JNJ-42756493 (Erdafitinib)   中文名称: 厄达替尼

FGFR1 (IC50 = 1.2 nM); FGFR2 (IC50 = 2.5 nM); FGFR3 (IC50 = 3.0 nM); FGFR4 (IC50 = 5.7 nM)

体外活性：JNJ-42756493 是一种有效的口服泛 FGFR 酪氨酸激酶抑制剂，对 FGFR 家族的所有成员（FGFR1 至 FGFR4）具有低纳摩尔范围内的半最大抑制浓度值，对血管内皮生长的活性极小因子受体 (VEGFR) 激酶与 FGFR 激酶相比（效力差异约 20 倍）。在体外，用 JNJ-42756493 处理的细胞增殖减少，与细胞凋亡增加和细胞存活率降低相关。激酶测定：Erdafitinib (JNJ-42756493) 是一种有效的口服 FGFR 家族抑制剂；抑制 FGFR1/2/3/4，IC50 分别为 1.2、2.5、3.0 和 5.7 nM。细胞测定：在 72 小时时，使用磺罗丹明 B (SRB) 测定法评估贴壁细胞（HCT116、HCA7、Caco2 和 NCI-H716 细胞）和台盼蓝染料排除悬浮液，评估不同药物浓度对细胞生长和存活的影响细胞，NCI-H716。

在体内，仅通过药物治疗，NCI-H716肿瘤的生长就会延迟5天，尽管当停止药物递送时，相对肿瘤体积与对照相比有所增加。 JNJ-42756493显示出良好的药物样特性，并且在肺、肝和肾组织中表现出高分布。 JNJ-42756493 在有效剂量下具有良好的耐受性，并产生有效的剂量依赖性抗肿瘤活性，同时对肿瘤 FGFR 和下游途径成分进行药效调节。

Time-resolved fluorescence kinase assays for FGFR1-4 and KDR/FGFR1-4和KDR的时间分辨荧光激酶测定[1]
在384孔黑色Optiplates中进行FGFR1-4和KDR的时间分辨荧光能量转移分析。 通过向含有化合物的混合物中加入酶（分别为0.1、0.8、0.8、0.4和0.7 nmol/L的FGFR 1、2、3、4和KDR）来引发激酶反应，ATP的米氏常数（Km）浓度为每种激酶（分别为5、0.4、25、5和3μmol/L）和500 nmol/L FLT3底物，最终测定体积为30μL。在室温下孵育FGFR1、FGFR3和KDR 60分钟、FGFR2 30分钟和FGFR4 45分钟后，通过加入10μL检测试剂停止酶反应。在室温下孵育1小时后，在Envision阅读器上以337nm的激发和620nm（Eu信号）和665nm（FRET信号）的双发射测量荧光。

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Kinase binding assays/激酶结合测定[1]
使用KINOMEscan平台评估JNJ-42756493与397种野生型激酶的结合亲和力。

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Cellular kinase assays/细胞激酶测定[1]
用编码TEL（ETV6）-激酶的pcDNA3.1质粒转染IL3依赖性（终浓度为10ng/mL）小鼠BaF3-pro-B细胞（20），并选择与遗传霉素的稳定整合。

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在DMSO中，Erdafitinib被溶解。Erdafitinib用于治疗KATO III、RT-112、A-204、RT-4、DMS-114、A-427和MDA-MB-453细胞（终浓度：2%DMSO；范围为10μM至0.01 nM）。MTT试剂用于评估4天孵育期后细胞的存活率。在540nm处进行光密度测量[1]。

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抑制FGFR家族受体磷酸化和下游信号传导[1]
将携带活化FGFR1、2、3或4的细胞系（分别为NCI-H1581、SNU-16、KMS-11和MDA-MB453）用不同浓度的JNJ-42756493处理4小时。移除培养基，用冰冷的磷酸盐缓冲盐水（PBS）洗涤细胞，并将其悬浮在裂解缓冲液中进行蛋白质印迹分析。在用含有FGF2（40ng/mL）的培养基替换之前，用含有100nmol/L JNJ-42756493或DMSO的培养基预处理NCI-H1581 NSCLC细胞系30分钟。用FGF2处理的细胞孵育0分钟（对照组，未用FGF2治疗）、5分钟、10分钟、30分钟、2小时、4小时或8小时。吸出培养基，用冰冷的PBS洗涤细胞，裂解并处理以进行蛋白质印迹分析。

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溶酶体化合物积累[1]
在530 nm成像之前，用50 nmol/L LysoTracker red和1μmol/L JNJ-42756493处理GAMG人胶质母细胞瘤细胞30分钟。GAMG细胞用巴非霉素（75 nmol/L）处理1小时，用PBS洗涤，然后在有或没有75 nmol/L巴非霉素时加入补充有1μmol/L JNJ-42756493或JNJ-42883919的培养基。在InCell Analyzer 2000仪器上，每5分钟在Texas Red和CFP通道中获得一次连续图像。将4幅不同图像的感兴趣区域（ROI）密度与T=0进行比较，并将平均差异绘制为百分比变化（%ROI）。

Mice: Erdafitinib at doses of 0, 3, 10, or 30 mg/kg is administered orally to mice with xenograft tumors of SNU-16 human gastric carcinoma (FGFR2 amplified). At 0.5, 1, 3, 7, 16, and 24 hours after dosing, tumor tissue and mouse plasma are extracted from three mice per time point[1].

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Human tumor cell lines were injected directly into the inguinal region of male nude mice (1 × 107 cells/200 μL/animal with Matrigel 1:1 in medium) on day 0. When tumors were established, mice were randomized according to tumor volume to either vehicle alone (10% HP-β-CD) or vehicle containing JNJ-42756493, administered in a volume of 5 mL/kg body weight for 21 days (8–10 mice/group). For PDX studies, Nu/Nu nude mice were used. Patient-derived tumor samples finely minced (∼1–2 mm3) were added to Matrigel and approximately 50 mm3 of minced tumor was implanted subcutaneously (s.c.) into flank of anaesthetized mice (Ketamine/Medatomidine). When the tumor volume reached 200 to 300 mm3 the mice were allocated to their treatment groups with uniform mean tumor volume and body weight between groups and treated according to protocol.[1]

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Pharmacodynamic and pharmacokinetic analysis of JNJ-42756493[1]
Mice-bearing SNU-16 human gastric carcinoma (FGFR2 amplified) xenograft tumors were dosed orally with 0, 3, 10, or 30 mg/kg JNJ-42756493. Tumor tissue and mouse plasma (3 mice per time point) were harvested at 0.5, 1, 3, 7, 16, and 24 hours after dosing. Tumor tissues were frozen in liquid nitrogen, crushed, and suspended in lysis buffer [25 mmol/L Tris-HCl (pH 7.5), 2 mmol/L EDTA (pH 8), 2 mmol/L EGTA (pH8), 1% Triton X-100, 0.1% SDS, 50 mmol/L disodium β-glycerophosphate, 2 mmol/L Na3VO4, 4 mmol/L Na-pyrophosphate, 2x Thermo protease/phosphatase inhibitor cocktail). After centrifugation (12,000 rpm for 15 minutes; RCF = 15,294), the supernatants were applied to SDS-PAGE and transferred onto PVDF membranes.
When tumors of lung cancer patient-derived xenograft reached approximately 400 mm3, mice were dosed orally with 12.5 mg/kg JNJ-42756493. Tumor and mouse plasma (3 mice per time point) were collected at 1, 2, 4, 8, and 24 hours post dose.

Absorption, Distribution and Excretion
Following administration of erdafitinib 8 mg once daily, the mean (coefficient of variation [CV%]) steady-state maximum observed plasma concentration (Cmax), area under the curve (AUCtau), and minimum observed plasma concentration (Cmin) were 1,399 ng/mL (51%), 29,268 ng·h/mL (60%), and 936 ng/mL (65%), respectively. Following single and repeated once-daily dosing, erdafitinib exposure (maximum observed plasma concentration [Cmax] and area under the plasma concentration-time curve [AUC]) increased proportionally across the dose range of 0.5 to 12 mg (0.06 to 1.3 times the maximum approved recommended dose). Steady-state was achieved after 2 weeks with once-daily dosing and the mean accumulation ratio was 4-fold. The median time to achieve peak plasma concentration (tmax) was 2.5 hours (range: 2 to 6 hours). No clinically meaningful differences with erdafitinib pharmacokinetics were observed following the administration of a high-fat and high-calorie meal (800 calories to 1,000 calories with approximately 50% of the total caloric content of the meal from fat) in healthy subjects.
After administering a single oral dose of radiolabeled erdafitinib, about 69% of the dose was recovered in feces (19% as unchanged) and 19% in urine (13% as unchanged).
The mean apparent volume of distribution determined for erdafitinib is about 26 to 29 L in patients.
The mean total apparent clearance (CL/F) documented for erdafitinib is about 0.362 L/h, while the oral clearance has been observed to be approximately 0.26 L/h.

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Metabolism / Metabolites
Erdafitinib is primarily metabolized by the cytochrome CYP2C9 and CYP3A4 isoenzymes in humans to form the O-demethylated major metabolite.. The contribution of CYP2C9 and CYP3A4 in the total clearance of erdafitinib is estimated to be 39% and 20% respectively. Unchanged erdafitinib was ultimately the predominant drug-related moiety found in the plasma - no circulating metabolites were observed.

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Biological Half-Life
The mean effective half-life documented for erdafitinib is 59 hours, although it has also been observed between 50 to 60 hours.

Hepatotoxicity
In the prelicensure clinical trials of erdafitinib in patients with urothelial carcinoma, liver test abnormalities were frequent although usually mild. Some degree of ALT elevation arose in up to 41% of erdafitinib treated patients, but were above 5 times the upper limit of normal in only 1% to 2%. In these trials that enrolled approximately 400 patients, there were no reports of serious or clinically apparent liver injury, or liver related deaths. Since the approval and more wide spread use of erdafitinib there have been no reports of liver injury attributed to its use. Nevertheless, the high rate of serum aminotransferase elevations during therapy suggests that rare instances of clinically apparent liver injury might occur.
Likelihood score: E* (unproven but suspected rare 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 erdafitinib during breastfeeding. Because erdafitinib is 99.8% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 59 hours in adults and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during erdafitinib therapy and for 1 month after the final 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
The protein binding recorded for erdafitinib is approximately 99.8%, and it was determined to be primarily bound to alpha-1-acid glycoprotein.

[1]. Discovery and pharmacological characterization of JNJ-42756493 (erdafitinib), a functionally selective small molecule FGFR family inhibitor. Mol Cancer Ther. 2017 Mar 24. pii: molcanther.0589.2016.

Pharmacodynamics
Upon administration, it was observed that erdafitinib increased serum phosphate levels as a consequence of FGFR inhibition. Erdafitinib should be increased to the maximum recommended dose to achieve target serum phosphate levels of 5.5– 7.0 mg/dL in early cycles with continuous daily dosing. Subsequently, in erdafitinib clinical trials, the use of drugs that could increase serum phosphate levels, such as potassium phosphate supplements, vitamin D supplements, antacids, phosphate-containing enemas or laxatives, and medications known to have phosphate as an excipient were prohibited unless no alternatives existed. To manage phosphate elevation, phosphate binders were utilized. Additionally, the concomitant use of agents that can alter serum phosphate levels before the initial erdafitinib dose increase period based on serum phosphate levels was also avoided. Furthermore, based on the evaluation of QTc interval in an open-label, dose escalation, and dose expansion study in 187 patients with cancer, erdafitinib had no large effect (i.e., > 20 ms) on the QTc interval.

C25H30N6O2

446.54

446.243

C, 67.24; H, 6.77; N, 18.82; O, 7.17

1346242-81-6

67462786

Yellow solid powder

1.2±0.1 g/cm3

662.3±55.0 °C at 760 mmHg

142°C

354.4±31.5 °C

0.0±2.0 mmHg at 25°C

1.618

3.6

77.33

1

7

9

33

583

0

O(C([H])([H])[H])C1C([H])=C(C([H])=C(C=1[H])N(C1C([H])=C([H])C2C(C=1[H])=NC(C1C([H])=NN(C([H])([H])[H])C=1[H])=C([H])N=2)C([H])([H])C([H])([H])N([H])C([H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])[H]

OLAHOMJCDNXHFI-UHFFFAOYSA-N

InChI=1S/C25H30N6O2/c1-17(2)26-8-9-31(20-10-21(32-4)13-22(11-20)33-5)19-6-7-23-24(12-19)29-25(15-27-23)18-14-28-30(3)16-18/h6-7,10-17,26H,8-9H2,1-5H3

N'-(3,5-dimethoxyphenyl)-N'-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]-N-propan-2-ylethane-1,2-diamine

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)

配方 1 中的溶解度: ≥ 2.75 mg/mL (6.16 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.75 mg/mL (6.16 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.33 mg/mL (5.22 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 23.3 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 2.08 mg/mL (4.66 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清的DMSO储备液加入400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 2.08 mg/mL (4.66 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100μL 20.8mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 6 中的溶解度: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 22.25mg/ml

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。