Hsp90 (Kd = 0.71 nM)

胶质瘤细胞迁移、存活和增殖受到乳酸 onalespib (0-0.4 µM；72 小时、48 小时) 的抑制 [2]。 AKT、P-AKT、ERK1/2、P-ERK1/2、S6 和 P-S6 蛋白表达均会因 onalespib 乳酸（0-0.4 µM；48 小时）而以剂量依赖性方式降低[2]。

在 6 周大的 ICR 小鼠中，onalespib milkate（30 mg/kg；腹腔注射；一次）穿过血脑屏障 (BBB)，导致 HSP90 的长期抑制 [2]。在斑马鱼胚胎中，onalespib milkate（0.5 µM；移植后 5 至 10 天）和 TMZ (10 µM) 抑制肿瘤生长并提高存活率 [2]。在HCT116和A431异种移植物中，乳酸onalespib（HCT116异种移植物为5、10 mg/kg，A431异种移植物为20 mg/kg；腹膜内注射；每天一次，持续3天）显示出抗癌功效[3]。

等温滴定量热法（ITC） [1]
ITC实验在25°C的mirlocal VP-ITC上进行，缓冲液包括25 mM Tris， 100 mM NaCl， 1mM MgCl2和1mM TCEP， pH为7.4。最终DMSO浓度在1-5%之间。所使用的蛋白质与两种x射线晶体学工作中使用的Hsp90 n端atp酶结构域结构相同。大多数ITC实验都是在样品细胞中设置蛋白质，在注射注射器中设置化合物，尽管在化合物溶解度有限的情况下，这是相反的。数据采用Origin 7.0软件拟合为单位点结合模型。所有数据分析的化学计量值在0.8 ~ 1.3范围内，为蛋白质和化合物的质量、纯度和稳定性提供了良好的内部控制。在Kd值大于蛋白浓度的情况下，化学计量参数固定为1使用上述程序，ADP和17-DMAG的解离常数分别为9.2µM和0.21µM。这些值与文献中人类Hsp90蛋白全长解离常数（ADP为11µM， 17-DMAG.32为0.35µM）一致为了准确测定化合物31的亲和力，必须采用竞争格式ITC。这需要在开始用化合物31滴定之前，将蛋白质与我们的一种中等效价的酚化合物在样品细胞中预孵养。采用竞争结合模型拟合数据，得到化合物31的Kd估计。

细胞增殖测定[2]
细胞类型： LN229、U251 和 A172 细胞
测试浓度： 0.1、0.2、0.4 µM
孵育时间：72小时
实验结果：以剂量依赖性方式抑制细胞增殖。

---

蛋白质印迹分析[2]
细胞类型： LN229、U251 和 A172 细胞
测试浓度： 0.1、0.2、0.4 µM
孵育时间：48小时
实验结果：减少EGFR、p-EGFR、AKT、P-AKT、ERK1/2、P-的表达ERK1/2、S6、P-S6蛋白呈剂量依赖性。

Animal/Disease Models: Female nu/nu balb/c (Bagg ALBino) mouse (HCT116 and A431 xenografts)[3]
Doses: 5, 10 mg/kg for HCT116 xenografts, 20 mg/kg for A431 xenografts
Route of Administration: Ip; on day 1, 2, and 3 days
Experimental Results: Inhibited tumor growth and had a median survival of 9.5 days and the maximum survival was 14 days in HCT116 xenografts, decreased the tumor size Dramatically by 32% in A431 xenografts.

---

In vivo Efficacy Study. [4]
HCT116 cells were injected SC into the right hind flank of male nude BALB/c mice. Tumours were apparent 7 to 10 days later. Mice were arranged into matched groups of 12 according to tumour volume giving a group mean of approximately 100 mm3 at initiation of dosing. Tumour volumes were measured S42 every 2 days. Statistical significance between groups was assessed using nonparametric one-way ANOVA. Mice were given the lactate salt of AT13387 (compound 35) using a repeated cycle of dosing of once per day for three days, no dose for three days, once per day for three days etc., for four dosing cycles at 60 mg/kg/dose (as free base equivalents) dissolved in 17.5% hydroxypropylβ-cyclodextrin via the IP route. Control mice received dose vehicle only via the same route. Tolerability was assessed by recording body weight, clinical observations and survival. AT13387 (compound 35) was well tolerated at the dose administered. Compound 1 and 17 (as the hydrochloride salt) were dosed qd (once daily) by the IP route and compound 18 (as the hydrochloride salt) was dosed q2d (every other day) by the same route, using the doses indicated below. An initial dose ranging tolerability study was performed prior to all in vivo efficacy experiments to select the most appropriate dose range. For all of the efficacy experiments carried out during the screening phase, the maximum doses used ranged between 40 and 80 mg/kg. [4]

---

Pharmacokinetic Study Methods. [4]
Plasma pharmacokinetic parameters of compounds 1, 17, 18 and 35 were determined after IV administration of individual compounds to BALB/c mice. Dosing details are given in Table A. AT13387 (compound 35) was also dosed by the oral route, formulated in 30% sterile water; 70% HPβCD (25% w/v aq) at a dose level of 50 mg free base equivalent/kg.

[1]. Erlotinib and Onalespib Lactate Focused on EGFR Exon 20 Insertion Non-Small Cell Lung Cancer (NSCLC): A California Cancer Consortium Phase I/II Trial (NCI 9878). Clin Lung Cancer. 2021 Nov;22(6):541-548.

[2]. Efficacy of Onalespib, a Long-Acting Second-Generation HSP90 Inhibitor, as a Single Agent and in Combination with Temozolomide against Malignant Gliomas. Clin Cancer Res. 2017 Oct 15;23(20):6215-6226.

[3]. The HSP90 inhibitor Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy: an in vitro and in vivo approach. Sci Rep. 2020 Apr 3;10(1):5923.

[4]. Discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment based drug design. J Med Chem. 2010 Aug 26;53(16):5956-69.

Onalespib Lactate is the lactate form of onalespib, a synthetic, orally bioavailable, small-molecule inhibitor of heat shock protein 90 (Hsp90) with potential antineoplastic activity. Onalespib selectively binds to Hsp90, thereby inhibiting its chaperone function and promoting the degradation of oncogenic signaling proteins involved in tumor cell proliferation and survival. Hsp90, a chaperone protein upregulated in a variety of tumor cells, regulates the folding, stability and degradation of many oncogenic signaling proteins.

C27H37N3O6

499.599187612534

499.268

C, 64.91; H, 7.47; N, 8.41; O, 19.21

1019889-35-0

Onalespib;912999-49-6

46915024

Typically exists as solid at room temperature

125

4

8

5

36

651

1

O=C(C1C(=CC(=C(C(C)C)C=1)O)O)N1CC2C=CC(=CC=2C1)CN1CCN(C)CC1.O[C@H](C(=O)O)C

VYRWEWHOAMGLLW-WNQIDUERSA-N

InChI=1S/C24H31N3O3.C3H6O3/c1-16(2)20-11-21(23(29)12-22(20)28)24(30)27-14-18-5-4-17(10-19(18)15-27)13-26-8-6-25(3)7-9-26;1-2(4)3(5)6/h4-5,10-12,16,28-29H,6-9,13-15H2,1-3H3;2,4H,1H3,(H,5,6)/t;2-/m.0/s1

(2,4-dihydroxy-5-isopropylphenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone (S)-2-hydroxypropanoate

Onalespib lactate; AT-13387 lactate; ATI 13387; AT-13387 lactate; 1019889-35-0; Onalespib lactate [USAN]; ATI-13387AU; ATI-13387A; Onalespib l-lactate; UNII-66226JUH2I; ATI-13387A; ATI-13387; ATI-13387AU; ATI13387.

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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。

---

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。

View More

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)

---

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。

View More

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

---

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。

---

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。