Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells.

BACKGROUND: The lifetime prevalence of degenerative disc disease is dramatically high. Numerous investigations on disc degeneration have been performed on cells from annulus fibrosus (AF) and nucleus pulposus (NP) of the intervertebral disc (IVD) in cell culture experiments utilising a broad variety of basal culture media. Although the basal media differ in nutrient formulation, it is not known whether the choice of the basal media itself has an impact on the cell's behaviour in vitro. In this study, we evaluated the most common media used for monolayer expansion of AF and NP cells to set standards for disc cell culture. METHODS: Human AF and NP cells were isolated from cervical discs. Cells were expanded in monolayer until passage P2 using six different common culture media containing alpha-Minimal Essential Medium (alpha-MEM), Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F-12 medium (Ham's F-12) as single medium or in a mixture of two media (alpha/F-12, DMEM/alpha, DMEM/F-12). Cell morphology, cell growth, glycosaminoglycan production and quantitative gene expression of cartilage- and IVD-related markers aggrecan, collagen type II, forkhead box F1 and keratin 18 were analysed. Statistical analysis was performed with two-way ANOVA testing and Bonferroni compensation. RESULTS: AF and NP cells were expandable in all tested media. Both cell types showed similar cell morphology and characteristics of dedifferentiation known for cultured disc cells independently from the media. However, proceeding culture in Ham's F-12 impeded cell growth of both AF and NP cells. Furthermore, the keratin 18 gene expression profile of NP cells was changed in alpha-MEM and Ham's F-12. CONCLUSION: The impact of the different media itself on disc cell's behaviour in vitro was low. However, AF and NP cells were only robust, when DMEM was used as single medium or in a mixture (DMEM/alpha, DMEM/F-12). Therefore, we recommend using these media as standard medium for disc cell culture. Our findings are valuable for the harmonisation of preclinical study results and thereby push the development of cell therapies for clinical treatment of disc degeneration.

Media-fill simulation tests in manual and robotic aseptic preparation of injection solutions in syringes.

OBJECTIVE: The purpose of this study was to evaluate the contamination rate of media-fill products either prepared automated with a robotic system (APOTECAchemo™) or prepared manually at cytotoxic workbenches in the same cleanroom environment and by experienced operators. Media fills were completed by microbiological environmental control in the critical zones and used to validate the cleaning and disinfection procedures of the robotic system. METHODS: The aseptic preparation of patient individual ready-to-use injection solutions was simulated by using double concentrated tryptic soy broth as growth medium, water for injection and plastic syringes as primary packaging materials. Media fills were either prepared automated (500 units) in the robot or manually (500 units) in cytotoxic workbenches in the same cleanroom over a period of 18 working days. The test solutions were incubated at room temperature (22℃) over 4 weeks. Products were visually inspected for turbidity after a 2-week and 4-week period. Following incubation, growth promotion tests were performed with Staphylococcus epidermidis. During the media-fill procedures, passive air monitoring was performed with settle plates and surface monitoring with contact plates on predefined locations as well as fingerprints. The plates got incubated for 5-7 days at room temperature, followed by 2-3 days at 30-35℃ and the colony forming units (cfu) counted after both periods. The robot was cleaned and disinfected according to the established standard operating procedure on two working days prior to the media-fill session, while on six other working days only six critical components were sanitized at the end of the media-fill sessions. Every day UV irradiation was operated for 4 h after finishing work. RESULTS: None of the 1000 media-fill products prepared in the two different settings showed turbidity after the incubation period thereby indicating no contamination with microorganisms. All products remained uniform, clear, and light-amber solutions. In addition, the reliability of the nutrient medium and the process was demonstrated by positive growth promotion tests with S. epidermidis. During automated preparation the recommended limits < 1 cfu per settle/contact plate set for cleanroom Grade A zones were not succeeded in the carousel and working area, but in the loading area of the robot. During manual preparation, the number of cfus detected on settle/contact plates inside the workbenches lay far below the limits. The number of cfus detected on fingertips succeeded several times the limit during manual preparation but not during automated preparation. There was no difference in the microbial contamination rate depending on the extent of cleaning and disinfection of the robot. CONCLUSION: Extensive media-fill tests simulating manual and automated preparation of ready-to-use cytotoxic injection solutions revealed the same level of sterility for both procedures. The results of supplemental environmental controls confirmed that the aseptic procedures are well controlled. As there was no difference in the microbial contamination rates of the media preparations depending on the extent of cleaning and disinfection of the robot, the results were used to adapt the respective standard operating procedures.

Stability of furosemide and chlorothiazide stored in syringes.

PURPOSE: The results of a study to determine the stability of solutions of furosemide and chlorothiazide over 96 hours are reported. METHODS: Chlorothiazide and furosemide were diluted in 5% dextrose USP to final concentrations of 10 and 1 mg/mL, respectively, and combined. In addition, sample solutions of chlorothiazide in dextrose, furosemide in dextrose, and dextrose alone were prepared for control purposes. The resulting solutions were analyzed immediately after preparation and 24, 48, 72, and 96 hours later using a liquid chromatography-tandem mass spectroscopy (LC-MS/MS) system with an electrospray ionization source. Mixtures and samples were diluted 10,000-fold prior to LC-MS/MS analysis so that concentrations of both drugs would be within the assay's linear range of detection. RESULTS: LC-MS/MS analysis showed that chlorothiazide typically eluted at 2.6 minutes and furosemide at 4.8 minutes. Each compound was degraded by exposure to strong ultraviolet light in a time-dependent manner. Both unmixed and mixed solutions retained over 90% of the original concentrations of chlorothiazide and furosemide for up to 96 hours. Furosemide and chlorothiazide are commonly used concomitantly to maximize diuresis in pediatric patients; the study findings suggest that solutions of furosemide and chlorothiazide can be combined in the same syringe without loss of stability for up to 96 hours. CONCLUSION: Solutions of chlorothiazide (10 mg/mL) and furosemide (1 mg/mL) stored either separately or together in polypropylene syringes remained stable for up to 96 hours at room temperature and protected from light.

Effects of outside air temperature on the preparation of antineoplastic drug solutions in biological safety cabinets.

PURPOSE: In Japan, biological safety cabinets are commonly used by medical staff to prepare antineoplastic agents. At the Division of Chemotherapy for Outpatients, Nagoya University Hospital, a class II B2 biological safety cabinet is used. The temperature inside this biological safety cabinet decreases in winter. In this study, we investigated the effect of low outside air temperature on the biological safety cabinet temperature, time required to admix antineoplastic agents, and accuracy of epirubicin weight measurement. METHODS: Studies were conducted from 1 January to 31 March 2008 (winter). The outside air temperature near the biological safety cabinet intake nozzle was compared with the biological safety cabinet temperature. The correlation between the outside air temperature and the biological safety cabinet temperature, time for cyclophosphamide and gemcitabine solubilization, and accuracy of epirubicin weight measurement were investigated at low and high biological safety cabinet temperatures. RESULT: The biological safety cabinet temperature correlated with the outside air temperature of 5-20℃ (p < 0.0001). Compared to cyclophosphamide and gemcitabine solubilization in the biological safety cabinet at 25℃, solubilization at 10℃ was significantly delayed (p < 0.01 and p < 0.0001, respectively). Measurement of epirubicin weight by using a syringe lacked accuracy because of epirubicin's high viscosity at low temperatures (p < 0.01). CONCLUSION: These results suggest that the biological safety cabinet temperature decreases when cool winter air is drawn into the biological safety cabinet, affecting the solubilization of antineoplastic agents. We suggest that a decrease in biological safety cabinet temperature may increase the time required to admix antineoplastic agents, thereby increasing the time for which outpatients must wait for chemotherapy.

Physico-chemical stability of busulfan in injectable solutions in various administration packages.

BACKGROUND AND OBJECTIVES: Busulfan is used as part of a conditioning regimen prior to hematopoietic stem cell transplantation for the treatment of certain cancers and immune deficiency syndromes. Due to its instability in aqueous preparations, busulfan for infusion is prepared from a concentrate and has a relatively short shelf life once prepared. The purpose of this study was to identify the most suitable storage container and temperature to maximize the shelf life of busulfan therapeutic infusions prepared from Busilvex(®). METHODS: Busilvex(®) 6 mg/mL was diluted to 0.55 mg/mL with 0.9 % NaCl and aliquots dispensed into polypropylene syringes, polyvinyl chloride bags, and glass bottles. Three storage temperatures were evaluated: 2-8 °C, 13-15 °C (thermostatically controlled chamber), and room temperature (20 ± 5 °C). At set time points, samples were analysed for busulfan content, using a high-performance liquid chromatography (HPLC) system with ultraviolet detection. The change in pH and osmolarity on storage was also determined, and solutions were inspected visually for formation of a precipitate or colour change. To determine the contribution of precipitation to loss of busulfan content on storage, samples from one time series were treated with the solvent dimethylacetamide prior to HPLC separation and quantitation of busulfan. RESULTS: The results of the active substance content monitoring study over a 48-h period demonstrate that busulfan solution is stable at a 5 % threshold, at 2-8 °C for 16 h in syringes, 14 h in glass bottles, and 6 h in bags. In addition, the period of stability decreases as the temperature increases (4 h at 20 ± 5 °C). The solution is considered to be stable, subject to precipitation liable to be observed regardless of the temperature. CONCLUSION: The best stability was observed for busulfan solutions placed at 2-8 °C in syringes. This study demonstrated that precipitation, in addition to hydrolysis, has a significant influence on the busulfan content.

Long-term stability study of clofarabine injection concentrate and diluted clofarabine infusion solutions.

PURPOSE: The aim of this study was to investigate the physicochemical stability of clofarabine (CAFdA) injection concentrate and ready-to-use CAFdA infusion solutions over a prolonged period of 28 days. METHODS: To determine the stability of CAFdA infusion solutions, the injection concentrate (Evoltra®, 1 mg/mL, Genzyme) was diluted either with 0.9% sodium chloride or 5% glucose infusion solution. The resulting concentrations of 0.2 mg/mL or 0.6 mg/mL, respectively, were chosen to represent the lower and upper limit of the ordinary concentration range. Test solutions were stored under refrigeration (2-8°C) or at room temperature either light protected or exposed to light. CAFdA concentrations and pH values were determined at different time intervals throughout a 28-day storage period. Compatibility of diluted CAFdA infusion solutions (0.1-0.4 mg/mL) with different container materials (polyvinyl chloride (PVC), glass, and polypropylene/polyethylene (PP/PE)) was tested over a 48-h storage period. CAFdA concentrations were measured by a stability-indicating reversed phase high-performance liquid chromatography (HPLC) assay with ultraviolet detection. RESULTS: CAFdA injection concentrate and CAFdA infusion solutions remained physicochemically stable (>90% CAFdA) for 4 weeks. Results are independent of storage conditions, drug concentrations (0.2, 0.6, and 1.0 mg/mL) and diluents (0.9% sodium chloride, 5% glucose infusion solution). Adsorption of CAFdA to container material can be excluded. CONCLUSIONS: CAFdA injection concentrate and diluted infusion solutions in commonly used vehicles are stable for at least 28 days either refrigerated or at room temperature. Physicochemical stability favors pharmacy-based centralized preparation. Due to microbiological reasons, strict aseptic handling and storage of the products under refrigeration is recommended.

Aluminum content in intravenous solutions for administration to neonates: role of product preparation and administration methods.

BACKGROUND: Aluminum loading can reach toxic levels depending on the amount of aluminum intake in intravenous solutions (IV). Premature infants are at a higher risk of aluminum toxicity because of their reduced urinary aluminum elimination. All steps involved in the preparation of intravenous solutions for premature neonates in intensive care units were evaluated to determine to what degree, if any, they increased the aluminum load and should be considered when assessing the daily aluminum intake (<5 mcg/kg) established by the U.S. Food and Drug Administration (FDA). METHODS: Products and medical devices used for the IV administration of solutions to preterm neonates were analyzed for their aluminum content. Commercial formulations, bags after compounding, and medications before and after their preparation, as well as infusion sets (including burettes) and syringes, were evaluated for their contribution to the aluminum levels in the final solution. The determination was carried out by atomic absorption spectrometry. RESULTS: Currently available products used to prepare parenteral nutrition solutions as well as injectable medications usually administered to premature neonates present aluminum contamination. Bags, burettes, and syringes were also contaminated by aluminum to some degree, which may be leached during use. CONCLUSIONS: Commercial products are the main source of aluminum in parenteral nutrition; nevertheless, manipulation, containers, and administration sets increased aluminum levels by about 40%. Because this is a significant rate, these sources should be taken into account when calculating the amount of aluminum delivered to the patient in order to comply with FDA standards.

Raman microscopic applications in the biopharmaceutical industry: in situ identification of foreign particulates inside glass containers with aqueous formulated solutions.

Particle identification is an important analytical procedure for quality control and assurance in the biopharmaceutical industry. Rapid and reliable identification of micro-particles helps in evaluating the nature of particle contamination and its consequences on the product quality regulated by internal and external standards. Raman microscopy is one of the microspectroscopic techniques that can be used to identify micro-particles with the advantage of in situ detection. In this paper we demonstrate that a visible laser Raman microscope was particularly useful to identify micro-particles that were inside glass containers such as glass syringes, vials, and test tubes, which are commonly used as containers for aqueous formulated drugs. The examples include the identifications of a droplet-like particle inside a pre-filled glass syringe, a fibrous particle inside a glass test tube, and a white particle inside a glass vial; all of these examples usually demand challenging or time-consuming sample manipulation for other techniques. The Raman microscopic technique was shown to be able to solve these challenging micro-particle identifications due to its ability to carry out detection in situ. Particularly in the example of micro-droplet identification, the Raman microscopic technique was the only choice for a fast and successful particle detection. For all three identifications, Raman in situ detection has significantly accelerated particle analysis and avoided potential sample secondary contamination or losses owing to none or minimal sample manipulation.

Effects of compounding and storage conditions on stability of pergolide mesylate.

OBJECTIVE: To determine the effects of temperature and light over a 35-day period on stability of pergolide mesylate after compounding in an aqueous vehicle. DESIGN: Evaluation study. PROCEDURES: Pergolide was compounded into a formulation with a final target concentration of 1 mg/mL. Aliquots of the formulation were then stored at -20 degrees, 8 degrees, 25 degrees, or 37 degrees C without exposure to light or at 25 degrees C with exposure to light for 35 days. Samples were assayed in triplicate by means of high-pressure liquid chromatography immediately after compounding and after 1, 7, 14, 21, and 35 days of storage. RESULTS: Mean+/-SD concentration of pergolide in the formulation immediately after compounding was 1.05+/-0.086 mg/mL. Samples exposed to light while stored at 25 degrees C had undergone excessive degradation by day 14, samples stored at 37 degrees C had undergone excessive degradation by day 21, and samples stored at 25 degrees C without exposure to light had undergone excessive degradation by day 35. The decrease in expected concentration corresponded with the appearance of degradation peaks in chromatograms and with a change in color of the formulation. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that pergolide mesylate was unstable after compounding in an aqueous vehicle and that storage conditions had an effect on stability of the compounded formulation. Compounded pergolide formulations in aqueous vehicles should be stored in a dark container, protected from light, and refrigerated and should not be used >30 days after produced. Formulations that have undergone a color change should be considered unstable and discarded.

Modeling of the solution interaction properties of plastic materials used in pharmaceutical product container systems.

Material/water equilibrium binding constants (Eb) were determined for 14 organic solutes and 17 plastic raw materials that could be used in pharmaceutical product container systems. Correlations between the measured binding constants and the organic solute's octanol/water and hexane/water partition coefficients were obtained. In general, while the materials examined exhibited a wide range of binding characteristics, the tested materials by and large fell within two broad classes: (1) those that were octanol-like in their binding characteristics, and (2) those that were hexane-like. Materials of the same class (e.g., polypropylenes) generally had binding models that were very similar. Rank ordering of the materials in terms of their magnitude of drug binding (least binding to most binding) was as follows: polypropylene < polyethylene < polyamide < styrene-ethylene-butylene-styrene < copolyester ether elastomer approximately equal to amine-terminated poly fatty acid amide polymer. The utilization of the developed models to estimate drug loss via sorption by the container is discussed.

Stability of a flavored formulation of acetylcysteine for oral administration.

PURPOSE: The chemical and physical stability of a flavored solution of acetylcysteine stored at room temperature or under refrigeration for up to 35 days in amber plastic prescription bottles were studied. METHODS: The flavored acetylcysteine solution was prepared by adding a sweetener and a strawberry creamsicle flavoring to acetylcysteine solution 10% to a final nominal acetylcysteine concentration of 86.5 mg/mL. Six identical samples of the formulation were prepared in amber plastic prescription bottles. Three bottles were stored at room temperature (23-25 degrees C) and the other three were stored in the refrigerator (3-5 degrees C). Immediately after preparation and at 7, 14, 21, 28, and 35 days, each sample was assayed in duplicate by high- performance liquid chromatography. Stability was defined as the retention of at least 90% of the initial drug concentration. RESULTS: On day 35, the refrigerated acetylcysteine samples retained 96.7% of their initial concentration. The samples stored at room temperature retained 92.5% of their initial concentration. CONCLUSION: A flavored oral formulation of acetylcysteine 86.5 mg/mL was stable for at least 35 days when stored at room temperature and in the refrigerator in amber plastic bottles.

Quality assessment of morphine hydrochloride solutions.

The therapeutic substances in solution prepared in pharmaceutical laboratories (prescribed drugs) must preserve their activity. Therefore, they must be stable throughout the period of storage in home conditions. The maintenance of stability is particularly difficult for morphine hydrochloride solutions administered orally to cancer patients at the last stage of the disease being at home. This study, aiming at the assessment of stability of morphine hydrochloride solutions, was performed on samples of 0.5% water solutions of the drug alone, 0.25% and 0.5% solutions of the drug in water with chloroform as well as injection solutions (Morphinum hydrochloricum, 20 mg, Polfa Warsaw). All the samples were kept at 20 degrees C for six months. Throughout this time observations were made to detect changes in their appearence and pH values. Their qualitative composition was determined by TLC and the content of morphine was checked by UV spectrophotometry in an environment of 0.1 mol/l of hydrochloric acid at 285 nm. Results of the kinetic study permitted drawing conclusions as to the mechanism of the decomposition of morphine hydrochloride in the solutions studied - according to a simple first order reaction and determination of the rate constants (k, s(-1)) of the process. Results of the chromatographic and spectrophotometric study did not show differences in the stability of water and chloroform/water solutions of morphine hydrochloride studied after 4 weeks and 6 months. After that time the decrease of morphine content was 10 and 25%, respectively.