Topoisomerase II
Topoisomerase II (TOP2). Etoposide Phosphate is a selective inhibitor of topoisomerase II. Topoisomerase II is an enzyme that manages DNA supercoiling and resolves DNA tangles during replication and transcription by creating transient double-strand breaks and re-ligating them. Etoposide stabilizes the topoisomerase II-DNA cleavage complex, preventing re-ligation and leading to accumulation of DNA breaks.

Etoposide phosphate is the solubility of etoposide and could be its prodrug; etoposide is identified by the presence of a phosphate group at the 4' position of its E ring [1]. The inhibitory effects of etoposide phosphate (0-1) on HCT116 FBXW+/+, FBXW-/-, and p53-/- are dose-dependent and have IC50 values of 0.945 μM, 0.375 µM, and 1.437 µM[2]. 6 hours) postpones the recovery of p53 in cells lacking FBXW7. Moreover, in FBXW7-/-cells, FBXW7 expression vanished [2].

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In vitro, Etoposide Phosphate demonstrates potent anti-cancer activity through topoisomerase II inhibition. The compound induces double-strand DNA breaks, inhibits DNA repair, and results in decreased DNA synthesis and tumor cell proliferation. It also induces cell cycle arrest (G2/M phase), apoptosis, and autophagy in cancer cell lines. As a prodrug, it is converted to etoposide in vivo.

In female CD-1 mice, a single dosage of mortgage phosphate (IV; 50, 100, or 150 mg/kg) results in gradual analgesic rescue, righting crises, torsional limb splay, and paralysis. On day 8, female CD-1 mice administered with etoposide phosphate (50, 100, or 150 mg/kg in a single dosage) exhibit increasing ataxia, decreased righting reflex, and splaying and paresis of the fore- and hindlimbs[3].

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The effects of high toxic doses of the anticancer drugs, etoposide and its phosphate derivative, BMY-40481, on the nervous system of female CD-1 mice were examined by light microscopy (LM) and transmission electron microscopy. Mice were euthanatized 4 wk following a single iv injection of either 0, 50, 100, or 150 mg/kg of BMY-40481 or 44 or 88 mg/kg of etoposide. Mice treated with 100 or 150 mg/kg of BMY-40481 or 88 mg/kg of etoposide had clinical symptomology of progressive ataxia, impaired righting reflex, and splaying and paresis of fore- and hindlimbs at day 8. Similar, dose-related LM changes were observed with both drugs at all doses and consisted of degeneration of dorsal root ganglion cells and axonal degeneration of their distal and proximal processes in peripheral nerves, dorsal spinal roots, and dorsal funiculi of spinal cord. Axonal degeneration was characterized by LM as shrinkage, swelling, and fragmentation of axon cylinders accompanied by secondary demyelination. Degenerative changes in ganglion cell bodies included eccentric nuclei, cytoplasmic vacuolation, central chromatolysis, and peripheral clumping of Nissl's bodies. Ultrastructurally, ganglion cell bodies had focally extensive dilation of the rough endoplasmic reticulum, mitochondrial swelling, increased numbers of phagolysosomes and prominent aggregations of microfilaments (globular filamentous bodies). Ultrastructural axonal changes occurred primarily in large, myelinated fibers and consisted of axonal swelling or loss, thinning of myelin sheaths, and a decrease in the number of organelles. This is the first report of etoposide-related sensory neuropathy in laboratory rats, a model that my be useful for the study of etoposide-related peripheral neuropathy in humans.[3]

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In vivo, Etoposide Phosphate is an orally active anti-cancer chemotherapy agent. The prodrug form provides improved water solubility compared to etoposide, facilitating administration. The compound has been used in the treatment of various cancers, including testicular cancer, lung cancer, and lymphomas. Its mechanism of DNA damage and cell cycle arrest contributes to its anti-tumor efficacy.

Cell-free topoisomerase II assays for Etoposide Phosphate use purified human topoisomerase II and a DNA substrate (e.g., supercoiled plasmid DNA). The compound is incubated with the enzyme and DNA at varying concentrations (0.01-1000 μM) for 30-60 minutes at 37°C. Topoisomerase II activity is measured by assessing DNA relaxation or cleavage using agarose gel electrophoresis. The formation of the topoisomerase II-DNA cleavage complex is detected by the appearance of linear DNA or specific cleavage products. IC50 values are calculated from dose-response curves.

Cell viability test[2]
Cell Types: FBXW+/+, FBXW-/- and p53-/- Cell
Tested Concentrations: 0.025 μM, 0.05 μM, 0.075 μM, 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibition of HCT116 FBXW+/+, FBXW-/- and p53-/- cell growth in concentrated form.

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Western Blot Analysis[2]
Cell Types: HCT116 FBXW7+/+ or FBXW7-/- Cell
Tested Concentrations: 25 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: It shows that the recovery of p53 levels after DNA damage is mediated by FBXW7.

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Cellular assays for Etoposide Phosphate are performed using cancer cell lines (e.g., HeLa, A549, MCF-7, or HL-60 cells). Cells are seeded in 96-well plates and treated with Etoposide Phosphate at concentrations ranging from 0.01-1000 μM for 24-72 hours. Cell viability is measured using MTT, CCK-8, or CellTiter-Glo assays. Cell cycle analysis is performed by flow cytometry after propidium iodide staining to assess G2/M arrest. Apoptosis is quantified using Annexin V/PI double staining or caspase-3/7 activity assays. DNA damage is assessed by measuring γ-H2AX foci by immunofluorescence. IC50 values for anti-proliferative activity are determined from dose-response curves.

Animal/Disease Models: Female CD-1 mice[3]
Doses: 50, 100 or 150 mg/kg
Route of Administration: intravenous (iv) (iv)injection; clinical symptoms[3]. Single dose
Experimental Results: Under light microscopy (LM), degeneration of dorsal root ganglion cells in peripheral nerves, dorsal roots and dorsal cord of the spinal cord and axonal degeneration of their distal and proximal processes were observed at all doses.

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In vivo efficacy studies are conducted in immunodeficient mice bearing subcutaneous xenografts of various cancer cell lines. Etoposide Phosphate is administered orally or intravenously at doses typically ranging from 1-50 mg/kg, on a scheduled regimen (e.g., daily for 5 days). Tumor volume is measured every 2-3 days using calipers. At study termination, tumors are excised and processed for immunohistochemistry or Western blot analysis to assess DNA damage markers, apoptosis, and cell cycle arrest.

Pharmacokinetic studies of Etoposide Phosphate demonstrate that the compound is a prodrug that is converted to etoposide in vivo. The phosphate ester improves the water solubility of etoposide, allowing for oral and intravenous administration. PK parameters such as Cmax, Tmax, AUC, half-life, and oral bioavailability are determined in preclinical species and in clinical studies. The compound's conversion to etoposide and its subsequent distribution and elimination are characterized.

Toxicology studies of Etoposide Phosphate were conducted as part of its clinical development program. As a topoisomerase II inhibitor, potential toxicities include bone marrow suppression (myelosuppression), gastrointestinal effects, and alopecia. Standard toxicology studies (acute, subchronic, and chronic) were performed in rodents and non-human primates. The compound's safety profile is well-established from its clinical use as an anti-cancer agent.

[1]. Etoposide phosphate, the water soluble prodrug of etoposide. Pharm World Sci. 1996 Oct;18(5):163-70.

[2]. FBXW7 Confers Radiation Survival by Targeting p53 for Degradation.Cell Rep. 2020 Jan 14;30(2):497-509.e4.

[3]. Etoposide- and BMY-40481-induced sensory neuropathy in mice.Toxicol Pathol. 1994 Sep-Oct;22(5):528-35.

[4]. SUMMARY OF PRODUCT CHARACTERISTICS.

Etoposide phosphate is a furanodioxane compound. Etoposide phosphate is the phosphate salt of a semi-synthetic derivative of podophyllotoxin. Etoposide binds to topoisomerase II, inducing DNA double-strand breaks and inhibiting DNA repair, thereby reducing DNA synthesis and promoting tumor cell proliferation. Cells in the S and G2 phases of the cell cycle are most sensitive to this drug. (NCI04) See also: Etoposide (active moiety).

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Etoposide Phosphate (BMY-40481) is a phosphate ester prodrug of etoposide that is used as an anti-cancer chemotherapy agent. It is a selective inhibitor of topoisomerase II. The prodrug form provides improved water solubility compared to etoposide, facilitating administration and formulation. Etoposide Phosphate has been used in the treatment of various cancers and is available for research purposes as well as for clinical use. Its mechanism of DNA damage and cell cycle arrest makes it a valuable tool for studying cancer biology and developing anti-cancer therapies.

C29H33O16P

668.53652

666.136

C, 52.10; H, 4.98; O, 38.29; P, 4.63

117091-64-2

Etoposide;33419-42-0;Etoposide phosphate disodium;122405-33-8

6918092

White to off-white solid powder

1.55 g/cm3

907.7±75.0 °C at 760 mmHg

502.7±37.1 °C

0.0±0.3 mmHg at 25°C

-1.28

4

16

7

46

1140

10

C[C@@H]1OC[C@@H]2[C@@H](O1)[C@@H]([C@H]([C@@H](O2)O[C@H]3[C@H]4COC(=O)[C@@H]4[C@@H](C5=CC6=C(C=C35)OCO6)C7=CC(=C(C(=C7)OC)OP(=O)(O)O)OC)O)O

LIQODXNTTZAGID-OCBXBXKTSA-N

InChI=1S/C29H33O16P/c1-11-38-9-20-27(42-11)23(30)24(31)29(43-20)44-25-14-7-17-16(40-10-41-17)6-13(14)21(22-15(25)8-39-28(22)32)12-4-18(36-2)26(19(5-12)37-3)45-46(33,34)35/h4-7,11,15,20-25,27,29-31H,8-10H2,1-3H3,(H2,33,34,35)/t11-,15+,20-,21-,22+,23-,24-,25-,27-,29+/m1/s1

[4-[(5S,5aR,8aR,9R)-5-[[(2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-8-oxo-5a,6,8a,9-tetrahydro-5H-[2]benzofuro[5,6-f][1,3]benzodioxol-9-yl]-2,6-dimethoxyphenyl] dihydrogen phosphate

Etopofos; Etophos; Etopophos; Vepeside; Etopophos Preservative Free; BMY 40481;

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.)