WO2010150082A2

WO2010150082A2 - Use of chromogranin a and of peptidic derivatives thereof in the treatment of inflammation

Info

Publication number: WO2010150082A2
Application number: PCT/IB2010/001522
Authority: WO
Inventors: Fabrizio Marcucci; Angelo Corti; Barbara Colombo; Anna Maria Gasparri; Cristiano Rumio; Marco Palazzo; Laura Zanobbio
Original assignee: Universita degli Studi di Milano; Ospedale San Raffaele SRL
Current assignee: Universita degli Studi di Milano; Ospedale San Raffaele SRL
Priority date: 2009-06-25
Filing date: 2010-06-24
Publication date: 2010-12-29
Anticipated expiration: 2011-12-25
Also published as: ITMI20091125A1; WO2010150082A3

Abstract

The invention concerns the use of chromogranin A, its peptidic derivatives and their non-peptidic peptidomimetic derivatives in the treatment and prevention of inflammatory pathologies of the digestive system, in the treatment and/or prevention of diseases such as Crohn's disease, ulcerative colitis and, more generally, chronic bowel disease and inflammatory diseases of the respiratory system.

Description

"Use of chromogranin A and of peptidic derivatives thereof in the treatment of inflammation"

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TECHNICAL BACKGROUND The present invention relates to the use of chromogranin A, its peptide fragments and their non-peptide peptidomimetic derivatives in the treatment and prevention of inflammatory pathologies of the digestive system and respiratory system. In particular, the invention concerns the use of active fragments of chromogranin A, for example vasostatin, in the treatment and/or prevention of diseases such as Crohn's disease, ulcerative colitis and, more generally, chronic bowel diseases and inflammatory diseases of the respiratory system such as asthma. Crohn's disease (below also referred to simply as CD) and ulcerative colitis (below also referred to simply as UC) are known collectively as chronic inflammatory bowel diseases (here IBD). Said pathologies are disorders characterised by chronic and idiopathic inflammation of the tissue of the bowel mucous membrane, which causes a series of symptoms including abdominal pain, serious diarrhoea, bleeding of the rectum and wasting.

IBDs tend to emerge in late infancy and are widespread mainly in the westernised regions of the world. CD and UC can be distinguished by the organs involved: CD can be located in any region of the alimentary tract discontinuously and transmurally, while in UC the pathology is limited to the mucous surface of the colon.

In Europe and the United States, the incidence of Crohn's disease is 6-7.1:100,000 persons/year while for ulcerative colitis there are approximately 10-12 cases every 100,000 persons/year. It has been observed that patients affected by these pathologies show a greater incidence of cancer of the colon.

The pharmacological treatments currently used comprise steroidal anti-inflammatory drugs, immunosuppressive drugs and anti-TNF antibodies. These treatments have not demonstrated any curative effect, however, and are suitable only for treating the inflammation.

Currently, in fact, there is no drug available that can cure these pathologies. Furthermore, as is known, corticosteroid and immunosuppressive treatments cause serious side effects which often lead to interruption of the treatment. In these cases, the only treatment possible is surgical removal of the damaged intestinal tract. Chromogranin A is an endogenous protein, the biological role of which is still little known.

Amino acid fragments of chromogranin A, in particular vasostatin I and vasostatin II, have also been described in literature.

OBJECTS OF THE INVENTION

The object of the present invention is to provide a curative and preventive treatment for chronic diseases of the digestive system, which overcomes the drawbacks of the treatments in the known art described above.

It has now surprisingly been found that it is possible to successfully treat inflammatory diseases of the digestive system including, in particular, chronic inflammatory diseases of the gastrointestinal tract, by administering chromogranin A and/or its active peptidic fragments, such as vasostatin and/or other peptidic fragments of chromogranin A with a lower molecular weight. It has also been found that the administration of chromogranin A and/or its active peptidic fragments such as vasostatin and/or other inferior peptidic fragments produces a preventive effect in subjects with a predisposition towards the development of chronic inflammatory diseases of the digestive system. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of the vasostatin 1 in damaged cells.

- Figure 2 shows the effect of the vasostatin 1 on cellular proliferation.

- Figure 3 shows the effect of the vasostatin 1 on weight loss induced by colitis.

- Figures 4 and 5 show the inhibition of the inflammation produced by the vasostatin 1 in colon cells.

- Figures 6-9 show the protective effect of the vasostatin and active fragments on lesions induced by interferon and TNF. - Figures 10 and 11 show the anti-chrornogranin A and anti- vasostatin antibodies that block the protective effects on lesions induced by interferon and TNF.

In the figures: min = minutes; hr = hours; Untr = untreated control. DISCLOSURE OF THE INVENTION

According to one of its aspects, the invention concerns the use of chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives for the preparation of a medicament for the treatment and/or prevention of inflammatory diseases of the digestive system.

According to another of its aspects, the invention concerns the use of chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives for the preparation of a medicament for the treatment and/or prevention of inflammatory diseases of the respiratory system.

As said above, chromogranin A is an endogenous peptide of 439 amino acids, described in literature (Konecki et al J Biol Chem. 1987 262 17026). The numbering of the sequences according to the present invention refers to the sequence reported by Konecki et al. as above.

According to the present invention, "active peptidic fragments" indicate the fragments of chromogranin A which are able to exercise its same effect in treatment of the pathologies indicated above. Examples of said fragments are vasostatin I, which is the fragment 1-76, and vasostatin II, which is the fragment 1-113, both described in literature.

Other examples of active peptidic fragments according to the invention are fragments of the vasostatin of variable length, for example the fragments consisting of 5-10, 10- 20, 20-40, 40-60 or 40-75 amino acid residues. The expression "active peptidic fragments" furthermore comprises the peptides having the amino acid sequences chosen from the sequences of the chromogranin A 1-76, 1-78, 7-57, 47-76, 47-91, 39-68 e 46-63.

The sequence 46-63 is new and constitutes a further subject-matter of the present invention. The preferred non-peptidic peptidomimetic derivatives are the derivatives of the sequences of the vasostatin I, vasostatin II and the peptidic sequences indicated above. According to the present invention, the expression "inflammatory diseases of the digestive system" indicates all the inflammatory pathologies that can affect the digestive system, in particular the chronic inflammatory diseases and more specifically the pathologies of the gastrointestinal system.

According to a preferred embodiment of the invention, said diseases are chosen from chronic inflammatory bowel disease, ulcerative colitis and Crohn's disease. According to the present invention, the expression "inflammatory diseases of the respiratory system" indicates all the inflammatory pathologies that can affect the respiratory system, in particular chronic inflammatory diseases, for example asthma and allergic asthma. It has been found, in fact, that chromogranin and the sequences indicated here are able to reduce the response of the respiratory system to allergic/inflammatory stimuli. According to another preferred aspects, the invention concerns the use of vasostatin I and/or vasostatin II and/or one of their active fragments and/or of their non-peptidic peptidomimetic derivatives for the treatment and/or prevention of chronic inflammatory bowel disease, ulcerative colitis and/or Crohn's disease. For the use according to the invention, chromogranin A and its active peptidic fragments can be administered orally. This represents a further surprising aspect of the invention, considering that peptides are not normally active orally.

According to a further aspect, the present invention concerns the use of a peptide which presents a homology of at least 60%, preferably at least 80%, with the chromogranin A or with the vasostatin I or vasostatin II, for the preparation of a medicament for the treatment and/or prevention of the above pathologies. The expression "peptide which presents a homology of at least 60%, preferably at least 80%" indicates a variation of the peptide, i.e. a peptidic sequence which differs in one or more amino acid positions with respect to that of the peptides described above, but which maintains at least 60 and preferably at least 80% amino acid identity with said peptides. For the use according to the invention, it is possible to administer the peptide(s) or their peptidomimetic derivatives as above, in oral formulations, prepared as known in the art. Tablets, capsules, granulates and liquid compositions can therefore be prepared comprising the chosen peptide(s), in combination with the excipients commonly used in the formulation of oral compositions and well known to a person skilled in the art. According to a preferred embodiment, the oral compositions of the invention as defined above are gastro-resistant compositions. Said compositions can be prepared as known to a person skilled in the art.

In evaluation assays in vitro of the capacity to restore a mechanically damaged intestinal epithelium, the peptides described above, and vasostatin in particular, proved very effective. In the same way, in vivo studies on murine models of acute and chronic colitis have shown that the above peptides, and vasostatin in particular, cure and protect against damaged induced by DSS (dextran sodium sulphate), thus demonstrating their effectiveness in the treatment and also prevention of inflammatory pathologies of the gastroenteric tract. The details of the experimentation conducted are provided in the experimental section of the present description.

According to a further aspects, the invention concerns a method for the treatment and/or prevention of inflammatory diseases of the digestive and/or respiratory system, in particular of chronic inflammatory diseases, which comprises administering orally, to a patient who requires it, an effective quantity of at least one peptide chosen from chromogranin A, its active peptidic fragments and the peptides which present a homology of at least 80%, advantageously 90%, with chromogranin A and/or its active peptidic fragments. Experimental section Example 1

Evaluation of the curative effect of vasostatin on intestinal epithelial cells Caco-2 cells were kept in a medium containing 0.1% serum for 18 hours before being treated for the assay. The culture of monolayer cells was punctured with the tip of a pipette and washed with PBS. One part of the culture was then incubated with a medium containing 0.1% serum, one part with a medium containing 0.1% serum and 3 μg/ml of vasostatin (I) and a third part with a medium containing 10% serum, at 37 °C. The cultures were examined under inversion microscope. The images were acquired with a digital camera 2, 4, 6 and 24 hours after the treatment, and healing of the wounds was calculated as a percentage of the damaged area covered by migrated cells. The results are shown in figures 1 and 2. Said results show that the cells exposed to vasostatin I, in a medium with very little FBS, cover the damaged area more quickly than the cells not exposed to vasostatin with a trend comparable to that obtained in a medium rich in FBS. The vasostatin I is therefore able to promote the physiological process of repair of the lesions. Example 2

Evaluation of the protective effect of vasostatin on a murine model of acute colitis Mice were fed with 2% dextran sodium sulphate (DSS) in water for 7 days, thus causing acute colitis. On the 8^th day, the solution of DSS was changed with water from an autoclave. From the 4^th day, the animals were treated with vasostatin (I ) (3 or 30 μg/mouse per day). The results obtained are reported in the figures from 3 to 5 and in Table 1.

Said results show that the vasostatin I administered to the mice in which acute colitis is induced has a curative effect. The weight loss measurable during the treatment with DSS is avoided by administering vasostatin, furthermore the mice treated with vasostatin I have a transepithelial resistance of the colon comparable to that of the healthy mice. The lowering of the resistance, measured in the animals treated with DSS, is an index of the damage to the tissue which is preserved by the administration of vasostatin 1. Evaluation of the protective effect of vasostatin on a murine model of chronic colitis 7 mice were divided into two groups. One group of three mice was given 2% dextran sodium sulphate (DSS) for 4 cycles according to the following scheme: 1 cycle of 7 days of DSS followed by 14 days of regular water. A second group of mice was given dextran sodium sulphate (DSS) for 4 cycles according to the scheme indicated above, followed by the administration of vasostatin (15 μg/mouse) orally, 5 times a day for 3 weeks. The results of the epithelial resistance are shown in Table 2. Said results show that the vasostatin I has a curative effect also in an experimental model of chronic colitis. Its administration, in fact, allows repair of the damaged tissue until a transepithelial resistance is obtained comparable to that of a healthy tissue.

Example 3

Evaluation of the protective effect of vasostatin on tight cell junctions in intestinal epithelial cells

Caco-2 cells were grown in a monolayer on Transwell supports made of polycarbonate membranes coated in collagen (Corning-Costar, Acton, MA) and used

17 to 20 days after the confluence.

The flow of dextran marked with fluorescein isothiocyanate (FITC) through the monolayers of the Caco-2 was assayed after 30 minutes and after 2 hours

(measurement of the fluorescence).

A flow of dextran through the monolayer indicates damage to the tight cell junctions.

The samples are treated with IFN and TNF.

In the assay results, shown in figures 6 and 7, in the presence of vasostatin at concentrations of 0.1, 1, 3 and 10 micrograms, the flow of dextran is significantly reduced, with dose-dependent effect, demonstrating that the vasostatin avoids damage to the cell junctions.

The same test was used to verify the effectiveness of the active fragments of the chromogranin (CgAl-76, CgA7-57 and CgA39-68) (figures 8 and 9), while the fragment CgAl -40 showed a modest activity.

To confirm the activity found, the same assay was conducted also in the presence of anti-chromogranin (aChrom) and anti-vasostatin (aVas) antibodies. The results are shown in figures 10 and 11.

Claims

1. Chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives, for use in the treatment and/or prevention of inflammatory diseases of the digestive system and/or of the respiratory system.

2. Chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives, as claimed in claim 1, for use in the treatment and/or prevention of inflammatory diseases of the digestive system.

3. Chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives, as claimed in claim 1 or 2, wherein said active peptidic fragments are vasostatin I or vasostatin II.

4. Chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives, as claimed in the claims from 1 to 3, wherein said active peptidic fragments are the amino acid sequences 1-76, 1-78, 7-57, 47-76, 47-91, 39-68 e 46-63 of the chromogranin A.

5. Chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives, as claimed in claim 3, wherein said active peptidic fragments are fragments of the vasostatin consisting of 5-10, 10-20, 20-40, 40-60 or 40-75 amino acid residues.

6. Chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives, as claimed in claim 1 or 2, wherein said non- peptidic peptidomimetic derivatives are derivatives of the vasostatin I, vasostatin II or of the derivatives of the amino acid sequences of claims 4 or 5.

7. Chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives, as claimed in any one of the preceding claims, wherein said inflammatory diseases of the digestive system are chosen from chronic inflammatory diseases, chronic inflammatory bowel disease, ulcerative colitis and Crohn's disease.

8. Use of a peptide which presents a homology of at least 60% with the chromogranin A or with the vasostatin I or vasostatin II, for the preparation of a medicament for the treatment and/or prevention of inflammatory diseases of the digestive system.

9. Use of chromogranin A, its active peptidic fragments or their non-peptidic peptidomimetic derivatives for the preparation of a medicament for the treatment and/or prevention of inflammatory diseases of the digestive system.

10. Amino acid sequence 46-63 of the chromogranin A.

11. Oral pharmaceutical composition comprising as the active ingredient a peptide sequence chosen from chromogranin A, one of its active peptidic fragments and one their non-peptidic peptidomimetic derivatives.

12. Oral pharmaceutical composition as claimed in claim 11, wherein said active ingredient is chosen from vasostatin I and vasostatin II.

13. Oral pharmaceutical composition as claimed in claim 11, wherein said active ingredient is chosen from the amino acid sequences 1-76, 1-78, 7-57, 47-76, 47-91, 39-68 e 46-63 of the chromogranin A.