Patent 6,765,104

The present invention provides transition metal complexes of N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane and related compositions and methods of synthesis and use in vitro and in vivo, such as a therapeutic agent or a delivery/imaging agent.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a transition metal complex of N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane. Preferably, the transition metal is copper, in particular copper II (Cu (II)). The trialkyl preferably comprises C₁ -C₆ alkyl groups, which may be the same or different. Preferably, the trialkyl is trimethyl or triethyl, with trimethyl being especially preferred. The transition metal can be radioactive, such as a positron emitter, e.g., Cu⁶⁴, or a .beta.-emitter, e.g, Cu⁶⁷.

The transition metal complex of N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane can be conjugated to a targeting agent. Preferably, the targeting agent is an immunological agent, a protein, a polypeptide, a peptide, a nucleic acid or a steroid.

Also provided by the present invention is a composition comprising a transition metal complex of N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane, or a conjugate thereof, and a carrier therefor.

In addition to the above, the present invention provides a method of synthesizing N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane. The method comprises derivatizing 1,3,5-cis,cis-triaminocyclohexane to a tris-sulfonamide, removing the sulfonamide proton with a base to generate a tris-anion, quenching the tris-anion with an alkylating agent, and cleaving the sulfonamide group with an acid to generate N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane as a protonated salt. The method can further comprise adding an equimolar amount of CuX₂ in water to the N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane, neutralizing the resulting solution with base solution (to form a deep-blue solution), and removing the hydroxides that form by filtration to yield a solution of the Cu (II) complex.

Further provided by the present invention is a method of cleaving a biological molecule. The method comprises contacting the biological molecule with an above-described complex, which cleaves the biological molecule. Preferably, the biological molecule is in vivo and the cleavage of the biological molecule has a therapeutic effect. Also preferably, the complex is targeted to an abnormally proliferating cell that comprises the biological molecule, such as a cancerous cell and the therapeutic effect is the treatment of cancer.

Still further provided by the present invention is a method of inhibiting expression of a nucleic acid. The method comprises contacting a nucleic acid with an above-described transition metal complex conjugated to an antisense nucleic acid specific for the nucleic acid, whereupon the antisense nucleic acid binds to the nucleic acid, thereby inhibiting expression of the nucleic acid.

In another embodiment, the present invention provides a method of radiation therapy. The method comprises administering to an animal in need of radiation therapy a therapeutically effective amount of an above-described radioactive complex, which is (i) conjugated to a targeting agent that binds to a molecule on the surface of a cell to be treated with radiation or (ii) encapsulated in a liposome comprising on its surface the targeting agent.

In yet another embodiment, a method of imaging is provided. The method comprises (i) administering to an animal an imaging effective amount of an above-described radioactive complex, which is either conjugated to a targeting agent that binds to a molecule on the surface of a cell to be imaged or encapsulated in a liposome comprising on its surface the targeting agent, and (ii) imaging the complex. Preferably, the targeting agent is an immunological agent.

In still yet another embodiment, the present invention provides a method of tracing a compound in an animal. The method comprises administering to an animal a mixture of the compound and an above-described complex and tracing the location of the complex in the animal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the surprising and unexpected discovery of transition metal complexes of N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane, which can cleave activated phosphate diesters at an unprecedented rate and demonstrate concentration-dependent cytotoxicity. The present invention is also predicated on the discovery of a method of synthesizing tri-alkyl-cis,cis-1,3,5-triaminocyclohexane, which is less complicated and provides higher yields than currently available methods of synthesis.

In view of the above, the present invention provides in one embodiment transition metal complexes of N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane. Preferably, the transition metal is copper, in particular copper II. The trialkyl preferably comprises C₁ -C₆ alkyl groups, which may be the same or different Trimethyl and triethyl are preferred as the trialkyl, with trimethyl being especially preferred.

In one embodiment of the transition metal complexes of N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane, the transition metal is preferably radioactive. A preferred radioactive transition metal is a positron emitter, such as Cu⁶⁴. Another preferred radioactive transition metal is a .beta.-emitter, such as Cu⁶⁷.

While N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane can be synthesized in accordance with methods known in the art (Stetter et al., Chem. Ber. 106: 2523-2529 (1973)), preferably N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane and transition metal complexes thereof are synthesized in accordance with the methods set forth herein.

The above-described complex can be conjugated to a targeting agent. By "targeting agent" is meant any means that enables specific interaction with a target The targeting agent can bind to a defined population of cells through a receptor, a substrate, an antigenic determinant or another binding site on the target cell population. Examples include an "immunological agent," which is used herein to refer to an antibody, such as a polyclonal antibody or a monoclonal antibody, an immunologically reactive fragment of an antibody, an engineered immunoprotein and the like; a protein (target is receptor, as substrate, or regulatory site on DNA or RNA); a polypeptide; a peptide (target is receptor); a nucleic acid, which is DNA or RNA, single-stranded or double-stranded, synthetic or isolated and purified from nature (target is complementary nucleic acid); a steroid (target is steroid receptor); and the like. Preferred targeting agents include an antibody or an immunologically reactive fragment thereof, a peptide, e.g., bombesin, gastrin-releasing peptide, RGD peptide, substance P, neuromedin-B, neuromedin-C, somatostatin, octreotide analogues, and metenkephalin, and a hormone, e.g., estradiol, neurotensin, melanocyte stimulating hormone, follicle analogues stimulating hormone, leutenizing hormone, and human growth hormone. Other suitable targeting agents include serum proteins, fibrinolytic enzymes, and biological response modifiers, such as interleukin, interferon, erythropoietin, and colony-stimulating factor. Analogs of targeting agents that retain the ability to bind to a defined target also can be used. In addition, synthetic targeting agents can be designed, such as to fit a particular epitope. The targeting agent can include any linking group that can be used to join a targeting agent to, in the context of the present invention, a complex. It will be evident to one skilled in the art that a variety of linking groups, including bifunctional reagents, can be used.

An above-described complex can be conjugated to a targeting agent by covalent or non-covalent bonding. If bonding is non-covalent, the conjugation can be through hydrogen bonding, ionic bonding, hydrophobic or van der Waals interactions, or any other appropriate type of binding.

In view of the above, the present invention provides in another embodiment a composition, e.g., a pharmaceutical composition, comprising an above-described complex, or conjugate thereof, and a carrier therefor, e.g., a pharmaceutically acceptable carrier. Suitable carriers for in vitro and in vivo use are known in the art. A biologically acceptable, normal saline solution can be appropriately employed. The carrier can include a minor amount of a carrier protein, such as human serum albumin, for example, to stabilize the targeting agent. Stabilizers, antioxidants, osmolality adjusting agents, buffers, pH adjusting agents, etc., can be included in the composition. The composition can be in the form of a solution, suspension or dispersion. Suitable additives include, for example, physiologically biocompatible buffers, additions of chelants or calcium chelate complexes, or optionally, additions of calcium or sodium salts.

Parenterally administrable forms, e.g., intravenous forms, should be sterile and free from physiologically unacceptable agents and should have low osmolality to minimize irritation or other adverse effects upon administration. Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions, such as sodium chloride injection, Ringer's injection, and dextrose injection. Lactated Ringer's injection and other solutions are as described in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co. (1975). The solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions and excipients and other additives that are compatible with the chelates and will not interfere with the manufacture, storage or use of products.

The concentration of complex or conjugate thereof in a composition will be a matter of choice. Levels of 0.5 mg/ml are readily attainable but the concentration may vary considerably depending upon the specifics of any given application. Appropriate concentrations of biologically active materials in a carrier are routinely determined in the art.

The effective dose of complex or conjugate thereof to be utilized for any application will also depend upon the particulars of that application. In treating tumors in the context of radioinmmunotherapy, for example, the dose will depend, inter alia, upon tumor burden, accessibility, route of administration, administration of other active agents, and the like. Generally, a therapeutically effective dose is from about 20 mCi to about 300 mCi.

The complexes and conjugates thereof can be administered in accordance with the present inventive methods by any suitable route. Such routes include intravenous, intraperitoneal, and the like, depending on the disease or cancer to be treated, respectively, the location of the diseased/cancerous cells, the extent of disease/cancer, and other factors. The determination of the appropriate route(s) of administration for a given application is within the skill in the art.

The present invention provides as another embodiment a method of synthesizing N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane. The method comprises the derivatization of 1,3,5-cis,cis-triaminocyclohexane to a tris-sulfonamide. The sulfonamide proton is then removed by a base (e.g., NaH) to generate the tris-anion. The tris-anion is then quenched with an alkylating agent (e.g., a dialkylsulphonate, although alkyl halides are not excluded). The sulfonamide group is then cleaved with an acid to generate the N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane as a protonated salt The method can further comprise the addition of an equimolar amount of CuX₂ in water to the N,N',N"-trialkyl-cis,cis1,3,5-triaminocyclohexane, which is then neutralized with base, e.g., 3 equivalents,to form a deep-blue solution. The hydroxides that form are removed by filtration to yield a solution of the Cu (II) complex.

In still yet another embodiment of the present invention, a method of cleaving a biological molecule is provided. The biological molecule comprises a peptide linkage or a phosphodiester bond and, thus, is preferably a peptide, polypeptide, protein or nucleic acid. The method comprises contacting a biological molecule with an above-described complex, whereupon the complex cleaves the biological molecule. Preferably, the biological molecule is in vivo and the cleavage of the biological molecule has a therapeutic effect, such as the treatment of disease, in particular a disease associated with abnormal cellular proliferation, such as cancer. Also preferably, the complex is targeted to an abnormally proliferating cell, which comprises the biological molecule to be cleaved. In this regard, the abnormally proliferating cell is preferably a cancerous cell and the therapeutic effect is the treatment of cancer.

It is also expected that the complex is advantageously suitable for cleavage of other agents having phosphodiester linkages or derivatives thereof, e.g., anticholinesterases, such as insecticides having oxygen-phosphorus linkages (see, generally, U.S. Pat. No. 5,739,022 (Burstyn et al.)).

The complex can be targeted to an abnormally proliferating cell, such as a cancerous cell, by conjugation to a targeting agent that binds to a molecule on the surface of the abnormally proliferating cell. Preferably, the targeting agent is an immunological agent, which can be generated in accordance with methods known in the art (see, generally, Pietersz et al., Adv. Exp. Med. Biol. 353: 169-179 (1994); Riethmuller et al., Cur. Opin. Immuno. 5: 732-739 (1993); Senter, FASEB J. 4: 188-193 (1990)). Examples of cell-surface molecules that can be targeted by the immunological agent include CD-20 in non-Hodgkins B-cell lymphoma, CD-33 in myelogenous leukemia, and the IL-2 receptor that is up-regulated in T-cell diseases, such as adult T-cell leukemia.

The complex also can be targeted to an abnormally proliferating cell, such as a cancerous cell, by encapsulation in a liposome comprising on its surface a targeting agent that binds to a molecule on the surface of the abnormally proliferating cell. When the complex is encapsulated in a liposome, the complex can be conjugated to an antisense nucleic acid that binds to a sense nucleic acid, which is expressed in an abnormally proliferating cell but not in a normally proliferating cell or which is expressed in an abnormally proliferating cell at a higher level than in a normally proliferating cell, whereupon expression of the sense nucleic acid is inhibited. The antisense nucleic acid can further comprise a ribozyme sequence.

Alternatively, the complex can be targeted to an abnormally proliferating cell, such as a cancerous cell, by direct administration to the abnormally proliferating cell. For example, when the abnormally proliferating cell is a tumor cell, the complex can be administered intratumorally or peritumorally, such as by injection.

The present inventive method can be used to treat any suitable cancer alone or in combination with any suitable anti-cancer therapy. Suitable cancers include cancers of the brain, lung (e.g., small cell and non-small cell), ovary, breast, prostate, and colon, as well as other carcinomas and sarcomas. A suitable anti-cancer therapy may include radiation and/or drug therapy, which includes those drugs given in treatment of the various conditions described above, examples of which can be found in the Physicians' Desk Reference (1998).

In the context of the method of cleaving a biological molecule in vivo, when the complex comprises a radioactive transition metal, such as radioactive copper, the radioactive copper is preferably cytotoxic to the abnormally proliferating cell. Cytotoxicity can be concentration-dependent. The determination of a cytotoxic concentration is a matter of routine experimentation, using methods known in the art.

A further embodiment of the present invention is a method of inhibiting expression of a nucleic acid. The method comprises contacting the nucleic acid with an above-described complex conjugated to an antisense nucleic acid specific for the nucleic acid to be inhibited. The antisense nucleic acid can further comprise a ribozyme sequence. The antisense nucleic acid binds to the nucleic acid, thereby inhibiting its expression (see, e.g., Senior, Bioteck Genet. Eng. Rev. 15: 79-119 (1998); Bird et al., Biotech. Genet. Eng. Rev. 9: 207-227(1991); Matzke et al., Trends Genet. 11(1): 1-3 (1995); Baulcombe, Plant Mol. Biol. 32(1-2): 79-88 (1996); Cstanatto et al., Crit. Rev. Eukaryot. Gene Exp. 2(4): 331-357 (1992); and Rossi, Trends Biotechnol. 13(8): 301-306 (1995)).

Antisense nucleic acids can be generated in accordance with methods known in the art. The nucleic acid sequence introduced in antisense inhibition generally is substantially identical to at least a portion, preferably at least about 20 continuous nucleotides, of the nucleic acid to be inhibited, but need not be identical. The complex can, thus, be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the nucleic acid. The introduced sequence also need not be full-length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective.

The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334: 585-591 (1988). Preferably, the ribozyme comprises at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.

A still further embodiment of the present invention is a method of radiation therapy. The method comprises administering to an animal in need of radiation therapy a therapeutically effective amount of an above-described complex. The complex can be (i) conjugated to a targeting agent that binds to a molecule on the surface of a cell to be treated with radiation or (ii) encapsulated in a liposome comprising on its surface the targeting agent. What constitutes a therapeutically effective amount can be determined in accordance with methods known in the art. The targeting agent preferably is an immunological agent.

A method of imaging is also provided by the present invention. The method comprises administering to an animal an imaging effective amount of an above-described complex and imaging the complex. The complex can be (i) conjugated to a targeting agent that binds to a molecule on the surface of a cell to be imaged or (ii) encapsulated in a liposome comprising on its surface the targeting agent. An imaging effective amount also can be determined in accordance with methods known in the art. The targeting agent preferably is an immunological agent. Imaging means are also known in the art and include positive emission tomography (PET), which is a highly specialized research imaging technique using short-lived radioactive substances, usually those made with a cyclotron, single photon emission computed tomography (SPECT), and gamma-camera scintigraphy, which produces photographs or cathode-ray tube images of the gamma-ray emissions from organs containing radionuclide tracers.

Further provided is a method of tracing a compound in an animal. The method comprises administering to the animal a mixture of the compound and an above-described complex and tracing the location of the complex in the animal. The location of the complex indicates the location of the compound. Methods of tracing a compound is this manner are known in the art and include determination by PET, SPECT, scintigraphic imaging and detailed necroscropy biodistribution methods of intrinsically radiolabelling complexes (using either tritium or C¹⁴).

Claim 1 of 35 Claims

What is claimed is:

1. A copper complex of N,N',N"-trialkyl-cis,cis-1,3,5-triaminocyclohexane.

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