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WO1996023527A1 - Procede de detection et de localisation des tumeurs malignes humaines - Google Patents

Procede de detection et de localisation des tumeurs malignes humaines Download PDF

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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
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Prior art keywords
peptide
group
pacap
labelled
lys
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PCT/US1996/001291
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English (en)
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WO1996023527A9 (fr
Inventor
Jean-Claude Reubi
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Mallinckrodt Medical, Inc.
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Publication of WO1996023527A1 publication Critical patent/WO1996023527A1/fr
Publication of WO1996023527A9 publication Critical patent/WO1996023527A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • 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.

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Abstract

La présente invention concerne un procédé permettant de détecter et de localiser, dans le corps d'un être humain, des tumeurs malignes humaines, y compris leurs métastases. Ce procédé consiste d'abord (i) à administrer à l'être humain considéré une composition comprenant, en quantité suffisant à l'imagerie externe, un peptide marqué appartenant au groupe constitué par le PACAP (Pituitary Adenylate Cyclase - Activating Polypeptide), c'est-à-dire le polypeptide activant l'adénylate cyclase hypophysaire, les agonistes des récepteurs du PACAP, les antagonistes des récepteurs du PACAP, les analogues des PACAP et les dérivés des PACAP. Le procédé consiste ensuite (ii) à soumettre l'être humain considéré à une imagerie externe, telle qu'une scintigraphie ou une IRM, pour déterminer les sites ciblés dans son corps. L'invention concerne en outre un procédé de traitement thérapeutique de ces tumeurs malignes humaines consistant en l'administration du peptide de la présente invention, marqué à cette fin. L'invention concerne également un procédé de marquage des composés du peptide, une composition pharmaceutique à utiliser pour la détection, une composition pharmaceutique à utiliser pour la thérapie et une trousse de préparation d'une composition radiopharmaceutique.
PCT/US1996/001291 1995-02-03 1996-02-02 Procede de detection et de localisation des tumeurs malignes humaines WO1996023527A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998033531A1 (fr) * 1997-02-03 1998-08-06 Mallinckrodt Medical, Inc. Procede servant a detecter et a localiser des tumeurs malignes humaines
WO1998056428A1 (fr) * 1997-06-13 1998-12-17 University Of New Mexico Composes marques a l'arsenic 72 pour l'imagerie medicale specifique a des tissus
US6630570B1 (en) * 1999-04-09 2003-10-07 Insitut für Diagnostikforschung GmbH Short-chain peptide-dye conjugates as contrast media for optical diagnosis
EP1154798B1 (fr) * 1999-02-24 2006-05-10 Universität Zürich Molecules permettant de traiter et de diagnostiquer des tumeurs
US7175953B2 (en) 1999-04-09 2007-02-13 Institute Fuer Diagnostik Forschung Short-warp peptide-dye conjugate as contrast agent for optical diagnostic
EP2110142A3 (fr) * 1999-09-23 2013-02-27 Syntaxin Limited Inhibition de la sécrétion de cellules non neuronales

Citations (3)

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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

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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

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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 (fr) * 1997-02-03 1998-08-06 Mallinckrodt Medical, Inc. Procede servant a detecter et a localiser des tumeurs malignes humaines
WO1998056428A1 (fr) * 1997-06-13 1998-12-17 University Of New Mexico Composes marques a l'arsenic 72 pour l'imagerie medicale specifique a des tissus
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 (fr) * 1999-02-24 2006-05-10 Universität Zürich Molecules permettant de traiter et de diagnostiquer des tumeurs
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 (fr) * 1999-09-23 2013-02-27 Syntaxin Limited Inhibition de la sécrétion de cellules non neuronales

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