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Labeling reactant

Labeling reactant description/claims

This application is a continuation of application Ser. No. 11/607,045, filed on Dec. 1, 2006, which claims priority to U.S. Provisional Application No. 60/748,195 filed on Dec. 8, 2005, the disclosure of which are incorporated herein in their entirety by reference. This application also claims priority under 35 U.S.C. § 119 to Finnish Patent Application No. 20055653, filed on Dec. 8, 2005, in the Finnish Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.

This invention relates to novel derivatives of diethylenetriaminepentaacetic acid which allow site specific introduction of the ligand of said derivatives to bioactive molecules on solid phase.

The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

Because of its excellent metal chelating properties diethylenetriaminepentaacetic acid (DTPA) is one of the most widely used organic ligands in magnetic resonance imaging (MRI) and positron emission tomography (PET) [Aime, S., Botta, M., Fasano, M. and Terrano, E. 1998, Chem. Soc. Rev., 27, 19, Caravan, P., Ellison, J. J., McMurry, T. J. and Lauffer, R. B., 1999, Chem. Rev., 99, 2293, Woods, M., Kovacs, Z. and Sherry, A. D., 2002, J. Supramol. Chem., 2, 1]. Indeed, the first FDA approved contrast agent in clinical use is the Gd3+ DTPA chelate [Runge, V. M., 2000, J. Magn. Res. Imaging, 12, 205.]. The corresponding 111 in and 68Ga chelates, in turn, are suitable for PET applications [Anderson, C. J. and Welch, M. J., 1999, Chem. Rev. 99, 2219], while Eu3+, Tb3+, Sm3+ and Dy3+chelates can be used in applications based on disassociation enhanced lanthanide fluorescence immunoassay (DELFIA) [PCT WO 03/076939A1]. 99mTc DTPA in turn, is suitable for single positron emission computed tomography (SPECT) [Lorberboym, M., Lampl, Y. and Sadeh, M., 2003, J. Nucl. Med. 44, 1898, Galuska, L., Leovey, A., Szucs-Farkas, Z., Garai, I., Szabo, J., Varga, J. and Nagy, E. V., 2002, Nucl. Med. Commun. 23, 1211]. Bioactive molecules labeled with 111In or 117mSn DTPA may find applications as target-specific radiopharmaceuticals [Volkert, W. A. and Hoffman, T. J., 1999, Chem. Rev. 99, 2269].

In several applications, covalent conjugation of DTPA to bioactive molecules is required. Often, isothiocyanato, N-hydroxysuccinimide or maleimide derivatives of the chelate are used in the labeling the target molecules such as oligonucleotides and oligopeptides. Several bifunctional DTPA derivatives are currently commercially available. Because in all of these cases the labeling reaction is performed in the presence of an excess of an activated label, laborious purification procedures cannot be prevented. Especially, when attachment of several label molecules is needed, purification and characterization of the desired biomolecule conjugate may be extremely difficult.

The purification problems can be avoided by performing the labeling reaction on solid phase. Hence, most of the impurities can be removed by washings when the biomolecule conjugate is still anchored to the solid support, and after release to the solution, only one chromatographic purification is needed. Several such blocks have been published. They include organic dyes [Loshe, J., Nielsen, P. E., Harrit, N. and Dahl, O., 1997, Bioconjugate Chem. 8, 503, McCafferty, D. G., Bishop, B. M., Wall, C. G., Hughes, S. G., Mecklenberg, S. L., Meyer, T. J., and Erickson, D. W., 1995, Tetrahedron, 51, 1093, WO 96/03409, Cuppoletti, A., Cho, Y., Park, J.-C., Strässler, G. and Kool, E. T. 2005, Bioconjugate Chem. 16, 528, Bethelot, T., Lain, G., Latxague, L. and Deleris, G., 2004, J. Fluorescence, 14, 671], derivatives of EDTA [Sluka, J. P., Griffin, J. H., Mack, D. P. and Dervan, P. B. 1990, J. Am. Chem. Soc, 112, 6369, Arya, R. and Gariepy, J. 1991, Bioconjugate Chem., 2, 323, Cuenoud, B. and Schepartz, A. 1991, Tetrahedron, 47, 2535, Rana, T. M., Ban, M. and Hearst, J. E., 1992, Tetrahedron Lett, 33, 4521, Song, A. I. and Rana, T. A., 1997, Bioconjugate Chem., 8, 249, Davies, J. C., Al-Jamri, L., 2002, J. peptide Sci., 8, 663, U.S. Pat. No. 5,637,759], DOTA [Bhorade, R., Weissleder, R., Nakakoshi, T., Moore, A. and Tung, C.-H., 2000, Bioconjugate Chem., 11, 301., Gallazzi, F., Wang, Y., Jia, F., Shenoy, N., Landon, L. A., Hannink, M., Lever, S. Z. and Lewis, M. R. 2003, Bioconjugate Chem., 14, 1083.] and luminescent and non-luminescent lanthanide chelates [U.S. Pat. No. 6,080,839; 6,949,696; Peuralahti, J., Hakala, H., Mukkala, V.-M., Hurskainen, P., Mulari, O. and Hovinen, J. 2002 Bioconjugate Chem. 13, 876.].

Although DTPA molecule is known for decades, and although reagents for solid phase oligonucleotide derivatization with DTPA has been demonstrated [U.S. Pat. No. 6,949,639], no reactants which allow its direct solid phase conjugation to oligopeptides have been synthesized. The solid phase methods published involve synthesis of oligopeptides, where one ε-amino group of lysine is selectively deprotected while the oligomer is still anchored to the resin [Handl, H. L., Vagner, J., Yamamura, H. I., Hruby, V. J. and Gilles, R. J. 2005, Anal. Biochem. 343, 299, Nagy, I. B., Vagra, I. and Hudecz, F, 2000, Anal. Biochem., 287, 17] Then, an activated DTPA molecule (as an anhydride or an HOBt ester) is coupled to the primary amino function, the oligopeptide is deprotected and converted to the appropriate DTPA chelate. However, this methodology has some drawbacks. First, practically only one DTPA molecule can be introduced. This may be problematic in applications were high detection sensitivity is required. Second, since one of the iminoacetic acid groups is used for conjugation, the resulting chelate is less stable than the parent DTPA molecule [Paul-Roth, C. and Raymond, K. N. 1995, Inorg. Chem. 34, 1408, Li, W. P., Ma, D. S., Higginbotham, C., Hoffman, T., Ketring, A. R., Cutler, C. S. and Jurisson, S. S. 2001, Nucl. Med. Biol. 28, 145.]. This may be a serious problem in vivo applications especially in MRI due to the high toxicity of free Gd(III) ion.

A schematic preparation of stable DTPA derivatives applicable to solid phase peptide incorporation have been proposed [U.S. Pat. No. 5,637,759], but the method of their preparation is challenging due to the carboxyl protecting strategy. There, selective deprotection of a single and specic carboxylic acid group out of six of similar reactivities is required. This problem can be avoided by changing the protecting group strategy, but the synthetic route will be considerably longer [WO 03/011115].

The main object of the present invention is to provide DTPA derivatives which allow solid phase introduction of the chelate to bioactive molecules using a standard oligopeptide synthesizer. The bioconjugates thus obtained are highly suitable for magnetic resonance imaging (MRI), positron emission tomography (PET), single positron emission computed tomography (SPECT) and dissossiation enhanced lanthanide fluorescence immunoassay (DELFIA) as well as target-specific radiopharmaceuticals. The major advantage of the present invention are:

(i) synthesis of the building block is simple and thus these molecules can be synthesized in large scale;

(ii) the blocks can be introduced to the biomolecule structure with standard oligopeptide synthesizer in high efficiency using normal procedures;

(iii) since the metal is introduced after the chain assembly is completed, the molecule synthesized can be used in various applications simply by changing the metal;

(iv) since none of the DTPA carboxylic acid residues are used for conjugation the stability of the chelate does not change;

(v) the chelate formation is considerably faster than in the case of DOTA (the other most commonly used chelator). This is advantageous while working with short-living radioisotopes.

(vi) the labeling reactant can be used in the labeling of a large variety of bioactive molecules such as oligopeptides, steroids and drugs.

In some applications it is advantageous that the chelate is neutral. Then, two of the acetate groups can be substituted with amides. Naturally, the stability of these chelates is lower than that of the corresponding acetates.

Thus, the present invention concerns a labeling reactant of formula (I) or (Ia) suitable for labeling of a biospecific binding reactant

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