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|Title: ||Therapeutic potential of BAC7(I-35), a Proline-rich Antimicrobial Peptide: in vitro and in vivo studies and Pegylation strategy to improve its bioavailability|
|Authors: ||Pelillo, Chiara|
|Supervisor/Tutor: ||Gennaro, Renato|
|Co-Advisor: ||Benincasa, Monica|
|Issue Date: ||28-Apr-2011|
|Publisher: ||Università degli studi di Trieste|
|Abstract: ||The antimicrobial peptides (AMPs) are an important component of the innate defense against invading microorganisms, are widespread in nature and may have multiple and diversified mechanisms of bactericidal action. In addition to their direct antimicrobial activity the are also involved in other biological processes.
The aim of this project was to investigate the in vivo activity of Bac7(1-35), a bovine proline-rich antimicrobial peptide, having in mind its possible use as a lead compound for the development of novel anti-infective agents. Before moving to animal models of infection, the in vitro stability of the peptide in the presence of murine and human serum or plasma as well as its biodistribution in mouse were investigated. Antibacterial activity assays against Salmonella enterica showed that the presence of murine blood components largely inhibits the antibacterial activity of the peptide. On the contrary, in human serum and plasma Bac7(1-35) maintains its efficacy. This is due to the more rapid degradation by proteases of murine blood. The in vivo biodistribution of Bac7(1-35) was investigated by using a time-domain optical imaging apparatus and a fluorescently-labeled Bac7(1-35) derivative. The compound reaches the kidney and the bladder respectively 1 and 3 hours after i.p. injection. The in vivo and ex vivo analyses performed after 24 h confirm that the compound has been totally excreted.
A mouse model of S. typhimurium infection was set up and used to test the therapeutic efficacy of Bac7(1-35). Treatment of infected mice with the peptide injected i.p. immediately after a lethal, intraperithoneal bacterial challenge, increased the mean survival time and reduced significantly the number of viable bacterial cells in liver and spleen of treated mice at 3 days post-inoculum. In 1/3 of the organ homogenates, the bacterial presence was undetectable and this result matches the percentage of cured animals (35%).
In an attempt to improve its pharmacokinetic profile, the peptide was conjugated with polyethylene glycol (PEG), a non-toxic, non-immunogenic and FDA-approved polymer. Different strategies of pegylation have been considered to find the best method in terms of chemical yield and of maintenance of biological activity. Pegylation via a thioether ligation resulted the best strategy to obtain a slow active peptide release in human blood components with a reduced renal clearance and an increased bioavailability of Bac7(1-35), as biodistribution analyses demonstrated.
Several important pathogens, such as S. enterica, cause disease by surviving and replicating within host cells. Since many AMPs have also immunomodulatory activities, we investigated the effect of Bac7(1-35) on the interaction between macrophages and Salmonella.
We carried out phagocytosis assays with macrophages and the results suggest that Bac7(1-35) plays a positive modulatory effect on this function. Phagocytosis assays were also performed to determine if Bac7(1-35) could inhibit survival and replication of intracellular Salmonella. The results show that the peptide inhibits the replication of intracellular Salmonella, suggesting that it can exert its antibacterial activity within eukaryotic cells. Further studies are required to fully understand the details of the Bac7(1-35) biological activities.
The results obtained provide encouraging evidence for future investigations on Bac7(1-35) and on the pegylated form Bac7(1-35)CAM-PEG20k also in other models of infection and with different intracellular pathogens.|
|PhD cycle: ||XXIII Ciclo|
|Keywords: ||antimicrobial PEPTIDES IN VIVO|
|Main language of document: ||en|
|Type: ||Tesi di dottorato|
|Scientific-educational field: ||BIO/10 BIOCHIMICA|
|Appears in Collections:||Scienze biologiche|
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