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Engineering a modular 44Ti/44Sc generator: eluate evaluation in preclinical models and estimation of human radiation dosimetry

EJNMMI Research volume 13, Article number: 17 (2023) Cite this article

Abstract

Background

44Sc/47Sc is an attractive theranostic pair for targeted in vivo positron emission tomographic (PET) imaging and beta-particle treatment of cancer. The 44Ti/44Sc generator allows daily onsite production of this diagnostic isotope, which may provide an attractive alternative for PET facilities that lack in-house irradiation capabilities. Early animal and patient studies have demonstrated the utility of 44Sc. In our current study, we built and evaluated a novel clinical-scale 44Ti/44Sc generator, explored the pharmacokinetic profiles of 44ScCl3, [44Sc]-citrate and [44Sc]-NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid) in naïve mice, and estimated the radiation burden of 44ScCl3 in humans.

Methods

44Ti/44Sc (101.2 MBq) in 6 M HCl solution was utilized to assemble a modular ZR resin containing generator. After assembly, 44Sc was eluted with 0.05 M HCl for further PET imaging and biodistribution studies in female Swiss Webster mice. Based on the biodistribution data, absorbed doses of 44/47ScCl3 in human adults were calculated for 18 organs and tissues using the IDAC-Dose software.

Results

44Ti in 6 M HCl was loaded onto the organic resin generator with a yield of 99.97%. After loading and initial stabilization, 44ScCl3 was eluted with 0.05 M HCl in typical yields of 82.9 ± 5.3% (N = 16), which was normalized to the estimated generator capacity. Estimated generator capacity was computed based on elution time interval and the total amount of 44Ti loaded on the generator. Run in forward and reverse directions, the 44Sc/44Ti ratio from a primary column was significantly improved from 1038 ± 440 to 3557 ± 680 (Bq/Bq) when a secondary, replaceable, ZR resin cartridge was employed at the flow outlet. In vivo imaging and ex vivo distribution studies of the reversible modular generator for 44ScCl3, [44Sc]-citrate and [44Sc]-NODAGA show that free 44Sc remained in the circulation significantly longer than the chelated 44Sc. The dose estimation of 44ScCl3 reveals that the radiation burden is 0.146 mSv/MBq for a 70 kg adult male and 0.179 mSv/MBq for a 57 kg adult female. Liver, spleen and heart wall will receive the highest absorbed dose: 0.524, 0.502, and 0.303 mGy/MBq, respectively, for the adult male.

Conclusions

A clinical-scale 44Ti/44Sc generator system with a modular design was developed to supply 44ScCl3 in 0.05 M HCl, which is suitable for further radiolabeling and in vivo use. Our data demonstrated that free 44ScCl3 remained in the circulation for extended periods, which resulted in approximately 10 times greater radiation burden than stably chelated 44Sc. Stable 44Sc/47Sc-complexation will be more favorable for in vivo use and for clinical utility.

Introduction

Positron emission tomography (PET) is well-established in oncologic radiological workflows to detect and monitor disease progression. The majority of investigations use the metabolic tracer 18F-fluorodeoxyglucose (18F-FDG). However, there is an increasing use of peptide ligands with radiometals for specific indications to delineate molecularly specific disease types. Replacement of positron emitters with therapeutic isotopes can then be used to localize cytotoxic treatments to sites of confirmed malignancy. This paradigm of radionuclide-based theranostics has attracted a great deal of research, clinical and pharmaceutical interest [1]. In particular 68Ga (t1/2 = 68 min, Emean (β+) = 830 keV (89%)) and 177Lu (t1/2 = 6.72 d, Emean(β−) = 134 keV)-labeled somatostatin receptor peptides and prostate-specific membrane antigen inhibitors have been approved for PET imaging and treatment of neuroendocrine tumor and metastatic castration-resistant prostate cancer, respectively.

Among the investigated theranostic pairs [2], 44Sc/47Sc is well-suited for targeted in vivo PET and beta-particle treatment, respectively [3, 4]. 44Sc has a suitable half-life of 4.04 h for centralized radiopharmaceutical production along with highly abundant positron decay (Emean (β+) = 632 keV (94%)) [5, 6]. 47Sc emits a low-energy β− particle (Emean (β−) = 162 keV) similar to 177Lu, with the potential for treating lesions with a half-life of 80.4 h. This is well-suited to the relatively fast pharmacokinetic profiles of small peptides [7].

44Sc can be obtained via either direction irradiation of natural or enriched calcium [8] or the decay of 44Ti (t1/2 = 60.6 ± 1.3 years) [4]. The potentially long utility of the 44Ti/44Sc generator system has many advantageous characteristics. It allows for daily elution over a long period of time, providing an attractive alternative for PET facilities that lack in-house cyclotron capabilities. The capability of 44Sc-labeled molecules have begun to be investigated in preclinical research. Multiple cancer xenografts models have been imaged, and several 44Sc-ligands have recently undergone initial clinical evaluation including [44Sc]-PSMA617 for imaging patients with metastatic prostate cancer [9, 10]. These in vivo studies have demonstrated that 44Sc-labeled ligands provide high contrast for disease delineation in pre-clinical xenografts and clinical patient studies.

In order to harness the potential for this isotope for theranostics, greater availability of the isotope and improved understanding of in vivo stability and pharmacodynamics is required [11,12,13]. In this work, we sought to produce and better understand how 44Sc is excreted in vivo, and to what degree this will impact the radiation burden in research and clinical use. To address these questions, we have engineered a clinical-scale 44Ti/44Sc generator using ZR resin [14]. Here, we use a reversible-flow modular column design with a disposable cartridge to recover any 44Ti breakthrough and have performed PET imaging and kinetic biodistribution studies with the eluted 44Sc material. These studies provide a comprehensive evaluation of the generator and produced material, including human dosimetry estimates for more widespread clinical use.

Material and methods

All chemicals were obtained from commercial sources and were used without further purification. 44Ti/44Sc solution (111.0 MBq, 421.8 MBq/mg titanium) was obtained from Brookhaven National Laboratory, Department of Energy. Both ZR resin and 0.43-mL ZR resin cartridges were obtained from TRISKEM International. Radioactivity amounts of 44Sc were measured with a dose calibrator (CAPINTEC, CRC-15R) or a 2480 WIZARD2 automatic γ-counter (PerkinElmer). Radiochemical purity was analyzed with high-purity germanium gamma ray detector (HPGe, ORTEC, GEM-50195-S), and spectral acquisitions were acquired and analyzed by Gamma-Vision Software (version 8.0, Ametek). PEEK columns were obtained from VICI precision sampling, Inc for assembly of the ZR resin column. Deionized water (18.2 MΩcm, Rephile) and 99.999% trace-metal HCl (37 wt% in H2O) were used for preparation of 44Sc elution. Female Swiss Webster mice (6–8 weeks from Charlies River Laboratories) were purchased for in vivo pharmacokinetic studies. All radioactive material handling and animal experimentation were conducted in compliance with institutional regulations and approved by Environmental Health and Safety Radioactive Materials protocol #1169-01 and Institutional Animal Care and Use Committee protocol #22-0023.

Design and assembly of 44Ti/44Sc generator

To construct the primary column approximately 200 mg of dry ZR resin was loaded in the PEEK column (50 × 4.0 mm), and the assembled ZR resin column was pre-conditioned with 3 × 2 mL of 6.0 M HCl. 44Ti/44Sc (101.2 MBq in 1.91 mL 6.0 M HCl) mixture. The initial washout solution was reloaded into the column, twice. After loading, two equivalently sized PEEK columns (pre-conditioned with 2 mL of 0.05 M HCl were attached to each end of the primary column to assemble the 44Ti/44Sc generator. 44Sc was eluted from the 44Ti/44Sc generator with 4 mL of 0.05 M HCl (flow rate:1 mL/min via syringe pump), and the elution profile was monitored in situ with a radiodetector (γ-RAM, IN/US) and recorded with Laura software (Lablogic). The radiochemical purity of the collected 44Sc was measured with HPGe and γ-counter immediately after elution, and 3 days later. Measurements with the γ-counter used an energy window of 430–580 keV for 44Sc, and 50–230 keV for 44Ti. Since the presence of 44Sc may significantly influence the measurement accuracy of 44Ti, aliquots of the elutions were stored for several days to afford time for 44Sc decay in order to calculate the ratio of 44Sc/44Ti in the eluted solution.

Preparation of free and chelated 44Sc-NODAGA or Citrate for in vivo evaluation

After approximately 74 MBq of 44Sc was eluted into a vial with 4 mL of 0.05 M HCl solution, the 44Sc solution was adjusted to pH 6–7 with 2 M Na2CO3 (or 4 M NaOH) to prepare the 44ScCl3 solution. For preparation of 44Sc-citrate, sodium citrate (10 μL, 38.7 mM) was added to the eluted 44ScCl3 solution; and the mixture was incubated at 97 °C for 10 min. 44Sc-NODAGA was prepared with a similar procedure, except that the pH of the 44Sc solution was further adjusted with 1.0 M of ammonium acetate adjusted to pH 5 for chelation with NODAGA (10 μL, 13.6 mM). After incubation and cooling down to room temperature, 44Sc-citrate or 44Sc-NODAGA was prepared in a 30G syringe for in vivo administration.

PET imaging of animals with free and chelated 44Sc (NODAGA, Citrate)

Female Naïve Swiss Webster mice (N = 4) were injected with 3.7 MBq/400 μL of 44ScCl3 (or 44Sc-NODAGA, or 44Sc-citrate) via tail vein catheterization under 2% isoflurane anesthesia. PET imaging was performed for an initial 0.5-h on-camera dynamic image acquisition, and for 10 min static scans at 1-, 2-, and 4-h post-injection using a microPET R4 rodent scanner (Siemens). The imaged mouse was centered in the field of view and maintained under 1–2% isoflurane anesthesia during PET imaging. The calibration factor of the PET scanner was determined with a mouse-sized phantom composed of a cylinder uniformly filled with an aqueous solution of 18F with a known activity concentration. Acquisitions were recorded using an energy window of 350–700 keV and coincidence-timing window of 6 ns. PET image data were corrected for detector non-uniformity, deadtime, random coincidences and physical decay and images were reconstructed by an iterative 3D maximum a priori algorithm.

The acquired PET images were analyzed using ASIPro software (Siemens). Volume of interest (VOI) analysis of the acquired images was performed using ASIPro software, and the observed value (percent injected activity/cubic centimeter, %IA/cc) represents the mean radiotracer accumulation in the organs. The sequential radioactivity measurements (%IA/cc) were plotted over time post-administration.

Kinetic biodistribution of 44ScCl3 in naïve mice

Animals were administered 3.7 MBq/400 μL of 44ScCl3 for kinetic biodistribution studies. Four animals at each time point 5, 30, 60, 120, 240 and 1440 min post-injection were submitted for CO2 asphyxiation prior to tissue dissection. The organs of interest were collected, rinsed of excess blood, blotted, weighed, and counted with a 2480 WIZARD2 automatic γ-counter. We computed the percent of injected activity per gram of tissue (%IA/g) by normalizing the activity of each tissue to an injection standard, and the sample mass.

Estimation of human radiation dose

Biodistribution data of 44ScCl3 in the Naïve Swiss Webster mice were extrapolated to human organs using the relative organ mass scaling method [15,16,17]. In this method, the animal organ data reported as percent of injected activity per gram of organ, \(\left( {\frac{{\% {\text{IA}}}}{{{\text{g}}_{{{\text{organ}}}} }}} \right)_{{{\text{mouse}}}}\), is extrapolated using the animal and human whole-body masses, \({\mathrm{kg}}_{\mathrm{TBweight}}\), and the human organs masses, \(\left({\mathrm{g}}_{\mathrm{organ}}\right)_{\mathrm{human}}\), employing the following equation:

$$\left( {\frac{{\% {\text{IA}}}}{{{\text{organ}}}}} \right)_{{{\text{human}}}} = \left[ {\left( {\frac{{\% {\text{IA}}}}{{{\text{g}}_{{{\text{organ}}}} }}} \right)_{{{\text{mouse}}}} \times \left( {{\text{kg}}_{{\text{TBweight}}} } \right)_{{{\text{mouse}}}} } \right] \times \left( {\frac{{{\text{g}}_{{\text{organ}}} }}{{{\text{kg}}_{{{\text{TBweight}}}} }}} \right)_{{\text{human}}}$$

The human organs masses were used as defined for adult male and female in the IDAC Dose 2.1 application [18]. This scaling was not applied to the organs of the gastrointestinal tract. Organ integrated time-activity were determined by numerical integration of time activity data. The cumulative activity, Ã, between time 0 and the first measured time point was calculated assuming a linear increase from 0 to the first measured activity. The à between the first measured time point and the last measured time point was integrated numerically using trapezoidal approximation. The à from the last measured time point to infinity was integrated considering only the physical decay. It was assumed that the radioisotope does not relocate following the last imaging point. For walled organs (heart, large intestine, small intestine, and stomach), the residence time was assigned entirely to the organ walls; with the large intestine, the residence time was divided evenly between the right and left colons. The bone residence time was likewise evenly divided between cortical and trabecular bone [19].

The cumulated activities for each organ were then used to compute the absorbed doses by IDAC Dose 2.1 [18]. The mean normal-organ absorbed doses (mGy/MBq administered) and the effective dose (mSv/MBq administered) for 44ScCl3 were calculated for standard human adults (female and male). Additionally, the biodistribution data of 44ScCl3 were used to model the absorbed doses for 47ScCl3. Time activity curves representing 47ScCl3 were calculated, taking into account the different half-life of the modeled radionuclide.

Statistical analysis

Data calculated using Microsoft Excel are expressed as mean ± SD. Student’s unpaired t test (GraphPad Prism 9) was used to determine statistical significance at the 95% confidence level. Differences with p values < 0.05 were considered to be statistically significant.

Results

Design, assembly and performance of a dual-direction multicolumn 44Ti/44Sc generator

Initial studies were performed to determine the ZR resin capacity for loading and retaining 44/natTi using a single-direction flow design (Fig. 1A). We determined that 25 mg of dry ZR resin is able to trap 23 μg of titanium. The provided material was specified as containing 87.7 μg/mCi of 44/natTi, indicating approximately 80.32 μg/mCi of natTi in excess of carrier-free 44Ti. We observed significant breakthrough of 44Ti from this initial generator after loading, with approximately 20% of the loaded 44/natTi was washed out from the micro-scale pilot generator through the first ten uses of this (one-way elution) system. Thus, a modular clinical-scale generator system was pursued. Here, 44Ti loaded on a primary column is further sequestered on additional columns in each flow direction, and 44Sc was eluted with 0.05 M HCl using an alternating bidirectional flow to minimize migration and loss of the parent isotope. Here, the direction of liquid flow is changed after each run. Figure 1B depicts the loading of 44Ti onto modular column generator and final assembly.

Fig. 1
figure 1

44Ti/44Sc generator and 44Sc elution. A Pilot micro-scale generator with uni-directional elution; B Clinical-scale modular generator with bidirectional elution; C Elution profiles of 44Sc with a mobile phase of 0.05 M HCl at a flow rate of 1.0 mL/min, which was monitored in situ with in-line gamma detector; D A consistent amount of 44Sc activity is collected after loading to the modular generator; E 44Sc/44Ti ratio in the eluted 44Sc solution. E4 to E11 elution is bidirectional without additional ZR resin cartridge; E12 to E19 elution is bidirectional with an additional ZR resin cartridge

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The primary (central) column of the clinical-scale generator system comprised of 200 mg ZR resin, loaded in a PEEK column with size of 50 × 4.0 mm containing 101.2 MBq (2.736 mCi) of 44Ti in 6.0 M HCl in 1.91 mL (Fig. 1B). The loading was performed by pumping of the 6 M HCl 44Ti/44Sc solution through the column. After loading the pass-through solution onto the column twice, 99.97% of the 44Ti was absorbed on the column, with 35 kBq detected in further passed through (measured after decay of the daughter). With 44Ti loaded on the primary column, two PEEK columns filled with 200 mg ZR resin were assembled at both terminals (Fig. 1B).

A lower concentration of HCl (0.05 M) was used to elute 44Sc (Fig. 1C) as it is amenable for radiopharmaceutical preparations. The initial first three column elutions were performed on the same day in the same direction with 0.05 M of HCl. As 44Ti was loaded under high concentration conditions of 6 M HCl, the shift to 0.05 M of HCl to elute 44Sc created a transient resin condition, resulting in higher initial radioactivities of 44Ti release. As expected, each of these initial elutions released decreasing amounts of 44Ti: 3.43% (3474 kBq); 0.66% (651 kBq); 0.39% (381 kBq). These values are significantly greater than those compared to the 44Ti breakthrough at later use (elution E4 and further; Fig. 1D). After transition and stabilization, 44Sc was eluted bidirectionally with 1 mL/min of 0.05 M HCl for 4 min to generate 44ScCl3 in a yield of 82.9 ± 5.3% (N = 16, elution sample: E4-E19), which was normalized to the estimated generator capacity. Estimated generator capacity is computed based upon the total amount of 44Ti loaded on the generator, and the time interval between sequential elutions.

To simulate daily clinical-use conditions, the elution interval of E4 to E19 was approximately 24 h. The 44Sc/44Ti ratio obtained was 1038 ± 440 (N = 8) from E4 to E11. From elution E12 and on, an additional disposable ZR resin cartridge (0.3 mL) was employed at the flow outlet to further recover 44Ti breakthrough; 44Sc/44Ti ratios were then improved to 3557 ± 680 (N = 8; Fig. 1E).

PET imaging of animals with free and chelated 44Sc

We next investigated the in vivo absorption, distribution and excretion of intravenously administered 44ScCl3 and chelated 44Sc (citrate and NODAGA). Dynamic PET imaging was performed initially on-camera (0.5 h) followed by static imaging at 1-, 2-, and 4-h post-injection. Representative acquisitions are shown in Fig. 2 and volume of interest analysis is presented in Fig. 3. PET imaging of 44ScCl3 showed significant cardiac signal in the acute post-injection phase, indicative of plasma binding and long circulation characteristics (Figs. 2 and 3). Within 1-h post-injection (Fig. 3A and D), renal accumulation of 44Sc was higher than that in the liver (P < 0.0001 at one half hour; 6.64 ± 0.84 vs 4.79 ± 0.81%IA/cc, P = 0.04 at 1 h), and there was no significant difference (Fig. 3D) at late time points (2 h: 6.31 ± 0.67 vs 5.28 ± 0.72%IA/cc, P = 0.16; 4 h: 6.11 ± 0.13 vs 5.68 ± 0.34%IA/cc, P = 0.14). A minor portion of 44Sc was eliminated via urinary excretion, resulting in visible bladder signal at 1-h post-administration (Fig. 2).

Fig. 2
figure 2

PET imaging of the in vivo distribution of 44ScCl3 and citrate or [44Sc]-NODAGA. Representative maximum intensity projections of the PET acquisitions at indicated times for each species. Later phase images reveal that 44Sc remained in circulation considerably longer than that of either chelated 44Sc formulations. The more stable [44Sc]-NODAGA is rapidly excreted via the urinary system

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Fig. 3
figure 3

Quantitative analysis of PET imaging of 44ScCl3 and citrate or NODAGA chelated 44Sc. Volumes of interest (VOI) analysis of dynamic imaging of free 44Sc (A), [44Sc]-citrate (B) and [44Sc]-NODAGA (C). VOI analysis of static images of free 44Sc (D), [44Sc]-citrate (E) and [44Sc]-NODAGA (F). Legends: (Blue filled circle) Kidney, (orange filled square) Heart, (green filled triangle) Liver. Error bars represent the standard deviation from N ≥ 4 subjects

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In contrast to free 44Sc ([44Sc]ScCl3), 44Sc complexed with citrate ([44Sc]-citrate) reveals a substantially higher kidney accumulation (Fig. 3B) out to 0.5-h post-injection (0.5 h: 9.21 ± 0.95 (44Sc) vs 11.85 ± 3.01%IA/cc ([44Sc]-citrate), P = 0.23), lower kidney accumulation at later time points (2 h: 6.31 ± 0.67 vs 5.07 ± 0.31%IA/cc, P = 0.05; 4 h: 6.11 ± 0.13 vs 4.72 ± 0.47%IA/cc, P = 0.006), and lower accumulation in the heart (2 h: 13.98 ± 1.52 vs 9.63 ± 0.63%IA/cc, P = 0.05; 4 h: 10.52 ± 1.16 vs 7.85 ± 0.81%IA/cc, P = 0.02) than that of free 44Sc (Fig. 3E). This indicates that the intact [44Sc]-citrate was rapidly excreted through the urinary system. The bladder was visible from 5-min to 4-h post-injection (Fig. 2). [44Sc]-citrate also displayed a lower accumulation in the liver than that of free 44Sc (Fig. 3D and 3E).

A stable chelation system was also evaluated. Here, [44Sc]-NODAGA demonstrated a short circulation in blood (t1/2 = 2.53 min with a range between 1.69 to 3.94 min), and the majority of the administered radiotracer was excreted into the bladder via the kidney within 5 min (Fig. 2). Kinetic PET imaging of 44Sc-NODAGA at 1-, 2-, and 4-h post-injection showed a lower and statistically significant accumulation in all organs evaluated in comparison with either free 44Sc or 44Sc-citrate (Fig. 3C and 3F; P < 0.05). However, 44Sc-NODAGA showed a varied accumulation in the kidneys within the 0.5-h post-injection (dynamic PET imaging), which resulted in mean renal values of uptake that are higher than that of free 44Sc and 44Sc-citrate (Fig. 3C).

Biodistribution of free 44Sc in healthy mice

To further confirm the observation of free 44ScCl3 in mice, a kinetic biodistribution study was performed. Results are shown in Fig. 4, and percent injected activity per gram values for each organ are included as Additional file 1: Table S1. The 44ScCl3 is rapidly distributed in blood, heart, aorta, cava, lung, liver, kidneys and spleen. High activity levels in the blood, heart, aorta and vena cava confirm the persistence of 44Sc in blood from the PET imaging. Analysis of the excretion profile show effective half-lives of 2.0 min (rapid phase) and 133 min (slow phase), respectively. The free 44Sc was then excreted primarily through the liver, and a minor portion through the kidneys. An increased uptake was found in the spleen, 27.6 ± 7.3%IA/g at 5 min, and 60.8 ± 13.5%IA/g at 24 h-post-injection, and peak accumulation is at 1-h post-injection (81.6 ± 39.7%IA/g). Low background levels of accumulation were measured in all other collected organs, including pancreas, bone, brain, salivary glands, stomach, intestine, fat, skin, and muscle.

Fig. 4
figure 4

Kinetic distribution of 44ScCl3 in Swiss Webster mice. Free 44Sc (ScCl3) circulates in the blood with an effective half-lives of 2.0 min (rapid phase) and 133 min (slow phase), respectively, and is excreted from the body through both urinary and hepatic systems. Low accumulation in all other organs is observed at extended time points. Small Intestine (SI), Large Intestine (LI), and Salivary Glands (SG)

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Estimation of human radiation dose

The estimated absorbed dose was extrapolated from the female murine data, and the results are listed in Table 1. The effective dose for a 70-kg adult male was 0.146 and 0.179 mSv/MBq, and 0.310 and 0.369 mSv/MBq for female for 44ScCl3 and 47ScCl3, respectively. For example, it would be 16.2–19.9 mSv and 34.4–41.0 mSv from an intravenously injected radioactivity of 111 MBq (3 mCi) of 44ScCl3 and 47ScCl3, respectively. The absorbed doses for 47ScCl3 are higher than the absorbed doses for 44ScCl3, except for the adrenal, heart wall and red bone marrow. Among all organs, the liver, spleen, and heart wall received the highest absorbed dose: 0.524, 0.502, and 0.303 mGy/MBq for the adult male and 44ScCl3, respectively. The majority of organs in an adult female will receive higher absorbed dose than that of a male, excepting the breast and colon wall, which indicate that gender difference may be a factor in the irradiation burden.

Table 1 Absorbed doses per unit activity administered for 44ScCl3 and 47ScCl3 (mGy/MBq)
Full size table

Discussion

There is an increased interest in the development and implementation of theranostic nuclear medicine approaches for personalized patient management. Access to radioisotopes with desirable characteristics for quantitative PET imaging that are chemically analogous to therapeutic isotopes is an area of particular focus. Recent preclinical and clinical studies have investigated 44Sc-labeled small molecules as a promising positron-emitting diagnostic and surrogate for 47Sc-based radiotherapy. In comparison with gallium-68 (1.13 h), the half-life of 44Sc (3.97 h) affords advantages for labeling, quality control evaluation, transport logistics and the biokinetics of many tracers. The imaging characteristics for emissions from 44Sc have also been shown to be well-suited for delineation of small lesions [20] [21]. Cyclotron production of 44Sc through irradiation of natural calcium metal or liquid targets enables tertiary medical centers and large production facilities to produce the isotope [22] [6]. Alternatively, distributed generator systems that separate parent 44Ti from would enable on-site production. In this study, we built and evaluated a modular 44Ti/44Sc generator, and further investigated the absorption, distribution, and excretion of the activity after a single intravenous injection of generator eluate in female mice.

Consistent with prior investigation [4, 13, 14], 44Ti was efficiently loaded on the resin, and we observed that this material can re-distribute on the column following repeated 44Sc elutions. This resulted in breakthrough of 44Ti and has the potential to contaminate the radiopharmaceutical and work space for compounding. To avoid 44Ti breakthrough from the generator, a bidirectional elution approach has been employed to delay the breakthrough by others, including Filosofov et al. [23] and Radchenko et al. [14]. A bidirectional elution approach cannot prevent 44Ti redistribution and breakthrough completely, and the 44Ti/44Sc generator must be re-assembled after a period of use. Therefore, we engineered the modular clinical-scale generator to allow us to: (1) recover 44Ti efficiently and conveniently; (2) replace the columns independently; and (3) load 44Ti to the ZR resin generator semi-automatically with a minimum radiation dose to the operation personnel.

It has been reported that 44Ti has a consistent absorption efficiency on ZR resin across a wide range of HCl concentrations [14]. The 44Ti/44Sc stock solution was provided dissolved in 6 M HCl solution. We therefore loaded to the primary ZR resin column under the condition of 6.0 M HCl with 44Ti/44Sc. More than 99.9% of 44Ti was trapped. While 44Sc can be eluted efficiently with 4.0–6.0 M HCl, the high concentration of HCl here would complicate safe-handling and requires additional adjustment of pH conditions to reach suitable conditions for radiolabeling. Thus, a lower concentration of 0.05 M HCl was chosen to elute 44Sc. A significantly higher 44Ti breakthrough was observed in the first three elutions using this lower concentration. We put forward that 44Ti3+ may be hydrolyzed into 44Ti(OH)x or 44TiO2 under the condition of less than 4 M HCl solution. During the transition from 6 M HCl to 0.05 M HCl, the absorbed 44Ti3+ may be quickly hydrolyzed and released from the resin. After elution with 0.05 M HCl for several days, 44Sc was obtained in a consistent yield with a high 44Sc/44Ti ratio. To further limit breakthrough of 44Ti from 44Sc elution, a disposable ZR resin cartridge (0.3 mL) was utilized at the flow outlet. Our results showed that 44Sc/44Ti ratio was further improved by 342%. Notably these small amounts of absorbed 44Ti on this disposable cartridge can be recovered by pass through of 6 M HCl/0.65% H2O2 [14], which can be combined, dried and redissolved in 0.05 M HCl solution for re-loading onto the center column of the generator.

PET imaging was performed to measure pharmacokinetic profiles of 44ScCl3 and chelated 44Sc with either citrate or NODAGA. After intravenously injection, dynamic PET imaging showed that free 44ScCl3 is distributed in blood (heart) and lung and that activity remained in circulation for an extended period. Ex vivo analyses recapitulated high radioactivity in the blood, heart, aorta and vena cava confirming 44Sc is mainly remained in blood with half-lives of 2.0 min (rapid phase) and 133 min (slow phase), respectively. Similar to 68GaCl3 PET imaging results in rats [24], 44ScCl3 in mice was slowly excreted through the kidney and liver. Our biodistribution results also show accumulation of 44ScCl3 in the spleen, with background levels of accumulation in all other tested organs. We hypothesize that similar to the Fe+3 and 45Ti [25] ion, a major portion of 44Sc+3 is bound to transferrin after intravenous administration. This results in extended circulation times and slow kinetics of excretion without specific accumulation in the heart wall or vasculature. Neither PET imaging nor biodistribution studies identified 44ScCl3 to be excreted via feces or the intestine. This is in contrast to the elimination of 68Ga or 64Cu, in which this gastrointestinal clearance presents a complication for interpreting preclinical imaging data. Together, these data motivate use of very high in vivo stability chelators for 44/47Sc targeted agents and for understanding of artifactual distributions.

44Sc-citrate showed a similar in vivo pharmacokinetic profile with an increased rate of clearance. The major difference identified was a higher kidney accumulation as 44Sc forms only a weak complex with citrate that may prevent rapid complexation by components in the blood. When the more stable 44Sc-NODAGA was used, activity was observed to transit into the bladder rapidly, which is consistent with the in vivo profiles of 44Sc-labeled peptides utilizing DOTA and NODAGA [26] or other novel chelate-conjugated ligands [27]. Further evaluation of the chemical identity of the generator output and its impact on radiotracer labeling and distribution will be conducted. This is of particular interest for future comparison of generator- and cyclotron-produced 44Sc.

Together, differences in distribution of free and chelated scandium imply that any unlabeled 44Sc/47Sc in the solution of 44Sc/47Sc-chelated ligand may cause a significant increase in the observed circulation time. To further clarify what amount of absorbed dose may be caused by the unlabeled 44Sc/47Sc, the data of a kinetic biodistribution in the mice was extrapolated to male and female adults. We observed a gender difference of irradiation dose and that the free-44Sc has a significantly higher irradiation burden than that of the conjugated 44Sc-ligands, as expected. For example, the mean effective dose of [44Sc]-PSMA617 in male patient is 0.0389 mSv/MBq [28], whereas the unconjugated 44Sc (current work) is 0.146 mSv/MBq. Similar results have been found in other critical organs, including spleen, liver and red marrow (0.185 vs 0.502; 0.107 vs 0.524; 0.0331 vs 0.124 mSv/MBq). These data indicate that a high purity for 44/47Sc-conjugated ligand is required for safe and effective radionuclide-based treatment in the future and provide insight into assessment of the radiation burden from decomplexed radioisotope in vivo.

Conclusion

A clinical-scale 44Ti/44Sc generator system with a modular design was been developed which can supply over 74 MBq (2 mCi) of 44Sc in 4 mL of 0.05 M HCl. The generator has consistent performance characteristics, and the eluted material was evaluated in animal models for imaging and distribution studies. Our data demonstrated that free 44ScCl3 (unchelated) remained in the circulation for extended periods and was excreted predominantly through the liver and spleen, which resulted in a significant absorbed dose difference over stably-chelated 44Sc. Our results reveal that highly in vivo stable 44Sc/47Sc-complexation will be more favorable for successful translation and clinical utility.

Availability of data and materials

The datasets generated and analyzed during the current study are available upon direct request to the authors upon reasonable request.

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Acknowledgements

The isotope used in this research was supplied by the US Department of Energy Isotope Program, managed by the Office of Science.

Funding

This work was supported in part by National Institutes of Health R01CA240711 (DLJT), R01CA229893 (DLJT), R01CA201035 (DLJT), and the Children’s Discovery Institute MC-II-2021–961 (DA). TRIUMF receives federal funding via a contribution agreement with the National Research Council of Canada.

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Authors and Affiliations

  1. Washington University School of Medicine, Mallinckrodt Institute of Radiology, 510 S. Kingshighway Boulevard, St. Louis, MO, 63110, USA

    Nadia Benabdallah, Hanwen Zhang, Ryan Unnerstall, Amanda Fears, Lucy Summer, Buck E. Rodgers, Diane Abou & Daniel L. J. Thorek

  2. Program in Quantitative Molecular Therapeutics, Washington University School of Medicine, St. Louis, MO, 63110, USA

    Nadia Benabdallah, Hanwen Zhang, Amanda Fears & Daniel L. J. Thorek

  3. Washington University School of Medicine, Siteman Cancer Center, St. Louis, MO, 63110, USA

    Hanwen Zhang, Buck E. Rodgers, Diane Abou & Daniel L. J. Thorek

  4. Chemistry Division, Los Alamos National Laboratory, PO Box 1663, Los Alamos, NM, 87545, USA

    Michael Fassbender

  5. Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, 63110, USA

    Buck E. Rodgers

  6. Mallinckrodt Cyclotron Facility, Washington University School of Medicine, St. Louis, MO, 63110, USA

    Diane Abou

  7. Life Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, BC, V6T 2A3, Canada

    Valery Radchenko

  8. Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1, Canada

    Valery Radchenko

  9. Department of Biomedical Engineering, Washington University, St. Louis, MO, 63110, USA

    Daniel L. J. Thorek

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  1. Nadia Benabdallah
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Contributions

HZ and DT conceived of the experiments. HZ, MF, BR, DA, VR, and DT assisted in generator concept and design. NB, HZ, RU, AF, and LS performed experiments and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Hanwen Zhang or Daniel L. J. Thorek.

Ethics declarations

Ethics approval and consent to participate

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All radioactive material handling and animal experimentation were conducted in compliance with institutional regulations and approved by Environmental Health and Safety protocol #Thorek 1169-01 and Institutional Animal Care and Use Committee protocol #22-0023, in compliance with ARRIVE guidelines.

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The authors declare no relevant conflicts of interest.

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Supplementary Information

Additional file 1

. Biodistribution of 44ScCl3 in healthy Swiss Webster mice. Description of data: Activity concentration per organ at time points after injection of 44ScCl3 in Naïve female mice.

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Benabdallah, N., Zhang, H., Unnerstall, R. et al. Engineering a modular 44Ti/44Sc generator: eluate evaluation in preclinical models and estimation of human radiation dosimetry. EJNMMI Res 13, 17 (2023). https://doi.org/10.1186/s13550-023-00968-5

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