Radiopharmacological characterisation of an 18F-labelled azadipeptide nitrile as novel probe for molecular imaging of the tumour-associated cathepsin activity

Objectives: The molecular processes leading to tumour invasion and metastasis are closely associated with a network of intra- and extracellular proteolysis. A prominent role in this network was assigned to the thioldependent cathepsins [1]. Among this class of cysteine proteases, the involvement in tumour pathology could be confirmed for the cathepsins L, S, B, and K.
Due to the vital role of these enzymes in tumour biology, molecular probes that enable their functional imaging in vivo by PET are highly desirable. To this end, an inhibitor of the azadipeptide nitrile chemotype [2] labelled with fluorine-18 was developed. Its radiopharmacological characterisation in normal and tumour-bearing animals to evaluate its potential for PET imaging was the objective of this study.
Methods: Compound 1 (Figure 1A) could be reliably prepared in its ¹⁸F-labelled version in a one-pot, two-step process from the corresponding compound bearing the free hydroxy group with intermediary 2-[¹⁸F]fluoroethyl nosylate. Its high affinity to the cathepsins L, S, B, and K is reflected by inhibition constants ranging from 0.17 nM (cathepsin K) to 2.4 nM (cathepsin B). The radiopharmacological behaviour of [¹⁸F]1 in normal rats was studied in vitro investigating its stability in rat blood, ex vivo by determining its biodistribution and metabolite analysis as well as in vivo by dynamic PET imaging. To evaluate the potential of [¹⁸F]1 for imaging of the tumour-associated cathepsin activity its fate in nude mice bearing the tumours derived from the human alveolar carcinoma cell line NCI-H292 was followed with dynamic PET. The expression of the target enzymes in these tumours was studied by immunohistochemistry.
Results: Biodistribution data for [¹⁸F]1 after 60 min indicated that renal filtration is the main elimination pathway. Another large part is remaining in the blood, which is obvious from dynamic PET imaging as well (Figure 1B).
This could be partly attributed to the reaction of [¹⁸F]1 with glutathione in the blood. The glutathione conjugate of [¹⁸F]1 can be also found in the urine and the gut besides the corresponding cysteine conjugate.
Evaluation of the kinetics of [¹⁸F]1 in NCI-H292 tumour bearing nude mice revealed its accumulation in the tumours with tumour to muscle ratios up to 10 after 2 h (not shown here) which indicates a specific tumour uptake.
This conclusion is further supported by the fact that all four cathepsins could be detected immunohistochemically in the tumour sections.
Conclusions: The in vivo behaviour of [¹⁸F]1 could be well characterised in normal rats as well as in NCI-H292 tumour mice. Despite suboptimal pharmacokinetics due to its inherent thiol reactivity, [¹⁸F]1 has the potential for imaging the tumour-associated cathepsin activity, which will be confirmed in further studies.
References: [1] Mason SD, et al. (2011) Trends Cell Biol, 21, 228-37.
[2] Löser R, et al. (2008) Angew Chem Int Ed, 47, 4331-4.