Claims
- 1. A compound of the formula: ##STR11## wherein: R represents independently hydrogen, C.sub.1 -C.sub.3 alkyl, or benzyl;
- R.sup.1 represents ##STR12## Q represents hydrogen, C.sub.1 -C.sub.3 alkyl or phenyl; R.sup.2 represents hydrogen, --CH.sub.2 CO.sub.2 H, --CH.sub.2 CH.sub.2 CO.sub.2 H, or --(CH.sub.2).sub.2 NH.sub.2 ;
- m and n are independently 2, 3, or 4;
- L is a linker/spacer group covalently bonded to, and replaces one hydrogen atom of the nitrogen atom to which it is joined said linker/spacer group being represented by the formula ##STR13## wherein: Y is selected from the group consisting of nitro, amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, bromoacetamido and maleimido;
- q is 1, 2, or 3; or
- a pharmaceutically acceptable salt thereof.
- 2. A compound of claim 1 wherein:
- m and n are 2 or b 3;
- R.sup.2 is hydrogen;
- R.sup.1 is ##STR14## Y is amino or isothiocyanato; or a pharmaceutically acceptable salt thereof.
- 3. A compound of claim 1 which is bis(2-hydroxybenzyl)-4-(p-nitrobenzyl)diethylenetriamine.
- 4. A compound of claim 1 which is bis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine.
- 5. A compound of claim 1 which is 3,3,11,11-tetramethyl-4,7,10-triazatridecane-7-(p-aminobenzyl)-2,12-dionedioxime.
- 6. A compound of claim 1 which is 1,7-bis(2-methylenepyrrole)-4-(p-nitrobenzyl)diethylenetriamine.
STATEMENT OF GOVERNMENT INTEREST
This invention was made with Government support under Grant No. DE-FG02-86ER60400 awarded by the Department of Energy. The Government has certain rights to this invention.
Functionalized chelants, or bifunctional coordinators, are known to be capable of being covalently attached to an antibody having specificity for cancer or tumor cell epitopes or anitgens. Radionuclide complexes of such antibody/chelant conjugates are useful in diagnostic and/or therapeutic applications as a means of conveying the radionuclide to a cancer or tumor cell. See, for example, Meares et al., Anal. Biochem. 142, 68-78, (1984); M. W. Brechbiel et al., Inorg. Chem. 25(16), 2772-2781 (1986); and Krejcarek et al., Biochem. and Biophys. Res. Comm. 77, 581-585 (1977).
Numerous bifunctional chelating agents based on aminocarboxylic acids have been proposed and prepared. For example the cyclic dianhydride of DTPA [Hnatowich et al. Science 220, 613-615, (1983); U.S. Pat. No. 4,479,930] and mixed carboxycarbonic anhydrides of DTPA [Gansow, U.S. Pat. Nos. 4,454,106 and 4,472,509; Krejcarek et al., Biochem. and Biophys. Res. Comm. 77, 581-585, (1977)] have been reported.
Some chelating agents based on functionalized triamines are known. For example, G. H. Searle et al., Aust. J. Chem. 32, 519-36 (1979) teach for the protection of the terminal nitrogens of linear triamines which allows the central nitrogen atom to be functionalized with a methyl group. These compounds were used for the chelation of cobalt ions. When the terminal nitrogen atoms of linear triamines are functionalized with moieties capable of binding to metal ions using the general method disclosed by R. J. Motekaitis et al. [R. J. Motekaitis et al., Inorg. Chem. 23(3), 275-283 (1984)], then a pentadentate chelant is prepared. Additionally the central nitrogen atom is shown to be substituted with a benzyl group. These compounds were also used for the chelation of cobalt ions. A. W. Addison et al., Inorg. Chimica Acta 147, 61-64 (1988) and F. Refosco et al., J. Chem. Soc. Dalton Trans. 611-615 (1988) teach the preparation of a salicylaldehyde Schiff base ligand with a linear triamine in which the central nitrogen atom is functionalized with a methyl group. These compounds are used for the chelation of iron metal ions, and technetium and rhenium metal ions, respectively. E. Chiotellis et al., Nucl. Med. Biol. 15(2), 215-223 (1988) teach the preparation of linear triamines in which the terminal nitrogen atoms are functionalized with alkylthiol moieties. The central nitrogen atom is functionalized with a propyl or a cyclohexyl moiety. The compounds were used for complexation of .sup.99m Tc.
Bifunctional chelating agents derived from triamines are also known. C. H. Paik et al., J. of Radioanal. Chem. 57(2), 553-564 (1980) teach a method to prepare functionalized terminal nitrogen atoms of linear triamines with moieties capable of binding to metal ions and having the central nitrogen atom substituted with a benzylamine group for conjugation to protein. These compounds were used for the chelation of .sup.111 In. Other linear triamine bifunctional chelating agents in which the central nitrogen atom is functionalized are disclosed by C. John et al. in J. Nucl. Med. 29, 914-815 (1988) in which the central nitrogen atom is functionalized with a benzylcarboxylic acid group. These compounds were used for the chelation of .sup.105 Rh and their conjugation with proteins and antibodies.
The invention includes the design and synthesis of novel bifunctional chelants, each containing a chelating functionality, and a chemically reactive group for covalent attachment to biomolecules. Also forming part of the invention are methods for preparing various BFC-metal complexes and the linking of the complexes to antibody to prepare radionuclide labeled antibody and/or fragments suitable for diagnostic and/or therapeutic applications.
The present invention is directed to novel bifunctional chelating agents (BFC) having a triamine functionality and derivatives thereof. The BFC's form complexes with "radioactive" metal ions. Preferred radioactive metal ions include .sup.99m Tc, .sup.105 Rh, .sup.109 Pd, .sup.57 Co, .sup.186 Re, .sup.188 Re, .sup.97 Ru, .sup.111 In, .sup.113m In, .sup.67 Ga, and .sup.68 Ga. The complexes so formed can be attached (covalently bonded) to an antibody or fragment thereof to form conjugates and used for therapeutic and/or diagnostic purposes. The complexes and/or conjugates can be formulated for in vivo or in vitro uses. A preferred use of the formulated conjugates is the treatment of cancer in animals, especially humans.
More specifically, the present invention is directed to a compound of the formula: ##STR1## wherein: R represents independently hydrogen, C.sub.1 -C.sub.3 alkyl, or benzyl;
Preferred features of the compounds of formula (I) are those where: R is hydrogen; R.sup.2 is hydrogen; m and n are independently 2 or 3; and L is a compound of the formula: ##STR4## wherein: Y is selected from the group consisting of nitro, amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, bromoacetamido and maleimido;
When a conjugate of the present invention is desired Y must be other than nitro.
Preferred compounds of formula (I) are those where m and n are 2 or 3, R.sup.2 is hydrogen, R.sup.1 is ##STR5## L is formula (A) where Y is amino or isothiocyanato, or a pharmaceutically acceptable salt thereof.
The present invention is also directed to radioactive metal ion complexes which have as their ligand the compounds of formula (I), especially radioactive metal ion complexes comprising .sup.99m Tc, .sup.105 Rh, .sup.109 Pd, .sup.57 Co, .sup.186 Rd, .sup.188 Re, .sup.97 Ru, .sup.111 In, .sup.113m In, .sup.67 Ga, and .sup.68 Ga, with the proviso that when R.sup.2 is --CH.sub.2 CO.sub.2 H or --CH.sub.2 CH.sub.2 CO.sub.2 H, then the radioactive metal ion is selected from the group consisting of .sup.186 Re, .sup.188 Re, .sup.99m Tc, .sup.111 In, .sup.113m In, .sup.67 Ga, and .sup.68 Ga.
Additionally, the present invention concerns conjugates which are formed with the aforementioned complexes and antibody or antibody fragments.
In addition, the present invention also includes formulations having the conjugates of the invention and a pharmaceutically acceptable carrier, especially formulations where the pharmaceutically acceptable carrier is a liquid. The invention also includes a method for the diagnosis or treatment of a disease state, especially cancer, in a mammal which comprises administering to the mammal an effective amount of the formulation.
As used herein, the following indicated terms have these meanings: with respect to the definition of Y, "electrophilic" moieties include, but are not limited to, isothiocyanate, bromoacetamide, maleimide, imidoester, thiophthalimide, N-hydroxysuccinimyl ester, pyridyl disulfide, and phenyl azide; suitable "nucleophilic" moieties include, but are not limited to, carboxyl, amino, acyl hydrazide, semicarbazide, and thiosemicarbazide; "synthetic linkers" include any synthetic organic or inorganic linkers which are capable of being covalently attached to an antibody or antibody fragment, preferred synthetic linkers are biodegradable synthetic linkers which are stable in the serum of a patient but which have a potential for enzymatic cleavage within an organ of clearance for the radioisotope, for example biodegradable peptides or peptide containing groups. Of the electrophilic moieties isothiocyanate is preferred and of the nucleophilic moieties amino, carboxyl, semicarbazide and thiosemicarbazide are preferred. It is desirable that the nature and/or position of Y be such that it does not appreciably interfere with the chelation reaction.
As used herein, the term "mammal" means animals that nourish their young with milk secreted by mammary glands, preferably warm blooded mammals, more preferably humans. "Antibody" refers to any polyclonal, monoclonal, chimeric antibody or heteroantibody, preferably a monoclonal antibody; "antibody fragment" includes Fab fragments and F(ab').sub.2 fragments, and any portion of an antibody having specificity toward a desired epitope or epitopes. When using the term "metal chelate/antibody conjugate" or "conjugate", the "antibody" portion is meant to include whole antibodies and/or antibody fragments, including semisynthetic or genetically engineered variants thereof.
As used herein, "complex" refers to a compound of the invention, e.g. formula (I), complexed with a radioactive metal ion, wherein at least one metal atom is chelated or sequestered; "radioactive metal ion chelate/antibody conjugate" or "radioactive metal ion conjugate" refers to a radioactive metal ion conjugate that is covalently attached to an antibody or antibody fragment; "radioactive" when used in conjunction with the work "metal ion" refers to one or more isotopes of the elements that emit particles and/or photons, such as .sup.99m Tc, .sup.105 Rh, .sup.109 Pd, .sup.57 Co, .sup.186 Re, .sup.97 Ru, .sup.111 In, .sup.113m In, .sup.67 Ga, and .sup.68 Ga; the terms "bifunctional coordinator", "bifunctional chelating agent" and "functionalized chelant" are used interchangeably and refer to compounds that have a chelant moiety capable of chelating a metal ion and a linker/spacer moiety covalently bonded to the chelant moiety that is capable of serving as a means to covalently attach to an antibody or antibody fragment.
As used herein, "pharmaceutically acceptable salt" means any salt of a compound of formula (I) which is sufficiently non-toxic to be useful in therapy or diagnosis of mammals. Thus, the salts are useful in accordance with this invention. Representative of those salts, which are formed by standard reactions, from both organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic, mucic, glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic acids and other suitable acids. Also included are salts formed by standard reactions from both organic and inorganic sources such as ammonium, alkali metal ions, alkaline earth metal ions, and other similar ions. Particularly preferred are the salts of the compounds of formula (I) where the salt is potassium, sodium, ammonium, H.sup.+ or mixtures thereof.
The bifunctional chelating agents described herein can be used to chelate or sequester the radioactive metal ions, so as to form metal ion chelates (also referred to herein as "complexes"). The complexes, because of the presence of the functionalizing moiety [represented by "Y" in formula (I)], can be attached to functionalized supports, such as functionalized polymeric supports, or preferably covalently attached to proteins or more specifically to antibodies or antibody fragments. Thus the complexes described herein complexed with radioactive metal ions may be covalently attached to a protein or an antibody or antibody fragment and are referred to herein as "conjugates". For example, human serum albumin (HSA), purchased from Sigma, was used to form conjugates with a complex.
The antibodies or antibody fragments which may be used in the conjugates described herein can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be produced by hybridization techniques well known in the art, see for example, Kohler and Milstein [Nature 256, 495-497 (1975); and Eur. J. Immunol. 6, 511-519 (1976)]. Such antibodies normally have a highly specific reactivity. In the radioactive metal ion conjugates, antibodies directed against any desired antigen or hapten may be used. Preferably the antibodies which are used in the radioactive metal ion conjugates are monoclonal antibodies, or fragments thereof having high specificity for a desired epitope(s). Antibodies used in the present invention may be directed against, for example, tumors, bacteria, fungi, viruses, parasites, mycoplasma, differentiation and other cell membrane antigens, pathogen surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules. Some examples of antibodies or antibody fragments are B72.3 and anti-human IgT. [The hybridome cell line B72.3 is deposited in the American Type Culture connection (ATCC) having the accession number HB 8108. The IgG was purchased from Sigma.] A more complete list of antigens can be found in U.S. Pat. No. 4,193,983, which is incorporated herein by reference. The radioactive metal ion conjugates of the present invention are particularly preferred for the diagnosis and treatment of various cancers.
The conjugates of this invention, and in some instances the complexes, are employed as a formulation. The formulation comprises a compound of formula (I) with the antibody and/or radioactive metal ion and a physiologically acceptable carrier, excipient or vehicle therefor. Thus, the formulations may consist of a physiologically acceptable carrier with a complex (metal ion+ligand), conjugate (metal ion+ligand+antibody). The methods for preparing such formulations are well known. A formulation may be in the form of a suspension, injectable solution or other suitable formulation. Physiologically acceptable suspending media, with or without adjuvants, may be used.
The formulations of the present invention are in the solid or liquid form containing the active radionuclide complexed with the ligand. These formulations may be in kit form such that the two components (i.e. ligand and metal, complex and antibody) are mixed at the appropriate time prior to use. Whether premixed or as a kit, the formulations usually require a pharmaceutically acceptable carrier.
Injectable compositions of the present invention may be either in suspension or solution form. In the preparation of suitable formulations it will be recognized that, in general, the water solubility of the salt is greater than the free base. In solution form the complex (or when desired the separate components) is dissolved in a physiologically acceptable carrier. Such carriers comprise a suitable solvent, preservatives such as benzyl alcohol, if needed, and buffers. Useful solvents include, for example, water, aqueous alcohols, glycols, and phosphonate or carbonate esters. Such aqueous solutions contain no more than 50 percent of the organic solvent by volume.
Injectable suspensions are compositions of the present invention requiring a liquid suspending medium, with or without adjuvants, as a carrier. The suspending medium can be, for example, aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or highly refined mineral oils, or aqueous carboxymethylcellulose. Suitable physiologically acceptable adjuvants, if necessary to keep the complex in suspension, may be chosen from among thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and the alginates. Many surfactants are also useful as suspending agents, for example, lecithin, alkylphenol, polyethylene oxide adducts, napthalenesulfonate, alkylbenzenesulfonates, and the polyoxyethylene sorbitan esters.
Many substances which effect the hydrophobicity, density, and surface tension of the liquid suspension medium can assist in making injectable suspensions in individual cases. For example, silicone antifoames, sorbitol, and sugars are all useful suspending agents.
An "effective amount" of the formulation is used for therapy. The dose will vary depending on the disease being treated. Although in vitro diagnostics can be performed with the formulations of this invention, in vivo diagnostics are also contemplated using formulations of this invention. The conjugates and formulations of this invention can also be used in radioimmunoguided surgery (RIGS); however, the metals which could be used for this purpose are .sup.99m Tc, .sup.111 In, .sup.113m In, .sup.67 Ga and .sup.68 Ga.
The present invention provides chelants, complexes, and antibody conjugates some of which are more stable, and/or have improved biodistribution, and/or have more rapid clearance from the body, than those known in the art.
All of the starting materials required for preparing the compounds of this invention are either available from commercial sources or can be made from a known literature reference description.
The electrophilic moiety ("Y" in the formula) can also be prepared by methods known to the art. Such methods may be found in Acc. Chem. Res. 17, 202-209 (1984).
General methods of preparation for the compounds of formula (I) are well known in the art. Some of the various method are shown in the following reaction schemes.
In the following Scheme I, the terminal amines of a symmetrical or unsymmetrical triamine are protected by the addition of phthalic anhydride using a modification of the procedure of G. H. Searle, et al., Aust. J. Chem. 32, 519-36 (1979). The central amine is then alkylated with the L moiety. ##STR6##
In Scheme II, the protecting phthalic moieties of the product from Scheme I are removed by hydrolysis with hydrochloric acid. The p-nitrobenzyl group is then reduced by conventional methods, i.e. Pd/C with H.sub.2. The compounds of formula (I) where R.sup.1 is an oxime are prepared by the addition of 3-chloro-3-methyl-2-butanone oxime. ##STR7##
In Scheme III, the protecting phthalic moieties are removed by hydrolysis with hydrochloric acid. The Schiff bases are then prepared by reaction with the appropriate aldehyde. Reduction of the Schiff bases proceeds with sodium borohydride. The nitro group is then reduced to the amine using Pd/C with H.sub.2. ##STR8##
In Scheme IV, the protecting phthalic moieties are removed by hydrolysis with hydrochloric acid. The compounds of formula (I) where Y is nitro and the R.sup.1 groups are the correspondingly protected aminoamide or thioamide are then formed by reaction with the corresponding alkyl amino ester (e.g. methyl 2-(tBOC)-aminoacetate) or alkylthio ester (e.g. methyl 2-mercaptoacetate). When the tertiary butylcarbamate (tBOC) is present, it is cleaved by trifluoroacetic acid.
For compounds having either the unprotected aminoamide or thioamide moiety, the nitro group is then reduced to the amine using Pd/C with H.sub.2.
Additionally, for compounds where Y is nitro having either the unprotected amino amide or thioamide moieties, reduction with borane leads to the corresponding alkyl amines or alkyl sulfhydryls. Subsequent reduction of the nitro group results in the corresponding amines. ##STR9##
In Scheme V, to prepare the compounds of formula (I) where R.sup.2 is other than hydrogen, the corresponding compounds of formula (I) where R.sup.2 is hydrogen are reacted in one of the following manners.
The compounds of formula (I) wherein R.sup.2 is alkyl amine are prepared by a modification of the Strecker reaction (aldehyde reaction with hydrogen cyanide, followed by reduction with lithium aluminum hydride.)
The compounds of formula (I) wherein R.sup.2 is a carboxylic acid are prepared by the addition of bromocarboxylic acid. See for example, J. F. Desreux, Inorg. Chem. 19, 1319-1324 (1980). ##STR10##
Radionuclides can be produced in several ways. For example, several preparations are detailed in an article by D. E. Troutner, Nucl. Med. Biol, 14(3), 171-176 (1987). The method of obtaining the nuclides employed in the present invention is not critical thereto.
The complexes of the present invention are generally prepared by mixing the ligand with the desired radioactive metal ion. However, when the radioactive metal ion is .sup.186 Re, .sup.188 Re or .sup.99m Tc, then a reducing agent must be added such as tin tartarate. When the radioactive metal ion is .sup.97 Ru or .sup.105 Rh, then the mixture must be heated.
Attachment of radionuclide to antibody can be carried out by conjugation of the antibody to preformed (ambient or elevated temperature) metal--BFC complex. [See European published application 0296522, published Dec. 28, 1988]. The conjugates of the present invention are prepared by first activating the complex with thiophosgene which converts the amine, where Y is NH.sub.2, to the corresponding isothiocyanate. The desired protein or antibody or antibody fragment, in presence of buffer, is added to the isothiocyanate compound. A covalent bond is formed (thiourea) by the reaction of the isothiocyanate with the alkylamine of a lysine group.
The invention will be further clarified by a consideration of the following examples, which are intended to be purely exemplary of the use of the invention.
In the following examples, the following terms and conditions were used unless otherwise specified:
Room temperature means about 25.degree. C.;
Bq means a Becquerel unit for measuring radioactivity. 1Curie=3.7.times.10.sup.10 By;
0.9% Saline solution was prepared by dissolving 9 g of NaCl in 1L of doubly distilled water;
.sup.105 Rh was prepared at the University of Missouri Research Reactor (MURR) and supplied as Na.sub.3 (RhCl.sub.6) or Na.sub.2 (RhCl.sub.5.H.sub.2 O) in approximately pH 4 HCl solution;
Rh carrier solutions were prepared by dissolving RhCl.sub.3.3H.sub.2 O in saline;
.sup.99m Tc was eluted from a Mallinckrodt technetium generator and diluted to 2 mCi/mL using 0.9% saline solution;
Human immunoglobulin (I-4506) was purchased from Sigma Chemical Company;
Sephadex.TM. G 75 was purchased from Sigma Chemical Company;
Anti human IgG (Y chain specific) Agarose (A-6656) was purchased from Sigma Chemical Company;
Thiophosgene was purchased from Aldrich Chemical Company;
RhCl.sub.3.3H.sub.2 O was purchased from Aldrich Chemical Company;
Flexible silica gel plates 7.5.times.2.5 cm, coating thickness 250 .mu.m were purchased from J. T. Baker Chemical Company;
Beckman series 320046 paper (30.times.2 cm) was used for paper electrophoresis and Gelman Science solvent saturation pads cut into 1.5.times.13.7 cm were used for paper chromatography;
.sup.1 H and .sup.13 C NMR spectra were recorded on a JEOL FX-90Q spectrometer with .delta. reported as ppm relative to TMs as an internal standard; and
Radioactivity was measured on a standard 5.times.5 cm NaI (T1) well scintillation counter.
US Referenced Citations (6)
Foreign Referenced Citations (1)
| Number |
Date |
Country |
| 0509940 |
Feb 1955 |
CAX |
Non-Patent Literature Citations (8)
| Entry |
| G. H. Searle et al., Aust. J. Chem. 32, 519-36 (1979). |
| R. J. Motekaitis et al., Inorg. Chem. 23(3), 275-283 (1984). |
| C. H. Paik et al., J. of Radioanal. Chem. 57(2), 553-564 (1980). |
| C. John et al., J. Nucl. Med. 29, 814-815 (1988). |
| M. W. Brechbiel et al., Inorg. Chem. 25(16), 2772-2781 (1986). |
| A. W. Addison et al., Inorg. Chimica Acta 147, 61-64 (1988). |
| F. Refosco et al., J. Chem. Soc. Dalton Trans. 611-615 (1988). |
| E. Chiotellis et al., Nucl. Med. Biol. 15(2), 215-223 (1988). |
Patent Grant
5101041
