Topoisomerase I ( IC50 = 0.8 μM ); Topoisomerase II ( IC50 = 2.67 μM ); Daunorubicins/Doxorubicins; HIV-1; - DNA topoisomerase II (inhibits enzyme activity by stabilizing the cleavable complex) [1]

In vitro activity: Doxorubicin, an antibiotic anthracycline, is widely believed to exhibit its anti-tumor activity on two basic levels: it modifies DNA and generates free radicals to cause DNA damage that causes cancer cells to undergo apoptosis. Doxorubicin inhibits DNA topoisomerase II (TOP2) and can intercalate into DNA strands to prevent DNA synthesis. When cells are multiplying quickly and expressing a lot of TOP2, doxorubicin works best. Additionally, doxorubicin can cause apoptosis by releasing cytochrome c from the mitochondria, ceramide (which activates p53 or other downstream pathways like JNK), the degradation of Akt by serine threonine proteases, an increase in the production of FasL (death receptor Fas/CD95 ligand) mRNA, and an increase in free radical production. The doxorubicin-resistant breast cancer cell line MCF7/Dx exhibits suppression of resistance upon pre-treatment with GSNO (nitrosoglutathione), which is accompanied by increased protein glutathionylation and doxorubicin accumulation in the nucleus. Elevated cyclin G2 (CycG2) expression and protein phosphorylation in the ATM, ATM, and Rad3-related (ATR) signaling pathways are responsible for doxorubicin-induced G2/M checkpoint arrest. In mouse embryonic fibroblasts (MEFs) and cardiomyocytes, doxorubicin inhibits AMP-activated protein kinase (AMPK), leading to SIRT1 dysfunction, p53 accumulation, and increased cell death. These effects can be further sensitized by pre-inhibition of AMPK. Doxorubicin causes a noticeable heat shock response, and in neuroblastoma cells, it increases the apoptotic effect by either inhibiting or silencing heat shock proteins. When administered in nanomolar doses to neuroblastoma cells, doxorubicin causes a dose-dependent over-ubiquitination of a particular set of proteins without any detectable proteasome inhibition. It also causes a decrease in the activity of ubiquitinated enzymes like lactate dehydrogenase and α-enolase, whose protein ubiquitination patterns resemble those of the proteasome inhibitor bortezomib, suggesting that Doxorubicin may also cause protein damage.

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- Doxorubicin inhibits DNA topoisomerase II by stabilizing the enzyme-DNA cleavable complex, leading to DNA double-strand breaks and subsequent cell cycle arrest and apoptosis. It shows cytotoxic activity against various cancer cell lines, with potency varying by cell type [1]
- Doxorubicin inhibits human DNA topoisomerase I with an IC50 of 16 μM, as measured by the relaxation of supercoiled DNA. This inhibition is weaker compared to its effect on topoisomerase II [3]
- In combination with simvastatin, Doxorubicin exhibits synergistic cytotoxicity against human cancer cell lines (MCF-7, HT-29, HepG2). The combination reduces cell viability more effectively than either agent alone, with the synergistic effect observed at various concentration ratios [4]
- Doxorubicin enhances the cytotoxicity of Apo2L/TRAIL in prostate cancer cell lines (PC-3, DU145). Co-treatment increases apoptosis, as indicated by increased caspase-3 activity and annexin V staining, compared to either agent alone [5]
- A doxorubicin-conjugated anti-HIV-1 envelope antibody inhibits HIV-1 replication in vitro, reducing viral p24 antigen levels in infected T cells. The conjugate selectively targets infected cells expressing the viral envelope protein, minimizing toxicity to uninfected cells [7]

The most effective method for reducing the size of MB231 tumors and extending the survival of mice in vivo is to combine doxorubicin with adenoviral MnSOD (AdMnSOD) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Doxorubicin is indispensable in the treatment of solid tumors in childhood, soft tissue sarcomas, osteosarcomas, Kaposi's sarcoma, oesophageal carcinomas, Hodgkin and non-Hodgkin lymphomas, and solid tumors of the breast and esophagus. However, its use is restricted by the toxic side effects, both acute and chronic.

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- In a prostate cancer xenograft model (PC-3 cells implanted in nude mice), combination treatment with Doxorubicin (2 mg/kg, intraperitoneal, once weekly) and Apo2L/TRAIL (15 mg/kg, intravenous, twice weekly) significantly reduces tumor growth compared to either agent alone. The combination also prolongs survival without increasing systemic toxicity [5]
- In rats, Doxorubicin induces cardiotoxicity characterized by left ventricular dysfunction, increased serum cardiac troponin I levels, and histopathological changes (myocardial vacuolization, fibrosis). These effects are dose-dependent, with higher cumulative doses (15 mg/kg over 3 weeks) causing more severe damage [6]
- A doxorubicin-conjugated anti-HIV-1 antibody reduces viral load in HIV-1-infected humanized mice. Administration of the conjugate (intraperitoneal) leads to a significant decrease in plasma HIV-1 RNA levels and viral replication in lymphoid tissues [7]

Purified human DNA topoisomerase I was assayed quantitatively by enzyme titrations with supercoiled pHC624 DNA in the presence of 0-2.0 microM doxorubicin. Supercoiled and relaxed DNAs were resolved by agarose gel electrophoresis in the presence of ethidium bromide, and the percentage of conversion of supercoiled DNA to relaxed DNA was quantified by scanning microdensitometry. The inhibition of DNA topoisomerase I activity was measured at varying concentrations of doxorubicin. Doxorubicin inhibited enzyme activity at an IC50 value (the concentration required to inhibit 50% of the total activity) of 0.8 microM. Similar inhibition was observed for daunomycin, a structurally related anthracycline antitumor drug. These results indicate that anthracyclines inhibit human DNA topoisomerase I activity at concentrations that cause DNA damage and cytotoxicity in vivo[3].

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- Topoisomerase II inhibition assay: Recombinant human topoisomerase II was incubated with supercoiled plasmid DNA and various concentrations of Doxorubicin. The reaction mixture was analyzed by agarose gel electrophoresis to assess DNA relaxation. The formation of cleavable complexes was detected by measuring DNA breakage after protein denaturation [1]
- Topoisomerase I inhibition assay: Human topoisomerase I was incubated with supercoiled DNA and Doxorubicin (0.1-100 μM). The extent of DNA relaxation was visualized by agarose gel electrophoresis, and IC50 was calculated based on the concentration required to inhibit 50% of enzyme activity [3]

Three 96-well U-bottom microplates with a suspension of Hela cells (3×10⁴ cell/mL) dispensed in 160 μL are then incubated for 24 hours at 37°C in a fully humidified atmosphere with 5% CO₂. In plate 1, 200 μL of final volume is filled with serial dilutions of doxorubicin (20 μL; final concentration, 0.1-2 μM) and simvastatin (20 μL; final concentration, 0.25-2 μM) before an additional 72 hours of incubation. 40 μL of each drug's serial dilution—doxorubicin or simvastatin—are added to plates 2 and 3. The medium is aspirated and the cells are cleaned in PBS after a 24-hour incubation period. Then, to reach a final volume of 200 μL, serial dilutions of the other medication (40 μL) are added, and the mixture is incubated for 48 hours. Separate positive controls (40 μL in each well) consisting of doxorubicin and simvastatin are employed, while the negative controls consist solely of solvent-treated cells. A 20 μL MTT solution (5 mg/mL in PBS) is added to each well, and the cells are incubated for three hours in order to assess cell survival. Afterwards, 150 μL of DMSO is added to the medium, and the solution is pipetted repeatedly to completely dissolve the formazan crystals. In the next step, an ELISA plate reader measures absorbance at 540 nm. Three assays are conducted, one for each drug concentration, using four or eight wells. Doxorubicin's cytotoxic/cytostatic effect is quantified and expressed as relative viability (% control). It is assumed that 100% of the cells in the negative control will survive. * Relative viability = (background absorbance - experimental absorbance) / (background absorbance - absorbance of untreated controls)× 100%[4].

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- Cytotoxicity assay (combination with simvastatin): Human cancer cells (MCF-7, HT-29, HepG2) were seeded in 96-well plates and treated with Doxorubicin (0.01-10 μM) alone, simvastatin (0.1-100 μM) alone, or their combinations. After 72 hours, cell viability was measured using a colorimetric assay, and combination index (CI) was calculated to determine synergism [4]
- Apoptosis assay (with Apo2L/TRAIL): Prostate cancer cells (PC-3, DU145) were treated with Doxorubicin (0.1-1 μM) and/or Apo2L/TRAIL (10-100 ng/ml) for 24-48 hours. Apoptosis was assessed by caspase-3 activity assay and annexin V-FITC staining followed by flow cytometry [5]
- HIV-1 inhibition assay: HIV-1-infected T cells were treated with doxorubicin-conjugated anti-envelope antibody (0.1-10 μg/ml) for 72 hours. Viral replication was measured by p24 antigen ELISA in culture supernatants [7]

Mice: Three to four-week-old male athymic nude mice are used. Subcutaneous injection of PC3 cells (4×10⁶) is administered to mice via the flanks. When the volumes of the xenografts reached approximately 100 mm³, the animals with tumors were randomly assigned to treatment groups, with five or six mice per group. Digital calipers are used to measure tumors and the formula Volume=Width²×Length×0.52 is used to calculate the volume of the tumor, with width denoting its shorter dimensions. Therapy is given as prescribed with vehicle (PBS with 0.1% BSA), Doxorubicin (2–8 mg/kg), Apo2L/TRAIL (500 μg/animal), or a mix of 4 mg/kg Doxorubicin and 500 μg Apo2L/TRAIL. Doxorubicin is delivered systemically, while Apo2L/TRAIL is delivered intra-tumorally or systemically. Every therapy is administered just once. Every day, mice are observed for indications of negative consequences, such as lethargic behavior and disheveled look. Looks like the treatments were well received. Every data point has its mean±SEM computed. Student t-tests are used to analyze differences between treatment groups. When P is less than 0.05, differences are deemed significant. Rats: Thirty-year-old man A total of ten Doxorubicin schedules—Doxorubicin schedule 1 (n = ten), Doxorubicin schedule 2 (n = ten), and Doxorubicin schedule 3 (n = ten)—are randomly assigned to Sprague-Dawley rats weighing 250–300 g. 10 mg/kg is the total dose of doxorubicin for all treatment regimens. One intraperitoneal injection of doxorubicin (10 mg/kg) is administered as part of Schedule 1. Doxorubicin injections intraperitoneally (10 mg/kg) for ten days in a row are part of Schedule 2. Schedule 2 calls for ten intraperitoneal injections of doxorubicin (1 mg/kg) spaced ten days apart. Schedule 3 calls for five weeks of weekly intraperitoneal injections of doxorubicin at a dose of two milligrams per kilogram. As long as there are at least three rats in each group, blood pressure and cardiac function are measured in all surviving animals prior to the first Doxorubicin treatment and once a week after the start of Doxorubicin treatment.

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- Prostate cancer xenograft model: Nude mice were subcutaneously implanted with PC-3 cells. Once tumors reached ~100 mm³, mice were treated with Doxorubicin (2 mg/kg, intraperitoneal, once weekly), Apo2L/TRAIL (15 mg/kg, intravenous, twice weekly), or their combination for 3 weeks. Tumor volume was measured twice weekly, and mice were monitored for survival [5]
- Cardiotoxicity model: Rats were administered Doxorubicin (2.5 mg/kg, intraperitoneal) once weekly for 6 weeks (cumulative dose 15 mg/kg) or saline (control). Cardiac function was assessed by echocardiography (left ventricular ejection fraction, fractional shortening) at 2, 4, and 6 weeks. Serum cardiac troponin I levels were measured, and heart tissues were collected for histopathological analysis [6]
- HIV-1-infected humanized mice: Mice were infected with HIV-1, then treated with doxorubicin-conjugated anti-envelope antibody (5 mg/kg, intraperitoneal) once weekly for 3 weeks. Plasma and lymphoid tissues were collected to measure viral RNA levels by RT-PCR [7]

Absorption, Distribution and Excretion
In patients with HIV-associated Kaposi's sarcoma, after administration of 10 mg/m² liposomal doxorubicin, the calculated Cmax and AUC values were 4.12 ± 0.215 μg/mL and 277 ± 32.9 μg/mL•h, respectively. Approximately 40% of the dose was found in bile within 5 days, while only 5% to 12% of the drug and its metabolites were found in urine during the same period. Less than 3% of the doxorubicin dose was recovered in urine within 7 days. The steady-state volume of distribution of doxorubicin ranged from 809 L/m² to 1214 L/m². The plasma clearance of doxorubicin ranged from 324 mL/min/m² to 809 mL/min/m², primarily through metabolism and bile excretion. There was also a sex difference in doxorubicin clearance, with males having a higher clearance than females (1088 mL/min/m² vs. 433 mL/min/m²). Following administration of doxorubicin hydrochloride at doses ranging from 10 mg/m² to 75 mg/m², plasma clearance was estimated at 1540 mL/min/m² in children over 2 years of age and 813 mL/min/m² in infants under 2 years of age. Unencapsulated doxorubicin hydrochloride is unstable in gastric acid, and animal studies have shown that the drug is almost not absorbed from the gastrointestinal tract. The drug is highly irritating to tissues and therefore must be administered intravenously. In patients with AIDS-associated Kaposi's sarcoma, after a single intravenous infusion of 10 or 20 mg/m² of liposomal doxorubicin hydrochloride, the mean peak plasma concentrations of doxorubicin (primarily bound to liposomes) were 4.33 or 10.1 μg/mL at 15 minutes post-infusion and 4.12 or 8.34 μg/mL at 30 minutes post-infusion. In adult patients with AIDS-associated Kaposi's sarcoma, the mean peak plasma concentration 15 minutes after intravenous infusion of 40 mg/m² doxorubicin hydrochloride was 20.1 μg/mL. Unencapsulated (conventional) doxorubicin hydrochloride exhibited linear pharmacokinetic characteristics; polyethylene glycol-stabilized liposomal doxorubicin hydrochloride also showed dose-proportional linear pharmacokinetic characteristics in the dose range of 10–20 mg/m². It has been reported that the pharmacokinetics of liposomally encapsulated doxorubicin at a dose of 50 mg/m² is non-linear. It is expected that at a dose of 50 mg/m², its elimination half-life will be longer, its clearance lower, and the increase in the area under the plasma concentration-time curve will be greater than that at a dose-proportional rate. Encapsulating doxorubicin hydrochloride in polyethylene glycol (PEG)-stabilized (stealthy) liposomes significantly alters its pharmacokinetics compared to conventional intravenous formulations (i.e., unencapsulated drugs), resulting in reduced distribution in peripheral tissues, increased distribution in Kaposi's sarcoma lesions, and decreased plasma clearance. Conventionally administered doxorubicin is widely distributed in plasma and tissues. It is detectable in the liver, lungs, heart, and kidneys within just 30 seconds of intravenous injection. Doxorubicin is absorbed by cells and binds to cellular components, especially nucleic acids. The volume of distribution for conventionally intravenously administered doxorubicin hydrochloride is approximately 700-1100 liters/m². Unencapsulated doxorubicin binds to plasma proteins at a rate of approximately 50-85%... Intravenous injection of liposome-encapsulated doxorubicin hydrochloride resulted in greater distribution in Kaposi's sarcoma lesions than in healthy skin. Following a single intravenous injection of 20 mg/m² liposome-encapsulated doxorubicin hydrochloride, doxorubicin concentrations in Kaposi's sarcoma lesions were 19 times higher (range: 3-53 times) than in healthy skin; however, this study did not consider blood drug concentrations in either the lesion or healthy skin. Furthermore, intravenous injection of liposome-encapsulated doxorubicin resulted in 5.2-11.4 times higher distribution in Kaposi's sarcoma lesions compared to intravenous injection of the same dose of conventional (unencapsulated) doxorubicin. The mechanism by which liposome encapsulation enhances doxorubicin distribution in Kaposi's sarcoma lesions is not fully elucidated, but previous studies have shown that polyethylene glycol (PEG)-like stable liposomes containing colloidal gold as a marker can penetrate Kaposi's sarcoma-like lesions in animals. Liposomes may also exude through the intercellular spaces of Kaposi's sarcoma endothelial cells. After drug entry into the lesion, liposome degradation makes them permeable in situ, potentially leading to local drug release. For more complete data on the absorption, distribution, and excretion of doxorubicin (16 in total), please visit the HSDB records page.

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Metabolism/Metabolites
Doxorubicin is metabolized via three pathways: single-electron reduction, two-electron reduction, and deglycosylation. However, approximately half of the dose is excreted unchanged. Two-electron reduction is the primary metabolic pathway of doxorubicin. In this pathway, doxorubicin is reduced to secondary doxorubicin alcohol by several enzymes, including alcohol dehydrogenase [NADP(+)], carbonyl reductase [NADPH]1, carbonyl reductase [NADPH]3, and aldehyde-ketone reductase 1 family member C3. Multiple oxidoreductases in the cytoplasm and mitochondria promote single-electron reduction, generating doxorubicin semiquinone radicals. These enzymes include mitochondrial and cytoplasmic NADPH dehydrogenases, xanthine oxidase, and nitric oxide synthase. This semiquinone metabolite can be reoxidized to doxorubicin, but this process generates reactive oxygen species (ROS) and hydrogen peroxide. The primary cause of doxorubicin-related adverse reactions (especially cardiotoxicity) is the reactive oxygen species (ROS) generated by this pathway, rather than the formation of doxorubicin semiquinone. Deglycosylation is a minor metabolic pathway, accounting for only 1% to 2% of doxorubicin metabolism. Doxorubicin can be reduced to doxorubicin deoxyaglycone or hydrolyzed to doxorubicin hydroxyaglycone by cytoplasmic NADPH quinone dehydrogenase, xanthine oxidase, and NADPH-cytochrome P450 reductase. Non-encapsulated doxorubicin is metabolized by NADPH-dependent aldehyde-ketone reductases to the hydrophilic 13-hydroxy metabolite doxorubicinol, which has antitumor activity and is the major metabolite of doxorubicin; these reductases are present in most cells, and possibly all cells, especially in erythrocytes, liver, and kidneys. Although not fully identified, doxorubicinol also appears to be a major culprit in the cardiotoxicity of this drug. Reports indicate that after a single intravenous injection of 10–50 mg/m² of polyethylene glycol-stabilized liposome-encapsulated doxorubicin hydrochloride, plasma doxorubicin alcohol concentrations are extremely low or undetectable (i.e., 0.8–26.2 ng/mL). It remains unclear whether these liposome-encapsulated anthracyclines are less cardiotoxic than conventional (unencapsulated) drugs, and whether current prophylactic measures for unencapsulated drugs should also apply to liposomal formulations. Following the use of polyethylene glycol (PEG)-stabilized liposome injections, a significant decrease or disappearance of plasma concentrations of the major doxorubicin metabolite has been observed, suggesting that the drug may not be released in large quantities from the liposomes during circulation, or that some doxorubicin may be released, but the elimination rate of doxorubicin alcohol is much higher than the release rate; doxorubicin hydrochloride not encapsulated in PEG-stabilized liposomes is metabolized to doxorubicin alcohol. Other non-therapeutic metabolites include poorly soluble aglycones such as doxorubicinone (doxorubicin) and 7-deoxydoxorubicinone (17-deoxydoxorubicin), and their conjugates. These aglycones are formed in microsomes via NADPH-dependent cytochrome reductase-mediated partial cleavage of aminoglycosides. The enzymatic reduction of doxorubicin to 7-deoxyaglycone is key to its cytotoxic effects, as this process generates hydroxyl radicals, leading to widespread cell damage and death. For unencapsulated doxorubicin, more than 20% of the drug in plasma is present as a metabolite within 5 minutes of administration; 70% after 30 minutes; 75% after 4 hours; and 90% after 24 hours. …At least six metabolites have been identified, the most prominent being doxorubicin alcohol. This product is generated by the reduction of the C13 ketone group by enzymes present in leukocytes and erythrocytes (and presumably malignant tissues).
Doxorubicin can be converted into doxorubicin alcohol, aglycones, and other derivatives.
For more complete data on the metabolism/metabolites of doxorubicin (a total of 6 metabolites), please visit the HSDB record page.
Doxorubicin is metabolized via three metabolic pathways: single-electron reduction, two-electron reduction, and deglycosylation. However, approximately half of the dose is excreted unchanged from the body. Two-electron reduction produces doxorubicin alcohol, a secondary alcohol. This pathway is considered the primary metabolic pathway. Single-electron reduction is catalyzed by various oxidoreductases, generating doxorubicin semiquinone radicals. These enzymes include mitochondrial and cytoplasmic NADPH dehydrogenases, xanthine oxidase, and nitric oxide synthase. Deglycosylation is a minor metabolic pathway (1-2% of the dose is metabolized via this pathway). The resulting metabolites are deoxyaglycones or hydroxyaglycones, formed through reduction or hydrolysis, respectively. Enzymes potentially involved in this pathway include xanthine oxidase, NADPH-cytochrome P450 reductase, and cytoplasmic NADPH dehydrogenase.
Excretion pathway: 40% of the dose is excreted via bile within 5 days. 5-12% of the drug and its metabolites are excreted via urine within the same time period. Less than 3% of the dose recovered in urine is doxorubicin alcohol.
Half-life: Terminal half-life = 20-48 hours.

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Biological Half-Life
The terminal half-life of doxorubicin is 20 to 48 hours. The distribution half-life of doxorubicin is approximately 5 minutes. For the liposomal formulation, in patients with AIDS-associated Kaposi's sarcoma, the first-phase half-life and second-phase half-life of 10 mg/m² doxorubicin were calculated to be 4.7 ± 1.1 hours and 52.3 ± 5.6 hours, respectively.
Plasma concentrations of unencapsulated doxorubicin and its metabolites exhibit a biphasic or triphasic decrease. In the first phase of the triphasic model, unencapsulated doxorubicin is rapidly metabolized, presumably through the first-pass effect in the liver. Most of the metabolism appears to be completed before administration. In the triphasic model, unencapsulated doxorubicin and its metabolites are rapidly distributed into the extravascular space, with a plasma half-life of doxorubicin of approximately 0.2–0.6 hours and that of its metabolites of approximately 3.3 hours. Subsequently, plasma concentrations of doxorubicin and its metabolites are maintained at relatively long levels, likely due to tissue binding. In the second phase, the plasma half-life of unencapsulated doxorubicin was 16.7 hours, while that of its metabolites was 31.7 hours. In the biphasic model, the average initial distribution half-life was reported to be approximately 5–10 minutes, and the average terminal elimination half-life was approximately 30 hours. The plasma concentrations of liposome-encapsulated doxorubicin hydrochloride appear to decrease in a biphasic manner. In patients with AIDS-associated Kaposi's sarcoma, following a single intravenous injection of 10–40 mg/m² doses of doxorubicin hydrochloride liposomes, the average initial plasma half-life (t1/2α) of doxorubicin was 3.76–5.2 hours, while the average terminal elimination half-life (t1/2β) was 39.1–55 hours. The approximately 5-minute initial distribution half-life indicates rapid tissue absorption of doxorubicin, while its slow elimination from tissue is reflected in the 20–48-hour terminal half-life. The plasma half-life of doxorubicin in patients is approximately 17 hours, while the plasma half-life of its metabolites is approximately 32 hours.

Toxicity Summary
Doxorubicin exerts its antimitotic and cytotoxic activity through several proposed mechanisms: it 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. Toxicity Data
LD50: 21800 ug/kg (subcutaneous injection, rat) (A308) Interactions Numerous reports in the literature describe increased cardiotoxicity when doxorubicin is used in combination with paclitaxel. Two published studies reported that infusion of paclitaxel 24 hours prior to doxorubicin 48 hours prior resulted in a significantly reduced doxorubicin clearance and more severe granulocytopenia and stomatitis compared to the reverse dosing order.
In a published study, patients with advanced malignant tumors (ECOG PS<2) were intravenously injected with high doses (up to 10 g over 24 hours) of progesterone, while simultaneously receiving a fixed dose of doxorubicin (60 mg/m²). Exacerbation of doxorubicin-induced neutropenia and thrombocytopenia was observed.
A study on the acute toxicity of verapamil to doxorubicin in mice showed that verapamil increased the initial peak concentration of doxorubicin in the heart and led to an increased incidence and severity of degenerative changes in cardiac tissue, thereby shortening the survival of mice.
Concomitant use of cyclosporine with doxorubicin may lead to an increase in the AUC of both doxorubicin and doxorubicin alcohol, possibly due to decreased maternal drug clearance and reduced metabolism of doxorubicin alcohol. Literature reports indicate that concomitant use of cyclosporine with doxorubicin results in more severe and longer-lasting hematologic toxicity than doxorubicin alone. Coma and/or seizures have also been reported.
For more complete data on interactions of doxorubicin (16 in total), please visit the HSDB records page.

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Non-human toxicity values
Rat intraperitoneal injection LD50 16 mg/kg
Rat intravenous injection LD50 12.6 mg/kg
Mouse oral LD50 570 mg/kg
Mouse intraperitoneal injection LD50 10,700 ug/kg
For more non-human toxicity values (complete) for doxorubicin (8 items in total), please visit the HSDB record page.

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- Doxorubicin can induce cardiotoxicity in rats, characterized by decreased left ventricular function, increased cardiac troponin I, and myocardial histopathological changes (vacuolation, fibrosis), which can be observed at a cumulative dose of 15 mg/kg[6]
- The plasma protein binding rate of doxorubicin is approximately 75-85%[1]

[1]. Targeting DNA topoisomerase II in cancer chemotherapy.Nat Rev Cancer. 2009 May;9(5):338-50.

[2]. Synthesis, cytotoxicity, and DNA topoisomerase II inhibitory activity of benzofuroquinolinediones. Bioorg Med Chem. 2007 Feb 15;15(4):1651-8.

[3]. Doxorubicin inhibits human DNA topoisomerase I. Cancer Chemother Pharmacol. 1992;30(2):123-5.

[4]. Cytotoxic evaluation of doxorubicin in combination with simvastatin against human cancer cells. Res Pharm Sci. 2010 Jul;5(2):127-33.

[5]. Doxorubicin increases the effectiveness of Apo2L/TRAIL for tumor growth inhibition of prostate cancerxenografts. BMC Cancer. 2005 Jan 7;5:2.

[6]. Doxorubicin cardiotoxicity in the rat: an in vivo characterization. J Am Assoc Lab Anim Sci. 2007 Jul;46(4):20-32.

[7]. Elimination of HIV-1 infection by treatment with a doxorubicin-conjugated anti-envelope antibody. AIDS. 2006;20(15):1911-1915.

Therapeutic Uses

Antibiotics; Antitumor Drugs
Doxorubicin has been successfully used to treat a variety of disseminated tumors, such as acute lymphoblastic leukemia, acute myeloid leukemia, nephroblastoma, neuroblastoma, soft tissue and osteosarcoma, breast cancer, ovarian cancer, bladder transitional cell carcinoma, thyroid cancer, gastric cancer, Hodgkin's lymphoma, malignant lymphoma, and bronchial carcinoma, with small cell histology showing the most significant response to doxorubicin. /US Product Label Includes/
Doxorubicin is also indicated for adjuvant therapy in women with axillary lymph node metastasis after primary breast cancer resection. /US Product Label Includes/
Doxorubicin liposomes (DOXIL) are an anthracycline topoisomerase inhibitor indicated for ovarian cancer after failure of platinum-based chemotherapy. /US Product Label Includes/
For more complete data on the therapeutic uses of doxorubicin (7 types), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Warning: Cardiomyopathy. Doxorubicin hydrochloride use may cause myocardial injury, including acute left ventricular failure. The risk of cardiomyopathy is proportional to the cumulative exposure, with an incidence of 1% to 20% when administered once every 3 weeks at a cumulative dose of 300 mg/m² to 500 mg/m². Concomitant use of cardiotoxic drugs further increases the risk of cardiomyopathy. Left ventricular ejection fraction (LVEF) should be assessed regularly before, during, and after treatment with doxorubicin hydrochloride.
/Black Box Warning/ Warning: Secondary Malignancies. The incidence of secondary acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) is higher in patients treated with anthracyclines, including doxorubicin hydrochloride.
/Black Box Warning/ Warning: Drug Extravasation and Tissue Necrosis. Extravasation of doxorubicin hydrochloride can cause severe local tissue damage and necrosis, requiring extensive excision of the affected area and skin grafting. Treatment should be discontinued immediately and the affected area should be iced. /Warning (Black Box)/ Warning: Severe bone marrow suppression. Severe bone marrow suppression may occur, leading to severe infection, septic shock, the need for blood transfusions, hospitalization, or even death. For more complete data on drug warnings for doxorubicin (65 total), please visit the HSDB record page.

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Pharmacodynamics
Doxorubicin is a cytotoxic, non-cell cycle-specific anthracycline antibiotic. It is generally believed to exert its antitumor effect by intercalating into DNA structures, disrupting DNA structure, thereby causing DNA strand breaks and damage. It not only alters the cell transcriptome, but the failure of DNA structure repair can also initiate the apoptosis pathway. Furthermore, doxorubicin intercalation can interfere with the activity of important enzymes such as topoisomerase II, DNA polymerase, and RNA polymerase, leading to cell cycle arrest. Finally, doxorubicin also produces cytotoxic reactive oxygen species, causing cell damage.

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- Doxorubicin is an anthracycline chemotherapy drug widely used in cancer treatment. Its main mechanism of action is the inhibition of DNA topoisomerase II, leading to DNA damage and cell death. It is effective against a variety of solid tumors and hematologic malignancies, but its application is limited by cardiotoxicity[1]. Doxorubicin can be conjugated with antibodies (e.g., anti-HIV-1 envelope antibodies) to achieve targeted delivery, thereby enhancing efficacy against specific cells (e.g., HIV-1 infected cells) while reducing off-target toxicity[7].

C27H29NO11

543.52

543.17

C, 59.66; H, 5.38; N, 2.58; O, 32.38.

23214-92-8

31703

Deep-red to black solid powder

1.61 g/cm3

205ºC

443.8ºC

9.64E-28mmHg at 25°C

1.709

1.503

6

12

5

39

977

6

[H][C@@]1(O[C@H]2C[C@](O)(C(CO)=O)CC(C2=C3O)=C(O)C4=C3C(C5=C(OC)C=CC=C5C4=O)=O)O[C@@H](C)[C@@H](O)[C@@H](N)C1

AOJJSUZBOXZQNB-TZSSRYMLSA-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,5S,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

Adriamycin; Hydroxydaunorubicin; ADR; DOX. Code name: FI106; chloridrato de doxorrubicina. Adriamycin; Adriacin; Adriblastina; Adriblastine; Adrimedac; DOXOCELL; Doxolem; Doxorubin; Farmiblastina; Rubex. Abbreviations: ADM; Adria;

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)

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.)