WO2010006635A1

WO2010006635A1 - Treatment of inflammatory bowel disease

Info

Publication number: WO2010006635A1
Application number: PCT/EP2008/008044
Authority: WO
Inventors: Karl-Uwe Petersen
Original assignee: Paion Deutschland Gmbh
Priority date: 2008-07-16
Filing date: 2008-09-23
Publication date: 2010-01-21

Abstract

Use of thrombomodulin or thrombomodulin analogues for the manufacture of a medicament for treatment of inflammatory bowel disease.

Description

"Treatment of inflammatory bowel disease"

The invention relates to the field of inflammatory intestinal disorders. More particularly, this invention relates to the treatment of inflammatory bowel disease such as Crohn^'s disease or ulcerative colitis.

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the large intestine and, in some cases, the small intestine. The two main forms of IBD are Crohn's disease and ulcerative colitis (UC). Other forms of IBD such as collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behget's syndrome, infective colitis and indeterminate colitis account for far fewer cases.

IBD affects approximately 1 million patients in the US and approx. 4 million patients worldwide.

Crohn^'s disease is an idiopathic chronic enteritis. Crohn^'s disease affects more than 500,000 patients in the US, of which approx. 50% are moderate to severe. This disease occurs most frequently in human in their twenties and becomes chronic. Both sexes are affected. It is a granulomatous lesion with fibrosis or ulceration and may be present within the entire alimentary tract from mouth to anus. The clinical symptoms of Crohn^'s disease are celialgia, general malaise, diarrhea, melena and occult bleeding positive, fervescence, loss of body weight, anemia, ileus, abdominal tumor and peritonitis. Finally, Crohn's disease presents with extra-intestinal manifestations (such as liver problems, arthritis, skin manifestations and eye problems such as episcleritis and uveitis). Ulcerative colitis is a disease of diffuse nonspecific inflammation of the colon, which attacks the mucous membrane and often forms an erosion or ulcer. UC affects an estimate of 500,000 patients in the US, 20% of which are moderate, 15% severe and 35% are refractory or in remission. The lesion is mainly submucosal. The clinical symptoms of this disease are viscous-hemafecia, celialgia, hemafecia, watery stool, fervescence, loss of appetite, nausea and vomiting. Also, ulcerative colitis may be accompanied with a lower incidence of certain syndromes such as arthritis, stricture of the large intestine and copious bleeding.

Depending on the level of severity, IBD may require immunosuppression to control the symptoms, such as prednisone, infliximab (Remicade), adalimumab (Humira), azathioprine (Imuran), methotrexate, or 6-mercaptopurine. More commonly, treatment of IBD requires a form of 5-aminosalicylic acid (INN: mesalazin; USAN: mesalamine). Often, systemic corticosteroids are used to control disease flares. Of this class, only budesonide, which has a very low oral availability, is used for this purpose.

For the treatment of Crohn's disease and, recently, of ulcerative colitis biologicals are used such as the intravenously administered infliximab. Severe cases may require surgery, such as bowel resection, strictureplasty or a temporary or permanent colostomy or ileostomy. Alternative medicine treatments for bowel disease exist in various forms, however such methods concentrate on controlling underlying pathology in order to avoid prolonged steroidal exposure or surgical excisement.

Usually the treatment is started by administering drugs with high anti-inflammatory affects, such as prednisone. Once the inflammation is successfully controlled, the treatment is usually changed to a lighter drug to keep the disease in remission, such as asacol, a mesalamine. If unsuccessful, a combination of the aforementioned immunosupression drugs with mesalamine may be administered.

Severe side effects are associated with the drugs commonly prescribed for IBD e.g. nausea, dizziness, changes in blood chemistry (including anemia and leukopenia), skin rashes and drug dependence; and the surgical treatments are radical procedures that often profoundly alter the everyday life of the patient. Thus the known treatment options do not meet the medical needs. Importantly, no drug is currently available which enables a remission of the symptoms and prevents a subsequent relapse with a good tolerability.

Thus, it is an objective of the present invention to provide novel means for treating inflammatory bowel disease, in particular Crohn^'s disease and ulcerative colitis; and to provide methods for manufacture of respective medicaments. This objective is solved by providing a medicament for the patient suffering from inflammatory bowel disease comprising a therapeutically effective amount of thrombomodulin (TM) or a TM analogue. According to one embodiment of the invention oxidation resistant thrombomodulin analogue can be administered.

As used herein the term "inflammatory bowel disease" shall refer to a chronic inflammatory physiological condition that negatively impacts the gastrointestinal tract of a mammal, particularly a human. Gastrointestinal conditions that may be treated by thrombomodulin include but may not limited to: Crohn's disease, ulcerative colitis (UC), collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, infective colitis and indeterminate colitis.

A "therapeutically effective amount" is defined as the amount of active ingredient that will reduce the symptoms associated with a chronic inflammatory gastrointestinal condition, such as IBD. "Therapeutically effective" also refers to any improvement in disorder severity or the frequency of incidences compared to no treatment. The term treatment encompasses either curing or healing as well as mitigation, remission or prevention.

In the natural environment thrombomodulin is a membrane protein that acts as a thrombin receptor on the endothelial cells lining the blood vessels. Thrombin is a central enzyme in the coagulation cascade, which converts fibrinogen to fibrin, the matrix clots are made of. Initially, a local injury leads to the generation of small amounts of thrombin from its inactive precursor, prothrombin. Thrombin, in turn, activates platelets and, second, certain coagulation factors including factors V and VIII. The latter action gives rise to the so-called thrombin burst, a massive activation of additional thrombin molecules, which finally results in the formation of a stable clot.

When bound to thrombomodulin, however, the activity of thrombin is changed as follows: A major feature of the thrombin-thrombomodulin complex is its ability to activate protein C, which then downregulates the coagulation cascade by proteolytically inactivating the essential cofactors Factor Va and Factor Villa (Esmon et al., Ann. N. Y. Acad. Sci. (1991 ), 614:30-43), thus affording anticoagulant activity. The thrombin- thrombomodulin complex is also able to activate the thrombin-activatable fibrinolysis inhibitor (TAFI), which then antagonizes fibrinolysis. Furthermore, activated protein C is known to exhibit anti-inflammatory properties.

Although earlier studies were negative, more recent studies have indicated that thrombomodulin is not only present in brain endothelial cells, but also is expressed on the surface of astrocytes, where it functions as in the vasculature, namely activating protein C after forming a complex with thrombin. Thrombomodulin is also upregulated in reactive astrocytes in the CNS, in response to mechanical injury. Other reports suggest that recombinant thrombomodulin is capable of blocking thrombin's activation of another receptor, the protease-activated receptor 1 (PAR-1 ) in cultured neuronal cells.

Mature TM is composed of five domains: an N-terminal lectin-like binding domain, an epidermal growth factor (EGF) domain which consists of 6 EGF-like-repeats, a Ser/Thr- rich region (O-linked glycosylation domain), a transmembrane domain and a cytoplasmic domain.

A domain is a three dimensional, self-assembling array of amino acids of a protein molecule, which contains structural elements necessary for a specific biological activity of that protein. While thrombin binding is thought to be linked to the EGF domain, the lectin-like domain is believed to be responsible for the direct anti-inflammatory properties of TM (van de Wouwer et. al: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis; in: Journal of Thrombosis and Haemostasis 4, 1813-1824, 2006).

(see CS. Yost, et al, (1983) Cell 34:759-766 and D. Wen, et al, (1987) Biochemistry 26:4350-4357, both herein incorporated by reference.)

The term "native" thrombomodulin refers to both the natural protein and its soluble form having substantially the same characteristic biological activities of membrane-bound or detergent solubilized (natural) thrombomodulin. These soluble peptides are also referred to as "wild-type" or "non-mutant" peptides. The biological activity include the ability to act as a receptor for thrombin, and therewith increase the activation of protein C₁ and other biological activities associated with native thrombomodulin, in particular anti-inflammatory activities such as suppression of the activation of the complement system. The assessment of the antithrombotic properties of TM analogues is known to the skilled person. Assessing the suppression of the complement system is disclosed e.g. in the above publication of van de Wouwer et al. The term "thrombomodulin analogue" in contrast refers to proteins which differ from the native TM by at least one amino acid, either by deletion, substitution, derivatisation or addition thereof, or any other kind of modification, whereas their activity substantially corresponds to the biological activities of native TM in terms of activation of protein C or activation of anti-inflammatory processes, in particular the suppression of the complement system. In one embodiment the TM analogues of the invention exhibit substantially the same properties as the native TM in both terms, i.e. regarding the activation of protein C and the induction of anti-inflammatory processes.

TM analogues applicable according to the invention can comprise one or more domains of the native TM. In one embodiment TM analogues can be used which are composed by the EGF and the lectin-like domain only. However, a TM analogue consisting of the EGF domain only can also be used. Such TM analogues are disclosed in WO 2008/073884 A1.

As outline above, in one embodiment of the invention the TM analogue has the amino acid amino sequence of the native sequence or parts thereof with one or more amino acids removed or replaced by one or more different amino acids. Specifically, the amino acids removed or replaced are either one or both of the methionine residues at positions 291 or 388 in the native TM protein sequence (SEQ ID No1 ). These methionines can be replaced with the amino acid alanine, leucine, isoleucine, glutamine or valin. Most preferred is the substitution with leucine.

Replacing these methionines results in an oxidation resistant TM analogue that retains its activity after exposure to oxidants, in particular the activation of protein C. Its antiinflammatory activities are not affected. It may also exhibit an increased specific activity when compared to an equivalent peptide not having an amino acid substitution. "Oxidation resistant TM analogue" thus are peptides which are resistant against inactivation by oxidants, such as oxygen radicals. Preferably these TM analogues are soluble.

Hence in one embodiment of the invention a thrombomodulin analogue can be used that has the characteristic antithrombotic and anti-inflammatory activity of native thrombomodulin but which is soluble in aqueous solution and is not inactivated after having been exposed to oxidants. According to this embodiment of the invention the peptides are lacking at least the membrane spanning and cytoplasmic domains of native thrombomodulin.

Various forms of soluble thrombomodulin or thrombomodulin analogues are known to the skilled person, e.g. the so called ART-123 developed by Asahi Corporation (Tokyo, Japan) or the recombinant soluble human thrombomodulin known as Solulin, currently under development by PAION Deutschland GmbH, Aachen (Germany). The recombinant soluble thrombomodulin, i.e. a soluble thrombomodulin without any modification of the amino acid sequence of the native, is subject of the EP 0 312 598. Both, ART-123 and Solulin can be used according to the invention.

Solulin is a soluble, as well as protease and oxidation-resistant analogue of human thrombomodulin and thus exhibits a long life in vivo. Solulin's main feature lies in its broad mechanism of action since it not exclusively inhibits thrombin. It also activates the natural protein C pathway, and therefore stops further generation of thrombin.

Solulin is inter alia subject of the European patent 0 641 215 B1, EP 0 544 826 B1 as well as EP 0 527 821 B1. Solulin contains modifications compared to the sequence of native human thrombomodulin (SEQ. ID NO. 1 ) at the following positions: G -3V, Removal of amino acids 1-3, M388L, R456G, H457Q, S474A and termination at P490. This numbering system is in accordance with the native thrombomodulin of SEQ. ID NO. 1. The sequence of Solulin as the preferred embodiment of the invention is shown in SEQ. ID NO. 2.

However, notably, according to the invention also thrombomodulin analogues can be utilized, which comprise only one or more of the above mentioned properties, or of the properties outlined in the above mentioned European patent documents EP 0 544 826 B1 , EP 0 641 215 B1 and EP 0 527 821 B1.

Other TM analogues are known, e.g. from WO 2008/073884 A2, which are modified as to have a reduced ability to activate protein C. In one embodiment of the invention also these analogues can be used to treat IBD.

Particularly preferred thrombomodulin analogues applicable according to the invention are those that have one or more of the following characteristics:

(i) oxidation resistance

(ii) protease resistance

(iii) homogeneous N- or C-termini

(iv) post-translational modification e.g., by glycosylation of at least some of the glycosylation sites of native thrombomodulin (SEQ ID NO: 1 ) (v) linear double-reciprocal thrombin binding properties (vi) solubility in aqueous solution in relatively low amounts of detergents and typically lack a transmembrane sequence (vii) lack of glycosaminoglycan chain. Most preferred is a molecule comprising all of these modifications, e.g. as shown by Solulin. The manufacture of these analogues is disclosed in the above mentioned European patent documents.

In a further embodiment of the invention, the thrombomodulin analogue known from the WO 01/98352 A2 or US 6,632,791 can be used. Also thrombomodulin derived by rabbits can be used. The 6EGF fragment of Solulin is an example of a thrombomodulin fragment with essentially the same biological activity regarding the formation of a complex with thrombin with the ability to activate human protein C. This fragment essentially consists of the six epidermal growth factors domain of native thrombomodulin.

In a further embodiment of the invention, thrombomodulin or thrombomodulin analogues can be used to treat patients with a distal-, left sided- or extensive pancolitis.

In yet another embodiment of this invention a treatment of mild, moderate, severe or fulminant IBD is possible.

In another embodiment of this invention thrombomodulin or the thrombomodulin analogues can be administered to an adult or paediatric patient in order to induce remission or to maintain the remission, i.e. to prevent a relapse.

In a further embodiment of the invention the treatment can be performed according to the localisation in ulcerative colitis patients with lesions in the terminal ileum or colon.

Thrombomodulin or the thrombomodulin analogues can be given non-orally as a parenteral formulation e.g. by intravenous or subcutaneous formulation. An intravenous or subcutaneous bolus application is possible. Thus, according to the invention a pharmaceutical composition is provided, which is suitable for a parenteral administration of thrombomodulin or thrombomodulin analogues.

Thrombomodulin or the thrombomodulin analogues can be administered in multiple doses. For multiple administration it can be given parenterally, preferably intravenously or subcutaneously once daily but also bidaily, or every thirth, fourth, fifth, sixth or seven day. Thus, according to one embodiment of the invention a pharmaceutical composition is provided, which is suitable for allowing a multiple administration of thrombomodulin or thrombomodulin analogues.

The effect of thrombomodulin or the thrombomodulin analogues underlying the present invention was found in an animal model of inflammatory bowel disease. In this model mice were treated with dextran sulfate which results in an acute progressive colitis. The mice received a daily bolus injection of soluble thrombomodulin which surprisingly ameliorates all clinical endpoints in comparison to an untreated control group, in particular the treatment resulted in an reduction of mortality.

I. EXAMPLE

Colitis model in mice - 1.5% DSS

Using a model of dextran sodium sulfate-induced colitis the ability of a soluble thrombomodulin analogue (Soluliή) to decrease colitis symptoms was tested. Solulin was given daily starting from day 0.

1. Test system

The study was conducted in male mice, C57BL/6 strain. The age of the animals was approximately 10 weeks.

2. Experimental procedures and study treatment

The mice were administered filter-purified and sterilized water containing 1.5% dextran sodium sulfate (DSS) for 10 days and gradually developed colitis. Eight animals (Group 1 ) received a daily intravenous injection of 3mg/kg Solulin, whereas the remaining eight animals of group 2 received no treatment.

The clinical disease activity index (DAI) as a scale ranging from 0 to 4 was calculated on a daily basis using the following parameters: weight loss, stool consistency and presence or absence of fecal blood (see the following table).

At the end of the treatment the mice were sacrificed and for each animal a histological quantification of the distal colon was performed. The three independent parameters measured were severity of inflammation, extent of inflammation and crypt damage. The colon length was measured as a marker of inflammation. 3. Results

DSS-Colitis: Administration of 1.5% DSS for 10 days resulted in severe acute colitis that was associated with a marked decrease of body weight and colon length and a histological evidence of colitis.

Body weight. In accordance with the literature C57BU6 mice exposed to 1.5% DSS in drinking water experienced a steady weight loss. As result of the Solulin treatment the DSS colitis animals displayed less severe weight loss during the last days of the observation period (see Figure 1 ).

Disease activity score. The progression of the acute colitis is mirrored by a steady increase in the DAI. Compared with the control animals, the Solulin treated mice showed a decreased DAI from day 5 to day 7 (see Figure 2).

Colon length: When measured, the average whole colon length of the Solulin- treatment group was notably longer than that of the Control Group (see Figure 3).

Histology score:

Histological analysis of colonic tissues revealed increased colitis scores in DSS-treated mice, whereas the Solulin treatment resulted in a prominent reduction of the histology score (see Figure 4).

3. Conclusion

Compared with the control animals the Solulin-treated mice displayed less severe colitis as demonstrated by reduced weight loss, increased colon length and improved DAI. In addition at the end ot the 10-day treatment schedule, the histological assessment showed that Solulin-treated mice displayed a marked decrease of inflammation compared with control animals.

Notably, despite the fact that Solulin is an anticoagulant and the DSS-colitis is accompanied with bleeding, the administration of Solulin failed to induce any bleeding events in the treated animals.

Legend

Fig. 1 Effect of Solulin on body weight in mice with DSS-colitis (1.5%DSS)

Fig. 2 Effect of Solulin on disease activity in mice with DSS-colitis (1.5% DSS) Fig. 3 Effect of Solulin on colon length in mice with DSS-colitis (1.5% DSS)

Fig. 4 Effect of Solulin on colon histology in mice with DSS-colitis (1.5% DSS) AMPLE

Colitis model in mice - 2% DSS

Using a model of dextran sulfate-induced colitis the ability of soluble thrombomodulin (Solulin) to decrease colitis symptoms was tested. In this experiment 2% instead of 1.5% DSS was used to induce a more severe colitis. Solulin was given daily starting from day 0.

1. Test system

2. Experimental procedures and study treatment

The mice were administered filter-purified and sterilized water containing 2% dextran sodium sulfate (DSS) for 10 days and gradually developed colitis. Six animals (Group 1 ) received a daily intravenous injection of 1mg/kg Solulin, whereas the remaining six animals of group 2 received no treatment.

The clinical disease activity index (DAI) as a scale ranging from 0 to 4 was calculated on a daily basis using the following parameters: weight loss, stool consistency and presence or absence of fecal blood.

At the end of the treatment the mice were sacrificed and for each animal a histological quantification of the distal colon was performed. The three independent parameters measured were severity of inflammation, extent of inflammation and crypt damage.

The colon length was measured as a marker of inflammation.

In addition a quantitative RT-PCR was performed in order to determine the mRNA levels of the cytokine interleukin-6 (IL-6) and the intercellular cell adhesion molecule-1

(ICAM-1 ).

3. Results DSS-Colitis: Administration of 2% DSS for 10 days resulted in severe acute colitis that was associated with a marked decrease of body weight and colon length, histological evidence of colitis and increased expression of cytokines.

Body weight. In accordance with the literature C57BL/6 mice exposed to 2.0% DSS in drinking water experienced a steady weight loss. As result of the Solulin treatment the DSS colitis animals displayed less severe weight loss, beginning at day 6 (see Figure 5).

Disease activity score. The progression of the acute colitis is mirrored by an steady increase in the DAI. Compared with the control animals, the Solulin treated showed a decreased DAI from day 4 to day 7 (see Figure 6).

Colon length: When measured, the average whole colon length of the Solulin- treatment group was notably longer than that of the Control Group (see Figure 7).

Histology score:

Histological analysis of colonic tissues revealed increased colitis scores in DSS-treated mice, whereas the Solulin treatment resulted in a prominent reduction of the histology score (see Figure 8).

Quantitative PCR analysis:

Expression analysis of colonic tissues revealed increased expression of mRNAs for the inflammatory cytokine interleukin-6 and the inflammation-associated cell adhesion molecule ICAM-1. Both mRNAs were found to be decreased after Solulin treatment (see Figure 9).

4. Conclusion

Compared with the control animals the Solulin-treated mice displayed less severe colitis, as demonstrated by reduced weight loss, increased colon length and improved DAI. In addition at the end of the 10-day treatment schedule, the histological assessment together with an increased colon length showed that Solulin-treated mice displayed a marked decrease of inflammation compared with control animals. The decrease of inflammation is consistent with the reduced expression observed with the inflammatory cytokine, interleukin-6, and the inflammation-associated cell adhesion molecule ICAM-1.

Notably, despite the fact that Solulin is an anticoagulant and the DSS-colitis is accompanied with bleeding, the administration of Solulin failed to induce any bleeding events in the treated animals. Legend

Fig. 5 Effect of Solulin on body weight in mice with DSS-colitis (2%DSS) Fig. 6 Effect of Solulin on disease activity in mice with DSS-colitis (2% DSS) Fig. 7 Effect of Solulin on colon length in mice with DSS-colitis (2% DSS) Fig. 8 Effect of Solulin on colon histology in mice with DSS-colitis (2% DSS)

Fig. 9 Effect of Solulin on IL-6 and ICAM-1on expression in mice with DSS-colitis (2% DSS)

Claims

1. Use of thrombomodulin or a thrombomodulin analogue for the manufacture of a medicament for the treatment of inflammatory bowel disease.

2. Use according to claim 1 , whereas the IBD is ulcerative colitis.

3. Use according to claim 1 , whereas the IBD is Crohn^'s disease.

4. Use according to claim 1 , whereas the IBD is selected from the group of diseases as follows: collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behςet's syndrome, infective colitis or indeterminate colitis.

5. Use according to any of the above claims, whereas the thrombomodulin analogue is oxidation resistant.

6. Use according to any of the above claims, whereas thrombomodulin is given in a multiple administration, preferable over a time period of two to four weeks, more preferably as a chronic administration.

7. Use according to any of the above claims, whereas the thrombomodulin analogue has an amino acid sequence corresponding to the amino acid sequence of the mature soluble thrombomodulin of SEQ ID No 1 and comprises one or more of the subsequent modifications: removal of amino acids 1-3

M388L

R456G

H457Q

S474A, and terminating at P490.