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]

Foxp3 [2]
DNA topoisomerase II [1][4]

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]

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Epirubicin hydrochloride (4'-Epidoxorubicin hydrochloride), like doxorubicin, interferes with the synthesis of DNA, RNA, and proteins and damages DNA as a means of achieving its antitumor effects. The integrity and functionality of cellular membranes may also be impacted by epirubicin hydrochloride. Epirubicin hydrochloride maximally kills cells during the S phase of the cell cycle. Effects are also observed in the early G2, G1, and M phases at higher concentrations[1].
Epirubicin hydrochloride exhibit antitumor activity against the majority of cancerous cells. With an IC50 of 1.6 μg/mL after 24 hours, epirubicin hydrochloride is cytotoxic to Hepatoma G2 cells. 1.6 μg/mL Hep G2 cells undergo apoptosis when exposed to epirubicin hydrochloride, 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%. NADPH-CYP 450 reductase is expressed more in cells when epirubicin hydrochloride is present, whereas GST-π expression is decreased[3].

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In human breast carcinoma R-27 cells, combined treatment with Epirubicin HCl and paclitaxel exhibited antitumor activity, showing inhibitory effects on cell proliferation [5]
- In human hepatoma G2 (HepG2) cells, Epirubicin HCl induced toxicity in a concentration-dependent manner, resulting in decreased cell viability and alterations in cellular functions [3]
- In regulatory T (Treg) cells, Epirubicin HCl identified as a Foxp3 inhibitor via luciferase reporter assay, suppressed Foxp3 expression and subsequent Treg cell activity, reducing the immunosuppressive function of Treg cells [2]
- In various human tumor cell lines (including breast, lung, colon carcinoma), Epirubicin HCl demonstrated antiproliferative activity by interfering with DNA synthesis and inducing DNA damage [1][4]

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]

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Epirubicin hydrochloride (4'-Epidoxorubicin hydrochloride) 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].
Epirubicin hydrochloride reduces the tumor mass of the human breast tumor xenograft R-27 by 74.4% when given at a dose of 3.5 mg/kg[5].

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In animal models bearing human breast carcinoma xenografts, Epirubicin HCl administered alone or in combination with paclitaxel showed significant antitumor efficacy, inhibiting tumor growth and reducing tumor volume [5]
- In preclinical animal models, Epirubicin HCl exhibited broad antitumor activity against multiple solid tumors, with therapeutic effects comparable to doxorubicin but with reduced cardiac toxicity [1][4]

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]

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A luciferase reporter assay was established to screen Foxp3 inhibitors; the assay utilized a reporter construct containing the Foxp3 promoter driving luciferase expression. Cells were transfected with the reporter construct and treated with Epirubicin HCl, followed by luciferase activity measurement to evaluate Foxp3 transcriptional activity. The results indicated that Epirubicin HCl inhibited luciferase activity, confirming its role as a Foxp3 inhibitor [2]
- DNA topoisomerase II activity assay: Isolated DNA topoisomerase II was incubated with supercoiled DNA substrate and Epirubicin HCl. The reaction products were separated by gel electrophoresis, and the inhibition of topoisomerase II-mediated DNA relaxation was analyzed. Epirubicin HCl was found to bind to DNA-topoisomerase II complex, preventing enzyme-mediated DNA strand passage [1][4]

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]

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A 96-well plate is plated with 500 monolayer Hep G2 cells per well. The following day, epirubicin is added to the medium and the cells are treated. 15% volume of MTT dye solution is added at the conclusion of the incubation times. An equal volume of solubilization/stop solution (dimethylsulfoxide) is added to each well for an additional hour of incubation at 37°C after the first hour of incubation. At 570 nm, the reaction solution's absorbance is measured.

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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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HepG2 cell toxicity assay: HepG2 cells were seeded in culture plates and allowed to adhere. Cells were treated with serial concentrations of Epirubicin HCl for a specified incubation period. Cell viability was assessed using a colorimetric assay, and cellular morphological changes were observed under a microscope. Toxicity parameters such as cell survival rate and IC50 values were calculated based on the assay results [3]
- Breast carcinoma R-27 cell antiproliferative assay: R-27 cells were plated in 96-well plates and treated with Epirubicin HCl alone or in combination with paclitaxel. After incubation, cell proliferation was measured using a thymidine incorporation assay, and the inhibitory rate of cell growth was determined [5]
- Treg cell function assay: Treg cells were isolated from peripheral blood or lymphoid tissues and cultured in vitro. Cells were treated with Epirubicin HCl at different concentrations, and Foxp3 expression was detected by flow cytometry. The suppressive capacity of Treg cells on effector T cell proliferation was evaluated using a co-culture assay [2]

Dissolved in saline; 3.5 mg/kg; i.v. injection
Human breast tumor xenograft R-27 Mouse in vivo assays [2]
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]

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Tumor-bearing animal model: Animals were subcutaneously inoculated with human breast carcinoma R-27 cells to establish xenograft tumors. When tumors reached a specified volume, animals were randomly divided into treatment groups. Epirubicin HCl was dissolved in an appropriate solvent and administered intravenously at a predetermined dose, with administration frequency set as once every few days for a total treatment course. Paclitaxel was co-administered in combination groups following the same schedule. Tumor volume and animal body weight were measured regularly throughout the experiment [5]
- Preclinical efficacy and toxicity evaluation: Animals were implanted with various tumor cell lines (breast, lung, colon carcinoma). Epirubicin HCl was administered intravenously at doses ranging from 5 to 20 mg/kg, with dosing intervals of 7-14 days. Animals were monitored for tumor growth, survival time, and signs of toxicity (e.g., weight loss, organ damage). At the end of the experiment, animals were euthanized, and tumors and major organs were collected for pathological analysis [1][4]

Absorption rate 100%
Elimination pathway: Epirubicin and its main metabolites are mainly eliminated through bile excretion, with a small amount eliminated through urine excretion. Volume of distribution
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]
Clearance
65 ± 8 L/hour [patients with solid tumors receiving intravenous epirubicin 60 mg/m²]
83 ± 14 L/hour [patients with solid tumors receiving intravenous epirubicin 75 mg/m²]
65 ± 13 L/hour [patients with solid tumors receiving intravenous epirubicin 120 mg/m²]
69 ± 13 L/hour [patient 1 with solid tumors receiving intravenous epirubicin 150 mg/m²] Metabolites/Metabolites
Epirubicin is extensively and rapidly metabolized in the liver. It can also be metabolized by other organs and cells, including erythrocytes. Its main metabolic pathways are fourfold: (1) C-13 keto reduction to produce the 13(S)-dihydro derivative epirubicinol; (2) both the original drug and epirubicinol are bound to glucuronic acid; (3) loss of the aminoglycoside via hydrolysis to produce doxorubicin and doxorubicin alcohol aglycone; (4) loss of the aminoglycoside via redox processes to produce 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 concentrations required to produce cytotoxic effects in vivo. Epirubicin is primarily metabolized rapidly and extensively in the liver, but it can also be 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 parent drug and epirubicinol are bound to glucuronic acid; (3) loss of the amino sugar moiety through hydrolysis to generate doxorubicin and doxorubicin alcohol aglycone; (4) loss of the amino sugar moiety through redox processes to generate 7-deoxydoxorubicin aglycone and 7-deoxydoxorubicin alcohol aglycone. The in vitro cytotoxic activity of epirubicinol is about one-tenth that of epirubicin. Because 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. National Institutes of Health; DailyMed. Latest Drug Information for Ellence (Epirubicin Hydrochloride) Injection (Updated: November 2014). As of June 16, 2015, it is available at: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=0a03c798-a652-4895-b29c-3b521a89ba42
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 cytoplasm reduced the carbonyl group on the tetracyclic side chain of doxorubicin, generating the secondary alcohol metabolite doxorubicinol (DOXol). A decrease in alcohol metabolite production was observed after replacing doxorubicin with epirubicin (EPI). Epirubicin is a low-cardiotoxic analog characterized by an axial-to-equatorial epimerization of the C-4 hydroxyl group of the amino sugar (daunorubicin) linked to the tetracyclic ring. A similar decrease was observed after replacing doxorubicin with the novel anthracycline antibiotic MEN. Preclinical evidence suggests that MEN has low 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 indicates that both the absence of the methoxy group and the presence of the disaccharide moiety limit the production of MEN metabolites. Studies of enzymatically generated or purified anthracycline secondary alcohols also show that the presence of the disaccharide moiety (rather than the absence of the methoxy group) reduces the reactivity of MEN metabolites with the cytoplasmic aconitase [4Fe-4S] cluster, manifested in limited reoxidation to the parent carbonyl anthracycline and 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 secondary alcohols in anthracyclines 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]. Minotti G et al.; Chemm Res Toxicol 13 (12): 1336-41 (2000)
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 formulation of cell inhibitors to detect the genotoxic effects that may result from occupational exposure to cell inhibitors under routine work conditions, as well as cell inhibitor-related effects that may occur in the event of accidental contamination. …Internal exposure levels of cell inhibitors were assessed by detecting 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 rounds of surveillance 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, respectively). In 12 cases of accidental workplace contamination, significantly elevated SCE or MN was detected in 7 cases, but no increases in blood platinum or plasma anthracyclines were observed in these cases. Two unreported cases of contamination were identified by detecting epirubicin in plasma. Smoking significantly increased SCE. No correlation was observed between individual SCE and MN scores. …/Author/ These findings support the possibility of transient increases in SCE or MN following exposure to relevant cytotoxic inhibitors in cases of accidental contamination. There were no significant differences in SCE and MN between hospital pharmacy staff and the unexposed control group, indicating high safety standards in the corresponding workplaces. PMID:11057412 Pilger A et al.; Int Arch Occup Environ Health 73 (7): 442-8 (2000)
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 to hemoglobin may affect their distribution in vivo. This study aimed to compare the pharmacokinetics and metabolism of doxorubicin and epirubicin based on plasma concentrations, the amount of drug bound to blood cells, and simultaneous monitoring of bile and urinary excretion of the parent drug and its metabolites after single and multiple injections. This study also determined the level of cardiac sarcoplasmic reticulum Ca2+ATPase 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; 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 pharmacokinetic parameters and constants were estimated by simultaneously fitting plasma and bile data to a compartmental model. Drug concentrations in blood cells were analyzed using a non-compartmental model. Bile and urine samples provided in vivo metabolic data. The level of Ca2+ATPase in the heart was determined using 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 hematopoietic cell-related AUC of doxorubicin 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 revealed 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 hematopoietic cell-related kinetics of epirubicin and doxorubicin are crucial. The linear correlation between the decrease in the net intensity of the biomarker and the area under the curve of doxorubicin binding to hematopoiesis confirms that the differences between the two compounds are related to their interaction with hematopoiesis. This observation, along with the observed metabolic differences, likely highlights the important role of hematopoiesis in the distribution and metabolism of doxorubicin and epirubicin. Ramanathan-Girish S, Boroujerdi M; J Pharmacol 53 (7): 987-97 (2001)
Epirubicin is extensively and rapidly metabolized in the liver. It is also metabolized by other organs and cells, including erythrocytes. Its four main metabolic pathways are: (1) reduction of the C-13 keto group to generate 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 form doxorubicin and doxorubicinol aglycone; (4) loss of the amino sugar moiety through redox processes to form 7-deoxydoxorubicin aglycone and 7-deoxydoxorubicinol aglycone. Epirubicinol has in vitro cytotoxic activity (approximately 10% of that of epirubicin), but it is unlikely to reach the concentration 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 by a three-compartment model, with median half-lives of 3.2 minutes, 1.2 hours and 32 hours for each phase, respectively. ……PMID: 8070217

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Epirubicinis mainly administered intravenously, with very low oral bioavailability. After intravenous injection, it is widely distributed in tissues, with high 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 about 40% of the dose excreted in urine within 7 days and 40% in feces [1][4]. Absorption: Oral epirubicin hydrochloride is poorly absorbed; intravenous injection is the preferred route of administration in clinical practice [1][4]. Distribution: After intravenous injection, epirubicin hydrochloride is widely distributed throughout the body, with higher concentrations in the liver, spleen, and tumor tissues. The large volume of distribution indicates its extensive tissue penetration [1][4]. Metabolism: Epirubicin hydrochloride is mainly metabolized in the liver through reduction and conjugation reactions. The main metabolites include epirubicinol, which retains some antitumor activity [1][4]. Excretion: The drug and its metabolites are mainly excreted via bile, with a small amount excreted via urine. Elimination half-life is 30 to 40 hours [1][4] - Plasma protein binding rate: Epirubicin hydrochloride binds to plasma proteins at a rate of approximately 77-89% [1][4]

Toxicity Overview
Identification and Use: Epirubicin is a red-orange crystal formulated for intravenous injection. It is used as adjuvant therapy in patients with axillary lymph node metastasis after primary breast cancer resection. Human Exposure and Toxicity: Higher doses of epirubicin have been reported, ranging from 150 to 250 mg/m². Adverse events observed in these patients were similar in nature to known epirubicin toxicities. Most patients recovered with 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), may 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 evaluating the carcinogenicity of epirubicin have not been conducted. However, 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 increased the incidence of subcutaneous fibromas in male rats over an 18-month observation period. Furthermore, subcutaneous injections of 0.75 or 1.0 mg/kg/day for 4 consecutive days on days 1 and 10 after birth for a total of 8 times increased the incidence of tumors compared to the control group over a 24-month observation period. Intravenous injection of 0.8 mg/kg/day epirubicin into rats between days 5 and 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 at this dose range. In rats, intravenous injection of epirubicin at 2 mg/kg/day on days 9-10 of gestation showed embryotoxicity (increasing late embryo resorption, post-implantation loss, and stillbirth, and reducing live births), leading to fetal growth retardation (fetal weight loss) and placental weight reduction. This dose also exhibited teratogenicity, 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 sacral deformities, rib abnormalities, and irregular ossification of the spine). In rabbits, intravenous injection of epirubicin at doses up to 0.2 mg/kg/day on days 6-18 of gestation showed no embryotoxicity or teratogenicity, but a maternally toxic dose of 0.32 mg/kg/day increased abortion rates and delayed ossification. Intravenous injection of epirubicin at a maternally toxic dose of 1 mg/kg/day on days 10-12 of gestation induced abortion, but no other signs of embryotoxicity or teratogenicity were observed. In rats administered epirubicin daily at doses up to 0.5 mg/kg from day 17 of gestation to day 21 postpartum, no permanent alterations in offspring development, functional activity, behavior, or reproductive capacity were observed. In rat fertility studies, male rats treated daily with epirubicin for 9 weeks were 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 effects 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 resulted in embryonic death. In these studies, a dose of 0.03 mg/kg/day was observed to increase the incidence of fetal growth retardation. Repeated daily injections of epirubicin in rabbits and dogs resulted in male reproductive organ atrophy. A single intravenous injection of 20.5 mg/kg and 12 mg/kg epirubicin induced testicular atrophy in mice and rats, respectively. A single intravenous injection of 16.7 mg/kg epirubicin induced 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 chromosomally break-inducing in vivo (mice bone marrow chromosomal aberrations). According to the Hazardous Substances Database (HSDB), 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.

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Protein binding rate 77%

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In vitro experiments showed that eprubicin could induce hepatotoxicity in HepG2 cells, manifested by increased lipid peroxidation and decreased glutathione reduction, indicating oxidative stress. Comet assays showed that it also caused DNA damage[3].
In vivo experiments showed that the main dose-limiting toxicity was cardiotoxicity, which was cumulative and could lead to congestive heart failure at high cumulative doses (> 900 mg/m²). Other toxicities included myelosuppression (leukopenia, thrombocytopenia), gastrointestinal reactions (nausea, vomiting) and hair loss[1][4].
Epirubicin has a high plasma protein binding rate (77-89%) and minimal displacement effect of other drugs in vitro[1].
Cardiotoxicity: Epirubicin hydrochloride showed dose-dependent cardiotoxicity, but its incidence and severity were lower than those of doxorubicin. High cumulative doses can lead to clinical manifestations such as decreased left ventricular ejection fraction and cardiomyopathy [1][4]
- Hepatotoxicity: In HepG2 cells, epirubicin hydrochloride can induce hepatocyte damage, manifested as elevated liver enzyme levels and impaired cell function [3]
- Bone marrow suppression: epirubicin hydrochloride can cause dose-related bone marrow suppression, with leukopenia being the most common hematologic toxicity, followed by thrombocytopenia and anemia [1][4]
- Gastrointestinal toxicity: Adverse reactions include nausea, vomiting, diarrhea, and mucositis, usually mild to moderate [1][4]
- Skin and hair toxicity: Hair loss is a common side effect, and in some cases, rash or hyperpigmentation may occur [1][4]

[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 hydrochloride is the hydrochloride salt of the 4'-epimer of the anthracycline antitumor antibiotic doxorubicin. Epirubicin intercalates into DNA and inhibits 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.
An anthracycline antibiotic, the 4'-epimer of doxorubicin. This compound exerts its antitumor effect by interfering with DNA synthesis and function.
See also: Epirubicin (with active moiety).

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4'-Epirubicin is an anthracycline antibiotic, the 4'-epimer of doxorubicin. It is an EC 5.99.1.3 [DNA topoisomerase (ATP hydrolysis)] inhibitor with antitumor and antibacterial activity. It is an anthracycline antibiotic belonging to the deoxyhexoside class, anthracycline antibiotics, aminoglycosides, monosaccharide derivatives, paraquinones, primary α-hydroxy ketones, and tertiary α-hydroxy ketones. It is functionally related to doxorubicin. It is the conjugate acid of 4'-epirarubicin. It is an anthracycline antibiotic and 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 be present in bovine bacteria, Coccidioides occulta, 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 the drug. It is an anthracycline antibiotic and the 4'-epirarubicin isomer of doxorubicin. This compound exerts its antitumor effect by interfering with DNA synthesis and function. Epirubicin possesses antimitotic and cytotoxic activities. It inhibits nucleic acid (DNA and RNA) and protein synthesis through multiple 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 in the topoisomerase II-catalyzed ligation-rejoining reaction. It also interferes with DNA replication and transcription by inhibiting DNA helicase activity.
An anthracycline antibiotic, the 4'-epiisomer of doxorubicin. This compound exerts its antitumor effect by interfering with DNA synthesis and function.
See also: Epirubicin hydrochloride (salt form).
Pharmaceutical Indications

For adjuvant treatment of patients with axillary lymph node metastases after primary breast cancer resection.
FDA Label
Mechanism of Action

Epirubicin possesses antimitotic and cytotoxic activities. 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 mechanisms of epirubicin's cytotoxic and/or antiproliferative properties are not 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, resulting in cytotoxicity. Epirubicin also inhibits DNA helicase activity, preventing the enzymatic dissociation of double-stranded DNA and interfering with replication and transcription. Epirubicin also participates in redox reactions by generating cytotoxic free radicals. Epirubicin's antiproliferative and cytotoxic activities are thought to originate from the aforementioned or other possible mechanisms. Epirubicin combats cancer by inhibiting topoisomerase II, thereby inducing 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 the 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 using high-performance liquid chromatography-coulometrics, and nucleic acid extraction was performed using the sodium iodide method. Vitamin A, vitamin E, and carotenoids were determined using high-performance liquid chromatography-spectrophotometry. Vitamin C was determined using 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 tail DNA percentage also increased from 3.47% to 3.94%. Both the median 8-oxo-dGuo/dGuo ratio and the tail DNA percentage remained within the normal range before and after chemotherapy. Only 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 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 demonstrates 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 research showed that the excretion of 8-oxo-dGuo in urine did not change significantly after doxorubicin treatment. However, due to the relatively slow DNA repair and subsequent renal clearance processes, the level of 8-oxo-dGuo 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. 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 a combination chemotherapy regimen [1][4]
Its mechanism of action includes DNA embedding, topoisomerase II inhibition and induction of cancer cell apoptosis. In addition, its ability to inhibit Foxp3 in Treg cells can enhance anti-tumor immunity, making it valuable in immunochemotherapy strategies [2]

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Epirubicin hydrochloride is an anthracycline antibiotic, an analogue of doxorubicin, and its development aims to reduce cardiotoxicity while maintaining anti-tumor activity [1][4]
- Mechanism of action:Epirubicin hydrochloride exerts anti-tumor effects by embedding in DNA, inhibiting DNA topoisomerase II, inducing DNA strand breaks and inhibiting DNA replication and transcription [1][4]
- Clinical indications: Used to treat a variety of solid tumors, including breast cancer, lung cancer, colorectal cancer and ovarian cancer [1][4]
- When used in combination with other chemotherapeutic drugs (such as paclitaxel), it shows synergistic anti-tumor effects [5]

C27H30CLNO11.HCL

579.98

579.15

C, 55.91; H, 5.21; Cl, 6.11; N, 2.42; O, 30.34

56390-09-1

56390-09-1(HCl salt); 56420-45-2

65348

Red solid powder

1.61g/cm3

810.3ºC at 760 mmHg

185ºC dec

443.8ºC

1.709

1.503

7

12

5

40

977

6

Cl[H].O([C@@]1([H])C([H])([H])[C@]([H])([C@]([H])(C([H])(C([H])([H])[H])O1)O[H])N([H])[H])[C@]1([H])C2C(=C3C(C4C(=C([H])C([H])=C([H])C=4C(C3=C(C=2C([H])([H])[C@@](C(C([H])([H])O[H])=O)(C1([H])[H])O[H])O[H])=O)OC([H])([H])[H])=O)O[H]

MWWSFMDVAYGXBV-FGBSZODSSA-N

InChI=1S/C27H29NO11.ClH/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;1H/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;hydrochloride

4'-epidoxorubicin HCl; 4'-Epidoxorubicin hydrochloride; IMI28; 4-epi DX; EPI; epi DX; 4-epiadriamycin; 4-epidoxorubicin; 4-epidoxorubicin HCl; epiADR; epidoxorubicin; epidorubicin; IMI 28; IMI-28; brand name: Ellence; Pharmorubicin PFS; Pharmorubicin; Farmorubicin; Farmorubicina; Epirubitec;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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 (4.31 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 2: ≥ 2.5 mg/mL (4.31 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 3: ≥ 2.08 mg/mL (3.59 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 20.8 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 4: ≥ 0.5 mg/mL (0.86 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 5: 1.1 mg/mL (1.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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