Using Milling To Explore Physical States: The Amorphous and

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Cite This: Cryst. Growth Des. XXXX, XXX, XXX−XXX

Using Milling To Explore Physical States: The Amorphous and Polymorphic Forms of Dexamethasone Published as part of a Crystal Growth and Design virtual special issue Honoring Prof. William Jones and His Contributions to Organic Solid-State Chemistry Paulo F. M. Oliveira,† Jean-François Willart,*,† Juergen Siepmann,§ Florence Siepmann,§ and Marc Descamps† †

Université Lille, UMET, UMR CNRS 8207, Villeneuve d’Ascq, France Université Lille, Inserm, Chu Lille, U1008, F-59000 Lille, France

§

ABSTRACT: This study aims to investigate the polymorphism, physical stability, and amorphization possibilities of dexamethasone (DEX) drug. Milling was found to be an advantageous mean to prepare amorphous DEX nonchemically degraded. It appears to be a very useful process that allowed generating crystalline polymorphic transformation, either from the milling induced amorphous sample or from a mechanically damaged polymorphic form. The paper illustrates the interest of milling as a complementary tool to screen crystal polymorphism and to determine the relative stability of polymorphs when the decision is difficult. Physical characterizations were mainly carried out using X-ray diffraction and differential scanning calorimetry.

1. INTRODUCTION

study of the coupling between molecular mobility and mutarotation. The study of the milling of crystalline dexamethasone (DEX) (Figure 1), which is an anti-inflammatory corticosteroid drug,

Milling is a widely used process during drug formulation. It may have many practical objectives: to improve the dissolution properties,1,2 to facilitate tablets compaction,3,4 to control the dosage accuracy, to facilitate the powder flowability, etc. This technique has recently become a more sophisticated chemical synthesis tool,5−8 especially for the synthesis of cocrystals.9,10 However, the mechanisms involved in these chemical modifications induced by milling remain unclear in detail. Jones and others11,12 have shown that milling-induced changes in physical state, either amorphization or conversions between polymorphic crystalline forms, can be a fundamental mediator of chemical reactions. Indeed, independently of the microstructural modifications, milling can induce structural modifications of the solids and, consequently, changes in the molecular dynamics.13−17 Molecular mobility can play a key role in the activation or nonactivation of chemical processes.18−22 This has been demonstrated recently in an investigation of the mutarotation occurring in the amorphous state of several isomerizable compounds such as glucose and lactose.23,24 Crossing the glass transition (at Tg) is associated with the freezing (upon cooling) or unfreezing (upon reheating) of the molecular relaxations controlling the viscosity of the amorphous compounds. The passage of Tg is also accompanied by a drastic modification of the effective activation energy of the mutarotation process. This shows a considerable change in the nature of the mechanisms controlling mutarotation: the physical mechanisms (molecular mobility) drive the process for T < Tg, whereas they are the chemical mechanisms for T > Tg. It may also be noted that milling can make it possible to modify the physical state without inducing any chemical modification. This has been used to carry out the aforementioned © XXXX American Chemical Society

Figure 1. Dexamethasone chemical structure.

presented in this article illustrates the variety of possibilities of manipulation of the physical state by milling. The two main aspects that will be developed concern: • The possibility of amorphizing DEX without chemical degradation. This enables determining the actual physical properties of the amorphous state. Specific strategies are used to flush out a glass transition hidden by recrystallizations. • The possibility of generating different polymorphic varieties by milling. This reveals that milling can be an interesting tool in the screening of polymorphs. An original aspect revealed by this DEX investigation is to show that a different polymorphic variety can be generated either from an amorphized sample or from a sample of the former polymorphic variety whose microstructure has been modified by milling. By forcing polymorphic Received: November 28, 2017 Revised: January 29, 2018 Published: February 1, 2018 A

DOI: 10.1021/acs.cgd.7b01664 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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points, and equivalent dissolution properties.25 However, there is no specific comparative study available. Therefore, in preliminary to milling studies, we give below some comparison of physical characteristics of the crystalline forms of the DEX. We completed a rapid characterization of the amorphous DEX conventionally obtained by quenching the liquid. This last point will be useful to compare with the amorphous state obtained by milling which will be identified hereafter. Polymorphic Crystalline Forms. Figure 2 presents the TGA and DSC heating scans recorded at 5 °C/min of the forms A (2a)

conversions, milling offers the unexpected possibility of determining the relative stability of different polymorphic forms, when the decision is difficult. Moreover, this study is, to our knowledge, the first detailed investigation of the polymorphism of solid dexamethasone.

2. EXPERIMENTAL SECTION Crystalline dexamethasone Form A (Discovery Fine Chemicals, Dorset, UK) was used as received. Form B was prepared from recrystallization of amorphous dexamethasone upon heating. Ball milling was performed with a high energy planetary mill (Pulverisette 7, Fritsch, Idar-Oberstein, Germany) at room temperature (RT). ZrO2 milling jars of 43 cm3 with seven balls (φ = 15 mm) of the same material were used. A total of 1.1 g of material was placed in the planetary mill, corresponding to a ball/sample weight ratio of 75:1. The rotation speed of the solar disk was set to 400 rpm, corresponding to an average acceleration of the milling balls of 5 g (g = 9.81 m s−2 is the acceleration of gravity). In order to limit the heating of the samples during milling, milling periods (15 min) were alternated with pause periods (5 min). Calorimetry experiments were performed with a Q200 DSC (differential scanning calorimetry) from TA Instruments (Guyancourt, France), equipped with a refrigerated cooling system. During all measurements the calorimeter head was flushed with highly pure nitrogen gas. Temperature and enthalpy readings were calibrated using pure indium at the same scan rates as used during the measurements of samples. In the modulated temperature mode, the amplitude and the period of modulation were respectively set to 0.663 °C and 50 s corresponding to “heat only” conditions. The samples were placed in open pans (pans with no lid) and annealed at 60 °C during 15 min to remove the small amount of water possibly adsorbed during the milling stage. Thermogravimetric analysis (TGA) were performed with a Q500 TGA from TA Instruments (Guyancourt, France). Samples were placed in open aluminum pans, and the furnace was flushed with a highly pure nitrogen gas (50 mL/min). The temperature reading was calibrated using the Curie points of alumel and nickel, while the mass reading was calibrated using balance tare weights provided by TA Instruments. All TGA scans were performed at 5 °C/min. Powder X-ray diffraction (PXRD) experiments were performed with a PanAlytical X’PERT PRO MPD (Almelo, The Netherlands) diffractometer (λCuKα = 1.5418 Å for combined Kα1 and Kα2), equipped with an X’celerator detector (Almelo, The Netherlands). Samples were placed into Lindemann glass capillaries (φ = 0.7 mm) and installed on a rotating sample holder to avoid any artifacts due to preferential orientations of crystallites. The diffractometer is equipped with a HUBER capillary furnace combined with the high temperature controller HTC 9634 for temperature experiments. Solution 1H NMR experiments were performed using a Bruker AVANCE-300 MHz NMR spectrometer with DMSO-d6 as solvent. Chemical shifts δ were expressed in parts per million (ppm) by frequency imposed by the spectrometer, relative to TMS. Solid-state 13C CP-MAS NMR experiments were performed in Bruker AVANCE-400 MHz NMR (9.4 T) spectrometer equipped with a 4 mm resonance probe operating 13C Larmor frequencies of 101 MHz. The sample was packed in a φ = 4 mm rotor and spun at νrot = 12.5 kHz. Chemical shifts δ were expressed in parts per million (ppm) by frequency imposed by the spectrometer.

Figure 2. DSC scan (5 °C/min), TGA scan (5 °C/min), and PXRD pattern of the initial materials: (a) DEX Form A, (b) DEX Form B.

and B (2b) of DEX. The insets show the room temperature X-ray diffractograms of the two forms. The forms were identified by references to the results of the published structure for Form B.26−28 For Form A, only the list of Bragg angles of the peaks is reported in a review article on the analytical profile of dexamethasone.25 It corresponds to the peaks shown in (2a). The DSC heating curves show that the endothermic melting peaks of the two forms occur in the same temperature range. The melting endotherm of Form B is sharp with an onset at TmB ≈ 250 °C. The one corresponding to the Form A is wider. The onset is located at slightly lower temperature (TmA = 242 °C). In a reproductive way, the endothermic massif shows a shoulder and ends with a peak located almost exactly at the position of the melting peak of Form B. This complex melting endotherm may be compatible with an entangled mechanism of melting (of Form

3. RESULTS AND DISCUSSION 3.1. The Initial Materials. Despite the current use of DEX, its solid-state physical properties are poorly documented. It has been reported that two crystalline forms (A and B) exist,25 but only the structure of phase B has been determined.26−28 DEX suppliers produce either form without providing any indication of the respective polymorph. These two forms are presumed to be of similar physical stability, with supposedly very close melting B



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degradation, mainly in the region in between δH = 5.5−3.5 ppm, which also comprises the CH2 (C21 protons). This indicates, as expected, that the side groups are the more affected ones. As a result, the strong chemical degradation upon melting revealed by both 1H NMR and TGA (Figure 2) makes it unreasonable to prepare amorphous DEX by the thermal path. 3.2. Milling Induced Amorphization. Strongly Milled Crystalline Samples. Long mechanical millings (12 h) were applied to the DEX forms A and B. For both experiments, DSC heating scans (5 °C/min) after milling, as well as PXRD patterns recorded after milling (at room temperature) and after partial heating (at 120 and 150 °C) of the milled samples, are shown in Figure 4a−d. After milling, the X-ray diffraction Bragg peaks have disappeared in both cases. Broad diffuse halos are observed, which are characteristic of amorphous materials. This suggests that each crystalline form of DEX has been highly amorphized during the milling process. When heated, the DSC curves exhibit pronounced exothermic components that develop between 95 and 140 °C. The PXRD patterns recorded during heating in this temperature range reveal the appearance of Bragg peaks, and thus the recrystallization of the amorphized samples. This confirms the amorphous character of the milled samples. Figure 3 (bottom) shows 1H NMR spectrum of the 12 h milled crystalline DEX. The spectrum of the milled sample is totally similar to that of the initial one. This shows that, contrary to melt quenching, a high-energy mechanical action is capable of amorphizing the sample without inducing chemical degradation. The bimodal aspect of the exothermic massifs seen on the DSC curve must be emphasized. This has already been observed for several molecular compounds amorphized by milling which have a high tendency to recrystallize upon heating.29−31 The lowtemperature component is then interpreted as due to surface recrystallization.32−34 The latter is faster than recrystallizations occurring in the core of the sample, which give rise to the hightemperature component. What must be noted is that the two diffractograms recorded after the two step recrystallization are totally identical. They correspond to that of Form B of DEX. This is not surprising for the amorphous material obtained by milling Form B, since seeding with crystalline residues of this form is conceivable. This is the situation most commonly observed. It is more surprising to observe that the amorphous DEX obtained by milling Form A recrystallizes toward Form B upon heating. In this case, the recrystallization cannot be simply triggered by crystalline nuclei of Form A which would have possibly escaped the milling. This polymorphic conversion will be investigated in more detail below. Tg Determination of the Amorphous Dexamethasone. PXRD has demonstrated that milling was able to amorphize the crystalline DEX, as confirmed, in the DSC thermogram, by the recrystallization observed upon heating. However, the DSC heating scans in Figure 4a,c do not show the characteristic Cp jump occurring at the glass transition temperature Tg of an amorphous material (the latter is usually expected to occur before recrystallization). In the present case, it may be hidden by recrystallization, as it sometimes happens.30,35 To determine the glass transition of amorphized DEX, we have used three different strategies, which aim to shift, or to remove, the recrystallization exotherm, in order to flush out the expected glass transition temperature range.

A) - recrystallization (of Form B) - melting (of Form B). This suggests that, at least near melting, Form A is metastable with respect to Form B. The two melting endotherms occur in the temperature range where there is a pronounced weight loss shown by the TGA scans. This is associated with a very severe degradation that makes it difficult to determine the melting enthalpies accurately. It is, a fortiori, impossible to compare the melting enthalpies of Forms A and B, and thus to conclude, with regard to the relation existing between these two forms, either being in a situation of monotropism or enantiotropism. Melt Quenched Amorphous DEX. Figure 2a,b (Run 2) shows the thermograms corresponding to the heating of the meltquenched DEX. In neither case is a recrystallization observed. However, both scans show a jump of specific heat (Cp) characterizing a glass transition situated at Tg ≈ 130 °C. DEX was therefore amorphized by quenching. Nevertheless, the degradation revealed by the TGA indicates that the amorphous state obtained is not that of the pure DEX. Moreover, the simple observation of the first upscan shows a level of heat flow after melting much lower than that preceding it. This is contrary to normal behavior, without degradation. To test further the degradation level, solution NMR analyses were performed with the as-received DEX and the quenched liquid. Figure 3 shows 1H NMR spectra of crystalline DEX and melt quenched amorphous DEX. It is clearly seen that the spectrum of

Figure 3. Solution 1H NMR (300 MHz) spectra of dexamethasone. From top to bottom: nonmilled sample, quenched melt and 12 h milled sample.

the thermally amorphized sample does not correspond to the initial crystalline one anymore. There are disappearances and shifts of several signals in the spectrum of the quenched melt, e.g., for chemical shifts δH = 5.28, 4.95, and 4.67 ppm, which are assigned to the O−H protons of the DEX molecule (from C11, C17, and C21, respectively). Additionally, many other chemical shifts disappeared or were altered as consequence of the C



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Figure 4. DSC thermograms and PXRD patterns recorded during the heating of 12 h milled samples: (a) and (b) DEX Form A; (c) and (d) DEX Form B.

• The first strategy consists of using high DSC scanning rates in the hope of separating the glass transition from the recrystallization. Both events are kinetic in nature but they may have different characteristic time scales. Recrystallization can be delayed, and even suppressed, upon fast scanning.30,31,35 Figure 5a shows fast DSC scans of the amorphized material. Heating is carried out at 100 and 200 °C/min, to be compared with the usual, much slower, DSC scan rate (5 °C/min, Figure 4a). This clearly induces a shift of the recrystallization exotherm toward the high temperatures. The shift enables revealing a Cp jump before recrystallization, and therefore, the Tg zone of amorphous DEX, which is around 120 °C for the high scan rates used. • The second strategy consists in taking into consideration the bimodal aspect of the recrystallization and in forcing a partial recrystallization of the sample to eliminate the low temperature crystallization exotherm. The partial recrystallization of the amorphized sample was here induced by first heating (at 5 °C/min) the sample to 120 °C which is a temperature in the range separating the two recrystallization exotherms (Figure 5b, Run 1). After being cooled down to RT, the sample was heated again throughout the recrystallization zone, in modulated DSC mode at 5 °C/ min (Figure 5b Run 2). A careful inspection of the total heat flow (Run 2) reveals the onset of a Cp jump, shouldering the beginning of the recrystallization exotherm of the remaining amorphous fraction. This Cp jump is more evident in the reversing heat flow signal and confirms the positioning of the glass transition (Tg ≈ 118 °C). In Run 1 the latter was masked by the first partial recrystallization exotherm. • The third strategy consists of compacting the amorphized sample expecting to reduce the interface effects which are at the origin of an acceleration of the recrystallization phenomenon.29 Figure 5c shows the DSC heating scan of

a compacted amorphous sample obtained by pressing the milled material at 1250 MPa. In these conditions, the observed bimodal recrystallization for the powder sample flips to a unimodal mode. The enthalpy of crystallization of the compacted amorphous sample gives ΔHc = 44.0 J/g. This value is slightly higher than that obtained by integration of the bimodal recrystallization curve (ΔHc = 40.0 J/g), but that is still very consistent because the unimodal recrystallization exotherm occurs at higher temperature. The unimodal recrystallization results from the reduction of the porosity and of the free surface.29 Hence, the surface recrystallization is no longer observed, and only an exotherm of recrystallization, coincident to the bulk recrystallization of the as-milled sample, is seen. As a result, the Cp jump can be clearly observed in both total and reversing heat flow. This confirms the existence and position of the glass transition revealed in the other experiments. The values of Tg = 116 °C, ΔCp = 0.30 J/g/ °C for amorphous DEX were determined. The results presented in Figure 5 clearly show that, if the DSC scanning rate is too small, the glass transition is masked by an efficient recrystallization process strikingly starting below Tg. Such an apparent lack of glass transition has already been observed for some amorphous molecular materials produced by milling, which recrystallize near or below Tg when scanned with conventional DSC scan rates of 5−10 °C/min.31,35 The previous strategies lead to values of Tg which are close (115 °C < Tg < 120 °C). The small differences observed are correlated with the differences in scanning rates imposed either in conventional DSC mode or in modulated temperature mode (MDSC). Interestingly enough, these Tg values differ by more than 10 °C from that recorded on the compound amorphized by cooling the liquid (cf. Figure 2). As shown by the NMR analysis of Figure 3, this is because the glass transition observed on the milled sample is that of chemically pure DEX, while that D



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Figure 6. (a) DSC heating scans (5 °C/min) of dexamethasone milled from zero (as received) to 36 h. The milling times are reported on the left-hand side of the figure, over the respective thermogram. (b) Amorphization kinetics of dexamethasone upon milling. The experimental data were fitted with an exponential law (dashed line). Figure 5. DSC heating scans of milling induced amorphous dexamethasone: (a) Fast DSC scans performed at 100 °C/min and 200 °C/min. (b) Run 1: heating (5 °C/min) to 120 °C. Run 2: rescan (5 °C/min) after run 1. (c) Temperature-modulated DSC signals (reversible and total heat flows) recorded upon heating (5 °C/min) after compaction of the milled sample at 1250 MPa.

presents the DSC heating scans (5 °C/min) of DEX (Form A) after different milling times, ranging from 0 min to 36 h. They show the progressive development and structuring of the broad bimodal recrystallization exotherm ranging between 80 and 140 °C. The smaller exotherm which peaks at about 150−160 °C does not correspond to a recrystallization of the amorphous state. Its origin will be discussed later. A recrystallization exotherm is already detectable near 125 °C after 5 min of milling. The low temperature component develops within 15 min. It gradually becomes larger and sharper. It also shifts slightly toward higher temperatures when the milling time increases. This reflects an increasing apparent stability of the amorphous fraction during milling. The thermogram becomes stationary after about 420 min of milling. The low temperature exotherm is the most

observed on the quenched melt arises from a chemically degraded DEX. This is a further illustration of the interest of mechanical activation to obtain chemically pure amorphous samples.23,24 Kinetics of Amorphization upon Milling. The amorphization kinetics of crystalline Form A of DEX upon milling has been investigated by DSC. Millings of different durations were carried out, each time with a fresh sample, and the DSC runs were recorded immediately after each milling procedure. Figure 6a E



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Figure 7. (a) DSC scans recorded upon heating (5 °C/min). Run 1: nonmilled Form A. Run 2: physical mixture (PM) Am DEX + nonmilled Form A. Run 3: 30 min-milled Form A. Run 4: 30 min-milled Form B. (b) Powder XRD of the form A partially amorphized after 30 min of milling and its evolution toward Form B upon heating. The arrows and colors in DSC thermogram (run 3) indicate the temperature at which the XRD patterns were recorded.

temperatures for increasing milling time so that it becomes hardly detectable, around 145 °C, after 12 h of milling. The structural evolution of the 30 min-milled Form A was followed by XRD upon heating. The diffractograms recorded at key temperatures separating the different thermal events (indicated in run 3 of Figure 7a) are shown in Figure 7b. At temperatures below the recrystallization exotherms (60 °C) there is a mixture of amorphous DEX and remaining crystalline Form A. The Bragg peaks of Form A which emerge from the amorphous X-ray halo are however very broad which puts forward the defective character and small size of milled crystallites of Form A (∼18 nm) not yet amorphized. Upon heating, the amorphous fraction recrystallizes toward Form B, and thus, at 130 °C there is a mixture of polymorphs A (stars) and B (squares). At 170 °C, after the complete development of the third exothermic event under consideration, the PXRD pattern only shows the Bragg peaks of Form B with increased intensities and the complete disappearance of peaks characteristic of Form A. This result indicates that the exothermic event at higher temperature is related to a polymorphic transformation of Form A toward Form B. This is consistent with the fact that the exotherm is more pronounced when the milling time is shorter (Figure 6a) because the residual fraction of Form A is larger. It also explains why the exotherm is seen upon heating the milled Form A and never seen upon heating the milled Form B. Interestingly, Figure 7a (Run 1) shows that the transformation from A to B does not appear in the DSC scan of the initial nonmilled material. The same applies for any physical mixture of A (microstructure unmodified by a milling) with either B or amophous DEX (the latter recrystallizes toward B upon heating) which do not lead to a transformation of the part A into B as seen upon heating the milled Form A in DSC. This indicates that Form B has no seeding effect during the heating of Form A if the latter is not made defective by the milling. Since, when Form B is milled, there is no polymorphic transformation upon heating (Run 4 in Figure 7a), this indicates that the polymorphic transformation occurs from the remaining defective Form A that has escaped amorphization. The sliding toward the low temperatures of the exothermic peak when the milling time

developed which indicates that the recrystallization takes place predominantly from the surface. Figure 6b shows the evolution of the amorphized fraction (Xam) in the course of the 12 h milling process (720 min). Xam has been determined from the ratio between the enthalpy of crystallization of any amorphous fraction (ΔHc(t)) obtained by integration of the respective recrystallization exotherms of DSC curves in Figure 6a, and that of the total enthalpy of crystallization of the highly amorphized sample (ΔHc(t = 36 h) = 40.0 J/g). The evolution shows a monotonically increase of the amorphous fraction which can be fitted by an exponential law (indicated in the Figure 6b) with a relaxation time (τ) close to 0.57 h. Upon milling, the mechanical action is a discontinuous and localized process. It consists of multiple short pulses which impact, randomly at different times, only a small fraction of the powder charge. It has been suggested that the shape of the amorphization curve of a material upon ball milling depends on the number of impacts required to amorphize a given elementary impacted fraction.36 With this hypothesis, which neglects the relaxation between the pulses, an exponential law is expected, if a single impact is sufficient to amorphize an elementary fraction of the material subjected to each shock. In spite of its simplifications, this approach suggests that DEX is readily amorphized under milling. 3.3. Polymorphic Transformation Triggered by Milling. The heating of the samples, partially amorphized by milling the DEX forms A and B, exhibit subtle differences that reveal unexpected effects of milling. These differences appear most clearly for samples which have undergone milling for short periods of time, from 15 to 60 min. On the DSC diagram it is associated with a small exotherm observed at temperatures higher than those of recrystallization, which exists for milled Form A, but not for the milled Form B. Figures 6a and 7a (Run 3) show that this exotherm peaks at about 160 °C for the Form A milled for 30 min, while it is totally absent for the Form B milled for the same milling period (Figure 7a, Run 4). Moreover the exotherm becomes smaller and slides toward the lower F



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previously mentioned, this seeding effect by the Form B, if it occurs, is efficient only if the Form A has been made defective. 3.4. Crystallite Coarsening. Whatever the starting form (A or B), DSC scans of 12 h-milled DEX samples (Figure 4a,c) show a last exothermic event occurring at higher temperature (190− 220 °C), just before melting. The nature of the associated modifications was first investigated by PXRD. Figure 9 shows recordings, carried out at RT, on milled samples, which have been brought to temperatures immediately

increases corroborates the fact that the more defective the Form A, the more rapid the subsequent transformation to the Form B. A real-time X-ray diffraction experiment makes possible to follow in situ the conversion of the Form A (damaged by the milling) to Form B. For this purpose, Form A has been partially amorphized (milling for 15 min, 30% amorphized), and the XRD monitoring was carried out at 150 °C, the temperature at which the “high temperature” exothermic signal starts to take place in these milling conditions. Figure 8 shows the isothermal evolution

Figure 8. PXRD diagram of the crystalline Form A milled 15 min at RT and its evolution during an annealing at 150 °C for some days, revealing a polymorphic transformation from Form A toward B. Figure 9. PXRD patterns of recrystallized dexamethasone recorded after heating at 180 °C (red) and 200 °C (black). The acquisitions were performed at RT.

as well as the diagram recorded at RT just after milling. It is observed that the freshly milled sample exhibits the considerably broadened Bragg peaks, characteristic of the Form A, which emerge from an amorphous halo. Heating up to 150 °C, some peaks of Form B can be detected (red squares), coming from the recrystallization of the amorphous fraction. These peaks continue to grow during the following days at 150 °C and become at the same time sharper. Simultaneously, the peaks of the Form A decrease. So that after 7 days the sample is predominantly of Form B. This result clearly demonstrates that the defective Form A converts to Form B. At this temperature Form B is therefore the most stable form. In addition, a sample of nonmilled crystalline Form A was also stored at 150 °C during 20 days, and no alteration on the crystalline structure was observed according to PXRD records (data not shown). These results confirm that, indeed, some extent of defect in the system is necessary to trigger the polymorphic transformation and, thus, only the defective Form A is able to undergo a solid-state polymorphic transformation toward crystalline Form B. Milling generally induces a severe reduction in the size of the crystallites to the 10 nm range and generates a complex microstructure with defects of any kind resulting from shearing and fractures.37 As a result, the most probable mechanism of the conversion from A to B is that of a heterogeneous nucleation coupled with a crystal growth that can be assisted by cracks.38−40 This transformation is facilitated by the increase in the number of interfaces and the expected molecular mobility at the surface of the crystallites. It is probable that the increase of the fraction of Form B generated from the amorphized portion can accelerate the conversion process further by a kind of seeding. However, as

before (red) and after (black) the thermal event under consideration. The samples were prepared by in situ heating, then cooling, of the capillary inside the PXRD instrument. On the two diagrams, only the Bragg peaks indicative of the Form B are observed. It is therefore possible to exclude that the upper exothermic event is associated with a phase transition between different polymorphic varieties. However, the Bragg peaks of Form B become more intense and sharper after the high temperature excursion. This could be an indication that the upper exotherm could correspond to the end of a recrystallization of the part of sample amorphized by milling. However, there is no trace of detectable glass transition during a second DSC scan for a milled sample carried previously just above the main recrystallization exotherms. It can therefore be assumed that the evolutions revealed by the PXRD correspond only to microstructural rearrangements of Form B. The latter are however sufficiently large to induce energy changes which can be detected with DSC. We have thus used the calorimetric test proposed by Chen and Spaepen41 to distinguish between (nucleation and growth) recrystallization and grain growth coarsening.42 These two processes have fundamentally different kinetics, enabling in principle to distinguish them. Figure 10 shows the isothermal enthalpy release rate of a 12 h-milled sample at 187.5 °C. This temperature is located at the very beginning of the exotherm seen in the Figure 4a,c. It has been chosen for the isothermal heat flow response to be sufficiently strong and slow to be respectively detected and followed by DSC. The enthalpy release consists of a G



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fact that, during the reheating, the initial recrystallization took place at low temperature, near Tg. This is a temperature domain where a high nucleation rate is expected, whereas the growth rate is relatively low.38,43 It should be noted, however, that during the evolution of the X-ray diffractogram, all the peaks are not affected in the same way. This can be an indication that the starting nanocrystalline Form B shows noticeable preferred orientation effects of the crystallites. Solid-state NMR spectra corresponding to the experiments under consideration are shown in Figure 11. Spectra of Form B below 180 °C and above 220 °C show similar aspects and clearly differ from that of Form A and the amorphous form. However, below 180 °C, broader lines are present systematically. That is strongly indicative of the existence of some kind of disorder which may have a variety of origins. The disorder can be generic or linked to the small size of crystallites and corresponds to a large volume of interfacial contribution where structural and dynamic disorder can be expected. It should be noticed that a couple of peaks present in the spectrum of Form B below 180 °C disappear in the high-temperature Form B, (e.g., those at about δc = 14 and 155 ppm). That could indicate some change in the conformational properties of the molecules, accompanied by a global coarsening process of the sample. Clearly, additional experiments are needed to clarify this interesting coarsening situation, which could include PDF analysis. It must be noticed that the 13C CP-MAS spectra of Forms A and B give some information related to the crystal structure of the respective polymorphs. While Form B presents a doublet for each nucleus, indicating that there are two molecules in the asymmetric unit cell in agreement with the reported structure,26−28 Form A has only one signal for each nucleus, which suggests that only one molecule integrates the asymmetric unit. This information combined with further PXRD investigations could help to resolve the unknown structure of polymorph A.

Figure 10. Isothermal DSC curve at 187.5 °C of a 12 h-milled sample.

monotonically increasing curve. The integration of the curve area gives 2.8 J/g, which is consistent with the value of 3.5 J/g obtained from the heating rate at 5 °C/min. The signal is clearly distinguishable of the calorimetric signal expected for a nucleation and growth Avrami process, which consists of an exothermic peak having an extremum at nonzero time. In a graincoarsening process, the energy evolution is linked to the decrease of the interfacial area. In a simple grain-coarsening process where the average grain size increases at a rate dr/dt = Mγ/rm, where M is the molecular mobility, γ is the interfacial surface tension, and m is an exponent in the range 0.5 < m < 3, the corresponding isothermal enthalpy release rate becomes41 −dH /dt = H(0)r(0)Mg /r m + 2

which has a monotonically evolution in time similar to that observed here. Obviously such an evolution can be detected with DSC if the interfacial energy changes are large enough. This is only possible if the crystallite sizes are originally nanometric, which is associated with a large interfacial surface. Such a small crystallite size is detected here, even with a conventional X-ray source, through the observed sharpening of the Bragg peaks. The initial nanometric size of the crystallites can be understood by the

4. CONCLUSIONS The results presented in this article give an example of the varieties of effects induced by milling on the physical state of a solid pharmaceutical compound. This allowed not only generating a chemically pure amorphous phase of DEX, which

Figure 11. 13C CP-MAS NMR (101 MHz) (νrot = 12.5 kHz) spectra of DEX: (a) Form A, (b) amorphous DEX, (c) Form B at 180 °C, and (d) Form B at 220 °C. H



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Figure 12. Gibbs free enthalpy diagrams showing the various situations that can be envisaged a priori for the relative stabilities of Forms A and B of dexamethasone. (a) Monotropism, (b) and (c) enantiotropism.

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ACKNOWLEDGMENTS The authors acknowledge the French National Research Agency (ANR, Grant ANR-15-CE19-0014) for financial support. We thank Professor Marco Geppi for fruitful discussion concerning ss-NMR.

was used to determine the Tg, but also to explore the crystalline polymorphism of this compound. The existence of two polymorphic varieties of DEX was suspected in the literature but had never been clearly documented. A fortiori, the relative stability of these phases was not known, and neither it is the monotropic nor the enantiotropic nature of their relative situation. Milling allowed generation, upon heating, of a polymorphic conversion (from A to B) mediated by the formation of an amorphous form. It has also been shown that it is possible to generate a direct transformation between these two phases, if a defective and nanometric crystalline form of A is initially prepared by milling. The calorimetric signatures of the various observed transformations enable discussing the nature of the polymorphism: (i) The DSC signal associated with the direct conversion of A (defective) to B is exothermic; (ii) the DSC analysis of the heating of the polymorphic forms shows that the melting temperature of the Form A (TmA) is slightly lower than that of the Form B (TmB). The relative situations that can be envisaged monotropic or enantiotropicof the polymorphic forms are illustrated schematically in Figure 12. Assuming an enantiotropic situation, case (b) would possibly be compatible with the exothermic nature of the A to B transformation. However, it is not compatible with the relative position of the melting temperatures. Moreover, no endothermic transformation from B to A was observed at higher temperatures. Case (c) is compatible with the relative position of the melting temperatures but is not compatible with the exothermic nature of transition A to B. At last, the situation of monotropism (a) is compatible with the DSC data with regard to both the melting temperatures and the nature of the transition from A to B. Therefore, it can be concluded that this situation is the most probable. The results reveal that, in unclear situations of amorphization and crystalline polymorphism, milling provides an additional possibility of exploration of physical states that may be useful for screening of polymorphs and their relative stabilities.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jean-François Willart: 0000-0002-7911-3918 Notes

The authors declare no competing financial interest. I



Article

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Using Milling To Explore Physical States: The Amorphous and

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