Optimization of a Raman Microscopy Technique to Efficiently Detect Amorphous–Amorphous Phase Separation in Freeze‐Dried Protein Formulations

A confocal Raman microscopic technique was optimized to more efficiently detect amorphous–amorphous phase separation in freeze‐dried protein formulations. A Renishaw Raman inVia confocal microscope was used to collect 100–200 μm line maps (2 μm step size) of freeze‐dried protein–excipient formulations. At each point across the line map, the composition was evaluated from the intensity of the nonoverlapping peaks representative of each component. Collection aperture, scan time, and line map length significantly contributed to the phase‐separation analysis, whereas different sample preparation methods did not affect the analysis. Using the optimized parameters (i.e., large aperture 5 s scan time, 200 μm line map), phase separation was successfully detected in binary polymer formulations and was comparable to the previously developed Raman method. However, the previous method required 2.5 h/sample, whereas the optimized method only requires 0.5 h/sample. Phase separation was detected in the following protein–excipient formulations: lysozyme–trehalose (1:1), lysozyme–isomaltose (1:1), β‐lactoglobulin–dextran (1:1), β‐lactoglobulin–dextran (1:3), and β‐lactoglobulin–trehalose (1:1). Phase separation was not detected in lysozyme–sucrose (1:1) and β‐lactoglobulin–sucrose (1:1) formulations. The optimized method successfully detected phase separation in several protein formulations, where phase separation was previously suspected, and promised to be a useful tool for detection of phase separation in amorphous therapeutic formulations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2749–2758, 2014     

Near‐Infrared Imaging for High‐Throughput Screening of Moisture Induced Changes in Freeze‐Dried Formulations

Evaluation of freeze‐dried biopharmaceutical formulations requires careful analysis of multiple quality attributes. The aim of this study was to evaluate the use of near‐infrared (NIR) imaging for fast analysis of water content and related physical properties in freeze‐dried formulations. Model formulations were freeze‐dried in well plates. Samples were imaged with a NIR hyperspectral camera after freeze‐drying and upon storage. On the basis of Karl Fischer titration reference values, a univariate quantification model was constructed and used to visualize the distribution of water within freeze‐dried samples. Differences observed between samples stored at 11% and 43% relative humidity (RH) were found to be related to the amount of amorphous component in the sample. When stored at 43% RH, the moisture content in samples with high sucrose content increased within 2 days and some degree of localized drying was observed within the samples after 3 days of storage. Further investigations with X‐ray powder diffraction confirmed this local drying to be related to crystallization of sucrose. The combination of fast analysis of water content and spatial solid‐state information makes NIR imaging a powerful tool for formulation development of freeze‐dried samples. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2839–2846, 2014     

Investigation of PEG Crystallization in Frozen PEG–Sucrose–Water Solutions: II. Characterization of the Equilibrium Behavior During Freeze-Thawing

Our objective was to characterize, by DSC and XRD, the equilibrium thermal behavior of frozen aqueous solutions containing polyethylene glycol (PEG) and sucrose. Aqueous solutions of (i) PEG (2.5–50% w/w), (ii) sucrose (10% w/v) with different concentrations of PEG (1–20% w/v), and (iii) PEG (2% or 10% w/v) with different concentrations of sucrose (2–20% w/v), were cooled to ? 70°C at 5°C/min and heated to 25°C at 2°C/min in a DSC. Annealing was performed for 2 or 6 h at temperatures, ranging from ? 50 to ? 20°C. Experiments under similar conditions, on select compositions, were also performed in a powder X-ray diffractometer. Two endotherms, observed during heating of a frozen PEG solution (10% w/v), were attributed to PEG–ice eutectic melting and ice melting, and were confirmed by XRD. At higher PEG concentrations (> 37.5% w/w), only the endotherm attributed to the PEG–ice eutectic melting was observed. Inclusion of sucrose decreased both PEG–ice melting and ice melting temperatures. In unannealed systems with a fixed sucrose concentration (10% w/v), the PEG–ice melting event was not observed at PEG concentration < 5% w/v. Annealing for 2–6 h facilitated PEG crystallization. In unannealed systems with a fixed PEG concentration (10% w/v), an increase in the sucrose concentration increased the devitrification but decreased the PEG–ice melting temperature. The PEG–ice melting temperatures obtained by DSC and XRD were in good agreement. In ternary systems at a fixed PEG to sucrose ratio, the T_g^′ as well as the PEG–ice melting temperature were unaffected by the total solute concentration. XRD confirmed the absence of a PEG–sucrose–ice ternary eutectic. When the PEG to sucrose ratio was systematically varied, the PEG–ice and ice melting temperatures decreased with an increase in the sucrose concentration. However, at a fixed PEG to sucrose ratio, the PEG–ice melting temperature, was unaffected by the total solute concentration.     

Crystallization of Amorphous Solid Dispersions of Resveratrol during Preparation and Storage—Impact of Different Polymers

The objective of this study was to investigate intermolecular interactions between resveratrol and polymers in amorphous blends and to study the potential correlations between compound–polymer interactions, manufacturability, and stability of the amorphous system to crystallization during storage. Polymers included two grades of poly (vinylpyrrolidone) (PVP), Eudragit E100 (E100), hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), carboxymethyl cellulose acetate butyrate, and poly (acrylic acid) (PAA). Amorphous blends (“solid dispersions”) were prepared by dissolving both resveratrol and polymer in a solvent followed by rotary evaporation. Crystallinity was evaluated using X‐ray powder diffraction and was studied as a function of time. Mid‐infrared (IR) spectroscopy was used to investigate resveratrol–polymer interactions. Polymer influence on the crystallization behavior of resveratrol varied and could be correlated to the polymer structure, whereby polymers with good hydrogen bond acceptor groups performed better as crystallization inhibitors. Resveratrol–polymer hydrogen bonding interactions could be inferred from the IR spectra. Somewhat surprisingly, E100 and resveratrol showed evidence of an acid–base reaction, in addition to intermolecular hydrogen bonding interactions. PVP K29/32 appeared to form stronger hydrogen bond interactions with resveratrol relative to HPMC, HPMCAS, and PAA, consistent with acceptor group chemistry. Long‐term stability of the systems against crystallization suggested that stability is linked to the type and strength of intermolecular interactions present. whereby resveratrol blended with E100 and PVP K29/32 showed the greatest stability to crystallization. In conclusion, amorphous resveratrol is unstable and difficult to form, requiring the assistance of a polymeric crystallization inhibitor to facilitate the formation of an amorphous solid dispersion. Polymers effective at inhibiting crystallization were identified, and it is rationalized that their effectiveness is based on the type and strength of their intermolecular interactions with resveratrol.     

Can Controlled Ice Nucleation Improve Freeze‐Drying of Highly‐Concentrated Protein Formulations?

The aim of this study was to investigate if controlled ice nucleation with our previouly published method is applicable to highly‐concentrated protein formulations of bovine serum albumin (100 mg/ml and 193.9 mg/ml) and a monoclonal antibody (161.2 mg/ml) and if positive effects on primary drying time as well as reconstitution times can be achieved. We observed that for both proteins, ice nucleation at a product temperature of ?5 °C significantly reduced primary drying time. Furthermore, reconstitution times of the lyophilized cakes could be shortened, especially for the monoclonal antibody formulation with a reconstitution time of 5 minutes instead of 15 minutes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3915–3919, 2013     

Direct Compression Tablet Containing 99% Active Ingredient—A Tale of Spherical Crystallization

Direct compression (DC) is the easiest and most cost-effective process for tablet manufacturing, because it only involves blending and compression. However, active pharmaceutical ingredients generally exhibit poor mechanical and micromeritic properties, which necessitate dilution and the use of high percentage of excipients to enable a robust DC manufacturing process. Consequently, drug loading in DC tablets is usually low (typically <30%, w/w). In this study, spherical crystallization by the quasi-emulsion solvent diffusion method was used to engineer a poorly flowing model compound, ferulic acid (FA), to attain superior mechanical properties, particle size distribution, and morphology. The engineered FA particles enabled the successful development of DC tablets containing 99% FA, which is in sharp contrast to the maximum 10% FA loading using as-received FA. The record high active pharmaceutical ingredient loading in this work illustrates the potential for spherical crystallization to enable high drug loading when developing a tablet product using the DC manufacturing process.     

Effect of Different “States” of Sorbed Water on Amorphous Celecoxib

Glass transition temperature (T_g) of an amorphous drug is a vital physical phenomenon that influences its visco-elastic properties, physical, and chemical stability. Water acts as a plasticizer for amorphous drugs thus increasing their recrystallization kinetics. This reduces the solubility advantage of an amorphous drug. Hence, there is an interest in understanding the relationship between water content and T_g of amorphous drug. We have studied the effect of “state” of sorbed water on T_g of amorphous celecoxib (ACLB). ACLB was allowed to sorb water at relative humidity of 33%, 53%, 75%, and 93%. ALCB showed biphasic sorption of water designated as “bound” and “solvent-like” state of water associated with ACLB. Molecular modeling studies provided deeper insights into the interaction of water with ACLB. A distinct co-relationship between the state of water and its plasticization capacity was observed. Bound state of water had a very profound effect on the fall in experimentally observed T_g (T_g-exp) value. Solvent-like state of water had little impact on T_g-exp value. T_g of ACLB–water mixture was predicted by Gordon–Taylor equation (T_g-pre). The deviations in T_g-exp and T_g-pre were correlated to volume non-additivity and non-ideal mixing. This study has implications on the development of formulations based on amorphous forms.     

Assessing the Impact of Polymers on the pH‐Induced Precipitation Behavior of Poorly Water Soluble Compounds using Synchrotron Wide Angle X‐Ray Scattering

The aim of this study was to investigate the pH‐induced precipitation behavior of four ionizable compounds (papaverine, dipyridamole, glyburide, and warfarin) in the absence and presence of polymers. Polymers selected included nonionic, anionic, and cationic polymers. Precipitates were analyzed immediately after formation using high‐energy radiation wide‐angle X‐ray scattering analysis and polarized light microscopy. Papaverine immediately crystallized to the original solid‐state form upon creation of a highly supersaturated solution and polymers were unable to prevent crystallization. Dipyridamole also crystallized rapidly, forming a metastable polymorph that was stabilized by several of the cellulosic polymers. For glyburide and warfarin, although the compounds readily crystallized in the absence of the polymers, several of the polymers were able to prevent crystallization for more than 6 h. In general, measurements of solution concentration immediately following precipitation corroborated the solid‐state analysis results, with the solution phase for the noncrystalline precipitates having a concentration considerably higher than that of the equilibrium solubility value, whereas for the crystalline precipitates, values were closer to the equilibrium solubility. Thus, precipitation to a noncrystalline solid was found to be promoted by the presence of some polymers, resulting in the formation of a supersaturated solution. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2724–2735, 2014     

Assessment of Crystallinity in Processed Sucrose by Near‐Infrared Spectroscopy and Application to Lyophiles

The objective of this study was to investigate the capability of near‐infrared spectroscopy (NIRS) to determine crystallinity in processed sucrose using a common set of calibration standards derived from binary physical mixtures. NIRS was applied as a primary method using binary mixtures of amorphous and crystalline standards to predict crystallinity in sucrose that was either rendered partially amorphous by milling, partially recrystallized from the amorphous phase, or amorphous lyophiles annealed to induce recrystallization. Crystallinity prediction in the case of milled crystalline and recrystallized amorphous sucrose was feasible using the two‐state binary calibration mixtures applying a univariate model. NIRS results for milled sucrose were comparable to those obtained using X‐ray powder diffraction. The changes in crystallinity after milling and recrystallization showed expected trends. However, the same NIRS univariate calibration method could not be successfully applied for directly through the vial. To overcome this complication, NIRS was applied as a secondary method relative to water vapor sorption (WVS) where a set of processed samples were measured using both NIRS and WVS and a partial least‐squares model applied. The NIRS secondary method was successfully applied and provided a standard error of calibration of 2.11% and standard error of prediction of 3.76%. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2884–2895, 2014     

Stabilized Amorphous State of Ibuprofen by Co‐Spray Drying With Mesoporous SBA‐15 to Enhance Dissolution Properties

A novel formulation process via co‐spray drying ibuprofen (IBU) with mesoporous SBA‐15 submicron particles exhibited excellence in production of stable amorphous IBU with significantly enhanced dissolution rate. With drug loading of IBU/SBA‐15 ratio being 50:50 (w/w) or below, most drug molecules were entrapped inside the straight mesoporous channels via the co‐spray drying and the morphology of SBA‐15 submicron particles remained unchanged. IBU confined inside the mesoporous structure was in the amorphous state shown by PXRD and DSC measurements. The amorphous state of IBU in the solid dispersion showed remarkable stability when subject to stress test condition of 40°C/75% RH in open pans for 12 months. The uniform pore walls were believed to prevent the re‐crystallization of the homogeneously dispersed drug molecules inside the mesoporous channels with confined nanospace. The dissolution rate of IBU from the co‐spray‐dried solid dispersion was significantly enhanced to achieve a rapid release. Even after the accelerated stability test, the rapid drug release property was well preserved. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1997–2007, 2010     

Crystallization from the Amorphous State: Nucleation–Growth Decoupling,Polymorphism Interplay,and the Role of Interfaces

The physical stability of the amorphous state is governed by crystallization, which results from the complex interplay of nucleation and growth processes. These processes can be further complicated by the preferred initial nucleation of less‐stable phases, and interpretation requires the evaluation of the relative roles of structure, dynamics, and thermodynamics on the kinetics of the recrystallization. As a contribution to this issue, we reanalyze data sets concerning recrystallization of two pharmaceutical compounds: l‐arabitol and RS ibuprofen. These compounds share the property of being good glass formers and present monotropic polymorphism. In the present analysis, we are mainly focusing on the localization of nucleation and growth zones and the role of a transient crystallization of the metastable phase. On the basis of the elementary theories, the results offer the opportunity to discuss the impact of interfacial energies, molecular mobility, crystal disorder, liquid short‐range order, and crack formation in the glass. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2615–2628, 2014     

Effect of 2-Hydroxypropyl-β-cyclodextrin on Crystallization and Polymorphic Transition of Nifedipine in Solid State

The glassy state of nifedipine (NP) was prepared in the absence and presence of 2-hydroxypropyl--cyclodextrin (HP--CyD), and its crystallization and polymorphic transition behavior was investigated by differential scanning calorimetry (DSC) and powder X-ray diffractometry. In DSC thermograms, the glassy NP exhibited an en-dothermic peak at 48°C representing the glass transition of NP, an exothermic peak at 105°C for the crystallization to a metastable form of NP (Form B), an exothermic peak at 125°C for the polymorphic transition of Form B to a stable form of NP (Form A), and an endothermic peak at 171°C for the melting of Form A. The powder X-ray diffractogram of Form B was apparently different from that of Form A. In the presence of HP--CyD, the exothermic peak at 125°C for the Form B to A transition disappeared and a new en-dothermic peak appeared at 163°C. This new peak was ascribed to the melting of Form B, and the conversion of Form B to Form A was significantly suppressed in HP--CyD matrix. Upon storage at 60°C, the glassy NP was converted to Form A with an activation energy of 18 kcal/mol. The apparent dissolution rate of the NP/HP--CyD (molar ratio 1:1) increased in the order of glassy NP < Form A < Form B, because the glassy NP was readily converted to Form A upon contact with water, resulting in a lower dissolution rate. The present data suggest that HP--CyD is useful for the preparation of a fast dissolving form of metastable NP through glassy NP.     

Effect of Fill Weight,Capsule Shell,and Sinker Design on the Dissolution Behavior of Capsule Formulations of a Weak Acid Drug Candidate BMS‐309403

Two strengths of BMS‐309403 capsules were developed from a common stock granulation. Dissolution testing of the capsules was conducted utilizing the USP apparatus 2 (paddle) with a neutral pH dissolution medium. Unexpectedly, the lower‐strength capsules exhibited slower dissolution than the higher‐strength capsules filled with the same stock granulation. Higher variability was also observed for the lower‐strength capsules. This was found to be mainly caused by a low fill weight in a relatively large size hard gelatin capsule shell. Instead of bursting open, some gelatin capsule shells softened and collapsed onto the granulation, which delayed the release of the active drug. The problem was aggravated by the use of coil sinkers which hindered the medium flow around the capsules. Switching from the gelatin capsule shells to the HPMC (hydroxypropyl methylcellulose) shells reversed the dissolution rate ranking between the two capsule strengths. However, both dissolved at a slower rate initially than the gelatin capsules due to the inherent dissolution rate of the HPMC shells at pH 6.8. Notably, the HPMC shells did not occlude the granulation as observed with the gelatin shells. The study demonstrated that the dissolution of capsule formulations in neutral pH media was significantly affected by the fill weight, sinker design, and capsule shell type. Careful selection of these parameters is essential to objectively evaluate the in vitro drug release.     

The Measurement of the β/α Anomer Composition Within Amorphous Lactose Prepared by Spray and Freeze Drying Using a Simple 1H-NMR Method

Purpose  

Reports of the anomeric composition of amorphous lactose are rare and state a highly variable range of composition (between 0% and 60% w/w β content). We aimed to develop a quantitative measurement by ¹H-NMR of α and β anomer content in amorphous lactose produced by different production methods.     

Co-Spray Dried Carbohydrate Microparticles: Crystallisation Delay/Inhibition and Improved Aerosolization Characteristics Through the Incorporation of Hydroxypropyl-β-cyclodextrin with Amorphous Raffinose or Trehalose

Pharmaceutical Research - To formulate and investigate the physicochemical properties, physical stability and aerosolization characteristics of nanoporous/nanoparticulate microparticles (NPMPs)...     

Effects of Inhibitor of κB Kinase Activity in the Nucleus Accumbens on Emotional Behavior

Inhibitor of κB kinase (IκK) has historically been studied in the context of immune response and inflammation, but recent evidence demonstrates that IκK activity is necessary and sufficient for regulation of neuronal function. Chronic social defeat stress of mice increases IκK activity in the nucleus accumbens (NAc) and this increase is strongly correlated to depression-like behaviors. Inhibition of IκK signaling results in a reversal of chronic social defeat stress-induced social avoidance behavior. Here, we more completely define the role of IκK in anxiety and depressive-like behaviors. Mice underwent stereotaxic microinjection of a herpes simplex virus expressing either green fluorescent protein, a constitutively active form of IκK (IκKca), or a dominant negative form of IκK into the NAc. Of all three experimental groups, only mice expressing IκKca show a behavioral phenotype. Expression of IκKca results in a decrease in the time spent in the non-periphery zones of an open field arena and increased time spent immobile during a forced swim test. No baseline differences in sucrose preference were observed, but following the acute swim stress we noted a marked reduction in sucrose preference. To determine whether IκK activity alters responses to other acute stressors, we examined behavior and spine morphology in mice undergoing an acute social defeat stress. We found that IκKca enhanced social avoidance behavior and promoted thin spine formation. These data show that IκK in NAc is a critical regulator of both depressive- and anxiety-like states and may do so by promoting the formation of immature excitatory synapses.     

Impacts of spinosad and λ-cyhalothrin on spider communities in cabbage fields in south Texas

Spiders are a principal arthropod group that preys on numerous pests of vegetables and other crops. In this study, we determined the effects of the two most commonly used insecticides, spinosad and λ-cyhalothrin, on diversity of spiders on cabbage in south Texas. In two seasons (fall 2008 and spring 2009), we collected a total of 588 spiders belonging to 53 species in 11 families from spinosad and λ-cyhalothrin-treated cabbages and the untreated control plants. A great majority of spiders were collected from the pitfall traps (554) where only a few (34) were collected from the blower/vacuum sampling. In the insecticide-treated plots, there were significantly fewer spider individuals, species and families than in untreated fields. Spinosad had significantly less effect on spiders in total individuals, number of species and families than λ-cyhalothrin. The effects of the two insecticides were further demonstrated by the Shannon–Weiner index (H′) and the hierarchical richness index (HRI). Spider diversity in the spinosad-treated plots were not significantly different from that in the untreated fields but were greater than those in λ-cyhalothrin-treated plots in both seasons when measured by H′ values. In contrast, the H′ values of spider’s diversity in the λ-cyhalothrin-treated plots were significantly lower than spinosad-treated and untreated plots. High values of HRI for spider richness in the spinosad-treated plots suggested that spinosad had less effect on spiders than λ-cyhalothrin. We concluded that spinosad was more compatible with spiders on cabbage compared to λ-cyhalothrin and that this information should be used when developing insecticide resistance management strategies.     

Protective effect of 23‐hydroxybetulinic acid on doxorubicin‐induced cardiotoxicity: a correlation with the inhibition of carbonyl reductase‐mediated metabolism

Background and Purpose

The clinical use of doxorubicin, an effective anticancer drug, is severely hampered by its cardiotoxicity. 23‐Hydroxybetulinic acid (23‐HBA), isolated from P ulsatilla chinensis, enhances the anticancer effect of doxorubicin while simultaneously reducing its cardiac toxicity, but does not affect the concentration of doxorubicin in the plasma and heart. As the metabolite doxorubicinol is more potent than doxorubicin at inducing cardiac toxicity, in the present study we aimed to clarify the role of doxorubicinol in the protective effect of 23‐HBA.

Experimental Approach

Doxorubicin was administered to mice for two weeks in the presence or absence of 23‐HBA. The heart pathology, function, myocardial enzymes and accumulation of doxorubicin and doxorubicinol were then analysed. A cellular pharmacokinetic study of doxorubicin and doxorubicinol, carbonyl reductase 1 (CBR1) interference and molecular docking was performed in vitro.

Key Results

23‐HBA alleviated the doxorubicin‐induced cardiotoxicity in mice, and this was accompanied by inhibition of the metabolism of doxorubicin and reduced accumulation of doxorubicinol selectively in hearts. In H9c2 cells, the protective effect of 23‐HBA was shown to be closely associated with a decreased rate and extent of accumulation of doxorubicinol in mitochondria and nuclei. siRNA and docking analysis demonstrated that CBR1 has a crucial role in doxorubicin‐mediated cardiotoxicity and 23‐HBA inhibits this metabolic pathway.

Conclusions and Implications

Inhibition of CBR‐mediated doxorubicin metabolism might be one of the protective mechanisms of 23‐HBA against doxorubicin‐induced cardiotoxicity. The present study provides a new research strategy guided by pharmacokinetic theory to elucidate the mechanism of drugs with unknown targets.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

23‐HBA

23‐hydroxybetulinic acid

AST

aspartate aminotransferase

CBR1

carbonyl reductase 1

CMC

carboxymethyl cellulose

CK

creatine kinase

CK‐MD

creatine kinase isoenzyme

EF

ejection fraction

FS

fractional shortening

i.g

intragastrically

LV

left ventricle

MDR

multidrug resistance

TCM

traditional Chinese medicine

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