Metallotexaphyrins as MRI-Active Catalytic Antioxidants for Neurodegenerative Disease: A Study on Alzheimer’s Disease

Summary The complex etiology of neurodegeneration continues to stifle efforts to develop effective therapeutics. New agents elucidating key pathways causing neurodegeneration might serve to increase our understanding and potentially lead to improved treatments. Here, we demonstrate that a water-soluble manganese(II) texaphyrin (MMn) is a suitable magnetic resonance imaging (MRI) contrast agent for detecting larger amyloid beta constructs. The imaging potential of MMn was inferred on the basis of in vitro studies and in vivo detection in Alzheimer’s disease C. elegans models via MRI and ICP-MS. In vitro antioxidant- and cellular-based assays provide support for the notion that this porphyrin analog shows promise as a therapeutic agent able to mitigate the oxidative and nitrative toxic effects considered causal in neurodegeneration. The present report marks the first elaboration of an MRI-active metalloantioxidant that confers diagnostic and therapeutic benefit in Alzheimer’s disease models without conjugation of a radioisotope, targeting moiety, or therapeutic payload.


General procedures
Starting materials were purchased from Fisher Scientific or Sigma Aldrich and used without further purification unless otherwise specified. Solvents were purified using a solvent purifier system (Vacuum Atmospheres). Dichloromethane (DCM) was freshly distilled after being dried over CaH2 under argon. Reaction progress was monitored by thin-layer chromatography (TLC silica gel 60 F254, Silicycle UltraPure Silica gels). Analytical RP-HPLC analyses for MMn and MLu were performed on a Thermo scientific Dionex Ultimate 3000 equipped with a PDA detector. The analytical column was a Syncronis C18 column, 5 µm, 4.6 x 250 mm (Thermo Scientific); the mobile phase consisted of a increasing gradient (from 10% to 99 % in 20 min) of acetonitrile into water, both containing 0.1% acetic acid. In this case the flow rate was 1.2 ml / min. Texaphyrins were monitored at 254, 470, and 740 nm. MMn, MGd, and MLu were purified on reverse phase-tC18 SPE (Waters Sep-Pak, Waters) columns containing 10 g of C-18 using an increasing gradient of acetonitrile in either 0.1 M ammonium acetate/ 1% acetic acid aqueous solution or 0.1 M potassium nitrate aqueous solution as the eluent, depending on which counter anion (AcOor NO3 -) was desired as the ancillary ligand. Mass spectrometric analyses were carried out in The University of Texas at Austin Mass Spectrometry Facility. Low-resolution and high-resolution electro-spray mass spectrometric (ESI-MS) analyses were carried out using Thermo Finnigan LTQ and Qq-FTICR (7 Telsa) instruments, respectively. Inductively coupled plasma mass spectrometry (ICP-MS) was carried out at the Bioimaging and Chemical Analysis Facilities Core of the Center for Environmental Health Sciences at MIT using an Agilent 7900 ICP-MS system. In general, samples were digested for 2 hours at 70 °C in concentrated nitric acid, before being diluted into 2% nitric acid to a working concentration of 10-100 ppb. Nine external standards (0-500 ppb) of the target metal were used to calibrate the instrument and 10 ppb Er was used as an internal standard in each sample. Samples were run in duplicate or triplicate to minimize systematic errors. 1 H-NMR spectra were recorded using a Varian 400 and 600 (solvent suppression) MHz as well as a Bruker AVII HD 500 with Prodigy liquid nitrogen cryoprobe (purchased with NIH grant 1-S10-OD021508-01). UV-Vis spectra were recorded using a Varian Cary 5000 spectrophotometer at room temperature. Fluorescence spectra were recorded on a Photon Technology International Fluorescence Master fluorimeter. The source was a 75 W Xenon short arc lamp. A cell length of 10 mm was used in all UV-Vis and fluorescence studies. Phantom MRI data were acquired in a 12 cm outer diameter birdcage transceiver for imaging in a 20 cm bore Bruker 7 T Avance III MRI scanner. Samples at varying concentrations (0 up to 500 µM), as determined by inductively coupled plasma mass spectrometry (ICP-MS) in HEPES (10 mM, pH 7.4) or Dulbecco's PBS (as supplied) were loaded into the wells of a 384-well clear polystyrene plate (Thermo Scientific TM Nunc TM ), which had been pre-cut in half to optimally fit the coil. Unused wells were filled with buffer. A 2 mm slice was imaged through the samples with the field of view of 5 x 5 cm and the data matrices were 256 x 256 points. Longitudinal (R1) and transverse (R2) relaxation measurements were acquired using multi-spin multi-echo (MSME) sequences (flip angle = 180°). R1 parameters; TE = 12 ms, TR = 100, 200 300, 400, 500, 600, 700 800, 900, 1000, 1200, 1500, 3000, 5000 ms. R2 parameters; TR = 5000 ms, TE = 12,24,36,48,60,72,84,96,108,120,132,144,156,168,280,192,204,216,228,240,252,264,276,288,300,312,324,336,348,360 ms. Custom routines written in Matlab (Mathworks, Natick, MA) were used to reconstruct the images and compute relaxation time constants by fitting image intensity data to exponential decay curves. Relaxivity values (r1 & r2) were generated from the gradient of the slope obtained by plotting the calculated relaxation rates against the concentration of the agent in each well. Electron Paramagnetic Resonance (EPR) spectroscopy was carried out using a Bruker EMX Plus spectrometer, X-band (9.5 GHz). Experimental settings were amplitude modulation = 10 G and microwave power = 2 mW. The observed spectra were taken as an average of four scans. Circular dichroism (CD) analyses were carried out using a Jasco J-815 CD spectrometer in The University of Texas at Austin Targeted Therapeutic Drug Discovery & Development Program (Ti3D). The data shown in this report was taken as an average of 3 scans from 260 to 195 nm using a 0.05 nm wavelength step. SEM analyses were prepared by drop casting 20 µM samples onto silica wafers (University Wafers) then gently dried with nitrogen. Images were taken on a FEI Quanta 650 ESEM in The University of Texas at Austin-Texas Materials Institute. There are inherent difficulties in working with Ab40 that are often ignored (e.g., aggregation state, solubility, left over additive to solubilize the peptide, etc.). The methods discussed in the ESI proved reproducible throughout the studies and when carried out by multiple authors.
Peptide synthesis. Aβ peptides were prepared via Fmoc amino solid-phase peptide synthesis using a Liberty Blue microwave peptide synthesizer. Preparative RP-HPLC purification of peptides was performed using an Agilent Zorbax SB-C18 Prep HT column 21.2 × 250 mm. Analytical RP-HPLC characterization of peptides was performed using an Agilent Zorbax column 4.6 × 250 mm. An Agilent Technologies 6530 Accurate Mass QT of LC/MS was used for high-resolution mass spectra of purified peptides. Solvents used were HPLC grade.

Synthesis and characterizations of metallotexaphyrins MGd, MLu and MMn.
Motexafin gadolinium (MGd) was a gift from Pharmacyclics, Inc., recently acquired by Abbvie. MLu and MMn were synthesized using literature protocols. S1,S2 The purity of MMn, MLu, and MGd was checked by reverse phase-HPLC before use.
1-Octanol: water partition experiments gave the following data: LogP(MGd) = -0.95; LogP(MMn) = 0.69 Solutions of Aβ40 peptide. Approximately 2 mg of Aβ40 (as a lyophilized powder) was dissolved in 1 mL aqueous NaOH (10 mM). After vortexing and sonication, the solution was filtered through a 0.22 μm PES syringe filter. PBS (pH = 7.15) was added to obtain a final concentration of 100 μM Aβ40. The concentration was checked by UV-vis spectrophotometry using the absorbance at 280 nm and a molar absorptivity of 1490 M -1 cm -1 . A similar procedure was used for the quantitative reactivity studies with the final dilution being in miliQ water to give a stock solution of approximately 600 μM.
Cell uptake experiments. Neuro-2A cells were grown until confluent in T-75 flasks. MMn was added (100 μM) and cells were incubated for 9 h, upon which cells were washed twice with PBS, incubated with trypsin (1X) for 2 minutes, diluted with supplemented EMEM, counted, and pelleted by centrifugation (3 min at 2000 rpm). After supernatant removal, the pellet was suspended/washed one more time with PBS (5 ml) and the pellet was reformed by centrifugation (3 min at 2000 rpm). The cell pellet was digested in concentrated nitric acid (100 µL) for 15 h at 60°C and diluted with miliQ water to a final concentration of 2% HNO3 before being analyzed by ICP-MS.
Caenorhabditis elegans. The C. elegans strains N2 and CL4176 were provided by CGC (nematodes shipped on 35 mm diameter plates), which is funded by the NIH Office of Research Infrastructure Programs (P40-OD010440). C. elegans were grown and maintained as described in "WormBook: The Online Review of C. elegans Biology" by Theresa Stiernagle (https://www.ncbi.nlm.nih.gov/books/NBK19649/). Nematode Growth Medium (NGM). NaCl (3 g), agar (17 g) and peptone (2.5 g) were added to 975 ml of H2O in a 2 L Erlenmeyer flask, which was then autoclaved for 1 h. All work after this was completed under sterile conditions. The flask was cooled and maintained at 60 °C in a water bath to prevent the solution from solidifying. To this heated solution was added CaCl2 (1 ml of a 1 M solution), cholesterol (1 ml of a solution in ethanol at 5 mg/ mL), MgSO4 (1 ml of 1 M solution), and KPO4 buffer (25 ml of 1 M solution). The NGM solution was dispensed into 10 mm diameter plates (15 ml/plate). The plates were left at room temperature for 1-2 days to allow excess moisture to evaporate. Plates showing contamination were discarded.

E. coli OP50 culture & plate.
Lysogeny broth (LB) was prepared by mixing 21 g LB powder (Sigma Aldrich) in 1 L of Milli Q water. LB agar was prepared by adding 17 g/ L of agar (Fisher Sci.) to the LB mixture. Both the broth and agar were autoclaved at 121 °C for 45 minutes. All work after autoclaving was conducted under sterile conditions. A bead of E. coli OP50 (BactoBeads, Fisher Sci.) was streaked on an LB agar plate and grown in a static incubator overnight at 37 °C. From this plate, a single colony was isolated and inoculated in 10 mL of LB. The mixture was placed in a shaking incubator heated to 37 °C overnight. From this NMS plates could be prepared by pipetting 1 mL of the overnight culture onto NMS agar. The plate was swirled to spread the culture and the seeded plates were incubated at room temperature overnight. NMR plates were found to have a lawn of E. coli OP50 and were stored inverted at 4 °C, with an up to 3-week shelf life.

MMn uptake experiment.
After receiving the C. elegans animals, N2 and CL4176, from the CGC (University of Minnesota) shipped on petri dishes, agar matrix was cut in small chunks (0.5 x 0.5 cm) with a razor blade and transferred on Nematode Growth Medium (NGM) agar plates previously seeded with E. coli strain OP50 as a food source. NGM plates were then placed at 16 °C in order to increase the population of C. elegans before drug exposure and prevent significant plaque formation in the CL4176 AD model. After 7 days, MMn was added to plates (1 ml/ plate of a solution at 100 µM), which were incubated at 25 °C for 48 hours. The animals were then transferred to Eppendorf tubes (previously weighted empty on analytical balance) using 1.5 ml of water. The tubes were put on ice for 5 min and centrifuged (14000 rpm for 1 min) and the supernatant was removed. The plate was washed again with 1.5 mL of water and centrifuged again. The resulting pellets were washed twice with 1 ml of DI water. C. elegans pellets were lyophilized over a period of 15 h before being weighed (dry pellet weights obtained by lyophilization were used to normalize the MRI and ICP-MS results).
Preparation of peroxynitrite solution. Three separate solutions of NaNO2 (0.6 M), HCl (0.68 M) with H2O2 (0.72 M), and NaOH (3.6 M) were prepared and stored in the freezer at -20 °C for thirty minutes. The solutions were transferred to an ice bath and allowed to sit for thirty minutes. The NaNO2 solution was added to a 100 mL beaker placed in an ice bath and stirred at > 500 rpm. The HCl/ H2O2 solution and NaOH solutions are added sequentially with the NaOH coming immediately after the HCl/ H2O2 solution. These solutions must be added in sequence (i.e. one then the other) with as little time between the two additions as possible. Best results were obtained by adding the two solutions with a very small offset. The mixture turned yellow and was allowed to stir in the ice bath for 5 minutes. MnO2 was added portion wise until gas evolution ceased. The mixture was split into aliquots, centrifuged, and the peroxynitrite solution was decanted using a glass pipette. The concentration could be calculated using lmax = 302 nm with e = 1670 M -1 cm -1 . Best results were obtained by centrifugation in a cold room at 4 °C.
Hemin-Serotonin Procedure. Serotonin (1 mM), H2O2 (1 mM), hemin (15 µM), and sodium ascorbate without and with MMn (120 µM) or MnTMPyP (120 µM) were incubated in PBS (pH 7.4, 1.5 mL total volume) at 37 °C for 2 hours in the dark. An analogous experiment without sodium ascorbate using the same concentrations of serotonin, H2O2, hemin, and MMn or MnTMPyP. The resulting mixture was passed through a reverse phase-tC18 SPE (Waters Sep-Pak, Waters) column containing 10 g of the C-18 substrate. The column was washed with 25 mL of 5% acetonitrile: H2O to separate the serotonin and 4,4'-serotonin dimer from paramagnetic species. The resulting solution was concentrated using a rotary evaporator. The mixture was taken up in D2O (0.7 mL) and analyzed via 1 H NMR spectroscopy. NMR spectroscopic yields were calculated by comparing the relative concentration of serotonin to the 4,4'serotonin dimer. Note: Additional washing of the column failed to yield additional serotonin or the 4,4'serotonin dimer.
Cu(II) Procedure. Serotonin (1 mM), H2O2 (1 mM), CuCl2 (100 µM) without and with MMn (120 µM) or MnTMPyP (120 µM) were incubated in PBS (pH 7.4, 1.5 mL total volume) at 37 °C for 20 hours in the dark. The resulting mixture was passed through a reverse phase-tC18 SPE (Waters Sep-Pak, Waters) column containing 10 g of the C-18 substrate. The column was washed with 25 mL of 5% acetonitrile: H2O to separate the serotonin and 4,4'-serotonin dimer from paramagnetic species. The resulting solution was concentrated using a rotary evaporator. The mixture was taken up in D2O (0.7 mL) and analyzed via 1 H NMR spectroscopy. NMR spectroscopic yields were calculated by comparing the relative concentration of serotonin to the 4,4'-serotonin dimer.
Studies of Aβ16-hemin-peroxynitrite. Hemin (15 µM), peroxynitrite (1 mM) and Aβ16 (300 µM) in PBS (200 µL total volume) were incubated together at 37 °C for 30 min. All compounds were added and then vortexed to provide a homogenous mixture. Immediately after adding peroxynitrite, the mixture was vortexed again and allowed to incubate at 37 °C for 30 minutes. The resulting mixtures were immediately subjected to HPLC-MS analysis. Vials were cooled to 4 °C inside the HPLC-MS queue to prevent additional reactivity.

HPLC-MS/MS analyses.
Analytical RP-HPLC characterization of peptides was performed using an Agilent Zorbax column 4.6 × 250 mm. An Agilent Technologies 6530 Accurate Mass QT of LC/MS was used for high-resolution mass spectra of purified peptides. Solvents used were HPLC grade. UV-vis spectra were recorded on a Hewlett Packard HP 8453 A diode array spectrophotometer equipped with a thermostated, magnetically stirred cuvette holder. The peptides were studied by direct HPLC-MS/MS analysis. LC-MS and LC-MS/MS data were obtained by using an LCQ DECA ion-trap mass spectrometer with an ESI ion source, coupled with an automatic injector Surveyor HPLC system and controlled by Xcalibur 1.3 software (Thermo-Finnigan, San Jose, CA). The system was run in an automated LC-MS/MS mode and by using a Surveyor HPLC system (Thermo-Finnigan, San Jose, CA, USA) equipped with a BIOBASIC C18 column (5 µM), 150 x 2.1 mm). For analysis of oxidation studies, the acquired MS/MS spectra were automatically searched against the human Aβ16 sequence by using the SEQUEST algorithm to identify the modified residues. This algorithm was incorporated into the Bioworks 3.1 software (Thermo-Finnigan, San Jose, CA). The elution was performed with a 0-55% linear gradient over 65 min with 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B with a flow rate of 0.2 mL/min. For analysis of oxidation studies, the acquired MS/MS spectra were automatically searched against the human Aβ16 sequence by using the SEQUEST algorithm to identify the modified residues. This algorithm was incorporated into the Bioworks 3.1 software (Thermo-Finnigan, San Jose, CA).
MS/MS spectrometric analyses of the Aβ16 ion series samples provided support for the conclusion that nitration occurs to Tyr10. MS/MS fragmentation of Aβ16 revealed that His13 or His14 are the major oxidized species with dimerization occurring at Tyr10. MS/MS fragmentation of Aβ40 revealed that oxidative modification is predominately comprised of oxidation at His13/His14 and Met35 with Tyr10 oxidation being minimal.
Cell culture. The neuroblastoma cell line Neuro-2A was bought from ATCC. Cells were grown in EMEM medium containing 10% heat inactivated fetal bovine serum (FBS) and antibiotics (100 mg/ ml streptomycin and 100 U/ ml penicillin) at 37 °C, under 5% CO2 atmosphere. The cells were sub-cultured once every 4 to 5 days.
MTT viability assay (determination of IC50 values for MMn, MnTMPyP, and MGd). The proliferation of exponential phase cultures of Neuro-2A was assessed by tetrazolium (MTT) dye reduction. In brief, tumor cells were seeded in 96-well microliter plates at 10,000 cells per well and allowed to adhere overnight in the culture medium described previously. After 15 h, agents were added via serial dilution (from 200 to 0.01 µM) and the plate was incubated at 37°C and 5% CO2. After 24 h incubation time with the agent in question (2 days overall) 50 µL of a solution of tetrazolium dye in EMEM without FBS (3 mg/ mL) was added to each well. The plate was incubated for 4 hours, whereupon the medium was removed and the formazan product dissolved in 50 µL of DMSO. The absorbance at 570 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA), corrected for background and then normalized to wells containing untreated cells to allow plate-to-plate comparisons. The growth inhibition data were fitted to a sigmoidal dose-response curve (logistic function, origin software) to generate IC50 values (defined as the concentrations of drug inhibiting cell growth by 50%). The IC50 is presented as mean +/-standard deviation.
24-well plate MTT experiments. Aβ40 (90 μM) was incubated in PBS alone or in the presence of H2O2 (1 mM), hemin (15 μM), NaNO2 (1 mM) (referred to as nitration mixture) for 3 h at 37 °C followed by 24 h at 4 °C. 250 μL of each of the solution was transferred into each well containing about 50,000 Neuro-2A cells (plated 24 h earlier) in 250 μL of supplemented EMEM medium without phenol red. After 24 h, the medium was removed and replaced with 500 μL of fresh EMEM not containing FBS and phenol red. To this was added, 200 μl of an MTT solution at 3 mg/ml in EMEM not containing phenol red and FBS. After 4 h at 37 °C, the supernatant was removed and 200 μl of DMSO was added to each well to dissolve the formazan purple crystals. Aliquot (50 μl) of each well were transferred into a 96-well plate well and the absorbance at 570 nm was read using a plate reader (see description above).