Preclinical Evaluation of ^99mTc-EC20 for Imaging Folate Receptor–Positive Tumors

General

N¹⁰-Trifluoroacetylpteroic acid was purchased from Eprova AG. Peptide synthesis reagents were purchased from NovaBiochem and Bachem. ^99mTc-Sodium pertechnetate was supplied by Syncor. Cellulose plates and diethylaminoethyl (DEAE) ion-exchange plates were purchased from J.T. Baker.

Synthesis, Purification, and Analytic Characterization of EC20 and EC53

EC20 is a folate-containing peptide consisting in sequence of pteroic acid (Pte), d-glutamic acid (d-Glu), β-l-diaminopropionic acid (βDpr), l-aspartic acid (l-Asp), and l-cysteine (l-Cys). EC20 was synthesized according to a previously described procedure (23). Briefly, using an acid-sensitive Wang resin loaded with fluorenylmethoxycarbonyl-trityl-l-cysteine (Fmoc-l-Cys(Trt)-OH), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP)-activated, protected amino acid monomers were sequentially applied. Fmoc protecting groups were removed after every coupling step under standard conditions (20% piperidine in dimethyl formamide). After the last assembly step, the peptide was cleaved from the polymeric support by treatment with 92.5% trifluoroacetic acid (TFA) containing 2.5% ethanedithiol, 2.5% triisopropylsilane, and 2.5% deionized water. This reaction also resulted in simultaneous removal of the t-butyl (t-Bu), t-butoxycarbonyl (t-Boc), and trityl protecting groups. Finally, the trifluoroacetyl moiety was removed in aqueous ammonium hydroxide to give EC20. EC53, the all-d-isomer of EC20, was similarly synthesized by replacing β-l-diaminopropionic acid (βDpr), l-Asp, and l-Cys with β-d-Dpr, d-Asp, and d-Cys, respectively. The crude EC20 and EC53 products were purified by high-performance liquid chromatography (HPLC) using an Xterra RP₁₈ 30 × 300 mm 7-μm column (Waters) with a gradient mobile phase of 32 mmol/L hydrochloric acid and methanol. The structures of EC20 and EC53 were confirmed by ¹H nuclear magnetic resonance (NMR) spectroscopy. Chemical shifts and J values for EC20 (23) were similar to those of EC53. EC20 and EC53 were also analyzed by electrospray-mass spectrometry. Major positive ion peaks (m/z, relative intensity): 746.1 (M+H)+, 100; 747.1, 44; 556.8, 32; 570.8, 16.

Preparation of ^99mTc-EC20/^99mTc-EC53

EC20/EC53 kits are used for the preparation of radioactive drug substances. Each kit contains a sterile, nonpyrogenic lyophilized mixture of 0.1 mg of EC20 or EC53, 80 mg of sodium α-d-glucoheptonate, 80 mg of tin (II) chloride dihydrate, and sufficient sodium hydroxide or hydrochloric acid to adjust the pH to 6.8 ± 0.2 before lyophilization. Lyophilized powder is then sealed in a 5-mL vial under an argon atmosphere. Chelation of ^99mTc to EC20 or EC53 is done by injecting 1 mL of ^99mTc-labeled sodium pertechnetate (<1,800 MBq) into this vial and heating it for ∼18 min in a boiling water bath. This solution can be stored at room temperature (15°C–25°C) protected from light, but it should be used within 6 h of preparation.

The radiochemical stability of the radioactive drug substances was determined by HPLC after storing at room temperature in a lead-shielded container for up to 24 h. Samples of ^99mTc-EC20 and ^99mTc-EC53 solutions were analyzed using an HPLC system consisting of a Waters 600E Multisolvent Delivery System and 490 ultraviolet (UV) detector, a Bioscan FC-3200 radiodetector, Laura 1.5 radiochromatogram software, and a Waters Nova-Pak C₁₈ (3.9 × 150 mm) column. Injected samples were eluted with a linear gradient using an aqueous mobile phase containing 1%–50% solvent B (solvent A = 0.1% TFA in water; solvent B = acetonitrile) at a flow rate of 1 mL/min. The radiochemical purities of both ^99mTc-EC20 and ^99mTc-EC53 remained >90% for at least 24 h.

Determination of Radiochemical Purity of ^99mTc-EC20/^99mTc-EC53 by Thin-Layer Chromatography

The major radiochemical impurities in the preparation of ^99mTc-EC20 and ^99mTc-EC53 are (a) ^99mTc-pertechnetate, (b) ^99mTc-glucoheptonate (ligand exchange precursor), (c) nonspecific-binding ^99mTc (^99mTc bound at a site other than the expected Dap-Asp-Cys chelating moiety of the EC20 molecule), and (d) hydrolyzed ^99mTc. Since ^99mTc-EC20 was being tested for possible clinical use, a 3-thin-layer chromatography (TLC)–based method had been developed to determine the amounts of each impurity and to estimate the overall radiochemical purity.

In the first system a cellulose plate is developed with deionized water. Here, ^99mTc-EC20/^99mTc-EC53, ^99mTc-glucoheptonate, nonspecific-binding ^99mTc, and ^99mTc- pertechnetate move to the solvent front (R_f = 1.0), while hydrolyzed ^99mTc remains at the origin (R_f = 0.0). The cellulose plate is cut into 2 pieces at R_f = 0.3, and each piece is counted using a dose calibrator. The percentage of hydrolyzed ^99mTc is calculated as follows: A = % hydrolyzed ^99mTc = (kBq in bottom piece/kBq in both pieces) × 100.

In the second system, a cellulose plate is developed with acetone and 0.9% NaCl (7:3, v/v). ^99mTc-Pertechnetate moves with R_f = 0.9, while ^99mTc-EC20/^99mTc-EC53, ^99mTc-glucoheptonate, nonspecific-binding ^99mTc, and hydrolyzed ^99mTc remain at the origin (R_f = 0.0). The cellulose/acetone-saline plate is cut into 2 pieces at R_f = 0.6, and each piece is counted using a dose calibrator. The percentage of ^99mTc-pertechnetate is calculated as follows: B = % ^99mTc-pertechnetate = (kBq in top piece/kBq in both pieces) × 100.

Finally, in the third system, a DEAE ion-exchange plate is developed with 0.3 mol/L Na₂SO₄. ^99mTc-Glucoheptonate moves to the solvent front (R_f = 1.0); nonspecifically bound ^99mTc moves with R_f = 0.6; and ^99mTc-EC20, hydrolyzed ^99mTc, and ^99mTc-pertechnetate remain near the origin (^99mTc-EC20/^99mTc-EC53, R_f = 0.1; hydrolyzed ^99mTc, R_f = 0.0; ^99mTc-pertechnetate, R_f = 0.3). The cellulose/Na₂SO₄ plate is cut into 2 pieces at R_f = 0.5, and each piece is counted using a dose calibrator. The percentage of ^99mTc-glucoheptonate and nonspecific-binding ^99mTc are calculated as follows: C = % (^99mTc-glucoheptonate + nonspecific-binding ^99mTc) = (kBq in top piece/kBq in both pieces) × 100. The overall radiochemical purity of ^99mTc-EC20/^99mTc-EC53 is then calculated as follows: Radiochemical purity = 100–(A + B + C). The radiochemical purity of each ^99mTc-labeled compound was determined by this 3-TLC–based method and, in all cases, was found to exceed 96% at the time of use.

Relative Affinity Assay

The relative affinity of EC53 was determined according to the method described by Westerhof et al. (24) with slight modification. Briefly, FR–positive KB cells were gently trypsinized in 0.25% trypsin in phosphate-buffered saline (PBS) at room temperature for 3 min and then diluted in folate-free RPMI medium (FFRPMI) containing 10% heat-inactivated fetal calf serum. After a 5-min 800g spin and one PBS wash, the final cell pellet was suspended in FFRPMI 1640 (no serum). Cells were incubated for 15 min on ice with 100 nmol/L of ³H-folic acid in the absence and presence of increasing concentrations of EC53. Samples were centrifuged at 10,000g for 5 min; cell pellets were suspended in buffer, transferred to individual vials containing 5 mL of scintillation cocktail, and then counted for radioactivity. Negative control tubes contained only the ³H-folic acid in FFRPMI (no competitor). Positive control tubes contained a final concentration of 1 mmol/L folic acid, and counts per minute (cpm) measured in these samples (representing nonspecific binding of label) were subtracted from all samples. Notably, relative affinities were defined as the inverse molar ratio of compound required to displace 50% of ³H-folic acid bound to the FR on KB cells, and the relative affinity of folic acid for the FR was set to 1.

Tissue Distribution Studies

Four- to 5-wk-old female mice (BALB/c strain) were purchased from Harlan Sprague–Dawley, Inc., and were maintained on a folate-free diet or leucovorin-supplemented folate-free diet for a total of 3 wk before the experiment. Syngeneic, FR–positive M109 tumor cells (1 × 10⁶ cells per animal) or 4T1 tumor cells (2 × 10⁵ cells per animal) were inoculated in the subcutis of the right axilla 3 wk before the experiment. Stock ^99mTc-EC20 or ^99mTc-EC53 solutions containing 100 μg of agent per milliliter were prepared on the day of use, and their radiochemical purities were >96%. All solutions were diluted with either saline alone or a saline solution containing 1 or 10 equivalents (eq.) of free folic acid such that the final radiopharmaceutical concentration was 10 μmol/mL.

Animals received an approximate 40 μmol/kg intravenous dose of test article in a 100-μL volume via a lateral tail vein during brief diethyl ether anesthesia. Alternatively, mice received intraperitoneal ^99mTc-EC20 with a single bolus administration of test article in saline. Four hours after injection, animals were killed by CO₂ asphyxiation and dissected. Selected tissues were removed and weighed, and their radioactivity content was measured in an automatic γ-counter to determine ^99mTc distribution. Uptake of the radiopharmaceutical in terms of the percentage injected dose of wet weight tissue (%ID/g) was calculated by reference to standards prepared from dilutions of the injected preparation.

FR Assay

A FR assay was performed according to Turk et al. (M.J. Turk, 2003), and all sample preparation procedures were performed at 4°C. Briefly, tissue samples were homogenized in homogenization buffer (10 mmol/L Tris, pH 8.0; 0.02 mg/mL each of leupeptin and aprotinin; 1 mL buffer per 50 mg of tissue). Membrane pellets were collected by centrifugation (40,000g for 60 min at 4°C) and resuspended in solubilization buffer (50 mmol/L Tris, pH 7.4; 150 mmol/L NaCl; 25 mmol/L n-octyl-β-d-glucopyranoside; 5 mmol/L ethylenediaminetetramethylene phosphonate; and 0.02% sodium azide). Insoluble material was removed by another centrifugation, and the total protein concentration of the supernatants was determined by the bicinchoninic acid protein assay method (Pierce Chemical Co.). Each sample was diluted to 0.25 mg/mL in solubilization buffer, and the solubilized FRs were concentrated by passing through Microcon-30 microconcentrators (30,000 molecular weight cutoff; Millipore Corp.). After a brief acid wash step to release endogenous folates, samples were neutralized and then allowed to bind ³H-folic acid or ³H-folic acid competing with 120 μmol/L unlabeled folic acid (25 mmol/L n-octyl-β-d-glucopyranoside; 350 mmol/L NaCl; 120 nmol/L ³H-folic acid). The concentrators were washed 3 times with a detergent-based buffer (50 mmol/L n-octyl-β-d-glucopyranoside; 0.7 mol/L NaCl in PBS, pH 7.4). After the final wash, the retentates containing the solubilized FRs were recovered with PBS containing 4% Triton X-100. The samples were then counted in a liquid scintillation counter (Packard Bioscience Co.). Radioactivity values (cpm) were converted to picomoles of FR using the reagent’s specific activity, and the final results were normalized with respect to the sample protein content.

Synthesis and Radiochemistry of EC20 and EC53, its All-d-Isomer

A peptide’s biodistribution pattern can dramatically be affected by peptidase (serum, interstitial fluid, and tissue)-mediated degradation. One technique for achieving resistance to peptidases is through the use of d-amino acid residues within the peptide molecule. Therefore, to prevent the possible degradation of EC20 catalyzed by endogenous peptidases, its all-d-enantiomeric counterpart (EC53) was synthesized. Both EC20 and EC53 were built on solid supports applying Fmoc and Boc protection strategies. After the last assembly step, pteroyl-containing peptides were cleaved from the solid support and then deprotected by TFA treatment. The purities of EC20 and EC53 were >96% when tested by analytic HPLC (Fig. 1). The products were also characterized by NMR and electrospray-mass spectrometry, and the data were consistent with the expected structures of EC20/EC53, as illustrated in Figure 2.

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FIGURE 1.

HPLC profiles of EC20 (Top) and EC53 (Bottom). Samples of EC20 or EC53 were analyzed on a Waters Nova-Pak C₁₈ (3.9 × 150 mm) column using a 30-min linear gradient from 10% to 50% solvent B (solvent A = 0.1% TFA in water; solvent B = methanol) at a flow rate of 1 mL/min. AU = absorbance units.

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FIGURE 2.

Formation of ^99mTc-EC20 and ^99mTc-EC53.

Labeling of EC20 and EC53 with ^99mTc was performed in alkaline aqueous medium using tin chloride. Incorporation of the radiotracer was practically quantitative within 20 min, and purities of >95% of ^99mTc-EC20 or ^99mTc-EC53 were commonly obtained. As shown in Figure 3, HPLC analysis of the ^99mTc-EC20 and ^99mTc-EC53 formulations showed 4 radiochemical components, designated as peaks A, B, C, and D. As described elsewhere (23), peak A was confirmed to be free ^99mTc, which was typically present at <1% in both formulations. Peak B was assumed to be a ^99mTc-EC20/^99mTc-EC53 product chelated at a site other than the Dap-Asp-Cys position, and this specie represented <6% of the mixture in both products. In addition, peaks C and D (which accounted for the majority of the formulations) were confirmed to be diastereomers possessing either a syn- or anti-configuration about the technetium-oxygen bond in the Dap-Asp-Cys chelating ring (23). Furthermore, TLC analysis indicated that <2% of the radioactivity in the formulation was attributed to reduced, hydrolyzed technetium. As a consequence, no further purification was needed before use.

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FIGURE 3.

HPLC profiles of ^99mTc-EC20 (Top) and ^99mTc-EC53 (Bottom). Samples of ^99mTc-EC20 or ^99mTc-EC53 were eluted on a Waters Nova-Pak C₁₈ (3.9 × 150 mm) column using a 30-min linear gradient from 1% to 50% solvent B (solvent A = 0.1% TFA in water; solvent B = acetonitrile) at a flow rate of 1 mL/min.

Binding Properties and Biodistribution of EC20 and EC53

Folate conjugates are typically analyzed using an in vitro relative affinity assay that measures a ligand’s ability to directly compete with folic acid for binding to cell surface FRs. Thus, ³H-folic acid is used as the radioligand, whereas unlabeled folic acid serves as the reference compound. We have previously reported that EC20 has a relative affinity value of 0.92 (23). As shown in Figure 4, EC20’s all-d-isomeric counterpart, EC53, was determined to have a relative affinity value of 0.23. Although EC53’s net affinity for the FR is lower compared with that of EC20, this new agent is still considered to be a moderately high-binding ligand.

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FIGURE 4.

Competitive blocking of ³H-folic acid binding to KB cells with EC53. KB cells were incubated for 15 min on ice with 100 nmol/L ³H-folic acid in the presence and absence of increasing EC53 concentrations. Error bars represent 1 SD (n = 3).

d-Amino acid replacement can possibly change the in vivo stability of the peptide portion because the cellular absorption, hepatic and renal clearance, and binding of protease to peptide may be affected by the interactions between peptide and various serum components. Hence, biodistributions of ^99mTc-EC20 and ^99mTc-EC53 were compared in BALB/c mice bearing subcutaneous FR–positive M109 tumors. Animals were dosed intravenously with the test articles, and selected organs were collected and counted for radioactivity 4 h later. Uptake was expressed as %ID/g tissue, and tumor-to-nontumor (T/NT) ratios were also calculated to assess the selective targeting of the agents.

As shown in Table 1, the highest tissue uptake with both agents was measured in the FR–positive tissues—namely, the tumors and kidneys. Relative to EC20, the d-amino acid substitutions in EC53 reduced its uptake in the kidneys and tumor by approximately 33%. T/NT organ ratios were also reduced with ^99mTc-EC53 in all cases except the muscle, heart, and lung. Tissue counting data from animals given either agent showed <5 %ID/g of gastrointestinal tract tissue, suggesting that the primary route of radioactivity elimination from the body in each case was urinary. Since EC53 did not promote increased tumor uptake or enhanced T/NT ratios in most organs relative to EC20, it was concluded that endogenous proteolytic activity on EC20 was probably insignificant. The fact that EC53 accumulated in kidney and tumor to a lesser degree than EC20 may have been due to EC53’s slightly reduced affinity for the FR (Fig. 4). Thus, all remaining experiments were performed using the EC20 reagent.

Effect of FR Expression Level on ^99mTc-EC20 Uptake by FR—Expressing Tumors

The mouse cell lines 4T1 and M109 reproducibly form tumors in BALB/c mice, and they express varying amounts of FR (1.6 and 95.5 pmol FR/mg membrane protein, respectively; Table 2). A third cell line was also generated by continuous culturing of 4T1 cells in growth medium containing very low levels (10 pmol/L) of folic acid. As expected, the growth of 4T1 cells in 10 pmol/L folic acid resulted in upregulated FR expression from 1.6 to 20.2 pmol FR/mg protein, and this new cell line was renamed 4T1-pico (Table 2).

When tumors of the 3 cell lines were generated in BALB/c mice and FR levels were quantified, FR expression was found to be the lowest in 4T1 tumors (1.5 pmol FR/mg protein) and the highest in M109 tumors (22.9 pmol FR/mg protein) as expected, whereas the 4T1-pico tumors expressed 7.2 pmol FR/mg of protein. Based on these differential levels of membrane-associated FR expression, 4T1, 4T1-pico, and M109 cells were used as tumor models for the assessment of ^99mTc-EC20 uptake.

As shown in Table 2, the net uptake of radiolabel in each of these tumors was proportional to their respective FR levels. Thus, M109 tumors accumulated the greatest amount (15.5 %ID/g), whereas the 4T1-pico tumors accumulated much less (8.71 %ID/g). The 4T1 tumors (derived from cells that express the lowest quantity of FR) accumulated the lowest amount of radiolabel (2.6 %ID/g). Importantly, uptake of ^99mTc-EC20 was reduced to background levels (<1 %ID/g) in all 3 tumor types when a competable excess of free folic acid was coinjected, thus indicating that tumor uptake was primarily due to the presence of the FR.

Effect of Tumor Size on ^99mTc-EC20 Uptake by M109 Tumors

A large tumor load is usually associated with decreased therapeutic efficacy. One reasonable explanation for this observation is that the tumor cells located deep within the malignant tissue become less accessible to parenterally administered drugs. In an attempt to relate tumor size with ^99mTc-EC20 uptake, we retrospectively analyzed the results of 4 different M109 biodistribution experiments. The majority of the tumors from these studies were found to be in the range of 50–400 mm³ (i.e., well established). Notably, mice bearing tumors with a size of <50 mm³ were excluded from evaluation since tumors that are barely palpatable are often difficult to excise without some associated normal connective tissue. The data from the various experiments were compiled, and the %ID of ^99mTc-EC20 within each tumor was plotted against the respective tumor size. As shown in Figure 5, the %ID of ^99mTc-EC20 in the tumors is very well correlated (r = 0.977) with the size of the tumor. From this analysis we concluded that as the tumor size increases, tumor-associated FRs remain readily accessible to blood-borne folate-drug conjugates. Interestingly, the data also suggest that the net amount of FR expressed per gram of tumor does not decrease as the disease progresses.

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FIGURE 5.

^99mTc-EC20 uptake in M109 tumors of varying sizes. BALB/c mice bearing M109 tumors received an intravenous dose of 50 μg/kg EC20 (67 nmol/kg) in approximately 0.1 mL PBS during brief diethyl ether anesthesia. At designated times after injection, each animal was killed by CO₂ asphyxiation, and tumors were collected and counted for associated radioactivity.

Uptake of ^99mTc-EC20 as a Consequence of Diet Modulation

Folic acid–deficient diets have traditionally been used on mouse models when investigating FR targeting. Although no apparent adverse effects occur with their use, many have wondered if alternative diets could be used. Within this study, we investigated the effect of feeding mice a leucovorin (5-formyltetrahydrofolate)-supplemented diet on the tumor-targeting ability of ^99mTc-EC20 (25). Thus, mice were maintained on a diet supplemented with 0.5 mg of leucovorin per kilogram of chow for a total of 5 wk before biodistribution studies were performed. Based on a food intake of 3-g chow per day for an average 20-g laboratory mouse, roughly 1.5 μg of leucovorin supplement would be ingested (75 μg/kg body weight, or 227 μg/m² of body-surface area). Notably, the U.S. Department of Agriculture (USDA)–recommended daily allowance for folate is ∼400 μg/d per human (5.7 μg/kg, or ∼235 μg/m² body-surface area). Thus, the 0.5-mg leucovorin/kg chow was expected to deliver the USDA–recommended folate equivalent to the mice, based on body-surface area normalization.

As shown in Table 3, the biodistribution pattern for mice on a leucovorin-supplemented diet was essentially identical with the distribution pattern in the folate-deficient diet (Table 1). Neither diet significantly affected the uptake of ^99mTc-EC20 in the FR–positive tumors or kidneys (17.7 vs. 17.2 %ID/g in tumor and 116.5 vs. 109.9 %ID/g in kidney from mice fed the folate-deficient and leucovorin-supplemented diets, respectively). These data suggest that neither leucovorin nor its metabolites substantially competed with ^99mTc-EC20 (a folate conjugate) for binding to the FR.

Effect of Dose Administration Route and Tumor Location on ^99mTc-EC20 Biodistribution

One major advantage of intraperitoneal versus intravenous dosing is that intraperitoneally resident tumors (e.g., ovarian cancer) can be quickly exposed to high localized drug concentrations. Although this benefit is obvious for a nontargeted drug whose entry into a cell is concentration dependent, the benefit for administering an FR–targeted (and saturable) molecule intraperitoneally has yet to be determined. To better understand the advantages and limitations of in vivo folate targeting, we believed it was necessary to study the uptake of a model folate conjugate (^99mTc-EC20) into FR–positive tumors as a function of tumor location as well as route of dose administration.

Using mice bearing either subcutaneous M109 tumor implants or clearly identifiable and excisable intraperitoneal M109 tumor nodules, an experiment was conducted to compare the tissue biodistribution of ^99mTc-EC20 after either intravenous or intraperitoneal administration. As shown in Table 4, uptake of the folate-targeted imaging agent into intraperitoneal tumors was independent of the dosing route. Thus, intravenous and intraperitoneal routes of administration enabled the capture of ^99mTc-EC20 at 17.1 and 16.2 %ID/g of intraperitoneal tumor, respectively. Other intraperitoneally resident tissues—such as intestine, stomach, spleen, liver, and kidney—displayed similar (but low) uptake of the targeted imaging agent after either intraperitoneal or intravenous dosing. Likewise, the biodistribution pattern of ^99mTc-EC20 in animals bearing subcutaneous tumors was apparently independent of the dosing route. Thus, intravenous and intraperitoneal routes of administration enabled the capture of EC20 at 17.2 and 18.8 %ID/g of subcutaneous tumor, respectively. We conclude from this study that FR–positive tumors in our murine model can be equally accessed by folate-targeted agents in a manner that is independent of both the tumor location and the route of dose administration.

Effect of Predosed and Codosed Folic Acid with ^99mTc-EC20

In an effort to improve the tumor-to-background ratio, we have investigated the effect of injecting unconjugated folic acid before or during the dosing of ^99mTc-EC20. Thus, 1 or 10 eq. of folic acid were injected either 15 min before the dosing of ^99mTc-EC20 or together as a single formulation. As shown in Table 5, the addition of folic acid decreased the uptake of ^99mTc-EC20 into tumors in a dose-dependent manner. Tumor uptake was reduced from 24.7 to 16.51 %ID/g with the codose and reduced to 16.47 %ID/g with the predose of 1 eq. of folic acid, whereas tumor uptake was reduced to 4.65 %ID/g with the codose and reduced to 5.0 %ID/g with the predose of 10 eq. of folic acid. Interestingly, T/NT ratios increased 1.6- to 2.7-fold for every nontarget organ when ^99mTc-EC20 was codosed with 1 eq. of folic acid. For example, the tumor-to-blood ratio increased from 79.6 to 213.6, and the tumor-to-muscle ratio increased from 13.6 to 36.7. T/NT ratios also increased in every organ when the mice were predosed with the 1 eq. of folic acid, albeit to a lesser extent, except in the case of spleen, where the ratio remained the same. When the mice were codosed with 10 eq. of folic acid, T/NT ratios were higher in the case of blood, heart, lung, and muscle as compared with similar ratios from animals dosed with ^99mTc-EC20 alone. However, since codosing ^99mTc-EC20 with 1 eq. of folic acid gave the highest T/NT ratios, especially with respect to all organs in the peritoneal region, we concluded that this dosing manipulation would yield the best tumor-to-background contrast at the typical radiopharmaceutical dose level used.