Synthesis, Purification, and Tumor Cell Uptake of 67Ga-Deferoxamine

S. Ben-David Makhluf , R. Arnon , C. R. Patra , D. Mukhopadhyay , A. Gedanken , P. Mukherjee and H. Breitbart. The Journal of ..... Ruth Röder , Erns...
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Bioconjugate Chem. 1996, 7, 56−62

56

Synthesis, Purification, and Tumor Cell Uptake of 67Ga-Deferoxamine-Folate, a Potential Radiopharmaceutical for Tumor Imaging Susan Wang,† Robert J. Lee,†,‡ Carla J. Mathias,§ Mark A. Green,§ and Philip S. Low*,† Department of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 47907. Received June 16, 1995X

The vitamin folic acid was covalently linked to the chelating agent deferoxamine (DF) via an amide bond using a simple carbodiimide coupling reaction. A mixture of two isomers, DF-folate(R) and DF-folate(γ), was produced involving the R- and γ-carboxyl group of folic acid, respectively. These two isomers were separated by anion-exchange chromatography using a NH4HCO3 gradient. Competitive binding studies revealed that only the DF-folate(γ) is recognized by the folate receptor on KB cells, interacting with an affinity comparable to unconjugated folic acid. The DF-folate conjugates were radiolabeled with the γ-emitting radionuclide 67Ga3+ and tested for uptake by cultured KB cells overexpressing the folate receptor. The cellular accumulation of 67Ga-DF-folate(γ) complex was found to be time-, temperature-, and concentration-dependent. The 67Ga-DF-folate(γ) tracer exhibited rapid uptake kinetics in cell culture with a t1/2 of ∼3 min. The KB cell association of 67GaDF-folate(γ) was competitively blocked by free folic acid, indicating that uptake of the 67Ga-DFfolate(γ) was specifically mediated by the folate receptor. Since the folate receptor is overexpressed on the surfaces of many neoplastic cells, these results suggest that 67Ga-DF-folate(γ) complex might be useful as a diagnostic agent for noninvasive imaging of folate receptor-positing tumors.

INTRODUCTION

Early detection and classification of human tumors is one of the major goals in the fight to overcome cancer. One strategy for noninvasively detecting the location and nature of neoplastic tissue involves imaging patients following administration of γ-emitting radiopharmaceuticals designed to localize specifically in the tumor mass. Consequently, substantial interest has developed in designing methods for selective delivery of radionuclides to tumors, for example, through the use of monoclonal antibodies directed to tumor-associated antigens and the use of peptides that are bound by tumor membrane receptors (Wilbur, 1992; Griffiths et al., 1992; Serafini, 1993; Fischman, 1993; Lamki, 1995). Recent studies have identified the folate receptor as a tumor marker that is overexpressed on many cancer cell surfaces but highly restricted in most normal tissues (Rettig et al., 1988; Campbell et al., 1991; Coney et al., 1991; Weitman et al., 1992; Garin-Chesa et al., 1993; Holm et al., 1994; Franklin et al., 1994; Ross et al., 1994). For example, immunohistochemical examination of ovarian carcinomas derived from coelomic epithelium showed that 52 out of 56 cases were strongly folate-receptorpositive (Garin-Chesa et al., 1993). For breast cancers, the corresponding frequency was 11 out of 53. Other * To whom correspondence should be addressed. Phone: (317) 494-5273. Fax: (317) 494-0239. † Department of Chemistry. ‡ Present address: Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. X Abstract published in Advance ACS Abstracts, October 15, 1995. 1 Abbreviations: acac, acetylacetone; DCC, dicyclohexylcarbodiimide; DEAE, (diethylamino)ethyl; DF, deferoxamine (desferrioxamine); FAB-MS, fast-atom bombardment mass spectroscopy; FDMEM, folate-deficient modified Eagle’s medium; HPLC, high-pressure liquid chromatography; PBS, phosphatebuffered saline (136.9 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4).

1043-1802/96/2907-0056$12.00/0

epithelial cancers showing folate-receptor-positive immunostaining included endometrial (10/11), colorectal (6/ 27), lung (6/18), and renal (9/18). Additionally, 4 out of 5 brain metastases derived from epithelial cancers were found to be strongly folate-receptor-positive. This measured folate receptor overexpression frequency might have increased even further if staining for other folate receptor isoforms had been conducted (Ross et al., 1994). The folate receptor is a glycosylphosphatidylinositolanchored glycoprotein (Elwood, 1989; Lacey et al., 1989; Verma et al., 1992) with high affinity for the vitamin folic acid (Kd ∼ 10-9 M) (McHugh and Cheng, 1979; Kamen and Capdevila, 1986; Kane and Waxman, 1989; Luhrs et al., 1992; Matsue et al., 1992). Membrane-associated folate receptor on cultured KB cells, a human nasopharyngeal epidermal carcinoma cell line, has been shown to mediate the specific internalization of folates at physiological concentrations (McHugh and Cheng, 1979; Antony et al., 1985). We have previously observed that when folate is covalently linked to a macromolecule via its γ-carboxylate moiety, its affinity for the cell surface receptors remains essentially unaltered (Leamon and Low, 1991, 1993). This property has been successfully exploited to deliver folate-conjugated protein toxins and drug/antisense oligonucleotide-carrying liposomes into cultured tumor cells overexpressing the folate receptor (Leamon and Low, 1992; Leamon et al., 1993; Lee and Low, 1994; Wang et al., 1995). Such folate-macromolecule conjugates were shown to be internalized via a nondestructive receptor-mediated endocytic pathway and delivered into the cytoplasm of the targeted cells in a biologically active form. In the present work, it was hypothesized that noninvasive diagnostic imaging of folate-receptor-bearing tumors might be feasible using radiolabeled folate conjugates obtained by folate derivatization of chelating agents that exhibit high affinity for γ-emitting metal radionuclides. We report here the synthesis, purification, and 67Ga-labeling of a deferoxamine-folate conjugate (DF© 1996 American Chemical Society

Folate Conjugate as Tumor Imaging Agent

folate) and the specific accumulation of the 67Ga-DFfolate in cultured tumor cells overexpressing the folate receptor. EXPERIMENTAL PROCEDURES

Materials. Folic acid, deferoxamine (DF) mesylate, DEAE-trisacryl anion-exchange resin, and carboxypeptidase G2 were purchased from Sigma (St. Louis, MO). Bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL). Acetonitrile (HPLC grade) and dicyclohexylcarbodiimide (DCC) were purchased from Aldrich (Milwaukee, WI). Gallium-67 chloride was purchased from Mallinckrodt Medical, Inc. (St. Louis, MO). Tissue culture products were obtained from GIBCO (Grand Island, NY), and cultured cells were received as a gift from the Purdue Cancer Center (West Lafayette, IN). Cell Culture. KB cells, a human nasopharyngeal epidermal carcinoma cell line that greatly overexpresses the folate binding protein, and WI38 cells, a human lung fibroblast primary cell line used as a control, were cultured continuously as a monolayer at 37 °C in a humidified atmosphere containing 5% CO2 in folatedeficient modified Eagle’s medium (FDMEM) (a folatefree modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal calf serum as the only source of folate) containing penicillin (50 units/mL), streptomycin (50 µg/mL), and 2 mM L-glutamine. The final folate concentration in the complete FDMEM is in the physiological range (∼2 nM). Forty-eight h prior to each experiment, the cells were transferred to 35 mm culture dishes at 5 × 105 cells per dish and grown to ∼80% confluence. Synthesis and Purification of Folate-Deferoxamine Conjugate. One hundred mg of DF mesylate was dissolved in 3 mL of dimethyl sulfoxide containing 200 µL of pyridine. A 10-fold excess of folic acid (672 mg) dissolved in 15 mL of warm (∼40 °C) dimethyl sulfoxide plus 5 molar equiv of DCC (157 mg) were then added. The reaction mixture was stirred at 40 °C in the dark, during which time ninhydrin assay and thin layer chromatography were used to follow the reaction progress. After the coupling was complete, the DF-folate conjugate and excess folic acid were precipitated with 200 mL of cold acetone and pelleted by centrifugation. The pellet was washed once with cold acetone, dried under vacuum, and then redissolved in 5 mL of deionized water. The pH of the solution was adjusted to 8.0 to facilitate dissolution of the solid. The crude product contained a mixture of folate linked to DF via either its R-carboxyl or γ-carboxyl group, as well as unreacted folic acid. The two isomers of DFfolate were isolated and purified on a weak anionexchange column. Briefly, the product mixture was loaded onto a 1.5 cm × 15 cm DEAE-trisacryl column preequilibrated in 10 mM NH4HCO3 buffer (pH 7.9). The column was washed with 50 mL of 10 mM NH4HCO3 and then eluted with a 500 mL gradient of 80-180 mM NH4HCO3 followed by 150 mL of 500 mM NH4HCO3. Three folate-containing peaks were obtained as detected by UV absorbance at 363 nm. Each peak was collected, lyophilized, and redissolved in deionized water. The purity of each component was confirmed by reversed-phase highpressure liquid chromatography (HPLC) with a 10 mm × 250 mm Licrosorb RP-18 column (Alltech, Deerfield, IL), and the molecular weights of the conjugates were evaluated by fast-atom bombardment mass spectroscopy (FAB-MS). The characteristic pKa values of the two DFfolate isomers were obtained by titration on a pH/ion

Bioconjugate Chem., Vol. 7, No. 1, 1996 57

analyzer (Corning, Corning, NY). Carboxypeptidase G2 analysis of the folate linker chemistry was performed as reported (Levy and Goldman, 1967; McCullough et al., 1971) using a UV-Vis spectrophotometer. Comparison of the Abilities of the Two DFFolate Isomers To Bind the Cell Surface Folate Receptors. The affinities of the DF-folate isomers for cell surface receptors were determined by adding different concentrations of tritium-labeled folate conjugates in 1 mL of FDMEM to cultured KB cells. The cells were incubated at 4 °C for 30 min, washed thoroughly with cold phosphate-buffered saline (PBS), and suspended in 1 mL of PBS by scraping. Cell-associated [3H]folate conjugates were measured by liquid scintillation counting, and the cellular protein content was evaluated by the BCA protein assay. The abilities of the two DF conjugates to interact with the folate receptors were also compared using a [3H]folic acid binding competition assay. Briefly, 100 pmol of [3H]folic acid and 100 pmol of either DF-folate(R) or DF-folate(γ) dissolved in 0.1 mL of PBS were added to KB cells in 1 mL of FDMEM. The cells were then processed and analyzed for folate binding by the same procedures. Preparation of the 67Ga-DF-Folate. A dilute HCl solution of 67Ga3+ was evaporated to dryness with heating under a stream of N2 and the tracer reconstituted in ∼300 µL of ethanol containing 0.002% acetylacetone (acac). The ethanolic 67Ga(acac)3 solution (3.2 mCi) was diluted with an equal volume of TRIS-buffered saline (pH 7.4) (Bates, 1978) followed by addition of 2.25 × 10-6 mol of aqueous DF-folate(γ) conjugate. Labeling was complete after the mixture stood at room temperature for 24 h. For control experiments, 67Ga(III)-citrate was prepared by evaporating a 67Ga-chloride solution to dryness and reconstituting with 0.1 mL of 3% sodium citrate (pH 7.4). A portion of the resulting 67Ga-citrate solution (50 µL) was mixed with 0.1 mg of deferoxamine to obtain 67Ga-deferoxamine (67Ga-DF), which was also employed in control experiments. The radiochemical purity of the 67Ga-tracers was determined by thin layer chromatography on commercial C18 reversed phase plates eluted with methanol and in all cases was found to exceed 98%. The radiochromatograms were evaluated using a Berthold (Wildbad, Germany) Tracemaster 20 Automatic TLC linear analyzer. Rf values of 0.93; 0.0; 0.1; and 0.74 were obtained for 67 Ga-DF-folate(γ) 67Ga(acac)3, 67Ga-DF, and 67Ga-citrate, respectively. All experiments employing the 67Ga-DFfolate(γ) tracer were performed within 1-2 days of preparation. Uptake of 67Ga-DF-Folate by Cultured KB Cells and WI38 Cells. In the time-dependent uptake study, 67Ga-DF-folate(γ) prepared as described was diluted in 0.5 mL of FDMEM, and 0.15 µCi (100 pmol) was added to monolayers of KB cells grown in 35 mm tissue culture dishes. The cells were incubated at 4 or 37 °C for different lengths of time, washed with 3 × 1 mL of cold PBS, and then suspended in 1 mL of PBS by scraping. The amount of cell-associated radioactivity was determined using an automatic γ-counter. Cellular protein content was measured by the BCA protein assay. To determine the concentration dependence of uptake, KB cells were incubated with various amounts of the 67GaDF-folate(γ) complex at 4 or 37 °C for 30 min. In free folate competition experiments, the same protocol was used except that different concentrations of free folic acid were also included in the incubation medium containing 200 nM DF-folate(γ) plus 67Ga-DF-folate(γ). To examine cell-type selectivity of the 67Ga-labeled conjugates, WI38 cells, as well as KB cells, were incubated with 100

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Wang et al.

Figure 1. Reaction scheme illustrating the synthesis of the DF-folate conjugates.

pmol of various complexes in 1 mL of FDMEM at 37 °C for 30 min, and cell-associated radioactivity was evaluated similarly. RESULTS

Synthesis and Purification of Folate-Deferoxamine Conjugate. The synthesis of folate-deferoxamine is outlined in Figure 1. The carboxyl groups of the folic acid were activated by DCC and the coupling reaction reached completion after 2 h incubation, as indicated by the disappearance of free amino groups in a ninhydrin assay and by the disappearance of DF analyzed by thin layer chromatography. The acetone-precipitated crude product can be purified by ion-exchange chromatography. Three folate-containing peaks were collected from the DEAE-trisacryl column as shown in Figure 2. The first two peaks were identified as DF-folate conjugates, both giving a molecular weight of 984.0 in their FAB-MS spectra (Figure 3), indicating the expected 1:1 ratio between folic acid and DF. Fragmentation of the molecules at the CH2-NH bond in folate resulted in a pteridinyl radical and complementary fragment of molecular weight 809. The third peak on the elution profile had the same molecular weight as free folic acid. Since the two isoforms of DF-folate conjugate retain either a free γ-carboxyl or free R-carboxyl, they can be distinguished from each other and from unreacted folic acid by their characteristic pKa values, which were determined by titration (Figure 4). The DF-folate(R) conjugate (pKa ) 4.5, constituting ∼20% of total DFfolate) eluted in 140-260 mL fractions (peak 1), the DFfolate(γ) conjugate (pKa ) 2.5, constituting ∼80% of total DF-folate) eluted in 340 to 420 mL fractions (peak 2), and the free folic acid (pKa1 ) 2.5, pKa2 ) 4.5) eluted between 580 and 680 mL (peak 3). Trace amounts of the unreacted DF and the side product bis(DF)-folate were removed during the 10 mM NH4HCO3 wash (Figure 2, inset). The purity of the two folate conjugates was

Figure 2. Purification of DF-folate by DEAE-trisacryl anionexchange chromatography. Thirty mg of the reaction product precipitated by acetone was dissolved in 1 mL of H2O (pH 9) and applied to the 1.5 × 15 cm column. The column was washed with 50 mL of 10 mM NH4HCO3, and the isomers were eluted with a gradient of 500 mL of 80-180 mM NH4HCO3. Unreacted folic acid was removed with 150 mL of 500 mM NH4HCO3. The inset contains a magnified elution profile of the first 500 mL off the column.

confirmed by reversed-phase HPLC. The R- and γisomers were eluted as single peaks at 7.0 and 7.8 min, respectively, with a linear gradient of 0-50% acetonitrile in 10 min. The assignment of the structures of the two DF-folate conjugates was further confirmed by experiments with carboxypeptidase G2, an enzyme which hydrolyzes folate compounds at the pteroate-glutamate linkage (Levy and Goldman, 1967). It has been shown that derivatization of the R-carboxyl of folate prevents hydrolysis by carboxypeptidase G2, but derivatization of the γ-carboxyl has little effect on enzyme activity (Fan et al., 1991). When the two isomers of DF-folate were tested as substrates (at 0.1 mM) for this enzyme (7.5 milliunits), peak 1 was inert while peak 2 was hydrolyzed at a rate of 0.029 ∆A303

Folate Conjugate as Tumor Imaging Agent

Bioconjugate Chem., Vol. 7, No. 1, 1996 59

Figure 3. FAB-MS spectrum of DF-folate(γ) conjugate. The m/z peak at 984.0 corresponds to the expected molecular weight of the complex, while the peak at 809 derives from the major fragmentation product (see text for details).

Figure 4. Determination of the pKa values of DF-folate(R), DF-folate(γ), and free folic acid by NaOH titration. Each folatecontaining peak eluted from the DEAE-trisacryl column was acidified to the indicated pH and titrated with 10 mM NaOH; (A) peak 1, pKa ) 4.5, identified as DF-folate(R); (B) peak 2, pKa ) 2.5, identified as DF-folate(γ); (C) peak 3, pKa1 ) 2.5, pKa2 ) 4.5, identified as free folic acid.

units/min, which is comparable to the rate for free folic acid (0.063 ∆A303 units/min). Determination of the Affinities of the Two DFFolate Isomers for Cell Surface Folate Receptors. The affinities of the DF-folate(R) and DF-folate(γ) conjugates for the KB cell folate-binding protein was both directly evaluated and also examined by a competitive binding assay using [3H]folic acid as the receptor ligand. As shown in Figure 5, binding of DF-folate(γ) by cultured KB cells was saturatable at 4 °C. Assuming a

Figure 5. Determination of the affinities of the DF-folate isomers for cell surface folate receptors. KB cells were incubated with different concentrations of tritium-labeled DF-folate(R) (1), DF-folate(γ) (b), or folic acid (O) at 4 °C for 30 min. The cells were then washed thoroughly by cold PBS and suspended in 1 mL of PBS by scraping. Cell-associated [3H]folates was measured by liquid scintillation counting, and the cellular protein content was evaluated by the BCA protein assay. Error bars represent standard deviations of three parallel experiments.

cellular protein content of 2.0 × 10-7 mg (Lee and Low, 1994), approximately 4.8 × 106 molecules were associated with each cell when maximum binding was achieved. Half-maximal binding was found at 2.0 nM, which is similar to that of free folic acid (1.8 nM). DF-folate(R), on the other hand, exhibited almost no affinity for the cell surface folate receptors. In competition assays, a 50% decrease in bound [3H]folic acid was observed in the presence of an equimolar amount of the DF-folate(γ) conjugate, while the DFfolate(R) conjugate displayed virtually no ability to compete with the radiolabeled vitamin (Table 1). Importantly, competition by DF-folate(γ) was similar to that of unlabeled folic acid, again indicating that covalent

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Table 1. Competitive Inhibition of [3H]Folic Acid Binding to KB Cells by r- and γ- Carboxylate DF-Folate Isomers amt of [3H]folic acid added (pmol)

amt of unlabeled folate compd added (pmol)

no. of bound [3H]folic acid per cell (106)

(%) inhibition

100 100 100 100

0 100 (R-isomer) 100 (γ-isomer) 100 (folic acid)

4.8 ( 0.4a 4.7 ( 0.5a 2.5 ( 0.3a 2.4 ( 0.2a

0 1.5 49 50

a

Data represent the average of three parallel experiments ( standard deviation. Labeled folic acid and unlabeled folate compound were added simultaneously to the KB cells.

Table 2. Cellular Uptake of Various Cultured KB Cells and WI 38 Cells

rel uptake by KB cells

% uptake by each WI38 cella

+

32.3 ( 2.4b 0.18 ( 0.05b

100 0.56

0.22 ( 0.04b 0.15 ( 0.02b

+

0.64 ( 0.07b 0.14 ( 0.03b

2.0 0.43

0.27 ( 0.05b 0.20 ( 0.02b

0.31 ( 0.05b 0.09 ( 0.02b

0.96 0.28

0.29 ( 0.04b 0.16 ( 0.03b

100 × folic acid

67Ga-DF-folate(R) 67Ga-DF-folate(R)

100 × folic acid

67Ga-citrate 67Ga-DF

Complexes by

% uptake by each KB cella 67Ga-DF-folate(γ) 67Ga-DF-folate(γ)

67Ga

a Percent uptake ) (cell-associated CPM per cell/total added CPM per cell) × 100%. b Data represent the average of three parallel experiments ( standard deviation.

Figure 6. Kinetics of association of 67Ga-DF-folate(γ) with cultured KB cells at 4 and 37 °C. KB cells were incubated for the indicated times with 200 nM 67Ga-DF-folate(γ) at 4 °C (O) or at 37 °C (b). Due to the high radioactivity and short half-life of the 67Ga-DF-folate(γ) complex, no attempt was made in this study to separate it from the nonradioactive metal-free conjugate. Thus, some competition with uncomplexed DF-folate(γ) conjugate will undoubtedly exist. After the cells were washed with 3 × 1 mL of cold PBS, the cells were suspended in 1 mL of PBS by scraping. The amount of cell-associated radioactivity was determined using a γ-counter, and cellular protein content was determined by BCA assay. Error bars represent standard deviations of three parallel experiments.

conjugation of DF to the γ-carboxyl of folic acid does not compromise the latter’s high affinity for the membraneassociated folate binding protein. These data demonstrate that the cell surface folate receptor recognizes folates derivatized at the γ- but not R-carboxylate. Uptake of 67Ga-DF-Folate Complex by Cultured KB Cells. Because folate and its conjugates bind to cell surface receptors at 4 °C, but are capable of endocytosis only at higher temperatures (Antony et al., 1985; Leamon and Low, 1991), it is possible to separately evaluate the kinetics of binding and internalization by measuring the rates of folate conjugate uptake at the two temperatures. As shown in Figure 6, half maximal binding of 67Ga-DFfolate(γ) was achieved in ∼3 min at 4 °C, suggesting rapid association of the conjugate with unoccupied receptors. By the end of the 30 min incubation, binding approached saturation with 18% of the initial radioactivity found associated with the cell surface. Incubation at 37 °C, a temperature which permits both binding and endocytosis, yielded similar kinetics, only maximal uptake reached 32% of the total conjugate added. Presumably, the difference in magnitude of the two cellular uptake curves reflects the ability of the folate receptor to internalize the conjugate at 37 °C and then recycle to the cell surface in its unoccupied form (Kamen et al., 1988). As controls, 67Ga-DF lacking the folate group did not show any significant cell association (Table

Figure 7. Competition for KB cell uptake between 67Ga-DFfolate(γ) and folic acid. KB cells were incubated for 30 min with 200 nM 67Ga-DF-folate(γ) as descried in Figure 6 in the presence of the indicated concentrations of free folic acid at 4 °C (O) or at 37 °C (b). The cells were then processed and analyzed for complex association as described in Figure 6. Error bars represent standard deviations of three parallel experiments.

2). Also, the 67Ga-DF-folate(R) complex was taken up with only 2% of the efficiency of 67Ga-DF-folate(γ), probably due to the inability of the R-conjugate to be recognized by the folate receptor (Figure 5 and Table 1). When 67Ga-citrate was added to the culture medium and incubated at 37 °C for 30 min, cell-associated 67Garadioactivity was 106-fold lower than observed with 67GaDF-folate(γ) (Table 2). Finally, the uptake of 67Ga-DFfolate(γ) was observed to be cell selective, since WI38 cells, a normal cell line that does not express the folate receptor, displayed no capacity to take up the complex (Table 2). To further verify the involvement of a cell surface folate receptor in mediating the uptake of 67Ga-DF-folate(γ), competition between the complex and free folic acid for uptake by KB cells was examined (Figure 7). As the concentration of the unligated vitamin was increased, binding of 67Ga-DF-folate(γ) decreased, falling to only ∼10% of its initial value by 800 nM free folate. In fact, only 0.5% of initial cellular uptake was retained in the presence of 100-fold molar excess of free folate (Table 2). Taken together, these results suggest that the 67Ga-DFfolate(γ) complex enters KB cells via the folate receptor in much the same manner as free folic acid. DISCUSSION

Gallium-67 has been used for many years as a radiopharmaceutical for detection of certain tumors, particu-

Folate Conjugate as Tumor Imaging Agent

larly broncogenic carcinoma, Hodgkin’s and non-Hodgkin’s lymphoma, and primary hepatomas (Hayes, 1978; Fritzberg et al., 1988; Front and Israel, 1995). In systemic circulation, 67Ga3+ administered in the form of the aqueous citrate complex is rapidly bound by the plasma protein, transferrin (Harris, 1983; Green and Welch, 1989), and slowly delivered to tissue and tumor cells expressing elevated levels of transferrin receptors (Hayes, 1978; van Leeuwen-Stok et al., 1993; Sohn et al., 1993). However, the clinical use of 67Ga-citrate for tumorimaging is plagued by slow tracer clearance from nontarget tissues, leading to delays in imaging, as well as poor tumor/background contrast (Hayes, 1978; Lentle, 1986). The linear trihydroxamate ligand, deferoxamine (DF), forms a highly-stable octahedral coordination complex with trivalent cations like ferric ion (Richardson and Baker, 1992) or 67Ga3+ (Weiner, 1979; Yokoyama et al., 1982; Smith-Jones et al., 1994) that can resist ligand exchange with plasma transferrin. A free amino group separated by five methylene units from the closest hydroxamate provides a site for covalent coupling of deferoxamine to targeting agents without interfering with its chelating properties. Thus, 67Ga-, 68Ga-, and/or 66Ga-labeled deferoxamine conjugates with antibodies and peptides have been frequently used for γ scintigraphy, positron emission tomography (PET), or other radioassays of receptor-positing tumors (Yokoyama et al., 1982; Presant et al., 1988; Furukawa et al, 1991; Vera, 1992; Green, 1993; Smith-Jones et al., 1994). In this study, we synthesized the DF-folate conjugate by a simple one-step coupling reaction. A large excess of folic acid and a controlled amount of DCC were used to avoid leaving unreacted DF in the product mixture and to also minimize the production of (DF)2-folate. (Contamination of the conjugate with unreacted DF must be avoided, since its presence will adversely affect the efficiency of conjugate radiolabeling with 67Ga and diminish the radiochemical purity of the 67Ga-labeled DFfolate.) By using DEAE-trisacryl chromatography, we were able to separate unreacted DF from the R- and γcarboxyl-linked conjugates and obtain pure DF-folate(γ) for use in tumor cell targeting. In vitro, the cellular uptake of 67Ga-citrate was less than 1/100 as efficient as uptake of the 67Ga-DF-folate(γ) complex, but was nevertheless higher than that of nontargeted 67Ga-DF. This uptake could be due to the transfer of 67Ga from citrate to transferrin present in the serum supplement to the cell culture medium, allowing tracer to be bound to cells via the transferrin receptor. As noted above, this targeting mechanism has been already exploited in the clinical use of 67Ga-citrate as a radiopharmaceutical for tumor imaging. The study of KB cell uptake of 67Ga-DF-folate(γ) revealed that the complex is specifically bound by the folate receptor and probably enters cells via folatereceptor-mediated endocytosis. The uptake kinetics are fast, reaching 50% maximal cell association in ∼3 min. This half-time is comparable to that reported for folateconjugated serum albumin (Mr ∼68 000), but much shorter than the 1-2 h half-life observed for folateconjugated liposomes (Leamon and Low, 1991; Lee and Low, 1994). Although barriers to effective tumor-specific drug delivery are more pronounced in vivo than in cell culture, our studies suggest that 67Ga-DF-folate(γ) might be useful as a radiopharmaceutical for tumor imaging. The affinity of the conjugate is comparable to that of folic acid, i.e., 10-9 M, for receptor-bearing cells. Furthermore, the complex is small in size, allowing for facile diffusion to receptor-bearing cells, and the conjugate shows little

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nonspecific adhesion to cell surfaces (e.g., free folate blocks cell surface binding almost quantitatively and the R-carboxyl derivative shows very low affinity for KB cells). Finally, the folate receptor is present in a large fraction of human tumors, suggesting the complex might be helpful in imaging numerous types of metastatic disease. ACKNOWLEDGMENT

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