Hancheng Cai

Tetrazine–trans-cyclooctene ligation for the rapid construction of 18F labeled probes

A radiolabeling method for bioconjugation based on the Diels-Alder reaction between 3,6-diaryl-s-tetrazines and an (18)F-labeled trans-cyclooctene is described. The reaction proceeds with exceptionally fast rates, making it an effective conjugation method within seconds at low micromolar concentrations.

Synthesis of a novel bifunctional chelator AmBaSar based on sarcophagine for peptide conjugation and 64Cu radiolabelling

Copper-64 shows promise as both a suitable PET imaging and therapeutic radionuclide due to its nuclear characteristics. Stable attachment of radioactive (64)Cu(2+) to targeted imaging probes requires the use of a bifunctional chelator. Sarcophagine (Sar) ligands coordinate the metal ion (64)Cu(2+) within the multiple macrocyclic rings comprising the cage structure, yielding extraordinarily stable complexes that are inert to dissociation of the metal ion in vivo. Several (64)Cu labelled RGD derivatives have been applied in imaging of the alpha(nu)beta(3) integrin receptor expression during tumour angiogenesis. In order to design and develop novel molecular imaging probes containing RGD and Sar ligands, we designed a novel versatile Sar cage-like bifunctional chelator named AmBaSar, synthesized using a conventional synthetic strategy. Conjugation with the cyclic peptide RGD, and subsequent labelling with (64)Cu, provided a new PET probe (64)Cu-AmBaSar-RGD for imaging the alpha(nu)beta(3) integrin receptor.

Evaluation of Copper-64 Labeled AmBaSar Conjugated Cyclic RGD Peptide for Improved MicroPET Imaging of Integrin αvβ3 Expression

Recently, we have developed a new cage-like bifunctional chelator 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) for copper-64 labeling and synthesized the positron emission tomography (PET) tracer (64)Cu-AmBaSar-RGD. In this study, we further evaluate the biological property of this new AmBaSar chelator by using (64)Cu-AmBaSar-RGD as the model compound. In vitro and in vivo stability, lipophilicity, cell binding and uptake, microPET imaging, receptor blocking experiments, and biodistribution studies of (64)Cu-AmBaSar-RGD were investigated, and the results were directly compared with the established radiotracer (64)Cu-DOTA-RGD. The (64)Cu-AmBaSar-RGD was obtained with high radiochemical yield (> or =95%) and purity (> or =99%) under mild conditions (pH 5.0-5.5 and 23-37 degrees C) in less than 30 min. For in vitro studies, the radiochemical purity of (64)Cu-AmBaSar-RGD was more than 97% in PBS or FBS and 95% in mouse serum after 24 h of incubation. The log P value of (64)Cu-AmBaSar-RGD was -2.44 +/- 0.12. For in vivo studies, (64)Cu-AmBaSar-RGD and (64)Cu-DOTA-RGD have demonstrated comparable tumor uptake at selected time points on the basis of microPET imaging. The integrin alpha(v)beta(3) receptor specificity was confirmed by blocking experiments for both tracers. Compared with (64)Cu-DOTA-RGD, (64)Cu-AmBaSar-RGD demonstrated much lower liver accumulation in both microPET imaging and biodistribution studies. Metabolic studies also directly supported the observation that (64)Cu-AmBaSar-RGD was more stable in vivo than (64)Cu-DOTA-RGD. In summary, the in vitro and in vivo evaluations of the (64)Cu-AmBaSar-RGD have demonstrated its improved Cu-chelation stability compared with that of the established tracer (64)Cu-DOTA-RGD. The AmBaSar chelator will also have general applications for (64)Cu labeling of various bioactive molecules in high radiochemical yield and high in vivo stability.

Design, synthesis and validation of integrin α2β1-targeted probe for microPET imaging of prostate cancer.

PurposeThe ability of PET to aid in the diagnosis and management of recurrent and/or disseminated metastatic prostate cancer may be enhanced by the development of novel prognostic imaging probes. Accumulating experimental evidence indicates that overexpression of integrin α2β1 may correlate with progression in human prostate cancer. In this study, 64Cu-labeled integrin α2β1-targeted PET probes were designed and evaluated for the imaging of prostate cancer.MethodsDGEA peptides conjugated with a bifunctional chelator (BFC) were developed to image integrin α2β1 expression with PET in a subcutaneous PC-3 xenograft model. The microPET images were reconstructed by a two-dimensional ordered subsets expectation maximum algorithm. The average radioactivity accumulation within a tumor or an organ was quantified from the multiple region of interest volumes.ResultsThe PET tracer demonstrated prominent tumor uptake in the PC-3 xenograft (integrin α2β1-positive). The receptor specificity was confirmed in a blocking experiment. Moreover, the low tracer uptake in a CWR-22 tumor model (negative control) further confirmed the receptor specificity.ConclusionThe sarcophagine-conjugated DGEA peptide allows noninvasive imaging of tumor-associated α2β1 expression, which may be a useful PET probe for evaluating the metastatic potential of prostate cancer.

Novel α2β1 Integrin-Targeted Peptide Probes for Prostate Cancer Imaging

Accumulating experimental evidence indicates that overexpression of α2β1 integrin may correlate with progression in human prostate cancer. The objective of this study was to design a novel imaging probe based on the Asp-Gly-Glu-Ala (DGEA) peptide for near-infrared-fluorescent (NIRF) imaging of α2β1, integrin expression in prostate cancer. The peptides were conjugated with appropriate fluorescent dyes, and the binding affinity of these probes was evaluated by flow cytometry in three human prostate cell lines (PC-3, CWR-22, and LNCaP). In vivo NIRF imaging of the α2β1-positive PC-3 xenograft model was performed to evaluate the α2β1 targeted probe. In vitro immunofluorescence staining was carried out to confirm the α2β1 integrin expression level. Flow cytometry analysis showed that PC-3 had the highest probe uptake, followed by CWR-22 and LNCaP tumor cells. In the subcutaneous PC-3 model, the tumor demonstrated prominent uptake with good tumor to background contrast. Immunohistochemistry staining also supported the in vivo optical imaging results. DGEA-based optical agents have been developed for specific imaging of α2β1, integrin expression. In vitro and in vivo localization demonstrated the potential of this agent to identify tumor subtypes amenable to anti-α2β1 integrin treatment and potentially provide prognostic information regarding tumor progression.

Biological characters of [18F]O-FEt–PIB in a rat model of Alzheimer's disease using micro-PET imaging

AbstractAim:To evaluate whether the newly-synthesized positron emission tomography (PET) tracer, [18F]2-(4′-(methylamino)phenyl)-6-fluoroethoxy- benzothiazole ([18F]O-FEt-PIB), could bind to β-amyloid aggregates in a rat model of Alzheimers disease (AD) using micro-PET.Method:[18F]O-FEt-PIB was synthesized and purified by radio HPLC. PET imaging was performed with a R4 rodent model scanner in 3 model and 3 control rats. Dynamic PET scans were performed for 40 min in each rat following an injection of approximately 37 MBq of [18F]O-FEt-PIB. Static scans were also performed for 15 min in each rat. PET data were reconstructed by a maximum posteriori probability algorithm. On the coronal PET images, regions of interest were respectively placed on the cortex, hemicerebrum [including the hippocampus and thalamus (HT)], and were guided by a 3-D digital map of the rat brain or the brain images of [18F]2-Deoxy-2-fluoro-D-glucose ([18F]FDG) in normal rats. Time-activity curves (TAC) were obtained for the cerebrum and cerebellum. The activity difference value (ADV) between 2 hemicerebrums was also calculated.Results:The TAC for [18F]O-FEt-PIB in the cerebrum or cerebellum peaked early (at approximately 2 min), but washed out a little slowly. In the dynamic and static micro-PET images, increased radioactivity was found in the area of the right HT in the model rats where infused with β-amyloid (1–40). No distinct difference of radioactivity was found between the right and left HT areas in the control rats. The ADV(HT) was approximately 14.6% in the AD model rats and approximately 4 times greater than that of the control rats (3.9%).Conclusion:To our knowledge, this study is the first to evaluate a small molecular PET probe for the β-amyloid deposits in vivo using micro-PET imaging in an AD-injected rat model. The suitable biological characters showed that the tracer had potential to be developed as a probe for detecting β-amyloid plaques in AD.

Radiolabeling and in vitro and in vivo Characterization of [18F]FB‐[R8,15,21, L17]‐VIP as a PET Imaging Agent for Tumor Overexpressed VIP Receptors

In an effort to develop a peptide‐based radiopharmaceutical for the detection of tumors overexpressed vasoactive intestinal peptide receptors with positron emission tomography, we have prepared a novel [R8,15,21, L17]‐VIP peptide for 18F‐labeling. This peptide inhibited 125I‐VIP binding to rats lung membranes with high affinity [half‐maximal inhibitory concentrations (IC50) of 0.12 nm]. Additionally, [R8,15,21, L17]‐VIP showed higher stability than native vasoactive intestinal peptide in vivo of mice. With N‐succinimidyl 4‐[18F] fluorobenzoate as labeling prosthetic group, [18F]FB‐[R8,15,21, L17]‐VIP was obtained in >99% radiochemical purity within 100 min in decay‐for‐corrected radiochemical yield of 33.6 ± 3% (n = 5) and a specific radioactivity 255 GBq/μmol at the end of synthesis. Stability of [18F]FB‐[R8,15,21, L17]‐VIP in vitro and in vivo were investigated. Biodistribution of this trace was carried out in mice with induced C26 colorectal tumor. Fast clearance of [18F]FB‐[R8,15,21, L17]‐VIP from non‐target tissues and specific uptakes by tumors realized higher tumor‐to‐muscle ratio (3.55) and tumor‐to‐blood ratio (2.37) 60 min postinjection. Clear difference was observed between the blocking and unblocking experiments in biodistribution and whole body radioautography. [18F]FB‐[R8,15,21, L17]‐VIP has demonstrated its potential for diagnosing tumors overexpressed vasoactive intestinal peptide receptors both in vitro and in vivo.

Radiosynthesis of 11C-Levetiracetam:A Potential Marker for PET Imaging of SV2A Expression

The multistep preparation of (11)C-levetiracetam ((11)C-LEV) was carried out by a one-pot radiosynthesis with 8.3 ± 1.6% (n = 8) radiochemical yield in 50 ± 5.0 min. Briefly, the propionaldehyde was converted to propan-1-imine in situ as labeling precursor by incubation with ammonia. Without further separation, the imine was reacted with (11)C-HCN to form (11)C-aminonitrile. This crude was then reacted with 4-chlorobutyryl chloride and followed by hydrolysis to yield (11)C-LEV after purification by chiral high-performance liquid chromatography (HPLC). Both the radiochemical and enantiomeric purities of (11)C-LEV were >98%.

Biological stability evaluation of the α2β1 receptor imaging agents: diamsar and DOTA conjugated DGEA peptide.

Robust chelating stability under biological condi-tions is critical for the design of copper-based radiopharmaceuticals. In this study, the stabilities of (64)Cu-DOTA and diamsar (two bifunctional Cu-64 chelators (BFCs)) conjugated DGEA peptides were evaluated. The in vitro stabilities of (64)Cu-DOTA-DGEA, (64)Cu-DOTA-Ahx-DGEA, and (64)Cu-Z-E(diamsar)-Ahx-DGEA were evaluated in PBS. A carboxyl-protected DOTA-DGEA was also synthesized to study the potential inter- and intramolecular interactions between DOTA and the carboxylate groups of DGEA peptide. microPET imaging of (64)Cu-DOTA-DGEA and (64)Cu-Z-E(diamsar)-Ahx-DGEA were performed in PC-3 prostate tumor model to further investigate the in vivo behavior of the tracers. DOTA-DGEA, DOTA-Ahx-DGEA, Z-E(diamsar)-Ahx-DGEA, and protected DOTA-DGEA peptides were readily obtained, and their identities were confirmed by MS. (64)Cu(2+) labeling was performed with high radiochemical yields (>98%) for all tracers after 1 h incubation. Stability experiments revealed that (64)Cu-DOTA-DGEA had unexpectedly high (64)Cu(2+) dissociation when incubated in PBS (>55% free (64)Cu(2+) was observed at 48 h time point). The (64)Cu(2+) dissociation was significantly reduced in the carboxyl-protected (64)Cu-DOTA-DGEA complex but not in the (64)Cu-DOTA-Ahx-DGEA complex, which suggests the presence of competitive binding for (64)Cu(2+) between DOTA and the carboxyl groups of the DGEA peptide. In contrast, no significant (64)Cu(2+) dissociation was observed for (64)Cu-Z-E(diamsar)-Ahx-DGEA in PBS. For microPET imaging, the PC-3 tumors were clearly visualized with both (64)Cu-DOTA-DGEA and (64)Cu-Z-E(diamsar)-Ahx-DGEA tracers. However, (64)Cu-DOTA-DGEA demonstrated 5× higher liver uptake than (64)Cu-Z-E(diamsar)-Ahx-DGEA. This biodistribution variance could be attributed to the chelating stability difference between these two tracers, which correlated well with the PBS stability experiments. In summary, the in vitro and in vivo evaluations of (64)Cu-Z-E(diamsar)-Ahx-DGEA and (64)Cu-DOTA-DGEA have demonstrated the significantly superior Cu-chelation stability for the diamsar derivative compared with the established DOTA chelator. The results also suggest that diamsar may be preferred for Cu chelation especially when multiple carboxylic acid groups are present. Free carboxyl groups may naturally compete with DOTA for (64)Cu(2+) binding and therefore reduce the complex stability.

64Cu labeled ambasar-RGD2 for micro-PET imaging of integrin αvβ3expression

Integrin αvβ3 plays a critical role in tumor-induced angiogenesis and metastasis. Previously, a 64Cu-AmBaSar- RGD monomer with high in vivo stability compared with 64Cu-DOTA-RGD was developed for integrin αvβ3 PET imaging. It has been established that dimeric RGD peptides have higher receptor-binding affinity and superior in vivo kinetics compared with monomeric RGD peptides due to the polyvalency effect. In this context, we synthesized and evaluated 64Cu-labeled AmBaSar dimeric RGD conjugates (64Cu-AmBaSar-RGD2) for PET imaging of integrin αvβ3 expression. The dimeric RGD peptide was conjugated with a cage-like chelator AmBaSar and labeled with 64Cu. Cell binding, microPET imaging, receptor blocking, and biodistribution studies of 64Cu-AmBaSar-RGD2 were conducted in the U87MG human glioblastoma xenograft model. AmBaSar-RGD2 conjugate was obtained in reasonable yield (45.0 ± 2.5%, n= 4) and the identity was confirmed by HPLC and MS (found 1779.8, calculated m/z for [M+H]+ M: C81H125N27O19 1779.9). 64Cu-AmBaSar-RGD2 was obtained with high radiochemical yield (92.0 ± 1.3%) and purity (≥ 98.0%) under mild conditions (pH 5.0∼5.5, 23∼37 °C) in 30 min. The specific activity of 64Cu-AmBaSar-RGD2 was estimated to be 15-22 GBq/μmol at the end of synthesis. Based on microPET imaging and biodistribution studies, 64Cu-AmBaSar-RGD2 has demonstrated higher tumor uptake at selected time points than 64Cu-AmBaSar-RGD. At 20 h p.i., the tumor uptake reached 0.65 ± 0.05 %ID/g for 64Cu-AmBaSar-RGD and 1.76 ± 0.38 %ID/g for 64Cu-AmBaSar-RGD2, respectively. The integrin αvβ3 targeting specificity was confirmed by blocking experiments. Therefore, the new tracer 64Cu-AmBaSar- RGD2 exhibited better tumor-targeting efficacy and more favorable in vivo pharmacokinetics than the 64Cu labeled RGD monomer due to the polyvalency effect.