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Evaluation of Human Thymidine Kinase 1 Substrates as New Candidates for Boron Neutron Capture Therapy

¹ Department of Molecular Biosciences, Division of Veterinary Medical Biochemistry, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden; and ² College of Pharmacy and ³ Department of Pathology, The Ohio State University, Columbus, Ohio

	ABSTRACT

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Thymidine analogs containing o-carboranylalkyl groups at the 3-position were screened as potential substrates for human thymidine kinase 1 (TK1), an enzyme that is selectively expressed in a variety of rapidly proliferating cells, including tumor cells. On the basis of previous studies, 12 of these were identified as potential delivery agents for boron neutron capture therapy, a therapeutic method used for the treatment of high-grade brain tumors. Compound 4 with a pentylene spacer between the o-carborane cage and the thymidine scaffold and compound 10, which has an additional dihydroxypropyl substituent at the o-carborane cage, were the best substrates for TK1 with k_cat/K_m values of 27% and 36% relative to that of thymidine, respectively. These compounds showed partial competitive inhibition for thymidine phosphorylation by TK1. Neither compound was a substrate of recombinant human thymidine phosphorylase nor were their respective 5'-monophosphates substrates of 5'-deoxynucleotidase 1, thereby indicating potential in vivo stability. The octanol/water partition coefficient for compound 10 was 2.09, suggesting that it has excellent physiochemical properties for crossing the blood brain barrier and penetrating brain tissue. The in vitro cytotoxic effect of the 12 analogs was moderate to low in mammalian cell cultures with IC₅₀ values between 10 and 160 µmol/L. Compounds 4 and 10 were taken up selectively and retained by the murine fibroblast L929 cell line, in contrast to its TK1-deficient variant. These findings suggest that compound 10 is a promising candidate for selective delivery of boron-10 to malignant cells, and additional in vivo studies are planned to evaluate it for boron neutron capture therapy of brain tumors.

	INTRODUCTION

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The treatment of high-grade gliomas and anaplastic astrocytomas remains refractory to conventional existing therapies, and new therapeutic modalities to improve the treatment are needed. Boron neutron capture therapy (BNCT) is a binary chemoradiotherapeutic system that has been used clinically to treat patients with primary brain tumors (1 , 2) and metastatic melanoma (3) . It is based on the nuclear capture and fission reactions that occur when boron-10 (¹⁰B) is irradiated with low-energy neutrons to produce high-linear energy-transfer particles (⁴He²⁺) and lithium-7 (⁷Li³⁺) ions, which can lethally damage cells (4 , 5) . Two drugs, boronophenylalanine and mercapto-closo-undecahydrododecaborate have been used clinically, but there is an urgent need for more effective ¹⁰B delivery agents. These should have low systematic cytotoxicity, selectively accumulate and be retained by malignant cells, and have sufficient bioavailability (4) .

Boronated analogs of pyrimidine nucleosides such as thymidine (dThd) and deoxyuridine potentially may be ideal vehicles for the selective delivery of ¹⁰B to malignant cells (6) . They can be phosphorylated to their corresponding 5'-monophosphates by thymidine kinase 1 (TK1), a cytosolic enzyme that catalyzes the transfer of -phosphate group from ATP to the 5'-hydroxyl group of dThd and deoxyuridine. The resulting boronated 5'-monophosphates would then be retained intracellularly because of the negatively charged phosphate group (6) . The expression of TK1 is cell cycle specific, and its enzymatic activity is absent in resting cells. Enzyme expression occurs in late G₁ cells, increases in S phase, coinciding with increased DNA synthesis, and disappears during mitosis (7) . Therefore, phosphate metabolites of carboranyl nucleosides should accumulate preferentially in tumor tissue.

Previously, we have reported the synthesis of several boronated dThd analogs as potential BNCT delivery agents (8 , 9) . These analogs are characterized by the attachment of a closo-carboranylalkyl group to the 3-position of dThd via a hydrocarbon tether of 2–7 methylene groups (Fig. 1) . The rationale for choosing the carborane cage as the boron moiety is its remarkable chemical and hydrolytical stability, its high ¹⁰B content (10 atoms/molecule), and its lipophilicity, which facilitates cellular penetration of agents to which the cage is attached (10) . Preliminary enzymatic studies have revealed that recombinant cytosolic human TK1, but not recombinant mitochondrial human thymidine kinase 2, tolerates bulky groups at 3-position of dThd (9 , 11) .

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Structure of closo-carboranylalkyl dThds: The syntheses of the compounds 1-12 is described in reference (9) , compound 13 in reference (8) , and compound 14 in Supplementary Data.

	MATERIALS AND METHODS

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Chemicals and Cell Lines.
Boronated dThd analogs (1-12) were synthesized as described previously (8 , 9) . Other natural and unnatural nucleosides and nucleotides were purchased from Sigma-Aldrich Corp (St. Louis, MO). The synthesis of compound 13 has been described previously (8) and that of compound 14 is described in Supplementary Data. The [methyl-³H]dThd (specific activity 25 Ci/mmol) was purchased from Amersham International (Arlington Heights, IL). L929 (ATCC-CCL-1) and LMTK^– (L929 TK1^–, ATCC-CCL-1.3; ref. 14 ) cell lines were purchased from The American Type Culture Collection (Manassas, VA). CCRF-CEM and CEM-TK^– cells were kindly provided by Dr. Buddy Ullman (The Oregon Health Sciences University, Portland, Oregon; ref. 15 ). Culture media was purchased from Invitrogen (Carlsbad, CA).

Expression and Purification of the Recombinant Human Enzymes.
TK1 (TK1^106Met sequence according to accession number NM_003258) was purified from bacterial expression system [BL 21 (DH3) pLys], as described previously (8) . The human TPase (NM_001953) cDNA was a gift from Dr. Carl-Henrik Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden; ref. 16 ). Following the manufacturer’s protocol, human dNT-1 (NM_032526) was cloned from a human fetal brain cDNA library (Stratagene, La Jolla, CA) by PCR methods using Advanced–GC cDNA PCR kit (Clontech, Palo Alto, CA) and the specific primers (5'-ggcagccatatgctcccaggctacgccttggc and 5'-gcagccggatcccaagggtgtggggggctagaa). The cDNAs of both TPase and dNT-1 were cloned into the NdeI and BamHI sites of the pET14b vector (Novagen, Madison, WI). The correct insertions were confirmed, and constructs were expressed in Escherichia coli, BL 21 (DH3) pLys host cells (8 , 13) , and the proteins were purified on Ni-NTA His bind resins column (Novagen).

TK Assays Using [-³²P]ATP as a Phosphate Donor.
The kinetic parameters for the boronated dThd analogs were determined using phosphoryl-transfer assays (9) . These were carried out in reaction mixtures containing 50 mmol/L Tris-HCl (pH 7.6), 5 mmol/L MgCl₂, 125 mmol/L KCl, 10 mmol/L DTT, 0.5% BSA, 1 mmol/L ATP, 0.3 µmol/L [-³²P]ATP (Amersham Pharmacia Biotech, Arlington Heights, IL), and various concentrations of boronated dThds ranging from 1 to 100 µmol/L. The DMSO concentration in all experiments was kept at 1%, and the reactions were initiated by the addition of pure recombinant TK1.

TK Assays Using Methyl-³H-Radiolabeled dThd.
Competition studies were performed after the detection of [methyl-³H]dThd phosphorylation in the presence of various concentrations of non-radiolabeled boronated dThd analogs. The standard reaction mixtures contained 50 mmol/L Tris-HCl (pH 7.6), 2 mmol/L DTT, 5 mmol/L MgCl₂, 10 mmol/L NaF, 5 mmol/L ATP, 0.5 mg/ml BSA, 0.5 µmol/L or 5 µmol/L [methyl-³H]dThd, and purified recombinant TK1 (8) . The samples were incubated at 37°C in the presence of various concentrations of boronated dThd analogs (1–80 µmol/L). The DMSO concentrations were kept at 4%. Aliquots of 10-µl each of the reaction mixtures were spotted onto DE-81 filter paper (Millipore, Billerica, MA) at time intervals of 0, 6, 12, and 18 minutes. The filters were washed three times with 5 mmol/L ammonium formate, 5 minutes for each wash, and the radioactivity was determined by -scintillation counting.

Kinetic Analysis.
The kinetic parameters were determined by nonlinear regression analysis using the Michaelis-Menten and Hill equations and were analyzed primarily by Eadie-Hofstee plots, as described previously (17) . The compound concentrations resulting in 50% inhibition of enzyme activity (IC₅₀) were determined by the equation v_I = v₀/(1 + [I]/IC₅₀), as described previously (17) . Data were analyzed by the Sigma Plot Enzyme Kinetic Module version 1.1 (SPSS Inc.).

5'-Deoxynucleotidase-1 Assay.
The dephosphorylation of compounds 13 and 14, as well as TMP, dGMP, UMP, 3'-azido-2', 3'-dideoxyuridine-monophosphate (AZT-MP) as controls, was measured by high performance liquid chromatography (HPLC) analysis. The reaction mixture containing 0.25 mmol/L sodium acetate (pH 5.5), 30 mmol/L KCl, 10 mmol/L MgCl₂, 5 mmol/L DTT, 0.2 mg/ml BSA, 10 µmol/L nucleoside-MP and 20 ng of purified recombinant dNT-1, was incubated at 37°C (13) . Nucleosides were separated from their monophosphates using a gradient system, 98% buffer E1 [50 mmol/L ammonium phosphate (pH 5.5) and 5 mmol/L tetrabutylammonium hydrogen sulfate] and 2% buffer E2 (5 mmol/L tetrabutylammonium hydrogen sulfate in 100% methanol) over 15 minutes. Subsequently, buffer E2 was increased over a period of 15 minutes from 2 to 20% and then from 20 to 50% over a 20-minute period. UV-based detection was performed at 267 and 280 nm.

Thymidine Phosphorylase Assay.
The phosphorolysis of boronated dThds (compounds 4 and 10) as well as dThd, AZT, and 5-(2-bromovinyl)-2'- deoxyuridine (BVDU) as controls by the bacterial E. coli TPase (Sigma-Aldrich Corp, St. Louis, MO) and the human TPase, was measured by HPLC analysis, as described previously (12) . Nucleosides were separated from their nucleoside bases and quantified by reversed-phase-C18 column chromatography (Re pro Sil-Pur, C18-AQ, Bischoff, Leonberg, Germany), using a linear gradient of 98% buffer A [1 mmol/L potassium phosphate buffer (pH 5.5)] and 2% buffer B [1 mmol/L potassium phosphate buffer (pH 5.5) in 80% methanol] to 20% buffer A and 80% buffer B. After injection of the samples, 98% buffer A and 2% buffer B were run for 10 minutes before the start of the gradient (5 minutes from 2–80% buffer B). UV-based detection was performed at 267 and 280 nm, as described previously (18) . Evaluation of the inhibitory effects of the compounds was performed as described previously (12) , and the conversion of dThd to thymine was quantified by HPLC.

Log P Values.
The physiochemical properties of the compounds were determined by HPLC experiments, as described by Teijeiro et al. (19) . The chromatographic analyses were performed on a reversed phase-18 (5 µm), LiChrosphere 100Å (250 mm/4 mm) column, using a Rainin HPLC instrument with UV detection at = 254 nm. Samples were dissolved in 20 µl of methanol for injection, and retention times were measured in different solvent systems (methanol dissolved in water at concentrations of 50, 60, 70, and 80%) at flow rate of 1 ml/minute. A plot of the retention time against the composition of mobile phase was generated for each compound to obtain the intercept of the plot, which corresponded to the retention time of the compound in 100% water (t_R). All measurements were carried out at ambient temperature. The log P was calculated using the following equation: Log P_o/w = 1.882 log k'_w – 1.346, where, k'_w = (t_R – t₀)/t_0. t₀ is the retention time of methanol (1.78 minutes); t_R is the retention time of the solute; and k'_w is the extrapolated "k" value at 0% methanol.

Cell Culture and Cytotoxicity Assay.
Cells were cultured in DMEM with glutamax-1 medium containing 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cytotoxicity assays were performed as described previously (20 , 21) . Cells were incubated in media containing the test compounds at final concentrations of 10–160 µmol/L in 0.5% DMSO. After 72 hours of incubation, 1 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (Sigma-Aldrich Corp.) was added. Formazan crystals were allowed to develop and were subsequently dissolved in 10% SDS/0.04 eq/L HCl solution for 1 hour at 37°C. Absorbance was measured at 540 nm using an ELISA plate reader (Labsystems Multiscan PLUS, software Delta Soft 3). Cell survival was expressed as the percentage of control cells [CS = (Mean A_{treated well}/Mean A_{control well}) x 100%].

Cellular Uptake and Retention of Boronated dThd Analogs.
L929 wild-type and TK1^– cells (1 x 10⁷) were cultured in DMEM (14) , as described above. Upon reaching semi-confluence, the cells were incubated in a fresh medium containing 17.5 µmol/L of compounds 4, 6, 10, or 12. After 24 hours of incubation, the boron-containing medium was decanted, and the cells were washed with PBS and counted, and their boron content was determined by direct current plasma-atomic emission spectroscopy. Values were normalized to micrograms of boron per gram of cells (10⁹ cells; ref. 22 ). For retention studies, after 24 hours of incubation with the boron-containing medium, the cells were washed with PBS and incubated for 12 hours with fresh boron-free medium. Then the cells were collected and counted, and their boron content was determined by direct current plasma atomic emission spectroscopy (22) .

	RESULTS AND DISCUSSION

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We have had a longstanding interest in boron-containing pyrimidine nucleosides as substrates for TK1 for application in BNCT (6, 7, 8, 9, 10, 11) . These compounds acquire a negative charge by phosphorylation and are primarily entrapped in rapidly proliferating malignant cells in which TK1 is highly expressed. closo-Carboranylalkyl dThd analogs (compounds 1–12, Fig. 1 ) are characterized by the attachment of the carborane cage to the 3-position at dThd via a hydrocarbon tether (2–7 methylene groups), which presumably enhances binding of the carboranyl dThds to the enzyme by reducing steric interference of the bulky carborane cluster with the active site of TK1 (8 , 23) .

The rational for choosing the 3-position to link the carborane cage was based on substrate activity studies with TK1 providing that 3-substituted dThd derivatives have TK1 substrate/inhibitor characteristics (24 , 25) . Previously, we had observed that TK1 tolerated substituents at the 5- and 3'-positions of deoxyuridine and dThd, respectively, which were comparable in size and physiochemical properties to a methyl and a hydroxyl group, respectively (26, 27, 28) , whereas the introduction of bulky carboranyl groups at these positions resulted in poor substrate characteristics (8 , 29) . In contrast, 3-methyl-, 3-ethyl-, and 3-isopropyl- dThd were reported to have high relative phosphorylation rates with TK1 (30) , prompting the synthesis and evaluation of 3-carboranylalkyl-substituted dThds in our laboratories. Previously, we have reported that recombinant mitochondrial thymidine kinase 2 can tolerate dThd analogs with small substituents at the 3-position, such as 3-methyl dThd, with moderate activity (11 , 30) . However, there was no indication for substantial phosphorylation of compounds 1-12 with thymidine kinase 2 (<0.3% relative to dThd), even when high substrate concentrations of 100 µmol/L were used (9) .

Kinetic Properties of Boronated dThds.
In general, the enzyme kinetics of boronated dThds (1-12) followed the Michaelis-Menten equation. TK1 phosphorylation of compounds 1-12 had 1.5- to 15-fold higher apparent K_m values compared with those of dThd (Table 1) . In the first series of compounds (1-6), lacking the dihydroxypropyl group, there was no strong correlation between tether lengths and kinetic parameters. In this series, compound 4 with a pentylene linker had the lowest K_m value (3.7 µmol/L), whereas compounds with propylene and heptylene spacers showed higher K_m values (26.1 and 36.5 µmol/L, respectively). The relative efficiencies (k_cat/K_m), expressed as percentage relative to that of dThd are shown in Table 1 . The relative catalytic efficiencies for this set of compounds decreased with increasing tether lengths except for compound 4 with 26.7% that of dThd.

Overall, the kinetic results were in accordance with relative phosphorylation rates reported previously (9) . In both cases, TK1 phosphorylation capacity decreased with increasing tether length. However, this tendency was not observed for all compounds. TK1 showed the best overall kinetic properties for compounds having a pentylene spacer (compounds 4 and 10). The results further indicate that the active site of TK1 has also sufficient space to accomodate a bulky carborane cage in closer proximity to the dThd scaffold as indicated by favorable relative k_cat/K_m values for compounds 1 and 7 (27.4 and 31.9%, respectively) having short ethylene spacers. Whereas the tether length increased, the carborane cluster appeared to interfere with the enzymatic activity. In the case of compounds with a pentylene linker, the carborane cluster presumably is located outside of the active site, which results in significantly improved activity. In the case of compounds with longer tether arms (5, 6, 11, and 12), the carborane cluster could interfere with substrate binding at the same active site by folding on itself or at active sites of other TK1 units of this tetrameric holoenzyme (9 , 11) .

We hypothesize that the substrate-binding pocket for pyrimidine nucleosides in TK1 is close to the enzyme surface and that the 3-position of dThd is directed toward an opening of the substrate pocket to the surface of the enzyme. This hypothesis is supported by the fact that carboranyl dThds with even longer linkers than in compounds 6 and 12 also were sufficiently phosphorylated by TK1.⁴ Several other kinases are known to have substrate-binding pockets near the surface of the protein, e.g., the crystal structure of the bacterial pantothenate kinase shows that CoA competitively binds to the ATP site as a feedback inhibitor at the enzymes surface. Furthermore, 5'-adenylimido-diphsophate, a nonhydrolyzable analog of ATP, was also found to bind to the enzyme surface of pantothenate kinase (31) .

Inhibition of dThd Phosphorylation by Boronated dThds.
Competition experiments were performed to determine the inhibition constants (K_i) of carboranyl dThds for the phosphorylation of dThd by TK1. However, the kinetic pattern of inhibition was very complex, indicating negative cooperativity (data not shown). Thus, we could not determine directly the K_i of compounds 1–12. The IC₅₀ values were determined to evaluate the degree of inhibition and are defined here as the concentration of carboranyl dThd that inhibited TK1 phosphorylation of dThd by 50%. As reported previously, the change in IC₅₀ values at different substrate concentrations is indicative of the type of inhibition (32) . When the boronated dThd analogs were tested, a slight increase in the IC₅₀ values was observed with increasing dThd concentrations, which indicated a competitive or predominantly competitive inhibition mechanism (Table 1) . The IC₅₀ values decreased with increasing tether lengths, suggesting a slower dissociation of the compounds with longer tether from the active site, and thus, less availability of active enzyme to phosphorylate dThd. These results are in accordance with enzyme kinetic data described above, in which the lowest k_cat/K_m values were found for compounds with heptylene linker (6 and 12).

Although both compounds 4 and 10 have similar biochemical properties, differences in inhibition of dThd phosphorylation were observed. The IC₅₀ values for compound 10 at both dThd concentrations were 2-fold lower than those for compound 4. This demonstrates the importance of the additional hydrophilic dihydroxypropyl group attached to the second carbon atom of the carborane moiety of compound 10. The increase of TK1 inhibitor capacity attributable to the dihydroxypropyl group was only observed in case of compounds 4 and 10 having pentylene tethers, indicating that only compound 10 had favorable TK1 inhibitor and substrate characteristics. Interestingly, compounds 1 and 7, both with ethylene spacer, had kinetic properties similar to those of compounds with pentylene spacers (4 and 10). The IC₅₀ values for both compounds, however, were 2- to 3-fold higher than those of compound 10, indicating a decreased capacity to compete with dThd for the active site of TK1. Kinetic studies outlined here show that compounds 4 and 10 have favorable kinetic properties, and both compounds are apparently partially effective competitive inhibitors of dThd phosphorylation. Therefore, both were chosen for an in depth biological evaluation.

Boronated dThd Monophosphates as Substrates for Human Cytosolic dNT-1.
Human dNT-1 presumably is the principle-degrading enzyme of deoxynucleoside monophosphates in mammalian cells (13) . Figure 2 summarizes the substrate specificity of pure recombinant dNT-1 (containing a His-tag attached to the NH₂-terminal) with various nucleoside 5'-monophosphates, including compounds 13 and 14 (the 5'-monophosphates of compounds 4 and 10, respectively). Similar to previous observation (33) , dNT-1 preferentially catalyzes the dephosphorylation of 5'-deoxyribonucleotides (dGMP and dTMP) rather than 5'-ribonucleotides (UMP). dGMP and dTMP were rapidly converted to 2'-deoxyguanosine (90%) and dThd (60%) after 30 minutes incubation with dNT-1. Almost 60% of the monophosphates of the antiviral dThd analog AZT were degraded to AZT under these conditions, whereas only a small amount (10%) of UMP was degraded to uridine (Fig. 2) . In contrast, no dephosphorylation of the 5'-monophosphates 13 and 14 was observed even after 120 minutes of incubation. Compounds 13 and 14 were not hydrolyzed by dNT-1 in vitro, which leads us to conclude that the same may be the case in vivo. However, in vivo, several additional types of 5'-dNTs may be operational (13) .

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Boronated dThds as substrates for human cytosolic 5'-doxynucleotidase 1. Natural nucleoside-MPs and dTMP analogs were incubated in reaction mixtures containing dNT-1. At the time points indicated, the nucleosides were separated from their MP derivatives by HPLC using the buffer system as described in Materials and Methods. Values represent the remaining dNMP as a percentage of the total dNMP in the reaction (20 µmol/L). The results represent the mean ± SDs of at least three independent experiments. dNMP, 5'-deoxyribonucleoside monophosphates

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Boronated-dThds as substrates or inhibitors for bacterial thymidine phosphorylase. Natural deoxynucleosides and dThd analogs were incubated in reaction mixtures containing TPase. At the time points indicated, the nucleosides were separated from their nucleoside bases by HPLC using the buffer system described in Materials and Methods. A, values represent the remaining deoxynucleoside as a percentage of the total deoxynucleosides in the reaction (20 µmol/L). B, inhibitory effects of dThd analogs (20 µmol/L) and BVDU (20 µmol/L) on dThd (10 µmol/L) hydrolysis. The results represent the mean ± SDs of at least three independent experiments.

Log P Values of the Boronated dThd Analogs.
The physicochemical properties of a drug are critical for passing the blood brain barrier and penetrating the brain to reach intracerebral targets (35) . One of the best physicochemical parameters to define these essential properties is the octanol/water partition coefficient log P (36) . The log P values of compounds 1-6 and 7-12 were determined by a reversed-phase HPLC method developed by Teijeiro et al. (19) . They increased as a function of the length of the methylene spacer except for compounds 1 and 7, which had slightly higher log P values than compounds 2 and 8 (Table 2) . The optimal log P value for compounds designed to penetrate into the central nervous system has been determined empirically to be 2 (35) . Interestingly, the log P value of compound 10 is 2.09, indicating that it might be a good candidate for the treatment of brain tumors by neutron capture therapy.

Cell growth inhibition was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium assay (21) and is expressed as the concentration of a compound that produces a 50% inhibition of cell growth over a 72-hour incubation period (IC₅₀). In case of L929 and L929 TK1^– cells, the cytotoxic effect of the boronated dThds was generally low. However, compounds 1-6 showed 2- to 3-fold more growth inhibition than compounds 7-12. Furthermore, the IC₅₀ values decreased with increasing tether lengths of the compounds. There were no obvious differences in growth inhibition of L929 as compared with L929 TK1^– cells, with the exception of compounds 4 and 10, in which cases the TK1^– cells were 2-fold more resistant than the wild-type cells.

For CCRF-CEM cells, the IC₅₀ values of all compounds were 2- to 3-fold lower than those observed for L929. Similarly, the cytotoxic effect of the compounds appeared to be dependent on the tether lengths, but this effect was not as pronounced as for L929 cells. The cytotoxic effects of the compounds were similar both in wild-type and TK1^– CCRF-CEM cells except for compounds 7-9, which had a lower growth-inhibitory effect. This probably was related to the higher hydrophilicity of these hydroxylated analogs with a shorter methylene spacer compared with compounds with a longer tether length.

Compounds with shorter tether lengths (1, 2, 7, and 8) had lower cytotoxicity and log P values (range 1.27–1.87) than those with long tethers (compounds 5, 6, 11, and 12), which showed some cytotoxicity and higher log P values (range 2.41–3.23). The compounds with intermediate tether length (compounds 3, 4, 9, and 10) had intermediate to low toxicity and their log P values ranged between 1.52 and 2.45. Thus, carborane-dependent lipophilicity most likely was involved in the growth inhibition that was observed. This conclusion was further supported by the fact that the attachment of additional hydrophilic groups (compounds 7-12) reduced the cytotoxic effect. Interestingly, the IC₅₀ values for compounds 4 and 10 observed in L929 wild-type cells were significantly lower compared with L929 TK^– cells. This finding is in agreement with the kinetic properties of both compounds and indicates higher metabolic activity than for all other compounds. Growth inhibition could have been related to significant differences in the intracellular accumulation of phosphorylated products of 4 and 10, which may have interfered with cellular processes, and this currently is under investigation.

In general, the compounds tested were only moderately cytotoxic. Furthermore, the cytotoxicity assay that was used in this study detected cell viability after 72 hours of exposure to the test compounds, a time course that is at least three times longer than that necessary to obtain sufficient intracellular boron accumulation. During the time period used for boron-uptake studies, no cytotoxic effect was detected (data not shown), indicating that the boronated dThds may be cytostatic rather than cytotoxic agents.

In vitro Uptake and Retention Studies.
Because the cellular uptake of sufficient amounts of ¹⁰B is a prerequisite for a boron delivery agent for neutron capture therapy, in vitro uptake and retention studies were performed as reported in a companion article in this issue (37) . Compounds 4, 6, 10, and 12 were evaluated in L929 wild-type and L929 TK1^– cell cultures (Table 3) . A significant 5- to 30-fold difference in uptake between L929 wild-type and TK1^– cells was observed. In addition, 29 to 46% of these compounds were retained for at least 12 hours in L929 wild-type but not in TK1^– cells. The uptake of compounds 10 and 12 occurred at 1.3- and 1.7-fold higher rates, respectively, in comparison to their counterparts lacking the dihydroxyl group. Compounds with a heptylene linker showed higher cellular uptake than compounds with a pentylene linker, most likely because of their increased lipophilicity. These data suggest that nucleoside phosphorylation by TK1 is a main factor for the uptake and retention of the carboranyl dThds in cells. However, we cannot exclude the possibility that cellular efflux mechanisms specific to L929 TK1 wild-type and TK1^– contribute to the observed uptake patterns.

	ACKNOWLEDGMENTS

 
The technical assistance of Michele R. Smith is gratefully acknowledged. The results described in this paper were partially presented at the (a) 10th International Symposium on Neutron Capture Therapy for Cancer; Essen Germany, September 8–13, 2002 and at the (b) Joint 11th International and 9th European Symposium on Purine and Pyrimidine Metabolism in Man. Egmond ann Zee, the Netherlands, June 9–13, 2003.

	FOOTNOTES

 
Grant support: From the United States Department of Energy Grant DE-FG02–90ER60972 and The Swedish Research Council.

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.

Note: Supplementary Data for this article can be found at Cancer Research Online (http://cancerres.aacrjournals.org).

Requests for reprints: Ashraf S. Al-Madhoun, Department of Molecular Biosciences, BMC, Box 575, SE–75 123 Uppsala, Sweden. Phone: 46-18-471-41-01; Fax: 46-18-55-07-62. E-mail: Ashraf.Al-Madhoun{at}bmc.uu.se

⁴ J. Johnsamuel, unpublished results.

Received 1/21/04. Revised 5/13/04. Accepted 5/25/04.

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Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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