100 μΐ) is prediluted in an equal volume of 0.5 M sodium acetate, and labeling is performed by mixing the ligand with InCl3 solution and homogenizing gently at room temperature.

10 HEPES buffer, pH 7.4 is then added to form a 10 "6 M solution.

EXAMPLE 6: 90Y-labeled DTPA-Phe-Cys-Phe-DTrp-Lys-i Thr-Cys-Thr-ol 90Y is obtained from a 90Sr-90Y radionuclide generator. Structure, elution and transformation of the developer The [Y] EDTA -> acetate complex is performed according to the method described in M. Chinol and D. J. Hna-20 towich J. Nucl. Med. 28, 1465-1470 (1987).

I I

Dissolve 1 mg of DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol in 5 ml of 0.01 M acetic acid and allow to warm to room temperature, and add 1.0 mCi Y 50 μΐ sterile 0.5 25 M acetate . The mixture is then left alone for 30 min - 1 h to maximize chelation.

One group of ligands of the invention are somatostatin peptides, e.g. somatostatin analogues, containing at least one amino acid unit 30 one chelating group attached to said amino group in an amide bond, in free or salt form; form.

One group of chelates according to the invention are the above-mentioned ligands, which form a complex with a detectable element, e.g. a metal ion in free or salt form.

Claims (6)

A somatostatin peptide derivative containing a chelating group, characterized in that it is constituted by CH2 ~ S-Y1 y2-s-ch2 I (I) RCO-NH-CH-CO-BC-Lys-E-NH-CH -G10 where RCO- is a) an L- or D-phenylalanine residue, optionally ring-substituted with F, Cl, Br, NO2 / NH2, OH, Chalky 1 and / or C1-3 alkoxy, or D-Trp or 3- (2-naphthyl) ) -D-Ala, Υχ and Y2 together are a direct bond or Υχ and Y2 is independently hydrogen, B is -Phe- optionally ring-substituted with halo gene, NO2, NH2, OH, C1-3 alkyl and / or C1-3 alkyl. Alkoxy (including pentafluoroalanine) or β-naphthyl-Ala, C is (L) -Trp- or (D) -Trp-, E is Thr, Ser, Vai, Phe or Ile, G is a group, of the formula CON <^ were 30 Ri! is hydrogen, C1-3 alkyl,,. R 12 is hydrogen, C 1-3 alkyl or a group, of the formula -CH (R 13) -X 1, R 13 is CH 2 OH, - (CH 2) 2-OH, - (CH 2) 3-OH, -CH (CH 3) OH, isobutyl or butyl or is one at the α-carbon atom of Trp or β-Nal solid substituent, is a group of the formula -Cl 2 ORxo or -CONRx4Rx5, wherein R10 is hydrogen or a physiologically acceptable, physiologically hydrolysable ester residue, R 14 is hydrogen or C 1 -C 3 alkyl and R 15 is hydrogen or C 1 -C 3 alkyl, provided that where R 2 is -CH (Rx 3) -Xx, then R 1 is hydrogen or methyl, the residues B and E being in the L configuration and those in 2- and 7-position residues are independently of each other in (L) or (D) configuration, the somatostatin peptide has at least one chelating group for a detectable element bound either directly or indirectly via a voluminous group of the formula (ai) H 2 N-R 35 -CO- wherein R 35 is C 1-6 alkylene or -CH-, wherein R 'R' is C 1-8 alkylalkyl, benzyl, naphthylmethyl or pyridylmethyl, to the N-terminal amino group of said peptide, and the chelating group is derived from ethylene diamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA) , ethylene glycol-O, O-bis (2-aminoethyl) - Ν, Ν, Ν ', N'-tetraacetic acid (EGTA), N, N'-bis (hydroxy-benzyl) ethylenediamine-N, N'-dietetic acid ( HBED), triethylenetetraamine hexacetic acid (TTHA), substituted EDTA or -DTPA 1,4,7,10-tetraazacyclododecane-N, N1, N ", N" 1 -tetraacetic acid (DOTA) and 1,4,8,11 tetraazacyclo-tetradecane-Ν, Ν ', N ", N"' - tetraacetic acid (ΤΕΤΑ), in free form or in the form of a salt, or complexed with the detectable element, which is a fluorescent element, a paramagnetic ion, a 5-positron radiant or γ-radionuclide radionuclide. A compound according to claim 1, which is a [DTPA-Dphe1] octreotide 10H / h. ij. Si 'llpf-jn-ns T - / 0g HN \ HO O (X) jini 0 Or 0 * 8 ^ 0 Ό ^ 20 in free form, in the form of a salt or complexed with a fluorescent element, a paramagnetic ion, a positron radiant or γ-radionuclide. A compound according to claim 1, wherein part CH 2 -S-Y 1 Y 2 -S-CH 2 II RCO-NH-CH-CO-BC-Lys-E-NH-CH-G is selected from the group to which 30 ' 1, 1 - "" - - (D) Phe-Cys-Thr- (D) Trp-Lys-Val-Cys-ThrNH2 1- (D) Phe-Cys-Tyr- (D) Trp-Lys -Val-Cys-TrpNH2 (D) Trp-Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 36 1 0 2 5 4 0 I —- 1 (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 I-1 3- (2-Naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val-Cys-ThrNH2, -1 3- (2- Naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val-Cys-β-Nal-NH 2, -1 3- (2-Naphthylyl) - (D) Ala-Cys-P-Nal - (D) Trp-Lys-Val-Cys-ThrNH2 / and II (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-p-Nal-NH2. A method for preparing a somatostatin peptide derivative according to claim 1, which is carbon-linked with a detectable element selected from the group of fluorescent element, paramagnetic ion, positrons radiating or γ-radiating radionuclide, characterized in that a somatostatin peptide according to formula I, with a chimeric tone, a chelating group according to claim 1, in free form or in the form of a complex or an uncomplexed form, reacts with a compound which contains a fluorescent element, a paramagnetic ion, a positrons radiating or γ-radiating radionuclide. Use of a compound according to claims 1-3, which is in complexed form with a detectable element, the detectable element being a fluorescent element, a paramagnetic ion, a positron radiant or γ-radiating radionuclide. , as a diagnostic substance. A diagnostic package for use as an imaging agent, characterized in that the diagnostic package contains unit doses of a somastostatin peptide derivative according to claims 1 to 3 in free form or in the form of a physiologically acceptable salt or in non-complex form and a detectable element, which is a fluorescent element, a paramagnetic ion, a positron radiation or γ-radiation radionuclide.