Use of tryptophan hydroxylase 1 (TPH1) inhibitors for the treatment of atherosclerosis

Abstract

Tryptophan (Trp) is one of the nine essential amino acids supplied by diet, and its metabolism appears to be a major metabolic gatekeeper of intestinal homeostasis; however, its systemic effects on atherosclerosis remain unclear. The inventors have shown that a high-fat diet (HFD), rather than a high-cholesterol diet (HCD), increases the activity of intestinal indoleamine 2,3-dioxygenase 1 (IDO), the main enzyme involved in Trp catabolism in the gastrointestinal tract, which shifts intestinal Trp metabolism from the production of indole metabolites and serotonin or 5-hydroxytryptamine (5-HT) produced by the microbiome to the production of kynurenine. Most importantly, the inventors have shown that inhibition of tryptophan hydroxylase 1 (TpH1) significantly reduces intestinal 5-HT production, thereby alleviating atherosclerosis and plaque inflammation. Therefore, the present invention relates to the use of tryptophan hydroxylase 1 (TpH1) inhibitors for the treatment of atherosclerosis.

Description

[Technical Field]

[0001] This invention relates to medicine, particularly to the field of cardiovascular disease. [Background technology]

[0002] The gastrointestinal tract has come to be recognized as a crucial link between diet and cardiovascular disease (CVD), including atherosclerosis (Cainzos-Achirica et al. 2020). In particular, the gastrointestinal microbiome has been identified as a potential mediator that may influence atherosclerosis. Although there is evidence explaining the relationship between gastrointestinal inflammation and CVD, little is known about the mechanisms by which the intestines may contribute to atherosclerosis. In particular, gastrointestinal metabolism may explain the link between local disruption of gastrointestinal homeostasis and the systemic development of atherosclerosis.

[0003] Tryptophan (Trp) is one of the essential amino acids supplied through diet, and its metabolism appears to be a major metabolic gatekeeper of intestinal homeostasis. Trp is involved in various physiological processes and contributes to the maintenance of intestinal and systemic homeostasis in health and disease (Zelante et al. 2013, Gao et al. 2018). In mice, dietary Trp deficiency leads to impaired intestinal immunity and enterotoxosis of the gastrointestinal microbiota, which causes enteritis and diarrhea (Hashimoto et al. 2012). In humans, patients with intestinal disease of the intestinal tract (IBD) exhibit disruption of tryptophan metabolism, likely due to alterations in the gastrointestinal microbiota (Lamas et al. 2016). However, despite the importance of intestinal Trp metabolism in gastrointestinal homeostasis, and with confirmation that enteritis is associated with CVD, the systemic effects of gastrointestinal Trp metabolism on atherosclerosis remain unclear.

[0004] Under homeostatic conditions, Trp catabolism in the intestinal tract follows three pathways: the kynurenine (Kyn) pathway via IDO, primarily in intestinal epithelial cells (IECs) (approximately 95% of Trp); the gastrointestinal microbiota pathway of direct conversion of Trp to indole metabolites (approximately 5% of Trp); and the serotonin or 5-hydroxytryptamine (5-HT) pathway via Trp hydroxylase 1 (TpH1) in enterochromaffin cells (ECs) (approximately 1-2% of Trp). This accounts for the majority of 5-HT production in the body (approximately 90% of 5-HT).

[0005] The inventors previously demonstrated the significant role of indoleamine 2,3-dioxygenase 1 (IDO), a Trp-degrading enzyme, in the fine-tuning of intestinal Trp metabolism under a high-fat diet (HFD), which causes obesity, along with a major causal relationship with metabolic syndrome (Laurans et al. 2018). However, the specific role of intestinal IDO in cardiometabolic diseases, including atherosclerosis, remains unclear. Furthermore, while recent studies have highlighted the local effects of Trp-derived metabolites on intestinal homeostasis, their systemic effects on atherosclerosis are largely unknown. [Overview of the Initiative]

[0006] The present invention is defined by the claims. In particular, the present invention relates to the use of tryptophan hydroxylase 1 (TpH1) inhibitors for the treatment of atherosclerosis.

[0007] Detailed description of the invention The present invention relates to a method for treating atherosclerosis in patients in need, comprising administering a therapeutically effective dose of a tryptophan hydroxylase 1 (TpH1) inhibitor to the patient.

[0008] As used herein, the terms “patient” or “subject” refer to any mammal, including rodents, felines, canines, and primates. In particular, in the present invention, the patient is a human. In some embodiments, the patient is a human who is prone to the atherosclerosis described above.

[0009] As used herein, the term “atherosclerosis” refers to a pathological process that leads to the abnormal accumulation of cholesterol and cholesteryl esters and related lipids in macrophages, smooth muscle cells, and other types of cells, resulting in narrowing and / or occlusion of one or more arteries and arterioles of the body and organs, including but not limited to the coronary arteries, aorta, renal arteries, carotid arteries, and arteries supplying blood to the limbs and central nervous system. The associated inflammatory responses and mediating factors of this pathological process are also included in this definition.

[0010] As used herein, the terms "treatment" or "treating" refer to both prophylactic or preventative treatment, as well as curative or disease-modifying treatment, including suppression of clinical recurrence, for patients at risk of or suspected of having a disease, and for patients diagnosed as being ill or having a disease or medical condition. Treatment can be administered to a patient having a medical disorder or who may ultimately acquire a disorder to prevent, cure, delay the occurrence of, reduce the severity of, or improve one or more symptoms of the disorder or recurring disorder, or to extend the patient's life expectancy beyond what is expected in the absence of such treatment. "Treatment regimen" means a pattern of treatment of a disease, such as a pattern of dosing used during therapy. A treatment regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a treatment regimen (or a portion of a treatment regimen) used for initial treatment of a disease. A general goal of an induction regimen is to provide a high level of drug to the patient during an initial period of the treatment regimen. An induction regimen can (partially or wholly) employ a "loading regimen" that includes administering more doses of a drug than a physician would employ during a maintenance regimen, administering the drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used for maintenance of a patient during treatment of a disease, e.g., to keep the patient in a remission state for a long period (for months or years). A maintenance regimen can employ continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at recurrence, or treatment upon achievement of certain predetermined criteria [e.g., disease signs, etc.]).

[0011] In particular, the TPH1 inhibitors of the present invention are particularly suitable for reducing the size of atherosclerotic plaques. As used herein, the term "atherosclerotic plaque" refers to an accumulation of cholesterol and triglycerides resulting from atherosclerosis.

[0012] As used herein, the terms “tryptophan hydroxylase” or “TPH” have their ordinary meaning in the art and refer to the enzyme (EC 1.14.16.4) involved in the synthesis of the neurotransmitter serotonin. TPH catalyzes the following chemical reaction: L-tryptophan + tetrahydrobiopterin + O2 ⇔ 5-hydroxytryptophan + dihydrobiopterin + H2O. It employs iron, an additional cofactor. In humans and other mammals, there are two separate TPH genes. In humans, these genes are located on chromosomes 11 and 12 and encode two different isoenzymes, TPH1 and TPH2 (sequence identity 71%) (Walther, Diego J., and Michael Bader. “A unique central tryptophan hydroxylase isoform.” Biochemical pharmacology 66.9 (2003): 1673-1680). TPH1 is expressed mostly in tissues that express serotonin (neurotransmitter) in the periphery (skin, gastrointestinal tract, pineal gland), although it is also expressed in the central nervous system. On the other hand, TPH2 is expressed only in neuronal cell types and is the dominant isoform in the central nervous system.

[0013] As used herein, the term “TPH1 inhibitor” is intended to encompass compounds that interact with TPH1 to substantially reduce or eliminate its catalytic activity, thereby increasing the concentration of its substrate. In particular, the term refers to a substance that reduces the amount of 5-hydroxytryptophan produced from tryptophan by TPH1 in a suitable assay compared to the amount of 5-hydroxytryptophan produced from tryptophan by TPH1 in the absence of the substance in the assay. Preferably, the reduction is at least about 10%. An assay for determining the level of TPH1 inhibition of an active substance is described in U.S. Patent Application Publication US2009 / 0029993. A TPH1 inhibitor can be any type of molecule that interferes with the activity of TPH1, for example, by reducing the transcription or translation of the TPH1-coding nucleic acid, or by inhibiting or blocking TPH1 activity, or both. Examples of TPH1 inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, TPH1-specific aptamers, anti-TPH1 antibodies, TPH1-binding fragments of anti-TPH1 antibodies, TPH1-binding small molecules, TPH1-binding peptides, and other polypeptides that specifically bind to TPH1 (including, but are not limited to, TPH1-binding fragments of one or more TPH1 ligands fused to one or more additional domains).

[0014] Examples of TPH1 inhibitors are well known in the art and include those disclosed in International Publication Nos. 2008073933, 2010056992, 2010062829, 2011053977, 2011056916, 2015035113, and WO2015075025.

[0015] Other examples of TPH1 inhibitors, - Camilleri, Michael. “LX-1031, a tryptophan 5-hydroxylase inhibitor that reduces 5-HT levels for the potential treatment of irritable bowel syndrome.” Idrugs: the Investigational Drugs Journal 13.12 (2010): 921-928. - Cianchetta, Giovanni, et al. “Mechanism of inhibition of novel tryptophan hydroxylase inhibitors revealed by co-crystal structures and kinetic analysis.” Current chemical genomics 4 (2010): 19. - Goldberg, Daniel R., et al. “Discovery of acyl guanidine tryptophan hydroxylase-1 inhibitors.” Bioorganic & Medicinal Chemistry Letters 26.12 (2016): 2855-2860. - Goldberg, Daniel R., et al. “Discovery of acyl guanidine tryptophan hydroxylase-1 inhibitors.” Bioorganic & Medicinal Chemistry Letters 26.12 (2016): 2855-2860. - Goldberg, Daniel R., et al. “Discovery of spirocyclic proline tryptophan hydroxylase-1 inhibitors.” Bioorganic & medicinal chemistry letters 26.4 (2016): 1124-1129. - Goldberg, Daniel R., et al. “Discovery of spirocyclic proline tryptophan hydroxylase-1 inhibitors.” Bioorganic & medicinal chemistry letters 26.4 (2016): 1124-1129. - Goldberg, Daniel R., et al. “Optimization of spirocyclic proline tryptophan hydroxylase-1 inhibitors.” Bioorganic & Medicinal Chemistry Letters 27.3 (2017): 413-419. - Liu, Qingyun, et al. “Discovery and characterization of novel tryptophan hydroxylase inhibitors that selectively inhibit serotonin synthesis in the gastrointestinal tract.” Journal of Pharmacology and Experimental Therapeutics 325.1 (2008): 47-55. - Liu, Qingyun, et al. “Discovery and characterization of novel tryptophan hydroxylase inhibitors that selectively inhibit serotonin synthesis in the gastrointestinal tract.” Journal of Pharmacology and Experimental Therapeutics 325.1 (2008): 47-55. - Pagire, Suvarna H., et al. “Identification of New Non-BBB Permeable Tryptophan Hydroxylase Inhibitors for Treating Obesity and Fatty Liver Disease.” Molecules 27.11 (2022): 3417. - Shi, Hailong, Yaya Cui, and Yifei Qin. “Discovery and characterization of a novel tryptophan hydroxylase 1 inhibitor as a prodrug.” Chemical biology & drug design 91.1 (2018): 202-212. - Specker, Edgar, et al. “Structure-based design of xanthine-benzimidazole derivatives as novel and potent tryptophan hydroxylase inhibitors.” Journal of Medicinal Chemistry 65.16 (2022): 11126-11149. This includes items listed below.

[0016] Specific examples of TPH1 inhibitors include the phenylalanine analogs p-chlorophenylalanine (phenclonin, PCPA) and p-ethynylphenylalanine (PEPA). Other specific examples include LP-923941, the active enantiomer of LP-521834;LP-534193;LP-533401, and its prodrugs LP-615819;LP-778902 or LX-1032, the prodrug of terotristat, or LP-920540;LX-1031; and LX-1033, the active enantiomers of terotristat ethyl. In some embodiments, the TPH1 inhibitor of the present invention is terotristat ethyl (also known as LX-1032 / LX-1606 / terotristat etiprate / Xermelo). In some embodiments, the TPH1 inhibitor of the present invention is KAR5417, and its oral prodrug KAR5585, also known as RVT-201 or rhodatristat ethyl.

[0017] In some embodiments, a TPH1 inhibitor is an inhibitor of TPH1 expression. “Inhibitor of expression” refers to a natural or synthetic compound that has a biological effect of inhibiting gene expression. In some embodiments, the inhibitor of gene expression is an siRNA, an antisense oligonucleotide, or a ribozyme. For example, antisense oligonucleotides, including antisense RNA and antisense DNA molecules, would act to directly block the translation of TPH1 mRNA by binding to them and thus inhibiting protein translation or increasing mRNA degradation, and thus reducing the level of TPH1 in the cell, and therefore its activity. For example, antisense oligonucleotides complementary to a specific region of the mRNA transcript sequence encoding TPH1, consisting of at least about 15 bases, can be synthesized, for example, by conventional phosphodiester techniques. Methods for using antisense techniques to specifically inhibit the gene expression of genes whose sequences are publicly known are well known in the art (see, for example, U.S. Patents 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as expression inhibitors for use in the present invention. TPH1 gene expression can be reduced (i.e., RNA interference or RNAi) by contacting a patient or cells with small double-stranded RNA (dsRNA) or a vector or construct that induces the production of small double-stranded RNA, so that TPH1 gene expression is specifically inhibited.

[0018] Those skilled in the art can easily determine the therapeutically effective dose of a TPH1 inhibitor to be administered to a given patient by taking into account factors such as the patient's size and weight; the degree of disease penetration; the patient's age, health status, and sex; the route of administration; and whether the administration is local or systemic. The effective dose of the compound may be based on the approximate or estimated body weight of the patient to be treated. Preferably, such an effective dose is administered parenterally or enterally as described herein. For example, the effective dose of the compound administered to a patient may range from about 5 to 10,000 micrograms / kg body weight, preferably about 5 to 3,000 micrograms / kg body weight, preferably about 700 to 1,000 micrograms / kg body weight, and more preferably above about 1,000 micrograms / kg body weight. Those skilled in the art can also easily determine an appropriate dosage regimen for administering the compound to a given patient. For example, the compound may be administered to the patient once (e.g., as a single injection or deposition).

[0019] The methods disclosed herein involve topical administration of TPH1 inhibitors to the intestines (i.e., the intestinal tract or colon). In some embodiments, the TPH1 inhibitors of the present invention are administered orally to the patient. In some embodiments, the TPH1 inhibitors of the present invention are administered rectally to the patient. Colonic drug delivery systems are well known in the art, including enemas; enema foams; and delayed oral-release formulations in the form of enteric-coated capsules that disintegrate at pH 7 in the terminal ileum.

[0020] The TPH1 inhibitor of the present invention is preferably formulated as a pharmaceutical composition before administration to a patient, according to techniques known in the art. The pharmaceutical composition of the present invention is characterized as being at least sterile and free of pyrogenic substances. As used herein, “pharmaceutical formulation” includes formulations for human and veterinary use. Methods for preparing the pharmaceutical composition of the present invention are within the scope of the skills in the art, for example, as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is incorporated herein by reference. The pharmaceutical formulation comprises a TPH1 inhibitor (e.g., 0.1 to 90% by weight) or a physiologically acceptable salt thereof, mixed with a pharmaceutically acceptable carrier. The pharmaceutical formulation of the present invention may also include a TPH1 inhibitor encapsulated in liposomes and a pharmaceutically acceptable carrier. Preferred pharmaceutically acceptable carriers include water, buffer water, ordinary saline, 0.4% saline, 0.3% glycine, hyaluronic acid, etc. The pharmaceutical compositions of the present invention may also include conventional pharmaceutically active ingredients and / or additives. Suitable pharmaceutically active ingredients include stabilizers, antioxidants, osmotic regulators, buffers, and pH adjusters. Suitable additives include, for example, the addition of physiologically biocompatible buffers (e.g., tromethamine hydrochloride), chelating agents (e.g., DTPA or DTPA-bisamide, etc.) or calcium chelate complexes (e.g., calcium DTPA, CaNaDTPA-bisamide, etc.), or optionally the addition of calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). The pharmaceutical compositions of the present invention may be packaged for use in liquid form or may be freeze-dried.

[0021] In some embodiments, the TPH1 inhibitor of the present invention is administered to the colon in the form of an enema formulation for rectal administration to the lower colon. A useful enema formulation comprises an effective amount of the TPH1 inhibitor dissolved or dispersed in a suitable fluid carrier vehicle such as water, alcohol, or aqueous alcohol solution. The carrier vehicle is preferably thickened with a natural or synthetic thickener such as gum, acrylate, or modified cellulose. The formulation may also contain an effective amount of a lubricant, such as a natural or synthetic fat or oil, i.e., trisglyceride or lecithin. Non-toxic nonionic surfactants may also be included as wetting and dispersing agents. The unit dose of the enema formulation may be administered from a pre-filled bag or syringe. The carrier vehicle may also contain an effective amount of a foaming agent, such as n-butane, propane, or i-butane. Such formulations may be delivered from a pre-loaded syringe pressurized container such that the vehicle is delivered to the colon as foam that inhibits its leakage from the target site.

[0022] In some embodiments, TPH1 inhibitors are administered via oral ingestion. An effective amount of TPH1 inhibitor may be locally administered to the patient's colon by oral ingestion of a unit dosage form, such as a pill, tablet, or capsule, containing an effective amount of TPH1 inhibitor that is enterically coated to be released from the unit dosage form within the patient's lower intestinal tract, for example, in the ileum and colon. The enteric coating remains intact in the stomach, but will dissolve and release the contents of the dosage form once it reaches a pH range that is optimal for the dissolution of the coating being used. The purpose of the enteric coating is to substantially delay the release of the TPH1 inhibitor until it reaches its target site of action in the ileum or colon. In particular, a useful enteric coating remains intact in the low pH environment of the stomach but dissolves immediately upon reaching the optimal dissolution pH for a given coating. This can vary between pH 3 and 7.5 depending on the chemical composition of the enteric coating. The thickness of the coating will depend on the dissolution characteristics of the coating material and the site to be treated. The most widely used polymer for enteric coatings is cellulose phthalate acetate (CAP). However, CAP has an optimal dissolution pH above 6, and therefore early drug release can occur. Another useful polymer is polyvinyl acetate phthalate (PVAP), which has low permeability to water and gastric juice, is more stable against hydrolysis, and dissolves at lower pH levels, which can also lead to early release of TPH1 inhibitors in the duodenum.

[0023] The present invention will be further illustrated by the following figures and embodiments. However, these embodiments and figures should not be construed as limiting the scope of the present invention. [Brief explanation of the drawing]

[0024] [Figure 1]Intestinal 5-HT plays an atherosclerotic role. A. 5-HT levels in small intestinal extracts (n=5 per group), B. Plaque size in the aortic sinus, C-D. Macrophage (MOMA-2+) and lymphocyte (CD3+) accumulation in plaques in the aortic sinus of Ldlr- / - mice that were injected daily with LP533401 (25 mg / kg) (n=6) or vehicle (n=8) and fed HFD+HCD for 8 weeks. Plaque size in the aortic sinus, lipocalin-2 (Lcn-2) levels in feces (n=5-10 per group), and histological analysis of the colon (n=5-11 per group) of Ldlr- / - IEC IDOKO mice and their littermates Ldlr- / - IEC IDO mice that were injected daily with E-G.LP533401 (25 mg / kg) or vehicle and fed HFD+HCD for 8 weeks. Individual data are presented as scatter point plots with mean and sem. **P<0.001, ***P<0.0001. [Figure 2] Effects of serotonin supplementation. A. Serum 5-hydroxytryptamine (HT) levels, B. Serum FITC-dextran levels, C. Plasma cholesterol, D. Representative images and plaque quantification in the aortic sinus (n=7-11 per group) of male ldlr- / - individuals fed a high-cholesterol diet (HCD) with or without 5-hydroxytryptophan (HTP) supplementation. E-F. Correlation between plaque size and serum FITC-dextran and serum 5-HT (Spearman correlation). Individual data are presented as scatter point plots with mean and sem. p-values ​​were determined using the two-sided Mann-Whitney test. **P<0.001, ***P<0.0001.

[0025] Examples: HFD has a significant effect on intestinal TRP catabolism in a mouse model of atherosclerosis. First, the inventors have identified low-density lipoprotein receptor (Ldlr) as a validated model of atherosclerosis. - / -We wanted to assess the effects of diet on the intestinal TRP catabolism pathway (i.e., the Kyn, 5-HT, and indole pathways) using mice. In particular, we investigated the effects of an arteriosclerosis-inducing high-cholesterol diet (HCD), as well as the effects of HCD, which has been previously shown to induce intestinal IDO activity in C57Bl / 6 mice (Laurans et al. 2018). For this purpose, Ldlr - / - Mice were fed either a normal diet (NCD), HFD, HCD, or a combination of HFD+HCD for 13 weeks. The inventors showed a significant increase in Kyn levels along with a decrease in intestinal Trp levels, and a substantial increase in intestinal IDO activity (assessed by Kyn / Trp ratio) was observed under HFD conditions (i.e., HFD, HFD+HCD) compared with NCD or HCD (data not shown). Consistent with previous reports (Laurans et al. 2018, Natividad et al. 2018), indole levels decreased under HFD conditions (with or without HCD) compared with HCD alone or NCD (data not shown). Intestinal 5-HT levels also decreased under HFD conditions (i.e., HFD, HFD+HC) compared with HCD or NCD (data not shown). These data indicate that HFD, not HCD, has a significant effect on intestinal TRP metabolism by promoting the Kyn pathway and adversely affecting the indole and 5-HT pathways.

[0026] Next, the inventors wanted to investigate the underlying mechanism of the increase in intestinal IDO activity observed under HFD conditions. Short-chain fatty acids (SCFAs) such as acetic acid and butyrate are the final products of anaerobic intestinal microbiota fermentation of dietary fiber (Schroeder et al. 2016). Recently, it has been shown that butyrate, a type of SCFA, negatively regulates IDO expression in IEC (Martin-Gallausiaux et al. 2018), suggesting a potential role of dietary fiber-inducible bacterial SCFA metabolites in regulating Trp metabolism. Consistent with previous reports, mice fed HFD (containing low levels of fiber) instead of HCD showed decreased levels of acetic acid and butyrate in their feces (data not shown). Interestingly, Ldlr fed HFD... - / - Supplementing mice with fiber as FOS (fructo-oligosaccharides) increased SCFA production, including acetic acid and butyrate (data not shown), leading to a significant decrease in fecal Kyn levels (data not shown) without a significant difference in fecal Trp levels, further suggesting the importance of fiber-mediated SCFA production in regulating intestinal IDO activity. In summary, the data indicate that feeding mice with HFD containing low levels of fiber, and therefore SCFAs including butyrate (a negative regulator of IEC IDO (Martin-Gallausiaux et al. 2018)), can at least partially explain the increase in intestinal IDO activity observed under these conditions.

[0027] IDO expressed in IEC plays a protective role in atherosclerosis under HFD conditions. The inventors then wanted to investigate the role of intestinal IDO in atherosclerosis. For this purpose, they used Ido-1 mice adjacent to loxP (Ido-1 flox / flox By crossing a mouse that expresses Cre recombinase under the control of a murine birin promoter (birin-cre+ / -), mice lacking IDO (Ido-1) were produced in the IEC. flox / flox We obtained bilin-cre. Next, the inventors obtained Ido-1 flox / floxBilirubin-cre (IEC IDOKO) mice and Ldlr - / - mice were mated. Male Ldlr - / - IEC IDOKO and littermate control mice (Ldlr - / - IEC IDO) were fed either NCD or HFD combined with HCD (named HFD + HCD) for 8 weeks to induce intestinal IDO activity and atherosclerosis, respectively. The absence of IDO in IEC led to a substantial decrease in Ido-1 mRNA expression in the small intestine compared to controls (data not shown), clearly demonstrating the importance of IDO expression in IEC in intestinal Trp metabolism. Furthermore, as shown above, Ldlr - / - IEC IDO mice fed HFD + HCD had an increase in intestinal IDO activity (assessed by the Kyn / Trp ratio) compared to Ldlr - / - IEC IDO mice fed NCD (data not shown). In addition, IDO activity (Kyn / Trp) in the small intestine was attenuated in the absence of IDO in IEC (data not shown). This was also true in circulation, assessed by an increase in the plasma Kyn / Trp ratio in HFD + HCD compared to NCD, with a marked decrease in plasma Kyn / Trp at the same level as in Ldlr - IEC IDOKO mice fed HFD + HCD (data not shown), highlighting the prominence of IEC IDO activity in contributing to whole-body Trp metabolism under HFD. It should be noted that the Kyn / Trp ratio was much higher in the small intestine than in the colon (data not shown), indicating the importance of the small intestine in contributing to the Kyn pathway. Notably, male Ldlr - / - IEC IDOKO mice fed HFD + HCD had no change in plasma cholesterol levels compared to littermate Ldlr - / - IEC IDO mice fed HFD + HCD and showed an increase in plaque size in the aortic sinus (data not shown), indicating that IEC IDO plays a protective role against atherosclerosis under HFD. ​

[0028] Next, the inventors wanted to know if the phenotype would be conserved under long-term HFD+HCD feeding. Therefore, male Ldlr - IEC IDOKO mice and their littermates / controls were fed HFD+HCD for 13 weeks. The inventors showed no significant difference in mouse body weight and observed metabolic parameters, including insulin resistance tests (ITT), oral glucose tolerance tests (OGTT), and insulin resistance index (HOMA-IR), between the two groups. Furthermore, the inventors found no significant differences in plasma cholesterol or plaque size (in the aortic sinus) between the two groups of mice after 13 weeks of HFD+HCD (data not shown). The absence of effect from long-term HFD+HCD feeding in males was likely due to a significant decrease in Ido-1 gene expression in the small intestine at 13 weeks compared to 8 weeks (data not shown). Next, the inventors examined plaque size in females with short-term (8 weeks) and long-term (13 weeks) HFD+HCD. In 8 weeks of HFD+HCD, despite a significant decrease in Ido-1 mRNA in the small intestine, the absence of IDO in the IEC did not significantly affect plaque size (in the aortic sinus) and plasma cholesterol levels (data not shown). We then analyzed atherosclerotic plaques after 13 weeks of HFD+HCD. No significant differences in body weight and ITT were observed between the two groups (data not shown). Notably, female mice with IDO deletion in the IEC had increased plaque size in the aortic sinus despite no significant change in plasma cholesterol levels (data not shown). Increased plaque size was also observed in the thoracic aorta (data not shown). Interestingly, Ido-1 mRNA increased at 13 weeks compared to 8 weeks of HFD+HCD feeding (data not shown), which may explain the effect of intestinal IDO on plaque size in females, not short-term but long-term, plaque size in atherosclerosis. In summary, the data demonstrate a protective role of IDO expressed in IEC against atherosclerosis in both males and females, in a sex-dependent temporal manner.

[0029] Consistent with the significant effect of HFD in inducing intestinal IDO activity, both male and female IEC IDO KO mice fed HCD alone showed no differences in aortic sinus plaque size or plasma cholesterol levels (data not shown), despite a marked decrease in Ido-1 mRNA in IEC IDOKO mice, further highlighting the importance of HFD in the intestinal IDO-mediated effects on atherosclerosis.

[0030] Intestinal IDO reduces local and systemic inflammation. Next, the inventors attempted to assess the mechanism explaining the atherosclerotic-inducing effect resulting from the absence of intestinal IDO. Gastrointestinal inflammation was associated with susceptibility to developing CVD (Cainzos-Achirica et al. 2020). This may be due to alterations in the intestinal barrier leading to increased permeability and translocation of microbial molecules such as lipopolysaccharides (LPS) into circulation, which in turn has been suggested to maintain a peripheral chronic inflammatory process (Cani et al. 2007). Interestingly, after 8 weeks of HFD+HCD feeding, male Ldlr showed increased vascular activity compared to controls. - / -IEC IDO KO mice exhibited broad characteristics of intestinal inflammation, including elevated levels of fecal lipocalin-2 (Lcn2), a highly sensitive marker of gastrointestinal inflammation (Chassaing et al. 2012), and histological scores of colon sections (data not shown). Furthermore, this was associated with increased intestinal expression of inflammatory factors such as tumor necrosis factor (TNF)α and interferon (IFN)γ, as well as chemokines and chemokine receptor coding genes XCL1, CXCR6, and CCR10 (data not shown). In addition, flow cytometry analysis of the small intestine showed a greater number of T cells (CD4+ and CD8+) without significant changes in T helper (Th) polarization, including T regulatory cells (Treg) expressing Th1-specific T-box transcription factor (T-bet), the RAR-associated orphan receptor gamma ROR-gt for Th17, and forkhead-box P3 (Foxp3) (data not shown). In addition, the inventors found a decrease in the expression of the tight junction occludin-1 gene (data not shown) and an increase in the level of serum antibodies against LPS (data not shown), suggesting increased intestinal permeability. Consistently, female Ldlr compared to controls fed HFD+HCD for 13 weeks showed increased intestinal permeability. - / - IEC IDOKO mice showed an increase in serum FITC-dextran 4,000 Da levels after oral forced administration (data not shown), further demonstrating increased intestinal permeability in the absence of intestinal IDO. Taken together, these data suggest increased gastrointestinal inflammation and altered intestinal permeability in the absence of intestinal IDO, which can lead to systemic inflammation.

[0031] Next, the inventors wanted to investigate systemic inflammation, particularly within atherosclerotic plaques. Compared with controls, male Ldlr - / - Analysis of lesion composition after 8 weeks of HFD+HCD in IEC IDOKO mice showed that CD3 + We identified a pro-inflammatory phenotype assessed by increased T cell accumulation (data not shown) and a large necrotic core (data not shown). MOMA2 +No differences in collagen content, as assessed by macrophages and Sirius Red staining, were observed between the two groups (data not shown).

[0032] To further evaluate whether the increase in T cells, known to be atherosclerotic (Taleb 2016), was involved in the atherosclerotic phenotype observed in IEC IDOKO mice, we developed a mouse model (Ldlr) lacking both intestinal IDO and lymphocytes. - / - Rag1 - / - We developed the IEC IDOKO mouse and used it in Ldlr - / - Rag1 - / - The mice were compared with IEC IDO littermates. Lymphocyte deficiency reversed the atherosclerotic-inducing effect observed in the absence of intestinal IDO, suggesting the involvement of T cells in this process (data not shown).

[0033] In summary, these data indicate that the absence of intestinal IDO is linked to gastrointestinal and plaque inflammation.

[0034] Intestinal 5-HT plays a role in inducing arteriosclerosis. The inventors then investigated 5-HT, which has been previously shown to play a detrimental role in other Trp-dependent pathways, particularly in several inflammatory diseases such as myocardial infarction (Mauler et al. 2019) and colitis (Ghia et al. 2009). After 8 weeks of dietary therapy, intestinal 5-HT levels did not change significantly in mice fed HFD+HCD compared to mice fed CD (data not shown). To investigate whether intestinal 5-HT is involved in atherosclerosis, the inventors specifically inhibited TpH1, which is involved in 5-HT production in the gastrointestinal tract (approximately 90% of total serotonin (Walther et al. 2003)). Ldlr mice were fed HFD+HCD treated with a TpH1 inhibitor (LP533401) for 8 weeks. - / -Mice exhibited a significant decrease in 5-HT production in the small intestine (Figure 1A) and in the blood (data not shown). Inhibition of TpH1 was associated with increased indole production, assessed by higher IAA and indole production, without any significant changes in intestinal IDO activity, assessed by the Kyn / Trp ratio (data not shown). This was accompanied by enhanced intestinal expression of the antimicrobial peptides regenerated islet-derived (Reg)3g, Reg3b, and the occludin-1 gene (data not shown). Notably, blockade of intestinal 5-HT production was associated with a significant reduction in plaque size in the aortic sinus (Figure 1B) and thoracic aorta (data not shown), without any changes in plasma cholesterol levels (data not shown). Furthermore, atherosclerotic plaques in the aortic sinus of TpH1 inhibitor-treated mice contained fewer inflammatory cells, including macrophages MOMA-2+ (Figure 1C) and CD3+ T cells (Figure 1D). In summary, these results indicate that intestinal 5-HT exerts pro-inflammatory and atherosclerotic effects.

[0035] Considering the observed atherosclerotic role of intestinal 5-HT, we hypothesized that a possible increase in 5-HT in the absence of intestinal IDO could explain the atherosclerotic phenotype in IEC IDOKO mice. Therefore, we investigated the effects of 8 weeks of HFD+HCD on male Ldlr - / - Intestinal 5-HT levels were examined in IEC IDOKO mice and littermates / controls. As expected, considering the availability of Trp for other catabolic pathways, the absence of IDO in IEC led to increased 5-HT production (data not shown). Interestingly, the inventors found a correlation between 5-HT levels in the small intestine and plaque size in these mice (data not shown). The inventors then sought to determine whether the gastrointestinal inflammation and plaque in the absence of intestinal IDO were attributable to the observed increase in 5-HT production. To this end, the inventors used male Ldlr mice fed HFD+HCD for 8 weeks. - / - IEC IDOKO and Ldlr - / -Inhibition of TpH1 in both IEC IDO mice. Consistent with the above results, Ldlr - / - IEC, IDO, and Ldlr - / - Inhibition of TpH1 in both IEC IDO KO mice led to a significant reduction in plaque size in the aortic sinus (Figure 1E) without any significant changes in plasma cholesterol levels (data not shown). Furthermore, TpH1 inhibition led to a reduction in intestinal inflammation observed in IEC IDO KO mice, as assessed by lower fecal Lcn2 levels (Figure 1F) and histological scores of colon sections (Figure 1G). HCD-fed male Ldlr - / - In mice, 5-HT supplementation increased intestinal permeability and enhanced atherosclerosis, as assessed by an increase in serum FITC-dextran 4kDa after oral forced administration, without any significant changes in plasma cholesterol levels between the two groups (Figure 2A-D). Consistent with the atherosclerotic role of 5-HT, we observed a significant correlation between blood 5-HT and plaque size in the aortic sinus (Figure 2E-F). TpH1 inhibition is associated with Ldlr - / - Although IEC IDO KO mice showed a significant reduction in plaque size, compared to littermates treated with a TpH1 inhibitor, Ldlr - / - The tendency towards larger plaque sizes persists in IEC IDO KO mice (Figure 1E), suggesting the involvement of other mechanisms that explain the atherosclerotic phenotype observed in IEC IDO KO mice.

[0036] Trp-dependent microbiome effects influence atherosclerosis. Accumulated evidence has shown that intestinal cells interact with the gastrointestinal microbiome along with immune cells to determine disease outcomes (Belkaid et al. 2014). Therefore, we hypothesized that intestinal IDO activity can form a gastrointestinal microbiome that may influence systemic inflammation and atherosclerosis. First, we fed male Ldlr with either HFD+HCD or CD for 8 weeks. - / -IEC IDOKO and Ldlr - / - The bacterial fecal composition of the microbiome was investigated using 16S rDNA sequencing of IEC IDO mice. Principal component analysis (PCA) based on genus composition revealed significant differences between mouse groups (data not shown). Consistent with the adverse effects of HFD on the microbiome, alpha-diversity analysis showed decreased diversity under HFD+HCD compared to CD, but no significant differences were observed according to mouse genotype (data not shown). Differences in microbiome composition were observed at the phylum level (data not shown). To address the importance of the microbiome, the inventors used a broad-spectrum antibiotic cocktail (ATB) supplemented in drinking water to study Ldlr, which was fed HFD+HCD. - / - IEC IDOKO and Ldlr - / - The gastrointestinal microbiome was depleted in IEC IDO male mice. Consistent with previous studies (Villette et al. 2020), microbiome depletion using ATB exacerbated atherosclerosis due to enhanced cholesterolemia (data not shown). Furthermore, ATB treatment affected Ldlr in male mice fed HFD+HCD. - / - Ldlr fed IEC IDO and HFD+HCD - / - We discarded the differences in plaque size and gastrointestinal inflammation assessed by Lcn-2 levels that had been previously observed between IEC IDOKO mice (data not shown). Next, we wanted to test whether the exchange of microbiomes between the two mouse genotypes affects atherosclerosis. For this purpose, we fed Ldlr mice HFD+HCD. - / - IEC, IDO, and Ldlr - / - IEC IDO KO mice were co-fed after weaning and compared with mice housed in cages isolated according to genotype. (Ldlr) - / - IEC IDO or Ldlr - / - Atherosclerotic plaque size in the aortic sinus (regardless of IEC IDO KO) was measured in Ldlr, which was housed in separate cages, with no significant change in plasma cholesterol levels between groups. - / -It is comparable to that of IEC IDO KO mice (data not shown), Ldlr - / - The dominant atherosclerotic-inducing effect of the microbiome derived from IEC IDO KO mice was demonstrated. Subsequently, since Trp is catabolized by indole metabolites produced by the microbiome, the inventors assessed whether the absence of IDO in IEC affects indole production. However, unexpectedly considering the availability of Trp, IEC IDOKO led to a decrease in indole production, as assessed by low levels of fecal IAA (data not shown), which may result from a decrease in bacteria that catabolize Trp into indole metabolites.

[0037] Trp is metabolized by gastrointestinal bacteria into indole derivatives, which activate the aryl hydrocarbon receptor (AhR) (Lamas et al. 2016). The inventors then isolated Ldlr by genotype. - / - IEC, IDO, and Ldlr - / - AhR agonists (IAA, indole-3-aldehyde (IAld), and tryptamine) were measured in the feces of IEC IDO KO mice, mice co-fed after weaning, and mice isolated by genotype but treated with ATB. - / Compared to IEC IDO mice, Ldlr - / - A decrease in AhR agonists was observed in IEC IDO KO mice, while this difference was discarded in co-bred mice. As expected, regarding bacterial indole derivative production, AhR agonists were compared with ATB-treated Ldlr - / - IEC IDO KO and Ldlr - / - It was significantly reduced in IEC IDO mice (data not shown).

[0038] To investigate the physiological importance of impaired microbiome AhR activity, Ldlr was fed HCD. - / -Mice were administered the AhR agonist 6-formylindro(3,2-b)carbazole (Ficz). Ficz treatment reduced atherosclerosis in the aortic sinus compared to the untreated counterpart, without any significant changes in plasma cholesterol levels (data not shown). This was associated with a decrease in CD3+ T cell accumulation in plaque, without any significant changes in macrophage MOMA-2+ surface (data not shown) (data not shown). In summary, these results highlight the importance of the microbiome and AhR-mediated effects in atherosclerosis.

[0039] References: Throughout this application, various references describe the state of the art to which the present invention relates. The disclosures of these references are incorporated herein by reference.

Claims

1. A method for treating atherosclerosis in a patient in need, comprising administering a therapeutically effective dose of a tryptophan hydroxylase 1 (TpH1) inhibitor to the patient.

2. The method according to claim 1, wherein a TPH1 inhibitor reduces the size of atherosclerotic plaques.

3. The method according to claim 1 or 2, comprising local administration of a TPH1 inhibitor to the intestine.

4. The method according to claim 3, wherein a TPH1 inhibitor is administered orally to the patient.

5. The method according to claim 3, wherein the TPH1 inhibitor of the present invention is administered rectally to a patient.

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