Fabrication and Characterization of Dissolving Microneedles for Transdermal Drug Delivery of Apomorphine Hydrochloride in Parkinson's Disease.

PURPOSE: We fabricated and characterized polyvinyl alcohol (PVA)-based dissolving microneedles (MNs) for transdermal drug delivery of apomorphine hydrochloride (APO), which is used in treating the wearing-off phenomenon observed in Parkinson's disease. METHODS: We fabricated MN arrays with 11 × 11 needles of four different lengths (300, 600, 900, and 1200 µm) by micromolding. The APO-loaded dissolving MNs were characterized in terms of their physicochemical and functional properties. We also compared the pharmacokinetic parameters after drug administration using MNs with those after subcutaneous injection by analyzing the blood concentration of APO in rats. RESULTS: PVA-based dissolving MNs longer than 600 µm could effectively puncture the stratum corneum of the rat skin with penetrability of approximately one-third of the needle length. Although APO is known to have chemical stability issues in aqueous solutions, the drug content in APO-loaded MNs was retained at 25°C for 12 weeks. The concentration of APO after the administration of APO-loaded 600-µm MNs that dissolved completely in skin within 60 min was 81%. The absorption of 200-µg APO delivered by MNs showed a Tmax of 20 min, Cmax of 76 ng/mL, and AUC0-120 min of 2,829 ng・min/mL, compared with a Tmax of 5 min, Cmax of 126 ng/mL, and AUC0-120 min of 3,224 ng・min/mL for subcutaneous injection. The bioavailability in terms of AUC0-120 min of APO delivered by MNs was 88%. CONCLUSION: APO-loaded dissolving MNs can deliver APO via skin into the systemic circulation with rapid absorption and high bioavailability.

Uniform Suspension of Heat-Killed Staphylococcus aureus for a Positive Control Used in the Monocyte-Activation Test.

Pyrogens, classified as bacterial endotoxins and non-endotoxin pyrogens (NEPs), induce fever or shock when released into the bloodstream or spinal fluid. Recently, a monocyte-activation test (MAT) involving human cell culture has been developed to detect pyrogens in injectable products. To evaluate the sensitivity of MAT, a reference standard endotoxin was used as a positive control; however, the reactivity differed between the endotoxins and NEPs, necessitating positive controls for NEPs. This study aimed to explore a preparation method for heat-killed Staphylococcus aureus (HKSA) as a positive control for NEPs in MAT. Because S. aureus forms grape-like clusters, nine types of glass filters with pore sizes of 0.5-2.7 µm were evaluated to obtain a uniform bacterial suspension. The suspension was then heat-treated to kill the bacteria, resulting in HKSA samples. Serial dilutions of HKSA were tested by MAT using peripheral blood mononuclear cells. The interleukin-6 concentrations in the culture supernatant were measured by enzyme-linked immuno-sorbent assay to assess pyrogenic activities of HKSA. The pore sizes of the glass filters affected the uniformity of HKSA, and GF/C filter was selected for HKSA preparation. Repeated filtration improved uniformity, and a uniform suspension of HKSA was obtained through double filtration using a GF/C filter. Despite the decrease in HKSA activity as filtration frequency increased, the detection limit remained consistently unchanged. This suggests that repeated filtration can adjust the activity of HKSA to a baseline level and that a uniform suspension of HKSA exhibiting low variation is suitable as a positive control in MAT.

Analysis of Factors Related to Variation in Dissolution Profiles Estimated from Continuously Conducted Dissolution Tests of Generic Products.

The development of generic pharmaceuticals involves a bioequivalence study to ensure the therapeutic equivalence of the test formulation to the original innovative product. The formulation characteristics of generic products are expected to be maintained in the long term after approval. This study analyzed the factors contributing to the changes in the dissolution profiles of approved products during their life cycles. Cumulative data on the dissolution similarity of 1675 products of 127 ingredients tested by official laboratories in Japan were assessed according to Japanese bioequivalence guidelines with slight modifications. The products showing dissimilarities in dissolution profiles were analyzed for reporting year, therapeutic category, co-development, physical properties of the active pharmaceutical ingredient (API), and suspected reasons for dissolution change. The increase in the number of dissimilar products is related to the co-development of generic products. Although the solubility of the API was not associated with the dissolution change in the analysis of the total dissolution data, control of the API particle size is suggested to be important for drugs with poorly soluble APIs. Additionally, a risk factor for dissolution changes in the test solutions at a certain pH was the presence of acidic or basic residues. These results indicate the importance of proper development through a thorough evaluation of the formulation and process factors affecting the dissolution properties throughout the product lifecycle.

Folded, undulating, and fibrous doxorubicin sulfate crystals in liposomes.

High-resolution cryogenic transmission electron microscopy (cryo-TEM) evidenced that doxorubicin sulfate crystals in liposomes (prepared by remote loading with ammonium sulfate) form folded, undulating, and fibrous crystals with a diameter of approximately 2.4 nm. An undulating, fibrous crystal considered to be undergrowth, in addition to bundles of fibrous crystals, was also observed in doxorubicin-loaded liposomes. This explains the validity of the formation of doxorubicin sulfate crystals of various shapes, e.g., curved, U-shaped, or circular, in addition to cylinder and/or rod-like crystals reported in the literature. Liposomes that do not contain crystals have inner aqueous phases with high electron density, suggesting that the doxorubicin is remotely loaded and remains as a solute without precipitation.

Effects of Apex Size on Dissolution Profiles in the USP II Paddle Apparatus.

The use of apex vessels may solve coning problems associated with dissolution testing. However, excessive dissolution acceleration can reduce the discriminatory power. This study aimed to clarify how different apex vessel sizes affect the dissolution behavior of cone-forming formulations. Five apex vessels with different heights, centralities, and compendial vessels were used. The paddle rotation speed at which the coning phenomenon resolved was measured using standard particles of different densities. Three model formulations-USP prednisone tablets, atorvastatin calcium hydrate tablets, and levofloxacin fine granules-were selected, and dissolution tests were conducted at 30-100 revolutions per minute (rpm). Compared to the compendial vessels, the disappearance of standard particles at the apex base at lower paddle speeds in apex vessels was observed. Standard particles tended to remain in the center of the apex vessels and disappear at rotational speeds comparable to those of the compendial vessels. Dissolution increased in an apex height-dependent manner in the model formulations, except for the atorvastatin calcium hydrate tablets at 50 rpm. For levofloxacin fine granules, dissolution was also improved by reducing the paddle agitation speed to 30 rpm in the compendial vessels. Differences in apex centrality by 3 mm did not affect the dissolution rate. Our results indicate that apex vessels with low apex heights have a mount-resolving effect, but the degree of dissolution improvement by avoiding the coning phenomenon depends on the formulation characteristics used in the dissolution tests.

Current Status and Challenges of Analytical Methods for Evaluation of Size and Surface Modification of Nanoparticle-Based Drug Formulations.

The present review discusses the current status and difficulties of the analytical methods used to evaluate size and surface modifications of nanoparticle-based pharmaceutical products (NPs) such as liposomal drugs and new SARS-CoV-2 vaccines. We identified the challenges in the development of methods for (1) measurement of a wide range of solid-state NPs, (2) evaluation of the sizes of polydisperse NPs, and (3) measurement of non-spherical NPs. Although a few methods have been established to analyze surface modifications of NPs, the feasibility of their application to NPs is unknown. The present review also examined the trends in standardization required to validate the size and surface measurements of NPs. It was determined that there is a lack of available reference materials and it is difficult to select appropriate ones for modified NP surface characterization. Research and development are in progress on innovative surface-modified NP-based cancer and gene therapies targeting cells, tissues, and organs. Next-generation nanomedicine should compile studies on the practice and standardization of the measurement methods for NPs to design surface modifications and ensure the quality of NPs.

In Vitro Sensitivity Analysis of the Gastrointestinal Dissolution Profile of Weakly Basic Drugs in the Stomach-to-Intestine Fluid Changing System: Explanation for Variable Plasma Exposure after Oral Administration.

An in vitro methodology for simulating the change in the pH and composition of gastrointestinal fluid associated with the transition of orally administered drugs from the stomach to the small intestine was developed (the stomach-to-intestine fluid changing system (the SIFC system)). This system was applied to in vitro sensitivity analysis on the dissolution of weakly basic drugs, and the obtained results were discussed in relation to the intrasubject variability in the plasma exposure in human bioequivalence (BE) study. Three types of protocols were employed (steep pH change: pH 1.6 FaSSGF → pH 6.5 FaSSIF, gradual pH change: pH 1.6 FaSSGF → pH 6.5 FaSSIF, and high gastric pH: pH 4.0 FaSSGF → pH 6.5 FaSSIF). Regardless of the protocols and the forms of drug applied in active pharmaceutical ingredient powder or formulation, dissolution profiles of pioglitazone after fluid shift were similar and the final concentrations in FaSSIF were approximately equal to the saturation solubility in FaSSIF, supporting its small intrasubject variance in human BE study. In contrast, dissolved concentration of terbinafine in the SIFC system became less than half in the high gastric pH protocol than that in other protocols, suggesting the fluctuation of gastric pH as one of the factors of high intrasubject variance of terbinafine in human. Plasma exposure of telmisartan was highly variable especially at the high dose. Although the dissolution of telmisartan in the SIFC system was greatly improved by formulation, it considerably fluctuated during fluid shift especially at the high dose, which corresponds well to in vivo results.

Evaluation of Drug Sorption on Laboratory Materials with Abraham Solvation Parameters of Drugs and its Prevention.

PURPOSE: Undesired drug sorption on laboratory material surfaces reduces the performance of analytical methods and results in the generation of unreliable data. Hence, we characterized the sorption of drugs and evaluated the sorption extent using a linear free energy relationship (LFER) model with Abraham solvation parameters of drugs. Furthermore, to prevent sorption, the effects of additives, such as organic solvents and salts, were evaluated. METHODS: The sorption of fifteen model drugs (concentration: 2 µM), with various physicochemical properties, on materials in 0.2% dimethyl sulfoxide aqueous solutions was evaluated. Drug sorption extent on the materials was determined using high-performance liquid chromatography. The obtained results were analyzed using an LFER model with Abraham solvation parameters of the drugs. The effect of additives on the sorption of itraconazole, one of the most hydrophobic drugs among those tested in this study, was investigated. RESULTS: Sorption was dependent on the physicochemical properties of drugs, rather than the type of materials used, and additives altered the rate of drug sorption. Equations were developed to evaluate the sorption extent (nmol) of drugs to glass and polypropylene using the Abraham solvation parameters of the drugs. CONCLUSIONS: LFER modeling with Abraham solvation parameters of drugs enabled us to evaluate drug sorption on materials. All the additives altered the rate of drug sorption, and some organic solvents effectively prevented sorption. The developed LFER model would be useful for assessment of the sorption properties of compounds in in vitro evaluations in drug discovery research and various other biochemical fields.

N-Nitrosodimethylamine (NDMA) Formation from Ranitidine Impurities: Possible Root Causes of the Presence of NDMA in Ranitidine Hydrochloride.

N-Nitrosodimethylamine (NDMA) is a probable human carcinogen. This study investigated the root cause of the presence of NDMA in ranitidine hydrochloride. Forced thermal degradation studies of ranitidine hydrochloride and its inherent impurities (Imps. A, B, C, D, E, F, G, H, I, J, and K) listed in the European and United States Pharmacopeias revealed that in addition to ranitidine, Imps. A, C, D, E, H, and I produce NDMA at different rates in a solid or an oily liquid state. The rate of NDMA formation from amorphous Imps. A, C, and E was 100 times higher than that from crystalline ranitidine hydrochloride under forced degradation at 110 °C for 1 h. Surprisingly, crystalline Imp. H, bearing neither the N,N-dialkyl-2-nitroethene-1,1-diamine moiety nor a dimethylamino group, also generated NDMA in the solid state, while Imp. I, as an oily liquid, favorably produced NDMA at moderate temperatures (e.g., 50 °C). Therefore, strict control of the aforementioned specific impurities in ranitidine hydrochloride during manufacturing and storage allows appropriate control of NDMA in ranitidine and its pharmaceutical products. Understanding the pathways of the stability related NDMA formation enables improved control of the pharmaceuticals to mitigate this risk.

Altered Media Flow and Tablet Position as Factors of How Air Bubbles Affect Dissolution of Disintegrating and Non-disintegrating Tablets Using a USP 4 Flow-Through Cell Apparatus.

This study investigated how air bubbles in media affect tablet dissolution in a flow-through cell system (USP 4) using disintegrating (USP prednisone) and non-disintegrating (USP salicylic acid) tablets. Cell hydrodynamics were studied using particle image velocimetry (PIV) and computational fluid dynamics (CFD). The PIV analysis showed periodic changes in the local flow corresponding to the discharge and suction of the pump cycles. The absence of prior deaeration induced small air bubbles in the media and lower maximum flow during the cycle, explaining the slower dissolution of the USP salicylic acid tablets. Bubbles, occurring during the USP prednisone tablets study, induced the transition of floating disintegrated particles towards the cell outlet, whereas the particles precipitated to form a white layer on the glass beads used in the study with prior deaeration. CFD analysis showed local flow variation in multiple positions of small (ID 12 mm) and large (ID 22.6 mm) cells, explaining the different rates of dissolution of prednisone tablet particles depending on their distribution. These results emphasize the importance of prior deaeration in dissolution studies using a flow-through system. Bubbles in the flow-through cell system affected tablet dissolution by reducing the area in contact with the media (wettability), lowering the maximum instantaneous flow (pressure buffering), and altering the position of disintegrated particles in the cell.

Physicochemical Characterization of Liposomes That Mimic the Lipid Composition of Exosomes for Effective Intracellular Trafficking.

Exosomes mediate communication between cells in the body by the incorporation and transfer of biological materials. To design an artificial liposome, which would mimic the lipid composition and physicochemical characteristics of naturally occurring exosomes, we first studied the physicochemical properties of exosomes secreted from HepG2 cells. The exosome stiffness obtained by atomic force microscopy was moderate. Some liposomes were then fabricated to mimic the representative reported lipid composition of exosomes. Their physicochemical properties and cellular internalization efficiencies were investigated to optimize the cellular internalization efficiency of the liposomes. A favorable internalization efficiency was obtained by incubating HeLa cells with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol)/1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) (40/40/20 mol %) liposomes, which have a similar stiffness and zeta potential to exosomes. A dramatic increase in internalization efficiency was demonstrated by adding DOPS to simple DSPC/Chol liposomes. We found that DOPS had a more desirable effect on cellular internalization than its saturated lipid counterpart, 1,2-distearoyl-sn-glycero-3-phospho-l-serine. Furthermore, it was shown that the phosphatidylserine-binding protein, T-cell immunoglobulin mucin protein 4, was largely involved in the intracellular transfer of DSPC/Chol/DOPS liposomes. Thus, DOPS was a key lipid to provide the appropriate stiffness, zeta potential, and membrane surface affinity of the resulting liposome. Our results may help develop efficient drug carriers aiming to internalize active substances into cells.

Utilization of Diluted Compendial Media as Dissolution Test Solutions with Low Buffer Capacity for the Investigation of Dissolution Rate of Highly Soluble Immediate Release Drug Products.

Research from the past decade has shown that the buffer capacities of intestinal fluids are much lower than those in the media used for dissolution test of many solid formulations. The purpose of this study was to elucidate the effect of buffer capacity on the dissolution profiles of highly soluble drug products, using metoclopramide (a biopharmaceutics classification system [BCS] class III drug) tablets as a model. The dissolution profiles of three metoclopramide products were obtained in Japanese pharmacopeia dissolution medium (pH 1.2 and 6.8), diluted medium with low buffer capacity comparable to that of gastrointestinal fluid, and other biorelevant media. One product showed slower dissolution in the medium with lower buffer capacity (bio-relevant, diluted compendial solution), but substantially similar dissolution in the compendial test solutions. Disintegration difference was implied to be involved in the different dissolution profiles depending on the medium buffer capacity. This study indicated the importance of media buffer capacity as a factor inducing different dissolution between products of highly soluble active pharmaceutical ingredients. The diluted compendial media would be a useful alternative to biorelevant media for the detection of the different formulation performances depending on the buffer capacities.

Morphological Analysis of Spherical Adsorptive Carbon Granules Using Three-Dimensional X-Ray Micro-computed Tomography.

The purpose of this study was to clarify applicability of three-dimensional X-ray micro-computed tomography (3D X-ray micro-CT) to elucidate interior morphology of spherical adsorptive carbon fine granules. Scanning of small single spherical granule hold on the rotating sample stage provided the structural information without particular preparation (e.g., slicing) that can affect the definite morphology. The three model formulations with similar appearance showed different internal structure in the 3D images, including large hollow in one of them. Other formulations showed some small empty or higher density area in the filled granules, suggesting uneven distribution of carbon. The results indicated relevance of the X-ray micro-CT analysis on the physical characterization of the spherical adsorptive carbon granule formulations.

Effect of Complex Coacervation with Hyaluronic Acid on Protein Transition in a Subcutaneous Injection Site Model System.

The occurrence of complex coacervation in an aqueous mixture of proteins (lysozyme, albumin, immunoglobulin G) and hyaluronic acid and its effect on protein transition in a model system was studied to elucidate factors determining the bioavailability of subcutaneously injected therapeutic proteins. Mixing of hyaluronic acid and the model proteins induced complex coacervation at solution pH close to or below the isoelectric point of the proteins. In vitro dialysis using membranes with large pore size tube represented a limitation in the protein transition of the coacervation mixture. Thermal analysis suggested there was retention of the protein conformation in the polymer complex.

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.

Relationship Between Geometric and Aerodynamic Particle Size Distributions in the Formulation of Solution and Suspension Metered-Dose Inhalers.

The relationship between the geometric particle size distribution (GPSD) and the aerodynamic particle size distribution (APSD) of commercial solution and suspension metered-dose inhaler (MDI) formulations was assessed to clarify the use of GPSD to estimate the APSD. The size distribution of particles discharged from four suspension and four solution MDIs was measured using the Inas®100 light-scattering spectrometer and a Next Generation Impactor. The conversion factor was calculated by measuring the GPSD and APSD of MDIs. The morphology and physical properties of MDIs were studied using scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). Six of the eight MDIs showed similar conversion factor profiles, irrespective of their composition and formulation types. Applying the conversion factor obtained from one of the six MDIs resulted in a particle size distribution comparable to each APSD except for some formulations. The two other solution MDIs, which contained citric acid, had much higher and variable conversion factors. SEM images and DSC scans of the solids obtained by nebulization of the solutions containing beclomethasone and/or citric acid showed the formation of a paste-like amorphous solid. These results indicated that APSD of solution and suspension MDIs that form rigid particles may be estimated by using the conversion factor and GPSD. Contrarily, the estimation is more difficult in formulations that tend to lose the particle structure during the measurement.

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.

Applications of Freezing and Freeze-Drying in Pharmaceutical Formulations.

Freeze-drying is a popular method to prepare pharmaceutical formulations containing structurally complex active ingredients and drug delivery system carriers. The solidification performed at lower temperature significantly improves storage stability of proteins, peptides, antibiotics, vaccines, and liposomes, which are marginally stable in aqueous solutions. Individual components of the freeze-drying process (freezing, primary drying, secondary drying), however, expose proteins to various stresses. Certain excipients, including disaccharides (e.g., sucrose, trehalose) and amino acids, can be added to protect the proteins and supramolecular drug delivery systems against physical stress associated with freezing and storage by substituting the molecular interactions provided by water molecules. Some excipients embed the active ingredients in glass-state solids with low molecular mobility, thereby reducing chemical reactivity. Thus, the use of appropriate excipients and process control is important to protect proteins during freeze-drying. This chapter describes the applications of freeze-drying in the pharmaceutical production process, mainly focusing on formulation and process optimization for protein therapeutics.