Probing Molecular Packing of Amorphous Pharmaceutical Solids Using X-ray Atomic Pair Distribution Function and Solid-State NMR.

The structural investigation of amorphous pharmaceuticals is of paramount importance in comprehending their physicochemical stability. However, it has remained a relatively underexplored realm primarily due to the limited availability of high-resolution analytical tools. In this study, we utilized the combined power of X-ray pair distribution functions (PDFs) and solid-state nuclear magnetic resonance (ssNMR) techniques to probe the molecular packing of amorphous posaconazole and its amorphous solid dispersion at the molecular level. Leveraging synchrotron X-ray PDF data and employing the empirical potential structure refinement (EPSR) methodology, we unraveled the existence of a rigid conformation and discerned short-range intermolecular C-F contacts within amorphous posaconazole. Encouragingly, our ssNMR 19F-13C distance measurements offered corroborative evidence supporting these findings. Furthermore, employing principal component analysis on the X-ray PDF and ssNMR data sets enabled us to gain invaluable insights into the chemical nature of the intermolecular interactions governing the drug-polymer interplay. These outcomes not only furnish crucial structural insights facilitating the comprehension of the underlying mechanisms governing the physicochemical stability but also underscore the efficacy of synergistically harnessing X-ray PDF and ssNMR techniques, complemented by robust modeling strategies, to achieve a high-resolution exploration of amorphous structures.

Engineering the glass structure of a discotic liquid crystal by multiple kinetic arrests.

X-ray scattering has been used to characterize the columnar packing and the π stacking in a glass-forming discotic liquid crystal. In the equilibrium liquid state, the intensities of the scattering peaks for π stacking and columnar packing are proportional to each other, indicating concurrent development of the two orders. Upon cooling into the glassy state, the π-π distance shows a kinetic arrest with a change in the thermal expansion coefficient (TEC) from 321 to 109 ppm/K, while the intercolumnar spacing exhibits a constant TEC of 113 ppm/K. By changing the cooling rate, it is possible to prepare glasses with a wide range of columnar and π stacking orders, including zero order. For each glass, the columnar order and the π stacking order correspond to a much hotter liquid than its enthalpy and π-π distance, with the difference between the two internal (fictive) temperatures exceeding 100 K. By comparison with the relaxation map obtained by dielectric spectroscopy, we find that the δ mode (disk tumbling within a column) controls the columnar order and the π stacking order trapped in the glass, while the α mode (disk spinning about its axis) controls the enthalpy and the π-π spacing. Our finding is relevant for controlling the different structural features of a molecular glass to optimize its properties.

Structures of glasses created by multiple kinetic arrests.

X-ray scattering has been used to characterize glassy itraconazole (ITZ) prepared by cooling at different rates. Faster cooling produces ITZ glasses with lower (or zero) smectic order with more sinusoidal density modulation, larger molecular spacing, and shorter lateral correlation between the rod-like molecules. We find that each glass is characterized by not one, but two fictive temperatures Tf (the temperature at which a chosen order parameter is frozen in the equilibrium liquid). The higher Tf is associated with the regularity of smectic layers and lateral packing, while the lower Tf with the molecular spacings between and within smectic layers. This indicates that different structural features are frozen on different timescales. The two timescales for ITZ correspond to its two relaxation modes observed by dielectric spectroscopy: the slower δ mode (end-over-end rotation) is associated with the freezing of the regularity of molecular packing and the faster α mode (rotation about the long axis) with the freezing of the spacing between molecules. Our finding suggests a way to selectively control the structural features of glasses.

Structures of glass-forming liquids by x-ray scattering: Glycerol, xylitol, and D-sorbitol.

Synchrotron x-ray scattering has been used to investigate three liquid polyalcohols of different sizes (glycerol, xylitol, and D-sorbitol) from above the glass transition temperatures Tg to below. We focus on two structural orders: the association of the polar OH groups by hydrogen bonds (HBs) and the packing of the non-polar hydrocarbon groups. We find that the two structural orders evolve very differently, reflecting the different natures of bonding. Upon cooling from 400 K, the O⋯O correlation at 2.8 Å increases significantly in all three systems, indicating more HBs, until kinetic arrests at Tg; the increase is well described by an equilibrium between bonded and non-bonded OH with ΔH = 9.1 kJ/mol and ΔS = 13.4 J/mol/K. When heated above Tg, glycerol loses the fewest HBs per OH for a given temperature rise scaled by Tg, followed by xylitol and by D-sorbitol, in the same order the number of OH groups per molecule increases (3, 5, and 6). The pair correlation functions of all three liquids show exponentially damped density modulations of wavelength 4.5 Å, which are associated with the main scattering peak and with the intermolecular C⋯C correlation. In this respect, glycerol is the most ordered with the most persistent density ripples, followed by D-sorbitol and by xylitol. Heating above Tg causes faster damping of the density ripples with the rate of change being the slowest in xylitol, followed by glycerol and by D-sorbitol. Given the different dynamic fragility of the three liquids (glycerol being the strongest and D-sorbitol being the most fragile), we relate our results to the current theories of the structural origin for the difference. We find that the fragility difference is better understood on the basis of the thermal stability of HB clusters than that of the structure associated with the main scattering peak.

Organic glasses with tunable liquid-crystalline order through kinetic arrest of end-over-end rotation: the case of saperconazole.

Liquid crystals (LCs) undergo fast phase transitions, almost without hysteresis, leading to the notion that it is difficult to bypass LC transitions. However, recent work on itraconazole has shown that a nematic-to-smectic phase transition can be frustrated or avoided at moderate cooling rates. At each cooling rate, the highest smectic order obtained is determined by the kinetic arrest of the end-over-end molecular rotation. We report that the same phenomenon occurs in the system saperconazole, an analog of itraconazole where each of the two Cl atoms is replaced by F. Saperconazole has a wider temperature range over which smectic order can develop before kinetic arrest, providing a stronger test of the previous conclusion. Together these results indicate a general principle for controlling LC order in organic glasses for electronic applications.

Polymer Nanocoating of Amorphous Drugs for Improving Stability, Dissolution, Powder Flow, and Tabletability: The Case of Chitosan-Coated Indomethacin.

As a result of its higher molecular mobility, the surface of an amorphous drug can grow crystals much more rapidly than the bulk, causing poor stability and slow dissolution of drug products. We show that a nanocoating of chitosan (a pharmaceutically acceptable polymer) can be deposited on the surface of amorphous indomethacin by electrostatic deposition, leading to significant improvement of physical stability, wetting by aqueous media, dissolution rate, powder flow, and tabletability. The coating condition was chosen so that the positively charged polymer deposits on the negatively charged drug. Chitosan coating is superior to gelatin coating with respect to stability against crystallization and agglomeration of coated particles.

Improving Stability and Dissolution of Amorphous Clofazimine by Polymer Nano-Coating.

PURPOSE: To inhibit the surface crystallization and enhance the dissolution of the basic amorphous drug clofazimine by polymer nano-coating. METHODS: The free surface of amorphous clofazimine was coated by dip coating in an alginate solution at pH 7. The stability of the coated amorphous drug against crystallization was evaluated by X-ray diffraction and light microscopy. The effect of coating on dissolution rate was measured in simulated gastric fluid in an USP-II apparatus at 37°C. RESULTS: At pH 7, the weak base clofazimine (pKa = 8.5) is positively charged, while the weak alginic acid (pKa = 3.5) is negatively charged, allowing coating by electrostatic deposition. Coated amorphous particles remain nearly amorphous after one year under the accelerated testing condition 40°C/75% R.H. and show faster dissolution than uncoated particles. In the first hour of dissolution, coated amorphous particles dissolve 50% faster than uncoated amorphous particles, and a factor of 3 faster than crystalline particles of the same size. CONCLUSIONS: A pharmaceutically acceptable polymer, alginate, is coated on amorphous clofazimine by electrostatic deposition and effectively inhibits its surface crystallization and enhances its dissolution rate. This is the first time the nano-coating technique is applied to a basic drug using the principle of electrostatic deposition, demonstrating the generality of the approach.

Effect of molecular size and hydrogen bonding on three surface-facilitated processes in molecular glasses: Surface diffusion, surface crystal growth, and formation of stable glasses by vapor deposition.

Recent work has shown that diffusion and crystal growth can be much faster on the surface of molecular glasses than in the interior and that the enhancement effect varies with molecular size and intermolecular hydrogen bonds (HBs). In a related phenomenon, some molecules form highly stable glasses when vapor-deposited, while others (notably those forming extensive HBs) do not. Here we examine all available data on these phenomena for quantitative structure-property relations. For the systems that form no HBs, the surface diffusion coefficient D s decreases with increasing molecular size d (d = Ω1/3, where Ω is the molecular volume); when evaluated at the glass transition temperature T g, D s decreases ∼5 orders of magnitude for 1 nm of increase in d. Assuming that center-of-mass diffusion is limited by the deepest part of the molecule in the surface-mobility gradient, these data indicate a mobility gradient in reasonable agreement with the Elastically Collective Nonlinear Langevin Equation theory prediction for polystyrene as disjointed Kuhn monomers. For systems of similar d, the D s value decreases with the extent of intermolecular HB, x (HB), defined as the fraction of vaporization enthalpy due to HB. For both groups together (hydrogen-bonded and otherwise), the D s data collapse when plotted against d/[1 - x(HB)]; this argues that the HB effect on D s can be described as a narrowing of the surface mobility layer by a factor [1 - x(HB)] relative to the van der Waals systems. Essentially the same picture holds for the surface crystal growth rate u s. The kinetic stability of a vapor-deposited glass decreases with x(HB) but is not better organized by the combined variable d/[1 - x(HB)]. These results indicate that surface crystal growth depends strongly on surface diffusion, whereas the formation of stable glasses by vapor deposition may depend on other factors.

Crystal nucleation rates in glass-forming molecular liquids: D-sorbitol, D-arabitol, D-xylitol, and glycerol.

The rate of crystal nucleation has been measured in four glass-forming molecular liquids: D-sorbitol, D-arabitol, D-xylitol, and glycerol. These polyalcohols have similar rates of crystal growth when compared at the same temperature relative to Tg (the glass transition temperature), peaking near 1.4 Tg, while the nucleation rates J are vastly different. In D-sorbitol and D-arabitol, J reaches a maximum of ∼108 m-3 s-1 near 1.1 Tg, whereas J < 10-2 m-3 s-1 in D-xylitol and <1 m-3 s-1 in glycerol based on no nucleation in large samples after long waits. This confirms the fundamentally different mechanisms for nucleation and growth. Near Tg, both nucleation and growth slow down with a similar temperature dependence, suggesting a similar kinetic barrier for the two processes. This temperature dependence is significantly weaker than that of viscosity η, approximately following η-0.75. This indicates that viscosity is a poor representative of the kinetic barrier for nucleation, and a better choice is the crystal growth rate. Under the latter assumption, the classical nucleation theory (CNT) describes our data reasonably well, yielding σ = 0.013 J/m2 for D-sorbitol and 0.026 J/m2 for D-arabitol, where σ is the critical nucleus/liquid interfacial free energy. There is no strong indication that the CNT fails as the length scale for corporative rearrangement exceeds the size of the critical nucleus, as recently suggested for lithium disilicate.

Miscibility of Amorphous Solid Dispersions: A Rheological and Solid-State NMR Spectroscopy Study.

Miscibility is critical in the prediction of stability against crystallization of amorphous solid dispersions (ASDs) in the solid state. However, currently available approaches for its determination are limited by both theoretical and practical considerations. Recently, a rheological approach guided by the polymer overlap concentration (c*) has been proposed for miscibility quantification of ASDs [J. Pharm. Sci., 112 (2023) 204-212] and shown to be useful in predicting both accelerated and long term physical stability in the absence of moisture. However, this approach can only be performed at high temperatures (slightly above the melting temperature, Tm, of drugs), and little is known about the difference in miscibility between high and low temperatures (e.g., below the glass transition temperature, Tg). Here we compare the miscibility of nifedipine (NIF)/polyvinylpyrrolidone (PVP) ASDs as determined by the rheological approach at 175°C (∼3°C above Tm of NIF) and solid state NMR (ssNMR) 1H T1 and T1ρ relaxation times at -20°C (∼66°C below Tg of NIF). Our results indicate agreement between the two methods. For low molecular weight (Mw) PVP, T1ρ measurements are more consistent with the rheological approach, while T1 measurements are closer for relatively high Mw PVP. Our findings support the use of the c* based rheological approach for inferring miscibility of deeply cooled ASDs.

Ultrafast magic angle spinning NMR characterization of pharmaceutical solid polymorphism: A posaconazole example.

Protons represent the most NMR-sensitive nucleus in pharmaceutical compounds. Therefore, proton-detected solid-state NMR techniques under fast magic angle spinning are among the few solutions to overcome the challenge of low sensitivity to analyze natural abundant drug substances and products. In this study, we report the structural characterization of crystal polymorphs of a commercial drug molecule, posaconazole, with a relatively large molecular weight of 700.8 g·mol-1 and at the natural abundance. The enhanced sensitivity and resolution at 100 kHz MAS enables the exploration of the distinct intermolecular packing in posaconazole forms I, III, and γ. These results demonstrate that proton-detected homo- and heteronuclear correlation methods can probe the structural details of pharmaceutical polymorphism.

Disrupting the phase separation of KAT8-IRF1 diminishes PD-L1 expression and promotes antitumor immunity.

Immunotherapies targeting the PD-1/PD-L1 axis have become first-line treatments in multiple cancers. However, only a limited subset of individuals achieves durable benefits because of the elusive mechanisms regulating PD-1/PD-L1. Here, we report that in cells exposed to interferon-γ (IFNγ), KAT8 undergoes phase separation with induced IRF1 and forms biomolecular condensates to upregulate PD-L1. Multivalency from both the specific and promiscuous interactions between IRF1 and KAT8 is required for condensate formation. KAT8-IRF1 condensation promotes IRF1 K78 acetylation and binding to the CD247 (PD-L1) promoter and further enriches the transcription apparatus to promote transcription of PD-L1 mRNA. Based on the mechanism of KAT8-IRF1 condensate formation, we identified the 2142-R8 blocking peptide, which disrupts KAT8-IRF1 condensate formation and consequently inhibits PD-L1 expression and enhances antitumor immunity in vitro and in vivo. Our findings reveal a key role of KAT8-IRF1 condensates in PD-L1 regulation and provide a competitive peptide to enhance antitumor immune responses.

Prolific Polymorph Generator ROY in Its Liquid and Glass: Two Conformational Populations Mirroring the Crystalline-State Distribution.

5-Methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile, dubbed ROY for its numerous crystal polymorphs of red, orange, and yellow colors, has been studied in its liquid and glassy state by infrared spectroscopy. Two populations of conformers are observed, whose equilibrium is characterized by ΔH = 2.4 kJ/mol and ΔS = 8.0 J/K/mol. The two populations correspond to the global and local minima of the torsional energy surface and to the conformational preference of the 13 crystal polymorphs. The local minimum features a more coplanar arrangement of the two aromatic rings, greater π conjugation, and lower CN stretch frequency. In the gas phase, the lowest-energy path between the two minima has an energy barrier 3.9 kJ/mol above the global minimum, consistent with the rapid equilibration between the two populations. The relevance of our result for understanding the prolific polymorphism of ROY is discussed.

Amorphous Drug-Polymer Salt with High Stability under Tropical Conditions and Fast Dissolution: The Challenging Case of Lumefantrine-PAA.

Lumefantrine (LMF), a high-mobility and easy-to-crystallize WHO drug for treating malaria, can form an amorphous salt with poly(acrylic acid) (PAA) that is remarkably stable against crystallization at high humidity and temperature and has fast dissolution rate. The amorphous salt up to 75% drug loading was synthesized under a mild slurry condition easily implemented in basic facilities for global health. Salt formation was confirmed by IR spectroscopy and the much elevated glass transition temperature. At 50% drug loading, the amorphous salt resists crystallization for at least 18 months under the highly stressful condition of 40 °C and 75% RH. In contrast, the dispersion containing neutral LMF in PVP fully crystallized in 4 d and the dispersion in HPMCAS, a weak polyelectrolyte of lower charge density than PAA, crystallized by 50% in 7 d. The amorphous salt at 50% drug loading showed much faster dissolution than crystalline LMF: In SGF, the area under the curve (AUC) was 30 times larger within the gastric emptying time (4 h); in FaSSIF, the enhancement was even larger - by 200 times. Nanodroplets were detected during the dissolution in SGF, possibly accounting for the apparent enhancement of dissolution rate. The LMF-PAA example as a challenging case, along with the previously reported clofazimine-PAA, demonstrates the general utility of amorphous drug-polymer salts to achieve high stability under tropical conditions and enhanced dissolution and bioavailability.

Using Deposition Rate and Substrate Temperature to Manipulate Liquid Crystal-Like Order in a Vapor-Deposited Hexagonal Columnar Glass.

We investigate vapor-deposited glasses of a phenanthroperylene ester, known to form an equilibrium hexagonal columnar phase, and show that liquid crystal-like order can be manipulated by the choice of deposition rate and substrate temperature during deposition. We find that rate-temperature superposition (RTS)-the equivalence of lowering the deposition rate and increasing the substrate temperature-can be used to predict and control the molecular orientation in vapor-deposited glasses over a wide range of substrate temperatures (0.75-1.0 Tg). This work extends RTS to a new structural motif, hexagonal columnar liquid crystal order, which is being explored for organic electronic applications. By several metrics, including the apparent average face-to-face nearest-neighbor distance, physical vapor deposition (PVD) glasses of the phenanthroperylene ester are as ordered as the glass prepared by cooling the equilibrium liquid crystal. By other measures, the PVD glasses are less ordered than the cooled liquid crystal. We explain the difference in the maximum attainable order with the existence of a gradient in molecular mobility at the free surface of a liquid crystal and its impact upon different mechanisms of structural rearrangement. This free surface equilibration mechanism explains the success of the RTS principle and provides guidance regarding the types of order most readily enhanced by vapor deposition. This work extends the applicability of RTS to include molecular systems with a diverse range of higher-order liquid-crystalline morphologies that could be useful for new organic electronic applications.