Clinical Cancer Research Grants Metabolism
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 14, 3529, June 1, 2008. doi: 10.1158/1078-0432.CCR-07-4885
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beumer, J. H.
Right arrow Articles by Egorin, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beumer, J. H.
Right arrow Articles by Egorin, M. J.

Cancer Therapy: Preclinical

Modulation of Gemcitabine (2',2'-Difluoro-2'-Deoxycytidine) Pharmacokinetics, Metabolism, and Bioavailability in Mice by 3,4,5,6-Tetrahydrouridine

Jan H. Beumer1,2, Julie L. Eiseman1,3, Robert A. Parise1, Erin Joseph1, Joseph M. Covey5 and Merrill J. Egorin1,3,4

Authors' Affiliations: 1 Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute; 2 Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy; 3 Department of Pharmacology and 4 Division of Hematology/Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania and 5 Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, Maryland

Requests for reprints: Jan H. Beumer, University of Pittsburgh Cancer Institute, Room G.27d, Hillman Research Pavilion, 5117 Centre Avenue, Pittsburgh, PA 15213-1863. Phone: 412-623-3216; Fax: 412-623-1212; E-mail: beumerjh{at}upmc.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Purpose: In vivo, 2',2'-difluoro-2'-deoxycytidine (dFdC) is rapidly inactivated by gut and liver cytidine deaminase (CD) to 2',2'-difluoro-2'-deoxyuridine (dFdU). Consequently, dFdC has poor oral bioavailability and is administered i.v., with associated costs and limitations in administration schedules. 3,4,5,6-Tetrahydrouridine (THU) is a potent CD inhibitor with a 20% oral bioavailability. We investigated the ability of THU to decrease elimination and first-pass effect by CD, thereby enabling oral dosing of dFdC.

Experimental Design: A liquid chromatography-tandem mass spectrometry assay was developed for plasma dFdC and dFdU. Mice were dosed with 100 mg/kg dFdC i.v. or orally with or without 100 mg/kg THU i.v. or orally. At specified times between 5 and 1,440 min, mice (n = 3) were euthanized. dFdC, dFdU, and THU concentrations were quantitated in plasma and urine.

Results: THU i.v. and orally produced concentrations >4 µg/mL for 3 and 2 h, respectively, whereas concentrations of >1 µg/mL have been associated with near-complete inhibition of CD in vitro. THU i.v. decreased plasma dFdU concentrations but had no effect on dFdC plasma area under the plasma concentration versus time curve after i.v. dFdC dosing. Both THU i.v. and orally substantially increased oral bioavailability of dFdC. Absorption of dFdC orally was 59%, but only 10% passed liver and gut CD and eventually reached the systemic circulation. Coadministration of THU orally increased dFdC oral bioavailability from 10% to 40%.

Conclusions: Coadministration of THU enables oral dosing of dFdC and warrants clinical testing. Oral dFdC treatment would be easier and cheaper, potentially prolong dFdC exposure, and enable exploration of administration schedules considered impractical by the i.v. route.

Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdC), Gemzar] is an important anticancer drug that has been approved for the treatment of non–small cell lung, pancreatic, bladder, and breast cancers (1). dFdC is extensively metabolized to 2',2'-difluoro-2'-deoxyuridine (dFdU) by cytidine deaminase (CD; EC 3.5.4.5),6 which abounds in the liver and gut (Fig. 1 ; refs. 2–4). This has limited dFdC use to the parenteral route. Oral dosing of dFdC would simplify administration and extend the systemic exposure time (e.g., time above a certain concentration) after a dose relative to an i.v. exposure profile, which has been correlated with increased activity of dFdC, both preclinically and clinically (1, 5). Furthermore, dFdU, although often considered inactive, may be anabolized to dFdU monophosphate, which is a thymidylate synthase inhibitor (6), has been shown to form dFdUTP (7), is incorporated into both DNA and RNA, and displays in vitro cytotoxicity (8). dFdU is produced in large amounts and has a human plasma terminal half-life of at least 65 h, which is substantially longer than the 17-min half-life of dFdC (9).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Metabolic and biochemical scheme of dFdC (gemcitabine). Other compounds involved are dFdC monophosphate (dFdCMP), dFdC diphosphate (dFdCDP), dFdC triphosphate (dFdCTP), dFdU, dFdU monophosphate (dFdUMP), dFdU diphosphate (dFdUDP), dFdU triphosphate (dFdUTP), UTP, CTP, CDP, dCDP, dCTP, and cytidylate deaminase (dCMP-D). THU blocks the conversion of dFdC to dFdU by CD. RR, ribonucleotide reductase; TS, thymidylate synthase.

 
3,4,5,6-Tetrahydrouridine (THU) is a potent inhibitor of CD (4, 10, 11). By inhibiting CD, THU can prolong the half-life of i.v. administered cytidine analogues, and by decreasing first-pass metabolism in the gut and liver, THU can increase the bioavailability of cytidine analogues administered orally, as has been shown for cytosine arabinoside, 5-azacytidine, 5-chloro-2'-deoxycytidine, and 5-fluoro-2'-deoxycytidine (FdCyd; refs. 12–17). We have recently shown that the bioavailability of THU in mice is sufficient to produce THU plasma concentrations that fully inhibit CD (18), and we have shown that oral THU increases plasma exposure to FdCyd in mice 58-fold (15), a finding that has resulted in initiation of investigations into the oral dosing of FdCyd and THU in humans. Administering dFdC orally would enable easier and cheaper treatment of patients, and it would allow exploration of administration schedules currently considered impractical. Furthermore, CD may be overexpressed in tumors (19, 20), conferring tumor resistance to dFdC treatment. Elevated levels of CD in leukemic cells were observed at the time of relapse following cytosine arabinoside or 5-azacytidine treatment (4), and high expression of CD in mononuclear cells was associated with a lower response rate and overall survival in patients with advanced pancreatic carcinoma (21). Codosing with THU will block CD and is expected to increase the antitumor effect of dFdC. Although THU may affect both the elimination and the oral bioavailability of dFdC, this has never been shown.

We aimed to study the effect of THU on the pharmacokinetics, metabolism, and oral bioavailability of dFdC in mice. dFdC was administered to mice orally or i.v. with or without THU, and plasma pharmacokinetics of dFdC, dFdU, and THU were characterized.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Chemicals and reagents
dFdC, dFdU, 5-fluoro-2'-deoxyuridine, and THU were provided by the Developmental Therapeutics Program, National Cancer Institute (Bethesda, MD). The clinical formulation of dFdC, obtained from Eli Lilly, was used for administration to mice. Formic acid was obtained from Sigma-Aldrich. Acetonitrile, ethyl acetate, and glacial acetic acid were obtained from Fisher Chemicals. All reagents were of analytic grade. Water was purified using a Q-gard 1 Gradient Milli-Q system (18.2 M{Omega} cm, Millipore). Control mouse plasma was obtained from Lampire Biological Laboratories.

Animals
Specific pathogen-free, adult CD2F1 male mice were purchased from Taconic. Mice were allowed to acclimate to the University of Pittsburgh Cancer Institute Animal Facility for 1 wk before the start of each study. To minimize infection, mice were maintained in microisolator cages in a separate room and handled in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and on a protocol approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Ventilation and airflow were set to 12 changes per hour. Room temperatures were regulated at 22 ± 1°C, and the rooms were kept on automatic 12-h light/dark cycles. Mice received Prolab IsoPro RMH 3000 Irradiated Lab Diet (PMI Nutrition International) and water ad libitum, except on the evening before dosing, when all food was removed. Mice were 6 to 8 wk old at the time of dosing. Sentinel animals were maintained in 1:5 dirty bedding in the same housing as the study mice and assayed at 3-mo intervals for specific murine pathogens by mouse antibody profile testing (Charles River). Sentinel animals remained free of specific pathogens, indicating that the study mice were pathogen-free.

Pharmacokinetics of dFdC, dFdU, and THU in mice
To investigate the plasma disposition of dFdC, mice of ~20 g body weight were dosed with 100 mg/kg of dFdC (~2 mg/mouse). dFdC was administered alone i.v. or orally. In addition, dFdC disposition studies were done with coadministration of 100 mg/kg of THU (~2 mg/mouse). The THU dose of 100 mg/kg corresponds to a human dose of 8.3 mg/kg or 300 mg/m2 (22), which is the approximate THU dose currently used in clinical trials (350 mg/m2; refs. 23, 24). This resulted in the following five dosing schedules: (a) dFdC i.v. alone, (b) dFdC orally alone, (c) dFdC i.v. and THU i.v. (formulated together), (d) dFdC orally 30 min after THU orally, and (e) dFdC orally and THU i.v. The rationale for separate dosing of THU orally 30 min before dFdC orally is the Tmax of THU orally of 30 min observed previously (18). The vehicle consisted of 5% dextrose (Baxter).

Mice were dosed (0.01 mL/g fasted body weight) by lateral tail vein injection or by oral gavage. Mice (three per time point) were euthanized with CO2 at 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, 180, 240, 360, 960, and 1,440 min after dosing. Blood was collected by cardiac puncture into heparinized (Baxter) syringes. Blood was divided into two aliquots, to one of which 70 µg of THU were added to prevent ex vivo dFdC deamination by CD in blood. Blood was centrifuged for 4 min at 13,000 x g to obtain plasma. The aspirated plasma was stored at –70°C until analysis. In the plasma with added THU, dFdC and dFdU were quantitated with a hydrophilic interaction chromatography (25) high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay (see below). THU in the other plasma aliquot was quantitated with a previously developed method (26) that had been validated according to the Food and Drug Administration guideline for Bioanalytical Method Validation.7.

To assess urinary excretion of dFdC, dFdU, and THU after administration of dFdC and/or THU in the various regimens, mice scheduled for euthanasia at 16 and 24 h after dosing were housed in metabolic cages. Urine was collected on ice, separately from feces. Quantitation of dFdC and dFdU, and THU in urine was accomplished by preparing a 10-fold, or higher, dilution of urine samples in plasma and analyzing those diluted samples with the respective hydrophilic interaction chromatography HPLC-MS/MS assay used for plasma samples.

Quantitation of dFdC and dFdU
Sample preparation. Calibration samples were prepared in mouse plasma at 10, 20, 50, 100, 200, 500, 1,000, 2,000, and 5,000 ng/mL. Initial sample preparation was done on ice. To 100 µL of plasma, or plasma-diluted sample, were added 10 µL of internal standard (10 µg/mL of 5-fluoro-2'-deoxyuridine in water), after which the mixture was vortexed for 20 s. A liquid-liquid extraction was done by adding 1 mL of ethyl acetate and vortexing the mixture for 5 min. The vortexed samples were centrifuged for 6 min at 10,000 x g, after which each supernatant layer was aspirated and evaporated to dryness under nitrogen at 37°C. The dried residues were reconstituted in 100 µL of acetonitrile and transferred to autosampler vials.

Chromatography. The HPLC system consisted of an Agilent 1100 autosampler and binary pump, a Shodex Asahipak NH2-50 2D column (5 µm, 150 mm x 2 mm inner diameter, Phenomenex), and a gradient mobile phase. Mobile phase solvent A consisted of 0.1% (v/v) formic acid in acetonitrile, and mobile phase solvent B consisted of 0.1% (v/v) formic acid in water. This resulted in a separation system based on hydrophilic interaction chromatography (25). The initial mobile phase composition was 98.0% solvent A and 2.0% solvent B pumped at a flow rate of 0.2 mL/min. From 0 to 10.0 min, solvent B was increased linearly from 2.0% to 20.0% and then held at this composition for 2 min. From 12.0 to 12.1 min, the flow was increased to 0.80 mL/min, and the gradient was increased to 60% B, and this composition was held until 14.0 min. From 14.0 to 14.1 min, the gradient was decreased to 0% B, and from 18.2 to 18.3 min, the gradient was returned to the initial setting of 2.0% solvent B. From 19.8 to 20.0 min, the flow was decreased to 0.2 mL/min, after which 5 µL of the next sample were injected.

Mass spectrometric detection. Mass spectrometric detection was carried out using a Waters Quattromicro triple-stage, bench-top quadrupole mass spectrometer with electrospray ionization alternating between positive and negative in the multiple reaction monitoring mode. The settings of the mass spectrometer were as follows: capillary voltage, 3.0 kV; cone voltage, 20 V; source temperature, 120°C; and desolvation temperature, 350°C. The cone and desolvation gas flows (nitrogen) were 110 and 550 L/h, respectively. The collision voltage used was 10 V. Quadrupoles 1 and 2 each had low-mass and high-mass resolution set at 12. The dwell time was 0.25 s, and the interscan delay was 0.3 s. The multiple reaction monitoring m/z transitions monitored for each analyte are shown in Table 1 . The HPLC system and mass spectrometer were controlled by Waters MassLynx software (version 4.0), and data were collected with the same software. The analyte-to-internal standard ratio was calculated for each standard by dividing the area of each analyte peak by the area of the respective internal standard peak for that sample. Standard curves of the analytes were constructed by plotting the analyte-to-internal standard ratio versus the known concentration of analyte in each sample. Standard curves were fit by linear regression with weighting by 1/y2 followed by back calculation of concentrations.


View this table:
[in this window]
[in a new window]

  		Table 1. Assay performance variables for dFdC and dFdU

 
Assay performance. Assay performance (precision and accuracy) was assessed for each compound by running a triplicate standard curve, and extraction recovery was assessed at 1,000 ng/mL. Precision was calculated as the relative SD of the triplicates; accuracy was calculated as the back-calculated concentration relative to the nominal concentration; recovery was calculated as the relative response of an extracted sample versus the response of an extracted blank to which the compounds had been added after reconstitution. Ion suppression was calculated as the absolute response of the respective compound added to a reconstituted extract of blank plasma, relative to the same amount of compound added to reconstitution solvent. The dynamic calibration range for both dFdC and dFdU was 10 to 5,000 ng/mL.

Pharmacokinetic analysis
The area under the plasma concentration versus time curve (AUC) and other pharmacokinetic variables were calculated noncompartmentally using WinNonlin Professional, version 4.1 (Pharsight Corp.) and the linear trapezoidal rule.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
The assay for dFdC and dFdU proved linear over the concentration ranges used. Assay performance variables and retention times are displayed in Table 1.

The plasma concentration versus time profiles of dFdC, dFdU, and THU are shown in Fig. 2A, B, and C , respectively. For the sake of comparison, Fig. 2C also contains the plasma concentration versus time profile of THU dosed alone i.v. or orally, as described earlier (18). Pharmacokinetic variables calculated noncompartmentally are displayed in Table 2 . Peak concentrations (Cmax) of dFdC were recorded in the earliest plasma samples (5 min) after i.v. administration and at later time points (30 or 75 min) after oral administration. As expected, peak concentrations of dFdU were observed after those of the parent compound. After administration of dFdC either i.v. or orally, dFdC plasma concentrations declined mono-exponentially with a half-life of approximately 30 to 40 min. Coadministration of i.v. THU to i.v. dFdC did not substantially increase the half-life nor the plasma AUC of dFdC. The slightly diverging profiles of dFdC plasma concentrations at later time points are reflected in the mean residence times after i.v. dFdC (22.6 min) and i.v. coadministration of dFdC and THU (38.1 min), respectively. Coadministration of i.v. or oral THU slightly increased the apparent plasma dFdC half-life only when dFdC was administered orally. Correspondingly, clearance and distribution volume of dFdC were not significantly affected by THU. Interestingly, coadministration of i.v. THU with i.v. dFdC did reduce dFdC conversion to dFdU 5-fold and was accompanied by a 60% increase in renal dFdC excretion. Most importantly, the oral bioavailability of dFdC alone was 10% but increased to ~40% when THU was administered orally 30 min before the oral administration of dFdC.


Figure 2
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Mean plasma concentration (±SD; n = 3) versus time curves of dFdC (A), dFdU (B), and THU (C) after dosing male CD2F1 mice with 100 mg/kg i.v. dFdC ({Delta}), 100 mg/kg oral dFdC ({circ}), 100 mg/kg i.v. dFdC with i.v. coadministration of 100 mg/kg THU ({blacktriangleup}), 100 mg/kg oral dFdC with i.v. coadministration of 100 mg/kg THU (x), 100 mg/kg oral dFdC with oral coadministration of 100 mg/kg THU (bullet), 100 mg/kg i.v. THU ({square}; data from ref. 18), and 100 mg/kg oral THU ({lozenge}; data from ref. 18). The oral THU curves in C were plotted starting at –30 min because THU was dosed 30 min before dFdC administration.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Pharmacokinetic variables of dFdC and dFdU after administration of 100 mg/kg dFdC to male CD2F1 mice, either i.v. or orally and with or without 100 mg/kg i.v. or oral THU

 
After 360 min, concentrations of dFdC fell below the lower limit of quantitation of the assay, except after oral dFdC with i.v. THU, which resulted in a mean dFdC plasma concentration of 18.5 (SD, 11.6) ng/mL at 960 min (data not shown in Fig. 2A). dFdU could be detected until the last sampling point (24 h). THU dosed i.v. or orally resulted in THU plasma concentrations above 1,000 ng/mL for over 6 h. By the next sampling time point (960 min), THU had fallen below the lower limit of quantitation. The plasma concentration versus time profile of oral THU seemed to be affected by codosing of oral dFdC, as indicated by the decrease in the THU plasma concentration shortly after dFdC administration.

Renal excretion of dFdC, dFdU, and THU in the 0- to 16-h and 0- to 24-h mouse urine is displayed in Table 2. Coadministration of THU consistently increased the recovery of dFdC in urine.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The present investigation aimed to characterize the pharmacokinetics, metabolism, and oral bioavailability of dFdC in mice with and without THU coadministration. The data generated in this study will aid in the development of an oral dosing schedule for dFdC, which should simplify its administration and provide more options to optimize its dosing schedule. We developed a highly sensitive HPLC-MS/MS assay to quantitate dFdC and its metabolite, dFdU, in murine plasma. After validation in human plasma, this assay may aid the further clinical development of the combination of dFdC and THU.

dFdC displayed a half-life of approximately 30 to 40 min, which is slightly longer than the half-life of other cytidine analogues, such as FdCyd (10 min; ref. 15), cytosine arabinoside (12 min; ref. 12), and 5-chloro-2'-deoxycytidine (10 min; ref. 12). Previously, the plasma half-life of dFdC in B6C3F1 mice was reported to be ~17 min (27).

The plasma AUC of both dFdC and dFdU after i.v. administration of dFdC agreed with previous reports (27). In contrast to what has been observed with FdCyd (15), 5-chloro-2'-deoxycytidine (12), and cytosine arabinoside (13), THU did not increase the plasma half-life, AUC, or clearance of dFdC after i.v. dosing. Intuitively, THU is expected to decrease the clearance of dFdC and, thereby, the AUC. However, previously, 10 mg/kg THU orally did not significantly increase the half-life of cytosine arabinoside in mice, although the AUC did increase 5-fold (17). The 5.4-fold reduction in dFdU AUC indicates that THU (assuming no interaction of THU with dFdU clearance) was indeed capable of decreasing the dFdC clearance via deamination. Apparently, there is a compensatory mechanism of dFdC clearance from plasma. This could reflect increased tissue uptake of dFdC or increased renal excretion of dFdC, which, according to our data, doubled with coadministration of THU. The increase in mean residence time from i.v. dFdC to i.v. dFdC combined with THU would indicate that THU does make dFdC stay around longer. However, mean residence time calculations are based on the area under the moment time curve, which tends to magnify differences in plasma profiles. Therefore, those results should be interpreted with caution.

The total urinary excretion of dFdC and dFdU after i.v. dFdC was approximately 35% to 54% of the dose, with only slightly more dFdU being excreted than dFdC. In contrast, previous reports suggest a total dFdC and dFdU excretion in urine of ~65% of the dose, with almost a 7:1 ratio of dFdU to dFdC (27). There are several possible explanations for this disparity. First, the plasma Cmax of ~100 µg/mL is 4-fold higher than the Km of CD (~25 µg/mL; ref. 28). Consequently, the metabolic pathway of dFdC to dFdU may have been approaching saturation. Perhaps more importantly, urinary collections from mice are generally associated with large experimental errors, gemcitabine may have been subjected to bacterial degradation, and the mouse kidney expresses high levels of CD (12, 29) and expresses the concentrative nucleoside transporter CNT1 and secretionary organic anion transporters (30). Consequently, our results should not be overinterpreted. After oral dosing of dFdC, only 1.7% of the dose was recovered in urine as dFdC, whereas this increased to 4.5% to 15% on coadministration of THU, either i.v. or orally. Conversely, excretion of dFdU was decreased by coadministration of THU, reflective of the inhibition of the deamination pathway by THU. It is conceivable that, at lower doses of dFdC, which result in tissue and plasma concentrations far below the Km of CD, the effect of THU on the metabolic conversion of dFdC will be more pronounced.

In general, pyrimidine nucleosides have a low oral bioavailability (15). Deamination by CD is the first metabolic step in the catabolism of dFdC, and using THU to block CD in gastrointestinal epithelium and liver should decrease first-pass metabolism of dFdC (19, 29). Because of the insensitivity of plasma dFdC half-life to THU, the increase in dFdC AUC after oral administration of dFdC, when codosed with THU, is expected to be less than the 58-fold increase in FdCyd AUC as observed when FdCyd was combined with THU (15). Indeed, oral THU increased the oral bioavailability of dFdC only 4-fold (from 10% to 40%). Although modest, this increase is nonetheless relevant; 40% of the oral dose reaches the plasma compartment. Naturally, clinical trials will have to establish the improvement of dFdC oral bioavailability by THU in humans. The dFdC AUC ratio of oral dFdC with i.v. THU to i.v. dFdC with i.v. THU is an indication of what fraction of a dFdC dose is actually absorbed from the gut lumen, as explained earlier (15). Our experiments show that, for dFdC, this seemed to be ~59%, comparable with the value of 81% reported for FdCyd (15).

dFdU is a major metabolite of dFdC, and THU can substantially reduce the exposure to dFdU. dFdU, although often considered inactive, may be anabolized to dFdU monophosphate, reported to be a thymidylate synthase inhibitor (6). In addition, production of dFdUTP (7), and its incorporation into both DNA and RNA, has been observed in vitro, with associated cytotoxicity (8). dFdU may add to the therapeutic effects of dFdC treatment but could very well cause undesired side effects. Reduction of dFdU production through THU coadministration results in pharmacologic effects more strictly related to the parent dFdC, with still undefined clinical relevance. In agreement with the >65 h terminal half-life of dFdU in humans (9), we observed a very long plasma half-life of ~24 h in mice, with dFdU being detected until the last sampling time point in all studies. Accurate determination of the dFdU half-life, which by convention requires following an analyte for at least 3 to 4 half-lives, was not possible because of the 24-h duration of the study.

Because we had previously developed an assay to quantitate THU in mouse plasma (26), we split the plasma samples and used one aliquot to quantitate THU. The pharmacokinetics of THU in this study were very similar to our earlier findings when investigating the pharmacokinetics and oral bioavailability of THU in mice (18). At a first glance, the coadministration of oral dFdC 30 min after oral THU seemed to reduce the absorption of THU from the gut. This would not be unlikely because THU has been reported to share the facilitated nucleoside diffusion carrier with cytidine analogues (31) and could compete with dFdC for nucleoside transporters that facilitate the absorption of nucleosides from the gut lumen into the systemic circulation (30, 32). However, on closer inspection, even before administration of dFdC, the two THU plasma concentration versus time curves are divergent, and the variability in the data points is too large to distinguish an effect of dFdC on THU absorption. The difference in the THU AUC after oral administration may only be due to inherent variability of the oral administration route for compounds with a moderately low oral bioavailability, such as THU at 20% (18).

As discussed previously, the safety of THU has been established both preclinically and clinically (12, 13, 33, 34). The ~20% oral bioavailability of THU in mice is obviously enough to enhance the bioavailability of cytidine analogues and reduce their metabolic elimination. In addition, THU can improve the exposure of organs with high CD activity to cytidine analogues and possibly increase the intracellular accumulation of the active CTPs (4, 31, 35–39). This will be relevant to both the effectiveness and toxicity of cytidine drugs codosed with THU.

Currently, dFdC administration is limited to the i.v. route. The availability of an oral formulation would have several benefits. First, it would be more convenient for patients to take a pill than to visit the clinic to receive their dFdC dose. It is much cheaper to dispense a pill than to administer an i.v. infusion. Oral dosing also allows exploration of different dosing schedules that would currently be considered too cumbersome, such as daily dosing or dosing every other day. Given the schedule-dependent activity of dFdC, alternative dosing schedules may yield better responses. The more prolonged exposure of an oral dose also mimics more closely the exposure to dFdC after a fixed dose rate schedule, which is receiving more interest of late (40, 41). Lastly, THU could sensitize tumors and organ environments to dFdC by blocking highly expressed CD. Conversely, the addition of THU may change the toxicity profile of dFdC by affecting organ systems, such as the gut and liver, which are otherwise protected by CD. Clinical trials are warranted to establish the relevance of these findings for human use of THU as a modifier of metabolism of dFdC and other cytidine analogues.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
We thank the University of Pittsburgh Cancer Institute, Hematology/Oncology Writing Group for constructive suggestions about the manuscript, Jeremy Hedges for administrative support, and Diane Mazzei and her colleagues in the University of Pittsburgh Animal Facility, without whose expert assistance these studies would not have been possible.


   Footnotes
 
Grant support: National Cancer Institute grants NO1-CM-52202 and P30-CA47904.

Note: WinNonlin software was provided as part of the Pharsight Academic Licensing Program.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 Ebeling C. BRENDA: the comprehensive enzyme information system. 2007. Available from: http://www.brenda.uni-koeln.de/index.php4. Back

7 U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, and Center for Veterinary Medicine. Guidance for industry—bioanalytical method validation. 2001. Available from: http://www.fda.gov/cder/guidance/index.htm. Back

Received 11/18/07; revised 2/12/08; accepted 2/12/08.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Toschi L, Finocchiaro G, Bartolini S, Gioia V, Cappuzzo F. Role of gemcitabine in cancer therapy. Future Oncol 2005;1:7–17.[CrossRef][Medline]
  2. Plunkett W, Huang P, Searcy CE, Gandhi V. Gemcitabine: preclinical pharmacology and mechanisms of action. Semin Oncol 1996;23:3–15.[Medline]
  3. Camiener GW, Smith CG. Studies of the enzymatic deamination of cytosine arabinoside. I. Enzyme distribution and species specificity. Biochem Pharmacol 1965;14:1405–16.[CrossRef][Medline]
  4. Laliberte J, Marquez VE, Momparler RL. Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2'-deoxycytidine by human cytidine deaminase. Cancer Chemother Pharmacol 1992;30:7–11.[Medline]
  5. Veerman G, Ruiz van Haperen V, Vermorken JB, et al. Antitumor activity of prolonged as compared with bolus administration of 2',2'-difluorodeoxycytidine in vivo against murine colon tumors. Cancer Chemother Pharmacol 1996;38:335–42.[CrossRef][Medline]
  6. Bergman AM, Pinedo HM, Peters GJ. Determinants of resistance to 2',2'-difluorodeoxycytidine (gemcitabine). Drug Resist Updat 2002;5:19–33.[CrossRef][Medline]
  7. Plunkett W, Huang P, Xu YZ, Heinemann V, Grunewald R, Gandhi V. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin Oncol 1995;22:3–10.[Medline]
  8. Veltkamp SA, Van Eijndhoven MA, Pluim D, et al. Gemcitabine metabolite, 2',2'-difluorodeoxyuridine (dFdU), can be phosphorylated and incorporated into nucleic acids. Proc Am Assoc Cancer Res 2007;48:366.
  9. Storniolo AM, Allerheiligen SR, Pearce HL. Preclinical, pharmacologic, and phase I studies of gemcitabine. Semin Oncol 1997;24:S7.[Medline]
  10. Stoller RG, Myers CE, Chabner BA. Analysis of cytidine deaminase and tetrahydrouridine interaction by use of ligand techniques. Biochem Pharmacol 1978;27:53–9.[CrossRef][Medline]
  11. Cohen RM, Wolfenden R. Cytidine deaminase from Escherichia coli. Purification, properties and inhibition by the potential transition state analog 3,4,5,6-tetrahydrouridine. J Biol Chem 1971;246:7561–5.[Abstract/Free Full Text]
  12. Hale JT, Bigelow JC, Mathews LA, McCormack JJ. Analytical and pharmacokinetic studies with 5-chloro-2'-deoxycytidine. Biochem Pharmacol 2002;64:1493–502.[CrossRef][Medline]
  13. Dareer SM, Mulligan LT, Jr., White V, Tillery K, Mellett LB, Hill DL. Distribution of [3H]cytosine arabinoside and its products in mice, dogs, and monkeys and effect of tetrahydrouridine. Cancer Treat Rep 1977;61:395–407.[Medline]
  14. Kreis W, Chan K, Budman DR, et al. Effect of tetrahydrouridine on the clinical pharmacology of 1-β-D-arabinofuranosylcytosine when both drugs are coinfused over three hours. Cancer Res 1988;48:1337–42.[Abstract/Free Full Text]
  15. Beumer JH, Eiseman JL, Parise RA, et al. Pharmacokinetics, metabolism, and oral bioavailability of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine in mice. Clin Cancer Res 2006;12:7483–91.[Abstract/Free Full Text]
  16. Neil GL, Moxley TE, Kuentzel SL, Manak RC, Hanka LJ. Enhancement by tetrahydrouridine (NSC-112907) of the oral activity of 5-azacytidine (NSC-102816) in L1210 leukemic mice. Cancer Chemother Rep 1975;59:459–65.[Medline]
  17. Neil GL, Moxley TE, Manak RC. Enhancement by tetrahydrouridine of 1-β-D-arabinofuranosylcytosine (cytarabine) oral activity in L1210 leukemic mice. Cancer Res 1970;30:2166–72.[Abstract/Free Full Text]
  18. Beumer JH, Eiseman JL, Parise RA, et al. Plasma pharmacokinetics and oral bioavailability of 3,4,5,6-tetrahydrouridine, a cytidine deaminase inhibitor, in mice. Cancer Chemother Pharmacol 2007 Epub ahead of print.
  19. Mekras JA, Boothman DA, Greer SB. Use of 5-trifluoromethyldeoxycytidine and tetrahydrouridine to circumvent catabolism and exploit high levels of cytidine deaminase in tumors to achieve DNA- and target-directed therapies. Cancer Res 1985;45:5270–80.[Abstract/Free Full Text]
  20. Boothman DA, Briggle TV, Greer S. Protective, tumor-selective dual pathway activation of 5-fluoro-2'-deoxycytidine provided by tetrahydrouridine in mice bearing mammary adenocarcinoma-755. Cancer Res 1987;47:2344–53.[Abstract/Free Full Text]
  21. Bengala C, Guarneri V, Giovannetti E, et al. Prolonged fixed dose rate infusion of gemcitabine with autologous haemopoietic support in advanced pancreatic adenocarcinoma. Br J Cancer 2005;93:35–40.[CrossRef][Medline]
  22. Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper HE. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep 1966;50:219–44.[Medline]
  23. Beumer JH, Parise RA, Newman EM, et al. Concentrations of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine (FdCyd) and its cytotoxic metabolites in plasma of patients treated with FdCyd and tetrahydrouridine (THU). Cancer Chemother Pharmacol 2007. Epub ahead of print.
  24. Newman EM, Longmate J, Lenz H, et al. Phase I and clinical pharmacokinetic evaluation of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine: a California Cancer Consortium study. Proc Am Soc Clin Oncol 2002;21:108a.
  25. Olsen BA. Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities. J Chromatogr A 2001;913:113–22.[CrossRef][Medline]
  26. Parise RA, Egorin MJ, Eiseman JL, Joseph E, Covey JM, Beumer JH. Quantitative determination of the cytidine deaminase inhibitor tetrahydrouridine (THU) in mouse plasma by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2007;21:1991–7.[CrossRef][Medline]
  27. Shipley LA, Brown TJ, Cornpropst JD, Hamilton M, Daniels WD, Culp HW. Metabolism and disposition of gemcitabine, and oncolytic deoxycytidine analog, in mice, rats, and dogs. Drug Metab Dispos 1992;20:849–55.[Abstract]
  28. Bouffard DY, Laliberte J, Momparler RL. Kinetic studies on 2',2'-difluorodeoxycytidine (gemcitabine) with purified human deoxycytidine kinase and cytidine deaminase. Biochem Pharmacol 1993;45:1857–61.[CrossRef][Medline]
  29. Kelly CJ, Gaudio L, Yesair DW, Schoenemann PT, Wodinsky I. Pharmacokinetic considerations in evaluating the effects of tetrahydrouridine on 5-azacytidine chemotherapy in L1210 leukemic mice. Cancer Treat Rep 1978;62:1025–32.[Medline]
  30. Clarke ML, Mackey JR, Baldwin SA, Young JD, Cass CE. The role of membrane transporters in cellular resistance to anticancer nucleoside drugs. Cancer Treat Res 2002;112:27–47.[Medline]
  31. Grant S, Bhalla K, McCrady C. Effect of tetrahydrouridine and deoxytetrahydrouridine on the interaction between 2'-deoxycytidine and 1-β-D-arabinofuranosylcytosine in human leukemia cells. Leuk Res 1991;15:205–13.[CrossRef][Medline]
  32. Mackey JR, Mani RS, Selner M, et al. Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines. Cancer Res 1998;58:4349–57.[Abstract/Free Full Text]
  33. Xiang TX, Niemi R, Bummer P, Anderson BD. Epimer interconversion, isomerization, and hydrolysis of tetrahydrouridine: implications for cytidine deaminase inhibition. J Pharm Sci 2003;92:2027–39.[CrossRef][Medline]
  34. Heinemann V, Plunkett W. Modulation of deoxynucleotide metabolism by the deoxycytidylate deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine. Biochem Pharmacol 1989;38:4115–21.[CrossRef][Medline]
  35. Furner RL, Mellett LB, Herren TC. Influence of tetrahydrouridine on the phosphorylation of 1-β-D-arabinofuranosyl-cytosine (ara-C) by enzymes from solid tumors in vitro. J Pharmacol Exp Ther 1975;194:103–10.[Abstract/Free Full Text]
  36. Riva C, Barra Y, Carcassonne Y, Cano JP, Rustum Y. Effect of tetrahydrouridine on metabolism and transport of 1-β-D-arabinofuranosylcytosine in human cells. Chemotherapy 1992;38:358–66.[Medline]
  37. North TW, Mathews CK. Tetrahydrouridine specifically facilitates deoxycytidine incorporation into herpes simplex virus DNA. J Virol 1981;37:987–93.[Abstract/Free Full Text]
  38. Chou TC, Arlin Z, Clarkson BD, Phillips FS. Metabolism of 1-β-D-arabinofuranosylcytosine in human leukemic cells. Cancer Res 1977;37:3561–70.[Abstract/Free Full Text]
  39. Ho DH, Carter CJ, Brown NS, et al. Effects of tetrahydrouridine on the uptake and metabolism of 1-β-D-arabinofuranosylcytosine in human normal and leukemic cells. Cancer Res 1980;40:2444–6.[Medline]
  40. Gandhi V. Questions about gemcitabine dose rate: answered or unanswered? J Clin Oncol 2007;25:5691–4.[Free Full Text]
  41. Grimison P, Galettis P, Manners S, et al. Randomized crossover study evaluating the effect of gemcitabine infusion dose rate: evidence of auto-induction of gemcitabine accumulation. J Clin Oncol 2007;25:5704–9.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beumer, J. H.
Right arrow Articles by Egorin, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beumer, J. H.
Right arrow Articles by Egorin, M. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online