+

WO1996023527A1 - Method for the detection and localization of malignant human tumours - Google Patents

Method for the detection and localization of malignant human tumours Download PDF

Info

Publication number
WO1996023527A1
WO1996023527A1 PCT/US1996/001291 US9601291W WO9623527A1 WO 1996023527 A1 WO1996023527 A1 WO 1996023527A1 US 9601291 W US9601291 W US 9601291W WO 9623527 A1 WO9623527 A1 WO 9623527A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
group
pacap
labelled
lys
Prior art date
Application number
PCT/US1996/001291
Other languages
French (fr)
Other versions
WO1996023527A9 (en
Inventor
Jean-Claude Reubi
Original Assignee
Mallinckrodt Medical, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mallinckrodt Medical, Inc. filed Critical Mallinckrodt Medical, Inc.
Publication of WO1996023527A1 publication Critical patent/WO1996023527A1/en
Publication of WO1996023527A9 publication Critical patent/WO1996023527A9/en

Links

Classifications

    • 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
    • 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

Definitions

  • the invention relates to a method of detecting and localizing malignant tumours in the body of a human being.
  • the invention further relates to the therapeutic treatment of these tumours in the body of said being.
  • the invention also relates to a pharmaceutical composition, to a labelled peptide to be used in this composition, and to a kit for preparing a pharmaceutical composition.
  • PACAP-(1-38) H 2 and PACAP-(1-27) H 2 in short PACAP-38 and PACAP-27, are known since a number of years.
  • PACAP is a member of a peptide family including VIP, PHI, PHV, etc.
  • PACAP has been studied by Christophe and coworkers. In this connection, Robberecht et al. (Am. J. Physiol. 2JL0, 1991, G97-G102) have studied PACAP and VIP receptors in rat liver membranes by using radioiodinated PACAP, viz.
  • octreotide a cyclic peptide containing 8 amino acid moieties
  • OctreoScan ® 111 This diagnosticum, labelled with indium-Ill, is specifically designed for tumour imaging, in particular of tumours in the abdomen (M-D-D-I Reports ("The Gray Sheet") Nov. 2, 1992, p.14). It has been observed, however, that various frequently occurring malignant tumours, such as endometrial and prostatic carcinoma as well as pancreatic and colonic adenocarcinoma, cannot well be detected and localized by using radiolabelled octreotide.
  • malignant human tumours are: (a) Carcinomas (Ca) , including adenocarcinomas and squamous cell carcinomas, such as: breast Ca, prostate Ca, ovarian Ca, endometrial Ca, bladder Ca, oesophageal Ca, stomach Ca, colon Ca, pancreas Ca, lung Ca (e.g.
  • nSCLC renal cell Ca
  • Neuroendocrine tumours such as: gastroenteropancreatic tumours, pituitary tumours, adrenocortical tumours, parathyroid tumours, pheochromacytomas, thyroid Ca, and all metastases thereof
  • Brain tumours such as: meningiomas, glioblastomas, astrocytomas, schwannomas, and all metastases thereof
  • Lymphomas and thymomas and all metastases thereof
  • Sarcomas and all metastases thereof (f) Melanomas and all metastases thereof;
  • ylms tumours and all metastases thereof are examples of the malignant tumours thereof.
  • Such a method would be a powerful tool, not only in diagnosing such tumours but also in supporting an effective therapy therefor.
  • the detection and localization of these tumours, and in particular of the metastases thereof, in an early stage of their development is of utmost importance.
  • Various requirements have to be imposed on an agent that is used in such a diagnostic method, such as non-toxic, no adverse influence on the host resistance and/or on the therapeutic treatment, well detectable and highly selective.
  • the required high selectivity means that the diagnostic agent, after having been introduced into the body, must accumulate more strongly in the target tumours to be detected or visualized than in surrounding tissues. This selectivity, i.e.
  • the diagnostic agent In order to be detectable from outside the body, the diagnostic agent should be labelled, preferably with a radionuclide or with a paramagnetic metal atom. In the former case, the radioactive radiation can be detected by using a suitable detector (scanning) .
  • a suitable detector scanning
  • Modern techniques in this field use emission tomography; when gamma radiating isotopes are used, the so-called single photon emission computerized tomography (SPECT) may be applied.
  • SPECT single photon emission computerized tomography
  • a method which comprises (i) administering to said being a composition comprising, in a quantity sufficient for external imaging, a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP) , PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, said peptide being labelled with (a) a radioactive metal isotope selected from the group consisting of "*Tc, 203 Pb, 66 Ga, 67 Ga, 68 Ga, 2 As, m In, 113m In, "Ru, 62 Cu, 64 Cu, 52 Fe, 52m Mn and 51 Cr, or (b) with a paramagnetic metal atom selected from the group consisting of Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er,
  • PACAP pituitary adenylate cycl
  • This objective can be achieved, according to a different aspect of the present invention, by (i) administering to said human being a composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP), PACAP-receptor agonists, PACAP- receptor antagonists, PACAP analogues and PACAP derivatives, said peptide being labelled with 161 Tb, 123 I or 125 I, and thereupon (ii) , after allowing the active substance to be taken up in said tumours and after blood clearance of radioactivity, subjecting said being to a radioimmunodetection technique in the relevant area of the body of said being, by using a gamma detecting probe. It is still another objective of the present invention to provide a method for the therapeutic treatment of malignant tumours in the body of a human being.
  • PACAP pituitary aden
  • composition comprising, in a quantity effective for combating or controling tumours, a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP) , PACAP-receptor agonists, PACAP-receptor antagonists,
  • PACAP pituitary adenylate cyclase - activating polypeptide
  • PACAP analogues and PACAP derivatives said peptide being labelled with an isotope selected from the group consisting of :86 Re, 188 Re, 77 As, 11m In, 90 Y, 67 Cu, 169 Er,
  • the method is especially usefull in the detection and therapeutic treatment of certain tumours and the metastasis thereof. Therefore the invention is also relating to a method for the detection and therapeutic treatment of tumours and the metastasis thereof, characterized in that the tumours and the metastasis thereof to be detected, localized or therapeutically treated are selected from the group consisting of Astrocytomas, Glioblastomas, Endometrial tumours, Ovarian tumours, Hemangiopericytomas and Pituitary adenomas.
  • the labelled peptide to be used according to the method of the invention is preferably derived from a compound of the general formula H -(((Xaa) p - Xbb) q - Xcc - Xdd - Il ⁇ ) r - Xee - Thr - Asp - Xff - Xgg 1 5 10
  • Ri is a (Ci-Cjjal anoyl group, an arylcarbonyl group, or an aryl-(C-Cjjalkanoyl group; or a lactam thereof, formed between a free NH 2 group of an amino acid moiety and a free C0 2 H group of another amino acid moiety; or a conjugate thereof with avidin or biotin; and wherein: Xaa is His or Phe;
  • Xbb is Ser, Ala, Arg, Phe or p-Cl-Phe;
  • Xcc is Asp or Glu;
  • Xdd is Gly or an aminoisobutyric acid moiety
  • Xee, Xgg, Xii and Xkk are each individually Phe, Tyr or Trp;
  • Xff is Ser or Asn Xhh is Arg or Lys
  • Xjj is Met or a norleucine moiety
  • n stands for 0 to 12 amino acid moieties which are equal or different and are selected from Leu, Asn, Gin, Val, Asp, Lys, Gly, Arg, Tyr, Trp, Phe, Ser, lie, Thr or Pro; p, q and r are each individually 0 or 1; and R 2 is a hydroxy group, an acetoxy group or an amino group.
  • Suitable examples of aryl groups in R x are: phenyl, substituted phenyl or indolyl; preferably phenyl, 4- fluorophenyl, 2- or 4-bromo-phenyl, 2-iodophenyl, 4- hydroxyphenyl, 3-iodo-4-hydroxyphenyl, 4-fluoro-2- bromophenyl and 4-fluoro-2-iodophenyl.
  • the label is attached subsequently by reaction with labelled biotin in the case of avidin- conjugated peptide as described by Kalofonos et al . (J. Nucl. Med. 1990, 3_1, 1791), or by reaction with labelled avidin in the case of biotin-conjugated peptide as described by Paganelli et al . (Int. J. Cancer 1988, 2 , 121) .
  • one or more of the amino acids may have the D-configuration instead of the normal L-configuration.
  • the labelled peptide compounds of the invention may also comprise so-called pseudo peptide bonds, viz. -CH 2 - NH- bonds, in addition to the natural amide bonds, viz. - CO-NH- bonds.
  • pseudo peptide bonds viz. -CH 2 - NH- bonds
  • Such modifications of the amino acids naturally occurring in peptides are within the scope of the present invention.
  • PACAP- (1-38)NH 2 (PACAP-38) PACAP- (1-27)NH 2 (PACAP-27), Ac-PACAP-27 (a PACAP receptor agonist) , PACAP(2-27) (a PACAP receptor agonist) , PACAP(2-38) (a PACAP receptor agonist), [Nle-17]PACAP-38 (a PACAP receptor agonist), PACAP(6-38) (a PACAP receptor antagonist), [Ala-2]PACAP-27 (a PACAP receptor agonist) and [p-Cl-Phe-2]PACAP-38 (a PACAP receptor antagonist) .
  • PACAP-38 H-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-
  • PACAP-27 H-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-
  • PACAP (2-27 ) H-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-
  • PACAP (2-38 ) H-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-
  • PACAP-38 H-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp- Ser-Tyr-Ser-Arg- Tyr-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys- Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-
  • PACAP 6-38 : H-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-
  • radioactive halogen atom is preferably selected from the group consisting of 13 I, 124 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br and 82 Br, said radioactive halogen isotope being attached to a Tyr or Trp moiety of the peptide, or to the aryl group of substituent R : .
  • metal atom is preferably selected from (a) the group consisting of the radioactive isotopes
  • R is a branched or non-branched, optionally substituted hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N, 0 and S and/or by one or more NH groups, and is a group which is capable of reacting with an amino group of the peptide and which is preferably selected from the group consisting of carbonyl, carbimidoyl, N- (Ci-Cgjalkylcarbimidoyl, N-hydroxy- carbimidoyl and N- (C 1 -C 6 )alkoxycarbimidoyl .
  • R 6 -R 20 are each individually hydrogen atoms or (C x - C 4 )alkyl groups, with the proviso that at least one of C 6 to C 9 is the symbol Y;
  • R 21 is a hydrogen atom or a C0 2 (C 1 -C 4 )alkyl group
  • R 22 and R 23 are each individually (C 1 -C 4 )alkyl groups or phenyl groups; v is 0 or 1; s is 2 or 3;
  • R 24 is CH 2 COOH or a functional derivative thereof;
  • A is (C 1 -C )alkylene, if desired substituted with C0 2 alkyl, CH 2 COalkyl, CONH 2 , CONHCH 2 C0 2 alkyl; phenylene, phenylene substituted by C0 2 alkyl, wherein the alkyl groups have 1 to 4 carbon atoms;
  • G is NH or S;
  • Y is a functional group capable of binding with a free amino group of the peptide or with the spacing group; and Z is S or 0.
  • Said functional group Y preferably comprises isocyanato, isothiocyanato, formyl, o-halonitrophenyl, diazonium, epoxy, trichloro-s-triazinyl, ethyleneimino, chlorosulfonyl, alkoxycarbimidoyl, (substituted or unsubstituted) alkylcarbonyloxycarbonyl, alkylcarbonylimidazolyl, succinimido-oxycarbonyl; said group being attached to a (C ⁇ C K ,)hydrocarbon biradical.
  • hydrocarbon biradicals are biradicals derived from benzene, (Cj-C alkanes, (C 2 - C 6 )alkenes and (Ci-C -alkylbenzenes.
  • Suitable chelators of the general formula II are described in the international patent application WO 89/07456, such as unsubstituted or substituted 2- imino-thiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4-mercaptomethylthiolane.
  • Suitable examples of spacing groups are groups of the general formula
  • R 3 is a C ⁇ C ⁇ , alkylene group, a C J -C K , alkylidene group or a C 2 -C 10 alkenylene group
  • X is a thiocarbo- nyl group or a group of the general formula
  • the invention further relates to a pharmaceutical composition to be used for the above-defined method, comprising in addition to a pharmaceutically acceptable carrier material, preferably a physiological saline solution, and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a labelled peptide as defined hereinbefore.
  • a pharmaceutically acceptable carrier material preferably a physiological saline solution
  • at least one pharmaceutically acceptable adjuvant as the active substance a labelled peptide as defined hereinbefore.
  • Suitable adjuvants are well-known in the art and include buffering agents such as HEPES buffer, TRIS buffer, etc., antioxidants and stabilizers such as ascorbic acid, gentisic acid or salts of these acids.
  • the pharmaceutical composition of the invention comprises preferably as the active substance a labelled peptide derived from a compound of the general formula I, wherein the symbols have the meanings given above.
  • the invention also relates to a labelled peptide to be used as an active ingredient in the above pharmaceutical composition, said peptide being labelled with a metal atom as defined hereinbefore. Suitable chelating agents for chelating said metal atom are described above.
  • the labelled peptide is preferably derived from a compound of the general formula I, wherein the symbols have the meanings given above.
  • the invention also relates to a method of preparing a metal atom - labelled peptide as defined above, by reacting a derivatized peptide, comprising a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP) , PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, derivatized with a chelating group bound by an amide bond or through a spacing group to the peptide molecule, with a metal atom as defined hereinbefore in the form of a salt or of a chelate, bound to a comparatively weak chelator, in order to form a complex.
  • PACAP pituitary adenylate cyclase - activating polypeptide
  • PACAP-receptor agonists PACAP-receptor antagonists
  • PACAP analogues PACAP derivatives
  • the metal-labelled peptides of the invention can be prepared in a manner known per se for related compounds.
  • the peptide molecule is derivatized with the desired chelating agent as defined hereinbefore, e.g. N t S (4 - t) , EDTA, DTPA, etc., directly or after introduction of a spacing group as defined above, after which the compound obtained is reacted with a metal isotope, as defined hereinbefore, in the form of a salt or of a chelate bound to a comparatively weak chelator, in order to form a complex.
  • the desired chelating agent as defined hereinbefore, e.g. N t S (4 - t) , EDTA, DTPA, etc.
  • Suitable examples of salts or chelates of the desired metal atom are: 111 In-oxinate, 99m Tc-tartrate, etc.
  • the complex-forming reaction can generally be carried out in a simple manner and under conditions that are not detrimental to the peptide.
  • the invention further relates to the results of the above preparation method, viz. a derivatized peptide, comprising a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP), PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, derivatized with a chelating group bound by an amide bond or through a spacing group to the peptide molecule.
  • PACAP pituitary adenylate cyclase - activating polypeptide
  • PACAP-receptor agonists PACAP-receptor antagonists
  • PACAP analogues PACAP derivatives
  • the invention also relates to a kit for preparing a radiopharmaceutical composition.
  • kit according to the present invention for preparing a radiopharmaceutical composition comprises (i) a derivatized peptide as defined above, to which derivatized peptide, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (ii) a solution of a salt or chelate of a metal isotope selected from the group consisting of the radioactive isotopes 203 Pb, 66 Ga , 67 Ga, 68 Ga , 72 As , n ⁇ In, 113m In, 114ra In , 97 Ru, 62 Cu, 6 Cu, 99m Tc , 186 Re , 188 Re, 52 Fe , 52m Mn, 51 Cr , 7 As , 90 Y, 67 Cu , l ⁇ 9 Er , U7m Sn, 121 Sn, 12 Te , 1 2 Pr, 143 Pr, 198 Au , 199 Au, 161 Tb, 109 Pd
  • the peptide compound to be used as an ingredient of the above kit has been derivatized by a reaction with a chelating agent as defined hereinbefore.
  • the resulting peptide conjugate provides a facility for firmly attaching the radionuclide in a simple manner.
  • Suitable chelating agents for modifying the peptide are described in detail hereinbefore.
  • N-containing di- or polyacetic acids or their derivatives, such as the compounds mentioned before, have proved to be pre ⁇ eminently suitable for attaching various metal radionuclides, such as In-Ill and In-113m, to the peptide molecules.
  • the kit to be supplied to the user may also comprise the ingredient(s) defined sub (i) above, together with instructions for use, whereas the solution of a salt or chelate of the radionuclide, defined sub (ii) above, which solution has a limited shelf life, may be put to the disposal of the user separately.
  • kits may comprise, in addition to the ingredient(s) defined sub (i) above, (ii) a reducing agent and, if desired, a chelator, and (iii) instructions for use with a prescription for reacting the ingredients of the kit with Tc-99m in the form of a pertechnetate solution, or with Re-186 or Re-188 in the form of a perrhenate solution.
  • the ingredients of the kit may be combined, provided they are compatible.
  • the kit should comprise a reducing agent to reduce the pertechnetate or perrhenate, for example, a dithionite, a metallic reducing agent or a complex-stabilizing reducing agent, e.g. SnCl 2 , Sn(II)-tartrate, Sn(II)-phosphonate or - pyrophosphate, or Sn(II)-glucoheptonate.
  • a reducing agent to reduce the pertechnetate or perrhenate for example, a dithionite, a metallic reducing agent or a complex-stabilizing reducing agent, e.g. SnCl 2 , Sn(II)-tartrate, Sn(II)-phosphonate or - pyrophosphate, or Sn(II)-glucoheptonate.
  • the pertechnetate or perrhenate solution can simply be obtained by the user from a suitable generator.
  • the complex forming reaction with the derivatized peptide can simply be produced by combining the components in a neutral medium and causing them to react.
  • the radionuclide may be presented to the derivatized peptide in the form of a chelate bound to a comparatively weak chelator, as described hereinbefore.
  • the kit comprises a derivatized peptide as defined hereinbefore and is intended for the preparation of a radiopharmaceutical composition, labelled with Tc- 99m, Re-186 or Re-188
  • the radionuclide will preferably be added separately in the form of a pertechnetate or perrhenate solution.
  • the kit will comprise a suitable reducing agent and, if desired, a chelator, the former to reduce the pertechnetate or the perrhenate.
  • a reducing agent may be used, for example, a dithionite or a metallic reducing agent.
  • the ingredients may optionally be combined, provided they are compatible.
  • Such a monocomponent kit, in which the combined ingredients are preferably lyophilized, is excellently suitable for being reacted, by the user, with the radionuclide solution.
  • a metallic reducing agent for example, Sn(II), Ce(III), Fe(II), Cu(I),
  • the peptide constituent of the above-mentioned kits i.e. preferably the derivatized peptide, may be supplied as a solution, for example, in the form of a physiological saline solution, or in some buffer solution, but is preferably present in a dry condition, for example, in the lyophilized condition.
  • a component for an injection liquid it should be sterile, in which, when the constituent is in the dry state, the user should preferably use a sterile physiological saline solution as a solvent.
  • the above-mentioned constituent may be stabilized in the conventional manner with suitable stabilizers, for example, ascorbic acid, gentisic acid or salts of these acids, or it may comprise other auxiliary agents, for example, fillers, such as glucose, lactose, mannitol, and the like.
  • suitable stabilizers for example, ascorbic acid, gentisic acid or salts of these acids, or it may comprise other auxiliary agents, for example, fillers, such as glucose, lactose, mannitol, and the like.
  • Receptor autoradiography is performed on 10- and 20-urn thick cryostat sections of the various tumour samples, as described by Reubi et al. (Cancer Res. 1990, 5_0, 5969- 5977) .
  • 125 I-labelled peptides are prepared via the lactoperoxydase procedure, according to procedures as reported earlier by Marchlonis (Biochemical Journal 1969, 113. 299-305) .
  • Tyr 10 labelled VIP and Tyr 22 labelled VIP are separated by HPLC, using a reverse phase RC 18 column and butane- sulphonic acid as the eluent.
  • the mono- 125 iodo- [Tyr 10 ]-VIP as well as the mono- 125 iodo-[Tyr 22 ]-VIP are each eluted as single peaks from the HPLC and analysed by mass- spectrometry. Specific activity: 2000 Ci/mmol. Both peaks can be used for binding experiments.
  • the tissues are cut on a cryostat, mounted on microscope slides, and then stored at -20°C for at least 3 days to improve adhesion of the tissue to the slide.
  • the slide-mounted tissue sections are allowed to reach room temperature and are incubated for 90 min in a solution of 50 mM Tris-HCl, pH 7.4, containing BSA (2%), EGTA (2 mM) , bacitracin (0.1 mM) , MgCl 2 (5 mM) , and 30 pM [ 125 I]-VIP, at room temperature, as described by Dietl et al. (Brain Res. 1990, 520., 14-26).
  • paired serial sections are incubated as described above, except that luM PACAP-27 or PACAP-38 are added to the incubation medium. After the incubation, the slides are rinsed with four washes of 30 sec each in ice- cold 50 mM Tris-HCl, pH 7.4, dipped in ice-cold water, and then quickly dried in a refrigerator under a stream of cold air. The sections are subsequently exposed to a 3 H-Ultrofilm for 1 week, to detect the precise location of the radioactivity.
  • A pancreatic adenocarcinoma
  • B colonic adenocarcinoma
  • C endometrial carcinoma
  • D prostatic carcinoma.
  • Tissue sections are incubated with 14,000 cpm/lOO ⁇ l [ 125 I]-VIP and increasing concentrations of unlabelled PACAP-27 (*), GRF ( ⁇ ) , somastotatin ( ⁇ ) or octreotide (o) .
  • Each point represents the optical density of binding measured in the tumour area. Non-specific binding is substracted from all values. In all cases, complete displacement of the ligand is achieved by PACAP, whereas GRF, somastotatin and octreotide are inactive in the nanomolar range.
  • Example 2
  • Receptor autoradiography and peptide labelling is performed as described in Example 1.
  • Tyr 10 labelled, Tyr 13 labelled and Tyr 22 labelled Ac- His 1 -PACAP are separated by HPLC, using a reverse phase RC 18 column and butane-sulphonic acid as the eluent.
  • the mono- 125 iodo-Ac-His 1 -PACAP's are each eluted as single peaks from the HPLC and analysed by mass-spectrometry. Specific activity: 2000 Ci/mmol.
  • the first peak is used for binding experiments, and assumed to be the the mono- 125 iodo- [Tyr 10 ]-Ac-His 1 -PACAP.
  • the tissues are cut on a cryostat, mounted on microscope slides, and then stored at -20°C for at least 3 days to improve adhesion of the tissue to the slide.
  • the slide-mounted tissue sections are allowed to reach room temperature and are incubated for 90 min in a solution of 50 mM Tris-HCl, pH 7.4, containing BSA (2%), EGTA (2 mM) , bacitracin (0.1 mM) , MgCl 2 (5 mM) , and 30 pM [ 125 I]-Ac-His 1 -PACAP, at room temperature, as described by Dietl et al. (Brain Res. 1990, 520, 14-26).
  • paired serial sections are incubated as described above, except that l ⁇ M PACAP-27 is added to the incubation medium. After the incubation, the slides are rinsed with four washes of 30 sec each in ice- cold 50 mM Tris-HCl, pH 7.4, dipped in ice-cold water, and then quickly dried in a refrigerator under a stream of cold air. The sections are subsequently exposed to a
  • Tissue sections are incubated with 14,000 cpm/lOO ⁇ l [ 125 I]-Ac- His ⁇ PACAP and increasing concentrations of unlabelled PACAP-27 ( ⁇ ), VIP ( ⁇ ) or somastotatin ( ⁇ ) .
  • Each point represents the optical density of binding measured in the tumour area. Non-specific binding is substracted from all values. In all cases, complete displacement of the ligand is achieved by PACAP, whereas VIP is active in Prostatic carcinoma and inactive in Astrocytoma and somastotatin is inactive in the nanomolar range.
  • SPPS Solid phase peptide synthesis
  • F oc (9-fluorenemethoxycarbonyl) strategy Solid phase peptide synthesis
  • the general principles and methods followed are well known in the art. For a description of the method see, “Fluorenemethoxycarbony-polyamide solid phase synthesis-General Synthesis and Development” - Chapter 3 in “Solid peptide synthesis- A practical approach” by E. Atherton and R.C. Sheppard, Information Press Ltd., Oxford, England (1989) .
  • Fmoc 9-fluorenemethoxycarbonyl
  • All the standard Fmoc-protected amino acids are purchased commercially.
  • Coupling with dicyclohexylcar- bodiimide/hydroxybenzotriazole using either p-hydroxy- methylphenoxy ethylpolystyrene for carboxyl terminus acids or Rink amide resin is used for carboxyl terminus amides.
  • the products are routinely cleaved using a solution comprised of trifluoroacetic acid:water:anisole:triisopropylsilane or trifluoracetic acid:ethanedithiol:thioanisole:water for 1-8 hours at room temperature.
  • the products are precipitated by ether and purified by C-18 reverse phase chromatography.
  • the N-terminal Fmoc-protecting group is removed in the synthesizer using the standard protocol of the synthesizer. Next 3-4 molar equivalents tri-t-butyl diethylenetriaminepentacetic acid is used for the condensation to the N-terminal. Cleavage and deprotection are carried out as outlined above.
  • the solid phase synthesis is carried out using commercially available Rink amide resin on 250 ⁇ mol scale.
  • Phe-6-condensation is completed, removal of the Fmoc-group, activation of tri-butyl-DTPA to the anhydride and coupling to the N-terminal Phe are completed in the SPPS in the synthesizer.
  • final piperidine wash is deleted from the synthesis protocol.
  • Starting material is 0.5 ml matrix solution containing 0.8 mg citric acid monohydrate, 11.2 mg sodium citrate trihydrate, 20 mg inositol and 4 mg gentisic acid/ml.
  • 10 ⁇ l of N-DTPA-PACAP(6-36) solution (1 mg/ml in water) is added.
  • 0.5 ml Indium-Ill chloride (111 Mbq/ l) is added.
  • the labelling yield is determined by instant thin layer chromatography using citrate lM/pH5 as eluent.
  • the radiochemical purity is determined by RP-HPLC using a C 18 /10 ⁇ column and a linear gradient 5% acetonitrile incl. 0.05% TFA-95% acetronitrile incl. 0.05% TFA in 30 minutes.
  • N-DTPA-PACAP(1-27)-piperidine amide The affinity of N-DTPA-PACAP(1-27)-piperidine amide towards PACAP receptors in gut carcinoid tumour and prostate tumour is determined as described in Example 1.
  • the figures G attached shows the mean of the displacement curves of [ 125 I]-VIP binding to tissue sections from two different tumours: Gut carcinoid tumour and prostate tumour. Tissue sections are incubated with 14,000 cpm/lOO ⁇ l [ 125 I]-VIP and increasing concentrations of unlabelled N-DTPA-PACAP(1-27) -piperidine amide ( ⁇ ) and VIP (•). Each point represents the optical density of binding measured in the tumour area. Non-specific binding is substracted from all values.
  • N-DTPA-PACAP(1- 27)-piperidine amide at a concentration of a factor of about 50 higher than VIP.
  • the specific binding of N-DTPA- PACAP(1-27)-piperidine shows that this compound or analogs thereof, after metal labelling, are promising candidates to visualize PACAP-R positive tumours in vivo.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a method of detecting and localizing malignant human tumours, including the metastases thereof, in the body of a human being, comprising the steps of: (i) administering to said being a composition comprising, in a quantity sufficient for external imaging, a labelled peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP), PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives; and thereupon (ii) subjecting said being to external imaging, by radioactive scanning or by magnetic resonance imaging, to determine the targeted sites in the body of said being. The invention further relates to a method for the therapeutic treatment of said malignant human tumours by administration of the above-defined peptide, labelled for this purpose, a method for labelling of the peptide compounds, a pharmaceutical composition to be used for detection, a pharmaceutical composition to be used for therapy and to a kit for preparing a radiopharmaceutical composition.

Description

Method for the detection and localization of malignant human tumours.
The invention relates to a method of detecting and localizing malignant tumours in the body of a human being. The invention further relates to the therapeutic treatment of these tumours in the body of said being. The invention also relates to a pharmaceutical composition, to a labelled peptide to be used in this composition, and to a kit for preparing a pharmaceutical composition.
Pituitary adenylate cyclase - activating polypeptide (PACAP) is a peptide having the ability to stimulate adenylate cyclase in pituitary cells. PACAP-(1-38) H2 and PACAP-(1-27) H2, in short PACAP-38 and PACAP-27, are known since a number of years. PACAP is a member of a peptide family including VIP, PHI, PHV, etc. PACAP has been studied by Christophe and coworkers. In this connection, Robberecht et al. (Am. J. Physiol. 2JL0, 1991, G97-G102) have studied PACAP and VIP receptors in rat liver membranes by using radioiodinated PACAP, viz. 125I-PACAP- 27. In a review (Biochim. Biophys. Acta 1154, 1993, 183- 199), Christophe demonstrates that there are at least two classes of receptors for PACAP, and that there is an abundant number of mammalian tissues and organs having these receptors. It will be evident that the great number of different mammalian tissues having PACAP receptors will make a selective use of this peptide in medicine very difficult.
Recently radiolabelled octreotide, a cyclic peptide containing 8 amino acid moieties, is commercialized under the brand name OctreoScan® 111. This diagnosticum, labelled with indium-Ill, is specifically designed for tumour imaging, in particular of tumours in the abdomen (M-D-D-I Reports ("The Gray Sheet") Nov. 2, 1992, p.14). It has been observed, however, that various frequently occurring malignant tumours, such as endometrial and prostatic carcinoma as well as pancreatic and colonic adenocarcinoma, cannot well be detected and localized by using radiolabelled octreotide. It is the objective of the present invention to provide a method of detecting and localizing malignant human tumours and the metastases thereof, in particular frequently occurring tumours, in the body of a human being. Examples of such malignant human tumours are: (a) Carcinomas (Ca) , including adenocarcinomas and squamous cell carcinomas, such as: breast Ca, prostate Ca, ovarian Ca, endometrial Ca, bladder Ca, oesophageal Ca, stomach Ca, colon Ca, pancreas Ca, lung Ca (e.g. nSCLC) and renal cell Ca, in particular also all metastases thereof; (b) Neuroendocrine tumours, such as: gastroenteropancreatic tumours, pituitary tumours, adrenocortical tumours, parathyroid tumours, pheochromacytomas, thyroid Ca, and all metastases thereof; (c) Brain tumours, such as: meningiomas, glioblastomas, astrocytomas, schwannomas, and all metastases thereof; (d) Lymphomas and thymomas, and all metastases thereof; (e) Sarcomas and all metastases thereof; (f) Melanomas and all metastases thereof; (g) Seminomas and all metastases thereof; (h) ylms tumours and all metastases thereof.
Such a method would be a powerful tool, not only in diagnosing such tumours but also in supporting an effective therapy therefor. As a matter of fact, in order to be able to achieve a specific therapy for the control of such tumours, the detection and localization of these tumours, and in particular of the metastases thereof, in an early stage of their development is of utmost importance. Various requirements have to be imposed on an agent that is used in such a diagnostic method, such as non-toxic, no adverse influence on the host resistance and/or on the therapeutic treatment, well detectable and highly selective. The required high selectivity means that the diagnostic agent, after having been introduced into the body, must accumulate more strongly in the target tumours to be detected or visualized than in surrounding tissues. This selectivity, i.e. a comparatively stronger concentration of the diagnostic agent in the target tumours compared with non-target tissues, enables the user to correctly diagnose the malignancy. In order to be detectable from outside the body, the diagnostic agent should be labelled, preferably with a radionuclide or with a paramagnetic metal atom. In the former case, the radioactive radiation can be detected by using a suitable detector (scanning) . Modern techniques in this field use emission tomography; when gamma radiating isotopes are used, the so-called single photon emission computerized tomography (SPECT) may be applied. The use of paramagnetic diagnostic agents enables a detection by means of imaging by magnetic resonance.
The above-defined objective can be achieved, according to the present invention, by a method, which comprises (i) administering to said being a composition comprising, in a quantity sufficient for external imaging, a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP) , PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, said peptide being labelled with (a) a radioactive metal isotope selected from the group consisting of "*Tc, 203Pb, 66Ga, 67Ga, 68Ga, 2As, mIn, 113mIn, "Ru, 62Cu, 64Cu, 52Fe, 52mMn and 51Cr, or (b) with a paramagnetic metal atom selected from the group consisting of Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er, or (c) with a radioactive halogen isotope, selected from 123I, 131I, 75Br, 76Br, 77Br and 82Br, and thereupon (ii) subjecting said being to external imaging, by radioactive scanning or by magnetic resonance imaging, to determine the targeted sites in the body of said being in relation to the background activity, in order to allow detection and localization of said tumours in the body.
The above labelled peptides have been tested in suitable model experiments that are predictive for in vivo application. In these model experiments human tumour tissue samples are used to mimic in vivo application. The experiments are described in the Example appended. From the results it will be evident that the tested labelled peptides have properties which make them pre-eminently suitable for the detection and localization of a great number of malignant human tumours.
This is indeed beyond expectation, in view of the abundance of PACAP receptors throughout the human body. It is another objective of the present invention to provide a method of intraoperatively detecting and localizing malignant tumours in the body of a human being.
This objective can be achieved, according to a different aspect of the present invention, by (i) administering to said human being a composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP), PACAP-receptor agonists, PACAP- receptor antagonists, PACAP analogues and PACAP derivatives, said peptide being labelled with 161Tb, 123I or 125I, and thereupon (ii) , after allowing the active substance to be taken up in said tumours and after blood clearance of radioactivity, subjecting said being to a radioimmunodetection technique in the relevant area of the body of said being, by using a gamma detecting probe. It is still another objective of the present invention to provide a method for the therapeutic treatment of malignant tumours in the body of a human being.
This objective can be achieved, according to a further different aspect of the present invention, by administering to said being a composition comprising, in a quantity effective for combating or controling tumours, a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP) , PACAP-receptor agonists, PACAP-receptor antagonists,
PACAP analogues and PACAP derivatives, said peptide being labelled with an isotope selected from the group consisting of :86Re, 188Re, 77As, 11mIn, 90Y, 67Cu, 169Er,
X1 -Sn 12_Sri f 127Tθ f 142pr / 14^ 198^ 199^ 149^ 161,^ 109pd ; 165Dy, 149Pm, 151Pm, 153Sm, 157Gd, 159Gd, 166Ho , 172Tm, 169Yb, 175Yb, 177Lu , 105Rh, nιAg , 124I and 131I .
The method is especially usefull in the detection and therapeutic treatment of certain tumours and the metastasis thereof. Therefore the invention is also relating to a method for the detection and therapeutic treatment of tumours and the metastasis thereof, characterized in that the tumours and the metastasis thereof to be detected, localized or therapeutically treated are selected from the group consisting of Astrocytomas, Glioblastomas, Endometrial tumours, Ovarian tumours, Hemangiopericytomas and Pituitary adenomas. The labelled peptide to be used according to the method of the invention is preferably derived from a compound of the general formula H -(((Xaa)p - Xbb)q - Xcc - Xdd - Ilβ)r - Xee - Thr - Asp - Xff - Xgg 1 5 10
-Ser - X h - Xii - Arg - Lys - Gin - Xjj - Ala - Val - Lys - Lys- Xkk
15 20
- Leu - Ala - Ala - Val - Leu - (Xll)n - R2 (I) or an acid amide thereof, formed between a free NH2-group of an amino acid moiety and RiCOOH, wherein Ri is a (Ci-Cjjal anoyl group, an arylcarbonyl group, or an aryl-(C-Cjjalkanoyl group; or a lactam thereof, formed between a free NH2 group of an amino acid moiety and a free C02H group of another amino acid moiety; or a conjugate thereof with avidin or biotin; and wherein: Xaa is His or Phe;
Xbb is Ser, Ala, Arg, Phe or p-Cl-Phe; Xcc is Asp or Glu;
Xdd is Gly or an aminoisobutyric acid moiety; Xee, Xgg, Xii and Xkk are each individually Phe, Tyr or Trp;
Xff is Ser or Asn Xhh is Arg or Lys
Xjj is Met or a norleucine moiety;
(Xll)n stands for 0 to 12 amino acid moieties which are equal or different and are selected from Leu, Asn, Gin, Val, Asp, Lys, Gly, Arg, Tyr, Trp, Phe, Ser, lie, Thr or Pro; p, q and r are each individually 0 or 1; and R2 is a hydroxy group, an acetoxy group or an amino group.
In the above formula I compounds a further preference is given to compounds of said formula, wherein g is 1 and (Xll)n stands for 10 to 12 amino acid moieties selected as defined above.
Suitable examples of aryl groups in Rx are: phenyl, substituted phenyl or indolyl; preferably phenyl, 4- fluorophenyl, 2- or 4-bromo-phenyl, 2-iodophenyl, 4- hydroxyphenyl, 3-iodo-4-hydroxyphenyl, 4-fluoro-2- bromophenyl and 4-fluoro-2-iodophenyl.
In the case of the use of a conjugate of the peptide with avidin or biotin, the label is attached subsequently by reaction with labelled biotin in the case of avidin- conjugated peptide as described by Kalofonos et al . (J. Nucl. Med. 1990, 3_1, 1791), or by reaction with labelled avidin in the case of biotin-conjugated peptide as described by Paganelli et al . (Int. J. Cancer 1988, 2 , 121) .
In the above labelled peptide compounds one or more of the amino acids may have the D-configuration instead of the normal L-configuration.
The labelled peptide compounds of the invention may also comprise so-called pseudo peptide bonds, viz. -CH2- NH- bonds, in addition to the natural amide bonds, viz. - CO-NH- bonds. Such modifications of the amino acids naturally occurring in peptides are within the scope of the present invention. Suitable examples of the above-defined peptides, which after labelling can be used in the method of the invention, are PACAP- (1-38)NH2 (PACAP-38) , PACAP- (1-27)NH2 (PACAP-27), Ac-PACAP-27 (a PACAP receptor agonist) , PACAP(2-27) (a PACAP receptor agonist) , PACAP(2-38) (a PACAP receptor agonist), [Nle-17]PACAP-38 (a PACAP receptor agonist), PACAP(6-38) (a PACAP receptor antagonist), [Ala-2]PACAP-27 (a PACAP receptor agonist) and [p-Cl-Phe-2]PACAP-38 (a PACAP receptor antagonist) . In formulas : (1) PACAP-38: H-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-
Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys- Gln-Arg-Val-Lys-Asn-Lys-NH2
(2) PACAP-27: H-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-
Ala-Ala-Val-Leu-NH2
(3) Ac-PACAP-27: Ac-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser- Tyr-Ser-Arg-Tyr- Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-
Leu-Ala-Ala-Val-Leu-NH2 (4 ) PACAP (2-27 ) : H-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-
Ala-Ala-Val-Leu-NH2
(5 ) PACAP (2-38 ) : H-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-
Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys- Gln-Arg-Val-Lys-Asn-Lys-NH2
( 6) [Nlβ-17] PACAP-38 : H-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp- Ser-Tyr-Ser-Arg- Tyr-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys- Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-
Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys-NH2
(7 ) PACAP ( 6-38 ) : H-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg- Lys-Gln-Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-
Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys- Asn-Lys-NH2
(8) [Ala-2]PACAP-27: H-His-Ala-Asp-Gly-Ile-Phe-Thr-Asp- Ser-Tyr-Ser-Arg- Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-
Tyr-Leu-Ala-Ala-Val-Leu-NH2 (Nle = norleucine) (9) [p-Cl-Phe-2]PACAP-38: H-His- (p-Cl-D-Phe)-Asp-Gly-Ile- Phe-Thr-Asp- Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-
Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly- Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys- NH2 If the peptide as defined above is labelled with a radioactive halogen atom, said radioactive halogen atom is preferably selected from the group consisting of 13I, 124I, 125I, 131I, 75Br, 76Br, 77Br and 82Br, said radioactive halogen isotope being attached to a Tyr or Trp moiety of the peptide, or to the aryl group of substituent R:. If the peptide as defined above is labelled with a metal atom, said metal atom is preferably selected from (a) the group consisting of the radioactive isotopes
"TC , 203pb j 66G3 67Qa / 68Ga ; 72As ^ lllj^ U3»In # l«»In f 97RU , 62Cu , 64Cu , 52Fe , 52mMn , 51Cr , 186Re , 188Re , 77As , 90Y , 67Cu , 169Er , 117mSn, 121Sn, 127Te, 142Pr, 143Pr, 198Au, 199Au, 149Tb, 161Tb, 109Pd, 165Dy, 149Pm, 151Pm, 153Sm, 157Gd, 166Ho, 172Tm, 169Yb, 175Yb, 17Lu, 105Rh and mAg or (b) the group consisting of the paramagnetic metal ions Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho, and Er; said metal atom being attached to the peptide by means of a chelating group chelating said atom, which chelating group is bound by an amide bond or through a spacing group to the peptide molecule. Suitable chelating groups for chelating said metal atom are NtS,4_t) tetradentate chelating agents, wherein t=2-4, or groups derived from ethylene diamine tetra- acetic acid (EDTA) , diethylene triamine penta-acetic acid (DTPA) , cyclohexyl 1,2-diamine tetra-acetic acid (CDTA) , ethyleneglycol-0, 0'-bis(2-aminoethyl)-N,N,N' , '-tetra- acetic acid (EGTA) , N,N-bis(hydroxybenzyl) -ethylenedia- mine-N,N'-diacetic acid (HBED) , triethylene tetramine hexa-acetic acid (TTHA) , 1,4, 7, 10-tetraazacyclododecane- N,N' ,N' ' ,N' ' '-tetra-acetic acid (DOTA) , hydroxyethyl- diamine triacetic acid (HEDTA), 1,4,8,11-tetra-azacyclo- tetradecane-N,N' ,N' ' ,N' ' '-tetra-acetic acid (TETA) , substituted DTPA, substituted EDTA, or from a compound of the general formula
Figure imgf000011_0001
wherein R is a branched or non-branched, optionally substituted hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N, 0 and S and/or by one or more NH groups, and is a group which is capable of reacting with an amino group of the peptide and which is preferably selected from the group consisting of carbonyl, carbimidoyl, N- (Ci-Cgjalkylcarbimidoyl, N-hydroxy- carbimidoyl and N- (C1-C6)alkoxycarbimidoyl .
NtS,4.t, chelating agents, wherein t=2-4, are preferably selected from
Figure imgf000012_0001
(VI) (VII)
Figure imgf000012_0002
Figure imgf000012_0003
wherein: R6-R20 are each individually hydrogen atoms or (Cx- C4)alkyl groups, with the proviso that at least one of C6 to C9 is the symbol Y;
R21 is a hydrogen atom or a C02(C1-C4)alkyl group; R22 and R23 are each individually (C1-C4)alkyl groups or phenyl groups; v is 0 or 1; s is 2 or 3;
R24 is CH2COOH or a functional derivative thereof; A is (C1-C )alkylene, if desired substituted with C02alkyl, CH2COalkyl, CONH2, CONHCH2C02alkyl; phenylene, phenylene substituted by C02alkyl, wherein the alkyl groups have 1 to 4 carbon atoms; G is NH or S; Y is a functional group capable of binding with a free amino group of the peptide or with the spacing group; and Z is S or 0. Said functional group Y preferably comprises isocyanato, isothiocyanato, formyl, o-halonitrophenyl, diazonium, epoxy, trichloro-s-triazinyl, ethyleneimino, chlorosulfonyl, alkoxycarbimidoyl, (substituted or unsubstituted) alkylcarbonyloxycarbonyl, alkylcarbonylimidazolyl, succinimido-oxycarbonyl; said group being attached to a (C^CK,)hydrocarbon biradical. Suitable examples of hydrocarbon biradicals are biradicals derived from benzene, (Cj-C alkanes, (C2- C6)alkenes and (Ci-C -alkylbenzenes.
Examples of suitable chelators of the general formula II are described in the international patent application WO 89/07456, such as unsubstituted or substituted 2- imino-thiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4-mercaptomethylthiolane.
Suitable examples of spacing groups, if present in the metal-labelled peptide molecule, are groups of the general formula
Figure imgf000014_0001
( III ) ( IV)
wherein R3 is a C^C^, alkylene group, a CJ-CK, alkylidene group or a C2-C10 alkenylene group, and X is a thiocarbo- nyl group or a group of the general formula
O NH
II II
—C—CH2-S—(CH2)m-C— (V)
wherein m is 1-5. Conjugates with avidin or biotin are formed as described by Paganelli et al . (Int. J. Cancer 1988, 2 , 121), Kalofonos et al . (J. Nucl. Med. 1990, 3_1, 1791) and Anderson et al. (FEBS LETT. 1991, 282/1. 35-40) .
The invention further relates to a pharmaceutical composition to be used for the above-defined method, comprising in addition to a pharmaceutically acceptable carrier material, preferably a physiological saline solution, and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a labelled peptide as defined hereinbefore. Suitable adjuvants are well-known in the art and include buffering agents such as HEPES buffer, TRIS buffer, etc., antioxidants and stabilizers such as ascorbic acid, gentisic acid or salts of these acids. The pharmaceutical composition of the invention comprises preferably as the active substance a labelled peptide derived from a compound of the general formula I, wherein the symbols have the meanings given above.
The invention also relates to a labelled peptide to be used as an active ingredient in the above pharmaceutical composition, said peptide being labelled with a metal atom as defined hereinbefore. Suitable chelating agents for chelating said metal atom are described above. The labelled peptide is preferably derived from a compound of the general formula I, wherein the symbols have the meanings given above.
The invention also relates to a method of preparing a metal atom - labelled peptide as defined above, by reacting a derivatized peptide, comprising a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP) , PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, derivatized with a chelating group bound by an amide bond or through a spacing group to the peptide molecule, with a metal atom as defined hereinbefore in the form of a salt or of a chelate, bound to a comparatively weak chelator, in order to form a complex.
The metal-labelled peptides of the invention can be prepared in a manner known per se for related compounds. For this purpose the peptide molecule is derivatized with the desired chelating agent as defined hereinbefore, e.g. NtS(4-t), EDTA, DTPA, etc., directly or after introduction of a spacing group as defined above, after which the compound obtained is reacted with a metal isotope, as defined hereinbefore, in the form of a salt or of a chelate bound to a comparatively weak chelator, in order to form a complex.
Suitable examples of salts or chelates of the desired metal atom are: 111In-oxinate, 99mTc-tartrate, etc. The complex-forming reaction can generally be carried out in a simple manner and under conditions that are not detrimental to the peptide.
The invention further relates to the results of the above preparation method, viz. a derivatized peptide, comprising a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP), PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, derivatized with a chelating group bound by an amide bond or through a spacing group to the peptide molecule.
In case a radioactive labelled peptide compound is used as a diagnostic agent, it is frequently impossible to put the ready-for-use composition at the disposal of the user, in connection with the often poor shelf life of the radiolabelled compound and/or the short half-life of the radionuclide used. In such cases the user will carry out the labelling reaction with the radionuclide in the clinical hospital or laboratory. For this purpose the various reaction ingredients are then offered to the user in the form of a so-called "kit". It will be obvious that the manipulations necessary to perform the desired reaction should be as simple as possible to enable the user to prepare from the kit the radioactive labelled composition by using the facilities that are at his disposal. Therefore the invention also relates to a kit for preparing a radiopharmaceutical composition.
Such a kit according to the present invention for preparing a radiopharmaceutical composition comprises (i) a derivatized peptide as defined above, to which derivatized peptide, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (ii) a solution of a salt or chelate of a metal isotope selected from the group consisting of the radioactive isotopes 203Pb, 66Ga , 67Ga, 68Ga , 72As , nιIn, 113mIn, 114raIn , 97Ru, 62Cu, 6 Cu, 99mTc , 186Re , 188Re, 52Fe , 52mMn, 51Cr , 7 As , 90Y, 67Cu , lδ9Er , U7mSn, 121Sn, 12 Te , 1 2Pr, 143Pr, 198Au , 199Au, 161Tb, 109Pd, 165Dy, 1 9Pm, 151Pm, 153Sm, 15 Gd, 166Ho , 172Tm, 169Yb, 175Yb, 177Lu , 105Rh and Ag, and (iii) instructions for use with a prescription for reacting the ingredients present in the kit. Preferably the peptide compound to be used as an ingredient of the above kit has been derivatized by a reaction with a chelating agent as defined hereinbefore. The resulting peptide conjugate provides a facility for firmly attaching the radionuclide in a simple manner. Suitable chelating agents for modifying the peptide are described in detail hereinbefore. N-containing di- or polyacetic acids or their derivatives, such as the compounds mentioned before, have proved to be pre¬ eminently suitable for attaching various metal radionuclides, such as In-Ill and In-113m, to the peptide molecules. The kit to be supplied to the user may also comprise the ingredient(s) defined sub (i) above, together with instructions for use, whereas the solution of a salt or chelate of the radionuclide, defined sub (ii) above, which solution has a limited shelf life, may be put to the disposal of the user separately. In case the kit serves to prepare a radiopharmaceutical composition labelled with Tc-99m, Re- 186 or Re-188, such a kit according to the present invention may comprise, in addition to the ingredient(s) defined sub (i) above, (ii) a reducing agent and, if desired, a chelator, and (iii) instructions for use with a prescription for reacting the ingredients of the kit with Tc-99m in the form of a pertechnetate solution, or with Re-186 or Re-188 in the form of a perrhenate solution. If desired, the ingredients of the kit may be combined, provided they are compatible. The kit should comprise a reducing agent to reduce the pertechnetate or perrhenate, for example, a dithionite, a metallic reducing agent or a complex-stabilizing reducing agent, e.g. SnCl2, Sn(II)-tartrate, Sn(II)-phosphonate or - pyrophosphate, or Sn(II)-glucoheptonate. The pertechnetate or perrhenate solution can simply be obtained by the user from a suitable generator.
When the radionuclide is present in the kit itself, the complex forming reaction with the derivatized peptide can simply be produced by combining the components in a neutral medium and causing them to react. For that purpose the radionuclide may be presented to the derivatized peptide in the form of a chelate bound to a comparatively weak chelator, as described hereinbefore. When the kit comprises a derivatized peptide as defined hereinbefore and is intended for the preparation of a radiopharmaceutical composition, labelled with Tc- 99m, Re-186 or Re-188, the radionuclide will preferably be added separately in the form of a pertechnetate or perrhenate solution. In that case the kit will comprise a suitable reducing agent and, if desired, a chelator, the former to reduce the pertechnetate or the perrhenate. As a reducing agent may be used, for example, a dithionite or a metallic reducing agent. The ingredients may optionally be combined, provided they are compatible. Such a monocomponent kit, in which the combined ingredients are preferably lyophilized, is excellently suitable for being reacted, by the user, with the radionuclide solution. As a reducing agent for the above- mentioned kits is preferably used a metallic reducing agent, for example, Sn(II), Ce(III), Fe(II), Cu(I),
Ti(III) or Sb(III); Sn(II) is excellently suitable. The peptide constituent of the above-mentioned kits, i.e. preferably the derivatized peptide, may be supplied as a solution, for example, in the form of a physiological saline solution, or in some buffer solution, but is preferably present in a dry condition, for example, in the lyophilized condition. When used as a component for an injection liquid it should be sterile, in which, when the constituent is in the dry state, the user should preferably use a sterile physiological saline solution as a solvent. If desired, the above-mentioned constituent may be stabilized in the conventional manner with suitable stabilizers, for example, ascorbic acid, gentisic acid or salts of these acids, or it may comprise other auxiliary agents, for example, fillers, such as glucose, lactose, mannitol, and the like.
The invention will now be described in greater detail with reference to the following specific Examples.
Examples
Example 1.
Test of the affinity of unlabelled PACA (1-27) to PACAP-
II receptors in different human tumours
Receptor autoradiography is performed on 10- and 20-urn thick cryostat sections of the various tumour samples, as described by Reubi et al. (Cancer Res. 1990, 5_0, 5969- 5977) . 125I-labelled peptides are prepared via the lactoperoxydase procedure, according to procedures as reported earlier by Marchlonis (Biochemical Journal 1969, 113. 299-305) .
Tyr10 labelled VIP and Tyr22 labelled VIP are separated by HPLC, using a reverse phase RC18 column and butane- sulphonic acid as the eluent. The mono-125iodo- [Tyr10]-VIP as well as the mono-125iodo-[Tyr22]-VIP are each eluted as single peaks from the HPLC and analysed by mass- spectrometry. Specific activity: 2000 Ci/mmol. Both peaks can be used for binding experiments. The mono-I25iodo-
[Tyr10]-VIP is used routinely in all experiments because of its superior selectivity.
The tissues are cut on a cryostat, mounted on microscope slides, and then stored at -20°C for at least 3 days to improve adhesion of the tissue to the slide. The slide-mounted tissue sections are allowed to reach room temperature and are incubated for 90 min in a solution of 50 mM Tris-HCl, pH 7.4, containing BSA (2%), EGTA (2 mM) , bacitracin (0.1 mM) , MgCl2 (5 mM) , and 30 pM [125I]-VIP, at room temperature, as described by Dietl et al. (Brain Res. 1990, 520., 14-26). To estimate non¬ specific binding, paired serial sections are incubated as described above, except that luM PACAP-27 or PACAP-38 are added to the incubation medium. After the incubation, the slides are rinsed with four washes of 30 sec each in ice- cold 50 mM Tris-HCl, pH 7.4, dipped in ice-cold water, and then quickly dried in a refrigerator under a stream of cold air. The sections are subsequently exposed to a 3H-Ultrofilm for 1 week, to detect the precise location of the radioactivity.
In all tumours, displacement experiments using successive sections of a tumour are performed with increasing concentrations of various biologically active or inactive peptides (see the above-mentioned publication by Reubi et al.). In comparison with PACAP, growth hormone releasing factor (GRF) , as well as somastotatin and octreotide are used.
The figures attached show displacement curves of [125I]-VIP binding to tissue sections from four different tumours: A = pancreatic adenocarcinoma, B = colonic adenocarcinoma, C = endometrial carcinoma and D = prostatic carcinoma. Tissue sections are incubated with 14,000 cpm/lOOμl [125I]-VIP and increasing concentrations of unlabelled PACAP-27 (*), GRF (■) , somastotatin (Δ) or octreotide (o) . Each point represents the optical density of binding measured in the tumour area. Non-specific binding is substracted from all values. In all cases, complete displacement of the ligand is achieved by PACAP, whereas GRF, somastotatin and octreotide are inactive in the nanomolar range. Example 2.
Test of the affinity of PACAP(1-27) to PACAP-I and -II receptors in different human tumours
Receptor autoradiography and peptide labelling is performed as described in Example 1.
Tyr10 labelled, Tyr13 labelled and Tyr22 labelled Ac- His1-PACAP are separated by HPLC, using a reverse phase RC18 column and butane-sulphonic acid as the eluent. The mono-125iodo-Ac-His1-PACAP's are each eluted as single peaks from the HPLC and analysed by mass-spectrometry. Specific activity: 2000 Ci/mmol. The first peak is used for binding experiments, and assumed to be the the mono- 125iodo- [Tyr10]-Ac-His1-PACAP. The tissues are cut on a cryostat, mounted on microscope slides, and then stored at -20°C for at least 3 days to improve adhesion of the tissue to the slide. The slide-mounted tissue sections are allowed to reach room temperature and are incubated for 90 min in a solution of 50 mM Tris-HCl, pH 7.4, containing BSA (2%), EGTA (2 mM) , bacitracin (0.1 mM) , MgCl2 (5 mM) , and 30 pM [125I]-Ac-His1-PACAP, at room temperature, as described by Dietl et al. (Brain Res. 1990, 520, 14-26). To estimate non-specific binding, paired serial sections are incubated as described above, except that lμM PACAP-27 is added to the incubation medium. After the incubation, the slides are rinsed with four washes of 30 sec each in ice- cold 50 mM Tris-HCl, pH 7.4, dipped in ice-cold water, and then quickly dried in a refrigerator under a stream of cold air. The sections are subsequently exposed to a
3H-Ultrofilm for 1 week, to detect the precise location of the radioactivity.
In all tumours, displacement experiments using successive sections of a tumour are performed with increasing concentrations of various biologically active or inactive peptides (see the above-mentioned publication by Reubi et al.) . In comparison with PACAP, VIP as well as somastotatin are used.
The figures attached show displacement curves of [125I]-Ac-His1-PACAP binding to tissue sections from two different tumours: E = Astrocytoma (PACAP-I receptor) and F = Prostatic carcinoma (PACAP-II receptor) . Tissue sections are incubated with 14,000 cpm/lOOμl [125I]-Ac- His^PACAP and increasing concentrations of unlabelled PACAP-27 (♦), VIP (■) or somastotatin (Δ) . Each point represents the optical density of binding measured in the tumour area. Non-specific binding is substracted from all values. In all cases, complete displacement of the ligand is achieved by PACAP, whereas VIP is active in Prostatic carcinoma and inactive in Astrocytoma and somastotatin is inactive in the nanomolar range.
Example 3
Synthesis of N-DTPA modified PACAP compounds
A. General method for the synthesis of DTPA-PACAP derivatives by solid phase synthesis
Solid phase peptide synthesis (SPPS) is carried out on an Applied Biosysterns Model 431 A or 432 A Peptide synthesizer using F oc (9-fluorenemethoxycarbonyl) strategy. The general principles and methods followed are well known in the art. For a description of the method see, "Fluorenemethoxycarbony-polyamide solid phase synthesis-General Synthesis and Development" - Chapter 3 in "Solid peptide synthesis- A practical approach" by E. Atherton and R.C. Sheppard, Information Press Ltd., Oxford, England (1989) .
In the following examples, 9-fluorenemethoxycarbonyl (Fmoc) amino terminus protected amino acids are used. All the standard Fmoc-protected amino acids are purchased commercially. Coupling with dicyclohexylcar- bodiimide/hydroxybenzotriazole using either p-hydroxy- methylphenoxy ethylpolystyrene for carboxyl terminus acids or Rink amide resin is used for carboxyl terminus amides. When the synthesis is completed, the products are routinely cleaved using a solution comprised of trifluoroacetic acid:water:anisole:triisopropylsilane or trifluoracetic acid:ethanedithiol:thioanisole:water for 1-8 hours at room temperature. The products are precipitated by ether and purified by C-18 reverse phase chromatography.
For the incorporation of DTPA (diethylenetriamine pentaacetic acid) the N-terminal Fmoc-protecting group is removed in the synthesizer using the standard protocol of the synthesizer. Next 3-4 molar equivalents tri-t-butyl diethylenetriaminepentacetic acid is used for the condensation to the N-terminal. Cleavage and deprotection are carried out as outlined above.
B. Synthesis of DTPA-PACAP ( 6-38 )
Figure imgf000024_0001
Lys-Gta-Met-Ala-V -Lys-Lys-Tyr-Leu-Ak-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-
Gln-Arg-Val-Lys-Asp-Lys 38-NH2
The solid phase synthesis is carried out using commercially available Rink amide resin on 250 μmol scale. When Phe-6-condensation is completed, removal of the Fmoc-group, activation of tri-butyl-DTPA to the anhydride and coupling to the N-terminal Phe are completed in the SPPS in the synthesizer. To prevent any side reaction, final piperidine wash is deleted from the synthesis protocol.
Cleavage and removal of the protecting groups from the solid phase is accomplished by using trifluoroacetic acid:ethanedithiol:thioanisole: water for 8 hours at room temperature. The product is isolated by preparative C-18 reverse phase chromatography. Molecular weight: Calculated: 4400.4 +0.5 Found: 2201.5 ((M+2)/2) .
C. Synthesis of DTPA-PACAP(1-27)-piperidine amide
Figure imgf000024_0002
Ser-Arg-Tyr-Arg-Lys-Gto-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH The solid phase synthesis is carried out using commercially available Rink amide resin on 250 umol scale. When His-1-condensation is completed, removal of the Fmoc-group, activation of tri-t-butyl-DTPA to the anhydride and coupling to the N-terminal Phe are completed in the SPPS in the synthesizer. After the coupling of tri-t-butyl DPTA, treatment with piperidine is used to form the amide.
Cleavage and removal of the protecting groups from the solid phase is accomplished by using trifluoroacetic acid: ethanedithiol:thioanisole: water for 8 hours at room temperature. The product is isolated by preparative C-18 reverse phase chromatography. Molecular weight: Calulated:3589.9±0.5 Found:1795.9 ((M+2)/2).
Example 4
A. Preparation of a suitable N-DTPA-PACAP(6-36) formulation and labelling with In-111 chloride
Starting material is 0.5 ml matrix solution containing 0.8 mg citric acid monohydrate, 11.2 mg sodium citrate trihydrate, 20 mg inositol and 4 mg gentisic acid/ml. To this solution 10 μl of N-DTPA-PACAP(6-36) solution (1 mg/ml in water) is added. Next 0.5 ml Indium-Ill chloride (111 Mbq/ l) is added.
After incubation at room temperature for 30 minutes the labelling yield is determined by instant thin layer chromatography using citrate lM/pH5 as eluent. The radiochemical purity is determined by RP-HPLC using a C18/10μ column and a linear gradient 5% acetonitrile incl. 0.05% TFA-95% acetronitrile incl. 0.05% TFA in 30 minutes.
The labelling yield is > 98%. The radiochemical purity is 74% after 20 hours. B. Preparation of a suitable N-DTPA-PACAP(1-27)- piperidine amide formulation and labelling with In-111 chloride
In the same way as described* above N-DTPA-PACPA(l-27) - piperidine amide is labelled.
Example 5
Test of the affinity of N-DTPA-PACAP(1-27)-piperidine amide to PACAP-II receptors in human tumours
The affinity of N-DTPA-PACAP(1-27)-piperidine amide towards PACAP receptors in gut carcinoid tumour and prostate tumour is determined as described in Example 1. The figures G attached shows the mean of the displacement curves of [125I]-VIP binding to tissue sections from two different tumours: Gut carcinoid tumour and prostate tumour. Tissue sections are incubated with 14,000 cpm/lOOμl [125I]-VIP and increasing concentrations of unlabelled N-DTPA-PACAP(1-27) -piperidine amide (■) and VIP (•). Each point represents the optical density of binding measured in the tumour area. Non-specific binding is substracted from all values. In both cases, complete displacement of the ligand is achieved by N-DTPA-PACAP(1- 27)-piperidine amide at a concentration of a factor of about 50 higher than VIP. The specific binding of N-DTPA- PACAP(1-27)-piperidine shows that this compound or analogs thereof, after metal labelling, are promising candidates to visualize PACAP-R positive tumours in vivo. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Mallinckrodt Medical, Inc.
(B) STREET: 675 McDonnell Blvd
(C) CITY: St. Louis (D) STATE: Missouri
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP) : 63134
(G) TELEPHONE: (314)8952414 (H) TELEFAX: (314)8952156
(ii) TITLE OF INVENTION: Method for the detection and localization of malignant human tumours
(iii) NUMBER OF SEQUENCES: 9
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)
( 2 ) INFORMATION FOR SEQ ID NO : 1 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 38 amino acids ( B ) TYPE : amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:1..38
(D) OTHER INFORMATION: /product= "OTHER" /note= "The peptide is labelled with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:38
(D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa is Lys-NH2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
His Ser Asp Gly lie Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin 1 5 10 15
Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys 20 25 30
Gin Arg Val Lys Asn Xaa 35
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATIO :1..27
(D) OTHER INFORMATION: /product= "OTHER" /note= "The peptide is labelld with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:27
(D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa is Leu-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
His Ser Asp Gly lie Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin
1 5 10 15
Met Ala Val Lys Lys Tyr Leu Ala Ala Val Xaa 20 25 INFORMATION FOR SEQ ID NO : 3 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 27 amino acids (B) TYPE : amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:1..27
(D) OTHER INFORMATION: /product= "OTHER" /note= "The peptide is labelled with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:1
(D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa is Ac-His"
(ix) FEATURE: (A) NAME/KEY: Modified-site
(B) LOCATION:27
(D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa is Leu-NH2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Xaa Ser Asp Gly lie Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin 1 10 15
Met Ala Val Lys Lys Tyr Leu Ala Ala Val Xaa 20 25
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE: (A) NAME/KEY: Modified-site
(B) LOCATION:1..26 (D) OTHER INFORMATION: /product= "OTHER"
/note= "The peptide is labelled with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:26
(D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa is Leu-NH2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Ser Asp Gly lie Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin Met 1 5 10 15
Ala Val Lys Lys Tyr Leu Ala Ala Val Xaa 20 25
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:1..37
(D) OTHER INFORMATION: /product= "OTHER"
/note= "The peptide is labelled with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:37
(D) OTHER INFORMATION: /product^ "OTHER" /note= "Xaa is Lys-NH2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Ser Asp Gly lie Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin Met 1 5 10 15
Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys Gin 20 25 30
Arg Val Lys Asn Xaa 35
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:1..38
(D) OTHER INFORMATION: /product^ "OTHER" /note= "The peptide is labelled with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:17 (D) OTHER INFORMATION : /product= "Nle "
( ix) FEATURE :
(A) NAME/KEY: Modified-site (B) LOCATION:38
(D) OTHER INFORMATION:/product= "OTHER" /note= "Xaa is Lys-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
His Ser Asp Gly lie Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin 1 5 10 15
Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys 20 25 30
Gin Arg Val Lys Asn Xaa 35
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE: (A) NAME/KEY: Modified-site
(B) LOCATION:1..33
(D) OTHER INFORMATION: /product= "OTHER"
/note= "The peptide is labelled with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:33 (D) OTHER INFORMATION: /product= "OTHER"
/note= "Xaa is Lys-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin Met Ala Val Lys Lys 1 5 10 15
Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys Gin Arg Val Lys Asn 20 25 30
Xaa
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:1..27
(D) OTHER INFORMATION:/product= "OTHER"
/note= "The peptide is labelled with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:27
(D) OTHER INFORMATION:/product= "OTHER" /note= "Xaa is Leu-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
His Ala Asp Gly lie Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin 1 5 10 15
Met Ala Val Lys Lys Tyr Leu Ala Ala Val Xaa 20 25
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide ( iii ) HYPOTHETICAL : NO
( iv) ANTI-SENSE : NO
( ix) FEATURE :
(A) NAME/KEY: Modified-site
(B) LOCATION:1..38
(D) OTHER INFORMATION: /product= "OTHER" /note= "The peptide is labelled with a radionuclide or with a paramagnetic metal isotope"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATIONS
(D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa is p-Cl-D-Phe"
(ix) FEATURE: (A) NAME/KEY: Modified-site
(B) LOCATION:38
(D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa is Lys-NH2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
His Xaa Asp Gly lie Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gin 1 5 10 15
Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys
20 25 30
Gin Arg Val Lys Asn Xaa 35

Claims

Claims
1. A method of detecting and localizing malignant tumours and their metastases in the body of a human being, which comprises (i) administering to said being a composition comprising, in a quantity sufficient for external imaging, a peptide selected from the group consisting of pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP-receptor agonists, PACAP- receptor antagonists, PACAP analogues and PACAP derivatives, said peptide being labelled with a radioactive metal isotope selected from the group consisting of "Tc, 203Pb, 66Ga, 67Ga, 68Ga, 72As, mIn, 113mIn, 97Ru, 62Cu, "Cu, 52Fe, 52πMn and 51Cr, or with a radioactive halogen isotope1, selected from 123I, 131I, 75Br, 76Br, 77Br and 82Br, and thereupon (ii) subjecting said being to external imaging by radioactive scanning to determine the targeted sites in the body of said being in relation to the background activity, in order to allow detection and localization of said tumours in the body.
2. A method of intraoperatively detecting and localizing malignant tumours in the body of a human being, which comprises (i) administering to said being a composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a peptide selected from the group consisting of pituitary adenylate cyclase- activating polypeptide (PACAP) , PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, said peptide being labelled with 161Tb, 123I or 125I, and thereupon (ii) , after allowing the active substance to be bound and taken up in said tumours and after blood clearance of radioactivity, subjecting said being to a radioimmunodetection technique in the relevant area of the body of said being, by using a gamma detecting probe .
3. A method for the therapeutic treatment of malignant tumours in the body of a human being, which comprises administering to said being a composition comprising, in a quantity effective for combating or controling tumours, a peptide selected from the group consisting of pituitary adenylate cyclase-activating polypeptide (PACAP) , PACAP- receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, said peptide being labelled with an isotope selected from the group consisting of 1B6Re, 188Re, 77As, 11 mIn, 90Y, 67Cu, 169Er, 117mSn, 121Sn, 127Te, 14Pr, 143Pr, 198Au, 199Au, 149Tb, 161Tb, 109Pd, 165Dy, 149Pm, 151Pm, 153Sm, 157Gd, 159Gd, 166Ho, 172Tm, 169Yb, 175Yb, 177Lu, 105Rh, ulAg, 124I and 131I.
4. A method as claimed in any one of the preceding claims, characterized in that the tumours and the metastasis thereof to be detected, localized or therapeutically treated are selected from the group consisting of Astrocytomas, Glioblastomas, Endometrial tumours, Ovarian tumours, Hemangiopericytomas and Pituitary adenomas.
5. A method as claimed in any one of the preceding claims, which comprises administering to said human being a composition comprising a labelled peptide, derived from a compound of the general formula
H - (((Xaa)p - Xbb)q - Xcc - Xdd - Ile)r - Xee - Thr - Asp - Xff - Xgg 1 5 10
- Ser- Xhh - Xii - Arg - Lys - Gin - Xjj - Ala - Val - Lys - Lys - Xkk
15 20
- Leu - Ala - Ala - Val - Leu - (Xll)n - R2 (I) 25 or an acid amide thereof, formed between a free NH2-group of an amino acid moiety and RiCOOH, wherein
Rx is a (Ci-Cjjalkanoyl group, an arylcarbonyl group, or an aryl- (C1-C3)alkanoyl group; or a lactam thereof, formed between a free NH2 group of an amino acid moiety and a free C02H group of another amino acid moiety; or a conjugate thereof with avidin or biotin; wherein: Xaa is His or Phe;
Xbb is Ser, Ala, Arg, Phe or p-Cl-Phe;
Xcc is Asp or Glu;
Xdd is Gly or an aminoisobutyric acid moiety;
Xee, Xgg, Xii and Xkk are each individually Phe, Tyr or Trp;
Xff is Ser or Asn
Xhh is Arg or Lys
Xjj is Met or a norleucine moiety;
(Xll)n comprises 0 to 12 amino acid moieties which are equal or different and are selected from Leu, Asn,
Gin, Val, Asp, Lys, Gly, Arg, Tyr, Trp, Phe, Ser, lie,
Thr or Pro; p, q and r are each individually 0 or 1; and
R2 is a hydroxy group, an acetoxy group or an amino group.
6. A method as claimed in claim 5, which comprises administering to said human being a composition comprising a labelled peptide derived from a compound of the general formula I, as defined in claim 5, wherein q is 1, (Xll)n comnprises 10 to 12 amino acid moieties selected as defined in claim 5, and the other symbols have the meanings as defined in claim 5.
7. A method as claimed in 5 which comprises administering to said human being a composition comprising a labelled peptide as defined in said preceding claims, wherein said peptide is labelled with a radioactive halogen isotope selected from the group consisting of 123I, 124I, 125I, 131I, 75Br, 76Br, 77Br and 82Br, said radioactive halogen isotope being attached to a Tyr or Trp moiety of the peptide, or to the aryl group of substituent
8 . A method as claimed in claims 1 , 2 or 3 which comprises administering to said human being a composition comprising a labelled peptide as defined in said preceding claims , wherein said peptide is labelled with a metal atom selected from the group consisting of the radioactive isotopes 99mTc , 203Pb, 66Ga, 67Ga , 68Ga , 72As , ιnIn, 113mIn,
114mIn, 97Ru, 62Cu, 64Cu, 52Fe , 52mMn, 51Cr , 186Re , 188Re , 77As , 90Y, 67Cu, 169Er, 117mSn, 121Sn, 127Te , 142Pr, 143Pr, 198Au, 199Au , 149Tb, 161Tb, 109Pd, 165Dy, 149Pm, 151Pm, 153Sm, 157Gd, 166Ho , 17 Tm, 169Yb, 175Yb, 177Lu, 105Rh and ιnAg ; said metal atom being attached to the peptide by means of a chelating group chelating said atom, which chelating group is bound by an amide bond or through a spacing group to the peptide molecule.
9. A method as claimed in claim 8, wherein said composition comprises a peptide labelled with a metal atom, chelated by an NtS,4.t) tetradentate chelating agent, wherein t=2-4, or by a chelating group derived from ethy- lene diamine tetra-acetic acid (EDTA) , diethylene tria ine penta-acetic acid (DTPA), cyclohexyl 1,2-diamine tetra- acetic acid (CDTA), ethyleneglycol-O, 0' -bis(2-aminoethyl) - N,N,N' ,N'-tetra-acetic acid (EGTA) , N,N-bis (hydroxybenz- yl)-ethylenediamine-N,N'-diacetic acid (HBED) , triethylene tetramine hexa-acetic acid (TTHA) , 1,4,7,10- tetraazacyclododecane-N,N' ,N' ' ,N' ' '-tetra-acetic acid (DO- TA) , hydroxyethyldiamine triacetic acid (HEDTA), 1,4,8,11- tetra-azacyclotetradecane-N,N' ,N' ' ,N' ' '-tetra-acetic acid (TETA) , substituted DTPA, substituted EDTA, or from a compound of the general formula
Figure imgf000043_0001
wherein R is a branched or non-branched, optionally substituted hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N, O and S and/or by one or more NH groups, and Q is a group which is capable of reacting with an amino group of the peptide and which is preferably selected from the group consisting of carbonyl, carbimidoyl, N- (Ci-CsJalkylcarbimidoyl, N-hydroxy- carbimidoyl and N- (Cα-C6)alkoxycarbimidoyl; and wherein said optionally present spacing group is a biotinyl moiety or has the general formula
O J v
—NH-R3-C— ∞ —CH2—(' N)—JNH-X— (III) (IV)
wherein R3 is a C^C^ alkylene group, a CJ-CK, alkylidene group or a C2-C10 alkenylene group, and X is a thiocarbo- nyl group or a group of the general formula O NH
II II
—C—CH2-S—(CH2)m-C— (V) wherein m is 1-5.
10. The use of a labelled peptide as defined in either claim 1 or claim 2 for preparing a diagnostic composition for detecting and localizing malignant human tumours in the body of a human being.
11. The use of a labelled peptide as defined in claim 3 for preparing a pharmaceutical composition for the therapeutic treatment of malignant human tumours in the body of a human being.
12. A pharmaceutical composition comprising as the active substance a labelled peptide as defined in claim 1, 2 or 3 and a pharmaceutically acceptable carrier material and, optionally, at least one pharmaceutically acceptable adjuvant.
13. A composition as claimed in claim 12, comprising as the active substance a labelled peptide derived from a compound of the general formula I, presented in claim 5, wherein the symbols have the meanings given in claim 5.
14. A labelled peptide to be used as an active ingredient in a composition as claimed in claim 12, said peptide being labelled with a metal atom as defined in claim 8.
15. A labelled peptide to be used as an active ingredient in a composition as claimed in claim 13 , said peptide being labelled with a metal atom as defined in claim 8.
16. A labelled peptide as claimed in claim 14, said peptide being derived from a compound of the general formula I, presented in claim 5, wherein the symbols have the meanings given in claim 5.
17. A labelled peptide as claimed in claim 15, said peptide being derived from a compound of the general formula I, presented in claim 5, wherein the symbols have the meanings given in claim 5.
18. A method of preparing a metal atom - labelled peptide as claimed in claim 14, characterized in that a derivatized peptide, comprising a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP), PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, derivatized with a chelating group bound by an amide bond or through a spacing group to the peptide molecule, is reacted with a metal atom as defined in claim 8 in the form of a salt or of a chelate, bound to a comparatively weak chelator, in order to form a complex.
19. A method of preparing a metal atom - labelled peptide as claimed in claim 15, characterized in that a derivatized peptide, comprising a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP), PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, derivatized with a chelating group bound by an amide bond or through a spacing group to the peptide molecule, is reacted with a metal atom as defined in claim 8 in the form of a salt or of a chelate, bound to a comparatively weak chelator, in order to form a complex.
20. A derivatized peptide as defined in claim 18, comprising a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide
(PACAP), PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, derivatized with a chelating group bound by an amide bond or through a spacing group to the peptide molecule.
21. A derivatized peptide as defined in claim 19, comprising a peptide selected from the group consisting of pituitary adenylate cyclase - activating polypeptide (PACAP) , PACAP-receptor agonists, PACAP-receptor antagonists, PACAP analogues and PACAP derivatives, derivatized with a chelating group bound by an amide bond or through a spacing group to the peptide molecule.
22. A kit for preparing a radiopharmaceutical composition, comprising (i) a derivatized peptide as claimed in claim 20, to which derivatized peptide, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (ii) a solution of a salt or chelate of a metal selected from the group consisting of the radioactive isotopes 203Pb, 66Ga, 6Ga, 68Ga, 72As, luIn, 113mIn, 114mIn, 97Ru, 62Cu, 6Cu, 99πTc, 186Re, 188Re, 52Fe, 52mMn, 51Cr, 77As, 90Y, 67Cu, 169Er, 11 mSn, 121Sn, 12Te, 12Pr, 13Pr, 198Au, 199Au, 161Tb, 109Pd, 165Dy, 149Pm, 151Pm, 153Sm, 157Gd, 166Ho, 172Tm, 169Yb, 175Yb, 177Lu, 105Rh and luAg, and (iii) instructions for use with a prescription for reacting the ingredients present in the kit.
23. A kit for preparing a radiopharmaceutical composition, comprising (i) a derivatized peptide as claimed in claim 21, to which derivatized peptide, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (ii) a solution of a salt or chelate of a metal selected from the group consisting of the radioactive isotopes 203Pb, 66Ga, 67Ga, 68Ga, 72As, nlIn, 113mIn, 11 mIn, 97Ru, 62Cu, 64Cu, 99mTc, 186Re, 188Re, 52Fe, 52πMn, 51Cr, 77As, 90Y, 67Cu, 169Er, 117mSn, 121Sn, 127Te, 12Pr, 13Pr, 198Au, 199Au, 161Tb, 109Pd, 165Dy, 19Pm, 151Pm, 153Sm, 157Gd, 166Ho, 172Tm, 169Yb, 175Yb, 177Lu, 105Rh and ιnAg, and (iii) instructions for use with a prescription for reacting the ingredients present in the kit.
24. A kit for preparing a radiopharmaceutical composition, comprising (i) a derivatized peptide as claimed in claim 20, to which derivatized peptide, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (ii) a reducing agent, and, if desired, a chelator, said ingredients (i) and (ii) optionally being combined, and (iii) instructions for use with a prescription for reacting the ingredients of the kit with 99mTc in the form of a pertechnetate solution or with 186Re or 188Re in the form of a perrhenate solution.
25. A kit for preparing a radiopharmaceutical composition, comprising (i) a derivatized peptide as claimed in claim 21, to which derivatized peptide, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (ii) a reducing agent, and, if desired, a chelator, said ingredients (i) and (ii) optionally being combined, and (iii) instructions for use with a prescription for reacting the ingredients of the kit with 99ιnTc in the form of a pertechnetate solution or with 186Re or 188Re in the form of a perrhenate solution.
26. A method of detecting and localizing malignant tumours and their metastases in the body of a human being, which comprises (i) administering to said being a composition comprising, in a quantity sufficient for external imaging, a peptide selected from the group consisting of pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP-receptor agonists, PACAP- receptor antagonists, PACAP analogues and PACAP derivatives, said peptide being labelled with a paramagnetic metal atom selected from the group consisting of Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er, and thereupon (ii) subjecting said being to external imaging by magnetic resonance imaging, to determine the targeted sites in the body of said being in relation to the background activity, in order to allow detection and localization of said tumours in the body.
27. A method as claimed in claim 26, characterized in that the tumours and the metastasis thereof to be detected or localized are selected from the group consisting of Astrocytomas, Glioblastomas, Endometrial tumours, Ovarian tumours, Hemangiopericytomas and Pituitary adenomas.
28. A method as claimed in claim 27, which comprises administering to said human being a composition comprising a labelled peptide, derived from a compound of the general formula
H - (((Xaa)p - Xbb)q - Xcc - Xdd - Ile)r - Xee - Thr - Asp - Xff - Xgg - 1 5 10
- Ser- Xhh - Xii - Arg - Lys - Gin - Xjj - Ala - Val - Lys - Lys - Xkk
15 20
- Leu - Ala - Ala - Val - Leu - (Xll)n - R- (I)
25 or an acid amide thereof, formed between a free NH2-group of an amino acid moiety and RiCOOH, wherein
R2 is a (C1-C3)alkanoyl group, an arylcarbonyl group, or an aryl-(Ci-CaJalkanoyl group; or a lactam thereof, formed between a free NH2 group of an amino acid moiety and a free C02H group of another amino acid moiety; or a conjugate thereof with avidin or biotin; wherein: Xaa is His or Phe;
Xbb is Ser, Ala, Arg, Phe or p-Cl-Phe;
Xcc is Asp or Glu;
Xdd is Gly or an aminoisobutyric acid moiety;
Xee, Xgg, Xii and Xkk are each individually Phe, Tyr or Trp;
Xff is Ser or Asn
Xhh is Arg or Lys
Xjj is Met or a norleucine moiety;
(Xll)n comprises 0 to 12 amino acid moieties which are equal or different and are selected from Leu, Asn,
Gin, Val, Asp, Lys, Gly, Arg, Tyr, Trp, Phe, Ser, lie,
Thr or Pro; p, q and r are each individually 0 or 1; and
R2 is a hydroxy group, an acetoxy group or an amino group.
29. A method as claimed in claim 28, which comprises administering to said human being a composition comprising a labelled peptide derived from a compound of the general formula I, as defined in claim 28, wherein q is 1, (Xll)n comnprises 10 to 12 amino acid moieties selected as defined in claim 28, and the other symbols have the meanings as defined in claim 28.
30. A method as claimed in claim 26 which comprises administering to said human being a composition comprising a labelled peptide as defined in said preceding claims, wherein said peptide is labelled with a metal atom selected from the group consisting of the paramagnetic metal atoms Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er; said metal atom being attached to the peptide by means of a chelating group chelating said atom, which chelating group is bound by an amide bond or through a spacing group to the peptide molecule.
31. The use of a labelled peptide as defined in claim 26 for preparing a diagnostic composition for detecting and localizing malignant human tumours in the body of a human being.
32. A pharmaceutical composition comprising as the active substance a labelled peptide as defined in claim 26 and a pharmaceutically acceptable carrier material and, optionally, at least one pharmaceutically acceptable adjuvant.
PCT/US1996/001291 1995-02-03 1996-02-02 Method for the detection and localization of malignant human tumours WO1996023527A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP95200263.2 1995-02-03
EP95200263 1995-02-03

Publications (2)

Publication Number Publication Date
WO1996023527A1 true WO1996023527A1 (en) 1996-08-08
WO1996023527A9 WO1996023527A9 (en) 1996-10-03

Family

ID=8219992

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/001291 WO1996023527A1 (en) 1995-02-03 1996-02-02 Method for the detection and localization of malignant human tumours

Country Status (1)

Country Link
WO (1) WO1996023527A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998033531A1 (en) * 1997-02-03 1998-08-06 Mallinckrodt Medical, Inc. Method for the detection and localization of malignant human tumours
WO1998056428A1 (en) * 1997-06-13 1998-12-17 University Of New Mexico Arsenic-72 labelled compounds for tissue specific medical imaging
US6630570B1 (en) * 1999-04-09 2003-10-07 Insitut für Diagnostikforschung GmbH Short-chain peptide-dye conjugates as contrast media for optical diagnosis
EP1154798B1 (en) * 1999-02-24 2006-05-10 Universität Zürich Molecules for the treatment and diagnosis of tumors
US7175953B2 (en) 1999-04-09 2007-02-13 Institute Fuer Diagnostik Forschung Short-warp peptide-dye conjugate as contrast agent for optical diagnostic
EP2110142A3 (en) * 1999-09-23 2013-02-27 Syntaxin Limited Inhibition of secretion from non-neuronal cells

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5217953A (en) * 1990-11-30 1993-06-08 The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services Vasoactive intestinal peptide antagonist
US5382654A (en) * 1992-02-05 1995-01-17 Mallinckrodt Medical, Inc. Radiolabelled peptide compounds
US5443816A (en) * 1990-08-08 1995-08-22 Rhomed Incorporated Peptide-metal ion pharmaceutical preparation and method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5443816A (en) * 1990-08-08 1995-08-22 Rhomed Incorporated Peptide-metal ion pharmaceutical preparation and method
US5217953A (en) * 1990-11-30 1993-06-08 The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services Vasoactive intestinal peptide antagonist
US5382654A (en) * 1992-02-05 1995-01-17 Mallinckrodt Medical, Inc. Radiolabelled peptide compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EUROPEAN JOURNAL OF BIOCHEMISTRY, Volume 204, issued 1992, PAULSSEN et al., "The Thyroliberin Receptor Interacts Directly With a Stimulatory Guanine-Nucleotide-Binding Protein in the Activation of Adenylyl Cyclase in GH3 Rat Pituitary Tumour Cells", pages 413-418. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998033531A1 (en) * 1997-02-03 1998-08-06 Mallinckrodt Medical, Inc. Method for the detection and localization of malignant human tumours
WO1998056428A1 (en) * 1997-06-13 1998-12-17 University Of New Mexico Arsenic-72 labelled compounds for tissue specific medical imaging
US5914096A (en) * 1997-06-13 1999-06-22 University Of New Mexico Arsenic-72 labeled compounds for tissue specific medical imaging
US6106804A (en) * 1997-06-13 2000-08-22 The University Of New Mexico Arsenic-72 labeled compounds for tissue specific medical imaging
EP1154798B1 (en) * 1999-02-24 2006-05-10 Universität Zürich Molecules for the treatment and diagnosis of tumors
US6630570B1 (en) * 1999-04-09 2003-10-07 Insitut für Diagnostikforschung GmbH Short-chain peptide-dye conjugates as contrast media for optical diagnosis
US7175953B2 (en) 1999-04-09 2007-02-13 Institute Fuer Diagnostik Forschung Short-warp peptide-dye conjugate as contrast agent for optical diagnostic
EP2110142A3 (en) * 1999-09-23 2013-02-27 Syntaxin Limited Inhibition of secretion from non-neuronal cells

Similar Documents

Publication Publication Date Title
EP0968001B1 (en) Method for the detection and localization of malignant human pancreatic tumours
JP3601827B2 (en) Somatostatin derivatives and their radiolabels
US5981477A (en) Peptide-metal chelate conjugates
EP0720621B1 (en) Radiolabeled somatostatin-derived peptides for imaging and therapeutic uses
US6866837B2 (en) Radiolabeled peptides for the diagnosis and treatment of breast and prostate tumors and metastases of such tumors
US6194386B1 (en) Labelled peptide compounds
US7147838B2 (en) Gastrin receptor-avid peptide conjugates
WO1998033531A9 (en) Method for the detection and localization of malignant human tumours
PL192734B1 (en) Radiotracer-labelled peptides for use in diagnostics and therapy
US7060247B2 (en) Gastrin receptor-avid peptide conjugates
JP2001523257A (en) Calcitonin receptor binding peptide
WO1996023527A1 (en) Method for the detection and localization of malignant human tumours
WO1996023527A9 (en) Method for the detection and localization of malignant human tumours
JP4295921B2 (en) Radiolabeled peptides for diagnosis and treatment of breast and prostate cancer and metastasis of such tumors
US20040185510A1 (en) Use of labelled CCK-B receptor ligands for the detection and localization of malignant human tumours
EP1208852B1 (en) Neuropeptide Y1 receptor binding compounds in the treatment and diagnosis of cancer
US5952464A (en) Labelled peptide compounds
US20050226813A1 (en) Labelled somatostatin analogs backbone cyclized through metal complexation
CZ279099A3 (en) Detection and localization method of human malignant tumors
Karacay et al. co), United States (), Reissued Patent
Storch et al. Evaluation of [^ sup 99m^ Tc/EDDA/HYNIC^ sup 0^] Octreotide Derivatives Compared with [^ sup 111^ In-DOTA^ sup 0^, Tyr^ sup 3^, Thr^ sup 8^] Octreotide and [^ sup 111^ In-DTPA^ sup 0^] Octreotide: Does Tumor or Pancreas Uptake Correlate with the Rate of Internalization?

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

COP Corrected version of pamphlet

Free format text: PAGES 1/2-2/2,DRAWINGS,REPLACED BY NEW PAGES BEARING THE SAME NUMBER;DUE TO LATE TRANSMITTAL BY THERECEIVING OFFICE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载