Nitrohydroxyquinoline for use in treatment or prevention of malignant peripheral nerve sheathing tumors

Abstract

The present invention relates to a composition comprising nitrooxquinoline or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of MPNST.

Description

Technical Field

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

[0002] Nitroxoline is used as an antibiotic in humans. It has not been widely used but has been on the market since the 1960s. It is used in the treatment or prevention of biofilm infections such as urinary tract infections. It is particularly effective in disrupting biofilms, and it is believed that the metal cation chelating properties are responsible for this effect. Nitroxoline is metabolized in the liver to the corresponding sulfate and glucuronide metabolites. There is evidence that both of these metabolites have antimicrobial activity. It has also been used in an anti-cancer context due to its anti-proliferative effects. Nitroxoline has the systematic name 5-nitroquinolin-8-ol.

[0003] Malignant peripheral nerve sheath tumor (MPNST) is a form of cancer in the connective tissue surrounding nerves. A sarcoma is defined as an MPNST when it meets at least one of the following criteria: it originates from a peripheral nerve, it originates from a pre-existing benign schwannoma (neurofibroma), or its histological examination shows Schwann cell differentiation. MPNSTs are considered aggressive and are associated with a low survival rate.

[0004] MPNSTs typically present as an enlarging palpable mass. Pain is a variable complaint. In the context of NF1, rapid enlargement is more common, and malignant degeneration of neurofibromas should be suspected. MPNSTs originating from peripheral nerves can cause a variety of clinical manifestations, including radicular pain, paresthesia, and motor weakness.

[0005] Many MPNSTs originate from plexiform neurofibromas (benign schwannomas), which themselves originate from cutaneous nerves during early development or from more internal nerve bundles such as cranial nerves or proximal large peripheral nerve sheaths. The incidence of transformation of plexiform neurofibromas to MPNSTs over a lifetime is 10% to 15%.

[0006] Many people with MPNSTs are also found to have neurofibromatosis type 1 (NF1), an autosomal dominant genetic disorder. NF1 is caused by a germline mutation in the NF1 tumor suppressor gene that encodes the protein neurofibromin. Neurofibromin acts as a GTPase-activating (GAP) protein and inactivates the intracellular signal transduction protein Ras by converting the active GTP-bound form to its inactive GDP-bound form. This in turn leads to a downregulation of Ras activity. Loss of neurofibromin activity increases Ras activity, which in turn promotes the transcription of many genes required for cell growth and proliferation.

[0007] Longitudinal imaging studies of patients with plexiform neurofibromas have identified a unique subset of nodular lesions that arise within existing plexiform neurofibromas, grow rapidly relative to the surrounding tumor, and are FDG-PET avid [Evans, D.G. et al., J Med Genet, 2002. 39(5): p. 311-4.]. Biopsies often reveal atypical neurofibromatous tumors of uncertain biological potential (ANNUBP), whose histopathological features overlap with MPNST and have been associated as potential MPNST precursors [Miettinen, M.M. et al., Hum Pathol, 2017. 67: p. 1-10]. Deletions of 9p21.3 encoding the entire CDKN2A / B locus (INK4 / ARF locus) have been identified as a single and highly recurrent genetic aberration in the majority of human ANNUBP (94%, n = 15 / 16). In addition, in two recent independent studies, haploinsufficiency or homozygous deletion of CDKN2A (p16 INK4A ) and its alternative reading frame p14 ARF were identified in 60% to 80% of MPNST [Brohl, A.S. et al., Sci Rep, 2017. 7(1): p. 14992; Lee, W. et al., Nat Genet, 2014. 46(11): p. 1227-32].

[0008] The Ink4a / Arf tumor suppressor plays a key role in suppressing plexiform neurofibroma progression by inducing widespread senescence features. Mice with conditional deletion of Nf1 and Ink4a / Arf in the embryonic neural crest spontaneously develop tumors that are histopathologically indistinguishable from human ANNUBP and subsequently progress to MPNST with high penetrance [Rhodes, S.D. et al., Hum MolGenet, 2019]. This tissue lineage-specific model has for the first time recapitulated the malignant transformation of pre-existing plexiform neurofibromas and ANNBUP precursor lesions observed in human patients.

[0009] Current MPNST therapies, including surgery and chemo- and radiotherapy, are both debilitating and mostly ineffective. The overall survival rate of patients with NF-1 related MPNST is only about 50% after 10 years. This has highlighted the need for new therapies. SUMMARY OF THE INVENTION

[0010] The inventors have found that nitroxoline inhibits cell proliferation and increases apoptosis in vitro in stem cells recapitulating transformed Nf1 MPNST. Thus, nitroxoline is expected to reduce, treat, and prevent MPNST.

[0011] Accordingly, the present invention is a composition comprising nitroxoline or a pharmaceutically acceptable salt thereof, which composition is for use in the treatment or prevention of MPNST.

[0012] A first aspect of the present invention is a composition comprising nitroxoline or a pharmaceutically acceptable salt thereof, which composition is for use in the treatment or prevention of MPNST.

[0013] A second aspect of the present invention is the use of nitroxoline or a pharmaceutically acceptable salt thereof for the preparation of a medicament for use in the treatment or prevention of MPNST.

[0014] A third aspect of the present invention provides a method for treating or preventing MPNST, the method comprising: administering to a patient a composition comprising nitroxoline or a pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shows the nitroxoline dose response in the proliferation and apoptosis of Nf1 - / - and Nf1 - / - ; Ink4a / Arf + / - DNCSCs. DETAILED DESCRIPTION

[0016] In the present invention, and as demonstrated by the data herein, nitroxoline inhibits proliferation and increases apoptosis in vitro in stem cells that recapitulate transformed Nf1 MPNSTs, and is thus an effective treatment for MPNST. Preferably, nitroxoline is for the treatment or prevention of MPNST, wherein the subject has neurofibromatosis type I.

[0017] As used herein, the term "treatment" or "treating" refers to therapeutic (curative) treatment, including reducing the size of MPNST. Biopsy can be used to diagnose MPNST. As used herein, the term "prevention" or "preventing" refers to "prophylactic" treatment, including administering nitroxoline to a patient to avoid the development of MPNST, such as a patient with plexiform neurofibroma who has not yet developed MPNST. Plexiform neurofibroma may have started to proliferate, such as rapid proliferation.

[0018] "Patient" and "subject" are used interchangeably and refer to a subject to whom nitroxoline is to be administered. Preferably, the subject is a human. Suitably, the subject has neurofibromatosis, preferably neurofibromatosis type I.

[0019] In one embodiment, nitroxoline is used for the treatment or prevention of MPNST, where the patient has had or will undergo surgery to remove some or all of the MPNST. This can be particularly advantageous when the MPNST is large and / or extends across tissue boundaries, such that it is difficult to remove all of it surgically and / or it is desirable / beneficial to rapidly remove at least some of it.

[0020] In one embodiment, nitroxoline is used for the prevention of MPNST, where the patient has had or will undergo surgery to remove some or all of the plexiform neurofibromas. Generally, in this embodiment, the plexiform neurofibromas have not yet progressed to MNPST.

[0021] The term "surgery" has its ordinary meaning in the art. Surgery is an invasive technique whose basic principle is physical intervention on an organ / organ system / tissue for diagnostic or therapeutic reasons.

[0022] In one aspect, the patient has a genetic aberration, such as a deletion of 9p21.3 encoding the entire CDKN2A / B locus (INK4 / ARF locus), or haploinsufficiency or homozygous deletion of CDKN2A (p16 INK4A ) and its alternative reading frame p14 ARF . Such genetic aberrations have been identified as being both solitary and highly recurrent in the majority of human ANNU BPs associated with MPNST.

[0023] As used herein, a pharmaceutically acceptable salt is a salt formed with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids and organic acids, 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 alkali metals (such as sodium or potassium) and alkaline earth metals (such as calcium or magnesium) hydroxides as well as organic bases (such as alkylamines, arylamines or heterocyclic amines).

[0024] The present invention relates to a composition comprising nitroxoline or a pharmaceutically acceptable salt thereof, which composition is for use in the treatment or prevention of MPNST.

[0025] In an alternative embodiment, the present invention relates to a composition comprising nitroxoline or a pharmaceutically acceptable salt thereof, which composition is for use in the treatment or prevention of MPNST, where nitroxoline is the sole active agent in the composition. By referring to the sole active agent, it is meant that the composition does not contain other components that can be used in the treatment or prevention of MPNST.

[0026] Alternatively, the composition comprising nitroxoline or a pharmaceutically acceptable salt thereof may further comprise one or more active agents in the composition, preferably one. The additional active agent may be an agent active against the treatment or prevention of MPNST.

[0027] The compositions of the present invention may contain a pharmaceutically acceptable carrier. The reference to "pharmaceutically acceptable carrier" means any diluent or excipient that is compatible with the other ingredients of the composition and harmless to the recipient, such as a filler or binder. The pharmaceutically acceptable carrier can be selected based on the desired route of administration according to standard pharmaceutical practice.

[0028] 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 by inhalation or by suppository.

[0029] The composition can be administered orally, for example as tablets, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules. Preferably, the composition is formulated such that it is suitable for oral administration, such as tablets and capsules. Tablets and capsules can be prepared with: binders, such as syrup, gum arabic, 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 can contain conventional additives, such as suspending agents, for example sorbitol syrup, methylcellulose, syrup, gelatin, carboxymethylcellulose or edible oils; emulsifying agents and surfactants, such as lecithin or gum arabic; vegetable oils, such as almond oil, coconut oil, cod liver oil or peanut oil; 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.

[0030] The composition can also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternal, transdermally or by infusion techniques.

[0031] The composition can also be administered by inhalation. Compared with many drugs taken by the oral route, the advantage of inhaled drugs is their direct delivery to areas with rich blood supply. Therefore, due to the large surface area and rich blood supply of the alveoli, the absorption is very fast and the first-pass metabolism is bypassed.

[0032] The present invention also provides an inhalation device containing the composition of the present invention. Generally, the device is a metered-dose inhaler (MDI), which contains a pharmaceutically acceptable chemical propellant to push the drug out of the inhaler.

[0033] The composition can also be administered by intranasal administration. The highly permeable tissues of the nasal cavity are very receptive to drugs and absorb drugs quickly and effectively. Compared with injection, nasal drug delivery is less painful and invasive, thus reducing patient anxiety. By this method, absorption is very fast and generally bypasses first-pass metabolism, thus reducing inter-patient variability. In addition, the present invention also provides an intranasal device containing the composition according to the present invention.

[0034] The composition can also be administered by transdermal administration. For topical delivery, transdermal and transmucosal patches, creams, ointments, gels, solutions or suspensions can be used. Therefore, the present invention also provides a transdermal patch containing the composition.

[0035] The composition can also be administered by sublingual administration. Therefore, the present invention also provides a sublingual tablet containing the composition.

[0036] The composition can also be formulated with an agent (such as an antibacterial agent or an inhibitor of a protease (which may be present in the patient or in symbiotic or parasitic organisms living on or in the patient's body surface or body and which is capable of degrading the compound)) that reduces the degradation of the substance by a process other than the patient's normal metabolism.

[0037] Liquid dispersions for oral administration can be syrups, emulsions and suspensions.

[0038] Suspensions and emulsions can contain, for example, natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol as carriers. Suspensions or solutions for intramuscular injection can contain a pharmaceutically acceptable carrier together with the active compound, such as sterile water, olive oil, ethyl oleate, ethylene glycol (such as propylene glycol) and, if desired, an appropriate amount of lidocaine hydrochloride.

[0039] Solutions for injection or infusion can contain, for example, sterile water as a carrier, or preferably they can be in the form of a sterile aqueous isotonic saline solution.

[0040] In an embodiment of the present invention, the composition is administered in an effective amount to treat or prevent MPNST. The effective dose will be apparent to those skilled in the art and depends on many factors, including age, sex, weight, and these factors can be determined by a medical practitioner.

[0041] 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, still more preferably 150 mg to 350 mg, and most preferably 200 mg to 300 mg of nitroxoline.

[0042] The composition can be administered once a day, twice a day, three times a day or four times a day.

[0043] In an 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, preferably 150 mg to 1500 mg, more preferably 300 mg to 1200 mg, still more preferably 450 mg to 1050 mg, and most preferably 600 mg to 900 mg of nitroxoline.

[0044] In an embodiment of the present invention, the composition is administered twice a day. Preferably, each dose is 45 mg to 900 mg, preferably 75 mg to 750 mg, more preferably 150 mg to 600 mg, still more preferably 225 mg to 525 mg, and most preferably 300 mg to 450 mg of nitroxoline.

[0045] In an embodiment of the present invention, the composition is administered three times a day. Preferably, each dose is 30 mg to 600 mg, preferably 50 mg to 500 mg, more preferably 100 mg to 400 mg, still more preferably 150 mg to 350 mg, and most preferably 200 mg to 300 mg of nitroxoline.

[0046] In an embodiment of the present invention, the composition is administered four times a day. Preferably, each dose is 15 mg to 500 mg, preferably 50 mg to 400 mg, more preferably 100 mg to 300 mg, still more preferably 125 mg to 225 mg, and most preferably 150 mg to 200 mg of nitroxoline.

[0047] Preferably, the dosing regimen is such that the total daily dose of nitroxoline does not exceed 1500 mg.

[0048] Suitably, the effective dose of nitroxoline results in a concentration in the cells of 1 μM to 150 μM, preferably 10 μM to 100 μM, more preferably 25 μM to 50 μM.

[0049] For the treatment or prevention of MPNST, the composition comprising nitroxoline is used in a chronic dosing regimen (i.e., chronic, long-term treatment). Suitably, the regimen lasts for at least one month, suitably at least two months, such as at least three months.

[0050] The present invention also relates to the use of nitroxoline or a pharmaceutically acceptable salt thereof for the preparation of a medicament for use in the treatment or prevention of MPNST. This embodiment of the present invention may have any of the preferred features described above.

[0051] The present invention also relates to a method of treating or preventing MPNST, the method comprising: administering to a patient a composition comprising nitroxoline or a pharmaceutically acceptable salt thereof. This embodiment of the invention may have any of the preferred features described above. The method of administration may be by any of the routes described above.

[0052] For the avoidance of doubt, the present invention also includes prodrugs that react in vivo to produce the compounds of the invention.

[0053] Experimental section

[0054] In vitro drug screening using Nf1 and Nf1-Ink4a / Arf mutant DNSC

[0055] Nf1-Ink4a / Arf-deficient cells contained in embryonic dorsal root ganglia (DRG) / dorsal nerve roots (DNSC) can be re-implanted into the nerve microenvironment and form tumors that are histologically indistinguishable from human ANNBUP within 6 weeks. By 3 to 4 months after implantation, these lesions further progress to high-grade MPNST with a 100% penetrance. By achieving efficient tumorigenesis within a defined latency period, this method will provide a robust platform for evaluating experimental therapies designed to treat, delay, and / or prevent the progression of ANNUBP and MPNST driven by Nf1 and Ink4 / Arf deletion [Chen, Z. et al., Cancer Cell, 2014. 26(5): p. 695-706].

[0056] Nf1-Ink4a / Arf mutant DNSC have increased cell cycle activity and proliferation in vitro, which is consistent with the in vivo phenotype. This study utilized Nf1 - / - and Nf1 - / - ; Ink4a / Arf + / - mutant DNSC, which were obtained by transiently infecting Nf1 flox / flox and Nf1 flox / flox ; Ink4a / Arf flox / + DNSC with adenovirus carrying Cre recombinase. The selectivity of nitroxoline was compared between Nf1 - / - and Nf1 - / - ; Ink4a / Arf + / - DNSC.

[0057] The above Nf1 - / - and Nf1 - / - ; Ink4a / Arf + / - DNSC were treated with nitroxoline serially diluted starting from 105 μM and incubated for 48 hours. After the incubation period, proliferation, viability, and apoptosis assays were performed as described below:

[0058] ● The proliferation and viability of DNSC were detected using the CellTiter-Glo assay (Promega) that measures ATP consumption. Briefly, Nf1 - / - and Nf1 - / - ; Ink4a / Arf + / - DNSC were plated at a concentration of 5,000 cells / well in 100 μl of DNSC growth medium supplemented with FGF and EGF in 96-well plates, in triplicate, and placed in a humidified incubator at 37 °C with 5% CO2, with or without the experimental compound, for 48 hours. After incubation, 100 μL of CellTiterGlo reagent was added to each well. After 10 minutes, the 96-well microplate photometer was used to read the plate.

[0059] ● To assess apoptosis, the Caspase-Glo 3 / 7 kit (Promega) was used according to the manufacturer's instructions. Nf1 - / - and Nf1 - / - ; Ink4a / Arf + / - DNSC were plated and treated with the experimental compound. After 48 hours, 100 μL of Caspase-Glo 3 / 7 reagent was added to each well. After 10 minutes, the 96-well photometer was used to read the plate to measure caspase 3 / 7 activity.

[0060] Results

[0061] As Figure 1 shown, niflumic acid inhibited cell proliferation and increased apoptosis in a dose-responsive manner. In Nf1- / - lnk4a / Arf- / - cells, an anti-proliferative effect was observed at concentrations greater than 3 μM, and in Nf1- / - cells, an anti-proliferative effect was observed at concentrations greater than 10 μM. In Nf1- / - lnk4a / Arf- / - cells, apoptosis induction was observed at concentrations greater than 10 μM, and in nf1- / - cells, apoptosis induction was observed at concentrations between 30 μM and 100 μM.

[0062] Conclusions

[0063] Niflumic acid inhibits cell proliferation and increases apoptosis in vitro in stem cells that recapitulate transformed Nf1 MPNST. Therefore, niflumic acid is expected to reduce, treat, and prevent MPNST.

Claims

1. A composition comprising nitroxoline or a pharmaceutically acceptable salt thereof, said composition for use in the treatment or prevention of malignant peripheral nerve sheath tumors (MPNSTs).

2. The composition for use according to claim 1, said composition for use in the treatment of MPNSTs.

3. The composition for use according to claim 1 or 2, wherein the subject to be treated or prevented has neurofibromatosis, preferably neurofibromatosis type I.

4. The composition for use according to any one of the preceding claims, wherein the subject to be treated or prevented is a human.

5. The composition for use according to any one of the preceding claims, wherein the composition comprises 30 mg to 600 mg, preferably 50 mg to 500 mg, more preferably 100 mg to 400 mg, still more preferably 150 mg to 350 mg, most preferably 200 mg to 300 mg of nitroxoline.

6. The composition for use according to any one of the preceding claims, 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, preferably 75 mg to 750 mg, more preferably 150 mg to 600 mg, still more preferably 225 mg to 525 mg, most preferably 300 mg to 450 mg of nitroxoline.

8. The composition for use according to any one of claims 1 to 4, 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, preferably 50 mg to 500 mg, more preferably 100 mg to 400 mg, still more preferably 150 mg to 350 mg, most preferably 200 mg to 300 mg of nitroxoline.

10. The composition for use according to any one of claims 1 to 4, 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, preferably 50 mg to 400 mg, more preferably 100 mg to 300 mg, still more preferably 125 mg to 225 mg, most preferably 150 mg to 200 mg of nitroxoline.

12. The composition for use according to any one of the preceding claims, said composition is administered orally or intravenously.

13. The composition for use according to any one of claims 1 to 11, said composition is administered by parenteral, transdermal, sublingual, rectal or inhalation administration.

14. A composition comprising nitroxoline or a pharmaceutically acceptable salt thereof, said composition for use in the treatment or prevention of MPNSTs, wherein nitroxoline is the sole active agent in the composition.

15. Use of nitroxoline or a pharmaceutically acceptable salt thereof for the preparation of a medicament for use in the treatment or prevention of MPNSTs.

16. The use according to claim 15, having any of the additional features of claims 2 to 14.

17. A method for treating or preventing MPNST, the method comprising: A composition comprising nitroxoline or a pharmaceutically acceptable salt thereof is administered to a patient.

18. The method according to claim 17, having any additional feature among the additional features of claims 2 to 14.

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