Nitroxoline for use in the treatment of cutaneous neurofibromas

Abstract

The present invention relates to a composition comprising nitroxoline or a pharma- ceutically acceptable salt thereof for use in the treatment or prevention of cutaneous neurofibromas.

Description

[Technical field]

[0001] The present invention relates to a new use of nitroxoline. [Background technology]

[0002] Neurofibromas are benign nerve sheath tumors of the peripheral nervous system. In 90% of cases, they are found as independent tumors, while the remainder are found in patients with neurofibromatosis type I (NF1), an autosomal dominant genetic disorder. Neurofibromas can cause a variety of symptoms, ranging from physical disfigurement and pain to cognitive impairment, and can transform into malignant tumors.

[0003] Cutaneous (or dermal) neurofibromas (cNFs) originate from the nerves of the skin and are usually associated with a single peripheral nerve. All neurofibromas consist of a mixture of NF1-mutated Schwann cells (SCs) and other neurofibrous elements such as axons, fibroblasts, mast cells, macrophages, and endothelial cells. Three types of cNFs are distinguished: 1) discontinuous cNFs: sessile or pedunculated masses on the skin that are fleshy and non-tender and of variable size; 2) discontinuous subcNFs: located beneath the skin, appearing as skin bumps and sometimes tender; and 3) deep nodular neurofibromas, which affect tissues and organs beneath the dermis but are otherwise similar to cutaneous and subcutaneous neurofibromas.

[0004] NF1 is caused by germline mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Neurofibromin functions as a GTPase-activating (GAP) protein, inactivating the intracellular signaling protein Ras by converting its active GTP-bound form to its inactive GDP-bound form. This in turn leads to downregulation of Ras activity. Loss of neurofibromin activity increases Ras activity, which promotes the transcription of many genes required for cell growth and proliferation. Cutaneous neurofibromas occur in at least 99% of NF1 patients and are derived from the Schwann cell lineage in the dermis.

[0005] cNFs usually develop during adolescence and tend to increase in number throughout life, so that their number can reach several thousand. Although these tumors are benign, they are disfiguring, often itchy and painful, and therefore have a significant impact on quality of life.

[0006] There have also been significant efforts to identify pharmacological targets to treat cutaneous neurofibromas. Cutaneous neurofibromas in particular have been frequent targets of repurposing efforts and repositioning of drugs in development. Many different standards and methods have been applied to this task. In many cases, repurposing candidates have been identified primarily based on clinical pattern matching, whereas in other cases, the basic disease mechanisms have been extensively studied to identify therapeutic targets, followed by thorough preclinical validation.

[0007] There are currently no approved drugs for cNF. Physical removal remains the most effective method for treating cNF, i.e., surgical excision or destruction with CO2 laser, electrodesiccation, and ablation. Challenges facing removal include tumor regrowth due to incomplete excision, significant scarring, and cost burden. A pilot phase II trial study of selumetinib in treating patients with neurofibromatosis type 1 and cutaneous neurofibromas is ongoing. However, it is currently unclear how successful this will be. Selumetinib is a selective inhibitor of mitogen-activated protein kinase kinases (MAPK kinases, MEK, MAP2K, and MAPKK) with the systematic name 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide.

[0008] Overall, efforts to treat cutaneous neurofibromas have offered some intriguing possibilities but have not achieved conclusive success despite many efforts, highlighting the need for new therapeutic approaches.

[0009] Nitroxoline has been used in humans as an antibiotic and, although not widely used, has been commercially available since the 1960s. It is used to treat or prevent biofilm infections, such as urinary tract infections. It is particularly effective at disrupting biofilms, and the cause of this action is thought to be its metal cation chelating properties. Nitroxoline is metabolized in the liver to the corresponding sulfate and glucuronide metabolites. There is evidence that both of these metabolites share antibacterial activity. It is also used in anticancer therapy via its antiproliferative effects. The systematic name for nitroxoline is 5-nitroquinolin-8-ol. Summary of the Invention

[0010] The present invention relates to a composition comprising nitroxoline or a pharma- ceutical acceptable salt thereof for use in the treatment or prevention of cutaneous neurofibroma. As is evident from the in vitro data presented below, nitroxoline is effective in the treatment and prevention of cutaneous neurofibroma.

[0011] A first aspect of the invention is a composition comprising nitroxoline, or a pharma- ceutically acceptable salt thereof, for use in the treatment or prevention of cutaneous neurofibroma.

[0012] A second aspect of the invention is the use of nitroxoline, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment or prevention of cutaneous neurofibroma.

[0013] A third aspect of the invention provides a method of treating or preventing cutaneous neurofibroma, the method comprising administering to a patient a composition comprising nitroxoline or a pharma- ceutically acceptable salt thereof. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1. Preparation and passaging of suspension sphere cultures for in vitro studies. [Diagram 2]FIG. 1 shows the dose-response effect of nitroxoline on the proliferation of Tom+NF1+ / − and Tom+NF1− / − cells. [Diagram 3] FIG. 1 shows the dose-response effect of nitroxoline in inducing cell death in Tom+NF1+ / − and Tom+NF1− / − cells. [Figure 4] Low magnification (5x) of fluorescent cNF tumor cells in mouse skin. A-D are representative images of Tom+ cells from two mice 1 month after vehicle treatment. E-H are representative images of Tom+ cells from two mice 1 month after nitroxoline treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In the present invention, as demonstrated by the following in vitro and in vivo data, nitroxoline inhibits cell proliferation and increases apoptosis in tumor cells isolated from cutaneous neurofibromas from NF1- / - mouse models, and is therefore an effective treatment for cutaneous neurofibromas. Preferably, nitroxoline is used for the treatment or prevention of cutaneous neurofibromas, and the subject has neurofibromatosis type I.

[0016] The term "treatment" or "treating" as used herein refers to curative (curative) and / or ameliorative treatment (improvement of the patient's condition), including reducing the size of cutaneous neurofibromas or the number of cNFs. The term "prevention" or "preventing" as used herein refers to "prophylactic" treatment to prevent the formation of cNFs. This includes administering the compositions of the invention to patients who have NF1 but have not developed cutaneous neurofibromas, or to patients who have cNFs but are aiming to prevent further development.

[0017] "Patient" and "subject" are used interchangeably and refer to a subject to which nitroxoline is administered. Preferably, the subject is a human. Suitably, the subject has neurofibromatosis type I. In one embodiment, the subject is an adult. "Adult" refers to a person aged 18 years or older. In another embodiment, the subject has reached puberty, e.g., the subject is between 8 and 18 years of age.

[0018] In one embodiment, nitroxoline is used for the treatment or prevention of cutaneous neurofibromas and the patient has undergone or will undergo surgery to remove some or all of the cutaneous neurofibroma. This may be particularly advantageous when the cutaneous neurofibroma is large and / or has spread beyond tissue boundaries such that it would be difficult to remove all of it by surgery and / or it would be desirable / beneficial to remove at least some of it quickly.

[0019] The term "surgery" has its usual meaning in the art. Surgery is an invasive technique based on physical intervention into an organ / system / tissue for diagnostic or therapeutic reasons.

[0020] As used herein, a pharma- ceutically acceptable salt is a salt with a pharma- ceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid, and organic acids such as citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, salicylic acid, stearic acid, benzenesulfonic acid or p-toluenesulfonic acid. Pharmaceutically acceptable bases include hydroxides of alkali metals (e.g., sodium or potassium) and alkaline earth metals (e.g., calcium or magnesium), and organic bases such as alkylamines, arylamines or heterocyclic amines.

[0021] The present invention is directed to a composition comprising nitroxoline or a pharma- ceutically acceptable salt thereof for use in the treatment or prevention of cutaneous neurofibromas.

[0022] In an alternative embodiment, the present invention is directed to a composition comprising nitroxoline or a pharma- ceutically acceptable salt thereof for use in the treatment or prevention of cutaneous neurofibroma, wherein nitroxoline is the only active agent in the composition. By only active agent, it is meant that the composition does not contain any other ingredient that can be used in the treatment or prevention of cutaneous neurofibroma. In an alternative embodiment, the composition further comprises a second active agent for treating cutaneous neurofibroma, preferably the second active agent is selumetinib or a pharma- ceutically acceptable salt thereof.

[0023] In an alternative embodiment, the present invention is directed to a composition comprising nitroxoline, or a pharma- ceutically acceptable salt thereof, for use in combination with a second composition comprising selumetinib, or a pharma- ceutically acceptable salt thereof, wherein the two compositions are administered to a subject simultaneously, separately or sequentially.

[0024] As used herein, "separate" administration means that the drugs are administered as part of the same overall dosing regimen (which may include several days), but preferably on the same day. As used herein, "concurrently" means that the drugs are taken together or formulated as a single composition. As used herein, "sequentially" means that the drugs are administered at about the same time, preferably within about one hour of each other. Preferably, the drugs are administered simultaneously, i.e., taken together or formulated as a single composition. Most preferably, the drugs are formulated as a single composition.

[0025] The composition of the present invention may contain a pharma- ceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant any diluent or excipient, such as a filler or binder, that is compatible with other components of the composition and is not harmful to the recipient. The pharma- ceutically acceptable carrier can be selected according to standard pharmaceutical practice based on the desired route of administration. In the present invention, the composition can be administered in various dosage forms. In one embodiment, the composition can be formulated in a form suitable for oral, rectal, parenteral, intranasal or transdermal administration, or for administration by inhalation or suppository.

[0026] The composition can be orally administered, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. Preferably, the composition is formulated to be suitable for oral administration, for example, tablets and capsules. Tablets and capsules can be prepared using binders such as syrup, acacia, gelatin, sorbitol, tragacanth, cellulose, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, or silica, and surfactants such as sodium lauryl sulfate. Liquid compositions may contain conventional additives such as suspending agents such as sorbitol syrup, methylcellulose, sugar syrup, gelatin, carboxymethylcellulose, or edible fats, emulsifiers and surfactants such as lecithin or acacia, vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil, and preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). The liquid composition can be encapsulated, for example, in gelatin to provide a unit dosage form.

[0027] The compositions can also be administered parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.

[0028] The composition can also be administered by inhalation.The advantage of inhalation is that it is delivered directly to areas with a rich blood supply, compared to many drugs taken by oral route.Therefore, absorption is very rapid, since the alveoli have a huge surface area and a rich blood supply, and first-pass metabolism is avoided.

[0029] The invention also provides an inhalation device comprising a composition of the invention, typically a metered dose inhaler (MDI) that includes a pharma- ceutically acceptable chemical propellant to propel the medicament out of the inhaler.

[0030] The composition can also be administered by intranasal administration. The highly permeable tissue of the nasal cavity is highly receptive to drugs and absorbs them quickly and efficiently. Nasal drug delivery is less painful and invasive than injections, reducing patient anxiety. Absorption is very fast in this method and first-pass metabolism is usually avoided, reducing inter-patient variability. The present invention also provides an intranasal device comprising the composition according to the present invention.

[0031] The composition can also be administered by transdermal administration. For topical delivery, transdermal and transmucosal patches, creams, ointments, jellies, solutions or suspensions, or microneedling can be used. Thus, the present invention also provides a transdermal patch comprising the composition.

[0032] The composition may also be administered by sublingual administration. Accordingly, the present invention also provides a sublingual tablet comprising the composition.

[0033] The compositions may also be formulated with agents that reduce degradation of the substance by processes other than the patient's normal metabolism, such as antibacterial agents or inhibitors of protease enzymes that may be present in the patient, or in commensals or parasites living on or within the patient, and which can degrade the compound.

[0034] Liquid dispersions for oral administration may be syrups, emulsions, and suspensions. Suspensions and emulsions may contain as a carrier, for example, a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharma- ceutically acceptable carrier, for example, sterile water, olive oil, ethyl oleate, glycols (e.g., propylene glycol), and, if desired, an appropriate amount of lidocaine hydrochloride.

[0035] The solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile aqueous isotonic saline solutions.

[0036] In one embodiment of the invention, the composition is administered in an amount effective to treat or prevent cutaneous neurofibroma. The effective amount will be apparent to one skilled in the art and will depend on many factors, including age, sex, and weight, and a physician will be able to determine the effective amount.

[0037] In a preferred embodiment, the composition contains 30 mg to 600 mg, preferably 50 mg to 500 mg, more preferably 100 mg to 400 mg, even more preferably 150 mg to 350 mg, and most preferably 200 mg to 300 mg of nitroxoline.

[0038] The compositions may be administered once daily, twice daily, three times daily, or four times daily.

[0039] In one embodiment of the present invention, the composition is administered at least once a day. Preferably, it is administered as a single daily dose. Preferably, the single daily dose is 90 mg to 1800 mg of nitroxoline, preferably 150 mg to 1500 mg, more preferably 300 mg to 1200 mg, even more preferably 450 mg to 1050 mg, and most preferably 600 mg to 900 mg.

[0040] In one embodiment of the invention, the composition is administered twice daily, preferably with each dose being between 45 mg and 900 mg of nitroxoline, preferably between 75 mg and 750 mg, more preferably between 150 mg and 600 mg, even more preferably between 225 mg and 525 mg, and most preferably between 300 mg and 450 mg.

[0041] In one embodiment of the invention, the composition is administered three times a day, preferably with each dose being 30 mg to 600 mg of nitroxoline, preferably 50 mg to 500 mg, more preferably 100 mg to 400 mg, even more preferably 150 mg to 350 mg, and most preferably 200 mg to 300 mg.

[0042] In one embodiment of the invention, the composition is administered four times a day, preferably with each dose being 15 mg to 500 mg of nitroxoline, preferably 50 mg to 400 mg, more preferably 100 mg to 300 mg, even more preferably 125 mg to 225 mg, and most preferably 150 mg to 200 mg.

[0043] Preferably, the dosing regimen is such that the total daily dose of nitroxoline does not exceed 1500 mg.

[0044] Suitably, an effective amount of nitroxoline results in an intracellular concentration of 1-75 μM, preferably 5-50 μM, more preferably 10-40 μM.

[0045] Preferably, the composition comprising nitroxoline and the second composition comprising a second active agent, preferably selumetinib, are administered once a day.Preferably, the two compositions are administered simultaneously, i.e., nitroxoline and selumetinib are taken together.The compositions can also be administered sequentially, i.e., at about the same time, preferably within about one hour of each other.

[0046] In embodiments where the composition comprises selumetinib, or where the composition is for use in combination with a second composition comprising selumetinib, suitably the composition comprising selumetinib comprises from 1 mg to 75 mg of selumetinib, preferably from 5 mg to 50 mg of selumetinib, more preferably from 10 mg to 35 mg of selumetinib, and most preferably from 15 mg to 30 mg of selumetinib.

[0047] Preferably, the effective amount of selumetinib administered to a subject is 1 mg / m 2 ~75mg / m 2 , preferably selumetinib 5 mg / m 2 ~50mg / m 2 , more preferably selumetinib 10 mg / m 2 ~35mg / m 2 , most preferably selumetinib 15 mg / m 2 ~30mg / m 2 It is.

[0048] To treat or prevent cutaneous neurofibromas, compositions containing nitroxoline are used in a chronic regimen, i.e., long-lasting, chronic treatment, preferably lasting for at least 1 month, preferably at least 2 months, for example at least 3 months.

[0049] The present invention also relates to a kit for simultaneous, separate or sequential use in the treatment or prevention of cutaneous neurofibroma, the kit comprising (i) at least one dose of nitroxoline or a pharma- ceutically acceptable salt thereof, and, optionally, (ii) at least one dose of selumetinib or a pharma- ceutically acceptable salt thereof.

[0050] The present invention also relates to the use of nitroxoline or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment or prevention of cutaneous neurofibroma. This embodiment of the invention may have any of the preferred features described above.

[0051] The present invention also relates to a method of treating or preventing cutaneous neurofibromas, the method comprising administering to a patient a composition comprising nitroxoline or a pharma- ceutically acceptable salt thereof. This embodiment of the invention may have any of the preferred features described above. The method of administration may follow any of the routes described above.

[0052] For the avoidance of doubt, the present invention also encompasses pro-drugs which react in vivo to produce the compounds of the present invention.

[0053] Experimental Section Example 1 - In vitro drug testing using Tomato (Tom)+ stem-like glial cells, the origin of cNF In this study, we use an Nf1-KO mouse line (Prss56Cre / +, R26tdTom / +, NF1fl / fl) that develops cutaneous neurofibromas (cNFs) that faithfully recapitulate the human disease (Radomska et al., 2019). In this model, simultaneous biallelic loss of Nf1 and expression of the Tomato fluorescent reporter were targeted to glial stem-like cells at the origin of cNFs. This assay was performed ex vivo using non-adherent cells capable of amplifying Tomato expressing Nf1- / - and Nf1+ / - stem-like cells from skin to further characterize their properties. In this experimental condition, all differentiated cells die rapidly (24 h), but glial stem-like cells at the origin of cNFs survive, proliferate, and form neurospheres (multicellular compact structures) that can proliferate for long periods, thus retaining their in vivo properties. In this system, Nf1- / - cells express high levels of p-ERK (as expected due to persistent activation of the RAS pathway), proliferate much faster compared to controls, and retain the ability to reform spheres after dissociation and replating. Furthermore, we observed that incubation of Nf1- / - and Nf1+ / - cells with selumetinib (a MEK1 / 2 inhibitor) reduced proliferation activity and promoted mutant death without affecting Nf1+ / - cells, supporting the robustness of our in vitro system for performing drug screening studies.

[0054] This model was used to evaluate the effect of nitroxoline on the proliferation and cell death of Nf1− / − tumor cells.

[0055] Model characterization In this in vitro culture system, we use Tomato (Tom)+ stem-like glial cells, which are the origin of cNFs that can amplify and proliferate for long periods of time. Briefly, skin from young mutant (NF1- / -) and control (NF1+ / -) mice was dissected and dissociated, and the cell suspension was incubated under non-adherent conditions in the presence of two mitogens, FGF and EGF. In these conditions, only stem-like cells survive and proliferate, forming dense multicellular floating structures called neurospheres. In addition to Tom+ glial stem-like cells, skin contains various other types of stem cells (Tom-), so neurospheres are composed of a mixture of Tom+ and Tom- cells. In mutant conditions, neurospheres contain Tom+, Nf1- / - and Tom-, Nf1+ / + cells, whereas in Nf1+ / - control conditions, neurospheres are composed of Tom+, Nf1+ / - and Tom-, Nf1+ / + cells. In this study, drug effects were analyzed in Tom+ cells derived from NF1- / - and NfF1+ / - mice.

[0056] Experimental design Cell preparation, clonal expansion and drug treatment In this study, we used floating sphere cultures of Nf1+ / - (Prss56Cre, R26Tom, Nf1flox / +) and Nf1- / - (Prss56Cre, R26Tom, Nf1flox / flox) glial cells isolated from the skin of newborn mice. They are then plated, treated and analyzed separately. For this purpose, Nf1+ / - and Nf1- / - spheres were amplified up to passage 3 (one passage corresponds to 7 days of culture). At the end of P0 (day 10), cells were split into four identical batches (clones 1-4) and amplified up to P3 (Figure 1).

[0057] At the end of P3, spheres from each clone were dissociated and cells were transferred to two 12-well plates. Drugs at six different concentrations (100 μm, 33 μm, 10 μm, 3 μm, 1 μm, and 0 μm) were added and cells were incubated for 72 h. An additional 12-well plate containing cells with DMSO (vehicle used for drug resuspension) was used as a reference to normalize the results. After 72 h, proliferation and cell death assays of non-fixed cells were analyzed.

[0058] result Proliferative activity Cell tracker lipophilic dye (CellTrace Proliferation Kit, Thermo Fishers) was used to analyze proliferation activity. The cell tracker was added simultaneously with the drug. The cells were incubated for 72 hours, and proliferation activity was measured by cytometric analysis using an LSR Fortessa X20 (BD Biosciences). This method measures the mean fluorescence intensity (MIF) of each condition. The condition with DMSO was used as a reference. Ratio: MIF(DMSO) / MIF(treatment) = Ratio < 1: reduced proliferation compared to DMSO Ratio > 1: Increased proliferation compared to DMSO

[0059] The highest concentration of nitroxoline tested in this assay, 100 μM, promoted massive nonspecific death of NF1+ / - and Nf1- / - cells and was therefore excluded from the analysis. Nitroxoline at concentrations ranging from 33 μM to 1 μM reduced the proliferation activity of NF1- / - cells (except for clone 3), whereas proliferation of NF1+ / - cells was unaffected (Figure 3).

[0060] At higher concentrations (mean 33 μM:MIF ratio=0.39+ / -0.11 and mean 10 μM:MIF ratio=0.5+ / -0.18), proliferation of NF1- / - cells was more affected compared to lower concentrations (mean 3 μM:MIF ratio=0.60+ / -0.22 and mean 1 μM:MIF ratio=0.7+ / -0.26), suggesting that the proliferation rate of NF1- / - cells is drug dose dependent and that concentrations of 33 μM and 10 μM show the best efficacy / toxicity ratio in this assay.

[0061] cell death After 72 h in culture, cells were labeled with DAPI (4',6-diamidino-2-phenylindole) to quantify cell death, and the labeled cells were analyzed by cytometry using an LSR Fortessa X20 (BD Biosciences). Ratio: Dead cells (treated)% / Dead cells (DMSO)%= Ratio > 1: more dead cells compared to DMSO Ratio < 1: Fewer dead cells compared to DMSO

[0062] Nitroxoline at 100 μM promoted massive cell death in NF1- / - and NF1+ / - cells, likely due to its toxic effects, and was therefore excluded from the analysis. Interestingly, nitroxoline at 33 μM promoted cell death in NF1- / - cells (mean dead cell ratio = 8.86 + / - 0.66) without affecting NF1+ / - cells. Lower concentrations of nitroxoline slightly promoted mutant cell death without affecting control conditions (10 μM:mean dead cell ratio = 2.04 + / - 0.63).

[0063] Example 2 - In vivo drug testing in Nf1-KO mice animal The study will use the same mouse model used for the in vitro study, Prss56Cre / +, R26tdTom / +, NF1fl / fl mice (Radomska et al., 2019). The study is a standard experimental design in the field, with end points being terminal rather than longitudinal throughout the study.

[0064] Prss56Cre / +, R26tdTom / +, Nf1fl / fl (herein referred to as Nf1-KO mice) develop cNFs after 1 year of age. However, the onset of cNFs occurs at an earlier age after skin damage induced by mouse fighting, which is a normal behavior in mice. In this experiment, at least five 6-week-old Nf1-KO male mice were caged together, and once the mice had sustained some degree of skin damage by fighting, they were removed from the cage and observed. At approximately 3 months of age, mice that had sustained damage from fighting developed some cNFs. Because a fluorescent reporter gene was turned on in cells with Nf1 deletion, the onset of cNFs could be monitored by fluorescent imaging of the skin using an epifluorescence microscope (Leica MZ75). After 1–2 months, mice with established mature cNFs were enrolled in a drug treatment study. Mice were treated with vehicle (20% DMSO in 80% corn oil) by oral gavage or 120 mg / kg nitroxoline by oral gavage once daily (5 days on, 2 days off) for 1 month.

[0065] Ex vivo analysis Mice were sacrificed one month after treatment. Epifluorescence imaging was used to guide the dissection of skin areas where cNFs were present. cNFs were excised by punch biopsy (5 mm), fixed overnight in 4% PFA, and then cryoprotected in 30% sucrose. Finally, samples were embedded in gelatin / sucrose (15% / 7.5%). cNFs were cryosectioned (14 μm) and fluorescent Tom+ cells were imaged using a fluorescence microscope (Leica M165FC).

[0066] result In vivo efficacy Schwann cells that are Nf1 null also express the fluorescent reporter tomato (Tom+). Because cNFs are derived from Nf1 null SCs that are Tom+, fluorescence can be used to identify tumors. Using low magnification (5x), we were able to visualize fluorescent cNF tumor cells within mouse skin (Figure 4). In these images, it can be seen that one month after treatment, tumors in mice treated with nitroxoline (E-H) are smaller than those in vehicle-treated mice (A-D). Thus, these images show that nitroxoline treatment at 120 mg / kg once daily reduces the overall abundance of cNF tumor cells within the skin.

[0067] conclusion Nitroxoline (10 μM to 33 μM) inhibits the proliferation and induces cell death of tumor cells isolated from cutaneous neurofibromas derived from NF1- / - mouse models. This effect is not observed in cells from NF1+ / - mice, suggesting that the effect of nitroxoline is dependent on the complete deletion of neurofibromin. In addition to the in vitro findings, we also showed that nitroxoline can reduce (in vivo) the number of tumor cells in cNFs that develop in mice. Thus, nitroxoline is expected to reduce, treat, and prevent cutaneous neurofibromas.

[0068] References Radomska KJ,Coulpier F,Gresset A,Schmitt A,Debbiche A,Lemoine S,Wolkenstein P,Vallat JM,Charnay P,Topilko P.Cellular Origin,Tumor Progression,and Pathogenic Mechanisms of Cutaneous Neurofibromas Revealed by Mice with Nf1 Knockout in Boundary Cap Cells.Cancer Discov.2019 Jan;9(1):130-147.

Claims

1. A composition comprising nitroxoline or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of cutaneous neurofibroma.

2. The composition for use according to claim 1, which is used in the treatment of cutaneous neurofibroma.

3. The composition for use according to claim 1, wherein the subject of the treatment or prevention has neurofibromatosis type I.

4. The composition for use according to claim 1, wherein the subject of the treatment or prevention is a human.

5. The composition for use according to claim 1, wherein the composition comprises 30 mg to 600 mg, preferably 50 mg to 500 mg, more preferably 100 mg to 400 mg, even more preferably 150 mg to 350 mg, and most preferably 200 mg to 300 mg of nitroxoline.

6. The composition for use according to claim 1, wherein the administration is carried out at a dose twice a day.

7. The composition for use according to claim 6, wherein the dose is 45 mg to 900 mg of nitroxoline, preferably 75 mg to 750 mg, more preferably 150 mg to 600 mg, even more preferably 225 mg to 525 mg, and most preferably 300 mg to 450 mg.

8. The composition for use according to claim 1, wherein the administration is carried out at a dose three times a day.

9. The composition for use according to claim 8, wherein the dose is 30 mg to 600 mg of nitroxoline, preferably 50 mg to 500 mg, more preferably 100 mg to 400 mg, even more preferably 150 mg to 350 mg, and most preferably 200 mg to 300 mg.

10. The composition for use according to claim 1, wherein the administration is carried out at a dose four times a day.

11. The composition for use according to claim 10, wherein the dose is 15 mg to 500 mg of nitroxoline, preferably 50 mg to 400 mg, more preferably 100 mg to 300 mg, even more preferably 125 mg to 225 mg, and most preferably 150 mg to 200 mg.

12. The composition for use according to claim 1, which is administered orally or intravenously.

13. The composition for use according to claim 1, which is administered by parenteral, transdermal, sublingual, rectal or inhalation administration.

14. The composition for use according to claim 1, wherein nitroxoline or a pharmaceutically acceptable salt is the only active agent in the composition.

15. The composition according to claim 1, further comprising selumetinib or a pharmaceutically acceptable salt thereof, for use as claimed.

16. For use in combination with a second composition comprising selumetinib or a pharmaceutically acceptable salt thereof, the two compositions being administered to the subject simultaneously, separately or sequentially, the composition for use as claimed in claim 1.

17. The amount of selumetinib is from 1 mg to 75 mg, preferably from 5 mg to 50 mg, more preferably from 10 mg to 35 mg, most preferably from 15 mg to 30 mg, the composition for use as claimed in claim 15.

18. Use of nitroxoline or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment or prevention of cutaneous neurofibroma.

19. Use as claimed in claim 18, having any of the additional features described in claims 2 to 17.

20. A method of treating or preventing cutaneous neurofibroma, comprising administering to a patient a composition comprising nitroxoline or a pharmaceutically acceptable salt thereof.

21. A method as claimed in claim 20, having any of the additional features described in claims 2 to 17.

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