The solute-rich mesoscopic precursors of crystal nuclei of olanzapine solid forms

Olanzapine (OZPN) is a BCS class II drug used to treat schizophrenia (bipolar disorder). OZPN exhibits rich solid-state diversity. To date, 60 distinct forms have been identified, including 3 polymorphs (I, II, III), 52 crystalline solvates, 3 dihydrates (DB, DD, DE), a disordered higher hydrate, plus an amorphous form. Atomic Force Microscopy (AFM) results suggest that the nucleation of OZPN DD on the surface of OZPN I in water may follow a non-classical mechanisms that includes formation of solute-rich mesoscopic clusters [1]. Since the solubility of OZPN I in water is very low, the kinetics of transformation are difficult to monitor. To increase the solubility of OZPN I, we added different ratios of a co-solvent, ethanol. AFM observations revealed that clusters similar to those seen in purely aqueous environments are present on the surface of OZPN:EtOH:H2O crystals in contact with both supersaturated and undersaturated EtOH/H2O solutions. To establish the mechanism of cluster formation, we monitored the dependence of the cluster size and volume fraction on time, OZPN concentration, and co-solvent concentration using Brownian Microscopy (BM). The characteristics of the cluster population were correlated with the standard enthalpy, entropy and free energy of crystallization obtained from temperature dependence of the solubility of OZPN:EtOH:H2O crystals. We verified, using small angle x-ray scattering, that the crystal form was preserved at all solvent compositions. We observed that the cluster radius was constant, at R ≈ 37 nm, in all solvent compositions tested and at all times. The volume of the cluster population φ mapped the non-monotonic dependence of the crystallization enthalpy on the EtOH content, indicating that φ is determined by the thermodynamics of the solute-solvent interactions. The decoupled behaviour of R suggests that, in contrast to φ, the cluster size is kinetically determined. These conclusions comply with the prediction of a model of mesoscopic solvent rich clusters, based on formation of transient solute oligomers in the solutions [3]. These are the first observations of solute-rich clusters in solutions of pharmaceutically active compounds and of their role in the nucleation of crystals and the transformations between crystal forms. The suggested cluster formation mechanism may point to means to control these behaviours that are crucial for the properties of pharmaceutical preparations.  References:[1] M. Warzecha, R. Guo, R. M. Bhardwaj, S. M. Reutzel-Edens, S. L. Price, D. Lamprou and A. J. Florence, In preparation 2017.[2] Gebauer, D., Kellermeier, M., Gale, J. D., Bergström, L. & Cölfen, H. Pre-nucleation clusters as solute precursors in crystallisation. Chem. Soc. Rev. 2014, 43, 2348 [3] Vekilov, P. G. The two-step mechanism of nucleation of crystals in solution. Nanoscale, 2010, 2, 2346

Early Onset of Kinetic Roughening due to a Finite Step Width in Hematin Crystallization

The structure of the interface of a growing crystal with its nutrient phase largely determines the growth dynamics. We demonstrate that hematin crystals, crucial for the survival of malaria parasites, transition from faceted to rough growth interfaces at increasing thermodynamic supersaturation Δμ. Contrary to theoretical predictions and previous observations, this transition occurs at moderate values of Δμ. Moreover, surface roughness varies nonmonotonically with Δμ, and the rate constant for rough growth is slower than that resulting from nucleation and spreading of layers. We attribute these unexpected behaviors to the dynamics of step growth dominated by surface diffusion and the loss of identity of nuclei separated by less than the step width w. We put forth a general criterion for the onset of kinetic roughening using w as a critical length scale.National Institutes of Health (U.S.) (Grant 1R21AI126215-01)National Science Foundation (U.S.) (Grant DMR-1710354)United States. National Aeronautics and Space Administration (Grant NNX14AD68G)United States. National Aeronautics and Space Administration (Grant NNX14AE79G)Robert A. Welch Foundation (Grant E-1794

Anisotropy of the Coulomb Interaction between Folded Proteins: Consequences for Mesoscopic Aggregation of Lysozyme

AbstractToward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation

Mesoscopic solute-rich clusters in olanzapine solutions

An organic molecule may crystallize in numerous distinct lattices, and the slow and unpredictable transitions between multiple crystal forms are a significant concern in its pharmaceutical application. Recent results indicate that the transformation of olanzapine (OZPN) from anhydrous to hydrate crystals is mediated by mesoscopic solute-rich clusters. Here we demonstrate the existence of such clusters in undersaturated OZPN solutions in purely aqueous and mixed EtOH/aqueous solvents. The clusters occupy about 10–8 to 10–7 of the solution volume and capture ca. 10–7 to 10–5 of the dissolved OZPN. The average cluster radius is steady in time at about 35 nm and independent of the OZPN concentration and the solvent composition, whereas the OZPN fraction captured in the clusters is dictated by the solution thermodynamics. Both behaviors are in dire conflict with classical theories of phase transformation and recent aggregation models. They are, however, consistent with the predictions of a model that assumes the formation of OZPN dimers and their decay upon exiting the clusters. We propose that a transient dimer, which may be akin to the centrosymmetric dimer present in all of the 60 known OZPN crystal structures, may underlie cluster formation. The finding of cluster formation in organic systems and the proposed cluster mechanism provide guidance toward enhanced control over nucleation, molecular transitions, and the solid forms in molecular systems

Confocal Depolarized Dynamic Light Scattering: a Novel Technique for the Characterization of Nanoparticles in Complex Fluids

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