Carrier peptide interactions with liposome membranes induce reversible clustering by surface adsorption and shape deformation.

The cell-penetrating peptide penetratin and its analogues shuffle and penetramax have been used as carrier peptides for oral delivery of therapeutic peptides such as insulin. Their mechanism of action for this purpose is not fully understood but is believed to depend on the interactions of the peptide with the cell membrane. In the present study, peptide-liposome interactions were investigated using advanced biophysical techniques including small-angle neutron scattering and fluorescence lifetime imaging microscopy. Liposomes were used as a model system for the cell membrane. All the investigated carrier peptides induced liposome clustering at a specific peptide/lipid ratio. However, distinctively different types of membrane interactions were observed, as the liposome clustering was irreversible for penetratin, but fully or partly reversible for shuffle and penetramax, respectively. All three peptides were found to adsorb to the surface of the lipid bilayers, while only shuffle and penetramax led to shape deformation of the liposomes. Importantly, the peptide interactions did not disrupt the liposomes under any of the investigated conditions, which is advantageous for their application in drug delivery. This detailed insight on peptide-membrane interactions is important for understanding the mechanism of peptide-based excipients and the influence of peptide sequence modifications.

Encapsulation of SAAP-148 in Octenyl Succinic Anhydride-Modified Hyaluronic Acid Nanogels for Treatment of Skin Wound Infections.

Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204-253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37-41% in 72 h) upon reconstitution. Lyophilized SAAP-148-loaded nanogels showed similar antimicrobial activity as SAAP-148 against planktonic and biofilm-residing AMR Staphylococcus aureus and Acinetobacter baumannii. Importantly, formulated SAAP-148 showed reduced cytotoxicity against human erythrocytes, primary human skin fibroblasts and human keratinocytes. Additionally, lyophilized SAAP-148-loaded nanogels eradicated AMR S. aureus and A. baumannii colonizing a 3D human epidermal model, without inducing any cytotoxicity in contrast to SAAP-148. These findings indicate that OSA-HA nanogels increase SAAP-148's therapeutic potential for treatment of skin wound infections.

Heterogeneous and Surface-Catalyzed Amyloid Aggregation Monitored by Spatially Resolved Fluorescence and Single Molecule Microscopy.

Amyloid aggregation is associated with many diseases and may also occur in therapeutic protein formulations. Addition of co-solutes is a key strategy to modulate the stability of proteins in pharmaceutical formulations and select inhibitors for drug design in the context of diseases. However, the heterogeneous nature of this multicomponent system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Using insulin as protein system and polysorbate 80 as co-solute, we combine a spatially resolved fluorescence approach with single molecule microscopy and machine learning methods to kinetically disentangle the different contributions from multiple species within a single aggregation experiment. We link the presence of interfaces to the degree of heterogeneity of the aggregation kinetics and retrieve the rate constants and underlying mechanisms for single aggregation events. Importantly, we report that the mechanism of inhibition of the self-assembly process depends on the details of the growth pathways of otherwise macroscopically identical species. This information can only be accessed by the analysis of single aggregate events, suggesting our method as a general tool for a comprehensive physicochemical characterization of self-assembly reactions.

Mucoadhesive chitosan- and cellulose derivative-based nanofiber-on-foam-on-film system for non-invasive peptide delivery.

Oromucosal administration is an attractive non-invasive route. However, drug absorption is challenged by salivary flow and the mucosa being a significant permeability barrier. The aim of this study was to design and investigate a multi-layered nanofiber-on-foam-on-film (NFF) drug delivery system with unique properties and based on polysaccharides combined as i) mucoadhesive chitosan-based nanofibers, ii) a peptide loaded hydroxypropyl methylcellulose foam, and iii) a saliva-repelling backing film based on ethylcellulose. NFF displays optimal mechanical properties shown by dynamic mechanical analysis, and biocompatibility demonstrated after exposure to a TR146 cell monolayer. Chitosan-based nanofibers provided the NFF with improved mucoadhesion compared to that of the foam alone. After 1 h, >80 % of the peptide desmopressin was released from the NFF. Ex vivo permeation studies across porcine buccal mucosa indicated that NFF improved the permeation of desmopressin compared to a commercial freeze-dried tablet. The findings demonstrate the potential of the NFF as a biocompatible drug delivery system.

Topical niclosamide (ATx201) reduces Staphylococcus aureus colonization and increases Shannon diversity of the skin microbiome in atopic dermatitis patients in a randomized, double-blind, placebo-controlled Phase 2 trial.

BACKGROUND: In patients with atopic dermatitis (AD), Staphylococcus aureus frequently colonizes lesions and is hypothesized to be linked to disease severity and progression. Treatments that reduce S. aureus colonization without significantly affecting the skin commensal microbiota are needed. METHODS AND FINDINGS: In this study, we tested ATx201 (niclosamide), a small molecule, on its efficacy to reduce S. aureus and propensity to evolve resistance in vitro. Various cutaneous formulations were then tested in a superficial skin infection model. Finally, a Phase 2 randomized, double-blind and placebo-controlled trial was performed to investigate the impact of ATx201 OINTMENT 2% on S. aureus colonization and skin microbiome composition in patients with mild-to-severe AD (EudraCT:2016-003501-33). ATx201 has a narrow minimal inhibitory concentration distribution (.125-.5 µg/ml) consistent with its mode of action - targeting the proton motive force effectively stopping cell growth. In murine models, ATx201 can effectively treat superficial skin infections of methicillin-resistant S. aureus. In a Phase 2 trial in patients with mild-to-severe AD (N = 36), twice-daily treatment with ATx201 OINTMENT 2% effectively reduces S. aureus colonization in quantitative colony forming unit (CFU) analysis (primary endpoint: 94.4% active vs. 38.9% vehicle success rate, p = .0016) and increases the Shannon diversity of the skin microbiome at day 7 significantly compared to vehicle. CONCLUSION: These results suggest that ATx201 could become a new treatment modality as a decolonizing agent.

Mucoadhesive Electrospun Nanofiber-Based Hybrid System with Controlled and Unidirectional Release of Desmopressin.

The sublingual mucosa is an attractive route for drug delivery, although challenged by a continuous flow of saliva that leads to a loss of drug by swallowing. It is of great benefit that drugs absorbed across the sublingual mucosa avoid exposure to the harsh environment of the gastro-intestinal lumen; this is especially beneficial for drugs of low physicochemical stability such as therapeutic peptides. In this study, a two-layered hybrid drug delivery system was developed for the sublingual delivery of the therapeutic peptide desmopressin. It consisted of peptide-loaded mucoadhesive electrospun chitosan/polyethylene oxide-based nanofibers (mean diameter of 183 ± 20 nm) and a saliva-repelling backing film to promote unidirectional release towards the mucosa. Desmopressin was released from the nanofiber-based hybrid system (approximately 80% of the loaded peptide was released within 45 min) in a unidirectional manner in vitro. Importantly, the nanofiber-film hybrid system protected the peptide from wash-out, as demonstrated in an ex vivo flow retention model with porcine sublingual mucosal tissue. Approximately 90% of the loaded desmopressin was retained at the surface of the ex vivo porcine sublingual mucosa after 15 min of exposure to flow rates representing salivary flow.

Cell-Penetrating Peptides as Carriers for Transepithelial Drug Delivery.

This chapter describes the use of cell-penetrating peptides (CPPs) as carriers for transepithelial delivery of therapeutic peptides. Assessment of transepithelial peptide permeation and the mechanisms of action that permeability enhancing drug carriers exert on the epithelium requires subtle sample preparation and analysis by orthogonal methods. Here, the preparation and use of CPP-insulin physical mixture samples including the quantification of insulin by enzyme-linked immunosorbent assay (ELISA) is described. In addition, effects of CPPs on the epithelium and its barrier properties immediately upon exposure and after a recovery period are evaluated by epithelial cell viability, transepithelial electrical resistance, immunostaining of the tight junction associated zonula occludens (ZO-1) protein, and actin cytoskeleton staining.

Revealing the importance of carrier-cargo association in delivery of insulin and lipidated insulin.

Delivery of therapeutic peptides upon oral administration is highly desired and investigations report that the cell-penetrating peptide (CPP) penetratin and its analogues shuffle and penetramax show potential as carriers to enhance insulin delivery. Exploring this, the specific aim of the present study was to understand the impact that their complexation with a lipidated or non-lipidated therapeutic cargo would have on the delivery, to evaluate the effect of differences in membrane interactions in vitro and in vivo, as well as to deduce the mode of action leading to enhanced delivery. Fundamental biophysical aspects were studied by a range of orthogonal methods. Transepithelial permeation of therapeutic peptide was evaluated using the Caco-2 cell culture model supplemented with epithelial integrity measurements, real-time assessment of the carrier peptide effects on cell viability and on mode of action. Pharmacokinetic and pharmacodynamic (PK/PD) parameters were evaluated following intestinal administration to rats and tissue effects were investigated by histology. The biophysical studies revealed complexation of insulin with shuffle and penetramax, but not with penetratin. This corresponded to enhanced transepithelial permeation of insulin, but not of lipidated insulin, when in physical mixture with shuffle or penetramax. The addition of shuffle and penetramax was associated with a lowering of Caco-2 cell monolayer integrity and viability, where the lowering of cell viability was immediate, but reversible. Insulin delivery in rats was enhanced by shuffle and penetramax and accompanied by a 10-20-fold decrease in blood glucose with immediate effect on the intestinal mucosa. In conclusion, shuffle and penetramax, but not penetratin, demonstrated to be potential candidates as carriers for transmucosal delivery of insulin upon oral administration, and their effect depended on association with both cargo and cell membrane. Interestingly, the present study provides novel mechanistic insight that peptide carrier-induced cargo permeation points towards enhancement via the paracellular route in the tight epithelium. This is different from the anticipated belief being that it is the cell-penetrating capability that facilitate transepithelial cargo permeation via a transcellular route.

Controlling the fractal dimension in self-assembly of terpyridine modified insulin by Fe2+ and Eu3+ to direct in vivo effects.

Metal ion-induced self-assembly (SA) of proteins into higher-order structures can provide new, dynamic nano-assemblies. Here, the synthesis and characterization of a human insulin (HI) analog modified at LysB29 with the tridentate chelator 2,2':6',2''-terpyridine (Tpy) is described. SA of this new insulin analog (LysB29Tpy-HI) in the presence of the metal ions Fe2+ and Eu3+ at different concentrations was studied in solution by fluorescence luminescence and CD spectroscopy, dynamic light scattering, and small-angle X-ray scattering, while surface assembly was probed by AFM. Unique oligomerization was observed in solution, as Fe2+ yielded small magenta-colored discrete non-native assemblies, while Eu3+ caused the formation of large fractal assemblies. Binding of both metal ions to Tpy was demonstrated spectroscopically, and emission lifetime experiments revealed a distinct Eu3+ coordination geometry that included two water molecules. SAXS suggested that LysB29Tpy-HI with Fe2+ oligomerized to a discrete, roughly octameric species, while LysB29Tpy-HI with Eu3+ gave very large assemblies that could be modelled as fractals. The fractal dimensionality increased with the Eu3+ concentration. We propose that this is a consequence of Eu3+ binding to both Tpy and to free carboxylic acid groups on the insulin surface. LysB29Tpy-HI maintained insulin receptor affinity, and showed extended blood glucose lowering and plasma concentration after subcutaneous injection in rats. The combination of metal ion directed SA and native SA provides control of nano-scale fractal dimensionality and points towards use in therapeutics.

Nanoparticle-mediated pulmonary drug delivery: state of the art towards efficient treatment of recalcitrant respiratory tract bacterial infections.

Recalcitrant respiratory tract infections caused by bacteria have emerged as one of the greatest health challenges worldwide. Aerosolized antimicrobial therapy is becoming increasingly attractive to combat such infections, as it allows targeted delivery of high drug concentrations to the infected organ while limiting systemic exposure. However, successful aerosolized antimicrobial therapy is still challenged by the diverse biological barriers in infected lungs. Nanoparticle-mediated pulmonary drug delivery is gaining increasing attention as a means to overcome the biological barriers and accomplish site-specific drug delivery by controlling release of the loaded drug(s) at the target site. With the aim to summarize emerging efforts in combating respiratory tract infections by using nanoparticle-mediated pulmonary delivery strategies, this review provides a brief introduction to the bacterial infection-related pulmonary diseases and the biological barriers for effective treatment of recalcitrant respiratory tract infections. This is followed by a summary of recent advances in design of inhalable nanoparticle-based drug delivery systems that overcome the biological barriers and increase drug bioavailability. Finally, challenges for the translation from exploratory laboratory research to clinical application are also discussed and potential solutions proposed.

Swelling of mucoadhesive electrospun chitosan/polyethylene oxide nanofibers facilitates adhesion to the sublingual mucosa.

Mucoadhesive chitosan-based electrospun nanofibers are promising candidates for overcoming challenges associated with sublingual drug delivery, yet studies focusing on evaluating the mucoadhesive properties of nanofibers for sublingual administration are limited. The aim was to elucidate the mucoadhesive properties of chitosan/polyethylene oxide (PEO) nanofibers focusing on how the degree of deacetylation (DDA, 53-96 %) of chitosan influenced their morphological and mucoadhesive properties. The mechanism of mucoadhesion was explained by the intermolecular interactions of chitosan with mucin from bovine submaxillary glands using quartz-crystal microbalance with dissipation monitoring and by adhesion of the nanofibers to ex vivo porcine sublingual mucosa. An increase in chitosan DDA improved the morphological stability of the nanofibers in water, but did not contribute to altered mucoadhesive properties. This study demonstrates excellent mucoadhesive properties of chitosan/PEO nanofibers and shows that the strong mucoadhesiveness of the nanofibers is attributed to their swelling ability.

Shuffled lipidation pattern and degree of lipidation determines the membrane interaction behavior of a linear cationic membrane-active peptide.

HYPOTHESIS: Permeation of macromolecular drugs across biological plasma membranes is a major challenge in drug delivery. Cationic cell-penetrating peptides (CPPs) are attractive functional excipient candidates for the delivery of macromolecules across membrane barriers, due to their membrane translocating ability. The properties of CPPs can be tailored by lipidation, a promising approach to facilitate enhanced membrane insertion, potentially promoting increased translocation of the CPP and cargo. EXPERIMENTS: To explore the impact that site and degree of lipidation have on the membrane interaction of a cationic CPP, we designed and investigated CPP conjugates with one or two fatty acid chains. FINDINGS: Compared to the parent CPP and the single-lipidated conjugates, the double-lipidated conjugate exhibited the most pronounced membrane perturbation effects, as measured by several biophysical techniques. The experimental findings were supported by molecular dynamics (MD) simulations, demonstrating that all CPP conjugates interacted with the membrane by insertion of the lipid chain(s) into the core of the bilayer. Moreover, membrane-thinning effects and induced membrane curvature were displayed upon CPP interaction. Our results demonstrate that the impact exerted by the CPP on the membrane is notably affected by positioning and especially the degree of lipidation, which might influence the properties of CPPs as functional excipients.

Membrane interactions in drug delivery: Model cell membranes and orthogonal techniques.

To generate the desired effect in the human body, the active pharmaceutical ingredient usually needs to interact with a receptor located on the cell membrane or inside the cell. Thus, understanding membrane interactions is of great importance when it comes to the development and testing of new drug molecules or new drug delivery systems. Nowadays, there is a tremendous selection of both model cell membranes and of techniques that can be used to characterize interactions between selected model cell membranes and a drug molecule, an excipient, or a drug delivery system. Having such a wide selection of model cell membranes and techniques available makes it sometimes challenging to select the optimal combination for a specific study. Furthermore, it is difficult to compare results obtained using different model cell membranes and techniques, and not all in vitro studies translate as well to an estimation of the in vivo biological activity or understanding of mode of action. This review provides an overview of the available lipid bilayer-based model cell membranes and of the most widely employed techniques for studying membrane interactions. Finally, the need for employing complimentary characterization techniques in order to acquire more reliable and in-depth information is highlighted.

In Vitro, Ex Vivo and In Vivo Evaluation of Microcontainers for Oral Delivery of Insulin.

Enhancing the oral bioavailability of peptides has received a lot of attention for decades but remains challenging, partly due to low intestinal membrane permeability. Combining a permeation enhancer (PE) with unidirectionally releasing microcontainers (MCs) has previously been shown to increase insulin permeation across Caco-2 cell monolayers. In the present work, this setup was further employed to compare three common PEs-sodium caprate (C10), sodium dodecyl sulfate (SDS), and lauroyl carnitine. The concept was also studied using porcine intestinal tissue with the inclusion of 70 kDa fluorescein isothiocyanate-dextran (FD70) as a pathogen marker. Moreover, a combined proteolysis and Caco-2 cell permeation setup was developed to investigate the effect of soybean trypsin inhibitor (STI) in the MCs. Lastly, in vivo performance of the MCs was tested in an oral gavage study in rats by monitoring blood glucose and insulin absorption. SDS proved to be the most potent PE without increasing the ex vivo uptake of FD70, while the implementation of STI further improved insulin permeation in the combined proteolysis Caco-2 cell setup. However, no insulin absorption in rats was observed upon oral gavage of MCs loaded with insulin, PE and STI. Post-mortem microscopic examination of their gastrointestinal tract indicated lack of intestinal retention and optimal orientation by the MCs, possibly precluding the potential advantage of unidirectional release.

Comparison of bulk and microfluidics methods for the formulation of poly-lactic-co-glycolic acid (PLGA) nanoparticles modified with cell-penetrating peptides of different architectures.

The efficient and reproducible production of nanoparticles using bulk nanoprecipitation methods is still challenging because of low batch to batch reproducibility. Here, we optimize a bulk nanoprecipitation method using design of experiments and translate to a microfluidic device to formulate surface-modified poly-lactic-co-glycolic (PLGA) nanoparticles. Cell-penetrating peptides (CPPs) with a short, long linear or branched architecture were used for the surface modification of PLGA nanoparticles. The microfluidics method was more time efficient than the bulk nanoprecipitation method and allowed the formulation of uniform PLGA nanoparticles with a size of 150 nm, a polydispersity index below 0.150 and with better reproducibility in comparison to the bulk nanoprecipitation method. After surface modification the size of CPP-tagged PLGA nanoparticles increased to 160-180 nm and the surface charge of the CPP-tagged PLGA nanoparticles varied between -24 mV and +3 mV, depending on the architecture and concentration of the conjugated CPP. Covalent attachment of CPPs to the PLGA polymer was confirmed with FTIR by identifying the formation of an amide bond. The conjugation efficiency of CPPs to the polymeric PLGA nanoparticles was between 32 and 80%. The development and design of reproducible nanoformulations with tuneable surface properties is crucial to understand interactions at the nano-bio interface.

Microcontainers for oral insulin delivery - In vitro studies of permeation enhancement.

Oral delivery of peptides is challenging due to their low uptake through the small intestinal epithelium. Tight junctions, connecting the enterocytes, impede permeability, often necessitating the use of permeation enhancers in the formulation. Loading of peptide and permeation enhancer into micro-scale devices, such as microcontainers, can potentially confine the effective absorptive area through unidirectional release and thereby enhance absorption. This concept is investigated by in vitro permeation studies of insulin across Caco-2 cell and Caco-2/HT29-MTX-E12 co-culture monolayers mimicking the intestinal absorption barrier. The importance of proximity between the microcontainers and the barrier is assessed, by keeping the amounts of insulin and sodium caprate fixed throughout all experiments, while collectively orienting the unidirectional release towards the cell monolayers. Increasing the distance is observed to have a negative effect on insulin permeation matching a one-phase exponential decay function, while no difference in insulin transport is observed between Caco-2 and co-culture monolayers. Although there are no signs of cytotoxicity caused by the microcontainer material, reversible cell deterioration, as a consequence of high local concentrations of sodium caprate, becomes evident upon qualitative assessment of the cell monolayers. These results both suggest a potential of increasing oral bioavailability of peptides by the use of microcontainers, while simultaneously visualising the ability of regaining monolayer integrity upon local permeation enhancer induced toxicity.

Mechanistic profiling of the release kinetics of siRNA from lipidoid-polymer hybrid nanoparticles in vitro and in vivo after pulmonary administration.

Understanding the release kinetics of siRNA from nanocarriers, their cellular uptake, their in vivo biodistribution and pharmacokinetics is a fundamental prerequisite for efficient optimisation of the design of nanocarriers for siRNA-based therapeutics. Thus, we investigated the influence of composition on the siRNA release from lipid-polymer hybrid nanoparticles (LPNs) consisting of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) (PLGA) intended for pulmonary administration. An array of siRNA-loaded LPNs was prepared by systematic variation of: (i) the L5 content (10-20%, w/w), and (ii) the L5:siRNA ratio (10,1-30:1, w/w). For comparative purposes, L5-based lipoplexes, L5-based stable nucleic acid lipid nanoparticles (SNALPs). and dioleoyltrimethylammoniumpropane (DOTAP)-modified LPNs loaded with siRNA were also prepared. Release studies in buffer and lung surfactant-containing medium showed that siRNA release is dependent on the presence of both surfactant and heparin (a displacing agent) in the release medium, since these interact with the lipid shell structure thereby facilitating decomplexation of L5 and siRNA, as evident from the retarded siRNA release when the L5 content and the L5:siRNA ratio were increased. This confirms the hypothesis that siRNA loaded in LPNs is predominantly present as complexes with the cationic lipid and primarily is located near the particle surface. Cellular uptake and tolerability studies in the human macrophage cell line THP-1 and the type I-like human alveolar epithelial cell line hAELVi, which together represents a monolayer-based barrier model of lung epithelium, indicated that uptake of LPNs was much higher in THP-1 cells in agreement with their primary clearance role. In vivo biodistributions of formulations loaded with Alexa Fluor® 750-labelled siRNA after pulmonary administration in mice were compared by using quantitative fluorescence imaging tomography. The L5-modified LPNs, SNALPs and DOTAP-modified LPNs displayed significantly increased lung retention of siRNA as compared to L5-based lipoplexes, which had a biodistribution profile comparable to that of non-loaded siRNA, for which >50% of the siRNA dose permeated the air-blood barrier within 6 h and subsequently was excreted via the kidneys. Hence, the enhanced lung retention upon pulmonary administration of siRNA-loaded LPNs represents a promising characteristic that can be used to control the delivery of the siRNA cargo to lung tissue for local management of disease.

The distribution of cell-penetrating peptides on polymeric nanoparticles prepared using microfluidics and elucidated with small angle X-ray scattering.

HYPOTHESIS: The distribution of three cell-penetrating peptides (CPPs) with different architectures (short, long linear and branched) on poly(lactic-co-glycolic) acid (PLGA) nanoparticles depends on the conjugation approach. Here, we explore the utilization of a zero-length crosslinking reaction for the covalent attachment of CPPs to PLGA nanoparticles and the translation of the reaction into a microfluidic platform. EXPERIMENTS: A microfluidic device with a staggered herringbone mixer was used for the formulation of CPP-tagged PLGA nanoparticles. CPP-tagged PLGA nanoparticles were labeled with gold nanoparticles (AuNPs) and transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) were used to elucidate the distribution of CPPs. FINDINGS: The SAXS scattering profiles for the CPP-tagged PLGA nanoparticles prepared with the in situ microfluidics conjugation approach indicated a distribution of the Au-labeled CPPs throughout the PLGA nanoparticles. For the post-microfluidics conjugation approach, the SAXS scattering profiles did not show the feature of the Au-labeled CPPs distributed throughout the PLGA nanoparticles and an arrangement of the Au-labeled CPP on the surface was support by TEM micrographs. The distribution of the CPPs was highly dependent on the conjugation approach and was not influenced by the architecture of the CPPs. The results provided insight for the rational design of CPP-tagged PLGA nanoparticles using microfluidics.

Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics.

Synthetic peptidomimetics may be designed to mimic functions of antimicrobial peptides, including potentiation of antibiotics, yet possessing improved pharmacological properties. Pairwise screening of 42 synthetic peptidomimetics combined with the antibiotics azithromycin and rifampicin in multidrug-resistant (MDR) Escherichia coli ST131 and Klebsiella pneumoniae ST258 led to identification of two subclasses of α-peptide/ß-peptoid hybrids that display synergy with azithromycin and rifampicin (fractional inhibitory concentration indexes of 0.03-0.38). Further screening of the best three peptidomimetics in combination with a panel of 21 additional antibiotics led to identification of peptidomimetics that potentiated ticarcillin/clavulanate and erythromycin against E. coli, and clindamycin against K. pneumoniae. The study of six peptidomimetics was extended to Pseudomonas aeruginosa, confirming synergy with antibiotics for five of them. The most promising compound, H-(Lys-ßNPhe)8-NH2, exerted only a minor effect on the viability of mammalian cells (EC50 ≥ 124-210 µM), and thus exhibited the highest selectivity toward bacteria. This compound also synergized with rifampicin and azithromycin at sub-micromolar concentrations (0.25-0.5 µM), thereby inducing susceptibility to these antibiotics at clinically relevant concentrations in clinical MDR isolates. This peptidomimetic lead and its analogs constitute promising candidates for efficient repurposing of rifampicin and azithromycin against Gram-negative pathogens.