All chemicals obtained commercially were of analytical grade and used without further purification. [64
was purchased from Isotrace, Inc (O'Fallon, MO). The peptide N
) was synthesized via solid-phase Fmoc method by Biomolecular Research Facility at the University of Virginia, and the structure was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy, as reported previously (9
). Bifunctional t
-butoxycarbonyl-protected PEG-succinimidyl ester (t
-Boc-PEG-NHS; molecular weight, 3.4 kD) was obtained from Laysan Bio, Inc.. 2,2',2”,2'”-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) was obtained from Macrocyclics, Inc.. N
-hydroxysulfosuccinimide and 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide were purchased from Pierce. All other chemical reagents and solvents were obtained from Sigma-Aldrich. Semipreparative reversed-phase (RP) high-performance liquid chromatography (HPLC) was performed on a Varian system with an ABI Spetroflow 783 ultraviolet detector and a Bioscan Flow Count Radio-HPLC detector with an Apollo C18 RP column (5μ, 250 × 10 mm). The mobile phase changed from 40% Solvent A (0.1% trifluoroacetic acid in 80% water) and 60% Solvent B (0.1% trifluoroacetic acid in 80% aqueous acetonitrile) to 100% solvent B at 30 min at a flow rate 3mL/min. MALDI-TOF mass spectroscopy analysis was performed on samples of peptide products at W.M. Keck biomedical mass spectrometry laboratory at the University of Virginia, and the data were obtained on a Bruker Daltonics system.
Human tumor necrosis factor-alpha (TNF-α) was procured from Peprotech and fMLF was purchased from Sigma. Aliquots of both samples were taken (TNF-α, 10 U/mL, and fMLP, 10 mM) and stored at -20°C. For every assay the solutions were thawed to ambient temperature and freshly diluted with hepatic arterial (HA) buffer before use. Multiscreen high throughput screening (HTS) with glass fiber filter (FC) 96-well plates, type C, with 1.2-μm glass filters were purchased from Millipore. Filtration from 96-well plates was performed under vacuum on Brandel filtration device. The membranes from each well were collected by punching with the Millipore Multiscreen punching instrument. The radioactivity from 64Cu-bound ligand was measured with either Minaxi (Packard), Autogamma 5000 series (Packard), or Wallac 1420 Wizard (Perkin-Elmer), γ-counters. Radioactivity was measured for 1 min per sample and was not corrected for decay.
Synthesis and radiolabeling
cFLFLFK-PEG-t-Boc was prepared by incubating a mixture of cFLFLFK(NH2) (10 mg, 10.6 μmol) dissolved in 2 mL of acetonitrile and t-Boc-PEG-NHS (30 mg, 8.8 μmol) dissolved in 2 mL of sodium borate buffer (0.1N, pH 8.5) overnight at 4°C. Removal of volatiles under reduced pressure using rotary evaporator afforded a residue which was dissolved in 2 mL of TFA and left at room temperature for 2 hr to remove t-Boc protecting group. Concentration of the mixture under reduced pressure followed by reconstitution in 50% actontrile:water (2.0 ml) yielded stock solution. This solution was subjected to multiple injections (~5-6) on semipreparative RP-HPLC to collect fractions containing pure cFLFLFK-PEG-NH2 (retention time 18.4 min.). The fractions were concentrated under reduced pressure to yield pure sample, which was further characterized by MALDI-TOF mass spectroscopy. The average molecular weight distribution of cFLFLFK-PEG-NH2 was centered at 4.3 kD and major m/z peaks were observed at 4240, 4284, 4372, and 4416. The average calculated mass was 4331.
(16.5 mg, 3.8 μmol) was dissolved in 1 mL of H2
O and the pH was adjusted to 8.5 with 0.1N NaOH. To this solution was added DOTA-NHS (19 μmol in 20 μL of water), prepared according to a previously reported method (9
). The mixture was incubated overnight at 4°C. The solution was subjected to HPLC purification (retention time 16.8 min.) to yield pure cFLFLF-PEG-DOTA (7.6 mg, 43%). Characterization of the peptide by MALDI-TOF revealed an average molecular weight distribution centered at 4.8 kD and major m/z
peaks were observed at 4644, 4688, 4776, and 4820. The average calculated mass was 4718.
The radiolabeling was accomplished by addition of 200-800 μCi (7.4 - 29.6 MBq) of 64CuCl2 to 5-20 μg of cFLFLF- PEG-DOTA in 0.1N ammonium acetate (pH 5.5) buffer and the mixture was incubated at 40°C for 30 minutes. The mixture was injected as is for RP-HPLC purification. The column eluate was monitored by UV absorbance at 215 nm and with a gamma detector. The collected product eluted at 17.2 min with a radiochemical yield higher than 95% and the specific activity of 1.1×106 MBq/mmol (yield > 90%). Pure fractions were concentrated under reduced pressure. The radiolabeled peptide was further characterized by comparing its chromatographic properties with non-radioactive copper labeled compound synthesized independently using copper chloride in the same process. Analysis by MALDI-TOF revealed an average MW distribution of about 4.8 kD and major m/z peaks were observed at 4692, 4736, 4778, and 4794. The average calculated mass was 4782, which is in strong agreement with experimental values.
To test for compound stability, we incubated the compound in serum at 37 °C for 1, 3, and 6 hrs. Following incubation, we monitored the compound with HPLC. To determine the partition coefficient of the pegylated and non-pegylated compound, we dissolved about 350 kBq of cFLFLF-PEG76-DOTA-64Cu (or cFLFLF-DOTA-64Cu) in 500μL of water and mixed the solution with 500μL of octanol in an Eppendorf microcentrifuge tube. The tube was sonicated for 10 min and then was centrifuged at 4,000 rpm for 5 min (model Fisher Scientific Marathon Micro-A). Radioactivity was measured in 100μL aliquots of both octanol and water layers in triplicate.
In vitro assays
Human neutrophils were prepared from normal heparinized (10 U/mL) venous blood by a one-step Ficoll-Hypaque separation procedure (10
), yielding approximately 98% neutrophils; greater than 95% viable as determined with trypan blue containing less than 50 pg·ml-1
of endotoxin. Following separation, neutrophils were washed with Hank's balanced salt solution (HBSS) with heparin (10 U/mL) three times. After the third wash, neutrophils were resuspended in HA Buffer, which was HBSS supplemented with 0.1% human albumin (Bayer Healthcare). Neutrophil experiments were completed in HA Buffer.
Freshly isolated human neutrophils (4×106 cells/mL) were treated with 10 U/mL of TNF-alpha (Peprotech) twenty minutes prior to binding studies and transferred to a 96 well plate (Multiscreen® HTS FC by Millipore, Billerica, MA. 1.2 μ glass filter type C, 50.0 μL,~ 2.0 × 105 cells/well). Saturation assays were carried out using eight different concentrations of cFLFLF-PEG-64Cu (specific activity = 139 mCi/μg or 0.89 μCi/mmol) ranging from 0.001 to 100 nM. Neutrophils were incubated with the radioligand at 25 °C for 90 minutes to obtain total binding. Following incubation, 96 well plates were filtered rapidly under vacuum using Brandel filtration device (Brandel Inc. Gaithersburg, MD), washed three times with cold Tris-Mg buffer (-5 °C, 10mM, 150 μL each time/well) to remove the unbound radioligand, and dried under vacuum. The membranes from each well were collected by Millipore multiscreen punching instrument (Billerica, MA). The bound radioactivity remaining on the membranes was measured in a gamma counter. Specific binding was calculated as the difference between total binding and nonspecific binding. Non-specific binding was assessed using the highest concentration of radiolabeled ligand used in the binding experiment following pre-incubation with cold compound (100 μM of cFLFLF-PEG-Cu). Binding parameters (Kd and Bmax values) were calculated using PRISM 4.0 (GraphPad).
Superoxide production assay
The biological activity of cFLFLF-PEG-Cu or fMLP was assessed by measuring the stimulated release of superoxide by neutrophils after exposure to a range of concentrations. The neutrophil oxidative activity (luminol-enhanced chemiluminescence) was measured using a microtitre polymorphonuclear (PMN) chemiluminescence assay (11
). Activated neutrophils emit light from unstable high-energy oxygen species produced by the plasma membrane associated NADPH oxidase, and release myeloperoxidase from primary granules. The light signal from activated neutrophils can be enhanced by the addition of luminol to the samples. Luminol-enhanced emission of light is stimulated by singlet oxygen, a reactive oxygen species, dependent on both the production of superoxide and mobilization of myeloperoxidase (12
To prime the PMNs, purified cells (2×106/ml) were incubated in a water bath (37°C) for 15 min with rhTNF (10 U/ml). Following priming, aliquots of the PMNs were transferred to a microtitre plate (white walled clear bottom 96 well tissue culture plates) containing luminol (100 μM) and (0.0001-10 μM) of cFLFLF-PEG-Cu or fMLF. Peak stimulated chemiluminescence was determined with a Victor 1420 Multilabel Counter set for chemiluminescence mode using Wallac Workstation software. Sigmoidal dose-response curves for fMLP and cFLFLF-PEG-Cu stimulation of PMN oxidative activity were fit using PRISM 4.0 (GraphPad). EC50 values were derived from concentration-response curves using PRISM software. We compared relative agonist potency (EC50) of cFLFLF-PEG-Cu to fMLF on PMN oxidative activity.
In vivo assays
Lung Inflammation model
Klebsiella pneumoniae strain 43816, serotype 2 (American Type Culture Collection) was grown in trypticase soy broth (TSB) overnight, then subcultured for 2 hr to log phase growth. Following extensive rinsing, bacteria were diluted in sterile normal saline for inoculation. C57BL/6 mice (male, 8-10 weeks old, Charles River) were inoculated by oropharyngeal aspiration of 50 μl of bacterial suspension (approximately 3×105 CFU) under light inhalational anesthesia with methoxyflurane. The size of the inoculum was quantitated by plating serial dilutions on MacConkey agar plates and counting colony-forming units (CFU) after overnight incubation. Mice showed signs of moderate illness 18-36 hr after inoculation, when imaging was performed.
Organ Distribution and Pharmacokinetics
Distribution of radioactivity in the body was determined in both control (n=4) and Klebsiella-infected (n=6) mice 18 hr post injection of cFLFLF- PEG-64
Cu. After taking a single blood sample from the tail vein, mice were euthanized by deep halothane anesthesia. The pulmonary circulation was flushed with 3 mL of sterile normal saline via the right ventricle and the following organs and tissues were removed and washed: heart, lungs, muscle, bone, liver, kidney, spleen, small intestine, and stomach. The dissected tissues were placed in a pre-weighed vial and later assayed in a gamma well counter. The measured radioactivity for each sample was decay corrected back to the time of tracer injection. Biodistribution values are expressed as a percentage of the injected activity (%ID) and normalized for body and organ/tissue mass (13
Blood kinetics of cFLFLF-PEG-64Cu was studied in three control mice. Approximately 50 μL of blood from the contralateral tail vein was collected in capillary tubes at 5, 15, 30, 60, 120, and 180 minutes after tracer injection (0.37 – 0.74 MBq). The capillary tubes were placed in a vial which was pre- and post-weighed. Activity in each blood sample was measured in a gamma counter, standardized for injected dose and animal body weight, and expressed as percent injected dose per gram of blood (%ID/g).
To estimate the number of intrapulmonary neutrophils, myeloperoxidase assays were performed. Immediately after being imaged, mice were euthanized by deep halothane anesthesia and their pulmonary circulation flushed with 3 mL of sterile normal saline via the right ventricle. The lungs were removed and snap frozen at -80°C until later assayed. Lungs were weighed and placed in homogenization buffer [hexadecyltrimethylammonium bromide], and homogenized followed by sonication and centrifugation. Five microliters of supernatant was added to assay buffer (o-dianisidine hydrochloride in potassium phosphate) in a 96 well plate and optical density kinetic measurements at wavelength 490 were made using a μQuant spectrophotometer. MPO activity is reported as change in OD/min/mg lung tissue.
Lung Immunohistochemistry Staining
Immunohistochemical (IHC) analysis was performed on harvested lung tissue 42 hrs post Klebsiella administration, which matches the time point of imaging post infection (24 hrs plus an additional 18 hrs of tracer clearance time). IHC was used to assess the relative amount and distribution of neutrophils compared to macrophages in the lungs of control versus infected mice. Prior to removal, the pulmonary circulation was flushed with saline via the right ventricle to eliminate non-adherent white blood cells. Lungs were then inflated with formalin to distend the alveolar spaces uniformly. The trachea was cannulated and 10% phosphate-buffered formalin infused at a pressure of 25 cm H20. After fixation, the lung was dissected coronally in the plane of the mainstem bronchus. Adjacent histological sections (3 μm thick) were specifically stained for either neutrophils with a monoclonal rat anti-mouse neutrophil IgG (MCA771G; Serotec) or for macrophages with anti MAC-2 IgG (ACL8942P; Accurate). Stained cells were observed under a light microscope (Microphot, Nikon, LRI Instruments AB, Tokyo, Japan).
PET/CT imaging study
24 hr after administration of Klebsiella pneumoniae, cFLFLF-PEG-64Cu (100 ~ 150 μCi) (3.7-5.5 MBq) in 200 μl of saline was injected via the tail vein. Lung SUVs were measured at several time points post injection and fit to a mono-exponential curve, allowing for the calculation of ligand clearance in the control and infected lung. This analysis provides us with an estimate of the time window post injection for which the signal difference between control and infected lungs is maximized.
For accurate image co-registration, mice were placed in the prone position in a custom designed portable imaging tray, facilitating precise positioning between scanners. Anesthesia (1-2% isoflurane in oxygen) was delivered throughout the duration of imaging. Micro-X-ray computed tomography (14
) images were acquired using a scanner developed in-house. Following CT acquisition, mice were transported to the PET scanner (Focus F-120, Siemens) and scanned for ~25 min. CT images were reconstructed with a 3D filtered back-projection algorithm using the COBRA software (Exxim, Inc.). The reconstructed pixel size was 0.15 × 0.15 × 0.15 mm on a 320 × 320 × 384 image matrix. Using MicroPET Manager (Siemens, version 188.8.131.52), PET data was reconstructed using the OSEM3D/MAP algorithm (zoom factor = 2.164). The reconstructed pixel size was 0.28 × 0.28 × 0.79 mm on a 128 × 128 × 95 image matrix. All microPET images were corrected for decay, but not attenuation.
CT-PET image co-registration was performed using ASIPRO (Siemens) and a transformation matrix previously obtained with an imaging phantom. To characterize the accumulation of the tracer in lungs, region-of-interest (ROI) analysis was performed. CT images were used to visualize lung boundaries and guide the placement of lung ROIs, which were drawn manually. 10 ± 2 contiguous transaxial lung ROIs were drawn to cover the entire lung volume. Lung ROIs were transferred to the PET images and the mean activity per milliliter of lung tissue was determined. Standardized uptake values (SUVs), defined as product of the mean lung ROI activity and the animal body weight divided by the injected dose were computed.
As a global index of lung inflammation, lung to muscle ratios were computed based on ROI analysis with the microCT image data sets. ROIs were drawn on transaxial slices, covering the entire lung volume. For every slice, the average pixel value was computed by dividing the total pixel values by the total number of pixels in that 2D ROI. The average pixel value in the whole lung is normalized to that of muscle to offset any potential fluctuations due to variability in CT acquisition parameters.
Group data are expressed as the mean ± SD. Student t-test was used to determine differences in SUV, %ID, microCT lung to muscle ratios, and MPO assay results among mice administered Klebsiella pneumonia and normal control mice. P value less than 0.05 was indicative of statistical significance. Sigma-Stat v3.0 (SPSS, Inc, Chicago, IL) was used for statistical calculations.