title: A dual inhibitor of matrix metalloproteinases and a disintegrin and metalloproteinases, [¹⁸F]FB-ML5, as a molecular probe for non-invasive MMP/ADAM-targeted imaging.
abstract: 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.
  
  
  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 ± 2.2 nM and 12.5 ± 3.1, 31.5 ± 13.7, 138.0 ± 10.9 and 24.7 ± 2.8 nM. 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 16 HBE cells, respectively, after co-incubation with 10 μM 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 90 min post injection (p.i.).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Matusiak
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Castelli
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Adriaan W
authors.initials: AW
authors.lastname: Tuin
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
authors.affiliation: Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.
authors.forename: Rosalina
authors.initials: R
authors.lastname: Wisastra
authors.affiliation: Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Dekker
authors.affiliation: Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.
authors.forename: Laurette M
authors.initials: LM
authors.lastname: Prély
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
authors.affiliation: Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.
authors.forename: Rainer P M
authors.initials: RP
authors.lastname: Bischoff
authors.affiliation: Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
authors.forename: Aren
authors.initials: A
authors.lastname: van Waarde
authors.affiliation: Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
authors.forename: Rudi A J O
authors.initials: RA
authors.lastname: Dierckx
authors.affiliation: Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands. Electronic address: p.h.elsinga@umcg.nl.
authors.forename: Philip H
authors.initials: PH
authors.lastname: Elsinga
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D000818:Animals; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D061986:MCF-7 Cells; D061965:Matrix Metalloproteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry; D020782:Matrix Metalloproteinases / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D015335:Molecular Probes; D049268:Positron-Emission Tomography / Q000379:methods*; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000737:chemistry; D013388:Succinimides / Q000138:chemical synthesis / Q000737:chemistry*; D023041:Xenograft Model Antitumor Assays
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D015335:Molecular Probes; D019275:Radiopharmaceuticals; D013388:Succinimides; D051722:ADAM Proteins; D020782:Matrix Metalloproteinases
keywords: ADAMs; HT1080 xenograft mouse model; Hydroxamate; MMP/ADAM inhibitor; MMPs; PET