WO2012000763A1 - 11c-marked peptide for detecting a diseased tissue that expresses an igf receptor - Google Patents

Abstract

The invention relates to the use of a peptide (1) for producing an agent for detecting a diseased tissue (18) that expresses an insulin-like growth factor receptor (IGF receptor) (4). The peptide (1) bonds to the IGF receptor (4) and has an ¹¹C carbon atom. The invention further relates to a radiopharmaceutical for locating a tumor (18) that expresses an IGF receptor (4). Said radiopharmaceutical comprises a peptide (1) that bonds to the IGF receptor (4) and has an ¹¹C carbon atom.

Description

description

¹¹ C-labeled peptide for detection of a pathological tissue ^¬ bes that expresses an IGF receptor

The invention relates to the use of a peptide for the manufacture ^¬ position of an agent for the detection of a diseased tissue, the insulin-like growth factor receptor (engl .: insulin-like growth factor-receptor) (= IGF receptor) expri- mized. It further relates to a radiopharmaceutical which comprises such a peptide, for the localization of a pathological tissue ^¬ bes that expresses an IGF receptor.

In modern diagnostics, biochemical analyzes of blood and other bodily fluids, as well as imaging techniques, for example, for the detection of tumors are used. Traditionally, X-ray, ultrasound, and magnetic resonance imaging have been used to localize diseased tissue and ectopic cell aggregates. Newer methods use 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 radioactive radiation of these molecules, for example, with a gamma camera, for so-called scintigraphy, taken and the Po ^¬ tion of the tumor detected. Biochemically, cancer diseases, as well as other diseases, are detected by 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 the body fluids, diseased cells but also molecules that remain anchored to their cell surface. These are mainly cell receptors, such as IGF receptors. On the basis of this surface It is also possible for biochemical detection of pathological cells in vivo by visualizing them using imaging techniques. IGF receptors are transmembrane tyrosine kinase receptors that are composed of four subunits. They are bound and activated by several ligands, including insulin and insulin-like growth factors (IGF) I and II.

The binding of the ligand leads to the phosphorylation of the tyrosine kinase, which activates various cellular signaling pathways. The IGF signaling system is important for the control of basic cell functions, such as cell proliferation, differentiation and apoptosis (Gualco E et al., 2009). Thus, IGF promotes the growth of cells and organs, both during early development and in the adult organism. Moreover, it was observed at a much ^¬ number of diseases, especially in cancer, ectopic activation of the IGF-signal system. In cancer cells, the overactivation of this signaling pathway leads to the fact that the affected cells do not die off and continue to proliferate. Accordingly, in biopsies of malignant tumors increased amounts of IGF receptors are regularly detected. Also, cancer therapies are being developed that specifically interfere with the IGF signaling system (Law J et al., 2008).

Excessive expression of IGF receptors is an indicator of both the malignancy of a tumor and the formation of metastases (Zhang C et al., 2010), and thus an unfavorable disease prognosis. Therefore, it is of great medical importance ^¬ SSSR determine early on whether and if so how many cells of a tumor expressing an IGF receptor. In addition, there is a need to be able to detect IGF receptor-positive metastases early. The invention is therefore based on the object, a cost-effective and well-tolerated for the patient agent for detecting a diseased tissue that expresses an IGF receptor provide. This task is done by the

Use of a peptide for the production of an agent for detecting a diseased tissue expressing an IGF receptor. By using a peptide which binds to the IGF receptor and having a ¹¹ C carbon atom is used, the agent can be produced inexpensively and, well comparable metabolized in the organisms ^¬ mechanism in which the diseased tissue is detected.

The term "peptide" refers to an organic compound of at least two linked via a peptide bond,

Amino acids. It includes both oligopeptides of up to about ten amino acids, polypeptides up to about 50 Aminosäu ^¬ reindeer, as well as 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 in the invention is chosen so that it binds to the IGF receptor. Suitable for this are antibodies, their fragments and other polypeptides which bind to the IGF receptor. By their specific binding to the IGF receptor, the peptides can be used to detect diseased tissues that form the IGF receptor. Preferably, the peptide is chosen so that the bond between the peptide and the receptor IGF-called a linear coefficient. KD value of <100 nM, preferably <10 nM, more be ^¬ vorzugt of 7.5 nM comprising , The peptide itself is acids from amino ^¬, that is constructed from autologous or body like molecules, making it very well comparable to the patient is tolerable. It is not toxic and can be natural

be metabolized, degraded and excreted.

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, hypertrophic 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 receptors for growth factors. These receptors are located on the cell surface and can be bound there by the peptide used according to the invention. IGF receptors are expressed by a variety, especially malignant, tumors. They were, among others, in tumors of the pancreas, lung and rectum, as well as among different ^¬'s brain tumors, especially in children, demonstrated (Gualco E et al., 2009, Kim SY et al., 2009). By the peptide used in the invention such tumors can be iden ^¬ tified localized and reliable, without an invasive procedure to biopsy requires. Similarly, metastases of such tumors can be detected, provided that they also form the IGF receptor.

The detection of the peptide and the bound IGF receptor via an integrated ¹¹ C-carbon atom. Upon decay of the ¹¹ C carbon isotope, positrons, also referred to as β ⁺ radiation, are formed. Push the positron on an electron, they form two photons away at an angle of 180 °, which is exactly opposite in ge ^¬ modifying the direction of each other. 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-carbon atom in the peptide used in the invention, makes it possible to avoid the use of chemical kör ^¬ perfremder substances. The direct incorporation of the ^ C carbon isotope into the peptide results in radioactive labeling without complexing agents, such as diethylenetriaminepentaacetate (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). or ethylenediamine tetraacetate (EDTA), possible. In addition, it can be avoided that a radioactive foreign substance, such as ¹⁸ fluorine, ¹³³ xenon, or ⁶⁸ 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 IGF 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 IGF 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 IGF 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. b

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 determine the half-life of a protein or peptide to ver ^¬ lengthen by even D-amino acids are used in addition to L-amino acids (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. Another way to influence the pharmacological clearance of the peptide is to replace some of the amino acids of the peptide with non-natural amino acids with similar chemical properties. The non-natural amino acids are metabolized more slowly because the body's proteolytic enzymes are specially adapted to the breakdown 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 modifications of individual amino acids of the peptide are possible in order to specifically influence the half-life of the peptide. In ^¬ play, the terminal amino group of the peptide may be replaced by an isonitrile group. Such modes ^¬ fication reduces, mediated by the amino group of an interaction with proteolytic enzymes without altering the bond between the peptide used in the invention and the IGF receptor. In an advantageous embodiment of the invention, the peptide is an antagonist of the IGF receptor. IGF receptors ^¬ the usually activated by the binding of insulin or insulin-like growth factors to produce approximately phosphorylation cascades are initiated in the cell. In order to avoid that it is controlled by the binding of the inventively used ^¬ th peptide to an activation of IGF signaling pathways, it is advantageous if the peptide binds to the IGF receptor without triggering its autophosphorylation. Particularly suitable for this purpose are peptides which function as antagonists of the IGF receptor. They show a specific bin ^¬ binding affinity to the receptor but do not lead to Akti ^¬ vation subsequent cellular signaling pathways. 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 most important fields of application are oncology, cardiology and neurology as well as drug research. When radionuclides are gamma or beta radiation emitting nuclides, for example Xenon ^133, "technetium, gallium ^68, fluorine ¹⁸ and used. They are usually about Kom ^¬ formers such as DOTA, DTPA or EDTA on mono- or polysaccharides The nuclides will be, depending on the nature of their

Radiation detected by scintigraphy, single photon emission com- puted tomography (SPECT) or positron emission tomography (PET). 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 this risk significantly, because neither the peptide itself, nor its Ab ^{¬ building} products are toxic. In addition, carbon is, in contrast to technetium or xenon, an element found in the body that can naturally be metabolized.

In a preferred embodiment, the diseased tissue expresses increased amounts of the IGF receptor. Compared to healthy tissue, for example, the cells Various ^¬ ner tumors carry particularly high levels of IGF receptors on their surface. These include, for example, lung, breast, and pancreatic cancers, sarcomas, and pediatric gastrointestinal stromal tumors.

In a preferred embodiment, the IGF receptor is an IGF-1 receptor. The family of IGF receptors includes the IGF-1 receptor, the IGF-2 receptor, as well as two insulin receptors (IR), IR-A and IR-B. The IGF-1 receptor is produced in increased amounts by many malignant tumor types and is not limited to ulcers of certain tissues. Therefore, an agent having a peptide which binds to the IGF-1 receptor is useful for the detection and localization of many different diseased tissues, particularly tumor tissues. The IGF-2 receptor has so far been mainly detected on adenocarcinomas of the esophagus and 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 from the peptide, as it would be possible at about a Seitenket ^¬ th one of the amino acids.

According to a further preferred embodiment of the invention, the C-carbon atom is the carbonyl carbon atom of N-terminal amino acid of the peptide. This embodiment is particularly preferred because the peptide immediately after the on ^¬ bring the ¹¹ C-labeled amino acid can be used. ¹¹ C-carbon has ten a half-life of only about 20 for minutes, so that the radiation dose to be selected the higher, the more time between the synthesis of the peptide and be ^¬ ner is situated. 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 Zvi ^¬'s processing of the ^C-11 carbon and the use of the peptide is reduced, so that the radiation loss is minimized during the manufacture of the peptide. 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 more cost-effective and thereby 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 an IGF receptor. By using a peptide which binds to the IGF receptor and ^X1 C- carbon atom, is used, the Radiophar ^¬ makon for the patient is well tolerated and can be produced inexpensively.

Therefore, the radiopharmaceutical invention provides an economical and medically beneficial agent to to determine the posi ^¬ tion of a tumor that expresses an IGF receptor in vivo. After the radiopharmaceutical has been administered to a patient, the peptides contained therein are distributed within the body and specifically bind to IGF receptors. There- They accumulate on the cells of the tumor where they are detected by the radioactive signal of the ¹¹ C carbon atom. In this way, the position of the tumor and ge ^¬ give if the true loading of metastases in the body of the patient.

According to an advantageous embodiment, the ¹¹ 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 by the annular configuration of the PET detectors a more precise spatial localization of the positron ^¬ nenemission and thus a substantially more accurate and detailed ^¬ profiled illustration of a diseased tissue or 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 in an organism expressing an IGF receptor, comprising the steps of a) providing a peptide, b) administering the peptide to the organism, and c) detecting the peptide in the organism using positron emission tomography (PET). The peptide binds to the IGF receptor and has an ¹¹ C carbon atom. With the present invention used a peptide IGF receptor in the interior of an organism is detected and lokali ^¬ Siert, so that the distribution of the IGF receptor may be observed in the body ei ^¬ nes patient. In this way, the size or extent of a tumor expressing the IGF 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. Hereinafter, preferred embodiments of the invention will be explained with reference to the accompanying schematic drawings.

FIG. 1 shows schematically the binding between a peptide 1 and an IGF receptor 4.

Peptide 1 comprises 27 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 (*). A portion of the peptide 1 is attached to the IGF receptor 4, which is located on the surface of a Tu ^¬ mors 18.

The ^ C-labeled peptide 1 binds specifically to the IGF receptor 4, but not to other molecules. Peptide 1 can therefore be used to detect the tumor expressing the IGF receptor 4. The emitted during the decay of the ^X1 C- carbon atom positrons are detected by positron emission tomography (PET). The location of the positron emission corresponds to the location of the peptide 1 and the IGF receptor 4 bound thereto. The peptide 1 can therefore be used to determine the position of the tumor 18 which forms the IGF 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. The peptide 1 binds specifically to the IGF receptor 4 and thus accumulates on the tumor 18, which forms the IGF receptor 4. This accumulation is visualized by PET and determines the distribution of the IGF receptor 4 or the location of the tumor 18 in the body of the patient. In addition, the medication of a therapeutic, for example, amount of drug and administration plan, according to the position, size and distribution of Tu ^¬ mors 18 are adjusted.

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 27 amino acids 2 of the following sequence: serine-phenylalanine-tyrosine-serine-cysteine-leucine-glutamic acid-serine-leucine-valine-asparagine-glycine-prolol-alanine-glutamic acid-lysine Serine-arginine-glycine-glutamine-tryptophan-aspartic acid-glycine-cysteine-arginine-lysine-lysine.

The N-terminal amino acids 2 serine and phenylalanine are represented by structural formula, the following amino acids 2 by their respective three-letter code. The sequence of the peptide is also given in SEQ ID NO: 1. The carbonyl carbon atom of the N-terminal serine is an ^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. Thereby, That the peptide synthesis is completed with the C-labeled amino acid 3, the peptide 1 can be used immediately after the radioactive label. The peptide of SEQ ID NO: 1 binds specifically to the IGF-1 receptor 4 (US 7,173,005 B2). This receptor is present in large quantities on the surface of cells of colon rectal tumors. The natural ligands of IGF-1 receptor 4 include insulin, IGF-I and IGF-II, among others. The peptide of SEQ ID NO: 1 also binds to the IGF-1 receptor 4 and is therefore suitable for the detection of an IGF-I receptor 4 expressing tissue. A mark by ^X1 C Carbon is particularly suitable because it does not affect the phy ^¬ siologische structure of the peptide 1 and neither the affected tissue distribution and tolerability of the peptide. 1

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 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 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 ^¬ . To the left of the center of the circulatory system 10 is additionally shown a pathological tissue 18 with IGF receptors 4, to which peptides 1 are increasingly attached. The distribution of 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 blood circulation system 10, the peptide 1 is transported into the organs 12, 13, 14, 15, and 16 of the organism.

Phase III: The circulating peptide 1 binds specifically to the IGF receptor 4 and accumulates on the diseased tissue 18 because it produces the IGF 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 lekülen Mo. 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

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

Gualco E, Wang JY, Del Valle L, Urbanska K, Peruzzi F,

Khalili K, Amini S, Reiss K; IGF-IR in neuroprotection and brain tumors; Front Biosci. 2009 Jan 1; 14: 352-75. Kirn SY, Toretsky JA, Scher D, Heiman LJ; The role of IGF-IR in pediatric malignancies; Onco logist. 2009 Jan; 14 (1): 83-91.

Law JH, Habibi G, Hu K, Masoudi H, Wang MY, Stratford AL,

Park E, Gee JM, Finlay P, Jones HE, Nicholson. RI, Carboni J, Gottardis M, Pollak M, Thin SE; Phosphorylated insulin-like growth factor-i / insulin receptor is present in all breast cancer subtypes and is related to poor survival; Cancer Res. 2008 Dec 15; 68 (24): 10238-46. 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.

Zhang C, Hao L, Wang L, Xiao Y, Ge H, Zhu Z, Luo Y, Zhang Y, Zhang Y; Elevated IGFIR expression regulating VEGF and VEGF-C predicts lymph node metastasis in human colorectal cancer; BMC Cancer. 2010 May 7; 10 (1): 184.

US 7, 173, 005 B2

Claims

claims Use of a peptide (1) for the production of an agent for the detection of a diseased tissue (18) which expresses an insulin-like growth factor receptor (IGF receptor) (4),  characterized , the peptide (1) binds to the IGF receptor (4) and has an ¹¹ C carbon atom. 2. Use according to claim 1,  characterized ,  the peptide (1) has at least one D-amino acid (2). 3. Use according to claim 1 or 2,  characterized ,  the peptide (1) is an antagonist of the IGF receptor (4). 4. Use according to one of the preceding claims,  characterized ,  that the agent is a radiopharmaceutical. 5. Use according to one of the preceding claims,  characterized , that the diseased tissue (18), compared to Gesun ^¬ the tissue, increased amounts of the IGF receptor (4) expri ^¬ mized. 6. Use according to one of the preceding claims,  characterized , the IGF receptor (4) is an IGF-1 receptor (4). Use according to one of the preceding claims, characterized 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 an IGF receptor (4), comprising a peptide (1),  characterized, the peptide (1) binds to the IGF receptor (4) and has an ¹¹ C carbon atom. Radiopharmaceutical according to claim 8,  characterized, 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). 10. radiopharmaceutical according to one of claims 8 or 9,  characterized,  that it is a positron emission tomography (PET) biomarker.