Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
2000-05-17
2003-08-05
Ceperley, Mary E. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C424S001530, C435S007940, C435S188000, C436S074000, C436S529000, C436S530000, C436S804000, C530S391100, C530S391500
Reexamination Certificate
active
06602989
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the production of binary and ternary protein-metal complexes and their utility as diagnostic and therapeutic agents and, more specifically, to the formation of protein conjugate-gallium(III) binary complexes and protein conjugate-gallium(III)-enzyme conjugate ternary complexes to provide in vitro or in vivo quantitative determination of gallium ion in body fluids and a means to transport therapeutic dosages of radioisotopes of gallium to localized, diseased body sites.
DEFINITIONS AND ABBREVIATIONS
To facilitate the understanding of this specification and the appended claims, definitions to various words and phrases are provided.
Binary Complex is a metal ion chelated with one ligand.
Chelators are part of a proteinaceous conjugate and are able to donate electrons and combine by coordinate bonding with a metal ion to form complexes.
Conjugate is an organic polymer, specifically a proteinaceous material having an exogenous ligand or chelating functionality covalently bound to the polymer.
Exogenous chelating group is a chelating group that is not present in the native protein molecule.
Ligand is an organic compound that has one or more sites to covalently bond to a metal ion (i.e., bidentate: two sites per ligand; tridentate: three sites per ligand).
Radioisotopes are radionuclide metal ions having a known half-life.
Ternary Complex is a complex composed of a metal ion chelated with two ligands, which may be identified or different.
BACKGROUND OF THE INVENTION
The use of radiopharmaceutical Ga-containing compositions to image and diagnose tumor formations by tumor tomography and to treat malignancies has been well documented. For example, Gallium-67, as the carrier-free citrate, is used routinely in clinical medicine in diagnosing, staging, and monitoring several neoplastic disease states. Anti-tumoral therapeutic effects of gallium(III) nitrate have also been widely demonstrated in laboratory animals; and the low degree of toxicity would appear to suggest its suitability for treating various tumors. Several workers have shown that when the concentration of Ga is increased, tumor mass is decreased.
Unfortunately, this agent is plagued with imaging problems which are related to plasma protein binding. Specifically, gallium has an affinity for blood and soft tissue proteins. When given in very low doses, it is extensively bound to non-target sites (see U.S. Pat. No. 4,448,763).
Furthermore, the mechanism of gallium uptake during tumor detection is still largely unknown. It appears that the nature and types of anionic species present are the determining factors responsible for the distribution of gallium in the various organs.
In addition, most of the available instrumental methods for determining gallium are expensive and unsuitable for gallium detection in biological samples. They exhibit poor selectivity due to interference from other metals. These include inductively-coupled plasma spectroscopy (ICP), atomic absorption spectroscopy (AAS), and neuron activation coupled with high-resolution spectrometry. A comparative investigation of the suitability of AAS, emission spectroscopy with arc and hollow cathode excitation sources for detecting gallium in biological samples indicated that all three methods are significantly influenced by matrix effects. Hollow cathode emission was found to be influenced, to a lesser extent, by matrix effects but was more precise than the other two detection methods.
In view of the widespread medical application, analytical methods are required which are capable of accurately determining gallium concentrations in different biological samples (e.g., at target sites and in the blood and soft tissue).
Gallium has been incorporated into binary protein conjugates. However, prior art processes often must overcome incompatibility between the uncomplexed gallium salt and the protein conjugate. At appropriate pH values that do not cause denaturization of the protein, gallium salts tend to precipitate as gallium hydroxide. Therefore, special procedures must be employed to keep the formulations homogeneous (see U.S. patent application, Ser. No. 650,127).
Several metals, including mercury, indium, and cadmium, can be detected by using antibodies formed against ethylenediamine tetra acetic acids (EDTA). These antibodies (Abs) are usually produced from haptens obtained from the derivatization of EDTA with large proteins such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) or synthetic peptides. However, such derivatization often significantly alters the properties of the antigen. The resultant Abs, particularly monoclonal antibodies, are likely to possess heterogeneous molecular composition comprising a number of “isoforms” which may be different in molecular structure, biological potency and physiological functions. In addition, the assay technologies largely depend on the availability of Abs, which are expensive to produce and limited by different antibody specificity and cross-reactivity patterns.
Therefore, non-antibody based detection methods provide an attractive alternative to immunochemical assays. U.S. Pat. No. 5,459,040 to Hammock, et al. describes a rapid and selective method for detecting and quantifying mercury. This process relies on the specific reaction of sulfur- and mercury-containing conjugates. The method in the Hammock et. al. patent requires no antibody; quantification depends on the amount of organometallic compound that is bound to a sulfur-mercury complex, and is inversely proportional to the quantity of metal ion present in the sample. The optical density response is also inversely proportional to the quantity of the metal ion present.
A recently issued patent, U.S. Pat. No. 5,925,570 to Nishidate, et al., describes a process for measuring the quantity of metals in samples of living bodies. That patent, which in many respects can be considered state-of-the-art, describes a process whereby quantification of an objective metal is determined after other metals are administered to release the objective metal. The quantification process involves the use of spectro optical measurements using color-generating indicator reagents that are metal specific.
Although that process can be used for diagnostic testing of metals in body fluids, it can not be used for therapeutic treatments requiring the transport of metal ions to a diseased site in the body.
The current state-of-the-art techniques for quantifying metals in vivo or in vitro, as hereinabove revealed, suffer from several deficiencies, namely: high cost, poor specificity if other metals are present (i.e., matrix interactions), and loss of metal due to non-target affinity.
SUMMARY OF THE INVENTION
Having described the current state-of-the-art and associated problems that still remain, it is an important aspect of the present invention to provide processes and materials that are used to deliver therapeutic medicines and to quantify concentrations of these medicines at the target and remote sites, in particular, the detection and quantification of gallium(III) ions in body fluids to assist in determining optimal dosage for gallium(III) radiotherapy of lesions and other illnesses.
Another aspect of the invention is to provide an analytical test for quantifying the level of gallium(III) with minimal interference from other metal ions that might be present, thereby eliminating the need for prior processing for removal of ancillary metal ions.
Another aspect of the invention is to provide a simple, rapid, and inexpensive process to quantify the level of gallium(III) in body fluids such as blood (whole blood, blood serum, blood plasma, blood cell), urine, feces, saliva, breast milk, and so on. Generally speaking, in clinical examination, metals contained mainly in blood or urine are measured in vivo and in vitro.
Another aspect of this invention is to provide a therapeutic ternary coordination complex from a protein conjugate-gallium(III) ion-enzyme conjugate where the protein has an affinity for an antigen from a cancerous lesion and whe
Sadik Omowunmi A.
Xu Hongwu
Ceperley Mary E.
Salzman & Levy
The Research Foundation of State University of New York
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