Identification of a Potential Conformationally Disordered Mesophase

Article pubs.acs.org/molecularpharmaceutics

Identification of a Potential Conformationally Disordered Mesophase in a Small Molecule: Experimental and Computational Approaches Paroma Chakravarty,*,† Simon Bates,‡ and Leonard Thomas§ †

Small Molecules Pharmaceutical Sciences, Genentech, Inc., 1 DNA way, South San Francisco, California 94080, United States Triclinic Laboratories, Inc., 1201 Cumberland Avenue, West Lafayette, Indiana 47906, United States § DSC Solutions LLC, 27 E. Braeburn Drive, Smyrna, Delaware 19977, United States ‡

S Supporting Information *

ABSTRACT: GNE068, a small organic molecule, was obtained as an amorphous form (GNE068-A) after isolation from ethanol and as a partially disordered form (GNE068-PC) from ethyl acetate. On subsequent characterization, GNE068-PC exhibited a number of properties that were anomalous for a two phase crystalline−amorphous system but consistent with the presence of a solid state phase having intermediate order (mesomorphous). Modulated DSC measurements of GNE068-PC revealed an overlapping endotherm and glass transition in the 135−145 °C range. ΔH of the endotherm showed strong heating rate dependence. Variable temperature XRPD (25−160 °C) revealed structure loss in GNE068-PC, suggesting the endotherm to be an “apparent melt”. In addition, gentle grinding of GNE068-PC in a mortar led to a marked decrease in XRPD peak intensities, indicating a “soft” crystalline lattice. Computational analysis of XRPD data revealed the presence of two noncrystalline contributions, one of which was associated with GNE068-A. The second was a variable component that could be modeled as diffuse scattering from local disorder within the associated crystal structure, suggesting a mesomorphous system. Owing to the dominance of the noncrystalline diffuse scattering in GNE068-PC and the observed lattice deformation, the mesomorphous phase exhibited properties consistent with a conformationally disordered mesophase. Because of the intimate association of the residual solvent (ethyl acetate) with the lattice long-range order, loss of solvent on heating through the glass transition temperature of the local disorder caused irrecoverable loss of the long-range order. This precluded the observation of characteristic thermodynamic mesophase behavior above the glass transition temperature. KEYWORDS: partially disordered, glass transition, lattice softness, mesomorphous, condis mesophase, solvent

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INTRODUCTION The material science concepts of “order” and “disorder”, as represented by the crystalline and amorphous states of a compound, are well known in the pharmaceutical community. Crystalline forms typically exhibit three-dimensional, longrange positional, orientational, and conformational order.1 Crystalline long-range order is, in general, the thermodynamic equilibrium form of a solid. In contrast, a liquid in its thermodynamic equilibrium state can be characterized by a complete absence of any long-range order. The absence of long-range order persists into metastable or kinetic glassy/ amorphous solids formed by rapid quench cooling of the associated liquid phase, although some short-range order may be induced by vitrification.2 As such, amorphous forms generally possess greater free energy than their crystalline counterparts, a property which is sometimes exploited advantageously to improve the dissolution and solubility of a poorly soluble, crystalline API (active pharmaceutical ingredient).3−5 Amorphization may also be induced inadvertently during formulation development, via pharmaceutical unit © 2013 American Chemical Society

operations such as milling (size reduction), drying, lyophilization, compression, etc. wherein temperature and mechanical stress destroy the lattice packing.6−10 Conventional characterization techniques such as X-ray powder diffractometry (XRPD), vibrational spectroscopy (Raman), and differential scanning calorimetry (DSC) are often adequate to satisfactorily identify and characterize these two forms. However, stresses induced by temperature, humidity, and mechanical agitation may also lead to the generation of intermediate states of order, which often require more rigorous characterization and greater in-depth understanding of the nature and type of disorder generated during processing. For example, solvent loss or milling might lead to partial loss of crystallinity in a material. The resultant order has been variously explained by, for example, (i) a simple, two-state model which implies the Received: Revised: Accepted: Published: 2809

October 1, 2012 May 13, 2013 June 24, 2013 July 9, 2013 dx.doi.org/10.1021/mp300558m | Mol. Pharmaceutics 2013, 10, 2809−2822

Molecular Pharmaceutics

Article

character. This is distinct from a mesophase which is an equilibrium thermodynamic system with mixed liquid and crystalline character, i.e., where the long-range order (crystalline property) exists independently of the static or dynamic disorder. The essential difference between the use of mesomorphic and mesophase in this work is the potential for liquid-like mobility for the local disordered components in a mesophase, while in a mesomorphic system, the local disorder is frozen into a metastable state. XRPD measurements alone can only determine if a system is potentially mesomorphic or not since laboratory X-ray powder diffraction is insensitive to whether the local disorder is frozen (solid) or dynamic (liquid). Determination of whether the mesomorphic character is attributable to an underlying thermodynamic mesophase requires detailed investigation of the nature of the disorder which can effectively be addressed by more exhaustive thermal analysis. For the reader’s convenience, these two terminologies (mesomorphous and mesophase) are explained via the schematic below. The definitions included in the schematic are meant to serve as general guidelines and are not exhaustive. For example, there may be other “mixed systems”, such as glassy mesophases (metastable with respect to the thermodynamically stable mesophase), where the local disorder, although independent of the long-range order, is kinetically “frozen”.

presence of distinct ordered (crystalline) and disordered (amorphous) domains in the compound or (ii) a more complex, one-state model which interprets the poor crystallinity to be representative of a single state of order, which is a continuum between the two extremes of complete order and disorder.11−13 Several explanations have been offered for such a single, intermediate ordered state, the most common one being loss of crystallinity owing to the increase in crystal lattice defect concentration with the resultant disorder being a distinct, intermediate kind.14−17 Partially ordered systems may also be indicative of the formation of a mesophase (liquid, plastic, or condis crystal), as was reported to occur upon dehydration of a calcium salt. In this case, the dehydrated material was characterized to be a liquid crystal, which is the most “liquidlike” of the mesophase family, possessing long-range orientational order, and, 1D- or 2D-positional order.18,19 Finally, in a recent study involving milling induced disorder generated in model pharmaceutical compounds, a third “model of crystallinity” was proposed since both the two- and one-state models could not satisfactorily explain the nature of disorder generated upon mechanical stress. As per the model, a more complex scenario may exist where two distinct one-phase systems (defected crystalline and amorphous domains) can coexist in the material, separated by a distinct phase transition as evident at longer milling times. In other words, partially disordered systems may very well consist of distinct phases of varying “disordered structures” unlike that assumed by a simplistic two-state model or a single continuum of order as proposed by a one-state model.20 Irrespective of the conclusions drawn by different authors as to the nature of stress generated disorder, it is obvious that the nature of the molecular order/disorder in such partially ordered systems is complex. Therefore, simple characterization techniques are often not sufficient, and more novel approaches (both analytical and computational) along with systematic and careful examination of the data are required to draw meaningful conclusions.14,21 Mesophase is a single phase material with at least one of the molecular degrees of freedom (conformation, position, and orientation) exhibiting crystalline-like long-range order, while the remaining degrees of freedom exhibit liquid-like motion/ mobility.22−25 As with a liquid, a mesophase can be quench cooled to ‘freeze’ the liquid-like mobility forming a glassy mesophase.23,26−28 A frozen glassy mesophase is considered to be a metastable kinetic state with the corresponding equilibrium thermodynamic state exhibiting the full liquid-like mobility. Because of their mixed nature, mesophases, the fourth phase of matter, will exhibit both “crystal-like” and “liquid-like” macroscopic properties.29 We hypothesize that the degrees of molecular freedom locked into long-range order in the mesophase will give sharp XRPD peaks and transition endotherms in a DSC measurement, while the disordered degrees of freedom (static or mobile) will give X-ray amorphous-like halos for XRPD and glass transition events in the DSC measurement. For single state partially ordered systems, a nomenclature known as “mesomorphous” exists in the literature where it is implied that a mesomorphic state is simply a mesophase (i.e., liquid crystal as mentioned in the articles).18,29,30 However, for this study, we will use the term mesomorphous to refer to a single solid-phase system that exhibits both long-range order and local disorder. In this definition, a mesomorphic system is a metastable solid system with both amorphous and crystalline

A condis crystal (conformationally disordered crystal) is the most “crystal-like” of the mesophase family since it possesses both long-range 3D positional and rotational order with partial or complete loss of long-range conformational order.23−25 As a result, condis crystals may be mistaken for crystalline polymorphs with lattice defects since they deviate only slightly in their properties from the fully ordered crystalline counterpart.22 For small molecules, the conformational disorder might be related to torsional degrees of freedom or be potentially due to the degrees of freedom in bond ordering. Condis crystal formers are often characterized as partially crystalline with the local changes in molecular conformation acting like point defects in the lattice. Because of the presence of a large number of these lattice defects, condis crystals may exhibit weak lattice strength and distort easily under light pressure.22 A molecule with a number of torsional degrees of freedom is typically a prerequisite for the formation of a condis crystal, making them relatively less common for smaller molecules. Owing to the presence of long-range 3D positional and orientational order, condis crystals have prominent X-ray diffraction peaks and show birefringence by microscopy.23 In addition, they also exhibit complex thermal behavior comprising multiple tran2810

dx.doi.org/10.1021/mp300558m | Mol. Pharmaceutics 2013, 10, 2809−2822


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over the 1−40° 2θ range. Data was analyzed using commercial software (JADE, version 9, Materials Data Inc., Livermore, CA). Variable Temperature XRPD. For VTXRPD, the diffractometer was configured using the symmetric Bragg−Brentano geometry. The GNE068-PC powder sample was packed into a 0.2 mm nickel-coated copper well. During the analysis, an ambient atmosphere was maintained around the specimen. The temperature was ramped at a rate of 10 °C/min from 25 to 240 °C, and diffraction patterns were collected in the reflection mode from 3.0 to 30° 2θ using the same step size of 0.017° 2θ and a scan speed of 1.3°/min. A single scan was collected at all temperatures. Heater power was supplied and controlled by Anton Paar TCU 100 interfaced with the data collector. Water Sorption Analysis. About 5−6 mg of powdered sample was placed in the sample pan of an automated water sorption analyzer (Q5000SA, TA Instruments, New Castle, DE) at 25 °C and a nitrogen flow rate of 200 mL/min. The sample was initially dried at 0% relative humidity (RH) for 300 min, following which it was subjected to a progressive increase in RH from 0 to 90%, in increments of 10% with a dwell time of 180 min at every RH. This was followed by a progressive decrease in RH in decrements of 10% back to 0% RH, with the same dwell time of 180 min at every RH. Raman Spectroscopy. Spectra of GNE068-A and GNE068PC were acquired using a MultiRAM FT-Raman spectrometer (Bruker Optics, Billerica, MA) with a 500 mW diode pumped Nd:YAG laser (1064 nm wavelength excitation source) and liquid nitrogen cooled germanium detector to collect the backscattered radiation. The spectra were collected over 4000− 100 cm−1 at a laser power of 301 mW with 32 scans per spectrum, at a resolution of 4 cm−1 and acquisition time of ∼1 min. The data was processed using commercial software (OPUS, version 7, Bruker Optics). Differential Scanning Calorimetry. Approximately 3−8 mg of powdered sample was analyzed using a DSC Q2000 (TA Instruments, New Castle, DE) equipped with a refrigerated cooling accessory. Samples were packed in open, nonhermetically crimped or hermetically crimped pans (Tzero, aluminum pans) and typically heated from 20 to 250 °C under dry nitrogen purge. The instrument was calibrated using sapphire (baseline) and indium (temperature and cell constant). The data was analyzed using commercial software (Universal Analysis 2000, version 4.7A, TA Instruments). The experimental conditions and pan configurations are as follows. (a) Nonhermetic crimped pan: For initial characterization, samples of GNE068-PC and GNE068-A were equilibrated at 20 °C, following which the temperature was increased to 250 °C at 10 °C/min. Experiments were also conducted in the modulated mode, where the samples were heated at 1 °C/min with a modulation of ±0.5 °C/min over 80 s. (b) Open pan: (i) In separate experiments, GNE068-PC samples were heated from 20 to 250 °C in open pans at heating rates of 1, 2.5, 5, 7.5, 10, 15, and 20 °C/min. (ii) A sample of GNE068-PC was annealed at 100 °C for 24 h (convective oven). This sample was then heated from 20 to 250 °C at 10 °C/min. (iii) In separate experiments, A sample of GNE068PC was heated to 160 °C at 10 °C/min and then cooled back to 20 °C at different cooling rates of 1 °C/min, 20 °C/min, and rapid equilibration (quench cooling). In all cases, the sample was removed from the pan and analyzed by polarized light microscopy.

sition events (exo- or endotherm) indicating solid−solid or solid−melt transitions which may be accompanied by a glass transition.26−28 Condis crystals have rarely been reported for pharmaceuticals, although it has been argued that there may be several developmental drug candidates which exist as such.22 The present study therefore focuses on the characterization of one such developmental drug candidate using routine in-house analytical techniques such as XRPD, polarized light microscopy (PLM), water sorption, Raman spectroscopy, and DSC along with computational analysis to explain its solid state makeup in terms of a mesomorphous form (single state of intermediate order) and the potential existence of a condis mesophase therein.

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EXPERIMENTAL SECTION Materials. GNE068, a small organic molecule in development, was chosen as the model compound. The critical steps of GMP synthesis and isolation of GNE068-PC are outlined as follows: A BOC-group (tert-Butyloxycarbonyl) protected API intermediate was obtained from the starting material, which was then dissolved in 2-propanol and acid (HCl) treated to yield GNE068·2HCl. This dihydrochloride salt was then resin treated till a solution pH of 5−7 was obtained. This solution was further treated with carbon, filtered, and concentrated. A solvent-exchange to ethyl acetate followed by vacuum drying at elevated temperature (80−85 °C) led to crystallization of the ethyl acetate solvate and isolation of GNE068-PC (monoHCl salt). GMP batches were typically produced at a 10−30 kg scale with a final purity ranging from 99.5 to 100.6%. Four GMP lots were analyzed. For all the lots, the ethyl acetate content, determined by head space gas chromatography, ranged from 0.2 to 0.5% w/w. The water content, determined by Karl Fischer titration, was determined to be 0.4−1.1%w/w. For GNE068-A, the final solvent of isolation was ethanol, and the API powder was obtained by rotary evaporation owing to the high solubility of the API in the isolating solvent. The ethanol and water contents were determined to be 0.6 and 2.1% w/w, respectively. A single GMP lot of GNE068-A was analyzed. Exposure to Mechanical Stress. A powder sample of GNE068-PC was triturated gently in a mortar for a minute. This gently ground sample was then characterized by PLM and XRPD. Annealing. A powder sample of GNE068-PC was annealed (stored isothermally) at 100 °C for 24 h over anhydrous CaSO4 in a convective oven. Post annealing, the sample was cooled to room temperature (RT) and analyzed by XRPD, DSC, and PLM. Methods. X-ray Powder Diffractometry (XRPD). XRPD patterns were collected with a PANalytical X’Pert PRO MPD diffractometer, using an incident beam of Cu Kα (1.541904 Å) radiation generated using an Optix long, fine-focus source (45 kV × 40 mA). Prior to the analysis, a standard silicon specimen (NIST SRM 640d) was used to verify the Si 111 peak position. GNE068 powder samples were packed between 3 μm thick films, and scans were collected in transmission geometry. A beam-stop, short antiscatter extension and an antiscatter knife were used to minimize background interference generated by air. Soller slits were used for both incident and diffracted beams to minimize axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X’Celerator) located 240 mm from the sample. A scan speed of 3.3°/ min and step size of 0.017° 2θ were employed to collect data 2811



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with the number of phase components fixed by the initial clustering results.34−37 Point Defects and Random Lattice Strain Models to Describe Noncrystalline Diffraction. Traditionally, the modeling of X-ray diffraction from disordered crystalline material has been based upon the change in crystalline peak width. As the microstructure of a crystalline material becomes more disordered (reduced crystalline size and increased microstrain), the diffraction peak width as measured in an X-ray powder pattern may increase. However, for a condis mesophase material the local molecular conformational disorder is inherent within the long-range position and orientation order and is not an example of kinetic microstructural disorder. More appropriate models of crystalline disorder where the disorder is within the coherent crystalline structure include point defects and random thermal motion.38 X-ray powder diffraction measurements average the state of the sample over time and over the measured sample volume; as such, random strain inherent within the crystal lattice (static) will be indistinguishable from random thermal motion (dynamic), and the X-ray diffraction from both sources can be described by traditional thermal vibration models.39,40 The presence of point defects and random strain within a crystalline lattice will not cause any observed diffraction peak broadening but will cause characteristic reductions in peak intensity. The loss in coherent diffraction intensity from the crystalline diffraction peaks will be balanced by the appearance of a corresponding increase in noncrystalline (X-ray amorphous) scattering. The X-ray amorphous profiles produced by point defects or random strain will have characteristic profile shapes which typically are different in appearance from the amorphous diffraction response from the same material. For an isotropic random strain field (static or dynamic), the loss in crystalline peak intensity and the corresponding X-ray amorphous profile can be described by the following expressions from Cowley:38

(c) Hermetically crimped pan: Initially, GNE068-PC samples were dried at 105 °C in an open pan for a few minutes, after which the sample-containing pan was crimped hermetically, equilibrated at 20 °C, and then subjected to a cycling temperature program, wherein it was heated through 125, 150, and 175 °C, with the sample being cooled to 20 °C in each of the cooling cycles. The heating and cooling rate for all the cycles was 1 °C/min, and a modulation of ±0.5 °C/min, 80 s was applied. Thermogravimetry. In a thermogravimetric analyzer (Q500 TGA, TA Instruments), 3−4 mg of GNE068-PC and GNE068A samples were heated in an open aluminum pan from RT to 250 °C at a heating rate of 10 °C/min and RT−250 °C at 1 °C/min, under dry nitrogen purge. Temperature calibration was performed using Alumel and Nickel. Standard weights of 100 mg and 1g were used for weight calibration. The TGA was hyphenated with a Nicolet 6700 FT-IR spectrometer (ETC EverGlo IR source, KBr beam splitter, DTGS detector) via an insulated, glass lined, stainless steel transfer line connected to the nickel plated, aluminum gas cell module of the spectrometer. A sample of GNE068-PC (∼15 mg) was placed on the TGA aluminum pan and heated to 250 °C at 20 °C/min with simultaneous data collection on both instruments. From the Gram-Schmidt plot recorded over the 20−250 °C temperature range, individual spectra (16 scans per spectrum with a resolution of 8 cm−1) were selected at desired time points and analyzed using commercial software (OMNIC, Thermo Fisher Scientific, Waltham, MA). Microscopy. Samples were dispersed in silicon oil and observed under cross-polarizers of a video-enhanced Leica DM 4000B microscope equipped with a high resolution CCD camera and motorized stage (Clemex Technologies Inc., Longueuil, Quebec, Canada) at 200× magnification. A hot stage microscopy experiment was conducted by dispersing the sample in silicon oil and heating from RT−200 °C at 10 °C/ min using a Linkam LTS 350 stage and TMS94 temperature controller (Linkam Scientific Instruments, Surrey, UK) to obtain images at 100× magnification. Photographs (for both ambient and hot stage microscopy) were acquired using the Clemex Vision PE software (Clemex Technologies Inc., Longueuil, Quebec, Canada). Computational Analysis. Chemometrics. A chemometric analysis was performed in order to characterize sample to sample variance in the crystalline and noncrystalline components observed by XRPD. Prior to the chemometric analysis, an estimated instrumental background contribution was scaled and removed from each measured powder pattern. The background stripped data files were then passed through a low pass multiloop digital filter to isolate the noncrystalline (X-ray amorphous) response from the crystalline response for each sample. The resulting noncrystalline components were then binned to a common step size in 2θ that gave the smallest number of data points required to retain the overall continuous profile. The binned noncrystalline and crystalline components were further scaled to a common integrated area to form the two input data ensembles for chemometric analysis. A traditional cluster analysis (partitioning around medoids) was initially performed using randomly generated decision trees to determine similarity (distance).31−33 To isolate the XRPD responses associated with each identified cluster and to provide a semiquantitative estimate of the relative amounts of each profile present for each sample, the input data ensemble was passed through an alternating least squares (ALS) procedure

2 I(Q )random − strain ∝ (1 − e−2W )|F(Q )|crystal

(1)

2 I(HKL)crystalline ∝ (e−2W )FHKL

(2)

2 2 FHKL = |F(Q )|crystal ∑∑∑δ H

K

L

(Q − (2π (Ha * + Kb * + Lc*)))

(3)

The important feature of the X-ray amorphous noncrystalline scattering intensity I(Q) (Q = 4π sin(θ)/λ) is that its profile shape depends on the continuous crystalline unit cell structure factor F(Q). The exponential exponent W plays the role of a traditional Debye−Waller factor and is given by

W = Q 2b2 /2

(4)

with b being the isotropic strain field distortion of the unit cell expressed in Å. For the simplest model of random point defects, the complete unit cells are randomly missing within the coherent crystalline lattice.38 The form of the incoherent noncrystalline scattering intensity will be directly related to the continuous unit cell structure factor F(Q) and for small defect concentrations will scale with the number of defects. 2 I(Q )point − defect ∝ |F(Q )|crystal

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(5)



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If a single crystal structure is available, then |F(Q)|2 can be directly calculated by applying the Debye−Menke calculation to a single crystal unit cell.39 Without a single crystal structure, the continuous structure factor |F(Q)|2 can be approximated from the discrete structure factor (FHKL)2 extracted from a measured crystalline powder pattern as expressed in eq 3. One approach to perform this approximate extraction is to Fourier transform (FHKL)2 giving the corresponding crystalline Patterson function in real space, P(r).

cumulative peak index determined from a measured powder pattern when plotted against S should approximately follow a power law with the power law exponent only dependent on the dimensionality of the long-range order. For measured XRPD data, a number of the diffraction peaks may not be observed due to overlap with other peaks, symmetry extinction, or weak intensity. The loss of peaks due to space group symmetry operators will not change the dimensionality of the cumulative peak index but will reduce the effective value of dE. The experimentally determined dE is referred to as a minimum value when the space group symmetry is unknown. However, the loss of peaks due to peak overlap and the overall loss of intensity at higher angles will change the apparent power law behavior of the cumulative peak index. The departure from ideal behavior will increase with increasing measurement angle. As such, the dimensionality analysis is usually performed using the low angle data and by modeling the power law as an asymptote of the small S cumulative peak index. The low angle data is usually sufficient to determine whether the long-range order is inherently 1D, 2D, or 3D in nature, provided that the unit cell dimensions are of a similar order of magnitude to each other. For unit cells with few symmetry operators, the value of dE can provide an estimate of the unit cell volume by interpreting dE as twice the radius of gyration of a hollow sphere with radius rS; rS = sqrt(3/8)dE. The occupied unit cell radius of gyration, Rcell, can be backed out by assuming the sphere to be uniformly filled with electron density; Rcell = sqrt(2/5)rS. Rcell will be directly related to the characteristic length scale of the Gaussian damping function required to estimate the continuous cell structure factor |F(Q)|2.

2 } = P(r ) = ρ0 (r ) × ρ0 ( −r ) × δ(r ) × δ( −r ) FT {FHKL

(6)

The continuous function ρ0(r) represents the electron density distribution within the crystalline unit cell (short distances), while the discrete delta functions δ(r) represent the placement of the unit cell according to the long-range lattice periodicity. In the Patterson function, the continuous and discrete electron density distribution functions are all convoluted together. Thus, by damping the Patterson function at larger distances to isolate the unit cell ρ0(r) at shorter distances, |F(Q)|2 can be approximately reconstructed by performing the inverse Fourier transform as follows: F(Q )2 = iFT {ρ0 (r ) × ρ0 ( −r )} ≈ iFT {Pdamped(r )}

(7)

with the simplest damping of the Patterson function being performed by a Gaussian function: Pdamped(r ) = P(r )e−r

2

/σ 2

(8)

To ensure that the damped Patterson function predominantly contains only atom−atom pair distances within a single unit cell, in this study, the characteristic length scale of the damping Gaussian was set to the radius of gyration, Rcell, of the unit cell itself. Determination of the Long-Range Order Dimensionality and Rcell (Unit Cell Radius of Gyration). When working with mesophase systems, the dimensionality of the long-range order is a significant component of the mesophase character. From the peak positions determined from an XRPD pattern, it is possible to estimate the dimensionality of the long-range order by a number of different approaches (such as indexing). For a first approximation, the functional form of the cumulative peak counts as a function of S (where S = 2 sin(θ)/λ, θ is half the diffraction angle 2θ, and λ is the X-ray wavelength in Å) and can provide a direct measure of effective dimensionality of longrange order. For a simple 1D lattice with periodicity d, from Bragg’s equation, we can express the cumulative peak index N as follows: sin θ = dS N = d2 λ

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RESULTS Preliminary Characterization. GNE068 structure is shown in Figure 1. XRPD patterns of both GNE068-A and

Figure 1. Structure of GNE068.

(9)

GNE068-PC are overlaid in Figure 2 along with the corresponding PLM images. GNE068-A, isolated from ethanol, was determined to be amorphous due to the presence of diffused halos in the XRPD pattern and absence of birefringence in the corresponding micrograph. In contrast, GNE068-PC (isolated from ethyl acetate), appeared to be partially crystalline since it showed the presence of both diffuse scattering (halo) and diffracted peaks in the XRPD pattern along with birefringence in the PLM image. Figures 3a and b show the overlaid DSC and TGA plots of GNE068-A and GNE068-PC, respectively. The MDSC profiles are shown in Figure 4. When samples were heated at 10 °C/ min, GNE068-A showed a shallow endotherm from 22 to 140

So a plot of cumulative peak index N against S gives a straight line passing through the origin with slope d. This expression can be generalized for crystalline space groups in higher dimensions D where the periodic order length scales in each dimension are similar but not equal. N = (dES)D

(10)

The single periodicity length dE is an effective distance corresponding to the radius of gyration of the individual basis vectors in each dimension about the origin, which for orthorhombic crystalline order is (sqrt ((a2 + b2 + c2)/3)) with a, b, and c being the usual unit cell dimensions). The 2813



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subtraction revealed the weight loss in the 130−145 °C range to be due to desolvation and vaporization of ethyl acetate (Figure 3c). MDSC profiles reveal the glass transition (Tg) events of GNE068-A and GNE068-PC (Figures 4a and b) at ∼125 and 135 °C (Tg midpoint), respectively. Overlaid Raman spectra and water sorption profiles of GNE068-A and GNE068-PC are shown in Figures 5 and 6, respectively. Barring minor differences, the spectra of GNE068A and GNE068-PC appeared to be very similar and consisted of mainly broad, defocused peaks (Figure 5). Water sorption behavior of both these forms was continuous as a function of RH, and the sorption profiles were near-superimposable (Figure 6). Both GNE068-PC and GNE068-A were found to deliquesce at ∼67% RH and RT (powdered sample converted to slush as examined visually). Effect of Temperature. Heating. In order to further investigate the nature of the second endotherm observed in the 135−150 °C range appearing in the DSC profile of GNE068PC, the sample was subjected to several controlled heating programs, and the associated enthalpy change (ΔH) was recorded. Figure 7 shows the effect of heating rate on ΔH. An open pan configuration was used since it provided a good separation between the first and second endotherms, unlike in the nonhermetic crimped pans where these two events overlapped at faster heating rates (≥5 °C/min). As evident from the figure, ΔH showed a prominent dependency on the heating rate, ranging from ∼11 J/g (20 °C/min) to ∼7 J/g (1 °C/min). Furthermore, as shown in Figure 8a, annealing the sample at 100 °C (Tg − 30) for 24 h also reduced ΔH to 4 J/g, as compared to the unannealed sample (ΔH = 10 J/g) heated at the same heating rate of 10 °C/min. The XRPD pattern and PLM image of the annealed sample, collected at RT, are

Figure 2. XRPD patterns and PLM images of GNE068-A and GNE068-PC. The GNE068-A diffraction pattern shows two diffused halos, whereas both amorphous halo and diffracted peaks are seen in the pattern of GNE068-PC. Birefringence is observed in GNE068-PC particles but is absent in GNE068-A.

°C, which corresponded well with the single step weight loss observed in its TGA profile (Figure 3a). This was attributed to the loss of residual solvent (water and ethanol) retained in the sample. For GNE068-PC, two endotherms were observed in the RT−250 °C range (Figure 3b). The first, shallow endotherm extended from 22 to 130 °C, while a second, sharp endotherm was observed in the 130−145 °C range. The DSC endotherms corresponded well with the 2-step weight loss observed in the TGA profiles. Since loss of residual water from the poorly crystalline lattice is expected to be almost instantaneous, the first endotherm/weight loss event was attributed to dehydration and vaporization of water. The small, second weight loss was investigated by hyphenated TGAIR, where spectra of the evolved volatile component were collected periodically as the sample was heated. Spectral

Figure 3. Overlaid DSC (nonhermetically crimped pan) and TGA plots of GNE068-A (a) and GNE068-PC (b). In the hyphenated TGA-IR plot (c), spectrum (collected at ∼165 °C) − spectrum (collected at ∼50 °C) yielded a spectrum which matched very well with the ethyl acetate standard spectrum (c). 2814



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Figure 4. MDSC traces of GNE068-A (a) and GNE068-PC (b) (1 °C/min heating rate, modulation of ±0.5 °C/min for 80 s, nonhermetically crimped pan). The reversing heat flow profiles show the corresponding glass transition (Tg) events. The Tg midpoints are indicated in the plots.

included in Figure 8b. The sample appeared to be X-ray amorphous but showed some birefringence in the PLM image. The reversing Cp (isobaric heat capacity) plots of GNE068PC are included in Figure 9, wherein the sample was heated to 125, 150, and 175 °C with consecutive cooling cycles in between, using a dried sample in a hermetically sealed pan, at a heating rate of 1 °C/min with a modulation of ±0.5 °C/min for 80 s. The prominent baseline shifts in the plot represent Cp change at Tg in both the heating and cooling cycles. As is evident from the plot, the Tg midpoint decreased from 119 to 116 °C in the cooling cycle after heating to 125 °C, remained unchanged in the second heating−cooling cycle from 150 to 20 °C, and reduced further to 109 °C in the cooling cycle upon heating to 175 °C and cooling to 20 °C. In order to investigate the second endotherm occurring in the 130−150 °C range, structural information was obtained by subjecting a sample of GNE068-PC to VTXRPD, where diffraction patterns were collected periodically from 25 to 240 °C. A plot of the VTXRPD patterns (Figure 10) showed loss of crystallinity in GNE068-PC with increase in temperature, with the most intense peak (7.2° 2θ) persisting up to 150 °C. At temperatures ≥150 °C, the poorly crystalline GNE068-PC appeared to be completely disordered. Similarly, the disappearance of birefringence was observed in hot stage microscopy images when the GNE068-PC sample was heated from RT-200 °C (the results are included in Supporting Information). Cooling. Figure 11 shows the DSC profiles of the GNE068PC samples heated to 160 °C/min and then cooled back to 20 °C at different cooling rates (quench cooling, fast cooling at 20 °C/min, and slow cooling at 1 °C/min). An exothermic event was observed at all cooling rates, the exotherm appearing at higher temperatures with increasing cooling rate. Upon cooling to 20 °C, the sample consisted of a glassy mass. Upon inspection under cross-polarizers, the glassy mass appeared to contain birefringent particles (inset). Effect of Mechanical Stress. The XRPD patterns of “as is” GNE068-PC and the triturated sample are compared in Figure 12, where the measured data has been background subtracted and normalized to the common area (integrated intensity). As is evident from the Figure, gentle trituration of GNE068-PC caused a decrease in all of the diffracted peak intensities, especially for the characteristic peak at 7.2° 2θ, which showed a >50% reduction in the integrated intensity. The loss of diffraction peak intensity is balanced by an overall increase in the level of the diffuse scattering contribution for the triturated sample. No significant peak broadening was observed on trituration.

Figure 5. Raman spectra of GNE068-A and GNE068-PC. The spectral range of 100−1700 cm−1 is shown. The Raman spectra of both forms are similar, with minor spectral differences.

Figure 6. Water sorption profiles of GNE068-A and GNE068-PC (0− 90% RH, 25 °C). Both forms show continuous weight gain upon exposure to moisture, with near identical sorption profiles.

Figure 7. Effect of heating rate on GNE068-PC (open pan). 2815



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Figure 8. Overlaid DSC traces of GNE068-PC (“as is”) and GNE068-PC annealed for 24 h, at 100 °C, in an open pan (panel a). The XRPD profile and PLM image of the annealed sample are shown in panel b.

Figure 9. Plots of reversing Cp vs temperature profiles of GNE068-PC heated through 125, 150, and 175 °C with corresponding cooling cycles. The sample was previously dried at 100 °C, crimped hermetically, and then heated and cooled in three consecutive heat−cool cycles at 1 °C/min. The Tg midpoints are indicated for each heating−cooling cycle. In the first cycle (heating to 125 °C and cooling), Tg is lowered by 4 °C in the cooling cycle (panel a), remains unchanged in the second cycle (heating to 150 °C and cooling, panel b), and further lowers by 7 °C in the third cycle (heating to 175 °C and cooling, panel c).

Computational Analysis. Analytical characterization of GNE068-PC suggested the presence of a complex, single state of order, possibly a mesophase. For a more complete understanding of the solid-state form of the compound, computational analysis was performed on 5 GMP lots (4 lots of GNE068-PC and 1 lot of GNE068-A). Prior to performing

computational analysis, each of the measured XRPD patterns were processed for chemometric analysis and passed through a digital filter to isolate the crystalline and noncrystalline diffraction components, for example, Figure 13a. A chemometric analysis of the noncrystalline X-ray amorphous responses revealed that at least three noncrystalline compo2816



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Figure 12. Comparison of XRPD patterns of “as is” GNE068-PC and gently triturated GNE068-PC. The measured data have been background corrected and normalized to a common area (integrated intensity). The triturated sample shows loss of crystallinity, as evident by a decrease in all diffracted peak intensities and a general increase in the diffuse noncrystalline contribution. No significant peak broadening was observed for the triturated sample.

Figure 10. Select VTXRPD patterns of GNE068-PC collected periodically from 25 to 240 °C at 10 °C/min. The sample loses crystallinity progressively, with the most intense peak at 7.2° 2θ (*) and the adjoining shoulder persisting up to 150 °C. Diffraction peaks were no longer detected >150 °C.

a plot of the cumulative peak index as a function of S shown in Figure 14. The power law behavior of the cumulative peak index was modeled using an asymptotic fit to the low angle (small S) data. The 3D asymptote gives the best description of the small S behavior and rises above the measured cumulative peak index at larger values of S as expected due to missing peaks. The corresponding effective d value, dE, is ∼16 Å and corresponds to the minimum radius of gyration of the 3D unit cell basis vectors about the origin. From the hollow sphere transformation, this corresponds to a unit cell volume of at least 4000 Å3, with Rcell ∼6.2 Å for an isotropic electron density distribution within the sphere. An initial visual inspection of the two noncrystalline components not representative of the amorphous GNE068-A (Figure 13) revealed that the lower angle halo position corresponded to a low angle peak cluster from the crystalline material. This suggested that the unidentified noncrystalline components may be related to the crystalline lattice. One model of noncrystalline diffraction tied to a parent crystalline lattice is random lattice strain. Within this model, the random strain occurs with the coherent crystal lattice (like thermal vibration) and does not destroy the long-range coherent order. To calculate the noncrystalline powder pattern arising from random strain requires that the continuous crystalline unit cell form factor |F(Q)|2 be known (eqs 1 and 5). In this study, the continuous form factor was estimated from a measured crystalline powder diffraction pattern (eq 7). The mean crystalline powder pattern of GNE068-PC was used as the input data to approximate |F(Q)|2 with the characteristic unit cell damping length scale for the Patterson function taken to be 6.2 Å as determined from the cumulative peak index. The resulting |F(Q)|2 was then used to investigate the form of the noncrystalline powder patterns arising from random strain within the GNE068-PC mean crystalline lattice using the RMS strain field extent as the primary variable. Figure 15, shows the calculated noncrystalline powder pattern for a 1 Å RMS strain field extent within the mean GNE068-PC crystalline lattice. The overall agreement between the calculated random strain induced noncrystalline diffraction and noncrystalline component 3 is good, although the position of the low angle halo is

Figure 11. DSC traces of GNE068-PC at different cooling rates with an exothermic event observed in all cooling cycles (open pan). Irrespective of the cooling rate, the cooled sample was found to be a glassy mass showing birefringence by PLM (inset).

nents were required to adequately describe the sample-tosample variance, as shown in Figure 13b. One of the identified noncrystalline XRPD components (component 1) matched the known noncrystalline XRPD profile of amorphous GNE068-A. The two remaining noncrystalline components (components 2 and 3) represent unidentified disordered components within the GMP batches of GNE068-PC. A chemometric analysis of the extracted crystalline powder patterns (i.e., the crystalline diffraction contribution from the partially crystalline XRPD pattern) indicated that only two components were required to fully describe the sample-tosample variance. A peak position analysis of the two identified crystalline components supported the conclusion that both crystalline components represented the same crystalline form, with the only variance being in relative peak intensities. As such, the two components were combined to give a mean crystalline powder pattern as representative of the GMP batches of GNE068-PC. The crystalline peak positions determined for the mean powder pattern were used to create 2817



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Figure 13. (a) Overlay of an example processed XRPD data file of GNE068-PC prepared for chemometric analysis and the corresponding low pass digital filter output used for noncrystalline contribution. (b) Overlay of the three noncrystalline components identified by chemometric analysis as being required to describe the sample to sample variance for the various GMP batches of GNE068-PC.

angles could be the origin of the observed offset in halo position. Noncrystalline diffraction responses arising from random strain will have a variable profile shape depending on the extent of the induced strain field (Figure 15). The variation in the calculated profile shapes is sufficient to incorporate both of the unidentified noncrystalline components within the same random strain model with a relatively small variation in the RMS strain field extent (Figure 16). The differences in the

Figure 14. Cumulative peak index plot N against S = 2 sin(θ)/λ for the mean crystalline powder pattern of GNE068-PC. The calculated asymptotic 3D and 2D solutions are displayed for comparative purposes.

Figure 16. Evolution in the calculated noncrystalline powder patterns based upon the random strain model with RMS strain field extents of 1.5, 1.0, and 0.7 Å. The inset shows the unidentified noncrystalline components 2 and 3 (scaled to match at higher measurement angles).

shape of the noncrystalline diffraction profiles between components 3 and 2 are similar to the differences in the calculated profiles for a 1.0 Å and 0.7 Å RMS strain field extent. There was no evidence of a noncrystalline profile arising from simple random strain as expressed in eq 5. With the crystalline and amorphous powder patterns taken as fixed profiles as identified by the chemometric analysis and taking a variable powder pattern profile as described by a random strain field, a simple semiquantitative analysis was performed using a least-squares refinement model. The variables in the model were the percentages of the individual profiles and the RMS strain field extent. The modeling was performed on the total XRPD diffraction signal measured for

Figure 15. Overlay of the unidentified noncrystalline component 3 with the calculated noncrystalline powder pattern for an RMS strain field extent of 1 Å.

shifted in 2θ by about 1.5°. At lower angles, the estimated | F(Q)|2 derived from a measured crystalline powder pattern exhibits the largest uncertainties due to the smaller number of crystalline peaks in this region. This increasing error at low 2818



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Experimental results, in addition to the computational analysis, provided adequate support to the mesomorphous makeup of the partially ordered compound. This partially ordered system exhibited a series of analytical responses that implied that a simple two phase crystalline−amorphous model was not a suitable description of this system. These are as follows: (a) Although a pure crystalline standard representative of the 3D order was never obtained for GNE068-PC, it is reasonable to assume that a two-phase system consisting of distinct crystalline and amorphous domains would likely exhibit sharp, well resolved peaks along with broad diffuse peaks in its spectra. In addition, the water sorption profile might be expected to exhibit a hygroscopicity intermediate to that of the pure crystalline and amorphous phases (the exception being isomorphic desolvates and nonstoichiometric hydrates; however, for GNE068-PC, no crystalline form could be isolated from water).41−44 In contrast, GNE068-A and GNE068-PC both exhibited near identical water sorption profiles. Furthermore, both GNE068-A and GNE068-PC gave essentially the same Raman spectra (Figures 5 and 6). This lack of specificity between the amorphous and partially crystalline materials with respect to hygroscopicity and vibrational spectroscopy suggests that the level of local disorder is similar for both materials and that the local molecular interrelationships may also be similar. Neither vibrational spectroscopy nor water sorption measurements proved to be sensitive to the presence of the long-range 3D crystalline-like order observed by XRPD. The mesomorphic nature of the partial crystalline phase as suggested by the XRPD modeling where there is significant local conformational disorder within the long-range ordered framework is also consistent with the water sorption and Raman results. A free-standing crystalline phase would likely impact the water sorption and/or the Raman spectroscopy data. (b) The DSC profile of GNE068-PC revealed the presence of an endotherm in the 130−150 °C range overlapping a glass transition (Figure 4b). This endotherm was different from a first order transition/true melting event owing to a marked dependence of the associated enthalpy (ΔH) on the heating rate (Figure 7). Since a true thermodynamic solid−liquid transition, i.e., melting of a crystalline form, involves addition of a fixed amount of thermal energy to dissociate the lattice, ΔH of melting should be unaffected by a change in heating rate.45 The observed enthalpy dependency on heating rate suggested that the 3D long-range ordered phase, producing the observed diffraction peaks, does not exhibit the typical thermal melting behavior expected for a standalone crystalline phase. With a glass transition and endothermic event occurring together, the possibility that the endotherm was an enthalpy recovery event has been explored. Often, pharmaceutical glassy materials undergo sufficient relaxation when stored at temperatures close to but below Tg due to the presence of residual mobility that causes the glass to evolve toward a lower state of enthalpy. When such an annealed glass is heated through its Tg, the glass recovers at this transition temperature and exhibits an endotherm in close proximity to Tg. The ΔH associated with such an endotherm is known as enthalpy of recovery and is used as a measure of the extent of relaxation of the glass below Tg.46,47 Enthalpic recovery is an irreversible event and is often separated from the reversible Tg in modulated DSC experiments. For GNE068-PC, the value of ΔH associated with a similar endotherm overlapping with the Tg, ranged from 7 to 11 J/g. This is substantially greater than that associated with

each of the GMP batches of GNE068-PC; see, for example, Figure 17. The most notable result was that for material

Figure 17. Total diffraction response (processed) from one of the representative GMP batch samples overlaid with the corresponding 3 component model derived through a simple least-squares model. The relative contributions of each component for this batch are mean crystalline, ∼21%; amorphous GNE068-A, ∼ 35%; and random strain noncrystalline, ∼44%.

showing crystalline diffraction peaks, the integrated intensity contribution of the random strain noncrystalline scattering profile was larger than the crystalline contribution. This would suggest that a significant portion of the electron density (atoms) within the molecule is associated with the local disorder. In general, for all of the GMP batches of GNE068-PC, the crystalline contribution was found to be ∼18−24%, the noncrystalline “random strain” contribution was ∼44−72%, and the noncrystalline “true amorphous”(GNE068-A) contribution ranged from ∼4−33%.

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DISCUSSION Computational analysis of the X-ray diffraction data indicates that in addition to a 3D crystalline-like contribution, there are two distinct types of noncrystalline contributions to the powder diffraction data. The first type of noncrystalline component matches the known amorphous response (GNE068-A), while the second type of noncrystalline component has a variable profile shape of which noncrystalline 2 and noncrystalline 3 are the extreme examples identified by chemometric analysis. The general shape of the XRPD profiles for noncrystalline 2 and noncrystalline 3 components and the relationship between them are best described in terms of a random strain field within the 3D crystalline lattice. It is the framework of long-range crystalline within which the local disorder occurs that imparts the crystalline unit cell intensity fingerprint to the noncrystalline 2 and 3 XRPD components. This suggests that GNE068PC is a mixed system consisting of an amorphous phase and a single state of intermediate order, i.e., a mesomorphic phase. The mesomorphic phase exhibits both long-range 3D crystalline-like order and local disorder. The disordered component of the mesomorphic phase generates the dominant XRPD intensity contribution for all batches of GNE068-PC studied, which suggests that a significant portion of the molecule is involved in the local disorder. However, to determine if the mesomorphic phase is indeed a condis crystal mesophase requires a comprehensive evaluation of thermal analysis results. 2819



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typical ΔHrecovery values reported for pharmaceutical glasses (∼2−3 J/g).47−49 Additionally, if an aged glass is further annealed close to the Tg for a certain duration, ΔHrecovery either increases or remains the same depending on the nature of the molecular motions and extent of relaxation (under the annealing conditions).46,47 In contrast, the annealing of GNE068-PC at Tg-30 °C, a temperature fairly close to Tg, led to a decrease in ΔH by >2 fold compared to the as-received, unannealed sample (Figure 8a). Such an anomalous behavior ruled out the possibility of the endotherm being entirely an enthalpy recovery event. (c) Consecutive heating and cooling experiments indicated an increase in sample mobility upon heating through the glass transition temperature (Figures 9a−c). In the first cycle, the Tg midpoint shifted to a lower temperature (120→116 °C) upon cooling the sample from 125 °C, which clearly suggested an increase in the mobility of the sample, thereby indicating the possibility of a structural change which would lead to such increased mobility. The sample mobility appeared to be unchanged in the second heat−cool cycle as no further changes in Tg was observed when the sample was heated to 150 °C. In the third heat−cool cycle, Tg in the cooling cycle further slipped to 109 °C as compared to 116 °C in the heating cycle. This drastic increase in sample mobility, as indicated by the marked shift of Tg to a lower temperature, was attributed to sample degradation occurring at 175 °C (confirmed by substantial weight loss by thermogravimetry conducted at 1 °C/min; data not shown). (d) In order to obtain structural information pertaining to the second endotherm, XRPD patterns were collected as the sample was heated from RT−200 °C. Upon exposure to high temperatures, GNE068-PC showed a progressive loss in crystallinity (Figure 10). The sharp endotherm was therefore deemed to be an apparent melting or decrystallization event. Enthalpy dependency on heating rates is characteristic of the apparent melting phenomenon and has been reported previously.45 (e) Gentle trituration of GNE068-PC for ∼1 min resulted in a marked reduction in all diffraction peak intensities (Figure 12) combined with an overall increase in the diffuse scattering intensity. Such ready loss of crystalline lattice integrity on application of low mechanical stress indicates a soft lattice, unlike that exhibited by a stand alone crystalline phase. Such loss of lattice integrity and lattice softness is a characteristic property of mesophases owing to the presence of the mobile molecular disorder, which renders them less rigid.22 Both experimental and computational data strongly suggest that the partially crystalline system is a single mesomorphic phase. Additionally, (i) due to the dominant contribution of the noncrystalline diffraction response (different from the ideal amorphous response) which shares the same local fingerprint with the 3D crystalline order and (ii) ready deformation of the lattice under stress, GNE068-PC exhibits some characteristic features of a condis mesophase. However, it is known that mesophases are equilibrium phases, and therefore, the ΔH of a transition, i.e., mesophase → isotropic liquid phase (isotropization temperature analogous to a melt) should ideally be independent of heating rates as well if the mesomorphous behavior is truly due to the presence of a thermodynamic condis mesophase. Taking the endothermic event in the 130− 150 °C range as the isotropization transition, our data has shown that the ΔH of this transition varies with heating rate. In addition, mesophase order (long-range order + disorder)

should be restored completely at its equilibrium temperature. Annealing the sample at temperatures just below the combined glass transition plus endothermic event led to the loss of all detectable long-range order by XRPD (Figure 8b). This was also observed by VTXRPD when the sample was heated to temperatures >140 °C. The samples (either annealed or exposed to high temperatures) always showed some birefringence at RT, but long-range order was no longer observed by XRPD. We believe that these somewhat contradictory results are due to the intimate role played by the residual solvent (ethyl acetate) in maintaining the longrange positional order in the mesomorphic form. This residual ethyl acetate is lost in the same temperature range (130−150 °C) where the endotherm and glass transition events are observed in the DSC profile of GNE068-PC. This delayed release of the ethyl acetate at ∼130−150 °C, along with the observation of a sharp endotherm, suggests that the solvent is in some way tied to the observed long-range order for GNE068-PC. In other words, any event that leads to irrecoverable solvent loss will also lead to a permanent structural alteration (collapse of long-range order) which limits the applicability of thermal methods to test for thermodynamic mesophase behavior. Although the total amount of ethyl acetate (∼0.5%w/w) in the mesophase at RT is not sufficient to justify the presence of a distinct solvated crystalline phase (for 20% crystalline material, a mono ethyl acetate corresponds to about 3% weight loss) to account for the crystalline order indicated by the diffracted peaks, it seems essential for the maintenance of long-range positional order. Loss of ethyl acetate at a higher temperature coincides with structural loss, as shown by TGA, DSC, and VTXRPD analysis from RT−160 °C and XRPD of the annealed sample at RT. Owing to this irreversible loss of long-range order at the vicinity of Tg, the endotherm in that temperature range represents an apparent melt and not a true thermodynamic transition event. Therefore, the associated ΔH shows a heating rate dependency. Slower heating rates (as well as annealing at 100 °C) allow greater residence time at higher temperatures, leading to early desolvation and increased loss in long-range order. Once the sample is heated beyond the Tg, solvent loss and associated structural collapse result in enhanced mobility of the material, as indicated by a decrease in the Tg temperature in the cooling cycle of the DSC experiment (Figure 9a). Upon cooling the sample to RT, birefringence is observed by PLM (Figures 8b and 11). This suggests that although long-range positional order is removed upon solvent loss, some orientational order may be regained. The exotherm observed in the cooling cycle is possibly indicative of such a molecular realignment (Figure 11). The exact nature of the molecular order formed on cooling from above the solvent loss event and a structural model of the local molecular disorder in the mesomorphous state are currently being investigated.

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CONCLUSIONS The study of GNE068-PC represents an in depth investigation of a small molecule pharmaceutical compound that potentially exhibits a mesophase during routine production. This partly disordered compound was characterized by both experimental and computational methods to determine the most selfconsistent description of the molecular order and disorder existing in this material. Computational analysis of XRPD data revealed the presence of three distinct contributions to the measured data, one of which was crystalline-like. The 2820



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Chemistry; Genentech, Inc.) are acknowledged for API supplies. Michael Dong, Kelly Louie, and Venkata Ravuri (Small Molecule Analytical Chemistry; Genentech, Inc.) are thanked for the GC and KF data. P.C. thanks Joseph Lubach (Small Molecule Pharmaceutical Sciences, Genentech, Inc.) for assistance with TGA-IR.

crystalline-like contribution was determined to correspond to a 3D ordered lattice. The first noncrystalline contribution was identified as matching the known amorphous response for this molecule, while the second noncrystalline response was determined to be directly related to the crystalline-like order. The simplest model consistent with the XRPD data is a binary amorphous plus mesomorphous system. The mesomorphic system is a single phase where the long-range 3D order provides a framework within which there is significant local molecular disorder. Both water sorption and Raman spectroscopy results were consistent with the proposed mesomorphic system where the long-range order and local disorder coexist within a single phase. Although GNE068 is a small molecule, the local disorder in the mesomorphous system is proposed to occur due to the conformational mobility of specific functional groups. This is consistent with the definition of a condis crystal mesophase (long-range positional and orientational order plus partial or full conformational disorder). Structurally, this appears plausible since the molecule consists of several functional groups/side chains (Figure 1), such as the substituted phenyl group or bicyclic cyclopentylpyrimidine group or the isopropyl amino group attached to the piperazine core by means of a single bond, which have the potential for torsional degrees of freedom, a precursor to conformational disorder. The overall mobility of the molecule as well as those of the individual functional groups has been systematically investigated by solidstate NMR in a subsequent manuscript, which indicates the bicyclic cyclopentylpyrimidine group to be a major contributor to the mobility in this mesomorphous system. Additionally, the crystalline-like lattice was easily deformed when subjected to light mechanical stress, which is a characteristic property of a mesophase in general. Thermal analysis was unable to confirm whether the observed mesomorphic behavior was indeed attributable to a thermodynamic condis crystal mesophase due to the intimate relationship between residual ethyl acetate solvent and the observed long-range order. Unlike their ordered counterparts, pharmaceutical molecules that exhibit mesophase or mesomorphic behavior (single state of intermediate order) possess unique properties which may pose a completely different set of challenges in formulation development and therefore require special attention.22

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ASSOCIATED CONTENT

S Supporting Information *

Select hot stage microscopy images of GNE068-PC as a function of temperature (RT-135 °C). This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Byrn, S. R.; Pfeiffer, R. R.; Stowell, J. G. Solid-State Chemistry of Drugs, 2nd ed.; SSCI, Inc.: West Lafayette, IN, 1999; p 5. (2) Yu, L. Amorphous pharmaceutical solids: preparation, characterization and stabilization. Adv. Drug Delivery Rev. 2001, 48 (1), 27−42. (3) Hancock, B. C.; Parks, M. What is the true solubility advantage for amorphous pharmaceuticals? Pharm. Res. 2000, 17 (4), 397−404. (4) Hancock, B. C.; Zografi, G. Characteristics and significance of the amorphous state in pharmaceutical systems. J. Pharm. Sci. 1997, 86 (1), 1−12. (5) Miller, D. A.; DiNunzio, J. C.; Yang, W.; McGinity, J. W.; Williams, R. O., III. Enhanced in vivo absorption of itraconazole via stabilization of supersaturation following acidic-to-neutral pH transition. Drug Dev. Ind. Pharm. 2008, 34 (8), 890−902. (6) Corrigan, O. I.; Sabra, K.; Holohan, E. M. Physicochemical properties of spray dried drugs: phenobarbitone and hydroflumethiazide. Drug Dev. Ind. Pharm. 1983, 9 (1−2), 1−20. (7) Matsunaga, Y.; Bando, N.; Yuasa, H.; Kanaya, Y. Effects of grinding and tableting on physicochemical stability of an anticancer drug, TAT-59. Chem. Pharm. Bull. 1996, 44 (10), 1931−1934. (8) Ohta, M.; Buckton, G. A study of the differences between two amorphous spray-dried samples of cefditoren pivoxil which exhibited different physical stabilities. Int. J. Pharm. 2005, 289 (1−2), 31−38. (9) Pyne, A.; Chatterjee, K.; Suryanarayanan, R. Crystalline to amorphous transition of disodium hydrogen phosphate during primary drying. Pharm. Res. 2003, 20 (5), 802−803. (10) Willart, J. F.; Caron, V.; Lefort, R.; Danede, F.; Prevost, D.; Descamps, M. Athermal character of the solid state amorphization of lactose induced by ball milling. Solid State Commun. 2004, 132 (10), 693−696. (11) Huettenrauch, R. Molecular galenics as the basis of modern drug formation. Acta Pharm. Technol., Suppl. 1978, 6, 55−127. (12) Suryanarayanan, R.; Mitchell, A. G. Evaluation of two concepts of crystallinity using calcium gluceptate as a model compound. Int. J. Pharm. 1985, 24 (1), 1−17. (13) Rani, M.; Govindarajan, R.; Surana, R.; Suryanarayanan, R. Structure in dehydrated trehalose dihydrate-evaluation of the concept of partial crystallinity. Pharm. Res. 2006, 23 (10), 2356−2367. (14) Bates, S.; Kelly, R. C.; Ivanisevic, I.; Schields, P.; Zografi, G.; Newman, A. W. Assessment of defects and amorphous structure produced in raffinose pentahydrate upon dehydration. J. Pharm. Sci. 2007, 96 (5), 1418−1433. (15) Chamarthy, S. P.; Pinal, R. The nature of crystal disorder in milled pharmaceutical materials. Colloids Surf., A 2008, 331 (1−2), 68−75. (16) Feng, T.; Pinal, R.; Carvajal, M. T. Process induced disorder in crystalline materials: differentiating defective crystals from the amorphous form of griseofulvin. J. Pharm. Sci. 2008, 97 (8), 3207− 3221. (17) Otte, A.; Zhang, Y.; Carvajal, M. T.; Pinal, R. Milling induces disorder in crystalline griseofulvin and order in its amorphous counterpart. CrystEngComm 2012, 14 (7), 2560−2570. (18) Atassi, F.; Byrn, S. R. General trends in the desolvation behavior of calcium salts. Pharm. Res. 2006, 23 (10), 2405−2412. (19) Terakita, A.; Byrn, S. R. Structure and physical stability of hydrates and thermotropic mesophase of calcium benzoate. J. Pharm. Sci. 2006, 95 (5), 1162−1172. (20) Luisi, B. S.; Medek, A.; Liu, Z.; Mudunuri, P.; Moulton, B. Milling-induced disorder of pharmaceuticals: one-phase or two-phase system? J. Pharm. Sci. 2012, 101 (4), 1475−1485.

AUTHOR INFORMATION

Corresponding Author

*Genentech, Inc., 1 DNA way, Mail stop 432A, South San Francisco, CA 94080. Phone: 650-467-6339. Fax: 650-2256238. E-mail: [email protected]. Author Contributions

P.C. and S.B. are cofirst authors of this article. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Francis Gosselin, Chong Han, Travis Remarchuk, Scott Savage, Frederic St-Jean, and Herbert Yajima, (Small Molecule Process 2821



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(21) Feng, T.; Bates, S.; Carvajal, M. T. Toward understanding the evolution of griseofulvin crystal structure to a mesophase after cryogenic milling. Int. J. Pharm. 2009, 367 (1−2), 16−19. (22) Cui, Y. A material science perspective of pharmaceutical solids. Int. J. Pharm. 2007, 339 (1−2), 3−18. (23) Wunderlich, B. A classification of molecules, phases, and transitions as recognized by thermal analysis. Thermochim. Acta 1999, 340−341, 37−52. (24) Wunderlich, B.; Grebowicz, J. Characterization of conformationally disordered crystals by DSC. Polym. Mater.: Sci. Eng. 1984, 50, 114−119. (25) Wunderlich, B.; Grebowicz, J. Do condis crystals exist? Polym. Sci. Technol. 1985, 28, 145−151. (26) Wunderlich, B. The detection of conformational disorder by thermal analysis. Pure Appl. Chem. 1989, 61 (8), 1347−1351. (27) Wunderlich, B. Glass transition of partially ordered macromolecules. Prog. Colloid Polym. Sci. 1994, 96, 22−28. (28) Wunderlich, B. Analysis of materials by temperature-modulated calorimetry using heat capacities, glass transitions and first-order transitions. Proc. NATAS Ann. Conf. Therm. Anal. Appl. 1999, 3−8. (29) Bunjes, H.; Rades, T. Thermotropic liquid crystalline drugs. J. Pharm. Pharmacol. 2005, 57 (7), 807−816. (30) Atassi, F.; Mao, C.; Masadeh, A. S.; Byrn, S. R. Solid-state characterization of amorphous and mesomorphous calcium ketoprofen. J. Pharm. Sci. 2010, 99 (9), 3684−3697. (31) Liaw, A.; Wiener, M. Classification and regression by randomForest. R News 2002, 2 (3), 18−22. (32) R Development Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2011. http://www.R-project.org/. (33) Breiman, L. Random Forests. Machine Learn. 2001, 45 (1), 5− 32. (34) van Stokkum, I. H. M.; Mullen, K. M.; Mihaleva, V. V. Global analysis of multiple gas chromatography-mass spectrometry (GC/MS) data sets: A method for resolution of co-eluting components with comparison to MCR-ALS. Chemom. Intell. Lab. Syst. 2009, 95 (2), 150−163. (35) Ivanisevic, I.; Bates, S.; Chen, P. Novel methods for the assessment of miscibility of amorphous drug-polymer dispersions. J. Pharm. Sci. 2009, 98 (9), 3373−3386. (36) Rumondor, A. C. F.; Ivanisevic, I.; Bates, S.; Alonzo, D. E.; Taylor, L. S. Evaluation of drug-polymer miscibility in amorphous solid dispersion systems. Pharm. Res. 2009, 26 (11), 2523−2534. (37) Teng, J.; Bates, S.; Engers, D. A.; Leach, K.; Schields, P.; Yang, Y. Effect of water vapor sorption on local structure of poly(vinylpyrrolidone). J. Pharm. Sci. 2010, 99 (9), 3815−3825. (38) Cowley, J. M. Diffraction Physics, 2nd ed.; Elsevier: Amsterdam, Holland, 1990; pp 139−157. (39) James, R. W. The Crystalline State. Vol. II. The Optical Principles of the Diffraction of X-rays; Ox Bow Press: Woodbridge, CT, 1962; pp 458−511. (40) Lovesey, S. W. Theory of Neutron Scattering from Condensed Matter; Clarendon Press: United Kingdom, 1984; Vol 2, pp 360 (with reference to quantum chemistry and scattering from electron plasmas and magnetic ions). (41) Chakravarty, P.; Berendt, R. T.; Munson, E. J.; Young, V. G., Jr.; Govindarajan, R.; Suryanarayanan, R. Insights into the dehydration behavior of thiamine hydrochloride (vitamin B1) hydrates: Part I. J. Pharm. Sci. 2010, 99 (2), 816−827. (42) Chakravarty, P.; Bhardwaj, S. P.; King, L.; Suryanarayanan, R. Monitoring phase transformations in intact tablets of trehalose by FTRaman spectroscopy. AAPS PharmSciTech 2009, 10 (4), 1420−1426. (43) Stephenson, G. A.; Groleau, E. G.; Kleemann, R. L.; Xu, W.; Rigsbee, D. R. Formation of isomorphic desolvates: creating a molecular vacuum. J. Pharm. Sci. 1998, 87 (5), 536−542. (44) Taylor, L. S.; Williams, A. C.; York, P. Particle size dependent molecular rearrangements during the dehydration of trehalose dihydrate. In situ FT-Raman spectroscopy. Pharm. Res. 1998, 15 (8), 1207−1214.

(45) Lee, J. W.; Thomas, L. C.; Schmidt, S. J. Investigation of the heating rate dependency associated with the loss of crystalline structure in sucrose, glucose, and fructose using a thermal analysis approach (Part I). J. Agric. Food Chem. 2011, 59 (2), 684−701. (46) Hancock, B. C.; Shamblin, S. L. Molecular mobility of amorphous pharmaceuticals determined using differential scanning calorimetry. Thermochim. Acta 2001, 380 (2), 95−107. (47) Hancock, B. C.; Shamblin, S. L.; Zografi, G. Molecular mobility of amorphous pharmaceutical solids below their glass transition temperatures. Pharm. Res. 1995, 12 (6), 799−806. (48) Kakumanu, V. K.; Bansal, A. K. Enthalpy relaxation studies of celecoxib amorphous mixtures. Pharm. Res. 2002, 19 (12), 1873−1878. (49) Kawakami, K.; Ida, Y. Direct observation of the enthalpy relaxation and the recovery processes of maltose-based amorphous formulation by isothermal microcalorimetry. Pharm. Res. 2003, 20 (9), 1430−1436.

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Identification of a Potential Conformationally Disordered Mesophase

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