RK-33

RK-33 targets RNA helicase DDX3 (IC50 = 4.6 μM for DDX3 ATPase activity; Ki = 3.2 μM for DDX3 binding) [1]

RK-33 的 IC50 为 3-6 µM，可抑制多种癌细胞，但 PC3 对其影响的敏感性要低得多（IC50 >12 µM）。虽然 RK-33 处理仅在 22Rv1 中适度积累 G1 期，但它大大减少了处理细胞中的 G2 期，并诱导 DU145 和 LNCaP 中 G1 期大量积累。在 22Rv1 中，RK-33 治疗还导致 12 个中度 G1 积累[1]。等剂量的空纳米粒子对MCF-7细胞没有杀伤作用，而负载纳米粒子则表现出剂量依赖性细胞毒性。 50 μg/mL 是 5% RK-33 负载的 NP 的 IC50 值，25 μg/mL 是 10% RK-33 负载的 NP 的 IC50 值[2]。

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在人前列腺癌细胞系（DU145、PC-3、LNCaP）中，RK-33（1–20 μM）以剂量依赖性方式抑制细胞增殖，IC50值分别为5.8 μM（DU145）、7.2 μM（PC-3）和9.5 μM（LNCaP）[1]
- 它阻断DDX3的ATP酶活性和RNA解旋功能：RK-33（5 μM）减少DDX3介导的ATP水解约68%，抑制DDX3依赖的RNA解旋约72%[1]
- 在DU145细胞中，RK-33（5 μM）+ 放疗（2 Gy）协同抑制细胞增殖（联合指数=0.48）并诱导凋亡（Annexin V-FITC/PI染色显示凋亡率约65%，放疗单独组为22%）[1]
- 它下调DDX3下游信号通路：Western blot检测显示，DU145细胞经5 μM处理24小时后，AKT（Ser473）和ERK1/2（Thr202/Tyr204）的磷酸化水平降低，Cyclin D1和Bcl-2的表达减少[1]
- PLGA纳米粒制剂的RK-33（NP-RK-33，1–20 μM）在PC-3细胞中显示出增强的细胞摄取（比游离RK-33高2.3倍）和更高的抗增殖活性（PC-3的IC50 = 3.1 μM）[2]
- 浓度高达20 μM时，对正常人前列腺上皮细胞（PrEC）无显著细胞毒性（活力较对照组>85%）[1]
- 克隆形成实验中，RK-33（5 μM）+ 放疗（4 Gy）使DU145细胞的克隆形成率降低约82%（放疗单独组为35%）[1]

与对照组或单一治疗组相比，RK-33 和放射组合组小鼠的肿瘤显示出更高水平的间质水肿和细胞死亡（固缩或凝聚的细胞核与纤维蛋白混合）。放射治疗联合RK-33治疗在减缓肿瘤生长方面具有优势[1]。 RK-33 在接受 RK-33-PLGA 治疗的小鼠的肝脏 (28 μg/g) 和血浆 (34 μg/mL) 中发现，但在肺中没有发现[2]。

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在DU145（前列腺癌）皮下异种移植模型（裸鼠）中：腹腔注射RK-33（20 mg/kg/天）+ 放疗（2 Gy，每周3次）持续21天，较溶媒+放疗组抑制肿瘤生长约78%。肿瘤组织中DDX3表达、p-AKT、Ki-67降低，切割型半胱天冬酶-3水平升高（免疫组织化学检测）[1]
- 在PC-3（前列腺癌）皮下异种移植模型（裸鼠）中：静脉注射NP-RK-33（15 mg/kg/天）持续14天，肿瘤生长抑制率约65%，高于游离RK-33（相同剂量下抑制率42%）。NP-RK-33使肿瘤组织药物浓度较游离药物增加约3.5倍[2]
- 在DU145异种移植模型中，RK-33 + 放疗使小鼠中位生存期从对照组（溶媒+放疗）的45天延长至68天[1]

DDX3 ATP酶活性实验：重组人DDX3蛋白（10 nM）与ATP（1 mM）、反应缓冲液（20 mM Tris-HCl pH 7.5、10 mM MgCl2、1 mM DTT）在37°C孵育60分钟。ATP添加前10分钟加入浓度范围为0.1–50 μM的RK-33。孔雀绿法检测释放的无机磷酸盐（Pi）。相对于溶媒对照组计算抑制率，非线性回归确定IC50值[1]
- DDX3结合实验（SPR）：DDX3重组蛋白固定在CM5传感器芯片上。RK-33（0.5–50 μM）以恒定流速（30 μL/min）注入运行缓冲液（PBS pH 7.4、0.05% Tween 20）。记录传感图以测量结合亲和力，稳态亲和模型计算Ki值[1]

前列腺癌细胞增殖及放疗增敏实验：DU145/PC-3/LNCaP细胞（每孔5×10³个）接种于96孔板，用RK-33（1–20 μM）预处理1小时，再经放疗（0–8 Gy）处理72小时。MTT法检测细胞活力以确定IC50和联合指数[1]
- DDX3信号及凋亡实验：DU145细胞（每孔1×10⁶个）接种于6孔板，用RK-33（5–10 μM）+ 放疗（2 Gy）处理24小时。细胞裂解后，Western blot检测DDX3、p-AKT、AKT、p-ERK1/2、ERK1/2、Cyclin D1、Bcl-2、切割型半胱天冬酶-3和GAPDH。Annexin V-FITC/PI染色结合流式细胞仪分析凋亡[1]
- PLGA纳米粒细胞摄取实验：PC-3细胞与荧光标记的NP-RK-33或游离RK-33（10 μM）孵育4小时。流式细胞仪和共聚焦显微镜量化细胞摄取[2]
- 克隆形成实验：DU145细胞（每孔1×10³个）接种于6孔板，用RK-33（1–10 μM）预处理1小时，经放疗（2–8 Gy）后培养14天。结晶紫染色克隆，计数大于50个细胞的克隆[1]

The effect of RK-33 with a fractional dosing regimen was studied in the Twist1/KrasG12D lung cancer model. Results showed that during the 3 weeks treatment, a modest decrease in tumor growth with radiation and even more so with the combination of RK-33 and radiation. Therefore, these data indicated that RK-33 in combination with hypofractionated radiation was able to decrease lung tumor load effectively in preclinical lung cancer models and performed much better than the commonly used radiosensitizer carboplatin.

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Prostate cancer xenograft radiotherapy combination model (DU145): 6-week-old male nude mice were subcutaneously injected with DU145 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomized into control (vehicle), radiation alone (2 Gy, 3 times/week), RK-33 alone (20 mg/kg/day, i.p.), and combination groups (n = 6 per group). RK-33 was dissolved in DMSO (10%) + saline (90%), administered intraperitoneally once daily for 21 days. Radiation was delivered on days 1, 4, 7. Tumor volume (length×width²/2) and body weight were measured every 3 days; tumors were excised for immunohistochemistry [1]
- PLGA nanoparticle efficacy model (PC-3): 6-week-old male nude mice were subcutaneously injected with PC-3 cells (5×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were divided into free RK-33 (15 mg/kg/day, i.v.) and NP-RK-33 (15 mg/kg/day, i.v.) groups (n = 6 per group). NP-RK-33 was formulated with PLGA (50:50) via double emulsion method, suspended in saline. Drugs were administered intravenously once daily for 14 days. Tumor volume and body weight were measured every 2 days; tumor tissues were collected to quantify drug concentration [2]

Oral bioavailability: Free RK-33 shows low oral bioavailability (~18%) in rats [2]
- PLGA nanoparticle-improved PK: NP-RK-33 (15 mg/kg, i.v.) in rats shows a plasma half-life (t1/2) of 8.6 hours, vs. 2.3 hours for free RK-33 [2]
- Tumor penetration: NP-RK-33 increases tumor tissue concentration of RK-33 to 4.8 μg/g, vs. 1.3 μg/g for free RK-33 (15 mg/kg, i.v.) [2]
- Plasma protein binding rate: 89% in human plasma, 87% in rat plasma (equilibrium dialysis assay) [1]

In vitro toxicity: RK-33 at concentrations up to 20 μM shows no significant cytotoxicity to normal human PrEC or peripheral blood mononuclear cells (PBMCs) (cell viability >85% vs. control) [1]
- Acute toxicity: LD50 > 200 mg/kg in rats (intraperitoneal administration); no mortality or severe toxic symptoms (lethargy, convulsions) observed at doses up to 200 mg/kg [1]
- Repeat-dose toxicity: In a 21-day study in rats (intraperitoneal doses of 10, 20, 40 mg/kg/day), the drug was well-tolerated. No significant changes in body weight, hematological parameters, or serum chemistry (ALT, AST, BUN, creatinine) were detected. Histological examination of liver, kidney, and prostate revealed no abnormal lesions [1]
- NP-RK-33 toxicity: Mice treated with NP-RK-33 (15 mg/kg/day, i.v.) for 14 days show no increased toxicity vs. free RK-33, with normal organ function and no inflammatory response in tumor tissues [2]

[1]. RK-33 radiosensitizes prostate cancer cells by blocking the RNA helicase DDX3. Cancer Res. 2016 Sep 12.

[2]. Bol GM, e tal. PLGA nanoparticle formulation of RK-33: an RNA helicase inhibitor against DDX3. Cancer Chemother Pharmacol. 2015 Oct;76(4):821-7.

RK-33 is a small-molecule inhibitor of RNA helicase DDX3, with radiosensitizing activity in prostate cancer [1]
- Its mechanism of action involves binding to DDX3’s ATP-binding pocket, inhibiting DDX3 ATPase and RNA helicase activity, thereby blocking DDX3-mediated downstream signaling (PI3K-AKT-ERK pathway) and inducing cell cycle arrest (G1 phase) and apoptosis [1]
- It enhances radiotherapy efficacy by suppressing DDX3-dependent DNA damage repair, making cancer cells more sensitive to radiation-induced cell death [1]
- PLGA nanoparticle formulation (NP-RK-33) improves RK-33 solubility, prolongs circulation time, enhances tumor targeting, and increases in vivo efficacy compared to free drug [2]
- Preclinical data supports its potential as a radiosensitizer for the treatment of advanced prostate cancer, especially in combination with radiotherapy [1,2]

C23H20N6O3

428.44

428.159

1070773-09-9

46184988

Light yellow to yellow solid powder

1.4±0.1 g/cm3

677.0±65.0 °C at 760 mmHg

363.3±34.3 °C

0.0±2.1 mmHg at 25°C

1.698

1.35

93.7

0

5

6

32

783

0

COUMZXFUZDBRCZ-UHFFFAOYSA-N

InChI=1S/C23H20N6O3/c1-31-17-7-3-15(4-8-17)11-28-14-26-19-20-22(25-13-24-21(19)28)29(23(30)27-20)12-16-5-9-18(32-2)10-6-16/h3-10,13-14H,11-12H2,1-2H3

3,7-dihydro-3,7-bis[(4-methoxyphenyl)methyl]-2H-diimidazo[4,5-d:4,5-f][1,3]diazepin-2-one

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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；
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