(4S)-4-(3-¹⁸F-Fluoropropyl)-l-Glutamate for Imaging of x_C^¯ Transporter Activity in Hepatocellular Carcinoma Using PET: Preclinical and Exploratory Clinical Studies

Radiopharmaceutical Preparation

¹⁸F-FSPG was radiolabeled as described previously (5). The investigational medicinal product ¹⁸F-FSPG for the patient study was batch-manufactured at Asan Medical Center with an automated module (Modular Lab System; Eckert & Ziegler). Each batch met the criteria listed in the specification for clarity, identity, purity, radioactive concentration, specific activity, pH, bacterial endotoxin level, and sterility. The final product was formulated for intravenous injection with a drug substance per injected unit of 300 ± 30 MBq and 100 μg or less of tracer mass and a specific activity of 18.2 GBq/μmol or more. The decay-corrected radiochemical yield was 28.6% ± 12.9% (range, 11.2%−44.8%), and the radiochemical purity was 91.8% ± 1.5% (range, 90.8%−94.3%).

In Vivo Animal Studies

Animal experiments were performed at Bayer Pharma AG in compliance with the current version of the German law concerning animal protection and welfare. The protocol was approved by the Institutional and Regional Animal Care and Use Committee. All animals used in the studies were kept under normal laboratory conditions at a temperature of 22°C ± 2°C and a dark–light rhythm of 12 h. The acclimation period was at least 7 d before the beginning of the studies. During this period, animals were examined daily to verify the absence of abnormal signs.

Huh7 liver cancer cells were obtained from the Japanese Collection of Research Bioresources, and MH3924a cells from Cell Line Services. Naval Medical Research Institute nude (NMRI-nu/nu) mice (Charles River) (female; age, 5−6 wk; weight, 20−25 g) were inoculated by orthotopic injection of 2 × 10⁶ human Huh7 liver cancer cells in a volume of 20 μL of Matrigel (BD Biosciences) plus medium without fetal calf serum. A small incision (1 cm) to the right of the linea alba was made, and the tumor cell suspension was injected into the left liver lobe. Wounds were closed using suture clips. PET studies with tumor-bearing mice were performed 12 d after tumor cell inoculation (n = 3). Three mice were injected with 125−170 μL of tracer solution containing an activity of 7.9−8.3 MBq of ¹⁸F-FSPG. The PET scan was started 50 min after injection and continued for 20 min, followed by CT scanning for 10 min.

August-Copenhagen Irish (ACI) rats (Harlan Laboratories; female; mean weight, 150 g) were inoculated 9 d before the PET study by intrahepatic injection of 3 × 10⁶ syngeneic Morris hepatoma MH3924a cells in a volume of 50 μL of Matrigel plus medium without fetal calf serum into the left liver lobe. PET studies with tumor-bearing rats were performed 9 d after tumor cell inoculation (n = 4). After the intravenous injection of 7.9−9.1 MBq of ¹⁸F-FSPG, animals were subjected to the PET scan (tracer distribution period, 60 min). PET was performed for 10 min.

All animal imaging studies were performed with a small-animal PET/CT scanner (Inveon PET; Siemens AG). Regions of interest were drawn over the tumor and normal liver, and an average value within the region of interest was generated. The results of the region-of-interest analysis were reported as the percentage injected dose per gram of tissue (%ID/g).

Exploratory Clinical Study Design

An open-label, nonrandomized, single-dose explorative study was performed to evaluate the safety, tolerability, and diagnostic performance of ¹⁸F-FSPG PET/CT in patients with HCC. The trial was registered at http://www.clinicaltrial.gov as NCT01103310. The primary outcome measure was the visual assessment of the tumor detection rate with ¹⁸F-FSPG, compared with that of ¹⁸F-FDG. The secondary outcome measures were quantitative analysis of ¹⁸F-FSPG uptake in tumors and assessment of safety variables including vital signs, laboratory findings, electrocardiogram, and adverse events. The study protocol was approved by both the Institutional Review Board of Asan Medical Center and the Korea Food and Drug Administration. This study was conducted in accordance with the Helsinki Declaration. Investigators disclosed potential conflicts to study participants. All patients provided written informed consent before participation in the study.

Patients

Patients were enrolled if they had HCC, ranged in age from 35 to 75 y, and had an ¹⁸F-FDG PET/CT scan that showed an iso- to hypermetabolic or heterogeneous metabolic liver lesion. Other inclusion criteria were an interval between ¹⁸F-FDG and ¹⁸F-FSPG PET/CT of 4 wk or less, adequate recovery (excluding alopecia) from potential previous anticancer treatment, an Eastern Cooperative Oncology Group performance status of 0–2, and adequate function of major organs. No chemotherapy, radiotherapy, or immune–biologic therapy or biopsy was allowed between the ¹⁸F-FDG and ¹⁸F-FSPG PET/CT scans. The patient had to have histologic confirmation of the tumor. However, histologic diagnosis of HCC was not necessary if the nodule was larger than 2 cm and if 2 dynamic imaging studies using contrast agents showed arterial hyperenhancement and washout in the venous phase. Patients were excluded from the study if any of the following conditions applied to them: being pregnant or lactating; having a concurrent, severe, or uncontrolled or unstable medical disease other than cancer; having a lifetime history of alcohol or drug abuse; being a relative or student of the investigator or otherwise dependent; or participating or having participated in another clinical study involving the administration of an investigational drug during the preceding 4 wk. Patients were recruited by referral from investigators.

PET/CT Procedure

¹⁸F-FDG and ¹⁸F-FSPG PET/CT images were obtained with a PET/CT scanner (Biograph True Point 40; Siemens). ¹⁸F-FDG PET/CT was performed for clinical reasons as described previously (13). ¹⁸F-FSPG PET/CT studies were performed during 3 time intervals (the first interval was from 0 to 45 min, the second was from 60 to 75 min, and the third was from 105 to 120 min). During each interval, 1 low-dose CT scan (80 kV, 31 mAs, CARE Dose4D; Siemens) without contrast medium administration was recorded for attenuation correction of the PET scan. Five consecutive images from the skull base to mid thigh were acquired for the first imaging interval of 0−45 min along with the injection of 300 ± 10 MBq of ¹⁸F-FSPG as follows: 30 s, 30 s, 1 min, 2 min, and 2 min per bed position. During the second and third imaging intervals, 1 image of the skull base to mid thigh was acquired per imaging interval with the same acquisition parameters as were applied for the previous scan.

Patients were asked to void their urinary bladder immediately after the first scan. Urinary bladder voiding was also encouraged before and after the second and third imaging sessions. Effective radiation doses for ¹⁸F-FSPG as extrapolated from biodistribution studies in mice to humans were 5.10 mSv for a male and 6.5 mSv for a female subject. The total radiation exposure from the 3 CT examinations did not exceed 3 mSv. Emission data were reconstructed by means of the Siemens TrueX reconstruction with 3 iterations and 21 subsets. No correction for partial-volume effects was performed.

Image Analysis of Clinical PET Studies

The PET/CT studies were assessed visually by the consensus of 2 board-certified nuclear medicine physicians who were informed of all available clinical and laboratory findings. The CT scans were assessed for the exact location of HCC lesions. For dynamic assessment of ¹⁸F-FSPG uptake, a spheric volume of interest with a diameter of 1.2 cm was placed on tumor, and a spheric volume of interest with a diameter of 1.5 cm was placed on the pancreas and kidneys. Volumes of interest on normal liver tissue and descending thoracic aorta (as the blood pool) were drawn as previously recommended (14). Mean standardized uptake value (SUVmean) of the volume of interest were obtained on each time frame to generate a time–activity profile of the ¹⁸F-FSPG uptake. All standardized uptake values (SUVs) were normalized to the injected dose and the patients’ body weight and were defined as follows:SUV=activity (Bq/g)/(injected activity [Bq]/body weight [g]).

The ¹⁸F-FSPG PET/CT image acquired 60 min after injection was used for visual and quantitative analysis. The number, location, size, and intensity of all abnormal uptake values were described relative to the background uptake in normal comparable tissues. Intensity features were classified as absent, minor, or major accumulation. Mildly increased uptake higher than that of the background was defined as minor accumulation. Moderately or markedly increased uptake, compared with the background, was considered major accumulation. Lesions with minor or major accumulation were regarded as positive. Selected lesions were visually compared with the uptake of ¹⁸F-FDG. A 3-step score was applied: visibility with ¹⁸F-FSPG was lower than, as good as, or better than that in the ¹⁸F-FDG PET image. The maximal standardized uptake value (SUVmax) of each selected tumor lesion was measured using the single maximum pixel count within the lesion. The SUV ratio (SUVR) was obtained by calculating the ratio of the SUVmax of the tumor lesion to the SUVmean of normal liver.

Safety Monitoring

Adverse events were continuously recorded, beginning with patient enrolment until the last patient contact on days 3–8 after ¹⁸F-FSPG administration. The safety of ¹⁸F-FSPG was assessed using blood parameters (hematology, clotting status, serum chemistry), semiquantitative urine dip-stick measurements, vital signs, electrocardiograms, and physical examinations at baseline and at about 2 and 24 h after the intravenous administration of ¹⁸F-FSPG.

Immunohistochemistry of x_C^¯ Transporter and CD44

Tumor tissues obtained during surgery for routine diagnostic pathologic examinations were used for immunohistochemistry studies of the xCT subunit of system x_C^¯ and CD44. Formalin-fixed, paraffin-embedded tissue sections were immunohistochemically stained for xCT and CD44 with a Benchmark automatic immunostaining device (Ventana Medical Systems). Five-micrometer-thick sections, obtained with a microtome, were transferred onto adhesive slides and dried at 62°C for 30 min. After incubation with primary antibodies against the xCT subunit (1:250 dilution; NB300-318, polyclonal antixCT antibody [Novus Biologicals]) and CD44 (1:100 dilution; Clone DF 1485 [DakoCytomation]), the slides were incubated using a biotinylated, antimouse secondary antibody. Then, antigens were detected using peroxidase-labeled streptavidin, a streptavidin–biotin kit, and the 3,3′-diaminobenzidine chromogen as a substrate. The level of xCT and CD44 was examined by an experienced pathologist who was completely masked to any patient and imaging information. The results of the immunoassay for xCT and CD44 were semiquantitatively assessed on a scale of 0, 1 (weak), 2 (medium), or 3 (strong), with a sample being reported as positive if more than 10% of the cells in the sample were positively stained.

Statistical Analysis

Data were reported as the mean ± SD unless otherwise specified. A P value of less than 0.05 was considered to be statistically significant. Quantitative parameters were compared using the Mann–Whitney U test. All statistical tests were performed with SPSS Statistics (version 19; SPSS, Inc., IBM Co.) for Windows (Microsoft).