Administration scheme and pharmaceutical application of AST-3424 for treating cancers and tumors

Abstract

The invention discloses an administration scheme and pharmaceutical application of AST-3424 for treating malignant tumors, relates to a malignant tumor treatment method, in particular to an administration scheme and pharmaceutical application of AKR 1C3 activated anti-cancer prodrug compound AST-3424 for treating malignant tumors, and belongs to the field of tumor treatment. The invention relates to an AST-3424 single drug administration scheme for treating human malignant tumors, which is characterized in that the interval time between two times of administration in one treatment cycle is 8-144 hours, preferably 12-72 hours, and the administration frequency in one treatment cycle is not less than 3 times. According to the pharmaceutical application of the AST-3424, the AST-3424 is used for preparing a medicine for treating human malignant tumors through a single medicine, and the AST-3424 is characterized in that the interval time between two times of administration in a treatment cycle is 8-144 hours, preferably 12-72 hours, and the number of times of administration in the treatment cycle is not less than 3.

Description

Dosage regimen and pharmaceutical use of AST-3424 for treating cancer and tumors Technical Field

[0001] The present invention relates to a method for treating human malignant tumors, in particular to a dosage regimen and pharmaceutical use of an AKR1C3 activated anticancer prodrug compound AST-3424 for treating malignant tumors, and belongs to the field of tumor treatment. Background Art

[0002] AST-3424 (WO2016145092, WO2017087428), a DNA alkylating agent prodrug targeting overexpressed aldehyde-keto reductase 1C3 (AKR1C3), has a CAS number of 2097713-69-2 and its structure is as follows:

[0003] Chemical structure of AST-3424

[0004] AST-3424 (also known as OBI-3424 and TH-3424) enters cancer cells and is activated by the overexpressed AKR1C3 enzyme, releasing the metabolite AST-2660 (also known as AST-2660). AST-3424 itself has minimal toxicity to cancer cells, and its pharmacological effects have been shown to correlate with AKR1C3 enzyme expression in animal models and in vitro pharmacological studies. The prodrug AST-3424 is metabolized to AST-2660 by the AKR1C3 enzyme and NADPH, and enzyme expression is positively correlated with drug efficacy (References 1; 2; 3; 4).

[0005] Chemical reaction formula for the metabolism of AST-3424 (OBI-3424) to AST-2660 (OBI-2660)

[0006] Currently, the drug has entered Phase I / II clinical trials in China and the United States, respectively (US NCT03592264, indications: liver cancer, pancreatic cancer and other solid tumors, applicant: OBIPharama Inc (4174), the drug is named OBI-3424; US NCT04315324, T-ALL / T-LBL (acute T-lymphocytic leukemia / T-lymphoblastic lymphoma), the drug is named OBI-3424; China CTR20191399, indications: various solid tumors, applicant: Ascentawits Pharmaceuticals, LTD., the drug is named AST-3424; CTR20201915, indications: acute T-lymphocytic leukemia and acute B-lymphocytic leukemia, applicant: Ascentawits Pharmaceuticals, LTD., the drug is named AST-3424).

[0007] In these Phase I / II clinical trials conducted in China and the United States, one of the dosing regimens was a 21-day treatment cycle, with the drug administered twice on days 1 and 8, and no dosing on days 2 to 7 and days 9 to 21.

[0008] The results of the Phase I clinical trial completed in the United States are summarized as follows (see the research poster in Reference 5 for details, which are extracted and translated from the Abstract section, Table 2, Figure 4, Figure 3, and Table 3 in the research poster in Reference 5. The applicant hereby translates the abstract as follows and correspondingly translates the above-mentioned figures into Figures 1, 2, 3, and 4 in this application):

[0009] Trial regimen: OBI-3424 at 1, 2, 4, 6, 8, or 12 mg / m 2 Doses of 8, 10, 12, or 14 mg / m2 were administered intravenously (on days 1 and 8 of a 21-day cycle, regimen A) or 14 mg / m2 2 (Dose is given only on day 1 of each 21-day cycle, schedule B). Dose escalation was performed using a "3+3" design. Patients received study treatment until disease progression, unacceptable toxicity, or after up to 2 years of treatment.

[0010] Results: A total of 39 adult patients were treated.

[0011] In patients receiving regimen A, the maximum tolerated dose (MTD) of OBI-3424 was determined to be 8 mg / m 2 Dose-limiting toxicities (DLTs) were reported at 12 mg / m 2 Adverse reactions occurred across dose levels, including thrombocytopenia (grade 3-4, 5 of 6 patients) and anemia (grade 3-4, 5 of 6 patients); platelet nadirs were observed on Day 15 or Day 22.

[0012] In patients receiving regimen B, at the maximum dose tested (14 mg / m 2 ) did not reach the MTD; 2 Among the 6 patients treated, 3 developed grade 3 or higher anemia; the recommended dose (RP2D) of the Phase II clinical trial was 12 mg / m 2 (One cycle is 21 days, with medication administered on day 1).

[0013] Treatment-related adverse events (TRAEs) occurred in 82% (32 / 39) of patients. The most common TRAEs were anemia (64%), thrombocytopenia (51%), nausea (26%), and fatigue (21%). No patients experienced fatal TRAEs, and 49% of patients reported grade ≥ 3 TRAEs, including 3 patients with severe TRAEs.

[0014] OBI-3424 at 1 to 14 mg / m 2 The drug showed linear pharmacokinetic characteristics with minimal accumulation after repeated dosing.

[0015] A retrospective immunohistochemistry (IHC) evaluation using validated methods demonstrated high AKR1C3 staining (H score ≥ 135) in 27% of patients. The best confirmed response was stable disease in 21 patients (54%).

[0016] Conclusion: We completed the dose-escalation portion of the OBI-3424 clinical study. The RP2D was determined to be 12 mg / m 2 , administered once every 3 weeks (total dose for one cycle is 12 mg / m 2 OBI-3424 was well tolerated. Dose-dependent, non-cumulative thrombocytopenia and anemia were the dose-limiting toxicities.

[0017] Similar to AST-3424, Vybio and WuXi AppTec / Medshine Discovery) developed TFX05-01 (patent application PCT / CN2020 / 120281, publication number WO2021068952A1, corresponding to Chinese application number 202080071652.8, publication number CN114555574A; PCT / CN2021 / 118597, publication number WO2022057838A1, corresponding to Chinese application number 202180064048.7, publication number CN116249698A). It is reported (see document 7 for details) that it is also an AKR1C3-activated DNA alkylating agent prodrug. At the AACR2022 annual meeting, it was demonstrated through a poster that the prodrug is metabolized to generate the DNA alkylating agent TFX05-01A, namely AST-2660, under the joint action of NADPH and AKR1C3. Based on the Phase I clinical trial protocols for AST-3424 and OBI-3424, this candidate drug is currently undergoing a Phase I clinical trial in China (Clinical Trial Registration Number CTR20220957) for injection of TFX05-01. The trial is evaluating the safety, tolerability, pharmacokinetics, efficacy, and correlation with AKR1C3 enzyme expression in subjects with advanced solid tumors. The dosing regimen is a three-week intravenous infusion on days 1 and 8 of each cycle. The trial plans to recruit 56 patients, and 10 have been enrolled to date.

[0018] Summary of the Invention

[0019] As the sponsor of the Phase I clinical trial of AST-3424 in China, the applicant has completed the Phase I clinical trial for solid tumors in China (CTR20191399). The applicant disclosed the results of the trial for the first time and summarized them as follows.

[0020] A total of 21 subjects were enrolled in this trial. Dosages of 1.0, 2.0, 4.0, 6.0, and 8.0 mg / m2 were administered on days 1 and 8 of each 21-day cycle. 2 The dose was escalated to 5 dose levels. After discussion by the Safety Review Committee (SRC), it was confirmed that every 21 days was a treatment cycle, and the MTD and RP2D of the drug administered on the 1st and 8th days were 6.0 mg / m 2 (The total dose for one cycle is 12 mg / m 2Safety and tolerability were slightly better in US participants than in Chinese participants (MTD / RP2D for the same dosing regimen was slightly higher than in the Chinese population); the safety profile trends were consistent (adverse events were mostly mild; anemia, decreased platelet count, fatigue, and vomiting were the most common adverse events). Other safety, PK, and efficacy results were generally similar to those in the OBI-3424 clinical trial.

[0021] In addition to the above-mentioned dose exploration of administration on days 1 and 8 of a 21-day cycle, the clinical trial in the United States also explored a dosing regimen of administration only on day 1 of a 21-day cycle (dosage: 8, 10, 12, or 14 mg / m 2 After comparison, the final dosing regimen for subsequent Phase II clinical trials in the United States was determined to be one treatment cycle every 21 days, with one dose administered on the first day of each cycle, and an RP2D of 12 mg / m 2 The Phase II clinical trial conducted in China, based on the results of Phase I, determined a dosing regimen of 21 days per treatment cycle, with the drug administered once on the first and eighth days of each cycle, and an RP2D of 6.0 mg / m 2 .

[0022] According to literature 5, the US Phase II clinical trial will use a higher dose (12 mg / m 2 ) and was only administered on the first day of a 21-day treatment cycle, which was obtained after comparing the administration on the first and eighth days of a 21-day treatment cycle: See Figure 1, at the same dosage (8, 12 mg / m 2 ) in patients who received only the 1st day dose at 12 mg / m 2 The probability of TE AEs greater than grade 3 was 33.3% (2 of 6 cases) at 12 mg / m2. 2 The probability of TEAEs greater than grade 3 at this dose was 100% (all 6 cases had TEAEs). The applicant speculated that in order to reduce more severe TEAEs, a high dose was used for RP2D and administered once in one treatment cycle.

[0023] The active ingredient of AST-3424 after activation of the AKR1C3 enzyme is AST-2660. AST-2660 is an anti-tumor compound similar to ifosfamide / DNA alkylating agents. Its efficacy is related to the dose. Higher doses can bring better efficacy but may also have more serious side effects and adverse reactions, because higher doses may cause the drug to accumulate in the body.

[0024] In order to maximize the total dosage of AST-3424 during the treatment cycle to improve efficacy, OBI adopted a strategy of reducing the number of dosing times while increasing the single dose. The applicant speculates that this is based on the fact that a high-dose single dose regimen can reduce more severe TEAEs.

[0025] However, the MTD and RP2D determined by the Phase I clinical trial completed in China (21 days per cycle, administration on the 1st and 8th days) were 6.0 mg / m 2 The MTD (8.0 mg / m 2 ) is slightly lower than .

[0026] Based on the above differences, in order to improve the therapeutic effect of AST-3424 in certain patients and minimize the occurrence of milder TEAEs, the approach should be to administer the drug at a high frequency and a low dose:

[0027] *The higher the total exposure to chemotherapy drugs per cycle, the better the efficacy;

[0028] *The larger the single dose of chemotherapy drugs, the greater the toxicity;

[0029] *To improve efficacy and reduce toxicity, low-dose, multiple-dose regimens are used.

[0030] Human pharmacokinetic data in China and the United States were reviewed to determine the dosing interval.

[0031] As shown in Figures 3 and 4, the clinical trial drug OBI-3424 in the United States quickly reached its peak blood concentration after intravenous injection. 1 / 2 The peak time of the original drug metabolite AST-2660 is delayed, T 1 / 2 About 2.3 hours.

[0032] Combined with the AST-3424 clinical trial conducted in China, the metabolism of AST-3424 (OBI-3424) and AST-2660 (OBI-2660) in patients from China and the United States is similar. 1 / 2 There was little difference, with both AST-3424 (OBI-3424) and AST-2660 (OBI-2660) being below 1 ng / ml almost after 8 hours, meaning that almost no drug (including AST-3424 and AST-2660) accumulated after 8 hours.

[0033] In particular, AST-3424 (OBI-3424) in blood C max The C level of AST-2660 (OBI-2660) in blood maxThis also indirectly shows that most of the AST-3424 (OBI-3424) metabolic process occurs in cells, and only a very small proportion of AST-3424 is activated and metabolized into AST-2660 (OBI-2660) in the blood.

[0034] The process of prodrug AST-3424 (OBI-3424) being activated by AKR1C3 to AST-2660 (OBI-2660) in humans is rapid in both Chinese and American patients.

[0035] Based on the blood pharmacokinetic experimental data obtained from clinical trials of patients in China and the United States, the inventors believe that the use of a new regimen of continuous multiple dosing with an interval of 8 hours or more between two doses is expected to increase the total dosage within a treatment cycle and thus enhance the efficacy of the drug. At the same time, based on the observed time pattern of platelet count decrease, the subjects will have a longer recovery period, which may reduce the incidence of related adverse reactions or reduce their severity.

[0036] To this end, the following novel dosing regimen and pharmaceutical use of AKR1C3-activated DNA alkylating agent prodrug compounds, including AST-3424 (OBI-3424) / TFX05-01, which metabolize to AST-2660, are proposed.

[0037] A dosing regimen for treating human tumors using a single agent or in combination with an AKR1C3-activated DNA alkylating agent prodrug compound that metabolizes AST-2660, characterized in that the interval between two doses within a treatment cycle is 8-144 hours, preferably 12-72 hours, and the number of doses within a treatment cycle is not less than 3 times, and the tumor is preferably a malignant tumor.

[0038] The pharmaceutical use of an AKR1C3-activated DNA alkylating agent prodrug compound that metabolizes AST-2660, which is used to prepare a drug for treating human tumors as a single drug or in combination, is characterized in that the interval between two doses within a treatment cycle is 8-144 hours, preferably 12-72 hours, and the number of doses within a treatment cycle is not less than 3 times, and the tumor is preferably a malignant tumor.

[0039] The AKR1C3 enzyme-activating prodrug is selected from the group consisting of compounds of formula (1)-(6):

[0040] Among them, X, Y, Z, R, A and X 10 The definition of is as described in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), T is

[0041] Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as set forth in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A);

[0042] in:

[0043] A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=CR 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, –O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 SO2R 100 、-SO2R 100 、-SO2NR 101 R 102 , C1-C6 alkyl, C3-C10 heterocyclic group;

[0044] Among them, R 100 、R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 together with the nitrogen atom to which it is attached, form a 5-7 membered heterocyclic ring;

[0045] wherein the alkyl group and the aryl group are each substituted with 1-3 halo groups or 1-3 C1-C6 alkyl groups;

[0046] R 1 and R 2 each independently phenyl or methyl;

[0047] X, Y and Z are each independently hydrogen or halo;

[0048] R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl;

[0049] Wherein, Rw is defined as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A);

[0050] Among them, the definitions of R1, R2, R3, R4, and T are as recorded in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1.

[0051] Formula (1) is an AKR1C3 enzyme-activated alkylating agent prodrug compound, more specifically, an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0052] Among them, X, Y, Z, R, T, A and X 10 The definition is as described in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), and the synthesis and preparation methods of specific compounds are also described in the above application, which are hereby incorporated into this application in their entirety, and are specifically defined as:

[0053] X 10 O, S, SO or SO2;

[0054] A is C6-C 10 Aryl, 5-15 membered heteroaryl or -N=CR 1 R 2 ;

[0055] R 1 and R 2 are independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0056] X, Y and Z are each independently hydrogen, CN, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0057] R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0058] R 13 and R 14 are independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle or ether;

[0059] T is and

[0060] These alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, and ether groups are substituted or unsubstituted.

[0061] Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as described in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A1, and the synthesis and preparation methods of specific compounds are also described in the above application, which are hereby incorporated into this application in their entirety, and are specifically defined as:

[0062] R1 is C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, 7-15 membered fused ring or Z-substituted fused ring;

[0063] R2 is hydrogen, a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a mercapto group, an amino group, OTs, OMS, a C1-C6 alkyl group or a Z-substituted alkyl group, a C2-C6 alkenyl group or a Z-substituted alkenyl group, a C2-C6 alkynyl group or a Z-substituted alkynyl group, a C3-C8 cycloalkyl group or a Z-substituted cycloalkyl group, a C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, ether of 1-6 carbon atoms or Z-substituted alkoxy of 1-6 carbon atoms, -CONR 6 R 7 、-SO2NR 6 R 7 、-SO2R 6 、-OCOO-R 6 、-COOR 6 、-NR 6 COR 7 、-OCOR6 、-NR 6 SO2R 7 、-NR 6 SO2NR 6 R 7 Or R2 and the atoms on the R1 group to which it is bonded together form a 7-15 membered fused ring or a Z-substituted fused ring;

[0064] R3 is hydrogen, halogen, cyano or isocyano, hydroxyl, thiol, amine, OTs, OMS, C1-C6 alkyl or Z substituted alkyl, C2-C6 alkenyl or Z substituted alkenyl, C2-C6 alkynyl or Z substituted alkynyl, C3-C8 cycloalkyl or Z substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, -CONR 6 R 7 、-SO2NR 6 R 7 、-SO2R 6 、-OCO-R 6 、-OCOO-R 6 、-COOR 6 、-NR 6 COR 7 , -OCOR 6 、-NR 6 SO2R 7 ;

[0065] R4 and R5 are each independently hydrogen, a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a mercapto group, an amino group, OTs, OLCMS, a C1-C6 alkyl group or a Z-substituted alkyl group, a C2-C6 alkenyl group or a Z-substituted alkenyl group, a C2-C6 alkynyl group or a Z-substituted alkynyl group, a C3-C8 cycloalkyl group or a Z-substituted cycloalkyl group, a C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, -CONR 6 R 7 、-SO2NR 6 R 7 、-SO2R 6 、-OCOO-R 6 、-COOR 6、 -NR 6 COR 6 、-OCOR 6 、-NR 6 SO2R 7Or R4, R5 and the atoms on the benzene ring to which they are bonded together form a 7-15 membered fused ring or a Z-substituted fused ring;

[0066] R 6 and R 7 Each is independently hydrogen, cyano or isocyano, C1-C6 alkyl or Z substituted alkyl, C2-C6 alkenyl or Z substituted alkenyl, C2-C6 alkynyl or Z substituted alkynyl, C3-C8 cycloalkyl or Z substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, or R 6 、R 7 The group together with the atoms to which it is bonded forms a 5-7 membered heterocyclic group or a Z-substituted 5-7 membered heterocyclic group;

[0067] R8, R 10 Each is independently hydrogen, deuterium, aryl or Z-substituted aryl, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, and at least one of them must be hydrogen or deuterium;

[0068] R9 is a substituted C6-C 10 aryl, substituted 4- to 15-membered heterocyclic ring having at least one fluorine atom or nitro group, substituted 5- to 15-membered heteroaryl group having at least one fluorine atom or nitro group.

[0069] The Z substituent is a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a thiol group, an amine group, OTs, OMS, a C1-C3 alkyl group or a substituted alkyl group, a C1-C3 alkoxy group or a substituted alkoxy group, a C2-C3 alkenyl group or a substituted alkenyl group, a C2-C3 alkynyl group or a substituted alkynyl group, a C3-C8 cycloalkyl group or a substituted cycloalkyl group, an aromatic ring, a heterocyclic ring, a heteroaromatic ring and a condensed ring or a substituted aromatic ring, a heterocyclic ring, a heteroaromatic ring and a condensed ring, and the substitution is monosubstitution or geminal disubstitution;

[0070] Substitution of C6-C in R9 10 The substituents of the aryl group, the substituted 4-15 membered heterocyclic ring, and the substituted 5-15 membered heteroaryl group are halogen atoms, nitro groups, cyano groups or isocyano groups, hydroxyl groups, amino groups, C1-C3 alkyl groups or alkoxy groups, alkenyl groups, alkynyl groups, cycloalkyl groups or benzene rings, substituted benzene rings, C1-C3 alkoxy groups, or halogen atom-substituted alkoxy groups.

[0071] Formula (4) is an AKR1C3 enzyme-activated alkylating agent prodrug compound, more specifically, an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0072] in:

[0073] A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=CR 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, –O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 SO2R 100 、-SO2R 100 、-SO2NR 101 R 10 2 , C1-C6 alkyl, C3-C10 heterocyclic group;

[0074] Among them, R 100 、R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 together with the nitrogen atom to which it is attached, form a 5-7 membered heterocyclic ring;

[0075] wherein the alkyl group and the aryl group are each substituted with 1-3 halo groups or 1-3 C1-C6 alkyl groups;

[0076] R 1 and R 2 each independently phenyl or methyl;

[0077] X, Y and Z are each independently hydrogen or halo;

[0078] R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl.

[0079] Wherein, Rw is defined as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1, and the synthesis and preparation methods of specific compounds are also described in the above-mentioned application, which are hereby incorporated into this application in their entirety, and are specifically defined as:

[0080] Rw is

[0081] R1 is H, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or phenyl, wherein the C1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl and phenyl are optionally substituted by 1, 2 or 3 R a replaced by;

[0082] Each R a are independently H, F, Cl, Br, I, -CN, -OH, C 1-3 Alkoxy or C 1-3 alkyl;

[0083] R2 is H or C 1-6 alkyl;

[0084] Or R1 and R2 are linked together to form a 4-6 membered heterocycloalkyl group, wherein the 4-6 membered heterocycloalkyl group is optionally substituted by 1, 2 or 3 R b replaced by;

[0085] Each R b are independently H, F, Cl, Br, I, -CN, -OH, -NH2, -OCH3, -OCH2CH3, -CH3 or -CH2CH3;

[0086] R3 is H, F, Cl, Br, I, -OH, -NH2, C 1-3 Alkoxy or C 1-3 alkyl;

[0087] Or R2 and R3 are connected together to make the structural unit for

[0088] T1 is -(CR c R d ) m -or-(CR c R d ) n -O-;

[0089] m is 1, 2, or 3;

[0090] n is 1 or 2;

[0091] T2 is N or CH;

[0092] R c and R d Each independently is H, F, C 1-3 Alkyl or C 1-3 alkoxy;

[0093] R4, R5 and R6 are each independently H, F, Cl, Br, I, C 1-3 Alkyl or C 1-3alkoxy;

[0094] T is N or CH;

[0095] R7 and R8 are each independently H, F, Cl, Br or I;

[0096] R9 and R 10 Each independently is H, F, Cl, Br, I, -CN or

[0097] The 4-6 membered heterocycloalkyl and 5-6 membered heteroaryl each contain 1, 2, 3 or 4 heteroatoms independently selected from N, -O- and -S-.

[0098] Wherein, the definitions of R1, R2, R3, R4 and T are as described in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1. The synthesis and preparation methods of specific compounds are also described in the above-mentioned application, which are hereby incorporated into this application in their entirety and are specifically defined as:

[0099] T is N or CH;

[0100] R1 and R2 are each independently H, F, Cl, Br, I or C 1-3 Alkyl, wherein the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R a replaced by;

[0101] Each R a independently F, Cl, Br, I, -CN, -OH or -NH2;

[0102] R3 and R4 are each independently H, F, Cl, Br, I, CN, C 1-3 Alkyl, C 1-3 Alkoxy, Among them, the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R e replaced by;

[0103] R b and R c Each independently represents H, -CH3, -CH2CH3, -(CH2)2CH3, -CH(CH3)2;

[0104] R d It is -CH3, -CH2CH3, -(CH2)2CH3, -CH(CH3)2;

[0105] Each R e independently F, Cl, Br, I, -CN, -OH or -NH2.

[0106] Particularly, the AKR1C3-activated DNA alkylating agent prodrug compounds that metabolize AST-2660 are selected from AST-3424 and TFX05-01.

[0107] TFX05-01 is selected from one of the following structural compounds:

[0108] Compound of structural formula (5)

[0109] Compound of structural formula (6)

[0110] The dosing regimen of AST-3424 as a single agent or in combination for the treatment of human malignant tumors is characterized in that the interval between two doses within a treatment cycle is 8-144 hours.

[0111] Preferably, the dosing interval is 12-72 hours, and more preferably, the number of administrations in one treatment cycle is no less than 3 times.

[0112] The pharmaceutical use of AST-3424 is to prepare a drug for treating human tumors as a single drug or in combination, characterized in that the interval between two doses within a treatment cycle is 8-144 hours.

[0113] Preferably, the dosing interval is 12-72 hours, and more preferably, the number of administrations in one treatment cycle is no less than 3 times.

[0114] The tumor is preferably a malignant tumor.

[0115] Tumors are further divided into two categories: benign and malignant, based on the cellular characteristics of the neoplasm and the degree of harm it poses to the body. Malignant tumors can be divided into carcinomas and sarcomas. Carcinomas are malignant tumors originating from epithelial tissue. Sarcomas are malignant tumors that arise from mesenchymal tissue, including fibrous connective tissue, fat, muscle, blood vessels, bone, and cartilage.

[0116] AST-3424 refers to a pharmaceutical agent containing AST-3424, i.e., a pharmaceutical preparation or drug that can be directly administered to human patients (as distinguished from an active pharmaceutical ingredient (API)).

[0117] The drug described herein refers to a medicine or preparation. The prepared medicine contains the active ingredient AST-3424 within a specific dosage range, and the prepared medicine is in a specific dosage form and is administered by a specific administration method.

[0118] The prepared medicine, drug, preparation may also contain pharmaceutically acceptable adjuvants or excipients. The medicine may be in any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small needles for injection, freeze-dried powder for injection, or large infusions. Depending on the specific dosage form and mode of administration, the pharmaceutically acceptable adjuvants or excipients in the medicine may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, adhesives, fillers, flavorings, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and pigments.

[0119] The currently available dosage form is an injection, namely the AST-3424 concentrated solution for injection disclosed in patent application PCT / CN2020 / 101870, publication number WO2021008520 (corresponding to Chinese application CN202080001484.5, publication number CN112469394A), which is diluted immediately before use and administered intravenously, preferably by intravenous drip. Other dosage forms may be developed in the future.

[0120] Monotherapy refers to single-agent treatment. In this document, monotherapy also requires that the drug contains only the active ingredient AST-3424 and no other active ingredients, meaning it is not a combination drug. Combination therapy refers to combined drug treatment. Monotherapy refers to the use of only one anticancer drug in a single course of treatment. Combination therapy refers to the simultaneous or sequential use of two or more anticancer drugs in a single course of treatment.

[0121] The drugs used in combination with AST-3424 can be other anticancer drugs. Existing literature has shown that it has a more obvious combination effect when used in combination with abiraterone, prednisolone, 5-fluorouracil, sunitinib (WO2022178821), immune checkpoint inhibitors such as PD-1 / L1 (WO2022231580), nelarabine (WO2019062919), oxaliplatin / 5-fluorouracil (Reference 6), etc.

[0122] The interval between two doses within a treatment cycle is 8-144 hours. The minimum value of 8 hours in this range is based on the blood drug metabolism data of the above-mentioned Phase I clinical trial of AST-3424 (OBI-3424) conducted in China and the United States. However, it is clear that the dosing interval cannot be infinite. The inventors recommend a maximum interval of 6 days, or 144 hours.

[0123] Since AST-2660, a DNA alkylating agent, can be observed in the blood and may have toxic side effects on the blood or other human tissues, it is important to prevent AST-2660 from accumulating in the blood to a high concentration. Based on the aforementioned drug metabolism results, AST-2660 essentially disappears from the blood after 8 hours, so the minimum dosing interval is set at 8 hours.

[0124] Chemotherapy is generally calculated in cycles, usually 21 days or 28 days as a cycle, of course there are also cycles of 14 days or even 7 days: that is, the time interval from the first day of medication to the start of the next cycle of chemotherapy is 21 days, 28 days, 14 days or even 7 days. Because chemotherapy not only kills tumor cells, but also some normal cells will be killed, the body needs a certain amount of recovery time, and chemotherapy can also cause bone marrow suppression, and blood indicators also need to recover to a certain level before the next cycle of treatment is feasible. Therefore, a chemotherapy treatment cycle can be divided into an administration period and a recovery period: continuous or intermittent administration during the administration period, and a certain time interval is required between two administrations. This time is related to the nature of the drug itself; and no administration is given during the recovery period. Continuous administration here refers to continuous administration every day during the administration period, and intermittent administration refers to administration with at least 1 day interval during the administration period.

[0125] The length of a treatment cycle is determined through clinical trials. For most chemotherapy drugs, 21 or 28 days is sufficient for the body to return to normal, while also preventing the tumor cells from proliferating or growing again. Therefore, chemotherapy cycles are typically 21 or 28 days.

[0126] The period from the first dose to the last dose in a treatment cycle is defined in this application as the dosing period, and the rest of the subsequent treatment cycle is the recovery period. Generally speaking, the recovery period of chemotherapy is greater than or equal to the dosing period, so as to ensure that there is enough time for the patient's body to recover on its own.

[0127] AST-3424 (OBI-3424) is a prodrug of the chemotherapy drug AST-2660, which is the chemotherapy drug that is active in the body. AST-2660 is a chemotherapy drug similar to ifosfamide, so its treatment is also compatible with the treatment cycle of chemotherapy drugs.

[0128] Preferably, the interval between two doses within a treatment cycle is 8, 12, 24, 48, 72, 96, 120, or 144 hours. This interval is determined based on the blood metabolism data from the Phase I clinical trial and the specific characteristics of the drug: this interval neither allows for significant drug accumulation in the blood nor allows for blood concentrations to be too low to create a concentration gradient between the intracellular and extracellular regions, preventing the drug from entering cancer cells and being activated by the AKR1C3 enzyme to exert its efficacy.

[0129] An interval of 8 hours means the drug is given 3 times a day; an interval of 12 hours means the drug is given twice a day; an interval of 24 hours means the drug is given once a day, and so on. This is a case of evenly spaced dosing.

[0130] In reality, given the differences in physical conditions of different patients or other circumstances, the actual dosing interval may not be so uniform and strict. Dosing may occur at intervals of 1, 2, 4, or 5 days. Therefore, the intervals between doses of 8, 12, 24, 48, 72, 96, 120, and 144 hours do not only include the uniform dosing intervals mentioned above, but also include unequal dosing intervals. The same applies to the intervals between doses within a treatment cycle of 8-144 hours.

[0131] The tumors include solid tumors and hematologic tumors. The solid tumors are selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, intrahepatic bile duct carcinoma, pancreatic cancer, submandibular gland carcinoma, prostate cancer, breast cancer, and melanoma. The hematologic tumors are selected from acute lymphoblastic leukemia, preferably acute T-lymphocytic leukemia and acute B-lymphocytic leukemia (T-ALL, B-ALL). All of the above tumor types have patients enrolled in Phase I clinical trials in China and the United States.

[0132] More preferably, a treatment cycle includes an administration period and a recovery period, during which the drug is administered continuously or intermittently with the interval between the two administrations being 12, 24, 48, or 72 hours. The remaining time is the recovery period and no drug is administered. The administration method is intravenous drip, and the dose for each administration is 1-8 mg / m 2 , preferably 1-6 mg / m 2 AST-3424 (OBI-3424) was administered via intravenous infusion in clinical trials over a 30-minute (±5-minute) infusion period. Specifically, the concentrated solution for injection described in patent application PCT / CN2020 / 101870, publication number WO2021008520 (corresponding to Chinese application CN202080001484.5, publication number CN112469394A) was used. The pH of the 5% glucose injection was adjusted to an isotonic solution of 7.4 using sodium bicarbonate solution.

[0133] A single dose is usually administered as an intravenous infusion over 20-60 minutes.

[0134] 1-8 mg / m 2 , corresponding to the average single dose for an average person, the body surface area can be calculated using the relevant body surface area calculation formula. The body surface area of ​​a Chinese person is generally between 1.5-2.0 square meters, and some thin elderly women have a body surface area of ​​approximately 1.3-1.4 square meters. The body surface area calculation result can be used to calculate the dose for each dose.

[0135] When a treatment cycle is 21 days, the specific regimen is selected from the following:

[0136] The drug was administered once a day for a total of three times on days 1, 2, and 3, and no drug was administered for the remaining 18 days as a recovery period;

[0137] The drug was administered once a day on days 1, 2, 3, and 4 for a total of 4 times, and no drug was administered for the other 17 days as a recovery period;

[0138] The drug was administered once a day on days 1, 2, 3, 4, and 5 for a total of 5 times, and no drug was administered for the other 16 days as a recovery period;

[0139] The drug was administered once a day on days 1, 2, 3, 4, 5, and 6 for a total of 6 times, and no drug was administered for the other 15 days as a recovery period;

[0140] The drug was administered once a day on days 1, 2, 3, 4, 5, 6, and 7 for a total of 7 times, and no drug was administered for the other 14 days as a recovery period;

[0141] The drug was administered once a day on days 1, 3, and 5 for a total of 3 times. The remaining 18 days (i.e., no drug administration on days 2 and 4, and days 6-21, a total of 18 days, the same calculation method below) were the recovery period, during which no drug administration was performed.

[0142] The drug was administered once a day on days 1, 3, 5, and 7 for a total of 4 times, and no drug was administered for the other 17 days as a recovery period;

[0143] The drug was administered once a day on days 1, 3, 5, 7, and 9 for a total of 5 times, and the other 16 days were a recovery period without drug administration;

[0144] The drug was administered once a day for a total of three times on days 1, 4, and 7, and no drug was administered for the remaining 18 days as a recovery period;

[0145] The drug was administered once a day for a total of three times on the 1st, 5th and 9th days respectively. The other 18 days were a recovery period during which no drug was administered.

[0146] When a treatment cycle is 14 days, the specific regimen is selected from the following:

[0147] The drug was administered once a day on days 1, 2, and 3 for a total of 3 times, and the other 11 days were a recovery period without drug administration;

[0148] The drug was administered once a day on days 1, 2, 3, and 4 for a total of 4 times, and the other 10 days were a recovery period without drug administration;

[0149] The drug was administered once a day on days 1, 2, 3, 4, and 5 for a total of 5 times, and the other 9 days were a recovery period without drug administration;

[0150] The drug was administered once a day on days 1, 2, 3, 4, 5, and 6 for a total of 6 times, and the other 8 days were a recovery period without drug administration;

[0151] The drug was administered once a day on days 1, 2, 3, 4, 5, 6, and 7 for a total of 7 times, and the other 7 days were a recovery period without drug administration;

[0152] The drug was administered once a day for a total of 3 times on days 1, 3, and 5, and no drug was administered for the other 11 days as a recovery period;

[0153] The drug was administered once a day on days 1, 3, 5, and 7 for a total of 4 times, and the other 10 days were a recovery period without drug administration;

[0154] The drug was administered once a day on days 1, 3, 5, 7, and 9 for a total of 5 times, and the other 9 days were a recovery period without drug administration;

[0155] The drug was administered once a day for a total of three times on the 1st, 4th and 7th days respectively. The other 11 days were a recovery period during which no drug was administered.

[0156] When a treatment cycle is 28 days, the specific regimen is selected from the following:

[0157] The drug was administered once a day on days 1, 2, and 3 for a total of 3 times, and no drug was administered for the other 25 days as a recovery period;

[0158] The drug was administered once a day on days 1, 2, 3, and 4 for a total of 4 times, and the other 24 days were a recovery period without drug administration;

[0159] The drug was administered once a day on days 1, 2, 3, 4, and 5 for a total of 5 times, and the other 23 days were a recovery period without drug administration;

[0160] The drug was administered once a day on days 1, 2, 3, 4, 5, and 6 for a total of 6 times, and the other 22 days were a recovery period without drug administration;

[0161] The drug was administered once a day on days 1, 2, 3, 4, 5, 6, and 7 for a total of 7 times, and no drug was administered for the other 21 days as a recovery period;

[0162] The drug was administered once a day on days 1, 2, 3, 4, 5, 6, 7, and 8 for a total of 8 times, and the other 20 days were a recovery period without drug administration;

[0163] The drug was administered once a day on days 1, 2, 3, 4, 5, 6, 7, 8, and 9 for a total of 9 times, and the other 19 days were a recovery period without drug administration;

[0164] The drug was administered once a day on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 for a total of 10 times, and no drug was administered for the other 18 days as a recovery period;

[0165] The drug was administered once a day for a total of 3 times on days 1, 3, and 5, and no drug was administered for the other 25 days as a recovery period;

[0166] The drug was administered once a day on days 1, 3, 5, and 7 for a total of 4 times, and the other 24 days were a recovery period without drug administration;

[0167] The drug was administered once a day on days 1, 3, 5, 7, and 9 for a total of 5 times, and the other 23 days were a recovery period without drug administration;

[0168] The drug was administered once a day for a total of 3 times on days 1, 4, and 7, and no drug was administered for the other 25 days as a recovery period;

[0169] The drug was administered once a day for a total of 4 times on days 1, 4, 7, and 10, and no drug was administered for the other 24 days;

[0170] The drug was administered once a day for a total of 5 times on days 1, 4, 7, 10, and 13, and no drug was administered on the other 23 days;

[0171] The drug was administered once a day for a total of 3 times on days 1, 5, and 9, and no drug was administered for the other 25 days as a recovery period;

[0172] The drug was administered once a day for a total of 4 times on days 1, 5, 9, and 13, and no drug was administered on the other 24 days.

[0173] In the above schemes, similar to the scheme of continuous daily administration such as 1, 2, 3, etc., the time interval between the two doses is 24 hours; similar to the scheme of alternate-day administration such as 1, 3, 5, etc., the time interval between the two doses is 48 hours; similar to the scheme of two-day administration such as 1, 4, 7, etc., the time interval between the two doses is 72 hours; similar to the scheme of three-day administration such as 1, 5, 9, etc., the time interval between the two doses is 96 hours, etc. However, due to various reasons, the actual administration may not necessarily be strictly in accordance with the 24, 48, 72, or 96-hour intervals, and may be advanced or delayed.

[0174] Based on Phase I clinical trials conducted in China and the United States, the above-mentioned administration dose is 1-8 mg / m 2 The specific dosage of monotherapy will be determined based on specific clinical trials and is related to the specific tumor type; the specific dosage of combination therapy will be determined based on specific clinical trials and is related to factors such as the specific tumor type, the type of combination drug, and the order of administration when combined.

[0175] In particular, unless otherwise specified, all numerical values ​​in the specification have a range of ±10%. That is, if a numerical range in the specification is 2-3, then in fact, due to the existence of measurement errors, accidental errors, and systematic errors, if the actual measurement or calculation results in 1.8-3.3, it should also be deemed to fall within the above-mentioned range of 2-3, which also meets the requirements.

[0176] The Phase I clinical trials of AST-3424 (OBI-3424) in China and the United States disclosed in the above invention content have all been approved by the local drug regulatory authorities, and the relevant clinical trials have been completed by clinical research institutions. The relevant clinical trial plans have all been approved by ethics agencies, and the clinical trial processes have complied with the relevant medical ethics requirements and laws and regulations of the locations where the clinical trials are conducted. BRIEF DESCRIPTION OF THE DRAWINGS

[0177] Figure 1 shows the TEAE statistics of patients enrolled in the Phase I solid tumor clinical trial of OBI-3424 (extracted from Table 2 in the research poster of Reference 5 and translated into Chinese).

[0178] Figure 2 shows the preliminary therapeutic efficacy statistics for patients enrolled in the Phase I solid tumor clinical trial of OBI-3424 (excerpted from figure 4 in the research poster of reference 5 and translated into Chinese. This figure is also figure 5 in the research article of reference 5).

[0179] Figure 3 shows the pharmacokinetic curves of OBI-3424 (A) and OBI-2660 (B) during the first cycle (combined days 1 and 8) in patients enrolled in the Phase I solid tumor clinical trial of OBI-3424 (excerpted from figure 3 in the research poster of reference 5 and translated into Chinese. This figure is also figure 4 in the research article of reference 5).

[0180] Figure 4 shows the pharmacokinetic parameters of OBI-3424 and OBI-2660 after intravenous infusion of OBI-3424 in patients enrolled in the Phase I solid tumor clinical trial of OBI-3424 (excerpted from Table 3 in the research poster of Reference 5 and translated into Chinese), where dose level represents the dose level.

[0181] Figure 5 Tumor growth curve of the LI6643 subcutaneous xenograft BALB / c nude mouse model after treatment with sorafenib and AST-3424.

[0182] Figure 6 Tumor growth curve of the BALB / c nude mouse model of subcutaneous hepatocellular carcinoma LI6664 after treatment with sorafenib and AST-3424.

[0183] Figure 7 Tumor growth curve of the BALB / c nude mouse model of lung cancer LU5173 subcutaneously transplanted with AST-3424.

[0184] Figure 8 Tumor growth curve of the BALB / c nude mouse model of lung cancer LU5161 subcutaneously transplanted with AST-3424.

[0185] Figure 9 Tumor growth curve of the pancreatic cancer PA1280 subcutaneous xenograft BALB / c nude mouse model after treatment with AST-3424.

[0186] Figure 10 Tumor growth curve of the pancreatic cancer PA2637 subcutaneous xenograft BALB / c nude mouse model after treatment with AST-3424.

[0187] Figure 11 Tumor growth curve of the pancreatic cancer PA9451 subcutaneous xenograft BALB / c nude mouse model after treatment with AST-3424.

[0188] Figure 12 Tumor growth curve of the pancreatic cancer PA1170 subcutaneous xenograft mouse model after treatment with AST-3424.

[0189] FIG13 shows the single-dose toxicokinetic parameters of AST-3424 and AST-2660 at two dosing schedules of D1+D8 / QD×5.

[0190] FIG14 shows the repeated-dose toxicology profile of AST-3424 at two dosing schedules of D1+D8 / QD×5, wherein ALT=alanine aminotransferase; AST=aspartate aminotransferase; MTD=maximum tolerated dose; HNSTD=highest non-severe toxic dose; RBC=red blood cells; #EOS=absolute eosinophil count; %EOS=%eosinophils; #RET=absolute reticulocyte count; %RETIC=% reticulocytes; #NEUT=absolute neutrophil count; #LYMPH=absolute lymphocyte count; %LYMPH=% lymphocytes; WBC=white blood cells; HGB=hemoglobin; HCT=hematocrit,

[0191] *: At a dose of 0.4 mg / kg / day, one male and one female animal became moribund on Day 10 and Day 12, respectively. Therefore, starting from the second cycle of dosing (i.e., Day 22 of the study), the dose of 0.4 mg / kg was reduced to 0.3 mg / kg. Example

[0192] General Instructions

[0193] To demonstrate that the new high-frequency, low-dose dosing regimen has acceptable safety and potentially better efficacy compared to the original regimen, the applicant conducted non-clinical animal experiments.

[0194] All animal experiments are completed by professional CROs, and the experimental process complies with relevant animal experiment ethical requirements.

[0195] The relevant tumor tissues used in the experiment were tested and found to have high expression of AKR1C3 enzyme.

[0196] 1. Non-clinical efficacy trials

[0197] The in vivo efficacy of AST-3424 was investigated in the human hepatocellular carcinoma (HepG2-GFP) nude mouse orthotopic CDX model. Liver cancer LI 6643 subcutaneous xenograft PDX model, Liver cancer LI6664 subcutaneous xenograft PDX model; human lung cancer (H460-GFP) nude mouse subcutaneous CDX model, Lung cancer LU5173 subcutaneous xenograft PDX model, Lung cancer LU5161 subcutaneous xenograft PDX model; Pancreatic cancer PA1280 subcutaneous xenograft PDX model, Pancreatic cancer PA2637 subcutaneous xenograft PDX model, Pancreatic cancer PA9451 subcutaneous xenograft PDX model, The pancreatic cancer PA1170 subcutaneous xenograft PDX model was evaluated.

[0198] For the experimental process and related evaluation indicators of the above-mentioned solid tumor animal efficacy model, please refer to Examples 7, 8, and 9 in Patent Application 1, PCT / US2016 / 062114, Publication No. WO2017 / 087428 (corresponding to Chinese application CN201680044608.1, Publication No. CN108290911B), and Examples 3 and 4 in Patent Application 2, PCT / CN2021 / 078115, Publication No. WO2022178821, and Research Document 1. The above-mentioned non-clinical animal efficacy models (PDX) mentioned in this application are similar to those in the above-mentioned documents, and their specific operation processes are not described here.

[0199] 1.1. Analysis and comparison of the efficacy of AST-3424 in liver cancer models

[0200] 1.1.1 Human Hepatocellular Carcinoma (HepG2-GFP) Orthotopic CDX Model in Nude Mice (QW×2 Protocol)

[0201] In the orthotopic CDX model of human liver cancer (HepG2-GFP) in nude mice, the relative tumor inhibition rate (TGI) of the negative control group (normal saline), the positive control group (sorafenib 30 mg / kg, QD×35, gavage), the low-dose AST-3424 group (1.25 mg / kg, QW×2, one week off, QW×2, a total of 4 doses, IV), the medium-dose group (2.5 mg / kg, QW×2, one week off, QW×2, a total of 4 doses, IV), and the high-dose group (5 mg / kg, QW×2, one week off, QW×2, a total of 4 doses, IV) were compared. The tumor burden of mice was non-invasively collected twice a week using the Fluor Vivo Mode l100 imaging system, indicating that AST-3424 had a strong inhibitory effect on the orthotopic mouse model of human liver cancer, and had a clear dose-response relationship. The data from this experiment are the same as those of the HepG2 model in Example 3-1 of the aforementioned patent application 2 (see Table 5 and Figures 4A and B of the application document). The dosing regimen of AST-3424 in this pharmacodynamic model is once a week for two consecutive weeks. This regimen is similar to the dosing regimen used in the Phase I clinical trials of AST-3424 (OBI-3424) conducted in China and the United States, and is referred to herein as the original regimen.

[0202] 1.1.2 Liver cancer LI6643 subcutaneous xenograft PDX model (QD×5 and QW regimens)

[0203] AST-3424 Significant tumor inhibition was demonstrated in the BALB / c nude mouse model of subcutaneous xenografts of liver cancer LI6643. The efficacy and safety of the study were evaluated by comparing the relative tumor inhibition rate (TGI) (%), body weight changes, and mortality in the vehicle negative control (7.5% anhydrous ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W (pH 7.4), sorafenib (30 mg / kg), AST-3424 (0.3 mg / kg), AST-3424 (1 mg / kg), and AST-3424 (1.25 mg / kg) treatment groups.

[0204] Modeling BALB / c nude mice were randomly divided into groups, with 6 mice in each group. Except for the sorafenib 30 mg / kg group (QD×21, a total of 21 doses), which was administered by gavage, the control group (7.5% anhydrous ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W (pH 7.4), QW×3, a total of 3 doses) and the AST-3424 low-dose, medium-dose, and high-dose groups (0.3 mg / kg, 1 mg / kg, 1.25 mg / kg) were all injected by tail vein. The AST-3424 low-dose and medium-dose groups were administered QD for 5 consecutive days, and after 16 days of drug withdrawal, they were administered for another 5 consecutive days, for a total of 10 doses; the AST-3424 high-dose group was administered QW for 2 consecutive weeks, and after one week of drug withdrawal, they were administered for another 2 consecutive weeks, for a total of 4 doses.

[0205] The results showed that on the 21st day after the first administration, the relative tumor inhibition rate TGI (%) of the AST-3424 low-dose group (QD) (0.3 mg / kg) was 60.16%, which was statistically different from the control group (P<0.05); the TGI (%) of the medium-dose (1 mg / kg) and high-dose groups (1.25 mg / kg) were 86.32% and 71.86%, respectively, which were statistically different from the control group (P<0.001 and P<0.01, respectively). In addition, the AST-3424 medium-dose group (QD) and high-dose group (QW) did not show a difference in TGI (P>0.05), but the TGI value of the medium-dose group (QD) was greater than that of the high-dose group (QW). See Figure 4 for details. Tumor growth curve of the LI6643 subcutaneous xenograft BALB / c nude mouse model after treatment with sorafenib and AST-3424.

[0206] 1.1.3 Liver cancer LI6664 subcutaneous xenograft PDX model (QD×5 and QW regimens)

[0207] AST-3424 The LI6664 subcutaneous xenograft BALB / c nude mouse model of hepatocellular carcinoma demonstrated significant tumor inhibition. The efficacy and safety of AST-3424 were evaluated by comparing the relative tumor inhibition rate (TGI) (%), body weight change, and mortality in the vehicle control group, positive control group, and AST-3424-treated group.

[0208] Modeling BALB / c nude mice were randomly divided into groups, with 6 mice in each group. Except for the sorafenib group (30 mg / kg, QD×21, a total of 21 doses), which was administered by gavage, the vehicle control group (7.5% anhydrous ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W (pH 7.4), QW×3, a total of 3 doses), and the AST-3424 low-dose, medium-dose, and high-dose groups (0.3 mg / kg, 1 mg / kg, and 1.25 mg / kg) were all administered by tail vein injection. The AST-3424 low-dose and medium-dose groups were administered once a day for 5 consecutive days. After 16 days of drug withdrawal, they were administered for another 5 consecutive days, for a total of 10 doses. The high-dose group was administered once a week for 2 consecutive weeks. After 1 week of drug withdrawal, they were administered for another 2 consecutive weeks, for a total of 4 doses.

[0209] The results showed that on the 28th day after the first administration, the relative TGI (%) of the AST-3424 low-dose group (QD) and high-dose group (QW) were 45.85% and 52.42%, respectively, which were not statistically different from the control group (P>0.05); the TGI (%) of the AST-3424 medium-dose group (QD) was 65.05%, which was statistically different from the control group (P<0.05). The AST-3424 medium-dose group (QD) and high-dose group (QW) did not show differences in TGI (P>0.05), but the TGI value of the medium-dose group (QD) was greater than that of the high-dose group (QW). Safety results showed that only a few mice in the control group and each treatment group had tumors exceeding 3000mm in volume. 3 Except for euthanasia, no euthanasia occurred due to severe weight loss. All treatment groups of AST-3424 were well tolerated during the experiment. Tumor growth curve of the BALB / c nude mouse model of subcutaneous hepatocellular carcinoma LI6664 after treatment with sorafenib and AST-3424.

[0210] The above research results show that AST-3424 has a significant inhibitory effect on the human liver cancer model, and under the premise of good tolerability, the new dosing regimen (QD×5) can increase the total dosage during the treatment cycle, thereby having a better therapeutic effect than the original regimen (QW×2) (the TGI value is larger after QD dosing cycle).

[0211] 1.2 Analysis and comparison of the efficacy of AST-3424 in lung cancer models

[0212] 1.2.1 Human lung cancer (H460-GFP) nude mouse orthotopic CDX model (QW×2 protocol)

[0213] In the study of the effect of AST-3424 on the subcutaneous CDX model of human lung cancer (H460-GFP) in nude mice, the tumor relative inhibition rate (TGI) of the negative control group, Taxol (15 mg / kg, BIWx4, IV) group, AST-3424 low-dose group (0.625 mg / kg, QW×2; 1 week off; QW×2, IV), AST-3424 medium-dose group (1.25 mg / kg, QW×2; 1 week off; QW×2, IV) and AST-3424 high-dose group (2.5 mg / kg, QW×2; 1 week off; QW×2, IV) were compared: tumor burden images of mice were non-invasively collected twice a week using the Fluor Vivo Model 100 imaging system, and the average tumor area of ​​each group was calculated based on the results of image analysis. The experimental results showed that the TGIs of the low, medium and high AST-3424 groups were 60.2%, 67.2% and 88% respectively, and 15 mg / kg of paclitaxel showed a TGI of 64%. The anti-tumor efficacy of 3424 was comparable to that of paclitaxel. Taxol and the current doses of AST-3424 had significant inhibitory effects on the subcutaneous model of human lung cancer (H460-GFP) in mice, with significant statistical differences compared to the control group (P < 0.01). The high-dose group of AST-3424 and the low-dose group of AST-3424 showed a dose-effect relationship. The data of this experiment are the data of the H460 model in Example 3-1 of the above-mentioned patent application 2 (see Table 5 and Figure 5 of the application document). The AST-3424 in this pharmacodynamic model was administered once a week for 3 or 6 consecutive doses. This regimen is similar to the dosing regimen in the Phase I clinical trial of AST-3424 (OBI-3424) conducted in China and the United States, and is referred to herein as the original regimen.

[0214] 1.2.2 Lung Cancer LU5173 Subcutaneous Xenograft PDX Model (QD×5 Protocol)

[0215] AST-3424 The anti-tumor effect of the drug was evaluated in the NOD / SCID nude mouse model of subcutaneous xenograft of lung cancer LU5173 in project E4354-B2116.

[0216] LU5173 was established from a lung cancer (small cell lung cancer) tumor of a female patient. Xenograft Model.

[0217] Modeling NOD / SCID mice were randomly divided into groups of 6 per group: a vehicle control group, an ifosfamide positive control group (6.0 mg / kg, QD x 5 / w, for 2 weeks), and an AST-3424 group (1 mg / kg, administered for 5 consecutive days, followed by a 16-day drug rest period and then 5 consecutive days, for a total of 10 doses, for 2 cycles). Efficacy was evaluated based on relative tumor inhibition (TGI) (%), and safety was assessed based on changes in animal body weight and mortality.

[0218] No animals died in the AST-3424 group and the treatment was well tolerated. On day 32 after the first dose, the average tumor volumes of mice in the vehicle control group, ifosfamide group, and AST-3424 group were 2276.68, 1229.15, and 503.91 mm, respectively. 3 The TGI (%) of the ifosfamide group was 44.45%, which showed no statistical difference compared with the control group (p>0.05); the TGI (%) of the AST-3424 group was 78.56%, which showed a significant statistical difference compared with the control group (P<0.001). Tumor growth curve of the BALB / c nude mouse model of lung cancer LU 5173 subcutaneously xenografted after treatment with AST-3424.

[0219] 1.2.3 Lung Cancer LU5161 Subcutaneous Xenograft PDX Model (QD×5 Protocol)

[0220] AST-3424 The anti-tumor effect of the drug was evaluated in the BALB / c nude mouse model of subcutaneous xenograft of lung cancer LU5161 in project E4354-B2131.

[0221] LU5161 was established from a lung cancer (small cell lung cancer) tumor of a female patient. Xenograft Model.

[0222] Modeling BALB / c nude mice were randomly divided into groups of six per group: a negative control group (glucose injection), an ifosfamide positive control group (60 mg / kg, QD x 5 / w, for 2 weeks), and an AST-3424 group (1 mg / kg, administered for 5 consecutive days, followed by a 16-day rest period and then 5 consecutive days, for a total of 10 doses, for 2 cycles). Efficacy was evaluated based on relative tumor inhibition (TGI) (%), and safety was assessed based on changes in animal body weight and mortality.

[0223] No animals died in the AST-3424 group and the treatment was well tolerated. On day 29 after the first administration, the average tumor volumes of mice in the negative control group, ifosfamide group, and AST-3424 group were 2238.97, 1636.39, and 94.42 mm, respectively. 3 The TGI (%) of the ifosfamide group was 27.39%, which was not statistically different from the control group (p>0.05); the TGI (%) of the AST-3424 group was 95.62%, which was statistically significantly different from the control group (P<0.001), and the tumor was cleared in one mouse. Tumor growth curve of the BALB / c nude mouse model of lung cancer LU5161 subcutaneously transplanted with AST-3424.

[0224] The above research results show that AST-3424 has a significant inhibitory effect on the human lung cancer model, and under the premise of good tolerability, the new dosing regimen (QD×5) can increase the total dosage during the treatment cycle, thereby having a better therapeutic effect than the original regimen (QW×2) (the TGI value is larger after QD dosing cycle).

[0225] 1.3. Analysis and comparison of efficacy of AST-3424 in pancreatic cancer models

[0226] 1.3.1 Pancreatic cancer PA1280 subcutaneous xenograft model (QW×3 regimen)

[0227] AST-3424 The anti-tumor effect of the drug was evaluated in the pancreatic cancer PA1280 subcutaneous xenograft BALB / c nude mouse model in project E4354-B1802.

[0228] PA1280 was established from a pancreatic cancer tumor in a female patient. Xenograft Model.

[0229] BALB / c nude mice were randomly divided into six groups: AST-3424 (2.5 mg / kg) and a control group (D5W (pH 7.4) vehicle). The mice were administered once weekly through the tail vein for three weeks, followed by one week of observation. Efficacy was evaluated based on relative tumor inhibition (TGI), and safety was assessed based on changes in body weight and mortality.

[0230] There was no animal death in the AST-3424 (2.5 mg / kg) treatment group, no obvious drug toxicity was shown, and the animals were well tolerated during the treatment period.

[0231] The average tumor volume of mice in the vehicle control group was 475.07 mm on day 7 (Day 27) after the end of the 3-week dosing cycle. 3 The average tumor volume of the AST-3424 (2.5 mg / kg) treatment group was 190.34 mm on Day 27 after the 3-week dosing cycle. 3 Compared with the control group, there was a statistically significant difference (P < 0.05), and the relative tumor inhibition rate TGI (%) was 60%. Please see the tumor growth curves of each group in Figure 9. Tumor growth curve of the pancreatic cancer PA1280 subcutaneous xenograft BALB / c nude mouse model after treatment with AST-3424.

[0232] The experimental results showed that AST-3424 at a dose of 2.5 mg / kg had an PA1280 showed statistically significant inhibition of tumor growth in pancreatic cancer. AST-3424 was well tolerated by tumor-bearing mice at all doses tested.

[0233] 1.3.2 Pancreatic cancer PA2637 subcutaneous xenograft model (QD×5 regimen)

[0234] AST-3424 The anti-tumor effect was evaluated in the pancreatic cancer PA2637 subcutaneous xenograft NOD.SCID mouse animal model in project E4354-B2131.

[0235] PA2637 was established from a patient-derived pancreatic cancer tumor. Xenograft Model.

[0236] Modeling mice were randomly divided into groups of six per group: a negative control group (glucose injection), an ifosfamide positive control group (60 mg / kg, QD x 5 / w, for two weeks), and an AST-3424 group (1 mg / kg, administered for five consecutive days, followed by a 16-day break and then five consecutive days, for a total of 10 doses, for two cycles). Efficacy was evaluated based on the relative tumor inhibition rate (TGI), and safety was assessed based on changes in animal body weight and mortality.

[0237] No animals died in the AST-3424 group, and no significant drug toxicity was observed. Treatment was well tolerated. On day 36 after the first dose, the average tumor volumes of mice in the vehicle control group, AST-3424 group, and ifosfamide group were 977.46, 186.08, and 938.33 mm, respectively. 3The TGI (%) of the ifosfamide group was 2.74%, which was not statistically different from the control group; the TGI (%) of the AST-3424 group was 80.78%, which was statistically significantly different from the control group (P<0.001), and the tumor was cleared in one mouse. Please refer to Figure 10 for the tumor growth curves of each group. Tumor growth curve of the pancreatic cancer PA2637 subcutaneous xenograft BALB / c nude mouse model after treatment with AST-3424.

[0238] 1.3.3 Pancreatic cancer PA9451 subcutaneous xenograft model (QD×5 and QW regimens)

[0239] AST-3424 The anti-tumor effect of PA9451 in a pancreatic cancer subcutaneous xenograft BALB / c nude mouse model was evaluated in project E4354-B2138.

[0240] PA9451 is established from a patient-derived pancreatic cancer tumor. Xenograft Model.

[0241] Modeling mice were randomly divided into groups of 6 per group: a vehicle control group (glucose injection); an olaparib positive control group (50 mg / kg, QD×30); a low-dose AST-3424 group (0.5 mg / kg, administered continuously for 5 days, followed by a 16-day break, followed by a 5-day break, for a total of 10 doses, for 2 cycles); a medium-dose group (1 mg / kg, administered continuously for 5 days, followed by a 16-day break, followed by a 5-day break, for a total of 10 doses, for 2 cycles); and a high-dose group (2 mg / kg, administered once a week for 2 weeks). Efficacy was evaluated based on relative tumor inhibition rate (TGI), and safety was evaluated based on changes in animal body weight and mortality.

[0242] There were no animal deaths in the AST-3424 group, no obvious drug toxicity was observed, and the drug was well tolerated during treatment.

[0243] On day 36 after the first dose, the average tumor volumes of mice in the vehicle control group and olaparib group were 661.61 and 344.84 mm3, respectively. The TGI (%) of the olaparib group was 47.88%, which was significantly different from the control group (P < 0.001). The average tumor volumes of the AST-3424 low (QD), medium (QD), and high dose (QW) groups were 54.83 mm3, respectively. 3 、46.05mm 3 and 76.78mm 3, TGI (%) were 91.77%, 92.99%, and 88.53%, respectively, which were statistically significantly different from the control group (P<0.001). There was no statistically significant difference between the AST-3424 groups. The tumor growth curves of each group are shown in Figure 11. Tumor growth curve of the pancreatic cancer PA9451 subcutaneous xenograft BALB / c nude mouse model after treatment with AST-3424.

[0244] The above research results show that AST-3424 has a significant inhibitory effect on the human pancreatic cancer model, and under the premise of good tolerability, the new dosing regimen (QD×5) can increase the total dosage during the treatment cycle, thereby having a better therapeutic effect than the original regimen (QW×2) (the TGI value is larger after QD dosing cycle).

[0245] 1.3.4 Pancreatic cancer PA1170 subcutaneous xenograft model (QD×5 and QW regimens)

[0246] AST-3424 The anti-tumor effect of the drug was evaluated in the pancreatic cancer PA1170 subcutaneous xenograft BALB / c nude mouse model under project E4354-B2135.

[0247] PA1170 is established from a patient-derived pancreatic cancer tumor. Xenograft Model.

[0248] Modeling mice were randomly divided into groups of 6 per group: a vehicle control group (10% anhydrous ethanol + 10% polyoxyethylene (35) castor oil + 80% glucose injection D5W (pH 7.4)); an olaparib positive control group (50 mg / kg, QD × 30); an AST-3424 administration group (1 mg / kg, continuous administration for 5 days, 16 days after drug withdrawal, and then continuous administration for 5 days, a total of 10 administrations, 2 cycles); and an AST-3424 administration group (2 mg / kg, once a week for 2 weeks). Efficacy was evaluated based on relative tumor inhibition rate (TGI) (%), and safety was evaluated based on animal body weight changes and mortality.

[0249] There were no animal deaths in the AST-3424 group, no obvious drug toxicity was observed, and the drug was well tolerated during treatment.

[0250] On the 30th day after the first administration, the average tumor volumes of mice in the vehicle control group and olaparib group were 1182.66 and 517.75 mm, respectively. 3The TGI (%) of the olaparib group was 57.66%, which was not significantly different from that of the control group (P>0.05). The average tumor volume of the AST-3424 (1 mg / kg, QD) and (2 mg / kg, QW) groups was 29.50 mm 3 and 64.50mm 3 , TGI (%) were 97.60% and 94.83%, respectively, which were statistically significantly different from the control group (P < 0.05). There was no statistically significant difference between the AST-3424 groups. The tumor growth curves of each group are shown in Figure 12. Tumor growth curve of the pancreatic cancer PA1170 subcutaneous xenograft BALB / c nude mouse model after treatment with AST-3424.

[0251] The above research results show that AST-3424 has a significant inhibitory effect on the human pancreatic cancer model, and under the premise of good tolerability, the new dosing regimen (QD×5) can increase the total dosage during the treatment cycle, thereby having a better therapeutic effect than the original regimen (QW×2) (the TGI value is larger after QD dosing cycle).

[0252] 1.4. AST-3424 in the treatment of different solid tumors: Efficacy and preliminary safety analysis and comparison of the new regimen (QD×5) and the old regimen (QW×2) in liver cancer, lung cancer, and pancreatic cancer

[0253] Overall, AST-3424 has a significant inhibitory effect on human liver cancer, lung cancer, and pancreatic cancer models. Under the premise of good tolerability, the new dosing regimen (QD×5) can increase the total dosage during the treatment cycle, thereby having a better therapeutic effect than the original regimen (QW×2) (the TGI value is larger after QD dosing cycle).

[0254] 2. Non-clinical safety experiments - toxicokinetic studies

[0255] 2.1 Toxicokinetics (D1+D8 / 21Days)

[0256] The toxicokinetics (TK) of AST-3424 were evaluated in SD rats (8 / dose / sex). AST-3424 was administered as a single injection at doses of 3, 10, and 30 mg / kg on D1, D8, D22, and D29, respectively. AST-3424 and AST-2660 were rapidly eliminated with mean half-lives of 0.145-0.373 h and 0.589-1.00 h, respectively. The exposure of AST-3424 (C max and AUC last) increased linearly but not in a dose-proportional manner with increasing doses. AST-3424 accumulated moderately on Day 29 compared to Day 1, but exposure to AST-2660 was reduced.

[0257] The toxicokinetics of AST-3424 were evaluated in cynomolgus monkeys. AST-3424 was administered as a single 30-minute intravenous infusion at doses of 0.1, 0.3, and 0.6 mg / kg on Days 1, 8, 22, and 29, respectively. max (maximum plasma peak concentration) and AUC last The area under the concentration-time curve (AUC) from the start of dosing to the last sample collection time) increased linearly but not dose-proportional with increasing doses in cynomolgus monkeys. The mean half-lives of AST-3424 and AST-2660 were 0.231h-0.592h and 0.919h-1.26h, respectively, independent of dose, time, or gender. The volume of distribution of AST-3424 was equal to or greater than the extracellular fluid volume (223-676ml / kg), with Cl (clearance) approaching the hepatic plasma flow rate (705-1090ml / h / kg). C was higher on day 29 compared to day 1. max and AUC last The ratios ranged from 0.90 to 1.21, and no accumulation of AST-3424 was observed. AST-2660 exposure was 27.2% and 11.0% of the AST-3424 exposure observed on Day 1 and Day 29, respectively.

[0258] 2.2 Toxicokinetics (QD×5 / 21Days)

[0259] This study evaluated the toxicokinetics of AST-3424 injection in cynomolgus monkeys administered once daily for 5 consecutive days via 30-minute intravenous infusion, followed by a 16-day drug-free period for 5 cycles. The experiment included a placebo control group (0 mg / kg), a low-dose group (0.1 mg / kg), a medium-dose group (0.2 mg / kg), and a high-dose group (0.4 / 0.3 mg / kg, with the first cycle dose being 0.4 mg / kg and the second cycle dose being adjusted to 0.3 mg / kg due to near-death euthanasia of two monkeys).

[0260] After repeated administration for 5 cycles in male and female cynomolgus monkeys, the exposure of the prototype drug AST-3424 and its metabolite AST-2660 (measured in AUC(0-t)) increased with increasing dose, with no significant gender differences or exposure accumulation. The plasma concentration of the prototype drug AST-3424 reached its peak immediately after the end of administration (T max , peak time), half-life (T 1 / 2) is approximately 0.20-0.78 hours; the metabolite AST-2660 reaches its peak at approximately 0.73-1.25 hours after the start of administration (T max ), half-life (T 1 / 2 ) is approximately 0.82-1.23 hours. The exposure of the metabolite AST-2660 accounts for 5.25%-12.27% of the parent drug.

[0261] FIG13 shows the single-dose toxicokinetic parameters of AST-3424 and AST-2660 at the above two dosing regimens.

[0262] The experiments on the above two dosing regimens show that the toxicokinetics of AST-3424 and its metabolite AST-2660 are not much different. It can be considered that the new daily dosing regimen has similar toxicokinetic parameters to the previous weekly dosing regimen, with little difference. In other words, the smaller dose of the daily dosing regimen results in less exposure to its metabolite AST-2660, which may lead to fewer or milder adverse reactions in human trials.

[0263] 3. Non-clinical safety experiments - repeated dose toxicology studies

[0264] 3.1 Repeated-dose toxicology study (D1+D8 / 21 days)

[0265] In a nonclinical Good Laboratory Practice (GLP) repeat-dose toxicity study in Sprague-Dawley rats, AST-3424 was administered intravenously at doses of 0, 3, 10, and 30 mg / kg on days 1, 8, 22, and 29 for two cycles according to the clinical dosing schedule. Rats developed dose-dependent, test article-related lesions at the injection site and tail tip. At the 10 mg / kg dose, there were mild, reversible changes in hematological and serum biochemical parameters. The no observed toxicity level (NOAEL) was established at 3 mg / kg in rats, and the dose at which 10% of animals experienced severe toxicity (STD10) was 30 mg / kg.

[0266] In a GLP repeated-dose toxicity study in cynomolgus monkeys, 0, 0.1, 0.3, or 0.6 mg / kg of AST-3424 were intravenously infused over 30 minutes on days 1, 8, 22, and 29. After multiple doses in cynomolgus monkeys, the most significant toxic reactions observed with AST-3424 were diarrhea and vomiting, and pathological findings showed intestinal necrosis / hyperplasia. At the same time, AST-3424 had a mild but reversible effect on red blood cell-related hematological indicators. The highest non-serious toxic dose (HNSTD) in monkeys was ultimately determined to be 0.6 mg / kg.

[0267] 3.2 Repeated-dose toxicology study (QD×5 / 21Days)

[0268] In a GLP-induced repeat-dose toxicity study in cynomolgus monkeys, AST-3424 was administered intravenously over 30 minutes on Days 1-5, 22-26, 43-47, 64-68, and 85-89. Following repeated dosing, AST-3424 primarily caused soft, watery stools. Pathology revealed mild to mild mucosal atrophy in the duodenum and jejunum, and mild to moderate mucosal atrophy in the cecum and colon. AST-3424 also had mild but reversible effects on red blood cell and white blood cell-related hematological parameters. Under these experimental conditions, the HNSTD in cynomolgus monkeys was determined to be 0.3 mg / kg, with target organs of toxicity being the duodenum, jejunum, colon, and cecum.

[0269] During the trial, decreases in #EOS (absolute eosinophil count), %EOS (% eosinophil count), #RET (absolute reticulocyte count), and %RETIC (% reticulocyte count) were primarily observed in the ≥0.1 mg / kg dose group. Decreases in #NEUT, #LYMPH (absolute lymphocyte count), and %LYMPH (% lymphocyte count) were also observed in the ≥0.2 mg / kg dose group. Decreases in WBC (white blood cells), HGB (hemoglobin), and HCT (hematocrit) were also observed in the 0.4 / 0.3 mg / kg dose groups. The decrease in eosinophils correlated with a decrease in eosinophils (%) on bone marrow smears obtained at the end of the dosing period, while the decrease in lymphocytes correlated with a decrease in thymic cortical lymphocytes on microscopic examination, suggesting a secondary stress response. All of these hematological changes resolved during the recovery period.

[0270] Figure 14 shows the repeated-dose toxicology profiles of AST-3424 for the above two dosing regimens.

[0271] The experiments on the above two dosing regimens show that the toxicological studies of AST-3424 show that the toxic reactions of the daily dosing regimen and the weekly dosing regimen are concentrated in the blood system and gastrointestinal tract, and the adverse reactions are similar. No new adverse reactions are caused by the high-frequency dosing regimen; the high-frequency dosing regimen reduces the HNSTD of cynomolgus monkeys to 0.3mg / kg, which suggests that the final dose of the daily dosing regimen in subsequent human clinical trials may be lower than the 6mg / m 2 .

[0272] Summary of Animal Model Experiments

[0273] Compared with the old dosing regimen of AST-3424 (OBI-3424), which used a 21-day cycle with dosing on days 1 and 8, the new dosing regimen has preliminarily verified its efficacy and preliminary safety and tolerability through multiple solid tumor PDX animal models. The new dosing regimen has further verified its safety and tolerability at specific doses through toxicokinetic studies in crab-eating macaques and repeated-dose toxicology studies. The new dosing regimen has further verified the safety and tolerability of a series of new specific dosing regimens through human clinical trial safety model predictions. Overall, the new dosing regimen may have advantages over the old dosing regimen in terms of efficacy and safety in the next phase of further human clinical trials of the drug, including better efficacy and milder adverse reactions.

[0274] Since the chemical structure of TFX05-01 is similar to that of AST-3424, and it also produces AST-2660 after AKR1C3 activation, thereby exerting a tumor inhibitory effect, the applicant speculates that the compound has similar metabolic properties and behaviors in the human body as AST-3424. Therefore, it can be predicted that TFX05-01 will have similar animal experiment / clinical trial data as AST-3424. Compared with the ongoing Phase I clinical trial adopting a 21-day cycle with dosing on days 1 and 8, the new dosing regimen designed in this application may have advantages in efficacy and safety: better efficacy and milder adverse reactions.

[0275] All patent applications cited in this application are incorporated into this specification in their entirety.

[0276] Non-patent literature cited in this application (academic journal articles, academic conference papers, etc.):

[0277] Document 1, Meng F, Li WF, Jung D, et al. A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity. Am J Cancer Res. 2021; 11(7): 3645-3659;

[0278] Reference 2, Evans K, Duan J, Pritchard T, et al. OBI-3424, a Novel AKR1C3-Activated Prodrug, Exhibits Potent Efficacy against Preclinical Models of T-ALL. Clin Cancer Res. 2019;25(14):4493-4503. doi:10.1158 / 1078-0432.CCR-19-0551;

[0279] Reference 3, Wang Y, Liu Y, Zhou C, et al. An AKR1C3-specific prodrug with potent anti-tumor activities against T-ALL. Leuk Lymphoma. 2020;61(7):1660-1668. doi:10.1080 / 10428194.2020.1728746;

[0280] Reference 4, He P, Wang C, Wang Y, et al. A Novel AKR1C3Specific Prodrug TH3424With Potent Antitumor Activity in Liver Cancer[retracted in: Clin Pharmacol Ther. 2021Jul;110(1):262]. Clin Pharmacol Ther. 2021, 110(1):229-237. doi:10.1002 / cpt.2171;

[0281] Reference 5,

[0282] Research poster: Tsimberidou, Apostolia & Verschraegen, Claire & Hsu, Pei & Pearce, Tillman. (2022). Safety, pharmacokinetics, and clinical activity of OBI-3424, an AKR1C3-activated prodrug, in patients with advanced or metastatic solid tumors: A phase 1 dose-escalation study. Journal of Clinical Oncology. 40.3030-3030.10.1200 / JCO.2022.40.16_suppl.3030. The poster can be downloaded from the OBIPharma Inc. website at https: / / www.obipharma.com / news / news-2022 / poster-presentations-at-the-2022-asco-annual-meeting-for-adagloxad-simolenin-obi-999-and-obi-3424 / .

[0283] And research article: Tsimberidou AM, Verschraegen CF, Wesolowski R, Shia CS, Hsu P, Pearce TE. Phase 1dose-escalation study evaluating the safety, pharmacokinetics, and clinical activity of OBI-3424 in patients with advanced or metastatic solid tumors. Br J Cancer.2023;129(2):266-274.doi:10.1038 / s41416-023-02280-4;

[0284] Literature 6: Zhang Y, Qin S, Chao J, Luo Y, Sun Y and Duan J (2022) The In-Vitro Antitumor Effects of AST-3424Monotherapy and Combination Therapy With Oxaliplatin or 5-Fluorouracil in Primary Liver Cancer.Front.Oncol.12:885139.doi:10.3389 / fonc.2022.885139;

[0285] Document 7: Charles Z Ding, Zhe Cai, Wei Sha. Preclinical evaluation of TFX05-01, a selective AKR1C3-targeted prodrug for solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5691. And the corresponding AACR2022 Poster.

[0286] Unless otherwise specified, the various English abbreviations in this application are based on the definitions in pharmaceutical and medical textbooks.

Claims

1. A dosing regimen for treating human tumors with an AKR1C3-activated DNA alkylating agent prodrug compound that metabolizes AST-2660, either alone or in combination, characterized in that The interval between two administrations in one treatment cycle is 8-144 hours, preferably 12-72 hours, and the number of administrations in one treatment cycle is not less than 3 times. The tumor is preferably a malignant tumor, and the AKR1C3 enzyme-activated prodrug is selected from the compound formula (1)-(6): Among them, X, Y, Z, R, A and X 10 As defined in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), T is Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as set forth in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A); in: A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=CR 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, -O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 S02R 100 、-SO2R 100 、-SO2NR 101 R 10 2 , C1-C6 alkyl, C3-C10 heterocyclic group; Among them, R 100 , R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 Together with the nitrogen atom to which it is attached, it forms a 5-7 membered heterocyclic ring; wherein the alkyl and aryl groups are each substituted with 1-3 halogen groups or 1-3 C1-C6 alkyl groups; R 1 and R 2 each independently is phenyl or methyl; X, Y and Z are each independently hydrogen or halo; R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl; Wherein, the definition of Rw is as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A); Among them, the definitions of R1, R2, R3, R4, and T are as recorded in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1.

2. The dosing regimen of AST-3424 alone or in combination for the treatment of human tumors is characterized by The interval between two administrations in one treatment cycle is 8-144 hours, preferably 12-72 hours, and the administration number in one treatment cycle is not less than 3 times. The tumor is preferably a malignant tumor.

3. The dosage regimen according to claim 1 or 2, characterized in that The interval between two administrations in one treatment cycle is 8, 12, 24, 48, 72, 96, 120, or 144 hours. The tumor includes solid tumors and blood tumors. The solid tumor is selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, intrahepatic bile duct cancer, pancreatic cancer, submandibular gland cancer, prostate cancer, breast cancer, and melanoma. The blood tumor is selected from acute lymphocytic leukemia, preferably acute T lymphocytic leukemia and acute B lymphocytic leukemia.

4. The dosage regimen according to claim 3, characterized in that The interval between two doses in one treatment cycle is 12, 24, 48, or 72 hours. The administration method is intravenous injection, preferably intravenous drip. The dose for each administration is 1-8 mg / m 2 .

5. The dosage regimen according to claim 3, wherein one treatment cycle is 21 days, specifically selected from the following regimens: The drug was administered once a day for a total of 3 times on days 1, 2, and 3, and no drug was administered for the other 18 days; The drug was administered once a day for a total of 4 times on days 1, 2, 3, and 4, and no drug was administered for the other 17 days; The drug was administered once a day for a total of 5 times on days 1, 2, 3, 4, and 5, and no drug was administered for the other 16 days; The drug was administered once a day for a total of 6 times on days 1, 2, 3, 4, 5, and 6, and no drug was administered for the other 15 days; The drug was administered once a day for a total of 7 times on days 1, 2, 3, 4, 5, 6, and 7, and no drug was administered for the other 14 days; The drug was administered once a day for a total of 3 times on days 1, 3, and 5, and no drug was administered for the other 18 days; The drug was administered once a day for a total of 4 times on days 1, 3, 5, and 7, and no drug was administered for the other 17 days; The drug was administered once a day for a total of 5 times on days 1, 3, 5, 7, and 9, and no drug was administered for the other 16 days; The drug was administered once a day for a total of 3 times on days 1, 4, and 7, and no drug was administered for the other 18 days; The drug was administered once a day for a total of 3 times on the 1st, 5th and 9th days, and no drug was administered for the other 18 days.

6. The dosage regimen according to claim 3, wherein one treatment cycle is 14 days, specifically selected from the following regimens: The drug was administered once a day for a total of 3 times on days 1, 2, and 3, and no drug was administered on the other 11 days; The drug was administered once a day for a total of 4 times on days 1, 2, 3, and 4, and no drug was administered for the other 10 days; The drug was administered once a day for a total of 5 times on days 1, 2, 3, 4, and 5, and no drug was administered on the other 9 days; The drug was administered once a day for a total of 6 times on days 1, 2, 3, 4, 5, and 6, and no drug was administered on the other 8 days; The drug was administered once a day for a total of 7 times on days 1, 2, 3, 4, 5, 6, and 7, and no drug was administered on the other 7 days; The drug was administered once a day for a total of 3 times on days 1, 3, and 5, and no drug was administered on the other 11 days; The drug was administered once a day for a total of 4 times on days 1, 3, 5, and 7, and no drug was administered for the other 10 days; The drug was administered once a day for a total of 5 times on days 1, 3, 5, 7, and 9, and no drug was administered on the other 9 days; The drug was administered once a day for a total of 3 times on the 1st, 4th and 7th days, and no drug was administered on the other 11 days.

7. The dosage regimen according to claim 3, wherein one treatment cycle is 28 days, specifically selected from the following regimens: The drug was administered once a day for a total of 3 times on days 1, 2, and 3, and no drug was administered for the other 25 days; The drug was administered once a day for a total of 4 times on days 1, 2, 3, and 4, and no drug was administered for the other 24 days; The drug was administered once a day for a total of 5 times on days 1, 2, 3, 4, and 5, and no drug was administered on the other 23 days; The drug was administered once a day for a total of 6 times on days 1, 2, 3, 4, 5, and 6, and no drug was administered on the other 22 days; The drug was administered once a day for a total of 7 times on days 1, 2, 3, 4, 5, 6, and 7, and no drug was administered on the other 21 days; The drug was administered once a day for a total of 8 times on days 1, 2, 3, 4, 5, 6, 7, and 8, and no drug was administered for the other 20 days; The drug was administered once a day on days 1, 2, 3, 4, 5, 6, 7, 8, and 9 for a total of 9 times, and no drug was administered on the other 19 days; The drug was administered once a day on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 for a total of 10 times, and no drug was administered for the other 18 days; The drug was administered once a day for a total of 3 times on days 1, 3, and 5, and no drug was administered for the other 25 days; The drug was administered once a day for a total of 4 times on days 1, 3, 5, and 7, and no drug was administered for the other 24 days; The drug was administered once a day for a total of 5 times on days 1, 3, 5, 7, and 9, and no drug was administered on the other 23 days; The drug was administered once a day for a total of 3 times on days 1, 4, and 7, and no drug was administered for the other 25 days; The drug was administered once a day for a total of 4 times on days 1, 4, 7, and 10, and no drug was administered for the other 24 days; The drug was administered once a day for a total of 5 times on days 1, 4, 7, 10, and 13, and no drug was administered on the other 23 days; The drug was administered once a day for a total of 3 times on days 1, 5, and 9, and no drug was administered for the other 25 days; The drug was administered once a day for a total of 4 times on days 1, 5, 9, and 13, and no drug was administered on the other 24 days.

8. Pharmaceutical use of an AKR1C3-activated DNA alkylating agent prodrug compound that metabolizes AST-2660, which is used to prepare a drug for treating human tumors alone or in combination, characterized in that The interval between two administrations in one treatment cycle is 8-144 hours, preferably 12-72 hours, and the number of administrations in one treatment cycle is not less than 3 times. The tumor is preferably a malignant tumor, and the AKR1C3 enzyme-activated prodrug is selected from the compound formula (1)-(6): Among them, X, Y, Z, R, A and X 10 As defined in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), T is Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as set forth in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A); in: A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=CR 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, -O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 S02R 100 、-SO2R 100 、-SO2NR 101 R 10 2 , C1-C6 alkyl, C3-C10 heterocyclic group; Among them, R 100 , R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 Together with the nitrogen atom to which it is attached, it forms a 5-7 membered heterocyclic ring; wherein the alkyl and aryl groups are each substituted with 1-3 halogen groups or 1-3 C1-C6 alkyl groups; R 1 and R 2 each independently is phenyl or methyl; X, Y and Z are each independently hydrogen or halo; R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl; Wherein, the definition of Rw is as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A); Among them, the definitions of R1, R2, R3, R4, and T are as recorded in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1.

9. Pharmaceutical use of AST-3424, which is used to prepare a drug for treating human tumors alone or in combination, characterized in that The interval between two administrations in one treatment cycle is 8-144 hours, preferably 12-72 hours, and the administration number in one treatment cycle is not less than 3 times. The tumor is preferably a malignant tumor.

10. The pharmaceutical use according to claim 8 or 9, characterized in that The interval between two administrations in one treatment cycle is 8, 12, 24, 48, 72, 96, 120 or 144 hours. The tumor includes solid tumors and blood tumors. The solid tumor is selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, intrahepatic bile duct cancer, pancreatic cancer, submandibular gland cancer, prostate cancer, breast cancer, and melanoma. The blood tumor is selected from acute lymphocytic leukemia, preferably acute T lymphocytic leukemia and acute B lymphocytic leukemia.

11. The pharmaceutical use according to claim 10, characterized in that The interval between two doses in one treatment cycle is 12, 24, 48, or 72 hours. The administration method is intravenous injection, preferably intravenous drip. The dose for each administration is 1-8 mg / m 2 .

12. The pharmaceutical use according to claim 10, wherein the treatment cycle is selected from 14 days, 21 days or 28 days, When a treatment cycle is 21 days, the specific regimen is selected from the following: The drug was administered once a day for a total of 3 times on days 1, 2, and 3, and no drug was administered for the other 18 days; The drug was administered once a day for a total of 4 times on days 1, 2, 3, and 4, and no drug was administered for the other 17 days; The drug was administered once a day for a total of 5 times on days 1, 2, 3, 4, and 5, and no drug was administered for the other 16 days; The drug was administered once a day for a total of 6 times on days 1, 2, 3, 4, 5, and 6, and no drug was administered for the other 15 days; The drug was administered once a day for a total of 7 times on days 1, 2, 3, 4, 5, 6, and 7, and no drug was administered for the other 14 days; The drug was administered once a day for a total of 3 times on days 1, 3, and 5, and no drug was administered for the other 18 days; The drug was administered once a day for a total of 4 times on days 1, 3, 5, and 7, and no drug was administered for the other 17 days; The drug was administered once a day for a total of 5 times on days 1, 3, 5, 7, and 9, and no drug was administered for the other 16 days; The drug was administered once a day for a total of 3 times on days 1, 4, and 7, and no drug was administered for the other 18 days; The drug was administered once a day for a total of 3 times on days 1, 5, and 9, and no drug was administered for the other 18 days; When a treatment cycle is 14 days, the specific regimen is selected from the following: The drug was administered once a day for a total of 3 times on days 1, 2, and 3, and no drug was administered on the other 11 days; The drug was administered once a day for a total of 4 times on days 1, 2, 3, and 4, and no drug was administered for the other 10 days; The drug was administered once a day for a total of 5 times on days 1, 2, 3, 4, and 5, and no drug was administered on the other 9 days; The drug was administered once a day for a total of 6 times on days 1, 2, 3, 4, 5, and 6, and no drug was administered on the other 8 days; The drug was administered once a day for a total of 7 times on days 1, 2, 3, 4, 5, 6, and 7, and no drug was administered on the other 7 days; The drug was administered once a day for a total of 3 times on days 1, 3, and 5, and no drug was administered on the other 11 days; The drug was administered once a day for a total of 4 times on days 1, 3, 5, and 7, and no drug was administered for the other 10 days; The drug was administered once a day for a total of 5 times on days 1, 3, 5, 7, and 9, and no drug was administered on the other 9 days; The drug was administered once a day for a total of 3 times on days 1, 4, and 7, and no drug was administered on the other 11 days; When a treatment cycle is 28 days, the specific regimen is selected from the following: The drug was administered once a day for a total of 3 times on days 1, 2, and 3, and no drug was administered for the other 25 days; The drug was administered once a day for a total of 4 times on days 1, 2, 3, and 4, and no drug was administered for the other 24 days; The drug was administered once a day for a total of 5 times on days 1, 2, 3, 4, and 5, and no drug was administered on the other 23 days; The drug was administered once a day for a total of 6 times on days 1, 2, 3, 4, 5, and 6, and no drug was administered on the other 22 days; The drug was administered once a day for a total of 7 times on days 1, 2, 3, 4, 5, 6, and 7, and no drug was administered on the other 21 days; The drug was administered once a day for a total of 8 times on days 1, 2, 3, 4, 5, 6, 7, and 8, and no drug was administered for the other 20 days; The drug was administered once a day on days 1, 2, 3, 4, 5, 6, 7, 8, and 9 for a total of 9 times, and no drug was administered on the other 19 days; The drug was administered once a day on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 for a total of 10 times, and no drug was administered for the other 18 days; The drug was administered once a day for a total of 3 times on days 1, 3, and 5, and no drug was administered for the other 25 days; The drug was administered once a day for a total of 4 times on days 1, 3, 5, and 7, and no drug was administered for the other 24 days; The drug was administered once a day for a total of 5 times on days 1, 3, 5, 7, and 9, and no drug was administered on the other 23 days; The drug was administered once a day for a total of 3 times on days 1, 4, and 7, and no drug was administered for the other 25 days; The drug was administered once a day for a total of 4 times on days 1, 4, 7, and 10, and no drug was administered for the other 24 days; The drug was administered once a day for a total of 5 times on days 1, 4, 7, 10, and 13, and no drug was administered on the other 23 days; The drug was administered once a day for a total of 3 times on days 1, 5, and 9, and no drug was administered for the other 25 days; The drug was administered once a day for a total of 4 times on days 1, 5, 9, and 13, and no drug was administered on the other 24 days.

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