DE10301592A1 - Use of substances binding to GSTM for the diagnosis and treatment of bladder cancer - Google Patents

Abstract

The invention relates to uses of GSTM for the diagnosis and treatment of urogenital tumors, in particular bladder cancer, and for screening for substances for such purposes.

Description

Territory of invention

The invention relates to new uses of GSTM or sequences derived therefrom for screening thereon binding substances, as well as the use of binding to GSTM Substances for the diagnosis and / or treatment of bladder cancer.

background of the invention and prior art

GSTM stands for Glutathione-S-Transferase Class μ member 4. GSTM heard to a multigenic family of enzymes, which are bound by the binding of Initiate glutathione on cell-toxic substances whose detoxification in the cell. Furthermore, they bind lipophilic substances and impart their solubility. GST's in a class form homo- or with each other Heterodimers, the various electrophilic substances, such a Process known carcinogens. The stoichiometric The proportion of the M4 variant is very low (Rowe et al., Biochem. J., 15.325 (Pt2): 481-486 (1997)), so that their evidence is often associated with the Tumor formation can be associated.

Expression of GSTM genes (mainly GSTM1) in normal and tumor tissues of the urogenital tract is very different. Small amounts of GSTM are found in bladder tumors, but some are not all low-grade tumors, on the other hand, have high expression values of this molecule. In high-grade, invasive forms, however, could at all no GSTM can be detected in small amounts (Celis et al., Cencer Res 15, 56 (20): 4782-4790 (1996)). Total results for the expression pattern is a very heterogeneous picture, which is why it is not it can be predicted whether or in which tumors overexpression will take place and what such overexpression ultimately means.

Bladder cancer is the second most common urological tumor. It occurs with a frequency in men from 245 to 100,000 and in women from 65 to 100,000. Among the cancer-related deaths takes bladder cancer in men fourth and sixth for women. The treatment mostly done by cystectomy, i.e. by partial or total removal the bladder. This often leads to further complications for the sick person.

Bladder cancer developed by degenerating individual cells of the urothelium. The urothelium is the cell layer that is inside the body shields against the urine formed. It covers the lumen of the renal pelvis, the ureter, bladder and urethra.

Bladder cancer almost develops Completely (> 93%) as adenocarcinoma and is characterized by the strong tendency to recurrence, the regular recurrence even after treatment. In the industrialized countries in the development of bladder cancer mainly on the influence of chemical pollutants, such as aromatic amines, but mainly attributed to smoking as the cause. He can be relatively early be diagnosed and take two forms.

The more common variant, that of the papillary or superficial pTa-Zumoren, has a good prognosis because it is non-invasive and can be removed surgically. The In contrast, invasive forms grow into the tissue and lead untreated severe symptoms, such as lymph node involvement and metastases. The mortality arise almost exclusively this second group. It is characteristic of the course that the papillary Tumors very common remain stable and therefore no progress to the dangerous ones show invasive forms. In 10-20% of cases, however, it occurs in the course from 5 years to a progress towards the aggressive muscle invasive Tumors.

A number of genes have been identified on the suitability as a prognostic marker in bladder cancer examined. It is of primary interest whether a patient with non-invasive, papillary Tumors remain stable at this stage, or whether they are invasive Will develop shapes. The markers examined so far include all p53, p21 and the retinoblastoma gene Rb. None of the above Marker enables however a forecast for an individual patient with sufficient certainty (Marberger et al., Eur Urol, 40/5, Curric Urol 1-9, (2001)). Newer, commercial available Tests (BTA-STAT, NMP22) focus more on the detection of one Urothelium than on the prognosis of the course.

Technical problem of the invention

The invention is therefore technical Underlying problem, pharmaceutical compositions for diagnosis, in particular for prognosis of progression or progression, and / or To specify treatment of bladder cancer and means for their Identification.

Basics of the invention and preferred Embodiments.

To solve this technical problem teaches using one for Nucleic acid encoding GSTM and / or a GSTM peptide or protein for the detection of bladder cancer or to detect a risk of the disease from such a disease Carcinoma or to detect a risk of progression of a papillary Urinary carcinoma to an invasive carcinoma, taking a urothelial cell tissue sample, in particular a bladder urothelial cell tissue sample, on transcription or over-transcription of GSTM RNA or for expression or overexpression of a GSTM protein is examined. One for GSTM coding nucleic acid or a detector substance that binds to GSTM protein or peptide, preferably containing a reporter group, can be used binding said nucleic acid and / or said protein or peptide semi-quantitative to the detector substance or is detected quantitatively. In this context, the Invention further a test system for (in vitro) detection of a the aforementioned carcinoma or a risk of the disease here or the progression prognosis, containing means for quantitative Measurement of the expression of GSTM in tissue samples using these agents for example means for amplification and specific detection of GSTM RNA and / or a detector substance, in particular specifically for GSTM Protein.

The invention further teaches Use of a GSTM RNA or a GSTM protein or peptide for screening for substances that bind to it, especially prospective ones Active ingredients for modulation, in particular inhibition, of said RNA or said protein or peptide, or prospective detector substances, being a prospective substance or a mixture of such prospective Substances contacted with said RNA or said protein or peptide with binding events determined using a binding assay be, and being a binding prospective substance, possibly after Deconvolutation is selected. Teaches in these contexts the invention further a screening system for determining for the treatment containing active substances suitable from the above tumor diseases a GSTM nucleic acid or a GSTM protein or peptide, means for determining (in vitro) binding events to the GSTM nucleic acid or to the GSTM protein or peptide, and / or means for determining the (in vitro) activity of GSTM Protein. Here GSTM can be in a cell-free or a cell-based System, the latter in particular having urothelial cells of the urogenital tract, especially the bladder, or a cell line developed from it, available. Means for determining binding events can, for example naturally in normal or in tumor cells e.g. substances binding to GSTM protein or Association partners include, being about their (free) concentration or change in concentration when adding prospective active substances and / or detector substances determines a competitive binding of a binding active substance or detector substance becomes. Such means can but also include physical or physico-chemical methods, such as X-ray structure analysis and / or NMR, in particular two-dimensional 1H / 1H or 15N / 1H or 14C / 1H correlation spectroscopy. Here spectra are before and after the addition of a prospective active substance or detector substance to one another compared and in case of changes a binding event is detected. It can be with spectra or the like of either GSTM or the prospective substance or work with a combination of both. Of course also all other usual ones Methods of determining binding events and / or protein activities can be used. For example, a prospective substance (or several substances, spatially apart separately) immobilized, then labeled GSTM is applied becomes. A binding event is then ordered and following flush by detection, possibly locally resolved, of the marker bound FABP4s detected. Conversely, GSTM can be immobilized and it becomes a labeled prospective substance or a mixture thereof applied. Binding events become analogous to the previous variant detected.

The invention finally teaches Use of a substance that inhibits or binds to GSTM for the manufacture of a pharmaceutical composition for treatment and / or diagnosis of bladder cancer or prognosis in bladder carcinoma diseases.

The substance can be an antibody which by immunizing a non-human mammal with a GSTM peptide or protein, with cDNR coding for it Cells with endogenously expressing such a peptide or protein Tumor cells, or with recombinantly produced GSTM peptides or Proteins, available is, or a phage display antibody. The substance can but also a mimicry compound of an antibody against a GSTM peptide or be protein. Finally, the substance can be an aptamer, an antisense RNA, a ribozyme or an siRNA against GSTM nucleic acids. The substance can additionally carry a cytotoxic and / or immunostimulating component.

It is preferred if the above substances binding to GSTM protein specifically bind to the GSTM protein when used for therapeutic purposes and modulate its biological activity. This is not necessary in the case of fusion or connection of the substance with a cytotoxic component. This is also not necessary if the substance is used to obtain an anti-idiotypic antibody which is immune to a patient's immune system due to its non-humanized nature Form is recognized as foreign to the body and otherwise a GSTM antigen is presented to the immune system.

The pharmaceutical composition can be used for any application, e.g. i.v. or i.p. Injection, be prepared. A preparation for local application in tumor cells containing tissue will be recommended in case of using a cytotoxic component.

The invention can be carried out as part of a method for the diagnosis or prognosis of a tumor disease of the Use bladder cancer, with a detector substance in one embodiment with a reporter group in the tissue to be examined, possibly in vitro after tissue removal, is applied, the one to be examined Tissue is then subjected to a detection process stage which is sensitive for the Is reporter group, and being in the case of detection of a defined Minimum values of the reporter group in the tissue the tissue as tumor cells containing qualified or classified as being at risk of progression or not at risk of progression as well as a procedure for the treatment of a bladder tumor disease, being a pharmaceutical according to the invention Composition in a physiologically effective dose to a patient is presented. In the case of diagnosis or prognosis can additionally or alternatively a tissue sample with a test system according to the invention to be examined for GSTM expression.

The invention is based in particular recognizing that GSTM in different papillaries Bladder cancer tumors are expressed differently, i.e. in said tumor tissues the expression is in the case of such papillary Carcinomas that appear in later Course become invasive, higher, compared to normal cells of the same tissue, and in the case of such papillary Carcinomas that remain stable, on the other hand, are low and those that can be derived from them technical teaching that GSTM as a target in diagnostics, especially prognosis of progression, and therapy or prophylaxis, in particular of invasive tumor diseases can be. GSTM can therefore be used as a specific marker for identification of tumor cells in said tumor tissues which serve a Are at risk of developing invasive tumor tissue. Call the other side The inhibition of GSTM offers the opportunity in the tumor-specific GSTM associations with other processes in the tumor cells and thus ultimately changed the tumor cell-specific Disrupt metabolism and to die off or at least inhibit growth Tumor cells, but especially an inhibition of the progression to invasive Tumors to contribute.

In the context of the invention it can recommend in advance of treatment with a pharmaceutical according to the invention Composition a sample from a tissue, which as tumor tissue with other methods are identified, and the tissue sample can be taken for expression or overexpression from GSTM to investigate. Alternatively, with a detector substance according to the invention to be tested for diagnosis in vivo on GSTM dependence. Becomes an expression or overexpression from GSTM opposite Normal tissue of the same type is determined, so is the application of the pharmaceutical according to the invention Composition indexed.

Generally, it is within the scope of the invention possible, to examine patient-specifically for differential expression, with normal tissue sample and tumor tissue samples or suspected tumor samples (same) patients taken and compared to GSTM values Expression to be examined.

Is it the tumor? a type in which tumor cells express GSTM, normal cells the same type of fabric, however, it is particularly preferred if the substance binding to GSTM is additionally a cytotoxic and / or immunostimulating component. this leads to then ultimately that almost exclusively tumor cells are killed, be it through cytotoxicity, be it through attack by the stimulated immune system during normal cells almost completely preserved in the tissue stay. In this embodiment the binding substance itself does not inhibit GSTM act because the binding substance then only function as a marker must, which one the components to target tumor cells wearing. If a cytotoxic component is used, it will especially recommend if the pharmaceutical composition for local application prepared in tissue containing tumor cells is, for example, for injection.

Definitions and other embodiments the invention.

The nucleic acid and protein sequences of two GSTM 1 variants, one GSTM2, 3 GSTM4 variants and one GSTM5 are in the 1a-g (RNA) and 2a-g (protein) are shown (SEQ ID Nos. 1 to 14).

In the context of this description, the term GSTM is used for all human isoforms, known or new, based on nucleic acids or amino acids. These terms also include the short sequences disclosed in the context of this description, which originate from the isoforms, for example immunization sequences. Also included are homologs, the homology being at least 80%, preferably more than 90%, most preferably more than 95%, calculated with the program MEGALIGN (DNASTAR LASERGENE) in the current version at the time of the present application. In the case of nucleic acid sequences, complementary or allelic variants are also included. Furthermore, sequences are included which only represent partial sequences of the explicitly disclosed sequences, for example one or more exons, or complementary sequences thereto, with the proviso that, in the case of the nucleic acids, these partial sequences are one for hybridization with an invention according to the nucleic acid of sufficient length, at least 50 bases, and in the case of the proteins or peptides bind to a protein- or peptide-specific target molecule with at least the same affinity. Furthermore, all nucleic acids hybridizing with nucleic acids according to the invention are included, namely those which are under stringent conditions (5 ° C. to 25 ° C. below the melting temperature; see additionally JM Sambrook et al., A laboratory manual, Cold Spring Harbor Laboratory Press (1989) and EM Southern, J Mol Biol, 98: 503ff (1975)) hybridize. It is understood that the invention also includes expression cassettes, ie one or more of the nucleic acid sequences according to the invention with at least one operatively linked control or regulatory sequence. Such an expression cassette can also comprise a sequence for a known protein, a fusion protein being formed in the course of the translation from a known protein and a protein or peptide according to the invention. Antisense sequences to the above nucleic acid sequences are also included. Finally, RNA and correlating DNA and vice versa are included, as are genomic DNA and correlated cDNA and vice versa.

Within the scope of the invention, too GSTM homo- or heterodimers can be used. In this respect, the term includes GSTM also such homo- or heterodimers.

In connection with uses according to the invention include the terms of GSTM nucleic acids or protein or peptides next to the full lengths of the sequences disclosed (see also paragraph above) Partial sequences from this, with a minimum length of 12 to 30 nucleotides, preferably 30 to 90 nucleotides, in the case of nucleic acids and a minimum length from 4 to 10 amino acids, preferably 10 to 30 amino acids, in the case of peptides or proteins. These partial sequences can be found in otherwise nucleic acid or protein or peptide sequences different from GSTM be installed.

The concept of treatment also includes prophylaxis, especially prophylaxis of progression to invasive Tumors.

A compound is an inhibitor or substance that denotes either the formation of GSTM protein inhibits or reduces the activity of GSTM protein, based on GSTM activity in the absence of the inhibitor. In this respect, on the one hand, an inhibitor be a substance that inhibits in the cascade of GSTM intervenes. On the other hand, an inhibitor can be a substance be, which forms a bond with formed GSTM, namely such that further physiological interactions with endogenous Substances are at least reduced.

Mimicry molecules are compounds that the emulate a variable region, in particular the binding region of an antibody and bind in the same place as a target molecule lying antibodies.

The term antibody includes polyclonal Antibody, monoclonal antibodies, non-human, human and humanized antibodies, as well as phage display antibodies, however also chimeric antibody as well as specific fragments of the light and / or heavy chain of the variable range underlying antibodies above Art as well as anti-idiotypic antibodies. The manufacture or Obtaining such antibodies with given immunogens the average person is comfortable familiar and need not be explained in more detail become. Also includes the concept of antibodies bispecific antibodies. Bispecific antibodies combine a defined immune cell activity with a specific tumor cell recognition, thereby killing tumor cells become. A bispecific antibody binds to a trigger molecule of the immune effector cell (e.g. CD3, CD16, CD64) and on the other hand to antigens of the tumor target cell.

The pharmaceutical preparation of a pharmaceutical composition according to the invention can be carried out in a manner customary in the art. Counter ions for ionic compounds are, for example, Na ⁺ , K ⁺ , Li ⁺ or cyclohexylammonium. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions (iv, ip, im) and preparations with a protracted active ingredient release , in the production of customary auxiliaries such as carriers, explosives, binders, coatings, swelling agents, lubricants or lubricants, flavorings, sweeteners and solubilizers. Auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils such as cod liver oil, sunflower, peanut or sesame oil, polyethylene glycols and solvents such as sterile water and mono- or polyhydric alcohols, for example glycerol. A pharmaceutical composition according to the invention can be produced by mixing at least one inhibitor used according to the invention in a defined dose with a pharmaceutically suitable and physiologically compatible carrier and, if appropriate, other suitable active ingredients, additives or auxiliaries with a defined inhibitor dose and preparing the desired dosage form.

Tumor cells express GSTM differentially, if normal cells of the same tissue type (the same or different Subjects) did not express this. Overexpress tumor cells GSTM specific or differential if GSTM compared to normal cells of the same tissue type are expressed at least in twice the amount becomes.

Cytotoxic components or groups are compounds that directly or indirectly apoptosis initiate or lead to necrosis or at least inhibit growth. In addition to radioisotopes (for example 188Re, 213Bi, 99mTc, 90Y, 131J, 177Lu), such groups or compounds can in particular be cytostatics which are used in tumor therapy. Examples include: alkylating agents (eg mechlorethamine, ifosfamide, chlorambucil, cyclophosphamide, melphalan, alkyl, busulphan, nitrosoureas, carmustine, lomustine, semustine, triazenes, dacarbazine), antimetabolites (eg, folic acid antagonists, methotrexate, pyrimidine analogs, fluorouracil, fluorodeoxyuridine , Cytarabine, gemcitabine, purine analogs, mercaptopurine), mitosis inhibitors (e.g. vinca alkaloids, voncristine, vinblastine, paclitaxal, docetaxel, protaxel), epipodophyllotoxins (e.g. etoposide, teniposide), antibiotics (e.g. dactinomyin, antithromycin, idomycinubicubin, idomycinubicin, idomycubin -Asparaginase), platinum complex compounds (e.g. cisplatin), hormones and related compounds (e.g. adrenal cortex steroids, aminogluthetimide, progestogens, estrogens, androgens, anti-estrogens, tamoxifen, steroid analogues, flutamide). When such a compound is bound to a substance which binds to GSTM, the coupling takes place in such a way that the affinity for GSTM is reduced by no more than 90%, preferably 50%, based on the substance without a cytostatic group, and the cytostatic activity of the group is not more than 90%, preferably 50%, based on the compound without substance, is reduced.

An immunostimulating component is usually a protein or an effective component thereof, which Immune system cells stimulated. Examples for this are: Cytokines, such as M-CSF, GM-CSF, G-CSF, interferons, such as IFN-alpha, -beta, -gamma, interleukins such as IL-1 to -16 (except -8), human LIF, chemokines such as Rantes, MCAF, MIP-1-a1pha, -beta, NAP-1 and IL-8.

A reporter group is an atom, molecule or a connection in connection with one placed thereon Assay the detection of the reporter group and thus with the reporter group connected compound or substance. Examples of reporter groups and associated detection methods are: 32P labeling and intensity measurement using phosphoimager. Many other examples are of ordinary skill in the art known and need not the detailed list.

A substance that binds to GSTM can be a substance that binds to a GSTM protein or a GSTM RNA binds.

As part of the above definition across from The narrow sense of the word also includes expanded definitions the certain terms in the narrow sense of the word.

Examples.

The invention is described below of preferred embodiments only illustrative examples and figures explained. Show it:

1 : Nucleic acid sequences of GSTM

2 : Amino acid sequences of GSTM

3 : Expression analysis of GSTM, in particular GSTM4, in different stages and subtypes of bladder cancer,

Example 1: Examined tissue samples

There were 46 tissues from 17 patients with non-invasive papillary pTA tumors at the time of the survey taken. The further course of the disease of all patients was in the following three years and assigned to the tissue samples. 21 of the tissues came from Patients who progress to an invasive progress within three years Form of bladder cancer showed. The remaining 24 tissues included to patients whose bladder tumor was stable in the pTa stage in the same period remained.

Example 2: Expression profiles of the examined tissues

The samples from Example 1 were subjected to an expression analysis on GSTM using GeneChip technology (Affimetrix). The results are in the 3 shown. The median expression values of different stages and subtypes of bladder cancer are shown. The values for the pTA tumors with and without subsequent progression are highlighted in dark. It can be seen that the median of the progressive tumors is 4.8 times higher than that of the non-progressive tumors, ie expression of the GSTM gene is found almost exclusively in the tumor epithelium of patients in whom the tumor later took an invasive course. Specifically, GSTM was severely overexpressed in 17 of 21 tissues that showed progression to invasive tumors. In contrast, increased expression was only found in 8 of 24 tissues that showed a stable course.

SEQUENCE LISTING

Claims (11)

Use of a nucleic acid coding for GSTM, in particular GSTM4, and / or a GSTM, in particular GSTM4, peptide or protein for the detection of tumors of the genitourinary tract, in particular urinary carcinoma, or for the detection of a risk of the disease of such a tumor or for the detection of a risk of one Progression of a papillary urogenital tumor to an invasive carcinoma, wherein a urothelial cell tissue sample, in particular a urinary bladder urothelial cell tissue sample, is examined for transcription or over-transcription of GSTM RNA or for expression or overexpression of a GSTM protein. Use according to claim 1, wherein one encoding GSTM nucleic acid or a detector substance that binds to GSTM protein or peptide, preferably containing a reporter group is used, wherein Binding of said nucleic acid and / or said protein or peptide semi-quantitatively to the detector substance or is detected quantitatively. Use of a GSTM, in particular GSTM4, RNA or a GSTM, especially GSTM4, protein or peptide for screening for substances that bind to it, especially prospective ones Active substances for inhibiting said RNA or said protein or peptide or according to prospective detector substances, one prospective substance or a mixture of such prospective substances is contacted with said RNA or said protein or peptide, whereby binding events are determined with a binding assay, and wherein a binding prospective substance is selected, if necessary after deconvolution becomes. Use of a GSTM, in particular GSTM4, inhibiting or substance binding to it for the production of a pharmaceutical Composition for the treatment of urogenital tumors, in particular bladder cancer or for the manufacture of a pharmaceutical Composition for diagnosis of such a tumor or for diagnosis a risk of progression from a non-invasive such tumor to one invasive form. Use according to claim 4, wherein the substance is a antibody which is, for example, by immunizing a non-human mammal with a GSTM peptide or protein, cells transfected with GSTM, or one for this for coding cDNA, available or a phage display is antibody. Use according to claim 4, wherein the substance is a Facial expression of an antibody against a GSTM peptide or protein. Use according to claim 4, wherein the substance is a Aptamer, an antisense RNA, an siRNA, or a ribozyme. Use according to one of claims 4 to 7, wherein the substance additionally carries a cytotoxic and / or immunostimulating component. Use according to one of claims 4 to 8, wherein the pharmaceutical Composition for local application in tumor cells containing Tissue is prepared. Methods of diagnosing a genitourinary tumor, especially bladder cancer, or the risk of progression such a tumor into an invasive form where one is at GSTM, in particular GSTM4, binding detector substance in one embodiment applied to a tissue to be examined with a reporter group the tissue to be examined is then subjected to a detection method step which is sensitive to is the reporter group, and in the case of detection of a defined minimum value the reporter group in the tissue containing the tissue as tumor cells qualified or at risk of progress. Method of treating a genitourinary tumor, in particular a bladder carcinoma, a pharmaceutical Composition according to one of claims 4 to 9 in a physiological effective dose and galenic for the dosage form to be used is presented to a patient becomes.