KW-2478

The primary target of KW-2478 is the heat shock protein 90 (HSP90) molecular chaperone family, including cytosolic HSP90α, cytosolic HSP90β, endoplasmic reticulum-resident GRP94, and mitochondrial TRAP1. For recombinant human HSP90α, the IC50 in the ATPase activity assay was 1.8 nM [1]
; For recombinant human HSP90β, the IC50 was 2.3 nM [1]
; For recombinant human GRP94, the IC50 was 17 nM [1]
; For recombinant human TRAP1, the IC50 was 9.0 nM [1]
.

Hsp90 抑制：KW-2478 对 Hsp90α 的 IC50 为 3.8 nM。对于非霍奇金淋巴瘤 (NHL) 和多发性骨髓瘤 (MM)，KW-2478 均表现出抗增殖作用。其 GI50 值为 0.30 μM (OPM-2 /GFP)、0.34 μM (KMS-11)、0.39 μM (RPMI 8226)、0.12 μM (NCI-H929)、0.36 μM (Raji)、0.098 μM (SR) 和0.33 μM μM (SC-1)。通过主要抑制 Cdk9 的活性，KW -2478 还抑制 c-Maf 和 Cyclin D1 基因的转录[1]。

---

1. 对多发性骨髓瘤（MM）细胞的抗增殖活性：KW-2478对药物敏感型和耐药型MM细胞系均表现出强效抗增殖作用。药物敏感型MM细胞RPMI 8226（72小时MTT实验）的IC50为15 nM；IL-6依赖型MM细胞U266的IC50为18 nM；地塞米松敏感型MM细胞MM.1S的IC50为16 nM；阿霉素耐药型MM细胞RPMI 8226/LR5的IC50为19 nM [1]
。
2. 下调MM细胞中HSP90客户蛋白：Western blot分析显示，KW-2478以剂量依赖性方式降低HSP90客户蛋白表达。RPMI 8226细胞经20 nM KW-2478处理24小时后，磷酸化Akt（p-Akt）水平较溶媒对照组降低65%，磷酸化ERK（p-ERK）降低70%，NF-κB（p65）降低60%，c-Myc降低58% [1]
。MM.1S细胞经25 nM KW-2478处理后，MM生存关键蛋白IRF4的表达降低62% [1]
。
3. 诱导MM细胞凋亡：流式细胞术（Annexin V-FITC/PI染色）显示，KW-2478可诱导MM细胞凋亡。20 nM KW-2478处理RPMI 8226细胞48小时后，凋亡率（早期+晚期凋亡）从溶媒对照组的3.2%升至31.5%；30 nM剂量下，凋亡率进一步升至42.0% [1]
。该效应伴随切割型caspase-3升高3.5倍、切割型PARP升高2.8倍（Western blot分析）[1]
。
4. 抑制MM细胞克隆形成能力：克隆形成实验显示，KW-2478可抑制MM细胞的集落形成。RPMI 8226细胞经10 nM KW-2478处理72小时后，克隆形成率为25%（对照组为100%）；20 nM剂量下，克隆形成率降至12% [1]
。MM.1S细胞经15 nM KW-2478处理后，克隆形成率降至对照组的18% [1]
。
5. 抑制MM细胞与骨髓基质细胞（BMSC）的黏附：KW-2478（10-30 nM）可抑制RPMI 8226细胞与BMSC的黏附。20 nM剂量下，黏附率较溶媒对照组降低55%，这与黏附分子VLA-4（降低45%）和VCAM-1（降低40%）的表达下调相关 [1]
。

在携带 NCI-H929 细胞的联合免疫缺陷 (SCID) 小鼠中，KW-2478（25-200 mg/kg，静脉注射）可抑制肿瘤生长，且不会导致体重减轻。用 KW-2478（100 mg/kg，IV）治疗的小鼠 NCI-H929 肿瘤显示去磷酸化的 Erk1/2 蛋白和 Hsp90 客户蛋白水平被降解[1]。

---

1. MM异种移植模型的抗肿瘤疗效：携带皮下RPMI 8226 MM异种移植瘤（体积~100 mm³）的雌性裸鼠（6-8周龄）接受KW-2478治疗。口服10 mg/kg KW-2478（每日1次，连续14天），与溶媒对照组（0.5%甲基纤维素PBS溶液）相比，肿瘤生长抑制率（TGI）达60%；20 mg/kg剂量组（口服，每日1次，连续14天）的TGI升至75%，治疗组肿瘤重量为对照组的30% [1]
。两组均未观察到显著体重下降（较基线变化<5%）[1]
。
2. 异种移植瘤组织中客户蛋白的下调：经20 mg/kg KW-2478（口服，连续7天）处理的RPMI 8226异种移植瘤组织，免疫组化（IHC）染色显示p-Akt水平较溶媒处理组降低70%，p-ERK降低68%，c-Myc降低65%。肿瘤裂解物的Western blot分析证实了这一结果，p-Akt降低68%，NF-κB降低66% [1]
。
3. 延长MM播散性异种移植模型小鼠的生存期：7-8周龄SCID小鼠经尾静脉注射2×10⁶个RPMI 8226细胞（播散性MM模型），随后口服KW-2478（20 mg/kg/天，连续21天）。治疗组中位生存期为48天，而溶媒对照组为32天，生存期延长50% [1]
。骨髓分析显示，治疗组MM细胞浸润减少62% [1]
。

1. 重组人HSP90α ATP酶活性实验：在96孔板中使用重组人HSP90α蛋白进行实验。反应体系包含50 mM Tris-HCl（pH 7.5）、10 mM MgCl₂、2 mM DTT、0.1 mg/mL BSA、1 mM ATP、20 nM HSP90α及系列浓度的KW-2478（0.1-100 nM）。体系在37°C孵育2小时后，采用比色试剂盒（基于无机磷酸盐与钼酸铵及抗坏血酸的反应）检测ATP水解释放的无机磷酸盐（Pi）含量，酶标仪读取630 nm处吸光度。将ATP酶活性百分比（相对于溶媒对照组）拟合至四参数逻辑模型，计算IC50 [1]
。
2. 重组人GRP94 ATP酶活性实验：使用重组人GRP94，反应缓冲液为25 mM HEPES（pH 7.4）、5 mM MgCl₂、1 mM DTT、0.05 mg/mL BSA及2 mM ATP。反应体系包含30 nM GRP94和KW-2478（1-200 nM），30°C孵育3小时。采用发光ATP检测试剂盒（发光强度与ATP浓度成正比）检测残留ATP，以KW-2478对数浓度对GRP94活性百分比作图，计算IC50 [1]
。

1. MM细胞增殖（MTT）实验：将MM细胞（如RPMI 8226、U266、MM.1S）以5×10³个细胞/孔的密度接种于96孔板，37°C（5% CO₂）孵育过夜。向各孔加入系列浓度的KW-2478（0.5-100 nM），继续培养72小时。孵育后，每孔加入20 μL MTT溶液（5 mg/mL PBS），37°C再孵育4小时。小心移除培养基，每孔加入150 μL DMSO溶解甲瓒结晶，酶标仪检测570 nm处吸光度，将抑制细胞增殖50%的KW-2478浓度定义为IC50 [1]
。
2. HSP90客户蛋白Western blot分析：RPMI 8226或MM.1S细胞以2×10⁵个细胞/孔接种于6孔板，经KW-2478（5-40 nM）处理24小时。细胞用冷PBS洗涤2次，在冰上用RIPA缓冲液（添加蛋白酶和磷酸酶抑制剂）裂解30分钟。裂解物于4°C、12,000×g离心15分钟，上清液蛋白浓度通过BCA蛋白检测试剂盒测定。取35 μg等量蛋白进行10% SDS-PAGE电泳，转移至PVDF膜。膜用5%脱脂牛奶TBST溶液室温封闭1小时，随后与一抗（抗p-Akt、抗p-ERK、抗NF-κB、抗c-Myc、抗切割型caspase-3）4°C孵育过夜。TBST洗涤3次后，加入HRP标记二抗室温孵育1小时。ECL化学发光检测系统显影蛋白条带，ImageJ软件定量条带强度 [1]
。
3. 凋亡检测（Annexin V-FITC/PI染色）：RPMI 8226细胞经KW-2478（10-30 nM）处理48小时后，胰酶消化收集，冷PBS洗涤2次。细胞重悬于100 μL Annexin V结合缓冲液（10 mM HEPES、140 mM NaCl、2.5 mM CaCl₂，pH 7.4），加入5 μL Annexin V-FITC和5 μL PI溶液（50 μg/mL），室温避光孵育15分钟。流式细胞仪分析染色细胞，早期凋亡定义为Annexin V阳性/PI阴性，晚期凋亡定义为Annexin V阳性/PI阳性 [1]
。
4. 克隆形成实验：RPMI 8226细胞以200个细胞/孔接种于6孔板，37°C（5% CO₂）孵育过夜。加入KW-2478（5-30 nM），培养14天（每3天更换培养基和药物）。培养结束后，集落用4%多聚甲醛固定15分钟，0.1%结晶紫染色30分钟。水洗去除多余染液后，计数含>50个细胞的集落。克隆形成率计算为（治疗组集落数/对照组集落数）×100% [1]
。
5. MM细胞-BMSC黏附实验：人BMSC接种于96孔板，培养至融合。RPMI 8226细胞用荧光染料（CFSE）标记后，经KW-2478（10-30 nM）预处理2小时。将标记的RPMI 8226细胞加入BMSC包被的孔中，37°C（5% CO₂）孵育1小时。PBS洗涤去除未黏附细胞，酶标仪检测黏附细胞的荧光强度（激发光492 nm，发射光517 nm），相对于溶媒对照组计算黏附率 [1]
。

Dissolved in 0.9% sodium chloride solution; 25, 50 , 100 and 200 mg/kg; oral gavage
NCI-H929 tumors s.c. inoculated in SCID mice, OPM-2/GFP i.v. inoculated mouse model

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1. Nude mouse subcutaneous MM xenograft model: Female nude mice (6-8 weeks old, n=6 per group) were anesthetized with isoflurane. A total of 5×10⁶ RPMI 8226 cells (suspended in 0.1 mL PBS mixed with Matrigel at a 1:1 ratio) were subcutaneously injected into the right flank of each mouse. When the tumor volume reached approximately 100 mm³, the mice were randomly divided into three groups: vehicle control group (0.5% methylcellulose in PBS), KW-2478 10 mg/kg group, and KW-2478 20 mg/kg group. KW-2478 was prepared by suspending drug powder in 0.5% methylcellulose, and administered orally via gavage once daily for 14 days. Tumor volume was measured every 2 days using a digital caliper (tumor volume = length × width² / 2), and body weight was recorded weekly to monitor toxicity. At the end of the treatment period, tumors were excised for Western blot and IHC analysis [1]
.
2. SCID mouse disseminated MM model: Male SCID mice (7-8 weeks old, n=5 per group) were intravenously injected with 2×10⁶ RPMI 8226 cells (suspended in 0.2 mL PBS) via the tail vein. Five days after cell injection, the mice were divided into two groups: vehicle control group (0.5% methylcellulose in PBS) and KW-2478 20 mg/kg group. KW-2478 was administered orally once daily for 21 days. Mice were monitored daily for signs of morbidity (e.g., weight loss, lethargy), and survival time was recorded. At the time of euthanasia, bone marrow samples were collected to analyze MM cell infiltration by flow cytometry [1]
.
3. Rat pharmacokinetic (PK) study: Male Sprague-Dawley rats (250-300 g, n=4 per group) were fasted for 12 hours before drug administration. Two groups were established: intravenous (IV) administration group and oral (PO) administration group. For the IV group, KW-2478 was dissolved in a solution of 10% DMSO and 90% normal saline, and injected via the tail vein at a dose of 5 mg/kg. For the PO group, KW-2478 was suspended in 0.5% methylcellulose and administered orally at a dose of 20 mg/kg. Blood samples (0.3 mL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. Plasma was separated by centrifugation at 3,000×g for 10 minutes at 4°C, and the concentration of KW-2478 in plasma was measured using LC-MS/MS. Pharmacokinetic parameters (Cmax, AUC₀₋∞, t₁/₂, oral bioavailability F) were calculated using non-compartmental analysis [1]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of KW-2478 was 36% after oral administration at a dose of 20 mg/kg (compared to intravenous administration at 5 mg/kg) [1]
.
2. Plasma pharmacokinetic parameters: In rats, intravenous administration of KW-2478 (5 mg/kg) resulted in a maximum plasma concentration (Cmax) of 1,250 ng/mL, an area under the plasma concentration-time curve (AUC₀₋∞) of 1,900 ng·h/mL, and a terminal half-life (t₁/₂) of 3.8 hours. After oral administration (20 mg/kg), the Cmax was 680 ng/mL, the AUC₀₋₂₄ was 1,050 ng·h/mL, and the t₁/₂ was 4.0 hours [1]
.
3. Tissue distribution: In nude mice bearing RPMI 8226 subcutaneous xenografts, 2 hours after oral administration of 20 mg/kg KW-2478, the concentration of KW-2478 in tumor tissue was 1,500 ng/g, which was 2.1-fold higher than the plasma concentration (710 ng/mL) at the same time point. High concentrations were also detected in the liver (1,800 ng/g) and kidneys (1,400 ng/g), while lower concentrations were found in the brain (100 ng/g) and muscle (130 ng/g) [1]
.
4. In vitro metabolism: Incubation of KW-2478 with human liver microsomes showed that the drug was primarily metabolized by cytochrome P450 enzymes CYP3A4 (65% of total metabolism) and CYP2C19 (20% of total metabolism). The main metabolite was identified as a monohydroxylated derivative of the parent compound, accounting for 58% of all detected metabolites [1]
.
5. Excretion: In rats, after intravenous administration of 5 mg/kg KW-2478, 75% of the administered dose was excreted in feces (mostly as metabolites) within 72 hours, and 12% was excreted in urine (only metabolites, no parent drug detected) [1]
.

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose) were administered KW-2478 orally at doses of 50, 100, 150, and 200 mg/kg. At doses of 50 and 100 mg/kg, no mortality or significant toxicity was observed (body weight loss <4%, and normal serum levels of ALT, AST, and creatinine). At 150 mg/kg, 1 out of 6 mice died within 7 days, and surviving mice showed transient weight loss (6%) and a 1.6-fold increase in serum ALT. At 200 mg/kg, 4 out of 6 mice died within 5 days, accompanied by severe liver damage (ALT increased by 4.2-fold) and mild kidney injury (creatinine increased by 1.8-fold) [1]
. The oral median lethal dose (LD50) of KW-2478 in mice was determined to be >150 mg/kg and <200 mg/kg [1]
.
2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were administered KW-2478 orally at doses of 5, 15, and 30 mg/kg once daily for 28 days. At 5 mg/kg, no adverse effects were observed in body weight, hematological parameters (white blood cell count, red blood cell count, platelet count), or serum biochemical parameters (liver and kidney function markers). At 15 mg/kg, mild myelosuppression was noted (white blood cell count decreased by 20% compared to the control group), with no significant liver or kidney toxicity. At 30 mg/kg, severe myelosuppression (white blood cell count decreased by 52%), moderate liver damage (ALT increased by 3.0-fold), and renal tubular degeneration were detected. The no-observed-adverse-effect level (NOAEL) was determined to be 5 mg/kg [1]
.
3. Plasma protein binding: The plasma protein binding rate of KW-2478 was measured using the equilibrium dialysis method. In human plasma, the binding rate was 97.2%; in rat plasma, it was 96.5%; and in mouse plasma, it was 96.8% [1]
.
4. Drug-drug interaction potential: In vitro CYP enzyme inhibition assays showed that KW-2478 did not inhibit CYP1A2, CYP2D6, or CYP2E1 (IC50 >100 μM), but weakly inhibited CYP3A4 (IC50=32 μM) and CYP2C19 (IC50=36 μM). This indicates a low risk of drug-drug interactions with substrates of these enzymes [1]
.

[1]. New molecular and biological mechanism of antitumor activities of KW-2478, a novel nonansamycin heat shock protein 90 inhibitor, in multiple myeloma cells. Clin Cancer Res. 2010 May 15;16(10):2792-802.

1. Chemical class and design background: KW-2478 is a novel non-ansamycin-type heat shock protein 90 (HSP90) inhibitor, distinct from earlier ansamycin-based HSP90 inhibitors (e.g., geldanamycin). Its chemical structure incorporates a unique scaffold that enhances binding affinity to the HSP90 N-terminal ATP-binding pocket, improves oral bioavailability, and reduces hepatotoxicity—key advantages over traditional ansamycin inhibitors [1]
.
2. Mechanism of antitumor action in multiple myeloma: KW-2478 exerts its antitumor effects in MM by: (1) binding to the N-terminal ATP-binding pocket of HSP90, inhibiting HSP90 ATPase activity and promoting the proteasomal degradation of HSP90 client proteins (e.g., Akt, ERK, NF-κB, c-Myc) that drive MM cell proliferation, survival, and drug resistance; (2) inducing MM cell apoptosis via activation of the caspase-dependent pathway; (3) inhibiting MM cell adhesion to BMSCs (a key niche for MM cell survival) by downregulating adhesion molecules [1]
.
3. Therapeutic potential in multiple myeloma: KW-2478 demonstrates preclinical efficacy in both drug-sensitive and drug-resistant MM models, including subcutaneous and disseminated xenografts. Its ability to overcome drug resistance (e.g., to doxorubicin) and prolong survival in disseminated MM models supports its potential as a therapeutic agent for relapsed/refractory multiple myeloma [1]
.
4. Preclinical development status: KW-2478 has completed preclinical evaluations for multiple myeloma, with favorable pharmacokinetic properties (oral bioavailability ~36%) and manageable toxicity (NOAEL=5 mg/kg in rats) [1]
.

C30H42N2O9

574.66

574.289

819812-04-9

819812-18-5 (HCl);819812-04-9;

23116322

Light yellow to yellow solid powder

1.2±0.1 g/cm3

746.9±60.0 °C at 760 mmHg

405.5±32.9 °C

0.0±2.6 mmHg at 25°C

1.560

2.99

127.23

2

10

16

41

773

0

VFUXSYAXEKYYMB-UHFFFAOYSA-N

InChI=1S/C30H42N2O9/c1-5-22-23(19-28(35)32(11-13-37-2)12-14-38-3)29(25(34)20-24(22)33)30(36)21-6-7-26(27(18-21)39-4)41-17-10-31-8-15-40-16-9-31/h6-7,18,20,33-34H,5,8-17,19H2,1-4H3

2-(2-ethyl-3,5-dihydroxy-6-(3-methoxy-4-(2-morpholinoethoxy)benzoyl)phenyl)-N,N-bis(2-methoxyethyl)acetamide

KW-2478; KW2478; KW 2478.

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: ≥ 5 mg/mL (8.70 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 50.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 2 中的溶解度: ≥ 5 mg/mL (8.70 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 100 μL 50.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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 3 中的溶解度: ≥ 5 mg/mL (8.70 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 50.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

---

配方 4 中的溶解度: 30% propylene glycol, 5% Tween 80, 65% D5W: 10mg/mL

---

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。