Tezacitabine targets ribonucleotide reductase (RNR) and DNA polymerase. RNR catalyzes the conversion of ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates, a necessary step for DNA synthesis. The diphosphate metabolite of Tezacitabine irreversibly inhibits RNR, depleting cellular deoxynucleotide pools. The triphosphate metabolite acts as a substrate for DNA polymerase and is incorporated into DNA during replication or repair, causing DNA chain termination. This agent is relatively resistant to metabolic deactivation by cytidine deaminase.

In the G1 and S phases of the cell cycle lag, tizacitabine (0.01-10 µM; 24 hours; CCRF-SB, KG-1, Jurkat, COLO-205, MCF-7, and PC-3 cells) promotes leakage resistance[1]. Tezacitabine (0.01-10 µM; 24 hours; CCRF-SB, KG-1, Jurkat, COLO-205, MCF-7, and PC-3 cells) acts concentration-dependently on apoptotic modes by inhibiting the caspase 3/7 pathway [1]. Tizacitabine possesses potent cytotoxic and cytostatic effects. Tezacitabine's cytotoxic effects include modifications in protein metabolism in addition to apoptosis [1].

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Tezacitabine demonstrates potent in vitro cytotoxic activity. In CCRF-SB, KG-1, Jurkat, COLO-205, MCF-7, and PC-3 cells, treatment with 0.01-10 μM Tezacitabine for 24 hours induces a leaky block of the G1 phase (at concentrations >10 nM) and S phase (at low concentrations) of the cell cycle. The compound concentration-dependently induces apoptotic cell death via the caspase 3/7 pathway. The cytotoxic effect is manifested not only in apoptosis but also in changes in protein metabolism. Tezacitabine has highly cytostatic and cytotoxic properties.

In HCT 116 tumor xenografts, tizacitabine treatment (100 mg/kg; i.p.; daily; female nude mice) suppresses tumor growth [2].

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Tezacitabine demonstrates in vivo anti-tumor activity. In a study using female nude mice (7-9 weeks old) injected with HCT 116 cells, treatment with Tezacitabine (100 mg/kg, intraperitoneal injection, daily) inhibited tumor growth in HCT 116 tumor xenografts over 14 days. The compound's ability to inhibit RNR and cause DNA chain termination underlies its anti-tumor efficacy. The agent is converted intracellularly into its active diphosphate and triphosphate metabolites. Further details on dose-response relationships and comparative efficacy are not extensively provided in the available literature.

In vitro enzyme assays for Tezacitabine involve measuring ribonucleotide reductase (RNR) activity. RNR enzyme is incubated with ribonucleoside diphosphate substrates and varying concentrations of Tezacitabine diphosphate (the active metabolite). The conversion of [³H]-CDP to dCDP is measured, or NADPH oxidation is monitored spectrophotometrically. IC50 values for RNR inhibition are calculated. For DNA polymerase assays, the enzyme is incubated with DNA template, primers, deoxynucleotide triphosphates, and Tezacitabine triphosphate. Incorporation of the analog into DNA is detected by measuring chain termination products using gel electrophoresis or sequencing.

Cell cycle analysis[1]
Cell Types: CCRF-SB, KG-1, Jurkat, COLO-205, MCF-7 and PC-3 Cell
Tested Concentrations: 0.01 µM, 0.1 µM, 1.0 µM and 10 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induces leakage block in G1 (concentrations above 10 nM) and S phase (low concentrations) of the cell cycle.

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Apoptosis analysis[1]
Cell Types: CCRF-SB, KG-1, Jurkat, COLO-205, MCF-7 and PC-3 Cell
Tested Concentrations: 0.01 µM, 0.1 µM, 1.0 µM and 10 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Apoptosis was induced through the caspase 3/7 pathway in a concentration-dependent manner.

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For in vitro cell-based assays, various cancer cell lines (CCRF-SB, KG-1, Jurkat, COLO-205, MCF-7, PC-3) are cultured in appropriate medium. Cells are treated with Tezacitabine at concentrations of 0.01, 0.1, 1.0, and 10 μM for 24 hours. Cell cycle analysis is performed by flow cytometry after propidium iodide staining to assess G1 and S phase block. Apoptosis is measured by caspase 3/7 activity assays or annexin V staining. Cell viability is assessed using MTT or similar assays. Protein metabolism changes are evaluated by proteomic analysis or Western blot.

Animal/Disease Models: Female nude mice (7-9 weeks old) injected with HCT 116 cells [2]
Doses: 100 mg/kg
Route of Administration: intraperitoneal (ip) injection; daily; 14 days
Experimental Results: Inhibition of tumors in HCT 116 tumor xenografts grow.

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In vivo animal studies for Tezacitabine use female nude mice (7-9 weeks old) implanted subcutaneously with HCT 116 human colon cancer cells. When tumors reach a measurable size, mice receive Tezacitabine at 100 mg/kg via intraperitoneal injection daily for 14 days. Tumor volume is measured using calipers twice weekly. Body weight is monitored to assess toxicity. At the end of the study, tumors are excised, weighed, and analyzed for histology and biomarkers. Control groups receive vehicle alone. Specific protocols and detailed results are not extensively provided in the available literature.

Tezacitabine is a nucleoside analogue with molecular formula C10H12FN3O4 and molecular weight 257.22. For in vitro use, it is soluble in DMSO. The compound should be stored as powder at -20°C for up to 3 years and in solvent at -80°C for up to 1 year. Tezacitabine is converted intracellularly into its active diphosphate and triphosphate metabolites. The agent is relatively resistant to metabolic deactivation by cytidine deaminase. Detailed pharmacokinetic parameters (absorption, distribution, half-life, bioavailability) are not extensively characterized in the available literature.

Specific toxicity data for Tezacitabine are not extensively detailed in the available literature. As a nucleoside analogue with cytotoxic and cytostatic properties, it would be expected to have dose-limiting toxicities similar to other antimetabolites, including myelosuppression, gastrointestinal toxicity, and hepatotoxicity. In animal studies at 100 mg/kg (i.p., daily for 14 days), the compound was tolerated in nude mice, with inhibition of tumor growth. Standard toxicology assessments would be required for therapeutic development, including acute, subchronic, and chronic toxicity studies, as well as genotoxicity and reproductive toxicity evaluations.

[1]. Tezacitabine Blocks Tumor Cells in G1 and S Phases of the Cell Cycle and Induces Apoptotic Cell Death. Acta Pol Pharm. May-Jun 2005;62(3):195-205.

[2]. Tezacitabine Enhances the DNA-directed Effects of Fluoropyrimidines in Human Colon Cancer Cells and Tumor Xenografts. Biochem Pharmacol. 2007 Jan 1;73(1):44-55.

Tezarcitabine is a hydroxypyrimidine compound, belonging to the synthetic purine nucleoside analogues, and possesses potential antitumor activity. After phosphorylation by cellular kinases, tezarcitabine is converted into its active metabolites, diphosphate and triphosphate. Tezarcitabine diphosphate binds to ribonucleotide reductase (RNR) and irreversibly inhibits its activity, thereby inhibiting DNA synthesis in tumor cells and inducing tumor cell apoptosis. Tezarcitabine triphosphate can serve as a substrate for DNA polymerase, further inhibiting DNA replication. This drug exhibits relative resistance to metabolic inactivation by cytidine deaminase. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotide 5'-diphosphate to deoxyribonucleotide 5'-diphosphate, required for DNA synthesis, and is overexpressed in various tumor types. Anhydrous tezarcitabine is the anhydrous form of tezarcitabine, a synthetic pyrimidine nucleoside analogue with potential antitumor activity. Tezarcitabine, upon phosphorylation by cellular kinases, is converted into its active diphosphate and triphosphate metabolites. Tezarcitabine diphosphate binds to ribonucleotide reductase (RNR) and irreversibly inhibits its activity, potentially leading to inhibition of DNA synthesis in tumor cells and ultimately inducing tumor cell apoptosis. Tezarcitabine triphosphate, as a substrate for DNA polymerase, further inhibits DNA replication. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleoside 5'-bisphosphate to deoxyribonucleoside 5'-bisphosphate, a necessary step in DNA synthesis and overexpressed in various tumor cell types. Drug Indications It has been investigated for the treatment of colorectal cancer, lung cancer, leukemia (not specified), and gastric cancer. Mechanism of Action Tezarcitabine, upon phosphorylation by cellular kinases, is converted into its active diphosphate and triphosphate metabolites. Tezacitabine diphosphate binds to ribonucleotide reductase (RNR) and irreversibly inhibits its activity, which may lead to suppression of DNA synthesis in tumor cells and induce tumor cell apoptosis. Tezacitabine triphosphate, as a substrate of DNA polymerase, further inhibits DNA replication. This drug is relatively resistant to the metabolic inactivation of cytidine deaminase. RNR catalyzes the conversion of ribonucleotide 5'-bisphosphate to deoxyribonucleotide 5'-bisphosphate, which is required for DNA synthesis, and is overexpressed in many tumor types.

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Tezacitabine is a cytostatic and cytotoxic antimetabolite and a purine nucleoside analogue with a dual mechanism of action. Its diphosphate metabolite irreversibly inhibits ribonucleotide reductase (RNR), depleting cellular deoxynucleotide pools, while its triphosphate metabolite acts as a substrate for DNA polymerase, resulting in DNA chain termination. This contrasts with gemcitabine. The compound blocks tumor cells in the G1 and S phases of the cell cycle, inducing apoptotic cell death via the caspase 3/7 pathway. Tezacitabine has potential applications in treating leukemia and solid tumors. No approved therapeutic status is reported.

C10H11FN2O4

242.2

275.091

130306-02-4

6435808

Off-white to light yellow solid powder

590.1ºC at 760 mmHg

310.7ºC

-2.3

3

5

2

18

448

3

C1=CN(C(=O)N=C1N)[C@H]2/C(=C/F)/[C@@H]([C@H](O2)CO)O

GFFXZLZWLOBBLO-ASKVSEFXSA-N

InChI=1S/C10H12FN3O4/c11-3-5-8(16)6(4-15)18-9(5)14-2-1-7(12)13-10(14)17/h1-3,6,8-9,15-16H,4H2,(H2,12,13,17)/b5-3+/t6-,8+,9-/m1/s1

4-amino-1-[(2R,3E,4S,5R)-3-(fluoromethylidene)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one

KW 2331; FMdC; MDL 101731

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)

H2O : ~200 mg/mL (~777.54 mM)
DMSO : ~200 mg/mL (~777.54 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (19.44 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 50.0 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: ≥ 5 mg/mL (19.44 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 50.0 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: ≥ 5 mg/mL (19.44 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (388.77 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.)