Design, (radio)synthesis and applications of radiolabelled matrix metalloproteinase inhibitors for PET

Ontregeling van de activiteit van ‘matrix metalloproteinases’ (MMPs) en ‘a disintegrin and metalloproteinases’ (ADAMs) treedt op bij een groot aantal ziekten, waaronder ontstekingen en kanker. Het meten van de activiteit van MMPs en ADAMs in vivo op een niet-invasieve wijze door middel van radioactief gemerkte synthetische remmers en nucleaire beeldvorming, zou de mogelijkheid bieden om ontstekingshaarden en kanker beter te karakteriseren. Het doel van dit proefschrift is het ontwerp, de bereiding en evaluatie van MMP/ADAM remmers met name voor Positron Emissie Tomografie (PET) om het profiel van MMP en ADAM aktiviteit in vivo te kunnen bepalen. Dit proefschrift beschrijft de bereiding van vier nieuwe MMP/ADAM remmers met een hydroxamaat groep die zijn gemerkt met fluor-18, en PET-studies met deze nieuwe stoffen in kleine proefdieren. Allereerst werd er een op een peptide gelijkende remmer bereid, te weten [18F]FB-ML5. Dit gebeurde via een directe acylering van de remmer ML5 met N-succinimidyl-4-[18F]fluorbenzoaat. Deze nieuwe tracer werd ge-evalueerd in tumor-dragende muizen (HT1080 fibrosarcoma) en in een muizenmodel van een chronische obstructieve longaandoening. Daarnaast werden er drie fluor-18 gemerkte MMP/ADAM remmers met een piperazine groep ontwikkeld. [18F]-1A en [18F]-2 werden bereid met behulp van een homoaromatische nucleofiele substitutie van hun corresponderende nitro uitgangsstoffen, terwijl [18F]-1B werd gemaakt met click chemie. Deze drie tracers werden ge-evalueerd in een HT1080 xenograft muizenmodel. Alle tracers gaven uitstekende in vitro resultaten (IC50 waarden voor binding aan MMP-9, -12 en ADAM-17 varieerden van 10-9 tot 10-7 M). Echter, de in vivo resultaten waren teleurstellend. Er kan geconcludeerd worden dat in dit proefschrift nieuwe lead structuren zijn ontwikkeld die verder moeten worden geoptimaliseerd om geschikte tracers te krijgen voor beeldvorming van MMPs/ADAMs

Probes for Non-invasive Matrix Metalloproteinase-targeted Imaging with PET and SPECT

Dysregulation of matrix metalloproteinase (MMP) activity can lead to a wide range of disease states such as atherosclerosis, inflammation or cancer. The ability to image MMP activity non-invasively in vivo, by radiolabelled synthetic inhibitors, would allow the characterization of atherosclerotic plaques, inflammatory lesions or tumors. Here we present an overview of radiolabelled MMP inhibitors (MMPIs) and MMP peptides for positron emission tomography (PET) and single photon emission computed tomography (SPECT) for the detection of proteolytic activity of MMPs. So far, most studies are at a preliminary stage; however, some hydroxamate-based tracers such as the peptidomimetics [In-111]-DTPA-RP782, [Tc-99m]-(HYNIC-RP805)(tricine)(TPPTS), or Marimastat-ArB[F-18]F-3 and the picolyl-benzenesulfonamide [I-123]I-HO-CGS 27023A identified specifically the enzymatic action of MMPs in animal models of various pathologies. The development of new compounds that may lead to novel tracers (e.g. modification of zinc-binding group, variation of substituents attached to the S1', S2' and S3' pockets of the MMP inhibitors) and the use of antibodies and cell penetrating peptides are also discussed. In general, preclinical studies with atherosclerosis models proved to be more successful than those with oncological models

MicroPET Evaluation of a Hydroxamate-Based MMP Inhibitor, [F-18]FB-ML5, in a Mouse Model of Cigarette Smoke-Induced Acute Airway Inflammation

Matrix metalloproteinases (MMPs) are the main proteolytic enzymes involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). A radiolabeled MMP inhibitor, [18F]FB-ML5, was prepared, and its in vivo kinetics were tested in a mouse model of pulmonary inflammation. BALB/c mice were exposed for 4 days to cigarette smoke (CS) or air. On the fifth day, a dynamic microPET scan was made with [18F]FB-ML5. Standardized uptake values (PET-SUVmean) were 0.19 ± 0.06 in the lungs of CS-exposed mice (n = 6) compared to 0.11 ± 0.03 (n = 5) in air-exposed controls (p < 0.05), 90 min post-injection MMP-9 levels in bronchoalveolar lavage fluid (BALF) were increased from undetectable level to 4615 ± 1963 pg/ml by CS exposure. Increased MMP expression in a COPD mouse model was shown to lead to increased retention of [18F]FB-ML5

Corrigendum to A dual inhibitor of matrix metalloproteinases and a disintegrin and metalloproteinases, [F-18]FB-ML5, as a molecular probe for non-invasive MMP/ADAM-targeted imaging (vol 23, pg 192, 2014)

BACKGROUND: Numerous clinical studies have shown a correlation between increased matrix metalloproteinase (MMP)/a disintegrin and metalloproteinase (ADAM) activity and poor outcome of cancer. Various MMP inhibitors (MMPIs) have been developed for therapeutic purposes in oncology. In addition, molecular imaging of MMP/ADAM levels in vivo would allow the diagnosis of tumors. We selected the dual inhibitor of MMPs and ADAMs, ML5, which is a hydroxamate-based inhibitor with affinities for many MMPs and ADAMs. ML5 was radiolabelled with (18)F and the newly obtained radiolabelled inhibitor was evaluated in vitro and in vivo. MATERIALS AND METHODS: ML5 was radiolabelled by direct acylation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB) for PET (positron emission tomography). The resulting radiotracer [(18)F]FB-ML5 was evaluated in vitro in human bronchial epithelium 16HBE cells and breast cancer MCF-7 cells. The non-radioactive probe FB-ML5 and native ML5 were tested in a fluorogenic inhibition assay against MMP-2, -9, -12 and ADAM-17. The in vivo kinetics of [(18)F]FB-ML5 were examined in a HT1080 tumor-bearing mouse model. Specificity of probe binding was examined by co-injection of 0 or 2.5mg/kg ML5. RESULTS: ML5 and FB-ML5 showed high affinity for MMP-2, -9, -12 and ADAM-17; indeed IC50 values were respectively 7.4±2.0, 19.5±2.8, 2.0±0.2 and 5.7±2.2nM and 12.5±3.1, 31.5±13.7, 138.0±10.9 and 24.7±2.8nM. Radiochemical yield of HPLC-purified [(18)F]FB-ML5 was 13-16% (corrected for decay). Cellular binding of [(18)F]FB-ML5 was reduced by 36.6% and 27.5% in MCF-7 and 16HBE cells, respectively, after co-incubation with 10μM of ML5. In microPET scans, HT1080 tumors exhibited a low and homogeneous uptake of the tracer. Tumors of mice injected with [(18)F]FB-ML5 showed a SUVmean of 0.145±0.064 (n=6) which decreased to 0.041±0.027 (n=6) after target blocking (p<0.05). Ex vivo biodistribution showed a rapid excretion through the kidneys and the liver. Metabolite assays indicated that the parent tracer represented 23.2±7.3% (n=2) of total radioactivity in plasma, at 90min post injection (p.i.). CONCLUSION: The nanomolar affinity MMP/ADAM inhibitor ML5 was successfully labelled with (18)F. [(18)F]FB-ML5 demonstrated rather low binding in ADAM-17 overexpressing cell lines. [(18)F]FB-ML5 uptake showed significant reduction in the HT1080 tumor in vivo after co-injection of ML5. [(18)F]FB-ML5 may be suitable for the visualization/quantification of diseases overexpressing simultaneously MMPs and ADAMs