HIV; DNA polymerase of HBV
AZT triphosphate tetraammonium targets HIV-1 reverse transcriptase, the viral enzyme responsible for converting viral RNA into DNA. As a chain-terminating nucleotide analog, it is incorporated into the growing viral DNA strand by reverse transcriptase, preventing further DNA chain elongation. The compound also inhibits HBV DNA polymerase. Its selectivity for viral reverse transcriptase over host DNA polymerases is the basis for its antiretroviral activity. The tetraammonium salt form enhances aqueous solubility for research applications.

In H9c2 cells, treatment with 100 µM zidovudine (AZT) for 48 hours causes AZT triphosphate (3'-Azido-3'-deoxythymidine-5'-triphosphate) tetraammonium buildup that disturbs the mitochondrial tubular network. Opa1 is downregulated and Drp1 is upregulated when AZT triphosphate tetraammonium accumulates. In the H9c2 cell model derived from rat embryonic myoblasts, AZT triphosphate tetraammonium disrupts the balance of the mitochondrial quality control system, results in mitochondrial malfunction, and enhances the generation of harmful reactive oxygen species (ROS)[1].

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In vitro studies demonstrate that AZT triphosphate tetraammonium is a potent inhibitor of HIV-1 reverse transcriptase, with the triphosphate form being the active species that competes with natural dTTP for incorporation into viral DNA. The compound inhibits HIV replication in infected cell cultures and also inhibits HBV DNA polymerase. Its in vitro activity is assessed using reverse transcriptase enzyme assays with radiolabeled nucleotides or using HIV-infected cell lines with p24 antigen production as a readout. The compound activates mitochondrial-mediated apoptosis pathways, which may contribute to its cytotoxicity.

Low-dose AZT administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced K562 cells suppressed tumor growth. This novel suicide gene therapy approach can thus be integrated as a safety switch into therapeutic vectors.[2]

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In vivo, AZT triphosphate is the active intracellular metabolite responsible for the antiviral activity of Zidovudine (AZT). Following administration of AZT, the drug is phosphorylated intracellularly to the mono-, di-, and triphosphate forms, with the triphosphate being the active antiviral agent. AZT triphosphate tetraammonium itself is not administered as a drug but is used in research to study the pharmacology of AZT and related nucleoside analogs. Its in vivo effects are primarily those of AZT, including antiviral activity and mitochondrial toxicity.

Acyclovir (ACV) triphosphate and azidothymidine (AZT) triphosphate inhibit the DNA polymerase of human hepatitis B virus (HBV) by 50% at submicromolar concentrations, but no effects of ACV or AZT treatment have been noted on the clinical manifestations of hepatitis B. We synthesized 1-O-octadecyl-sn-glycero-3-phospho-acyclovir (ODG-P-ACV), 1-O-hexadecylpropanediol-3-phospho-acyclovir (HDP-P-ACV), and 1-O-octadecyl-sn-glycero-3-phospho-azidothymidine (ODG-P-AZT), and evaluated their antiviral activity in human hepatoma cells that constitutively produce HBV (2.2.15 cells). ACV and AZT up to 100 microM caused only slight inhibition of HBV replication in 2.2.15 cells. However, HDP-P-ACV and ODG-P-ACV inhibited viral replication by 50% at 0.5 and 6.8 microM, respectively. ODG-P-AZT also showed increased antiviral activity, with a 50% reduction in HBV replication at 2.1 microM. Based on the EC50, HDP-P-ACV, ODG-P-ACV, and ODG-P-AZT were > 200, > 14.7, and > 48 times more active than their free nucleosides in reducing HBV replication in 2.2.15 cells. To evaluate the biochemical basis for the increased antiviral activity, we studied the uptake and metabolism of 1-O-octadecyl-sn-glycero-3-phospho-[3H]acyclovir (ODG-P-[3H]ACV) in HepG2 cells. Cellular uptake of ODG-P-[3H]ACV was found to be substantially greater than that of [3H]ACV, and cellular levels of ACV-mono-, -di-, and -triphosphate were much higher with ODG-P-ACV. ODG-P-[3H]ACV was well absorbed orally. Based on urinary recovery of tritium after oral or parenteral administration of the radiolabeled compounds, oral absorption of ODG-P-ACV in mice was 100% versus 37% for ACV. ODG-P-ACV plasma area under the curve was more than 7-fold greater than that of ACV. Lipid prodrugs of this type may be useful orally in treating viral diseases.[3]

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In vitro enzyme/receptor binding assays for AZT triphosphate tetraammonium involve measuring its inhibition of HIV-1 reverse transcriptase or HBV DNA polymerase. The assay typically uses purified recombinant reverse transcriptase, a template-primer (e.g., poly(rA)-oligo(dT)), and radiolabeled dTTP. The compound is incubated with the enzyme and substrates at 37degC, and incorporation of labeled nucleotide into the growing DNA chain is quantified by scintillation counting or filter binding. IC₅0 values are calculated from dose-response curves. The compound's ability to inhibit viral replication in cell culture is assessed using HIV-1-infected cell lines.

Gene therapy and stem cell transplantation safety could be enhanced by control over the fate of therapeutic cells. Suicide gene therapy uses enzymes that convert prodrugs to cytotoxic entities; however, heterologous moieties with poor kinetics are employed. We describe a novel enzyme/prodrug combination for selectively inducing apoptosis in lentiviral vector-transduced cells. Rationally designed variants of human thymidylate kinase (tmpk) that effectively phosphorylate 3'-azido-3'-deoxythymidine (AZT) were efficiently delivered. Transduced Jurkat cell lines were eliminated by AZT. We demonstrate that this schema targeted both dividing and non-dividing cells, with a novel killing mechanism involving apoptosis induction via disruption of the mitochondrial inner membrane potential and activation of caspase-3. Primary murine and human T cells were also transduced and responded to AZT. [2]

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Cellular assays for AZT triphosphate tetraammonium are conducted using HIV-1-susceptible cell lines such as MT-2, CEM, or primary CD4+ T cells. Cells are infected with HIV-1 and treated with varying concentrations of the compound for 3-7 days. Viral replication is measured by p24 antigen ELISA, reverse transcriptase activity in culture supernatants, or by using reporter viruses expressing luciferase or GFP. Cytotoxicity is assessed in parallel using MTT or similar assays to ensure selectivity. The compound's effects on mitochondrial function may also be evaluated by measuring mitochondrial membrane potential or oxygen consumption.

In vivo animal studies for AZT triphosphate tetraammonium are not typically conducted because the compound is a metabolite rather than a therapeutic agent itself. Instead, studies focus on AZT (the parent drug) in animal models of retroviral infection. Pharmacokinetic studies in rodents or monkeys evaluate the formation of AZT triphosphate in various tissues following AZT administration. The compound's intracellular phosphorylation and accumulation are assessed using tissue homogenates and LC-MS/MS analysis. Efficacy studies are conducted in murine models of HIV or feline immunodeficiency virus (FIV) infection using AZT treatment.

Pharmacokinetic properties of AZT triphosphate tetraammonium are characteristic of nucleotide analogs. The compound has a molecular weight of 575.30 g/mol and a molecular formula of C10H2₈N₉O13P3. The tetraammonium salt form enhances aqueous solubility. As a triphosphate, the compound is highly charged and membrane-impermeant, requiring intracellular phosphorylation from the parent nucleoside for cellular uptake. Following administration of AZT, the triphosphate metabolite is formed intracellularly and has an intracellular half-life that determines the duration of antiviral activity. The compound is typically stored at -20degC.

Toxicological data for AZT triphosphate tetraammonium are related to the toxicity of AZT and its metabolites. AZT is associated with bone marrow suppression (anemia, neutropenia), mitochondrial toxicity, and hepatotoxicity. The triphosphate metabolite can inhibit mitochondrial DNA polymerase gamma, leading to mitochondrial dysfunction and apoptosis. This mitochondrial toxicity is responsible for some of the adverse effects of AZT, including myopathy and lactic acidosis. In cell-based assays, the compound's cytotoxicity is assessed alongside its antiviral activity to determine the therapeutic index.

[1]. Azidothymidine-triphosphate Impairs Mitochondrial Dynamics by Disrupting the Quality Control System. Redox Biol. 2017 Oct;13:407-417.

[2]. Engineered Human tmpk/AZT as a Novel Enzyme/Prodrug Axis for Suicide Gene Therapy. Mol Ther. 2007 May;15(5):962-70.

[3]. Enhanced Oral Absorption and Antiviral Activity of 1-O-octadecyl-sn-glycero-3-phospho-acyclovir and Related Compounds in Hepatitis B Virus Infection, in Vitro. Biochem Pharmacol. 1997 Jun 15;53(12):1815-22.

See also: Lamivudine; Zidovudine (notes moved to).

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AZT triphosphate tetraammonium is the active triphosphate metabolite of Zidovudine (AZT), a nucleoside reverse transcriptase inhibitor used for the treatment of HIV infection. It exhibits antiretroviral activity by inhibiting HIV-1 reverse transcriptase and also inhibits HBV DNA polymerase. The compound activates mitochondrial-mediated apoptosis pathways. It is used as a biochemical reagent in virology and nucleotide metabolism studies. The compound is not a therapeutic agent itself but is a research tool for studying AZT pharmacology and mitochondrial toxicity. It is available in high purity (≥98%) and is typically stored at -20degC.

C10H28N9O13P3

575.30

524.022

106060-92-8

AZT triphosphate;92586-35-1;AZT triphosphate TEA

72187

Off-white to light yellow solid powder

-3.2

5

15

9

31

973

3

CC1=CN(C(=O)NC1=O)[C@H]2C[C@@H]([C@H](O2)COP(=O)(O)OP(=O)(O)OP(=O)(O)O)N=[N+]=[N-]

GLWHPRRGGYLLRV-XLPZGREQSA-N

InChI=1S/C10H16N5O13P3/c1-5-3-15(10(17)12-9(5)16)8-2-6(13-14-11)7(26-8)4-25-30(21,22)28-31(23,24)27-29(18,19)20/h3,6-8H,2,4H2,1H3,(H,21,22)(H,23,24)(H,12,16,17)(H2,18,19,20)/t6-,7+,8+/m0/s1

[[(2S,3S,5R)-3-azido-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate

AZT triphosphate tetraammonium; 106060-92-8

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : 100 mg/mL (173.82 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (8.69 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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 (Please use freshly prepared in vivo formulations for optimal results.)