LC-MS/MS assay for the quantitation of FdCyd and its metabolites FdUrd and FU in human plasma.

The hypomethylating agent 5-fluoro-2'-deoxycytidine (FdCyd, NSC 48006) is being evaluated clinically both via the intravenous route and via the oral route in combination with 3,4,5,6-tetrahydrouridine (THU), a potent inhibitor of FdCyd catabolism. To determine the pharmacokinetics of FdCyd and downstream metabolites, we developed and validated an LC-MS/MS assay for the quantitation of FdCyd, 5-fluoro-2'-deoxyuridine (FdUrd), and 5-fluorouracil (FU) in 0.2mL human plasma. After acetonitrile protein precipitation, the sample was split and separate chromatography was achieved for FdCyd with a Synergi Polar-RP column and for FdUrd and FU with a Shodex Asahipak NH2P-50 2D column. Gradients of 0.1% acetic acid in acetonitrile and water were used. Detection with a Quattromicro quadrupole mass spectrometer with electrospray ionization in positive-ion (FdCyd) or negative-ion (FdUrd and FU) multiple reaction monitoring (MRM) mode. The assay was linear from 5 to 3000ng/mL for all three analytes and proved to be accurate (96.7-105.5%) and precise (<8.1%CV), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring FdCyd and metabolites FdUrd and FU in plasma from a patient who was administered 120mg PO FdCyd 30min after 3000mg THU. Our LC-MS/MS assay will be an essential tool to further define the pharmacology of FdCyd in ongoing and future studies.

Validation of a hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine.

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine-(13)C, (15)N2 used as an internal standard, were extracted from human plasma treated with THU using a 96-well Hybrid SPE-Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2'-2'-difluoro-2'-deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra-batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter-batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC-MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies.

Stability of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in combination.

In vivo, the DNA methyltransferase inhibitor, 5-fluoro-2'-deoxycytidine (FdCyd, NSC-48006), is rapidly converted to its unwanted metabolites. Tetrahydrouridine (THU, NSC-112907), a cytidine deaminase inhibitor can block the first metabolic step in FdCyd catabolism. Clinical studies have shown that co-administration with THU can inhibit the metabolism of FdCyd. The National Cancer Institute is particularly interested in a 1:5 FdCyd/THU formulation. The purpose of this study was to investigate the in vitro pH stability of FdCyd and THU individually and in combination. A stability-indicating high-performance liquid chromatography method for the quantification of both compounds and their degradants was developed using a ZIC(R)-HILIC column. The effect of THU and FdCyd on the in vitro degradation of each other was studied as a function of pH from 1.0 to 7.4 in aqueous solutions at 37 degrees C. The degradation of FdCyd appears to be first-order and acid-catalyzed. THU equilibrates with at least one of its degradants. The combination of FdCyd and THU in solution does not affect the stability of either compound. The stability and compatibility of FdCyd and THU in the solid state at increased relative humidity and at various temperatures are also evaluated.

Synthesis of deoxytetrahydrouridine.

The alpha-hydroxyamido functionality of 2'-deoxytetrahydrouridine (dTHU) makes this seemingly simple and generally useful compound difficult to obtain. Reported synthetic strategies produce extremely poor yields and multiple products, and full characterization data is not available. Described herein is a two-step approach for synthesizing dTHU in increased yields and purity; stability concerns are also addressed. Catalytic reduction (5% Rh/alumina) of 2'-deoxyuridine, followed by reduction with sodium borohydride as a limiting reagent, produces dTHU and limits formation of side products. Evidence was obtained for formation of a methoxy-substituted analogue during purification. By this strategy, dTHU of >95% purity can be obtained in 40% yield on a 150 mg scale.

Quantitative determination of the cytidine deaminase inhibitor tetrahydrouridine (THU) in mouse plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

A number of anticancer drugs are cytidine analogues that undergo metabolic deactivation catalyzed by cytidine deaminase (CD). 3,4,5,6-Tetrahydrouridine (THU) is a potent inhibitor of CD, by acting as a transition-state analogue of its natural substrate cytidine. However, to date its pharmacokinetic properties have not been fully characterized, which has impaired its optimal preclinical evaluation and clinical use. We report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for the sensitive, accurate and precise quantitation of THU in mouse plasma. Validation was performed according to FDA guidelines. The assay employed deuterated THU as the internal standard and an acetonitrile protein precipitation step. Separation, based on hydrophilic interaction chromatography, was achieved with an amino column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water followed by a wash. Chromatographic separation was followed by positive-mode electrospray ionization MS/MS detection in the multiple reaction monitoring (MRM) mode. The assay was accurate (92.5-109.9%) and precise (2.1-9.0%) in the concentration range of 0.2-50 microg/mL. Recovery from plasma was near-complete (92.9-119.3%) and ion suppression was negligible (-17.5 to -0.2%). Plasma freeze/thaw stability (93.1-102.1%), stability for 3 months at -80 degrees C (99.5-110.9%), and stability for 4 h at room temperature (92.1-102.4%) were all in order. This assay is currently being used to quantitate THU in ongoing pharmacokinetic studies. In addition, the assay is expected to be a useful tool in any future studies involving co-administration of THU with cytidine analogues.

The 1.48 A resolution crystal structure of the homotetrameric cytidine deaminase from mouse.

Cytidine deaminase (CDA) is a zinc-dependent enzyme that catalyzes the deamination of cytidine or deoxycytidine to form uridine or deoxyuridine. Here we present the crystal structure of mouse CDA (MmCDA), complexed with either tetrahydrouridine (THU), 3-deazauridine (DAU), or cytidine. In the MmCDA-DAU complex, it clearly demonstrates that cytidine is distinguished from uridine by its 4-NH(2) group that acts as a hydrogen bond donor. In the MmCDA-cytidine complex, cytidine, unexpectedly, binds as the substrate instead of the deaminated product in three of the four subunits, and in the remaining subunit it binds as the product uridine. Furthermore, the charge-neutralizing Arg68 of MmCDA has also exhibited two alternate conformations, I and II. In conformation I, the only conformation observed in the other structurally known homotetrameric CDAs, Arg68 hydrogen bonds Cys65 and Cys102 to modulate part of their negative charges. However, in conformation II the side chain of Arg68 rotates about 130 degrees around the Cgamma-Cdelta bond and abolishes these hydrogen bonds. The lack of hydrogen bonding may indirectly weaken the zinc-product interaction by increased electron donation from cysteine to the zinc ion, suggesting a novel product-expelling mechanism. On the basis of known structures, structural analysis further reveals two subclasses of homotetrameric CDAs that can be identified according to the position of the charge-neutralizing arginine residue. Implications for CDA-RNA interaction have also been considered.

Epimer interconversion, isomerization, and hydrolysis of tetrahydrouridine: implications for cytidine deaminase inhibition.

Tetrahydrouridine (THU) is an inhibitor of cytidine deaminase (CDA), the enzyme responsible for the deactivation of ara-C and other cytidine analogues in vivo, and therefore is capable of improving the therapeutic efficacy of these antitumor agents. In aqueous solution formulations, THU exists as a mixture of epimers differing in stereochemistry of the 4-OH substituent. The aims of this study were to investigate the interconversion kinetics of the epimers of THU, the CDA inhibitory effects of these epimers, and the stability and degradation mechanisms of THU epimer mixtures in aqueous solution with the ultimate goal of developing optimal conditions for a parenteral formulation of THU. A stability indicating HPLC assay utilizing a derivatized beta-cyclodextrin column was developed to separate the two epimers of THU and to monitor their reversible isomerization to their beta-ribopyranosyl counterparts and their hydrolysis to form N-glycosidic bond cleavage products. MS and one- and two-dimensional (1)H- and (13)C-NMR measurements were conducted to identify THU epimers and degradation products and to quantitatively model the degradation kinetics. The interconversion reaction between the two THU epimers is acid catalyzed with a first-order rate constant for conversion of epimer 1(1) to epimer 1(2) of (7.4 +/- 0.3) x 10(-3) h(-1) and an equilibrium constant ([1(2)]/[1(1)] of 1.7 +/- 0.1 at pH 7.4 and 25 degrees C. Epimer interconversion was therefore sufficiently slow at pH 7.4 to allow the isolation of each and evaluation of their CDA inhibitory activities utilizing 1% (w/v) mouse kidney homogenates as a source for cytidine deaminase and cytidine as a substrate. Inhibition constants for the two THU epimers (1(1) and 1(2)) were determined to be 8 +/- 1 x 10(-7) M and 6.2 +/- 0.2 x 10(-8) M, respectively. Studies at elevated temperature suggested that THU degradation from epimer mixtures is biphasic with the initial rate of disappearance being acid catalyzed and first order in initial THU concentration, thus ruling out dimerization as a potential reaction mechanism. NMR/MS analyses revealed that the major degradation products included the beta-ribopyranosyl THU isomers (two epimers), the reduced pyrimidinone base (tetrahydrouracil), and various anomers of D-ribose formed through N-glycosidic bond cleavage, and the products of subsequent reactions of the base. Kinetic modeling of the data obtained from both HPLC and NMR measurements indicated that in an acidic solution THU beta-ribofuranosyl --> beta-ribopyranosyl isomerization is a rapid equilibrium reaction, which proceeds through an intermediate observable in 1H-NMR, and is followed by slower N-glycosidic bond hydrolysis. All the reactions between THU, its ribopyranosyl isomers, the intermediate, and the base are acid catalyzed and appear to proceed through the same sugar ring-opened intermediate (carbinolamine), consistent with previous literature.