Ribociclib and NS1643 combined composition and application thereof

Abstract

The invention provides a Ribociclib and NS1643 combined composition and an application of the Ribociclib and NS1643 combined composition. In the Ribociclib and NS1643 combined composition, the ratio of Ribociclib to NS1643 is 50 mg: (0.1-10 mg). According to the invention, NS1643 is used for activating hERG current inhibited by Ribociclib, shortening the action potential of myocardial cells prolonged by Ribociclib, and prolonging the QT interval, so that the QT interval is prolonged without stopping using ribociclib to treat breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and further belongs to the field of medicine for breast cancer, and particularly relates to a Ribociclib combined with NS1643 composition and its application. Background Art

[0002] Ribociclib is a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor used for the treatment of HR+ / HER2- advanced breast cancer. Although the treatment effect is good, there is certain cardiotoxicity during use, manifested as a certain probability of QT interval prolongation in users, and even syncope or sudden death caused thereby. However, when a user has QT interval prolongation, the usual solution is to stop using Ribociclib and conduct symptomatic treatment. And stopping the drug has the risk of aggravating the breast cancer condition of the patient. Therefore, it is very necessary to develop a product and method that can solve the above technical problems. Summary of the Invention

[0003] The first object of the present invention is to provide a Ribociclib combined with NS1643 composition; the second object is to provide the application of the Ribociclib combined with NS1643 composition.

[0004] The first object of the present invention is achieved as follows. In the Ribociclib (ribociclib) combined with NS1643 composition, the ratio of Ribociclib to NS1643 is 50 mg: (0.1 - 10 mg); the structure of the NS1643 is: .

[0005] In the present invention, NS1643 is used to activate the hERG current inhibited by Ribociclib, shorten the action potential of cardiomyocytes prolonged by Ribociclib, and prolong the QT interval, achieving the prolongation of the QT interval while not stopping the treatment of breast cancer with ribociclib. Brief Description of the Drawings

[0006] Figure 1 It is the dose-effect relationship between NS1643 and the hERG channel in Example 1 of the present invention; Figure 2 It is the influence of NS1643 on the action potential duration of hiPSC-CMs in Example 1 of the present invention; Figure 3 It is the influence of NS1643 on the QT interval of guinea pigs in Example 1 of the present invention. Detailed Embodiments

[0007] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited in any way. Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0008] In the Ribociclib combined with NS1643 composition described in the present invention, the ratio of Ribociclib to NS1643 is 50 mg: (0.1 - 10 mg).

[0009] The ratio of Ribociclib to NS1643 is 50 mg: (1 - 10 mg).

[0010] The ratio of Ribociclib to NS1643 is 50 mg: (5 - 10 mg).

[0011] The application of the present invention is the application of the Ribociclib combined with NS1643 composition in the preparation of a pharmaceutical composition for treating breast cancer.

[0012] The following is a further description of the present invention with specific implementation cases: Example 1 The NS1643 is used to prepare a drug that activates the hERG current inhibited by Ribociclib, shortens the action potential of cardiomyocytes prolonged by Ribociclib (Ribociclib), and prolongs the QT interval; the structure of the NS1643 is .

[0013] Method: Record the electrical activity of cell membrane ion channels in the whole-cell mode 1. Preparation of cell slides Cells transfected with the target channel (HEK293T cells transfected with the hERG channel, hiPSC-CMs, guinea pig cardiomyocytes) are inoculated in a cell culture dish paved with round glass slides, and electrophysiological recording is carried out 1 hour later.

[0014] 2. Recording of ion channel electrical activity Place the cell bath containing an appropriate amount of the corresponding external solution on the stage of the microscope. Put the prepared cell slide into the cell bath. Fix the electrode on the electrode holder and connect it to the amplifier (EPC 10 USB). Apply positive pressure to the electrode and use the motorized micromanipulator to control the electrode to enter the solution. After the electrode contacts the cell, release the positive pressure to seal the cell. After the holding is stable, compensate for the fast capacitance. Apply negative pressure to the sealed cell, and then slowly lift the whole cell using the micromanipulator. Switch the recording mode to the whole-cell recording mode (Whole-Cell), and perform compensation for the cell capacitance, series resistance, and leakage current. Slowly move the cell to the front of the pipeline of the drug delivery system RSC-200 using the micromanipulator. Open the pipeline of the external solution of the electrode, stimulate the cell with the corresponding stimulation mode of the recorded ion channel, and record the current signal. Change the drug or concentration to be detected by switching different pipelines. The pipeline switching data of the drug delivery system is transmitted to the amplifier in real time and processed by the Patch Master software, and the current change data corresponding to the drugs in each pipeline is recorded in real time. The following are the involved stimulation modes: The hERG current refers to the rapidly activating delayed rectifier potassium channel current (IKr) encoded by the human ether-á-go-go-related gene (hERG), which plays an important role in the cardiac repolarization process. The protein encoded by the hERG gene is the α subunit of the potassium ion channel, mainly mediating the repolarization phase of the delayed rectifier potassium current (IKr) in the cardiac action potential. The uniqueness of the hERG channel lies in its voltage-dependent and time-dependent inactivation characteristics. In the cardiac action potential, the hERG channel is responsible for mediating the delayed rectifier potassium current, which is an important component of the repolarization phase. When the myocardial cell is depolarized to a certain voltage, the hERG channel opens, allowing potassium ions to flow out, thereby ending the action potential and restoring the resting state of the cell. Many drugs prolong the cardiac repolarization time by blocking the hERG channel current, which may lead to QT interval prolongation in some cases and may trigger fatal arrhythmias. Therefore, the detection of the inhibitory activity of the hERG channel is very important in new drug research and development.

[0015] Method for recording the hERG channel: Clamp the voltage at -80 mV, hyperpolarize to -100 mV to monitor the change in series resistance, and then stimulate with a baseline current of -50 mV to analyze the final result. Activate the hERG channel at +50 mV (800 ms), then stimulate with -50 mV to record the outward tail current, and then continue to hyperpolarize to -120 mV (200 ms), mainly for the hERG channel to recover from the inactivated state.

[0016] The recording method of cardiomyocyte action potential is to digitally record the action potential in the 1 kHz C-Clamp mode. The stimulation current is 800 - 1000 pA, the frequency is 1 Hz, the injection current is 50 pA, and the duration is 10 ms. Perfuse with different concentrations of ribociclib and different concentrations of NS1643, and record the effects of the drugs on the action potential. Statistically analyze the time required for 50%, 70%, and 90% repolarization of the action potential, namely APD50 / 70 / 90.

[0017] (1) Heterologously express the hERG gene in HEK293T cells using the lipo3000 transfection reagent. Record the hERG current in HEK293T cells overexpressing the hERG channel through the whole-cell patch clamp recording mode. When the current recording is stable, use 25 μM Ribociclib to inhibit the hERG current. When the inhibitory effect is stable, give different concentrations of NS1643 (0.1, 0.3, 1, 3, 5, 10 μM). The inhibited hERG current can be activated by NS1643, and as the dosage of NS1643 increases, the hERG current gradually increases. As Figure 1 shown, NS1643 can activate the hERG current inhibited by Ribociclib. Figure 1 A is the current graph of NS1643 at 0.1 μM - 10 μM acting on hERG. Figure 1 B is the dose-effect curve graph of NS1643 and the hERG channel (the activation rate of the hERG current when the NS1643 concentration is 0 is set to 100%). Its EC 50 is 9.62 ± 0.83 μM, indicating that NS1643 can activate the hERG current inhibited by Ribociclib in a concentration-dependent manner and with high efficiency.

[0018] (2) In cardiomyocytes, after recording the stable action potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMS) using the patch clamp, give 30 μM Ribociclib. After the prolongation effect is stable, give different concentrations of NS1643. The results show that using Ribociclib alone can prolong the action potential duration (APD) of hiPSC-CMS, which means that the repolarization time of the myocardium is prolonged, which is an important manifestation in cardiomyocytes during QT interval prolongation. At this time, simultaneously give hiPSC-CMS cells Ribociclib and different concentrations of NS1643, and the prolonged hiPSC-CMS action potential can be shortened. As Figure 2 shown, NS1643 can shorten the hiPSC-CMS action potential prolonged by Ribociclib.

[0019] Figure 2 A-2C represents the control group (Vehicle), Ribociclib, and the groups of Ribociclib combined with NS1643 at different concentrations. The action potential duration (APD) of hiPSC-CMS was quantitatively analyzed in different groups (APD50, APD70, and APD90 from left to right). The results showed that as the concentration of NS1643 used with 30 μM Ribociclib increased, the action potential duration (APD) of hiPSC-CMS gradually shortened. And when the concentration of NS1643 increased to 3 μM, there was a statistically significant difference compared with the APD of the 30 μM Ribociclib group. APD50: the time when the action potential repolarizes to 50%; APD70: the time when the action potential repolarizes to 70%; APD90: the time when the action potential repolarizes to 90%. ns represents no significant difference; * represents p less than 0.05; ** represents p less than 0.01; **** represents p less than 0.0001.

[0020] (3) Guinea pigs were randomly divided into a solvent control group and an experimental group. The dosing regimens are shown in Table 1 below. The dissolution regimens for Ribociclib and NS1643 were 5% DMSO + 40% PEG300 + 5% Tween80 + 50% normal saline. After 30 min of dosing, isoflurane was administered by a small animal anesthesia machine (RWD: R540IP) for gas inhalation anesthesia, and the electrocardiogram was recorded. The positive electrode of the guinea pig was connected to the left upper limb, the negative electrode was connected to the right upper limb, and the ground wire was connected to the left lower limb. The electrode needle was inserted subcutaneously, taking care not to insert it into the muscle to avoid electromyogram interference. The electrocardiogram was recorded for 30 min using a BL420F biological signal acquisition and processing system (Tanon).

[0021] Table 1 Dosing regimens for acute animal experiments Previous exploration at the cellular level suggested that NS1643 had the potential to intervene in ribociclib-induced QT prolongation. Therefore, we verified it again at the animal level. Guinea pigs were given ribociclib in combination with different concentrations of NS1643 (1 mg / kg, 5 mg / kg, 10 mg / kg), and electrocardiograms were recorded. The QT interval was statistically analyzed. The results showed that when guinea pigs were given 50 mg / kg ribociclib, the QT interval was prolonged. When ribociclib was used in combination with NS1643, the QT interval of guinea pigs was shortened, and it was found that as the concentration of NS1643 increased, the shortening of the QT interval became more obvious. These results indicate that NS1643 can treat ribociclib-induced QT interval prolongation.

[0022] Figure 3 In A, guinea pigs were administered by intravenous injection, and their electrocardiograms were recorded under isoflurane inhalation anesthesia 30 minutes later. Figure 3 In A, from top to bottom are the representative electrocardiograms of guinea pigs in the control group, Ribociclib group, and Ribocicliib group with different concentrations of NS1643 (1 mg / kg, 5 mg / kg, 10 mg / kg).

[0023] Figure 3 In B, for Figure 3 In A, the QT interval in the electrocardiograms of guinea pigs was statistically analyzed. NS1643 could dose-dependently shorten QTc, that is, when NS1643 was used in combination with 50 mg / kg Ribociclib, as the dose of NS1643 increased, the QT interval in the electrocardiograms of guinea pigs gradually shortened. When 10 mg / kg of NS1643 was used, compared with using 50 mg / kg Ribociclib alone, there was a significant difference in the shortening of the QT interval in guinea pigs.

[0024] The main mechanism of ribociclib-induced LQTS (long QT syndrome) is that this drug inhibits the hERG ion channel. For this mechanism of occurrence, NS1643 was formulated into a drug so that the QT interval prolongation could be treated in patients without stopping the use of ribociclib.

[0025] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent variations made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A composition of Ribociclib combined with NS1643, characterized in that: The ratio of Ribociclib to NS1643 in the Ribociclib combined with NS1643 composition is 50 mg: (0.1~10 mg).

2. The Ribociclib combined with NS1643 composition according to claim 1, characterized in that: The ratio of Ribociclib to NS1643 is 50mg:(1~10mg).

3. The Ribociclib combined with NS1643 composition according to claim 1, characterized in that: The ratio of Ribociclib to NS1643 is 50mg:(5~10mg).

4. Use of the Ribociclib combined with NS1643 composition according to any one of claims 1 to 3 in the preparation of a pharmaceutical composition for treating breast cancer.

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