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  Non-invasive enzyme screen for tissue remodeling associated conditionsUSPTO Application #: 20070015227Title: Non-invasive enzyme screen for tissue remodeling associated conditions Abstract: Methods and kits for diagnosing the presence of and prognosing the appearance of tissue remodelling-associated conditions, involving the presence of enzymes in a biological sample, are disclosed. In particular, the method pertains to diagnosing the presence of or prognosing appearance of cancer, metastatic cancer, and obstructive and degenerative conditions. (end of abstract)
Agent: David S. Resnick - Boston, MA, US Inventors: Marsha A. Moses, Michael R. Freeman, Dmitri Wiederschain USPTO Applicaton #: 20070015227 - 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 20070015227. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a continuation-in-part application of Ser. No. 08/639,373 filed on Apr. 26, 1996, now pending. The contents of the aforementioned application is expressly incorprated herein by reference. BACKGROUND OF THE INVENTION [0002] A class of disorders may be characterized as tissue remodelling-associated conditions, and includes cancers, arthritic conditions, obstructive disorders, degenerative disorders, and problematic wound-healing and ulcerative disorders. Paradigmatic among these is prostate cancer (CaP), the leading source of new cases of cancer in men in this country, and the second leading cancer cause of cancer death after lung cancer. Over 40,000 Americans are estimated to have died of CaP in 1995, and about 244,000 new cases of prostate cancer were detected (Cancer Facts and Figures-1995, American Cancer Society, Inc., 1995) and these numbers have increased annually at an alarming rate. Further, the rate of appearance of prostate cancer in African-American men is 37% higher than for their white counterparts (Jaroff, L. (Apr. 1, 1996), Time). [0003] The current primary diagnostic tool for disorders of the prostate is measurement of the level of prostate-specific antigen (PSA) in blood, which in normal men ranges from 0 to 4 nanograms/milliliters. Prostate enlargement, a condition known as benign prostatic hyperplasia (BPH), is found in about half of men over age 45. With BPH, PSA levels rise in proportion to prostate size, possibly obscuring diagnosis of CaP. In addition, a significant proportion of men with CaP have normal PSA levels. The PSA test is somewhat non-specific for distinguishing CaP and BPH, and produces a degree of false negative results (Garnick, M., (1993), Am. Inst. Med, 118:804-818). Further, the PSA test is somewhat invasive, requiring the subject to give a blood sample, a procedure that requires trained personnel, in the setting of a doctor's office or clinic. The PSA test, a major advance over previous procedures, thus leaves much to be desired. [0004] CaP is treated by surgery, radiation therapy, cryotherapy or implantation of radioactive seeds, or a combination of these procedures. In choosing one of these treatments, consideration is also taken of possible sequelae that impact negatively on quality of life, such as temporary or long-term incontinence and impotence. Further, following surgical or chemotherapeutic treatment, production of testosterone is suppressed hormonally, to discourage metastases in the CaP patient. Hormonal suppression is found to be effective for several years duration, however cancer cells that have metastasized eventually become resistant to the drugs of hormonal suppression. CaP metastasis to other sites is inevitably fatal. Only a small percent of men with microscopically-detectable CaP progress to metastatic cancer and actually die of this disease. A current medical approach, particularly for the elderly, is "watchful waiting", wherein tumors are not treated but rather monitored for progression. SUMMARY OF THE INVENTION [0005] The present invention provides biological markers to non-invasively monitor the diagnosis and prognosis of prostate disorders and other tissue remodelling-associated conditions. This invention provides methods and kits using non-invasive procedures for detection of tissue remodelling-associated conditions in subjects and patients, for diagnosis of diseases such as prostate cancer, breast cancer, ovarian cancer, brain tumors, arthritic conditions, obstructive conditions, and ulcerative conditions. The primary screens use biological fluid samples that may be obtained by personnel without medical training, and do not require visiting a clinic or hospital. The statistical association between positive results and occurrence of tissue remodelling-associated conditions are applied to early diagnoses of the appearance of these conditions, and to prognoses of changes in these conditions. [0006] The present invention features non-invasive methods for facilitating diagnosis of a subject for a tissue remodelling-associated condition. The method involves obtaining a biological sample from that subject and detecting an enzyme in that sample, facilitating the diagnosis. The non-invasive method was based at least in part, on the observation of full length, active intact enzymes that are normally associated with the process of tissue remodelling, in urine samples from patients with certain conditions. For example, gelatin-degrading matrix metalloproteinases and other proteases have been found in the urine of cancer patients. Further, high statistical associations between presence of prostate cancer and appearance of certain enzymes in urine, and between metastatic cancer and certain enzymes in urine, have been found. [0007] The tissue remodelling conditions that can be monitored by the methods of this invention include a variety of types of cancer; moreover, the enzymes are suitable for diagnosis of other tissue remodelling conditions, such as arthritis, degenerative conditions, and obstructive conditions. The invention provides non-invasive methods for diagnosing these conditions by assay for enzymes in biological fluids. [0008] More preferably, the methods of this invention embody detection of enzymes in urine, for diagnosis and prognosis of cancer, and most preferably, prostate cancer. The invention also relates to diagnosis and prognosis of metastatic prostate cancer. The varieties of cancer suitable for diagnosis by the methods of this invention include, among others, cancers of epithelial origin, for example, cancers of the nervous system, breast, retina, lung, skin, kidney, liver, pancreas, genito-urinary tract, ovarian, uterine and vaginal cancers, and gastrointestinal tract cancers, which form in cells of epithelial origin. Using the methods described here, cancers of mesodermal and endodermal origin, for example, cancers arising in bone or in hematopoietic cells, are also diagnosed. [0009] In a preferred embodiment, the enzymes that are detected are matrix-digesting enzymes, more preferably, enzymes that are proteinases, and most preferably, enzymes that are metalloproteinases. In a different aspect, the methods of this invention involve enzymes that are full-length active enzymes, and they are matrix metalloproteinases. In another aspect, the method involves removal of low molecular weight contaminants from urine prior to the detection step; preferably, the urine is dialyzed to remove low molecular contaminants prior to the detection step. [0010] Another aspect of the methods features a fully non-invasive means for facilitating the diagnosis of a subject for a disorder of the prostate. A urine sample is obtained from the subject, and a prostate disorder-associated enzyme is detected in the urine sample, facilitating the diagnosis of that subject for the prostate disorder. More preferably, the prostate disorder-associated enzyme is a matrix-digesting enzyme, most preferably, a proteinase which is a metalloproteinase. The disorders of the prostate include benign prostatic hyperplasia, "problematic" prostatic hyperplasia, organ-defined prostate cancer, this cancer which may previously have been treated surgically or chemically, and particularly, situations in which metastatic cancer is suspected. The method encompasses diagnosis of subjects who are being treated hormonally with agents that block testosterone. [0011] The invention facilitates diagnosis of subjects for prostate cancer, using a urine sample from such subjects, and detecting one or more prostate cancer-associated enzymes. Enzymes of the matrix metalloproteinase class are among those that are diagnostic, and in the case of prostate cancer, the method involves detection of gelatinase A, gelatinase B, and related activities. More preferably, the detected metalloproteinase enzyme has a molecular weight approximately equal to 72 kDa, 92 kDa, or equal to or greater than approximately 150 kDa. Yet another feature of the invention is a method for prognosis of metastatic prostate cancer, by obtaining a biological sample from a subject and detecting a metastatic prostate cancer-associated enzyme in that biological sample facilitating the prognosis of metastatic prostate cancer. In the preferred embodiment for detecting these enzymes, low molecular weight contaminants are removed from the urine prior to the detection step. Detection of enzymes in biological fluids may be by electrophoresis, and a preferred method of analysis of the electrophoretogram is to develop a pattern of enzyme migration mobilities as a zymogram. The zymogram involves incorporating an enzymatic substrate into the inert matrix in which the enzyme species migrate. Examples of suitable substrates are type IV collagen or a derivative of a type IV collagen, and in the Examples used here, the substrate is the collagen derivative gelatin. Other convenient protein substrates, e.g., casein, are also encompassed by the methods of the invention. Other methods of enzyme detection may be immunochemical, for example, the enzymes may be detected by radio-immune assay or by enzyme-linked immunosorbant assay. [0012] The invention features kits for facilitating diagnosis and prognosis of tissue remodelling-associated conditions, which have a container with a reagent for detecting an enzyme in a urine sample, and instructions. In a preferred embodiment of the kit, the tissue remodelling-associated conditions being detected are one or more types of cancer, for example, organ-confined prostatic cancer, metastatic cancer, and prognosis of metastasis in a prostate cancer patient. In a different embodiment, the tissue remodelling-associated condition is an arthritic, obstructive, or degenerative condition. DETAILED DESCRIPTION OF THE INVENTION [0013] The present invention provides non-invasive methods, between presence of enzymes in biological fluids, and diagnosis and prognosis of tissue remodelling-associated conditions (TRACs), especially cancers, obstructive and degenerative conditions, and arthritic conditions, and kits for use for such diagnosis and prognosis. Diagnoses and prognoses for TRACs have been developed based on observed statistical associations between these conditions and the presence of a pattern of enzymes in biological fluids. For convenience, certain terms employed in the specification, examples and appended claims are collected here. [0014] The term "subject," as used herein, refers to a living animal or human in need of diagnosis or prognosis for, or susceptible to, a condition, in particular an "tissue remodelling-associated condition" as defined below. The subject is an organism capable of responding to tissue remodelling signals such as growth factors, under some circumstances, the subject is susceptible to cancer and to arthritis. In preferred embodiments, the subject is a mammal, including humans and non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. In the most preferred embodiment, the subject is a human. The term "subject" does not preclude individuals that are entirely normal with respect to tissue remodelling-associated conditions or normal in all respects. The subject may formerly have been treated surgically or by chemotherapy, and may be under treatment by hormone therapy or have been treated by hormone therapy in the past. [0015] The term "patient," as used herein, refers to a human subject who has presented at a clinical setting with a particular symptom or symptoms suggesting one or more diagnoses. A patient may be in need of further categorization by clinical procedures well-known to medical practitioners of the art (or may have no further disease indications and appear to be in any or all respects normal). A patient's diagnosis may alter during the course of disease progression, such as development of further disease symptoms, or remission of the disease, either spontaneously or during the course of a therapeutic regimen or treatment. In the invention here, a patient described in the Examples is listed with other patients according to the most recent diagnosis of the medical condition, and any previous diagnoses, if different, are described in the text. Thus, the term "diagnosis" does not preclude different earlier or later diagnoses for any particular patient or subject. The term "prognosis" refers to assessment for a subject or patient of a probability of developing a condition associated with or otherwise indicated by presence of one or more enzymes in a biological sample, preferably in urine. [0016] The term "biological sample" includes biological samples obtained from a subject. Examples of such samples include urine, blood taken from a prick of the finger or other source such as intravenous, blood fractions such as serum and plasma, feces and fecal material and extracts, saliva, cerebrospinal fluid, amniotic fluid, mucus, and cell and tissue material such as cheek smear, Pap smear, fine needle aspiration, sternum puncture, and any other biopsied material taken during standard medical and open surgical procedures. [0017] The term "invasiveness" as used here with respect to metastatic cancer (Darnell, J. (1990), Molecular Cell Biology, Third Ed, W.H. Freeman, NY) is distinct from the use of the term "invasive" to describe a medical procedure, and the distinction is made in context. "Invasive" for a medical procedure pertains to the extent to which a particular procedure interrupts the integrity of the body. "Invasiveness" ranges from fully non-invasive, such as collection of urine or saliva; to mildly invasive, for example a Pap smear, a cheek scrape or blood test, which requires trained personnel in a clinical setting; to more invasive, such as a sternum marrow collection or spinal tap; to extensively invasive, such as open surgery to detect the size and nature of tumors by biopsy of material, taken for example during brain surgery, lung surgery, or transurethral resection in the case of prostate cancer. [0018] The term "invasive" is also used with respect to proclivity of a tumor for expanding beyond its boundaries into adjacent tissue, or to the characteristic of the tumor with respect to metastasis (Darnell, J. (1990), Molecular Cell Biology, Third Ed., W.H. Freeman, NY). For example, a basal cell carcinoma of the skin is a non-invasive or minimally invasive tumor, confined to the site of the primary tumor and expanding in size, but not metastasizing. In contrast, the cancer melanoma is highly invasive of adjacent and distal tissues. The invasive property of a tumor is often accompanied by the elaboration of proteolytic enzymes, such as collagenases, that degrade matrix material and basement membrane material to enable the tumor to expand beyond the confines of the capsule, and beyond confines of the particular tissue in which that tumor is located. Elaboration of such enzymes may be by endogenous synthesis within the tumor cells, or may be elicited from adjacent cells or by circulating neutrophils, in which cases the elicitation by the tumor results from chemical messengers elaborated by the tumor and expression of the enzymes occurs at the tumor site or proximal to the tumor. The enzymes of the present invention are not intended to be limited to those produced exclusively as endogenous tumor products, but are found in biological samples in patients in need of or subjects in need of prognosis or diagnosis of TRACs. [0019] Cancer or neoplasia is characterized by deregulated cell growth and division. A tumor arising in a tissue originating from endoderm or exoderm is called a carcinoma, and one arising in tissue originating from mesoderm is known as a sarcoma (Darnell, J. (1990), Molecular Cell Biology, Third Ed., W.H. Freeman, NY). A current model of the mechanism for the origin of a tumor is by mutation in a gene known as an oncogene, or by inactivation of a second tumor-suppressing genes (Weinberg, R. A., (September 1988), Scientific Amer., 44-51). The oncogenes identified thus far have arisen only in somatic cells, and thus have been incapable of transmitting their effects to the germ line of the host animal. In contrast, mutations in tumor-suppressing genes can be identified in germ line cells, and are thus transmissible to an animal's progeny. Examples of cancers include cancers of the nervous system, breast, retina, lung, skin, kidney, liver, pancreas, genito-urinary tract, gastrointestinal tract, cancers of bone, and cancers of hematopoietic origin such as leukemias and lymphomas. In one embodiment of the present invention, the cancer is not a cancer of the bladder. [0020] An arthritic condition such as rheumatoid arthritis is an example of a TRAC since the disease when chronic is characterized by disruption of collagenous structures (J. Orten et al., (1982), Human Biochemistry, Tenth Ed., C. V. Mosby, St. Louis, Mo.). Excess collagenase is produced by cells of the proliferating synovium. Other TRAC conditions such as ulcerative, obstructive and degenerative diseases are similarly characterized by alterations in the enzymes of metabolism of structural proteins. Continue reading... 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