Microbial challenge test of a novel epoprostenol sodium formulation.

AIM: The aim of the current study was to present a comprehensive display of antimicrobial activity of a novel epoprostenol sodium formulation with respect to seven different microorganisms, two levels of inoculation (102-103 colony forming units [CFU]/mL and 105-106 CFU/mL), two diluents (sterile water for injection [SWI] and sterile saline [sodium chloride 0.9%] for injection [SSI]), two concentrations (3,000 ng/mL and 15,000 ng/mL), and seven different storage time points at two temperatures (up to 10 days at 2°C-8°C and 20°C-25°C). MATERIALS AND METHODS: Antimicrobial activity was evaluated for, 1) solutions at 3,000 ng/mL inoculated with 102-103 CFU/mL and 105-106 CFU/mL; and 2) solutions at 15,000 ng/mL inoculated with 102-103 CFU/mL and 105-106 CFU/mL. All solutions were stored for up to 10 days at 2°C-8°C and 20°C-25°C. Solutions were prepared by reconstitution and further dilution of an epoprostenol sodium formulation using SWI or SSI. Antimicrobial activity was measured after inoculation with seven species of bacteria, yeast, and mold. RESULTS: For all solutions, after 10 days, no microbial growth with respect to initial inoculum was observed, with the exception of a few early time points when using SWI as diluent. Some microorganisms died off completely, whereas others remained stable overall or returned to initial levels. Prior to decreasing, some microorganisms displayed a slight initial increase, presumed to be caused by breakup of clusters. Storage temperature had a negligible influence on the results, whereas choice of diluent (SSI or SWI) impacted growth kinetics in that SSI had a greater antimicrobial effect than SWI. CONCLUSION: Upon reconstitution and further dilution of the novel epoprostenol formulation to concentrations of 3,000 ng/mL and 15,000 ng/mL with SWI or SSI, the resulting solutions did not support growth of the tested microorganisms when stored at 2°C-8°C or 20°C-25°C for up to 10 days.

Stability of refrigerated miglustat after preparation in InOrpha(®) flavored suspending excipient for compounding of oral solutions and suspensions.

BACKGROUND: Miglustat (Zavesca(®)) is an oral treatment for type 1 Gaucher disease and Niemann-Pick disease type C. Patients with Niemann-Pick disease type C often have difficulties swallowing, and miglustat has an unpleasant taste. The stability of miglustat at 2°C-8°C prepared in InOrpha(®) suspending vehicle, a liquid taste-masking agent, was assessed. METHODS: The contents of Zavesca(®) 100 mg capsules (a powder blend comprising miglustat and several excipients) were transferred into InOrpha(®). Although miglustat was soluble in InOrpha(®) at all concentrations tested, some of the excipients were not. An InOrpha(®) suspension containing 20 mg/mL miglustat was investigated initially. Subsequently, a pH-adjusted suspension of 20 mg/mL, and non-adjusted 10 and 5 mg/mL suspensions were evaluated. All suspensions were stored under refrigerated conditions. Physicochemical and microbiological challenge testing was performed at 0 hours and after 14 and 28 days. Degradation was assessed by high-performance liquid chromatography, appearance was assessed visually, and pH was recorded. Suspensions were inoculated with seven species of bacteria, yeast, and mold, and growth evaluated using membrane filtration. RESULTS: Miglustat 20 mg/mL suspension changed from yellow (0 hours) to brown (days 14 and 28); pH remained stable at 7.4-7.6. Pure InOrpha(®) (pH 4.6) remained yellow throughout the study. Pure InOrpha(®) adjusted to pH 7.5 displayed a brownish discoloration after 9 days. Miglustat 5 and 20 mg/mL suspensions, adjusted to pH 6.5 and 4.4, respectively, remained yellow at days 14 and 28. Miglustat 10 mg/mL suspension (pH 7.3) changed from yellow to brown on day 9. No degradates were detected for any of the concentrations tested. There was no proliferation of microorganisms over the study period; in all cases the level of contamination was clearly reduced. CONCLUSION: InOrpha(®) suspensions containing miglustat 5 mg/mL (without pH adjustment) and 20 mg/mL (with pH adjusted to 4.4) display stable physicochemical and microbiological properties over 28 days.

Stability and preservation of a new formulation of epoprostenol sodium for treatment of pulmonary arterial hypertension.

BACKGROUND: The aim of this study was to evaluate the stability and microbiological properties of a formulation of epoprostenol sodium with L-arginine and sucrose excipients (epoprostenol AS). METHODS: The stability of the reconstituted solutions after storage at 5°C and 25°C, diluted solutions (3000-60,000 ng/mL) at controlled room temperature, and diluted solutions (3000-60,000 ng/mL) stored at 5°C and then at room temperature were evaluated. Solutions were prepared using sterile water for injection or sterile saline (sodium chloride 0.9%) for injection. Shelf-life was assessed by determining potency over time relative to initial potency. In this context, potency is synonymous with content. The antimicrobial activity of reconstituted (100,000 ng/mL for 0.5 mg vial, 300,000 ng/mL for 1.5 mg vial) and diluted (3000 ng/mL) epoprostenol AS was measured using an antimicrobial effectiveness test after inoculation with six species of bacteria, yeast, and mold. RESULTS: Reconstituted epoprostenol AS was stable for up to one day's storage at 25°C or 7 days' storage at 5°C. Epoprostenol AS was stable for up to 72 hours when diluted, depending on temperature and concentration. The maximum shelf-life of the diluted solution if the reconstituted solution had been stored for up to one day at room temperature or up to 7 days at 5°C, was between 24 and 72 hours, depending on concentration. Following storage of diluted solutions at 5°C for up to 8 days, maximum shelf-life was between one and 2 days, depending on temperature and concentration. Potency was not dependent on diluents. Preservative testing confirmed no microbial growth for any of six organisms tested for at least 14 days at 5°C or 25°C for the reconstituted solution and for at least 16 days at 5°C followed by one day at 25°C for the diluted solutions. CONCLUSION: Epoprostenol AS has favorable thermal stability and does not support the growth of any micro-organism tested for up to 17 days. This extended stability under ambient conditions has the potential to improve convenience further for patients.

Stability and microbiological properties of a new formulation of epoprostenol sodium when reconstituted and diluted.

PURPOSE: Epoprostenol, used for the treatment of pulmonary arterial hypertension (PAH), has a number of limitations related to its short half-life in aqueous solution. The aim of this study was to evaluate the stability and microbiological properties of a new formulation, namely epoprostenol sodium with arginine and mannitol excipients (epoprostenol AM; Veletri®; Actelion Pharmaceuticals Ltd, Allschwil, Switzerland). METHODS: Stability and microbiological properties of epoprostenol AM were investigated at 5°C, 25°C, and 30°C over a range of concentrations (3000-30,000 ng/mL) when reconstituted and immediately diluted with sterile water for injection (SWI) or sterile saline (sodium chloride 0.9%) for injection (SSI). Stability (change in potency over time) for up to 72 hours at 25°C and 30°C was measured immediately following dilution and after storage at 5°C. Shelf-life was assessed by determining the maintenance of potency over time relative to initial potency. For microbiological testing, diluted samples of epoprostenol AM were inoculated with a range of bacteria, yeasts, and molds for up to 14 days at 5°C or 4 days at 25°C. RESULTS: Epoprostenol AM reconstituted and immediately diluted to the required concentration with SWI or SSI was stable for up to 3 days at 25°C and up to 7 days at 5°C depending on the concentration. None of the diluted epoprostenol AM solutions supported microbial growth for any of the six organisms tested for up to 14 days. CONCLUSIONS: Epoprostenol AM has improved thermal stability and does not support the growth of any microorganism tested for up to 14 days. This extended stability under ambient conditions has the potential to improve convenience for patients.

Near-infrared spectroscopy of blood plasma for diagnosis of sporadic Alzheimer's disease.

There are currently no accepted blood-based biomarkers of sporadic Alzheimer's disease (AD). Augmented oxidative stress has been implicated in both neural and peripheral AD tissues. In this study, we determined whether short-wavelength near-infrared (NIR) spectrophotometry of blood plasma differentiates mild sporadic AD from normal aging. NIR analysis was conducted on 75 microl plasma samples from 19 AD, 27 amnestic MCI, and 17 normal elderly control (NEC) persons using an optical fiber-coupled, holographic grating-based NIR spectrograph. Five spectral bands associated with heme, R-CH, R-OH, H2O, and R-NH functional groups were sensitive to oxidative modification in pre-clinical studies and were pre-selected to develop a logistic regression model for sample classification. This model differentiated AD from NEC samples with a sensitivity of 80% and specificity of 77%. Fifteen and twelve MCI patients were classified with the NEC and AD groups, respectively. The spectra were not influenced by age, gender, exposure to cholinesterase inhibitors or vitamin E, or sample storage time. The NIR data further implicate oxidative stress in the systemic pathophysiology of sporadic AD and differentiate mild (and possibly pre-clinical) AD from NEC individuals with moderate-high accuracy. The procedure is minimally-invasive, rapid, relatively-inexpensive, and may provide a useful biological marker of sporadic AD.

Spectroscopy of human plasma for diagnosis of idiopathic Parkinson's disease.

BACKGROUND: No established chemical biomarkers of idiopathic Parkinson's disease (PD) currently exist. Augmented oxidative stress (OS) has been implicated in both neural and peripheral PD tissues. METHODS: In this study, Raman scattering and near-infrared spectroscopy were used to detect and quantify oxidative substrate modifications in blood plasma samples from PD and normal elderly control (NEC) subjects. RESULTS: Hypothesis-driven preselection of OS-sensitive bandwidths distinguished PD from NEC subjects with approximately 75% sensitivity and specificity using both complementary spectroscopic techniques. CONCLUSION: Biospectroscopy of plasma may provide a rapid, minimally invasive and inexpensive chemical biomarker of idiopathic PD.

Characterization of UV-transparent capillaries for CEC and CE.

This short communication describes features of UV-transparent capillaries employed for CEC and CE. A waveguide effect was observed when using UV-transparent capillaries. Through imaging with SEM, the UV-transparent coating was found to be highly porous unlike polyimide coating, which did not exhibit any porosity at all. Prolonged exposure to several commonly employed solvents with elevated pH caused abrasion of the coating at the capillary tip but no swelling of the UV-transparent coating was observed. Lastly, four different cutting techniques were compared to obtain smooth capillary tips.

Pore size characterization of monolith for electrochromatography via atomic force microscopy studies in air and liquid phase.

This paper investigates the use of scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the characterization of monoliths used in capillary electrochromatography (CEC) while focusing on the nature of the information available from both techniques. SEM imaging revealed a compact structure of non-porous micrometer sized particles homogeneously agglomerated. With a simple AFM methodology, we found by direct observation that the same material exhibits mesopores in the nanometer range while SEM showed non-porous surfaces. These results obtained by AFM clearly showed that micrometer sized particles shrank and micrometer sized pores increased in the monolith when wetted. Thus, AFM was capable of demonstrating the morphological differences between wet and dried monolithic materials that are not possible by other imaging methods at micrometer resolution.

Capillary electrochromatography of peptides and proteins.

This review surveys the accomplishments in the separation of peptides and proteins by capillary electrochromatography (CEC) over the last decade. A significant number of research articles have been published on this topic since the last review. Peptide and proteins separations have been carried out in all three formats of CEC, i.e., packed bed, continuous bed and open-tubular (OT) format. In addition to electrophoresis, different chromatographic modes have been successfully exploited with the most prevalent being reversed-phase mode followed by ion-exchange. Although many researchers continue to use model proteins and peptides primarily to evaluate the performance of novel stationary phases some researchers have also applied CEC to the analysis of real-life samples. The potential of CEC to yield complementary information and sometimes a superior separation with respect to established techniques, i.e., microbore HPLC and capillary electrophoresis has been demonstrated. Instrumental modifications in order to facilitate coupling of CEC to mass spectrometry have further upgraded the value of CEC for proteomic analysis. Capillaries are still the separation vehicle of choice for most researchers yet the microfluidic platform is gaining momentum, propelled particularly by its potential for multitasking, e.g., performing different chromatographic modes in series.

Protein separation by monolithic capillary electrochromatography.

This work presents the separation of model proteins by capillary electrochromatography involving a monolithic stationary phase with C4 functionality. The monolith was fabricated in UV-transparent capillaries by employing a slight modification of a recently published photopolymerization procedure. With the number of theoretical plates per column ranging between 11000 and 33000, the separation efficiency proved to be lower than capillary zone electrophoresis where plate numbers ranged between 18000 and 66000. However, higher resolution was obtained due to the additional chromatographic separation mechanism. Inter- and intra-column reproducibility were evaluated, the latter could be significantly improved when using a rinsing procedure that contained 0.05% sodium dodecylsulfate in the mobile phase. Plate heights became nearly independent of mobile phase velocities higher than 0.5 mm/s indicating that high velocities can be applied without sacrificing efficiency. Furthermore, peak heights showed a dependence on injection times. For proteins, an increase in capacity factors was found when increasing the percentage of organic solvent in the mobile phase.