FIELD OF THE INVENTION

The present invention relates to an aminoquinoline compound for use in the treatment of mast cell activation diseases. More specifically, it concerns the use of hydroxychloroquine for the treatment of mastocytosis and mast cell activation syndromes (MCAS). BACKGROUND OF THE INVENTION

Mast cell activation diseases (MCAD) include benign and malignant pathologic mast cell (MC) states ¹ . MCAD are classically divided in aberrant MC activation disorders classified as mast cell activation syndromes (MCAS) and in proliferation and/or abnormal accumulation of MC in various organs, classified as mastocytosis ² . Mastocytosis can be divided between cutaneous mastocytosis (CM) if only skin involvement is present and in systemic mastocytosis if at least one internal organ is involved ³ . There are internationally approved diagnosis criteria for MCAS, CM and systemic mastocytosis ³ ' ⁴ ' ⁵ ' ⁶ . No treatment is specifically approved for CM and MCAS. HI anti-histamines are recommended by international guidelines for the treatment of clinical manifestations associated with MCAD.

Accordingly, there is a medical need to identify specific therapeutical tools to treat mast cell activation diseases (MCAD).

SUMMARY OF THE INVENTION

Inventors have discovered that aminoquinoline compound (such hydroxychloroquine (HCQ) can improve clinical symptoms and signs present in MCAD patients.

Accordingly, the present invention relates to a method of treating MCAD patients using aminoquinoline compound (such HCQ). More specifically, it concerns the discovery of the unexpected properties of an aminoquinoline compound to modulate mast cell functions, useful for the treatment of mast cell activation diseases (MCAD) such as mastocytosis and MCAS.

The present invention relates to an aminoquinoline compound for use in the treatment of mast cell activation diseases (MCAD).

DETAILED DESCRIPTION OF THE INVENTION In the present study, in all patients, six months after HCQ treatment initiation, a significant improvement of MC activation symptoms was observed (see Table 1 and Fig 2).

These clinical observations prompted inventors to investigate the biological effects of HCQ treatment on human MCs in vitro. The results indicate that HCQ treatment modifies key features of MC biology and induces a profound alteration of MC granules homeostasis. Namely, it leads to the storage of inactive tryptase and to the decreased expression of key MC inflammatory mediator, such as IL-8. Taken together, the HCQ induced dramatic reduction of MC inflammatory capabilities contributing to the observed efficacy of HCQ in MCAD patients.

Aminoquino lines are derivatives of quinoline (heterocyclic aromatic organic compound with the chemical formula C9H7N) well known by the man skilled in the art, most notable for their roles as antimalarial drugs (modulator of endosomal signalling). Depending upon the location of the amino group, they can be divided into: 4-Aminoquinoline (PubChem CID: 68476) and 8-Aminoquinoline (PubChem CID: 11359). For a review on aminoquinolines, see Egan, TJ (2001) Expert Opinion on Therapeutic Patents;, Vol. 11 Issue: 2 pl85-209; Ridley, RG et al (1998), Expert Opinion on Therapeutic Patents; Vol. 8 Issue: 2 pl21-136, WO 2005/007672. Aminoquinolines have been recognized as useful not only as anti-malarial agents but also as anti-inflammatory agents. Although its mechanism of action is not well understood, Aminoquinolines chloroquine has been used effectively in the treatment of various autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). (see Wallace DJ (1996) Lupus 5 Suppll :859-64.).

4-Aminoquinoline is a form of aminoquinoline with the amino group at the 4-position of the quinoline. A variety of derivatives of 4-aminoquinoline are antimalarial agents useful in treating erythrocytic plasmodial infections. Examples of 4-Aminoquinoline include but not limited to

Amodiaquine chloroquine hydroxychloroquine 8-Aminoquinoline is a form of aminoquinoline with an amine at the 8-position of quinoline. They may be used to eradicate malaria hypnozoites from the liver and have both been used for malaria prophylaxis. The 8-aminoquinoline family of drugs contains three main members,

primaquine, pamaquine tafenoquine.

Hydroxychloroquine (HCQ), sold under the brand names Plaquenil (hydroxychloroquine sulphate) among others, is a medication used for the prevention and treatment of certain types of malaria, specifically it is used for chloroquine sensitive malaria (Hamilton, Richart (2015). Tarascon Pocket Pharmacopoeia 2015 Deluxe Lab-Coat Edition. Jones & Bartlett Learning, p. 463) Other uses include rheumatoid arthritis, lupus, and porphyria cutanea tarda. Hydroxychloroquine is on the World Health Organization's List of Essential Medicines, the most effective and safe medicines needed in a health system. The term «Mast cell activation diseases)) (or MCAD) refers to a heterogeneous group of disorders linked to benign and malignant pathologic mast cell (MC) states. MCAD are classically divided in aberrant MC activation disorders classified as mast cell activation syndromes (MCAS) and characterized by aberrant release of variable subsets of MC mediators and in proliferation and/or abnormal accumulation of MC in various organs, classified as mastocytosis. Sometimes in MCAD and always in mastocytosis the MC are abnormal morphological, phonotypical and are monoclonal. Mastocytosis can be divided in cutaneous mastocytosis (CM) if only skin involvement is present and in systemic mastocytosis if at least one internal organ is involved. Rarely MC can induce severe diseases as MC leukemia (MCL) or MC sarcoma. Clinical signs and symptoms in MCAD vary depending on disease subtype and result from excessive mediator release by MCs and, in aggressive forms, from organ failure related to MC infiltration. In most cases, treatment of MCAD is directed primarily at controlling the symptoms associated with MC mediator release. In advanced forms, such as aggressive SM and MCL, agents targeting MC proliferation such as tyrosine kinase inhibitors may be provided. Targeted therapies aimed at blocking mutant protein variants and/or downstream signalling pathways are currently being developed. Other targets, such as specific surface antigens expressed on neoplastic MCs, might be considered for the development of future therapies (see the review Molderings, G.J. et al Arch Pharmacol (2016) 389: 671) .

In a particular embodiment "Mast cell activation diseases" according to the invention are selected from the group consisting of Mast cell activation syndrome [MCAS] and mastocytosis.

In a particular embodiment mastocytosis is selected from the group consisting of cutaneous mastocytosis (CM) and systemic mastocytosis.

In a particular embodiment mastocytosis is cutaneous mastocytosis (CM).

The present invention also relates to a method for treating mast cell activation diseases in a patient, such method involving the step of administering to a patient in need thereof a therapeutically effective amount of aminoquinoline.

As used herein, the term "patient" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human.

By a "therapeutically effective amount" is meant a sufficient amount to be effective, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient in need thereof will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, the duration of the treatment; drugs used in combination or coincidental with the and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

In a particular embodiment of the invention, said MACD patient is treated every day during at least , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22; 23; 24 months. In a particular embodiment of the invention, when aminoquinoline is hydroxychloroquine (HCQ) the dose administered is 4.5 mg/kg/day and can be increased to 6.8 mg/kg/day to reach a blood HCQ level of 1000 μ^.

The aminoquinoline can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, rectal, nasal, buccal, ocular, sublingual, transdermal, rectal, topical, vaginal, parenteral (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intraperitoneal. It will be appreciated that the preferred route may vary with for example the condition of the recipient. In a preferred embodiment aminoquinoline is administered by oral way.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

Typically medicaments according to the invention comprise a pharmaceutically-acceptable carrier. A person skilled in the art will be aware of suitable carriers. Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known text such as Remington; The Science and Practice of Pharmacy.

The compounds of the invention may be used or prepared in a pharmaceutical composition. In one embodiment, the invention relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the treatment of mast cell activation diseases in a subject of need thereof.

Typically, the compound of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, sub-dermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Preferably, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being administered by oral-route forms.

In addition to the compounds of the invention formulated for oral administration, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time-release capsules; and any other form currently used.

Vehicles used usually for hydroxychloroquine (HCQ) may contain lactose, which can be advantageously substituted by cellulose, mannitol or hypromellose, because lot of MCAD patient suffers from lactose intolerance.

Pharmaceutical compositions of the invention may include any further compound, which is used in the treatment of mast cell activation diseases. For example, the anti-MACD therapy may include compounds used anti HI, anti H2, cromolyn sodium, montelukast and/or all tyrosine kinase inhibitor (i.e. masitinib and midostautrin).

In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of mast cell activation diseases in a subject in need thereof.

The invention also provides kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.

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

Figure 1. : Hydroxychloroquine impairs pro-inflammatory capabilities of human mast cells. A-B: CD63 expression and localization. C: tryptase and chymase expression as detected by flow cytometry (isotype control mAb). D: mast cell intracellular beta-hexosaminidase, chymase and tryptase activities. E: IL-8 production following PMA/ionomycin stimulation. P values were determined using unpaired t test, ns P>0.05, * P<0.05, ** P<0.01, **** PO.0001. Figure 2. : Improvement of mast cell mediator-related symptoms in patients treated with hydroxychloroquine. The Severity of mast cell mediator-related symptoms was evaluated on the visual analog score of 0 to 100. EXAMPLE

METHODS (of in vitro study)

Reagents. Anti-mast cell tryptase (clone G-12, Santa Cruz Biotechnology, Santa Cruz, CA), Anti-CD 117-APC (clone 104D2), anti-IL8-PE (clone AS14), anti-CD63-PE (clone H5C6), all from BD Biosciences, San Jose, CA. Anti-mast cell chymase (clone B7, Millipore, Billerica, MA), Anti-FcsRI-FITC (clone AER-37, eBioscience, San Diego, CA). All the secondary antibodies used (Alexa Fluor-conjugated) were from Molecular Probes, Inc., Eugene, OR. Mast cell granule matrix was stained using avidin-sulforhodamine 101 (Av. SRho, Sigma- Aldrich). Hydroxychloroquine-sulfate, Phorbol 12-myristate 13-acetate (PMA) and Ionomycin were from Sigma- Aldrich.

Human mast cells (hMCs). Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats (Etablissement Francais du Sang). CD34+ precursors cells were isolated from PBMCs (EasySep™ Human CD34 Positive Selection Kit, STEMCELL Technologies) and grown under serum-free conditions using StemSpanTM medium (STEMCELL Technologies) supplemented with recombinant human IL-6 (50 ng/mL; Peprotech), human IL-3 (10 ng/mL; Peprotech) and 3% supernatant of CHO transfectants secreting murine SCF (a gift from Dr. P. Dubreuil, Marseille, France, 3% correspond to ~50 ng/mL SCF) for one week. Cells were next grown in IMDM Glutamax I, sodium pyruvate, 2-mercaptoethanol, 0.5% BSA, Insulin-transferrin selenium (all from Invitrogen), ciprofloxacin (10 μg/mL; Sigma Aldrich), IL-6 (50 ng/mL) and 3% supernatant of CHO transfectants secreting murine SCF for 8 weeks then tested phenotypically (Tryptase+, CD117+, Fc8RI+) and functionally (β-hexosaminidase release in response to FcsRI crosslinking) before use for experiments. Only primary cell lines showing more than 95% CD117+/Fc8RI+ cells were used for experiments. Some hMC were cultured with culture medium plus 2.5, 5 or 10 μΜ HCQ for 3 to 5 weeks. Culture medium containing HCQ was replaced weekly.

Confocal microscopy analysis. 5x104 hMCs treated or not with HCQ were transferred to poly-L-lysine-coated slides then fixed, permeabilized and stained with anti-CD63 mAb followed by secondary antibodies and 2 μg/mL avidin-sulforhodamine 101 (highly cationic glycoprotein that selectively stains mast cell granules, Sigma).The samples were mounted and examined using a Zeiss LSM 710 confocal microscope and ZEN software with a 63x Plan- Apochromat objective (1.4 oil), electronic zoom 3. Scoring of the slides was performed in a blinded fashion by evaluating for each condition at least 50 cells in randomly selected fields from 2 independent experiments.

Quantitation of intracellular enzymes. To quantify cellular granule enzymes contents, 10x105 hMCs treated or not with HCQ were disrupted with 100 μΐ. 1 % Triton X-100 in PBS and the cell lysates were immediately processed. Enzymatically active tryptase was measured spectrophotometrically by adding 50 μΐ _^ of 0.5 mmol/L Tosyl-Gly-Pro-Lys-p-nitroanilide (Sigma, T6140) 100 mmol/L Tris-HCl, pH 8, 100 mmol/L NaCI, to 50 cell lysate. Enzymatically active chymase was quantified by adding 50 of 1 mmol/L Succinyl-Ala- Ala-Pro-Phe-p-nitroanilide (Sigma, S7388) 300 mmol/L Tris, pH 8, 1.5 mol/L NaCI to 50μί cell lysate. Changes in absorbances at 405 nm were immediately registered for 5 minutes at 30-second intervals at room temperature. Enzymatically active β-hexosaminidase was quantified by incubating 50 μΐ _^ of 1.3 mg/mL para-nitrophenyl-N-acetyl-P-D-glucosaminide (Sigma, N9376), 0.1 mol/L sodium citrate, pH 4.5 plus 10 μΐ, cell lysate for 30 min. at 37°C. The enzymatic reaction was stopped by adding 150 μΐ, of 0.2 mol/L glycine, pH 10.7. Absorbance was read at 405 nM.

Intracellular chemokine staining. 5x104 hMCs were stimulated with PMA (100 ng/mL) and ionomycin (^g/mL) for 12 hours in the presence of brefeldin A (10 μg/mL). Cells were washed, fixed (2% PFA), permeabilized (0.1% saponin in PBS containing 1% BSA, Sigma- Aldrich), and stained with anti-IL8-PE mAbs for 45 min. Flow cytometric data were acquired on a MACSQuant® Analyzer 10 (Miltenyi Biotec) and were analyzed by using Flow Jo software (Tree Star, Inc, Ashland, Ore).

Viability assays. MTT assay was used to measure HCQ toxicity. Briefly, cells were seeded into 96-well plates (3x104 cells in ΙΟΟμί culture medium) and treated for 16 hours with HCQ. 20 μΐ, of MTT solution (5 mg/mL in PBS, Sigma) were added to each well. After 3 hours, supernatant was carefully discarded and 150 μΐ, DMSO (Sigma) were added. Absorption was measured at 570 nm. The viability of hMCs cultured in presence of HCQ was regularly checked by flow cytometry by using a calcein assay. Briefly, hMCs (5x104 cells) were incubated with 1 μιηοΙ/L of calcein-AM (Invitrogen) at 37°C for 30 min. Cells were next washed with PBS and analyzed by flow cytometry.

RESULTS Interestingly, we have observed that hydroxychloroquine (HCQ) can improve clinical symptoms and signs present in MCAD patients. Our preliminary data from four patients are summarized in Table 1 (and figure 2).

The first patient was a 57 year-old man with a four-year history of CM (Fig 1, Λ) without D816V mutation of KIT. He experienced moderate intensity abdominal pain and diarrhea as

MC mediator-related symptoms. He also presented with unclassified inflammatory peripheral arthritis for 2 years. Arthritis was not controlled by oral corticosteroids and methotrexate.

HCQ (4.5 mg/kg/day) was introduced to alleviate joint symptoms. At six months HCQ dose was increased to 6.8 mg/kg/day to reach a blood HCQ level of 1000 μg/L. Twelve months after the introduction of HCQ treatment, a clear improvement of CM lesions was observed.

Two years after the introduction of HCQ complete disappearance of CM was observed.

Histological examination of a skin biopsy of a lesion of CM showed a significant reduction in the number of MC in the skin as shown by anti-tryptase antibodies staining (Mast Cell

Tryptase, FL-275 antibody, Santa Cruz Biotechnology, Inc., Dallas, USA). The number of skin MC per mm2 decreased from 110/mm2 at baseline to 50/mm2 at 12 months and 42/mm2 at 24 months. The MC mediator-related symptoms were controlled at six months of treatment.

The level of serum tryptase was stable (baseline: 5.^g/L versus 4.85μg/L at 24 months).

HCQ was discontinued after 42 months of treatment. No relapse of CM was observed with a follow-up of 16 months. Other three MCAD patients with unclassified inflammatory rheumatism were treated with HCQ. It was about of diffuse joint pain with sensation of stiffness the morning at wake up, during 30 to 60 minutes. The imaging exams showed a non-erosive arthritis and the immunological tests - anti-citrullinated cyclic peptide antibodies and rheumatoid factor - were negative. All three patients had concomitant severe and uncontrolled MC mediator-related symptoms. In all patients, six months after HCQ treatment initiation, a significant improvement of MC activation symptoms was observed (Table 1 and Fig 2).

These clinical observations prompted us to investigate the biological effects of HCQ treatment on human MCs in vitro. We used primary human MC lines (hMCs) derived from CD34+ peripheral blood progenitor cells. hMCs were treated with HCQ concentrations ranging from 2.5 to 10 μιηοΙ/L. These concentrations are clinically relevant because HCQ concentration in the blood of patients treated with 400 mg/day HCQ is about 3 μιηοΙ/L ⁷ and HCQ is expected to accumulate in tissues. This range of concentrations did not exhibit cellular toxicity as tested with a classical toxicity assay after 18 hours treatment. We observed that such HCQ concentrations induced hMC death after 3 to 4 weeks of treatment. This effect of HCQ was not observed on human fibroblasts treated for 18 hours.

Because HCQ is known to modify lysosome homeostasis, we analyzed the impact of HCQ treatment on hMC granule compartment (i.e. secretory lysosomes). We first investigated the expression and the localization of the lysosomal molecule CD63. We found that prolonged treatment with HCQ for three to five weeks decreased the intracellular expression level of CD63 and modified the expression pattern of CD63. While untreated hMCs showed an homogeneous cytoplasmic localization of CD63, hMCs treated with HCQ exhibited a more discrete and punctuated expression (Fig 1, A-B). We next analyzed the expression and the function of key enzymes stored on MC granules such as β-hexosaminidase, chymase and tryptase. We found that HCQ treatment did not alter the intracellular levels of tryptase and chymase (as detected by flow cytometry) but dramatically decreased tryptase enzymatic activity. Conversely, the β-hexosaminidase and chymase activities were barely affected (Fig 1, C-D). These results indicate that prolonged HCQ treatment interferes with lysosome function and leads to the accumulation of non- functional tryptase in mast cell granules.

Because cytokine production is an important component of the mast cell-mediated inflammation, we next investigated the production of IL-8 by flow cytometry. This analysis showed that two weeks treatment with HCQ decreased IL-8 expression by hMC. (Fig 1, E). These results indicate that HCQ treatment modifies key features of MC biology and induces a profound alteration of MC granules homeostasis. Namely, it leads to the storage of inactive tryptase and to the decreased expression of key MC inflammatory mediator, such as IL-8.

Taken together, we may say, the HCQ induced dramatic reduction of MC inflammatory capabilities contributing to the observed efficacy of HCQ in MCAD patients. Table 1. Characteristics and treatment of 4 patients

* dosage searched to have a serum level of hydroxychloroquine of 1 mg/dL; MCAD: mast cell activation disease; CM: cutaneous mastocytosis; MCAS: mast cell activation syndrome; mcas: mast cell activation symptoms; HCQ: hydroxychloroquine; mg: milligrams; ng: nanograms; mL: milliliter; L: liter; kg: kilogram REFERENCES

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