WO2025248089A1 - Haloquinolines for treating immune-mediated inflammatory diseases - Google Patents

Abstract

The invention relates to a clioquinol for use in the prevention or treatment of psoriasis or of inflammatory bowel disease (IBD).

Description

HALOQUINOLINES FOR TREATING IMMUNE-MEDIATED INFLAMMATORY DISEASES

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions consisting of haloquinoline PHGDH activators like clioquinol to treat Immune-mediated inflammatory diseases (IMIDs) like psoriasis and Inflammatory Bowel Disease.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD), is an IMID that describes the recurrent inflammation of the GI tract due to failure of intestinal barrier resolution. This leads to diarrhoea, weight loss, and chronic fatigue in patients. There are two types, Crohn's disease (CD), characterized by transmural patchy lesions, and ulcerative colitis (UC), characterized by more superficial, central lesions. Together, they accounted for 8 million cases in 2017 globally. On the other hand, psoriasis is an IMID of the skin that is frequently associated with IBD, with almost a two-fold increase in co-occurrence rate. In psoriasis, erythematous scaling plaques form on the skin due to hyperproliferation of keratinocytes. The reported prevalence of psoriasis ranges between 0.09% and 11.4%, with cases increasing in Western countries.

For the first-line treatment of psoriasis, local corticosteroid application is still the norm [Del Rosso (2020) J Clin Aesthet Dermatol 13, 22-29]. Here, however, long term use is not recommended due to possible adverse effects such as burning sensations, atrophy, and persistent erythema. Steroids, such as prednisolone, are also used in IBD flare-up management as a first-line treatment. They are potent suppressors of inflammation however, while effective they increase the risk of opportunistic infections and toxic systemic side effects. Therefore, steroids cannot be used for long-term management of IMIDs. Methotrexate is another immunomodulator that is used in severe cases however due to its side effects its use in the clinic is negligible.

Clioquinol is known as an antifungal and antiprotozoal drug.

Locacorten-Vioform™ is a cream comprising clioquinol as an anti-infective agent agents fungi, and the corticosteroid flumethasone as an anti-inflammatory and antiitching agent, and is used for a variety of skin conditions including psoriasis. In the past decennia, biologicals have changed the therapeutical landscape for IBD and psoriasis management. With the introduction of anti-tumour necrosis factor alpha (TNFa) biologicals Infliximab and Adalimumab, IBD and psoriasis patients got access to safe and efficient treatment, alleviating symptoms in 20 to 30% of patients. Soon after, anti-integrin, anti-interleukin 12/23, and Janus kinase (JAK) inhibitors were approved for use in IBD. This has led to fewer surgical interventions, mortality, and colorectal cancer rates. Despite all this success, the therapeutic success rates remain below expectations. Around 48% of biologies failed within eight months in psoriasis management, mostly due to a loss of efficacy. This so-called non-response rate for biologicals in IBD management is even higher, around 50-60%, with up to 70% of the CD patient population requiring surgery within 10 years after diagnosis. Not only does this cause a substantial societal and economic burden, it also underlies the high unmet need for novel therapeutical strategies for the management of psoriasis and IBD.

SUMMARY OF THE INVENTION

Haloquinolines like clioquinol (CQ), broxyquinoline and chloroxine activate the enzyme phosphoglycerate dehydrogenase (PHGDH) (WO2021185791A1, WO2022258629) which steers anti-inflammatory responses. Low doses of haloquinolines, provide profound anti-inflammatory activity in mouse models for various IMIDs like psoriasis and IBD.

Cumulative evidence supports the novel idea that the accumulation and differentiation of pro-resolving monocyte-derived macrophages (Mcps) is essential to limit chronic inflammation present during inflammation [Bain & Mowat (2014) Cell Immuno! 291, 41-48; Bain et al. (2013) Mucosal Immunol. 6, 498-510; Na et al. (2019) Nat Rev Gastroenterol Hepatol 16, 531-543]. Hence, steering Mcps towards a pro-resolving phenotype may represent a novel therapeutic approach to limit inflammation and promote tissue repair. Recently, the enzyme phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in the de novo serine biosynthesis, was identified as a key metabolic checkpoint in steering Mcps towards an anti-inflammatory phenotype [Wilson et al. (2020) Cell Rep 30, 1542-1552]. PHGDH is required for the expression of key anti-inflammatory molecules, such as Arginase 1 (Argl) and Resistin-like molecule alpha (Retnla). Hence, compounds that activate/potentiate PHGDH enzymatic activity, like the haloquinolines (WO202118579), can in theory be used to steer Mcps toward a pro-resolving phenotype.

While clioquinol binds zinc, PHGDH is a zinc independent enzyme.

In the present invention is shown that there was a significant increase wound healing under clioquinol treatment as compared to the vehicle control in a macrophage- dependent -and independent manner in human intestinal and skin cells. Wound healing is important to heal the damaged skin and gut barrier, thereby closing the cycle of continuous inflammation caused by infiltration of harmful pathogens. The present invention also identified a novel treatment for psoriasis-like lesions. More specifically, a cream consisting of a low dose (0.3%) of haloquinolines was found more effective in reducing psoriasis-like lesions in an imiquimod-induced psoriasis murine model. In addition, oral administration of clioquinol at 62.5 mg/kg/day in an acute colitis mouse model was more effective in reducing IBD readouts (diarrhoea, blood loss in stool, weight loss, and colonic architecture) as compared to its vehicle control and standard of care, prednisolone. This is also shown in a zebrafish colitis model, where colitis-related outcomes were improved under clioquinol treatment.

The invention is further summarised in the following statements.

1. Clioquinol or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of psoriasis or of inflammatory bowel disease.

2. The clioquinol or salt thereof for use according to statement 1, in the prevention or treatment of psoriasis.

3. The clioquinol or salt thereof for use according to statement 1 or 2, in the prevention or treatment of psoriasis, wherein the clioquinol is applied topically.

4. The clioquinol or salt thereof for use according to any one of statements 1 to 3, in a therapy without pharmaceutically active compounds comprising a sulfonamidegroup, or without adrenal corticosteroids.

Pharmaceutically active compounds comprising a sulfonamide-group include sulfanilamide, sulfadiazine, sulfamethazine, sulfamethoxazole, sulfasalazine, acetazolamide, furosemide, celecoxib, zonisamide, and tamsulosin.

Adrenal corticosteroids include cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, flumethasone, aldosterone and fludrocortisone.

5.The clioquinol or salt thereof for use according to any one of statements 1 to 4, in a therapy without sulfasalazine.

6.The clioquinol or salt thereof for use according to any one of statements 1 to 5, in a therapy without flumethasone.

7. The clioquinol or salt thereof for use according to an one of statements 1 to 6, in a therapy without sulfasalazine and without flumethasone.

8.The clioquinol or salt thereof for use according to any one of statements 1 to 7, in a therapy without adrenal corticosteroids, wherein clioquinol is applied as a cream comprising between 0,1 and 0,5 % (w/w), or between 0,2 and 0,4 % (w/w) clioquinol.

9.The clioquinol or salt thereof for use according to any one of statements 1 to 8, in a therapy without adrenal corticosteroids, wherein clioquinol is applied as a cream comprising between 0,3 % (w/w) clioquinol.

10. The clioquinol or salt thereof for use according to statement 1, in the prevention or treatment of inflammatory bowel disease.

11.The clioquinol or salt thereof for use according to statement 10, wherein clioquinol is formulated in a sirop.

12. The clioquinol or salt thereof for use according to statement 10 or 11, wherein clioquinol is administered at a dose of 2-6 mg/kg/day or 3-5 mg/kg/ day.

13. The clioquinol or salt thereof for use according to any one of statements 1 to 12, wherein clioquinol is administered at a dose of 4 mg/kg/day.

14. A pharmaceutical formulation for application on the skin, comprising clioquinol, characterised in the absence of pharmaceutically active compounds comprising a sulfonamide group, and/or without adrenal corticosteroids.

15. The pharmaceutical formulation according to statement 14, wherein the composition comprises between 0,1 and 0,5 % (w/w) clioquinol.

16.The pharmaceutical formulation according to statement 14 or 15, wherein the composition comprises 0,3 % (w/w) clioquinol.

17. A haloquinoline or pharmaceutically acceptable salt thereof for use in the prevention or treatment of an immune-mediated inflammatory diseases.

18. The haloquinoline or salt thereof for use according to statement 17, which is a 8- hyd roxyquinoline.

19. The haloquinoline or salt thereof for use according to statement 17 or 18, wherein the haloquinoline is selected from the group consisting of clioquinol, chloroxine and broxyquinoline.

20. The haloquinoline or salt thereof for use according to any one of statements 17 to 19, wherein the haloquinoline is clioquinol.

21. The haloquinoline or salt thereof for use according to any one of statements 17 to 20, wherein the immune-mediated inflammatory disease is psoriasis. Herein the haloquinoline can be topically applied.

22. The haloquinoline or salt thereof for use according to any one of statements 17 to 20, wherein the immune-mediated inflammatory disease is inflammatory bowel disease. Herein the haloquinoline can be administered orally, including controlled release formulation, releasing the compound in the intestine.

DETAILED DESCRIPTION

Figure legends

Figure 1. Indirect and direct effects of Clioquinol (CQ) on barrier wound healing in vitro. A) Visual representation showing the impact of conditioned medium from M2- and Ml-polarized macrophages treated with clioquinol on the wound healing capacities of murine intestinal epithelial MODE-K cells. The medium from M2-polarized macrophages with clioquinol demonstrates enhanced wound healing compared to that from Ml-polarized macrophages. B) Confluence of human intestinal epithelial CaCo2 cells treated with clioquinol compared to vehicle control over time in a wound healing assay (Incucyte ®). The data indicate a significant increase in cell confluence under clioquinol treatment, suggesting direct wound healing properties of clioquinol independent of macrophage mediation. C) The confluence of HaCaT cells under conditions of clioquinol treatment versus vehicle control, highlighting clioquinol's capability to enhance wound healing in skin cells independently of macrophage influence. All data are presented as mean ± SEM. **** = P < 0.0001.

Figure 2. Beneficial effect of oral clioquinol administration as compared to the vehicle control on colitis severity in mice. After a two week acclimatization period, mice were given 2.25% DSS in drinking water for 5 consecutive days to induce colitis symptoms. The disease activity index (DAI), assessing stool consistency, presence of occult blood, and weight loss, was recorded daily. The results show a significant reduction in DAI scores with clioquinol treatment as compared to the vehicle control, indicating improved colitis outcomes, n = 10/group.

Figure 3. Micro -and seemingly macroscopic improvement of murine DSS- colitis related manifestations under clioquinol treatment. A) Mice treated with clioquinol had a borderline significantly better Mouse Colitis Histology index as compared to vehicle control mice. The Mouse Colitis Histology index consists of the scores for goblet cell loss (P < 0.05), crypt density loss (P < 0.05), hyperplasia score (ns), and the submucosal infiltrate score (ns). B) Representative dl5 images of haematoxylin and eosin-stained DSS-treated colon of vehicle control and clioquinol treated mice. C) Colon length of vehicle and clioquinol treated mice. D) Macroscopical parameters for inflammation assessment. Data are presented as mean ± SD.

Figure 4. clioquinol improves DSS-related outcomes in a zebrafish larvae model of colitis. A) Co-administration of clioquinol, vehicle control DMSO and 0.70% of DSS shows an improved mortality under clioquinol, but not vehicle control, treatment in a zebrafish larvae model of DSS colitis (n = 30/group). B) Hindgut length is improved under clioquinol treatment. C) Eye diameter development remains unchanged under DSS treatment. D) WGA staining for goblet cells quantified by the integrated density on Image! shows a rescue of the loss of goblet cells in the hindgut in zebrafish larvae treated clioquinol under DSS regimen, but not in the vehicle control. Data are presented as mean ± SD. * = P < 0.05.

Figure 5. Beneficial effect of oral clioquinol administration as compared to the vehicle control and prednisolone on colitis severity in mice.

After a two week acclimatization period, mice were given 2.25% DSS in drinking water for 5 consecutive days to induce colitis symptoms. The disease activity index (DAI), assessing stool consistency, presence of occult blood, and weight loss, was recorded daily. The results show a significant reduction in DAI scores with clioquinol treatment as compared to the vehicle control and prednisolone, indicating improved colitis outcomes, n = 10/group. Data are presented as mean ± SEM. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Figure 6. Clioquinol, but not prednisolone, prevents colonic monocyte infiltration and thereby reduces inflammation in an acute DSS colitis model. Flow cytometric gating strategy was as follows: after doublet discrimination live immune cells (7AAD- CD45+) were gated further for myeloid populations (CDllb+, Ly6G-). CD64+ cells were further divided based on macrophage markers Ly6C and MHCII into the so-called monocyte-to-macrophage waterfall. Data are presented as mean ± SEM.

Figure 7. Efficacy of Low-Dose Topical Clioquinol in an Imiquimod-Induced Psoriasis Mouse Model

A) Schematic diagram illustrating the application protocol of imiquimod (IMQ) and clioquinol (CQ). B) Mice were exposed to IMQ for 5 days, followed by topical application of 200 mg of 0.3% clioquinol cream every other day. clioquinol was applied using a Finn chamber patch to localize treatment to the lesion site and prevent exposure to the control area. Starting from day 5, clioquinol application alternated with ongoing IMQ treatment. C) Daily scores for erythema, scaling, and thickness, measured across groups (n = 5 mice/group, with two technical replicates). D) The visual Psoriasis Area and Severity Index (PAST) score comprised the sum of erythema and scaling scores, while the comprehensive PAST score also included thickness measurements (n = 5 mice/group, with two technical replicates). E) Ratio of visual and comprehensive PAST scores comparing the control area to the clioquinol- treated area. Each mouse served as its own internal control (n = 5). Black arrows denote the points of clioquinol or control treatment. All data are presented as mean ± SEM.

Figure 8. Gene expression of Ml/M2-like murine bone marrow-derived macrophages treated with clioquinol/vehicle. All data are expressed as mean ± SEM. All data is normalized to rp!32.

Figure 9 : Gene expression of phgdh of control, Ml and M2-like murine bone- marrow derived macrophages. All data are expressed as mean ± SEM. All data is normalized to rp!32.

Figure 10: Gene expression of argl of control, Ml and M2-like murine bone- marrow derived macrophages. All data are expressed as mean ± SEM. All data is normalized to rp!32.

Figure 11 : Gene expression of retina of control, Ml and M2-like murine bone- marrow derived macrophages. All data are expressed as mean ± SEM. All data is normalized to rp!32.

Figure 12: Macrophage-specific CsflRiCre Phgdh knockout (phgdh ko) performs worse in a chronic DSS induced colitis compared to its wild-type littermate controls (phgdh wt). After a two week acclimatization period, mice were fed a tamoxifen containing chow for 5 weeks. Tamoxifen chow was continued and mice were given 2.25% DSS in drinking water for 5 consecutive days to induce colitis symptoms. After a washout period, DSS was introduced again in increments, and this was repeated for three cycles to induce a chronic phenotype. The disease activity index (DAI), assessing stool consistency, presence of occult blood, and weight loss, was recorded daily. The results show a significant increase in DAI scores in phgdh ko as compared to phgdh wt, indicating improved colitis outcomes, n = 5/group. Data are presented as mean ± SEM. * = P < 0.05, ** = P < 0.01, **** = P < 0.0001.

Figure 13: Spectral analysis of macrophages from the lamina propria of the colon of phgdh ko vs. phgdh wt mice subjected to chronic DSS colitis shows an increase of pro-inflammatory Ly6C+MHCII- monocytes and a decrease of TREM2+CD206+ anti-inflammatory macrophages in phgdh ko vs. phgdh wt. Cells were isolated from the lamina propria of the colon of mice subjected to chronic DSS colitis 10 days after the last DSS cycle and analysed by spectral flow cytometry. Data are presented as mean ± SEM. * = P < 0.05, ** = P < 0.01.

Immune Mediated Inflammatory Diseases (IMIDs) are a broad group of disorders characterised by inflammation that are found within the fields of gastroenterology, rheumatology and dermatology.

IMIDs include inflammatory bowel disease (IBD) (including the subtypes Crohn's and ulcerative colitis), uveitis, rheumatoid arthritis (R.A), psoriatic arthritis (PsA), psoriasis, systemic lupus erythematosus (SLE), ankylosing spondylitis, hidrosadenitis suppurativa, sarcoidosis, atopic dermatitis (AD), connective tissue disorders, asthma, and some neurological diseases, such as multiple sclerosis (MS).

The examples of the present invention show the involvement of macrophages in IMIDS such as IDB and psoriasis. Macrophages can be affected by a variety of factors to change their phenotype and thus affect their function. Activated macrophages are usually divided into two categories, Ml-like macrophages and M2-like macrophages. Both Ml macrophages and M2 macrophages are closely related to inflammatory responses, among which Ml macrophages are mainly involved in pro-inflammatory responses and M2 macrophages are mainly involved in anti-inflammatory responses. Modulating the activation state of macrophages is an effective method for the treatment of diseases [Yunna et al. (2020) Eur J Pharmacol 877, 173090]. The examples of the present invention demonstrate the effect of clioquinol activation state of macrophages. This provides evidence for the therapeutic effect of clioquinol and other haloquinolines on IMIDS in general.

One aspect of the invention relates to the use of clioquinol in the treatment of psoriasis. The prior art creams for psoriasis comprise corticosteroids as active ingredient against psoriasis and clioquinol for protecting the vulnerable skin against infections. It is a surprising finding that psoriasis can be treated with clioquinol only in the absence of corticosteroids, and this even at 10-fold lower doses of clioquinol compared with the prior art cream.

A second aspect of the invention relates to use of clioquinol in the treatment of inflammatory bowel disease (IBD). In IDB the microbiome of the gut is disturbed (dysbiosis), and use of antibiotics generally has a negative effect on IDB by further disturbing the microbiome of IDB patent. Considering the use of an antimicrobial against IDB is contra-intuitive. The fact that clioquinol, which is an antimicrobial has a positive effect on IDB is an unexpected finding.

The finding that clioquinol in the below mouse experiments is also effective at lower doses allows the use of clioquinol in the treatment of IBD.

Clioquinol is a lipophilic compound that passes the stomach with limited absorption, and is absorbed in the small intestine, especially the duodenum and jejunum. The microbiome which is present in the large intestine is thus not or minimally affected by the oral ingestion of clioquinol.

"haloquinoline" as used in the present invention relates to halogenated quinolines, typically quinolines with halogen groups at position 5 and 7.

Most particular it related to halogenated 8-hydroxyquinoline as depicted in formula I or pharmaceutically acceptable salts thereof:

Herein Ri and R.2 are a halogen, or Ri is a halogen and R2 is H, or Ri is H and R2 is a halogen.

Typically in dihalogenated compounds, Ri and R2 are each independently selected from the group consisting of fluorine (F), chlorine (Cl), bromine (Br), iodine (I).

More typically Ri and R2 are each independently selected from the group consisting of chlorine (Cl) iodine (I).

In specific embodiments at least one of Ri or R2 is chlorine.

In other specific embodiments at Ri is chlorine.

In specific embodiments at least one of Ri or R2 is bromine.

In preferred haloquinolines in the context of the present invention Ri= Cl and R2 is I (Clioquinol (iodochlorhydroxyquin)), or Ri= Cl and R2 = Cl (chloroxine), Ri= Br and R2 = Br (broxyquinoline), or Ri = Cl and R2 is H (cloxyquin) Clioquinol was considered safe and efficacious for many years. It was used as an antifungal and an antiprotozoal drug until it was linked to an outbreak of subacutemyelo-optic neuropathy (SMON), a debilitating disease almost exclusively confined to Japan.

Humans have been treated with Clioquinol at a usual dose of 1.5-2 g/day (-25-30 mg/(kg day)), with reports of patients receiving 3.5g (-50 mg/(kg day))/day without toxicity.

Ritchie et al. (2003) Arch Neurol. 60, 1685-1691 describes the use of clioquinol against Alzheimer, herein no adverse effects were apparent when using 750 mg/day for weeks (dose of 10 mg/kg/day). Moreover, dose-limiting neurotoxicity and abdominal pain were observed at a dose of 3200 mg/day (dose of 42.5 mg/kg/day). Thus in embodiments of the prevention or treatment of IMID concentration of clioquinol doses may range from 10 and 25 mg/kg/day to 50, 75, or 100 mg/kg/day (including all ranges with the above lower and upper limits).

In Japanese humans, administration of daily clioquinol has been associated with neurological side effects. Evidence was gathered that clioquinol inhibits cAMP- transporting ABC pumps (reviewed in Perez et al. (2019) Pharmacol Ther. 199, 155- 163). A key concept is the ability of clioquinol to block cAMP efflux from cells and thus, to trigger the phosphorylation of CREB Serl33, a classical cAMP effector that activates target genes. This finding provided a connection to possible targets of clioquinol: ABCC4 and ABCC11, transporters that normally efflux numerous endogenous substrates, including cAMP. A further analysis revealed the presence of SNPs in both ABCC4 and ABCC11, capable of reducing transporter function and at the same time present with a high frequency in the Japanese population. Modern studies showed that these SNPs are critical for patient sensitivity to cancer and immunosuppressor nucleotide-like drugs, substrates of ABCC4 and ABCC11 transporters. Thus, this line of research provides a plausible explanation for the SMON phenomenon: patients that carry SNPs in ABC transporters that dramatically affect nucleotide efflux are expected to be more sensitive to clioquinol. Since these SNPs are geographically restricted to Japan, this also accounts for the specific distribution of the disease. Thus in specific embodiments, the treatment or prevention of the present invention is not used for persons having SNPS in ABCC4 or ABCC11, or is more generally not used for persons or Japanese origin.

Clioquinol has been described as a chelator for zinc and copper. Hence it been uses as a shuttle for conditions with a zinc deficiency, as well as chelators for scavenging excess metals.

As PHGDH, the target for claimed conditions, is metal ion independent, both the use of haloquinolines without complexed metals and haloquinolines with complexed metals are envisaged.

EXAMPLES

Example 1: Clioquinol induces increased wound healing in both a macrophage-dependent and independent manner in human intestinal epithelial cells and keratinocytes in vitro.

In patients with IBD and psoriasis, there is an impairment in barrier wound closure leading to a cycle of inflammation. To assess the barrier closing potential of clioquinol, an in vitro wound healing assay was employed. Murine intestinal epithelial cells (MODE-K) were seeded at a density of 15,000 cells per well (96-well plate; Imagelock Incuyte). After 24h, a uniform scratch was made in the cell monolayer using the IncuCyte® Wound Maker tool. Cells were treated with pre-conditioned bone-marrow derived macrophage (BMDM) medium that were previously isolated and cultured with either LPS from E. Coli and IFNy (100 ng/mL; Sigma-Aldrich L6529, 50 ng/mL; Peprotech 315-05) in the presence or absence of clioquinol (Sigma, 233165). Human intestinal epithelial cells (CaCo2) and human keratinocytes (HaCaT) cells were seeded at a density of 25,000 cells per well (96-well plate; Imagelock Incuyte) for the wound healing assay. 24h after seeding, a uniform scratch was made. Subsequently, cells were incubated with increasing concentrations of clioquinol in medium and 0.16% DMSO (Sigma, D2650) or control (0.16% DMSO). Images at baseline were taken immediately after creating the open wound area and cells were imaged every 4h using the IncuCyte live cell imaging system at 10X magnification. For each time point, relative wound closure (confluence) was calculated using the Scratch Wound analyses pipeline of the IncuCyte ZOOM™ software (Figure 1A). In MODE-K cells, a direct effect on wound healing capacities of M2-polarized macrophage medium treated with clioquinol was observed, as opposed to Mi- polarized macrophage medium (Figure 1A). Interestingly, clioquinol on its own also seemed to exert wound healing functions. Therefore, the effect of clioquinol directly on CaCo2 and HaCaT cells was tested. Here, there was a significant increase in confluence under clioquinol conditions as compared to the vehicle control indicating macrophage-dependent -and independent manners of clioquinol-driven wound healing in the intestines and skin (Figure 1B-C).

Example 2: Oral administration of Clioquinol leads to an improvement of colitis symptoms in a murine and zebrafish model of colitis

Female C57BL/6J (8-10 weeks, 18-20g, ENVIGO®) were maintained at the KU Leuven animal facility on a 12h hour light-dark cycle and had ad libitum access to tap water and commercially available chow (Ssniff®). All experimental procedures were approved by the Animal Care and Animal Experiments Ethical Committee of KU Leuven (188/2019). Colitis was induced by administration of 2.25% DSS (TbB Consultancy AB) in drinking water for 5 days followed by normal drinking water. After 10 or 15 days, mice were sacrificed by CO2 overdose. Spleen and colon length/weight were recorded. During the whole duration of the experiment, mice were scored daily using the disease activity index (DAI) score. The DAI consists of the following parameters: stool consistency (0-4), presence or absence of occult blood in the stool (0-4) (Hemoccult, Beckman Coulter), and weight loss (0-4) [Cooper et al. (1993) Lab Invest 69, 238-249]. Mice treated with clioquinol orally had less diarrhoea and blood loss in the stool compared to the vehicle control (Figure 2). Overall, clioquinol significantly improved colitis-related disease outcomes, as reflected in the DAI average score.

After sacrificing, colon length and weight as well as spleen weight were assessed as a direct measure for the severity of the inflammation. In addition, (part of) the colon was immediately fixed in 4% formalin, dehydrated in 100% ethanol, and embedded in paraffin. A hematoxylin and eosin (H&E) staining was performed to determine the rate of damage and inflammation in the colon. Each section was blindly scored using a validated scoring system [Koelink et al. (2018) J Crohns Colitis 12, 794-803] and the total score is reflected in the Mouse Histology Index (MHI). Mice treated with clioquinol had significantly less goblet cell loss and a lower crypt density (Figure 3 A- B). Overall, clioquinol treatment improved gut barrier-related healing, as reflected in the lower MCHI score (borderline), clioquinol-treated mice also had a seemingly improved colon weigh/length ratio and lower spleen weight, indicating less immune cell infiltration in the gut possibly (Figure 3 C-D). All zebrafish experiments were performed according to the rules and regulations of the Ethical Committee of KU Leuven, in compliance with the regulations of the European Union (EU) concerning the welfare of laboratory animals as declared in Directive 2010/63/EU. Zebrafish larvae were used from 72 hpf (3 dpf) until a maximum of 120 hpf (5 dpf). Wild type AB adult zebrafish were maintained in the aquatic facility of the KU Leuven on a 14/10 h light/dark cycle at 28°C. Zebrafish larvae were placed in a solution of Danieau's containing 0.70% of DSS, with or without clioquinol/vehicle (DMSO), from 3 dpf until 5 dpf. The solution was replaced daily and moribund larvae were removed, clioquinol treatment was able to reverse colitis-induced mortality as well as hindgut shortening as compared to the vehicle control (Figure 4 A-B). The eye diameter was the same in every group, indicating no impairment of larvae development, which could be a possible confounder for gut length (figure 4 C). A wheat germ agglutin (WGA) staining was used to stain mucusproducing cells in the gut. A decrease of WGA signal was observed in larvae treated with DSS, which was reversed by clioquinol treatment (Figure 4 D). These results indicate that clioquinol can improve colitis-related outcomes in zebrafish larvae and additionally shows that zebrafish larvae are a good platform for further drug screening.

Example 3: Oral administration of Clioquinol leads to an improvement of colitis-induced symptoms as compared to Prednisolone

The same setup as in example 2 was used, only this time a third group of mice were treated with prednisolone, which is used in the clinical as a first-line standard of care. Mice were given DSS through drinking water and at the peak of their symptoms (around day 5), DSS was replaced with water and treatment with either clioquinol, prednisolone or vehicle was started daily through oral gavage. Mice treated with clioquinol performed significantly better in terms of body weight gain, less diarrhoea and blood loss in the stool (Figure 5). This all is reflected in the DAI average, in which clioquinol mice have a significant improvement of colitis-related outcomes as compared to the standard of care and vehicle control.

The colon of mice that were sacrificed on day 10 was used for further flow cytometric analysis. The lamina propria was digested and a single cell suspension was acquired. Stained cells were analysed on the BD FACSymphony A5 (California Mountain Snake). First, all events were gated, followed by doublet discrimination and live immune cells (7AAD- CD45+). Live immune cells were gated further for myeloid populations (CDllb+, Ly6G-). CD64+ cells were further divided based on macrophage markers Ly6C and MHCII. There seems to be less overall immune cell infiltration (7AAD- CD45+) in clioquinol treated mice as compared to the vehicle control and prednisolone-treated mice. This difference could be driven by less mononuclear infiltration into the lamina propria as shown by a seemingly decreased numbers of monocytes (MHCII-, Ly6C+) and intermediate macrophages (MHCII+, LyC6+).

Example 4: Low-dose topical Clioquinol administration ameliorates clinical lesion outcomes in an Imiquimod-induced psoriasis mouse model

Female Balb/c mice (8-10 weeks, 18-20g) were maintained at the KU Leuven animal facility on a 12h hour light-dark cycle and had ad libitum access to tap water and commercially available chow (Ssniff®). WT Balb/cAnNCrl (Charles River Laboratories) were bred at the KU Leuven animal facility and all experimental procedures were approved by the Animal Care and Animal Experiments Ethical Committee of KU Leuven (080/2022). Dorsal hair was removed by electrical shaving followed by hair removal cream (Veet®) under sedation with isoflurane (Iso-Vet, one day prior to the application of Imiquimod (Aldara®, Meda Pharma). Two Finn chambers (8 mm FinnChambers on Scanpor, SmartPractice, USA) were placed on the dorsal skin of each mouse, filled with 25 mg Aldara according to the protocol described previously by Horvath et al. (2019) Sci Rep, 9. 3685. Imiquimod treatment was performed daily for five consecutive days, with the Finn chambers being replaced daily (Figure 8A). All procedures were done under isoflurane (Iso-Vet) anaesthesia. After five days, 0.3% clioquinol cream was administered to one of the lesions using a Finn chamber. The other lesion was covered with an empty Finn chamber. Every other day, Imiquimod treatment was repeated to ensure lesion progression. Mice were sacrificed after 10 days. During the whole duration of the experiment, mice were scored daily using the PASI score [Neu et al. (2021) Antioxidants 10, 1338]. The colour of the back skin (erythema) was scored on a scale from 0-3 with 0 (no redness, pale pink), 1 (pale pink with <20% red patches), 2 (pink with 20-60% red patches), and 3 (pink/red with >60% red patches). Scaling score (0-3) was based on the percentage of the area that had patches (0; 0%, 1; <20%, 2; 20-60%, 3; 60%). The thickness score (0-4) was based on the increase of two-fold skin thickness compared with day 0 (0; <20%, 1; >20-39%; 2; 40-59%, 3; 60-79%, 4; >80%). Scores were cumulatively compiled and a visual PASI score (sum of erythema and scaling) and PASI score (sum of erythema, scaling, and thickness) were calculated. For the ratio calculation, 1 point was added to each score and then the ratio of the untreated area over the treated area was taken. Mice subjected to topical clioquinol treatment had a rapid significant improvement of the psoriasis-like lesion as compared to the internal controls, which is reflected in a decreased erythema and scaling score (Figure 8 B-C). There was a significant improvement in both the PAST and visual PAST score already one day after clioquinol treatment and after repeated administration (Figure 8 D). By taking the ratio of the (visual) PAST scores for the clioquinol-treated area over the control area internally, a beneficial effect of clioquinol treatment was observed (Figure 8 E).

Example 5: Clioquinol (CQ) drives Ml to M2 transition and inhibits M2 to M l transition of macrophages

Bone marrow-derived macrophages (BMDM) were isolated from healthy wild type C57BL mice as described in Toda et al. (2020) STAR Protoc 2, 100246. After 7 days of culture in DMEM supplemented with L929 serum, macrophages were harvested and seeded into a 6-well plate with 1x10^6 cells per well. After attaching, cells were pre-incubated for 1 hour with different concentrations of clioquinol or vehicle (0.1 DMSO%) after which an M2 (IL-4; 20 ng/mL) or Ml (LPS; 20 ng/mL and IFNy; 50 ng/mL) stimulus was added on top for 6h before cell lysis and RIMA extraction for further processing with the RNAeasy Mini Kit (Qiagen). In case of a double-hit, macrophages were stimulated overnight with IL-4 followed by washing, preincubation of lh with clioquinol or vehicle, and LPS/IFNy stimulation for 6h. qPCR was performed using primer sets for Phgdh, il6, ccl5, nos2, illb, retnla and argl.

Results: clioquinol stimulates the anti-inflammatory macrophage phenotype, steering the phenotype away from the inflammatory phenotype toward a more pro-resolving phenotype

Increasing concentrations of clioquinol result in increased phgdh expression in M2- like macrophages (Fig. 8 A). In M2-like macrophages, challenged with a Ml stimulus (LPS/IFNy), clioquinol is able to prevent a strong induction of the Ml-like phenotype, depending on the concentration of clioquinol, as seen in a seemingly lower 116 expression (Fig. 8 B), cc/5 expression (3 pM clioquinol, Fig. 8 C) and nos2 expression (Fig. 8 D). When clioquinol is administered to Ml-like macrophages directly, it reduces the expression of Ml-like associated inflammatory genes illb (Fig. 8 E), cc/5 (Fig. 8 F), and nos2 (Fig. 8 G) as compared to the vehicle control. On top, clioquinol is able to, in increasing concentrations, boost argl expression in Ml-like macrophages (Fig. 8 H). Arginasel (Argl) is a hallmark feature for anti-inflammatory M2-like macrophages.

Example 6. clioquinol induces PHGDH expression and macrophage polarisation toward anti-inflammatory M2 status.

Figure 9 shows that phgdh expression is a hallmark of IL-4 treated anti-inflammatory M2 macrophages, characterized by the expression of key anti-inflammatory molecules Arginase-1 (Argl) and resistin-like molecule alpha (Retnla) as shown in Figure 10 and Figure 11 respectively (BMDM). Figure 8 shows that phgdh expression is elevated in clioquinol treated macrophages and that CQ downregulates Ml-related genes (Nos2, IL6) and upregulates M2-related genes (argl and retnla in supplementary figure 8F and 8G.

Example 7: macrophage-derived phgdh plays a crucial role in the resolution of inflammation in a mouse model of colitis

Macrophage-specific Phgdh-knockout mice phgdhⁿⁱⁿ CsflR\Cre) and wild-type littermate controls were subjected to 5 weeks of tamoxifen chow diet (SNIFF, BioServices) to induce timely deletion of Phgdh in macrophages specifically. Mice were then subjected to a chronic DSS colitis setup, with three cycles of DSS with a washout period for recovery in between. Macrophage-specific Phgdh-knockouts had a significantly worse disease activity outcome compared to their wild-type littermates (Figure 12). This difference is likely driven by changes in the macrophage population, as is seen by a reduction in increase in Ly6c+ MHCII- pro-inflammatory monocytes and a decrease of TREM2+ CD206+ anti-inflammatory macrophages in the knockout (Figure 13).

Claims

1. Clioquinol or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of psoriasis or of inflammatory bowel disease.

2. The clioquinol or salt thereof for use according to claim 1, in the prevention or treatment of psoriasis.

3. The clioquinol or salt thereof for use according to claim 1 or 2, in the prevention or treatment of psoriasis, wherein the clioquinol is applied topically.

4. The clioquinol or salt thereof for use according to any one of claims 1 to 3, in a therapy without pharmaceutically active compounds comprising a sulfonamidegroup, or without adrenal corticosteroids.

5. The clioquinol or salt thereof for use according to any one of claims 1 to 4, in a therapy without sulfasalazine.

6. The clioquinol or salt thereof for use according to any one of claims 1 to 5, in a therapy without flumethasone.

7. The clioquinol or salt thereof for use according to an one of claims 1 to 6, in a therapy without sulfasalazine and without flumethasone.

8. The clioquinol or salt thereof for use according to any one of claims 1 to 7, in a therapy without adrenal corticosteroids, wherein clioquinol is applied as a cream comprising between 0,1 and 0,5 % (w/w) clioquinol.

9. The clioquinol or salt thereof for use according to any one of claims 1 to 8, in a therapy without adrenal corticosteroids, wherein clioquinol is applied as a cream comprising between 0,3 % (w/w) clioquinol.

10. The clioquinol or salt thereof for use according to claim 1, in the prevention or treatment of inflammatory bowel disease.

11. The clioquinol or salt thereof for use according to claim 10, wherein clioquinol is formulated in a sirop.

12. The clioquinol or salt thereof for use according to claim 10 or 11, wherein clioquinol is administered at a dose of 2-6 mg I kg/ day.

13. The clioquinol or salt thereof for use according to any one of claims 1 to 12, wherein clioquinol is administered at a dose of 4 mg / kg/ day.

14. A pharmaceutical formulation for application on the skin, comprising clioquinol, characterised in the absence of pharmaceutically active compounds comprising a sulfonamide group, and/or without adrenal corticosteroids.

15. The pharmaceutical formulation according to claim 14, wherein the composition comprises between 0,1 and 0,5 % (w/w) clioquinol.

16. The pharmaceutical formulation according to claim 14 or 15, wherein the composition comprises 0,3 % (w/w) clioquinol.