A simple and rapid method to simultaneously analyze ciclesonide and its impurities in a ciclesonide metered-dose inhaler using on-line supercritical fluid extraction/supercritical fluid chromatography/quadrupole time-of-flight mass spectrometry.

A simple and rapid on-line SFE/SFC/quadrupole TOF-MS method to simultaneously analyze active pharmaceutical ingredients and impurities from metered-dose inhalers (MDIs) was developed using ciclesonide MDI (CIC-MDI) as an example. CIC-MDI, as drug Alvesco®, has been approved for the treatment of bronchial asthma, and its major impurities are listed in the European Pharmacopoeia and in the supplementary package inserts of Alvesco® (called as "Pharmaceutical interview form" in Japan). In the developed method, CIC-MDI was manually sprayed only once on a glass disc prior to the SFE/SFC/quadrupole TOF-MS. In the SFE, CIC and its impurities and other impurities having various polarities and hydrophobicity, were extracted in 3.5 min and subsequently separated on a CHIRALPAK IE-3 column to be detected by quadrupole TOF-MS in 6.5 min. This method would be applicable to the analysis of other inhalable pharmaceutical products whose sample preparation requires complicated procedures, as well as to the analysis of general pharmaceutical products for profiling impurities.

Regulatory landscape of nanotechnology and nanoplastics from a global perspective.

Nanotechnology and more particularly nanotechnology-based products and materials have provided a huge potential for novel solutions to many of the current challenges society is facing. However, nanotechnology is also an area of product innovation that is sometimes developing faster than regulatory frameworks. This is due to the high complexity of some nanomaterials, the lack of a globally harmonised regulatory definition and the different scopes of regulation at a global level. Research organisations and regulatory bodies have spent many efforts in the last two decades to cope with these challenges. Although there has been a significant advancement related to analytical approaches for labelling purposes as well as to the development of suitable test guidelines for nanomaterials and their safety assessment, there is a still a need for greater global collaboration and consensus in the regulatory field. Furthermore, with growing societal concerns on plastic litter and tiny debris produced by degradation of littered plastic objects, the impact of micro- and nanoplastics on humans and the environment is an emerging issue. Despite increasing research and initial regulatory discussions on micro- and nanoplastics, there are still knowledge gaps and thus an urgent need for action. As nanoplastics can be classified as a specific type of incidental nanomaterials, current and future scientific investigations should take into account the existing profound knowledge on nanotechnology/nanomaterials when discussing issues around nanoplastics. This review was conceived at the 2019 Global Summit on Regulatory Sciences that took place in Stresa, Italy, on 24-26 September 2019 (GSRS 2019) and which was co-organised by the Global Coalition for Regulatory Science Research (GCRSR) and the European Commission's (EC) Joint Research Centre (JRC). The GCRSR consists of regulatory bodies from various countries around the globe including EU bodies. The 2019 Global Summit provided an excellent platform to exchange the latest information on activities carried out by regulatory bodies with a focus on the application of nanotechnology in the agriculture/food sector, on nanoplastics and on nanomedicines, including taking stock and promoting further collaboration. Recently, the topic of micro- and nanoplastics has become a new focus of the GCRSR. Besides discussing the challenges and needs, some future directions on how new tools and methodologies can improve the regulatory science were elaborated by summarising a significant portion of discussions during the summit. It has been revealed that there are still some uncertainties and knowledge gaps with regard to physicochemical properties, environmental behaviour and toxicological effects, especially as testing described in the dossiers is often done early in the product development process, and the material in the final product may behave differently. The harmonisation of methodologies for quantification and risk assessment of nanomaterials and micro/nanoplastics, the documentation of regulatory science studies and the need for sharing databases were highlighted as important aspects to look at.

Visualizing the spatial localization of ciclesonide and its metabolites in rat lungs after inhalation of 1-µm aerosol of ciclesonide by desorption electrospray ionization-time of flight mass spectrometry imaging.

Inhaled ciclesonide (CIC), a corticosteroid used to treat asthma that is also being investigated for the treatment of corona virus disease 2019, hydrolyzes to desisobutyryl-ciclesonide (des-CIC) followed by reversible esterification when exposed to fatty acids in lungs. While previous studies have described the distribution and metabolism of the compounds after inhalation, spatial localization in the lungs remains unclear. We visualized two-dimensional spatial localization of CIC and its metabolites in rat lungs after administration of a single dose of a CIC aerosol (with the mass median aerodynamic diameter of 0.918-1.168 µm) using desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI). In the analysis, CIC, des-CIC, and des-CIC-oleate were imaged in frozen lung sections at high spatial and mass resolutions in negative-ion mode. MSI revealed the coexistence of CIC, des-CIC, and des-CIC-oleate on the airway epithelium, and the distribution of des-CIC and des-CIC-oleate in peripheral lung regions. In addition, a part of CIC independently localized on the airway epithelium. These results suggest that distribution of CIC and its metabolites in lungs is related to both the intended delivery of aerosols to pulmonary alveoli and peripheral regions, and the potential deposition of CIC particles on the airway epithelium.

Temperature-Dependent Formation of N-Nitrosodimethylamine during the Storage of Ranitidine Reagent Powders and Tablets.

The purpose of this study was to elucidate the effect of high-temperature storage on the stability of ranitidine, specifically with respect to the potential formation of N-nitrosodimethylamine (NDMA), which is classified as a probable human carcinogen. Commercially available ranitidine reagent powders and formulations were stored under various conditions, and subjected to LC-MS/MS analysis. When ranitidine tablets from two different brands (designated as tablet A and tablet B) were stored under accelerated condition (40 °C with 75% relative humidity), following the drug stability guidelines issued by the International Conference on Harmonisation (ICH-Q1A), for up to 8 weeks, the amount of NDMA in them substantially increased from 0.19 to 116 ppm and from 2.89 to 18 ppm, respectively. The formation of NDMA that exceeded the acceptable daily intake limit (0.32 ppm) at the temperature used under accelerated storage conditions clearly highlights the risk of NDMA formation in ranitidine formulations when extrapolated to storage under ambient conditions. A forced-degradation study under the stress condition (60 °C for 1 week) strongly suggested that environmental factors such as moisture and oxygen are involved in the formation of NDMA in ranitidine formulations. Storage of ranitidine tablets and reagent powders at the high temperatures also increased the amount of nitrite, which is considered one of the factors influencing NDMA formation. These data indicate the necessity of controlling/monitoring stability-related factors, in addition to controlling impurities during the manufacturing process, in order to mitigate nitrosamine-related health risks of certain pharmaceuticals.

Evaluation of off-target effects of gapmer antisense oligonucleotides using human cells.

Antisense oligonucleotide (ASO) has the potential to induce off-target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. Conventional animal studies cannot be used to assess toxicity induced by off-target effects because of differences in the genome sequence between humans and other animals. Consequently, the assessment of off-target effects with in silico analysis using a human RNA database and/or in vitro expression analysis using human cells has been proposed. Our previous study showed that the number of complementary regions of ASOs with mismatches in the human RNA sequences increases dramatically as the number of tolerated mismatches increases. However, to what extent the expression of genes with mismatches is affected by off-target effects at the cellular level is not clear. In this study, we evaluated off-target effects of gapmer ASOs, which cleave the target RNA in an RNase H-dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off-target effects depending on the degree of complementarity between the ASO and off-target candidate genes. Based on our results, we also propose a scheme for the assessment of off-target effects of gapmer ASOs.

Rapid and efficient high-performance liquid chromatography analysis of N-nitrosodimethylamine impurity in valsartan drug substance and its products.

In July 2018, certain valsartan-containing drugs were voluntary recalled in Japan owing to contamination with N-nitrosodimethylamine (NDMA), a probable human carcinogen. In this study, an HPLC method was developed for the quantitative detection of NDMA simultaneously eluted with valsartan. Good linearity with a correlation coefficient (R2) > 0.999 was achieved over the concentration range of 0.011-7.4 µg/mL. The limits of detection and quantification were 0.0085 µg/mL and 0.0285 µg/mL, respectively. When the recalled valsartan samples were subjected to this method, the observed NDMA contents were in agreement with the reported values, indicating that our method achieved sufficient linearity, accuracy, and precision to detect NDMA in valsartan drug substances and products. Moreover, six samples (valsartan drug substances and tablet formulations), which had a possibility for NDMA contamination, were analyzed; none of the samples contained NDMA at detectable levels. Our method would be useful for the rapid screening and quantification of NDMA impurity in valsartan drug substances and products.

Analysis of an Impurity, N-Nitrosodimethylamine, in Valsartan Drug Substances and Associated Products Using GC-MS.

Valsartan products, commonly used to treat high blood pressure and heart failure, have been recalled in many countries due to the presence of an impurity, N-nitrosodimethylamine (NDMA), in the recalled products. We present and evaluate a GC-MS-based analytical method for the determination of NDMA levels and attempt an investigation of NDMA concentrations in valsartan drug substances and associated products. The limit of detection and limit of quantification for the method were estimated to be 0.1 and 0.5 µg/g, respectively, when testing a 0.5-g sample. A good trueness (99%) with a small relative standard deviation (1.9%) was obtained for a valsartan product spiked with NDMA at a concentration of 1.0 µg/g. Additionally, a valsartan drug substance and the associated product, which were previously determined to have NDMA contamination, were analyzed by the method. The NDMA content by our method was very close to previously determined values. Finally, six samples, including valsartan drug substances and associated, commercially available products in Japan, all of which were derived from the company implicated in the NDMA contamination, were analyzed by our method, revealing that none of these samples contained detectable concentrations of NDMA. Overall, the data indicate that the present method is reliable and useful for determination of NDMA in valsartan drug substances and associated products.

Detailed Morphological Characterization of Nanocrystalline Active Ingredients in Solid Oral Dosage Forms Using Atomic Force Microscopy.

The characterization of nanocrystalline active ingredients in multicomponent formulations for the design and manufacture of products with increased bioavailability is often challenging. The purpose of this study is to develop an atomic force microscopy (AFM) imaging method for the detailed morphological characterization of nanocrystalline active ingredients in multicomponent oral formulations. The AFM images of aprepitant and sirolimus nanoparticles in aqueous suspension show that their sizes are comparable with those measured using dynamic light scattering (DLS) analysis. The method also provides information on a wide-sized range of particles, including small particles that can often only be detected by DLS when larger particles are removed by additional filtration steps. An expected advantage of the AFM method is the ability to obtain a detailed information on particle morphology and stiffness, which allows the active pharmaceutical ingredient and excipient (titanium dioxide) particles to be distinguished. Selective imaging of particles can also be achieved by varying the surface properties of the AFM solid substrate, which allows to control the interactions between the substrate and the active pharmaceutical ingredient and excipient particles. AFM analysis in combination with other methods (e.g., DLS), should facilitate the rational development of formulations based on nanoparticles.

Emerging technologies for food and drug safety.

Emerging technologies are playing a major role in the generation of new approaches to assess the safety of both foods and drugs. However, the integration of emerging technologies in the regulatory decision-making process requires rigorous assessment and consensus amongst international partners and research communities. To that end, the Global Coalition for Regulatory Science Research (GCRSR) in partnership with the Brazilian Health Surveillance Agency (ANVISA) hosted the seventh Global Summit on Regulatory Science (GSRS17) in Brasilia, Brazil on September 18-20, 2017 to discuss the role of new approaches in regulatory science with a specific emphasis on applications in food and medical product safety. The global regulatory landscape concerning the application of new technologies was assessed in several countries worldwide. Challenges and issues were discussed in the context of developing an international consensus for objective criteria in the development, application and review of emerging technologies. The need for advanced approaches to allow for faster, less expensive and more predictive methodologies was elaborated. In addition, the strengths and weaknesses of each new approach was discussed. And finally, the need for standards and reproducible approaches was reviewed to enhance the application of the emerging technologies to improve food and drug safety. The overarching goal of GSRS17 was to provide a venue where regulators and researchers meet to develop collaborations addressing the most pressing scientific challenges and facilitate the adoption of novel technical innovations to advance the field of regulatory science.

Evaluating the Properties of Poly(lactic-co-glycolic acid) Nanoparticle Formulations Encapsulating a Hydrophobic Drug by Using the Quality by Design Approach.

We applied the Quality by Design (QbD) approach to the development of poly(lactic-co-glycolic acid) (PLGA) nanoparticle formulations encapsulating triamcinolone acetonide, and the critical process parameters (CPPs) were identified to clarify the correlations between critical quality attributes and CPPs. Quality risk management was performed by using an Ishikawa diagram and experiments with a fractional factorial design (ANOVA). The CPPs for particle size were PLGA concentration and rotation speed, and the CPP for relative drug loading efficiency was the poor solvent to good solvent volume ratio. By assessing the mutually related factors in the form of ratios, many factors could be efficiently considered in the risk assessment. We found a two-factor interaction between rotation speed and rate of addition of good solvent by using a fractional factorial design with resolution V. The system was then extended by using a central composite design, and the results obtained were visualized by using the response surface method to construct a design space. Our research represents a case study of the application of the QbD approach to pharmaceutical development, including formulation screening, by taking actual production factors into consideration. Our findings support the feasibility of using a similar approach to nanoparticle formulations under development. We could establish an efficient method of analyzing the CPPs of PLGA nanoparticles by using a QbD approach.

Atomic Force Microscopic Analysis of the Effect of Lipid Composition on Liposome Membrane Rigidity.

Mechanical rigidity of the liposome membrane is often defined by the membrane bending modulus and is one of the determinants of liposome stability, but the quantitative experimental data are still limited to a few kinds of liposomes. Here, we used atomic force microscopy to investigate the membrane bending moduli of liposomes by immobilizing them on bovine serum albumin-coated glass in aqueous medium. The following lipids were used for liposome preparation: egg yolk phosphatidylcholine, dioleoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol, and N-(carbonylmethoxypoly(ethylene glycol) 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine. By using liposomes of various compositions, we showed that the thermodynamic phase state of the membrane rather than the electric potential or liposome surface modification with poly(ethylene glycol) is the predominant determinant of the bending modulus, which decreased in the following order: solid ordered > liquid ordered > liquid disordered. By using the generalized polarization value of the Laurdan fluorescent probe, we investigated membrane rigidity in terms of membrane fluidity. Atomic force microscopic analysis was superior to the Laurdan method, especially in evaluating the membrane rigidity of liposomes containing hydrogenated soybean phosphatidylcholine and cholesterol. Positively charged liposomes with a large bending modulus were taken up by cells more efficiently than those with a small bending modulus. These findings offer a quantitative method of analyzing the membrane rigidity of nanosized liposomes with different lipid compositions and will contribute to the control of liposome stability and cellular uptake efficiency of liposomal formulations intended for clinical use.

Observation of liposomes of differing lipid composition in aqueous medium by means of atomic force microscopy.

Liposomes present a challenge for atomic force microscopy (AFM) observation in aqueous medium because they easily collapse. Here, we demonstrate that bovine serum albumin coating of a glass substrate enables AFM observation of various liposomes in aqueous medium. With this AFM system, liposomes can be systematically observed and morphologically analyzed regardless of their surface charge, phase state, degree of lipid acyl chain unsaturation or PEG modification. This system thus has the potential to reveal the mechanical properties of liposomes of various lipid types and contents.

Analysis of Distribution of Ingredients in Commercially Available Clarithromycin Tablets Using Near-Infrared Chemical Imaging with Principal Component Analysis and Partial Least Squares.

The aim of this study was to evaluate pharmaceuticals using a near-infrared chemical imaging (NIR-CI) technique for visualizing the distribution of ingredients in solid dosage forms of commercially available clarithromycin tablets. The cross section of a tablet was measured using the NIR-CI system for evaluating the distribution of ingredients in the tablet. The chemical images were generated by performing multivariate analysis methods: principal component analysis (PCA) and partial least squares (PLS) with normalized near-infrared (NIR) spectral data. We gained spectral and distributional information related to clarithromycin, cornstarch, and magnesium stearate by using PCA analysis. On the basis of this information, the distribution images of these ingredients were generated using PLS analysis. The results of PCA analysis enabled us to analyze individual components by using PLS even if sufficient information on the products was not available. However, some ingredients such as binder could not be detected using NIR-CI, because their particle sizes were smaller than the pixel size (approximately 25×25×50 µm) and they were present in low concentrations. The combined analysis using both PCA and PLS with NIR-CI was useful to analyze the distribution of ingredients in a commercially available pharmaceutical even when sufficient information on the product is not available.

Interaction kinetics of serum proteins with liposomes and their effect on phospholipase-induced liposomal drug release.

We used surface plasmon resonance (SPR) to measure the affinity and kinetics of the interaction between serum proteins and both conventional and PEGylated liposomes. The effect of the interactions on secretory phospholipase A2 (sPLA2)-induced release of a model drug from liposomes was also assessed. SPR analysis of 12 serum proteins revealed that the mode of interaction between serum proteins and liposomes greatly varies depending on the type of protein. For example, albumin bound to liposomes at slower association/dissociation rates with higher affinity and prevented sPLA2-induced drug release from PEGylated liposomes. Conversely, fibronectin bound at faster association/dissociation rates with lower affinity and demonstrated little impact on the drug release. These results indicate that the effect of serum proteins on sPLA2 phospholipid hydrolysis varies with the mode of interaction between proteins and liposomes. Understanding how the proteins interact with liposomes and impact sPLA2 phospholipid hydrolysis should aid the rational design of therapeutic liposomal formulations.

Effects of lipid composition on the properties of doxorubicin-loaded liposomes.

BACKGROUND: The liposomal lipid composition of doxorubicin-loaded liposome likely will influence its pharmacological activity. Results & methodology: We prepared 18 formulations of doxorubicin-loaded liposomes in which the lipid composition was varied. It was indicated that the intracellular uptake of doxorubicin is the primary property of doxorubicin-loaded liposome that affects its cytotoxicity in vitro. Furthermore, the release rate of doxorubicin from liposome and the biological activity of the lipid itself also affected the cytotoxicity. SUMMARY: These findings provide an insight into how lipid composition influences the cytotoxicity of the doxorubicin-loaded liposomes. Our results provide valuable information that should help to enhance the therapeutic efficacy of liposomal anticancer drug products by optimizing their formulations.

Effect of Knockout of Mdr1a and Mdr1b ABCB1 Genes on the Systemic Exposure of a Doxorubicin-Conjugated Block Copolymer in Mice.

We previously elucidated that ATP-binding cassette subfamily B member 1 (ABCB1) mediates the efflux of doxorubicin-conjugated block copolymers from HeLa cells. Here, we investigated the role of ABCB1 in the in vivo behavior of a doxorubicin-conjugated polymer in Mdr1a/1b(-/-) mice. The area under the curve for intravenously administered polymer in Mdr1a/1b(-/-) mice was 2.2-fold greater than that in wild-type mice. The polymer was mostly distributed in the liver followed by spleen and less so in the brain, heart, kidney, and lung. The amount of polymer excreted in the urine was significantly decreased in Mdr1a/1b(-/-) mice. The amounts of polymers excreted in the feces were similar in both groups despite the higher systemic exposure in Mdr1a/1b(-/-) mice. Confocal microscopy images showed polymer localized in CD68(+) macrophages in the liver. These results show that knockout of ABCB1 prolonged systemic exposure of the doxorubicin-conjugated polymer in mice. Our results suggest that ABCB1 mediated the excretion of doxorubicin-conjugated polymer in urine and feces. Our results provide valuable information about the behavior of block copolymers in vivo, which is important for evaluating the pharmacokinetics of active substances conjugated to block copolymers or the accumulation of block copolymers in vivo.

General considerations regarding the in vitro and in vivo properties of block copolymer micelle products and their evaluation.

Block copolymer micelles are nanoparticles formed from block copolymers that comprise a hydrophilic polymer such as poly(ethylene glycol) and a poorly soluble polymer such as poly(amino acids). The design of block copolymer micelles is intended to regulate the in vivo pharmacokinetics, stability, and distribution profiles of an entrapped or block copolymer-linked active substance. Several block copolymer micelle products are currently undergoing clinical development; however, a major challenge in the development and evaluation of such products is identification of the physicochemical properties that affect the properties of the drug product in vivo. Here we review the overall in vitro and in vivo characteristics of block copolymer micelle products with a focus on the products currently under clinical investigation. We present examples of methods suitable for the evaluation of the physicochemical properties, non-clinical pharmacokinetics, and safety of block copolymer micelle products.

Investigation of factors affecting in vitro doxorubicin release from PEGylated liposomal doxorubicin for the development of in vitro release testing conditions.

Establishing appropriate drug release testing methods of liposomal products for assuring quality and performance requires the determination of factors affecting in vitro drug release. In this study, we investigated the effects of test conditions (human plasma lot, pH/salt concentration in the test media, dilution factor, temperature, ultrasound irradiation, etc.), and liposomal preparation conditions (pH/concentration of ammonium sulfate solution), on doxorubicin (DXR) release from PEGylated liposomal DXR. Higher temperature and lower pH significantly increased DXR release. The evaluation of DXR solubility indicated that the high DXR release induced by low pH may be attributed to the high solubility of DXR at low pH. Ultrasound irradiation induced rapid DXR release in an amplitude-dependent manner. The salt concentration in the test solution, human plasma lot, and dilution factor had a limited impact on DXR-release. Variations in the ammonium sulfate concentration used in solutions for the formation/hydration of liposomes significantly affected DXR release behavior, whereas differences in pH did not. In addition, heating condition in phosphate-buffered saline at lower pH (<6.5) exhibited higher discriminative ability for the release profiles from various liposomes with different concentrations of ammonium sulfate than did ultrasound irradiation. These results are expected to be helpful in the process of establishing appropriate drug release testing methods for PEGylated liposomal DXR.