Flt3 (Kd = 1.6±0.7 nM)

体外活性：AC220 是一种独特、有效、选择性的 FLT3 抑制剂，对 FLT3 具有高亲和力，Kd 值为 1.6 nM。 AC220 抑制人白血病细胞系 MV4-11 中 FLT3 的自磷酸化，MV4-11 具有纯合 FLT3-ITD 突变且依赖于 FLT3，而 RS4;11 则表达野生型 FLT3，IC50 值分别为 1.1 nM 和 4.2 nM。 AC220 是最有效的细胞 FLT3-ITD 抑制剂，与 IC50 值范围为 0.87 nM 至 64 nM 的所有其他 FLT3 抑制剂相比，对 MV4-11 细胞增殖具有最显着的抑制作用，IC50 为 0.56 nM。 AC220 对 A375 细胞的增殖没有抑制活性，该细胞在 BRAF 中具有激活突变，并且不依赖于 FLT3，这表明 FLT3 抑制和一般细胞毒性作用之间存在很大的窗口。激酶测定：为了测量 FLT3 自磷酸化的抑制，将 MV4-11 或 RS4;11 细胞在低血清培养基 (0.5% FBS) 中培养过夜，并于第二天以每孔 400 000 个细胞的密度接种到 96 孔板中。将细胞与不同浓度的 AC220 在 37°C 下孵育 2 小时。为了在 RS4;11 细胞中诱导 FLT3 自磷酸化，在 AC220 孵育 2 小时后添加 100 ng/mL FLT3 配体 15 分钟。制备细胞裂解物并在预涂有总 FLT3 捕获抗体的 96 孔板中孵育。将包被板与抗 FLT3 的生物素化抗体一起孵育以检测总 FLT3，或与抗磷酸酪氨酸的抗体一起孵育以检测 FLT3 自磷酸化。在这两种情况下，SULFO 标记的链霉亲和素二抗用于在 Meso Scale Discovery 平台上进行电化学发光检测。 AC220抑制FLT3-ITD或TLT3-WT自磷酸化50%的浓度代表IC50值。细胞测定：细胞（MV4-11 和 RS4;11 细胞）在低血清培养基（0.5% FBS）中培养过夜，以每孔 40 000 个细胞接种到 96 孔板中，并在 37° 下暴露于 AC220 72 小时C。使用 Cell Titer-Blue Cell Viability Assay 测量细胞活力。

在 FLT3-ITD 依赖性 MV4-11 肿瘤异种移植小鼠模型中，口服 AC220（10 mg/kg）可诱导 FLT3 自磷酸化的时间依赖性抑制； 2小时时抑制率为90%，24小时时抑制率为40%。 AC220 每天口服一次，剂量低至 1 mg/kg，可显着延长 FLT3-ITD AML 小鼠模型的生存期。使用 10 mg/kg AC220 治疗 28 天，所有小鼠的肿瘤均快速完全消退，并且在治疗后 60 天期间没有肿瘤再生长。与舒尼替尼治疗相比，AC220 显示出更显着的疗效，舒尼替尼治疗使除一只小鼠外的所有小鼠的肿瘤缓慢缩小并在停止治疗后立即恢复生长。

生化激酶结合试验[1]
KinomeScan激酶结合测定如前所述进行。对于FLT3测定，我们使用了仅跨越催化结构域的激酶构建体（NP_004110.2中的氨基酸592至969）。该构建体不包括膜旁结构域，旨在测量开放FLT3活性位点对抑制剂的内在结合亲和力。

细胞检测[1]
MV4-11和RS4；分别在含有10%胎牛血清（FBS）的Iscove培养基和含有10%FBS的RPMI中培养11个细胞。对于增殖试验，细胞在低血清培养基（0.5%FBS）中培养过夜，然后以每孔40000个细胞的速度接种在96孔板上。向细胞中加入抑制剂，并在37°C下孵育72小时。使用Promega的Cell Titer Blue细胞活力测定法测量细胞活力。为了测量FLT3自磷酸化的抑制作用，将细胞在低血清培养基（0.5%FBS）中培养过夜，并在第二天以每孔400000个细胞的密度接种在96孔板上。将细胞与抑制剂在37°C下孵育2小时。诱导RS4中FLT3自磷酸化；在化合物孵育2小时后加入100ng/mL FLT3配体15分钟。制备细胞裂解物，并在预涂有总FLT3捕获抗体的96孔板中孵育。将涂覆的板与抗FLT3的生物素化抗体一起孵育以检测总FLT3，或与抗磷酸酪氨酸的抗体一起孵育来检测FLT3自磷酸化。在这两种情况下，在Meso Scale Discovery平台上使用SULFO标记的链霉抗生物素二抗进行电化学发光检测。

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原代细胞分析1]
白血病细胞标本由约翰·霍普金斯肿瘤和细胞采购银行的Sidney Kimmel癌症中心提供，并得到区域肿瘤研究中心拨款2 P30 CA 006973-44的支持。使用Ficoll-Hypaque密度梯度离心从全血或骨髓中分离出单核细胞，并将其储存在含有10%二甲亚砜的FBS中的液氮中。使用时，冷冻样品迅速解冻，在培养基中孵育过夜，然后进行另一轮密度离心（添加DNA酶），以消除冻融循环中发生凋亡的细胞。FLT3突变状态如所述确定。46使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑（MTT）分析评估细胞毒性。19为了通过蛋白质印迹评估FLT3磷酸化，将患者来源的白血病母细胞在磷酸缓冲盐水中洗涤，然后在摇动的同时将其重新悬浮在裂解缓冲液（20 mM Tris，pH 7.4，100 mM NaCl，1%Igepal，1 mM EDTA，2 mM NaVO4，加完全蛋白酶抑制剂鸡尾酒）中30分钟进行裂解。通过在18000g下离心澄清裂解物，并测定上清液中的蛋白质（Bio-Rad）。将抗FLT3（S18）抗体加入提取物中，孵育过夜；然后再加入蛋白A琼脂糖2小时。十二烷基硫酸钠聚丙烯酰胺电泳并转移到Immobilon膜上后，用抗磷酸酪氨酸抗体（4G10）进行免疫印迹以检测磷酸化的FLT3，然后用抗FLT3抗体剥离和重定以测量总FLT3。使用增强化学发光法对蛋白质进行可视化。为了定量磷酸化FLT3水平，如“细胞测定”所述，通过ELISA测定细胞裂解物中的磷酸化FLT 3和总FLT3

Mice: The mice used are female nu/NU or severe combined immunodeficient mice. Quizartinib (hydrochloride salt) is formulated in 22% hydroxypropyl-β-cyclodextrin, CEP-701 is formulated in 20% gelucire 44/14 in water (vol/vol), MLN-518 and SU 11248 are formulated in 10 mM sodium citrate (pH 3.5), PKC-412 is formulated in 3:1 gelucire 44/14-propylene glycol (vol/vol), and Bay 43-9006 is formulated in 80% PEG-400. Compound concentrations are selected in a volume of 10 mL/kg to deliver the intended dose. Oral gavage is used to administer compounds, and plasma samples are taken 0,25,0.5,1,2,4,6, and 24 hours after dosing. In order to obtain three independent plasma concentration time courses, eye bleeds (150 μL) are obtained semilongitudinally using three groups of three animals each, taking two to three time points per animal. Using four volumes of acetonitrile containing an internal standard, plasma samples and controls (25 μL) are extracted, and liquid chromatography tandem mass spectrometry is used for analysis.

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Pharmacokinetic studies[1]
Female NU/NU or severe combined immunodeficient mice were purchased from Charles River Laboratories or Harlan. AC220 (hydrochloride salt) was formulated in 22% hydroxypropyl-β-cyclodextrin, CEP-701 was formulated in 20% gelucire 44/14 in water (vol/vol), MLN-518 and sunitinib were formulated in 10 mM sodium citrate (pH 3.5), PKC-412 was formulated in 3:1 gelucire 44/14–propylene glycol (vol/vol), and sorafenib (toluene sulfonate salt) was formulated in 80% PEG-400. Compound concentrations were chosen to deliver the desired dose in a volume of 10 mL/kg. Compounds were administered by oral gavage and plasma samples collected 0.25, 0.5, 1, 2, 4, 6, and 24 hours after dosing. To collect plasma samples, eye bleeds (150 μL) were taken semilongitudinally using 3 groups of 3 animals each, taking 2 to 3 time points per animal to obtain a total of 3 independent plasma concentration time courses. Plasma samples and controls (25 μL) were extracted with 4 volumes of acetonitrile containing an internal standard and analyzed by liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were obtained by fitting the normalized liquid chromatography tandem mass spectrometry peak areas to a noncompartmental model using the linear trapezoidal estimation method in the WinNonlin software package. Mouse studies at Ambit complied with the recommendations of the “Guide for Care and Use of Laboratory Animals”45 with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care.

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Animal efficacy studies[1]
Subcutaneous xenograft model.[1]
This model was performed at Ambit to measure in vivo inhibition of FLT3, and by Piedmont Research Center LLC to determine antitumor efficacy, following published procedures. Compounds were formulated and administered as described for pharmacokinetic studies. To measure FLT3 inhibition, tumors were harvested at 2 or 24 hours after compound administration, weighed, and lysed by mechanical dissociation. Tumor lysates were cleared of protein and tissue fragments by centrifugation at 835g for 15 minutes. Cleared lysates were assayed for total and phosphorylated FLT3 using the electrochemiluminescence-based enzyme-linked immunoassay (ELISA) described in “Cellular assays.”

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Bone marrow engraftment model.[1]
The model was performed according to published procedures.20 For intravenous bone marrow engraftment, nonobese diabetic/severe combined immunodeficient mice were acclimated for 2 weeks before pretreatment with 150 mg/kg cyclophosphamide delivered intraperitoneally once a day for 2 days. After a 48-hour rest period, animals were given an intravenous injection of 5 × 106 MV4-11 cells into the tail vein. AC220 was formulated and delivered as described for pharmacokinetic studies.

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Formulated in 22% hydroxypropyl-β-cyclodextrin; 10 mg/kg; Oral gavage

Female NU/NU or severe combined immunodeficient mice implanted with MV4-11 cells

Absorption, Distribution and Excretion
The mean (SD) absolute bioavailability of quizartinib from the tablet formulation was 71% (±7%) in healthy subjects. After oral administration under fasted conditions, time to peak concentration (median Tmax) of quizartinib and AC886 measured post dose was approximately 4 hours (range 2 to 8 hours) and 5 to 6 hours (range 4 to 120 hours), respectively, in healthy subjects. Following the administration of 35.4 mg quizartinib once daily in patients with newly diagnosed acute myeloid leukemia, the Cmax and AUC0-24h were calculated to be 140 ng/mL (71%) and 2,680 ng.h/mL (85%) respectively during the induction therapy and 204 ng/mL (64%) and 3,930 ng.h/mL (78%) respectively during the consolidation therapy. For the metabolite AC886, the Cmax and AUC0-24h were estimated to be 163 ng/mL (52%) and 3,590 ng.h/mL (51%) respectively during the induction therapy and 172 ng/mL (47%) and 3,800 ng.h/mL (46%) respectively during the consolidation therapy. Increasing the once daily dose of quizartinib to 53 mg also increases the Cmax and AUC0-24h of quizartinib to 529 ng/mL (60%) and 10,200 ng.h/mL (75%) respectively at steady state. The Cmax and AUC0-24h of the metabolite AC886 also increases to 262 ng/mL (48%) and 5,790 ng•h/mL (46%) respectively. No clinically significant differences in the pharmacokinetics of quizartinib were observed when administered with a high-fat, high-calorie meal.
Following a single radiolabeled dose of quizartinib 53 mg to healthy subjects, 76.3% of the total radioactivity was recovered in feces (4% unchanged) and 1.64% in urine.
Volume of distribution at steady state in healthy subjects was estimated to be 275 L (17%).
Total body clearance of quizartinib in healthy subjects was estimated to be 2.23 L/hour (29%).

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Metabolism / Metabolites
In vitro quizartinib is primarily metabolized via oxidation by CYP3A4/5 and AC886 is formed and metabolized by CYP3A4/5.

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Biological Half-Life
The mean (SD) effective half-lives (t1/2) in patients with newly diagnosed AML for quizartinib and AC886 during maintenance therapy are 81 hours (±73) and 136 hours (±113), respectively.

Hepatotoxicity
In the prelicensure clinical trials of quizartinib in patients with AML, ALT elevations were arose in 10% to 16% of patients and were above 5 times the upper limit of normal (ULN) in 1% to 3%. However, similar rates were reported in subjects receiving chemotherapy without quizartinib and in most instances the elevations were transient, asymptomatic, and not associated with elevations in serum bilirubin. Intermittent elevations in liver enzymes are not uncommon in patients with untreated AML due to bacterial, viral and opportunistic infections. In the registration trials of quizartinib there were uncommon instances of acute liver injury and hepatic failure, but all were attributable to other comorbidities and factors (multiorgan failure), and none were considered due to quizartinib. Since its approval in the United States, there have been no reported cases of clinically apparent liver injury associated with quizartinib therapy.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of quizartinib during breastfeeding. Because quizartinib is more than 99% bound to plasma proteins, the amount in milk is likely to be low. However, the manufacturer recommends that breastfeeding be discontinued during quizartinib therapy and for 1 month after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
In vitro plasma protein binding of quizartinib and AC886 is 99% or greater. In vitro blood-to-plasma ratio for quizartinib and AC886 ranges from 0.79-1.30 and 1.36-3.19, respectively.

[1]. AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood, 2009, 114(14), 2984-2992.

[2]. SYK is a critical regulator of FLT3 in acute myeloid leukemia. Cancer Cell. 2014 Feb 10;25(2):226-42.

[3]. PROTACs: great opportunities for academia and industry. Signal Transduct Target Ther. 2019 Dec 24;4:64.

Pharmacodynamics
Quizartinib showed antitumor activity in a mouse model of FLT3-ITD-dependent leukemia. In vitro, studies have shown that quizartinib is a predominant inhibitor of the slow delayed rectifier potassium current, IKs. In AML patients receiving quizartinib at a dose of 90 mg/day for females and 135 mg/day for males on a 28-day schedule, the median levels of phospho-FLT3 (pFLT3) and total FLT3 (tFLT3) decreased from 3312 RLU or 5639 RLU respectively at day 1 to 1235 RLU and 142 RLU respectively at day 8. Additionally, pFLT3 levels are statistically significantly higher (p < 0.0001, Mann Whitney test) for the ITD+ subjects on day 1; however, pFLT3 levels was reduced to a similar level in patients with or without the ITD mutation. The exposure-response analysis predicted a concentration-dependent QTcF interval median prolongation of 18 and 24 ms [upper bound of 2-sided 90% confidence interval (CI): 21 and 27 ms] at the median steady-state Cmax of quizartinib at the 26.5 mg and 53 mg dose level during maintenance therapy.

C29H34CL2N6O4S

633.59

632.174

C, 54.98; H, 5.41; Cl, 11.19; N, 13.26; O, 10.10; S, 5.06

1132827-21-4

1132827-21-4 (HCl);950769-58-1;

24889392

Typically exists as White to off-white solid at room temperature

6.893

137.63

2

8

8

40

849

0

CC(C)(C)C1=CC(=NC(=O)NC2=CC=C(C=C2)C3=CN4C5=C(C=C(C=C5)OCCN6CCOCC6)SC4=N3)NO1.Cl.Cl

CVWXJKQAOSCOAB-UHFFFAOYSA-N

InChI=1S/C29H32N6O4S/c1-29(2,3)25-17-26(33-39-25)32-27(36)30-20-6-4-19(5-7-20)22-18-35-23-9-8-21(16-24(23)40-28(35)31-22)38-15-12-34-10-13-37-14-11-34/h4-9,16-18H,10-15H2,1-3H3,(H2,30,32,33,36)

N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride

AC220 HCl or AC010220 HCl; AC220 diHCl; AC 220; AC-220 dihydrochloride; AC010220; Quizartinib dihydrochloride; 1132827-21-4; AC-220 dihydrochloride; vanflyta; quizartinib hydrochloride; AC 010220 (dihydrochloride); AC010220.2HCL; WK7Q6ZIZ10; AC-010220; AC 010220; AC010220

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)