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Chem. Res. Toxicol. 2002, 15, 922-926
Quantification of 2′-Fluoro-2′-deoxyuridine and 2′-Fluoro-2′-deoxycytidine in DNA and RNA Isolated from Rats and Woodchucks Using LC/MS/MS Frank C. Richardson,*,† Chenghong Zhang,‡ S. Russ Lehrman,§ Hasan Koc,| James A. Swenberg,| Katherine A. Richardson,⊥ and Raymond A. Bendele† OSI Pharmaceuticals, Inc., Boulder, Colorado 80301, Pfizer, Inc., Groton, Connecticut 06340, Inhale Therapeutic Systems, Inc., San Carlos, California 94070, University of North Carolina, Chapel Hill, North Carolina 27509, and Molecular Biology Experimental Design, Louisville, Colorado 80027 Received February 14, 2002
Apatmers are synthesized using 2′-fluoropyrimdines in place of normal pyrmidines to stabilize them against enzymatic degradation, and thereby improve their therapeutic efficacy. Despite this stabilizing effect, the apatmers can still be degraded by nucleases in the blood. Primer template extension studies have demonstrated that mammalian DNA polymerases can incorporate these 2′-fluoropyrimidines into growing strands of DNA. The toxicologic effects of these compounds have been examined in rats and woodchucks, animals known to be susceptible to the toxic effects of other modified pyrimidines. Whether these nucleosides can be incorporated into DNA in vivo has not been established. These studies report the development of methodologies and the results of studies designed to determine if and to what extent 2′-fluoropyrimidines are incorporated into tissue DNA following long-term treatment. Rats were dosed intravenously with either 2′-fluorouridine (2′-FU) or 2′-fluorocytidine (2′-FC) at doses of 5, 50, and 500 mg/kg/day for 90 days. Woodchucks were dosed intravenously with either 2′-FU or 2′-FC at doses of 0.75 or 7.5 mg/kg/day for 90 days. The amounts of 2′-FU or 2′-FC in DNA and RNA were quantified using newly developed LC/MS/MS methodologies. Administration of 2′-FU to rats and woodchucks resulted in incorporation of the compound into DNA from liver, spleen, testis, muscle, and kidney. Incorporation also occurred in RNA from rat liver (only tissue examined). Similarly, administration of 2′-FC to rats and woodchucks resulted in incorporation into liver DNA (only tissue examined). These data demonstrate that 2′-fluoropyrimidines are incorporated into DNA and RNA of various tissues of rats and woodchucks following long-term administration.
Introduction Nucleoside analogues have been used in the development of therapeutic aptamers. In particular, 2′-deoxy2′-fluorouridine (2′-FU), 2′-deoxy-2′-fluorocytidine (2′-FC), 2′-O-methylguanosine, and 2′-O-methyladenosine have been used to increase the in vivo half-life of aptamers by reducing their degradation by endogenous nucleases (1, 2), thereby making them more amenable for use as therapeutics. While degradation has been slowed, it has not been eliminated, and the biological fate of these nucleoside analogues has not been studied. Nuclear and/or mitochondrial DNA incorporation has been implicated or correlated with the toxicities of several antiviral and anticancer nucleoside analogues, including fludarabine, araC, gemcitabine, 2′,2′-difluorodeoxyguanosine, ddC, AZT, and fialuridine (3-9). Work in this laboratory evaluated the toxicities of 2′-FC and 2′-FU and the potential of 2′-FC and 2′-FU to be incorporated into * To whom correspondence should be addressed at OSI Pharmaceuticals, Inc., 2860 Wilderness Place, Boulder, CO 80301. Phone: 303546-7764, FAX: 303-444-0672. † OSI Pharmaceuticals, Inc. ‡ Pfizer, Inc. § Inhale Therapeutic Systems, Inc. | University of North Carolina, Chapel Hill. ⊥ Molecular Biology Experimental Design.
DNA using polymerase extension assays. Results from 90-day toxicity studies in rats and woodchucks showed that 2′-FU and 2′-FC were relatively nontoxic and did not induce delayed toxicities associated with other fluoronucleosides (10). Polymerase extension/incorporation studies demonstrated that several DNA polymerases could incorporate 2′-fluoropyrimidines (11-13) into DNA (but not 2′-O-methylpurines), suggesting that similar incorporation could occur in nuclear and mitochondrial DNA. Whether the 2′-fluoropyrimidines could be incorporated into DNA in vivo following long-term exposure remained unknown. The studies described here sought to answer this question. Radiolabeled nucleosides and antibodies have been used to detect and quantify nucleoside incorporation into DNA. In long-term studies, the use of radiolabeled tracers is impractical. Antibody detection systems require the development of antibodies to each nucleoside with sufficient specificity and sensitivity to allow quantification at low concentrations of the modified nucleosides among large concentrations of normal nucleosides; this can be overcome by labor-intensive preseparation methods. LC/ MS techniques offer flexibility, the potential to quantify numerous analytes in a single analysis, and have been used to measure and detect chemical modifications in DNA (14-16) (a complete review is beyond the scope of
10.1021/tx020014d CCC: $22.00 © 2002 American Chemical Society Published on Web 06/14/2002
2′-FU and 2′-FC in DNA of Rats and Woodchucks
this paper) and to quantify plasma concentrations of lamivudine and zidovudine (17). We report development of LC/MS/MS methods to detect and quantify the nucleosides 2′-FU and 2′-FC in enzymatically hydrolyzed DNA. These methods were used to determine whether, and to what extent, 2′-FU and 2′-FC are incorporated into DNA of rats or woodchucks following chronic treatment with either 2′-FC or 2′-FU.
Materials and Methods Test Articles. 2′-FU (purity >98.5%, MW ) 246.2, formula ) C9H11N2O5F) and 2′-FC hydrochloride (purity >98.5%, MW ) 272.57, formula ) C9H12N3O4F‚0.75 HCl) were manufactured by JBL Scientific, Inc., San Luis Obispo, CA. Animals and Dosing. DNA was isolated from animals that were used in studies evaluating the safety of 2′-FU and 2′-FC (10). Rats were chosen because they are routinely used in assessing the long-term safety of drug candidates. Woodchucks were chosen because recent studies have indicated that they may be particularly sensitive to chronic nucleoside toxicity (18). Compounds were administered parenterally because it is the anticipated route of administration for most aptamer-based therapeutics. All animals were maintained in compliance with the Animal Welfare Act, the Institutional Animal Care and Use Committee, and the Guide for Care and Use of Laboratory Animals (19). Rats were fed Purina 5001 Rat Chow, and woodchucks were fed Purina Rabbit Chow ad libitum. Water was provided ad libitum. Rats were maintained under a 12-h light/dark cycle, and woodchucks were maintained on a light/ dark cycle that corresponded to seasonal lighting patterns. Male Fisher 344 rats (Taconic Farms, Hanover, NY), 125175 g, were dosed with either 2′-FU or 2′-FC via the lateral tail vein at doses of 0, 5, 50, or 500 mg/kg/day for 90 consecutive days. Captive bred woodchucks (Marmota monax) (Marmotech Inc., Ithaca, NY) were dosed by the femoral vein with either 2′-FU or 2′-FC at doses of 0, 0.75, or 7.5 mg/kg/day for 90 consecutive days. Tissue Collection, Storage, DNA Extraction, and Hydrolysis. All animals were humanely euthanized prior to tissue collection in accordance with the 1993 AVMA Guidelines (20). Tissues were removed from animals, immediately frozen in liquid nitrogen, and stored at or below -70 °C until processed for DNA or RNA extraction. DNA was isolated from selected tissues using the Genomic ASAP DNA Isolation Kit (Boehringer Mannheim, Indianapolis, IN). The DNA (75 µL, 1.33 mg/mL solution) was enzymatically hydrolyzed to component nucleosides by adding the following: 7.5 µL of 10 mM MgCl2; 10 µL of 10 mM Tris-HCl, pH 6.5; and 3.5 µL of DNase I (2000 units/ mL) (Sigma Chemical Co., St. Louis, MO) and heating at 37 °C for 2 h. This was followed by adding the following: 7.5 µL of 100 mM Tris-HCl, pH 8.3; 3.5 µL of snake venom phosphodiesterase (100 units/mL, Worthington Enzymes, Freehold, NJ); 3.5 µL of bacterial alkaline phosphatase (200 units/mL, Sigma Chemical Co.), and heating at 37 °C for an additional 12-16 h. RNA was extracted from selected tissues using the RNAgents Kit (Promega, Madison, WI). The RNA, 100 µL of 1 µg/µL, was enzymatically hydrolyzed to component ribonucleosides using DNase-free RNase (Boehringner-Mannheim, Indianapolis, IN) and bacterial alkaline phosphatase (200 units/mL, Sigma Chemical Co.). Enzymatic hydrolyses of DNA and RNA were verified, and thymidine (dThd) and guanosine (Gua) concentrations were quantified using high-pressure liquid chromatography (HPLC). HPLC conditions were as follows: 1100 Hewlett-Packard HPLC (Wilmington, DE); Supelco Supelcosil 5 µm LC-18S column, 4 mm × 25 cm; flow rate 1.5 mL/min; isocratic buffer of 12% methanol in 50 mM KPO4; monitor at 260 nm with an 1100 Hewlett-Packard Diode Array Detector. LS/MS/MS Quantification of 2′-FU. One hundred microliters of a 100 ng/mL internal standard of 2′-deoxy-2′-fluoro-
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 923 uridine-5,6-D2 (2′-FUIS) (CDN Isotopes, Quebec, Canada) was added to each DNA or RNA hydrolysate as well as standard and quality control samples. The upper and lower limits of quantification for 100 µL of sample were 0.1 and 400 ng/mL, respectively. Subsequently, 100 µL of 65 mM acetic acid was added, and each sample was extracted on a Gilson ASPEC XL4 (Gilson, Middleton, WI) using Isolute MF C18 SPE extraction cartridges (Burdick and Jackson, Muskegon, MI.) The analytes were eluted with 0.5 mL of methanol and dried under nitrogen. The dried extracts were dissolved in 100 µL of NANOpure water (Barnstead model D7331, Dubuque, IA) for subsequent LC/MS/ MS analysis. Analyses were performed with a Hewlett-Packard 1090 pump and autosampler. Twenty microliters of the extracted sample was injected onto a J’Sphere M80, 4 µm column (100 × 2.0 mm i.d.). The mobile phases consisted of NANOpure water as solvent A and methanol as solvent B. Each analysis was performed using a mobile phase flow rate of 0.2 mL/min and a gradient that started from 8/92 B/A and ended at 20/80 of B/A over 4.5 min, ramped to 100% B over 3.7 min, held at 100% B for 1 min, and equilibrated at 8/92 B/A for 2 min. A PE SCIEX API III (Concord Ontario, Canada) atmospheric pressure ionization (API) triple quadrupole mass spectrometer was used for 2′-FU detection and quantification. Nitrogen was used as the nebulizing gas and was maintained at 50 psi for the TurboIonSpray LC/MS interface. Rodapex mass calibration solution (Rhone Poulenc) was used for time and mass-axis calibration for each mass-resolving quadrupole. Both Q1 and Q3 mass analyzers were operated under unit mass resolution conditions. Ultrapure argon was used as the collision gas in the collision cell (Q2). The mass spectrometer was programmed to focus the deprotonated molecule ions [M-H]- at m/z of 245 and 247 for 2′-FU and the internal standard 2′-FUIS, respectively, via the first quadrupole mass filter (Q1) with collision-induced fragmentation in Q2 and monitoring of the product ions at m/z of 111 (2′-FU) and 113 (2′-FUIS) in Q3 using the selected reaction monitoring (SRM) mode. 2′-FU was monitored in the negative ion mode. The collision gas thickness was maintained at approximately 2.5 × 1014 atoms/cm2, which produced a collision energy of 12 eV. The dwell time for each monitored transition was 333 ms. Data were collected energy in PE-SCIEXRAD software. Peak area ratios obtained from SRM of the analyte (m/z of 245-111) and the internal standard (m/z of 247113) were computed using SCIEX’s MacQuan software from the corresponding chromatographic peak areas. The calibration curve was fit by a weighted (1/x2) linear regression. LS/MS/MS Quantification of 2′-FC. Microcon-10 filters were washed with 500 µL of water. The equivalent of 20 µg of DNA hydrolysate was mixed with 20 µL of 2.42 µM 2′-FUIS, placed into the Microcon-10 filter, and centrifuged for 20 min. The filters where subsequently washed with water sufficient to create a final filtrate volume of 100 µL. Ten microliters of filtrate was injected into the LC/ESI/MS/MS for 2′-FC quantification. Sample analysis was conducted on a Finnigan TSQ 7000 triple quadrupole mass spectrometer with an API2 electrospray source and a Magic 2002 micro HPLC unit (Michrom Bioresources) A Supelcosil LC-18-DB 5 µm reverse phase column (2.1 × 250 mm i.d.) was used to separate the analytes. The mobile phase consisted of NANOpure water as solvent A and methanol as solvent B. The analysis was performed using a mobile phase flow rate of 0.2 mL/min and a gradient that started at 2/98 B/A for 15 min, ramp to 97/3 B/A over 10 min, held at 97/3 B/A for 8 min, and reequilibrated at 2/98 B/A for 20 min. The electrospray ionization was performed in the positive ion mode. Spray voltage was set at 4.5 kV. Sheath and auxiliary gases were set at 80 and 40 psi, respectively. The heated transfer capillary was maintained at 350 °C for desolvation. Tandem mass spectrometric detection was done in the selected reaction monitoring (SRM) mode. The SRM transitions used for 2′-FC and 2′-FUIS were m/z 255.9-111.7 and m/z 248.9-114.9, respectively. A collision offset voltage of -25 V for collison-
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Table 1. Inter-Assay Accuracy and Precision of 2′-FU in a Mixture of Hydrolyzed Rat Liver and Spleen DNA theor concn of 2′-FU (ng/mL)
mean concn of 2′-FU (ng/mL)
standard deviation
precision (%)
accuracy (%)
0.1 0.25 0.75 1 10 100 200 400
0.094 0.259 0.696 1.023 10.05 100.8 202.5 397.5
0.010 0.007 0.039 0.050 0.278 1.711 2.000 14.30
10.6 2.7 5.6 4.9 2.8 1.7 1.0 3.6
94 104 93 102 100 101 101 99
induced dissociation was used for both 2′-FC and 2′-FUIS. Data collection was done using Xcalibur software running on a Windows NT operating system. Calculation of DNA Incorporation. Standard curves generated for 2′-FU and 2′-FC were used to calculate the concentration (pmol/mL) of analyte in each sample. The picomoles of either 2′-FU or 2′-FC were then divided by the concentration of thymidine (DNA) or guanosine (RNA) (µmol/ mL) in the same sample to provide final concentrations of incorporation in picomoles of 2′-fluoropyrimidine/µmol of thymidine for DNA and in picomoles of 2′-fluoropyrimidine/µmol of guanosine for RNA.
Results This study reports the use of LC/MS/MS to detect and quantify 2′-FU and 2′-FC in DNA and RNA. Table 1 provides the inter-assay accuracy and precision of the 2′FU assay when samples were spiked into a mixture of DNA from rat liver and spleen. Using the methods described, lower limits of detection of 2-6 pmol of 2′-FU/ µmol of dThd in DNA were achieved. This was based on a 1 mg/mL DNA solution with 20 µL injection volume and the fact that unidentified interference (see control rats in Table 2) made results below this concentration unreliable. Quantification of 2′-FC was achieved using a separate method of separation and a standard curve generated using spiked enzymatically hydrolyzed DNA. A standard curve was generated with standards ranging from oncolumn injections of 125 pmol of 2′-FC, generating a peak area of 1335-15 000 pmol of 2′-FC, which generated a peak area of 160 049. Least-squares fit of the data
generated a calibration curve of: picomoles ) (peak area)(0.2478) - 0.0867. The standard errors for the x coefficient and the constant were 0.0098 and 0.1298, respectively, and the R2 value was 0.9922. The on-column detection limit was estimated to be ∼60 fmol based on the peak area generated by the lowest concentration calibration standard prepared by spiking the standard into DNA hydrolysate. Water appeared to form an adduct with dC that had the same m/z as 2′-FC during the electrospray ionization process; a longer HPLC retention time was therefore required to provide adequate separation between this water adduct and 2′-FC. Because an internal standard of isotopically labeled 2′-FC could not be obtained, 2′-FUIS was used as an internal standard for the 2′-FC assay; this may have limited the sensitivity of this method. 2′-FU was present in DNA of tissues from rats and woodchucks given the molecule by the intravenous route for 90 days (Table 2). In the rat, the mean concentration of 2′-FU in DNA was dose-dependent and greatest in the liver and smallest in the testis. While mean 2′-FU concentrations in the liver DNA were dose-dependent, they were not dose-proportional, increasing only 5-fold over a 100-fold dose range. 2′-FU was also incorporated into RNA from rat liver tissue at mean concentrations approximately 5-10-fold less than DNA. Mean 2′-FU concentrations in liver DNA from woodchucks were dosedependent and, in contrast to the rat, were doseproportional over the 10-fold range in doses used in this study. 2′-FC was present in the hepatic DNA of rats and woodchucks that received 2′-FC by an intravenous route for 90 days (Table 3). In contrast to 2′-FU, the concentrations of 2′-FC in the liver of rats were considerably greater than in woodchucks, although not when adjusted for administered dose.
Discussion These studies used newly developed LC/MS/MS procedures to determine if 2′-fluoropyrmidines, used to stabilize aptamers against enzymatic degradation, were incorporated into tissue DNA following long-term administration and whether administration and degrada-
Table 2. Incorporation of 2′-FU into DNA and RNA from Rat Tissues and DNA from Woodchuck Liver DNAa treatment rat 0 mg of 2′-FU/kg/day 5 mg of 2′-FU/kg/day 50 mg of 2′-FU/kg/day 500 mg of 2′-FU/kg/day woodchuck 0 mg of 2′-FU/kg/day
kidney 2.7 ( 2.0 [3]b 18.3 ( 1.4 [3] NDd 150.8 ( 71.0 [3] ND
0.75 mg of 2′-FU/kg/day
ND
7.5 mg of 2′-FU/kg/day
ND
liver
muscle
RNAa spleen
testes
liver
2.8 ( 0.3 [3] 48.4 ( 5.1 [3] 128.6 ( 17.1 [3] 205.0 ( 33.4 [3]
BLQc [3] 53.5 ( 4.5 [3] ND
5.8 ( 3.0 [3] 24.3 ( 2.0 [3] ND
4.2 ( 2.4 [3] 22.3 ( 1.4 [3] ND
2.1 [1] 4.3 ( 0.8 [3] ND
168.0 ( 16.2 [3]
161.7 ( 14.9 [2]
43.1 ( 13.7 [3]
42.7 ( 6.3 [3]
BLQ [3] 35.3 ( 12.5 [3] 407.7 ( 124.3 [3]
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
a DNA incorporation is pmol of 2′-FU/µmol of dThd. RNA incorporation is pmol of 2′-FU/µmol of Gua. b Number of animals is in brackets. BLQ ) below limit of quantitation, which is approximately 2-6 pmol of 2′-FU/µmol of dThd, depending on amount of DNA hydrolysate analyzed. d ND ) not determined. c
2′-FU and 2′-FC in DNA of Rats and Woodchucks Table 3. Incorporation of 2′-FC into DNA of Rat and Woodchucka treatment liver
500 mg/kg/day rat
7.5 mg/kg/day woodchuck
2301.3 ( 545.1 [5]
171.3 ( 77.5 [3]
a DNA incorporation is pmol of 2′-FC/µmol of dThd. b Number of animals in brackets.
tion of aptamers containing these nucleosides could result in their incorporation into DNA. The LC/MS/MS assay for 2′-FU described in this paper was able to quantify 2′-FU concentrations as low as 0.1 ng/mL (∼400 pM) in spiked DNA hydrolysate samples. With an injection volume of 20 µL, the on-column detection limit was approximately 0.002 pmol. Based on an injection volume of 20 µL and DNA concentration of 1 µg/µL (0.003 µmol of dThd/µL), the theoretical limit of detection of this assay should have been approximately 0.03 pmol of 2′-FU/µmol of dThd. However, interference from unknown sources occurred occasionally. Because of this interference, the limit of detection of 2′-FU in DNA was set at 2-6 pmol/µmol of dThd. Additional efforts to improve sample cleanup and modify separation conditions could bring the limit of the detection for 2′-FU closer to its theoretical limit. The assay for 2′-FC, with an oncolumn limit of detection of approximately 60 pmol, was less developed, and further work is necessary to optimize the sensitivity of this assay. While dependent on the molecule, current on-column limits of detection for DNA adducts and incorporated modified nucleosides appear to be in the low femtomole range (20-22). Thus, the LC/ MS/MS procedures reported in this paper appear to provide rapid and sensitive assays for quantifying 2′-FU and 2′-FC in DNA comparable to currently reported assays. DNA polymerases R and γ can incorporate 2′-FU and 2′-FC into DNA in in vitro systems. Results reported here demonstrate that administration of 2′-FU and 2′-FC to woodchucks and rats results in their incorporation into the DNA of various tissues with the liver having the greatest concentrations of 2′-FU and woodchucks incorporating 2′-FU into DNA with greater efficiency (based on administered dose) than rats. These findings are similar to those observed when fialuridine, a nucleoside once under development for hepatitis B, was administered to woodchucks and rats at similar concentrations (9) by the oral route. However, in toxicology studies, fialuridine caused delayed mitochondrial toxicity in both species, whereas 2′-FC and 2′FU did not cause any observed effects. While it may be that the inherent toxicities of these nucleosides are different, it is also critical to compare their molecular doses to ensure that vastly different incorporation rates did not occur. In this case, fialuridine concentrations in DNA were 10 times greater than the concentrations of 2′-FU observed here, correlating with DNA polymerase extension experiments (11), and no fialuridine was found in RNA. The greater amount of DNA incorporation may therefore play a role in the greater toxicity of fialuridine compared to 2′-FC and 2′-FU. These studies also demonstrated that these compounds reached potential target tissues, were phosphorylated, and were incorporated into DNA. This further supports the conclusions of the earlier safety assessment studies
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 925
by verifying that the compounds were adequately absorbed into the tissues of and phosphorylated by the test models. In summary, LC/MS/MS provided a sensitive method to detect and quantify 2′-FU and 2′-FC in DNA from tissues and with further validation could be usable for monitoring DNA in a clinical setting. Using this method, it was shown that 2′-FU and 2′-FC are incorporated into tissue DNA and RNA when administered chronically in safety assessment studies. These studies strongly suggest that administration and degradation of aptamer-based therapeutics containing these 2′-fluoropyrimidines could also lead to their incorporation into cellular DNA. Coupled with the data from previous safety assessment studies, the results would also suggest that the incorporation of these 2′-fluoropyrimidines into tissue DNA is an event with marginal toxicological consequences.
Acknowledgment. We thank Dr. Jack Henion and Dr. Steven Lowes of Advance Bioanalytical Systems, Ithaca, NY, and Anna Mazurkeiwicz for their assistance in the conduct of these studies. In addition, a portion of this research was supported, in part, by NIH Grants CA83369, P30-CA16086, and P30 ES10126.
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(13) Ono, T., Scalf, M., and Smith, L. M. (1997) 2′-Fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry. Nucleic Acids Res. 25, 4581-4588. (14) Dizdaroglu, M., Jaruga, P., and Rodriguez, H. (2001) Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry. Nucleic Acids Res. 29, 1-8. (15) Andrews, C. L., Vouros, P., and Harsch, A. (1999) Analysis of DNA adducts using high-performance separation techniques coupled to electrospray ionization mass spectrometry. J. Chromatogr., A 856 (1-2), 515-526. (16) Koc, H., and Swenberg, J. A. (2001) A review of applications of mass spectrometry to quantitation of DNA adducts. J. Chromatogr., B (in press). (17) Kenney, K. B., Wring, S. A., Carr, R. M., Wells, G. N., and Dunn, J. A. (2000) Simultaneous determination of zidovudine and lamivudine in human serum using HPLC with tandem mass spectrometry. J. Pharm. Biomed. Anal. 22, 967-983. (18) Tennant, B. C., Baldwin, B. H., Graham, L. A., Ascenzi, M. A., Hornbuckle, W. E., Rowland, P. H., Tochkov, I. A., Yeager, A. E., Erb, H. N., Colacino, J. M., Lopez, C., Engelhardt, J. A., Bowsher, R. R., Richardson, F. C., Lewis, W., Cote, P. J., Korba, B. E., and Gerin, J. L. (1998) Antiviral Activity and Toxicity of Fialuridine
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