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Synthesis of an In vivo MRI-detectable Apoptosis Probe
Published on: July 31, 2012
Development of caspase-3-selective activity-based probes for PET imaging of apoptosis
Louis Lauwerys1,2, Lucas Beroske1,2, Angelo Solania3
1Molecular Imaging and Radiology, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Antwerp, Belgium.
Background:
The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging.
Results:
A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-βhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far.
Conclusions:
In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.
Insights
Researchers developed novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging. While the PET tracer [18F]MICA-316 showed promise in vitro, it had limited in vivo tumor uptake, highlighting the need for improved probes for cancer treatment response evaluation.
Area of Science:
- Biomedical Imaging
- Molecular Imaging
- Cancer Research
Background:
- Caspase-3 is a key executioner protease in apoptosis, making it a potential biomarker for cancer therapy response.
- Previous PET tracers for caspase-3 lacked sufficient tumor uptake or selectivity for clinical use.
- Developing selective activity-based probes (ABPs) is crucial for effective apoptosis imaging in cancer patients.
Purpose of the Study:
- To develop and preclinically evaluate novel caspase-3-selective ABPs for apoptosis imaging.
- To identify a PET tracer capable of accurately assessing treatment response by detecting tumor cell death.
Main Methods:
- Synthesis and 18F-labeling of a library of caspase-3-selective ABPs.
- In vitro evaluation of binding kinetics and selectivity.
- Preclinical testing of promising tracers in in vitro apoptosis models and in vivo colorectal cancer models.
- Comparison with a known cell death imaging agent, [99mTc]Tc-duramycin.
Main Results:
- A second-generation ABP based on Ac-ATS010-KE showed improved binding kinetics over the first generation.
- [18F]MICA-316 demonstrated increased uptake in apoptotic cells in vitro.
- In vivo, [18F]MICA-316 exhibited limited tumor uptake and could not differentiate treated from untreated tumors.
- [99mTc]Tc-duramycin effectively detected increased cell death, outperforming the developed PET tracers.
Conclusions:
- A novel library of caspase-3-binding PET tracers was developed with binding kinetics similar to the parent inhibitor.
- [18F]MICA-316 selectively bound caspase-3 in apoptotic cells in vitro but showed insufficient in vivo performance.
- Future generations of caspase-3-selective ABPs require enhanced tumor accumulation and in vivo stability for effective cancer treatment response assessment.
