CA2004532C - Peptide derivatives - Google Patents
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- CA2004532C CA2004532C CA002004532A CA2004532A CA2004532C CA 2004532 C CA2004532 C CA 2004532C CA 002004532 A CA002004532 A CA 002004532A CA 2004532 A CA2004532 A CA 2004532A CA 2004532 C CA2004532 C CA 2004532C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/575—Hormones
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- C07K14/6555—Somatostatins at least 1 amino acid in D-form
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- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
Somatostatin peptides bearing at least one chelating group for a detectable element, said chelating group being linked to an amino group of said peptide, and said amino group having no significant binding affinity for somatostatin receptors, in free or salt form, are complexed with a detectable element and are useful as a pharmaceutical, e.g. a radiopharmaceutical for in vivo imaging of somatostatin receptor positive tumors or for therapy.
Description
a PEPTIDB DERIVATIVES
The present invention relates to polypeptides, process for their production, pharmaceutical preparations containing them and their use as a pharmaceutical, e.g. for treatment of somatostatin receptor positive tumors or as in vivo diagnostic imaging agents.
In the last few years a high incidence of somatostatin receptors has been demonstrated in a variety of human tumors, e.g.
pituitary tumors, central nervous system tumors, breast tumors, gastro-enteropancreatic tumors and their metastases. Some of them are small or slow-growing tumors which are difficult to precisely localize by conventional diagnosis methods.
In vitro visualization of somatostatin receptors has been per-formed through autoradiography of~.tumoral tissues using radio-iodinated somatostatin or somatostatin analogues, e.g.
[izsl-Tyrii] somatostatin-14 (Taylor, J.E. et al., Life Science (1988) 43: 421), or [1~SI-Tyr3] SMS 201-995 also called [lisl]
204-090 (Reubi, J.C. et al., Brain Res. (1987 ) 406: 891; Reubi, J.C. et al., J. Clin. Endocr. Metab. (1987) 65: 1127; Reubi, J.C.
et al., Cancer Res. (1987) 47: 551; Reubi, J.C. et al., Cancer Res. (1987) 47: 5758).
The present invention relates to polypeptides, process for their production, pharmaceutical preparations containing them and their use as a pharmaceutical, e.g. for treatment of somatostatin receptor positive tumors or as in vivo diagnostic imaging agents.
In the last few years a high incidence of somatostatin receptors has been demonstrated in a variety of human tumors, e.g.
pituitary tumors, central nervous system tumors, breast tumors, gastro-enteropancreatic tumors and their metastases. Some of them are small or slow-growing tumors which are difficult to precisely localize by conventional diagnosis methods.
In vitro visualization of somatostatin receptors has been per-formed through autoradiography of~.tumoral tissues using radio-iodinated somatostatin or somatostatin analogues, e.g.
[izsl-Tyrii] somatostatin-14 (Taylor, J.E. et al., Life Science (1988) 43: 421), or [1~SI-Tyr3] SMS 201-995 also called [lisl]
204-090 (Reubi, J.C. et al., Brain Res. (1987 ) 406: 891; Reubi, J.C. et al., J. Clin. Endocr. Metab. (1987) 65: 1127; Reubi, J.C.
et al., Cancer Res. (1987) 47: 551; Reubi, J.C. et al., Cancer Res. (1987) 47: 5758).
New somatostatin peptides useful in therapeutic and which can be labelled for in vivo diagnostic and therapeutic applications have now been found.
According to the invention, there is provided a somatostatin peptide bearing at least one chelating group for a detectable element, this chelating group being linked to an amino group of said peptide, and this amino group having no significant binding affinity for somatostatin receptors.
These compounds are referred to thereafter as LIGANDS OF THE
INVENTION. They possess one chelating group capable of reacting with a detectable element, e.g. a radionuclide, a radioopaque element or a paramagnetic ion, to form a complex and further are capable of binding to somatostatin receptors, e.g. expressed or overexpressed by tumors or metastases.
The chelating group is linked by a covalent bond 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 group of LIGANDS is that wherein the chelating group is at-tached directly to the amino group of the somatostatin peptide.
Another group of LIGANDS is that wherein the chelating group is attached indirectly by a bridging or a spacer group to the amino group of the somatostatin peptide.
According to the invention, there is provided a somatostatin peptide bearing at least one chelating group for a detectable element, this chelating group being linked to an amino group of said peptide, and this amino group having no significant binding affinity for somatostatin receptors.
These compounds are referred to thereafter as LIGANDS OF THE
INVENTION. They possess one chelating group capable of reacting with a detectable element, e.g. a radionuclide, a radioopaque element or a paramagnetic ion, to form a complex and further are capable of binding to somatostatin receptors, e.g. expressed or overexpressed by tumors or metastases.
The chelating group is linked by a covalent bond 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 group of LIGANDS is that wherein the chelating group is at-tached directly to the amino group of the somatostatin peptide.
Another group of LIGANDS is that wherein the chelating group is attached indirectly by a bridging or a spacer group to the amino group of the somatostatin peptide.
- 3 - loa-7382 Preferably the chelating group is attached by an amide bond to the peptide.
The term somatostatin peptides includes the naturally occurring somatostatin (tetradecapeptide) and its analogues or derivatives.
By derivatives or analogues as used herein is meant any straight-chain or cyclic polypeptide derived from that of the naturally occurring tetradecapeptide somatostatin wherein one or more amino acid units have been omitted and/or replaced by one or more other amino acid radicals) and/or wherein one or more functional groups have been replaced by one or more other functional groups and/or one or more groups have been replaced by one or several other isosteric groups. In general, the term covers all modified derivatives of a biologically active peptide which exhibit a qualitatively similar effect to that of the unmodified soma-tostatin peptide, e.g. they bind to somatostatin receptors and decrease hormone secretion.
Cyclic, bridge cyclic and straight-chain somatostatin analogues are known compounds. Such compounds and their preparation are described e.g. in European Patent Specifications EP-A-1295;
29,579; 215,171; 203,031; 214,872; 298,732; 277,419.
Preferred LIGANDS OF THE INVENTION are those derived from the following somatostatin analogues:
A. Analogues of formula I
A' CHZ-S-Yl Yz-S-CHz jN-CH-CO-B-C-D-E-NH-CH-G I
A
The term somatostatin peptides includes the naturally occurring somatostatin (tetradecapeptide) and its analogues or derivatives.
By derivatives or analogues as used herein is meant any straight-chain or cyclic polypeptide derived from that of the naturally occurring tetradecapeptide somatostatin wherein one or more amino acid units have been omitted and/or replaced by one or more other amino acid radicals) and/or wherein one or more functional groups have been replaced by one or more other functional groups and/or one or more groups have been replaced by one or several other isosteric groups. In general, the term covers all modified derivatives of a biologically active peptide which exhibit a qualitatively similar effect to that of the unmodified soma-tostatin peptide, e.g. they bind to somatostatin receptors and decrease hormone secretion.
Cyclic, bridge cyclic and straight-chain somatostatin analogues are known compounds. Such compounds and their preparation are described e.g. in European Patent Specifications EP-A-1295;
29,579; 215,171; 203,031; 214,872; 298,732; 277,419.
Preferred LIGANDS OF THE INVENTION are those derived from the following somatostatin analogues:
A. Analogues of formula I
A' CHZ-S-Yl Yz-S-CHz jN-CH-CO-B-C-D-E-NH-CH-G I
A
wherein A is C1_lZalkyl, C~_lophenylalkyl or a group of formula RCO-, whereby i) R is hydrogen, Cl_llalkyl, phenyl or C~_lo_ phenylakyl, or ii) RCO- is a) an L- or D-phenylalanine residue optio-nally ring-substituted by F, C1, Br, NOz, NHa, OH, C1_;alkyl and/or C1_3alkoxy;
b) the residue of a natural or synthetic a-amino acid other than defined under a) above or of a corresponding D-amino acid, or c) a dipeptide residue in which the indivi-dual amino acid residues are the same or different and are selected from those defined under a) and/or b) above, the a-amino group of amino acid residues a) and b) and the N-terminal amino group of dipeptide residues c) being optionally mono- or di-C1_l2alkylated or substituted by C1_ealkanoyl, A' is hydrogen, C1_izalkyl or C~_lophenylalkyl, Y1 and Yi represent together a direct bond or each of Y1 and YZ is independently hydrogen or a radical of formulae (1) to (5) 2C104.i32 I . ~ CHz -C0-C-(CHz)"-H -CO-CH -CO-NHR~
Rb \(CHz )n (1) (2) (3) R ' a Re -CO-NH-CH-COOR, -CO-(NH)p- C -(CHz)r Ra Rb~ q (4) (5) wherein R, is methyl or ethyl Rb is hydrogen, methyl or ethyl m is a whole number from 1 to 4 n is a whole number from 1 to 5 R~ is (C1_6)alkyl Ra represents the substituent attached to the a-carbon atom of a natural or synthetic a-amino acid (including hydrogen) R, is (C1_s)alkyl R,' and are independently hydrogen, methyl or Rb' ethyl, Rs and are independently hydrogen, halogen, R9 (C1_3)alkyl or (C1_3)alkoxy, p is 0 or 1, q is 0 or 1, and r is 0, 1 or 2, B is -Phe- optionally ring-substituted by halogen, NOz, NHz, OH, C1_3alkyl and /or Cl_3alkoxy (in-cluding pentafluoroalanine), or S--naphthyl-Ala C is (L)-Trp- or (D)-Trp- optionally a-N-methyl-ated and optionally benzene-ring-substituted by halogen, NOz, NHz, OH, C1_jalkyl and/or C1_3 alkoxy, D is Lys, Lys in which the side chain contains 0 or S in ~-position, YF-Lys or bF-Lys, optionally a-N-methylated, or a 4-amfnocyclohexylAla or 4-aminocyclohexylGly residue E is Thr, Ser, Val, Phe, Ile or an aminoisobutyric or aminobutyric acid residue G is a group of formula Ris /Ri i ~
-COORS, -CHiORlo, -CON or -CO-N--LXl ~ Ri z wherein R~ is hydrogen or C1_3alkyl,.
Rlo is hydrogen or the residue of a physiologically acceptable, physiologically hydrolysable ester, Ril is hydrogen, C1_3alkyl, phenyl or C~_lophenyl-alkyl, Riz is hydrogen, C1_3alkyl or a group of formula -CH(Ri3)-Xi, R13 is CHZOH, -(CHi)i-OH, -(CHZ)3-OH, or -CH(CH3)OH
or represents the substituent attached to the a-carbon atom of a natural or synthetic a-amino acid (including hydrogen) and 200~3~
Xi is a group of formula -COORS, -CHzORio or /Ri a -CO-N
~Ri s wherein R~ and Rlo have the meanings given above, Ria is hydrogen or C1_3alkyl and Ris is hydrogen, C1_3alkyl, phenyl or C~_iophenyl-alkyl, and Ris is hydrogen or hydroxy, with the proviso that when Rli is -CH(R13)-X1 then R11 is hydrogen or methyl, wherein the residues B, D and E have the L-configuration, and the residues in the 2-and 7-position and any residues Y1 4) and YZ 4) each independently have the (L)- or (D)- configu-ration.
The significances of A and A' in formula I are preferably selected so that the compound contains a terminal -NH- group capable of being linked to a chelating agent.
In the compounds of formula I, the following significances are preferred either individually or in any combination or sub-combination:
1. A is C~_lo phenylalkyl, especially phenethyl, or a group of formula RCO. Preferably A is a group of formula RCO.
1.1. Preferably R is C1_ii alkyl or C~_lo phenylalkyl, especially C~_lo phenylalkyl, more especially phenethyl, or RCO has the meanings a), b) or c).
200~~32 1.2. When RCO has the meanings a), b) or c), the «-amino group of amino acid residues a) and b) and the N-terminal amino group of dipeptide residues c) is preferably non-alkylated or mono-C1_li alkylated, especially -C1_e alkylated, more especially -methylated. Most preferably the N-terminal is non-alkylated.
1.3. When RCO has the meaning a) this is preferably a') an L-or D-phenylalanine or -tyrosine residue optionally mono-N-C1_lz alkylated. More preferably a') is an L- or D-phenylala-nine residue.
1.4. When RCO has the meaning b) or c) the defined residue is preferably lipophilic. Preferred residues b) are thus b') a-amino acid residues having a hydrocarbon side chain, e.g.
alkyl with 3, preferably 4, or more C atoms, e.g. up to 7 C-atoms, naphthyl-methyl or heteroaryl, e.g. 3-(2- or 1-naph-thyl)-alanine, pyridyl-methyl or tryptophane residue, said residues having the L- or D-configuration, and preferred residues c) are dipeptide residues in which the individual amino acid residues are the same or different and are selected from those defined under a') and b') above.
Example of a residue c) is e.g. 3-(2-naphthyl)-alanine resi-due.
1.5. Most preferably RCO has the meaning a) especially the meaning a').
2. B is B', where B' is Phe or Tyr.
3. C is C', where C' is (D)Trp.
.~ 2fl~4 i3~
4. D is D', where D' is Lys, MeLys or Lys(E-Me), especially Lys.
5. E is E', where E' is Val or Thr, especially Thr.
~Rl 1 6. G is G', where G' is a group of formula -CO-N.\ , especially a group of formula -CO-N/
\CH(R13)-X1 (in which case R11=H or CH3). In the latter case the moiety -CH(R13)-X1 preferably has the L-configuration.
6.1. R11 is preferably hydrogen.
6.2. As the substituent attached to the a-carbon atom of a natural amino acid (i.e. of formula H2N-CH(R13)-COOH), R13 is preferably -CHZOH, -CH(CH3)-OH, isobutyl or butyl, or R13 is -(CHz)2-OH or -(CH2)3-OH. It is especially -CH~OH or -CH(CH3)OH.
' ~1 4 6.3. X1 is preferably a group of formula -CO-N\
\Rl s or -CHZ-ORIO, especially of formula -CHI-ORIO and Rlo is pre-ferably hydrogen or has 'the meaning given under 7 below. Most preferably Rlo is hydrogen.
The following individual compounds are illustrative of com-pounds of formula I:
-lo- 2004532 H-(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-of also known as octreotide (D)Phe-Cys-Thr-(D)Trp-Lys-Val-Cys-ThrNHZ
(D)Phe-Cys-Tyr-(D)Trp-Lys-Val-Cys-TrpNHi (D)Trp-Cys-Phe-(D)Trp-Lys-Thr-Cys-ThrNHi (D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-ThrNHi 3-(2-Naphthyl)-(D)Ala-Cys-Tyr-(D)Trp-Lys-Val-Cys-ThrNHz 3-(2-Naphthyl)-(D)Ala-Cys-Tyr-(D)Trp-Lys-Val-Cys-S-Nal-NHz 3-(2-Naphthyl)-(D)Ala-Cys- ~-Nal-(D)Trp-Lys-Val-Cys-ThrNHi (D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-~Nal-NH=
B. Analogues of formula II
H-Cys-Phe-Phe-(D)-Trp-Lys-Thr-Phe-Cys-of II
[see Vale et al., Metabolism, 27, Supp. 1, 139, (1978)]
H-Cys-His-Hfs-Phe-Phe-(D)Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH III
(see EP-A-200,188) A
w- 2004532 Particular preferred LIGANDS are those derived from H-(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-ol.
Suitable chelating groups are physiologically acceptable chela-ting groups capable of complexing a detectable element.
Preferably the chelating group has substantially a hydrophilic character. Examples of chelating groups include e.g. iminodi-carboxylic groups, polyaminopolycarboxylic groups, e.g. those derived from non cyclic ligands e.g. ethylene diaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), ethylene glycol-0,0'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N'-bis(hydroxybenzyl)ethylenediamine-N, N'-diacetic acid (HBED) and triethylenetetramine hexaacetic acid (TTHA), those derived from substituted EDTA or -DTPA, e.g. p-isothiocyanato-benzyl-EDTA or -DTPA those derived from macrocyclic ligands, e.g.
1,4,7,10-tetra-azacyclododecane-N,N',N " ,N " '-tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane-N,N',N " ,N " '-tetra-acetic acid (TETA), those derived from N-substituted or C-substi-tuted macrocyclic amines including also cyclames, e.g. as dis-closed in EP 304,780 A1 and in WO 89/01476-A, groups of formula IV or V
R1-C-S-(CHZ)n~-C-(TT)i-C- IV
n n RZ-C-S-(CHZ)n.-C-NH-CHI 0 n CH-C- V
R3-C-S-(CHZ)".-C-NH
i n wherein each of R1, Rz and Rj independently is C1_salkyl, C5_earyl or C~_9arylalkyl, each optionally substituted by OH, C1_4alkoxy, COOH or S03H, n' is 1 or 2, i is an integer from 2 to 6, and TT are independently a or S amino acids linked to each other by amide bonds, groups derived from bis-aminothiol derivatives, e.g. compounds of formula VI
~CH2 HN NH
R ~~ X22 vI
21 C~ i~ ~R
R2a wherein each of Rzo, Rsi, Riz and Rz3 independently is hydrogen or C1_4alkyl, Xz is a group capable of reacting with the N-amino group of the peptide, and m' is 2 or 3, groups derived from dithiasemicarbazone derivatives, e.g.
compounds of formula VII
HN ~ N N ~ H VII
HN i ' SH HS NH
wherein XZ is as defined above, groups derived from propylene amine oxime derivatives, e.g.
compounds of formula VIII
~ ~cN2 HN NH
R26~ R27 VIII
R25~ _R28 R24 ~ ~ R29 OH OH
wherein each of RZ,, Rzs, Rzs, Rz~, Ria and Rz9 independently are hydrogen or C1-4alkyl, and Xz and m' are as defined above, groups derived from diamide dimercaptides, e.g. compounds of formula IX
20~4~3~:
Y S S IX
H5C6C0 COC64~
wherein X3 is a divalent radical optionally substituted and bearing a group capable of reacting with the N-amino group of the peptide, e.g. C1_4alkylene or phenylene bearing a group Xz, and YS is hydrogen or COZR3o, wherein R3o is C1_4alkyl, or groups derived from porphyries, e.g. N-benzyl-5,10,15,20-tetrakis-(4-carboxyphenyl)porphine or TPP bearing a group Xi as defined above.
Aryl is preferably phenyl. Arylalkyl is preferably benzyl.
Examples of X~ include radicals of formula -(X,)" " -Xs wherein X4 is C1_salkylene; or C1_6alkylene optionally attached to the carbon atom by an oxygen atom or -NH-, n " is 0 or 1 and X5 is -NCS, a carboxy group or a functional derivative thereof, e.g.
acid halide, anhydride or hydrazide. It is understood that XZ is attached to one of the carbon atom of -[CHij~.- or =CH-CH. in replacement of an hydrogen atom.
The chelating group may be attached either directly or indirectly to the N-amino group of the somatostatin peptide. When it is attached indirectly, it is preferably linked through a bridging or spacer group, for example a group of formula (al) ._ ~~~i Z-R35-CO- (ai) R3s is C1_l alkylene, Cz_llalkenylene or -CH(R')- wherein R' is the residue attached in a to a natural or synthetic a-amino acid, e.g. hydrogen, C1_llalkyl, benzyl, optionally substi-tuted benzyl, naphthyl-methyl, pyridyl-methyl, Z is a functional moiety capable of covalently reacting with the chelating agent.
Z may be for example a group which can form an ether, ester or amide bonding with the chelating group. Z is preferably amino.
The chelating groups, when comprising carboxy, -S03H and/or amino groups may exist in free form or in salt form.
Preferred chelating groups are those derived from polyamino-poly-carboxylic groups, e.g. those derived from EDTA, DTPA, DOTA, TETA
or substituted EDTA or DTPA. Chelating groups derived from DTPA
are most preferred.
In the LIGANDS OF THE INVENTION the chelating group, when poly-functional, may be linked either to a single somatostatin peptide molecule or to more than one somatostatin peptide molecules e.g.
to two somatostatin peptide molecule's.
The LIGANDS OF THE INVENTION may exist e.g. in free or salt form.
Salts include acid addition salts with e.g. organic acids, poly-meric acids or inorganic acids, for example hydrochlorides and acetates, and salt forms obtainable with the carboxylic or sul-phonic acid groups present in the chelating group, e.g. alkali metal salts such as sodium or potassium, or substituted or un-substituted ammonium salts.
The present invention also includes a process for the production of the LIGANDS OF THE INVENTION. They may be produced by analogy to known methods.
The LIGANDS OF THE INVENTION may be produced for example as follows:
a) removing at least one protecting group which is present in a somatostatin peptide bearing a chelating group, or b) linking together by an amide bond two peptide fragments each of them containing at least one amino acid or amino alcohol in protected or unprotected form and one of them containing the chelating group, wherein the amide bond is in such a way that the desired amino acid sequence is obtained, and stage a) of the process is then optionally effected, or c) linking together a chelating agent and the desired somato-statin peptide in protected or unprotected form in such a way that the chelating group is fixed on the desired N-amino group of the peptide, and stage a) is then optionally effected or, d) removing a functional group of an unprotected or a protected peptide bearing a chelating group or converting it into another functional group so that another unprotected or protected peptide bearing a chelating group is obtained and in the latter case stage a) of the process is effected, or e) oxidising a somatostatin peptide modified by a chelating group in which the mercapto groups of Cys radicals exist in free form so as to produce an analogue in which two Cys ra-dicals are joined by an S-S-bridge 2004 i3 c;
and recovering the LIGAND thus obtained in free form or in salt form.
The above reactions may be effected in analogy with known me-thods, e.g. as described in the following examples, in particular processes a) and c). When the chelating group is attached by an amide bond, this may be carried out analogously to the methods used for amide formation. Where desired, in these reactions, protecting groups which are suitable for use in peptides or for the desired chelating groups may be used for functional groups which do not participate in the reaction. The term protecting group may also include a polymer resin having functional groups.
When it is desired to attach the chelating group to the terminal N-amino group of a peptide or peptide fragment used as starting material, and which comprises one or more side chain amino groups, these side chain amino groups are conveniently protected with a protecting group , e.g. as used in peptide chemistry.
When it is desired to attach the chelating group to a side chain amino group of a peptide or peptide fragment used as starting ma-terial, and the peptide comprises a free terminal N-amino group, the latter is preferably protected with a protecting group.
The peptide fragment bearing the chelating group and used in sta-ge b) may be prepared by reacting the peptide fragment comprising at least one amino acid or amino alcohol in protected or unpro-tected form with the chelating agent. The reaction may be perfor-med in analogy with stage c).
The chelating groups of formula IV or V may be linked to a peptide by reacting a chelating agent of formula IV' or V' R1-C-S-(CH2)n.-C-(TT)i-C-X IV' n a RZ-C-S-(CHZ)n.-C-NH- HC'z 0 CH-C-X V' R3-C-S-(CHz)n.-C-NH
wherein X is an activating group capable of forming an amide bond with the N-amino group of the peptide. The reaction may be per-formed as disclosed in EP 247,866 A1.
The chelating agent used in process step c) may be known or pre-pared in analogy with known procedures. The compound used is such that it allows the introduction of the desired chelating group on the somatostatin peptide, e.g. a polyaminopolycarboxylic acid as disclosed, a salt or anhydride thereof.
In the above process, when in the amino-acids, peptide fragments or peptides used as starting materials, the chelating group is attached through a bridging or spacer group to the peptide, e.g.
a radical of formula (al) as defined above, such amino-acids, peptide fragments or peptides may be prepared by reacting in conventional manner the corresponding amino-acids or peptides free of bridging or spacer group with a corresponding bridging-or spacer-yielding compound, for example an acid or reactive acid derivative comprising the bridging or spacer group, e.g. an acid of formula Z-R35-COON or a reactive acid derivative thereof such as an active ester. Examples of active ester groups or carboxy activating groups are e.g. 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, succinimidyl or 1-hydroxy-benzotriazolyl.
Alternatively the chelating agent may first be reacted with a bridging or spacer group-yielding compound, in order to bear the bridging or spacer group and then be reacted in conventional manner with the peptide, peptide fragment or amino-acid.
The LIGANDS OF THE INVENTION may be purified in 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 in free form or in the form of phar-maceutically acceptable salts are valuable compounds.
According to a further embodiment, the LIGANDS OF THE INVENTION
can be complexed with a detectable element.
Accordingly, the present invention also provides the LIGANDS OF
THE INVENTION as defined above which are complexed with a detec-table element (hereinafter referred to as CHELATES OF THE INVEN-TION), in free form or in salt form, their preparation and their use for in vivo diagnostic and therapeutic treatment.
By detectable~element is meant any element, preferably a metal ion which exhibits a property detectable in therapeutic or in vivo diagnostic techniques, e.g. a metal ion which emits a de-tectable radiation or a metal ion which is capable of influencing NMR relaxation properties.
Suitable detectable metal iohs include for example heavy elements or rare earth ions, e.g. as used in CAT scanning (Computer axial tomography), paramagnetic ions, e.g. Gd3+, Fe3+, MnZ+ and Cr2+, fluorescent metal ions, e.g. Eu3+, and radionuclides, e.g.
Y-emitting radionuclides, S-emitting radionuclides, a-emitting radionuclides, positron-emitting radionuclides e.g. 68Ga.
Suitable Y-emitting radionuclides include those which are useful in diagnostic techniques. The r-emitting radionuclides advanta-geously have a half-life of from 1 hour to 40 days, preferably from 5 hours to 4 days, more preferably from 12 hours to 3 days.
Examples are radionuclides derived from Gallium, Indium, Tech-netium, Ytterbium, Rhenium and Thallium e.g. s~Ga, illln, 99'"Tc, issyb and lBSRe. Preferably the Y-radionuclide is selected depen-ding on the metabolism of the LIGAND OF THE INVENTION or somato-statin peptide used. More preferably the LIGAND OF THE INVENTION
is chelated with a Y-radionuclide having a longer half-life than the half-life of the somatostatin peptide on the tumor.
Further radionuclides suitable for use in imaging are positron-emitting radionuclides, e.g. as mentioned above.
Suitable ~-emitting radionuclides include those which are useful in therapeutic applications, for example Soy, s~_Cu, iasRe, iaeRe~ is9Er~ izign~ iz~Te~ 143pr~ 198Au~ io9pd~ issDY~ 3zP~
iazpr. The ~-radionuclide advantageously have a half-life of from 2.3 hrs to 14.3 d, preferably from 2.3 to 100 hrs. Preferably the remitting radionuclide is selected in order to have a longer half-life than the half-life of the somatostatin peptide on the tumor.
Suitable a-emitting radionuclides are those which are used in therapeutic treatments, e.g. zllAt, zizgi.
The CHELATES OF THE INVENTION may be prepared by reacting the LIGAND with a corresponding detectable element yielding compound, e.g. a metal salt, preferably a water-soluble salt. The reaction may be carried out by analogy with known methods, e.g. as disclo-sed in Perrin, Organic Ligand, Chemical Data Series 22. NY Perga-mon Press (1982); in Krejcarit and Tucker, Biophys. Biochem. Res.
Com. 77: 581 (1977) and in Wagner and Welch, J. Nucl. Med. _20:
428 (1979).
Preferably the complexing of the LIGAND is effected at a pH at which the LIGAND OF THE INVENTION is physiologically stable.
Alternatively the detectable element may also be provided to the solution as a complex with an intermediate chelating agent, e.g.
a chelating agent which forms a chelate complex that renders the element soluble at the physiological pH of the LIGAND but is less thermodynamically stable than the CHELATE. Example of such an intermediate chelating agent is 4,5-dihydroxy-1,3-benzene-di-sulfonic acid (Tiron). In such a process, the detectable element exchanges the ligand.
The CHELATES OF THE INVENTION may also be produced by linking together a chelating agent complexed with the detectable element, and a somatostatin peptide in protected or unprotected form and if desired removing at least one protecting group which is present. The same reaction may be performed with a chelating agent complexed with a non detectable metal ion and then in the resulting complexed peptide the metal ion may be replaced by the desired detectable element.
The CHELATES OF THE INVENTION may also be produced by linking together a chelating agent complexed with the detectable element, and a somatostatin peptide fragment comprising at least one amino acid in protected or unprotected form and then continuing the peptide synthesis step by step until the final peptide sequence is obtained and if desired removing at least one protecting group which is present. Instead of the detectable element the chelating agent may be complexed with a non detectable metal and this metal 2004,53:
may then be replaced by the detectable element in the resulting complexed somatostatin peptide.
Where the chelating group is attached through a bridging or spacer group to the somatostatin peptide, e.g. through a radical of formula (al) as defined above, either the somatostatin peptide or peptide fragment or the chelating agent may bear said bridging or spacer group.
The above mentioned reactions may be effected in analogy to known methods. Depending on the chelating group present, the labeling efficiency may approach 100X so that purification is not requi-red. Radionuclides such as for example Technetium-99m may be used in oxidized form, e.g. Tc-99m pertechnetate, which may be com-plexed under reducing conditions.
The above mentioned reactions are conveniently effected under conditions avoiding trace metal contamination. Preferably dis-tilled de-ionized water, ultrapure reagents, chelation-grade radioactivity etc..are used to reduce the effects of trace metal.
The CHELATES OF THE INVENTION may exist e.g. in free or salt form. Salts include acid addition salts with e.g. organic acids, polymeric acids or inorganic acids,~for example hydrochlorides and acetates, and salt forms obtainable with the carboxylic acid groups present in the molecule which do not participate to the chelate formation, e.g. alkali metal salts such as sodium or potassium, or substituted or unsubstituted ammonium salts.
The CHELATES OF THE INVENTION and their pharmaceutical acceptable salts exhibit pharmaceutical activity and are therefore useful either as an imaging agent, e.g. visualisation of somatostatin receptor positive tumors and metastases when complexed with a paramagnetic, a Y-emitting metal ion or a positron-emitting radionuclide, or as a radiopharmaceutical for the treatment in vivo of somatostatin receptor positive tumors and metastases when complexed with a a- or ~-radionuclide, as indicated by standard tests.
In particular, the CHELATES OF THE INVENTION possess affinity for somatostatin receptors expressed or overexpressed by tumors and metastases, as indicated in standard in vitro binding assays.
A somatostatin receptor positive tumor originating from the human gastro intestinal tract is removed from a patient and immediately put on ice and within a maximal delay of 30 min frozen at - 80 ° C. For further autoradiography this frozen material is cut on a cryostat (Leitz 1720) in 10 um sections, mounted on pre-cleaned microscope slides and stored at - 20 ° C for at least 3 days to improve adhesion of the tissue to the slide. The sections are preincubated in Tris-HC1 buffer (50 mM, pH 7.4), containing CaCl2 (2mM) and KC1 (5mM), for 10 min at ambient temperature and then washed twice for 2 min in the same buffer without additional salts added. The sections are then incubated with a CHELATE OF THE INVENTION for 2 hours at ambient tempe-rature in Tris-HC1 buffer (170 mM, pH 7.4), containing bovine serum albumin.(10 g/1), bacitracin (40 mg/1) and MgCl~ (5 mM~
to inhibit endogenous proteases. Non-specific binding is determi-ned by adding the corresponding non-labelled, hon-modified soma-tostatin peptide at a concentration of 1 uM. Incubated sections are washed twice for 5 min in cold incubation buffer containing 0.25 g/1 BSA. After a brief dip in distilled water to remove ex-cess salts, the sections are dried quickly and apposed to [3H]-LKB films. After a time exposure of about 1 week in X-ray casset-tes, it is observed that the CHELATES OF THE INVENTION, e.g. a radionuclide CHELATE, give very good results in labeling the tumoral tissue without labeling the surrounding healthy tissue when added at a concentration of about 10'1° to 10'3 M.
2Q04~32 The affinity of the CHELATES OF THE INVENTION for somatostatin receptors can also be shown by in vivo testing.
Rats bearing transplantable exocrine pancreatic somatostatin receptor positive tumors are treated with an intravenous injec-tion of a CHELATE OF THE INVENTION. Injection site is the penis vein. Immediately after administration, the animals are positio-ned on the collimator of a gamma-camera and the distribution of radioactivity is monitored at various time intervals.
Biodistribution of radioactivity may also be determined through serial sacrifice of a number of such treated rats and determi-nation of the organ radioactivity.
After administration of a CHELATE OF THE INVENTION, e.g. a radio-nuclide CHELATE, for example a Y-emitting CHELATE, at a dosage of from 1 to 5 ug/kg of LIGAND labeled with 0.1 to 2 mCi radionucli-de the tumor site becomes detectable together with the organs where excretion essentially takes place.
Accordingly, in a series of specific or alternative embodiments, the present invention also provides:
1. A method for in vivo detection of somatostatin receptor positive tumors or metastases in a subject which comprises a) administering a CHELATE OF THE INVENTION to said subject and b) recording the localisation of the receptors targeted by said CHELATE.
CHELATES OF THE INVENTION for use in the in vivo detection method of the invention are the CHELATES which are complexed with a Y-emitting radionuclide, a positron-emitting radio-nuclide or a paramagnetic metal ion, e.g. as indicated above.
The CHELATES OF THE INVENTION for use as an imaging agent in method (1) may be administered parenterally, preferably in-travenously, e.g. in the form of injectable solutions or sus-pensions, preferably in a single injection. The appropriate dosage will of course vary depending upon, for example, the LIGAND and the type of detectable element used, e.g. the radionuclide. A suitable dose to be injected is in the range to enable imaging by photoscanning procedures known in the art. When a radiolabeled CHELATE OF THE INVENTION is used, it may advantageously be administered in a dose having a radio-activity of from 0.1 to 50 mCi, preferably 0.1 to 30 mCi, mo-re preferably 0.1 to 20 mCi. An indicated dosage range may be of from 1 to 200 ug LIGAND labeled with 0.1 to 50 mCi, pre-ferably 0.1 to 30 mCi, e.g. 3 to 15 mCi, Y-emitting radionu-clide, depending on the Y-emitting radionuclide used. For example with In, it is preferred to use a radioactivity in the lower range, whereas with Tc, it is preferred to use a radioactivity in the upper range.
The enrichment in the tumorigenic sites with the CHELATES may be followed by the corresponding imaging techniques, e.g.
using nuclear medicine imaging instrumentation, for example a scanner, Y-camera , rotating Y-camera, each preferably com-puter assisted; PET-scanner (Positron emission tomography);
MRI equipment or CAT scanning equipment.
The CHELATES OF THE INVENTION, e.g. a major part of the Y-emitting CHELATES is substantially excreted through the kidneys and does not significantly accumulate in the liver.
2. A method for in vivo treatment of somatostatin receptor posi-tive tumors and metastases in a subject in need of such a treatment which comprises administering to said subject a therapeutically effective amount of a CHELATE OF THE INVEN-2U04S3~
TION.
CHELATES OF THE INVENTION for use in the in vivo treatment method of the invention are the CHELATES complexed with a a-or ~-radionuclide as defined above.
Dosages employed in practising the therapeutic method of the present invention will of course vary depending e.g. on the particular condition to be treated, for exemple the volume of the tumor, the particular CHELATE employed, for exemple the half-life of the CHELATE in the tumor, and the therapy desi-red. In general, the dose is calculated on the basis of ra-dioactivity distribution to each organ and on observed target uptake. For example the CHELATE may be administered at a dai-ly dosage range having a radioactivity of from 0.1 to 3mCi/kg body weight, e.g. 1 to 3 mCi, preferably 1 to 1.5 mCi/kg body weight. An indicated daily dosage range is of from 1 to 200 ug LIGAND labeled with 0.1 to 3 mCi/kg body weight, e.g.
0.1 to 1.5/kg body weight a- or S-emitting radionuclide, conveniently administered in divided doses up to 4 times a day.
The a- or ~--emitting CHELATES OF THE INVENTION may be admini-stered by any conventional route, in particular parenterally, e.g. in the form of injectable solutions or suspensions. They may also be administered advantageously by infusion, e.g. an infusion of 30 to 60 min. Depending on the site of the tumor, they may be administered as close as possible to the tumor site, e.g. by means of a'catheter. The mode of administration selected may depend on the dissociation rate of the CHELATE
used and the excretion rate.
The CHELATES OF THE INVENTION may be administered in free form or in pharmaceutically acceptable form. Such salts may be prepared ~00453~
in conventional manner and exhibit the same order of activity as the free compounds.
The CHELATES OF THE INVENTION for use in the method of the pre-sent invention may preferably be prepared shortly before the ad-ministration to a subject, i.e. the radiolabeling with the desi-red detectable metal ion, particularly the desired a-, S- or Y-radionuclide, may be performed shortly before the administration.
The CHELATES OF THE INVENTION may be suitable for imaging or treating tumors such as pituitary, gastroenteropancreatic, central nervous system, breast, prostatic, ovarian or colonic tumors, small cell lung cancer, paragangliomas, neuroblastomas, pheochromocytomas, medullary thyroid carcinomas, myelomas, etc.
and metastases thereof.
According to a further embodiment of the invention, the Y-emitting CHELATES OF THE INVENTION may also be used as imaging agent for the evaluation of the kidney function.
Groups of five mice are used. Each mouse is injected intra-venously through a tail vein with 0.1 ml containing 1 mCi of a CHELATE OF THE INVENTION. The mice are then placed in metabolic cages for the collection of excreted urine. At 10 or 120 min.
post-injection, the urethras are ligated and the mice anestheti-zed with chloroform. Imaging of the uropoietic system is carried out using the usual imaging technique. In this test, the Y-emit-ting CHELATES OF THE INVENTION improves imaging of renal excre-tion when administered at a dosage of from 0.1 to 30 mCi.
Accordingly, the present invention also provides a method for in vivo evaluation of the kidney function in a subject which comprises administering to said subject an effective amount of a Y-emitting CHELATE and recording the kidney function.
2004x32 According to a further aspect of the invention, there is pro-vided:
i. a pharmaceutical composition comprising a LIGAND OF THE
INVENTION in free or in pharmaceutically acceptable salt form, together with one or more pharmaceutically acceptable carriers or diluents therefor;
ii. a pharmaceutical composition comprising a CHELATE according to the invention in free or in pharmaceutically acceptable salt form, together with one or more pharmaceutically accep-table carriers or diluents therefor.
Such compositions may be manufactured in conventional manner.
A composition according to the invention may also be presented in separate package with instructions for mixing the LIGAND with the metal ion and for the administration of the resulting CHELATE. It may also be presented in twin-pack form, that is, as a single package containing separate unit dosages of the LIGAND and the detectable metal ion with instructions for mixing them and for administration of the CHELATE. A diluent or carrier may be pre-sent in the unit dosage forms. .
In the following examples, all temperatures are in ° C and [a]2~-values uncorrected. The following abbreviations are employed: D
Boc tert.-butoxycarbonyl TFA trifluoroacetic acid DTPA diethylenetriamine-pentaacetic acid BgAMPLB 1: DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of 1.1 g of DPhe-Cys-Phe-DTrp-Lys(E-Boc)-Thr-Cys-Thr-of in free base (1 mM), are dissolved in 5 1 of dioxan/H~0 1/1 (v/v) and reacted with 5 g NaHCb3. The 520 mg of DTPA dianhydride is slowly added with stirring. The reaction mixture is stirred for a further 30 min and dry-frozen. The residue is dissolved in 250 ml water and the pH is adjusted to pH 2.5 with concentrated HC1. The pre-cipitated product is filtered out, washed and dried over phosp-horus pentoxide. After chromatography on a silica-gel column, the following products are isolated: 230 mg of DTPA-DPhe-Cys-Phe-DTrp-Lys(E-Boc)-Thr-Cys-Thr-of and 500 mg of the corresponding dimer DTPA-(DPhe-Cys-Phe-DTrp-Lys(e-Boc)-Thr-Cys-Thr-ol)~.
3 ml of TFA are mixed with 200 mg of DTPA-DPhe-Cys-Phe-DTrp-Lys(E-Boc)-Thr-Cys-Thr-ol. After 5 min at room temperature, the mixture is precipitated with diisopropylether, filtered out and dried. The residue is desalted over Duolite and lyophilised to yield 150 mg of the title compound:
[a]D° _ - 26,6 ° (c = 1 95 % AcOH)..
The starting material may be produced as follows:
a) H-DPhe~Cys-Phe-DTrp-Lys(Boc) Thr-Cys=Thr=of 2.25 g of di-tert.butyl-pyrocarbonate, dissolved in 30 ml of DMF, are slowly added in drops at room temperature to a r solution of 10 g of H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol-acetate in 100 ml of DMF. After two hours at room tempe-A
rature, the solvent is drawn off under vacuum, and 200 ml of diisopropylether are added to the residue. The deposit which is being formed is filtered off, washed with diisopropylether and dried. The crude product is purified by chromatography over silica gel (eluant: CHZClz/MeOH 9/1) and is then iso-lated as a white amorphous powder.
[a]D° = 29.8 ° (c = 1.28 in DMF).
BgAMPLB 2: DTPA-(DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol)2 The fraction containing the intermediate product DTPA-DPhe-Cys-Phe-DTrp-Lys(s-Boc)-Thr-Cys-Thr-ol)2 as obtained in example 1 is treated as described above for the corresponding monomeric form, the Boc protecting groups being removed to yield the title compound:
[a]D° _ - 24,5 ° (c = 0,55 95 Y AcOH).
BgAMPLB 3: H2N- CHz $-CO-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of a. 0.56 g of~H-DPhe-Cys-Phe-DTrp-Lys(BOC)-Thr-Cys-Thr-ol, 0.5 mmole of Fmoc-E-aminocaproic acid and 115 mg of hydroxy-benzotriazole are dissolved in 10 ml of DMF and cooled to -30° C. To this solution is added a solution of 115 mg of dicyclohexylcarbodiimide in 5 ml of DMF (cooled to -10° C).
After a reaction time of'24 hours, during which the mixture warms to the room temperature, the resulting dicyclohexylurea is filtered off and the filtrate is diluted with water to ten times its volume. The precipitated reaction product is filte-red off, washed and dried over phosphorus pentoxide. The crude product is used without further purification for the next step.
b. Fmoc-cleavage 0.5 g of crude product from coupling reaction (a) are treated for 10 minutes at room temperature with 5 ml of DMF/piperi-dine 4/1 v/v (clear solution) and subsequently mixed with 100 ml of diisopropylether. The reaction product which is thus precipitated is filtered off, washed and dried. This crude product is used without further purification in the next step.
c. BOC cleavage 300 mg of crude product obtained in (l.b) are treated for 5 minutes at room temperature with 5 ml of 100 X TFA (complete-ly dissolved) and subsequently mixed with 50 ml of diisopro-pylether. After addition of 2 ml of HCI/diethylether, the resulting deposit is filtered off, washed and dried in a high vacuum.
The end product is purified by chromatography on silica gel (CHC13/MeOH/H20/AcOH 7/3/0.5/0.5), with subsequent de-salting over Duolite (gradient: Hz0/AcOH 95/5)---H20/dioxane/AcOH
45/50/5).
The title~compound is obtained as an acetate (white lyophili-sate).
[a]D° _ - 32 ° (c = 0.5 95 9: AcOH).
The resulting compound may be used for reaction with DTPA in accordance with the procedure of Examples 1 and 2.
RYAItDT.R 4 By following the procedure disclosed in Examples 1 and 3, the following LIGAND can be prepared:
DTPA-SAla-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol.
[a]DO _ - 14,8 ° (c = 0.5 95 X AcOH).
EgAHPLg 5: iiuIn labeled DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of 1 mg DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of is dissolved in ml O.O1M acetic acid. The resulting solution is passed through a 0.22u Millex-GV filter and dispensed in 0.1 ml portions and stored at -20°C. liilnCl3 (Amersham~, 1 mCi/100 ul) is prediluted in an equal volume of 0.5M sodium acetate and labeling is carried out by mixing the ligand with the InCl3 solution and gentle homo-genisation at room temperature.
HEPES buffer, pH 7.4, is then added to make a solution 10-6 M.
EgAIiPLB 6: 9°Y labeled DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of 9°Y is obtained from a 9°Sr-9oY radionuclide generator. The cons-truction of the generator, its elution and the conversion of the [9oY]EDTA to the acetate complex are performed in accordance with the method disclosed by M.Chinol and D.J. Hnatowich in J. Nucl.
Med. 28, 1465-1470 (1987). l~mg of DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of dissolved in 5m1 O.O1M acetic acid is allowed to warm to room temperature and 1.0 mCi of 9oY in 50 ul sterile 0.5M
acetate is added. The mixture is then left undisturbed for 30 min to 1 hr to maximize chelation.
One group of LIGANDS OF THE INVENTION are somatostatin peptides, e.g. somatostatin analogues, which contain at least on one of the amino acid units a chelating group which is attached to said amino group by an amide bond, in free form or in salt form.
One group of CHELATES OF THE INVENTION are the LIGANDS just men-tioned above complexed with a detectable element, e.g. a metal ion, in free form or in salt form.
b) the residue of a natural or synthetic a-amino acid other than defined under a) above or of a corresponding D-amino acid, or c) a dipeptide residue in which the indivi-dual amino acid residues are the same or different and are selected from those defined under a) and/or b) above, the a-amino group of amino acid residues a) and b) and the N-terminal amino group of dipeptide residues c) being optionally mono- or di-C1_l2alkylated or substituted by C1_ealkanoyl, A' is hydrogen, C1_izalkyl or C~_lophenylalkyl, Y1 and Yi represent together a direct bond or each of Y1 and YZ is independently hydrogen or a radical of formulae (1) to (5) 2C104.i32 I . ~ CHz -C0-C-(CHz)"-H -CO-CH -CO-NHR~
Rb \(CHz )n (1) (2) (3) R ' a Re -CO-NH-CH-COOR, -CO-(NH)p- C -(CHz)r Ra Rb~ q (4) (5) wherein R, is methyl or ethyl Rb is hydrogen, methyl or ethyl m is a whole number from 1 to 4 n is a whole number from 1 to 5 R~ is (C1_6)alkyl Ra represents the substituent attached to the a-carbon atom of a natural or synthetic a-amino acid (including hydrogen) R, is (C1_s)alkyl R,' and are independently hydrogen, methyl or Rb' ethyl, Rs and are independently hydrogen, halogen, R9 (C1_3)alkyl or (C1_3)alkoxy, p is 0 or 1, q is 0 or 1, and r is 0, 1 or 2, B is -Phe- optionally ring-substituted by halogen, NOz, NHz, OH, C1_3alkyl and /or Cl_3alkoxy (in-cluding pentafluoroalanine), or S--naphthyl-Ala C is (L)-Trp- or (D)-Trp- optionally a-N-methyl-ated and optionally benzene-ring-substituted by halogen, NOz, NHz, OH, C1_jalkyl and/or C1_3 alkoxy, D is Lys, Lys in which the side chain contains 0 or S in ~-position, YF-Lys or bF-Lys, optionally a-N-methylated, or a 4-amfnocyclohexylAla or 4-aminocyclohexylGly residue E is Thr, Ser, Val, Phe, Ile or an aminoisobutyric or aminobutyric acid residue G is a group of formula Ris /Ri i ~
-COORS, -CHiORlo, -CON or -CO-N--LXl ~ Ri z wherein R~ is hydrogen or C1_3alkyl,.
Rlo is hydrogen or the residue of a physiologically acceptable, physiologically hydrolysable ester, Ril is hydrogen, C1_3alkyl, phenyl or C~_lophenyl-alkyl, Riz is hydrogen, C1_3alkyl or a group of formula -CH(Ri3)-Xi, R13 is CHZOH, -(CHi)i-OH, -(CHZ)3-OH, or -CH(CH3)OH
or represents the substituent attached to the a-carbon atom of a natural or synthetic a-amino acid (including hydrogen) and 200~3~
Xi is a group of formula -COORS, -CHzORio or /Ri a -CO-N
~Ri s wherein R~ and Rlo have the meanings given above, Ria is hydrogen or C1_3alkyl and Ris is hydrogen, C1_3alkyl, phenyl or C~_iophenyl-alkyl, and Ris is hydrogen or hydroxy, with the proviso that when Rli is -CH(R13)-X1 then R11 is hydrogen or methyl, wherein the residues B, D and E have the L-configuration, and the residues in the 2-and 7-position and any residues Y1 4) and YZ 4) each independently have the (L)- or (D)- configu-ration.
The significances of A and A' in formula I are preferably selected so that the compound contains a terminal -NH- group capable of being linked to a chelating agent.
In the compounds of formula I, the following significances are preferred either individually or in any combination or sub-combination:
1. A is C~_lo phenylalkyl, especially phenethyl, or a group of formula RCO. Preferably A is a group of formula RCO.
1.1. Preferably R is C1_ii alkyl or C~_lo phenylalkyl, especially C~_lo phenylalkyl, more especially phenethyl, or RCO has the meanings a), b) or c).
200~~32 1.2. When RCO has the meanings a), b) or c), the «-amino group of amino acid residues a) and b) and the N-terminal amino group of dipeptide residues c) is preferably non-alkylated or mono-C1_li alkylated, especially -C1_e alkylated, more especially -methylated. Most preferably the N-terminal is non-alkylated.
1.3. When RCO has the meaning a) this is preferably a') an L-or D-phenylalanine or -tyrosine residue optionally mono-N-C1_lz alkylated. More preferably a') is an L- or D-phenylala-nine residue.
1.4. When RCO has the meaning b) or c) the defined residue is preferably lipophilic. Preferred residues b) are thus b') a-amino acid residues having a hydrocarbon side chain, e.g.
alkyl with 3, preferably 4, or more C atoms, e.g. up to 7 C-atoms, naphthyl-methyl or heteroaryl, e.g. 3-(2- or 1-naph-thyl)-alanine, pyridyl-methyl or tryptophane residue, said residues having the L- or D-configuration, and preferred residues c) are dipeptide residues in which the individual amino acid residues are the same or different and are selected from those defined under a') and b') above.
Example of a residue c) is e.g. 3-(2-naphthyl)-alanine resi-due.
1.5. Most preferably RCO has the meaning a) especially the meaning a').
2. B is B', where B' is Phe or Tyr.
3. C is C', where C' is (D)Trp.
.~ 2fl~4 i3~
4. D is D', where D' is Lys, MeLys or Lys(E-Me), especially Lys.
5. E is E', where E' is Val or Thr, especially Thr.
~Rl 1 6. G is G', where G' is a group of formula -CO-N.\ , especially a group of formula -CO-N/
\CH(R13)-X1 (in which case R11=H or CH3). In the latter case the moiety -CH(R13)-X1 preferably has the L-configuration.
6.1. R11 is preferably hydrogen.
6.2. As the substituent attached to the a-carbon atom of a natural amino acid (i.e. of formula H2N-CH(R13)-COOH), R13 is preferably -CHZOH, -CH(CH3)-OH, isobutyl or butyl, or R13 is -(CHz)2-OH or -(CH2)3-OH. It is especially -CH~OH or -CH(CH3)OH.
' ~1 4 6.3. X1 is preferably a group of formula -CO-N\
\Rl s or -CHZ-ORIO, especially of formula -CHI-ORIO and Rlo is pre-ferably hydrogen or has 'the meaning given under 7 below. Most preferably Rlo is hydrogen.
The following individual compounds are illustrative of com-pounds of formula I:
-lo- 2004532 H-(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-of also known as octreotide (D)Phe-Cys-Thr-(D)Trp-Lys-Val-Cys-ThrNHZ
(D)Phe-Cys-Tyr-(D)Trp-Lys-Val-Cys-TrpNHi (D)Trp-Cys-Phe-(D)Trp-Lys-Thr-Cys-ThrNHi (D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-ThrNHi 3-(2-Naphthyl)-(D)Ala-Cys-Tyr-(D)Trp-Lys-Val-Cys-ThrNHz 3-(2-Naphthyl)-(D)Ala-Cys-Tyr-(D)Trp-Lys-Val-Cys-S-Nal-NHz 3-(2-Naphthyl)-(D)Ala-Cys- ~-Nal-(D)Trp-Lys-Val-Cys-ThrNHi (D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-~Nal-NH=
B. Analogues of formula II
H-Cys-Phe-Phe-(D)-Trp-Lys-Thr-Phe-Cys-of II
[see Vale et al., Metabolism, 27, Supp. 1, 139, (1978)]
H-Cys-His-Hfs-Phe-Phe-(D)Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH III
(see EP-A-200,188) A
w- 2004532 Particular preferred LIGANDS are those derived from H-(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-ol.
Suitable chelating groups are physiologically acceptable chela-ting groups capable of complexing a detectable element.
Preferably the chelating group has substantially a hydrophilic character. Examples of chelating groups include e.g. iminodi-carboxylic groups, polyaminopolycarboxylic groups, e.g. those derived from non cyclic ligands e.g. ethylene diaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), ethylene glycol-0,0'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N'-bis(hydroxybenzyl)ethylenediamine-N, N'-diacetic acid (HBED) and triethylenetetramine hexaacetic acid (TTHA), those derived from substituted EDTA or -DTPA, e.g. p-isothiocyanato-benzyl-EDTA or -DTPA those derived from macrocyclic ligands, e.g.
1,4,7,10-tetra-azacyclododecane-N,N',N " ,N " '-tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane-N,N',N " ,N " '-tetra-acetic acid (TETA), those derived from N-substituted or C-substi-tuted macrocyclic amines including also cyclames, e.g. as dis-closed in EP 304,780 A1 and in WO 89/01476-A, groups of formula IV or V
R1-C-S-(CHZ)n~-C-(TT)i-C- IV
n n RZ-C-S-(CHZ)n.-C-NH-CHI 0 n CH-C- V
R3-C-S-(CHZ)".-C-NH
i n wherein each of R1, Rz and Rj independently is C1_salkyl, C5_earyl or C~_9arylalkyl, each optionally substituted by OH, C1_4alkoxy, COOH or S03H, n' is 1 or 2, i is an integer from 2 to 6, and TT are independently a or S amino acids linked to each other by amide bonds, groups derived from bis-aminothiol derivatives, e.g. compounds of formula VI
~CH2 HN NH
R ~~ X22 vI
21 C~ i~ ~R
R2a wherein each of Rzo, Rsi, Riz and Rz3 independently is hydrogen or C1_4alkyl, Xz is a group capable of reacting with the N-amino group of the peptide, and m' is 2 or 3, groups derived from dithiasemicarbazone derivatives, e.g.
compounds of formula VII
HN ~ N N ~ H VII
HN i ' SH HS NH
wherein XZ is as defined above, groups derived from propylene amine oxime derivatives, e.g.
compounds of formula VIII
~ ~cN2 HN NH
R26~ R27 VIII
R25~ _R28 R24 ~ ~ R29 OH OH
wherein each of RZ,, Rzs, Rzs, Rz~, Ria and Rz9 independently are hydrogen or C1-4alkyl, and Xz and m' are as defined above, groups derived from diamide dimercaptides, e.g. compounds of formula IX
20~4~3~:
Y S S IX
H5C6C0 COC64~
wherein X3 is a divalent radical optionally substituted and bearing a group capable of reacting with the N-amino group of the peptide, e.g. C1_4alkylene or phenylene bearing a group Xz, and YS is hydrogen or COZR3o, wherein R3o is C1_4alkyl, or groups derived from porphyries, e.g. N-benzyl-5,10,15,20-tetrakis-(4-carboxyphenyl)porphine or TPP bearing a group Xi as defined above.
Aryl is preferably phenyl. Arylalkyl is preferably benzyl.
Examples of X~ include radicals of formula -(X,)" " -Xs wherein X4 is C1_salkylene; or C1_6alkylene optionally attached to the carbon atom by an oxygen atom or -NH-, n " is 0 or 1 and X5 is -NCS, a carboxy group or a functional derivative thereof, e.g.
acid halide, anhydride or hydrazide. It is understood that XZ is attached to one of the carbon atom of -[CHij~.- or =CH-CH. in replacement of an hydrogen atom.
The chelating group may be attached either directly or indirectly to the N-amino group of the somatostatin peptide. When it is attached indirectly, it is preferably linked through a bridging or spacer group, for example a group of formula (al) ._ ~~~i Z-R35-CO- (ai) R3s is C1_l alkylene, Cz_llalkenylene or -CH(R')- wherein R' is the residue attached in a to a natural or synthetic a-amino acid, e.g. hydrogen, C1_llalkyl, benzyl, optionally substi-tuted benzyl, naphthyl-methyl, pyridyl-methyl, Z is a functional moiety capable of covalently reacting with the chelating agent.
Z may be for example a group which can form an ether, ester or amide bonding with the chelating group. Z is preferably amino.
The chelating groups, when comprising carboxy, -S03H and/or amino groups may exist in free form or in salt form.
Preferred chelating groups are those derived from polyamino-poly-carboxylic groups, e.g. those derived from EDTA, DTPA, DOTA, TETA
or substituted EDTA or DTPA. Chelating groups derived from DTPA
are most preferred.
In the LIGANDS OF THE INVENTION the chelating group, when poly-functional, may be linked either to a single somatostatin peptide molecule or to more than one somatostatin peptide molecules e.g.
to two somatostatin peptide molecule's.
The LIGANDS OF THE INVENTION may exist e.g. in free or salt form.
Salts include acid addition salts with e.g. organic acids, poly-meric acids or inorganic acids, for example hydrochlorides and acetates, and salt forms obtainable with the carboxylic or sul-phonic acid groups present in the chelating group, e.g. alkali metal salts such as sodium or potassium, or substituted or un-substituted ammonium salts.
The present invention also includes a process for the production of the LIGANDS OF THE INVENTION. They may be produced by analogy to known methods.
The LIGANDS OF THE INVENTION may be produced for example as follows:
a) removing at least one protecting group which is present in a somatostatin peptide bearing a chelating group, or b) linking together by an amide bond two peptide fragments each of them containing at least one amino acid or amino alcohol in protected or unprotected form and one of them containing the chelating group, wherein the amide bond is in such a way that the desired amino acid sequence is obtained, and stage a) of the process is then optionally effected, or c) linking together a chelating agent and the desired somato-statin peptide in protected or unprotected form in such a way that the chelating group is fixed on the desired N-amino group of the peptide, and stage a) is then optionally effected or, d) removing a functional group of an unprotected or a protected peptide bearing a chelating group or converting it into another functional group so that another unprotected or protected peptide bearing a chelating group is obtained and in the latter case stage a) of the process is effected, or e) oxidising a somatostatin peptide modified by a chelating group in which the mercapto groups of Cys radicals exist in free form so as to produce an analogue in which two Cys ra-dicals are joined by an S-S-bridge 2004 i3 c;
and recovering the LIGAND thus obtained in free form or in salt form.
The above reactions may be effected in analogy with known me-thods, e.g. as described in the following examples, in particular processes a) and c). When the chelating group is attached by an amide bond, this may be carried out analogously to the methods used for amide formation. Where desired, in these reactions, protecting groups which are suitable for use in peptides or for the desired chelating groups may be used for functional groups which do not participate in the reaction. The term protecting group may also include a polymer resin having functional groups.
When it is desired to attach the chelating group to the terminal N-amino group of a peptide or peptide fragment used as starting material, and which comprises one or more side chain amino groups, these side chain amino groups are conveniently protected with a protecting group , e.g. as used in peptide chemistry.
When it is desired to attach the chelating group to a side chain amino group of a peptide or peptide fragment used as starting ma-terial, and the peptide comprises a free terminal N-amino group, the latter is preferably protected with a protecting group.
The peptide fragment bearing the chelating group and used in sta-ge b) may be prepared by reacting the peptide fragment comprising at least one amino acid or amino alcohol in protected or unpro-tected form with the chelating agent. The reaction may be perfor-med in analogy with stage c).
The chelating groups of formula IV or V may be linked to a peptide by reacting a chelating agent of formula IV' or V' R1-C-S-(CH2)n.-C-(TT)i-C-X IV' n a RZ-C-S-(CHZ)n.-C-NH- HC'z 0 CH-C-X V' R3-C-S-(CHz)n.-C-NH
wherein X is an activating group capable of forming an amide bond with the N-amino group of the peptide. The reaction may be per-formed as disclosed in EP 247,866 A1.
The chelating agent used in process step c) may be known or pre-pared in analogy with known procedures. The compound used is such that it allows the introduction of the desired chelating group on the somatostatin peptide, e.g. a polyaminopolycarboxylic acid as disclosed, a salt or anhydride thereof.
In the above process, when in the amino-acids, peptide fragments or peptides used as starting materials, the chelating group is attached through a bridging or spacer group to the peptide, e.g.
a radical of formula (al) as defined above, such amino-acids, peptide fragments or peptides may be prepared by reacting in conventional manner the corresponding amino-acids or peptides free of bridging or spacer group with a corresponding bridging-or spacer-yielding compound, for example an acid or reactive acid derivative comprising the bridging or spacer group, e.g. an acid of formula Z-R35-COON or a reactive acid derivative thereof such as an active ester. Examples of active ester groups or carboxy activating groups are e.g. 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, succinimidyl or 1-hydroxy-benzotriazolyl.
Alternatively the chelating agent may first be reacted with a bridging or spacer group-yielding compound, in order to bear the bridging or spacer group and then be reacted in conventional manner with the peptide, peptide fragment or amino-acid.
The LIGANDS OF THE INVENTION may be purified in 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 in free form or in the form of phar-maceutically acceptable salts are valuable compounds.
According to a further embodiment, the LIGANDS OF THE INVENTION
can be complexed with a detectable element.
Accordingly, the present invention also provides the LIGANDS OF
THE INVENTION as defined above which are complexed with a detec-table element (hereinafter referred to as CHELATES OF THE INVEN-TION), in free form or in salt form, their preparation and their use for in vivo diagnostic and therapeutic treatment.
By detectable~element is meant any element, preferably a metal ion which exhibits a property detectable in therapeutic or in vivo diagnostic techniques, e.g. a metal ion which emits a de-tectable radiation or a metal ion which is capable of influencing NMR relaxation properties.
Suitable detectable metal iohs include for example heavy elements or rare earth ions, e.g. as used in CAT scanning (Computer axial tomography), paramagnetic ions, e.g. Gd3+, Fe3+, MnZ+ and Cr2+, fluorescent metal ions, e.g. Eu3+, and radionuclides, e.g.
Y-emitting radionuclides, S-emitting radionuclides, a-emitting radionuclides, positron-emitting radionuclides e.g. 68Ga.
Suitable Y-emitting radionuclides include those which are useful in diagnostic techniques. The r-emitting radionuclides advanta-geously have a half-life of from 1 hour to 40 days, preferably from 5 hours to 4 days, more preferably from 12 hours to 3 days.
Examples are radionuclides derived from Gallium, Indium, Tech-netium, Ytterbium, Rhenium and Thallium e.g. s~Ga, illln, 99'"Tc, issyb and lBSRe. Preferably the Y-radionuclide is selected depen-ding on the metabolism of the LIGAND OF THE INVENTION or somato-statin peptide used. More preferably the LIGAND OF THE INVENTION
is chelated with a Y-radionuclide having a longer half-life than the half-life of the somatostatin peptide on the tumor.
Further radionuclides suitable for use in imaging are positron-emitting radionuclides, e.g. as mentioned above.
Suitable ~-emitting radionuclides include those which are useful in therapeutic applications, for example Soy, s~_Cu, iasRe, iaeRe~ is9Er~ izign~ iz~Te~ 143pr~ 198Au~ io9pd~ issDY~ 3zP~
iazpr. The ~-radionuclide advantageously have a half-life of from 2.3 hrs to 14.3 d, preferably from 2.3 to 100 hrs. Preferably the remitting radionuclide is selected in order to have a longer half-life than the half-life of the somatostatin peptide on the tumor.
Suitable a-emitting radionuclides are those which are used in therapeutic treatments, e.g. zllAt, zizgi.
The CHELATES OF THE INVENTION may be prepared by reacting the LIGAND with a corresponding detectable element yielding compound, e.g. a metal salt, preferably a water-soluble salt. The reaction may be carried out by analogy with known methods, e.g. as disclo-sed in Perrin, Organic Ligand, Chemical Data Series 22. NY Perga-mon Press (1982); in Krejcarit and Tucker, Biophys. Biochem. Res.
Com. 77: 581 (1977) and in Wagner and Welch, J. Nucl. Med. _20:
428 (1979).
Preferably the complexing of the LIGAND is effected at a pH at which the LIGAND OF THE INVENTION is physiologically stable.
Alternatively the detectable element may also be provided to the solution as a complex with an intermediate chelating agent, e.g.
a chelating agent which forms a chelate complex that renders the element soluble at the physiological pH of the LIGAND but is less thermodynamically stable than the CHELATE. Example of such an intermediate chelating agent is 4,5-dihydroxy-1,3-benzene-di-sulfonic acid (Tiron). In such a process, the detectable element exchanges the ligand.
The CHELATES OF THE INVENTION may also be produced by linking together a chelating agent complexed with the detectable element, and a somatostatin peptide in protected or unprotected form and if desired removing at least one protecting group which is present. The same reaction may be performed with a chelating agent complexed with a non detectable metal ion and then in the resulting complexed peptide the metal ion may be replaced by the desired detectable element.
The CHELATES OF THE INVENTION may also be produced by linking together a chelating agent complexed with the detectable element, and a somatostatin peptide fragment comprising at least one amino acid in protected or unprotected form and then continuing the peptide synthesis step by step until the final peptide sequence is obtained and if desired removing at least one protecting group which is present. Instead of the detectable element the chelating agent may be complexed with a non detectable metal and this metal 2004,53:
may then be replaced by the detectable element in the resulting complexed somatostatin peptide.
Where the chelating group is attached through a bridging or spacer group to the somatostatin peptide, e.g. through a radical of formula (al) as defined above, either the somatostatin peptide or peptide fragment or the chelating agent may bear said bridging or spacer group.
The above mentioned reactions may be effected in analogy to known methods. Depending on the chelating group present, the labeling efficiency may approach 100X so that purification is not requi-red. Radionuclides such as for example Technetium-99m may be used in oxidized form, e.g. Tc-99m pertechnetate, which may be com-plexed under reducing conditions.
The above mentioned reactions are conveniently effected under conditions avoiding trace metal contamination. Preferably dis-tilled de-ionized water, ultrapure reagents, chelation-grade radioactivity etc..are used to reduce the effects of trace metal.
The CHELATES OF THE INVENTION may exist e.g. in free or salt form. Salts include acid addition salts with e.g. organic acids, polymeric acids or inorganic acids,~for example hydrochlorides and acetates, and salt forms obtainable with the carboxylic acid groups present in the molecule which do not participate to the chelate formation, e.g. alkali metal salts such as sodium or potassium, or substituted or unsubstituted ammonium salts.
The CHELATES OF THE INVENTION and their pharmaceutical acceptable salts exhibit pharmaceutical activity and are therefore useful either as an imaging agent, e.g. visualisation of somatostatin receptor positive tumors and metastases when complexed with a paramagnetic, a Y-emitting metal ion or a positron-emitting radionuclide, or as a radiopharmaceutical for the treatment in vivo of somatostatin receptor positive tumors and metastases when complexed with a a- or ~-radionuclide, as indicated by standard tests.
In particular, the CHELATES OF THE INVENTION possess affinity for somatostatin receptors expressed or overexpressed by tumors and metastases, as indicated in standard in vitro binding assays.
A somatostatin receptor positive tumor originating from the human gastro intestinal tract is removed from a patient and immediately put on ice and within a maximal delay of 30 min frozen at - 80 ° C. For further autoradiography this frozen material is cut on a cryostat (Leitz 1720) in 10 um sections, mounted on pre-cleaned microscope slides and stored at - 20 ° C for at least 3 days to improve adhesion of the tissue to the slide. The sections are preincubated in Tris-HC1 buffer (50 mM, pH 7.4), containing CaCl2 (2mM) and KC1 (5mM), for 10 min at ambient temperature and then washed twice for 2 min in the same buffer without additional salts added. The sections are then incubated with a CHELATE OF THE INVENTION for 2 hours at ambient tempe-rature in Tris-HC1 buffer (170 mM, pH 7.4), containing bovine serum albumin.(10 g/1), bacitracin (40 mg/1) and MgCl~ (5 mM~
to inhibit endogenous proteases. Non-specific binding is determi-ned by adding the corresponding non-labelled, hon-modified soma-tostatin peptide at a concentration of 1 uM. Incubated sections are washed twice for 5 min in cold incubation buffer containing 0.25 g/1 BSA. After a brief dip in distilled water to remove ex-cess salts, the sections are dried quickly and apposed to [3H]-LKB films. After a time exposure of about 1 week in X-ray casset-tes, it is observed that the CHELATES OF THE INVENTION, e.g. a radionuclide CHELATE, give very good results in labeling the tumoral tissue without labeling the surrounding healthy tissue when added at a concentration of about 10'1° to 10'3 M.
2Q04~32 The affinity of the CHELATES OF THE INVENTION for somatostatin receptors can also be shown by in vivo testing.
Rats bearing transplantable exocrine pancreatic somatostatin receptor positive tumors are treated with an intravenous injec-tion of a CHELATE OF THE INVENTION. Injection site is the penis vein. Immediately after administration, the animals are positio-ned on the collimator of a gamma-camera and the distribution of radioactivity is monitored at various time intervals.
Biodistribution of radioactivity may also be determined through serial sacrifice of a number of such treated rats and determi-nation of the organ radioactivity.
After administration of a CHELATE OF THE INVENTION, e.g. a radio-nuclide CHELATE, for example a Y-emitting CHELATE, at a dosage of from 1 to 5 ug/kg of LIGAND labeled with 0.1 to 2 mCi radionucli-de the tumor site becomes detectable together with the organs where excretion essentially takes place.
Accordingly, in a series of specific or alternative embodiments, the present invention also provides:
1. A method for in vivo detection of somatostatin receptor positive tumors or metastases in a subject which comprises a) administering a CHELATE OF THE INVENTION to said subject and b) recording the localisation of the receptors targeted by said CHELATE.
CHELATES OF THE INVENTION for use in the in vivo detection method of the invention are the CHELATES which are complexed with a Y-emitting radionuclide, a positron-emitting radio-nuclide or a paramagnetic metal ion, e.g. as indicated above.
The CHELATES OF THE INVENTION for use as an imaging agent in method (1) may be administered parenterally, preferably in-travenously, e.g. in the form of injectable solutions or sus-pensions, preferably in a single injection. The appropriate dosage will of course vary depending upon, for example, the LIGAND and the type of detectable element used, e.g. the radionuclide. A suitable dose to be injected is in the range to enable imaging by photoscanning procedures known in the art. When a radiolabeled CHELATE OF THE INVENTION is used, it may advantageously be administered in a dose having a radio-activity of from 0.1 to 50 mCi, preferably 0.1 to 30 mCi, mo-re preferably 0.1 to 20 mCi. An indicated dosage range may be of from 1 to 200 ug LIGAND labeled with 0.1 to 50 mCi, pre-ferably 0.1 to 30 mCi, e.g. 3 to 15 mCi, Y-emitting radionu-clide, depending on the Y-emitting radionuclide used. For example with In, it is preferred to use a radioactivity in the lower range, whereas with Tc, it is preferred to use a radioactivity in the upper range.
The enrichment in the tumorigenic sites with the CHELATES may be followed by the corresponding imaging techniques, e.g.
using nuclear medicine imaging instrumentation, for example a scanner, Y-camera , rotating Y-camera, each preferably com-puter assisted; PET-scanner (Positron emission tomography);
MRI equipment or CAT scanning equipment.
The CHELATES OF THE INVENTION, e.g. a major part of the Y-emitting CHELATES is substantially excreted through the kidneys and does not significantly accumulate in the liver.
2. A method for in vivo treatment of somatostatin receptor posi-tive tumors and metastases in a subject in need of such a treatment which comprises administering to said subject a therapeutically effective amount of a CHELATE OF THE INVEN-2U04S3~
TION.
CHELATES OF THE INVENTION for use in the in vivo treatment method of the invention are the CHELATES complexed with a a-or ~-radionuclide as defined above.
Dosages employed in practising the therapeutic method of the present invention will of course vary depending e.g. on the particular condition to be treated, for exemple the volume of the tumor, the particular CHELATE employed, for exemple the half-life of the CHELATE in the tumor, and the therapy desi-red. In general, the dose is calculated on the basis of ra-dioactivity distribution to each organ and on observed target uptake. For example the CHELATE may be administered at a dai-ly dosage range having a radioactivity of from 0.1 to 3mCi/kg body weight, e.g. 1 to 3 mCi, preferably 1 to 1.5 mCi/kg body weight. An indicated daily dosage range is of from 1 to 200 ug LIGAND labeled with 0.1 to 3 mCi/kg body weight, e.g.
0.1 to 1.5/kg body weight a- or S-emitting radionuclide, conveniently administered in divided doses up to 4 times a day.
The a- or ~--emitting CHELATES OF THE INVENTION may be admini-stered by any conventional route, in particular parenterally, e.g. in the form of injectable solutions or suspensions. They may also be administered advantageously by infusion, e.g. an infusion of 30 to 60 min. Depending on the site of the tumor, they may be administered as close as possible to the tumor site, e.g. by means of a'catheter. The mode of administration selected may depend on the dissociation rate of the CHELATE
used and the excretion rate.
The CHELATES OF THE INVENTION may be administered in free form or in pharmaceutically acceptable form. Such salts may be prepared ~00453~
in conventional manner and exhibit the same order of activity as the free compounds.
The CHELATES OF THE INVENTION for use in the method of the pre-sent invention may preferably be prepared shortly before the ad-ministration to a subject, i.e. the radiolabeling with the desi-red detectable metal ion, particularly the desired a-, S- or Y-radionuclide, may be performed shortly before the administration.
The CHELATES OF THE INVENTION may be suitable for imaging or treating tumors such as pituitary, gastroenteropancreatic, central nervous system, breast, prostatic, ovarian or colonic tumors, small cell lung cancer, paragangliomas, neuroblastomas, pheochromocytomas, medullary thyroid carcinomas, myelomas, etc.
and metastases thereof.
According to a further embodiment of the invention, the Y-emitting CHELATES OF THE INVENTION may also be used as imaging agent for the evaluation of the kidney function.
Groups of five mice are used. Each mouse is injected intra-venously through a tail vein with 0.1 ml containing 1 mCi of a CHELATE OF THE INVENTION. The mice are then placed in metabolic cages for the collection of excreted urine. At 10 or 120 min.
post-injection, the urethras are ligated and the mice anestheti-zed with chloroform. Imaging of the uropoietic system is carried out using the usual imaging technique. In this test, the Y-emit-ting CHELATES OF THE INVENTION improves imaging of renal excre-tion when administered at a dosage of from 0.1 to 30 mCi.
Accordingly, the present invention also provides a method for in vivo evaluation of the kidney function in a subject which comprises administering to said subject an effective amount of a Y-emitting CHELATE and recording the kidney function.
2004x32 According to a further aspect of the invention, there is pro-vided:
i. a pharmaceutical composition comprising a LIGAND OF THE
INVENTION in free or in pharmaceutically acceptable salt form, together with one or more pharmaceutically acceptable carriers or diluents therefor;
ii. a pharmaceutical composition comprising a CHELATE according to the invention in free or in pharmaceutically acceptable salt form, together with one or more pharmaceutically accep-table carriers or diluents therefor.
Such compositions may be manufactured in conventional manner.
A composition according to the invention may also be presented in separate package with instructions for mixing the LIGAND with the metal ion and for the administration of the resulting CHELATE. It may also be presented in twin-pack form, that is, as a single package containing separate unit dosages of the LIGAND and the detectable metal ion with instructions for mixing them and for administration of the CHELATE. A diluent or carrier may be pre-sent in the unit dosage forms. .
In the following examples, all temperatures are in ° C and [a]2~-values uncorrected. The following abbreviations are employed: D
Boc tert.-butoxycarbonyl TFA trifluoroacetic acid DTPA diethylenetriamine-pentaacetic acid BgAMPLB 1: DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of 1.1 g of DPhe-Cys-Phe-DTrp-Lys(E-Boc)-Thr-Cys-Thr-of in free base (1 mM), are dissolved in 5 1 of dioxan/H~0 1/1 (v/v) and reacted with 5 g NaHCb3. The 520 mg of DTPA dianhydride is slowly added with stirring. The reaction mixture is stirred for a further 30 min and dry-frozen. The residue is dissolved in 250 ml water and the pH is adjusted to pH 2.5 with concentrated HC1. The pre-cipitated product is filtered out, washed and dried over phosp-horus pentoxide. After chromatography on a silica-gel column, the following products are isolated: 230 mg of DTPA-DPhe-Cys-Phe-DTrp-Lys(E-Boc)-Thr-Cys-Thr-of and 500 mg of the corresponding dimer DTPA-(DPhe-Cys-Phe-DTrp-Lys(e-Boc)-Thr-Cys-Thr-ol)~.
3 ml of TFA are mixed with 200 mg of DTPA-DPhe-Cys-Phe-DTrp-Lys(E-Boc)-Thr-Cys-Thr-ol. After 5 min at room temperature, the mixture is precipitated with diisopropylether, filtered out and dried. The residue is desalted over Duolite and lyophilised to yield 150 mg of the title compound:
[a]D° _ - 26,6 ° (c = 1 95 % AcOH)..
The starting material may be produced as follows:
a) H-DPhe~Cys-Phe-DTrp-Lys(Boc) Thr-Cys=Thr=of 2.25 g of di-tert.butyl-pyrocarbonate, dissolved in 30 ml of DMF, are slowly added in drops at room temperature to a r solution of 10 g of H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol-acetate in 100 ml of DMF. After two hours at room tempe-A
rature, the solvent is drawn off under vacuum, and 200 ml of diisopropylether are added to the residue. The deposit which is being formed is filtered off, washed with diisopropylether and dried. The crude product is purified by chromatography over silica gel (eluant: CHZClz/MeOH 9/1) and is then iso-lated as a white amorphous powder.
[a]D° = 29.8 ° (c = 1.28 in DMF).
BgAMPLB 2: DTPA-(DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol)2 The fraction containing the intermediate product DTPA-DPhe-Cys-Phe-DTrp-Lys(s-Boc)-Thr-Cys-Thr-ol)2 as obtained in example 1 is treated as described above for the corresponding monomeric form, the Boc protecting groups being removed to yield the title compound:
[a]D° _ - 24,5 ° (c = 0,55 95 Y AcOH).
BgAMPLB 3: H2N- CHz $-CO-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of a. 0.56 g of~H-DPhe-Cys-Phe-DTrp-Lys(BOC)-Thr-Cys-Thr-ol, 0.5 mmole of Fmoc-E-aminocaproic acid and 115 mg of hydroxy-benzotriazole are dissolved in 10 ml of DMF and cooled to -30° C. To this solution is added a solution of 115 mg of dicyclohexylcarbodiimide in 5 ml of DMF (cooled to -10° C).
After a reaction time of'24 hours, during which the mixture warms to the room temperature, the resulting dicyclohexylurea is filtered off and the filtrate is diluted with water to ten times its volume. The precipitated reaction product is filte-red off, washed and dried over phosphorus pentoxide. The crude product is used without further purification for the next step.
b. Fmoc-cleavage 0.5 g of crude product from coupling reaction (a) are treated for 10 minutes at room temperature with 5 ml of DMF/piperi-dine 4/1 v/v (clear solution) and subsequently mixed with 100 ml of diisopropylether. The reaction product which is thus precipitated is filtered off, washed and dried. This crude product is used without further purification in the next step.
c. BOC cleavage 300 mg of crude product obtained in (l.b) are treated for 5 minutes at room temperature with 5 ml of 100 X TFA (complete-ly dissolved) and subsequently mixed with 50 ml of diisopro-pylether. After addition of 2 ml of HCI/diethylether, the resulting deposit is filtered off, washed and dried in a high vacuum.
The end product is purified by chromatography on silica gel (CHC13/MeOH/H20/AcOH 7/3/0.5/0.5), with subsequent de-salting over Duolite (gradient: Hz0/AcOH 95/5)---H20/dioxane/AcOH
45/50/5).
The title~compound is obtained as an acetate (white lyophili-sate).
[a]D° _ - 32 ° (c = 0.5 95 9: AcOH).
The resulting compound may be used for reaction with DTPA in accordance with the procedure of Examples 1 and 2.
RYAItDT.R 4 By following the procedure disclosed in Examples 1 and 3, the following LIGAND can be prepared:
DTPA-SAla-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol.
[a]DO _ - 14,8 ° (c = 0.5 95 X AcOH).
EgAHPLg 5: iiuIn labeled DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of 1 mg DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of is dissolved in ml O.O1M acetic acid. The resulting solution is passed through a 0.22u Millex-GV filter and dispensed in 0.1 ml portions and stored at -20°C. liilnCl3 (Amersham~, 1 mCi/100 ul) is prediluted in an equal volume of 0.5M sodium acetate and labeling is carried out by mixing the ligand with the InCl3 solution and gentle homo-genisation at room temperature.
HEPES buffer, pH 7.4, is then added to make a solution 10-6 M.
EgAIiPLB 6: 9°Y labeled DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of 9°Y is obtained from a 9°Sr-9oY radionuclide generator. The cons-truction of the generator, its elution and the conversion of the [9oY]EDTA to the acetate complex are performed in accordance with the method disclosed by M.Chinol and D.J. Hnatowich in J. Nucl.
Med. 28, 1465-1470 (1987). l~mg of DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-of dissolved in 5m1 O.O1M acetic acid is allowed to warm to room temperature and 1.0 mCi of 9oY in 50 ul sterile 0.5M
acetate is added. The mixture is then left undisturbed for 30 min to 1 hr to maximize chelation.
One group of LIGANDS OF THE INVENTION are somatostatin peptides, e.g. somatostatin analogues, which contain at least on one of the amino acid units a chelating group which is attached to said amino group by an amide bond, in free form or in salt form.
One group of CHELATES OF THE INVENTION are the LIGANDS just men-tioned above complexed with a detectable element, e.g. a metal ion, in free form or in salt form.
Claims (17)
1. A modified somatostatin peptide modified with a physiologically acceptable chelating group for a detectable element covalently linked directly or indirectly to the N-terminal amino group of a somatostatin peptide, in free form or in pharmaceutically acceptable salt form.
2. A modified somatostatin peptide according to claim 1, wherein the chelating group is covalently linked indirectly to the N-terminal amino group of the somatostatin peptide through a spacer or bridging group.
3. A modified somatostatin peptide according to claim 1, wherein the chelating group is covalently linked by an amide bond to the somatostatin peptide.
4. A modified somatostatin peptide according to claim 1, wherein the somatostatin peptide is a compound of formula I
wherein A is a group of formula RCO-, where RCO- is a) an L- or D-phenylalanine residue optionally ring-substituted by F, Cl, Br, NO2, NH2, OH, C1-3 alkyl and/or C1-3 alkoxy;
b) the residue of a natural or synthetic .alpha.-amino acid other than defined under a) above or of a corresponding D-amino acid, or c) a dipeptide residue in which the individual amino acid residues are the same or different and are selected from those deffined under a) and/or b) above;
A' is hydrogen;
Y1 and Y2 represent together a direct bond or each of Y1 and Y2 is independently hydrogen;
B is -Phe- optionally ring-substituted by halogen, NO2, NH2 OH, C1-3 alkyl and/or C1-3alkoxy, or .beta.-naphthyl-Ala;
C is (L)-Trp- or (D)-Trp- optionally .alpha.-N-methylated and optionally benzene-ring-substituted by halogen, NO2, NH2, OH, C1-3allkyl and/or C1-3 alkoxy;
D is Lys, or a 4-aminocyclohexylAla or 4-aminocyclohexylGly residue;
E is Thr, Ser, Val, Phe, Ile or an aminoisobutyric or aminobutyric acid residue;
G is a group of formula wherein R7 is hydrogen or C1-3alkyl;
R10 is hydrogen or the residue of a physiologically acceptable, physiologically hydrolysable ester;
R11 is hydrogen, C1-3alkyl, phenyl or C7-10phenyl-alkyl;
R12 is hydrogen, C1-3alkyl or a group of formula -CH(R13)-X1 R13 is CH~OH, - (CH2) 2-OH, -(CH2) 3-OH, or -CH(CH3)OH or represents the substituent attached to the .alpha.-carbon atom of a natural or synthetic .alpha.-amino acid (including hydrogen) and X1 is a group of formula -COOR7 -CH2OR10 or -CONR14R15 wherein R7 and R10 have the meanings given above;
R14 is hydrogen or C1-3alkyl;
R15 is hydrogen, C1-3alkyl, phenyl or C7-10phenylalkyl; and R16 is hydrogen or hydroxy;
with the proviso that when R12 is -CH(R13)-X1 then R11 is hydrogen or methyl, wherein the residues B, D and E have the L-configuration, and the residues in the 2- and 7-position independently have the (L)- or (D)- configuration.
wherein A is a group of formula RCO-, where RCO- is a) an L- or D-phenylalanine residue optionally ring-substituted by F, Cl, Br, NO2, NH2, OH, C1-3 alkyl and/or C1-3 alkoxy;
b) the residue of a natural or synthetic .alpha.-amino acid other than defined under a) above or of a corresponding D-amino acid, or c) a dipeptide residue in which the individual amino acid residues are the same or different and are selected from those deffined under a) and/or b) above;
A' is hydrogen;
Y1 and Y2 represent together a direct bond or each of Y1 and Y2 is independently hydrogen;
B is -Phe- optionally ring-substituted by halogen, NO2, NH2 OH, C1-3 alkyl and/or C1-3alkoxy, or .beta.-naphthyl-Ala;
C is (L)-Trp- or (D)-Trp- optionally .alpha.-N-methylated and optionally benzene-ring-substituted by halogen, NO2, NH2, OH, C1-3allkyl and/or C1-3 alkoxy;
D is Lys, or a 4-aminocyclohexylAla or 4-aminocyclohexylGly residue;
E is Thr, Ser, Val, Phe, Ile or an aminoisobutyric or aminobutyric acid residue;
G is a group of formula wherein R7 is hydrogen or C1-3alkyl;
R10 is hydrogen or the residue of a physiologically acceptable, physiologically hydrolysable ester;
R11 is hydrogen, C1-3alkyl, phenyl or C7-10phenyl-alkyl;
R12 is hydrogen, C1-3alkyl or a group of formula -CH(R13)-X1 R13 is CH~OH, - (CH2) 2-OH, -(CH2) 3-OH, or -CH(CH3)OH or represents the substituent attached to the .alpha.-carbon atom of a natural or synthetic .alpha.-amino acid (including hydrogen) and X1 is a group of formula -COOR7 -CH2OR10 or -CONR14R15 wherein R7 and R10 have the meanings given above;
R14 is hydrogen or C1-3alkyl;
R15 is hydrogen, C1-3alkyl, phenyl or C7-10phenylalkyl; and R16 is hydrogen or hydroxy;
with the proviso that when R12 is -CH(R13)-X1 then R11 is hydrogen or methyl, wherein the residues B, D and E have the L-configuration, and the residues in the 2- and 7-position independently have the (L)- or (D)- configuration.
5. A modified somatostatin peptide according to claim 4, wherein the somatostatin peptide is also known as octreotide.
6. A modified somatostatin peptide according to claim 4, wherein the somatostatin peptide is
7. A modified somatostatin peptide according to claim 4, wherein the chelating group is selected from the group consisting of iminodicarboxylic groups, polyaminopoly-carboxylic groups, groups derived from macrocyclic amines, groups of formula IV or V
wherein each of R1, R2 and R3 independently is C1-6alkyl, C6-8aryl or C7-9arylalkyl, each optionally substituted by OH, C1-4alkoxy, COOH or SO3H, n' is 1 or 2, i is an integer from 2 to 6, and TT are independently .alpha. or .beta. amino acids linked to each other by amide bonds, groups derived from bis-aminothiol derivatives, from dithiasemicarbazone derivatives, from propylene amine oxime derivatives, from diamide dimercaptides or from porphyrins, in free form or in pharmaceutically acceptable salt form.
wherein each of R1, R2 and R3 independently is C1-6alkyl, C6-8aryl or C7-9arylalkyl, each optionally substituted by OH, C1-4alkoxy, COOH or SO3H, n' is 1 or 2, i is an integer from 2 to 6, and TT are independently .alpha. or .beta. amino acids linked to each other by amide bonds, groups derived from bis-aminothiol derivatives, from dithiasemicarbazone derivatives, from propylene amine oxime derivatives, from diamide dimercaptides or from porphyrins, in free form or in pharmaceutically acceptable salt form.
8. A modified somatostatin peptide according to claim 4, wherein the chelating group is derived from ethylene diaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), ethylene glycol-0,0'-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-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), in free form or in pharmaceutically acceptable salt form.
9. A modified somatostatin peptide according to claim 4, which is in free form or in pharmaceutically acceptable salt form.
10. A pharmaceutical composition comprising a modified somatostatin peptide according to any one of claims 1 to 9, in free form or in pharmaceutically acceptable salt form in association with a pharmaceutically carrier or diluent.
11. A modified somatostatin peptide according to any one of claims 1 to 9, which is complexed with a detectable element, in free form or in pharmaceutically acceptable salt form.
12. A modified somatostatin peptide according to claim 1, which is complexed with a .alpha.-, .beta.-, .gamma.- or positron-emitting radionuclide.
13. A modified somatostatin peptide according to claim 12, wherein the radionuclide is selected from 68Ga, 67Ga, 111In, 99mTC, 169Yb, 186Re, 90Y, 67Cr, 186Re, 188Re, 169Er, 121Sn, 127Te 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, 211At and 212Bi.
14. A modified somatostatin peptide according to claim 4, which is 111In or 90Y labelled or in salt form.
15. A pharmaceutical composition comprising a modified somatostatin peptide as defined in claim 11, in free form or in pharmaceutically acceptable salt form in association with a pharmaceutically carrier or diluent.
16. A package comprising separate unit dosages of a composition according to claim 10, and of a metal ion emitting a radiation detectable by diagnostic techniques with instructions for mixing them.
17. A method for in vivo detection of somatostatin receptor positive tumors or metastases in a subject which comprises a) administering a modified somatostatin peptide as defined in claim 11, the detectable element being selected from the group consisting of .gamma.-emitting radionuclide, a positron-emitting radionuclide and a paramagnetic metal ion, to said subject and b) recording the localisation of the receptors targeted by the somatostatin peptide.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB888828364A GB8828364D0 (en) | 1988-12-05 | 1988-12-05 | Improvements in/relating to organic compounds |
| GB8828364 | 1988-12-05 | ||
| GB8916115 | 1989-07-13 | ||
| GB898916115A GB8916115D0 (en) | 1989-07-13 | 1989-07-13 | Improvements in or relating to organic compounds |
| GB8916761 | 1989-07-21 | ||
| GB898916761A GB8916761D0 (en) | 1989-07-21 | 1989-07-21 | Improvements in or relating to organic compounds |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2004532A1 CA2004532A1 (en) | 1990-06-05 |
| CA2004532C true CA2004532C (en) | 2000-02-22 |
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ID=27264222
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002004532A Expired - Lifetime CA2004532C (en) | 1988-12-05 | 1989-12-04 | Peptide derivatives |
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| KR (1) | KR0156541B1 (en) |
| AT (1) | AT403476B (en) |
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| BE (1) | BE1002296A5 (en) |
| CA (1) | CA2004532C (en) |
| CH (1) | CH678329A5 (en) |
| DE (1) | DE3991505B4 (en) |
| DK (1) | DK175338B1 (en) |
| ES (1) | ES2023533A6 (en) |
| FI (1) | FI102540B (en) |
| FR (1) | FR2639947B1 (en) |
| GB (1) | GB2225579B (en) |
| HK (1) | HK189995A (en) |
| HU (2) | HUT53375A (en) |
| IE (1) | IE62091B1 (en) |
| IL (1) | IL92534A (en) |
| LU (1) | LU87633A1 (en) |
| MY (1) | MY106120A (en) |
| NL (1) | NL194828C (en) |
| PT (1) | PT92487B (en) |
| SA (1) | SA96160495B1 (en) |
| SE (1) | SE508799C2 (en) |
| WO (1) | WO1990006949A2 (en) |
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| US5443816A (en) * | 1990-08-08 | 1995-08-22 | Rhomed Incorporated | Peptide-metal ion pharmaceutical preparation and method |
| GB9004017D0 (en) * | 1990-02-22 | 1990-04-18 | Krenning Eric P | Improvements in or relating to organic compounds |
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| DK0542934T3 (en) * | 1991-02-08 | 1999-11-22 | Biomeasure Inc | Use of somatostatin analogues for the treatment of melanomas |
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| US5225180A (en) * | 1991-09-10 | 1993-07-06 | Diatech, Inc. | Technetium-99m labeled somatostatin-derived peptides for imaging |
| US5783170A (en) * | 1991-11-27 | 1998-07-21 | Diatide, Inc. | Peptide-metal chelate conjugates |
| US5556609A (en) * | 1992-02-20 | 1996-09-17 | Rhomed Incorporated | YIGSR peptide radiopharmaceutical applications |
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| WO1993015770A1 (en) * | 1992-02-05 | 1993-08-19 | Mallinckrodt Medical, Inc. | Radiolabelled peptide compounds |
| US5643549A (en) * | 1992-02-20 | 1997-07-01 | Rhomed Incorporated | Leukostimulatory agent for in vivo leukocyte tagging |
| AU3967593A (en) * | 1992-03-25 | 1993-10-21 | Mallinckrodt Medical, Inc. | Method of intraoperatively detecting and locating tumoral tissues |
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| US6017512A (en) * | 1992-06-23 | 2000-01-25 | Diatide, Inc. | Radiolabeled peptides |
| NZ250653A (en) * | 1993-01-12 | 1995-12-21 | Sandoz Ltd | Somatostatin derivatives and pharmaceutical or diagnostic imaging compositions |
| US5650134A (en) * | 1993-01-12 | 1997-07-22 | Novartis Ag (Formerly Sandoz Ltd.) | Peptides |
| WO1994017829A1 (en) * | 1993-02-02 | 1994-08-18 | Neorx Corporation | Directed biodistribution of small molecules |
| US5879657A (en) * | 1993-03-30 | 1999-03-09 | The Dupont Merck Pharmaceutical Company | Radiolabeled platelet GPIIb/IIIa receptor antagonists as imaging agents for the diagnosis of thromboembolic disorders |
| US5932189A (en) * | 1994-07-29 | 1999-08-03 | Diatech, Inc. | Cyclic peptide somatostatin analogs |
| ATE337338T1 (en) * | 1993-06-23 | 2006-09-15 | Diatide Inc | RADIOACTIVE-LABELED PEPTIDE DERIVATIVES OF SOMATOSTATIN FOR IMAGE-FORMING AND THERAPEUTIC USE |
| CA2190727C (en) * | 1994-05-19 | 2006-07-18 | Sudhakar Kasina | Aromatic amine substituted bridged nitrogen and sulfur donor atom ligands for imaging |
| US6051206A (en) * | 1994-06-03 | 2000-04-18 | Diatide, Inc | Radiolabeled somatostatin-derived peptides for imaging and therapeutic uses |
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| US5556939A (en) * | 1994-10-13 | 1996-09-17 | Merck Frosst Canada, Inc. | TC or RE radionuclide labelled chelate, hexapeptide complexes useful for diagnostic or therapeutic applications |
| US5632969A (en) * | 1994-10-13 | 1997-05-27 | Merck & Co., Inc. | N3 S2 chelating ligands optionally radiolabelled with Tc or Re, useful for diagnostic or therapeutic applications |
| US5830431A (en) * | 1995-06-07 | 1998-11-03 | Mallinckrodt Medical, Inc. | Radiolabeled peptide compositions for site-specific targeting |
| GB9708265D0 (en) * | 1997-04-24 | 1997-06-18 | Nycomed Imaging As | Contrast agents |
| FI965181A7 (en) * | 1996-12-20 | 1998-06-21 | Map Medical Tech Oy | Polyalcohol-peptide derivatives |
| US7175953B2 (en) | 1999-04-09 | 2007-02-13 | Institute Fuer Diagnostik Forschung | Short-warp peptide-dye conjugate as contrast agent for optical diagnostic |
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| CA1222691A (en) * | 1981-12-29 | 1987-06-09 | Wilhelmus T. Goedemans | Method of preparing radionuclide-labelled proteins, in particular antibodies or antibody fragments |
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| US5073541A (en) * | 1987-11-18 | 1991-12-17 | Administrators Of The Tulane Educational Fund | Treatment of small cell lung cancer with somatostatin analogs |
| FR2638968B1 (en) * | 1988-11-11 | 1994-10-07 | Sandoz Sa | NEW THERAPEUTIC USE OF SOMATOSTATIN AND ITS ANALOGS AND DERIVATIVES |
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1989
- 1989-11-29 MY MYPI89001655A patent/MY106120A/en unknown
- 1989-11-30 WO PCT/EP1989/001448 patent/WO1990006949A2/en not_active Ceased
- 1989-11-30 AT AT0901789A patent/AT403476B/en active
- 1989-11-30 DE DE3991505A patent/DE3991505B4/en not_active Expired - Lifetime
- 1989-11-30 CH CH2578/90A patent/CH678329A5/de not_active IP Right Cessation
- 1989-12-01 GB GB8927255A patent/GB2225579B/en not_active Expired - Lifetime
- 1989-12-04 AU AU45871/89A patent/AU633859B2/en not_active Expired
- 1989-12-04 CA CA002004532A patent/CA2004532C/en not_active Expired - Lifetime
- 1989-12-04 IL IL9253489A patent/IL92534A/en not_active IP Right Cessation
- 1989-12-04 IE IE386689A patent/IE62091B1/en not_active IP Right Cessation
- 1989-12-04 SE SE8904087A patent/SE508799C2/en unknown
- 1989-12-04 FR FR8915993A patent/FR2639947B1/en not_active Expired - Lifetime
- 1989-12-04 KR KR1019890018033A patent/KR0156541B1/en not_active Expired - Lifetime
- 1989-12-04 JP JP1315124A patent/JP2726320B2/en not_active Expired - Lifetime
- 1989-12-04 PT PT92487A patent/PT92487B/en active IP Right Grant
- 1989-12-04 NL NL8902981A patent/NL194828C/en not_active IP Right Cessation
- 1989-12-04 HU HU896359A patent/HUT53375A/en unknown
- 1989-12-04 FI FI895809A patent/FI102540B/en active IP Right Grant
- 1989-12-05 DK DK198906126A patent/DK175338B1/en not_active IP Right Cessation
- 1989-12-05 ES ES8904151A patent/ES2023533A6/en not_active Expired - Lifetime
- 1989-12-05 BE BE8901294A patent/BE1002296A5/en not_active IP Right Cessation
- 1989-12-05 LU LU87633A patent/LU87633A1/en unknown
-
1995
- 1995-06-15 HU HU95P/P00214P patent/HU211468A9/en unknown
- 1995-12-21 HK HK189995A patent/HK189995A/en not_active IP Right Cessation
-
1996
- 1996-01-01 SA SA96160495A patent/SA96160495B1/en unknown
-
1997
- 1997-08-04 JP JP20891597A patent/JP3686503B2/en not_active Expired - Lifetime
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