Using milling to explore physical states: The amorphous and

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Using milling to explore physical states: The amorphous and polymorphic forms of dexamethasone Paulo Filho Marques, Jean-Francois Willart, Juergen Siepmann, Florence Siepmann, and Marc Descamps Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01664 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Crystal Growth & Design

Using milling to explore physical states: The amorphous and polymorphic forms of dexamethasone Paulo F. M. Oliveira†, Jean-François Willart†, Juergen Siepmann§ Florence Siepmann§ and Marc Descamps†

†

Univ. Lille, UMET, UMR CNRS 8207, Villeneuve d'Ascq, France

§

Univ. Lille, Inserm, CHU Lille, U1008, F-59000 Lille, France

*corresponding author: [email protected]

Abstract. This study aims to investigate the polymorphism, physical stability and the amorphization possibilities of dexamethasone (DEX) drug. Milling was found to be an advantageous mean to prepare amorphous DEX non-chemically 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 XRD and DSC.

Keywords: polymorphism, amorphous drug, dexamethasone, milling, physical stability.

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1. INTRODUCTION 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 co-crystals.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 non-activation 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. 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 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, presented in this article illustrates the variety of


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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 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.

Figure 1. Dexamethasone chemical structure



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 (ø = 15 mm) of the same material were used. 1.1 g of material were 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 ms-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 sec 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


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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 (ø = 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 13

C CP-MAS NMR experiments were performed in Bruker AVANCE-400 MHz NMR (9.4

T) spectrometer equipped with a 4 mm resonance probe operating

13

C Larmor

frequencies of 101 MHz. The sample was packed in a ø = 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.



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 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 complete by 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) 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 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 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


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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.

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.



Melt quenched amorphous DEX Figures 2a and 2b (Run 2) show the thermograms corresponding to the heating of the melt-quenched 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.

As received

Intensity / a.u.

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Quenched melt

12 h - milled

8

7

6

5

4

3

2

1

0

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


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Figure 3 shows 1H NMR spectra of crystalline DEX and melt quenched amorphous DEX. It is clearly seen that the spectrum of 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 4.67ppm, 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 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°C 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 °C 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 hours 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 low-temperature 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 high-temperature component.

(a)

(b) XRD

-1 5 °C min XRD

∆Hc = 40.0 J g

-1

150°C

Intensity / a. u.

120°C

Heat flow / a. u.

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(c) XRD

5 °C min

-1

Milled 12 h, RT (Int x 2.5 )

(d)

XRD

∆Hc = 40.0 J g

-1

150°C 120°C

Exo

Milled 12 h, RT

(Int x 2.5 )

60

80

100 120 140 160 180 200 220

8

10

Temperature / °C

12

14

16

18

20

22

24

2θ / degrees

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


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What must be noted is that the two diffractograms recorded after the two step recrystallization are totally identical. They correspond to that of the 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.



(a) Fast DSC

200 °C min-1 100 °C min-1

Cp jump

(b) Partial recrystallization Run 1 Heat flow

Heat flow / a.u.

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Run 2 Rev Heat flow Run 2 Heat flow 5 °C min-1

(c) Compaction Tg = 116 °C

Rev Heat flow Compacted

∆Cp = 0.30 J g-1 °C-1

Heat flow Compacted

∆Hc = 44 J g-1

Exo

5 °C min-1

60

80

100

120

140

160

180

Temperature / °C

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.


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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 Figures 4a and 4c 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.

- The first strategy consists in 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 4 a). This clearly induces a shift of the recrystallization exotherm towards 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 cooling 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 in 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 ∆Hc = 44.0 J/g. This value is slightly higher than that obtained by integration of the bimodal recrystallization curve (∆Hc = 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, ∆Cp = 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

Using milling to explore physical states: The amorphous and

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