Bone metastases that can cause extreme pain, fractures, neurological complications, and hypercalcemia are a serious complication of many oncological diseases. Relief of pain from metastasis to the skeleton is one of the most important challenges of clinical medicine. Available methods for palliative therapy of bone pain include the use of analgesics (including narcotics), bisphosphonates, chemotherapy, and external beam therapy [1]. However, radionuclide therapy (RNT) with osteotropic radiopharmaceuticals (RPs) is most interesting because they are tolerated well and highly efficacious with simultaneous systemic action at all metastatic sites [2, 3].

Osteotropic RPs can be divided into two groups, i.e., calcimimetics and phosphonates. Calcimimetics include 32P, 89Sr, and 223Ra, which are calcium analogs so that their in vivo distribution can be extremely unpredictable [3]. Phosphonates are enzyme-resistant analogs of natural pyrophosphate, possess high affinity for hydroxyapatite of bone tissue, and are widely used to design osteotropic RPs, e.g., 153Sm lexidronam (153Sm-EDTMP, Quadramet®), Samarium 153Sm oxabiphor, etc. [4, 5].

The main problem with development of effective medicines for palliative therapy of bone pain due to metastasis of a primary tumor to the skeleton is the need to ensure delivery of an adequate dose of ionizing radiation to sites of bone metastases with minimal radiation-induced damage to bone marrow. Therefore, the choice of radionuclide with the optimal nuclear-physical properties is extremely important.

Lutetium-177 (177Lu) has great potential for therapeutic applications, as has been demonstrated in many works dedicated to targeted RNT of metastatic castrate-resistant prostate cancer and neuroendocrine tumors [6,7,8,9]. The possibility of using 177Lu in practically all compound classes used for RNT is currently being studied [10]. It was proposed that 177Lu could become a key therapeutic radionuclide for targeted RNT [11]. Owing to the optimal decay characteristics of 177Lu [T1/2 = 6.65 d; Eβmax = 498 keV (78.6%), 384 keV (9.1%), and 176 keV (12.2%); Eγ = 113 keV (6.6%), 208 keV (11%); and moderate penetration of soft tissues of ~0.6 mm] and the ability to produce it in mediumand high-flux research reactors, the number of which in the world is rather large, 177Lu can be considered a promising radionuclide for palliative therapy of bone metastases [10, 12].

N,N,N′,N′-Ethylenediaminetetrakis(methylenephosphonic acid) (EDTMP) is one of the most frequently used ligands because it can form stable complexes with various radiometals (153Sm, 177Lu, 166Ho, 68Ga, etc.) [13,14,15]. This work was aimed at studying the in vivo biodistribution of the novel osteotropic compound 177Lu-EDTMP in intact laboratory animals.

Experimental Chemical Part

Preparation and quality control of 177Lu-EDTMP. Radioactive 177LuCl3 was obtained from State Scientific Center, Research Institute or Atomic Reactors (SSC RIAR, Dimitrovgrad, Russia). Other reagents [NaOH, HCl, NaOAc, NH4OAc, N,N,N′,N′-ethylenediaminetetrakis(methylenephosphonic acid)] were purchased (Sigma-Aldrich, Germany).

The labeled RP was prepared by placing EDTMP (25 mg) into a 10-mL vial, adding NaOH solution (1 mL, 0.1 M), and stirring until the solid was completely dissolved. Then, the vial with the EDTMP solution was treated with sodium-acetate buffer (1.5 mL, 0.4 M, pH 4.6), stirred for 5 min, and treated with 177LuCl3 (37 MBq, 1.0 mCi) in HCl (0.2 mL, 0.1 M). The reaction mixture was held at 95°C for 30 min, cooled to room temperature, treated with deionized H2O (1.0 mL), and filtered through a 0.22-μm syringe filter. 177Lu bound to EDTMP and free 177Lu (not bound to EDTMP) were quantitatively determined by paper chromatography on Whatman-1 paper (Sigma-Aldrich, USA). The mobile phase was NH4OAc solution (0.1 M). 177Lu-EDTMP migrated to the solvent front (Rf = 0.85 – 0.95) while free 177Lu remained at the origin (Rf = 0) upon elution by the mobile phase. Hydrolyzed unbound 177Lu and bound 177Lu were quantitatively determined by radiometric counting of the bands on the chromatography paper. The radiometry used a Wizard 2480 automated gamma-counter (PerkinElmer/Wallac, Finland).

The obtained RP was intended for intravenous injections. Radiochemical impurities in the 177Lu-EDTMP RP were ≤3.0%. The pH was 4.5.

Experimental Biological Part

Pharmacokinetic studies used 177Lu-EDTMP and 177LuCl3 . Solutions of 177Lu-EDTMP and 177LuCl3 were prepared immediately before biological tests.

The pharmacokinetics of 177Lu-EDTMP were studied in intact female Wistar rats (160 ± 40 g). A total of 20 animals were used. They were injected intravenously (i.v.) (into a tail vein) with 177Lu-EDTMP (0.1 mL, 0.37 MBq, 4.81 mg/kg of 177Lu-EDTMP or 0.77 mg/rat).

The distribution of free Lu as 177LuCl3 in intact rats (20 females) was also studied to evaluate the in vivo stability of 177Lu-EDTMP. They were injected i.v. with 177LuCl3 (0.1 mL, 0.37 MBq, 0.37 MBq/rat). The animals were isolated after injection of the radioactive compounds in a room specially designed for working with RPs. All manipulations with experimental animals were conducted in compliance with a handbook [16].

Animals were euthanized 5 min and 1, 3, 24, and 48 h after injection (four animals at each time point) by decapitation. Internal organs and tissues were collected. The obtained samples were placed into plastic tubes, weighed on a Sartorius electronic balance (Germany), and assayed for radioactivity. Samples (0.1 mL) of 177Lu-EDTMP and 177LuCl3 were placed into separate tubes at the time of injection for use as standards of the injected dose.

The content of 177Lu-EDTMP or 177LuCl3 per gram of organ or tissue was radiometrically assayed in percent of the injected amount (%/g) at each time point. The total content of 177Lu-EDTMP and 177LuCl3 in the skeleton was calculated considering that the rat skeleton mass was 10% of the body mass [17]. The coefficients of differential accumulation (CDA) were also calculated as the ratio of the 177Lu-EDTMP or 177LuCl3 concentration in bone to that in other organs and tissues.

The radiometric results were statistically processed using Microsoft Excel 2010 with calculation of the arithmetic means (M) and standard errors of the mean (m). Concentrations of 177Lu-EDTMP and 177LuCl3 in groups were compared using the Student t-criterion. Differences were considered statistically significant for p < 0.05.

Results and Discussion

Figure 1 shows results for binding of 177Lu to EDTMP. The binding of 177Lu to the ligand was observed to be 98% after heating of the reaction mixture was finished. This value practically did not change for 72 h. These data were indicative of a highly stabile RP. Radiochemical impurities in the RP solution were ≤3.0% after 72 h.

Fig. 1
figure 1

Radiochemical yield of 177Lu-EDTMP.

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The analytical results for the biodistribution found that the 177Lu-EDTMP concentration was highest in bone over the whole study time (Table 1). For example, 177Lu-EDTMP accumulated in knee joint from 1.683 ± 0.151 %/g to 4.793 ± 0.223 %/g. The 177Lu-EDTMP concentration in femur varied from 1.590 ± 0.342 %/g to 3.758 ± 0.323 %/g. The RP accumulated in other bones to ~1.5 – 2 times less than in femur. The maximum concentrations of 177Lu-EDTMP in cranium, rib, and spine reached 1.561 ± 0.286 %/g, 1.800 ± 0.477 %/g, and 1.599 ± 0.345 %/g, respectively (Table 1). It is noteworthy that the maximum concentration of 177Lu-EDTMP in all studied bones occurred 24 h after i.v. injection of the RP (Table 1).

Table 1 Concentration of 177Lu-EDTMP and 177LuCl3 in Organs and Tissues of Intact Wistar Rats After Intravenous Injection (% of Injected Dose per Gram of Organ or Tissue)
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The distribution of 177LuCl3 in bones was practically the same as that of 177Lu-EDTMP. The concentration of 177Lu-EDTMP in bones except for spine was statistically significantly greater than 177LuCl3 only at the initial times after injection (5 min and 1 h) (Table 1).

177Lu-EDTMP was the first osteotropic RP with 177Lu to be studied [18]. The radioactive label was introduced by heating 177LuCl3 with EDTMP in a boiling-water bath for 30 min. 177Lu-EDTMP accumulated primarily in the skeleton (maximum content in femur of intact rats reached 7.5%/g in 24 h) [18].

Accumulation of high levels of 177Lu-EDTMP in bone was also reported in other studies [13, 14, 19,20,21]. 177Lu-EDTMP was prepared at room temperature in >98% radiochemical yield in one study [14]. Rapid accumulation in the skeleton, rapid elimination from blood, and minimal accumulation in internal organs were demonstrated during biodistribution studies of 177Lu-EDTMP in Wistar rats. The RP concentration in femur was 1.74 ± 0.30%/g in 3 h and reached a maximum (2.05 ± 0.48%/g) 24 h after injection [14]. It is worth noting that the time to reach the peak concentration of 177Lu-EDTMP in bone (24 h) was analogous in the present work.

The biodistributions of 177Lu-EDTMP and 177LuCl3 in wild-type rats were comparatively analyzed [20]. 177Lu-EDTMP rapidly accumulated in bone (~2%/g 4 h after injection), remaining practically unchanged for the next 24 h. Then, the concentration of 177Lu-EDTMP in bone gradually increased (to 7%/g 7 d after injection). In turn, 177LuCl3 also accumulated in bone, practically not differing from 177Lu-EDTMP. However, high concentrations of 177LuCl3 were observed in liver (up to 3%/g), spleen, intestines, and muscle (up to 1%/g) [20].

The total content of 177Lu-EDTMP in skeleton already 5 min after injection was 22.67 ± 3.74% of the injected dose (Fig. 2). The content of 177Lu-EDTMP in skeleton doubled after 1 h to 47.13 ± 3.11% of the injected dose and remained at that level (46.15 ± 2.37% of the injected dose) up to 3 h. The maximum content of the RP in skeleton occurred 24 h after injection and was 54.89 ± 4.88% of the injected dose. The total content of 177Lu-EDTMP in skeleton 48 h after injection decreased to 26.48 ± 0.89% of the injected dose. The total content of 177LuCl3 in skeleton was statistically significantly less than that of 177Lu-EDTMP only 5 min and 1 h after injection while its distribution was practically the same as 177Lu-EDTMP upon further accumulation (Fig. 2). The maximum content of 177LuCl3 in skeleton reached 63.80 ± 2.87% of the injected dose 24 h after i.v. injection.

Fig. 2
figure 2

Total content of 177Lu-EDTMP and 177LuCl3 in skeleton of intact Wistar rats after intravenous injection (% of injected dose); * statistically significant differences between groups (p < 0.05).

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Similar contents of 177Lu-EDTMP in skeleton were found before [22]. The studies used intact Wistar rats. The accumulation of 177Lu-EDTMP in skeleton after 30 min was already 40.48 ± 7.48%. The total RP content in skeleton after 3 h increased to 43.50 ± 4.25%. The maximum amount of the injected dose (46.25 ± 3.48%) was noted after 24 h [22].

The biodistribution of 177Lu-EDTMP prepared from a kit of reagents identical to those used to prepare 153Sm-EDTMP (Quadramet®) was studied [21]. Each vial contained a lyophilized mixture of EDTMP (35 mg), NaOH (14.1 mg), and CaCO3 (5.8 mg). The studies used Wistar rats. Significant accumulation of 177Lu-EDTMP by bone (56.68 – 66.35% of the injected dose) was observed during 7 d after injection [21]. Moreover, accumulation of 177Lu-EDTMP was found to be greater than that of 153Sm-EDTMP, 90Y-EDTMP, and 166Ho-EDTMP [13].

The concentration of 177Lu-EDTMP in internal organs and tissues was statistically significantly less than that of 177LuCl3 . The maximum content of 177Lu-EDTMP in blood was 0.283 ± 0.043%/g 5 min after injection, decreasing toward the end of the study to 0.001 ± 0.001%/g. The 177LuCl3 blood concentration was 5.5 – 59 times greater than that of 177Lu-EDTMP and varied from 0.011 ± 0.003%/g to 1.882 ± 0.340%/g (Table 1).

Kidneys had the highest concentration of 177Lu-EDTMP of all organs. This was due to renal excretion of labeled phosphonates, which has been reported in many works [15, 23, 24]. The 177Lu-EDTMP content in kidneys during 48 h decreased from 0.906 ± 0.148%/g to 0.109 ± 0.026%/g (Table 1). Conversely, the 177LuCl3 concentration in kidneys increased to the maximum of 0.751 ± 0.063%/g 3 h after injection, after which it decreased (Table 1).

Thyroid concentrations of 177Lu-EDTMP were also characterized by lower values than 177LuCl3 . However, statistically significant differences were noted only at individual time points (5 min and 24 h) (Table 1).

The 177LuCl3 content in other internal organs was significantly greater than that of 177Lu-EDTMP. This agreed with results from studies comparing the biodistributions of 177Lu-EDTMP and 177LuCl3 [13, 20].

An analysis of the femur/internal-organs CDA data found that the 177Lu-EDTMP concentration in femur was much greater than in other organs and tissues over the whole study time. This was consistent with the high numerical values of the CDA (Table 2). The maximum CDA values were recorded toward the end of the study (24 – 48 h after injection of 177Lu-EDTMP).

Table 2 Coefficients of Differential Accumulation of 177Lu-EDTMP and 177LuCl3 in Femur of Intact Wistar Rats After Intravenous Injection
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The femur/internal-organs values for 177LuCl3 were also greater than unity at practically all time points except for the femur/blood and femur/lungs ratios at 5 min after injection (Table 2). However, the femur/internal-organs CDA values for 177LuCl3 were statistically significantly less than the corresponding CDA values for 177Lu-EDTMP

Thus, 177Lu-EDTMP demonstrated high affinity for bone tissue with minimal accumulation in internal organs. This was confirmed by the high femur/internal-organs CDA values at all time points. The 177LuCl3 content in bone tissue was practically the same as that of 177Lu-EDTMP although the 177LuCl3 concentration in internal organs was significantly greater than that of 177Lu-EDTMP.