Antimicrobial Peptide P60.4Ac-Containing Creams and Gel for Eradication of Methicillin-Resistant Staphylococcus aureus from Cultured Skin and Airway Epithelial Surfaces

Abstract

We previously found the LL-37-derived peptide P60.4Ac to be effective against methicillin-resistant Staphylococcus aureus (MRSA) on human epidermal models (EMs). The goal of this study was to identify the preferred carrier for this peptide for topical application on skin and mucosal surfaces. We prepared P60.4Ac in three formulations, i.e., a water-in-oil cream with lanolin (Softisan 649), an oil-in-water cream with polyethylene glycol hexadecyl ether (Cetomacrogol), and a hydroxypropyl methylcellulose (hypromellose) 4000 gel. We tested the antimicrobial efficacy of the peptide in these formulations against mupirocin-resistant and -sensitive MRSA strains on EMs and bronchial epithelial models (BEMs). The cytotoxic effects of formulated P60.4Ac on these models were determined using histology and WST-1 and lactate dehydrogenase assays. Moreover, we assessed the stability of the peptide in these formulations with storage for up to 3 months. Killing of MRSA by P60.4Ac in the two creams was less effective than that by P60.4Ac in the hypromellose gel. In agreement with those findings, P60.4Ac in the hypromellose gel was highly effective in eradicating the two MRSA strains from EMs. We found that even 0.1% (wt/wt) P60.4Ac in the hypromellose gel killed >99% of the viable planktonic bacteria and >85% of the biofilm-associated bacteria on EMs. Hypromellose gels containing 0.1% and 0.5% (wt/wt) P60.4Ac effectively reduced the numbers of viable MRSA cells from BEMs by >90%. No cytotoxic effects of P60.4Ac in the hypromellose gel with up to 2% (wt/wt) P60.4Ac on keratinocytes in EMs and in the hypromellose gel with up to 0.5% (wt/wt) P60.4Ac on epithelial cells in BEMs were observed. High-performance liquid chromatography analysis showed that P60.4Ac was stable in the Softisan cream and the hypromellose gel but not in the Cetomacrogol cream. We conclude that P60.4Ac formulated in hypromellose gel is both stable and highly effective in eradicating MRSA from colonized EMs and BEMs.A bout 10 to 35% of humans are persistent carriers of Staphylococcus aureus, while 20 to 75% are intermediate carriers (1-3). The primary sites for colonization of humans are the nasal cavity, pharynx, perineum, and skin (3). Colonization of healthy individuals with this opportunistic bacterium causes no serious health problems; however, the risk of developing an S. aureus infection is increased in hospitalized patients or those undergoing surgery (4-6). Common infection problems include surgical site infections (7), wound and burn wound infections (5), bloodstream infections (8), catheter-related bacteremia (9, 10), and catheter (intubation)-related infections, such as pneumonia (11). Moreover, the symptoms of atopic eczema patients are often aggravated by the actions of S. aureus and S. epidermidis on their lesion sites (12, 13).Strategies currently used to eradicate methicillin-resistant S. aureus (MRSA) from carriers involve treatment with antibiotics, such as vancomycin, mupirocin, bacitracin, rifampin, and retapamulin (14-18). It has been show...

MRSA decolonization failure—are biofilms the missing link?

“…Our data provide evidence that OCT and CHG are effective components for disinfection of MRSA biofilms in vitro and that exposure to MUP at the standard concentrations in topical preparations up to 2% does not effectively inhibit the metabolic activity of MRSA biofilms, even after the prolonged exposure of 3.5 h. The limited efficacy of MUP against the bacteria in biofilms has previously been described [35]. The biofilm probably provides a physical barrier for MUP so that only insufficient concentrations are reached in the bacteria themselves.…”

Identification of an IDR peptide formulation candidate that prevents peptide aggregation and retains immunomodulatory activity

“…This is likely due to the cationic peptide binding to negatively charged functional groups on the polymers and limiting its availability for activity against the bacteria. Conversely, a previous study found that a gel formulation containing HPMC and a synthetic LL‐37 derivative exhibited antimicrobial activity in a human epidermal model of S. aureus infection without causing significant toxicity towards keratinocytes or epithelial cells . However, both of these studies assessed direct antimicrobial killing by the formulated peptides rather than immunomodulatory activity.…”

Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application

“…By administering peptides to infected wounds in viscous gel-like polymer delivery systems, the peptides will become localized at the site of infection and exhibit increased resistance to exogenous proteases [56]. For example, topical delivery of the LL-37 derivative P60.4Ac in a water-based hypromellose gel decreased the peptide's cytotoxic effects and allowed for the killing of greater than 85% of MRSA biofilm in a human epidermal model [57]. The peptide-gel formulation retained antimicrobial activity and had increased ability to eradicate biofilm on epidermal cells, while reducing cytotoxicity, as compared to peptide dissolved in phosphate buffered saline [57].…”

“…For example, topical delivery of the LL-37 derivative P60.4Ac in a water-based hypromellose gel decreased the peptide's cytotoxic effects and allowed for the killing of greater than 85% of MRSA biofilm in a human epidermal model [57]. The peptide-gel formulation retained antimicrobial activity and had increased ability to eradicate biofilm on epidermal cells, while reducing cytotoxicity, as compared to peptide dissolved in phosphate buffered saline [57]. This same gel formulation was successfully used by de Breij et al [24] to administer antibiofilm peptide SAAP-148 to MRSA and A. baumannii biofilms in human skin and murine models without any systemic toxicity or irritation.…”