Hsp90 (Kd = 0.71 nM)

Compound 35, onalespib, has a Kd of 0.71 nM, making it a strong inhibitor of Hsp90. In HCT116 cells, onalespib has strong antiproliferative activity with an IC50 of 31 nM. Additionally, onalespib exhibits substantial inhibition of a panel of human tumor cell lines' growth, with an IC50 of less than 100 nM[1]. Against numerous PPTP cell lines, onalespib demonstrates cytotoxic action, with a median IC50 of 41 nM[2].

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In vitro testing: The median AT13387 IC50 value (concentration inhibiting growth 50% relative to controls) for the PPTP cell lines was 41 nM, (range 14 nM - 201 nM). The ratio of the median EC50 of the entire panel to that of each cell line was calculated Table I. The median EC50 (concentration causing 50% maximum effect) for the Ewing sarcoma panel was significantly lower (p=0.015) than that of the remaining PPTP cell lines. AT13387 demonstrated an activity pattern consistent with cytotoxic activity for many of the PPTP cell lines with minimum (Ymin) T/C% values approaching 0%.[2]

Onalespib (60 mg/kg, intraperitoneal; 3 days on, 3 days off) exhibits anticancer efficacy in BALB/c nude mice expressing human colon carcinoma xenografts in the early stage [1]. In 17% of evaluable solid tumor xenografts, onalespib (40 or 60 mg/kg, ip) causes substantial variations in the EFS distribution with respect to controls, but not in any of the ALL xenografts[2].

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In vivo testing[2]
AT13387 was tested in vivo using a 40 mg/kg dose administered IP twice-weekly (Mon-Thurs) repeated weekly for 6 weeks. All 43 xenograft models studied were considered evaluable for efficacy. A complete summary of results is provided in Supplemental Table I. AT13387 induced significant differences in EFS distribution compared to control in 6 of 35 (17%) evaluable solid tumor xenografts, Table II. AT13387 did not induce high or intermediate (EFS T/C>2) activity in any solid tumor xenografts evaluable. For the ALL panel, no xenografts showed a significant difference in EFS distribution between treated and control animals. AT13387 did not induce objective responses (PR or CR) in the PPTP solid tumor panels. The best response in the solid tumor panel was PD2 (progressive disease with growth delay), which was observed in 4 of 35 xenografts (11%).

Isothermal Titration Calorimetry (ITC) [1]
ITC experiments were performed on a MircoCal VP-ITC at 25°C in a buffer comprising 25 mM Tris, 100 mM NaCl, 1 mM MgCl2 and 1mM TCEP at pH 7.4. The final DMSO concentration was between 1-5%. The protein used was the same Hsp90 N-terminal ATPase domain construct used in both X-ray crystallography work. The majority of ITC experiments were set up with protein in the sample cell and compound in the injection syringe although in cases where compound solubility was limiting this was reversed. Data were fit to a single site binding model using Origin 7.0 software. All the stoichiometry values from the data analysis were in the range 0.8-1.3, providing an excellent internal control for the quality, purity and stability of both the protein and the compounds. The stoichiometry parameter was fixed at 1 in cases where the Kd value was greater than the protein concentration.53 Using the procedure outlined above, ADP and 17-DMAG had measured dissociation constants of 9.2 µM and 0.21 µM. These values are in good agreement with literature dissociation constants with the full length human Hsp90 protein of 11 µM for ADP, and 0.35 µM for 17-DMAG.32 A competition format ITC was necessary to accurately determine the affinity of compound 31. 54 This required pre-incubating the protein with one of our moderately potent phenol compounds in the sample cell prior to initiating the titration with compound 31. A competition binding model was used to fit the data and obtain a Kd estimate for compound 31.

In vitro testing[2]
In vitro testing was performed using DIMSCAN, as previously described. Cells were incubated in the presence of AT13387 for 96 hours at concentrations from 1 nM to 10 μM and analyzed as previously described.

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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+ddH2O: 10 mg/mL

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Solubility in Formulation 5: 16.67 mg/mL (40.71 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)