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Basic Science Investigations |
Endocyte, Inc., West Lafayette, Indiana
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: folate receptor • tumor targeting • endocytosis • 99mTc • tumor imaging
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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In contrast to 111In-containing agents, 99mTc-based imaging agents are currently dominating the field of diagnostic nuclear medicine owing to their lower production costs and favorable nuclear characteristics (6-h half-life, 141-keV 
-rays) (19). The wide availability and cost effectiveness of 99mTc are also of major importance for routine clinical applications. Furthermore, it is now generally recognized that the rapid blood clearance and fast target localization properties of small molecule–based imaging agents are best used when short-lived radionuclides, such as 99mTc, are used (19). Thus, the impact of this clinical transition from 111In to 99mTc prompted the creation of a variety of folate conjugate 99mTc-chelators, such as 99mTc-6-hydrazinonicotinamide (HYNIC) (20), 99mTc-ethylenedicysteine (21), and 99mTc-DTPA (22).
Our group previously reported on the development of an alternative peptide-based 99mTc-folate conjugate, referred to as 99mTc-EC20 (23). As a continuation of this work, we now report the results of more detailed preclinical performance evaluations. Thus, we investigated the effect of an all-D-amino acid substitution in the peptide fragment of EC20 on its in vivo distribution in a mouse model. The effect of tumor FR density and tumor size on 99mTc-EC20 tumor uptake was also explored. Further experiments were conducted to compare the biodistribution of intraperitoneal versus intravenous 99mTc-EC20 and on the accessible targeting of both subcutaneous and intraperitoneal FR–positive tumors. Finally, the ability to improve 99mTc-EC20’s tumor-to-background contrast with coinjected versus preinjected folic acid was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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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), ß-L-diaminopropionic acid (ßDpr), 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 ß-L-diaminopropionic acid (ßDpr), L-Asp, and L-Cys with ß-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 x 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 
-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 x 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 (Rf = 1.0), while hydrolyzed 99mTc remains at the origin (Rf = 0.0). The cellulose plate is cut into 2 pieces at Rf = 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) x 100.
In the second system, a cellulose plate is developed with acetone and 0.9% NaCl (7:3, v/v). 99mTc-Pertechnetate moves with Rf = 0.9, while 99mTc-EC20/99mTc-EC53, 99mTc-glucoheptonate, nonspecific-binding 99mTc, and hydrolyzed 99mTc remain at the origin (Rf = 0.0). The cellulose/acetone-saline plate is cut into 2 pieces at Rf = 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) x 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 (Rf = 1.0); nonspecifically bound 99mTc moves with Rf = 0.6; and 99mTc-EC20, hydrolyzed 99mTc, and 99mTc-pertechnetate remain near the origin (99mTc-EC20/99mTc-EC53, Rf = 0.1; hydrolyzed 99mTc, Rf = 0.0; 99mTc-pertechnetate, Rf = 0.3). The cellulose/Na2SO4 plate is cut into 2 pieces at Rf = 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) x 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 x 106 cells per animal) or 4T1 tumor cells (2 x 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 -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-ß-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-ß-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-ß-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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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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.
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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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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.
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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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99mTc-EC20 and 99mTc-EC53 were evaluated in M109 tumor-bearing BALB/c mice, and both agents were found to have similar patterns of tissue biodistribution. Unexpectedly, the net uptake of 99mTc-EC53 in FR–positive tumor and kidney was actually 33% lower compared with 99mTc-EC20. However, this finding was attributed to the measured lower affinity that EC53 has for the FR (Fig. 4). From these results we concluded that D-amino acid substitutions (in the ß-Dpr-Asp-Cys locus of EC20) did not afford any biologic advantage for in vivo FR targeting.
It has been previously demonstrated that the uptake of folate-targeted biomolecules in cell culture is roughly proportional to the FR density on the cell membranes (9,26). However, in vivo tumor uptake of a folate ligand presumably depends not only on FR expression levels but also on the tumor microenvironment. To address this issue, we first examined the uptake of 99mTc-EC20 into murine tumors that expressed various levels of the FR protein. We found that the net uptake of radiolabel in each of these tumors was proportional to their respective FR expression levels. Since tumor size can possibly influence drug accumulation, we next studied the relationship between 99mTc-EC20 uptake and tumor mass. Notably, a large tumor load could decrease the therapeutic efficacy of drugs by various mechanisms. In fact, many solid tumors have poorly formed vasculatures with intermittent blood flow and large distances between functional blood vessels (27). Further, in the center of large tumors, increased interstitial pressure gradients cause radial fluid flow from the center to the periphery (28). To reach all of the cells in a tumor, the agent must penetrate through multiple layers of solid tissue, and this requirement may represent a formidable barrier to many therapeutics. For 99mTc-EC20, we found that its tumor penetration was not significantly different among tumors of widely varying sizes. More specifically, there was a consistent increase in the relative amount of 99mTc-EC20 in the tumor as a function of tumor size over the range of 50–400 mm3. Taken together, these data confirm that folate conjugates can readily penetrate deeply into solid FR–positive tumors, and the net uptake is apparently proportional to the tumor’s size.
It has been shown that nontargeted drugs, such as methotrexate and cisplatin, are retained for prolonged periods of time within the peritoneal cavity after intraperitoneal administration (29). This enables an intraperitoneal tumor to be exposed to higher drug concentrations, thus increasing the potential for enhanced antitumor activity. When dosed at 50 nmol/kg, the amount of 99mTc-EC20 found in the tumor was very similar when the agent was administered either intraperitoneally or intravenously (irrespective of the tumor location). There were also no significant differences in uptake of 99mTc-EC20 within the kidneys and other abdominally localized tissues after either intraperitoneal or intravenous administration. These results indicate that there is no advantage in administering 99mTc-EC20 intraperitoneally compared with intravenously and further suggests that the main entry of this agent into tissues is vascular.
It is noteworthy that standard laboratory chows are extensively supplemented with folic acid (typically 6 mg/kg of chow). This causes laboratory animals to have high basal plasma and tissue folate levels (16). In fact, mice fed such diets have plasma folate levels of 300–800 nmol/L, which is 15- to 40-fold higher than that measured in human plasma. These supraphysiologic levels of plasma folate will compete with folate-drug conjugates for FR binding and possibly tumor uptake. Thus, most laboratories that study folate/antifolate or folate-conjugate pharmacology have often used a nonfolate–supplemented laboratory chow. In some cases, 1% of the succinylsulfathiazole antibiotic is included into the chow formulation to kill folate-producing intestinal flora. However, we have noted that mice fed such chows are not as healthy or active as those animals that are fed diets lacking the antibiotic.
To the best of our knowledge and experience, use of a folate-free chow (antibiotic free) enables the lowering of serum folate levels to more physiologically relevant levels (50 nmol/L) without causing any concomitant adverse effects (16). However, because folate deficiency has been implicated in multiple disorders—such as atherosclerotic cardiovascular disease, neurologic disorders, congenital defects, and carcinogenesis (30–32)—and the safety of animals kept on such diets for long periods is unknown, we searched for an alternative chow that might maintain tissue folate levels without significantly interfering with a folate conjugate’s ability to target tumor-associated FRs. We hypothesized that substituting folic acid in the chow with a compound such as leucovorin (folinic acid) should maintain similar plasma folate pools as folic acid (25) and, therefore, satisfy the tissue folate needs in animals enduring long-term FR–targeted therapy. The difference, though, is that leucovorin (and other reduced folates) is a good substrate for the reduced folate transport carrier, but it has low affinity for the FR; thus, leucovorin—and any quantity thereof that may have been methylated within the intestine before release into the plasma—will not effectively compete with folate-conjugate binding to the FR (33). Therefore, we custom formulated a nonfolate–supplemented chow with 0.5 mg leucovorin per kilogram, which incidentally was expected to deliver the USDA–recommended folate equivalent to the mice (based on body-surface area normalization). Indeed, our experimental results indicated that mice fed the leucovorin-supplemented diet looked and behaved normally, and their serum folate levels were measured at 62 nmol/L after 3 wk. In addition, the net 99mTc-EC20 uptake in FR–expressing tumors was nearly identical to the tumor uptake levels of mice fed the common nonfolate–supplemented chow. Although it was not specifically assessed here, these results suggested that leucovorin-supplemented diets could be beneficial to the animal’s well being when treated for extended periods of time with folate-drug conjugates. Likewise, clinical applications are possible since leucovorin is commonly administered to cancer patients, albeit for a different purpose (34,35).
Recent evidence has shown that the presence of a critical mass of unlabeled peptide significantly improves the uptake of peptide-based radioligands at the target tissue in both experimental animals and patients (1). Although we predicted that a similar approach would have a negative effect on the uptake of 99mTc-EC20 in tumors, we elected to coadminister 99mTc-EC20 to tumor-bearing animals in various combinations with free folic acid. As expected, our results confirmed that the percentage uptake of 99mTc-EC20 in FR–positive tumors was optimal when folic acid was absent from the formulation. However, we did discover that higher T/NT uptake ratios were obtained when a single equivalent of free folic acid was coadministered with the 99mTc-EC20. This finding might be the result of low levels of FRs in blood and some nontarget tissues that become at least partially saturated by the coformulated free folic acid; thus, only the more highly expressing FR–positive organs (tumor and kidney) would be available to bind 99mTc-EC20 under these dosing conditions. The implication of this result is that the sensitivity (contrast) of detecting FR–positive tumors might be improved by varying the mass of free folic acid in the radiotracer formulation.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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For correspondence or reprints contact: Christopher P. Leamon, PhD, Endocyte, Inc., 1205 Kent Ave., West Lafayette, IN 47906.
E-mail: Leamon{at}endocyte.com
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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