WO2013013826A1 - Necroptosis inhibitors for the treatment of inflammatory diseases of the gastrointestinal tract - Google Patents

Abstract

The invention relates to necroptosis inhibitors for the treatment of inflammatory diseases of the gastrointestinal tract. Particularly, the invention relates to inhibitors of the receptor-interacting protein-1 /receptor-interacting protein-3 (RIP1/RIP3) complex, for the treatment of inflammatory diseases or conditions of the gastrointestinal tract, in particular inflammatory bowel disease.

Description

Necroptosis inhibitors for the treatment of inflammatory diseases of the gastrointestinal tract

Field of the Invention

The invention relates to necroptosis inhibitors for the treatment of inflammatory diseases of the gastrointestinal tract. Particularly, the invention relates to inhibitors of the receptor-interacting protein-1 /receptor-interacting protein-3 (RIP1/RIP3) complex, for the treatment of inflammatory diseases or conditions of the gastrointestinal tract, in particular inflammatory bowel disease.

Background to the invention

Having an area of about 200 m², the intestinal epithelium represents the most important internal surface of the human body. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defense functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium.

As a semipermeable membrane, the intestinal epithelium has a dual function. The membrane allows for the translocation and thus uptake of essential nutrients, electrolytes and water from the lumen into the circulation. On the other hand, the intestinal epithelium represents a barrier, and thus protection, against pathogenic agents and dangerous substances, such as antigens or microorganisms and their toxins. The intestinal epithelium is subject to constant immunological stimulation and the need to build a homeostatic balance between defense mechanisms and immunological tolerance. It is known that excessive infiltration of bacteria or antigens leads to a dysregulated intestinal immune response and may represent an important factor in the development of gastrointestinal diseases and conditions.

However, etiology of inflammatory bowel disease is still not fully understood. It is believed that inflammatory bowel disease results from an interaction of genetic, immunologic and environmental factors. During inflammation processes, immune cells such as T cells, macrophages and neutrophils migrate into their corresponding areas and produce a number of proinflammatory cytokines, such as TNF-a, IFN-γ, IL-6 and IL-22, which might lead to tissue damage. A key mechanism leading to inflammation processes in patients with inflammatory bowel disease is the dysregulation of the immune response towards bacterial antigens related with physiological intestinal flora. Since the intestinal epithelium represents the barrier between these antigens and the immune system, it is hence crucially involved in the activation of the immune response. Furthermore, the intestinal epithelium exhibits a barrier function and separates the intestinal lumen and its microbial flora against the immune system. This function is ensured by so-called tight-junctions between intestinal epithelial cells. Specialized epithelial cells perform an innate immune response. Goblet cells secrete mucus, and Paneth cells secrete antimicrobial peptides into the lumen, and may thus prevent a direct contact between bacteria and epithelium.

A reduced control against, and thus an insufficient elimination of microorganisms, is a characteristic of Crohn^'s disease. In the vast majority of patients, this defect is due to a reduced number of Paneth cells. It is further known that instability of the barrier function of the intestinal wall may lead to a translocation of bacteria from the lumen into subepithelial areas, which in turn causes a non-regulated activation of the immune system. Obviously, such dysregulated immune response represents an important factor in the development of inflammatory bowel disease.

The integrity of the intestinal epithelium is extremely important for homeostasis and functionality of the intestine. Consistently, a balance between activation of proliferation and cell death is very important. Dysregulation of cell death activation can lead to cancer, and further destabilize the intestinal epithelium.

Epithelial cell death is a characteristic of inflammatory bowel disease. However, neither the causes nor the exact mechanism are known today. The best analyzed apoptotic way is the extrinsic signaling pathway which is activated via TNF receptor signaling. Dependent on cell type and surrounding conditions, TNF-a administration leads to survival or death of a cell. TNF-a seems to be one of the key factors in pathogenesis of inflammatory bowel disease. Patients suffering from Crohn^'s disease usually have an elevated TNF-a level. Inflammatory bowel disease is an incurable idiopathic disease with chronic inflammation or ulceration at the mucous membrane of the large and small intestine. Diarrhea or bloody stool continues over a long period with repeated recurrence. Inflammatory bowel disease includes two major types of diseases, that is, Crohn's disease and ulcerative colitis. These diseases impair quality of life of more than 4 million patients worldwide. Moreover IBD patients frequently develop extraintestinal manifestations and are predisposed for the development of colorectal cancer. Crohn's disease, which is also called regional enteritis, granulomatous ileitis or ileocolitis, is a chronic inflammation developed on the intestinal wall, or even on any site of the digestive tract. Ulcerative colitis is a chronic disease wherein inflammation occurs in the large intestine to produce an ulcer, resulting in bloody diarrhea, severe abdominal pain, or an attack accompanied with fever.

At present, no medical options are available for curing Crohn^'s disease, neither by pharmacological nor by surgical means. Conventional therapeutic agents for diarrhea and the like are not effective in a satisfactory manner. Therapeutic options are limited to controlling symptoms, maintaining remission and avoiding relapses. Currently, the treatment of choice for chronic inflammatory gastrointestinal diseases is an immunosuppressive and/or anti-inflammatory medication at sustained high doses. However, such treatment leads, at best, to long-lasting regression of symptoms.

Currently, several classes of medicaments are administered to patients suffering from inflammatory bowel disease. However, most drugs display adverse effects and the attending physician has to evaluate benefit and risk of a certain treatment option in each specific case. Furthermore, it is well known that individual patients respond differently to different drugs.

For the treatment of inflammatory bowel disease, anti-inflammatory drugs, such as corticosteroids and aminosalicylic acid preparations, such as sulfasalazine and 5- aminosalicylic acid, have been widely used from the past as first-line and second-line drugs. Corticosteroids reduce inflammation but have lots of side effects such as swollen face, facial hair, night sweats, insomnia and hyperactivity. Further side effects are hypertension, type II diabetes, osteoporosis, bone fractures, cataract and increased susceptibility to infections. Long-term treatment in children with corticosteroids may result in delayed development. Given for a short period (about 3- 4 months) corticosteroids may alleviate symptoms, however, they are not intended for long-term treatment.

The corticosteroid sulfasalazine may be beneficial for the alleviation of inflammatory conditions in the colon, however it is not always effective in the treatment of Crohn^'s disease. Another drawback is that possible side effects such as nausea, heartburn and headache may occur. Further, sulfasalazine is contra-indicated in patients with known allergy to sulfa drugs.

The corticosteroid mesalamine exhibits fewer side effects, however, nausea, heartburn and headache may also occur. Depending on the part of the colon which is affected, mesalamine may be administered orally or rectally. Generally, mesalamine is not effective for diseases of the small intestine. Bundesonide, belonging to a newer generation of corticosteroids, exibits less side effects and a faster effect as conventional corticosteroids. However, the beneficial effect of bundesonide is limited to Crohn^'s disease relating to the terminal part of the small intestine or the upper colon. In general, corticosteroids are not indicated for use in Crohn^'s patients. The treatment option of inflammatory bowel disease with corticosteroids is limited to severe inflammation conditions after other medical options have failed.

Immunosuppressants reduce inflammation processes by suppressing the immune system. Azathioprin and mercaptopurin are the drugs of this class which are most frequently administered to inflammatory bowel disease patients. Even though it may take up to 2-4 months until these drugs show an effect, they may alleviate symptoms of the disease, e.g. heal fistulae in Crohn^'s patients.

Infliximab, which is a chimeric monoclonal anti-TNF-a antibody, is approved for adults and children with Crohn^'s disease with medium to severe disease, who do not tolerate or respond to other treatment options. Infliximab inhibits TNF in the blood so that it cannot contribute to inflammation of the gastrointestinal tract. Infliximab is contra-indicated in patients with known cardiac insufficiency, multiple sclerosis and tumors. Further, dormant infections such as tuberculosis may be reactivated by taking the drug. A number of adverse effects of infliximab are known, such as hypertension, nausea, rash, fever, headache, and eczema. Furthermore, since infliximab is a chimeric antibody, it may show antigenicity and sometimes cause acute ultra-hypersensitive reaction. Similar to infliximab, the monoclonal anti-TNF-a antibody adalimumab is approved for severe Crohn^'s disease and takes effect by blocking TNF-a. Also the profile of side effects is similar to that of infliximab. Adalimumab is administered subcutaneously. Certolizumab is a PEGylated Fab^' fragment of a humanized monoclonal anti-TNF-a antibody. Also certolizumab exhibits similar properties as the previously mentioned antibodies. Presently, treatment with TNF blocking agents seems to be one of the most effective means for Crohn^'s patients. However, treatment of TNF-a induced cell death is still limited.

Although indicated primarily for therapy of psoriasis and rheumatoid arthritis, methotrexate may also be used in therapy of refractory Chrohn^'s disease. Side effects of methotrexate are nausea, fatigue and diarrhea, as well as life-threatening complications such as severe pneumonis, cirrhosis of the liver and cancer.

Cyclosporine is considered as a reserve drug, and is primarily effective in the treatment of fistulae in Crohn^'s patients. Side effects are liver and kidney damages, hypertension and an increased risk for the development of lymphoma.

Natalizumab is approved for the treatment of medium and severe progression of Crohn^'s disease. The drug leads to an anti-inflammatory effect by inhibiting so-called integrins. Although a rare side effect, multifocal leukoencephalopathy can occur and cause serious disabilities or even death of the patient.

Another class of drugs is antibiotics. Antibiotics may be beneficial for healing fistulae and abscesses in Crohn^'s patients. Possibly, antibiotics help to reduce the number of harmful enterobacteria and thus, strengthen the immune system. Antibiotics which are often prescribed are metronidazol and ciprofloxacin. Metronidazol is effective against anaerobes. Severe side effects may occur occasionally, e.g. numbness and tingling of hands and feet, muscle pain or weakness. Further side effects might be a metallic taste in the mouth, headache and lack of appetite. Today, ciprofloxacin is preferred over metronidazol, even if it may cause nausea, vomiting, headache and rarely tendon rupture.

Further medicaments are administered to improve the general condition of patients suffering from inflammatory bowel disease.

Medication against diarrhoe improves liquid bowel movement. Loperamid belongs to this class of drugs. Extreme care has to be taken with such medication and administration should be coordinated with a physician. The most feared complication is the so-called toxic megacolon, which is a live-theatening condition.

Laxative drugs, e.g. bifiteral, may be administered to alleviate constipation. Since these drugs interfere with the water and electrolyte balance, a physician should be consulted before taking said drugs.

For pain medication, different types of substances with different spectra of adverse effects are available. Mostly used are the so-called non-steroidal anti-inflammatory drugs (NSAID), e.g. Aspirin and Ibuprofen. If regularly taken, these drugs have to be combined with proton pump inhibitors due to their ulcerogenic effect.

Chronic intestinal blood loss leads to iron deficiency. Iron is essential for blood formation, and blood formation in insufficient when iron deficiency occurs in a patient. Thus, anemia gets worse. Iron supplementation remedies this deficiency and allows the body for formation of new blood cells.

In case of malnutrition in inflammatory bowel disease patients several possibilities are available, e.g. enteral or parenteral nutrition. The caloric requirement depends on the individual patient. The calculation considers body height and weight, laboratory parameters such as albumin, and pre-existing diseases, e.g. infectious diseases.

Vitamin B12 may be administered to patients with vitamin B12 deficiency. Reasons for vitamin B12 deficiency are insufficient supply by nutrition and/or insufficient resorption of vitamin B12. The latter is caused by a deficiency in intrinsic factor, a glycoprotein produced by parietal cells, required for the absorption of vitamin B12 from the gastrointestinal tract. The intrinsic factor binds cobalamin within a complex protected from digestive enzymes for the transport into intestinal cells. Vitamin B12 deficiency may lead to pernicious anemia and funicular myelosis.

A main side effect of steroids, which are administered very often to inflammatory bowel disease patients, is development of osteoporosis. To prevent this side effect, administration of calcium and vitamin D is beneficial.

Surgical therapy might be an option for inflammatory bowel disease patients in case of insufficient efficacy, severe side effects of drugs and/or serious complications of inflammation. Surgery is used mainly in case of fistulae, abscesses and stenosis, as well as in therapy-refractory courses of the disease. During surgery, the affected parts of the intestine are resected. Healthy parts are maintained as best as possible, however, frequently a new artificial anus (anus praeter) has to be constructed to unburden the sutures. Normally, the anus praeter can be relocated after few months. However, recurrences are frequent and many patients require repeated surgical intervention. For example, about 20-25% of ulcerative colitis patients experience therapy failure and surgery (proctocolectomy).

The above described drawbacks of the state of the art medication clearly show the need for novel treatment options for inflammatory bowel disease.

Description of the Invention

The invention relates to inhibitors of the RIP1/RIP3 complex, in particular to inhibitors of the activation or expression of the RIP1/RIP3 complex, for the treatment of inflammatory diseases or conditions of the gastrointestinal tract, in particular inflammatory bowel disease.

Historically, cell death has been subdivided into regulated (apoptosis, programmed cell death) and unregulated (necrosis) forms. Apoptosis has always been recognized to be a pathway of highly coordinated signaling events which is a naturally occurring cause of cellular death and can often provide beneficial effects to the organism. For a long time TNF-a receptor activated caspase-dependent apoptosis has believed to be the primary way of programmed cell death. Caspase-8 is a cystein protease critically involved in regulating cellular apoptosis. Upon activation of death receptors, including TNF-receptor and Fas, caspase-8 is activated by limited autoproteolysis and the processed caspase-8 subsequently triggers the caspase cascade which finally leads to apoptotic cell death.

Historically, necrosis has been considered an accidental cell death and not set to determined pathways or cellular regulation. Necrotic cell death is defined by an increase in cell volume, swelling of organelles, plasma membrane rupture and eventual leakage of intracellular components.

Newly research is determining that necrosis is not just a series of unregulated, uncontrollable processes but may in fact be a series of 'programmed necrosis' named necroptosis. Recent findings have shown that after inhibition of caspase activity in genetic models or by using specific caspase inhibitors an apoptosis-independent type of necroptosis can occur, i.e. TNF receptor signaling can result in necroptosis (Vandenabeele, P. et al., Nat Rev Mol Cell Biol 1 1 (10), 700-714, 2010). Thus, necroptosis is currently considered as a specialized biochemical pathway of programmed necrosis. Necroptosis has been shown to be mediated by the kinase activity of receptor-interacting proteins 1 and 3 (RIP1 and RIP3) (Cho, Y.S. et al., Cell 137 (6), 11 12-1 123, 2009). Phosphorylation-driven assembly of the RIP1 -RIP3 necrosis complex seems to regulate necroptosis. Specifically, RIP1 kinase activation is required as an upstream regulator of necroptotic death pathway. In summary, caspase-8 has a critical function in regulating intestinal homeostasis and in protecting intestinal epithelial cells (lECs) from TNF-a induced necroptotic cell death. Normally, TNF receptor signalling leads to activation of the classical caspase signalling pathway, by cleavage of the initiator caspase-8, which in turn activates the classical caspase cascade. Furthermore, it cleaves RIP1 and RIP3 and thus, inactivates the necroptosis signalling pathway. In case of inhibition of caspase-8, e.g. by pharmacological or genetic factors, RIP3 is recruited to RIP1 to establish a necroptosis inducing protein complex, both proteins are being phosphorylated and consequently activated and constitute the effector molecules for the necroptosis signalling pathway. For the activation of necroptosis, kinase activity of both RIP1 and RIP3 is required.

RIP3 seems to be essential for the molecular mechanisms driving necroptosis, and expression of RIP3 has been demonstrated to correlate with the sensitivity of cells towards necroptosis (He, S. ef al., Cell 137 (6), 1 100-1 1 1 1 , 2009).

Activation of the necroptosis inducing protein complex including RIP1 and RIP3 can be inhibited or blocked by specific inhibitors, e.g. necrostatin-1 (nec-1 ), an allosteric small-molecule inhibitor of the RIP1 kinase. Nec-1 inhibits necroptosis by blocking kinase activity of RIP1 , which in turn inhibits phosphorylation of RIP3.

The present inventors have surprisingly found that inhibition of necroptosis activated via the RIP1/RIP3 complex leads to an effective reduction of the symptoms of inflammatory processes in the gastrointestinal tract.

Further, the present invention discloses the unexpected function of caspase-8 in regulating necroptosis of intestinal epithelial cells and in maintaining immune homeostasis in the gut.

Caspase-8 deficient mice have no defect in overall gut morphology, demonstrating that cell death independent from the extrinsic apoptosis pathway can regulate intestinal turn over. However, caspase-8 deficient mice completely lacked Paneth cells, suggesting that these cell types are highly susceptible to necroptosis. This lack of Paneth cells results in a diminished expression of anti-microbial peptides and the development of spontaneous intestinal inflammation in the terminal ileum, similar to features of Crohn^'s disease.

It has been shown that epithelial cell death induced by tumor necrosis factor TNF-a, was associated with increased expression of RIP3. An inhibitor of the RIP1/RIP3 complex is able to block TNF-a induced necroptotic cell death in caspase-8 deficient intestinal epithelial cells in vivo after injection of TNF-a in caspase-8 deficient mice.

Crohn's disease patients frequently show reduced Paneth and goblet cell numbers and reduced expression of Paneth cell derived defensins in areas of acute inflammation. The present invention provides data demonstrating a constitutive expression of RIP3 in human intestinal epithelial cells especially in Paneth cells at the base of the crypts of Liberkiihn in the small intestine. Furthermore Crohn^'s disease patients show high levels of the necroptosis mediator TNF-a in the terminal ileum, and anti-TNF-a treatment is successful! in the therapy of IBD patients. Thus human Paneth cells might be susceptible to TNF-a-induced and RIP-mediated necroptosis. In line with this, cells undergoing necroptosis can be found at the crypt base in patients with Crohn's disease. Paneth cell death could be inhibited by blocking necroptosis. These findings demonstrate the role of necroptosis in the pathogenesis of inflammatory bowel disease, e.g. Crohn^'s disease and ulcerative colitis.

Thus, the inhibitor according to the invention provides a novel and targeted treatment option for inflammatory diseases or conditions of the gastrointestinal tract, e.g. inflammatory bowel disease. This opens up a novel therapeutic possibility not only to reduce or suppress the symptoms of inflammatory bowel disease, but also to inhibit the underlying molecular processes and causes of this disease.

The therapeutic possibilities of the inventive inhibitors have already been proven as efficient in animal models for acute and/or chronic inflammation of the gastrointestinal tract. Further, a therapeutic effect could be shown in vitro in human studies.

The inhibitors of the present invention are inhibitors of the RIP1/RIP3 complex. In the context of the present invention, the term "inhibitor of the RIP1/RIP3 complex" refers to a biochemical or chemical compound which preferably inhibits, suppresses or reduces the function, activity and/or abundance of the RIP1/RIP3 complex. For example, the meaning of this term comprises the inhibition of the activity of the RIP1/RIP3 complex and the inhibition of the expression of the RIP1 and/or RIP3 genes. In one embodiment, the phosphorylation-driven assembly of the RIP1/RIP3 complex in the intestinal epithelial, which regulates necroptosis, is inhibited. In another embodiment, the inhibition of the RIP1/RIP3 complex might be due to inhibition of RIP3 phosphorylation.

In a preferred embodiment, the inhibition of the complex is due to inhibition of RIP1 , in particular to inhibition of the kinase activity of RIP1. Other necroptosis inhibitors do not directly target RIP-1 but may target an additional regulatory molecule in the pathway of necroptosis. For example, Necrostatin-7 does not inhibit RIP1 kinase but may target an additional regulatory molecule in the pathway of necroptosis. Furthermore, the murin cytomegalovirus M45 protein is an example for a compound which functions as a viral inhibitor of RIP1 -mediated signalling in response to TLR3 stimulation.

In a preferred embodiment of the invention, the inhibition of the RIP1/RIP3 complex might be due to inhibition of the activity of the RIP1/RIP3 complex, for example but without limitation by reducing the expression, inducing enzymatic cleavage, altering posttranslational modifications (e.g. ubiquitination, phosphorylation) of the RIP1/RIP3 complex, or by interfering with the recruitment of other cellular proteins to RIP1 or RIP3. In a further preferred embodiment, the inhibition of the RIP1/RIP3 complex may be due to the inhibition of the expression of the RIP1 and/or RIP3 gene.

Examples of the inhibitors of the invention are binding proteins or binding peptides directed against RIP1 and/or RIP3, in particular against the active site of RIP1 and/or RIP3, nucleic acids directed against the RIP1 and/or RIP3 gene or RIP1 and/or RIP3 itself, and a chemical molecule, preferably a small molecule.

In one embodiment of the invention, the inhibitor is a binding protein or binding peptide directed against RIP1 and/or RIP3, in particular against the active site of RIP1 and/or RIP3. The term "binding protein" or "binding peptide" refers to a class of proteins or peptides which bind and inhibit RIP1 and/or RIP3 including, without limitation, polyclonal or monoclonal antibodies, antibody fragments and protein scaffolds directed against RIP1 and/or RIP3. The procedure for preparing an antibody or antibody fragment is effected in accordance with methods which are well known to the skilled person. According to the present invention the term antibody or antibody fragment is also understood as meaning antibodies or antigen-binding parts thereof, which have been prepared recombinantly and, where appropriate, modified, such as chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-stranded antibodies and F(ab) or F(ab)2 fragments, which are all well known in the art.

In another embodiment of the invention, the inhibitor is a chemical molecule. The term "chemical molecule" encompasses non-polymeric organic compounds, lipids, carbohydrates and peptides. Especially preferred are small chemical molecules, in particular non-polymeric organic compounds.

In a further embodiment of the invention, the inhibitor is a nucleic acid directed against the RIP1 and/or RIP3 gene or RIP1 and/or RIP3 itself. The term "nucleic acids against the RIP1 and/or RIP3 gene or RIP1 and/or RIP3 itself refers to double- stranded or single stranded DNA or RNA which, for example, inhibit the expression of the RIP1 and/or RIP3 gene or the activity of RIP1 and/or RIP3. Techniques for inhibition of gene expression are well known in the art, and may for example be without limitation microRNA, shRNA, RNAi, siRNA, antisense nucleic acid, aptamer and ribozyme techniques.

In a preferred embodiment, the necroptosis inhibitor is an RIP1 inhibitor. One example for an RIP-1 inhibitor is necrostatin-1 (nec-1 ), also known as 5-(1 H-indol-3- ylmethyl)-3-methyl-2-thioxo-4-imidazolidinone or 5-(lndol-3-ylmethyl)-3-methyl-2-thio- Hydantoin or methylthiohydanthoin-DL-thryptophan. Other Necroptosis inhibitors are, for example, Necrostatin-3 and Necrostatin-5 [Nec-5; 3-p-Methoxyphenyl-5.6-tet.ra- methylenothieno[2,3-d] pyrimidin-4-one-2-mercaptoethylcyanide].

The inhibitor of the present invention is beneficial for the inhibition of necroptosis, in particular necroptosis in epithelial cells of the gastrointestinal tract.

As used in the context of the invention the term "necroptosis" includes any "RIP1 or RIP3-dependent cell death". The term "RIP1 or RIP3-dependent cell death" comprises any cell death which is related to the function, activity and/or abundance of the RIP1/RIP3 complex, for example, but not limited to the assembly and activation of the RIP1-RIP3 complex and/or the activation of RIP1 kinase.

Epithelial cells of the gastrointestinal tract comprise different cell types, e.g. epithelial stem cells, progenitor cells located within the crypt region as well as differentiated epithelial cells, e.g. enterocytes, Paneth cells, goblet cells or enteroendocrine cells. In a preferred embodiment of the invention, the epithelial cells are Paneth cells. Dysfunction of Paneth cells and the related defects of antimicrobial peptides is one characteristic of inflammatory bowel disease. TNF blocking medication has been used for the treatment of Crohn^'s disease for a long time. However, the exact mode of action has not been known until now. It is assumed that Paneth cells are more susceptible to TNF-a induced cell death (apoptosis and necroptosis). Without wishing to be bound by theory, it is thought that the underlying mechanism of the therapeutic effect is the inhibition of TNF-a induced, RIP mediated necroptosis in Paneth cells of patients.

In another embodiment, the inhibitor of the present invention is beneficial for the treatment of inflammatory diseases or conditions related to necroptosis in the gastrointestinal tract.

As used herein, "treatment", "treat" or "therapy" refers to any and all uses which remedy a disease state or symptoms, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way. Thus, the term treatment shall include alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized state of disease, i.e. not worsening, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, partial or total regulation of a disease and remission. The term treatment further comprises the prevention of a disease, e.g. stopping, hindering, and/or slowing down the onset of symptoms.

The inflammatory disease of interest in the present invention includes acute and chronic inflammatory diseases or conditions of the gastrointestinal tract. Inflammatory diseases or conditions of the gastrointestinal tract include a number of diseases or conditions which are characterized by an inflammation of parts of the intestine or the entire intestins. Non-limiting examples are inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, coeliac condition, food allergies, infectious gastritis or enterocolitis, necrotizing enterocolitis, ischemic colitis, inflammation due to therapeutic regimens such as chemo- or radiotherapy, collagenous colitis, lymphocytic colitis, and bypass colitis.

Particularly, the disease is selected from Crohn's disease and ulcerative colitis. In a preferred embodiment, the disease is Crohn's disease.

Further, the present invention relates to a method for inhibiting necroptosis in epithelial cells of the gastrointestinal tract, comprising administering a therapeutically effective amount of an inhibitor of the receptor-interacting protein-I/receptor- interacting protein-3 complex or a pharmaceutically acceptable salt thereof, or a solvate thereof to a patient.

In a preferred embodiment of the method for inhibiting necroptosis in epithelial cells of the gastrointestinal tract, the inhibitor inhibits the activity of the RIP1/RIP3 complex. In another embodiment, the necroptosis inhibitor is an RIP1 inhibitor, e.g. nec-1.

Preferably, said inflammatory disease or condition is inflammatory bowel disease, in particular Crohn^'s disease or ulcerative colitis.

In another embodiment, the present invention relates to a method for treating an inflammatory disease or condition of the gastrointestinal tract, comprising administering a therapeutically effective amount of an inhibitor of the receptor- interacting protein-1 /receptor-interacting protein-3 complex or a pharmaceutically acceptable salt thereof, or a solvate thereof to a patient.

In a preferred embodiment of the method for preventing or treating an inflammatory disease or condition of the gastrointestinal tract, the inhibitor inhibits the activity of the RIP1/RIP3 complex. In another embodiment, the necroptosis inhibitor is an RIP1 inhibitor, e.g. nec-1.

Preferably, said inflammatory disease or condition is inflammatory bowel disease, in particular Crohn^'s disease or ulcerative colitis.

The term "therapeutically effective amount" of necrosis inhibitor as used herein means a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, a therapeutically effective amount or synonym thereto depends upon the context in which it is being applied. The amount of inhibitor that will correspond to a therapeutically effective amount will vary depending upon various factors, such as the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject, and the like, but can nevertheless be routinely determined by one skilled in the art.

Further, the invention relates to the use of an inhibitor of the receptor-interacting protein-1 /receptor-interacting protein-3 complex for the treatment of an inflammatory disease or condition of the gastrointestinal tract. In one embodiment, the inhibitor inhibits the activity of the RIP1/RIP3 complex. In another embodiment, the inhibitor inhibits the expression of the RIP1/RIP3 complex.

In another embodiment, the inhibitor is a RIP-1 inhibitor, e.g. necrostatin.

In a preferred embodiment, the inflammatory disease or condition is inflammatory bowel disease. In a preferred embodiment, the disease or condition is Crohn^'s disease or ulcerative colitis.

The necroptosis inhibitor of the invention is suitable for therapeutic administration in vivo. Preferably, the administration is intended for mammals, more preferably for humans.

The administration method of the necroptosis inhibitor of the present invention is not particularly limited, but suitably determined depending on dosage forms, the age, sex, and other conditions of a patient, severity of symptoms of the patient, and the like.

The administration may be orally, nasally, parenterally intraveneously, intra- peritoneally, subcutaneaouly, intramuscularly and transepithelially. For example, tablets, pills, powders, granules, capsules, solutions, suspensions and emulsions are orally administered. Injections may be administered intravenously, or may be administered intraarterially, intramuscularly, intracutaneously, subcutaneously, or intraperitoneally. Suppositories are administered intra recta I ly.

The dose of the active ingredient of the medication of the present invention can be suitably selected depending on the dosing regimen, the age, sex, and other conditions of a patient, severity of the disease, and the like and may be readily determined by one of ordinary skill by routine methods known in the art.

The necroptosis inhibitor of the present invention may be administered once daily or in several divided doses per day, e.g. 2 to 4 doses. For example, infusions are administerd continuously, or within specified time intervals. Preferably, the administration is a long-term administration, e.g. for blocking necroptosis for a time interval of several hours, days, weeks months or even years.

Further, the necroptosis inhibitor of the invention can be administered alone or in combination with other drugs or therapies, which are not particularly limited, but suitably determined by the attending physician depending on age, sex, and other conditions of a patient, severity of symptoms of the patient, and the like. In certain embodiments of the invention the inhibitor is administered in combination with one or more other therapeutic agents. In a preferred embodiment, the necroptosis inhibitor is administered in combination with an anti-TNF medication. Preferred examples for anti-TNF medication are infliximab, etanercept and/or adalimumab. Anti-TNF medication is a drug, which targets TNF-a. By binding to TNF- a, these drugs might for example block the action of tumor necrosis factor TNF-a. Preferred Examples for anti-TNF medication are infliximab, etanercept and adalimumab.

In another embodiment of the invention the inhibitor is administered in combination with an antibiotic. Preferred examples for antibiotics are metronidazole and ciprofloxacin. Antibiotics might help to reduce harmful intestinal bacteria and suppress the intestine's immune system, which can trigger symptoms of IBD patients. Furthermore necroptosis can be activated via different Toll-like-receptors (TLR), e.g. TLR4, that detects lipopolysaccharide from Gram-negative bacteria.

In another embodiment of the invention, the inhibitor is administered in combination with a corticosteroid.

In another embodiment of the invention, the inhibitor is administered in combination with an immunosuppressant, for example azathioprin.

Brief Description of the Figures

Figure 1 shows survival of Casp8 ^iEC mice and wild type mice after TNF-a induced epithelial necroptosis with or without pre-treatment with necrostatin-1.

Figure 2 shows hematoxylin and eosin (H&E) stained small intestine cross sections of wild type mice and Casp8^^EC mice after TNF-a induced epithelial necroptosis with and without pre-treatment with necrostatin-1.

Figure 3 shows inhibition of TNF-a induced epithelial necroptosis with or without pre- treatment with necrostatin-1 in organoids generated from small intestine of Casp8 ^iEC mice. ATP level was used as cell viability marker.

Figure4 shows representative microscopic pictures of Casp^⁰ organoids treated with TNF-a with or without necrostatin-1.

Figure 5 shows H&E stained biopsies from the small intestine of control patients stimulated in vitro with either DMSO (mock), TNF-a alone or in combination with necrostatin-1. Figure 6 shows quantitative expression levels of the Paneth cell marker lysozyme and the enterocyte cell marker sucrose isomaltase relative to the housekeeping gene HPRT in treated biopsies in control patients stimulated in vitro with either DMSO (mock), TNF-a alone or in combination with necrostatin-1.

Figure 7 shows electron microscopy images of the terminal ileum of a Crohn's disease patient.

Figure 8 shows representative immunofluorescence staining images for TUNEL and cleaved caspase-3 in crypts of the terminal ileum of a Crohn's disease patient.

Figure 9 shows inhibition of TNF-a induced Paneth cell necroptosis in small intestinal of Casp8^AIEC mice in a long term experiment.

Examples

The following examples are provided to further illustrate the invention.

Statistical analysis

Data were analyzed by Student's t-test using Microsoft Excel. ^* p<0.05, ^** p<0.01 , ^*** p<0.001.

Generation of Casp8 ^mc mice

To study the function of caspase-8 in the gut, mice with an intestinal epithelial cell specific deletion of caspase-8 (Casp^⁰) were generated. Accordingly, mice with floxed caspase-8 alleles were bred with mice expressing the Cre-recombinase under the control of the lEC-specific villin promoter (Villin-Cre or Villin-CreERT2 mice). Mice carrying a /oxP-flanked caspase-8 allele (Casp *) and Villin-Cre mice were described earlier (Beisner, D.R. et al., J Immunol 175 (6), 3469-3473, 2005; Madison, B.B. ei al., J Biol Chem 277 (36), 33275-33283, 2002; El Marjou, F. et al., Genesis 39 (3), 186-193, 2004. In all experiments, littermates carrying the /oxP-flanked alleles but not expressing Cre recombinase were used as controls. Cre mediated recombination was genotyped by polymerase chain reaction on tail DNA. Specific deletion of caspase-8 in IEC was confirmed by PCR and Western blotting (Data now shown). Caspe^^⁰ mice were born at the expected Mendelian ratios and developed normally, although weighing on average slightly less than control littermates at 8 weeks of age (data not shown). Despite the paradigm that apoptosis is important for regulating epithelial cell numbers⁴, histological and morphometrical analysis of the jejunum and colon of Casp8 ^lEC mice showed no overt changes of tissue architecture or apoptosis dysregulation (Data not shown). While this suggested that caspase-8 is not essential for the structural development of the gut, high resolution endoscopy showed erosions in the terminal ileum but not in the colon of Casp8^ ^BC mice. Histological analysis demonstrated marked destruction of the architecture and signs of inflammation including bowel wall thickening, crypt loss and increased cellularity in the lamina propria in more than 80% of all ileal specimens. This finding of spontaneous ileitis in the absence of caspase-8 in lEC was further supported by increased expression of the inflammation markers S100A9 and TNF-a and by elevated infiltration of the lamina propria with CD4⁺ T cells and granulocytes.

To investigate whether caspase-8 deficiency sensitizes mice to experimental intestinal inflammation in the large bowel, Casp8 ^lEC and control mice were subjected to dextran sodium sulfate (DSS), an established model of experimental colitis. Experimental colitis was induced by treating mice with 1-1.5% dextrane sodium sulfate (DSS, MP Biomedicals) in the drinking water for 5-10 day. DSS was exchanged every other day. Mice were examined by measuring body temperature, weight loss and monitoring development of diarrhea. Colitis development was monitored by analysis of rectal bleeding and high resolution mouse video endoscopy. Mice were anesthetized with 2-2.5% isoflurane in oxygen during endoscopy. Mice were routinely screened for pathogens according to FELASA guidelines. Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Erlangen.

Caspf ^⁰ mice but not control mice showed high lethality and lost significantly more weight than control mice. All Casp^⁰ but none of the control mice developed rectal bleeding, endoscopic and histological signs of very severe colitis with epithelial erosions, a finding which was confirmed by quantitative PCR for the I EC marker villin. Together, the data indicate that a lack of caspase-8 in lEC renders mice highly susceptible to spontaneous ileitis and experimentally induced colitis.

Effect of TNF-a signaling in Casp8^^EC mice

TNF-a stimulated death receptor signalling has been described to promote necroptosis in a number of different target cell types, especially when apoptosis was blocked using caspase-inhibitors. Therefore, the effect of TNF-a in absence of caspase-8 was examined. Mice were intravenously (i.v.) administered with TNF-a using a dose which is not lethal to normal mice. While all control mice were still alive after 5 hours, Casp8 ^iE0 mice showed significantly more pronounced hypothermia and very high lethality. Histological analysis demonstrated villous atrophy and severe destruction of the small bowel of Casp8 *^EC mice as compared to control littermates and an increased number of dying epithelial cells, as indicated by the pyknotic nuclei seen in the H&E stain of crypts and cells in the crypt lumen (Data not shown). Similar to unchallenged mice, dying crypt cells were negative for active caspase-3 but positive for TUNEL staining, showing that in the absence of caspase-8, TNF drives excessive necrosis of epithelial cells.

Inhibition of TNF- a induced epithelial necroptosis in small intestinal organoid cultures from Casp8 ^lEC mice

Organ culture of freshly isolated human small intestinal biopsies was performed in RPMI medium (Gibco). For organoid culture, crypts were isolated from the small intestine of mice and cultured for a minimum of 7 days as previously described (Sato, T. et a/., Nature 459 (7244), 262-265, 2009). In brief, crypts were isolated by incubating pieces of small intestine in isolation buffer (phosphate buffered saline without calcium and magnesium (PBSO), 2 mM EDTA). Crypts were then transferred into matrigel (BD Bioscience) in 48 well plates and 350 μΙ culture medium (Advanced DMED/F12 (Invitrogen), containing HEPES (10 mM, PAA), GlutaMax (2 mM, Invitrogen), Penicillin (100 U/ml, Gibco), Streptomycin (100 pg/ml, Gibco), murine EGF (50 ng/ml, Immunotools), recombinant human R-spondin (1 pg/ml, R&D Systems), N2 Supplement 1x (Invitrogen), B27 Supplement 1x (Invitrogen), 1 mM N- acetylcystein (Sigma-Aldrich) and recombinant murine Noggin (100 ng/ml, Peprotech)). Organoid growth was monitored by light microscopy. In some experiments, human biopsies or organoids were treated with recombinant mouse TNF-a (25 ng/ml, Immunotools), recombinant human TNF-a (50 ng/ml, Immunotools), necrostatin-1 (30 μΜ, Enzo) or caspase-8 inhibitor (50 μΜ, Santa Cruz). Cell Viability of organoids was analyzed indirectly by quantification of relative ATP level with the CellTiter-Glo assay from Promega according to the manufacturer^'s instructions. Luminescence was measured on the microplate reader infinite M200 (Tecan).

In vitro, small intestinal organoid cultures from Casp8^^iE0 mice but not from control mice exhibited necrosis within 24 hours after addition of TNF-a to the tissue culture. However, when cell cultures were pre-treated with nec-1 , organoid necrosis was blocked (Figure 3, 4). Figure 3 shows cell viability data of Casp8 ^iE0 organoids treated for 24 h with TNF-a +/- necrostatin-1 as well as mock treated organoids. Figure 4 shows Representative microscopic pictures of Casp ^⁰ organoids treated for 24 h with TNF-a +/- necrostatin-1 as well as mock treated. Three replicate experiments were conducted. ATP was used as indicator of cell viability and assessed with the Cell Viability Assay (Promega).

Inhibition of TNF-a induced epithelial necroptosis in small intestinal of Casp8 ^iEC mice

Casp8 ^iEC mice were injected i.v. with rm-TNF-a (200 ng/g body weight, Immunotools) +/- necrostatin-1 (1.65 pg/g body weight, Enzo). Necrostatin-1 was dissolved in DMSO or ethanol. Control mice were injected with the same amount of DMSO or ethanol. Survival and hematoxylin and eosin (H&E) stained small intestine cross sections of control (n=5), Casp8 ^iE0 (mock pretreated, n=7) and Casp8^^iEC Casp^^E0 (nec-1 pretreated, n=8) mice after intravenous injection of TNF-a were examined. Pre-treatment with nec-1 significantly reduced TNF-a induced lethality and small intestinal tissue destruction in Casp8^^iEC mice. Figure 1 shows survival of Casp8 *^EC mice and wild type mice after TNF-a induced epithelial necroptosis with or without pre-treatment with necrostatin-1. The axis of abscissas depicts hours after TNF-a i.v. administration. The asterisks designate statistical relevancy of the data. Figure 2 shows representative H&E stained small intestine cross sections of wild type mice and Casp^^iEC mice after TNF-a induced epithelial necroptosis with and without pre-treatment with necrostatin-1. Asterisks show significance level relative to Casp8^^lEC without nec-1. All experiments were performed at least 3 times with similar results.

Collectively, the experimental data suggest that deficient caspase-8 expression renders intestinal epithelial cells, especially Paneth cells susceptible to TNF-a induced necroptosis highlighting a regulatory role of caspase-8 in antimicrobial defence and in maintaining immune homeostasis in the gut.

Inhibition of TNF-a induced Paneth cell necroptosis in small intestinal of Casp8^AIEC mice in a long term experiment

The inhibition of TNF-a induced Paneth cell necroptosis in small intestinal of Casp8^AIEC mice has been examined in a chronic inflammation model in a long term experiment. Caspase8 deficient mice were treated for 7 days, twice per day, with a TNF-α medication (Enbrel) (group a); or for 10 days, twice per day, with combined therapy with a TNF-a medication (Enbrel) and nec-1 (group b).

Figure 9 shows the inhibition of TNF-a induced Paneth cell necroptosis in small intestinal of Casp8^AIEC mice in this long term experiment. From Figure 9 a) it can be seen that in mice of group a RT-PCR for Paneth cell marker Ang4 showed no rescue of Paneth cell death. From Figure 9 b) it can be seen that that in mice of group b) RT- PCR showed increased level of Paneth cell marker Ang4 after 10 days of Enbrel/Nec-1 therapy. Figure 9 c) shows representative immunofluorescence staining for the Paneth cell marker lysozyme in crypts of the terminal ileum of Caspase-8 deficient mice under placebo or Enbrel/nec-1 therapy (group b).

Analysis of histological human samples

To further investigate RIP mediated necroptosis of Paneth cells in patients with Crohn's disease, samples derived from human patients undergoing endoscopic examination were examined. Paraffin-embedded specimens from the terminal ileum of control patients and patients with active Crohn's disease were obtained from the Institute of Pathology of the University Clinic Erlangen. The specimens had been taken from routine diagnostic samples and patient data had been made anonymous. EM and tissue culture experiments were performed with endoscopic biopsy specimens collected in the endoscopy ward of the Department of Medicine I. The collection of samples was approved by the local ethical committee and the institutional review board of the University of Erlangen-Nuremberg and each patient gave written informed consent.

Histopathological analysis was performed on formalin-fixed paraffin embedded tissue after H&E staining. Immunofluorescence of cryosections was performed using the TSA Cy3 system as recommended by the manufacturer (PerkinElmer). Fluorescence microscopy (Olympus, Hamburg, Germany) and confocal microscopy (Leica TCS SP5, Wetzlar, Germany) was used for analysis. The following primary antibodies were used: The nuclei were stained with Hoechst 3342 (Invitrogen). Cell death was analyzed using CaspACE FITC-VAD-FMK (Promega) for early apoptosis and the in situ cell death detection kit (Roche) for TUNEL. For electron microscopy, glutaraldehyde fixed material was used. After embedding in Epon Araldite, ultrathin sections were cut and analyzed using a Zeiss EM 906 (Zeiss, Oberkochen, Germany). Figure 5 shows H&E staining of biopsies from the small intestine of control patients stimulated in vitro for 8 h with either DMSO (mock), TNF-a (50ng/ml) alone or in combination with necrostatin-1 (30μΜ).

Gene expression analyses were performed as follows. Total RNA was extracted from gut tissue or isolated lECs using an RNA isolation kit (Nucleo Spin RNA II, Macherey Nagel). cDNA was synthesized by reverse transcription (iScript cDNA Synthesis Kit, Bio Rad) and analysed by real-time PCR with SsoFast EvaGreen (Bio-Rad) reagent and QuantiTect Primer assays (Qiagen). Experiments were normalized to the level of the housekeeping gene HPRT.

Figure 6 shows a graph depicting the quantitative expression level of the Paneth cell marker lysozyme and the enterocyte marker sucrose isomaltase relative to HPRT. Data from one representative experiment out of 2 is shown.

It has been found that immunohistochemistry of samples derived from the terminal ileum of human patients undergoing endoscopic examination revealed high expression of RIP3 exclusively in Paneth cells, but not in other intestinal epithelial cell types. Importantly, analysis of histological samples from the terminal ileum of control patients and patients with active Crohn's disease showed a significant decrease in the number of Paneth cells and high numbers of dying cells with shrunken eosinophilic cytoplasm at the crypt base, similarly to Casp8 ^iEC mice. Electron microscopy of the Paneth cell area in the terminal ileum of patients with CD, showed increased necrotic cell death, as indicated by abundant organelle swelling, vacuole formation and the lack of blebbing. Figure 7 shows electron microscopy images of the terminal ileum of a Crohn's disease patient showing dying crypt cells with necrotic features. Asterisks highlight Paneth cell granules; arrows indicate mitochondrial swelling; n designates the nucleus. Moreover, crypt epithelial cells in areas of acute inflammation usually were TUNEL positive but lacked staining for active caspase-3. Figure 8 shows representative immunofluorescence staining images for TUNEL and cleaved caspase-3 in crypts of the terminal ileum of a Crohn's disease patient. Scale bars represent 50 mm; arrows indicate Paneth cells. Finally, ileal biopsies from control patients showed Paneth cell loss in the presence of high levels of exogenous TNF-a, an effect that was reversible by co-incubation with necrostatin-1 (Figure 5 and 6). Thus, the experimental data show that necroptosis of Paneth cells is a feature of Crohn's disease. The present data suggest that human Paneth cells are be susceptible to TNF-a induced necroptosis.

The present data for the first time demonstrate necroptosis in the terminal ileum of patients with Crohn's disease and show that regulating necroptosis in the intestinal epithelium is critical for the maintenance of intestinal immune homeostasis. Thus, with the targeting of necroptotic cellular mechanisms the present invention provides a promising novel therapeutic option in treating patients with inflammatory diseases of the gastrointestinal tract.

Claims

Claims

1. Inhibitor of the receptor-interacting protein-1 /receptor-interacting protein-3 complex for use in the treatment of an inflammatory disease of the gastrointestinal tract.

2. Inhibitor of the receptor-interacting protein-1 /receptor-interacting protein-3 (RIP1/RIP3) complex for use in the inhibition of cell death in epithelial cells of the gastrointestinal tract, in particular for the inhibition of necroptosis.

3. Inhibitor according to claim 1 or 2, wherein the inhibitor inhibits the activity of the RIP1/RIP3 complex.

4. Inhibitor according to claim 1 or 2, wherein the inhibitor inhibits the expression of the RIP1/RIP3 complex

5. Inhibitor according to any of the previous claims, wherein the inhibitor is a receptor-interacting protein-1 inhibitor, in particular necrostatin-1.

6. Inhibitor according to any of the previous claims for use in treatment of inflammatory bowel disease, in particular Crohn^'s disease and ulcerative colitis.

7. Inhibitor according to any of the previous claims, wherein said inhibitor is administered in combination with one or more other therapeutic agents, in particular selected from the group consisting of anti-TNF medication, antibiotics, corticosteroids and immunosuppressants.

8. Inhibitor according to claim 7, wherein said inhibitor is administered in combination with infliximab, etanercept, adalimumab and/or azathioprin.

9. Use of an inhibitor of the receptor-interacting protein-1/receptor-interacting protein-3 complex for the treatment of an inflammatory disease of the gastrointestinal tract.

10. Use according to claim 9, wherein the inhibitor inhibits the activity of the RIP1/RIP3 complex.

11. Use according to claim 9, wherein the inhibitor inhibits the expression of the RIP1/RIP3 complex.

12. Use according to any of claims 9 to 1 1 , wherein the inhibitor is a receptor- interacting protein-1 inhibitor, in particular necrostatin-1.

13. Use according to any of claims 9 to 12, wherein the inflammatory disease or condition is inflammatory bowel disease, in particular Crohn^'s disease or ulcerative colitis.

14. Use according to any of claims 9 to 13, wherein said inhibitor is administered in combination with one or more other therapeutic agents, in particular selected from the group consisting of anti-TNF medication, antibiotics, corticosteroids and immunosuppressants.

15. Use according to claim 14, wherein said inhibitor is administered in combination with infliximab, etanercept, adalimumab and/or azathioprin.