EP4739314A1 - Use of tryptophan hydroxylase 1 (tph1) inhibitors for the treatment of atherosclerosis - Google Patents

Abstract

Tryptophan (Trp) is one of the nine essential amino acids supplied by the diet, whose metabolism appears as a key metabolic gatekeeper of intestinal homeostasis, although its systemic effects, particularly on atherosclerosis, remain unknown. The inventors show that the high-fat diet (HFD) but not the high-cholesterol diet (HCD) increases intestinal indoleamine 2, 3-dioxygenase 1 (IDO) activity, the main enzyme involved in Trp catabolism in the gut, which shifts intestinal Trp metabolism from microbiota-produced indole metabolites and serotonin or 5-hydroxytryptamine (5-HT) production towards Kynurenine production. Most importantly the inventors showed that inhibition of tryptophan hydroxylase 1 (TpH1), markedly reduces intestinal 5-HT production and alleviates atherosclerosis as well as plaque inflammation. Accordingly, the present invention relates to the use of tryptophan hydroxylase 1 (TpH1) inhibitors for the treatment of atherosclerosis.

Description

USE OF TRYPTOPHAN HYDROXYLASE 1 (TPH1) INHIBITORS FOR THE TREATMENT OF ATHEROSCLEROSIS

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular cardiovascular diseases.

BACKGROUND OF THE INVENTION:

The gut has become recognized as an important link between diet and cardiovascular diseases (CVDs), including atherosclerosis (Cainzos-Achirica et al. 2020). Particularly, the gut microbiota has been identified as a potential mediator that could impact atherosclerosis. Although evidence has been put forth to account for the relationship between gut inflammation and CVDs, little is known about the mechanisms whereby the intestine might contribute to atherosclerosis. Particularly, the gut metabolism could account for the links between local disruption of gut homeostasis and systemic development of atherosclerosis.

Tryptophan (Trp) is one of the essential amino acids supplied by the diet, whose metabolism appears as a key 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(Zc7a///c et al. 2013, Gao et al. 2018). In mice, dietary lack of Trp leads to impaired intestinal immunity and gut microbiota dysbiosis, which causes intestinal inflammation and diarrhea (Hashimoto et al. 2012). In humans, patients with intestinal bowel disease (IBD) exhibit disturbed tryptophan metabolism, likely due to altered gut microbiota (Lamas et al. 2016). However, despite the importance of intestinal Trp metabolism in gut homeostasis along with the ascertainment that intestinal inflammation is associated with CVD, the systemic impact of gut Trp metabolism on atherosclerosis is still unclear.

Under homeostatic conditions, Trp catabolism in the intestine follows three pathways, which consist in the Kynurenine (Kyn) pathway mainly in intestinal epithelial cells (TECs) via IDO (~95 % of Trp); the gut microbiota pathway of direct transformation of Trp into indole metabolites (~5 % of Trp); and the serotonin or 5-hydroxytryptamine (5-HT) pathway in enterochromaffin cells (EC) via Trp hydroxylase 1 (TpHl) (~l-2 % of Trp). This represents the majority of body 5-HT production (~90 % of 5-HT). We have previously shown a critical role for the Trp-degrading enzyme, indoleamine 2, 3- dioxygenase 1 (IDO) in the fine-tuning of intestinal Trp metabolism under the obesogenic high- fat diet (HFD), with major consequences on metabolic syndrome (Laurans et al. 2018). However, the specific role of intestinal IDO in cardiometabolic diseases, including atherosclerosis, is still unknown. Moreover, although recent studies highlighted the local effects of Trp-derived metabolites in intestinal homeostasis, their systemic impact on atherosclerosis is poorly known.

SUMMARY OF THE INVENTION:

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

DETAILED DESCRIPTION OF THE INVENTION:

The present invention relates to a method of treating atherosclerosis in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a tryptophan hydroxylase 1 (TpHl) inhibitor.

As used herein, the term “patient” or “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the patient is a human. In some embodiments, the patient is a human who is susceptible to have atherosclerosis as described above.

As used herein, the term “atherosclerosis” refers to the pathologic processes that leads to abnormal accumulation of cholesterol and cholesteryl esters and related lipids in macrophages, smooth muscle cell and other types of cells leading to narrowing and/or occlusion of one or several arteries and arterioles of the body and bodily organs, including but not limited to, the coronary arteries, aorta, renal arteries, corotid arteries, and arteries supplying blood to the limbs and central nervous system. The associated inflammatory reactions and mediators of this pathologic process also are included in this definition.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a 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 therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In particular, the TPH1 inhibitor of the present invention is particularly suitable for reducing the size of the atherosclerotic plaques. As used herein, the term “atherosclerotic plaque” refers to the build up of cholesterol and triglycerides due to atherosclerosis.

As used herein, the term “tryptophan hydroxylase” or “TPH” has its general meaning in the art and refers to an enzyme (EC 1.14.16.4) involved in the synthesis of the neurotransmitter serotonin. TPH catalyzes the following chemical reaction L-tryptophan + tetrahydrobiopterin + 02 ^Arightleftharpoons 5-Hydroxytryptophan + dihydrobiopterin + H20. It employs one additional cofactor, iron. In humans, as well as in other mammals, there are two distinct TPH genes. In humans, these genes are located on chromosomes 11 and 12 and encode two different homologous enzymes 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 mostly expressed in tissues that express serotonin (a neurotransmitter) in the periphery (skin, gut, pineal gland) but it is also expressed in the central nervous system. On the other hand, TPH2 is exclusively expressed in neuronal cell types and is the predominant isoform in the central nervous system.

As used herein, the term “TPH1 inhibitor” is intended to encompass a compound that interacts with TPH1 to substantially reduce or eliminate its catalytic activity, thereby increasing the concentrations of its substrate(s). In particular, the term refers to a substance that reduces the amount of 5-hydroxytryptophan produced from tryptophan by TPH1 in a suitable assay, as compared to the amount of 5-hydroxytryptophan produced from tryptophan by TPH1 in the assay in the absence of the substance. Preferably, the decrease is at least about 10%. Assays for determining the level of TPH1 inhibition of an agent are described in U.S. Patent Application Publication US 2009/0029993. A TPH1 inhibitor can be a molecule of any type that interferes with the activity of TPH1 for example, either by decreasing transcription or translation of TPHl-encoding 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-TPHl antibodies, TPH1- binding fragments of anti-TPHl antibodies, TPH1 -binding small molecules, TPH1 -binding peptides, and other polypeptides that specifically bind TPH1 (including, but not limited to, TPH1 -binding fragments of one or more TPH1 ligands, optionally fused to one or more additional domains), such that the interaction between the TPH1 inhibitor and TPH1 results in a reduction or cessation of TPH1 activity or expression.

Examples of TPH1 inhibitors are well known in the art and include those disclosed in W02008073933, W02010056992, W02010062829, WO2011053977, WO2011056916, W02015035113, and W02015075025.

Other examples of TPH1 inhibitors include those described in:

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, Suvama 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., and,

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.

Specific examples of TPH1 inhibitors include the phenylalanine analogs p-chlorophenylalanine (Fenclonine, PCPA) and p-ethynylphenylalanine (PEPA). Other specific examples include LP- 521834; LP-534193; LP-923941, the active enantiomer of LP-533401 and its prodrug LP- 615819; LP-920540, the active enantiomer of LX-1032 or telotristat ethyl, which is the prodrug ofLP-778902 or telotristat; LX-1031; and LX-1033). In some embodiments, the TPH1 inhibitor of the present invention is telotristat ethyl (also known as LX-1032/LX-1606/telotristat etiprate/Xermelo). In some embodiments, the TPH1 inhibitor of the present invention is KAR5417 and its oral prodrug KAR5585 also knowns as RVT-201 or rodatristat ethyl. In some embodiments, the TPH1 inhibitor is an inhibitor of TPH1 expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of TPH1 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of TPH1, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding TPH1 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 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 inhibitors of expression for use in the present invention. TPH1 gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that TPH1 gene expression is specifically inhibited (i.e. RNA interference or RNAi).

One skilled in the art can readily determine a therapeutically effective amount of the TPH1 inhibitor to be administered to a given patient, by taking into account factors such as the size and weight of the patient; the extent of disease penetration; the age, health and sex of the patient; the route of administration; and whether the administration is regional or systemic. An effective amount of said compound can be based on the approximate or estimated body weight of a patient to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described herein. For example, an effective amount of the compound administered to a patient can range from about 5-10000 micrograms/kg of body weight and is preferably between about 5-3000 micrograms/kg of body weight, and is preferably between about 700- 1000 micrograms/kg of body weight, and is more preferably greater than about 1000 micrograms/kg of body weight. One skilled in the art can also readily determine an appropriate dosage regimen for the administration of the compound to a given patient. For example, the compound can be administered to the patient once (e.g., as a single injection or deposition). The methods herein disclosed include the local administration to the bowel (i.e. intestine or colon) of the TPH1 inhibitor. In some embodiments, the TPH1 inhibitor of the present invention is orally administered to the patient. In some embodiments, the TPH1 inhibitor of the present invention is rectally administered to the patient. Colonic drug delivery systems are well known in the art, including enemas; rectal foam and delayed oral release formulations in the form of enteric-coated capsules which disintegrate at pH 7 in the terminal ileum.

The TPH1 inhibitors of the present invention are preferably formulated as pharmaceutical compositions, prior to administering to a patient, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the present invention are within the skill 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 herein incorporated by reference. The present pharmaceutical formulations comprise TPH1 inhibitor (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier. The pharmaceutical formulations of the present invention can also comprise TPH1 inhibitor which are encapsulated by liposomes and a pharmaceutically-acceptable carrier. Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. Pharmaceutical compositions of the present invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the present invention can be packaged for use in liquid form or can be lyophilized.

In some embodiments, the TPH1 inhibitor of the present invention is administered to the colon in the form of an enema formulation, which is rectally administered to the lower colon. Useful enema formulations comprise an effective amount of the TPH1 inhibitor dissolved or dispersed in a suitable flowable carrier vehicle, such as water, alcohol or an aqueous-alcoholic fluid. The carrier vehicle is preferably thickened with natural or synthetic thickeners such as gums, acrylates or modified celluloses. The formulation can also comprise an effective amount of a lubricant such as a natural or synthetic fat or oil, i.e., a tris-fatty acid glycerate or lecithin. Nontoxic nonionic surfactants can also be included as wetting agents and dispersants. Unit dosages of enema formulations can be administered from prefilled bags or syringes. The carrier vehicle may also comprise an effective amount of a foaming agent such as n-butane, propane or i-butane. Such formulations can be delivered from a preloaded syringe pressurized container, so that the vehicle is delivered to the colon as a foam, which inhibits its escape from the target site.

In some embodiments, the TPH1 inhibitor is administered via oral ingestion. The effective amount of the TPH1 inhibitor can be locally administered to the colon of the patient by oral ingestion of a unit dosage form such as a pill, tablet or capsule, comprising an effective amount of the TPH1 inhibitor which is enterically coated so as to be released from the unit dosage form in the lower intestinal tract, e.g., in the ileum and in the colon of the patient. Enteric coatings remain intact in the stomach, but will dissolve and release the contents of the dosage form once it reaches the region where the pH is optimal for dissolution of the coating used. The purpose of an 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 is one that remains intact in the low pH environment of the stomach, but readily dissolved when the optimum dissolution pH of the particular coating is reached. This can vary between pH 3 to 7.5 depending upon the chemical composition of the enteric coating. The thickness of the coating will depend upon the solubility characteristics of the coating material and the site to be treated. The most extensively used polymer for enteric coating is cellulose acetate phthalate (CAP). However, CAP has an optimum dissolution pH greater than 6, thus early drug release may occur. Another useful polymer is polyvinyl acetate phthalate (PVAP) which is less permeable to moisture and gastric fluid, more stable to hydrolysis and able to dissolve at a lower pH, which could also result in early release of the TPH1 inhibitor in the duodenum.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES: Figure 1. Intestinal 5-HT exerts a pro-atherogenic role. A. 5-HT levels in the extracts of the small intestine (n=5 per group), B. plaque size in the aortic sinus, C-D. macrophage (MOMA- 2+) and lymphocyte (CD3+) accumulation within plaques in the aortic sinus of Ldlr-/- mice daily injected with LP533401 (25mg/kg) (n=6) or vehicle (n=8) and fed HFD+HCD for 8 weeks. E-G. plaque size in the aortic sinus, lipocalin-2 (Lcn-2) levels in feces (n=5-10 per group), and histological analysis in the colons (n=5-l 1/per group) of Ldlr-/- IEC IDOKO mice and littermates Ldlr-/- IEC IDO daily injected with LP533401 (25mg/kg) or vehicle and fed HFD+HCD for 8 weeks. Individual data are presented as scattered dot plots, with the mean and s.e.m. **P<0.001, ***P<0.0001.

Figure 2: effects of serotonin supplementation. A. blood 5-hydroxytryptamine (HT) levels, B. serum FITC-dextran levels, C. plasma cholesterol, D. representative images and plaque quantifications in the aortic sinus of male ldlr ' fed high-cholesterol diet (HCD) supplemented or not with 5-hydroxytryptophan (HTP) (n=7-l 1 per group). E-F. correlations between plaque size with serum FITC-dextran and blood 5-HT (Spearman correlation). Individual data are presented as scattered dot plots, with the mean and s.e.m. The p values were determined using the two-tailed Mann-Whitney test. ** <0.001, ***P<0.0001.

EXAMPLE:

HFD has a major effect on intestinal Trp catabolism in a mouse model of atherosclerosis First, we wanted to assess the effect of the diet on intestinal Trp catabolic pathways (i.e. Kyn, 5-HT, and indole pathway) using low-density lipoprotein receptor Ldlr⁷) mice, as a validated model of atherosclerosis. In particular, we examined the effect of the pro-atherogenic, high- cholesterol diet (HCD) as well as the effect of HFD, which has previously been shown to induce intestinal IDO activity in C57B1/6 mice(Laurans et al. 2018). To this purpose, Zt//r^/_mice were fed with either a normal chow diet (NCD), HFD, HCD, or the combination of HFD+HCD, for 13 weeks. We showed that intestinal Trp levels decrease along with a marked increase in Kyn levels, indicating a substantial increase in intestinal IDO activity (as assessed by Kyn/Trp ratio), under HFD conditions (i.e. HFD, HFD+HCD) compared to NCD or HCD (data not shown). In agreement with previous reports(Laurans et al. 2018, Natividad et al. 2018), indole levels decreased under HFD conditions (with or without HCD) compared to HCD alone or NCD (data not shown). Intestinal 5-HT levels also decreased in HFD conditions (i.e. HFD, HFD+HC) compared to HCD or NCD (data not shown). These data indicate that HFD rather than HCD, has a major effect on intestinal Trp metabolism through promoting the Kyn pathway to the detriment of the pathways of indole and 5-HT. Then we wanted to investigate the mechanisms underlying the observed increase in intestinal IDO activity under HFD. Short-chain fatty acids (SCFAs), such as acetate and butyrate, are the end products of the fermentation of dietary fibers by the anaerobic intestinal microbiota(Schroeder et al. 2016). Recently, it has been shown that butyrate, one of the SCFA, negatively regulates IDO expression in IECs(Martin-Gallausiaux et al. 2018), suggesting a potential role of dietary fibers-induced bacterial SCFA metabolites in the regulation of Trp metabolism. In agreement with previous reports, mice fed HFD (containing a low level of fibers) but not HCD, exhibited a decrease in acetate and butyrate levels in feces (data not shown). Interestingly, the supplementation with fibers as FOS (fructo-oligosaccharids) oLdlr ^/_mice fed HFD, increased SCFAs production including acetate and butyrate (data not shown) and led to a significant decrease in fecal Kyn levels without significant differences in fecal Trp levels (data not shown), further suggesting the importance of fibers-mediated SCFA production in the regulation of the intestinal IDO activity. Taken together, the data indicate that feeding mice with HFD, which contains a low levels of fibers, and therefore SCFAs including that of butyrate (a negative regulator of IEC IDO(Martin-Gallausiaux et al. 2018), could explain, at least in part, the observed increase in intestinal IDO activity under this condition.

IDO expressed in lECs has a protective role in atherosclerosis under HFD

We next wanted to explore the role of intestinal IDO in atherosclerosis. For this, mice devoid of IDO in lECs (Ido-l^Jk‘^:'^:Jk‘^:'^: villin-cre) were obtained by crossing the /ox -flanked Ido-1 mice (Ido-l^flox/flox) with mice expressing Cre recombinase under the control of the murine villin promoter (yillin-cre+/-). Then, we crossed Ido- ^ox/^^ox Villin-cre (IEC IDOKO) mice withZ /r mice. Male Ldlr IEC IDOKO and the littermate control mice (Ldlr ~ IEC IDO) were fed for 8 weeks with either NCD or HFD combined with HCD (named HFD+HCD) to induce, respectively, intestinal IDO activity and atherosclerosis. The absence of IDO in lECs led to a substantial decrease in Ido-1 mRNA expression in small intestines compared to controls (data not shown), underscoring the importance of IDO expression in lECs in intestinal Trp metabolism. Moreover, as shown above, Ldlr IEC IDO mice fed HFD+HCD had increased intestinal IDO activity (as assessed by Kyn/Trp ratio) compared to Ldlr ~ IEC IDO mice fed NCD (data not shown). In addition, IDO activity (Kyn/Trp) in small intestines was dampened in absence of IDO in lECs (data not shown). This was also true in the circulation as assessed by an increase in plasma Kyn/Trp ratio in HFD+HCD compared to NCD, and a marked decrease in plasma Kyn/Trp was observed in Ldlr IEC IDOKO mice fed HFD+HCD at the same level as Ldlr IEC IDOKO mice fed NCD (data not shown), emphasizing the prominence of IEC IDO activity in the contribution to the systemic Trp metabolism under HFD. Of note, Kyn/Trp ratio was much higher in the small intestines than in the colons (data not shown), indicating the importance of the small intestine for contributing to the Kyn pathway. Remarkably, male Ldlr ' IEC IDOKO mice fed HFD+HCD exhibited an increase in plaque size in the aortic sinus compared to the littermate Ldlr ' IEC IDO mice fed HFD+HCD without any changes in plasma cholesterol levels (data not shown), indicating that IEC IDO exerts a protective role against atherosclerosis under HFD.

Then we wanted to know whether the phenotype was conserved under a long time of HFD+HCD feeding. Thus, male Ldlr IEC IDOKO mice and their littermate controls were fed HFD+HCD for 13 weeks. We showed no major differences in mouse body weight, and metabolic parameters including insulin tolerance test (ITT), oral glucose tolerance test (OGTT) and insulin-resistance index (HOMA-IR) were observed between the two groups. Moreover, we found no significant differences in plasma cholesterol and plaque size (in the aortic sinus) between the two groups of mice, after 13 weeks of HFD+HCD (data not shown). The absence of effects at a long time of HFD+HCD feeding in males was likely due to a significant decrease in Ido- 1 gene expression in small intestines at 13 weeks compared to 8 weeks (data not shown). Then we examined plaque size in females at short (8 weeks) and long time (13 weeks) of HFD+HCD. At 8 weeks of HFD+HCD, the absence of IDO in lECs did not significantly impact plaque size (in the aortic sinus) and plasma cholesterol levels, despite a significant decrease in Ido-1 mRNA in small intestines (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 2 groups (data not shown). Remarkably, female mice with IDO deletion in lECs had increased plaque size in the aortic sinus despite no significant changes in plasma cholesterol levels (data not shown). The increase in plaque size was also observed in the thoracic aorta (data not shown). Interestingly, Ido-1 mRNA was increased at 13 weeks compared to 8 weeks of HFD+HCD feeding (data not shown), which may explain the impact of intestinal IDO on plaque size at the long but not at the short time of atherosclerosis, in femal es. Taken together, the data show a protective role of IDO expressed in IEC on atherosclerosis in both males and females in a sex-dependent time manner.

In agreement with a major effect of HFD in inducing intestinal IDO activity, both male and female IEC IDO KO mice fed with only HCD did not display any differences in plaque size in the aortic sinus and plasma cholesterol levels despite a marked decrease m Ido-1 mRNA in IEC IDOKO (data not shown), further pointing to the importance of HFD in intestinal IDO- mediated effects on atherosclerosis.

Intestinal IDO decreases local and systemic inflammation

Then we sought to assess the mechanisms that accounted for the pro-atherogenic effects due to the absence of intestinal IDO. Gut inflammation was associated with susceptibility to develop CVDs(Cainzos-Achirica et al. 2020). This may be due to alterations of the intestinal barrier, leading to enhanced permeability and translocation of microbial molecules such as lipopolysaccharides (LPS) to the circulation, which in turn, has been suggested to sustain the peripheral chronic inflammatory processes(Cani et al. 2007). Interestingly, after 8 weeks of HFD+HCD feeding, male Ldlr ’ TEC IDO KO mice compared to controls displayed a range of features of intestinal inflammation including elevated levels of fecal lipocalin-2 (Lcn2), a sensitive marker for gut inflammation (Chassaing et al. 2012), as well as, a histological scoring of colonic sections (data not shown). Moreover, this was associated with increased intestinal expression of inflammatory factors such as tumor necrosis factor (TNF)a and interferon (IFN)y as well as the chemokine and chemokine receptors-encoding genes XCL1, CXCR6, and CCR10 (data not shown). Also, flow cytometry analysis within the small intestine showed a higher number of T cells CD4+ and CD8+ cells (data not shown), without significant changes in T helper (Th) polarizations, including Th 1 -specific T box transcription factor (T-bet) and RAR- related orphan receptor gamma ROR-gt for Thl7 as well as T regulatory cells (Treg) expressing forkhead box P3 (Foxp3) (data not shown). In addition, we found a decrease in the expression of the tight junction occludin-1 gene (data not shown) as well as elevated levels of serum antibodies to LPS (data not shown), suggesting increased intestinal permeability. Consistently, female Ldlr EC IDOKO KO mice compared to controls fed HFD+HCD for 13 weeks, showed increased serum FITC-dextran 4,000 Da levels following oral gavage (data not shown), further indicating an increase in intestinal permeability in absence of intestinal IDO. Collectively, these data indicate an increase in gut inflammation and alteration in intestinal permeability in the absence of intestinal IDO, which may cause systemic inflammation.

Then, we wanted to examine systemic inflammation, particularly within the atherosclerotic plaques. Analysis of lesion composition after 8 weeks of HFD+HCD in male Ldlr ' IEC IDOKO mice compared to the controls revealed a pro-inflammatory phenotype as assessed by increased accumulation of CD3⁺ T cells (data not shown), and large necrotic cores (data not shown). No differences in M0MA2⁺ macrophages and collagen content as assessed by Sirius Red staining were observed between the 2 groups (data not shown).

To further evaluate whether increased T cells known to be pro-atherogenic (Taleb 2016), was involved in the pro-atherogenic phenotype observed in IEC IDOKO mice, we developed a mouse model deficient for both intestinal IDO and lymphocytes (Ldlr^Ragf^IEC IDOKO mice) and compared them to Ldlr^Ragf^IEC IDO littermate mice. Deficiency in lymphocytes abrogated the pro-atherogenic effects observed in the absence of intestinal IDO, suggesting the involvement of T cells in this process (data not shown).

Taken together these data indicate that the absence of intestinal IDO leads to gut and plaque inflammation.

Intestinal 5-HT exerts a pro-atherogenic role

We then investigated the other Trp-dependent pathways, particularly 5-HT, which was previously shown to exert a deleterious role in several inflammatory diseases such as myocardial infarction (Mauler et al. 2019) and colitis (Ghia et al. 2009). Intestinal 5-HT levels were not significantly changed in mice fed HFD+HCD compared to mice fed CD, after 8 weeks of diet (data not shown). To examine whether intestinal 5-HT is involved in atherosclerosis, we specifically inhibited TpHl, which is responsible for 5-HT production within the gut (-90% of total serotonin (Walther et al. 2003)). Ldlr mice fed HFD+HCD for 8 weeks treated with TpHl inhibitor (LP533401) exhibited a marked decrease in 5-HT production in the small intestine (Figure 1A) as well as in the blood (data not shown). The inhibition of TpHl is associated with an increase in indole production, as assessed by higher IAA and indole production, without any significant changes in intestinal IDO activity as assessed by Kyn/Trp ratio (data not shown). This is accompanied by an enhanced intestinal expression of antimicrobial peptides, regenerating islet-derived (Reg)3g, Reg3b as well as occludin-1 genes (data not shown). Remarkably, blockage of intestinal 5-HT production was associated with an important reduction in plaque size in the aortic sinus (Figure IB) and in the thoracic aorta (data not shown), without any changes in plasma cholesterol levels (data not shown). Moreover, atherosclerotic plaques within the aortic sinus of TpHl inhibitor-treated mice, contain fewer inflammatory cells including macrophages M0MA-2+ (Figure 1C) and CD3+ T cells (Figure ID). Taken together, these results indicate that intestinal 5-HT exerts pro-inflammatory and pro-atherogenic effects. Considering the observed pro-atherogenic role of intestinal 5-HT, we hypothesized that the possible increase in 5-HT in the absence of intestinal IDO may explain the pro-atherogenic phenotype of IEC IDOKO mice. We, therefore, examined intestinal 5-HT levels in male Ldlr ~ IEC IDOKO mice and littermate controls after 8 weeks of HFD+HCD. As expected, considering the availability of Trp for the other catabolic pathways, the absence of IDO in lECs led to an increase in 5-HT production (data not shown). Interestingly, we found a correlation between 5-HT levels within the small intestine and plaque size in these mice (data not shown). Then we seek to know whether inflammation within the gut and plaques in the absence of intestinal IDO was due to the observed increase in 5-HT production. To this end, we inhibited TpHl in both male Ldlr ~ IEC IDOKO and Ldlr ~ IEC IDO mice fed HFD+HCD for 8 weeks. In agreement with the above results, TpHl inhibition in both Ldlr ~ IEC IDO and Ldlr ~ IEC IDO KO mice led to a significant decrease in plaque size in the aortic sinus (Figure IE), without any significant changes in plasma cholesterol levels (data not shown). Moreover, TpHl inhibition led to a decrease in intestinal inflammation observed in IEC IDOKO mice, as assessed by lower fecal Lcn2 levels (Figure IF) and histological scoring (Figure 1G) of colonic sections. 5-HT supplementation in HCD-fed male Ldlr '~ mice increased intestinal permeability, as assessed by augmented serum FITC-dextran 4KDa after oral gavage, and enhanced atherosclerosis, without any significant changes in plasma cholesterol levels between the 2 groups (Figure 2A-D). In agreement with the 5-HT pro-atherogenic role, we observed a significant correlation between blood 5-HT with plaque size in the aortic sinus (Figure 2E-F). Although TpHl inhibition significantly reduced plaque size in Ldlr ~ IEC IDO KO mice, there is still a trend towards higher plaque size in Ldlr ^ IEC IDO KO mice treated with TpHl inhibitor compared to littermate controls treated with TpHl inhibitor (Figure IE), suggesting the involvement of other mechanisms explaining the pro-atherogenic phenotype observed in IEC IDO KO mice.

Trp-dependent microbiota effects impact atherosclerosis

Accumulating evidence showed that intestinal cells along with immune cells interact with gut microbiota to determine disease outcomes (Belkaid et al. 2014). We, therefore, hypothesized that intestinal IDO activity may shape gut microbiota that can impact systemic inflammation and atherosclerosis. We first explored the bacterial fecal composition of the microbiota by the use of 16S rDNA sequencing of male Ldlr ~ IEC IDOKO and Ldlr ~ IEC IDO mice fed either HFD+HCD or CD for 8 weeks. Principal component analysis (PCA) based on genus composition revealed significant differences between the groups of mice (data not shown). In agreement with the deleterious effects of HFD on microbiota, the alpha-diversity analysis showed decreased diversity under HFD+HCD compared to CD, but no significant differences were observed according to the mice genotypes (data not shown). At phylum levels, there are differences in microbiota composition (data not shown). To address the importance of the microbiota, we depleted gut microbiota in Ldlr ~ IEC IDOKO and Ldlr ~ IEC IDO male mice fed HFD+HCD using a broad spectrum antibiotic cocktail (ATB) supplemented in drinking water. In agreement with previous studies (Villette et al. 2020), depletion of microbiota with ATB aggravated atherosclerosis due to enhanced cholesterolemia (data not shown). Moreover, ATB treatment abrogated the differences in plaque size as well as gut inflammation as assessed by Lcn-2 levels, previously seen between male HFD+HCD-fed Ldlr^ IEC IDO and HFD+HCD-fed Ldlr ^ IEC IDOKO mice (data not shown). Then, we wanted to test whether microbiota exchange between the two mouse genotypes impacts atherosclerosis. To this end, HFD+HCD-fed Ldlr ~ IEC IDO and Ldlr ~ IEC IDO KO mice were co-housed after weaning and compared to mice housed in cages separated by genotype. Atherosclerosis plaque size in the aortic sinus of co-housed animals (whether Ldlr ^ IEC IDO or Ldlr ^ IEC IDO KO) was similar to those of Ldlr ^ IEC IDO KO mice housed in separate cages without significant changes in plasma cholesterol levels between the groups (data not shown), indicating a dominant pro-atherogenic effect of microbiota from Ldlr ~ IEC IDO KO mice. Then, as Trp is catabolized through microbiota-generated indole metabolites, we assessed whether the absence of IDO in IEC impacts 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 likely decrease in bacteria-catabolizing Trp to indole metabolites.

Trp is metabolized by gut bacteria into indole derivatives that activate aryl hydrocarbon receptor (AhR)(Lamas et al. 2016). We then measured AhR agonists (IAA, Indole-3 -aldehyde (lAld), and tryptamine) in feces of Ldlr ^ IEC IDO and Ldlr ^ IEC IDO KO mice separated by genotype and mice co-housed after weaning as well as in mice separated by genotype but treated with ATB. A decrease in AhR agonists was observed in Ldlr ^ IEC IDO KO compared to Ldlr ' IEC IDO mice, whereas this difference was abrogated in the cohoused mice. Expectedly regarding the production of indole derivative by bacteria, AhR agonists markedly decreased in Ldlr ~ IEC IDO KO and Ldlr ~ IEC IDO mice treated with ATB (data not shown). To investigate the physiological importance of impaired microbiota AhR activity, 6- formylindolo(3,2-b)carbazole (Ficz), an AhR agonist, was administered to HCD-fed Ldlr^ mice. The Ficz treatment, as compared to the untreated counterparts alleviated atherosclerosis in the aortic sinus without any significant changes in plasma cholesterol levels (data not shown). This was associated with a decrease in CD3+ T cell accumulation within plaques (data not shown), without significant changes in macrophage M0MA-2+ surface (data not shown). Taken together, these results pointed to the importance of microbiota and AhR-mediated effects in atherosclerosis. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of treating atherosclerosis in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a tryptophan hydroxylase 1 (TpHl) inhibitor.

2. The method of claim 1 wherein the TPH1 inhibitor reduces the size of the atherosclerotic plaques.

3. The method of claim 1 or 2 that involves the local administration to the bowel of the TPH1 inhibitor.

4. The method of claim 3 wherein the TPH1 inhibitor is orally administered to the patient.

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