Sensitivity evaluation of [C-11]TMP for PET reporter gene imaging

Objectives Trimethoprim (TMP) is a highly specific small molecule inhibitor of E. coli dihydrofolate reductase (Ec DHFR). Radiotracer derivatives of TMP such as [C-11]TMP have great potential as PET probes when paired with an Ec DHFR reporter gene and we have recently characterized the biodistribution of [C-11]TMP in mice with Ec DHFR xenografted tumors.¹ However, it is critical to quantify the number of cells detectable with a probe-reporter gene pair to show the sensitivity of the system.² In these experiments, we determined imaging sensitivity (lower limit of the number of DHFR cells detectable) using [C-11]TMP as well as evaluated excretion and metabolism pattern of the radiotracer.

Methods  [C-11]TMP was radiolabeled from phenol precursor using [C-11]methyl iodide in the presence of NaOH and the precursor was prepared via a known method.³  Sensitivity experiments with [C-11]TMP to report the location and number of cells were performed using HEK 293 reporter cells at concentrations of 3x10⁶, 3x10⁵, and 3x10⁴ cells suspended in 0.1 mL of matrigel and injected into the sub-cutaneous tissues of Nu/Nu mice. Small animal PET was performed the next day, and images were acquired from 60-90 min post-injection of [C-11]TMP. The xenografts were harvested and assayed ex vivo on a gamma counter. To confirm the identity of the activity in the bladder and renal excretion, urine was collected and analyzed by radio-TLC and phosphor-imaging and autoradiography of the gastrointestinal (GI) tract with [C-11]TMP was performed.

Results  [C-11]TMP can detect as few as 300,000 cells as assayed by excised matrigel/tissue, and shows over 2-fold increased uptake of [C-11]TMP in 300,000 cells compare to muscle (p<0.05). Autoradiography of GI tract shows accumulation of radioactivity in the small bowel, likely related to hepatobiliary excretion of tracer, with relatively low levels of uptake in the cecum and the large bowel. No metabolites were seen in the urine, the primary route of metabolism. CONCLUSIONS: [C-11]TMP is a highly sensitive radiotracer for Ec DHFR cells (limit was 300,000 cells), which is comparable to hsvTK/[F-18]FEAU at 4h.² Additionally, we have characterized the favorable routes of metabolism of [C-11]TMP, showing no metabolites in the urine and little off target uptake in the large bacterial compartment located in the cecum and large bowel. REFERENCES: [1] J Nucl Med, 2015; 56: no. supplement 3, 615; [2] J Nucl Med, 2015; 56:1055-1060; [3] ChemBioChem. 2007; 8: 767-774