Absorption, distribution and excretion of intravenously injected ⁶⁸Ge/⁶⁸Ga generator eluate in healthy rats, and estimation of human radiation dosimetry

The latest research has produced a series of new compounds labeled with ⁶⁸Ga, which is a positron-emitting metal especially suitable for labeling of peptides and PET. In this study, we investigated the absorption, distribution, and excretion of ⁶⁸Ga/⁶⁸Ge radioactivity after a single intravenous injection of ⁶⁸Ge/⁶⁸Ga generator eluate in female and male rats over a period of 3 h.

The observed differences between the female and male biodistribution data are probably, at least in part, due to the small number of animals. However, higher concentrations of some elements, including iron, have been observed in the organs of female rats than in male rats, and since Ga³⁺ is transported into tissues in a similar way as Fe³⁺, this may explain part of the gender difference observed in this study [5]. This, in addition to higher injected radioactivity dose per gram, might in part explain higher plasma values of female rats. Interestingly, the estimated human radiation dose was higher when using the female rat data than when using the male rat data. Possible explanations for this include the small sample size and/or actual gender dependent differences in handling certain elements [5]. The body weight of female rats was approximately 100 g lower than that of male rats. However, both genders received an identical 50-MBq dose of ⁶⁸Ga eluate. This may also be one source of difference.

When the studies started, the ⁶⁸Ge/⁶⁸Ga generator was already used for 7 month; the studies lasted up to 5 month. According to manufacturer’s metals screen by inductively coupled plasma mass spectrometry (ICP-MS), the ⁶⁸Ge/⁶⁸Ga generator (no. 1484-7, with 1850-MBq nominal radioactivity) had antimony 0.004 ppm, boron 0.17 ppm, sodium 0.56 ppm, titanium 0.069 ppm, and zinc 0.006 ppm, which reflects the typical metal impurities in the eluate. In the respective Ph. Eur. monograph, only zinc and iron are mentioned as metal impurities, because they might interfere as 3+ metals with the Ga-68 during the labeling. The limit of zinc and iron is 10 μg/GBq. The generator has a zinc level of 0.02 μg/GBq, which is well below the limit, and iron was not detected by ICP-MS by a detection limit of 0.006 ppm, which also correlates to a value way below the limit. The ⁶⁸Ge breakthrough was 0.000017 % correlated to the ⁶⁸Ga radioactivity of the eluate at the reference date. This value is well below the limit of ⁶⁸Ge-breakthrough mentioned in the respective Ph. Eur. monograph. Thus, it was estimated that these levels of metal impurities and ⁶⁸Ge breakthrough had no effect on the results. However, the chemical form of ⁶⁸Ga used in this study (⁶⁸Ga eluate was mixed with a phosphate-buffered saline) might be different from ⁶⁸Ga-species present as by-products in ⁶⁸Ga-labeled radiopharmaceuticals. In radiolabeling peptides, ⁶⁸Ga is reacted with chelate-conjugated peptides at elevated temperature, which should accelerate the hydrolysis reaction of Ga that does not form complex with peptide-chelate conjugate.

The whole-body distribution of ⁶⁸Ga radioactivity reported here is in line with previous publications. Velikyan and co-workers studied biodistribution of ⁶⁸GaCl₃ in healthy Sprague-Dawley rats in order to control the organs where the accumulation would occur in case of impure tracer or in vivo release of ⁶⁸Ga from the tracer (⁶⁸Ga-DOTATOC and ⁶⁸Ga-DOTATATE). The ⁶⁸GaCl₃ was acetate buffered to pH of 4.6 and formulated with phosphate-buffered saline (pH 7.4) for i.v. injection. The ⁶⁸Ga radioactivity concentration at 75-min post-injection was the highest in the blood, and the accumulation in the heart, lung, liver, and spleen was considerably higher as compared to that of peptide tracers [6]. Previously, we i.v. injected NaOH neutralized ⁶⁸Ga-chloride (12 MBq from Cyclotron Co. ⁶⁸Ge/⁶⁸Ga generator, Obninsk, Russia) in anesthetized, athymic, male Hsd/RH-rnu/rnu rats having subcutaneous tumor xenografts and reported the following SUVs at 90 min after injection (the values at 120 min of the present report are given in the parentheses): blood 2.7 ± 0.3 (3.074 ± 0.337), liver 5.9 ± 3.3 (2.280 ± 0.918), lung 1.9 ± 0.8 (1.445 ± 0.195), muscle 0.2 ± 0.03 (0.478 ± 0.067), and skin 0.5 ± 0.1 (0.662 ± 0.233) [7]. Subsequently, we also studied distribution of ⁶⁸GaCl₃ (Cyclotron Co., Obninsk, Russia) in healthy C57BL/6 N mice. The ⁶⁸GaCl₃ was neutralized with 1 mol/l sodium hydroxide to pH of 7, and the final product contained 13 % of colloidal forms of ⁶⁸Ga as determined by ultrafiltration. Still, the biodistribution of ⁶⁸Ga radioactivity was quite similar to the present study. The highest level of ⁶⁸Ga radioactivity at 3-h post-injection was found in the blood and liver followed by spleen, kidneys, bone with bone marrow, and lung, respectively [8]. Nanni and co-workers have studied ⁶⁸Ga-citrate in patients with infectious diseases [9]. Since citrate is only a weak chelator of ⁶⁸Ga, the radionuclide is rapidly released in vivo and subsequently binds to transferrin and some other plasma proteins. The biodistribution of ⁶⁸Ga-citrate may actually resemble that of free ⁶⁸Ga or the eluate of the ⁶⁸Ge/⁶⁸Ga generator. In clinical whole-body PET scanning, ⁶⁸Ga-citrate showed relatively high vascular radioactivity, moderate hepatic uptake, mild bone marrow radioactivity, and no bowel radioactivity. The relatively high vascular radioactivity, which is not seen in ⁶⁷Ga-citrate scintigraphy, was a particularly interesting finding. In the current rat study, the elimination of radioactivity in urine and feces at each time point and the overall mass balance as a percentage of administered radioactivities could not be determined since urine or feces were sampled, not collected in their entirety. The observed high plasma values in rats supports that the ⁶⁸Ga radioactivity is bound to transferrin.

⁶⁸Ga has been used extensively for the labeling of synthetic peptides. However, there are only few human dosimetry reports available, including, for example, those on peptide analogues that bind to somatostatin receptors (Table 7) [10‐15]. The effective dose of the ⁶⁸Ge/⁶⁸Ga generator eluate reported here is somewhat higher than that of ⁶⁸Ga-DOTANOC and ⁶⁸Ga-DOTATOC [10, 11]. The higher dose of the ⁶⁸Ge/⁶⁸Ga generator eluate can be explained by the slow clearance from the blood and the retention in the liver. The biodistribution of ⁶⁸Ga-labeled complexes is determined by the pharmacokinetics of the complexing molecules, such as peptides, and not by the incorporated Ga³⁺.

The radiation dose resulting from the i.v. injection of ⁶⁸Ga-citrate has been estimated from ⁶⁷Ga-citrate data [16]. The estimated absorbed dose for total body, calculated assuming a uniform distribution of radioactivity, was 0.052 rads/mCi. For ⁶⁸Ge/⁶⁸Ga generator eluate, the total-body radiation dose estimates for female and male rats were 0.02560 and 0.01730 mSv/MBq, respectively. However, the ⁶⁸Ga-citrate and ⁶⁸Ga-eluate values are not directly comparable because they are expressed with different units (rads/mCi vs. mSv/MBq), but assuming that only gamma radiation is taken into account for the energy dose, the rad value can be converted into the equivalent dose value rem, thus giving an absorbed dose of 0.052 rem/mCi = 0.014 mSv/MBq. There are limited number of studies comparing the PET tracer dosimetry in animals and humans. Table 8 contains a list of reference studies in which human effective doses derived from preclinical studies are reported and compared to effective doses from human measurements [17‐24]. The absorbed doses and effective doses from the in vivo studies in rats may be different from those obtained in human studies because of the dissimilar physiology of rodents and humans. For example, the blood flow rate has a remarkable influence on the time-activity curve shape, the area under the curve, and the measured number of disintegrations.

In this study, scaling between rat and human data was performed using the overall non-organ-specific weight. In general, interspecies extrapolation of biokinetic data is based on the fact that the cellular structures and biochemistry are remarkably alike across the entire animal kingdom. Despite these similarities, however, the extrapolation of biokinetic data from laboratory animals to humans entails uncertainty, particularly for the liver, due to the qualitative differences among various species in the handling of many elements by this organ. Allometric scaling from laboratory animals to humans on the basis of body weight or surface area is the most commonly used method. It is based on the assumption that the biokinetics of compounds primarily depends on the metabolic rate of the animal and that the metabolic rate is a function of the body weight or body surface area of the animal. Yet, several other scaling methods have been proposed [25], based on, for example, the modeling of pharmacokinetic parameters where the variation of serum protein binding between species is taken into account.