Application of combination of chrysoeriol and Jinnofen in preparation of anti-melanoma drugs

Abstract

The invention relates to the technical field of biological medicines, and discloses an application of combination of chrysoeriol and Jinnofen in preparation of an anti-melanoma drug. The pharmaceutical composition comprises Chrysoeriol and Auranofin, the Chrysoeriol and the Auranofin are combined according to the weight part ratio of (3: 1) to (8: 1), and the preferable ratio is 5: 1. According to the composition, oxidative stress is induced through a synergistic effect, the activity of thioredoxin reductase is inhibited, and the level of Thiol substances in cells is reduced, so that proliferation and migration of melanoma cells are effectively inhibited. Particularly, when the weight ratio is 5: 1, the cell viability of a drug combination group can be remarkably reduced to about 30%, the synergistic inhibition effect of the drug combination is far better than that of a single drug, and the strong inhibition effect of combination of chrysoeriol and Jinnofen on melanoma cell proliferation is clearly shown.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of chrysoeriol combined with auranofin in the preparation of an anti - melanoma drug. Background Art

[0002] Melanoma is a highly malignant tumor originating from melanocytes, which is characterized by rapid progression, strong metastasis, and high drug resistance, and is one of the skin tumors with the highest fatality rate. With the aging of the global population and the increasing exposure to ultraviolet rays, the incidence of melanoma is rising continuously. Especially in high - incidence regions such as Europe and America, the annual growth rate is as high as 3% - 7%. The current mainstream clinical treatments include surgery, chemotherapy, targeted therapy, and immune checkpoint inhibitor therapy. Although immunotherapies such as PD - 1 / CTLA - 4 / LAG - 3 and targeted drugs such as BRAF / MEK bring short - term remission, the treatment effect is limited by the rapidly developing drug resistance mechanisms and phenotypic plasticity. Especially in patients with advanced melanoma, tumor cells often exhibit multiple escape mechanisms such as phenotypic heterogeneity, immunosuppressive microenvironment, and enhanced antioxidant barrier, resulting in the gradual attenuation of the curative effect.

[0003] Existing treatment methods are difficult to solve the problem of "multi - phenotypic melanoma drug resistance". Research shows that melanoma cells have strong metabolic adaptability, can escape treatment through phenotypic drift (such as the transformation from high expression of MITF to high expression of AXL), and enhance their antioxidant capacity by up - regulating the intracellular Thiols level (such as glutathione GSH), thus developing drug resistance to targeted drugs and oxidative stress drugs. In addition, the ROS level in melanoma is generally low, and the level of thiol - reducing substances (GSH, Trx system) increases, forming a powerful endogenous antioxidant barrier, which limits the apoptotic effect of chemotherapeutic drugs and radiotherapy. Therefore, it is difficult to eradicate the melanoma cell population with multi - phenotypes and high antioxidant expression only by single - target drugs.

[0004] The "chrysoeriol + auranofin" combination strategy proposed by the present invention breaks drug resistance and escape at multiple levels. The present invention first proposes that by combining the natural multi - target flavonoid molecule "chrysoeriol" with the thioredoxin reductase (TrxR) inhibitor "auranofin", continuous inhibition of melanoma cells can be achieved from multiple key pathways. Different from existing combined drugs, the uniqueness of the present invention lies in:

[0005] Multi - phenotypic regulation: Chrysoeriol can induce melanogenesis. By promoting the expression of pigment - synthesis - related factors (such as MITF, TYR), it makes tumor cells return to the "high - differentiation" phenotype and enter the "senescence - like" state, thereby weakening their migration and division abilities. This process not only inhibits the invasiveness of the tumor, but may also cause tumor cells to enter a "functional quiescence" state by promoting the accumulation of melanin, thereby reducing their pathogenicity.

[0006] Induction of melanin accumulation = induction of senescence: Melanin accumulation is considered an important marker of cellular senescence and stable differentiation. In this invention, by promoting melanin synthesis, tumor cells enter a "functional quiescence" state under "non-lethal" conditions. Although the tumor cells in this state survive, their proliferation and invasion abilities are significantly reduced, thus providing new ideas for clinical treatment.

[0007] ROS-mediated immune activation: Auranofin induces ROS burst after inhibiting TrxR, activating oxidative stress-related signaling pathways (such as NF-κB, p38 / MAPK), which not only induces apoptosis but also helps enhance T cell infiltration and antigen presentation, improving the sensitivity of immunotherapy. The discovery of this mechanism provides a new target for the immunotherapy of melanoma. By regulating the ROS level, the body's immune system can be effectively activated to combat tumor cells.

[0008] Inhibiting the Thiols pathway and improving the anti-drug resistance effect: The Thiols antioxidant network (such as GSH, GSR, GCLM) in melanoma cells is a typical basis for drug resistance. In this invention, Chrysoeriol significantly downregulates the expression of related proteins, and then combines with auranofin to precisely target TrxR, forming a "dual inhibition" strategy to break the antioxidant defense from the source. This strategy can effectively overcome the antioxidant defense mechanism of tumor cells and improve the drug sensitivity.

[0009] The combined mechanism is independent of traditional mutation pathways (such as BRAF, NRAS): Being independent of specific gene mutations makes this invention particularly suitable for patients with wild-type BRAF, mutant NRAS, and those resistant to immune checkpoint inhibitors. This feature makes the treatment strategy of this invention have a wider applicability, capable of covering more types of melanoma patients, especially those ineffective to existing treatment methods.

[0010] In summary, the root causes of drug resistance, immune escape, and treatment failure in current melanoma treatment lie in the multi-phenotypic plasticity and strong antioxidant adaptability of tumors. The combined drug strategy proposed in this invention no longer relies on single-target intervention, but starts from multi-dimensional mechanisms such as induction of differentiation, oxidative attack, immune regulation, and inhibition of anti-drug resistance pathways, providing a new solution for highly efficient and low-toxic melanoma treatment, especially suitable for patients with refractory, metastatic, or recurrent melanoma.

[0011] Through the "Chrysoeriol + Auranofin" combination strategy of the present invention, multi-target and multi-level inhibition of melanoma cells can be achieved, thereby overcoming the limitations of existing treatment methods. Chrysoeriol induces melanogenesis and promotes the expression of factors related to pigment synthesis, causing tumor cells to return to a highly differentiated phenotype and enter a functional quiescent state. At the same time, Auranofin induces ROS bursts by inhibiting thioredoxin reductase (TrxR), activates oxidative stress-related signaling pathways, promotes apoptosis, and enhances the sensitivity of immunotherapy. In addition, the combination strategy of the present invention also breaks the antioxidant defense line of tumor cells from the source by inhibiting the Thiols pathway, improving the anti-drug resistance effect. Since the present invention does not rely on specific gene mutations, it has a wider applicability and can provide new treatment options for patients with BRAF wild-type, NRAS mutant, and resistant to immune checkpoint inhibitors. This innovative treatment strategy brings new hope to melanoma patients, especially in cases where traditional treatment methods are difficult to work, and is expected to significantly improve the prognosis and quality of life of patients. Summary of the Invention

[0012] To solve the key problems such as strong drug resistance, immune escape, and high antioxidant defense barriers in existing melanoma treatment methods, the present invention provides a combination scheme of Chrysoeriol and Auranofin for preparing an anti-melanoma drug. This scheme precisely intervenes in the proliferation, migration, immune escape, and phenotypic plasticity of melanoma cells through multi-pathway and multi-target synergistic effects.

[0013]

I

[0014] The present invention relates to a pharmaceutical composition, and the composition comprises the following active ingredients: Chrysoeriol: a natural polyhydroxy flavonoid compound; Auranofin: a metal thiosaccharide complex, a TrxR inhibitor. The two active ingredients are combined in a weight ratio of 3:1 to 8:1, preferably 5:1, and can be prepared into pharmaceutical preparations such as gels, injections, sprays, or oral liquids, which are suitable for oral, intravenous injection, or topical administration.

[0015]

II

[0016] (1) Inducing melanogenesis, promoting differentiation and inhibiting malignant transformation

[0017] Chrysoeriol significantly upregulates the expression of the melanin synthesis pathway (MITF, TYR, DCT, etc.), induces melanin accumulation, makes cells present a senescent-like phenotype, and reduces their migration and proliferation abilities;

[0018] (2) Inhibiting the Thiol antioxidant system and weakening the drug resistance mechanism

[0019] Chrysoeriol can effectively inhibit the expression of Thiols regulatory proteins (such as GSR, GCLC, GCLM), and auranofin potently inhibits TrxR activity. The two act synergistically to disrupt the cellular antioxidant defense line and enhance oxidative stress sensitivity;

[0020] (3) Induce ROS accumulation and activate cell death and immune responses

[0021] Combined drug use leads to a significant increase in ROS, a significant decrease in intracellular GSH and total Thiols content, promotes the activation of programmed cell death pathways such as apoptosis and ferroptosis, and at the same time can enhance T cell infiltration and antigen presentation ability, improving the effect of immunotherapy;

[0022] (4) Strike multiple pathway targets to overcome phenotypic drift and treatment escape

[0023] The present invention covers the typical biological escape paths of melanoma from three directions of epigenetic regulation, redox regulation, and melanin synthesis regulation, including pathways such as NF-κB, Nrf2, PI3K / AKT, and MAPK.

Three

[0024] (1) The synergistic inhibitory effect is significantly better than that of single drugs: when chrysoeriol and auranofin are used in combination and the weight ratio is 5:1, the cell viability is significantly decreased to below 30%, which is significantly better than either single drug;

[0025] (2) The said pharmaceutical composition induces oxidative stress, inhibits thioredoxin reductase, and reduces the level of intracellular Thiol substances through a synergistic mechanism, thereby inhibiting the proliferation and migration of melanoma cells;

[0026] (3) The said pharmaceutical composition enhances the anti-melanoma effect by inducing the accumulation of reactive oxygen species (ROS), reducing the intracellular glutathione (GSH) and total Thiol levels;

[0027] (4) The said chrysoeriol can induce melanogenesis, provide a physical barrier and antioxidant protection at the precancerous stage of melanocytes, and inhibit malignant transformation;

[0028] (5) The action targets are independent of the BRAF and NRAS mutation backgrounds, and the applicable population range is wide;

[0029] (6) The dosage form is flexible, can be administered locally or systemically, has relatively low toxic and side effects, and has good clinical transformation potential.

[0030] In summary, through the scientific combination of natural flavonoids and metal complex drugs, the present invention achieves synergistic intervention in multiple key aspects such as induced differentiation, immune regulation, antioxidant barrier disruption, and ROS synergistic killing, providing a novel treatment strategy with a clear mechanism and remarkable effect for melanoma, especially BRAF drug-resistant, NRAS mutant, and metastatic melanoma.

[0031]

IV

[0032] The present invention provides an innovative drug combination strategy with multi-pathway and multi-mechanism synergy in the field of melanoma drug resistance treatment. Compared with the prior art, its core technological innovation points and the contents that should be key protected include:

[0033] 1. Novelty of the synergistic mechanism of combined drug use

[0034] In traditional melanoma treatment, there are very few reports on the strategy of combining "metal complex drugs" with "natural flavonoids". The present invention first proposes to combine auranofin and chrysoeriol in a specific ratio, with complementary targets for the two, and form a dual antioxidant defense inhibition effect by inhibiting the TrxR enzyme system + GSH / Thiols system.

[0035] Innovation Point 1: First achieve dual-targeted combined inhibition of the Trx / GSH system, improving the killing efficiency against drug-resistant melanoma.

[0036] 2. Intervention mechanism of promoting differentiation and senescence-like state by "inducing melanogenesis"

[0037] Different from the traditional "poisoning" idea, the present invention upregulates the melanogenesis pathway (such as MITF / TYR), induces tumor cells to turn into a "senescence-like" state of high differentiation and low migration, and reduces their malignant potential from the source.

[0038] Innovation Point 2: Establish a new treatment model of "promoting melanin accumulation → inducing tumor differentiation → inhibiting migration and proliferation".

[0039] 3. Precise dose ratio matching

[0040] The invention clearly proposes that the optimal combination ratio is chrysoeriol:auranofin = 5:1, and its synergistic killing effect is verified to be the best in multiple rounds of cell viability experiments.

[0041] Innovation Point 3: Clearly propose and verify a specific drug ratio to ensure the maximization of the synergistic effect.

[0042] 4. The mechanism of action covers multiple pathways and is applicable to various mutation backgrounds

[0043] Different from existing targeted drugs that rely on gene mutations such as BRAF and NRAS, the mechanism of action of the present invention does not depend on specific driver genes and is applicable to BRAF wild-type, NRAS mutant, and drug-resistant melanoma, expanding the scope of indications.

[0044] Innovation Point 4: The mechanism is independent of the mainstream mutation pathway, has a wide range of applicable populations, and has universality.

[0045] 5. Flexible design of multiple dosage forms and administration methods

[0046] The pharmaceutical composition of the present invention can be in various preparation forms such as oral, injection, topical gel, or spray according to the condition of the disease, and has a good basis for clinical application transformation.

[0047] Innovation Point 5: It can be prepared into various forms of drugs such as nano-gel, injection solution, and topical spray to meet the needs of different treatment scenarios. Description of the Drawings

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained based on the provided drawings.

[0049] The structures, ratios, sizes, etc. disclosed in this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they do not have technical substantive significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the efficacy and purpose that the present invention can achieve, should still fall within the scope covered by the technical content disclosed in the present invention.

[0050] Figure 1 Shows the effects of different concentrations of Chrysoeriol on the viability of melanoma cells;

[0051] Figure 2 Shows the effects of different concentrations of Auranofin on the viability of melanoma cells;

[0052] Figure 3 Shows the effects of different concentrations of Auranofin combined with 10 μM Chrysoeriol on the viability of melanoma cells;

[0053] Figure 4 Shows the cell state diagrams under treatment with different concentrations of Auranofin and a fixed concentration of Chrysoeriol for comparison.

[0054] Figure 5 Shows the induction of melanin accumulation by Chrysoeriol

[0055] Figure 6 Effect of Chrysoeriol at Different Concentrations on the Expression Levels of Thiols-Regulated Proteins

[0056] Figure 7 Effect of Chrysoeriol at Different Concentrations on ROS, GSH and Total Thiols in Melanoma Cells

[0057] Figure 8 Mechanism Diagram of the Combined Inhibition of Melanoma by Chrysoeriol and Auranofin Detailed Implementation Modes

[0058] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0059] The weight ratio of chrysoeriol to auranofin is 3:1 to 8:1, preferably 5:1; the pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient, including but not limited to starch, lactose, microcrystalline cellulose, polyethylene glycol or phospholipid;

[0060] Example 1: Effect of Chrysoeriol on the Viability of Melanoma Cells

[0061] The names and treatment conditions of each group in this example are as follows: (1) blank control group; (2) chrysoeriol (2 μM) group; (3) chrysoeriol (4 μM) group; (4) chrysoeriol (6 μM) group; (5) chrysoeriol (8 μM) group; (6) chrysoeriol (10 μM) group; (7) chrysoeriol (12 μM) group. After 24 h of drug treatment, 20 μL of CCK8 solution was added to each well, and incubation was continued for 1 - 4 h. The absorbance (OD value) at 450 nm was measured with an enzyme-labeled instrument. The results are as Figure 1 shown. As the concentration increased (2 μM - 12 μM), the cell viability decreased slightly, and it still reached about 90% at 12 μM, and the inhibitory effect was not significant.

[0062] Example 2: Effect of Auranofin on the Viability of Melanoma Cells

[0063] The names and treatment conditions of each group in this example are as follows: (1) blank control group; (2) auranofin (0.05 μM) group; (3) auranofin (0.1 μM) group; (4) auranofin (0.2 μM) group; (5) auranofin (0.4 μM) group; (6) auranofin (0.8 μM) group; (7) auranofin (1 μM) group. After 24 hours of drug treatment, 20 μL of CCK8 solution was added to each well, and incubation was continued for 1 - 4 hours. The absorbance (OD value) at 450 nm was measured using an enzyme - linked immunosorbent assay (ELISA) reader. The results are as Figure 2 shown. Low - concentration auranofin (such as 0.05 μM - 0.8 μM) has little effect on cell viability, and cell viability drops to about 80% at 1 μM.

[0064] Example 3: Effect of Chrysoeriol combined with Auranofin on the viability of melanoma cells

[0065] The names and treatment conditions of each group in this example are as follows: (1) blank control group; (2) 10 μM chrysoeriol group + auranofin (0.05 μM); (3) 10 μM chrysoeriol group + auranofin (0.1 μM); (4) 10 μM chrysoeriol group + auranofin (0.2 μM); (5) 10 μM chrysoeriol group + auranofin (0.4 μM); (6) 10 μM chrysoeriol group + auranofin (0.8 μM); (7) 10 μM chrysoeriol group + auranofin (1 μM). After 24 hours of drug treatment, 20 μL of CCK8 solution was added to each well, and incubation was continued for 1 - 4 hours. The absorbance (OD value) at 450 nm was measured using an enzyme - linked immunosorbent assay (ELISA) reader. The results are as Figure 3 shown. When 10 μM chrysoeriol is combined with auranofin, as the concentration of auranofin increases (0.05 μM - 1 μM), cell viability decreases significantly. The cell viability of the 10 μM chrysoeriol group + 1 μM auranofin (mass ratio is about 5:1) drops to about 30%, and the synergistic inhibitory effect is significantly better than that of single drugs.

[0066] Example 4: Effect of Chrysoeriol combined with Auranofin on the morphology of melanoma cells

[0067] The names and treatment conditions of each group in this example are as follows: ((1) blank control group; (2) 10 μM chrysoeriol group + auranofin (0.05 μM); (3) 10 μM chrysoeriol group + auranofin (0.1 μM); (4) 10 μM chrysoeriol group + auranofin (0.2 μM); (5) 10 μM chrysoeriol group + auranofin (0.4 μM); (6) 10 μM chrysoeriol group + auranofin (0.8 μM); (7) 10 μM chrysoeriol group + auranofin (1 μM). The results are as Figure 4As shown, when 10 μM chrysoeriol is combined with auranofin, as the concentration of auranofin increases (0.05 μM - 1 μM), the cell morphology gradually becomes elliptical and the cell quantity significantly decreases. Moreover, the inhibitory effect on tumor proliferation is the best in the group of 10 μM chrysoeriol + 1 μM auranofin (mass ratio is approximately 5:1).

[0068] Example 5: Chrysoeriol induces melanin accumulation in a time-dependent manner

[0069] The names and treatment conditions of each group in this example are as follows: (1) blank control group; (2) 16 μM chrysoeriol (24 h) group; (2) 16 μM chrysoeriol (48 h) group; (2) 16 μM chrysoeriol (72 h) group. The results are as Figure 5 (B, D) shown, the melanin content increases with time, and the melanin content at 72 h is about twice that of the blank group.

[0070] Example 6: Chrysoeriol induces melanin accumulation in a dose-dependent manner

[0071] The names and treatment conditions of each group in this example are as follows: (1) blank control group; (2) positive group (8-MOP); (3) chrysoeriol (4 μM) group; (4) chrysoeriol (8 μM) group; (5) chrysoeriol (16 μM) group. The results are as Figure 5 (A, C, E) shown, the melanin content increases with the dose, and the melanin content of 16 μM chrysoeriol is about twice that of the blank group and significantly higher than that of the positive group.

[0072] Example 7: Effect of chrysoeriol on tyrosinase activity

[0073] The names and treatment conditions of each group in this example are as follows: (1) blank control group; (2) positive group (8-MOP); (3) chrysoeriol (4 μM) group; (4) chrysoeriol (8 μM) group; (5) chrysoeriol (16 μM) group. After 72 h of drug treatment, the tyrosinase activity was detected by the L-DOPA oxidation method. The results are as Figure 5 (F) shown, chrysoeriol promotes tyrosinase activity in a dose-dependent manner. The tyrosinase activity of 16 μM chrysoeriol is about 1.5 times that of the blank group and significantly higher than that of the positive group. In summary, chrysoeriol can induce melanin production, thereby providing a physical barrier and antioxidant protection at the pre-cancerous stage of melanocytes and inhibiting malignant transformation.

[0074] Example 8: Effect of chrysoeriol on the expression of proteins related to thiol regulation

[0075] The names and treatment conditions of each group in this example are as follows: (1) blank control group; (2) positive group (8-MOP); (3) chrysoeriol (4 μM) group; (4) chrysoeriol (8 μM) group; (5) chrysoeriol (16 μM) group. After 72 h of drug treatment, Western blotting was used to detect the expression of sulfhydryl regulation-related proteins. The results are as Figure 6 shown. Compared with the blank group, the expressions of GSR, GCLC, and GCLM in the chrysoeriol administration groups were significantly decreased, indicating that chrysoeriol can inhibit the proliferation of tumors by inhibiting the expression of sulfhydryl regulation-related proteins, reducing the intracellular sulfhydryl level, and inducing oxidative stress.

[0076] Example 9: Effects of Chrysoeriol on ROS and Thiols

[0077] The names and treatment conditions of each group in this example are as follows: (1) blank control group; (2) chrysoeriol (4 μM) group; (3) chrysoeriol (8 μM) group; (4) chrysoeriol (16 μM) group. After 24 h of drug treatment, DCFH-DA, MCB, and CMF-DA probes were used to detect ROS, GSH, and total thiol, respectively. The results are as Figure 7 shown. Chrysoeriol can significantly increase the ROS content and significantly decrease the GSH and thiol contents, further indicating that chrysoeriol can induce oxidative stress.

[0078] In summary, through the cell viability detection experiment, the present invention confirmed that the combined use of chrysoeriol and auranofin has a significantly better synergistic inhibitory effect on the viability of melanoma cells than single drugs; with the help of molecular mechanism experiments such as sulfhydryl regulation and ROS accumulation, the mechanism of action of the combined drug in synergistically inhibiting the proliferation and migration of melanoma cells was deeply analyzed.

[0079] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. The use of Jinshengcao flavin combined with auranofin in the preparation of anti-melanoma drugs, characterized in that: The active ingredients of the pharmaceutical composition include Chrysoeriol and Auranofin, wherein the weight ratio of Chrysoeriol to Auranofin is 3:1 to 8:1, preferably 5:

1.

2. The use according to claim 1, characterized in that: The pharmaceutical composition induces oxidative stress, inhibits thioredoxin reductase, and reduces the level of intracellular Thiol substances through a synergistic mechanism, thereby inhibiting the proliferation and migration of melanoma cells.

3. The use according to claim 1, characterized in that: The pharmaceutical composition enhances the anti-melanoma effect by inducing the accumulation of reactive oxygen species (ROS) and reducing the levels of intracellular glutathione (GSH) and total Thiol.

4. The use according to claim 1, characterized in that: The chrysanthemum yellow can induce melanin production, provide a physical barrier and antioxidant protection in the precancerous stage of melanocytes, and inhibit malignant transformation.

5. The use according to claim 1, characterized in that: The pharmaceutical composition is suitable for oral administration, intravenous injection, local gel application or spray inhalation.

6. The use according to claim 5, characterized in that: The pharmaceutical composition is in the form of nano-sized particle gel, cream, injection or spray, and can be prepared by freeze drying or spray drying technology.

7. The use according to claim 6, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, and the carrier or excipient includes starch, lactose, microcrystalline cellulose, polyethylene glycol, phospholipids or other commonly used pharmaceutical excipients.

8. The use according to claim 1, characterized in that: The pharmaceutical composition is used for treating patients with BRAF mutant, NRAS mutant, BRAF inhibitor-resistant, and advanced metastatic melanoma.

Images