WO2012000861A1 - 11c-labelled aptamer for detecting a diseased tissue - Google Patents

11c-labelled aptamer for detecting a diseased tissue Download PDF

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Publication number
WO2012000861A1
WO2012000861A1 PCT/EP2011/060421 EP2011060421W WO2012000861A1 WO 2012000861 A1 WO2012000861 A1 WO 2012000861A1 EP 2011060421 W EP2011060421 W EP 2011060421W WO 2012000861 A1 WO2012000861 A1 WO 2012000861A1
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Prior art keywords
aptamer
amino acid
carbon atom
diseased tissue
tissue
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PCT/EP2011/060421
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German (de)
French (fr)
Inventor
Hartmuth C. Kolb
Ursus KRÜGER
Oliver Lade
Arno Steckenborn
Tanja Weil
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Siemens Aktiengesellschaft
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO2012000861A1 publication Critical patent/WO2012000861A1/en

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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
    • A61K51/0491—Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes

Definitions

  • the invention relates to the use of an aptamer for Her ⁇ position of an agent for detecting a diseased tissue. It further relates to a radiopharmaceutical for the localization of a diseased tissue comprising such an aptamer.
  • biochemical analyzes of blood, other body fluids and tissue samples are used to characterize diseases. It examines the presence and amount of molecules that are typical of a particular disease. In addition to foreign substances also endogenous substances are detected, which are ⁇ example, only formed in an infection by viruses or bacteria.
  • tumor cells frequently form large amounts of certain proteins, in particular cellular receptors whose expression is specific for a type of tumor.
  • proteins that are made specifically from diseased cells are surface molecules that are anchored in the membrane which he ⁇ diseased cells. These surface molecules can be detected by appropriate diagnostic procedures. For this purpose, usually cells from tissue or blood samples are examined with antibodies that bind to specific, specific for a disease surface molecules. Such in vitro studies can diagnose the presence of a disease.
  • ectopic cell aggregates such as tumors or swellings of individual organs, can be used with them. locate gane.
  • a diseased tissue shows no marked morphological abnormalities, or is relatively small, it can easily be overlooked in traditional studies.
  • the invention is therefore based on the object, an agent be ⁇ riding determine by which a diseased tissue can be specifically and regardless of its size detected.
  • This object is achieved by the use of an aptamer for the manufacture of an agent for detecting a lung diseased tissue ge ⁇ dissolves.
  • aptamer refers to short, single-stranded nucleic lein Textre oligomers that can accommodate both RNA and DNA molecules to ⁇ . Depending on their respective sequence aptamers form diverse structures and bind to Zielmo ⁇ leküle of various classes. This results in a specific structural compatibility between an aptamer and its target molecule, similar to antigen-antibody binding.
  • the structure compatibility of the molecules occurs via electrostatic interactions, ionic Bin ⁇ compounds, van der Waals interactions, hydrogen bonds and so-called. Stacking interactions between the aromatic rings of the bases of nucleic acids.
  • Aptamers that bind to a specific target molecule are identified and produced through in vitro selection and amplification techniques known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) processes.
  • Aptamers comprise regularly 8-220 nucleic ⁇ otide, preferably 20 to 60 nucleotides. However, it is also possible to use aptamers with up to 500 nucleotides. she can be synthesized or obtained by enzymatic degradation of genomic DNA (Kulbachinskiy AV, 2007).
  • aptamers can be identified that bind a specific Zielmole ⁇ kül. This can be both larger biomolecules, such as proteins, as well as to individual chemical ele ⁇ ments. In addition, aptamers bind almost all classes of substances, so that aptamers can also be identified that recognize and bind to specific protein modifications, such as fatty acid or sugar modifications.
  • Each cell carries on its surface, anchored in its cell membrane, a multitude of different molecules, most of which belong to the class of proteins. Besides Mole ⁇ cules with basic biological functions that are found in almost every cell, many molecules only by cells of a particular tissue or a particular cell type can be expressed. In addition, cells that are infested with pathogens or have impaired cell functions, such as tumor cells, form their own molecules. Many of these disease-specific molecules are membrane bound and Queen ⁇ NEN be detected on the surface of the respective cell.
  • an aptamer is selected that interacts with one, indicative of an abnormal tissue molecule, am ⁇ det the aptamer specifically to the pathological tissue.
  • the aptamer is selected so that the bond between the aptamer and the target molecule is a linear coefficient called. KD value of ⁇ 100 nM, preferably ⁇ 10 nM, which most preferably of 7.5 nM. With such a
  • Aptamer can be specifically detected even a few cells of a diseased tissue.
  • diseased tissue refers to cells, parts of organs or whole organs that do not or not fully fulfill their physiological function. These include, for example, viruses or bacteria infected cells hy pertrophes tissue, inflamed tissue and organs, hyperplasti ⁇ MOORISH and neoplastic tissue, such as ulcers, tumors and cancers.
  • Diseased cells often form proteins whose expression is indicative of a particular disease, for example, because they are derived from the genetic material of a virus or bacterium. When these molecules are cell surface molecules, they are anchored to the cell membrane. By specifically binding a surface molecule of a diseased tissue, the aptamer enables a reliable localization of this tissue.
  • the aptamer is coupled to an amino acid, which in turn has an 11 C carbon atom.
  • positrons also referred to as ß + radiation, are formed. Push the positrons on
  • Electron they form two photons, which at an angle of 180 °, so exactly in the opposite direction, from each other.
  • the photons can be detected and from this the position of the positron emission, or of the 11 C carbon atom, can be calculated.
  • the Men are ⁇ ge of aptamers, which is located at a certain point, quantified.
  • the coupling of an amino acid with an 11 C carbon atom to the aptamer used in the invention makes it possible to detect and image both the presence and the position of the aptamer.
  • the processes described in patent applications DE 10 2009 035 648.7 and DE 10 2009 035 645.2 are particularly suitable.
  • the labeling the aptamer with a C-carbon atom via an amino acid is particularly advantageous because there ⁇ by any of the usual chelating agents such as diethylenetriamine aminpentaacetat (DTPA), 1, 4, 7, 10-tetraazacyclododecane-
  • DTPA diethylenetriamine aminpentaacetat
  • 1, 4, 7, 10-tetraacetic acid must be used.
  • the resulting complex of aptamer and amino acid comprises kör ⁇ pereigene molecules, whereby it is particularly compatible for the organism.
  • Both the aptamer and its single nucleic acids, as well as the amino acid are non-toxic. They can of course be metabolized, broken down and excreted.
  • By using an integrated X1 C carbon atom it is also possible to prevent a ra- 1
  • diooxider impurity such as fluorine, xenon, or 68 gallium, must be introduced into the organism.
  • a further advantage of the labeled via an amino acid with 1X C- carbon aptamer is in the low-Sig nal ⁇ / background ratio during detection of the aptamer.
  • the aptamer binds to the diseased tissue, whereas free, unbound aptamers are rapidly metabolised and excreted from the organism because they are rapidly degraded by endogenous enzymes. This creates a strong and specific signal at the location of the diseased tissue, and the background signal is minimized.
  • the agent is a radiopharmaceutical.
  • radiopharmaceuticals refers to medicines containing radionuclides whose radiation is used for diagnosis and therapy.
  • the main applications are in oncology, Kar ⁇ ogy and neurology, as well as pharmaceutical research.
  • radionuclides gamma or beta-emitting nuclides, for example 133 xenon, "technetium, 68 gallium, and fluorine, used. They are usually about Kom ⁇ formers such as DTPA, DOTA or ethylenediaminetetraacetate (EDTA) bound to mono- or polysaccharides.
  • the nuclides are detected by scintigraphy, single photon emission computed tomography (SPECT) or positron emission tomography (PET), depending on the nature of their radiation.
  • SPECT single photon emission computed tomography
  • PET positron emission tomography
  • conventional diet drugs can cause side effects, such as anaphylactic or allergic reactions, in the body of a patient.
  • the use of an aptamer from the body's own nucleic acids significantly reduces this risk because neither the aptamer itself nor its degradation products are toxic.
  • carbon is an element found in the body that naturally can be metabolized.
  • the amino acid is glycine, alanine, valine or serine.
  • the use of one of these amino acids to couple the 11 C-carbon isotope to the aptamer is particularly advantageous because these amino acids are relatively small and have no reactive side chains. Therefore, they neither affect the conformation nor the binding affinity of the aptamer, and the specificity of the aptamer for its target molecule is retained.
  • the amino acid is coupled via a peptide bond to a free amino group of a nucleotide of the aptamer.
  • the Apt ⁇ mer forth ⁇ tional methods of peptide synthesis such as solid phase synthesis, the coupling of the amino acid.
  • the preparation of the complex is therefore possible without expensive additional synthesis process, where ⁇ is reduced by the technical and financial effort.
  • the complex of aptamer and amino acid can be can be used directly after attaching the 11 C-labeled amino acid. 11C carbon has a half-life of only about 20 minutes, so the longer the time between synthesis of the complex and its use, the higher the radiation dose must be.
  • the aptamer can then be used immediately ⁇ the. In this way, the time between the proces ⁇ processing of the C-11 carbon and the use of the aptamer is re- pokerd so that the radiation loss is minimized during the herstel ⁇ development of the complex. Therefore, the radiation dose ⁇ which must be ⁇ sets in the processing of the 11 C-carbon can to ensure a certain strength of the radiation Pro ⁇ domestic product may be correspondingly lower. The manufacturer's position is more cost-effective and thereby the radiation exposure for the technical staff that provides the agent ago ⁇ reduced.
  • the 11 C carbon atom is the carbonyl carbon atom of the peptide bond.
  • the peptide bond is relatively protected inside the complex of aptamer and amino acid. This ensures that the 11 C carbon atom is not split off from the amino acid, as would be possible with an exposed side chain of the amino acids.
  • the amino acid is a D-amino acid.
  • D-amino acid With the exception of glycine besit ⁇ zen all amino acids at its C-alpha-carbon atom is a chiral center and may therefore as configurational isomers, namely as D- or L-amino acid present.
  • Endogenous peptides and proteins are largely composed of amino acids in L configuration.
  • most natural proteases and peptidases work stereoselectively and metabolise mainly L-amino acids. Therefore, the degradation of D-amino acids by endogenous enzymes takes longer than that of L-amino acids.
  • This fact can be used to prolong the half-life of the complex between aptamer and an amino acid by a D-amino acid ⁇ is used instead of an L-amino acid (Neundorf I et al., 2008).
  • a D-amino acid ⁇ is used instead of an L-amino acid
  • Another way to delay the cleavage is to use a non-natural amino acid.
  • the non-natural amino acids are metabolized more slowly because the body's own proteolytic enzymes are specially adapted to the breakdown of natural amino acids.
  • other chemical modifications of the amino acid are possible.
  • the terminal amino group of the amino acid can be replaced by an isonitrile group. Such a modification reduces the amino group mediated interaction with proteolytic enzymes without altering the binding specificity.
  • Another object of the invention is a radiopharmaceutical for the localization of a tumor comprising an aptamer.
  • the aptamer binds to the tumor and is gekop ⁇ pelt to an amino acid, which in turn comprises a C-11 carbon atom gron- NEN even a few cells of the tumor are located.
  • the radiopharmaceutical of the present invention provides a sensitive and specific agent for determining the position of a tumor in vivo.
  • the radiopharmaceutical is administered to the patient and the aptamers contained therein, which are coupled to the 11 C-labeled amino acids, are distributed quickly and efficiently in the body due to their size. They bind to the tumor and collect on its surface.
  • the accumulation of radioactively labeled aptamers is detected by positron emission tomography (PET) to determine the exact location of the tumor in the patient's body.
  • PET positron emission tomography
  • the amino acid is glycine, alanine, valine or serine, so that neither the conformation nor the binding affinity of the aptamer is influenced by the amino acid.
  • the C-carbon atom is the carbon atom of the terminal ⁇ -carboxyl group of the amino acid.
  • the X1 C carbon atom can not be split off from the amino acid, as would be possible with an exposed side chain of the amino acids.
  • the radiopharmaceutical is a PET biomarker.
  • PET is an established method for detecting the radiation of radioactive elements and determining their position (Massoud TF, Gambhir SS, 2003). With the aid of detector devices arranged annularly around the patient, sectional images are created on which the decay events in their spatial distribution in the interior of the body are represented. The PET also makes it possible to determine the amount of mar ⁇ -labeled molecules quantitatively in a tissue.
  • a method for localization of a diseased tissue comprising the steps of: a) providing an aptamer, which is coupled to an amino acid ⁇ ; b) administering the aptamer to the Or ⁇ organism; and c) detecting the aptamer in the organism by positron emission tomography (PET).
  • PET positron emission tomography
  • aptamer according to the invention to which a 11 C-labeled amino acid is coupled to a morbid Ge ⁇ tissue is detected in the interior of an organism and localized, and can thus be in the body of a patient observed. In this way, for example, the size or extent of an infection or a tumor can be determined.
  • the aptamer According to the invention used is therefore ideal for Be ⁇ observation of progress and success of a treatment, so-called. Therapy monitoring, appropriate.
  • FIG. 1 shows schematically an aptamer 1 with 14 nucleotides 3, which is bound to a pathological tissue 18.
  • the aptamer 1 is coupled to an amino acid 2 which is linked to an X1 C-
  • Carbon atom is radiolabeled.
  • the radioactive label is represented by an asterisk (*).
  • the specific binding affinity between the aptamer 1 and the pathological tissue 18 comes off due to chemical exchange ⁇ effects between the aptamer 1 and the surface of the diseased tissue 18th
  • the pathological tissue 18 forms surface molecules that are characteristic of the disease of the tissue ⁇ esp.
  • an aptamer 1 is identified that interacts with a surface molecule of diseased tissue 18.
  • an amino acid labeled with a C-carbon atom is coupled to the aptamer 1.
  • the aptamer 1 can then be replaced by the positrons emitted upon the decay of the ⁇ C carbon atom be detected by positron emission tomography (PET).
  • PET positron emission tomography
  • the aptamer 1 coupled to an 11 C-labeled amino acid 2 is administered to a patient in the form of a drug. It binds to the pathological tissue 18 and collects on its cells. This accumulation becomes visible in a PET, so that the distribution of the aptamer 1 or the position of the diseased tissue 18 in the body of the patient can be determined.
  • FIG. 2 shows a representation of an aptamer by means of a chemical formula.
  • the aptamer has the sequence SEQ ID No .: 1 and is gekop ⁇ pelt to a 11 C-labeled amino acid, namely, glycine.
  • the aptamer comprises 70 nucleic acids of the following sequence: GGG AGG ACG AUG CGG ACC GAA AAA GAC CUG ACU UCU AUA CUA AGU CUA CGU UCC CAG ACG ACU CGC CCG A.
  • the 3 'terminal adenosine of the aptamer and it gekop ⁇ pelte glycine are represented by structural formula, and the remaining nucleic acids 3 by their respective letter code.
  • the sequence of the aptamer is also given in SEQ ID NO: 1.
  • the carbonyl carbon glycine is an X1 C carbon atom represented by the number 11 above the carbonyl carbon atom.
  • the aptamer of SEQ ID NO .: 1 has a specific binding affinity Bin ⁇ specific to the prostate membrane antigen (PSMA) on.
  • PSMA prostate membrane antigen
  • This protein is mainly expressed in prostate tissue and comes in prostate carcinomas in particularly high Quantities.
  • PSMA is a membrane protein present in healthy prostate cells but in a particular cytoplasmic form. In prostate cancer, it is mainly located on the cell membrane of tumor cells.
  • the aptamer 1 is selected and amplified by its binding specificity to PSMA with SELEX methods from an aptamer library. Subsequently, the 11 C-labeled amino acid ⁇ 2 is coupled to a conventional peptide synthesis methods to the aptamer. 1 The aptamer 1 is administered to the patient and thus the prostate carcinoma by PET Darge ⁇ presents. In this way, metastases which also express membrane-bound PSMA can be localized.
  • Figure 3 shows a schematic representation (greatly simplified by Faller A, Schünke M, The human body, Thieme, 2008) of a circulatory system 10 of an organism and the distribution of an aptamer 1 therein.
  • the circulation system 10 includes various organs schematically represented, such as the lungs 12, heart 13, liver 14, 15 intestine and kidney 16 and the main wires 11 which these organs ver ⁇ bind.
  • the aptamer 1 is represented by triangles along the wires 11.
  • the degradation products 17 of the aptamer 1 are represented by individual lines within the outline of the kidney 16 Darge ⁇ .
  • Left of center of the circulatory system 10 is additionally ⁇ a diseased tissue 18, for example, a tumor or an inflammation, shown, are attached to the increased aptamers. 1
  • Phase I The aptamer 1 is injected into the circulatory system 10 of the organism.
  • Phase II is via the blood circulation system 10, the aptamer 1 in the organs 12, 13, 14, 15, and 16 of the ⁇ organism transported advantage.
  • Phase III The circulating aptamer 1 binds specifically to the diseased tissue 18.
  • Phase IV Unbound aptamer 1 is rapidly metabolised and enzymatically degraded.
  • the organism not failed ⁇ det between the body's own molecules and the aptamer 1, because it is constructed from nucleic acids 3 and an amino acid 2, which correspond to the body's own molecules.
  • the degradation products 17 of the aptamer 1 and the nucleic acids 3 and the amino acid 2 accumulate predominantly in the kidney 16, from where they are excreted via the bladder and the ureter.
  • Massoud TF, Ga bhir SS Molecular imaging in living subjects: seeing fundamental biological processes in a new light; Genes Dev. 2003 Mar 1; 17 (5): 545-80.

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Abstract

There is described the use of an aptamer (1) for the preparation of an agent for detecting a diseased tissue (18). The aptamer (1) binds to the diseased tissue (18) and is coupled to an amino acid (2) which, in turn, includes an 11C carbon atom. There is furthermore described a radiopharmaceutical for locating a tumour (18), which radiopharmaceutical comprises such an aptamer (1).

Description

Beschreibung description
C-markiertes Aptamer zur Detektion eines krankhaften Gewebes C-labeled aptamer for detection of diseased tissue
Die Erfindung betrifft die Verwendung eines Aptamers zur Her¬ stellung eines Agens zur Detektion eines krankhaften Gewebes. Sie betrifft ferner ein Radiopharmakon zur Lokalisation eines krankhaften Gewebes, das ein solches Aptamer umfasst. The invention relates to the use of an aptamer for Her ¬ position of an agent for detecting a diseased tissue. It further relates to a radiopharmaceutical for the localization of a diseased tissue comprising such an aptamer.
In der modernen Diagnostik werden zur Charakterisierung von Krankheiten vor allem biochemische Analysen von Blut, anderen Körperflüssigkeiten und Gewebeproben eingesetzt. Dabei wird die Anwesenheit und Menge von Molekülen untersucht, die für eine bestimmte Krankheit typisch sind. Neben Fremdstoffen werden auch körpereigene Stoffe nachgewiesen, die beispiels¬ weise nur bei einer Infektion durch Viren oder Bakterien gebildet werden. Insbesondere Tumorzellen bilden häufig große Mengen bestimmter Proteine, insbesondere zelluläre Rezepto- ren, deren Expression für eine Tumorart spezifisch ist. Viele der Proteine, die speziell von krankhaften Zellen gebildet werden, sind Oberflächenmoleküle, die in der Membran der er¬ krankten Zellen verankert werden. Diese Oberflächenmoleküle können mit entsprechenden diagnostischen Verfahren nachgewie- sen werden. Dazu werden üblicherweise Zellen aus Gewebe- oder Blutproben mit Antikörpern untersucht, die an bestimmte, für eine Krankheit spezifische Oberflächenmoleküle binden. Durch derartige in vitro Untersuchungen kann das Vorliegen einer Krankheit diagnostiziert werden. Es ist aber nicht möglich, auch den genauen Ort des erkrankten Gewebes festzustellen. Zu diesem Zweck werden in der Regel bildgebende Verfahren, wie beispielsweise Röntgen, Ultraschall und Kernspinntomographie verwendet. Mit ihnen lassen sich vor allem ektopische Zellansammlungen, wie etwa Tumore, oder Schwellungen einzelner Or- gane lokalisieren. Zeigt ein krankhaftes Gewebe jedoch keine deutlichen morphologischen Auffälligkeiten, oder ist es verhältnismäßig klein, kann es bei traditionellen Untersuchungen leicht übersehen werden. In modern diagnostics, biochemical analyzes of blood, other body fluids and tissue samples are used to characterize diseases. It examines the presence and amount of molecules that are typical of a particular disease. In addition to foreign substances also endogenous substances are detected, which are ¬ example, only formed in an infection by viruses or bacteria. In particular, tumor cells frequently form large amounts of certain proteins, in particular cellular receptors whose expression is specific for a type of tumor. Many of the proteins that are made specifically from diseased cells are surface molecules that are anchored in the membrane which he ¬ diseased cells. These surface molecules can be detected by appropriate diagnostic procedures. For this purpose, usually cells from tissue or blood samples are examined with antibodies that bind to specific, specific for a disease surface molecules. Such in vitro studies can diagnose the presence of a disease. But it is not possible to determine the exact location of the diseased tissue. For this purpose, imaging techniques such as X-ray, ultrasound, and nuclear spin tomography are typically used. In particular, ectopic cell aggregates, such as tumors or swellings of individual organs, can be used with them. locate gane. However, if a diseased tissue shows no marked morphological abnormalities, or is relatively small, it can easily be overlooked in traditional studies.
Der Erfindung liegt daher die Aufgabe zugrunde ein Agens be¬ reitzustellen, mit dem ein krankhaftes Gewebe spezifisch und unabhängig von seiner Größe detektiert werden kann. Diese Aufgabe wird durch die Verwendung eines Aptamers zur Herstel- lung eines Agens zur Detektion eines krankhaften Gewebes ge¬ löst. Indem das Aptamer an das krankhafte Gewebe bindet und an eine Aminosäure gekoppelt ist, die wiederum ein X1C- Kohlenstoffatom aufweist, können selbst wenige Zellen eines krankhaften Gewebes an Hand des radioaktiven Signals lokali- siert werden. The invention is therefore based on the object, an agent be ¬ riding determine by which a diseased tissue can be specifically and regardless of its size detected. This object is achieved by the use of an aptamer for the manufacture of an agent for detecting a lung diseased tissue ge ¬ dissolves. By binding the aptamer to the diseased tissue and being coupled to an amino acid, which in turn has an X1 C carbon atom, even a few cells of a diseased tissue can be localized by the radioactive signal.
Der Begriff "Aptamer" bezeichnet kurze, einzelsträngige Nuk- leinsäure-Oligomere, die sowohl RNA als auch DNA Moleküle um¬ fassen können. In Abhängigkeit von ihrer jeweiligen Sequenz bilden Aptamere vielfältige Strukturen und binden an Zielmo¬ leküle der verschiedensten Stoffklassen . Dabei kommt es zu einer spezifischen Strukturkompatibilität zwischen einem Aptamer und seinem Zielmolekül, ähnlich einer Antigen- Antikörper-Bindung . Die Strukturkompatibilität der Moleküle erfolgt über elektrostatische Wechselwirkungen, ionische Bin¬ dungen, van-der-Waals Wechselwirkungen, Wasserstoffbrücken und sog. Stacking Interactions zwischen den aromatischen Ringen der Basen der Nukleinsäuren. Aptamere, die ein bestimmtes Zielmolekül binden, werden durch in vitro Selektionsverfahren und Amplifikationstechniken, sog. SELEX-Prozesse (Systematic Evolution of Ligands by Exponential Enrichment) identifiziert und produziert. Aptamere umfassen regelmäßig 8 bis 220 Nukle¬ otide, vorzugsweise 20 bis 60 Nukleotide. Es können aber auch Aptamere mit bis zu 500 Nukleotiden verwendet werden. Sie können synthetisch hergestellt oder durch enzymatischen Abbau genomischer DNA gewonnen werden (Kulbachinskiy AV, 2007) . The term "aptamer" refers to short, single-stranded nucleic leinsäure oligomers that can accommodate both RNA and DNA molecules to ¬. Depending on their respective sequence aptamers form diverse structures and bind to Zielmo ¬ leküle of various classes. This results in a specific structural compatibility between an aptamer and its target molecule, similar to antigen-antibody binding. The structure compatibility of the molecules occurs via electrostatic interactions, ionic Bin ¬ compounds, van der Waals interactions, hydrogen bonds and so-called. Stacking interactions between the aromatic rings of the bases of nucleic acids. Aptamers that bind to a specific target molecule are identified and produced through in vitro selection and amplification techniques known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) processes. Aptamers comprise regularly 8-220 nucleic ¬ otide, preferably 20 to 60 nucleotides. However, it is also possible to use aptamers with up to 500 nucleotides. she can be synthesized or obtained by enzymatic degradation of genomic DNA (Kulbachinskiy AV, 2007).
Unter Verwendung umfangreicher Aptamer Bibliotheken können Aptamere identifiziert werden, die ein spezifisches Zielmole¬ kül binden. Dabei kann es sich sowohl um größere Biomoleküle, beispielsweise Proteine, als auch um einzelne chemische Ele¬ mente handeln. Darüber hinaus binden Aptamere nahezu alle Stoffklassen, so dass auch Aptamere identifiziert werden kön- nen, die spezielle Proteinmodifikationen, wie beispielsweise Fettsäure- oder Zuckermodifikationen erkennen und binden. Using extensive aptamer libraries aptamers can be identified that bind a specific Zielmole ¬ kül. This can be both larger biomolecules, such as proteins, as well as to individual chemical ele ¬ ments. In addition, aptamers bind almost all classes of substances, so that aptamers can also be identified that recognize and bind to specific protein modifications, such as fatty acid or sugar modifications.
Jede Zelle trägt auf ihrer Oberfläche, verankert in ihrer Zellmembran, eine Vielzahl unterschiedlicher Moleküle, wobei die meisten zur Stoffklasse der Proteine gehören. Neben Mole¬ külen mit grundlegenden biologischen Funktionen, die in nahezu jeder Zelle vorkommen, werden viele Moleküle nur von Zellen eines bestimmten Gewebes oder eines bestimmten Zelltyps exprimiert. Außerdem bilden Zellen, die von Krankheitserre- gern befallen sind oder gestörte Zellfunktionen aufweisen, wie beispielsweise Tumorzellen, eigene Moleküle. Viele dieser krankheitsspezifischen Moleküle sind membranständig und kön¬ nen auf der Oberfläche der jeweiligen Zelle nachgewiesen werden. Indem ein Aptamer gewählt wird, das mit einem, für ein krankhaftes Gewebe kennzeichnenden Molekül interagiert, bin¬ det das Aptamer spezifisch an das krankhafte Gewebe. Vorzugs¬ weise wird das Aptamer dabei so gewählt, dass die Bindung zwischen dem Aptamer und dem Zielmolekül einen linearen Koeffizient, sog. kD-Wert, von < 100 nM, bevorzugt von < 10 nM, am meisten bevorzugt von 7,5 nM aufweist. Mit einem solchenEach cell carries on its surface, anchored in its cell membrane, a multitude of different molecules, most of which belong to the class of proteins. Besides Mole ¬ cules with basic biological functions that are found in almost every cell, many molecules only by cells of a particular tissue or a particular cell type can be expressed. In addition, cells that are infested with pathogens or have impaired cell functions, such as tumor cells, form their own molecules. Many of these disease-specific molecules are membrane bound and Queen ¬ NEN be detected on the surface of the respective cell. By an aptamer is selected that interacts with one, indicative of an abnormal tissue molecule, am ¬ det the aptamer specifically to the pathological tissue. Preference ¬ example the aptamer is selected so that the bond between the aptamer and the target molecule is a linear coefficient called. KD value of <100 nM, preferably <10 nM, which most preferably of 7.5 nM. With such a
Aptamer können selbst wenige Zellen eines krankhaften Gewebes spezifisch nachgewiesen werden. Der Begriff "krankhaftes Gewebe" bezeichnet Zellen, Teile von Organen oder ganze Organe, die ihre physiologische Funktion nicht oder nicht in vollem Umfand erfüllen. Dazu zählen beispielsweise mit Viren oder Bakterien infizierte Zellen, hy- pertrophes Gewebe, entzündete Gewebe und Organe, hyperplasti¬ sches und neoplastisches Gewebe, etwa Geschwüre, Tumore und Karzinome. Krankhafte Zellen bilden häufig Proteine, deren Expression für eine bestimmte Erkrankung kennzeichnend ist, beispielsweise weil sie vom genetischen Material eines Virus oder eines Bakteriums abstammen. Handelt es sich bei diesen Molekülen um Zelloberflächenmoleküle, werden sie auf der Zellmembran verankert. Indem das Aptamer speziell ein Oberflächenmolekül eines krankhaften Gewebes bindet, ermöglicht es eine zuverlässige Lokalisation dieses Gewebes. Aptamer can be specifically detected even a few cells of a diseased tissue. The term "diseased tissue" refers to cells, parts of organs or whole organs that do not or not fully fulfill their physiological function. These include, for example, viruses or bacteria infected cells hy pertrophes tissue, inflamed tissue and organs, hyperplasti ¬ MOORISH and neoplastic tissue, such as ulcers, tumors and cancers. Diseased cells often form proteins whose expression is indicative of a particular disease, for example, because they are derived from the genetic material of a virus or bacterium. When these molecules are cell surface molecules, they are anchored to the cell membrane. By specifically binding a surface molecule of a diseased tissue, the aptamer enables a reliable localization of this tissue.
Um das an ein Gewebe gebundene Aptamer zu detektieren, wird das Aptamer an eine Aminosäure gekoppelt, die wiederum ein 11C-Kohlenstoffatom aufweist. Beim Zerfall des 11C-Kohlen- stoffisotops werden Positronen, die auch als ß+-Strahlung be- zeichnet werden, gebildet. Stoßen die Positronen auf einTo detect the tissue-bound aptamer, the aptamer is coupled to an amino acid, which in turn has an 11 C carbon atom. Upon decay of the 11 C carbon isotope, positrons, also referred to as ß + radiation, are formed. Push the positrons on
Elektron, bilden sie zwei Photonen, die sich in einem Winkel von 180°, also genau in entgegen gesetzter Richtung, von einander entfernen. Die Photonen können detektiert und daraus die Position der Positronenemission, bzw. des 11C-Kohlen- Stoffatoms, berechnet werden. Des Weiteren kann auch die Men¬ ge an Aptameren, die sich an einer bestimmten Stelle befindet, quantifiziert werden. Die Kopplung einer Aminosäure mit einem 11C-Kohlenstoffatom an das erfindungsgemäß verwendete Aptamer ermöglicht es, sowohl das Vorhandensein, als auch die Position des Aptamers nachzuweisen und abzubilden. Zur Herstellung einer 11C-markierten Aminosäure und zur Kopplung der Aminosäure an das Aptamer sind insbesondere die Verfahren, die in den Patentanmeldungen DE 10 2009 035 648.7, und DE 10 2009 035 645.2 beschrieben werden, geeignet. Die Markierung das Aptamers mit einem C-Kohlenstoffatom über eine Aminosäure ist insbesondere vorteilhaft, weil da¬ durch keine der üblichen Komplexbildner, wie Diethylentri- aminpentaacetat (DTPA), 1, 4, 7, 10-tetraazacyclododecane-Electron, they form two photons, which at an angle of 180 °, so exactly in the opposite direction, from each other. The photons can be detected and from this the position of the positron emission, or of the 11 C carbon atom, can be calculated. Furthermore, the Men are ¬ ge of aptamers, which is located at a certain point, quantified. The coupling of an amino acid with an 11 C carbon atom to the aptamer used in the invention makes it possible to detect and image both the presence and the position of the aptamer. For the preparation of an 11 C-labeled amino acid and for the coupling of the amino acid to the aptamer, the processes described in patent applications DE 10 2009 035 648.7 and DE 10 2009 035 645.2 are particularly suitable. The labeling the aptamer with a C-carbon atom via an amino acid is particularly advantageous because there ¬ by any of the usual chelating agents such as diethylenetriamine aminpentaacetat (DTPA), 1, 4, 7, 10-tetraazacyclododecane-
1, 4, 7, 10-tetraacetic acid (DOTA) verwendet werden müssen. Der entstehende Komplex aus Aptamer und Aminosäure umfasst kör¬ pereigene Moleküle, wodurch er für den Organismus besonders verträglich ist. Sowohl das Aptamer und seine einzelnen Nuk- leinsäuren, als auch die Aminosäure sind nicht toxisch. Sie können natürlich verstoffwechselt , abgebaut und ausgeschieden werden. Durch die Verwendung eines integrierten X1C- Kohlenstoffatoms kann außerdem vermieden werden, dass ein ra- 1 1, 4, 7, 10-tetraacetic acid (DOTA) must be used. The resulting complex of aptamer and amino acid comprises kör ¬ pereigene molecules, whereby it is particularly compatible for the organism. Both the aptamer and its single nucleic acids, as well as the amino acid, are non-toxic. They can of course be metabolized, broken down and excreted. By using an integrated X1 C carbon atom, it is also possible to prevent a ra- 1
dioaktiver Fremdstoff, wie beispielsweise Fluor, Xenon, oder 68Gallium, in den Organismus eingebracht werden muss. dioaktiver impurity such as fluorine, xenon, or 68 gallium, must be introduced into the organism.
Ein weiterer Vorteil des über eine Aminosäure mit 1XC- Kohlenstoff markierten Aptamers liegt in dem günstigen Sig¬ nal/Hintergrund Verhältnis während der Detektion des Apta- mers . Das Aptamer bindet an das krankhafte Gewebe, wohingegen freie, ungebundene Aptamere rasch verstoffwechselt und aus dem Organismus ausgeschieden werden, weil sie von endogenen Enzymen zügig abgebaut werden. Dadurch entsteht ein starkes und spezifisches Signal an der Position des krankhaften Gewe- bes, und das Hintergrundsignal wird minimiert. A further advantage of the labeled via an amino acid with 1X C- carbon aptamer is in the low-Sig nal ¬ / background ratio during detection of the aptamer. The aptamer binds to the diseased tissue, whereas free, unbound aptamers are rapidly metabolised and excreted from the organism because they are rapidly degraded by endogenous enzymes. This creates a strong and specific signal at the location of the diseased tissue, and the background signal is minimized.
In einer vorteilhaften Weiterbildung der Erfindung ist das Agens ein Radiopharmakon . Der Begriff "Radiopharmaka" bezeichnet Arzneimittel, die Radionuklide enthalten, deren Strahlung zur Diagnostik und Therapie verwendet wird. Die wichtigsten Anwendungsgebiete sind dabei die Onkologie, Kar¬ diologie und Neurologie, aber auch die Arzneimittelforschung. Als Radionuklide werden Gamma- bzw. Beta-Strahlen emittierende Nuklide, zum Beispiel 133Xenon, "Technetium, 68Gallium, und Fluor, verwendet. Sie werden üblicherweise über Kom¬ plexbildner wie DTPA, DOTA oder Ethylendiamintetraacetat (EDTA) an Mono- oder Polysaccharide gebunden. Die Nuklide werden, je nach der Art ihrer Strahlung, mittels Szintigraphie, Single Photon Emission Computed Tomography (SPECT) oder Positronen-Emissions-Tomographie (PET) detektiert. Aufgrund ihrer unphysiologischen Bestandteile können herkömmliche Ra- diopharmaka jedoch Nebenwirkungen, wie anaphylaktische oder allergische Reaktionen, im Körper eines Patienten verursachen. Die Verwendung eines Aptamers aus körpereigenen Nukleinsäuren reduziert diese Gefahr deutlich, weil weder das Aptamer selbst, noch seine Abbauprodukte toxisch sind. Zudem ist Kohlenstoff, im Gegensatz zu Technetium oder Xenon, ein im Körper vorkommendes Element, das natürlich verstoffwechselt werden kann. In an advantageous embodiment of the invention, the agent is a radiopharmaceutical. The term "radiopharmaceuticals" refers to medicines containing radionuclides whose radiation is used for diagnosis and therapy. The main applications are in oncology, Kar ¬ ogy and neurology, as well as pharmaceutical research. As radionuclides, gamma or beta-emitting nuclides, for example 133 xenon, "technetium, 68 gallium, and fluorine, used. They are usually about Kom ¬ formers such as DTPA, DOTA or ethylenediaminetetraacetate (EDTA) bound to mono- or polysaccharides. The nuclides are detected by scintigraphy, single photon emission computed tomography (SPECT) or positron emission tomography (PET), depending on the nature of their radiation. However, because of their nonphysiological components, conventional diet drugs can cause side effects, such as anaphylactic or allergic reactions, in the body of a patient. The use of an aptamer from the body's own nucleic acids significantly reduces this risk because neither the aptamer itself nor its degradation products are toxic. In addition, unlike technetium or xenon, carbon is an element found in the body that naturally can be metabolized.
Gemäß einer vorteilhaften Weiterbildung der Erfindung ist die Aminosäure Glycin, Alanin, Valin oder Serin. Die Verwendung einer dieser Aminosäuren, um das 11C-Kohlenstoffisotop an das Aptamer zu koppeln, ist besonders vorteilhaft, weil diese Aminosäuren verhältnismäßig klein sind und keine reaktiven Seitenketten aufweisen. Sie beeinflussen daher weder die Konformation noch die Bindungsaffinität das Aptamers, und die Spezifität des Aptamers für sein Zielmolekül bleibt erhalten. According to an advantageous development of the invention, the amino acid is glycine, alanine, valine or serine. The use of one of these amino acids to couple the 11 C-carbon isotope to the aptamer is particularly advantageous because these amino acids are relatively small and have no reactive side chains. Therefore, they neither affect the conformation nor the binding affinity of the aptamer, and the specificity of the aptamer for its target molecule is retained.
Gemäß einer vorteilhaften Weiterbildung der Erfindung ist die Aminosäure über eine Peptidbindung an eine freie Aminogruppe eines Nukleotids des Aptamers gekoppelt. Dadurch können her¬ kömmliche Peptidsyntheseverfahren, wie beispielsweise eine Festphasensynthese, zur Koppelung der Aminosäure an das Apta¬ mer verwendet werden. Die Herstellung des Komplexes ist daher ohne aufwendige zusätzliche Syntheseverfahren möglich, wo¬ durch der technische und finanzielle Aufwand reduziert wird. Des Weiteren kann der Komplex aus Aptamer und Aminosäure di- rekt nach dem Anbringen der 11C-markierten Aminosäure verwendet werden kann. 11C-Kohlenstoff hat eine Halbwertszeit von nur ca. 20 Minuten, so dass die Strahlungsdosis desto höher gewählt werden muss, je mehr Zeit zwischen der Synthese des Komplexes und seiner Verwendung liegt. Wird die Aminosäure mit der 11C-Markierung im letzten Schritt der Synthese angebracht, kann das Aptamer anschließend sofort verwendet wer¬ den. Auf diese Weise wird die Zeitspanne zwischen der Verar¬ beitung des 11C-Kohlenstoffs und dem Einsatz des Aptamers re- duziert, so dass der Strahlungsverlust während der Herstel¬ lung des Komplexes minimiert wird. Deshalb kann die Strahlen¬ dosis, die bei der Verarbeitung des 11C-Kohlenstoffs einge¬ setzt werden muss um eine bestimmte Strahlungsstärke des Pro¬ dukts zu gewährleisten, entsprechend geringer sein. Die Her- Stellung wird dadurch kostengünstiger und die Strahlenbelastung für das technische Personal, welches das Agens her¬ stellt, verringert. According to an advantageous embodiment of the invention, the amino acid is coupled via a peptide bond to a free amino group of a nucleotide of the aptamer. Thereby can be used in the Apt ¬ mer forth ¬ tional methods of peptide synthesis such as solid phase synthesis, the coupling of the amino acid. The preparation of the complex is therefore possible without expensive additional synthesis process, where ¬ is reduced by the technical and financial effort. Furthermore, the complex of aptamer and amino acid can be can be used directly after attaching the 11 C-labeled amino acid. 11C carbon has a half-life of only about 20 minutes, so the longer the time between synthesis of the complex and its use, the higher the radiation dose must be. Is the amino acid attached to the 11 C-label in the last step of the synthesis, the aptamer can then be used immediately ¬ the. In this way, the time between the proces ¬ processing of the C-11 carbon and the use of the aptamer is re- duced so that the radiation loss is minimized during the herstel ¬ development of the complex. Therefore, the radiation dose ¬ which must be ¬ sets in the processing of the 11 C-carbon can to ensure a certain strength of the radiation Pro ¬ domestic product may be correspondingly lower. The manufacturer's position is more cost-effective and thereby the radiation exposure for the technical staff that provides the agent ago ¬ reduced.
Gemäß einer vorteilhaften Weiterbildung der Erfindung ist das 11C-Kohlenstoffatom das Carbonylkohlenstoffatom der Peptid- bindung. Die Peptidbindung liegt verhältnismäßig geschützt im Inneren des Komplexes aus Aptamer und Aminosäure. Dadurch ist gewährleistet, dass das 11C-Kohlenstoffatom nicht von der Aminosäure abgespalten wird, wie es etwa bei einer exponier- ten Seitenkette der Aminosäuren möglich wäre. According to an advantageous development of the invention, the 11 C carbon atom is the carbonyl carbon atom of the peptide bond. The peptide bond is relatively protected inside the complex of aptamer and amino acid. This ensures that the 11 C carbon atom is not split off from the amino acid, as would be possible with an exposed side chain of the amino acids.
In einer vorteilhaften Weiterbildung der Erfindung ist die Aminosäure eine D-Aminosäure. Mit Ausnahme des Glycins besit¬ zen alle Aminosäuren an ihrem alpha-C-Kohlenstoffatom ein chirales Zentrum und können daher als Konfigurationsisomere, nämlich als D- oder L-Aminosäure, vorliegen. Endogene Peptide und Proteine sind weitgehend aus Aminosäuren in L-Konfigu- ration aufgebaut. Zudem arbeiten die meisten natürlichen Proteasen und Peptidasen stereoselektiv und verstoffwechseln hauptsächlich L-Aminosäuren . Daher dauert der Abbau von D- Aminosäuren durch körpereigene Enzyme länger als der von L- Aminosäuren. Dieser Umstand kann verwendet werden, um die Halbwertszeit des Komplexes aus Aptamer und einer Aminosäure zu verlängern, indem statt einer L-Aminosäure eine D-Amino¬ säure verwendet wird (Neundorf I et al . , 2008) . Dadurch kann die Zeit bis die Aminosäure von dem Aptamer, durch proteoly¬ tischen Verdau, abgespalten wird, positiv beeinflusst werden. Eine weitere Möglichkeit, die Abspaltung zu verzögern, be- steht darin, eine nicht natürliche Aminosäure zu verwenden. Die nicht natürlichen Aminosäuren werden langsamer verstoff- wechselt, weil die körpereigenen proteolytischen Enzyme speziell an den Abbau natürlicher Aminosäuren angepasst sind. Darüber hinaus sind auch andere chemische Modifikationen der Aminosäure möglich. Beispielsweise kann die endständige Ami- nogruppe der Aminosäure durch eine Isonitrilgruppe ersetzt werden. Eine solche Modifikation reduziert die von der Ami- nogruppe vermittelte Interaktion mit proteolytischen Enzymen, ohne die Bindungsspezifität zu verändern. In an advantageous embodiment of the invention, the amino acid is a D-amino acid. With the exception of glycine besit ¬ zen all amino acids at its C-alpha-carbon atom is a chiral center and may therefore as configurational isomers, namely as D- or L-amino acid present. Endogenous peptides and proteins are largely composed of amino acids in L configuration. In addition, most natural proteases and peptidases work stereoselectively and metabolise mainly L-amino acids. Therefore, the degradation of D-amino acids by endogenous enzymes takes longer than that of L-amino acids. This fact can be used to prolong the half-life of the complex between aptamer and an amino acid by a D-amino acid ¬ is used instead of an L-amino acid (Neundorf I et al., 2008). Thereby, the time until the amino acid of the aptamer that is cleaved by Proteolytic digestion ¬ tables, are positively influenced. Another way to delay the cleavage is to use a non-natural amino acid. The non-natural amino acids are metabolized more slowly because the body's own proteolytic enzymes are specially adapted to the breakdown of natural amino acids. In addition, other chemical modifications of the amino acid are possible. For example, the terminal amino group of the amino acid can be replaced by an isonitrile group. Such a modification reduces the amino group mediated interaction with proteolytic enzymes without altering the binding specificity.
Ein weiterer Gegenstand der Erfindung ist ein Radiopharmakon zur Lokalisation eines Tumors, das ein Aptamer umfasst. Indem das Aptamer an den Tumor bindet und an eine Aminosäure gekop¬ pelt ist, die wiederum ein 11C-Kohlenstoffatom aufweist, kön- nen selbst wenige Zellen des Tumors lokalisiert werden. Another object of the invention is a radiopharmaceutical for the localization of a tumor comprising an aptamer. By the aptamer binds to the tumor and is gekop ¬ pelt to an amino acid, which in turn comprises a C-11 carbon atom kön- NEN even a few cells of the tumor are located.
Auf Grund der Vorteile des enthaltenen Komplexes aus Aptamer und Aminosäure bietet das erfindungsgemäße Radiopharmakon ein sensitives und spezifisches Agens, um die Position eines Tu- mors in vivo zu bestimmen. Das Radiopharmakon wird dem Patienten verabreicht und die darin enthaltenen Aptamere, die an die 11C-markierte Aminosäuren gekoppelt sind, verteilen sich auf Grund ihrer Größe schnell und effizient im Körper. Sie binden an den Tumor und sammeln sich an dessen Oberfläche. Die Häufung der radioaktiv markierten Aptamere wird mittels Positronen-Emissions-Tomographie (PET) nachgewiesen und so die genaue Position des Tumors im Körper des Patienten bestimmt . Because of the advantages of the contained aptamer-amino acid complex, the radiopharmaceutical of the present invention provides a sensitive and specific agent for determining the position of a tumor in vivo. The radiopharmaceutical is administered to the patient and the aptamers contained therein, which are coupled to the 11 C-labeled amino acids, are distributed quickly and efficiently in the body due to their size. They bind to the tumor and collect on its surface. The accumulation of radioactively labeled aptamers is detected by positron emission tomography (PET) to determine the exact location of the tumor in the patient's body.
Gemäß einer vorteilhaften Weiterbildung der Erfindung ist die Aminosäure Glycin, Alanin, Valin oder Serin, so dass weder die Konformation noch die Bindungsaffinität das Aptamers durch die Aminosäure beeinflusst werden. According to an advantageous development of the invention, the amino acid is glycine, alanine, valine or serine, so that neither the conformation nor the binding affinity of the aptamer is influenced by the amino acid.
Gemäß einer vorteilhaften Weiterbildung der Erfindung ist das ^C-Kohlenstoffatom das Kohlenstoffatom der endständigen α-Carboxylgruppe der Aminosäure. Dadurch kann das X1C- Kohlenstoffatom nicht von der Aminosäure abgespalten werden, wie es etwa bei einer exponierten Seitenkette der Aminosäuren möglich wäre. According to an advantageous embodiment of the invention, the C-carbon atom is the carbon atom of the terminal α-carboxyl group of the amino acid. As a result, the X1 C carbon atom can not be split off from the amino acid, as would be possible with an exposed side chain of the amino acids.
In einer bevorzugten Ausführungsform ist das Radiopharmakon ein PET Biomarker. Die PET ist ein etabliertes Verfahren um die Strahlung radioaktiver Elemente zu erfassen und ihre Position zu bestimmen (Massoud TF, Gambhir SS, 2003) . Mit Hilfe von ringförmig um den Patienten angeordneten Detektorgeräten werden Schnittbilder erstellt, auf denen die Zerfallsereig- nisse in ihrer räumlichen Verteilung im Körperinneren darge- stellt werden. Die PET ermöglicht es auch, die Menge an mar¬ kierten Molekülen in einem Gewebe quantitativ zu bestimmen. In a preferred embodiment, the radiopharmaceutical is a PET biomarker. PET is an established method for detecting the radiation of radioactive elements and determining their position (Massoud TF, Gambhir SS, 2003). With the aid of detector devices arranged annularly around the patient, sectional images are created on which the decay events in their spatial distribution in the interior of the body are represented. The PET also makes it possible to determine the amount of mar ¬-labeled molecules quantitatively in a tissue.
Außerdem wird ein Verfahren zur Lokalisation eines krankhaften Gewebes in einem Organismus offenbart, umfassend die Schritte: a) Bereitstellen eines Aptamers, das an eine Amino¬ säure gekoppelt ist; b) Verabreichen des Aptamers an den Or¬ ganismus; und c) Detektieren des Aptamers in dem Organismus mittels Positronen-Emissions-Tomographie (PET) . Indem das Ap- tamer an den Tumor bindet und die Aminosäure ein C-Kohlenstoffatom aufweist, kann das krankhafte Gewebe im Organismus des Patienten spezifisch lokalisiert werden. In addition, a method for localization of a diseased tissue is disclosed in an organism, comprising the steps of: a) providing an aptamer, which is coupled to an amino acid ¬; b) administering the aptamer to the Or ¬ organism; and c) detecting the aptamer in the organism by positron emission tomography (PET). By attaching the ap- ter to the tumor and inserting the amino acid Having C-carbon atom, the diseased tissue in the patient's organism can be specifically localized.
Mit dem erfindungsgemäß verwendeten Aptamer, an das eine 11C- markierte Aminosäure gekoppelt ist, wird ein krankhaftes Ge¬ webe im Inneren eines Organismus detektiert und lokalisiert, und kann so, im Körper eines Patienten, beobachtet werden. Auf diese Weise kann beispielsweise die Größe oder Ausdehnung einer Infektion oder eines Tumors bestimmt werden. Das erfin- dungsgemäß verwendete Aptamer ist daher hervorragend zur Be¬ obachtung von Verlauf und Erfolg einer Behandlung, sog. Therapiemonitoring, geeignet. With the aptamer according to the invention, to which a 11 C-labeled amino acid is coupled to a morbid Ge ¬ tissue is detected in the interior of an organism and localized, and can thus be in the body of a patient observed. In this way, for example, the size or extent of an infection or a tumor can be determined. The aptamer According to the invention used is therefore ideal for Be ¬ observation of progress and success of a treatment, so-called. Therapy monitoring, appropriate.
Im Folgenden werden bevorzugte Ausführungsformen der Erfin- dung anhand der beigefügten schematischen Zeichnungen erläutert . In the following, preferred embodiments of the invention are explained with reference to the attached schematic drawings.
Figur 1 zeigt schematisch ein Aptamer 1 mit 14 Nukleotiden 3, das an ein krankhaftes Gewebe 18 gebunden ist. Das Aptamer 1 ist an eine Aminosäure 2 gekoppelt, die mit einem X1C-FIG. 1 shows schematically an aptamer 1 with 14 nucleotides 3, which is bound to a pathological tissue 18. The aptamer 1 is coupled to an amino acid 2 which is linked to an X1 C-
Kohlenstoffatom radioaktiv markiert ist. Die radioaktive Markierung ist durch einen Stern (*) dargestellt. Carbon atom is radiolabeled. The radioactive label is represented by an asterisk (*).
Die spezifische Bindungsaffinität zwischen dem Aptamer 1 und dem krankhaften Gewebe 18 kommt auf Grund chemischer Wechsel¬ wirkungen zwischen dem Aptamer 1 und der Oberfläche des krankhaften Gewebes 18 zustande. Das krankhafte Gewebe 18 bildet Oberflächenmoleküle, die für die Erkrankung des Gewe¬ bes kennzeichnend sind. Aus einer Aptamer-Bibliothek wird ein Aptamer 1 identifiziert, das mit einem Oberflächenmolekül des krankhaften Gewebes 18 interagiert. Anschließend wird eine Aminosäure, die mit einem ^C-Kohlenstoffatom markiert ist, an das Aptamer 1 gekoppelt. Das Aptamer 1 kann dann durch die beim Zerfall des ^C-Kohlenstoffatoms abgegebenen Positronen mittels Positronen-Emissions-Tomographie (PET) nachgewiesen werden. Der Ort der Positronenemission entspricht dem Ort des Aptamers 1 und somit dem des krankhaften Gewebes 18, an wel¬ ches das Aptamer 1 gebunden ist. The specific binding affinity between the aptamer 1 and the pathological tissue 18 comes off due to chemical exchange ¬ effects between the aptamer 1 and the surface of the diseased tissue 18th The pathological tissue 18 forms surface molecules that are characteristic of the disease of the tissue ¬ esp. From an aptamer library, an aptamer 1 is identified that interacts with a surface molecule of diseased tissue 18. Subsequently, an amino acid labeled with a C-carbon atom is coupled to the aptamer 1. The aptamer 1 can then be replaced by the positrons emitted upon the decay of the ^ C carbon atom be detected by positron emission tomography (PET). The location of the positron emission corresponding to the location of the aptamer 1, and thus that of the diseased tissue 18 to wel ¬ ches bound aptamer. 1
Das an eine 11C-markierte Aminosäure 2 gekoppelte Aptamer 1 wird einem Patienten in Form eines Arzneimittels verabreicht. Es bindet an das krankhafte Gewebe 18 und sammelt sich an dessen Zellen. Diese Anhäufung wird bei einer PET sichtbar, so dass die Verteilung des Aptamer 1 bzw. die Position des krankhaften Gewebes 18 im Körper des Patienten bestimmt werden können. The aptamer 1 coupled to an 11 C-labeled amino acid 2 is administered to a patient in the form of a drug. It binds to the pathological tissue 18 and collects on its cells. This accumulation becomes visible in a PET, so that the distribution of the aptamer 1 or the position of the diseased tissue 18 in the body of the patient can be determined.
Figur 2 zeigt eine Darstellung eines Aptamers mittels chemi- scher Formel. Das Aptamer hat die Sequenz SEQ ID Nr.: 1 und ist an eine 11C-markierte Aminosäure, nämlich Glycin, gekop¬ pelt. FIG. 2 shows a representation of an aptamer by means of a chemical formula. The aptamer has the sequence SEQ ID No .: 1 and is gekop ¬ pelt to a 11 C-labeled amino acid, namely, glycine.
Das Aptamer umfasst 70 Nukleinsäuren der folgenden Sequenz: GGG AGG ACG AUG CGG ACC GAA AAA GAC CUG ACU UCU AUA CUA AGU CUA CGU UCC CAG ACG ACU CGC CCG A. The aptamer comprises 70 nucleic acids of the following sequence: GGG AGG ACG AUG CGG ACC GAA AAA GAC CUG ACU UCU AUA CUA AGU CUA CGU UCC CAG ACG ACU CGC CCG A.
Das 3' terminale Adenosin des Aptamers und das daran gekop¬ pelte Glycin sind mittels Strukturformel dargestellt, die restlichen Nukleinsäuren 3 durch ihren jeweiligen Buchstaben Code. Die Sequenz des Aptamers ist auch in SEQ ID Nr.: 1 angegeben. Das Carbonylkohlenstoffatom Glycins ist ein X1C- Kohlenstoffatom, dargestellt durch die Ziffer 11 oberhalb des Carbonylkohlenstoffatoms . The 3 'terminal adenosine of the aptamer and it gekop ¬ pelte glycine are represented by structural formula, and the remaining nucleic acids 3 by their respective letter code. The sequence of the aptamer is also given in SEQ ID NO: 1. The carbonyl carbon glycine is an X1 C carbon atom represented by the number 11 above the carbonyl carbon atom.
Das Aptamer mit der SEQ ID Nr.: 1 weist eine spezifische Bin¬ dungsaffinität zu dem Prostata spezifischen Membran Antigen (PSMA) auf. Dieses Protein wird vor allem in Prostatagewebe exprimiert und kommt in Prostatkarzinomen in besonders hohen Mengen vor. PSMA ist ein Membranprotein, das in gesunden Prostatazellen aber in einer besonderen zytoplasmatischen Form vorliegt. Bei Prostatakarzinomen ist es vor allem auf der Zellmembran der Tumorzellen lokalisiert. The aptamer of SEQ ID NO .: 1 has a specific binding affinity Bin ¬ specific to the prostate membrane antigen (PSMA) on. This protein is mainly expressed in prostate tissue and comes in prostate carcinomas in particularly high Quantities. PSMA is a membrane protein present in healthy prostate cells but in a particular cytoplasmic form. In prostate cancer, it is mainly located on the cell membrane of tumor cells.
Das Aptamer 1 wird anhand seiner Bindungsspezifität zu PSMA mit SELEX Verfahren aus einer Aptamer-Bibliothek ausgewählt und amplifiziert . Anschließend wird die 11C-markierte Amino¬ säure 2 mit einem herkömmlichen Peptidsyntheseverfahren an das Aptamer 1 gekoppelt. Das Aptamer 1 wird dem Patienten verabreicht und so das Prostatakarzinom mittels PET darge¬ stellt. Auf diese Weise können auch Metastasen, die ebenfalls membranständiges PSMA exprimieren, lokalisiert werden. Figur 3 zeigt eine schematische Darstellung (stark vereinfacht nach Faller A, Schünke M, Der Körper des Menschen, Thieme, 2008) eines Blutkreislaufsystems 10 eines Organismus und die Verteilung eines Aptamers 1 darin. Das Blutkreislaufsystem 10 umfasst verschiedene schematisch dargestellte Organe, wie Lunge 12, Herz 13, Leber 14, Darm 15 und Niere 16 und die Hauptadern 11, welche diese Organe ver¬ binden. Das Aptamer 1 ist durch Dreiecke entlang der Adern 11 dargestellt. Die Abbauprodukte 17 des Aptamers 1 sind durch einzelne Striche innerhalb der Umrisse der Niere 16 darge¬ stellt. Links der Mitte des Blutkreislaufsystems 10 ist zu¬ sätzlich ein krankhaftes Gewebe 18, zum Beispiel ein Tumor oder eine Entzündung, dargestellt, an das vermehrt Aptamere 1 angelagert sind. The aptamer 1 is selected and amplified by its binding specificity to PSMA with SELEX methods from an aptamer library. Subsequently, the 11 C-labeled amino acid ¬ 2 is coupled to a conventional peptide synthesis methods to the aptamer. 1 The aptamer 1 is administered to the patient and thus the prostate carcinoma by PET Darge ¬ presents. In this way, metastases which also express membrane-bound PSMA can be localized. Figure 3 shows a schematic representation (greatly simplified by Faller A, Schünke M, The human body, Thieme, 2008) of a circulatory system 10 of an organism and the distribution of an aptamer 1 therein. The circulation system 10 includes various organs schematically represented, such as the lungs 12, heart 13, liver 14, 15 intestine and kidney 16 and the main wires 11 which these organs ver ¬ bind. The aptamer 1 is represented by triangles along the wires 11. The degradation products 17 of the aptamer 1 are represented by individual lines within the outline of the kidney 16 Darge ¬ . Left of center of the circulatory system 10 is additionally ¬ a diseased tissue 18, for example, a tumor or an inflammation, shown, are attached to the increased aptamers. 1
Die Verteilung des Aptamers 1 im Blutkreislaufsystem 10 umfasst vier Phasen, die entlang der Darstellung von oben nach unten aufgeführt sind. Phase I: Das Aptamer 1 wird in das Blutkreislaufsystem 10 des Organismus injiziert. The distribution of the aptamer 1 in the circulatory system 10 includes four phases listed along the top-to-bottom illustration. Phase I: The aptamer 1 is injected into the circulatory system 10 of the organism.
Phase II: Über das Blutkreislaufsystem 10 wird das Aptamer 1 in die Organe 12, 13, 14, 15, und 16 des Organismus transpor¬ tiert . Phase II: is via the blood circulation system 10, the aptamer 1 in the organs 12, 13, 14, 15, and 16 of the ¬ organism transported advantage.
Phase III: Das zirkulierende Aptamer 1 bindet spezifisch an das krankhafte Gewebe 18. Phase III: The circulating aptamer 1 binds specifically to the diseased tissue 18.
Phase IV: Nicht gebundenes Aptamer 1 wird schnell verstoff- wechselt und enzymatisch abgebaut. Der Organismus unterschei¬ det nicht zwischen körpereigenen Molekülen und dem Aptamer 1, weil es aus Nukleinsäuren 3 und einer Aminosäure 2 aufgebaut ist, die den körpereigenen Molekülen entsprechen. Die Abbauprodukte 17 des Aptamer 1 und der Nukleinsäuren 3 bzw. der Aminosäure 2 sammeln sich vorwiegend in der Niere 16, von wo aus sie über die Blase und den Harnleiter ausgeschieden werden . Phase IV: Unbound aptamer 1 is rapidly metabolised and enzymatically degraded. The organism not failed ¬ det between the body's own molecules and the aptamer 1, because it is constructed from nucleic acids 3 and an amino acid 2, which correspond to the body's own molecules. The degradation products 17 of the aptamer 1 and the nucleic acids 3 and the amino acid 2 accumulate predominantly in the kidney 16, from where they are excreted via the bladder and the ureter.
Referenzen : References :
Faller A, Schünke M; Der Körper des Menschen; Thieme-Verlag; 2008 Faller A, Schünke M; The body of man; Thieme-Verlag; 2008
Ja ier DJ, Nitin N, Levy M, Ellington A, Richards-Kortum R; Aptamer-targeted gold nanoparticles as molecular-specific contrast agents for reflectance imaging; Bioconjug Chem. 2008 Jun; 19(6) : 1309-12. Yes DJ, Nitin N, Levy M, Ellington A, Richards-Kortum R; Aptamer-targeted gold nanoparticles as molecular-specific contrast agents for reflectance imaging; Bioconjug Chem. 2008 Jun; 19 (6): 1309-12.
Ku1bachinskiy AV; Methods for selection of aptamers to pro¬ tein targets; Biochemistry (Mose) ; 2007 Dec; 72 (13) : 1505-18. Ku1bachinskiy AV; Methods for selection of aptamers to pro ¬ tain targets; Biochemistry (Moses); 2007 Dec; 72 (13): 1505-18.
Massoud TF, Ga bhir SS; Molecular imaging in living subjects: seeing fundamental biological processes in a new light; Genes Dev. 2003 Mar 1; 17 (5) : 545-80. Massoud TF, Ga bhir SS; Molecular imaging in living subjects: seeing fundamental biological processes in a new light; Genes Dev. 2003 Mar 1; 17 (5): 545-80.
Neundorf I, Rennert R, Franke J, Közle I, Bergmann R; De- tailed analysis concerning the biodistribution and metabolism of human calcitonin-derived cell-penetrating peptides; Bio¬ conjug Chem. 2008 Aug; 19 (8) : 1596-603. Neundorf I, Rennert R, Franke J, Közle I, Bergmann R; Detailed analysis concerning the biodistribution and metabolism of human calcitonin-derived cell-penetrating peptides; Bio ¬ conjug Chem. 2008 Aug; 19 (8): 1596-603.

Claims

Verwendung eines Aptamers (1) zur Herstellung eines Agens zur Detektion eines krankhaften Gewebes (18), d a d u r c h g e k e n n z e i c h n e t, Use of an aptamer (1) for the production of an agent for the detection of a diseased tissue (18), d a d u r c h e c e n e c e n e,
dass das Aptamer (1) an das krankhafte Gewebe (18) bin¬ det, an eine Aminosäure (2) gekoppelt ist und die Amino¬ säure (2) ein 11C-Kohlenstoffatom aufweist, wobei die Aminosäure (2) über eine Peptidbindung an eine freie Aminogruppe eines Nukleotids (3) des Aptamers (1) gekop¬ pelt ist. that the aptamer (1) to the diseased tissue (18) am ¬ det, to an amino acid (2) is coupled, and the amino ¬ acid (2) having a 11 C carbon atom, wherein the amino acid (2) via a peptide bond to a free amino group of a nucleotide (3) of the aptamer (1) is gekop ¬ pelt.
Verwendung eines Aptamers (1) nach Anspruch 1, Use of an aptamer (1) according to claim 1,
d a d u r c h g e k e n n z e i c h n e t, characterized,
dass das Agens ein Radiopharmakon ist. that the agent is a radiopharmaceutical.
Verwendung eines Aptamers (1) nach Anspruch 1 oder 2, d a d u r c h g e k e n n z e i c h n e t, Use of an aptamer (1) according to claim 1 or 2, a d a c e c e s e c e n e c e n e,
dass die Aminosäure (2) ausgewählt ist aus der Gruppe bestehend aus Glycin, Alanin, Valin und Serin. the amino acid (2) is selected from the group consisting of glycine, alanine, valine and serine.
Verwendung eines Aptamers (1) nach einem der vorangehenden Ansprüche, Use of an aptamer (1) according to one of the preceding claims,
d a d u r c h g e k e n n z e i c h n e t, characterized,
dass das 11C-Kohlenstoffatom das Carbonylkohlenstoffatom der Peptidbindung ist. the 11 C carbon atom is the carbonyl carbon of the peptide bond.
Radiopharmakon zur Lokalisation eines Tumors (18) umfassend ein Aptamer (1), Radiopharmaceutical for the localization of a tumor (18) comprising an aptamer (1),
d a d u r c h g e k e n n z e i c h n e t, characterized,
dass das Aptamer (1) an den Tumor (18) bindet, an eine Aminosäure (2) gekoppelt ist und die Aminosäure (2) ein 11C-Kohlenstoffatom aufweist, wobei die Aminosäure (2) über eine Peptidbindung an eine freie Aminogruppe eines Nukleotids (3) des Aptamers (1) gekoppelt ist. the aptamer (1) binds to the tumor (18), is coupled to an amino acid (2) and the amino acid (2) has an 11 C carbon atom, the amino acid (2) is coupled via a peptide bond to a free amino group of a nucleotide (3) of the aptamer (1).
6. Radiopharmakon nach Anspruch 5, 6. radiopharmaceutical according to claim 5,
d a d u r c h g e k e n n z e i c h n e t,  characterized,
dass die Aminosäure (2) ausgewählt ist aus der Gruppe bestehend aus Glycin, Alanin, Valin und Serin.  the amino acid (2) is selected from the group consisting of glycine, alanine, valine and serine.
7. Radiopharmakon nach Anspruch 5 oder 6, 7. radiopharmaceutical according to claim 5 or 6,
d a d u r c h g e k e n n z e i c h n e t,  characterized,
dass das 11C-Kohlenstoffatom das Kohlenstoffatom der endständigen α-Carboxylgruppe der Aminosäure (2) ist. the 11 C carbon atom is the carbon atom of the terminal α-carboxyl group of amino acid (2).
8. Radiopharmakon nach einem der Ansprüche 5 bis 7, 8. radiopharmaceutical according to one of claims 5 to 7,
d a d u r c h g e k e n n z e i c h n e t,  characterized,
dass es ein Positronen-Emissions-Tomographie (PET) Bio- marker ist.  that it is a positron emission tomography (PET) biomarker.
PCT/EP2011/060421 2010-06-30 2011-06-22 11c-labelled aptamer for detecting a diseased tissue WO2012000861A1 (en)

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