Combination of calcium channel blockers and BRAF and / or MEK inhibitors to treat melanoma

Abstract

The present invention concerns the use of an L-type calcium channel (LTCC) blocker in combination with a BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi) for the treatment of melanoma in a patient.

Description

[0001] Combination of calcium channel blockers and BRAF and / or MEK inhibitors to treat melanoma

[0002] FIELD OF THE INVENTION

[0003] The invention concerns the treatment of melanoma in a patient.

[0004] TECHNOLOGICAL BACKGROUND

[0005] Melanoma is a malignant tumor of the pigmentary system (melanocytes) that occurs either initially in healthy skin, most often (70 to 80% of cases), or by degeneration of a pre-existing nevus. Melanoma is the leading cause of death from skin cancer worldwide. With 17,922 new cases of cutaneous melanoma in 2023 in metropolitan France (9,109 men and 8,813 women) and 1,920 deaths in 2021 (1,080 men and 840 women), this cancer represents approximately 4% of all incident cancers and 1.2% of cancer deaths, all sexes combined. It is one of the cancers whose incidence and mortality have increased significantly over the past 40 years. It is the leading cause of cancer death in young adults. In the European Union (EU-27), cutaneous melanoma represented approximately 4% of all new cancer diagnoses (excluding non-melanoma skin cancers) and about 1.3% of all cancer deaths in 2020. Incidence and mortality vary widely between member states, with markedly higher age-standardized incidence rates in several Western and Northern European countries; overall trends show increasing incidence and, in many countries, increasing melanoma-specific mortality. Globally, recent GLOBOCAN / IARC estimates indicate on the order of -330,000 new melanoma cases and -59,000 deaths per year (2022 estimates), with large geographical variation (highest incidence in Australia / New Zealand and parts of North America and Western Europe; lowest rates in many Asian and South American countries). These global figures underscore melanoma’s substantial public-health burden and the unmet need for improved therapeutic strategies.

[0006] The prognosis of the disease is generally good, with a high cure rate if melanoma is detected early (Breslow index < 2 mm and no metastases) and treated. Melanomas are then cured, most often by surgical excision. However, for advanced forms and especially metastatic forms, the prognosis remains poor, although it has improved with the introduction in the late 2010s of complementary treatments such as immune checkpoint inhibitors and targeted therapies. Survival depends on the stage at diagnosis: According to the American SEER program, the relative survival at 5 years is 98% at the localized stage, 62% with locoregional extension and 15% at the metastatic stage. From a molecular viewpoint, deregulation of the mitogenic intracellular signaling pathway RAS / MAPK (mitogen-activated protein kinase) is responsible for the majority of cutaneous melanomas. Typically, this results from the appearance of gain-of-function mutations in NRAS kinases (15-20% of cutaneous melanomas) or BRAF kinases (40-50%) or a loss of function of the tumor suppressor gene NF 1.

[0007] Since 2011, first and second line therapeutic strategies for inoperable stage III or IV cutaneous melanomas are based on two categories of treatment: targeted therapies and immunotherapy. The therapeutic decision depends on the BRAF V600 mutational status determined by genotyping tumor samples (biopsy of an accessible metastasis or, failing that, lymph nodes from dissection or primary tumor). This mutation is present in 40-60% of patients.

[0008] Targeted therapies aim to block the growth or spread of the tumor by interfering with the molecular changes that cause cancer cells to develop and / or spread. In metastatic cutaneous melanoma, the molecules used are BRAF inhibitors (BRAFi or anti-BRAF: e.g., vemurafenib (ZELBORAF®), dabrafenib (TAFINLAR®) or encorafenib (BRAFTOVI®)) that target mutated BRAF kinase. These are now combined with inhibitors of MEK, a kinase that is located downstream of BRAF in the MAPK signaling pathway (MEKi or anti -MEK: e.g., trametinib (MEKINIST®), cobimetinib (COTELLIC®) or binimetinib (MEKTOVI®). Clinical prescription of these BRAFi+MEKi dual therapies leads to partial or complete tumor regression in responder patients. However, a certain number of patients do not respond to these treatments. For the others, despite a high efficacy at the start, a residual disease persists and a relapse is almost systematically observed (development of resistance). The disease is more aggressive in escaping with a poor prognosis related to multiple brain metastases and a low response rate to second therapeutic lines. Recent preclinical studies and translational efforts have provided insight into the mechanisms of initial and acquired resistance to BRAF and MEK inhibitors. Acquired resistance to targeted drugs involves genetic and non-genetic changes that are largely due to tumor cell plasticity and phenotype change processes. Under the pressure of the drug, melanoma cells have the ability to reactivate developmental programs and move to a dedifferentiated mesenchymal-like state characterized by upregulation of receptor tyrosine kinases (RTKs) such as PDGFR, NGFR or AXL and downregulation of melanocyte differentiation proteins such as the transcription factor MITF. This cellular state is associated with greater metastatic potential and protection against both pharmacological agents (BRAF and MEK inhibitors) and immunotherapies. It is important to note that such adaptive responses to inhibition of the BRAF oncogenic pathway precede the emergence of acquired resistance induced by mutations. Immunotherapy is a treatment based on monoclonal antibodies directed against immune system checkpoints (immune checkpoint inhibitors (ICIs): anti-PDl or anti-CTLA4 such as ipilimumab (YERVOY®)). Nivolumab (OPDIVO®) and pembrolizumab (KEYTRUDA®) are the first two representatives of a new class of immunotherapy, humanized monoclonal antibodies, PD-1 inhibitors, which have been approved for marketing in advanced, unresectable or metastatic melanoma, regardless of its B-RAF mutational status. Binding of PD-1 to the ligands PD-L1 and PD-L2, which are expressed on antigen presenting cells and may be expressed by tumor cells or other cells of the tumor microenvironment, results in inhibition of T-cell proliferation and cytokine secretion. Anti -PD Is potentiate T-cell responses, including antitumor responses, by blocking PD-1 binding to PD-L1 and PD-L2 ligands. By restoring anti-tumor immunity, these cancer immunotherapies can have remarkable clinical effectiveness and lead to total remission, but unfortunately, response rates remain low and resistance mechanisms and the presence of metastases can also limit their effects.

[0009] There is therefore a real medical need to develop new therapeutic strategies for an effective and sustainable treatment of melanoma, and especially metastatic melanoma.

[0010] SUMMARY OF THE INVENTION

[0011] To meet this need, the inventors propose treating a melanoma with a blocker of L-type calcium channels (LTCCs) combined with a BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi).

[0012] An object of the invention is an L-type calcium channel (LTCC) blocker for use in the treatment of melanoma in a patient, in combination with a BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi), wherein said LTCC blocker is selected from a dihydropyridine (DHP), a phenylalkylamine or a benzothiazepine, or a pharmaceutically acceptable salt or derivative thereof.

[0013] Another object of the invention is a blocker specific for L-type calcium channels (LTCCs) for use in the treatment of melanoma in a patient, in combination with a BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi).

[0014] The LTCC-specific blocker may be selected from a dihydropyridine (DHP), a phenylalkylamine or a benzothiazepine, or a pharmaceutically acceptable salt or derivative thereof.

[0015] Another object of the invention is therefore an LTCC-specific blocker for use in the treatment of melanoma in a patient, in combination with a BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi), wherein said LTCC-specific blocker is selected from a dihydropyridine (DHP), a phenylalkylamine or a benzothiazepine, or a pharmaceutically acceptable salt or derivative thereof.

[0016] Preferably, the LTCC blocker or LTCC-specific blocker is a dihydropyridine selected from amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivatives thereof.

[0017] Advantageously, said BRAFi may be selected from vemurafenib, dabrafenib or encorafenib, or a pharmaceutically acceptable salt or derivative thereof, and said MEKi may be selected from cobimetinib, trametinib or binimetinib, or a pharmaceutically acceptable salt or derivative thereof.

[0018] The combination of the LTCC blocker or LTCC-specific blocker with BRAFi and / or MEKi may be adapted for systemic (i.e., enteral or parenteral) or local administration, preferably for oral administration. The combination according to the invention can also be adapted for simultaneous, sequential or separate administration.

[0019] Another object of the invention is a pharmaceutical composition comprising the combination of the LTCC blocker or LTCC-specific blocker with a BRAFi and / or MEKi as well as a pharmaceutically acceptable carrier or excipient.

[0020] Preferably, the invention targets the treatment of metastatic or unresectable forms of melanoma.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1 illustrates the classification (A) and structure (B-C) of voltage-dependent calcium channels with high activation threshold (HVA) and low activation threshold (LVA), specifying the pharmacology of LTCCs (A).

[0023] Figure 2 demonstrates that CACNA1C (CaV1.2) is overexpressed in melanoma cells / tumors refractory / resistant to targeted therapies. (A) Gene expression (Heatmap) of voltage-gated calcium channel (VGCC) subunits (al, P, a25, and y) in human melanoma cell lines M229, M238, and SK-Mel-28 (P = parental / sensitive, BRAFi2D = 2 days after BRAFi (vemurafenib) treatment, DTP = BRAFi -tolerant persister cells, DTPP = BRAFi-tolerant persister cells that have reverted to proliferation, SDR = cells that have acquired resistance to BRAFi, and DDR = cells that have acquired dual resistance to BRAFi and MEKi (gene expression is quantified as Log2 (FPKM+1) from RNAseq data GSE75299 of Song et al., Cancer Discov 2017 Nov; 7(11): 1248-1265). (B) Expression of CACNA1C and CACNA1D in melanoma tumors of patients progressing on BRAFi treatment (ON) compared to baseline (“Pre”, expression in the tumor at diagnosis before treatment).

[0024] Figure 3 demonstrates that CACNA1C (CaV1.2) is overexpressed in melanoma cells / tumors refractory / resistant to targeted therapies. (A) Expression of CACNA1C analyzed by quantitative PCR in a panel of melanoma cell lines classified according to their phenotype (melanocytic or mesenchymal). (B) Correlation analysis of CACNA1C expression and activity of MAP kinase inhibitors (BRAFi and MEKi) in a panel of melanoma cell lines never previously exposed to these drugs (CCLE / CTRP database).

[0025] Figure 4 shows that LTCC-specific blockers make it possible to overcome in vitro the resistance to BRAFi of melanoma cells. (A) Dose-response curves showing the viability of BRAFi -resistant M229 melanoma cells (M229R) treated with increasing doses of BRAFi (PLX4032 / vemurafenib) with or without increasing doses of a specific calcium channel blocker (calcium antagonist family): amlodipine (dihydropyridine), diltiazem (benzothiazepine) or verapamil (phenylalkylamine) (as indicated in the graphs). The cells were exposed to BRAFi + / - calcium antagonists for 3 days before measuring the cell viability by the CellTiter-Glo test. The graphs are representative of 4 independent experiments. Panels on the right, 3D representation of synergy scores (Bliss methods) calculated from dose-response matrices established previously. Synergy scores greater than 10 (in red) are considered interactions with a significant synergistic effect (potentiation). (B) Tests of clonogenicity (ability to form colonies) showing colony formation after 10 days of BRAFi + / - calcium antagonist treatment. Cells were treated with BRAFi (10 pM, vemurafenib), amlodipine (Amlo.) (5 pM), verapamil (10 pM), diltiazem (20 pM) or combinations thereof as indicated. The colony-forming cells were stained with crystal violet after 10 days of treatment.

[0026] Figure 5 shows that LTCC-specific blockers make it possible to overcome in vitro the resistance to BRAFi / MEKi of melanoma cells. (A and B). Same experiments as in Figure 4 A and B but carried out with the BRAFi / MEKi combination (vemurafenib / trametinib, ratio 10: 1) on M229 melanoma cells resistant to these two types of inhibitors (M229DDR).

[0027] Figure 6 shows that potentiation of BRAFi by amlodipine induces the death of melanoma cells resistant to targeted therapies. (A) Effects of BRAFi and amlodipine (Amlo.) treatments, alone or in combination (BRAFi+Amlo.), on cell death. M229R cells were treated with vemurafenib (10 pM), amlodipine (10 pM) or combotherapy for 3 days and analyzed by flow cytometry after Annexin V-FITC (AnnV) / propidium iodide (PI) labeling. The quadrants indicate the percentage of cells detected in each area. Quadrant 4 (Q4): living cells (Annexin V- / PI-); Q3: early apoptotic cells (Annexin V+ / PI-); QI : necrotic cells (Annexin V- / PI+); Q2: late apoptotic cells (Annexin V+ / PI+). (B) Cell death quantifications for BRAFi and / or amlodipine-treated M229 (parental) and M229R (BRAFi-resistant) cells. The bars represent the mean ± SEM of 3 independent experiments.

[0028] Figure 7 shows the synergistic effect between the LTCC-specific blocker (amlodipine) and BRAFi (vemurafenib; PLX4032) (A). No synergistic effect is found between BRAFi (vemurafenib) and another antihypertensive agent (e.g. perindopril being an angiotensin converting enzyme inhibitor) (B). The synergistic effect is maintained between the LTCC blocker (amlodipine) and the BRAFi (vemurafenib) when the LTCC blocker (amlodipine) is combined with another hypertensive agent such as perindopril (as a combination formulation of amlodipine and perindopril, i.e., COVERAM®) (C).

[0029] Figure 8 shows that the expression of CACNA1C is increased in mouse melanoma tumors YLTMM1.7, which progress on BRAFi, and that amlodipine delays the onset of relapse on MAPKi treatment (BRAFi+MEKi) in the preclinical model of melanoma in mice, i.e., the syngeneic model YLTMMM1.7. (A) Presentation of the preclinical melanoma model used. (B) Gene expression profiles of CACNA1C and CACNA1D in the MAPKi resistance stages of YLTMM1.7 melanoma tumors. The data were analyzed from RNAseq data of tumors collected at different stages (GSE10372, Song et al., Cancer Discov 1, November 2017; 7 (11): 1248-1265. (C) Effect of amlodipine and MAPKi (BRAFi+MEKi) treatments on the growth of YUMM1.7 tumors in a syngeneic mouse model. Cells were injected subcutaneously into C57BL / 6J mice and tumors greater than 0.1 cm3were treated 3 times weekly with 30 mg / kg PLX2032 (vemurafenib) + 0.3 mg / kg trametinib (oral gavage) + / - 7.5 mg / kg amlodipine (Amlo.) administered 7 times weekly by intraperitoneal injection. Therapeutic effects were assessed by regular measurements of tumor volume (top) and Kaplan-Meier survival curve (bottom).

[0030] Figure 9 shows that LTCC-specific blockers reduce the migratory and invasive capacities of BRAFi-resistant melanoma lines. (A) BRAFi-resistant M238 cells (M238R) were inoculated at low density on fibronectin, treated or not with 5 pM amlodipine and followed by video microscopy for 24 h. The panel on the left shows the Spider plots of the X and Y migration of untreated cells (M238R) or cells treated with amlodipine (M238R+Amlo). The graph on the right shows the quantifications of total single cell displacements (Euclidean distances traveled) for 30 cells (untreated cells: Amlo. -; treated cells: Amlo. +) represented with medians (in red) and quartiles (in blue) (one-way ANOVA, p*<0.05). (B) BRAFi-resistant UACC62 cell spheroids (UACC62R) were included in a collagen matrix and treated with amlodipine or not. The capacity of the cells to invade this matrix was quantified after 48 h.

[0031] Figure 10 shows that LTCC blockers reduce cell migration across dihydropyridine generations. BRAFi-resistant M238 cells (M238R) were inoculated at low density on fibronectin, treated or not with 5 pM of nicardipine (first generation), 5 pM of amlodipine (third generation), or 5 pM of lercanidipine (fourth generation) and cell movements were monitored by time-lapse videomicroscopy for 24 hours. The graphs show the quantification of total single-cell displacement (Euclidean distance traveled) for more than 50 individual cells per condition, with means shown in red (t test, ****p < 0.0001).

[0032] Figure 11 shows the broad synergistic activity of DHP-type LTCC blockers with MAPK- pathway inhibitors in overcoming resistance by inducing cell death. (A) Human BRAF-mutant melanoma cells resistant to targeted therapy (M229R cell line) were treated for 72 hours with increasing concentrations of the drugs, alone and in combination, in double dose-response assays as described in Figures 4 and 5. The BRAF inhibitors tested were vemurafenib (Vemu.), dabrafenib (Dabra.), and encorafenib (Enco.); the MEK inhibitors tested were cobimetinib (Cobi.), trametinib (Tra.), and binimetinib (Bini.); the BRAFi / MEKi combinations tested were vemurafenib / cobimetinib,, dabrafenib / trametinib, and encorafenib / binimetinib (all at a 10:1 ratio); the DHP-type LTCC blockers were nicardipine (Nicar., first generation), amlodipine (Amlo., third generation), and lercanidipine (Lerca., fourth generation). Cell viability was assessed using the CellTiter-Glo luminescent assay, allowing the generation of full interaction matrices for each pairwise combination. The resulting data were analyzed using the SynergyFinder software to calculate synergy scores. On graphs are displayed the Most Synergistic Area (MSA) score for each drug combinations, representing the highest synergy observed within each 3-by-3 dose window of the dose-response matrix. (B) Effects of BRAFi (Vemurafenib) and amlodipine, nicardipine or lercanidipine treatments, alone or in combination on cell death. M229R cells were treated for 3 days with vemurafenib (10 pM), the DHP-type LTCC blocker (10 pM), or their association, and analyzed by flow cytometry after Annexin V- FITC (AnnV) / propidium iodide (PI) labeling. Graphs represents cell death quantification (Annexin V- / PI- Live cells and V+ / PI-, V- / PI+ and V+ / PI+ dead cells).

[0033] Figure 12 shows that LTCC blockers treatment re-sensitizes refractory BRAF-mutant melanoma cells in an in vivo setting. The anti-tumor efficacy of Lercanidipine alone and in combination with Dabrafenib and Trametinib was evaluated in the AVI-CellDX™ model transplanted with BRAF / MEK inhibitor-resistant GLO-R melanoma cells (Jarrosson L, et al.. EMBO Mol Med. 2023 Mar 8;15(3):el6629). (A) GLO-R cells were engrafted at HH14 stage (E2). Twenty-four hours later, a single intravenous injection of Lercani dipine and / or Dabrafenib and Trametinib was administered at stage E3. The drugs were used at the maximum tolerated dose of their combination (as indicated in B). The experiment was terminated at HH25 (E4), followed by quantitative 3D analysis of tumor volume in viable embryos using light sheet microscopy 24 h post-treatment (48 h post-engraftment). (B) Graphs represent the tumor volume normalized to body surface area for each treated embryo and the bars represent the mean ± SEM. Statistical analysis was performed using unpaired t-tests.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The invention aims to treat melanoma, and especially advanced melanoma (unresectable or metastatic) using new therapeutic strategies to increase the effectiveness of current treatments.

[0036] In particular, the inventors sought to develop a treatment for melanoma using an L-type calcium channel (LTCC) blocker. To date, no therapeutic strategy for the management of melanoma includes an ion channel blocker such as the LTCC.

[0037] Ion channels are a family of specialized proteins that ensure the passage of ions through cell membranes. L-type calcium channels (LTCCs) are part of the family of high-voltage activated voltage-dependent calcium channels (HVA). “L” means long duration with reference to the activation time. This subfamily has four isoforms: Cavl. l, Cavl.2, Cavl.3 and Cavl.4. L-type calcium channels are responsible for the excitation-contraction coupling of skeletal and smooth cardiac muscle, as well as aldosterone secretion in the endocrine cells of the adrenal cortex. They are also found in neurons where they regulate neurohormones and neurotransmitters. These channels also play a role, for example, in gene expression, mRNA stability, cell survival, synaptic efficiency, and the activation and deactivation of other ion channels.

[0038] CaV1.2 (encoded by the CACNA1C gene) and CaV1.3 (encoded by the CACNA1D gene) are channel proteins expressed on the surface of cells. They are alpha- 1 subunits (forming the pore that passes calcium ions through the membranes) of the L-type calcium channel (LTCC) subfamily. Functional channels are macromolecular complexes composed of an ion-conducting protein (the al subunit) and several helper proteins with regulatory function called a25-, pi-4-, and y subunits (Figure 1).

[0039] L-type calcium channel blockers (also known as calcium antagonists or calcium channel inhibitors) are used as cardiac antiarrhythmics or antihypertensive agents, depending on whether the drugs have a higher affinity for the heart (phenylalkylamines, such as verapamil) or for blood vessels (dihydropyridines, such as nifedipine or amlodipine).

[0040] The present invention thus proposes the repositioning of particular calcium antagonists (i.e. LTCC blockers such as LTCC-specific blockers) for the treatment of melanoma.

[0041] The inventors have therefore developed a new combination therapy to treat melanoma, which increases the effectiveness of current treatments, both in terms of response rates and durations.

[0042] First, the inventors have demonstrated that some LTCCs are found expressed in tumors and melanoma cell lines. They showed, among others, that the CaV1.2 channel is, for example, particularly overexpressed in dedifferentiated cells with a mesenchymal phenotype that is associated with resistance to targeted therapies and that in patients, the expression of CaV1.2 (encoded by the CACNA1C gene) and / or CaV1.3 (encoded by the CACNA1D gene) is found to be increased in certain tumors that progress on treatment with targeted therapy (Figures 2 and 3).

[0043] Next, the inventors surprisingly established that the combination of a particular calcium antagonist with targeted therapies makes it possible to suppress in vitro the resistance of cells to targeted therapies and to eliminate them by a synergistic effect inducing cell death (Figures 4, 5, 6 and 11).

[0044] The inventors also demonstrated in a pre-clinical melanoma model that CACNA1D expression is increased during the residual disease phase and CACNA1C expression is greatly increased in tumors progressing on targeted therapy (Figure 8). In a similar preclinical model, the combination of an LTCC blocker with a BRAF inhibitor and a MEK inhibitor significantly delays the onset of resistance and the resumption of tumor growth.

[0045] The inventors have also demonstrated by various in vitro migration tests that the pharmacological inhibition of LTCCs by a dihydropyridine, such as amlodipine, lercani dipine or nicardipine, reduces the migratory capacities of different melanoma lines (e.g. M238R, UACC62R) with a dedifferentiated phenotype associated with tolerance / resi stance to targeted therapies (Figures 9 and 10). The inventors have therefore shown that LTCC blockers have an anti -metastatic action.

[0046] Finally, the inventors have also validated the in vivo therapeutic efficacy of the combination therapy according to the invention as a second-line treatment for refractory or relapsed BRAF- mutant and / or MEK-mutant melanoma (Figure 12). Thus, the inventors propose new synergistic therapeutic combinations enabling the treatment of melanoma, in particular metastatic or unresectable melanoma, and to prevent or at least delay the appearance of resistance to targeted therapies during the treatment of melanoma.

[0047] Definitions

[0048] Within the meaning of the present invention, the term “treatment” and “treat” refers to a curative or symptomatic treatment aimed at alleviating or slowing down the appearance or development of melanoma, or of these symptoms or one of its complications. The term “treatment” also means slowing down the growth of cancer cells, reducing the number of cancer cells, inhibiting the proliferation of cancer cells or destroying cancer cells. The term “ treatment ” also refers to reducing the number of metastases and / or reducing the risk of developing metastases. In the context of the treatment of metastases, the objective is especially to reduce the size and number of metastases and to prevent or delay relapse for the patient. The term “treat” also means preventing or delaying the onset of resistance to targeted therapies.

[0049] The term “targeted therapy or therapies” is used here to designate any therapy that relies on treatments developed on the basis of a better understanding of the molecular mechanisms leading to the appearance and development of tumors and more particularly therapies already known to treat melanoma, especially therapies based on MAPK pathway inhibitors, and in particular, therapies based on the use of anti-BRAF (also called BRAFi or BRAF inhibitors or BRAF antagonists) and / or anti-MEK (also called MEKi or MEK inhibitors or MEK antagonists).

[0050] In the context of the present invention, the term “L-type calcium channel (LTCC) blocker” or “L- type calcium channel (LTCC) inhibitor” refers to a molecule that inhibits LTCC channels. Preferably, LTCC blocker according to the invention is selected from three major structural families of LTCC modulators, namely dihydropyridines, phenylalkylamines, and benzothiazepines. LTCC blockers according to the invention also include derivatives of dihydropyridines, phenylalkylamines, or benzothiazepines, said derivatives having blocking activity on LTCCs.

[0051] In the context of the invention, the term “L-type calcium channel (LTCC)-specific blocker” or “L-type calcium channel (LTCC)-specific inhibitor” means a molecule that selectively inhibits LTCC channels. More particularly, a specific or selective blocker of LTCCs is defined here as a compound which at a concentration x inhibiting 50% of the maximum activity of L-type calcium channels (such as CaV1.2) (IC50) does not inhibit more than 25% of the maximum activity of T, N, R or P / Q type voltage-dependent calcium channels measured under the same experimental conditions. The reference test is a measure of channel activity by patch-clamp on recombinant channel complexes (human alpha- 1 subunit + / - accessory subunits) overexpressed in eukaryotic cells (e.g. HEK or CHO) under the same experimental conditions (patch system, intra- and extracellular solutions, etc.) as described by Kuryshev et al., Assay Drug Technol. 2014 Mar 1; 1252): 110-119. Preferably, a specific blocker of LTCCs is selected from dihydropyridines, phenylalkylamines, and benzothiazepines.

[0052] The term “effective dose” means a dose sufficient to obtain the desired therapeutic effect. The person skilled in the art is able to adapt this dose according to the stage and severity of the melanoma to be treated.

[0053] The term “and / or” as used herein is to be considered a specific description of each of the two specified characteristics or components, with or without the other. For example, “A and / or B” should be considered a specific disclosure of each of the following: (i) A, (ii) B and (iii) A and B, as if each were presented individually.

[0054] Therapeutic combination according to the invention

[0055] The present invention proposes a novel therapy for treating melanoma, and more particularly metastatic or unresectable melanoma, by repositioning particular calcium antagonists, i.e. LTCC blockers, such as LTCC-specific blockers and combinations thereof with at least one targeted therapy. More particularly, the invention relates to a particular therapeutic combination corresponding to the combination of at least one LTCC blocker, for example LTCC-specific blocker, with at least one targeted therapy selected from anti-BRAF therapies and anti-MEKi therapies. This therapeutic combination according to the invention provides greater therapeutic efficacy in the treatment of melanoma than targeted anti-BRAF and / or anti-MEK therapies alone. In the context of the invention, the terms “therapeutic combination”, “combination” or “combotherapy” are interchangeable.

[0056] The LTCC blocker or LTCC-specific blocker may be selected from a dihydropyridine (DHP), a phenylalkylamine or a benzothiazepine, or a pharmaceutically acceptable salt or derivative thereof.

[0057] Preferably, the LTCC blocker or LTCC-specific blocker is the Cavl.2 channel blocker (CACNA1C) and / or Cavl.3 channel blocker (CACNA1D).

[0058] Advantageously, said dihydropyridine (DHP) is selected from amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof. Preferably, the LTCC blocker or LTCC-specific blocker is amlodipine, lercanidipine or nicardipine, or salts or derivatives thereof.

[0059] Advantageously, said phenylalkylamine is selected from fendiline, gallopamil or verapamil (Calan, Isoptin), or a pharmaceutically acceptable salt or derivative of these compounds.

[0060] Advantageously, said benzothiazepine is diltiazem (Cardizem, Bi-Tildiem, Mono-Tildiem, Tildiem), or a pharmaceutically acceptable salt or derivative thereof.

[0061] Advantageously, said BRAFi is selected from vemurafenib, dabrafenib or encorafenib, or a pharmaceutically acceptable salt or derivative thereof.

[0062] Advantageously, said MEKi is selected from cobimetinib, trametinib or binimetinib, or a pharmaceutically acceptable salt or derivative thereof.

[0063] In a particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0064] In a other particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0065] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0066] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0067] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, bamidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0068] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0069] In a particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0070] In a particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0071] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, bamidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof. In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0072] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, bamidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0073] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0074] In a particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with encorafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0075] In a particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with encorafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0076] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, bamidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with encorafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0077] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with encorafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0078] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, aranidipine, azelnidipine, bamidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with encorafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0079] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker selected from amlodipine, lercanidipine, and nicardipine, or a pharmaceutically acceptable salt or derivative thereof for use in the treatment of melanoma in a patient, in combination with encorafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0080] The preferred therapeutic combination based on a dihydropyridine according to the invention is one of the following combinations: amlodipine, vemurafenib and cobimetinib, amlodipine, dabrafenib and trametinib, amlodipine, encorafenib and binimetinib, amlodipine, vemurafenib and trametinib, amlodipine and vemurafenib, lercanidipine, vemurafenib and cobimetinib, lercanidipine, dabrafenib and trametinib, lercanidipine, encorafenib and binimetinib, lercanidipine, vemurafenib and trametinib, lercanidipine and vemurafenib, nicardipine, vemurafenib and cobimetinib, nicardipine, dabrafenib and trametinib, nicardipine, encorafenib and binimetinib, nicardipine, vemurafenib and trametinib, nicardipine and vemurafenib, amlodipine, vemurafenib and binimetinib, amlodipine, dabrafenib and cobimetinib, amlodipine, dabrafenib and binimetinib, amlodipine, encorafenib and cobimetinib, amlodipine, encorafenib and trametinib, lercanidipine, vemurafenib and binimetinib, lercanidipine, dabrafenib and cobimetinib, lercanidipine, dabrafenib and binimetinib, lercanidipine, encorafenib and cobimetinib, lercanidipine, encorafenib and trametinib, nicardipine, vemurafenib and binimetinib, nicardipine, dabrafenib and cobimetinib, nicardipine, dabrafenib and binimetinib, nicardipine, encorafenib and cobimetinib, nicardipine, encorafenib and trametinib, amlodipine and dabrafenib, amlodipine and encorafenib, amlodipine and cobimetinib, amlodipine and trametinib, amlodipine and binimetinib, lercanidipine and dabrafenib, lercanidipine and encorafenib, lercanidipine and cobimetinib, lercanidipine and trametinib, lercanidipine and binimetinib, nicardipine and dabrafenib, nicardipine and encorafenib, nicardipine and cobimetinib, nicardipine and trametinib, or nicardipine and binimetinib, or pharmaceutically acceptable salts or derivative thereof. In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0081] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0082] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0083] In a particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0084] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0085] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0086] In a particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with encorafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0087] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with encorafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0088] In another particular embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being a phenylalkylamine selected from fendiline, gallopamil or verapamil, or a pharmaceutically acceptable salt or derivative of these compounds, for use in the treatment of melanoma in a patient, in combination with encorafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0089] The preferred therapeutic combination based on a phenylalkylamine according to the invention is one of the following combinations: verapamil, vemurafenib and cobimetinib, verapamil, dabrafenib and trametinib, verapamil, encorafenib and binimetinib, verapamil, vemurafenib and trametinib, verapamil and vemurafenib, verapamil, vemurafenib and binimetinib, verapamil, dabrafenib and cobimetinib, verapamil, dabrafenib and binimetinib, verapamil, encorafenib and cobimetinib, verapamil, encorafenib and trametinib, verapamil and dabrafenib, verapamil and encorafenib, verapamil and cobimetinib, verapamil and trametinib, or verapamil and binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0090] In another embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0091] In another embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0092] In another embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with vemurafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0093] In an embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0094] In another embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0095] In another embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with dabrafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0096] In an embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with encorafenib and / or cobimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0097] In another embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with encorafenib and / or trametinib, or pharmaceutically acceptable salts or derivatives thereof.

[0098] In another embodiment, the invention concerns an LTCC blocker or LTCC-specific blocker being diltiazem or a pharmaceutically acceptable salt or derivative thereof, for use in the treatment of melanoma in a patient, in combination with encorafenib and / or binimetinib, or pharmaceutically acceptable salts or derivatives thereof.

[0099] As illustrated in the experimental part, the inventors have clearly demonstrated that the therapeutic combinations according to the invention produce remarkable synergistic effects.

[0100] Thus, in a particular embodiment, the invention concerns a synergistic combination of an LTCC blocker or LTCC-specific blocker, as described in the present application, with a BRAFi and / or a MEKi, as described in the present application, for use in the treatment of melanoma in a patient.

[0101] In other particular embodiments, the combination according to the invention may comprise one or more LTCC blockers or LTCC-specific blockers, one or more BRAFis and / or one or more MEKis among those described above.

[0102] Melanoma treatment

[0103] The invention proposes using the combinations described above for the management of melanomas, and more particularly advanced melanomas.

[0104] In a preferred embodiment, the present invention aims to use the combinations described above to treat metastatic or unresectable melanoma, or to prevent or treat one of its complications, in a patient. Typically, metastatic or unresectable forms of melanoma correspond to stages III and IV according to the classification of the AJCC international TNM (tumor, nodes, metastasis) system.

[0105] A method for treating a melanoma, in particular a metastatic or unresectable melanoma, or for preventing or treating one of its complications, in a patient, in which the combination or pharmaceutical composition according to the invention is administered to the patient, is also described.

[0106] Preferably, the therapeutic strategies for the management of metastatic or unresectable melanoma according to the invention are based on therapeutic targeting of CaV1.2 (CACNA1C) and / or CaV1.3 (CACNA1D) in order to block the tumor progression of melanoma and improve the response to treatments. For example, the combinations according to the invention can be applied to the population of patients with non-operable cutaneous metastatic melanoma, or as an adjunct treatment to the first / second line standard of care, aimed at eliminating tumor cells. Thus, according to particular embodiments, the combination according to the invention can be applied for the treatment of melanoma in the first or second therapeutic line.

[0107] In a particular embodiment, the invention relates to the treatment of metastatic or unresectable stage III or stage IV melanoma. In another particular embodiment, the invention relates to the treatment of stage III or stage IV inoperable melanoma. In another particular embodiment, the invention relates to the management of BRAF -mutated and / or MEK-mutated melanomas.

[0108] In another particular embodiment, the invention also makes it possible to prevent the development of certain types of resistance to metastatic cutaneous melanoma treatments (especially resistance to targeted therapies) and / or makes it possible to eliminate cells that have become resistant. The invention also prevents the development of cross-resistance to targeted therapies and immunotherapies.

[0109] In a particular embodiment, the therapeutic combination according to the invention can be used in combination with other cancer therapies, and / or any other treatment or surgery. Said other cancer treatments may be drug-based or non-drug-based. Drug-based cancer treatments especially include chemotherapy and immunotherapy (anti-PDl, anti-CTLA-4, vaccines, etc.), and non-drug-based treatments include radiotherapy.

[0110] According to another particular embodiment, the combination according to the invention can be used to delay the appearance of resistance to at least one targeted therapy selected from anti- BRAF therapies and anti-MEK therapies.

[0111] The invention also relates to increasing the therapeutic window by preventing recurrence and increasing recurrence-free survival.

[0112] The invention would thus increase the survival of patients with advanced melanoma, and the chances of remission by filling a medical need for which no therapeutic option exists.

[0113] Pharmaceutical compositions

[0114] The present invention also relates to pharmaceutical compositions comprising at least one LTCC blocker or LTCC-specific blocker and at least one BRAFi and / or at least one MEKi, as described in the present application, formulated in combination with a pharmaceutically acceptable carrier or excipient.

[0115] In a particular embodiment, the invention therefore also concerns a pharmaceutical composition comprising a LTCC blocker or LTCC-specific blocker, in combination with a BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi), as described in the present application, and a pharmaceutically acceptable carrier or excipient, for use in the treatment of melanoma in a patient.

[0116] Pharmaceutically acceptable carrier or excipient is understood to mean any carrier or excipient which does not interfere with the efficacy of the biological activity of the therapeutic active agent(s) present in the pharmaceutical composition and which is not toxic for the patient to whom the composition is administered.

[0117] The pharmaceutical compositions according to the invention advantageously comprise one or more pharmaceutically acceptable carriers or excipients. The compositions may contain one or more agents selected from binders or emulsifiers, lubricants, disintegrants, gelling agents, colorants, flavoring agents, dispersants, solubilizers, stabilizers, preservatives, solvents, etc.

[0118] Furthermore, the composition according to the invention may also comprise at least one other therapeutic agent or active ingredient, preferably selected from a peptide, a protein, a nucleic acid, an antibody, a nanobody, a synthetic molecule or a toxin. For example, the combination according to the invention can optionally be used in combination with other LTCC blocker (such as synthetic molecules, therapeutic antibodies, nanobodies or other biologies), and / or antihypertensive agents (e.g., angiotensin converting enzyme (ACE) inhibitors such as perindopril, angiotensin II receptor antagonist such as irbesartan or candesartan, beta-blockers such as Prolol, or bisoprolol, etc., and / or diuretics such as hydrochlorothiazide or furosemide.

[0119] Thus, the present invention also concerns an LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or a MEKi, or a pharmaceutical composition comprising such a combination, for use in the treatment of melanoma in a patient, as described in the present application, in combination with at least one additional active compound, preferably selected from a peptide, a protein, a nucleic acid, an antibody, a nanobody, a synthetic molecule, a toxin and / or a chemotherapeutic, immunotherapeutic, radiotherapeutic or surgical treatment.

[0120] Routes of administration and dosage

[0121] The therapeutic combination according to the invention (i.e., combination of at least one LTCC blocker or LTCC-specific blocker with at least one BRAFi and / or at least one MEKi), or the pharmaceutical composition comprising such a combination, can be administered to the patient in various forms and by any type of routes known to the person skilled in the art.

[0122] In particular embodiments, the therapeutic combination according to the invention can be adapted for simultaneous, sequential or separate administration. Thus, the active ingredients of the combination according to the invention can be administered to the patient simultaneously, sequentially or separately. For example, the LTCC blocker LTCC-specific blocker may be administered together with a BRAFi or MEKi or BRAFi and MEKi dual therapy, but it may also be administered separately or sequentially, especially before or after BRAFi or MEKi therapy or BRAFi and MEKi dual therapy. The pharmaceutical compositions which are useful according to the invention can, for example, be administered enterally (such as orally, perlingually, etc.) or parenterally (such as intravenously, intramuscularly, subcutaneously, intradermally, etc.). For injections, the compounds are generally packaged in the form of liquid suspensions, which can be injected by means of syringes or infusions, for example. The preferred route of parenteral administration is intravenous administration.

[0123] The administration of the LTCC blocker or LTCC-specific blocker is preferably oral but may also be parenteral, preferably intravenous (for example for nicardipine).

[0124] Preferably, the administrations of BRAFi or MEKi monotherapy or BRAFi and MEKi dual therapy are oral and more specifically in the form of capsules or tablets with specific dosing schedules depending on each therapeutic active agent. In a particular embodiment, drinkable or liquid nasogastric tube solutions are used for the administration of BRAFi and / or MEKi.

[0125] In a preferred embodiment, all the compounds of the combination according to the invention (i.e., the LTCC blocker or LTCC-specific blocker and the BRAFi and / or MEKi) are suitable for oral administration. Preferred oral dosage forms include tablets, capsules, gelcaps, solutions, suspensions, and orodispersible films. Tablets and capsules offer controlled release and facilitate dosing, while solutions and suspensions allow faster absorption, particularly for pediatric or geriatric populations. In addition, the integration of encapsulation and nanoparticle technologies improves the bioavailability of the active ingredients, thus allowing better control of the therapeutic effects while reducing adverse effects.

[0126] Typically, the active compounds of the combination according to the invention are administered at doses which can vary between approximately 1 mg and approximately 2000 mg / per administration.

[0127] According to one embodiment of the invention, the LTCC blocker or LTCC-specific blocker or one of its pharmaceutically acceptable salts or derivative is suitable for a daily administration of 1 to 1200 mg.

[0128] In particular embodiments, the active compounds of the combination according to the invention are administered orally at the following daily dose: from 1 to 10 mg for amlodipine, preferably from 5 to 10 mg per day, from 5 to 20 mg for lercani dipine, preferably from 10 to 20 mg per day, from 40 to 120 mg for nicardipine, preferably from 50 to 120 mg per day, from 480 to 1920 mg for vemurafenib, preferably 1920 mg per day, from 40 to 300 mg for dabrafenib, preferably 300 mg per day, from 300 to 450 mg for encorafenib, preferably 450 mg per day, from 20 to 60 mg for cobimetinib, preferably 60 mg per day, from 1 to 2 mg for trametinib, preferably 2 mg per day, from 60 to 90 mg for binimetinib, preferably 90 mg per day.

[0129] The administration of the active ingredients may be daily as a single dose or repeated several times a day, for example: vemurafenib: 960 mg twice daily, dabrafenib: 150 mg twice daily, binimetinib: 45 mg twice daily, nicardipine: 20-40 mg three times daily, or 30-60 mg twice daily (preferably for the sustained-release form).

[0130] According to a particular example, the administration and dosing modalities of the combination according to the invention are as follows:

[0131] AMLODIPINE: from 5 to 10 mg amlodipine per day orally;

[0132] DABRAFENIB 75 mg: 150 mg morning and evening orally (i.e. 2 morning gelcaps and 2 evening gelcaps);

[0133] TRAMETINIB 2 mg: 1 tablet per day.

[0134] According to another particular example, the administration and dosage modalities of the combination according to the invention are as follows:

[0135] AMLODIPINE: from 5 to 10 mg amlodipine per day orally;

[0136] ENCORAFENIB 75 mg: 450 mg once daily;

[0137] BINIMETINIB 15 mg: 45 mg morning and evening.

[0138] It is understood that the therapeutically effective dose of each active ingredient and the dosage regimen may be adapted by a person skilled in the art according to the patient, the age and weight of the patient, the route of administration and the stage of development of the melanoma, etc.

[0139] The examples below illustrate the invention without limiting its scope.

[0140] EXAMPLES

[0141] Example 1: Overexpression of calcium channels in melanoma tumor cells resistant to targeted therapies

[0142] Voltage-dependent calcium channels are a family of channels permeable to calcium ions normally expressed in so-called excitable cells (neurons, muscle cells, etc.). The entry of physiological calcium that they allow into these cells is of paramount importance in many cellular processes: contraction, secretion, gene activation, etc. These channels can also be found overexpressed in certain cancers where they promote tumor progression. The expression of these channels in melanoma was analyzed either by exploring public sequencing data, or by quantitative PCR or western blotting approaches. This example shows that some LTCCs are found expressed in tumors and melanoma lines. CaV1.2 (CACNA1C) is more particularly expressed in dedifferentiated cells with a mesenchymal phenotype that is associated with resistance to targeted therapies (Figures 2 and 3).

[0143] There is a negative correlation between the expression of CACNA1C in a cell type (line) and the sensitivity of this line to BRAF or MEK inhibitors (Figure 3B). During chronic treatment of melanoma cells bearing the BRAF V600E mutation with a BRAF inhibitor, CACNA1C and CACNA1D expression increases with the development of resistance. The expression of CACNA1D is higher in drug-tolerant persister (DTP) and drug-tolerant proliferating persister (DTPP) cells. The expression of CACNA1C is higher in DTPP cells and / or cells that have acquired resistance to therapies targeted by the phenotypic plasticity mechanism (BRAFi alone = SDR or BRAFi+MEKi = DDR) (Figures 2 and 3). In patients, the expression of CACNA1C and / or CACNA1D is increased in some tumors that progress on treatment (Figure 2B).

[0144] In this example, the inventors have demonstrated that LTCC-type calcium channels (CACNA1C / CaV1.2 or CACNA1D / CaV1.3) are overexpressed in human melanoma cell lines (especially M229, M238 and SK-Mel-28) that are refractory / tolerant / resistant to targeted therapies and in melanoma tumors that progress on targeted therapies (see Figures 2 and 3).

[0145] Example 2: Synergistic effect of combining an LTCC blocker with a BRAFi and / or MEKi

[0146] LTCCs are the target of a family of drugs called calcium antagonists, of which there are 3 classes: dihydropyridines (e.g. amlodipine, nifedipine, etc.), phenylalkylamines (e.g. verapamil) and benzothiazepines (e.g. diltiazem). These active ingredients are used in the management of cardiovascular diseases such as hypertension. We therefore used calcium antagonists to analyze the effect of an inhibition of an LTCC-type calcium channel, especially Cavl.2, on the response of melanoma cells to BRAFi and / or MEKi targeted therapies.

[0147] M229R cells (M229 cells that were made resistant to BRAFi by chronic in vitro exposure) were treated for 72 h with increasing doses of each type of calcium antagonist (amlodipine, verapamil, or diltiazem) combined with increasing doses of BRAFi (PLX4032 / vemurafenib). Parental M229 (M229S) cells, treated only with a BRAFi range, are used as a positive control for cell death. The cell viability was measured by CellTiter-Glo®. The double dose-response graphs obtained validate the resistance of the M229Rs to BRAFi, with an IC50 of 20 pM versus 740 nM for M229s. Treatment of M229Rs with a calcium antagonist range crossed with a BRAFi range induces a re-sensitization of the cells which results in a decrease in the BRAFi IC50 (Figure 4A). Indeed, the IC50 of the BRAFi goes from 20 pM for the control M229R to 6.67 pM when the cells are treated simultaneously with 12.5 pM of amlodipine. To achieve the same transition from the BRAFi IC50 with diltiazem and verapamil, cells require 40 pM and 20 pM of each of these calcium antagonists, respectively. Then, in order to determine whether the observed effects on cell viability result from an additive effect of the action of the BRAFi and that of the LTCC blockers, or rather from a synergistic effect of the two combined molecules, a synergy analysis was performed with the Bliss method, which allows calculating synergy scores for each of the 3 BRAFi-LTCC-specific blocker combinations. The results of these scores are presented in the form of 3-D maps obtained with SynergyFinder 2.0 software in Figures 4 and 5. Scores below - 10 indicate an antagonistic effect of the two compounds. Scores between -10 and 10 indicate that the decrease in cell viability results from a simple addition of the effects of each of the 2 molecules. Scores above 10 indicate a synergistic relationship between the BRAFi and the LTCC-specific blocker.

[0148] In the case of synergy, the decrease in cell viability results from an interaction of the 2 molecules that potentiate each other, and the combinatorial effect on cell viability is therefore greater than that of an additive effect. The combination of BRAFi and LTCC-specific blockers of this example reveals areas of very strong synergy for BRAFi concentrations of [2.2 - 20 pM] coupled with doses of [10 - 15 pM] amlodipine or [15 - 50 pM] verapamil. For diltiazem, the zone of maximum synergy corresponds to concentrations of [6.67-20 pM] BRAFi combined with [40-120 pM] diltiazem.

[0149] We also confirmed these synergistic effects by a clonogenicity test (or clonogenic survival test) (see Figure 4B and Figure 5B). Given that the cells, inoculated at very low density, are treated for 10 days to allow the formation of cell clones (visualized by violet crystal staining), the treatments (BRAFi and LTCC-specific blocker) were renewed 2 times during the test. The M229Rs were treated with 10 pM BRAFi combined or not with 5 pM amlodipine, 10 pM verapamil or 20 pM diltiazem. These are the optimal concentrations with little or no effect on the formation of clones when used as monotherapy over a period of 10 days, which makes it possible to better appreciate the effect of a potential synergy.

[0150] The results show that the BRAFi-amlodipine combination results in an average decrease of 90% of the cellular clones compared to each of the molecules used alone (Figure 4B and Figure 5B). The combination of BRAFi with verapamil or diltiazem resulted in a decrease in clones of 62% and 55% on average, respectively.

[0151] This example demonstrates that the combination of an LTCC blocker with MEKi and / or BRAFi targeted therapies, allows to suppress in vitro the resistance of cells to targeted therapies and to eliminate resistant cells by a synergistic effect inducing cell death (see Figures 4, 5 and 6). Thus, the inventors have clearly demonstrated that the combination of an LTCC-specific blocker (such as amlodipine) and targeted anti-BRAF and / or anti-MEK therapy has a very strong synergistic effect on the elimination of tumor cells and thus allows to obtain a better therapeutic effectiveness in the treatment of melanoma.

[0152] Example 3: Synergistic effect of combining amlodipine with BRAFi (vemurafenib) and an antihypertensive agent (perindopril)

[0153] The drugs commonly prescribed to control blood pressure include calcium antagonists that facilitate the relaxation of the arteries (mainly those belonging to the family of dihydropyridines such as amlodipine marketed under the name Amlor®); angiotensin converting enzyme (ACE) inhibitors, such as perindopril) and angiotensin II receptor antagonists (ARA II) that act on certain hormones (renin and angiotensin) that regulate blood pressure by decreasing the contraction of vessels; or thiazide diuretics which act on the kidneys and promote the elimination of salt (but which have been associated with an increase in the incidence of melanomas). If one of these modalities alone is not sufficient to control arterial hypertension (AH) in a patient, combinations may be prescribed such as Coveram which combines amlodipine and perindopril. Therefore, we compared the effects of amlodipine, perindopril and their combination (Coveram) on the response of cells to BRAFi. Figure 7 shows dose-response curves demonstrating viability of M229R melanoma cells treated with increasing doses of BRAFi (PLX4032 / vemurafenib) combined with increasing doses of an antihypertensive agent: amlodipine (A), perindopril (B), Coveram (C), as shown in the graphs. The cells were exposed to BRAFi+ / - antihypertensive agents for 3 days before measuring the cell viability with the CellTiter-Glo test. The graphs are representative of 3 independent biological replicas. The panels on the right show synergy analyses by the Bliss method. Cell viability data obtained on M229R indicate that amlodipine exhibits strong synergy with BRAFi (vemurafenib), whereas perindopril has no effect on the response of M229R cells to BRAFi. Coveram (amlodipine + perindopril) has a synergistic effect with BRAFi similar to that of amlodipine, indicating that there is no drug interaction negatively affecting the action of amlodipine. These data show the significant synergistic effect for the combination of the LTCC-specific blocker being also an antihypertensive agent (amlodipine) with BRAFi (vemurafenib) as well as for the combination of the LTCC-specific blocker (amlodipine) with BRAFi (vemurafenib) and another antihypertensive (perindopril). However, there is no synergistic effect between BRAFi (vemurafenib) and another antihypertensive agent that is not an LTCC-specific blocker (perindopril).

[0154] Example 4: The effect of the therapeutic combination according to the invention in vivo

[0155] In a pre-clinical model of melanoma, CACNA1D expression is increased during the residual disease phase and CACNA1C expression is greatly increased in tumors progressing under targeted therapy (Figure 8B). In a similar preclinical model, the combination of an LTCC- specific blocker (amlodipine) with a BRAF inhibitor (vemurafenib) and a MEK inhibitor (trametinib) significantly delays the onset of resistance and the resumption of tumor growth. This establishes an in vivo proof of concept of the benefit of the combination according to the invention (Figure 8C).

[0156] In order to confirm the interest of the association between BRAFi targeted therapy and the LTCC-specific blocker, we used a syngeneic model of murine melanoma tumors carrying the BRAF V600E mutation induced from YUMM1.7 cell transplants (Figure 8 A). When induced in mice, YUMM1.7 melanoma tumors are initially susceptible to targeted therapies but eventually develop resistance during treatment (Erkes et al., Cancer Discov 2020 10 (2): 254-269; Song et al., Cancer Discov 1 November 2017; 7 (11): 1248-1265). This model therefore mimics the development of resistance observed in patients treated with a BRAF inhibitor alone or, as is currently the case in clinical practice, with a BRAF inhibitor combined with a MEK inhibitor. In their study exploiting this preclinical model in vivo, Song et al. carried out a study of the transcriptome of tumors at the different stages of their treatment with a BRAF inhibitor (untreated, regression phase, residual disease, relapse). We then analyzed the expression profile of CACNA1C and CACNA1D at different stages of acquisition of therapeutic resistance of YUMM1.7 from the data of this study (Figure 8B). These analyses show that when cells are still sensitive to therapies and the tumor regresses, there is no difference in expression between CACNA1C and CACNA1D.

[0157] During the so-called “residual” phase, melanoma cells acquire tolerance to treatments; the tumor no longer regresses but does not evolve. This treatment tolerance phase then corresponds to the “DTP” (drug-tolerant persister). During this residual phase the expression of CACNA1D is greater compared to earlier stages. Finally, when the cells become resistant to MAPKi and the tumor progresses, it is the expression of CACNA1C that is greatly increased compared to the early stages while CACNA1D is found underexpressed. These expression profiles are therefore consistent with those observed during the different in vitro resistance acquisition phases for human lines M229 and M238. They reinforce the hypothesis of a central role played by these channels in the development of resistance to targeted melanoma therapy and validate the model of subcutaneous tumors of YUMM1.7 cells to establish proof of concept. The YUMM1.7 cells were therefore injected into C57BL / 6 mice subcutaneously to induce tumors. Vemurafenib / BRAFi + trametinib / MEKi and / or amlodipine treatments were then administered to mice as shown in the diagram (Figure 8C).

[0158] Monitoring of tumor volume over time, as well as Kaplan-Meier survival analysis, show that tumors in mice receiving amlodipine monotherapy progressed at the same rate as tumors in control mice. For the mice which received the MAPKi monotherapy, we observed an initial phase of control of the tumor growth before these tumors resumed their growth as expected (Figure 8C). In return, tumors treated with the amlodipine-BRAFi / MEKi combination therapy have a significantly longer tolerance phase than other groups before resumption of tumor progression. These results indicate that the combination of amlodipine with targeted therapies delays the onset of resistance (by approximately 10 days in this model) compared to BRAFi / MEKi combination therapy.

[0159] In conclusion, targeting voltage-dependent calcium channels through an LTCC-specific blocker such as amlodipine, makes it possible to resensitize BRAF V600E melanoma to targeted therapy.

[0160] Thus, it has been demonstrated here that the inhibition of calcium channels by amlodipine suppresses the resistance of melanoma to its treatment with BRAFi (vemurafenib) and MEKi (trametinib), leading to cell death. Similar synergistic activity was also observed with other LTCC blockers that were not of the dihydropyridine family, such as phenylalkylamine (e.g. verapamil) or benzothiazepine (e.g. diltiazem).

[0161] In addition, testing the effect of amlodipine in combination with vemurafenib / trametinib in a pre- clinical melanoma model demonstrated that targeting the L-type voltage-dependent calcium channel (LTCC) pathway delays the onset of resistance to treatment.

[0162] These results demonstrate that the combination of an LTCC blocker with agents targeting the MAPK pathway such as BRAFi and / or MEKi, allows to delay or even prevent the resistance of cancer cells to treatment targeting melanoma. These in vivo data clearly prove the benefit of the therapeutic combination according to the invention over existing targeted therapies, as well as its therapeutic efficacy in the treatment of melanoma.

[0163] Example 5: The anti-metastatic effect of an LTCC-specific blocker

[0164] BRAFi-resistant M238 cells (M238R) were inoculated at low density on fibronectin, treated or not with 5 pM amlodipine and followed by video microscopy for 24 h. The results of this experiment are shown in Figure 9A. The panel on the left of Figure 9 A shows the Spider plots of the X and Y migration of untreated cells (M238R) or cells treated with amlodipine (M238R+Amlo). The graph on the right of Figure 9A shows the quantifications of total displacements of single cell (Euclidean distances traveled) for 30 cells (untreated cells: Amlo. treated cells: Amlo. +) represented with medians (in red) and quartiles (in blue) (one-way ANOVA, p*<0.05). These results show that BRAFi-resistant M238 (M238R) cells that have been treated with amlodipine migrate less than untreated M238R cells. Thus, amlodipine significantly reduces the migratory capacity of BRAFi-resistant M238 cells (M238R). Next, another cell line associated with resistance to targeted therapies was tested, such as the BRAFi- resistant UACC-62 cell line (UACC62R). Spheroids of UACC62R cells were included in a collagen matrix and treated or not with amlodipine. The capacity of the cells to invade this matrix was quantified after 48 h. As shown in Figure 9B, amlodipine significantly reduces the invasive capabilities of UACC62R cells.

[0165] In conclusion, the inventors have clearly demonstrated that LTCC blockers, and in particular LTCC-specific blockers such as amlodipine, exhibit anti-metastatic effects by significantly reducing the migratory and invasive capacities of different melanoma lines resistant to targeted therapies.

[0166] Example 6: LTCC inhibitors reduce cell migration across dihydropyridine generations

[0167] Among the calcium channel blockers (CCBs) most widely used in clinical practice, the 1,4- dihydropyridine (DHP) derivatives represent the predominant class, mainly prescribed for the management of hypertension due to their marked vascular selectivity and good tolerability. Nowadays, DHP -type CCBs are among the most frequently prescribed antihypertensive drugs.

[0168] New generations of DHP CCBs have been developed, showing higher vascular selectivity, slower onset, and longer duration of hypotensive action compared with the prototype compound nifedipine. Four generations of DHP CCBs are currently available. The first-generation agents (e.g., nifedipine and nicardipine) are effective against hypertension but, due to their rapid onset and short half-life, are often associated with adverse effects. The second generation includes slow-release nifedipine and short-acting preparations, offering improved control of therapeutic response and reduced baroreflex activation. The third-generation compounds, such as amlodipine, are more lipophilic, display stable pharmacokinetics with prolonged action, and are well tolerated in patients with heart failure or chronic kidney disease. The fourth-generation DHPs, including lercanidipine, are highly lipophilic and provide improved therapeutic comfort, stable activity, fewer adverse effects, and a broad therapeutic spectrum.

[0169] To assess whether the anti -migratory effect of amlodipine described in the previous example is conserved across the DHP family of CCBs, cell migration assays were performed using the highly invasive BRAFi -resistant M238 melanoma cells (M238R). Cells were seeded at low density on fibronectin and treated or not with 5 pM of nicardipine (first generation), amlodipine (third generation), or lercanidipine (fourth generation). Cell movements were monitored by timelapse videomicroscopy for 24 hours. The results are presented in Figure 10. The graphs show the quantification of total single-cell displacement (Euclidean distance traveled) for more than 50 individual cells, with means shown in red (t test, ****p < 0.0001).

[0170] The data demonstrate that M238R cells treated with nicardipine, amlodipine, or lercanidipine exhibited significantly reduced migration compared with untreated controls. Thus, all tested generations of DHP -type CCBs markedly decreased the migratory capacities of highly invasive and BRAFi-resistant melanoma cells.

[0171] No significant morphological alterations or signs of cytotoxicity were observed under these experimental conditions, indicating that the reduced motility was not due to cell death or detachment.

[0172] In conclusion, these data demonstrate that inhibition of L-type calcium channels using clinically approved LTCC blockers, and in particular DHP -type calcium channel blockers consistently impairs the migratory behavior of targeted therapy -resistant melanoma cells across multiple DHP generations.

[0173] Example 7: Broad synergistic activity of DHP-type CCBs with MAPK-pathway inhibitors

[0174] As shown in Example 2, the combination of a LTCC blocker, and in particular a DHP-type CCB, with a BRAF inhibitor (BRAFi), alone or in association with a MEK inhibitor (MEKi), produced a synergistic effect in in vitro assays by re-sensitizing melanoma cells that had become resistant to such targeted therapies.

[0175] Several BRAF inhibitors (e.g., vemurafenib, dabrafenib, encorafenib) and MEK inhibitors (e.g., cobimetinib, trametinib, binimetinib) are currently available, and three BRAFi / MEKi combinations are used in clinical management of metastatic melanoma: vemurafenib / cobimetinib, dabrafenib / trametinib, and encorafenib / binimetinib.

[0176] To further exemplify in vitro the potential synergistic interactions between different DHP-type CCBs and MAPK-pathway-targeting agents, three DHP-type CCBS from different generations were tested for their ability to potentiate the effects of various BRAFi, MEKi, and clinically used BRAFi / MEKi combinations.

[0177] Human BRAF-mutant melanoma cells resistant to targeted therapy (M229R cell line) were treated for 72 hours with increasing concentrations of each drug, alone and in combination, in double dose-response assays. The BRAF inhibitors tested were vemurafenib, dabrafenib, and encorafenib; the MEK inhibitors tested were cobimetinib, trametinib, and binimetinib, the BRAFi / MEKi combinations tested were vemurafenib / cobimetinib, dabrafenib / trametinib, and encorafenib / binimetinib. Cell viability was assessed using the CellTiter-Glo luminescent assay, allowing the generation of full interaction matrices for each pairwise combination. The resulting data were analyzed using the SynergyFinder software to calculate synergy scores and identify the most synergistic drug pairs. The analysis was based on the Most Synergistic Area (MSA) score, representing the highest synergy observed within each 3-by-3 dose window of the dose-response matrix (Figure 11 A).

[0178] All three DHP-type CCB tested exhibited synergistic effects with each of the BRAFi, MEKi, and BRAFi / MEKi combinations evaluated.

[0179] Complementary apoptosis assays using Annexin V / Propidium Iodide staining confirmed that the combination of each DHP-type CCB with a BRAF inhibitor (vemurafenib) induced significant cell death in resistant melanoma cells.

[0180] These results demonstrate that the ability of DHP-type CCBs, to overcome drug resistance by inducing melanoma cell death through synergistic action with targeted therapies is a shared property across the DHP-type CCB family. Moreover, this synergistic effect is independent of the specific targeted therapy used, but rather depends on the targeting of the MAPK signaling pathway itself.

[0181] The data clearly indicate that DHP-type CCBs act synergistically with all tested agents targeting the MAPK pathway. Thus, the synergistic potential of DHP-type CCBs extends to any therapeutic agent that interferes with the MAPK signaling cascade, supporting their broad applicability in combination strategies designed to overcome resistance in BRAF-mutant and / or MEK-mutant melanoma cancers. Example 8 : In vivo demonstration of the re-sensitization effect of a DHP CCB in resistant melanoma models

[0182] The results described in Example 4 demonstrated that the combination of a LTCC specific blocker, and in particular a DHP -type CCB, with agents targeting the MAPK pathway, such as BRAFi and / or MEKi, delayed or even prevented the emergence of resistance in melanoma cells treated with targeted therapy. These findings highlight the therapeutic interest of this novel combination as a first-line treatment for BRAF-mutant metastatic melanoma. Furthermore, Examples 2, 3, and 7 showed that this combination, or combinations including other LTCC blockers, and in particular other DHP -type CCB, re-sensitized in vitro melanoma cells that had acquired resistance to targeted therapies, suggesting potential applications of these new combinations in second-line treatment for melanomas progressing under such therapies.

[0183] To further evaluate this effect in vivo, studies were conducted using an avian model of human melanoma (AVLCellDX™), a validated preclinical system for the rapid and robust evaluation of novel combination strategies, particularly those aimed at overcoming therapeutic resistance in melanoma (EMBO Mol Med, 2023, Jarrosson et al.). In this model, drug efficacy was assessed using GLO-R cells, derived from a patient whose melanoma had progressed under dabrafenib / trametinib treatment (courtesy of Julie Caramel, CRCL, Lyon).

[0184] Using this BRAF / MEK inhibitor-resistant GLO-R AVI-CellDX™ model we have evaluated the anti-tumor efficacy of the DHP -type CCB Lercanidipine, as well as that of the combination of Dabrafenib and Trametinib (as example of standard-of-care, SOC), both as monotherapies and in combination. The maximum tolerated dose (MTD) of Lerceni dipine was first determined individually in toxicity assays, followed by the MTD of its combination with the SOC regimen, in avian embryos. All compounds were subsequently used at the determined MTDs for the association, SOC efficacy at these doses having been independently validated in a BRAF / MEK - sensitive AVI-CellDX™ model. For Efficacy assays, the anti-tumor efficacy of Lercanidipine alone and in combination with Dabrafenib and Trametinib was evaluated in the AVI-CellDX™ model transplanted with BRAF / MEK inhibitor-resistant GLO-R melanoma cells as follow: GLO-R cells were engrafted at HH14 stage (E2). Twenty-four hours later, a single intravenous injection of Lercanidipine and / or Dabrafenib and Trametinib was administered at stage E3. The experiment was terminated at HH25 (E4), followed by quantitative 3D analysis of tumor volume in viable embryos using light sheet microscopy 24 h post-treatment (48 h post-engraftment) (Figure 12A). Statistical analysis was performed using an unpaired / -test. 3D light sheet microscopy revealed that, in the resistant GLO-R AVI-CellDX™ model, treatment with Dabrafenib and Trametinib induced only minor and non-significant changes in tumor volume (+11%), consistent with the resistant phenotype (Figure 12B). Lercanidipine alone produced a slight but non-significant tumor growth reduction (-4%). Remarkably, the combination of Lercanidipine with Dabrafenib and Trametinib led to a significant normalized tumor volume reduction (-29%, normalized to body surface area). Synergy analysis confirmed a strong synergistic effect according to the Bliss independence model, further supported by HSA, Loewe, and ZIP synergy scores.

[0185] These results indicate that Lercanidipine treatment re-sensitizes melanoma cells resistant to BRAF / MEK inhibition in an in vivo setting. Overall, while Lercanidipine monotherapy showed only a mild trend toward tumor reduction, its combination with Dabrafenib and Trametinib demonstrated a strong capacity to overcome therapeutic resistance in the GLO-R context by synergizing with BRAF / MEK inhibition. These in vivo results clearly demonstrate the therapeutic advantage of the combination therapy according to the invention over existing targeted therapies. The data validate the in vivo therapeutic interest of this repositioning strategy, supporting its potential use as a first-line treatment for refractory BRAF -mutant melanoma or as a second-line therapy for BRAF-mutant melanoma progressing under targeted therapy.

Claims

CLAIMS1. An L-type calcium channel (LTCC) blocker or LTCC-specific blocker for use in the treatment of melanoma in a patient, in combination with a BRAF inhibitor (BRAFi) and / or a MEK inhibitor (MEKi), wherein said LTCC blocker or LTCC-specific blocker is selected from a dihydropyridine (DHP), a phenylalkylamine or a benzothiazepine, or a pharmaceutically acceptable salt or derivative thereof.

2. The LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or MEKi for use according to claim 1, said LTCC blocker or LTCC-specific blocker being a dihydropyridine selected from amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine or pranidipine, or a pharmaceutically acceptable salt or derivative thereof.

3. The LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or MEKi for use according to claim 1 or 2, said BRAFi being selected from vemurafenib, dabrafenib or encorafenib, or a pharmaceutically acceptable salt or derivative thereof.

4. The LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or MEKi for use according to any one of claims 1 to 3, said MEKi being selected from cobimetinib, trametinib or binimetinib, or a pharmaceutically acceptable salt or derivative thereof.

5. The LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or MEKi for use according to any one of claims 1 to 4, said combination being one of the following combinations:- amlodipine, vemurafenib and cobimetinib,- amlodipine, dabrafenib and trametinib,- amlodipine, encorafenib and binimetinib,- amlodipine, vemurafenib and trametinib,- amlodipine and vemurafenib,- lercanidipine, vemurafenib and cobimetinib,- lercanidipine, dabrafenib and trametinib,- lercanidipine, encorafenib and binimetinib,- lercanidipine, vemurafenib and trametinib,- lercanidipine and vemurafenib,- nicardipine, vemurafenib and cobimetinib,35- nicardipine, dabrafenib and trametinib,- nicardipine, encorafenib and binimetinib,- nicardipine, vemurafenib and trametinib,- nicardipine and vemurafenib,- amlodipine, vemurafenib and binimetinib,- amlodipine, dabrafenib and cobimetinib,- amlodipine, dabrafenib and binimetinib,- amlodipine, encorafenib and cobimetinib,- amlodipine, encorafenib and trametinib,- lercanidipine, vemurafenib and binimetinib,- lercanidipine, dabrafenib and cobimetinib,- lercanidipine, dabrafenib and binimetinib,- lercanidipine, encorafenib and cobimetinib,- lercanidipine, encorafenib and trametinib,- nicardipine, vemurafenib and binimetinib,- nicardipine, dabrafenib and cobimetinib,- nicardipine, dabrafenib and binimetinib,- nicardipine, encorafenib and cobimetinib,- nicardipine, encorafenib and trametinib,- amlodipine and dabrafenib,- amlodipine and encorafenib,- amlodipine and cobimetinib,- amlodipine and trametinib,- amlodipine and binimetinib,- lercanidipine and dabrafenib,- lercanidipine and encorafenib,- lercanidipine and cobimetinib,- lercanidipine and trametinib,- lercanidipine and binimetinib,- nicardipine and dabrafenib,- nicardipine and encorafenib,- nicardipine and cobimetinib,- nicardipine and trametinib, or- nicardipine and binimetinib,or pharmaceutically acceptable salts or derivatives thereof.

6. The LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or MEKi for use according to claim 5, said LTCC blocker or LTCC-specific blocker, BRAFi and / or MEKi being adapted for daily administration:- from 1 to 10 mg for amlodipine, preferably 5 to 10 mg per day,- from 5 to 20 mg for lercani dipine, preferably from 10 to 20 mg per day,- from 40 to 120 mg for nicardipine, preferably from 50 to 120 mg per day,- from 480 to 1920 mg for vemurafenib, preferably 1920 mg per day,- from 40 to 300 mg for dabrafenib, preferably 300 mg per day,- from 300 to 450 mg for encorafenib, preferably 450 mg per day,- from 20 to 60 mg for cobimetinib, preferably 60 mg per day,- from 1 to 2 mg for trametinib, preferably 2 mg per day,- from 60 to 90 mg for binimetinib, preferably 90 mg per day.

7. The LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or MEKi for use according to any one of claims 1 to 6, said LTCC blocker or LTCC-specific blocker, BRAFi and / or MEKi being adapted for simultaneous, sequential or separate administration.

8. The LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or MEKi for use according to any one of claims 1 to 7, said LTCC blocker or LTCC-specific blocker, BRAFi and / or MEKi being adapted for systemic or local administration, preferably for oral administration.

9. The LTCC blocker or LTCC-specific blocker in combination with a BRAFi and / or MEKi for use according to any one of claims 1 to 8, said melanoma being a metastatic or unresectable melanoma.

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