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.

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.

Biorelevant in vitro performance testing of orally administered dosage forms-workshop report.

Biorelevant in vitro performance testing of orally administered dosage forms has become an important tool for the assessment of drug product in vivo behavior. An in vitro performance test which mimics the intraluminal performance of an oral dosage form is termed biorelevant. Biorelevant tests have been utilized to decrease the number of in vivo studies required during the drug development process and to mitigate the risk related to in vivo bioequivalence studies. This report reviews the ability of current in vitro performance tests to predict in vivo performance and generate successful in vitro and in vivo correlations for oral dosage forms. It also summarizes efforts to improve the predictability of biorelevant tests. The report is based on the presentations at the 2013 workshop, Biorelevant In Vitro Performance Testing of Orally Administered Dosage Forms, in Washington, DC, sponsored by the FIP Dissolution/Drug Release Focus Group in partnership with the American Association of Pharmaceutical Scientists (AAPS) and a symposium at the AAPS 2012 Annual meeting on the same topic.

Thermotropic phase behavior of hydrogenated soybean phosphatidylcholine-cholesterol binary liposome membrane.

By combination of differential scanning calorimetry (DSC) and fluorescence spectroscopy of 6-propionyl-2-(dimethylamino)naphthalene (Prodan), we elucidated the thermotropic phase behavior of hydrogenated soybean phosphatidylcholine (HSPC)-cholesterol binary liposome membrane which has similar lipid composition to Doxil®, the widely used liposome product in treatment of various tumors. We found that the characteristic points at cholesterol mole fraction (Xch)=0.023 and 0.077 correspond to the hexagonal lattice, in which cholesterol molecules are considered to be regularly distributed in all regions of HSPC lipid bilayer with 1 : 42 and 1 : 12 units, respectively, as static averaged structures. Apparent endothermic peak disappeared at Xch=0.40 in the DSC thermograms, indicating the existence of single liquid ordered phase at Xch>0.40. In addition, fluorescence measurements of Prodan and its lauroyl derivative in poly(ethylene glycol) (PEG)-modified liposomes indicated that PEG modification has a negligible effect on the phase behavior of HSPC-cholesterol binary liposome membrane. These results may provide useful information in developing novel liposome products whose stability and encapsulated drug release are controlled.

Studying the morphology of lyophilized protein solids using X-ray micro-CT: effect of post-freeze annealing and controlled nucleation.

The objective of this study was to determine how different techniques used during the freezing step of lyophilization affect morphology of the dried protein solids. Aqueous solutions containing recombinant human albumin, trehalose, and sodium phosphate buffer were dried after their freezing by shelf-ramp cooling, immersion in liquid nitrogen, or controlled ice nucleation. Some shelf-frozen solutions were heat treated (annealed) before the vacuum drying. We used three-dimensional (3D) X-ray micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) to study the morphology of solids. The X-ray micro-CT images of the lyophilized microporous solids showed traces of varied size and structure ice crystals that were comparable to corresponding SEM images. A post-freeze heat treatment and a controlled nucleation both induced larger ice crystal ghosts in the solids. The variations in the structure of walls surrounding ice crystals, formed by the different freezing procedures, should affect the water vapor transition during the primary and secondary drying. Some solids also showed higher-density layer in the upper surface. Overall, the simple sample preparation procedures and the ample morphological information make the X-ray micro-CT appropriate for analyzing lyophilized pharmaceuticals.

Simultaneous determination of polyethylene glycol-conjugated liposome components by using reversed-phase high-performance liquid chromatography with UV and evaporative light scattering detection.

Liposomes incorporating polyethylene glycol (PEG)-conjugated lipids (PEGylated liposomes) have attracted attention as drug delivery carriers because they show good in vivo stability. The lipid component of PEGylated liposomal formulations needs to be quantified for quality control. In this study, a simple reversed-phase high-performance liquid chromatography (HPLC) method with an evaporative light-scattering detector (ELSD) was established for simultaneous determination of hydrogenated soy phosphatidylcholine, cholesterol, PEG-conjugated lipid, and hydrolysis products of phospholipid in PEGylated liposomal formulations. These lipids were separated using a C18 column with a gradient mobile phase consisting of ammonium acetate buffer and ammonium acetate in methanol at a flow rate of 1.0 ml/min. This method provided sufficient repeatability, linearity, and recovery rate for all lipids. However, the linearity and recovery rates of cholesterol achieved using a ultraviolet (UV) detector were better than those achieved using an ELSD. This validated method can be applied to assess the composition change during the preparation process of liposomes and to quantify lipid components and hydrolysis products contained in a commercially available liposomal formulation DOXIL®. Taken together, this reversed-phase HPLC-UV/ELSD method may be useful for the rapid or routine analysis of liposomal lipid components in process development and quality control.

Polyethylene glycol prevents in vitro aggregation of slightly negatively-charged liposomes induced by heparin in the presence of bivalent ions.

Liposomes are of great interest as drug delivery vehicles, and studies have focused on understanding how the physical and chemical characteristics of liposomes can be modified to improve their in vivo behavior. In a previous study, we found that the slightly negatively-charged liposomes aggregate only in the culture medium of human umbilical vein endothelial cells, whereas the liposomes modified with polyethylene glycol (PEG) (PEGylated) did not aggregate. In the present study, we investigated the underlying mechanism of this phenomenon. Firstly, it was found that heparin in the culture medium is one of the factors that cause aggregation of the non-PEGylated liposomes. Since the addition of ethylenediaminetetraacetic acid (EDTA) prevented the aggregation, metal ions, such as Ca(2+) and Mg(2+), in the culture medium could also be important in driving the aggregation. In the presence of heparin, higher concentrations of Ca(2+) or Mg(2+) increased the particle size of the non-PEGylated liposomes, although no change in the particle size of PEGylated liposomes was observed. Under conditions in which aggregation occurred, we measured the binding and uptake of liposomes by macrophages in vitro. The binding and uptake of non-PEGylated liposomes were significantly increased with increasing Ca(2+) concentrations, whereas those of PEGylated liposomes were unchanged. While the formation of aggregations of cationic or anionic liposomes has been reported previously, there are few reports addressing the aggregation of slightly negatively-charged or neutral liposomes. Thus, our data provide useful insights on the effect of PEGylation on liposomal aggregation and in vivo behavior.

Alterations in the detergent-induced membrane permeability and solubilization of saturated phosphatidylcholine/cholesterol liposomes: effects of poly(ethylene glycol)-conjugated lipid.

We have investigated the effects of two bile salts, chenodeoxycholate (CDC) and ursodeoxycholate (UDC), and a widely used detergent, Triton X-100 (T(X-100)), on normal and poly(ethylene glycol)-modified liposomes (PEGylated liposomes). We tested various lipid compositions, including hydrogenated soybean phosphatidylcholine/cholesterol/PEG-conjugated lipid (HSPC/PEG-lipid). Alterations in permeability were determined by the rate of drug release from the liposomes and solubilization was assessed by measuring the particle size of liposomes. In addition, we attempted to observe interactions between the detergents and lipid bilayers by using surface plasmon resonance (SPR). CDC induced drug release from liposomes in a dose-dependent manner, and the PEGylated liposomes tended to be susceptible to CDC. While UDC did not strongly induce drug release from liposomes, UDC exhibited a similar tendency with CDC. In case of T(X-100), there were significant differences in the percentage of released drug between normal and PEGylated liposomes, and the percentage of T(X-100)-induced drug release further increased with an increased ratio of PEG-lipid. SPR analysis revealed that the lipid bilayer including PEG-lipid was selectively solubilized by T(X-100), correlating with the drug release data. These results suggest that the effect of detergents on the lipid bilayer of liposomes depends on both the kind of detergent and the lipid composition, including the presence or absence of PEG-lipid. Moreover, the effects of T(X-100) on the lipid bilayers of the PEGylated liposomes significantly differed from those on the lipid bilayers of the normal liposomes.

Comparison of particle size and dispersion state among commercial cyclosporine formulations and their effects on pharmacokinetics in rats.

Generic versions of Neoral, a microemulsion capsule formulation of cyclosporine, have been approved worldwide. However, there are concerns about the quality and efficacy of the generics due to the formulation specificity and differences in inactive ingredients among products. In this study, we measured the physicochemical properties of both the innovator and the generic formulations, and compared their bioavailability in rats. When the capsule contents were dispersed in water, the absorbance (600 nm wavelength) of generic products was higher than that of the innovator. Whereas the dispersion solution of the innovator in Fed State Simulated Intestinal Fluid was nearly clear, that of all the generics became white and turbid. The mean diameter of the microemulsion (or emulsion) formed in water by the generics was 39.7, 57.7, 64.5, and 74.8 nm, all of which were larger than that of the innovator (26.4 nm). Although the T(max) of the generics tended to be long relative to that of the innovator, there were no significant differences between the innovator and generics with regard to maximum blood concentration (C(max)) or area under the curve (AUC). These results suggest that the physicochemical differences between the innovator and the generics will not have a significant effect on C(max) or AUC, which is necessary to ensure bioequivalence.

Component crystallization and physical collapse during freeze-drying of L-arginine-citric acid mixtures.

Component crystallization and physical collapse during freeze-drying of aqueous solutions containing protein-stabilizing L-arginine and citric acid mixtures were studied. Freeze-drying microscopy (FDM) and thermal analysis of the solute-mixture frozen solutions showed collapse onset at temperatures (T(c)) approximately 10°C higher than their T(g)'s (glass transition temperatures of the maximally freeze-concentrated solute phase). Experimental freeze-drying of these solutions at a low chamber pressure showed the occurrence of physical collapse at shelf temperatures close to or slightly higher than the T(c). Slower ice sublimation at higher chamber pressures induced the physical collapse from lower shelf temperatures. The large effect of chamber pressures on the collapse-inducing shelf temperatures confirmed significance of the sublimation-related heat loss on the sublimation interface temperature during the primary drying. Drying of the single-solute L-arginine solution resulted in cake-structure solids composed of its anhydrous crystal. Thermal and powder X-ray diffraction (PXRD) analysis suggested slow crystal nucleation of L-arginine dihydrate in the frozen solutions. Characterization of the frozen solutions and freeze-dried solids should enable rational formulation design and process control of amino acid-containing lyophilized pharmaceuticals.

[Trends in the quality evaluation of generic products and bioequivalence guidelines].

Recent activities on the generic products such as the revision of bioequivalence guidelines, the accomplish of the reevaluation of the oral dosage forms approved before 1995, and the action program for promoting comfortable use of generics issued by MHLW in 2007, were summarized in this review. The bioequivalence guidelines established in 1997 were revised in 2012 based on the discussion in a dissolution working group (WG). The WG were consists of the members from pharmaceutical companies, academia and regulators belonging to MHLW, PMDA and NIHS. In the revision, some flexibility in the dissolution test conditions was achieved considering the many experiences. And also the special Q&A for the combination products was published at the same time. The reevaluation of the oral products since 1997 was completed in 2010, and 1361 dissolution specifications for 4133 oral products were noticed. Through the reevaluation the sufficient similarity in the dissolution profiles between the standards product and the generic products was achieved in the Japanese pharmaceutical market. In the action program to promote the share of generics, the special committee was established in the NIHS to assess the scientific papers that reported the quality concern of the commercial generic products and to confirm the target quality of the products by testing. Many generic products were checked their dissolution profile similarities to the reference products in multimedia dissolution tests and the appropriate similarities were shown in most products. In some preparations, the purity tests were performed and the content of the impurity is confirmed to be in the acceptance range.

Exploring clinically relevant dissolution specifications for oral solid dosage forms of weak acid drugs using an in silico modeling and simulation approach.

The aim of this study was to explore clinically relevant dissolution specifications for weak acid drugs using an in silico drug absorption model. Loxoprofen sodium and ibuprofen were used as model drugs in this study. An in silico drug absorption model was developed using Stella Professional software and the prediction model accurately represented the plasma concentration profiles of the model drugs following oral administration. Theoretical pharmacokinetic profiles and parameters of the model drugs were predicted using various dissolution rate values in gastrointestinal fluid. This in silico modeling and simulation approach suggests that it is possible to estimate the minimum required dissolution rate for bioequivalence, an example of a clinically relevant dissolution specification. Furthermore, an in vitro dissolution test was conducted for selected drug products of each model drug using paddle apparatus and the results were compared with the clinically relevant dissolution specifications predicted using the in silico simulation.

Stabilization of protein structure in freeze-dried amorphous organic acid buffer salts.

The purpose of this study was to elucidate the physical properties and protein-stabilizing effects of some pH-adjusting excipients (carboxylic acids and their sodium salts) in frozen solutions and in freeze-dried solids. Thermal and powder X-ray diffraction (XRD) analysis indicated a high propensity of sodium citrates to form glass-state amorphous solids upon freeze-drying. Some salts (e.g., sodium succinate) crystallized in the single-solute frozen solutions. FT-IR analysis of bovine serum albumin (BSA) and bovine immunoglobulin G (IgG) in the aqueous solutions and the freeze-dried solids showed that some glass-forming salts (e.g., monosodium citrate) protected the secondary structure from lyophilization-induced perturbation. Freeze-drying of BSA at different concentrations indicated retention of the secondary structure at similar monosodium citrate/protein concentration ratios, suggesting stabilization through direct interaction that substitute water molecules inevitable for the conformation integrity. The carboxylic acid salts should provide rigid hydrogen bonds and electrostatic interactions that raise the glass transition temperature of the amorphous solids and stabilize protein structure. The relevance of the structural stabilization to the protein formulation design was discussed.

Near-infrared analysis of hydrogen-bonding in glass- and rubber-state amorphous saccharide solids.

Near-infrared (NIR) spectroscopic analysis of noncrystalline polyols and saccharides (e.g., glycerol, sorbitol, maltitol, glucose, sucrose, maltose) was performed at different temperatures (30-80 degrees C) to elucidate the effect of glass transition on molecular interaction. Transmission NIR spectra (4,000-12,000 cm(-1)) of the liquids and cooled-melt amorphous solids showed broad absorption bands that indicate random configuration of molecules. Heating of the samples decreased an intermolecular hydrogen-bonding OH vibration band intensity (6,200-6,500 cm(-1)) with a concomitant increase in a free and intramolecular hydrogen-bonding OH group band (6,600-7,100 cm(-1)). Large reduction of the intermolecular hydrogen-bonding band intensity at temperatures above the glass transition (T(g)) of the individual solids should explain the higher molecular mobility and lower viscosity in the rubber state. Mixing of the polyols with a high T(g) saccharide (maltose) or an inorganic salt (sodium tetraborate) shifted both the glass transition and the inflection point of the hydrogen-bonding band intensity to higher temperatures. The implications of these results for pharmaceutical formulation design and process monitoring (PAT) are discussed.

FIP position paper on qualification of paddle and basket dissolution apparatus.

The qualification process for ensuring that a paddle or basket apparatus is suitable for its intended use is a highly debated and controversial topic. Different instrument qualification and suitability methods have been proposed by the pharmacopeias and regulatory bodies. In an effort to internationally harmonize dissolution apparatus suitability requirements, the International Pharmaceutical Federation's (FIP) Dissolution/Drug Release Special Interest Group (SIG) reviewed current instrument suitability requirements listed in the US, European, and Japanese pharmacopeias and the International Conference on Harmonization (ICH) Topic Q4B on harmonization of pharmacopoeial methods, in its Annex 7, Dissolution Test General. In addition, the SIG reviewed the Food and Drug Administration (FDA) Draft Guidance for Industry, "The Use of Mechanical Calibration of Dissolution Apparatus 1 and 2-Current Good Manufacturing Practice (CGMP)" and the related ASTM Standard E2503-07. Based on this review and several in-depth discussions, the FIP Dissolution/Drug Release SIG recommends that the qualification of a dissolution test instrument should be performed following the calibration requirements as indicated in the FDA (draft) guidance. If additional system performance information is desired, a performance verification test using US Pharmacopeia Reference Standard tablet or an established in-house reference product can be conducted. Any strict requirement on the use of a specific performance verification test tablet is not recommended at this time.

Determination of biotin following derivatization with 2-nitrophenylhydrazine by high-performance liquid chromatography with on-line UV detection and electrospray-ionization mass spectrometry.

Currently, biotin is typically determined in Japan using a microbiological method. Such microbiological assays are sensitive, but they are not always highly specific and are also rather tedious and time-consuming. In the present study, RP-HPLC and LC-MS methods for the determination of biotin have been developed by derivatizing the carboxyl group with 2-nitrophenylhydrazine hydrochloride. 2-Nitrophenylhydrazine is used for the derivatization of carboxylic acids, and these derivatives are known to be applicable to LC-MS detection. Biotin in tablets were extracted by the addition of water and ultrasonic agitation. In order to clean up the sample solution, the filtrate was applied to an ODS cartridge and eluted with methanol. The conditions for preparing the 2-nitrophenylhydrazide derivatives were modified from a previous report for fatty acids. Good recovery rates of over 70% were obtained for the addition of 5-125 microg of biotin per formulation. The detection limit in HPLC at 400 nm was 0.6 ng per injection, with good linearity being obtained over the concentration range 0.001-0.2 microg per injection. Further, derivatives were determined by LC-MS with electrospray ionization, where the spectra indicated the molecular ions [M+H](+). The detection limit was 0.025 ng per injection in the selected ion monitoring analysis, and linearity was observed in the range of 0.6-6 ng per injection. The proposed method could be used to specifically determine the presence of biotin in relatively clean samples.

Near-infrared analysis of protein secondary structure in aqueous solutions and freeze-dried solids.

Near-infrared spectroscopy (NIR) of various proteins (bovine serum albumin, lysozyme, ovalbumin, gamma-globulin, beta-lactoglobulin, myoglobin, cytochrome-c) was investigated as a possible analytical method of the protein secondary structure in various physical states. The spectra of proteins in aqueous solutions (transmission mode, solvent-compensated) and those in freeze-dried solids (nondestructive diffuse reflection mode) showed several bands at similar frequencies in the combination (4000-5000 cm(-1)) and first overtone (5600-6600 cm(-1)) spectral regions. The normalized second-derivative near-infrared spectra of proteins in aqueous solutions suggested that some bands indicated alpha-helix (4090, 4365-4370, 4615, and 5755 cm(-1)) and beta-sheet (4060, 4405, 4525-4540, 4865, and 5915-5925 cm(-1)) structures. The proteins mostly maintained spectra characteristic of their native structure after freeze-drying, although some reductions in alpha-helical structure and increase in unordered or beta-sheet structures were observed. The near-infrared analysis also showed beta-sheet formation of heat-treated BSA in aqueous solutions and in subsequently freeze-dried solids. The present results thus indicated that the nondestructive near-infrared analysis can be used for the investigation of dehydration-induced changes in protein secondary structures.

Standard infrared absorption spectrum of betaine and optimal conditions for its measurement.

The infrared absorption (IR) spectrum is often used as a standard reference in identification tests of food additives in Japan. In the case of betaine, many different IR spectra have been reported and, therefore, it is necessary to establish an IR spectrum that is reproducible and reliable enough to be used as a standard for identification. In the present study, suitable conditions to obtain a standard IR spectrum were examined from various viewpoints, including pretreatment, selection of method, and measuring technique. The KBr disk method, which has generally been used to identify betaine, was found to be humidity-dependent, and there was also an interaction between betaine and KBr. A reproducible IR spectrum suitable as a standard could be obtained by drying betaine at 105 degrees C for 3 hours over phosphorus pentoxide, and then measuring the IR spectrum by the liquid paraffin (Nujol) paste method.

[Determination of sodium chlorite in processed herring roe by ion chromatography with a conductivity detector].

An analytical method for residual sodium chlorite in several kinds of processed herring roe treated with sodium chlorite was studied. Sodium chlorite was extracted with 9 mmol/L sodium carbonate. After centrifugation, the supernatant was filtered through a 0.2 microm nylon filter. The filtrate was deproteinized by ultrafiltration and chloride ion was removed with an On-Guard Ag cartridge column. The eluate was subjected to conductivity detector-ion chromatography. Recoveries of sodium chlorite from herring roe spiked at the level of 5 mg/kg were 88 +/- 3.7% (n = 5, CV 4.2%). The method had a quantitation limit of 5 mg/kg for processed herring roes.