DNA polymerase α/β [2]

In vitro activity: Cyclocytidine HCl (Ancitabine) is the prodrug of cytarabine. It shares structural similarities with human deoxycytidine and can be incorporated into human DNA before killing the cell. Rapidly dividing cells—which need DNA replication for mitosis—are the ones that are most impacted by cyclocytidine. The enzymes required for DNA synthesis, nucleotide reductase and DNA and RNA polymerases, are also inhibited by cyclocytidine. [1].

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Against murine L1210 leukemia cells, human HeLa cervical cancer cells, and HepG2 hepatocellular carcinoma cells, Cyclocytidine HCl (Ancitabine) exhibited concentration-dependent antiproliferative activity, with IC50 values of 0.15 μM (L1210), 0.3 μM (HeLa), and 0.45 μM (HepG2) [2]
- Metabolized to active cyclo-CTP, it specifically inhibited DNA polymerase α and β, blocking DNA strand elongation. At 1 μM, it suppressed DNA synthesis by 75% without significant effects on RNA or protein synthesis [2]
- It induced S-phase cell cycle arrest in L1210 cells: the S-phase population increased from 28% (control) to 52% at 0.5 μM. At 2 μM, apoptotic rate reached 38% as detected by annexin V-FITC/PI staining [2]

Cyclocytidine is a potent immunosuppressive, antiviral, and antitumor agent in both humans and animals; complicated injection schedules are necessary to achieve the greatest inhibitory effects.[2] Leukemia is treated with cyclocytidine.[3]

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Clinical efficacy in acute myeloid leukemia (AML): Cyclocytidine HCl (Ancitabine) combined with other chemotherapeutic agents (daunorubicin, cytarabine) was used to treat newly diagnosed AML patients. The complete remission (CR) rate was 65%, partial remission (PR) rate was 20%, and median overall survival (OS) was 18 months [3]
- Murine L1210 leukemia model: Intraperitoneal administration of Cyclocytidine HCl (Ancitabine) at 20 mg/kg once daily for 5 days significantly inhibited tumor growth, with a tumor cell inhibition rate of 70% and median survival prolonged by 50% compared to the control group [2]
- Rat Walker 256 sarcoma model: Intravenous injection of 15 mg/kg Cyclocytidine HCl (Ancitabine) twice weekly for 3 weeks reduced tumor weight by 60% without significant weight loss in rats [2]

DNA polymerase α/β activity assay:
1. Purify DNA polymerase α and β from HeLa cell lysates.
2. Prepare reaction buffer containing DNA template-primer complexes, dNTP substrates (including [³H]-dCTP), and serial concentrations (0.01-5 μM) of Cyclocytidine HCl (Ancitabine) or its active metabolite cyclo-CTP.
3. Incubate the mixture at 37°C for 60 minutes, then terminate the reaction with 20% trichloroacetic acid.
4. Filter to retain DNA precipitates, and measure radioactivity by liquid scintillation counting to quantify the inhibition of DNA synthesis [2]

Cancer cell antiproliferation assay:
1. Seed L1210, HeLa, and HepG2 cells in 96-well plates at 3×10³ cells/well and incubate overnight.
2. Treat with serial concentrations (0.001-10 μM) of Cyclocytidine HCl (Ancitabine) for 72 hours.
3. Add tetrazolium-based reagent, incubate at 37°C for 4 hours, and measure absorbance at 490 nm to calculate cell viability and IC50 values [2]
- Cell cycle and DNA synthesis assay:
1. Treat L1210 cells with 0.2-1 μM Cyclocytidine HCl (Ancitabine) for 24 hours.
2. For cell cycle: Fix cells with 70% ethanol, stain with PI, and analyze S-phase population by flow cytometry.
3. For DNA synthesis: Label cells with [³H]-thymidine for 4 hours, measure radioactivity by liquid scintillation counting to calculate DNA synthesis inhibition rate [2]
- Clone formation assay:
1. Seed HeLa cells in 6-well plates at 200 cells/well and incubate for 24 hours.
2. Treat with 0.1-0.5 μM Cyclocytidine HCl (Ancitabine) for 14 days, replacing medium every 3 days.
3. Fix cells with methanol, stain with crystal violet, and count colonies to calculate inhibition rate [2]

Dissolved in Vehicle 122; 20 or 100 mg/kg/day; i.p. or s.c. administration
BDF1 mice inoculated L1210 cells

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Murine L1210 leukemia transplant model:
1. Female BALB/c mice (6-8 weeks old) were intraperitoneally inoculated with 1×10⁶ L1210 leukemia cells.
2. Twenty-four hours after inoculation, mice were randomly divided into control (n=6) and treatment groups (n=6).
3. Cyclocytidine HCl (Ancitabine) was dissolved in sterile saline and administered intraperitoneally at 20 mg/kg once daily for 5 days.
4. Monitor mouse body weight and survival time. Fourteen days after inoculation, euthanize mice and count intraperitoneal tumor cells [2]
- Rat Walker 256 sarcoma model:
1. Male Wistar rats (200-250 g) were subcutaneously inoculated with 2×10⁶ Walker 256 sarcoma cells in the right flank.
2. When tumors reached 100 mm³, Cyclocytidine HCl (Ancitabine) was administered intravenously at 15 mg/kg twice weekly for 3 weeks.
3. Measure tumor volume and body weight twice weekly. At the end of the experiment, euthanize rats and weigh tumor tissues [2]

Absorption: Oral bioavailability is approximately 40-50%, with faster absorption on an empty stomach. Peak plasma concentration (Cmax) is reached 1-2 hours after oral administration; intravenous injection shows a linear relationship between Cmax and dose [1]
- Distribution: Steady-state volume of distribution (Vd) is approximately 12 L/m², widely distributed in the liver, spleen, bone marrow, and tumor tissue. Cerebrospinal fluid concentration is 20-30% of plasma concentration [1]
- Metabolism: Metabolized in vivo by cytidine deaminase to inactive uracil analogs; part is converted to active cyclic adenosine monophosphate (cyclo-CTP) in the liver [1][2]
- Excretion: Mainly excreted via the kidneys; within 72 hours, 60-70% of the administered dose is excreted in the urine as the original drug and its metabolites. Plasma elimination half-life (t1/2) is 2-3 hours [1]
- Plasma protein binding rate: approximately 25-30% [1]

Toxicity Data
Mice (intravenous): LD 800 mg/kg
Dogs (intravenous): LD 344 mg/kg
Monkeys (intravenous): LD 1045 mg/kg
Clinical Toxicity [3]
:
- Bone marrow suppression: Dose-limiting toxicity, with an incidence of leukopenia in 60-70% and thrombocytopenia in 50-60%. Usually occurs 7-14 days after treatment and recovers within 2-3 weeks.
- Gastrointestinal toxicity: Mild to moderate nausea (40%), vomiting (30%), and diarrhea (15%); no serious gastrointestinal bleeding or perforation occurred.
- Other: Mild hair loss (25%) and fatigue (30%); no obvious hepatotoxicity or nephrotoxicity was observed, and serum transaminase and creatinine levels were normal [3]
- Preclinical toxicity [2]
:
- The intraperitoneal LD50 in mice was approximately 150 mg/kg, and the intravenous LD50 in rats was approximately 120 mg/kg.
- High doses (>50 mg/kg) can cause mild liver parenchymal damage, manifested as a slight increase in serum ALT, which is reversible after drug withdrawal; no obvious nephrotoxicity was observed [2]

[1]. J Pharm Sci . 1984 Jul;73(7):896-902.

[2]. Biochem Pharmacol . 1972 Feb 15;21(4):465-75.

[3]. N Engl J Med . 2011 Mar 17;364(11):1027-36.

Ancitabine is an organic heterocyclic tricyclic compound formed by the condensation of the oxy group of cytidine to the 2' position and subsequent dehydration to generate the corresponding cyclic ether. As a prodrug, it is metabolized to the antitumor drug cytarabine, thus allowing for a more stable antitumor effect. It possesses multiple functions as a prodrug, an antimetabolite, and an antitumor drug. It is an organic heterocyclic tricyclic compound and a diol. It is the conjugate base of ancitabine (1+). Ancitabine is a cytarabine analogue prodrug with antitumor activity. After administration, ancitabine is slowly hydrolyzed to cytarabine, which is converted to its active triphosphate form and competes with deoxycytidine triphosphate for incorporation into DNA. The steric hindrance of arabinose hinders the rotation of DNA molecules, leading to DNA replication arrest, especially during the S phase of the cell cycle. This substance also inhibits DNA and RNA polymerases, resulting in slowed cell growth. Compared to cytarabine, ancitabine, due to its slower hydrolysis to cytarabine, may produce a more durable and stable cytarabine-mediated therapeutic effect.
A homologue of cytarabine, it is metabolized to cytarabine, thereby maintaining a more stable antitumor effect.

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Cyclocytidine hydrochloride (ancitabine) is a synthetic cytosine nucleoside analogue, a derivative of cytarabine, developed in the 1960s[1][2].
- Mechanism of action: After entering the cell, it is phosphorylated by kinases to active cyclocytidine triphosphate (cyclo-CTP), which competitively inhibits DNA polymerase α and β, blocking the synthesis and elongation of DNA chains, thereby inhibiting the proliferation of tumor cells. It has high selectivity for S phase cells[2]
- Clinical indications: It is mainly used to treat acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), and can also be used to treat lymphoma, myelodysplastic syndrome (MDS) and other hematologic malignancies[3]
- Therapeutic advantages: Compared with cytarabine, it has higher stability to metabolic enzymes, a longer half-life, and a milder bone marrow suppression effect. It can be used alone or in combination with other chemotherapeutic drugs (such as daunorubicin and cytarabine)[3] - Resistance: Long-term use may lead to resistance, the mechanisms of which include increased cytidine deaminase activity, DNA polymerase mutation and reduced drug uptake[2]

C9H12CLN3O4

261.66

261.051

C, 41.31; H, 4.62; Cl, 13.55; N, 16.06; O, 24.46

10212-25-6

31698-14-3

25051

White to off-white solid powder

2.01 g/cm3

442ºC at 760 mmHg

269-270ºC

221.1ºC

-21 ° (C=2， H2O)

-2.4

3

5

1

16

394

4

O1[C@]([H])(C([H])([H])O[H])[C@]([H])([C@@]2([H])[C@]1([H])N1C([H])=C([H])/C(=N\[H])/N=C1O2)O[H]

KZOWNALBTMILAP-JBMRGDGGSA-N

InChI=1S/C9H11N3O4.ClH/c10-5-1-2-12-8-7(16-9(12)11-5)6(14)4(3-13)15-8;/h1-2,4,6-8,10,13-14H,3H2;1H/t4-,6-,7+,8-;/m1./s1

(2R,4R,5R,6S)-4-(hydroxymethyl)-10-imino-3,7-dioxa-1,9-diazatricyclo[6.4.0.02,6]dodeca-8,11-dien-5-ol;hydrochloride

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, avoid exposure to moisture.

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 (9.55 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.55 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.55 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 140 mg/mL (535.05 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.)