Towards Optimized Bioavailability of 99mTc-Labeled Barbiturates for Non-invasive Imaging of Matrix Metalloproteinase Activity

Introduction
 Dysregulated activity of matrix metalloproteinases (MMPs) drives a variety of pathophysiological conditions. Non-invasive imaging of MMP activity in vivo promises diagnostic and prognostic value. However, current targeting strategies by small molecules are typically limited with respect to the bioavailability of the labeled MMP binders in vivo. To this end, we here introduce and compare three chemical modifications of a recently developed barbiturate-based radiotracer with respect to bioavailability and potential to image MMP activity in vivo. Methods Barbiturate-based MMP inhibitors with an identical targeting unit but varying hydrophilicity were synthesized, labeled with technetium-99m, and evaluated in vitro and in vivo. Biodistribution and radiotracer elimination were determined in C57/BL6 mice by serial SPECT imaging. MMP activity was imaged in a MMP-positive subcutaneous xenograft model of human K1 papillary thyroid tumors. In vivo data were validated by scintillation counting, autoradiography, and MMP immunohistochemistry. Results We prepared three new 99mTc‐labeled MMP inhibitors, bearing either a glycine ([99mTc]MEA39), lysine ([99mTc]MEA61), or the ligand HYNIC with the ionic co-ligand TPPTS ([99mTc]MEA223) yielding gradually increasing hydrophilicity. [99mTc]MEA39 and [99mTc]MEA61 were rapidly eliminated via hepatobiliary pathways. In contrast, [99mTc]MEA223 showed delayed in vivo clearance and primary renal elimination. In a thyroid tumor xenograft model, only [99mTc]MEA223 exhibited a high tumor-to-blood ratio that could easily be delineated in SPECT images. Conclusion Introduction of HYNIC/TPPTS into the barbiturate lead structure ([99mTc]MEA223) results in delayed renal elimination and allows non-invasive MMP imaging with high signal-to-noise ratios in a papillary thyroid tumor xenograft model. Supplementary Information The online version contains supplementary material available at 10.1007/s11307-021-01668-z.

based on a barbiturate lead structure reported so far [27]. All these examples are characterized by fast blood clearance and therefore short availability for binding to the target enzymes which is mainly caused by their pharmacokinetic profile rather than by their binding properties to the active MMPs. Introduction of ionic charges like in [ 99m Tc]RP805, a cyclic hydroxamate-based MMP-radiotracer, gave promising results in imaging of MMP activation [28].
Therefore, we developed and evaluated three MMPIs presenting a novel series of 99m Tc-labeled barbiturates with gradually increasing hydrophilicity. Starting with a glycine-spacer ([ 99m Tc]MEA39), we increased hydrophilicity by changing the spacer to lysine ([ 99m Tc]MEA61) and finally introduced HYNIC as a bifunctional coupling agent for 99m Tc-labeling, together with TPPTS and tricine as co-ligands, resulting in high hydrophilicity with regard to the final radiotracer ([ 99m Tc]MEA223). We evaluated the impact of altered MMPI hydrophilicity on tracer dynamics and clearance in wild-type mice in vivo. Furthermore, we investigated the potential of these 99m Tc-labeled barbiturates to non-invasively assess tumor-associated MMP activity, specifically whether the altered tracer hydrophilicity and dynamics leads to improved tumor/blood contrast.

Chemistry
All chemicals, reagents, and solvents for the syntheses of the compounds were analytical grade, purchased from commercial sources and used without further purification unless otherwise specified. For radiosynthesis, only solvents of pharmaceutical purity (Reag.Ph.Eur.) were used.
The labeling of compounds was accomplished by using two different kits which both are prepared in house. Briefly, one labeling kit to produce a triaquatricarbonylcomplex [ 99m Tc(CO) 3 (OH 2 ) 3 ] + was used for radiosynthesis of [ 99m Tc]MEA39 and [ 99m Tc]MEA61; the second one was used for the HYNIC-derivative [ 99m Tc]MEA223 (see SI). The precursors and corresponding kits were heated to 100 °C for 16-20 min. After cooling to room temperature, the crude reaction mixtures were purified by HPLC. Products were collected, diluted with water, and filtered through Sep-Pak® C-18 Plus cartridges. Solvents were removed under reduced pressure without heating, and the residues were solved in 0.9% NaCl-solution containing 1.6 vol% of Tween80® (100-500 µL). For quality control, all injectable solutions were analyzed determining the radiochemical purity (RCP), the pH-value, and osmolality.

Introduction
Matrix metalloproteinases (MMPs) comprise a subfamily of the metzincins and belong to the zinc-and calcium depending endopeptidases. MMPs are involved in a variety of physiological processes but also play a crucial role in different pathophysiological conditions, e.g., in cancer, joint disorders (including rheumatoid arthritis and osteoarthritis), neurodegenerative diseases, respiratory disorders, cardiovascular disease, and many more [1].
MMPs are capable to enzymatically cleave the protein components of the extracellular matrix (ECM) with overlapping substrate specificities. Moreover, MMPs are involved in processing bioactive molecules such as proteinase inhibitors, growth factors, cytokines, and chemokines [2,3].
The non-invasive detection and assessment of locally upregulated and activated matrix metalloproteinases (MMPs) in vivo using MMP inhibitor-based radiotracers for positron emission tomography (PET) or single photon emission computed tomography (SPECT) is still a challenge [4]. However, if successful, the visualization of MMP activity by means of aforementioned scintigraphic technologies would become a breakthrough by improving diagnosis and assessment of disease progression [5].
Several groups are working on the design, improvement, and evaluation of MMP inhibitor-based radiotracers aiming at the non-invasive imaging of MMP-associated diseases by means of SPECT or PET [4,[6][7][8][9][10][11][12]. For this purpose, different classes of radiolabeled MMP inhibitors (MMPIs) have been developed and explored as radiotracers. Radiolabeled hydroxamate-based MMPIs have been successfully applied to image MMP activity, for example in preclinical models of atherosclerosis [13,14] and stroke [15] and to visualize MMP activity in patients with multiple sclerosis [16]. These lead structures mainly behave like so called combined or right-hand side MMP inhibitors depending on the substituents occupying both the S1-S3 and S1'-S3' enzyme pockets (combined) or S1'-S3' enzyme pockets (right hand side). Via such MMP inhibitor radiotracer approaches, it is putatively feasible to follow locally upregulated MMPs in their activated forms in vivo [5,13,[17][18][19][20]. Disadvantages of hydroxamates in MMP imaging are their broad inhibition spectrum, metabolic instability, as well as interactions with other metalloproteinases due to their high transition-metal binding potential [21]. Non-hydroxamate-based MMPIs like substituted pyrimidine-2,4,6-triones (barbiturates) often possess higher specificity for the gelatinases MMP-2 and MMP-9 [22] and were the basis for the development of C-5-disubstituted barbiturates with improved MMP specificity and potency [23]. We have introduced 18 F-labeled C5-disubstituted barbiturates as potential MMP-targeted radiotracers, putatively binding to the zinc ion at the active site via the enolic tautomer of the barbiturate moiety [24][25][26]. Moreover, we have also presented a first 68 Ga-labeled version of a barbiturate which was synthesized by azide-alkyne cycloaddition. This potential PET tracer was the first radiometal-labeled MMP inhibitor The serum stability of all radiolabeled compounds was evaluated by incubation in human and murine serum at 37 °C for up to 120 min and analyzed by HPLC. The distribution coefficients (logD exp ) were determined in a two-phase system consisting of 1-octanol and PBS-buffer (pH = 7.4) according to the literature [29].

Animals
All animal experiments performed in the study were in accordance with the German Law on the Care and Use of Laboratory Animals and approved by the local authorizing agency of North Rhine-Westphalia.
C57/BL6 mice (female, 12-15 weeks, 20-23 g) were anesthetized with 2% isoflurane (Abbott Animal Health) in 100% O 2 , and a lateral tail vein catheter was placed using a 27G needle connected to 15-cm polyethylene tubing. 80-100 MBq of the respective tracer was injected as a bolus (100 μL compound flushed with 100 μL saline) via the tail vein, and subsequent SPECT imaging was performed.

SPECT/CT Imaging
SPECT experiments were carried out using a small-animal SPECT/CT scanner (NanoScan, Mediso). For biodistribution studies, dynamic SPECT scans were acquired over the course of 90 min p.i. (9 × 10 min frames, field of view 108 mm). Following the acquisition, CT contrast agent (Ultravist ® -370, 5 µl/g bw) was injected via the tail vein catheter, and a CT image was obtained. Mice underwent subsequent SPECT/ CT scans 4 h p.i. (1 × 30 min frame) and 24 h p.i. (1 × 60 min frame). For in vivo tumor uptake studies, mice were imaged 0-60 min and 4 h p.i. of tracer with a reduced field of view (1 × 30 min frame, 26 mm).
wEx Vivo Validation Following the last SPECT/CT acquisition, mice were euthanized by cervical dislocation and a necropsy was performed. Ex vivo biodistribution of radioactivity was analyzed by scintillation counting (Wizard2 gamma counter, Perkin Elmer), and the radioactivity in respective organs was decay-corrected and calculated as %ID per Gram tissue (% ID/g).
In-text values are mentioned as mean ± standard deviation.

Results
The synthesis of the three different 99m Tc-labeled MMPIs starts with the preparation of the barbiturate 11 with an azido PEG-chain for installing the different Tc-chelators in all cases [30]. This azide-functionalized derivative was also characterized by crystal structure which is outlined together with the synthetic procedures in the supporting information. For the first two 99m Tc-labeled tracers [ 99m Tc]MEA39 and [ 99m Tc]MEA61, we applied the "click to chelate" concept [31]. In contrast to the literature, the corresponding precursors for labeling which are usually prepared in situ were isolated because direct labeling failed. We assume that this is likely being caused by the two very basic tertiary amines of the piperazine ring interfering with the amino acid. This makes an in situ formation of the necessary precursors impossible or at least unfavorable. For [ 99m Tc]MEA39, the azide 11 was used in a click reaction with the commercially available fully functionalized (S)-2-[(tert-butoxycarbonyl)amino]pent-4-ynoic acid to get the protected precursor 13. After deprotection and purification (as potassium salt), the corresponding Re-complex MEA39 was prepared as cold reference using [NEt 4 ] 2 [ReBr 3 (CO) 3 ][32] (Scheme 1). For radiolabeling an in-house prepared [ 99m Tc(CO) 3 (OH 2 ) 3 ] + -kit was used yielding [ 99m Tc]MEA39 with a radiochemical yield of 21 ± 11% (n = 6) and reproducible radiochemical purity of over 99%. The total time for synthesis was 131 min ± 9 min (n = 6). The identity of the product was verified by HPLC by co-injection of the analogous Rhenium-complex MEA39 as well as by mass spectrometry. For [ 99m Tc]MEA61, we installed the more polar lysine-derivative 8 which was prepared according to the literature [33]. After forming the triazole and subsequent deprotection, the resulting precursor 17 was converted into the corresponding rhenium complex (MEA61) and radiolabeled with technetium-99 m ([ 99m Tc]MEA61) in the same way as mentioned before. The radiochemical yield was comparable with 23 ± 11% (n = 3) and radiochemical purities of over 99% (130 min total synthesis time).
In order to further improve solubility and hydrophilicity of the envisioned tracers, a HYNIC-functionalized

In vitro Characterization
In order to evaluate the affinity of the new barbituratebased compounds towards MMPs, an in vitro inhibition study was performed. The IC 50 -values were determined for MMP-2, MMP-8, MMP-9, MMP-13, and MMP-15 using the protocol from Huang et al. [37] The rhenium-labeled non-radioactive counterparts MEA39 and MEA61 were used in this assay, and the results are summarized in Table 1. Additionally, the corresponding precursors were also measured and outlined in the SI for comparison. For [ 99m Tc]MEA223 the determination of IC 50 -values was not possible because of lacking the Re-labeled non-radioactive derivative (see above). The amino acid-based barbiturates MEA39 and MEA61 show high affinity towards the tested MMPs in the nanomolar range. The glycine-based MEA39 has some specificity towards the gelatinases MMP-2 and MMP-9 over MMP-8, while the lysine-based MEA61 shows selectivity for the gelatinases over MMP-13 and MMP-14.
Experimental logD value were determined [29] and showed decreasing lipophilicity from [ 99m Tc]MEA39 (0.79 ± 0.03) to [ 99m Tc]MEA61 (0.35 ± 0.23) and [ 99m Tc]MEA223 (0.15 ± 0.07). in vitro stability tests were performed by incubating tracers in human and murine blood serum followed by HPLC analysis for 2 h. All tracers proved to be stable with no detectable radiometabolites or decomposition products in both human and murine serum after 120 min. (see SI).

In vivo Imaging of Tumor MMP Activity
We applied the three radiotracers for in vivo imaging of MMP activity in a subcutaneous xenograft model of human K1 papillary thyroid tumors which are known for high MMP activity. MMP-2 and MMP-9 activity in patients with papillary thyroid tumors are linked to tumor cell invasion and metastasis, and high gelatinase activity has been associated with poor prognosis [38,39].
Representative SPECT images (axial sections) of radiotracer uptake at 4 h p.i. coregistered to a CT are shown in Fig. 3A (Fig. 3D). Additionally, ex vivo autoradiography confirmed that only [ 99m Tc]MEA223 showed relevant radiotracer accumulation throughout the tumor, even though all tumors were highly positive for the target in MMP immunohistochemistry (Fig. 4).

Conclusion
We successfully prepared two new [ 99m Tc]Tc(CO) 3labeled MMP inhibitors, bearing either a glycine or a lysine residue within the 99m Tc-chelator. In a third compound, HYNIC was used together with TPPTS and tricine as co-ligands. All tracers were prepared with high radiochemical purities and reproducible radiochemical yields.
In vitro experiments showed high stability of all tracers in human and murine blood serum, and excellent affinity of the amino acid-based reference compounds MEA39 and MEA61 towards targeted matrix metalloproteinases.