Simultaneous determination of Ziagen and its phosphorylated metabolites by ion-pairing high-performance liquid chromatography–tandem mass spectrometry

Fung, Eliza N.; Cai, Zongwei; Burnette, Thimysta C.; Sinhababu, Achintya K.

doi:10.1016/s0378-4347(00)00619-8

Cited by 72 publications

(95 citation statements)

References 29 publications

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“…Thus, the first criterion in selecting an appropriate ion-pairing agent was to ensure that the nucleotides were retained and separated on a reversed-phase HPLC column. According to the literature (2,8,17), the best results for ion pairing are obtained with DMHA. First, the pure compounds were infused into the eluent mixture (ratio of mobile phase A to mobile phase B, 40:60 [vol/vol]; 1 g/ml) at 10 ml/min to determine the optimum ionization mode.…”

Section: Methodsmentioning

confidence: 95%

Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N ⁴ -Hydroxycytidine in Different Liver Cells

Hernandez-Santiago

Beltran

Stuyver

et al. 2004

Antimicrob Agents Chemother

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␤-D-N4 -Hydroxycytidine (NHC) was found to have selective anti-hepatitis C virus (HCV) activity in the HCV replicon system (clone A). The intracellular metabolism of tritiated NHC was investigated in the HCV replicon system, Huh-7 cells, HepG2 cells, and primary human hepatocytes. Incubation of cells with 10 M radiolabeled NHC demonstrated extensive and rapid phosphorylation in all liver cells. Besides the 5-mono, -di-, and -triphosphate metabolites of NHC, other metabolites were characterized. These included cytidine and uridine mono-, di-, and triphosphates. UTP was the predominant early metabolite in Huh-7 cells and primary human hepatocytes, suggesting deamination of NHC as the primary catabolic pathway. The intracellular half-lives of radiolabeled NHC-triphosphate and of CTP and UTP derived from NHC incubation in Huh-7 cells were calculated to be 3.0 ؎ 1.3, 10.4 ؎ 3.3, and 13.2 ؎ 3.5 h (means ؎ standard deviations), respectively. Studies using monkey and human whole blood demonstrated more-rapid deamination and oxidation in monkey cells than in human cells, suggesting that NHC may not persist long enough in plasma to be delivered to liver cells.Hepatitis C virus (HCV) causes liver disease and is spread primarily by contact with the blood of an infected person. Globally, an estimated 170 million persons are chronically infected with HCV (20). An estimated 3.9 million Americans (1.8%) have been infected with HCV, and cirrhosis will eventually develop in at least 15 to 20 percent of them (1, 12). HCV is one of the most important causes of chronic liver disease and seems to progress more rapidly to liver damage in human immunodeficiency virus-infected persons than in noninfected persons (13). Interferon, alone and in combination with ribavirin, is approved for the treatment of persons with chronic hepatitis C. Treatment with interferon for genotype 1-infected individuals is effective in about 15 to 20% of patients (6, 16), but when combined with ribavirin, its effectiveness increases to almost 42%; ribavirin alone, however, is ineffective (6). The combination of ribavirin and interferon is 80% effective against genotypes 2 and 3 (12, 16). Therefore, we need to develop new antiviral agents active against all genotypes of HCV, particularly genotype 1, which is commonly found in the United States and China (11).␤-D-N 4 -Hydroxycytidine (NHC), a base-modified ribonucleoside analogue, was identified as a potent and selective anti-HCV candidate (19). In a bovine viral diarrhea virus infection system and in HCV replicon RNA in Huh-7 cells, NHC had 90% effective concentrations (EC 90 ) of 2 and 5 M, respectively (19). NHC was nontoxic (50% inhibitory concentration, Ͼ100 M) in the HCV replicon system (clone A cells, genotype 1b), Huh-7 cells, HepG2 cells, and human peripheral blood mononuclear cells (19). Levels of mitochondrial DNA and RNA or lactic acid in HepG2 cells treated with NHC did not change, suggesting no delayed toxicity (19). Although the antiviral mechanism of action of NHC is not completely understood, it...

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Section: Methodsmentioning

confidence: 95%

Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N ⁴ -Hydroxycytidine in Different Liver Cells

Hernandez-Santiago

Beltran

Stuyver

et al. 2004

Antimicrob Agents Chemother

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“…As reported previously [5], DMHA is a better ion-pairing agent than tetra-alkyl ammonium salts for high-performance liquid chromatography-electrospray mass spectrometry (HPLC-MS) analysis of nucleosides and nucleotides. So we selected DMHA as an ion-pairing agent to analyze cAMP.…”

Section: Demonstration Of Camp With Hplc-msmentioning

confidence: 68%

Determination of Trace Level of cAMP in Locusta Migratoria Manilensis Meyen by HPLC with Fluorescence Derivation

Zhang

Wang

et al. 2006

IJMS

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A sensitive and rapid method was developed for the determination of cAMP in Locusta migratoria manilensis Meyen by high-performance liquid chromatography with fluorescence detection. The cAMP was derivatized using chloroacetaldehyde and TBAS buffer/methanol was used as the mobile phase. A detection quantification of 40 fmol/ml could be achieved when using fluorescence detection. An HPLC-MS method using DMHA as an ion-pair agent to analyze cAMP was also demonstrated. We studied the effect of dopamine and other stimulants on cAMP levels from isolated locust central nervous systems. The new method is well suited for the analysis of cAMP in small biological samples.

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“…Following centrifugation to clarify the homogenate, aliquots of the supernatants (100 l) were evaporated to dryness in a Savant Speed-Vac Plus (1 h; room temperature). The resulting dried residue was reconstituted with 100 l of mobile phase and then analyzed for nucleotides by a modification (8) of an LC-MS/MS method as described below.…”

Section: Methodsmentioning

confidence: 99%

Antiviral Efficacy upon Administration of a HepDirect Prodrug of 2′- C -Methylcytidine to Hepatitis C Virus-Infected Chimpanzees

Carroll

Koeplinger

Vavrek

et al. 2011

Antimicrob Agents Chemother

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. One approach to increase the antiviral efficacy of 2-C-methylcytidine is to increase the concentration of the active inhibitory species, the 5-triphosphate, in infected hepatocytes. HepDirect prodrug technology can increase intracellular concentrations of a nucleoside triphosphate in hepatocytes by introducing the nucleoside monophosphate into the cell, bypassing the initial kinase step that is often rate limiting. Screening for 2-C-methylcytidine triphosphate levels in rat liver after oral dosing identified 1-[3,5-difluorophenyl]-1,3-propandiol as an efficient prodrug modification. To determine antiviral efficacy in vivo, the prodrug was administered separately via oral and intravenous dosing to two HCV-infected chimpanzees. Circulating viral loads declined by ϳ1.4 log 10 IU/ml and by >3.6 log 10 IU/ml after oral and intravenous dosing, respectively. The viral loads rebounded after the end of dosing to predose levels. The results indicate that a robust antiviral response can be achieved upon administration of the prodrug.Chronic hepatitis C virus (HCV) infection is associated with increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation in the United States (13). The current standard for treatment is a combination of pegylated interferon alpha and ribavirin, which results in sustained viral response (SVR) (undetectable virus 6 months after the end of treatment) in 50% or 80% of treated patients harboring a genotype 1 or a genotype 2 or 3 infection, respectively (25). Thus, novel therapies resulting in higher SVR rates, particularly in the treatment of genotype 1 infections, are needed.Nucleoside analogs inhibiting the HCV RNA polymerase have been investigated as potential therapeutics to treat HCV infections. 2Ј-C-Methylcytidine inhibits HCV RNA replication in the replicon assay (consensus 1 50% effective concentration [EC 50 ], ϳ1 M) via inhibition of the HCV RNA polymerase, which is inhibited by the 2Ј-C-methylcytidine triphosphate in vitro in cell-free biochemical assays (50% inhibitory concentration [IC 50 ], ϳ0.2 M) (15). NM283, the 3Ј-O-valinyl ester prodrug of 2Ј-C-methylcytidine, has improved oral bioavailability compared to 2Ј-C-methylcytidine (21) and has been investigated in HCV-infected patients in clinical studies. In a phase IIb study, patients receiving NM283 in combination with pegylated interferon alpha and ribavirin at 24 weeks of therapy experienced mean viral load reductions of up to 3.3 log 10 IU/ml compared to 2.3 log 10 IU/ml reductions for patients receiving pegylated interferon-ribavirin alone (2). Due to the high incidence of gastrointestinal side effects, which required reduction of the highest daily dose of 800 mg to 200 or 400 mg, further development of NM283 was placed on hold.The HepDirect prodrug modifications of nucleoside 5Ј-monophosphates (NMP) are substituted cyclic 1,3-propanyl esters that offer an opportunity to bypass the initial phosphorylation step that is often rate limiting for the formation of...

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Simultaneous determination of Ziagen and its phosphorylated metabolites by ion-pairing high-performance liquid chromatography–tandem mass spectrometry

Cited by 72 publications

References 29 publications

Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N ⁴ -Hydroxycytidine in Different Liver Cells

Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N ⁴ -Hydroxycytidine in Different Liver Cells

Determination of Trace Level of cAMP in Locusta Migratoria Manilensis Meyen by HPLC with Fluorescence Derivation

Antiviral Efficacy upon Administration of a HepDirect Prodrug of 2′- C -Methylcytidine to Hepatitis C Virus-Infected Chimpanzees

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Simultaneous determination of Ziagen and its phosphorylated metabolites by ion-pairing high-performance liquid chromatography–tandem mass spectrometry

Cited by 72 publications

References 29 publications

Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N 4 -Hydroxycytidine in Different Liver Cells

Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N 4 -Hydroxycytidine in Different Liver Cells

Determination of Trace Level of cAMP in Locusta Migratoria Manilensis Meyen by HPLC with Fluorescence Derivation

Antiviral Efficacy upon Administration of a HepDirect Prodrug of 2′- C -Methylcytidine to Hepatitis C Virus-Infected Chimpanzees

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Product

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Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N ⁴ -Hydroxycytidine in Different Liver Cells

Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N ⁴ -Hydroxycytidine in Different Liver Cells