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Selective Delivery of CB300638, a Cyclopenta[g]quinazoline-based Thymidylate Synthase Inhibitor into Human Tumor Cell Lines Overexpressing the -Isoform of the Folate Receptor¹

Section of Medicine [D. S. T., D. G., A. L. J.] and the Cancer Research United Kingdom Centre for Cancer Therapeutics [V. B., L. A. S., J. T.], the Institute of Cancer Research, Sutton, Surrey, SM2 5NG, United Kingdom, and Department of Oncology/Rheumatology, University Hospital Vrije Universiteit, Amsterdam, 1081 HV the Netherlands [G. J.]

	ABSTRACT

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The -isoform of the glycosylphosphatidylinositol cell membrane tethered folate receptor (-FR) is overexpressed in some carcinomas (notably ovarian carcinomas) relative to normal tissues. The nonpolyglutamatable folate-based thymidylate synthase (TS) inhibitor, CB300638 (TS K_i = 0.24 nM) displayed an IC₅₀ of 0.0028 µM for the inhibition of the growth of human A431-FBP cells transfected with the -FR. In contrast, the IC₅₀ for the neotransfected A431 cells was 0.81 µM (300-fold higher). Similarly, this compound inhibited the growth of human KB cells that constitutively overexpress the -FR with an IC₅₀ of 0.0036 µM. These data were derived from cells grown in a physiological concentration of folate (20 nM R,S-leucovorin). Incubation of KB cells with a 1 µM excess of folic acid (FA), to selectively block uptake via the -FR, increased the CB300638 IC₅₀ to 0.39 µM. The relatively low potency of CB300638 under these conditions, or in cell lines not expressing the -FR, is ascribed to its low affinity for the ubiquitously expressed folate transporter, the reduced-folate carrier (K_i for inhibition of [³H]methotrexate transport >100 µM). The high potency of CB300638 in -FR-overexpressing cell lines is attributable to high affinity of the -FR (53% of FA) and efficient endosomal trafficking mediated by the -FR. Sixteen-h exposure to CB300638 inhibited the rate of ³H₂O release from 5-[³H]dUrd (in situ TS assay) in A431, A431-FBP, and KB cells with IC₅₀ values of 0.1 µM, 0.005 µM, and 0.002 µM, respectively. The coaddition of 1 µM FA increased the IC₅₀s for A431-FBP and KB cells to 0.1 µM consistent with -FR-mediated transport of CB300638. In conclusion, -FR-mediated uptake of CB300638 leads to TS and growth inhibition that is highly selective for -FR overexpressing tumor cell lines. The low expression of the -FR in normal tissues, particularly those sensitive to TS inhibitors, together with the low affinity of CB300638 for the reduced-folate carrier, suggests that the compound may have potential as an antitumor agent with a high therapeutic index.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The -FR³ (membrane-associated folate-binding protein) is a glycosylphosphatidylinositol-anchored cell membrane protein that has very high affinity for FA and the more biologically relevant reduced-folates (K_d 0.1 nM; Refs. 1, 2, 3 ). The -FR can function as a high-affinity, low-capacity folate transporter, and the mechanism of folate internalization is receptor-mediated endocytosis (1, 2, 3, 4, 5) . After release from the endosome, the membrane-bound -FR is recycled to the cell membrane. A second proposed mechanism, potocytosis, involves -FR clustered in caveolae (reviewed in Refs. 1 , 3 ; Ref. 6 ). Significant expression of the -FR is largely restricted to kidney proximal tubules and choroid plexus (7, 8, 9, 10) , although it is suggested that the -FR is localized to the apical membrane surface in these organs and, therefore, may not play a significant role in folate uptake from blood (11) . There may be a specialized function of the -FR in the kidney to scavange folates from the glomerular filtrate. The -FR is highly overexpressed in many carcinomas (7 , 8 , 12 , 13) particularly those of ovarian and uterine origin where it is homogeneously overexpressed in up to 90% of cases (7 , 14, 15, 16) . Furthermore, high -FR expression is associated with high grade, platinum resistance, and poor prognosis (17) .

The ß-isoform of the folate receptor is expressed at low levels in several adult normal tissues, and has lower affinity than the -FR for folates and antifolates (18, 19, 20) . It is expressed in CD34-positive hemopoietic stem cells but does not function as a folate transporter (21) . The ß-FR is widely expressed in tumors of epithelial and nonepithelial origin with expression levels being generally low/moderate and high, respectively (Ref. 7 ; reviewed in Ref. 11 ). Expression of a third isoform () is restricted to hemopoietic tissues (22 , 23) . Lack of an efficient signal for glycosylphosphatidylinositol modification leads to continuous shedding from cells, and it is therefore a secretory form of the FR.

The ubiquitously expressed, high-capacity RFC is considered the principal cell membrane transporter that transfers reduced folates from the blood into tissues (reviewed in Ref. 1 ), whereas functional folate receptors have been hypothesized to play a secondary role in sequestering effluxed folates back into the cell (2) . The -FR may also be positioned in cell signaling pathways. For example, in IGROV-1 ovarian carcinoma cells, immunoprecipitation experiments have shown that the -FR is associated in membranes with the G protein G_1–3, and the nonreceptor kinase lyn (24) .

High FR expression in some tumors relative to normal tissues is being exploited as a means of delivering conjugates of FA and toxins, liposomes, imaging, or cytotoxic agents to tumors (11 , 25 , 26) . For example, FA-deferroxamine-¹¹¹indium conjugates are detected only in FR-expressing tumors and not normal tissues of mice, with the exception of kidney cells (27) . The high selectivity of this approach resides in the very low affinity of FA (not a major component of plasma) for the RFC and high affinity for the FR, respectively (1) . Thus, antifolate drugs with similarly low and high affinity for the RFC and -FR, respectively, could be highly selective for -FR overexpressing tumors relative to normal tissues.

A number of antifolates with intracellular targets such as TS and glycinamide ribonucleotide formyltransformylase are transported into cells primarily via the RFC (K_m 1 to 5 µM). These include raltitrexed (Tomudex), ZD9331 (TS), pemetrexed (Alimta; LY231514; TS and other targets), and lometrexol (glycinamide ribonucleotide formyltransformylase; Refs. 28, 29, 30, 31, 32 ). However, they also have high affinity for the -FR, and in vitro studies suggest that this transporter may become functionally relevant when overexpressed, particularly under conditions of very low RFC expression and/or low extracellular folate (1 , 33 , 34) . CB3717, a potent TS inhibitor (K_i = 3 nM) that is no longer used clinically, displays a comparatively low affinity for the RFC (K_m 20–50 µM) and consequently low growth inhibitory activity in most tumor cell lines (IC₅₀ 1 µM; Refs. 33 , 35, 36, 37 ). However, CB3717 was shown to have high affinity for the -FR and high activity (1 nM IC₅₀s) in some -FR overexpressing tumor cell lines, although this was observed largely under conditions of low (subphysiological) extracellular folate (33 , 37 , 38) . The low water solubility of CB3717, particularly in the acidic environment of the kidney tubules, was identified as the probable cause of some nephrotoxicity seen in clinical trials (35) . This suggested to us that a more water-soluble antifolate with similar or improved properties could be developed with -FR targeting properties in physiological folate conditions. Clinical activity would be confined to -FR-overexpressing tumors but, in contrast with conventional antifolates, normal tissue toxicity should be spared because of low uptake into cells via the ubiquitously expressed RFC.

Recently, we reported a series of highly water-soluble cyclopenta[g]quinazoline-based compounds that have properties that partly overlap with CB3717. They display high affinity for TS, low affinity for the RFC, and low growth-inhibitory potency in L1210 cells (39, 40, 41, 42) . However, members of this series are intrinsically more potent TS inhibitors than CB3717 and are not substrates for folylpolyglutamyl synthetase, the enzyme that metabolizes several antifolates to more active polyglutamate forms. Thus, they should overcome antifolate resistance attributable to low folylpolyglutamyl synthetase expression. Later studies described their high affinity for mouse -FR and identified 6-R,S-CB300638 as a lead compound transported selectively into -FR-overexpressing tumor cell lines (41) . The present study is concerned with the additional in vitro evaluation of CB300638 (Fig. 1) in the 6S-chirally pure form. In particular, the activity is described in human tumor cell lines overexpressing the receptor but expressing functionally normal RFC. These are the human A431-FBP (transfected with the human -FR) and human KB cell lines. Parental A431 cells (neotransfected) were used as a control non--FR-expressing cell line. It was necessary to grow the cell lines in folate-free medium supplemented with the reduced-folate cofactor, R,S-5,formyl-tetrahydrofolic acid (folinic acid; Leucovorin; R,S-LV). Standard commercial medium contain a very high concentration of FA (2–8 µM) that blocks -FR-mediated drug uptake and can down-regulate receptor expression (43) . CB300638 is the first example of a highly water-soluble antifolate that is selectively transported via the -FR rather than ubiquitously expressed RFC.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Structures of 6S-CB300638 and CB3717.

	MATERIALS AND METHODS

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Inhibition of Isolated TS.
The partial purification of L1210 TS, the inhibition assay, and methods for determining K_{i apparent} have been described previously (36 , 39) . The assay measures, over 1 h, the release of ³H₂O from [5-³H]dUMP at 37°C with 200 µM (6R,S)-5,10-methylene tetrahydrofolate as the cofactor (Schircks Laboratories, Jona, Switzerland). The K_i was determined for CB300638 by determining K_iapps over a range of (6RS)-5,10-methylene tetrahydrofolate concentrations and the K_i estimated from the intersection of the Y-axis as described by Henderson et al. (45) .

Cell Culture.
A431 epidermoid vulval (neotransfected) and A431-FBP cells (transfected with the human -FR) were a generous gift from Dr. Antonella Tomassetti (Instituto Tumori, Milan, Italy). The human -FR cDNA derived from IGROV-1 ovarian carcinoma cells was cloned into the pcDNAI/neo vector and used to transfect A431 cells (46) . These were cultured in DMEM without FA (Life Technologies, Inc., Paisley, Scotland) containing 10% dialyzed FCS (heat inactivated for 30 min at 60°C; PAA Laboratories, Yeovil, United Kingdom), 0.02 mg/ml gentamicin, 2 µg/ml amphotericin B, 2 mM L-glutamine, 1% nonessential amino acids (all purchased from Life Technologies, Inc.), 4 µg/ml bovine insulin, and 0.4 µg/ml hydrocortisone (Sigma, Poole, United Kingdom). Twenty nM or 1 nM R,S-LV was added as the folate source. Cells were selected for two passages once every 6 weeks with 0.8 mg/ml G418 (Sigma) and subsequently grown without this antibiotic for 2 weeks before use in assays.

Human KB cells were cultured in RPMI 1640 without FA (Life Technologies, Inc.) containing 10% dialyzed, heat-inactivated FCS, 0.02 mg/ml gentamicin, 0.5 µg/ml amphotericin B, and 2 mM L-glutamine (Life Technologies, Inc.). Twenty nM or 1 nM R,S-LV was added as the folate source.

Human W1L2 lymphoblastoid cells were grown as suspension cultures as described previously (36) .

Cell Surface -FR Expression in the A431 and A431-FBP Cells.
Single cell suspensions from nonconfluent A431 and A431-FBP cells grown in 1 nM or 20 nM R,S-LV were incubated for 30 min at 4°C with either the human MOv-18 anti--FR (47) or mouse IgG negative isotype IgG1 monoclonal antibodies (Sigma). After two washes in 0.5 ml ice-cold PBA (PBS with 2% FCS and 0.02% sodium azide) cell surface-bound antibodies were detected with a secondary FITC goat antimouse IgG1 monoclonal antibody (Sigma) also by incubating for 30 min at 4°C. All of the antibodies were used at a volume of 0.1 ml at 1:100 dilutions in PBA. After two additional washes in PBA, cells were counterstained with PI (0.01 ml of a 0.4 mg/ml solution in water; Sigma). Cells were analyzed on the same day on a Coulter EPICS Elite ESP (Beckman Coulter, Buckinghamshire, United Kingdom) to collect green (FITC) and red (PI) fluorescence at 525 nm and 630 nm, respectively, after excitation with an argon-ion laser at 488 nm. PI-positive staining cells were assessed to be dead and, therefore, excluded from the analysis. The mean fluorescence from negative and positive antibody isotypes was used as an indication of the level of cell surface -FR expression.

Cell Surface [³H]FA Binding Capacity.
The surface binding of [³H]FA was used to estimate the concentration of -FR expressed by the different cell lines in the conditions described above. This method has been published elsewhere but incorporates some important modifications (43) . Briefly, KB, A431, and A431-FBP cells were detached from tissue culture flasks (60% confluency) with 10 mM EDTA (Sigma) in PBS. All of the cell lines were washed with ice-cold HEPES-buffered saline solution (HBSS; pH 7.4), and then washed rapidly with acidic HBSS (pH 3.5) to remove surface-bound folates. Cells were resuspended in ice-cold HBSS (pH 7.4) at a concentration of 1–3 x 10⁶/ml. One-ml aliquots of cell suspension were incubated in HBSS (pH 7.4) at 4°C with 100 pmol [³H]FA (specific activity 0.5 Ci/mmol) for 10 min and then washed once with 0.5 ml ice-cold HBSS (pH 7.4) before scintillation counting. Cell surface binding was expressed as pmol [³H]FA bound/10⁷ cells.

Affinity for the -FR Relative to FA.
The method used is essentially a whole cell [³H]FA competitive binding assay of Westerhof et al. (37) that has been adapted for adherent cells. A431-FBP cells were detached from tissue culture flasks (60% confluent) with 10 mM EDTA (Sigma) in PBS before being washed with ice-cold HBSS. Cells were resuspended at a concentration of 1–2 x 10⁶/ml and the assay performed as above. One-ml aliquots were incubated with various concentrations of FA or antifolate compound (50–1000 nM) and 100 pmol of [³H]FA (specific activity 0.5 Ci/mmol) for 10 min at 4°C. Relative affinities are defined as the inverse molar ratio of compound required to inhibit [³H]FA binding to the -FR by 50%. The relative affinity of FA is set at 1. Compounds with a lower or higher affinity for the -FR have values of <1 and >1, respectively.

Compound Affinities for the RFC.
This method has been described elsewhere (30) . Briefly, human W1L2 cells [suspended in HBSS (pH 7.4) at 5 x 10⁶/ml] were incubated for 5 min at 37°C in the presence of various compound dilutions and 0.5 µM [³H]MTX (2.5Ci/mmol) and the cellular associated radioactivity measured so that the K_i for inhibition of [³H]MTX could be determined.

Growth Inhibition Studies.
A431 and A431-FBP cells were seeded at 1500 cells/well (0.16 ml volume) in Falcon 96-well plates (Becton Dickinson Labware Europe, Meylan Cedex France), and after 24 h various compound dilutions were added to quadruplicate wells (0.02 ml volume). The well volume was made up to 0.2 ml with 0.02 ml of supplemented DMEM. For FA and dThd protection studies 0.02 ml of 10 µM and 100 µM, respectively, (Sigma) in unsupplemented medium was added to give a final concentration of 1 µM FA and 10 µM dThd. They were added 15 min before compound addition. A431 and A431-FBP cells were incubated for 72 h and 96 h, respectively (equivalent to 4 control cell doublings). Growth inhibition was estimated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as a measure of cell viability (48 , 49) .

KB cells were seeded at 15 x 10³ cells/well (1 ml volume) in 24-well plates (Triple Red Laboratories, Oxfordshire, United Kingdom), and after 24 h various compound dilutions were added in duplicate (0.11 ml volume). In protection studies, 1 µM FA or 10 µM dThd (0.02 ml of 56.5 µM and 565 µM, respectively) was added 15 min before compound addition. Cells were incubated for 72 h (equivalent to four control cell doublings) after which time they were detached with trypsin-EDTA (Life Technologies, Inc.) resuspended in medium and counted using a Coulter particle counter (Z2 model; Coulter Electronics Ltd., Luton, United Kingdom). The population doubling times were 15 h irrespective of folate concentration.

In Situ TS Assay.
This assay measures the rate of ³H release (as ³H₂0) from 5-[³H]dUrd over 1 h and is a semiquantitative measure of TS inhibition in cultured cells (50) . The method, originally described for suspension cells, has now been adapted for use with adherent cell lines. A431, A431-FBP, and KB tumor cells growing in tissue culture medium with 20 nM LV as the folate source were seeded in the same medium at 5–7.5 x 10⁵ cells/5 ml in T-25 tissue culture flasks (Corning Inc. Lutterworth, United Kingdom). After 24–30 h, increasing concentrations of CB300638 (0.3–300 nM) were added for 16 h (cells remained largely attached). Some flasks were coincubated with an excess concentration of FA (1 µM added 10 min before CB300638) to block uptake of CB300638 via the -FR. After the 16-h incubation, 0.05 ml of 30 µM 5-[³H]dUrd (3.3 Ci/mmol; Moravek Biochemicals; 22 Ci/mmol) was added to the flasks, giving a final concentration of 0.3 µM. After 20, 40, and 60 min, 3 x 0.4 ml aliquots of medium were removed into 1.5-ml microfuge tubes and mixed with an equal volume of ice-cold 1 M perchloric acid followed by 0.5 ml of an ice-cold charcoal suspension (200 mg/ml activated charcoal and 10 mg/ml dextran; Sigma). After 15 min the microfuge tubes were centrifuged at 13,000 rpm for 5 min and 0.5 ml of supernatant removed, mixed with 10 ml of Ultima Gold scintillation fluid (Canberra Packard) and radioactivity measured by liquid scintillation counting. After the assay, the flasks containing the cells were drained, the cells detached with trypsin, and the cell number determined by counting on a hemocytometer using trypan blue exclusion. The rate of ³H₂O formation was calculated by fitting the data to a straight line by linear regression, the data for controls is given as pmol of ³H₂O formed/min/10⁶ cells, and the effect of treatment is given as percentage of control.

	RESULTS

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Characterization of the Human A431 and A431-FBP Isogenic Cell Line Pair.
The cell population doubling times for the A431 (neotransfected) and the A431-FBP (transfected) were 18 h and 21 h, respectively, when cultured in medium containing either 1 nM or 20 nM R,S-LV as the folate source.

Cell surface -FR expression was determined using the -FR-specific MOv-18 antibody and flow cytometry. A431-FBP cells grown in 1 nM R,S-LV had mean fluorescence intensities an order of magnitude higher than the negative isotype control and A431 cells (Fig. 2) . The expression level in 20 nM R,S-LV was the same (data not shown). The cell surface binding capacity of [³H]FA is an alternative and more quantitative measure of -FR expression in cell lines. In medium supplemented with 1 nM R,S-LV the surface binding was <1 pmol/10⁷ cells and 211 ± 65 pmol/10⁷ cells for A431 and A431-FBP cells, respectively. No significant difference was seen when the cells were grown in 20 nM R,S-LV (A431-FBP = 171 ± 42 pmol/10⁷ cells). For reference, KB cells grown in 1 nM R,S-LV had a surface binding capacity of 123 ± 43 pmol/10⁷ cells (91 ± 17 in 20 nM R,S-LV).

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Cell surface -FR expression in the A43A431-FBP isogenic pair of cell lines. The A431 is neotransfected, and A431-FBP cells are transfected with the human -FR. Cells were grown in medium with 1 nM R,S-LV as the folate source. The same results were obtained with cells cultured in 20 nM R,S-LV. Cells were incubated with either the MOv-18 anti--FR or mouse IgG negative isotype IgG1 monoclonal antibodies, and binding detected by a FITC-goat antimouse IgG1 antibody and flow cytometry.

The A431 and A431-FBP cell lines were similarly sensitive to 5-fluorodeoxyuridine, a pyrimidine-based TS inhibitor (51) transported into cells via nucleoside transporters (0.0063 ± 0.0013 µM and 0.0042 ± 0.001 µM IC₅₀s, respectively, in 20 nM R,S-LV as the folate source). The IC₅₀ of the nonpolyglutamatable TS inhibitor, ZD9331 (30) , was similar for the two cell lines when an excess of FA (1 µM) was added to block -FR-mediated uptake (A431 = 0.067 ± 0.029 µM and A431-FBP = 0.034 ± 0.0087 µM). This suggests that RFC-mediated transport is functionally similar in the transfected cell line.

Together, these data are consistent with the isogenic cell line pair being genetically similar except for -FR expression.

Inhibition of Isolated TS.
A K_i for CB300638 was determined and found to be 0.24 nM (Fig. 3) . In common with CB3717 (K_i = 3 nM), the mechanism of TS inhibition by CB300638 was mixed, noncompetitive, but tending toward competitive at low and physiologically relevant 5,10-methylene tetrahydrofolate concentrations (36) . Computer graphics modeling suggested that the conformational restriction introduced by the presence of the pentocycle together with the dipeptide ligand favored binding to the enzyme (39 , 42) .

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Henderson plot for the Ki for inhibition of L1210 TS by CB300638. Kiapps were determined by fitting the data to the Goldstein equation for Zone-B binding by nonlinear least-squares regression analysis. The Kiapps over a range of (6R,S)-5,10-methylene tetrahydrofolate concentrations are plotted with their 95% confidence limits. Results represent a single experiment in which the (6R,S)-5,10-methylene tetrahydrofolate Km = 33 ± 8.1 µM (plotted as -Km on X-axis). The Ki for CB300638 = 0.24 nM; bars, ± 95% confidence limits.

Relative Affinity for the -Folate Receptor.

Growth Inhibition in Human A431 (Neo-transfected) and A431-FBP (-FR-transfected) Cell Lines.
In 1 nM LV the IC₅₀ for CB300638 in A431 and A431-FBP cells was 0.67 µM and 0.0021 µM, respectively. Similar results were seen with CB3717 (IC₅₀ = 0.60 and 0.0038 µM, respectively). However, in contrast with CB3717 the potency of CB300638 was preserved in A431-FBP cells grown in 20 nM LV (CB300638 IC₅₀ = 0.0028 µM; CB3717 IC₅₀ = 0.25 µM). Thus, CB300638 is 300-fold more selective for A431-FBP compared with A431 cells in physiological folate conditions (Table 2 ; Fig. 4 ). The addition of 1 µM FA reduced the selectivity to 2-fold, demonstrating the role of the -FR in the effects observed. Although the exposure time is different for the two cell lines to give approximately four control doublings (72 h and 96 h for A431 and A431-FBP, respectively), increasing the A431 exposure time to 96 h did not affect the IC₅₀ or selectivity (data not shown). The IC₅₀ of CB300638 in both cell lines in the presence of 10 µM dThd was >30 µM. dThd can circumvent TS inhibition induced by drugs such as CB3717 by providing dTMP through the dThd salvage pathway (52) . Thus, these results support TS being the growth rate-limiting locus of action of CB300638 in these cells (Fig. 4) .

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Growth inhibition curves for CB300638 in human A431 and A431-FBP cells (±10 µM dThd). Cells were incubated with increasing concentrations of CB300638 in medium containing 20 nM R,S-LV as the folate source. After 72 h (A431) and 96 h (A431-FBP) incubation (approximately four control population doublings) an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed as a surrogate for cell viability, and the absorbance at 540 nm presented as percentage control. A431-FBP cells - CB300638 alone (, ———), CB300638 + 10 µM dThd (, ----). A431 cells - CB300638 alone (, ———), and CB300638 + 10 µM dThd (, ----).

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Growth inhibition curve for CB300638 in human KB cells in the absence and presence of 1 µM FA. Cells were incubated with increasing concentrations of CB300638 in medium containing 20 nM R,S-LV as the folate source. Cells were counted after a 72 h incubation and presented as percentage of control cell counts. CB300638 alone (———), CB300638 + 1 µM FA (----).

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Inhibition of the rate of 3H2O-release from 5-[3H]deoxyuridine in tumor cells exposed to CB300638 for 16 h. This is an indirect measurement of the effect of increasing concentrations of CB300638 on the flux through TS in A431-FBP cells in the absence () and presence () of 1 µM FA. The cells were grown in medium containing 20 nM R,S-LV as the folate source and exposed to CB300638 for 16 h. After this time, the rate of 3H2O release from 5-[3H]dUrd was measured over 1 h. Control rates, measured after the same exposure time, were 2 pmol/min/106 cells for all cell lines ±1 µM FA. Results are presented as percentage of control. Data pooled from more than one experiment; bars, ± SD. Inset, concentration of CB300638 to inhibit flux through TS by 50% in human A431, A431-FBP, and KB cells in the absence () and presence () of 1 µM FA.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

These data have demonstrated that a nonpolyglutamatable, but nonetheless highly potent TS inhibitor, can be a potent inhibitor of growth of -FR-expressing cells. Previously, polyglutamation of CB3717 and other antifolates was thought to be important for its -FR-mediated activity, because relatively slow accumulation via this endocytotic mechanism would be compensated by the formation of highly potent polyglutamates (33) . CB300638 (TS K_i = 0.24 nM) is 2 orders of magnitude more active in A431-FBP and KB cells than in the non--FR-expressing A431 tumor cell line. Additional work is evaluating the activity of CB300638 in cell lines displaying lower -FR expression. The pharmacokinetic, tissue distribution, and antitumor properties of CB300638 are being evaluated in mice. Although the compound is predicted to display low normal tissue toxicity, particular attention will be paid to the effects in tissues expressing moderate levels of the -FR i.e., kidney and choroid plexus. However, these tissues do not have a high proliferative index and are relatively insensitive to TS inhibition. Therefore, in contrast with other antifolates, CB300638 may display a high therapeutic index.

	ACKNOWLEDGMENTS

 
We thank Dr. Antonella Tomassetti for the generous gift of the A431 and A431-FBP isogenic pair of cell lines, and Dr. Michael Ormerod for very helpful advice.

	FOOTNOTES

 
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.

¹ Supported by a grant from the Cancer Research Campaign, United Kingdom (now Cancer Research United Kingdom).

² To whom requests for reprints should be addressed, at Haddow Laboratories, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, United Kingdom. E-mail: annj{at}icr.ac.uk

³ The abbreviations used are: -FR, -isoform of the folate receptor; FA, folic acid; RFC, reduced-folate carrier; TS, thymidylate synthase; PI, propidium iodide; MTX, methotrexate; dThd, thymidine; dUrd, 2'-deoxyuridine.

Received 5/10/02. Accepted 4/24/03.

	REFERENCES

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