WO2013101276A2 - Diagnosis and detection of disease conditions modulated by activation of the sphingolipid pathway - Google Patents

Abstract

The present invention provides a method of detecting whether a subject has a disease condition modulated by activation of the sphingolipid pathway. This method comprises selecting a subject, detecting ceramidase level in a bodily fluid sample from the subject, and comparing the detected ceramidase level in the bodily fluid sample to a ceramidase level standard for a subject not having a disease condition modulated by activation of the sphingolipid pathway. The present invention also provides for a method of monitoring effectiveness of a therapy in a subject having a disease condition modulated by activation of the sphingolipid pathway, a method of monitoring progression of a disease condition modulated by activation of the sphingolipid pathway in a subject, and a kit with a reagent to detect ceramidase level in a bodily fluid sample and instructions for measuring ceramidase level in the sample using the reagent.

Description

DIAGNOSIS AND DETECTION OF DISEASE CONDITIONS MODULATED BY ACTIVATION OF THE SPHINGOLIPID PATHWAY [0001] This application claims benefit of U.S. Provisional Patent Application

Serial No. 61/470,869, filed on April 1, 2011, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the diagnosis and detection of disease conditions modulated by activation of the sphingolipid pathway.

BACKGROUND OF THE INVENTION

[0003] Sphingo lipids comprise a group of lipids having ceramide, i.e., N- acylsphingosine as the basic group. Ceramide (N-acylsphingosine) is a lipid metabolite and is an important intracellular messenger which is released inside a cell. Ceramide is regarded as a second messenger in the context of the sphingomyelin signal transduction pathway. Ceramides are released by sphingomyelin as a result of the enzymatic effect of sphingomyelinases, which are forms of phospholipase C specific for sphingomyelin.

Inside cells, ceramide can influence growth and differentiation, regulate protein secretion, induce DNA fragmentation and apoptosis, and increase the synthesis and secretion of cytokines. Ceramide is an essential, ubiquitous lipid found in small quantities in all cells, but when elevated above threshold levels disrupts cell membranes, inducing signaling cascades that frequently lead to cell cycle arrest and/or cell death.

[0004] A ceramidase is an enzyme which hydrolyzes a ceramide into a sphingoid base (sphingosine) and a fatty acid. The sphingosine generated by hydrolyzing the ceramide with the ceramidase possesses various physiological activities such as inhibition of protein kinase C, activation of phospholipase D and inhibition of a calmodulin- dependent enzyme. Sphingosine is an important substance which is thought to be acting on the regulation of the cell functions because it is involved in proliferation of cells and intracellular signal transduction. After stimulation, ceramidases may then hydrolyze ceramide into the individual fatty acid and sphingosine components (Gatt, "Enzymic Hydrolysis and Synthesis of Ceramide," J. Biol. Chem. 238:3131-3 (1963); Gatt, "Enzymatic Hydrolysis of Sphingolipids. 1. Hydrolysis and Synthesis of Ceramides by an Enzyme from Rat Brain," J. Biol. Chem. 241 :3724-31 (1966); Hassler & Bell,

"Ceramidase: Enzymology and Metabolic Roles," Adv. Lip. Res. 26:49-57 (1993)). There is no de novo pathway for cells to generate sphingosine, and it is therefore only generated by the hydrolysis of ceramide by ceramidases. Sphingosine is further modified in cells to the phosphorylated form, sphingosine- 1 -phosphate (SIP) which is another bioactive sphingo lipid with strong proliferative and mitogenic effects.

[0005] Ceramide is produced in response to various stimuli and extrinsic factors, including serum deprivation and treatment with many chemotherapy drugs, as well as in many human diseases (Hannun, "Function of Ceramide in Coordinating Cellular

Responses to Stress," Science 274:1855-9 (1996); Spiegel et al, "Signal Transduction Through Lipid Second Messengers," Curr. Opin. Cell. Biol. 8:159-67 (1996)). Normally present in low amounts, in response to these factors, ceramide is rapidly produced at the cell surface, leading to membrane re-organization and downstream signaling that results in apoptosis.

[0006] Because ceramide degradation is the only source of intracellular sphingosine (Rother et al, "Biosynthesis of Sphingolipids: Dihydro ceramide and Not Sphinganine is Desaturated by Cultured Cells," Biochem. Biophys. Res. Commun.

189: 14-20 (1992)), these enzymes may also be rate-limiting steps in determining the intracellular levels of this compound. Importantly, SIP can counteract the apoptotic effects of ceramide (Cuvillier et al, "Suppression of Ceramide-mediated Programmed Cell Death by Sphingosine- 1 -phosphate," Nature 381 :800-3 (1996)), leading to the suggestion that ceramidases can be "rheostats" that maintain a proper balance between cell growth and death (Spiegel & Merrill, "Sphingolipids Metabolism and Cell Growth Regulation," FASEB J. 10: 1388-97 (1996)). Both sphingosine and SIP are important signaling lipids, and SIP in particular has numerous trophic effects that stimulate cell proliferation.

[0007] Dysfunctional sphingolipid metabolism and modulation of the

sphingolipid pathway are found in a multitude of disease conditions in the human body. For example, ceramide is a mediator of proinflammatory cytokine signaling and induces cartilage degradation by reducing synthesis of type II collagen in cartilage. Accumulation of ceramide is associated with arthritis in Farber's disease (Gilbert et al.,

"Sphingomyelinase Decreases Type II Collagen Expression in Bovine Articular Cartilage Chondrocytes via the ERK Signaling Pathway," Arthritis & Rheumatism 58(l):209-220 (2008)), and cancer. Sphingolipids, specifically SIP, are also elevated in the presence of type 1 diabetes (Fox et al., "Circulating Sphingolipid Biomarkers in Models of Type 1 Diabetes," J. Lipid Res. (Published Online November 10, 2010)). In addition, because sphingolipid metabolism is a process that modulates the formation of bioactive metabolites, it is essential that the human brain contain a proper balance of sphingolipids. In some brain disorders, including Alzheimer's Disease, sphingolipids are altered (Mielke et al., "Alterations of the Sphingolipid Pathway in Alzheimer's Disease: New Biomarkers and Treatment Targets?," Neuromol. Med. 12:331-340 (2010)).

[0008] Acid ceramidase ("AC") (N-acylsphingosine deacylase, I.U.B.M.B.

Enzyme No. EC 3.5.1.23) is one particular ceramidase responsible for the catabolism of ceramide. Due to its involvement in the human genetic disorder Farber

Lipogranulomatosis, AC is the most extensively studied member of the ceramidase enzyme family. The protein has been purified from several sources, and the human and mouse cDNAs and genes have been obtained (Bernardo et al., "Purification,

Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem.

270: 11098-102 (1995); Koch et al, "Molecular Cloning and Characterization of a Full- length Complementary DNA Encoding Human Acid Ceramidase. Identification of the First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 2711 :33110-5 (1996); Li et al., "Cloning and Characterization of the Full-length cDNA and Genomic Sequences Encoding Murine Acid Ceramidase," Genomics 50:267-74 (1998); Li et al, "The Human Acid Ceramidase Gene (ASAH): Chromosomal Location, Mutation Analysis, and Expression," Genomics 62:223-31 (1999)). Growing interest in the biology of this and other ceramidases stems from the fact that these enzymes play such a central role in ceramide metabolism.

[0009] Current methods of detecting and treating disease conditions of the sphingolipid pathway have low sensitivity with respect to particular disease conditions and are not approved by the FDA. Currently available biomarkers are, therefore, unreliable and ineffective at detecting disease conditions of the sphingolipid pathway in serum.

[0010] The present invention is directed to overcoming these deficiencies in the art. SUMMARY OF THE INVENTION

[0011] One aspect of the present invention is directed to a method of detecting a disease condition modulated by activation of the sphingolipid pathway. The method includes selecting a subject, detecting ceramidase level in a bodily fluid sample from the selected subject, and comparing the detected ceramidase level in the bodily fluid sample to a ceramidase level standard for a subject not having a disease condition modulated by activation of the sphingolipid pathway.

[0012] Another aspect of the present invention relates to a method of monitoring effectiveness of a therapy in a subject having a disease condition modulated by activation of the sphingolipid pathway. The method includes selecting a subject, providing a baseline ceramidase level in a bodily fluid sample from the selected subject before the therapy, and treating a disease condition modulated by activation of the sphingolipid pathway in the selected subject with the therapy. The method further includes detecting a post-therapy ceramidase level in a bodily fluid sample from the selected subject following the therapy, comparing the baseline ceramidase level with the post-therapy ceramidase level, and identifying whether the therapy has been effective based on the comparing.

[0013] Another aspect of the present invention relates to a method of monitoring progression of a disease condition modulated by activation of the sphingolipid pathway in a subject. The method includes selecting a subject, providing a baseline ceramidase level in a bodily fluid sample taken from the selected subject at an initial time, and detecting a later ceramidase level in a bodily fluid sample taken from the selected subject at a time after the initial time when the bodily fluid sample corresponding to the baseline ceramidase level is taken. The method further includes comparing the baseline ceramidase level to the later ceramidase level and identifying whether the disease condition modulated by activation of the sphingolipid pathway has undergone progression or regression, between when the initial and later bodily fluid samples were taken, based on the comparing.

[0014] Another aspect of the present invention relates to a kit which has a reagent suitable to detect ceramidase level in a bodily fluid sample and instructions for measuring ceramidase level in bodily fluid sample using the reagent.

[0015] The present invention is based on applicant's discovery that enough active

AC and/or other ceramidases could be secreted from tissue to make the ceramidase stable enough to be detected in bodily fluids, such as blood. Also, it was not known that the ceramidase activity levels in such bodily fluids would correlate with the stage of cancer and/or the aggressiveness of the tumor.

[0016] The present invention may use a commercially available, fluorescent AC substrate, however, the substrate has never been used before for serum determination of AC. This method, which is the first to describe a serum assay for ceramidase, preferably utilizes a highly sensitive method with a new HPLC system from Waters ("UPLC"), as well as pH adjustments to obtain reliable values.

[0017] Ceramidase protein and/or mR A levels are elevated in numerous cancer cells, including prostate, breast, melanoma, ovarian and others. Ceramidase levels are also elevated in the presence of a benign growth, arthritis, cardiovascular disease, diabetes, and Alzheimer's Disease. Using a highly sensitive AC assay, the present invention evaluates AC and other ceramidases as a biomarker for ovarian cancer, as well as other disease conditions modulated by activation of the sphingolipid pathway.

Analysis was performed in two parts, first to assess the usefulness of AC for detecting early stage cancers (pre-surgery), and second to determine its usefulness post-surgery for patient management. Correlations with and without CA125 levels were studied.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows the activity of AC in the serum of ovarian cancer patients pre-surgery. Dot plots show the activity of AC in serum samples from healthy controls ("Normal", n = 19), patients with benign gynecologic conditions ("Benign", n = 9), stage I and II ovarian cancer patients ("Stage Ι/Π", n = 6), stage III and IV ovarian cancer patients ("Stage III/IV", n = 23), or all cancer patients combined ("Stage I-IV", n = 29). The solid benchmark lines (red) indicate the median levels of AC in each group. One Unit of AC activity is defined as the amount of the enzyme that catalyzes the formation of 1 pmol of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l ,3-diazol-4-yl)) ("NBD")-conjugated C12-fatty acid (produced by hydrolysis of NBD-C 12-ceramide) in 17 hours. Final data is expressed as U/microgram of total protein. Significance was assessed by t-test and marked by asterisk (*P<0.05, ** P<5E-5, as compared to "normal control"). The solid horizontal line (green) indicates the highest value of the healthy control group. [0019] Figure 2 illustrates the activity of AC in the serum of ovarian cancer patients post-surgery and during chemotherapy. Dot plots illustrate the activity of AC in serum samples from healthy controls ("Normal", n = 19), patients with ovarian cancer ("Stage I-IV", pre-surgery, n = 29), patients with ovarian cancer post-surgery during chemotherapy ("During Chemo", n = 28), and patients with recurrent ovarian cancer

("Recurrent", n = 5). The solid benchmark lines (red) indicate the median levels of AC in each group. For the "During Chemo" group, the plotted data represents the mean of several samples collected from each individual over time. One Unit of AC activity is defined as the amount of the enzyme that catalyzes the formation of 1 pmol of NBD- conjugated C12-fatty acid (produced by hydrolysis of NBD-C12-ceramide) in 17 hours. Final data is expressed as U/microgram of total protein. Significance was assessed by t- test and marked by asterisk (*P<0.05, ** P<5E-4, as compared to "normal control"). The solid horizontal line (green) indicates the highest value of the healthy control group.

[0020] Figure 3 shows the AC activity in ovarian cancer tissue. Bar graph illustrates AC activity in tissue samples from patients with benign gynecologic conditions (n = 4) or with ovarian cancer (n = 2). One Unit of AC activity is defined as the amount of the enzyme that catalyzes the formation of 1 pmol of NBD-conjugated C12-fatty acid (produced by hydrolysis of NBD-C12-ceramide) in 17 hours. Final data is expressed as U/microgram of total protein.

[0021] Figure 4 illustrates the AC activity level of a cell line sample as compared to the AC protein level of the same sample. The top panel shows a representative western blot of seven different cancer cell lines. The AC lane was developed using a specific antibody against AC. GAPDH was used as a loading control. The bottom panel shows the activity in the cell extract from the same cell lines. There is no correlation of AC protein levels (Western Blot) with activity (i.e. compare protein and activity in 231L, a breast cancer cell line, to SKMel, a melanoma cell line).

[0022] Figures 5 A-B show AC activity in synovial fluid (Figure 5 A) and serum

(Figure 5B) of normal, MPS Type I, and MPS Type VII dog model subjects. AC activity was determined using NBD-C12 ceramide and a UPLC system. P values compare the activities in MPS I and MPS VII dogs to normal dogs. Both the serum and synovial fluid AC activities were significantly elevated in the two different MPS animal models.

[0023] Figure 6 illustrates AC activity in the intra-articular space of MPS I dog subjects. AC activity after monthly injections of 1 mg of recombinant alpha iduronidase into the intra-articular space of the right elbows and knees of 4 MPS I dogs for 6 months is shown (black bars). AC in the left elbows and knees that were untreated controls is shown (white bars). Synovial fluid was collected monthly from each joint for AC activity assays using NBD-C12 ceramide and a UPLC system. The data represents average (combined for 6 time-points) for the 4 dogs. The synovial fluid from the untreated joints had significantly higher AC activity than the treated joints.

[0024] Figure 7 shows sphingosine levels in the intra-articular space of MPS I dogs. Sphingosine levels after monthly injections of 1 mg of recombinant alpha iduronidase into the intra-articular space of the right elbows and knees of 4 MPS I dogs for 6 months is shown (black bars). Sphingosine levels in the left elbows and knees that were untreated controls is shown (white bars). Synovial fluid was collected monthly from each joint, and sphingosine assays were carried out using a UPLC system. The data shown in Figure 7 represents average values for the 4 dogs after 3 months of treatment. The synovial fluid from the untreated joints had significantly higher sphingosine levels than the treated joints. The reduction in sphingosine levels in the treated animals is consistent with the reduction in AC shown in Figure 6.

DETAILED DESCRIPTION OF THE INVENTION

[0025] One aspect of the present invention is directed to a method of detecting a disease condition modulated by activation of the sphingolipid pathway. The method includes selecting a subject, detecting ceramidase level in a bodily fluid sample from the selected subject, and comparing the detected ceramidase level in the bodily fluid sample to a ceramidase level standard for a subject not having a disease condition modulated by activation of the sphingolipid pathway.

[0026] Sphingo lipids are a class of lipids typically found in membranes and possess a polar head and two nonpolar tails. Sphingo lipids are derived ultimately from palmitoyl-CoA and serine. They are composed of one molecule of the long-chain amino alcohol sphingosine (4-sphingenine) or one of its derivatives, optionally one molecule of a long-chain acid, a polar head alcohol and optionally phosphoric acid in the diester linkage at the polar head group. Ceramides are sphingolipids containing two acyl- moieties. [0027] AC is an enzyme that catalyzes the hydrolysis of ceramide to sphingosine and free fatty acid (Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem. 270(19): 11098-102 (1995), which is hereby incorporated by reference in its entirety). Mature AC is a ~50kDa protein composed of an a-subunit (~13kDa) and a β-subunit (~40kDa) (Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem.

270(19): 11098-102 (1995), which is hereby incorporated by reference in its entirety). It is produced through cleavage of the AC precursor protein (Ferlinz et al., "Human Acid Ceramidase: Processing, Glycosylation, and Lysosomal Targeting," J. Biol. Chem.

276(38):35352-60 (2001), which is hereby incorporated by reference in its entirety), which is the product of the Asahl gene (NCBI UniGene GenelD No. 427, which is hereby incorporated by reference in its entirety).

[0028] As used herein, the term "assay" refers to an assay for detecting the presence or absence of ceramidase in a given sample of a bodily fluid. Also included is a quantitative assay, which measures the amount of a substance in a sample.

[0029] A powerful approach to discovery of a treatment for and detection of disease is to screen large samples in functional assays to identify compounds that detect the target protein. The assay should be insensitive to most chemicals in the concentration range normally used in the drug discovery process. These assays should also be selective and not show inhibition by antibiotics known to target proteins in processes outside of replication.

[0030] Ceramidase level can refer to the amount of ceramidase present or the ceramidase functional activity. The amount of ceramidase present in a sample is determined by the amount of ceramidase protein in the sample and may be measured by western blotting techniques. Alternatively, the functional activity of ceramidase in a sample is determined by the amount of activity of the ceramidase, as detected by an activity assay.

[0031] Assays suitable for determining ceramidase level are apparent to the skilled artisan. Suitable methods include, for example, ceramidase activity assays (Eliyahu et al., "Acid Ceramidase is a Novel Factor Required for Early Embryo

Survival," FASEB J. 21(7): 1403-9 (2007), which is hereby incorporated by reference in its entirety), western blotting to determine the relative amount of ceramidase present in the sample (where a higher amount of ceramidase protein correlates to a higher ceramidase activity level) (Eliyahu et al., "Acid Ceramidase is a Novel Factor Required for Early Embryo Survival," FASEB J. 21(7): 1403-9 (2007), which is hereby

incorporated by reference in its entirety), and RIA (Ferlinz et al., "Human Acid

Ceramidase: Processing, Glycosylation, and Lysosomal Targeting," J. Biol. Chem.

276(38):35352-60 (2001), which is hereby incorporated by reference in its entirety).

[0032] For binding assays, while native binding targets may be used, it is frequently preferred to use portions (i.e., peptides) thereof so long as the portion provides binding affinity and avidity to the subject replication protein conveniently measurable in the assay. Generally, a plurality of assay mixtures are run in parallel with different agents to obtain a response to various chemical structures. Typically, one of these serves as a negative control (i.e. at zero chemicals or below the limits of assay detection). Additional controls are often present such as a positive control, a dose response curve, use of known inhibitors, use of control heterologous proteins, etc. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably they are small organic compounds and are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins (i.e., albumin, detergents, etc.), which may be used to facilitate optimal binding and/or reduce nonspecific or background interactions, etc. Also, reagents that otherwise improve the efficiency of the assay (i.e., protease inhibitors, nuclease inhibitors, antimicrobial agents, etc.) may be used. In one embodiment of the present invention, the detecting ceramidase level in the bodily fluid sample comprises contacting the bodily fluid with ceramidase assay reagents and determining the ceramidase level in the bodily fluid sample based on the contacting.

[0033] One particularly effective non-specific assay used to detect total proteins is the Bradford protein assay (Bradford, M. M., "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Proteins Utilizing the Principle of Protein-Dye Binding," Anal. Biochem. 72:248-254 (1976), which is hereby incorporated by reference in its entirety).

[0034] The Bradford protein assay uses a dye stock of Coomassie Blue G (C.I.#

42655) (100 mg), which is dissolved in 50 mL of methanol. The solution is added to 100 mL of 85% H₃PO₄, and diluted to 200 mL with water, resulting in a dark red and a pH of -0.01. The final reagent concentrations of the assay are 0.5 mg/mL Coomassie Blue G, 25% methanol, and 42.5% H ₃PO₄. The assay reagent of the Bradford assay is prepared by diluting 1 part dye stock with 4 parts distilled H₂0. The resulting color should be brown with a pH of 1.1. A series of protein standards are prepared in the same buffer as the samples to be assayed, using bovine serum albumin ("BSA") with concentrations of 0, 250, 500, 750, and 1500 μg/mL for a standard assay. The absorbance was read at 595 nm for standard assay procedure and 450 nm for a micro assay (Dynex Technologies, Chantilly, VA), and the ratio of the absorbances, 595 nm over 450 nm, was used for standard curve calculations (Zor, et al., "Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies," Anal. Biochem.

236:302-308 (1996), which is hereby incorporated by reference in its entirety).

[0035] Examples of assays that monitor binding between two components include, but are not limited to, Biacore Surface Plasmon Resonance, ELISA antibody based assays, proximity assays using FRET, and antibody pull down assays.

[0036] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from biological samples. The bodily fluid sample is selected from the group consisting of serum, synovial fluid, cerebrospinal fluid, and peritoneal fluid. Of particular interest are samples that are serum. Those skilled in the art will recognize that plasma or whole blood or a sub-fraction of whole blood may also be used. Biological fluid samples may be obtained from animals (including humans) and include blood products, such as plasma, serum and the like.

[0037] The body fluid sample described may be obtained by use of a standard blood draw, as disclosed in U.S. Patent No. 4,263,922 to White, which is hereby incorporated by reference in its entirety. Generally, in a standard blood draw, blood is drawn through a needle assembly and handle system into a collection tube. Subsequent to the blood draw, the needle assembly and the handle are removed from an end of the tube and a separate cap is fitted over each end of the tube to retain the blood sample in the tube for analysis. In the case of humans, a finger prick with a lancet or a blood draw via standard venipuncture are also convenient methods to obtain a body fluid sample.

[0038] Upon obtaining a blood sample from a subject, the drawn blood is preferably exposed immediately to an anticoagulant to preclude coagulation thereof. Known anticoagulants include without limitation heparin, EDTA, D-Phe-Pro-Arg chloromethyl ketone dihydrochloride ("PPACK"), and sodium citrate. Other

anticoagulants may also be used. [0039] In one embodiment, the disease condition modulated by activation of the sphingolipid pathway is a cancer. The cancer may be acute lymphocytic leukemia, acute myelogenous leukemia, biliary cancer, breast cancer, cervical cancer, chronic

lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, renal cancer, prostate cancer, glial and other brain and spinal cord tumors, pancreatic cancer, melanoma, ovarian cancer, liver cancer, or urinary bladder cancer.

[0040] Ovarian cancer is the most common cause of death among gynecological malignancies. Approximately twenty percent of women will be diagnosed with a pelvic mass in their lifetime, and nearly 300,000 women undergo surgery each year for this indication in the United States alone. Between fifteen and twenty percent of these women will be diagnosed with an epithelial ovarian cancer ("EOC"). The current standard of care for EOC patients is primary cytoreductive surgery followed by chemotherapy.

[0041] The ability to identify early stage malignant masses pre-operatively is essential to provide optimal care for women with EOC, since numerous studies have shown that if such women are identified and undergo surgery at specialized care centers they have decreased morbidity and improved survival. For this reason, many laboratories have investigated heavily in finding serum biomarkers for EOC. In addition, appropriate post surgery treatment could best be managed if suitable biomarkers were available to predict the treatment course and efficacy throughout the procedure, as well as the risk of reoccurrence.

[0042] The most well known and commonly used serum biomarker for the evaluation of a woman with a pelvic mass and for management of EOC is CA125— a glycoprotein that is expressed in mesothelial cells that line the peritoneum, pleural cavity, and pericardium. While serum CA125 levels are elevated in about eighty percent of patients with EOC, serum concentrations can also be elevated by a number of common benign gyneocological and medical conditions, including endometriosis, leiomyomas, congestive heart failure, and others. In addition, mean serum concentrations vary according to menopausal status, with higher levels noted in premenopausal women with functional reproductive systems. These factors result in impaired sensitivity and specificity for CA125, especially among premenopausal women, a population where the prevalence of EOC is low and the likelihood of false positives is high. Thus, CA125 has not been approved by the Food and Drug Administration ("FDA") and/or recommended by the major cancer organizations as a screening test for EOC. It is used, however, to assist physicians in the management of patients post surgery with varying success.

[0043] For this reason, other serum biomarkers are being actively pursued, and some (i.e., HE4) have recently become available to clinicians for the use in management of women with EOC. Serum HE4 levels are elevated in at least a third of EOC patients who do not have tumors that overexpress CA125, suggesting a complementary use of the two tests. Serum biomarkers, such as CA125, are unreliable and not approved by the FDA, creating a lack of effective serum biomarkers available for use in the detection of ovarian cancer. It has been published previously that AC is over expressed in various cancer cell lines and tissues. However, before the method of the current invention, it was never known that enough active AC and/or other ceramidase could be secreted from the tissues to make it stable enough for detection in the blood. This invention is the first to identify that serum activity levels correlate with the stage of cancer and/or the aggressiveness of a tumor.

[0044] In a further embodiment, the disease condition modulated by activation of the sphingolipid pathway is a benign growth. The benign growth can be an ovarian fibroid or a cyst.

[0045] Ovarian fibroids consist of solid growths, as compared to ovarian cysts, which are generally fluid-filled. Fibroids are made of smooth muscular tissues that are bound by fibrous tissues. Patients with fibroids may be required to undergo surgical removal in the presence of pain or discomfort, such as abdominal pain, cramping, bleeding, or bloating.

[0046] Cysts are the most common ovarian pathology. Most of cysts are benign, however some are malignant. An ovarian cyst is formed when part of the ovary is filled with fluid while the ovarian tissue is compressed to the remaining volume. The fluid within the cyst may be clear or turbid. Cysts may also contain small regions of ovarian tissue penetrating from the cyst's boundary into its volume. Such projections are called papillations. A cyst may be divided into several parts to form a multilocular cyst by septations, which are narrow stripes of ovarian tissue. These septations can be either complete (thus forming several separated cystic lumens) or incomplete. The thickness of the wall, i.e., the layer between the cyst and the external ovarian boundary (containing ovarian tissue), the size of the cyst, and the regularity (i.e., smoothness) of the cyst wall are all important parameters for diagnosis. See U.S. Patent No. 6,858,007 to Akselrod et al., which is hereby incorporated by reference in its entirety.

[0047] Additional exemplary pathological reproductive processes are

endometriosis, adenomyosis, dysfunctional uterine bleeding, uterine leiomyoma

(fibroids), and choriocarcinoma. Another pathological reproductive disorder is polycystic ovarian disease ("POD"), by bilaterally enlarged ovaries with numerous follicular cysts, causing infertility. POD is treated with hormonal regulation or surgical intervention.

[0048] In another embodiment, the method is carried out repeatedly in spaced intervals over a period of time.

[0049] Exemplary spaced intervals include carrying out the method of detection prior to, during, or subsequent to, a treatment for a disease condition of the sphingolipid pathway. Medical providers could, therefore, use this method of detection to assess levels of ceramidase, and in turn, the presence or absence of disease conditions modulated by the sphingolipid pathway before, during, and after a treatment protocol.

[0050] In one embodiment, the method is carried out where the subject selected is undergoing surgery. In yet a further embodiment, the method is carried out where the subject selected is undergoing a biopsy.

[0051] Exemplary surgeries for carrying out this method of detection of disease conditions of the sphingolipid pathway include surgeries with varying degrees of invasiveness. Surgeries may be minimally invasive, involving a smaller outer incision used to insert miniaturized instruments within a body cavity or structure, such as laparoscopic surgery. Surgeries may also be open procedures, requiring a larger incision to the area of interest. The surgery may also be by laser as opposed to a traditional scalpel instrument, or by microsurgery which uses an operating microscope to view minute structures. Local or general anesthesia may be used. Types of surgeries included in this method are breast, cardiothoracic, colorectal, endocrine, gynecological, neurosurgery, ophthalmological, oral and maxillofacial, orthopedic, pediatric, podiatric, skin, transplant, urological, and vascular.

[0052] Detection methods used to confirm presence of a fibroid, cyst, or cancer may include ultrasounds or other diagnostic imaging tests, such as x-rays, CT scans, nuclear scans, and MRI scans. If cancer is suspected, a biopsy may be necessary, where a sample of tissue from the mass or the entire mass itself is removed and examined for cancerous cells. Biopsies may vary with respect to both type and invasiveness. Exemplary types of biopsies include needle biopsies, CT-guided biopsies, ultrasound- guided biopsies, bone biopsies, bone marrow biopsies, liver biopsies, kidney biopsies, aspiration biopsies, prostate biopsies, skin biopsies, and surgical biopsies. Most biopsies access suspicious tissue by needle. Some, such as surgical biopsies, are invasive and occur in a hospital or specialized physician's office. Invasive biopsies require several doctor appointments and result in temporary discomfort. The detection method of the present invention may avoid the use of invasive biopsies or surgeries, by detecting ceramidase levels in a subject and comparing those detected levels to a standard sample not having a disease condition.

[0053] In a further embodiment, when the method of comparing indicates that the ceramidase level in the bodily fluid sample is greater than the ceramidase level standard, the selected subject has a disease condition modulated by activation of the sphingo lipid pathway.

[0054] Exemplary methods of comparing ceramidase levels between a subject and a standard include comparing differences in detected ceramidase levels, based on results of protein assays. Ceramidase levels are higher in the presence of a disease condition modulated by activation of the sphingo lipid pathway, as illustrated by Figures 1-3 and 5. A subject with a higher detected ceramidase level in his or her bodily fluid than ceramidase levels in a ceramidase level standard would indicate presence of a disease condition modulated by activation of the sphingolipid pathway.

[0055] Exemplary disease conditions modulated by activation of the sphingolipid pathway include cancer, arthritis, cardiovascular disease, diabetes, and Alzheimer's Disease. Ceramide is a mediator of proinflammatory cytokine signaling and induces cartilage degradation and reduces type II collagen synthesis in articular cartilage. The accumulation of ceramide is associated with arthritis in Farber's disease (Gilbert et al.,

"Sphingomyelinase Decreases Type II Collagen Expression in Bovine Articular Cartilage Chondrocytes via the ERK Signaling Pathway," Arthritis & Rheumatism 58(l):209-220 (2008), which is hereby incorporated by reference in its entirety), and cancer.

Sphingo lipids, specifically sphingosine-1 -phosphate (SIP), are also elevated in the presence of type 1 diabetes (Fox et al, "Circulating Sphingolipid Biomarkers in Models of Type 1 Diabetes," J. Lipid Res. (Published Online November 10, 2010), which is hereby incorporated by reference in its entirety). In addition, because sphingolipid metabolism is a process that modulates the formation of bioactive metabolites, it is essential that the brain contain a proper balance of sphingo lipids. In some brain disorders, including Alzheimer's Disease, sphingo lipids are altered and the present invention can be used to detect such alterations. See Mielke et al., "Alterations of the Sphingolipid Pathway in Alzheimer's Disease: New Biomarkers and Treatment

Targets?," Neuromol. Med. 12:331-340 (2010), which is hereby incorporated by reference in its entirety.

[0056] In another embodiment, when the method of comparing indicates that the ceramidase level in the bodily fluid sample is the same or less than the ceramidase level standard, the selected subject does not have a disease condition modulated by activation of the sphingolipid pathway.

[0057] Exemplary methods of comparing ceramidase levels between a subject and a standard include comparing differences in detected ceramidase levels, based on results of protein assays. Ceramidase levels are higher in the presence of a disease condition modulated by activation of the sphingolipid pathway, as illustrated by Figures 1-3 and 5- 7. A subject with a lower detected ceramidase level in his or her bodily fluid than ceramidase levels in a ceramidase level standard would indicate the subject dies not have a disease condition modulated by activation of the sphingolipid pathway.

[0058] Exemplary disease conditions modulated by activation of the sphingolipid pathway are the same as those described above.

[0059] In yet another embodiment, the selected subject is an individual who previously had a disease condition modulated by activation of the sphingolipid pathway and the comparing is used to determine risk of reoccurrence of the disease condition modulated by activation of the sphingolipid pathway in the subject.

[0060] Exemplary disease conditions modulated by activation of the sphingolipid pathway are the same as those described above. Exemplary disease conditions at high risk of reoccurrence include various types of cancer and benign disease conditions such as fibroids and cysts, as described above.

[0061] Examples of methods used to compare levels of the selected subject to the standard, in order to determine the risk of reoccurrence, are the same as those described above.

[0062] In a further embodiment, the selected subject is an individual with a predisposition to a disease condition modulated by activation of the sphingolipid pathway and the method is used for early detection of the disease condition. [0063] An individual with a predisposition to a disease condition modulated by activation of the sphingolipid pathway includes any person who inherits a genetic defect or deviation that would increase their chances of developing a disease condition.

Exemplary disease conditions modulated by activation of the sphingolipid pathway are the same as those described above.

[0064] For example, inheritance of the Apolipoprotein Ε-ε4 (ΑΡΟΕ-ε4) allele is a risk factor for Alzheimer's Disease. Corder et al., "Gene Dose of Apolipoprotein E Type 4 Allele and the Risk of Alzheimer's Disease in Late Onset Families," Science 261 :921— 3 (1993), which is hereby incorporated by reference in its entirety. Nevertheless, epidemiological studies estimate that 42-68% of Alzheimer's Disease sufferers do not present the ΑΡΟΕ-ε4 allele, suggesting that additional genetic or environmental factors could play essential roles in the disease. Daw et al., "The Number of Trait Loci in Late- Onset Alzheimer Disease," Am. J. Hum. Genet. 66:196-204 (2000), which is hereby incorporated by reference in its entirety. A fact consistent with this observation is that genome-wide screens have identified several regions that show significant linkage to Alzheimer's Disease, of which the most likely to harbor new risk factors are

chromosomes 10 and 12. Pericak- Vance et al., "Complete Genome Screen in Late-Onset Familial Alzheimer Disease," JAMA 278:1237-41 (1997); Rogaeva et al, "Evidence for an Alzheimer Disease Susceptibility Locus on Chromosome 12 and for Further Locus Heterogeneity," JAMA 280:614-8 (1998); Wu et al, "Genetic Studies on Chromosome 12 in Late-Onset Alzheimer Disease," JAMA 280:619-22 (1998); Kehoe et al, "A Full Genome Scan For Late Onset Alzheimer's Disease," Hum. Mol. Genet. 8:237-45 (1999); Scott et al., "Fine Mapping of the Chromosome 12 Late-Onset Alzheimer Disease Locus: Potential Genetic and Phenotypic Heterogeneity," Am. J. Hum. Genet. 66:922-32 (2000); Myers et al., "Full Genome Screen For Alzheimer Disease: Stage II Analysis," Am. J. Med. Genet. 114:235-44 (2002); Mayeux et al, "Chromosome- 12 Mapping of Late- Onset Alzheimer Disease Among Caribbean Hispanics," Am. J. Hum. Genet. 70:237-43 (2002); Blacker et al., "Results of a High-Resolution Genome Screen of 437 Alzheimer's Disease Families," Hum. Mol. Genet. 12:23-32 (2003), which are hereby incorporated by reference in their entireties. Low density lipoprotein receptor related protein 6 (LRP6), a co-receptor for Wnt signaling, is located on chromosome 12. Brown et al., "Isolation and Characterization of LRP6, A Novel Member of the Low Density Lipoprotein Receptor Gene Family," Biochem. Biophys. Res. Commun. 248:879-88 (1998); Pinson et al, "An LDL-Receptor-Related Protein Mediates Wnt Signalling in Mice," Nature 407:535-8 (2000); Tamai et al., "LDL-Receptor-Related Proteins in Wnt Signal Transduction," Nature 407:530-5 (2000); Wehrli et al, "Arrow Encodes an LDL-Receptor-Related Protein Essential For Wingless Signalling," Nature 407:527-30 (2000), which are hereby incorporated by reference in their entireties.

[0065] In yet a further embodiment, the method of detecting a disease condition modulated by activation of the sphingolipid pathway is used with biomarkers other than ceramidase level to identify a particular disease condition modulated by activation of the sphingolipid pathway.

[0066] Biomarkers of the current invention can include traceable substances introduced into the subject as a means to examine whether a change in expression or state of a protein correlates with the risk of or progression of a disease condition modulated by activation of the sphingolipid pathway. Other useful biomarkers include substances whose detection indicates a particular disease state, for example, the presence of an antibody. Presence of disease conditions can also be determined by measuring levels of a biomarker in the biological fluids (i.e. blood, urine) of a patient. Exemplary forms include cancer marker proteins such as, calcitonin, PSA, thymosin β-15, thymosin β-16, and matrix metalloproteinase (MMP).

[0067] In an additional embodiment, the disease condition modulated by the sphingolipid pathway is selected from the group consisting of arthritis, cardiovascular disease, diabetes, and Alzheimer's Disease, as described above.

[0068] Another aspect of the present invention relates to a method of monitoring effectiveness of a therapy in a subject having a disease condition modulated by activation of the sphingolipid pathway. The method includes selecting a subject, providing a baseline ceramidase level in a bodily fluid sample from the selected subject before the therapy, and treating a disease condition modulated by activation of the sphingolipid pathway in the selected subject with the therapy. The method further includes detecting a post-therapy ceramidase level in a bodily fluid sample from the selected subject following the therapy, comparing the baseline ceramidase level with the post-therapy ceramidase level, and identifying whether the therapy has been effective based on the comparing.

[0069] This method is carried out with regard to samples and diseases as described above. Exemplary methods of treatment include surgery, radiation, chemotherapy, and enzyme replacement therapy. Treatment according to the present invention may be modified based on whether the therapy is effective.

[0070] Exemplary forms of surgery include those described above. Additional examples of surgery include hysterectomies, which are currently used for the treatment of a variety of disorders including ovarian fibroids and cysts, as well as cancer of the uterus, ovaries or cervix. A hysterectomy, or surgical removal of the uterus, can occur in three types. A radical hysterectomy is the complete removal of the uterus, cervix, upper vagina, and parametrium. Radical hysterectomies are used to treat cancer. In the presence of advanced stage cancer, lymph nodes, ovaries and fallopian tubes may also usually removed. A second type, the total hysterectomy is a complete removal of the uterus and cervix. Third, a subtotal hysterectomy requires removal of the uterus, leaving the cervix in situ.

[0071] Exemplary forms of radiation therapy include ionizing radiation, such as

X-rays, gamma rays, and charged particles, all which emit high-energy radiation to shrink tumors and kill cancer cells. Radiation may be delivered by external-radiation therapy or internal-radiation therapy). It may also come in the form of systemic radiation therapy or non-ionizing radiation, such as neutron radiation, electromagnetic radiation, visible light, infrared, microwave, radiowaves, very low frequency, extremely low frequency, or thermal radiation. The source of radiation can be either external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiation therapy (EBRT). When the source of radiation is internal to the patient, the treatment is called brachytherapy (BT).

[0072] Radiation according to the present invention is administered with standard equipment manufactured for this purpose, such as AECL Theratron and Varian Clinac. The dose of radiation will depend on numerous factors as is well known in the art. Such factors include the organ being treated, the healthy organs in the path of the radiation that might inadvertently be adversely affected, the tolerance of the patient for radiation therapy, and the area of the body in need of treatment. The dose will typically be between 1 and 100 Gy, and more particularly between 2 and 80 Gy. Some doses that have been reported include 35 Gy to the spinal cord, 15 Gy to the kidneys, 20 Gy to the liver, and 65-80 Gy to the prostate. It should be emphasized, however, that the present invention is not limited to any particular radiation dose. The dose will be determined by the treating physician in accordance with the particular factors in a given situation, including the factors mentioned above.

[0073] The distance between the source of the external radiation and the point of entry into the patient may be any distance that represents an acceptable balance between killing target cells and minimizing side effects. Typically, the source of the external radiation is between 70 and 100 cm from the point of entry into the patient.

[0074] Brachytherapy is generally carried out by placing the source of radiation in the patient. Typically, the source of radiation is placed approximately 0-3 cm from the tissue being treated. Known techniques include interstitial, intercavitary, and surface brachytherapy. The radioactive seeds can be implanted permanently or temporarily. Some typical radioactive atoms that have been used in permanent implants include iodine-125 and radon. Some typical radioactive atoms that have been used in temporary implants include radium, cesium- 137, and iridium- 192. Some additional radioactive atoms that have been used in brachytherapy include americium-241 and gold- 198.

[0075] The dose of radiation for brachytherapy can be the same as that mentioned above for external beam radiation therapy. In addition to the factors mentioned above for determining the dose of external beam radiation therapy, the nature of the radioactive atom used is also taken into account in determining the dose of brachytherapy.

[0076] Exemplary forms of chemotherapy of the present invention include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents. Particular examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-flurouracil, vinblastine, vincristine, bleomycin, paclitaxel

(taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, magastrol melphalan, mercaptopurine, oxaloplatin, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, taxol, and combinations thereof.

[0077] Enzyme Replacement Therapy ("ERT") according to the present invention involves administration, preferably intravenous, of an exogenously-produced natural or recombinant enzyme. ERT proof-of-principle has been established in a Hurler animal model (Shull et al., "Enzyme Replacement in a Canine Model of Hurler Syndrome," Proc. Natl. Acad. Sci. USA 91 :12937-12941 (1994), which is hereby incorporated by reference in its entirety). Others have developed effective methods for cell culture expression of recombinant enzyme in sufficient quantities to be collected for therapeutic use (Kakkis et al., "Overexpression of the Human Lysosomal Enzyme a-L-Iduronidase in Chinese Hamster Ovary Cells," Prot. Express. Purif. 5:225-232 (1994), which is hereby incorporated by reference in its entirety). ERT used in the present invention includes, but is not limited to, Alglucerase, Imiglucerase, Velaglucerase Alfa, Laronidase, Agalsidase Beta, Galsulfase, Algluscosidase Alfa, N-acetylgalactosamine-6 sulfatase, and

Idursulfase.

[0078] According to the present invention, the therapy begins at the time before initial treatment, as well as at the time before any subsequent treatments. Therefore, the sample may be obtained prior to initial treatment and prior to any or all subsequent treatments. In addition, the present invention may be used to treat patients after primary surgery who may not otherwise receive treatment, i.e. those patients with primary complete resection without evidence of residual or distant disease in order to prevent metastatic spread. Furthermore, the present invention may be used as a diagnostic tool as well to determine those patients at highest risk for metastatic spread.

[0079] Effectiveness of treatment may be monitored by comparing detected levels of ceramidase in a sample at various times, including during initial testing and prior diagnosis, as well as prior to and after an initial treatment, and/or prior to and after each subsequent treatment. Effectiveness may also be determined based on measurements after treatment ends.

[0080] In one embodiment, when the comparing indicates that the baseline ceramidase level is greater than the post-therapy ceramidase level, the therapy is effective. In yet a further embodiment, when the comparing indicates that the baseline ceramidase level is less than the post-therapy ceramidase level, the therapy is not effective.

[0081] Exemplary methods of comparing are the same as those described above.

[0082] In another embodiment, the method comprises developing a follow-up treatment regimen based on the comparing and the identifying. [0083] The follow-up treatment regimen may be based on factors including genetics, environment, and habitual behavior, such as smoking, among others. A follow- up treatment regimen may be individually tailored to a subject, based on the subject's disease condition of the sphingolipid pathway and the risk of reoccurrence to the subject.

[0084] In a further embodiment, the therapy is carried out in spaced intervals over a period of time, where the baseline ceramidase level is obtained at an intermediate point within the period of time and the post-therapy ceramidase level is obtained after that intermediate point.

[0085] The determination of whether the method of monitoring effectiveness of a therapy in a subject with a disease condition modulated by activation of the sphingolipid pathway can be completed immediately prior to, during, or subsequent to, treatment for the disease condition, or at any time thereafter. In certain embodiments, the

determination of disease condition modulated by activation of the sphingolipid pathway is completed by obtaining body fluid samples as soon as possible or immediately after exposure to treatment, e.g., within the first hour after the treatment. In other

embodiments, the body fluid sample may be obtained from the subject up to 24 hours after the treatment, preferably within about six hours after the treatment occurs.

Additional body fluid samples may be further obtained within hours, days, or weeks after exposure to the treatment, i.e., as a means to monitor recovery from the disease condition modulated by activation of the sphingolipid pathway.

[0086] In another embodiment, the method is used with biomarkers other than ceramidase level, as described above, to identify a particular disease condition modulated by activation of the sphingolipid pathway.

[0087] Exemplary disease conditions that are modulated by the sphingolipid pathway include arthritis, cardiovascular disease, cancer, diabetes, and Alzheimer's Disease, as described above.

[0088] Another aspect of the present invention relates to a method of monitoring progression of a disease condition modulated by activation of the sphingolipid pathway in a subject. The method includes selecting a subject, providing a baseline ceramidase level in a bodily fluid sample taken from the selected subject at an initial time, and detecting a later ceramidase level in a bodily fluid sample taken from the selected subject at a time after the initial time when the bodily fluid sample corresponding to the baseline ceramidase level is taken. The method further includes comparing the baseline ceramidase level to the later ceramidase level and identifying whether the disease condition modulated by activation of the sphinglolipid pathway has undergone progression or regression, between when the initial and later bodily fluid samples were taken, based on the comparing.

[0089] This method is carried out with regard to samples and diseases described above.

[0090] In yet another embodiment, when the comparing indicates that the baseline ceramidase level is greater than the later ceramidase level, the disease condition modulated by activation of the sphingolipid pathway has regressed.

[0091] For example, tumor regression is triggered by the death of transformed cells. Other disease conditions of the sphingolipid pathway, such as arthritis and

Alzheimer's Disease, show regression by a reduction in symptoms. Disease conditions of the sphingolipid pathway like diabetes and cardiovascular disease show regression by reduced blood sugar levels in a subject with such a disease condition, among others.

[0092] In a further embodiment, when the comparing indicates that the baseline ceramidase level is less than the later ceramidase level, the disease condition modulated by activation of the sphingolipid pathway has progressed.

[0093] According to the present invention, disease conditions of the sphingolipid pathway may progress several ways. For example, cancer cells proliferate as a result of DNA mutations or loss of tumor suppressor gene function in normal cells. These genetic alterations cause many new protein products, such as overexpression of tumor-associated growth factors or chemokines or receptors, that can stimulate other cells (i.e., endothelial cells) to proliferate and form the new blood vessels within the tumor for continued growth and promotion of metastasis. Until such time as metastasis occurs, a tumor, although it may be malignant, is confined to one area of the body. This may cause discomfort and/or pain, or even lead to more serious problems including death, but if it can be located, it may be surgically removed and, if done with adequate care, be treatable. However, once metastasis sets in, cancerous cells have invaded the body and while surgical resection may remove the parent tumor, this does not address other tumors.

[0094] In yet a further embodiment, the method develops a follow-up treatment regimen based on the comparing and the identifying.

[0095] Exemplary forms of follow up treatment include methods of treatment described herein, but are not limited to those described. [0096] Another aspect of the present invention relates to a kit which has a reagent suitable to detect ceramidase level in a bodily fluid sample and instructions for measuring ceramidase level in bodily fluid sample using the reagent. The kit may include an adjuvant.

[0097] The amount of reagent to be administered will, of course, vary depending upon the particular conditions. The amount required to obtain the desired effect may vary depending on the cell type and conditions. Effective amounts can be determined empirically by those of skill in the art. For example, this may involve assays in which varying amounts of ceramidase are administered to cells in culture and the concentration effective for obtaining the desired result is calculated.

[0098] The detection kit according to the invention may contain reporting material in the form of notebooks and writing materials, detection stickers, pouches and the like for taking samples, as well as instructions for use. The detection kit may further comprise sampling tubes. The detection kit will be constructed, for example, in a container, in which all the ingredients required for the detection are inside and which may also have a carrying handle and/or means for attachment to a belt or sling. For completion of the instructions, the detection kit may comprise an electronic time warning system which after each operation indicates to the user that an operation has been completed by means of an optical or acoustical signal.

[0099] In another embodiment, the kit further comprises additional biomarkers to detect the presence or absence of a particular disease condition modulated by the sphingolipid pathway selected from the group consisting of a benign growth, cancer, arthritis, cardiovascular disease, diabetes, and Alzheimer's Disease, as described above.

[00100] The use of ceramidase as a biomarker may also be useful in the detection of other conditions where the sphingolipid pathway, and AC in particular, are abnormally elevated. These include, but are not limited to benign growths, cancer, arthritis, cardiovascular disease, diabetes, Alzheimer's disease, and others.

[00101] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. EXAMPLES

Example 1 - Serum samples.

[00102] Serum samples were collected from healthy individuals, patients with benign gynecologic conditions, or ovarian cancer patients using tiger top tubes containing a clot activator and serum separator gel. Tubes were centrifuged at 2,000x g in a 581 OR centrifuge from Eppendorf (USA). After centrifugation the serum samples were frozen at -20°C for up to 4 weeks prior to determining AC activity. For measurement of the total protein concentration samples were diluted 1 :50 in PBS. Total protein was measured by the Bradford assay.

Example 2 - Tissue Samples.

[00103] Ovarian tissue from patients with benign gynecologic conditions or ovarian cancer was homogenized in a lysis buffer containing 20mM Hepes, 150mM NaCl, 0.2% Igepal pH 7.5, using a tissue tearor homogenizer (model 985370, Biospec Products, inc., USA). Total protein was measured by the Bradford assay.

Example 3 - AC Activity Assay.

[00104] Sixteen microliters of serum were incubated for 17h at 37°C with 16 microliters of 0.2mM NBD-C12-ceramide in 0.1M citrate/phosphate buffer, pH 4.5, 150mM NaCl, and 0.1 % Igepal CA-630. The reactions were stopped by ethanol (100 ml), centrifuged at 16,000 x g, and the supernatants were analyzed using a high performance liquid chromatography system (Acquity UPLC H-Class, Waters).

Fluorescence was quantified using a Waters fluorescence detector set to excitation and emission wavelengths of 435 and 525nm. Separation of the substrate (NBD-C12- ceramide) and product (NBD-C 12-fatty acid) was achieved using an Acquity UPLC BEH CI 8, 1.7 um, 2 x 30 mm column. The amount of product was calculated using a regression equation that was established from a standard curve using NBD-conjugated C12 fatty acid. One unit of AC activity was defined as the amount of enzyme that catalyzes formation of 1 pmol of NBD-conjugated C 12-fatty in 17 hours. Final data was expressed as U/microgram total protein. Example 4 - Design of Study Comparing AC Activity between EOC patients and Healthy Patients.

[00105] The usefulness of determining serum AC activity was determined in 29 women with EOC, as compared to 19 healthy women and 9 women with benign gynecological disease. Within the EOC group, 23 women had late stage (III/IV) cancer, while 6 had early stage (I/II). The analysis was stratified into two groups: evaluation of serum AC activities pre-surgery, and evaluation post-surgery and during chemotherapy. Comparison to CA125 levels was performed, and correlations with surgical and chemotherapy outcomes were assessed, alone and in combination with CA125. Other assessments included correlations with age, menopausal status, and cancer recurrence.

Example 5 - Increased Levels of AC in EOC Patients Pre-surgery and Upon Cancer

Recurrence.

[00106] In pre-surgery assay screens, highly significant differences were observed between the AC levels of healthy control and the EOC groups. Importantly, even the early stage cancer group could be readily discriminated from the normal group (p value <5E-5). As shown in Figure 1, the activity of AC in the serum of ovarian cancer patients pre-surgery was measured from healthy controls, patients with benign gynecological conditions, and cancerous conditions. Samples from patients with advanced stage ovarian cancer exhibited a higher level of AC activity. There was also a significant difference between women with benign gynecological disease and the normal controls. Overall, the serum AC levels in the EOC patients were higher than the benign group. The overall cancer detection rate was 83% (i.e., 24/29 EOC patients were above the highest normal value). AC serum activity levels also were determined at the time of surgery and during chemotherapy for 28 EOC patients. Notably, the average AC values for the patients during chemotherapy were markedly reduced compared to patients pre-treatment.

Moreover, in a small number of treated patients in whom the cancer had recurred, the values were increased. Finally, AC activity levels were assessed in primary cancer tissue from two patients with EOC as compared to 4 individuals with benign disease who underwent surgery (i.e., two with ovarian fibroids and two with ovarian cysts). In both EOC cases the AC activity was elevated, and in one patient with an extremely aggressive and metastatic tumor the values were more than 4 times the other EOC patient with more typical disease. Figure 2 illustrates reduced AC activity in the serum of EOC patients post-surgery and during treatment of chemotherapy. AC activity in ovarian cancer tissue samples or samples of benign gynecological conditions is shown in Figure 3. The highest AC activity level was exhibited by aggressive EOC, as compared to ovarian fibroids and cysts which illustrated low AC activity.

Example 6 - AC Serum Activity Level Used as a Screen for Early Stage EOC.

[00107] The data suggests that the determination of AC serum activity levels may be a useful screening test for the diagnosis of early stage EOC. Moreover, the data suggests that AC levels may be used to assist in the management of patients post-surgery by evaluation of the activity in the tumor tissue and/or by monitoring the activity during and after chemotherapy.

Example 7 - Acid Ceramidase Activity Compared to Acid Ceramidase Protein

Level.

[00108] Seven different cancer cell lines (6 human and 1 mouse) were plated at the same cell density (10⁵) and grown in T-75 culture flasks (DMEM media containing 10% fetal bovine serum) until they were confluent. These included four liver cancer cells lines (HCC), Hepala, Huh7, Hep3b and HepG2, two breast cancer lines (231L and BT474), and one melanoma line (SKMel). The cells were then trypsinized and cell extracts prepared for AC western blotting and activity assays. For western blotting, a specific anti-AC antibody from Santa Cruz Biotechnology Corporation was used. For activity assays NBD C12-ceramide was used as the substrate. Activity assays were carried out at 37 °C for 3 hours, and then analyzed by UPLC. As illustrated in Figure 4, despite large variations in the amount of AC protein by western blotting, there was no correlation with AC activity. This revealed that for AC (one ceramidase) the activity must be determined.

Example 8 - Activity of Acid Ceramidase and Level of Sphingosine in Serum and

Synovial Fluid is Reduced in Dogs Treated for MPS.

[00109] Acid Ceramidase Assay - Samples (dog serum or synovial fluid) were incubated at 37 °C with equal volume of substrate buffer (0.2 mM NBD-C12 ceramide, 0.2M citrate/phosphate buffer, pH 4.5, 0.3 MNaCl, 10% FBS, and 0.2% Igepal CA-630) for up to 6 hours. The reaction was stopped by ethanol and centrifuged, and the supernatant was analyzed using an Acquity H-Class UPLC system (Waters). The separation was achieved on Waters Acquity UPLC BEH CI 8 column (2.0x30 mm, 1.7 μιη). The fluorescent product (NBD-C12 fatty acid) was monitored at the excitation wavelength of 435 nm and the emission wavelength of 525 nm. Quantification of the product peak was calculated using the Waters Empower software according to a standard curve derived from commercial NBD-C12 fatty acid.

[00110] Sphingosine Assay— Prior to quantifying the sphingosine, a total lipid extract was prepared from the dog serum or synovial fluid by chloroform/methanol extraction. The dried lipid was dissolved in ethanol and mixed with a fluorogenic reaction mixture (25 mM sodium borate buffer, pH 9, 1.25 mM sodium cyanide, and 1.25 mM Naphthalene-2,3-dicarboxyaldehyde). After incubation at 50°C for 10 min, the reaction mixture was centrifuged, and the supernatant was analyzed using an Acquity H- Class UPLC system (Waters). The separation was achieved on Waters Acquity UPLC BEH RP18 column (2.0x50 mm, 1.7 μιη). The fluorescent sphingosine-derivative was monitored at the excitation wavelength of 252 nm and the emission wavelength of 483 nm. Quantification of the sphingosine peak was calculated using the Waters Empower software according to a standard curve derived from commercial, purified sphingosine.

[00111] Results - As illustrated by Figures 5A-B, AC activity is significantly elevated in the synovial fluid (Figure 5A) and serum (Figure 5B) of two different dog models of the mucopolysaccharidoses (MPS). MPS diseases are due to specific lysosomal enzyme deficiencies, in this case enzymes that degrade extracellular degenerative joint and bone disease. Animals, in particular the dog models shown in Figure 5, are excellent models of human arthritis.

[00112] In data collected from 4 dogs with MPS I that were treated by intraarticular injection of enzyme replacement therapy (with the commercial enzyme therapy product used to treat MPS I children), synovial fluid from untreated joints had significantly higher AC activity than in treated joints, as shown in Figure 6. In this example, the enzyme (1 mg of recombinant alpha iduronidase) was injected into the right joints (i.e., knees and elbows) of 4 MPS I dogs for 6 months. The left joints (i.e., knees and elbows) were not injected and were used as internal controls. AC activity in the treated joints was significantly lower than in the untreated joints. Similarly to the levels detected of AC, the levels of the lipid sphingosine (a product of acid ceramidase) was reduced in treated joints of dogs with MPS I as illustrated in Figure 7. The reduction of AC activity in Figure 6 was consistent with the reduction of sphingosine levels, the product of this enzyme, in Figure 7. Figures 5-7 show that AC activity can be used to (a) detect MPS diseases and serve as a biomarker for disease progression, and (b) to monitor treatment response (in this example, enzyme therapy). The results illustrated in Figures 5-7 further indicate a correlation of AC with disease severity and with treatment response, when tested animals received ERT.

[00113] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various

modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED:

1. A method of detecting a disease condition modulated by activation of the sphingo lipid pathway, said method comprising:

selecting a subject;

detecting ceramidase level in a bodily fluid sample from said selected subject; and

comparing said detected ceramidase level in said bodily fluid sample to a ceramidase level standard for a subject not having a disease condition modulated by activation of the sphingolipid pathway.

2. The method of claim 1, wherein said detecting comprises:

contacting said bodily fluid with ceramidase assay reagents and determining the ceramidase level in the bodily fluid sample based on said contacting.

3. The method of claim 2, wherein the ceramidase assay is selected from the group consisting of western blot, radioimmuno assay, Bradford protein assay, Biacore Surface Plasmon Resonance, ELISA antibody-based assay, proximity assay, and antibody pull down assay.

4. The method of claim 1, wherein said bodily fluid sample is selected from the group consisting of serum, synovial fluid, cerebrospinal fluid, and peritoneal fluid. 5. The method of claim 1, wherein said disease condition modulated by activation of the sphingolipid pathway is a cancer.

6. The method of claim 5, wherein said cancer is acute lymphocytic leukemia, acute myelogenous leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, renal cancer, prostate cancer, glial and other brain and spinal cord tumors, pancreatic cancer, melanoma, ovarian cancer, liver cancer, or urinary bladder cancer.

7. The method of claim 6, wherein said cancer is ovarian cancer.

8. The method of claim 1, wherein said disease condition modulated by activation of the sphingolipid pathway is a benign growth.

9. The method of claim 8, wherein said benign growth is an ovarian fibroid.

10. The method of claim 8, wherein said benign growth is a cyst.

11. The method of claim 1 , wherein said method is carried out repeatedly in spaced intervals over a period of time.

12. The method of claim 1, wherein said subject selected is undergoing surgery. 13. The method of claim 1, wherein said subject selected is undergoing a biopsy.

14. The method of claim 1, wherein, when said comparing indicates that said ceramidase level in said bodily fluid sample is greater than said ceramidase level standard, said selected subject has a disease condition modulated by activation of the sphingolipid pathway.

15. The method of claim 1, wherein, when said comparing indicates that the ceramidase level in said bodily fluid sample is the same or less than said ceramidase level standard, said selected subject does not have a disease condition modulated by activation of the sphingolipid pathway.

16. The method of claim 1, wherein said selected subject is an individual who previously had a disease condition modulated by activation of the sphingolipid pathway and said comparing is used to determine risk of reoccurrence of the disease condition modulated by activation of the sphingolipid pathway in the subject.

17. The method of claim 1, wherein said selected subject is an individual with a predisposition to a disease condition modulated by activation of the sphingolipid pathway and said method is used for early detection of the disease condition. 18. The method of claim 1 further comprising:

using biomarkers other than ceramidase level to identify a particular disease condition modulated by activation of the sphingolipid pathway.

19. The method of claim 1, wherein said disease condition modulated by the sphingolipid pathway is selected from the group consisting of arthritis,

cardiovascular disease, diabetes, and Alzheimer's Disease.

20. A method of monitoring effectiveness of a therapy in a subject having a disease condition modulated by activation of the sphingolipid pathway comprising:

selecting a subject;

providing a baseline ceramidase level in a bodily fluid sample from said selected subject before said therapy;

treating a disease condition modulated by activation of the sphingolipid pathway in said selected subject with said therapy;

detecting a post-therapy ceramidase level in a bodily fluid sample from said selected subject following said therapy;

comparing said baseline ceramidase level with said post-therapy ceramidase level; and

identifying whether said therapy has been effective based on said comparing.

21. The method of claim 20, wherein said detecting comprises: contacting said bodily fluid with ceramidase assay reagents and determining the ceramidase level in the bodily fluid based on said contacting. 22. The method of claim 21, wherein the ceramidase assay is selected from the group consisting of western blot, radioimmuno assay, Bradford protein assay, Biacore Surface Plasmon Resonance, ELISA antibody-based assay, proximity assay, and antibody pull down assay. 23. The method of claim 20, further comprising:

modifying said therapy based on said identifying.

24. The method of claim 20, wherein said bodily fluid sample is selected from the group consisting of serum, synovial fluid, cerebrospinal fluid, and peritoneal fluid.

25. The method of claim 20, wherein said disease condition modulated by activation of the sphingolipid pathway is a cancer. 26. The method of claim 25, wherein said cancer is acute lymphocytic leukemia, acute myelogenous leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, renal cancer, prostate cancer, glial and other brain and spinal cord tumors, pancreatic cancer, melanoma, ovarian cancer, liver cancer, or urinary bladder cancer.

27. The method of claim 26, wherein said cancer is ovarian cancer. 28. The method of claim 20, wherein said disease condition modulated by activation of the sphingolipid pathway is a benign growth. The method of claim 28, wherein said benign growth is an ovarian fibroid.

30. The method of claim 28, wherein said benign growth is a cyst.

31. The method of claim 20, wherein said method is carried out repeatedly in spaced intervals over a period of time.

32. The method of claim 20, wherein said therapy comprises surgery.

33. The method of claim 20, wherein said therapy comprises taking a biopsy.

34. The method of claim 20, wherein, when said comparing indicates that the baseline ceramidase level is greater than the post-therapy ceramidase level, said therapy is effective.

35. The method of claim 20, wherein, when said comparing indicates that the baseline ceramidase level is less than the post-therapy ceramidase level, said therapy is not effective.

36. The method of claim 20 further comprising:

developing a follow-up treatment regimen based on said comparing and said identifying.

37. The method of claim 20 further comprising:

administering a follow-up treatment regimen comprising surgery, radiation, enzyme replacement therapy, or chemotherapy based on said comparing and said identifying.

38. The method of claim 20, wherein said therapy is carried out in spaced intervals over a period of time, wherein the baseline ceramidase level is obtained at an intermediate point within the period of time and the post-therapy ceramidase level is obtained after that intermediate point.

39. The method of claim 20 further comprising:

using biomarkers other than ceramidase level to identify a particular disease condition modulated by activation of the sphingolipid pathway.

40. The method of claim 20, wherein said disease condition modulated by the sphingolipid pathway is selected from the group consisting of arthritis,

cardiovascular disease, diabetes, and Alzheimer's Disease.

41. A method of monitoring progression of a disease condition modulated by activation of the sphingolipid pathway in a subject comprising:

selecting a subject;

providing a baseline ceramidase level in a bodily fluid sample taken from said selected subject at an initial time;

detecting a later ceramidase level in a bodily fluid sample taken from said selected subject at a time after said initial time when said bodily fluid sample

corresponding to said baseline ceramidase level is taken;

comparing said baseline ceramidase level to said later ceramidase level; and

identifying whether said disease condition modulated by activation of the sphinglolipid pathway has undergone progression or regression, between when said initial and later bodily fluid samples were taken, based on said comparing.

42. The method of claim 41, wherein said detecting comprises:

contacting said bodily fluid with ceramidase assay reagents and

determining the ceramidase level in the bodily fluid based on said contacting.

43. The method of claim 42, wherein the ceramidase assay is selected from the group consisting of western blot, radioimmuno assay, Bradford protein assay, Biacore Surface Plasmon Resonance, ELISA antibody-based assay, proximity assay, and antibody pull down assay.

44. The method of claim 41, further comprising:

treating a disease condition modulated by activation of the sphingolipid pathway in the selected subject with a therapy and

modifying said therapy based on said identifying.

45. The method of claim 41 , wherein said bodily fluid sample is selected from the group consisting of serum, synovial fluid, cerebrospinal fluid, and peritoneal fluid.

46. The method of claim 41, wherein said disease condition modulated by activation of the sphingolipid pathway is a cancer.

47. The method of claim 46, wherein said cancer is acute lymphocytic leukemia, acute myelogenous leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, renal cancer, prostate cancer, glial and other brain and spinal cord tumors, pancreatic cancer, melanoma, ovarian cancer, liver cancer, or urinary bladder cancer.

48. The method of claim 47, wherein said cancer is ovarian cancer.

49. The method of claim 41, wherein said disease condition modulated by activation of the sphingolipid pathway is a benign growth.

50. The method of claim 49, wherein said benign growth is an ovarian fibroid.

51. The method of claim 49, wherein said benign growth is a cyst.

52. The method of claim 41, wherein said method is carried out repeatedly in spaced intervals over a period of time.

53. The method of claim 41, wherein said selected subject has undergone surgery.

54. The method of claim 41, wherein said selected subject has undergone a biopsy.

55. The method of claim 41, wherein, when said comparing indicates that the baseline ceramidase level is greater than the later ceramidase level, said disease condition modulated by activation of the sphingolipid pathway has regressed.

56. The method of claim 41, wherein, when said comparing indicates that the baseline ceramidase level is less than the later ceramidase level, said disease condition modulated by activation of the sphingolipid pathway has progressed.

57. The method of claim 44 further comprising:

developing a follow-up treatment regimen based on said comparing and said identifying.

58. The method of claim 44 further comprising:

administering a follow-up treatment regimen comprising surgery, radiation, enzyme replacement therapy, or chemotherapy based on said comparing and said identifying.

59. The method of claim 41, wherein said selected subject is an individual who previously had a disease condition modulated by activation of the sphingolipid pathway and said comparing and said identifying are used to determine risk of reoccurrence of said disease condition modulated by activation of the sphingolipid pathway in said selected subject.

60. The method of claim 41, wherein said selected subject is an individual with a predisposition to said disease condition modulated by activation of the sphingo lipid pathway in said selected subject and said method is used for early detection of the disease condition.

61. The method of claim 41 further comprising:

using biomarkers other than ceramidase level to identify a particular disease condition modulated by activation of the sphingolipid pathway to detect the presence or absence of the disease condition.

62. The method of claim 41, wherein said disease condition modulated by the sphingolipid pathway is selected from the group consisting of arthritis,

cardiovascular disease, diabetes, and Alzheimer's Disease. 63. A kit comprising:

a reagent suitable to detect ceramidase level in a bodily fluid sample and instructions for measuring ceramidase level in said bodily fluid sample using said reagent. 64. The kit of claim 63, wherein said kit further comprises an adjuvant.

65. The kit of claim 63, wherein said kit further comprises additional biomarkers to detect the presence or absence of a particular disease condition modulated by the sphingolipid pathway selected from the group consisting of a benign growth, cancer, arthritis, cardiovascular disease, diabetes, and Alzheimer's Disease.