CN115381954B

CN115381954B - Combined medicine for treating osteosarcoma

Info

Publication number: CN115381954B
Application number: CN202110560718.6A
Authority: CN
Inventors: 赵子建; 许丽君; 周素瑾; 李芳红; 赵正刚
Original assignee: Shenzhen Hanhui Pharmaceutical Technology Co ltd
Current assignee: Shenzhen Hanhui Pharmaceutical Technology Co ltd
Priority date: 2021-05-21
Filing date: 2021-05-21
Publication date: 2023-12-05
Anticipated expiration: 2041-05-21
Also published as: CN115381954A

Abstract

The application provides a combined medicament for treating malignant tumors, in particular osteosarcoma, which contains a PDE4 inhibitor ZL-n-91 and chloroquine phosphate. The combined medicine can effectively treat osteosarcoma, has the effect obviously superior to that of PDE4 inhibitor ZL-n-91 and chloroquine phosphate which are singly used, shows that the two have obvious synergistic effect after being matched for use, has strong anti-tumor activity and small side effect, and provides a new treatment option for patients who lose clinical operation indication and cannot operate or have poor effects of radiotherapy and chemotherapy.

Description

Combined medicine for treating osteosarcoma

Technical Field

The application belongs to the field of antitumor drugs, and in particular provides a combined drug for treating osteosarcoma, which contains a PDE4 inhibitor ZL-n-91 and chloroquine phosphate.

Background

Osteosarcoma (OS) is one of the most common malignant bone tumors, and the World Health Organization (WHO) defines Osteosarcoma as a primary malignant bone tumor consisting of mesenchymal cells that produce osteoid and immature bone. Osteosarcoma is often found in active growth sites such as the lower femur, upper tibia, upper humerus, etc., and its clinical manifestations include: pain in bones or joints, local tumor and lameness. When the condition further worsens to a general condition, it manifests as fever, discomfort, weight loss, anemia and failure. In most countries osteosarcoma has an age-related rate (ASR) of 0.20-0.35/10 ten thousand, accounting for 20-40% of bone tumors, with a 20% ratio in China. Osteosarcoma is highly prevalent in adolescents, with male morbidity being slightly higher than female, and the overall survival rate of osteosarcoma patients has changed little, about 70%, over the last three decades. However, for patients with metastatic disease and those who do not respond well to initial treatment, the 5-year survival rate is only 25% for people aged 2-68. Therefore, the search for new molecular targets has great significance for early diagnosis and targeted therapy of osteosarcoma patients. The current treatment mode of osteosarcoma is pre-operative neoadjuvant chemotherapy, surgical resection and post-operative adjuvant chemotherapy. Chemotherapeutic drugs include Methotrexate (MTX), doxorubicin (ADM), cisplatin (DDP), ifosfamide (IFO) and the like, but high doses of chemotherapeutic drugs have significant side effects, and can cause rare toxic effects such as cardiotoxicity, acute and chronic nephrotoxicity, neurotoxicity, hearing loss, infertility and the like in addition to common complications such as alopecia, myelosuppression, mucositis, nausea and vomiting. Thus, there is a need for further research to identify effective drugs and to develop new therapeutic strategies with fewer side effects to treat this fatal disease.

The research shows that phosphodiesterase 4 inhibitor (PDE 4 i) Roflumilast (Roflumilast) can remarkably inhibit the growth of ovarian cancer, lung cancer and B cell lymphoma; another study found that PDE4i Rolipram (Rolipram) inhibited proliferation of the human mandibular OS cell line HOSM-1, indicating potential anti-OS action of PDE4 i. Rolipram is the first prototype PDE4 inhibitor, but was not first marketed due to its serious adverse effects and was generally used as a positive control in subsequent studies of PDE4 inhibitors. In addition to Rolipram, researchers have further developed a new generation of PDE4 inhibitors, of which Roflumilast, apremilast and Crisborole are continuously marketed in batches. The european union and the united states approved oral Roflumilast for the treatment of severe COPD and asthma symptoms, respectively, in 2010 and 2011, apremilast for active psoriatic arthritis patients and moderate to severe plaque psoriasis patients (2014), and crisabaole for the topical treatment of mild to moderate atopic dermatitis in patients 2 years and older (2016). However, the therapeutic window of the PDE4 inhibitors described above is narrow and is accompanied by adverse effects such as nausea, vomiting, etc. during the course of treatment. Various novel PDE4 selective inhibitors have been developed for the above side effects, such as ZL-n-91 developed by university of north carolina Ke Hengming:

ZL-n-91 is a novel selective PDE4i which is thousands of times more selective than other PDE family molecules. Studies by Ruihong Ma in 2008 indicate that ZL-n-91 has an inhibitory effect on the production of IL-17 by memory T helper (Th) 17 cells, but has no effect on cell viability. Studies by Hui-Fang Tang et al in 2010 found that ZL-n-91 could ameliorate the inflammatory response of the smoking (CS) -induced male Sprague-Dawley rat COPD-like model, and subsequently the authors further found that ZL-n-91 could be used to treat lipopolysaccharide-induced Acute Lung Injury (ALI) in mice. In addition, ZL-n-91 has been found to inhibit the proliferation of PC-3, but no practical research has been conducted to verify the efficacy of other cancers.

Chloroquine phosphate, chloroquine diphosphate salt, CAS number 50-63-5, having the structural formula:

chloroquine is a classical antimalarial drug, and recent researches find that chloroquine has antitumor activity and has various action mechanisms on tumors, such as induction of apoptosis of tumor cells, inhibition of autophagy and the like. Chloroquine phosphate as a weak alkaline medicine can selectively enter tumor cell acid lysosome, effectively inhibit the action of the tumor cell acid lysosome to inhibit autophagy, and has the advantages of low cost, and no toxicity _p The effect of H normal cells is weak, which lays a good microenvironment foundation for better effect of chloroquine on tumor cells and reduction of adverse reaction of normal tissues. The autophagy can reduce the sensitivity of chemotherapeutics by regulating energy metabolism in a stress environment, and promote the survival of tumor cells. Autophagy inhibitor chlorineThe quindox can effectively prevent autophagy in the tumor toxicity chemotherapy process, and the blocking autophagy can promote tumor cell death and increase the sensitivity of the tumor cell to chemotherapeutics. Thus, targeted drugs and bi-directional intervention to regulate autophagy may be an effective cancer treatment strategy.

Disclosure of Invention

The application aims to provide a combination medicament for treating osteosarcoma, which is used for solving the problem that the administration effect is affected due to intolerance of organisms caused by the fact that the single treatment concentration of a PDE4 inhibitor ZL-n-91 is too high. The application utilizes in vitro tumor cell culture, and researches the combined action effect of ZL-n-91 and chloroquine through cell proliferation experiments, plate clone formation experiments and apoptosis experiments. Experiments prove that: chloroquine phosphate (CQ) has the capacity of synergistically inhibiting MNNG/HOS, U2OS and Saos-2 proliferation of osteosarcoma cells of ZL-n-91, and can enhance the apoptosis-promoting effect mediated by ZL-n-91.

In one aspect, the application provides a combination for the treatment of malignancy comprising a PDE4 inhibitor and chloroquine phosphate.

In another aspect, the application provides the use of a PDE4 inhibitor and chloroquine phosphate in the manufacture of a combination for the treatment of malignancy.

Further, the malignancy is osteosarcoma.

Further, wherein the PDE4 inhibitor is ZL-n-91.

Further, the medicament inhibits tumor cell proliferation.

Further, the medicament induces apoptosis of tumor cells.

Further, the molar ratio of PDE4 inhibitor to chloroquine phosphate in the medicament is 40:1-5:1.

Further, the molar ratio of PDE4 inhibitor to chloroquine phosphate in the medicament is 20:1-5:1.

Further, the molar ratio of PDE4 inhibitor to chloroquine phosphate in the medicament is 10:1.

Further, the PDE4 inhibitor and the chloroquine phosphate in the medicament are administered simultaneously or separately.

Further, the medicament is in an injection form.

The chloroquine phosphate, chloroquine, chloroquinoline phosphate, chloroquine phosphate, chloroquinoline phosphate and the like can be used interchangeably.

PDE4 inhibitors described in the present application include, but are not limited to, known and studied PDE4 inhibitors such as theophylline, rolipram, roflumilast, cilomilast, aplastt, ZL-n-91, and the like

Osteosarcoma described in the present application includes various types of osteosarcoma of various characteristics including, but not limited to, traditional osteosarcoma, intramedullary hyperdifferentiated osteosarcoma, periosteosarcoma, periosteal osteosarcoma, capillary expanded osteosarcoma, and small cell osteosarcoma.

The formulation and specification of ZL-n-91 and chloroquine phosphate in the application can be the same or different.

The ZL-n-91 and chloroquine phosphate in the application are preferably injection formulations, and can also be oral formulations, and specific formulations can be oral formulations or injection formulations, including but not limited to tablets, capsules, oral liquid preparations, pills, granules, powder, injection, oil injection, emulsion injection, powder injection and the like.

In addition to oral or injectable dosage forms, other dosage forms known or studied such as transdermal, aerosol, targeted carrier dosage forms may be routinely selected and designed by those skilled in the pharmaceutical arts depending on the circumstances.

ZL-n-91 may be used in combination with other known or studied agents for treating osteosarcoma or prepared into the same formulation, including but not limited to chemotherapeutic and targeted agents.

The phosphodiesterase 4 inhibitor ZL-n-91 of the application can be purchased directly, synthesized by reference to the existing literature or by entrusting, for example, can be prepared synthetically by reference to the text, such as Ruihong Ma, bin-yan Yang, chang-you Wu A selective phosphodiesterase 4 (PDE 4) inhibitor Zl-n-91 supports IL-17production by human memory Th17 cells.International Immunopharmacology,2008,8 (10): 1408-1417.

The beneficial effects are that: the selective PDE4 inhibitor ZL-n-91 provided by the application can obviously inhibit the proliferation of tumor cells, and is predicted to be an important target point for anti-osteosarcoma proliferation research; meanwhile, chloroquine phosphate has the function of inhibiting autophagy, can enhance the killing effect of ZL-n-91 on osteosarcoma cells, and has good application prospect on the treatment of osteosarcoma.

Drawings

FIG. 1 is a graph showing proliferation inhibition effects of ZL-n-91 and CQ in combination on osteosarcoma cells MNNG/HOS, saos-2, U2 OS: CQ concentration was set to 10. Mu.M, ZL-n-91 concentration was set to 200. Mu.M, the drug alone was treated for 48 hours in the group, the combination was pretreated with CQ for 2 hours and then ZL-n-91 was treated for 48 hours, and cell proliferation activity was measured by a living cell count method. All data are expressed as mean ± standard deviation. (n=3), P <0.05, P <0.01, P <0.001.

FIG. 2 is a graph showing interaction effects of ZL-n-91 and CQ in combination on osteosarcoma cells MNNG/HOS, saos-2, U2 OS: the CQ single group concentration was 0,2.5,5,10,20. Mu.M, the ZL-n-91 single group concentration was 0,25,50,100,200. Mu.M, the drug concentration ratio of the combined groups was 1:10, and 4 drug concentration groups were set, namely group one (2.5. Mu.M CQ+25. Mu.M ZL-n-91), group two (5. Mu.M CQ+50. Mu.M ZL-n-91), group three (10. Mu.M CQ+100. Mu.M ZL-n-91) and group four (20. Mu.M CQ+200. Mu.MZL-n-91), were pretreated with CQ for 2 hours, and then treated with ZL-n-91 for 48 hours, respectively, and proliferation activity of various cells was examined by a viable cell count method. The CI values of CQ in combination with ZL-n-91 were analyzed using CalcuSyn software. Synergy is provided when the CI values are all less than 1, additive is provided when the CI is equal to 1, and antagonism is provided when the CI is greater than 1.

FIG. 3 is a graph showing proliferation inhibition effects of ZL-n-91 and CQ in combination on osteosarcoma cells MNNG/HOS, saos-2, U2 OS: taking cells in logarithmic growth phase, inoculating the cells in a six-hole plate at a proper concentration, and adding medicines for treatment after the cells adhere to the wall. CQ concentration was set to 10. Mu.M, ZL-n-91 concentration was set to 200. Mu.M, the drug alone was treated for 48h with the combination group pretreated with CQ for 2h and then ZL-n-91 for 48h, and fresh medium was changed. The culture was terminated when macroscopic cell clones appeared, and the number of cell clone formations was counted for each group by fixed staining observation. (n=3), P <0.05, P <0.01, P <0.001.

FIG. 4 is a graph showing pro-apoptotic effects of ZL-n-91 and CQ combinations on osteosarcoma cells MNNG/HOS, saos-2, U2 OS: CQ concentration was set to 10. Mu.M, ZL-n-91 concentration was set to 200. Mu.M, the drug alone was treated for 48h in the group, the combination was pretreated with CQ for 2h, then ZL-n-91 was treated for 48h, and flow cytometry was used to detect apoptosis. All data are expressed as mean ± standard deviation. (n=3), P <0.05, P <0.01, P <0.001.

Detailed Description

The present application will be further described with reference to the accompanying drawings for a clear and intuitive understanding to those skilled in the art.

Example 1

Living cytometry to detect the effect of ZL-n-91 in combination with CQ on osteosarcoma cell proliferation

1) Cell U2OS, saos-2 and MNNG/HOS in logarithmic growth phase were used to prepare single cell suspension. 2ml of cell suspension per well was inoculated into six well plates and divided into 4 groups: solvent control group (added with solvent EtOH of the same volume), CQ group (10. Mu.M), ZL-n-91 group (200. Mu.M), CQ+ZL-n-91 group, 3 sub-wells each;

2) After the cells are attached, CQ is added for pretreatment for 2 hours, ZL-n-91 is added for treatment for 48 hours;

3) After distressing the medium and washing twice with PBS, cells were collected by pancreatin digestion and counted.

The results are shown in FIG. 1: CQ and ZL-n-91 treatment can inhibit proliferation of osteosarcoma cells MNNG/HOS, U2OS and Saos-2 cells, and cell viability is further reduced by the combination of the two.

Example 2

Calculation of CQ and ZL-n-91 Joint action index

1) Cell U2OS, saos-2 and MNNG/HOS in logarithmic growth phase were used to prepare single cell suspension. 2ml of cell suspension per well was inoculated into six well plates, and 3 groups (CQ-n-91 group alone, ZL-n-91 group alone and CQ+ZL-n-91 group) of 3 sub-wells per group were set;

2) The CQ working concentration was 0,2.5,5,10,20. Mu.M, ZL-n-91 single-use group concentration was 0,25,50,100,200. Mu.M, and the drug concentration ratio of the combined group was 1:10.

3) After the cells are attached, the combined group is pretreated with CQ for 2 hours, and then ZL-n-91 is added for 48 hours.

4) After distressing the medium and washing twice with PBS, cells were collected by pancreatin digestion and were counted for cell viability. The data was entered into the CalcuSyn software to calculate the combination index (Combination Index, CI). Wherein the CI value is less than 1, and the CI value is equal to 1, and the CI value is greater than 1, and the CI value is equal to 1.

The results are shown in FIG. 2: CQ has the capacity of synergistic ZL-n-91 to inhibit osteosarcoma cell proliferation

Experimental example 3

Plate clone formation assay to examine the effect of ZL-n-91 in combination with CQ on the clonogenic potential of osteosarcoma cells

2) After the cells are attached, CQ is added for pretreatment for 2 hours, ZL-n-91 is added for treatment for 48 hours, and fresh culture medium is replaced;

3) After 10d of culture, macroscopic cell clones appear, namely, the culture is stopped, cells are fixed by paraformaldehyde solution, crystal violet staining is carried out, after PBS washing, more than or equal to 50 cell clone groups are marked as 1 cell clone, the number of cell clone formation of each group is observed and counted, and the result is expressed as the number of cell clone formation.

The results are shown in FIG. 3: CQ can obviously enhance the inhibition effect of ZL-n-91 on the clonogenic capacity of osteosarcoma cells.

Experimental example 4

Flow cytometry detection of induction of osteosarcoma apoptosis by combination of Zl-n-91 and CQ

3) After 48h, cells were harvested, washed 2 times with cold PBS, 100. Mu.l 1 XBindingBuffer was added to prepare cell suspension in flow tubes, 5. Mu.l 7AA-D and 5. Mu.L PE were added for staining according to kit instructions, the gently vortexed cells were incubated at room temperature for 15min in the dark, 200u1 XBindingBuffer was added to the tubes, and flow cell detection was performed within 1h, flowJoV10 analysis software analysis results.

The results are shown in FIG. 4: CQ can significantly enhance the apoptosis promotion effect mediated by ZL-n-91.

The research results show that the phosphodiesterase 4 inhibitor ZL-n-91 and CQ combined action can inhibit the proliferation of osteosarcoma cells, and the effect is superior to that of CQ and ZL-n-91 which are independently used, and the application has good anti-tumor effect.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present application. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present application is not limited to the embodiments described herein, and those skilled in the art, based on the present disclosure, should make improvements and modifications within the scope of the present application.

Claims

Use of a PDE4 inhibitor and chloroquine phosphate for the manufacture of a medicament for the treatment of osteosarcoma, wherein the PDE4 inhibitor is ZL-n-91; the molar concentration of PDE4 inhibitor in the drug was 200. Mu.M and the molar concentration of chloroquine phosphate was 20. Mu.M.
2. The use according to claim 1, wherein the osteosarcoma is a classical osteosarcoma, an intramedullary hyperdifferentiated osteosarcoma, a paraosteosarcoma, a periosteal osteosarcoma, a capillary expanded osteosarcoma or a small cell osteosarcoma.
3. The use of claim 1, wherein the medicament inhibits osteosarcoma cell proliferation.
4. The use of claim 1, wherein the medicament induces apoptosis of osteosarcoma cells.
5. The use according to claim 1, wherein the medicament is in an oral dosage form.
6. The use according to claim 5, wherein the medicament is a tablet, capsule, oral liquid, pill, granule or powder.
7. The use according to claim 1, wherein the medicament is in the form of an injection.
8. The use according to claim 7, wherein the medicament is in the form of a water injection, an oil injection, a milk injection or a powder injection.
9. The use according to claim 1, wherein the medicament is used in combination with or comprises other medicaments for the treatment of osteosarcoma.
10. The use according to claim 9, wherein the other drug for the treatment of osteosarcoma is a chemotherapeutic drug.
11. The use according to claim 9, wherein the other drug for the treatment of osteosarcoma is a targeting drug.