Topoisomerase II

In an in vivo assay, tumor growth of ISOS-1 was significantly inhibited by more than 2.5 mg/kg of ETO dose-dependently, and by more than 30 mg/kg of TNP-470, and 100 mg/kg of PSL individually. Combination treatments of ETO+TNP-470 and TNP-470+PSL showed synergistic enhancement of inhibition (% control inhibition: ETO vs. TNP-470 vs. ETO+TNP-470: 55 versus 55 vs. 16%) (% control inhibition: TNP-470 vs. PSL vs. TNP-470+PSL: 41 vs. 86 vs. 21%). ETO+PSL combination treatment, however, failed to show significant enhancement of anti-tumor effects. In conclusion, our results indicated that TNP-470 may be a very effective drug for angiosarcoma treatment, especially in combination with ETO or PSL. We eagerly anticipate the use of TNP-470 in clinical treatment of angiosarcoma.[2]

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These results support the hypothesis that in addition to its antineoplastic cytotoxic effect, VP-16 induces changes in 3LL cells which are recognized by the host immune system resulting in immune rejection of 3LL. often immunosuppressive and therapeutic advantage is generally based on the tumor cytotoxicity of individual drugs or combinations of drugs [13]. Our earlier work showed a link between the use of cytotoxic chemotherapy with etoposide (VP-16) and the induction of an immune response against syngeneic murine leukemia in the intact host [16]. VP-16 is an immunosuppressive topoisomerase II-inhibiting drug which induces tumor cell apoptosis and is frequently used clinically to treat a variety of tumors [1, 3, 9, 10]. We have noted that the addition of cyclosporin A to VP-16 produces CD8 T lymphocyte-mediated tumor-specific immunity in mice bearing L1210 leukemia [17]. We have extended these experiments to a spontaneously arising non-carcinogen-induced neoplasm, Lewis lung cancer (3LL), and now report that surviving mice successfully treated with VP-16, in the absence of cyclosporin A, reject challenge with 3LL. In addition, results are presented to show that VP-16 modifies 3LL cells rendering them immunogenic. These findings are submitted to support the hypothesis that VP-16-induced cytotoxic changes include cellular membrane alterations in 3LL cells which are recognized by the immune system and cause rejection of this syngeneic lung tumor.[4]

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Etoposide administered as a single agent has been shown to be ineffective in the growth of many xenografts, including human neuroblastoma xenograft[7], human gastrointestinal cancer xenograft [8], and heterotransplanted hepatoblastoma NMHB1, and NMHB 2[6]. However, the dose of 10 mg/kg i.p. Etoposide inhibits 36% of controls' murine angiosarcoma cell ISOS-1 tumors. Lewis lung cancer is treated with etoposide to induce tumor immunity. Lewis lung cancer cell (3LL) injections in C57B1/6 mice result in a 60% survival rate after a single 50 mg/kg intraperitoneal injection. This survival rate lasts for 60 days. While none of the control mice survive for more than 30 days, about 40% of these surviving mice reject a subsequent challenge with 3LL. Seventy-five percent of recipient mice are killed by 3LL cells that have withstood a 90% lethal concentration of etoposide in vitro; however, sixty percent of surviving mice reject challenge with 3LL. When naive mice are injected with 3LL, spleenocytes taken from tumor-rejecting mice provide protection.[9]

Nuclei are isolated and nuclear extracts are prepared. The percentage of decatenation obtained is used to calculate the activity of topoisomerase II. The substrate is tritiated kinoplast DNA (KDNA 0.22 μg). After 30 minutes of incubation at 37 °C, etoposide and topoisomerase II are stopped with 100 μg/mL of proteinase K and 1% sodium dodecyl sulfate (SDS). We obtain the percentages of topoisomerase II decatenation and inhibition by etoposide.

Cells treated with etoposide are removed from the dish and diluted into culture dishes in an amount sufficient to produce 20–200 colonies. The phosphate-buffered saline (PBS) solution contains 0.03% trypsin and 0.27 mM ethylenediaminetetraacetic acid (EDTA). Methanol-acetic acid is used to fix the cultures after 12 days, crystal violet is used for staining, and colonies with more than 50 cells are scored. Unless otherwise specified, standard errors are usually less than 15% of the mean value.

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To develop effective therapies for angiosarcoma, we investigated the anti-tumor effects of etoposide (ETO), TNP-470 and prednisolone (PSL) using an established murine angiosarcoma cell line (ISOS-1). We examined the direct anti-tumor and anti-angiogenic effects of these drugs on ISOS-1 cells and normal murine microvascular endothelial cells (mECs) in vitro. Cell growth of ISOS-1 was inhibited significantly by ETO, moderately by TNP-470, and not at all by PSL (IC(50): 0.25 microg/ml, 10 microg/ml, >8000 microg/ml, respectively). One the other hand, cell growth of mECs was inhibited significantly by TNP-470, slightly by PSL, and negligibly by ETO (IC(50): 0.85 ng/ml, 0.7 microg/ml, 10 microg/ml, respectively). [2]

Of C57B1/6 mice injected with 10(6) Lewis lung cancer (3LL) cells followed by treatment with a single 50 mg/kg dose of etoposide (VP-16), 60% survived over 60 days, in contrast to untreated control mice which died within 30 days. Approximately 40% of surviving mice rejected a subsequent challenge with 3LL. Their splenocytes protected naive mice injected with 3LL. To test if VP-16 treatment produced alterations in 3LL cells, which induce host immunity, leading to tumor rejection, C57B1/6 mice were injected with 3LL cells that had survived an 80-90% lethal concentration of VP-16 in vitro. These cells killed 75% of recipient mice but 60% of the surviving mice rejected challenge with 3LL. Splenocytes harvested from tumor-rejecting mice protected naive mice injected with 3LL.[4]

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Murine angiosarcoma xenografts ISOS-1; 10 mg/kg; i.p. every day for 5 days from day 7

Murine angiosarcoma xenografts ISOS-1

Absorption, Distribution and Excretion
Absorbed well, time to peak plasma concentration is 1-1.5 hrs. Mean bioavailability is 50% (range of 25% - 75%). Cmax and AUC values for orally administered etoposide capsules display intra- and inter-subject variability. There is no evidence of first-pass effect for etoposide.
Etoposide is cleared by both renal and nonrenal processes, i.e., metabolism and biliary excretion. Glucuronide and/or sulfate conjugates of etoposide are also excreted in human urine. Biliary excretion of unchanged drug and/or metabolites is an important route of etoposide elimination as fecal recovery of radioactivity is 44% of the intravenous dose. 56% of the dose was in the urine, 45% of which was excreted as etoposide.
The disposition of etoposide is a biphasic process with a distribution half-life of 1.5 hours. It does not cross into cerebrospinal fluid well. Volume of distribution, steady state = 18 - 29 L.
Total body clearance = 33 - 48 mL/min [IV administration, adults]
Mean renal clearance = 7 - 10 mL/min/m^2
Excretion of etoposide in breast milk was demonstrated in a woman with acute promyelocytic leukemia receiving daily doses of 80 mg/sq m (route not stated). Peak concentrations of 0.6 to 0.8 ug/mL were measured immediately after dosing but had decreased to undetectable levels by 24 hr.
Thirty minutes after intravenous administration of etoposide to rats, the highest concentrations were found in the liver, kidneys and small intestine. By 24 hr after the dose, the tissue concentrations were negligible.
After intravenous infusion (5 min) of etoposide phosphate to beagle dogs at doses of 57-461 mg/sq m, a dose-proportional increase was seen in the maximal plasma concentration and AUC for etoposide. The total plasma clearance rate (342-435 mL/min per sq m) and the distribution volume (22-27 L/sq m) were not dose-dependent. The peak plasma concentration occurred at the end of the infusion of etoposide phosphate, indicating rapid conversion of the pro-drug to etoposide.
Less than 4% of a dose was recovered in the bile after 48 hr in patients with biliary drainage tubes. The fecal recovery of radiolabel after intravenous administration of 3(H)etoposide (130-290 mg/sq m) was variable, representing 0-16% of dose, but the collections were known to be incomplete because of fecal retention and other difficulties associated with the poor general condition of many of the patients). In a study reported as an abstract in four patients with small-cell lung cancer given 14(C)-glucopyranoside etoposide, 56% of the radiolabel was recovered in urine and 44% in feces over five days, for a total recovery of 100 +/- 6%.
For more Absorption, Distribution and Excretion (Complete) data for ETOPOSIDE (18 total), please visit the HSDB record page.

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Metabolism / Metabolites
Primarily hepatic (through O-demethylation via the CYP450 3A4 isoenzyme pathway) with 40% excreted unchanged in the urine. Etoposide also undergoes glutathione and glucuronide conjugation which are catalyzed by GSTT1/GSTP1 and UGT1A1, respectively. Prostaglandin synthases are also responsible for the conversion of etoposide to O-demethylated metabolites (quinone).
The proposed hydroxy acid metabolite of etoposide, formed by opening of the lactone ring, has been detected in human urine, but only at low concentrations, accounting for 0.2-2.2% of the administered dose.
The major urinary metabolite of etoposide in humans is reported to be the glucuronide conjugate. Although urinary glucuronide and/or sulfate conjugates were reported to account for 5-22% of an intravenous dose of etoposide, other studies suggest that the glucuronide predominates. Etoposide glucuronide in the urine of treated patients accounted for 8-17% of a dose of 0.5-3.5 g/sq m etoposide and 29% of a dose of 100-800 mg/sq m etoposide, with no other metabolites other than etoposide glucuronide detected in the latter study. In patients with renal or liver impairment given somewhat lower doses of 70-150 mg/sq m, 3-17% of the dose was excreted in the urine within 72 hr as etoposide glucuronide.
Etoposide appears to be metabolized principally at the D ring to produce the resulting hydroxy acid (probably the trans-hydroxy acid); this metabolite appears to be pharmacologically inactive. The picrolactone isomer of etoposide has been detected in two concentrations in the plasma and urine of some patients but not in others. The aglycone of etoposide and/or its conjugates have not been detected to date in patients receiving the drug. In vitro, the picrolactone isomer and aglycone of etoposide have minimal cytotoxic activity.
Generally, few or no etoposide metabolites have been detected in plasma. Etoposide is administered as the trans-lactone, but cis-etoposide can also be detected in human urine. This might be a storage phenomenon, since isomerization sometimes occurs during freezing of plasma samples under slightly basic conditions. The cis isomer accounts for < 1% of the dose. The catechol metabolite has also been reported in patients receiving 600 mg/sq m etoposide, with an AUC of around 2.5% that of etoposide. In patients given 90 mg/sq m etoposide, the catechol metabolite represented 1.4-7.1% of the urinary etoposide and < 2% of the administered dose.
In rat liver homogenates, liver microsomes and in rats in vivo, etoposide was extensively metabolized to only one major metabolite, which was not formally identified. In perfused isolated rat liver incubated with etoposide, the total recovery in bile was 60-85%, with roughly equal amounts of etoposide and two glucuronide metabolites, confirmed as glucuronide species by liquid chromatography and mass spectrometry. After intravenous injection of 3(H)etoposide to rabbits, the total urinary excretion of radiolabel was 30% after five days, with very little thereafter. A single glucuronide metabolite was identified in rabbit urine, which was present in larger amounts than etoposide. No hydroxy acid was identified in either species.
Primarily hepatic (through O-demethylation via the CYP450 3A4 isoenzyme pathway) with 40% excreted unchanged in the urine. Etoposide also undergoes glutathione and glucuronide conjugation which are catalyzed by GSTT1/GSTP1 and UGT1A1, respectively. Prostaglandin synthases are also responsible for the conversion of etoposide to O-demethylated metabolites (quinone).
Route of Elimination: Etoposide is cleared by both renal and nonrenal processes, i.e., metabolism and biliary excretion. Glucuronide and/or sulfate conjugates of etoposide are also excreted in human urine. Biliary excretion of unchanged drug and/or metabolites is an important route of etoposide elimination as fecal recovery of radioactivity is 44% of the intravenous dose. 56% of the dose was in the urine, 45% of which was excreted as etoposide.
Half Life: 4-11 hours

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Biological Half-Life
4-11 hours
... In adults with normal renal and hepatic function, the half-life of etoposide averages 0.6-2 hours ... in the initial phase and 5.3-10.8 hours ... in the terminal phase. In one adult with impaired hepatic function, the terminal elimination half-life was reportedly 78 hours. In children with normal renal and hepatic function, the half-life of etoposide averages 0.6-1.4 hours in the initial phase and 3-5.8 hours in the terminal phase.
... Elimination half-life of 3 to 7 hr in children and 4 to 8 hr in adults.

Toxicity Summary
Etoposide inhibits DNA topoisomerase II, thereby inhibiting DNA re-ligation. This causes critical errors in DNA synthesis at the premitotic stage of cell division and can lead to apoptosis of the cancer cell. Etoposide is cell cycle dependent and phase specific, affecting mainly the S and G2 phases of cell division. Inhibition of the topoisomerase II alpha isoform results in the anti-tumour activity of etoposide. The drug is also capable of inhibiting the beta isoform but inhibition of this target is not associated with the anti-tumour activity. It is instead associated with the carcinogenic effect.

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Most sources consider breastfeeding to be contraindicated during maternal antineoplastic drug therapy. It might be possible to breastfeed safely during intermittent therapy with etoposide after an appropriate period of breastfeeding abstinence. A period of at least 24 hours is required after a dose of 80 mg/sq. m. or less. Others have suggested an abstinence period of 72 hours after etoposide use. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ Effects in Breastfed Infants
One mother received with 5 daily doses of etoposide 80 mg/sq. m. and cytarabine 170 mg/sq. m. intravenously as well as 3 daily doses of 6 mg/sq. m. of mitoxantrone intravenously. She resumed breastfeeding her infant 3 weeks after the third dose of mitoxantrone at a time when mitoxantrone was still detectable in milk. The infant had no apparent abnormalities at 16 months of age.
◉ Effects on Lactation and Breastmilk
A telephone follow-up study was conducted on 74 women who received cancer chemotherapy at one center during the second or third trimester of pregnancy to determine if they were successful at breastfeeding postpartum. Only 34% of the women were able to exclusively breastfeed their infants, and 66% of the women reported experiencing breastfeeding difficulties. This was in comparison to a 91% breastfeeding success rate in 22 other mothers diagnosed during pregnancy, but not treated with chemotherapy. Other statistically significant correlations included: 1. mothers with breastfeeding difficulties had an average of 5.5 cycles of chemotherapy compared with 3.8 cycles among mothers who had no difficulties; and 2. mothers with breastfeeding difficulties received their first cycle of chemotherapy on average 3.4 weeks earlier in pregnancy. Of the 9 women who received a taxane-containing regimen, 7 had breastfeeding difficulties.

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Protein Binding
97% protein bound.

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Interactions
Additive bone marrow depression may occur; dosage reduction may be required when two or more bone marrow depressants, including radiation, are used concurrently or consecutively.
Multidrug resistance is one of the mechanisms of resistance to multiple cytotoxic drugs and is mediated by the expression of a membrane pump called the P-glycoprotein. Nifedipine is one of the calcium channel blocking agents which reverses multidrug resistance in vitro. Fifteen patients with various malignancies received nifedipine at three dose levels: 40 mg, 60 mg and 80 mg orally twice daily for 6 days. Etoposide was administered intravenously on day 2 in a dose of 150-250 mg/sq m and orally 150-300 mg twice daily on days 3 and 4. Cardiovascular effects of nifedipine were dose limiting and the maximum tolerated dose was 60 mg twice a day. Mean area under the plasma concentration curve and plasma half-life of nifedipine and its major metabolite MI at the highest dose level were 7.87 uM.hr, 7.97 hr and 4.97 uM.hr, 14.0 hr respectively. Nifedipine did not interfere with the pharmacokinetics of etoposide.
Dipyridamole has chemical characteristics similar to other known modulators of etoposide, doxorubicin, and vinblastine sensitivity. When compared to verapamil, dipyridamole was as efficacious but twice as potent in its synergistic enhancement of etoposide sensitivity. These results demonstrate that dipyridamole can markedly increase the cytotoxicity of etoposide, doxorubicin, and vinblastine and suggest possible clinical applications.
Enhanced antineoplastic action of etoposide in the presence of cyclosporin A, was investigated in several in vitro and in vivo tumor systems. Macromolecular DNA damage induced by etoposide at drug levels comparable to plasma area under the curve values achieved in patients was increased not only in leukemic peripheral blood cells from patients but also in mononuclear peripheral blood cells from a healthy donor. Intracellular retention of radioactivity from (3)H etoposide was increased by a factor of 1.5 at the most in the presence of cyclosporin A. The cytotoxicity of etoposide and adriamycin to L 1210 leukemic cells was clearly enhanced, whereas cyclosporin A had no effect on the action of cisplatin or ionizing irradiation. At cyclosporin A blood levels not exceeding 1.44 ug/ml, increased tumor inhibition of etoposide was observed in a human embryonal cancer xenograft, but there was also higher lethality in normal mice. With respect to chemosensitization the effects of cyclosporin A resemble those of calcium channel blockers or anticalmodulin agents. In contrast to calcium channel blockers, however, adequate plasma levels of cyclosporin A can well be achieved in patients.
For more Interactions (Complete) data for ETOPOSIDE (7 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Mouse IV 118 mg/kg bw
LD50 Rat IV 68 mg/kg bw
LD50 Rabbit IV > 80 mg/kg bw
LD50 Mouse ip 108 mg/kg bw

[1]. J Natl Cancer Inst . 1988 Dec 7;80(19):1526-33.

[2]. J Dermatol Sci . 2000 Nov;24(2):126-33.

[3]. Cancer Res . 1983 Apr;43(4):1592-7.

[4]. Cancer Chemother Pharmacol . 2001 Oct;48(4):327-32.

[5]. Cancer Chemother Pharmacol . 1998;41(2):93-7.

[6]. Cancer . 1998 Dec 1;83(11):2400-7.

[7]. Gan To Kagaku Ryoho . 1991 Jun;18(7):1155-61.

[8]. J Surg Oncol . 1993 Dec;54(4):211-5.

[9]. Cancer Chemother Pharmacol . 2001 Oct;48(4):327-32.

Therapeutic Uses
Antineoplastic Agents, Phytogenic; Nucleic Acid Synthesis Inhibitors
Etoposide injection is indicated, in combination with other antineoplastics, for first-line treatment of testicular tumors (Evidence rating: 1A). /Included in US product labeling/
Etoposide is indicated in combination with other agents as first-line treatment of small cell lung carcinoma. /Included in US product labeling/
Etoposide also is indicated, alone and in combination with other agents, for treatment of Hodgkin's and non-Hodgkin"s lymphomas and acute nonlymphocytic (myelocytic) leukemia. /NOT included in US product labeling/
For more Therapeutic Uses (Complete) data for ETOPOSIDE (13 total), please visit the HSDB record page.

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Drug Warnings
The major and dose-limiting adverse effect of etoposide is hematologic toxicity. Myelosuppression, which is dose related, is manifested mainly by leukopenia (principally granulocytopenia). Myelosuppression resulting in death has been reported in patients receiving etoposide. Thrombocytopenia occurs less frequently, and anemia may also occur; pancytopenia has occurred in some patients. Myelosuppression apparently is not cumulative but may be more severe in patients previously treated with other antineoplastic agents or radiation therapy. Leukopenia has reportedly occurred in 60-91% of patients receiving etoposide and was severe (leukocyte count less than 1000/cu mm) in 3-17% of patients. Neutropenia (less than 2000 cu mm) occurred in 88% of patients treated with etoposide phosphate; severe neutropenia has reportedly occurred in 22-41% of patients receiving the drug and was severe (platelet count less than 50,000/cu mm) in 1-20% of patients. Anemia has occurred in up to 33% of patients receiving etoposide. Anemia (hemoglobin less than 11 g/dL) occurred in 72% of patients treated with etoposide phosphate; severe anemia (hemoglobin less than 8 g/dL) occurred in 19% of patients treated. Granulocyte and platelet nadirs usually occur within 7-14 and 9-16 days, respectively, after administration of etoposide, and within 12-19 and 10-15 days, respectively, after administration of etoposide phosphate; leukocyte nadir has been reported to occur within 15-22 days after administration of etoposide, phosphate. Bone marrow recovery is usually complete within 20 days after administration, but may occasionally require longer periods. Fever and infection have been reported in patients with drug-induced neutropenia.
Pregnancy risk category: D /POSITIVE EVIDENCE OF RISK. Studies in humans, or investigational or post-marketing data, have demonstrated fetal risk. Nevertheless, potential benefits from the use of the drug may outweigh the potential risk. For example, the drug may be acceptable if needed in a life-threatening situation or serious disease for which safer drugs cannot be used or are ineffective./
Reversible alopecia, sometimes progressing to complete baldness, has occurred in 8-66% of patients receiving etoposide. The degree of alopecia may be dose related. Stevens-Johnson syndrome has been reported infrequently in patients receiving etoposide. Rash, pigmentation, urticaria, and severe pruritus have occurred infrequently, and cutaneous radiation-recall reactions associated with etoposide have been reported. ...
Anaphylactoid reactions consisting principally of chills, rigors, diaphoresis, pruritus, loss of consciousness, nausea, vomiting, fever, bronchospasm, dyspnea, tachycardia, hypertension, and/or hypotension have occurred during or immediately after administration of etoposide or etoposide phosphate in 0.7-3% of patients receiving the drug. Other manifestations have included flushing, rash, substernal chest pain, lacrimation, sneezing, coryza, throat pain, back pain, generalized body pain, abdominal cramps, and auditory impairment.
For more Drug Warnings (Complete) data for ETOPOSIDE (24 total), please visit the HSDB record page.

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Pharmacodynamics
Etoposide is an antineoplastic agent and an epipodophyllotoxin (a semisynthetic derivative of the podophyllotoxins). It inhibits DNA topoisomerase II, thereby ultimately inhibiting DNA synthesis. Etoposide is cell cycle dependent and phase specific, affecting mainly the S and G2 phases. Two different dose-dependent responses are seen. At high concentrations (10 µg/mL or more), lysis of cells entering mitosis is observed. At low concentrations (0.3 to 10 µg/mL), cells are inhibited from entering prophase. It does not interfere with microtubular assembly. The predominant macromolecular effect of etoposide appears to be the induction of DNA strand breaks by an interaction with DNA-topoisomerase II or the formation of free radicals.

C29H32O13

588.56

588.184

C, 59.18; H, 5.48; O, 35.34

33419-42-0

117091-64-2

36462

White to off-white solid powder

1.6±0.1 g/cm3

798.1±60.0 °C at 760 mmHg

236-251ºC

263.6±26.4 °C

0.0±3.0 mmHg at 25°C

1.662

0.3

3

13

5

42

969

10

O=C1OC[C@]2([H])[C@H](O[C@H]3[C@@H]([C@H]([C@@H]4O[C@H](C)OC[C@H]4O3)O)O)C5=C(C=C6OCOC6=C5)[C@@H](C7=CC(OC)=C(O)C(OC)=C7)[C@]21[H]

VJJPUSNTGOMMGY-MRVIYFEKSA-N

InChI=1S/C29H32O13/c1-11-36-9-20-27(40-11)24(31)25(32)29(41-20)42-26-14-7-17-16(38-10-39-17)6-13(14)21(22-15(26)8-37-28(22)33)12-4-18(34-2)23(30)19(5-12)35-3/h4-7,11,15,20-22,24-27,29-32H,8-10H2,1-3H3/t11-,15+,20-,21-,22+,24-,25-,26-,27-,29+/m1/s1

(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]-9-(4-hydroxy-3,5-dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5H-[2]benzofuro[6,5-f][1,3]benzodioxol-8-one

Demethyl Epipodophyllotoxin; Ethylidine Glucoside; epipodophyllotoxin; trans-Etoposide; (-)-Etoposide; Lastet; Zuyeyidal; US brand names: Toposar; VePesid. Foreign brand name: Lastet. Abbreviation: EPEG Code names: VP16; VP16213;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (4.25 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 (4.25 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 3: ≥ 2.5 mg/mL (4.25 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: ≥ 2.5 mg/mL (4.25 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 0.5 mg/mL (0.85 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL

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