FI101967B - Method for preparing a ligand complexed with a pharmaceutically acceptable radionuclide - Google Patents

Description

101967

METHOD FOR THE PREPARATION OF A PHARMACEUTICALLY USABLE LIGAND COMPLEXED WITH RADIONUCLE-DIN

The invention relates to a process for the preparation of a pharmaceutically useful ligand complexed with a radionuclide for use in radiotherapy.

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 accurately localize by conventional diagnostic methods.

In vitro visualization of somatostatin receptors has been performed by autoradiography of tumor tissue using radioiodinated somatostatin or somatostatin analogs and, e.g., [125 I-Tyr11] somatostatin-20 14 (Taylor, JE et al., Life Science (1988) 43: 421). [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.

25 (1987) 47: 551; Reubi, J.C. you do not. ai., Cancer Res.

(1987) 47: 5758).

Novel somatostatin peptides useful as therapeutic agents that can be labeled for therapeutic use have now been developed.

: According to the invention there is provided a somatostatin peptide having at least one chelating group with a detectable element, this chelating group being linked to the amino group of said peptide, and this amino group has no significant binding affinity for somatostatin receptors.

101967 2 These compounds are hereinafter referred to as ligands according to the invention. They have a single chelating group that can react with a detectable element, e.g., a radionuclide, to form a complex behind it, and can further bind to somatostatin receptors that are, for example, 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 somatostatin 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 ligand group is one in which the chelating group is directly attached to the amino group of the somatostatin peptide.

Another ligand moiety is one in which the chelating group 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 radicals and / or or 35 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. Generally, the term encompasses all modified derivatives of a biologically active peptide that exhibit qualitatively similar potency to unmodified somatostatin peptide-5 di, 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; 29.579; 215.171; 203.031; 214.872; 298.732; 277.419.

Suitable 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

20 RCO-N-CH-CO-B-C-Lys-E-NH-CH-G I

wherein RCO- is L- or D-Phe, -Tyr, -Trp or -β-Nal 25 • Yx and Y2 together are a direct bond or Yx and Y2 are independently hydrogen B is Phe, Tyr or B-naphthyl-Ala, C is (L) -Trp- or (D) -Trp- E is Thr, Ser or Val «G is a group of formula -CO-NH-CH 2 R 2 J 1 wherein R 13 is CH 2 OH, ~ (CH 2) 2 -OH, - (CH 2) 3 -OH, CH (CH 3) OH, isobutyl or butyl or 101967 4 is a substituent attached to the α-carbon atom of Trp or P-Nal

Xx is -COOH, -CH2OH or -CONH2 wherein radicals B, Lys and E are in the L-configuration and residues at the 2- and 7-positions are each 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 semi-combination: 1. B is B ', in which B' is Phe or Tyr.

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

3. E is E ', where E' is Val or Thr, especially Thr.

4. R13 is preferably -CH2OH, -CH (CH3) -OH, isobutyl or butyl or R13 is - (CH2) 2-OH or - (CH2) 3-OH. In particular, it is -CH2OH or -CH (CH3) OH.

The following individual compounds describe compounds of formula I: 20, -1H- (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-Thr-ol is also known as octreotide I-1 (D) Phe- Cys-Thr- (D) Trp-Lys-Val-Cys-ThrNH2 25 I-, (D) Phe-Cys-Tyr- (D) Trp-Lys-Val-Cys-TrpNH2

I I

(D) Trp-Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 30, -1 (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 1 -1 3 - (2-Naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val-Cys-ThrNH2 35 | -1 3- (2-Naphthyl) - (D) Ala-Cys-Tyr- ( D) Trp-Lys-Val-Cys-β-

Nal-NH 2

I I

3- (2-Naphthyl) - (D) Ala-Cys-B-Nal (D) Trp-Lys-Val-Cys

ThrNH2 5 101967

I I

5 (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-B-Nal-NH 2 B. Analogs of formula II l l

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

10 [see Vale et. al., Metabolism, 27, Supp, 1, 139, (1978)]

I I

H-Cys-His-His-Phe-Phe- (D) Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH III

15 (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 that are derived

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, (2-aminoethyl) -N, N, N1, N'-tetraacetic acid (EGTA), N, N'-bis (hydroxybenzyl) ethylenediamine-N, N'-diethylacetic acid (HBED) and triethylenetetraamine hexacetic acid ( TTHA), those derived from substituted EDTA or DTPA, e.g. p-isothiocyanato-101967 6 benzyl EDTA or -DTPA derived from macrocyclic ligands, e.g. 1,4,7,10- tetraazacyclododedecane-N, N ', N' ', N' '' tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane-5 N, NNN '' 'tetraacetic acid (TETA), and the like Derived from N-substituted or C-substituted macrocyclic amines, including cyclames, e.g. as described in EP 304,780 A1 and WO 89/01476-A. groups of formula IV or V 10 O 0 0

Il II II

Ri-C-S-iCHaJn.-C-fTTJi-C-IV

15

O O

Il II

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

20 \ Il

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

Il II

wherein R 1, R 2 and R 3 are each independently C 1-6 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 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, 1 groups derived from bis-aminothiol derivatives, e.g. compounds of formula VI 7 101967 ΗΝ ΝΗ νι 5 R21 ^ r "R ^ '' SH US 'wherein each R20 / R21 / R 22 and R 23 are independently hydrogen or C 1-4 alkyl, X 2 is a group capable of reacting with the N-amino group of the peptide, and m 'is 2-3 groups derived from dithiasemicarbazone derivatives, e.g. compounds of formula VII X, A \

^ n

HN MH

HN ^ SH HS ^ NH

25 VII

wherein X2 is as defined above, 30 groups derived from propylene amine oxime derivatives, e.g. compounds of formula VIII 35 8 "Γ <r r 7 χ2

HN NH

Ά? VIII

5 25 ^ s A, 28 324 j1 p ^ 29

OH OH

wherein each of R24, R25, R26 * R27 / R28 and R29 independently of one another is hydrogen or C1-4alkyl and X2 and m 'have the same meaning as above, groups derived from diamide dimaptaptides, e.g. compounds of formula IX axa

° Y "V

yA ^ s 20 5 | j H, C, CO COCl 3 -H- 3 0 0 3 wherein X 3 is a divalent radical optionally substituted and has a group which can react with the N-amino group of the peptide, e.g. C 1-4 alkylene or phenylene having a group X 2, and Y 5 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-carboxyphenyl) porphine or TPP , having a group X2 as defined above.

Aryl is preferably phenyl. Aralkyl is preferably benzyl.

Examples of X2 groups contain radicals of the formula - (X4) n ..- X5, where X4 is C1-4 galkylene, -9 101967 or C1-4alkylene optionally bonded to a carbon atom by an oxygen atom or -NH-, n '' is O or 1 and X5 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 bulky group, for example via a group of formula (ai).

Z-R35-CO- (ai) 15. R 35 is C 1-11 alkylene, C 2-11 alkenylene or - CH (R ') -, wherein R' is a residue attached to the natural or synthetic α-amino acid of the α-carbon, e.g. hydrogen, C 1-11 alkyl, benzyl, optionally substituted benzyl, naphthylmethyl, pyridylmethyl, Z is a functional group that can react covalently with a chelating agent.

For example, Z may be a group that can form an ether, ester or amide bond with the chelating group. Z is preferably amino.

The chelating group, when containing carboxy, -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 popular are chelating groups derived from DTPA.

In the ligands of the invention, the chelating group, when polyfunctional, may be attached to either a single 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 refer to 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 sulfonic 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, for example, 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 manner. or in unprotected form, and one of these 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) by linking the chelating agent and the desired somatostatin peptide in protected or unprotected form. in such a way that the chelating group is attached to the desired N-amino group of the peptide, and optionally then performing step a), or d) removing the functional group from the unprotected or protected peptide having the chelating group or converting it to another functional group. nii n to obtain another unprotected or protected peptide having a chelating group, and in the latter case carrying out step a), or 11 101967 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 analogue in which 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 bond, this can be done analogously to the methods used to prepare the amide. If desired, protecting groups suitable for use with peptides may be used in these reactions. or in the desired chelating groups as functional groups which do not participate in the reaction. The term protecting group also includes a polymeric resin having functional groups.

If it is desired to link the chelating group terminal 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 12 101967 can be attached to a peptide by introducing chelating agents of formulas IV or V, 0 0 0

Il II II

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

o o

Il II

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

10 I

___ CH-C-X V

R3-C-S- (CH2) n, -C-NH

Il II

o o 15. 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 into the somatostatin peptide, e.g. a polyaminopolycarboxylic acid as described, a salt or anhydride thereof.

In the above method, if in amino acids, peptide fragments or peptides used as starting materials, the chelating group is 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 or peptides may be prepared by reacting the corresponding amino acids or peptides without a bridging or bulking group in a conventional manner with the corresponding bridging or bulking compound, for example an acid or a reactive acid derivative containing a bridging or bulking group, 13 101967 e.g. -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-nitrophenyl, pentachloro-5-phenyl, pentafluorophenyl, succinimidyl or 1-hydroxy-benzotriazolyl.

Alternatively, the chelating agent may first be reacted with a bridging or bulking agent to form a bridging or bulking group, and then reacted with a 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 free of chelating groups.

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 for therapeutic purposes. .

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

Suitable detectable metal ions 35 are e.g. heavy elements or rare earth metal ions, e.g. as used in CAT (Computer axial tomography) scanning, and radionuclides, e.g. β-14 101967 radiating radionuclides and α-radiating radionuclides .

Examples of β-radiating radionuclides include 90Y, 67-Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 5 198Au, 109Pd, 165Dy, 32P, 142Pr. The β-radionuclide preferably has a half-life of 2.3 h to 14.3 days, suitably 2.3 to 100 h. The β-radiating radionuclide may have a longer half-life than the somatostatin peptide in the tumor.

Suitable α-emitting radionuclides are those used in therapeutic therapy, e.g. 2UAt, 212Bi.

In addition, radionuclides emitting Auger electrons suitable for therapeutic treatment include radionuclide-15. dit.

The chelates of the invention may be prepared by reacting a ligand with a corresponding detectable element-giving compound, e.g. a metal salt, preferably 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 25 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 auxiliary 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 15 101967 changes the ligand.

The chelates of the invention may also be prepared by combining a chelating agent, complexed with a detectable element, and a somatostatin peptide in protected or unprotected form, and, if desired, removing at least one protecting group present. The same reaction can be carried out 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.

The chelates of the invention can also be prepared by combining a chelating agent, complexing-15. and a somatostatin peptide fragment containing at least one 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 chelating agent may form a complex with a non-detectable metal, 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 somatostatin peptide via a bridging bond or a spacer group, e.g., 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 coil. in the active substance.

The above reactions can be carried out analogously according to known methods. Depending on the chelating group 35 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, chelation ester radioactivity, 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, 15. obtained from carboxylic acid groups present in the molecule which do not participate in chelating, e.g. alkali metal salts such as sodium or potassium, or substituted or unsubstituted ammonium salts.

The chelates of the invention and their pharmaceutically acceptable salts exhibit pharmaceutical activity and are therefore useful as a radiopharmaceutical for the in vivo treatment of somatostatin receptor-positive tumors and metastases by forming complexes with α- or β-radionuclides, as indicated by standard assays.

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 frozen within a maximum delay of 30 min at -80 ° C. In addition, for autoradiography, this frozen material is cut into 10 μπ \ 17 101967 slices with a cryostat (Leitz 1720), placed on a pre-cleaned microscope slide, 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 in a concentration of 1 μΜ. 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, to remove excess salts, the slices are quickly dried and checked for [3H] -LKB film. After about a week of exposure to X-ray cassettes, it is found that the chelates of the invention, e.g. the radionuclide chelate, give very good results in labeling tumor tissue without the surrounding healthy tissue being degraded when added at a concentration of 10'10 to 10 ”3 25 M .

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

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 18 101967 rats treated in this way and determining the radioactivity of the organs.

After a dose of 1-5 μς / kg of ligand labeled with 0.1-2 mCi of radionuclide of a chelate according to the invention, e.g. a radionuclide chelate, e.g.

Thus, in specific or alternative embodiments, the invention also provides methods:

An in vivo method for treating somatostatin receptor-positive tumors and metastases in a subject in need of such treatment, wherein 15. the chelate of the invention is administered to the subject in a therapeutically effective amount.

The chelates of the invention used in an in vivo method of treatment are chelates that form a complex with an α, β or Auger electron emitting radionuclide as defined above.

In the therapeutic method according to the invention, the doses will, of course, vary depending on e.g. the specific condition being treated, e.g. the size of the tumor, the particular chelate used, e.g. the chelation half-life in the tumor, and the desired therapy. Usually, the dose is calculated based on the radioactivity received by the organ distributed and detected. For example, the chelate may be administered in a daily dose of 30 to 3 mCi / kg, e.g. 1-3 mCi, suitably 1 to 1.5 mCi / kg body weight. The indicated daily dose range is 1 to 200 μg of ligand labeled with 0.1 to 3 mCi / kg body weight, e.g. 0.1 to 1.5 mCi / kg body weight of 35 with α- or β-radiating radionuclide, usually the dose is divided up to 4 times daily doses.

The α- or β-radiating chelates of the invention can be administered by all conventional means, in particular parenterally, e.g. as injectable solutions or suspensions. They can also advantageously be administered as infusions, e.g. as a 30-60 minute infusion. Depending on the location of the tumor, they may be administered as close to the location of the tumor as possible, e.g. by means of a catheter. The mode of administration chosen depends on the dissociation rate and the specific rate of chelate used.

The chelates of the invention may be administered in free form or in the form of a pharmaceutically acceptable salt. Such salts can be prepared by conventional methods and show activity. which is 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, particularly preferred α- or β-radionuclides, may be performed immediately prior to dosing.

The chelates of the invention may be useful in the treatment of tumors such as pituitary, gastrointestinal, pancreatic, central nervous, breast, prostate, ovarian or colon tumors, small cell lung cancer, paraganglionic tumor tumors, neuroblastoma, peochromocytoma me-30 dollar thyroid carcinoma, myeloma, etc. as well as these metastases.

»

According to a further embodiment of the invention there is provided: i. A pharmaceutical composition comprising a ligand of the invention in free or pharmaceutically acceptable salt form together with one or more pharmaceutically acceptable carriers or diluents therefor; ii. a pharmaceutical 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 pack, i.e. a single pack containing the ligand and the detectable metal ion in separate unit doses together with instructions, for mixing them and for dosing the chelate. The diluent or carrier may be present in unit dosage form.

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

Boc tert-butoxycarbonyl TFA trifluoroacetic acid 25 DTPA diethylenetriaminepentaacetic acid I-1 EXAMPLE 1: DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol I-1 30 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 the dry mixture is 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

5 230 mg DTPA-DPhe-Cys-Phe-DTrp-Lys (ε-Boc) -Thr-Cys-Thr-ol and 500 mg of the corresponding dimer DTPA- (DPhe-Cys-Phe-DTrp- -1 10 Lys (eBoc) -Thr-Cys-Thr-ol) 2.

3 ml of TFA are mixed with 200 mg of DTPA-DPhe-Cys-Phe-DTrp-

With Lys (ε-Boc) -Thr-Cys-Thr-ol. After 5 minutes at room temperature, the mixture is precipitated with diisopropyl ether, filtered and dried. The residue is desalted with Duolite and lyophilized, yield 150 mg of the title compound: [α] D 20 = -26.6 ° (c = 95% AcOH).

20 The starting material can be prepared as follows: a) H-DPhe-Cys-Phe-DTrp-Lys (Boc) -Thr-Cys-Thr-ol 2.25 g of di-tert-butyl pyrocarbonate, dissolved in 25 ml of DMF, are slowly added dropwise at room temperature to a solution of I-1 10 g of H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol acetate and 100 ml of DMF. After 2 h at room temperature, solution 30 is removed in vacuo and 200 ml of diisopropyl ether. is added to the residue. The precipitate which forms is filtered off, washed with diisopropyl ether and dried. The crude product is purified by chromatography on silica gel (eluent: CH 2 Cl 2 / MeOH 9/1) and then isolated as a white amorphous powder.

[α] D 20 = 29.8 ° (c = 1.28 in DMF).

22 101967

I I

EXAMPLE 2: DTPA- (DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol) 2 5 Fraction containing 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: [α] D 20 = -24.5 ° (c = 0, 55 95% AcOH).

I- EXAMPLE 3: H2N- (CH2) 5-CO-DPhe-Cys-Phe-DTrp-Lys-Thr- 15. Cys-Thr-ol I-1 a. 0.56 g H-DPhe-Cys-Phe-DTrp-Lys (BOC) -Thr-Cys-Thr-ol, 0.5 mM Fmoc-s-aminocaproic acid and 115 mg hydroxy -Benzotriazole is dissolved in 10 ml of DMF and cooled to 20-30 ° C. A solution of 115 mg of dicyclohexylcarbodiimide and 5 ml of DMF (cooled to -10 ° C) is added to the solution. 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 25 times. The precipitated reaction product was filtered; washed, washed and dried over phosphorus pentoxide. The crude product is used for the next step without further purification.

b. Fmoc cleavage 30 0.5 g of crude product obtained from the coupling reaction. (a), 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. BOC cleavage 23 101967 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 the 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, 10 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 15. according to the methods of Examples 1 and 2.

EXAMPLE 4:

The following ligand can be prepared following the procedure described in Examples 1 and 3: 11 DTPA-LAla-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol.

[α] D 20 = -14.8 ° (c = 0.55% AcOH).

I- EXAMPLE 5: InIn Labeled DTPA-DPhe-Cys-Phe-DTrp-Lys- 25 -1

Thr-Cys-Thr-ol I-1 Dissolve 1 mg of DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol in 5 ml of 0.01 M acetic acid. The resulting solution is passed through a 0.22 μ Millex-GV filter and divided by 0.1. ml doses and stored at -20 ° C. ulInCl3 (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 gently homogenizing at 35 room temperature.

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

I- 24 101967 an

EXAMPLE 6: SUY labeled DTPA-Phe-Cys-Phe-DTrp-Lys-I

Thr-Cys-Thr-ol 90 90 90 5 Y is obtained from the Sr-Y 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-towich J. Nucl. Med. 28, 1465-1470 (1987).

10, -1 1 mg of DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol is dissolved in 5 ml of 0.01 M acetic acid and allowed to warm to room temperature, and 1.0 mCi Y 50 μΐ sterile 0.5 M acetate is added. The mixture is then left alone for 15 minutes to 1 hour to maximize chelation.

One group of ligands of the invention are somatostatin peptides, e.g. somatostatin analogues, which contain at least one amino acid unit in the free or salt form of one chelating group attached to said 20 amino groups by an amide bond.

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

Claims (5)

101967 A process for the preparation of a pharmaceutically usable ligand complexed with a radionuclide for use in radiotherapy, which comprises administering a ligand comprising a somatostatin peptide of formula I CH 2 -S-Y! Y2-S-CH2 I I 10 RCO-N-CH-CO-BC-Lys-E-NH-CH-G I wherein RCO- is L- or D-Phe, -Tyr, -Trp or -β-Nal 15 Y1 and Y2 together are a direct bond or Y 3 and Y 2 are independently hydrogen B is -Phe-, Tyr or fi-Nal, 20. is (L) -Trp- or (D) -Trp- E is Thr, Ser or Val is a group of formula -CO-NH-CH (R13) X3 wherein R13 is CH2OH, - (CH2) 2-OH, - (CH2) 3-OH, CH (CH3) OH, isobutyl or butyl or is Trp or β-Nal a substituent attached to the α-carbon atom of X3 is -COOH, -CH2OH or -CONH2 wherein residues B, Lys and E are in the L-configuration and residues in the 2- and 7-positions are each independently in the (L) or (D) configuration, somatostatin has at least one chelating group attached either directly or indirectly via a space-occupying group of formula (ai) H2N-R35-CO- wherein R35 is C1-4alkylene or -CH- wherein R 'R' is C1-11alkyl, benzyl, naphthylmethyl or pyridylmethyl to the N-terminal amino group of said peptide, the chelating group being derived from ethylenediaminetetraacetate (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 (HBED), triethylenetetramine hexaacetic acid (TTHA), substituted EDTA or -DTPA 1,4,7,10-20 tetraazacyclododecane-N, N ' , N '', N '' '- tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclotetra-decane-N, N', N '', N '' '- tetraacetic acid (TETA), react in radiotherapy with the radionuclide 25 used. The method of claim 1, wherein the ligand comprises a somatostatin residue selected from the group consisting of 30. 1 (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-Thr-ol II (D) Phe-Cys-Thr- (D) Trp-Lys-Val-Cys-ThrNH2 II 35 (D) Phe-Cys-Tyr- (D) Trp-Lys-Val-Cys-TrpNH2 II (D) Trp -Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 101967 II (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 I 3- (2-naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val-Cys-5 ThrNH2 I-1 3- (2-naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val -Cys-β-Nal-NH2 II 10 3- (2-naphthyl) - (D) Ala-Cys-fi-Nal- (D) Trp-Lys-Val-Cys-ThrNH2 and II (D) Phe-Cys- Phe- (D) Trp-Lys-Thr-Cys-fi-Nal-NH 2 Process according to Claim 1 or 2, characterized in that the ligand is reacted with an α-, β- or Auger-electron-emitting radionuclide. Process according to Claim 1, characterized in that ii DTPA-DPhe-Cys-Che-Phe-Dtrp-Lys-Thr-Cys-Thr-ol is prepared which is complexed with an α-, β- or Auger-electron emitter. with a radionuclide. A method according to claim 4, characterized in that the radionuclide is Y. 101967