USE OF THE RHO GDP DISSOCIATION INHIBITOR 2 PROTEIN AS A DIAGNOSTIC AND PROGNOSTIC MARKER OF INTESTINAL INFLAMMATORY DISEASES

Abstract

Disclosed is a method that uses Rho GDP dissociation inhibitory 2 protein as a diagnostic and prognostic marker of inflammatory bowel diseases by way of the ex vivo detection of the protein level in faecal samples of patients.

Claims

1. A method for diagnosing/prognosing of intestinal inflammatory diseases by ex vivo detection of the level of human Rho GDP-Dissociation Inhibitor 2 protein in faecal samples of patients wherein an increased level of Rho GDP-dissociation inhibitor 2 detected in the faeces of the subject is associated with intestinal inflammation.

2. The method according to claim 1 wherein the concentration value of the human Rho GDP-Dissociation Inhibitor 2 protein can be obtained by a technique for qualitatively and quantitatively determining the protein in a faecal sample.

3. The method according to claim 2, wherein the technique for qualitatively and quantitatively determining the protein present in the faecal sample is selected from the group consisting of: two-dimensional electrophoresis, proteomics, chromatography, Western blot and immunoblotting and enzyme-linked immunosorbent assay (ELISA).

4. The method according to claim 1, wherein the concentration value of said protein in the faecal sample is detected by immunoblotting and enzyme-linked immunosorbent assay (ELISA).

5. The method according to claim 4, comprising the following steps: (a) weighing the fecal sample and suspension in an extraction buffer to obtain a concentration of 500 mg/ml; (b) homogenizing the sample, mixing and centrifuging for 5 minutes at 5000 RPM at a temperature of 4 C. twice; (c) detection of human Rho GDP-Dissociation Inhibitor protein level in the faecal extract obtained in (b).

6. The method according to claim 5 wherein the extracting buffer used in (a) is a saline buffer pH 7.2 comprising Triton X-100 and sodium azide.

7. The method according to claim 5, wherein the determination of the protein level in the faecal extract is obtained by means of a specific antibody-specific immunosorbent assay (ELISA).

8. The method according to claim 1, wherein the concentration value of the Rho GDP-Dissociation Inhibitor protein in the faecal sample of a subject suffering from intestinal inflammatory diseases is higher than 42 pg/ml.

9. The method according to claim 1, wherein intestinal inflammatory disease is: Crohn's disease and ulcerative colitis.

10. The method according to claim 2, wherein the concentration value of said protein in the faecal sample is detected by immunoblotting and enzyme-linked immunosorbent assay (ELISA).

11. The method according to claim 3, wherein the concentration value of said protein in the faecal sample is detected by immunoblotting and enzyme-linked immunosorbent assay (ELISA).

12. The method according to claim 2, wherein the concentration value of the Rho GDP-Dissociation Inhibitor protein in the faecal sample of a subject suffering from intestinal inflammatory diseases is higher than 42 pg/ml.

13. The method according to claim 3, wherein the concentration value of the Rho GDP-Dissociation Inhibitor protein in the faecal sample of a subject suffering from intestinal inflammatory diseases is higher than 42 pg/ml.

14. The method according to claim 4, wherein the concentration value of the Rho GDP-Dissociation Inhibitor protein in the faecal sample of a subject suffering from intestinal inflammatory diseases is higher than 42 pg/ml.

15. The method according to claim 10, wherein the concentration value of the Rho GDP-Dissociation Inhibitor protein in the faecal sample of a subject suffering from intestinal inflammatory diseases is higher than 42 pg/ml.

16. The method according to claim 11, wherein the concentration value of the Rho GDP-Dissociation Inhibitor protein in the faecal sample of a subject suffering from intestinal inflammatory diseases is higher than 42 pg/ml.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0029] With reference to the attached figures, it should be noted that the expression RGDI2 corresponds to the acronym of Rho GDP Dissociation Inhibitor 2.

[0030] FIG. 1 shows the graph that represents the concentration of the Rho GDP dissociation inhibitor 2 in the faecal sample of healthy subjects and of subjects suffering from Crohn's disease (A); in (B) the protein concentration is presented in the same affected subjects, subdivided according to the degree of severity of the disease (e.g. inactive, moderate and severe) assessed by endoscopy.

[0031] FIG. 2 shows the graphs which represents the concentration of Rho GDP dissociation inhibitor 2 in the faecal sample of healthy subjects and of subjects suffering from ulcerative colitis (A); in (B) the protein concentration is presented in the same affected subjects, subdivided according to the degree of disease severity (e.g. inactive, moderate and severe) evaluated by endoscopy.

[0032] FIG. 3 shows the graph presenting the correlation between the level of calprotectin and that of Rho GDP dissociation inhibitor 2 in the faecal sample of subjects affected by Crohn's disease.

[0033] FIG. 4 shows the graph showing the correlation between the level of calprotectin and that of Rho GDP dissociation inhibitor 2 in the faecal sample of subjects suffering from ulcerative colitis.

[0034] FIG. 5 shows the graph showing the correlation between the level of Rho GDP dissociation inhibitor 2 in subjects affected by Crohn's disease and the endoscopic index (SES-CD), normally used for this disease, which reflects the severity of the disease.

[0035] FIG. 6 shows the graph showing the correlation between the level of Rho GDP dissociation inhibitor 2 in subjects suffering from ulcerative colitis and the endoscopic index (Mayo), normally used to express the degree of severity of the disease.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The invention consists in using the level of Rho GDP dissociation inhibitor 2 present in a faecal sample of a subject as a diagnostic marker of the intestinal inflammatory pathologies and, therefore, in the determination of the level of such protein in the stool of a subject, and in its comparison to a predetermined value identified in the faeces of a group of subjects wherein a diagnosis of inflammatory bowel disease has been placed with clinical and laboratory techniques known to skilled in the field, such as the endoscopic investigation.

[0037] According to the invention the subject on which the level of Rho GDP dissociation inhibitor 2 is determined is a mammal, preferably it is a primate, even more preferably it is a human being. The value of the concentration of the Rho GDP dissociation inhibitor 2 can be obtained by any conventional technique suitable for qualitatively and quantitatively determining a protein present in a faecal sample, known to those skilled in the art, such as Western blot, two-dimensional electrophoresis, proteomics, chromatographic techniques; however, according to the invention, preferably the concentration of the Rho GDP dissociation inhibitor 2 is determined by immuno-absorbing assay bound to specific antibody (ELISA).

[0038] The value of concentration of the Rho GDP dissociation inhibitor 2 identified in the faeces of a group of healthy subjects, i.e. subjects that at the time of detection do not show signs of the disease and in which a diagnosis of inflammatory bowel disease posed with clinical and laboratory techniques known to skilled in the field has been ruled out, ranges from 0 to 42 pg/ml (average value 20 pg/ml), the value of 42 pg/ml is therefore the threshold value below which the subject is considered healthy, while above which the subject presents the disease.

[0039] As shown in FIGS. 1 and 2, it was observed that in subjects suffering from inflammatory bowel disease, particularly in individuals with Crohn's disease and ulcerative colitis, the level of Rho GDP dissociation inhibitor 2 detected in a faecal sample is significantly higher than in healthy subjects.

[0040] Furthermore, the concentration of Rho GDP dissociation inhibitor 2 in faeces finds excellent correlation with parameters obtained by other methods of diagnosis, both invasive and non-invasive. For example, a correlation was made between the faecal levels of Rho GDP dissociation inhibitor 2 with the levels of faecal calprotectin, currently considered the non-invasive diagnostic marker of choice for inflammatory bowel diseases. The comparison of faecal samples of affected individuals from both Crohn's disease and ulcerative colitis showed a positive and statistically significant correspondence between the two markers, as shown in FIGS. 3 and 4.

[0041] The correlation is maintained even when faecal levels of Rho GDP dissociation inhibitor 2 are compared with the endoscopic SES-CD index used for Crohn's disease, which classifies the degree of severity of the disease as a function of the size of ulcers present in the mucosa, the ulcerated surface, the extension of the surface of the affected mucosa and the presence of stenosis [Daperno M. et al., Gastrointest Endosc 2004; 60: 505-512. Moskovitz D. N., Disease. Gastroenterology 2007; 132: S1097]. Furthermore, SES-CD is related to clinical parameters and to the serum level of C-reactive protein. The correlation analysis data is shown in FIG. 5.

[0042] The correlation was also demonstrated for ulcerative colitis, in fact the correspondence between the endoscopic index Mayo Score, evaluating the stage of ulcerative colitis based on the four components: frequency of defecation, rectal bleeding, endoscopic evaluation and overall judgment [Schroeder K W et al., N Eng J Med 1987; 317: 1625-1629; Rutgeerts P. et al., N Engl J Med. 2005; 353: 2462-2476], and faecal levels of Rho GDP dissociation inhibitor 2 was observed. As shown in FIG. 6, there is a correspondence between the score attributed by the Mayo Score endoscopic index, which varies from zero (normal value or inactive pathology) to 3 (severe activity) and the protein concentration in the faeces.

[0043] The determination of the faecal concentration of the Rho GDP dissociation inhibitor 2, besides being useful as a diagnostic marker, has proved to be a valid prognostic tool, useful in assessing the real course of the disease and the response to therapy. In fact, when the disease enters remission, the level of protein returns to a physiological level, that is comparable with that of healthy subjects. Therefore, the fecal Rho GDP dissociation inhibitor 2 can be used not only to diagnose the condition, but also to confirm in the patient the actual state of remission of the disease, and, when an increase in protein values occurs, predict an imminent relapse and to assess whether and to what extent the patient responds to the therapy. Thus, the invention provides a method for determining whether a subject will benefit from continuing with therapy, also providing indications on the appropriateness of the dosage of the therapy in place, or by a change of therapy. The benefit typically results in a reduction in the level of protein concentration in the faecal sample that correlates with a reduction in signs, symptoms and typical manifestations of the disease, or in a more rapid recovery from such signs, including, for example, diarrhea and abdominal pain, presence of blood and mucus in the stool, which is often associated with anemia, weight loss, fatigue, loss of appetite and fever. It is therefore clear that this method has important implications for the treatment of patients and for the clinical development of new possible therapies, in fact to be able to determine whether a person will benefit by continuing with therapy or changing doses or changing the therapy itself is of absolute clinical relevance and allows to identify those patients that are most likely to respond to the chosen and undertaken therapeutic approach.

[0044] Actually, the experimentation has shown that the detection of the Rho GDP dissociation inhibitor 2 is also possible in the faeces of patients with disease defined as inactive on the basis of standard clinical indexes and which however still show a certain amount of intestinal inflammation, according to the evaluation based on the endoscopic score.

[0045] Thus, the present invention clearly demonstrates the usefulness and advantage deriving from the use of the Rho GDP dissociation inhibitor 2 detected in the fecal sample as a potential molecular prognostic parameter to predict possible recurrences in patients with IBD with apparent remission disease. In addition, due to the direct correlation between protein levels and disease severity, detection of the Rho GDP dissociation inhibitor 2 protein in faeces can be used as a prognostic marker of response to therapy. According to the invention, the determination of the level of Rho GDP dissociation inhibitor 2 is obtained by using conventional methods known to those skilled in the art, preferably obtained by an ELISA assay, or other immunological assays or conventional techniques for determining the presence of Rho GDP dissociation inhibitor 2, with appropriate modifications necessary to conduct the immunological assay on a biological sample as complex as the fecal matrix, which has very different characteristics compared to those of a body fluid such as saliva, urine, lymph, plasma, serum.

[0046] Currently, various kits comprising the reagents necessary for the determination of Rho GDP dissociation inhibitor 2 protein by a specific antibody-linked immuno-absorbent assay (ELISA) in a biological sample are commercially available. In a particularly preferred embodiment of the present invention for the determination of Rho GDP dissociation inhibitor 2 in the faecal sample, the SEE330Hu 96 Tests Enzyme-linked Immunosorbent Assay Kit for Rho GDP Dissociation Inhibitor Beta (ARHGDIb) product and commercialized by Cloud-Clone Corporation USA was used, but it will be evident to the skilled in the field that the choice of the kit is not limiting since any immuno-absorbent test linked to antibody specific for the Rho GDP dissociation inhibitor 2 protein can be used.

CONCLUSION

[0047] The present invention has shown that the level, and in particular the increased level, of Rho GDP dissociation inhibitor 2 present in a faecal sample of a subject affected by inflammatory bowel pathology constitutes a reliable faecal diagnostic, prognostic and of response to therapies marker, able to correlate with the degree of severity of intestinal inflammation. The information obtained from the use of the protein as described is particularly valuable for clinicians who such way have more tools to monitor the condition of the patient and can, therefore, provide the most appropriate therapies.

[0048] Furthermore, the use of Rho GDP dissociation inhibitor protein as a faecal marker of intestinal inflammation allows to overcome the technical problems still present in the use of some diagnostic and prognostic markers, in particular the technical problems resulting from the use of other non-invasive diagnostic markers, such as faecal concentration of HMGB1.

[0049] In fact, compared to the use of the determination of the level of HMGB1, for which at present there is no specific antibody functioning in an ELISA test, the advantage resulting from the use of the Rho GDP dissociation inhibitor 2 is evident, since this determination can be carried out in a simple, rapid and reproducible manner by means of an ELISA assay in the analysis laboratory. Therefore, the introduction of the use of the determination of the faecal Rho GDP dissociation inhibitor 2 protein as a diagnostic and prognostic marker of intestinal inflammation does not simply represent a valid alternative to the diagnostic and prognostic methods of the state of the art, but contributes to a technological advancement overcoming technical problems still related to their use.

Experimental Part

[0050] Preparation of the Faecal Sample

[0051] Fecal samples were obtained from 57 patients with Crohn's disease and 60 patients with ulcerative colitis (Table 1), with different level of disease severity, and 31 healthy controls, recruited at the Unit of Gastroenterology and Pediatric Hepatology of University La Sapienza of Rome-Policlinico Umberto I. Patients with an acute-severe form of the disease, suffering from toxic megacolon, subjects with complications requiring immediate surgery were excluded from the study. All the subjects who took part in the study were informed of the trial and gave their consent in writing. Samples, collected in sterile containers for faeces, were stored at 20 C. until molecular analysis.

TABLE-US-00001 TABLE 1 Characteristics of patients enrolled in the clinical study n = 117 IBD Patient group Crohn's Ulcerose Disease Colitis Patients n = 57 n = 60 Age (mean, range) 46 (21-76) 49 (24-83) Gender (male/female) 27/30 33/27 Disease features (n) Inactive 27 17 Ulcerative colitis 3 Left Colitis 3 Extense Colitis 37 Ileo 7 Colonic 6 Ileocolonic 16 Inflammation localized in 1 (3%) the upper gastrointestinal tract Endoscopic activity of the disease (n, %) Inactive 27 (47%) 17 (28%) Moderate 16 (28%) 26 (44%) Severe 14 (25%) 17 (28%) Endoscopic remission Istologic Inflammation 9 (33%) 5 (29%) Istologic remission 18 (67%) 12 (71%)

[0052] Stool Weighing and Suspension in the Buffer

[0053] Each stool sample (about the size of an azelnut, equivalent to the contents hold by the little teaspoon inside a standard container for faeces) was taken with a sterile tip from the container, placed in a 1.5 ml eppendorf tube and weighed. The sample was resuspended in extraction buffer (salt phosphate buffer pH 7.2) containing detergent, Triton X-100, and sodium azide (ScheBo Biotech), to obtain a concentration of 500 mg/ml.

[0054] Stool Homogenization and Extraction

[0055] The sample was vigorously mixed with vortex for one minute at room temperature (RT); the sample was then placed in orbital excitement for an hour at RT. Then, it was mixed and centrifuged for 5 minutes at 5000 RPM at a temperature of 4 C. The supernatant was then taken and centrifugation was repeated. The supernatant called faecal extract was taken. At this point, the sample can be analyzed immediately by an ELISA test or stored at 80 C. and successively analyzed.

[0056] Analysis of Faecal Extracts by ELISA

[0057] All ELISA assays were performed using commercial kits following the instructions for use provided by the manufacturer.

[0058] The microplate provided in the kit used according to the invention is pre-coated with an antibody specific for the Rho GDP dissociation inhibitory 2 protein. The faecal samples were diluted to 1:50 using the dilution buffer provided in the kit. A volume of 100 l of standard or sample is then added to the appropriate wells of the microtiter plate with a reagent containing the antibody specific for the Rho GDP dissociation inhibitory 2 protein conjugated with biotin. Subsequently, a second reagent containing avidin conjugated to horseradish peroxidase (HRP) is added to each well of the microplate and incubated. When the substrate solution, 3,3,5,5-tetramethylbenzidine or TMB is added, only in those wells containing Rho GDP dissociation inhibitory 2 protein, the biotin conjugated antibody and the avidin conjugate enzyme will show a color change. The reaction of the enzymatic substrate is terminated by addition of a sulfuric acid solution provided in the kit and the color change is spectrophotometrically measured at the wavelength of 450 nm10 nm. The concentration of the Rho GDP dissociation inhibitor 2 in the samples is then determined by comparing the O.D. of the sample to the standard curve.

[0059] The reading of the O.D. in each sample it was conducted in duplicate.

[0060] Results

[0061] FIGS. 1-6 show the graphical representations of the data obtained in the experiment conducted to validate the use of the determination of the level of the Rho GDP dissociation inhibitory 2 in a faecal sample as a non-invasive diagnostic and prognostic marker, for intestinal inflammations.

[0062] FIG. 1 shows the data of the analysis performed by means of an ELISA test performed on faecal samples of patients with Crohn's disease and healthy controls. The results show that the Rho GDP dissociation inhibitory 2 in the faecal sample are significantly higher in patients compared to controls. Furthermore, when patients are divided according to disease severity evaluated by endoscopy, such as: inactive, moderate and severe disease, it is observed that the levels of the fecal Rho GDP dissociation inhibitory 2 protein increase significantly as the severity of the disease increases.

[0063] Subject healthy vs. CD: P<0.001 (***)

[0064] Subject healthy vs. inactive disease: P<0.001 (***)

[0065] Inactive disease vs. moderate: P<0.075 not significative.

[0066] Moderate disease vs. severe: P<0.05 (*)

[0067] FIG. 2 shows the data of the same analysis carried out on faecal samples of patients suffering from ulcerative colitis. Also in this case the analysis showed that the levels the fecal Rho GDP dissociation inhibitory 2 protein are significantly higher in patients affected than in controls. Furthermore, even in the case of ulcerative colitis, the levels of the fecal Rho GDP dissociation inhibitory 2 protein increase significantly with the increase in the severity of the pathology evaluated by endoscopy. Statistical analysis conducted by Mann Whytney test, [0068] Subject healthy vs. UC *** P<0.001 [0069] Subject healthy vs. inactive disease *** P<0.001 [0070] Inactive disease vs. moderate disease ** P<0.01 [0071] Moderate disease vs. severe. ** P<0.01.

[0072] FIG. 3 shows the positive and statistically significant correlation between the fecal calprotectin level and the level of the Rho GDP dissociation inhibitory 2 in patients affected by Crohn's disease, fecal dissociation protein inhibitor 2 of the Rho GDP vs. Fecal calprotectin: Spearman r=0.61, p<0.001 (***).

[0073] FIG. 4 shows the data of the same analysis carried out on fecal samples of patients suffering from ulcerative colitis, fecal Rho GDP dissociation inhibitory 2 protein vs. fecal calprotectin: Spearman r=0.34, p<0.01 (***).

[0074] FIG. 5 shows the positive and statistically significant correlation between the level of Rho GDP dissociation inhibitory 2 in patients suffering from the Crohn's disease and the endoscopic index used for the classification of disease severity, fecal Rho GDP dissociation inhibitory 2 protein vs. SES-CD: Spearman r=0.64, p<0.001 (***).

[0075] FIG. 6 shows the correlation between the level of Rho GDP dissociation inhibitory 2 protein in patients with rectal ulcerative colitis and the endoscopic index used for the classification of disease severity, fecal Rho GDP dissociation inhibitory 2 protein vs. Mayo: Spearman r=0.63, p<0.001 (***).