Andreas Faust

Molecular Imaging of Matrix Metalloproteinases In Vivo Using Small Molecule Inhibitors for SPECT and PET

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent secreted or membrane anchored endopeptidases. MMPs are involved in many physiological processes but also take part in the pathophysiological mechanisms responsible for a wide range of diseases. Pathological expression and activation of MMPs are associated with cancer, atherosclerosis, stroke, arthritis, periodontal disease, multiple sclerosis and liver fibrosis. Thus, noninvasive visualisation and quantification of MMP activity in vivo are of great interest in basic research and clinical application. This can be achieved by scintigraphic molecular imaging techniques such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) provided suitable radiolabelled tracers exist, e.g. radioactive inhibitors of matrix metalloproteinases (MMPIs). The approach to monitor MMP activity in vivo using radiolabelled small molecule inhibitors suitable for SPECT and PET is summarised in this review. Briefly, latest advances in scintigraphic imaging are introduced and followed by a report about the enzyme class of MMPs. The involvement of MMPs in cancer and atherosclerosis is exemplified and small molecule MMPIs are classified. Subsequently, the development of radiolabelled small molecule MMPIs, their synthesis and in vitro and in vivo evaluation is reviewed. Finally, an outlook on the clinical potential of labelled MMPIs in diagnostic algorithms is given.

Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity.

Inflammation has a key role in the pathogenesis of various human diseases. The early detection, localization and monitoring of inflammation are crucial for tailoring individual therapies. However, reliable biomarkers to detect local inflammatory activities and to predict disease outcome are still missing. Alarmins, which are locally released during cellular stress, are early amplifiers of inflammation. Here, using optical molecular imaging, we demonstrate that the alarmin S100A8/S100A9 serves as a sensitive local and systemic marker for the detection of even sub-clinical disease activity in inflammatory and immunological processes like irritative and allergic contact dermatitis. In a model of collagen-induced arthritis, we use S100A8/S100A9 imaging to predict the development of disease activity. Furthermore, S100A8/S100A9 can act as a very early and sensitive biomarker in experimental leishmaniasis for phagocyte activation linked to an effective Th1-response. In conclusion, the alarmin S100A8/S100A9 is a valuable and sensitive molecular target for novel imaging approaches to monitor clinically relevant inflammatory disorders on a molecular level.

Fluorinated isatin derivatives. Part 2. New N-substituted 5-pyrrolidinylsulfonyl isatins as potential tools for molecular imaging of caspases in apoptosis.

Caspases are responsible for the execution of the cell death program and are potentially suitable targets for the specific imaging of apoptosis in vivo. A series of N-1-substituted analogues of the small molecule nonpeptide caspase inhibitor (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (1), which may be useful for the development of caspase-targeted radioligands, were synthesized and their inhibition potencies were evaluated in vitro. Two of the most powerful techniques to introduce fluorine into organic compounds, viz, bromofluorination of olefins and fluorohydrin synthesis by ring-opening of epoxides, were used. Most of the target compounds are potent inhibitors of the two effector caspases-3 and -7. Furthermore, the (18)F-radiolabeled model compound (S)-1-[4-(1-[(18)F]fluoro-2-hydroxyethyl)benzyl]-5-[1-(2-methoxymethyl-pyrrolidinyl)sulfonyl]isatin ([(18)F]37), a putative tracer for the noninvasive imaging of apoptosis by positron emission tomography (PET) was synthesized by nucleophilic epoxide ring-opening of its precursor 36. The radiochemistry utilized in the (18)F-fluorination reverted to carrier-added [(18)F]Et(3)N.3HF, a new fluorine-18 source for radiolabeling.

A new 18F-labelled derivative of the MMP inhibitor CGS 27023A for PET: Radiosynthesis and initial small-animal PET studies

The CGS 27023A derivative (R)-2-(N-((6-fluoropyridin-3-yl)methyl)-4-methoxyphenyl-sulphonamido)-N-hydroxy-3-methylbutanamide 1a was identified as a very potent matrix metalloproteinase inhibitor. Here, we describe a one-step radiosynthesis of the target compound [(18)F]1a. The syntheses of [(18)F]1a resulted in a radiochemical yield of 12.1+/-5.9% (decay-corrected), a radiochemical purity of 98.8+/-0.6%, and a specific activity of 39+/-27 GBq/micromol at the end of synthesis within 160+/-18 min from the end of radionuclide production (n=5). Initial small-animal PET studies in wild-type mice (C57/BL6) showed no unfavourable tissue accumulation of [(18)F]1a.

Synthesis and evaluation of a novel fluorescent photoprobe for imaging matrix metalloproteinases.

The measurement of matrix metalloproteinase (MMP) activity in diseases like inflammation, oncogenesis, or atherosclerosis in vivo is highly desirable. Fine-tuned pyrimidine-2,4,6-triones (barbiturates) offer nonpeptidyl lead structures for developing imaging agents for specifically visualization of activated MMPs in vivo. The aim of this study was to modify a C-5-disubstituted barbiturate and thus design a highly affine, nonpeptidic, optical MMP inhibitor (MMPI)-ligand for imaging of activated MMPs in vivo. A convergent 10 step synthesis was developed, starting with a malonic ester and (4-bromophenoxy)benzene to generate 5-bromo-pyrimidine-2,4,6-trione as the key intermediate. To minimize the interactions between activated MMPs and the dye of the conjugate 6, a PEGylated piperazine derivative was used as a spacer and an azide as a protected amino function. After linking both building blocks, reducing the azide ( Staudinger reaction) and labeling with Cy 5.5, we obtained the nonhydroxamate MMP inhibitor 6 with high affinity (IC 50-value: 48 nM for MMP-2) measured in a fluorogenic assay using commercially available MMP-substrates and the purified enzyme. Zymography revealed an efficient blocking of enzyme activity of purified MMP-2 and MMP-9 and of MMP-containing cell supernatants (HT-1080), (A-673) using the PEGylated barbiturate 5. Fluorescence microscopy studies using a highly (A-673) and a moderate (HT-1080) MMP-2 secreting cell line showed efficient binding of the Cy 5.5 labeled tracer 6 to the MMP-2 positive cells while MMP-2 negative cells (MCF-7) did not bind. Therefore, this new barbiturate-based MMP-probe has a high affinity and specificity toward MMP-2 and -9 and is thus a promising candidate for sensitive MMP detection in vivo.

Radiofluorinated pyrimidine-2,4,6-triones as molecular probes for noninvasive MMP-targeted imaging.

Matrix metalloproteinases (MMPs) are zinc‐ and calcium‐dependent endopeptidases. Representing a subfamily of the metzincin superfamily, MMPs are involved in the proteolytic degradation of components of the extracellular matrix. Unregulated MMP expression, MMP dysregulation and locally increased MMP activity are common features of various diseases, such as cancer, atherosclerosis, stroke, arthritis, and others. Therefore, activated MMPs are suitable biological targets for the specific visualization of such pathologies, in particular by using radiolabeled MMP inhibitors (MMPIs). The aim of this work was to develop a radiofluorinated molecular probe for noninvasive in vivo imaging for the detection of up‐regulated levels of activated MMPs in the living organism. Fluorinated MMPIs (26, 31 and 38) based on the pyrimidine‐2,4,6‐trione lead structure RO 28‐2653 (1) were synthesized, and their MMP inhibition potency was evaluated in vitro. The radiosynthesis and the in vivo biodistribution of the first 18F‐labeled prototype, MMP‐targeted tracer [18F]26, suitable for molecular imaging by means of positron emission tomography (PET) were realized.

Fluorinated isatin derivatives. Part 1: Synthesis of new N-substituted (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatins as potent caspase-3 and -7 inhibitors

A series of new N-substituted (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin derivatives has been synthesized and tested as inhibitors of caspases-3 and -7, which are known to be downstream enzymes critical in the execution of apoptosis. N-Propyl- and N-butyl isatins, as well as the corresponding terminal alcohols and regioisomeric fluorobutyl derivatives were shown to be excellent inhibitors having different binding potencies for caspases-3 and -7. In contrast, the corresponding fluoroethyl and fluoropropyl compounds were about 100-1000 times less active. Fluorinated N-benzyl isatins as well as trifluoroalkyl and difluoroalkyl derivatives were moderate inhibitors. However, isatins bearing different alkylether groups at N-1 are very weak or not active as inhibitors of caspases-3 and -7.

Synthesis and evaluation of a novel hydroxamate based fluorescent photoprobe for imaging of matrix metalloproteinases.

The assessment of matrix metalloproteinase (MMP) activity in vivo is highly desirable in various human diseases such as cancers. Hydroxamic acids based on CGS27023A or CGS25966 are nonpeptidyl lead structures that specifically target activated MMPs in vivo. The aim of this study was the modification and fluorescent labeling of these lead structures to develop a highly affine, nonpeptide MMP inhibitor (MMPI)-ligand for molecular optical imaging of activated MMPs. An 11 step synthesis was developed involving a PEGylated benzyl derivative as a spacer to minimize the interactions between the activated MMP and the dye of conjugate 11 with an azide as a protected amino function. After reducing the azide (Staudinger reaction) and labeling with Cy5.5, we obtained a CGS-based MMP inhibitor 11 with a fluorescent signaling flag. To evaluate the biological properties of this photoprobe, three human cancer cell lines (A-673, HT-1080 and BT-20) were characterized with respect to their MMP-2 and -9 (gelatinases) expression levels (real-time PCR) and protein levels (Western blotting). Initially, fluorogenic inhibition assays were used to assess the MMP inhibition potential. The PEGylated CGS 10 showed complete inhibition of MMP-2 and MMP-9 activities in vitro both for purified MMP-2/-9 (active and pro-forms) and MMP-2/-9 containing cell culture supernatants. To test the imaging potential in biological tissues, gelatinase activity was measured on tumor cryostat sections of the above-mentioned tumor cells using FITC-labeled dye-quenched gelatin. Gelatinase positive tumors revealed strong binding of CGS-Cy5.5 11, while gelatinase negative tumors were not targeted. In conclusion, this new CGS-based MMP photoprobe has a high affinity for MMP-2 and -9 and is thus a promising candidate for sensitive imaging of MMP activity in various diseases in patients.

Imaging matrix metalloproteinase activity in multiple sclerosis as a specific marker of leukocyte penetration of the blood-brain barrier

A method to visualize MMPs reveals penetration of the blood-brain barrier by leukocytes early in multiple sclerosis. Seeing the earliest signs of multiple sclerosis In multiple sclerosis and similar diseases in animals, the brain becomes inflamed and, ultimately, neurons degenerate. Previous work from the Sorokin lab found two enzymes that are absolutely required for this process (MMP-2 and MMP-9). Investigating further, they found that MMP-9 in immune cells is required for the entry of these cells into the brain, the beginning of disease. With the aid of fluorescent- and radioactive-labeled MMP inhibitors, Gerwien and colleagues visualized this initial stage of multiple sclerosis and its mouse equivalent, as immune cells began their inflammatory infiltration of the brain. This imaging method allows noninvasive monitoring of lesion formation and resolution in multiple sclerosis patients and animal models. The enzymes gelatinase A/matrix metalloproteinase-2 (MMP-2) and gelatinase B/MMP-9 are essential for induction of neuroinflammatory symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS); in the absence of these enzymes, the disease does not develop. We therefore investigated the cellular sources and relative contributions of MMP-2 and MMP-9 to disease at early stages of EAE induction. We demonstrated that MMP-9 from an immune cell source is required in EAE for initial infiltration of leukocytes into the central nervous system and that MMP-9 activity is a reliable marker of leukocyte penetration of the blood-brain barrier. We then developed a molecular imaging method to visualize MMP activity in the brain using fluorescent- and radioactive-labeled MMP inhibitors (MMPis) in EAE animals and used the radioactive MMP ligand for positron emission tomography (PET) imaging of MMP activity in patients with MS. In contrast to traditional T1-gadolinium contrast-enhanced MRI, MMPi-PET enabled tracking of MMP activity as a unique feature of early lesions and ongoing leukocyte infiltration. MMPi-PET therefore allows monitoring of the early steps of MS development and provides a sensitive, noninvasive means of following lesion formation and resolution in murine EAE and human MS.

Metabolite identification of a radiotracer by electrochemistry coupled to liquid chromatography with mass spectrometric and radioactivity detection.

Radioligands, which specifically bind to a receptor or enzyme (target), enable molecular imaging of the target expression by positron emission tomography (PET). One very promising PET tracer is (S)-1-(4-(2-[(18)F]-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (isatin), a caspase-3 inhibitor, which has been developed at the University Hospital of Münster to image cell death (apoptosis). The translation of this novel tracer from preclinical evaluation to clinical examinations requires biodistribution studies, which characterize the pharmakodynamics and metabolic fate of the compound. This information is used to further optimize the radioligands and to interpret radioactive signals from tissues upon injection of the radioligand in vivo with respect to their specificity. The analysis of the metabolism of radioligands is hampered by the low amount of the compound being typically injected (nano/picomolar amount per injection). In the present study, electrochemistry (EC) is applied to elucidate the oxidative metabolism pathway of the radiotracer. Previous studies have demonstrated that EC can be utilized as a complementary tool to conventional in vitro approaches in drug metabolism studies. Thereby, potential oxidative metabolites of the isatin are determined by EC coupled to electrospray ionization mass spectrometry (EC/ESI-MS). Moreover, using EC/liquid chromatography (LC) and ESI-ion trap MS(n), structural elucidation of the oxidation products is performed. Comparatively to EC, in vitro metabolism studies with rat liver microsomes are conducted. Finally, the developed LC/ESI-MS method is applied to determine metabolites in body fluids and cell extracts from in vivo studies with the nonradioactive ((19)F) and radioactive isatin ((18)F). On the basis of the electrochemically generated oxidation products of the radioligand, the major radioactive metabolite occurring in vivo was successfully identified.