CA2102633A1 - Radiolabelled somatostatin derivatives, their preparation and use - Google Patents
Radiolabelled somatostatin derivatives, their preparation and useInfo
- Publication number
- CA2102633A1 CA2102633A1 CA002102633A CA2102633A CA2102633A1 CA 2102633 A1 CA2102633 A1 CA 2102633A1 CA 002102633 A CA002102633 A CA 002102633A CA 2102633 A CA2102633 A CA 2102633A CA 2102633 A1 CA2102633 A1 CA 2102633A1
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- amino acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/655—Somatostatins
- C07K14/6555—Somatostatins at least 1 amino acid in D-form
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/083—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being octreotide or a somatostatin-receptor-binding peptide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Radioactive-technetium-labelled and radioactive-rhenium-labelled somatostatin derivatives are disclosed for the selective detection/localisation or for the selective therapeutic treatment of tumours with somatostatin receptors. The labelled polypeptides are characterised by the general formula:
R1-S1-A1-A2-(D)Trp-A3-A4-A5-Thr-S2-R2, wherein R1 is a hydrogen atom or a C1-C4 alkylcarbonyl group, R2 is an amino group, a hydroxy group or a C1-C4 alkoxy group, A1 and A5 each independently are Phe, MePhe, EtPhe, Tyr, Trp, Nal (naphthylalanyl), or Cys, A2 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, A3 is Lys, MeLys or (.epsilon.-Me)Lys, A4 is Thr or Val, S1 is an amino acid sequence of 1 to 6 amino acids, selected from the group consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys, (.epsilon.-Me)Lys, Thr, Val, Asn and Ser, and S2 is an amino acid sequence of 0 to 3 amino acids selected from the group mentioned sub S1, with the proviso that the polypeptide comprises two cysteine amino acid radicals, the metal-radionuclide being selected from a radioactive Tc or Re isotope which, as a cation, is bound covalently to the mercapto groups of the cysteine amino acid radicals.
R1-S1-A1-A2-(D)Trp-A3-A4-A5-Thr-S2-R2, wherein R1 is a hydrogen atom or a C1-C4 alkylcarbonyl group, R2 is an amino group, a hydroxy group or a C1-C4 alkoxy group, A1 and A5 each independently are Phe, MePhe, EtPhe, Tyr, Trp, Nal (naphthylalanyl), or Cys, A2 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, A3 is Lys, MeLys or (.epsilon.-Me)Lys, A4 is Thr or Val, S1 is an amino acid sequence of 1 to 6 amino acids, selected from the group consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys, (.epsilon.-Me)Lys, Thr, Val, Asn and Ser, and S2 is an amino acid sequence of 0 to 3 amino acids selected from the group mentioned sub S1, with the proviso that the polypeptide comprises two cysteine amino acid radicals, the metal-radionuclide being selected from a radioactive Tc or Re isotope which, as a cation, is bound covalently to the mercapto groups of the cysteine amino acid radicals.
Description
wo 9~/21383 PCr/US92~'04559 21 02~3 RADIOLABELLED SOMATOSTATIN DERIYATI\iES, THEIR PREPARATION AND USE
The invention relates to a metal radionuclide-labelled polypeptide intended for diagnostic or therapeu~ic applications.
Radionuclide-labelled compounds may be used for diagnostic ~xaminations, for example, into deviations in shape and ~5 functions of internal organs and into the presence and location of pathological processes in the body. For t~is p~rpose a composition in which the radioactive compound is present is administered to the patient, for example, in the form of an in~ection liquid. By means of a sui~able detection apparatus, for example, a ga~ma cameral images of, ~or example, the organ or the pathological process in which the acti~e compound is incorporated can be obtained by detecting the emitted radiation ("scannin~").
.. ~ . . . .
Radioactive-lab~lied biological ~acromolecules, in particular polypeptides,~present int~re ting perspec~ives ~or diagnostic applications. Certain polypeptides have a very large target organ specificity ~nd, after ha~ing been intxoduced into the body of the patient, can react very selectively with biological macromolecules present therein.
Binding studies have demonstrated that certain endocrine-related tumours comprise large numbers of binding sites with a high affinity to somatostatin and somatostatin-related polypeptides. Examples of such tumours having large numbers o~ somatostatin-receptors are pituitary tumours, tumours of .
21 02~33 2 the central nervous system, malignant breast tumours, gastro entero-pancreatic tumours, and metastases hereof.
Various metal radionuclides, provided they are bound to tumour-selective polypeptides, may successfully be used for controlling tumours and hence form a pOwërful tool in radiotherapy. The polypeptides used hence serve as vehicles to transport the desired radiation dose, viz. the metal radionuclide, to the tumour to be exposed to the radiation.
The direct labelling of a polypeptide with a metal radionuclide has two disadvantages. First, the biologically active site of the peptide necessary for a good target organ specificity or selectivity, is easily blocked by this reaction, so that the normal behaviour of the biological macromolecule is disturbed. In addition, the affinity between metal-radionuclide and macromolecule often is unsatisfactory, so that the formed bond i8 not sufficiently stable to remain intact under physiological conditions~ The administered material then is no longer useful - neither as a diagnostic -it cannot be detected any more how the polypeptide behaves in the body - nor as a therapeutic - the radiation dose is no longer transported to the desired target but cause~ an undesired radi~tion ~urden elsewhere!
` In or~er to m~tigate th~se disadvantages it is suggested in European Patent Specification 237150 to tre~t proteinaceous materials which comprise disulphide bonds first with a disulphide reducing agent, for example, dithiothreitol, and to react then the reduced proteinaceous substance which now comprises free mercapto groups, specifically with radionuclide species, for example, with Tc-99m-tartrate or -glucoheptonate. It has been found, however, that in this reductive treatment of the protein, in which the protein is "unfolded" by cleavage of the disulphide bonds to the desired mercapto groups, damage to WO92/21383 PC~/US92/04~59 3 210263~
- the protein molecules may easily occur, as a result of which the selertivity is disturbed.
S In the past few years a large number of publications have .
appeared in which biological macromolecules, usually proteins or proteinaceous substances, are described which comprise chelating groups for a bond with the desired metal-radionuclide. For example, International Patent Application WO 90/06949 describes somatostatin analogues which compri~e chelating groups, preferably derived from TDPA and the like, for a bond with a detectable element. As an example is describad DTPA-modified octreotide which may be labelled radioactive with In-lll or with Y-gO, for diagnostic or therapeutic purposes, respectively.
However, better suitable isotopes for these applications are radioact~ve technetium, ~n particular Tc-99m, and radioactive rhenium, in particular Re-186 and Re-188, becau~e these radioisotopes have better radiation characteristics and are more readily available. However, it has 80 far not succeeded to label the somatostatin analogues mentioned hereinbe~ore with these radioisotopes to compositions wh`ich are ~uitable for in vivo applications and which are~-sufficiently stable. Somatostatin it~elf is ~r~pi~ly biologically converted in the body and i8 therefore generally considered not su~table for xadioactiva labelling.
Therefore, one has so far resorted to stabilised somatostatin derivatives in which the two cysteine amino acid radicals, which apparently ar~ responsible for the instability, are oxidised together to a cystine group. ~oth the commercially available somatostatin, and the octreotide described in Patent Application W0 90/06949 mentioned hereinbefore, comprise cystine bridges, formed by oxidation from two cysteine amino acid radicals.
210263~ 4 It is the object of the present invention to provide an easily accessible radioactive-technetium-labelled or radio-active-rhenium-labelled polypeptide for the selective detection/localisation or for the selective therapeutic treatment of tumours with somatostatin r~ceptors, which is sufficiently stable for in ViVQ application. This object can be achieved with a labelled polypeptide which according to the present invention is characterised by the general formula Rl~Sl~Al~A2~(D)TrP-R3-A4-A5-Thr-s2-~2 (I~
wherein Rl is a hydrogen atom or a Cl-C4 alkylcarbonyl group, R2 is an amino group, a hydroxy group or a Cl-C4 alkoxy group, Al and A5 each independently are Phe, MePhe, EtPhe, Tyr, Trp, Nal or Cys, A2 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, A3 is Lys, MeLys or (-Me) Lys, is Thr or Val, Sl ~s an amino acid ~equence of 1 to 6 amino acids, selected from the group consisting of Ala, Cys, . Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, 25 - MeLys, (-Me)Lys, Thr, Val, Asn and Sar, and S2 i8 an a~no ~cid~sequence of 0 to 3 ~ino acids, selected from the group mentioned ~ub Sl, with the pro~iso that the polypeptide comprises two cysteine amino acid radicals, the metal-radionuclide being selected from a radioactive Tc-isotope or Re-isotope which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.
(Nal = naphthylalanyl).
It has been found surprisingly that the labelled polypeptide can simply be prepared and is sufficiently stable in ViYo .
5 21~ 33 for performing the desired examinations and the desired therapeutic treatment, respectively. It has been established that the labelled compound remained stable at least cne hour after injection. The selectivity is not adversely influenced by the labelling with radioactive technetium ~ rhenium. For example, Tc-99m-labelled polypeptide according to the invention is bound specifically to som~tostatin receptor-sites.
Suitable labelled polypeptides according to the invention are derived from the previously mentioned octreotide and analogues thereof, and may then be represented by the general formula Rl-A6-cys-A2-(D)Trp-A3-A4-cys-Thr-R4 (II) wherein Rl, R2, A2, A3 and A4 have the meanings given here~nbefore, and A6 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, the met~l radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 wh~ch as a cation is bound covalently to the mercapto groups of the cysteine amino acid rad~cals.
``l Other excellently suitable labelled polypeptides according to the invention are derived from so~atostatin and analogues thereof, and may be represented by the general formula Rl S 1 A l~A2~(D)TrP~A3~A4-A'5-Thr-s'2-R (III) wherein Rl, A2, A3, A4 and R2 have the meanings given hereinbefore, A'l and A'5 each independently represent Phe, MePhe, EtPhe, Tyr, Trp or Nal, S'1 is an amino acid sequence of 1 to 6 amino acids, preferably of 5 amino acids, selected from the group ~. . .
: :
.. ..
~2~33 6 consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, NeLys, (~-Me)Lys, ~hr, Val, Asn and Ser, with the proviso that S'l comprises a cysteine amino acid radical, and S'2 is an amino acid sequence of 1 to 3 am~n~ acids, preferably of 2 amino acids, selected from the group mentioned sub Sl', with the proviso that S2' comprises a cysteine amino acid radical, the metal radionuclide being selected from the group consisting of Tc-99m, Re-186 and R~-188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.
In connection with the excellent properties of the .labelled product and the ready availability of comatostatin as a starting peptide, a labelled polypeptide is preferred of the general formula Rl-Ala-Gly-Cy~-Lys-Asn-Phe-Phe-(D)Trp-Lys-Thr-Phe-Thr-Ser-Cys-R2 (IV) the metal radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound co~alentl~y to the mercapto groups of the cysteine amino ac~d radicals.
The invention also relates to a method of preparing a metal-radionuclide-labelled po~ypeptide according to the invention by startin~ from a cyclised polypeptide, in which the cysteine amino acid radicals together are oxidised to a cystine group. Examples of such cyclised polypeptides are the already mentioned somatostatin commercial product and analogues thereof. Analogues are to be understood to mean polypeptides having corresponding biological activity, i.e.
specific somatostatin-receptor binding afinity, but with modifications in the amino acid sequence. It has been found ." ., , - .
..
WO92/21383 PCT/US92/04~59 - 21021;~.~3 .
that the said cyclised polypeptides can be excellently reduced and may then be labelled under reducing conditions without the polypeptide molecule being attacked.
S AS a reducing agent are preferably chosen5zinc ions or metallic zinc, the latter, for example, in the form of zinc powder, because such reducing agents are suitable both for the preparation of the polypeptide from the cyclised material, and also for the reduction of pertechnetate or perrhenate. An excellent example of use of the metalllc zinc powder in the so-called SPED (Solid Phase Electron Donor) technique, in which the cyclised polypeptide is incubated by means of zinc powder, for example, on a 0.22 ~ filter, after which addition of a Tc-99m pertechnetate solution immediately provides a solution of the pure, Tc-99m-labelled polypeptide in the filtrate. Contaminations and non-reacted starting material remain on the filter, so that the filtrate is immediately ready for use. Metallic zinc may also be provided excellently as a zinc mirror on the inner wall of a tube or other reaction vessel and thus produce the desired conversions in the tube or reaction vessel.
., :
~. The. invention~- further .relates to a pharmaceutical composition::which comprises the metal-radionuclide-labelled polypeptide according to the invention, and to the use of the said c.omposition for diagnostic or therapeutic purposes. For diagnostic purposes, i.e. for detecting and localising certain tumour tissues, as defined hereinbefore, the active substance should be labelled with radioactive technetium, for therapeutic purposes, the active substance should be labelled with radioactive rhenium. All this is defined in more detail in Claims 8 and 9.
The invention finally relates to a kit for preparing a W 0 92/21383 P ~ /US9ZJ04~59 .
The invention relates to a metal radionuclide-labelled polypeptide intended for diagnostic or therapeu~ic applications.
Radionuclide-labelled compounds may be used for diagnostic ~xaminations, for example, into deviations in shape and ~5 functions of internal organs and into the presence and location of pathological processes in the body. For t~is p~rpose a composition in which the radioactive compound is present is administered to the patient, for example, in the form of an in~ection liquid. By means of a sui~able detection apparatus, for example, a ga~ma cameral images of, ~or example, the organ or the pathological process in which the acti~e compound is incorporated can be obtained by detecting the emitted radiation ("scannin~").
.. ~ . . . .
Radioactive-lab~lied biological ~acromolecules, in particular polypeptides,~present int~re ting perspec~ives ~or diagnostic applications. Certain polypeptides have a very large target organ specificity ~nd, after ha~ing been intxoduced into the body of the patient, can react very selectively with biological macromolecules present therein.
Binding studies have demonstrated that certain endocrine-related tumours comprise large numbers of binding sites with a high affinity to somatostatin and somatostatin-related polypeptides. Examples of such tumours having large numbers o~ somatostatin-receptors are pituitary tumours, tumours of .
21 02~33 2 the central nervous system, malignant breast tumours, gastro entero-pancreatic tumours, and metastases hereof.
Various metal radionuclides, provided they are bound to tumour-selective polypeptides, may successfully be used for controlling tumours and hence form a pOwërful tool in radiotherapy. The polypeptides used hence serve as vehicles to transport the desired radiation dose, viz. the metal radionuclide, to the tumour to be exposed to the radiation.
The direct labelling of a polypeptide with a metal radionuclide has two disadvantages. First, the biologically active site of the peptide necessary for a good target organ specificity or selectivity, is easily blocked by this reaction, so that the normal behaviour of the biological macromolecule is disturbed. In addition, the affinity between metal-radionuclide and macromolecule often is unsatisfactory, so that the formed bond i8 not sufficiently stable to remain intact under physiological conditions~ The administered material then is no longer useful - neither as a diagnostic -it cannot be detected any more how the polypeptide behaves in the body - nor as a therapeutic - the radiation dose is no longer transported to the desired target but cause~ an undesired radi~tion ~urden elsewhere!
` In or~er to m~tigate th~se disadvantages it is suggested in European Patent Specification 237150 to tre~t proteinaceous materials which comprise disulphide bonds first with a disulphide reducing agent, for example, dithiothreitol, and to react then the reduced proteinaceous substance which now comprises free mercapto groups, specifically with radionuclide species, for example, with Tc-99m-tartrate or -glucoheptonate. It has been found, however, that in this reductive treatment of the protein, in which the protein is "unfolded" by cleavage of the disulphide bonds to the desired mercapto groups, damage to WO92/21383 PC~/US92/04~59 3 210263~
- the protein molecules may easily occur, as a result of which the selertivity is disturbed.
S In the past few years a large number of publications have .
appeared in which biological macromolecules, usually proteins or proteinaceous substances, are described which comprise chelating groups for a bond with the desired metal-radionuclide. For example, International Patent Application WO 90/06949 describes somatostatin analogues which compri~e chelating groups, preferably derived from TDPA and the like, for a bond with a detectable element. As an example is describad DTPA-modified octreotide which may be labelled radioactive with In-lll or with Y-gO, for diagnostic or therapeutic purposes, respectively.
However, better suitable isotopes for these applications are radioact~ve technetium, ~n particular Tc-99m, and radioactive rhenium, in particular Re-186 and Re-188, becau~e these radioisotopes have better radiation characteristics and are more readily available. However, it has 80 far not succeeded to label the somatostatin analogues mentioned hereinbe~ore with these radioisotopes to compositions wh`ich are ~uitable for in vivo applications and which are~-sufficiently stable. Somatostatin it~elf is ~r~pi~ly biologically converted in the body and i8 therefore generally considered not su~table for xadioactiva labelling.
Therefore, one has so far resorted to stabilised somatostatin derivatives in which the two cysteine amino acid radicals, which apparently ar~ responsible for the instability, are oxidised together to a cystine group. ~oth the commercially available somatostatin, and the octreotide described in Patent Application W0 90/06949 mentioned hereinbefore, comprise cystine bridges, formed by oxidation from two cysteine amino acid radicals.
210263~ 4 It is the object of the present invention to provide an easily accessible radioactive-technetium-labelled or radio-active-rhenium-labelled polypeptide for the selective detection/localisation or for the selective therapeutic treatment of tumours with somatostatin r~ceptors, which is sufficiently stable for in ViVQ application. This object can be achieved with a labelled polypeptide which according to the present invention is characterised by the general formula Rl~Sl~Al~A2~(D)TrP-R3-A4-A5-Thr-s2-~2 (I~
wherein Rl is a hydrogen atom or a Cl-C4 alkylcarbonyl group, R2 is an amino group, a hydroxy group or a Cl-C4 alkoxy group, Al and A5 each independently are Phe, MePhe, EtPhe, Tyr, Trp, Nal or Cys, A2 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, A3 is Lys, MeLys or (-Me) Lys, is Thr or Val, Sl ~s an amino acid ~equence of 1 to 6 amino acids, selected from the group consisting of Ala, Cys, . Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, 25 - MeLys, (-Me)Lys, Thr, Val, Asn and Sar, and S2 i8 an a~no ~cid~sequence of 0 to 3 ~ino acids, selected from the group mentioned ~ub Sl, with the pro~iso that the polypeptide comprises two cysteine amino acid radicals, the metal-radionuclide being selected from a radioactive Tc-isotope or Re-isotope which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.
(Nal = naphthylalanyl).
It has been found surprisingly that the labelled polypeptide can simply be prepared and is sufficiently stable in ViYo .
5 21~ 33 for performing the desired examinations and the desired therapeutic treatment, respectively. It has been established that the labelled compound remained stable at least cne hour after injection. The selectivity is not adversely influenced by the labelling with radioactive technetium ~ rhenium. For example, Tc-99m-labelled polypeptide according to the invention is bound specifically to som~tostatin receptor-sites.
Suitable labelled polypeptides according to the invention are derived from the previously mentioned octreotide and analogues thereof, and may then be represented by the general formula Rl-A6-cys-A2-(D)Trp-A3-A4-cys-Thr-R4 (II) wherein Rl, R2, A2, A3 and A4 have the meanings given here~nbefore, and A6 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, the met~l radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 wh~ch as a cation is bound covalently to the mercapto groups of the cysteine amino acid rad~cals.
``l Other excellently suitable labelled polypeptides according to the invention are derived from so~atostatin and analogues thereof, and may be represented by the general formula Rl S 1 A l~A2~(D)TrP~A3~A4-A'5-Thr-s'2-R (III) wherein Rl, A2, A3, A4 and R2 have the meanings given hereinbefore, A'l and A'5 each independently represent Phe, MePhe, EtPhe, Tyr, Trp or Nal, S'1 is an amino acid sequence of 1 to 6 amino acids, preferably of 5 amino acids, selected from the group ~. . .
: :
.. ..
~2~33 6 consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, NeLys, (~-Me)Lys, ~hr, Val, Asn and Ser, with the proviso that S'l comprises a cysteine amino acid radical, and S'2 is an amino acid sequence of 1 to 3 am~n~ acids, preferably of 2 amino acids, selected from the group mentioned sub Sl', with the proviso that S2' comprises a cysteine amino acid radical, the metal radionuclide being selected from the group consisting of Tc-99m, Re-186 and R~-188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.
In connection with the excellent properties of the .labelled product and the ready availability of comatostatin as a starting peptide, a labelled polypeptide is preferred of the general formula Rl-Ala-Gly-Cy~-Lys-Asn-Phe-Phe-(D)Trp-Lys-Thr-Phe-Thr-Ser-Cys-R2 (IV) the metal radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound co~alentl~y to the mercapto groups of the cysteine amino ac~d radicals.
The invention also relates to a method of preparing a metal-radionuclide-labelled po~ypeptide according to the invention by startin~ from a cyclised polypeptide, in which the cysteine amino acid radicals together are oxidised to a cystine group. Examples of such cyclised polypeptides are the already mentioned somatostatin commercial product and analogues thereof. Analogues are to be understood to mean polypeptides having corresponding biological activity, i.e.
specific somatostatin-receptor binding afinity, but with modifications in the amino acid sequence. It has been found ." ., , - .
..
WO92/21383 PCT/US92/04~59 - 21021;~.~3 .
that the said cyclised polypeptides can be excellently reduced and may then be labelled under reducing conditions without the polypeptide molecule being attacked.
S AS a reducing agent are preferably chosen5zinc ions or metallic zinc, the latter, for example, in the form of zinc powder, because such reducing agents are suitable both for the preparation of the polypeptide from the cyclised material, and also for the reduction of pertechnetate or perrhenate. An excellent example of use of the metalllc zinc powder in the so-called SPED (Solid Phase Electron Donor) technique, in which the cyclised polypeptide is incubated by means of zinc powder, for example, on a 0.22 ~ filter, after which addition of a Tc-99m pertechnetate solution immediately provides a solution of the pure, Tc-99m-labelled polypeptide in the filtrate. Contaminations and non-reacted starting material remain on the filter, so that the filtrate is immediately ready for use. Metallic zinc may also be provided excellently as a zinc mirror on the inner wall of a tube or other reaction vessel and thus produce the desired conversions in the tube or reaction vessel.
., :
~. The. invention~- further .relates to a pharmaceutical composition::which comprises the metal-radionuclide-labelled polypeptide according to the invention, and to the use of the said c.omposition for diagnostic or therapeutic purposes. For diagnostic purposes, i.e. for detecting and localising certain tumour tissues, as defined hereinbefore, the active substance should be labelled with radioactive technetium, for therapeutic purposes, the active substance should be labelled with radioactive rhenium. All this is defined in more detail in Claims 8 and 9.
The invention finally relates to a kit for preparing a W 0 92/21383 P ~ /US9ZJ04~59 .
2 i ~6~3 radiopharmaceutical composition, comprising, in an optionally dry condition, a cyclised polypeptide, as defined hereinbefore, a reducing agent, preferably metallic zinc or zinc ions, and directives for reacting the ingredients of the kit and of the resulti~g~-product with Tc-99m pertechnetate or with Re-186 or Re-188 perrhenate.
In this manner the user of the kit himself can prepare in a clinical laboratory the labelled polypeptide according to the in~ention in the form of a composition to be administered: reduction of the cyclised polypeptide to the polypeptide to be labelled, as well as the required labelling. The use of one reaction agent for the two reactions simplifies the method of preparation.
The invention will now be described in greater detail with reference to the following examples.
Exam~le 1 Commercially available somatostatin is treated for 30 minutes at a pH~of 8 on a 0.22~ filter by means of the so-called SPED technique as descrîbed hereinbefore. A freshly 2S ;eluted sodiwm~pertèchnetate solution is then added and the mixture is incubated at room temperature for 15 minutes.
The labelled polypeptide may be obtained as a filtrate.
Precipitate and liquid can be separated without a filter by decanting and extracting the liquid by means of a syringe.
A labelling efficiency of 90% is obtained. Free technetium is bound to the SPED and cannot contaminate the product.
- Labelling is confirmed by means of thin-layer chromatography and ion exchange column chromatography. The labelled compound is stable in vitro up to 4 hours.
A quantity of 22 MBq of the labelled compound is W092/21383 PCT/US92/~4559 . ~
- 21~2~33 `
9, administered t~ rats suffering from colorectal carcinoma and the biodistribution is determined by dynamic gamma camera scintigraphy up to one hour after injection.
The tumour take-up reaches a maximum at a,pproximately 4 minutes after injection. During the determination period no sig~ificant acti~ity reduction from the tumour can be observed. The labelled compound is stable in vivo during the whole of the determination period, as appears from the absence of thyroid gland and stomach activities.C
Scintigrams of the tumours are made four minutes after injection. The uptake ratios tumour:muscle t~ssue are favourable, namely 5:1.
That the tumour accummulation of technetium is related to the somatostatin binding to receptors in the tumour has been checked by treating one group of experîmental animals having tumours prior to administration of the labelled compound with Sursamine~ to block the receptor sites. No tumour uptake is observed in these animals, so that it is confirmed that the binding takes place at somatostatin receptors in the tumour.
; , .
25 : Examole 2;
.
A group of adult female rats were implanted with CC531 Colon Carcinoma which is known to have somatostatin receptors. When the subcutaneous tumour implants had reached a size of ~1.5cm diameter, one group was injected with Technetium Somatostatin complex by intravenous injection and a second group was injected in a similar manner with Indium-111 Octreotide complex for comparison.
The animals were anaesthetised by means of nembutal and serial scintigrams were made. The Technetium Somatostatin - ~; . .
In this manner the user of the kit himself can prepare in a clinical laboratory the labelled polypeptide according to the in~ention in the form of a composition to be administered: reduction of the cyclised polypeptide to the polypeptide to be labelled, as well as the required labelling. The use of one reaction agent for the two reactions simplifies the method of preparation.
The invention will now be described in greater detail with reference to the following examples.
Exam~le 1 Commercially available somatostatin is treated for 30 minutes at a pH~of 8 on a 0.22~ filter by means of the so-called SPED technique as descrîbed hereinbefore. A freshly 2S ;eluted sodiwm~pertèchnetate solution is then added and the mixture is incubated at room temperature for 15 minutes.
The labelled polypeptide may be obtained as a filtrate.
Precipitate and liquid can be separated without a filter by decanting and extracting the liquid by means of a syringe.
A labelling efficiency of 90% is obtained. Free technetium is bound to the SPED and cannot contaminate the product.
- Labelling is confirmed by means of thin-layer chromatography and ion exchange column chromatography. The labelled compound is stable in vitro up to 4 hours.
A quantity of 22 MBq of the labelled compound is W092/21383 PCT/US92/~4559 . ~
- 21~2~33 `
9, administered t~ rats suffering from colorectal carcinoma and the biodistribution is determined by dynamic gamma camera scintigraphy up to one hour after injection.
The tumour take-up reaches a maximum at a,pproximately 4 minutes after injection. During the determination period no sig~ificant acti~ity reduction from the tumour can be observed. The labelled compound is stable in vivo during the whole of the determination period, as appears from the absence of thyroid gland and stomach activities.C
Scintigrams of the tumours are made four minutes after injection. The uptake ratios tumour:muscle t~ssue are favourable, namely 5:1.
That the tumour accummulation of technetium is related to the somatostatin binding to receptors in the tumour has been checked by treating one group of experîmental animals having tumours prior to administration of the labelled compound with Sursamine~ to block the receptor sites. No tumour uptake is observed in these animals, so that it is confirmed that the binding takes place at somatostatin receptors in the tumour.
; , .
25 : Examole 2;
.
A group of adult female rats were implanted with CC531 Colon Carcinoma which is known to have somatostatin receptors. When the subcutaneous tumour implants had reached a size of ~1.5cm diameter, one group was injected with Technetium Somatostatin complex by intravenous injection and a second group was injected in a similar manner with Indium-111 Octreotide complex for comparison.
The animals were anaesthetised by means of nembutal and serial scintigrams were made. The Technetium Somatostatin - ~; . .
3 PCTtUS92/04~59 21 ~js33 ` `~
was clearly visible in the tumour within ~-5 minutes post injection. At six minutes post injection the animals were sacrificed by cervical dislocation and tissue samples were taken and counted. Approximately ll~ of the injected dose was found in the total tumour tissue with ~ tumour to soft tissue uptake ratio of 4.2/l. The blood concentration was relatively high tumour to blood ratios being 0.23. The bulk of the activity was recovered in the stomach, liver, spleen and kidneys.
In the case of animals injected with indium Octreotide complex the visualisation of the tumour was similar in pattern to that of the technetium complex. At 24 hours post injection, the optimal scanning time in humans, the animals were sacrificed and tissue samples were counted.
An average concentration of 4.9% of the injected dose was found in the total tumour with a tumour to soft tissue ratio of 9.8/l this higher value in relation to the technetium somatostatin was prim~rily due to the low blood concentration. As with the technetium complex the bulk of the activity was recovered from the liver, spleen, GI tract and kidneys.
The results show a slight difference in the biodistribution characteristics of the two complexes but the kinetics during the first lS minutes post injection were similar, and during ~his period good scintigraphic images of the tumour could be obtained with the technetium complex using a gamma camera.
was clearly visible in the tumour within ~-5 minutes post injection. At six minutes post injection the animals were sacrificed by cervical dislocation and tissue samples were taken and counted. Approximately ll~ of the injected dose was found in the total tumour tissue with ~ tumour to soft tissue uptake ratio of 4.2/l. The blood concentration was relatively high tumour to blood ratios being 0.23. The bulk of the activity was recovered in the stomach, liver, spleen and kidneys.
In the case of animals injected with indium Octreotide complex the visualisation of the tumour was similar in pattern to that of the technetium complex. At 24 hours post injection, the optimal scanning time in humans, the animals were sacrificed and tissue samples were counted.
An average concentration of 4.9% of the injected dose was found in the total tumour with a tumour to soft tissue ratio of 9.8/l this higher value in relation to the technetium somatostatin was prim~rily due to the low blood concentration. As with the technetium complex the bulk of the activity was recovered from the liver, spleen, GI tract and kidneys.
The results show a slight difference in the biodistribution characteristics of the two complexes but the kinetics during the first lS minutes post injection were similar, and during ~his period good scintigraphic images of the tumour could be obtained with the technetium complex using a gamma camera.
Claims (10)
1. A metal-radionuclide-labelled polypeptide of the general formula R1-S1-A1-A2-(D)Trp-A3-A4-A5-Thr-S2-R2 (I) wherein R1 is a hydrogen atom or a C1-C4 alkylcarbonyl group, R2 is an amino group, a hydroxy group or a C1-C4 alkoxy group, A1 and A5 each independently are Phe, MePhe, EtPhe, Tyr, Trp, Nal or Cys, A2 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, A3 is Lys, MeLys or (.epsilon.-Me)Lys, A4 is Thr or Val, S1 is an amino acid sequence of 1 to 6 amino acids, selected from the group consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys, (.epsilon.-Me)Lys, Thr, Val, Asn and Ser, and S2 is an amino acid sequence of 0 to 3 amino acids, selected from the group mentioned sub S1, with the proviso that the polypeptide comprises two cysteine amino acid radicals, the metal-radionuclide being selected from a radioactive Tc-isotope or Re-isotope which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.
2. A labelled polypeptide as claimed in Claim 1, having general formula R1-A6-Cys-A2-(D)Trp-A3-A4-Cys-Thr-R4 (II) wherein R1, R2, A2, A3 and A4 have the meanings given in Claim 1, and A6 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, the metal radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.
3. A labelled polypeptide as claimed in Claim 1, having the general formula R1-S1'-A1'-A2-(D)Trp-A3-A4-A5'-Thr-S2'-R2 (III), wherein R1, A2, A3, A4 and R2 have the meanings given in Claim 1, A1' and A4' each independently are Phe, MePhe, EtPhe, Tyr, Trp or Nal, S1' is an amino acid sequence of 1 to 6 amino acids, preferably of 5 amino acids, selected from the group consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys, (.epsilon.-Me)Lys, Thr, Val, Asn and Ser, with the proviso that S1' comprises a cys-teine amino acid radical, and S2' is an amino acid sequence of 1 to 3 amino acids, preferably of 2 amino acids, selected from the group mentioned sub S1', with the proviso that S2' comprises a cysteine amino acid radical, the metal-radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.
4. A labelled polypeptide as claimed in Claim 3, having the general formula R1-Ala-Gly-Cys-Lys-Asn-Phe-Phe-(D)Trp-Lys-Thr-Phe-Thr--Ser-Cys-R2 (IV) the metal-radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound to the mercapto groups of the cysteine amino acid radicals.
5. A method of preparing a metal-radionuclide-labelled polypeptide as claimed in any of the preceding Claims, characterised in that a polypeptide of the general formula I, wherein the symbols have the meanings given in Claim 1, is prepared by reducing the corresponding cyclised polypeptide, in which the cysteine amino acid radicals have been oxidized together to a cystine group, with a suitable reducing agent, and the resulting polypeptide is reacted under reducing conditions with Tc-99m in the form of a pertechnetate solution or with Re-186 or Re-188 in the form of a perrhenate solution.
6. A method as claimed in Claim 5, characterised in that metallic zinc or zinc ions is/are used as a reducing agent for the cyclised polypeptide.
7. A pharmaceutical composition which, in addition to a pharmaceutically acceptable liquid carrier material and optionally one or more equally pharmaceutically acceptable adjuvants, comprises a metal-radionuclide-labelled polypep-tide as claimed in any of the Claims 1 to 4 as the active substance.
8. A method of detecting and localising tumour tissues in the body of a warmblooded living being, charac-terised in that a composition as claimed in Claim 7 optionally after dilution with a pharmaceutically acceptable liquid, is administered to the being in a quantity which is sufficient for external imaging and that the being is then subjected to a technique of external imaging to determine the radioactive-labelled sites in the body in relation to the background-activity.
9. A method of treating tumours in the body of a warmblooded living being, characterised in that a composi-tion as claimed in Claim 7, optionally after dilution with a pharmaceutically acceptable liquid, is administered to the being in a quantity effective for controlling tumours.
10. A kit for the preparation of a radiopharmaceutical composition comprising (1) in an optionally the dry condition a cyclised polypeptide of the general formula I, wherein the symbols have the meanings given in Claim 1 but the cysteine-amino acid radicals together are oxidised to a cystine group, (2) a reducing agent and (3) directives for reacting the ingredients mentioned sub (1) and (2) and the resulting reaction product with Tc-99m in the form of a pertechnetate solution or with Re-186 or Re-188 in the form of a perrhenate solution.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL9100954 | 1991-06-03 | ||
| NL9100954 | 1991-06-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2102633A1 true CA2102633A1 (en) | 1992-12-04 |
Family
ID=19859317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002102633A Abandoned CA2102633A1 (en) | 1991-06-03 | 1992-06-02 | Radiolabelled somatostatin derivatives, their preparation and use |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0588905A1 (en) |
| JP (1) | JPH06508357A (en) |
| AU (1) | AU657770B2 (en) |
| CA (1) | CA2102633A1 (en) |
| WO (1) | WO1992021383A1 (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5382654A (en) * | 1992-02-05 | 1995-01-17 | Mallinckrodt Medical, Inc. | Radiolabelled peptide compounds |
| US5849261A (en) * | 1991-02-08 | 1998-12-15 | Diatide, Inc. | Radiolabeled vasoactive intestinal peptides for diagnosis and therapy |
| US5443815A (en) * | 1991-11-27 | 1995-08-22 | Diatech, Inc. | Technetium-99m labeled peptides for imaging |
| 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 |
| JPH08504166A (en) * | 1992-02-05 | 1996-05-07 | マリンクロッド・メディカル・インコーポレイテッド | Radiolabeled peptide compound |
| US5716596A (en) * | 1992-06-23 | 1998-02-10 | Diatide, Inc. | Radioactively labeled somatostatin-derived peptides for imaging and therapeutic uses |
| US6017512A (en) * | 1992-06-23 | 2000-01-25 | Diatide, Inc. | Radiolabeled peptides |
| US5871711A (en) * | 1992-06-23 | 1999-02-16 | Diatide, Inc. | Radioactively-labeled somatostatin-derived peptides for imaging and therapeutic uses |
| ES2114209T3 (en) * | 1993-07-19 | 1998-05-16 | Resolution Pharm Inc | HYDRAZINO TYPE RADIONUCLID CHELATORS THAT HAVE A N3S CONFIGURATION. |
| US5574140A (en) * | 1993-09-03 | 1996-11-12 | Resolution Pharmaceutical Inc. | Hydrazino-type N2 S2 chelators |
| US5858327A (en) * | 1993-09-03 | 1999-01-12 | Resolutions Pharmaceuticals, Inc. | Hydrazino-type N2 S2 radionuclide chelating compounds |
| US6051206A (en) * | 1994-06-03 | 2000-04-18 | Diatide, Inc | Radiolabeled somatostatin-derived peptides for imaging and therapeutic uses |
| JPH11507623A (en) * | 1995-06-07 | 1999-07-06 | イムノメディクス，インコーポレイテッド | Thiolation of peptides for radionuclide-based radiation detection and radiotherapy |
| IT1277391B1 (en) * | 1995-07-28 | 1997-11-10 | Romano Deghenghi | CYCLIC PEPTIDES ANALOGUE OF SOMATOSTATIN TO ACTIVITY INHIBITING THE GROWTH HORMONE |
| US6355613B1 (en) | 1996-07-31 | 2002-03-12 | Peptor Limited | Conformationally constrained backbone cyclized somatostatin analogs |
| FI965181A (en) | 1996-12-20 | 1998-06-21 | Map Medical Technologies Oy | Polyalcohol peptide derivatives |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1107273A (en) * | 1978-05-19 | 1981-08-18 | Chester A. Meyers | Somatostatin analogs having a substituted tryptophyl residue in position eight |
| JPH0725691B2 (en) * | 1988-06-03 | 1995-03-22 | ソシエテ ドゥ コンセイユ ドゥ ルシエルシエ エ ダプリカシオン サイエンティフィック | Vascular blockage inhibitor |
| MY106120A (en) * | 1988-12-05 | 1995-03-31 | Novartis Ag | Peptide derivatives. |
| DK0486622T3 (en) * | 1989-08-09 | 1999-07-19 | Rhomed Inc | Direct radiolabelling of antibodies and other proteins with technetium or rhenium |
-
1992
- 1992-06-02 AU AU20174/92A patent/AU657770B2/en not_active Expired - Fee Related
- 1992-06-02 WO PCT/US1992/004559 patent/WO1992021383A1/en not_active Application Discontinuation
- 1992-06-02 EP EP92912727A patent/EP0588905A1/en not_active Withdrawn
- 1992-06-02 CA CA002102633A patent/CA2102633A1/en not_active Abandoned
- 1992-06-02 JP JP5500596A patent/JPH06508357A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| AU657770B2 (en) | 1995-03-23 |
| WO1992021383A1 (en) | 1992-12-10 |
| AU2017492A (en) | 1993-01-08 |
| EP0588905A1 (en) | 1994-03-30 |
| JPH06508357A (en) | 1994-09-22 |
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Legal Events
| Date | Code | Title | Description |
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| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 19951203 |