Development and validation of assays for the quantification of ß-D-N4-hydroxycytidine in human plasma and ß-D-N4-hydroxycytidine-triphosphate in peripheral blood mononuclear cell lysates.

The novel antiviral prodrug molnupiravir is under evaluation for the treatment of SARS-CoV-2. Molnupiravir is converted to ß-D-N4-hydroxycytidine (NHC), which is the primary form found in systemic circulation. ß-D-N4-hydroxycytidine-triphosphate (NHCtp) is the bioactive anabolite produced intracellularly. Sensitive and accurate bioanalytical methods are required to characterize NHC and NHCtp pharmacokinetics in clinical trials. Human K2EDTA plasma or peripheral blood mononuclear cell (PBMC) lysates were spiked with NHC (plasma) or NHCtp (PBMC), respectively. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or lysate dilution, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations. NHC can be quantified in plasma with a lower limit of quantification (LLOQ) of 1 ng/mL; the primary linearity of the assay is 1-5000 ng/mL. Assay precision and accuracy were ≤ 6.40% and ≤ ± 6.37%, respectively. NHC is unstable in whole blood and has limited stability in plasma at room temperature. The calibration range for NHCtp in PBMC lysates is 1-1500 pmol/sample, and the assay has an LLOQ of 1 pmol/sample. Assay precision and accuracy were ≤ 11.8% and ≤± 11.2%. Ion suppression was observed for both analytes; isotopically-labeled internal standards showed comparable ion suppression, resulting in negligible (<5%) relative matrix effects. Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the quantification of NHC in plasma and NHCtp in PBMC lysates. The described methods are appropriate for use in clinical trials.

Use of Metabolomics to Trend Recovery and Therapy After Injury in Critically Ill Trauma Patients.

IMPORTANCE: Metabolomics is the broad and parallel study of metabolites within an organism and provides a contemporaneous snapshot of physiologic state. Use of metabolomics in the clinical setting may help achieve precision medicine for those who have experienced trauma, where diagnosis and treatment are tailored to the individual patient. OBJECTIVE: To examine whether metabolomics can (1) distinguish healthy volunteers from trauma patients and (2) quantify changes in catabolic metabolites over time after injury. DESIGN, SETTING, AND PARTICIPANTS: Prospective cohort study with enrollment from September 2014 to May 2015 at an urban, level 1 trauma center. Included in the study were 10 patients with severe blunt trauma admitted within 12 hours of injury with systolic blood pressure less than 90 mm Hg or base deficit greater than 6 mEq/L and 5 healthy volunteers. Plasma samples (n = 35) were obtained on days 1, 3, and 7, and they were analyzed using mass spectrometry. MAIN OUTCOMES AND MEASURES: Principal component analyses, multiple linear regression, and paired t tests were used to select biomarkers of interest. A broad-based metabolite profile comparison between trauma patients and healthy volunteers was performed. Specific biomarkers of interest were oxidative catabolites. RESULTS: Trauma patients had a median age of 45 years and a median injury severity score of 43 (interquartile range, 34-50). Healthy fasting volunteers had a median age of 33 years. Compared with healthy volunteers, trauma patients showed oxidative stress on day 1: niacinamide concentrations were a mean (interquartile range) of 0.95 (0.30-1.45) relative units for trauma patients vs 1.06 (0.96-1.09) relative units for healthy volunteers (P = .02), biotin concentrations, 0.43 (0.27-0.58) relative units for trauma patients vs 1.21 (0.93-1.56) relative units for healthy volunteers (P = .049); and choline concentrations, 0.17 (0.09-0.22) relative units for trauma patients vs 0.21 (0.18-0.22) relative units for healthy volunteers (P = .004). Trauma patients showed lower nucleotide synthesis on day 1: adenylosuccinate concentrations were 0.08 (0.04-0.12) relative units for trauma patients vs 0.15 (0.14-0.17) relative units for healthy volunteers (P = .02) and cytidine concentrations were 1.44 (0.95-1.73) relative units for trauma patients vs 1.74 (1.62-1.98) relative units for healthy volunteers (P = .05). From trauma day 1 to day 7, trauma patients showed increasing muscle catabolism: serine levels increased from 42.03 (31.20-54.95) µM to 79.37 (50.29-106.37) µM (P = .002), leucine levels increased from 69.21 (48.36-99.89) µM to 114.16 (92.89-143.52) µM (P = .004), isoleucine levels increased from 20.43 (10.92-27.41) µM to 48.72 (36.28-64.84) µM (P < .001), and valine levels increased from 122.56 (95.63-140.61) µM to 190.52 (136.68-226.07) µM (P = .004). There was an incomplete reversal of oxidative stress. CONCLUSIONS AND RELEVANCE: Metabolomics can function as a serial, comprehensive, and potentially personalized tool to characterize metabolism after injury. A targeted metabolomics approach was associated with ongoing oxidative stress, impaired nucleotide synthesis, and initial suppression of protein metabolism followed by increased nitrogen turnover. This technique may provide new therapeutic and nutrition targets in critically injured patients.

Metabolomics analysis identifies novel plasma biomarkers of cystic fibrosis pulmonary exacerbation.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by infection, inflammation, lung function decline, and intermittent pulmonary exacerbations. However, the link between pulmonary exacerbation and lung disease progression remains unclear. Global metabolomic profiling can provide novel mechanistic insight into a disease process in addition to putative biomarkers for future study. Our objective was to investigate how the plasma metabolomic profile changes between CF pulmonary exacerbation and a clinically well state. METHODS: Plasma samples and lung function data were collected from 25 CF patients during hospitalization for a pulmonary exacerbation and during quarterly outpatient clinic visits. In collaboration with Metabolon, Inc., the metabolomic profiles of matched pair plasma samples, one during exacerbation and one at a clinic visit, were analyzed using gas and liquid chromatography coupled with mass spectrometry. Compounds were identified by comparison to a library of standards. Mixed effects models that controlled for nutritional status and lung function were used to test for differences and principal components analysis was performed. RESULTS: Our population had a median age of 27 years (14-39) and had a median FEV1 % predicted of 65% (23-105%). 398 total metabolites were identified and after adjustment for confounders, five metabolites signifying perturbations in nucleotide (hypoxanthine), nucleoside (N4-acetylcytidine), amino acid (N-acetylmethionine), carbohydrate (mannose), and steroid (cortisol) metabolism were identified. Principal components analysis provided good separation between the two clinical phenotypes. CONCLUSIONS: Our findings provide putative metabolite biomarkers for future study and allow for hypothesis generation about the pathophysiology of CF pulmonary exacerbation.

Quantification of 2'-deoxy-2'-ß-fluoro-4'-azidocytidine in rat and dog plasma using liquid chromatography-quadrupole time-of-flight and liquid chromatography-triple quadrupole mass spectrometry: Application to bioavailability and pharmacokinetic studies.

2'-Deoxy-2'-ß-fluoro-4'-azidocytidine (FNC) is a novel pyrimidine analog that inhibits not only the replication of the hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV but also the replication of lamivudine-resistant HBV, 4'-azidocytidine or 2'-ß-methylcytidine-resistant HCV, and nucleoside reverse-transcriptase inhibitor-resistant HIV variants. The present study was undertaken to evaluate the absolute oral bioavailability of FNC in rats and the pharmacokinetic properties of FNC after intragastric administration of single and multiple doses in rats and dogs. A sensitive high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) method and a reliable high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC/QqQ MS/MS) method were established for the determination of FNC in the rat and dog plasmas, respectively. The sample preparation involved a protein-precipitation method with methanol after the addition of lamivudine as an internal standard. FNC was analyzed by LC using a YMC-Pack Pro C18 column (150mm×4.6mm, 3µm) with methanol (containing 0.3% formic acid): 10mM ammonium acetate (containing 0.3% formic acid, pH 2.8) (35:65, v/v) as the mobile phase. Both mass spectrometers were equipped with an electrospray ionization interface in the positive-ion mode. The linear range was from 2.00 to 2000.00ngmL(-1) in rat plasma and 0.50 to 400.00ngmL(-1) in dog plasma. The intraday and interday precision were less than 10.55%, and the accuracy was in the range of -5.86 to 5.13%. The mean recoveries were greater than 82.70% and 82.97% for FNC and IS, respectively. The HPLC/Q-TOF MS and HPLC/QqQ MS/MS methods were both successfully applied in the pharmacokinetic studies of FNC in rats and dogs.

Plasma metabolite profiles of Alzheimer's disease and mild cognitive impairment.

Previous studies have demonstrated altered metabolites in samples of Alzheimer's disease (AD) patients. However, the sample size from many of them is relatively small and the metabolites are relatively limited. Here we applied a comprehensive platform using ultraperformance liquid chromatography-time-of-flight mass spectrometry and gas chromatography-time-of-flight mass spectrometry to analyze plasma samples from AD patients, amnestic mild cognitive impairment (aMCI) patients, and normal controls. A biomarker panel consisting of six plasma metabolites (arachidonic acid, N,N-dimethylglycine, thymine, glutamine, glutamic acid, and cytidine) was identified to discriminate AD patients from normal control. Another panel of five plasma metabolites (thymine, arachidonic acid, 2-aminoadipic acid, N,N-dimethylglycine, and 5,8-tetradecadienoic acid) was able to differentiate aMCI patients from control subjects. Both biomarker panels had good agreements with clinical diagnosis. The 2 panels of metabolite markers were all involved in fatty acid metabolism, one-carbon metabolism, amino acid metabolism, and nucleic acid metabolism. Additionally, no altered metabolites were found among the patients at different stages, as well as among those on anticholinesterase medication and those without anticholinesterase medication. These findings provide a comprehensive global plasma metabolite profiling and may contribute to making early diagnosis as well as understanding the pathogenic mechanism of AD and aMCI.

Correlations of creatine and six related pyrimidine metabolites and diabetic nephropathy in Chinese type 2 diabetic patients.

OBJECTIVES: The assessment of the clinical significance of creatine, cytosine, cytidine, uridine, thymine, thymidine, and 2'-deoxyuridine concentrations in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) for the detection of the relationship between pyrimidine metabolites and disease. DESIGN AND METHODS: The study group consisted of 119 subjects, which were divided to three groups: control (n=31), type 2 diabetes without nephropathy (DM, n=23), and with nephropathy (DN, n=65). Levels of related metabolites were measured in plasma of all participants. RESULTS: There is a significant increase in levels of cytosine (P<0.001), cytidine (P<0.001), and thymidine (P=0.016) with DN compared to DM. The levels of uridine, thymine, 2'-deoxyuridine, and creatine did not change. CONCLUSIONS: The levels of cytosine, cytidine, and thymidine may be useful for monitoring the progression of DM and evaluating the treatment.

Quantitative analysis of the experimental cytotoxic drug cyclopentenyl cytosine and its metabolite in plasma with HPLC tandem mass spectrometry.

The cytotoxic drug cyclopentenyl cytosine (CPEC) is currently being investigated in early clinical trials. Monitoring of plasma levels is required for pharmacokinetic analysis and management of toxicity. This paper describes the analysis of CPEC and cyclopentenyl uracil (CPEU) in plasma by HPLC-electrospray ionization tandem mass spectrometry. The calibration curves for CPEC and the metabolite CPEU were linear up to 10 microm with correlation coefficients of 0.997 (SD = 0.002, n = 10) and 0.997 (SD = 0.004, n = 10), respectively. The detection limit for CPEC was 0.03 and 0.12 microM for CPEU. The intra- and interassay coefficients of variation for CPEC and CPEU were less then 10%. The usefulness of the method was demonstrated by analyzing CPEC and CPEU in plasma of a patient treated with CPEC. HPLC with electrospray ionization tandem mass spectrometry allowed rapid and sensitive determination of CPEC and CPEU levels in plasma.

R1626 plus peginterferon Alfa-2a provides potent suppression of hepatitis C virus RNA and significant antiviral synergy in combination with ribavirin.

UNLABELLED: R1626, a prodrug of the hepatitis C virus (HCV) RNA polymerase inhibitor R1479, showed time-dependent and dose-dependent reduction of HCV RNA levels in a previous study. The present study evaluated the efficacy and safety of R1626 administered for 4 weeks in combination with peginterferon alfa-2a +/- ribavirin in HCV genotype 1-infected treatment-naive patients. Patients were randomized to: DUAL 1500 (1500 mg R1626 twice daily [bid] + peginterferon alfa-2a; n = 21); DUAL 3000 (3000 mg R1626 bid + peginterferon alfa-2a; n = 32); TRIPLE 1500 (1500 mg R1626 bid + peginterferon alfa-2a + ribavirin; n = 31); or standard of care (SOC) (peginterferon alfa-2a + ribavirin; n = 20). At 4 weeks HCV RNA was undetectable (<15 IU/mL) in 29%, 69%, and 74% of patients in the DUAL 1500, DUAL 3000, and TRIPLE 1500 arms, respectively, compared with 5% of patients receiving SOC, with respective mean reductions in HCV RNA from baseline to week 4 of 3.6, 4.5, 5.2, and 2.4 log(10) IU/mL. Synergy was observed between R1626 and peginterferon alfa-2a and between R1626 and ribavirin. There was no evidence of development of viral resistance. Adverse events (AEs) were mainly mild or moderate; seven patients had nine serious AEs (including one patient with one serious AE in SOC). The incidence of Grade 4 neutropenia was 48%, 78%, 39%, and 10% in DUAL 1500, DUAL 3000, TRIPLE 1500, and SOC, respectively, and was the main reason for dose reductions. CONCLUSION: A synergistic antiviral effect was observed when R1626 was combined with peginterferon alfa-2a +/- ribavirin; up to 74% of patients had undetectable HCV RNA at week 4. Dosing of R1626 was limited by neutropenia; a study of different dosages of R1626 in combination with peginterferon alfa-2a and ribavirin is underway.

Bisphosphonate derivatives of nucleoside antimetabolites: hydrolytic stability and hydroxyapatite adsorption of 5'-beta,gamma-methylene and 5'-beta,gamma-(1-hydroxyethylidene) triphosphates of 5-fluorouridine and ara-cytidine.

Kinetics of the hydrolytic reactions of four bisphosphonate derivatives of nucleoside antimetabolites, viz., 5-fluorouridine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 4), 5-fluorouridine 5'-beta,gamma-methylene triphosphate ( 5), ara-cytidine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 6), and ara-cytidine 5'-beta,gamma-methylene triphosphate ( 7), have been studied over a wide pH range (pH 1.0-8.5) at 90 degrees C. With each compound, the disappearance of the starting material was accompanied by formation of the corresponding nucleoside 5'-monophosphate, the reaction being up to 2 orders of magnitude faster with the beta,gamma-(1-hydroxyethylidene) derivatives ( 4, 6) than with their beta,gamma-methylene counterparts ( 5, 7). With compound 7, deamination of the cytosine base competed with the phosphate hydrolysis at pH 3-6. The measurements at 37 degrees C (pH 7.4) in the absence and presence of divalent alkaline earth metal ions (Mg (2+) and Ca (2+)) showed no sign of metal ion catalysis. Under these conditions, the initial product, nucleoside 5'-monophosphate, underwent rapid dephosphorylation to the corresponding nucleoside. Hydrolysis of the beta,gamma-methylene derivatives ( 5, 7) to the corresponding nucleoside 5'-monophosphates was markedly faster in mouse serum than in aqueous buffer (pH 7.4), the rate-acceleration being 5600- and 3150-fold with 5 and 7, respectively. In human serum, the accelerations were 800- and 450-fold compared to buffer. In striking contrast, the beta,gamma-(1-hydroxyethylidene) derivatives did not experience a similar decrease in hydrolytic stability. The stability in human serum was comparable to that in aqueous buffer (tau 1/2 = 17 and 33 h with 4 and 6, respectively), and on going to mouse serum, a 2- to 4-fold acceleration was observed. To elucidate the mineral-binding properties of 4- 7, their retention on a hydroxyapatite column was studied and compared to that of zoledronate ( 1a) and nucleoside mono-, di-, and triphosphates.

Hypotensive effects of intravenously administered uridine and cytidine in conscious rats: involvement of adenosine receptors.

In the present study, we investigated the cardiovascular effects of intravenously injected uridine or cytidine, and the role of adenosine receptors in mediating these effects, in conscious normotensive rats. Intravenous (i.v.) administration of uridine (124, 250, 500 mg/kg) dose-dependently decreased arterial pressure and heart rate. Cytidine (124, 250, 500 mg/kg; i.v.) produced slight dose-related hypotension without changing heart rate. Plasma uridine and cytidine concentrations increased time- and dose-dependently while plasma adenosine levels did not change after injection of the respective nucleosides. Pretreatment with intravenous caffeine (20 mg/kg), 8-phenyltheophylline (8-PT) (1 mg/kg), nonselective adenosine receptor antagonists, or 8-p-sulfophenyltheophylline (8-SPT) (20 mg/kg), a nonselective adenosine receptor antagonist which does not cross the blood-brain barrier, abolished the cardiovascular effects of uridine (250 mg/kg; i.v.) or cytidine (250 mg/kg; i.v.). Intracerebroventricular (i.c.v.) caffeine (200 microg) or 8-SPT (50 microg) pretreatment did not change the magnitude of the cardiovascular responses induced by nucleosides. Intravenous 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX) (5 mg/kg), a selective adenosine A(1) receptor antagonist, greatly attenuated the cardiovascular responses to uridine and cytidine. Pretreatment with 3,7,-dimethyl-1-propargylxanthine (DMPX) (2 mg/kg), an adenosine A(1)/A(2) receptor antagonist, attenuated hypotension induced by uridine and blocked the arterial pressure decrease in response to cytidine. Uridine-induced bradycardia was blocked by DMPX. 4-(2-[7-amino-2-(2-furyl[1,2,4]-triazolo[2,3-a[1,3,5]triazin-5-yl-aminoethyl)phenol (ZM241385) (1 mg/kg; i.v.), a selective adenosine A(2A) receptor antagonist, pretreatment produced an only very small blockade in the first minute of the hypotensive effects of uridine without affecting the bradycardia. ZM241385 pretreatment completely blocked cytidine's hypotensive effect. In Langendorff-perfused rat heart preparation, uridine (10(-3) M), but not cytidine, decreased the heart rate. Our results show that intravenously injected uridine or cytidine is able to decrease arterial pressure by activating peripheral adenosine receptors. The data also implicates that the mainly adenosine A(1) receptor activation is involved in the uridine-induced cardiovascular effects, while both adenosine A(1) and A(2A) receptor activations mediate the cytidine's effects.

Quantitative determination of zebularine (NSC 309132), a DNA methyltransferase inhibitor, and three metabolites in murine plasma by high-performance liquid chromatography coupled with on-line radioactivity detection.

The metabolism of zebularine (NSC 309132), a novel agent that inhibits DNA methyltransferases, is still uncharacterized. To examine the in vivo metabolism of zebularine, an analytical method was developed and validated (based on FDA guidelines) to quantitate 2-[(14)C]-zebularine and its major metabolites in murine plasma. Zebularine and its metabolites uridine, uracil and dihydrouracil were baseline-separated based on hydrophilic interaction chromatography by using an amino column. The assay was accurate and precise in the concentration ranges of 5.0-100 microg/mL for zebularine, 2.5-50 microg/mL for uridine, 1.0-10 microg/mL for uracil and 0.5-5.0 microg/mL for dihydrouracil. This assay is being used to quantitate zebularine and its metabolites in ongoing pharmacokinetic studies of zebularine.

Determination of ribavirin in human serum and plasma by capillary electrophoresis.

The electrophoretic separation of ribavirin and 5-methylcytidine (internal standard) by capillary electrophoresis was examined. Separation was achieved using reverse polarity in a 100 mM borate electrolyte, pH 9.1, with 5 mM spermine added to reduce the electroosmotic flow. Sample preparation based on acetonitrile protein precipitation was found to be unsuitable for ribavirin analysis in patient samples due to insufficient sensitivity and interferences. Solid-phase extraction employing phenyl boronic acid cartridges provided cleaner separations. Using this approach with 500 microL sample and reconstitution of the dried extract into 100 microL of 33% v/v 100 mM phosphate buffer, pH 6.4 / 67% v/v acetonitrile, the detection and quantitation limits were determined to be 0.05 and 0.10 microg/mL, respectively, a sensitivity that is suitable for therapeutic drug monitoring of ribavirin in human plasma and serum samples. The method was validated and compared to a high-performance liquid chromatography (HPLC) method, showing excellent agreement between the two for a set of samples that stemmed from patients being treated with ribavirin and interferon-alpha-2b for a hepatitis C virus infection.

Rapid determination of rat plasma uridine levels by HPLC-ESI-MS utilizing the Captiva plates for sample preparation.

A rapid, accurate and precise HPLC-ESI-MS method for the determination of rat plasma uridine concentrations was developed and is described here. Sample preparation involves methanol precipitation of plasma proteins in a 96-well Captiva protein precipitation filter plate. A clear extract is drawn through the filter plate with vacuum, followed by evaporation of the extract and subsequent reconstitution prior to chromatography on a reversed-phase column with an aqueous mobile phase [0.1% (v/v) glacial acetic acid]. Detection was accomplished by positive-ion electrospray ionization mass spectrometry. A calibration curve ranging in concentration from 0.78 to 25 microM was constructed by best-fit, 1/x weighting linear regression analysis of the calibration standard concentrations vs peak height ratios of analyte with internal standard. The correlation coefficient was >0.995. The overall assay accuracy as shown by the back-calculated concentrations of the calibration curve ranged from 96.6 to 103% with RSD ranging from 4.5 to 20%. While this assay method was developed for the determination of uridine in rat plasma, it could be readily adapted for determination of uridine in plasma from other species, such as human.

Determination of 3'-C-ethynylcytidine in human plasma and urine by liquid chromatographic-electrospray ionization tandem mass spectrometry.

A liquid chromatographic-electrospray ionization tandem mass spectrometric (LC-ESI/MS/MS) method was developed for the quantitative analysis of a novel anticancer drug, 3'-C-ethynylcytidine (I) in human plasma and urine. I and its stable isotope-labeled internal standard (II) were extracted from human plasma and urine samples using a polymer-based cation-exchange cartridge, and LC-ESI/MS/MS analysis was performed by monitoring the positive fragment ions of I and II. The linear ranges are 1-500 ng/ml in plasma and 10-5000 ng/ml in urine. The limits of quantitation for I were 1 ng/ml in plasma and 10 ng/ml in urine. The relative errors (RE) for I ranged from -8.4 to 3.0% in plasma and from 0.8 to 4.4% in urine. The relative standard deviations (RSD) for I ranged from 1.2 to 8.9% in plasma and from 0.7 to 2.8% in urine. This validated analytical method is demonstrated to be useful for the analysis of I in human plasma and urine in clinical studies.

Improvement in precision of the liquid chromatographic-electrospray ionization tandem mass spectrometric analysis of 3'-C-ethynylcytidine in rat plasma.

During the development of the liquid chromatography with electrospray ionization-tandem mass spectrometry for the quantitative determination of 3'-C-ethynylcytidine (I) in rat plasma, ion suppression caused by the matrix components was observed for I and its structural analogue, 3'-C-ethylcytidine (II) as the internal standard. In the method initially designed, I/II peak area ratios varied according to the degree of matrix effect, which led to the poor precision of the assay. From the examination of the ion suppression behavior for I and II, it was assumed that this phenomenon is attributed to the difference in the retention time between I and II. Based on this assumption, therefore, the methanol content in the mobile phase was changed from 5 to 25% so as to make I and II the same retention time. As a result of this modification of the initial method, the precision expressed as relative standard deviation was improved from 5.2-16.2 to 2.7-4.2% in intra-assay and from 6.8-14.9 to 3.5-7.2% in inter-assay validations.

Effect of oral CDP-choline on plasma choline and uridine levels in humans.

Twelve mildly hypertensive but otherwise normal fasting subjects received each of four treatments in random order: CDP-choline (citicoline; 500, 2000, and 4000 mg) or a placebo orally at 8:00 a.m. on four different treatment days. Eleven plasma samples from each subject, obtained just prior to treatment (8:00 a.m.) and 1-12 hr thereafter, were assayed for choline, cytidine, and uridine. Fasting terminated at noon with consumption of a light lunch that contained about 100 mg choline. Plasma choline exhibited dose-related increases in peak values and areas under the curves (AUCs), remaining significantly elevated, after each of the three doses, for 5, 8, and 10 hr, respectively. Plasma uridine was elevated significantly for 5-6 hr after all three doses, increasing by as much as 70-90% after the 500 mg dose, and by 100-120% after the 2000 mg dose. No further increase was noted when the dose was raised from 2000 to 4000 mg. Plasma cytidine was not reliably detectable, since it was less than twice blank, or less than 100 nM, at all of the doses. Uridine is known to enter the brain and to be converted to UTP; moreover, we found that uridine was converted directly to CTP in neuron-derived PC-12 cells. Hence, it seems likely that the circulating substrates through which oral citicoline increases membrane phosphatide synthesis in the brains of humans involve uridine and choline, and not cytidine and choline as in rats.

Reversed-phase high-performance liquid chromatographic determination of the new antitumor agent cyclopentenyl cytosine in biological fluids.

Cyclopentenyl cytosine (CPE-C) is a synthetic carbocyclic nucleoside that possesses diverse antitumor and antiviral activity. CPE-C has been studied extensively at the preclinical level and has been evaluated in a Phase I clinical trial involving patients with solid tumors. A narrow-bore, reversed-phase HPLC method that has been developed for the sensitive measurement of CPE-C in plasma and urine in order to carry out these studies is described. Covalent solid-phase extraction based on an immobilized phenylboronic acid ligand is employed to isolate both CPE-C and endogenous ribonucleosides from the biological matrix selectively and efficiently. This is followed by isocratic elution of the extract with pH 5.0, 0.1 M ammonium formate buffer at 0.150 ml/min on a tandem, switchable, C18 narrow-bore (2.1 mm I.D.) column system in which the precolumn is automatically backflushed to eliminate late-eluting components. UV detection at 278 nm provides a limit of quantitation of 0.1 microM for CPE-C in rat and human plasma with a precision better than 4% for the range 1-20 microM in rat plasma. Application of this assay to the determination of the bolus dose plasma kinetics and disposition of 2 mg/kg CPE-C in rats is illustrated. This method is amenable to partial automation and is well-suited for the analysis of clinical samples.

Separation of aracytidine and cytidine by capillary electrophoretic techniques.

Aracytidine (cytarabine, 1-beta-D-arabinofuranosylcytosine) is a synthetic analog of cytidine in which ribose is substituted by arabinose; it is used as a drug for the treatment of leukemia. A fast and reliable capillary electrophoretic method for the analysis of cytarabine and cytidine is described. The procedure utilizes the interactions with sodium dodecyl sulfate (SDS) micelles and borate, present in the background electrolyte, for the mobilization and selective separation of the analytes. The detection is carried out by UV absorbance at 275 nm. The method was applied both to pharmaceutical preparations and human serum. Analysis of an untreated serum requires 15 min; the detection limit is 0.8 microgram/mL and the relative standard deviation (RSD) is 5.3%.

Evidence that 5'-cytidinediphosphocholine can affect brain phospholipid composition by increasing choline and cytidine plasma levels.

We examined the effects of orally administered 5'-cytidinediphosphocholine (CDP-choline) on arterial plasma choline and cytidine levels and on brain phospholipid composition in rats. Animals receiving a single oral dose of 100, 250, or 500 mg/kg showed peak plasma choline levels 6-8 h after drug administration (from 12 +/- 1 to 17 +/- 2, 19 +/- 2, and 24 +/- 2 microM, respectively). The area under the plasma choline curve at > 14 microM, i.e., at a concentration that induces a net influx of choline into the brain, was significantly correlated with CDP-choline dose. In rats receiving 500 mg/kg this area was 2.3 times that of animals consuming 250 mg/kg, which in turn was 1.8 times that of rats receiving 100 mg/kg. Plasma cytidine concentrations increased 5.4, 6.5, and 15.1 times baseline levels, respectively, 8 h after each of the three doses. When the oral CDP-choline treatment was prolonged for 42 and 90 days, brain phosphatidylcholine concentrations increased significantly (by 22-25%; p < 0.05) in rats consuming 500 mg/kg/day. Brain phosphatidylethanolamine and phosphatidylserine concentrations also increased significantly under some experimental conditions; levels of other phospholipids were unchanged.

Reversal by cytidine of cyclopentenyl cytosine-induced toxicity in mice without compromise of antitumor activity.

Among nine compounds surveyed, cytidine was found to be the most effective in reversing the antiproliferative effects of cyclopentenyl cytosine (CPEC) on human T-lymphoblasts (MOLT-4) in culture. Cytidine, at concentrations of 1-25 microM, enabled cells to maintain normal logarithmic growth when added up to 12 hr after exposure to a 200 nM concentration of the oncolytic nucleoside, CPEC. The most abundant CPEC metabolite, CPEC-5'-triphosphate, is a potent [K1 approximately 6 microM] inhibitor of CTP synthetase (EC 6.3.4.2). Accumulation of this inhibitor resulted in a depletion of CTP levels to 17% of their original cellular concentration. Exogenous cytidine reversed CPEC-induced cellular cytotoxicity by suppressing the formation of CPEC-5'-triphosphate by 70%, and by partially replenishing intracellular CTP to at least 60-70% of its original concentration. In vivo, cytidine (500 mg/kg) administered intraperitoneally 4 hr after each daily dose of CPEC (LD10-LD100) for 9 days reduced the toxicity and abolished the lethality of CPEC to non-tumored mice. Of greater practical importance is the finding that, under these experimental conditions, cytidine did not curtail the antineoplastic properties of CPEC in L1210 tumor-bearing mice. Moreover, the concentration range over which CPEC exhibited antineoplastic activity was extended with cytidine administration.