^99mTc-Labeled Duramycin as a Novel Phosphatidylethanolamine-Binding Molecular Probe

High-Performance Liquid Chromatography (HPLC) Method 1

A C₁₈ column (pore size, 90 Å; 250 × 4.6 mm) (Jupiter; Phenomenex) was used with a mobile phase system consisting of acetonitrile and water at a flow rate of 1.0 mL/min at room temperature. A baseline of 5 min at 90% of buffer A (water with 0.1% trifluoroacetic acid, v/v) and 10% of buffer B (acetonitrile with 0.1% trifluoroacetic acid, v/v) was followed by a linear gradient to 10% of buffer A and 90% of buffer B in 35 min.

HPLC Method 2

A Jupiter C₁₈ column (pore size, 90 Å; 250 × 4.6 mm) was used with a buffered mobile phase system consisting of a phosphate buffer (10 μM of sodium phosphate, pH 6.7) and acetonitrile at a flow rate of 1.0 mL/min at room temperature. A baseline of 5 min at 90% of buffer A (phosphate buffer, pH 6.7) and 10% of buffer B (acetonitrile) was followed by a linear gradient to 10% of buffer A and 90% of buffer B in 30 min. The level of radioactivity in the HPLC eluate was monitored by γ-counting at an energy window of 140 ± 15 keV.

Radiolabeling of Duramycin

Duramycin was labeled with ^99mTc after HYNIC modification. Specifically, the HYNIC-conjugated duramycin was synthesized by reacting HYNIC with duramycin at a molar ratio of 8:1, in the presence of 4 equivalents of triethylamine. The reaction was performed in dimethylformamide for 18 h at room temperature with gentle stirring. HYNIC-conjugated duramycin was purified using HPLC method 1. The fractions that contained HYNIC-conjugated duramycin were pooled, aliquoted into 15-μg fractions, freeze-dried, and stored under argon at −80°C until use. The MW of the final product was confirmed using matrix-assisted laser desorption/ionization mass spectrometry. To synthesize a control peptide, which does not bind PtdE but has a minimally altered structure and the MW of duramycin, we inactivated duramycin by modifying a carboxylate group of Asp15 in the binding pocket. The blocking reaction was performed in dimethylformamide at room temperature, with duramycin, ethanolamine, and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride at a molar ratio of 1:10:40. The inactivated compound, duramycin^I, was purified by C₁₈ reversed-phase HPLC. For radiolabeling, duramycin^I was derivatized with HYNIC, purified by C₁₈ reversed-phase HPLC, and aliquoted as described earlier.

HYNIC-conjugated duramycin was labeled with ^99mTc using the tricine–phosphine coligand system. Specifically, 15 μg of HYNIC-conjugated duramycin was mixed with 40 mg of tricine, 1 mg of trisodium triphenylphosphine-3,3′,3″-trisulfonate (TPPTS), and 20 μg of SnCl₂ in 0.8 mL at pH 5.3. The labeling was initiated with the addition of about 37 MBq of ^99mTc. The incubation was allowed to proceed for 40 min at room temperature. The final radiopharmaceutical, ^99mTc-duramycin, was analyzed and purified using HPLC method 2. After purification, acetonitrile was removed by evaporation under nitrogen, and ^99mTc-duramycin was reconstituted in saline for use. ^99mTc-labeled duramycin^I was synthesized in the same fashion. For stability tests, cysteine challenge was performed at a cysteine–to–^99mTc-duramycin ratio of 100:1, in 0.01 μM of phosphate buffer (pH 6.8) for 2 h at room temperature. The sample was analyzed with HPLC radiodetection (radio-HPLC) (method 2).

Preparation of Liposomes

Liposomes with the following chemical compositions were made: phosphatidylcholine (PtdC) (100%), PtdC/PtdE (50/50, w/w), PtdC/PtdS (50/50, w/w), and PtdC/phosphatidylglycerol (PtdG) (50/50, w/w). Solid phospholipids were weighed (2 mg) for each type of lipid in separate glass test tubes (10 × 140 mm). One milliliter of chloroform was used to completely dissolve the phospholipid in each tube. The chloroform was evaporated under a stream of nitrogen, and the dried phospholipids formed a thin film of residue on the test tube wall. The samples were thoroughly dried under vacuum overnight. The next morning, each sample was resuspended in 2 mL of N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer (15 μM of HEPES, 120 μM of sodium chloride; pH 7.4) at 45°C. Each suspension was sonicated for 2–5 min at 15 s/cycle until the mixture turned opaque, which indicated the formation of multilaminar liposomes. The samples were kept on ice until use.

In Vitro Binding Tests

The PtdE-binding activity of ^99mTc-duramycin was examined using binding assays in vitro. A good source of exposed PtdE was apoptotic Jurkat lymphocytes after incubation with a final concentration of camptothecin of 5 μM at 37°C in a humidified atmosphere, with 5% of CO₂. Nontreated control or treated cells were harvested by centrifugation at 800g for 5 min and resuspended at 2 × 10⁶/mL. Radiolabeled duramycin or duramycin^I was added to each milliliter of cells at a final concentration of 100 nM, and the binding was allowed to proceed for 5 min at room temperature. The mixture was then loaded onto a layer of Histopaque (Sigma-Aldrich Co.). After centrifugation at 800g for 5 min, the cell pellet was collected at the bottom of the tube below the Histopaque layer (the medium that contained the nonbound radioactivity remained above the Histopaque). The aqueous supernatant was removed by gentle aspiration. The tip of the tube that contained the pellet was cut off using a hot scalpel. Bound radioactivity was determined by direct γ-counting at an energy window of 140 ± 15 keV.

Competition Assay

The retention of the binding activity was determined using a competition assay in the presence of PtdE-containing liposomes. PtdC/PtdE liposomes were prepared as described earlier. A binding assay was performed in triplicate, in the presence of increasing amounts of PtdC/PtdE liposomes, from 1 to 10 μM of final concentration, in 4-fold increments. The radioactivity bound to the cell pellet was determined by γ-counting as described earlier. The assay was repeated using liposomes containing other species of closely related phospholipids, including PtdC, PtdG, and PtdS.

Blood Half-Life and Pharmacokinetic Studies

The animal protocol was approved by the Institutional Animal Care and Use Committee under the National Institutes of Health guidelines. Healthy Sprague–Dawley rats were injected with radiolabeled duramycin (2.3 nmol) via the tail vein. At 0, 1, 5, 15, 30, and 60 min, 50 μL of blood were withdrawn from a preinstalled femoral artery catheter and collected in heparinized tubes. The blood sample was centrifuged at 5,000g, and the radioactivity in the cellular and plasma fractions was measured separately by direct γ-counting using an energy window of 140 ± 15 keV. The data output included total blood activity with time and the activity associated with blood cells and the plasma, respectively. The blood half-life was measured in triplicate. To investigate the pharmacokinetics of the radiopharmaceutical, 10 μL of the plasma or urine sample were analyzed using HPLC method 2.

Biodistribution Studies

Healthy Sprague–Dawley rats were injected with radiolabeled duramycin (2.3 nmol). A dynamic whole-body biodistribution profile was acquired using an in-house γ-camera (XRT; GE Healthcare). The anesthetized animal was continually imaged at an energy window of 140 ± 15 keV; field of view, 22.5 × 22.5 cm; and matrix, 128 × 128, at 1 min per image for up to 60 min after injection.

For quantitative biodistribution at each designated time point after intravenous injection of radiolabeled duramycin, a group of 4 rats was sacrificed. Various tissues were dissected, rinsed in saline, and weighed, and the level of radioactivity was determined using direct γ-counting (140 ± 15 keV).

In Vivo Imaging of Acute Cardiac Cell Death Using a Rat Model of Ischemia and Reperfusion

Sprague–Dawley rats were intraperitoneally anesthetized with sodium pentobarbital (50 mg/kg of body weight). After tracheotomy and intubation, respiration was maintained using a rodent ventilator. The proximal left anterior descending coronary artery (LAD) was occluded using a 6.0 suture at 1 mm below the left atrial appendage. For the sham operation, the suture was passed underneath the LAD without ligation. The presence of acute ischemia or reperfusion was confirmed by paleness in the area at risk and changes in electrocardiogram profiles including immediate elevation of ST segment and significant increase in the QRS complex amplitude and width. The coronary ligation was continued for 30 min. After reperfusion, the chest wall was closed by suturing in layers, and ventilation was maintained until the rat could regain spontaneous respiration.

After 2 h of reperfusion, ^99mTc-duramycin or ^99mTc-duramycin^I (2.3 nmol, ∼7.4 MBq) was injected via the tail vein. Dynamic in vivo planar imaging was performed using a γ-camera (energy window, 140 ± 15 keV; matrix size, 128 × 128; field of view, 22.5 × 22.5 cm) at 5 min/image for 60 min. A static image with 500 K counts was acquired after dynamic imaging.

At the end of the imaging study, the animal was sacrificed. The heart was excised and quickly rinsed in saline. About 2 mL of 0.5% triphenyl tetrazolium chloride in a HEPES buffer (w/v) was infused retrograde into the aorta to stain the entire myocardium. After 15 min of incubation at 37°C, the heart was fixed in 4% formaldehyde. Short-axis sections of the heart with a thickness of about 500 μm were cut and exposed to film (BMX MS; Kodak) overnight at −80°C. After the film was developed, each corresponding slice was differentiated overnight in 1% of formaldehyde in phosphate-buffered saline (v/v). Digitized autoradiography and histology section images were visually inspected. For measuring the radioactivity uptake in the infarcted tissues, the heart was harvested and stained by triphenyl tetrazolium chloride as described earlier. Infarcted myocardium was dissected from the viable tissue. After weighing, the radioactivity in the specimen was measured by γ-counting with an energy window at 140 ± 15 keV. The data were converted to percentage injected dose per gram (%ID/g).

Bioconjugation and Radiolabeling

After conjugation, the MW of HYNIC-conjugated duramycin was confirmed using mass spectrometry (both expected and actual MW, 2,188.4 g/mol). At this MW, 1 HYNIC covalently attaches to each molecule of duramycin. The primary sequence of duramycin has 19 amino acids in the order of CKQSCSFGPFTFVCDGNTK (Fig. 1A). Two primary amines are available for conjugation at the N-terminal and the side chain of Lys2 (Fig. 1A). It is likely that HYNIC-conjugated duramycin is in a form of isomers, where the HYNIC moiety is present on either Cys1 or Lys2. The duramycin conjugate with 2 HYNIC moieties was removed by HPLC purification and confirmed by mass spectrometry. Figure 1B demonstrates a typical radiochromatogram, in which ^99mTc-duramycin was eluted with a retention time of 24 min. At the current labeling condition, the labeling efficiency was 80%–85%, the radiochemical purity was between 78% and 89% before HPLC purification, and the specific activity was 54 GBq. Although column chromatography purification (Figs. 1B and 1C) is necessary to obtain the final radiopharmaceutical for further experiments, the current radiolabeling protocol is sufficiently amenable to conduct our preliminary studies. Once purified, ^99mTc-duramycin is stable, and no significant levels of detached ^99mTc were detected for at least 24 h in aqueous solution. This result is consistent with the prior report that a phosphine coligand substantially improves the stability of ^99mTc coordination, compared with tricine alone (17,18).

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FIGURE 1. 

(A) Diagram illustrating primary structure of duramycin with cross-linking bridges. (B and C) Radio-HPLC chromatograms of ^99mTc-duramycin before and after purification are shown in B and C, respectively. Low, but significant, level of ^99mTc-pertechnetate is present before purification with retention time of 2 min. (D) Representative competition curve of 3 independent experiments. ^99mTc-duramycin binding of apoptotic cells is competitively diminished by presence of PtdE-containing liposomes (•) but not other phospholipid species, including PtdC (▪), PtdG (□), or PtdS (○). cpm = counts per minute.

When the native form of duramycin was labeled with ^99mTc without HYNIC, the radiochemical yield was about 10%. In a cysteine challenge experiment, the radioactivity associated with the native duramycin was diminished by another 90% at a cysteine-to-duramycin ratio of 100:1. In contrast, under the same condition the radioactivity bound to HYNIC-conjugated duramycin was reduced by less than 3%. The native form of duramycin can indeed take up a limited amount of ^99mTc but does so only loosely, without, perhaps, forming a well-defined coordination complex. The presence of HYNIC, or another form of chelation core, is essential for the stable labeling of duramycin with ^99mTc.

In Vitro Binding Tests

The cellular binding of ^99mTc-duramycin was dramatically enhanced in apoptotic versus viable control cells, and the binding was competitively abolished in the presence of PtdE-containing liposomes.

Specifically, ^99mTc-duramycin was incubated with 2 × 10⁶ viable Jurkat lymphocytes as control or apoptotic cells induced by camptothecin treatment. Although the radioactivity uptake in viable cells remained near background, the binding of ^99mTc-duramycin to apoptotic cells was elevated by a factor of 32 ± 6 (n = 3). According to Figure 1D, this binding to the apoptotic cells was exclusively and competitively diminished in the presence of increasing concentrations of PtdE-containing liposomes. However, liposomes consisting of PtdC, PtdG, or PtdS were unable to competitively reduce the radioactivity uptake of ^99mTc-duramycin in apoptotic cells. ^99mTc-duramycin^I bound to apoptotic cells at background levels only, and this binding was not competitively diminished in the presence of PtdE-containing liposomes (data not shown).

Pharmacokinetics and Biodistribution of ^99mTc-Duramycin in Rats

After intravenous injection, ^99mTc-duramycin exhibited rapid clearance, with a blood half-life of less than 4 min (Fig. 2A). Of the radioactivity in the blood, greater than 95% was associated with the plasma fraction at all time points, confirming that ^99mTc-duramycin has minimal interactions with normal blood cells. According to radio-HPLC analysis of plasma samples, the majority of the injected radioactivity remains as the parent ^99mTc-duramycin (Figs. 2C and 2D). A minor peak is present with a longer retention time (27 min), which appears to be in equilibrium with an unknown blood component (Figs. 2C and 2D). The chemical species of this minor peak was not excreted by the renal function because it was not detected in the urine. Radio-HPLC analysis of ^99mTc-duramycin in the presence of rat serum albumin did not result in any complex formation between the tracer and albumin. It is likely that after intravenous injection, ^99mTc-duramycin is associated to a degree with the lipoproteins, which are known to contain PtdE. The intravenously injected ^99mTc-duramycin showed no sign of metabolic degradation and was recovered intact from urine samples, in which neither additional radioactive derivatives nor ^99mTc-pertechnetate was detected (Fig. 2E).

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FIGURE 2. 

(A) Blood clearance of ^99mTc-duramycin in healthy rats (n = 3). (B–E) Radio-HPLC chromatograms of ^99mTc-duramycin standard (B), serum at 1 min after injection (C), serum at 5 min after injection (D), and urine at 60 min after injection (E).

For biodistribution, ^99mTc-duramycin renal excretion is the predominant route of clearance, with a low uptake in the hepatic and gastrointestinal regions (Table 1). The agent does not seem to cross the blood–brain barrier, because the brain uptake was low (Table 1). The presence of radioactivity in the lungs and the normal myocardium promptly approached background levels with time because of a rapid blood clearance, thus paving the way for an early and clear detection of acute cardiac cell death. This biodistribution profile was confirmed by in vivo dynamic studies using dynamic anterior planar scintigraphic imaging (Fig. 3A). At the current dosage tested, no signs of toxicity or other adverse effects were observed.

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FIGURE 3. 

(A) Dynamic planar imaging of ^99mTc-duramycin distribution in healthy rat. Note rapid renal clearance of radiotracer and low hepatic uptake. (B) Whole-body dynamic planar imaging of ^99mTc-duramycin uptake in rat with acute myocardial infarction. (C) Non–color-enhanced, raw counts of static planar images of sham-operated rat (left) and infarcted rat (right) acquired at 120 min after intravenous injection of ^99mTc-duramycin. Infarct site is marked by arrows. In autoradiography, radioactivity uptake in myocardium colocalizes with infarct with excellent infarct-to-noninfarct ratio (inset). B = bladder; K = kidney.

In Vivo Imaging of Acute Cardiac Cell Death

In dynamic images, a focal uptake was seen at the infarct as early as 10 min after injection (Fig. 3B). The prompt blood clearance and low hepatic uptake facilitated an early and conspicuous appearance of the infarct. At 2 h after injection, the infarct-to-lung ratio was 4.8 ± 0.4 (n = 3) and the infarct-to-muscle ratio was 12.2 ± 1.3 (n = 3). The distribution of radioactivity in the autoradiography colocalized with the infarct in the histology of the myocardium (Fig. 3C, inset). The level of radioactivity remained persistent, with no washout over time. In contrast, the presence of ^99mTc-duramycin^I in the infarcted heart was accompanied by a washout, a result consistent with the compromised PtdE-binding activity. At 1 h after injection, the average radioactivity at the infarct site was above 4.0 %ID/g for ^99mTc-duramycin, whereas that for ^99mTc-duramycin^I was less than 1.0 %ID/g.