Profiling antimicrobial peptides from the medical maggot Lucilia sericata as potential antibiotics for MDR Gram-negative bacteria.

Background: The ability of MDR Gram-negative bacteria to evade even antibiotics of last resort is a severe global challenge. The development pipeline for conventional antibiotics cannot address this issue, but antimicrobial peptides (AMPs) offer an alternative solution. Objectives: Two insect-derived AMPs (LS-sarcotoxin and LS-stomoxyn) were profiled to assess their suitability for systemic application in humans. Methods: The peptides were tested against an extended panel of 114 clinical MDR Gram-negative bacterial isolates followed by time-kill analysis, interaction studies and assays to determine the likelihood of emerging resistance. In further in vitro studies we addressed cytotoxicity, cardiotoxicity and off-target interactions. In addition, an in vivo tolerability and pharmacokinetic study in mice was performed. Results: LS-sarcotoxin and LS-stomoxyn showed potent and selective activity against Gram-negative bacteria and no cross-resistance with carbapenems, fluoroquinolones or aminoglycosides. Peptide concentrations of 4 or 8 mg/L inhibited 90% of the clinical MDR isolates of Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii and Salmonella enterica isolates tested. The 'all-d' homologues of the peptides displayed markedly reduced activity, indicating a chiral target. Pharmacological profiling revealed a good in vitro therapeutic index, no cytotoxicity or cardiotoxicity, an inconspicuous broad-panel off-target profile, and no acute toxicity in mice at 10 mg/kg. In mouse pharmacokinetic experiments LS-sarcotoxin and LS-stomoxyn plasma levels above the lower limit of quantification (1 and 0.25 mg/mL, respectively) were detected after 5 and 15 min, respectively. Conclusions: LS-sarcotoxin and LS-stomoxyn are suitable as lead candidates for the development of novel antibiotics; however, their pharmacokinetic properties need to be improved for systemic administration.

Biological Profiling of Coleoptericins and Coleoptericin-Like Antimicrobial Peptides from the Invasive Harlequin Ladybird Harmonia axyridis.

The spread of antibiotic-resistant human pathogens and the declining number of novel antibiotics in the development pipeline is a global challenge that has fueled the demand for alternative options. The search for novel drug candidates has expanded to include not only antibiotics but also adjuvants capable of restoring antibiotic susceptibility in multidrug-resistant (MDR) pathogens. Insect-derived antimicrobial peptides (AMPs) can potentially fulfil both of these functions. We tested two coleoptericins and one coleoptericin-like peptides from the invasive harlequin ladybird Harmonia axyridis against a panel of human pathogens. The AMPs displayed little or no activity when tested alone but were active even against clinical MDR isolates of the Gram-negative ESKAPE strains when tested in combination with polymyxin derivatives, such as the reserve antibiotic colistin, at levels below the minimal inhibitory concentration. Assuming intracellular targets of the AMPs, our data indicate that colistin potentiates the activity of the AMPs. All three AMPs achieved good in vitro therapeutic indices and high intrahepatic stability but low plasma stability, suggesting they could be developed as adjuvants for topical delivery or administration by inhalation for anti-infective therapy to reduce the necessary dose of colistin (and thus its side effects) or to prevent development of colistin resistance in MDR pathogens.

Novel nicotinamide analog as inhibitor of nicotinamide N-methyltransferase.

Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by ∼80% at 2 h when dosed in mice orally at 50 mg/kg.

Biorelevant subcutaneous in vitro test predicts the release of human and fast acting insulin formulations.

The administration of insulins by subcutaneous injection is nowadays widely prevalent. The injection site is located below the dermis and composed of cells and the extracellular matrix formed of a network of macromolecules such as hyaluronic acid and collagen. Following an injection, the insulins from the formulated products are timely released as drug molecules from the injection site into systemic circulation. In this publication, we show the development of an in vitro setup utilizing a hydrogel composed of a special collagen-hyaluronic acid mixture that mimics the extracellular matrix. Another setup was used for differentiation of the commercially available and research insulin formulations by determining the in vitro permeation characteristics with the results that were correlated with the human in vivo data. Significant differentiation was achieved at 90 % confidence level between the permeation curves of insulin glulisine containing formulations (U100 and a concentrated research formulation), while in case of the insulin lispro containing formulations (U100 and U200) the permeation curves showed similarity. These results demonstrated that the in vitro setup may be used as a tool for formulation development and drug candidate profiling as it is able to differentiate or show similarities between the agglomeration states and concentration of the active pharmaceutical ingredients.

Full Profiling of GE81112A, an Underexplored Tetrapeptide Antibiotic with Activity against Gram-Negative Pathogens.

After the first total synthesis combined with structure revision, we performed thorough in vitro and in vivo profiling of the underexplored tetrapeptide GE81112A. From the determination of the biological activity spectrum and physicochemical and early absorption-distribution-metabolism-excretion-toxicity (eADMET) properties, as well as in vivo data regarding tolerability and pharmacokinetics (PK) in mice and efficacy in an Escherichia coli-induced septicemia model, we were able to identify the critical and limiting parameters of the original hit compound. Thus, the generated data will serve as the basis for further compound optimization programs and developability assessments to identify candidates for preclinical/clinical development derived from GE81112A as the lead structure. IMPORTANCE The spread of antimicrobial resistance (AMR) is becoming a more and more important global threat to human health. With regard to current medical needs, penetration into the site of infection represents the major challenge in the treatment of infections caused by Gram-positive bacteria. Considering infections associated with Gram-negative bacteria, resistance is a major issue. Obviously, novel scaffolds for the design of new antibacterials in this arena are urgently needed to overcome this crisis. Such a novel potential lead structure is represented by the GE81112 compounds, which inhibit protein synthesis by interacting with the small 30S ribosomal subunit using a binding site distinct from that of other known ribosome-targeting antibiotics. Therefore, the tetrapeptide antibiotic GE81112A was chosen for further exploration as a potential lead for the development of antibiotics with a new mode of action against Gram-negative bacteria.

Antimicrobial Peptides from Rat-Tailed Maggots of the Drone Fly Eristalis tenax Show Potent Activity against Multidrug-Resistant Gram-Negative Bacteria.

The spread of multidrug-resistant Gram-negative bacteria is an increasing threat to human health, because novel compound classes for the development of antibiotics have not been discovered for decades. Antimicrobial peptides (AMPs) may provide a much-needed breakthrough because these immunity-related defense molecules protect many eukaryotes against Gram-negative pathogens. Recent concepts in evolutionary immunology predict the presence of potent AMPs in insects that have adapted to survive in habitats with extreme microbial contamination. For example, the saprophagous and coprophagous maggots of the drone fly Eristalis tenax (Diptera) can flourish in polluted aquatic habitats, such as sewage tanks and farmyard liquid manure storage pits. We used next-generation sequencing to screen the E. tenax immunity-related transcriptome for AMPs that are synthesized in response to the injection of bacterial lipopolysaccharide. We identified 22 AMPs and selected nine for larger-scale synthesis to test their activity against a broad spectrum of pathogens, including multidrug-resistant Gram-negative bacteria. Two cecropin-like peptides (EtCec1-a and EtCec2-a) and a diptericin-like peptide (EtDip) displayed strong activity against the pathogens, even under simulated physiological conditions, and also achieved a good therapeutic window. Therefore, these AMPs could be used as leads for the development of novel antibiotics.

IMI - oral biopharmaceutics tools project - evaluation of bottom-up PBPK prediction success part 1: Characterisation of the OrBiTo database of compounds.

Predicting oral bioavailability (Foral) is of importance for estimating systemic exposure of orally administered drugs. Physiologically-based pharmacokinetic (PBPK) modelling and simulation have been applied extensively in biopharmaceutics recently. The Oral Biopharmaceutical Tools (OrBiTo) project (Innovative Medicines Initiative) aims to develop and improve upon biopharmaceutical tools, including PBPK absorption models. A large-scale evaluation of PBPK models may be considered the first step. Here we characterise the OrBiTo active pharmaceutical ingredient (API) database for use in a large-scale simulation study. The OrBiTo database comprised 83 APIs and 1475 study arms. The database displayed a median logP of 3.60 (2.40-4.58), human blood-to-plasma ratio of 0.62 (0.57-0.71), and fraction unbound in plasma of 0.05 (0.01-0.17). The database mainly consisted of basic compounds (48.19%) and Biopharmaceutics Classification System class II compounds (55.81%). Median human intravenous clearance was 16.9L/h (interquartile range: 11.6-43.6L/h; n=23), volume of distribution was 80.8L (54.5-239L; n=23). The majority of oral formulations were immediate release (IR: 87.6%). Human Foral displayed a median of 0.415 (0.203-0.724; n=22) for IR formulations. The OrBiTo database was found to be largely representative of previously published datasets. 43 of the APIs were found to satisfy the minimum inclusion criteria for the simulation exercise, and many of these have significant gaps of other key parameters, which could potentially impact the interpretability of the simulation outcome. However, the OrBiTo simulation exercise represents a unique opportunity to perform a large-scale evaluation of the PBPK approach to predicting oral biopharmaceutics.