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Radiosynthesis and pre-clinical evaluation of [68Ga] labeled antimicrobial peptide fragment GF-17 as a potential infection imaging PET radiotracer
Applied Radiation and Isotopes 149 (2019) 9–21
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Radiosynthesis and pre-clinical evaluation of [68Ga] labeled antimicrobial peptide fragment GF-17 as a potential infection imaging PET radiotracer
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Shalini Chopraa, Baljinder Singha,∗, Ashwani Koulb, Anil K. Mishrac, Stephanie Robud, Amritjyot Kaura, Anchal Ghaie, Neena Caplashf, Hans-Jürgen Westerd a
Department of Nuclear Medicine & PET, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh, 160012, India Department of Biophysics, Panjab University, Sector 25, Chandigarh, 160014, India c Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, DRDO, Brig. S.K. Mazumdar Road, Timarpur, Delhi, 110054, India d Department of Nuclear Medicine, Klinikum Rechts Der Isar, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany e Department of Radiology, School of Medicine, Washington University, 510 South Kingshighway Boulevard, St. Louis, Missouri, 63110-107, USA f Department of Biotechnology, Panjab University, Sector 25, Chandigarh, 160014, India b
H I GH L IG H T S
peptide fragment GF-17 was synthesized in-house and conjugated with DOTA and characterization of the peptide-conjugate was carried out. • Antimicrobial The peptide conjugate was radiolabeled successfully with [ Ga] achieving radiolabeling efficiency of 95.0% using the optimized peptide conjugate amount of • 20.0 nmol. uptake studies demonstrated that [ Ga]DOTA-peptide had significant binding in the two bacterial strains studied. • The animal bio-distribution and PET imaging studies demonstrated renal mode of excretion for [ Ga]DOTA-GF-17. • The biodistribution studies in infection and inflammation models had shown significant upake of radiolabeled peptide conjugate at the infection sites. • The • [ Ga]DOTA-GF-17 peptide holds good potential for further undertaking the tracer for detailed pre-clinical evaluation to assess its translational relevance. 68
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Keywords: [68Ga]DOTA-GF-17 AMP Infection PET imaging Preclinical evaluation
The antimicrobial peptide fragment GF-17 was synthesized in-house and conjugated with DOTA and measured molecular mass of DOTA-GF-17 conjugate was 2489 Da. The peptide conjugate was purified and labeled with [68Ga]. The best radiolabeling efficiency (95.0%) of [68Ga]DOTA-GF-17 was achieved at pH 4 with peptide conjugate amount of 20.0 nmol with 30 min of heating at 95 °C. The product remained stable for up to 3 h. The plasma protein binding and lipophilicity for [68Ga]DOTA-GF-17 were 80.98% and −3.12 respectively. The uptake studies with [68Ga]DOTA- GF-17 in S.aureus and P.aeruginosa bacterial strains demonstrated binding of 69.08% and 43.69% respectively. The animal bio-distribution and PET imaging studies were in agreement showing similar pattern for organs’ tracer distribution and renal excretion. The tracer had rapid blood clearance and uptake in bone marrow and muscles was very low. The highest uptake of [68Ga]DOTA-GF-17 was observed at 45 min and 120 min in S.aureus and P.aeruginosa infections respectively. [68Ga]DOTA-GF-17 could be a promising PET tracer and holds a great scope for taking the product further to perform extensive PET studies in animal infection (using gram negative/positive strains) models to prove the diagnostic utility of this novel PET tracer for futuristic clinical applications.
1. Introduction Infection and inflammation are two closely related and clinically highly relevant phenomena. Infection occurs due to invasion of pathogens in tissues or organs, whereas, inflammation often occurs as a
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response of host immune system towards the pathogen invasion and toxins produced by these organisms (Brouwer et al., 2008). An early detection of active bacterial infection and differentiation from inflammation provides important information relevant for selecting appropriate treatment option. Although, noninvasive imaging techniques
Corresponding author. Department of Nuclear Medicine PGIMER, Chandigarh, 160012, India. E-mail address: [email protected] (B. Singh).
https://doi.org/10.1016/j.apradiso.2019.04.008 Received 19 August 2018; Received in revised form 4 April 2019; Accepted 5 April 2019 Available online 10 April 2019 0969-8043/ © 2019 Elsevier Ltd. All rights reserved.
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pre-clinical testing for evaluation of its translational clinical utility in PET imaging of human bacterial infections.
such as ultrasound (US), computed tomography (CT) and magnetic imaging resonance (MRI) are available, but they suffer from low specificity for the accurate diagnosis of infection. Further, these techniques pick only the morphological and structural changes which are often the late manifestations of the disease patho-physiology (Becker and Meller, 2001). On the other hand, nuclear medicine techniques i.e. single photon emission computed tomography (SPECT) and positron emission tomography (PET) provide biochemical/physiological information and further allow detection of subtle changes of physiological processes at a very early stage even before the onset of morphological changes. Previously, many SPECT based tracers have been developed and used extensively for the detection of human infections (Love and Palestro, 2004). These SPECT tracers include technetium-hexamethylpropyleneamine oxime ([99mTc] HMPAO), Indium ([111In])-oxine, Gallium-67 ([67Ga])-citrate and [99mTc] labelled antibiotics (Palestro, 2016; Singh et al., 2008). Amongst these, radiolabeled leukocytes based SPECT imaging is considered as ‘gold standard’ imaging technique for the detection of human infections. However, the labeling procedure is cumbersome, requires GMP facility for in vitro blood radiolabeling and re-injection of the radiolabeled leukocytes in the patients is not always safe. SPECT imaging using radiolabeled antibiotics is easier to perform but have the limitations of not picking up the resistant bacterial infections and thus, have compromised sensitivity and specificity (Welling et al., 2009). PET radiotracer 2-deoxy-2-[fluorine-18] fluoro-D-glucose ([18F] FDG) has also been used for the imaging of infection and found to be very sensitive but lacks specificity (Glaudemans et al., 2013; Gotthardt et al., 2013; Becker and Meller, 2001). [18F]FDG labeled leukocyte PET imaging is superior to [18F]FDG imaging for detection of infection as well as for the response assessment to antibiotic therapy. Like [99mTc] HMPAO, radiolabeling leukocytes with [18F] FDG is also a cumbersome and time consuming process, and often have low radiolabeling efficiency (Dumarey et al., 2006; Rini et al., 2006]. Over the last decade, the use of Gallium-68 ([68Ga]) (half-life t1/2 = 68 min; positron emission intensity = 87%) in various PET clinical applications has increase (Schultz et al., 2013b; Singh et al., 2013; Buchmann et al., 2007; Ghai et al., 2018). This PET radionuclide has the potential for an on-demand production via generator technologies that provide reliable and highpurity [68Ga] in sufficient quantities for routine radiopharmaceutical production without the need for cyclotron operations (Prata, 2012; Roesch, 2012). Many [68Ga] labeled compounds have shown very encouraging pre-clinical results, but their clinical validation is under evaluation (Ghai et al., 2015; Jaswal et al., 2017). To further expand the applications of [68Ga] PET imaging, labeling of [68Ga] with antimicrobial peptides (AMPs) is viewed as a very promising approach for the detection of bacterial infections. AMPs are short sequences of amino acids and are reported to kill a wide range of microorganisms (Chan et al., 2006; Shai, 1999; Dürr et al., 2006). A large number of microorganisms causing common human infections have been reported not to develop resistance to this new class (AMPs) of therapeutic agents (Zasloff, 2002; Hancock and Diamond, 2000). Therefore, in this study, we have explored the potential of GF-17, an antimicrobial peptide fragment of LL-37 to be developed as a promising PET probe for imaging infection. GF-17 corresponds to residues 17 to 32 of peptide LL-37, has been studied and found to have the major antimicrobial region of the parent peptide. This smaller peptide has killing activity against Escherichia coli and Staphylococcus aureus (Li et al., 2006; Wang et al., 2012). In the present study, GF-17 was synthesized in the laboratory by the process of solid phase peptide synthesis (SPPS). This peptide was conjugated with a bi-functional chelator i.e. 1,4,7,20-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and further purification and characterization of peptide DOTA conjugate was performed. The peptide conjugate was radiolabeled with [68Ga]. The resultant [68Ga] labeled peptide conjugate was subjected to a detailed
2. Materials All the reagents and chemicals used in the study used were of analytical grade. All the fluorenylmethyloxycarbonyl (fmoc) protected amino acids were purchased from Bachem, Bubendorf, Switzerland and Biotech, Marktredwitz, Germany. 2-Chlorotrityl chloride (2-CTC) resin was procured from PepChem, Tubingen, Germany. Coupling agents hydroxybenzotriazole (HOBt), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIPEA) used for the SPPS were procured from Sigma Aldrich (Munich, Germany). Bifunctional chelator DOTA was procured from Chematech (Dijon, France). All the remaining reagents used were procured from Sigma Aldrich (Munich, Germany and India). Dichloromethane (DCM) was used for swelling and washing resins during SPPS. Dimethylformamide (DMF), n-methyl-2-pyrrolidone (NMP) and methanol were used for washing resin during SPPS. A cleavage solution containing DCM, 2,2,2-trifluoroethanol (TFE), and acetic acid (v/v/v; 6/1/3) was used as for cleaving GF-17 from resin. Nhydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCl) and DIPEA were used for DOTA coupling reaction. A solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and distilled water (H2O) (v/v/v; 95.00%, 2.5%, 2.5%) was used to cleave DOTA conjugated AMP fragment from resin and removal of protecting groups. Piperidine (20.00%) was used for the removal of fmoc group. Reverse phase high performance liquid chromatography (RP-HPLC) was done for the purification of DOTA-GF-17 post synthesis with different gradients of 0.1% TFA (v/v) in water (solvent A) and 0.1% TFA (v/v) in acetonitrile (CH3CN) (solvent B) with constant flow rate of 1.0 mL/min (for analytical RP-HPLC) and 5.0 mL/min (for preparative RP-HPLC). Analytical RP-HPLC was done on Shimadzu Prominence HPLC system (Shimadzu Germany, Neufahrn, Germany) (having a diode array detector) using Nucleosil 100 C 18 (5 μm, 125 × 4.00 mm) column. Preparative RP-HPLC was done on Sykam Gradient HPLC System (Sykam GmBH, Ersing, Germany) (having a 206 PHD UV-VIS detector) using Multospher 100 RP 18 (250 × 20.00 mm) column (CS GmbH, Langerwehe, Germany).Electron spray ionosation mass spectrometry (ESI-MS) of GF-17 and DOTA conjugated GF-17 was done by using a 500-MS IT mass spectrometer (Varian, Agilent Technologies, Santa Clara, USA). For radiolabeling, sodium acetate buffer of different pH (3.0,4.0,5.0) was prepared using 0.2 M sodium acetate and 0.2 M glacial acetic acid. For instant thin layer chromatograph (ITLC), silica gel coated plates, 0.2 M sodium citrate (mobile phase) and solution of 1.0 M ammonium acetate and methanol (1:1) (mobile phase) were used. [68Ga] was eluted from germanium-68/gallium-68 ([68Ge]/ [68Ga]) generator (ITG, Munich, Germany) with 0.05 M hydrochloric acid (HCl). The radioactivity was measured by using a dose calibrator (CRC-15, Capintec Inc., USA) and well type gamma counter (CAPTUS 3000, Capintec Inc., USA). Purification of radiolabeled DOTA-AMPs was done using C-8 SPE light cartridge procured from Waters Corporation Milford, Massachusetts, USA. Radio-HPLC was performed using a Nucleosil 100 C 18 column (5 μm, 125 × 4.00 mm) connected to a NaI (Tl) well type scintillation counter (EG & G Ortect, Munich, Germany). Trichloroacetic acid (TCA) (5.00%) was used to separate plasma proteins. N-octanol and distilled H2O were used to determine lipophilicity of [68Ga]DOTA-GF-17. Phosphate buffered saline (PBS) and normal saline were used to form suspension of bacterial cultures. Ethyl ether and isoflurane were used as anesthesia during animal studies. For the uptake/binding studies and infection models, live strains of Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa) (2 × 107 CFU) were used. PBS was used for performing periodic washing in the binding studies. Turpentine oil was used for 10
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Fig. 1. Chemical structure of antimicrobial peptide fragment GF-17.
Fig. 2. HPLC chromatogram of crude GF-17 showing peak for GF-17 at 8.8 min (gradient 10%–90% of solution B, flow rate 1.00 mL/min).
Fig. 3. ESI-MS spectrum of fraction containing GF-17 showing m/z ratios of 701.8 ([M+3H]3+), 1052.6 ([M+2H]2+), 1063.6 ([M+2H + Na]2+) and 1402.3 (2[M +3H]3+). Fig. 4: Chemical structure of peptide conjugate DOTA-GF-17.
Fig. 4. Chemical structure of peptide conjugate DOTA-GF-17.
3. Methods
inducing sterile inflammation in animals. For the biodistribution studies, female Wistar rats (6–9 weeks, 200.0 ± 20.0 g) and male Balb/c mice (8–10 weeks, 30.0 ± 5.0 g)were used. For animal PET imaging specific pathogen–free Balb/c mice (male/female, 20.0–25.0 g) were used. The animals were procured and housed in the central animal house facility at PGIMER, Chandigarh, India (for biodistribution) and Klinikum Rechts Der Isar, Technical University of Munich, Munich, Germany (for PET imaging). All the animal studies were performed in compliance with the Institute Animal Ethical Committee.
3.1. Synthesis AMP fragment GF-17 was synthesized by SPPS on 2-CTC resin by coupling fmoc protected amino acids. The resin (1.0 eq) was swelled in DCM and fmoc protected valine (fmoc-val-OH) (2.4 eq) was loaded on resin in the presence of DIPEA (4.5 eq). Capping of the resin was done with methanol. Fmoc deprotection was done with 20.0% piperidine in DMF followed by washing with NMP. Next fmoc protected amino acid was coupled in the presence of HOBt (1.5 eq), TBTU (1.5 eq) and DIPEA (4.5 eq). After the coupling of last amino acid i.e. fmoc-glycine-OH (fmoc-gly-OH) and removal of last fmoc group, the peptide was cleaved 11
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Fig. 5. HPLC chromatogram for HPLC chromatogram of crude DOTA-GF-17 showing peak for unconjugated GF-17 at 8.5 min and DOTA-GF-17 at 9.2 min (gradient 37%–55% of solution B, flow rate 5.00 mL/min).
Fig. 6. ESI-MS spectrum of purified DOTA-GF-17 showing m/z ratios of 831.0 ([M+3]3+), 1245.5 ([M+2]2+) and 1660.2 (2[M+3]3+).
pH, heating time, amount of peptide conjugate. Sodium acetate buffer of different pH (3.0,4.0,5.0) was used for radiolabeling. Peptide conjugate in different amounts (5 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol) was added to 800.0 μL of the buffer. Briefly, 185.00 MBq of [68Ga] was added to the reaction solution and heated at 95 °C for different time periods (10 min, 15 min, 30 min, and 45 min). The radiolabeling efficiency of each reaction solution was analyzed by performing ITLC. Radiochemical purity was also checked for freshly radiolabeled peptide conjugate by performing radio HPLC (flow rate 2.0 mL/min, 37–45% gradient of solution B in 25 min). The radiolabeled peptide conjugate was purified using a C-8 light cartridge, preconditioned with 4.0 mL ethanol (70.00%) and 4.0 mL of distilled water. The purified radiolabeled peptide conjugate was subjected to different quality control tests.
from resin using a cleavage solution containing DCM, TFE and acetic acid (6:1:3). The peptide was lyophilized and subjected to analytical RP-HPLC, and mass spectroscopy for characterization. DOTA (4.0 eq) was conjugated to the amino (N) terminal of the peptide by activation with NHS (5.0 eq) in the presence of EDCl (5.0 eq) and DIPEA (8.0 eq). The reaction was allowed to occur for 12 h at room temperature. All the protecting groups were cleaved from the DOTA-peptide conjugate using a cleavage solution containing TFA, TIPS and water (95%:2.5%:2.5%). The newly synthesized DOTA-GF-17 was purified by RP-HPLC and characterized by mass spectroscopy.
3.2. Radiolabeling Radiolabeling of peptide conjugate was performed with freshly eluted [68Ga] (in 5.0 mL of HCl; 0.05M) from [68Ge]/[68Ga] generator. Optimization of radiolabeling conditions was done by varying buffer 12
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checked by performing ITLC as described in section 3.3.1. The solvent system used was 1.0 M ammonium acetate and methanol (1:1). 3.3.3. In vitro stability To check the in vitro degradation of [68Ga]DOTA-GF-17 as a function of time, RCP was checked for the purified [68Ga]DOTA-GF-17 at different time intervals (30 min, 45 min, 60 min, 90 min, 120 min and 180 min). 3.3.4. In vitro serum stability To check the degradation of [68Ga]DOTA-GF-17 in serum, in vitro serum stability of [68Ga]DOTA-GF-17 was determined by incubating purified radiolabeled peptide conjugate in serum. Precisely, 200.00 μL of purified [68Ga]-DOTA-GF-17 was added to 800.00 μL of serum and incubated at 37 °C for different time periods of 30 min, 45 min, 60 min, 90 min, 120 min. RCP was determined for each sample. 3.3.5. Plasma protein binding The in vitro protein binding for purified [68Ga]DOTA-GF-17 was determined by incubating the radiolabeled peptide conjugate in human plasma. Briefly, 100.0 μL of purified [68Ga]DOTA-GF-17 was incubated in 900.0 μL plasma at 37 °C for 1 h. The plasma proteins were precipitated after 1 h by adding 1.00 mL of 5.00% TCA followed by centrifugation at 3000 rpm for 5min. The supernatant was collected separately and precipitate was re-suspended in 1.00 mL TCA (5.00%). The radioactivity was measured in both precipitates and supernatant by using a well type gamma counter. Protein binding was determined by calculating binding fraction of the total radioactivity.
Fig. 7. Radiolabeling efficiency (%) of [68Ga] DOTA-GF-17 at different amounts (nmol) of peptide conjugate DOTA-GF-17.
3.3. Quality control tests of [68Ga]DOTA-GF-17 3.3.1. Radiolabeling efficiency ITLC was done to determine the radiolabeling efficiency of the freshly prepared radilolabeled peptide conjugate using two solvent systems: a) 0.2 M sodium citrate and b) 1.0 M ammonium acetate and methanol (1:1). Briefly, 4.00 μL of freshly prepared radiolabeled peptide conjugate was applied to the ITLC strip (8.00 cm) and the strip was kept in a solvent chamber. The strips were dried, cut into small pieces measuring 1 cm and measured for radioactivity in a well type gamma counter. Free [68Ga] was also subjected to ITLC strip. The percentage radiolabeling efficiency and retention factors (Rf) were calculated. Radio HPLC was also performed for the freshly labeled peptide conjugate.
3.3.6. Lipophilicity In a test tube, 100.00 μL of purified [68Ga]DOTA-GF-17 and 400.00 μL of distilled water were mixed. To this solution 500.00 μL of noctanol was added. The mixture was mixed thoroughly for 3 min. The mixture was then centrifuged at 6000 rpm for 5 min to separate the aqueous and organic phases. Precisely, 100.00 μL of aqueous phase and 100.00 μL of organic phase were carefully collected in separate test tubes. The radioactivity was measured in both phases using a well type gamma counter. The lipophilicity for [68Ga]DOTA-GF-17 was measured in terms of log P value by using the following formula:
3.3.2. Radiochemical purity Radiochemical purity (RCP) of purified [68Ga]DOTA-GF-17 was
Fig. 8. Radiolabeling efficiency (%) of [68Ga] DOTA-GF-17 at different pH of sodium acetate buffer. 13
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Fig. 9. Radiolabeling efficiency (%) of [68Ga] DOTA-GF-17 at different heating time points.
Fig. 10. In-vitro stability of [68Ga] DOTA-GF-17 as a function of time.
log Poct / wat = log[counts in octanol ÷ counts in water]
were collected separately and radioactivity was counted. The radioactivity (counts/min) in supernatants and pellet was measured. The percent uptake/binding of [68Ga]DOTA-GF-17 was calculated by using following formula:
3.4. Uptake/binding studies Uptake/binding of [68Ga]DOTA-GF-17 was studied in live strains of S.aureus and P.aeruginosa. Microbial strains (2 × 107) were suspended in 200.00 μL of PBS and approximately 7.4 MBq of radiolabeled peptide conjugate was added to the suspension. The microbial suspensions were incubated at 37 °C for different time intervals (30 min, 60 min, 120 min). The suspensions were centrifuged at 10000 rpm for 10 min. The supernatant was collected separately and pellet was suspended in PBS. The suspension was again centrifuged; supernatants and pellets
%Uptake = [Counts in Pellet ÷ Total counts(Pellet + Supernantants)] × 100
3.5. Blood kinetics studies The blood kinetics study of [68Ga]DOTA-GF-17 was carried out in female Wistat rats (n = 3). Briefly, 7.4 MBq of [68Ga]DOTA-GF-17 was 14
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Fig. 11. In-vitro serum stability of [68Ga] DOTA-GF-17 as a function of time.
Fig. 12. Radio HPLC of freshly prepared [68Ga] DOTA-GF-17 showing retention time, tr of 19.39 min.
3.7. PET imaging in normal animals
injected intravenously into each animal. The blood samples were drawn from the ocular vein of the rat at different time intervals (15 min, 30 min, 45 min, 1 h, 2 h). The radioactivity in each blood sample was measured in a well type gamma counter. The data was expressed as percent injected dose per gram (% ID/g) at each time point.
PET Imaging was done in normal Balb/c mice (n = 3) using an animal PET scanner (Inveon Seimens PET scanner) at TUM, Munich, Germany. Each mouse was injected with 3.7 MBq of radiolabeled peptide conjugate under anesthesia (isoflurane) into the tail vein. Dynamic PET imaging was done following the injection for 90 min. Reconstruction of images was done using 2 dimensional ordered subset expectation maximum algorithm (2D-OSEM) and analyzed with the help of Inveon software.
3.6. Biodistribution studies in normal animals Biodistritbution of [68Ga]DOTA-GF-17 was studied in female Wistar rats (n = 15, weighing between 200.0 and 250.0 g). A dose of 7.40 MBq of [68Ga] labeled peptide conjugate was injected in the tail vein of each animal. Venous blood was collected from each animal. The animals were sacrificed at different time points (15 min, 30 min, 45 min, 60 min, and 120 min). The various organs/tissues such as bone, muscle, heart, thyroid, lung, liver, spleen, stomach, small intestine, large intestine, kidney and brain were excised and collected. The organs/tissues were washed in saline, dried and kept in pre-weighted test tubes. The radioactivity in blood was counted and expressed as the percentage of the injected dose per mL (%ID/mL) of blood. On the other hand, the radioactivity counted in organs was expressed as injected dose per gram (%ID/g) of the organ.
3.8. Biodistribution in infection and inflammation models Infection model was developed in Balb/c mice (n = 30) using S.aureus and P.aeruginosa. Briefly, 2 × 107 CFUs of S.aureus were suspended in 200.00 μL of normal saline and injected subcutaneously into right thigh of each mouse. Similarly, infection model for P.aeruginosa was developed in mice. The animals were housed in Central Animal House Facility in PGIMER for one week and development of infection was monitored daily. Sterile inflammation was induced in Balb/c mice by injecting 50.0 μL of turpentine oil into left thigh of the animal. The 15
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Fig. 13. ITLC of purified [68Ga] DOTA-GF-17 with 0.2 M sodium citrate as mobile phase.
Fig. 14. ITLC of purified [68Ga] DOTA-GF-17 with 1.0 M ammonium acetate and methanol (1:1) as mobile phase.
chromatogram (Fig. 2) showed three prominent peaks at time 8.8 min, 12.3 min and 14.3 min. The ESI-MS analysis of fractions collected during HPLC demonstrated that GF-17 was present in fraction collected at 8.8 min with the m/z ratios showing m/z ratios of 701.8 ([M +3H]3+), 1052.6 ([M+2H]2+), 1063.6 ([M+2H + Na]2+) and 1402.3 (2[M+3H]3+) (Fig. 3) corresponding to molecular mass of 2102.4 Da. The analytical RP-HPLC data for GF-17 demonstrated its retention time, tr = 8.8 min (10%–90% gradient of solution B in 20 min). The purification of newly synthesized DOTA-GF-17 (Fig. 4)
organ biodistribution of radiotracer in infection and inflammation models was performed as described in section 3.6. Tissues from infection and inflammation sites were also collected along with other organs. The percent injected doses for all organs including infection and inflammation sites were calculated. 4. Results Analytical HPLC was done for crude GF-17 (Fig. 1). The HPLC 16
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Fig. 15. Uptake/binding values (%) of [68Ga] DOTA-GF-17 in strains of S.aureus (red) and P.aeruginosa (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 16. Biodistribution pattern of [68Ga] DOTA-GF-17 in normal Wistar rats at different time intervals.
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Fig. 17. Time activity curves of [68Ga] DOTA-GF-17 obtained from dynamic PET imaging in normal Balb/c mouse.
stable for up to 3 h (Fig. 10) and up to 2 h in serum (Fig. 11). The ITLC of the radiolabeled peptide conjugate demonstrated Rf value of 0.1 in 0.2 M sodium citrate and 0.7 in 1.0 M ammonium acetate and methanol. Radio-HPLC (Fig. 12) demonstrated RCP of 93.28% for [68Ga] DOTA-GF-17 with retention time, tr = 19.39 min. ITLC of purified [68Ga]DOTA-GF-17 demonstrated high RCP of 95.0% (Figs. 13–14). The plasma protein binding and lipophilicity for [68Ga]DOTA-GF-17 were 80.98% and −3.12 respectively. The uptake binding assays for the radiolabeled peptide conjugate were performed in live stains of S.aureus and P.aeruginosa. The results demonstrated that the uptake/binding of [68Ga]DOTA-GF-17 in live S.aureus after 30 min of incubation was 67.57% and increased to 69.08% after 120 min of incubation. The results for live strain of P.aeruginosa demonstrated the uptake binding of 32.56% at 30 min post incubation with [68Ga]DOTA- GF-17 and increased to 43.69% at 120 min post incubation (Fig. 15). The results of blood kinetics demonstrated (Fig. 16) that [68Ga] DOTA-GF-17 cleared from the systematic circulation with only 0.17% of the injected activity remaining at 30 min. The biodistribution studies were performed using [68Ga]DOTA-GF-17 in normal animals showed initial blood pool activity, uptake in kidney, heart, liver and lung. The results indicated a decrease in uptake of [68Ga]DOTA-GF-17 in all the organs with time. In kidneys the uptake increased with time. At all the time points, the radioactive uptake in organs like liver, spleen, bone, muscle and brain was lower than that in kidneys (Fig. 16). The reconstructed [68Ga] DOTA-GF-17 PET images showed initial uptake in heart, liver, kidneys, in the region of thyroid gland and low grade uptake in bone marrow and muscles. The dynamic images showed significant clearance of tracer from the heart and liver during subsequent imaging (Fig. 17). However, the clearance from the kidneys is minimal (Fig. 18). Results for biodistribution of [68Ga] DOTA-GF-17 in infection and inflammation models have been presented in Figs. 19 and 20. The biodistribution studies in mice bearing S.aureus infection (Fig. 19) and inflammation demonstrated that 0.78% of the ID/g accumulated in infection at 15 min. The maximum uptake was observed at infection site at 45 min with 0.89% of the ID/g. Initial uptake of the at inflammation site was maximum (0.86% of ID/g at 15 min) but the tracer washed out with time. In mice bearing P.aeruginosa infection (Fig. 20), the uptake of radiotracer at 15 min was 0.45% of ID/g at the infection site. The maximum uptake was observed at 120 min with accumulation of 0.75% of ID/g. It was observed that initial uptake of tracer in inflammation was more as compared to that in infection site but the tracer showed significant clearance from the inflammation site with time.
Fig. 18. Reconstructed PET image showing uptake pattern of [68Ga] DOTA-GF17 in normal Balb/c mouse.
was done by performing preparative RP-HPLC. The gradient of solution B was optimized to 37%–55% in 20 min for purification of DOTA-GF-17 with tr = 9.2 min (Fig. 5). The presence of DOTA-GF-17 in fraction collected at 9.2 min was confirmed by performing ESI-MS. The mass spectroscopy analysis demonstrated the m/z ratios of 831.0 ([M +3]3+), 1245.5 ([M+2]2+) and 1660.2 (2[M+3]3+) (Fig. 6) corresponding to molecular mass of 2489 Da. The radiolabeling efficiency for [68Ga]DOTA-GF-17 was evaluated and found to be 90.95% ± 2.61 with an amount of 20 nmol of DOTAGF-17 (Fig. 7). The best radiolabeling was observed with buffer of pH 4.0 (Fig. 8) and heating time of 30 min (Fig. 9). Highest radiolabeling efficiency of 95.0% was achieved by optimizing the radiolabeling conditions for [68Ga]DOTA-GF-17. The radiolabeled product remained 18
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Fig. 19. Biodistribution pattern of [68Ga] DOTA-GF-17 in normal Balb/c mice bearing S.aureus infection and sterile inflammation at different time intervals.
Fig. 20. Biodistribution pattern of [68Ga] DOTA-GF-17 in normal Balb/c mice bearing P.aeruginosa infection and sterile inflammation at different time intervals.
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5. Discussion
with no vital conformational changes in the molecule that would have affected the binding of DOTA-GF-17 to the bacterial cell membrane. The high binding values in live S.aureus at greater incubation time period also suggested that the amount of antimicrobial peptide was not enough to kill the bacterial cells as the radiolabeling required only nanomolar concentration of peptide conjugate. [68Ga]DOTA-GF-17 can be developed as diagnostic tracer for live S.aureus infection imaging and follow up cases. However, lower binding values of [68Ga]DOTA- GF-17 in live strains of P. aeroginosa were observed. Biodistribution studies demonstrated an initial increase in the uptake of [68Ga]DOTA-GF-17 in blood, heart, lung, liver and kidney due to the perfusion. We observed that there was no specific uptake in any organ as the uptake decreased in each organ with time. Kidneys were the only organs to show increased uptake with time. This indicated renal mode of excretion for [68Ga]DOTA-GF-17. Results from PET imaging done in normal animals demonstrated initial uptake of [68Ga]DOTA- GF-17 in heart, liver and kidneys which was in agreement with the data from biodistribution studies. The increased uptake in kidneys in later images again confirmed the renal mode of exertion for [68Ga]DOTA-GF-17. No soft tissue and bone uptake indicated absence of specific uptake of this radiotracer. The biodistribution studies performed in infection and inflammation models demonstrated that the uptake of [68Ga]DOTA-GF-17 in case of S.aureus infection was highest at 45 min post injection. However, the highest uptake of [68Ga]DOTA-GF-17 in case P.aeruginosa infection was observed at 120 min post injection. It was also observed that the uptake of [68Ga]DOTA-GF-17 was comparatively less in case of P.aeruginosa infection than that in S.aureus infection. The uptake of [68Ga]DOTA-GF17 in inflammation site was initially high as compared to that in both types of infection but decreased with time. The present study describes the synthesis of antimicrobial peptide fragment GF-17 and conjugation with bifunctional chelator DOTA. A successful radiolabeling of DOTA conjugated GF-17 with [68Ga] was achieved with high radiolabeling efficiency and stability. The normal biodistribution pattern of [68Ga] labeled DOTA-GF-17 in bone, muscles and fast clearance from liver may be useful for targeting bacterial infections in these areas. However the potential of [68Ga]DOTA-GF-17 for infection imaging requires further pre-clinical validation before translation to clinical use.
Currently, many bacterial strains have developed resistance towards the conventional antibiotics. This growing problem of bacterial resistance has created a need to develop alternate therapeutic agents for bacterial infections. Antimicrobial peptides have emerged as new diagnostic and therapeutic agents against bacterial infections. In the past, antimicrobial peptides have been studied for the potency against various bacterial strains (Gordon et al., 2005). Defensins, peptide fragments of lactoferrin and ubiquicidin have been radiolabeled and evaluated for the diagnostic and therapeutic efficacy (Welling et al., 2001). UBI 29–41 is a peptide fragment of ubiquicidin that has been radiolabeled with [99mTc] to detect animal as well as human bacterial infections (Nibbering et al., 2004; Akhtar et al., 2005).The studies have shown that [99mTc]UBI 29–41 was found to be very effective to locate infection sites in bones and soft tissues. In a recent study done by Ebenhan et al. (2017), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA) conjugated UBI was radiolabeled with [68Ga] and evaluated for imaging infection in non human primates. Our study is the first attempt where GF-17 has been conjugated with DOTA and radiolabeled with [68Ga] for the purpose of PET imaging. In the present study, we synthesized GF-17, a peptide fragment of LL-37 with antimicrobial property by SPPS. It was further conjugated with DOTA in the presence of NHS in solution phase and radiolabeled with [68Ga]. GF-17 is a cationic peptide fragment that is active against both gram positive and gram negative bacteria (Wang et al., 2012) It has been reported that the antimicrobial activity of this peptide fragment is due to the presence of cationic amino acids lysines and arginines in its structure. These amino acids play a crucial role in recognition of bacterial membrane, binding to the bacterial surface and killing the bacterial cells (Epand et al., 2009; Wang et al., 2012; Mishra et al., 2013). The side chains of arginines, lysines, isoleucine and glycine presented potential site for DOTA coupling. Due to the active involvement of cationic amino acid in antibacterial activity of this peptide and to avoid any conformational changes due to the incorporation of DOTA in the centre of the peptide, glysine present at the N terminal was chosen for the conjugation of DOTA. The synthesized peptide conjugate DOTA-GF17 was purified and characterized. The mass spectroscopy of synthesized peptide conjugate demonstrated m/z ratios of 831.0 ([M +3H]3+), 1245.5 ([M+2H]2+) and 1660.2 92[M+3]3+) which corresponded to the molecular mass of 2489 Da. DOTA-GF-17 was further radiolabeled with [68Ga] and the radiolabeling conditions such as buffer pH, amount of peptide conjugate, reaction time were optimized by performing a series of experiments. The highest radiolabeling efficiency of 95.0% was achieved although the observed range of radiolabeling was from 60% to 95%. In a recent study, authors have radiolabeled various fragments of ubiquicidin with [68Ga] and demonstrated radiolabeling efficiency that ranged from 51% to 85% and 46%–78% for NOTA-UBI 29–41 and NOTA-UBI-30-41, respectively (Ebenhan et al., 2014). The best radiolabeling efficiency was observed at pH 4. Buffer plays an important role in radiolabeling as it avoids hydrolysis of Ga3+ and formation of insoluble metal hydroxide [Ga (OH)3] that occur at pH 3.0–7.0 due to the absence of ligands (Bartholomä et al., 2010). Radiolabeling of DOTA conjugated peptides require heating at high temperature (90°-98 °C) for 20–30 min depending on the radioisotope and peptide (Sosabowski and Mather, 2006). We achieve maximum radiolabeling efficiency by heating the reaction solution at 95 °C for 30 min. We observed that [68Ga]DOTAGF-17 was stable for up to 3 h indicating that post preparation, the radiolabeled peptide conjugate preparation is safe for injecting till 3 h. Wang et al. (2012) reported that GF-17 has the essential sequence of LL-37 peptide that is active against both gram positive and gram negative bacteria. We performed uptake binding studies in live strains of S.aureus and P. aeroginosa. Our study demonstrated high uptake binding values in live strains of S.aureus. High binding values suggested that DOTA conjugate GF-17 retained the property of unconjugated GF-17
Acknowledgements The author B. Singh is thankful to the Indian Council of Medical Research (ICMR), New Delhi, India (Grant No. 5/13/89/2013-NCD-III) for award of this research project as an extramural grant to him as Principal Investigator. The author S. Chopra acknowledges the award of six months Deutscher Akademischer Austasch Dienst (DAAD) fellowship to her to undertake part of this work at Technical University of Munich, Munich, Germany under the stewardship of Prof. Hans J Wester. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apradiso.2019.04.008. References Akhtar, M.S., Qaisar, A., Irfanullah, J., Iqbal, J., Khan, B., Jehangir, M., Nadeem, M.A., Khan, M.A., Afzal, M.S., Ul-Haq, I., Imran, M.B., 2005. Antimicrobial peptide 99mTcubiquicidin 29-41 as human infection-imaging agent: clinical trial. J. Nucl. Med. 46 (4), 563–573. Bartholomä, M.D., Louie, A.S., Valliant, J.F., Zubieta, J., 2010. Technetium and gallium derived radiopharmaceuticals: comparing and contrasting the chemistry of two important radiometals for the molecular imaging era. Chem. Rev. 110, 2903–2920. Becker, W., Meller, J., 2001. The role of nuclear medicine in infection and inflammation. Lancet Infect. Dis. 1, 326 233. Brouwer, C.P., Wulferink, M., Welling, M.M., 2008. The pharmacology of radiolabeled
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Love, C., Palestro, C.J., 2004. Radionuclide imaging of infection. J. Nucl. Med. Technol. 32, 47–57. Mishra, B., Epand, R.F., Epand, R.M., Wang, G., 2013. Structural location determines functional roles of the basic amino acids of KR-12, the smallest antimicrobial peptide from human cathelicidin LL-37. RSC Adv. 14. https://doi.org/10.1039/ C3RA42599A. Nibbering, P.H., Welling, M.M., Paulusma-Annema, A., Brouwer, C.P., Lupetti, A., Pauwels, E.K., 2004. 99mTc-Labeled UBI 29-41 peptide for monitoring the efficacy of antibacterial agents in mice infected with Staphylococcus aureus. J. Nucl. Med. 45, 321–326. Palestro, C.J., 2016. Radionuclide imaging of musculoskeletal infection: a review. J. Nucl. Med. 57 (9), 1406–1412. Prata, M.I., 2012. Gallium-68: a new trend in PET radiopharmacy. Curr. Radiopharm. 5, 142–149. Rini, J.N., Bhargava, K.K., Tronco, G.G., Singer, C., Caprioli, R., Marwin, S.E., Richardson, H.L., Nichols, K.J., Pugliese, P.V., Palestro, C.J., 2006. PET with FDG-labeled leukocytes versus scintigraphy with [111In]oxine-labeled leukocytes for detection of infection. Radiology 238, 978–987. Roesch, F., 2012. Maturation of a key resource-the germanium-68/gallium-68 generator: development and new insights. Curr. Radiopharm. 5, 202–211. Schultz, M.K., Donahue, P., Musgrave, N.I., Zhernosekov, K., Naidoo, K., Razbash, A., Tworovska, I., Dick, D.W., Watkins, G.L., Graham, M.M., Runde, W., Clanton, J.A., Sunderland, J.J., 2013b. An increasing role for 68Ga-PET imaging: a perspective on the availability of parent 68Ge material for generator manufacturing in an expanding market. Postgrad. Med. Educ. Res. 47, 26–30. Shai, Y., 1999. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membranelytic peptides. Biochim. Biophys. Acta 1462, 55–70. Singh, B., Prasad, V., Schuchardt, C., Kulkarni, H., P Baum, R.P., 2013. Can the standardized uptake values derived from diagnostic 68Ga-dotatate PET/CT imaging predict the radiation dose delivered to the metastatic liver NET lesions on 177Ludotatate peptide receptor radionuclide therapy? J. Postgrad. Med. Educ. Res. 47, 7–13. Singh, B., Sunil, H.V., Sharma, S., Prasad, V., Kashyap, R., Bhattacharya, A., Mittal, B.R., Taneja, A., Rai, R., Goni, V.G., Aggarwal, S., Gill, S.S., Bhatnagar, A., Singh, A.K., 2008. Efficacy indigenously developed single vial kit preparation of [99mTc]ciprofloxacin in the detection of bacterial infection: an Indian experience. Nucl. Med. Commun. 29 (12), 1123–1129. Sosabowski, J.K., Mather, S.J., 2006. Conjugation of DOTA-like chelating agents to peptides and radiolabeling with trivalent metallic isotopes. Nat. Protoc. 2, 972–976. Wang, G., Epand, R.F., Mishra, B., Lushnikova, T., Thomas, V.C., Bayles, K.W., Epand, R.M., 2012. Decoding the functional roles of cationic side chains of the major antimicrobial region of human cathelicidin LL-37. Antimicrob. Agents Chemother. 56, 845–856. Welling, M.M., Flores, G.F., Pirmettis, I., Brouwer, C.P.J.M., 2009. Current status of imaging infection with radiolabeled anti-infective agents. Anti-Infect. Agents Med. Chem. 8, 272–287. Welling, M.M., Lupetti, A., Balter, H.S., Lanzzeri, S., Souto, B., Rey, A.M., Savio, E.O., Paulusma-Annema, A., Pauwels, E.K., Nibbering, P.H., 2001. 99mTc-labeled antimicrobial peptides for detection of bacterial and Candida albicans infections. J. Nucl. Med. 42, 788–794. Zasloff, M., 2002. Antimicrobial peptides of multicellular organisms. Nature 415, 389–395.
cationic antimicrobial peptides. J. Pharm. Sci. 97, 1633–1651. Buchmann, I., Henze, M., Engelbrecht, S., Eisenhut, M., Runz, A., Schafer, M., Schilling, U., Haufe, S., Herrmann, T., Haberkorn, U., 2007. Comparison of 68Ga-DOTATOC PET and 111In-DTPAOC (Octreoscan) SPECT in patients with neuroendocrine tumors. Eur. J. Nucl. Med. Mol. Imaging 34 (10), 1617–1626. Chan, D.I., Prenner, E.J., Vogel, H.J., 2006. Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action. Biochim. Biophys. Acta 1758, 1184–1202. Dumarey, N., Egrise, D., Blocklet, D., Stallenberg, B., Remmelink, M., del Marmol, V., Van Simaeys, G., Jacobs, F., Goldman, S., 2006. Imaging infection with [18F]FDG-labeled leukocyte PET/CT: initial experience in 21 patients. J. Nucl. Med. 47, 625–632. Dürr, U.H., Sudheendra, U.S., Ramamoorthy, A., 2006. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim. Biophys. Acta 1758, 1408–1425. Ebenhan, T., Chadwick, N., Sathekge, M.M., Govender, P., Govender, T., Kruger, H.G., Marjanovic-Painter, B., Zeevaart, J.R., 2014. Peptide synthesis, characterization and 68 Ga-radiolabeling of NOTA-conjugated ubiquicidin fragments for prospective infection imaging with PET/CT. Nucl. Med. Biol. 41, 390–400. Ebenhan, T., Sathekge, M.M., Lenagana, T., Koole, M., Gheysens, O., Govender, T., Zeevaart, J.R., 2017. 68Ga-NOTA-functionalized ubiquicidin: cytotoxicity, biodistribution, radiation dosimetry and first-in-human positron emission tomography/ computed tomography imaging of infections. J. Nucl. Med. 59 (2) jnumed.117.200048. Epand, Raquel F., Wang, Guangshun, Berno, Bob, Epand, Richard M., 2009. Lipid segregation explains selective toxicity of a Series of fragments derived from the human Cathelicidin LL-37. Antimicrob. Agents Chemother. 53, 3705–3714. Ghai, A., Singh, B., Li, M., Daniels, T.A., Coelho, R., Orcutt, K., Watkins, G.L., Norenberg, J.P., Cvet, D., Schultz, M.K., 2018 oct. Optimizing the radiosynthesis of [68Ga]DOTAMLN6907 peptide containing three disulfide cyclization bonds – a GCC specific chelate for clinical radiopharmaceuticals. Appl. Radiat. Isot. 140, 333–341. https:// doi.org/10.1016/j.apradiso.2018.08.006. Ghai, A., Singh, B., Panwar Hazari, P., Schultz, M.K., Parmar, A., Kumar, P., Sharma, S., Dhawan, D., Kumar Mishra, A., 2015. Radiolabeling optimization and characterization of (68)Ga labeled DOTA-polyamido-amine dendrimer conjugate - animal biodistribution and PET imaging results. Appl. Radiat. Isot. 105, 40–46. Glaudemans, A.W., de Vries, E.F., Galli, F., Dierckx, R.A., Slart, R.H., Signore, A., 2013. The use of (18) F-FDG-PET/CT for diagnosis and treatment monitoring of inflammatory and infectious diseases. Clin. Dev. Immunol. 623036. https://doi.org/10. 1155/2013/623036. Gordon, Y.J., Romanowski, E.G., McDermott, A.M., 2005. A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs. Curr. Eye Res. 30, 505–515. Gotthardt, M., Bleeker-Rovers, C.P., Boerman, O.C., Oyen, W.J.G., 2013. Imaging of inflammation by PET, conventional scintigraphy, and other imaging techniques. J. Nucl. Med. Technol. 41, 157–169. Hancock, R.E., Diamond, G., 2000. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 8, 402–410. Jaswal, A.P., Meena, V.K., Prakash, S., Pandey, A., Singh, B., Mishra, A.K., Hazari, P.P., 2017. [68Ga/118Re] complexed [CDTMP] trans-1,2-cyclohexyldinitrilotetraphosphonic acid as a theranostiv agent for skeletal metastases. Front. Med. 4, 72. https:// doi.org/10.3389/fmed.2017.00072. Li, X., Li, Y., Han, H., Miller, D.W., Wang, G., 2006. Solution structures of human LL-37 fragments and NMR-based identification of a minimal membrane-targeting antimicrobial and anticancer region. J. Am. Chem. Soc. 128, 5776–5785.
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Radiosynthesis and pre-clinical evaluation of [68Ga] labeled antimicrobial peptide fragment GF-17 as a potential infection imaging PET radiotracer
T
Shalini Chopraa, Baljinder Singha,∗, Ashwani Koulb, Anil K. Mishrac, Stephanie Robud, Amritjyot Kaura, Anchal Ghaie, Neena Caplashf, Hans-Jürgen Westerd a
Department of Nuclear Medicine & PET, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh, 160012, India Department of Biophysics, Panjab University, Sector 25, Chandigarh, 160014, India c Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, DRDO, Brig. S.K. Mazumdar Road, Timarpur, Delhi, 110054, India d Department of Nuclear Medicine, Klinikum Rechts Der Isar, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany e Department of Radiology, School of Medicine, Washington University, 510 South Kingshighway Boulevard, St. Louis, Missouri, 63110-107, USA f Department of Biotechnology, Panjab University, Sector 25, Chandigarh, 160014, India b
H I GH L IG H T S
peptide fragment GF-17 was synthesized in-house and conjugated with DOTA and characterization of the peptide-conjugate was carried out. • Antimicrobial The peptide conjugate was radiolabeled successfully with [ Ga] achieving radiolabeling efficiency of 95.0% using the optimized peptide conjugate amount of • 20.0 nmol. uptake studies demonstrated that [ Ga]DOTA-peptide had significant binding in the two bacterial strains studied. • The animal bio-distribution and PET imaging studies demonstrated renal mode of excretion for [ Ga]DOTA-GF-17. • The biodistribution studies in infection and inflammation models had shown significant upake of radiolabeled peptide conjugate at the infection sites. • The • [ Ga]DOTA-GF-17 peptide holds good potential for further undertaking the tracer for detailed pre-clinical evaluation to assess its translational relevance. 68
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A B S T R A C T
Keywords: [68Ga]DOTA-GF-17 AMP Infection PET imaging Preclinical evaluation
The antimicrobial peptide fragment GF-17 was synthesized in-house and conjugated with DOTA and measured molecular mass of DOTA-GF-17 conjugate was 2489 Da. The peptide conjugate was purified and labeled with [68Ga]. The best radiolabeling efficiency (95.0%) of [68Ga]DOTA-GF-17 was achieved at pH 4 with peptide conjugate amount of 20.0 nmol with 30 min of heating at 95 °C. The product remained stable for up to 3 h. The plasma protein binding and lipophilicity for [68Ga]DOTA-GF-17 were 80.98% and −3.12 respectively. The uptake studies with [68Ga]DOTA- GF-17 in S.aureus and P.aeruginosa bacterial strains demonstrated binding of 69.08% and 43.69% respectively. The animal bio-distribution and PET imaging studies were in agreement showing similar pattern for organs’ tracer distribution and renal excretion. The tracer had rapid blood clearance and uptake in bone marrow and muscles was very low. The highest uptake of [68Ga]DOTA-GF-17 was observed at 45 min and 120 min in S.aureus and P.aeruginosa infections respectively. [68Ga]DOTA-GF-17 could be a promising PET tracer and holds a great scope for taking the product further to perform extensive PET studies in animal infection (using gram negative/positive strains) models to prove the diagnostic utility of this novel PET tracer for futuristic clinical applications.
1. Introduction Infection and inflammation are two closely related and clinically highly relevant phenomena. Infection occurs due to invasion of pathogens in tissues or organs, whereas, inflammation often occurs as a
∗
response of host immune system towards the pathogen invasion and toxins produced by these organisms (Brouwer et al., 2008). An early detection of active bacterial infection and differentiation from inflammation provides important information relevant for selecting appropriate treatment option. Although, noninvasive imaging techniques
Corresponding author. Department of Nuclear Medicine PGIMER, Chandigarh, 160012, India. E-mail address: [email protected] (B. Singh).
https://doi.org/10.1016/j.apradiso.2019.04.008 Received 19 August 2018; Received in revised form 4 April 2019; Accepted 5 April 2019 Available online 10 April 2019 0969-8043/ © 2019 Elsevier Ltd. All rights reserved.
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pre-clinical testing for evaluation of its translational clinical utility in PET imaging of human bacterial infections.
such as ultrasound (US), computed tomography (CT) and magnetic imaging resonance (MRI) are available, but they suffer from low specificity for the accurate diagnosis of infection. Further, these techniques pick only the morphological and structural changes which are often the late manifestations of the disease patho-physiology (Becker and Meller, 2001). On the other hand, nuclear medicine techniques i.e. single photon emission computed tomography (SPECT) and positron emission tomography (PET) provide biochemical/physiological information and further allow detection of subtle changes of physiological processes at a very early stage even before the onset of morphological changes. Previously, many SPECT based tracers have been developed and used extensively for the detection of human infections (Love and Palestro, 2004). These SPECT tracers include technetium-hexamethylpropyleneamine oxime ([99mTc] HMPAO), Indium ([111In])-oxine, Gallium-67 ([67Ga])-citrate and [99mTc] labelled antibiotics (Palestro, 2016; Singh et al., 2008). Amongst these, radiolabeled leukocytes based SPECT imaging is considered as ‘gold standard’ imaging technique for the detection of human infections. However, the labeling procedure is cumbersome, requires GMP facility for in vitro blood radiolabeling and re-injection of the radiolabeled leukocytes in the patients is not always safe. SPECT imaging using radiolabeled antibiotics is easier to perform but have the limitations of not picking up the resistant bacterial infections and thus, have compromised sensitivity and specificity (Welling et al., 2009). PET radiotracer 2-deoxy-2-[fluorine-18] fluoro-D-glucose ([18F] FDG) has also been used for the imaging of infection and found to be very sensitive but lacks specificity (Glaudemans et al., 2013; Gotthardt et al., 2013; Becker and Meller, 2001). [18F]FDG labeled leukocyte PET imaging is superior to [18F]FDG imaging for detection of infection as well as for the response assessment to antibiotic therapy. Like [99mTc] HMPAO, radiolabeling leukocytes with [18F] FDG is also a cumbersome and time consuming process, and often have low radiolabeling efficiency (Dumarey et al., 2006; Rini et al., 2006]. Over the last decade, the use of Gallium-68 ([68Ga]) (half-life t1/2 = 68 min; positron emission intensity = 87%) in various PET clinical applications has increase (Schultz et al., 2013b; Singh et al., 2013; Buchmann et al., 2007; Ghai et al., 2018). This PET radionuclide has the potential for an on-demand production via generator technologies that provide reliable and highpurity [68Ga] in sufficient quantities for routine radiopharmaceutical production without the need for cyclotron operations (Prata, 2012; Roesch, 2012). Many [68Ga] labeled compounds have shown very encouraging pre-clinical results, but their clinical validation is under evaluation (Ghai et al., 2015; Jaswal et al., 2017). To further expand the applications of [68Ga] PET imaging, labeling of [68Ga] with antimicrobial peptides (AMPs) is viewed as a very promising approach for the detection of bacterial infections. AMPs are short sequences of amino acids and are reported to kill a wide range of microorganisms (Chan et al., 2006; Shai, 1999; Dürr et al., 2006). A large number of microorganisms causing common human infections have been reported not to develop resistance to this new class (AMPs) of therapeutic agents (Zasloff, 2002; Hancock and Diamond, 2000). Therefore, in this study, we have explored the potential of GF-17, an antimicrobial peptide fragment of LL-37 to be developed as a promising PET probe for imaging infection. GF-17 corresponds to residues 17 to 32 of peptide LL-37, has been studied and found to have the major antimicrobial region of the parent peptide. This smaller peptide has killing activity against Escherichia coli and Staphylococcus aureus (Li et al., 2006; Wang et al., 2012). In the present study, GF-17 was synthesized in the laboratory by the process of solid phase peptide synthesis (SPPS). This peptide was conjugated with a bi-functional chelator i.e. 1,4,7,20-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and further purification and characterization of peptide DOTA conjugate was performed. The peptide conjugate was radiolabeled with [68Ga]. The resultant [68Ga] labeled peptide conjugate was subjected to a detailed
2. Materials All the reagents and chemicals used in the study used were of analytical grade. All the fluorenylmethyloxycarbonyl (fmoc) protected amino acids were purchased from Bachem, Bubendorf, Switzerland and Biotech, Marktredwitz, Germany. 2-Chlorotrityl chloride (2-CTC) resin was procured from PepChem, Tubingen, Germany. Coupling agents hydroxybenzotriazole (HOBt), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and N,N-diisopropylethylamine (DIPEA) used for the SPPS were procured from Sigma Aldrich (Munich, Germany). Bifunctional chelator DOTA was procured from Chematech (Dijon, France). All the remaining reagents used were procured from Sigma Aldrich (Munich, Germany and India). Dichloromethane (DCM) was used for swelling and washing resins during SPPS. Dimethylformamide (DMF), n-methyl-2-pyrrolidone (NMP) and methanol were used for washing resin during SPPS. A cleavage solution containing DCM, 2,2,2-trifluoroethanol (TFE), and acetic acid (v/v/v; 6/1/3) was used as for cleaving GF-17 from resin. Nhydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCl) and DIPEA were used for DOTA coupling reaction. A solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and distilled water (H2O) (v/v/v; 95.00%, 2.5%, 2.5%) was used to cleave DOTA conjugated AMP fragment from resin and removal of protecting groups. Piperidine (20.00%) was used for the removal of fmoc group. Reverse phase high performance liquid chromatography (RP-HPLC) was done for the purification of DOTA-GF-17 post synthesis with different gradients of 0.1% TFA (v/v) in water (solvent A) and 0.1% TFA (v/v) in acetonitrile (CH3CN) (solvent B) with constant flow rate of 1.0 mL/min (for analytical RP-HPLC) and 5.0 mL/min (for preparative RP-HPLC). Analytical RP-HPLC was done on Shimadzu Prominence HPLC system (Shimadzu Germany, Neufahrn, Germany) (having a diode array detector) using Nucleosil 100 C 18 (5 μm, 125 × 4.00 mm) column. Preparative RP-HPLC was done on Sykam Gradient HPLC System (Sykam GmBH, Ersing, Germany) (having a 206 PHD UV-VIS detector) using Multospher 100 RP 18 (250 × 20.00 mm) column (CS GmbH, Langerwehe, Germany).Electron spray ionosation mass spectrometry (ESI-MS) of GF-17 and DOTA conjugated GF-17 was done by using a 500-MS IT mass spectrometer (Varian, Agilent Technologies, Santa Clara, USA). For radiolabeling, sodium acetate buffer of different pH (3.0,4.0,5.0) was prepared using 0.2 M sodium acetate and 0.2 M glacial acetic acid. For instant thin layer chromatograph (ITLC), silica gel coated plates, 0.2 M sodium citrate (mobile phase) and solution of 1.0 M ammonium acetate and methanol (1:1) (mobile phase) were used. [68Ga] was eluted from germanium-68/gallium-68 ([68Ge]/ [68Ga]) generator (ITG, Munich, Germany) with 0.05 M hydrochloric acid (HCl). The radioactivity was measured by using a dose calibrator (CRC-15, Capintec Inc., USA) and well type gamma counter (CAPTUS 3000, Capintec Inc., USA). Purification of radiolabeled DOTA-AMPs was done using C-8 SPE light cartridge procured from Waters Corporation Milford, Massachusetts, USA. Radio-HPLC was performed using a Nucleosil 100 C 18 column (5 μm, 125 × 4.00 mm) connected to a NaI (Tl) well type scintillation counter (EG & G Ortect, Munich, Germany). Trichloroacetic acid (TCA) (5.00%) was used to separate plasma proteins. N-octanol and distilled H2O were used to determine lipophilicity of [68Ga]DOTA-GF-17. Phosphate buffered saline (PBS) and normal saline were used to form suspension of bacterial cultures. Ethyl ether and isoflurane were used as anesthesia during animal studies. For the uptake/binding studies and infection models, live strains of Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa) (2 × 107 CFU) were used. PBS was used for performing periodic washing in the binding studies. Turpentine oil was used for 10
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Fig. 1. Chemical structure of antimicrobial peptide fragment GF-17.
Fig. 2. HPLC chromatogram of crude GF-17 showing peak for GF-17 at 8.8 min (gradient 10%–90% of solution B, flow rate 1.00 mL/min).
Fig. 3. ESI-MS spectrum of fraction containing GF-17 showing m/z ratios of 701.8 ([M+3H]3+), 1052.6 ([M+2H]2+), 1063.6 ([M+2H + Na]2+) and 1402.3 (2[M +3H]3+). Fig. 4: Chemical structure of peptide conjugate DOTA-GF-17.
Fig. 4. Chemical structure of peptide conjugate DOTA-GF-17.
3. Methods
inducing sterile inflammation in animals. For the biodistribution studies, female Wistar rats (6–9 weeks, 200.0 ± 20.0 g) and male Balb/c mice (8–10 weeks, 30.0 ± 5.0 g)were used. For animal PET imaging specific pathogen–free Balb/c mice (male/female, 20.0–25.0 g) were used. The animals were procured and housed in the central animal house facility at PGIMER, Chandigarh, India (for biodistribution) and Klinikum Rechts Der Isar, Technical University of Munich, Munich, Germany (for PET imaging). All the animal studies were performed in compliance with the Institute Animal Ethical Committee.
3.1. Synthesis AMP fragment GF-17 was synthesized by SPPS on 2-CTC resin by coupling fmoc protected amino acids. The resin (1.0 eq) was swelled in DCM and fmoc protected valine (fmoc-val-OH) (2.4 eq) was loaded on resin in the presence of DIPEA (4.5 eq). Capping of the resin was done with methanol. Fmoc deprotection was done with 20.0% piperidine in DMF followed by washing with NMP. Next fmoc protected amino acid was coupled in the presence of HOBt (1.5 eq), TBTU (1.5 eq) and DIPEA (4.5 eq). After the coupling of last amino acid i.e. fmoc-glycine-OH (fmoc-gly-OH) and removal of last fmoc group, the peptide was cleaved 11
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Fig. 5. HPLC chromatogram for HPLC chromatogram of crude DOTA-GF-17 showing peak for unconjugated GF-17 at 8.5 min and DOTA-GF-17 at 9.2 min (gradient 37%–55% of solution B, flow rate 5.00 mL/min).
Fig. 6. ESI-MS spectrum of purified DOTA-GF-17 showing m/z ratios of 831.0 ([M+3]3+), 1245.5 ([M+2]2+) and 1660.2 (2[M+3]3+).
pH, heating time, amount of peptide conjugate. Sodium acetate buffer of different pH (3.0,4.0,5.0) was used for radiolabeling. Peptide conjugate in different amounts (5 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol) was added to 800.0 μL of the buffer. Briefly, 185.00 MBq of [68Ga] was added to the reaction solution and heated at 95 °C for different time periods (10 min, 15 min, 30 min, and 45 min). The radiolabeling efficiency of each reaction solution was analyzed by performing ITLC. Radiochemical purity was also checked for freshly radiolabeled peptide conjugate by performing radio HPLC (flow rate 2.0 mL/min, 37–45% gradient of solution B in 25 min). The radiolabeled peptide conjugate was purified using a C-8 light cartridge, preconditioned with 4.0 mL ethanol (70.00%) and 4.0 mL of distilled water. The purified radiolabeled peptide conjugate was subjected to different quality control tests.
from resin using a cleavage solution containing DCM, TFE and acetic acid (6:1:3). The peptide was lyophilized and subjected to analytical RP-HPLC, and mass spectroscopy for characterization. DOTA (4.0 eq) was conjugated to the amino (N) terminal of the peptide by activation with NHS (5.0 eq) in the presence of EDCl (5.0 eq) and DIPEA (8.0 eq). The reaction was allowed to occur for 12 h at room temperature. All the protecting groups were cleaved from the DOTA-peptide conjugate using a cleavage solution containing TFA, TIPS and water (95%:2.5%:2.5%). The newly synthesized DOTA-GF-17 was purified by RP-HPLC and characterized by mass spectroscopy.
3.2. Radiolabeling Radiolabeling of peptide conjugate was performed with freshly eluted [68Ga] (in 5.0 mL of HCl; 0.05M) from [68Ge]/[68Ga] generator. Optimization of radiolabeling conditions was done by varying buffer 12
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checked by performing ITLC as described in section 3.3.1. The solvent system used was 1.0 M ammonium acetate and methanol (1:1). 3.3.3. In vitro stability To check the in vitro degradation of [68Ga]DOTA-GF-17 as a function of time, RCP was checked for the purified [68Ga]DOTA-GF-17 at different time intervals (30 min, 45 min, 60 min, 90 min, 120 min and 180 min). 3.3.4. In vitro serum stability To check the degradation of [68Ga]DOTA-GF-17 in serum, in vitro serum stability of [68Ga]DOTA-GF-17 was determined by incubating purified radiolabeled peptide conjugate in serum. Precisely, 200.00 μL of purified [68Ga]-DOTA-GF-17 was added to 800.00 μL of serum and incubated at 37 °C for different time periods of 30 min, 45 min, 60 min, 90 min, 120 min. RCP was determined for each sample. 3.3.5. Plasma protein binding The in vitro protein binding for purified [68Ga]DOTA-GF-17 was determined by incubating the radiolabeled peptide conjugate in human plasma. Briefly, 100.0 μL of purified [68Ga]DOTA-GF-17 was incubated in 900.0 μL plasma at 37 °C for 1 h. The plasma proteins were precipitated after 1 h by adding 1.00 mL of 5.00% TCA followed by centrifugation at 3000 rpm for 5min. The supernatant was collected separately and precipitate was re-suspended in 1.00 mL TCA (5.00%). The radioactivity was measured in both precipitates and supernatant by using a well type gamma counter. Protein binding was determined by calculating binding fraction of the total radioactivity.
Fig. 7. Radiolabeling efficiency (%) of [68Ga] DOTA-GF-17 at different amounts (nmol) of peptide conjugate DOTA-GF-17.
3.3. Quality control tests of [68Ga]DOTA-GF-17 3.3.1. Radiolabeling efficiency ITLC was done to determine the radiolabeling efficiency of the freshly prepared radilolabeled peptide conjugate using two solvent systems: a) 0.2 M sodium citrate and b) 1.0 M ammonium acetate and methanol (1:1). Briefly, 4.00 μL of freshly prepared radiolabeled peptide conjugate was applied to the ITLC strip (8.00 cm) and the strip was kept in a solvent chamber. The strips were dried, cut into small pieces measuring 1 cm and measured for radioactivity in a well type gamma counter. Free [68Ga] was also subjected to ITLC strip. The percentage radiolabeling efficiency and retention factors (Rf) were calculated. Radio HPLC was also performed for the freshly labeled peptide conjugate.
3.3.6. Lipophilicity In a test tube, 100.00 μL of purified [68Ga]DOTA-GF-17 and 400.00 μL of distilled water were mixed. To this solution 500.00 μL of noctanol was added. The mixture was mixed thoroughly for 3 min. The mixture was then centrifuged at 6000 rpm for 5 min to separate the aqueous and organic phases. Precisely, 100.00 μL of aqueous phase and 100.00 μL of organic phase were carefully collected in separate test tubes. The radioactivity was measured in both phases using a well type gamma counter. The lipophilicity for [68Ga]DOTA-GF-17 was measured in terms of log P value by using the following formula:
3.3.2. Radiochemical purity Radiochemical purity (RCP) of purified [68Ga]DOTA-GF-17 was
Fig. 8. Radiolabeling efficiency (%) of [68Ga] DOTA-GF-17 at different pH of sodium acetate buffer. 13
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Fig. 9. Radiolabeling efficiency (%) of [68Ga] DOTA-GF-17 at different heating time points.
Fig. 10. In-vitro stability of [68Ga] DOTA-GF-17 as a function of time.
log Poct / wat = log[counts in octanol ÷ counts in water]
were collected separately and radioactivity was counted. The radioactivity (counts/min) in supernatants and pellet was measured. The percent uptake/binding of [68Ga]DOTA-GF-17 was calculated by using following formula:
3.4. Uptake/binding studies Uptake/binding of [68Ga]DOTA-GF-17 was studied in live strains of S.aureus and P.aeruginosa. Microbial strains (2 × 107) were suspended in 200.00 μL of PBS and approximately 7.4 MBq of radiolabeled peptide conjugate was added to the suspension. The microbial suspensions were incubated at 37 °C for different time intervals (30 min, 60 min, 120 min). The suspensions were centrifuged at 10000 rpm for 10 min. The supernatant was collected separately and pellet was suspended in PBS. The suspension was again centrifuged; supernatants and pellets
%Uptake = [Counts in Pellet ÷ Total counts(Pellet + Supernantants)] × 100
3.5. Blood kinetics studies The blood kinetics study of [68Ga]DOTA-GF-17 was carried out in female Wistat rats (n = 3). Briefly, 7.4 MBq of [68Ga]DOTA-GF-17 was 14
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Fig. 11. In-vitro serum stability of [68Ga] DOTA-GF-17 as a function of time.
Fig. 12. Radio HPLC of freshly prepared [68Ga] DOTA-GF-17 showing retention time, tr of 19.39 min.
3.7. PET imaging in normal animals
injected intravenously into each animal. The blood samples were drawn from the ocular vein of the rat at different time intervals (15 min, 30 min, 45 min, 1 h, 2 h). The radioactivity in each blood sample was measured in a well type gamma counter. The data was expressed as percent injected dose per gram (% ID/g) at each time point.
PET Imaging was done in normal Balb/c mice (n = 3) using an animal PET scanner (Inveon Seimens PET scanner) at TUM, Munich, Germany. Each mouse was injected with 3.7 MBq of radiolabeled peptide conjugate under anesthesia (isoflurane) into the tail vein. Dynamic PET imaging was done following the injection for 90 min. Reconstruction of images was done using 2 dimensional ordered subset expectation maximum algorithm (2D-OSEM) and analyzed with the help of Inveon software.
3.6. Biodistribution studies in normal animals Biodistritbution of [68Ga]DOTA-GF-17 was studied in female Wistar rats (n = 15, weighing between 200.0 and 250.0 g). A dose of 7.40 MBq of [68Ga] labeled peptide conjugate was injected in the tail vein of each animal. Venous blood was collected from each animal. The animals were sacrificed at different time points (15 min, 30 min, 45 min, 60 min, and 120 min). The various organs/tissues such as bone, muscle, heart, thyroid, lung, liver, spleen, stomach, small intestine, large intestine, kidney and brain were excised and collected. The organs/tissues were washed in saline, dried and kept in pre-weighted test tubes. The radioactivity in blood was counted and expressed as the percentage of the injected dose per mL (%ID/mL) of blood. On the other hand, the radioactivity counted in organs was expressed as injected dose per gram (%ID/g) of the organ.
3.8. Biodistribution in infection and inflammation models Infection model was developed in Balb/c mice (n = 30) using S.aureus and P.aeruginosa. Briefly, 2 × 107 CFUs of S.aureus were suspended in 200.00 μL of normal saline and injected subcutaneously into right thigh of each mouse. Similarly, infection model for P.aeruginosa was developed in mice. The animals were housed in Central Animal House Facility in PGIMER for one week and development of infection was monitored daily. Sterile inflammation was induced in Balb/c mice by injecting 50.0 μL of turpentine oil into left thigh of the animal. The 15
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Fig. 13. ITLC of purified [68Ga] DOTA-GF-17 with 0.2 M sodium citrate as mobile phase.
Fig. 14. ITLC of purified [68Ga] DOTA-GF-17 with 1.0 M ammonium acetate and methanol (1:1) as mobile phase.
chromatogram (Fig. 2) showed three prominent peaks at time 8.8 min, 12.3 min and 14.3 min. The ESI-MS analysis of fractions collected during HPLC demonstrated that GF-17 was present in fraction collected at 8.8 min with the m/z ratios showing m/z ratios of 701.8 ([M +3H]3+), 1052.6 ([M+2H]2+), 1063.6 ([M+2H + Na]2+) and 1402.3 (2[M+3H]3+) (Fig. 3) corresponding to molecular mass of 2102.4 Da. The analytical RP-HPLC data for GF-17 demonstrated its retention time, tr = 8.8 min (10%–90% gradient of solution B in 20 min). The purification of newly synthesized DOTA-GF-17 (Fig. 4)
organ biodistribution of radiotracer in infection and inflammation models was performed as described in section 3.6. Tissues from infection and inflammation sites were also collected along with other organs. The percent injected doses for all organs including infection and inflammation sites were calculated. 4. Results Analytical HPLC was done for crude GF-17 (Fig. 1). The HPLC 16
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Fig. 15. Uptake/binding values (%) of [68Ga] DOTA-GF-17 in strains of S.aureus (red) and P.aeruginosa (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 16. Biodistribution pattern of [68Ga] DOTA-GF-17 in normal Wistar rats at different time intervals.
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Fig. 17. Time activity curves of [68Ga] DOTA-GF-17 obtained from dynamic PET imaging in normal Balb/c mouse.
stable for up to 3 h (Fig. 10) and up to 2 h in serum (Fig. 11). The ITLC of the radiolabeled peptide conjugate demonstrated Rf value of 0.1 in 0.2 M sodium citrate and 0.7 in 1.0 M ammonium acetate and methanol. Radio-HPLC (Fig. 12) demonstrated RCP of 93.28% for [68Ga] DOTA-GF-17 with retention time, tr = 19.39 min. ITLC of purified [68Ga]DOTA-GF-17 demonstrated high RCP of 95.0% (Figs. 13–14). The plasma protein binding and lipophilicity for [68Ga]DOTA-GF-17 were 80.98% and −3.12 respectively. The uptake binding assays for the radiolabeled peptide conjugate were performed in live stains of S.aureus and P.aeruginosa. The results demonstrated that the uptake/binding of [68Ga]DOTA-GF-17 in live S.aureus after 30 min of incubation was 67.57% and increased to 69.08% after 120 min of incubation. The results for live strain of P.aeruginosa demonstrated the uptake binding of 32.56% at 30 min post incubation with [68Ga]DOTA- GF-17 and increased to 43.69% at 120 min post incubation (Fig. 15). The results of blood kinetics demonstrated (Fig. 16) that [68Ga] DOTA-GF-17 cleared from the systematic circulation with only 0.17% of the injected activity remaining at 30 min. The biodistribution studies were performed using [68Ga]DOTA-GF-17 in normal animals showed initial blood pool activity, uptake in kidney, heart, liver and lung. The results indicated a decrease in uptake of [68Ga]DOTA-GF-17 in all the organs with time. In kidneys the uptake increased with time. At all the time points, the radioactive uptake in organs like liver, spleen, bone, muscle and brain was lower than that in kidneys (Fig. 16). The reconstructed [68Ga] DOTA-GF-17 PET images showed initial uptake in heart, liver, kidneys, in the region of thyroid gland and low grade uptake in bone marrow and muscles. The dynamic images showed significant clearance of tracer from the heart and liver during subsequent imaging (Fig. 17). However, the clearance from the kidneys is minimal (Fig. 18). Results for biodistribution of [68Ga] DOTA-GF-17 in infection and inflammation models have been presented in Figs. 19 and 20. The biodistribution studies in mice bearing S.aureus infection (Fig. 19) and inflammation demonstrated that 0.78% of the ID/g accumulated in infection at 15 min. The maximum uptake was observed at infection site at 45 min with 0.89% of the ID/g. Initial uptake of the at inflammation site was maximum (0.86% of ID/g at 15 min) but the tracer washed out with time. In mice bearing P.aeruginosa infection (Fig. 20), the uptake of radiotracer at 15 min was 0.45% of ID/g at the infection site. The maximum uptake was observed at 120 min with accumulation of 0.75% of ID/g. It was observed that initial uptake of tracer in inflammation was more as compared to that in infection site but the tracer showed significant clearance from the inflammation site with time.
Fig. 18. Reconstructed PET image showing uptake pattern of [68Ga] DOTA-GF17 in normal Balb/c mouse.
was done by performing preparative RP-HPLC. The gradient of solution B was optimized to 37%–55% in 20 min for purification of DOTA-GF-17 with tr = 9.2 min (Fig. 5). The presence of DOTA-GF-17 in fraction collected at 9.2 min was confirmed by performing ESI-MS. The mass spectroscopy analysis demonstrated the m/z ratios of 831.0 ([M +3]3+), 1245.5 ([M+2]2+) and 1660.2 (2[M+3]3+) (Fig. 6) corresponding to molecular mass of 2489 Da. The radiolabeling efficiency for [68Ga]DOTA-GF-17 was evaluated and found to be 90.95% ± 2.61 with an amount of 20 nmol of DOTAGF-17 (Fig. 7). The best radiolabeling was observed with buffer of pH 4.0 (Fig. 8) and heating time of 30 min (Fig. 9). Highest radiolabeling efficiency of 95.0% was achieved by optimizing the radiolabeling conditions for [68Ga]DOTA-GF-17. The radiolabeled product remained 18
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Fig. 19. Biodistribution pattern of [68Ga] DOTA-GF-17 in normal Balb/c mice bearing S.aureus infection and sterile inflammation at different time intervals.
Fig. 20. Biodistribution pattern of [68Ga] DOTA-GF-17 in normal Balb/c mice bearing P.aeruginosa infection and sterile inflammation at different time intervals.
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5. Discussion
with no vital conformational changes in the molecule that would have affected the binding of DOTA-GF-17 to the bacterial cell membrane. The high binding values in live S.aureus at greater incubation time period also suggested that the amount of antimicrobial peptide was not enough to kill the bacterial cells as the radiolabeling required only nanomolar concentration of peptide conjugate. [68Ga]DOTA-GF-17 can be developed as diagnostic tracer for live S.aureus infection imaging and follow up cases. However, lower binding values of [68Ga]DOTA- GF-17 in live strains of P. aeroginosa were observed. Biodistribution studies demonstrated an initial increase in the uptake of [68Ga]DOTA-GF-17 in blood, heart, lung, liver and kidney due to the perfusion. We observed that there was no specific uptake in any organ as the uptake decreased in each organ with time. Kidneys were the only organs to show increased uptake with time. This indicated renal mode of excretion for [68Ga]DOTA-GF-17. Results from PET imaging done in normal animals demonstrated initial uptake of [68Ga]DOTA- GF-17 in heart, liver and kidneys which was in agreement with the data from biodistribution studies. The increased uptake in kidneys in later images again confirmed the renal mode of exertion for [68Ga]DOTA-GF-17. No soft tissue and bone uptake indicated absence of specific uptake of this radiotracer. The biodistribution studies performed in infection and inflammation models demonstrated that the uptake of [68Ga]DOTA-GF-17 in case of S.aureus infection was highest at 45 min post injection. However, the highest uptake of [68Ga]DOTA-GF-17 in case P.aeruginosa infection was observed at 120 min post injection. It was also observed that the uptake of [68Ga]DOTA-GF-17 was comparatively less in case of P.aeruginosa infection than that in S.aureus infection. The uptake of [68Ga]DOTA-GF17 in inflammation site was initially high as compared to that in both types of infection but decreased with time. The present study describes the synthesis of antimicrobial peptide fragment GF-17 and conjugation with bifunctional chelator DOTA. A successful radiolabeling of DOTA conjugated GF-17 with [68Ga] was achieved with high radiolabeling efficiency and stability. The normal biodistribution pattern of [68Ga] labeled DOTA-GF-17 in bone, muscles and fast clearance from liver may be useful for targeting bacterial infections in these areas. However the potential of [68Ga]DOTA-GF-17 for infection imaging requires further pre-clinical validation before translation to clinical use.
Currently, many bacterial strains have developed resistance towards the conventional antibiotics. This growing problem of bacterial resistance has created a need to develop alternate therapeutic agents for bacterial infections. Antimicrobial peptides have emerged as new diagnostic and therapeutic agents against bacterial infections. In the past, antimicrobial peptides have been studied for the potency against various bacterial strains (Gordon et al., 2005). Defensins, peptide fragments of lactoferrin and ubiquicidin have been radiolabeled and evaluated for the diagnostic and therapeutic efficacy (Welling et al., 2001). UBI 29–41 is a peptide fragment of ubiquicidin that has been radiolabeled with [99mTc] to detect animal as well as human bacterial infections (Nibbering et al., 2004; Akhtar et al., 2005).The studies have shown that [99mTc]UBI 29–41 was found to be very effective to locate infection sites in bones and soft tissues. In a recent study done by Ebenhan et al. (2017), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA) conjugated UBI was radiolabeled with [68Ga] and evaluated for imaging infection in non human primates. Our study is the first attempt where GF-17 has been conjugated with DOTA and radiolabeled with [68Ga] for the purpose of PET imaging. In the present study, we synthesized GF-17, a peptide fragment of LL-37 with antimicrobial property by SPPS. It was further conjugated with DOTA in the presence of NHS in solution phase and radiolabeled with [68Ga]. GF-17 is a cationic peptide fragment that is active against both gram positive and gram negative bacteria (Wang et al., 2012) It has been reported that the antimicrobial activity of this peptide fragment is due to the presence of cationic amino acids lysines and arginines in its structure. These amino acids play a crucial role in recognition of bacterial membrane, binding to the bacterial surface and killing the bacterial cells (Epand et al., 2009; Wang et al., 2012; Mishra et al., 2013). The side chains of arginines, lysines, isoleucine and glycine presented potential site for DOTA coupling. Due to the active involvement of cationic amino acid in antibacterial activity of this peptide and to avoid any conformational changes due to the incorporation of DOTA in the centre of the peptide, glysine present at the N terminal was chosen for the conjugation of DOTA. The synthesized peptide conjugate DOTA-GF17 was purified and characterized. The mass spectroscopy of synthesized peptide conjugate demonstrated m/z ratios of 831.0 ([M +3H]3+), 1245.5 ([M+2H]2+) and 1660.2 92[M+3]3+) which corresponded to the molecular mass of 2489 Da. DOTA-GF-17 was further radiolabeled with [68Ga] and the radiolabeling conditions such as buffer pH, amount of peptide conjugate, reaction time were optimized by performing a series of experiments. The highest radiolabeling efficiency of 95.0% was achieved although the observed range of radiolabeling was from 60% to 95%. In a recent study, authors have radiolabeled various fragments of ubiquicidin with [68Ga] and demonstrated radiolabeling efficiency that ranged from 51% to 85% and 46%–78% for NOTA-UBI 29–41 and NOTA-UBI-30-41, respectively (Ebenhan et al., 2014). The best radiolabeling efficiency was observed at pH 4. Buffer plays an important role in radiolabeling as it avoids hydrolysis of Ga3+ and formation of insoluble metal hydroxide [Ga (OH)3] that occur at pH 3.0–7.0 due to the absence of ligands (Bartholomä et al., 2010). Radiolabeling of DOTA conjugated peptides require heating at high temperature (90°-98 °C) for 20–30 min depending on the radioisotope and peptide (Sosabowski and Mather, 2006). We achieve maximum radiolabeling efficiency by heating the reaction solution at 95 °C for 30 min. We observed that [68Ga]DOTAGF-17 was stable for up to 3 h indicating that post preparation, the radiolabeled peptide conjugate preparation is safe for injecting till 3 h. Wang et al. (2012) reported that GF-17 has the essential sequence of LL-37 peptide that is active against both gram positive and gram negative bacteria. We performed uptake binding studies in live strains of S.aureus and P. aeroginosa. Our study demonstrated high uptake binding values in live strains of S.aureus. High binding values suggested that DOTA conjugate GF-17 retained the property of unconjugated GF-17
Acknowledgements The author B. Singh is thankful to the Indian Council of Medical Research (ICMR), New Delhi, India (Grant No. 5/13/89/2013-NCD-III) for award of this research project as an extramural grant to him as Principal Investigator. The author S. Chopra acknowledges the award of six months Deutscher Akademischer Austasch Dienst (DAAD) fellowship to her to undertake part of this work at Technical University of Munich, Munich, Germany under the stewardship of Prof. Hans J Wester. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apradiso.2019.04.008. References Akhtar, M.S., Qaisar, A., Irfanullah, J., Iqbal, J., Khan, B., Jehangir, M., Nadeem, M.A., Khan, M.A., Afzal, M.S., Ul-Haq, I., Imran, M.B., 2005. Antimicrobial peptide 99mTcubiquicidin 29-41 as human infection-imaging agent: clinical trial. J. Nucl. Med. 46 (4), 563–573. Bartholomä, M.D., Louie, A.S., Valliant, J.F., Zubieta, J., 2010. Technetium and gallium derived radiopharmaceuticals: comparing and contrasting the chemistry of two important radiometals for the molecular imaging era. Chem. Rev. 110, 2903–2920. Becker, W., Meller, J., 2001. The role of nuclear medicine in infection and inflammation. Lancet Infect. Dis. 1, 326 233. Brouwer, C.P., Wulferink, M., Welling, M.M., 2008. The pharmacology of radiolabeled
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