DNA synthesis; metabolite of Gemcitabine
DNA polymerase and ribonucleotide reductase. Gemcitabine triphosphate (dFdCTP) competes with the natural nucleotide deoxycytidine triphosphate (dCTP) for incorporation into DNA. Once incorporated, it allows the addition of one more nucleotide before terminating DNA chain elongation (masked chain termination). This inhibits DNA synthesis and leads to apoptosis. Additionally, dFdCTP inhibits ribonucleotide reductase, reducing the pool of deoxynucleotides available for DNA synthesis and further potentiating its cytotoxic effects.

Gemcitabine is a nucleoside analogue with excellent clinical activity against solid tumors. Within the cell, gemcitabine is rapidly phosphorylated to its active di- and triphosphate metabolites. Cytotoxicity with gemcitabine appears to be related to multiple effects on DNA replication, where gemcitabine triphosphate can serve as both an inhibitor and substrate for DNA synthesis. Gemcitabine diphosphate inhibits ribonucleotide reductase, producing decreases in cellular dNTP pool levels in a cell-specific manner. These two major characteristics of gemcitabine, reduction in cellular dNTP pools and incorporation into DNA, are features of other antimetabolites antitumor agents which also exhibit radiosensitizing properties. Based on these favorable metabolic characteristics and the clinical activity of gemcitabine in tumor types which are commonly treated with radiation, the ability of gemcitabine to enhance X-radiation induced cytotoxicity was evaluated. Gemcitabine has been shown to be a potent radiosensitizer in a variety of tumor cell lines, including HT-29 colorectal carcinoma, pancreatic cancer, breast, non-small cell lung and head and neck cancer cell lines. Gemcitabine was most effective as a radiosensitizer when administered at least 2 hours prior to irradiation. For most cell lines, radiosensitization was evident at non-cytotoxic concentrations. The extent of radiosensitization increased with both increasing gemcitabine concentration and duration of exposure. Radiosensitization did not require redistribution of cells into a more radiosensitive phase of the cell cycle. The major metabolic effects observed under radiosensitizing conditions were the accumulation of high levels of gemcitabine triphosphate, and a selective decrease in the cellular dATP pool. The pattern of dATP decrease paralleled the increase in radiosensitization, whereas the level of gemcitabine triphosphate was not associated with the enhanced sensitivity to radiation. Compared to other radiosensitizers, the advantage of gemcitabine is that is can induce radiosensitization at concentrations that are 1000 times lower than typical plasma levels obtained with this drug. These studies will be used as guidelines for developing clinical trials of gemcitabine with radiation[1].

---

In vitro, Gemcitabine triphosphate trisodium is a potent cytotoxic agent that inhibits processes required for DNA synthesis. It is used as a standard in radiolabeled probe imaging studies to identify tumors sensitive to gemcitabine and to evaluate gemcitabine uptake and retention by cells. It inhibits T-araCTP and araCTP, which are incorporated into DNA and terminate DNA chain extension. Its activity is assessed in various cancer cell lines, showing potent antiproliferative effects. It is the active metabolite responsible for gemcitabine's antitumor activity.

In vivo, gemcitabine triphosphate is the active intracellular metabolite responsible for the antitumor activity of the prodrug gemcitabine. It is not administered directly as a drug due to its charged nature and poor membrane permeability. Instead, gemcitabine is administered, and dFdCTP is formed intracellularly. In animal models, gemcitabine shows antitumor activity in various xenograft models, which is attributed to the formation of dFdCTP. The pharmacokinetics and pharmacodynamics of gemcitabine are often studied by measuring dFdCTP levels in tumor and normal tissues.

In vitro enzyme/receptor binding assays for Gemcitabine triphosphate involve measuring its inhibition of DNA polymerases. The compound is incubated with purified DNA polymerase, a DNA template, and radiolabeled nucleotides. The incorporation of dFdCTP into the growing DNA strand is measured, and the extent of chain termination is assessed. Inhibition of ribonucleotide reductase is measured by quantifying the conversion of CDP to dCDP. These assays are performed in cell-free systems using enzyme preparations. The Ki values for these interactions are determined.

In vitro cell-based assays for Gemcitabine triphosphate are performed in cancer cell lines. Cells are treated with varying concentrations of gemcitabine or directly with dFdCTP using specialized delivery methods (e.g., electroporation) to bypass uptake limitations. Cytotoxicity is assessed using MTT or CellTiter-Glo assays. DNA synthesis inhibition is measured by incorporation of radiolabeled thymidine. Apoptosis is assessed by flow cytometry using Annexin V/PI staining. The intracellular concentration of dFdCTP is measured by LC-MS/MS to correlate with pharmacological effects.

In vivo animal experiments are conducted using the prodrug gemcitabine, not the triphosphate metabolite directly. In mouse xenograft models, tumor-bearing mice are administered gemcitabine via intraperitoneal or intravenous injection. Tumor volume and body weight are monitored. After treatment, tumors and normal tissues are harvested, and intracellular dFdCTP levels are measured by LC-MS/MS. The antitumor efficacy and pharmacokinetics of gemcitabine are assessed. These studies establish the relationship between dFdCTP formation and therapeutic response.

Pharmacokinetic properties are applicable to the prodrug gemcitabine, not directly to the triphosphate metabolite. Gemcitabine is administered intravenously and has a short plasma half-life due to rapid metabolism. It is phosphorylated intracellularly to dFdCTP, which is the active moiety. The intracellular half-life of dFdCTP can be prolonged. The compound is a trisodium salt and is soluble in water. Its stability in solution is characterized for research use. No direct PK studies are performed on the triphosphate as it is not administered as a drug.

Toxicological data are applicable to the prodrug gemcitabine, not directly to the triphosphate metabolite. Gemcitabine is a cytotoxic chemotherapeutic agent with known toxicities including myelosuppression, nausea, and hepatotoxicity. The triphosphate metabolite is responsible for the cytotoxic effects. In vitro, dFdCTP shows potent cytotoxicity. No specific toxicology studies are performed on the isolated triphosphate. Its handling requires appropriate safety precautions due to its potent biological activity.

[1].Gemcitabine and radiosensitization in human tumor cells. Invest New Drugs. 1996;14(3):257-63.

Gemcitabine triphosphate trisodium (dFdCTP trisodium) is a gemcitabine derivative that is cytotoxic and inhibits processes required for DNA synthesis. Its molecular formula is C9H13F2N3O13P3 (trisodium salt). It is one of the two nucleoside metabolites of gemcitabine; the other is the active diphosphate (dFdDTP). It is used as a standard in research studies to evaluate gemcitabine uptake and retention. It is intended for research use only.

C9H11F2N3NA3O13P3

569.08

White to off-white solid powder

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)

H2O :~125 mg/mL (~219.65 mM; with sonication)

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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