WO2020058968A1

WO2020058968A1 - Circadian clock gene expression as a diagnostic tool for inflammatory bowel disease (ibd) and for differentiating between ibd and irritable bowel syndrome (ibs)

Info

Publication number: WO2020058968A1
Application number: PCT/IL2019/051012
Authority: WO
Inventors: Oren FROY; Yael WEINTRAUB
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.; The Medical Research Infrastructure And Health Services Fund Of The Tel Aviv Medical Center
Priority date: 2018-09-17
Filing date: 2019-09-10
Publication date: 2020-03-26

Abstract

The present invention relates to methods and kits for the diagnosis of inflammatory bowel disease (IBD) by detecting the expression of one or more circadian clock gene, or by detecting the expression of proteins encoded by said circadian clock gene. The methods and kits of the invention are further useful in differentiating between IBD and inflammatory bowel syndrome (IBS), for differentiating between the remission or exacerbation states of IBD, and for grading the severity of IBD. In an embodiment, the one or more clock gene biomarker is selected from the group consisting of: Circadian Locomotor Output Cycles Kaput (CLOCK), Aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), Cryptochrome 1 (CRY1), Cryptochrome 2 (CRY2), Period 1 (PERI), and Period 2 (PER2).

Description

CIRCADIAN CLOCK GENE EXPRESSION AS A DIAGNOSTIC TOOL FOR INFLAMMATORY BOWEL DISEASE (IBD) AND FOR DIFFERENTIATING BETWEEN IBD AND IRRITABLE

BOWEL SYNDROME (IBS)

FIELD OF THE INVENTION

The present invention relates to the detection and measurement of biomarkers. More specifically, the present invention relates to methods and kits for the detection and measurement of biomarkers for diagnosing and monitoring inflammatory bowel disease (IBD).

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic or relapsing immune activation and inflammation within the gastrointestinal (Gl) tract. The etiology of IBD is incompletely understood. Infectious agents, genetic predisposition, environmental factors, immune response and adhesion molecule expression on intestinal epithelial cells all play a role in disease pathogenesis.

Sleep dysfunction has been implicated as a potential trigger of IBD flares. Sleep disturbances affect immune function, and activation of the immune system disturbs sleep patterns. This duality is mediated by cytokines, such as tumor necrosis factor alpha (TNFa), interleukin-1 (IL-1) and IL-6, known to be involved in the pathogenesis of IBD. Indeed, increased levels of plasma TNF-a, IL6 and C reactive peptide (CRP) were found in subjects with sleep disturbances.

Sleep dysfunction also alters the synchrony among clock genes leading to disruption of overall circadian regulation. Specifically, in the intestine, it is manifested by increased gut cellular permeability. The circadian clock regulates the activity of the immune system by modulating the number of circulating lymphocytes, natural killer (NK) cells, antibody production, complement levels, cytokine production, host-pathogen interactions and the activation of innate and adaptive immunity. Diurnal variations were also found in the expression of adhesion molecules and chemokines on endothelial cells influencing trafficking of leukocytes into the intestinal tissue.

The central circadian clock, located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus, generates endogenous ~24-h rhythms. Similar clocks are found in peripheral tissues, such as the liver, muscle and white blood cells (WBC). Little is known about the interaction between the circadian clock and IBD.

Current IBD diagnosis requires a multi-faceted workup: blood and stool samples, invasive endoscopy and bowel imaging. Ultimate diagnosis is based on macroscopic and histopathological findings obtained through colonoscopy. However, colonoscopy poses risks, such as sedation complications, bleeding from biopsy sites and tears in the intestinal wall (perforation).

Fecal calprotectin (Fcal), a stool inflammatory marker, is commonly used to support IBD diagnosis and follow disease activity. Nevertheless, it is a non-specific marker whose levels increase in other intestinal pathologies, such as infection, appendicitis, celiac and even non-steroidal anti-inflammatory (NSAID), including aspirin, drug use.

As IBD is considered a systemic disorder, many patients suffer from extra-intestinal manifestations involving joint, eyes, skin, etc.

Thus, to reliably and specifically diagnose IBD, there is an urgent need to develop specific, non-invasive tools relying on systemic rather than local markers. The present invention provides such a systemic marker, which is the expression of the circadian clock genes or proteins in a subject. It is therefore an object of the present invention to provide a method and kit for the rapid and reliable screening of patients suffering from gastrointestinal disorders, and to distinguish between IBD and non-IBD patients.

Another object of the present invention is to provide a method and kit for the specific diagnosis of IBD, particularly at an early stage of the disease presentation, at preliminary stages of symptoms, prior to evolution of complications.

It is a further object of the invention to provide a method and kit for monitoring the response of an IBD patient to treatment.

It is a still further object of the invention to provide a method and kit for accurately predicting imminent flares in an IBD patient by identifying an alteration in the disease state of the patient.

Other objects and advantages of the invention will become apparent as the description proceeds.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a method for detecting inflammatory bowel disease (IBD) in a subject. In another embodiment, the invention provides a method for the treatment of IBD in a subject suspected of having IBD. In a further embodiment, the invention provides a method of differentiating between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) in a subject. In a still further embodiment, the invention provides a method for determining the severity of an inflammatory bowel disease. In yet another embodiment, the invention provides a method for identifying an alteration in the disease state of an IBD patient. In a further embodiment, the invention provides a method for monitoring the response of a subject diagnosed with IBD to treatment. All the above and other characteristics and advantages of the invention will be further understood through the following illustrative and non-limitative description of embodiments thereof, with reference to the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1F show relative mRNA expression levels of clock genes in intestinal biopsies of newly diagnosed IBD patients.

Fig. 1A shows relative mRNA levels of Circadian Locomotor Output Cycles Kaput (CLOCK) gene in intestinal biopsies of newly diagnosed IBD patients and in healthy subjects; data are means ± SE; groups designated with the letter B are statistically different from the group designated with the letter A in a comparison between the 3 groups; asterisks denote significant differences between the two indicated groups, as analyzed using Student's t-test; *P<0.05.

Fig. IB shows relative mRNA levels of Aryl hydrocarbon receptor nuclear translocator like protein 1 or Brain and muscle Arnt-like protein-1 (BMAL1) in intestinal biopsies of newly diagnosed IBD patients and in healthy subjects; data are means ± SE; groups designated with the letter B are statistically different from the group designated with the letter A in a comparison between the 3 groups.

Fig. 1C shows relative mRNA levels of Cryptochrome 1 (CRY1) in intestinal biopsies of newly diagnosed IBD patients and in healthy subjects; data are means ± SE; the letter A denotes no significant difference between the 3 groups.

Fig. ID shows relative mRNA levels of Cryptochrome 2 (CRY2) in intestinal biopsies of newly diagnosed IBD patients and in healthy subjects; data are means ± SE; groups designated with the letter B are statistically different from the group designated with the letter A in a comparison between the 3 groups; asterisks denote significant differences between the two indicated groups, as analyzed using Student's t-test; **P<0.01.

Fig. IE shows relative mRNA levels of Period 1 (PERI) in intestinal biopsies of newly diagnosed IBD patients and in healthy subjects; data are means ± SE; groups designated with the letter B are statistically different from the group designated with the letter A in a comparison between the 3 groups; asterisks denote significant differences between the two indicated groups, as analyzed using Student's t-test; *P<0.05.

Fig. IF shows relative mRNA levels of Period 2 (PER2) in intestinal biopsies of newly diagnosed IBD patients and in healthy subjects; data are means ± SE; groups designated with the letter B are statistically different from the group designated with the letter A in a comparison between the 3 groups; asterisks denote significant differences between the two indicated groups, as analyzed using Student's t-test; ***P<0.001.

Abbreviations: HC (biopsies of healthy controls); HS (healthy sick, non-inflamed biopsies of IBD patients); SS (sick sick, inflamed biopsies of IBD patients).

Figs. 2A-2F show relative mRNA expression levels of clock genes in white blood cells (WBCs) of newly diagnosed IBD patients.

Fig. 2A shows relative mRNA levels of CLOCK gene in WBCs of newly diagnosed IBD patients (IBD) and in healthy control subjects (HC); *P<0.05.

Fig. 2B shows relative mRNA levels of BMAL1 in WBCs of newly diagnosed IBD patients (IBD) and in healthy control subjects (HC); *P<0.05.

Fig. 2C shows relative mRNA levels of CRY1 in WBCs of newly diagnosed IBD patients (IBD) and in healthy control subjects (HC); *P<0.05.

Fig. 2D shows relative mRNA levels of CRY2 in WBCs of newly diagnosed IBD patients (IBD) and in healthy control subjects (HC); ***P<0.001.

Fig. 2E shows relative mRNA levels of PERI in WBCs of newly diagnosed IBD patients (IBD) and in healthy control subjects (HC); ***P<0.001.

Fig. 2F shows relative mRNA levels of PER2 in WBCs of newly diagnosed IBD patients (IBD) and in healthy control subjects (HC); **P<0.01.

Figs. 3A-3F show relative mRNA expression levels of clock genes in WBCs of newly diagnosed ulcerative colitis patients, Crohn's disease patients and healthy control subjects. Fig. 3A shows relative mRNA levels of CLOCK gene in WBCs of newly diagnosed ulcerative colitis (UC) patients, Crohn's disease (CD) patients and healthy control subjects (HC); data are means ± SE; the group designated with the letter B is statistically different from the group designated with the letter A in a comparison between the 3 groups, while the group designated with the letters AB is not significantly different from the group designated with either A or B; asterisks denote significant differences between the two indicated groups, as analyzed using Student's t-test; *P<0.05.

Fig. 3B shows relative mRNA levels of BMAL1 in WBCs of newly diagnosed ulcerative colitis (UC) patients, Crohn's disease (CD) patients and healthy control subjects (HC); data are means ± SE; the group designated with the letter B is statistically different from the group designated with the letter A in a comparison between the 3 groups, while the group designated with the letters AB is not significantly different from the group designated with either A or B; asterisks denote significant differences between the two indicated groups, as analyzed using Student's t-test; *P<0.05.

Fig. 3C shows relative mRNA levels of CRY1 in WBCs of newly diagnosed ulcerative colitis (UC) patients, Crohn's disease (CD) patients and healthy control subjects (HC); data are means ± SE; the group designated with the letter B is statistically different from the group designated with the letter A in a comparison between the 3 groups, while the group designated with the letters AB is not significantly different from the group designated with either A or B; asterisks denote significant differences between the two indicated groups, as analyzed using Student's t-test; **P<0.01.

Fig. 3D shows relative mRNA levels of CRY2 in WBCs of newly diagnosed ulcerative colitis (UC) patients, Crohn's disease (CD) patients and healthy control subjects (HC); data are means ± SE; the group designated with the letter B is statistically different from the group designated with the letter A in a comparison between the 3 groups, while the group designated with the letters AB is not significantly different from the group designated with either A or B.

Fig. 3E shows relative mRNA levels of PERI in WBCs of newly diagnosed ulcerative colitis (UC) patients, Crohn's disease (CD) patients and healthy control subjects (HC); data are means ± SE; the group designated with the letter B is statistically different from the group designated with the letter A in a comparison between the 3 groups, while the group designated with the letters AB is not significantly different from the group designated with either A or B.

Fig. 3F shows relative mRNA levels of PER2 in WBCs of newly diagnosed ulcerative colitis (UC) patients, Crohn's disease (CD) patients and healthy control subjects (HC); data are means ± SE; the group designated with the letter B is statistically different from the group designated with the letter A in a comparison between the 3 groups, while the group designated with the letters AB is not significantly different from the group designated with either A or B; asterisks denote significant differences between the two indicated groups, as analyzed using Student's t-test; **P<0.01.

Fig. 4 shows the ratio between the expression levels of CLOCK, BMAL1, CRY1, CRY2, PERI and PER2 genes in WBCs of treated IBD patients compared to the same patients before treatment (at the time of diagnosis); value >1 represents higher expression levels of clock genes in patients undergoing treatment than the expression levels at the time of diagnosis; data are means ± SE.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have surprisingly found that inflammatory bowel disease (IBD) can be accurately detected, diagnosed and monitored by analyzing the circadian clock gene expression in a sample obtained from a subject. The invention is based on the identification of biomarker genes, belonging to the circadian clock gene family, that enable the specific and rapid detection of IBD, and the differentiation between IBD and other gastrointestinal disorders, such as irritable bowel syndrome (IBS).

Accordingly, the quantification of the expression levels of several clock genes or proteins, obtained from a biological sample of a subject can be used to determine whether the subject is suffering from IBD. The sample can be obtained from the blood, white blood cells, serum, plasma or is a biopsy from an inflamed or non-inflamed tissue of the intestines.

According to one specific embodiment of the invention, rapid detection of IBD can be carried out by routine means, such as a simple blood test, while relying on systemic markers rather than local indicators, for example, genes expressed in white blood cells (WBCs) compared to intestinal biopsies.

More specifically, the present invention relates to methods that allow the detection and monitoring of IBD based on the measurement of six clock gene biomarkers, either alone, together or in sub-combinations, using gene specific oligonucleotides (i.e., mRNA) or antibodies for the quantification of the expression levels of said biomarkers. The oligonucleotides or antibodies are designed to recognize specifically the gene sequence or biomarker protein.

The biomarker genes, belonging to the circadian clock gene family, which are encompassed by the present invention, are (accession numbers within parenthesis): Circadian Locomotor Output Cycles Kaput (CLOCK) (NM_001267843, NM_004898), Aryl hydrocarbon receptor nuclear translocator-like protein 1 or Brain and muscle Arnt-like protein-1 (BMAL1) (NM_001351813.1, NM_001351814.1, NM_001351807.1,

NM_001351810.1, 001351804.1, NM_001351811.1, NM_001351824.1, NM_001351815.1, NM_001351821.1, NM_001351806.1, NM_001351816.1, NM_001351812.1, NM_001351820.1, NM_001351809.1, NM_001351805.1, NM_001351819.1, NM_001351818.1, NM_001351808.1, NM_001351817.1, NM 001351822.1, NM 001351823.1, NM 001297724.1, NM 001297722.1,

NM_001297719.1, NM_001030273.2, NM_001030272.2, NM_001178.5), Cryptochrome 1 (CRY1) (NM_004075.4), Cryptochrome 2 (CRY2) (NM_021117.4, NM_001127457.2), Period 1 (PERI) (NM_002616.2) and Period 2 (PER2) (NM_022817.2). The clock mechanism in both suprachiasmatic nucleus (SCN) neurons and peripheral tissues consists of CLOCK and BMAL1 (brain-muscle-Arnt-like 1) that heterodimerize and bind to E-box sequences to mediate transcription of a large number of genes, including Periods (Perl, Per2, Per3) and Cryptochromes (Cryl, Cry2). PERs and CRYs constitute part of the negative feedback loop and when they are produced in the cytoplasm, they oligomerize and translocate to the nucleus to inhibit CLOCK:BMALl-mediated transcription.

The biomarkers and the biomarker panels, which include any combination of the six biomarkers disclosed herein, can be used in methods to screen and diagnose subjects that have or are at risk for having IBD; to monitor individuals who are undergoing therapies for IBD in order to evaluate their responsiveness for treatment and adjust the therapeutic regime; to differentially diagnose disease states associated with IBD; to evaluate the severity or progression of IBD in a patient; or to select or modify therapies or interventions for use in treating subjects with the disease. The methods of the invention include measuring the levels of clock biomarkers in an individual and comparing those to reference levels of biomarkers in a normal control, a population with IBD, and/or levels taken at a previous time point from the individual being monitored.

In one aspect, the invention provides a method of detecting inflammatory bowel disease (IBD) in a subject, comprising obtaining a biological sample from the subject, measuring the expression level of at least one clock biomarker and comparing the measurement with a reference value of the biomarker. Specifically, the method comprises:

a. obtaining a biological sample from a subject;

b. determining the expression level of one or more clock gene or protein biomarker; c. comparing the expression level of the biomarker with at least one reference value of the biomarker; and

d. determining the deviation of said expression level from said reference value, to decide whether such a deviation is indicative of the existence of IBD.

In another aspect, the invention provides a method for the treatment of IBD in a subject suspected of having IBD, the method comprising:

a. detecting if the subject has IBD as specified above; and

b. administering to a subject tested positive in (a) an anti-IBD treatment.

According to one embodiment, the method further comprises:

c. monitoring the response of the subject to the anti-IBD treatment, if the expression levels of the tested one or more clock biomarker is lower than the at least one reference value.

In a specific embodiment, the invention provides a method for the treatment of IBD in a subject suspected of having IBD, the method comprising:

a. detecting if the subject has IBD as specified above; and

b. administering to a subject tested positive in (a) an anti-IBD treatment selected from aminosalicylates compounds such as 5-aminosalicylic acid (5-ASA), oral and topical enemas, systemic corticosteroids, topical corticosteroids, thiopurines, methotrexate, calcineurin inhibitors selected from cyclosporine and tacrolimus, anti-tumor necrosis factor alpha (anti-TNFa), anti-a4 integrin subunit antibody, anti-p7 integrin subunit antibody, anti-intercellular adhesion molecule 1 (ICAM- 1) antisense oligonucleotide, IL12/23 pathway blockage agent, anti-p40 antibody, anti-pl9 antibody, anti-IL6 antibody, a nti-l L6 receptor antibody, Janus Kinase inhibitors (JAK) selected from JAK1, JAK2, and JAK3 inhibitors, anti- Madcam antibodies, SMAD7 antisense oligonucleotide, laquinimod, sphingosine-l-phosphate (SIP) pathway modulation drugs, S1P1/S1P5 agonists, Treg cell therapy, gd T cell therapy, modulation of mucosal immunity by helminths, probiotic agents, prebiotics, synbiotics, granulocyte/monocyte apharesis, fecal microbiota transplantation, antibiotics, anti-mycobacterial treatment, curcumin, herbs and plants with immune-regulating characteristics, vitamin and mineral supplementations, exclusive enteral nutrition, partial enteral nutrition, specific exclusion diets, proton pump inhibitors, surgical resection and/or endoscopic dilatation, complementary and alternative medicine, and any combination thereof; and optionally,

c. changing the dose, timing, or type of treatment according to said detection.

It should be noted that treatment efficiency can be further monitored by measuring clock expression at various time points, such as after each change of dose, timing or type of treatment.

In another aspect, the invention provides a method of differentiating between IBD and other gastrointestinal disorders, specifically irritable bowel syndrome (IBS), using the clock biomarkers.

In some embodiments, the method comprises obtaining a biological sample from a subject, determining the expression level of one or more clock gene biomarker, comparing the expression level of the biomarker with at least one reference value of the biomarker, and making a diagnosis of IBD if the expression level of the biomarker is lower than the reference value level, or making a diagnosis of IBS if the expression level of the biomarker is equal or higher than the reference value level.

According to a specific embodiment, the invention provides a method of differentiating between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) in a subject, comprising:

a. obtaining a biological sample from a subject; b. determining the expression level of one or more clock gene biomarker in said biological sample;

c. comparing the expression level of the biomarker with at least one reference value of the biomarker;

d. differentiating between a diagnosis of IBD or of IBS, according to the result of said comparison; and optionally

e. changing the dose, timing, or type of treatment according to said differentiation.

According to another specific embodiment, the invention provides a method of differentiating between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) in a subject, comprising:

a. determining the expression level of one or more clock gene biomarker in a biological sample obtained from the subject;

b. comparing the expression level of the biomarker with at least one reference value of the biomarker; and

c. differentiating between a diagnosis of IBD or of IBS according to the result of said comparison.

In cases the reference value pertains to a healthy individual, if the expression level of the biomarker is lower than the reference value level, making a diagnosis of IBD. Alternatively, if the expression level of the biomarker is equal or higher than the reference value level, making a diagnosis of IBS.

In a further aspect, the invention provides a method of treating a subject with a disease selected from IBD and IBS. The method comprises determining if the subject has IBD or IBS according to the method of differentiating between IBD and IBS as specified above, and if the subject has IBS, providing an IBS treatment. The IBS therapy may be selected from general management including exercise, and dietary modifications such as gluten free and low in fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP diet); medical management including antidiarrheal medications such as peristalsis inhibitors and serotonin agents; antispasmodics such as nutritional supplements, anticholinergics, and calcium channel blockers; fiber supplements; laxative agents such as osmotic and stimulant laxatives; prosecretory drugs; modification of the microbiota with probiotics or antibiotics; central acting treatments selected from antidepressants such as TCA-tricyclic antidepressants, SSRI-selective serotonin reuptake inhibitors, and SNRI-serotonin norepinephrine reuptake inhibitors; psychological and behavioral therapies; complementary and alternative medicine. The IBS therapy may include any combinations of the above.

Alternatively, if the patient has IBD, the method comprises providing an IBD treatment selected from: aminosalicylates/5-ASA compounds such as oral and topical enemas, systemic corticosteroids, topical corticosteroids, immunomodulators such as thiopurines, methotrexate, and calcineurin inhibitors (cyclosporine, tacrolimus), anti- TNFa, anti-adhesion therapy such as anti-a4 integrin subunit antibody, anti-p7 integrin subunit antibody, anti-intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, IL12/23 pathway blockage agent such as anti-p40 antibody, and anti- pl9 antibody, a nti-l L6 antibody, anti-IL6 receptor antibody, Janus Kinase inhibitors (JAK) selected from JAK1, JAK2, and JAK3 inhibitors, anti-Madcam antibodies, SMAD7 antisense oligonucleotide, laquinimod, sphingosine-l-phosphate (SIP) pathway modulation drugs such as S1P1/S1P5 agonists, Treg cell therapy, y6 T cell therapy, modulation of mucosal immunity by helminths, probiotic agents, prebiotics and synbiotics, granulocyte/monocyte apharesis, fecal microbiota transplantation, antibiotics and anti-mycobacterial treatment, curcumin and other herbs and plants with immune-regulating characteristics, vitamin and mineral supplementations, exclusive enteral nutrition, partial enteral nutrition, specific exclusion diets, proton pump inhibitors, surgical resection and/or endoscopic dilatation, complementary and alternative medicine. In the methods of the invention, IBD is considered to be progressive, or at the exacerbation stage ("flare-up"), if the level of at least one clock gene biomarker decreases from a reference level over time. In contrast, the disease is in remission, if the level of at least one clock gene biomarker remains constant over time, or approaches the reference level of a healthy individual or of the subject itself.

In a still further aspect, the present invention provides a method for determining the severity of an inflammatory bowel disease (IBD) in a subject.

In some embodiments, the method comprises obtaining a biological sample from a subject, determining the expression level of one or more clock gene biomarker in said sample, comparing the expression level of the biomarker with at least one reference value. A decrease or increase in the expression level relative to the reference value indicates the severity of the disease. The method further optionally comprises the step of changing the dose, timing, or type of treatment according to the determination.

In other embodiments, invention provides a method for determining the severity of an IBD in a biological sample obtained from subject, the method comprising determining the expression level of one or more clock biomarker in the sample, and comparing the expression level of the biomarker with at least one reference value. A decrease or increase in the expression level relative to the reference value indicates the severity of the disease.

In another aspect, the present invention provides a method for identifying an alteration in the disease state of an IBD patient, from remission or exacerbation, or vice versa. The method enables the identification of whether a flare-up of IBD in a subject is imminent.

In some embodiments, the method comprises the following:

a. obtaining two or more biological samples from a subject at different times; b. determining the expression level of one or more clock gene biomarker in a first biological sample obtained from the patient at a first time point, to create a first biomarker profile;

c. determining the expression level of one or more clock gene biomarker in a second biological sample obtained from the patient at a second time point, to create a second biomarker profile; and

d. comparing the first biomarker profile and the second biomarker profile, wherein a decrease or increase in the second biomarker profile relative to the first biomarker indicates that the disease state is altered; and optionally

e. changing the dose, timing, or type of treatment according to said identification.

In other embodiments, the invention provides a method for identifying an alteration in the disease state of an I BD patient by analyzing two or more biological samples taken at different times, comprising:

a. determining the expression level of one or more clock gene biomarker in a first biological sample obtained from the patient at a first time point, to create a first biomarker profile;

b. determining the expression level of one or more clock gene biomarker in a second biological sample obtained from the patient at a second time point, to create a second biomarker profile; and

c. comparing the first biomarker profile and the second biomarker profile, wherein a decrease or increase in the second biomarker profile at a second time point relative to the first biomarker profile at a first time point indicates that the disease state is altered.

In a another aspect, the invention provides a method for monitoring the response of a subject diagnosed with I BD to treatment by analyzing two or more biological samples taken at different times. In some embodiments, the method comprises:

a. obtaining a first biological sample from a subject;

b. determining the expression level of one or more clock gene biomarker in said first biological sample, to create a first biomarker profile;

c. treating the subject for IBD;

d. obtaining a second biological sample from said subject at a time after treatment; e. determining the expression level of one or more clock gene biomarker in said second biological sample, to create a second biomarker profile;

f. comparing the first biomarker profile and the second biomarker profile, wherein a decrease or increase in the second biomarker profile relative to the first biomarker indicates responsiveness of the subject to the treatment; and optionally

g. changing the dose, timing, or type of treatment according to said analysis.

In other embodiments, the invention provides a method for monitoring the response of a subject diagnosed with IBD to treatment by analyzing two or more biological samples taken at different times, comprising:

a. determining the expression level of one or more clock gene biomarker in a first biological sample obtained from the subject, to create a first biomarker profile; b. treating the subject for IBD;

c. determining the expression level of one or more clock gene biomarker in a second biological sample obtained from the subject a time point after said treating, to create a second biomarker profile; and

d. comparing the first biomarker profile and the second biomarker profile, wherein a decrease or increase in the second biomarker profile relative to the first biomarker indicates responsiveness of the subject to the treatment.

Specifically, a decrease in the second biomarker profile relative to the first biomarker indicates that the IBD patient is not responding well to the treatment, and the treatment should be replaced or the therapeutic regime should be adjusted. I n contrast, an increase in the second biomarker profile relative to the first biomarker indicates that the IBD patient is responding well to the treatment.

The use of the clock biomarkers allows for real-time testing to diagnose and monitor the disease state and treatment of IBD. Measurement of any combination of biomarkers described herein may be used to assemble a biomarker panel. The combination may refer to the measurement of an entire set or any su bset or sub-combination of biomarkers. Specifically, the detection of a plurality of biomarkers in a sample can increase the sensitivity and/or specificity of the test. Thus, in some embodiments, a one or more clock gene/protein biomarker or a biomarker panel as described herein may be used to measure and determine the expression level of 1, 2, 3, 4, 5, or 6, clock gene/protein biomarkers. Accordingly, the one or more clock gene biomarker is selected from the group consisting of: two biomarkers, three biomarkers, four biomarkers, five biomarkers and six biomarkers.

Relevant biomarkers according to the invention are selected from the group consisting of: Clock, Bmall, Cryl, Cry2, Perl and Per2. I n some embodiments a single biomarker may be measured. In other embodiments a biomarker panel may be used to measure one or more of the listed biomarkers in any combination. Specifically, a combination of at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, or preferably all six biomarkers is used according to the invention.

The clock biomarkers of the invention show a statistically significant difference in subjects with IBD compared to healthy controls as shown in Examples 4 and 5, below.

Biomarkers in a biological sample, such as whole blood, white blood cells, serum, plasma or a tissue sample can generally be measured and detected through a variety of assays and methods known to those of skill in the art. The term "measuring" or "detecting" refers to a quantitative or qualitative determination of the amount or concentration of the biomarker in a particular sample. The term "concentration" or "level" can refer to an absolute or relative quantity.

The biomarkers used herein to diagnose, monitor, and grade the severity of IBD may be measured using any process known to those of skill in the art. In some embodiments, the determining step is performed by nucleic acid hybridization, nucleic acid amplification, or an immunological method.

In some embodiments, the determining step comprises the step of obtaining nucleic acid molecules from a biological sample. The nucleic acids molecules are selected from mRNA molecules, DNA molecules and cDNA molecules. In some embodiments, the cDNA molecules are obtained by reverse transcribing the mRNA molecules. In some embodiments, the expression level of a clock gene biomarker can be determined by detecting or measuring the level of the mRNA transcript in a biological sample.

Methods for measuring the expression level of a given mRNA are known to one of skill in the art, and can include but are not limited to, PCR procedures, RT-PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray based analysis, next-generation sequencing; hybridization methods, etc.

In one embodiment, the invention provides a method and kit for the quantification of RNA based on PCR assays. According to a specific embodiment of the invention, the biomarker gene expression is measured by Real-Time PCR.

In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In one embodiment, mRNA level of clock gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.

The nucleic acid sequences of the clock genes described herein have been assigned NCBI accession numbers. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.

In some embodiments, the level of a clock gene biomarker can be determined by detecting or measuring the level of the expressed clock polypeptide. The protein or polypeptide of interest can be determined by any of a number of means well known to those of skill in the art. Such methods include, but are not limited to, immunodiffusion, Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like. Protein levels can also be determined by constructing an antibody microarray in which binding sites comprise immobilized monoclonal antibodies specific to a combination of clock proteins.

Accordingly, the invention provides an antibody-based assay for the detection of said biomarker proteins, including, but not limited to, enzyme linked immunosorbent assay (ELISA). For example, the biomarkers may be identified by an ELISA test specific for the biomarker or set of biomarkers of interest. The term "antibody" as used herein encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multi-specific antibodies formed from antibody fragments.

The methods used herein include establishing reference levels or values for the clock gene/protein biomarkers. The reference values may be established from a healthy individual not known to have a gastrointestinal disorder, a group of healthy individuals, an individual who has IBD (i.e., IBD patient), a group of IBD patients, or from the subject of interest itself. These reference levels may be used as a comparison for biomarker levels in samples (such as whole blood or WBCs) obtained from an individual at risk for IBD, suspected of having IBD, or under treatment for IBD.

The subject is determined to be at risk for or have IBD if the clock gene biomarker levels are statistically different (i.e., lower) in relative amounts from the biomarkers in the biological sample of a healthy control or a group of healthy controls.

The subject is determined to be at risk for or have IBD if the biomarker levels are statistically equivalent to the biomarker levels in a population previously diagnosed with

IBD. The subject is determined to be responding to treatment for I BD if the relative amounts of the biomarkers in the biological sample have altered from the biomarkers in a biological sample taken at an earlier time point from the subject.

The disease state of the subject may be progressing if the biomarker levels in a biological sample are changing relative to the levels in the subject taken at an earlier time point.

The disease state of the subject may be altered from remission to exacerbation (flare up), or vice versa, if the biomarker levels in a biological sample are changing relative to the levels in the subject taken at an earlier time point.

As used herein, the term "inflammatory bowel disease" or "IBD" includes gastrointestinal disorders such as, e.g., Crohn's disease (CD), ulcerative colitis (UC), indeterminate colitis (1C), and I BD that is inconclusive for CD vs. UC ("I nconclusive") or undefined IBD.

The term "biological sample" as used herein includes any biological specimen obtained from a subject. Suitable samples for use in the present invention include but are not limited to, whole blood, white blood cells, plasma, serum, saliva, urine, stool (i.e., feces), buccal mucosa, or any other bodily fluid, or an intestinal mucosal biopsy such as esophagus, stomach, small intestine or colon sample. The term "biological sample" also encompasses any material derived by processing a biological sample. Derived materials include, but are not limited to, cells isolated from the sample, proteins or nucleic acid molecules extracted from the sample.

In some embodiments of the invention, the biological sample is, or is derived from, whole blood, white blood cells, serum, plasma, or an intestinal tissue biopsy of a subject. It should be noted that when the sample is a biopsy of an intestinal tissue, the same pattern of clock gene expression was found in both healthy (i.e., non-inflamed) and inflamed tissue of I BD patients. Accordingly, the methods and kits of the invention enable the diagnosis and monitoring of an IBD patient regardless of whether the tissue of the intestines is inflamed or non-inflamed at the time the sample is obtained.

The terms "biomarker" and "marker" are used herein interchangeably. They refer to a substance that is a distinctive indicator of a biological process, biological event and/or pathologic condition.

As used herein, a "reference value" of a biomarker can be an absolute value, a relative value, a value that has an upper and/or lower limit, a range of values, an average value, a median value, a mean value, a value as compared to a particular control or baseline value or a combination thereof. A reference value can be based on an individual sample value; for example, a value obtained from a sample from the individual with I BD, but at an earlier point in time, or a value obtained from a sample from an I BD patient other than the individual being tested, or a healthy subject, that is an individual not diagnosed with I BD. The reference value can be based on a large number of samples, such as a group of IBD patients or a group of normal individuals.

As used herein, "biomarker panel" refers to a set of biomarkers that can be used alone, together, or in sub-combinations for the detection, diagnosis, prognosis, staging, or monitoring of IBD, based on detection values for the set of biomarkers. The biomarkers within the panel of biomarkers used herein include Clock, Bmall, Cryl, Cry2, Perl and

Per2. The terms "subject", "individual" or "patient" typically refers to humans, but also to other animals including, e.g., other primates, rodents, canines, felines, equines, and the like.

The terms "normal" and "healthy" are used herein interchangeably. They refer to an individual or group of individuals who have not shown any symptoms of gastrointestinal disorder. The term "normal" is also used herein to qualify a sample (e.g., a blood sample) obtained from a healthy individual.

In the context of the present invention, the term "control", when used to characterize a subject, refers to a subject that is healthy. The term "control sample" refers to one, or more than one, sample that has been obtained from a healthy subject.

The present invention further provides a kit for detecting, diagnosing and monitoring inflammatory bowel disease (I BD). The kit enables to determine the expression level of one or more clock gene biomarker in a biological sample. The kit comprises a reagent for detecting and quantifying the mRNA encoded by a clock gene, a corresponding polypeptide, or any combination or fragment thereof. The kit comprises one or more molecules capable of specifically binding a nucleic acid sequence (DNA or RNA) of 1, 2, 3, 4, 5, or all 6 clock genes, or their corresponding polypeptides. It should be noted that the kit optionally further comprises instruction for use, and pre-determined calibration curves or pre-determined standards providing standard expression values of the one or more biomarker.

In some embodiments, the kit comprises reagents adapted to detect and determine the expression level of a clock gene or polypeptide, or any combination of the six clock genes or polypeptides. The reagents are selected from mRNA or cDNA hybridization or amplification reagents, nucleic acid specific probes, and amplification primers, as well as buffers, nucleotide bases, and other compositions to be used in hybridization and/or amplification reactions. In some embodiments, the kit further comprises detectable tags or labels, solutions for rendering a nucleic acid susceptible to hybridization, solutions for lysing cells, or solutions for the purification of nucleic acids.

In some embodiments, the reagents comprise an antibody that specifically binds to one, two, or more clock polypeptide or a fragment thereof. In some embodiments, the kit further comprises one or more of: detectable tags or labels, solutions for rendering a polypeptide susceptible to the binding of an antibody, solutions for lysing cells, or purification solutions.

The antibody may be directly linked to an indicator reagent, wherein said indicator reagent is selected from the group consisting of fluorescent, colorimetric, immunoperoxidase and isotopic reagents. Alternatively, the kit may further include a second indicator antibody linked to an indicator reagent, wherein said indicator reagent is selected from the group consisting of fluorescent, calorimetric, immunoperoxidase and isotopic reagents.

In some embodiments, the antibodies are provided immobilized on solid support, which is a solid inert surface or body to which the antibody can be immobilized. Non-limiting examples of a solid support include plastic, nitrocellulose, and membranes chips. In a specific embodiment, the solid support is an antibody chip, which allows the detection of multiple biomarkers with a single chip.

The kits can further comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition is effective for diagnosing IBD. The label or package insert indicates that the composition is used for in vitro diagnosis of IBD. Thus, in some embodiments, the invention provides a kit comprising reagents adapted to determine the expression level two or more of the six clock genes or polypeptides in a biological sample, for detecting or monitoring IBD.

In other embodiments, the invention provides a kit comprising reagents adapted to determine the expression level of one or more clock gene or polypeptide in a biological sample.

In some embodiments, the kit comprises reagents for the detection and quantification of two or more clock genes in the RNA level. The reagents are selected from nucleic acid hybridization or amplification reagents, nucleic acid specific probes, amplification primers, buffers, nucleotide bases, and other compositions to be used in hybridization and/or amplification reactions. In some embodiments, the kit further comprises detectable tags or labels, solutions for rendering a nucleic acid susceptible to hybridization, solutions for lysing cells, or solutions for the purification of nucleic acids.

In other embodiments, the kit comprises reagents for the detection and quantification of two or more clock gene polypeptides by enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunological assay (RIA), sandwich assay, immunohistological staining, radioimmunometric assay, or immunofluoresence assay. The reagents comprise antibodies specific for two or more clock polypeptides or fragments thereof. The kit may further comprise detectable tags or labels, solutions for rendering the polypeptides susceptible to the binding of antibodies, solutions for lysing cells, and purification solutions.

According to a specific embodiment, the kit is clinical, comprising immobilized probes, reference data and optionally clinical instructions. The present invention shows for the first time that clock gene expression is reduced in the blood, as well as inflamed and non-inflamed intestinal tissues in newly diagnosed, naive to treatment, young IBD patients.

More specifically, gene expression of Circadian Locomotor Output Cycles Kaput (CLOCK), Aryl hydrocarbon receptor nuclear translocator-like protein 1 or Brain and muscle Arnt- like protein-1 (BMAL1), Cryptochrome 2 (CRY2), Period 1 (PERI) and Period 2 (PER2) is reduced in inflamed intestinal biopsies of IBD patients compared to healthy subjects. Surprisingly, non-inflamed tissue samples of IBD patients show a similar reduction in clock gene expression.

Moreover, the clock gene reduction in IBD subjects is not limited to the intestines, as evident by the reduction of CLOCK, CRY1, CRY2, PERI and PER2 expression in white blood cells of young naive to treatment IBD patients. Importantly, the reduction in clock gene expression correlates with the disease activity of IBD patients.

The circadian clock dictates daily gene expression rhythms in peripheral tissues. Thus, the time of tissue sampling reflects a specific gene expression levels. The absence of difference in the time of endoscopy further reinforces the clock gene reduction found in both inflamed and non-inflamed intestinal tissue of IBD patients.

The results of the study specified in the Examples herein below demonstrate that central and peripheral oscillators are disrupted at early stages of the inflammatory bowel disease. The reduced clock gene expression in non-inflamed intestinal biopsies and systemic white blood cells (WBCs) of IBD patients indicates that initial clock disruption has a pathogenic role in the disease inception. Furthermore, after medical treatment of IBD patients with 5-aminosalicylic acid (5-ASA) or anti-tumor necrosis factor alpha (anti-TNFa), they were considered in remission and their clock genes expression was also restored. In conclusion, at the time of primary disease diagnosis, central and peripheral clocks are already disrupted, presenting alterations in clock gene expression in both inflamed and non-inflamed intestinal tissues as well as in WBCs. Accordingly, the present invention links, for the first time, between the circadian clock and IBD, and enables the detection, diagnosis, and monitoring of the disease state, for example, by a simple, non-invasive blood test.

The invention will now be described with reference to specific examples and materials. The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of specific embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

EXAMPLES

Materials and methods

Study population

Patients were prospectively recruited at the pediatric and adult gastroenterology unit at the Sourasky Tel Aviv Medical Center from August 2016 to August 2017. Patients (at ages between 6 to 25 years), who were evaluated by colonoscopy due to gastrointestinal complaints, were considered eligible for recruitment. Exclusion criteria included former diagnosis of IBD, current immunomodulatory or immunosuppressive treatment, chronic inflammatory or autoimmune disease, genetic abnormalities, congenital malformations and pregnancy. Group (IBD or control) assignment was based on the results of histopathologic diagnosis. The control group comprised of patients with histopathological intact ileal and colonic mucosa and the IBD group were defined according to characteristic histopathological findings and accepted diagnostic criteria (as described below). The study was approved by the Tel Aviv Medical Center Institutional Review Board (IRB). Participants over 18 years of age or legal guardians of those under 18 years provided written informed consent.

Study design

On the day of colonoscopy, patients completed a questionnaire that included demographic, clinical and sleep habits information. Stool samples were collected prior to nutritional and medical preparation for colonoscopy and preserved at -20 °C for fecal calprotectin (Fcal). Blood was drawn prior to initiation of anesthesia for analysis of clock gene expression, hemoglobin (Hb), albumin and C - reactive protein (CRP) levels. During diagnostic colonoscopy, additional biopsies were snap frozen and kept at -80 °C for RNA extraction.

Clinical assessment

Clinical disease activity was determined using the Pediatric Crohn's Disease Activity Index (PCDAI) or Pediatric UC Activity Index (PUCAI) for 6-16 years old patients and the Harvey Bradshau Index (HBI) or Modified Mayo score for >16 years old patients. Endoscopic scores were calculated according to macroscopic findings: Crohn's disease index of severity (CDEIS) for Crohn's disease (CD) and Mayo score for ulcerative colitis (UC). Sleep evaluation was performed based on several validated questionnaires and included information on sleep duration and habits, the Morningness-Eveningness questionnaire, the Sleep Disturbance Scale for Children (SDSC, for 6-12 years old subjects), the Adolescent Sleep-Wake Scale (ASWS, for 12-18 years old subjects) and the Pittsburgh Sleep Quality Index for adults of >18 years. Data on height and weight were obtained from each participant and calculated for the body mass index (BMI) and the BMI-z score. CD and UC diagnosis were determined according to accepted diagnostic criteria. Analysis of gene expression in white blood cells (WBC)

Blood for gene expression was collected in Tempus tubes (Applied Biosystems, Foster City, CA, USA) and total RNA extracted according to the manufacturer's instructions. Total RNA was DNase l-treated using RQ1 DNase (Promega, Madison, Wl, USA) for 2 hours at 37 °C. 2 pg of DNase l-treated RNA were reverse-transcribed using MMuLV reverse-transcriptase and random hexamers (Promega). One twentieth of the reaction was then subjected to quantitative real-time PCR using primers spanning exon-exon boundaries and the ABI Prism 7300 Sequence Detection System (Applied Biosystems). The fold change in target gene expression was calculated by the 2^_DDa relative quantification method (Applied Biosystems).

Sample size and power analysis

A sample size of n=15 controls and n=15 IBD patients was determined to have more than 90% power to detect a true, inter-group difference of 40±25% relative mRNA expression levels of clock genes with a p value=0.05. Forty-two participants were recruited.

Statistical analysis

Statistical analysis for demographic and clinical characteristics was performed by SPSS software version 23 (SPSS Inc., Chicago, IL, USA). RNA statistical analysis was performed with JMP software (version 12, SAS Institute Inc., Cary, NC, USA). Continuous variables with normal distribution were compared using independent student t-test. Continuous variables without normal distribution were compared using the non-parametric Kruskal- Wallis test. Categorical variables were explored by Pearson's chi-square test. P values <0.05 were considered significant.

Example 1:

Participants show similar demographic characteristics Forty patients were recruited. Fourteen were newly diagnosed IBD patients and 18 were controls. Eight patients were excluded from the analyses, 3 due to technical problems and 5 due to inconclusive histopathological findings. As shown in Table 1, there were no significant demographic differences between control and IBD groups.

Table 1. Participants' demographic characteristics and clinical parameters.

*BMI Z score was calculated for participants under 18 years of age. Each group included one adult, above 18 years with normal weight BMI (18.5-24.9).

Example 2:

Clinical and endoscopic scores are used to diagnose IBD severity

Of the 14 newly diagnosed IBD patients, 8 were diagnosed with Crohn's disease (CD), 5 with Ulcerative Colitis (UC) and one with IBD unclassified (IBD-U). According to clinical scores, 3 of the CD patients were diagnosed with mild disease and 5 with moderate disease. Endoscopic scores revealed 4 mild CD patients, 2 moderate, 1 severe and one inconclusive severity due to suboptimal preparation. Among the UC patients, 1 patient was diagnosed according to clinical scores with mild UC, 3 moderate and 1 severe. Endoscopic scores revealed 3 moderate and 2 severe UC.

Example 3:

Participants show similar sleeping habits

As presented in Table 2, no significant differences were found in the Morningness- Eveningness questionnaire scores and the percentage of snoring between IBD patients compared to controls. Sleep timing and duration did not differ between the two groups. Notably, later weeknight bed and rise time trend was observed in the study group compared to controls, however, it did not reach statistical significance.

Table 2. Sleep measures in patients with new-onset IBD and controls.

Example 4:

Clock gene expression in intestinal biopsies of newly diagnosed IBD patients is reduced

Analyses of clock gene expression revealed that Circadian Locomotor Output Cycles Kaput (CLOCK), Aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), Cryptochrome Circadian Regulator 2 (CRY2), Period Circadian Regulator 1 (PERI) and Period Circadian Regulator 2 (PER2) were reduced (p<0.05) in inflamed biopsies of IBD patients compared to healthy controls (Figs. 1A-1F). Interestingly, non-inflamed biopsies of IBD patients showed a similar reduction in clock gene expression, except for PER2 that was induced (p<0.05) compared to controls.

Taken together, the results show that clock gene expression is reduced in both inflamed and non-inflamed intestinal tissues in IBD patients compared to intestinal tissues of healthy subjects. Furthermore, these results point to a systemic clock gene reduction in IBD patients.

Example 5:

Clock gene expression in the WBCs of newly diagnosed IBD patients is reduced

Clock gene expression in systemic blood was analyzed in order to evaluate the systemic manifestation of IBD. Clock gene expression in white blood cells (WBCs) revealed that CLOCK, BMAL1, CRY1, CRY2, PERI and PER2 were significantly reduced in IBD patients compared to healthy controls (Figs. 2A-2F).

The expression of these clock genes, except for BMAL1, correlated with the high levels of Fcal (Table 3). Fcal is a stool inflammatory marker, commonly used in IBD diagnosis and to follow disease activity. Fcal is known to be significantly higher in IBD patients compared to healthy subjects. Correlations of Fcal and clock gene expression in WBCs as presented in Table 3 indicate that a reduction in the expression of each clock gene (except for BMAL1) can serve as a valid marker for IBD diagnosis.

Table 3. Correlations of Fcal and clock gene expression in WBCs.

In a separate analysis of Crohn's disease (CD) and ulcerative colitis (UC) patients, CLOCK, BMAL1, CRY1, CRY2, PERI and PER2 were significantly reduced in UC patients and only CLOCK was significantly reduced in CD patients compared to healthy controls (Figs. BA SF). CLOCK, BMAL1 and CRY1 were significantly reduced in UC patients compared to CD patients.

Example 6:

Clock gene expression is restored in remitted IBD patients

A follow up of a cohort of IBD patients treated with 5-aminosalicylic acid (5-ASA) or anti tumor necrosis factor alpha (anti-TNFa) revealed that while the patients' clinical score improved so that the patients were considered in remission, the expression of clock genes in WBCs was also elevated compared to the levels of clock genes expression at the time of diagnosis, namely, before treatment (Fig. 4).

Taken together, the inventors found that the expression level of clock genes correlates with active inflammation of IBD, such that said expression level decreases with the exacerbation/ flare-up of IBD and is restored during remission. Accordingly, alterations in clock genes expression levels can serve as an indicator that enables monitoring the clinical state of IBD patients by identifying alterations in the disease state, such as remission or exacerbation of the disease.

Claims

1. A method of differentiating between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) in a subject, comprising:

c. differentiating between a diagnosis of I BD or of IBS according to the result of said comparison.

2. A method according to claim 1, wherein the reference value pertains to a healthy individual, and wherein diagnosis of IBD is made when the expression level of the biomarker is lower than the reference value level; and diagnosis of I BS is made when the expression level of the biomarker is equal or higher than the reference value level.

3. A method for determining the severity of an inflammatory bowel disease (I BD) in a biological sample obtained from a subject, comprising:

a. determining the expression level of one or more clock gene biomarker in said sample; and

b. comparing the expression level of said biomarker with at least one reference value, wherein a decrease or increase in the expression level relative to the reference value indicates the severity of the disease.

4. A method for identifying an alteration in the disease state of an IBD patient by analyzing two or more biological samples taken at different times, comprising:

a. determining the expression level of one or more clock gene biomarker in a first biological sample obtained from the patient at a first time point , to create a first biomarker profile; b. determining the expression level of one or more clock gene biomarker in a second biological sample obtained from the patient at a second time point , to create a second biomarker profile; and

c. comparing the first biomarker profile and the second biomarker profile, wherein a decrease or increase in the second biomarker profile at the second time point relative to the first biomarker profile at the first time point indicates that the disease state is altered.

5. A method for monitoring the response of a subject diagnosed with IBD to treatment by analyzing two or more biological samples taken at different times, comprising: a. determining the expression level of one or more clock gene biomarker in a first biological sample obtained from the subject, to create a first biomarker profile; b. treating the subject for IBD;

6. The method of any one of claims 1-5, wherein the biological sample obtained from the subject is whole blood, white blood cells, serum, plasma, or a biopsy sample.

7. The method of claim 6, wherein the biopsy sample is an inflamed or non-inflamed tissue of the intestines.

8. The method of any one of claims 1-5, wherein the one or more clock gene biomarker is selected from the group consisting of: Circadian Locomotor Output Cycles Kaput (CLOCK), Aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), Cryptochrome 1 (CRY1), Cryptochrome 2 (CRY2), Period 1 (PERI), and Period 2 (PER2).

9. The method of claim 8, wherein the one or more clock gene biomarker is a combination of at least two biomarkers, optionally all six biomarkers.

10. The method of any one of claims 1-5, wherein the level of the one or more clock gene biomarker is determined by measuring the level of the mRNA transcript in the sample.

11. The method of claim 10, wherein the level of the mRNA transcript is determined using an assay selected from the group consisting of: PCR, RT-PCR; quantitative RT- PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization.

12. The method of any one of claims 1-5, wherein the level of the one or more clock gene biomarker is determined by measuring the level of the polypeptide.

13. The method of claim 12, wherein the level of the polypeptide is determined using an assay selected from the group consisting of: enzyme-linked immunosorbent assay (ELISA); Western blot; immunoprecipitation; radioimmunological assay (RIA); sandwich assay; immunohistological staining; radioimmunometric assay; immunofluoresence assay.

14. The method of any one of claims 1-5, wherein the clock expression is detected by ELISA.

15. The method of any one of claims 1 and 3-5, wherein the reference value is established from a healthy individual, an IBD patient, or from the subject itself.

16. The method of claim 4, wherein the disease state is remission or exacerbation.

17. A kit comprising at least one detecting molecule and reagents adapted to determine the expression level of one or more clock gene or polypeptide biomarker, or any combination thereof, in a biological sample, optionally with instruction for use and pre-determined calibration curves or pre-determined standards providing standard expression values of the one or more clock biomarker.

18. The kit of claim 17, wherein said reagents are selected from nucleic acid hybridization or amplification reagents, nucleic acid specific probes, and amplification primers.

19. The kit of claim 17, wherein said reagents comprise an antibody that specifically binds to a clock polypeptide or a fragment thereof.

20. The kit of any one of claims 17-19, further comprising one or more of:

a. detectable tags or labels;

b. solutions for rendering a nucleic acid susceptible to hybridization;

c. solutions for rendering a polypeptide susceptible to the binding of an antibody; d. solutions for lysing cells;

e. purification solutions;

f. instructions for use; and

g. pre-determined calibration curves.

21. A method of detecting inflammatory bowel disease (IBD), comprising:

a. obtaining a biological sample from a subject;

22. A method for the treatment of IBD in a subject suspected of having IBD, the method comprising:

a. detecting if the subject has IBD according to the method of claim 1; and b. administering to a subject tested positive in (a) an anti-IBD treatment.

23. The method according to claim 22, further comprising:

c. monitoring the response of the subject to the anti-IBD treatment, and, if needed.

24. The method according to claim 22, wherein the anti-IBD treatment is selected from aminosalicylates/5-ASA compounds; oral and topical enemas; systemic corticosteroids; topical corticosteroids; thiopurines; methotrexate; calcineurin inhibitors selected from cyclosporine and tacrolimus; anti-TNFa; anti-a4 integrin subunit antibody; anti-p7 integrin subunit antibody; anti-intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide; IL12/23 pathway blockage agent; anti-p40 antibody; and anti-pl9 antibody; anti-IL6 antibody; anti-l L6 receptor antibody; Janus Kinase inhibitors (JAK) selected from JAK1, JAK2, and JAK3 inhibitors; anti -Mad cam antibodies; SMAD7 antisense oligonucleotide; laquinimod; sphingosine-l-phosphate (SIP) pathway modulation drugs; S1P1/S1P5 agonists; Treg cell therapy; y6 T cell therapy; modulation of mucosal immunity by helminths; probiotic agents; prebiotics; synbiotics; granulocyte/monocyte apharesis; fecal microbiota transplantation; antibiotics; anti-mycobacterial treatment; curcumin; herbs and plants with immune-regulating characteristics; vitamin and mineral supplementations; exclusive enteral nutrition; partial enteral nutrition; specific exclusion diets; proton pump inhibitors; surgical resection and/or endoscopic dilatation; complementary and alternative medicine; and any combination thereof; and optionally

25. The method of claim 21, wherein the reference value is established from a healthy individual, an IBD patient, or from the subject itself.

26. The method of claim 21 or claim 22, wherein the biological sample obtained from the subject is whole blood, white blood cells, serum, plasma, or a biopsy sample.

27. The method of claim 26, wherein the biopsy sample is an inflamed or non-inflamed tissue of the intestines.

28. The method of claim 21 or claim 22, wherein the one or more clock gene biomarker is selected from the group consisting of: Circadian Locomotor Output Cycles Kaput (CLOCK), Aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), Cryptochrome 1 (CRY1), Cryptochrome 2 (CRY2), Period 1 (PERI), and Period 2 (PER2).

29. The method of claim 21 or claim 22, wherein the one or more clock gene biomarker is selected from the group consisting of: two biomarkers, three biomarkers, four biomarkers, five biomarkers and six biomarkers.

30. The method of claim 21 or claim 22, wherein the level of the one or more clock gene biomarker is determined by measuring the level of the mRNA transcript in the sample.

31. The method of claim 30, wherein the level of the mRNA transcript is determined using an assay selected from the group consisting of: PCR; RT-PCR; quantitative RT- PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization.

32. The method of claim 21 or claim 22, wherein the level of the one or more clock gene biomarker is determined by measuring the level of the clock polypeptide.

33. The method of claim 32, wherein the level of the clock polypeptide is determined using an assay selected from the group consisting of: enzyme-linked immunosorbent assay (ELISA); Western blot; immunoprecipitation; radioimmunological assay (RIA); sandwich assay; immunohistological staining; radioimmunometric assay; immunofluoresence assay.

34. The method of claim 33, wherein the level of the clock polypeptide is determined by

ELISA.

35. A method for detecting inflammatory bowel disease (IBD), comprising obtaining a biological sample from a subject and determining the expression level of two or more clock gene or polypeptides using a kit according to claim 17.

36. A method of differentiating between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) in a subject, comprising:

a. obtaining a biological sample from a subject;

b. determining the expression level of one or more clock gene biomarker in said biological sample;

c. comparing the expression level of the biomarker with at least one reference value of the biomarker; and

d. differentiating between a diagnosis of IBD or of IBS, according to the result of said comparison.

37. A method according to claim 36, further comprising:

38. A method of treating a subject with a disease selected from IBD or IBS, comprising: a. determining if the subject has IBD or IBS according to the method of claim 36; b. if the subject has IBS, providing an IBS treatment selected from: exercise; dietary modification such as gluten free and low in fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP diet); antidiarrheal medication selected from peristalsis inhibitors and serotonin agents; antispasmodics selected from nutritional supplements, anticholinergics and calcium channel blockers; fiber supplements; laxative agents selected from osmotic and stimulant laxatives; prosecretory drugs; probiotics; antibiotics; antidepressants selected from TCA-tricyclic antidepressants, SSRI-selective serotonin reuptake inhibitors, and SNRI-serotonin norepinephrine reuptake inhibitors, psychological and behavioral therapies, complementary and alternative medicine; and any combination thereof; or

c. if the subject has IBD, providing an IBD treatment selected from: aminosalicylates/5-ASA compounds; oral and topical enemas; systemic corticosteroids; topical corticosteroids; thiopurines; methotrexate; calcineurin inhibitors selected from cyclosporine and tacrolimus; anti-TNFa; anti-a4 integrin subunit antibody; anti-p7 integrin subunit antibody; anti-intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide; IL12/23 pathway blockage agent; anti-p40 antibody; and anti-pl9 antibody; anti-l L6 antibody; anti-IL6 receptor antibody; Janus Kinase inhibitors (JAK) selected from JAK1, JAK2, and JAK3 inhibitors; anti-Madcam antibodies; SMAD7 antisense oligonucleotide; laquinimod; sphingosine-l-phosphate (SIP) pathway modulation drugs; S1P1/S1P5 agonists; Treg cell therapy; y6 T cell therapy; modulation of mucosal immunity by helminths; probiotic agents; prebiotics; synbiotics; granulocyte/monocyte apharesis; fecal microbiota transplantation; antibiotics; anti-mycobacterial treatment; curcumin; herbs and plants with immune- regulating characteristics; vitamin and mineral supplementations; exclusive enteral nutrition; partial enteral nutrition; specific exclusion diets; proton pump inhibitors; surgical resection and/or endoscopic dilatation; complementary and alternative medicine; and any combination thereof.

39. A method according to claim 36, wherein the reference value pertains to a healthy individual and wherein:

a. If the expression level of the biomarker is lower than the reference value level, making a diagnosis of IBD; and

b. If the expression level of the biomarker is equal or higher than the reference value level, making a diagnosis of IBS.

40. A method for monitoring the response of a subject diagnosed with IBD to treatment by analyzing two or more biological samples taken at different times, comprising: a. obtaining a first biological sample from a subject;

c. treating the subject for IBD;

d. obtaining a second biological sample from said subject at a time after treatment; e. determining the expression level of one or more clock gene biomarker in said second biological sample, to create a second biomarker profile; and

f. comparing the first biomarker profile and the second biomarker profile, wherein a decrease or increase in the second biomarker profile relative to the first biomarker indicates responsiveness of the subject to the treatment.

41. The method according to claim 40, further comprising:

g. changing the dose, timing, or type of treatment according to said analysis.