Patent ID: 12233047

DETAILED DESCRIPTION OF THE EMBODIMENT

The following examples further illustrate the invention, but the invention is not limited thereto.

Embodiment 1. Synthesis of Compound C

Compound C was synthesized according to WO2015161032A1.

Embodiment 2. Synthesis of Compound 23, 3-(2-(1H-pyrazolo[3,4-b]pyridin-5-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide

Compound 23 was synthesized according to embodiment 23 of WO2012000304A1.

Embodiment 3. Synthesis of Compound 41, 3-(2-(1H-pyrazolo[3,4-b]pyridin-5-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide Dimesylate

Compound 41 was synthesized according to embodiment 41 of WO2012000304A1.

Embodiment 4. Evaluation of Activity of Compound 23 on Various Type of FLT3 Kinase

The kinase assay was conducted using KINOMEscan™.

Kinase-tagged T7 phage strains were grown in parallel in 24-well blocks in anE. colihost derived from the BL21 strain.E. coliwere grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection=0.4) and incubated with shaking at 32° C. until lysis (90-150 minutes). The lysates were centrifuged (6,000×g) and filtered (0.2 μm) to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compound (Compound 23) in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compound were prepared as 40× stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR. The result was shown in Table 1.

TABLE 1Compound 23 inhibits FLT3 kinases with nanomolar potencyCompound 23kinase (gene symbol)% Ctrl@10 nM% Ctrl@100 nMFLT3 (wild type)2.40.75FLT3 (D835H)182.2FLT3 (D835Y)4217FLT3 (ITD)582.9FLT3 (K663Q)50.6FLT3 (N841I)2.90.05FLT3 (R834Q)2315Note:The initial screening result for binding is indicated by “% Ctrl”. A lower % Ctrl value indicates stronger binding of the compound to the protein.
% Ctrl value=[(test compound signal−positive control signal)/(negative control signal−positive control signal)]×100. Negative control=DMSO (100% Ctrl); positive control=control compound (0% Ctrl).

Embodiment 5. Study of the Combination of Compound C and Compound 23 on Human MOLM-13 AML Mouse Xenograft Model

1.1. Preparation of Test Compound Solution

The formulation of Compound C was prepared as homogenous suspension in 0.2% HPMC using a mortar. The drug suspension was prepared every three days or once a week and placed at 4° C. when not in use. Before use, the drug suspension was placed at room temperature and shaken to mix well. The formulation of Compound C was administered to mice via p.o. in a volume of 10 mL/kg.

The formulation of Compound 23 was prepared in 0.2% HPMC in a mortar to generate homogenous suspension. The drug suspension was prepared once every other day and placed at 4° C. when not in use. Before use, the drug suspension was placed at room temperature and shaken well. The formulation of Compound 23 was administered to mice via p.o. in a volume of 10 mL/kg.

1.2. Cell Lines

MOLM-13 cell lines (Human AML cell line MOLM-13 (FLT3-ITD positive and wild type TP53 gene)) were maintained in RPMI-1640 (Cat. C11875500BT, GIBCO) with 10% fetal bovine serum (Cat. 10099-141, GIBCO), 1% L-glutamine, 100 U/mL penicillin G and 100 μg/mL streptomycin (Cat. GNM15140, Hangzhou Geno Biomedical Technology Co., Ltd). Cells were incubated at 37° C. in a humidified incubator with 5% CO2.

1.3. Animals

Six to eight weeks old, female, NOD SCID and BALB/c nude mice were used. Animals were obtained from Vital River Laboratories (VRL, Beijing, China, license number SCXK(J) 2012-0001 and SCXK(J) 2016-0006). The body weights were 17-24 g.

1.4. Animal Housing and Care

Animals were housed in the SPF animal laboratory of Experiment Animal Center in Suzhou GenePharma Co., Ltd. The daily care of animals were conducted by full-time employee of the Experimental Animal Center of GenePharma. Ascentage staff were responsible for conducting the experiments.

1.5. Animal Identification

Each cage was identified by a cage card with the study name, study number, study group, species, and sex. Individual mice were identified by ear tags.

1.6. Randomization

Animals were randomized based on the body weight. However, more than 5 animals were included in each treatment group to minimize the variation between animals.

1.7. Operation and Management Specifications

The protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of GenePharma prior to conduct. During the study, the care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

1.8. Housing Conditions

Housing: Animals were housed in cages with not more than 5 mice per cage.

Range of daily temperature: 20-26° C.

Range of daily humidity: 40%-70%.

Light: 12 hours on and 12 hours off.

Diet and Water

Radiation-sterilized dry granule food was offered ad libitum during the study period. Autoclaved second-level osmotic water was made freely available from water bottles. Reverse osmosis (RO) water was autoclaved before use. Animals had free access to sterile drinking water.

Bedding

Wood shavings were used for animal bedding, which were sterilized by autoclave before use. Beddings were changed once a week.

Acclimation

Upon arrival, mice were acclimated to the environment for at least three days prior to initiate the study.

1.9. Experimental Procedure

MOLM-13 systemic tumor model were established by injecting tumor cells into the tail vein (10×106cells/mouse) under the sterile condition. To establish the MOLM-13 systemic model, all animals were intraperitoneally injected (i.p.) with cyclophosphamide (150 mg/kg) for two consecutive days before the inoculation of MOLM-13 cells. On the third day after cell inoculation, animals were randomized into 10 mice per group and treatment was initiated (day 1) according to Table 2.

TABLE 2Groups and dosage regimenDoseGroupAnimalsTreatment(mg/kg)RouteSchedule110Vehicle—p.o.q2d × 21 d210Compound 2310p.o.q2d × 21 d310Compound C20p.o.q2d × 21 d410Compound 2310p.o.q2d × 21 dCompound C20p.o.q2d × 21 d

The animals were euthanized when the experimental animals had symptoms such as weight loss and weight loss greater than 20%, limb paralysis, tumor metastasis to the eye causing ocular protrusion, tumor metastasis to the abdominal cavity causing abdominal enlargement. These severe health issues were considered as the same as animal death caused by tumor growth and the affected animals were sacrificed immediately.

1.10. Results

The date of the last mouse died (the last measurement) was used for analysis of median overall survival and the generation of Kaplan-Meier plots. Survival curves of different treatment groups were compared using log-rank test with Bonferroni's multiple test. All data were analyzed in SPSS version 18.0 (IBM, Armonk, NY, U.S.A.). Prism version 6 (GraphPad Software Inc., San Diego, CA, U.S.A.) was used for graphic presentation.

The results were shown inFIG.1(* means P<0.05) and Table 3.

TABLE 3Median survival time of mice of each groupGroupVehicleCompound CCompound 23CombinationMedian1925.52935survival timeof mice (days)Prolonged—6.51016survival timeof mice (days)

The mice in the vehicle group began to die on the 17th day after the start of treatment (i.e., 19 days after cell inoculation), and all mice died on the 20th day after the start of treatment (i.e., 22 days after cell inoculation), showing a median survival of 19 days. The median survival time of mice of the Compound C group was 25.5 days (P<0.05 compared with the vehicle control group), showing an excellent therapeutic effect. Compound 23 as a single drug also significantly prolonged the survival time of mice, showing a median survival time of 29 days (P<0.05, compared with the vehicle group). The combination of Compound C and Compound 23 further prolonged the survival time of mice, and the median survival time reached 35 days (P<0.05 compared with the vehicle group, compared with the Compound C group or compared with the Compound 23 group).

Embodiment 6. Study of the Combination of Compound C and Compound 23 on Human MV-4-11 AML Mouse Xenograft Model

1.1. Preparation of Test Compound Solution

The formulation of Compound C was prepared as homogenous suspension in 0.2% HPMC using a mortar. The drug suspension was prepared every three days or once a week and placed at 4° C. when not in use. Before use, the drug suspension was placed at room temperature and shaken to mix well. The formulation of Compound C was administered to mice via p.o. in a volume of 10 mL/kg.

The formulation of Compound 23 was prepared in 0.2% HPMC in a mortar to generate homogenous suspension. The drug suspension was prepared once every other day and placed at 4° C. when not in use. Before use, the drug suspension was placed at room temperature and shaken well. The formulation of Compound 23 was administered to mice via p.o. in a volume of 10 mL/kg.

1.2. Cell Line

MV-4-11 cells (Human AML cell line MV-4-11 (FLT3-ITD positive and wild type TP53 gene)) were maintained in IMDM (Cat. C12440500BT, GIBCO), with 10% fetal bovine serum (Cat. 10099-141, GIBCO), 1% L-glutamine, 100 U/mL penicillin G and 100 μg/mL streptomycin (Cat. GNM15140, Hangzhou Geno Biomedical Technology Co., Ltd). Cells were incubated at 37° C. in a humidified incubator with 5% CO2.

1.3. Animals

Six to eight weeks old, female, NOD SCID and BALB/c nude mice were used. Animals were obtained from Vital River Laboratories (VRL, Beijing, China, license number SCXK(J) 2012-0001 and SCXK(J) 2016-0006). The body weights were 17-24 g.

1.4. Animal Housing and Care

Animals were housed in the SPF animal laboratory of Experiment Animal Center in Suzhou GenePharma Co., Ltd. The daily care of animals were conducted by full-time employee of the Experimental Animal Center of GenePharma.

1.5. Animal Identification

Each cage was identified by a cage card with the study name, study number, study group, species, and sex. Individual mice were identified by ear tags.

1.6. Randomization

Randomization was conducted when the size of xenograft tumors reached 100-150 mm3. Based on the size of the tumor and body weight, animals were randomly assigned into experimental groups. Difference between mean tumor volume of each group and mean tumor volume of all test animals did not exceed ±10%.

1.7. Operation and Management Specifications

The protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of GenePharma prior to conduct. During the study, the care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

1.8. Housing Conditions

Housing: Animals were housed in cages with not more than 5 mice per cage.

Range of daily temperature: 20-26° C.

Range of daily humidity: 40%-70%.

Light: 12 hours on and 12 hours off.

Diet and Water

Radiation-sterilized dry granule food was offered ad libitum during the study period. Autoclaved second-level osmotic water was made freely available from water bottles. Reverse osmosis (RO) water was autoclaved before use. Animals had free access to sterile drinking water.

Bedding

Wood shavings were used for animal bedding, which were sterilized by autoclave before use. Beddings were changed once a week.

Acclimation

Upon arrival, mice were acclimated to the environment for at least three days prior to initiate the study.

1.9. Experimental Procedure

MV-4-11 subcutaneous xenograft models were established by subcutaneously injecting tumor cells (1×107cells (50% Matrigel)/mouse) into the right flank of the mouse under the sterile condition. When the tumors reached an appropriate size (100-200 mm3), mice were randomized into 6 mice per group and treatment was initiated (day 1) according to Table 4.

TABLE 4Groups and dosage regimenDoseGroupAnimalsTreatment(mg/kg)RouteSchedule16Vehicle—p.o.q2d × 22 d26Compound 2310p.o.q2d × 22 d36Compound C30p.o.q2d × 22 d46Compound 2310p.o.q2d × 22 dCompound C30p.o.q2d × 22 d

The tumors sizes and animal body weights were measured twice a week. Tumor volumes were estimated from measurements of two diameters of the individual tumors as follows:
Tumor Volume (mm3)=(a×b2)/2

Relative tumor volume (RTV) were calculated according to the follows:
RTV=Vt/V1

where V1and Vtwere respectively the average tumor volume on the first day of treatment (day 1) and the average tumor volume on a certain time point (day T).

Standard NCI procedures were used to calculate tumor parameters (Teicher, B. A. (2011). “Tumor Models in Cancer Research.” Second Edition, Human Press). Percent tumor growth inhibition (% T/C) was calculated as the mean RTV of treated tumors (T) divided by the mean RTV of control tumors (C)×100%. The percentage T/C value is an indication of antitumor effectiveness: a value of T/C<42% is considered significant antitumor activity by the NCI. A T/C value <10% is considered to indicate highly significant antitumor activity, and is the level used by the NCI to justify a clinical trial if toxicity and certain other requirements are met (termed DN-2 level activity). A body weight loss (mean of group) of greater than 20%, or greater than 20% of drug deaths are considered to indicate an excessively toxic dosage.

The synergistic analysis was performed using the following formula described in Clarke R. Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models[J].Breast Cancer Research&Treatment,1997, 46(2-3):255-278, which is incorporated by reference in its entirety: expected value=(A/C)×(B/C); actual value=(AB)/C; A: RTV of drug A; B: RTV of drug B; C=RTV of the vehicle control group; AB=RTV of the combination group. Ratio=expected/actual value. A ratio >1 indicates that the two drugs have a synergistic effect. A ratio=1 indicates the two drugs have additive effects. A ratio <1 indicates the two drugs may have antagonist effects.

According to the earlier publication (Gao, H., et al. (2015). “High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response”Nat Med21: 1318), additional measurements of response included stable disease (SD), partial tumor regression (PR), and complete regression (CR) were determined by comparing tumor volume change at day t to its baseline: tumor volume change (%)=(Vt−V1)/V1. The best response was the minimum value of tumor volume change (%) for t≥10. For each time t, the average of tumor volume change from t=1 to t was also calculated. Best average response was defined as the minimum value of this average for t≥10. The criteria for response (mRECIST) were adapted from RECIST criteria (Gao, H., et al. (2015). “High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response”Nat Med21: 1318; Therasse, P., et al. (2000). “New Guidelines to Evaluate the Response to Treatment in Solid Tumors”J Natl Cancer Inst92(3): 205-216) and defined as follows: mCR, best response <−95% and best average response <−40%; mPR, best response <−50% and best average response <−20%; mSD, best response <35% and Best average response <30%; mPD, not otherwise categorized. SD, PR, and CR were considered responders and used to calculate response rate (%). Body weight of animals were monitored simultaneously. The change in body weight was calculated based on the animal weight of the first day of dosing (day 1). Tumor volume and changes in body weight (%) were represented as the mean±standard error of the mean (SEM).

1.10. Results

One-way ANOVA followed by Games-Howell's post-test was applied to assess the statistical significance of differences between multiple treatment groups. All data were analyzed in SPSS version 18.0 (IBM, Armonk, NY, U.S.A.). Prism version 6 (GraphPad Software Inc., San Diego, CA, U.S.A.) was used for graphic presentation.

Tumor growth curves were plotted with the treatment duration (i.e., days) on the X-axis and corresponding tumor volume (geometric mean) on the Y-axis, which was shown inFIG.2.

The tumor weight on day 22 was shown in Table 5 andFIG.3.

TABLE 5Tumor weight on the day 22Tumor weight onTreatmentD 22 (Mean ± SEM)Vehicle control2.29 ± 0.29Compound 23 10 mg/kg0.67 ± 0.11***Compound C 30 mg/kg1.45 ± 0.32Compound 23 + Compound C0.29 ± 0.10****##***P < 0.001,****P < 0.0001 versus vehicle control;##P < 0.01 versus Compound C monotherapy.

The result of RTV, T/C (%) values, synergy scores and response rate on the day 22 was shown in Table 6.

TABLE 6RTV, T/C (%) values, synergy scoresand response rate on the day 22ResponseRTV@T/C(%)@Synergy @rate @TreatmentD 22D 22D 22D 22Vehicle18.2 ± 3.3———Compound 235.2 ± 0.928.6——10 mg/kgCompound C10.8 ± 1.659.6——30 mg/kgCompound 232.4 ± 0.9*#13.41.27—10 mg/kg +Compound C30 mg/kg*p < 0.05;#p < 0.05 vs. Compound C 30 mg/kg group;Ratio > 1, Synergistic;Ratio = 1, Additive;Ratio < 1, Antagonistic

Synergistic effect between Compound C and Compound 23 was demonstrated as synergy score was larger than 1 in the treatment group of Compound 23 10 mg/kg+Compound C 30 mg/kg.

It is to be understood that the foregoing description of the preferred embodiments is intended to be purely illustrative of the principles of the invention, rather than exhaustive thereof, and that changes and variations will be apparent to those skilled in the art, and that the invention is not intended to be limited other than expressly set forth in the following claims.