Controlled Sublimation Rate of Guest Drug from Polymorphic Forms of a Cyclodextrin-Based Polypseudorotaxane Complex and Its Correlation with Molecular Dynamics as Probed by Solid-State NMR.

Encapsulation of active pharmaceutical ingredients (APIs) in confined spaces has been extensively explored as it dramatically alters the molecular dynamics and physical properties of the API. Herein, we explored the effect of encapsulation on the molecular dynamics and physical stability of a guest drug, salicylic acid (SA), confined in the intermolecular spaces of γ-cyclodextrin (γ-CD) and poly(ethylene glycol) (PEG)-based polypseudorotaxane (PPRX) structure. The sublimation tendency of SA encapsulated in three polymorphic forms of the γ-CD/PEG-based PPRX complex, monoclinic columnar (MC), hexagonal columnar (HC), and tetragonal columnar (TC), was investigated. The SA sublimation rate was decreased by 3.0-6.6-fold and varied in the order of MC form > HC form > TC form complex. The 13C and 1H magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectra and 13C spin-lattice relaxation time (T1) indicated that the encapsulated SA molecules existed as the monomeric form, and its molecular mobility increased in the order of MC form > HC form > TC form complex. In the complexes, a rapid chemical exchange between two dynamic states of SA (free and bound) was suggested, with varying adsorption/desorption rates accounting for its distinct molecular mobility. This adsorption/desorption process was influenced by proton exchange at the interaction site and interaction strength of SA in the complexes, as evidenced by 1H MAS spectra and temperature dependency of the 13C carbonyl chemical shift. A positive correlation between the molecular mobility of SA and its sublimation rate was established. Moreover, the molecular mobility of γ-CD and PEG in the complexes coincided with that of SA, which can be explained by fast guest-driven dynamics. This is the first report on the stability improvement of an API through complexation in polymorphic supramolecular host structures. The relationship between the molecular dynamics and physical properties of encapsulated API will aid in the rational design of drug delivery systems.

Quantitative Investigation of Intestinal Drug Absorption Enhancement by Drug-Rich Nanodroplets Generated via Liquid-Liquid Phase Separation.

Drug-rich droplets formed through liquid-liquid phase separation (LLPS) have the potential to enhance the oral absorption of drugs. This can be attributed to the diffusion of these droplets into the unstirred water layer (UWL) of the gastrointestinal tract and their reservoir effects on maintaining drug supersaturation. However, a quantitative understanding of the effect of drug-rich droplets on intestinal drug absorption is still lacking. In this study, the enhancement of intestinal drug absorption through the formation of drug-rich droplets was quantitatively evaluated on a mechanistic basis. To obtain fenofibrate (FFB)-rich droplets, an amorphous solid dispersion (ASD) of FFB/hypromellose (HPMC) was dispersed in an aqueous medium. Physicochemical characterization confirmed the presence of nanosized FFB-rich droplets in the supercooled liquid state within the FFB/HPMC ASD dispersion. An in situ single-pass intestinal perfusion (SPIP) assay in rats demonstrated that increased quantities of FFB-rich nanodroplets enhanced the intestinal absorption of FFB. The effective diffusion of FFB-rich nanodroplets through UWL would partially contribute to the improved FFB absorption. Additionally, confocal laser scanning microscopy (CLSM) of cross sections of the rat intestine after the administration of fluorescently labeled FFB-rich nanodroplets showed that these nanodroplets were directly taken up by small intestinal epithelial cells. Therefore, the direct uptake of drug-rich nanodroplets by the small intestine is a potential mechanism for improving FFB absorption in the intestine. To quantitatively evaluate the impact of FFB-rich droplets on the FFB absorption enhancement, we determined the apparent permeabilities of the FFB-rich nanodroplets and dissolved FFB based on the SPIP results. The apparent permeability of the FFB-rich nanodroplets was 110-130 times lower than that of dissolved FFB. However, when the FFB-rich nanodroplet concentration was several hundred times higher than that of dissolved FFB, the FFB-rich nanodroplets contributed significantly to FFB absorption improvement. The present study highlights that drug-rich nanodroplets play a direct role in enhancing drug absorption in the gastrointestinal tract, indicating their potential for further improvement of oral absorption from ASD formulations.

Mechanistic Analysis of Temperature-Dependent Curcumin Release from Poly(lactic-co-glycolic acid)/Poly(lactic acid) Polymer Nanoparticles.

In this study, we investigated the mechanism of curcumin (CUR) release from poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) nanoparticles (NPs) by evaluating the temperature-dependent CUR release. NPs were prepared by the nanoprecipitation method using various PLGA/PLA polymers with different lactic:glycolic ratios (L:G ratios) and molecular weights. Increasing the polymer molecular weight resulted in a decrease in the particle size of NPs. The wet glass transition temperature (Tg) of PLGA/PLA NPs was lower than the intrinsic polymer Tg, which can be derived from the water absorption and nanosizing of the polymer. The reduction in Tg was more significant for the PLGA/PLA NPs with lower polymer L:G ratios and lower polymer molecular weight. The greater decrease of Tg in the lower polymer L:G ratios was possibly caused by the higher water absorption due to the more hydrophilic nature of the glycolic acid segment than that of the lactic acid segment. The efficient water absorption in PLGA/PLA NPs with lower molecular weight could cause a significant reduction of Tg as it has lower hydrophobicity. CUR release tests from the PLGA/PLA NPs exhibited enhanced CUR release with increasing temperatures, irrespective of polymer species. By fitting the CUR release profiles into mathematical models, the CUR release process was well described by an initial burst release followed by a diffusion-controlled release. The wet Tg and particle size of the PLGA/PLA NPs affected the amount and temperature dependence of the initial burst release of CUR. Above the wet Tg of NPs, the initial burst release of CUR increased sharply. Smaller particle sizes of PLGA/PLA NPs led to a higher fraction of initial CUR burst release, which was more pronounced above the wet Tg of NPs. The wet Tg and particle sizes of the PLGA/PLA NPs also influenced the diffusion-controlled CUR release. The diffusion rate of CUR in the NPs increased as the wet Tg values of the NPs decreased. The diffusion path length of CUR was affected by the particle size, with larger particle size resulting in a prolonged diffusion-controlled release of CUR. This study highlighted that for the formulation development of PLGA/PLA NPs, suitable PLGA/PLA polymers should be selected considering the physicochemical properties of PLGA/PLA NPs and their correlation with the release behavior of encapsulated drugs at the application temperature.

Quantitative Analysis of Drug Supersaturation Region by Temperature-Variable Nuclear Magnetic Resonance Measurements, Part 2: Effects of Solubilizer.

This study utilized temperature-variable nuclear magnetic resonance (NMR) spectroscopy to investigate the effects of a solubilizing agent on the ketoprofen (KTP) supersaturation region. Quantitative NMR analysis showed that the solubilizing agent cetyltrimethylammonium bromide (CTAB) increased both the crystalline and amorphous solubilities of KTP, shifting the KTP supersaturation region to a higher KTP concentration range. The amorphous solubility of KTP was found to be independent of the enantiomeric composition of KTP, even in the presence of CTAB. However, the supersaturation region of the S-enantiomer of KTP (s-KTP) in CTAB solutions was smaller than that of the racemic form of KTP (rac-KTP), likely because of the higher crystalline solubility of s-KTP. When KTP formed a KTP-rich phase via liquid-liquid phase separation from KTP-supersaturated solutions, CTAB was observed to be distributed into the KTP-rich phase, decreasing the chemical potential of KTP and the maximum thermodynamic activity of KTP in the aqueous phase. Additionally, the incorporation of CTAB into the KTP-rich phase diminished the solubilization effect of CTAB micelles in the aqueous phase, narrowing the KTP supersaturation region to a greater extent at higher KTP dose concentrations. Furthermore, the upper-temperature limit of the supersaturated dissolvable region of KTP was lowered in the presence of CTAB, which was rationalized by the melting point depression of the KTP crystal upon mixing with CTAB. The findings of this study highlight the importance of considering the molecular-level impact of solubilizing agents on the drug supersaturation region to fully exploit the potential benefits of supersaturated formulations.

Quantitative Analysis of Drug Supersaturation Region by Temperature-Variable Nuclear Magnetic Resonance Measurements, Part 1: Effects of Polymer and Drug Chiralities.

We examined the effects of the polymer-additive and drug chiralities on the ketoprofen (KTP) supersaturation region using temperature-variable nuclear magnetic resonance (NMR). Quantitative NMR analysis revealed that the racemic KTP and corresponding S-enantiomer (rac- and s-KTP) exhibited similar amorphous solubilities in a buffer, while the crystalline solubility of s-KTP was higher than that of rac-KTP. Therefore, rac-KTP exhibited a larger supersaturation region than s-KTP. In contrast, polyvinylpyrrolidone (PVP) reduced the amorphous solubility of both rac- and s-KTP, whereas the crystalline solubility of KTP remained unchanged. Partitioning PVP into the KTP-rich phase reduced the chemical potential of KTP in the KTP-rich phase and the amorphous solubility of KTP. At higher temperatures, the distribution of PVP into the KTP-rich phase became more significant, which considerably reduced the amorphous solubility. Because the upper limit of the KTP supersaturation decreased, PVP narrowed the KTP supersaturation region. The maximum KTP supersaturation ratio decreased with increasing temperature, and the supersaturated dissolvable area of KTP finally disappeared. The maximum temperature at which KTP can form the supersaturation was lowered by replacing rac- with s-KTP and the addition of PVP. The maximum supersaturation temperature was dominated by the melting behavior of crystalline KTP in an aqueous solution. The present study highlighted that a quantitative understanding of the supersaturation region is essential to determine whether supersaturated formulations are beneficial for improving the oral absorption of poorly water-soluble drugs.

Designing a Novel Coamorphous Salt Formulation of Telmisartan with Amlodipine to Enhance Permeability and Oral Absorption.

Coamorphous formulation is a useful approach for enhancing the solubility of poorly water-soluble drugs via intermolecular interactions. In this study, a hydrogen-bonding-based coamorphous system was developed to improve drug solubility, but it barely changed the apparent permeability (Papp) of the drug. This study aimed to design a novel coamorphous salt using ionic interactions to improve drug permeability and absorption. Telmisartan (TMS), with an acidic group, was used to form a coamorphous salt with basic amlodipine (AML). Evaluation of the physicochemical properties confirmed the formation of a coamorphous salt via ionic interactions between the amine group of AML and the carboxyl group of TMS at a molar ratio of 1:1. The coamorphous salt of TMS/AML enhanced the partitioning of both drugs into octanol, indicating increased lipophilicity owing to the interaction between TMS and AML. The coamorphous salt dramatically enhanced TMS solubility (99.8 times that of untreated TMS) and decreased AML solubility owing to the interaction between TMS and AML. Although the coamorphous salt showed a decreased Papp in the permeation study in the presence of a thicker unstirred water layer (UWL) without stirring, Papp increased in the presence of a thinner UWL with stirring. The oral absorption of TMS from the coamorphous salt increased by up to 4.1 times compared to that of untreated TMS, whereas that of AML remained unchanged. Although the coamorphous salt with increased lipophilicity has a disadvantage in terms of diffusion through the UWL, the UWL is thin in human/animal bodies owing to the peristaltic action of the digestive tract. Dissociation of the coamorphous salt on the membrane surface could contribute to the partitioning of the neutral form of drugs to the membrane cells compared with untreated drugs. As a result, coamorphous salt formation has the advantage of improving the membrane permeation and oral absorption of TMS, owing to the enhanced solubility and supply of membrane-permeable free TMS on the surface of the membrane.

Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

DDOST-CDG: Clinical and molecular characterization of a third patient with a milder and a predominantly movement disorder phenotype.

Congenital disorders of glycosylation (CDG) are a group of heterogeneous inherited metabolic disorders affecting posttranslational protein modification. DDOST-CDG, caused by biallelic pathogenic variants in DDOST which encodes dolichyl-diphospho-oligosaccharide-protein glycosyltransferase, a subunit of N-glycosylation oligosaccharyltransferase (OST) complex, is an ultra-rare condition that has been described in two patients only. The main clinical features in the two reported patients include profound developmental delay, failure to thrive, and hypotonia. In addition, both patients had abnormal transferrin glycosylation. Here, we report an 18-year-old male who presented with moderate developmental delay, progressive opsoclonus, myoclonus, ataxia, tremor, and dystonia. Biochemical studies by carbohydrate deficient transferrin analysis showed a type I CDG pattern. Exome sequencing identified compound heterozygous variants in DDOST: a maternally inherited variant, c.1142dupT (p.Leu381Phefs*11), and a paternally inherited variant, c.661 T > C (p.Ser221Pro). Plasma N-glycan profiling showed mildly increased small high mannose glycans including Man0-5 GlcNAc2, a pattern consistent with what was previously reported in DDOST-CDG or defects in other subunits of OST complex. Western blot analysis on patient's fibroblasts revealed decreased expression of DDOST and reduced intracellular N-glycosylation, as evident by the biomarkers ICAM-1 and LAMP2. Our study highlights the clinical variability, expands the clinical and biochemical phenotypes, and describes new genotype, which all are essential for diagnosing and managing patients with DDOST-CDG.

Polymorphism at the nestling stage and host-specific mimicry in an Australasian cuckoo-host arms race.

Decades of research have shown that the coevolutionary arms race between avian brood parasites and their hosts can promote phenotypic diversification in hosts and brood parasites. However, relatively little is known about the role of brood parasitism in promoting phenotypic diversification of nestlings. We review field data collected over four decades in Australia, New Caledonia and New Zealand to assess potential for coevolutionary interactions between the shining bronze-cuckoo (Chalcites lucidus) and its hosts, and how diversification at the nestling stage may be generating different subspecies. The shining bronze-cuckoo is a specialist parasite of a few hosts in the family Acanthizidae. It has diversified into subspecies, of which the nestlings closely mimic the respective host nestlings in each region. Additionally, some cuckoo subspecies have polymorphic nestlings. The Acanthizidae hosts have similar breeding and nesting habits and only moderately effective frontline defences against parasitism at cuckoo egg laying or at the egg stages. However, some hosts have developed highly effective defences at the nestling stage by recognising and ejecting cuckoo nestlings from the nest. As with the cuckoo nestlings, some hosts have polymorphic nestlings. The coevolutionary interactions in each region suggest different evolutionary stages of the arms race in which either the parasite or the host is currently in the lead. The presence of moderately effective defences at the egg laying and egg stages might explain why some hosts do not have defences at the nestling stage. The south-Pacific cuckoo - host systems are excellent models to explore the evolutionary mechanisms driving the diversification at the nestling stage in the coevolutionary arms race between avian brood parasites and their hosts.

Dystonia in individuals with spastic cerebral palsy and isolated periventricular leukomalacia.

AIM: To determine the prevalence of dystonia in individuals with periventricular leukomalacia (PVL) and spastic cerebral palsy (CP), but without basal ganglia and thalamic injury (BGTI) on brain magnetic resonance imaging (MRI). METHOD: This was a retrospective study of individuals with spastic CP and PVL on MRI evaluated between 2005 and 2018 in a CP center. Individuals with non-PVL brain lesions on MRI, including BGTI, were excluded. Dystonia was assessed via blinded review of neurological exam videos by pediatric movement disorders specialists. RESULTS: Eighty-five participants (45 males, 40 females; mean age at videotaping 12 years [standard deviation 5 years 6 months], range 4-26 years) met inclusion and exclusion criteria. Of these participants, 50 (59%) displayed dystonia in their exam videos. The most common locations of dystonia were the fingers and hip adductors. The prevalence of dystonia was unaffected by the gestational age or severity of PVL, and was affected by Gross Motor Function Classification System level. INTERPRETATION: Dystonia is common in individuals with spastic CP and PVL, even without BGTI on MRI. Our findings suggest vigilance for dystonia in individuals with spastic CP should remain high, even without MRI evidence of BGTI. WHAT THIS PAPER ADDS: Individuals with spastic cerebral palsy and isolated periventricular leukomalacia on magnetic resonance imaging commonly display dystonia. Common sites of dystonia are in the fingers and hip adductors.

Determinants of gait dystonia severity in cerebral palsy.

AIM: To determine the movement features governing expert assessment of gait dystonia severity in individuals with cerebral palsy (CP). METHOD: In this prospective cohort study, three movement disorder neurologists graded lower extremity dystonia severity in gait videos of individuals with CP using a 10-point Likert-like scale. Using conventional content analysis, we determined the features experts cited when grading dystonia severity. Then, using open-source pose estimation techniques, we determined gait variable analogs of these expert-cited features correlating with their assessments of dystonia severity. RESULTS: Experts assessed videos from 116 participants (46 with dystonia aged 15 years [SD 3] and 70 without dystonia aged 15 years [SD 2], both groups ranging 10-20 years old and 50% male). Variable limb adduction was most commonly cited by experts when identifying dystonia, comprising 60% of expert statements. Effect on gait (regularity, stability, trajectory, speed) and dystonia amplitude were common features experts used to determine dystonia severity, comprising 19% and 13% of statements respectively. Gait variables assessing adduction variability and amplitude (inter-ankle distance variance and foot adduction amplitude) were significantly correlated with expert assessment of dystonia severity (multiple linear regression, p < 0.001). INTERPRETATION: Adduction variability and amplitude are quantifiable gait features that correlate with expert-determined gait dystonia severity in individuals with CP. Consideration of these features could help optimize and standardize the clinical assessment of gait dystonia severity in individuals with CP.

Multistep Crystallization of Pharmaceutical Amorphous Nanoparticles via a Cognate Pathway of Oriented Attachment: Direct Evidence of Nonclassical Crystallization for Organic Molecules.

Crystallization of organic molecules is important in a wide range of scientific disciplines. However, in contrast to maturely studied crystallization of inorganic materials, the crystallization mechanisms of organic molecules involving nucleation and crystal growth are still poorly understood. Here, we used time-resolved cryogenic transmission electron microscopy to directly map the morphological evolution of amorphous cyclosporin A (CyA) nanoparticles during CyA crystallization. We successfully observed its initial nucleation and found that the amorphous CyA nanoparticles crystallized via a pathway cognate with oriented attachment, which is the nonclassical crystallization mechanism usually reported for inorganic compounds. Crystalline mesostructured intermediates (mesocrystals) were formed during crystallization. This study revealed clear and direct evidence of mesocrystal formation and oriented attachment in organic pharmaceuticals, providing new insights into the crystallization of organic molecules and theories of nonclassical crystallization.

Transition from Amorphous Cyclosporin A Nanoparticles to Size-Reduced Stable Nanocrystals in a Poloxamer 407 Solution.

Amorphous drug nanoparticles usually exhibit low storage stability. A comprehensive understanding of the molecular states and physicochemical properties of the product is indispensable for designing stable formulations. In the present study, an amorphous cyclosporin A (CyA) nanosuspension with a mean particle size of approximately 370 nm was prepared by wet bead milling with poloxamer 407 (P407). Interestingly, the prepared amorphous CyA nanoparticles were transformed into uniform CyA nanocrystals with a reduced mean particle size of approximately 200 nm during storage at 25 °C. The CyA nanocrystals were stably maintained for at least 1 month. The particle morphologies and molecular structures of the CyA nanosuspensions before and after storage were thoroughly characterized by cryogenic transmission electron microscopy and magic-angle spinning nuclear magnetic resonance spectroscopy, respectively. They revealed that the freshly prepared amorphous CyA nanoparticles (∼370 nm) were secondary particles composed of aggregated primary particles with an estimated size of 50 nm. A portion of P407 was found to be entrapped at the gaps between the primary particles due to aggregation, while most of P407 was dissolved in the solution either adsorbing at the solid/liquid interface or forming polymeric micelles. The entrapped P407 is considered to play an important role in the destabilization of the amorphous CyA nanoparticles. The resultant CyA nanocrystals (∼200 nm) were uniform single crystals of Form 2 hydrate and showed corner-truncated bipyramidal features. Owing to the narrow particle size distribution of the CyA nanocrystals, the rate of Ostwald ripening was slow, giving long-term stability to the CyA nanocrystals. This study provides new insights into the destabilization mechanism of amorphous drug nanoparticles.

Unusual Correlation between the Apparent Amorphous Solubility of a Drug and Solubilizer Concentration Revealed by NMR Analysis.

Herein, we investigated the effect of the solubilizers, cetyltrimethylammonium bromide (CTAB) and amino methacrylate copolymer (Eudragit E PO, EUD-E), on the apparent amorphous solubility of ketoprofen (KTP) and free KTP concentrations in an aqueous phase when a KTP-rich phase was generated by liquid-liquid phase separation. Quantitative analysis by solution nuclear magnetic resonance (NMR) revealed that the apparent amorphous solubility of KTP increased with increasing EUD-E concentrations by the solubilization of KTP into the EUD-E micelles; this was reminiscent of the improvement in the apparent crystalline solubility of KTP observed when EUD-E was added. In contrast, the apparent amorphous solubility of KTP decreased with increasing CTAB concentrations, although the solubilizing ability of CTAB was stronger than that of EUD-E when the KTP-rich phase was absent. NMR analysis revealed that CTAB was distributed into the KTP-rich phase to a relatively large extent. This resulted in a significant reduction of the chemical potential of KTP in the KTP-rich phase in the CTAB solution. Thus, the maximum free KTP concentration in the aqueous phase was reduced more significantly in the CTAB solution than in the EUD-E solution. Moreover, the solubilization effect of KTP by the CTAB micelles in the aqueous phase was drastically diminished due to the distribution of CTAB into the KTP-rich phase. As a result, the apparent amorphous solubility of KTP reached a minimum at a CTAB concentration of 200 µg/mL. A further increase in the CTAB concentration resulted in an improvement in the apparent amorphous solubility of KTP due to the solubilization effect of CTAB remaining in the aqueous phase. The present study highlights the impact of solubilizer selection on the apparent amorphous solubility and attainable supersaturation of the drug, which should be considered during the development of supersaturating formulations to obtain preferable oral absorption.

Variable-Temperature NMR Analysis of the Thermodynamics of Polymer Partitioning between Aqueous and Drug-Rich Phases and Its Significance for Amorphous Formulations.

We previously reported that the polymers used in amorphous solid dispersion (ASD) formulations, such as polyvinylpyrrolidone (PVP), polyvinylpyrrolidone/vinyl acetate (PVP-VA), and hypromellose (HPMC), distribute into the drug-rich phase of ibuprofen (IBP) formed by liquid-liquid phase separation, resulting in a reduction in the maximum drug supersaturation in the aqueous phase. Herein, the mechanism underlying the partitioning of the polymer into the drug-rich phase was investigated from a thermodynamic perspective. The dissolved IBP concentration in the aqueous phase and the amount of polymer distributed into the IBP-rich phase were quantitatively analyzed in IBP-supersaturated solutions containing different polymers using variable-temperature solution-state nuclear magnetic resonance (NMR) spectroscopy. The polymer weight ratio in the IBP-rich phase increased at higher temperatures, leading to a more notable reduction of IBP amorphous solubility. Among the polymers, the amorphous solubility reduction was the greatest for the PVP-VA solution at lower temperatures, while HPMC reduced the amorphous solubility to the greatest extent at higher temperatures. The change in the order of polymer impact on the amorphous solubility resulted from the differences in the temperature dependency of polymer partitioning. The van't Hoff plot of the polymer partition coefficient revealed that both enthalpy and entropy changes for polymer transfer into the IBP-rich phase from the aqueous phase (ΔHaqueous→IBP-rich and ΔSaqueous→IBP-rich) gave positive values for most of the measured temperature range, indicating that polymer partitioning into the IBP-rich phase was an endothermic but entropically favorable process. The polymer transfer into the IBP-rich phase was more endothermic for HPMC than for PVP and PVP-VA. The solid-state NMR analysis of the IBP/polymer ASD implied that the newly formed IBP/polymer interactions in the IBP-rich phase upon polymer incorporation were weaker for HPMC, providing a rationale for the larger positive transfer enthalpy for HPMC. The change in Gibbs free energy for polymer transfer (ΔGaqueous→IBP-rich) showed negative values across the experimental temperature range, decreasing with an increase in temperature, indicating that the distribution of the polymer into the IBP-rich phase is favored at higher temperatures. Moreover, ΔGaqueous→IBP-rich for HPMC showed the greatest decrease with the temperature, likely reflecting the temperature-induced dehydration of HPMC in the aqueous phase. This study contributes fundamental insights into the phenomenon of polymer partitioning into drug-rich phases, furthering the understanding of achievable supersaturation levels and ultimately providing information on polymer selection for ASD formulations.

Drug-Loaded Nanocarriers Composed of Cholesteryl Oleate Crystal Cores and Multiple-Nanosheet Shells of γ-Cyclodextrin Inclusion Complex Crystals.

In this study, we prepared drug-loaded nanocarriers made of cholesteryl oleate (ChO) and γ-cyclodextrin (γ-CD). A nanosuspension (nanosuspension-I, NS-I) containing nanoparticles with a mean size of approximately 170 nm was obtained through the solvent-diffusion method using ethanol. A second nanosuspension (nanosuspension-II, NS-II), which was prepared by freeze-drying and redispersion of NS-I, exhibited an increased particle size of approximately 210 nm. Cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM) force-distance curves indicated that the nanoparticles in NS-I were oblong and soft. However, those in NS-II were angular and stiff, and, interestingly, multiple nanosheets covered the solid-liquid interface. Synchrotron wide-angle X-ray diffraction (WAXD) analysis of NS-II indicated that the nanoparticles in it had a core-shell structure, where the ChO crystal in the inner core was covered by multiple nanosheets of ChO/γ-CD inclusion complex crystals. The X-ray peak analysis suggested that the γ-CD columns of the nanosheets were vertically stacked onto the ChO crystal interface. It was found that the nanosheets on the nanoparticle interface were formed during the freezing process. A model drug carbamazepine (CBZ) was loaded into the ChO/γ-CD nanoparticles by pre-dissolving CBZ in ethanol during the solvent-diffusion process. Cryo-TEM, 1H NMR, ζ-potentials, and synchrotron WAXD indicated that CBZ was unexpectedly loaded into the shell as a CBZ/γ-CD inclusion complex crystalline nanosheet. The specific nanosheet structure, where ChO and CBZ coexisted in the same crystal of γ-CD, could achieve CBZ loading in the nanoparticles. ChO/γ-CD nanoparticles with the unique core-shell structure are expected to perform as practical carriers for drug delivery.

Impact of Surfactants on the Performance of Clopidogrel-Copovidone Amorphous Solid Dispersions: Increased Drug Loading and Stabilization of Nanodroplets.

PURPOSE: Surfactants are increasingly being added to amorphous solid dispersion (ASDs) formulations to enhance processability and release performance. The goal of the current work was to investigate the impact of cationic, anionic and non-ionic surfactants on the rate and extent of clopidogrel (CPD) release from copovidone-based ASDs. METHODS: CPD release was evaluated for ASDs with different drug loadings using a surface normalized intrinsic dissolution apparatus. Studies were also carried out using dynamic light scattering, zeta potential measurements, and nuclear magnetic resonance spectroscopy to probe the impact of surfactants on drug-rich nanodroplet physical stability and clopidogrel-surfactant interactions. RESULTS: CPD ASDs showed good release for drug loadings as high as 40%, before the release fell off a cliff at higher drug loadings. Only sodium dodecyl sulfate, added at a 5% level, was able to improve the release at 50% drug loading, with other surfactants proving to be ineffective. However, some of the surfactants evaluated did show some benefits in improving nanodroplet stability against size enlargement. Ionic and non-ionic surfactants were observed to interact differently with CPD-rich nanodroplets, and variations in the kinetics and morphology of water-induced phase separation were noted in the presence and absence of surfactants in ASD films. CONCLUSIONS: In summary, addition of surfactants to ASD formulations may lead to some improvements in formulation performance, but predictive capabilities and mechanisms of surfactant effect still require further studies.

Amorphous Drug Solubility and Maximum Free Drug Concentrations in Cyclodextrin Solutions: A Quantitative Study Using NMR Diffusometry.

Cyclodextrin (CD) has been widely used as a solubilizing agent for poorly water-soluble drugs. In the present study, the effect of CD on the amorphous drug solubility and the maximum thermodynamic activity of the drug in the aqueous phase when the drug concentration exceeded the liquid-liquid phase separation (LLPS) concentration was investigated using three chemically diverse CDs, ß-cyclodextrin (ß-CD), dimethyl-ß-CD (DM-ß-CD), and hydroxypropyl-ß-CD (HP-ß-CD). The amorphous solubility of ibuprofen (IBP) increased substantially linearly with the increase in the CD concentration due to IBP/CD complex formation. Surprisingly, although the crystalline solubility of IBP in the ß-CD solution reached a plateau at ß-CD concentrations above 3 mM (BS-type solubility diagram) because of the limited crystalline solubility of the IBP/ß-CD complex, the amorphous solubility of IBP increased linearly even when the ß-CD concentration was higher than 3 mM. The amorphous solubility of IBP in CD solutions was influenced primarily by the phase separation of the IBP-supersaturated solution to the aqueous phase and the other phase mainly composed of IBP, namely, the IBP-rich phase, via LLPS. NMR spectroscopy revealed that DM-ß-CD was distributed into the IBP-rich phase when the IBP concentration exceeded its amorphous solubility, while ß-CD and HP-ß-CD showed minimal mixing with the IBP-rich phase. NMR diffusometry showed that the maximum free IBP concentration was reduced in the DM-ß-CD solution compared to that in the buffer. The mixing of DM-ß-CD with the IBP-rich phase reduced the chemical potential of IBP in the IBP-rich phase, which in turn reduced the maximum thermodynamic activity of IBP in the aqueous phase. In contrast, the maximum free IBP concentration was unchanged when ß-CD or HP-ß-CD was present. The hydrophobic nature of the DM-ß-CD substituent may contribute to its partitioning into the IBP-rich phase. The present study highlights the impact of CD on the maximum thermodynamic activity of drugs as well as the apparent amorphous solubility of the drug. This aspect should be considered for improving the effective absorption of poorly water-soluble drugs.

Effect of Polymer Species on Maximum Aqueous Phase Supersaturation Revealed by Quantitative Nuclear Magnetic Resonance Spectroscopy.

The polymer used in an amorphous solid dispersion (ASD) formulation impacts the maximum achievable drug supersaturation. Herein, the effect of dissolved polymer on drug concentration in the aqueous phase when a drug-rich phase was generated by liquid-liquid phase separation (LLPS) was investigated for different polymers at various concentrations of drug and polymer. Solution nuclear magnetic resonance (NMR) spectroscopy revealed that polyvinylpyrrolidone (PVP), polyvinylpyrrolidone/vinyl acetate (PVP-VA), and hypromellose (HPMC) distributed into the ibuprofen (IBP)-rich phase formed by LLPS when the amorphous solubility of IBP was exceeded. The amount of polymer in the drug-rich phase increased for higher-molecular-weight grades of PVP and HPMC. Moreover, PVP-VA showed a greater extent of distribution into the IBP-rich phase compared to PVP, and this is attributed to its reduced hydrophilicity resulting from the incorporation of vinyl acetate monomers. Direct quantification by NMR measurements indicated that the IBP concentration in the aqueous phase decreased as the amount of polymer in the IBP-rich phase increased. This can be attributed to a reduction of the chemical potential of IBP in the IBP-rich phase. The reduction in dissolved IBP concentration was greater for the IBP/PVP-VA system compared to the IBP/HPMC system, as a result of more extensive drug-polymer interactions in the former system. The present study highlights the impact of polymer selection on the attainable supersaturation of the drug and the factors that need to be considered in the formulation of ASDs to obtain optimized in vivo performance.

Nanostructure and Molecular-Level Characterization of Aminoalkyl Methacrylate Copolymer and the Impact on Drug Solubilization Ability.

The effects of pH changes and saccharin (SAC) addition on the nanostructure and mobility of the cationic aminoalkyl methacrylate copolymer Eudragit E PO (EUD-E) and its drug solubilization ability were investigated. Small-angle X-ray scattering performed using synchrotron radiation and atomic force microscopy showed that the EUD-E nanostructure, which has a size of approximately several nanometers, changed from a random coil structure at low pH (pH 4.0-5.0) to a partially folded structure at high pH (pH 5.5-6.5). The EUD-E also formed a partially folded structure in a wide pH range of 4.5-6.5 when SAC was present, and the coil-to-globule transition was moderate with pH increase, compared with that when SAC was absent. The equilibrium solubility of the neutral drug naringenin (NAR) was enhanced in the EUD-E solution and further increased as the pH increased. The enlargement of the hydrophobic region of EUD-E in association with the coil-to-globule transition led to efficient solubilization of NAR. The interaction with SAC enhanced the mobility of the EUD-E chains in the hydrophobic region of EUD-E, resulting in changes in the drug-solubilizing ability. 1H high-resolution magic-angle spinning NMR measurements revealed that the solubilized NAR in the partially folded structure of EUD-E showed higher molecular mobility in the presence of SAC than in the absence of SAC. This study highlighted that solution pH and the presence of SAC significantly changed the drug solubilization ability of EUD-E, followed by changes in the EUD-E nanostructure, including its hydrophobic region.