A. Production and quality of ¹⁵³SmCl₃, ¹⁷⁷LuCl₃ and ¹⁶⁶LuCl₃ solutions

¹⁵³Sm, ¹⁷⁷Lu and ¹⁶⁶Ho were produced by neutron irradiation of 100 μg of the ¹⁵²Sm₂O₃, Lu₂O₃ and ¹⁶⁵Ho₂O₃ targets according to reported procedure [14] at a thermal neutron flux of 4×10¹³ n/(cm² s) for 5 days. The irradiated targets were dissolved in 200 μL of 1.0 mol/L HCl and diluted to the appropriate volume with ultra-pure water, to produce a stock solution. The mixture was filtered through a 0.22 μm biological filter and sent for use in the radiolabeling step. Radionuclidic purity of the solution was checked using beta and HPGe spectroscopy to detect various interfering beta- and gamma-emitting radionuclides. The radiochemical purity was checked by instant thin layer chromatography (ITLC) method.

B. Preparation of radio-labeled BLM

Radiolabeling of BLM using cation solution was performed according to the previously reported literature [10]. A stock solution was prepared by dissolving 1 mg of BLM in 1 ml normal saline. 100 μL of the stock solution was added to 100–200 MBq of the radionuclide solution. The mixture was maintained for 120 min at room temperature and the radiochemical purity was checked by ITLC method on Whatman No. 3 paper with 10 mmol L^-1 DTPA solution as mobile phase every 15 min. HPLC of the final preparation was also performed on the reverse stationary phase using a mixture of water:acetonitrile 1:1 (V/V) as the eluent (flow rate: 1 mL/min). The final solution was then passed through a 0.22 μm filter and pH was adjusted to 5.5 by the addition of 1 mol/L sodium acetate buffer.

A. Radionuclide production

Radionuclidic purity of the solutions was checked by counting the samples on an HPGe detector for 5 h. All of the radionuclides were prepared with the radionuclidic purity of over 99.98%. The radiochemical purity was checked by the two solvent systems. In 10 mmol L^-1 DTPA aqueous solution (Solvent 1), free cation was complexed to more lipophilic LuDTPA form and migrated to higher R_f. On the other hand, in 10% ammonium acetate: methanol mixture (1: 1) (Solvent 2), free cation remains at the origin, while other ionic species would migrate to higher R_f.

B. Preparation of radio-labeled BLM

In order to achieve maximum complexation yield, various parameters such as the ligand concentration, pH of the reaction mixture and incubation time were optimized. A satisfactory labeling yield of 94%–97% was obtained at room temperature using 0.15–0.3 mg of BLM. The best pH for the labeling step was 5.5–7.

The ratio of the peaks of the radiolabeled-BLM complexes at R_f =0.1 to free cation radiopeak (R_f=0.8) was considered as the radiochemical yield using 10 mmol L^-1 DTPA solution (Fig. 2). HPLC analysis showed that the fast eluting compound was hydrophilic free cation (2.9 min), while the radiolabeled-BLM was eluted after 15.51 min (Fig. 3).

Fig. 2.Full-screen View

(Color online) Radio chromatogram of free cation (a) and radiolabeled-BLM (b) in 10 mmol/L DTPA solution (pH 3) in optimum conditions.

Fig. 3.Full-screen View

(Color online) HPLC chromatogram for free cation (a), and radiolabeled-BLM (b).

C. Stability studies

Stability of the complexes prepared under optimized reaction conditions were studied at room temperature and in presence of human serum at 37 ℃. It was observed that the complexes showed excellent stability both at room temperature and in presence of human serum at 37 ℃ even after 48 h.

D. Biodistribution studies in wild-type rats

The animals were sacrificed by CO₂ asphyxiation at selected hours after injection. Dissection began by drawing blood from the aorta, followed by collecting heart, spleen, muscle, brain, bone, kidneys, liver, intestine, stomach, lung and skin samples. Tissue uptakes of the complexes were calculated as the percentage of area under the curve of the related photo peak per gram of tissue (% ID/g). The non-decay corrected clearance curves for the main organ sources of the rats are shown in Fig. 4.

Fig. 4.Full-screen View

(Color online) The clearance curves for ¹⁷⁷Lu-BLM (a), ¹⁵³Sm-BLM (b) and ¹⁶⁶Ho-BLM from each organ of the rats.

The liver and kidney were the major accumulation sites of the radiolabeled BLMs. Most of the injected dose was excreted from kidneys, which is similar to the free BLM biokinetic [19]. Significant accumulation was observed in the lung, too, due to the reported side effects for this antibiotic [20, 21]. Slight bone uptake can be attributed to the small amounts of free cation in the injected sample or complex dissociation.

For comparing behaviors of the radiolabeled BLMs, tissue uptakes in the bone, kidney, liver and lung in 2–48 h after injection were studied. As demonstrated in Fig. 5, biokinetics of the radiolabeled BLMs in bone and lung follow a similar pattern. However, accumulation of ¹⁵³Sm-BLM in lung is significantly low and can be disregarded. The biokinetic pattern of the radiolabeled BLMs in kidney differ from that of the radiolabeled BLMs in liver. For ¹⁶⁶Ho-BLM, the renal excretion reaches its maximum at 48 h, accumulation of ¹⁵³Sm-BLM and ¹⁷⁷Lu-BLM in kidney decreases with time. Liver uptake for ¹⁵³Sm-BLM is considerably higher than the other complexes (Fig. 5).

Fig. 5.Full-screen View

Activities of ¹⁶⁶Ho-BLM, ¹⁵³Sm-BLM and ¹⁷⁷Lu-BLM in bone, lung, kidney and liver of wild-type rats.

Since BLMs are tumour-seeking antibiotics, biodistribution studies of the radiolabeled BLMs are suggested in tumour bearing rodents. In this study, due to the inaccessibility to the large number of tumour bearing rats, biodistribution of the radiolabeled BLMs are studied in normal rodents.

E. Dosimetric studies

Biological study is necessary to investigate effect of radiopharmaceutical in treatment of the disease, but due to the direct relationship between absorbed dose and response in terms of cell killing/survival, calculation of the radiation absorbed dose is an important parameter to be considered [22]. Therefore, absorbed dose of the radiolabeled BLMs for each human organ was calculated by RADAR method based on biodistribution data in rat organs (Table 1). Calculation of the absorbed dose in human organs from biodistribution in small animals can be useful for determining the injected activity and is a common first step, consistent with the recommendations of ICRP 62 [23].

As indicated in Table 1, most of the organs receive the minimum absorbed dose after injection of ¹⁷⁷Lu-BLM, while the maximum absorbed dose was observed after injection of ¹⁶⁶Ho-BLM. In addition to the different biodistribution of the radiolabeled BLMs, the difference in absorbed dose is attributed to the decay characteristics of the radionuclides. ¹⁶⁶Ho (E_{β max} = 1.84 MeV, T_1/2 = 26.8 h), ¹⁵³Sm (E_{β max} = 0.81 MeV, T_1/2 = 46.3 h) and ¹⁷⁷Lu (E_{β max} = 0.497 MeV, T_1/2 = 161.52 h) are interesting radionuclides for targeted therapy modalities for treatment of the large, medium and small sized tumors, respectively. Therefore, the higher amount of the absorbed dose of ¹⁶⁶Ho-BLM can be considered as a result of the high energy beta particles.

Generally, the organs with the maximum absorbed dose after injection of the radiolabeled BLMs are the kidney, liver, lung, red marrow and bones. In the case of ¹⁶⁶Ho-BLM, the kidneys and lungs are the critical organs which can restrict the injected activity. However, for ¹⁷⁷Lu-BLM and ¹⁵³Sm-BLM, liver receive higher absorbed dose than lungs and it can be considered as a critical organ. Furthermore, for all the radiolabeled BLMs, due to the slight bone uptake, significant absorbed dose was observed in red marrow. However, with respect to the high energy beta particles emitted from ¹⁶⁶Ho, red marrow absorbed dose is considerably higher (0.362 versus 0.208 and 0.143 mSv/MBq for ¹⁵³Sm-BLM and ¹⁷⁷Lu-BLM, respectively). These calculated radiation absorbed dose can be used for the treatment planning of radiotherapy with the radiolabeled BLMs as a preliminary dose data.