WO2012000865A1

WO2012000865A1 - 11c-marked peptide for detecting a tumor that expresses an her2/neu receptor

Info

Publication number: WO2012000865A1
Application number: PCT/EP2011/060447
Authority: WO
Inventors: Friedrich Herberg; Hartmuth C. Kolb; Ursus KRÜGER; Oliver Lade; Arno Steckenborn
Original assignee: Siemens Aktiengesellschaft; Universität Kassel
Priority date: 2010-06-30
Filing date: 2011-06-22
Publication date: 2012-01-05
Also published as: DE102010026061A1

Abstract

The invention relates to the use of a peptide (1) for producing an agent for detecting a tumor (18) that expresses an HER2/neu receptor (4). The peptide (1) bonds to the HER2/neu receptor (4) and has an ¹¹C carbon atom. The invention further relates to a radiopharmaceutical for locating a tumor (18) that expresses an HER2/neu receptor (4). Said radiopharmaceutical comprises a peptide (1) that bonds to the HER2/neu receptor (4) and has an ¹¹C carbon atom.

Description

description

¹¹ C-labeled peptide for the detection of a tumor expressing a Her2 / neu receptor

The invention relates to the use of a peptide for the manufacture ^¬ position of an agent for detecting a tumor expressing a Her2 / neu receptor. It further relates to a radiopharmaceutical comprising such a peptide for the localization of a tumor expressing a Her2 / neu receptor.

In modern cancer diagnostics, both biochemical analyzes of blood and other body fluids, as well as imaging techniques for the detection of tumors are used. Traditionally, X-ray, ultrasound and Kernspintomogra ^¬ phy be used to take advantage of ectopic cell clusters to localize ^¬ ren. Newer methods, the increased metabolic activity of tumor cells compared to healthy tissue. The patient is injected with radioactively labeled sugar molecules that accumulate in the tumor cells. Subsequently, the radioactivity of these molecules, taken at ^¬ game as with a gamma camera, the so-called Szinti ^¬ chromatography, and the position of the tumor Festge provides ^¬. Biochemically, cancers are detected using tumor-specific molecules. The presence and amount of these substances in blood or tissue samples of the patient is determined. In addition to soluble substances that are released into body fluids, tumor cells also produce molecules that remain anchored to their cell surface. This is mainly to cell receptors, such as receptors of the epidermal growth factor, insulin-like growth factor or wax ^¬ tumshormons. On the basis of these surface molecules, a biochemical chemical detection of tumor cells in vivo by visualizing them using imaging techniques.

The Her2 / neu receptor, also known as ErbB2, is expressed by a large number of different cancers. It belongs to the family of epidermal growth factor (EGF) receptors. Unlike other receptors of this family, the Her2 / neu receptor itself does not bind ligands, but forms heterodimers with other EGF receptors after they have been ligated. The Her2 / neu receptor thereby contributes to the enhancement of the, via the tyrosine kinase function of the EGF receptors mediated intracellular signal, which is triggered by the binding of the ligand. To detect Her2 / neu receptors in vivo, suitable ligands were radiolabeled so that they could be detected by scintigraphy / positron emission tomography (PET) in the patient's body. For this they were provided with radionuclides via large chelator molecules, for example 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) (Smith-Jones PM et al., 2006). The

However, preparation of such radiolabeled molecules is very expensive, since the three components, ligand, chelator and radionuclide, must first be provided individually and then connected. In addition, conventional radioactive-labeled Her2 / neu receptor ligands cause side effects such as malaise or allergies in many patients due to their foreign radioactive elements and chemical chelator molecules. The invention is therefore based on the object, a cost-effective and well-tolerated for the patient agent for the detection of a tumor expressing a Her2 / neu receptor to provide. This object is achieved by the use of a peptide for the production of an agent for detection of a tumor expressing a Her2 / neu receptor. By using a peptide which binds to the Her2 / neu receptor and has an ¹¹ C carbon atom, the agent can be inexpensively produced and metabolized well in the organism in which the tumor is detected.

The term "peptide" refers to an organic compound of at least two amino acids linked via a peptide bond. It includes both oligopeptides of up to about ten amino acids, polypeptides up to about 50 Aminosäu ^¬ ren and proteins of up to 150 amino acids, regardless of their primary, secondary or tertiary structure. In this case, both naturally occurring and biotechnologically or synthetically produced compounds are included. The peptide used according to the invention is chosen so that it binds to the Her2 / neu receptor. Suitable are, antibodies and fragments thereof, such as trastuzumab, affibodies (US 2010/0048868), Nanobodies (WO 2009/068625), and other Po ^¬ lypeptide, (as described in WO 2009/080810 described) to the Her2 / neu receptor tie. By their specific binding to the Her2 / neu receptor, the peptides can be used to detect tumors that form the Her2 / neu receptor. Preferably, the peptide is chosen so that the Bin ^¬ connection between the peptide and the Her2 / neu receptor, a so-called lithium-linear coefficient. KD value of <100 nM, preferably <10 nM, most preferably of 7 , 5 nM. The peptide itself is made of amino acids, that is constructed so that it is very well tolerated by the patient from körpereige ^¬ nen or body similar molecules. It is non-toxic and can be naturally metabolized, broken down and excreted ^¬ to be.

The term "tumor" refers to a local increase in Vo ^¬ lumens of tissue, such as an inflammatory Anschwel- or a spontaneous, unrestrained new formation of cells. Tumor cells often express certain receptor molecules that sit on the cell surface and are bound by specific ligands. These receptors also belong

Her2 / neu receptors, which are strongly expressed in breast cancers, among others. Many of the tumors expressing the Her2 / neu receptor show aggressively invasive behavior, resulting in a correspondingly unfavorable disease prognosis. In most cases, the extremely increased expression of the Her2 / neu receptor is due to amplification of the

Her2 / neu gene (c-erbB2) returned to tumor cells. Tumor cells expressing Her2 / neu receptor can therefore be distinguished well from healthy tissue by means of the peptide used according to the invention, since the tumor cells carry substantially larger amounts of Her2 / neu receptors and therefore more

Bind peptides to them. This allows a reliable Loka ^¬ lization of the tumor.

Detection of the peptide and the Her2 / neu receptor bound thereto is via an integrated ¹¹ C carbon atom. Upon decay of the ¹¹ C carbon isotope, positrons, also referred to as β ⁺ radiation, are formed. If the positrons hit an electron, they form two photons which move away from each other at an angle of 180 °, ie exactly in the opposite direction. The photons can be detected and used to calculate the position of the positron emission, or of the ¹¹ C carbon atom. The Integra ^¬ tion of a ^C-11 carbon atom in the peptide used in the invention, makes it possible to avoid the use of chemical, physi- perfremder substances. By the direct incorporation of ¹¹ C-carbon isotope in the peptide is the radioactive Mar ^¬ kierung without complexing agents such as diethylene triamine pentaacetate (DTPA), 1,4,7, 10-tetraazacyclododecanes-l, 4,7, 10-tetraacetic acid (DOTA) or ethylenediamine tetraacetate (EDTA), possible. In addition, it can be avoided that a radioactive foreign substance, such as fluorine, xenon, or gallium, must be introduced into the organism. For the preparation of a peptide to be used according to the invention, the processes described in patent applications DE 10 2009 035 648.7 and DE 10 2009 035 645.2 are particularly suitable. Thus, by the use of the peptide according to the invention, both the presence and the position of the Her2 / neu receptor can be detected and mapped. Furthermore, the amount of peptides located at a particular site can also be quantified.

Another advantage of the peptide directly labeled with ^X1 C lies in the favorable signal / background ratio during detection. The peptide binds specifically to the Her2 / neu receptor and forms a stable complex with it. Free, unbound peptides, on the other hand, are rapidly metabolized and excreted from the organism because they can be rapidly degraded by endogenous enzymes. This creates a strong and specific signal at the position of the Her2 / neu receptor, and the background signal is minimized.

In an advantageous embodiment of the invention, the peptide has at least one D-amino acid. With the exception of glycine, amino acids have a chiral center at their alpha carbon atom and can therefore exist as configurational isomers, namely as the D or L amino acid. Endogenous peptides and proteins are largely made up of amino acids in ^¬ L-configuration. In addition, most natural proteases and peptidases work stereoselectively and mainly metabolize 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 predict the half-life of a protein or peptide. b longer, in addition to L-amino acids and D-amino acids are used (Neundorf I et al., 2008). As a result, the pharmacological clearance, ie the time until the peptide is eliminated from the organism, can be positively influenced. However, when replacing individual L-amino acids with their D-configuration, care must be taken not to alter the binding specificity of the peptide. A ^further possibility to influence the pharmacological clearance of the peptide is to replace some of the amino acids of the peptide with non-natural amino acids having similar chemical properties. The non-natural amino acids are metabolized more slowly because the körperei ^¬-related proteolytic enzymes are specially adapted to the exploitation of natural amino acids. However, when modifying the peptide, the non-natural amino acids should be chosen so that the binding affinity of the peptide is not altered. In addition, other chemical Mo ^¬ difikationen individual amino acids of the peptide are possible to influence the half-life of the peptide specifically. For example, the terminal amino group of the peptide can be replaced by an isonitrile group. Such modes ^¬ fication reduces, mediated by the amino group, in ^¬ teraktion with proteolytic enzymes, without the bond between the peptide used in the invention and the

To change Her2 / neu receptor.

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 attached via Kom ^¬ formers such as DOTA, DTPA or EDTA mono- or polysaccharides. The nuclides are detected by scintigraphy, single photon emission com- puted tomography (SPECT) or positron emission tomography (PET), depending on the nature of their radiation. However, due to their unphysiologi ^¬ rule ingredients conventional radiopharmaceuticals can cause side effects such as anaphylactic or allergic Reaktio ^¬ nen, in the body of a patient. The use of a peptide from the body's own amino acids reduces these

Danger clearly, because neither the peptide itself, nor its Ab ^{¬ building} products are toxic. In addition, unlike technetium or xenon, carbon is an element found in the body that naturally can be metabolized.

In a preferred embodiment, the tumor expresses increased levels of the Her2 / neu receptor. In comparison to healthy tissue, the cells of different tumors carry particularly high amounts of Her2 / neu receptors on their surface. These include, for example, lung, breast, and ovarian cancer, carcino ^¬ me, such as breast cancer, and tumors of the gastrointestinal tract.

According to an advantageous development of the invention, the C-carbon atom is the carbonyl carbon atom of an amino acid. The carbonyl groups are part of the peptide bonds between the amino acids and are located inside the peptide. This ensures that the ^ C carbon atom is not cleaved off the peptide, as would be possible with a side chain of one of the amino acids.

In a further preferred embodiment of the invention, the C-carbon atom is the carbonyl carbon atom of the N-terminal amino acid of the peptide. This embodiment is particularly preferred because the peptide can be used directly after the on ^¬ bring the ¹¹ C-labeled amino acid. ¹¹ C-carbon has a half-life of only about 20 Minu ^¬ th, so that the radiation dose must be chosen the higher, the more time is between the synthesis of the peptide and its ^¬ ner use. If the ¹¹ C-labeling with the N-terminal amino acid and thus in the last step of the synthesis is applied, the peptide can be used immediately after its synthesis. In this way, the time between the processing of the ¹¹ C carbon and the use of the peptide is reduced, so that the radiation loss during the preparation of the peptide is minimized. Therefore, the radiation dose that must be used in the processing of the ¹¹ C carbon to ensure a certain radiation intensity of the product, be correspondingly lower. The

Production is less expensive and thus the radiation exposure for the technical staff that forth ^¬ represents the peptide reduced. Another object of the invention is a radiopharmaceutical comprising a peptide having an ¹¹ C carbon atom for the localization of a tumor expressing a Her2 / neu receptor. By using a peptide that binds to the Her2 / neu receptor and has an ¹¹ C carbon atom, the radiopharmaceutical is well tolerated by the patient and can be produced inexpensively.

Therefore, the radiopharmaceutical invention provides a host ^¬ economically and medically beneficial agent to the posi- tion of a tumor that expresses a Her2 / neu receptor to determine in vivo. After the radiopharmaceutical has been administered to a patient, the peptides contained therein are distributed into the body and specifically bind to Her2 / neu receptors. As a result, they accumulate on the cells of the tumor where they are detected by the radioactive signal of the C carbon atom. In this way, the position of the tumor in the body of the patient is determined. In a preferred embodiment of the tumor expressed in comparison to healthy tissue, increased amounts of the Her2 / neu receptor, as WUR observed for various tumor types ^¬ de. According to an advantageous embodiment, the ^X1 C-

Carbon atom is a carbonyl carbon atom of an amino acid, preferably the carbonyl carbon atom of the N-terminal amino acid of the peptide. 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 are represented in their spatial distribution in the interior of the body. In contrast to the usual scintigraphic chromatography method is nenemission a more precise spatial localization of the positron by the annular configuration of the PET detectors and hence a much more precise and detailed ^¬ profiled image of the tumor possible. PET also makes it possible to quantify the amount of labeled molecules in a tissue. Also disclosed is a method of localizing a tumor expressing a Her2 / neu receptor in an organism, comprising the steps of a) providing a peptide, b) administering the peptide to the organism, and c) detecting the peptide in the organism Organism using positron Emission tomography (PET). The peptide binds to the Her2 / neu receptor and has an ¹¹ C carbon atom.

With the peptide used in the invention is a Her2 / neu receptor in the interior of an organism is detected and lokali ^¬ Siert, so that the distribution of the Her2 / neu receptor in Kör ^¬ can be observed by a patient. In this way, for example, the size or extent of an infection or of a tumor expressing the Her2 / neu receptor can be determined. The peptide used according to the invention is ^therefore outstandingly suitable for observing the course and success of a treatment, so-called therapy monitoring.

In the following, preferred embodiments of the invention are explained with reference to the attached schematic drawings.

FIG. 1 shows schematically the binding between a peptide 1 and a Her2 / neu receptor 4.

Peptide 1 comprises 19 amino acids 2, of which the N-terminal amino acid 3 is radioactively labeled with an ¹¹ C carbon atom. The radioactive label is represented by an asterisk (*). Part of the peptide 1 is attached to the Her2 / neu receptor 4, which is located on the surface of a tumor 18.

The ¹¹ C-labeled peptide 1 binds specifically to the Her2 / neu receptor 4, but not to other molecules. The peptide 1 can therefore be used to detect the Her2 / neu receptor 4 ^¬ . The positrons released upon decay of the ¹¹ C carbon are detected by positron emission tomography (PET). The location of the positron emission corresponds to the location of peptide 1 and the bound Her2 / neu- Receptor 4. Peptide 1 can therefore be used to determine the position of a tumor 18 that forms the Her2 / neu receptor 4. To localize a tumor 18 as part of a cancer diagnosis, a patient is administered a radiopharmaceutical containing the ¹¹ C-labeled peptide 1. Peptide 1 binds specifically to the Her2 / neu receptor 4 and thus accumulates on the tumor 18, whose cells form the Her2 / neu receptor 4. This accumulation is represented by PET and the distribution of the Her2 / neu receptor 4 or the localization of the tumor 18 in the body of the patient is determined. In this way, newly formed metastases carrying the Her2 / neu receptor can be identified by PET. In addition, the medication of a tumor therapeutic, for example, drug amount and

Administration schedule, according to the position, size and distribution of the tumor 18 adapted.

FIG. 2 shows a representation of a peptide having the sequence SEQ ID NO: 1 by means of a chemical formula.

The peptide of SEQ ID NO: 1 comprises 35 amino acids 2 of the following sequence: homocysteine proline aspartic acid aspartic acid tyrosine isoleucine serine arginine isoleucine lysine alanine arginine lysine glutamine glutamine asparagine asparagine Leucine-asparagine-proline-aspartic acid-leucine-alanine-alanine-glutamic acid-tryptophan-tyrosine-arginine-asparagine-arginine-methionine-glutamic acid-lysine-asparagine homocysteine.

The N-terminal amino acids 2 proline and homocysteine are represented by structural formula, the following amino acids 2 by their respective three-letter code. The amino acid homocysteine is designated Cys _H. The sequence of the peptide is also given in SEQ ID NO: 1. The carbonyl carbon atom of the N-terminal homocysteine is a ^X1 C carbon atom, represented by the number 11 above the carbonyl carbon atom.

Peptide 1 is prepared by conventional protein synthesis methods and the ¹¹ C-labeled N-terminal amino acid 3 is added in the last step because the half-life of the ¹¹ C carbon isotope is only about 20 minutes. Characterized in that the peptide synthesis is closed with the ¹¹ C-labeled amino acid from ^¬, Peptide 1 can be used immediately after labeling.

SEQ ID NO: 1 corresponds to the Her2 / neu receptor-binding 2-helix protein MUT-DS (Ren G et al., 2009). The peptide of the sequence SEQ ID NO: 1 belongs to the so-called Affibodies. These are artificial peptides or proteins derived from one of the IgG-binding domains of protein A of the bacterium Staphylococcus aureus. They regularly include 3 alpha helices and are about 7 kDa in size. You do not trigger immunological reactions and are therefore geeig ^¬ net as biomolecules for preparing a radiopharmaceutical. To find a Her2 / neu receptor-binding affibody, corresponding libraries are screened by phage display technology (Wikman M et al., 2004). Small molecules that show a better distribution and clearance in the body are preferred for Ver ^¬ application for preparing a radiopharmaceutical. Therefore, the peptide of sequence SEQ ID NO: 1 comprises only the two alpha helices actually involved in binding to the Her2 / neu receptor 4 (Ren G et al., 2009).

Peptide 1 binds specifically to the Her2 / neu receptor 4, which is formed in large quantities by some tumor cells, and it is used to localize ^¬ ren these tumor cells. A mark by ¹¹ C-carbon is particular ^¬ DERS suitable because it does not affect the physiological structure of the Pep ^¬ TIDS 1 and neither the tissue distribution and tolerability of the peptide 1 impaired.

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 a peptide 1 therein.

The circulatory system 10 comprises various schematically represented organs, such as lung 12, heart 13, liver 14, intestine 15 and kidney 16, and the main arteries 11, which connect these organs. The peptide 1 is represented by triangles along the wires 11. The degradation products 17 of the peptide 1 are represented by individual lines within the outline of the kidney 16 Darge ^¬ . Left of center of the circulatory system 10, a tumor is to be ^¬ additionally shown 18, are attached to the increased Her2 / neu receptors 4 and peptides thereon. 1

The distribution of the peptide 1 in the circulatory system 10 comprises four phases, which are listed along the top-down view.

Phase I: Peptide 1 is injected into the circulatory system 10 of the organism.

Phase II: Via the circulatory system 10, the peptide 1 is transpo ted into the organs 12, 13, 14, 15, and 16 of the organism. Phase III: The circulating peptide 1 binds specifically to the Her2 / neu receptor 4 and accumulates on the tumor 18 because it produces the Her2 / neu receptor 4. Phase IV: Unbound peptide 1 is rapidly metabolised and enzymatically degraded. The organism not failed ^¬ det between own peptides and the peptide 1, because it is composed of amino acids 2, 3, which correspond to the body's own molecules. The degradation products 17 of the peptide of amino acids 1 and 2, 3 collect predominantly they are over the bladder and the ureter excreted ^¬ in the kidney 16 from where.

References :

Cheng Z, De Jesus GP, Namavari M, De A, Levi J, Webster JM, Zhang R, Lee B, Syud FA, Gambhir SS Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F- labeled protein scaffold molecules; J Nucl Med, 2008 May; 49 (5): 804-13.

Faller A, Schünke M; The body of man; Thieme-Verlag; 2008

Massoud TF, Gambhir 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; Detailed analysis concerning the biodistribution and metabolism of human calcitonin-derived cell-penetrating peptides; Bioconjug Chem. 2008 Aug; 19 (8): 1596-603.

Ren G, Zhang R, Liu Z, Webster JM, Miao Z, Gambhir SS, Syud FA, Cheng Z; A 2-helix small protein labeled 68Ga for PET imaging of HER2 expression .; J Nucl Med. 2009

Sep; 50 (9): 1492-9.

Smith-Jones PM, Solit D, Afroze F, Rosen N, Larson SM; Early tumor response to Hsp90 therapy using HER2 PET: comparison with 18F-FDG PET; J Nucl Med. 2006 May; 47 (5): 793-6. Wikman M, Steffen AC, Gunneriusson E, Tolmachev V, Adam's GP, Carlsson J, Stahl S; Selection and characterization of

HER2 / neu-binding affibody ligands; Protein Eng Des Sei. 2004 May; 17 (5): 455-62. US 2010/0048868 WO 2009/068625 WO 2009/080810

Claims

Use of a peptide (1) for the production of an agent for the detection of a tumor (18) expressing a Her2 / neu receptor (4),

characterized ,

the peptide (1) binds to the Her2 / neu receptor (4) and has an ¹¹ C carbon atom.

Use according to claim 1,

characterized ,

the peptide (1) has at least one D-amino acid (2).

Use according to claim 1 or 2,

characterized ,

that the agent is a radiopharmaceutical.

Use according to one of the preceding claims, characterized in that

the tumor (18) expresses increased levels of the Her2 / neu receptor (4) compared to healthy tissue.

Use according to one of the preceding claims, characterized in that

the ¹¹ C carbon atom is the carbonyl carbon atom of an amino acid (2), preferably of the N-terminal amino acid (3) of the peptide (1).

A radiopharmaceutical for the localization of a tumor (18) expressing a Her2 / neu receptor (4), comprising a peptide (1),

characterized , the peptide (1) binds to the Her2 / neu receptor (4) and has an ¹¹ C carbon atom.

7. radiopharmaceutical according to claim 6,

characterized ,

8. radiopharmaceutical according to claim 6 or 7,

characterized ,

9. radiopharmaceutical according to any one of claims 6 to 8,

characterized ,

that it is a positron emission tomography (PET) biomarker.