The rapid emergence of antibiotic-resistant microbes worldwide and the urgent need of new antimicrobial agents have stimulated interest in antimicrobial peptides (AMPs) as new therapeutics for treatment of infectious diseases. AMPs are present in all living species and constitute an important part of the innate immune system in multicellular organisms, including humans. AMPs display a remarkably broad spectrum of antimicrobial activity covering both Gram-positive and Gram-negative bacteria, including many antibiotic-resistant strains, as well as fungi, viruses, and protozoa. Further, in contrast to many conventional antibiotics, AMPs rapidly kill bacteria instead of just inhibiting bacterial growth. In addition, AMPs act as modulators of the innate immune system and, importantly, bacteria seem less efficient in developing resistance towards AMPs than towards conventional antibiotics. Together these properties make AMPs highly attractive as a new class of antimicrobials, with clinical potential also extending to diseases where inflammation is part of the pathology. The aim of this thesis was to study novel AMPs with respect to their mechanism of action (MOA), antimicrobial spectrum, propensity to select for resistance, and in vivo efficacy and safety. To achieve this, we used a number of in vitro and in vivo assays, together generating a comprehensive preclinical evaluation of the peptides. The hypothesis was that the AMPs in this thesis have potential to be developed as therapeutic agents for several infectious and inflammatory conditions, including treatment of skin and soft tissue infections and prevention of postsurgical adhesion formation. The results showed that all AMPs tested (i.e. PXL03, PXL150, HLR1r, and five variants of CEN1 HC-Br) had broad antimicrobial spectra in vitro with varying sensitivity to salt and serum. Furthermore, PXL150 caused a rapid permeabilization of bacterial membrane in vitro, indicating that this is at least one part of the MOA of this peptide. Under selection pressure in vitro, bacteria did not develop resistance to the peptides tested, i.e. PXL150 and CEN1 HC. Interestingly, all peptides showed anti-inflammatory activity by inhibiting the secretion of proinflammatory mediators from stimulated human cell lines. In addition, PXL01, PXL150, and HLR1r demonstrated fibrinolytic ability in vitro by suppressing the release of plasminogen activator inhibitor-1 (PAI-1). In ex vivo and in vivo skin/wound infection models, the peptides reduced the number of viable bacteria and yeast cells. Further, PXL01 decreased postsurgical adhesion formation in vivo. Notably, nonclinical safety studies showed that PXL150 was safe and well tolerated. In conclusion, several of the peptides evaluated in this thesis demonstrated a promising preclinical efficacy and safety profile motivating further development as drug candidates for local treatment of infectious and inflammatory conditions.

In the past 15 years, close to 1000 of new psychoactive substances (NPS) have been reported in Europe and globally. At the time of identification, data on safety, toxicity and carcinogenic potential of many NPS are not available or very limited. To work more efficiently, a strategy and collaboration between the Public Health Agency of Sweden (PHAS) and the National Board of Forensic Medicine was established involving in vitro receptor activity assays to demonstrate neurological activity of NPS. This report summarizes the first results on the synthetic cannabinoid receptor agonists (SCRAs), and subsequent actions taken by PHAS. A total of 18 potential SCRAs were selected by PHAS for in vitro pharmacological characterization. 17 compounds could be acquired and investigated for their activity on the human cannabinoid-1 (CB1) receptors expressed together with the AequoScreen system in CHO-K1 cells. Dose-response curves were established using eight different concentrations in triplicates at three occasions with JWH-018 as reference. For the MDMB-4en-PINACA, MMB-022, ACHMINACA, ADB-BUTINACA, 5F-CUMYL-PeGACLONE, 5C-AKB48, NM-2201, 5F-CUMYL-PINACA, JWH-022, 5Cl-AB-PINACA, MPhP-2201, 5F-AKB57 the half maximal effective concentration values ranged from 2.2 nM (5F-CUMYL-PINACA) to 171 nM (MMB-022). EG-018 and 3,5-AB-CHMFUPPYCA were none-active. The results contributed to 14 of these compounds being scheduled as narcotics in Sweden. In conclusion, many of the emerging SCRAs are potent activators of the CB1 receptor in vitro, although some lack activity or are partial agonists. The new strategy proved useful when data on psychoactive effects of the SCRAs under investigation were not available or limited. © 2023 The Authors

There is a strong need for innovative and efficient drug delivery systems for ocular therapy development. However, testing intravitreal drug delivery systems without using live animals is challenging. Ex vivo animal models offer an interesting alternative. We analyzed the potential of using fresh porcine eyes obtained from the local slaughterhouse as a model for testing the intravitreal biodistribution and retention of liposomes with or without polyethylene glycol (PEG) conjugation and with different surface charges. The histology of the eyes was analyzed to localize the liposomes, and it was found that liposomes with PEG absorbed rapidly on the retina (within 1 h), with positively charged and PEG-coated liposomes being retained for at least 24 h. In parallel, fluorophotometry was employed on intact eyes, to determine the pharmacokinetics of the fluorophore calcein, as a substitute for a small hydrophilic therapeutic compound. We found a 4.5-fold increase in the vitreous half-life of calcein loaded in liposomes, compared with the free solution. Retinal toxicity was addressed using murine-derived retinal explant cultures. Liposomes were non-toxic up to 500 µg/mL. Toxicity was observed at 5 mg/mL for anionic and cationic liposomes, with 2-fold and 2.5-fold increased photoreceptor cell death, respectively. Overall, we could show that important ocular drug delivery considerations such as pharmacokinetics and biodistribution can be estimated in ex vivo porcine eyes, and may guide subsequent in vivo experiments.

Nanomaterials are small and the small size and corresponding large surface area of nanomaterials confers specific properties, making these materials desirable for various applications, not least in medicine. However, it is pertinent to ask whether size is the only property that matters for the desirable or detrimental effects of nanomaterials? Indeed, it is important to know not only what the material looks like, but also what it is made of, as well as how the material interacts with its biological surroundings. It has been suggested that guidelines should be implemented on the types of information required in terms of physicochemical characterization of nanomaterials for toxicological studies in order to improve the quality and relevance of the published results. This is certainly a key issue, but it is important to keep in mind that material characterization should be fit-for-purpose, that is, the information gathered should be relevant for the end-points being studied.

Most metal nanoparticles (NPs), except noble metal NPs, rapidly form a thin surface oxide in ambient conditions. The protective properties of these oxides improve or worsen depending on the environment, e.g., the human lung. Several properties, including the chemical/electrochemical stability and defect density, determine the capacity of these surface oxides to hinder the bulk metal from further oxidation (corrosion). The aim of this study was to investigate whether electrochemical surface oxide characterization of non-functionalized base metal NPs of different characteristics (Al, Mn and Cu) can assist in understanding their bioaccessibility (metal release) in cell media (DMEM+) and their cytotoxic properties following exposure in lung epithelial (A549) cells. The composition and valence states of surface oxides of metal NPs and their electrochemical activity were investigated using an electrochemical technique based on a graphite paste electrode to perform cyclic voltammetry in buffer solutions and open circuit potential measurements in DMEM+. The electrochemical surface oxide characterization was complemented and verified by Raman spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The open circuit potential trends in DMEM+ correlated well with metal release results in the same solution, and provided information on the kinetics of oxide dissolution in the case of Cu NPs. Extensive particle agglomeration in cell medium (DMEM+) was observed by means of photon-cross correlation spectroscopy for all metal NPs, with sedimentation taking place very quickly. As a consequence, measurements of the real dose of added non-functionalized metal NPs to cell cultures for cytotoxicity testing from a sonicated stock solution were shown necessary. The cytotoxic response was found to be strongly correlated to changes in physico-chemical and electrochemical properties of the surface oxides of the metal NPs, the most potent being Cu NPs, followed by Mn NPs. No cytotoxicity was observed for Al NPs. The electrochemical surface oxide characterization corresponded well with other tools commonly used for nanotoxicological characterization and provided additional information.

Herein, we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMC) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze-drying and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wound was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG+hPBMC as compared to untreated and hyaluronic acid treated controls (p<0.05). Addition of the hPBMC to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG+hPBMC were completely closed as compared to controls. Thus, the gel facilitated generation of new skin with well arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.

The marine secondary metabolite stryphnusin (1) was isolated from the boreal sponge Stryphnus fortis, collected off the Norwegian coast. Given its resemblance to other natural acetylcholinesterase antagonists, it was evaluated against electric eel acetylcholinesterase and displayed inhibitory activity. A library of twelve synthetic phenethylamine analogs, 2a-7a and 2b-7b, containing tertiary and quaternary amines respectively were synthesized to investigate the individual structural contributions to the activity. Compound 7b was the strongest competitive inhibitor of both acetylcholinesterase and butyrylcholinesterase with IC50 values of 57 and 20 μM, respectively. This inhibitory activity is one order of magnitude higher than the positive control physostigmine, and is comparable with several other marine acetylcholinesterase inhibitors. The physiological effect of compound 7b on muscle function and neuromuscular transmission was studied and revealed a selective mode of action at the investigated concentration. This data is of importance as the interference of therapeutic acetylcholinesterase inhibitors with neuromuscular transmission can be problematic and lead to unwanted side effects. The current findings also provide additional insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

Alternative ways to prevent and treat infectious diseases are needed. Previously, we identified a fungal peptide, NZX, that was comparable to rifampicin in lowering M. tuberculosis load in a murine tuberculosis (TB) infection model. Here we assessed the potential synergy between this cationic host defence peptide (CHDP) and the current TB drugs and analysed its pharmacokinetics. We found additive effect of this peptide with isoniazid and ethambutol and confirmed these results with ethambutol in a murine TB-model. In vivo, the peptide remained stable in circulation and preserved lung structure better than ethambutol alone. Antibiotic resistance studies did not induce mutants with reduced susceptibility to the peptide. We further observed that this peptide was effective against nontuberculous mycobacteria (NTM), such as M. avium and M. abscessus, and several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In conclusion, the presented data supports a role for this CHDP in the treatment of drug resistant organisms.

Systems for studying the toxicity of metal aggregates on the airways are normally not suited for evaluating the effects of individual particle characteristics. This study validates a set-up for toxicological studies of metal aggregates using an air–liquid interface approach. The set-up used a spark discharge generator capable of generating aerosol metal aggregate particles and sintered near spheres. The set-up also contained an exposure chamber, The Nano Aerosol Chamber for In Vitro Toxicity (NACIVT). The system facilitates online characterization capabilities of mass mobility, mass concentration, and number size distribution to determine the exposure. By dilution, the desired exposure level was controlled. Primary and cancerous airway cells were exposed to copper (Cu), palladium (Pd), and silver (Ag) aggregates, 50–150 nm in median diameter. The aggregates were composed of primary particles &lt;10 nm in diameter. For Cu and Pd, an exposure of sintered aerosol particles was also produced. The doses of the particles were expressed as particle numbers, masses, and surface areas. For the Cu, Pd, and Ag aerosol particles, a range of mass surface concentrations on the air–liquid interface of 0.4–10.7, 0.9–46.6, and 0.1–1.4 µg/cm2, respectively, were achieved. Viability was measured by WST-1 assay, cytokines (Il-6, Il-8, TNF-a, MCP) by Luminex technology. Statistically significant effects and dose response on cytokine expression were observed for SAEC cells after exposure to Cu, Pd, or Ag particles. Also, a positive dose response was observed for SAEC viability after Cu exposure. For A549 cells, statistically significant effects on viability were observed after exposure to Cu and Pd particles. The set-up produced a stable flow of aerosol particles with an exposure and dose expressed in terms of number, mass, and surface area. Exposure-related effects on the airway cellular models could be asserted.