Gøril Eide Flaten

A Synthetic Antimicrobial Peptidomimetic (LTX 109): Stereochemical Impact on Membrane Disruption

LTX 109 is a synthetic antimicrobial peptidomimetic (SAMP) currently in clinical phase II trials for topical treatment of infections of multiresistant bacterial strains. All possible eight stereoisomers of the peptidomimetic have been synthesized and tested for antimicrobial effect, hemolysis, and hydrophobicity, revealing a strong and unusual dependence on the stereochemistry for a molecule proposed to act on a general membrane mechanism. The three-dimensional structures were assessed using nuclear magnetic resonance spectroscopy (NMR) and molecular dynamics (MD) simulations in aqueous solution and in phospholipid bilayers. The solution structures of the most active stereoisomers are perfectly preorganized for insertion into the membrane, whereas the less active isomers need to pay an energy penalty in order to enter the lipid bilayer. This effect is also found to be reinforced by a significantly improved water solubility of the less active isomers due to a guanidyl-π stacking that helps to solvate the hydrophobic surfaces.

Gene Transfer Potential of Outer Membrane Vesicles of Acinetobacter baylyi and Effects of Stress on Vesiculation

ABSTRACT Outer membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negative Acinetobacter baylyi as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of Escherichia coli and A. baylyi, including naturally transformation-deficient mutants of A. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT to A. baylyi and E. coli at transfer frequencies ranging from 10−6 to 10−8, with transfer efficiencies of approximately 103 and 102 per μg of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs from A. baylyi interact with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released from A. baylyi.

In vitro skin models as a tool in optimization of drug formulation

(Trans)dermal drug therapy is gaining increasing importance in the modern drug development. To fully utilize the potential of this route, it is important to optimize the delivery of active ingredient/drug into/through the skin. The optimal carrier/vehicle can enhance the desired outcome of the therapy therefore the optimization of skin formulations is often included in the early stages of the product development. A rational approach in designing and optimizing skin formulations requires well-defined skin models, able to identify and evaluate the intrinsic properties of the formulation. Most of the current optimization relies on the use of suitable ex vivo animal/human models. However, increasing restrictions in use and handling of animals and human skin stimulated the search for suitable artificial skin models. This review attempts to provide an unbiased overview of the most commonly used models, with emphasis on their limitations and advantages. The choice of the most applicable in vitro model for the particular purpose should be based on the interplay between the availability, easiness of the use, cost and the respective limitations.

In-vitro permeability of poorly water soluble drugs in the phospholipid vesicle-based permeation assay: the influence of nonionic surfactants

Objectives  The aim of this study was to determine the influence of nonionic surfactants on drug permeability using the phospholipid vesicle‐based permeation assay (PVPA), which excludes other than trans‐membrane diffusion pathways.

Synthesis of Cationic Antimicrobial β2,2-Amino Acid Derivatives with Potential for Oral Administration

We have prepared a series of highly potent achiral cationic β(2,2)-amino acid derivatives that fulfill the Lipinskis rule of five and that contain the basic structural requirements of short cationic antimicrobial peptides. Highest antimicrobial potency was observed for one of the smallest β(2,2)-amino acid derivatives (M(w) 423.6) exhibiting a MIC of 3.8 μM against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and Staphylococcus aureus, and 7.7 μM against Escherichia coli. The β(2,2)-amino acid derivatives were shown to have similar absorption properties as several commercially available drugs, and the results implied a resembling membrane disrupting mechanism of action as reported for much larger cationic antimicrobial peptides. By their high potency, nontoxicity, absorption properties, and ease of synthesis, the β(2,2)-amino acid derivatives demonstrate a way to modify a vastly investigated class of cationic antimicrobial peptides into small drug-like molecules with high commercial potential.

Drug permeability across a phospholipid vesicle-based barrier 4. The effect of tensides, co-solvents and pH changes on barrier integrity and on drug permeability.

In this study the integrity of the recently developed phospholipid vesicle-based permeability barrier in the presence of a variety of co-solvents and tensides has been investigated. Also included are studies of the influence of these additives on drug permeation and the effect of pH changes on the permeability of ionogenic drug compounds. Permeability experiments using the hydrophilic model compound calcein together with polysorbate 80 (Tween 80), polyoxyl 35 castor oil (Cremophor EL), macrogol lauryl ether (Brij 35), sorbitan monolaurate (Span 20), polyethylene glycol 400 (PEG 400), ethanol and dimethylsulphoxide (DMSO) were performed to determine whether the barriers were affected by the presence of these additives in the donor compartment. It was found that the integrity of the phospholipid vesicle-based barriers did not seem to be influenced by Span 20 up to a concentration of 5mg/ml, PEG 400 up to a concentration of 40mg/ml and ethanol and DMSO up to a concentration of 20mg/ml, respectively. Brij 35, Tween 80 and Cremophor EL were however found to be incompatible with the model at all concentrations as the barriers became leaky. Appearance of phospholipid in the donor chamber in presence of these three tensides indicated that the loss of integrity was due to partial dissolution of the phospholipid vesicles from the barrier. The permeability of testosterone was not significantly improved by the presence of the different co-solvents, except for 40 mg/ml PEG 400 and 20 mg/ml DMSO where the permeability was increased. In the pH study the permeability of metoprolol and naproxen was shown to decrease with increasing degree of ionisation according to the pH partition hypothesis. This renders the permeability model suitable for using pH-shift as a factor to influence solubility of drugs as well as to predict segmental absorption in the gastrointestinal tract.

In-vitro permeability screening of melt extrudate formulations containing poorly water-soluble drug compounds using the phospholipid vesicle-based barrier.

Objectives  The phospholipid vesicle‐based barrier has recently been introduced as an in‐vitro permeation model mimicking gastro‐epithelial barriers in terms of passive diffusion of drugs. The aim of this study was to investigate whether the phospholipid vesicle‐based barrier was suitable for permeability screening of complex formulations such as solid dispersions.

A method to determine the incorporation capacity of camptothecin in liposomes.

The purpose of this study was to establish a new experimental approach to determine the maximum amount of campothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultra-centrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubili ty of CPT in the buffer was improved by approximately a factor of 10 (from ∼2.7 to 15–50 μg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ∼100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ∼20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrisic water solubility of the drug.

Altered activity and physicochemical properties of short cationic antimicrobial peptides by incorporation of arginine analogues.

The incorporation of nongenetically encoded amino acids is a well established strategy to alter the behavior of several types of promising cationic antimicrobial peptides. Generally, these elements have been improved mimics of the hydrophobic amino acids yielding peptides with increased stability and potency. In this initial study, the effect of systematic replacement of Arg in a well-defined moderately antimicrobial tripeptide library is described. It is shown that the arginine analogues need to display a strong basicity to produce active peptides. It is further revealed that the hydrophobic units needed for activity in these peptides can be effectively incorporated in the direct vicinity of the cationic charge to produce compounds with improved antibacterial properties. A well-defined facial amphiphilic structure, which remains intact upon introduction of hydrophobic elements in the cationic side chains, is seen for the majority of the tested peptides. Microcalorimetric studies revealed a peptide binding to large anionic unilamellar vesicles (LUVs) mimicking the Gram-positive bacterial membrane as well as a potentially competitive binding to human serum albumin in the low- to mid-micromolar range. No considerable alterations in binding to either albumin or the LUVs were seen for the analogue containing peptides. A neutral LUV mimicking the eukaryotic cell membrane showed no significant binding to any of the peptides. The oral absorption of this class of short lactoferricin based peptides was investigated for the first time and revealed that incorporation of weaker bases than Arg produced peptides with much improved permeability in a recently developed permeation model, the phospholipid vesicle based barrier assay. Collectively, the results presented here show that there is ample room to toggle the activity and physical properties of short cationic antimicrobial peptides by incorporation of arginine analogues.

New Applications of Phospholipid Vesicle-Based Permeation Assay: Permeation Model Mimicking Skin Barrier

The phospholipid vesicle-based permeation assay (PVPA), based on a tight barrier composed of liposomes mimicking cells, is providing an opportunity to predict passive drug permeability through biological membranes. Although it was originally developed to mimic the intestinal epithelia, this study focuses on its potential as a simple and affordable skin model for transdermal permeation of drug candidates and evaluation of various drugs and formulations at an early development stage. The changes induced in lipid composition of the lipid-based barriers to better mimic the in vivo stratum corneum lipid composition required optimization of liposomal properties and manufacturing conditions applied in barrier formation. The preparation conditions could be modified to prepare lipid-based barriers of different degrees of leakiness, potentially representing different degree of intact and compromised skin. The different PVPA models developed in this study appeared to be able to distinguish between drugs with different degrees of lipophilicity and penetration potential. Moreover, the PVPA can be produced in controlled and reproducible manner with different degree of leakiness. The model could therefore be applied in both pharmaceutical and cosmeceuticals manufacturing and also has the potential to provide deeper insight on safety of nanodelivery systems administered onto the skin.