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99mTc-Labeled poly(ethyleneglycol)–N-(N-(3-diphenylphosphinopropionyl)glycyl)-S-tritylcysteine (PEG-PN2S)-linked ubiquicidin (UBI)

[99mTc]-PEG-PN2S-UBI29-41
, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: October 28, 2011.

Chemical name:99mTc-Labeled poly(ethyleneglycol)–N-(N-(3-diphenylphosphinopropionyl)glycyl)-S-tritylcysteine (PEG-PN2S)-linked ubiquicidin (UBI)
Abbreviated name:[99mTc]-PEG-PN2S-UBI29-41
Synonym:
Agent Category:Peptide
Target:Bacterial cell membrane
Target Category:Other
Method of detection:Single-photon emission computed tomography (SPECT); gamma planar imaging
Source of signal / contrast:99mTc
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.

Background

[PubMed]

Infections are the most common cause of patient morbidity in hospitals, and noninvasive imaging modalities such as X-ray/computed tomography (CT) or magnetic resonance imaging that are used to detect an infected site(s) in a patient are not able to distinguish between inflammation, tumors, and microbial infections (1). The early detection of infection in a patient can help healthcare providers devise a suitable treatment plan to control and monitor recovery from the disease. 18F-Labeled fluorodeoxy glucose ([18F]-FDG) has often been used with positron emission tomography to detect infections in the clinical setting, but the diagnosis obtained with this radiochemical must be confirmed with CT before any therapy can be initiated (2). Investigators have also used 67Ga-labeled citrate and 111In- or 99mTc-labeled human immunoglobulins and monoclonal antibodies to detect infections; however, these agents show non-specific accumulation in the infected and inflamed areas of the body (3).

Researchers have shown that a thirteen-amino-acid (aa) cationic antimicrobial peptide (AMP) derived from ubiquicidin (UBI; for the aa sequence and other characteristics of UBI, see Brouwer et al. (4)) and radiolabeled directly with 99mTc ([99mTc]-UBI29-41; the aa sequence of UBI29-41 is: Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg) can be used to detect and distinguish infection from inflammation with scintigraphy in animals and humans (5). The mode of action of AMPs has been schematically presented and explained by Brouwer et al. (1). This labeled peptide was demonstrated to be suitable for the visualization of infections in the soft tissues, bone, bone prosthesis, and the spine (6). However, the chemical structure of the directly labeled peptides is not well understood, and the 99mTc labeling of UBI29-41 is assumed to be due to the coordination of the radionuclide with the Lys and Arg7 residues in the peptide sequence as described elsewhere (7). In an effort to clearly define the chemical structure of [99mTc]-UBI29-41, the peptide was radiolabeled with an indirect method as described by Meléndez-Alafort et al. (6). For this, UBI29-41 was first conjugated to a poly(ethyleneglycol) (PEG)–bearing bifunctional metal chelating agent (N-(N-(3-diphenylphosphinopropionyl)glycyl)-S-tritylcysteine (PN2S)) and then labeled with 99mTc ([99mTc]-PEG-PN2S-UBI29-41). The biodistribution and the ability of [99mTc]-PEG-PN2S-UBI29-41 to visualize infection in mice with single-photon emission computed tomography (SPECT) were then compared to that of the directly labeled [99mTc]-UBI29-41 (6).

Synthesis

[PubMed]

The synthesis of PEG-PN2S-UBI29-41 and its indirect labeling with 99mTc has been described by Meléndez-Alafort et al. (6). Using the Snyder assay, the PEG-PN2S-UBI29-41 molecule was determined to be monoPEGylated (6). The radiochemical purity of [99mTc]-PEG-PN2S-UBI29-41 was 98 ± 1.2% (n = 20 reactions) as determined with reversed-phase high-performance liquid chromatography (RP-HPLC). The radiochemical yield and specific activity of the labeled peptide were not reported.

The direct labeling of UBI29-41 with 99mTc was performed as described elsewhere, with a radiochemical purity of 97 ± 1.5% (n = 5 reactions) as determined with RP-HPLC (8). The radiochemical yield and specific activity of [99mTc]-UBI29-41 were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

HPLC analysis showed that at least 95 ± 1% of [99mTc]-PEG-PN2S-UBI29-41 was intact for up to 6 h in a 1:50 dilution with 0.9% sodium chloride or 0.1 M phosphate buffer (pH 7.0) (6). When incubated with human serum, 72 ± 2.7% of the radiolabel from [99mTc]-PEG-PN2S-UBI29-41 was reported to bind to high molecular weight serum proteins by 1.5 h, and the binding increased to 93 ± 1.5% after 24 h (6). The stability of [99mTc]-PEG-PN2S-UBI29-41 was also evaluated in different dilutions of cysteine (cysteine:[99mTc]-PEG-PN2S-UBI29-41 ratios were 5:1, 50:1, and 500:1, respectively) after incubating the solutions for 1 h at 37°C (6). RP-HPLC analysis of the solutions at the end of the incubation showed that 20%, 34%, and 45% of the radioactivity was associated with cysteine in the 5:1, 50:1, and 500:1 solutions, respectively. The stability of [99mTc]-PEG-PN2S-UBI29-41 was not compared to that of [99mTc]-UBI29-41 stored under the same conditions described above.

Animal Studies

Rodents

[PubMed]

The biodistribution of [99mTc]-PEG-PN2S-UBI29-41 was compared to that of [99mTc]-PEG-PN2S in healthy mice (n = 6 animals/group) as described by Meléndez-Alafort et al. (6). The animals (under anesthesia) were injected with the tracers through the tail vein, and SPECT images of the mice were acquired every 5 min for the next 2 h. After the imaging session, the animals were euthanized to retrieve the major organs and determine the amount of label accumulated in the various tissues. The level of radioactivity in each organ was reported as a percentage of injected dose per gram (% ID/g). Data obtained from this study showed that radioactivity from [99mTc]-PEG-PN2S-UBI29-41 accumulated primarily in organs rich in reticuloendothelial cells (e.g., liver (~40% ID/g), spleen (~25% ID/g), and lungs (~5% ID/g)) and was excreted through the urinary system. With [99mTc]-PEG-PN2S, <5% ID/g uptake of radioactivity was observed in all of the organs. The biodistribution data obtained with [99mTc]-PEG-PN2S-UBI29-41 correlated well with the scintigraphic images obtained from animals injected with this radiochemical.

The biodistribution of [99mTc]-PEG-PN2S-UBI29-41 was also compared to that of [99mTc]-UBI29-41 in mice (n = 6 animals/group) infected with Staphylococcus aureus in the right thigh muscle (6). The animals were injected with the radiochemicals as described above. SPECT images were obtained every 5 min for up to 2 h after injection, and the amount of label in the various tissues was determined as before. Radioactivity from [99mTc]-UBI29-41 was present mainly in the kidneys (27.45 ± 3.81% ID/g), gall bladder (4.12 ± 0.22% ID/g), liver (1.46 ± 0.07% ID/g), and at the site of infection (2.29 ± 0.51% ID/g). All other organs showed an uptake of <0.5% ID/g tracer. With [99mTc]-PEG-PN2S-UBI29-41, the label was present in the liver (14.96 ± 0.99% ID/g), kidney (14.38 ± 1.80% ID/g), spleen (6.96 ± 2.19% ID/g), gallbladder (3.52 ± 1.40% ID/g), blood (3.81 ±0.12% ID/g), lungs (2.02 ± 0.15% ID/g), and the site of infection (2.19 ± 0.40% ID/g). All other organs showed an uptake of <1.0% ID/g tracer with this radiochemical. These results indicated that [99mTc]-PEG-PN2S-UBI29-41 had a longer circulation half-life (attributed by the investigators to PEGylation (6)) than [99mTc]-UBI29-41, which was cleared rapidly from the system. From SPECT images of the animals, it was clear that both tracers could detect the infection site in the animals as observed during the biodistribution study (6).

From these studies, the investigators concluded that, although [99mTc]-PEG-PN2S-UBI29-41 can detect infections with SPECT imaging, it is not suitable for the visualization of infections in the abdomen because radioactivity from this tracer tends to accumulate in organs within the abdominal area (6).

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

No supplemental information is currently available.

References

1.
Brouwer C.P., Sarda-Mantel L., Meulemans A., Le Guludec D., Welling M.M. The use of technetium-99m radiolabeled human antimicrobial peptides for infection specific imaging. Mini Rev Med Chem. 2008;8(10):1039–52. [PubMed: 18782056]
2.
Israel O., Keidar Z. PET/CT imaging in infectious conditions. Ann N Y Acad Sci. 2011;1228:150–66. [PubMed: 21718330]
3.
Signore A., Mather S.J., Piaggio G., Malviya G., Dierckx R.A. Molecular imaging of inflammation/infection: nuclear medicine and optical imaging agents and methods. Chem Rev. 2010;110(5):3112–45. [PubMed: 20415479]
4.
Brouwer C.P., Wulferink M., Welling M.M. The pharmacology of radiolabeled cationic antimicrobial peptides. J Pharm Sci. 2008;97(5):1633–51. [PubMed: 17786940]
5.
Arteaga de Murphy C., Gemmel F., Balter J. Clinical trial of specific imaging of infections. Nucl Med Commun. 2010;31(8):726–33. [PubMed: 20526222]
6.
Melendez-Alafort L., Nadali A., Pasut G., Zangoni E., De Caro R., Cariolato L., Giron M.C., Castagliuolo I., Veronese F.M., Mazzi U. Detection of sites of infection in mice using 99mTc-labeled PN(2)S-PEG conjugated to UBI and 99mTc-UBI: a comparative biodistribution study. Nucl Med Biol. 2009;36(1):57–64. [PubMed: 19181269]
7.
Ferro-Flores G., de Maria Ramirez F., Melendez-Alafort L., de Murphy C.A., Pedraza-Lopez M. Molecular recognition and stability of 99mTc-UBI 29-41 based on experimental and semiempirical results. Appl Radiat Isot. 2004;61(6):1261–8. [PubMed: 15388119]
8.
Melendez-Alafort L., Ramirez Fde M., Ferro-Flores G., Arteaga de Murphy C., Pedraza-Lopez M., Hnatowich D.J. Lys and Arg in UBI: a specific site for a stable Tc-99m complex? Nucl Med Biol. 2003;30(6):605–15. [PubMed: 12900286]

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