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.).

Focused ultrasound enhanced molecular imaging and gene therapy for multifusion reporter gene in glioma-bearing rat model.

The ability to monitor the responses of and inhibit the growth of brain tumors during gene therapy has been severely limited due to the blood-brain barrier (BBB). A previous study has demonstrated the feasibility of noninvasive in vivo imaging with 123I-2'-fluoro-2'-deoxy-5-iodo-1-ß-D-arabinofuranosyluracil (123I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here, we tested the enhancement of SPECT with 123I-FIAU and ganciclovir (GCV) treatment in brain tumors after BBB disruption induced by focused ultrasound (FUS) in the presence of microbubbles. We established an orthotopic F98 glioma-bearing rat model with trifusion reporter genes. The results of this study showed that the rat model of HSV1-tk-expressing glioma cells could be successfully detected by SPECT imaging after FUS-induced BBB disruption on day 10 after implantation. Compared to the control group, animals receiving the GCV with or without sonication exhibited a significant antitumor activity (P < 0.05) of glioma cells on day 16 after implantation. Moreover, combining sonication with GCV significantly inhibited tumor growth compared with GCV alone. This study demonstrated that FUS may be used to deliver a wide variety of theranostic agents to the brain for molecular imaging and gene therapy in brain diseases.

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.

Comparison of [14C]FMAU, [3H]FEAU, [14C]FIAU, and [3H]PCV for monitoring reporter gene expression of wild type and mutant herpes simplex virus type 1 thymidine kinase in cell culture.

PURPOSE: To assess the optimal reporter probe/reporter gene combination for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression, we compared the cellular uptake of 1-(2'-fluoro-2'-deoxy-D-arabinofuranosyl)-5-methyluracil (FMAU), 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), 2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl-5-iodouracil (FIAU) and penciclovir (PCV) in both HSV1-tk and HSV1-sr39tk expressing cells. PROCEDURES: For stably transfected cell studies, C6 rat glioma cells, C6 HSV1-tk transfectant, C6 mutant HSV1-sr39tk transfectant, rat Morris hepatoma cells (MH3924A), and MH3924A HSV1-tk transfectant cells were used. For adenoviral infection studies, C6 rat glioma cells were exposed to serial titers of AdCMV-HSV1-tk, AdCMV-HSV1-sr39tk, or AdCMV-fluc for 24 hours. These cells were incubated with [(14)C]FMAU, [(3)H]FEAU, [(14)C]FIAU, and [(3)H]PCV, and cellular uptake of radioactivity was measured. RESULTS: [(3)H]FEAU exhibited the highest or second highest accumulation and the most selectivity regardless of the mode of gene transfer for both HSV1-tk and mutant HSV1-sr39tk reporter genes. CONCLUSION: This combination of high accumulation and high selectivity for both HSV1-tk and HSV1-sr39tk makes suitably radiolabeled FEAU a promising candidate as a radiotracer for imaging HSV1-tk/HSV1-sr39tk gene expression in living subjects.

Pharmacokinetics of netivudine, a potent anti-varicella zoster virus drug, in patients with renal impairment.

The pharmacokinetics of a single oral 200 mg dose of netivudine (1-(beta-D-arabinofuranosyl)-5-(1-propynyl)uracil), a nucleoside analogue under development for use in varicella zoster virus infections, were studied in 12 renal failure (RF) subjects (creatinine clearance 15 +/- 7 mL/min) and 12 age-matched healthy subjects with normal creatinine clearance. Blood and urine samples were collected up to nine days after drug administration. Concentrations of netivudine and of its main metabolite, the pyrimidine base 5-(1-propynyl)uracil (5 PU), were determined by a specific high performance liquid chromatography assay. The mean peak plasma concentrations of netivudine, Tmax, and volume of distribution were not significantly affected by RF. The elimination half-life of netivudine was approximately 15 h in subjects with normal renal function and 60 h in RF patients. Plasma and renal clearances of netivudine were significantly reduced in RF patients and AUC was three to four times higher in these patients. Cmax and AUC of 5 PU were higher in RF patients, and the half-life was also significantly longer. However, the half-life of this metabolite was much lower than that of the parent compound. Tmax and the lag time were similar in the two groups. There were highly significant correlations for netivudine and 5 PU between half-life and creatinine clearance and between renal clearance and creatinine clearance. These findings suggest that netivudine dosage may need to be reduced in patients with severe renal failure, and confirm that formation of the 5 PU is independent of the elimination of netivudine from plasma.

Sensitive and specific radioimmunoassay for fialuridine: initial assessment of pharmacokinetics after single oral doses to healthy volunteers.

Fialuridine (FIAU) is a halogen-substituted analog of thymidine that was undergoing clinical investigation as a drug for the treatment of chronic hepatitis B viral infection. However, clinical trials of FIAU were terminated after adverse events occurred following chronic oral administration. Prior to the termination of clinical trials, a sensitive assay was needed for the measurement of FIAU because of the anticipated low dose administered to patients. We therefore undertook the development of a radioimmunoassay (RIA). A specific antiserum was raised in rabbits following immunization with a 5'-O-hemisuccinate analog of FIAU coupled to keyhole limpet hemocyanin. Radiolabeled FIAU was synthesized by a destannylation procedure by using sodium [125I]iodide. We developed a competitive-binding procedure and used precipitation with polyethylene glycol as the method for separating the bound and free forms of FIAU. The RIA is sensitive (0.2 ng/ml), specific (negligible interference from known metabolites and endogenous nucleosides), and reproducible (interassay coefficients of variation range from 5 to 19.7% for serum controls). We used the RIA to assess the pharmacokinetics of FIAU in healthy adult volunteers following administration of a single 5-mg oral dose. The sensitivity of the RIA permitted the detection of a prolonged elimination phase for FIAU in healthy volunteers and dogs, with mean elimination half-lives of 29.3 and 35.3 h, respectively. We conclude the RIA is a valid method for the quantification of FIAU in biological fluids.

A radioimmunoassay for the new antiviral agent 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil.

A specific and sensitive radioimmunoassay (RIA) for the measurement of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) in biological fluids has been developed. The assay has a range of 2.5-1,000 ng/ml and 10-1,000 ng/ml for serum and urine, respectively, and has the sensitivity to detect 2.5 and 25 ng/ml of BV-araU in serum and urine, respectively. A satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free ligand was achieved by employing polyethylene glycol-goat anti-rabbit gamma globulin separant. A quantitative recovery of the exogenous analyte was obtained at all concentrations of BV-araU tested. The assay is specific for the parent drug and is not affected by the presence of its metabolite, BV-U (bromovinyl uracil) or serum components (nucleotides, nucleosides, or sugars). Intraassay coefficients of variation were 3.1-4.4% and 2.5-4.2% for serum and urine controls, respectively. Interassay variability was < 8.6% for all serum and urine controls. Linear regression analysis showed that the correlation between RIA and high-pressure liquid chromatography was excellent (r = 0.997). The ascending dosage studies have been analyzed by the BV-araU RIA, and results indicate that the values of area under the serum concentration-time curve increased proportionally with the administered dose of BV-araU up to 80 mg. Cumulative urinary excretion data showed that approximately 50% of unchanged BV-araU was excreted in the urine within 24 h.

Determination of 3H-labelled cytosine arabinoside and eight metabolites in cell extracts by high-performance liquid chromatography and scintillation spectrometry.

We have developed a method for the separation and quantitation of radiolabeled cytosine arabinoside and its eight metabolites in cell extracts by anion-exchange gradient high-performance liquid chromatography. Baseline separation of cytosine arabinoside and uracil arabinoside and their respective 5'-mono-, di- and triphosphates, as well as cytosine arabinoside diphosphocholine was obtained with the shortest interval between peaks being 3 min. This degree of separation was found to be essential for quantitation of 3H-labeled metabolites by scintillation counting of 1-min fractions. Application of this procedure to the quantitation of [3H] cytosine arabinoside and its metabolites from HL60 human leukemia cells is demonstrated.

Quantitative determination of antiviral nucleoside analog in DNA.

A technique for the analysis of the amount of an antiviral nucleoside analog incorporated into DNA, utilizing enzymatic digestion of DNA, followed by high-performance liquid chromatography is described. The cells or tissue samples were treated with perchloric acid to inactivate the nucleases, then digested with pronase in the presence of EDTA. DNA was purified by CsCl centrifugation followed by Sephadex chromatography and treatment with deoxyribonuclease 1 and venom phosphodiesterase. The deoxyribonucleoside monophosphates and the monophosphate of the nucleoside analog liberated from DNA were separated and quantitated by HPLC analysis and measurement of radioactivity. This assay is more sensitive, specific, and precise than the determination of DNA density shift. It is also applicable for nucleoside analogs which do not change the density of DNA either because of their structure or their very small degree of incorporation.

Sensitive radioimmunoassay for cytarabine and uracil arabinoside in plasma.

A sensitive and specific radioimmunoassay for cytarabine (ara-C) or uracil arabinoside (ara-U) was established by using [3H]ara-C and anti-ara-C antiserum or [3H]ara-U and anti-ara-U antiserum. The antiserum was prepared by immunizing rabbits with ara-C or ara-U hapten conjugated to human albumin. In the present assay, as low as 2.5 ng/ml (0.01 microM) of ara-C or 5 ng/ml (0.02 microM) of ara-U in blood plasma samples could be detected. The cross-reaction of ara-C or ara-U structurally related compounds with each antiserum was so small that the nucleosides could be determined without any purification procedure. The plasma concentration of ara-C and ara-U in mice following oral administration of an antileukemic agent, cytosine arabinoside-5'-stearylphosphate (C18PCA) (100 mg/kg), or ara-C (48 mg/kg) was determined by using this method. In the case of C18PCA administration, ara-C was detectable in blood for at least 24 hours longer than in the case of ara-C administration. This method is suitable for the analysis of ara-C and ara-U, the metabolites of depot drugs of ara-C such as C18PCA.

High-performance liquid chromatographic assay for cytosine arabinoside, uracil arabinoside and some related nucleosides.

A novel, dual-column high-performance liquid chromatographic method for determination of the anti-cancer drug cytosine arabinoside (Ara-C) and its major metabolite uracil arabinoside (Ara-U) has been developed. The analytical procedure is sensitive (25 ng/ml) and specific for Ara-C, Ara-U and the endogenous nucleosides that may influence response to Ara-C therapy, cytidine and deoxycytidine. Conventional and high dose calibration curves were linear and the method precise with the assay coefficient of variation for Ara-C and Ara-U not greater than 9.1% over the range of 0.1-10 micrograms/ml. Accuracy was determined to be within +/- 3 to 9% over this concentration range. Using this method, patient plasma samples from both conventional dose (100-200 mg/m2 per day) and high dose (3500-6500 mg/m2 per day) Ara-C can be simultaneously analyzed for Ara-C, Ara-U and nucleosides so that comparative pharmacokinetic and pharmacodynamic studies can be conducted.

A radioimmunoassay for 1-beta-D-arabinofuranosyluracil with reference to cross-reactivity of 1-beta-D-arabinofuranosylcytosine with an antibody.

Antibodies directed against 1-beta-D-arabinofuranosyluracil have been produced in rabbits by immunization with a conjugate of 1-(5-O-succinyl-beta-D-arabinofuranosyl)uracil with human serum albumin. Two of four antibodies so obtained showed high specificity for 1-beta-D-arabinofuranosyluracil and allowed the development of a sensitive and reliable radioimmunoassay for this substrate. On the other hand, one antibody had a high affinity for 1-beta-D-arabinofuranosylcytosine. The binding of 1-beta-D-arabinofuranosylcytosine to this antibody was practically constant between pH 5.2 and 9.0, whereas 1-beta-D-arabinofuranosyluracil binding was affected drastically by pH. The pH-binding profile for 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosyluracil was reminiscent of the specificity of ara-C-specific antibodies, which we previously obtained after immunization of rabbits with 1-(5-O-succinyl-beta-D-arabinofuranosyl)cytosine as a hapten.

Extraction and analytic procedures for cytosine arabinoside and 1-beta-D-arabinofuranosyluracil and their 5'-mono-, di-, and tri-phosphates.

A qualitative and quantitative comparison of perchloric acid (PCA) and trichloroacetic acid (TCA) extraction procedures of biologic material containing cytosine arabinoside (Ara-C) and 1-beta-D-arabinofuranosyluracil (Ara-U) and their biologic 5'-phosphate derivatives revealed definite superiority of the PCA procedure over the TCA procedure. High-pressure liquid chromatography provides rapid and accurate quantitative and qualitative separation of both Ara-C and Ara-U and their 5'-mono-, di-, and tri-phosphates (mu Bondapak NH2 column), or their 5'-mono-, di-, and tri-phosphates without nucleosides (ABX Permaphase columns).

A radioimmunoassay method for 1-beta-D-arabinofuranosyluracil using antibodies directed against 1-beta-D-arabinofuranosylcytosine.

Above pH 7.0 1-beta-D-arabinofuranosyluracil (ara-U) shows marked pH-dependent cross-reactivity with antibodies directed towards 1-beta-D-arabinofuranosylcytosine. Since this peculiar phenomenon has not been observed with other nucleosides and nucleotides thus far tested, it is probably the result of base-catalyzed tautomerism of ara-U to its enolic form which renders it more structurally similar to 1-beta-D-arabinofuranosylcytosine. By performing the radioimmunoassay at both pH 6.2 and 8.6 we could determine 1-beta-D-arabinofuranosylcytosine and ara-U simultaneously. This method for ara-U assay is simple, fairly reliable, and applicable to blood level studies.

Separation of 5'-mono-, di- and triphosphates of 1-beta-D-arabinofuranosylcytosine and of 1-beta-D-arabinofuranosyluracil by thin-layer and high pressure liquid chromatography.

The separation of all the 5'-mono-, di- and triphosphates of ara-C and ara-U by thin-layer (TLC) and high pressure liquid chromatography (HPLC) is described. Whereas for the TLC two different systems are necessary, HPLC allows a rapid separation of all the nucleotides with high resolution, reproducibility, and sensitivity within 15 minutes. An example of an HPLC separation of ara-C and ara-U nucleotides derived from a biological sample is presented.