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[^99mTcOAADT]-(CH₂)₂-NEt₂: A Potential Small-Molecule Single-Photon Emission Computed Tomography Probe for Imaging Metastatic Melanoma

¹ Department of Radiology, Harvard Medical School, Boston, Massachusetts and ² Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts

Requests for reprints: Ashfaq Mahmood, Department of Radiology, Harvard Medical School, Armenise Building, 200 Longwood Avenue, Boston, MA 02115. Phone: 617-432-3995; Fax: 617-432-2419; E-mail: amahmood{at}hms.harvard.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Evaluation of [^99mTc]oxotechnetium(V) complexes of the amine-amide-dithiol (AADT) chelates containing tertiary amine substituents as small-molecule probes for the diagnostic imaging of metastatic melanoma has shown that technetium-99m–labeled AADT-(CH₂)₂-NEt₂ (^99mTc-1) has the highest tumor uptake and other favorable biological properties. We have, therefore, assessed this agent in a more realistic metastatic melanoma model in which, after i.v. tail injection, a highly invasive melanoma cell line, B16F10, forms pulmonary tumor nodules in normal C57BL6 mice. Small melanotic lesions develop in the lungs and, on histologic examination, appear as small black melanoma colonies, increasing in size and number with time after tumor cell injection. Groups of mice received tumor cell inocula of 2 x 10⁵, 4 x 10⁵, or 8 x 10⁵ B16F10 cells; 14 days later, 2 hours after ^99mTc-1 administration, lung uptake of 2.83 ± 0.21%, 3.63 ± 1.07%, and 4.92 ± 1.61% injected dose per gram of tissue (% ID/g), respectively, was observed, compared with normal lung uptake of 2.13 ± 0.2% ID/g (P < 0.05). Additionally, a higher level of ^99mTc-1 accumulation was seen 17 days after tumor cell inoculation as the lung lesions grew. These in vivo studies coupled with additional in vitro and ex vivo assessment show that ^99mTc-1 has high and specific uptake in melanoma metastases in lungs and can potentially follow the temporal growth of these tumors.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Malignant melanoma is a public health challenge with a rising incidence rate and estimates of 47,700 new cases to be diagnosed annually (1–3). The significant mortality of this cancer is associated with its high cellular proliferation rate and the early occurrence of metastases. Because early detection of the melanoma and its associated metastases considerably improves management and prognosis of the disease, there has been intense interest in the search for melanoma-specific diagnostic imaging probes and therapeutic agents (4, 5).

2-[¹⁸F]Fluoro-2-deoxy-D-glucose ([¹⁸F]FDG) is widely used in positron emission tomographic (PET) imaging of various tumors including melanoma. In patients with stage III or in-transit melanoma, prospective whole-body [¹⁸F]FDG PET studies display a sensitivity of 87.3% with a positive predictive value of 78.6%, which can be further improved with the help of other pertinent clinical information (6). Other prospective clinical studies have also shown the utility of whole-body [¹⁸F]FDG PET imaging in stage II to IV melanoma with sensitivity and specificity of 94.2% and 83.3%, respectively, for lesions in the soft tissue, lymph nodes, and liver (7); false-positives are due to the accumulation of [¹⁸F]FDG in surgical wounds, pneumonia, and other etiologies including infection/inflammation (6–9).

A number of radiolabeled, single-photon emission computed tomography (SPECT) metabolic tracers, peptides, and monoclonal antibodies have also been investigated for melanoma detection, staging, and follow-up (4, 5). Among these SPECT agents, the most promising are the radioiodinated benzamides (10–14). Although the nature of the affinity of iodobenzamides for melanoma is not clearly understood, it has been shown that radiolabeled iodobenzamides possess a considerable in vivo affinity for melanotic melanoma lesions compared with amelanotic lesions (10). Animal and human studies of several radioiodobenzamide derivatives indicate a promising profile for their use in melanoma diagnosis. A scintigraphic phase II clinical study in 110 patients for detecting malignant melanoma and its metastases with N-(2-diethylaminoethyl)-4-[¹²³I]iodobenzamide ([¹²³I]BZA; Fig. 1) found 81% diagnostic sensitivity, 87% accuracy, and 100% specificity (11) for the compound. Additional studies to optimize the pharmacokinetic characteristics of [¹²³I]BZA derivatives have shown that N-(2-diethylaminoethyl)-3-[¹²³I]iodo-4-methoxybenzamide ([¹²³I]IMBA; Fig. 1) has an 8-fold higher melanoma/nontarget tissue ratio than [¹²³I]BZA at 1 hour postinjection and a 4-fold higher ratio at 4 hours in the C57BL6 mouse model (12, 13). Preliminary scintigraphic studies in patients with melanoma metastases have confirmed the efficacy of this compound (12). More recently, a single-center clinical trial in 25 patients using another radioiodinated iodobenzamide derivative, N-(2-diethylaminoethyl)-2-[¹²³I]iodobenzamide ([¹²³I]BZA₂; Fig. 1), showed 100% sensitivity, 95% specificity, a positive predictive value of 86%, and a negative predictive value of 100% on a per patient basis (14).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Iodobenzamide and technetium complexes with melanoma affinity.

Our attempts to design technetium-based, small molecules that possess structural elements of the benzamides are based on the strategy of replacing the aromatic moiety in the benzamide structure with a chelated metal-oxo core linked to the tertiary amine substituent. Applying this strategy via a "3 + 1" tridentate/monodentate chelating approach has produced ^99mTcO(SNS)(S-C₂-NEt₂) (Fig. 1) and ^99mTcO(SNS)(S-C₂-NBu₂) complexes with significantly higher tumor accumulation (3.1% and 5% ID/g, respectively, at 60 minutes postinjection; ref. 18) compared with the benzamide-containing technetium complex TcO-Cf. Because the use of these "3 + 1" complexes as imaging agents could be hampered by their known tendency to form transchelation products with glutathione in vivo (19), we have modified the strategy and have synthesized a focused library of complexes based on the tetradentate chelate, amine-amide-dithiol (AADT), containing only tertiary amine substituents [AADT-CH₂-[CH₂]_n-NR₂ (n = 1, 2; R = Et, n-Bu, piperidino, morpholino); refs. 20–22]; most of these complexes display considerable affinity for melanoma both in vitro and in vivo, with the [^99mTcOAADT]-(CH₂)₂-NEt₂ derivative (^99mTc-1) having a high in vivo melanoma uptake of 7.6% ID/g (melanoma/nontarget tissue = 9.5) at 1 hour postinjection in the s.c. C57BL6/B16F0 mouse model (20). Described herein is a study of this small, neutral complex ^99mTc-1 (Fig. 1) in a more realistic lung metastatic melanoma model with a temporal tumor growth pattern that illustrates the potential use of ^99mTc-1 as a small-molecule SPECT imaging probe for the in vivo diagnosis of metastatic sites in malignant melanoma.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
General. All chemicals and reagents were purchased from commercial sources (Sigma-Aldrich, St. Louis, MO; Gibco Life Technologies, Gaithersburg, MD). Technetium-99m pertechnetate was obtained via a ⁹⁹Mo-^99mTc generator (DuPont NEN, Bilricca, MA). High-performance liquid chromatography (HPLC) purification was done on a Waters Millennium Chromatography System equipped with a 996 UV-Vis diode-array detector attached in series to a -detector consisting of a shielded photomultiplier powered by a Canberra voltage amplifier and connected to a ratemeter. For the analytic and preparative HPLC purification of the radiolabeled complex, a reverse-phase C8 column equipped with a C18 guard was eluted with methanol (solvent A) and 5 mmol/L PBS, pH 7.4 (solvent B, Gibco Life Technologies) using a 30-minute linear gradient from 15:85 (A/B) to 90:10 (A/B) at a 1.0 mL/min flow rate.

Synthesis of [^99mTcOAADT]-(CH₂)₂-NEt₂. The AADT-(CH₂)₂-NEt₂ ligand and its technetium-99m complex were synthesized following procedures described previously (20). Briefly, 5.0 mg trityl-protected ligand was dissolved in 10 mL trifluoroacetic acid (TFA), and the resulting yellow-colored solution was titrated with triethylsilylhydride until it turned colorless. TFA was completely evaporated from the colorless solution on a rotary evaporator followed by additional drying via high vacuum. The dry residue was later redissolved in argon-saturated methanol and distributed into 10 vials that were dried and kept under vacuum for later preparation of the technetium-99m–labeled compound. ^99mTc-1 was synthesized using a vial containing 0.5 mg of the above thiol-deprotected ligand which was dissolved in 0.25 mL PBS (5 mmol/L, pH 7.4). To this was added the required ^99mTc activity in the form of a ^99mTc-glucoheptonate solution (0.3 mL), and the reaction was heated at 75°C for 25 minutes in a water bath. HPLC analysis of the reaction mixture typically indicated >90% radiochemical yield of the ^99mTc-1 complex (Fig. 2). In addition, coinjection of the structurally characterized rhenium complex (20) with the analogous ^99mTc-1 complex resulted in coelution of the radioactive species with the corresponding UV-active rhenium complex. All in vitro and in vivo experiments were conducted with the ^99mTc-1 complex isolated via preparative HPLC. The solution was subsequently evaporated to complete dryness and the complex reconstituted in sterile distilled water to the required radioactivity concentration.

View larger version (7K): [in this window] [in a new window] [Download PPT slide]   Figure 2. HPLC chromatogram of 99mTc-1 reaction mixture with radioactivity detection.

At the time of the cell uptake assay, all cells were harvested from the culture flasks by trypsinization with 1 mL trypsin-EDTA solution (0.25% trypsin, 1 mmol/L EDTA·4Na; Gibco Life Technologies). After being washed twice with 12 mL cold PBS (Gibco Life Technologies), the cells were counted and resuspended in Ca²⁺-free MEM with reduced Mg²⁺ content (S-MEM, Gibco Life Technologies) to a final concentration of 5.0 x 10⁶ cells/mL. The effect of inhibition of melanin synthesis on the uptake of ^99mTc-1 was studied by substituting DMEM containing 500 µmol/L N-butyldeoxynojirimycin (NBdNJ; ref. 23) as the growth medium 48 hours before the assay, and replenishing the NBdNJ medium every 24 hours. Similarly, the effect of increased melanin synthesis/content on the uptake of ^99mTc-1 was assessed by stimulating melanin synthesis in the B16F10 cells with DMEM supplemented with 2 mmol/L L-tyrosine 24 hours before the assay.

For in vitro cell uptake studies of ^99mTc-1, 2.5 x 10⁶ B16F10 tumor cells were incubated at 37°C or 4°C in polypropylene test tubes with 2 to 5 µCi (10 µL) ^99mTc-1 in a total volume of 500 µL S-MEM with intermittent agitation. At appropriate time intervals, the tubes were vortexed and triplicate samples of 15 µL were layered in individual 400-µL Eppendorf microcentrifuge tubes containing 350 µL cold calf serum. After centrifugation at 12,000 rpm for 1 minute, the tubes were frozen in a dry ice-acetone bath and, while still frozen, cut into two sections (a bottom section containing the cell pellet and an upper section with the supernatant) that were placed in individual counting tubes. Both fractions were counted for radioactivity in a -counter (WALLAC, 1480 WIZARD 3). The percentage of ^99mTc-1 uptake was calculated as % cell uptake = 100 x [cpm (pellet)] / [cpm (pellet) + cpm (supernatant)].

In vivo studies in murine melanoma C57BL6/B16F10 mouse models. Murine B16F10 cells grown in DMEM were harvested from cell culture flasks by trypsinization and washed as described above. The cells were counted and resuspended in PBS to final concentrations of 2.0 x 10⁵, 4.0 x 10⁵, or 8.0 x 10⁵ cells/mL and were maintained at 4°C for immediate i.v. or s.c. inoculation in C57BL6 male mice. The cell viability always exceeded 95%, as determined by trypan blue dye exclusion.

Pulmonary melanoma metastases in C57BL6 mice resulting from the i.v. inoculation of B16F10 melanoma cells were produced using two independent groups of C57BL6 male mice as previously described (24–26). One group was given 4 x 10⁵ viable B16F10 melanoma cells (0.1 mL) via lateral tail vein injection. On 11, 14, and 17 days after tumor cell inoculation, these mice received an i.v. injection of ^99mTc-1 (25–40 µCi in 0.1 mL). At 2 and 3 hours after injection of ^99mTc-1, the mice were weighed and sacrificed. Various organs and tumor-bearing lungs were harvested and placed in preweighed tubes, with the excised lungs, liver, spleen, and brain collected in tubes containing 15% formalin buffer. Radioactivity in the various organs was determined in a -counter along with a known standard of technetium-99m, and subsequently the tissues/organs were weighed. The excised lung tissues were then prepared for ex vivo radiophosphor imaging (see below). In the second group of mice, similar biodistribution studies were done 14 days after i.v. injection in three subgroups of 2 x 10⁵, 4 x 10⁵, and 8 x 10⁵ B16F10 melanoma cells. After the counting of radioactivity and measurement of organ weights, the tubes were stored for radioactivity decay. Subsequently the tissues/organs were processed for histopathologic examination. In vivo biodistribution studies with ^99mTc-1 were also done in normal (control) mice that did not receive an i.v. tumor cell inoculation.

Subcutaneous tumors were created using 2.5 x 10⁵ B16F10 cells (0.1 mL) transplanted s.c. on the left hind flank of C57BL6 male mice. After 10 to 14 days, the animals developed palpable tumor nodules 0.5 to 1.0 cm in diameter. The biodistribution studies were carried out in these mice at 2, 3, and 6 hours after tail vein injection of 25 to 40 µCi (0.1 mL) ^99mTc-1. The organs and tumors were harvested and counted in a -counter along with technetium-99m standards of the injected dose. The results are expressed as % ID/g.

All animal experiments were done in compliance with the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985) and under approved institutional animal protocols.

Radiophosphor imaging. Excised lung tissue collected from tumor-bearing mice was placed in preweighed tubes containing 15% formalin buffer solution and subjected to ex vivo radiophosphor imaging. The formalin-fixed lung tissues were blotted dry on tissue paper and placed on a clean microscope slide and photographed with a digital camera. The slides were then placed in a phosphor imaging cassette (Molecular Dynamics, Inc., Sunnyvale, CA). In the closed imaging cassette, the lung tissues were compressed to a thickness of 1 to 2 mm. The imaging plates were exposed overnight to the lung tissues to accumulate counts. These counts were then processed on a Molecular Dynamics Phosphor Imager (Storm 860 Scanner, Scanner Control Version 4.1, Molecular Dynamics) to obtain the radiophosphor images. Image analysis was carried out with the software program ImageQuant 1.2 (Molecular Dynamics).

Histology. The excised lung, liver, spleen, and brain tissues collected in 15% formalin buffer were processed for histopathologic studies after the complete decay of radioactivity. The tissue was embedded in paraffin and 5-µm-thin sections were cut on a microtome and stained with H&E. Slides of all the sample tissues were examined under light microscopy to determine the presence, extent, and size of melanoma metastases.

Statistical methods. Statistical analysis was done using Student's t test for unpaired data. A 95% confidence level was chosen to determine the significance between days, with P < 0.05 being significantly different.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Small technetium-labeled molecules based on the structural elements of the iodobenzamides currently in clinical trials for melanoma detection (11, 12, 14) have also been identified as possessing high in vivo melanoma accumulation. [^99mTc]oxotechnetium(V) complexes of tetradentate N₂S₂ (AADT) chelates containing only tertiary amine substituents have considerable melanoma affinity (18, 20–22), with ^99mTc-1 displaying an in vivo melanoma uptake of 7.6% ID/g (melanoma/nontarget tissue = 9.5) at 1 hour postinjection in the s.c. C57BL6/B16F0 mouse model (20). This in vivo melanoma uptake of ^99mTc-1 is comparable with that of the radioiodinated IMBA and BZA molecules, which have 6% to 8% ID/g in the same s.c. mouse model (13).

In vitro cell uptake studies. B16F10 cell uptake studies over a 1-hour incubation period at 37°C (Fig. 3A) show rapid uptake of ^99mTc-1, with a 30% maximum being reached within 20 minutes. Because L-tyrosine is the main substrate in the synthesis of melanin via the oxidative enzyme tyrosinase, melanin synthesis/content is enhanced by growing B16F10 cells in DMEM medium supplemented with additional L-tyrosine (2.0 mmol/L). There is a significant darkening of the cells/medium compared with control cells cultured in DMEM alone. These tyrosine-stimulated cells have a rapid and significantly enhanced cellular accumulation of the ^99mTc complex at 37°C, such that within 5 minutes of incubation they display a 70% uptake of ^99mTc-1, which reaches a maximum of 78% in 60 minutes. To distinguish an active accumulation component from passive diffusion, simultaneous experiments were done at 4°C (Fig. 3B). Under these conditions a significant reduction in cellular uptake is observed in the cells cultured in DMEM alone, with a maximum of only 8% observed over a 60-minute period, indicating that a significant fraction of the cellular uptake of ^99mTc-1 by B16F10 melanoma cells is due to active, temperature-dependent processes. At 4°C, the cellular accumulation in tyrosine-stimulated cells is reduced from a maximum of 78% to 50%; moreover, the uptake is higher than that observed for control cells at 37°C, suggesting that the uptake and accumulation of the ^99mTc-1 complex is related to the melanin synthesis/content of the cells, a phenomenon also observed with the radioiodinated benzamides (10, 22, 27).

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 3. In vitro cell uptake of 99mTc-1 by intact B16F10 cells at 37°C (A) and 4°C (B).

In vivo biodistribution of [^99mTcOAADT]-(CH₂)₂-NEt₂ in B16F10 metastatic melanoma models. In vivo distribution of ^99mTc-1 was examined in the s.c. mouse model bearing metastatic B16F10 tumors. The biodistribution values in Table 1 show high in vivo tumor uptake of ^99mTc-1 at 2 hours after its administration. This tumor uptake is similar to that observed previously (20) and is also comparable with that obtained with the iodobenzamides in the B16F0 tumor model (13). The biodistribution data over an extended 6-hour period also indicate that tumor uptake of ^99mTc-1 remains high whereas a relatively faster washout from normal organs results in increasing tumor/nontumor ratios (Table 1).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Comparison of in vivo tumor accumulation of 99mTc-1 (Tc-1), [18F]FDG (FDG), [18F]DOPA (F-DOPA), and [123I]IMBA (IMBA) in s.c. B16-melanoma tumor model. Tumor uptake expressed as differential absorption ratio (DAR) as reported by Ishiwata et al. (28) for [18F]FDG and [18F]DOPA. Data for IMBA calculated from Eisenhut et al. (13).

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Uptake at 2 hours after administration of 99mTc-1 in the lung of C57BL6 mice bearing metastatic melanoma lesions as a function of tumor growth post i.v. tumor cell inoculation (A) and tumor dose (x 106 B16F10 cells) injected 14 days before 99mTc-1 administration (B).

View larger version (137K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Histology of lung and brain tissues of C57BL6 mice 14 days after i.v. inoculation of B16F10 melanoma cells. A, 2 x 105 B16F10 cells at x10 magnification; arrows, tumors. B, 2 x 105 B16F10 cells at x50 magnification of A. C, 4 x 105 B16F10 cells at x5 magnification. D, 8 x 105 B16F10 cells at x5 magnification. E, 14-day-old tumor brain tissue after i.v. dose of 4 x 105 B16F10 cells at x25 magnification. F, 14-day-old tumor brain tissue after i.v. dose of 4 x 105 B16F10 cells at x150 magnification.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Ex vivo radiophosphor images of 11-, 14-, and 17-day-old tumors.

^99mTc-1, a small, neutral technetium complex, displays high melanoma uptake in both s.c. and pulmonary metastatic melanomas. The use of a more realistic model for evaluating metastatic melanoma-targeting agents shows that ^99mTc-1 not only has high and specific uptake by melanoma metastases in the lungs but also can potentially provide a means to assess noninvasively both temporal growth and extent of metastatic tumor burden in vivo.

	Acknowledgments

 
Grant support: NIH grant R37 CA34970.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Alice D. Carmel for her excellent technical support and Roderick T. Bronson and his staff in the Rodent Histopathology Core, Dana-Farber/Harvard Cancer Center.

Received 10/ 1/03. Revised 2/ 3/05. Accepted 3/16/05.

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