Use of selective cdK9 modulators in the manufacture of a medicament for adrenocortical carcinoma

Abstract

The application relates to application of a selective CDK9 modulator in preparation of an adrenocortical carcinoma (ACC) drug. The application proves that the selective CDK9 modulator can significantly inhibit ACC tumor growth and hormone secretion through in-vitro and in-vivo experiments, and CDK9 is an important target point for ACC treatment; especially in steroidogenic factor-1 (SF-1) positive cells, the selective CDK9 modulator shows obvious anti-tumor and anti-secretion effects at a nanomolar level, and has a good market application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tumor treatment, and particularly relates to CDK9 as a target for ACC treatment and application of a selective CDK9 modulator in preparation of an adrenocortical carcinoma drug. BACKGROUND

[0002] Adrenocortical carcinoma (ACC) is a rare endocrine malignancy originating from the adrenal cortex. ACC has the characteristics of strong invasiveness, high malignancy and poor prognosis.

[0003] The treatment of ACC is very limited, and surgical resection is still the main treatment. However, even after complete resection (R0 resection), more than half of the patients will relapse, and the postoperative recurrence rate is as high as 60-80%. For patients with advanced and unresectable tumors, Mitotane (trade name: Lysodren) is the only drug officially approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of ACC, and is recommended as the first-line palliative treatment for ACC, either alone or in combination with platinum-based chemotherapy. Mitotane, also known as o,p'-DDD, is a chemical name of 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane, which was isolated from the insecticide dichlorodiphenyltrichloroethane (DDT) in 1940. However, the efficacy of Mitotane is very limited, and the objective response rate (ORR) is only 20%-35%. In addition, in most cases, the maintenance time of Mitotane treatment response is limited, and the patient will progress, with a median PFS of only about 4.1 months. Currently, the recommended daily dose of Mitotane is usually between 2-10g to achieve the desired plasma concentration (14-20mg / L) or maximum tolerance. However, it usually takes at least 3 months for most patients to achieve the therapeutic plasma concentration (14-20mg / L) of Mitotane on a daily basis, with a median of about 6 months. At the same time, a considerable proportion of patients cannot achieve the desired therapeutic plasma concentration even if the dose is increased and the medication time is prolonged, which to some extent affects the therapeutic effect of Mitotane. In addition, ~80% of patients will have adverse reactions during Mitotane treatment, mainly gastrointestinal symptoms (nausea, vomiting, and diarrhea, etc.), nervous system symptoms (including dizziness, headache, ataxia, paresthesia, etc.), and studies have shown that blood system abnormalities, skin rash, liver damage, high cholesterol, high triglycerides, male breast development, etc. Adverse reactions, and even severe neurotoxicity and severe liver function damage can occur. Therefore, the side effects and toxicity of Mitotane have greatly limited its application.

[0004] In addition, it is worth noting that since the first approval of Mitotane for the treatment of ACC in 1970, the FDA has not approved any new drugs for patients with advanced or inoperable ACC. Currently, the research of ACC drug therapy is mainly limited to preclinical studies or relatively small-scale clinical studies. Among them, clinical trials such as ACC targeted drug therapy include insulin-like growth factor 1 receptor (IGF-1R) inhibitors (such as Linsitinib), tyrosine kinase inhibitors (TKI) (such as Sorafenib, Sunitinib and Cabozantinib), and immunotherapy including anti-PD-1 / PD-L1 therapy (such as Avelumab and Nivolumab), and the therapeutic effect is not satisfactory. The development of ACC drug research is lagging behind, and there are mainly the following difficulties: Although genomics research reveals that ACC tumors have extensive mutations, including TP53, CTNNB1, ZNRF3, CDKN2A, RB1, telomere maintenance genes (such as TERT, ATRX) and epigenetic regulators (such as MEN1, genes encoding SWI / SNF complex), etc., but most of the mutant genes are currently non-druggable targets (such as TP53, RB1), and the development of targeted drugs has certain difficulties. In addition, ACC is a rare tumor, with an annual incidence of about 0.7-2 person-time per million people. Even if it is a multi-center study, the time span required to recruit patients for clinical trials is large and difficult to develop, resulting in a lack of ACC clinical trials. The preclinical research of ACC also faces the problem of limited available models.

[0005] Therefore, ACC treatment has not made breakthrough progress in the past 50 years, and it is basically in a state of no available drugs, so it is urgent to research new ACC treatment drugs to break through the current difficulties. SUMMARY

[0006] The technical problem to be solved by the present application is to provide the application of selective CDK9 modulators in the preparation of adrenocortical carcinoma drugs, and to explore new targets or new drugs for ACC treatment.

[0007] The present application provides the application of selective CDK9 modulators in the preparation of adrenocortical carcinoma drugs, and proves that CDK9 is an important target for ACC treatment.

[0008] Preferably, the selective CDK9 modulator includes CDK1 / 2 / 5 / 9 modulator Dinaciclib, highly selective CDK9 modulator AZD4573 and VIP152.

[0009] Further, the selective CDK9 modulator has a significant inhibitory effect on adrenal cortex cancer cells such as H295R cells.

[0010] Preferably, the medicine further comprises a pharmaceutically acceptable carrier and / or adjuvant.

[0011] Preferably, the pharmaceutically acceptable carrier and / or adjuvant comprises at least one of a diluent, a binder, a surface active agent, a wetting agent, an adsorption carrier, a lubricant, a filler, a disintegrant.

[0012] The present application also provides a pharmaceutical composition for adrenal cortex cancer, which comprises a therapeutically effective amount of a selective CDK9 modulator.

[0013] Preferably, the dosage form of the pharmaceutical composition comprises at least one of a tablet, a pill, a powder, a solution, a suspension, an emulsion, a granule.

[0014] Advantageous effects

[0015] The present application proves by in vitro and in vivo experiments that the selective CDK9 modulator can significantly inhibit ACC tumor growth and hormone secretion, and CDK9 is an important target for ACC treatment; especially in steroidogenic factor-1 (SF-1) positive cells, the selective CDK9 modulator shows significant anti-tumor and anti-secretion effects at the nanomolar level, which has good market application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A is the HTS flow chart;

[0017] Figure 1 B is the primary screening showing that Dinaciclib has a significant inhibitory effect on H295R cells and SW13 cells;

[0018] Figure 1 C is the CCK8 method for detecting cell activity after H295R cells and SW13 cells are treated with different concentrations of Dinaciclib for 6d and 4d respectively, the dose-response curve is drawn using GraphPad Prism 8, and the IC50 is calculated;

[0019] Figure 1 D is a clonogenic assay to evaluate the inhibitory effect of Dinaciclib on the clonogenic ability of H295R cells and SW13 cells. Figure 2 A is the Caspase-3 / 7 activity detection after H295R cells and SW13 cells are treated with Dinaciclib 50nM, 100nM; 10nM, 20nM for 24h respectively;

[0020] Figure 2 B is a scratch assay to evaluate the effect of Dinaciclib on the migration ability of H295R and SW13 cells.

[0021] Figure 2 C represents H295R cells treated with Dinaciclib 50 nM for 48 h. The supernatant was collected after 12 h for steroid hormone detection, and the results were corrected using cellular protein levels. The results showed that the secretion levels of cortisol and aldosterone were significantly reduced (top). The heatmap shows the changes in steroid hormones in H295R cells after Dinaciclib 50 nM treatment (bottom).

[0022] Figure 3 AB represents the effect of Dinaciclib on the ACC cell cycle.

[0023] Figure 4 A represents the growth curve of H295R-Xenograft tumors. Vehicle, control group; DIN, Dinaciclib group (40 mg / kg); MIT, Mitotane group (200 mg / kg); MIT + DIN, Dinaciclib (40 mg / kg) and Mitotane (200 mg / kg) combination therapy group;

[0024] Figure 4 Five weeks after administration of H295R-Xenograft, nude mice were sacrificed, tumors were dissected, photographed, and weighed. Tumor growth inhibition rate (TGI) was also calculated.

[0025] Figure 4 C represents the weight gain curve of H295R-Xenograft nude mice;

[0026] Figure 5 A represents the effect of Dinaciclib treatment on H295R cells for 24 h and 48 h on the phosphorylation level of the downstream target protein p27 at the 187th threonine (Thr) site [p-P27(Thr 187)] and the phosphorylation level of the 2nd serine (Ser2) site on the C-terminal domain of RNA polymerase II [p-RNPII(Ser 2)].

[0027] Figure 5 B represents the apoptosis level detection in H295R cells after CDK9 knockdown using shRNA;

[0028] Figure 5 C represents the plate colony formation assay used to evaluate the inhibitory effect of CDK9 knockdown on the colony formation ability of H295R cells;

[0029] Figure 5 D represents a plate colony formation assay to evaluate the inhibitory effect of highly selective CDK9 regulators (AZD4573, VIP152) on the colony formation ability of H295R cells.

[0030] Figure 5 The anti-secretion effect was assessed by detecting the cortisol level in the supernatant of H295R cells after treatment with highly selective CDK9 regulators (AZD4573, VIP152).

[0031] Figure 5 F represents the responsiveness of SW13 cells and H295R cells to treatment with CDK7 inhibitor THZ1, CDK12 / 13 inhibitor THZ531, and CDK8 inhibitor BI-1347 (comparison of antiproliferative IC50). Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0033] Example 1

[0034] High-throughput screening (HTS) is an important strategy for drug development and the discovery of new targets and bioactive molecules. In this embodiment, HTS was performed using ACC cell lines (H295R and SW13 cells), evaluating a total of 7310 compounds. The criteria for evaluation were a proliferation inhibition rate ≥70% at 2 μM and ≥50% at 0.4 μM; simultaneously, a proliferation inhibition rate >0.4 μM at 2 μM. A total of 142 potentially sensitive compounds, i.e., candidate compounds, that showed significant killing effects on both H295R and SW13 cells were identified. Figure 1 A).

[0035] Among them, the cyclin-dependent kinase (CDK) 1 / 2 / 5 / 9 inhibitor Dinaciclib showed significant inhibitory effects on both H295R and SW13 cells: 2 μM inhibition rate >95%, 0.4 μM inhibition rate >70%. Figure 1B). Further validation experiments demonstrated that both H295R and SW13 cells were highly sensitive to Dinaciclib, which dose-dependently inhibited the proliferation of both H295R and SW13 cells, with IC50 values ​​of 39.9±6.8 nM and 10.0±2.3 nM, respectively; and the inhibitory effect was significantly better than that of Mitotane. Figure 1 C). Simultaneously, plate colony experiments showed that Dinaciclib significantly inhibited cell colony formation ability, confirming that Dinaciclib can effectively inhibit ACC proliferation for a long time. Figure 1 D).

[0036] Further analysis of Caspase-3 / 7 activity revealed that Caspase 3 / 7 was significantly activated in H295R and SW13 cells after 24 hours of treatment with Dinaciclib, indicating that Dinaciclib can rapidly and significantly induce apoptosis in ACC cells. Figure 2 A). Scratch assay results showed that Dinaciclib also inhibited the migration of H295R and SW13 ( Figure 2 B). Further investigation using H295R cells with hormone-secreting function revealed that Dinaciclib significantly inhibited the secretion of steroid hormones from H295R cells. Figure 2 C).

[0037] Given that Dinaciclib can inhibit the activity of multiple CDKs, including CDK1 and CDK2, which are closely related to the cell cycle, it is speculated that it may induce cell cycle arrest in ACC cells, thereby exerting its anti-tumor effect. The effect of Dinaciclib on the ACC cell cycle was assessed by detecting DNA content in cells using flow cytometry after propidium iodide (PI) staining. It was observed that after 24 h of Dinaciclib treatment, SW13 cells underwent significant changes in cell cycle: the proportion of cells in G2 phase was significantly increased compared to the control group (DMSO 13.7±3.8%) (Dinaciclib 10 nM: 24.3±3.8%, P = 0.0043; Dinaciclib 20 nM: 38.1±7.9%, P = 0.0002). Figure 3 B) suggests that the cell transition from G2 to M phase is blocked, i.e., G2 / M phase arrest. However, in H295R cells, only a slight increase in the proportion of G2 phase cells was observed after 48 h of Dinaciclib treatment (DMSO: 20.7±3.3%, Dinaciclib 50nM: 26.4±2.8%; Dinaciclib 100nM: 27.0±5.7%). Figure 3A) suggests that in H295R cells, Dinaciclib may have a cell cycle-independent mechanism involved in anti-tumor effects, in addition to interfering with other cell cycles.

[0038] In vivo evaluation using the ACC model showed that Dinaciclib significantly inhibited tumor growth, with effects superior to Mitotane. Furthermore, all mice tolerated the treatment well, and there was no significant difference in body weight between the treatment group and the control group (P > 0.05); indicating the safety of Dinaciclib in vivo. Figure 4 AC).

[0039] Next, further target analysis was conducted. As mentioned earlier, cell cycle assessment after treatment with the CDK1 / 2 / 5 / 9 regulator Dinaciclib revealed no significant cell cycle changes in H295R cells, suggesting a cell cycle-independent mechanism involved in the anti-tumor effect. Western blot results showed that after Dinaciclib treatment, the phosphorylation level at threonine 187 (Thr) of the CDK2 downstream target protein p27 did not change significantly; however, the phosphorylation level at serine 2 (Ser2) on the C-terminal domain of CDK9 downstream RNA polymerase II was significantly inhibited, suggesting that CDK9 inhibition plays a major role. CDK9 knockdown in H295R cells inhibited cell colony formation and significantly increased apoptosis. Figure 5 AC).

[0040] Further evaluation using highly selective CDK9 regulators (AZD4573, VIP152) revealed that ACC cells were highly sensitive to CDK9 regulators and exhibited both antitumor and antisecretory effects, suggesting that CDK9 is an important target for ACC treatment. Compared to SF-1 negative SW13 cells, SF-1 expressing H295R cells were highly sensitive only to CDK9 regulators; however, their response to CDK7 or CDK12 / 13 inhibitors was significantly inferior to that of SW13 cells, suggesting that highly selective CDK9 regulators hold promise as therapeutic agents for ACC, especially for SF-1 positive ACC. Figure 5 DF).

Claims

1. The application of selective CDK9 modulators in the preparation of drugs for treating adrenocortical carcinoma, characterized in that: The selective CDK9 modifier is selected from CDK1 / 2 / 5 / 9 modifier Dinaciclib, highly selective CDK9 modifier AZD4573, or VIP152.

2. The application according to claim 1, characterized in that: The selective CDK9 modulator has a significant inhibitory effect on adrenocortical cancer cells.

3. The application according to claim 1, characterized in that: The drug also contains pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that: The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

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