WO2021130232A1 - Bile acid derivatives in the medical intervention of anxieties and/or stress symptoms - Google Patents

Abstract

The invention relates to nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use in the treatment or the prevention of a behavioral disorder. Equally, the invention relates to method of treatment of a behavioral disorder comprising the step of administering to a patient in need of medical intervention nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof. For example, the behavioral disorder may be selected from the group consisting of an anxiety disorder, a depression-disorder/a depression-related symptom, a mood disorder and/or stress/stress symptoms. The invention may further comprise a non-medical use of nor-ursodeoxycholic acid, a prodrug or an ester thereof or an acceptable salt thereof as mood enhancer, as mood alleviator, as relaxation agent, as stress releasing agent, as weight-reduction supplement and/or for the amelioration of a depressed mental state. Particularly, said nor-ursodeoxycholic acid may be 24-nor-urs-odeoxycholic acid.

Description

Bile acid derivatives in the medical intervention of anxieties and/or stress symptoms

The invention relates to nor-ursodeoxycholic acid/“norucholic acid”, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use in the treatment or the prevention of a behavioral disorder. Equally, the invention relates to a method of treatment of a behavioral disorder comprising the step of administering to a patient in need of medical intervention nor- ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof. For example, the behavioral disorder may be selected from the group consisting of an anxiety disorder, a depression-disorder/a depression-related symptom, a mood disorder and/or stress/stress symptom(s). The invention may further comprise a non-medical use of nor- ursodeoxycholic acid, a prodrug or an ester thereof or an acceptable salt thereof as mood enhancer, as mood alleviator, as relaxation agent, as stress releasing agent, as weight-reduction supplement and/or for the amelioration of a depressed mental state. Particularly, said nor- ursodeoxycholic acid may be 24-nor-urs-odeoxycholic acid.

Today, anxiety, depression and obesity pose significant medical problems in Western societies. Anxiety disorders as a group have the greatest prevalence among all mental disorders (Craske et al., Nature reviews. Disease primers 3, 17024, doi:10.1038/nrdp.2017.24 (2017)). Worldwide, about one in four adults are expected to experience any type of anxiety disorder during their lifetime (Kessler, R. C. et al., World psychiatry: official journal of the World Psychiatric Association 6, 168-176 (2007)), but estimated regional percentages range significantly from as low as 4.8% in China to as high as 31% in the USA (Baxter et al., Psychological medicine 43, 897-910 (2013)). The lifetime prevalence of depressive disorders is also high, estimated at 9% in developing countries (Baxter et al., Psychological medicine 43, 897-910 (2013)) and at 18% in the USA and Europe (Kessler, et al., Archives of general psychiatry 68, 90-100, (2011)). Additionally, anxiety and depressive disorders show a high rate of comorbidity, and depression has been linked to all well-known anxiety disorders (Alonso et al., J Clin Psychiatry 68 Suppl 2, 3-9 (2007); Kessler, et al., Arch Gen Psychiatry 62, 593-602, (2005); Kessler, et al. Depress Anxiety 27, 351-364, doi:10.1002/da.20634 (2010)). The prevalence of both anxiety disorders (Gariepy, et al., Int J Obes (Lond) 34, 407-419, (2010)) and depressive disorders (Milaneschi, et al. Mol Psychiatry, doi:10.1038/s41380-018-0017-5 (2018); Dixon, et al., Arch Intern Med 163, 2058-2065, doi:10.1001/archinte 163.17.2058 (2003)) increase significantly in obese individuals. Especially in Western societies, high rates of obesity create a large population vulnerable to anxiety and depressive disorders. Indeed, in the US alone up to 35% of men and up to 40.4% of women are considered obese (Flegal, et al., JAMA 315, 2284-2291, doi:10.1001/jama.2016.6458 (2016)).

The high levels of comorbidity of anxiety, depression and obesity together with the high prevalence of each disorder by itself highlight the need for novel therapeutic strategies.

Bile acids (BAs) are well-known mediators of dietary fat absorption. NorUDCA/Nor- ursodeoxy cholic acid is a derivative of UDCA (ursodeoxycholic acid, a naturally occurring bile acid) used for the treatment of biliary disorders in humans (see e.g., Beuers U, et al. 2015 Apr; 62(lSuppl): S25-37; Kowdley KV. 2000 Apr 15; 108(6):481 -6. Review. Erratum in: Am J Med 2000 Jun 1;108(8):690; Trauner M, et al. Aliment Pharmacol Ther. 1999 Aug;13(8):979-96).). NorUDCA is currently under clinical evaluation in humans for treatment of PSC and NAFLD/NASH (Traussnigg S., et al. Austrian/German NAFLD-norUDCA study group. Norursodeoxycholic acid versus placebo in the treatment of non-alcoholic fatty liver disease: a double-blind, randomised, placebo-controlled, phase 2 dose-finding trial. Lancet Gastroenterol Hepatol. 2019 Oct;4(10):781-793; Fickert P, et al. European PSC norUDCA Study Group. norUrsodeoxycholic acid improves cholestasis in primary sclerosing cholangitis. J Hepatol. 2017 Sep;67(3):549-558; Steinacher D, et al. Therapeutic Mechanisms of Bile Acids and Nor- Ursodeoxycholic Acid in Non-Alcoholic Fatty Liver Disease. Dig Dis. 2017;35(3):282-287; Halilbasic E, et al. Nor-Ursodeoxycholic Acid as a Novel Therapeutic Approach for Cholestatic and Metabolic Liver Diseases. Dig Dis. 2017;35(3):288-292). Because of its different pharmacokinetics, norUDCA has shown promise in the treatment of primary sclerosing cholangitis (PSC) in humans, and it ameliorates non-alcoholic steatohepatitis (NASH) in humans . W02006/119803 proposed the use of nor-ursodeoxycholic acid in the treatment and/or prevention of liver diseases, in particular chronic cholestatic liver disease, like primary biliary cirrhosis, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, liver carcinomas, autoimmune hepatitis, (chronic) viral hepatitis, and the like. In WO 2009/013334, nor-UDCA/ norUDCA is proposed for the treatment of arteriosclerosis and EP-B1 2392336 discloses the use of nor-UDCA/ norUDCA in the treatment of autoimmune disease, in particular autoimmune hepatitis (for human clinical trials on nor-UDCA/norUDCA see, e.g., Traussnigg S., et al. Austrian/German NAFLD-norUDCA study group. Norursodeoxycholic acid versus placebo in the treatment of non-alcoholic fatty liver disease: a double-blind, randomised, placebo-controlled, phase 2 dose-finding trial. Lancet Gastroenterol Hepatol. 2019 Oct;4(10):781-793; Fickert P, et al. European PSC norUDCA Study Group. norUrsodeoxycholic acid improves cholestasis in primary sclerosing cholangitis. J Hepatol. 2017 Sep;67(3):549-558).

As discussed herein above, some therapies for behavioral disorders are available to the skilled artisan. Such medicaments comprise, inter alia, anti-depressants, anti-anxiety drugs or beta- blockers. Yet, such drugs are known to have severe side effects and may even alter the brain structure. For example, benzodiazepines may cause drowsiness as well as impaired memory function and withdrawal symptoms have often been reported. Beta-blockers may prevent certain physical symptoms associated with anxieties, but have undesired side effects due their influence on the heart rhythm Monoamine oxidase inhibitors, being the oldest antidepressants, can cause confusion, seizures or even undesired blood pressure events. Selective serotonine reuptake inhibitors sometime produce nausea or jitters and there are several reports on sexual dysfunction experiences.

There is still a need for medical intervention in behavioral disorders, in particular for anxiety disorders and in depressions.

These problems have been solved by the embodiments provided herein and as characterized in the appended claims.

Accordingly, the present invention relates to nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use in the treatment or the prevention of a behavioral disorder, preferably selected from the group consisting of an anxiety disorder, a depression-disorder/a depression-related symptom, a mood disorder and/or stress/stress symptoms.

Equally, the invention relates to a method of treatment of a behavioral disorder, said behavioral disorder in particular selected from the group consisting of an anxiety disorder, a depression- disorder/a depression-related symptom, a mood disorder and/or stress/stress symptoms, said method comprising the step of administering to a patient in need of medical intervention nor- ursodeoxy cholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof.

Further, the invention relates to a non-medical use of norUDCA/nor-UDCA/nor-ursodeoxycholic acid as mood enhancer, as mood alleviator, as relaxation agent, as stress releasing agent, as weight-reduction supplement and/or for the amelioration of a depressed mental state, In this context, said nor-ursodeoxycholic acid may also be of use in not-yet diseased individuals, for example for preventive purposes.

As illustrated in the appended example, it was surprisingly be found that norUDCA/nor-UDCA has distinct, ameliorating effects on anxieties and/or depression like behavior. In particular, and without being limiting, the appended Examples document in relevant mouse models an unexpected effect of norUDCA/nor-UDCA/nor-ursodeoxycholic acid on the brain. A surpringing effect of “norUDCA” on the control of “high fat diet” (HFD)-related associated stress, anxiety and depression is documented in the appended Examples. Furthermore, it could be shown that ameliorating HFD induced- fatty liver disease in these mice could be improved with norUDCA. [NJorUDCA treatment in mice reduced food consumption and body weight as well as anhedonia, fear and anxiety. A strong decrease of classical stress, anxiety and depression read-outs following norUDCA treatment (in particular in mice that receive such a high fat diet) was observed.

The hallmark, for example of anxiety disorders, is a marked, persistent and excessive or unreasonable fear, see, inter alia American Psychiatric Association (editor), “Diagnostic and Stastical Manual of Mental Disorders”, DSM-.5 or Cryan (2005), Nature ReviewsDrug Discovery 4, pages 775-790. One of the symptoms of these mental disorders is a markedly diminished interest or pleasure in everyday activities (anhedonia), see also Cryan (2005) loc cit. . As surprisingly shown in the appended example, norUDCA/nor-UDCA/ "Nor-Urso" interacts with the brain circuitry and modulates anxiety and depression-like behavior in mice. In particular, in an sucrose preference test (SPT) it can be documented that non-treated diet-induced obesity (DIO) animals showed decreased sucrose consumption, indicating increased anhedonia. This behavior was absent in the nor-UDCA cohort, which greatly preferred sucrose solution over water. Furthermore, it could surprisingly be found that in arena-based anxiety tests, that non treated DIO mice behaved more anxiously than DIO mice treated with nor-UDCA.

In enrichment gene expression analyses as illustrated in the appended examples, regular Chow diet and high-fat diet (HFD) mice treated with norUDCA surprisingly showed modulation of genes associated with neuronal function in the brain as indicated by differential enrichment in synaptic signaling (normalized enrichment score, NES), suggesting action on brain neurotransmission (see appended Figure 9a). Moreover, a detailed analyses of these data demonstrates modulation of gene sets associated with Xenobiotics, Neurogenic Inflammation and particularly Mood and Mood disorders within the amygdala-hypothalamus-insula-prefrontal cortex (PFC) - ventral hippocampus (vHPC) brain network (see Figure 9b). Thus, from these results, it can be reasonably concluded that norUDCA interacts with the brain circuitry in a diet- independent manner and modulates genes associated with mood and mood disorders. Furthermore, as illustrated in the appended Examples and shown in appended Figure 10a to j, the influence of selected metabolites, such as neurotransmitters, vitamins, tryptophan and prostaglandins, of Chow and HFD mice treated with norUDCA were assessed in the brain of said mice. The results showed that levels of selected metabolites implicated with depression and mood disorders were altered in mice treated with norUDCA compared to mice which were not treated with norUDCA and the levels were altered in treated mice independent of regular or high- fat diet.

Metabolites that known in the art to be implicated with depression and mood disorders include, but are not limiting vitamins, (neuro-)transmitters), hormones and hormone-like compounds (for example prostaglandins), etc. Corresponding examples may comprise: vitamin E associated with depression (see, e.g., Anderson, George, and Michael Maes. 2014; Current Pharmaceutical Design 20 (23): 3812-47); vitamine B1 associated with depression (see, e.g., Dhir. 2019; Frontiers in Psychiatry / Frontiers Research Foundation 10. https://doi.org/10.3389/ fpsyt.2019.00207), vitamin B5 associated with depression (see, e.g., Rubio-Lopez, 2016; International Journal of Environmental Research and Public Health 13 (3). https://doi.org/); dopamine associated with depression (see, e.g., Grace, Anthony A. 2016; Nature Reviews. Neuroscience 17 (8): 524-32), acetylcholine associated with depression (see, e.g., Higley, Michael J., and Marina R. Picciotto. 2014; Current Opinion in Neurobiology 29 (December): 88-95), serotonin associated with depression and gamma- aminobutyric acid (GABA) associated with anxiety and depression (see, e.g., Hilal-Dandan, 2012; Neuropharmacology 62 (1): 42-53), prostaglandin associated with inflammation, particularly neuroinflammation, and depression (see, e.g., Leonard, Brian E. 2018; Acta Neuropsychiatrica 30 (1): 1-16).

Thus, whereas the herein shown experimental behavioral data are related to high-fat-diet induced anhedonia (see also the control cohorts), a diet-independent impact by the treatment with norUDCA can reasonably be assumed from the results of the experimental data relating to gene expression enrichment and analyses of selected metabolites and, thus, that the present findings have also impact on general behavioral disorders.

Accordingly, the present invention relates to the medical use of 24-nor-ursodeoxycholic acid/norUDCA/nor-UDCA in the treatment/prevention/amelioration of behavioral disorders, like anxiety disorders, depressions, depression-related symptoms, mood disorders and/or stress/stress symptoms. In one embodiment of the present invention, these behavioral disorders are linked to and/or related to impaired food intake/eating behaviors. Accordingly, in one specific embodiment of the present invention the behavioral disorders are induced in the patient to be treated by a high energy intake, like a high fat diet, western diet, fast food, high caloric diet, beverages containing high fructose. Therefore, and in one aspect of the invention, the present invention also relates to the treatment of behavioral disorders, like anxiety disorders, depressions, depression-related symptoms, mood disorders and/or stress/stress symptoms in particular in obese patients. Especially in such patients the medical intervention with 24-nor- ursodeoxycholic acid/norUDCA/nor-UDCA can support and/or facilitate (sustained) weight loss, potential reduced weight gain and/or reduce food intake, as also illustrated in the appended example. Accordingly, the present invention also provides for the medical use of 24-nor- ursodeoxycholic acid/norUDCA/nor-UDCA in the treatment/prevention/amelioration of diet- induced behavioral disorders, like anxiety disorders, depressions, depression-related symptoms, mood disorders.

The present invention in particular relates to nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use, wherein said anxiety disorder is selected from the group consisting of acute stress disorder, agoraphobia, panic attacks, social phobia, post-traumatic stress disorder, obsessive-compulsive disorder, generalized anxiety disorder, anxiety based sleep disturbances/insomnia, hyperarousal and separation anxiety; said depression-disorder/said depression-related symptom is selected from the group consisting of anhedonia, chronic fatigue or loss of energy, indecisiveness/diminished ability to think or concentrate, recurrence of death thoughts or suicide, inappropriate guilt, drug-induced or intoxication-induced depression, depression, in particular interferon-induced, contraceptive- induced, hormonal-induced depression and abuse-induced depression; said mood-disorder is selected from the group consisting of depressive mood, manic mood, bipolar conditions/bipolar disorders, seasonal affective disorder, mania, hypomania, cyclothymic disorders, premenstrual dysphoric disorders, persistent depressive disorders and disruptive mood dysregulation disorder.

In one embodiment, the behavioral disorder, anxiety disorder, a depression-disorder/a depression-related symptom, a mood disorder and/or stress/stress symptoms to be treated and/or prevented by the means and methods herein is diet-related, related to an unfavorable diet, and/or related to the negative eating habits of the individual in need of treatment/prevention. Said individual may, inter alia , without being limiting, suffer from hyperphagia, addictive eating, compulsive (over-)eating, “binge eating”, etc.. More preferably, said unfavorable diet is a high fat diet, a high carb diet, a high caloric diet and/or a mixed form of such unfavorable diets.

Accordingly, said nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof is to be administered to a patient in need of treatment in form of an adjunct and/or co-therapy. Such a co-therapy may comprise the administration of anxiolytics, sedative, anti-depression agents and the like.

Preferably, said nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof is formulated for oral, parenteral, sub-cutaneous, intravenous, intramuscular, nasal, intrathekal, vaginal, rectal or topical administration. Most preferred is an oral dosage form. In particular, an optional oral preparation is disclosed in WO 2009/013334.

Preferably, said nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof is to be administered to a patient in need of treatment in an amount of 25mg to 5g, preferably lg to 5g, in particular lOOmg to 2,5g, most preferably 800mg to l,5g per dayPreferably, the nor-ursodeoxycholic acid to be used in context of the present invention is in form of an oral dosage form, like a tablet, a capsule and/or a suspension. According to a preferred embodiment of the present invention the medicament comprises 10 to 8000 mg, preferably 25 to 5000 mg, more preferably 50 to 1500 mg, in particular 500mg-1500 mg, of 24-nor- ursodeoxycholic acid. In a further preferred embodiment, the daily dosage of the compound to be used in accordance with this invention, comprises 800mg to 1500 mg, more preferably, lOOOmg to 1500mg. The person skilled in the art is readily in a position to obtain the herein used 24-nor-ursodeoxycholic acid. Corresponding synthesis are provided, e.g. in EP 2 468 762 or in US9512167B2. A common, yet not llimiting, dosage for other indications, which may also be used in context of this invention is 500mg to 1500mg/d.

Preferably, said nor-ursodeoxycholic acid to be used in context of this invention is 24-nor-urs odeoxycholic acid.

As used herein, the terms 24-nor-ursodeoxycholic acid, norUDCA/nor-UDCA are equivalent and are employed interchangeably. In particular, said norUDCA can be represented by the following formula:

A corresponding formula is provided, e.g. in W02006/119804.

According to the present invention the dosage forms comprising 24-nor-ursodeoxycholic acid can further include conventional excipients, preferably pharmaceutically acceptable organic or inorganic carrier substances which do not react with the active compound. Suitable pharmaceutically acceptable carriers include, for instance, water, salt solutions, alcohol, oils, preferably vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, surfactants, perfume oil, fatty acid mono-glycerides and diglycerides, petroethral fatty acid esters, hy-droxymethyl-cellulose, polyvinylpyrrolidone and the like. The pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, like lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. For parenteral application, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Various delivery systems are known and can be used to administer 24-nor-ursodeoxycholic acid, including, for example, encapsulation in liposomes, emulsions, microparticles, microcapsules and microgranules. The required dosage can be administered as a single unit or in a sustained release form.

The bioavailability of 24-nor-ursodeoxycholic acid can be enhanced by micronization of the formulations using conventional techniques such as grinding, milling and spray drying in the presence of suitable excipients or agents such as phospholipids or surfactants According to the invention, 24-nor-ursodeoxycholic acid can be formulated in a pharmaceutically acceptable salt or ester form. Pharmaceutically acceptable salts of 24-nor- ursodeoxycholic acid include preferably metal salts, in particular alkali metal salts, or other pharmaceutically acceptable salts. Suitable pharmaceutically acceptable acid addition salts may be prepared from inorganic acids, like hydrochloric, hydrobromic, hy-droiodic, nitric, carbonic, sulfuric and phosphoric acid, or organic acids, like aliphatic, cycloaliphatic, aromatic, heterocyclic, carboxylic and sulfonic classes of organic acids, such as, formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, ethanesulfonic, anthranilic, mandelic, mesylic, salicylic, p- hydroxybenzoic, phenylacetic, methanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, algenic, sulfanilic, stearic, p-hydroxybutyric, cyclohexylaminosulfonic, galactaric and galacturonic acid and the like. Pharmaceutically acceptable base addition salts include metallic salts made from lithium, aluminum, calcium, magnesium, potassium, sodium and zinc or organic salts made from primary, secondary and tertiary amines and cyclic amines. All 24-nor-ursodeoxycholic acid salts can be prepared by methods known in the state of the art (e.g. by reacting 24-nor-ursodeoxycholic acid with the appropriate acid or base) . 24-nor-ursodeoxycholic acid esters are non-toxic esters, prefer- ably alkyl esters such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl esters, or aryl esters. Esterification of carboxylic acids, such as 24-nor-ursodeoxycholic acid, is performed by a variety of conventional procedures, including reacting the carboxylic group with an appropriate alcohol. These reactions are known to the person skilled in the art.

Methods for the manufacture of a drug according to the present invention comprising 24-nor- ursodeoxycholic acid and formulated for the administration as outlined herein can be found, for instance, in the "Handbook of Pharmaceutical Manufacturing Formulations" (Sarfaraz K Niazi, CRC Press LLC, 2004) . The drug comprises preferably an effective amount of 24-nor- ursodeoxycholic acid and a pharmaceutically acceptable carrier and/or excipient . Exemplary dosage forms for the administration of norUDCA can be found, for instance, in WO 2009/013334.

As discussed herein above, the present invention also relates to the non-medical/non- therapeutical use of norUDCA/nor-ursodeoxycholic acid as mood enhancer, as mood alleviator, as relaxation agent, as stress releasing agent, as weight-reduction supplement and/or for the amelioration of a depressed mental state, etc. The appended Examples the present invention also surprisingly illustrate an unexpected effect of norETDCA (nor-ursodeoxycholic acid) on anhedonia (inability to feel pleasure) as well as on anxiety and anxiety-like behavior. This is in particular documented with corresponding mouse models. Due to this surprising effect of norETDCA on anhedonia, the present invention also provides for the above described medical uses in in the treatment or the prevention of a behavioral disorder/diseases, like anxiety disorder, a depression-disorder/a depression-related symptom, a mood disorder and/or (pathological) stress/stress symptoms.

Yet, the present invention and the research results shown herein render also a non-therapeutic use of norCTDCA/nor-ursodeoxycholic acid plausible and encompassed by the present invention. Accordingly, norETDCA/nor-ursodeoxycholic acid may also be used, for example as (mild) mood enhancer, as stress reliever etc.. The present invention is also characterized by the surprising finding (as also illustrated in the appended examples) that norETDCA treatment in mice (which received a high-fat diet) leads to reduced food consumption and body weight in the treated test individuals. Accordingly, the present invention also provides for means and methods to improve also slightly depressed mood situations and/or to (also non-pathological) bad mood situations in individuals in need of such an intervention. Accordingly, the present invention also provides for norETDCA as mood enhancer or as stress reliever in non-pathological stress situations.

The surprising effect documented herein and illustrated in the appended examples on food consumption in mice receiving a high-fat diet render it also plausible that norETDCA may be employed in situations where weight loss of an individual is desired. Accordingly, said norETDCA may also be used in supporting weight loss endeavors, like corresponding diets, etc. Therefore it is also envisaged as part of this invention that norETDCA is helpful in non-therapeutic and/or non-medical intervention, for example in generally healthy individuals that desire to lose weight and/or that desire to improve anhedonic, yet not-pathologic, mental periods, to improve (slight) mood swings, to improve non-pathological depressive moods, or to improve listlessness. In this context of non-medical/non-therapeutic uses, it is also disclosed herein that the present invention relates to invention norUDCA comprised in food and/or dietary supplement and/or corresponding foods or beverages comprising norUDCA. Accordingly, the present invention also relates to (food and/or dietary) supplements or “nutriceuticals” comprising said norUDCA/nor-ursodeoxycholic acid. Such non-therapeutic uses may also comprise the support of diets for weight loss (see, as mentioned above, e.g. in form of a weight-reduction supplement).

Also in this context of non-medical/non-therapeutic, the terms “norUDCA”, “nor- ursodeoxycholic acid” comprise correponding an esters thereof or acceptable salts thereof. In this embodiment of the invention, the nor UDCA may be 24-nor-urs-odeoxycholic acid.

In these non-medical/non-therapeutic supplements, the norUDCA may be employed in lower dosages that in the above described medical settings and/or as comprised in a pharmaceutical for use in the above recited diseases/disorders.

FIGURE LEGEND

The Invention is illustrated by the following Figures.

Figure 1. Effect of norUDCA and HFD on anhedonia and anxiety behavior

(a) HFD-control animals showed a markedly decreased intake of sucrose solution, indicative of an anhedonic state. Note that this was not seen in the HFD-norUDCA cohort (two-way ANOVA Finteraction (1, 56) = 11.01, P = 0.0016; Fdmg (1, 56) = 48.55, P < 0.0001; Fdiet (1, 56) = 40.51, P < 0.0001; Tukey’s multiple comparison test; n = 15). (b) A longer latency to feed was observed in the HFD-control cohort, in line with greater anxiety (two-way ANOVA Finteraction (1, 56) = 12.6, P = 0.0008; Fdmg (l, 56) = 1.511, P = 0.2241; Fdiet (1, 56) = 9.628, P = 0.003; Tukey’s multiple comparison test; n = 15). (c, d) Both the HFD-control and the chow-norUDCA spent less time in the open arms of the EPM than chow-control animals, indicative of anxiety (c; two-way ANOVA Finteraction (1, 54) = 14.11, P = 0.0004 ; Fdrug (1, 54) = 0.00117, P = 0.9728; Fdiet (1, 54) = 0.06331, P = 0.8023; Tukey’s multiple comparison test; n = 14-15). In the open field test (OFT), a similar pattern was seen, but no significant differences were found in post-hoc analysis (d, two-way ANOVA Finteraction (1, 56) = 5.83, P = 0.019 ; Fdmg (1, 56) = 0.2333, P = 0.631; Fdiet (1, 56) = 0.3126, P = 0.5783; Tukey’s multiple comparison test; n = 15).

Bars are means ± standard deviation. Significance levels between groups at * P < 0.05, ** p < 0.01, *** P < 0.001, **** p < 0.0001. For a and b, asterisks show significance compared to the HFD-control group. For c, asterisks show significance compared to the chow-control group.

Figure 2. Effect of norUDCA and HFD on conditioned fear behavior

(a) Overview of conditioning paradigm. (b,c) Neither diet nor drug significantly affected freezing to the sound paired with a foot shock (b, CS+; two-way ANOVA Finteraction (1, 56) = 1.656, P = 0.2032; Fdmg (1, 56) = 1.629, P = 0.2071; Fdiet (1, 56) = 1.302, P = 0.2587; Tukey’s multiple comparison test; n = 15) or to the sound associated with safety (c, CS-; two-way ANOVA Finteraction (1, 56) = 0.1293, P = 0.7205; Fdmg (1, 56) = 0.1092, P = 0.7423; Fdiet (1, 56) = 1.216, P = 0.2749; Tukey’s multiple comparison test; n = 15). (d) HFD-control mice showed stronger freezing during the ambiguous inter-trial-interval (ITI), which was reversed by norUDCA treatment (two-way ANOVA Finteraction (1, 56) = 12.62, P = 0.0008; Fdmg (1, 56) = 1.248, P = 0.2687; Fdiet (1, 56) = 1.716, P = 0.1956; Tukey’s multiple comparison test; n = 15). (e) When mice were re-exposed to the conditioning context, a main drug effect was found in the second half of the session (data for first half not shown), with both the norUDCA-control and norUDC A-HFD groups freezing less than HFD-control mice (two-way ANOVA Finteraction (1 , 56) = 1.911, P = 0.1724; Fdmg (l, 56) = 13.46, P = 0.0005; Fdiet (1, 56) = 0.195, P = 0.6605; Tukey’s multiple comparison test; n = 15).

Bars are means ± standard deviation. Significance levels between groups at * P < 0.05, ** p < 0.01.

Figure 3. Effect of norUDCA and HFD on blood corticosterone levels, brain myelin content and bone density

(a) Blood corticosterone levels under baseline conditions. HFD-control mice showed significantly higher levels than all other groups (two-way ANOVA Finteraction (1, 56) = 11.2, P = 0.0015; Fdmg (1, 56) = 8.204, P = 0.0059; Fdiet (1, 56) = 5.49, P = 0.0227; Tukey’s multiple comparison test; n = 15). (b) Blood corticosterone levels under stressed conditions (two-way ANOVA Finteraction (1, 55) = 4.085, P 0.0481; Fdmg (1, 55) = 0.7249, P 0.3984; Fdiet (1, 55) = 31.82, P< 0.0001; Tukey’s multiple comparison test; n= 14-15). (c) Magnetization transfer ratio as a proxy of brain myelin content (two-way ANOVA Finteraction (1, 16) = 0.06149, P = 0.8073; Fdmg (1, 16) = 0.6495, P = 0.4321; Fdiet (1, 16) = 0.5534, P = 0.4677; Tukey’s multiple comparison test; n = 5). (d) Bone volume between brain and masseter muscle, taken from MRI scans (two-way ANOVA Finteraction (1, 16) = 8.079, P = 0.0118; Fdmg (1, 16) = 37.56, P < 0.0001; Fdiet (1, 16) = 18.94, P = 0.0005; Tukey’s multiple comparison test; n = 5).

Bars are means ± standard deviation. Significance levels between groups at * P < 0.05, ** p < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 4. Body weight and food and water consumption over time

(a) Analysis of body weight showed a clear weight increase in the HFD-control, but not the HFD-norUDCA group, which showed lower body weight compared to both the chow-control and chowchow-norUDCA groups. (RM two-way ANOVA Finteraction (84, 1568) = 172.8, P < 0.0001; Ftime (28, 1568) = 887.6, P < 0.0001; Ftreatment (3, 56) = 85.63, P < 0.0001; after week 15, all groups differed significantly from each other in Tukey’s multiple comparison test; n = 15). (b, c) Cage-based data on food and water consumption (b) After week 15, food consumption was significantly different between the HFD-control and HFD-norUDCA groups, while other group comparisons showed no such a reliable pattern (RM two-way ANOVA Finteraction (81, 216) = 1.467, P = 0.0154; Ftime (27, 216) = 11.12, P < 0.0001; Ftreatment (3, 8) = 26.6, P = 0.0002; Tukey’s multiple comparison test; n = 3). (c) No treatment effect was found on water consumption over time (RM two-way ANOVA Finteraction (81, 216) = 1.226, P = 0.1257; Ftime (27, 216) = 9.256, P < 0.0001; F_treatment (3, 8) = 2.6, P = 0.1244; n = 3).

Graphs show means ± standard deviation.

Figure 5. norUDCA binding to brain.

Chronic norUDCA in chow resulted in significantly higher levels of norUDCA measured in brain tissue by mass spectrometry. Brains were perfused with saline before measurements to avoid contamination with norUDCA in the blood.

Figure 6. norUDCA impacts neuromodulation.

Chronic norUDCA in chow resulted in significantly higher levels of dopamine measured in brain tissue by mass spectrometry. Brains were perfused with saline before measurements.

Figure 7. HFD and norUDCA shape transcriptomes in mPFC and vHC top down modulators of anxiety and depression.

7a norUDCA overrules dietary effects in liver and various brain region, shown by the drastically lower number of DE genes when comparing Chow norUDCA and HFD norUDCA groups (n = 5, excl denotes that some animals had to be excluded after quality control. For Insula, 1 animal in the Chow Ctrl group was excluded, for Hypothalamus, 2 animals in the HFD norUDCA group were excluded). No, or only very low numbers of, DE genes were found in Amygdala, Hypothalamus and Insula. Thus, these regions were not analyzed further. 7b Volcano plot of DE genes in Prefrontal Cortex comparing HFD norUDCA to Chow Ctrl. Gpr88 and Cyp3al 1 were among the most upregulated genes. 7c Volcano plot of DE genes in ventral Hippocampus comparing HFD Ctrl to Chow Ctrl. The most upregulated gene was Th, strongly implicating an effect on catecholamine synthesis.

7d,e Volcano plot of DE genes in ventral Hippocampus comparing norUDCA to corresponding Ctrl groups. In both cases, Cyp3al 1 was among the most upregulated genes.

Figure 8. Chronic norUDCA treatment in mice which received a high-fat diet reduced food consumption and body weight as well as anhedonia, fear and anxiety.

Mass spectrometry showed that norUDCA is present in the brain tissue of norUDCA-treated mice, and that norUDCA treatment also alters neurotransmitter makeup. Using transcriptome analysis, 23 differentially expressed (DE) genes were induced by HFD in both the liver and the prefrontal cortex (PFC) (Fig. 8a), and 1081 DE genes were induced in both the liver and PFC in the HFD norUDCA group when compared to Chow Ctrl group (Fig. 8b).

Figure 9. Effect of NorUDCA and HFD on gene expression in the mouse brain.

Next generation sequencing (NGS) brain analysis reveals differential enrichment of relevant genes across different brain regions (a) Different diets as well as NorUDCA change gene expression of brain relevant GO categories when compared to each other. Values of bar graphs represent significant NES score enrichments of selected specific-gene categories from the gene ontology network. (b) Region specific diet/drug effects in Harmonizome (https://maayanlab.cloud/Harmonizome/) categories Mood, Neurogenic Inflammation and Xenobiotics (left) and for The Comparative Toxicogenomics Database (CTD) (http://ctdbase.org/ ) category Mood Disorder. Heatmap values are the NES scores of one of the four shown gene sets. Significance levels of significant categories are FDR corrected at P < 0.1.

Figure 10a to lOj. Effect of NorUDCA and HFD on brain metabolites

NorUDCA influences brain metabolites. (a) NorUDCA was detectable in flushed mouse brains and there was a significant difference in NorUDCA levels between Chow and HFD animals (two-way ANOVA Finteraction (1, 36) = 19.00, P = 0.0001; Fdmg (1, 36) = 648.9, P < 0.0001; Fdiet (1, 36) = 19.00, P = 0.0001; Tukey’s multiple comparison test; n = 10).

(b-d) NorUDCA treatment influences vitamin levels in flushed mouse brain tissue.

(b) VitBl was reduced in NorUDCA-HFD animals (two-way ANOVA Finteraction (1, 36) = 29.74, P < 0.0001; Fdmg (1, 36) = 12.48, P = 0.0011; Fdiet (1, 36) = 7.938, P = 0.0078; Tukey’s multiple comparison test; n = 10), while

(c) NorUDCA treatment greatly increased Vit B5 levels regardless of diet (two-way ANOVA Finteraction (1, 36) = 0.8856, P = 0.3529; Fdmg (1, 36) = 287.3, P < 0.0001; Fdiet (1, 36) = 7.225, P = 0.0108; Tukey’s multiple comparison test; n = 10).

(d) Vit E levels were not significantly changed by either diet or drug (two-way ANOVA Finteraction (1, 36) = 0.7502, P = 0.3921; Fdmg (1, 36) = 0.2297, P = 0.6347; Fdiet (1, 36) = 8.597, P = 0.0058; Tukey’s multiple comparison test; n = 10).

(e-h) NorUDCA influenced the levels of neurotransmitters in flushed mouse brain tissue.

(e) Acetylcholine is reduced by NorUDCA treatment regardless of diet (two-way ANOVA Finteraction (1, 36) = 0.6127, P = 0.4389; Fdmg (1, 36) = 27.37, P < 0.0001; Fdiet (1, 36) = 7.335, P = 0.0103; Tukey’s multiple comparison test; n = 10).

(f) Serotonin levels were changed by neither drug nor diet (two-way ANOVA Finteraction (1, 35) = 1.763, P = 0.1929; Fdmg (1, 35) = 2.029, P = 0.1632; Fdiet (1, 35) = 0.09048, P = 0.7653; Tukey’s multiple comparison test; n = 9 - 10).

(g) Dopamine was increased by NorUDCA treatment (two-way ANOVA Finteraction (1, 35) = 0.6483, P = 0.4261; Fdmg (1, 35) = 9.272, P = 0.0044; Fdiet (1, 35) = 1.919, P = 0.1748; Tukey’s multiple comparison test; n = 9 - 10).

(h) GABA levels were increased by HFD in the control setting but decreased in NorUDCA-HFD animals (two-way ANOVA Finteraction (1, 36) = 5.180, P = 0.0289; Fdmg (1, 36) = 0.0006063, P = 0.9805; Fdiet (1, 36) = 0.02679, P = 0.8709; Tukey’s multiple comparison test; n = 10).

(i) NorUDCA treatment increased tryptophan levels in Chow-fed but not in HFD-fed animals (two-way ANOVA Finteraction (1, 36) = 9.022, P = 0.0048; Fdmg (1, 36) = 19.41, P < 0.0001; Fdiet (1, 36) = 0.04607, P = 0.8313; Tukey’s multiple comparison test; n = 10).

(j) Prostaglandin levels were reduced by NorUDCA regardless of diet (two-way ANOVA Finteraction (1, 36) = 0.3874, P = 0.5376; Fdmg (1, 36) = 30.97, P < 0.0001; Fdiet (1, 36) = 0.7231, P = 0.4008; Tukey’s multiple comparison test; n = 10). Bars are means ± standard deviation. Significance levels between groups at * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

EXAMPLES

The invention is further illustrated, but not limited by the following examples.

Background and Aims

Chronic liver diseases, such as non-alcoholic fatty liver disease, and metabolic conditions like obesity and their association with behavioral disorders like anxiety and depression was assessed in context of this invention. Also, the use of a bile acid 24-nor-ursodeoxycholic acid (norUDCA) was assessed in the treatment of anxiety disorders and/or depression. Corresponding mouse models were used and employed. The underlying pathophysiology of the interaction of high-fat diet induced liver disease and obesity with brain structures involved in emotion processing is assessed herein since it was not well understood. In order to evaluate the potential of targeting a putative shared underlying disease mechanism, the effect of [chronic] 24-nor-ursodeoxycholic acid (norUDCA) on anxiety and depression-like behavior in mice was evaluated. In context of this invention, a “chronic 24-nor-ursodeoxycholic acid (norUDCA)” means treatment for extended periods, like more than 1 week, more than 2 weeks, more than 3 weeks, preferably at least one month or more.

Methods

Two cohorts of C57BL/6 mice (n = 15) were fed a standard high-fat diet (HFD, 36% fat from butter, HF260, Safe Diets, SAFE A04 Rats Mice Hamsters maintenance diet; Code number: U8220G10R obtained from scientific animal food & engineering (SAFE) Route de Saint Bris 89290 AUGY - France; http://www.safe-diets.com/wp-content/uploads/2017/Q5/SAFE-DS- A04.pdf; ). with or without 0.5% NorUDCA, mixed into the food. Another two cohorts of equal size received a regular chow (chow) diet (A04, Safe Diets, SAFE 260 HF Rat & Mouse Diet; Code number: U8978P Version 0019 obtained from scientific animal food & engineering (SAFE) Route de Saint Bris 89290 AUGY - France; http://www.safe-diets.com/wp- content/uploads/2017/04/DS- -260-HF- -U8978-version-0019.pdf). with or without 0.5% NorUDCA, mixed into the food.

Feeding began when mice were 6 weeks old and lasted for 39 weeks. After eight weeks of feeding, the animals’ behavior was assessed in standard behavioral assays. Starting at week 8, all groups were tested in standard behavioral assays while feeding continued.

Sucrose preference test (SPT)

For the assessment of depression-like behavior, anhedonia in the sucrose preference test (SPT) was measured.

Novelty-Suppressed Feeding (NSF)

For the assessment of anxiety diet and treatment effects were measured in the Novelty- Suppressed Feeding (NSF)

Behavior assays

Behavior was assessed using standard mouse arenas, fear conditioning boxes and the phenomaster system (TSE systems). In particular, two arena-based anxiety assays, the Elevated Plus Maze (EPM) (Fi. lc) and the Open Field Test (OFT) (Fig. Id) were performed. Further, to further explore the effects of NorUDCA on fear and anxiety, all cohorts were subjected to a fear conditioning paradigm (Fig. 2a). After fear conditioning, mice were exposed to the learned cues in a novel context. Functional magnetic resonance imaging was used to evaluate brain activity.

Measurement of blood corticosterone

Levels of blood corticosterone in both baseline (Fig. 3a) and stressed (Fig. 3b) conditions were assessed to identify metabolic markers of stress.

Magnetic resonance imaging (fMRI)

Functional magnetic resonance imaging (fMRI) of the brain was used in both baseline (unstressed) as well as stressed conditions and compared brain-wide blood oxygen-level dependent (BOLD) imaging pattern as a proxy for neuronal activity.

Gene expression analysis mRNA was extracted from livers and brains (n = 5 per group). A Tn5 enzyme-based RNA-seq library prep was performed and underwent Ilumina seq. Genes were ranked by log2 fold change (FC) and a p-adjusted threshold of < 0.05 was applied. Ingenuity Pathway Analysis (IP A, Quiagen) and PANTHER were used for further analysis. Mass spectrometry

To address the potential mechanism of action of the effect of norUDCA on depression and anxiety behavior, it was investigated whether norUDCA is present in the brain using mass spectrometry. Liquid-chromatography based mass spectral analysis was used for the measurement of neurotransmitter and bile acid species.

Results

Sucrose preference test (SPT)

In the sucrose preference test (SPT), HFD-control cohort showed decreased sucrose consumption, indicating increased anhedonia. This behavior was absent in the HFD-norUDCA cohort, which greatly preferred sucrose solution over water (Fig. la). No difference was observed between the two chow cohorts. Overall, these data indicate that HFD-induced anhedonia is reverted by chronic norUDCA treatment, highlighting its potential to treat obesity-induced depression-like behavior.

Novelty-Suppressed Feeding (NSF)

The Novelty-Suppressed Feeding (NSF) assay revealed delayed feeding time in the HFD-control cohort, but not the HFD-norUDCA cohort, while norUDCA treatment had no effect on chow mice. This is in line with a norUDCA-induced reversal of increased anxiety caused by a HFD (Fig. lb).

Behavior assays

Elevated Plus Maze (EPM) and the Open Field Test ( OFT)

In both assays, the Elevated Plus Maze (EPM) (Fi. lc) and the Open Field Test (OFT) (Fig. Id), a 2x2 ANOVA showed a significant interaction of drug and diet in the time spent in the anxiogenic zone (center for OFT, open arms for EMP). In both arena-based anxiety tests, HFD- control mice behaved more anxiously than HFD-norUDCA mice. However, chow-norUDCA mice were also more anxious than chow-control animals. This indicates that norUDCA treatment has a greater potential in an obese population, while there might be inverse effects in a non- pathological population. Fear conditioning boxes

While no effect was seen on either learned fear (cue paired to shock, CS+) (Fig. 2b), nor on learned safety (cue paired to no-shock, CS-) (Fig. 2c), an absence of any cue in the inter trial interval (ITI) (Fig. 2d), i.e. when the animals had no indicator of what would happen next, resulted in a strong interaction effect, with the HFD-control freezing significantly more than either the chow-control or HFD-norUDCA animals. Furthermore, re-exposure to the conditioning context revealed a reduction in freezing in both drug cohorts (Fig. 2e).

These results indicate that norUDCA influences anxiety related behaviors, as shown by the results in freezing during the ITIs and context re-exposure. While the results from ITI freezing are in line with the results from the novelty suppressed freezing and sucrose preference tests described above (reversal of anxiogenic HFD effect by norUDCA), the results from the fear- context re-exposure experiment show a strong drug effect independent of group, i.e., context re exposure induced freezing was reduced regardless of diet. As this test can be seen as a paradigm to model post-traumatic stress disorder (PTSD; VanElzakker, et ak, Neurobiol Learn Mem 113, 3-18, (2014)), (indeed, re-exposure to environmental cues reminiscent of a traumatic event is often a trigger for PTSD-linked panic attacks in humans (Pitman et ak, Nat Rev Neurosci 13, 769-787, (2012); Shalev et ak, The New England journal of medicine 376, 2459-2469, (2017)), these results seem especially promising, and might lead to novel insights in the pathophysiology and treatment of anxiety independent of obesity.

Measurement of blood corticosterone

In the baseline condition, only the HFD-control group had significantly increased levels of serum levels of corticosterone indicating that HFD/obesity induced stress is ameliorated by chronic norUDCA treatment.

For the stressed condition, mice were re-exposed to the conditioning context of the fear conditioning paradigm, and blood samples were taken thereafter. Here, HFD control mice again showed greater levels of blood corticosterone than chow control mice, while such a difference was not found between the HFD-norUDCA and chow-norUDCA groups.

Together, these results indicate that chronic norUDCA treatment serves to mitigate chronically increased blood corticosterone caused by HFD/obesity. Functional magnetic resonance imaging (fMRI)

Significant differences in several brain regions (see table 1), all of them part of the limbic system, were detected. In particular, norUDCA appeared to affect the amygdala independent of dietary setting in the baseline state. The directional activity change of the hypothalamus (chow-control > HFD-control < HFD-norUDCA) parallels the corticosterone measurements (chow-control < HFD-control; > HFD norUDCA; see Fig. 4a), whereas in the stressed condition, norUDCA appeared to affect the hypothalamus independent of diet. Thus, the BOLD activation of the hypothalamus in the fMRI corresponded to the increased corticosterone baseline serum levels in HFD-control group.

No effect was found between both norUDCA groups indicating that norUDCA can revert HFD induced changes in brain accooltivity. Myelin content was not different within any of the groups reflected by magnetization transfer ratio (Fig. 3c), indicating that there is no severe malnutrition causing these differences in fMRI.

Table 1: Effect of norUDCA and HFD on fMRI BOLD signal. Regions shown were found to be significantly different in post-hoc comparisons after brain-wide analysis in BOLD signal in baseline (top) or stressed (bottom) conditions.

Metabolomic and genetic analysis by Mass spectrometry

Mass spectrometry showed that norUDCA is present in the brain tissue of norUDCA-treated mice, and that norUDCA treatment also alters neurotransmitter makeup (Fig. 5). Next, mass spectrometry was used to check for the effect of norUDCA on canonical neurotransmitters. Chronic norUDCA treatment significantly increased dopamine levels in brain tissue (Fig. 6). Next, NGS was used to investigate transcription patterns in the liver as well as in five brain regions of mice treated chronically with norUDCA and/or HFD implicated in emotional processing. Differentially expressed (DE) genes between Chow norUDCA and HFD norUDCA groups were drastically lower than in other comparisons, indicating that norUDCA overrules dietary effects in both the liver and brain. No or only very low numbers of, DE genes were found in Amygdala, Hypothalamus and Insula.

In the Prefrontal Cortex, CYP3al l and GPR 88 were among the most highly expressed genes when comparing HFD norUDCA to Chow Ctrl (Fig. 7b). In the ventral Hippocampus, CYP3al 1 was among the most highly expressed DE genes when comparing HFD norUDCA to HFD Ctrl and Chow norUDCA to Chow Ctrl (Fig. d,e). Further, in the ventral Hippocampus, HFD induced the expression of tyrosine hydrolase (Th), strongly suggesting an effect on catecholamine synthesis in this condition.

The increased expression of Cyp 3al l is indicative for increased de-toxifi cation induced by norUDCA.

In sum, sing transcriptome analysis, 23 differentially expressed (DE) genes were induced by HFD in both the liver and the prefrontal cortex (PFC) (Fig. 8a), and 1081 DE genes were induced in both the liver and PFC in the HFD norUDCA group when compared to Chow Ctrl group (Fig. 8b).

Furthermore, enrichment of gene expression in the mouse brain of three different groups of mice were compared, wherein one group denoted as “Chow / HFD” refers to a comparison of HFD (high-fat diet) mice not treated with norUDCA with Chow (regular Chow diet) mice also not treated with norUDCA; and two groups denoted as “Brain Chow / Chow NorUDCA” and “Brain HFD / HFD NorUDCA” which refer to a comparison of Chow and HFD mice treated with norUDCA compared to said mice not treated with norUDCA; see Figure 9a.

As shown in Figure 9a, enriched gene expression (mRNA level) in norUDCA treated and untreated mice was correlated to the corresponding most enriched pathways relevant for neuronal function, denoted as “GO-term” and shown in Figure 9a. As shown in Figure 9a, norUDCA treated mice under regular Chow diet or high-fat diet (HFD) (Chow/ChowNorUDCA and HFD/HFDNorUDCA) showed high enrichment in the same pathways (i.e. “GO-terms”) and, in addition, enrichment to a similar extend as indicated by the normalized enrichment score (NES in Figure 9a reflecting the degree to which the genes in a gene set are overrepresented at the top or bottom of the entire ranked list of genes.). In contrast, the most enriched pathways in mice (Chow/HFD) not treated with norUDCA differed from the ones in mice treated with norUDCA. Since regions/pathways (“Go-terms” as shown in Figure 9a) relevant for neuronal function were highly enriched and showed a similar enriched pattern in nor UDCA treated regularly Chow fed (Chow) and high-fat diet (HFD) mice, this further indicates that norUDCA interacts with the brain circuitry in a diet-independent manner.

Moreover, as shown in Figure 9b, brain region-specific gene expression modulation was assessed in mice treated with norUDCA (“Brain Chow / Chow NorUDCA” and “Brain HFD / HFD NorUDCA”) in comparison to mice not treated with norUDCA (“Chow / HFD”). Particularly, analyses of gene expression in mice treated with norUDCA independent of their diet demonstrated that expression of genes associated with xenobiotics, neurogenic inflammation and particularly mood and/or mood disorders is modulated within the amygdala-hypothalamus- insula-prefrontal cortex (PFC) - ventral hippocampus (vHPC) brain network (see Figure 9b). Thus, from these results, it is to be concluded that norUDCA interacts with the brain circuitry also in a diet-independent manner and modulates genes associated with mood and mood disorders.

Furthermore, in mice treated with norUDCA (Chow/ChowNorUDCA and HFD/HFDNorUDCA), gamma- aminobutyric acid (GABA) levels appeared to be altered in contrast to the untreated mice (Chow/HFD) (data not shown). Therefore, the influence of GABA and further selected metabolites, such as neurotransmitters, vitamins, tryptophan and prostaglandins, were assessed in the brain of Chow and HFD mice treated with norUDCA via mass spectrometry and as shown in Figure 10a to lOj.

Fig. 10a shows that NorUDCA is detectable within brain tissue after chronic feeding. It was further found that NorUDCA treatment profoundly lowers Vit B1 (thiamin) levels (Fig. 10b). In contrast, Vit B5 (pantothenic acid) was increased by the drug treatment (Fig. 10c). Vit E levels were raised by HFD, but were not influenced by NorUDCA treatment (Fig. lOd). Furthermore, significant drug effects were found on the neurotransmitters acetylcholine and dopamine (Fig. lOe and lOg), as well as an interaction effect on the neurotransmitter GABA (Fig. lOh); see also figure legend for details. No significant effect was found on serotonin levels (Fig. lOf). These findings indicated that drug profoundly alters available neurotransmitters in the mouse brain. Further, an increase in tryptophan levels was found, an important mediator in depression and mood disorder (Fig. lOi). Next to neurotransmitter modulation, a decrease of prostaglandins was found (Fig. 102j), indicating a direct suppression of neuroinflammatory signalling and associated aversive neurocognitive states. Body weight analysis

Weight, food and water intake were recorded weekly and analyzed for the first 29 weeks, after which animals were separated into smaller cohorts (Fig. 4). norUDCA treated mice gained significantly less weight compared to controls, regardless of fat content in the diet (Fig. 4a). Nevertheless, the HFD-norUDCA cohort showed the lowest weight gain and food consumption of all cohorts (Fig. 4b). Water consumption however did not vary significantly among cohorts (Fig. 4c).

Conclusion

This example shows an effect of a bile acid derivative on the brain to control HFD associated stress, anxiety and depression as well as ameliorating HFD induced- fatty liver disease in mice. Chronic norUDCA treatment in mice which received a high-fat diet reduced food consumption and body weight as well as anhedonia, fear and anxiety. A strong decrease of classical stress, anxiety and depression readouts following norUDCA treatment in mice given a HFD was achieved as illustrated in Table 2. Therefore, norUDCA could counteract the negative effects of obesity and/or high fat diet on mood and stress and is, accordingly, proposed indicating to be used in the treatment and/or the medical intervention of stress, anhedonia, fear, fear symptoms, anxiety disorder and/or depression, in particular in high-fat diet induced stress and/or stress symptoms and in particular obesity-induced anxiety disorder and/or depression in humans.

Table 2: Summary of key readouts. Arrows indicate either an increase (†) or decrease (j) or no change (1) of anxiety/fear, anhedonia/depression, bodyweight and plasma corticosterone, compared to the chow-control cohort, which was defined as (1). In addition to significant differences in post hoc testing, strong trends were included in this table. CD-control HFD-control CD-NorUDCA HFD-NorUDCA

sucrose preference i ttt 1 1 baseline corticosterone i t tt 1 1 stressed corticosterone i ttt 1 tt bodyweight over time i ttt

Further, enrichment gene expression and metabolic analyses as illustrated in the appended Examples above and shown in Figure 9a and b as well as Figure 10a to j demonstrated a diet- independent drug effect of norUDCA on neuronal processing in the brain.

Furthermore, the results showed that levels of selected metabolites in mice implicated with depression and mood disorders were altered in mice treated with nor UDCA compared to mice which were not treated with norUDCA and independent of regular or high-fat diet in these mice. Particularly, the altered levels of selected metabolites as shown in Figure 10a to j reflect the difference seen in the behavioral assays as demonstrated in the appended experiments above, with the GABA expression pattern resembling the arena-based anxiety assays (open field test and elevated plus maze) and the acetylcholine levels resembling the freezing levels in the fear conditioning context-re-exposure assay. Thus, these findings support that the behavioral findings are mediated by a direct NorUDCA effect in the brain.

Further, brain-wide NGS analysis uncovered an alteration of gene ontology pathways relevant for neuronal activity and/or transmission in both norUDCA conditions and thus a modulatory effect on brain function (Fig 9a). Additional brain-wide analysis by fMRI highlighted the amygdala and the hypothalamus as functional hotspots in both chow and HFD conditions (Table 1). To gain added information on the effect of diet and norUDCA on the limbic system, several brain regions known to be involved in mood processing and mood disorders were analyzed separately by NGS. Here, we could find a strong modulation of the prefrontal cortex (PFC) by norUDCA. Because the top-down modulation of the amygdala by the PFC is well established (doi: 10.1038/nn.4101/ https://doi.org/10.1038/nm4028), this is in line with the fMRI results presented above. The data provided herein and illustrated in the appended Figures demonstrate molecular-to- systems-level effects of norUDCA on mood, attenuating translational models of anxiety disorders, depression disorders and depression-related symptoms as well as stress and stress symptoms.

Claims

Claims

1. Nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use in the treatment or the prevention of a behavioral disorder, preferably selected from the group consisting of an anxiety disorder, a depression-disorder/a depression-related symptom, a mood disorder and/or stress/stress symptoms.

2. Nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use of claim 1, wherein said anxiety disorder is selected from the group consisting of acute stress disorder, agoraphobia, panic attacks, social phobia, post-traumatic stress disorder, obsessive-compulsive disorder, generalized anxiety disorder, anxiety-based sleep disturbances/insomnia, hyperarousal and separation anxiety; wherein said depression-disorder/said depression-related symptom is selected from the group consisting of anhedonia, chronic fatigue or loss of energy, indecisiveness/diminished ability to think or concentrate, recurrence of death thoughts or suicide, inappropriate guilt, drug-induced or intoxication-induced depression, depression, in particular interferon-induced, contraceptive-induced, hormonal-induced depression and abuse-induced depression; wherein said mood-disorder is selected from the group consisting of depressive mood, manic mood, bipolar conditions/bipolar disorders, seasonal affective disorder, mania, hypomania, cyclothymic disorders, premenstrual dysphoric disorders, persistent depressive disorders and disruptive mood dysregulation disorder.

3. Nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use of claim 1 or two wherein said behavioral disorder, anxiety disorder, a depression-disorder/a depression-related symptom, a mood disorder and/or stress/stress symptoms is diet-related.

4. Nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use of claim 4, wherein said diet is a high fat diet, a high carb diet, a high caloric diet and/or any unfavorable diet leading to undesired weight gain.

5. Nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use of anyone of claims 1 to 4, wherein said Nor-ursodeoxycholic acid, a prodrug thereof or a pharmaceutically acceptable salt thereof is to be administered to a patient in need of treatment in form of an adjunct and/or co-therapy.

6. Nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use of anyone of claims 1 to 5 wherein said is formulated for oral, parenteral, sub-cutaneous, intravenous, intramuscular, nasal, intrathekal, vaginal, rectal or topical administration.

7. Nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use of anyone of claims 1 to 6, wherein said Nor-ursodeoxycholic acid, a prodrug thereof or a pharmaceutically acceptable salt thereof is to be administered to a patient in need of treatment in an amount of 25mg to 5g, preferably lg to 5g, in particular lOOmg to 2,5g, most preferably 800mg to l,5g per day.

8. Method of treatment of a behavioral disorder, said behavioral disorder in particular selected from the group consisting of an anxiety disorder, a depression-disorder/a depression-related symptom, a mood disorder and/or stress/stress symptoms, said method comprising the step of administering to a patient in need of medical intervention nor- ursodeoxycholic acid, a prodrug thereof or a pharmaceutically acceptable salt thereof.

9. Non-medical use of nor-ursodeoxycholic acid as mood enhancer, as mood alleviator, as relaxation agent, as stress releasing agent, as weight-reduction supplement and/or for the amelioration of a depressed mental state.

10. Nor-ursodeoxycholic acid, a prodrug or an ester thereof or a pharmaceutically acceptable salt thereof for use of anyone of claims 1 to 7, the method of treatment of claim 8 or the non-medical use of claim 9, wherein said nor-ursodeoxycholic acid is 24-nor-urs- odeoxycholic acid.