WO2006056227A1

WO2006056227A1 - Conjugates comprising the active agent and flanking or branched-chain peptides

Info

Publication number: WO2006056227A1
Application number: PCT/EP2004/013471
Authority: WO
Inventors: Stefan Heckl; Thomas NÄGELE; Ulrich Vogel
Original assignee: Eberhard-Karls- Universität Tübingen Universitätsklinikum
Priority date: 2004-11-27
Filing date: 2004-11-27
Publication date: 2006-06-01
Also published as: WO2006056227A8

Abstract

The present invention relates to a peptide conjugate that is useful in diagnostics and therapy, to the use of said conjugate for preparing a diagnostic or therapeutic composition or a composition for the observation of the functioning of angio-genese inhibitory substances in a patient, to a pharmaceutical or/and diagnostic composition, to a method for the diagnostic or analytical treatment of biological material or a living being, and to methods for the therapeutic treatment of a living being. The conjugate comprises a therapeutically and/or diagnostically active agent and at least two peptides flanking said agent or/and at least one branched-chain peptide. Preferred peptides are derived from nuclear localisation signals, and Gd-chelates are among the preferred active agents for nuclear magnetic imaging or neutron capture therapy.

Description

CONJUGATES COMPRISING THE ACTIVE AGENT AND FLANKING OR BRANCHED-CHAIN PEPTIDES

The present invention relates to a peptide conjugate that is useful in diagnostics and therapy, to the use of said conjugate for preparing a diagnostic or therapeutic composition or a composition for the observation of the functioning of angio- genese inhibitory substances in a patient, to a pharmaceutical or/and diagnostic composition, to a method for the diagnostic or analytical treatment of biological material or a living being, and to methods for the therapeutic treatment of a living being.

Conjugates which are useful in diagnostics and therapy are generally known in the art.

An aim of modern medicine and pharmaceutics is to develop im¬ proved therapeutic and diagnostic compounds or compositions which can be applied in a targeted way, i.e. in a way that once they are administered to a living being or to biological mate¬ rial, they will independently find the areas of the organism or the biological material, where they are supposed to display their properties and/or accumulate in them.

Contrast media, e.g. gadolinium complexes, which are commonly used in routine clinical magnetic resonance imaging (MRI) ex¬ aminations, such as Magnevist (Schering), when administered to a patient will accumulate in the extracellular space and cannot enter the cytoplasm. This has the disadvantage that, e.g., the margins of a tumor cannot be sharply outlined but are imaged in a diffused way, since the gadolinium complex does not accumu¬ late in the tumor cells but only in the interstitium. This characteristic of a common contrast medium is especially prob¬ lematic if the latter is administered during or directly after a surgery. In this case the interstitium is opened by the sur¬ geon resulting in an leakage of the contrast medium along the interstitium. Therefore, the development of a cytoplasmatic or even an intranuclear contrast medium for the use in imaging methods is essential.

Kobayashi et al. (2001), Avidin-dendrimer-(1B4M-Gd)_2s3: A Tumor- Targeting Therapeutic Agent for Gadolinium Neutron Capture Therapy of Intraperitoneal Disseminated Tumor Which Can Be Monitored by MRI, Bioconjugate Chem. 12, pages 587 to 593, describe a gadolinium complex coupled to avidin, referred to as avidin-G6-(lB4M-Gd)₂₅₄, that was internalized in ovarian cancer cells and accumulated in the cytoplasm. The preparation of these complexes is very time-consuming and costly and, there¬ fore, such complexes are not suitable for a large-scale produc¬ tion of routinely usable contrast media. All the more, these known complexes cannot enter the nucleus.

Shihata et al. (2002), In Vitro Cellular Accumulation of Gado¬ linium Incorporated Into Chitosan Nanoparticles Designed for Neutron-Capture Therapy of Cancer, Eur. J. Pharm. Biopharm. 53(1), pages 57 to 63, describe particles consisting of gado¬ linium coupled to chitosan, referred to as Gd-nanoCPs, which are able to enter L929 fibroblast cells and various tumor cells, and accumulate in the cytoplasm. Also these particles are very expensive and have to be produced in an intricately way. Furthermore, they are likewise not able to enter the nu¬ cleus.

Allen, M.J. and Meade, T.J. (2003), Synthesis and Visualization of a Membrane-Permeable MRI Contrast Agent, J. Biol. Inorg. Chem. 8, pages 746-750, describe a complex consisting of a terminal gadolinium compound coupled to a linear polyarginine chain. This conjugate was able to cross the cell membrane of NIH 3T3 cells, but failed to enter the nucleus. Additionally, these conjugates are produced by a time-consuming and expensive method.

De Stasio et al. (2001), Gadolinium in Human Glioblastoma Cells for Gadolinium Neutron Capture Therapy, Cancer Research 61, pages 4272 to 4277, describe a study in which, allegedly, pure Gd-diethylenetriaminepentaacetic acid (Gd-DTPA) was internal¬ ized and accumulated in cell nuclei. Therefor Gd-DTPA concen¬ trations of up to 25 mg/ml in cell culture were used and the cells were incubated for up to three days. At such high concen¬ trations it is highly probable that an unspecific uptake of the Gd-DTPA has taken place, e.g. due to a degradation of the cel¬ lular and nuclear membranes. Under such conditions an applica¬ tion of this method in a living being or even a human is irre¬ sponsible. Bhorade et al. (2000), Macrocyclic Chelators with Paramagnetic Cations are Internalized into Mammalian Cells via a HIV-tat Derived Membrane Translocation Peptide, Bioconjug. Chem. 11, pages 301-305, describe a study in which gadolinium was trans¬ ported across the cell membrane by means of a HIV-tat peptide that was coupled to the contrast agent. Unfortunately, the HIV- tat peptide possesses transactivating properties and induce apoptosis in the hippocampal neurons. The application of such a conjugate to a living being is, therefore, unjustificable.

The EP 1 424 343 Al and Heckl et al. (2003), CNN-Gd³⁺ Enables Cell Nucleus Molecular Imaging of Prostate Cancer Cells: The Last 600 nm, Cancer Research 62, pages 7018 to 7024, disclose a peptide conjugate consisting of an amphiphilic transport pep¬ tide of human origin as a transmembrane module, realized by a member of the penetratin family, or transportan, a nuclear localization sequence, and a signalling and/or drug carrying module, realized preferably by gadolinium (Gd) . As the authors describe, this conjugate is able to enter the nucleus of bio¬ logical cells. However, the preparation of these conjugates is also very time-consuming and costly. Additionally, the trans¬ port peptide, i.e. the penetratin, is of high molecular weight and, therefore, does negatively influence the imaging signal in MRI examinations due to the unfavorable ratio of the transport peptide unit to the signal emitting Gd complex.

Pharmaceutical substances, e.g. cytotoxic agents which are currently used in the treatment of tumor diseases are largely non-specific and directed to all fast-dividing cells. This results in serious side-effects, e.g. the injury of healthy tissues or cells, such as the blood-forming system, the gonads or the hair follicles, etc. The first step for the development of a cellular-targeted and tumor-specific agent, therefore, would be to provide a compound that can enter the cytoplasm or even the nucleus of a biological cell, and that can be easily modified in such a way, that it largely only will be internal¬ ized by a tumor cell but not by a healthy cell.

Against this background it is an object of the present inven¬ tion to provide a diagnostic or/and therapeutic valuable sub¬ stance by which the disadvantages of the known substances can be avoided. Especially an improved contrast medium or/and a targeted active agent should be provided, that is capable of entering the cell and also the nucleus, capable of emitting a distinctly detectable signal or/and can be easily altered for ensuring, e.g., specificity for transformed, infected or apop- totic cells, and which can be cost-efficiently prepared on a large scale.

This object is achieved by providing a conjugate comprising a therapeutically and/or diagnostically active agent and at least two peptides flanking said agent or/and at least one branched- chained peptide coupled to said agent.

A diagnostically agent refers to any chemical, biochemical, organic, inorganic compound, composition, element or substance that can be used as an image creating agent in a diagnostic method, whereby its nature depends on the desired diagnostic method such as imaging methods, optical methods, computer tomo¬ graphy, positron emission tomography, magnetic resonance imag¬ ing, single photon emission computed tomography, gamma-camera, SPECT, etc. Such an agent encompasses any contrast medium, like metals, semi-metals, or corresponding compositions, etc.

A therapeutically active agent refers to any chemical, bio¬ chemical, organic, inorganic compound, composition, element or substance that is designated or can be used for the treatment of diseases, disorders, malfunctions, etc. of a living being or of biological material in general. Such an agent can, e.g., be realized by a neutron-absorbing substance, a so-called "small molecule", a cytostatic or cytotoxic substance, any substance that interacts with nucleic acids or cellular factors control¬ ling cell division or cellular proliferation, but also by any other drug.

Flanking peptides means according to the invention that at least two amino acid chains are coupled to the agent in such a way, that the agent is more or less positioned in the center of the entire conjugate. Flanking peptides means also according to the invention that at least two peptides are positioned lin- earily in the conjugate in respect of the agent. A conceivable structure of the conjugate according to the invention, e.g., is: N-terminus - peptide 1 - therapeutically/diagnostically active agent - peptide 2 - C-terminus. Other structures are also possible.

The inventors have found out that the problem underlying the invention can also be solved if at least one branched-chained peptide is coupled to said agent. This means according to the invention that one first amino acid chain is coupled to the agent and another amino acid chain is coupled to said first amino acid chain, e.g. via lysine residues, to said first amino acid chain, constituting a branched-chained peptide. In such a conjugate, the agent can be positioned on the end of the whole molecule. However, it is also possible to provide an additional peptide on the side of the agent that is opposite to said branched-chained peptide. A conceivable structure of this re¬ alization of a conjugate according to the invention, e.g., is: N-terminus - branched-chained peptide - C-terminus - therapeu- tically/diagnostically active agent, or C-terminus - branched- chained peptide - N-terminus - therapeutically/diagnostically active agent. Other structures are also possible.

The peptides can be coupled to the agent by any method known in the art, e.g. via a covalent bond. Instead of proteinogenic amino acids for providing the at least two peptides, also non- proteinogenic or any modified amino acids can be used.

Astonishingly, comparable conjugates comprising merely one single non-branched peptide or linear peptides which do not flank said agent, were neither able to enter the cytoplasm nor the nuclei of the cells.

The inventors have realized that a conjugate of such a simple structure is not just able to penetrate the cellular membrane and to enter the cytoplasm, but is also able to enter the nu¬ cleus. This was very surprising and totally unexpected, since it was neither necessary to provide a cellular membrane trans¬ port peptide, like penetratin, nor to use a specific amino acid sequence, like a nuclear localization sequence (NLS), in order to enable the conjugate entering the nucleus. To the contrary, the inventors have prepared a complex merely consisting of gadolinium (Gd) as a representative diagnostically/therapeu- tically active agent, and two peptides of randomized amino acid sequences flanking said Gd, and succeeded in providing a cellu¬ lar membrane- and nuclear envelope-permeable substance.

The conjugate according to the invention has several advan¬ tages. It has a small size, consists of merely at least a pep¬ tide compound and a diagnostic/therapeutic compound, and can, therefore, be easily and cost-effectively produced in large amounts. Such a conjugate is largely resistant against prote¬ ases and nucleases, so that a degradation in the blood of a human being after the administration is essentially avoided.

Furthermore, the favorable ratio between the small peptide compound and the diagnostic/therapeutic compound ensures an improved emitting signal in the case where the conjugate ac¬ cording to the invention is designed as a contrast medium.

Another advantageous feature of the conjugate according to the invention is, that said conjugate enters the nucleus and dis¬ plays its diagnostic or/and therapeutic property within the nucleus, but then in turn exits the nucleus and the cytoplasm and can be excreted by the organism, even without a specific nuclear export signal amino acid sequence. This characteristic favors the conjugate for a therapeutic or diagnostic applica¬ tion in a human being since it is ensured, that no accumulation of exogenous or, as the case may be, toxic compounds within the organism occurs.

That property of the conjugate according to the invention was totally unexpected since the conjugate disclosed in EP 1 424 343 Al and in Heckl et al. (cit. loc. ) penetrates the nuclear envelope, but remains within the nucleus without being cleared out of the cell and the organism. The known conjugate was, therefore, unsuited for an application in a living being. This property was also surprising in view of the studies presented by Allen, M.J. and Meade, T.J. (cit. loc. ) since the conjugate analysed within the frame of these studies was also of small molecular weight like the conjugate according to the invention, however failed to enter the nucleus.

Furthermore, the inventors have realized that the conjugate according to the invention is not only internalized by the cells or the nucleus just because of passive diffusion, but due to the at least two peptides or the branched-chained peptide via a mechanism that has not been elucidated in detail so far. This has the advantage that a targeted entering of the cell and the nucleus by the conjugate according to the invention can be controlled by the provision and, where applicable, an appropri¬ ate design of the flanking peptides or the branched-chained peptide. It is, e.g. possible to provide specially designed peptides which ensure an internalization of the conjugate ex¬ clusively in tumor, infected or apoptotic cells, whereas healthy cells cannot be penetrated.

According to a preferred embodiment said at least two peptides or/and said at least one branched-chained peptide are largely positively charged.

A largely positively charged peptide refers to an amino acid chain comprising a largely positive net charge. This can be realized by the introduction of basic amino acids, such as arginine, lysine, histidine, or modified variants thereof. As the inventors have found out, the provision of at least two largely positively charged peptides or a corresponding branched-chained peptide enables the internalization of the conjugate according to the invention through the cellular mem¬ brane into the cytoplasm and through the nuclear pore complex into the nucleus. This phenomenon was verified by experiments where, beside the positively charged peptide component, further charge-neutralizing amino acid chains were provided resulting in a conjugate that was no longer able to enter the cell or the nucleus, respectively. The same result was obtained by peptides comprising largely neutral or negatively charged amino acid chains.

In this connection it was surprising that for a targeted trans¬ port of the conjugate into the nucleus, it was not crucial to choose a specific amino acid sequence, e.g. a NLS, rather than to provide peptides having a positive net charge. This is in contradiction to the general doctrine among experts. Up to now it was assumed that a specific and targeted import of molecules into the nucleus can only be attained via specific peptides consisting of a defined sequence of certain amino acids, that is recognized by the nuclear pore complex, mediating the inter¬ nalization of said molecule attached thereto. This hypothesis was also supported by the authors of EP 1 424 343 Al and Heckl et al (cit. loc.) resulting in the provision of a .peptide com¬ prising an NLS at the conjugate described therein.

The fact that at least two peptides or a branched-chained pep¬ tide of largely any amino acid sequence can be used, as long as a positive net charge is ensured, results in the advantage that the preparation of the conjugate according to the invention is once more simplified and gives the synthesizing chemist more independence and options in performing an appropriate synthesis reaction.

According to a preferred embodiment said at least two peptides consist of about two to fifteen, preferably of about six to eight amino acids, or/and said at least one branched-chained peptide consisting of about four to thirty, preferably of about twelve to sixteen amino acids.

As the inventors have found out, the attachment of at least two peptides or/and one branched-chained peptide, respectively, of such a length is especially advantageous and result in a conju¬ gate that can enter the cell and the nucleus and also can exit the nucleus and the cell in a satisfactory manner.

It is preferred if said at least two peptides or/and said at least one branched-chained peptide comprise nuclear localiza¬ tion sequences (NLS) or sequences which are derived therefrom.

As stated before, for a functioning of the conjugate according to the invention it is not necessary to design the peptide component so that it comprises nuclear localization sequences. However, the inventors have found out that the uptake of the conjugate according to the invention into the cell and the nucleus is also well enabled if the peptide component comprises an NLS or if the corresponding amino acid sequence is derived therefrom.

A preferred NLS is the NLS of the SV 40 T antigen [proline(P)- lysine(K)-lysine(K)-lysine(K)-arginine(R)-lysine(K)-valine(V); cf. Kalderon et al. (1984), A Short Amino Acid Sequence Able to Specify Nuclear Location, Cell 39, pages 499 to 509] or of the ALL-I protein [arginine(R)-lysine(K)-arginine(R)-lysine(K)- arginine(R)-lysine(K) ; cf. Lorenzen et al. (1995), COOH- Terminal Sequence Motifs Target the T Cell Protein Tyrosine Phosphatase to the ER and Nucleus, J. Cell. Biol. 131, pages 631 to 643, and Yano et al. (1997), Nuclear Punctate Distribu¬ tion of ALL-I is conferred by distinct elements at the N termi¬ nus of the protein, Proc. Nat. Acad. Sci. USA 94, pages 7286- 7291], the NLS of the ALL-I protein is harboring motifs of several other nuclear localization sequences.

As the inventors have realized an especially well functioning conjugate is provided if the at least two peptides or the at least one branched-chained peptide comprise the before-mention¬ ed NLS. Further suitable NLS are originating from the following proteins: NF-kappaB (VQRKRQKLMP) , TFIIE-β (SKKKKTKV), Oct-6 (GRKRKKRT), TCF-1-alpha (GKKKKRKREKL) , HATF-3 (ERKKRRRE), C. elegans SDC₃ (FKKFRKF). However all NLS can be used that are known by the skilled person, or sequences derived therefrom.

In this connection the inventors have, e.g., surprisingly found out that a sequence that is derived from the SV 40 T antigen NLS (VRKKPKK instead of PKKKRKV) or a sequence that is derived from the ALL-I NLS (RRRRRR instead of RKRKRK), so-called non- NLS random sequences, are equally effective in mediating cellu¬ lar and nuclear membrane permeability to the conjugate accord¬ ing to the invention. According to another preferred embodiment said diagnos¬ tic/therapeutic agent comprise an image creating substance, preferably Gd, Ga, I, Fe, Mn and F.

This measure has the advantage that herewith a conjugate is provided, that can be used as a contrast medium, e.g. in mag¬ netic resonance imaging (MRI) examinations. Compared to com¬ monly used Gd-containing contrast media the conjugate according to the invention accumulates in the nucleus of the treated cells, instead of accumulating in the interstitium. This has the advantage that cellular structures, as well as, e.g., the margins of a tumor, can be displayed with high resolution. Furthermore, an operative MRI examination can be performed without running the risk that the contrast medium will leak along the interstitium. Moreover, unlike membrane-impermeable contrast media with the new conjugate it is no longer required to employ huge amounts of medium in order to obtain an imaging signal, since the nuclear accumulation enables the administer¬ ing of low concentrations, while at the same time the contrast effect is maintained.

Unlike the Gd-containing contrast medium known from EP 1 424 343 Al and Heckl et al (cit. loc.) without any efflux proper¬ ties, rapid nuclear uptake and efflux of the conjugate accord¬ ing to the invention is ensured. This characteristic strongly favors the use of the new conjugate in a human being.

It is preferred if in the conjugate according to the invention said diagnostic/therapeutic agent comprise a cell damaging property. A cell damaging property refers to any property that causes a negative effect on a cell, e.g. the inhibition of cell prolif¬ eration, of cell division, the induction of apoptosis, necro¬ sis, oncosis in the cell, the modification, e.g. alkylation, or destruction of the DNA, the activation of endogenous factors like tumor suppressors, such as p53, pRB, or inhibitors of cyclin-dependend kinases, such as members of the INK4 family, p21^cipl or p27^Kipl, p57^Kip2, or mechanisms, like the immune system resulting in the attack and degradation of the affected cell.

This measure has the advantage that herewith a substance is provided that is valuable in the treatment of malignant trans¬ formed cells, as this can be observed in several diseases, such as cancer. Such a substance is also advantageous in the treat¬ ment of infected or otherwise degenerated cells which have to be damaged for the well-being of healthy cells or the whole organism.

The provision of such a cell damaging property can be realized, e.g., by the usage of a neutron-absorbing substance, such as boron, gadolinium, and any other substance known by the skilled person, which can be used in Neutron Capture Therapy (NCT), as the therapeutically active agent; for an overview see Hawthorne et al. (1998), New Horizons for Therapy Based on the Boron Neutron Capture Reaction, MoI. Med. Today 4, pages 174 to 181, or Sauerwein, W. (1993), Principles and History of Neutron Capture Therapy, Strahlenther. Onkol. 169, pages 1 to 6. Such a conjugate can be locally administered into a tumor, e.g. a brain tumor, accumulates in the tumor cells, whereas the blood- brain barrier prevents said conjugate to be distributed over the whole body or even to be delivered into healthy parenchymal brain tissue via the blood stream. Subsequently, the organism is irradiated with a neutron beam resulting in the conversion or activation, respectively, of the non-toxic neutron-absorbing substance into a radiotoxic substance. Alternatively, the con¬ jugate can be administered systemically followed by a locally restricted irradiation of the affected area, i.e. the area where the tumor is located.

The cell-damaging property can also be realized by the usage of a cytotoxic drug as the therapeutically active agent of the conjugate, such as an alkylating drug, like Temodal, BCNU or any other cytostatic drugs known in the art. Such a "conven¬ tional" cytotoxic drug can be used as sole therapeutically active agent without any further neutron-absorbing substance or image creating compound, like gadolinium, but also in combina¬ tion with afore-mentioned substances or compounds, respec¬ tively, and vice versa.

With this conjugate it is ensured that the cytostatic drug is in fact in direct contact to the DNA to be affected or alky¬ lated, since the drug accumulates in the cell nucleus. Side effects are herewith further reduced.

According to a preferred embodiment the conjugate according to the invention further comprises at least one segment or/and compound establishing specificity for a tumor cell, or/and an apoptotic cell, or/and a cell of a thrombosis area, or/and a cell infected by a virus.

Such a segment or compound coupled to or included in the conju¬ gate according to the invention, can be realized in form of an antibody or/and an aptamer, having a specificity or an affinity for such a cell, e.g. via an interaction with a tumor marker or an infection or apoptosis marker that is specifically expressed on the surface of a tumor or infected cell. Such a segment can also be realized by any other e.g. synthetic ligand having a specificity for such cells, as well as by means of viruses or components thereof coupled to the conjugate according to the invention, mediating specificity for such cells.

Such a segment can also be provided by an amino acid sequence recognizable and cleavable by an enzyme specific for above- mentioned cells, optionally coupled to a negatively charged further peptide. Said enzyme is preferably selected from the group consisting of: matrix metallo-proteinase 2 (MMP2), cathepsin, prostate-specific antigen (PSA), herpes simplex virus-protease, human immunodeficiency virus-protease, cyto- megalovirus-protesase, caspase, interleukin-lβ converting en¬ zyme, thrombin.

This measure has the advantage that herewith specificity for such cells is established for the conjugate according to the invention in a very effective way. It is known in the art that different tumor or carcinoma cells, respectively, express char¬ acteristic enzymes and secrete the latter into their cellular environment, e.g. in order to digest connective tissue for enabling their invasion of so far healthy tissue or organs. Glioblastoma, e.g., mainly express and secrete metallo- proteinase 2 (MMP2). Mamma carcinoma, e.g., mainly express and secrete cathepsins which recognize and cleave a specific amino acid sequence. Prostate carcinoma predominantly express and secrete prostate-specific antigen (PSA). It is also known in the art that cells infected by viruses express and secrete virus-specific proteases. Herpes simplex virus-(HSV)infected cells secrete herpes simplex virus prote¬ ase. Cells which have been infected by the human immunodefi¬ ciency virus (HIV) express and secrete HIV protease. Cells infected by the cytomegalovirus express and secrete a protease that is specific for this kind of virus.

Apoptotic cells are characterized by a high expression level of caspase enzymes and interleukin converting enzymes, which can also be found in the extracellular environments and, therefore, have access to the administered conjugate according to the invention.

Furthermore, it is known from cells of thrombosis areas that they highly express and secrete the thrombin enzyme.

All enzymes have a substrate specificity based on a defined amino acid sequence. MMP2 recognizes the sequence PLGVR, whereas cathepsin B recognizes the specific sequences KK and/or RR. Cathepsin D recognizes the sequence PIC(Et)FF, where "Et" refers to an ester branching side. Cathepsin K recognizes the specific sequence GGPRGLPG. The PSA, however, recognizes the sequence HSSKLQ. Other tumor cell-specific enzymes and their specific recognition and cleavage sites are well described in the art. An overview of this issue is given in Hahn, W.C. and Weinberg, R.A. (2002), Rules for Making Human Tumor Cells, N. Engl. J. Med. 347, pages 1593-1603, the content of this publi¬ cation is incorporated herein by reference. The HSV protease recognizes the sequence LVLA3SFGY, the HIV protease recognizes the sequence GVSQNYPIVG, the cytomegalo¬ virus protease, however, recognizes the sequence GWQASCRLA.

Caspase-3 recognizes and cleaves the amino acid sequence DEVD, another apoptosis-specific enzyme interleukin 1-β converting enzyme recognizes the sequence GWEHDG.

Thrombin recognizes and cleaves the sequence dFPipR, whereby Pip stands for pipecolic acid.

All before-mentioned sequences can be used as a compound of the segment establishing specificity or as such a compound itself.

The negatively charged further peptide that is optionally cou¬ pled to the conjugate according to the invention, has the func¬ tion to neutralize the largely positive charge of the at least two peptides. As a result of this neutralization of charge the so-modified conjugate accumulates in the interstitium and is no longer able to enter the cytoplasm or the nucleus of non- transformed, healthy cells. The situation is different with, e.g., tumor cells. Here tumor cell-specific extracellular pro¬ teases are present, which cause a cleavage of the conjugate at their specific recognition sites, resulting in a loss of the negatively charged further peptide. Consequently, again the positive charge of the at least two peptides predominates medi¬ ating cell membrane and nucleus envelope permeability of the conjugate. As a result of this, the conjugate will be internal¬ ized by the tumor cell and enter the nucleus, whereas at nor¬ mal, healthy, non-transformed cells the conjugate remains in the interstitium and can be easily eliminated from the organ¬ ism.

The negatively charged further peptide can be coupled to the conjugate according to the invention at any conceivable posi¬ tion as long as it is coupled to the conjugate via said amino acid sequence recognizable and cleavable by an enzyme that is specific for above-mentioned cells.

A suitable negatively charged further peptide comprise e.g. glutamic acid (E) or asparaginic acid (D) residues.

It is preferred if said at least one segment that establishes tumor cell specificity, comprises a nucleic acid molecule capa¬ ble of hybridizing to tumor cell specific molecules, preferably to oncogenes or/and genes involved in the growth of the tumor cell.

Such a nucleic acid molecule can be provided at the conjugate according to the invention by methods known in the art, in addition to said amino acid sequence recognizable and cleavable by a tumor cell specific enzyme. The nucleic acid can be lo¬ cated anywhere in the conjugate according to the invention, i.e. coupled to one of the peptides or even coupled to the therapeutically and/or diagnostically active agent. This nu¬ cleic acid molecule or molecules further increase the tumor cell specificity of the conjugate by means of so-called au- tisense-technology. The nucleic acid sequence of said mole- cule(s) is to be chosen is such a way that it is largely com¬ plementary to the nucleic acid sequence of tumor cell specific molecules. Such a nucleic acid molecule is capable of hybridiz- ing to oncogenes or growth involved genes in the tumor cells and therewith acts as a kind of "anchor" ensuring concentrated and maintaining activity of the conjugate in the desired trans¬ formed cell. To the contrary, the conjugate in non-transformed normal cells cannot either enter the latter due to the before- mentioned measures, or after a short time will again exit the cells since no oncogenes or growth involved genes are present and/or no or merely a negligible hybridizing reaction takes place. An overview of known oncogenes and growth involved genes from which the sequences of such nucleic acid molecules can be derived from is given in Vogelstein, B. and Kinzler, K.W. (2004), Cancer Genes and Pathways they Control, Nat. Med. 10, pages 789-799.

Genes involved in the growth of tumor cells refer to genes encoding for factors which are at least partially responsible for the growth of a tumor cell, such as growth factors like transforming growth factor β (TGF-β) or epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), FGF-3, -4, -5, int-1, etc.

According to the invention, such a nucleic acid molecule can be designed in such a way, that the conjugate according to the invention is able to hybridize to an mRNA as well as to a DNA largely complementary to said nucleic acid molecule.

In this connection use can also be made of peptides modified by nucleic acid molecules, so-called 'modified peptide nucleic acid¹ (PNA) as, e.g., described in WO 2004/24757 A2, WO 2004/01055 A2, or WO 2002/42316 A2. According to another preferred embodiment the conjugate accord¬ ing to the invention further comprises a detectable marker, preferably a fluorescent marker.

Appropriate fluorescent markers are: fluorescein (FITC), rhoda- mine, dansyl chloride, fluorescamine, green fluorescent protein (GFP), ethidium bromide, 4',6-Diamidino-2-phenylindole (DAPI), etc. Further conceivable marker are radioactive marker, like ³⁵S, ³²P, ¹²⁵I, ¹³¹I, ¹⁴C, ³H, non-radioactive marker like biotin, digoxygenine, luminescent marker, the BCIP/NBT system, etc.

By this measure a tool is provided that is useful in cell biol¬ ogy research, e.g. in order to study mechanisms of the cyto- plasmatic or nuclear import of molecules in vitro or even in vivo. This marker enables the determination of the localization of the conjugate by means of well-established methods in the art, such as fluorescence microscopy that can also be performed operatively in vivo, autoradiography, etc.

It is also preferred if at least one of the coordination sites of said image creating substance within the conjugate, prefera¬ bly Gd, is blocked by a substrate of a cellular enzyme.

For generating Tl weighted images it is necessary that the ninth coordination site of the gadolinium complex within the conjugate according to the invention is able to react with water protons. If all of the nine coordination sites are occu¬ pied or blocked, than the conjugate cannot function as a con¬ trast medium. This ninth coordination site can be blocked by a substrate for an enzyme in the nucleus resulting in an inactive contrast medium. When the substrate is removed by the intranu- clear enzyme water protons can interact with the gadolinium resulting in a signal in the Tl weighted image. The brighter the signal is, the more activity of said intranuclear enzyme can be concluded. By this measure a tool is provided by which the activity of enzymes in the nucleus can be analyzed via MRI.

According to a preferred embodiment the conjugate of the pre¬ sent invention further comprises an amphiphilic transport pep¬ tide.

By this measure the membrane permeability through the cell membrane of the conjugate according to the invention is addi¬ tionally ensured. Appropriate amphiphilic transport peptides are described in EP 1 424 343 Al, and encompass for example: members of the penetratin family, transportans, homeobox pro¬ teins, or fragments, derivatives, parts thereof having the same biological activity, i.e. still mediate membrane permeability.

Preferred structures of the conjugate according to the inven¬ tion are:

PKKKRKV or VRKKPKK - agent or agent/marker - RKRKRK or RRRRRR; the sequences at the N terminus (left) or the C terminus (right) are preferred amino acid sequences (one letter code) of the at least two flanking peptides.

Said agent preferably comprises the following structure: K(Gd-DTPA) resulting in the following entire structure of the conjugate: PKKKRKV/VRKKPKK-K(Gd-DTPA)-RKRKRK/RRRRRR. In case where an additional marker, e.g. FITC, is provided in the conjugate according to the invention, the following struc¬ ture is preferred: PKKKRKV/VRKKPKK-K(Gd-DTPA)-R-K(FITC)-RKRKRK/ RRRRRR.

In case where a recognition and cleavage site for a tumor cell- specific enzyme, e.g. PLGVR, and a negatively charged further peptide, e.g. EEEEEEEEEE, has to be provided, the following general structure is preferred: PKKKRKV/VRKKPKK-K(Gd-DTPA)- RKRKRK/RRRRRR-PLGVR-EEEEEEEEEE. It goes without saying that the negatively charged further peptide can also consist of more or less negatively charged amino acids; optionally the FITC unit can be included as described above, resulting in the following Structure: PKKKRKV/VRKKPKK-K(Gd-DTPA)-R-K(FITC)-RKRKRK/RRRRRR- PLGVR-EEEEEEEEEE.

It is also thought of a conjugate that does not comprise gadolinium or any other substance that can be used as a contrast medium or neutron-absorbing substance, but of a conjugate comprising a "conventional" cytostatic drug, such as a alkylating substance, and at least two peptides flanking said substance, as well as, if applicable, a recognition and cleavage site for a tumor cell-specific enzyme and a negatively charged further peptide: PKKKRKV/VRKKPKK-σytostatiσ drug- RKRKRK/RRRRRRf-PLGVR-EEEEEEEEEE]. Of course combinations are also possible: PKKKRKV/VRKKPKK-K(Gd-DTPA)-cytostatic drug- RKRKRK/RRRRRR[-PLGVR-EEEEEEEEEE] .

It goes without saying, that all the units of these conjugates can be modified, e.g. by adding further amino acids to their N termini or C termini, by the exchange of certain amino acids against amino acids having comparable properties, i.e. belong¬ ing to the same group of amino acids (polar, non-polar, acid, basic), by chemically modifying the units, by altering the positions of the units within the conjugate, as long as their specific functions which are explained further above, are not essentially affected.

Against this background, another subject-matter of the present invention is the use of the explained conjugate according to the invention, for preparing a diagnostic composition, prefera¬ bly a contrast medium for use in MRI examination.

The invention also relates to the use of the conjugate accord¬ ing to the invention, for preparing a therapeutic composition, preferably a cellular targeted cytotoxic substance. In this connection the present invention also relates to a pharmaceuti¬ cal or/and diagnostic composition comprising the conjugate according to the invention, and, if applicable, a pharmaceuti¬ cally or/and diagnostiσally acceptable carrier.

Pharmaceutically or/and diagnostically acceptable carriers are well known in the art, and e.g. described in Kibbe A. (2000), Handbook of Pharmaceutical Excipients, American Pharmaceutical Association and Pharmaceutical Press; this publication is in¬ corporated herein by reference. Of course, such a pharmaceuti¬ cal composition can also comprise further active agents, such as cytostatics or other anti-cancer drugs.

The present invention furthermore relates to the use of the conjugate according to the invention for preparing a composi- tion for the observation of the functioning of angiogenese inhibitory substances in a patient.

Several anti-cancer drugs are angiogenese-blocking substances, like Prinomastat that is inhibiting MMP2. By the use of MMP2- sensitive conjugate according to the invention, the action of such a drug can be monitored via the uptake of the conjugate into the cells or the nuclei, respectively.

The present invention also relates to a method for the diagnos¬ tic or analytical treatment of biological material or a living being, comprising the steps of: (a) Incubation or administering of the conjugate according to the invention with biological material or to a living being, and, (b) performing an imaging method.

The incubation can be performed under common laboratory condi¬ tion well known in the art, e.g. by the usage of conventional buffer systems, like Tris or HEPES buffer, etc., or salts as well as further supplements, as this is, e.g., described in Sambrook and Russell (2001), Molecular Cloning - A Laboratory Handbook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; this publication is incorporated herein by reference.

Biological material refers to any material that is derived or originates from a living being, e.g. a biological cell, parts thereof, cell compartments like nuclei, tissues, organs, pro¬ teins, nucleic acids, lipids, viruses, whole organisms, etc.

Suitable imaging methods are described further above. Another subject-matter of the present invention relates to a method for the therapeutic treatment of a living being, com¬ prising the step of: (a) Administering of a conjugate according to the invention comprising a neutron-absorbing substance, and (b) performing a neutron capture irradiation.

Suitable neutron-absorbing substances are explained in more detail further above.

Another subject of the present invention is a method for the therapeutic treatment of a living being comprising the step of: Administering of a conjugate according to the invention, comprising a cytotoxic agent, to a living being.

It will be understood that the features mentioned before and still to be mentioned in the following are not only applicable in the respective combination mentioned, but also in other combinations or in isolation, without leaving the scope of the present invention.

The present invention is now explained in more detail by means of preferred embodiments which are purely illustrative and do not restrict the invention, resulting in further features and advantages of the invention.

In the attached figures it is depicted:

Fig. 1 The nuclear localization of different conjugates according to the invention in nuclei of healthy kid¬ ney and lung cells demonstrated by confocal laser scanning microscopy (CLSM); CLSM of the kidney (three left-hand columns) and the lung (three right-hand columns) in nude mice 1 hour after intraperitoneal administration of istonic saline solution alone (row 1), isotonic saline plus FITC (row 2), and isotonic saline plus conjugates 1 - 6 (rows 3 - 8). Left-hand column for each organ and row: nuclei are demon¬ strated by TO-PRO-3 (specific staining of cell nu¬ clei) . Right-hand column for each organ and row: Lo¬ calization of the FITZ-labeled conjugates 1 - 6 or of the FITC alone. Middle column for each organ and row: Superimposed FITC and TO-PRO-3 images clearly demon¬ strate the nuclear localization of conjugates 2 - 5. Note the nuclear areas not showing fluorescence in rows 1 - 3 and 8.

Fig. 2 The efflux properties of a representative conjugate according to the invention shown by CLSM in kidney cells of nude mice; CLSM of the kidneys of nude mice 40 minutes [row 1 (magnification x40), row 2 (magni¬ fication xlOO)], 2,5 hours [row 3 (magnification xlOO)], and 3 days [row 4 (magnification xlOO)] after intraperitoneal injection of conjugate 2. Left-hand column: staining of the cell nuclei with FITC. Right- hand column: staining of the cell nuclei with FITC. Middle column: superimposed TO-PRO-3 and FITC images.

Fig. 3 CLSM image of LN-229 human gliomas one hour after intraperitoneal injection of isotonic saline plus a representative conjugate according to the invention (conjugate 2). (a) Tl weighted fat-suppressed MR im¬ age (TR 600 ms, TE 18 ms) showing the tumor mass situated on the left side of the falx cerebri; (b) corresponding H&E section (magnification xl2.5); (c) CLSM image of the tumor margin (upper area with stained cell nuclei). The cell nuclei in the healthy brain parenchyma with an intact blood-brain barrier did not stain (lower, dark area); (d) left-hand col¬ umn: staining of the cell nuclei of the LN-229 glioma with TO-PRO-3, right-hand column: staining of the cell nuclei with FITC, middle column: superimposed TO-PRO-3 and FITC images.

Fig. 4 MRI signal intensity of renal parenchyma before and after intraperitoneal administration of a representa¬ tive conjugate according to the invention (Tl weighted images, TR 800 ms, TE 9.1 ms).

Fig. 5 Electron microscopic images of kidney cell nuclei; A: section covered with DAB(3,3 '-diamino benzidine tetra hydrochloride) . The nuclei remained unstained. B: section covered with DAB and then incubated with per- oxidase-conjugated anti-FITC antibody for two minutes at 37 ⁰C. The nuclei stained dark.

Fig. 6 Sagittale and axial 3D MRI images (TR 300 ms, TE 15 ms) of 6 million LN-299 glioma cells demonstrating tumor cell specificity of conjugate 8 (C8) according to the invention comprising a recognition and cleav¬ age site for a tumor cell-specific enzyme and a nega¬ tively charged further peptide (upper image); confo- cal laser scanning images of LN 229 glioma cells in- cubated with tumor cell-specific conjugates according to the invention ( lower image ) ;

Fig. 7 Selective cleavage of conjugate 8 (C8) according to the invention comprising a recognition and cleavage site for metallo-proteinase 2 (MMP2), demonstrated by High Performance Liquid Chromatography (HPLC) analy¬ sis .

Embodiments

I. Material and methods

Synthesis of the gadolinium conjugates

For the solid phase synthesis of peptides 1-8 the Fmoc proce¬ dure (Merrifield, R.B.J. (1963), Amer. Chem. Soc . 85, 2149- 2154) was employed in a fully automated synthesizer (ABI 431) on 0.05 mmol Fmoc-Lys( BoC)-TCP Resin (Trityl-Resin) and on a 0.052 mmol Fmoc-Glu(OBut)-TCP Resin (Trityl-Resin). As coupling agent 2- ( lH-Bensotriazole-1-yl ) -1 , 1 , 3 , 3-tetramethyluronium hexafluorophosphate (HBTU) was used. The side chains of tri- functional amino acids were protected as follows: Arg(Pbf), Lys(Boc), Lys(Mmt), Lys(ivDde), and Glu(OBut) [ ivDde = 4,4- dimethyl-2,6-dioxocyclohexyl-l-ylidine)-3-metylbutyl; Mmt = 4- Methoxytrityl ; Pbf = 2, 2,4 , 6, 7-Pentamethyldihydrobenzofurane-5- sulfonyl; Boc=t-Butyloxycarbonyl; OBut=t-Butoxy ] . For coupling of the N-terminal amino acid Boc-Pro was used. The so synthesized conjugates according to the invention are depicted in the following table I.

conjugate Structure/Sequence #

Cl K(GdDTPA)-R-K(FITC)-RKRKRK C2 PKKKRKV-K(GdDTPA)-R-K(FITC)-RKRKRK C3 VRKKPKK-K(GdDTPA)-R-K(FITC)-RKRKRK C4 PKKKRKV-K(GdDTPA)-R-K(FITC)-RRRRRR C5 VRKKPKK-K(GdDTPA)-R-K(FITC)-RRRRRR C6 PKKKRKV-K(GdDTPA)-R-K(FITC)-R-K(GdDTPA)-RKRKRKRKRK Cl PKKKRKV-K(GdDTPA)-R-K(FITC)-RKRKRK-GVRPL-EEEEEEEEEE C8 PKKKRKV-K(GdDTPA)-R-K(FITC)-RKRKRK-PLGVR-EEEEEEEEEE

Table I formulas of conjugates 1-8 (Cl-8); K, lysine; R, arginine; P, proline; V, valine; Gd-DTPA; gadolinium- diethylenetriaminepentaacetate

Tumor Implantation

Animal experiments were approved by the Committee for Animal Experiments of the Regional Council (Rsgisrungsprasidium) of Tubingen.

Athymic female nude mice (CDl Nu/Nu) (weight: 25 g, age: 7 weeks) were purchased from Charles River, Sulzfeld, Germany. Intracerebral implantation of human LN-229 gliomas was per¬ formed as described previously, cf. Wick, W. et al. (2001), J Neuroscience 21(10): 3360-8. Confocal Laser Scanning Microscopy

The brain with the LN-229 glioma, all the organs (heart, lung, liver, kidneys, spleen) , the muscle tissue, peritoneum, and skin were excised and snap-frozen in Tissue Tek OCT in liquid nitrogen.

Cell nuclei were counterstained with TO-PRO-3 iodide.

The localization of the FITC-labeled gadolinium constructs, FITC alone, and TO-PRO was documented with a confocal laser scanning microscope (410 UV, Carl Zeiss, Jena, Germany). For the fluorescence excitation of FITC and TO-PRO we used the 488 and the 633 nm line, respectively, of an argon ion laser and appropriate beam splitters and barrier filters. The confocal aperture diameter was adjusted for an optical slice thickness of 400 nm.

Superimposed images of FITC- and TO-PRO stained samples were created by overlaying coincident views.

Hematoxylin and Eosin (HSE) stained sections of these specimens were also prepared.

Electron microscopy

Specimens were freeze-substituted at -30 ⁰C and then embedded in Lowicryl K4M (Polysciences, Eppelheim, Germany) . Ultrathin sections (70 nm) were obtained. Sections were first covered with DAB (3,3'-diaminobenzidine tetra hydrochloride) (Roche, Mannheim, Germany) and then incubated with peroxidase-conju- gated anti-FITC antibody (Roche, Mannheim, Germany) for two minutes at 37 ⁰C. Afterwards the sections were rinsed three times with PBS. Specimens were analyzed and documented with an EM 1OA electron microscope (Zeiss, Oberkochen, Germany).

Influx Studies

0.5 mg FITC (389 Dalton) alone, 2.8 mg conjugate 1 (2148 DaI- ton), 4 mg conjugates 2-5 (3036, 3013, 3097, 3097 Dalton) and 5.7 mg conjugate 6 (4388 Dalton) each were dissolved in 0.5 ml isotonic saline.

60 minutes after intraperitoneal administration of 0.5 ml iso¬ tonic saline solution alone (n=2), FITC (n=2), conjugate 1 (n=2), conjugate 2 (n=2), conjugate 3 (n=2), conjugate 4 (n=2), conjugate 5 (n=2), and conjugate 6 (n=2) and 90 minutes after intraperitoneal administration of conjugate 2 (n=2) and conju¬ gate 3 (n=2) the animals were killed and the organs, the brain, and the LN-229 brain tumors were frozen, as described above.

Efflux studies

To determine whether conjugate 2 also flows out of the cell nuclei, it was injected intraperitoneally into 6 animals, and the animals subsequently killed: 2 after 40 min, 2 after 2.5 hours, and another 2 after 3 days. Organs were excised and snap-frozen in Tissue Tek OCT in liquid nitrogen. The animals were not anesthetized for this procedure in order to be able to observe their behavior. MRI measurements

MRI was performed using a clinical 3 Tesla Siemens whole body MRI (Trio) with the nude mice in prone position in a standard circular polarized wrist coil. The imaging protocol consisted of:

TI weighted fat-suppressed transverse images: slice thickness 2 mm, field of view read 31 mm, field of view phase 82,3 %, voxel seize 0.2 x 0.2 x 2 mm, TR 600 ms, TE 18 ms, flip angle 180, number of slices 12, distance factor 0, scan time 11:35 min.

TI weighted transverse images: slice thickness 2 mm, field of view read 60 mm, field of view phase 87.5 %, voxel seize 0.2 x 0.2 x 2 mm, TR 800 ms, TE 9.1 ms, flip angle 90,2 acquisitions, number of slices 25, distance factor 0, scan time 06:02 min.

12 weighted transverse images: slice thickness 2 mm, field of view read 31 mm, field of view phase 82.3 %, voxel seize 0.2 x 0.2 x 2 mm, TR 3000 ms, TE 91 ms, flip angle 180, number of slices 12, distance factor 0, scan time 10:53 min.

3D sequence: slice thickness 0.3125 mm, field of view read 63 mm, field of view phase 100.0 %, base resolution 256, phase resolution 100 %, slice resolution 100 %, voxel seize 0.2 x 0.2 x 0.3 mm, slab group 1, slabs 1, slices per slab 16, TR 300 ms, TE 15 ms, flip angle 70, distance factor 50, scan time 12:02 min. The animals were sedated by intraperitoneal injection of keta- mine plus xylazine.

Non-contrast enhanced imaging studies were first performed in all of the nude mice (n=12). After intraperitoneal administra¬ tion of 4 mg conjugate 2 (n=4), 4 mg conjugate 3 (n=4), and 0.7 mg Gd-DTPA (532 Dalton) (n=4), MRI was performed every 6-12 minutes.

After 60 minutes [conjugate 2 (n=2), conjugate 3 (n=2), and Gd- DTPA (n=4)] and 90 minutes [conjugate 2 (n=2), conjugate 3 (n=2)] the animals were killed and the organs, brain, and the LN-229 brain tumors were excised and snap-frozen in Tissue Tek OCT in liquid nitrogen.

II. Results

Nuclear localization and accumulation of different conjugates according to the invention

In order to exclude that the intraperitoneal administration of a fluorochroine dye (FITG) alone increases the fluorescence in the cell nuclei of nude mice with LN-229 gliomas, the CLSM images of lung, heart, liver, spleen, intestinal tract, skin, smooth and striate muscle, peritoneum, brain, and brain tumor in nude mice in which only 0.5 ml of isotonic saline solution was injected intraperitoneally, were compared with those from mice that had received 0.5 mg FITC (dissolved in 0.5 ml iso¬ tonic saline solution) intraperitoneally (organs excised 1 hour after injection) . In both cases the fluorescent signal in the cell nuclei is only very weak on the CLSM images (Fig. 1).

No increase in fluorescence in the cell nuclei could be ob¬ served after intraperitoneal injection of the gadolinium com¬ plex that was only bound to the nuclear localization sequence of the ALL-I protein either (conjugate 1, Fig. 1, Table I).

The fluorescence did not increase considerably in the cell nuclei until a gadolinium complex was injected in which not only the nuclear localization sequence of the ALL-I protein was bound to the one side but where the NLS of the SV 40 T antigen was also bound to the other side (conjugate 2, Fig. 1, Table I).

Strong fluorescence in the cell nuclei could also be observed after intraperitoneal injection of gadolinium complexes in which one of the NLS sequences was altered [correct NLS se¬ quence of the ALL-I protein but a random NLS of the SV 40 T antigen (conjugate 3, Fig. 1, Table 1) or a random NLS of the ALL-I protein but correct NLS of the SV 40 T antigen (conjugate 4, Fig. 1, Table I)]. Surprisingly, an increase in fluorescence was also seen after intraperitoneal administration of a gado¬ linium complex to which not only the wrong SV 40 T antigen NLS but also the wrong NLS of the ALL-I protein was bound (conju¬ gate 5, Fig. 1, Table I). Only after a second gadolinium com¬ plex was bound to a conjugate with the correct NLS of the SV 40 T antigen and the correct nuclear localization sequence of the ALL-I protein extended by 4 amino acids (Arginine-Lysine- Arginine-Lysine) was no increase in fluorescence seen in the cell nuclei after intraperitoneal administration (conjugate 6, Fig. 1, Table 1).

The efflux properties and further advantages of the conjugates according to the invention

Figure 2 illustrates the strong fluorescence seen in renal cell nuclei 40 minutes after intraperitoneal injection of conjugate 2 with the two correct NLS in non-anesthetized nude mice. By 2.5 hours after the injection, only weak fluorescence is seen in the cell nuclei. Two other nude mice were observed for 3 days and the clinical and histopathological findings in these mice were normal. Conjugates were also detectable in MRI (in¬ crease in signal intensity in all the organs and the LN-229 gliomas except for the healthy brain parenchyma, Fig. 3 and were rapidly excreted via the gall bladder and kidneys (Fig. 4).

After intraperitoneal administration of the standard gadolinium complex (corresponding to the amount of the gadolinium complex in conjugate 2), no increase in signal intensity was seen in the LN 229 gliomas, however. That the LN-229 gliomas could be better visualized using conjugate 2 may be a result of the intranuclear localization of the gadolinium complex but also of higher resorption of conjugate 2 by the peritoneum.

The individual conjugates 2-5 were not quantitatively compared regarding the nuclear uptake because differences in resorption by the peritoneum might already exist. Conjugates 2-5 (each with 16 amino acids and a gadolinium com¬ plex) may also be taken up by cell nuclei by passive diffusion. Gadolinium conjugates of comparable size (gadolinium complex bound to 16 arginine) were synthesized (Allen, M.J. & Meade T.J. (2003), J. Biol. Inorg. Chem. 8, 746-50); however, these conjugates remained in the cytoplasm in vitro and could not permeate the cell nucleus.

In conclusion, by binding two nuclear localization sequences to the gadolinium complex, the substance could not only be trans¬ ported across the external cell membrane but also across the nuclear membrane. The conjugates according to the invention avoid using large transmembrane transport units, which in turn has a positive effect on the relationship between the unit giving the signal on MRI (gadolinium complex) and the peptide components.

Changing the order of the amino acids within the nuclear local¬ ization sequences did not prevent uptake of the gadolinium complex in the cell nucleus.

Tumor Cell Specificity of Specific Conjugates according to the invention

Six million L-229 glioma cells were incubated for one hour in Eppendorf tubes with conjugate 2 (0.2 mg/0.3 ml, Fig. 6, left), conjugate 7 (0.35 mg/0.3 ml, Fig. 6 middle), and conjugate 8 (0.35 mg/0.3 ml, Fig. 6 right) in the presence of the tumor cell specific enzyme matrix metallo-proteinase-2 (MMP 2). Each tube was washed with culture medium and centrifuged. Fig. 6 shows sagittal and axial 3D MRI images (TR 300 ms, DE 15 mf) of the cells.

As can be seen in the upper image of Fig. 6 conjugate 2 (left) is uptaken into the nuclei. Therefore, the cells appear hyper- intense after the centrifugation in the Tl weighted MRI images (pellet), since the gadolinium complex remains in the nuclei after the centrifugation and washing of the cells.

Conjugate 7 (upper image of Fig. 6, middle) is not internalized by the cells, since the matrix metallo-proteinase-2 that is present in the incubation medium, cannot cleave the sequence GVRPL. After three times washing and a centrifugation step, the glioma cells maintain their original brightness in the MRI images, i.e. no increase of the intensity of the signal can be observed.

Conjugate 8 (upper image of Fig. 6, right) is uptaken into the nuclei, since the matrix metallo-proteinase-2 that is present in the incubation medium, is able to recognize and cleave the sequence PLGVR, resulting in a positively charged conjugate 2. Therefore, the cells appear hyperintense in the Tl weighted MRT images after the centrifugation step, like in the left tube, since the gadolinium complex remains in the nuclei even after the centrifugation and washing of the cells.

The lower images show confocal laser scanning images of LN 229 glioma cells which grow at the bottom of the culture bottle (one hour of incubation with conjugates 8 and 7, concentrations as given further above, followed by three times washing without centrifugation, the cells remain adherent on the bottom) . As can be seen on the left image of Fig. 6, lower part, conju¬ gate 7 cannot be detected in the nuclei, i.e. is not able to enter the nuclei of the cells. In contrast, conjugate 8 accumu¬ lates in the nuclei of the tumor cells; cf. Fig. 6 lower im¬ ages, right.

Cleavage of a tumor cell-specific conjugate by matrix metallo- proteinase-2 (MMP2) (Enzyme cleavage assay)

In an enzyme cleavage assay it was verified that conjugate 8 comprising a MMP2 sensitive sequence (PLGVR) was recognized and cleaved by MMP2, whereas conjugate 7 comprising a MMP2 random sequence (GVRPL) was not cleaved.

Therefor both conjugates were incubated with 5 μg of active human MMP2 (Cat # PF023) . The reaction products were analyzed by high performance liquid chromatography (HPLC) .

As can be seen in Fig. 7A, for MMP2 sensitive conjugate 8 the initial peak (upper diagram) disappears, resulting in several smaller peaks (lower diagram). This means that conjugate 8 was cleaved by MMP2 into two smaller main peptides.

As can be seen in Fig. 7B MMP2 non-sensitve conjugate 7 cannot be cleaved by MMP2, so that this conjugate remains in its ini¬ tial form: Both peaks in upper and lower diagram are equal.

This assay additionally demonstrates the enablement of a tumor cell specific conjugate according to the invention.

Claims

1. A conjugate comprising a therapeutically and/or diagnosti- cally active agent and at least two peptides flanking said agent or/and at least one branched-chained peptide coupled to said agent.

2. The conjugate according to claim 1, characterized in that said at least two peptides or/and said at least one branched-chained peptide are largely positively charged.

3. The conjugate according to claim 1 or 2, characterized in that said at least two peptides consisting of about two to fifteen, preferably of about six to eight amino ac¬ ids, or/and said at least one branched-chained peptide consisting of about four to thirty, preferably of about twelve to sixteen amino acids.

4. The conjugate according to any of claims 1 to 3, charac¬ terized in that said at least two peptides or/and said at least one branched-chained peptide comprise a nuclear localization sequence (NLS) or a sequence that is derived therefrom.

5. The conjugate according to any of claims 1 to 4, charac¬ terized in that one of said at least two peptides com¬ prises the NLS of the SV 40 T antigen, and another one of said at least two peptides comprises the NLS of the ALL-I protein, and/or said at least one branched-chained peptide comprise the NLS of the SV 40 T antigen and the NLS of the ALL-I protein.

6. The conjugate according to any of claims 1 to 5, charac¬ terized in that said agent comprise an image creating substance.

7. The conjugate according to claim 6, characterized in that said image creating substance is selected from the group consisting of: Gd, Ga, I, Fe, Mn and F.

8. The conjugate according to any of claims 1 to 7, charac¬ terized in that said agent comprise a cell damaging property.

9. The conjugate according to claim 8, characterized in that said agent is a neutron-absorbing substance that can be used in the Neutron Capture Therapy (NCT) .

10. The conjugate according to any of claims 1 to 9, characterized in that said agent comprise a cytotoxic drug.

11. The conjugate according to any of claims 1 to 10, charac¬ terized in that it further comprises at least one seg¬ ment or/and a compound establishing specificity for a tu¬ mor cell, or/and an apoptotic cell, or/and a cell of a thrombosis area, or/and a cell infected by a virus.

12. The conjugate according to claim 11, characterized in that said at least one segment comprises an amino acid sequence recognizable and cleavable by an enzyme specific for a tumor cell, or/and an apoptotic cell, or/and a cell of a thrombosis area, or/and a cell infected by a virus, said segment is optionally coupled to a negatively charged further peptide.

13. The conjugate according to claim 12, characterized in that said enzyme is selected from the group consisting of: matrix metallo-proteinase 2 (MMP2), cathepsin, pros¬ tate-specific antigen (PSA) , herpes simplex virus- protease, human immunodeficiency virus-protease, cytomega- lovirus-protease, caspase, interleukin-lβ converting en¬ zyme, thrombin.

14. The conjugate according to any of claims 11 to 13, characterized in that said at least one segment comprises a nucleic acid molecule capable of hybridizing to tumor cell specific molecules, preferably to oncogenes or/and genes involved in the growth of the tumor cell.

15. The conjugate according to any of claims 1 to 14, charac¬ terized in that it further comprises a detectable marker, preferably a fluorescent marker.

16. The conjugate according to any of claims 7 to 15, charac¬ terized in that at least one of the coordination sites of said image creating substance, preferably of Gd, is blocked by an substrate of a cellular enzyme.

17. The conjugate according to any of claims 1 to 16, charac¬ terized in that it further comprises an amphiphilic transport peptide.

18. The conjugate according to any of claims 1 to 17, charac¬ terized in that it comprises the following structure: PKKKRKV or VRKKPKK - agent or agent/marker - RKRKRK or RRRRRR.

19. The conjugate according to claim 18, characterized in that said agent comprises the following structure: K(Gd-DTPA) .

20. The conjugate according to claim 18 or 19, characterized in that said agent/marker comprises the following struc¬ ture: K(Gd-DTPA)-R-K(FITC).

21. The conjugate according to any of claims 18 to 20, further comprising at its N terminus the following structure: tu¬ mor enzyme-recognition amino acid sequence - negatively charged amino acid sequence, preferably PLGVR-EEEEEEEEE or KK-EEEEEEEEE or RR-EEEEEEEEE or PIC(Et)FF-EEEEEEEEE or GGPRGLPG-EEEEEEEEE or HSSKLQ-LVLASSSFGY.

22. The conjugate according to any of claims 18 to 20, further comprising at its N terminus the following structure: vi¬ rus enzyme-recognition amino acid sequence - negatively- charged amino acid sequence, preferably LVLASSSFGY- EEEEEEEEE or GVSQNYPIVG-EEEEEEEEE or GWQASCRLA-EEEEEEEEE.

23. The conjugate according to any of claims 18 to 20, further comprising at its N terminus the following structure: apoptosis enzyme-recognition amino acid sequence - nega¬ tively charged amino acid sequence, preferably DEVD- EEEEEEEEE or GWQASCRLA-EEEEEEEEE.

24. The conjugate according to any of claims 18 to 20, further comprising at its N terminus the following structure: thrombin recognition amino acid sequence - negatively charged amino acid sequence, preferably dFPipR-EEEEEEEEE.

25. Use of the conjugate according to any of claims 1 to 24 for preparing a diagnostic composition, preferably a con¬ trast medium for use in MRI examination.

26. Use of the conjugate according to any of claims 1 to 24 for preparing a therapeutic composition, preferably a cel¬ lular targeted cytotoxic substance.

27. Use of the conjugate according to any of claims 1 to 24 for preparing a composition for the observation of the functioning of angiogenese inhibitory substances in a pa¬ tient.

28. A pharmaceutical or/and diagnostic composition comprising the conjugate according to any of claims 1 to 24, and, if applicable, a pharmaceutically or/and diagnostically ac¬ ceptable carrier.

29. A method for the diagnostic or analytical treatment of biological material or a living being, comprising the steps of:

(a) Incubation or administering of a conjugate according to any of claims 1 to 9, or 11 to 24 with biological material or to a living being, and

(b) Performing an imaging method.

30. A method for the therapeutic treatment of a living being, comprising the step of:

(a) Administering of a conjugate according to any of claims 9, or 11 to 24, to a living being, local or systemic, and

(b) Performing a neutron capture irradiation.

31. A method for the therapeutic treatment of a living being comprising the step of: Administering of a conjugate according to any of claims 10 to 24 to a living being.