Applications of sulfasalazine and pharmaceutical composition thereof in drug-resistant head and neck malignant tumors

Abstract

The invention provides application of sulfasalazine and a combined drug thereof in drug-resistant head and neck malignant tumors, which comprises the following steps: when the sulfasalazine is independently used, the sulfasalazine has a treatment effect on the drug-resistant head and neck malignant tumors and can delay the growth of the drug-resistant head and neck malignant tumors; combined use of sulfasalazine and chemotherapeutic drugs has a synergistic effect, and the sulfasalazine can be used for treating drug-resistant head and neck malignant tumors. The technical problem to be solved by the invention is to explore the drug activity of sulfasalazine in the treatment of drug-resistant head and neck malignant tumors and solve the limitation of drug resistance to treatment in the drug treatment process.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically, to the application of sulfasalazine and its combination drugs in drug-resistant head and neck malignancies. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, and oral squamous cell carcinoma (OSCC) is one of its main classifications. Due to the critical location and complex anatomical structure of head and neck malignancies, the existing treatment methods mainly based on surgery impose a huge burden, and there is an urgent need for safe and effective drug treatment strategies. The existing drug treatment strategies mainly rely on broad-spectrum radiotherapy and chemotherapy, and the first-line treatment drugs are mainly based on platinum drugs or 5-fluorouracil. However, the occurrence of tumor drug resistance during the existing drug treatment severely limits the effectiveness and durability of treatment, which is one of the urgent problems to be solved currently.

[0003] Sulfasalazine (SAS) is a mature drug that has been clinically approved for the treatment of rheumatoid arthritis. In recent years, it has been reported to have the ability to induce ferroptosis. By inhibiting the cystine / glutamate antiporter (system Xc-), it can cause the depletion of the intracellular reducing agent glutathione, thereby leading to ferroptosis in cells. However, the drug activity and safety of sulfasalazine in the treatment of drug-resistant head and neck malignancies are still unclear. Summary of the Invention

[0004] The technical problem solved by the present invention is to explore the drug activity of sulfasalazine in the treatment of drug-resistant head and neck malignancies and to solve the limitation of treatment caused by the occurrence of drug resistance during the drug treatment process.

[0005] To solve the above problems, the present invention provides an application of sulfasalazine in drug-resistant head and neck malignancies. Sulfasalazine alone has a therapeutic effect on drug-resistant head and neck malignancies and can delay the growth of drug-resistant head and neck malignancies. When sulfasalazine is used in combination with chemotherapy drugs, it has a synergistic effect and can be used to treat drug-resistant head and neck malignancies.

[0006] In one example of the present invention, the drug-resistant head and neck malignancy is drug-resistant oral squamous cell carcinoma.

[0007] The present invention also provides a combination drug that can be used to treat drug-resistant head and neck malignancies. The combination drug includes sulfasalazine and chemotherapy drugs.

[0008] In one example of the present invention, the chemotherapeutic drugs include 5-fluorouracil and platinum-based drugs.

[0009] In one example of the present invention, the chemotherapeutic drug is a platinum-based drug, and the platinum-based drug is cisplatin.

[0010] In one example of the present invention, the combination drug includes sulfasalazine and cisplatin, and the combination drug is made of sulfasalazine and cisplatin at different ratio concentrations. Among them, the concentration range of sulfasalazine is 0-800 μM, and the concentration range of cisplatin is 0-50 μM.

[0011] In one example of the present invention, the ratio concentrations with synergistic effects in drug-resistant head and neck malignancies include any one of the following: 50:2, 50:10, 50:50, 100:0.08, 100:2, 100:10, 100:50, 200:0.08, 200:0.4, 200:2, 200:10, 200:50, 400:0.4, 400:2, 400:10, 800:0.08, 800:0.4, 800:2, 800:10, 800:50.

[0012] In one example of the present invention, the preferred ratio concentrations in the ratio concentrations include 200:2 or 200:10.

[0013] In one example of the present invention, the combination drug can be used to treat oral squamous cell carcinoma resistant to cisplatin chemotherapy.

[0014] After adopting the technical solution of the present invention, the following technical effects can be achieved: (1) The present invention discovers for the first time the therapeutic activity of sulfasalazine in drug-resistant head and neck malignancies; (2) The present invention provides the use of sulfasalazine in the preparation of drugs for drug-resistant head and neck malignancies, which is expected to overcome the chemotherapy resistance dilemma of clinical head and neck malignancies and help clinicians reasonably select treatment plans; (3) The sulfasalazine drug proposed by the present invention is mature and is expected to achieve rapid clinical transformation. Combining it with other existing anti-cancer active drug ingredients can further achieve anti-cancer synergistic effects and reduce side effects at the same time. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings to be used in the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts; Figure 1Drug-resistant oral squamous cell carcinoma cells are sensitive to sulfasalazine treatment; Figure 2 Sulfasalazine has therapeutic ability on the patient-derived organoids (PDOs) of drug-resistant oral squamous cell carcinoma; Figure 3 Sulfasalazine can delay the growth of drug-resistant OSCC tumors; Figure 4 The combination of sulfasalazine and cisplatin has a significant synthetic lethality in drug-resistant OSCC cells; Figure 5 Sulfasalazine has therapeutic ability on the patient-derived organoids (PDOs) of drug-resistant oral squamous cell carcinoma; Figure 6 Sulfasalazine can delay the growth of drug-resistant OSCC tumors. Detailed implementation mode

[0016] To make the above objects, features and advantages of the present invention more obvious and understandable, the following specific embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0017] The present invention provides an application of sulfasalazine in drug-resistant head and neck malignancies. Sulfasalazine alone has a therapeutic effect on drug-resistant head and neck malignancies and can delay the growth of drug-resistant head and neck malignancies; the combined use of sulfasalazine and chemotherapeutic drugs has a synergistic effect and can be used to treat drug-resistant head and neck malignancies.

[0018] In one example of the present invention, the drug-resistant head and neck malignancy is drug-resistant oral squamous cell carcinoma.

[0019] The present invention also provides a combined drug that can be used to treat drug-resistant head and neck malignancies. The combined drug includes sulfasalazine and a chemotherapeutic drug.

[0020] In one example of the present invention, the chemotherapeutic drug includes 5-fluorouracil and platinum drugs.

[0021] In one example of the present invention, the chemotherapeutic drug is a platinum drug, and the platinum drug is cisplatin.

[0022] In one example of the present invention, the combined drug includes sulfasalazine and cisplatin. The combined drug is made of sulfasalazine and cisplatin at different ratio concentrations. Among them, the concentration range of sulfasalazine is 0-800 μM, and the concentration range of cisplatin is 0-50 μM.

[0023] In one example of the present invention, the ratio concentrations having a synergistic effect in drug-resistant head and neck malignancies include any one of the following: 50:2, 50:10, 50:50, 100:0.08, 100:2, 100:10, 100:50, 200:0.08, 200:0.4, 200:2, 200:10, 200:50, 400:0.4, 400:2, 400:10, 800:0.08, 800:0.4, 800:2, 800:10, 800:50.

[0024] In one example of the present invention, the preferred ratio concentrations in the ratio concentrations include 200:2 or 200:10.

[0025] In one example of the present invention, the combined drug can be used to treat oral squamous cell carcinoma resistant to cisplatin chemotherapy.

[0026] Example 1 Drug-resistant oral squamous cell carcinoma cells are more sensitive to sulfasalazine The parental and drug-resistant CAL27 cells were seeded in 96-well plates at 3000 cells / well. Subsequently, sulfasalazine (SAS) with a final concentration range of 0 - 1000 μM was added to the culture medium, and after treatment for 72 hours, cell viability was detected using the CCK8 method.

[0027] Experimental results See Figure 1 , (A) Oral squamous cell carcinoma CAL27 parental cells (Parental) and drug-resistant cells (Resistant) were treated with different concentrations of sulfasalazine (SAS) respectively, and then cell viability was detected. The results showed that at the same sulfasalazine (SAS) concentration, the cell viability of drug-resistant cells was lower, indicating that drug-resistant cells are more sensitive to sulfasalazine (SAS); (B) Parental cells and drug-resistant cells were treated with sulfasalazine (SAS) and ferroptosis inhibitor (Fer-1) respectively, and cell death was detected. The results showed that compared with parental cells, sulfasalazine (SAS) could significantly increase the mortality of drug-resistant cells, and the killing effect of sulfasalazine (SAS) on cells could be rescued after treatment with Fer-1, indicating that sulfasalazine (SAS) can induce ferroptosis in drug-resistant cells and drug-resistant cells are more sensitive.

[0028] Example 2 Drug-resistant human organoids are more sensitive to sulfasalazine Construct a drug-resistant patient-derived organoids (PDO) model. Digest and dissociate freshly obtained surgically resected tumor tissues. After filtering the formed cell suspension through a sieve, wash the sieve with sterile PBS. Wash the precipitated cells once with PBS, add an appropriate amount of cell culture medium to make a single-cell suspension, and then centrifuge and resuspend. Mix the resuspended cells with BME matrix gel and drop them on the bottom of a preheated culture plate. After incubating for a certain time, add the culture medium for cultivation. Continuously add cisplatin at a concentration of 3 μM to the culture medium for drug screening. Perform drug sensitivity tests on the PDOs that survived after 120 days of drug screening. As shown by Figure 2 B, the construction of drug-resistant PDOs was successful. (2) Compare the drug sensitivities of drug-resistant PDOs and parental PDOs to sulfasalazine (SAS). Continuously add sulfasalazine (SAS) at a concentration of 100 μM to the culture media of drug-resistant PDOs and parental PDOs. After 7 days, photograph the PDOs in the well plates and compare the area sizes of the PDOs at the start on the first day. As shown by Figure 2 C, compared with parental PDOs, sulfasalazine (SAS) has a more significant growth inhibitory effect on drug-resistant PDOs.

[0029] Experimental results See Figure 2 , (A) Schematic diagram of the PDO model construction process. Obtain tumor samples from clinical patients and construct PDO models. Subsequently, give clinical chemotherapy drugs to construct drug-resistant PDO models, and give empty vectors to the control group to construct chemotherapy-sensitive PDO models. (B) The construction of the drug-resistant PDO model was successful. Treat drug-resistant and parental PDO models with different concentrations of the chemotherapy drug cisplatin (DDP) and detect cell viability. The results show that at the same cisplatin (DDP) concentration, the cell viability of drug-resistant PDOs is significantly higher than that of parental PDOs, indicating that the construction of the PDO drug-resistant model was successful. (C) Drug-resistant PDOs are more sensitive to sulfasalazine (SAS). Observe the sizes of PDOs after treating parental and drug-resistant PDOs with sulfasalazine (SAS) for 7 days. The results show that compared with no drug treatment, the volume of drug-resistant PDOs treated with sulfasalazine (SAS) decreases significantly, suggesting that sulfasalazine (SAS) significantly slows down the growth of drug-resistant PDOs, while the growth inhibitory effect of sulfasalazine (SAS) on parental PDOs is not significant.

[0030] Example 3 Sulfasalazine can significantly delay the growth of drug-resistant tumors (1) Construct a mouse xenograft tumor model. Twelve immunodeficient Nude / Nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.), female, 5 weeks old. Respectively inoculate them with 2×10 6200 μL of DMEM medium containing CAL27 parental cells and CAL27 drug-resistant cells was injected subcutaneously into the bilateral flanks of mice, and the tumor volume was measured with a vernier caliper. (2) Establish a mouse PDOX model. 18 severely immunodeficient NSG mice (Jiangsu Jicui Yakang Biotech Co., Ltd.), female, 5 weeks old. The cultured drug-resistant PDOs were transplanted subcutaneously into 6 NCG mice by injection. After transplantation, the organoid cells grew in the mice and formed tumors. Subsequently, the subcutaneous tumors were surgically removed, divided into tissue blocks with a volume of about 20 mm 3 and inoculated subcutaneously into the bilateral flanks of 12 new NCG mice, and the tumor volume was measured with a vernier caliper. (3) Experimental treatment. When the average tumor volume reached 100 mm 3 , the mice were randomly divided into a control group and a sulfasalazine (SAS) treatment group. The control group was given the corresponding solvent, and the sulfasalazine (SAS) treatment group was intraperitoneally injected with a dose of 200 mg / kg per day. The body weight and tumor volume of the mice were measured and recorded every three days. After the experiment, the tumors were taken, fixed in 4% paraformaldehyde, and then sectioned and stained with H&E.

[0031] Experimental results See Figure 3 , taking the tumor growth volume as the evaluation criterion. (A) Growth curve of parental CAL27 xenografts under sulfasalazine SAS treatment. The results showed that sulfasalazine (SAS) alone could not significantly inhibit the growth of parental CAL27 xenografts. (B) Growth curve of drug-resistant CAL27 xenografts under sulfasalazine (SAS) treatment. The results showed that sulfasalazine (SAS) alone could significantly inhibit the growth of drug-resistant CAL27 xenografts. (C) Growth curve of drug-resistant PDOX under sulfasalazine SAS treatment. The results showed that sulfasalazine (SAS) alone could significantly inhibit the growth of drug-resistant PDOX, indicating that sulfasalazine (SAS) has therapeutic activity against drug-resistant tumors. The above results prove that sulfasalazine (SAS) alone has an inhibitory effect on drug-resistant oral squamous cell carcinoma.

[0032] Example 4 Sulfasalazine and cisplatin combination has a synergistic lethal effect in drug-resistant oral squamous cell carcinoma Experimental method: Parental and drug-resistant CAL27 cells were seeded at 3000 cells / well in 96-well plates. Subsequently, cisplatin with a final concentration range of 0 - 50 μM and SAS with a final concentration range of 0 - 800 μM were added to the medium and treated for 72 hours. Then, the cell viability was detected by the CCK8 method. By Figure 4It can be seen that the synergistic effect of the combination of SAS and cisplatin in drug-resistant CAL27 cells is stronger than that in parental CAL27 cells. Among them, the drug ratio concentrations (μM) with synergistic effects in drug-resistant cells are SAS: Cisplatin (50:2, 50:10, 50:50, 100:0.08, 100:2, 100:10, 100:50, 200:0.08, 200:0.4, 200:2, 200:10, 200:50, 400:0.4, 400:2, 400:10, 800:0.08, 800:0.4, 800:2, 800:10, 800:50), the optimal ratio concentration is SAS: Cisplatin = 200:2, 200:10, and the preferred ratio concentration is 200:2.

[0033] Experimental results See Figure 4 , the combination of sulfasalazine and cisplatin has significant synthetic lethality in drug-resistant OSCC cells. Parental CAL27 cells (A) and drug-resistant CAL27 cells (B) were treated with different concentrations of sulfasalazine (SAS) and cisplatin (Cisplatin) separately or simultaneously and the cell viability was detected. The left heat map represents the cell viability under different treatments, and the right is the synergy index analysis. The red area indicates a synergistic effect, the white area is an additive effect, and the blue area indicates an antagonistic effect. The results show that sulfasalazine (SAS) and cisplatin have a stronger synergistic effect in drug-resistant cells.

[0034] Example 5 The combination of sulfasalazine and cisplatin has therapeutic activity against drug-resistant human organoids Experimental method: (1) Construct a drug-resistant PDO model. The freshly obtained surgically resected tumor tissue was digested and dissociated, and the formed cell suspension was filtered through a sieve and the sieve was washed with sterile PBS. The precipitated cells were washed once with PBS, an appropriate amount of cell culture medium was added to make a single-cell suspension, and then centrifuged and resuspended. The resuspended cells were mixed with BME matrix gel and dropped on the bottom of a preheated culture plate. After incubation for a certain time, the culture medium was added for culture. Cisplatin with a concentration of 3 μM was continuously added to the culture medium for drug screening. (2) Verify the effectiveness of the combination of SAS and cisplatin in drug-resistant PDOs and parental PDOs. Cisplatin with a concentration of 3 μM and sulfasalazine (SAS) with a concentration of 100 μM were continuously added to the culture media of drug-resistant PDOs and parental PDOs respectively. After 7 days, the PDOs in the well plates were photographed and the area sizes of the PDOs at the start on the first day were compared.

[0035] Experimental results See Figure 5, Sulfasalazine (SAS) has therapeutic ability against the PDOs of drug-resistant oral squamous cell carcinoma. (A) Schematic diagram of the PDO model construction process. Tumors were collected from clinical patients to construct PDO models, and then clinical chemotherapy drugs were administered to construct drug-resistant PDO models, while the control group was given the corresponding solvent to construct chemotherapy-sensitive PDO models. (B) and (C) The sizes of PDOs were observed after treatment with sulfasalazine (SAS) and / or cisplatin for 7 days in parental and drug-resistant PDOs respectively. The results showed that both pairs of parental PDOs were sensitive to cisplatin treatment, and the combination of cisplatin and sulfasalazine (SAS) could inhibit the growth of PDOs; drug-resistant PDOs were insensitive to cisplatin but more sensitive to sulfasalazine (SAS), and the inhibitory effect on drug-resistant PDOs was more significant when sulfasalazine (SAS) and cisplatin were used in combination; the above results proved that the combination of sulfasalazine (SAS) and cisplatin had therapeutic activity against drug-resistant PDOs.

[0036] Example 6 The combination of sulfasalazine and cisplatin can inhibit the growth of drug-resistant oral squamous cell carcinoma Experimental method: (1) Establish a mouse xenograft tumor model. 12 immunodeficient Nude / Nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.), female, 5 weeks old. 200 μL of DMEM medium containing 2×10 6 drug-resistant CAL27 cells were injected subcutaneously into the bilateral flanks of the mice, and the tumor volume was measured with a vernier caliper. (2) Establish a mouse PDOX model. 18 severely immunodeficient NSG mice (Jiangsu Jicui Yakang Biotech Co., Ltd.), female, 5 weeks old. The cultured drug-resistant PDOs were transplanted subcutaneously into 6 NCG mice by injection. After transplantation, the organoid cells grew in the mice to form tumors. Subsequently, the subcutaneous tumors were surgically removed, divided into tissue blocks with a volume of about 20 mm 3 and inoculated subcutaneously into the bilateral flanks of 12 new NCG mice, and the tumor volume was measured with a vernier caliper. (3) Experimental treatment. When the average tumor volume reached 100 mm 3 , the mice were randomly divided into a control group, a sulfasalazine (SAS) treatment group, a cisplatin treatment group, and a sulfasalazine (SAS)-cisplatin combination group. The control group was given the corresponding solvent, the sulfasalazine (SAS) treatment group was intraperitoneally injected at a dose of 200 mg / kg per day, the cisplatin treatment group was intraperitoneally injected at a dose of 3 mg / kg every three days, and the body weight and tumor volume of the mice were measured and recorded every three days. After the experiment, the tumors were removed, fixed in 4% paraformaldehyde, and then sectioned and stained with H&E.

[0037] Experimental results See Figure 6, taking the tumor growth volume as the evaluation criterion, sulfasalazine (SAS) can delay the growth of drug-resistant OSCC tumors. (A) In the drug-resistant PDX model, cisplatin alone cannot inhibit the growth of drug-resistant tumors, while sulfasalazine (SAS) alone can delay the growth of drug-resistant tumors, and when sulfasalazine (SAS) is combined with cisplatin, the tumors can regress. (B) In the drug-resistant PDOX model, cisplatin alone cannot inhibit tumor growth, while sulfasalazine (SAS) alone can delay tumor growth, and when sulfasalazine (SAS) is combined with cisplatin, the tumors can regress; whether in the drug-resistant tumor cell xenograft model or in the drug-resistant PDOX model, the drug-resistant tumors are not sensitive to the treatment with cisplatin alone, while sulfasalazine (SAS) alone can delay the growth of drug-resistant tumors, and when sulfasalazine (SAS) is combined with cisplatin, the tumor growth is significantly inhibited, and a reduction in the volume of all tumors was observed in the combination treatment group of the PDOX model. The above results prove that the combination of sulfasalazine (SAS) and cisplatin has a significant inhibitory effect on drug-resistant oral squamous cell carcinoma.

[0038] Although the present invention has been disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. Use of sulfasalazine in drug-resistant head and neck malignancies, characterized in that, Said sulfasalazine alone has a therapeutic effect on said drug-resistant head and neck malignancies and can delay the growth of said drug-resistant head and neck malignancies; Said sulfasalazine combined with a chemotherapeutic drug has a synergistic effect and can be used to treat said drug-resistant head and neck malignancies.

2. The use according to claim 1, characterized in that, Said drug-resistant head and neck malignancies are drug-resistant oral squamous cell carcinomas.

3. A combined drug capable of treating the drug-resistant head and neck malignancies according to any one of claims 1-2, characterized in that, Said combined drug comprises sulfasalazine and a chemotherapeutic drug.

4. The combined drug according to claim 3, characterized in that, Said chemotherapeutic drug comprises 5-fluorouracil and platinum drugs.

5. The combined drug according to claim 4, characterized in that, Said chemotherapeutic drug is said platinum drug, and said platinum drug is cisplatin.

6. The combined drug according to claim 5, characterized in that, Said combined drug comprises said sulfasalazine and said cisplatin, and said combined drug is made from said sulfasalazine and said cisplatin at different ratio concentrations, wherein the concentration range of said sulfasalazine is 0-800 μM, and the concentration range of said cisplatin is 0-50 μM.

7. The combined drug according to claim 6, characterized in that, The ratio concentrations with synergistic effects in said drug-resistant head and neck malignancies include any one of the following: 50:2, 50:10, 50:50, 100:0.08, 100:2, 100:10, 100:50, 200:0.08, 200:0.4, 200:2, 200:10, 200:50, 400:0.4, 400:2, 400:10, 800:0.08, 800:0.4, 800:2, 800:10, 800:

50.

8. The combined drug according to claim 7, characterized in that, The preferred ratio concentrations in said ratio concentrations include 200:2 or 200:

10.

9. The combined drug according to claim 5, characterized in that, Said combined drug can be used to treat oral squamous cell carcinomas resistant to said cisplatin chemotherapy.

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