EP0726910A1 - Peptides capables de liaison avec les metaux et sans cysteine pour le diagnostic et le traitement, procedes pour leur preparation et compositions pharmaceutiques contenant ces composes - Google Patents

Peptides capables de liaison avec les metaux et sans cysteine pour le diagnostic et le traitement, procedes pour leur preparation et compositions pharmaceutiques contenant ces composes

Info

Publication number
EP0726910A1
EP0726910A1 EP95900063A EP95900063A EP0726910A1 EP 0726910 A1 EP0726910 A1 EP 0726910A1 EP 95900063 A EP95900063 A EP 95900063A EP 95900063 A EP95900063 A EP 95900063A EP 0726910 A1 EP0726910 A1 EP 0726910A1
Authority
EP
European Patent Office
Prior art keywords
gly
met
group
amino
compounds
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP95900063A
Other languages
German (de)
English (en)
Inventor
Jürgen CONRAD
Ludger Dinkelborg
Sebastian Erber
Cornelius FRÖMMEL
Wolfgang Höhne
Wolfgang Kramp
Gabriele KÜTTNER
Reinhard Malin
Hans Martin Schier
Jens Schneider-Mergener
Renate Steinbrecher
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Pharma AG
Original Assignee
Institut fuer Diagnostikforschung GmbH
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 Institut fuer Diagnostikforschung GmbH filed Critical Institut fuer Diagnostikforschung GmbH
Publication of EP0726910A1 publication Critical patent/EP0726910A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to metal-complexing, cysteine-free peptides, which are optionally coupled directly or via a linker to an organ-specific probe and thus accumulate specifically as a conjugate in tumors, organs, tissues or inflammation centers.
  • Antibodies or partial sequences of antibodies against tumor-associated antigens are used, for example, as organ-specific probes, such as, for. B. the Carcinoembryonic Antigen (CEA), which thus accumulate specifically in tumors.
  • CEA Carcinoembryonic Antigen
  • the invention further relates to methods for producing the metal-complexing cysteine-free peptides and their conjugates.
  • the present invention further relates to the use of the conjugates as components of a kit for in-vivo diagnosis or in-vivo therapy and radiopharmaceuticals which contain these conjugates together with radioisotopes.
  • the organ-specific conjugates are used for imaging tumors, organs or inflammation centers.
  • malignant cancer is the most common cause of death due to uncontrolled growth, since even if the primary tumor is successfully removed, the often very small metastases cannot be localized and therefore cannot be removed.
  • Therapy control after surgical interventions or when chemotherapeutic agents are administered includes imaging methods such as CT or MR on the one hand, and, in particular in the case of colorectal tumors, the determination of the content of tumor-associated antigens such as CEA in the serum of the patient.
  • a disadvantage of these imaging methods is the lack of differentiation between scar and local recurrence.
  • the disadvantage of determining the level of CEA in the serum, in addition to the lack of tumor localization, is the lack of sensitivity.
  • metastasis formation cannot be ruled out even with CEA values in the normal range, and on the other hand, non-malignant diseases such as inflammatory processes can also lead to increased CEA values.
  • radioisotopes in nuclear medicine diagnostics today are 1-131, 1-123, In-111 and Tc-99m, which are covalently bound (1-131, 1-123) or as a complex compound (In-111, Tc-99m ) are available.
  • 1-131, 1-123 and In-111 have various disadvantages.
  • 1-131 with a ⁇ -energy of 364 KeV and a half-life of 8 d emits ß-radiation, which is highly tissue-damaging.
  • the radioisotope of choice for diagnostics is Tc-99m, which with a short half-life of 6 h and a radiation energy of 140 KeV, which is favorable for imaging in vivo examinations, is available at any time and extremely inexpensively via a molybdenum generator in the form of pertechnetate and after reduction is present in an oxidation state suitable for complexing organ-specific substances.
  • the complexation of metals with organ-specific substances can take place directly or indirectly.
  • bifunctional chelates such as. B. DTPA derivatives are used, which after activation on the one hand covalently to organ-specific carriers such as Antibodies coupled, on the other hand, can be complexed with metals (Meares, Nucl. Med. Biol., 13, 311-318, 1986).
  • Substance is a complex protection group technique, and cleaning of the complex conjugate is also essential.
  • the protein derivatization of the organ-specific substance with a bifunctional chelating agent can lead to an impairment of the specific binding with the target tissue due to intermolecular interactions.
  • the in vivo localization of tumors with the aid of isotope-labeled murine antibodies against tumor-associated antigens is associated with various disadvantages.
  • a parameter for biodistribution is the molecular weight. Due to the high molecular weight (150 kDalton) there is an inhomogeneous distribution with slow blood clearance, especially in solid tumors.
  • HAMA reaction an allergic reaction due to the formation of human antibodies against murine antibodies
  • the allergic reaction is attributed to the constant antibody regions, so that a further development to reduce the Hama reaction is the production of chimeric antibodies with the variable, antigen-recognizing region of murine origin and the constant region of human origin
  • Increasing blood clearance can reduce the molecular weight by forming antibody fragments such as F (ab) 2 (100 kDalton) and F (ab) (50 kDalton) (Andrew et al., Eur. J. Nucl. Med., 12 , 168-172, 1986), where antibody fragments with respect to an isotope Marking have the disadvantages already mentioned.
  • a further reduction in molecular weight is achieved by the representation of "single chain fragments" (sFv).
  • Single-chain fragments have a molecular weight of 27 kDaltons and consist of the variable region of the light chain of the antibody, which is linked via a linker to the variable region of the heavy chain of the antibody (Bird et al., Science, 242, 423 -426, 1988).
  • Milenic et al. (Cancer Res., 51, 6363-6371, 1991) comparably described the affinity, specificity and biodistribution of an iodinated monoclonal antibody against the tumor-associated antigen TAG-72 with the corresponding F (ab) 2 -, F (ab) - and sFv fragments.
  • the sFv fragment Due to the low molecular weight, the sFv fragment shows pharmacokinetics suitable for imaging diagnostics. In contrast to the complete antibody, the lower affinity of the monovalent sFv fragment stands for the much faster one
  • Radioisotope could not be shown.
  • S-protected cysteine residues interrupted by an amino acid act as Tc-99m-binding peptides, which by coupling to organ-specific
  • Polypeptides should enable enrichment and localization.
  • the disadvantage of this method lies in the time-consuming and cost-intensive production of the Tc-binding peptides or the conjugates, since the labeling tion of cysteine-rich sequences requires complex protection group technology.
  • Nedelmann et al. J. Nucl. Med., 34, 234-241, 1993 describes in the animal model the imaging of infarcts with a "single chain fragment" directed against myosin, which is coupled to a Tc-99m-complexing bifunctional chelating agent. Due to the complex coupling of the chelating agent with the sFv fragment and the protective group technology required to avoid oxidation processes of the conjugate, inexpensive production and simple handling in clinical use are not possible.
  • R 1 -XR 2 (I) wherein X is a chain of up to 20 identical or different ⁇ -, ⁇ - and / or ⁇ -amino acid residues, this chain being at least one amino acid from the Group contains methionine, arginine, lysine and asparagine and no cysteine and at the N-terminus of which there is a free valence or a radical R 1 is substituted by a hydrogen atom and at whose C-terminus there is a free valence or a radical is substituted by a hydroxyl group R 2 is bound, wherein
  • R 1 represents a hydrogen atom, a branched or straight-chain alkyl, aryl, alkylaryl, aralkyl, alkylcarbonyl or arylcarbonyl group with up to 10 carbon atoms, which are each optionally substituted by a hydroxyl, amino or carboxy group,
  • R 2 is a hydroxyl group, a branched or straight-chain alkoxy or aryloxy group with up to 10 carbon atoms, which are optionally substituted with a hydroxyl, amino or carboxy group, an amino group, an N (R a R b ) group,
  • R a and R b are the same or different and represent branched alkyl or acyl radicals having up to 10 carbon atoms, which with
  • R 1 -XR 2 (I) are further characterized in that R 1 represents a single bond or a hydrogen atom or that R 2 denotes a single bond, a hydroxyl group or an amino group.
  • conjugates of peptides, proteins, bio- or macromolecules and substances binding to receptors with compounds of the general formula I are also provided.
  • Conjugates are preferred, the peptides, proteins, bio- or macromolecules as well as substances binding to receptors being characterized by the fact that the conjugates are present selectively in diseased tissue or in substances accumulating in tumors, one between them and the compounds of general formula I. there is a covalent bond and this is present in the case of substances containing amino groups such as peptides, proteins and antibodies or their fragments, amidically or in the case of substances containing hydroxyl groups such as alcohols, ester-like or in the case of substances containing aldehyde groups imidically.
  • These substances which accumulate selectively in diseased tissue are preferably hormones, neurohormones, neurotransmitters, growth factors, plant-based
  • Hormones pheromones, enzyme cofactors, enzyme substrates, drugs that bind specifically to receptors or oligonucleotides.
  • substances accumulating in diseased tissue are oxytocins, vasopressins, angiotensins, melanocyte-stimulating hormones, somatostatins, tyrotropin-releasing hormones, gona, dotropin-releasing hormones, testosterones, estradiols, progesterones, cortisols, aldosterone, aldosterone Secretins, somatropins, prostaglandins, neurotensins, insulins, glucagons, calcitonins, growth hormone-releasing hormones, prolactins, enkephalins, Endorphins, dopamine, norepinephrine, glutamate, serotonine, acetylcholine, epinephrine, interleukine,
  • Angiogenins thymopoetins, erythropoietins, zeatins, gibberelic acid derivatives, yeast factors, insect pheromones, coenzyme A, fibrinogens, angiotensinogens,
  • Mecamylamine, ranitidine, cimetidine, lovastatine or isoproterenol derivatives Mecamylamine, ranitidine, cimetidine, lovastatine or isoproterenol derivatives.
  • the substances accumulating in diseased tissue or in tumors mean peptides or proteins, in particular antibodies or antibody fragments such as F (ab) 2 , F (ab), single-chain antibodies or CDRs.
  • Particularly preferred compounds are characterized in that the antibody accumulating in diseased tissue or tumors is an ⁇ -CEA antibody, the hybrid cell clone used for its production has been deposited under number B4-F / C3 at the Central Institute for Molecular Biology (list 1988),
  • compounds according to the invention are characterized in that the substances accumulating in diseased tissue mean peptides such as endotheline, partial sequences of endothelin, endothelin analogues, endothelin derivatives or endothelin antagonists.
  • the compounds according to the invention are distinguished in that the peptides which accumulate in diseased tissue have the following sequences
  • the compounds according to the invention can exist as complexes with metal ions. Complexes in which the metal ion is a radionuclide are preferred. Preferred radionuclides are isotopes of the elements Tc, Re, At, In or Ga. The radionuclide Tc-99m is particularly preferred.
  • the present invention furthermore relates to a process for preparing the compounds of the general formula I according to the invention,
  • R 1 -XR 2 (I) wherein X is a chain of up to 20 identical or different ⁇ -, ⁇ - and / or ⁇ -amino acids, this chain being at least one amino acid from the group consisting of methionine, arginine, lysine and asparagine and none Contains cysteine and has a free valence at its N-terminus or an R 1 radical is attached to replace a hydrogen atom and has a free valence at its C-terminus or an R 2 residue is attached to replace a hydroxyl group, where
  • R 1 is a hydrogen atom, a branched one or
  • alkyl straight-chain alkyl, aryl, alkylaryl, aralkyl, alkylcarbonyl or arylcarbonyl group with up to 10 carbon atoms, which are optionally substituted with one hydroxyl, amino or carboxy group,
  • R 2 is a hydroxy group, a branched or
  • R a and R * 3 are the same or different and represent branched alkyl or acyl radicals having up to 10 carbon atoms, which are optionally with a hydroxyl, amino or
  • the process according to the invention is characterized in that a) amino acids are coupled one after the other amidically to synthetic resin-coupled amino acids in the desired order and in a manner known per se and are cleaved from the synthetic resin after the peptide synthesis has been completed,
  • the compounds thus prepared are conjugated selectively to diseased tissue or to substances accumulating in tumors, a covalent bond being formed between them, which in the case of substances containing amino groups such as peptides, proteins and antibodies, amidically or in the case of hydroxyl groups containing substances such as alcohols, ester-like or, in the case of substances containing aldehyde groups, imidically, and that the compounds and conjugates thus prepared are reacted with a metal in the form of a salt, optionally in the presence of a reducing agent and optionally an auxiliary ligand.
  • Another object of the present invention is a kit for the production of radiopharmaceuticals, consisting of a compound of general formula I according to the invention or a conjugate of this compound with peptides, proteins, bio- or macromolecules and from receptor-binding substances and optionally a reducing agent and optionally an auxiliary - Ligands that are in the dry state or in solution, an instruction for use with a reaction instruction for the reaction of the described compounds with Tc-99m or Re in the form of a pertechnetate solution or perrhenate solution.
  • radiopharmaceutical compositions for radiotherapy or radio diagnosis characterized in that they contain a compound according to the invention of the type described above.
  • the radiopharmaceutical compositions can contain conventional pharmaceutical excipients and carriers
  • the production of the peptides according to the invention and their coupling to organ-specific probes can be carried out without complex protective group technology and with little expenditure of time.
  • organ-specific probes Antibodies or partial sequences of antibodies, peptide structures such as endotheline, endothelin derivatives, partial sequences of endothelin, endothelin analogs or endothelin antagonists, hormones, neurohormones, neurotransmitters, growth factors, plant hormones, pheromones, enzyme cofactors, enzyme substrates, specifically binding to receptors Medicines or oligonucleotides can be used.
  • Compounds from the abovementioned groups can be, for example, oxytocins, vasopressins, angiotensins, melanocyte-stimulating hormones, somatostatins, tyrotropin-releasing hormones, gonadotropin-releasing hormones, testosterones, estradiols, progesterones, cortisols, aldosterones, vitamin D, progranomatins, secretrins , Neurotensins, insulins, glucagones, calcitonins, growth hormone-releasing hormones, prolactins, enkephalins, endorphins, dopamines, norepinephrines, glutamates, serotonins, acetylcholines, epinephrines, interleukins, angiogenins, zyphropinietins, erythropoietins, erythropoietins, erythrop
  • conjugates consisting of the peptides according to the invention and the organ-specific probe, can also be genetically represented as a whole unit.
  • Site-directed mutagenesis can be used in these potentially binding sites to replace nucleotides which code for non or only poorly metal-complexing amino acids with nucleotides which code for metal-complexing amino acids.
  • the generation of a metal-complexing site can involve the exchange of one or more amino acids, it being possible for the amino acids involved in the metal complexation to be interrupted by one or more "loops" which are not on the
  • a metal-complexing site is generated by known molecular biological methods, such as via double-strand plasmid DNA mutagenesis with T4 polymerase and selection primer (Deng et al., Anal. Bioch., 200, 81-88, 192) or via double-strand
  • Plasmid DNA mutagenesis by means of PCR (Landt et al., Gene, 96, 125-128, 1990). The so manipulated and
  • modified organ-specific probe can then be genetically expressed.
  • biomolecules per se can be changed in such a way that they retain their
  • Biomolecule areas generated which correspond to the general formula I, wherein the radicals R 1 and R 2 by the respective segments of the modified biomolecules are formed.
  • biomolecules can be changed by the site-directed mutagenesis in such a way that in the
  • Biomolecules are preserved, especially theirs
  • the peptides according to the invention have a defined metal binding site and have excellent, metal complexing properties.
  • peptides show high in vivo stability.
  • Table 1 shows the data determined according to Example 5 for the stability of Tc-peptide complexes in
  • Tc-peptide complexes are stable for 4 hours. This period is sufficient for therapy and diagnosis using the compounds according to the invention.
  • Table 1 Stability of Tc-peptide complexes in bovine plasma
  • conjugates e.g. B. consisting of partial sequences of antibodies against CEA and the peptides according to the invention, can be genetically expressed while maintaining the immunogenicity, since the use of cysteine-free peptides prevents the formation of undesirable disulfide bridges and thus an incorrect folding of the conjugate during expression.
  • a radioactively labeled, organ-specific probe which is derived from the ⁇ -CEA antibody B4F / C3 or its fragments such as, B. Fab2, Fab or sFv and a cysteine-free, Tc-99m-complexing peptide, specifically and stably binds to tumors, the specific accumulation of the tumor taking place very quickly.
  • the hybrid cell clone used to produce the antibody was deposited in the Central Institute for Microbiology in the former GDR (list 1988) under number B4-F / C3.
  • the light region variable region and the heavy chain variable region were sequenced.
  • the nucleotide sequence and the associated amino acid sequence of these regions and the synthetic linker are shown in Fig. 1.
  • the use of the conjugates according to the invention is characterized in that, due to the high, specific accumulation in the tumor and the favorable pharmacokinetics, the duration of the examination for optimal tumor localization is short, and thus the burden, in particular the radiation exposure, is minimized for the patient.
  • cysteine-free metal-complexing peptides according to the invention can be used per se as a liver function diagnostic.
  • Tables 2, 3 and 4 show the organ distribution and excretion kinetics of compounds according to the invention. The experiments were carried out as in Examples 2.3., 3.3. and 6.3. described, carried out. A large proportion of the radiation dose is then found in the liver. This effect can be used for liver function tests.
  • mice Tc-99 m-Arg-Gly-Met-Gly-Met-Gly in mice
  • FIGS 2 and 3 show the accumulation of the Tc-complexed compounds according to the invention, which were conjugated to the endothelin partial sequence His-Leu-Asp-Ile-Ile-Trp (cf. Example 7).
  • peptides according to the invention and the conjugates obtained by coupling to organ-specific probes are particularly suitable for use as diagnostic or therapeutic agents in the form of a kit.
  • Fmoc-protected (9-fluorenylmethoxycarbonyl) derivatives are predominantly used as N-protected amino acids.
  • the compounds of general formula (I) are synthesized from the carboxy termini to the amino terminus on the synthetic resin.
  • the compounds covalently bonded to the synthetic resin are cleaved from the synthetic resin at room temperature (2-4 h) with 97% trifluoroacetic acid / 2% triisobutylsilane / l% water and purified by preparative HPLC (RP-18, gradient from 0.1% trifluores- acetic acid after 60% acetonitrile / 0.1% trifluoroacetic acid within 30 minutes, flow rate 10 ml / min).
  • the peptides were synthesized and purified analogously to the general procedure described above.
  • H 2 N-Met-Gly-Met-Gly-His-Gly-His-COOH (1.2.2) A solution of 0.5 mg (0.69 ⁇ mol) H 2 N-Met-Gly-Met-Gly-His -Gly-His-CONH 2 or H 2 N-Met-Gly-Met-Gly-His-Gly-His-COOH in 300 ⁇ l phosphate buffer (Na 2 HPO 4 , 0.5 mol / l, pH 8.5) 50 ⁇ l of a 0.15 molar trisodium citrate dihydrate solution and 2.5 ⁇ l of a 0.2 molar tin (II) chloride dihydrate solution are added. After thorough mixing, the reaction mixture is mixed with a pertechnetate solution (0.4-0.9 mCi) from a Mo-99 / Tc-99m generator, incubated for 10 minutes at room temperature and then filtered (0.2 ⁇ m filter).
  • a pertechnetate solution 0.4-0.9 mCi
  • the radiochemical purity of the Tc-99m complexes is more than 95%.
  • the peptides were synthesized and purified analogously to the general procedure described above.
  • 150 ⁇ l Arg-Gly-Met-Gly-Met-Gly-Gly-Gly-Gly were mixed with 750 ⁇ l phosphate buffer, 150 ⁇ l citrate solution, 7.5 ⁇ l SnCl 2 and 150 ⁇ l Tc-99m pertechnetate and incubated for 20 min at room temperature. This labeling mixture was then diluted with 2793 ⁇ l PBS (pH 7.4). Total volume: 4000 ⁇ l (pH 7.4) with a total activity of 6.2 MBq (168 ⁇ Ci).
  • the radiochemical yield of the Tc-99m complex was 100%.
  • Dosage: 100 ⁇ l per animal, corresponding to 54 nmol Arg-Gly-Met-Gly-Met-Gly-Gly-Gly with an activity of 155.4 kBq (4.2 ⁇ Ci), specific activity 2.88 kBq / nmol (77.8 nCi / nmol).
  • Time points 0.25 h, 1 h, 3 h, 5 h after application (three animals per time).
  • Parameters radioactivity in the blood, liver, kidneys, muscle, heart, brain, spleen, intestine, skin, lungs, bones and residual body as well as in urine and faeces.
  • 150 ⁇ l Arg-Gly-Met-Gly-Met-Gly were mixed with 750 ⁇ l phosphate buffer, 150 ⁇ l citrate solution, 7.5 ⁇ l SnCl2 and 200 ⁇ l Tc-99m pertechnetate and incubated for 20 min at room temperature. This labeling mixture was then diluted with 2742.5 ⁇ l PBS (pH 7.4). Total volume: 4000 ⁇ l (pH 7.4) with a total activity of 6.2 MBq (168 ⁇ Ci).
  • the radiochemical yield of the Tc-99m complex was 100%.
  • Tc-99m-Arg-Gly-Met-Gly-Met-Gly should be examined after a single intravenous application to mice.
  • Time points 0.25 h, 1 h, 3 h, 5 h after application (three animals per time).
  • Residual body as well as in urine and faeces.
  • the substance dissolved in PBS was applied into a caudal vein. After application, the animals were kept in metabolic cages and urine and faeces were collected. Three animals were killed and prepared at the times indicated. In blood, liver, kidneys, muscle, heart, brain, spleen, intestine, skin, lungs, bones and residual bodies as well as in urine and faeces the radioactivity was measured in the gamma counter (program routine No. 9).
  • the radiochemical purity of the Tc-99m complex is more than 95%.
  • the stability of the Tc-peptide complexes not bound to protein was determined as follows: The peptides were synthesized, purified and labeled as described in Example 1 in accordance with the general procedure described above. 0.5 ml (corresponding to 0.5 mCi) of the labeling mixture was mixed with 4.5 ml bovine plasma (from Kraeber) or PBS. Immediately after mixing and after 1h, 2h, and 4h, 1 ml of sample is taken from each of the two batches and the protein is separated off by ultrafiltration (Centricon 30,000 from Amicon). The filtrate is separated on the HPLC: MERCK Nucleosil column, 125 ⁇ 44 mm, 5 ⁇ m;
  • the protein binding of the Tc-peptide complexes was calculated from the difference in the peak areas of the complex incubated with bovine plasma and with PBS. When calculating the stability, the total of the peak areas was assumed to be 100% and the percentage of the peak area of the complex was calculated therefrom. The peptide complexes examined are stable for 4 hours (see Table 1). Example 6
  • the peptide was synthesized and purified analogously to the general procedure described above.
  • the radiochemical yield of the Tc-99m complex was 95%.
  • Time points 0.25 h, 1 h, 3 h, 5 h after application (three animals per time).
  • Parameters radioactivity in the blood, liver, kidneys, muscle, heart, brain, spleen, intestine, skin, lungs, bones and residual body as well as in urine and faeces.
  • the substance dissolved in PBS was applied into a caudal vein. After application, the animals were kept in metabolic cages and urine and faeces were collected. Three animals were killed and prepared at the times indicated. In blood, liver, kidneys, muscle, heart, brain, spleen, intestine, skin, lungs, bones and residual bodies as well as in urine and faeces, the radioactivity was measured in the gamma counter (routine routine no. 9).
  • Atherosclerotic vascular lesions were detected on the gamma camera using Tc-99m-Glu-Met-Gly-Asn-Gly-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile-Trp after a single intravenous application depicted in WHHL rabbits.
  • the preparation and labeling was carried out as indicated in Examples 6.1 and 6.2. Execution : Species: Froxfield rabbit HH 047560, Emsicon-Jung, female, 4.3 kg, born October 15, 1992.
  • the serum cholesterol level on May 3, 1993 was 24.6 mmol / l,
  • WHHL rabbits 1 ml of Tc-99m-Glu-Met-Gly-Asn-Gly-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile-Trp was administered to the WHHL rabbit via an ear vein.
  • WHHL rabbits have high LDL levels in the blood due to a missing or defective LDL receptor and therefore spontaneously develop atherosclerotic vascular changes.
  • a left lateral admission of 0.5 h pi showed high activity in Heart, liver, kidneys and bladder and little activity in the bones.
  • the atherosclerotic lesions in the area of the abdominal aorta can be seen (see Fig. 2).
  • the rabbit was sacrificed 5 hours after application and both Sudan III staining and autoradiography of the aorta were carried out (see FIG. 3).
  • the r-specific activity was 353 cpm / mm2 in the plaque and 42 cpm / mm2 in the normal aortic wall
  • mRNA was isolated according to the standard protocol and cDNA was produced therefrom, which serves as a template for a PCR with suitable primer combinations for generating the complete VL and VH-coding regions of the antibody.
  • a synthetic oligonucleotide coding for (GGGGS) 3 was used for the linker peptide, and a synthetic oligonucleotide coding for the sequence PPMGMGHG was also used for the Tc-binding peptide (Tc peptide).
  • Tc peptide Tc-binding peptide
  • pHEN derivative modified vector
  • the periplasmic fraction was obtained by osmotic shock and from it the soluble single-chain Fv fragment via affinity chromatography
  • Single-chain Fv fragment with Tc binding site in the framework The construct for the single-chain Fv fragment was produced as described in Example 1, but by site-specific mutagenesis in the framework area of VL (loop of positions L: 36 to L: 43 ) or in the framework area of VH (loop of positions H: 38 to H: 45) using synthetic primers to produce Tc-binding variants, with the loop sequences MGMGHG in the named VH or VL area or in both areas for improvement the Tc mark.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Optics & Photonics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Vascular Medicine (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des peptides sans cystéine formant des complexes avec des métaux, qui sont le cas échéant couplés directement ou indirectement par un élément de liaison à une sonde spécifique d'un organe et qui s'enrichissent ainsi en tant que conjugués spécifiquement dans des tumeurs, des organes, des tissus ou des foyers d'inflammation. Comme sondes spécifiques d'organes on emploie par exemple des anticorps ou des séquences partielles d'anticorps dirigés contre des antigènes associés à des tumeurs, par exemple l'antigène carcino-embryonnaire (CEA), qui s'enrichissent ainsi de manière spécifique dans des tumeurs. L'invention concerne également des procédés pour la préparation des peptides sans cystéine formant des complexes avec les métaux et de leurs conjugués. La présente invention concerne également l'utilisation des conjugués comme éléments d'une trousse pour le diagnostic in vivo ou le traitement in vivo, ainsi que des produits radiopharmaceutiques contenant ces conjugués avec des radioisotopes. les conjugués spécifiques d'un organe sont utilisés pour l'imagerie concernant des tumeurs, des organes ou des foyers d'inflammation.
EP95900063A 1993-11-01 1994-10-27 Peptides capables de liaison avec les metaux et sans cysteine pour le diagnostic et le traitement, procedes pour leur preparation et compositions pharmaceutiques contenant ces composes Withdrawn EP0726910A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4337599 1993-11-01
DE4337599A DE4337599A1 (de) 1993-11-01 1993-11-01 Metallbindende cysteinfreie Peptide für Diagnose und Therapie, Verfahren zu ihrer Herstellung und diese Verbindungen enthaltende pharmazeutische Zusammensetzungen
PCT/DE1994/001302 WO1995012613A1 (fr) 1993-11-01 1994-10-27 Peptides capables de liaison avec les metaux et sans cysteine pour le diagnostic et le traitement, procedes pour leur preparation et compositions pharmaceutiques contenant ces composes

Publications (1)

Publication Number Publication Date
EP0726910A1 true EP0726910A1 (fr) 1996-08-21

Family

ID=6501749

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95900063A Withdrawn EP0726910A1 (fr) 1993-11-01 1994-10-27 Peptides capables de liaison avec les metaux et sans cysteine pour le diagnostic et le traitement, procedes pour leur preparation et compositions pharmaceutiques contenant ces composes

Country Status (12)

Country Link
US (1) US6291639B1 (fr)
EP (1) EP0726910A1 (fr)
JP (1) JPH09508352A (fr)
KR (1) KR960705847A (fr)
CN (1) CN1073116C (fr)
AU (1) AU688407B2 (fr)
CA (1) CA2173845A1 (fr)
DE (1) DE4337599A1 (fr)
HU (1) HUT75867A (fr)
NO (1) NO961744D0 (fr)
WO (1) WO1995012613A1 (fr)
ZA (1) ZA948410B (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101592668B (zh) * 2008-05-31 2012-10-24 刘秀梅 定量测定植物中草药提取成分中雌二醇含量的方法

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19652374A1 (de) * 1996-12-04 1998-06-10 Schering Ag Verwendung von Endothelin-Konjugaten in der Therapie, neue Endothelin-Konjugate, diese enthaltende Mittel, sowie Verfahren zu deren Herstellung
EP0968001B1 (fr) * 1997-02-03 2003-11-05 Mallinckrodt Inc. Procede servant a detecter et a localiser des tumeurs malignes humaines du pancreas
WO2007000978A1 (fr) * 2005-06-27 2007-01-04 Nippon Kayaku Kabushiki Kaisha Substance physiologiquement active nk13650p3, procédé de production de celle-ci et utilisation de celle-ci
KR100716802B1 (ko) 2005-12-30 2007-05-14 (주)아모레퍼시픽 산성도 민감성 고분자를 포함한 고분자-리포좀 나노복합체제조 및 이를 함유하는 피부 외용제 조성물
CN114401749B (zh) * 2020-06-29 2024-04-16 北京拓界生物医药科技有限公司 一种放射性核素标记物及其应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4348376A (en) * 1980-03-03 1982-09-07 Goldenberg Milton David Tumor localization and therapy with labeled anti-CEA antibody
US5196510A (en) * 1988-12-29 1993-03-23 Cytogen Corporation Molecular recognition units
GB8914020D0 (en) * 1989-06-19 1989-08-09 Antisoma Ltd Synthetic peptides for use in thrombus detection
US5196404B1 (en) * 1989-08-18 1996-09-10 Biogen Inc Inhibitors of thrombin
US5443815A (en) * 1991-11-27 1995-08-22 Diatech, Inc. Technetium-99m labeled peptides for imaging
JPH0570484A (ja) * 1991-09-12 1993-03-23 Hitachi Chem Co Ltd ペプチドおよびその塩
US5639860A (en) * 1991-12-27 1997-06-17 Kyowa Hakko Kogyo Co., Ltd. Endothelin-antagonizing peptide
DK0641222T3 (da) * 1992-05-21 2000-12-11 Diatide Inc Peptider mærket med technetium-99m til trombeafbildning

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9512613A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101592668B (zh) * 2008-05-31 2012-10-24 刘秀梅 定量测定植物中草药提取成分中雌二醇含量的方法

Also Published As

Publication number Publication date
AU688407B2 (en) 1998-03-12
ZA948410B (en) 1995-06-27
KR960705847A (ko) 1996-11-08
HU9601139D0 (en) 1996-07-29
NO961744L (no) 1996-04-30
NO961744D0 (no) 1996-04-30
AU8104094A (en) 1995-05-23
JPH09508352A (ja) 1997-08-26
CN1134159A (zh) 1996-10-23
WO1995012613A1 (fr) 1995-05-11
US6291639B1 (en) 2001-09-18
HUT75867A (en) 1997-05-28
CN1073116C (zh) 2001-10-17
CA2173845A1 (fr) 1995-05-11
DE4337599A1 (de) 1995-05-04

Similar Documents

Publication Publication Date Title
DE69635080T2 (de) Radiometall-bindende analoge von lhrh
DE69837038T2 (de) Tetraaza- oder n2s2-komplexanten, und deren verwendung in der radiodiagnostik oder radiotherapie
DE3728599A1 (de) Verfahren zur herstellung einer mit technetium-99m-markierten organspezifischen substanz
DE19845798A1 (de) Verwendung von Neoangiogenese-Markern für Diagnose und Therapie von Tumoren, diese enthaltende Mittel, sowie Verfahren zu deren Herstellung
DE602004007371T2 (de) Mikrowellenverfahren zur herstellung von radiomarkierten gallium komplexe
EP3074407B1 (fr) Liaison et procédé de radiomarquage sélectif de polypeptides par synthèse en phase solide
DE19536783A1 (de) Bifunktionelle Nicotinamid-Chelatbildner vom Typ N¶2¶S¶2¶ für radioaktive Isotope
DE19536781A1 (de) Bifunktionelle sulfidhaltige Sulfonamid-Chelatbildner vom Typ XSNS für radioaktive Isotope
EP0726910A1 (fr) Peptides capables de liaison avec les metaux et sans cysteine pour le diagnostic et le traitement, procedes pour leur preparation et compositions pharmaceutiques contenant ces composes
DE69230287T2 (de) Verbindungen und pharmazeutische zusammensetzungen zum nachweis und zur lokalisierung von geweben mit neurokinin-1-rezeptoren
DE19536785A1 (de) Bifunktionelle sulfidhaltige Sulfonamid-Chelatbildner vom Typ S¶2¶NY für radioaktive Isotope
DE60014375T2 (de) Uebergangsmetallkomplexe der vii-nebengruppe mit multidentaten aminopolycarboxylat-liganden und ein kit zu deren herstellung
AT405906B (de) Radiomarkierte somatostatin rezeptor-liganden zur diagnose und therapie
DE69839050T2 (de) Bilderzeugungs verfahren und zusammensetzungen
DE60032175T2 (de) Markierte ascorbinsäurederivate
DE69507408T2 (de) Peptide mit Affinität zu Tumoren und diese enthaltende radioaktive diagnostische und therapeutische Zusammensetzungen
EP0772633B1 (fr) Complexes destines au diagnostic de maladies vasculaires
DE69831158T2 (de) Hydroxymethyl phosphine derivate zur verwendung als diagnostische und therapeutische arzneimittel und deren verfahren
EP0588229A2 (fr) Agents chélatants macrocycliques pour la préparation de complexes de Thechnetium - ou Rhénium
DE4408729A1 (de) Nichtcyclische Chelatbildner auf Basis von Aminodialkylphosphoroxiden zur Herstellung von Technetium- oder Rheniumkomplexen
DE69327760T2 (de) Hydroxyaryl enthaltenden aminocarboxylsäure-chelatbildnern
Guzik Investigation of Folate-Based Radiopharmaceuticals for Theragnostic Application and Combination Therapy
DE112011105133T5 (de) Octapeptid zur Herstellung von radiopharmazeutischen Mitteln, das auf dessen Basis hergestellte Arzneimittel und Diagnoseverfahren von Somatostatin-Rezeptor exprimierenden Tumoren
WO2012000763A1 (fr) Peptide marqué 11c pour la détection d'un tissu malade exprimant un récepteur igf
Müller Synthesis and preclinical evaluation of novel ⁹⁹mTc-and ¹⁸⁸Re-folate radiopharmaceuticals for potential use in diagnostic and therapeutic nuclear medicine

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19960423

AK Designated contracting states

Kind code of ref document: A1

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

17Q First examination report despatched

Effective date: 19971111

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 20020418