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  5-fluoro-uracil immunoassayUSPTO Application #: 20060177883Title: 5-fluoro-uracil immunoassay Abstract: Novel conjugates of 5-fluoro-uracil and novel 5-fluoro-uracil immunogens and monoclonal antibodies generated by these immunogens which are useful in immunoassays for the quantification and monitoring of 5-fluoro-uracil in biological fluids. (end of abstract)
Agent: Gibbons, Del Deo, Dolan, Griffinger & Vecchione - Newark, NJ, US Inventors: Salvatore J. Salamone, Jodi Blake Courtney, Dennis Stocker USPTO Applicaton #: 20060177883 - Class: 435007920 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Assay In Which An Enzyme Present Is A Label, Heterogeneous Or Solid Phase Assay System (e.g., Elisa, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20060177883. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] This invention relates to the field of immunological assays for determining the presence and/or quantifying the amount of 5-fluoro-uracil [5-FU] in human biological samples in order to rapidly determine optimal drug concentrations during chemotherapy. BACKGROUND OF THE INVENTION [0002] Cancer is a term used to describe a group of malignancies that all share the common trait of developing when cells in a part of the body begin to grow out of control. Most cancers form as tumors, but can also manifest in the blood and circulate through other tissues where they grow. Cancer malignancies are most commonly treated with a combination of surgery, chemotherapy, and/or radiation therapy. The type of treatment used to treat a specific cancer depends upon several factors including the type of cancer malignancy and the stage during which it was diagnosed. [0003] 5-FU is one of the more commonly used cytotoxic agents that are used for the treatment of Breast and Colorectal cancer. This chemotherapeutic agent has the formula: [0004] This compound has been associated with debilitating side effects such as bone marrow density loss, mucositis, nausea and vomiting. By monitoring the levels of 5-FU in the body and adjusting the dose these side effects can be better controlled and limited in patients. [0005] At the same time, there is often a highly variable relationship between the dose of 5-FU and the resulting serum drug concentration that affects therapeutic effect. The degree of intra- and inter-individual pharmacokinetic variability of 5-FU can be as high as 10-fold (Diasio et. al. J. Clin. Invest. 81: pp 47-51, 1988, Wei et. al. J. Clin. Invest. 98: pp610-615, 1996) and is impacted by many factors, including: [0006] Organ function [0007] Genetic regulation [0008] Disease state [0009] Age [0010] Drug-drug interaction [0011] Time of drug ingestion, [0012] Mode of drug administration, and [0013] Technique-related administration. As a result of this variability, equal doses of the same drug in different individuals can result in dramatically different clinical outcomes (Hon et. al. Clinical Chemistry 44, pp 388-400,1998). The effectiveness of the same 5-FU dosage varies significantly based upon individual drug clearance and the ultimate serum drug concentration in the patient. Therapeutic drug management would provide the clinician with insight on patient variation in both oral and intravenous drug administrations. With therapeutic drug management, drug dosages could be individualized to the patient, and the chances of effectively treating the cancer without the unwanted side effects would be much higher (Nieto, Current Drug Metabolism 2: pp 53-66, 2001). [0014] In addition, therapeutic drug management of 5-FU would serve as an excellent tool to ensure compliance in administering chemotherapy with the actual prescribed dosage and achievement of the effective serum concentration levels. It has been found that variability in serum concentration is not only due to physiological factors, but can also result from variation in administration technique and ability of the body to absorb 5-FU. As a chemotherapeutic agent, 5-FU can be administered in its pro-drug form as tegafur which has the structure: Tegafur, when administered to a patient, is generally absorbed and metabolized into 5-FU by the patient at different rates. Therefore, in monitoring the level of 5-FU in patients by means of an immunoassay, it is important that the immunoassay be able to distinguish between tegafur, the inactive substance, and 5-FU, the active substance, into which tegafur metabolizes. The problem with antibodies to 5-FU is that they could be cross-reactive with tegafur making these immunoassays not useful. [0015] Routine therapeutic drug management of 5-FU would require the availability of simple automated tests adaptable to general laboratory equipment. Tests that best fit these criteria are immunoassays. Currently there are no immunoassays for 5-FU available and monitoring levels of this drug is conducted by physical methods like high pressure liquid chromatography (HPLC) (Escoriaza et. al. J. of Chromatography B: Biomedical Sciences and applications, 736 (1+2): pp 97-102, 1999). In order to be most effective in monitoring drug levels the antibody should be most specific to 5-FU and display very low cross-reactivity to no cross-reactivity to related pyrimidine bases, particularly tegafur. SUMMARY OF INVENTION [0016] In accordance with this invention, a new class of antibodies have been produced which are substantially selectively reactive to 5-FU so as to bind to 5-FU without any substantial cross reactivity to tegafur, as well as, to other interfering pyrimidine bases, uracil and cytosine. By selectively reactive it is meant that this antibody reacts with the 5-FU molecule and does not substantially react with the other interfering pyrimidine bases such as analogues of 5-FU, the most important blocking pyrimidine base being tegafur. By providing an antibody that does not substantially cross-react with tegafur, allows one to provide an immunoassay for 5-FU which can accurately monitor levels of 5-FU for therapeutic management of patients being treated with 5-FU. [0017] It has been found that by using immunogens which are conjugates of an immunogenic polyamine polymer with a compound of the formula: [0018] wherein Y is an organic spacing group; [0019] X is a terminal functional group capable of binding to a polyamine polymer; and [0020] p is an integer from 0 to 1. produce antibodies which are specific for 5-FU and do not substantially react with or bind to tegafur, as well as other pyrimidine bases such as uracil, and cytosine. The provision of these antibodies which substantially selectively react with 5-FU and do not cross react with tegafur allows one to produce an immunoassay which can specifically detect and monitor 5-FU in the fluid samples of patients being treated with 5-FU. Also included within this invention are reagents and kits for said immunoassay. The presence of tegafur is the major cause for false positive readings which have made immunoassays for 5-FU unsuitable. DETAILED DESCRIPTION [0021] In accordance with this invention, a new class of antibodies is provided which substantially selectively reacts with 5-FU and does not substantially react or cross react with tegafur, as well as other interfering pyrimidine bases such as uracil, and cytosine. It has been discovered that through the use of the 3-substituted 5-FU derivative of formula II-A as an immunogen, this new class of antibodies of this invention are provided. It is through the use of these antibodies that an immunoassay, including reagents and kits for such immunoassay for detecting and/or quantifying 5-FU in blood, plasma or other body fluid samples has been developed. By use of this immunoassay, the presence and amount of 5-FU in body fluid samples, preferably a blood or plasma sample, can be detected and/or quantified. In this manner, a patient being treated with 5-FU can be monitored during therapy and his treatment adjusted in accordance with said monitoring. By means of this invention one achieves the therapeutic drug management of 5-FU in cancer patients being treated with 5-FU as a chemotherapeutic agent. [0022] The reagents utilized in the immunoassay of this invention are conjugates of a carrier, preferably containing polyamine functional groups, with the 1-substituted 5-FU compound of formula II-B: [0023] wherein p, X and Y are as above. [0024] In the immunoassay of this invention, these conjugates are competitive binding partners with the 5-FU present in the sample for the binding with the antibodies of this invention. Therefore, the amount of conjugate reagent which binds to the antibody will be inversely proportional to the amount of 5-FU in the sample. In accordance with this invention, the assay utilizes any conventional measuring means for detecting and measuring the amount of said conjugate which is bound or unbound to the antibody. Through the use of said means, the amount of the bound or unbound conjugate can be determined. Generally, the amount of 5-FU in a sample is determined by correlating the measured amount of the bound or unbound conjugate produced by the 5-FU in the sample with values of the bound or unbound conjugate determined from standard or calibration curve samples containing known amounts of 5-FU, which known amounts are in the range expected for the sample to be tested. These studies for producing calibration curves are determined using the same immunoassay procedure as used for the sample. [0025] The conjugates are prepared from the compound of formula II-B whereas immunogens are prepared from compounds of the formula II-A. In performing the immunoassay in accordance with this invention, it is important that the conjugate be formed from the compound of formula II-B and the immunogen be formed from the compound of formula II-A. In the immunogens, the polyamine polymer is linked to the ligand portion which has the formula: [0026] wherein Y and p are as above; and [0027] X is --CH.sub.2-- or a functional linking group; and in the conjugate the carrier is linked to the ligand portion which has the formula: [0028] wherein X', Y and p are as above. [0029] These ligand portions may be linked to one or more active sites on the carrier of the conjugate or polyamine polymer. Definitions [0030] Throughout this description the following definitions are to be understood: [0031] The terms "immunogen" and "immunogenic" refer to substances capable of eliciting, producing, or generating an immune response in an organism. [0032] The term "conjugate" refers to any substance formed from the joining together of two parts. Representative conjugates in accordance with the present invention include those formed by the joining together of a small molecule, such as the compound of formula II-B, and a large molecule, such as a carrier or a polyamine polymer, particularly protein. In the conjugate the small molecule may be joined at one or more active sites on the large molecule. [0033] "Haptens" are partial or incomplete antigens. They are protein-free substances, mostly low molecular weight substances, which are not capable of stimulating antibody formation, but which do react with antibodies. The latter are formed by coupling a hapten to a high molecular weight immunogenic carrier and then injecting this coupled product, i.e., immunogen, into a human or animal subject. The hapten of this invention is 5-FU. Continue reading... Full patent description for 5-fluoro-uracil immunoassay Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this 5-fluoro-uracil immunoassay patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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