Autophagy; cell cycle (IC50 = 0.4 nM); DNA repair (IC50 = 0.42 μM)
Dactinomycin acts as an antagonist to the cellular membrane-permeable SH2 domain, inhibiting the Shc/Grb2 interaction, and interferes with RNA polymerase activity by forming stable complexes with DNA. [1]

Actinomycin D inhibits BrdU incorporation at 80 nM, which significantly reduces SMC proliferation. Using a flowcytometric analysis, the G1-phase arrest provides more evidence for this. Actinomycin D suppresses the levels of focal adhesion kinase (FAK), proliferating cell nuclear antigen (PCNA), and Raf protein expression. Actinomycin D is found to increase extracellular signalregulated kinases (Erk) involved in cell-cycle arrest.[1]

---

Dactinomycin markedly reduced vascular smooth muscle cell (SMC) proliferation by inhibiting BrdU incorporation at a concentration of 80 nM. It induced a significant G1-phase arrest in SMC, with an IC50 for inhibition of S-phase entry at 0.4 nM. The lethal dose (LD50) for SMC was 260 µM. Western blot analysis showed that Dactinomycin dose-dependently down-regulated the protein expression levels of proliferating cell nuclear antigen (PCNA) and Raf, and inhibited focal adhesion kinase (FAK) protein levels (maximal inhibition reached at 80 nM). In contrast, the phosphorylated form of extracellular signal-regulated kinases (Erk 1/2) was significantly up-regulated by increasing concentrations of Dactinomycin, with peak phosphorylation observed 30 minutes after stimulation. [1]

Actinomycin D is active in two distinct mouse models that are typified by either an unmutated B-cell receptor or inactive p53 function, both of which are recognized adverse prognostic factors in CLL. Actinomycin D targets the survival proteins TOSO, BCL2, and MCL1.[3]

---

Local application of Dactinomycin in pluronic gel to the adventitial surface of balloon-injured rat carotid arteries significantly reduced neointimal formation. Two weeks after injury, treatment with 80 nM Dactinomycin reduced neointimal thickness by 45%, and treatment with 80 µM Dactinomycin reduced it by 55%, compared to the balloon-injured control group without Dactinomycin. [1]

Actinomycin D is co-incubated for three hours at thirty degrees Celsius with a reaction mixture that has the following contents: 120 mg of a whole-cell extract of HeLa cells, 70 mM KCl, 0.4 mM of dGTP, dCTP, dATP, and digoxygenylated-dUTP in reaction buffer that has 40 mM Hepes-KOH (pH 7.6), 5 mM MgCl₂, 0.5 mM Dithiotreitol, 2 mM EGTA, 10 mM phosphocreatine, 50 mg/mL creatine phosphate, and 360 mg/mL of bovine serum albumin combined. DNA damage is identified during this reaction, and neosynthesized DNA fragments replace the removed patches. Digoxygenylated-dUMPs are integrated during this DNA synthesis. Three washes halt the DNA repair reaction.

Actinomycin D at different doses is incubated at 37°C after cultured SMC are starved for 24 hours. 18 to 24 hours are spent on drug treatment. Since actinomycin D dissolves in 0.1% DMSO, DMSO-containing vehicle control is also provided.

---

Cell Cycle and Viability Assay: Vascular smooth muscle cells (A10 line) were subcultured in 6-well plates at a density of 3.5 × 10^5 cells per well. Synchronized cells, after 12 hours of serum starvation, were cultured in medium containing 15% fetal bovine serum (FBS) with or without various doses of Dactinomycin (0.8 pM to 8 µM) for 18 hours. Cells were collected, washed with PBS, resuspended in a DNA-staining solution containing propidium iodide and RNase, and then analyzed by flow cytometry to determine the percentage of cells in each phase of the cell cycle. Another set of identically treated cells were trypsinized and subjected to hematocytometric analysis to determine the number of viable cells for LD50 assessment. [1]
BrdU Incorporation Assay: Vascular SMC were subcultured in 96-well plates at a density of 1 × 10^4 cells per well. Serum-starved cells were grown in medium containing 15% FBS with various doses of Dactinomycin for an additional 20 hours. Cells were then labeled with BrdU for 4 hours at 37°C. After removing the labeling medium, cells were fixed with a commercial FixDenat solution for 30 minutes at room temperature. Anti-BrdU antibody was added and incubated for 90 minutes at room temperature. Following washing, a substrate solution was added, and the optical density (representing DNA synthesis) was measured at 370 nm using an ELISA reader. [1]
Western Blot Analysis: Dactinomycin-treated cells were lysed using a buffer containing 2% SDS, 50 mM DTT, and 62.5 mM Tris-HCl (pH 6.8), followed by incubation at 95°C for 5 minutes. Total protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. After blocking, the membrane was incubated overnight at 4°C with primary antibodies against PCNA, FAK, Raf, or Erk. Following washing, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized using an enhanced chemiluminescence (ECL) reaction and exposure to X-ray film. [1]

Mice: For more than nine generations, the original Eμ-TCL1a transgenic mice have been backcrossed to C57BL/6 mice.The Eμ-TCL-1 transgenic mice's tumor cells are engrafted into C57BL/6 wild-type mice. Mice are regularly given blood from the tail vein, which is then subjected to flow cytometry analysis to determine the percentage of CD5+/CD19+ cells in the peripheral blood. Treatment is initiated when 40–60% of the tumor cells in the peripheral blood are present. IV injections of actinomycin D (0.06 mg/kg over 10 days) are administered every day.

---

Thirty-two male Sprague-Dawley rats weighing 350-400 g were used. Rats were anesthetized with 3.6% chloral hydrate (1 ml/100 g, i.p.). Balloon angioplasty injury was induced in the carotid artery using a 2F Fogarty balloon catheter. The inflated balloon (1.3 kg/cm²) was pushed and pulled through the vessel lumen three times to cause injury. Immediately after injury, Dactinomycin was applied topically to the adventitia of the injured carotid artery segment. The drug was formulated as a suspension in 30% (w/v) pluronic gel at two concentrations: 80 nM (low dose) and 80 µM (high dose). Rats were randomly divided into four groups: sham control (no injury, n=8), balloon-injured control (injury, no drug, n=8), low-dose Dactinomycin-treated (injury + 80 nM gel, n=8), and high-dose Dactinomycin-treated (injury + 80 µM gel, n=8). Animals were sacrificed two weeks after balloon injury with an overdose of pentobarbital. Injured carotid arteries were harvested, sectioned (5 µm thickness), stained using Weigert's method (Iron Hematoxylin, Resorcin-Fuchsin, and Van Gieson's solutions) to visualize elastic fibers and neointimal layers. Morphometric analysis of neointimal thickness was performed using a digital imaging system. [1]

Absorption, Distribution and Excretion
Gastrointestinal Absorption Actinomycin D is poorly absorbed in the gastrointestinal tract. It is highly irritating to tissues and therefore must be administered intravenously. Actinomycin D is rapidly distributed to tissues, with higher concentrations in bone marrow and nucleated cells (including granulocytes and lymphocytes). It appears to have difficulty crossing the blood-brain barrier, and may even be unable to cross it at all. The plasma protein binding rate of actinomycin D is 5%. Actinomycin D appears to cross the placenta. It is unclear whether actinomycin D is excreted into breast milk. For more complete data on the absorption, distribution, and excretion of actinomycin D (6 types), please visit the HSDB records page. Metabolism/Metabolites Hepatic Metabolism Actinomycin D appears to be metabolized very little; small amounts of the drug's monolactone have been detected in urine.

---

Biological Half-Life
36 hours
The terminal plasma half-life of radioactive materials is approximately 36 hours.
The terminal elimination half-life of actinomycin D is 36 to 48 hours.

Hepatotoxicity
Actinomycin D combined with other chemotherapy drugs can cause elevated serum enzymes in a significant proportion of patients, but the exact percentage depends on the dose, other medications used, monitoring frequency, and the definition criteria for the elevated indicators. Elevated ALT is usually asymptomatic and transient, resolving spontaneously without dose adjustment. In many cases, due to concurrent exposure to other potentially hepatotoxic drugs, it is difficult to attribute abnormal liver function to actinomycin D. Actinomycin D can also cause a unique, clinically significant liver injury called hepatopathy-thrombocytopenia syndrome (HTS), which can be severe and even life-threatening. This syndrome appears to be caused by sinusoidal obstruction, but direct damage to the liver and bone marrow may also be a factor. In large studies, 1% to 5% of children with cancer treated with actinomycin D regimens developed acute liver injury and thrombocytopenia, suggesting possible high-fatal actinomycin syndrome (HTS). This syndrome is more common in younger children and occurs more frequently with higher doses of actinomycin D. Onset typically occurs within 3 to 6 weeks after the first dose, often 5 to 10 days after the second or third cycle of actinomycin D chemotherapy. Symptoms have a sudden onset, with children usually presenting with right upper quadrant pain or tenderness, hepatomegaly, abnormal liver function, and signs of excessive bleeding, such as nosebleeds or bruising. Early serum transaminase levels are significantly elevated (10 to 100 times the upper limit of normal), but decrease rapidly and return to normal within 7 to 14 days. Platelet counts are usually below 25,000/μL and also return to normal rapidly. Serum alkaline phosphatase is usually normal, and bilirubin levels are slightly elevated unless the condition progresses rapidly and is life-threatening. Serum ammonia and the international normalized ratio (INR) may also be elevated, and ascites is common in the acute phase. The overall pattern of damage resembles acute hepatic necrosis, with liver histology showing evidence of centrilobular necrosis and sinusoidal obstruction. Recovery is rapid and usually complete. Probability score: C (Clinically significant cause of liver injury).

---

Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the use of actinomycin D during lactation. Most sources suggest that pregnant women should avoid breastfeeding while receiving antitumor drug treatment. The manufacturer recommends discontinuing breastfeeding during actinomycin D treatment and for 14 days after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

---

Protein binding rate
5%Interactions
Radiation therapy, cyclophosphamide, or vincristine may enhance the response to actinomycin D.
Actinomycin D may increase serum uric acid levels; dosage adjustments to antigout medications may be necessary to control hyperuricemia and gout; allopurinol may be preferred due to the risk of uric acid nephropathy associated with uricosuric antigout medications. If actinomycin D is used concurrently or recently with medications that cause hematologic disorders and also cause leukopenia and/or thrombocytopenia, the leukopenic and/or thrombocytopenic effects of actinomycin D may be enhanced; dosage adjustments based on blood cell counts should be made if necessary. Concomitant use with actinomycin D may enhance the side effects of other myelosuppressants or radiation therapy, including gastrointestinal toxicity, myelosuppression, and skin erythema and tanning; dosage reduction for each medication is recommended. Actinomycin D alone may cause recurrence of erythema induced by previous radiation therapy. For more complete data on interactions with actinomycin D (10 items in total), please visit the HSDB record page.

---

Non-human toxicity values
Oral LD50 in rats: 7200 μg/kg
Intraperitoneal LD50 in rats: 100 μg/kg
Intravenous LD50 in rats: 460 μg/kg
Subcutaneous LD50 in rats: 800 μg/kg
For more complete (6) non-human toxicity data for actinomycin D, please visit the HSDB record page.

---

In vitro, the median lethal dose (LD50) of actinomycin D for vascular smooth muscle cells was determined to be 260 µM by counting viable cells after drug treatment. The half-maximal inhibitory concentration (IC50) for inhibiting cell cycle arrest in the G1 phase (preventing entry into the S phase) was 0.4 nM. The difference between the LD50 and IC50 is approximately five orders of magnitude, indicating a high therapeutic window for its antiproliferative effect. An unacceptable incidence of adverse events was reported in a multicenter clinical trial (ACTION) using actinomycin-eluting scaffolds. [1]

[1]. J Biomed Sci . 2005;12(3):503-12.

[2]. Carcinogenesis . 1997 Dec;18(12):2441-5.

[3]. Leukemia . 2012 Dec;26(12):2508-16.

Therapeutic Uses

Antitumor drug; Antibacterial drug; Nucleic acid synthesis inhibitor; Protein synthesis inhibitor. Actinomycin D is indicated as part of combination chemotherapy and/or multimodal therapy for the treatment of nephroblastoma, pediatric rhabdomyosarcoma, Ewing's sarcoma, and metastatic non-seminomatous testicular cancer. /Included on US product label/
Actinomycin D is indicated as monotherapy or as part of combination chemotherapy for the treatment of gestational trophoblastic tumors. /Included on US product label/
Actinomycin D is indicated as part of regional perfusion therapy for palliative and/or adjuvant therapy of locally recurrent or localized solid malignancies. /Included on US product label/
For more complete data on the therapeutic uses of actinomycin D (7 types), please visit the HSDB record page.

---

Drug Warnings
/Black Box Warning/ COSMEGEN (Actinomycin D for Injection) should only be used under the guidance of a physician experienced in the use of cancer chemotherapy drugs. /Warning (Black Box)/ This drug is highly toxic. Both the powder and solution must be handled and used with care. Inhalation of dust or vapors, and contact with skin or mucous membranes, especially the eyes, must be avoided. Pregnant women should avoid contact. Due to the toxicity of actinomycin D (e.g., corrosiveness, carcinogenicity, mutagenicity, teratogenicity), special operating procedures should be carefully read and strictly followed before handling. Actinomycin D is extremely corrosive to soft tissues. Extravasation during intravenous injection can cause severe soft tissue damage. In at least one case, this resulted in arm contracture. COSMEGEN is a toxic drug, and patients must be monitored very carefully and frequently for adverse reactions. These reactions can affect any tissue in the body, most commonly the hematopoietic system, leading to bone marrow suppression. Therefore, live virus vaccines should not be administered during COSMEGEN treatment. The possibility of anaphylactic reactions should be noted. Patients may be more susceptible to infection due to suppression of normal immune mechanisms. Severe systemic illness, sometimes even fatal, may occur if actinomycin D is taken during or before/after a varicella infection. For more complete data on drug warnings for actinomycin D (24 in total), please visit the HSDB records page.

---

Pharmacodynamics
Generally, actinomycins are inhibitory against Gram-positive bacteria, Gram-negative bacteria, and certain fungi. However, the toxicity of actinomycins (including actinomycin D) compared to their antibacterial activity makes them unsuitable for use as antibiotics to treat infectious diseases. Actinomycins possess cytotoxicity and therefore antitumor activity, which has been demonstrated in laboratory animals implanted with various types of tumors. This cytotoxic effect is the basis for their use in treating certain types of cancer. Actinomycin D is believed to exert its cytotoxic effect by binding to DNA and inhibiting RNA synthesis.

---

Actinomycin D is a pigment peptide lactone with potent antitumor activity. Its mechanism of action in preventing restenosis is believed to be the arrest of vascular smooth muscle cell proliferation during the G1 phase of the cell cycle. This is primarily achieved by downregulating key proliferation-related proteins (PCNA, FAK, Raf) and upregulating phosphorylated Erk 1/2, which may contribute to DNA damage response and cell cycle arrest. The drug was previously used in the ACTION clinical trial to evaluate its efficacy in preventing coronary restenosis via drug-eluting stents, but the trial was discontinued due to an excessively high incidence of adverse events. This study suggests that the method of local administration (Pranic gel applied to the adventitia) and drug concentration may be key factors influencing efficacy and safety. [1]

C62H86N12O16

1255.43

1254.628

C, 59.32; H, 6.90; N, 13.39; O, 20.39

50-76-0

457193

Orange to red solid powder

1.4±0.1 g/cm3

1386.0±65.0 °C at 760 mmHg

251-253 °C

792.1±34.3 °C

0.0±0.3 mmHg at 25°C

1.656

Streptomyces

-4.03

5

18

8

90

3030

10

O=C(C1=C(N)C(C(C)=C2OC3=C(N=C21)C(C(N[C@@H]4C(N[C@@H](C(C)C)C(N5[C@](CCC5)([H])C(N(C)CC(N(C)[C@@H](C(C)C)C(O[C@@H]4C)=O)=O)=O)=O)=O)=O)=CC=C3C)=O)N[C@@H]6C(N[C@@H](C(C)C)C(N7[C@](CCC7)([H])C(N(C)CC(N(C)[C@@H](C(C)C)C(O[C@@H]6C)=O)=O)=O)=O)=O

RJURFGZVJUQBHK-IIXSONLDSA-N

InChI=1S/C62H86N12O16/c1-27(2)42-59(84)73-23-17-19-36(73)57(82)69(13)25-38(75)71(15)48(29(5)6)61(86)88-33(11)44(55(80)65-42)67-53(78)35-22-21-31(9)51-46(35)64-47-40(41(63)50(77)32(10)52(47)90-51)54(79)68-45-34(12)89-62(87)49(30(7)8)72(16)39(76)26-70(14)58(83)37-20-18-24-74(37)60(85)43(28(3)4)66-56(45)81/h21-22,27-30,33-34,36-37,42-45,48-49H,17-20,23-26,63H2,1-16H3,(H,65,80)(H,66,81)(H,67,78)(H,68,79)/t33-,34-,36+,37+,42-,43-,44+,45+,48+,49+/m1/s1

2-amino-4,6-dimethyl-3-oxo-1-N,9-N-bis[(3R,6S,7R,10S,16S)-7,11,14-trimethyl-2,5,9,12,15-pentaoxo-3,10-di(propan-2-yl)-8-oxa-1,4,11,14-tetrazabicyclo[14.3.0]nonadecan-6-yl]phenoxazine-1,9-dicarboxamide

DACT; ACTD; actinomycin C1; actinomycin D; actinomycin I1; actinomycin IV; actinomycin X 1; actinomycinthrvalprosarmeval; dactinomycine; meractinomycin; actinomycin D; Actinomycin C1; Actinomycin IV; Meractinomycin; 50-76-0; Cosmegen; Actinomycin I1; US brand names: Cosmegen; Lyovac.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 27 mg/mL (~21.5 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (1.66 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.

---

Solubility in Formulation 2: 2.08 mg/mL (1.66 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

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