Synthesis of Specific SPECT-Radiopharmaceutical for Tumor Imaging

Bioconjugate Chem. 2010, 21, 229–239

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Synthesis of Specific SPECT-Radiopharmaceutical for Tumor Imaging Based on Methionine: 99mTc-DTPA-bis(methionine) Puja Panwar Hazari, Gauri Shukla, Vijay Goel, Krishna Chuttani, Nitin Kumar, Rajnish Sharma, and Anil Kumar Mishra* Division Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig SK Mazumdar Road, Delhi-110054, India. Received May 2, 2009; Revised Manuscript Received January 6, 2010

Methionine-diethylenetriaminepentaaceticacid-methionine [DTPA-bis(Met)] was synthesized by covalently conjugating two molecules of methionine (Met) to DTPA and was labeled with 99mTc in high radiochemical purity and specific activity (166-296 MBq/µmol). Kinetic analysis showed Km of 12.95 ( 3.8 nM and a maximal transport rate velocity (Vmax) of 80.35 ( 0.42 pmol µg protein-1 min-1 of 99mTc-DTPA-bis(Met) in U-87MG cells. DTPA-bis(Met) had dissociation constants (Kd) of 0.067 and 0.077 nM in U-87MG and BMG, respectively. 35 S-methionine efflux was trans-stimulated by 99mTc-labeled DTPA conjugate demonstrating concentrative transport. The blood kinetic studies showed fast clearance with t1/2 (F) ) 36 ( 0.5 min and t1/2 (S) ) 5 h 55 min ( 0.85 min. U-87MG and BMG tumors saturated at ∼2000 ( 280 nmol/kg of 99mTc-DTPA-bis(Met). Initial rate of transport of 99mTc-DTPA-bis(Met) in U-87MG tumor was found to be 4.68 × 10-4 µmol/kg/min. The tumor (BMG cell line, malignant glioma) grafted in athymic mice were readily identifiable in the gamma images. Semiquantitative analysis from region of interest (ROI) placed over areas counting average counts per pixel with maximum radiotracer uptake on the tumor was found to be 11.05 ( 3.99 and compared ROI with muscle (0.55 ( 0.13). The tumor-to-contralateral muscle tissue ratio of 99mTc-DTPA-bis(Met) was found to be 23 ( 3.3. Biodistribution revealed significant tumor uptake and good contrast in the U-87MG, BMG, and EAT tumorbearing mice. In clinical trials, the sensitivity, specificity, and positive predictive values were found to be 87.8%, 92.8%, and 96.6%, respectively. 99mTc-DTPA-bis(Met) showed excellent tumor targeting and has promising utility as a SPECT-radiopharmaceutical for imaging methionine-dependent human tumors and to quantify the ratio of MET+/HCY-.

INTRODUCTION Development of a highly specific and sensitive noninvasive imaging modality that can enhance the ability to closely correlate diagnosis with pathology, distinguish inflammation from tumors, differentiate tumor grades, accurately delineate tumor volume, monitor treatment responses, and identify residual tumor/ recurrence remains a desirable goal to improve the current clinical management of cancers. The natural amino acids are transported inside the cells by specific carrier-mediated transport systems and get incorporated into proteins and intermediary metabolites to different extents. Several transporters are based on system L such as LAT1, LAT2, LAT3, and LAT4, which have been identified at the molecular level (1-5). Among the identified transporters, LAT1 is consistently expressed at high levels in cancer cells as LAT1 transporter, and therefore is a candidate substrate for transport into cancer cells (6). Many synthetic derivatives, such as methylaminoisobutyric acid (MeAIB), and various halogenated derivatives of phenylalanine and tyrosine have also been shown to enter the cells using similar transport systems but are not metabolized. The role of overexpression of transporter systems of neutral amino acid has been reported in literature (7). Therefore, many natural amino acids (such as methionine, glycine, tyrosine, phenylalanine, and leucine) and their synthetic analogues have been labeled with radioactive isotopes and are being explored as tumor imaging agents for PET/SPECT (8-13). * Corresponding author. Dr. Anil Kumar Mishra, Brig. S. K. Mazumdar Road, Delhi-110054, India, Phone: +91-11-2390-5118, Fax: +91-11-2391-9509, E-mail: [email protected].

The major metabolic functions of methionine are in protein synthesis and conversion to S-adenosylmethionine (Adomet), which is required in multiple metabolic pathways. Methionine (Met) dependence has been shown in vitro in a number of human cell lines of different cellular origin, and the dependence may reflect the overall imbalance in the transmethylation (14-17). Metabolic defects in cancer cells often manifest in the inability to grow in media where Met has been replaced by its precursor HCY (18, 19). Increase in amino acid uptake is one of the earliest events associated with in vitro transformation (20) thus inducing upregulation of the amino acid transporter expression to enhance the facilitated transport (21). Because the uptake of amino acids (AA) in normal tissue is low and increases across glioma capillaries and tumors, the contrast between tumor and normal tissue is generally better with amino acid scanning as compared to FDG-PET or any other imaging modality (22-25). Currently, PET using L-[methyl-11C]-methionine (MET) is the most popular AA-imaging modality for tumors, although its use is restricted to PET centers with an in-house cyclotron facility for successful use of isotope in amino acid labeling. Polydentate ligands are of particular interest because of their capability to adopt an organized conformation in the complexed form. Entrapment of metal ion into a framework of multidentate poly(aminocarboxylates) such as DTPA covalently attached to mAbs with some metallic radionuclides, especially 111In, and various other lanthanides has been proven to provide thermodynamically stable compounds suitable for clinical trials (26). The anhydride derivatives of DTPA are generally easier to synthesize because of the commercial availability of DTPA. In particular, complexation of DTPA moiety with 99mTc showed excellent results as a metallopharmaceutical for medical imaging.

10.1021/bc900197n  2010 American Chemical Society Published on Web 01/28/2010

230 Bioconjugate Chem., Vol. 21, No. 2, 2010

The work reported in the literature has shown that the conjugated DTPA [DTPA-B-(SLex)A] forms stable complexes with lanthanides and transition metals (27). The main objective of our study was to design and synthesize a novel amino acid derivative based on DTPA, which forms stable complexes with most lanthanides and transition metals in the periodic table. Second, to introduce a chelating group without compromising the biological integrity of the amino acid for diagnosis of cancer using nuclear medicine and MR techniques. As a part of our efforts to explore the application of targeted radiopharmaceuticals for site-directed diagnosis and cancer therapy, (28-31) we hereby report the synthesis, characterization, and radiochemistry of 99mTc labeled DTPA-bis(Met) conjugate and its evaluation in nude mice bearing human malignant glioma cell lines (U-87MG and BMG). Covalent attachment of a strong chelating agent, DTPA, with methionine provides binding sites to which reduced 99mTc is strongly bound. Radiolabeling of the purified methionine DTPA-conjugate with 99m Tc, stability studies in human serum under physiological conditions, and blood kinetics in rabbits are reported. Cytotoxicity was determined by MTT and clonogenic assays. The efficiency of DTPA-bis(Met) in vitro was assessed by cell uptake assays in tumor cell lines (U-87MG and BMG) and human embryonic kidney cell line and in vivo by biodistribution studies with athymic mice bearing subcutaneous U-87MG and BMG cell line. Clinical studies in patients were carried out to establish the specificity and sensitivity of the new SPECT radiopharmaceutical in patients for better detection and delineation of metabolic tumors of different histological grades.

MATERIALS AND METHODS Chemicals. Acetonitrile, methanol, and water (HPLC grade) were obtained from Merck Germany Ltd. L-Methionine, diethylenetriaminepentaacetic acid, DTPA bisanhydride, triethylamine, dimethylformamide, methanol, ethanol, stannous chloride dihydrated (SnCl2 · 2H2O), trifluoroacetic acid, MTT, 2-methylaminoisobutyric acid (MeAIB), 2-amino-bicyclo-(2,2,1)heptane-2-carboxylic acid (BCH), L-arginine, L-valine, L-alanine, L-leucine, and L-glutamine were purchased from Sigma-Aldrich Co. 99mTc and 35S-L-methionine were procured from Regional Center for Radiopharmaceuticals (Northern Region), Board of Radiation and Isotope Technology (BRIT), Department of Atomic Energy, India. Cell Culture. Monolayer cultures of human malignant glioma cells, U-87MG (obtained from NIMHANS, Bangalore), normal embryonic kidney cells, HEK, and BMG were maintained at 37 °C in a humidified CO2 incubator (5% CO2, 95% air) in DMEM (Sigma, USA) supplemented with 10% fetal bovine serum (GIBCO), 50 U/mL penicillin, 50 µg/mL streptomycin sulfate, and 2 µg/mL nystatin. Cells were routinely subcultured twice a week using 0.05% Trypsin (Sigma, USA) in 0.02% EDTA. Instrumentation. 1H NMR spectra were determined by using Bruker Avance II 400. Mass spectrum (ES+MS) was recorded on a Aligent 6310 ion trap. HPLC analyses were performed on a Waters Chromatograph efficient with 600s coupled to a Waters 2487 photodiode array UV detector. The C-18 RP Beckman column (5 µ, 1 mm × 125 cm) column was used applying the elution system described in the text. C-18 Cartridges were purchased from Waters. Radioactive samples were counted using Capintech automated well-type counter. Beta scintillation counts were obtained by mixing samples in optiphase scintillation cocktail using Wallac Micro beta Trilux 1450 LSC, PerkinElmer (ACBR, Delhi University). Radioimaging and biodistribution studies were done using a planner gamma camera HAWKEYE. The clinical scintigraphs were obtained using a rectangular large field of view gamma camera (HAWKEYE dual

Hazari et al. Scheme 1. Synthesis of Diethylenetriaminepentaacetic Acid bis(methionine)a

a (i) DTPA-bis anhydride, (ii) triethylamine.

L-methionine,

(iii) DMF, (iv)

head, GE medical systems, Milwaukee, Wisconsin, USA) with a low-energy all-purpose collimator. Animal Models. All animal experiments were performed in accordance with guidelines of INMAS animal ethics committee. New Zealand rabbits, athymic mice, and albino balb/c mice were used for blood clearance, imaging, and biodistribution. Mice and rabbits were housed under conditions of controlled temperature of 22 ( 2 °C and normal diet. Athymic mice were inoculated subcutaneous with 0.1 mL of cell suspension (5 × 106 BMG/U-87MG cells) just above the right hind leg under sterilized conditions. Balb/c mice were inoculated subcutaneously EAT (15 × 106 cells) in the right hind leg.

PATIENTS AND METHODS Patients. Human protocols have been approved by Institutional human ethics committee. Scintigraphy. Whole body images were obtained using dual head gamma camera. 99mTc-DTPA-bis(Met) imaging was performed after i.v. administration of 15 mCi (555 MBq) radiotracer. Whole body scan was done after 1 h, 3 h, 6 h, and 24 h injection of radiotracer as a standard protocol and 5 patients were also imaged at 5, 10, 30, 45, 60, and 180 min. Semiquantitative analysis was generated from region of interest (ROI) placed over areas counting average counts per pixels with maximum radiotracer uptake on the lesions and compared to symmetric counterparts with ROI in soft tissues. Thirty-three breast cancer patients underwent in-house FDG scan. Brain cancer patients also underwent MDP scan. Statistical Methods. Data is reported as mean ( standard deviation (S.D.). Data were analyzed by the two-tailed Student’s t test for comparison with the control. Value of P < 0.05 was considered statistically significant.

EXPERIMENTAL SECTION Synthesis of Diethylenetriaminepentaacetic Acid Bis(methionine) Scheme 1. DTPA anhydride (500 mg; 1.4 mmol) and L-methionine (522 mg; 3.50 mmol) were dissolved in 5 mL of

SPECT-Radiopharmaceutical for Radiodiagnostic Imaging

DMF (anhyd.), and 15 mol equiv of triethylamine (2.35 mL) was then added. The reaction was allowed to proceed for 48 h at 55 °C. Completion of the reaction was checked by running TLC plates in water (0.01% TFA) and methanol (7:3; v/v) [Rf for Met: 0.89; Rf for DTPA-bis(Met): 0.75]. Triethylamine and DMF were evaporated under reduced pressure using a rotaevaporator. The chemical purity of DTPA-bis(Met) conjugate was examined by analytical HPLC on Beckman C-18 reverse-phase column (4.6 mm × 250 mm). The mobile phase was 0.05% TFA in water (solvent A) and methanol (solvent B) with a flow rate of 0.5 mL/min. Gradient 0-14 min A/B 90/10-50/50, 14-23 min A/B 50/50-0/100, 23-28 min A/B 0/100-90/10. Yield: 758 mg,1.16 mmol, 82.66%. IR (ν, cm-1) 3437, 2817, 1610. 1H NMR (400 MHz, D2O): δ ) 4.42-4.19 (m, NH-CH2, 2H), 3.685 (br, s, CH2, 4H), 3.7-2.37 (m, CH2-CH2, 22H), 2.026, 2.005 (br, s, 2H) ppm. 13C NMR (400 MHz, D2O): δ ) 178.70, 178.35 (CdO), 173.18, 174.48 (COOH), 58.40, 58.09, 57.09, 56.16, 54.01, 53.89, 52.45, 49.77, 49.37 (CH2-CH2, 11C), 30.86, 29.87, 29.76, 28.79 (CH2-S, 4C), 14.20, 13.87 (S-CH3, 2C) ppm. Calculated mass [M+H]+ 656.2 found m/z ) 656.6 [M+H]+, 678.2 [M+Na]+ (see Supporting Information). Elemental analysis calcd for C24H41N5O12S2: C, 43.96; H, 6.30; N, 10.68; O, 29.28; S, 9.78. Found C, 43.95; H, 6.31; N, 10.67; O, 29.29; S, 9.78. Radiolabeling of DTPA-bis(Met) with 99mTechnetium. DTPA-bis(Met) (2 mg) was dissolved in a shielded vial and stannous chloride (300 µL; 1 mg dissolved in N2 purged 1 mL of 10% acetic acid) was added followed by addition of freshly eluted (99%. Phosphor imaging showed a single spot indicating formation of only one species. Radiolabeled DTPAbis(Met) conjugate was eluted in g99% radiochemical purity with specific activity of 4500-8000 µCi/µmol (166-296 MBq/ µmol) through C-18 extraction cartridge. Radiochromatogram showed a single peak eluting at 6.4 min by C-18 reverse-phase column and came through flow using solvent system of 10 mM ammonium acetate and acetonitrile. Human Serum Stability Evaluation. The purified 99mTcDTPA-bis(Met) was incubated in freshly extracted human serum under physiological conditions. Radiolabeled conjugates were challenged with the proteins (albumin and transferrin) present in the serum to test the stability of the radiolabeled DTPAbis(Met). Major transcomplexation of 99mTc was observed for unmodified Met to be 66% after 1 h. DTPA-bis(Met) showed 2.9% transcomplexation of 99mTc in serum. Approximately more than 94% of the radioactivity remains associated with the conjugate after 2 h challenge at 37 °C. It was sufficiently stable up to 24 h, as only 4% of the radiolabeled drug dissociated in serum at 24 h. Cytotoxicity Studies of DTPA-bis(Met). By analysis of the MTT assay data at different concentrations (nM to mM range), it was found that, at a concentration of 1 mM, 2 h exposure resulted in lyses of 60% of U-87MG cells. It was observed that,

when 10 µM of DTPA-bis(Met) was incubated with BMG cell line for 2 h, only 5% of the cells were killed. The two cell lines U-87MG and BMG showed concentration-dependent cytotoxicity. U-87MG showed survival value of 0.47 ( 0.028 at 1 mM for unlabeled DTPA-bis(Met). A surviving fraction of 0.54 ( 0.015 at 1 mM for the BMG cell line was observed in the clonogenic assay (Figure 2). The time-dependent curve showed a regain of the metabolic viability of the BMG cell line at 72 h post-treatment. Cell Uptake Studies. Time Course and Kinetics. Time course experiments revealed that transport of 99mTc-DTPA-bis(Met) into cells was rapid and linear for up to 10 min, saturating thereafter, and reaching a plateau after 30 min of incubation. Kinetic analysis revealed that transport of 99mTc-DTPA-bis(Met) occurs via a saturable high-affinity carrier with a Michaelis constant (Km) of 12.95 ( 3.8 nM and a maximal transport rate velocity (Vmax) of 80.35 ( 0.42 pmol. µg protein-1 min-1 (Figure 3). A single linear regression line was obtained in the Eadie-Hofstee transformation (inset Figure 3B) indicative of the function of a saturable system or systems with comparable functional characteristics. When repeated in the absence of Na+, transport of 99m Tc-DTPA-bis(Met) was indistinguishable from that observed in normal Krebs buffer, since rates measured in Na+-free Krebs solution were only marginally lower than those determined in the presence of Na+. Cell Binding Studies. The ability of DTPA-bis(Met) conjugate to bind LAT1 transporters on the surface of tumor cell lines U-87MG, BMG, and HEK was examined by binding assay using 99mTc- DTPA-bis(Met) as the labeled ligand. Nonspecific binding was determined using 100-fold excess of unlabeled methionine. Examination of binding curves showed significant external binding of the labeled DTPAbis(Met) conjugate. Scatchard plot analysis revealed affinity of the labeled DTPA-bis(Met) on tumor cell lines. Kd was

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Hazari et al.

Figure 2. Cytotoxicity of DTPA-bis(Met) conjugate in U-87MG and BMG cell line: (a) concentration-dependent and (b) time-dependent curve as determined by clonogenic survival assay.

Figure 3. Time course and kinetics of 99mTc-DTPA-bis(Met). (A) Uptake was monitored from 30 s to 60 min. (B) Transport of 99mTc-DTPAbis(Met) was monitored over 10 min period in U-87MG Cells. The inset shows an Eadie-Hofstee plot of saturable transport. Values are the mean ( SD of 3 replicate measurements in 3 separate experiments.

Figure 4. (A) Displacement using increasing concentrations of unlabeled methionine and labeled 99mTc-DTPA-bis(Met) was conducted in binding buffer The plot is representative of three independent experiments. (B) Scatchard plot of the specific binding data to the ratio of hound to free (B/F) for U-87MG, BMG, and HEK cell lines.

found to be 0.067 and 0.077 nM in U-87MG and BMG, respectively. In human embryonic kidney cell line, Kd was found to be 0.10 nM (Figure 4B). SelectiVity of 99mTc-DTPA-bis(Met) Transport. Cross inhibition studies were carried out in the presence and absence of Na+ to evaluate the substrate specificity of the transporter associated with 99mTc-DTPA-bis(Met) uptake by U-87MG cells. Under these conditions, transport of 99mTc-DTPA-bis(Met) was not affected by a 100-fold excess of either 2-methylaminoisobutyric acid (MeAIB), a model substrate for system A, or L-valine and L-alanine, which are known substrates for the ASC system but was inhibited by 68 ( 1.9%, 52 ( 1.2%, and 36 ( 2.1% in

the presence of 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), L-phenylalanine, and L-leucine, respectively. Trans-Stimulation of 35S-L-methionine Efflux in U-87MG Cells. The efflux of 35S-methionine was measured in the presence and absence of 1 mM (10 mCi) of 99mTc-labeled DTPA-bis(methionine). The amount of 35S-L-methionine incorporated during the loading and subsequent washing protocol was less than 10 µM. It was evident from the experiment that 35 S-L-methionine efflux was trans-stimulated by extracellular 1 mM of DTPA-bis(Met). The magnitude of effect was observed to be higher at 30 min (0.32 ( 0.04 µg/mL). It was evident from the experiment that the presence of 99mTc-labeled DTPA-

SPECT-Radiopharmaceutical for Radiodiagnostic Imaging

Figure 5. Trans-stimulation of 35S-methionine efflux by extracellular 99m Tc-labeled DTPA-bis(methionine). Efflux was measured for 90 min at 25 °C. Values mean ( SD, n ) 3, each performed in duplicate P < 0.05(0.0016) compared with control.

bis(Met) in the culture medium significantly (P < 0.05) transstimulated efflux of 35S-methionine (Figure 5). Blood Kinetics. In vivo clearance in rabbits revealed that there was a rapid clearance of DTPA-bis(Met) from the circulation, as only 17.6% of injected activity remained in the circulation at 30 min. After 30 min, the clearance followed a slow pattern, and at 24 h, approximately 0.76% activity persisted in the blood (Figure 6). The biological half-life was found to be t1/2 (F) ) 36 ( 0.5 min and t1/2 (S) ) 5 h 55 min ( 0.85 min. Biodistribution. Dose escalation studies were performed to determine the concentration of 99mTc-DTPA-bis(Met) that would saturate U-87MG and BMG tumors. U-87MG tumor grafted nude mice were injected with increasing concentrations in the range 10-4000 nmol/kg, and after allowing 4 h for clearance of unbound conjugates from the tissues, tumor and normal tissue were dissected. U-87MG and BMG tumors were saturated at ∼2000 ( 280 nmol/kg of 99mTc-DTPA-bis(Met). In contrast, soft tissue (muscles) accumulated negligible quantities of the conjugate. Biodistribution studies in mice conducted to quantify localization of the radiolabeled DTPA conjugate on U-87MG, BMG, and EAT tumors are shown in Table 1. Athymic mice implanted with U-87MG and BMG cell line exhibited accumulation in kidneys (2.21 ( 0.08% ID/g) and 3.24 ( 0.52% ID/g, respectively, at 1 h showing that the complex is excreted

Figure 6. Blood clearance of

99m

Bioconjugate Chem., Vol. 21, No. 2, 2010 235

by renal routes. Biodistribution revealed high tumor uptake in the U-87MG tumor bearing nude mice; tumor to muscle ratio reached 52.5 at 4 h postinjection. Biodistribution studies in nude mice xenographed with U-87MG and BMG human malignant glioma cell lines have shown that tumor uptake of 99mTc was found to be 2.14 ( 0.9% ID/ g and 1.08 ( 0.11% ID/ g at 1 h. Comparative biodistribution was carried out for 99mTc-labeled methionine and 99mTc-DTPA-bis(Met) in EAT tumor. It was evident from the study that directly labeled methionine accumulated in liver and a major part of the radiolabeled methionine cleared through the hepatobiliary route (Figure 7). Localization of the radioactivity in the liver, stomach, and small intestine was low, less than 2% ID/g at 1 h for 99mTc-DTPAbis(Met). Good clearance of the radiolabeled conjugate was also observed from these organs. EAT tumor uptake at 1 h was 2.8 ( 0.12% ID/g for 99mTc-DTPA-bis(Met) at 4 h. 99m Tc-DTPA-bis(Met) was administered through the ear vein in U-87MG grafted athymic mice with and without coadministration of a 250 µg blocking dose of methionine, which showed significant blocking of the receptors at the tumor site with uptake of 0.36 ( 0.06% ID/g as compared to 2.14 ( 0.9% ID/ g for 99m Tc-DTPA-bis(Met) at 1 h (Figure 8). In Vivo Tumor Kinetics and Scintigraphy. In vivo tumor kinetics was studied in U-87MG cells over a time period ranging from 1 to 270 min. Initial rate of transport of 99mTc-DTPAbis(Met) was found to be 4.68 × 10-4 µmol/kg/min (Figure 9). Further, the rate was decreased to 6.77 × 10-5 µmol/kg/ min over 30 min and found to saturate at 1.02× 10-5 µmol/ kg/min. However, the initial rate of soft tissue uptake was 3.23 × 10-5 µmol/kg/min. After 30 min, the rate of transport further increases 100-fold as compared to the accumulation in the soft tissue. Imaging of animals was carried out at different time intervals after administering labeled compound intravenously. The mice depicted the beginning of accumulation of activity in tumor at 30 min, which reached a maximum at 4 h and remained almost stable for 6 h. The tumors (BMG cell line) grafted in athymic mice were readily identifiable in the γ images (Figure 10). Semiquantitative analysis generated from region of interest (ROI) placed over areas counting average counts per pixel with maximum radiotracer uptake on the tumor was found to be 11.05 ( 3.99 and compared to ROI with muscle (0.55 ( 0.13). The tumor-to-contralateral muscle tissue ratio of 99m Tc-DTPA-bis(Met) was found to be 23 ( 3.3. In the mice that received coadministration of 250 µg blocking dose of

Tc-DTPA-bis(Met) (11.1 MBq activity) administered through ear vein in normal rabbit.

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Figure 7. Comparative biodistribution of grafted Balb/c mice.

99m

Tc-Met and

Hazari et al.

99m

Tc-DTPA-bis(Met) (11.1 MBq activity) administered through ear vein in EAT tumor-

and compared to symmetric counterparts with ROI in soft tissues (target to nontarget ratios are shown in Table 2).

DISCUSSION

Figure 8. Tumor, blood, liver, and kidney activity of 99mTc-DTPAbis(Met) administered through the ear vein in U-87MG grafted athymic mice with and without a coadministration of a blocking dose.

methionine, the uptake in the tumor was 0.46 ( 0.06% ID/ g, significantly lower at 1 h postinjection (Figure 9). Comparative evaluation of biodistribution of 99mTc-labeled methionine and 99mTc-DTPA-bis(Met) in normal rabbits was also conducted (see Supporting Information). It was found that directly labeled methionine accumulated in the liver, whereas negligible uptake was seen for 99mTc-DTPA-bis(Met). Clinical Studies. Clinical studies in normal volunteers showed fast renal clearance in patients. Two hours after administration of 99mTc-DTPA-bis(Met), a good percentage of the injected activity was cleared through the kidneys. Insignificant tracer accumulation was observed in the hepatic region. Semiquantitative analysis was generated from region of interest (ROI) placed over areas counting average counts per pixel with maximum radiotracer uptake at the tumor site

The polyaminopolycarboxylate ligand having two reactive sites (bisanhydride of DTPA) for the conjugation of amino acid favors the formation of complex with only one ligand per metal atom, which is required for in vivo stability and biological activity. The conjugate was labeled with 99mTc and evaluated in vitro and in vivo for its competence in clinical diagnosis of metabolic tumors. Radiochemical analysis showed that the drug is specifically coordinated with 99mTc radionuclide. In vitro human serum stability studies under physiological conditions suggested that there was least transcomplexation of the labeled drug. This targeted agent was tested for the ability to induce cytotoxicity in the cancer cell lines using both an assay of mitochondrial activity (in a MTT assay) and a clonogenic assay. The unlabeled conjugate showed concentration-dependent cytotoxicity. A direct correlation was observed between MTT assay and clonogenic assay. Receptor binding experiments were performed to verify the biological integrity upon modification of methionine. Scatchard analysis confirmed good binding affinity to its target, as Kd values on malignant cancer cell lines were found to be in sub-picomolar range. The expression and function of L-type amino acid transporter 1 (LAT1), a major catalytic subunit of system L, in human glioma cells have been reported in the literature (33). Several of the amino acid transport systems can be distinguished by their dependence on Na+, response to other competitive AA, and response to specific nonmetabolizable AA analogues. System A, for example, is inhibited by R-(methylamino)isobutyric acid (MeAIB), whereas system L is inhibited by BCH. A combination of treatments was applied to incubated skin strips to define the AA transport systems operating for L-Ala, L-Leu,

SPECT-Radiopharmaceutical for Radiodiagnostic Imaging

Bioconjugate Chem., Vol. 21, No. 2, 2010 237

Figure 9. In vivo U-87MG tumor kinetics of 99mTc-DTPA-bis(Met) as a function of time after i.v. administration of a saturating dose of 2500 nmol/kg in tumor-bearing nude mice. (A) Rate of transport at an interval of 1 min over 30 min time period (inset: initial rate up to 5 min). (B) Rate of transport over 270 min time period.

Figure 10. Whole-body γ image of female athymic mice with subcutaneous BMG tumor above the right hind leg at 1 h with and without coadministration of blocking dose. Table 2. Semiquantative ROI Analysis Showing Lesion Target to Nontarget Ratios (T/NT) in Patients Expressed as Mean ( S.D. no. of patients

patients

ROI (T/NT)

results

47

palpable breast masses, median age 44 (range 28-68 years), 33 breast carcinoma, 14 benign masses

3.7 ( 0.29 (range 2.73-6.06)

3

brain cancer, median age 65 (range 45-82) 2 brain malignancy, 1 suspected recurrence regional lymph node cancer osteosarcoma

4.72 ( 0.48 (range 4.38-5.06)

true positive DTPA-bis(Met) uptake in 29/33 patients, negative in 4 patients. 13 benign masses came out to be negative and 1 positive. Sensitivity ) 87.8%; Specificity ) 92.8% positive in brain malignancies, recurrence not observed for 1 patient in both DTPA-bis(Met) and FDG scan

2 3

3.47 ( 2.05 (range 2.01-4.92) 5.31 ( 0.93 (range 4.64-6.38)

L-Cys, and L-Lys. The L system for which there is strong evidence of its involvement in the uptake process of 99mTcDTPA-bis(Met) is the Na+-insensitive system L, which shows high specificity for branched-chain and aromatic amino acids and utilizes the nonmetabolizable bicyclic BCH analogue as a system-specific substrate. Thus, since BCH was found to block the transport of 99mTc-DTPA-bis(Met) in competitive binding studies, it is reasonable to conclude that this pathway does contribute to the uptake of 99mTc-DTPA-bis(Met) to a significant extent. Moreover, the system L substrates such as L-leucine and L-phenylalanine caused marked inhibition of 99mTc-DTPAbis(Met) transport. The inhibition was found to be independent

DTPA-bis(Met) scan positive in all cases DTPA-bis(Met) scan positive in all cases

of the presence of Na+, which is a characteristic of the L transporter. Thus, the marked inhibition caused by L-leucine and BCH strongly supports the idea that system L is being utilized by 99mTc-DTPA-bis(Met) for entry into cells. Moreover, 99mTcDTPA-bis(Met) demonstrated trans-stimulation of 35S-L-methionine indicated for an exchange mechanism of transport. The data provide direct evidence for transport against a concentration gradient and not just via passive diffusion only. The pharmacokinetic profile of 99mTc-DTPA-bis(Met) showed its high target uptake with the diagnostically useful target-tonontarget ratio over a short period of time. The labeled drug

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showed fast clearance that resulted in a decrease in the background activity. The high specificity of results in selective uptake and distribution of the radiolabeled receptor ligand at the tissues, which are known to contain a substantial concentration of the target receptor that can be visualized in the high-quality images, obtained 1 h after administration of 99mTc-DTPA-bis(Met) in animal models with and without coadministration of blocking dose, which allows the advantage of comparing in vitro receptor binding studies and tumor-bearing animal studies. Dynamic study as a function of time revealed the rate of transport of 99m Tc-DTPA-bis(Met) at the tumor site and soft tissue. The biodistribution studies showed that 99mTc-labeled methionine persisted in the liver up to 4 h, which indicates its hepatobiliary clearance, and the radiolabeled DTPA-conjugate cleared through renal routes. Accurate interpretation of SPECT scans in suspected tumors relies on comparison of the amount of methionine taken up by the lesion with the normal value for that region of soft tissue. A normal value is typically determined by measuring uptake in the corresponding region of the contralateral tissue.99mTc-DTPAbis(Met) accumulated in nearly all cancers with varying histology and histological grades. 99mTc-DTPA-bis(Met) imaging with SPECT is an effective method for evaluating head and neck cancer and, apparently, other types of human cancer. Furthermore, SPECT images of the tumors appeared to correlate well with the extent of cancers determined by other methods. It can provide useful information with reasonably high sensitivity and specificity in prognostic value for determining tumor masses. Normal tissues such as the salivary glands, however, accumulate 99m Tc-DTPA-bis(Met), which may impair the detection of small tumors with a low uptake. In summary, we have synthesized a new imaging agent 99mTcDTPA-bis(Met) showing substantial promise for tumor scintigraphy, as significant accumulation is observed in athymic mice bearing the subcutaneous BMG cell line (malignant glioma). Breast, head, and neck cancers of varying histology can be imaged with 99mTc-DTPA-bis(Met). Although accumulation of the tracer in the salivary and lachrymal glands may occasionally interfere with image interpretation, 99mTc-DTPA-bis(Met) SPECT is a cost-effective method for imaging breast, head, and neck cancer. It can detect malignancy with reasonably high sensitivity and specificity. It may also be useful in the assessment of tumor extent when management of cancer is planned. This system has an added advantage over 11C-methionine, as there are logistical hindrances in its use at centers that do not have an on-site cyclotron. Therefore, considering the apparent usefulness of new SPECT radiopharmaceutical 99mTc DTPA-bis(Met), future clinical study is warranted with attractive radionuclides and metal ions used in diagnosis and therapy.

ACKNOWLEDGMENT We thank Dr. R. P. Tripathi, Director INMAS, for providing necessary facilities. The work was supported by Defence Research and Development Organization, Ministry of Defence, under R&D project INM -311. Supporting Information Available: 1H, 13C NMR, and mass spectra of DTPA-bis(Met). Comparative evaluation of biodistribution of 99mTc-labeled methionine and 99mTc-DTPA-bis(Met) in normal rabbit. Clinical scintigraphs of 99mTc-DTPA-bis(Met) depicting breast carcinoma, regional lymph node cancer, and osteosarcoma. This material is available free of charge via the Internet at http://pubs.acs.org.

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