Phase I Clinical Trial Using [99mTc]Tc-1-thio-D-glucose for Diagnosis of Lymphoma Patients

Similar to [18F]-FDG, [99mTc]Tc-1-thio-D-glucose ([99mTc]Tc-TG) also binds to GLUT receptors. The aim of this Phase I study was to evaluate the safety, biodistribution and dosimetry of [99mTc]Tc-TG. Twelve lymphoma patients were injected with 729 ± 102 MBq [99mTc]Tc-TG. Whole-body planar imaging was performed in 10 patients at 2, 4, 6 and 24 h after injection. In all 12 patients, SPECT/CT (at 2 h) and SPECT (at 4 and 6 h) imaging was performed. Vital signs and possible side effects were monitored during imaging and up to 7 days after injection. [99mTc]Tc-TG injections were well-tolerated and no side effects or alterations in blood and urine analyses data were observed. The highest absorbed dose was in the kidneys and urinary bladder wall, followed by the adrenals, prostate, bone marrow, lungs, myocardium, ovaries, uterus, liver and gall bladder wall. [99mTc]Tc-TG SPECT/CT revealed foci of high activity uptake in the lymph nodes of all nine patients with known nodal lesions. Extranodal lesions were detected in all nine cases. In one patient, a lesion in the humerus head, which was not detected by CT, was visualized using [99mTc]Tc-TG. Potentially, [99mTc]Tc-TG can be considered as an additional diagnostic method for imaging GLUT receptors in lymphoma patients.


Introduction
Positron emission tomography (PET), and more recently PET/CT, using [ 18 F]-2-fluoro-2-deoxy-D-glucose ([ 18 F]-FDG), is the most sensitive and specific imaging technique currently available for patients with lymphoma. Numerous studies indicate the utility of [ 18 F]-FDG PET/CT for staging lymphomas, predicting their response to therapy and evaluating their treatment effectiveness [1,2]. Despite the advantages of this technology for patients' management, the use of PET in many developing countries is limited by the high cost of studies and low number of PET/CT facilities. However, SPECT scanners are installed in many hospitals around the world and, therefore, the use of tracers labeled with gamma-emitting nuclides for the imaging of hypermetabolic lesions might be a solution for these countries. Several glucose analogues have been preclinically evaluated for the SPECT imaging of tumor metabolisms [3][4][5][6][7], and [ 99m Tc]Tc-1-thio-D-glucose ([ 99m Tc]Tc-TG) is one Pharmaceutics 2022, 14, 1274 2 of 12 of such tracers ( Figure 1). The mechanism of how [ 99m Tc]Tc-TG binds to malignant cells was elucidated using colorectal carcinoma (HCT-116) and human lung adenocarcinoma (A549) cells [4]. The [ 99m Tc]Tc-TG and [ 18 F]-FDG accumulation level in HCT-116 cells was very close; however, the [ 18 F]-FDG uptake in the A549 cell line was almost double that of [ 99m Tc]Tc-TG. The cellular accumulation of both [ 99m Tc]Tc-TG and [ 18 F]-FDG decreased with an increase of the glucose competitor concentration, and was increased in the presence of insulin. These observations implied that both [ 18 F]-FDG and [ 99m Tc]Tc-TG bound to cells with the participation of glucose transport proteins. Furthermore, cellular uptake of both [ 18 F]-FDG and [ 99m Tc]Tc-TG was reduced in the presence of cytochalasin B, which blocks the GLUT1-5 channels. On the contrary, no reduction of cellular uptake was observed for either [ 99m Tc]Tc-TG or [ 18 F]-FDG when SGLT1-3 was blocked by phloretin, suggesting that these transporters are not involved in the uptake process [4]. This lead to the conclusion that these agents are taken up by the cells mainly via GLUT transporters, which was in agreement with the data demonstrating a substantial overexpression of GLUT1 in the tested cell lines [8][9][10]. Importantly, [ 99m Tc]Tc-TG accumulates predominantly in cell membranes, whereas [ 18 F]-FDG is predominantly localized in the cytoplasm. This feature was explained by the large size of the [ 99m Tc]Tc-TG molecule, which might hamper its transport into cells, and the tracer remains attached to membrane-bound transporter proteins [4]. Overall, this study suggested that [ 99m Tc]Tc-TG could be used for the mapping of sites with elevated GLUT expression, including malignant tumors.
There were three primary objectives of this first-in-human study: firstly, to obtain initial information concerning the safety and tolerability of [ 99m Tc]Tc-TG after a single intravenous injection; secondly, to assess the distribution of [ 99m Tc]Tc-TG in normal tissues and in tumors over time; and thirdly, to evaluate the dosimetry of [ 99m Tc]Tc-TG.
The secondary objective was to evaluate the possibility of using [ 99m Tc]Tc-TG SPECT/CT for visualizing GLUT expression in the nodal and extranodal lesions of lymphoma patients. In preparation for clinical studies, a single-vial kit for labeling 1-thio-D-glucose and the analytical methods for characterizing the labeled product were developed [11]. Further preclinical studies demonstrated that [ 99m Tc]Tc-TG does not have any acute or cumulative toxicity and is not allergenic. Accumulation of [ 99m Tc]Tc-TG in tumor cells, including the RMA lymphoma cell line, was demonstrated in vivo and in vitro [11,12].
There were three primary objectives of this first-in-human study: firstly, to obtain initial information concerning the safety and tolerability of [ 99m Tc]Tc-TG after a single intravenous injection; secondly, to assess the distribution of [ 99m Tc]Tc-TG in normal tissues and in tumors over time; and thirdly, to evaluate the dosimetry of [ 99m Tc]Tc-TG.
The secondary objective was to evaluate the possibility of using [ 99m Tc]Tc-TG SPECT/CT for visualizing GLUT expression in the nodal and extranodal lesions of lymphoma patients.

Patients
This was a prospective, open-label, non-randomized diagnostic study in patients with untreated lymphoma (ClinicalTrials.gov Identifier: NCT04292626). (4) negative pregnancy test for female patients of childbearing potential; and (5) capability to undergo the diagnostic investigations planned in the study.
Patients with lymphoma were not accepted based one or more of the following exclusion criteria: (1) a previous diagnosis with an autoimmune disease; (2) an active infection or history of severe infection; (3) known to be HIV positive or have a chronically active hepatitis B or C infection; or (4) had been administered other investigational medicinal products.
Twelve patients (four male and eight female) were enrolled (Table 1, Figure S1) and, as a local standard of care, chest and abdomen CT (Siemens Somatom Emotions 16 ECO) and ultrasound (GE LOGIQ E9) imaging was performed along with biopsy sampling for all patients. Biopsy samples of nodal and extranodal lesions were collected and the diagnosis was verified by immunohistochemistry (IHC).
It was demonstrated during the tracer development that the labelled compound is stable under challenge with a 1000-fold excess of L-cysteine during 4 h (no measurable release of free 99m Tc).
For a release of the clinical batch, a test labelling was performed using 10 kit vials. [ 99m Tc]Tc-TG was tested according to State Pharmacopoeia of the Russian Federation, XIII ed. (SP XIII) for the authenticity of all reagents, as well as for pH, volume activity, radiochemical purity, bacterial endotoxin content (LAL test) and sterility using thioglycollate fluid medium and Sabouraud Dextrose Broth. [ 99m Tc]Tc-TG met all Pharmacopoeia requirements, was sterile and pyrogen-free.

Imaging Protocol
The [ 99m Tc]Tc-TG, in a dose of 729 ± 102 MBq, was injected as an intravenous bolus. A Siemens Symbia Intevo Bold scanner was used for imaging. A high-resolution low-energy collimator was used to images acquisition. Anterior and posterior whole-body planar imaging (at a scan speed of 12 cm/min, 1024 × 256 pixel matrix) was carried out in patients 1-10 at 2, 4, 6 and 24 h after injection. All 12 patients received SPECT/CT scans (SPECT: 60 projections, 20 s each, 256 × 256 pixel matrix; CT: 130 kV, effective 36 mAs) at 2 h and only SPECT scans (60 projections, 20 s each, 256 × 256 pixel matrix) 4 and 6 h after injection. For SPECT reconstruction, the xSPECT (Siemens) protocol based on the ordered subset conjugate gradient (OSCG) method (24 iterations, 2 subsets) was used. The 3D Gaussian FWHM 10 mm filter (Soft Tissue) was used. For processing of obtained images, the proprietary software package syngo.via (Siemens) was used.
Vital signs were monitored before, during and after imaging. Parameters of blood biochemistry were analyzed before injections of [ 99m Tc]Tc-TG and 24, 48 h and 7 days after injections.

Evaluation of Distribution and Dosimetry
Regions of interest (ROI) were drawn over organs of interest and the body contour on the anterior and posterior whole-body images. A geometric mean of counts at 2, 4, 6 and 24 h was calculated for each ROI. A known activity of 99m Tc in a water-filled phantom was measured and used for quantification. Chang's correction was used for attenuation correction. To assess the activity in the blood, the data from a POI placed over the heart were used. Data were fitted to single exponential functions and residence times were calculated as areas under fitted curves using Prism 9 (version 9.3.1, GraphPad Software, LLC, San Diego, CA, USA). Absorbed doses were calculated by OLINDA/EXM 1.1 using an adult female and male phantoms.
To calculate tumor-to-background ratios at 2, 4 and 6 h, a one cm 3 volume of interest (VOI) was drawn on tomograms centered on the highest tumor uptake, and counts were recorded. Thereafter, this VOI was copied to the contralateral side to obtain reference counts. The maximal standard uptake value (SUVmax) in nodal or extranodal lesions with the highest [ 99m Tc]Tc-TG accumulation was calculated 2 h after injection when CT data was available. Tumor sizes were defined by CT as the maximum size of the largest lesion.

Statistics
Values are reported as mean ± standard deviation. The significance of differences between uptakes in organs at different time points was analyzed using 1-way ANOVA.

Safety and Tolerability
The intravenous bolus administration of [ 99m Tc]Tc-TG was safe and well-tolerated by all patients. At all time points of objective control over the somatic state of patients after intravenous administration of [ 99m Tc]Tc-TG, no changes in vital signs or adverse reactions were registered. Patients did not actively present any complaints. The functional state of the main organs and systems did not have significant differences before and after the administration of [ 99m Tc]Tc-TG, which was confirmed by the results of the performed instrumental and laboratory tests. No changes in blood or urine samples were found (Table S1).

Evaluation of Distribution and Dosimetry
The kinetics of [ 99m Tc]Tc-TG elimination from blood is shown in Figure S2. The elimination half-lives were 3.1 h (95% CI 1.3 to 16 h) in female patients and 3.6 h (95% CI 1.6 to 19 h) in male patients. The kidneys, liver and lungs were the organs with the highest accumulation of activity (Table 2; Figure 2). Noticeable activity was also observed in the gall bladder, spleen, thyroid, small intestines, testes and stomach. No significant difference between male and female patients was found. Table 2. Decay-corrected uptake of 99m Tc in the highest-uptake organs based on planar imaging of tumor-free areas. The data are presented as average %ID ± SD per organ for six females and four males at different time points after injection with [ 99m Tc]Tc-TG.

Kidney
Liver Lung 1.6 to 19 h) in male patients. The kidneys, liver and lungs were the organs with the highest ac activity (Table 2; Figure 2). Noticeable activity was also observed in th spleen, thyroid, small intestines, testes and stomach. No significant diffe male and female patients was found.   The calculated absorbed doses is shown in Table 3. The highest absorbed dose was in the kidneys and urinary bladder wall, followed by the adrenals, prostate, osteogenic cells, lungs, heart wall, ovaries, uterus, liver and gall bladder wall. The effective doses were 0.0072 ± 0.0036 and 0.0135 ± 0.0091 mSv/MBq for female and male patients, respectively. For a typical injected activity of 730 MBq, which was used in this study, an expected effective dose would be 5.0-9.8 mSv.

[ 99m Tc]Tc-TG SPECT/CT Imaging of Nodal and Extranodal Lesions
According to standard diagnostic methods (clinical examination, CT and ultrasound), lymph node lesions were known in 9 of the 12 examined lymphoma cases and extranodal lesions also in 9 of the patients. The [ 99m Tc]Tc-TG SPECT/CT scans revealed foci of elevated radiopharmaceutical uptake in the lymph nodes of all nine patients with nodal lesions (example in Figure 3). Extranodal lesions were also detected by [ 99m Tc]Tc-TG SPECT in all nine of the known cases. [ 99m Tc]Tc-TG SPECT/CT correctly visualized brain (Figure 4), neck, chest ( Figure 5) and abdominal lesions ( Figure 6). In one patient, according to [ 99m Tc]Tc-TG SPECT/CT, a lesion in the humerus head was visualized, which was not detected by CT ( Figure 7A). Based on this finding, an MRI of the left shoulder joint was performed and a lymphoma lesion in the metaepiphysis of the humerus was confirmed ( Figure 7B) and the stage of the disease was changed from IIIB to IVB. In the highest [ 99m Tc]Tc-TG accumulating lesions, SUVmax was 2.6 ± 1.1 at 2 h after injection ( Table 4). The tumor/contralateral background ratios for extracranial lesions reached 3.3 ± 1.2 at 2 h after injection, decreased to 2.3 ± 1.4 after 4 h and did not change significantly after that (Table 4).  Table 4). The tumor/contralateral background ratios for extracranial lesions reached 3.3 ± 1.2 at 2 h after injection, decreased to 2.3 ± 1.4 after 4 h and did not change significantly after that (Table 4).     Table 4). The tumor/contralateral background ratios for extracranial lesions reached 3.3 ± 1.2 at 2 h after injection, decreased to 2.3 ± 1.4 after 4 h and did not change significantly after that (Table 4).                In our study, patient 5 had cholecystitis as a concomitant diagnosis. In this case, a high [ 99m Tc]Tc-TG uptake (SUVmax = 20.8 vs. 4.4 ± 1.6 in normal patients) in the gallbladder was found (Figure 8, Table 4). In the rest of the patients, elevated [ 99m Tc]Tc-TG accumulation in the gallbladder was not visualized (Figure 8, Table 4).
Pharmaceutics 2022, 14, 1274 9 of 12 *-Mean ± SD was calculated for normal [ 99m Tc]Tc-TG gallbladder uptake, ** values for extracranial lesions; *** the significance of the differences is shown in comparison with the tumor/background value for extracranial lesions at 2 h.
In our study, patient 5 had cholecystitis as a concomitant diagnosis. In this case, a high [ 99m Tc]Tc-TG uptake (SUVmax = 20.8 vs. 4.4 ± 1.6 in normal patients) in the gallbladder was found (Figure 8, Table 4). In the rest of the patients, elevated [ 99m Tc]Tc-TG accumulation in the gallbladder was not visualized (Figure 8, Table 4).

Discussion
This study demonstrated that the injection of [ 99m Tc]Tc-TG is well-tolerated and not associated with any adverse effects. The favorable dosimetry properties of 99m Tc ensured a moderately effective dose. In the current study, typical equivalent doses were 5.0-9.8 mSv. However, the injected activity in this study was specifically selected to obtain good counting statistics 24 h after injection for dosimetry calculations. For routine use, the injected activity could be reduced at least three-fold (see below) with a proportional reduction in the effective dose. For comparison, a typical effective dose from [ 18 F]-FDG PET is 3.5 mSv when 185 MBq is administered [13].
[ 99m Tc]Tc-TG SPECT allowed us to identify foci corresponding to all nodal lesions found according to the standard diagnostic methods in this study. There was also an excellent correlation between visualizing extranodal lesions using [ 99m Tc]Tc-TG SPECT and the standard diagnostic methods in all cases of brain (Figure 4), neck, chest ( Figure 5) and abdominal lesions ( Figure 6). A major advantage of [ 18 F]-FDG PET/CT over CT is the possibility to detect bone marrow lesions in lymphomas, since CT allows visualization only in the case of bone destruction [14]. The use of [ 99m Tc]Tc-TG SPECT enabled detection of a bone lesion, which was not visualized by CT ( Figure 7A). This finding prompted an additional MRI examination, which confirmed a lymphoma lesion in the humerus ( Figure  7B). For this patient, the stage of the disease was changed based on [ 99m Tc]Tc-TG SPECT imaging followed by MRI confirmation.

Discussion
This study demonstrated that the injection of [ 99m Tc]Tc-TG is well-tolerated and not associated with any adverse effects. The favorable dosimetry properties of 99m Tc ensured a moderately effective dose. In the current study, typical equivalent doses were 5.0-9.8 mSv. However, the injected activity in this study was specifically selected to obtain good counting statistics 24 h after injection for dosimetry calculations. For routine use, the injected activity could be reduced at least three-fold (see below) with a proportional reduction in the effective dose. For comparison, a typical effective dose from [ 18 F]-FDG PET is 3.5 mSv when 185 MBq is administered [13].
[ 99m Tc]Tc-TG SPECT allowed us to identify foci corresponding to all nodal lesions found according to the standard diagnostic methods in this study. There was also an excellent correlation between visualizing extranodal lesions using [ 99m Tc]Tc-TG SPECT and the standard diagnostic methods in all cases of brain (Figure 4), neck, chest ( Figure 5) and abdominal lesions ( Figure 6). A major advantage of [ 18 F]-FDG PET/CT over CT is the possibility to detect bone marrow lesions in lymphomas, since CT allows visualization only in the case of bone destruction [14]. The use of [ 99m Tc]Tc-TG SPECT enabled detection of a bone lesion, which was not visualized by CT ( Figure 7A). This finding prompted an additional MRI examination, which confirmed a lymphoma lesion in the humerus ( Figure 7B). For this patient, the stage of the disease was changed based on [ 99m Tc]Tc-TG SPECT imaging followed by MRI confirmation.
Because the tumor-to-background ratio reached 3.3 ± 1.2 at 2 h after injection, then decreased to 2.30 ± 1.42 after 4 h and after that did not change significantly, the optimal time for SPECT imaging is 2 h after the injection. At this time point, the [ 99m Tc]Tc-TG SUVmax was 2.6 ± 1.1, which is considerably lower than the typical [ 18 F]-FDG SUVmax in lymphoma lesions according to the literature [15]. A possible explanation for this is that [ 99m Tc]Tc-TG is not internalized after binding to GLUT1 to the same extent as [ 18 F]-FDG [4] and does not undergo metabolic trapping. Since [ 99m Tc]Tc-TG images likely GLUT-receptor expression rather than glucose metabolism, it will be essential for future clinical applications to confirm that the uptake of [ 99m Tc]Tc-TG in the rest of the human body is also GLUT-mediated. It is well known that GLUT receptors are overexpressed in inflammatory cells leading to an active uptake of [ 18 F]-FDG in inflammatory foci [16]. In particular, an accumulation of [ 18 F]-FDG in cholecystitis has been previously demonstrated [17][18][19]. For the study herein, imaging of patient 4 with documented chronic cholecystitis showed a much higher activity accumulation in the gallbladder, compared to other patients (Table 4; Figure 8). This suggests that [ 99m Tc]Tc-TG uptake is dependent on GLUT-receptor expression, similar to [ 18 F]-FDG, which might support its use for the imaging of hypermetabolic lesions overexpressing these receptors. Unfortunately, this also means that all pitfalls and artifacts known when using [ 18 F]-FDG for the imaging of hematologic malignancies [20] might also apply when using [ 99m Tc]Tc-TG.
Currently, [ 18 F]-FDG PET is applied for post-therapy-response assessment in "[ 18 F]-FDG-avid" lymphomas [21,22]. Investigating the use of [ 99m Tc]Tc-TG for this application might also be worthwhile, taking into account that current SPECT/CT technology permits the semiquantitative assessment of uptake.
Another interesting finding is that [ 99m Tc]Tc-TG is not accumulated in the brain, despite GLUT1 being the main transporter for glucose through the intact blood-brain barrier [23]. This might be because the transport of [ 99m Tc]Tc-TG through cellular membranes is impaired [4]. This phenomenon might facilitate the use of [ 99m Tc]Tc-TG for the visualization of lymphoma-associated brain lesions ( Figure 4) and brain tumors, where the blood-brain-barrier is typically disrupted.
Obviously, [ 99m Tc]Tc-TG SPECT/CT cannot compete with [ 18 F]-FDG PET/CT in countries where PET is available. First, the accumulation of [ 18 F]-FDG in lymphoma lesions is higher, which is a prerequisite for better imaging contrast. Second, sensitivity and quantification accuracy of PET is better. Still, [ 99m Tc]Tc-TG might be the only tracer for molecular imaging of lymphoma for patients living in countries with poor or no access to PET. Therefore, we believe that further clinical development of this tracer is warranted.

Conclusions
Injections of [ 99m Tc]Tc-TG appear safe and the radiation burden was comparable to the burden from [ 18 F]-FDG. The [ 99m Tc]Tc-TG showed an elevated uptake in the nodal and extranodal lesions in malignant lymphomas patients. Further studies concerning the use of [ 99m Tc]Tc-TG SPECT/CT for lymphoma staging and therapy monitoring are justified.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14061274/s1, Figure S1: Standards for Reporting of Diagnostic Accuracy Studies (STARD) flow diagram; Figure S2: Kinetics of elimination of [ 99m Tc]Tc-TG from blood. Data are based on count rates in regions of interest placed over hearts. %ID = percentage injected dose; Table S1: Parameters of blood biochemistry in patients before intravenous administration of a radiopharmaceutical and after 24, 48 h and 7 days post administration.