Preparation and Biologic Evaluation of a Novel Radioiodinated Benzylpiperazine, ¹²³I-MEL037, for Malignant Melanoma

General

1-(4-Iodobenzyl)piperazine (2a) was purchased from ACB Blocks Ltd. All other chemicals were purchased from Sigma-Aldrich and used without further purification. ¹²³I was produced by the National Medical Cyclotron (Sydney, Australia), using the ¹²⁴Xe (p, 2n) reaction and was delivered as ¹²³I-NaI in 0.02 mol/L NaOH at a concentration of 37 GBq/mL. ³H-1,3-Di-O-tolylguanidine (³H-DTG) (2.2 TBq/mmol) and ³H-(+)-pentazocine (2.2 TBq/mmol) were purchased from Perkin Elmer Life Sciences Australia. Melting points were determined in open capillary tubes using a Gallenkamp melting point apparatus and are uncorrected. Nuclear magnetic resonance (NMR) spectra were performed on a Bruker Avance DPX 400 operating at 400 MHz for ¹H NMR spectra and 100 MHz for ¹³C NMR spectra. Elemental analysis was done at The Campbell Microanalytical Laboratory (Department of Chemistry, University of Otago, Otago, New Zealand). Low-resolution mass spectrometry (LRMS) was performed on a Micromass ZMD Quadrupole Mass Spectrometer whereas high-resolution mass spectrometry (HRMS) was performed using a Bruker Daltonics BioApex-II 7T FTICR mass spectrometer equipped with an off-axis Analytic electrospray (ES) ionization source. Preparative high-performance liquid chromatography (HPLC) was performed on a Waters Empower system, equipped with a 600 gradient pump and a 486 UV(ultraviolet) detector using a C18 Alltech Econosphere column (10 μm, 22 × 250 mm). After radiolabeling, ¹²³I-MEL037 was purified by an HPLC system equipped with linear variable UV, set at 254 nm, and a γ-detector using a reverse-phase C18 Phenomenex Bondclone column (10 μm, 7.8 × 300 mm) eluting with acetonitrile/0.1 mol/L ammonium acetate (70:30) at 1.5 mL/min. For radiochemical purity and stability measurements, the radioiodinated tracer solution was ascertained on an analytic HPLC system using a C18 Phenomenex Luna column (5 μm, 4.6 × 150 mm) eluting with acetonitrile/0.1 mol/L ammonium acetate (45:55) at 1 mL/min. Measurement of unmetabolized radiotracer was performed by thin-layer chromatography (TLC) analysis on silica gel plates with a preconcentration zone (Merck). The TLC plates were developed with ethyl acetate/methanol/concentrated NH₄OH (85:15:0.2), and the radioactivity distribution was measured using a static radiochromatogram analyzer (Berthold Co).

Chemistry

Lipophilicity

The lipophilicity of MEL037 and its bromo analog was assessed using HPLC by determining the logP_7.5 value using a Waters C18 column (X-Terra; 5 μm, 4.6 × 150 mm) eluting with a mobile phase consisting of methanol and phosphate buffer (0.1 mol/L, pH 7.5), 50:50, at 1 mL/min at pH 7.5 representing physiologic conditions. The logP_7.5 of the studied compounds was estimated by a comparison of its retention time with that of standards of known log P values (18).

Radiolabeling

To a solution of the trimethylstannyl precursor (3) (0.25 mg, 0.5 μmol) in ethanol (200 μL), ¹²³I-NaI (1 GBq, 30 μL), chloramine-T (100 μg, 0.45 μmol in 100 μL H₂O), and HCl (100 μL, 1 mol/L) were successively added. After 5 min at room temperature, Na₂S₂O₅ (5 mg, 30 μmol in 100 μL H₂O), NaHCO₃ (5 mg, 60 μmol in 100 μL H₂O), and 350 μL of HPLC mobile phase were added. The resulting mixture was purified on the semipreparative HPLC system. The radiolabeled compound (retention time = 15 min) was collected and dried in vacuo. The radioiodinated tracer was recovered with 100 μL ethanol and then formulated in 10 mL saline for biologic evaluation.

Ligand-Binding Assays

The percentage inhibition of unlabeled MEL037 to σ₁- and σ₂-receptors was determined in in vitro competitive binding assays at 10⁻⁶ and 10⁻⁵ mol/L concentrations. The assays were performed according to literature methods with minor modification (19). Briefly, the percentage of inhibition was determined by incubating, in triplicate, aliquots of diluted rat brain membrane (300 μg of protein) with a 10⁻⁵ mol/L concentration of MEL037 in 50 mmol/L Tris-HCl (pH 8.0) with ³H-(+)-pentazocine (3 nmol/L) at 37°C for 2.5 h for σ₁-receptors or with ³H-DTG (10 nmol/L) and (+)-pentazocine (1 μmol/L) at 25°C for 1.5 h for σ₂-receptors. In both cases nonspecific binding was determined in the presence of haloperidol (10 μmol/L). After incubation, the reaction was terminated by rapid filtration using a Brandel 48-well cell harvester (Brandel) over Whatman GF/B glass fiber filters that were soaked in a solution of 0.5% polyethyleneimine at 4°C for at least 2 h before use. Filters were washed 3 times with 5 mL of ice-cold wash buffer (50 mmol/L Tris-HCl, pH 7.4). The filters were collected and the amount of bound radioactivity was measured using a β-scintillation counter. The percentage inhibition of unlabeled MEL037, at a 10⁻⁵ mol/L concentration, was also determined for other receptors using specific radioligands and tissue membranes (Novascreen Biosciences) according to literature methods (Table 1). The 50% inhibitory concentration (IC₅₀) values were then converted to apparent K_i (inhibition constant) values using the Cheng–Prussof equation and radioligand K_d (dissociation constant) values (³H-(+)-pentazocine K_d σ₁ = 2.5 nmol/L; ³H-DTG K_d σ₂ = 77 nmol/L).

In Vivo Studies

Animal experiments were performed in compliance with the National Health and Medical Research Council Australian Code of Practice for the care and use of animals for scientific purposes. Female C57BL/6J black and BALB/c nude albino mice (15–18 g) of 5 wk age were obtained from the Animal Resources Centre, Western Australia. B16F0 murine melanoma cells were originally obtained from the European Collection of Cell Cultures and A375 human amelanotic melanoma cells were originally obtained from the American Type Culture Collection. Before transplantation, B16F0 and A375 melanoma cells were maintained as a monolayer in RPMI culture medium supplemented with 10% fetal calf serum and antibiotics and passaged with trypsinization. Early passages were frozen and stored in liquid nitrogen. Cells were passaged to P = 10 and then discarded. Frozen aliquots were grown in a monolayer culture to between 80% and 95% confluence and for transplantation were trypsinized and washed with Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS). For inoculation, B16F0 melanoma cells were resuspended in Ca²⁺- and Mg²⁺-free PBS at 3 or 5 × 10⁶ viable cells per milliliter, and 0.1 mL was subcutaneously injected at the left flank of 6- to 7-wk-old C57BL/6J mice. Eleven days later, tumors could be palpated in >98% of inoculated animals. A375 human melanoma cells were resuspended at 1 × 10⁷ viable cells per milliliter, and 0.1 mL was injected subcutaneously at the left flank of 6-wk-old BALB/c nude mice; 25–26 d later tumors could be palpated in ∼60% of inoculated animals developing tumors.

Biodistribution

Eleven days (B16F0 melanoma) and 25 d (A375 human melanoma) after tumor transplantation, ¹²³I-MEL037 (0.37–0.74 MBq, 100 μL) was injected intravenously into the mice via the tail vein. At defined times after injection (1, 3, 6, 24, and 48 h), groups of mice (n = 5) were sacrificed by CO₂ administration, which was followed by cervical dislocation, weighing, and dissection. Selected organs were weighed, and their radioactivity was measured with a γ-counter (Wallac model 1480). The remaining activity in the carcass was also determined to obtain the total activity in the mouse at a defined time point. The fraction of injected activity (%ID) in the organs was calculated by comparison with suitable dilutions of the injected dose. The radioactivity concentration in the organ and the mean concentration in the mouse (%ID/g) were found by dividing the %ID for each organ by the weight of the organ. A mean contrast ratio in the tumor at time of sacrifice (MCR_t) was calculated for each animal by dividing the tumor concentration by the radioactive concentration in the mouse excluding the tumor according to the following formula: MCR_t = (activity/tissue_g)/(remaining activity at time t/animal weight_g).

Competition Studies

In competition experiments, groups of mice bearing B16F0 murine melanoma (n = 5) were injected intravenously with 1 mg/kg haloperidol 5 min before the radiotracer (0.37–0.74 MBq) dose. The mice were sacrificed 1 or 24 h after injection, and tissues were handled as described for the biodistribution study. Radioactive concentrations in organs and tumor of treated animals were compared with that of control animals. Statistical significance was evaluated using the 1-way ANOVA test. The criteria for significance were P < 0.05 and P < 0.01.

Metabolite Studies

For metabolite studies, animals were injected with 3–5 MBq of ¹²³I-MEL037. Unmetabolized radiotracer in the plasma, brain, and tumors was determined at 0.25, 1, 3, and 24 h after injection by radio-TLC analysis. Samples of the brain and tumor (100–200 mg minced in small pieces) and plasma (0.2 mL) were added to unlabeled MEL037 (20 nmol, 10 μL of 1 mg/mL acetonitrile solution) and acetonitrile (1 mL), exposed for 2 min to an ultrasonic probe designed for cell disruption (Sonicator; Misonix Inc., USA), and then centrifuged. The radioactivity of the precipitate was measured to quantify the acetonitrile extraction efficiency. If necessary, a second extraction with acetonitrile (1 mL) was performed to ensure maximum recovery of the radioactivity. The supernatant was evaporated to dryness, redissolved in 50 μL methanol, and applied to TLC plates.

SPECT

SPECT was performed using a high-resolution γ-camera (X-SPECT; Gamma Medica Inc.), designed for laboratory animals, equipped with an array of discrete 2 × 2 × 6 mm NaI(Tl) crystals optically isolated from each other and a high-resolution, parallel-hole collimator that has a 12.5 × 12.5 cm field of view. The mouse was anesthetized via inhalant isoflurane (Forthane; Abbott) in 200 mL/min oxygen using a nose cone fitted to the animal bed and imaged for 10–40 min at 1, 24, and 48 h after intravenous injection of 7–10 MBq ¹²³I-MEL037.

Chemistry and Radiochemistry

Compound MEL037 was synthesized from commercially available 1 and 2a using EDC and HOBT with NMM in DMF (yield = 43%) (Fig. 2). Similarly, condensation of piperazine 2b with the acid 1 by the same route afforded Br-MEL037 (yield = 69%). Subsequent treatment of the bromo precursor Br-MEL037 with hexamethylditin in the presence of catalytic palladium tetrakistriphenylphosphine in refluxing toluene yielded the stannane 3 (yield = 37%). The log P values of MEL037 and Br-MEL037 were 3.6 and 3.3, respectively. The regioselectivity of the labeling of MEL037 in the 4 position on the benzene ring was achieved by electrophilic substitution of a trimethyltin leaving group using chloramine-T as oxidant. The radiochemical yield of ¹²³I-MEL037 preparation was 65% ± 9% (n = 12). Radiochemical purity as assessed by analytic HPLC was >99% and the specific activity was >2 GBq/nmol based on the limit of detection of the HPLC system. The radiochemical purity was still >98% when ¹²³I-MEL037 was stored at −20°C for 24 h and 97% when kept in 1% ethanol saline solution at room temperature for 4 h.

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FIGURE 2. 

Preparation of MEL037 and radiolabeling of ¹²³I-MEL037. RT = room temperature; CAT = chloramine-T.

Ligand-Binding Assays

The potency of unlabeled MEL037 to inhibit the specific binding of ³H-(+)-pentazocine to σ₁-receptors and of ³H-DTG to σ₂-receptors from rat brain was determined in competitive binding assays at 10⁻⁶ and 10⁻⁵ mol/L concentration of unlabeled compound. In spite of structural similarities with IBP, very low affinities for σ₁- and σ₂-receptors were obtained for MEL037. At 10⁻⁵ and 10⁻⁶ mol/L concentrations, MEL037 was found to inhibit 95% and 54%, respectively, of σ₁-binding of ³H-(+)-pentazocine (K_i σ₁ = 0.5 μmol/L) and at 10⁻⁵ mol/L, 60% of σ₂-binding of ³H-DTG (K_i σ₂ = 10 μmol/L) (Table 1). Similarly, the bromo analog Br-MEL037 did not present any σ-receptor potency. As the studied compound is not an inhibitor for σ-receptors, its potency on other receptor systems was assayed. MEL037 did not display any affinity for other receptors studied.

Biodistribution

To evaluate ¹²³I-MEL037 as a tumor marker, the biodistribution of the radiotracer was studied in black mice bearing the B16F0 murine melanoma tumors and in nude mice bearing the A375 human amelanotic melanoma. Six hours after injection of the radiotracer, the highest uptake (20–30 %ID) was found in the gastrointestinal tract (Table 2). At 24 h after injection, <3% of injected dose was found in the intestine. In the liver, the uptake amounted to 15 %ID that cleared with a biologic half-life of 5 h to reach a value of 0.7–1.3 %ID at 24 h according to the animal species. In kidney, lung, spleen, and heart, the uptake values of ¹²³I-MEL037 were <2 %ID at 1 h and 0.2 %ID at 24 h after injection. In the spleen of C57BL/6J black mice, large variations of the ¹²³I-MEL037 uptake values were observed in groups of animals at each time point, which correlated with the extent of pigmentation in the organ. In the BALB/c nude mice, the uptake values in this organ were homogeneous for each group. The thyroid uptake of the ¹²³I-MEL037 was particularly low and increased slightly during the study from 0.05 %ID at 1 h to ∼0.2 %ID from 6 to 24 h. In C57BL/6J black mice, a significant uptake of ¹²³I-MEL037 was observed in the eyes (2.3 %ID). In BALB/c nude mice, the eye uptake was 100 times lower than that in black mice. As a consequence of the simultaneous low organ uptake and rapid clearance, the amount of radioactivity that remained in the black and nude mouse body at 24 and 48 h after injection was less than 5 and 2 %ID, respectively.

The values of tumor and organ concentration, expressed in %ID/g, are summarized in Table 3. In the B16 melanoma tumor, ¹²³I-MEL037 exhibited a high uptake at 1 h (13 %ID/g) that increased with time and reached 25 %ID/g at 6 h. The tumor uptake was still 10 %ID/g at 24 h. In the A375 amelanotic tumor, the uptake was very low, decreasing from 1.8 to 0.4 %ID/g between 1 and 24 h after injection. For each of the 2 mice species, the uptake values in heart, lung, and kidney ranged from 3 to 9 %ID/g at 1 h after injection to values <1 %ID/g at 24 h. In the brain, at 1 and 24 h, the uptake values of ¹²³I-MEL037 were 2 and 0.2 %ID/g, respectively. The tumor-to-body contrast of ¹²³I-MEL037 was ascertained by calculating the MCR_t. These values are reported in Table 3. Because of the reduction of the radioactive concentration in the body and the slower decrease in target tissues such as tumor and the eyes, MCR₂₄ was 35 and MCR₄₈ was 60 in black mice. The corresponding MCR₂₄ was <1 in nude mice.

Competition Studies

The blocking effect of haloperidol on tracer uptake in vivo was examined over 24 h in selected organs and tumor tissue to assess the uptake mechanism in the B16 tumor. As seen in Table 4, at 1 h after injection, the radioactive concentration in the tumor was significantly reduced in treated animals compared with that in control animals (−23%; P < 0.05). Simultaneously, significant increases of 10%–15% in lung, heart, blood, and brain uptakes were observed. At 24 h after injection, significant decreases in uptake values were observed in the tumor (−50%; P < 0.01), thyroid (−40%; P < 0.05), eyes, and liver (−16%, P < 0.05). At this time, the uptake in lung, heart, blood, and brain was found to be unchanged.

Metabolite Studies

After ultrasonic disruption and protein elimination, >96% of the radioactivity in the brain and plasma was recovered in acetonitrile. In the tumor, the recovery was 85%, 84%, and 75% at 15 min, 1 h, and 3 h, respectively. In plasma, at 1 h after injection, 60% of the radioactivity was related to unmetabolized ¹²³I-MEL037. The remaining 40% of the radioactivity represented polar radioactive metabolites. In the brain and tumor at 3 h, the metabolite study indicated that >90% of the recovered radioactivity represented unmetabolized ¹²³I-MEL037 (Fig. 3). Metabolite measurements were unreliable at 24 h because of the very low activity present in the brain and plasma, whereas in the tumor only a recovery of 37% recovery was possible.

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FIGURE 3. 

Fraction of unmetabolized ¹²³I-MEL037 in plasma, brain, and tumor at 0.25, 1, and 3 h after injection.

SPECT Studies

The whole-body distribution of ¹²³I-MEL037 in mice bearing the B16F0 melanotic and the A375 amelanotic melanoma was monitored over 48 h using SPECT. Figure 4 shows typical SPECT images of mice acquired 24 h after the radiotracer injection. In the image of the mouse bearing the B16F0 melanoma tumor, the highest radioactivity concentration was observed in the tumor. Lower concentrations of radioactivity were seen in the eyes and in the abdomen. No tumor or eye uptake was observed in the mouse bearing A375 amelanotic melanoma tumors. These observations confirmed the quantitative results obtained in the biodistribution studies.

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FIGURE 4. 

SPECT planar images obtained 24 h after injection of 10 MBq ¹²³I-MEL037 in C57BL/6J mouse bearing B16F0 murine melanoma tumor and in BALB/c nude mouse bearing A375 human amelanotic melanoma tumor. Scale indicates the highest value in counts/pixel in image.