Deoxythymidine (FT) - Ingenta Connect

38

Current Radiopharmaceuticals, 2012, 5, 38-46

Biochemistry and Biology of 2′-Fluoro-2′-Deoxythymidine (FT), A Putative Highly Selective Substrate for Thymidine Kinase Type 2 (TK2) Leonard I. Wiebe*,1, William Sun1,†, Aihua Zhou1, Jennifer Yang1, Elena V. Sjuvarsson2, Staffan Eriksson2, Robert J. Paproski1, Carol E. Cass1, Piyush Kumar1 and Edward E. Knaus3 1

Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada; 2Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, S-753 24 Uppsala, Sweden; 3Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8, Canada Abstract: 2′-Deoxy-2′-fluorothymidine (FT) is a bioisostere of both thymidine (TdR), in which F replaces H at C-2′ in the ribosyl configuration, and methyluridine, in which F replaces OH at C-2′ in the ribosyl configuration. Fluorine is bioisosteric with H with respect to atomic radius and is bioisosteric with OH with respect to polarity and H-bonding as an H acceptor. The consequences of this C-2′ F for H substitution on cytotoxicity, nucleoside transporter affinity, phosphorylation by thymidine kinases (TK1, TK2), cell uptake and biodistribution of FT in a murine tumor model are now reported. FT toxicity against a bank of murine and human cells was seen only at very high (~1 mM) concentrations, although the cellular uptake of [3H]FT in these cells was comparable to that of [3H]TdR over a 24 h period. Human equilibrative nucleoside transporters (hENT1, hENT2) displayed weaker affinity for FT than for TdR, but the concentrative transporters (hCNT1, hCNT2, hCNT3) had much higher affinities for FT. FT was phosphorylated by both mitochondrial thymidine kinase (TK2) (58 % of TdR) and cytosolic thymidine kinase (TK1) (39 % of TdR). Preliminary in vivo imaging with [18F]FT in mice bearing implanted KBALB and contralateral KBALB-STK tumors showed highly selective uptake, with a tumor:blood ratio of 33 in a small herpes simplex type 1 (HSV-1 TK) expressing tumor. In conclusion, [18F]FT appears to be a strong candidate for PET imaging of viral TK transgene imaging, based on its TK1:TK2 phosphorylation differential, its selective uptake by an HSV-TK expressing murine tumor model, its interaction with nucleoside transporters and its low toxicity.

Keywords: Nucleoside transport, thymidine kinase (TK), 2′-fluoro-2′-deoxythymidine (FT), PET imaging. INTRODUCTION Herpes simplex type 1 thymidine kinase (HSV-1 TK) has been exploited as a target for anti-herpesvirus therapy, based on its ability to phosphorylate certain nucleosides that are not good substrates for mammalian cytosolic thymidine kinase (TK1) [1]. This discovery spurred research into HSV1 TK gene transfer, and intensified the search for novel antiviral nucleoside analogs [2] and for selective substrates to exploit HSV-1 TK expression in genetically transformed cell systems for gene therapy [3]. Most experimental antiviral and anti-cancer nucleosides have failed as therapeutics ─ they were simply not efficacious or they were too toxic following metabolic activation [4]. Nonetheless, nucleoside analogs, including fluorinated nucleosides (Fig. 1), remain prime candidates for antiviral and anti-cancer therapy [5]. HSV-TK as a target for imaging herpes simplex encephalitis with radioiodinated substrate nucleosides proved equivocal [6, 7], but nuclear imaging of HSV-1 TK transgene expression using a variety of radiolabelled nucleosides in experi*Address correspondence to this author at the Division of Oncological Imaging, 1807 Cross Cancer Institute, Edmonton, T6G 1Z2, Canada; Tel: +1 780-432-8524; E-mail: [email protected] † Medimage Biopharmaceutical Technology Co. Ltd., Zhangjiang Pharma Valley Park, Shanghai 201203, P.R. China 1874-4710/12 $58.00+.00

mental tumor models has been efficacious [8-12] albeit with limited clinical success [13]. Cell proliferation imaging with radiolabelled nucleosides, which is dependent on nucleoside phosphorylation by cytosolic thymidine kinase (TK1), began with radioiodinated iododeoxyuridine (IUdR) [14], which was initially developed as an anticancer drug but was soon shown to have antiviral properties. Unfortunately, IUdR has low therapeutic efficacy and weak selectivity as a proliferation marker (imaging) because it is rapidly catabolised in vivo and has low solubility, making it difficult to attain effective intracellular concentrations. It is a good substrate for mammalian cytosolic nucleoside kinases, and when phosphorylated, interacts with many enzymes in a thymidine-like manner. Its facile incorporation into the cellular DNA of normal cells, cancer cells and infected cells expressing viral nucleoside kinases does contribute some therapeutic effect, but this is not disease-specific. Moreover, its accumulation in sub-cellular molecular fractions other than DNA imply that its uptake may not reflect cell proliferation. Interested readers are referred to early [15] and recent reviews [2] for more detail. Fluorosugar pyrimidine nucleosides such as 2′-fluoro-2′deoxyarabinothymidine (FMAU)[16] labelled with C-11 [17] and 3′-fluoro-2′,3′-dideoxythymidine (FLT)[18] labelled © 2012 Bentham Science Publishers

Biochemistry of 2′-Fluoro-2′-Deoxythymidine (FT)

H3C

NH N

N

O

N

O

FMAU

F O

N

The reported specificity of several 2′-fluororibonucleoside analogues for HSV-TK [26] prompted the current work, which includes studies of the transport, phosphorylation, in vitro uptake and in vivo biodistribution of FT. EXPERIMENTAL Materials FT and the radiolabelling precursor, 3-t-butoxycarbonyl1-(3′,5′-di-O-benzoyl-2′-O-p-nitrophenylsulfonyl-β-Darabinofuranosyl)thymine, were synthesized using literature procedures [27]. [3H]Methyl-2′-fluorothymidine [3H]FT; sp. act. 1 Ci/mmol; 1 mCi/mL) and [3H]methylthymidine [3H]TdR; sp. act. 25 Ci/mmol; 1 mCi/mL) were purchased (Moravek Biochemicals and Radiochemicals, Brea, CA). [18F]FT was prepared via SN2 displacement of 2′-O-nosyl on the protected precursor by [18F]fluoride, followed by deprotection, using a GE TracerLab FX automated synthesis unit (ASU; GE Medical Systems; Milwaukee, WI) [27]. [18F]FT was recovered with radiochemical purity >94% and an average overall radiochemical yield of 5.5 %. The human 143B osteosarcoma cell line, obtained from American Type Culture Collection (ATCC, Manassas, VA), is a TK- mutant of R-970-5 that is tumorigenic in nude mice. The 143B-LTK cell line was derived from parental 143B cells by transduction with a replication incompetent amphotropic Moloney murine leukemia virus vector (LSNtk) with the HSV-1 TK gene being directed by the 5′-LTR and neoR gene under control of a SV-40 early promoter/enhancer [28]. The murine KBALB cell line was obtained from ATCC, and the KBALB-STK cell line, transduced with a retroviral vector possessing the HSV-1 TK gene under direction of the SV-40 early promoter/enhancer and neoR gene driven by the 5′-LTR, was obtained from Dr. Scott Freeman (Tulane University). Cell lines were maintained as suspen-

O

F

O

HO FLT

Fig. (1). Structures of thymidine (TdR) and the fluorinated nucleosides 2′-deoxy-2′-fluorothymidine deoxyarabinothymidine (FMAU), 3′-fluoro-2′,3′-dideoxythymidine (FLT) and 5-fluoro-2′-deoxyuridine (FUdR).

with F-18 [19] are among the leading radiotracers for cell proliferation imaging. [18F]FLT has achieved almost universal acceptance as the clinical imaging agent of choice for cell proliferation when used with positron emission tomography (PET) [20, 21]. 2′-Deoxy-2′-fluorothymidine (FT; CAS 122799-38-6; Fig. 1) [22, 23], a 2′-fluororibonucleoside, is virtually devoid of antiviral activity [24], but has been used as a backbone stabilizer in oligonucleotide synthesis [25]. Chemical structures of several fluoropyrimidine nucleosides are shown in Fig. (1).

NH

HO O

OH HF

39

O NH

N

O F F

FT

O HO

O

OH TdR

O H3C

NH

HO

O

OH

H3C

NH

HO

HO

O

O

O H3C

Current Radiopharmaceuticals, 2012, Vol. 5, No. 1

FUdR

(FT),

2′-fluoro-2′-

sions in Dulbecco′s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL). 143B cells were maintained in medium containing 5-bromo-2′-deoxyuridine (15 µg/mL). KBALB-STK and 143B-LTK cells were maintained in medium that contained geneticin (G-418; 1 mg/mL) in addition to FBS and antibiotics. EMT-6, a transplantable mouse mammary tumor cell line, was obtained from ATCC. Cells were cultured at 37 °C in a 5 % CO2 atmosphere. FT Cytotoxicity by MTT Assay Cells (EMT-6, KBALB, KBALB-STK, 143B, 143BLTK; 8 x 102 cells/mL) suspended in DMEM supplemented with 10 % FBS were plated (100 µL) into 96-well microplates and incubated for 24 h at 37° C in 5 % CO2. Various concentrations of test nucleosides in DMEM were added to the cultures to give a final volume of 200 µL per well. The plates were then incubated for three days, after which 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, Mo) was added to each well (50 µL of a 1 mg/mL solution). The plates were then incubated at 37 °C for four h, after which the medium was aspirated and dimethyl sulfoxide (150 µL) was added to each well. After shaking for 15 min, the plates were read at 540 nm on an ELISA plate reader (Vmax kinetic microplate reader, Molecular Devices Corp., Sunnyvale, Ca). Nucleoside Transporter Affinity for FT Two human equilibrative nucleoside transporters (hENT1, hENT2) and three human concentrative nucleoside transporters (hCNT1, hCNT2, hCNT3) produced in Saccharomyces cerevisiae (Fui1::TRP1) [29] were used to test the apparent affinities of nucleoside transporters for FT. The capacity of FT to inhibit the uptake of [3H]uridine was assessed by the inhibitor-sensitivity assay as follows: Yeast cell cultures, each producing one of the recombinant hENTs or hCNTs, were incubated (10 min) with graded concentrations of the test nucleoside in the presence of [3H]uridine (1 µM) in complete minimal medium (CMM) containing 0.67% yeast nitrogen base (Difco, Detroit, MI), amino acids and 2% glucose (CMM/GLU; pH 7.4) medium. Transport was arrested by collecting yeast cells with a 96-well plate cell harvester (Micro96 Harvester; Skatron Instruments, Lier, Norway). The amount of [3H]uridine associated with yeast in the presence of excess non-radioactive uridine (10 mM) was also determined to quantify nonspecifically associated radioactiv-

40 Current Radiopharmaceuticals, 2012, Vol. 5, No. 1

Wiebe et al.

ity, which was subtracted from the total radioactivity for each transport assay. The concentration of test nucleoside that inhibited [3H]uridine by 50 % (IC50) was determined from plots in which uptake was expressed as a function of the concentrations of test compound [30]. FT Phosphorylation by TK1 and TK2 Phosphotransferase Assay The adenosine 5′-triphosphate transfer assay was performed with [γ 32P]ATP (10 mCi/mL; 0.05 mM), ATP (100 mM), Tris-HCl (50 mM; pH 7.6), MgCl2 (5 mM), KCl (100 mM), bovine serum albumin (0.5 mg/mL), dithiothreitol (10 mM) and nucleoside substrate in a total volume of 25 mL. The reaction was initiated by adding enzyme (25 ng) followed by incubation at 37 °C and terminated after 20 min by boiling for one min. The mixture was centrifuged and 2 mL of the supernatant was applied to a polyethyleniminemodified cellulose thin layer plate (PEI-cellulose F; Merck KGaA, Darmstadt, Germany). Chromatography was performed for 8-12 h using 99 % isobutyric acid: NH4OH:H2 O (66:1:33) (v/v) as the mobile phase. The products of the kinase reaction were detected by autoradiography and quantified by phosphoimaging (Fuji BAS 2500/LAS 1000, I &IImaging & Information; Image Reader V 1.7E; Fujifilm Corp., Tokyo, Japan). The relative values obtained with 100 µM TdR for TK1 and TK2 were set as 100% and phosphorylation of other substrates was expressed relative to these values. For TK1 studies, concentrations used were 10 and 100 µM; for TK2, concentrations were 50, 25, 12 µM (FT and TdR) and 100, 200, 400 µM (FLT). Data are expressed as activity relative to TdR, and specific activity in nmol/mg/min. [3H]FT Uptake in Cell Culture Cells (KBALB, KBALB-STK; 143B, 143B-LTK, EMT6) were seeded (8000 cells/well) into 96-well plates. Following a two day incubation, test compounds [3H]TdR or [3H]FT; 0.2 mCi/well; n=3 for each nucleoside) in culture medium were added to each well and incubations were continued for pre-determined intervals, after which cells were harvested and transferred onto Filtermats (Perkin Elmer, Inc., Waltham, MA) on a TOMTEC™ harvester (Tomtec,

USA). The Filtermats were dried at room temperature (RT), then FilterWax (Perkin Elmer, Inc., Waltham, MA) was melted onto the Filtermat. Once the wax had cooled to room temperature (RT), the radioactivity was measured using a 1450 Wallac Microbeta liquid scintillation & luminescence counter (Perkin Elmer, Inc., Waltham, MA). [18F]FT Biodistribution and Positron Emission Tomography (PET) Imaging Experiments were performed on BALB/c mice (20-24 g, Charles River, Saint-Constant, Quebec, Canada) bearing contralateral, subcutaneously seeded (5x106 cells in 100 µL PBS) KBALB and KBALB-STK tumors. Animal experiments were carried out in accordance with guidelines of the Canadian Council on Animal Care under a protocol approved by the local animal care committee (Cross Cancer Institute, Alberta Health Services). Mice were imaged seven days after tumor implant. The mice were anaesthetized with isoflurane in 40% oxygen/60% nitrogen (gas flow, 1 L/min). Each mouse was positioned and immobilized in the prone position with its medial axis parallel to the axial axis of the scanner, with its head, thorax and upper abdomen in the centre of the field of view of the microPET® R4 scanner (Siemens, Knoxville, TN). The injected dose of radioactivity was determined by radiometry of the loaded syringe using a dose calibrator. [18F]FT (~18 MBq) in saline (100-150 µL) was injected via a tail vein. MicroPET images were reconstructed using filtered back projection. The mice were euthanized by CO2 after completion of the imaging studies and tissues were immediately collected for quantitative measurement of radioactivity. RESULTS The cytotoxicity of FT was compared to that of FUdR, a highly cytotoxic fluoronucleoside, in five cell lines. FT was minimally toxic at concentrations below 0.1 mM; 50 % survival was observed only in 143B-LTK cells at 1 mM FT. The lack of FT toxicity was in marked contrast to that of FUdR, which was toxic to EMT-6 and KBALB cells at 20 % survival) (Fig. 1). 1.2

1.0

Surviving fraction

Surviving fraction

1.0

0.8

0.6

0.4 KBALB KBALB-STK 143B-LTK 143B EMT-6

0.2

0.8

0.6

0.4

KBALB KBALB-STK 143B-LTK 143B EMT-6

0.2

0.0

0.0 -4

-3

-2

[FT] log10(mM)

-1

0

-10

-9

-8

-7

-6

-5

-4

-3

-2

-1

[FUdR] log10(mM)

Fig. (2). MTT in vitro cytotoxicity data for FT (left) and FUdR (right) in murine EMT-6 and KBALB / KBALB-STK cells, and human 143B / 143B-LTK cells. The 50% inhibitory concentrations for FT were above 1 mM, whereas for FUdR they were 1 µM or less for murine cells and approximately 10 µM for the human cells.


Table 1.


41

Concentrations of TdR, FLT and FT Required for 50 % Inhibition (IC50) of [3H]uridine Uptake by Recombinant Human Equilibrative Nucleoside Transporters (hENT1, hENT2) and Human Concentrative Nucleoside Transporters (hCNT1, hCNT2, hCNT3) Produced in Saccharomyces Cerevisiae. Data are Means ± SEM, n = 5 Thymidine [30]

FLT [31]

FT

IC50 (µM)

IC50 (µM)

IC50 (µM)

hENT1

76 ± 18

886 ± 136

107 ± 13

hENT2

158 ± 10

541 ± 89

342 ± 13

hCNT1

22 ± 3

115 ± 20

1.1 ± 0.14

hCNT2

1225 ± 208

>10000

326 ± 49

hCNT3

35 ± 5

499 ± 67

9.7 ± 1.1

NT

The apparent affinities of nucleoside transporters for FT were assessed by determining the concentration dependence of inhibition of uridine transport using recombinant human nucleoside transporters produced individually in yeast cells. The results (IC50 values for inhibition of transport) are shown in Table 1, together with previously published results from similar experiments with TdR [30] and FLT [31] for comparison. With the exception of hCNT1 and hCNT2, the transporters were only slightly less sensitive to inhibition by FT than by the natural substrate TdR. All five transporters exhibited higher affinities for FT than for FLT. Qualitatively, all three test nucleosides appear to be phosphorylated facilely (Fig. 3). Quantitatively, however, FT phosphorylation by TK1 was approximately 60 percent of TdR and FLT values, whereas FT phosphorylation by TK2 was about 6-fold greater than that of FLT. Data are expressed relative to TdR, and in absolute terms (nmol/mg/min), in Table 2. FT and TdR accumulations in four cell lines in cell culture are presented in Fig. (4). KBALB and KBALB-STK cells had similar uptakes for FT and TdR throughout the 24 h study, although FT levels were considerably higher in KBALB-STK cells. The levels of both FT and TdR in 143B-

LTK cells, however, were much higher than in 143B cells; again both tracers were accumulated to similar concentrations in the respective 143B/143B-LTK lines. PET images of [18F]FT biodistribution in mice are presented in Fig. (5). Each mouse carried an implanted KBALB tumor on its right flank (left side on image) and a KBALBSTK tumor on the contralateral flank. In one mouse (left image), the tumors were large relative to the respective tumors in the second animal (right image), double the size for KBALB tumor and about ten-fold larger for the KBALBSTK tumor. In both animals, the KBALB tumor showed faint but discernable uptake, whereas the KBALB-STK tumors were well demarcated. Quantitative radiometric data for most tissues/organs in each mouse, upon necropsy, are presented in Table 3. These data are indicative of extensive renal and hepatobiliary clearance, large uptake differentials between the two tumor lines (KBALB:KBALB-STK = 5:1 and 20:1, left & right images, respectively), and strong accumulation in the KBALB-STK tumors (8:1 and 33.5:2 tumor:blood, left & right images, respectively). The tumor:blood concentration ratios for the KBALB tumor were only 1.4 and 1.6 in the left & right images, respectively, underscoring the high avidity of the HSV-1 TK expressing tu-

Fig. (3). Autoradiography of phosphotransferase assay plates for TK1 (left; six lanes) and TK2 (right; nine lanes). Substrates (TdR, FLT, FT) and their concentrations (µM) are depicted for their respective lanes.


Table 2.

Wiebe et al.

Phosphorylation of TdR, FT and FLT. The Relative Values Obtained with TdR for TK1 and TK2 at 100 µM Concentration was Set as 100% and Phosphorylation of Other Substrates were Expressed Relative to these Values. The Specific Activities are Expressed as Nmol/mg/min. Comparative Literature Data are Presented for TdR and FMAU TK1

Substrate

Activity

TdR

Reference

100

100

nmol/mg/min

563

917

TdR [32]

Vmax (pmol/min/mg)

2100±300

FT

Relative to TdR

58

39

nmol/mg/min

327

377

Relative to TdR

89

6

nmol/mg/min

499

57

Relative to TdR

82

219

nmol/mg/min

---

---

Vmax (pmol/min/mg)

1000±300

FLT

FMAU [33]

FMAU [32]

TK1

TK2

Vmax /Km

670

150±40

31

265±30

TK2 Vmax /Km

150

38

Uptake (pmol/mg protein)

100

10

FT K FT STK TdR K

1

TdR STK

0.1 1

6

12

24

Incubation time (h)

Uptake (pmol/mg protein)

100

10

FT 143B FT LTK 1

TdR 143B TdR LTK

0.1 1

6

12

24

Incubation time (h)

Fig. (4). Accumulation of [3H]FT and [3H]TdR in KBALB (K) and KBALB-STK (STK) cells (upper graph) and 143B and 143B-LTK (LTK) cells (lower graph) in cell culture. Data, expressed as functions of cell protein (pmol/mg protein), are means ± SEM, n=3.

mor for [18F]FT. A PET image of 18FLT in this tumor model (top centre inset, Fig. 5) showed good uptake by the KBALB tumor, and strong uptake by the KBALB-STK tumor. DISCUSSION Radionucleosides used for imaging cell proliferation and HSV-1 TK transgene expression must be phosphorylated by

thymidine kinases (TKs), but while proliferation imaging relies on expression of cytosolic kinases (TK1) present in differentiating cells, phosphorylation by only the transgene (HSV-1 TK) is essential for gene therapy imaging. Substrate overlap between HSV-1 TK, TK1 and mitochondrial thymidine kinase (TK2) can produce misleading results. Nucleosides for HSV-1 TK transgene imaging and cell prolif-



43

Fig. (5). PET images of three EMT-6 mice bearing KBALB and KBALB-STK murine tumors. Mouse #1 (left) was injected with [18F]FT (18 MBq and a 20 min image was acquired 2.5 h post injection (i.e., 150-170 min p.i.). Mouse #2 (right) was injected with [18F]FT (18 MBq), and a 20 min image was acquired 2 h post injection (i.e., 120-140 min p.i.). Quantitative biodistribution data obtained by necropsy after the imaging procedures are presented in Table 3. Inset shows a 5-minute 18FLT image (top centre) acquired 53 min after injection. Table 3.

Tissue Biodistributions of Radioactivity in Two Balb/c Mice Bearing Implanted KBALB and KBALB-STK Murine Tumors, 3 h (Mouse #1) and 2.5 h (Mouse #2) after i.v. (Tail Vein) Injection of [18F]FT Weight (g)

Tissue

cpm/mg tissue

#1

#2

Blood

0.71

KBALB

Tissue:blood ratio

#1

#2

#1

#2

0.74

431

604

1.0

1.0

0.77

0.39

597

992

1.4

1.6

KBALB-STK

0.43

0.03

3452

20210

8.0

33.5

Gall blabber*

0.02

0.02

1830

8515

4.2

14.1

Liver

1.06

0.92

442

759

1.0

1.3

Stomach*

0.52

0.27

248

434

0.6

0.7

Spleen

0.27

0.19

520

3337

1.2

5.5

Kidneys

0.43

0.41

1152

606

2.7

1.0

Bladder*

0.09

0.03

19178

1842

44.5

3.0

Intestines*

1.95

1.89

878

2566

2.0

4.2

*Organ plus residual contents at necropsy.

eration imaging have tended to be F-18 or I-124 labeled PET tracers [12], particularly the 2′-fluoro-2′-deoxyarabinonucleosides (e.g. FIAU [9]) and the acyclopurine nucleosides (e.g., FHBG) [34], whereas the PET proliferation imaging literature is primarily based on FLT [20] with less focus on the thymidine analogue FMAU [17]. Investigations of the 2′-fluoro-2′deoxyribosides for PET imaging are limited to recent papers on the radiosynthesis of 2′-[18F]fluoro-2′-deoxyribouridine [35] and 2′-[18F]fluoro-2′deoxyribothymidine [27], and their radioiodinated 5-iodoanalogue 5-iodo-2′-fluoro-2′-deoxyribouridine ([124/125/131I] FIRU) [26, 36]. Classical biodistribution and metabolism

studies of their 5-substituted-2′-fluoro-2′,3′-dideoxy analogues have utilized H-3 and C-14 labels [37, 38], or ′nonPET′ radiohalogens, especially radioiodine (123/125/131I) [7, 37, 39, 40]. Exceptions include (E)-5-(2-iodovinyl)-1-(2′-fluoro2′deoxyribofuranosyl)uracil (IVFRU) [41] which has antiherpesvirus activity, and a fluororibocytosine analogue that interacts with the subgenomic HCV replicon system to generate anti-viral activity [42]. The 2′-fluoro-2′-deoxyribofuranosyl pyrimidine nucleosides, unlike their fluoroarabinosyl analogues, are not toxic and are virtually devoid of antiviral activity [24]. There is only one report documenting the biodistribution and tumor uptake of [3H]FT in a murine tumor model [38].


Expression of HSV-1 TK by the transduced cell lines (KBALB-STK; 143B-LTK) used in this study is consistent with their high sensitivities to the cytotoxic effects of ganciclovir (IC50 0.030-0.065 µM [28]). The low cytotoxicity (IC50 > 0.1 mM) of FT against all five cell lines (Fig. 2) was similar to that reported for FMAU against HL-60 cells (136 µM) [43] and marginally lower than that reported for FLT (361 µM) against 2.2.15 cells [44]. All three sugarfluorinated nucleosides (FT, FIAU, FLT) are much less toxic than FUdR, a C-5 fluoropyrimidine deoxynucleoside (Fig. 1) that is readily phosphorylated to its mononucleotide (FdUMP). FdUMP is cytotoxic via complex mechanisms based on thymidylate synthase (TS) inhibition and the generation of other antimetabolic effects along the DNA and RNA pathways [45]. FUdR was highly toxic to all cell lines tested (Fig. 2). FT and TdR were concentrated by all four cell lines (KBALB, KBALB-STK, 143B, 143-LTK) in cell culture, with approximately ten-fold increases in uptake over 24 h (Fig. 4). In each case, the HSV-TK expressing cells acquired more of the labeled compound, but quantitatively this was much greater with the 143B-LTK line. The retention of radioactivity by these cells implies monophosphorylation by both normal and transgene-expressing cells, and possibly further anabolism, but no attempt was made to identify the chemical nature of the retained radioactivity. The current work compared the binding of TdR, FLT and FT to cloned human equilibrative (hENT1; hENT2) and concentrative (hCNT1; hCNT2; hCNT3) nucleoside transporters by determining IC50 values for inhibition of [3H]uridine transport into yeasts expressing these recombinant hNTs (Table 1). A comparison of IC50 values provides an indication of the rank order of apparent binding affinities of the transporters for potential permeants, and for many nucleosides also predicts transportability [29]. The concentrative transporters have a particularly high affinity for FT; for hCNT1 (IC50 1.1±0.14 µM) it was forty-fold greater than TdR and over one hundred-fold greater than FLT) and for hCNT3 (IC50 9.7±1.1 µM) it was three-fold greater than TdR; fifty-fold greater than FLT). Affinity of the equilibrative transporters for FT was intermediate between TdR and FLT the (IC50 107±13 and 342±13 µM for hENT1 and hENT2, respectively). hCNT2 had low affinity for all compounds, although its affinity for FT was higher than for either TdR or FLT. Contrary to the situation for FLT, where F for OH substitution at C-3′ decreased the affinities of all 5 hNTs for FLT (relative to TdR) [31], F for OH substitution at C-2′ in the ribo configuration only marginally decreased the affinities of the equilibrative transporters for FT but significantly increased the affinities of the concentrative hNTs for FT. The lack of antiviral activity by 2′-fluororibonucleosides has been attributed at least in part to lack of phosphorylation by cellular enzymes [46]. New data (Fig. 3; Table 2) confirm that FT is indeed a good substrate for human TK2, albeit a weaker substrate for TK1. FLT and FMAU are also phosphorylated by both TK1 and TK2 (Table 2), but in contrast to FT, they are reported to be incorporated into DNA [47]. Other studies have provided evidence for pooling of FLT at the mononucleotide stage, but reported that there was no

Wiebe et al.

evidence of incorporation of FLT into DNA of human lung adenocarcinoma cells (A549 cells) despite the formation of mono-, di- and tri-phosphates [48, 49]. Cellular phosphorylation, with subsequent metabolic elaboration, would explain the reported in vivo incorporation of [3H]FT into RNA, DNA and other metabolites (4.4, 0.4 and 94.1 %, respectively, of tumor radioactivity) in mammalian EMT-6 cells after a single dose of [3H]FT in BALB/c mice bearing implanted EMT-6 tumors [38]. The higher incorporation into RNA relative to DNA would be expected on the basis that fluorine mimics hydroxyl in terms of electronegativity and hydrogen bonding, even though fluorine is closer to hydrogen in terms of atomic (Van der Vaal) radii. 2′-Deoxy-2′-fluororibonucleosides may also be more like ribonucleosides than 2′-deoxyribonucleosides, since 2′deoxy-2′-fluororibo- and ribo- moieties both adopt a 3′-endo conformation [50, 51]. The high toxicity of FMAU relative to FT has been observed in other analogue ribo/arabino pairs, e.g., FIAU and FIRU [25, 35], and appears to be more a function of downstream metabolism, e.g., incorporation into DNA, than phosphorylation to the respective mononucleotides. Much of the transgene imaging literature addresses the use of 2′-fluoroarabinonucleosides, despite their known phosphorylation by both TK1 and TK2. For example, the Vmax/Km ratios for FIAU and FMAU for TK1 are reported to be 2 and 5 % of that for deoxythymidine, respectively, and for TK2, 30 % of that for deoxythymidine, respectively [32]. The critical test of any putative imaging or therapeutic agent lies in the in vivo evaluation. With [18F]FT, KBALB tumors retained very little radioactivity at sacrifice (~2.5 h post-injection), whereas the HSV-1 TK expressing tumors were very radioactive, and the smaller of the two HSV-TK expressing tumors provided a tumor:blood radioactivity ratio of 33:1. The FLT image used for reference clearly delineated both tumors. Images (Fig. 5) and dissection biodistribution data (Table 3) for FT and FLT reflected in vitro phosphorylation data. A full PET imaging and biodistribution study is underway, based on the promising biodistribution data from two mice bearing contralateral KBALB and KBALB-STK tumors. In summary, FT was shown to be minimally toxic to a range of cell lines, including two lines engineered to express HSV-1 TK. In vitro cell uptake was higher in non-HSV-1 TK expressing cells than in the engineered cells, and of the same magnitude as TdR accumulation. Facile phosphorylation of FT by TK1 and TK2 enzyme preparations was reflected in the cell culture studies, in PET images, and in vivo biodistribution experiments. Additional studies comparing FT with FMAU are warranted to provide a better understanding of the influence of 2’-fluoro substitution geometry (i.e., ribo vs. arabino) on their monophosphorylation by TK1 and TK2 and their subsequent metabolic processing that leads to their respective imaging properties. [18F]FT is a strong candidate for PET imaging of viral TK transgene imaging. This conclusion is based on its strong interaction with nucleoside transporters, especially the concentrative nucleoside transporters, its facile phosphorylation



by TK2, and its selective in vivo uptake by an HSV-TK expressing murine tumor model.

[18]

ACKNOWLEDGEMENTS

[19]

This work was supported in part through grants from the Canadian Cancer Society Research Institute (C Cass), the Swedish Research Council (S Eriksson) and the Canadian Institutes of Health Research (L Wiebe & E Knaus). We thank Dr. J Wilson, Cross Cancer Institute, for providing F18 for the radiofluorinations, and Ms. Gail Hipperson, Cross Cancer Institute Vivarium, for assistance with animal studies.

[20]

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Accepted: July 26, 2011