Radiosynthesis and Preliminary Evaluation of [11C]SSI-4 for the Positron Emission Tomography Imaging of Stearoyl CoA Desaturase 1.

Stearoyl CoA desaturase 1 (SCD1) is the rate-limiting enzyme for converting saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs) and plays a key role in endogenous (de novo) fatty acid metabolism. Given that this pathway is broadly upregulated across many tumor types with an aggressive phenotype, SCD1 has emerged as a compelling target for cancer imaging and therapy. The ligand 2-(4-(2-chlorophenoxy)piperidine-1-carboxamido)-N-methylisonicotinamide (SSI-4) was identified as a potent and highly specific SCD1 inhibitor with a strong binding affinity for SCD1 at our laboratory. We herein report the radiosynthesis of [11C]SSI-4 and the preliminary biological evaluation including in vivo PET imaging of SCD1 in a human tumor xenograft model. Radiotracer [11C]SSI-4 was labeled at the carbamide position via the direct [11C]CO2 fixation on the Synthra MeIplus module in high molar activity and good radiochemical yield. In vitro cell uptake assays were performed with three hepatocellular carcinoma (HCC) cell lines and three renal cell carcinoma (RCC) cell lines. Additionally, in vivo small animal PET/CT imaging with [11C]SSI-4 and the biodistribution were carried out in a mouse model bearing HCC xenografts. Radiotracer [11C]SSI-4 afforded a 4.14 ± 0.44% (decay uncorrected, n = 10) radiochemical yield based on starting [11]CO2 radioactivity. The [11C]SSI-4 radiosynthesis time including HPLC purification and SPE formulation was 25 min from the end of bombardment to the end of synthesis (EOS). The radiochemical purity of [11C]SSI-4 was 98.45 ± 1.43% (n = 10) with a molar activity of 225.82 ± 33.54 GBq/µmol (6.10 ± 0.91 Ci/µmol) at the EOS. In vitro cell uptake study indicated all SSI-4 responsive HCC and RCC cell line uptakes demonstrate specific uptake and are blocked by standard compound SSI-4. Preliminary small animal PET/CT imaging study showed high specific uptake and block of [11C]SSI-4 uptake with co-injection of cold SSI-4 in high SCD1-expressing organs including lacrimal gland, brown fat, liver, and tumor. In summary, novel radiotracer [11C]SSI-4 was rapidly and automatedly radiosynthesized by direct [11C]CO2 fixation. Our preliminary biological evaluation results suggest [11C]SSI-4 could be a promising radiotracer for PET imaging of SCD1 overexpressing tumor tissues.

Improving Circulation Half-Life of Therapeutic Candidate N-TIMP2 by Unfolded Peptide Extension.

Matrix Metalloproteinases (MMPs) are drivers of many diseases including cancer and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are human proteins that inhibit MMPs and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties of a drug candidate, such as complete MMP inhibition, low toxicity and immunogenicity, high tissue permeability and others. A major challenge with TIMPs, however, is their formulation and delivery, as these proteins are quickly cleared from the bloodstream due to their small size. In this study, we explore a new method for plasma half-life extension for the N-terminal domain of TIMP2 (N-TIMP2) through appending it with a long intrinsically unfolded tail containing a random combination of Pro, Ala, and Thr (PATylation). We design, produce and explore two PATylated N-TIMP2 constructs with a tail length of 100- and 200-amino acids (N-TIMP2-PAT100 and N-TIMP2-PAT200, respectively). We demonstrate that both PATylated N-TIMP2 constructs possess apparent higher molecular weights compared to the wild-type protein and retain high inhibitory activity against MMP-9. Furthermore, when injected into mice, N-TIMP2-PAT200 exhibited a significant increase in plasma half-life compared to the non-PATylated variant, enhancing the therapeutic potential of the protein. Thus, we establish that PATylation could be successfully applied to TIMP-based therapeutics and offers distinct advantages as an approach for half-life extension, such as fully genetic encoding of the gene construct, mono-dispersion, and biodegradability. Furthermore, PATylation could be easily applied to N-TIMP2 variants engineered to possess high affinity and selectivity toward individual MMP family members, thus creating attractive candidates for drug development against MMP-related diseases.