WO1997032590A1

WO1997032590A1 - Compositions and method for evaluating tissue for the presence of cancer cells

Info

Publication number: WO1997032590A1
Application number: PCT/US1997/003367
Authority: WO
Inventors: Juan Carlos Vera; William G. Bornmann; Pilar Zamora-Leon; David W. Golde; Maria Spassova
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 1996-03-04
Filing date: 1997-03-04
Publication date: 1997-09-12

Abstract

Breast cancer cells, melanoma cells and other types of cancer cells express the GLUT5 transporter and thus can accumulate significant amounts of fructose within the cell. GLUT5 is not expressed in normal breast cells or melanocytes, and may be differentially expressed in other tissue types. This permits the use of novel fructose-based imaging and therapeutic agents for the diagnosis and treatment of cancer.

Description

Compositions and Method for Evaluating Tissue for the Presence of Cancer Cells

DESCRIPTION

This work was performed with support from the United States Government, National Institutes of Health Grants Nos. CA30388 and ROl HL42107. The US Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION This application relates to the use of derivatives of fructose in the detection, localization and treatment of cancer, including in particular breast cancer.

Breast cancer is the most frequent malignancy among women in the western hemisphere, and its incidence is increasing. As with most cancers, early detection may be the key to successful treatment. Thus, there is a continuing need for improved methods for detecting the presence of breast cancer, and localizing cancerous tissues within the a patient to increase the chances of complete and effective therapeutic intervention

It has previously been observed that cancer cells in general, including breast cancer cells, have an increased ability to transport glucose through the cell membrane based upon an overexpression of GLUT 1 transporters. This fact has been used to localize tumors in patients and to assess tumor metabolism and response to therapy by detecting

¹⁸fluorodeoxyglucose by positron emission tomography (PET). Glucose transport occurs in both cancerous and normal cells, however, and in cells throughout the body. Thus, information obtained using glucose uptake always must be evaluated against a background of the glucose transport occurring in normal cells. It has now been found that at least two different types of cancer cells, namely breast cancer cells and melanoma cells, also have active GLUT5 transporters which transport fructose through the cell membrane, resulting in an observable accumulation of fructose within the cells. In contrast, normal breast cells and normal epidermal melanocytes do not express the GLUT5 transporter Thus, fructose transport provides the ability to mark cancer cells without significant marking of noncancerous cells, and to introduce therapeutic agents selectively into cancer cells. It is an object of the present invention to exploit this newly found characteristic of breast cancer and melanoma cells to provide methods and compositions for the detection, localization and treatment of breast cancer and other cancers which exhibit increased expression of GLUT5 transporters

SUMMARY OF THE INVENTION The present invention provides a method for evaluating tissue for the presence of cancer cells which express GLUT5 transporter at levels greater than non-cancerous cells of the same type, comprising the steps of

(a) exposing the tissue to a detectably-labeled compound selected from among fructose and derivatives of fructose for a period of time sufficient to accumulate the detectably-labeled compound in cancer cells expressing GLUT5 transporter, and

(b) observing the tissue for accumulation of the detectably-labeled compound in cells, wherein an increased accumulation of the detectably-labeled compound relative to the accumulation in normal cells is indicative of the presence of cancer cells The method of the invention is particularly applicable to detection of breast cancer cells in mammary tissue, for example using positron emission tomography (PET)

The present invention also provides a method for specifically introducing a selected chemical moiety into breast cancer cells, comprising the steps of

(a) combining the selected chemical moiety with a molecule of fructose, to form a fructose compound which is transportable by GLUT5 transporters; and (b) exposing tissue containing breast cancer cells to the fructose derivative for a period of time sufficient to permit accumulation of cytotoxic levels of the fructose compound within the breast cancer cells This method takes advantage of the fact that GLUT5 transporters are selectively expressed in breast cancer cells, and not in normal breast cells to introduce chemical entities, such as radioactive nuclei or cytotoxic groups, which may have therapeutic benefits for a patient suffering from breast cancer

BRIEF DESCRIPTION OF THE DRAWINGS Figs 1 A-1G show the results of deoxyglucose uptake experiments using human cancer cell lines, Figs 2A-21 show the results of fructose uptake experiments using human cancer cell lines. Fig 3 shows a reaction scheme for the synthesis of fluorofructose compounds, Fig 4 shows a reaction scheme for the synthesis of fluorofructose compounds, Fig 5 shows a reaction scheme for the synthesis of fluorofructose compounds, Fig 6 shows a reaction scheme for the synthesis of a fluorescent ascorbic acid derivative, and

Fig 7 shows a reaction scheme for the synthesis of a fluorescent ascorbic acid derivative

DETAILED DESCRIPTION OF THE INVENTION The present invention relies on the discovery that breast cancer cells and melanoma cells express the GLUT5 transporter and thus can accumulate significant amounts of fructose within the cell GLUT5 is not expressed in normal breast cells or melanocytes

GLUT5 is one of six known facilitative hexose transporter isoforms, and is a high affinity transporter of fructose Prior to the present invention, GLUT5 expression had been identified in only the small intestine and sperm cells Kayano et al , J. Biol. Chem. 265 13276-13282 (1990), Burant et al , J. Biol. Chem. 267 14523-14526 (1992) Furthermore,

GLUT5 is not expressed at detectable levels in normal breast cells It was therefore surprising to find that GLUT5 is expressed at significant levels, and effectively transports fructose into breast cancer cells This finding, however, makes available a new set of possibilities for the detection, localization, and treatment of breast cancer In addition, these same approaches are applicable to other cancer types which may be identified as expressing GLUT5 differentially from normal cells of the same type Thus, for example immunolocalization experiments have shown that GLUT5 is expressed in serninoma, embryonal carcinoma of the testis and colon carcinoma In the case of melanoma and colon carcinoma where cell lines are available, fructose uptake presumably mediated by the GLUT5 transporter has also been observed Identification of cancer types which express GLUT5 can be done using an immunoassay with anti-hexose transporter antibodies The specific details of the processes used in identifying the expression of GLUT5 in breast cancer cells or melanoma cells are set forth in Examples 1 -3 and 10 below In general, however, the process involves incubation of a sample with primary antibodies against GLUT5, for example rabbit anti-GLUT5 which is available from East-Acres Biologicals If GLUT5 is present, the antibody binds to the sample

Unbound antibody is then washed away, and a secondary antibody which binds to the rabbit anti-GLUT5 and contains a detectable moiety, for example horseradish peroxidase-goat anti- rabbit IgG (available from Amersham) is then added. If GLUT5 was present in the sample, the detectable moiety becomes bound to the sample Thus, routine screening permits identification of additional cell types to which the present invention is applicable. Mammary tissue and other tissue types which are found to express GLUT5 transporter in cancer cells at levels greater than non-cancerous cells of the same type can be evaluated in accordance with the present invention for the present invention by exposing the tissue to a compound which is a detectable form of fructose such as a radiolabeled fructose molecule, or a detectably-labeled derivative of fructose for a period of time sufficient to accumulate the compound in cancer cells expressing GLUT5 transporter, and observing the tissue for accumulation of the detectably-labeled derivative of fructose in cells. Suitable detectable labels will be generally small chemical moieties which do not interfere with the transport of fructose by GLUT5, but which are readily detectable, particularly using imaging techniques Specific examples of detectable labels useful in the invention include radiolabels incorporated into the fructose molecule which can be detected using techniques such as scintillation counting and autoradiography; fluorofructose derivatives, which can be detected using techniques such as magnetic resonance imaging (MRI) using ^I9F-fructose derivatives or PET using ^,8F-fructose derivatives; and fluorescent derivatives such as a 4-chloro-7-nitro-2- oxa-l ,3,-diazole (NBO chloride) derivative. Compounds of this type can be synthesized in good yield using the methods shown in Examples 4 - 9. In general, these methods involve either ( 1 ) reacting fructose with a sulfonic acid to form a fructose derivative having a sulfonyloxy group attached thereto; and the displacing the sulfonyloxy group with a fluorine containing reagent to form the fluorofructose compound, or (2) reacting fructose with DAST reagent to form the fluorofructose compound by direct fluorination.

In each of these techniques, the tissue to be evaluated is exposed to detectably- labeled fructose derivative for a period of time sufficient to allow accumulation of a detectable amount of the detectable label within cancerous cells. Based upon uptake of fluorinated glucose derivatives for PET scan experiments, the time required will generally be around 10 to 60 minutes. The tissue is then observed for accumulation of the detectably-labeled compound, permitting the detection of cancerous cells Furthermore, where imaging techniques are employed, cancerous cells can be localized within a mass of normal tissue

In addition to the detection and localization of cancerous cells, the presence of an enhanced fructose transport system in cancer cells can be used to deliver therapeutic agents selectively to these cells For example, deoxyfructose or a therapeutically effective radio- labeled fructose could be used to kill or inhibit cells in which the fructose was accumulated Fructose might also be coupled with known cytotoxic agents such a mitomycin C, to provide a targeted therapeutic for use in cancer cells which express elevated levels of GLUT5 transporter The therapeutic agent is formulated in a pharmaceutically acceptable carrier for example an iηjectable solution or an oral preparation for treatment of breast cancer or as a topical solution or patch for treatment of melanoma

Coupling of fructose to detectable groups such as fluorophores or to chemotherapeutic agents such as mitomycin C can be carried out by first converting the fructose to a reactive derivative such as 1 -amino-fructose (Garcia Martin, et al , Carbohydr. Res. 199 139-51 ( 1990) ), and then using the reactive group and an appropriate coupler to link to the desired substituent to the fructose For example, in the case of mitomycin C, a diacid coupler is connected to amino group of fructose and mitomycin C In the case where the compound to be coupled itself has an appropriate leaving group, the linker can be omitted Figs 6 and 7 show synthesis of ascorbic acid derivatives of this type for ascorbic acid which could be modified by substitution of fructose for ascorbic acid to make compounds useful in the present invention

The invention will now be further described with reference to the following non-limiting examples

EXAMPLE 1

IDENTIFICATION OF GLUT5 PRODUCING CELLS USING IMMUNOBLOTTING

Cancer cells which express GLUT5 can be identified using immunoblotting techniques as exemplified using MCF-7 and MDA-468 breast cancer cell lines The cell lines were obtained from the American Type Culture Collection and grown in a mixture of Dulbecco's Modified Eagle Medium containing high glucose (17 5 mM) and F-12 medium

(1 1, v/v), supplemented with 10% fetal bovine serum and 1% penicillin streptomycin For immunoblotting, MCF-7 and MDA-468 cells were homogenized in Tris- HCl (pH 7 4) containing 1 5% deoxycholic acid, 1 5 % Triton X-100, 1 mM EDTA, 10 mM EGTA, 25 mM dithiothreitol, 0 1 mM phenylmethylsulfonyl fluoride and 10 μg/ml each of aprotonin, leupeptin, pepstatin A and soybean trypsin inhibitor and centrifuged at 4,000 X g for 15 minutes at 4°C Protein samples ( 100 μg) were separated by SDS-polyacrylamide gel

(10%) electrophoresis and transferred to polyvinylidene fluoride membranes (lmmobilon-P, Millipore) After transfer, the membranes were blocked with phosphate buffered saline (PBS) containing 5% bovine serum albumin (BSA) and incubated with 5-10 μl of primary rabbit antibodies purchased from East-Acres Biologicals at dilutions of 1 1000 for anti-GLUTl and 1 500 for anti-GLUT2 and anti-GLUT5 (final volume 5 mL) for 60 minutes The membranes were then washed with PBS containing 1% BSA prior to the addition of secondary antibodies

Goat anti-rabbit IgG coupled to horseradish peroxidase was purchased from Amersham and used as the secondary antibody 1 μl of the secondary antibody (diluted 1 5000, final volume 5 mL) was incubated with the washed membranes for 60 minutes The membranes were then washed with PBS containing 1% BSA to remove any secondary antibody which had not bound to the membrane and evaluated using enhanced chemiluminescence

Evaluation of the membranes treated in this way revealed the presence of several overlapping bands which react with anti-GLUTl and anti-GLUT2 antibodies In addition, bands having apparent molecular weights of 50 and 70 kilodaltons in the case of

MCF-7 cells and 50 and 85 kilodaltons in the case of MDA-468 cells were found to react with anti-GLUT5

EXAMPLE 2 IDENTIFICATION OF GLUT5 PRODUCING

CELLS USING IMMUNOCYTOCHEMISTRY

Cancer cells which express GLUT5 can be identified using immunocytochemical procedures as exemplified using MCF-7 and MDA-468 breast cancer cell lines The cells were grown in 8-well microscope slides for 48 hours, fixed with buffered formaldehyde-acetone for 30 seconds and washed twice with PBS Fixed cells were incubated in a humid chamber for 1 hour in PBS containing 5% BSA, followed by incubation for 1 hour at room temperature in the same buffer containing 1% BSA, 0 3% Triton X-100 and one type of rabbit anti-GLUT antibody (dilution 1 : 100) or rabbit preimmune serum. After washing, the cells were then incubated with fluorescein isothiocyanate-goat anti-rabbit IgG (Life Technologies) at a dilution of 1 40 for 1 hour, mounted and analyzed by fluorescence microscopy Fluorescence was observed when anti-GLUTl , anti-GLUT2 and anti-GLUT5 primary antibodies were used, with the fluorescence being most intense when anti-GLUTl and anti-GLUT5 antibodies were used No reactivity was observed using preimmune serum.

EXAMPLE 3 IDENTIFICATION OF GLUT5 PRODUCING CELLS USING IMMUNOHISTOCHEMICAL ANALYSIS

Breast tissue expression of GLUT 5 was also determined by immunohistochemical analysis of a set of thin sections prepared from archived paraffin tissue blocks from 20 breast tissue samples of normal and neoplastic primary human beast tissue Paraffin was removed by incubating the sections in xylene, followed by absolute alcohol and afterwards the sections were hydrated by immersion in graded alcohol solutions. The hydrated sections were incubated in PBS containing 5% skim milk for I hour at room temperature, washed with PBS and incubated with a 1 :100 dilution of rabbit anti-GLUT5 antibodies for 2 hours. After extensive washing with PBS, the sections were incubated with anti-rabbit IgG coupled to alkaline phosphatase ( 1 .500) Color development was performed using 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.

All twenty primary breast cancer tissues tested were positive for the presence of GLUT 5 with strong straining being observed in the perinuclear region, cytoplasm and cell membrane of tumor cells. Staining was also seen in malignant cells invading the fibroadipose tissue No staining was observed for the 4 normal mammary tissue samples tested, indicating that GLUT5 is not expressed in breast cells.

EXAMPLE 4 UPTAKE OF RADIOLABELED FRUCTOSE INTO BREAST CANCER CELLS

Human breast cell cancer lines MCF-7 and MDA-468 were grown as monolayer cultures in six-well plates to a density of approximately 1 X 10⁶ cells per well.

Cultures were examined by microscopy to ensure that only plates showing uniformly grown cells were used Two wells in each plate were used to determine the number of cells, and the other four wells were used for uptake assays

For uptake assays, the cells were washed with incubation buffer containing 15 mM HEPES, 135 mM NaCl, 5 mM KCl, 1 8 mM CaCl₂, and 0 8 mM MgCl₂ and incubated in the same medium for 30 minutes at 37 °C Uptake assays were performed at room temperature in 1 ml incubation buffer containing either 0 2 mM deoxyglucose, and analog of glucose transported only by the facilitative glucose transporters, and 2-4 μCi of 2-[l,2- Η](N)-deoxy-D-glucose (30 Ci per mmole, NEN-DuPont) or 1 mM fructose and 0 8 μCi/ml of D-[U^l4C]-fructose (285 mCi per mmole, Amersham) Uptake was stopped by washing the cells with three 1 -ml aliquots of ice-cold PBS Cells were then dissolved in 0 5 ml of lysis buffer (10 mM Tris-CI pH 8 0, - 2% SDS), and the incorporated activity was measured by liquid scintillation spectrometry Competitors or inhibitors were added in some instances

The results of these uptake experiments are summarized in Figs 1 A-H and 2 A-I Both cell lines showed a notable capacity to take up deoxyglucose (DOG) in a ten minute period (Fig 1A) When this data was replotted in a Lineweaver-Burk plot (Figs IB and IC), both cell types evidenced a high affinity component with an apparent Km for transport of 2 mM and a second, lower affinity component with an apparent Km for transport of lO nrM

To further refine the rate of uptake of deoxyglucose, measurements were taken at very short intervals from 5 seconds to 2 minutes after initiation of uptake Uptake was linear throughout the observation period (Fig I D) Double-reciprocal Lineweaver-Burk plots confirmed the presence of two functionally distinct glucose transporters in breast cancer cells (Fig IE and IF) Using the Michaelis expression for a single-substrate reaction under conditions of initial velocity v=(S x V_ma /(S+Km), in which v represents the velocity of the forward reaction, Km represents the Michaelis constant, S represents the concentration of substrate and V max represents the maximum velocity of the reaction, and assuming a physiologic concentration of glucose of 5 5 mM, this data suggests that the high affinity transporter comprises three quarters of the capacity of the breast cancer cell lines to take up glucose (Figs 1 G and 1H) Figs 2A-2I show the results when fructose uptake is measured Fig 2 A shows that both types of cells take up fructose This transport could have been due to GLUT2 which is known to occur in breast cells, but the linear double reciprocal Lineweaver-Burk plots (Figs 2B and 2C) revealed an apparent Km for transport of fructose of 10 mM This value is approximately an order of magnitude lower than the values previously described for the transport of fructose by GLUT2 which were obtained after subtracting from the date the contribution of a low-affinity pathway that showed no saturation at concentrations of fructose as high as 50 mM (Okuno et al Biυchim. Biophys Ada 862 329-334 (1986), Colville et al , Biochem. J. 290 701 -706 (1993) ) Analysis of transport at concentrations of fructose from 1 to 50 mM revealed that, at concentrations of fructose lower than 10 mM the high affinity pathway contributed greater than 90% of the capacity of the breast cancer cell lines MCF-7 (Fig 2D) and MDA-469 (Fig 2E) to transport fructose

Because these data suggested the possibility that an additional fructose transporter might be present, we tested the cell lines for the presence of GLUT5 GLUT5 has a Km for transport of fructose of 6 mM, is not inhibited by cytochalsin B, a specific inhibitor of GLUT! and GLUT2, and does not transport deoxyglucose Measurement of the uptake of fructose in the presence of cytochalsin B therefore provides evidence of the presence of

GLUT5 transporters Cytochalsin E, an analog of cytochalsin B which does not inhibit deoxyglucose transport was used as a control

As shown in Fig 2F, transport of deoxyglucose in MCF-7 cells was completely inhibited by cytochalsin B at concentrations above about 100 μM Fifty percent inhibition was observed at 0 4 μM cytochalsin B, a value which falls between the value of Ki for GLUT1

(-0.2 μM) and Ki for GLUT2 (~2 μM) On the other hand, 100 μM cytochalsin B inhibited less than 40 % of the fructose uptake, and less than 20% inhibition was observed at 1 μM cytochalsin B (Fig 2G) This residual fructose transport activity even at levels of cytochalsin B that completely inhibit deoxyglucose transport can be attributed to the presence of GLUT 5 Further evidence for the presence of GLUT 5 in human breast cancer cells was found by observing the effect of deoxyglucose on the uptake of fructose Previous data indicated that deoxyglucose was able to inhibit completely the uptake of fructose mediated by GLUT2, but does not affect fructose uptake mediated by GLUT5 Burant et al , J. Biol. Chem. 267 14523-14526 (1992) As shown in Fig 21, 100 mM deoxyglucose inhibited less than 20% of fructose uptake, in contrast to the effect of 100 mM unlabeled fructose which inhibited uptake completely EXAMPLE 5 Fig 3 shows a synthetic route for the synthesis of 1 -deoxy- 1 fluorofructopyranose In the first step of the reaction, D-fructose (13 5 g) was combined with anhydrous ZnCl₂ ( 18 g) and acetone (90 ml) A solution of P₂O₅ (0 75 g) in 85% H,PO₄ ( 1 5 g) was added and the resulting mixture was stirred overnight The solution was neutralized with Amberlite IRA 400)OH) to pH 7, concentrated, and extracted with chloroform (3 X 250 ml) The solvent was evaporated from product chloroform phase on a rotary evaporator at reduced pressure The resulting residue was dissolved on 0 1 N H₂SO₄ (100 ml) and stirred at room temperature overnight The product was then extracted with chloroform (3X250 ml), dried over MgSO₄ and evaporated to give 1 1 622g (60% yield) of

2,3 4,5-di-O-isopropylidene-β-D-fructopyranose (I) as a crystalline solid

Trifluoromethane sulfonic anhydride (1 975 gm 7 mmoles) and 2,6-di-tert- butyl-4-methylpyridine (1 45 g, 7 1 mmoles) were dissolved in anhydrous CH₂C1₂ (40 ml) The solution was cooled to 0°C and a solution of 2,3 4,5-di-O-isopropyIidene-β-D- fructopyranose (I) (1 35 g, 5 mmoles in 15 ml CH₂C1₂) was added dropwise The temperature of the reaction mixture was allowed to rise to 25°C where it was maintained with stirring for 1 hour, at which time the solution was poured into ice water (200 ml) containing 5 g NaHCO₃ with vigorous stirring The layers were separated, and the aqueous layer was extracted with CH₂CI₂ (2 X 70 ml) The combined organic extracts were dried over MgSO₄ and solvent evaporated to give 2 g of a crude product material This crude product was separated on an

SiO₂ column (Merck Silica 60, 40-60 μm) using 10% diethylether in hexane as the eluent to yield 0 98 g (99% yield) of 2,3 4,5-di-O-isopropylidene-l-trifluoromethylsulfonyl fructopyranose (II)

2,3 4,5-Di-O-isopropylidene-l-trifluoromethylsulfonyl fructopyranose (II) was converted to 2,3 4,5-Di-O-isopropylidene-l-deoxy-l -fluorofructopyranose (III) by reaction with tetrabutylammonium fluoride Tetrabutylammonium fluoride hydrate (2 5 g, 9 56 mmoles) was refluxed in 40 ml benzene with a Dean-Stark water separator for two hours The triflate compound (II, lg, 2 55 mmoles) was and Molecular sieve 4A (3g) were added to this solution, and the mixture was refluxed for 1 hour The molecular sieve was filtered off, and the solvent evaporated The resulting residue was taken up in 100 ml CH₂C1₂ and washed with a saturated solution of NaHCO, The organic layer was dried over MgSO₄ and evaporated to dryness The product was purified on a SiO₂ column (SiO₂ Sigma, Type H, 10- 40 μm) using a 5% diethyl ether in hexane eluent to yield 389 g (58%) of the 1 -fluorofructose intermediate (III) The isopropylidene groups were then removed by combining the 1- fluorofructose intermediate (III, 0 547 g, 2 09 mmoles) with Amberlite GR 120 H⁺ Resin in water and refluxing for 1 hour, resulting in 0 343 g (94% yield) of α-1- fluorofructopyranoside This material was further purified by chromatography to yield 0 328 g of product (89% yield)

EXAMPLE 6 Fig 4 shows two alternative routes to the synthesis of fluorofructose derivatives in accordance with the invention a) D-fructose (9 g, 0.05 mol) and H₂SO₄ (1 125ml) were added to a mixture of absolute methanol (150 ml) and CaSO₄ (3 375 g) to form a suspension The suspension was stirred for 12 hours at room temperature The resulting suspension containing unreacted fructose and two reaction products was filtered and the filtrate was worked up with IRA 400

(OH) resin to pH 7 The resin was then filtered off and washed with methanol (3 X 50 ml) The filtrate and the washings were combined and evaporated to dryness to give an oily residue Flash chromatography of the residue (SiO₂ 800 ml, eluent 3% methanol in ethyl acetate and 5 % methanol) resulted in 2 91 g (30% yield) of α-methyl fructofuranoside (IV), 0 97 g (10%) of β-methyl fructofuranoside (V) and mixed fractions of IV and V, and V and fructose The mixed fractions were rechromatographed to improve the yield of the products IV and V b) Alternatively, products IV and V can be made by reacting D-fructose with 0.2N HC1 in methanol After work-up, this process yields 41% product IV and 34% product V

A solution of α-methyl fructofuranoside (IV) (1 48 g, 7 6 mmol) in CH₂CI₂ (30 ml) and DMF (2 ml) was added dropwise with a syringe through a rubber cap to a solution of trifluoromethane sulfonic anhydride (6 04 g, 3 6 ml, 21 4 mmol) and 2,6-di-tert-butyl-4- methylpyridine (4.45 g, 21 7 mmol) in CH₂C1₂(40 ml) under argon The reaction mixture was stirred at room temperature while monitoring the triflation of the fructofuranoside (IV) by

TLC (solvent system EtOAc/Hexane 1 1 , visualization with a solution of anisaldehyde in ethanol). After 2 hours the reaction mixture was poured with stirring into 300 ml of ice-water containing NaHCO₃ (8 g) The solution was extracted with CH₂C1₂ (3 X 200 ml), and the combined organic extracts were dried over MgSO₄, and evaporated to dryness The crude reaction product was separated by flash silica gel chromatography (SiO₂ Sigma, Type H, 10- 40 μm, eluent 10 % ethyl acetate in diethyl ether) to give 0 700 g (20% yield) of methyl- 1,6-

O-di[(trifluoromethyl)sulfonyl]-α-D-fructofuranoside (VII)

The triflate (VII) was then converted to methyl-6-deoxy-6-fluoro-α-D- fructofuranoside (VIII) Tetrabutylammonium fluoride hydrate (0 434 g, 1 6 mmol) was refluxed in dichlorobenzene (70 ml) with 1 teaspoon of molecular sieve for 2 hours The solution was decanted and the triflate (VII, 0 30 g, 0 4 mmol) and Vi teaspoon of fresh molecular sieve was added The resulting suspension was refluxed for an additional 6 hours The solvent was then evaporated under reduced pressure and the residue was partitioned between saturated NaHCO₃ and CH₂C1₂ The aqueous phase was collected, washed with CH₂C1, and then separated on a silica gel column (SiO₂ Merck, 15-40 μm flash) to give 0 4 g (21%) of methyl-6-deoxy-6-fluoro-α-D-fructofuranoside (VIII) Essentially no difluoro product was formed using this method

EXAMPLE 7 As shown in Fig 5, a solution of β-methyl fructofuranoside (V) (0 9 g, 4 63 mmol) in CH₂C1₂ (20 ml) was added dropwise with a syringe through a rubber cap to a solution of trifluoromethane sulfonic anhydride (3 66 g, 13 mol, 2 19 ml) and 2,6-di-tert-butyl- 4-methylpyridine (2 1 g, 13 2 mmol) in CH₂C1₂ (25 ml) under argon The reaction mixture was stirred at room temperature while monitoring the triflation of the fructofuranoside (V) by TLC (solvent system EtOAc/Hexane 1 1 , visualization with a solution of anisaldehyde in ethanol) After 2 hours the reaction mixture was poured with stirring into 250 ml of ice-water containing NaHCO, (7 g) The solution was extracted with CH₂C1₂ (3 X 200 ml), and the combined organic extracts were dried over MgSO₄, and evaporated to dryness The crude reaction product was separated by flash silica gel chromatography (SiO₂ Sigma, Type H, 10- 40 μm eluent 10 % ethyl acetate in diethyl ether) to give 0 483 g (25% yield) of the triflate product, methyl- l,6-O-di[(trifluoromethyl)sulfonyl]-β-D-fructofuranoside (IX) The triflate (IX) was then converted to methyl-6-deoxy-6-fluoro-β-D- fructofiiranoside (X) Tetrabutylammonium fluoride hydrate (0 434 g, 1 6 mmol) was refluxed in dichlorobenzene (70 ml) with 1 teaspoon of molecular sieve for 2 hours The solution was decanted and the triflate (IX, 0 30 g, 0 4 mmol) and Vi teaspoon of fresh molecular sieve was added The resulting suspension was refluxed for an additional 6 hours The solvent was then evaporated under reduced pressure and the residue was partitioned between saturated NaHCO, and CH₂C1₂ The aqueous phase was evaporated to dryness and the residue was separated on a silica gel column (SiO₂, Sigma H-Type, 10-40 μm, eluent 3% methanol in CHCI,) to give 0 198 g (10% yield) of methyl-6-deoxy-6-fluoro-β-D-fructofuranoside (X) Essentially no difluoro product was formed using this method

EXAMPLE 8 As shown in Figs 4, direct fluorination of methyl-α-D-fructofuranoside yields a mixture of 1 ,6 difluoro and 6 fluoro products Methyl-α-D-fructofuranoside (0 80 g, 4 12 mmol) was dissolved in CH₂CI₂ (45 ml) and DMF (2 5 ml) The solution was cooled to -15

°C and DAST (6 60 g, 41 20 mmol, 5 45 ml) was added in five portions with vigorous stirring and protection of argon The reaction mixture was stirred at this temperature for 2 hours, then the excess DAST was quenched with methanol (30 ml) The pH was adjusted to 7.0 with Amberlite IRA 400(OH) and the solvent evaporated The residue was separated on an SiO₂ column (SiO, Merck 15-40 μ , eluent CHCI, and 3% MeOH/CHCl, to give 0 015 g (2% yield) of methyl- l,6-dideoxy-l ,6-difluoro-α-D-fructofuranoside and 0 250 g (31% yield) of methyl-6-deoxy-6-difluoro-α-D-fructofuranoside

EXAMPLE 9 Direct fluorination of methyl-β-D-fructofuranoside yields a mixture of 1,6- difluoro and 6-fluoro products Methyl-β-D-fructofuranoside (0 80 g, 4 12 mmol) was dissolved in CH₂C1₂ (45 ml) and DMF (2 8 ml) The solution was cooled to -15 °C and DAST (6.60 g, 41 20 mmol, 5 45 ml) was added in five portions over 1 hour with vigorous stirring and protection of argon The reaction mixture was stirred at this temperature for 2 hours, then the excess DAST was quenched with methanol (30 ml) The pH was adjusted to 7 0 with Amberlite IRA 400(OH) and the solvent evaporated The residue was separated on an SiO₂ column (SiO₂ Merck 15-40 μm, eluent CHCI, and 3% MeOH/CHCl, to give 0 054 g (6% yield) of methyl- l ,6-dideoxy-l,6-difluoro-β-D-fructofuranoside and 0 30 g (30% yield) of methyl-6-deoxy-6-difluoro-β-D-fructofuranoside

EXAMPLE 10

IDENTIFICATION OF GLUT TRANSPORTERS IN MELANOMA CELLS USING IMMUNOCYTOCHEMISTRY

Normal human epidermal melanocytes (Clonetics, San Diego, CA) and melanoma cells (Spielholz et al , Blood 85 973-980 (1995)) were grown in 8-well microscope slides for 48 hours, fixed with buffered 4% paraformaldehyde-40% acetone for 15 minutes and washed twice with PBS Fixed cells were incubated in a humid chamber for 15 minutes at room temperature in 0 1% H₂O₂ in PBS to inhibit endogenous peroxidase, washed twice with PBS, and incubated for 30 minutes in PBS containing 4% BSA After incubation, the cells were washed twice in incubation buffer containing 8 4 mM Na₂HPO₄, 3 5 mM KH₂PO₄, 120 mM NaCl, 10 mM Tris pH 7.8, followed by incubation overnight at room temperature in the same buffer containing 1% BSA and one type of rabbit anti-GLUT antibody (dilution 1 100) or rabbit preimmune serum After washing with incubation buffer, the cells were incubated in the same buffer containing 1% BSA and swine anti-rabbit IgG antibody (DAKO Corporation, Carpenteria, CA) at a dilution of 1 50 for 2 hours After incubation the cells were washed three times in incubation buffer containing 1% BSA, followed by incubation for 30 minutes at room temperature in the same buffer containing peroxidase-antiperoxidase rabbit complex (DAKO) diluted 1 50 After washing three times in incubation buffer, the cells were incubated for 10 minutes in incubation buffer containing a metal-enhanced diaminobenzidine substrate kit (Pierce, Rockford, IL), washed, mounted and analyzed by light microscopy In total, six different melanoma cell lines as listed in Table 1 were analyzed As shown in the table, a positive immune response was observed for all melanoma cells lines tested when anti-GLUT- 1, anti-GLUT-2 and anti-GLUT5 primary antibodies were used (+++. cells highly immunoreactive with the anti-GLUT antibody, ++. cells showing an intermediate level of immunoreactivity, +. cells showing a weak level of immunoreactivity, - cells showing no immunoreactivity with the anti-GLUT antibody) No reactivity was observed in the melanoma cell lines with anti-GLUT3 and anti-GLUT4 antibodies, or when the primary antibody was absent in the reaction Only a weak immunoreactivity was observed in the normal human melanocytes when anti-GLUTl was used as the primary antibody, and no immunoreactivity was observed with anti-GLUT2, anti-GLUT3, anti-GLUT4 or anti-GLUT5.

TABLE 1

Cell Type GLUT1 GLUT2 GLUT3 GLUT4 GLUT5

SK MEL 131, +++ ++ - - +++ clone 1 -5

SK MEL 131 , +++ ++ - - +++ clone 3-44

SK MEL 29 +++ ++ - - +++

SK MEL 22A +++ ++ - - +++

SK MEL 23 +++ ++ - - +++

SK MEL 181 +++ ++ - - +++

Normal +- - - - - Melanocytes

Claims

I s 1 A method for evaluating tissue for the presence of cancer cells which express GLUT5 transporter at levels greater than non-cancerous cells of the same type, comprising the steps of (a) exposing the tissue to a detectably-labeled compound selected from

5 among detectably-labeled fructose and detectably-labeled derivatives of fructose for a period

6 of time sufficient to accumulate the detectably-labeled compound in cancer cells expressing GLUT5 transporter, and

8 (b) observing the tissue for accumulation of the detectably-labeled

9 compound in cells, wherein an increased accumulation of the detectably-labeled compound 0 relative to the accumulation in normal cells is indicative of the presence of cancer cells

1 2 The method according to claim 1 , wherein the tissue is mammary tissue

1 3 The method according to claim 2, wherein the detectably-labeled compound is detected using positron emission tomography

1 4 The method according to claim 3, wherein the detectably-labeled compound is an ^,8F-fructose

1 5 The method according to claim 1, wherein the tissue is epidermal tissue

1 6 A method for specifically introducing a selected chemical moiety into cancer cells which express GLUTS transporter at levels greater than non-cancerous cells of the same type, , comprising the steps of (a) combining the selected chemical moiety with a molecule of fructose, to form a fructose compound, and (b) exposing tissue containing cancer cells to the fructose compound for a period of time sufficient to permit accumulation of the fructose compound within the breast cancer cells 7 The method according to claim 6, wherein the selected moiety is a cytotoxic agent

8 The method according to claim 6, wherein the cancer cells are breast cancer cells

9 The method according to claim 6, wherein the cancer cells are melanoma cells

10 A pharmaceutical composition for treatment of cancer comprising a therapeutically effective amount of a cytotoxic derivative of fructose in a pharmaceutically acceptable carrier

1 1 A method for synthesizing a fluorofructose compound comprising the steps of (a) reacting fructose with a sulfonic acid to form a fructose derivative having a sulfonyloxy group attached thereto, and (b) displacing the sulfonyloxy group with a fluorine containing reagent to form the fluorofructose compound

12 A method for synthesizing a fluorofructose compound comprising the step of reacting fructose with DAST reagent to form the fluorofructose compound

13 A composition comprising a cytotoxic derivative of fructose