Combination therapy of receptor tyrosine kinase inhibitors with biphenylcyclooctyl lignans and their uses

Abstract

This invention proposes a combined use of a receptor tyrosine kinase inhibitor and biphenylcyclooctyl lignan, and its application therein. This application includes the preparation of a pharmaceutical composition comprising: a compound of formula I or a derivative thereof, and biphenylcyclooctyl lignan. The pharmaceutical composition according to embodiments of the present invention can effectively prolong the duration of action of the compound of formula I in vivo, significantly reduce the formation of metabolites of formula II, increase the brain penetration dose of the compound of formula I, and improve efficacy, thereby more effectively treating or preventing cancer.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the combined use of receptor tyrosine kinase inhibitors and biphenylcyclooctanine lignans and their applications, and more specifically, to a pharmaceutical composition, a single dosage form, a pharmaceutical combination, a pillbox, a method of administration of a compound of Formula I or a salt thereof, a method of prolonging the half-life of a compound of Formula I or a salt thereof, and a method of reducing the half-life of a compound of Formula I or a salt thereof to generate a compound of Formula II. Background Technology

[0002] (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate and its salts are not limited to oral administration and are inhibitors of epidermal growth factor receptor tyrosine kinase activity with activating mutations (EGFR-TKIs). They were initially developed for the treatment of non-small cell lung cancer (NSCLC) with central nervous system metastases (CNS). The main CYP450 enzymes that metabolize (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate and its salts in the human body are the CYP3A4 and CYP3A5 subtypes. Its efficacy increases with increasing dosage, but adverse reactions limit its further clinical application. These adverse reactions may be related to the formation of metabolites after action by CYP450 enzymes, such as its subtype CYP3A, with some metabolites being produced at a rate higher than the administered dose. Current research indicates that (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride can cross the blood-brain barrier at 100% concentration. However, the equally active metabolites generated during metabolism have a significantly lower ability to cross the blood-brain barrier than this drug, resulting in reduced intracranial efficacy. Furthermore, drug interactions may occur when different CYP450 enzyme inhibitors are combined with (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate or its salts. Therefore, further research is needed to explore a safe and reliable method of administration that can enhance the efficacy of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate and its salts. Summary of the Invention

[0003] This application is based on the inventor's discovery of the following problems and facts:

[0004] (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester or its salt is an effective central nervous system-penetrating EGFR inhibitor. Biphenylcyclooctyl lignan is a CYP450 enzyme inhibitor and also lowers serum alanine aminotransferase, thus playing a hepatoprotective role. The inventors studied (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester or its salt, alone or in combination with various CYP450 enzyme inhibitors, such as schisandrin, silymarin, and ketoconazole. They found that schisandrin significantly inhibited human liver microsomal cytochrome CYP450 enzymes compared to other inhibitors, and the degree of inhibition was moderate. Compared to (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester or its salt, it showed a more effective inhibitory effect. When used alone or in combination, biphenylcyclooctyl lignans reduces the metabolism of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl-2,4-dimethylpiperazine-1-carboxylate or its salts by inhibiting CYP450 enzymes, thus prolonging its retention time in the body and enhancing its efficacy. At the same time, it reduces the concentration or content of metabolites by inhibiting CYP450 cytochrome metabolic enzymes, thereby improving both efficacy and safety.

[0005] In a first aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: a compound of formula I or a salt thereof and biphenylcyclooctanene lignan. The pharmaceutical composition according to embodiments of the present invention can reduce the level of alanine aminotransferase in serum, protect the liver, and, compared with the use of the compound of formula I or a salt thereof alone, can effectively prolong the duration of action of the compound of formula I or a salt thereof in vivo, significantly reduce the concentration of metabolites of formula II during metabolism, increase the exposure of the compound of formula I or a salt thereof in vivo and the dose in brain tissue, and reduce adverse reactions caused by metabolites of formula II, thereby improving the efficacy and safety of the compound of formula I or a salt thereof, and thus effectively treating or preventing cancer.

[0006] According to embodiments of the present invention, the above-described pharmaceutical composition may further include at least one of the following additional technical features:

[0007] According to embodiments of the present invention, the salts of the compounds represented by Formula I include at least one of the following: sulfates, phosphates, citrates, tartrates, fumarates, benzoates, adipates, succinates, methanesulfonates, and maleates.

[0008] According to an embodiment of the present invention, the salt of the compound represented by Formula I is a hydrochloride salt.

[0009] According to an embodiment of the present invention, the salt of the compound represented by Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride.

[0010] According to embodiments of the present invention, the biphenylcyclooctanene lignan includes at least one of the following: schisandrin A, schisandrin A, schisandrin B, schisandrin A, schisandrin B, and schisandrol.

[0011] According to an embodiment of the present invention, the biphenylcyclooctanyl lignan is schisandrin A.

[0012] According to embodiments of the present invention, the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctylene lignan is (1:31) to (4:3). When the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctylene lignan is (1:31) to (4:3), the half-life of the compound or its salt represented by Formula I is prolonged, and the content of the metabolite represented by Formula II during metabolism is lower than that of the compound or its salt represented by Formula I alone, exhibiting strong antitumor activity and high safety. According to the description of the embodiments of this application, the dosage for mice and humans can be converted, and the addition of the two substances using the converted values ​​can effectively protect humans from or alleviate tumors.

[0013] According to embodiments of the present invention, the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctylene lignan is (1:10) to (5:6). When the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctylene lignan is (1:10) to (5:6), the half-life of the compound or its salt represented by Formula I is significantly prolonged, and the content of the metabolite represented by Formula II during metabolism is significantly lower than that of the compound or its salt represented by Formula I alone, exhibiting strong antitumor activity and high safety. According to the description of the embodiments of this application, the dosage for mice and humans can be converted, and the addition of the two substances using the converted values ​​can effectively protect humans from or alleviate tumors.

[0014] According to embodiments of the present invention, the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctanyl lignan is (7:50) to (4:5). When the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctanyl lignan is (7:50) to (4:5), the half-life of the compound or its salt represented by Formula I is significantly prolonged, and the content of the metabolite represented by Formula II during metabolism is significantly lower than that of the compound or its salt represented by Formula I alone. The pharmaceutical composition exhibits strong antitumor activity and high safety.

[0015] According to embodiments of the present invention, the mass ratio of the compound of Formula I or its salt to biphenylcyclooctanyl lignan is 3:20, 1:5, 81:250, or 81:125. When the mass ratio of the compound of Formula I or its salt to biphenylcyclooctanyl lignan is 3:20, 1:5, 81:250, or 81:125, the half-life of the compound of Formula I or its salt is significantly prolonged, and the content of the metabolite of Formula II during metabolism is significantly lower than that of the compound of Formula I or its salt alone. Therefore, the pharmaceutical composition exhibits strong antitumor activity and high safety.

[0016] According to embodiments of the present invention, the pharmaceutical composition is an oral formulation. The pharmaceutical composition may be an oral formulation, an injectable formulation, a pill, a sustained-release formulation, an implant, or an aerosol, and is not particularly limited thereto. According to specific embodiments of the present invention, when the pharmaceutical composition is an oral formulation, the single-dose form has a longer half-life, and the content of the metabolite shown in Formula II during metabolism is significantly lower than that of the compound shown in Formula I or its salt when used alone. This is beneficial for increasing the brain penetration dose of the compound shown in Formula I or its salt, further improving efficacy. Therefore, the pharmaceutical composition possesses stronger antitumor activity and higher safety.

[0017] According to some specific embodiments of the present invention, the pharmaceutical composition comprises a compound of Formula I provided herein, or a salt thereof, schisandrin A, and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc. Specific examples may be water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, etc., and one or more combinations thereof. In many cases, the pharmaceutical composition includes isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Of course, a pharmaceutically acceptable carrier may also include trace amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, to prolong the shelf life or potency of the antibody.

[0018] For example, the compounds of Formula I of the present invention, or their salts, and biphenylcyclooctanene lignans can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms, such as liquids, semi-solids, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of oral formulations, injection solutions, or infusion solutions. These pharmaceutical compositions can be administered orally, intravenously, by injection, or by intramuscular or subcutaneous injection.

[0019] In a second aspect, the present invention proposes a single-dose formulation. According to embodiments of the present invention, it comprises: a compound of Formula I or a salt thereof, and biphenylcyclooctanyl lignan. The inventors have discovered that biphenylcyclooctanyl lignan, such as schisandrin A, not only has hepatoprotective functions but also significantly inhibits hepatic microsomal cytochrome CYP450 enzymes. Furthermore, by inhibiting CYP450 enzymes, the metabolism of the compound of Formula I or its salt is reduced, prolonging its retention time in the body and enhancing efficacy. Simultaneously, the concentration or content of metabolites of Formula II produced by metabolic reactions in serum is reduced, increasing the exposure of the compound of Formula I or its salt in the body and the dose in brain tissue, and reducing adverse reactions caused by metabolites of Formula II. Therefore, the efficacy and safety of the compound of Formula I or its salt are both improved. Thus, the single-dose formulation possesses strong antitumor activity and high safety. According to the description of the embodiments of this application, by converting the dosage between mice and humans, using this ratio for conversion can effectively protect humans from or alleviate tumors.

[0020] According to embodiments of the present invention, the above-mentioned single dosage form may further include at least one of the following additional technical features:

[0021] According to an embodiment of the present invention, the mass ratio of the compound of Formula I or its salt to biphenylcyclooctyl lignan is (1:31) to (4:3).

[0022] According to an embodiment of the present invention, the mass ratio of the compound of Formula I or its salt to biphenylcyclooctyl lignan is (1:10) to (5:6).

[0023] According to an embodiment of the present invention, the mass ratio of the compound or its salt shown in Formula I to biphenylcyclooctanyl lignan is (7:50) to (4:5). When the mass ratio of the compound or its salt shown in Formula I to biphenylcyclooctanyl lignan is (7:50) to (4:5), the half-life of the single-dose formulation is significantly prolonged, and the content of the metabolite shown in Formula II during metabolism is significantly lower than that of the compound or its salt shown in Formula I alone, thereby increasing the brain penetration dose of the compound or its salt shown in Formula I and improving the efficacy. Therefore, the single-dose formulation has strong antitumor activity and high safety.

[0024] According to embodiments of the present invention, the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctanyl lignan is 3:20, 1:5, 81:250, or 81:125. When the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctanyl lignan is 3:20, 1:5, 81:250, or 81:125, the half-life of the single-dose formulation is significantly prolonged, and the content of the metabolite represented by Formula II during metabolism is significantly lower than that of the compound or its salt represented by Formula I alone, thereby increasing the brain penetration dose of the compound or its salt represented by Formula I and improving the efficacy. Therefore, the single-dose formulation possesses strong antitumor activity and high safety.

[0025] According to embodiments of the present invention, the single-dose formulation comprises 200-300 mg of the compound represented by Formula I or its salt. As described in the embodiments of this application, the above-mentioned single-dose formulation can be used 2, 3, or 4 times daily, preferably 2 times. Through conversion between mouse and human dosages, this dosage conversion demonstrates its effectiveness in protecting humans from or alleviating tumors. Those skilled in the art will understand that the specifications of the single-dose formulation are not particularly limited. For example, the specifications of the single-dose formulation can be set according to the characteristics of different target populations, such as according to the weight of the target population (e.g., 40 kg, 60 kg, 70 kg, 80 kg, 90 kg, 100 kg, etc.), or according to the mass ratio of the compound represented by Formula I or its salt to biphenylcyclooctyl lignan, such as 3:20, 1:6, 5:6, 4:5, 81:250, or 81:125, etc., or, alternatively, according to the characteristics of the target population and the mass ratio of the compound represented by Formula I or its salt to biphenylcyclooctyl lignan.

[0026] According to an embodiment of the present invention, the single-dose formulation is an oral preparation. The single-dose formulation can be an oral preparation, an injectable preparation, a pill, a sustained-release preparation, an implant, or an aerosol, and is not particularly limited thereto. According to a specific embodiment of the present invention, when the single-dose formulation is an oral preparation, the half-life of the single-dose formulation is longer, and the content of the metabolite shown in Formula II during metabolism is significantly lower than that of the compound shown in Formula I or its salt when used alone, thereby increasing the brain penetration dose of the compound shown in Formula I or its salt and improving efficacy. Therefore, the single-dose formulation possesses stronger anti-tumor ability and higher safety.

[0027] In a third aspect, the present invention proposes a drug combination. According to embodiments of the present invention, it comprises: a compound of Formula I or a salt thereof as a first active ingredient and biphenylcyclooctanene lignan as a second active ingredient. As previously mentioned, the inventors have discovered that schisandrin not only has hepatoprotective functions, but also enhances efficacy by inhibiting CYP450 enzymes, such as subtype CYP3A, thereby reducing the metabolism of the compound of Formula I or its salt, prolonging its retention time in the body, and simultaneously reducing the metabolites of Formula II produced by demethylation reactions in serum at the same time, increasing the exposure of the compound of Formula I or its salt in the body and the dose in brain tissue, and reducing adverse reactions caused by the metabolites of Formula II. This improves both the efficacy and safety of the compound of Formula I or its salt. Therefore, the drug combination according to embodiments of the present invention can significantly prolong the half-life and brain-penetrating dose of the compound of Formula I or its salt, effectively inhibit tumor growth, and has high safety.

[0028] According to embodiments of the present invention, the above-described drug combination may further include at least one of the following additional technical features:

[0029] According to embodiments of the present invention, the salts of the compounds represented by Formula I include at least one of the following: sulfates, phosphates, citrates, tartrates, fumarates, benzoates, adipates, succinates, methanesulfonates, and maleates.

[0030] According to an embodiment of the present invention, the salt of the compound represented by Formula I is a hydrochloride salt.

[0031] According to an embodiment of the present invention, the salt of the compound represented by Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride.

[0032] According to embodiments of the present invention, the biphenylcyclooctanene lignan includes at least one of the following: schisandrin A, schisandrin A, schisandrin B, schisandrin A, schisandrin B, and schisandrol.

[0033] According to an embodiment of the present invention, the biphenylcyclooctanyl lignan is schisandrin A.

[0034] According to embodiments of the present invention, the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctylene lignan is (1:31) to (4:3). When the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctylene lignan is (1:31) to (4:3), the half-life of the compound or its salt represented by Formula I is prolonged, and the content of the metabolite represented by Formula II during metabolism is lower than that of the compound or its salt represented by Formula I alone, exhibiting strong antitumor activity and high safety. According to the description of the embodiments of this application, the dosage for mice and humans can be converted, and the addition of the two substances using the converted values ​​can effectively protect humans from or alleviate tumors.

[0035] According to embodiments of the present invention, the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctanyl lignan is (1:10) to (5:6). When the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctanyl lignan is (1:10) to (5:6), the half-life of the compound or its salt represented by Formula I is significantly prolonged, and the content of the metabolite represented by Formula II during metabolism is significantly lower than that of the compound or its salt represented by Formula I alone, thereby increasing the brain penetration dose of the compound or its salt represented by Formula I, improving efficacy, and the compound or its salt represented by Formula I after drug combination possesses stronger antitumor ability and higher safety. According to the description of the embodiments of this application, by converting the dosage for mice and humans, using this ratio for conversion can effectively protect humans from or alleviate tumors.

[0036] According to embodiments of the present invention, the mass ratio of the compound or its salt shown in Formula I to biphenylcyclooctanyl lignan is (7:50) to (4:5). When the mass ratio of the compound or its salt shown in Formula I to biphenylcyclooctanyl lignan is (7:50) to (4:5), the half-life of the compound or its salt shown in Formula I is significantly prolonged, and the content of the metabolite shown in Formula II is significantly lower than that of the compound or its salt shown in Formula I alone, thereby increasing the brain penetration dose of the compound or its salt shown in Formula I, improving efficacy, and the compound or its salt shown in Formula I after drug combination has stronger antitumor ability and higher safety.

[0037] According to embodiments of the present invention, the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctanyl lignan is 3:20, 1:5, 81:250, or 81:125. When the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctanyl lignan is 3:20, 1:5, 81:250, or 81:125, the half-life of the compound or its salt represented by Formula I is significantly prolonged, and the content of the metabolite represented by Formula II is significantly lower than that of the compound or its salt represented by Formula I alone, thereby increasing the brain penetration dose of the compound or its salt represented by Formula I, improving efficacy, and the compound or its salt represented by Formula I exhibits stronger antitumor activity and higher safety after drug combination.

[0038] According to embodiments of the present invention, the compound represented by Formula I or its salt and the biphenylcyclooctyl lignan are prepared together or separately.

[0039] According to embodiments of the present invention, the compound represented by Formula I or its salt and the biphenylcyclooctyl lignan may be used simultaneously or separately.

[0040] It should be noted that the drug combination includes combinations that are separate in time and / or space, provided that the compound represented by Formula I or its salt can work synergistically with biphenylcyclooctanene lignans to achieve the objectives of the present invention. For example, the components contained in the drug combination may be administered to the subject or test sample as a whole, or separately. When the components contained in the drug combination are administered separately to the subject or test sample, the individual components may be administered simultaneously or sequentially to the subject or test sample.

[0041] According to embodiments of the present invention, the biphenylcyclooctyl lignan is administered prior to the compound represented by Formula I or its salt. Those skilled in the art will understand that the order of administration of the biphenylcyclooctyl lignan and the compound represented by Formula I or its salt is not particularly limited, as long as the biphenylcyclooctyl lignan can exert its effect within the metabolic cycle of the compound represented by Formula I or its salt. For example, the administration of the biphenylcyclooctyl lignan prior to the compound represented by Formula I or its salt, or the administration of the biphenylcyclooctyl lignan subsequently to the compound represented by Formula I or its salt, or the simultaneous administration of the biphenylcyclooctyl lignan and the compound represented by Formula I or its salt, are all within the scope of protection of this application. According to some specific embodiments of the present invention, when the biphenylcyclooctanene lignan is administered before the (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid hydrochloride, the half-life of the compound shown in Formula I or its salt can be significantly prolonged, and the content of the metabolite shown in Formula II produced during its metabolism is significantly reduced, resulting in a higher brain penetration rate of the compound shown in Formula I or its salt, an increased brain penetration dose, and further improved efficacy. The drug combination has good antitumor effects and safety.

[0042] According to an embodiment of the present invention, the second active ingredient is an oral dosage form. The second active ingredient can be an oral formulation, an injectable formulation, a pill, a sustained-release formulation, an implant, or an aerosol, and is not particularly limited thereto. According to a specific embodiment of the present invention, when the second active ingredient is an oral formulation, the single dosage form has a longer half-life, and the content of the metabolite shown in Formula II during metabolism is significantly lower than that of the compound shown in Formula I or its salt when used alone. The compound shown in Formula I or its salt possesses stronger antitumor activity and higher safety.

[0043] In a fourth aspect, the present invention provides a medicine box. According to an embodiment of the present invention, the medicine box comprises: a compound of Formula I or a salt thereof as a first active ingredient and biphenylcyclooctanyl lignan as a second active ingredient. As previously stated, the inventors have discovered that biphenylcyclooctanyl lignan enhances efficacy by inhibiting CYP450 enzymes, such as its subtype CYP3A enzyme, thereby reducing the metabolism of the compound of Formula I or its salt thereof, prolonging its retention time in the body, and simultaneously reducing the concentration or content of the metabolite of Formula II in serum at the same time, increasing the exposure of the compound of Formula I or its salt thereof in the body and the dose in brain tissue, and reducing the adverse reactions caused by the metabolite of Formula II. This results in improved efficacy and safety of the compound of Formula I or its salt thereof, effectively inhibiting tumors and exhibiting high safety.

[0044] According to embodiments of the present invention, the above-mentioned medicine box may further include at least one of the following additional technical features:

[0045] According to embodiments of the present invention, the salts of the compounds represented by Formula I include at least one of the following: sulfates, phosphates, citrates, tartrates, fumarates, benzoates, adipates, succinates, methanesulfonates, and maleates.

[0046] According to an embodiment of the present invention, the salt of the compound represented by Formula I is a hydrochloride salt.

[0047] According to an embodiment of the present invention, the salt of the compound represented by Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride.

[0048] According to embodiments of the present invention, the biphenylcyclooctanene lignan includes at least one of the following: schisandrin A, schisandrin A, schisandrin B, schisandrin A, schisandrin B, and schisandrol.

[0049] According to an embodiment of the present invention, the biphenylcyclooctanyl lignan is schisandrin A.

[0050] According to embodiments of the present invention, the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctylene lignan is (1:31) to (4:3). When the mass ratio of the compound or its salt represented by Formula I to biphenylcyclooctylene lignan is (1:31) to (4:3), the half-life of the compound or its salt represented by Formula I is prolonged, and the content of the metabolite represented by Formula II during metabolism is lower than that of the compound or its salt represented by Formula I alone, exhibiting strong antitumor activity and high safety. According to the description of the embodiments of this application, the dosage for mice and humans can be converted, and the addition of the two substances using the converted values ​​can effectively protect humans from or alleviate tumors.

[0051] According to embodiments of the present invention, the mass ratio of the compound or its salt shown in Formula I to biphenylcyclooctylene lignan is (1:10) to (5:6). When the mass ratio of the compound or its salt shown in Formula I to biphenylcyclooctylene lignan is (1:10) to (5:6), the half-life of the compound or its salt shown in Formula I is significantly prolonged, and the content of the metabolite shown in Formula II during metabolism is significantly lower than that of the compound or its salt shown in Formula I alone, exhibiting strong antitumor activity and high safety. According to the description of the embodiments of this application, the dosage for mice and humans can be converted, and the addition of the two substances using the converted values ​​can effectively protect humans from or alleviate tumors.

[0052] According to an embodiment of the present invention, the mass ratio of the compound or its salt shown in Formula I to biphenylcyclooctylene lignan is (7:50) to (4:5). When the mass ratio of the compound or its salt shown in Formula I to biphenylcyclooctylene lignan is (7:50) to (4:5), the half-life of the compound or its salt shown in Formula I is significantly prolonged, and the content of the metabolite shown in Formula II during metabolism is significantly lower than that of the compound or its salt shown in Formula I alone. The drug kit possesses strong antitumor activity and high safety.

[0053] According to embodiments of the present invention, the mass ratio of the compound of Formula I or its salt to biphenylcyclooctanyl lignan is 3:20, 1:5, 81:250, or 81:125. When the mass ratio of the compound of Formula I or its salt to biphenylcyclooctanyl lignan is 3:20, 1:5, 81:250, or 81:125, the half-life of the compound of Formula I or its salt is significantly prolonged, and the content of the metabolite of Formula II during metabolism is significantly lower than that of the compound of Formula I or its salt alone. The pharmaceutical composition thus possesses strong antitumor activity and high safety.

[0054] According to embodiments of the present invention, the compound represented by Formula I or its salt and the biphenylcyclooctadiene lignan can be prepared together or separately. Those skilled in the art will understand that the preparation method of the compound represented by Formula I or its salt and the biphenylcyclooctadiene lignan is not particularly limited; they can be prepared separately or together.

[0055] According to embodiments of the present invention, the compound of Formula I or its salt and the biphenylcyclooctyl lignan are used simultaneously or separately. It should be noted that the method includes the separate use of the compound of Formula I or its salt and the biphenylcyclooctyl lignan in time and / or space, provided that the compound of Formula I or its salt and the biphenylcyclooctyl lignan salt can work together to achieve the purpose of the present invention. For example, the compound of Formula I or its salt and the biphenylcyclooctyl lignan can be applied as a whole to the subject or the test sample, or applied separately to the subject or the test sample. When the compound of Formula I or its salt and the biphenylcyclooctyl lignan are applied separately to the subject or the test sample, the individual components can be applied simultaneously or sequentially to the subject or the test sample.

[0056] According to embodiments of the present invention, the biphenylcyclooctyl lignan is administered prior to the compound represented by Formula I or its salt. Those skilled in the art will understand that the order of administration of the biphenylcyclooctyl lignan and the compound represented by Formula I or its salt is not particularly limited, as long as the biphenylcyclooctyl lignan can exert its effect within the metabolic cycle of the compound represented by Formula I or its salt. For example, the biphenylcyclooctyl lignan may be administered prior to the compound represented by Formula I or its salt, or subsequently after the compound represented by Formula I or its salt, or simultaneously with the compound represented by Formula I or its salt; all of these are within the scope of protection of this application. According to some specific embodiments of the present invention, when the biphenylcyclooctyl lignan is administered prior to the compound represented by Formula I or its salt, the half-life of the compound represented by Formula I or its salt can be significantly prolonged, and the content of the metabolite represented by Formula II produced during its metabolism is significantly reduced, exhibiting good antitumor effects and safety.

[0057] According to an embodiment of the present invention, the second active ingredient is an oral dosage form. The second active ingredient can be an oral formulation, an injectable formulation, a pill, a sustained-release formulation, an implant, or an aerosol, and is not particularly limited thereto. According to a specific embodiment of the present invention, when the second active ingredient is an oral formulation, the half-life of the drug cartridge is longer, and the content of the metabolite shown in Formula II during metabolism is significantly lower than that of the compound shown in Formula I or its salt when used alone. The drug cartridge possesses strong anti-tumor activity and high safety.

[0058] In a fifth aspect, the present invention provides a method for administering a compound of Formula I or a salt thereof. According to an embodiment of the invention, the method comprises administering the compound of Formula I or a salt thereof in combination with biphenylcyclooctanene lignan.

[0059] According to embodiments of the present invention, the above method further includes at least one of the following additional technical features:

[0060] According to embodiments of the present invention, the salts of the compounds represented by Formula I include at least one of the following: sulfates, phosphates, citrates, tartrates, fumarates, benzoates, adipates, succinates, methanesulfonates, and maleates.

[0061] According to an embodiment of the present invention, the salt of the compound represented by Formula I is a hydrochloride salt.

[0062] According to an embodiment of the present invention, the salt of the compound represented by Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride.

[0063] According to embodiments of the present invention, the biphenylcyclooctanene lignan includes at least one of the following: schisandrin A, schisandrin A, schisandrin B, schisandrin A, schisandrin B, and schisandrol.

[0064] According to an embodiment of the present invention, the biphenylcyclooctanyl lignan is schisandrin A.

[0065] According to embodiments of the present invention, the combined drug administration includes simultaneously or separately administering the compound of Formula I or its salt with biphenylcyclooctanene lignan.

[0066] According to embodiments of the present invention, the mass ratio of the compound of Formula I or its salt to biphenylcyclooctanene lignan is (1:31) to (4:3). As described in the embodiments of this application, by converting the dosage for mice to humans, this ratio can effectively protect humans from or alleviate tumors.

[0067] According to an embodiment of the present invention, during the medication process, the mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctanene lignan is (1:10) to (5:6).

[0068] According to an embodiment of the present invention, during the medication process, the mass ratio of the compound of formula I or its salt to biphenylcyclooctanene lignan is (7:50) to (4:5).

[0069] According to embodiments of the present invention, during the medication process, the mass ratio of the compound represented by Formula I or its salt to biphenylcyclooctanene lignan is 3:20, 1:5, 81:250 or 81:125.

[0070] According to embodiments of the present invention, the biphenylcyclooctyl lignan is administered prior to the compound represented by Formula I or its salt. Those skilled in the art will understand that the order of administration of the biphenylcyclooctyl lignan and the compound represented by Formula I or its salt is not particularly limited, as long as the biphenylcyclooctyl lignan can exert its effect within the metabolic cycle of the compound represented by Formula I or its salt. For example, the administration of the biphenylcyclooctyl lignan prior to the compound represented by Formula I or its salt, or the administration of the biphenylcyclooctyl lignan subsequently to the compound represented by Formula I or its salt, or the simultaneous administration of the biphenylcyclooctyl lignan and the compound represented by Formula I or its salt, are all within the scope of protection of this application.

[0071] According to embodiments of the present invention, the biphenylcyclooctanyl lignan is administered orally. Those skilled in the art will understand that the biphenylcyclooctanyl lignan can be an oral formulation, an injectable formulation, a pill, a sustained-release formulation, an implant, or an aerosol, and is not particularly limited thereto. According to specific embodiments of the present invention, when the biphenylcyclooctanyl lignan is an oral formulation, the compound represented by Formula I or its salt has a longer half-life, and the content of metabolite 1 during metabolism is significantly lower than when using the compound represented by Formula I or its salt alone. This method of administration has stronger antitumor activity and higher safety.

[0072] According to an embodiment of the present invention, the dosage of the biphenylcyclooctanene lignan is 0-25 mg / kg.

[0073] According to an embodiment of the present invention, the dosage of the biphenylcyclooctanyl lignan is 5–25 mg / kg. According to a specific embodiment of the present invention, when the dosage of the biphenylcyclooctanyl lignan is 5–25 mg / kg, the half-life of the compound represented by Formula I or its salt can be significantly prolonged, the content of the metabolite represented by Formula II produced during its metabolism is significantly reduced, the exposure of the compound represented by Formula I or its salt in vivo and the dose in brain tissue are increased, and the adverse reactions caused by the metabolite represented by Formula II are reduced. This improves both the efficacy and safety of the compound represented by Formula I or its salt, and the administration method can more effectively inhibit tumor growth and has higher safety.

[0074] In a sixth aspect, the present invention provides a method for prolonging the half-life of a compound of Formula I or a salt thereof. According to embodiments of the invention, the method comprises co-administering the compound of Formula I or a salt thereof with biphenylcyclooctyl lignan. As previously mentioned, the inventors have found that biphenylcyclooctyl lignan enhances therapeutic efficacy by inhibiting the metabolism of the compound of Formula I or a salt thereof by inhibiting CYP450 enzymes, such as its subtype CYP3A enzyme, thereby prolonging its retention time in vivo. Therefore, the method according to embodiments of the present invention can effectively prolong the half-life of the compound of Formula I or a salt thereof.

[0075] According to embodiments of the present invention, the above method further includes at least one of the following additional technical features:

[0076] According to embodiments of the present invention, the salts of the compounds represented by Formula I include at least one of the following: sulfates, phosphates, citrates, tartrates, fumarates, benzoates, adipates, succinates, methanesulfonates, and maleates.

[0077] According to an embodiment of the present invention, the salt of the compound represented by Formula I is a hydrochloride salt.

[0078] According to an embodiment of the present invention, the salt of the compound represented by Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride.

[0079] According to embodiments of the present invention, the biphenylcyclooctanene lignan includes at least one of the following: schisandrin A, schisandrin A, schisandrin B, schisandrin A, schisandrin B, and schisandrol.

[0080] According to an embodiment of the present invention, the biphenylcyclooctanyl lignan is schisandrin A.

[0081] According to embodiments of the present invention, the combined drug administration includes simultaneously or separately administering the compound of Formula I or a salt thereof with biphenylcyclooctanene lignan. The manner of combined drug administration will not be elaborated further, but can be referred to the combined drug administration method in the administration method of the compound of Formula I or a salt thereof described in the fifth aspect.

[0082] According to embodiments of the present invention, the mass ratio of the compound of Formula I or its salt to biphenylcyclooctanene lignan is (1:31) to (4:3). As described in the embodiments of this application, by converting the dosage for mice to humans, this ratio can effectively prolong the duration of drug efficacy in protecting humans from or alleviating tumors.

[0083] According to an embodiment of the present invention, during the medication process, the mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctanene lignan is (1:10) to (5:6).

[0084] According to an embodiment of the present invention, during the medication process, the mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctanene lignan is (7:10) to (4:5).

[0085] According to embodiments of the present invention, during the medication process, the mass ratio of the compound represented by Formula I or its salt to biphenylcyclooctanene lignan is 3:20, 1:5, 81:250 or 81:125.

[0086] According to embodiments of the present invention, the biphenylcyclooctyl lignan is administered prior to the compound represented by Formula I or its salt. As mentioned above, the order of administration of the biphenylcyclooctyl lignan and the compound represented by Formula I or its salt is not particularly limited, as long as the biphenylcyclooctyl lignan can exert its effect within the metabolic cycle of the compound represented by Formula I or its salt. For example, the biphenylcyclooctyl lignan may be administered prior to the compound represented by Formula I or its salt, or the biphenylcyclooctyl lignan may be administered subsequently to the compound represented by Formula I or its salt, or the biphenylcyclooctyl lignan may be administered simultaneously with the compound represented by Formula I or its salt, all of which are within the scope of protection of this application.

[0087] According to an embodiment of the present invention, the biphenylcyclooctanil lignan is administered orally.

[0088] According to an embodiment of the present invention, the dosage of the biphenylcyclooctanene lignan is 0-25 mg / kg.

[0089] According to an embodiment of the present invention, the dosage of the biphenylcyclooctanyl lignan is 5–25 mg / kg. According to a specific embodiment of the present invention, when the dosage of the biphenylcyclooctanyl lignan is 5–25 mg / kg, the half-life of the compound represented by Formula I or its salt can be significantly prolonged, and the content of the metabolite represented by Formula II produced during its metabolism is significantly reduced. This method can more effectively prolong the half-life of the compound represented by Formula I or its salt and improve its brain penetration rate, thereby enhancing efficacy.

[0090] In a seventh aspect, the present invention provides a method for reducing the production of a compound of Formula I or a salt thereof to form a compound of Formula II. According to an embodiment of the present invention, the method includes co-administering the compound of Formula I or a salt thereof with biphenylcyclooctanyl lignan. As previously described, the inventors have found that biphenylcyclooctanyl lignan inhibits CYP450 enzymes, such as its subtype CYP3A enzyme, thereby reducing the metabolism of the compound of Formula I or a salt thereof, and decreasing the concentration or content of the metabolite of Formula II in serum, increasing the exposure of the compound of Formula I or a salt thereof in vivo and the dose in brain tissue, and reducing adverse reactions caused by the metabolite of Formula II, thus improving both the efficacy and safety of the compound of Formula I or a salt thereof.

[0091] According to embodiments of the present invention, the above method further includes at least one of the following additional technical features:

[0092] According to embodiments of the present invention, the salts of the compounds represented by Formula I include at least one of the following: sulfates, phosphates, citrates, tartrates, fumarates, benzoates, adipates, succinates, methanesulfonates, and maleates.

[0093] According to an embodiment of the present invention, the salt of the compound represented by Formula I is a hydrochloride salt.

[0094] According to an embodiment of the present invention, the salt of the compound represented by Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride.

[0095] According to embodiments of the present invention, the biphenylcyclooctanene lignan includes at least one of the following: schisandrin A, schisandrin A, schisandrin B, schisandrin A, schisandrin B, and schisandrol.

[0096] According to an embodiment of the present invention, the biphenylcyclooctanyl lignan is schisandrin A.

[0097] According to embodiments of the present invention, the combined drug administration includes simultaneously or separately administering the compound of Formula I or its salt with biphenylcyclooctanene lignan.

[0098] According to an embodiment of the present invention, the mass ratio of the compound of Formula I or its salt to biphenylcyclooctyl lignan is (1:31) to (4:3).

[0099] According to an embodiment of the present invention, during the medication process, the mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctanene lignan is (1:10) to (5:6).

[0100] According to an embodiment of the present invention, during the medication process, the mass ratio of the compound of formula I or its salt to biphenylcyclooctanene lignan is (7:50) to (4:5).

[0101] According to embodiments of the present invention, during the medication process, the mass ratio of the compound represented by Formula I or its salt to biphenylcyclooctanene lignan is 3:20, 1:5, 81:250 or 81:125.

[0102] According to embodiments of the present invention, the biphenylcyclooctyl lignan is administered prior to the compound represented by Formula I or its salt. As mentioned above, the order of administration of the biphenylcyclooctyl lignan and the compound represented by Formula I or its salt is not particularly limited, as long as the biphenylcyclooctyl lignan can exert its effect within the metabolic cycle of the compound represented by Formula I or its salt. For example, the biphenylcyclooctyl lignan may be administered prior to the compound represented by Formula I or its salt, or the biphenylcyclooctyl lignan may be administered subsequently to the compound represented by Formula I or its salt, or the biphenylcyclooctyl lignan may be administered simultaneously with the compound represented by Formula I or its salt, all of which are within the scope of protection of this application.

[0103] According to an embodiment of the present invention, the biphenylcyclooctanil lignan is administered orally.

[0104] According to an embodiment of the present invention, the dosage of the biphenylcyclooctanene lignan is 0-25 mg / kg.

[0105] According to an embodiment of the present invention, the dosage of the biphenylcyclooctanene lignan is 5–25 mg / kg. According to a specific embodiment of the present invention, when the dosage of the schisandrin A is 5–25 mg / kg, the concentration or content of the metabolite shown in Formula II produced in serum can be more effectively reduced during the metabolism of the compound shown in Formula I or its salt. Therefore, the method according to an embodiment of the present invention can more significantly reduce the content of the metabolite shown in Formula II during the metabolism of the compound shown in Formula I or its salt. Detailed Implementation

[0106] The embodiments of the present invention are described in detail below. These embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0107] In the process of describing this invention, the terms used herein have been explained and described. These explanations and descriptions are only for the purpose of facilitating the understanding of the solution and should not be regarded as a limitation on the protection of this invention.

[0108] In this application, "the compound shown in Formula I" refers to compounds with the following structures:

[0109]

[0110] In this application, the molecular formulas of "the hydrochloride salt of the compound shown in Formula I", "(R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride", "test drug", and "original drug" are C 22 H 23 The chemical structure of ClFN5O3 is:

[0111]

[0112] In this application, the molecular formula of "metabolite shown in Formula II" and "metabolite 1" is C 21 H 21 ClFN5O3 has the following chemical structure:

[0113]

[0114] In this application, "schisandrin A" refers to the compound with the molecular formula C. 24 H 32 O6, a substance with a molecular weight of 416.507, has the following chemical structure:

[0115]

[0116] The molecular formula of "Schisandra ester A" is C 30 H 32 O9, with a molecular weight of 536.577, has the following chemical structure:

[0117]

[0118] The molecular formula of "Schisandra chinensis alcohol" is C 23 H 28 O7, with a molecular weight of 416.47, has the following chemical structure:

[0119]

[0120] The molecular formula of "Schisandrin A" is C 24 H 32 O7, with a molecular weight of 432.51, has the following chemical structure:

[0121]

[0122] The molecular formula of schisandrin B is C 23 H 28 O6, with a molecular weight of 400.46, has the following chemical structure:

[0123]

[0124] The molecular formula of schisandrol is C2 23 H 30 O6, with a molecular weight of 402.48, has the following chemical structure:

[0125]

[0126] In this application, "single dosage form" refers to a dosage form in which a single preparation is used up in a single dose.

[0127] In this application, "drug combination" refers to the simultaneous or sequential use of two or more drugs to achieve a therapeutic purpose, with the main goal of increasing the efficacy of the drugs or reducing their toxic side effects.

[0128] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0129] Example 1: Detection of the inhibitory ability of CYP450 enzyme inhibitors on CYP450 enzymes

[0130] In this application, the inventors have studied the use of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride alone and in combination with CYP450 enzyme inhibitors. The chemical structure of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride is as follows:

[0131] The specific experimental procedures are as follows:

[0132] The experimental system used human liver microsomes, with four groups of liver microsomes in each group: 1) (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride group (test drug used alone); 2) (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride group combined with schisandrin (WWZ) group (…). The test drug + WWZ group); 3)(R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride combined with silybin group (test drug + silybin group); 4)(R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride combined with ketoconazole group (test drug + ketoconazole group). The specific experimental procedures are as follows:

[0133] Accurately measure 1 mg / mL of the stock solution (Stock0009) of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride and dilute with PBS buffer to a standard working solution with a molar concentration of 10 μM. Accurately measure 1 mg / mL of the stock solution (Stock0009) of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride and dilute with PBS buffer containing 5 μM each of schisandrin, silymarin, or ketoconazole to a standard working solution with a molar concentration of 20 μM. This yields a 10 μM mixed working solution containing 5 μM of various CYP450 enzyme inhibitors, 2. Weigh approximately 7 mg of NADPH (reduced form) and dilute with PBS buffer to a working solution with a molar concentration of 10 mM. The initial concentration of liver microsomes (HLM) was 20 mg / mL, which was diluted with PBS buffer to a working solution with a molar concentration of 2 mg / mL. The working solutions were prepared according to the measurements in Table 1.

[0134] Table 1: Sample preparation method for liver microsomal incubation system

[0135]

[0136] Mix the four test working solutions, HLM, and PBS buffer solution in triplicate in the incubation tubes according to the volumes shown in the table, and preheat at 37°C for 5 minutes. Simultaneously, preheat 10 mM NADPH at 37°C. After thorough preheating, add 30 μL of NADPH to the liver microsome incubation tube, gently agitate the tube, and begin incubation. At 2 min, 15 min, 1 h, 2 h, and 4 h of incubation, add 160 μL of acetonitrile solution (propranolol 20 ng / mL) to each 40 μL incubation tube. After shaking, centrifuge for 10 min (20-200 g), and use the supernatant for LC / MS / MS analysis.

[0137] The experimental results are shown in Table 2. By comparing the clearance effect of drug (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride alone and in combination with various CYP450 enzyme inhibitors, the inventors found that schisandrin A has a moderate degree of inhibition on CYP450 enzymes compared with other high and low strength CYP450 enzyme inhibitors. In this study, the moderate-strength inhibitor WWZ was selected for further research.

[0138] Table 2:

[0139] Test drug 12.85 Test drug + WWZ 7.08 Test drug + silymarin 10.36 Test drug + ketoconazole 1.54

[0140] Example 2: In vitro metabolic stability study of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride in liver microparticles.

[0141] Based on Example 1, this experiment mainly investigated the differences in metabolic stability of the original drug (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride in a liver microsome incubation system, and the changes in metabolic stability after co-administration with the inhibitor WWZ. The experimental system used human liver microsomes, with three groups of liver microsomes in each group: 1) (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride group (test drug alone); 2) (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride group combined with WWZ (test drug + WWZ group); 3) positive control drug (verapamil, VPL) group. The specific experimental procedures are as follows:

[0142] Accurately measure 1 mg / mL of the stock solution (Stock0009) of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride and dilute with PBS buffer to a 10 μM standard working solution. Accurately measure 1 mg / mL of the stock solution (Stock0009) of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride and dilute with PBS buffer containing 5 μM schisandrin to a 20 μM standard working solution, thus obtaining a 10 μM mixed working solution containing 5 μM schisandrin. Measure 100 μL of the stock solution (StockVPL) of verapamil (1 mg / mL) and dilute with PBS buffer to a 10 μM working solution as a positive control. Weigh approximately 7 mg of NADPH (reduced form) and dilute with PBS buffer to a working solution with a molar concentration of 10 mM. The initial concentration of liver microsomes (HLM) is 20 mg / mL, which is then diluted with PBS buffer to a working solution with a molar concentration of 2 mg / mL. Prepare the working solutions according to the quantities specified in Table 3.

[0143] Table 3: Sample preparation method for liver microsome incubation system

[0144]

[0145] Mix the three test working solutions, HLM, and PBS buffer solution in triplicate in the incubation tubes according to the volumes shown in the table, and preheat at 37°C for 5 minutes. Simultaneously, preheat 10 mM NADPH at 37°C. After thorough preheating, add 30 μL of NADPH to the liver microsome incubation tube, gently agitate the tube, and begin incubation. At 2 min, 15 min, 1 h, 2 h, and 4 h of incubation, add 160 μL of acetonitrile solution (propranolol 20 ng / mL) to each 40 μL incubation tube. After shaking, centrifuge for 10 min (20-200 g), and use the supernatant for LC / MS / MS analysis.

[0146] Verapamil was used as a positive control (sampling times were 30 min and 2 h at the start of incubation), and verapamil without NADPH (replaced with PBS) was used as a negative control. The drug-free group (replaced with PBS) served as a blank control.

[0147] The in vitro metabolic stability of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride in human liver microsomes is shown in Table 4. Verapamil, used as a positive control, showed 10% of the original drug remaining after 2 hours of incubation, indicating normal enzyme metabolic activity in this batch of incubation system. Metabolism occurred in human liver microsomes after incubation with the test drug, and the concentration of the original drug decreased with increasing incubation time. Simultaneously, the metabolic rate of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride was slowed after co-administration with WWZ.

[0148] Table 4:

[0149]

[0150] Example 3: A study of the metabolic kinetics of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride in SD rats after combined use with schisandrin A.

[0151] This example is based on Examples 1 and 2, and mainly examines the metabolic kinetics of the test drug ((R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride) in SD rats after combined administration with schisandrin A. Nine healthy male rats aged 6-8 weeks, weighing 220-300g, were divided into three groups: a group receiving the test drug (5 mg / kg) via gavage; a group receiving 25 mg / kg of schisandrin A via gavage followed by 5 mg / kg of the test drug (A0009+WWZ(PO)); and a group receiving 10 mg / kg of schisandrin A via intravenous injection followed by 5 mg / kg of the test drug via gavage (A0009+WWZ(IV)).

[0152] Whole blood was collected from the three groups of rats mentioned above at the following time points: before drug administration, and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 36 h after drug administration. The blood collection site and volume were as follows: approximately 0.3 mL of blood was collected from the posterior ocular venous plexus. The obtained whole blood samples were centrifuged (2000 g, 4℃) for 10 min, and the supernatant plasma was collected. The plasma samples were treated using the organic solvent protein precipitation method. 100 μL of plasma was added to 400 μL of internal standard acetonitrile solution (propranolol, 20 ng / mL), vortexed, and centrifuged for 10 min (20200 g, 4℃). The supernatant was then analyzed by LC / MS / MS. The Winnonlin pharmacokinetic program was used to analyze the measured data and calculate the main pharmacokinetic parameters.

[0153] Table 5 shows the changes in plasma drug concentration-time for the test drug in each group of animals, and Tables 6 and 7 show the main pharmacokinetic parameters. The combined in vivo metabolism results indicate that the in vivo PK behavior of the test drug compound was affected after co-administration with WWZ. This effect is related to the administration route of WWZ; intravenous co-administration did not change the clearance rate of the test drug, while oral co-administration increased its bioavailability. Therefore, the inventors hypothesize that the drug-drug interaction of co-administered WWZ cannot be simply explained by the inhibitory effect of WWZ on hepatic metabolic enzymes, but is mainly manifested in the absorption phase. WWZ mainly acts on the intestine and liver in the absorption phase, improving bioavailability by increasing the absorption of the test drug or reducing enterohepatic first-pass metabolism. After oral co-administration with WWZ, the exposure AUCinf and half-life t of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid hydrochloride were... 1 / 2 The average h value was significantly higher than that of the group using (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride alone.

[0154] Table 5:

[0155]

[0156] Table 6: Pharmacokinetic parameters of the test drugs in monotherapy and combined WWZ (gavage, intravenous) trials.

[0157]

[0158] Example 4: Metabolic transformation of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride in SD rats after co-administration with WWZ.

[0159] To further observe the pharmacokinetic characteristics of metabolite 1 (the compound shown in Formula II) in vivo after administration of WWZ, this part of the study used plasma samples from SD rats obtained in Example 3 to detect the concentration-time changes of metabolite 1 and calculate pharmacokinetic parameters. The specific experimental procedures are as described in Example 2.

[0160]

[0161] The concentration-time changes of metabolite 1 in plasma of SD rats in each group are shown in Table 7. Tables 7 and 8 show that the metabolic formation pattern of metabolite 1 was altered after combined administration of WWZ. Regardless of whether WWZ was administered orally or intravenously, the concentration of metabolite 1 was lower in the group receiving the test drug alone within 8 hours after administration, indicating that combined administration of WWZ can effectively inhibit the formation of metabolite 1. In the group receiving the test drug alone, the blood exposure level (Cmax) of metabolite 1 reached as high as 261 ng / mL at 4 hours after administration. After oral or intravenous administration of WWZ, the blood exposure level of metabolite 1 was first reduced, with Cmax at 190 ng / mL and 118 ng / mL, respectively; secondly, the time to peak concentration of metabolite 1 was prolonged, reaching Cmax at 8 hours and 12 hours after administration, respectively. The data on the exposure level and time to peak concentration of metabolite 1 indicate that combined administration of WWZ reduces the production of metabolite 1.

[0162] Table 7: Plasma concentrations of metabolite 1 in SD rat plasma samples

[0163]

[0164]

[0165] Table 8:

[0166]

[0167] Example 5: Growth inhibitory effect of (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride in combination with WWZ on human lung cancer PC9 xenografts in nude mice.

[0168] This embodiment, based on Examples 1-4, continues the study of the test drug and WWZ. PC9 tumor tissue was extracted under aseptic conditions, cut into 2.0mm × 2.0mm × 2.0mm pieces, and evenly inoculated subcutaneously into the left axillary dorsal region of male BALB / cAnSlacNifdc-nu mice weighing 16.0-18.0g. The day of inoculation was recorded as D0. When the tumor volume reached 200.0mm... 3At approximately 6:00 AM (D6), tumors were randomly grouped according to their volume into two groups: a solvent control group (25%) and a control group (25%). Seven groups were established, with 10 animals in each group. The treatment consisted of PEG400 + pH 5.5 water, solvent control (pH 5.5 water), WWZ (25.0 mg / kg), test drug [(R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride] (8.1 mg / kg), test drug [(R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride] (16.2 mg / kg), WWZ (25.0 mg / kg) + test drug (8.1 mg / kg), and WWZ (25.0 mg / kg) + test drug (16.2 mg / kg). On the day of grouping, all animals were administered the drug via gavage once daily for 28 days, with 28 administrations in total.

[0169] Dosage Instructions: Studies have shown that the tested drug, calculated based on free base, had good antitumor effects in in vivo antitumor studies in mouse xenograft tumor models, with doses of 7.5 mg / kg and 15 mg / kg. This dose was continued in this study. According to CoA, the free base content of this batch of compound (batch number: CPo121798-04-05-02-34-01-RS) was 91.85%, the free base molecular weight was 459.9, and the hydrochloride molecular weight was 496.4. Based on the hydrochloride content in CoA, the mouse doses used in this study were 8.1 mg / kg and 16.2 mg / kg, respectively. These pharmacodynamic doses, converted to human doses based on body surface area, are 0.7 mg / kg and 1.4 mg / kg. WWZ Dosage Instructions: Currently, WWZ is clinically used in humans under the name Wuzhi Capsules (Traditional Chinese Medicine), and its formulation is capsules, each containing 11.25 mg of WWZ. The clinical dosage is two tablets each time, three times a day, with a daily dose of 67.5 mg. This dose is the highest effective dose used clinically for adults (60 kg) per day. This highest effective dose was converted to the dose used in mice in this study based on body surface area. In this study, mice were given a dose group WWZ, with the highest effective dose being 25.0 mg / kg.

[0170] Tumor volume and body weight were measured and recorded twice weekly during the experiment. At the end of the experiment, the tumor growth inhibition rate (TGI) and relative tumor proliferation rate (T / C%) were calculated based on the tumor volume. Tumor volume was measured twice weekly using calipers, measuring both the long and short diameters. The volume calculation formula was: Tumor volume = 0.5 × long diameter × short diameter 2 .

[0171] Tumor growth inhibition rate (TGI, %) = (1 - T(tumor volume in the treatment group) / C(tumor volume in the solvent control group)) × 100.

[0172] Relative tumor proliferation rate (T / C, %) = RTV of treatment group / RTV of solvent control group.

[0173] The experimental results are shown in Table 9.

[0174] Table 9:

[0175]

[0176] Note: Evaluation criteria: T / C (%) > 40% is ineffective; T / C (%) ≤ 40%; TGI: tumor inhibition rate; T / C %: relative tumor proliferation rate.

[0177] The data in Table 9 show that, at day 34, except for the WWZ (25.0 mg / kg) group, the tumor growth inhibition rate (calculated by tumor volume and weight) of the gavage administration groups (WWZ (25.0 mg / kg) + test drug (8.1 mg / kg) and WWZ (25.0 mg / kg) + test drug (16.2 mg / kg) was greater than 60%, and the T / C ratio was less than 40%. This suggests that gavage administration of the test drug (8.1 mg / kg), test drug (16.2 mg / kg), WWZ (25.0 mg / kg) + test drug (8.1 mg / kg), and WWZ (25.0 mg / kg) + test drug (16.2 mg / kg) significantly inhibited tumor growth in human lung cancer PC9 xenografts in nude mice.

[0178] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0179] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, include: The compound of Formula I or its salt thereof serves as the first active ingredient, and biphenylcyclooctanyl lignan serves as the second active ingredient. The mass ratio of the compound of Formula I or its salt to biphenylcyclooctanyl lignan is (1:31) to (4:3). The salt of the compound of Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride, and the biphenylcyclooctanyl lignan is schisandrin A. The pharmaceutical composition is an oral formulation. Ⅰ。 2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is (1:10) to (5:6).

3. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is (7:50) to (4:5).

4. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is 3:20, 1:5, 81:250 or 81:

125.

5. A single-dose formulation, characterized in that, include: The compound shown in Formula I or its salt is used as the first active ingredient, and biphenylcyclooctanyl lignan is used as the second active ingredient. The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctanyl lignan is (1:31) to (4:3). The salt of the compound shown in Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride, and the biphenylcyclooctanyl lignan is schisandrin A. The single-dose formulation is an oral preparation, and the single-dose formulation comprises 200-300 mg of the compound shown in Formula I or its salt. Ⅰ。 6. The single-dose formulation according to claim 5, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is (1:10) to (5:6).

7. The single-dosage form according to claim 5, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is (7:50) to (4:5).

8. The single-dosage form according to claim 5, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is 3:20, 1:5, 81:250 or 81:

125.

9. A drug combination, characterized in that, include: The compound shown in Formula I or its salt is used as the first active ingredient, and biphenylcyclooctanyl lignan is used as the second active ingredient. The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctanyl lignan is (1:31) to (4:3). The salt of the compound shown in Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride, and the biphenylcyclooctanyl lignan is schisandrin A. The second active ingredient is in an oral dosage form. Ⅰ。 10. A medicine box, characterized in that, include: The compound shown in Formula I or its salt is used as the first active ingredient, and biphenylcyclooctanyl lignan is used as the second active ingredient. The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctanyl lignan is (1:31) to (4:3). The salt of the compound shown in Formula I is (R)-4-(3-chloro-2-fluorophenylamino)-7-methoxyquinazoline-6-yl 2,4-dimethylpiperazine-1-carboxylic acid ester hydrochloride, and the biphenylcyclooctanyl lignan is schisandrin A. The second active ingredient is in an oral dosage form. Ⅰ。 11. The drug combination according to claim 9 or the pillbox according to claim 10, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is (1:10) to (5:6).

12. The drug combination according to claim 9 or the pillbox according to claim 10, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is (7:50) to (4:5).

13. The drug combination according to claim 9 or the pillbox according to claim 10, characterized in that, The mass ratio of the compound shown in Formula I or its salt to biphenylcyclooctyl lignan is 3:20, 1:5, 81:250 or 81:125.

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