Vemurafenib: A Tetramorphic System Displaying Concomitant

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Vemurafenib: A Tetramorphic System Displaying Concomitant Crystallization from the Supercooled Liquid Ming Lu, and Lynne S. Taylor Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b01066 • Publication Date (Web): 29 Aug 2016 Downloaded from http://pubs.acs.org on August 30, 2016

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Crystal Growth & Design

Vemurafenib: A Tetramorphic System Displaying Concomitant Crystallization from the Supercooled Liquid Ming Lu1,2 and Lynne S. Taylor2* 1.

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China

2.

Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States

ABSTRACT: The current interest in amorphous forms of active pharmaceutical ingredients stems from their increasing use in enabling formulations for the delivery of poorly water-soluble compounds. Because amorphous drugs are metastable and have a tendency to revert to crystalline forms, their crystallization behavior is an important topic. This study reports the observation of concomitant polymorphs of vemurafenib following crystallization from the supercooled liquid, and their subsequent phase transformations. The crystallization behavior of amorphous vemurafenib was evaluated between the glass transition temperature and melting temperature. Six characteristic crystal morphologies were observed. Based on X-ray diffraction and Raman spectroscopic studies, these morphologies were associated with four polymorphic forms, designated α, β, γ and δ. The thermodynamic stability is α > β > γ or δ. Isothermal crystallization between 120 and 240oC resulted in concomitant polymorphs with samples containing either 2 or 3 of the 4 polymorphs depending on the specific temperature, with the α-form or β-form typically dominating. Crystallization at even higher temperature yielded only the α-form. A monotropic relationship was inferred between the α- and β-forms based on calorimetric data. β-to-α, γ-to-β and δ-to-β phase transformations were observed using hot-stage polarized light microscopy, Raman microscopy and powder X-ray diffraction. The 1

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discovery of three new concomitant polymorphs of vemurafenib following crystallization from the supercooled liquid of vemurafenib highlights the rich polymorphic landscape that can be accessed by evaluating crystallization in supercooled liquids, and underscores the complexity in evaluating the crystallization of amorphous drug forms.

INTRODUCTION Based on the biopharmaceutical classification system, 90% of compounds in development and 40% of marketed drugs are considered as poorly water-soluble1. Amorphous drugs are an important strategy for achieving an enhancement of solubility and dissolution rate, since they lack a crystal lattice, reducing the energy barrier for the dissolution process. However, amorphous drugs are thermodynamically un/metastable and are apt to recrystallize during storage or during the dissolution process, with a subsequent decrease in the amorphous solubility advantage. Therefore, inhibition of recrystallization is an important issue for amorphous drug formulations. In turn, a fundamental understanding of the kinetics and crystallization behavior of amorphous drugs is necessary. Solid-state crystallization can be divided into melt crystallization and cold crystallization, which refers to crystallization during cooling of the melt and crystallization following heating of the glass/supercooled liquid, respectively. Cold crystallization has been widely investigated in polymers2, 3, and occurs from the re-arrangement of amorphous regions of the polymer chains into a crystalline phase4. For small molecules, cold crystallization involves the rearrangement of molecules in the amorphous material into the crystal lattice. Cold crystallization can occur in the glass or the supercooled liquid formed by heating the glass, whereby the glass transition temperature (Tg) separates the glassy region from the supercooled region. Thus the amorphous compound below Tg is a glass, while for temperatures between the Tg and melting point (Tm), it exists as a supercooled liquid. Research on amorphous pharmaceutical materials has focused on the role of kinetic 2


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factors especially molecular mobility additives

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, as well as the influence of polymeric

and water 9. However, the mechanisms and crystallization behavior of

amorphous systems are still poorly understood. Understanding the evolution of different crystal phases from amorphous systems of pharmaceutical relevance is of fundamental importance, however, relatively few studies that address this topic can be found 10-16. Vemurafenib is a water insoluble compound with an aqueous solubility of less than 0.1 µg/ml

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. Preliminary studies suggested that this compound has a strong

crystallization tendency and a very fast maximum crystal velocity (106±4 µm/s at 250oC) compared with values reported for other compounds in the literature18. Therefore, amorphous vemurafenib might be anticipated to be physically unstable as the amorphous form. However, the commercial formulation contains the drug in amorphous form, as an amorphous solid dispersion. The goal of the current study was to evaluate the crystallization kinetics of amorphous vemurafenib as a function of temperature, as well as to assess the polymorphic landscape.

MATERIALS AND METHODS Materials. Vemurefenib (Figure 1, >99% purity, α-form) was purchased from Attix Pharmaceuticals (Toronto, Canada).

Figure 1 Molecular structure of vemurafenib.

Preparation of amorphous samples. Amorphous vemurafenib was prepared by heating the as received material (placed between two coverslips) at 285oC for 1 min to ensure complete melting, and then quenching to room temperature by placing the

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sample on metal block. Following heating and cooling using this protocol, no change in color of the sample was observed. Analysis of samples after heating using high performance liquid chromatography indicated that no detectable degradation had occurred. The amorphous state of the quenched sample was confirmed using a polarizing microscopy (Nikon Eclipse E600 POL microscope, Nikon Corp, Tokyo, Japan).

Determination of morphology, crystal growth and phase transformation. The morphology, crystal growth rate and phase transformations following isothermal crystallization at the cold crystallization temperatures (Tcc), which ranged from 120oC (slightly above the glass transition temperature) to 270oC (close to the melting temperature) were investigated using hot-stage microscopy. The temperature of the sample was controlled using a hot stage (Linkam THMS 600, Surrey, U.K.). Images were captured during the crystallization process at regular intervals using time-lapse photography, using a digital camera (Nikon DS-Fi1, Nikon Corp, Tokyo, Japan) interfaced with a polarizing light microscope (Nikon Eclipse E600 POL microscope, Nikon Corp, Tokyo, Japan). The size of the crystals as a function of time and temperature was measured from the images using Elements software (v 300, Nikon Corp, Tokyo, Japan). The crystal size was plotted against time using Prism software (GraphPad Software Inc., San Diego, CA) and the growth rate was determined from the slope. Non-isothermal cold-crystallization was also investigated to better understand phase transformations between the various polymorphs of vemurafenib.

Raman Microscopy. A DXR™ Raman confocal Microscope (Thermo Scientific, Madison, WI, USA) was used in this study to evaluate the polymorphism of vemurafenib crystallites presenting with different morphologies. A 532 nm excitation laser coupled to a microscope and spectrometer with a high resolution grating were used for spectral collection. A 10× objective was used to visualize the morphology of vemurafenib and obtain Raman spectra from specific locations in the sample. The estimated laser spot diameter was 2.1 µm and the spectral resolution was around 2.0 cm-1. This spatial resolution is sufficiently high to obtain a spectrum from each spherulite or other morphology. The laser power used was 8.0 mW with a 25 µm 4


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pinhole. Spectra were collected and analyzed using the Thermo Scientific OMNIC™ Software. The wavelength scale was calibrated using polystyrene.

Differential scanning calorimetry (DSC). DSC thermograms were recorded using a TA Instruments system (DSC Q2000, Delware, USA). 1-2 mg of the α-form was accurately weighed into a Tzero pan and then sealed. The sample was heated to 280oC at a rate of 10oC/min. After holding for 2 min, the sample was cooled to 25oC at a cooling rate of 10oC/min and then reheated to 280oC at 10oC/min. Amorphous samples were prepared by heating the sample in a DSC pan at 285oC for 1 min on a hot stage and then quenching to room temperature on a metal block. The resultant amorphous sample was heated from -40oC to 285oC at the rate of 10oC/min. The data was analyzed using the Advantage software.

Powder X-ray diffraction (PXRD). PXRD patterns were recorded using a Rigaku SmartLAB system using Cu Kα radiation with λ= 1.542 Å. The samples were loaded onto a glass sample holder and scanned from 4.5o to 30o (2θ) using a scanning speed of 5o/min.

Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy was performed in attenuated total reflectance (ATR) mode using a Bruker Vertex 70 spectrometer (Bruker, Germany) with a Golden Gate ATR accessory (Specac Ltd., England). The amorphous sample was prepared on the surface of a Kapton film by melting at 285oC for 1 min and then quenching to room temperature on a metal block. The sample was removed from the Kapton film and transferred to the ATR crystal. The as-received vemurafenib powder was used to obtain the spectrum of the α-form sample. The spectra were obtained by co-adding 128 scans over the wavenumber region of 4000– 400 cm−1 with a resolution of 4 cm-1.

RESULTS AND DISCUSSION Differential Scanning Analysis. The as-received vemurafenib exhibited a sharp endothermic peak with a peak temperature of 271.3oC with an enthalpy of melting (∆Hm) of 55.9 kJ/mol (Figure 2) and is this form is designated the 5



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α-form. During cooling of the melt at 10oC/min, crystallization occurred, commencing at 248.9oC, with a crystallization enthalpy (∆Hc) value of 48.7 kJ/mol. The degree of supercooling achieved prior to crystallization was only ~25oC, suggesting that vemurafenib is a relatively poor glass former. Reheating of the sample indicated that the polymorph that crystallized during cooling of the melt was the α-form and this was confirmed using XRPD.

Figure 2. DSC curves of vemurafenib obtained during (A) heating to 285oC followed by a 1 min isothermal hold; (B) cooling to 20oC ; (C) reheating to 285oC. Heating/cooling rate was 10oC/min.

With more rapid cooling of the melt, crystallization was avoided and a glass could be formed. The glass transition temperature of this sample was 108°C when evaluated at a heating rate of 10°C/min (Figure 3A). Above the Tg, recrystallization took place with the occurrence of a sharp exotherm at 157°C, which was followed by a broad exotherm, a convoluted exothermic/endothermic event, and a final melting peak around 271.5oC with a ∆Hm value of 51.0 kJ/mol, indicating melting of the α-crystal. This complex thermogram suggests that a different polymorph may crystallize from this sample during heating of the glass, with subsequent phase transformation to the α-form. An extensive evaluation of the crystallization behavior following isothermal heating of glassy vemurafenib was therefore carried out to evaluate the polymorphic landscape.

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A

B o

Figure 3. (A) DSC curve of amorphous vemurafenib heated at 10 C/min. The insets show different crystal morphologies and phase transformations observed during hot stage experiments. The spherulites marked by yellow rings in Figure 3-A were the δ-form. (B) PXRD patterns of vemurafenib samples quenched from 236oC and 170oC after heating at 10oC/min.

Isothermal Crystallization Studies. Based on microscopic observations, four polymorphs (α, β, γ and δ) which crystallized with six morphologies were observed during the cold crystallization of amorphous vemurafenib in the temperature range 120-270oC (Figure 4). The occurrence and postulated stability relationships between the various forms are summarized in Figure 5.

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Actinomorphic needle (α) o

Diamond spherulite (α)

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Brown feather (α) o

Growth region: 200-270 C

Growth region: 170-200 C

Growth region: 150-240oC

Melting temperature: 271oC



A

B

C





Transform to β at 200-225oC

Transform to β at 170-225oC

D

E

F

Figure 4. POM images and Raman spectra of the various vemurafenib polymorphs. (A) α-actinomorphic needles; (B) α-diamond spherulites; (C) α-brown feather crystals; (D) β-form; (D) γ-form; (E) δ-form.

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Figure 5. Schematic illustrating the temperature regions where different polymorphs were observed to form and grow, and phase transformations between different forms. The β-polymorph was metastable relative to the α-polymorph between room temperature and the melting point. The γ- and δ-forms were metastable with respect to the β- and α-forms over the temperature ranges indicated. The stability relationship between the γ- and δ-forms could not be established.

α-form (Tm=272oC). The α-form was observed to nucleate and grow over a very wide temperature range (170-270oC). The as-received material is also the α-form and the XRPD pattern of this material is shown in Figure 6. Interestingly, the α-form exhibited three different morphologies depending on the crystallization conditions, and these three morphologies were termed diamond spherulite, brown feather, and needle crystals where representative examples are shown in Figure 4A-). Although the three morphologies have a very different appearance, the crystals had the same melting point and Raman spectra, suggesting the same crystal structure. The conditions where the three morphologies were observed are described in detail below.

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A

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B

Figure 6. PXRD patterns of (A) vemurafenib as-received and samples prepared by isothermal crystallization at different temperatures, and (B) α-form and β-form prepared by cold-isothermal crystallization at 260oC and melt-isothermal crystallization at 190oC.

α-Diamond spherulite. For Tccs of 170-200oC, the α-crystals were observed to nucleate from the supercooled liquid and grow as spherulites. These spherulites had a shiny appearance when formed at lower Tccs (170-180oC) (Figure 4C) and hence, were termed “diamond” spherulites. The growth rate of diamond spherulites accelerated with increasing temperature as summarized in Figure 7. The diamond spherulites always crystallized concomitantly with the β-form, and at lower temperatures, the γ-form was also present. Due to the slower growth rate of the α-diamond spherulites, they always appeared embedded in a matrix of the β-crystal, however at higher temperatures, they were able to grow larger before all of the liquid phase was consumed. Interestingly, the presence of the diamond 10


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spherulites in the β-phase triggered the surface transformation of the β-crystals to α-form (Figure 8). The newly formed α-crystals had a feathery texture with brown color and this morphology was termed “brown feather”.

Figure 7. Crystal growth rate as a function of temperature for various vemurafenib polymorphs.

Figure 8. POM images, Raman spectra and PXRD patterns showing the β-to-α phase transformation at a Tcc=170oC. Only the POM images were obtained at the crystallization temperature; Raman and PXRD information was obtained by cooling samples.

α-Brown feather. The α-brown feather crystals are the product of a β-to-α phase conversion. The progression of a growth front of the brown feather crystals from the diamond spherulite:β-form interface made visualization of this phase transformation possible. This solid-solid phase transformation is very slow, presumably due to a high 11



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activation barrier. The transformation accelerates with increasing temperature, as shown in Figure 7. However, it can be seen that the evolution of the α-crystals via phase transformation of the β-form is considerably slower than the growth of α-crystals from the supercooled liquid (Figure 7). Above 250oC, the β-form does not nucleate, and hence the brown feather morphology is not observed. Figures 8 and S1, further illustrate how the α-brown feather always commenced growth from the interface between α- and β-crystals, either the diamond spherulites or the needles (discussed below), whereas no transformation was observed in regions of β-crystals which lacked contact with the α-crystalline phase.

α-Needles. As the crystallization temperature increased to 200oC, a new morphology, designated α-needles (Figure 4-C), appeared together with the aforementioned diamond spherulites (Figure S2). Above 210oC, the diamond spherulites ceased to appear, and the α-form that nucleated from the supercooled liquid grew exclusively with a needle-like morphology. The needle crystals exhibited a much smoother surface compared with other two morphologies of α-form, and a more distinct interface with the melt. Such changes in growth morphology have been discussed extensively by Jackson19. For organic compounds, he noted that at low undercoolings, the growth morphology often showed faceted or spikey crystals, while at larger undercoolings, isotropic growth, in the form of spherulites was observed. The change in morphology was attributed to the phenomenon of kinetic roughening which becomes more predominant at large undercoolings. Figure 7 demonstrates that the temperature dependence of the growth rate for the needle and spherulite morphologies follow the same trend, in contrast to the α-brown feathers, which grow much more slowly, as discussed above. The grow rate of α-needles increased with increasing temperature up to 250°C, reaching the maximum value at this temperature (Figure 7). At higher temperatures, the growth rate slowed, as the melting point was approached. The bell-shaped growth profile can be readily explained by considering the thermodynamic and kinetic factors that influence the growth rate, as discussed in 12


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detail by Okui.20 . Close to the melting point, although molecular mobility is high, the thermodynamic driving force for crystal growth is low. As the temperature is lowered, the thermodynamic driving force increases, but the molecular mobility decreases. The interplay of these two factors leads to a maximum in the growth rate, at a temperature intermediate to the melting and glass transition temperatures. Naito has suggested that the maximum in growth rate, termed the maximum crystal velocity typically occurs at a value corresponding to 0.94 of the melting temperature21. For the α-polymorph of vemurafenib, this corresponds to a temperature of 240°C, which is close to the experimentally observed maximum observed at 250°C. Furthermore, if the maximum crystal velocity is compared to values reported in the literature for other drug-like molecules, it is apparent that vemurafenib is able to grow very rapidly relative to comparable small organic molecules18. All three morphologies of the α-crystals maintain crystallinity without any apparent phase transformations until the melting point is attained.

β-form (Tm=250oC). The β-form crystallizes as a spherulite and has a distinct Raman spectrum (Figure 4-D). This form nucleates over the temperature region, Tcc=120-240oC. At low crystallization temperatures (Tcc150oC, the growth rate accelerates and becomes much faster than other crystal forms. Consequently, this polymorph is the dominant form observed in the temperature range 150-190°C. Beyond 200oC, the α-form also shows a significant acceleration in the crystal growth rate and also nucleates more readily, whereas, the β-form, while still having a faster growth rate, nucleates less readily At temperatures between 220 and 240oC, the nucleation of β-form is erratic, with a low frequency. Melting of the β-form was observed at 250-255°C. As mentioned above, the β-form can transform to the α-form, and the transformation will be discussed in more detail below. Hence this polymorph is a metastable form over this temperature range.

γ-form. The γ-form also crystallizes as spherulites (Figure 4-E) and was observed to nucleate between 170-190oC and then to grow slowly (Figure 7). Generally, the 13



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γ-form is always surrounded by the fast-growing β-form, and transforms to the β-form during both isothermal and non-isothermal heating. Therefore, no melting point could be determined for this form. Figure 9 illustrates the γ-to-β phase transformation during heating. The sample shown here was generated via isothermal crystallization at 190oC, leading to the concomitant crystallization of α, β and γ-forms, and was then was heated at 10oC/min. The γ-crystal was observed to convert to a new phase when the temperature exceeded 200oC. The phase conversion was complete around 220oC. The newly formed phase melts at 254-255oC together with the original β-form, indicating that the new phase has the same melting point as the β-form. Raman microscopy confirmed that the new phase was the β-form. Figure S3 illustrates that the conversion also occurs when the sample is held at 190oC, albeit much more slowly, whereby 60 min was required for the transformation process. Therefore, it can be concluded that, at this temperature, the γ-form is metastable with respect to both the α- and β-forms.

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Figure 9. POM images showing the γ-to-β phase transformation during heating at 10oC/min. I: α-diamond spherulite; II: β-form; III: γ-form; IV: new phase (β-form) transformed from γ-form; V: newly formed phase (α-needle crystal). Raman spectra were used to determine the polymorphs observed under the microscope during heating.

δ-form. The δ-form (Figure 4-F) nucleates at lower temperatures (110-165oC), appearing concomitantly with the β-form. It grows as dense, slightly irregular, circular brown clusters of crystals. However, the growth rate of the δ-form is extremely slow and does not increase very much with temperature (Figure 7). Therefore, the δ-form was always “engulfed” by the faster-growing β-form (shown in the inserted image in

Figure 3A) due to the slow growth. In regions of the liquid where there are no β-nuclei, as shown in Figure 10 (Tcc=150oC), the δ-crystals can grow to a larger size. We observed a small area separated from the bulk sample, where only δ-nuclei existed (Tcc=150oC). When this sample is subsequently heated, phase conversion to the β-form is observed at around 220oC. Similar to γ-form, the melting point of δ-form cannot be determined because of the δ-to-β phase conversion. The δ-form is also metastable with respect to the α- and β-forms over the temperature range 170-225 oC.

Figure 10. POM images showing the δ-to-β phase transformation during heating at 10oC/min.

Raman and FTIR of vemurafenib polymorphs. Raman and FTIR spectra were

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used to help confirm which morphologies corresponded to different polymorphs. Raman spectra were obtained from each of the morphologies shown in Figure 4 using a Raman microscope, taking advantage of the high spatial resolution of this technique. For FTIR measurements, only the amorphous form, α-form and β-form could be prepared in sufficient amount to enable evaluation using ATR mode (Figure 11 and Table 1). Comparison between Raman and IR spectra of the amorphous form, α-form and β-form was useful for peak assignment. In particular, the Raman spectra of the various forms exhibited differences in peaks between 1550-1700 cm-1.

A

B

C

D

Figure 11. FTIR (A) and Raman (B-D) of the amorphous, α- and β-forms of vemurafenib.

The Raman peaks at 1597 cm-1 and 1608 cm-1 (amorphous form), 1597 cm-1 and 1613 cm-1 (α-form), 1597 cm-1 and 1609 cm-1 (β-form), and the IR peaks at 1589 cm-1 (amorphous form), 1590 cm-1 and 1589 cm-1 (β-form), were attributed to the aromatic carbon-carbon stretching vibrations of benzene ring. The peaks at 1640 cm-1 and 1639 16


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cm-1 were assigned to the C=O stretching vibration. These vibrations are active in both in the IR and Raman spectra, but the C=O peaks show a higher intensity in the IR spectra while the aromatic carbon stretching vibrations are more dominant in the Raman spectra. For the various morphologies (Figure 4A-C), identical Raman spectra were obtained for the diamond spherulites, needles and brown feathers, confirming that these three morphologies all correspond to the α-form, while different spectra were observed for the other morphologies. The peaks attributed to the C=O stretching vibration were significantly different, suggesting different intermolecular interactions between the C=O and N-H groups in the different crystal structures. The Raman spectra obtained from the different morphologies provide strong evidence to support the existence of a tetramorphic system.

Table 1. Raman and Bulk-IR peaks observed for different forms of vemurafenib forms

Solid state

Raman (cm-1) Amorphous 1597 s 1608 sh 1640 w α-form 1597 s 1613 w 1640 m β-form 1597 s 1609 w, sh 1640 w 1655 w γ-form 1600 s, sh 1610 s, sh δ-form 1597 s 1612 m, sh 1624 m, sh

Infrared (cm-1) 1589 m 1640 s 1590 m 1618 w, sh 1640 s 1589m 1640s 1655w,sh

Assignment v(C=C) in benzene ring v(C=C) in benzene ring v(C=O) v(C=C) in benzene ring v(C=C) in benzene ring v(C=O) v(C=C) in benzene ring v(C=C) in benzene ring v(C=O) v(C=O) v(C=C) in benzene ring v(C=C) in benzene ring v(C=C) in benzene ring v(C=C) in benzene ring v(C=O)

vs-very strong; s-strong; m-medium; w-weak; sh-shoulder; v-stretch.

Phase transformations during non-isothermal cold-crystallization. The phase transformations of vemurafenib during non-isothermal cold-crystallization were investigated using a combination of DSC, POM and PXRD. As briefly discussed above, amorphous vemurafenib exhibited complex thermal events during the 17



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non-isothermal heating process (Figure 3A). First, a glass transition temperature was observed at 108oC. Then, crystallization occurred, as evidenced by a sharp exothermic peak which commenced at ~152oC with a ∆Hc value of 23.6 kJ/mol. A second, broad exothermic event commenced at ~200oC, with a peak temperature of 217oC and a ∆H of 19.6 KJ/mol. This thermal event was then followed by a very small endothermic peak at 250oC that was immediately followed by a small exothermic peak. Finally, a sharp endothermic peak occurred at 271.5oC with a ∆Hm value of 53.8 kJ/mol. POM and PXRD data were used to characterize the various phase transitions. POM images clearly indicated that the first exotherm can be attributed to crystallization to predominantly the β-form with a few δ-form crystals also apparent. The δ-crystals gradually transformed to the β-form (Figure 10). No obvious peak for the δ-to-β phase transformation can be observed in the DSC curve, probably due to the low amount present. A new phase (α-brown feather) appeared at 200oC, covering the original β-from. The PXRD data indicated that the β-form gradually transformed to α-form (Figure 3B), although this transformation was not complete. This solid-solid phase transformation is an exothermic process, which occurs quite slowly (the growth rate of brown feather on the β-crystals is 0.3±0.1 µm/s at 220oC), thus producing a very board exothermic peak. As shown in the PXRD patterns, there are still several diffraction peaks arising from the β-crystal in the sample quenched after heating to 236oC (close the endpoint of the second peak in DSC curve, 237oC). This indicates that the β-crystals don’t completely transform to the α-crystals, and visually the transformation appears to occur at the surface of the β-crystal crystals. The residual β-crystals melted at 250oC and this is thought to result in the small endothermic peak in the DSC curve, which was immediately followed by a small exothermic peak. The POM images in Figure 9-D suggest that as the β-crystals melt, α-needle crystals begin to grow from the edge of the α-diamond spherulites. Melting of the β-crystals provide a supply of drug to enable growth of the α-needles. By 277oC, all of the crystals with a morphology characteristic of the α-form had melted, confirming that the final endotherm observed in the DSC is due to 18


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melting of this polymorph. The small endothermic and exothermic peaks at 250°C are thus attributed to melting of the small amount of β-crystal that did not undergo a solid-solid phase transformation to the α-form, followed by crystallization of α-needle crystal from the liquid phase. Since these two events occur almost simultaneously, it is likely that the endothermic melting is offset by the exothermic crystallization event. To further explore this, samples were analyzed at a much faster heating rate (100°C) in order to better capture the melting of the β form. The amorphous sample was first annealed at 150oC for 20 min to crystallize the β-form (with very little δ-form), and then, heated to 285oC at the heating rate of 100oC. As shown in Figure S4, the β-form melting peak is much more visible, but is still convoluted by the exothermic recrystallization peak. However, this experiment confirms the observations made with hot stage microscopy that the β-polymorph melts at 250°C, followed by immediate conversion to the α-polymorph.

Competition Between Thermodynamic and Kinetic Factors. According to Ostwald’s law of stages22, the least stable polymorph crystallizes first, followed by conversion to the second least stable polymorph, and so on until the most stable polymorph is produced. However, to explain concomitant polymorphism, i.e. where more than one polymorph is observed to crystallize at the same time, it has been suggested that the two polymorphs have similar nucleation rates, leading to an equal probability of appearance

23-25

. However, Yu has pointed out that considering only

relative nucleation rates may have flaws in terms of understanding which polymorphs are actually observed. He noted that the fast nucleating polymorph may be slow growing, allowing a slow nucleating, but fast growing polymorph to be observed or even dominate the apparent crystallization outcome15. He also reported on an interesting phenomenon whereby a fast growing polymorph could heterogeneously nucleate from a fast nucleating polymorph, a phenomenon termed cross-nucleation13, 16

. Based on the phase transformations observed in this study, the α-polymorph

appears to be the most thermodynamically stable polymorph, at least at the higher temperatures studied, since all of the other polymorphs eventually transform to this 19



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phase. Additional studies were conducted at room temperature, where a solvent-mediated β-to-α phase conversion was observed (data not shown), confirming that the α-form is also the room temperature stable form. The α-polymorph shows the same diffractogram as the crystalline form of vemurafenib reported by Shah et al.17. Two additional solid state forms of vemurafenib have been reported in a patent, prepared using a solvent method 26. In this instance, Form B was prepared by drying of Form A, and it appears that Form A is a solvate, while Form B is the product of desolvation. Neither the diffraction peaks nor the melting points observed for these forms matched any of the forms evaluated in this study (Table 2). Based on the data presented herein, it thus appears that vemurafenib is at least a tetramorphic system, which shows a high tendency for concomitant polymorphism during crystallization from the supercooled liquid.

Table 2. PXRD peaks and DSC data of vemurafenib solid-state forms Crystal form Form α Form β Form γ Form δ Form A26 Form B26

2θ (o) Tm(oC) 6.8, 9.3, 13.5, 14.2, 14.6, 15.1, 18.3, 19.2, 19.8, and 24.4 271 5.0, 8.4, 9.9, 12.0, 14.9, 16.7, 20.8, and 27.2 248 6.5, 9.7, 18.0, 19.5, and 23.7 268.5 5.9, 9.1, 14.2, 18.3, 19.7, and 23.7 270.9

ΔHm(J/g) 114 68 96.5 128.6

* λ=1.542 (CuKa)

Although the α-crystal is the most thermodynamically stable form, at lower temperatures other forms predominate. This can be explained by considering that different polymorphs will have different nucleation and growth zones27. The temperature overlap between the nucleation and growth zones, as well as the magnitude of the two process, combined with the crystallization conditions (i.e. temperature), will determine the type and amount of each polymorph observed at each crystallization temperature. Based on the DSC thermogram shown in Figure 2, it is clear that the α-form has a sufficiently high nucleation rate in the temperature region of maximum crystal growth (250°C), for spontaneous crystallization to occur during cooling. Given that the maximum in the nucleation rate occurs at a lower temperature than the maximum in the growth rate curve20, 20


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combined with the observation that the α-form is not seen when samples are crystallized at temperatures of 165°C and below, it appears that the favorable nucleation zone for the α-form is between approximately 170 and 270°C. More rapid cooling of the melt leads to glass formation, with subsequent devitrification upon non-isothermal heating to give predominantly the β-polymorph (Figure 3). The XRPD pattern of this polymorph is shown in Figure 6. The crystallization peak for this form is seen around 160°C in the DSC thermogram. At this temperature, the β-polymorph shows the fastest growth rate among the four polymorphs (Figure 7), so even if there is concomitant nucleation of other forms, this polymorph would be expected to grow faster than the other forms and be the dominant polymorph in the non-isothermal experiment. Even though this polymorph has a much faster growth rate, in particular at higher temperatures, other polymorphs do crystallize concomitantly; the δ-form at lower temperatures (120-165 °C), the γ-polymorph over the temperature range 170-190°C and the α-form between 170 and 250°C. This is due to the balance between nucleation and growth rate of the different forms. Based on the microscope observations, nucleation appears to be the limiting step for crystallization of β-form, whereby a low nucleation density is observed above 150oC. However, once nucleation has occurred, the growth rate is rapid leading to large regions of the β-form at temperatures less than 200°C. Above 200oC, the nucleation of β-form becomes less favorable, and even though the grow rate continues to be much faster than the α-form in this temperature regimen, the α-form starts to be the predominant form due to more favorable nucleation. The γ-form represents an example of a polymorph that nucleates rapidly but grows slowly. It crystallizes concomitantly with the β- and α-forms between Tcc=170-190oC but exists as a low mass fraction of the crystallized sample due to a slow growth rate. Consequently, the diffraction peaks of γ-form are difficult to observe in XRD patterns of samples crystallized at these temperatures (Figure 6A), although it could be readily observed as discrete regions with a different morphology by microscopic analysis. Because the γ-form spontaneously transforms to the β-form it is clearly metastable with respect to both the β and α-forms. 21



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The δ-form exhibits a fast nucleation rate and hence a high nucleation density. At 120oC, only the β-form and δ-form can nucleate and at this temperature, they have a similar growth rate. The PXRD of a sample crystallized at this temperature exhibited diffraction peaks specific to the δ-form at 2θ=8.8, 9.3, 11.4, 11.7, 16.3 and 24.4o (Figure 6A). Although the peaks at 9.3 and 24.4o also can be found in the diffractogram of the α-form, other peaks at 8.8, 11.4, 11.7 and 16.3o were specific for δ-form, and the Raman spectrum of the δ-form was very different from that of α-form (Figure 4), suggesting that they have different crystal structures. This form clearly has favorable nucleation in a lower temperature region than other forms, whereby an increase in the crystallization temperature leads to a decreased nucleation density and no δ-form was observed for samples crystallized at 165oC (confirmed by POM and Raman microscopy). Since the δ-form also spontaneously converts to the β-form, it is less stable than both the β and α-forms. For the cold-crystallization of vemurafenib, the α- and β-forms were the two dominant polymorphs. In addition, considering the difficulties in obtaining phase pure samples of the γ- and δ-forms and their rapid conversion to other forms, only the thermodynamic relationship between α-form and β-form could be evaluated in more detail. The α-form has a higher Tm (271oC) with a larger ∆Hm (53.8 kJ/mol) relative to the β-form (Tm of 248oC and ∆Hm of 33.3 kJ/mol). According to the heat-of-fusion rule

28

, the α- and β-form are therefore monotropically related. This analysis is

consistent with our experimental data, where the β-form always converts to the α-form over the temperature range 25-250°C and also with the observation that the β-to-α phase transformation is exothermic (Figure 3A).

22


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Figure 12. DSC thermogram showing the melting of the α-form (obtained by cooling the melt at o

10 C/min) and the β-form (obtained by isothermal crystallization at 190oC for 20 min following melting at 280oC) of vemurafenib.

Figure 13. Free energy versus temperature for different forms of vemurafenib based on predictions of the Hoffman equation.

The Hoffman equation (Eq.1) can be used to approximate the difference in free energy between the liquid and crystalline phases (∆Gv)29, and therefore gain insight into the thermodynamic driving force for transformation to the crystalline phases:

1

where T is the crystallization temperature and ∆Hm is the enthalpy of fusion at the melting temperature, Tm. As shown in Figure 13, the α-form always has a larger thermodynamic driving force for transformation from the liquid to the crystal than the β-form, consistent with this form being the most thermodynamically stable form over 23



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the temperature range of interest. The system is monotropic based on these calculations with a virtual transition temperature of 319 oC. Further, the G-T plot also supports observations that the β-to- α phase transformation is spontaneous due to the lower free energy of α-form across the entire temperature range studied. However, in spite of the difference in the thermodynamic driving force, the β-form dominates the crystallization outcome between 120oC and 190oC. These observations indicate that the cold-crystallization of amorphous vemurafenib is controlled by factors other than the thermodynamic driving force. Instead, the results of our study are consistent with literature observations that at larger undercoolings, metastable forms are more probable30.

CONCLUSION Herein,

the

concomitant

polymorphism

of

vemurafenib

following

cold-crystallization was investigated, and we report on morphology, crystal growth kinetics, phase transformation and relative thermodynamic stability. Three new metastable polymorphs (designated β, γ and δ) were identified and observed during cold-crystallization, together with the previously reported α-form. Both melt-mediated (β-to-α) and solid-solid (β-to-α, γ-to-β, and δ-to-β) phase transformations were observed. The α- and β-forms were the dominant polymorphs observed following cold-crystallization, and had a monotropic relationship. The α-form has the highest thermodynamic stability, followed by the β, γ and δ forms. The discovery of three new polymorphs of vemurafenib following crystallization from the supercooled liquid of vemurafenib highlights the rich polymorphic landscape that can be accessed by evaluating crystallization in supercooled liquids, and underscores the complexity in evaluating the crystallization of amorphous drug forms.

AUTHOR INFORMATION *(L.S.T.) Email: [email protected]. Phone: 765-496-6614. 24


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Present Address * 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United states Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We are grateful to Thermofisher Scientific for the help in Raman Microscopy characterization. The present work was partially supported by China Scholarship Council (Grant No. 201406385060). The authors also acknowledge the U.S. Food and

Drug

Administration

for

financial

support

under

grant

award

1U01FD005259-01.

Supporting Information The Supporting Information is available free of charge at the ACS Publications website at DOI: Additional polarized light microscope images showing transformations, Raman spectra of different crystal morphologies and DSC thermogram showing β-form melting.

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(10) Zhang, S.; Lee, T. W.; Chow, A. H., Cryst. Growth Des. 2016, 16, 3791-3801. (11) Yoshioka, M.; Hancock, B. C.; Zografi, G., J. Pharm. Sci. 1994, 83, 1700-1705. (12) Andronis, V.; Zografi, G., J. Non-Cryst. Solids 2000, 271, 236-248. (13) Tao, J.; Jones, K. J.; Yu, L., Cryst. Growth Des. 2007, 7, 2410-2414. (14) Wu, T.; Yu, L., J. Phys. Chem. B 2006, 110, 15694-15699. (15) Yu, L., CrystEngComm 2007, 9, 847-851. (16) Chen, S.; Xi, H.; Yu, L., J. Am. Chem. Soc. 2005, 127, 17439-17444. (17) Shah, N.; Iyer, R. M.; Mair, H. J.; Choi, D. S.; Tian, H.; Diodone, R.; Fähnrich, K.; Pabst‐Ravot, A.; Tang, K.; Scheubel, E., J. Pharm. Sci. 2013, 102, 967-981. (18) Trasi, N. S.; Baird, J. A.; Kestur, U. S.; Taylor, L. S., J. Phys. Chem. B 2014, 118, 9974-9982. (19) Jackson, K. A., Kinetic Processes: Crystal Growth, Diffusion, and Phase Transformations in Materials. 1st ed; Wiley-VCH: Weinheim, 2004. (20) Okui, N., J. Mater. Sci. 1990, 25, 1623-1631. (21) Naito, K., Chem. Mater. 1994, 6, 2343-2350. (22) Ostwald, W., Z. Phys. Chem 1897, 22, 289-330. (23) Bernstein, J., Polymorphism in molecular crystals. Oxford University Press: Oxford, 2002; p P43. (24) Bernstein, J.; Davey, R. J.; Henck, J. O., Angew. Chem. Int. Ed. 1999, 38, 3440-3461. (25) Etter, M. C., J. Phys. Chem. 1991, 95, 4601-4610. (26) Davis, D.; Ridvan, L.; Klvana, R.; Dammer, O. Crystalline forms of vemurafenib, WO 2015078424 A1. (27) Woo, E. M.; Sun, Y.-S.; Yang, C.P., Prog. Polym. Sci. 2001, 26, 945-983. (28) Burger, A.; Ramberger, R., Microchim. Acta 1979, 72, 259-271. (29) Andronis, V.; Zografi, G., J. Non-Cryst. Solids 2000, 271, 236-248. (30) Ishihara, K.; Maeda, M.; Shingu, P., Acta Metall. 1985, 33, 2113-2117.

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For Table of Contents Use Only

Vemurafenib: A Tetramorphic System Displaying Concomitant Crystallization from the Supercooled Liquid Ming Lu and Lynne S. Taylor

TOC Graphic

Synopsis Vemurafenib forms four different polymorphs during crystallization from the supercooled liquid. One of these polymorphs exhibits three morphologies.

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Vemurafenib: A Tetramorphic System Displaying Concomitant

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