Efficient synthesis of chelators for nuclear imaging and radiotherapy: compositions and applications
10925977 ยท 2021-02-23
Assignee
- Ceil>Point, LLC (Spring, TX, US)
- The Board Of Regents Of The University Of Texas System (Austin, TX)
Inventors
Cpc classification
A61K49/0002
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
A61K49/06
HUMAN NECESSITIES
A61K51/0491
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
International classification
Abstract
Novel methods of synthesis of chelator-targeting ligand conjugates, compositions comprising such conjugates, and therapeutic and diagnostic applications of such conjugates are disclosed. The compositions include chelator-targeting ligand conjugates optionally chelated to one or more metal ions. Methods of synthesizing these compositions in high purity are also presented. Also disclosed are methods of imaging, treating and diagnosing disease in a subject using these novel compositions, such as methods of imaging a tumor within a subject and methods of diagnosing myocardial ischemia.
Claims
1. A method of synthesizing a chelator-targeting ligand conjugate, comprising: (a) conjugating, in an organic medium, a protected ethylenedicysteine of the following formula: ##STR00010## to at least one unprotected glucosamine, wherein: A and D are each a protected thiol; B and C are each a tertiary amine, wherein the tertiary amine is a protected secondary amine; and the conjugation is via an amide bond formed between a COOH group of the protected ethylenedicysteine and the amino group of the unprotected glucosamine to form a protected ethylenedicysteine glucosamine; and (b) removing each protecting group, in one or more steps, from the protected ethylenedicysteine glucosamine to form a chelator-targeting ligand conjugate, wherein the chelator-targeting ligand conjugate is ethylenedicysteine glucosamine (EC-G), wherein the purity of the chelator-targeting ligand conjugate is between about 80% (w/w) and about 99.9% (w/w), as measured by HPLC using ELSD detection.
2. The method of claim 1, wherein the organic medium comprises a polar solvent.
3. The method of claim 1, wherein the organic medium is dimethylformamide, dimethylsulfoxide, dioxane, methanol, ethanol, hexane, methylene chloride, acetonitrile, tetrahydrofuran, or a mixture thereof.
4. The method of claim 1, further comprising at least one purification step, wherein the purification step is silica gel column chromatography, HPLC, or a combination thereof.
5. The method of claim 4, wherein the purity of the chelator-targeting ligand conjugate is between about 85% (w/w) and about 99.9% (w/w), as measured by HPLC using ELSD detection.
6. The method of claim 5, wherein the purity of the chelator-targeting ligand conjugate is between about 90% (w/w) and about 99.9% (w/w), as measured by HPLC using ELSD detection.
7. The method of claim 1, further comprising chelating a metal ion to the chelator-targeting ligand conjugate to generate a metal ion labeled-chelator-targeting ligand conjugate.
8. The method of claim 7, wherein the purity of the metal ion labeled-chelator-targeting ligand conjugate is between about 80% w/w and about 99.9% w/w, as measured by HPLC using ELSD detection.
9. The method of claim 8, wherein the purity of the metal ion labeled-chelator-targeting ligand conjugate is between about 85% w/w and about 99.9% w/w, as measured by HPLC using ELSD detection.
10. The method of claim 9, wherein the purity of the metal ion labeled-chelator-targeting ligand conjugate is between about 90% w/w and about 99.9% w/w, as measured by HPLC using ELSD detection.
11. The method of claim 7, wherein the metal ion is selected from the group consisting of a technetium ion, a copper ion, an indium ion, a thallium ion, a gallium ion, an arsenic ion, a rhenium ion, a holmium ion, a yttrium ion, a samarium ion, a selenium ion, a strontium ion, a gadolinium ion, a bismuth ion, an iron ion, a manganese ion, a lutecium ion, a cobalt ion, a platinum ion, a calcium ion and a rhodium ion.
12. The method of claim 7, wherein the metal ion is .sup.187Re, .sup.99mTc, platinum, or .sup.188Re.
13. The method of claim 7, wherein the metal ion is a radionuclide.
14. The method of claim 13, wherein the radionuclide is selected from the group consisting of .sup.99mTc, .sup.188Re, .sup.186Re, .sup.153Sm, .sup.166Ho, .sup.90Y, .sup.89Sr, .sup.67Ga, .sup.68Ga, .sup.111In, .sup.183Gd, .sup.59Fe, .sup.225Ac, .sup.212Bi, .sup.211At, .sup.45Ti, .sup.60Cu, .sup.61Cu, .sup.67Cu, .sup.64Cu and .sup.62Cu.
15. The method of claim 14, further comprising the addition of a reducing agent.
16. The method of claim 1, wherein the protected thiol is protected using a thiol protecting agent selected from a group consisting of an alkyl halide, a benzyl halide, a benzoyl halide, a sulfonyl halide, benzhydrol, a triphenylmethyl halide, a methoxytriphenylmethyl halide and cysteine.
17. The method of claim 1, wherein the protected tertiary amine is protected using an amine protecting agent selected from the group consisting of benzylchloroformate, p-nitro-chlorobenzylformate, ethylchloroformate, di-tert-butyl-dicarbonate, triphenylmethyl chloride and methoxytriphenylmethyl chloride.
18. The method of claim 1, wherein the protected ethylenedicysteine glucosamine is EC-Benzhydrol-Cbz-Glucosamine.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
(15) The present inventors have identified novel synthetic methods for the preparation of chelator-targeting ligand conjugates optionally chelated to one or more metal ions. The present invention further provides syntheses of chelators, such as unconjugated chelators, protected chelators (that is, chelators wherein one or more functional groups are protected using a protecting agent) and metal ion labeled-chelators (that is, chelators that are chelated to one or more metal ions). These synthetic methods comprise, generally, the use of organic solvents and synthetic organic procedures and purification methods. Methods based on wet (aqueous) chemistry are also provided. Compounds of the present invention resulting from such organic chemistry methods are high in purity, especially when compared to compounds prepared by wet chemistry. A preferred chelator is ethylenedicysteine. The targeting ligand can be, for example, a tissue-targeting moiety, a diagnostic moiety, or a therapeutic moiety. The metal ions chelated to compounds of the present invention may further render the compound useful for imaging, diagnostic, or therapeutic use. Compounds of the present invention, methods of their synthesis and use are further described below.
A. Chelators
(16) Persons of skill in the art will be familiar with compounds capable of chelating one or more metal ions (chelators). Chelators employed in the method of the present invention generally comprise one or more atoms capable of chelating to one or more metal ions. Chelators comprising three or four atoms available for chelation are preferred. Typically, a chelator chelates to one metal ion.
(17) Chelation of a metal ion to a chelator can be by any method known to those of ordinary skill in the art. Methods of chelation (also called coordination) are described in more detail below. Atoms available for chelation are known to those of skill in the art, and typically comprise O, N or S. In preferred embodiments, the atoms available for chelation are selected from the group consisting of N and S. In certain preferred embodiments, the metal ion is chelated to a group of atoms, referred to herein as chelates, selected from the group consisting of NS.sub.2, N.sub.2S, S.sub.4, N.sub.2S.sub.2, N.sub.3S and NS.sub.3. Chelation can also occur among both the chelator and the targeting ligandi.e., both the chelator and the targeting ligand may contribute atoms that chelate the same metal ion.
(18) In certain embodiments, the chelator comprises compounds incorporating one or more amino acids. Amino acids will typically be selected from the group consisting of cysteine and glycine. For example, the chelator may comprise three cysteines and one glycine or three glycines and one cysteine. As discussed below, a spacer may connect one amino acid to another.
(19) It is well known to those of ordinary skill in the art that chelators, in general, comprise a variety of functional groups. Non-limiting examples of such functional groups include hydroxy, thiol, amine, amido and carboxylic acid.
(20) 1. Bis-Aminoethanethiol (BAT) Dicarboxylic Acids
(21) Bis-aminoethanethiol (BAT) dicarboxylic acids may constitute a chelator employed in the method of the present invention. In preferred embodiments, the BAT dicarboxylic acid is ethylenedicysteine (EC). BAT dicarboxylic acids are capable of acting as tetradentate ligands, and are also known as diaminodithiol (DADT) compounds. Such compounds are known to form stable Tc(V)O-complexes on the basis of efficient binding of the oxotechnetium group to two thiol-sulfur and two amine-nitrogen atoms. The .sup.99mTc labeled diethylester (.sup.99mTc-L,L-ECD) is known as a brain agent. .sup.99mTc-L,L-ethylenedicysteine (.sup.99mTc-L,L-EC) is its most polar metabolite and was discovered to be excreted rapidly and efficiently in the urine. Thus, .sup.99mTc-L,L-EC has been used as a renal function agent. (Verbruggen et al. 1992). Other metals such as indium, rhenium, gallium, copper, holmium, platinum, gadolinium, lutetium, yttrium, cobalt, calcium and arsenic may also be chelated to BAT dicarboxylic acids such as EC.
(22) 2. Spacers
(23) Chelators of the present invention may comprise one or more spacers. For example, amino acids and their derivatives may be joined by one or more spacers. An example of two amino acids joined by a spacer includes ethylenedicysteine, described above. Such spacers are well known to those of ordinary skill in the art. These spacers, in general, provide additional flexibility to the overall compound that may facilitate chelation of one or more metal ions to the chelator. Non-limiting examples of spacers include alkyl groups of any length, such as ethylene (CH.sub.2CH.sub.2), ether linkages, thioether linkages, amine linkages and any combination of one or more of these groups. It is envisioned that multiple chelators (that is, two or more) linked together are capable of forming an overall molecule that may chelate to one or more, or more typically two or more, metal ions. That is, each chelator that makes up the overall molecule may each chelate to a single separate metal ion.
B. Protecting Groups
(24) When a chemical reaction is to be carried out selectively at one reactive site in a multifunctional compound, other reactive sites often must be temporarily blocked. A protecting group, as used herein, is defined as a group used for the purpose of this temporary blockage. Thus, the function of a protecting group is to protect one or more functional groups (e.g., NH.sub.2, SH, COOH) during subsequent reactions which would not proceed well, either because the free (in other words, unprotected) functional group would react and be functionalized in a way that is inconsistent with its need to be free for subsequent reactions, or the free functional group would interfere in the reaction. Persons of skill in the art recognize that the use of protecting groups is typical in synthetic organic chemistry.
(25) During the synthesis of the compounds of the present invention, various functional groups must be protected using protecting agents at various stages of the synthesis. A protecting agent is used to install the protecting group. Thus, in a typical procedure, a protecting agent is admixed with a compound featuring a functional group that is to be protected, and the protecting agent forms a covalent bond with that functional group. In this manner, the functional group is protected by a protecting group (and effectively rendered unreactive) by the covalent bond that formed with the protecting agent. Multiple functional groups can be protected in one or more steps using properly selected protecting agents. Such proper selection is understood by those of skill in the art. Such selection is often based upon the varying reactivity of the functional groups to be protected: thus, more reactive groups (such as sulfur/thiol) are typically protected before less reactive groups (such as amine) are protected.
(26) There are a number of methods well known to those skilled in the art for accomplishing such a step. For protecting agents, their reactivity, installation and use, see, e.g., Greene and Wuts (1999), herein incorporated by reference in its entirety. The same protecting group may be used to protect one or more of the same or different functional group(s). Non-limiting examples of protecting group installation are described below.
(27) Use of the phrase protected hydroxy or protected amine and the like does not mean that every such functional group available to be protected is protected. Similarly, a protected chelator, as used herein, does not imply that every functional group of the chelator is protected.
(28) Compounds of the present invention, including compounds used and made during the practice of the method of the present invention, are contemplated both in protected and unprotected (or free) form. Persons of ordinary skill in the art will understand that functional groups necessary for a desired transformation should be unprotected.
(29) When a protecting group is no longer needed, it is removed by methods well known to those skilled in the art. For deprotecting agents and their use, see, e.g., Greene and Wuts (1999). Agents used to remove the protecting group are typically called deprotecting agents. Protecting groups are typically readily removable (as is known to those skilled in the art) by methods employing deprotecting agents that are well known to those skilled in the art. For instance, acetate ester and carbamate protecting groups may be easily removed using mild acidic or basic conditions, yet benzyl and benzoyl ester protecting groups need much stronger acidic or basic conditions. It is well known that certain deprotecting agents remove some protective groups and not others, while other deprotecting agents remove several types of protecting groups from several types of functional groups. For instance, Birch reduction reactions using liquid ammonia and sodium (as described below) deprotect benzyl groups from thiols (or sulfur, more particularly) or carbamate groups from nitrogen, but not acetate groups from oxygen. Thus, a first deprotecting agent may be used to remove one type of protecting group, followed by the use of a second deprotecting agent to remove a second type of protecting group, and so on.
(30) Persons of ordinary skill in the art will be familiar with the proper ordering of protective group removal using deprotecting agents. See e.g., Greene and Wuts (1999). Non-limiting examples of protecting group removal are discussed below.
(31) Amine protecting groups are well known to those skilled in the art. See, for example, Greene and Wuts (1999), Chapter 7. These protecting groups can be installed via protecting agents well known to those of skill in the art. Removal of these groups is also well known to those of skill in the art.
(32) In some embodiments, the amine protecting group may be selected from the group consisting of t-butoxycarbonyl, benzyloxycarbonyl, formyl, trityl, acetyl, trichloroacetyl, dichloroacetyl, chloroacetyl, trifluoroacetyl, difluoroacetyl, fluoroacetyl, benzyl chloroformate, 4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl, 4-ethoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, 2-(4-xenyl)isopropoxycarbonyl, 1,1-diphenyleth-1-yloxycarbonyl, 1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl, 2-(p-toluoyl)prop-2-yloxycarbonyl, cyclopentanyloxycarbonyl, 1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl, 1-methylcyclohexanyloxycabonyl, 2-methylcyclohexanyloxycarbonyl, 2-(4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl, 2-(triphenylphosphino)ethoxycarbonyl, fluorenylmethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, 1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl, 5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl, cyclopropylmethoxycarbonyl, 4-(decyloxyl)benzyloxycarbonyl, isobornyloxycarbonyl, 1-piperidyloxycarbonyl and 9-fluorenylmethyl carbonate.
(33) In some embodiments, the protecting agent for amine protection is selected from the group consisting of benzylchloroformate, p-nitro-chlorobenzylformate, ethylchloroformate, di-tert-butyl-dicarbonate, triphenylmethyl chloride and methoxytriphenylmethyl chloride. In a preferred embodiment, the protecting group is benzyloxycarbonyl, installed by the protecting agent benzyloxychloroformate.
(34) Thiol protecting groups are well known to those skilled in the art. See, for example, Greene and Wuts (1999), Chapter 6. These protecting groups can be installed via protecting agents well known to those of skill in the art. Removal of these groups is also well known to those of skill in the art.
(35) In some embodiments, a thiol protecting group may be selected from the group consisting of acetamidomethyl, benzamidomethyl, 1-ethoxyethyl, benzoyl, triphenylmethyl, t-butyl, benzyl, adamantyl, cyanoethyl, acetyl and trifluoroacetyl.
(36) In some embodiments, the protecting agent for thiol protection is selected from the group consisting of an alkyl halide, a benzyl halide, a benzoyl halide, a sulfonyl halide, a triphenylmethyl halide, a methoxytriphenylmethyl halide and cysteine. Non-limiting examples of these protecting agents include ethyl halides, propyl halides and acetyl halides. Halides may comprise chloro, bromo or iodo, for example. In a preferred embodiment, the protecting group is benzyl, installed by the protecting agent benzyl chloride.
(37) Hydroxy (or alcohol) protecting groups are well known to those skilled in the art. See, for example, Greene and Wuts (1999), Chapter 2. These protecting groups can be installed via protecting agents well known to those of skill in the art. Removal of these groups is also well known to those of skill in the art.
(38) A suitable hydroxy protecting group may be selected from the group consisting of esters or ethers. Esters such as acetate, benzoyl, tert-butylcarbonyl and trifluoroacetyl groups are removable by acidic or basic conditions. Ethers such as methoxy, ethoxy and tri-benzylmethyl are removable by stronger acidic or basic conditions. A preferred protecting group is an acetate ester.
(39) Carbonyl protecting groups are well known to those skilled in the art. See, for example, Greene and Wuts (1999), Chapter 4. Such protecting groups may protect, for example, ketones or aldehydes, or the carbonyl present in esters, amides, esters and the like. These protecting groups can be installed via protecting agents well known to those of skill in the art. Removal of these groups is also well known to those of skill in the art.
(40) In some embodiments, a carbonyl protecting group may be selected from the group consisting of dimethylacetal, dimethylketal, diisopropylacetal, diisopropylketal, enamines and enol ethers.
(41) Carboxylic acid protecting groups are well known to those skilled in the art. See, for example, Greene and Wuts (1999), Chapter 5. Removal of these groups is also well known to those of skill in the art.
(42) A suitable carboxylic acid protecting group may be selected from the group consisting of amides or esters, for example. Amides such as sulfonamide, para-nitroaniline, benzylamide and benzolyamide may be hydrolyzed in acidic conditions. Esters such as methyl ester, ethyl ester and benzyl ester may be hydrolyzed by acidic or basic conditions. A preferred protecting group is an amide.
C. Metal Ions
(43) As set forth above, certain embodiments of the present invention pertain to compositions that will function to chelate one or more metal ions. The targeting ligands of the present invention may also participate in chelating one or more metal ions. A metal ion is defined herein to refer to a metal ion that is capable of forming a bond, such as a non-covalent bond, with one or more atoms or molecules. The other atom(s) or molecule(s) may be negatively charged.
(44) Any metal ion known to those of ordinary skill in the art is contemplated for inclusion in the compositions of the present invention. One of ordinary skill in the art would be familiar with the metal ions and their application(s). In some embodiments, the metal ion may be selected from the group consisting of Tc-99m, Cu-60, Cu-61, Cu-62, Cu-67, In-111, Tl-201, Ga-67, Ga-68, As-72, Re-186, Re-187, Re-188, Ho-166, Y-90, Sm-153, Sr-89, Gd-157, Bi-212, Bi-213, Fe-56, Mn-55, Lu-177, a iron ion, a arsenic ion, a selenium ion, a thallium ion, a manganese ion, a cobalt ion, a platinum ion, a rhenium ion, a calcium ion and a rhodium ion. For example, the metal ion may be a radionuclide. A radionuclide is an isotope of artificial or natural origin that exhibits radioactivity. In some embodiments, the radionuclide is selected from the group consisting of .sup.99mTc, .sup.188Re, .sup.186Re, .sup.153Sm, .sup.166Ho, .sup.90Y, .sup.89Sr, .sup.67Ga, .sup.68Ga, .sup.111In, .sup.178Gd, .sup.55Fe, .sup.225Ac, .sup.212Bi, .sup.211At, .sup.45Ti, .sup.60Cu, .sup.61Cu, .sup.67Cu, and .sup.64Cu. In preferred embodiments, the metal ion is rhenium or a radionuclide such as .sup.99mTc, .sup.188Re, or .sup.68Ga. As described below, a reducing agent may need to accompany one of the radionuclides, such as .sup.99mTc. Non-limiting examples of such reducing agents include a dithionite ion, a stannous ion and a ferrous ion.
(45) Due to better imaging characteristics and lower price, attempts have been made to replace the .sup.123I, .sup.131I, .sup.67Ga and .sup.111In labeled compounds with corresponding .sup.99mTc labeled compounds when possible. Due to favorable physical characteristics as well as extremely low price ($0.21/mCi), .sup.99mTc has been preferred to label radiopharmaceuticals.
(46) A number of factors must be considered for optimal radioimaging in humans. To maximize the efficiency of detection, a metal ion that emits gamma energy in the 100 to 200 keV range is preferred. A gamma emitter is herein defined as an agent that emits gamma energy of any range. One of ordinary skill in the art would be familiar with the various metal ions that are gamma emitters. To minimize the absorbed radiation dose to the patient, the physical half-life of the radionuclide should be as short as the imaging procedure will allow. To allow for examinations to be performed on any day and at any time of the day, it is advantageous to have a source of the radionuclide always available at the clinical site. .sup.99mTc is a preferred radionuclide because it emits gamma radiation at 140 keV, it has a physical half-life of 6 hours, and it is readily available on-site using a molybdenum-99/technetium-99m generator. One of ordinary skill in the art would be familiar with methods to determine optimal radioimaging in humans.
(47) In certain particular embodiments of the present invention, the metal ion is a therapeutic metal ion. For example, in some embodiments, the metal ion is a therapeutic radionuclide that is a beta-emitter. As herein defined, a beta emitter is any agent that emits beta energy of any range. Examples of beta-emitters include Re-188, Re-187, Re-186, Ho-166, Y-90, Bi-212, Bi-213, and Sn-153. The beta-emitters may or may not also be gamma-emitters. One of ordinary skill in the art would be familiar with the use of beta-emitters in the treatment of hyperproliferative disease, such as cancer.
(48) In further embodiments of the compositions of the present invention, the metal ion is a therapeutic metal ion that is not a beta emitter or a gamma emitter. For example, the therapeutic metal ion may be platinum, cobalt, copper, arsenic, selenium, calcium or thallium. Compositions including these therapeutic metal ions may be applied in methods directed to the treatment of diseases such as hyperproliferative diseases, cardiovascular disease, infections, and inflammation. Examples of hyperproliferative diseases include cancers. Methods of performing dual chemotherapy and radiation therapy that involve the compositions of the present invention are discussed in greater detail below.
D. Targeting Ligands
(49) A targeting ligand is defined herein to be a molecule or part of a molecule that binds with specificity to another molecule. One of ordinary skill in the art would be familiar with the numerous agents that can be employed as targeting ligands in the context of the present invention.
(50) Examples of targeting ligands include disease cell cycle targeting compounds, angiogenesis targeting ligands, tumor apoptosis targeting ligands, disease receptor targeting ligands, gene expression markers, drug-based ligands, antimicrobials, tumor hypoxia targeting ligands, an antisense molecule, an agent that mimics glucose, amifostine, angiostatin, EGF receptor ligands, capecitabine, COX-2 inhibitors, deoxycytidine, fullerene, herceptin, human serum albumin, lactose, leuteinizing hormone, pyridoxal, quinazoline, thalidomide, transferrin, and trimethyl lysine.
(51) In further embodiments of the present invention, the targeting ligand is an antibody. Any antibody is contemplated as a targeting ligand in the context of the present invention. For example, the antibody may be a monoclonal antibody. One of ordinary skill in the art would be familiar with monoclonal antibodies, methods of preparation of monoclonal antibodies, and methods of use of monoclonal antibodies as ligands. In certain embodiments of the present invention, the monoclonal antibody is an antibody directed against a tumor marker. In some embodiments, the monoclonal antibody is monoclonal antibody C225, monoclonal antibody CD31, or monoclonal antibody CD40.
(52) A single targeting ligand, or more than one such targeting ligand, may be conjugated to a chelator of the present invention. In these embodiments, any number of targeting ligands may be conjugated to the chelators set forth herein. In certain embodiments, a conjugate of the present invention may comprise a single targeting ligand. In other embodiments, a conjugate may comprise only two targeting ligands. In further embodiments, a targeting ligand may comprise three or more targeting ligands. In any situation where a conjugate comprises two or more targeting ligands, the targeting ligands may be the same or different.
(53) The targeting ligands can be bound to the chelator in any manner, including for example covalent bonds, ionic bonds and hydrogen bonds. For example, the targeting ligand may be bound to the chelator in an amide linkage, an ester linkage, or a carbon-carbon bond linkage of any length. If two or more targeting ligands are bound to a chelator, the modes of binding may be the same or different. In other embodiments, the linkage comprises a linker. Non-limiting examples of such linkers include peptides, glutamic acid, aspartic acid, bromo ethylacetate, ethylene diamine, lysine and any combination of one or more of these groups. One of ordinary skill in the art would be familiar with the chemistry of these agents, and methods to conjugate these agents as ligands to the chelators of the claimed invention. Methods of synthesis of the compounds of the present invention, including modes of conjugation, are discussed in detail below.
(54) Information pertaining to targeting ligands and conjugation with compounds is provided in U.S. Pat. No. 6,692,724, U.S. patent application Ser. No. 09/599,152, U.S. patent application Ser. No. 10/627,763, U.S. patent application Ser. No. 10/672,142, U.S. patent application Ser. No. 10/703,405, and U.S. patent application Ser. No. 10/732,919, each of which is herein specifically incorporated by reference in their entirety for this section of the specification and all other sections of the specification.
(55) In some embodiments of the compositions of the present invention, the targeting ligand is a tissue-specific ligand, which is conjugated to the chelator. A tissue-specific ligand is defined herein to refer to a molecule or a part of a molecule that can bind or attach to one or more tissues. The binding may be by any mechanism of binding known to those of ordinary skill in the art. Examples include therapeutic agents, antimetabolites, apoptotic agents, bioreductive agents, signal transductive therapeutic agents, receptor responsive agents, or cell cycle specific agents. The tissue may be any type of tissue, such as a cell. For example, the cell may be the cell of a subject, such as a cancer cell. In certain embodiments, the tissue-targeting ligand is a tissue-targeting amino acid sequence that is conjugated to a chelator that is capable of binding to a metal ion.
(56) Representative examples of targeting ligands are discussed below.
(57) 1. Drugs
(58) In some embodiments of the compositions of the present invention, a targeting ligand is a drug, or therapeutic ligand, which is defined herein to refer to any therapeutic agent. A therapeutic agent or drug is defined herein to include any compound or substance that can be administered to a subject, or contacted with a cell or tissue, for the purpose of treating a disease or disorder, or preventing a disease or disorder, or treating or preventing an alteration or disruption of a normal physiologic process. For example, the therapeutic ligand may be an anti-cancer moiety, such as a chemotherapeutic agent. In certain embodiments of the present invention, the therapeutic ligand is a therapeutic amino acid sequence that is conjugated to the therapeutic amino acid sequence. Such conjugates are discussed further in other parts of this specification.
(59) a. Chemotherapeutic Agents
(60) Examples of anti-cancer ligands include any chemotherapeutic agent known to those of ordinary skill in the art. Examples of such chemotherapeutic agents include, but are not limited to, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing. In certain particular embodiments, the anti-cancer ligand is methotrexate.
(61) A wide variety of chemotherapeutic agents may be used in accordance with the present invention. The term chemotherapy refers to the use of drugs to treat cancer. A chemotherapeutic agent is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
(62) Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and calicheamicin omegaI1; dynemicin, including dynemicin A); bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2,2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
(63) Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines such as gene therapy vaccines and pharmaceutically acceptable salts, acids or derivatives of any of the above.
(64) Additional examples of anti-cancer agents include those drugs of choice for cancer chemotherapy listed in Table 1:
(65) TABLE-US-00001 TABLE 1 Drugs of Choice for Cancer Chemotherapy The tables that follow list drugs used for treatment of cancer in the USA and Canada and their major adverse effects. The Drugs of Choice listing based on the opinions of Medical Letter consultants. Some drugs are listed for indications for which they have not been approved by the U.S. Food and Drug Administration. Anti-cancer drugs and their adverse effects follow. For purposes of the present invention, these lists are meant to be exemplary and not exhaustive. DRUGS OF CHOICE Cancer Drugs of Choice Some alternatives Adrenocortical** Mitotane Doxorubicin, streptozocin, Cisplatin etoposide Bladder* Local: Instillation of BCG Instillation of mitomycin, Systemic: Methotrexate + vinblastine + doxorubicin + cisplatin doxorubicin or thiotape (MVAC) Pacitaxel, substitution of Cisplatin + Methotrexate + vinblastine carboplatin for cisplatin in (CMV) combinations Brain Anaplastic astrocytoma* Procarbazine + lomustine + vincristine Carmustine, Cisplatin Anaplastic oligodendro- Procarbazine + lomustine + vincristine Carmustine, Cisplatin Glioma* Glioblastoma** Carmustine or lomustine Procarbazine, cisplatin Medulloblastoma Vincristine + carmustine mechlorethamine methotrexate Etoposide Mechiorethamine + vincristine + procarbazine + prednisone (MOPP) Vincristine + cisplatin cyclophosphamide Primary central nervous Methotrexate (high dose Intravenous and/or system lymphoma Intrathecal) cytarabine (Intravenous and/or Intrathecal) Cyclophosphamide + Doxorubicin + vincristine + prednisone (CHOP) Breast Adjuvant.sup.1: Cyclophosphamide + methotrexate + fluorouracil Paclitaxel; thiotepa + Doxorubicin + vinblastine; (CMF); mitomycin + vinblastine; Cyclophosphamide + Doxorubicin fluorouracil mitomycin + methotrexate + mitoxantrone; (AC or CAF); Tamoxifen fluorouracil Metastatic: Cyclophosphamide + methotrexate + fluorouracil by continuous infusion; (CMF) or Bone marrow transplant.sup.3 Cyclophosphamide + doxorubicin fluorouracil (AC or CAF) for receptor- negative and/or hormone-refractory; Tamoxifen for receptor-positive and/or hormone-sensitive.sup.2 Cervix** Cisplatin Chlorambucil, vincristine, Ifosfamide with means fluorouracil, Doxorubicin, Bleomycin + ifosfamide with means + cisplatin methotrexate, altretamine Choriocarcinoma Methotrexate leucovorin Methotrexate + Dactinomycin dactinomycin + cyclophosphamide (MAC) Etoposide + methotrexate + dactinomycin + cyclophosphamide + vincristine Colorectal* Adjuvant colon.sup.4: Fluorouracil + levamisole; Hepatic metastases: fluorouracil + leucovorin Intrahepatic-arterial Metastatic: fluorouracil + leucovorin floxuridine Mitomycin Embryonal rhabdomyosarcoma.sup.5 Vincristine + dactinomycin cyclophosphamide Same + Doxorubicin Vincristine + ifosfamide with means + etoposide Endometrial** Megastrol or another progestin fluorouracil, tamoxifen, Doxorubicin + cisplatin cyclophosphamide altretamine Esophageal* Cisplatin + fluorouracil Doxorubicin, methotraxate, mitomycin Ewing's sarcoma.sup.5 Cyclophosphamide (or ifosfamide with CAV + etoposide means) + Doxorubicin + vincristine (CAV) dactinomycin Gastric** Fluorouracil leucovorin Cisplatin, Doxorubicin, etoposide, methotrexate + leucovorin, mitomycin Head and neck squamous cell* Cisplatin + fluorouracil Bleomycin, carboplatin, Methotrexate paclitaxel Islet cell** Streptozocin + Doxorubicin Streptozocin + fluorouracil; chlorozotocin.sup.; octreotide Kaposi's sarcoma* (Aids-related) Etoposide or interferon alfa or vinblastine Vincristine, Doxorubicin, Doxorubicin + bleomycin + vincristine or bleomycin vinblastine (ABV) Leukemia Acute lymphocytic leukemia Induction: Vincristine + Induction: same high- (ALL).sup.6 prednisone + asparaginase daunorubicin dose methotrexate cytarabine; CNS prophylaxis: Intrathecal methotrexate systemic high-dose methotrexate with pegaspargase leucovorin Intrathecal cytarabine Intrathecal instead of asparaginese hydrocortisone Teniposide or etoposide Maintenance: Methotrexate + mercaptopurine High-dose cytarabine Bone marrow transplant..sup.3 7 Maintenance: same + periodic vincristine + prednisone Acute myeloid leukemia (AML).sup.8 Induction: Cytarahine + either daunorubicin Cytarabine + mitoxentrone or idarubicin High-dose cytarabine Post Induction: High-dose cytarabine other drugs such as etoposide Bone marrow transplant.sup.3. Chronic lymphocytic leukemia Chlorambucil prednisone Cladribine, (CLL) Fludarabin cyclophosphamide, pentostatin, vincristine, Doxorubicin Chronic myeloid leukemia (CML).sup.9 Chronic phase Bone marrow transplant.sup.3 Busulfan Interferon alfa Hydroxyurea Accelerated.sup.10 Bone marrow transplant.sup.3 Hydroxyures, busulfan Blast crisis.sup.11 Lymphoid: Vincristine + prednisone + L- Tretinoln.sup. asparaginase + intrathecal methotrexate ( maintenance Amsecrine,.sup. azacitidine with methotrexate + 8- Vincristine plicamycin mercaptopurine) Hairy cell Leukemia Pentostatin or cladribine Interferon alfa, chlorambucil, fludarabin Liver** Doxorubicin Intrahepatic-arterial Fluorouracil floxuridine or claplatin Lung, small cell (cat cell) Cisplatin + etoposide (PE) Ifosfamide with means + carboplatin + etoposide Cyclophosphamide + doxorubicin + vincristine (ICE) (CAV) Daily oral etoposide PE alternated with CAV Etoposide + ifosfamide Cyclophosphamide + etoposide + cisplatin with means + claplatin (CEP) (VIP Doxorubicin + cyclophosphamide + etoposide Paclitaxel (ACE) Lung Cisplatin + etoposide Cisplatin + fluorouracil + leucovorin (non-small cell)** Cisplatin + Vinblastine mitomycin Carboplatin + paclitaxel Cisplatin + vincristine Lymphomas Hodgkin's.sup.11 Doxorubicin + bleomycin + vinblastine + dacarbazine Mechlorethamine + vincristine + (ABVD) procarbazine + prednisone (MOPP) ABVD alternated with MOPP Chlorambusil + vinblastine + Mechlorethamine + vincristine + procarbazine + prednisone carmustine procarbazine ( prednisone) + Etoposide + doxorubicin + bleomycin + vinblastine vinblastine + doxorubicin (MOP[P]-ABV) Bone marrow transplant.sup.3 Non-Hodgkin's Cyclophosphamide + vincristine + methotrexate Ifosfamide with means Burkitt's lymphoma Cyclophosphamide + high-dose cytarabine methotrexate Cyclophosphamide + doxorubicin + with leutovorin vincristine + prednisone (CHOP) Intrathecal methotrexate or cytarabine Diffuse large-cell lymphoma Cyclophosphamide + doxorubicin + vincristine + prednisone Dexamethasone (CHOP) sometimes substituted for prednisone Other combination regimens, which may include methotrexate, etoposide, cytarabine, bleomycin, procarbazine, ifosfamide and mitoxantrone Bone marrow transplant.sup.3 Follicular lymphoma Cyclophosphamide or chlorambusil Same vincristine and prednisone, etoposide Interferon alfa, cladribine, fludarabin Bone marrow transplant.sup.3 Cyclophosphamide + doxorubicin + vincristine + prednisone (CHOP) Melanoma** Interferon Alfa Carmustine, lomustine, Dacarbazine cisplatin Dacarbazine + clapletin + carmustine + tamoxifen Aldesleukin Mycosis fungoides* PUVA (psoralen + ultraviolet A) Isotretinoin, topical Mechlorethamine (topical) carmustine, pentosistin, Interferon alfa fludarabin, cladribine, Electron beam radiotherapy photopheresis (extra- Methotrexate corporeal photochemitherapy), chemotherapy as in non- Hodgkin's lymphoma Myloma* Melphalan (or cyclophosphamide) + prednisone Interferon alfa Melphalan + carmustine + Bone marrow transplant.sup.3 cyclophosphamide + prednisone + vincristine High-dose dexamethasone Dexamethasone + doxorubicin + vincristine (VAD) Vincristine + carmustine + doxorubicin + prednisone (VBAP) Neuroblastoma* Doxorubicin + cyclophosphamide + cisplatin + teniposide Carboplatin, etoposide or etoposide Bone marrow transplant.sup.3 doxorubicin + cyclophosphamide Claplatin + cyclophosphamide Osteogenic sarcoma.sup.5 Doxorubicin + cisplatin etoposide ifosfamide Ifosfamide with means, etoposide, carboplatin, high-dose methotrexate with leucovorin Cyclophosphamide + etoposide Ovary Cisplatin (or carboplatin) + paclitaxel Ifosfamide with means, Cisplatin (or carboplatin) + cyclophosphamide paclitaxel, tamoxifen, (CP) doxorubicin melphalan, altretamine (CAP) Pancreatic** Fluorouracil leucovorin Prostate Leuprolide + flutamide Estramustine + vinblastine, aminoglutethimide + hydrocortisone, estramustine + etoposide, diethylstilbestrol, nilutamide Renal** Aldesleukin Vinblastine, floxuridine Inteferon alfa Retinoblastoma.sup.5* Doxorubicin + cyclophosphamide + Carboplatin, etoposide, cisplatin + etoposide + vincristine Ifosfamide with means Sarcomas, soft tissue, adult* Doxorubicin + dacarbazine + cyclophosphamide + Ifosfamide Mitornyeln + doxorubicin + cisplatin with means Vincristine, etoposide Testicular Cisplatin + etoposide + bleomycin (PEB) Vinblastine (or etoposide) + Ifosfamide with means + cisplatin (VIP) Bone marrow transplant.sup.3 Wilms' tumor.sup.5 Dactinomycin + vincristine + doxorubicin + cyclophosphamide Ifosfamide with means, etoposide, carboplatin *Chemotherapy has only moderate activity. **Chemotherapy has only minor activity. .sup.1Tamoxifen with or without chemotherapy is generally recommended for postmenopausal estrogen-receptor-positive, mode-positive patients and chemotherapy with or without tamoxifen for premenopausal mode-positive patients. Adjuvant treatment with chemotherapy and/or tamoxifen is recommended for mode-negative patients with larger tumors or other adverse prognostic indicators. .sup.2Megastrol and other hormonal agents may be effective in some patients with tamoxifen fails. .sup.3After high-dose chemotherapy (Medical Letter, 34: 79, 1982). .sup.4For rectal cancer, postoperative adjuvant treatment with fluorouracil plus radiation, preceded and followed by treatment with fluorouracil alone. .sup.5Drugs have major activity only when combined with surgical resection, radiotherapy or both. .sup.Available in the USA only for investigational use. .sup.6High-risk patients (e.g., high counts, cytogenetic abnormalities, adults) may require additional drugs for induction, maintenance and intensificiation (use of additional drugs after achievement of remission). Additional drugs include cyclophosphamida, mitoxantrone and thloguanine. The results of one large controlled trial in the United Kingdom suggest that intensificiation may improve survival in all children with ALL (Chasselle et al, 1995). .sup.7Patients with a poor prognosis initially or those who relapse after remission. .sup.8Some patients with acute promyelocytic leukemia have had complete responses to tratinoin. Such treatment can cause a toxic syndrome characterized primarily by fever and respiratory distress (Warrell, Jr et al, 1993). .sup.9Allogeneic HLA-identical sibling bone marrow transplantation can cure 40% to 70% of patients with CML in chronic phase, 18% to 28% of patients with accelerated phase CML, and <15% patients in blast crisis. Disease-free survival after bone marrow transplantations adversely influenced by age >50 years, duration of disease >3 years from diagnosis, and use of one-antigen-mismatched or matched-unrelated donor marrow. Interferon also may be curative in patients with chronic phase CML who achieve a complete cytogenetic response (about 10%); it is the treatment of choice for patents >80 years old with newly diagnosed chronic phase CML and for all patients who are not candidates for an allgensic bone marrow transplant. Chemotherapy alone is palliative. .sup.10If a second chronic phase is achieved with any of these combinations, allogeneic bone marrow transplant should be considered. Bone marrow transplant in second chronic phase may be curative for 30% to 35% of patients with CML. .sup.11Limited-stage Hodgkin's disease (stages 1 and 2) is curable by radiotherapy. Disseminated disease (stages 3b and 4) require chemotherapy. Some intermediate stages and selected clinical situations may benefit from both. + Available in the USA only for investigational use.
(66) b. Cardiovascular Drugs
(67) A cardiovascular drug is defined herein to refer to any therapeutic agent that can be applied in the treatment or prevention of a disease of the heart and/or blood vessels.
(68) In certain embodiments, the cardiovascular drug is an agent that lowers the concentration of one of more blood lipids and/or lipoproteins, known herein as an antihyperlipoproteinemic, which can be applied in the treatment of atherosclerosis and thickenings or blockages of vascular tissues. Examples include an aryloxyalkanoic/fibric acid derivative, a resin/bile acid sequesterant, a HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a miscellaneous agent or a combination thereof. Non-limiting examples of aryloxyalkanoic/fibric acid derivatives include beclobrate, benzafibrate, binifibrate, ciprofibrate, clinofibrate, clofibrate (atromide-S), clofibric acid, etofibrate, fenofibrate, gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrate and theofibrate. Non-limiting examples of resins/bile acid sequestrants include cholestyramine (cholybar, questran), colestipol (colestid) and polidexide. Non-limiting examples of HMG CoA reductase inhibitors include lovastatin (mevacor), pravastatin (pravochol) or simvastatin (zocor). Non-limiting examples of nicotinic acid derivatives include nicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid. Non-limiting examples of thyroid hormones and analogs thereof include etoroxate, thyropropic acid and thyroxine. Non-limiting examples of miscellaneous antihyperlipoproteinemics include acifran, azacosterol, benfluorex, -benzalbutyramide, camitine, chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, 5,8,11,14,17-eicosapentaenoic acid, eritadenine, furazabol, meglutol, melinamide, mytatrienediol, ornithine, -oryzanol, pantethine, pentaerythritol tetraacetate, -phenylbutyramide, pirozadil, probucol (lorelco), -sitosterol, sultosilic acid-piperazine salt, tiadenol, triparanol and xenbucin.
(69) Non-limiting examples of an antiarteriosclerotic include pyridinol carbamate.
(70) In certain embodiments, the cardiovascular drug is an agent that aids in the removal or prevention of blood clots. Non-limiting examples of antithrombotic and/or fibrinolytic agents include anticoagulants, anticoagulant antagonists, antiplatelet agents, thrombolytic agents, thrombolytic agent antagonists or combinations thereof. Examples of antithrombotic agents include aspirin and wafarin (coumadin. Examples of anticoagulant include acenocoumarol, ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide, tioclomarol and warfarin. Non-limiting examples of antiplatelet agents include aspirin, a dextran, dipyridamole (persantin), heparin, sulfinpyranone (anturane) and ticlopidine (ticlid). Non-limiting examples of thrombolytic agents include tissue plasminogen activator (activase), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase (streptase), antistreplase/APSAC (eminase).
(71) In some embodiments, the cardiovascular drug is a blood coagulant. Non-limiting examples of a blood coagulation promoting agent include thrombolytic agent antagonists and anticoagulant antagonists. Non-limiting examples of anticoagulant antagonists include protamine and vitamin K1.
(72) Non-limiting examples of thrombolytic agent antagonists include aminocaproic acid (amicar) and tranexamic acid (amstat). Non-limiting examples of antithrombotics include anagrelide, argatroban, cilostazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.
(73) The cardiovascular drug may be an antiarrythmic agent. Non-limiting examples of antiarrhythmic agents include Class I antiarrythmic agents (sodium channel blockers), Class II antiarrythmic agents (beta-adrenergic blockers), Class II antiarrythmic agents (repolarization prolonging drugs), Class IV antiarrhythmic agents (calcium channel blockers) and miscellaneous antiarrythmic agents. Non-limiting examples of sodium channel blockers include Class IA, Class IB and Class IC antiarrhythmic agents. Non-limiting examples of Class IA antiarrhythmic agents include dispyramide (norpace), procainamide (pronestyl) and quinidine (quinidex). Non-limiting examples of Class IB antiarrhythmic agents include lidocaine (xylocaine), tocamide (tonocard) and mexiletine (mexitil). Non-limiting examples of Class IC antiarrhythmic agents include encamide (enkaid) and flecaimide (tambocor). Non-limiting examples of a beta blocker, otherwise known as a -adrenergic blocker, a -adrenergic antagonist or a Class II antiarrhythmic agent, include acebutolol (sectral), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol, propranolol (inderal), sotalol (betapace), sulfinalol, talinolol, tertatolol, timolol, toliprolol and xibinolol. In certain aspects, the beta blocker comprises an aryloxypropanolamine derivative. Non-limiting examples of aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propranolol, talinolol, tertatolol, timolol and toliprolol. Non-limiting examples of an agent that prolong repolarization, also known as a Class III antiarrhythmic agent, include amiodarone (cordarone) and sotalol (betapace). Non-limiting examples of a calcium channel blocker, otherwise known as a Class IV antiarrythmic agent, include an arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil, phenylamine, terodiline, verapamil), a dihydropyridine derivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) a piperazine derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a miscellaneous calcium channel blocker such as bencyclane, etafenone, magnesium, mibefradil or perhexyline. In certain embodiments a calcium channel blocker comprises a long-acting dihydropyridine (nifedipine-type) calcium antagonist. Non-limiting examples of miscellaneous antiarrhymic agents include adenosine (adenocard), digoxin (lanoxin), acetamide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, diisopyramide, hydroquinidine, indecamide, ipratropium bromide, lidocaine, lorajmine, lorcamide, meobentine, moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate and viquidil.
(74) Other examples of cardiovascular drugs include antihypertensive agents. Non-limiting examples of antihypertensive agents include sympatholytic, alpha/beta blockers, alpha blockers, anti-angiotensin II agents, beta blockers, calcium channel blockers, vasodilators and miscellaneous antihypertensives. Non-limiting examples of an alpha blocker, also known as an -adrenergic blocker or an -adrenergic antagonist, include amosulalol, arotinolol, dapiprazole, doxazocin, ergoloid mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin and yohimbine. In certain embodiments, an alpha blocker may comprise a quinazoline derivative. Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazocin, prazosin, terazosin and trimazosin. In certain embodiments, an antihypertensive agent is both an alpha and beta adrenergic antagonist. Non-limiting examples of an alpha/beta blocker comprise labetalol (normodyne, trandate). Non-limiting examples of anti-angiotension II agents include angiotensin converting enzyme inhibitors and angiotension II receptor antagonists. Non-limiting examples of angiotension converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (vasotec), captopril, cilazapril, delapril, enelaprilat, fosinopril, lisinopril, moveltopril, perindopril, quinapril and ramipril. Non-limiting examples of an angiotensin II receptor blocker, also known as an angiotension II receptor antagonist, an ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS), include angiocandesartan, eprosartan, irbesartan, losartan and valsartan. Non-limiting examples of a sympatholytic include a centrally acting sympatholytic or a peripherally acting sympatholytic. Non-limiting examples of a centrally acting sympatholytic, also known as an central nervous system (CNS) sympatholytic, include clonidine (catapres), guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet). Non-limiting examples of a peripherally acting sympatholytic include a ganglion blocking agent, an adrenergic neuron blocking agent, a -adrenergic blocking agent or a alpha1-adrenergic blocking agent. Non-limiting examples of a ganglion blocking agent include mecamylamine (inversine) and trimethaphan (arfonad). Non-limiting of an adrenergic neuron blocking agent include guanethidine (ismelin) and reserpine (serpasil). Non-limiting examples of a -adrenergic blocker include acenitolol (sectral), atenolol (tenormin), betaxolol (kerlone), carteolol (cartrol), labetalol (normodyne, trandate), metoprolol (lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken), propranolol (inderal) and timolol (blocadren). Non-limiting examples of alpha1-adrenergic blocker include prazosin (minipress), doxazocin (cardura) and terazosin (hytrin). In certain embodiments a cardiovasculator therapeutic agent may comprise a vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or a peripheral vasodilator). In certain preferred embodiments, a vasodilator comprises a coronary vasodilator. Non-limiting examples of a coronary vasodilator include amotriphene, bendazol, benfurodil hemisuccinate, benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole, droprenilamine, efloxate, erythritol tetranitrane, etafenone, fendiline, floredil, ganglefene, herestrol bis(-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin, lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin, pentaerythritol tetranitrate, pentrinitrol, perhexyline, pimefylline, trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine. In certain aspects, a vasodilator may comprise a chronic therapy vasodilator or a hypertensive emergency vasodilator. Non-limiting examples of a chronic therapy vasodilator include hydralazine (apresoline) and minoxidil (loniten). Non-limiting examples of a hypertensive emergency vasodilator include nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten) and verapamil.
(75) Non-limiting examples of miscellaneous antihypertensives include ajmaline, -aminobutyric acid, bufeniode, cicletainine, ciclosidomine, a cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, a protoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodium nitroprusside, ticrynafen, trimethaphan camsylate, tyrosinase and urapidil.
(76) In certain aspects, an antihypertensive may comprise an arylethanolamine derivative, a benzothiadiazine derivative, a N-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative, a guanidine derivative, a hydrazines/phthalazine, an imidazole derivative, a quanternary ammonium compound, a reserpine derivative or a sulfonamide derivative. Non-limiting examples of arylethanolamine derivatives include amosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol and sulfinalol. Non-limiting examples of benzothiadiazine derivatives include althizide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythiazide, tetrachlormethiazide and trichloromethiazide. Non-limiting examples of N-carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril, cilazapril, delapril, enalapril, enelaprilat, fosinopril, lisinopril, moveltipril, perindopril, Non-limiting examples of dihydropyridine derivatives include amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitrendipine. Non-limiting examples of guanidine derivatives include bethanidine, debrisoquin, guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan. Non-limiting examples of hydrazines/phthalazines include budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine and todralazine. Non-limiting examples of imidazole derivatives include clonidine, lofexidine, phentolamine, tiamenidine and tolonidine. Non-limiting examples of quanternary ammonium compounds include azamethonium bromide, chlorisondamine chloride, hexamethonium, pentacynium bis(methylsulfate), pentamethonium bromide, pentolinium tartrate, phenactropinium chloride and trimethidinium methosulfate. Non-limiting examples of reserpine derivatives include bietaserpine, deserpidine, rescinnamine, reserpine and syrosingopine. Non-limiting examples of sulfonamide derivatives include ambuside, clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide.
(77) Other examples of cardiovascular drugs include vasopressors. Vasopressors generally are used to increase blood pressure during shock, which may occur during a surgical procedure. Non-limiting examples of a vasopressor, also known as an antihypotensive, include amezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine.
(78) Other examples of cardiovascular drugs include agents that can be applied in the treatment or prevention of congestive heart failure. Non-limiting examples of agents for the treatment of congestive heart failure include anti-angiotensin II agents, afterload-preload reduction treatment, diuretics and inotropic agents. Examples of afterload-preload reduction agents include hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitrate). Non-limiting examples of a diuretic include a thiazide or benzothiadiazine derivative (e.g., althiazide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythiazide, tetrachloromethiazide, trichloromethiazide), an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurous chloride, mersalyl), a pteridine (e.g., furtherene, triamterene), purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom, protheobromine, theobromine), steroids including aldosterone antagonists (e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative (e.g., acetazolamide, ambuside, azosemide, bumetamide, butazolamide, chloraminophenamide, clofenamide, clopamide, clorexolone, diphenylmethane-4,4-disulfonamide, disulfamide, ethoxzolamide, furosemide, indapamide, mefruside, methazolamide, piretanide, quinethazone, torasemide, tripamide, xipamide), a uracil (e.g., aminometradine, amisometradine), a potassium sparing antagonist (e.g., amiloride, triamterene) or a miscellaneous diuretic such as aminozine, arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine, isosorbide, mannitol, metochalcone, muzolimine, perhexyline, ticmafen and urea. Non-limiting examples of a positive inotropic agent, also known as a cardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline, aminone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin, strphanthin, sulmazole, theobromine and xamoterol. In particular aspects, an intropic agent is a cardiac glycoside, a beta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limiting examples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin (crystodigin). Non-limiting examples of a -adrenergic agonist include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex), dopamine (intropin), dopexamine, ephedrine, etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol and xamoterol. Non-limiting examples of a phosphodiesterase inhibitor include aminone (inocor). Antianginal agents may comprise organonitrates, calcium channel blockers, beta blockers and combinations thereof. Non-limiting examples of organonitrates, also known as nitrovasodilators, include nitroglycerin (nitro-bid, nitrostat), isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol, vaporole).
(79) 2. Disease Cell Cycle Targeting Compounds
(80) Disease cell cycle targeting compounds refers to compounds that target agents that are upregulated in proliferating cells. Compounds used for this purpose can be used to measure various parameters in cells, such as tumor cell DNA content.
(81) Many of these agents are nucleoside analogues. For example, pyrimidine nucleoside (e.g., 2-fluoro-2-deoxy-5-iodo-1--D-arabinofuranosyluracil [FIAU], 2-fluoro-2-deoxy-5-iodo-1--D-ribofuranosyl-uracil [FIRU], 2-fluoro-2-5-methyl-1--D-arabinofuranosyluracil [FMAU], 2-fluoro-2-deoxy-5-iodovinyl-1--D-ribofuranosyluracil [IVFRU]) and acycloguanosine: 9-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]guanine (GCV) and 9-[4-hydroxy-3-(hydroxy-methyl)butyl]guanine (PCV) (Tjuvajev et al., 2002; Gambhir et al., 1998; Gambhir et al., 1999) and other .sup.18F-labeled acycloguanosine analogs, such as 8-fluoro-9-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]guanine (FGCV) (Gambhir et al., 1999; Namavari et al., 2000), 8-fluoro-9-[4-hydroxy-3-(hydroxymethyl)butyl]guanine (FPCV) (Gambhir et al., 2000; Iyer et al., 2001), 9-[3-fluoro-1-hydroxy-2-propoxy methyl]guanine (FHPG) (Alauddin et al., 1996; Alauddin et al., 1999), and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) (Alauddin and Conti, 1998; Yaghoubi et al., 2001) have been developed as reporter substrates for imaging wild-type and mutant (Gambhir et al., 2000) HSV1-tk expression. One or ordinary skill in the art would be familiar with these and other agents that are used for disease cell cycle targeting.
(82) 3. Angiogenesis Targeting Ligands
(83) Angiogenesis targeting ligands refers to agents that can bind to neovascularization or revascularization of tissue. For example, the neovascularization of tumor cells or revascularization of myocardium tissue. Agents that are used for this purpose are known to those of ordinary skill in the art for use in performing various measurements, including measurement of the size of a tumor vascular bed and measurement of tumor volume. Some of these agents bind to the vascular wall. One of ordinary skill in the art would be familiar with the agents that are available for use for this purpose.
(84) Throughout this application, angiogenesis targeting refers to the use of an agent to bind to neovascular tissue. Some examples of agents that are used for this purpose are known to those of ordinary skill in the art for use in performing various tumor measurements, including measurement of the size of a tumor vascular bed, and measurement of tumor volume. Some of these agents bind to the vascular wall. One of ordinary skill in the art would be familiar with the agents that are available for use for this purpose. A tumor angiogenesis targeting ligand is a ligand that is used for the purpose of tumor angiogenesis targeting as defined above. Examples of angiogenesis targeting ligands include COX-2 inhibitors, anti-EGF receptor ligands, herceptin, angiostatin, C225 and thalidomide. COX-2 inhibitors include, for example, celecoxib, rofecoxib, etoricoxib and analogs of these agents.
(85) 4. Tumor Apoptosis Targeting Ligands
(86) Tumor apoptosis targeting refers to use of an agent to bind to a cell that is undergoing apoptosis or at risk of undergoing apoptosis. These agents are generally used to provide an indicator of the extent or risk of apoptosis, or programmed cell death, in a population of cells, such as a tumor and cardiac tissue. One of ordinary skill in the art would be familiar with agents that are used for this purpose. A tumor apoptosis targeting ligand is a ligand that is capable of performing tumor apoptosis targeting as defined in this paragraph. The targeting ligand of the present invention may include TRAIL (TNF-related apoptosis inducing ligand) monoclonal antibody. TRAIL is a member of the tumor necrosis factor ligand family that rapidly induces apoptosis in a variety of transformed cell lines. The targeting ligand of the present invention may also comprise a substrate of caspase-3, such as peptide or chelator that includes the 4 amino acid sequence aspartic acid-glutamic acid-valine-aspartic acid. caspase-3 substrate (for example, a peptide or chelator that includes the amino acid sequence aspartic acid-glutamic acid-valine-aspartic acid), and any member of the Bcl family. Examples of Bcl family members include, for example, Bax, Bcl-xL, Bid, Bad, Bak and Bcl-2. One of ordinary skill in the art would be familiar with the Bcl family, and their respective substrates.
(87) Apoptosis suppressors are targets for drug discovery, with the idea of abrogating their cytoprotective functions and restoring apoptosis sensitivity to tumor cells (Reed, 2003).
(88) 5. Disease Receptor Targeting Ligands
(89) In disease receptor targeting, certain agents are exploited for their ability to bind to certain cellular receptors that are overexpressed in disease states, such as cancer, neurological diseases and cardiovascular diseases. Examples of such receptors which are targeted include estrogen receptors, androgen receptors, pituitary receptors, transferrin receptors and progesterone receptors. Examples of agents that can be applied in disease-receptor targeting include androgen, estrogen, somatostatin, progesterone, transferrin, luteinizing hormone and luteinizing hormone antibody.
(90) The radiolabeled ligands, such as pentetreotide, octreotide, transferrin and pituitary peptide, bind to cell receptors, some of which are overexpressed on certain cells. Since these ligands are not immunogenic and are cleared quickly from the plasma, receptor imaging would seem to be more promising compared to antibody imaging.
(91) The folate receptor is included herein as another example of a disease receptor. Folate receptors (FRs) are overexposed on many neoplastic cell types (e.g., lung, breast, ovarian, cervical, colorectal, nasopharyngeal, renal adenocarcinomas, malignant melanoma and ependymomas), but primarily expressed only several normal differentiated tissues (e.g., choroid plexus, placenta, thyroid and kidney) (Weitman et al., 1992a; Campbell et al., 1991; Weitman et al., 1992b; Holm et al., 1994; Ross et al., 1994; Franklin et al., 1994; Weitman et al., 1994). FRs have been used to deliver folate-conjugated protein toxins, drug/antisense oligonucleotides and liposomes into tumor cells overexpressing the folate receptors (Ginobbi et al., 1997; Leamon and Low, 1991; Leamon and Low, 1992; Leamon et al., 1993; Lee and Low, 1994). Furthermore, bispecific antibodies that contain anti-FR antibodies linked to anti-T cell receptor antibodies have been used to target T cells to FR-positive tumor cells and are currently in clinical trials for ovarian carcinomas (Canevari et al., 1993; Bolhuis et al., 1992; Patrick et al., 1997; Coney et al., 1994; Kranz et al., 1995).
(92) Examples of folate receptor targeting ligands include folic acid and analogs of folic acid. Preferred folate receptor targeting ligands include folate, methotrexate and tomudex. Folic acid as well as antifolates such as methotrexate enter into cells via high affinity folate receptors (glycosylphosphatidylinositol-linked membrane folate-binding protein) in addition to classical reduced-folate carrier system (Westerhof et al., 1991; Orr et al., 1995; Hsueh and Dolnick, 1993).
(93) 6. Cardiac Ischemia Markers
(94) In some embodiments, the targeting ligand is a cardiac ischemia marker. A cardiac ischemia marker is a ligand that is relatively selective for ischemic cardiac tissue. Non-limiting examples of cardiac ischemia markers include interleukin-6, tumor necrosis factor alpha, matrix metalloproteinase 9, myeloperoxidase, intercellular and vascular adhesion molecules, soluble CD40 ligand, placenta growth factor, high sensitivity C-reactive protein (hs-CRP), ischemia modified albumin (IMA), free fatty acids, and choline.
(95) 7. Viability Cardiac Tissue Markers
(96) In some embodiments, the targeting ligand is a viability cardiac tissue marker. A viability cardiac tissue marker refers to a ligand that is relatively selective for viable cardiac tissue compared to nonviable cardiac tissue. Non-limiting examples of cardiac viability tissue markers include those selected from the group consisting of phospholipase C, myosin light-chain phosphatase, nitric oxide, prostacyclin, endothelin, thromboxane, L-arginine and L-citrulline.
(97) 8. Congestive Heart Failure Markers
(98) In some embodiments, the targeting ligand is a congestive heart failure marker. A congestive heart failure marker is a ligand that is relatively selective for cardiac tissue of a heart in congestive heart failure compared to normal healthy heart tissue. Non-limiting examples of congestive heart failure markers include those selected from the group consisting of interleukin-1, cardiotrophin-1, insulin-like growth factor, epidermal growth factor, tyrosine kinase receptor and angiotensin II.
(99) 9. Rest/Stress Cardiac Tissue Markers
(100) In some embodiments, the targeting ligand is a rest/stress cardiac tissue marker. A rest/stress cardiac tissue marker is a ligand that is relatively selective for cardiac tissue that is stressed compared to non-stressed (at rest) cardiac tissue, or vice versa. Non-limiting examples of rest/stress cardiac tissue markers include those selected from the group consisting of mitogen-activated protein kinase, cyclic adenosine monophosphate, phospholipase C, phosphatidylinositol bisphosphate, isositol trisphosphate, diacylglycerol and tyrosine kinases.
(101) 10. Drug Assessment
(102) Certain drug-based ligands can be applied in measuring the pharmacological response of a subject to a drug. A wide range of parameters can be measured in determining the response of a subject to administration of a drug. One of ordinary skill in the art would be familiar with the types of responses that can be measured. These responses depend in part upon various factors, including the particular drug that is being evaluated, the particular disease or condition for which the subject is being treated, and characteristics of the subject. Examples of drug-based ligands include carnitine, puromycin, verapamil, digoxin, prazosin, quinidine, diisopyramide, theophylline, protease inhibitors nifedipine, diltiazem, flecaimide, amiodarone, sotalol, adenosine, dopamine dobutamine, inamrinone, milrinone, spironolactone, prazosin, aspirin and warfarin.
(103) 11. Antimicrobials
(104) Any antimicrobial is contemplated for inclusion as a targeting ligand. Preferred antimicrobials include ampicillin, amoxicillin, penicillin, clindamycin, gentamycin, kanamycin, neomycin, natamycin, nafcillin, rifampin, tetracycline, vancomycin, bleomycin, doxycyclin, amikacin, netilmicin, streptomycin, tobramycin, loracarbef, ertapenem, imipenem, meropenem, cefadroxil, cefazolin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, teicoplanin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, aztreonam, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole, demeclocycline, minocycline, oxytetracycline, arsphenamine, chloramphenicol, ethambutol, fosfomycin, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin, dalfopristin, spectinomycin, and telithromycin.
(105) Antifungals include natamycin, rimocidin, filipin, nystatin, amphotericin B, miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butocanazole, finticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole, ravuconazole, posaconazole, voriconazole, terconazole, terbinafine, amorolfine, naftifine, butenafine, anidulafungin, caspofungin, micafungin, ciclopirox, flucytosine, griseofulvin, gentian violet, haloprogin, tolnaftate, undecyclenic acid, amantadine, polymycin, acyclovir and ganciclovir for fungi. One of ordinary skill in the art would be familiar with the various agents that are considered to be antimicrobials.
(106) 12. Agents that Mimic Glucose
(107) Agents that mimic glucose are also contemplated for inclusion as targeting ligands. Such agents can also be considered glucose analogs or glucose derivatives.
(108) Glucose is utilized by living organisms through the glycolysis pathway. Compounds such as neomycin, kanamycin, gentamycin, amikacin, tobramycin, netilmicin, ribostamycin, sisomicin, micromicin, lividomycin, dibekacin, isepamicin, and astromicin belong to a group called aminoglycosides.
(109) In terms of structure, agents that mimic glucose typically have a glucose ring structure. Exceptions exist, however, such as puromycin, which has a pentose ring structure, but which can still be considered an agent that mimics glucose.
(110) In terms of function, aminoglycosides are used as antibiotics that block the glycolysis pathway by their property of being structurally similar to glucose and thus, they are functionally considered as agents that mimic glucose. When these aminoglycosides are used in imaging studies, there are no detectable pharmacological effects.
(111) The word mimic, as defined by the American Heritage Dictionary fourth edition, means to resemble closely or simulate. Aminoglycosides are functionally utilized through the glycolytic pathway by virtue of their structural similarity to glucose and block the glycolysis pathway. Hence, aminoglycosides are considered to mimic or simulate glucose in structural and functional manner.
(112) Non-limiting examples of chemical structures with their PubChem Database (NCBI) identifier CID number are as follows: Amikacin CID 37768; Aminoglycoside CID 191574; Astromicin CID 65345; Deoxy-glucose CID 439268; D-glucosamine CID 441477; Dibekacin CID 3021; Gentamicin CID 3467; Glucose CID 5793; Isepamicin CID 456297; Kanamycin CID 5460349; Lividomycin CID 72394; Micromicin CID 107677; Neomycin CID 504578; Netilmicin CID 441306; Puromycin CID 439530; Ribostamycin CID 33042; Sisomicin CID 36119; and Tobramycin CID 36294.
(113) References which describe the glycolysis blocking by aminoglycosides include, for example, Tachibana et al., 1976; Borodina et al., 2005; Murakami et al., 1996; Hoelscher et al., 2000; Yang et al., 2004; Michalik et al., 1989; Murakami et al., 1997; Diamond et al., 1978; Hostetler and Hall, 1982; Benveniste and Davies, 1973; Hu, 1998; Yanai et al., 2006; Myszka et al., 2003; Nakae and Nakae, 1982; Ozmen et al., 2005; and Tod et al., 2000.
(114) Preferred agents that mimic glucose, or sugars, include neomycin, kanamycin, gentamycin, paromycin, amikacin, tobramycin, netilmicin, ribostamycin, sisomicin, micromicin, lividomycin, dibekacin, isepamicin, astromicin, and aminoglycosides glucose and glucosamine.
(115) 13. Hypoxia Targeting Ligands
(116) In some embodiments of the present invention, the targeting ligand is a tumor hypoxia targeting ligand. For example, tumor cells are more sensitive to conventional radiation in the presence of oxygen than in its absence; even a small percentage of hypoxic cells within a tumor could limit the response to radiation (Hall, 1988; Bush et al., 1978; Gray et al., 1958). Hypoxic radioresistance has been demonstrated in many animal tumors but only in few tumor types in humans (Dische, 1991; Gatenby et al., 1988; Nordsmark et al., 1996). The occurrence of hypoxia in human tumors, in most cases, has been inferred from histology findings and from animal tumor studies. In vivo demonstration of hypoxia requires tissue measurements with oxygen electrodes and the invasiveness of these techniques has limited their clinical application.
(117) Misonidazole, an example of a tumor hypoxia targeting ligand, is a hypoxic cell sensitizer, and labeling MISO with different radioisotopes (e.g., .sup.18F, .sup.123I, .sup.99mTc) may be useful for differentiating a hypoxic but metabolically active tumor from a well-oxygenated active tumor by PET or planar scintigraphy. [.sup.18F]Fluoromisonidazole (FMISO) has been used with PET to evaluate tumors hypoxia. Recent studies have shown that PET, with its ability to monitor cell oxygen content through [.sup.18F]FMISO, has a high potential to predict tumor response to radiation (Koh et al., 1992; Valk et al., 1992; Martin et al, 1989; Rasey et al., 1989; Rasey et al., 1990; Yang et al., 1995). PET gives higher resolution without collimation, however, the cost of using PET isotopes in a clinical setting is prohibitive.
(118) 14. Antisense Molecules
(119) Antisense molecules interact with complementary strands of nucleic acids, modifying expression of genes.
(120) Some regions within a double strand of DNA code for genes, which are usually instructions specifying the order of amino acids in a protein along with regulatory sequences, splicing sites, noncoding introns and other complicating details. For a cell to use this information, one strand of the DNA serves as a template for the synthesis of a complementary strand of RNA. The template DNA strand is called the antisense strand and the RNA is said to be sense (the complement of antisense). Because the DNA is double-stranded, the strand complementary to the antisense strand is also called sense and has the same base sequence as the mRNA (though T bases in DNA are substituted with U bases in RNA). For example:
(121) DNA strand 1: sense strand
(122) DNA strand 2: antisense strand (copied to).fwdarw.RNA strand (sense).
(123) Many forms of antisense have been developed and can be broadly categorized into enzyme-dependent antisense or steric blocking antisense. Enzyme-dependent antisense includes forms dependent on RNase H activity to degrade target mRNA, including single-stranded DNA, RNA, and phosphorothioate antisense. Double stranded RNA acts as enzyme-dependent antisense through the RNAi/siRNA pathway, involving target mRNA recognition through sense-antisense strand pairing followed by target mRNA degradation by the RNA-induced silencing complex (RISC). Steric blocking antisense (RNase-H independent antisense) interferes with gene expression or other mRNA-dependent cellular processes by binding to a target sequence of mRNA and getting in the way of other processes. Steric blocking antisense includes 2-O alkyl (usually in chimeras with RNase-H dependent antisense), peptide nucleic acid (PNA), locked nucleic acid (LNA) and Morpholino antisense. Cells can produce antisense RNA molecules naturally, which interact with complementary mRNA molecules and inhibit their expression.
(124) Antisense nucleic acid molecules have been used experimentally to bind to mRNA and prevent expression of specific genes. Antisense therapies are also in development; the FDA has approved a phosphorothioate antisense oligo, fomivirsen (Vitravene), for human therapeutic use.
(125) 15. Imaging Moieties
(126) In certain embodiments of the compositions of the present invention, the targeting ligand is an imaging moiety. As defined herein, an imaging moiety is a part of a molecule that is a agent or compound that can be administered to a subject, contacted with a tissue, or applied to a cell for the purpose of facilitating visualization of particular characteristics or aspects of the subject, tissue, or cell through the use of an imaging modality. Imaging modalities are discussed in greater detail below. Any imaging agent known to those of ordinary skill in the art is contemplated as an imaging moiety of the present invention. Thus, for example, in certain embodiments of compositions of the present invention, the compositions can be applied in multimodality imaging techniques. Dual imaging and multimodality imaging are discussed in greater detail in the specification below.
(127) In certain embodiments, the imaging moiety is a contrast media. Examples include CT contrast media, MRI contrast media, optical contrast media, ultrasound contrast media, or any other contrast media to be used in any other form of imaging modality known to those of ordinary skill in the art. Examples include diatrizoate (a CT contrast agent), a gadolinium chelate (an MRI contrast agent) and sodium fluorescein (an optical contrast media). Additional examples of contrast media are discussed in greater detail in the specification below. One of ordinary skill in the art would be familiar with the wide range of types of imaging agents that can be employed as imaging moieties in the chelators of the present invention.
E. Methods of Synthesis
(128) 1. Source of Reagents for the Compositions of the Present Invention
(129) Reagents for preparation of the compositions of the present invention can be obtained from any source. A wide range of sources are known to those of ordinary skill in the art. For example, the reagents can be obtained from commercial sources such as Sigma-Aldrich Chemical Company (Milwaukee, Wis.), from chemical synthesis, or from natural sources. For example, one vendor of radionuclides is Cambridge Isotope Laboratories (Andover, Mass.). The reagents may be isolated and purified using any technique known to those of ordinary skill in the art, as described herein. The free unbound metal ions can be removed with, for example, ion-exchange resin or by adding a transchelator (e.g., glucoheptonate, gluconate, glucarate, or acetylacetonate).
(130) 2. Use of an Intermediate Product as the Active Pharmaceutical Ingredient (API)
(131) Disulfide formation and nucleophilic attack of the anomeric center in the glucosamine moiety of certain compounds of the present invention can be problematic. For example, these unwanted reactions may occur at the thiol groups and/or the amino groups in EC-glucosamine (EC-G): these are the major side reactions that may cause the instability of EC-G. Furthermore, the typically low yield of the deprotection step with Na/NH.sub.3 to get the primary product of EC-G may yield low purity (see
(132) ##STR00008## ##STR00009##
(133) 3. Purification Procedures and Determinations of Purity
(134) As mentioned above, persons of ordinary skill in the art will be familiar with methods of purifying compounds of the present invention. As used herein, purification refers to any measurable increase in purity relative to the purity of the material before purification. Purification of every compound of the present invention is generally possible, including the purification of intermediates as well as purification of the final products. The purification step is not always included in the general methodologies explained below, but one of ordinary skill in the art will understand that compounds can generally be purified at any step. Examples of purification methods include gel filtration, size exclusion chromatography (also called gel filtration chromatography, gel permeation chromatography or molecular exclusion), dialysis, distillation, recrystallization, sublimation, derivatization, electrophoresis, silica gel column chromatography and high-performance liquid chromatography (HPLC), including normal-phase HPLC and reverse-phase HPLC. In certain embodiments, size exclusion chromatography and/or dialysis are specifically excluded as forms of purification of compounds of the present invention. Purification of compounds via silica gel column chromatography or HPLC, for example, offer the benefit of yielding desired compounds in very high purity, often higher than when compounds are purified via other methods. Radiochemical purity of compounds of the present invention can also be determined. Methods of determining radiochemical purity are well-known in the art and include chromatographic methods in conjunction with radioactivity detection methods (e.g., autoradiography analyses). Examples of comparisons of purity of compounds made via organic and wet methodologies and purified by varying methods are provided below.
(135) Methods of determining the purity of compounds are well known to those of skill in the art and include, in non-limiting examples, autoradiography, mass spectroscopy, melting point determination, ultra violet analysis, calorimetric analysis, (HPLC), thin-layer chromatography and nuclear magnetic resonance (NMR) analysis (including, but not limited to, 1H and 13C NMR). In some embodiments, a calorimetric method could be used to titrate the purity of a chelator or chelator-targeting ligand conjugate. For instance, generation of a thiol-benzyl adduct (that is, a thiol functional group protected by a benzyl group) or the performance of an oxidation reaction by using iodine could be used to determine the purity of chelator or chelator-targeting ligand conjugate. In one embodiment, the purity of an unknown compound may be determined by comparing it to a compound of known purity: this comparison may be in the form of a ratio whose measurement describes the purity of the unknown. Software available on varying instruments (e.g., spectrophotometers, HPLCs, NMRs) can aid one of skill in the art in making these determinations, as well as other means known to those of skill in the art.
(136) The following non-limiting parameters may be used, in certain embodiments, to determine the purity of compounds of the present invention:
(137) Column: Primesep100, 4.6150 mm, 5 m, ambient temperature
(138) Mobile phase (A): H.sub.2O with 0.025% TFA
(139) Mobile phase (B): acetonitrile with 0.025% TFA
(140) Isocratic run: A/B (50/50) at 1.0 ml/min
(141) Detection: ELSD, SEDEX75, 50C, 4.5 bar
(142) In certain embodiments of the present invention, purification of a compound does not remove all impurities. In some embodiments, such impurities can be identified.
(143) 4. Obtaining a Chelator
(144) Methods of preparing and obtaining chelators are well known to those of skill in the art. For example, chelators may be obtained from commercial sources, chemical synthesis, or natural sources.
(145) In one embodiment, the chelator may comprises ethylenedicysteine (EC). The preparation of ethylenedicysteine (EC) is described in U.S. Pat. No. 6,692,724. Briefly, EC may be prepared in a two-step synthesis according to the previously described methods (Ratner and Clarke, 1937; Blondeau et al., 1967; each incorporated herein by reference). The precursor, L-thiazolidine-4-carboxylic acid, was synthesized and then EC was then prepared. It is often also important to include an antioxidant in the composition to prevent oxidation of the ethylenedicysteine. The preferred antioxidant for use in conjunction with the present invention is vitamin C (ascorbic acid). However, it is contemplated that other antioxidants, such as tocopherol, pyridoxine, thiamine, or rutin may also be useful.
(146) Chelators may also comprise amino acids joined together by spacers. Such a spacer may comprise, as described above, an alkyl spacer such as ethylene.
(147) Amide bonds may also join one or more amino acids together to form a chelator. Examples of synthetic methods for the preparation of such chelators include solid-phase synthesis and solution-phase synthesis. Such methods are described, for example, in Bodansky, 1993 and Grant, 1992.
(148) 5. Organic Synthesis of Chelator-Targeting Ligand Conjugates
(149) In a preferred embodiment, the present invention further provides a method of organically synthesizing chelator-targeting ligand conjugates. The method includes obtaining, for example, a chelator such as ethylenedicysteine (EC) as described above and admixing the EC with a thiol protecting group in an organic medium in order to protect both free thiols, resulting in an SS-bis-protected-EC, which is then admixed with an amino protecting group in an organic/aqueous medium in order to protect both free amines, resulting in an SS-bis-protected-N,N-bis-protected-EC. Thiol groups are more reactive than nitrogen groups; thus, thiol groups are typically protected first. As described above, persons of skill in the art will be familiar with the proper ordering of the installation of protecting groups depending on the types of functional groups present on the chelator. This protected EC is then conjugated to a targeting ligand of any type described herein via any mode of conjugation described herein followed by removal of the thiol and amino protecting groups, which results in a chelator-targeting ligand conjugate.
(150) In certain embodiments, conjugation between a chelator and a targeting ligand takes place in one step. In particular embodiments, the conjugation comprises a covalent attachment of a chelator to a targeting ligand, wherein the covalent attachment occurs in one step. As mentioned, such one-step procedures are preferable as they minimize time, reagents, waste and loss of product.
(151) Chelator-targeting ligand conjugates synthesized by this method may next be chelated to a metal ion of any type described herein. Such methods of chelation are well known to those of ordinary skill in the art and are described herein. Examples of methods of chelation of metal ions to chelator-targeting ligand conjugates are described, for example, in U.S. Pat. No. 6,692,724. Methods described herein where a metal ion is chelated to a chelator may also serve as examples of how to chelate a metal ion to a chelator-targeting ligand conjugate.
(152) Benefits of synthesizing chelator-targeting ligand conjugates via methods of the present invention using organic synthesis include, for example, obtaining conjugates of high purity relative to conjugates obtained via aqueous synthesis, and the efficient synthesis and purification of small-molecule compounds (e.g., 1000 g/mol or less). These benefits allow for conjugates that can be utilized in imaging, diagnostic, and/or therapeutic experiments and/or clinical trials.
(153) 6. Organic Synthesis of Chelator-Targeting Ligand Conjugates Chelated to a Metal Ion
(154) In another preferred embodiment, the present invention further provides a method of organically synthesizing chelator-targeting ligand conjugates chelated to a metal ion for imaging, diagnostic, or therapeutic use. The method includes, for example, first obtaining a chelator, such as EC. EC may then admixed with a metal ion, which may be a radionuclide or any other metal ion as described herein, in an organic medium in order to chelate to the EC via an N.sub.2S.sub.2 chelate. See, e.g.,
(155) Benefits of synthesizing metal ion-labeled chelator-targeting ligand conjugates via methods of the present invention using organic synthesis include, for example, obtaining conjugates of high purity relative to conjugates obtained via aqueous synthesis, and the efficient synthesis and purification of small-molecule compounds (e.g., 1000 g/mol or less). These benefits allow for conjugates that can be utilized in imaging, diagnostic, and/or therapeutic experiments and/or clinical trials.
(156) 7. Aqueous Synthesis of Chelator-Targeting Ligand Conjugates
(157) The present invention further provides a method of synthesizing chelator-targeting ligand conjugates in an aqueous medium. Chelator-targeting ligand conjugates were prepared, in general, as a means of comparing the relative purity of such or similar products when synthesized in organic mediums. The method includes, for example, first obtaining a chelator, such as EC. EC is then dissolved in a basic aqueous solution and coupling agents of any type described herein are added. The targeting ligand is then added to this solution to generate the chelator-targeting ligand conjugate.
(158) 8. Aqueous Synthesis of Chelator-Targeting Ligand Conjugates Chelated to a Metal Ion
(159) The present invention further provides a method of synthesizing, in an aqueous medium, chelator-targeting ligand conjugates chelated to a metal ion. Like the aqueous synthesis mentioned above, chelator-targeting ligands conjugates chelated to a metal ion were prepared as a means of comparing the relative purity of such or similar products when synthesized in organic mediums. The method commences, in one embodiment, with obtaining a chelator chelated to a metal ion as described above (Organic Synthesis of Chelator-Targeting Ligand Conjugates Chelated to a Metal Ion). This chelator chelated to a metal ion may be, for example, chelated EC as described above. Chelation may occur by any method described herein. Chelated EC may be dissolved in a basic aqueous solution and coupling agents, as described herein, are added along with a targeting ligand of any type described herein in order to generate a chelator-targeting ligand conjugate chelated to a metal ion.
(160) 9. Conjugation of a Chelator with a Targeting Ligand
(161) The present invention contemplates methods for conjugating a targeting ligand to a chelator (optionally chelated to a metal ion). The targeting ligand may be of any type described herein. One of ordinary skill in the art will be familiar with the means of conjugating targeting ligands to various functional groups. Most commonly, as between the chelator and the targeting ligand, one acts as the nucleophile and one acts as the electrophile such that conjugation takes place via a covalent bond. Non-limiting examples of such covalent bonds include an amide bond, an ester bond, a thioester bond and a carbon-carbon bond. In preferred embodiments, the conjugation takes place via an amide or ester bond. In some embodiments, the conjugation takes place at one or more functional groups of the chelator selected from the group consisting of carboxylic acid, amine and thiol. When acting as electrophiles, chelators and targeting ligands may comprise functional groups such as halogens and sulfonyls which act as leaving groups during conjugation. Targeting ligands may also comprise nucleophilic groups, such as NH.sub.2, which may participate in conjugation with an electrophilic chelator.
(162) Coupling agents, as used herein, are reagents used to facilitate the coupling of a chelator to a targeting ligand. Such agents are well known to those of ordinary skill in the art and may be employed in certain embodiments of methods of the present invention. Examples of coupling agents include, but are not limited to, sulfo-N-hydroxysuccinimide (sulfo-NHS), dimethylaminopyridine (DMAP), diazabicyclo[5.4.0]undec-7-ene (DBU), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and dicyclohexylcarbodiimide (DCC). Other carbodiimides are also envisioned as coupling agents. Coupling agents are discussed, for example, in Bodansky, 1993 and Grant, 1992. These coupling agents may be used singly or in combination with each other or other agents to facilitate conjugation. Once the targeting ligand is conjugated using a coupling agent, urea is typically formed. The urea by-product may be removed by filtration. The conjugated product may then be purified by, for example, silica gel column chromatography or HPLC.
(163) In general, the ligands for use in conjunction with the present invention will possess functional groups that are able to conjugate to one or more functional groups of a chelator, such as EC. For example, a targeting ligand may possess a halogenated position that will react with a free amine of a chelator to form the conjugate. If functional groups are not available, or if an optimal functional group is not available, a desired ligand may still be conjugated to a chelator, such as EC, by adding a linker, such as ethylenediamine, amino propanol, diethylenetriamine, aspartic acid, polyaspartic acid, glutamic acid, polyglutamic acid, cysteine, glycine or lysine. For example, U.S. Pat. No. 6,737,247 discloses several linkers which may be used with the present invention and is hereby incorporated by reference in its entirety without disclaimer. U.S. Pat. No. 5,605,672 discloses several preferred backbones which may be used as linkers in the present invention and is hereby incorporated by reference in its entirety. In certain embodiments, the chelator may be conjugated to a linker, and the linker is conjugated to the targeting ligand. In other embodiments more than one linker may be used; for example, a chelator may be conjugated to a linker, and the linker is conjugated to a second linker, wherein the second linker is conjugated to the targeting ligand. In certain embodiments, two, three, four, or more linkers that are conjugated together may be used to conjugate a chelator and targeting ligand. However, it is generally preferable to only use a single linker to conjugate a chelator and a targeting ligand.
(164) Some chelators, such as EC, are water soluble. In some embodiments, the chelator-targeting ligand conjugate chelated to a metal ion of the invention is water soluble. Many of the targeting ligands used in conjunction with the present invention will be water soluble, or will form a water soluble compound when conjugated to the chelator. If the targeting ligand is not water soluble, however, a linker which will increase the solubility of the ligand may be used. Linkers may attach to, for example, an aliphatic or aromatic alcohol, amine, peptide or to a carboxylic acid. Linkers may be, for example, either poly amino acids (peptides) or amino acids such as glutamic acid, aspartic acid or lysine. Table 2 illustrates preferred linkers for specific drug functional groups.
(165) Benefits of synthesizing chelator-targeting ligand conjugates optionally chelated to one or more valent metal ions via methods of the present invention using organic synthesis include, for example, obtaining conjugates of high purity relative to conjugates obtained via aqueous synthesis, and the efficient synthesis and purification of small-molecule compounds (e.g., 1000 g/mol or less). These benefits allow for conjugates that can be utilized in imaging, diagnostic, and/or therapeutic experiments and/or clinical trials.
(166) TABLE-US-00002 TABLE 2 Linkers Drug Functional Group Linker Example Aliphatic or EC-poly(glutamic acid) estradiol, topotecan, phenolic-OH (MW 750-15,000) or EC paclitaxel, raloxifen poly(aspartic acid) (MW etoposide 2000-15,000) or bromo ethylacetate or EC-glutamic acid or EC-aspartic acid. Aliphatic or EC-poly(glutamic acid) doxorubicin, aromatic-NH.sub.2 (MW 750-15,000) or EC- mitomycin C, or peptide poly(aspartic acid) (MW endostatin, annexin V, 2000-15,000) or EC- LHRH, octreotide, glutamic acid (mono- or VIP diester) or EC-aspartic acid. Carboxylic acid or Ethylene diamine, lysine methotrexate, folic peptide acid
(167) 10. Chelation of a Metal Ion
(168) The present invention further contemplates methods for the chelation (also called coordination) of one or more metal ions to a chelator or a chelator-targeting ligand conjugate. Such chelation steps may take place in organic media. In other embodiments, chelation takes place in aqueous media. In certain embodiments, the chelator and the targeting ligand may each contribute to the chelation of the metal ion. In preferred embodiments, the metal ion is chelated only to the chelator. The chelated metal ion may be bound via, for example, an ionic bond, a covalent bond, or a coordinate covalent bond (also called a dative bond). Methods of such coordination are well known to those of ordinary skill in the art. In one embodiment, coordination may occur by admixing a metal ion into a solution containing a chelator. In another embodiment, coordination may occur by admixing a metal ion into a solution containing a chelator-targeting ligand conjugate. In one embodiment, chelation occurs to the chelator, with or without a targeting ligand, via an N.sub.2S.sub.2 chelate formed by the chelator, such as ethylenedicysteine (EC). The chelator and the targeting ligand may each be protected by one or more protecting groups before or after chelation with the metal ion.
(169) Chelation may occur at any atom or functional group of a chelator or targeting ligand that is available for chelation. The chelation may occur, for example, at one or more N, S, O or P atoms. Non-limiting examples of chelation groups include NS.sub.2, N.sub.2S, S.sub.4, N.sub.2S.sub.2, N.sub.3S and NS.sub.3, and O.sub.4. In preferred embodiments, a metal ion is chelated to three or four atoms. In some embodiments, the chelation occurs among one or more thiol, amine or carboxylic acid functional groups. The chelation, in particular embodiments, may be to a carboxyl moiety of glutamate, aspartate, an analog of glutamate, or an analog of aspartate. These embodiments may include multiple metal ions chelated to poly(glutamate) or poly(aspartate) chelators. In some embodiments, chelation of the metal ion is to a targeting ligand, such as to carboxyl groups of a tissue-specific ligand. In preferred embodiments, the chelation is between one or more thiol groups and one or more amine groups of the chelator.
(170) In some non-limiting examples, the metal ion may be technetium, indium, rhenium, gallium, copper, holmium, platinum, gadolinium, lutetium, yttrium, cobalt, calcium, arsenic, or any isotope thereof. Any metal ion described herein may be chelated to a compound of the present invention.
(171) 11. Reducing Agents
(172) For purposes of the present invention, when the metal ion is technetium it is preferred that the Tc be in the +4 oxidation state. The preferred reducing agent for use this purpose is stannous ion in the form of stannous chloride (SnCl.sub.2) to reduce the Tc to its +4 oxidation state. However, it is contemplated that other reducing agents, such as dithionate ion or ferrous ion may be useful in conjunction with the present invention. It is also contemplated that the reducing agent may be a solid phase reducing agent. The amount of reducing agent can be important as it is necessary to avoid the formation of a colloid. It is preferable, for example, to use from about 10 to about 100 g SnCl.sub.2 per about 100 to about 300 mCi of Tc pertechnetate. The most preferred amount is about 0.1 mg SnCl.sub.2 per about 200 mCi of Tc pertechnetate and about 2 mL saline. This typically produces enough Tc-EC-targeting ligand conjugate for use in 5 patients.
F. Examples of Imaging Modalities
(173) 1. Gamma Camera Imaging
(174) A variety of nuclear medicine techniques for imaging are known to those of ordinary skill in the art. Any of these techniques can be applied in the context of the imaging methods of the present invention to measure a signal from the reporter. For example, gamma camera imaging is contemplated as a method of imaging that can be utilized for measuring a signal derived from the reporter. One of ordinary skill in the art would be familiar with techniques for application of gamma camera imaging (see, e.g., Kundra et al, 2002, herein specifically incorporated by reference). In one embodiment, measuring a signal can involve use of gamma-camera imaging of a 111-In-octreotide-SSRT2A reporter system.
(175) 2. PET and SPECT
(176) Radionuclide imaging modalities (positron emission tomography (PET); single photon emission computed tomography (SPECT)) are diagnostic cross-sectional imaging techniques that map the location and concentration of radionuclide-labeled radiotracers. Although CT and MRI provide considerable anatomic information about the location and the extent of tumors, these imaging modalities cannot adequately differentiate invasive lesions from edema, radiation necrosis, grading or gliosis. PET and SPECT can be used to localize and characterize tumors by measuring metabolic activity.
(177) PET and SPECT provide information pertaining to information at the cellular level, such as cellular viability. In PET, a patient ingests or is injected with a slightly radioactive substance that emits positrons, which can be monitored as the substance moves through the body. In one common application, for instance, patients are given glucose with positron emitters attached, and their brains are monitored as they perform various tasks. Since the brain uses glucose as it works, a PET image shows where brain activity is high.
(178) Closely related to PET is single-photon emission computed tomography, or SPECT. The major difference between the two is that instead of a positron-emitting substance, SPECT uses a radioactive tracer that emits low-energy photons. SPECT is valuable for diagnosing coronary artery disease, and already some 2.5 million SPECT heart studies are done in the United States each year.
(179) PET radiopharmaceuticals for imaging are commonly labeled with positron-emitters such as .sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.82Rb, .sup.62Cu, and .sup.68Ga. SPECT radiopharmaceuticals are commonly labeled with positron emitters such as .sup.99mTc, .sup.201Tl, and .sup.67Ga. Regarding brain imaging, PET and SPECT radiopharmaceuticals are classified according to blood-brain-barrier permeability (BBB), cerebral perfusion and metabolism receptor-binding, and antigen-antibody binding (Saha et al., 1994). The blood-brain-barrier SPECT agents, such as .sup.99mTcO4-DTPA, .sup.201 Tl, and [.sup.67 Ga]citrate are excluded by normal brain cells, but enter into tumor cells because of altered BBB. SPECT perfusion agents such as [.sup.123I]IMP, [.sup.99mTc]HMPAO, [.sup.99mTc]ECD are lipophilic agents, and therefore diffuse into the normal brain. Important receptor-binding SPECT radiopharmaceuticals include [.sup.123I]QNE, [.sup.123I]IBZM, and [.sup.123I]iomazenil. These tracers bind to specific receptors, and are of importance in the evaluation of receptor-related diseases.
(180) 3. Computerized Tomography (CT)
(181) Computerized tomography (CT) is contemplated as an imaging modality in the context of the present invention. By taking a series of X-rays, sometimes more than a thousand, from various angles and then combining them with a computer, CT made it possible to build up a three-dimensional image of any part of the body. A computer is programmed to display two-dimensional slices from any angle and at any depth.
(182) In CT, intravenous injection of a radiopaque contrast agent can assist in the identification and delineation of soft tissue masses when initial CT scans are not diagnostic. Similarly, contrast agents aid in assessing the vascularity of a soft tissue or bone lesion. For example, the use of contrast agents may aid the delineation of the relationship of a tumor and adjacent vascular structures.
(183) CT contrast agents include, for example, iodinated contrast media. Examples of these agents include iothalamate, iohexyl, diatrizoate, iopamidol, ethiodol and iopanoate. Gadolinium agents have also been reported to be of use as a CT contrast agent (see, e.g., Henson et al., 2004). For example, gadopentate agents has been used as a CT contrast agent (discussed in Strunk and Schild, 2004).
(184) 4. Magnetic Resonance Imaging (MRI)
(185) Magnetic resonance imaging (MRI) is an imaging modality that is newer than CT that uses a high-strength magnet and radio-frequency signals to produce images. The most abundant molecular species in biological tissues is water. It is the quantum mechanical spin of the water proton nuclei that ultimately gives rise to the signal in imaging experiments. In MRI, the sample to be imaged is placed in a strong static magnetic field (1-12 Tesla) and the spins are excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. By collecting and analyzing these signals, it is possible to compute a three-dimensional image which, like a CT image, is normally displayed in two-dimensional slices.
(186) Contrast agents used in MR imaging differ from those used in other imaging techniques. Their purpose is to aid in distinguishing between tissue components with identical signal characteristics and to shorten the relaxation times (which will produce a stronger signal on T1-weighted spin-echo MR images and a less intense signal on T2-weighted images). Examples of MRI contrast agents include gadolinium chelates, manganese chelates, chromium chelates, and iron particles.
(187) Both CT and MRI provide anatomical information that aid in distinguishing tissue boundaries and vascular structure. Compared to CT, the disadvantages of MRI include lower patient tolerance, contraindications in pacemakers and certain other implanted metallic devices, and artifacts related to multiple causes, not the least of which is motion (Alberico et al, 2004). CT, on the other hand, is fast, well tolerated, and readily available but has lower contrast resolution than MRI and requires iodinated contrast and ionizing radiation (Alberico et al., 2004). A disadvantage of both CT and MRI is that neither imaging modality provides functional information at the cellular level. For example, neither modality provides information regarding cellular viability.
(188) 5. Optical Imaging
(189) Optical imaging is another imaging modality that has gained widespread acceptance in particular areas of medicine. Examples include optical labelling of cellular components, and angiography such as fluorescein angiography and indocyanine green angiography. Examples of optical imaging agents include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green derivative, rhodamine green, a derivative of rhodamine green, an eosin, an erythrosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, or dapoxyl dye.
(190) 6. Ultrasound
(191) Another biomedical imaging modality that has gained widespread acceptance is ultrasound. Ultrasound imaging has been used noninvasively to provide realtime cross-sectional and even three-dimensional images of soft tissue structures and blood flow information in the body. High-frequency sound waves and a computer to create images of blood vessels, tissues and organs.
(192) Ultrasound imaging of blood flow can be limited by a number of factors such as size and depth of the blood vessel. Ultrasonic contrast agents, a relatively recent development, include perfluorine and perfluorine analogs, which are designed to overcome these limitations by helping to enhance grey-scale images and Doppler signals.
(193) 7. Procedure for Dual Imaging
(194) Certain embodiments of the present invention pertain to methods of imaging a site within a subject using two imaging modalities that involve measuring a first signal and a second signal from the imaging moiety-chelator-metal ion complex. The first signal is derived from the metal ion and the second signal is derived from the imaging moiety. As set forth above, any imaging modality known to those of ordinary skill in the art can be applied in these embodiments of the present imaging methods.
(195) The imaging modalities are performed at any time during or after administration of the composition comprising the diagnostically effective amount of the composition of the present invention. For example, the imaging studies may be performed during administration of the dual imaging composition of the present invention, or at any time thereafter. In some embodiments, the first imaging modality is performed beginning concurrently with the administration of the dual imaging agent, or about 1 sec, 1 hour, 1 day, or any longer period of time following administration of the dual imaging agent, or at any time in between any of these stated times.
(196) The second imaging modality may be performed concurrently with the first imaging modality, or at any time following the first imaging modality. For example, the second imaging modality may be performed about 1 sec, about 1 hour, about 1 day, or any longer period of time following completion of the first imaging modality, or at any time in between any of these stated times. In certain embodiments of the present invention, the first and second imaging modalities are performed concurrently such that they begin at the same time following administration of the agent. One of ordinary skill in the art would be familiar with performance of the various imaging modalities contemplated by the present invention.
(197) In some embodiments of the present methods of dual imaging, the same imaging device is used to perform a first imaging modality and a second imaging modality. In other embodiments, a different imaging device is used to perform the second imaging modality. One of ordinary skill in the art would be familiar with the imaging devices that are available for performance of a first imaging modality and a second imaging modality, and the skilled artisan would be familiar with use of these devices to generate images.
G. Radiolabeled Agents
(198) As set forth above, certain embodiments of the compositions of the present invention include a metal ion chelated to a chelator as set forth above. In some embodiments, the metal ion is a radionuclide. Radiolabeled agents, compounds, and compositions provided by the present invention are provided having a suitable amount of radioactivity. For example, in forming .sup.99mTc radioactive complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 millicurie (mCi) to about 300 mCi per mL.
(199) Radiolabeled imaging agents provided by the present invention can be used for visualizing sites in a mammalian body. In accordance with this invention, the imaging agents are administered by any method known to those of ordinary skill in the art. For example, administration may be in a single unit injectable dose. Any of the common carriers known to those with skill in the art, such as sterile saline solution or plasma, may be utilized after radiolabeling for preparing the compounds of the present invention for injection. Generally, a unit dose to be administered has a radioactivity of about 0.01 mCi to about 300 mCi, preferably 10 mCi to about 200 mCi. The solution to be injected at unit dosage is from about 0.01 mL to about 10 mL.
(200) After intravenous administration of a diagnostically effective amount of a composition of the present invention, imaging can be performed. Imaging of a site within a subject, such as an organ or tumor can take place, if desired, in hours or even longer, after the radiolabeled reagent is introduced into a patient. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour. As set forth above, imaging may be performed using any method known to those of ordinary skill in the art. Examples include PET, SPECT, and gamma scintigraphy. In gamma scintigraphy, the radiolabel is a gamma-radiation emitting radionuclide and the radiotracer is located using a gamma-radiation detecting camera. The imaged site is detectable because the radiotracer is chosen either to localize at a pathological site (termed positive contrast) or, alternatively, the radiotracer is chosen specifically not to localize at such pathological sites (termed negative contrast).
H. Kits
(201) Certain embodiments of the present invention are generally concerned with kits for preparing an imaging or diagnostic agent. For example, in some embodiments the kit includes one or more sealed containers that contain a predetermined quantity of a chelator-targeting ligand conjugate. In some embodiments, the kit further includes a sealed container containing a metal ion. For example, the metal ion may be a radionuclide or a cold metal ion.
(202) A kit of the present invention may include a sealed vial containing a predetermined quantity of a chelator of the present invention and a sufficient amount of reducing agent to label the compound with a metal ion. In some embodiments of the present invention, the kit includes a metal ion that is a radionuclide. In certain further embodiments, the radionuclide is .sup.99mTc. In further embodiments of the present invention, the chelator is conjugated to a targeting ligand that can be any of those targeting ligands discussed elsewhere in this application.
(203) The kit may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like.
(204) In certain embodiments, an antioxidant is included in the composition to prevent oxidation of the chelator moiety. In certain embodiments, the antioxidant is vitamin C (ascorbic acid). However, it is contemplated that any other antioxidant known to those of ordinary skill in the art, such as tocopherol, pyridoxine, thiamine, or rutin, may also be used. The components of the kit may be in liquid, frozen, or dry form. In a preferred embodiment, kit components are provided in lyophilized form.
(205) The cold (that is, non-radioactivity containing) instant kit is considered to be a commercial product. The cold instant kit could serve a radiodiagnostic purpose by adding pertechnetate to vial with API and bulking agents (agents which have not been tested yet). The technology is known as the shake and shoot method to those of skill in the art. The preparation time of radiopharmaceuticals would be less than 15 min. The same kit could also encompass chelators or chelator-targeting ligand conjugates that could be chelated with different metals for different imaging applications. For instance, copper-61 (3.3 hrs half life) for PET; gadolinium for MRI. The cold kit itself could be used for prodrug purposes to treat disease. For example, the kit could be applied in tissue-specific targeted imaging and therapy.
I. Hyperproliferative Disease
(206) Certain aspects of the present invention pertain to compositions wherein a therapeutic moiety is conjugated to a chelator of the present invention. When a metal ion is chelated to a chelator or to both a chelator and its conjugated targeting ligand, the composition of the present invention may, in certain embodiments, be useful in dual imaging and therapy. In certain particular embodiments, the therapeutic moiety is a moiety that is an agent known or suspected to be of benefit in the treatment or prevention of hyperproliferative disease in a subject. The subject may be an animal, such as a mammal. In certain particular embodiments, the subject is a human.
(207) In other embodiments of the present invention, the metal ion is a therapeutic metal ion (e.g., Re-188, Re-187, Re-186, Ho-166, Y-90, Sr-89, and Sm-153), and the chelator-metal ion chelate is an agent that is a therapeutic agent (rather than an imaging agent) that can be applied in the treatment or prevention of a hyperproliferative disease.
(208) A hyperproliferative disease is herein defined as any disease associated with abnormal cell growth or abnormal cell turnover. For example, the hyperproliferative disease may be cancer. The term cancer as used herein is defined as an uncontrolled and progressive growth of cells in a tissue. A skilled artisan is aware other synonymous terms exist, such as neoplasm or malignancy or tumor. Any type of cancer is contemplated for treatment by the methods of the present invention. For example, the cancer may be breast cancer, lung cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia. In other embodiments of the present invention, the cancer is metastatic cancer.
J. Dual Chemotherapy and Radiation Therapy (Radiochemotherapy)
(209) In certain embodiments of the present invention, the compositions of the present invention are suitable for dual chemotherapy and radiation therapy (radiochemotherapy). For example, the chelator as set forth herein may be chelated to a metal ion that is a therapeutic metal ion, as well as a targeting ligand that is a therapeutic moiety (such as an anti-cancer moiety). As another example, a therapeutic metal ion may be chelated to both a chelator and its targeting ligand conjugate.
(210) For example, the metal ion may be a beta-emitter. As herein defined, a beta emitter is any agent that emits beta energy of any range. Examples of beta emitters include Re-188, Re-187, Re-186, Ho-166, Y-90, and Sn-153. One of ordinary skill in the art would be familiar with these agents for use in the treatment of hyperproliferative disease, such as cancer.
(211) One of ordinary skill in the art would be familiar with the design of chemotherapeutic protocols and radiation therapy protocols that can applied in the administration of the compounds of the present invention. As set forth below, these agents may be used in combination with other therapeutic modalities directed at treatment of a hyperproliferative disease, such as cancer. Furthermore, one of ordinary skill in the art would be familiar with selecting an appropriate dose for administration to the subject. The protocol may involve a single dose, or multiple doses. The patient would be monitored for toxicity and response to treatment using protocols familiar to those of ordinary skill in the art.
K. Pharmaceutical Preparations
(212) Pharmaceutical compositions of the present invention comprise a therapeutically or diagnostically effective amount of a composition of the present invention. The phrases pharmaceutical or pharmacologically acceptable or therapeutically effective or diagnostically effective refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of therapeutically effective or diagnostically effective compositions will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.
(213) As used herein, a composition comprising a therapeutically effective amount or a composition comprising a diagnostically effective amount includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the present compositions is contemplated.
(214) The compositions of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The compositions of the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art.
(215) The actual required amount of a composition of the present invention administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the tissue to be imaged, the type of disease being treated, previous or concurrent imaging or therapeutic interventions, idiopathy of the patient, and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
(216) In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of the chelator-metal ion chelate. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 0.1 mg/kg/body weight to about 1000 mg/kg/body weight or any amount within this range, or any amount greater than 1000 mg/kg/body weight per administration.
(217) In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including, but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
(218) The compositions of the present invention may be formulated in a free base, neutral or salt form. Pharmaceutically acceptable salts include the salts formed with the free carboxyl groups derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
(219) In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.
(220) Sterile injectable solutions may be prepared using techniques such as filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO (dimethylsulfoxide) as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
(221) The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
(222) In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
L. Combinational Therapy
(223) Certain aspects of the present invention pertain to compositions comprising a chelator that is conjugated to a targeting ligand that is a therapeutic moiety. In other embodiments, the chelator includes an amino acid sequence that is a therapeutic amino acid sequence.
(224) These compositions can be applied in the treatment of diseases, such as cancer and cardiovascular disease, along with another agent or therapy method. Treatment with these compositions of the present invention may precede or follow the other therapy method by intervals ranging from minutes to weeks. In embodiments where another agent is administered, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell. For example, it is contemplated that one may administer two, three, three or more doses of one agent substantially simultaneously (i.e., within less than about a minute) with the compositions of the present invention. In other aspects, a therapeutic agent or method may be administered within about 1 minute to about 48 hours or more prior to and/or after administering a therapeutic amount of a composition of the present invention, or prior to and/or after any amount of time not set forth herein. In certain other embodiments, a composition of the present invention may be administered within of from about 1 day to about 21 days prior to and/or after administering another therapeutic modality, such as surgery or gene therapy. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several weeks (e.g., about 1 to 8 weeks or more) lapse between the respective administrations.
(225) Various combinations may be employed, as demonstrated below, wherein a conjugate of the present invention is designated A and the secondary agent, which can be any other therapeutic agent or method, is B:
(226) TABLE-US-00003 A/B/AB/A/BB/B/AA/A/BA/B/BB/A/AA/B/B/B B/A/B/BB/B/B/AB/B/A/BA/A/B/BA/B/A/BA/B/B/A B/B/A/AB/A/B/AB/A/A/BA/A/A/BB/A/A/AA/B/A/A A/A/B/A
(227) Administration of the compositions of the present invention to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of these agents. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described agent. These therapies include but are not limited to additional pharmacotherapy (such as chemotherapy for cancer), additional radiotherapy, immunotherapy, gene therapy and surgery.
(228) 1. Chemotherapy
(229) Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.
(230) 2. Radiotherapy
(231) Other factors that cause DNA damage and have been used extensively include what are commonly known as -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. The terms contacted and exposed, when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
(232) 3. Immunotherapy
(233) Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.
(234) Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with gene therapy. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.
(235) 4. Genes
(236) In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic composition is administered before, after, or at the same time as the therapeutic agents of the present invention. Delivery of a therapeutic amount of a composition of the present invention in conjunction with a vector encoding a gene product will have a combined anti-hyperproliferative effect on target tissues.
(237) 5. Surgery
(238) Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
M. Other Embodiments of the Present Invention
(239) In one aspect, the present invention generally pertains to a method of synthesizing a chelator, such as EC, comprising at least three functional groups, the method comprising obtaining a chelator and either: (a) protecting at least one first functional group of the chelator with a first protecting agent to generate a firstly protected chelator; or (b) chelating the chelator to a metal ion to generate a metal ion-labeled chelator.
(240) Any method of synthesis as described herein, such as this, may take place in an organic medium, as described herein. The method may further comprise at least one purification step, as described herein. Chelators, functional groups, metal ions and modes of chelation and conjugation that may be used in the methods of the present invention are familiar to those of ordinary skill in the art and are described herein. The chelator may further comprise a spacer as described herein, such as ethylene. Such chelators are useful intermediates for the preparation of chelator-targeting ligand conjugates.
(241) In some embodiments, the method comprises protecting at least one first functional group of the chelator with a first protecting agent to generate a firstly protected chelator. In certain embodiments, the first functional group is a thiol functional group. In certain embodiments, the first protecting agent is a thiol protecting agent. In further embodiments, the thiol protecting agent is selected from a group consisting of an alkyl halide, a benzyl halide, a benzoyl halide, a sulfonyl halide, a triphenylmethyl halide, a methoxytriphenylmethyl halide and cysteine.
(242) The method may, in some embodiments, comprise protecting a second functional group with a second protecting agent to generate a secondly protected chelator. In certain embodiments, the first functional group comprises at least one thiol functional group and the second functional group comprises at least one amine functional group. In some embodiments, a thiol functional group is first protected with a thiol protecting agent and then an amine functional group is protected with an amine protecting agent. In further embodiments, an amine protecting agent is selected from the group consisting of benzylchloroformate, p-nitro-chlorobenzylformate, ethylchloroformate, di-tert-butyl-dicarbonate, triphenylmethyl chloride and methoxytriphenylmethyl chloride. An example of a chelator that may be prepared comprises ethylenedicysteine, wherein the two thiol groups of ethylenedicysteine are protected with two equivalents of a thiol protecting agent followed by protection of the two amine groups of ethylenedicysteine with two equivalents of an amine protecting agent. Since thiol groups are more reactive than amine groups, thiol groups will typically be protected before amine groups are protected.
(243) In other embodiments, the method further comprises removing one or more protecting groups from any composition described herein comprising one or more protecting groups. The protecting groups may be removed, for example, from the chelator moiety, the targeting ligand moiety, or both moieties in one or more steps before or after a chelator-targeting ligand conjugate is chelated to a metal ion, as described herein. Protecting groups are described in more detail herein, including their installation and removal.
(244) Any composition of the present invention may be purified via any method known to those of skill in the art. Methods of purification are described in more detail herein. In some embodiments, the firstly protected chelator is between about 90% and about 99.9% pure. In some embodiments, the secondly protected chelator is between about 90% and about 99.9% pure.
(245) In some embodiments, methods of the present invention further comprise conjugation of a chelator to a targeting ligand, wherein the targeting ligand and/or the chelator comprises at least one functional group to form a chelator-targeting ligand conjugate. In some embodiments, a functional group of the targeting ligand is protected by at least one protecting agent prior to conjugation to the chelator. In some embodiments, at least one functional group is a carboxylic acid functional group. In some embodiments, the functional groups of the chelator and the targeting ligand together form a chelate. Chelation of the metal ion to the chelator can be by any method known to those of ordinary skill in the art.
(246) A chelator-targeting ligand conjugate of the present invention may further comprise a linker between the chelator and the targeting ligand, as described herein. As mentioned, the targeting ligand may of any type known to those of skill in the art, and such ligands are discussed in more detail herein.
(247) Other general aspects of the present invention contemplate a method of synthesizing a metal ion labeled-chelator-targeting ligand conjugate, comprising: (a) obtaining a protected chelator comprising at least three functional groups protected by at least one protecting agent; (b) conjugating the protected chelator to a targeting ligand to generate a chelator-targeting ligand conjugate; (c) removing at least one protecting group from the chelator-targeting ligand conjugate; (d) chelating a metal ion to the chelator of the chelator-targeting ligand conjugate; and (e) removing any remaining protecting groups.
(248) The chelator, protecting agents, functional groups, mode of conjugation, targeting ligand, method of removing a protecting group, mode of chelation and metal ion may be that of any type described herein. The method may take place in an organic medium, as described herein. The method may comprise one or more purification steps, as described herein. In some embodiments, at least one functional group of the targeting ligand is protected by at least one protecting agent prior to conjugation. In preferred embodiments, three or four atoms of the chelator are available for chelation.
(249) Other general aspects of the present invention contemplate a method of synthesizing a metal ion labeled-chelator-targeting ligand conjugate comprising: (a) obtaining a chelator comprising at least three functional groups; (b) chelating a metal ion to the chelator to generate a metal ion labeled-chelator; (c) conjugating the metal ion labeled-chelator to a targeting ligand.
(250) The chelator, functional groups, mode of conjugation, targeting ligand, mode of chelation and metal ion may be that of any type described herein. The method may take place in an organic medium, as described herein. The method may comprise on or more purification steps, as described herein. In some embodiments, at least one functional group of the targeting ligand is protected by at least one protecting agent prior to conjugation. The method may further comprise the removal of all protecting groups from the metal ion labeled-chelator-targeting ligand conjugate. The method also contemplates, in certain embodiments, at least one functional group of the targeting ligand being protected by at least one protecting agent prior to conjugation.
(251) The present invention also contemplates kits for preparing an imaging agent, a chemotherapeutic agent, or a radio/chemotherapeutic agent, comprising one or more sealed containers, and a predetermined quantity of any composition as described herein in one or more of the sealed containers. The present invention also contemplates, in some embodiments, an imaging, chemotherapeutic, or radio/chemotherapeutic agent, comprising any composition as described herein.
(252) In some embodiments, the present invention contemplates a method of imaging or treating a subject, comprising administering to the subject a pharmaceutically effective amount of any composition as described herein. The subject may be a mammal, such as a human.
N. Methods of Diagnosis, Treatment, or Imaging in a Subject with Known or Suspected Heart Disease
(253) Embodiments of the present invention also generally pertain to methods of diagnosis, treatment, or imaging in a subject with known or suspected heart disease. The subject can be any subject, such as a mammal or avian species. The mammal, for example, may be a dog, cat, rat, mouse, or human. In preferred embodiments, the subject is a human with known or suspected cardiovascular disease.
(254) The cardiovascular disease can be any disease of the heart or of a blood vessel. The blood vessel may be a coronary vessel, or may be a vessel other than a coronary vessel. The vessel may be an artery, vein, arteriole, venule, or capillary.
(255) Examples of cardiovascular diseases include diseases of the heart, such as myocardial infarction, myocardial ischemia, angina pectoris, congestive heart failure, cardiomyopathy (congenital or acquired), arrhythmia, or valvular heart disease. In particular embodiments, the subject is known or suspected to have myocardial ischemia.
(256) The subject, for example, may be a patient who presents to a clinic with signs or symptoms suggestive of myocardial ischemia or myocardial infarction. Imaging of the heart of the subject to diagnose disease may involve administering to the subject a pharmaceutically effective amount of a metal ion labeled chelator-targeting ligand conjugate synthesized using any of the methods set forth herein. Imaging can be performed using any imaging modality known to those of ordinary skill in the art. In particular embodiments, imaging involves use radionuclide-based imaging technology, such as PET or SPECT. In particular embodiments, the metal ion-labeled radionuclide-targeting ligand conjugate is 99m-Tc-EC-glucosamine. Glucosamine is actively taken up by viable myocardial tissue. Areas of ischemic myocardium would take up less or no conjugate. Severity of ischemia can be visually assessed or graded depending on magnitude of the signal that is measured using any method known to those of ordinary skill in the art. In some embodiments, imaging using any of the conjugates set forth herein is performed before, during, or after imaging of the heart using a second imaging modality. For example, the second imaging modality may be thallium scintigraphy.
(257) Myocardial Perfusion SPECT (MPS) consist of a combination of a stress modality (exercise or pharmacologic) with rest and stress administration and imaging of radiopharmaceuticals. Thallium has excellent physiologic properties for myocardial perfusion imaging. Being highly extracted during the first pass through the coronary circulation, a linear relationship between blood flow to viable myocardium and thallium uptake has been shown during exercise; however, at very high levels of flow, a roll-off in uptake occurs. As an unbound potassium analogue, thallium redistributes over time. Its initial distribution is proportional to regional myocardial perfusion and at equilibrium, the distribution of thallium is proportional to the regional potassium pool, reflecting viable myocardium. The mechanisms of thallium redistribution are differential washout rates between hypoperfused but viable myocardium and normal zones and wash-in to initially hypoperfused zones. The washout rate of thallium is the concentration gradient between the myocardial cell and the blood. There is slower blood clearance of thallium following resting or low-level exercise injection. Diffuse slow washout rates, mimicking diffuse ischemia, may be observed in normal patients who do not achieve adequate levels of stress. Hyperinsulinemic states slow redistribution, leading to an underestimation of viable myocardium; thus fasting is recommended prior to and for 4 hrs following thallium injection. This is why if EC-G is used as an viable agent in combination with thallium it will target the precise area of interest which would be the viable area (Angello et al., 1987; Gutman et al., 1983; Pohost et al., 1977).
(258) Imaging using any of the metal ion-labeled chelator-targeting ligand conjugates of the present invention may also be performed in conjunction with other diagnostic methods, such as measurement of cardiac isozymes, or cardiac catheterization. The imaging may be performed at various intervals following onset of symptoms, or can be performed to assess for changes in myocardial perfusion over time.
O. Examples
(259) The following examples are included to demonstrate certain non-limiting aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
(260) The following figures, chemical structures and synthetic details provide certain compounds of the present invention.
Example 1
(261) Non-Limiting Example of an Organic Synthesis of N,N-Ethylenedicysteine-Glucosamine (EC-G). See
Step 1: Synthesis of S,S-Bis-benzyl-N,N-ethylenedicysteine (Bz-EC)
(262) Cysteine-HCl (30 g) was dissolved in water (100 mL). To this, 37% formaldehyde (22.3 mL) was added and the reaction mixture was stirred overnight at room temperature. Pyridine (25 mL) was then added and a precipitate formed. The crystals were separated and washed with ethanol (50 mL), then filtered with a Buchner funnel. The crystals were triturated with petroleum ether (150 mL), again filtered and dried. The precursor, L-thiazolidine-4-carboxylic acid (m.p. 195 C., reported 196-197 C.) weighed 23.408 g. The precursor (22 g) was dissolved in liquid ammonia (200 mL) and refluxed. Sodium metal was added until a persistent blue color appeared for 15 min. Ammonium chloride was added to the blue solution, the solvents were evaporated to dryness. The residue was dissolved in water (200 mL) and the pH was adjusted to 2 with concentrated HCl. A precipitate was formed, filtered and washed with water (500 mL). The solid was dried in a calcium chloride dessicator. EC was then prepared 10.7 g (m.p. 237 C., reported 251-253 C.). The structure of EC was confirmed by H-1 and C-13 NMR. EC (2.684 g, 10 mmol) was dissolved in 1N NaOH (40 mL). Benzyl chloride (5.063 g, 40 mmol) was dissolved in dioxane (30 mL) and stirred. The reaction was stirred for 30 min. The pH of the solution was adjusted to 2 with concentrated HCl. The precipitate was filtered and washed with water and recrystallized from trifluoroacetic acid, yielding 79.0% (3.5454 g), m.p. 227-229 C. (dec.) (reported 229-230 C.). The structure of Bz-EC was confirmed by H-1 and C-13 NMR.
Step 2: Synthesis of S,S-Bis-benzyl-N,N-bis-CBZ ethylenedicysteine (Cbz-Bz-EC)
(263) Bz-EC (2.243 g, 5 mmol) was dissolved in sodium carbonate (1.20 g, 11.2 mmol) solution and the pH was adjusted to 10 using 1N NaOH. The final aqueous volume was 30 mL. Benzyl chloroformate (233 mL, 16.5 mmol) was dissolved in dioxane (0.75 mL) and stirred. The pH was adjusted to 10 by adding solid Na.sub.2CO.sub.3. The reaction mixture was stirred for 2 hours and extracted with diethyl ether to remove the excess benzyl chloroformate (CBZ). The pH of the aqueous layer was adjusted to 2 with 1N HCl and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated. The residue was chromatographed on a silica gel column column eluted with CH.sub.2Cl.sub.2:acetic acid (99:1) to CH.sub.2Cl.sub.2:methanol:acetic acid (94:5:1) to yield the desired product 87.2% (3.127 g). The structure of Cbz-Bz-EC was confirmed by H-1 and C-13 NMR.
Step 3: Synthesis of S,S-Bis-benzyl-N,N-bis-CBZ ethylenedicysteine-glucosamine (tetra acetate) Conjugate (Cbz-Bz-EC-G-4-Ac)
(264) To a stirred flask of dichloromethane (22 mL), Cbz-Bz-EC (2.1467 g, 3 mmol) was added. This was followed by dicyclohexylcarbodiimide (DCC) (2.476 g, 12 mmol) and dimethylaminopyridine (1.466 g, 12 mmol). Tetraacetylated glucosamine hydrochloride (2.533 g, 6.6 mmol) (4-Ac-G-HCl) (Oakwood Products Inc., West Columbia, S.C.) was added to the mixture and stirred until completely dissolved. The structure of 4-Ac-G-HCl was confirmed by H-1 and C-13 NMR. The reaction was stirred at room temperature overnight. Water (0.5 mL) was added and the solid was filtered. The filtrate was dried over magnesium sulfate and the solvent was evaporated. The product was purified by silica gel column chromatography using dichloromethane:methanol:acetic acid (9.9:0:0.1) to 56.4:3:0.6 as a mobile phase. The product was isolated 66.4% yield (2.7382 g). H-1 and C-13 NMR of Cbz-Bz-EC-G-4-Ac provided confirmation as well as mass spectrometry.
Step 4: Synthesis of N,N-ethylenedicysteine-glucosamine (EC-G)
(265) Cbz-Bz-EC-G-4-Ac (687.7 mg, 0.5 mmol) was dissolved in liquid ammonia (20 mL) and pieces of sodium (223 mg, 10 mmol) were added. After adding all of the sodium, the reaction mixture sustained a dark blue color for 20 minutes. Ammonium chloride (641.9 mg, 12 mmol) was added slowly and the dark blue color solution turned colorless. The liquid ammonia was removed by nitrogen. The residual solid was dissolved in water and dialyzed overnight using MW<500. The crude product weighed 206.7 mg (yield: 70%). H-1 and C-13 NMR of the crude EC-G bis-acetylated compound were obtained along with mass spectra. The molecular ion was 861 which contains the matrix 187 and parent ion 674 (EC-G bis-acetylated). The major ion (100%) was 656 which was from the loss of water. EC-G bis-acetylated compound (200 mg) was further purified by dissolving in sodium carbonate and stirring for 2 hours. The product, EC-G, was then lyophilized, yielding a weight of 70 mg. H-1 NMR and C-13 NMR of EC-G were then obtained. C-13 NMR of EC-G showed 16 major carbon peaks. The mass spectra of EC-G was difficult to obtain due to its hydrophilicity and its tendency to be retained on the mass spectrometry column. However, EC-G bis-acetylated compound is less hydrophilic than EC-G; thus, mass spectra of EC-G bis-acetylated could be obtained. Mass spectra of EC-G showed that there was small impurity from EC-G bis-acetylated compound resulting from incomplete hydrolysis procedure. H-1 and C-13 NMR of EC-G were close to the predicted values of EC-G. Although 10 carbon peaks are expected for the symmetric structure of EC-G, glucosamine has 12 carbons instead of 6 carbons, suggesting that glucosamine has two configurations. H-1 NMR experimental values appeared to have a somewhat different profile than the predicted values; however, C-13 NMR experimental values of glucosamine were close to the predicted values of glucosamine. Thus, EC-G appears to have two configurations.
Example 2
Non-Limiting Example of an Organic Synthesis of .SUP.187.Re-EC-G Using Re-EC and Protected Glucosamine See FIG. 2
(266) .sup.187Re-EC-G was used as a reference standard for .sup.99mTc-EC due to the similarity in structure and lipophilicity. Synthesis of cold Re-EC-G is shown in
(267) To a stirred dimethylformamide (4 mL) solvent, Re-EC (116.9 mg, 0.25 mmol) was added followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (150 mL, 1.0 mmol). Next, dicyclohexylcarbodiimide (DCC) (123.8 mg, 0.6 mmol) was added. The reaction mixture was stirred for 1 hour. Tetraacetylated glucosamine (4-Ac-G-HCl) (184.9 mg, 0.5 mmol) was added and then the reaction was stirred at room temperature overnight. Water (1 mL) was added and the reaction stirred for additional 1 hour at room temperature. The reaction mixture was evaporated under reduced pressure. Water (5 mL) was added, followed by chloroform (5 mL). The water layer was separated and lyophilized to yield a crude dark-brown solid. The solid was purified by column chromatography using Sephadex G-50 to yield cold Re-EC-G (128.4 mg, 65% yield). The structure of cold Re-EC-G was confirmed by H-1 and C-13 NMR and mass spectrometry. Again, the Re-complex distinctively shows two parent ions with 40:60 ratios.
(268) Elemental analysis of cold Re-EC-G showed C.sub.20H.sub.35N.sub.4O.sub.13ReS.sub.2 (C, H, N) with the calculated value C, 30.41; H, 4.47; N, 7.09; found value C, 30.04; H, 4.93; N, 6.09. H-1 and C-13 NMR of cold Re-EC-G was similar to the predicted NMR spectrometry. EC-G (5 mg) was labeled with .sup.99mTc (pertechnetate) (1 mCi) in the presence of tin(II) chloride (0.1 mg). HPLC analysis showed that cold Re-EC-G had a similar retention time to that of .sup.99mTc-EC-G.
Example 3
Synthesis of EC-G Using EC and Glucosamine in an Aqueous Reaction
(269) EC (107 mg, 0.4 mmol) was dissolved in NaHCO.sub.3 (1N, 12 mL). To this colorless solution, sulfo-N-hydroxysuccinimide (sulfo-NHS, 173.7 mg, 0.8 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDAC) (Aldrich Chemical Co, Milwaukee, Wis.) (153.4 mg, 0.8 mmol) were added. D-Glucosamine hydrochloride salt (Sigma Chemical Co., St Louis, Mo.) (345 mg, 1.6 mmol) was then added. A pH of 8 was measured. The mixture was stirred at room temperature for 16 hours and then dialyzed for 24 hours using Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was filtered by a 0.45 m Nylon-filter and then freeze-dried using a lyophilizer (Labconco, Kansas City, Mo.). The crude product weighed 300-400 mg. H-1 NMR of EC-G showed similar patterns; however, it appears that the mixture is not as pure when compared to the organic EC-G. Elemental analysis showed EC-G purity was 63-77% using different reaction ratios between EC and glucosamine. Prep-HPLC (7.8300 mm C-18 column, Waters) (flow rate: 0.5 mL/min, 100% water, UV 235 nm) was used to purify the crude product, 180-240 mg (yield 60%). H-1 and C-13 NMR of EC-G after prep-HPLC showed additional peaks suggesting impurities from mono EC-G or EC-glucosamine, sulfo-NHS and EDAC. Prep-HPLC purification of the raw EC-G yielded some incremental improvement to the chemical purity; however, when the raw EC-G is labeled with .sup.99mTc in the presence of tin(II) chloride, a greater than 95% radiochemical purity of .sup.99mTc-EC-G can be achieved using gluconate as a transchelator (as shown in radio-TLC and HPLC analysis).
Example 4
Cellular Uptake Study Comparing Products Synthesized Via the Aqueous Method and the Organic Method
(270) To further validate EC-G biological activity, in vitro cell culture assays were performed. Briefly, the cellular uptake was determined in tumor cells (50,000 cells/well) incubated with .sup.99mTc-EC-G (2 Ci/well) at various time intervals. The cellular uptake assay showed no marked difference between raw (unpurified) EC-G and prep-HPLC purified EC-G (
Example 5
Synthesis of Cold Re-EC-G Using Re-EC and Glucosamine in an Aqueous Reaction
(271) Re-EC (255.8 mg, 0.5 mmol) (from Example 2) was dissolved in NaOH (1N, 4.5 mL). Added to this dark-purple color solution were sulfo-NHS (217.1 mg, 1 mmol) and D-glucosamine hydrochloride salt (Sigma Chemical Co., St. Louis, Mo.) (431.3 mg, 2 mmol). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDAC) (Aldrich Chemical Co., Milwaukee, Wis.) (191.7 mg, 1 mmol) was then added. The pH measured greater than 8. The mixture was stirred at room temperature for 16 hours. The mixture was dialyzed for 24 hours using Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, the product was filtered and then freeze-dried using a lyophilizer (Labconco, Kansas City, Mo.). The crude product weighed 276 mg. H-1 NMR of aqueous Re-EC-G showed a similar pattern; however, there appears to be some evidence of impurities when compared to the organic Re-EC-G compound. HPLC analysis of the organic cold Re-EC-G compound showed one peak at 272 nm; however, aqueous cold Re-EC-G had two peaks. One of the peaks in the aqueous cold Re-EC-G corresponds to the organic cold Re-EC-G compound (peaks 12.216 and 12.375, respectively). The remaining peaks were sulfo-NHS and other minor impurities.
Example 6
Quantitative Analysis of Glucosamine (Active Pharmaceutical Ingredient)
(272) D-Glucosamine was derivatized for colorimetric assays. Briefly, to a solution of D-glucosamine hydrochloride (25 g, 0.12 mol) in a freshly prepared aqueous solution of 1N NaOH (120 mL) under stirring was added p-anisaldehyde (17 mL, 0.14 mol). After 30 min., crystallization began and the mixture was refrigerated overnight. The precipitated product was then filtered and washed with cold water (60 mL), followed by a mixture of EtOH-Et.sub.2O (1:1) to give 2-deoxy-2-[p-methoxybenzylidene(amino)]-D-glucopyranose (D-glucosamine-anisaldehyde, 32.9408 g, 110.8 mmol, 95.5% yield) m.p. 165-166 C. H-1 NMR confirmed the structure.
(273) Raw EC-G (50 mg) was hydrolyzed using 1N NaOH. Anisaldehyde was added to the reaction solution. After 2 hours, the reaction mixture was extracted with chloroform. The chloroform layer, which contained unreacted anisaldehyde, was evaporated under nitrogen. The reacted anisaldehyde weight was used to determine the amount of glucosamine in the D-glucosamine-anisaldehyde adduct.
Example 7
(274) Quantitative Analysis of EC in EC-G
(275) Raw EC-G (50 mg) was hydrolyzed using 1N NaOH. Benzyl chloride was dissolved in dioxane (30 mL) and then added in to the stirred mixture. The reaction was stirred for 2 hours and then extracted with chloroform. The chloroform layer, which contained unreacted benzyl chloride, was evaporated under nitrogen. The reacted benzyl chloride weight was used to determine the amount of EC in EC-G (Table 3).
Example 8
Quantitative Analysis of Sulfo-NHS and EDAC in EC-G
(276) A standard curve of sulfo-NHS was generated at UV 272 nm. Raw EC-G was dissolved in water. From the standard curve, the amount of sulfo-NHS in EC-G was determined at UV 272 nm. The amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDAC) was calculated by subtracting EC, glucosamine and sulfo-NHS from total EC-G weight shown in Table 3.
Example 9
Quantitative Analysis of Glucose Phosphorylation Assay
(277) An in vitro hexokinase assay was used to assess the glucose phosphorylation process of EC-G. Using a kit (Sigma Chemical Company, MO), fluorodeoxyglucose (FDG, 1.0 mg), EC-G (1.0 mg), D-glucosamine (G, 1.0 mg) and D-glucose (2.5 mg) were dissolved in 1 mL (EC-G, G) or 2.5 mL (D-glucose) of water. From there, 200 L was removed and diluted in 2.5 mL of water. A 100 L aliquot was then removed and combined in solution with 900 L of Infinity Glucose Reagent and incubated at 37 C. for three min. The phosphorylated glucose and NADH were then assayed at a wavelength of 340 nm. The peaks of FDG (340 and 347 nm), glucose (301 and 342 nm), EC-G (303 and 342 nm) and G (302 and 342 nm) were obtained.
Example 10
Chemical Identity Assay of Glucosamine (Active Pharmaceutical Ingredient) in EC-G (Synthesized from the Aqueous Reaction)
(278) A colorimetric assay was used to determine the amount of glucosamine. A solution of copper sulfate (6.93 g in 100 mL water) and sodium potassium tartrate (34.6 g in 100 mL water containing 10 g NaOH) was prepared. EC-G (25 mg) and glucosamine (standard) were added with basic copper tartrate solution until no visualization of copper oxide red precipitate existed. The amount of glucosamine in EC-G was 8.7 mg (35% w/w) determined from titration volume (Table 3).
(279) Alternatively, as described in Example 5, D-glucosamine hydrochloride (25 g, 0.12 mol) was added to a freshly prepared aqueous solution of 1N NaOH (120 mL) under stirring and then p-anisaldehyde (17 mL, 0.14 mol) was added to the mixture. After 30 min., the crystallization began and the mixture was refrigerated overnight. The precipitated product was filtered and washed with cold water (60 mL), followed by a mixture of EtOH-Et.sub.2O (1:1) to yield 2-deoxy-2-[p-methoxybenzylidene(amino)]-D-glucopyranose (D-glucosamine-anisaldehyde, 32.9408 g, 110.8 mmol, 95.5% yield) m.p. 165-166 C. Raw EC-G (50 mg) was hydrolyzed using 1N NaOH. Anisaldehyde was added to the reaction solution. After 30 min., the crystallization began and the mixture was refrigerated overnight. The precipitated product was filtered and washed with cold water and the melting point was determined to be 165-166 C. (containing 18 mg glucosamine).
(280) TABLE-US-00004 TABLE 3 Qualitative Analysis of Glucosamine and EC in EC-G (synthesized from the aqueous reaction) Theoretical Value Percentage (weight/weight) Compound Molecular Weight (100%) (65%) EC-G 591 EC 268 Glucosamine (G) 179 EC in EC-G 39% (234/591) 25% G in EC-G 60% (356/591) 39% Experimental Value Percentage Compound (weight/weight) Method EC in EC-G 30% colorimetric G in EC-G 35% colorimetric Sulfo-NHS in EC-G 34% UV (268 nm) EDAC 1% calculation
Example 11
Chemical Identity Assay of Ethylenedicysteine (Chelator) in EC-G (Synthesized from the Aqueous Reaction)
(281) Two methods were used to determine the purity of EC-G. In the first method, a colorimetric assay was used to determine the amount of EC. A solution of iodine (0.1 mol/L) (13 g along with 36 g KI in 1000 mL water) was prepared and EC-G (25.2 mg) and EC (25 mg) (standard) were added to the iodine solution. In the standard EC, a pale white solid was precipitated, but no precipitate was noted in the EC-G. A titration method was used (yellowish color (persists more than 5 min.)) to determine the amount of EC in the EC-G. Each 1 mL of iodine solution that was used equals 13.4 mg of EC. The amount of EC in the EC-G was 7.6 mg (30.2% w/w).
(282) In the second method, measurement the melting point of a thiol-EC-G adduct was performed. Example 1 outlined the synthesis of S,S-Bis-benzyl-N,N-ethylenedicysteine (Bz-EC). Briefly, EC (2.684 g, 10 mmol) was dissolved in 1N NaOH (40 mL). Benzyl chloride (5.063 g, 40 mmol) was dissolved in dioxane (30 mL) and added to a stirred mixture. After 30 min., the pH of the solution was adjusted to 2 with concentrated HCl. The precipitate was filtered and washed with water and recrystallized from trifluoroacetic acid. The yield was 79.0% (3.5454 g), m.p. 227-229 C. (dec.) (reported 229-230 C.). Raw EC-G (50 mg) was then hydrolyzed using 1N NaOH, and benzyl chloride (40 mg) was added. The reaction mixture was stirred for 30 min. The pH of the solution was adjusted to 2 with concentrated HCl. The precipitate was filtered and washed with water to give EC-benzyl adduct, m.p. 227-229 C. (containing EC 16 mg).
Example 12
Chemical Identity Assay of Sulfo-N-Hydroxysuccinimide (Sulfo-NHS) in EC-G (Synthesized from the Aqueous Reaction)
(283) The assay for N-hydroxysulfosuccinimide (sulfo-NHS) was determined by UV (268 nm). A standard curve of sulfo-NHS was produced at UV 268 nm. Under this UV absorbance, poor absorbance was observed for EC-G and EDAC. Raw EC-G (50 g/mL) was dissolved in water and the absorbance was measured at 268 nm. The estimated sulfo-NHS was 355% (w/w).
Example 13
Radiochemical Purity and Identity Assay
(284) Thin-layered chromatography (TLC) and high performance liquid chromatography (HPLC) were used to determine radiochemical identity. For the TLC assay, both aqueous and organic synthesized EC-G were labeled with .sup.99mTc and spotted on a TLC strip impregnated with silica gel column (ITLC-SG) and scanned using a radio-TLC scanner. The retention factor (Rf) values of .sup.99mTc-EC-G (from the aqueous synthesis) and the reference standard (.sup.99mTc-EC-G from the organic synthesis) were 0.8 (determined by ammonium acetate (1M):methanol; 4:1) or saline. For the HPLC assay, the chemical purity of the organic and aqueous synthesized EC-G were 95.64% and 90.52%, respectively. EC-G synthesized from the organic reaction was more pure than EC-G synthesized from the aqueous reaction. Both the organic and aqueous synthesized EC-G were labeled with .sup.99mTc and loaded (20 L, 1 mg/mL EC-G) on a C-18 reverse phase column (Waters, semi-prep, 7.8300 mm). The retention time (Rt) values of .sup.99mTc-EC-G and cold Re-EC-G (the reference standard from the organic synthesis) were between 11.7-13.5 min. (determined by 100% water @ 0.5 mL/min, UV at 210 nm). Both the organic and aqueous synthesized .sup.99mTc-EC-G were detected by UV wavelength (210 nm) and the matched radioactive detector findings were within the above stated ranges. In vitro cell culture assays showed that Re-EC-G produced a dose response curve (
(285) Summary:
(286) The radiochemical purity of the .sup.99mTc-EC-G measured by HPLC and TLC is greater than 95% for the aqueous synthesized EC-G, which closely approximates the radiochemical purity for the organic synthesized EC-G. The chemical purity of the unlabeled aqueous EC-G measured by colorimetric and elemental analysis falls in the range of 60-70%. All impurities contained in the EC-G compound (whether the aqueous or organic synthesis) have been clearly identified through calorimetric assays and UV spectrometry as glucosamine (35%), EC (30%), sulfo-NHS (34%) and EDAC (1%) on a % w/w basis. When measured by HPLC at UV 210 nm, the chemical purity of the unlabeled aqueous EC-G compares very favorably to the unlabeled organic EC-G at 90.52% vs. 95.64%, respectively.
(287) Retention time of the aqueous .sup.99mTc-EC-G is in the range of cold Re-EC-G measured by HPLC at 272 nm. NMR (.sup.1H, .sup.13C) of aqueous EC-G is in the range of cold Re-EC-G. Unlabeled organic EC-G, labeled organic EC-G and cold Re-EC-G are used as reference standards. Biologic assays (in vitro uptake and in vivo imaging) showed no marked difference between aqueous and organic synthesized EC-G.
Example 14
Purity Analysis of .SUP.68.Ga-EC-G
(288) .sup.68Ga-EC-G synthesized by both organic and aqueous means were analyzed via radio-TLC.
Example 15
Stability Analysis of .SUP.68.Ga-EC-G
(289)
(290)
(291) The protein binding rate increased from 18.6% to 51.5% after 2 hrs, suggesting the targeting potential of .sup.68Ga-EC-G.
Example 16
In Vitro Update Study of 68Ga-Labeled Compounds in Breast Cancer Cell Line 13762
(292)
Example 17
Imaging of Cardiovascular Disease
(293)
Example 18
Non-Limiting Example of Organic Synthesis of Ec-G Via an EC-Benzhydrol-Cbz-Glucosamine Intermediate (see FIG. 13)
(294) EC-Benzhydrol-Cbz-Glucosamine can be dissolved in ethyl acetate and precipitated out by adding MTBE or n-Hexane. This was envisioned as a method of obtaining pure a penultimate species in a method of obtaining EC-G. The purity (HPLC) of EC-Benzhydrol-Cbz-Glucosamine before this trituration treatment was about 64%. After trituration, the purity was about 68% (MTBE) or 65%-80% (n-Hexane). Another envisioned method for purifying the product is through use of a biotage cartridge, as since the silica gel in these cartridges is more active than flash grade silica gel.
(295) Other purification techniques and procedures were also attempted using different solvent systems as an alternative to chromatography, the results of which are shown in Table 4 below. Precipitation was attempted in different solvent systems. The EC-Benzhydrol-Cbz was dissolved in a selected solvent (A), and slowly charged to a larger volume of co-solvent (B). However, the results did not indicate this approach would be as effective as other methods, as the purity changes were negligible. Triturations were also attempted using the selected solvent systems in various ratios for precipitation. The results for the triturations also suggest the material is not pure enough for certain applications. Column chromatography was also attempted, and the conditions were modified from the previous week (15:1 silica:crude, loaded dry on silica). This method did allow for moderate clean up of the material (from 55A % to 75A %).
(296) TABLE-US-00005 TABLE 4 EC-Benzyhydrol-Cbz Purification by Precipitation and Trituration Solvent A Solvent B Precipitation Result Trituration Result Ethyl Acetate Hexane Sticky solid Oil Methanol Water Sticky oil Oil DCM Hexane Sticky oil Oil Ethanol Water Sticky oil Oil
(297) All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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