Simultaneous determination of cytosine arabinoside and its metabolite uracil arabinoside in human plasma by LC-MS/MS: Application to pharmacokinetics-pharmacogenetics pilot study in AML patients.

Purine analogs like aracytine (AraC) are a mainstay for treating acute myeloid leukemia (AML). There are marked differences in drug dosing and scheduling depending on the protocols when treating AML patients with AraC. Large inter-patient pharmacokinetics variability has been reported, and genetic polymorphisms affecting cytidine deaminase (CDA), the liver enzyme responsible for the conversion of Ara-C to inactive uracil arabinoside (AraU) could be a culprit for either life-threatening toxicities or poor efficacy related to substantial changes in plasma exposure levels among patients. The quantitative determination of Ara-C in plasma is challenging due the required sensitivity because of the short half-life of this drug (i.e., <10 min) and the metabolic instability in biological matrix upon sampling possibly resulting in erratic values. We developed and validated a liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) for the simultaneous determination of Ara-C and Ara-U metabolite in human plasma. After simple and rapid precipitation, analytes were successfully separated and quantitated over a 1-500 ng/ml range for Ara-C and 250-7500 ng/ml range for AraU. The performance and reliability of this method was tested as part of an investigational study in AML patients treated with low dose cytarabine and confirmed marked differences in drug exposure levels and metabolic ratio, depending on the CDA status of the patients. Overall, this new method meets the requirements of current bioanalytical guidelines and could be used to monitor drug levels in AML patients with respect to their CDA phenotypes.

Integrating Dynamic Positron Emission Tomography and Conventional Pharmacokinetic Studies to Delineate Plasma and Tumor Pharmacokinetics of FAU, a Prodrug Bioactivated by Thymidylate Synthase.

FAU, a pyrimidine nucleotide analogue, is a prodrug bioactivated by intracellular thymidylate synthase to form FMAU, which is incorporated into DNA, causing cell death. This study presents a model-based approach to integrating dynamic positron emission tomography (PET) and conventional plasma pharmacokinetic studies to characterize the plasma and tissue pharmacokinetics of FAU and FMAU. Twelve cancer patients were enrolled into a phase 1 study, where conventional plasma pharmacokinetic evaluation of therapeutic FAU (50-1600 mg/m2 ) and dynamic PET assessment of 18 F-FAU were performed. A parent-metabolite population pharmacokinetic model was developed to simultaneously fit PET-derived tissue data and conventional plasma pharmacokinetic data. The developed model enabled separation of PET-derived total tissue concentrations into the parent drug and metabolite components. The model provides quantitative, mechanistic insights into the bioactivation of FAU and retention of FMAU in normal and tumor tissues and has potential utility to predict tumor responsiveness to FAU treatment.

HPLC-MS/MS method for the simultaneous determination of MB07133 and its metabolites, cytarabine and arabinofuranosyluracil, in rat plasma.

MB07133 is an intravenously administered cytarabine mononucleotide (araCMP) prodrug, for the treatment of hepatocellular carcinoma (HCC). A simple, selective and sensitive HPLC-MS/MS method using high pressure liquid chromatography (HPLC) coupled to triple-quadrupole mass spectrometer, was developed and validated for the detection of prodrug MB07133 and its metabolites, cytarabine (araC) and arabinofuranosyluracil (araU) in rat plasma. Protein precipitation using 3% trichloroacetic acid (TCA) was employed to extract analytes from 100µL rat plasma. Adequate separation of araC and araU from their endogenous compounds was achieved on the Synergi(®) fusion-RP column (150mm×4.6mm, 4µm) by a gradient-elution with a mobile phase consisting of ammonium formate (1mM) and methanol at a flow rate of 1mL/min. Multiple reaction monitoring mode (MRM) was applied in the detection of MB07133, araC, araU and Ganciclovir (internal standard) with ion pairs 441.2/330.2, 244.2/112.2, 245.2/113.2 and 256.1/152.2, respectively. The assays were validated with respect to specificity, linearity (100-50000ng/mL for MB07133, 2-1000ng/mL for araC and araU), accuracy and precision, extraction recovery, matrix effect and stability. The validated method has been successfully applied to an intravenous bolus pharmacokinetic study of MB07133 in male Sprague-Dawley rats (18mg/kg i.v.).

Simultaneous determination of 1-ß-d-Arabinofuranosylcytosine and two metabolites, 1-ß-d-Arabinofuranosyluracil and 1-ß-d-Arabinofuranosylcytosine triphosphate in leukemic cell by HPLC-MS/MS and the application to cell pharmacokinetics.

A specific and reliable HPLC-MS/MS method was developed and validated for the simultaneous determination of 1-ß-d-Arabinofuranosylcytosine (ara-C), 1-ß-d-Arabinofuranosyluracil (ara-U) and 1-ß-d-Arabinofuranosylcytosine triphosphate (ara-CTP) in the leukemic cells for the first time. The analytes were separated on a C18 column (100mm×2.1mm, 1.8µm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The ion-pairing reagent, NFPA, was added to the mobile phase to retain the analytes in the column. The cell homogenates sample was prepared by the simple protein precipitation. The calibration curves were linear over a concentration range of 3.45-3450.0ng/mL for ara-C, 1.12-1120.0ng/mL for ara-U and 4.13-4130.0ng/mL for ara-CTP. The intra-day and inter-day precision was less than 15% and the relative error (RE) were all within ±15%. The validated method was successfully applied to assess the disposition characteristics of ara-C and support cell pharmacokinetics after the patients with leukemia were intravenously infused with SDAC and HiDAC. The result of the present study would provide the valuable information for the ara-C therapy.

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for determination of 1-ß-d-arabinofuranosyluracil in human plasma and application to therapeutic drug monitoring in patient with leukemia.

A specific and reliable HPLC-MS/MS method was developed and validated for the determination of ara-U in human plasma. The analyte was separated on a C18 column (50 mm × 2.1mm, 1.7 µm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. The plasma sample was prepared by a simple protein precipitation pretreatment and the recovery was about 80%. The calibration curves were linear over a concentration range of 1.0-7000.0 ng/mL for ara-U. The intra-day and inter-day precision was less than 15% and the relative error (RE) was all within ± 15%. It was successfully applied to assess the disposition characteristics of ara-U and support the therapeutic drug monitoring after the patients with leukemia were infused with ara-C.

Enhancing the efficacy of Ara-C through conjugation with PAMAM dendrimer and linear PEG: a comparative study.

1ß-d-Arabinofuranosylcytosine (Cytarabine, Ara-C) is a key drug in the treatment of acute myeloid leukemia. Ara-C has a number of limitations such as a rapid deactivation by cytidine deaminase leading to the formation of a biologically inactive metabolite, Ara-U (1ß-d-arabinofuranosyluracil), a low lipophilicity, and fast clearance from the body. To address these problems, we developed a conjugate in which hydroxyl-terminated PAMAM dendrimer, G4-OH ["D"] and PEG were used as carriers for the drug (Ara-C). The conjugates were synthesized using an efficient multistep protection/deprotection method resulting in the formation of a covalent bond between the primary hydroxyl group of Ara-C and dendrimer/PEG. The structure, physicochemical properties, and drug release kinetics were characterized extensively. (1)H NMR and MALDI-TOF mass spectrometry suggested covalent attachment of 10 Ara-C molecules to the dendrimer. The release profile of Ara-C in human plasma and in PBS buffer (pH 7.4) showed that the conjugates released the drug over 14 days in PBS, with the release sped up in plasma. In PBS, while most of the drug is released from PEG-Ara-C, the dendrimer continues to release the drug in a sustained fashion. The results also suggested that the formation of the inactive form of Ara-C (Ara-U) was delayed upon conjugation of Ara-C to the polymers. The inhibition of cancer growth by the dendrimer-Ara-C and PEG-Ara-C conjugates was evaluated in A549 human adenocarcinoma epithelial cells. Both dendrimer- and PEG-Ara-C conjugates were 4-fold more effective in inhibition of A549 cells compared to free Ara-C after 72 h of treatment.

Simultaneous determination of cytosine arabinoside and its metabolite, uracil arabinoside, in human plasma using hydrophilic interaction liquid chromatography with UV detection.

A practical high-performance liquid chromatography using a Cosmosil HILIC column and UV detection was developed for determining the concentrations of cytosine arabinoside (Ara-C) and uracil arabinoside (Ara-U), which is a major metabolite of Ara-C, in human plasma. This method was used to determine the plasma concentrations of Ara-C and Ara-U in a patient treated with high-dose Ara-C therapy for end-stage renal failure.

Simultaneous determination of 1-(2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl) uracil (FAU) and 1-(2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl) 5-methyluracil (FMAU) in human plasma by liquid chromatography/tandem mass spectrometry.

A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of 1-(2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl) uracil (FAU) and its active metabolite 1-(2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl) 5-methyluracil (FMAU) in human plasma. FAU and FMAU were extracted from plasma samples using solid-phase extraction with Waters Sep-Pak® Vac C18 cartridge. Chromatographic separation was achieved on a Waters Atlantis T3 C18 column with a gradient mobile phase consisting of methanol and water with 0.45% formic acid (v/v) running at a flow rate of 0.2 ml/min. The analytes were monitored by triple quadrupole mass spectrometer under positive ionization mode. The lower limit of quantitation (LLOQ) was 10 and 2 ng/ml for FAU and FMAU in plasma, respectively. Calibration curves were linear over FAU and FMAU plasma concentration range of 10-2000 and 2-1000 ng/ml, respectively. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the plasma pharmacokinetics of FAU and FMAU in cancer patients receiving 1-h intravenous infusion of FAU 50 mg/m².

Normal-phase liquid chromatography coupled with electrospray ionization mass spectrometry for chiral separation and quantification of clevudine and its enantiomer in human plasma.

A new, simple and enantioselective normal-phase liquid chromatography-mass spectrometry method was presented for the quantification of clevudine and its enantiomer in human plasma. A C18 cartridge was used in this method to extract the enantiomers in 200µL plasma followed by a chiral separation on a cellulose-based LC column with mobile phase consisted of hexane, methanol and ethanol (62:28:10, V/V/V). The eluate was directed to a mass spectrometry through an electrospray ionization interface. A transition of m/z 261.0 to m/z 126.8 was used for monitoring of clevudine and its enantiomer. This method showed good linearity (R>0.997), precision (<9.6%) and accuracy (within 95.48-105.9%) within a range of 10-1000ng/mL for the enantiomers and has been applied to the pharmacokinetics study of clevudine capsules in human plasma.

Effective clearance of Ara-U the major metabolite of cytosine arabinoside (Ara-C) by hemodialysis in a patient with lymphoma and end-stage renal failure.

PURPOSE: We report that hemodialysis clears Ara-U from the blood after high-dose Ara-C treatment in a patient with lymphoma and end-stage renal failure. METHODS: The patient received two doses of Ara-C 1 g/m(2) 24 h apart and was hemodialyzed at about 6 h after each dose and subsequently as per her usual dialysis schedule. Multiple blood samples were collected after dosing. Blood and dialyzate were also collected from the dialysis circuit during a second identical treatment cycle. Ara-C and its metabolite Ara-U in plasma and dialyzate were measured chromatographically, and the data subjected to pharmacokinetic analysis. RESULTS: The distribution and elimination half-lives, steady-state volume of distribution and clearance values were 0.5 h, 7 h, 181 L and 307 l/h for Ara-C and 4.1 h, 34 h, 118 L and 2.64 l/h for Ara-U, respectively. The dialysis sessions immediately after the first and second doses cleared 39 and 52% (as Ara-U) of the respective Ara-C doses. Some 63% of Ara-U in plasma was extracted by dialysis. The patient showed no signs of neurotoxicity or other drug-related adverse effects. CONCLUSION: Hemodialysis is very effective in clearing Ara-U from the plasma in renal failure, and this maneuver could easily be used routinely to prevent Ara-U accumulation and minimize adverse effects in patients with renal failure.

5-[18F]Fluoroalkyl pyrimidine nucleosides: probes for positron emission tomography imaging of herpes simplex virus type 1 thymidine kinase gene expression.

INTRODUCTION: The preliminary in vivo evaluation of novel 5-[(18)F]fluoroalkyl-2'-deoxyuridines ([(18)F]FPrDU, [(18)F]FBuDU, [(18)F]FPeDU; [(18)F]1a-c, respectively) and 2'-fluoro-2'-deoxy-5-[(18)F]fluoroalkyl-1-beta-d-arabinofuranosyl uracils ([(18)F]FFPrAU, [(18)F]FFBuAU, [(18)F]FFPeAU; [(18)F]1d-f, respectively) as probes for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression is described. METHODS: [(18)F]1a-f were successfully synthesized by a rapid and efficient two-step one-pot nucleophilic fluorination reaction using 5-O-mesylate precursors and [(18)F]F(-). For in vivo studies, tumor xenografts were grown in nude mice by implanting RG2 cells stably expressing HSV1-tk (RG2TK+) and wild-type cells (RG2). RESULTS: Biodistribution studies at 2 h pi revealed that the uptake of [(18)F]1a-b and [(18)F]1d-e in RG2TK+ tumors was not significantly different from control tumors. However, [(18)F]1c and [(18)F]1f had an average 1.6- and 1.7-fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [(18)F]1c and [(18)F]1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [(18)F]1c and [(18)F]1f showed higher initial uptake (3.5- and 1.4-fold, respectively) in RG2TK+ tumors than in control tumors, with continued washout of activity from both tumors over time. CONCLUSIONS: Biological evaluations suggest that [(18)F]1c and [(18)F]1f may have limited potential for imaging HSV1-tk gene expression due to fast washout of activity from the blood, thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1-tk-expressing tumors.

Clevudine: a potent inhibitor of hepatitis B virus in vitro and in vivo.

Clevudine (CLV) is a nucleoside analog of the unnatural L-configuration that has potent anti-hepatitis B virus (HBV) activity in vitro and in vivo with a favorable toxicity profile in all species tested. In cell culture, CLV is readily phosphorylated to the corresponding 5'-triphosphate form of the compound. The mechanism of action of CLV involves the inhibition of the HBV polymerase by CLV 5'-triphosphate. In vivo efficacy studies performed in the duck and woodchuck models showed marked, rapid inhibition of virus replication and no significant toxicity. In the woodchuck model, there was a dose-dependent delay in viral recrudescence and a reduction or loss of covalently closed circular DNA. In Phase II clinical studies, CLV was well tolerated and exhibited potent antiviral activity at all doses investigated. In Phase III studies in both hepatitis B e antigen (HBeAg)-positive and -negative patients, CLV 30 mg administered once daily demonstrated potent antiviral efficacy and significant biochemical improvement after only 24 weeks of therapy. These effects were sustained in a significant portion of the patients when therapy was stopped after 6 months with no viral rebound occurring in approximately 3 and 16% in HBeAg-positive and -negative patients, respectively. There have been no significant safety or tolerance issues associated with the drug in these studies. Future studies will investigate the safety and tolerance of CLV 30 mg given once daily over 48 weeks and longer.

Pharmacokinetics of the thymidine analog 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FMAU) in tumor-bearing rats.

The thymidine analog 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FMAU) is incorporated into DNA and is resistant to catabolism. We performed pharmacokinetic measurements with [(14)C]FMAU and PET studies with [(11)C]FMAU using rats bearing several different syngeneic tumors. Among normal tissues, FMAU uptake reflected relative cell turnover rates. Among tumors, the highest uptake occurred in a rapidly growing colon carcinoma, but was similarly low in both rapidly and slowly growing prostate tumors. FMAU was not catabolized and was rapidly incorporated into DNA by small intestine and colon tumors. Results indicate that FMAU may be useful for imaging tissue DNA synthesis, although tumor uptake was modest and not well correlated with growth rate among the models examined.

Imaging [18F]FAU [1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl) uracil] in dogs.

We have studied the biodistribution of [(18)F]FAU [(1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)uracil], which previous work has shown is incorporated into DNA and functions as an inhibitor of DNA synthesis. It is being tested as a potential antineoplastic agent and imaging agent for PET. We have produced [(18)F]FAU and injected the tracer into 3 normal dogs and imaged them for up to 4 hours and removed tissues along with blood and urine samples for HPLC and activity analysis. The results showed that [(18)F]FAU evenly distributed to most of organs. In sharp contrast to our prior experience with thymidine and its analogs, marrow had less retention of [(18)F]FAU than the non-proliferating tissues.

Micellar electrokinetic capillary chromatography quantification of cytosine arabinoside and its metabolite, uracil arabinoside, in human serum.

Cytosine arabinoside (Ara-C) is widely used to induce remission in adult granulocytic leukemia. High doses can be infused in refractory leukemia or in relapse. After injection, Ara-C is quickly metabolized to uracil arabinoside (Ara-U), the main inactive metabolite. We here described a micellar electrokinetic capillary chromatography (MECC) method to simultaneously determine Ara-C/Ara-U in human serum using 6-O-methylguanine as an internal standard. The assay was linear from 6.25 to 200 microg/ml with a quantification limit between 3 and 6 microg/ml. The analytical precision was satisfactory between 2 and 4.3% (within-run) and 3.7 and 7.3% (between-runs). This assay was applied to the analysis of serum from acute granulocytic leukemia patient treated by high doses cytarabine (3 g/m2 body surface).

19F NMR study of the uptake of 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil in erythrocytes: evidence of transport by facilitated and nonfacilitated pathways.

The 19F NMR resonances of intra- and extracellular 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU) in suspensions of human erythrocytes are well resolved. This phenomenon allows its transport behavior to be monitored in a 19F NMR time-course experiment. The rate of L-FMAU uptake at 25 degrees in a suspension containing L-FMAU at an initial extracellular concentration of 4 mM was 7.6 +/- 1.0 x 10(-7) pmol cell(-1) sec(-1) (N = 5). Concentration-dependent uptake studies of L-FMAU indicate the existence of both saturable and nonsaturable transport mechanisms, with a Km for the saturable uptake of approximately 1 mM. Although the transport of L-FMAU at 25 degrees was inhibited significantly (54-65%) by nitrobenzylthioinosine (NBTI) and dipyridamole, consistent with the participation of the nucleoside transporter, these inhibitors did not achieve complete blockage of L-FMAU uptake. The participation of the nucleobase transporter in L-FMAU uptake was ruled out by the absence of competition with uracil uptake, and by the lack of inhibition by papaverine. In addition, the NBTI-insensitive uptake of L-FMAU was not affected by pretreatment of the cells with the sulfhydryl reagent, p-chloromercuriphenylsulfonic acid (pCMBS). However, the NBTI- and dipyridamole-insensitive transport of L-FMAU was found to increase upon treatment of the erythrocytes with butanol, an agent that affects membrane fluidity. The partition coefficient of L-FMAU in octanol/phosphate-buffered saline determined by absorption spectrophotometry was 0.31. These data indicate that under the conditions of the studies, L-FMAU uptake by erythrocytes proceeds by both the nucleoside transporter and nonfacilitated membrane diffusion.

The effect of sorivudine on dihydropyrimidine dehydrogenase activity in patients with acute herpes zoster.

OBJECTIVE: Bromovinyl-uracil (BVU) is the principal metabolite of sorivudine, a potent anti-zoster nucleoside. BVU binds to, and irreversibly inhibits, the enzyme dihydropyrimidine dehydrogenase (DPD). The objective of this study was to assess the time course of recovery of DPD activity after oral administration of sorivudine in patients with herpes zoster and to correlate restoration of DPD activity and levels of uracil with the elimination of sorivudine and its metabolite BVU from the circulation. METHODS: Sorivudine was given orally as 40 mg once-daily doses for 10 consecutive days to a total of 19 patients with herpes zoster. Serum sorivudine, BVU, and circulating uracil and DPD activity in peripheral blood mononuclear cells (PBMCs) were determined before, during, and after administration of sorivudine. RESULTS: BVU was eliminated from the circulation within 7 days after the last sorivudine dose. DPD activity in PBMCs, which was completely suppressed in 18 of the 19 subjects and markedly suppressed in the remaining subject during administration of sorivudine, recovered to baseline levels within 19 days after the last dose of sorivudine in all subjects and within 14 days in all but one of the subjects. The restoration of DPD activity was temporally associated with elimination of BVU from the circulation. The elevated uracil concentrations produced by inhibition of DPD activity fell rapidly after cessation of sorivudine administration and also were temporally associated with elimination of BVU from the circulation. The time course of recovery of DPD activity in three patients with renal impairment was similar to that of the other subjects. CONCLUSIONS: This study indicates that sorivudine therapy is associated with a profound depression of DPD activity. Recovery of DPD activity occurred within 4 weeks of the completion of sorivudine therapy, which indicates that fluorinated pyrimidines may be safely administered 4 weeks after completion of sorivudine therapy.

Optimizing therapy for acute myeloid leukemia based on differences in intracellular metabolism of cytosine arabinoside between leukemic blasts and normal mononuclear blood cells.

The increasing insights into the pharmacokinetics and the metabolism of arabinoside C (AraC) have improved the rationale for its application in leukemia therapy and have led to a pharmacologically directed design of antileukemic treatment. The current study aims at adding to this approach by detecting differences in the intracellular metabolism of AraC 5'-triphosphate (AraCTP) between leukemic and normal mononuclear blood cells. Measurements of intracellular AraCTP levels were complemented by determinations of plasma AraC and arabinoside uridine (AraU) concentrations and were performed in 26 patients with acute myeloid leukemia (AML) who were undergoing combination therapy, including high-dose (1.0 or 3.0 g/m2 x 2/day) AraC. Plasma AraC concentrations showed a linear relationship to the applied AraC dose but did not correlate with intracellular AraCTP levels. Substantial differences in AraCTP retention times were revealed, during 3-h infusions of either 1.0 or 3.0 g/m2 AraC in leukemic blasts from 10 patients with t1/2 values of 1.60-7.63 h (median, 2.42 h). In addition, AraCTP levels declined in only one patient by > 10% within the first hour after the end of therapy and remained constant or even increased up to 1.5-fold during a posttreatment period of 1-2.5 h in the other nine cases. In contrast, AraCTP retention times were relatively uniform in normal mononuclear blood cells from 11 patients, with t1/2 values of 3.34-5.29 h (median, 3.85 h). More importantly, AraCTP levels dropped by > 10% within the first hour after the end of the high-dose AraC infusion in eight of 11 cases. A posttherapeutic increase of > 10% was not observed in any patient. These differences in AraCTP pharmacokinetics between leukemic and normal blood cells provided the basis for a modified timing of AraC administration with the aim of selectively maintaining cytotoxic AraCTP levels in leukemic blasts while allowing an intermittent drop of AraCTP levels in normal cells. This modification may result in higher antileukemic activity without increasing the damaging effect on normal cells and may, thus, improve the therapeutic index for AraC.