Prodrug of ganetespib

Continuous HSP90 inhibition led to durable destabilization of client proteins in vitro; however, transient exposure required >10× drug for comparable effects[1].

In vivo, KIT was rapidly degraded following a single dose of HSP90i but returned to baseline levels within a day. HSP90 levels increased and stabilized 16 hours after HSP90i and were not elevated following a subsequent near-term exposure, providing a functional pool of chaperone to stabilize proteins and a means for greater therapeutic activity upon HSP90i reexposure. HSP90i administered on days 1 and 2 (D1/D2) demonstrated increased biologic activity compared with D1 treatment in KIT or EGFR-driven murine tumor models. In a trial of dogs with MCT, D1/D2 dosing of HSP90i was associated with sustained KIT downregulation, 50% objective response rate and 100% clinical benefit rate compared with D1 and D1/D4 schedules.

HSP90 binding assay. [1]
H1975 cells, cultured in RPMI-1640 and 10% FBS, were seeded at a density of 3 × 105 cells per well in 6 well plates. Twenty-four hours later, cells were treated with ganetespib as indicated and incubated at 37°C. Cells were washed twice in cold PBS then lysed in cold HSP90 binding buffer (20mM HEPES pH 7.3, 1mM EDTA, 100mM KCl, 5mM MgCl, 0.01% v/v NP-40, 0.5mg/mL bovine gamma globulin, 1mM TCEP) by incubation on ice for 10 minutes followed by three freeze/thaw cycles. Lysates were clarified by centrifugation at 14,000 x g. To remove unbound ganetespib, lysates were passed over 40K MWCO size exclusion columns. To titrate unoccupied HSP90 binding sites, 10 μM of a deuterated form of ganetespib (D3-ganetespib) was added to eluates and incubated at 4°C for 2 hours then passed over a size exclusion column to remove unbound D3-ganetespib. Total protein from flow through was quantified by BCA protein assay and all samples diluted to 1 mg/mL. The concentrations of ganetespib and D3-ganetespib were measured by LC/MS-MS. A Phenomenex Kinetex column (C18, 30 × 2.1 mm, 2.6 µm) was used with a run time of 3.5 minutes per sample. The following equation was used to calculate the percent of ganetespib bound to HSP90 (HSP90 occupancy): [ganetespib]/([ganetespib] + [D3-ganetespib]) x 100 [1].

Western blotting[1]
Following in vitro assays, tumor cells were disrupted in lysis buffer on ice for 10 minutes. For the pharmacodynamic analysis, xenograft tumors (average volume of 100–200 mm3) were excised, cut in half, and flash frozen in liquid nitrogen. Each tumor fragment was lysed in 0.5 mL of lysis buffer using a FastPrep-24 homogenizer and Lysing Matrix A. Lysates were clarified by centrifugation and equal amounts of protein resolved by SDS-PAGE before transfer to nitrocellulose membranes. Membranes were blocked with Starting Block T20 Blocking Buffer and immunoblotted with the indicated antibodies. Antibody–antigen complexes were visualized using an Odyssey system (LI-COR).

Female CB.17 (SCID) mice at 7–12 weeks of age were maintained in a pathogen-free environment and all in vivo procedures were approved by the Synta Pharmaceuticals Corp. Institutional Animal Care and Use Committee. Human GIST882 cells were provided by Dr. Jonathan Fletcher (Dana Farber Cancer Institute) and implanted subcutaneously at 10 × 106 into mice. Mice bearing established tumors (~110 mm3) were randomized into treatment groups of 8 and dosed intravenously with vehicle or ganetespib, formulated in DRD (10% DMSO, 18% Cremophor RH 40, 3.6% dextrose), using the schedules indicated. Human H1975 NSCLC cells were purchased from the ATCC, selected to stably express a HIF-1α-LUC reporter and implanted at 10 × 106 into mice. Mice bearing established tumors (~143 mm3) were randomized into treatment groups of 4 and dosed intravenously with vehicle or ganetespib, formulated in DRD, using the schedules indicated. Tumor volumes (V) were calculated by caliper measurements of the width (W), length (L) and thickness (T) of each tumor using the formula: V=0.5236(LWT). Tumor growth inhibition was determined as described previously.[1]

Absorption, Distribution and Excretion
Bioavailability is 58-70% following oral administration, compared to parenteral forms (i.v. and i.m. = 100%).

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Biological Half-Life
t1/2-alpha = minutes (10-20 minutes in rat); t1/2-beta = a few hours

Hepatotoxicity
Defibrotide therapy has not been linked to serum aminotransferase elevations or with instances of clinically apparent liver injury separate from the features of SOS for which it is given. In a trial of defibrotide as prophylaxis against SOS conducted in 356 children undergoing HCT, rates of severe adverse events such as hemorrhage, gastrointestinal complaints and liver injury were similar in those receiving defibrotide as in untreated children.
Likelihood score: E (unlikely cause of clinically apparent acute liver injury).

[1]. Consecutive Day HSP90 Inhibitor Administration Improves Efficacy in Murine Models of KIT-Driven Malignancies and Canine Mast Cell Tumors. Clin Cancer Res. 2018 Dec 15;24(24):6396-6407.

Defibrotide is the sodium salt of a mixture of single-stranded oligodeoxyribonucleotides derived from porcine mucosal DNA. It has been shown to have antithrombotic, anti-inflammatory and anti-ischemic properties (but without associated significant systemic anticoagulant effects). It is marketed under the brand names Dasovas (FM), Noravid, and Prociclide in a variety of countries. In the USA it is was approved in March, 2016 as Defitelio.
Defibrotide is a complex mixture of single stranded polydeoxyribonucleotides derived from porcine intestinal mucosa that has antithrombotic and profibrinolytic activity and is used in the treatment of severe sinusoidal obstruction syndrome (SOS) after hematopoietic cell transplantation (HCT). Defibrotide is used in patients with severe liver injury and has not been associated with worsening of serum aminotransferase elevations during therapy and has not been linked to cases of clinically apparent, idiosyncratic liver injury.
Defibrotide is a mixture of single-stranded oligodeoxyribonucleotides derived from the intestinal mucosa of pigs, with anti-thrombotic, thrombolytic, and fibrinolytic activities. Upon administration, and although the exact mechanism of action has yet to be fully elucidated, defibrotide induces the release of prostaglandin I2 (PGI2), E2 (PGE2), and prostacyclin and reduces the expression of adhesion molecules on endothelial cells. This relaxes the smooth muscle of blood vessels and prevents platelets from adhering to each other and to the endothelium. This protects the endothelium lining bloods vessels. Defibrotide increases tissue plasminogen activator (t-PA) and decreases plasminogen activator inhibitor-1 activity. This increases the activity of plasmin, prevents blood clot formation and dissolves blood clots.
See also: Defibrotide Sodium (annotation moved to).

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Drug Indication
Indicated for the treatment of severe hepatic veno-occlusive disease (VOD), also known as sinusoidal obstruction syndrome (SOS), with renal or pulmonary dysfunction following hematopoietic stem-cell transplantation (HSCT).
FDA Label

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Mechanism of Action
The drug appears to prevent the formation of blood clots and to help dissolve blood clots by increasing levels of prostaglandin I2, E2, and prostacyclin, altering platelet activity, increasing tissue plasminogen activator function, and decreasing activity of tissue plasminogen activator inhibitor. Prostaglandin I2 relaxes the smooth muscle of blood vessels and prevents platelets from adhering to each other. Prostaglandin E2 at certain concentrations also inhibits platelet aggregation. Moreover, the drug provides additional beneficial anti-inflammatory and antiischemic activities as recent sudies have shown. It is yet unclear, if the latter effects can be utilized clinically (e.g., treatment of ischemic stroke).

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Pharmacodynamics
Defibrotide is a deoxyribonucleic acid derivative extracted from mammalian organs, which has been developed for the treatment of a number of vascular disorders. It appears to increase fibrinolysis and may possess antithrombotic, antiatherosclerotic and anti-ischaemic actions, probably due to its ability to selectively increase prostaglandin I2 and E2 levels and to increase tissue plasminogen activator and decrease plasminogen activator inhibitor function. Defibrotide is available as an intravenous and intramuscular preparation, and also as an oral formulation for long term use.

C20H21N4O6P

444.378

444.12

C, 54.06; H, 4.76; N, 12.61; O, 21.60; P, 6.97

1118915-78-8

1402907-09-8 (disodium);1118915-79-9 (monosodium);1118915-78-8 (free acid);83712-60-1(sodium);

135565962

Typically exists as solid at room temperature

1.987

4

7

5

31

773

0

CC(C)C1=CC(=C(C=C1OP(=O)(O)O)O)C2=NN=C(N2C3=CC=C4C(=C3)C=CN4C)O

JNWFIPVDEINBAI-UHFFFAOYSA-N

InChI=1S/C20H21N4O6P/c1-11(2)14-9-15(17(25)10-18(14)30-31(27,28)29)19-21-22-20(26)24(19)13-4-5-16-12(8-13)6-7-23(16)3/h4-11,25H,1-3H3,(H,22,26)(H2,27,28,29)

5-hydroxy-2-isopropyl-4-(4-(1-methyl-1H-indol-5-yl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl dihydrogen phosphate

STA-1474; ganetespib-prodrug; STA 1474; STA-9090 prodrug; STA1474; STA9090 prodrug

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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