WO2022063979A1 - Spermidine in treatment of dysbiosis and associated inflammatory conditions of the gastrointestinal tract - Google Patents

Abstract

The invention relates to the use of spermidine or its pharmaceutically acceptable salt in prevention or treatment of an inflammatory disease of the intestine, particularly with inflammatory bowel disease, ulcerative colitis, Crohn's disease, or non-alcoholic steatohepatitis, wherein the inflammatory disease of the intestine is associated with intestinal dysbiosis; or of intestinal dysbiosis.

Description

SPERMIDINE IN TREATMENT OF DYSBIOSIS AND ASSOCIATED INFLAMMATORY

CONDITIONS OF THE GASTROINTESTINAL TRACT

The present invention relates to the medical use of spermidine in treatment of inflammatory conditions of the intestinal tract, particularly such conditions as are associated with dysbiosis, i.e. a deviation of the gut microbiome’s distribution of bacterial phylae, classes or genera.

Background of the Invention

Spermidine (CAS 124-20-9) is a naturally occurring polyamine (C7N3H19). Its hydrochloride has the CAS No. 334-50-9.

PTPN2 tyrosine-protein phosphatase non-receptor type 2 (UniProtKB - P17706 (PTN2_HUMAN)) is a ubiquitously expressed enzyme, with functions in T-cell receptor signalling through dephosphorylation of FYN and LCK to control T-cells differentiation and activation. PTPN2 dephosphorylates CSF1 R, negatively regulating its downstream signalling and macrophage differentiation, and negatively regulates interleukin-2 and interferon- mediated signalling through dephosphorylation of the cytoplasmic kinases JAK1 , JAK3 and their substrate STAT 1 , that propagate signalling downstream of the cytokine receptors.

The presence of PTPN2 variant rs1893217 is associated with increased risk for IBD, type-1 diabetes, rheumatoid arthritis and multiple sclerosis. The presence of the variant can be determined as described in Scharl et al., Inflammatory Bowel Diseases, Volume 18, Issue 5, 1 May 2012, Pages 900-912.

Activation of PTPN2 by spermidine exerts anti-inflammatory effects in vitro as well as in an in vivo model of colitis. The inventors have demonstrated that treatment of PTPN2 wild-type human mononuclear cells with spermidine not only induced PTPN2 mRNA and protein levels, but also enhances PTPN2 enzymatic activity (Niechcial et al., Inflamm. Bowel Dis. 2020 Jun 18;26(7):1038-1049). This results in inhibition of IFNy- and TNF-induced activation of pro- inflammatory signal transduction events and cytokine secretion. Existing pre-clinical data were up to now not deemed sufficient to initiate clinical trials using spermidine for the treatment of IBD.

Niechcial et al. (ibid.) exposed commonly used intestinal epithelial cell lines in vitro to micromolar (1 pM to 10OpM) concentrations of spermidine.

Kiechl et al. (American Journal of Clinical Nutrition, 108(2), 2018, 371-380) have found that higher spermidine intake is linked to lower mortality in a prospective population-based study. Of note, the study participants’ daily intake of spermidine through their diet ranged from 50 to 90 pMol/day (or 7,3 mg to 13 mg). Suppliers of dietary supplements recommend a daily dose of 1 ,2 mg.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to restore a colon microbiome associated with, or conducive to the absence of inflammation. Another problem solved by the present invention is the absence of data on a safe and effective dosing regimen to enable the use of spermidine as a treatment of inflammatory conditions of the intestinal tract, particularly such conditions as are associated with dysbiosis.

This objective is attained by the subject-matter of the independent claims of the present specification.

Summary of the Invention

In one aspect, the invention relates to the use of spermidine, or a pharmaceutically acceptable salt thereof, in prevention or treatment of an inflammatory disease of the intestine, particularly with Inflammatory Bowel Disease (IBD), ulcerative colitis (UC), Crohn’s disease (CD), or Nonalcoholic steatohepatitis (NASH), wherein the IBD, UC, CD or NASH is associated with intestinal dysbiosis.

In an alternative of this aspect, the invention relates to the use of spermidine, or a pharmaceutically acceptable salt thereof, in prevention of relapse of relapsing-remitting forms of IBD, UC, CD, or NASH associated with dysbiosis.

In an alternative of this aspect, the invention relates to the use of spermidine, or a pharmaceutically acceptable salt thereof, in prevention or treatment of intestinal dysbiosis.

Intestinal dysbiosis may be characterized by the prevalence in a patient’s intestinal microbiome of certain bacterial phylae, classes, genera, or even finer grained categories of biological distinction. The characterization may be made on the basis of the presence of phylae, classes, genera etc. of bacteria not present in patients that are deemed non-dysbiotic, or it may be assigned on the absence, or scarcity of bacteria usually found in healthy patients’ microbiomes.

On the level of phylae, the diagnosis of dysbiosis may be assigned to a patient, and the patient be subsequentially assigned to a treatment according to the invention, if bacterial ribosomal RNA sequences representing the phylum Firmicutes represent less than (<) 30% of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the phylum Bacteroidetes represent less than (<) 15% of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome.

Similarly, the diagnosis of dysbiosis may be assigned to a patient, and the patient be subsequentially assigned to a treatment according to the invention, if bacterial ribosomal RNA sequences representing the phylum Proteobacteria represent more than (>) 15%, particularly > 20% of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome.

One aspect of the invention relates to the dosing of spermidine or its pharmaceutically acceptable salt at a dose of >50 mg, particularly at a dose of >100mg.

In another aspect, the invention relates to a pharmaceutical composition formulated for oral administration, said pharmaceutical composition comprising >50 mg per dosing unit, particularly >100mg spermidine per dosing unit, even more particularly >200mg per dosing unit.

Further aspects of the invention relate to a method of treating an inflammatory disease of the intestine, particularly Inflammatory Bowel Disease (IBD), Ulcerative Colitis (UC), Crohn’s disease (CD), or Non-alcoholic steatohepatitis (NASH), more particularly when the inflammatory disease of the intestine is associated with dysbiosis as defined herein, with an effective dose of spermidine or its salt. Alternatively, the invention may be defined as a method to manufacture a medicament for the treatment of intestinal disease or dysbiosis as specified herein.

The data presented in the present specification demonstrate that spermidine can completely normalize colitis-induced dysbiosis. In the transfer colitis mouse model employed in the examples, an intestinal dysbiosis comparable to the dysbiosis of an IBD patient can be detected in mice. The administration of spermidine in this mouse model leads to almost complete regression of the intestinal dysbiosis, and to restoration of an almost normal intestinal flora.

E. coli have been shown to be responsible for the induction of pro-inflammatory M1 macrophages in humans (IBD patients). Thus, the change induced in the intestinal flora, particularly a significant reduction of E. coli observed as a consequence of the treatment according to the invention, has high medical relevance.

In addition, the administration of spermidine increases the amount of intestinal bacteria, such as butyrate-producing Clostridiales bacteria, associated with reduction of inflammatory outcomes.

The spermidine treatment according to the invention reverses intestinal dysbiosis induced by an inflammation of the intestine, e.g. IBD, and normalizes the intestinal bacterial composition. A further aspect of the present invention relates to a pharmaceutical composition comprising spermidine or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

Detailed Description of the Invention

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term dysbiosis in the context of the present specification relates to a significant, and disease-associated deviation of the intestinal bacterial flora, when compared to an average, or sample representative of healthy patients of similar nutritional status and eating habits.

Dysbiosis, for the purpose of defining the invention, is determined by obtaining a plurality of microbial nucleic acid sequences allowing the determination of frequencies of phylae, classes or genera of bacteria representative of the patient’s gut microbiome, particularly from a stool sample obtained from the patient.

The identity of the bacteria constituting the patient’s microbiome can be ascertained using 16S RNA sequencing or by metagenomic shotgun sequencing.

Spermidine for use in treatment or prevention of intestinal disease

A first aspect of the invention relates to the use of spermidine, or a pharmaceutically acceptable salt thereof, for use in prevention, or treatment of an inflammatory disease of the intestine, where the inflammatory disease of the intestine is associated with intestinal dysbiosis (intestinal dysbiosis as defined below in the section Spermidine for use in treatment or prevention of dysbiosis).

In certain particular embodiments, the inflammatory disease is Inflammatory Bowel Disease (IBD). In other particular embodiments, the inflammatory disease is Ulcerative Colitis (UC). In still others, the inflammatory disease is Crohn’s disease (CD). In further particular embodiments, the inflammatory disease is non-alcoholic steatohepatitis (NASH).

In an alternative of this aspect, the invention relates to the use of spermidine, or a pharmaceutically acceptable salt thereof, in prevention of relapse, also referred to as a ‘flare- up’, of relapsing remitting forms of IBD, UC, CD, or NASH associated with dysbiosis. According to certain embodiments of this aspect of the invention, the patient may begin a course of administration of spermidine during a flare up, in order to reduce the severity of dysbiosis and inflammation of the current outbreak. In other embodiments, the patient is administered spermidine after the resolution of an acute episode of inflammation in order to delay, or reduce the severity of future flare-ups. In particular embodiments, the patient with intestinal inflammation is administered spermidine continuously, through periods of health and relapse, in order to sustain microbial biodiversity, and prevent, or reduce the severity of further flare- ups of the disease.

In certain particular embodiments, the treatment according to the invention is administered to a patient having been determined to carry the PTPN2 variant rs1893217. for use in treatment or

In an alternative aspect of the invention, spermidine, or a pharmaceutically acceptable salt thereof, is used in prevention or treatment of intestinal dysbiosis, without prior diagnosis of IBD, CD, UC, or NASH, and not necessarily concurrent with these diseases.

While IBD or NASH may often be co-diagnosed with dysbiosis, it is at present unclear whether one is the cause of the other. Current knowledge suggests that IBD, CD, UC, and NASH, present together with intestinal dysbiosis. However, IBD, CD, UC, and NASH, themselves create specific conditions that will promote intestinal dysbiosis (e.g. by affecting immune- metabolism, intestinal pH levels, antimicrobial immunity, etc.). On the other hand, presence of intestinal dysbiosis affects key factors that promote the onset of the diseases (e.g. aberrant activation of the immune system, production of pro-inflammatory metabolites/reduced production of anti-inflammatory metabolites, alterations in nutrient digestion or vitamin production, bile acid metabolism, etc.). Therefore, IBD, CD, UC, and NASH, as well as intestinal dysbiosis, constitute separate conditions that influence each other. However, presence of intestinal dysbiosis does not always result in the onset of IBD, CD, UC, and NASH, but is also associated with the onset of other e.g. chronic inflammatory, malignant or neuropsychiatric diseases. The treatment of intestinal dysbiosis per se therefore represents a novel therapeutic approach which is distinct and different from treating IBD, CD, UC, or NASH, by targeting inflammation.

In certain embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the prevalence in the patient’s intestinal microbiome of both Firmicutes and Bacteroidetes together is less than (<) 85% of the bacteria in the intestinal microbiome. In particularly pronounced cases of dysbiosis, prevalence in the patient’s intestinal microbiome of both Firmicutes and Bacteroidetes together is <75%, in even more particular cases <50%, and in even more pronounced cases < 45%.

In certain embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the prevalence in the patient’s intestinal microbiome of bacterial ribosomal RNA sequences representing the phylum Firmicutes is < 30% of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the phylum Bacteroidetes is < 15% of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome.

In certain embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the prevalence in the patient’s intestinal microbiome of bacterial ribosomal RNA sequences representing the phylum Proteobacteria is more than (>) 15%, particularly >20% of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome.

In certain particular embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the dysbiosis diagnostic criteria for prevalence of Firmicutes, Bacteroidetes and Proteobacteria are all fulfilled at the same time.

In certain embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the prevalence in the patient’s intestinal microbiome of bacterial ribosomal RNA sequences representing the class Clostridia is less than (<) 25% [particularly <20%, more particularly <15%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the class Bacteroidia is less than (<) 20% [particularly <15%, more particularly <10%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the class Verrucomicrobiae is less than (<) 4% [particularly <2%, more particularly <1 %] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the class Gammaproteobacteria is more than (>) 5% [particularly >15%, more particularly >50%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; and/or bacterial ribosomal RNA sequences representing the class Bacilli is more than (>) 3% [particularly >5%, more particularly >7,5%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome;

In certain particular embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the criteria for prevalence of at least three, particularly four, even more particularly all of the classes mentioned above are all fulfilled at the same time.

In certain embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the prevalence in the patient’s intestinal microbiome of bacterial ribosomal RNA sequences representing the genera Shigella ssp. and Escherichia coll is more than (>) 30% [particularly >35%, more particularly >40%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the genus Enterococcus is more than (>) 3% [particularly >4%, more particularly >6%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; In certain particular embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the prevalence in the patient’s intestinal microbiome of bacterial ribosomal RNA sequences representing the genus Lachnospiraceae represent less than (<) 15% [particularly <12%, more particularly <10%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the genus Odoribacter represent less than (<) 8% [particularly <5%, more particularly <2%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the genera of the Rikenellaceae RC9 gut group represent less than (<) 8% [particularly <5%, more particularly <2%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; bacterial ribosomal RNA sequences representing the genus Faecalibaculum represent less than (<) 2% [particularly <1 ,5%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome; and/or bacterial ribosomal RNA sequences representing the genus Ruminiclostridium 5 represent less than (<) 1 % [particularly <0,3%] of all bacterial ribosomal RNA sequences in the patient’s intestinal microbiome.

In certain particular embodiments, dysbiosis is diagnosed and the patient is administered a treatment according to the invention if the criteria for prevalence of at least three, particularly four, even more particularly all of the genera mentioned above are all fulfilled at the same time.

Pharmaceutical Compositions, Dosing, and Administration

Another aspect of the invention relates to a pharmaceutical composition comprising spermidine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handled product.

The pharmaceutical composition can be formulated for oral administration. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).

The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily. In particular embodiments, the spermidine, it’s pharmaceutical salt, or a pharmaceutical composition comprising same, is formulated to provide an optimal dose as described in the section Dosing, in a single daily administration.

The therapeutically effective dosage of spermidine, or the pharmaceutical composition, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

The spermidine, or the pharmaceutical compositions according to the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

The means by which the spermidine or its pharmaceutically acceptable salt is prepared is not particularly limited according to the invention. In some embodiments, the spermidine is prepared using an industrial process, for example, a synthesised in recombinant yeast or bacteria. In other embodiments, the spermidine is purified, or extracted from a natural source rich in spermidine, including, but not limited to plant products such as wheat germ, or soy bean.

In certain embodiments, spermidine or its pharmaceutically acceptable salt is administered by enteral administration, particularly by oral administration.

In certain particular embodiments, spermidine or its pharmaceutically acceptable salt is formulated for enteral administration, particularly as tablet or capsule.

As the inventors have demonstrated that the site of impact of the treatment is the intestinal microbiome, administration as a tablet or capsule for delivery of the spermidine to the intestine (gastro resistant tablet or capsule) is provided as a particularly advantageous form of administration. In particular embodiments, the spermidine is administered in a slow, delayed, or extended- release format, formulated for release specifically within the intestinal microenvironment.

Dosina

In certain particular embodiments, spermidine, the salt thereof, or the pharmaceutical composition comprising spermidine, is administered at a dose least (>) 30 mg, particularly at a dose of >50mg, more particularly >100mg, still more particularly >200mg. In particular embodiments, the spermidine, or salt thereof, is administered at a dose of 250 to 300mg. In still more particular embodiments, the doses specified above are provided as a single daily dose.

One daily dose administered to the mice is estimated to be around 1 ,3 mg spermidine, leading to a dose of 60mg/kg. Applying a conversion factor of 0,08 (Nair and Jacob J Basic Clin Pharm. March 2016-May 2016; 7(2): 27-31 ), this leads to a human dose of 4,8 mg/kg or ca. 290 mg spermidine per day per patient.

Assuming that one tenth of that dose is still expected to elicit a significant effect, and is approx, three times the dietary input of spermidine has led the inventors to suggest 30 mg as the lower daily dose range to test.

Another aspect of the invention relates to a pharmaceutical composition formulated for oral administration, said pharmaceutical composition comprising >30 mg per dosing unit.

In a particular embodiment, the pharmaceutical composition formulated for oral administration comprises >100mg spermidine per dosing unit, or even >200mg per dosing unit.

In one embodiment, the pharmaceutical composition formulated for oral administration comprises 30 to 50 mg spermidine per dosing unit. In one embodiment, the pharmaceutical composition formulated for oral administration comprises 50 to 70 mg spermidine per dosing unit. In one embodiment, the pharmaceutical composition formulated for oral administration comprises 50 to 100 mg spermidine per dosing unit.

In one embodiment, the pharmaceutical composition formulated for oral administration comprises 90 to 150 mg spermidine per dosing unit. In one embodiment, the pharmaceutical composition formulated for oral administration comprises 100 to 200 mg spermidine per dosing unit. In one embodiment, the pharmaceutical composition formulated for oral administration comprises 150 to 250 mg spermidine per dosing unit.

In one embodiment, the pharmaceutical composition formulated for oral administration comprises 250 to 300 mg spermidine per dosing unit.

In one embodiment, the pharmaceutical composition formulated for oral administration is a tablet or capsule. In one embodiment, the pharmaceutical composition formulated for oral administration is a tablet or capsule for delivery of the spermidine to the intestine (gastro resistant tablet or capsule).

In one set of embodiments, the spermidine, or the pharmaceutical composition is formulated for oral delivery into the small or large intestines of the subject, where the majority of the gut microbiota reside. One such embodiment relates to enteric coatings that protect the spermidine from high pH in the stomach, and dissolve on reaching the intestines. Examples of such coatings include, without being limited to polymers and copolymers such as eudragit (Evonik).

In certain embodiments, the spermidine, or the pharmaceutical composition may be delivered into a specific region of the intestines in the form of buffered sachets, or with a coating that dissolves in a pH range specific to a certain portion of this intestine. For example, a formulation which decomposes in the pH range from 6.8 to 7.5, will favour delivery to the colon (for a full description of targeted delivery to regions of the gastrointestinal tract see Villena et al. 2015. Int J. Pharm. 487 (1-2):314-9.)

In yet another similar embodiment, the spermidine, or the pharmaceutical composition can be administered specifically to the intestines by means of a time-delay delivery method, which considers the time it takes to transit through the stomach, small intestine and colon. Delayed release formulations include hydrogel preparations, and biodegradable, water-soluble, hydrolysable or enzyme degradable polymers. Examples of coating materials that are suitable for delayed-release formulations include, but are not limited to, cellulose-based polymers, acrylic acid polymers, and vinylpolymers.

In another embodiment where the spermidine, or the pharmaceutical composition is formulated for delivery to the colon, the formulation includes a coating which can be removed by an enzyme present in the human gut, for example a carbohydrate reductase. Examples of enzyme-sensitive coatings include amylose, xanthan gum and azopolymers.

In embodiments of the invention that relate to rectal administration the spermidine, or the pharmaceutical composition may be formulated for delivery as a suppository, enema or through topical application as part of an endo- or colonoscopy procedure. In certain embodiments, the spermidine, or the pharmaceutical composition can be targeted to a particular site through intubation of an orifice, or with a surgical intervention.

The pharmaceutical composition according to the invention may be used in prevention or treatment of an inflammatory disease of the intestine or NASH. In particular embodiments, the inflammatory disease of the intestine is IBD, UC, or CD. In more particular embodiments, the IBD, UC, CD or NASH is associated with intestinal dysbiosis. The pharmaceutical composition according to the invention may be used in treatment of intestinal dysbiosis, irrespective of any association with IBD, UC, CD, or NASH. The dysbiosis may be characterized as specified in any one of the preceding embodiments.

In planning and conducting the experiments leading up to the results presented herein, inventors aimed to determine the optimal dosage and interval of spermidine treatment by oral administration.

One major achievement of the invention is the proof that long-term administration of spermidine is safe and exerts no relevant side effects in vivo. Further, the determination of well tolerated treatment intervals and dosages for spermidine treatment in the setting of colitis in vivo has been obtained, paving the way to subsequent clinical trials.

The data presented herein confirm that spermidine treatment is a successful anti-inflammatory treatment in an immune-mediated and an epithelial damage-mediated mouse model of chronic colitis. This is of particular importance, since the onset of intestinal inflammation in DSS colitis (barrier damage) and T-cell transfer colitis (immune-mediated colitis) are due to very different pathological mechanisms. Spermidine treatment is effective in three different well-established colitis models.

Another aspect of the invention is the demonstration that the IBD-associated PTPN2 variant does not determine therapy response in IBD patients, which opens a larger patient group to this novel treatment.

Medical treatment. Dosage Forms and Salts

Further provided is a method of treating an inflammatory disease of the intestine, particularly with Inflammatory Bowel Disease (IBD), Ulcerative Colitis or Crohn’s Disease, or Non-alcoholic steatohepatitis (NASH), said method comprising providing to the patient an effective dose of spermidine, or a pharmaceutically acceptable salt thereof, as laid out above.

The scope of the present invention encompasses a method or treating intestinal dysbiosis in a patient in need thereof, comprising administering to the patient spermidine or a salt thereof, according to the above description.

Likewise, spermidine, or a pharmaceutically acceptable salt thereof, may be employed in a method for manufacturing a pharmaceutical for the treatment of an inflammatory disease of the intestine, particularly with IBD, CD, UC, or NASH.

Similarly, a dosage form for the prevention or treatment of intestinal dysbiosis is provided, comprising spermidine or a salt thereof according to any of the above aspects or embodiments of the invention. The skilled person is aware that spermidine may be present as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate.

Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as a suppository. Alternatively, particularly in a surgical setting, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.

Topical administration is also within the scope of the advantageous uses of the invention, particularly in a surgical setting, or via rectal administration. The skilled artisan is aware of a broad range of possible recipes for providing topical formulations, as exemplified by the content of Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1 st Edition, Wiley 2011 , ISBN-13: 978-0470450291 ); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2^nd Ed., CRC Press 2002, ISBN- 13: 978-0824708610); Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1^st Ed. CRC Press 1989; ISBN-13: 978-0824781835).

Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention further encompasses the following items:

A. Spermidine, or a pharmaceutically acceptable salt thereof, for use in prevention or treatment a. of an inflammatory disease of the intestine, particularly with Inflammatory Bowel Disease (IBD), Colitis Ulcerosa or Morbus Crohn, or Non-alcoholic steatohepatitis (NASH), wherein the IBD, colitis, or NASH is associated with intestinal dysbiosis; or b. of intestinal dysbiosis.

B. Spermidine, or a pharmaceutically acceptable salt thereof, for use according to item A, wherein the intestinal dysbiosis is characterized by a prevalence in a patient’s intestinal microbiome of a. the phylum Firmicutes of less than (<) 30% of the patient’s intestinal microbiome; and/or b. the phylum Bacteroidetes of less than (<) 15% of the patient’s intestinal microbiome; and/or c. the phylum Proteobacteria of more than (>) 20% of the patient’s intestinal microbiome.

C. Spermidine, or a pharmaceutically acceptable salt thereof, for use according to item A or B, wherein the intestinal dysbiosis is characterized by a prevalence in a patient’s intestinal microbiome of a. the class Clostridia of less than (<) 40% [particularly <35%, more particularly <30%] of the patient’s intestinal microbiome; b. the class Bacteroidia of less than (<) 30% [particularly <20%, more particularly <15%] of the patient’s intestinal microbiome; c. the class Verrucomicrobiae of less than (<) 4% [particularly <2%, more particularly <1 %] of the patient’s intestinal microbiome; d. the class Gammaproteobacteria of more than (>) 5% [particularly >15%, more particularly >50%] of the patient’s intestinal microbiome; and/or e. the class Bacilli of more than (>) 3% [particularly >5%, more particularly >7,5%] of the patient’s intestinal microbiome.

D. Spermidine for use according to any one of the preceding items, wherein the intestinal dysbiosis is characterized by a prevalence in a patient’s intestinal microbiome of a. the genera Shigella ssp. and Escherichia coll of more than (>) 30% [particularly >35%, more particularly >40%] of the patient’s intestinal microbiome; and/or b. the genus Enterococcus of more than (>) 3% [particularly >4%, more particularly >6%] of the patient’s intestinal microbiome.

E. Spermidine for use according to any one of the preceding items, wherein the intestinal dysbiosis is characterized by a prevalence in a patient’s intestinal microbiome of a. the genus Lachnospiraceae of less than (<) 15% [particularly <12%, more particularly <10%] of the patient’s intestinal microbiome; and/or b. the genus Odoribacter of less than (<) 2% [particularly <5%, more particularly <8%] of the patient’s intestinal microbiome; and/or c. the genera of the Rikenellaceae RC9 gut group of less than (<) 8% [particularly <5%, more particularly <2%] of the patient’s intestinal microbiome; and/or d. the genus Faecalibaculum of less than (<)2% [particularly <1 ,5%] of the patient’s intestinal microbiome; and/or e. the genus Ruminiclostridium 5 of less than (<)1% [particularly <0,3%] of the patient’s intestinal microbiome. F. Spermidine for use according to any one of the preceding items, wherein the spermidine is administered to a patient having been determined to carry the PTPN2 variant rs1893217.

G. Spermidine for use according to any one of the preceding items, wherein the spermidine is administered by enteral administration, particularly by oral administration.

H. Spermidine for use according to any one of the preceding item A to G, wherein the spermidine is administered at a dose of >30 mg, particularly at a dose of >1 OOmg, even more particularly at a dose of 250 to 300 mg.

I. Spermidine for use according to any one of the preceding items A to H, wherein the spermidine is formulated for enteral administration, particularly as tablet or capsule, more particularly as a tablet or capsule for delivery of the spermidine to the intestine (gastro resistant tablet, capsule, suppository or enema).

J. A pharmaceutical composition formulated for oral administration, said pharmaceutical composition comprising >50 mg spermidine per dosing unit, particularly >100mg per dosing unit, even more particularly >200mg per dosing unit.

K. The pharmaceutical composition according to item J, formulated for enteral administration, particularly as tablet, capsule, suppository or enema, more particularly as a tablet or capsule for delivery of the spermidine to the intestine (gastro resistant tablet or capsule).

L. The pharmaceutical composition according to item J or K, for use in prevention or treatment of a. an inflammatory disease of the intestine, particularly with IBD, UC or CD, or NASH particularly wherein the IBD, UC, CD or NASH is associated with intestinal dysbiosis; b. intestinal dysbiosis.

M. The pharmaceutical composition for use in prevention or treatment a. of IBD or NASH associated with intestinal dysbiosis, or b. of dysbiosis, according to item L, wherein the dysbiosis is characterized as specified in any one of items C to E.

N. A method of treating an inflammatory disease of the intestine, particularly an inflammatory disease of the intestine selected from IBD, UD, CD, or NASH, said method comprising providing to the patient an effective dose of spermidine, or a pharmaceutically acceptable salt thereof. O. Spermidine, or a pharmaceutically acceptable salt thereof, for use in a method for manufacturing a pharmaceutical for the treatment of an inflammatory disease of the intestine, particularly IBD, UD, CD, or NASH.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures

Fig. 1 shows PTPN2 phosphatase activity in mouse colon (A) and histology of protein levels of PTPN2 (B) 8h, 24h, 48h and 72h post application of 1 mg/kg, 10 mg/kg and 100 mg/kg of spermidine. **p<0.01 , ***p<0.001

Fig. 2 shows levels of spermidine measured by mass spectrometry from BI6 wild type mice after oral gavage of 3mM SPD, in (A) mouse serum and (B) faeces 15 min, 30min, 1 h, 2h, 4h, 8h and 24h post application.

Fig. 3 shows that long-term spermidine treatment has no toxic effects in vivo. (A) Liver and kidney function serum markers alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (CREA) are normal after 13 weeks of oral administration of 3 mM or 10 mM spermidine, as is histology of colon, kidney and liver (B) in a long-term spermidine administration in mice fed with standard diet (SD) or low polyamine diet (LPD).

Fig. 4 shows that spermidine treatment ameliorates T cell transfer colitis, prevents weight loss and shortening of mouse colon, and macroscopic inflammation in the T cell transfer colitis model. 5 x 10⁵ splenic CD4⁺ CD62L^hl CD44^|OW CD25' naive T cells were injected intraperitoneal into Rag2'^/_ immunodeficient mice, and 3mM spermidine (SPD) was administered in drinking water from the day of T-cell transfer. The graphs show (A) weight development, (B) colon length, (C) murine endoscopic index of colitis severity (MEICS) score, (D) colon histology score, and (E) MPO activity. Supplying drinking water with 3mM, 0.3mM, or 11 ,5uM spermidine supplementation confirmed that doses below 3mM do not ameliorate dysbiosis and inflammation in a model of T cell transfer colitis as in A, in terms of (F) weight development, (G) colon length, (H) murine endoscopic index of colitis severity (MEICS) and (I) H&E histology pathology score.

Fig. 5 shows naive T cells injected into Rag2'^/_ immunodeficient mice as above, with 3mM spermidine in the drinking water from the day of naive T cell transfer (treatment), or for 7 days prior to the transfer (pre-treatment), or throughout the experiment (continuous). The graphs show (A) weight development, (B) colon length, (C) murine endoscopic index of colitis severity (MEICS) score (D) histology score of sections of H&E stained distal colon tissue. *p<0.05, **p<0.01 ***p<0.001 Fig. 6 shows that spermidine ameliorates T cell transfer colitis regardless of PTPN2 presence in transferred T cells. The graphs show (A) weight development, (B) colon length, (C) MEICS score and representative pictures of mouse endoscopy, (D) myeloperoxidase activity in colon, (E) histological score and representative pictures of H&E-stained section of the terminal colon. *p<0.05, **p<0.01 , ***p<0.001

Fig. 7 shows spermidine has a prominent effect on immunity and promotes anti-inflammatory (M2-like) macrophages. Graphs show (A) relative abundance of CD3⁺ T cells measured by flow cytometry, (B) number of CD3⁺ T cells and % of proliferating (Ki67⁺) CD3⁺ T cells assessed by immunofluorescence staining, (C) relative abundance of CD4⁺ T cells producing IFN-y, IL- 17 and both IFN-y and IL-17, (D) relative abundance of myeloid cells and (E) mRNA expression of macrophage-related genes within the lamina propria. *p<0.05, **p<0.01 , ***p<0.001 .

Fig. 8 shows that spermidine reduces severity of chronic DSS colitis. DSS (dextran sodium sulphate) was administrated in the drinking water in 4 cycles of 7-days exposition followed by 10 days of recovery, and 3mM spermidine (SPD) was administered in drinking water. Graphs show (A) colon length, (B) murine endoscopic index of colitis severity (MEICS) and (C) histology scores from H&E-stained section of the terminal colon.

Fig. 9 shows 16S ribosomal DNA analysis of the T cell transfer colitis faecal samples. Graphs show (A) Alpha rarefaction and (B) Alpha diversity, based on Faith’s phylogeny diversity (PD), (C) Beta diversity based on weighted Unifrac principal (T cell alone group circled).

Fig. 10 shows the relative abundance of gut microbiota at the level of phylum and class in the T cell transfer colitis.

Fig. 11 shows that the presence of the PTPN2 risk allele (CT, SNP rs1893217) ameliorates PTPN2 phosphatase activity in PBMC from CD patients. Activation of PTPN2 by spermidine prevents the IFN-y-induced increase in STAT phosphorylation and expression of inflammatory markers in human PBMC. Graphs show (A) enzymatic activity of PTPN2, (B) mRNA expression of IFN-y and (C) representative Western blots and densitometry of STAT1 and STAT3 phosphorylation levels after the treatment with 100 ng/mL IFN-y and/or 10 pM spermidine. PBMC were isolated from healthy controls (HC, homozygous for the T allele, n = 5), or IBD patients homozygous for the T allele (TT, n = 6) or heterozygous (CT, risk variant, n = 5) for the PTPN2 SNP rs1893217. *p<0.05

Table 1 . Relative abundance of bacteria phylogeny in IBD patients with dysbiosis and in T cell transfer colitis as in Fig. 4. Adapted from Liu et al. Protein Cell (2020), Alam et al. Gut Pathog 12, 1 (2020); Lloyd-Price et al. Nature 569, 655-662 (2019); Matsuoka and Kanai Immunopathol 37, 47-55 (2015); Arumugam et al. Nature 473, 174-180 (2011 ). The inventors first determined the optimal interval and dosage of spermidine administration in vivo, which was indicated by the lowest dose that still resulted in convincing biological effects. For this purpose, spermidine was first administered in different doses (1 , 10, 100 mg/kg of body weight) via oral gavage to wild-type mice followed by analysis of PTPN2 activity 8h, 24h, 48h and 72h later. Highest PTPN2 activity in colon tissue of mice was observed 24h after spermidine application, what suggests, that once daily application might be a relatively promising dosing interval in vivo. Moreover, 24h post application similar PTPN2 activity levels upon administration of 10 mg/kg and 100 mg/kg of spermidine (clearly higher than with 1 mg/kg spermidine) were observed. This leads to the second conclusion, that 10 mg/kg might be the optimal concentration to achieve biological activity in vivo (Fig. 1 ).

Furthermore, the inventors evaluated levels of spermidine in mouse serum and faeces using liquid chromatography UPLC, in collaboration with the Swiss Institute of Asthma and Allergy in Davos. Spermidine reached the highest serum concentration within 30 minutes and returned to baseline levels within 4h (Fig. 2A), whereas in the faeces the highest levels were observed after 4h and returned to the baseline after 24h (Fig. 2B).

The inventors then studied long-term safety of spermidine administration in their mouse models in vivo. For this purpose, they determined whether long-term spermidine treatment might exert any adverse effects in vivo. They applied spermidine in drinking water for 13 weeks at two different concentrations (3mM and 10mM, corresponding to 10 mg/kg/day and 30 mg/kg/day, respectively). Serum markers for liver and kidney damage, e.g. alanine aminotransferase, aspartate aminotransferase or creatinine, were not elevated at the end of long-term spermidine treatment at week 13 suggesting no detrimental effect of spermidine on liver or kidney function (Fig. 3A). Histologic assessment by a pathologist confirmed that no morphological changes in liver, kidney and intestinal tissue in response to long-term spermidine treatment were detectable (Fig. 3B). Mass spectrometry nevertheless confirmed the uptake of spermidine in the tissue in a time- and dose-dependent manner.

Those data demonstrate that a working dosage and treatment interval for oral spermidine application has been determined, and that spermidine administration over an extended time is safe.

It was next investigated whether spermidine treatment is effective in the T cell transfer colitis model. 5 x 10⁵ splenic CD4⁺ CD62L^hl CD44^|OW CD25' naive T cells were injected intraperitoneal into Rag2'^/_ immunodeficient mice. Mice were kept on AIN-93M low polyamine diet throughout the experiment and spermidine was applied directly after T cell transfer. Spermidine treatment significantly alleviated colitis symptoms, as demonstrated by reduced weight loss, prevention of colon shortening, inhibited infiltration/activation of myeloid cells, as demonstrated by significantly lower myeloperoxidase activity within the colonic tissue, and prevented mucosal damage assessed macroscopically by mouse endoscopy and MEICS score (Fig. 4A-E) as well as microscopically by colon histology (H&E staining) and statistical scoring. Spermidine administration had no effect in control animals that did not receive naive T cells.

To identify the lowest effective dose, a range of spermidine was administered daily for the duration of a T cell transfer colitis model (3mM corresponding to 10 mg/kg/day, and in addition 0.3mM and 11.5uM, corresponding to 1 mg/kg/day and 0.30 mg/kg/day, respectively). The lowest two doses, 0.3mM, and 11 ,5uM, representing previous studies, or the dosage currently recommended for use as a food supplement product (Moron et al. 2013: PLoS One 8(9):e73703; https://spermidinelife.com), had no effect on tissue integrity, suggesting again that a minimum of 3mM, equivalent to roughly 10mg/kg/day is required for spermidine to be effective as a treatment for dysbiosis and/or intestinal inflammation (Fig. 4F-I). Furthermore, in order to determine the optimal spermidine treatment window, mice were subjected to the T cell transfer colitis and received 3mM spermidine in the drinking water from the day of naive T cell transfer (treatment), or for 7 days prior to the transfer (pre-treatment), or throughout the experiment (continuous). Treatment group as well as the group receiving spermidine continuously was protected from the weight loss (Fig. 5), showed reduction of colitis severity scores and histology scores comparing to the non-spermidine treated mice, while colon length was not affected. Mice that were only pre-treated with spermidine showed no alleviation of colitis symptoms. These results suggests that spermidine may reduce the symptoms of patients diagnosed with intestinal inflammation, and applied continuously to patients diagnosed with IBD, or ulcerative colitis, may decrease the severity of disease relapse, or flare- ups.

Having demonstrated the efficacy of spermidine treatment as anti-inflammatory approach for treatment of colitis in vivo, the inventors next studied whether the spermidine-mediated effect was dependent on PTPN2 activity in the T cells. For this purpose, naive T cells derived from T cell specific PTPN2 knock-out mice (PTPN2-CD4Cre mice) were used in the T cell transfer colitis model. Again, strong anti-inflammatory effects of spermidine were observed in all clinical, endoscopic and histologic read-outs of the distal part of colon (Fig. 6). However, the spermidine-induced anti-inflammatory effect was not altered in mice that had received PTPN2 deficient naive T cells. Those data suggest that the anti-inflammatory effect of spermidine is independent from PTPN2 activity in CD4+ T-cells.

When analysing immune cell populations in the colonic lamina propria in groups that received T cell transfer, the inventors found that spermidine reduced abundance of CD3⁺ T cells and their proliferation rate (Fig. 7A-B) but had no significant effect on IFN-y , or IL-17 producing T cells (Fig. 7C). In contrast, among myeloid cells reduction of infiltrating neutrophils (in line with reduced MPO levels), monocytes and macrophages in colonic lamina propria was observed (Fig. 7D) along with the relative reduction of pro-inflammatory M1-like macrophages and increase of anti-inflammatory M2-like macrophages upon spermidine administration (Fig. 7D). In line with the flow cytometry data, mRNA expression of genes encoding for intestinal macrophages markers F4/80 and CD64 was decreased together with the pro-inflammatory macrophage marker gene Nos2, while M2-specific marker CD206 was increased by spermidine supplementation in the colonic tissue mice subjected to colitis (Fig. 7E).

Similar anti-inflammatory effects of spermidine were observed in a chronic dextran sodium sulphate (DSS)-induced colitis model, where spermidine treatment reduced endoscopic and histologic colitis scores (Fig. 8). These data confirm the colitis alleviating effect of spermidine and the absence of adverse effects in control animals.

The investigators found that spermidine treatment was also able to recover the intestinal microbiome composition of mice undergoing T cell transfer. 16S sequencing of faecal samples demonstrated comparable microbiome compositions in the intestine of control mice as well as mice receiving T cell transfer and spermidine treatment (Fig. 9, 10). The microbiome alterations observed in mice receiving naive T-cells only featured a dysbiotic microbiome composition comparable to that observed in IBD patients (Table 1 ). Samples from colitis mice without spermidine treatment showed significantly lower bacterial richness and phylogenic diversity, in contrast to those that received T cells and spermidine treatment. Taxonomy analysis revealed that Firmicutes and Bacteroides - two dominant phyla present in healthy mice, were almost completely replaced by Proteobacteria in colitis mice, however that shift did not take place when spermidine was supplied (Fig. 10). Of note an increase in relative abundance of Verrucomicrobia was the only shift induced by spermidine in the healthy controls. Also, clear differences were present in the classes of bacteria observed with a massive increase in the abundance of Gammaproteobacteria in the T cell group, whereas in the spermidine treated colitis mice the classes found in Clostridia and Bacteroidia were most abundant - similarly to healthy mice treated with and without spermidine (Table 1 ).

Finally, the inventors evaluated the effect of spermidine on patient-derived peripheral blood mononuclear cells (PBMCs) carrying the IBD- associated PTPN2 single nucleotide polymorphism (SNP) rs1893217 (CT-variant). Interestingly the anti-inflammatory effect of spermidine is enhanced in the presence of the CT-variant comparing to the wild-type (TT) PTPN2, and the enzymatic activity of PTPN2 is decreased. However in IFN-y challenged PBMCs, spermidine more efficiently downregulates the expression of pro-inflammatory markers and phosphorylation of STATs molecules when C-allele is present (Fig. 11 ) Methods

Animal experiments

All animal experiments were approved by the local animal welfare commission (Veterinary Office of the Canton Zurich) and performed according to the Swiss animal welfare legislation. Wild type C57BL/6 mice were obtained from Janvier (France), while Rag2'^/_ mice were bred inhouse. All animals were kept in a specific pathogen-free (SPF) facility and received food and water ad libitum. For T-cell transfer colitis, naive T-cells were isolated from the spleen of wild type mice or PTPN2^fl/fl-CD4Cre mice using a CD4⁺ T-cell isolation kit (Stemcell Technologies) followed by FACS sorting (Aria III cell sorter, BD Bioscience) for CD4⁺CD62L^hl9hCD44^l0WCD25‘ cells and subsequent intraperitoneal injection of 5 x 10⁵ cells. Acute colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) (MP Biomedicals, France) in the drinking water for 7 days, followed by 3 days of recovery with the normal drinking water. Chronic colitis was induced by administration of four cycles of 2% dextran sodium sulphate (DSS) (MP Biomedicals, France) in the drinking water, each lasting for 7 days, followed by 10 days of recovery with the normal drinking water. Sterile filtered spermidine solution (Sigma Aldrich, USA) was administrated either via oral gavage (1 mg/kg, 10 mg/kg or 100 mg/kg) or diluted in the drinking water (0.0115, 0.3, 3, or 10 mM). In all studies 8-10-week-old females were used, receiving either standard chow or a low polyamine diet (M/R EXP AIN 93M, Granovit AG, Switzerland).

Colitis assessment

Colonoscopy was performed using a mouse endoscope (Karl Storz, Germany) and evaluated using the murine endoscopic index of colitis severity (MEICS) according to Becker et al. (Becker et al., 2006). For histological assessment, paraffin-embedded, 5pM sections of the most distal part of the colon were stained with hematoxylin-eosin (H&E) according to standard protocols, and scored for epithelial damage and inflammatory infiltration as described by Obermeier et al., Clin Exp Immunol 1999; 116:238-245.

Analysis of liver and kidney damage markers

Blood samples were collected into BD Microtainer SST tubes (Becton, Dickinson and Company, USA) and centrifuged 30 min after collection for 5 min at maximum speed. Obtained serum was used for the measurement of alanine aminotransferase (GPT/ALT-P III, Fuji Dri- Chem, Japan), aspartate aminotransferase (GOT/AST-P III, Fuji Dri-Chem, Japan), and creatinine (CRE-P III, Fuji Dri-Chem, Japan), using a Fuji Dri-Chem 4000i reader. Histological analysis was performed using paraffin embedded and haematoxylin-eosin (H&E) stained 5pM sections of colon, kidney and liver tissues according to standard procedures. MPO assay

Colon samples were homogenized in 50 mM phosphate buffer containing 0.5% hexadecyl trimethyl-ammonium bromide (Sigma Aldrich, USA) using a gentleMACS tissue dissociator (Miltenyi Biotec, Germany). Following three freeze and thaw cycles, homogenates were centrifuged and the supernatant mixed with assay buffer (50 mM phosphate buffer, 0.02% dianisidine, 0.0005% H2O2, Sigma Aldrich, USA) and incubated for 20 min. Absorbance was measured at 460 nm using a microplate reader (Synergy H1 , BioTek, USA) and Gene5 V1 .11 software. Protein concentration was determined using a bicinchoninic acid protein assay (BCA) according to the manufacturer’s instructions. Myeloperoxidase activity was calculated as a mean absorbance (460 nm) per incubation time (min) per protein concentration (g).

Isolation of lamina propria lymphocytes

Colon tissue was rinsed with PBS and stored in HBSS (Hank’s Balanced Salts, Sigma Aldrich, USA) supplemented with 2% FCS (Biowest, France) until processing. The tissue was cut into 0.5 cm pieces, incubated in HBSS containing 2mM EDTA (Carl Roth, Germany) at 37°C, 300 rpm for 15 min, washed with HBSS and again incubated in HBSS containing 2mM EDTA at 37°C, 300 rpm for 30 min to release epithelial cells. After removal of epithelial cells by vigorous shaking, the tissue pieces were digested using 0.6 mg/ml dispase II (Gibco, Life Technologies, USA) and 0.4 mg/ml collagenase IV (Sigma Aldrich) dissolved in RPMI 1640 Medium (Gibco, Life Technologies, USA) containing 1 % FCS (Biowest, France) at 37°C, 300 rpm for 30min. Finally, the digested tissue was passed through a 18G needle several times and filtered through a 70 pM strainer to remove undigested tissue.

Flow cytometry

For intracellular cytokines staining, cells were stimulated with 50 ng/ml PMA (Sigma Aldrich, USA), 10pg/ml brefeldin A (Sigma Aldrich, USA) and 1 pg/ml ionomycin (Sigma Aldrich, USA) for 3h at 37°C in 5% CO2 incubator. Cells were washed with PBS (Sigma Aldrich, USA), stained with antibodies against surface marker for 30 min at 4°C, washed with PBS and fixed in fixation I permeabilization buffer (Cytofix I Cytoperm, BD Bioscience, USA) for 30 min at 4°C. For intracellular staining, cells were then washed with wash buffer (Cytofix I Cytoperm, BD Bioscience, USA) and stained with intracellular antibodies for 30 min at 4°C. For the intranuclear staining, cells were processed using the FoxP3 Transcription Factor Staining Buffer Set (eBioscience, USA) according to the manufacturer’s instructions and stained with anti-FoxP3 antibody for 30 min at 4°C. All analyses were performed using a BD LSR Fortessa cytometer (BD Bioscience, USA). Patient Samples

Whole blood samples were obtained from Crohn’s disease (CD) and ulcerative colitis (UC) patients from the Swiss IBD cohort and from healthy volunteers. All patients, including healthy controls (HCs), signed informed consent before study inclusion. To isolate peripheral blood mononuclear cells (PBMCs), the blood was diluted 1 :2 in Dulbecco’s Phosphate Buffer Saline (PBS, Sigma-Aldrich, USA) and overlaid on Ficoll (Histopaque-1077, Sigma-Aldrich, USA) before density gradient centrifugation. Peripheral blood mononuclear cells were collected from the interphase, washed twice in PBS, and resuspended in RPMI Medium 1640 (Thermo Fisher Scientific, USA) or frozen in FCS (Biowest, France) with 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich).

Cell stimulation

Peripheral blood mononuclear cells were seeded on 12-well culture plates (TPP, Buchs, Switzerland) at a density of 1.0 x 10⁶ cells per well and left to adhere for 24 hours. The cells were then treated with IFN-y (100 ng/mL, Sigma-Aldrich, USA) and I or 10pM spermidine (Sigma-Aldrich, USA) for 30 minutes for protein collection and for 24 hours for mRNA isolation. In each experiment, a group of untreated cells was included as a reference control.

Protein isolation, immunoprecipitation and phosphatase activity assay

Prior to protein isolation, colon pieces were homogenized in Mammalian Protein Extraction Reagent (M-PER™, ThermoFisher Scientific, USA) containing complete™ mini protease inhibitor cocktail (Roche, Switzerland) using gentleMACS Octo Dissociator (Miltenyi Biotec, Germany) and incubated for 30min on ice, whereas PBMCs were washed twice with PBS (Sigma-Aldrich, USA) and directly incubated in mentioned buffer for 30 minutes on ice. Tissue I cell lysates where centrifuged, and protein-containing supernatant was collected. Protein concentration was measured at 280 nm wavelength using a NanoDrop ND1000 spectrophotometer (Marshall Scientific, USA). For immunoprecipitation of PTPN2, protein lysates were precleared with 50% (v/v) protein G-Sepharose 4 Fast Flow beads (Ge Healthcare Life Science, USA) and supernatants subsequently incubated with anti-PTPN2 antibody (Merck, Germany) overnight at 4°C. Sepharose 4 Fast Flow beads were then added to each sample and left to incubate for 1 hour at 4°C. Pellets containing beads-antibody- antigen complex were washed 3 times with ice-cold PBS and re-suspended in reaction buffer from the EnzChek-Phosphatase assay Kit (ThermoFisher Scientific, USA). PTPN2 phosphatase activity was measured using the EnzChek-Phosphatase assay Kit (ThermoFisher Scientific, USA) according to the manufacturer’s instructions. Each sample was applied in triplicate to a 96-well black plate and mixed with 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), which emits a fluorescent signal at 562 nm upon dephosphorylation. Fluorescence was measured on a microplate reader (Synergy HT and Synergy H1 , BioTek, USA) using Gene5 V1.11 software.

Western Blotting

Equal amounts of proteins were loaded on to 10% sodium dodecyl sulphate polyacrylamide gels and separated by electrophoresis (SDS-PAGE) and then transferred on to nitrocellulose membranes (0.45 pm pore size, Membrane Solution, Auburne, Washington, USA). Further, membranes were blocked in 3% milk powder (Carl Roth, Karlsruhe, Germany) and 1 % bovine serum albumin (BSA, PAN Biotech, Regensburg, Germany) and incubated with primary antibodies and corresponding horseradish peroxidase (HRP)-labelled secondary antibodies. Detection was performed using the WesternBright (Advansta, USA) or WesternBright Sirius (Advansta, USA) ECL kit. Pictures were taken with a Fusion Solo S imager (Vilber Lourmat, Paris, France) using the Vision Capture Fusion Solo 3 v16.15 software. Densitometry measurements were performed using NIH Imaged software. For phosphorylated STAT1 and STAT3, normalization was conducted to total STAT1 and STAT3 protein, respectively. The following antibodies were used for protein detection: anti-STAT-1 (9172S, Cell Signaling, Danvers, Massachusetts, USA), anti-pSTAT1 (7649S, Cell Signaling), anti-STAT3 (9139S, Cell Signaling), anti-pSTAT3 (9131 S, Cell Signaling).

RNA Isolation and RT-PCR

Before mRNA isolation, cells were washed twice with PBS and snap frozen in RLT buffer (Qiagen, Germany) supplemented with 40 mM 1 ,4-dithiothreitol (DTT, Sigma-Aldrich, USA), and RNA was isolated using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Total RNA concentration was quantified using a Nanodrop ND1000 spectrophotometer (Marshall Scientific, USA) at a wavelength of 260 nm. Complementary DNA (cDNA) synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) was performed using FAST qPCR MasterMix for Taqman Assays (Thermo Fisher Scientific, USA) on a Fast HT7900 Real-Time PCR system using SDS Software (Thermo Fisher Scientific, USA) or on a QuantStudio 6 System (Thermo Fisher Scientific, USA). Measurements were performed in triplicates, human Actb was used as endogenous control, and results were analysed by the AACT method. The used gene expression assay was obtained from Thermo Fisher Scientific.

Polyamine measurements

Assessment of spermidine content in mouse serum and faeces was performed in a collaboration with Swiss Institute of Asthma and Allergy in Davos. Standard stock solution of spermidine was prepared at 1000 mg/l in distilled water and further diluted to generate standard curves. Serum sample, faeces or standard solution was mixed with 1 ,7- diaminoheptane (Sigma Aldrich, USA), 2M NaOH, saturated sodium bicarbonate solution and dansyl chloride (Sigma Aldrich, USA) solution in acetone, and incubated for 45 min at 40°C, 200 rpm. Residual dansyl chloride was removed by adding 25% ammonium hydroxide (Merck, Germany) and incubating at 25°C for 30min. Afterwards the volume was adjusted to 2.5 ml with acetonitrile (Biosolve Chimie, France) and centrifuged for 5 min at 3500 rpm. Supernatants were collected and filtered (0.22 pm) before ultra-performance liquid chromatography (UPLC) analysis. Duplicate samples were analysed in parallel. Separation was carried out by UPLC on an ACQUITY UPLC H-Class Bio System (Waters Corp, USA) and the data was analysed using MassLynx v. 4.1 (Waters Corp, USA).

16S rRNA Gene Sequencing Microbiome analysis

The QIIME2 pipeline was used for the analysis of 16S rRNA. After checking data quality, DAAD2 merged the paired reads, and removed noise them selecting 8000 feature depth. Alpha rarefaction module was used to ensure sufficient depth to capture the majority of features. Both alpha and beta diversity were calculated using the core-metrics-phylogenetic module. For alpha analysis, the faith phylogenetic matrix computed the richness and incorporated features' phylogenetic relations. For Beta diversity, a weighted UniFrac distance matrix was used to quantify the dissimilarity between communities. Principle coordinate analysis (PoCA) was used for better visualization of beta diversity. A pre-trained Naive Bayes silva-132-99-nb-classifier trained against Silva (release 132) full-length sequences was used to explore the taxonomic composition. Taxa-barplot module was used for visualization of the different taxonomy composition.

Statistical Analysis

All data are presented as means ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8 (Graph Pad Software, La Jolla, CA), by using analysis of variance (ANOVA), followed by Tukey post-hoc test or by non-parametric Kruskal-Wallis test followed by multiple comparison test with Benjamini- Hochberg correction. P values < 0.05 were considered significant.

Claims

Claims

1 . Spermidine, or a pharmaceutically acceptable salt thereof, for use a. in treatment, or prevention of relapse of an inflammatory disease of the intestine, particularly an inflammatory disease of the intestine selected from Inflammatory Bowel Disease (IBD), ulcerative colitis (UC), Crohn’s disease (CD), or Non-alcoholic steatohepatitis (NASH), wherein the IBD, UC, CD, or NASH is associated with intestinal dysbiosis; or b. in treatment of intestinal dysbiosis.

2. Spermidine, or a pharmaceutically acceptable salt thereof, for use according to claim

1 , wherein the intestinal dysbiosis is characterized by a prevalence in a patient’s intestinal microbiome of a. the phylum Firmicutes of less than (<) 30% of the patient’s intestinal microbiome; and/or b. the phylum Bacteroidetes of < 15% of the patient’s intestinal microbiome; and/or c. the phylum Proteobacteria of more than (>) 15% [particularly > 20%] of the patient’s intestinal microbiome.

3. Spermidine, or a pharmaceutically acceptable salt thereof, for use according to claim 1 or 2, wherein the intestinal dysbiosis is characterized by a prevalence in a patient’s intestinal microbiome of a. the class Clostridia of < 25% [particularly <15%, more particularly <10%] of the patient’s intestinal microbiome; b. the class Bacteroidia of < 30% [particularly <20%, more particularly <15%] of the patient’s intestinal microbiome; c. the class Verrucomicrobiae of < 4% [particularly <2%, more particularly <1 %] of the patient’s intestinal microbiome; d. the class Gammaproteobacteria of > 5% [particularly >15%, more particularly >50%] of the patient’s intestinal microbiome; and/or e. the class Bacilli of > 3% [particularly >5%, more particularly >7,5%] of the patient’s intestinal microbiome;

4. Spermidine for use according to any one of the preceding claims, wherein the intestinal dysbiosis is characterized by a prevalence in a patient’s intestinal microbiome of a. the genera Shigella ssp. and Escherichia coll of more than (>) 30% [particularly >35%, more particularly >40%] of the patient’s intestinal microbiome; and/or

27 b. the genus Enterococcus of > 3% [particularly >4%, more particularly >6%] of the patient’s intestinal microbiome. Spermidine for use according to any one of the preceding claims, wherein the intestinal dysbiosis is characterized by a prevalence in a patient’s intestinal microbiome of a. the genus Lachnospiraceae of <15% [particularly <12%, more particularly <10%] of the patient’s intestinal microbiome; and/or b. the genus Odoribacter of <8% [particularly <5%, more particularly <2%] of the patient’s intestinal microbiome; and/or c. the genera of the Rikenellaceae RC9 gut group of <8% [particularly <5%, more particularly <2%] of the patient’s intestinal microbiome; and/or d. the genus Faecalibaculum of <2% [particularly <1 ,5%] of the patient’s intestinal microbiome; and/or e. the genus Ruminiclostridium 5 of <1 % [particularly <0,3%] of the patient’s intestinal microbiome. Spermidine for use according to any one of the preceding claims, wherein the spermidine is administered to a patient having been determined to carry the PTPN2 variant rs1893217. Spermidine for use according to any one of the preceding claims, wherein the spermidine is administered by enteral administration, particularly by oral administration. Spermidine for use according to any one of the preceding claims 1 to 7, wherein the spermidine is administered at a dose of at least (>) 30 mg, particularly at a dose of > 100mg, even more particularly at a dose of 250 to 300 mg. Spermidine for use according to any one of the preceding claims 1 to 8, wherein the spermidine is formulated for enteral administration, particularly as tablet, capsule, suppository or enema, more particularly as a tablet or capsule for delivery of the spermidine to the intestine (gastro resistant tablet, or capsule). A pharmaceutical composition formulated for enteral administration, said pharmaceutical composition comprising > 50 mg spermidine per dosing unit, particularly > 100mg per dosing unit, even more particularly > 200mg per dosing unit. The pharmaceutical composition according to claim 10, formulated for enteral administration, particularly as tablet, capsule, suppository, or enema, more particularly as a tablet, or capsule for oral delivery of the spermidine to the intestine (gastro resistant tablet, or capsule). The pharmaceutical composition according to claim 10 or 11 , for use a. in treatment, or prevention of relapse of an inflammatory disease of the intestine, particularly an inflammatory disease of the intestine selected from IBD, UC, CD, or NASH, particularly wherein the IBD, UC, CD, or NASH is associated with intestinal dysbiosis; b. treatment of intestinal dysbiosis. The pharmaceutical composition for use a. in treatment, or prevention of relapse of IBD, UC, CD, or NASH associated with intestinal dysbiosis, or b. treatment of intestinal dysbiosis, according to claim 11 , wherein the dysbiosis is characterized as specified in any one of claims 3 to 5. A method of treating, or preventing relapse of an inflammatory disease of the intestine, particularly an inflammatory disease of the intestine selected from IBD, UC, CD, or NASH, or a method of treating intestinal dysbiosis, said method comprising providing to the patient an effective dose of spermidine, or a pharmaceutically acceptable salt thereof. Spermidine, or a pharmaceutically acceptable salt thereof, for use in a method for manufacturing a pharmaceutical for the treatment, or prevention of relapse of an inflammatory disease of the intestine, particularly an inflammatory disease of the intestine selected from IBD, UC, CD, or NASH, or for the treatment of intestinal dysbiosis.