Improved Pharmacokinetics and Reduced Antibody Reactivity of Lysostaphin Conjugated to Polyethylene Glycol

Walsh, S. M.; Shah, Anjali; Mond, James J.

doi:10.1128/aac.47.2.554-558.2003

Cited by 100 publications

(86 citation statements)

References 12 publications

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“…PEG moieties are inert, long-chain amphiphilic molecules produced by linking repeating units of ethylene oxide (33). PEGylation can decrease clearance, retain biologic activity, obtain a stable linkage, and enhance water solubility without significantly altering bioavailability (34). Moreover, PEG spacers are nontoxic and unreactive.…”

Section: Discussionmentioning

confidence: 99%

microPET of Tumor Integrin v 3 Expression Using 18F-Labeled PEGylated Tetrameric RGD Peptide (18F-FPRGD4)

Wu¹,

Li²,

Chen³

et al. 2007

Journal of Nuclear Medicine

194

275

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In vivo imaging of a v b 3 expression has important diagnostic and therapeutic applications. Multimeric cyclic RGD peptides are capable of improving the integrin a v b 3 -binding affinity due to the polyvalency effect. Here we report an example of 18 F-labeled tetrameric RGD peptide for PET of a v b 3 expression in both xenograft and spontaneous tumor models. Methods: The tetrameric RGD peptide EfE[c(RGDyK)] 2 g 2 was derived with amino-3,6,9-trioxaundecanoic acid (mini-PEG; PEG is poly(ethylene glycol)) linker through the glutamate a-amino group. NH 2 -mini-PEG-EfE[c(RGDyK)] 2 g 2 (PRGD4) was labeled with 18 F via the N-succinimidyl-4-18 F-fluorobenzoate ( 18 F-SFB) prosthetic group. The receptor-binding characteristics of the tetrameric RGD peptide tracer 18 F-FPRGD4 were evaluated in vitro by a cell-binding assay and in vivo by quantitative microPET imaging studies. Results: The decay-corrected radiochemical yield for 18 F-FPRGD4 was about 15%, with a total reaction time of 180 min starting from 18 F-F 2 . The PEGylation had minimal effect on integrin-binding affinity of the RGD peptide. 18 F-FPRGD4 has significantly higher tumor uptake compared with monomeric and dimeric RGD peptide tracer analogs. The receptor specificity of 18 F-FPRGD4 in vivo was confirmed by effective blocking of the uptake in both tumors and normal organs or tissues with excess c(RGDyK). Conclusion: The tetrameric RGD peptide tracer 18 F-FPRGD4 possessing high integrin-binding affinity and favorable biokinetics is a promising tracer for PET of integrin a v b 3 expression in cancer and other angiogenesis related diseases.

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Section: Discussionmentioning

confidence: 99%

microPET of Tumor Integrin v 3 Expression Using 18F-Labeled PEGylated Tetrameric RGD Peptide (18F-FPRGD4)

Wu¹,

Li²,

Chen³

et al. 2007

Journal of Nuclear Medicine

194

275

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“…However, the 40-mg/kg dose resulted in peak levels of only 0.02 g/ml. The lysostaphin pharmacokinetics for adults appears different than that for neonates (26).…”

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confidence: 85%

“…Pups remained with their dams throughout the experiment. The Baylor College of Medicine Institutional Animal Use Committee approved the research.Three-day-old pups received 1 mg/kg of body weight/dose of lysostaphin at 1, 6, 24, and 30 h. Blood was obtained by cardiac puncture at 1,2,3,7,8,23,25,26,27, 31, 32, 72, and 96 h after the first dose. Three pups' blood was pooled for each data point, and each time point had two data points.…”

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confidence: 99%

Lysostaphin in Treatment of Neonatal Staphylococcus aureus Infection

Oluola

Kong

Fein

et al. 2007

Antimicrob Agents Chemother

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This study describes lysostaphin's effect against methicillin-sensitive Staphylococcus aureus in suckling rats. Standard techniques determined minimal inhibitory and bactericidal concentrations, pharmacokinetics, and efficacy. The numbers of surviving rats after vancomycin, oxacillin, and lysostaphin treatment were comparable and were different from that of controls (P < 0.00001). Lysostaphin appears effective in the treatment of neonatal S. aureus infection.Additional antibacterial agents for staphylococcal infection are needed (3,9,10,12,13,21,27). Lysostaphin, first identified in 1944 (20), is an endopeptidase which specifically cleaves the cell wall cross-linking pentaglycine bridges of Staphylococcus aureus (25) and is active against nondividing organisms (17,19). We describe the effect of lysostaphin against S. aureus in a neonatal animal model.A clinical methicillin-sensitive S. aureus (MSSA) serotype 5 strain (Hernandez) was stored in tryptic soy broth at Ϫ80°C and grown before each experiment. An MSSA strain allowed comparison of lysostaphin to oxacillin and vancomycin. Minimum bactericidal concentrations (MBCs) and MICs of vancomycin, oxacillin, and lysostaphin were determined (7, 18).Fourteen-day timed-pregnancy Wistar rats (Charles River Laboratories) received antibiotic-free water and food ad libitum and delivered at 21 to 22 days of gestation. Pups remained with their dams throughout the experiment. The Baylor College of Medicine Institutional Animal Use Committee approved the research.Three-day-old pups received 1 mg/kg of body weight/dose of lysostaphin at 1, 6, 24, and 30 h. Blood was obtained by cardiac puncture at 1,2,3,7,8,23,25,26,27, 31, 32, 72, and 96 h after the first dose. Three pups' blood was pooled for each data point, and each time point had two data points. Lysostaphin serum concentration was determined by enzyme-linked immunosorbent assay (26).Plates were coated with 100 l (1 g/ml) of rabbit antilysostaphin for 2 h at room temperature and washed with phosphate-buffered saline plus 0.01% Tween 20, and 100 l of standard or sera was added. After 60 min, plates were washed and incubated with biotinylated rabbit antilysostaphin and ExtrAvidin (catalog no. 81K4875; Sigma) for 30 min separately. Substrate tetramethylbenzidine (catalog no. TMBW0100-01; BioFx) was added. Absorbances were measured and converted to concentrations.Three-day-old pups received 0.2 ml subcutaneously (1 ϫ 10 7 CFU/ml) of S. aureus cephalad to the tail. The bacterial concentration was determined by optical density and confirmed by quantitative culture. In the first experiment, littermate pups were randomly assigned to either earlier treatment at 0.5, 6.5, 24, and 30 h after infection or later treatment at 6, 24, 30, and 48 h after infection. Each pup received 0.2 ml intraperitoneally of lysostaphin (1 mg/kg) or an equal volume of normal saline.In the second experiment, littermate pups randomly received 0.2 ml intraperitoneally of lysostaphin (1 mg/kg), vancomycin (15 mg/kg), oxacillin (50 mg/kg), or normal saline ...

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“…Protein PEGylation, the covalent attachment of PEG chains to protein, is an effective method to reduce or overcome these problems (46,47). It has been shown that PEGylation of lysostaphin, conjugating PEG to the ε-amino groups of lysine residues, improves in vivo half-life and reduces immunogenicity, while bacteriolytic activity of PEGylated lysostaphin is diminished (48). Nonspecific reaction of PEG with lysostaphin at different sites produces a number of isomers, most of which have decreased binding affinity to the big network of cell wall peptidoglycan (see Fig.…”

Section: Discussionmentioning

confidence: 99%

Hydrogen/Deuterium Exchange Mass Spectrometry and Site-Directed Disulfide Cross-Linking Suggest an Important Dynamic Interface between the Two Lysostaphin Domains

Huang

et al. 2013

Antimicrob Agents Chemother

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Lysostaphin is a peptidoglycan hydrolase secreted by Staphylococcus simulans. It can specifically lyse Staphylococcus aureus and is being tested as a novel antibacterial agent. The protein contains an N-terminal catalytic domain and a C-terminal cell wall targeting domain. Although the two domains from homologous enzymes were structurally determined, the structural organization of lysostaphin domains remains unknown. We used hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and sitedirected disulfide cross-linking to probe the interface between the lysostaphin catalytic and targeting domains. H/DX-MS-mediated comparison of peptides from full-length lysostaphin and the separated domains identified four peptides of lower solvent accessibility in the full-length protein. Cross-linking analysis using cysteine pair substitutions within those peptides showed that two pairs of cysteines can form disulfide bonds, supporting the domain association role of the targeted peptides. The crosslinked mutant exhibited a binding capacity to S. aureus that was similar to that of the wild-type protein but reduced bacteriolytic activity probably because of restraint in conformation. The diminished activity was further reduced with increasing NaCl concentrations that can cause contractions of bacterial peptidoglycan. The lytic activity, however, could be fully recovered by reducing the disulfide bonds. These results suggest that lysostaphin may require dynamic association of the two domains for coordinating substrate binding and target cleavage on the elastic peptidoglycan. Our study will help develop site-specific PEGylated lysostaphin to treat systemic S. aureus infections.

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Improved Pharmacokinetics and Reduced Antibody Reactivity of Lysostaphin Conjugated to Polyethylene Glycol

Cited by 100 publications

References 12 publications

microPET of Tumor Integrin v 3 Expression Using 18F-Labeled PEGylated Tetrameric RGD Peptide (18F-FPRGD4)

microPET of Tumor Integrin v 3 Expression Using 18F-Labeled PEGylated Tetrameric RGD Peptide (18F-FPRGD4)

Lysostaphin in Treatment of Neonatal Staphylococcus aureus Infection

Hydrogen/Deuterium Exchange Mass Spectrometry and Site-Directed Disulfide Cross-Linking Suggest an Important Dynamic Interface between the Two Lysostaphin Domains

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