Sibaprasad Bhattacharyya

Synthesis of Clinical-Grade [18F]-Fluoroestradiol as a Surrogate PET Biomarker for the Evaluation of Estrogen Receptor-Targeting Therapeutic Drug

16α-[18F]-fluoroestradiol ([18F]FES), a steroid-based positron emission tomography (PET) tracer, has emerged as a dependable tracer for the evaluation and management of estrogen receptor-positive (ER+) breast cancer patients. We have developed a fully automatic, one-pot procedure for the synthesis of [18F]FES using the Eckert & Ziegler (E & Z) radiomodular system. After [18F]fluorination, the intermediate was hydrolyzed with 2.0 M HCl twice and neutralized with sodium bicarbonate. After high-performance liquid chromatography (HPLC) purification, the decay-corrected radiochemical yield and purity of [18F]FES were 40 ± 5.0% (n = 12) and >97%, respectively. The product was stable up to 10 h. Total synthesis time including HPLC purification was 80 min. This new, fully automated rapid synthetic procedure provided high and reproducible yields of [18F]FES. Quality control (QC) tests showed that the [18F]FES produced by this method met all specifications for human injection.

Synthesis and biological evaluation of panitumumab–IRDye800 conjugate as a fluorescence imaging probe for EGFR-expressing cancers

To investigate panitumumab-IRDye800 as an intraoperative optical imaging agent for epidermal growth factor receptor (EGFR)-expressing cancers, we developed clinical-quality panitumumab-IRDye800 and evaluated its specificity and sensitivity to visualize tumors by fluorescence imaging in a variety of mouse xenograft models with different levels of EGFR-expression. Panitumumab was chemically conjugated to NIR-dye (Li-COR 800CW) at well-defined and limited substitution ratio (1:1-2) for the characterization of fluorescence signals. Yield and purity of the conjugate was 80±5% and 95±2% respectively (n= 6). Quality control (QC) tests showed that product was suitable for clinical development. Female athymic nude xenograft tumor bearing mice (n=5 per tumor model) with very low (BT-474), moderate (MDA-MB-231), and high (MDA-MB-468) EGFR-expression levels were administered panitumumab-IRDye800 formulations (100 μg of mAb in 100 μL of 0.9% saline) via tail-vein injection. Animal imaging and biodistribution experiments were conducted on the FMT 2500 (Perkin Elmer) fluorescence scanner at 24, 48, 72, 96, and 144 hours post injection. Immuno-fluorescence images of panitumumab-IRDye conjugate recorded in mouse xenograft models showed a good correlation (R2 = 0.91) between EGFR-expression level and tumor uptake. Uptake of panitumumab labeled with IR-Dye or [89Zr] in different tumor xenografts with high, medium, and low EGFR expression, as measured by fluorescence or radioactive counts are highly correlated (r2= 0.99). This preclinical in-vivo study proved that panitumumab-IRDye800 is specific and optical imaging in conjunction with this probe is sensitive enough to detect EGFR-expressing tumors.

Imaging the Met Receptor Tyrosine Kinase (Met) and Assessing Tumor Responses to a Met Tyrosine Kinase Inhibitor in Human Xenograft Mouse Models with a [99mTc] (AH-113018) or CY 5** (AH-112543) Labeled Peptide

Developing an imaging agent targeting the hepatocyte growth factor receptor protein (Met) status of cancerous lesions would aid in the diagnosis and monitoring of Met-targeted tyrosine kinase inhibitors (TKIS). A peptide targeting Met labeled with [99mTc] had high affinity in vitro (Kd = 3.3 nM) and detected relative changes in Met in human cancer cell lines. In vivo [99mTc]-Met peptide (AH-113018) was retained in Met-expressing tumors, and high-expressing Met tumors (MKN-45) were easily visualized and quantitated using singlephoton emission computed tomography or optical imaging. In further studies, MKN-45 mouse xenografts treated with PHA 665752 (Met TKI) or vehicle were monitored weekly for tumor responses by [99mTc]-Met peptide imaging and measurement of tumor volumes. Tumor uptake of [“mTc]-Met peptide was significantly decreased as early as 1 week after PHA 665752 treatment, corresponding to decreases in tumor volumes. These results were comparable to Cy5**-Met peptide (AH-112543) fluorescence imaging using the same treatment model. [99mTc] or Cy5**-Met peptide tumor uptake was further validated by histologic (necrosis, apoptosis) and immunoassay (total Met, p Met, and plasma shed Met) assessments in imaged and nonimaged cohorts. These data suggest that [99mTc] or Cy5**-Met peptide imaging may have clinical diagnostic, prognostic, and therapeutic monitoring applications.

Rapid synthesis of [18F]fluoroestradiol: remarkable advantage of microwaving over conventional heating

16α-[(18)F]fluoroestradiol ([(18)F]FES) is known as a clinically important tracer in nuclear medicine as an estrogen receptor ligand for investigating primary and metastatic breast cancers. Synthesizing [(18)F]FES is a two-step process associated with [(18)F]fluoride incorporation to the precursor (3-methoxymethyl 16β,17β-epiestriol-O-cyclic sulfone) and subsequent hydrolysis of the [(18)F]fluorinated intermediate with 2 N HCl. The impact of microwave (MW) heating on both fluorination and hydrolysis reactions was investigated. The duration and temperatures of the fluorination reaction were varied for both MW heating and conventional heating (CH) methods. Chemical and radiochemical purity and radiochemical yields were investigated for CH and compared with MW-assisted radiosyntheses. Quality control tests of MW-assisted [(18)F]FES were performed following US Pharmacopeia procedures for clinical-grade positron emission tomography pharmaceuticals. The results demonstrate that microwaving not only improves the (18)F-fluoride incorporation (~55% improvement at 110°C for 4 min) but also significantly reduces hydrolysis time (approximately sevenfold reduction at 120°C) in comparison with CH under similar conditions. The overall isolated radiochemical yield of purified [(18)F]FES was significantly higher (~90% improvement) with MW, and side products were notably fewer. Quality control test results demonstrated that [(18)F]FES produced by microwaving was suitable for human injection.