Analysis of enzyme activity, antibacterial activity, antiparasitic activity and physico-chemical stability of skin mucus derived from Amphiprion clarkii.

Recently, mucosal surfaces, especially fish skin and its secreted mucus, have attracted significant interest from immunologists. Amphiprion clarkii, a member of the family Pomacentridae, lives symbiosis with sea anemones and has a good resistance to common seawater bacterial diseases and parasites owing to the protection from its abundant skin mucus. In the present work, the activity of immune-related enzymes (lysozyme, protease, antiprotease, cathepsin B, alkaline phosphatase and peroxidase), the antibacterial activity against two Gram-positive bacteria and five Gram-negative bacteria, the antiparasitic activity against the pathogen of marine white spot disease (Cryptocaryon irritans theronts) and the physico-chemical stability (to pH and heat) of the skin mucus of A. clarkii were analysed. The results showed that the levels of lysozyme and peroxidase were very similar (from 2 to 4 U mg-1 protein). However, cathepsin B was detected of 63.32 U mg-1 protein and alkaline phosphatase was only 0.12 U mg-1 protein. Moreover, protease showed a higher percentage of activity than antiprotease. A. clarkii skin mucus showed a strong antibacterial activity against Gram-negative bacteria, particularly against Aeromonas hydrophila and Vibrio parahaemolyticus but showed no effect on Gram-positive bacteria at the tested concentrations. The bactericidal activity functioned within a short time in a distinct time- and dose-dependent manner. SEM showed that after treated with A. clarkii skin mucus, the V. parahaemolyticus cells distorted and piled together, and the filaments appeared and became into cotton-shaped or quasi-honeycomb texture to adhere cells. Meanwhile, A. clarkii skin mucus showed an apparent antiparasitic activity against C. irritans theronts with a distinct dose- and time-dependent relationship. LM and SEM observation showed that after treated with skin mucus, the theronts quickly stopped their swimming and cilia movement, cells became rounded, cilia shed, small bubbles formed on the surface, cell nucleolus enlarged, cytoskeleton deformed, cell membranes ruptured and cell content leaked out. Antibacterial activity was not affected by 30-90 °C heat treatment but was slightly suppressed by 100 °C. In the pH treatment groups, antibacterial activity was not affected by the moderate pH treatment of 5.0-8.0, but slightly suppressed by weak acid and weak base. Therefore, we speculated that the skin mucus of A. clarkii might be a potential source of novel antibacterial and antiparasitic components for fish or human health-related applications. This study broadened our understanding of the role of skin mucus in the innate immune system and provided a basis for the further isolation and purification of active substances.

Development of a novel physico-chemically and microbiologically stable oral solution of flecainide for pediatrics.

There is as yet no commercialized preparation for oral administration of flecainide acetate (FA) to children. In such cases, manipulation of commercial tablets is the usual practice in pharmacy services of hospitals and compounding pharmacies, to provide a suitable dosage form for this vulnerable pediatric population group. In this study, we have formulated FA as an oral solution, as an alternative to the suspension elaborated from commercial tablets. Due to this sensitivity of young patients, we have used the pure active pharmaceutical ingredient (API) and the lowest permitted levels of pediatric excipients. Despite being a highly soluble API, only one of the formulations appears as a transparent solution due to complete FA solubilization. The proposed formulation is physico-chemically and microbiologically stable and the mass and dose uniformity is appropriate for 30 days' storage at 25 °C.

Formulation design of oral pediatric Acetazolamide suspension: dose uniformity and physico-chemical stability study.

CONTEXT: The formulation of an active pharmaceutical ingredient (API) as oral solution or suspension in pediatrics is a habitual practice, due to the non-existence of many commercialized medicines in pediatric doses. It is also the simplest way to prepare and administer them to this vulnerable population. The design of a formulation that assures the dose and the system stability depends on the physico-chemical properties of the API. OBJECTIVE: In this study, we formulate a class IV API, Acetazolamide (AZM) as suspension for oral administration to pediatric population. The suspension must comply attributes of quality, safety and efficacy for this route of administration. MATERIALS AND METHODS: We use simple compounding procedures, as well as fewer pure excipients, as recommended for children. Mass and uniformity content assays and physical and chemical stability studies were performed. To quantify the API an UPLC method was used. RESULTS AND DISCUSSION: We verified the physico-chemical stability of the suspensions and that they passed the mass test of the European Pharmacopeia (EP), but not the dose uniformity test. CONCLUSIONS: This reveals that AZM must be formulated as liquid forms with a more complex system of excipients (not usually indicated in pediatrics), or otherwise solid forms capable of assuring uniformity of mass and dose for every dosage unit.

Physico-chemical stability of eribulin mesylate containing concentrate and ready-to-administer solutions.

OBJECTIVES: The aim of this study was to determine the stability of commercially available eribulin mesylate containing injection solution as well as diluted ready-to-administer solutions stored under refrigeration or at room temperature. METHODS: Stability was studied by a novel developed stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) assay with ultraviolet detection (detection wavelength 200 nm). Triplicate test solutions of eribulin mesylate containing injection concentrate (0.5 mg/mL) and with 0.9% sodium chloride solution diluted ready-to-administer preparations (0.205 mg/mL eribulin mesylate in polypropylene (PP) syringes, 0.020 mg/mL eribulin mesylate in polypropylene/polyethylene (PE) bags) were stored protected from light either at room temperature (25) or under refrigeration (2-8). Samples were withdrawn on day 0 (initial), 1, 3, 5, 7, 14, 21 and 28 of storage and assayed. Physical stability was determined by measuring the pH value once a week and checking for visible precipitations or colour changes. RESULTS: The stability tests revealed that concentrations of eribulin mesylate remained unchanged over a period of 28 days irrespective of concentration, container material or storage temperature. Neither colour changes nor visible particles have been observed. The pH value varied slightly over time but remained in the stability favourable range of 5-9. CONCLUSION: Eribulin mesylate injection (0.5 mg/mL) is physico-chemically stable over a period of 28 days after first puncture of the vial. After dilution with 0.9% NaCl vehicle solution, ready-to-administer eribulin mesylate injection solutions (0.205 mg/mL in PP syringe) and infusion solutions (0.02 mg/mL in prefilled PP/PE bags) are physico-chemically stable for a period of at least four weeks either refrigerated or stored at room temperature. For microbiological reasons storage under refrigeration is recommended.

Evaluation of the Stability of Newborn Hospital Parenteral Nutrition Solutions.

(1) Background: parenteral nutrition (PN) solutions are an extremely complex mixture. It is composed of a multitude of chemical elements that can give rise to a large number of interactions that condition its stability and safety. The aim of this study was to evaluate the stability of PN solutions for preterm infants. (2) Methods: eight samples were prepared according to the protocol for prescribing PN in preterm infants. Samples PN1-PN7 had the normal progression of macronutrients and standard amounts of micronutrients for a 1 kg preterm infant. The PN8 sample had a high concentration of electrolytes, with the idea of forcing stability limits. Samples were stored both at room temperature and under refrigeration. Measurements of globule size, pH, density, and viscosity were performed in both storage protocols on different days after processing. (3) Results: the changes in the composition of the samples did not affect the evolution of the stability at the different measurement times and temperatures. Viscosity was affected by the compositional changes made in the PN samples, but no alterations due to time or temperature were observed. Density and pH remained stable, without significant changes due to time, storage temperature, or different composition. (4) Conclusion: all samples remained stable during the study period and did not undergo significant alterations due to compositional changes or different experimental conditions.

One-chamber and two-chamber parenteral nutrition admixtures for pediatric and adult patients: An evaluation of physico-chemical stability at room and cold temperature.

OBJECTIVES: The aim of this study was to evaluate the physico-chemical stability of compounded total parenteral nutrition admixtures through peroxidation assay and ultraviolet-visible spectroscopy, high-performance liquid chromatography analysis, nuclear magnetic resonance spectrometry, pH meter, and dynamic light scattering. METHODS: The present study considered parenteral nutrition (PN) admixtures for pediatric and adult patients. The admixtures were characterized by a high content of vitamins and trace elements. They were prepared in one- or two-chamber bags in the hospital pharmacy using an automatic compounding system in a sterile room with laminar airflow at different temperature conditions and light exposure. The experiment setup comprised fat emulsions, lipid-free PN solutions, and single-chamber bags before and after adding vitamins and trace elements. The stability at room temperature (+25°C) and cold temperature (+2-8°C) was assessed by various means. RESULTS: Two-compartment admixtures, single-chamber bags, and all-in-one PN supplemented with vitamins and trace elements are stable up until 35, 9, and 7 d, respectively, when protected from light and stored at +2 to 8°C. Also, the supplemented single-chamber PN was found to be stable up to 48 h when stored at +25°C with light exposure. CONCLUSIONS: The results obtained will help improve PN management at the compounding center and in hospital wards, because they allow for the extension of the validity time frame provided so far by the different formulations and, therefore, therapy scheduling over several days.

Quantitative Monitoring of Cocrystal Polymorphisms in Model Tablets Using Transmission Low-Frequency Raman Spectroscopy.

Cocrystallization is a technique for improving the physical properties of active pharmaceutical ingredients. However, cocrystals can transform into more stable polymorphs as well as dissociate to original materials. Therefore, an analytical technique is required to determine the polymorphic transformation quickly and accurately in tablets. The purpose of this study is to develop a method to monitor cocrystal polymorphs in model tablets using transmission low-frequency Raman spectroscopy. The tablets, consisting of only metastable polymorphs of caffeine-glutaric acid cocrystals, were stored under various relative humidity levels. The composition of the cocrystal polymorphs were calculated from a calibration curve relating the actual composition to the predicted values calculated by partial least squares regression processing of low-frequency Raman spectra. The metastable form gradually converted to a stable form, and polymorphic phase transformation occurred with increasing relative humidity. Ninety-six percent of the metastable form converted into a stable form stored at 25 °C after 3 h at 95% RH. In conclusion, transmission low-frequency Raman spectroscopy can be used to quantitatively monitor cocrystal polymorphs. This technique is one of the candidate techniques to quantifiably evaluate the physico-chemical stability of cocrystal polymorphs in tablets.

Physical and chemical stability of a generic etoposide formulation as an alternative to etoposide phosphate.

The generic Mylan® etoposide (ETP) has been investigated as an alternative for Etopophos®, in part due to a global shortage of the latter. The generic alternative is different both in its formulation and in its very limited stability (6 h at 25 °C against 4 days for Etopophos®) once reconstituted in ready-to-use chloride or glucose solutions. Its intrinsic stability has been thoroughly studied under various conditions. Two degradation products resulting from hydrolysis were characterized by LC-HR-MSn and supported by density functional theory calculations of the frontier molecular orbitals energies, molecular electrostatic potential mapping, and Mulliken charge analysis. Chemical degradation increases with temperature and can be fitted to a zero order kinetic model with a half-life of 119 days and a kinetic constant of 0.0028 mM day-1. Precipitation was only observed in solutions at 5 °C and -20 °C indicating that at these temperatures the reconstituted solutions are thermodynamically metastable. In conclusion, ETP at concentrations of 0.68 and 1 mM prepared and stored at 25 °C under good manufacturing practices remained unchanged over a period of 21 days irrespective of the nature of the solvents or the type of container.

Assessment of the Antimicrobial Potentiality and Functionality of Lactobacillus plantarum Strains Isolated from the Conventional Inner Mongolian Fermented Cheese Against Foodborne Pathogens.

Lactobacillus plantarum are amongst the diversified lactic acid bacteria (LAB) species which are being utilized abundantly in the food industry. Numerous L. plantarum strains have been reported to produce several antimicrobial compounds. Diacetyl, hydrogen peroxide, organic acids, as well as bacteriocins can also be exemplified by a variable spectrum of actions. The current study was intended to conduct the screening and characterization of antimicrobial prospective of L.plantarum from traditional Inner Mongolian fermented hard cheese. Foodborne pathogens, Salmonellatyphimurium, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcusaureus, were examined by using the Oxford cup technique and the mixed culture inhibition assays. The resulting analyses disclosed that L. plantarum KLDS1.0344 indicated broad antimicrobial spectrum against all selected pathogens as compared to other LAB used in this study. Additionally, the decrement of the pathogen population was observed up to 3.47 logs in mixed culture inhibition assays. L. plantarum KLDS 1.0344 acid production was recorded up to 71.8 ± 3.59 °D in mixed culture while antimicrobial particles released in cell free supernatants demonstrated bacteriocin-like characteristics showing substantial pH stability (2.0-6.0), proteolytic enzyme reduced the antibacterial activity (15.2 ± 0.6 mm-20.4 ± 0.8 mm), heat stability (20 min at 120 °C) against selected pathogens. Moreover, the spectrum range of antimicrobial peptides after the partial purification was decreased as compared to the crude bacteriocin-like compound. The SDS-PAGE analysis showed the molecular weight range of partially purified bacteriocin from 12 to 45 kDa. After analyzing the obtained data from the current experimentation showed that the capability of L. plantarum KLDS 1.0344 to oppose the pathogen growth in vitro relies on the occurrence of organic acids along with bacteriocin-like compounds proving L. plantarum KLDS 1.0344 as a potentially appropriate candidate as an alternative bio-control agent against foodborne pathogens.

Does controlled nucleation impact the properties and stability of lyophilized monoclonal antibody formulations?

This study provides the first systematic investigation of the impact of the nucleation protocol during freeze-drying on physico-chemical properties and long-term stability of two IgG1 antibodies in sugar formulations. We hypothesized that the lower specific surface area (SSA) generated by controlled nucleation could be beneficial for the stability of interface sensitive proteins. The study compares controlled nucleated (CN) and randomly nucleated (RN) lyophilizates with high and low antibody concentrations stored at different temperatures. Formulations with and without polysorbate (PS) were included. In the "high concentration" study the formulation without PS showed reduced particle formation for CN samples compared to RN samples. PS containing formulations had an overall lower particle level with no further advantage of CN on stability. Besides the intended comparison of CN and RN samples, we observed that PS promoted sucrose crystallization in both low concentration antibody studies during storage. Additionally, our results indicate that the nucleation temperature (TN) was not the only determining factor for the resulting ice crystal size and consequently the product`s SSA. Overall, the application of CN had neither a positive nor a negative impact on the product's physico-chemical stability. The surfactant had a much higher stabilizing effect than the reduction of the SSA by CN.