Baseline health parameters of rhinoceros auklets (Cerorhinca monocerata) using serum protein electrophoresis, acute phase proteins, and biochemistry.

Clinical metrics of baseline health in sentinel seabird species can offer insight into marine ecosystem dynamics, individual and population health, and assist in wildlife rehabilitation and conservation efforts. Protein electrophoresis is useful for detecting changes in acute phase proteins and immunoglobulin levels that may indicate subtle inflammatory responses and/or infectious disease. Serum biochemistry can highlight nutritional status, metabolic derangements, and organ injury and function. However, baseline values for such health parameters are largely unknown for many seabird species. Therefore, the objective of this study is to establish baseline clinical health reference intervals for serum protein electrophoresis, acute phase proteins including serum amyloid A and haptoglobin, and biochemistry parameters in the rhinoceros auklet (Cerorhinca monocerata), a key sentinel species in the North Pacific. From 2013 to 2019, 178 wild, apparently healthy breeding adult rhinoceros auklets were captured across four breeding colonies in British Columbia, Canada (Lucy Island, Pine Island, Triangle Islands, and SGang Gwaay) and from one colony in Washington, United States (Protection Island). Reference intervals were calculated for protein electrophoresis fractions and acute phase proteins (n = 163), and serum biochemistry (n = 35) following established guidelines by the American Society of Veterinary Clinical Pathology. Animals were also assessed for the presence of antibodies to the influenza A virus. Approximately 48% (70/147) of sampled birds were seropositive for influenza A virus, with a prevalence of 50% (6/12) in 2013, 75% (47/63) in 2014, and 24% (17/72) in 2019. This work provides clinical baseline health metrics of a key North Pacific sentinel species to help inform marine ecosystem monitoring, recovery, and rehabilitation efforts in the Pacific Northwest.

C5a peptidase (ScpA) activity towards human type II and type III interferons.

C5a peptidase, also known as ScpA, is a surface associated serine protease derived from Streptococcus pyogenes and has been described as an important factor in streptococcus virulence, capable of cleaving complement components C5a, C3 and C3a. Although the interactions of ScpA with complement components is well studied, extensive screening of ScpA activity against other pro-inflammatory cytokines is lacking. Here, ScpA's ability to cleave human pro-inflammatory cytokines was tested, revealing its ability to cleave human IFNγ, IFNλ1, IFNλ2, C5, IL-37 but with significantly reduced activities. The functional consequence of ScpA's cleavage of IFNγ in its signalling through the Jak-Stat pathway has also been evaluated in an in vitro RPE1 cell model. These newly identified targets for ScpA highlight the complexity of streptococcus infections and indeed, the potential for ScpA to have a therapeutic role in the progression of inflammatory diseases involving these cytokines.

Anionic liposome formulation for oral delivery of thuricin CD, a potential antimicrobial peptide therapeutic.

Thuricin CD is a two-peptide antimicrobial produced by Bacillus thuringiensis. Unlike previous antibiotics, it has shown narrow spectrum activity against Clostridioides difficile, a bacterium capable of causing infectious disease in the colon. However, peptide antibiotics have stability, solubility, and permeability problems that can affect their performance in vivo. This work focuses on the bioactivity and bioavailability of thuricin CD with a view to developing a formulation for delivery of active thuricin CD peptides through the gastrointestinal tract (GIT) for local delivery in the colon. The results indicate that thuricin CD is active at low concentrations only when both peptides are present. While thuricin CD was degraded by proteases and was unstable and poorly soluble in gastric fluid, it showed increased solubility in intestinal fluid, probably due to micelle encapsulation. Based on this, thuricin CD was encapsulated in anionic liposomes, which showed increased activity compared to the free peptide, maintained activity after exposure to pepsin in gastric fluid and intestinal fluid, was stable in suspension for over 21 days at room temperature and for 60 days at 4 °C, and exhibited no toxicity to epithelial intestinal cells. These findings suggest that an anionic lipid-based nano formulation may be a promising approach for local oral delivery of thuricin CD.

Pre-formulation of an additive combination of two antimicrobial agents, clofazimine and nisin A, to boost antimicrobial activity.

According to the World Health Organization, antimicrobial resistance is one of the top ten issues that pose a major threat to humanity. The lack of investment by the pharmaceutical industry has meant fewer novel antimicrobial agents are in development, exacerbating the problem. Emerging drug design strategies are exploring the repurposing of existing drugs and the utilization of novel drug candidates, like antimicrobial peptides, to combat drug resistance. This proactive approach is crucial in fighting global health threats. In this study, an additive combination of a repurposed anti-leprosy drug, clofazimine, and an antimicrobial peptide, nisin A, are preformulated using liquid antisolvent precipitation to generate a stable amorphous, ionized nanoparticle system to boost antimicrobial activity. The nanotechnology aims to improve the physicochemical properties of the inherently poorly water-soluble clofazimine molecules while also harnessing the previously unreported additive effect of clofazimine and nisin A. The approach transformed clofazimine into a more water-soluble salt, yielding amorphous nanoparticles stabilized by the antimicrobial peptide; and combined the two drugs into a more soluble and more active formulation. Blending pre-formulation strategies like amorphization, salt formation, and nanosizing to improve the inherent low aqueous solubility of drugs can open many new possibilities for the design of new antimicrobial agents. This fusion of pre-formulation technologies in combination with the multi-hurdle approach of selecting drugs with different effects on microbes could be key in the design platform of new antibiotics in the fight against antimicrobial resistance.

Mesoscale Clusters in the Crystallisation of Organic Molecules.

The early stages of the molecular self-assembly pathway leading to crystal nucleation have a significant influence on the properties and purity of organic materials. This mini review collates the work on organic mesoscale clusters and discusses their importance in nucleation processes, with a particular focus on their critical properties and susceptibility to sample treatment parameters. This is accomplished by a review of detection methods, including dynamic light scattering, nanoparticle tracking analysis, small angle X-ray scattering, and transmission electron microscopy. Considering the challenges associated with crystallisation of flexible and large-molecule active pharmaceutical ingredients, the dynamic nature of mesoscale clusters has the potential to expand the discovery of novel crystal forms. By collating literature on mesoscale clusters for organic molecules, a more comprehensive understanding of their role in nucleation will evolve and can guide further research efforts.

Impact of carrier particle surface properties on drug nanoparticle attachment.

HYPOTHESIS: The stabilization and isolation to dryness of drug nanoparticles has always been a challenge for nano-medicine production. In the past, the use of montmorillonite (MMT) clay carrier particles to adsorb drug nanoparticles and maintain their high surface area to volume ratio after isolation to dryness has proven to be effective. We hypothesise that the distribution of hydrophilic and hydrophobic patches on the clay's surface as well as its porosity/roughness, hinder the agglomeration of the drug nanoparticles to the extent that they retain their high surface area to volume ratio and display fast dissolution profiles. EXPERIMENTS: In this work, the distribution of hydrophobicity and hydrophilicity, and the porosity/roughness, of the surface of selected silica carrier particles were varied and the impact of these variations on drug nanoparticle attachment to the carrier particle and subsequent dissolution profiles was studied. FINDINGS: The fastest dissolution profiles at the highest drug nanoparticle loadings were obtained with a periodic mesoporous organosilane carrier particle which had a homogeneous distribution of hydrophobic and hydrophilic surface properties. Carrier particles with rough/porous surfaces and a combination of hydrophobic and hydrophilic patches resulted in nanocomposite powders with faster dissolution behaviour than carrier particles with predominantly either a hydrophobic or hydrophilic surface, or with non-porous/smoother surfaces.

New Solid Forms of Griseofulvin: A Solvate and a Relict Polymorph Related to Reported Solvates.

This work presents two new solid forms, a polymorph and a solvate, of the antifungal active pharmaceutical ingredient griseofulvin (GSF). The novel forms were characterized by powder X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis, and their crystal structures were determined by single-crystal X-ray diffraction. The new polymorphic form (GSF Form VI) was obtained upon drying at room temperature the GSF-acetonitrile solvate. GSF Form VI is a relict structure related to reported solvates of GSF. Thermal stability studies show that Form VI is metastable and monotropically related to the stable GSF Form I. The new GSF-n-butyl acetate solvate was obtained by crystallization from an n-butyl acetate solution. The stoichiometry of the n-butyl acetate solvate is 1:0.5. The solvate loses the solvent from the crystal lattice at a temperature between 363.15 and 374.15 K.

Impact of Additives on Drug Particles during Liquid Antisolvent Crystallization and Subsequent Freeze-Drying.

The impact of single or combinations of additives on the generation of nanosuspensions of two poorly water-soluble active pharmaceutical ingredients (APIs), fenofibrate (FF) and dalcetrapib (DCP), and their isolation to the dry state via antisolvent (AS) crystallization followed by freeze-drying was explored in this work. Combinations of polymeric and surfactant additives such as poly(vinyl alcohol) or hydroxypropyl methyl cellulose and sodium docusate were required to stabilize nanoparticles (∼200-300 nm) of both APIs in suspension before isolation to dryness. For both FF and DCP, multiple additives generated the narrowest, most-stable particle size distribution, with the smallest particles in suspension, compared with using a single additive. An industrially recognized freeze-drying process was used for the isolation of these nanoparticles to dryness. When processed by the liquid AS crystallization followed by freeze-drying in the presence of multiple additives, a purer monomorphic powder for FF resulted than when processed in the absence of any additive or in the presence of a single additive. It was noted that all nanoparticles freeze-dried in the presence of additives had a flat, flaky habit resulting in large surface areas. Agglomeration occurred during freeze-drying, resulting in micron-size particles. However, after freeze-drying, powders produced with single or multiple additives showed similar dissolution profiles, irrespective of aging time before drying, thus attenuating the advantage of multiple additives in terms of size observed before the freeze-drying process.

Impact of Propeptide Cleavage on the Stability and Activity of a Streptococcal Immunomodulatory C5a Peptidase for Biopharmaceutical Development.

Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group AStreptococcus pyogenesC5a peptidase (ScpA) is a multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group AS. pyogenesknown to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed inEscherichia colicells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.

Identification of monoclonal antibody drug substances using non-destructive Raman spectroscopy.

Monoclonal antibodies provide highly specific and effective therapies for the treatment of chronic diseases. These protein-based therapeutics, or drug substances, are transported in single used plastic packaging to fill finish sites. According to good manufacturing practice guidelines, each drug substance needs to be identified before manufacturing of the drug product. However, considering their complex structure, it is challenging to correctly identify therapeutic proteins in an efficient manner. Common analytical techniques for therapeutic protein identification are SDS-gel electrophoresis, enzyme linked immunosorbent assays, high performance liquid chromatography and mass spectrometry-based assays. Although effective in correctly identifying the protein therapeutic, most of these techniques need extensive sample preparation and removal of samples from their containers. This step not only risks contamination but the sample taken for the identification is destroyed and cannot be re-used. Moreover, these techniques are often time consuming, sometimes taking several days to process. Here, we address these challenges by developing a rapid and non-destructive identification technique for monoclonal antibody-based drug substances. Raman spectroscopy in combination with chemometrics were used to identify three monoclonal antibody drug substances. This study explored the impact of laser exposure, time out of refrigerator and multiple freeze thaw cycles on the stability of monoclonal antibodies. and demonstrated the potential of using Raman spectroscopy for the identification of protein-based drug substances in the biopharmaceutical industry.

The development of a solid lipid nanoparticle (SLN)-based lacticin 3147 hydrogel for the treatment of wound infections.

Chronic wounds affect millions of people globally. This number is set to rise with the increasing incidence of antimicrobial-resistant bacterial infections, such as methicillin-resistant Staphylococcus aureus (MRSA), which impair the healing of chronic wounds. Lacticin 3147 is a two-peptide chain bacteriocin produced by Lactococcus lactis that is active against S. aureus including MRSA strains. Previously, poor physicochemical properties of the peptides were overcome by the encapsulation of lacticin 3147 into solid lipid nanoparticles. Here, a lacticin 3147 solid lipid nanoparticle gel is proposed as a topical treatment for S. aureus and MRSA wound infections. Initially, lacticin 3147's antimicrobial activity against S. aureus was determined before encapsulation into solid lipid nanoparticles. An optimised gel formulation with the desired physicochemical properties for topical application was developed, and the lacticin-loaded solid lipid nanoparticles and free lacticin 3147 aqueous solution were incorporated into separate gels. The release of lacticin 3147 from both the solid lipid nanoparticle and free lacticin gels was measured where the solid lipid nanoparticle gel exhibited increased activity for a longer period (11 days) compared to the free lacticin gel (9 days). Both gels displayed potent activity ex vivo against S. aureus-infected pig skin with significant bacterial eradication (> 75%) after 1 h. Thus, a long-acting potent lacticin 3147 solid lipid nanoparticle gel with the required physicochemical properties for topical delivery of lacticin 3147 to the skin for the potential treatment of S. aureus-infected chronic wounds was developed.

Osmolality of Excipients for Parenteral Formulation Measured by Freezing Point Depression and Vapor Pressure - A Comparative Analysis.

PURPOSE: To investigate the difference in methods to determine the osmolality in solutions of stabilizers used for long-acting injectable suspensions. METHODS: The osmolality was measured by freezing point depression and vapor pressure for 11 different polymers and surfactants (PEG 3350, 4000, 6000, 8000, 20,000, PVP K12, K17 and K30, poloxamer 188, 388 and 407, HPMC E5, Na-CMC, polysorbate 20 and 80, vitamin E-TPGS, phospholipid, DOSS and SDS) in different concentrations. RESULTS: Independently of the measuring method, an increase in osmolality with increasing concentration was observed for all polymers and surfactants, as would be expected due to the physicochemical origin of the osmolality. No correlation was found between the molecular weight of the polymers and the measured osmolality. The osmolality values were different for PVPs, PEGs, and Na-CMC using the two different measurement methods. The values obtained by the freezing point depression method tended to be similar or higher than the ones provided by vapor pressure, overall showing a significant difference in the osmolality measured by the two investigated methods. CONCLUSIONS: For lower osmolality values (e.g. surfactants), the choice of the measuring method was not critical, both the freezing point depression and vapor pressure could be used. However, when the formulations contained higher concentrations of excipients and/or thermosensitive excipients, the data suggests that the vapor pressure method would be more suited.

The impact of varying dextran oxidation levels on the inhibitory activity of a bacteriocin loaded injectable hydrogel.

In the design of injectable antimicrobial dextran-alginate hydrogels, the impact of dextran oxidation and its subsequent changes in molecular weight and the incorporation of glycol chitosan on (i) gel mechanical strength and (ii) the inhibitory profile of an encapsulated bacteriocin, nisin A, are explored. As the degree of oxidation increases, the weight average molecular mass of the dextran decreases, resulting in a reduction in elastic modulus of the gels made. Upon encapsulation of the bacteriocin nisin into the gels, varying the dextran mass/oxidation level allowed the antimicrobial activity against S. aureus to be controlled. Gels made with a higher molecular weight (less oxidised) dextran show a higher initial degree of inhibition while those made with a lower molecular weight (more oxidised) dextran exhibit a more sustained inhibition. Incorporating glycol chitosan into gels composed of dextran with higher masses significantly increased their storage modulus and the gels' initial degree of inhibition.

Impact of Excipients and Seeding on the Solid-State Form Transformation of Indomethacin during Liquid Antisolvent Precipitation.

Long-acting injectables are a unique drug formulation strategy, providing a slow and sustained release of active pharmaceutical ingredients (APIs). In this study, a novel approach that combines liquid antisolvent precipitation with seeding to obtain a stable form of the API indomethacin while achieving the desired particle size distribution is described. It was proven that when a metastable form of indomethacin was initially nucleated, the rate of its transformation to the stable form was influenced by the presence of excipients and seeds (17.10 ± 0.20 µm), decreasing from 48 to 4 h. The final particle size (D50) of the indomethacin suspension produced without seeding was 7.33 ± 0.38 µm, and with seeding, it was 5.61 ± 0.14 µm. Additionally, it was shown that the particle size distribution of the seeds and the time point of seed addition were critical to obtain the desired solid-state form and that excipients played a crucial role during nucleation and polymorphic transformation. This alternative, energy-efficient bottom-up method for the production of drug suspensions with a reduced risk of contamination from milling equipment and fewer processing steps may prove to be comparable in terms of stability and particle size distribution to current industrially accepted top-down approaches.

Long acting injectables for therapeutic proteins.

Biotherapeutic development presents a myriad of challenges in relation to delivery, in particular for protein therapeutics. Protein delivery is complicated due to hydrophilicity, size, rate of degradation in vivo, low permeation through biological barriers, pH and temperature sensitivity, as well as the need to conserve its quaternary structure to retain function. To preserve therapeutic levels in vivo, proteins require frequent administration due to their short half-lives. Formulation strategies combining proteins with lipid carriers for parenteral administration show potential for improving bioavailability, while preserving protein activity and bypassing the mucosal barriers of the body. Encapsulating protein in long acting injectable delivery systems can improve therapeutic indices by prolonging and controlling protein release and reducing the need for repeat interventions. Two lyotropic crystal forming lipids, monoolein and phytantriol, have been formulated to produce lipidic cubic phases and assessed for their use as long acting protein eluting injectables. Three soluble proteins, cytochrome c, glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase and one membrane protein, cytochrome c oxidase, were incorporated into bulk cubic phase formulations of each lipid system to comparatively assess protein release kinetics. The activity of the soluble proteins was measured upon release from a phytantriol bulk cubic phase and phytantriol cubosomes, produced using a liquid precursor method.

Single versus double occupancy solid lipid nanoparticles for delivery of the dual-acting bacteriocin, lacticin 3147.

The bacteriocin lacticin 3147 (lacticin) has shown activity against clinically relevant and antimicrobial-resistant bacteria such as Listeria monocytogenes and Clostridioides difficile. It is composed of two peptides, Ltnα and Ltnß, which work together to form pores in the membrane of Gram-positive bacteria. Lacticin possesses poor aqueous solubility and is degraded by intestinal proteases. In a previous study, peptides encapsulated into solid lipid nanoparticles (SLNs) displayed activity in aqueous media and were protected from enzyme degradation but showed a low encapsulation efficiency (EE%) for Ltnα. In this study, however, lacticin was encapsulated into SLNs both individually (single occupancy, SLNα + SLNß) and together (double occupancy SLNαß) via a nanoprecipitation technique. This achieved SLNs of uniform size with an EE% above 87% for both peptides at loadings of 9 or 18 mg/g of lipid under single occupancy or double occupancy respectively. SLNαß dispersions displayed more potent activity at 3.13 and 1.56 µg/ml lacticin than SLNα + SLNß dispersions. Thus, the SLNαß dispersion was chosen for further analysis. SLNαß dispersions showed no cytotoxicity to endothelial cells. The SLN release media (fasted state simulated intestinal fluid; FaSSIF) retained activity at 1 h and 3 h indicating that lacticin may be sufficiently protected from proteases present in the duodenum. Finally, a reconstituted freeze-dried SLNαß dispersion was stable and achieved 99.99% bacterial killing at 3.125 µg/ml lacticin. Thus, an SLN based lacticin delivery system was developed, potentially enabling oral administration of the bacteriocin to the colon to treat local infections such as C. difficile.

The influence of poly(allylamine hydrochloride) hydrogel crosslinking density on its thermal and phosphate binding properties.

Sevelamer hydrochloride (SH) or Renagel® is an effective phosphate binder prescribed to prevent the absorption of phosphate in end stage renal disease (ESRD) patients. The relationship between SH structure and binding capacity and affinity is very important and can be used in characterising the sensitivity of the hydrogel to binding conditions. Thus, a series of hydrogels were prepared by varying the amount of crosslinker, whilst the other hydrogel components were kept constant. Variation of this parameter influenced the hydrogel structure as shown by swelling data, differential scanning calorimetry and solid state nuclear magnetic resonance spectroscopy. The hydrogels' physical characteristics were found to correlate with the number of phosphate binding sites and affinity obtained from the Langmuir-Freundlich Isotherm (L-FI) and affinity distribution spectra (AD). The hydrogels formed using lower amounts of crosslinker showed a slight increase in binding capacity but with lower affinity. However, the influence of the pH of the binding media on the binding parameters of sevelamer hydrochloride was significant. This is the first report on the use of AD spectra generated from L-FI binding parameters in hydrogels, which demonstrates the sensitivity of the affinity and binding site numbers to changes in hydrogel physical properties and the pH of the binding media.

Bird protection treatments reduce bird-window collision risk at low-rise buildings within a Pacific coastal protected area.

Background: In North America, up to one billion birds are estimated to die annually due to collisions with glass. The transparent and reflective properties of glass present the illusion of a clear flight passage or continuous habitat. Approaches to reducing collision risk involve installing visual cues on glass that enable birds to perceive glass as a solid hazard at a sufficient distance to avoid it. Methods: We monitored for bird-window collisions between 2013 and 2018 to measure response to bird protection window treatments at two low-rise buildings at the Alaksen National Wildlife Area in Delta, British Columbia, Canada. After 2 years of collision monitoring in an untreated state, we retrofitted one building with Feather Friendly® circular adhesive markers applied in a grid pattern across all windows, enabling a field-based assessment of the relative reduction in collisions in the 2 years of monitoring following treatment. An adjacent building that had been constructed with a bird protective UV-treated glass called ORNILUX® Mikado, was monitored throughout the two study periods. Carcass persistence trials were conducted to evaluate the likelihood that carcasses were missed due to carcass removal between scheduled searches. Results and Conclusions: After accounting for differences in area of glass between the two buildings, year, and observer effects, our best-fit model for explaining collision risk included the building's treatment group, when compared to models that included building and season only. We found that the Feather Friendly® markers reduced collision risk at the retrofitted building by 95%. Collision incidence was also lower at the two monitored façades of the building with ORNILUX® glass compared to the building with untreated glass. Although more research is needed on the effectiveness of bird-protection products across a range of conditions, our results highlight the benefit of these products for reducing avian mortality due to collisions with glass.