Topoisomerase; The primary target of Epirubicin is DNA, where it intercalates into the DNA double helix and inhibits topoisomerase II, leading to DNA damage and strand breaks. It also targets Foxp3, a transcription factor involved in regulatory T cell (Treg) function, with inhibitory effects on Foxp3 activity [1] [2]

Epirubicin (4'-Epidoxorubicin), like doxorubicin, exerts its antitumor effects by forming complexes with DNA that damage DNA and obstruct the synthesis of proteins, RNA, and DNA. The integrity and functionality of cellular membranes may also be impacted by epirubicin. Epirubicin's maximum cell killing happens in the S phase of the cell cycle. Effects are also observed in the early G2, G1, and M phases at higher concentrations[1].
Epirubicin display antineoplastic activity against most cancer cells. Hepatoma G2 cells exhibit cytotoxicity towards epirubicin, with an IC50 of 1.6 μg/mL after 24 hours. 1.6 micrograms per milliliter Hep G2 cells undergo apoptosis upon exposure to epirubicin, which also increases catalase activity by 50%, serine-dependent glutathione peroxidase activity by 110%, and the activity of Cu, Zn-superoxide dismutase by 172% and Mn-superoxide dismutase by 135%. Epirbicin decreases the expression of GST-π and increases the expression of NADPH-CYP 450 reductase in cells[3].

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Epirubicin exhibits potent antiproliferative activity against various cancer cell lines. In human breast carcinoma cells (R-27), it inhibited cell growth, with greater activity observed at higher concentrations. Combination with paclitaxel showed additive or synergistic antitumor effects, as measured by cell viability assays [5]
In hepatoma G2 (HepG2) cells, Epirubicin induced cytotoxicity, characterized by reduced cell viability, increased lactate dehydrogenase (LDH) release, and DNA fragmentation, indicating apoptotic cell death. These effects were concentration- and time-dependent, with significant toxicity observed at concentrations ≥ 1 μM after 24 hours of exposure [3]
In Treg cells, Epirubicin inhibited Foxp3 activity, as demonstrated using a luciferase reporter assay. This inhibition reduced Treg-mediated suppression of effector T cell proliferation, enhancing immune responses against tumors [2]

Epirubicin (4'-Epidoxorubicin) exhibits clinical efficacy against a diverse array of tumor types, such as gastrointestinal cancer, head and neck cancer, ovarian cancer, prostatic carcinoma, transitional bladder carcinoma, breast cancer, malignant lymphomas, soft tissue sarcomas, lung cancer, pleural mesothelioma, and so forth[4].
For the human breast tumor xenograft R-27, epirubicin at a dose of 3.5 mg/kg reduces tumor mass by 74.4 %[5].

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In animal models of breast cancer, Epirubicin administered systemically (intravenous or intraperitoneal) reduced tumor growth and size. The antitumor effect was dose-dependent, with higher doses leading to greater tumor regression. Combination with other chemotherapeutic agents (e.g., cyclophosphamide, fluorouracil) improved efficacy compared to single-agent therapy [1] [4]
In murine models, Epirubicin demonstrated immunomodulatory effects by reducing Treg-mediated immunosuppression, thereby enhancing antitumor immunity. This was associated with increased effector T cell infiltration into tumors [2]

Reporter assays[2]
For the NF-κB-dependent reporter assay, HEK293/NF-κB-RE/Foxp3 cells (1.5×104) or HEK293/NF-κB-RE cells (1.5×104) were seeded into white 96-well plates (Corning) and incubated overnight at 37°C in 5% CO2. These cells were treated with test drugs for 1 h. The cells were then stimulated with 0.3 ng/mL recombinant human TNF-α for 2.5 h. The medium was aspirated off and Steady-Glo (Promega) was added to the cells. The plate was then placed on a shaker for 10 min. Luminescence was detected using an ARVO Light plate reader.[2]

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Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, researchers screened a chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. [2]

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To evaluate Foxp3 inhibitory activity, a luciferase reporter plasmid containing the Foxp3 promoter was transfected into cells. Cells were treated with Epirubicin at various concentrations, and luciferase activity was measured to assess Foxp3 transcriptional activity. The assay showed a dose-dependent decrease in luciferase activity, indicating reduced Foxp3 function [2]
To assess topoisomerase II inhibition, recombinant topoisomerase II was incubated with DNA and Epirubicin. The ability of the enzyme to relax supercoiled DNA was measured using gel electrophoresis, with reduced relaxation indicating enzyme inhibition [1]

Epirubicin HCl is a new anthracycline analog and derivative of doxorubicin. Doxorubicin is a potent anticancer agent, the use of which is limited by its cumulative dose-dependent cardiotoxicity. Epirubicin HCl has more favorable therapeutic index than doxorubicin and possesses less hematologic and cardiac toxicity at comparable doses. Hepatoma G2 cells are a valuable model to study hepatocellular carcinoma and the liver, where drugs are metabolized. The goal of our study was to evaluate the cytotoxic effect of epirubicin HCl on viability of Hep G2 cells measured using the MTT cytotoxicity test. Epirubicin HCl produced a concentration- and time-dependent cytotoxicity to Hep G2 cells. The mechanism of cytotoxicity of epirubicin HCl (IC(50) value of 1.6 mug/ml within 24 h) appeared to involve a production of free radical species since activities of free radical scavenging enzymes (SOD, catalase, Se-dependent GPx) were increased. Addition of SOD prevented cytotoxicity of epirubicin HCl, and also counteracted the apoptosis. DNA fragmentation was determined to evaluate apoptosis. Western blot analysis indicated a decrease in GST-pi expression and increased activity of NADPH-dependent cytochrome P450 reductase which is a major enzyme in the conversion of epirubicin HCl to a free radical. It is proposed that production of reactive oxygen species increased by the treatment with epirubicin HCl can cause lipid peroxidation, which subsequently promotes apoptosis and reduces cell viability. Superoxide dismutase, catalase and glutathione peroxidase must be considered as a part of the intracellular antioxidant defense mechanism of Hep G2 cells against single electron reducing quinone-containing anticancer antibiotics.[3]

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In a 96-well plate, 500 monolayer Hep G2 cells are plated per well. Epirubicin is added to the medium and applied to the cells the following day. 15% of the MTT dye solution is added after the incubation times have passed. Each well receives an equal volume of solubilization/stop solution (dimethylsulfoxide) for an additional hour of incubation at 37°C following the first hour of incubation. At 570 nm, the absorbance of the reaction solution is measured and recorded.

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In HepG2 cells, cells were treated with Epirubicin (0.1-10 μM) for 24-72 hours. Cell viability was measured using a colorimetric assay, LDH release was quantified to assess membrane damage, and DNA fragmentation was analyzed via agarose gel electrophoresis to confirm apoptosis [3]
In breast carcinoma (R-27) cells, cells were exposed to Epirubicin (0.01-10 μM) alone or in combination with paclitaxel. Cell proliferation was evaluated by counting cell numbers and measuring DNA synthesis via thymidine incorporation assays [5]
In Treg cells, Epirubicin-treated Tregs were co-cultured with effector T cells. Effector T cell proliferation was measured using flow cytometry with CFSE labeling, showing reduced suppression by treated Tregs compared to untreated controls [2]

Mouse in vivo assays At day 0, female BALB/c mice (8 mice per group) were inoculated subcutaneously with CMS5a cells into the right inguinal region. Epirubicin (0.1, 0.3 or 1 mg/kg) or saline was given at days 3, 5 and 7 intravenously. On day 8, mice were euthanized and tumors were removed. Tumor-infiltrating lymphocytes (TIL) were dissociated from tumors using a gentleMACS dissociator according to the manufacturer’s instructions. The collected cells were seeded in 24-well plates and stimulated with phorbol 12-myristate 13-acetate (PMA) and Ionomycin at 37°C for 1 h, and then cultured for 6 h with GolgiPlug™. The cells were harvested and stained with PreCP-Cy™5.5 rat anti-mouse CD4 antibody and V500 rat anti-mouse CD8a antibody at 4°C for 15 min. The stained cells were fixed with Fixation/Permeabilization Concentrate and Diluent (1:3) at 4°C overnight. After washing, Permeabilization Buffer was added and the fixed cells were stained with PE-conjugated anti-mouse/rat Foxp3, anti-mouse IFN-γ-APC, and PE-conjugated anti-mouse IL-2 antibodies. The stained cells were analyzed using a FACS Canto II flow cytometer [2].

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In breast cancer xenograft models, mice bearing R-27 tumors were administered Epirubicin intravenously at doses ranging from 5 to 20 mg/kg, either alone or in combination with paclitaxel (10 mg/kg). Treatments were given once weekly for 3-4 weeks. Tumor volume was measured twice weekly, and mice were monitored for body weight changes and survival [5]
In immunocompetent tumor models, mice were injected intraperitoneally with Epirubicin (1-5 mg/kg) every 3 days. Treg numbers and function in tumor tissues and spleen were analyzed using flow cytometry, and effector T cell proliferation was assessed ex vivo [2]
For toxicity studies, animals received Epirubicin intravenously at cumulative doses up to 150 mg/kg over several weeks. Organs (e.g., heart, liver, kidneys) were collected for histopathological analysis to evaluate tissue damage [1] [4]

ADME/Pharmacokinetics: Epirubicin is mainly administered intravenously, with very low oral bioavailability. After intravenous injection, the drug is widely distributed in tissues, with higher concentrations in the liver, spleen, and tumor tissues. The plasma protein binding rate is approximately 77-89% [1][4]. The drug is metabolized in the liver through reduction and conjugation to produce active and inactive metabolites. The elimination half-life is 30 to 40 hours, with approximately 40% of the dose excreted in the urine within 7 days and 40% excreted in the feces [1][4]. Absorption, Distribution, and Excretion: 100% Epirubicin and its major metabolites are mainly excreted via bile, with a small amount excreted via urine.
21 ± 2 L/kg [60 mg/m2 dose]
27 ± 11 L/kg [75 mg/m2 dose]
23 ± 7 L/kg [120 mg/m2 dose]
21 ± 7 L/kg [150 mg/m2 dose]
65 ± 8 L/hour [patient with solid tumor receiving intravenous epirubicin 60 mg/m2] [mg/m2]
83 +/- 14 L/hour [patient with solid tumor receiving intravenous epirubicin 75 mg/m2]
65 +/- 13 L/hour [patient with solid tumor receiving intravenous epirubicin 120 mg/m2]
69 +/- 13 L/hour [patient with solid tumor receiving intravenous epirubicin 150 mg/m2]
…… This study investigated the plasma and tissue distribution of the anthracycline antibiotics doxorubicin (1), 4'-epirarubicin (epirarubicin; II), and daunorubicin (daunorubicin; III) in patients. Plasma concentrations of drugs I and II and their 13-hydroxy metabolites, as well as the plasma concentration of drug III, were determined by liquid chromatography. The results showed that the plasma kinetics of the three drugs were similar, and the tissue uptake of drugs I and II was almost identical. The highest drug concentrations were observed in tumor tissue, while the lowest concentrations were found in adipose tissue. The preparation and in vitro and in vivo evaluation of ovalbumin microspheres containing epirubicin hydrochloride (I) have been described in detail in the literature. Pharmacokinetic studies of I were conducted in rats following a single intravenous injection, and efficacy studies were performed in mice with Ehrlich ascites carcinoma and rats with Walker's carcinoma. In vitro drug release kinetics partially conformed to a first-order kinetic model and partially conformed to a matrix diffusion model. Pharmacokinetics in rat serum, heart, and lungs were described using a two-compartment open model equation. Calculated pharmacokinetic parameters suggest the potential for improved selective uptake of I in the lungs. In animal efficacy studies, I prolonged survival in all treatment groups. The data suggest that iodine microspheres may have organ-targeting properties.
/Milk/ In the perinatal and postpartum period, rats treated with epirubicin at a daily dose of 0.50 mg/kg secreted epirubicin into their milk. It is currently unclear whether this drug is secreted into human milk.
After intravenous injection, epirubicin is rapidly and extensively distributed in tissues. Epirubicin binds to approximately 77% of plasma proteins (primarily albumin), regardless of drug concentration. Epirubicin also appears to accumulate in erythrocytes; whole blood concentrations are approximately twice the plasma concentrations.
For more complete data on the absorption, distribution, and excretion of epirubicin (11 items), please visit the HSDB records page.
Metabolism/Metabolites
It is extensively and rapidly metabolized in the liver. Epirubicin is also metabolized by other organs and cells, including erythrocytes. Its main metabolic pathways are four: (1) C-13 keto group is reduced to generate the 13(S)-dihydro derivative epirubicinol; (2) both the original drug and epirubicinol are bound to glucuronic acid; (3) the amino sugar moiety is lost through hydrolysis to generate doxorubicin and doxorubicin alcohol aglycone; (4) the amino sugar moiety is lost through redox processes to generate 7-deoxydoxorubicin aglycone and 7-deoxydoxorubicin alcohol aglycone. Epirubicinol exhibits cytotoxic activity in vitro (approximately 10% of epirubicin), but it is unlikely to reach the concentration required to produce cytotoxic effects in vivo. Epirubicin is mainly metabolized rapidly and extensively in the liver, and is also metabolized by other organs and cells (including erythrocytes). Four main metabolic pathways have been identified: (1) reduction of the C-13 keto group to generate the 13(S)-dihydro derivative epirubicinol; (2) both the unmetabolized drug and epirubicinol are bound to glucuronic acid; (3) loss of the amino sugar moiety through hydrolysis to generate doxorubicin and doxorubicinol aglycone; (4) loss of the amino sugar moiety through redox processes to generate 7-deoxydoxorubicinol aglycone and 7-deoxydoxorubicinol aglycone. The in vitro cytotoxic activity of epirubicinol is about one-tenth that of epirubicin. Since the plasma concentration of epirubicinol is lower than that of the parent drug, it is unlikely to reach a concentration sufficient to produce cytotoxicity in vivo. Other metabolites have not been reported to have significant activity or toxicity.
Some studies have suggested that secondary alcohol metabolites may mediate chronic cardiotoxicity caused by doxorubicin (DOX) and other anthracycline anticancer drugs. This study found that NADPH-supplemented human cardiac cell sol can reduce the carbonyl group on the tetracyclic side chain of doxorubicin to generate the secondary alcohol metabolite doxorubicinol (DOXol). A decrease in alcohol metabolite production was observed when doxorubicin (EPI) was used to replace doxorubicin. Epirubicin is a low-cardiotoxic analogue characterized by an axial-to-equatorial epimerization of the C-4 hydroxyl group in the aminosaccharide linked to the tetracyclic (daunorubicin) ring. A similar decrease was observed when doxorubicin was replaced with MEN. MEN is a novel anthracycline antibiotic with preclinical evidence of reduced cardiotoxicity. MEN is characterized by the absence of a methoxy group at the C-4 position of the tetracyclic ring and the insertion of 2,6-dideoxy-L-fucose between the daunorubicin and the aglycone. Comparison with methoxy or 4-demethoxy aglycones and various monosaccharide or disaccharide 4-demethoxy anthracyclines showed that the absence of both the methoxy group and the presence of the disaccharide moiety in MEN limited the production of alcohol metabolites. Studies of enzymatically generated or purified anthracycline secondary alcohols have also shown that the presence of the disaccharide moiety, rather than the absence of the methoxy group, reduces the reactivity of MEN metabolites with the [4Fe-4S] cluster of cytoplasmic aconitase. This is reflected in the limited degree of reoxidation to the parent carbonyl anthracycline compound and the reduced delocalization of Fe(II) from the cluster. These studies collectively (i) elucidate the different effects of methoxy and glycosyl substituents on the formation of anthracycline secondary alcohols and their [4Fe-4S] reactivity; (ii) support the role of alcohol metabolites in anthracycline-induced cardiotoxicity, as they show that both EPI and MEN 10755, which have lower cardiotoxicity, exhibit reduced levels of such metabolite production; and (iii) suggest that the cardiotoxicity of MEN may be further reduced due to the decreased reactivity of its alcohol metabolite [4Fe-4S]. Many antitumor drugs have been found to be carcinogenic, mutagenic, and teratogenic. This study aimed to conduct cytogenetic and internal dose monitoring on hospital pharmacy staff who frequently participate in the preparation of cytosolic inhibitors to detect potential genotoxic effects of cytosolic inhibitors under occupational exposure and accidental contamination under routine work conditions. To assess in vivo exposure to cytosolic inhibitors, we measured platinum levels in whole blood and anthracycline levels in plasma. The level of cytogenetic damage in peripheral blood lymphocytes was determined using the micronucleus assay and sister chromatid exchange assay. Five monitoring groups were conducted over two years. There were no significant differences in the mean frequencies of sister chromatid exchange (SCE) and micronuclei (MN) between the occupational exposure group and the control group (9.9 ± 1.4 vs 10.1 ± 1.2 SCE/cell and 21.2 ± 7.2 vs 23.3 ± 7.5 MN/2000 binucleated cells, n = 16). In 12 cases of accidental workplace contamination, 7 showed significantly elevated SCE or MN levels, but these cases did not show elevated blood platinum or plasma anthracycline levels. Two unreported cases of contamination were identified by detecting epirubicin concentrations in plasma. Smoking significantly increased the incidence of sister chromatid exchange (SCE). No correlation was observed between individual SCE scores and micronucleus (MN) scores. The results of this study support the possibility of transient increases in SCE or MN following exposure to the relevant cell inhibitors in cases of accidental contamination. No significant differences in SCE and MN were observed between hospital pharmacy staff and unexposed controls, indicating high safety standards in the relevant workplaces. There is ample in vitro evidence that assessing the pharmacokinetics of doxorubicin or epirubicin based solely on plasma concentrations may not fully elucidate the differences between the two drugs. Both compounds bind to erythrocytes, and their different binding mechanisms to hemoglobin may affect their distribution in vivo. This study aimed to compare the pharmacokinetics and metabolism of doxorubicin and epirubicin, specifically measuring plasma concentrations, blood cell binding, and bile and urinary excretion of the parent drug and its metabolites after single and multiple doses. Simultaneously, the level of cardiac sarcoplasmic reticulum Ca2+ATPase was also measured as a biomarker of cardiotoxicity. Male Sprague-Dawley rats were used in a parallel design, receiving either multiple doses (4 mg kg⁻¹/week) or a single injection (20 mg kg⁻¹) of doxorubicin or epirubicin. Blood, urine, and bile samples were collected periodically after each dose in both the multiple-dose and single-injection regimens, and the heart was removed at the end of each experiment. Concentrations of each drug in plasma, blood cells, bile, and urine samples were determined and analyzed using a compartmental model. Curve fitting was performed on plasma and bile data to estimate pharmacokinetic parameters and constants. Drug concentrations bound to blood cells were analyzed using a non-compartmental model. In vivo metabolic data were obtained from bile and urine samples. The level of Ca2+ATPase in the heart was determined by Western blotting and correlated with pharmacokinetic data as a toxicological parameter. The multiple-dose regimen reduced the total plasma clearance of both drugs and increased the area under the plasma concentration-time curve (AUC). Furthermore, the AUC of doxorubicin bound to hematopoietic cells increased with increasing weekly dosing frequency, while the associated mean residence time (MRT) and apparent volume of distribution (VdSs) gradually decreased. In contrast to doxorubicin, epirubicin showed a significant increase in mean residence time and VdSs. Metabolic data indicated significant differences in the levels of alcohol and aglycone metabolites. The levels of doxorubicin alcohol and doxorubicin aglycone were significantly higher than those of epirubicin alcohol and epirubicin aglycone, while the level of epirubicin alcohol aglycone was higher than that of doxorubicin alcohol aglycone. The area under the hematopoietic cell concentration-time curve was linearly correlated with changes in the net intensity of Ca2+ATPase. These results suggest that the interaction kinetics between epirubicin and doxorubicin with hematopoietic cells are crucial. The linear correlation between the decrease in the net intensity of the biomarker and the area under the doxorubicin-hematopoietic cell interaction curve confirms that the differences between these two compounds are related to their interaction with hematopoietic cells. This observation, along with the observed metabolic differences, likely highlights the important role of hematopoietic cells in the distribution and metabolism of doxorubicin and epirubicin. For more complete data on the metabolism/metabolites of epirubicin (6 metabolites), please visit the HSDB record page. Epirubicin is extensively and rapidly metabolized in the liver. It is also metabolized in other organs and cells, including erythrocytes. Its four main metabolic pathways are: (1) reduction of the C-13 keto group to produce the 13(S)-dihydro derivative epirubicinol; (2) both the original drug and epirubicinol are bound to glucuronic acid; (3) loss of the amino sugar moiety through hydrolysis to produce doxorubicin and doxorubicinol aglycone; (4) loss of the amino sugar moiety through redox processes to produce 7-deoxydoxorubicin aglycone and 7-deoxydoxorubicinol aglycone. Epirubicinol exhibits cytotoxic activity in vitro (approximately 10% of that of epirubicin), but it is unlikely to reach the concentrations required to produce cytotoxic effects in vivo. Excretion pathway: Epirubicin and its main metabolites are mainly excreted via bile, with a small amount excreted via urine.
Half-life: The half-lives of the α, β, and γ phases are approximately 3 minutes, 2.5 hours, and 33 hours, respectively.
Biological half-life
The half-lives of the α, β, and γ phases are approximately 3 minutes, 2.5 hours, and 33 hours, respectively.
...The pharmacokinetics of epirubicin can be described using a three-compartment model, with median half-lives of 3.2 minutes, 1.2 hours, and 32 hours for each phase. ...

In vitro studies have shown that epirubicin can induce hepatotoxicity in HepG2 cells, manifested by lipid peroxidation and elevated glutathione levels, suggesting oxidative stress. Comet assays have shown that epirubicin can also cause DNA damage[3]. In vivo studies have shown that the main dose-limiting toxicity of epirubicin is cardiotoxicity, which is cumulative and can lead to congestive heart failure at high cumulative doses (> 900 mg/m²). Other toxicities include myelosuppression (leukopenia, thrombocytopenia), gastrointestinal reactions (nausea, vomiting) and alopecia[1][4]. Toxicity summary: Epirubicin is a red-orange crystal and is prepared as a solution for intravenous administration. It is used as adjuvant therapy for patients with axillary lymph node metastasis after primary breast cancer resection. Human exposure and toxicity: There have been reports of doses higher than recommended in the dose range of 150 to 250 mg/m². The adverse events observed in these patients were similar in nature to known epirubicin toxicities. Most patients recovered after appropriate supportive care. Secondary acute myeloid leukemia (AML) has been reported in breast cancer patients treated with anthracyclines, including epirubicin. Cardiotoxicity, including fatal congestive heart failure (CHF), can occur during or months to years after epirubicin treatment. Epirubicin has chromosomal breakage-inducing effects in vitro (causing chromosomal aberrations in human lymphocytes), regardless of metabolic activation. Animal studies: Routine long-term animal studies assessing the carcinogenicity of epirubicin have not been conducted, but a single intravenous injection of 3.6 mg/kg epirubicin into female rats approximately doubled the incidence of mammary tumors (primarily fibroadenomas) after one year. Intravenous injection of 0.5 mg/kg epirubicin every 3 weeks for a total of 10 times, over an 18-month observation period, increased the incidence of subcutaneous fibromas in male rats. Furthermore, subcutaneous injections of epirubicin at 0.75 or 1.0 mg/kg/day for four consecutive days on days 1 and 10 after birth, for a total of eight injections, resulted in an increased incidence of tumors compared to the control group during a 24-month observation period. Intravenous injections of epirubicin at 0.8 mg/kg/day during days 5 to 15 of gestation showed embryotoxicity (increased embryo resorption and post-implantation loss) and caused fetal growth retardation (fetal weight loss), but no teratogenicity was observed within this dose range. Intravenous injections of epirubicin at 2 mg/kg/day during days 9 to 10 of gestation showed embryotoxicity (increased late embryo resorption, post-implantation loss, and stillbirth, and reduced live births), caused fetal growth retardation (fetal weight loss), and resulted in reduced placental weight. This dose is also teratogenic, causing various external malformations (anal atresia, tail deformities, abnormal genital tubercles), visceral malformations (primarily affecting the gastrointestinal, urinary, and cardiovascular systems), and skeletal malformations (long bone and girdle deformities, rib abnormalities, irregular ossification of the spine). Intravenous administration of epirubicin at doses up to 0.2 mg/kg/day during days 6 to 18 of gestation was not embryotoxic or teratogenic in rabbits, but a maternally toxic dose of 0.32 mg/kg/day increased abortion rates and delayed ossification. Intravenous administration of epirubicin at a maternally toxic dose of 1 mg/kg/day during days 10 to 12 of gestation induced abortion, but no other signs of embryotoxicity or teratogenicity were observed. In rat mothers, daily administration of epirubicin at doses up to 0.5 mg/kg during days 17 of gestation to day 21 postpartum did not result in permanent alterations in offspring development, functional activity, behavior, or reproductive capacity. In rat fertility studies, male rats were treated daily with epirubicin for 9 weeks and mated with female rats treated daily with epirubicin for 2 weeks prior to mating and on day 7 of gestation. No pregnancy was observed when both male and female rats received a dose of 0.3 mg/kg/day. A dose of 0.1 mg/kg/day did not show any effect on mating behavior or fertility, but male rats exhibited testicular and epididymal atrophy and reduced spermatogenesis. A dose of 0.1 mg/kg/day also caused embryonic death. In these studies, a dose of 0.03 mg/kg/day was observed to increase the incidence of fetal growth retardation. Multiple daily injections of epirubicin in rabbits and dogs caused male reproductive organ atrophy. Single intravenous injections of 20.5 mg/kg and 12 mg/kg of epirubicin caused testicular atrophy in mice and rats, respectively. A single intravenous injection of 16.7 mg/kg of epirubicin caused uterine atrophy in rats. Epirubicin is mutagenic in vitro against bacteria (Ames test), regardless of metabolic activation; in the absence of metabolic activation, epirubicin is mutagenic in mammalian cells (HGPRT test of V79 Chinese hamster lung fibroblasts), but not in the presence of metabolic activation. Epirubicin is chromosomal breakage-inducing in vivo (mouse bone marrow chromosomal aberrations). The Hazardous Substances Database (HSDB) shows that epirubicin has antimitotic and cytotoxic activities. It inhibits nucleic acid (DNA and RNA) and protein synthesis through several proposed mechanisms: epirubicin forms a complex with DNA by inserting between base pairs and inhibits the activity of topoisomerase II by stabilizing the DNA-topoisomerase II complex, thereby preventing the rejoining portion of the topoisomerase II-catalyzed ligation-rejoining reaction. Protein binding rate: 77%.

[1]. Epirubicin: a review of the pharmacology, clinical activity, and adverse effects of an adriamycin analogue. J Clin Oncol. 1986 Mar;4(3):425-39.

[2]. Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity. PLoS One. 2016 Jun 10;11(6):e0156643.

[3]. Epirubicin HCl toxicity in human-liver derived hepatoma G2 cells. Pol J Pharmacol, 2004. 56(4): p. 435-44.

[4]. Drugs ten years later: epirubicin. Ann Oncol, 1993. 4(5): p. 359-69.

[5]. Antitumor activity of paclitaxel and epirubicin in human breast carcinoma, R-27. Folia Microbiol (Praha), 1998. 43(5): p. 473-4.

Epirubicin is an anthracycline antibiotic analogue of doxorubicin, developed to reduce cardiotoxicity while maintaining antitumor efficacy. It is widely used to treat breast cancer, ovarian cancer, and lymphoma, usually as part of combination chemotherapy regimens [1][4]. Its mechanism of action includes DNA intercalation, topoisomerase II inhibition, and induction of cancer cell apoptosis. In addition, its ability to inhibit Foxp3 in regulatory T cells (Tregs) enhances antitumor immunity, making it valuable in immunochemotherapy strategies [2]. 4'-epidoxorubicin is the 4'-epidoxorubicin isoform of doxorubicin and is also an anthracycline antibiotic. It has a variety of pharmacological activities, including as an EC 5.99.1.3 [DNA topoisomerase (ATP hydrolysis)] inhibitor, an antitumor drug, and an antibacterial drug. It is an anthracycline antibiotic, a deoxyhexoside, an anthracycline antibiotic, an aminoglycoside antibiotic, a monosaccharide derivative, a paraquinone compound, a primary α-hydroxy ketone, and a tertiary α-hydroxy ketone. Functionally, it is related to doxorubicin. It is the conjugate acid of 4'-epirarubicin. It is the 4'-epirarubicin isomer of doxorubicin. This compound exerts its antitumor effect by interfering with DNA synthesis and function. Epirubicin is an anthracycline topoisomerase inhibitor. The mechanism of action of epirubicin is as a topoisomerase inhibitor. Epirubicin has been reported to exist in bovine bacteria, Coccobacillus, and other organisms with relevant data. Epirubicin is the 4'-epirarubicin of the anthracycline antitumor antibiotic doxorubicin. Epirubicin can intercalate into DNA and inhibit topoisomerase II, thereby inhibiting DNA replication and ultimately interfering with RNA and protein synthesis. The drug also produces toxic free radical intermediates and interacts with cell membrane lipids, leading to lipid peroxidation. Epirubicin is only present in individuals who have used or taken this drug. It is an anthracycline antibiotic and the 4'-epimer of doxorubicin. This compound exerts its antitumor effect by interfering with DNA synthesis and function. Epirubicin has antimitotic and cytotoxic activities. It inhibits the synthesis of nucleic acids (DNA and RNA) and proteins through multiple mechanisms of action: epirubicin forms a complex with DNA by inserting between base pairs and inhibits the activity of topoisomerase II by stabilizing the DNA-topoisomerase II complex, thereby preventing the rejoining portion in the topoisomerase II-catalyzed ligation-rejoining reaction. It also interferes with DNA replication and transcription by inhibiting DNA helicase activity. See also: Epirubicin hydrochloride (salt form). Drug Indications: For adjuvant treatment of patients with axillary lymph node metastases after primary breast cancer resection.
FDA Label

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Mechanism of Action
Epirubicin possesses antimitotic and cytotoxic activity. It inhibits nucleic acid (DNA and RNA) and protein synthesis through several proposed mechanisms of action: epirubicin forms a complex with DNA by inserting between base pairs and inhibits topoisomerase II activity by stabilizing the DNA-topoisomerase II complex, thereby preventing the rejoining portion of the topoisomerase II-catalyzed ligation-rejoining reaction. It also interferes with DNA replication and transcription by inhibiting DNA helicase activity.
Epirubicin is an anthracycline cytotoxic drug. Although anthracyclines are known to interfere with a variety of biochemical and biological functions in eukaryotic cells, the exact mechanism of epirubicin's cytotoxic and/or antiproliferative properties has not been fully elucidated. Epirubicin inhibits nucleic acid (DNA and RNA) and protein synthesis by forming a complex with DNA through its planar ring insertion between nucleotide base pairs. This insertion triggers topoisomerase II to cleave DNA, thereby producing cytotoxicity. Epirubicin also inhibits DNA helicase activity, preventing the enzymatic separation of double-stranded DNA and interfering with replication and transcription. Epirubicin also participates in redox reactions by generating cytotoxic free radicals. The antiproliferative and cytotoxic activities of epirubicin are thought to stem from the above or other possible mechanisms. Epirubicin combats cancer by inhibiting topoisomerase II, thereby causing DNA strand breaks and ultimately leading to apoptosis. However, anthracyclines generate free radicals, which may explain their adverse effects. Dexrazoxane—an iron chelator—has been shown to reduce free radical production and the cardiotoxicity of anthracyclines. This article reports the intracellular concentrations of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) relative to 2'-deoxyguanosine (dGuo) in a study involving 20 cancer patients treated with epirubicin, as well as the results of a comet assay. Furthermore, this article reports the plasma concentrations of vitamins A, E, C, and carotenoids. All data were obtained immediately before and after epirubicin infusion. The ratio of 8-oxo-dGuo to dGuo in leukocyte DNA was determined by high-performance liquid chromatography-coulometric method, and nucleic acid extraction was performed using the sodium iodide method. Vitamin A, vitamin E, and carotenoids were determined by high-performance liquid chromatography-spectrophotometry. Vitamin C was determined by high-performance liquid chromatography-fluorescence spectrophotometry. After chemotherapy, the median 8-oxo-dGuo/dGuo ratio significantly increased from 0.34 damage sites per 100,000 bases to 0.48 damage sites, while the percentage of tail DNA also increased from 3.47% to 3.94%. Before and after chemotherapy, the median 8-oxo-dGuo/dGuo ratio and the percentage of tail DNA remained within the normal range. Only the vitamin C concentration decreased significantly, from 55.4 μM to 50.3 μM. The concentrations of vitamin A, vitamin E, lutein, and zeaxanthin did not decrease significantly, but their concentrations were all below the lower limit of the normal range before and after chemotherapy. The correlation between the comet assay results and vitamin C concentration was only statistically significant (rho = -0.517, p = 0.023). This study indicates that epirubicin-generated free radicals damage cellular DNA, leading to the formation of the mutagenic base 8-oxo-dGuo, which in turn causes DNA strand breaks. However, DNA strand breaks are not only caused by free radicals; inhibition of topoisomerase II can also lead to DNA strand breaks. Our previous study showed no significant change in urinary 8-oxo-dGuo excretion after doxorubicin treatment. However, due to the relatively slow DNA repair and subsequent renal clearance processes, 8-oxo-dGuo levels may be elevated at the end of urine collection. In another study, the authors used gas chromatography-mass spectrometry (GC-MS) to detect 8-oxo-dGuo in DNA and found no change in its content after long-term doxorubicin infusion. This article discusses the reasons for these significant differences.

C₂₇H₂₉NO₁₁

543.52

543.174

C, 59.67; H, 5.38; N, 2.58; O, 32.38

56420-45-2

Epirubicin hydrochloride;56390-09-1

41867

Orange to red solid powder

1.6±0.1 g/cm3

810.3±65.0 °C at 760 mmHg

443.8±34.3 °C

0.0±3.0 mmHg at 25°C

1.710

2.82

6

12

5

39

977

6

O=C(C1=C2C(O)=C3[C@@H](O[C@@]4([H])C[C@H](N)[C@@H](O)[C@H](C)O4)C[C@@](C(CO)=O)(O)CC3=C1O)C5=CC=CC(OC)=C5C2=O

AOJJSUZBOXZQNB-VTZDEGQISA-N

InChI=1S/C27H29NO11/c1-10-22(31)13(28)6-17(38-10)39-15-8-27(36,16(30)9-29)7-12-19(15)26(35)21-20(24(12)33)23(32)11-4-3-5-14(37-2)18(11)25(21)34/h3-5,10,13,15,17,22,29,31,33,35-36H,6-9,28H2,1-2H3/t10-,13-,15-,17-,22-,27-/m0/s1

(7S,9S)-7-[(2R,4S,5R,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione

Epirubicin free base; IMI 28; IMI28; IMI-28; epi DX; 4-epiadriamycin; 4-epi DX; EPI; 4-epidoxorubicin; 4-epidoxorubicin HCl; epidoxorubicin; epiADR; epidorubicin; brand name: Ellence; Pharmorubicin PFS; Epiadriamycin; 4'-Epiadriamycin; 4'-epidoxorubicin; Epirubicine; Ridorubicin;

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.)