Preparation and evaluation of modified composition for lyophilized kits of [Cu(MIBI)4]BF4 for [99mTc] technetium labeling

NUCLEAR PHARMACY

Preparation and evaluation of modified composition for lyophilized kits of [Cu(MIBI)₄]BF₄ for [^99mTc] technetium labeling

Marisa de Jesus de C. Lima; Fabio Luiz Navarro Marques^*; Miriam Roseli Yoshie Okamoto; Alexandre Tales Garcez; Marcelo Tatit Sapienza; Carlos Alberto. Buchpiguel

Centro de Medicina Nuclear; Departamento de Radiologia; Faculdade de Medicina; Universidade de São Paulo; Trav. R. Dr. Ovídio Pires de Campos s/n; [e-mail]; 054030-010; São Paulo - SP - Brasil

ABSTRACT

The [Cu(MIBI)₄]BF₄ complex was synthesized and different formulations for lyophilized kits that could be cost-effectively used with different routines in nuclear medicine laboratories were investigated. In one preparation the kit components were kept similar to the Cardiolite® , except that the SnCl₂.2H₂O concentration was increased to 0.150 mg. In a second formulation, component concentrations were reduced to 1/5 of the original value and the SnCl₂.2H₂O concentration was adjusted to 0.04 mg. These products were labeled with maximum activities of 55.5 GBq and 8.14 GBq, respectively, and have shown an average radiochemical purity of 95 %. Biodistribution of the products was assessed by dissection in mice and in rabbits, and did not show any statistical difference when compared to Cardiolite®. In the synthesis of [Cu(MIBI)₄]BF₄ a new procedure was introduced for the synthesis of N-(2-methyl-propenyl)-formamide, with the use of microwave radiation as heat source. This modification reduced the reaction time to 25 seconds, while maintaining a yield of 68%.

Key words: [^99mTc]technetium, [[^99mTc](MIBI)₆]⁺, radiopharmaceutical, biodistribution

RESUMO

Neste trabalho são apresentados dados relativos a síntese do complexo [Cu(MIBI)₄]BF₄ e à utilização do mesmo para produção de kit liofilizado para obtenção do radiofármaco [[^99mTc](MIBI)₆]⁺. No processo de síntese foi utilizado radiação de microondas como fonte de aquecimento em uma etapa do processo, permitindo que a reação fosse executada em 25 segundos, obtendo rendimento de 68 %. Na preparação dos kits liofilizados, um deles denominado FULL, nos quais a quantidade dos componentes foi mantida igual ao produto comercial Cardiolite^®, e outro denominado FRACTION, nos quais as quantidades foram reduzidas a 1/5 do total, foram adicionadas quantidades extras de SnCl₂.2H₂O. Deste modo, tornou-se possível marcar os kits liofilizados com atividades máximas de 55,5 GBq e 8,14 GBq, respectivamente. A biodistribuição dos produtos marcados, em camundongos e coelhos, não mostrou diferença estatística (p>0,05), para o teste t de Student, quando comparada ao produto obtido pela reconstituição do Cardiolite^®.

INTRODUCTION

Nuclear Medicine had a great development in the 1980s, when new classes of compounds, such as the tetraamines, aminethiols and isonitriles, were synthesized and evaluated as possible new [^99mTc]technetium radiopharmaceuticals (Jurrisson et al., 1993). In the isonitrile class prepared by Jones (Jones et al., 1984), 1-isociane-2-methoxy-2-methyl-propane, currently named MIBI, exhibited excellent characteristics for use in myocardial perfusion studies (Piwnica-Worms et al., 1989), with a quality superior to that of (²⁰¹Tl) thallium (Taillefer et al., 1997). Also, it was indicated for use in breast cancer detection and parathyroid scanning (Bagni et al., 2003; Geatti et al., 1994). Because of these important applications and the high cost of the commercial product Cardiolite®, several nuclear medicine laboratories have introduced routines to make the product cost-effective, such as labeling the product with higher activities than that established by the manufacturer or using a cold kit fractionation technique, allowing labeling of fractions in different times (Lazarus et al., 1998; Millar, 1999). However, because of the low concentration of stannous ion (Sn²⁺) in the formulation (max 0.075 mg), the use of these procedures can lead to a failure in the labeling process, resulting in a product with low radiochemical purity (Fleming et al., 1992; Decristoforo et al., 1996; Kumar, 1997). An alternative for those procedures is the production of an in-house kit, with kit components being adjusted to the clinical demand according to the number of studies performed per day. A limitation in this case is that MIBI, the ligand to [^99mTc]technetium, is a non-commercial product and needs to be synthesized (Van Wyk et al., 1991; Muradian et al., 1992; Ferro Flores et al., 1994; Lee et al., 1996; Deicas et al., 1997), a task that cannot be easily accomplished in all localities. In this paper a modified synthesis of MIBI is presented, introducing the use of microwave radiation in one step of the synthesis. Two formulations will also be presented, one preparation being named FULL, to be labeled with high activities, and another named FRACTION, to be labeled with low activities. This procedure furnishes a cost-effective product to be used in the routine of nuclear medicine laboratories of various sizes.

MATERIALS AND METHODS

Domestic microwave (Panasonic-model NN 5556B) was used as a heat source in the synthesis step. ¹H-NMR spectra were recorded (Brucker AC-200 system) using TMS as internal reference and the chemical shifts were registered in d (ppm). Lyophilizations was performed in a Lyophilizator Supermodoly (Edwards-BOC). Radioactive samples were measured in a dose calibrator (Victoreen - mod 34-056) and in a well counter (Gamma Nuclear). Animal imaging was acquired in a planar gamma-camera (Siemmens - mod LEM) using a low energy all purpose (LEAP) collimator. The [^99mTc]O₄^- was obtained from ⁹⁹Mo/^99mTc generator (IPEN/CNEN-Brazil). All the chemical products were used under analytical grade and without purification, except POCl₃ that was destillated. Animal experiments were performed in compliance with regulations of this Institution and approved by the Ethical Committee. Statistical analyses were performed using the Student t test with 5 % level of significance.

Synthesis of [Cu(MIBI)₄]BF₄

The synthesis of 1-isociane-2-methoxy-2-methyl-propane (compound 3), as a cooper complex (compound 4), was performed using a previously published synthetic route (Van Wyk et al., 1991) and adaptations published later (Muradian et al., 1992; Deicas et al., 1997), as shown in fig 1. A modification was introduced in the preparation of [2-methyl-propenyl]-formamide (Step 1) as described below.

In a round bottomed pyrex glass flask, fitted with a dry ice condenser, 2-methyl-propenylamine hydrochloride (5.35 g – 50 mmols) and formamide (3.38 g – 75 mmols) were mixed and irradiated at the center of a domestic microwave oven at 240 W for 25 seconds. The flask was cooled in a water bath, 10 mL of CHCl₃ was added, the mixture was filtered, and the solid was washed with 5 mL of CHCl₃. The filtrate was concentrated in a rotary evaporator, the crude product distilled at reduced pressure (0.9 mmHg) and the product collected at 72-6º C, yielding 3.36 g (68.0%). ¹H-NMR (200 MHz, CDCl₃/d pm) 1.72 (s, 3H, CH₃); 3.77 (d, 2H, J_NH 6.05 Hz, CH₂); 4.85 (d, 2H, HC=); 6.94 (br s, 1H, NH); 8.19 (s, lH, CHO).

Kit preparation

L-Cysteine hydrochloride monohydrate, [Cu(MIBI)₄]BF₄, SnCl₂.2H₂O, sodium citrate and mannitol were dissolved in water previously flushed with nitrogen, and the pH was adjusted to 6.0 using NaOH (0.lN) to obtain two types of solution with the final concentration (per mL) presented below. FULL content: 1.0 mg of [Cu(MIBI)₄]BF₄, 0.15 mg of SnCl₂.2H₂O, 1.0 mg of L-cysteine hydrochloride monohydrate, 2.6 mg of sodium citrate and 20.0 mg of mannitol. FRACTION content: 0.2 mg of [Cu(MIBI)₄]BF₄, 0.04 mg of SnCl₂.2H₂O, 0.2 mg of L-cysteine monohydrate hydrochloride, 0.41 mg of sodium citrate and 4.0 mg of mannitol. The solutions were sterilized by membrane filtration (0.22 µm) and lyophilized for 24 hours.

Radiochemical labeling

The lyophilized products were reconstituted using fresh [^99mTc]O₄^- solution from generators eluted previously at 24, 48 or 72 h, using activities below 55.5 GBq, for FULL content, and below 8.14 GBq for FRACTION content. The vials were heated on a boiling water bath during 10 minutes. Cardiolite® was labeled according to the manufacturer’s instructions, except for the activities that reached 18.5 GBq.

Labeling efficiency assessment

Labeling efficiencies (LE) of [[^99mTc](MIBI)₆]⁺ preparations were determined by ascendant chromatography or by solvent extraction. In the former procedure, a drop of labeled product was applied to the TLC-Al₂O₃ Baker-Flex chromatographic strip (7.5x2.0 cm) and the product was developed using ethanol as mobile phase. The strips were dried, cut in three equal parts and the radioactivity was measured in a well counter. The [[^99mTc](MIBI)₆]⁺ was found at Rf = 0.3-0.6. In the latter procedure, a drop of labeled product was added to a test tube containing 3 mL CHCl₃ and 3 mL NaCl 0.9 % solution. The mixture was shaken and 1 mL of each phase was removed and transferred to two new tubes and radioactivity was measured in a dose calibrator (the radiopharmaceutical must be in the CHCl₃ phase).

Animal biodistribution

Quantitative biodistribution studies were performed in Swiss mice (male, seven weeks old, about 45 g). Unanesthetized animals were injected with 0.37 MBq of [[^99mTc](MIBI)₆]⁺ via the tail vein. At 5, 30 and 60 minutes post-injection, the animals were anesthetized with ether and sacrificed by decapitation. The organs of interest (heart, liver, lungs, kidneys and blood) were removed and weighed. The radioactivity was assayed in a well counter and expressed as mean injected dose/gram ± SD (n = 6 for each time point).

Also, dynamic imaging studies were performed in male, three-year-old, New Zeland white rabbits (about 3.5 kg). Immobilized animals were injected with 18.5-37.0 MBq/kg of [[^99mTc](MIBI)₆]⁺ in the marginal ear vein and 56 serials images (30 sec/frame - matrix 256x256) were immediately collected in a planar gamma-camera. A ROI was drawn over the heart, lung and liver and time-activity curves were obtained with background subtraction.

RESULTS

Ligand synthesis

The complex [Cu(MIBI)₄]BF₄ was synthesized using a route established (Van Wyk et al, 1991) with previously described modifications (Muradian et al., 1992; Deicas et al., 1997) in an overall yield of 9.38%. A new approach introduced in the reaction between N-(2-methyl-propenyl)-amine hydrochloride and formamide permitted the attainment of a product in 68% yield, with a maximum reaction time of 25 seconds. Spectral data and physical chemical characteristics of all compounds were in agreement with previously published results.

Kit preparation, labeling and quality control.

The change in kit composition was focused on Sn²⁺ augmentation. Compared to the Cardiolite® kit, the FULL content kit had twice the maximum concentration of Sn²⁺ the reducing agent and the FRACTION content kit had the equivalent of 1.6 times more. These modifications permitted labeling with activities that ranged from 3.7 GBq to 55.5 GBq for FULL content and 1.11 GBq to 8.14 GBq for FRACTION content, giving a labeling efficiency yield, in 40 samples each, of 96.66± 1.48% and 96.45± 1.67%, respectively. The maximum activities were achieved using fresh eluate from 24 h previously eluated generator. The maximum activities for other eluition profile are shown in table 1. Chromatographic and extraction methods for determination of labeling efficiency were compared in 40 samples, obtaining values of 95.46± 1.38% and 96.72± 1.71%, respectively.

Animal biodistribution

Quantitative biodistribution studies performed in mice showed no statistical difference, at the p<0.05 significance level, for the concentration of the three products in the organs of interest (Table 2). Compounds showed fast clearance from the lung and blood, considered background compartments, and concentration in the heart, liver and kidneys, these last two being organs of excretion. The ratio of heart/liver activities (around 1.09% ID/g organ) and heart/lung activities (around 11.07% ID/g organ) at 30 minutes after product administration showed a product with good characteristics for image purposes.

A dynamic gamma-camera imaging study, performed in rabbits, permitted a direct comparison of pharmacokinetics for different products. The time-activity curve (Fig. 2) from the rabbits image showed a fast accumulation of the compounds in heart, liver and kidneys, followed by a moderate flushing from the hepatobiliary system and kidneys and a fast transit of the material through the lungs. After the dynamic study, a static acquisition evaluated the relationship between heart and the background organs, liver and lungs, as expressed in Table 3. The heart/liver ratio of around 1.1 was identical for rabbits and mice. The same was not true for the heart/lung ratio, whose value in mice was around 11.6 and, in rabbits, was 4.1.

DISCUSSION

Although MIBI synthesis is not an original problem, since it has been performed by several routes, our contribution in this work was to introduce the microwave radiation as a heat source, in the reaction between N-(2-methyl-propenyl)-amine hydrochloride and formamide in a system without solvent. This modification reduced the reaction time to 25 seconds, with a yield of 68%, a value similar to those obtained in previous works, where the reaction between N-(2-methyl-propenyl)-amine hydrochloride and formamide was performed in refluxing toluene for three hours (Van Wyk et al., 1991) and in a non-solvent system, heating the sample for 30 minutes in an oil bath (Muradian et al., 1992). Although the results have shown a great advantage in the use of microwave radiation over other heating techniques or reaction conditions, care must be taken when microwave radiation is used in organic synthesis, because several variables need to be considered, for instance, the power of the equipment, the position of the sample inside of the microwave oven, the sample geometry, among others (Stone-Elander and Elander, 1991). Thus, to scale up the synthesis from that established in this work, new reactions conditions need to be found. Other reaction steps were performed according to the previously published procedure (Muradian et al., 1992), except for the copper complex [Cu(MIBI)₄]⁺ that, instead of using Cl^- as the counterion, was obtained as the BF₄^- salt (Deicas et al., 1997). This modification furnished a product with better characteristics and stability.

A critical issue with the Cardiolite^® lyophilized kit is the low Sn²⁺ content (maximum 75 µg and minimum 25 µg) that could permit the labeling of the product using activities below 5.55 GBq, as established by the Cardiolite^® manufacturer, although it is known that activities such as 18.5 GBq could be used with relative security (Hung et al., 1996). However, care needs to be taken since, depending on the generator elution intervals, age of eluted solution and activity used, the Sn²⁺ content might not be sufficient to completely reduce the [^99mTc]O₄^- (Marques et al., 2001), and the Sn²⁺ content may be diminished even more during the cold fractionation technique. Thus, the addition of extra Sn²⁺ in an "in-house" prepared kit, permitted labeling of the products using activities as high as 55.5 GBq with fresh eluate from a generator eluted after 24 hours, and this maximum activity decreased to 33.3 GBq when the generator was eluted after 72 hours. This activity is related to the increase in [⁹⁹Tc] technetium isomer content, which does not contribute with gamma radioactivity, but is chemically equal to [^99mTc]technetium and reacts with kit components. Although the content of the FRACTION product had been reduced five-fold, an additional quantity of Sn²⁺ was necessary to permit the use of an activity equivalent to that used in the FULL product (Table 3). The final concentration of Sn²⁺ in the FRACTION product (40 µg) was similar that presented in the literature for other fractioned preparation procedures (Chowdhury and Hung, 1995). Labeling efficiency evaluated by chromatography and by the extraction procedure confirmed that the latter furnished results similar to the chromatographic method, indication that both could be used in nuclear medicine laboratories. However, when using the extraction procedure, extra care needs to be taken, due the personal and place contamination risk.

Modification of the kit content and use of large amounts of [⁹⁹Tc + ^99mTc]technetium could give a product with different biological characteristics (Ikeda et al, 1977). Thus, the [[^99mTc](MIBI)₆]⁺ complex was assessed by two distinct techniques and animal models, since it is known that biodistribution of the [^99mTc]technetium isonitrile complex can change in different animal species (Kronauge et al., 1992; Barbarics et al., 1994). The assessment of biodistribution in mice permitted absolute quantification of the concentration of the [[^99mTc](MIBI)₆]⁺ in the target organs demonstrating that the products obtained from modified formulations behaved similarly to that obtained from Cardiolite^®. This result was in agreement with results obtained by Barbarics (Barbarics et al, 1994), but with characteristics different from that obtained by Bouquillon (Bouquillon et al., 1995). Imaging assessment permitted comparison of the pharmacokinetic behaviour of the radiopharmaceuticals and quality of diagnostic with these compounds. The heart/liver ratio observed in this work was similar that established in the literature (Barbarics et al., 1994), around 0.9-1.4, but the heart/lung ratio was different. Barbarics et al. (1994) observed a ratio of 1.6 to 1.8, a ratio of approximately 4 was observed in this study. This difference could be related to a lack of background correction in Barbaric's work.

CONCLUSION

The introduction of microwave radiation in MIBI synthesis was an interesting approach since the reaction time in that step could be decreasec drastically. Changes in kit composition introduced in this work did not alter the biological behaviour, when compared with Cardiolite^®, but presented the advantage of being labeled with high activities or of being used as fractionated a preparation.

ACKNOWLEDGEMENTS

The authors would like to thank Profa. Dra. Liliana Marzorati for laboratorial support and to FUNDAP for the fellow to Marisa J.C. Lima.

Received: July 02, 2005;

Revised: July 14, 2005;

Accepted: August 01, 2005

• Bagni, B.; Franceschetto, A.; Casolo, A.; De Santis, M.; Bagni, I.; Pansini, F. and Di Leo, C. (2003), Scintimammography with ^99mTc-MIBI and magnetic resonance imaging in the evaluation of breast cancer. Eur. J. Nucl. Med. Mol. Imag., 30, 1383-1388.
• Barbarics, E.; Kronauge, J. F.; Costello, C. E.; Janoki, G. A.; Holman, B. L.; Davison, A. and Jones, A. G. (1994), In vivo metabolism of the technetium isonitrile complex [Tc(2-ethoxy-2-methyl-1-isocyanopropane)₆]⁺ Nucl. Med. Biol., 21, 583-591.
• Bouquillon, S.; Coulais, Y.; Dartiguenave, M.; Tafani, J. A. M. and Guiraud, R. (1995), Synthesis, characterization and biodistribution of a new technetium-99m complex with trimethylsilylmethy-lisonitrile. Comparison with ^99mTc-TBI and ^99mTc-MIBI. Nucl. Med. Biol., 22, 585-588.
• Chowdhury, S. and Hung, J. C. (1995), Reconstitution and fractionationation of Cardiolite^® lit into multi-dose cold kits for cost saving. J. Nucl. Med., 36, 80.
• Decristoforo, C. and Riccabona, G. (1996), Problems with fractionated cold kits. J. Nucl. Med., 37, 1912-1913.
• Deicas, B.; Gambino, D.; Kremer, C.; Kremer, E.; Mombru, A.; Suescun, L.; Mariezcurrena, R.; Gonzalez, O.; Rey, A.; Mallo, L. and Leon, A. (1997), Synthesis, characterization and spectroscopic properties of [Cu-I(alkylisocyanide)₄]BF₄ complexes. X-ray crystal structures of [Cu(MIBI)₄]BF₄ Polyhedron, 16, 2397-2403.
• Ferro-Flores, G.; Ramirez-Garcia, L.; Lezama, J.; Garcia-Garcia, P. and Tendilla, J. I. (1994), An improved procedure for the synthesis of 2-methoxyisobutylisonitrile - an efficient complexing agent for Tc-99m. J. Radioanal. Nucl. Chem. Letters, 188, 409-415.
• Fleming, W. K.; Jay, M. and Ryo, U. Y. (1992), Reconstitution and fractionation of radiopharmaceuticals kits. J. Nucl. Med., 33, 1915.
• Geatti, O.; Shapiro, B.; Orsolon, P. G.; Proto, G.; Guerra, U. P.; Antonucci, F. and Gasparini, D. (1994), Localization of parathyroid enlargement - experience with ^99mTc-methoxyisobutylisonitrile and ²⁰¹Tl scintigraphy, ultrasonography and computed-tomography. Eur. J. Nucl. Med, 21, 17-22.
• Hung, J. C.; Herold, T. J. and Gibbons, R. J. (1996), Optimal conditions of ^99mTc eluate for the radiolabeling of ^99mTc-sestamibi. Nucl. Med. Biol., 23, 599-603.
• Ikeda, I.; Inoue, O. and Kurata, K. (1977), Preparation of various Tc-99m dimercaptosuccinate complex and their evaluation as radiotracers. J. Nucl. Med, 18, 1222-1229.
• Jones, A. G.; Abrams, M. J.; Davison, A.; Brodack, J. W.; Toothaker, A. K.; Adelstein, S. J. and Kassis, A. I. (1984), Biological studies of a new class of technetium complex: the hexakis(alkylisonitrile) technetium(I) cations. J. Nucl. Med. Biol., 11, 225-34.
• Jurrisson, S.; Berning, D.; Jia, W. and Ma, D. (1993), Coordination compounds in Nuclear Medicine. Chem. Rev., 93, 1137-1156.
• Kronauge, J. F.; Noska, M. A.; Davison, A.; Holman, B. L. and Jones, A. G. (1992), Interspecies variation in biodistribution of technetium(2-carbomethoxy-2-isocyanopropane)₆⁺ J. Nucl. Med., 33, 1357-1365.
• Kumar, V. (1997), Fractionated cold-kits: Address the critical issues to obviate problems. J. Nucl. Med., 38, 1664.
• Lazarus, C.; Palmer, A. M. and Hesslewood, S. R. (1998), Radiopharmaceutical kit modification. Nucl. Med. Comm., 19, 613-614.
• Lee, T. W.; Su, C. S. and Ting, G. (1996), Synthesis, reactivity and ^99mTc labelling of 2-alkoxyisobutylisonitrile. Appl. Radiat. Isot., 47, 207-210.
• Marques, F. L. N.; Okamoto, M. R. Y. and Buchpiguel, C. A. (2001), Technetium-99m generators and radiopharmaceuticals and quality control tests. Radiol. Bras., 34, 233-239.
• Millar, A. M. (1999), Radiopharmaceutical kit modification. Nucl. Med. Comm., 20, 105-106.
• Muradian, J.; Marques, F. L. N.; Rockmann, T. M. B. and Dias Neto, A. L. (1992), Synthesis of the 2-methoxyisobutylisonitrila (MIBI) for medical use. Radiol. Bras., 25, 171-172.
• Piwnica-Worms, D.;. Kronauge, J. F.; Holman, B. L.; Davison, A. and Jones, A. G. (1989), Comparative myocardial uptake characteristics of hexakis (alkylisonitrile) technetium(I) complex. Effect of lipophilicity. Invest. Radiol., 24, 25-29.
• Stone-Elander, S. and Elander, N. (1991), Microwave cavities - some parameters affecting their use in radiolabeling. Appl. Radiat. Isot, 42, 885-887.
• Taillefer, R.; DePuey, E. G.; Udelson, J. E.; Beller, G. A.; Latour, Y. and Reeves, F. (1997), Comparative diagnostic accuracy of Tl-201 and Tc-99m sestamibi SPECT imaging (perfusion and ECG-gated SPECT) in detecting coronary artery disease in women. J. Am. Coll. Cardiol., 29, 69-77.
• Van Wyk, A. J.; Van Aswegen, A.; Knoesen, O.; Fourie, P. J.; Koekemoer, J.; Herbst, C. P.; Otto, A. C. and Lotter, M. G. (1991), Synthesis and ^99mTc labelling of MMI (MIBI) and its ethyl analogue EMI. Appl. Radiat. Isot., 42, 687-689.

*

Author for correspondence

Publication Dates

• Publication in this collection
  09 Dec 2005
• Date of issue
  Oct 2005

History

• Accepted
  14 July 2005
• Received
  02 July 2005