Introducing the Alba® Primary Packaging Platform. Part 2: Inorganic Extractables Evaluation.

Sensitivity of drugs to one or more elements of the primary packaging is a serious concern for the pharmaceutical industry. Biologics in particular are highly sensitive, leading to a higher risk of incompatibility and stability test failure as worst-case scenario.This potential incompatibility-and the consequent formulation instability due to the interactions between the drug and the primary container surface-may have multiple causes: the intrinsic nature of the container surface, leachables coming from the materials used, substances coming from the production process, or silicone oil droplets or other particles.The Alba primary packaging platform was designed to have the same interface between the drug and the glass container surface on the different primary packaging containers in order to minimize the emergence of instabilities at later stages of formulation development. Alba containers are internally treated with an innovative cross-linked coating based on silicone oil lubricant, and the additional rubber components have been selected to minimize the possible differences between the container typologies.This paper shows in great detail the reduction of the inorganic extractables released and the comparability of the performances of the different containers obtained using Alba technology.The improvement has been demonstrated by stressing the containers with different extract solutions; Alba-coated containers show a strong reduction of inorganic extractables and of corrosion degree compared to spray-on siliconized and bulk products. The containers included in the Alba platform present comparable results, and this represents a strong advantage during the drug formulation development by facilitating the transition from one container to another.LAY ABSTRACT: The sensitivity of drugs to one or more elements of the primary packaging is a serious concern for the pharmaceutical industry. Biologics in particular are highly sensitive, leading to a higher risk of incompatibility and stability test failure worst-case scenario.This potential incompatibility-and the consequent formulation instability due to the interactions between the drug and the primary container surface-may have multiple causes: the intrinsic nature of the container surface, leachables coming from the materials used, substances coming from the production process, or silicone oil droplets or other particles.The Alba primary packaging platform was designed to minimize these problems associated with the interaction between the drug and its primary packaging. This paper shows in great detail and with robust data the inorganic extractables release reduction and the delamination risk mitigation obtained using the Alba technology.

Assessing and Mitigating the Hazard Potential of Two-Dimensional Materials.

The family of two-dimensional (2D) materials is comprised of a continually expanding palette of unique compositions and properties with potential applications in electronics, optoelectronics, energy capture and storage, catalysis, and nanomedicine. To accelerate the implementation of 2D materials in widely disseminated technologies, human health and environmental implications need to be addressed. While extensive research has focused on assessing the toxicity and environmental fate of graphene and related carbon nanomaterials, the potential hazards of other 2D materials have only recently begun to be explored. Herein, the toxicity and environmental fate of postcarbon 2D materials, such as transition metal dichalcogenides, hexagonal boron nitride, and black phosphorus, are reviewed as a function of their preparation methods and surface functionalization. Specifically, we delineate how the hazard potential of 2D materials is directly related to structural parameters and physicochemical properties and how experimental design is critical to the accurate elucidation of the underlying toxicological mechanisms. Finally, a multidisciplinary approach for streamlining the hazard assessment of emerging 2D materials is outlined, thereby providing a pathway for accelerating their safe use in a range of technologically relevant contexts.

Boron phenylalanine and related impurities: HPLC analysis, stability profile and degradation pathways.

Boron phenylalanine is one of the lead drug candidates in the field of Boron Neutron Capture Therapy. Its inherent low toxicity allows large doses to be administered, but this makes it important to identify, rationalise and quantify impurities. Here we report a chromatographic assay method, the conditions under which the parent compound is unstable, and the suggested degradation mechanisms.

A novel hybrid mode of sample injection to enhance CZE sensitivity for simultaneous determination of a pyridine-triphenylborane anti-fouling agent and its degradation products.

We developed a novel hybrid sample injection mode (HSIM) that presents the combination of electrokinetic injection and vacuum injection to enhance detection sensitivity in CZE. Samples were introduced using both vacuum and electrokinetic injections simultaneously, with a water plug injected into the capillary prior to sample introduction (i.e. similarly to field-amplified sample injection, FASI). Using a sample mixture containing an anti-fouling agent applied to ship hulls, pyridine-triphenylborane and its degradation products (diphenylborinic acid, phenylboronic acid, and phenol) dissolved in ACN, the length of water plug, time, and voltage for sample introduction were optimized. The signal intensity (peak height) was found to be up to a 30-fold increased using HSIM by applying 4 kV for 4 s at the inlet end of the capillary as the cathode with supplementary vacuum in comparison with only vacuum injection for 4 s. The LODs (at a S/N of 3) for pyridine-triphenylborane, diphenylborinic acid, phenylboronic acid, and phenol were 0.88, 1.0, 21, and 23 µg/L, respectively. At the level of 0.04 mg/L, the RSDs (n=4, intra-day) for the above analytes were in the ranges of 1.9-11, 4.3-9.2, and 0.34-0.66% for peak area, peak height, and migration time, respectively. The HSIM is a simple and promising procedure useful for enhancing the sensitivity for both low-and high-mobility ions in CZE.

Surface-directed structure formation of ß-lactoglobulin inside droplets.

The morphology of ß-lactoglobulin structures inside droplets was studied during aggregation and gelation using confocal laser scanning microscopy (CLSM) equipped with a temperature stage and transmission electron microscopy (TEM). The results showed that there is a strong driving force for the protein to move to the interface between oil and water in the droplet, and the ß-lactoglobulin formed a dense shell around the droplet built up from the inside of the droplets. Less protein was found inside the droplets. The longer the ß-lactoglobulin was allowed to aggregate prior to gel formation, the larger the part of the protein went to the interface, resulting in a thicker shell and very little material being left inside the droplets. The droplets were easily deformed because no network stabilizes them. When 0.5% emulsifier, polyglycerol polyresinoleat (PGPR), was added to the oil phase, the ß-lactoglobulin was situated both inside the droplets and at the interface between the droplets and the oil phase; when 2% PGPR was added, the ß-lactoglobulin structure was concentrated to the inside of the droplets. The possibility to use the different morphological structures of ß-lactoglobulin in droplets to control the diffusion rate through a ß-lactoglobulin network was evaluated by fluorescence recovery after photobleaching (FRAP). The results show differences in the diffusion rate due to heterogeneities in the structure: the diffusion of a large water-soluble molecule, FITC-dextran, in a dense particulate gel was 1/4 of the diffusion rate in a more open particulate ß-lactoglobulin gel in which the diffusion rate was similar to that in pure water.

I-FABP expression alters the intracellular distribution of the BODIPY C16 fatty acid analog.

To investigate the structure-function relationships of intestinal fatty acid-binding protein (I-FABP) in cellular fatty acid (FA) trafficking, we compared the distribution of a fluorescent FA analog (BODIPY FL C16) in Cos-1 cells transiently transfected with the wild type protein (wt I-FABP) to that of a variant deleted of the alpha helical domain (HL I-FABP). In vector-only cells, BODIPY fluorescence was distributed throughout the cytoplasm. In the absence of added FA, wt I-FABP was found largely in the perinuclear region with some cytoplasmic staining as well. Addition of BODIPY FL C16 to transfected cells showed that the fluorescent FA was essentially completely colocalized with the protein in the cytoplasmic and perinuclear regions as well as in cytoplasmic clusters that are not observed in the absence of wt I-FABP. For HL I-FABP, the distribution of the protein in the absence of FA was diffusely cytoplasmic, in marked contrast to the wt protein. Addition of BODIPY led to less extensive colocalization than that observed for wt I-FABP. In particular, no localization to the perinuclear region was found. Organelle colocalization studies showed that both proteins colocalized with mitochondria and endoplasmic reticulum/golgi markers, but little with a lysosomal marker. The perinuclear localization for wt I-FABP and BODIPY did not show colocalization with any of the markers tested. Taken together, these results indicate that I-FABP binds FA in vivo and that the helical domain may be important for targeting I-FABP to a perinuclear domain but not, perhaps, to the endoplasmic reticulum, golgi apparatus or mitochondria.

Fluorescent detection of lipid droplets and associated proteins.

Most eukaryotic cells can store excess lipid in cytosolic lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets with nile red and BODIPY 493/503 are included. The differences in the spectral properties of these two lipophilic dyes and advantages of each are discussed. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of adipophilin, a broadly expressed, lipid droplet-associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included.

Synthesis of ethyleneoxide modified 3-carboranyl thymidine analogues and evaluation of their biochemical, physicochemical, and structural properties.

Eleven 3-carboranyl thymidine analogues (3CTAs) containing highly hydrophilic and flexible ethyleneoxide moieties were synthesized as potential agents for boron neutron capture therapy (BNCT) and their biochemical and physicochemical properties were evaluated. Based on specific structural features, this library of 3CTAs was divided into three subgroups. The first group contained 3CTAs with 1-4 ethyleneoxide units between the thymidine (Thd) scaffold and a carborane cluster. The second group of 3CTAs contained a pentylene spacer between Thd and the carborane and 2-4 ethyleneoxide units additionally attached to the carborane cluster. The third group contained three 3CTAs all with pentylene spacers and four ethylene units but with different carborane cages. The ethyleneoxide modified 3CTAs were good substrates of thymidine kinase 1 (TK1) and poor substrates of human mitochondrial thymidine kinase 2 (TK2) as determined in phosphoryl transfer assays. In the first group of 3CTAs, all the compounds were efficiently phosphorylated regardless of varying spacer lengths (37-42% of the activity of Thd). The second group of 3CTAs was less effectively phosphorylated (17-26% of the activity of Thd) probably due to a less favorable sterical orientation of Thd within the active site of TK1 and/or an increased lipophilicity compared with the first group. In the third group of structural isomers, no significant differences in phosphorylation rates were observed (17-25%). A structure-function hypothesis explaining these results is presented.

Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis.

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.

Spectromicroscopy of boron in human glioblastomas following administration of Na2B12H11SH.

Boron neutron capture therapy (BNCT) is an experimental, binary treatment for brain cancer which requires as the first step that tumor tissue is targeted with a boron-10 containing compound. Subsequent exposure to a thermal neutron flux results in destructive, short range nuclear reaction within 10 microm of the boron compound. The success of the therapy requires than the BNCT agents be well localized in tumor, rather than healthy tissue. The MEPHISTO spectromicroscope, which performs microchemical analysis by x-ray absorption near edge structure (XANES) spectroscopy from microscopic areas, has been used to study the distribution of trace quantities of boron in human brain cancer tissues surgically removed from patients first administered with the compound Na2B12H11SH (BSH). The interpretation of XANES spectra is complicated by interference from physiologically present sulfur and phosphorus, which contribute structure in the same energy range as boron. We addressed this problem with the present extensive set of spectra from S, B, and P in relevant compounds. We demonstrate that a linear combination of sulfate, phosphate and BSH XANES can be used to reproduce the spectra acquired on boron-treated human brain tumor tissues. We analyzed human glioblastoma tissue from two patients administered and one not administered with BSH. As well as weak signals attributed to BSH, x-ray absorption spectra acquired from tissue samples detected boron in a reduced chemical state with respect to boron in BSH. This chemical state was characterized by a sharp absorption peak at 188.3 eV. Complementary studies on BSH reference samples were not able to reproduce this chemical state of boron, indicating that it is not an artifact produced during sample preparation or x-ray exposure. These data demonstrate that the chemical state of BSH may be altered by in vivo metabolism.