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
The bioavailability after oral administration is 58-70%, while the bioavailability after parenteral administration (intravenous and intramuscular injection) is 100%. Biological Half-Life t1/2-α = minutes (10-20 minutes in rats); t1/2-β = several hours

Hepatotoxicity
Diffluride treatment was not associated with elevated serum transaminases or clinically significant liver injury other than the SOS characteristics associated with its treatment. In a trial of diffluride for SOS prevention in 356 children who underwent hematopoietic stem cell transplantation (HCT), the incidence of serious adverse events such as bleeding, gastrointestinal upset, and liver injury was similar between children treated with diffluride and those who did not. Probability Score: E (Unlikely to be a cause of clinically significant 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 a sodium salt of a mixture of single-stranded oligodeoxynucleotides derived from porcine mucosal DNA. It has been shown to possess antithrombotic, anti-inflammatory, and anti-ischemic properties (but no significant systemic anticoagulant activity). It is marketed in several countries under the trade names Dasovas (FM), Noravid, and Prociclide. In the United States, it was approved in March 2016 under the trade name Defitelio. Defibrotide is a complex mixture of single-stranded oligodeoxynucleotides derived from porcine intestinal mucosa with antithrombotic and fibrinolytic activities, used to treat severe sinusoidal obstruction syndrome (SOS) following hematopoietic stem cell transplantation (HCT). Defibrotide has been used to treat patients with severe liver injury, and no worsening of serum transaminase elevations or clinically significant specific liver injury cases were observed during treatment. Defibrotide is a mixture of single-stranded oligodeoxynucleotides derived from porcine intestinal mucosa with antithrombotic, thrombolytic, and fibrinolytic activities. Following administration, although its exact mechanism of action is not fully elucidated, difrubitide induces the release of prostaglandin I2 (PGI2), E2 (PGE2), and prostacyclin, and reduces the expression of adhesion molecules on endothelial cells. This relaxes vascular smooth muscle and prevents platelets from adhering to each other and to endothelial cells, thereby protecting the vascular endothelium. Difrubitide increases tissue-type plasminogen activator (t-PA) activity and decreases plasminogen activator inhibitor-1 activity. This product enhances plasmin activity, prevents thrombus formation, and dissolves thrombi.
See also: Difrubitide sodium (note moved to).

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Indications
Indications for the treatment of severe hepatic venous occlusion (VOD), also known as sinusoidal obstruction syndrome (SOS), following hematopoietic stem cell transplantation (HSCT) with renal or pulmonary dysfunction.
FDA Label

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Mechanism of Action
This product appears to prevent thrombosis and help dissolve thrombi by increasing the levels of prostaglandin I2, E2, and prostacyclin, altering platelet activity, enhancing the function of tissue plasminogen activator (TPA), and decreasing the activity of TPA inhibitors. Prostaglandin I2 relaxes vascular smooth muscle and prevents platelets from adhering to each other. Prostaglandin E2 also inhibits platelet aggregation at certain concentrations. Furthermore, recent studies have shown that this drug also possesses additional anti-inflammatory and anti-ischemic activities. It is currently unclear whether the latter efficacy can be used clinically (e.g., for the treatment of ischemic stroke).

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Pharmacodynamics
Diffubuxin is a deoxyribonucleic acid derivative extracted from mammalian organs and has been developed for the treatment of various vascular diseases. It appears to enhance fibrinolysis and may possess antithrombotic, anti-atherosclerotic, and anti-ischemic effects, possibly due to its ability to selectively increase prostaglandin I2 and E2 levels and enhance the activity of tissue plasminogen activator (tPA) while decreasing the activity of plasminogen activator inhibitors (PAIs). Difubutrie is available in various dosage forms, including intravenous, intramuscular, and oral formulations, for long-term treatment.

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.)