WO2014182689A1

WO2014182689A1 - Method of using biomarkers in predicting inflammatory bowel disease

Info

Publication number: WO2014182689A1
Application number: PCT/US2014/036958
Authority: WO
Inventors: Bruce R. YACYSHYN; Mary Beth YACYSHYN; Feng Wang
Original assignee: Yacyshyn Bruce R; Yacyshyn Mary Beth; Feng Wang
Priority date: 2013-05-06
Filing date: 2014-05-06
Publication date: 2014-11-13

Abstract

The method and system of the subject invention is directed to a more effective, individual based treatment regimen which is built on clinical identified target biomarkers. In a preferred embodiment of the invention, the biomarkers identified herein establishes a foundation of IBD target biomarkers can be used to screen and test patients that are susceptible to IBD prior to the outbreak of symptoms.

Description

METHOD OF USING BIOMARKERS IN PREDICTING INFLAMMATORY

BOWEL DISEASE

Technical Field

[0001] The subject invention is a method and process of using specific biomarkers in predicting inflammatory bowel diseases.

Background

[0002] Inflammatory bowel diseases (IBD) are chronic disorders and are viewed as unpredictable diseases and include a heterogeneous group of conditions affecting the gastrointestinal (Gl) tract. Crohn's disease (CD or CR) and ulcerative colitis (UC) are the two main recognized diseases. While the two diseases share much in common, CD typically affects any part of the Gl tract while UC tends to be limited to the large intestine (colon), further the symptoms of CD tend to be more subtle than those of UC and include abdominal pain, diarrhea, and recurrent fever. Symptoms for CD can also include various complications such as inflammation of the eyes, joints, and skin, liver disease, kidney stones, and amyloidosis. Symptoms associated with UC include chronic diarrhea with cramping, abdominal pain, rectal bleeding, loose discharges of blood. Symptoms can also include various complications such as arthritis, inflammation of the eyes, skin ulcers, and liver disease.

[0003] UC may occur in people of any age, but most often starts between ages 15 and 30, or less frequently between ages 50 and 70. UC affects men and women equally and appears to run in families. A thorough physical exam and a series of tests are usually required to diagnose UC. Blood tests are often done to determine anemia, which could mean bleeding in the colon or rectum. The blood tests can also be used to determine white blood cell count for determining the presence of inflammation. Stool samples can also be taken to detect bleeding or infection in the colon or rectum. A colonoscopy or sigmoidoscopy can be performed to examine for inflammation, friability, granularity or bleeding, or ulcerations of the colon wall. Biopsies, barium enema X-rays of the colon can also be taken to provide a view of the colon and return to visualize any radiologic abnormalities supportive of UC that may exist.

[0004] Treatment for UC depends on the severity of the disease and most people are treated with medication. For severe, life-threatening or complicated UC, surgery may be required to remove most or all of colon. Since UC affects individuals differently, treatments must be tailored specifically for each patient. In some cases, symptoms are so severe that the person must be hospitalized, such as an individual experiencing severe bleeding or diarrhea. Treatments typically include the use of Aminosalicylates drugs that contain 5-aminosalicyclic acid (5-ASA), to reduce inflammation. Sulfasalazine is a combination of sulfapyridine and 5-ASA and is used to induce and maintain remission. The sulfapyridine component is linked to the anti-inflammatory 5-ASA until these molecules reach the intestine at that time their azo-bond is cleaved by bacterial enzymes naturally found in the colon and they become independent molecules acting in separate ways. However, sulfapyridine as a whole may lead to side effects such as include nausea, vomiting, heartburn, diarrhea, and headache. Other 5-ASA agents such as olsa!azine, mesa!amine, and balsa!azide, have a different carrier, offer fewer side effects; and may be used by people who cannot take sulfasalazine. 5- ASAs are given orally, through an enema, or in a suppository, depending on the location of the inflammation in the colon. Most people with mild or moderate ulcerative colitis are treated with this group of drugs first. Corticosteroids, such as prednisone and hydrocortisone are also used to reduce inflammation and are typically used with patients who have moderate to severe UC or who do not respond to 5-ASA drugs. These drugs can cause side effects such as weight gain, acne, facial hair, hypertension, mood swings, and an increased risk of infection and are not recommended for long- term use. Immununomoduiators, such as azathioprine and 6-mercapto-purine (6-MP) reduce inflammation by affecting the immune system and are used for patients who have not responded to 5-ASAs or corticosteroids or who are dependent on corticosteroids. However, immunomodulators are slow-acting and may take up to 6 months before the full benefit is seen. Patients taking these drugs must be monitored for complications including pancreatitis and hepatitis, a reduced white blood cell count, and an increased risk of infection. Cyclosporine A may be used with 6-MP or azathioprine to treat active, severe UC in people who do not respond to intravenous corticosteroids. Other drugs may be given to relax the patient or to relieve pain, diarrhea, or infection.

[0005] Crohn's disease (CD) is generally classified as being mild, intermediate, or severe and is the failure of the intestinal mucosal barrier of the Gl tract. Treatment often includes the use of anti-inflammatory drugs, such as 5-aminosalicylates (e.g., mesalamine) or corticosteroids, but they have been found not to be always effective. Immunosuppression with cyclosporine is sometimes beneficial for patients resistant to or intolerant of corticosteroids. However, immunosuppressive drugs, such as cyclosporine, tacrolimus, and mesalamine have been used to treat corticosteroid-resistant cases of CD with mixed success. Treatments have also been proposed for the treatment of CD that include the use of various cytokine antagonists (e.g., IL- 1ra), inhibitors (e.g., of IL-.beta. converting enzyme and antioxidants) and anti-cytokine antibodies. In particular, monoclonal antibodies against TNF- .alpha. have been tried with some success in the treatment of CD.

[0006] Accordingly, predicting which patients are more susceptible to developing severe IBD, and the type of IBD (UC or CD) is important to a clinician in choosing an individualized course of therapy. Further, early aggressive treatment often results in better outcomes than less aggressive treatments, however such aggressive therapies are not appropriate for all patients as not all of them will develop complicated or severe disease. Early use of such therapies can lead to a greater chance of toxicity and adverse effects, such as infections and malignancy, and their high cost prohibits their universal use. Thus, aggressive treatment regimens should be avoided for many patients.

[0007] Better methods and systems of predicting patients that would be susceptible to severe IBD prior to the outbreak of symptoms, therefore having the greatest need for aggressive therapy, will reduce the likelihood of a patient receiving unnecessary aggressive treatments, such as a patient in remission who has a low probability of relapse, that could lead to adverse effects. A reliable method and system to predict which patients will develop more aggressive disease, particularly prior to the patients exhibiting symptoms, allows clinicians to choose strategies that will most likely benefit the patient and create an improved monitoring strategy for each patient thereby reducing the need for prolonged hospitalization and associated costs. Further, predicting outcomes can also be useful in adjusting treatment regimens for patients being treated for IBD.

[0008] Biomarkers have been developed for inflammatory bowel disease (IBD) including the use of fecal calprotectin and lactoferrin proteins for identifying patients with IBD, assessing disease severity and for predicting relapses; the use of Serum anti-Saccharomyces cerevisiae antibody (ASCA) and perinuclear antoneutriphil cytoplasmic antibody (pANCA) biomarkers to differentiate CD from UC; and the use of serum anti-OmpC IgA anti-CBir1 biomarkers with ASCA and other biomarker assays for IBD diagnosis as well as UC and CD differentiation. IBD disease biomarkers including anti-G -CSF antibody, CD11 b, TNF-a, CRP, aldo-keto reductase family 1 B10 (AKR1B10), perforin, NF-kB, CXC-chemokines, Aquaporins, kinesins, adaptor protein-1 (AP-1), C5a, iL-2R, integrins, HCC-4, IL-7, MCP-1 , MSP protein, IL-11 , G- CSF, adrenoreceptors, ST2, E-cadhein, KC, IL-12/23p40, IL-17, chiorotyrosine, PAP/REG3, MIF, DMBT1 , LCN2, IL-22, haptoglobin, CCL20, IL-6, IL-33, CAP37, E4A (UBE4A), CXCL16, resistin, apolipoprotein A-IV, beta-defensin, NOD2/CARD15, NOD1/CARD4, toll-like receptors (TLR) 2 and 4, teptin, adiponectin, IL-10, DPP-IV, and CXCR4 have also been identified. Such biomarkers have also been used for determining the responsiveness of steroid and biological treatments. However, until now, diagnoses and treatments have been based largely on clinical symptoms being exhibited by the patient and there have been no biomarkers identified and systems developed for predicting patients who are most iikeiy susceptible to IBD prior to the patient developing symptoms, or can be used to predict the development of CD or UC, and can be used to determine patients who have the greatest need for early aggressive therapy. It is also desirable to have a method and system that can be used to predict iBD risks, the presence of IBD in an individual, or to tailor a treatment regimen for an individual suffering from IBD prior to the patient exhibiting symptoms.

Disclosure of the Invention

[0009] The method and system of the subject invention is directed to a more effective, individual based treatment regimen which is buitt on clinical identified target biomarkers. In a preferred embodiment of the invention, the biomarkers identified herein establish a basis whereby the onset of IBD can be predicted before clinical changes (symptoms) appear.

[0010] A preferred embodiment of the subject invention is a method and system that utilizes target biomarkers to effectively develop new diagnostics and diagnostics standards for IBD therapeutic strategies.

[0011] Another preferred embodiment of the subject invention is a method and system that utilizes target biomarkers to permit personalized clinical IBD medication regimens based on an individual patient's biomarker profiles.

[0012] Another preferred embodiment of the subject invention is a method and system that operates to identify relatively early stage of IBD using biomarkers which allows for the development of a treatment regimen having greater clinical success while minimizing the likelihood of toxicity and adverse effects, such as infections and malignancy. [0013] Another preferred embodiment of the subject invention is a method and system that operates to identify the onset of IBD, prior to the patient exhibiting symptoms, using biomarkers which allows faster and effective disease control with alternative treatments.

[0014] Another preferred embodiment of the subject invention is a method and system that uses target biomarkers to open more potential targets or target pathways for IBD drug development and provides insights in the understanding of IBD mechanisms.

[0015] Another preferred embodiment of the subject invention is a method and system for patient susceptible to IBD comprising the steps of obtaining a sample from a patient that is susceptible to IBD but prior to the patient exhibiting symptoms of IBD; examining the sample to identify one or more target biomarkers and create a marker profile; using the marker profile to predict IBD; and administering a treatment regimen based on the prediction of IBD. [

[0016] A preferred embodiment of the invention the target biomarkers are selected from a panel consisting of Resistin, C-reactive protein (CRP), Granulocyte colony stimulating factor (G-CSF), pANCA, and Angiotensin converting enzyme (ACE) (CD 143).

[0017] In a preferred embodiment of the invention the method and system includes comparing the levels of one or more of the target biomarkers forming the marker profile for patients that may be suffering from IBD to the levels of the target biomarkers of individuals not suffering from IBD, and for patients having elevated or reduction in levels of one or more of the target biomarkers administrating preventative medication and performing a treatment regimen.

[0018] In a preferred embodiment of the invention, the target biomarker is Resistin and wherein an elevated level is about 52% greater than control (the level of the target biomarker in patients not suffering from UC).

[0019] In a preferred embodiment of the invention, the target biomarker is Resistin and wherein an elevated level is about 26% greater than control (over the level of the target biomarker in patients not suffering from CD).

[0020] In a preferred embodiment of the invention, the target biomarker is C-reactive protein (CRP) and wherein an elevated level is about 5% less than control (the level of the target biomarker in patients not suffering from UC).

[0021] In a preferred embodiment of the invention, the target biomarker is C-reactive protein (CRP) and wherein an elevated level is about 111% greater than control over (the level of the target biomarker in patients not suffering from CD).

[0022] In a preferred embodiment of the invention, the target biomarker is Granulocyte colony stimulating factor (G-CSF) and wherein an elevated level is about 36% greater than control (the level of the target biomarker in patients not suffering from UC).

[0023] In a preferred embodiment of the invention, the target biomarker is Granulocyte colony stimulating factor (G-CSF) and wherein an elevated level is about 49% greater than control (the level in patients not suffering from CD). [0024] In a preferred embodiment of the invention, the target biomarker is pANCA and wherein an elevated level is about 26% greater than control (the level in patients not suffering from UC).

[0025] In a preferred embodiment of the invention, the target biomarker is pANCA and wherein an elevated level is about 2% greater than control (the level in patients not suffering from CD).

[0026] In a preferred embodiment of the invention, the target biomarker is Angiotensin converting enzyme (ACE) (CD 143) and wherein an elevated level is about 5% less than control (the level in patients not suffering from UC).

[0027] In a preferred embodiment of the invention, the target biomarker is Angiotensin converting enzyme (ACE) (CD 143) and wherein an elevated level is a 15% less than control (the level in patients not suffering from CD).

[0028] In another preferred embodiment of the invention, the system and method includes the steps of comparing the level of target biomarkers suffering from IBD and using the target biomarkers to determine if the patient is suffering from UC or CD.

[0029] In a preferred embodiment of the invention the method and system further comprises the step of identifying patients that may be susceptible to IBD from a group of individuals consisting of individuals associated with the military that have been deployed to isolated or distant locations from the United States of America, relatives of patients currently suffering from IBD or has suffered from IBD, and household contacts or friends of patients currently suffering from IBD or has suffered from IBD.

[0030] A preferred embodiment of the invention is a method or system that provides means whereby target biomarkers operate as drug targets that are conventionally used to develop new medications and therapies effective for the treatment of IBD patients.

[0031] Another preferred embodiment of the invention is a method and system that uses target biomarkers as a screening mechanism to conventionally identify therapeutic compounds that may have a therapeutic benefit and potential use for the treatment of IBD patients.

[0032] Another preferred embodiment of the invention, the method and system compares changes in the level of one or more target markers to monitor the therapeutic effectiveness of medications being administered to IBD patients.

[0033] Another preferred embodiment of the invention the method and system is used to identify other proteins, both upstream and downstream, of the disease pathway that may be utilized as additional target biomarkers or as other proteins that may be used for creating medications for treating IBD patients.

[0034] Another preferred embodiment of the invention, the method and system uses target biomarkers to monitor the therapeutic efficacy of the medication being administered to a IBD patient.

[0035] Other embodiments, advantages and objects of the invention will be apparent from the following description and the appended claims.

Brief Description of the Drawings

[0036] To provide a more complete understanding of the present invention and further features and advantages thereof, reference is now made to the following description taken in conjunction with the accompanying drawings, in which:

[0037] FIG. 1 is a schematic representation illustrating the general methodology of the system of the subject invention showing the selection of individuals from one or more populations that may be susceptible to iBD, obtaining samples from the individuals, and developing a panel of target markers that may be used to predict or determine if one or . more of the individuals have IBD prior to diagnosis or symptoms;

[0038] FIG. 2 is a graphical representation showing a significant increase in baseline Resistin levels of CD (CR) and UC patients compared to controls at T1 and only in UC at T2;

[0039] FIG. 3 is a graphical representation showing a significant increase in baseline CRP levels of CD (CR) and UC patients compared to controls at T1 and T2;

[0040] FIG. 4 is a graphical representation showing a significant increase in baseline G-CSF levels of CD (CR) patients compared to controls at T1 and T2;

[0041] FIG. 5 is a graphical representation showing an increase in baseline pANCA levels of CD (CR) patients compared to controls at T1 and a significant increase at T2;

[0042] FIG. 6 is a graphical representation showing an increase in baseline ACE and significantly increased in controls and CD (CR) and decreased in UC over time;

[0043 FIG. 7 is an illustration showing steps comprising the method of a preferred embodiment of the subject invention for predicting patients having IBD; [0044] FIG. 8 is an illustration showing the steps comprising the method of another preferred embodiment of the subject invention for screening, diagnosing, and treating patients diagnosed with IBD; and

[0045] FIG. 9 is an illustration showing the steps comprising the method of another preferred embodiment of the subject invention showing the use of target biomarkers to open target pathways for IBD drug development and for providing insights in the understanding of IBD disease mechanisms. Best Mode for Carrying Out the Invention

[0046] The subject invention identifies panels of protein biomarkers ("target biomarkers") and a method developed to predict patients that may be suffering from IBD prior to the onset of symptoms. Using these target biomarkers as described herein, IBD can be identified earlier. Further, using such target biomarkers a new clinical medication approach has been developed having greater efficacy, and is faster and more effective for disease control while allowing for the development of alternative treatment regimes.

[0047] The method and system of the subject invention is effective for the screening and testing of patients that are susceptible to IBD prior to the outbreak of symptoms. The target biomarker profiles defined herein can be used to determine the propensity of individuals to develop IBD and for the evaluation of treatment response in patients being treated with various pharmaceutical or biological agents. While one or more of the biomarkers may have been previously associated with a disease state on an individual basis, the use of the target biomarkers or profiles have not been used to predict IBD or to diagnose and evaluate treatment responses. Using the target biomarkers and/or profiles provided herein, patients identified as being most susceptible to severe IBD (or diagnosed with severe IBD) can be treated with a more aggressive treatment regimen that will reduce the likelihood of more aggressive procedures, such as surgery. Further, patients that are found to be less likely to have severe IBD, such as a patient in remission who has a low probability of relapse, will receive a less aggressive treatment regimen and a reduce probability of adverse effects.

[0048] Descriptions of well-known starting materials, processing techniques, components and equipment are omitted herein so as not to unnecessarily obscure the invention in detail. It should be- understood, however, that the detailed description and the specific examples, while indicating preferred embodiments if the invention, are given by way of illustration only and not by way of limitation. Various substitutions, modifications, additions and/or rearrangements within the spirit and/or scope of the underlying inventive concept will become apparent to those skilled in the art from this disclosure.

[0049] It should be understood that as used herein the term "sample" means any biological specimen taken from an individual and includes blood, plasma, urine, stool, saliva, and tissue specimen. The term "profile" as used herein means one or more of the target biomarkers as identified herein. The terms "effective amount" or "effective dosage" of medication means the amount or dosage of a drug, medication, or agent that is effective for achieving a therapeutic effect that reduces the severity or prevents one or more of the symptoms associated with IBD (such as those associated with UC or CD). [0050] Resistin is a cysterine-rich adipose-derived peptide hormone that is encoded by the RETN gene. Resistin increases the expression of several pro-inflammatory cytokines including (but not limited to) interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (JL-12), and tumor necrosis factor-a (TNF-a) in an NF-KB-mediated fashion. It has also been shown that Resistin upreguiates intercellular adhesion molecule-1 (ICAM1) vascular cell-adhesion molecule-1 (VCAM1) and CCL2, all of which are occupied in chemotactic pathways involved in leukocyte recruitment to sites of infection. Resistin itself can be upregulated by interleukins and also by microbial antigens such as lipopolysaccharide, which are recognized by leukocytes. Taken together, because Resistin is reputed to contribute to insulin resistance, suggest that Resistin may be a link in the well-known association between inflammation and insulin resistance. It is beiieved that Resistin has features of a proinflammatory cytokine, and could act as a key node in inflammatory diseases.

[0051] C-reactive protein (CRP) is a pentameric protein found in the blood plasma, the levels of which rise in response to inflammation, its physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system via the C1Q complex. CRP levels are often measured for use in screening for infectious and inflammatory diseases where marked increases in CRP indicate the presence of inflammation, infection, trauma and tissue necrosis, malignancies, or autoimmune disorders. Because there are many disparate conditions that can increase levels of CRP, specific disease diagnose cannot be made. CRP is used mainly as a marker of inflammation. Apart from liver failure, there are few known factors that interfere with CRP production. Measuring and charting CRP values can prove useful in determining disease progress or the effectiveness of treatments. Blood, usually collected in a serum-separating tube, is analyzed in a medical laboratory or at the point of care. Various analytical methods are available for CRP determination, such as ELISA, immunoturbidimetry, rapid immunodiffusion, and visual agglutination. A high-sensitivity CRP (hs-CRP) test measures low levels of CRP using laser nephelometry. The test gives results in 25 minutes with a sensitivity down to 0.04 mg/L. Normal concentration in healthy human serum is usually lower than 10 mg/L, slightly increasing with aging. Higher levels are found in late pregnant women, mild inflammation and viral infections (10- 0 mg/L), active inflammation, bacterial infection (40-200 mg/L), severe bacterial infections and burns (>200 mg/L). Several studies investigated differential diagnostic values of CRP in a series of inflammatory disease {including inflammatory bowel disease, Intestinal Lymphoma, Intestinal Tuberculosis and Behcet's Syndrome), and compared CRP to other inflammatory biomarkers, such as ESR and WBC.

[0052] Granulocyte-Colony Stimulating Factor (G-CSF) is secreted by monocytes, macrophages and neutrophils after cell activation or by stromal cells, fibroblasts, and endothelial cells. Epithelial carcinomas, acute myeloid leukemia cells and various tumor cell lines (bladder carcinomas, medulloblastomras), also express this factor. The synthesis of G-CSF can be induced by bacterial endotoxins, TN, IL-1 , and GM-CSF. Prostaglandin E2 inhibits the synthesis of G-CSF. In epithelial, endothelial, and fibroblastic ceils secretion of G-CSF is induced by IL-17. One general effect of treatment with G-CSF appears to be a marked reduction of severe infections and episodes of fever, which are normally observed to occur in patients with ostrann syndrome. G-CSF treatment also allows dose intensification with various antitumor drug regimes.

[0053] p-ANCA, or Perinuclear Anti-Neutrophil Cytoplasmic Antibodiesis is associated with several medical conditions. It is specific, but not sensitive for CD (not UC), so is not useful as a sole diagnostic test. When measured together with Anti-saccharomyces cerevisiae antibodies (ASCA), p- ANCA has been estimated to have a specificity of 97% and a sensitivity of 48% in differentiating patients with UC from normal controls. p-ANCA with specificity to other antigens are associated with inflammatory bowel disease, rheumatoid arthritis, drug-induced vasculitis, autoimmune liver disease, drug induced syndromes and parasitic infections.

[0054] Angiotensin-converting enzyme also known as ACE indirectly increases blood pressure by causing blood vessels to constrict. It does that by converting angiotensin I to angiotensin II, which constricts the vessels. For this reason, drugs known as ACE inhibitors are used to lower blood pressure. ACE, angiotensin I and angiotensin I! are part of the renin-angiotensin system (RAS), which controls blood pressure by regulating the volume of fluids in the body. ACE is secreted in the lungs and kidneys by cells in the inner layer of blood vessels. Elevated levels of ACE are found in sarcoidosis, and are used in diagnosing and monitoring this disease. Elevated levels of ACE are also found in leprosy, hyperthyroidism, acute hepatitis, primary biliary cirrhosis, diabetes mellitus, multiple myeloma, osteoarthritis, amyloidosis, Gaucher disease, pneumoconiosis, histoplasmosis, miliary tuberculosis. Serum levels are decreased in renal disease, obstructive pulmonary disease, and hypothyroidism.

[0055] In developing one or more panels consisting of one or more target biomarkers, serum specimens were obtained from the U.S. Department of Defense Serum Repository (DODSR) which was established in 1989 to store serum following mandatory HIV testing. Referring to FIG. 1, serum specimens were selected from active duty military personnel at two time points (T1 - 2 years before diagnosis, T2 - within 1 year after diagnosis) which had been stored at -30 degrees Celsius and comprised 0.5cc aliquots from 50 UC, 50 CD and 50 controls (25 UC-matched, 25 CD-matched) and de-identified at the time of the analysis. ICD-9 codes for each disease was used to identify CD (555.x) and UC (556.x), and only cases in the last 3 years were included to limit degradation of protein. Both inpatient and outpatient encounters were included requiring 2 visits with the same ICD-9 codes to confirm diagnosis. Controls included age, gender, race and time matched. Candidate target biomarkers were pre-identified with work from ASCEND 3 biomarker sub-study (Mesalamine for mild-moderate UC). . Accordingly, 659/772 UC points consent for proteomic/genetic; serum proteins obtained at baseline and week 6; and 1243 serum samples (284 females, 375 males) screened and quantified by a panel of 212 antibodies (Multi-Anatyte Profiling assay developed by Rules-Based medicine, Inc.). In the analysis, 186/212 proteins were analyzed. Table 1 shows the original set of proteins that were selected and tested by conventional Rules based medicine and was used to establish potential proteins of interest. TABLE 1

PROTEINS FOR ANALYSIS

Protein Sub-Category Results ASCEND III Historical

Discovery

Resistin Hormone Positive Trend Discovery

SICAM-1 inflammatory Statistically Significant Discovery

CRP Inflammatory Statistically Significant Historical

G-CSF Cytokines Statistically Significant Discovery

SCL-70 Auto-antibody No association Discovery

P-ANCA Auto-antibody Statistically Significant Historical

ACE-CD143 Inflammatory Statistically Significant Discovery

Tetanus Toxoid Bacterial proteins Statistically Significant Discovery

Haptoglobin Inflammatory Statistically Significant Discovery

Five subgroups were then established consisting of Inflammatory Discovery Markers (Table 2), Hormones (Table 3), Chemokine/Cytokines (Table 4), Bacterial/Viral Proteins (Table 5), and Autoantibodies (Table 6).

TABLE 2

INFLAMMATORY DISCOVERY MARKERS

TABLE 3

TABLE 4

IL12p40 IL.3 RANTES

TABLE 5

BACTERIAIJVIRAL PROTEINS

TABLE 6

AUTOANTIBODIES

[0056} The various serum samples were analyzed for specific protein expressions for IBD diagnosed patients using a multiplexed Luminex and traditional single ELISAs for 9 antibodies (preclinical and post clinical) and compared to a group of controls. The protein expressions were further compared to levels before and after diagnosis of IBD. A Ko!mogorov-Smirnov analysis was used to test data for normality and due to significant deviation from normality, data was transformed using the natural log to normalize data prior to analysis. One-way ANOVA was used to compare between group baseline differences at time 1 (T1) - preclinical and time 2 (T2) - post clinical in biomarkers. Pairwise comparisons were made using LSD for multiple comparisons and repeated measure ANOVA and paired t-test was used for within group comparisons for each biomarker over 2 time points. Natural log mean data was then converted back to original scale for graphic display. It was also determined that a probability of p<0.05 is considered statistically significant. Table 7 provides a summary of biomarkers (proteins) summarizing changes in levels for UC pre-clinical (before diagnosis) and post- clinical (after diagnosis) from control and percent change from T1 and T2.

Similarly, Table 8 provides a summary of biomarkers (proteins) summarizing changes in levels for CD pre-clinical (before diagnosis) and post-clinical (after diagnosis) from control and percent change from T1 and T2.

TABLE 7

TABLE 8

CD PROTEIN SUMMARY

[0057] The study revealed a number of unexpected results. As shown in FIG. 2, a significant increase in baseline levels was observed for UC and CD patients compared to controls at T1 (prior to diagnosis) and only for UC patients at T2 (after diagnoses) while Resistin levels remained similar over time for each group. As shown in FIG. 3, CRP was significantly higher in CD and UC patients compared to controls at T1 and 12. CRP levels slightly increased for UC patients and decreased in CD but the change was not significant. Referring to FIG. 4, G-CSF was significantly increased in CD compared to controls at T1 but not at T2. Within each group, G-CSF did not significantly change over time. As shown in FIG. 5, pANCA significantly increased in CD patients. Over time and became significant to controls at T2. pANCA was unchanged in controls and UC over time. As shown, pANCA decreased 2.3% in controls while there was a 34.3% increase in patients with CD. ACE in patients diagnosed with UC was significantly greater than that of controls and decreased 3.6% in patients diagnosed with UC and only decreased 13% in controls (FIG. 6).

[0058] Based on the results of the analysis, a panel of target biomarkers have been created that can be used to screen and test patients that are susceptible to IBD prior to the outbreak of symptoms. The target biomarker panel defined in Table 8 can be used to determine the propensity of individuals to develop !BD or diagnose patients having IBD before the outset of symptoms and can be used for the evaluation of treatment response in patients being treated with various pharmaceutical or biological agents. TABLE 8

TARGET BIOMARKER PANEL

Resistin

C-reactive protein (CRP)

Granulocyte colony stimulating factor (G-CSF)

Panca

Angiotensin converting enzyme (ACE) (CD 1 3).

[0059] The method of the subject invention utilizes the panel of target biomarkers as individual markers alone or in any combinations of target biomarkers forming additional panels. The target biomarkers are examined based on their significant difference in levels between individuals not suffering from IBD and individuals that are suffering from IBD prior to the onset of symptoms, in a preferred embodiment of the invention the method and system includes comparing the levels of one or more of the target biomarkers forming the marker profile for patients that may be suffering from IBD to the levels of the target biomarkers of individuals not suffering from IBD, and for patients having elevated or lower levels compared to controls of one or more of the target biomarkers administrating preventative medication and performing a treatment regimen. It has been surprising found for patients suffering from UC, the target biomarker of Resistin, the level of Resistin is about 52% greater than control of patients not suffering from UC and for patients suffering from CD, the level of Resistin is about 26% greater than control over the level of the target biomarker in patients not suffering from CD. The target biomarker of C-reactive protein (CRP) the level is about 5% less than control from the level of the target biomarker in patients not suffering from UC and the target biomarker is about 111% greater than control over the level of the target biomarker in patients not suffering from CD. The target biomarker of Granulocyte colony stimulating factor (G-CSF) is about 36% greater than control over the level of the target biomarker in patients not suffering from UC and about 49% greater than control over the level in patients not suffering from CD. The level of target biomarker of pANCA is about 26% greater than control over the level in patients not suffering from UC and about 2% greater than control over the level in patients not suffering from CD. The target biomarker of Angiotensin converting enzyme (ACE) (CD 143) is about 5% less than control over the level in patients not suffering from UC and is about 15% less than control from the level in patients not suffering from CD.

[0060] The method and system of the subject invention utilizes the panel of target biomarkers as unique tools allowing physicians to identify individuals who may be suffering from IBD prior to the onset of symptoms thereby allowing for the implementation of early optimal personalized IBD therapy strategies. The method system further utilizes the target biomarkers to determine patients that are more likely suffering from CD or from UC. The target biomarkers can also be used to identify and validate new IBD drug targets or can be new drug targets themselves, or used in understanding IBD mechanisms. The target biomarkers can also be used as a new tool for screening IBD therapeutic compounds.

[0061] Personalized medicine promises more effective, individual based treatment regimen which is based on clinical biomarker development. Panels of protein target biomarkers (each panel comprising one or more of the identified target biomarkers) have now been identified as part of the subject invention and have been found effective in diagnosing patients with IBD prior to the onset of symptoms. Using these target biomarkers or panels, patients having IBD can be identified earlier. Further, using such target biomarkers or panels new clinical medication approaches can be more fully developed having greater efficacy, and faster and effective disease control with alternative less aggressive treatments for those not likely to experience severe IBD and more aggressive treatments being given to those who are likely to experience severe IBD without such aggressive early stage treatments.

[0062] By way of a non-limiting example, the subject invention provides a method and system for creating outcomes that predict the likelihood of an individual having IBD and the expected severity of the disease. In a preferred embodiment of the invention as shown in FIG. 7, the method and system includes identifying a population that has been identified as having a higher than average rate of IBD (step 200). A clinician obtains one or more samples (step 202) from individuals ("patients") within the population. The samples are then analyzed and the levels of one or more target biomarkers are obtained (step 204). The levels of the target biomarkers are then compared to levels of controls (individuals not having IBD) (step 206) and an outcome is generated (step 208) that is a prediction as to the likelihood that the patient has IBD.

[0063] It should be understood that by examining the levels of the target biomarkers compared to control can also be used to predict CD or UC. Further, by taking additional samples at a preclinical time (T1) and later at a post clinical time (T2) (step 210), the clinician can determine the likelihood of IBD becoming severe and differentiate between CD and UC. In a preferred embodiment, the levels of the one or more of the panel of target biomarkers obtained from sample(s) are compared in relation to levels for individuals having CD and patients having UC (step 212) and an additional outcome is generated (step 214) that is a prediction as to the likelihood that the patient has CD or UC, The appropriate treatment regimen can then be established (step 216). It should also now be apparent that the panel of target biomarkers identified herein can be conventionally applied as targets of the early diagnosis of !BD, for the development of new personalized IBD drugs, for the measurement of the response to drug therapy, or for assays for compound screening therapeutics.

[0064] In another preferred embodiment of the invention as shown in FIG. 8, the subject invention uses the panel of target biomarkers identified herein for patients with a particular susceptibility for !BD. In this illustrative example, the one or more populations of individuals that are susceptible to IBD. Such populations are military personnel, particularly individuals that have been deployed to isolated or distant locations outside the United States of America, individuals who have previously been diagnosed with IBD as well as family members, household contacts and friends of individuals currently suffering from IBD or has suffered from IBD. In this particular example, the method includes identifying individual within such populations (step 300). A clinician runs one or more samples, such as by the use of blood tests and creates a serum, (step 302) and determines the levels of target biomarkers (step 304) prior to treatment of this patient's IBD. The target biomarkers are compared to controis (step 306) and the appropriate treatment based on the comparisons is provided to the patient (step 308). A clinician obtains additional samples (step 310). The levels of the target biomarkers are then compared to controis, such as for individuals not having IBD (step 312) and depending on the changes in the levels of the target biomarkers, an outcome is created that predicts the efficacy of the administered medication (step 314). For example, if the patient shows significant difference in levels, of the target biomarkers it can be determined that the proscribed medication is improving the patient's condition or not. It should now be apparent to one skilled in the art that the subject invention allows for the development of new personalized IBD drugs, for the measurement and monitoring of a patient's response to drug therapy, or for assays for compound screening therapeutics.

[0065] It should now be apparent to one skilled in the art that the present invention provides a method or process whereby target biomarkers are used for risk assessment and predict with a high degree of reliability the treatment outcome with respect to a patient expressing higher than normal levels of one or more of the disclosed targeted biomarkers and thus provides substantive value in various aspects of patient care management. It should also now be apparent to one skilled in the art that the method and system of the subject invention will prevent or reduce the likelihood of treatment using ineffective medications as well as reducing the possibility of the patient experiencing un-necessary side effects of aggressive treatments whens such treatments are not necessary [0066] It should now be understood that the target biomarkers identified herein and in conjunction with other clinical factors, can be used to tailor treatments for individual patients including selecting specific drug treatments and administration regimes, as well used for developing treatments, therapies and medications. In a preferred embodiment, as shown in FIG. 9, the system and method of the subject invention comprises the steps of identifying patients diagnosed with IBD for the study (step 400). It should be understood that as used in this example, patients can be human or animal subjects for use in the study. One or more samples are obtained from each of the patients (step 402) and the levels of target biomarkers are determined (step 404). New IBD therapeutic agents are identified and administered to the patients (step 406). After the new IBD therapeutic agents have been administered to the patients and given time to react, additional samples are taken from the patients and the levels of target biomarkers are determined and compared to controls (step 408). Based on the comparisons, the effectiveness of the new therapeutic agents are assessed (step 412). For example, if the treatment alters one or more of the target biomarkers the efficacy of the administered medication can be determined and if the treatment is not effective, changes can be made to the therapy. If the subject shows significant difference in levels of the target biomarkers it can be determined that the proscribed medication is improving the subject's condition or not. Using the method and the appropriate target biomarkers, the effective dosage of the medication can also be determined using conventional methods for determining dosages.

[0067] The method and system of the subject invention is directed to a more effective, individual (persona!ized-medicine) based treatment regimen which is built on clinical identified biomarkers. It should now be apparent that the method and system of the subject invention provides an accurate and easy to administer process that can be used for the diagnosis, prognosis, and therapy alternatives for the treatment of IBD. In a preferred embodiment of the invention the method of system of the subject invention provides means whereby the target biomarkers identified herein operate as drug targets that are conventionally used to develop new medications and therapies effective for the treatment of IBD patients. For example, if a patient shows significant difference in levels of the target biomarkers it can be determined that the proscribed medication is improving or not improving the patient's condition.

[0068] In another preferred embodiment of the invention the method and system uses the target biomarkers identified herein as a screening mechanism to conventionally identify therapeutic compounds that may have a therapeutic benefit and potential use for medications to treat IBD patients. For example, by examining target biomarkers, compounds can be identified that are known to effect, produce, modify, or change one or more of the target biomarkers. Such compounds can then be used to create medications for that particular IBD condition.

[0069] In another preferred embodiment of the invention, the method and system uses the target biomarkers identified herein, such as comparing changes in the level of one or more of the target markers as described above, to monitor the therapeutic effectiveness of medications being administered to IBD patients.

[0070] In another preferred embodiment of the invention the method and system of the subject invention operates to identify additional proteins, both upstream and downstream, of the disease pathway for a particular IBD condition. Such proteins are then used as additional target biomarkers for creating medications for the particular IBD condition. Further, after such proteins are identified changes therein (and their effect on target biomarkers) are determined, such information is used to provide insight into the disease mechanism of the particular IBD condition.

[0071] In another preferred embodiment of the invention, the method and system of subject invention uses the target biomarkers identified herein to monitor the therapeutic efficacy of the medication being administered to a IBD patient.

[0072] Although the foregoing invention has been described in some detail for purposes of clarity of understandings, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. It should now be apparent that the various embodiments presented can be easily modified while keeping within the scope and spirit of the subject invention. Accordingly, it should be understood that the present disclosure is to be considered as exemplary of the principals of the invention and is not intended to limit the invention to the embodiments and the specific examples illustrated and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the descriptions and examples contained herein.

Claims

1. A method for treating a patient susceptible to IBD comprising the steps of:

Identifying a population of individuals susceptible to IBD;

obtaining a sample from one or more individuals in the population prior to the individual exhibiting symptoms of IBD;

examining the sample to identify one or more target biomarkers and create a marker profile;

using the marker profile to make a IBD prediction in the one or more individuals; and administering a treatment regimen to each of the one or more individuals based on the prediction of IBD.

2. The method of Claim 1 wherein the one or more target biomarkers is from a panel of target biomarkers consisting of Resistin, C-reactive protein (CRP), Granulocyte colony stimulating factor (G-CSF), pANCA, and Angiotensin converting enzyme (ACE) (CD 143).

3. The method of Claim 1 further comprising the step of comparing the levels of one or more of the target biomarkers forming the marker profile for patients that may be suffering from IBD to the levels of the target biomarkers of individuals not suffering from IBD; and for patients having elevated levels of one or more of the target biomarkers, administrating preventative medication and performing a treatment regimen.

4. The method of Claim 1 wherein the target biomarker is Resistin and wherein an elevated level is about 52% greater than control over the level of the target biomarker in patients not suffering from UC.

5. The method of Claim 1 wherein the target biomarker is Resistin and wherein an elevated level is about 26% greater than control over the level of the target biomarker in patients not suffering from CD.

6. The method of Claim 1 wherein the target biomarker is C-reactive protein (CRP) and wherein an elevated level is about 5% reduction than control from the level of the target biomarker in patients not suffering from UC.

7. The method of Claim 1 wherein the target biomarker is C-reactive protein (CRP) and wherein an elevated level is about 111% greater than control over the level of the target biomarker in patients not suffering from CD.

8. The method of Claim 1 wherein the target biomarker is Granulocyte colony stimulating factor (G-CSF) and wherein an elevated level is about 36% greater than control over the level of the target biomarker in patients not suffering from UC

9. The method of Claim 1 wherein the target biomarker is Granulocyte colony stimulating factor (G-CSF) and wherein an elevated level is about 49% greater than control over the level in patients not suffering from CD.

10. The method of Claim 1 wherein the target biomarker is pANCA and wherein an elevated level is about 26% greater than control over the level in patients not suffering from UC.

11. The method of Claim 1 wherein the target biomarker is pANCA and wherein an elevated level is about 2% greater than control over the level in patients not suffering from CD.

12. The method of Claim 1 wherein the target biomarker is Angiotensin converting enzyme (ACE) (CD 143) and wherein an elevated level is about 5% reduction than control over the level in patients not suffering from UC,

13. The method of Claim 1 wherein the target biomarker is Angiotensin converting enzyme (ACE) (CD 143) and wherein an elevated level is a 15% reduction than control from the level in patients not suffering from CD.

14. The method of Claim 1 further comprising the steps of comparing the level of target biomarkers suffering from IBD and using the target biomarkers to determine if the patient is suffering from UC or CD.

15. the method of Claim 1 wherein the population of individuals susceptible to IBD comprises a group of individuals consisting of individuals associated with the military that have been deployed to isolated or distant locations from the United States of America, relatives of patients currently suffering from IBD or has suffered from IBD, and household contacts or friends of patients currently suffering from iBD or has suffered from IBD.

16. The method of Claim 1 further comprising the step of using one or more of the target biomarkers as drug targets that are conventionally used to develop new medications and therapies effective for the treatment of IBD patients.

17. The method of Claim 1 further comprising the step of using the target biomarkers as a screening mechanism to conventionally identify therapeutic compounds that may have a therapeutic benefit and potential use for the treatment of IBD patients.

18. The method of Claim 1 further comprising the step of comparing changes in the level of one or more of the target biomarkers to monitor the therapeutic effectiveness of medications being administered to IBD patients.

19. The method of Claim 1 further comprising the step of using the one or more target biomarkers to identify other proteins, both upstream and downstream, of the disease pathway that may be utilized as additional target biomarkers or as other proteins that may be used for creating medications for treating IBD patients.

20. The method of Claim 1 further comprising the step of using the one or more target biomarkers to monitor the therapeutic efficacy of the medication being administered to a IBD patient.