Hsp90 (Kd = 0.71 nM)

化合物 35，onalespib，Kd 为 0.71 nM，使其成为 Hsp90 的强抑制剂。在 HCT116 细胞中，onalespib 具有很强的抗增殖活性，IC50 为 31 nM。此外，onalespib 对一组人类肿瘤细胞系的生长具有显着抑制作用，IC50 低于 100 nM[1]。针对多种 PPTP 细胞系，onalespib 表现出细胞毒性作用，中值 IC50 为 41 nM[2]。

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体外测试：PPTP细胞系的AT13387 IC50中值（相对于对照组抑制生长50%的浓度）为41 nM，范围为14 nM - 201 nM。计算整个组EC50中位数与各细胞系EC50中位数之比表1。Ewing肉瘤组EC50中位数（引起50%最大效应的浓度）显著低于其余PPTP细胞系（p=0.015）。AT13387在许多PPTP细胞系中表现出与细胞毒活性一致的活性模式，其最小（Ymin） T/C%值接近0%。[2]

Onalespib（60 mg/kg，腹腔注射；用药 3 天，停药 3 天）在表达早期人类结肠癌异种移植物的 BALB/c 裸鼠中表现出抗癌功效 [1]。在 17% 的可评估实体瘤异种移植物中，onalespib（40 或 60 mg/kg，腹膜内注射）会导致 EFS 分布相对于对照发生显着变化，但在任何 ALL 异种移植物中均未出现这种情况[2]。

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体内试验[2]
AT13387采用40 mg/kg剂量的IP进行体内试验，每周两次（周一至周四），每周重复6周。所有43种异种移植模型的疗效均可评估。补充表1提供了完整的结果摘要。 在35例可评估的实体瘤异种移植物中，AT13387诱导的EFS分布与对照组相比有显著差异（17%），表2。AT13387在任何可评估的实体瘤异种移植物中均未诱导高或中等（EFS T/C>2）活性。对于ALL组，没有异种移植物在治疗动物和对照动物之间显示出显著的EFS分布差异。AT13387在PPTP实体瘤组中没有诱导客观反应（PR或CR）。实体瘤组的最佳反应是PD2（进展性疾病伴生长延迟），35例异种移植物中有4例（11%）出现PD2（进展性疾病伴生长延迟）。

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。

体外试验[2]
如前所述，使用DIMSCAN进行体外测试。细胞在AT13387的存在下，以1 nM至10 μM的浓度孵育96小时，并按先前描述的方法进行分析。

In vivo Efficacy Study. [1]
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. [1]

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Pharmacokinetic Study Methods. [1]
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.

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CB17SC scid−/− female mice, were used to propagate subcutaneously implanted kidney/rhabdoid tumors, sarcomas, neuroblastoma, and non-glioblastoma brain tumors, while BALB/c nu/nu mice were used for glioma models, as previously described. Human leukemia cells were propagated by intravenous inoculation in female non-obese diabetic (NOD)/scid−/− mice as described previously. Female mice were used irrespective of the patient gender from which the original tumor was derived. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee of the appropriate consortium member. Eight to ten mice were used in each control or treatment group.
AT13387 was provided to the Pediatric Preclinical Testing Program by Astex Therapeutics, through the Cancer Therapy Evaluation Program (NCI). Powder was stored at 4°C, protected from light. Drug was formulated in 17.5% hydroxy-propyl-β-cyclodextrin, in sterile water for injection, and made fresh prior to administration. AT13387 was administered intraperitoneally using a twice-weekly schedule for 6 weeks at a dose of 40 mg/kg, or 60 mg/kg for 3 weeks. AT13387 was provided to each consortium investigator in coded vials for blinded testing.[2]

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Dissolved in 17.5% cyclodextrin; 80 mg/kg; i.p. injection

Athymic BALB /c mice

[1]. 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.

[2]. Initial testing (Stage 1) of AT13387, an HSP90 inhibitor, by the pediatric preclinical testing program. Pediatr Blood Cancer. 2012 Jul 15;59(1):185-8.

Onalespib is a member of the class of isoindoles that is isoindole in which the amino group has been acylated by a 2,4-dihydroxy-5-isopropylbenzoyl group and in which position 5 of the isoidole moiety has been substituted by a (4-methylpiperazin-1-yl)methyl group. A second-generation Hsp90 inhibitor. It has a role as a Hsp90 inhibitor and an antineoplastic agent. It is a member of resorcinols, a member of benzamides, a tertiary carboxamide, a member of isoindoles and a N-alkylpiperazine.
Onalespib is 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.

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Drug Indication
Investigated for use/treatment in cancer/tumors (unspecified).

C24H31N3O3

409.52

409.236

C, 70.39; H, 7.63; N, 10.26; O, 11.7

912999-49-6

Onalespib lactate;1019889-35-0

11955716

White to off-white solid powder

1.2±0.1 g/cm3

605.7±55.0 °C at 760 mmHg

320.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.633

1.52

67.25

2

5

4

30

592

0

IFRGXKKQHBVPCQ-UHFFFAOYSA-N

InChI=1S/C24H31N3O3/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/h4-5,10-12,16,28-29H,6-9,13-15H2,1-3H3

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

Onalespib lactate; AT13387; AT-13387; 912999-49-6; (2,4-Dihydroxy-5-isopropylphenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone; Onalespib (AT13387); AT 13387; ATI-13387X; AT 13387; Onalespib;

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: ≥ 2.5 mg/mL (6.10 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (6.10 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 25.0 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: ≥ 2.5 mg/mL (6.10 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 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2% DMSO+30% PEG 300+ddH2O: 10 mg/mL

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配方 5 中的溶解度: 16.67 mg/mL (40.71 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。