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An Investigation into the Polymorphism and Crystallization of Levetiracetam and the Stability of its Solid Form
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
An Investigation into the Polymorphism and Crystallization of Levetiracetam and the Stability of its Solid Form KAILIN XU, XINNUO XIONG, LIUQI GUO, LILI WANG, SHANSHAN LI, PEIXIAO TANG, JIN YAN, DI WU, HUI LI College of Chemical Engineering, Sichuan University, Chengdu 610065, China Received 9 June 2015; revised 21 July 2015; accepted 11 August 2015 Published online 1 September 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24628 ABSTRACT: Levetiracetam (LEV) crystals were prepared using different solvents at different temperatures. The LEV crystals were systematically characterized by X-ray powder diffraction (XRPD) and morphological analysis. The results indicated that many kinds of crystal habits exist in a solid form of LEV. To investigate the effects of LEV concentration, crystallization temperature, and crystallization type on crystallization and solid phase transformation of LEV, multiple methods were performed for LEV aqueous solution to determine if a new solid form exists in solid-state LEV. However, XRPD data demonstrate that the LEV solid forms possess same spatial arrangements that are similar to the original solid form. This result indicates that the LEV concentration, crystallization temperature, and crystallization type in aqueous solution have no influence on the crystallization and solid phase transformation of LEV. Moreover, crystallization by sublimation, melt cooling, and quench cooling, as well as mechanical effect, did not result in the formation of new LEV solid state. During melt cooling, the transformation of solid form LEV is a direct process from melting amorphous phase to the original LEV crystal phase, and the conversion C 2015 Wiley rate is very quick. In addition, stability investigation manifested that LEV solid state is very stable under various conditions. Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:4123–4131, 2015 Keywords: levetiracetam; crystals; morphology; X-ray powder diffractometry; solid state; stability
INTRODUCTION The solid phase diversity of active pharmaceutical ingredients (APIs), including pharmaceutical polymorphs,1–4 pseudo polymorphs (solvates and hydrates),5,6 salts,7,8 co-crystals,9,10 and amorphous solids,11–15 has become one of the most investigated areas in contemporary drug research because of the unique physicochemical properties, efficacy, and toxic side effects existing in different solid forms of drugs.16–18 Pharmaceutical solid form screening has rapidly developed into a general approach to improve the physicochemical properties of drugs, which can remarkably affect the solubility, bioavailability, hygroscopicity, melting point, stability, compressibility, and other performance characteristics of pharmaceutical products.2,17,19–21 A large number of methods can be applied to produce polymorphs of APIs, such as high-temperature-triggered transformation,16,22 cooling crystallization,23–25 pressure-induced transformation,22 milling,26,27 humidity,28 and solution crystallization by changing the crystallization temperature, solution concentration, cooling rate, solvent types, pH, and additive.19,29–34 Studies aimed at obtaining solid forms of relevant drug are greatly essential and meaningful to develop optimal drug crystals and to control the quality of the final state of the drug.35,36 In general, the stable polymorph should be used in the marketed formulation to prevent polymorphic transformation during manufacturing, delivery, or storage. Therefore, to examine the stabilities of crystal forms, investigating the polymorph transformation and understanding the process of solid-state transformation at different conditions are highly important. Levetiracetam (LEV), whose chemical name is (S)-2-(2oxopyrrolidin-1-yl) butanamide, is an ethyl analog of the Correspondence to: Hui Li (Telephone: +86-028-85405149; Fax: +86-02885401207; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 104, 4123–4131 (2015) C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
nootropic and dementia drug piracetam, and is used as an anticonvulsant in the treatment of epilepsy.37–39 Compared with other antiepileptic drugs, LEV has a unique mechanism for the treatment of epilepsy, and it has satisfactory pharmacokinetic characteristics and efficiency with minimal side effects.40 Moreover, LEV could prevent the progression to Alzheimer’s disease by reducing hippocampal hyperactivity in patients with cognitive impairment.41 Polymorphism and the solid state of drugs can have a significant impact on the clinical efficacy, toxic side effects, and quality of medicines. Thus, studies on polymorphism, polymorph transformation, and stability of solid state of LEV are very significant. The racemic compound of LEV, etiracetam, was reported to have three different solid-state phases, including two polymorphs and one hydrate phase.38 Moreover, the features of LEV molecular structure, including five freely rotatable bonds, four hydrogen acceptors, and two strong Hbond donor, make it very attractive in investigating whether chemically identical yet conformationally flexible LEV molecule exist in multiple crystal forms and conversion between poly˚ morphism. The crystal structure of LEV [a = 9.199 A, ˚ c = 6.272 A, ˚ $ = 108.65°, unit-cell volume V = b = 7.993 A, ˚ 3 , Z = 2, and space group P21 ] has been reported,42 436.962 A and systematic studies on LEV co-crystal were reported in several literatures.43–45 Two patents46,47 claim four polymorphs of LEV, as well as the corresponding preparation methods. But in our primary experiments, it was found that only one solid form of LEV was obtained after performing the same preparation methods multiple times. This work aims to search for various solid forms of LEV, and to investigate the stability of solid forms of LEV by changing the crystallization condition and mechanisms of solid-state transformation. The methods of potentially obtaining LEV polymorphs, including changing solvent type, crystallization temperature, LEV concentration in solution, humidity, physical effects, crystallization types, high temperature effects, and
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so on, were systematically investigated. The solid forms of LEV were prepared and characterized by X-ray powder diffraction (XRPD), and morphological analysis. Moreover, the effects of LEV concentration, crystallization temperature, and crystallization type in water on the formation of LEV polymorphs were also investigated in detail. The stability of LEV solid state was discussed under various conditions.
MATERIALS AND METHODS Materials The title compound, LEV (99% purity), in powder form was obtained from Zhejiang Jingxin Pharmaceutical Company, Ltd. (Shaoxing, China). Ultrapure water (18 M resistivity from a Millipore system; Massachusetts, USA) was used throughout the experiment. All analytical grade solvents were purchased from Kelong Company, Ltd. (Chengdu, China), and used without further purification. Methods Preparation of LEV Crystals In the solvent evaporation method, the LEV crystals were obtained by slow solvent evaporation and crystallization from different solvents of LEV at 25°C. Most of these crystals easily crystallized from a number of organic solvents, such as ethanol, methanol, 2-propanol, dimethylsulfoxide, acetone, acetonitrile, N, N-dimethyl formamide, chloroform, 1, 4-dioxane, methyl tertbutyl ether, ethyl acetate, and tetrahydrofuran. In the cooling crystallization method, the LEV crystals were crystallized and obtained from the LEV saturated solution of different solvents (25°C) by slowly decreasing the temperature from 25°C to 4°C. Crystallization Experiments in Water To investigate the effects of LEV concentration and crystallization temperature in water on the crystallization of LEV, the different concentrations of LEV aqueous solution from 0.01 to 2.5 g/mL was prepared. The samples were then placed in a thermotank to obtain crystals via slow solvent evaporation and crystallization at 4°C, 25°C, 50°C, 75°C, and 90°C. The obtained LEV crystals were then immediately analyzed by XRPD detection to avoid the effects caused by other factors.
periods (typically 2 min) were applied to limit the mechanical heating of the sample. The samples were milled for 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h to evaluate the effect of milling on the LEV. The milled samples were then immediately analyzed after the end of the milling process. Crystallization by Sublimation, Melt Cooling, and Quench Cooling Sublimation experiment was performed using a sublimation unit after the LEV was melted and the temperature was maintained at 125°C for 30 min. The crystals were then collected and detected as soon as possible. To investigate the effect of melt cooling on the solid-state transition of LEV, the LEV APIs samples were placed in an oven and subjected to melting at 125°C. The melted samples were cooled at 25°C and then to 4°C to obtain solid-state LEV. The samples were immediately monitored via XRPD to determine if the crystal phase was changed in this process. Quench cooling is a process in which the drug is first heated and melted at its melting point and then cooled at −20°C. In this study, 1 g of LEV was placed in a crucible and heated up to 125°C. The melted product was immediately covered with aluminum foil and kept at −20°C in a deep freezer for 24 h. The quench-cooled samples were analyzed as soon as possible. Analytical Techniques Single-Crystal X-Ray Diffraction Single-crystal data were collected using an Oxford Diffraction ˚ Xcalibur Nova system with Mo K" radiation (8 = 0.71073 A) at room temperature, and the 2 from 3.13° to 26.37°. The program package Olex2–1.1 (SHELX-97) was used for structure solution and refinement. Simulated XRPD patterns based on the single-crystal X-ray diffraction (SXRD) data were obtained using Mercury 2.4 software. Optical Microscopy The LEV crystals were placed in a transparent glass holder. The shape and surface morphology were recorded by an optical microscope (CX21; Olympus; Tokyo, Japan) equipped with a digital CCD camera and a thermostatic stage system (TS62, Instec; USA).
Freeze-Drying Experiments Different concentrations of LEV in aqueous solution (0.01, 0.05, 0.50, 1.00, 1.50, and 2.00 g/mL) were frozen and freeze-dried for 48 h (FD-1A-50; Biocool Instruments Company, Ltd., Beijing, China). The freeze-dried LEV was used for shape and surface morphology analysis and XRPD detection as soon as possible after drying. Milling Experiments Milling experiments were performed using a planetary mill (LNMN-QM 0.4L; Heishan Xinlitun Agate Handicrafts Company, Ltd.; Jinzhou, China) at room temperature. Four agate ball milling jars of 50 cm3 with 10 balls (ø = 10 mm) of the same material were used. Each milling was performed with 2 g of the powder to ensure homogeneous milling and reproducible results. The rotation speed of the solar disk was set to 400 rpm, and alternate milling periods (typically 10 min) with pause Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
Figure 1. XRPD pattern of LEV (red represents experimental pattern, and black belongs to the simulated pattern from LEV singlecrystal structure). DOI 10.1002/jps.24628
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Figure 2. Photos or photomicrograph of LEV crystals obtained from different solvents by slow solvent evaporation at 25°C (the scale bar in the bottom right corner of the photos represents 0.5 cm, and the photomicrograph was magnified 4×). (a) Water, (b) methanol, (c) ethanol, (d) isopropanol, (e) acetone, (f) acetonitrile, (g) tetrahydrofuran, (h) N,N-dimethyl formamide, (i) chloroform, (j) ethyl acetate, (k) 1,4-dioxane, (l) dimethylsulfoxide, (m) methyl tertbutyl ether, and (n) photomicrograph of the crystal obtained from methyl tertbutyl ether.
XRPD X-ray powder diffraction data were collected at room temperature using an X’Pert PRO diffractometer (PANalytical; Almelo, Netherlands) with a PIXcel 1D detector and Cu K" radiation ˚ generator setting: 40 kV and 40 mA). The (8 = 1.54056 A, diffraction data were collected in the 22 range from 5° to 50° with a step size of 0.01313° and a counting time of 30 ms/step.
RESULTS AND DISCUSSION SXRD Analysis The LEV crystal used for SXRD detection was obtained from methanol, and the SXRD analytical results are similar to the reported data.42 Namely, the LEV crystal produced in methanol has a monoclinic structure with space group P21 and unit-cell ˚ b = 8.006(3) A, ˚ c = 6.285(2) A, ˚ $= parameters: a = 9.211(3) A, ˚ 3 , and Z = 2. 108.371(5)°, unit-cell volume V = 439.8(3) A XRPD Analysis The experiment and simulated XRPD patterns of the LEV crystal were obtained and are illustrated in Figure 1. The DOI 10.1002/jps.24628
diffraction peak positions of the crystal obtained from the supplier and those simulated by the Mercury 2.4 software were similar, as shown in the XRPD results. The result indicated that the spatial arrangement of the LEV from methanol is similar to that of the LEV from the supplier. From the XRPD patterns, the LEV crystal has major peaks at 22 values of 10.10°, 14.96°, 18.52°, 20.48°, 22.17°, 23.29°, 23.84°, 24.43°, 26.77°, 28.86°, 30.30°, and 31.97°. Effect of Solvent Type and Crystallization Temperature on Solid-State LEV Crystallization can cause either changes in internal structure, leading to polymorphism and solvation, or, more frequently, changes in external properties, such as crystal habit, elongation ratio, mean particle size, and so on.48 Figure 2 shows the shape and surface morphology of LEV crystals obtained by slow solvent evaporation and crystallization from different solvents at 25°C. Most of the crystals have a prismatic and transparent crystal structure, including the crystals produced from methanol, isopropanol, acetonitrile, N, N-dimethyl formamide, chloroform, and ethyl acetate. However, particular crystals display isodimensional characteristics, and the crystals will grow Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
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Figure 3. Photos or photomicrograph of LEV crystals obtained from the LEV saturated solution of different solvents (25°C) by slowly decreasing the temperature from 25°C to 4°C. (The scale bar in the bottom right corner of the photos represents 0.5 cm, and the photomicrograph was magnified 4×). (a) Water, (b) methanol, (c) ethanol, (d) isopropanol, (e) acetone, (f) acetonitrile, (g) tetrahydrofuran, (h) N,N-dimethyl formamide, (i) chloroform, (j) ethyl acetate, (k) dimethylsulfoxide, and (l) photomicrograph of the crystal obtained from methyl tertbutyl ether.
around a central point to produce an agglomerate of tabular and prismatic crystals with a larger radius, such as crystals obtained from water, ethanol, 1,4-dioxane, and dimethylsulfoxide. The crystal produced from methyl tertbutyl ether is significantly smaller than the others probably because the solubility of LEV in methyl tertbutyl ether solution is obviously lower than in other solvents. The shape and surface morphology of LEV crystals obtained via cooling crystallization method is displayed in Figure 3. The result indicates that most of crystals also have a prismatic and transparent crystal structure, which is similar to the structure of the crystals obtained via slow solvent evaporation method at 25°C. However, the characteristic of the crystals produced from tetrahydrofuran and ethyl acetate is significantly different from the rest, where prismatic crystals usually agglomerate, as shown in Figures 3g and 3j. In addition, the crystals from dimethylsulfoxide are isodimensional with an irregular shape and have a tendency to form particle agglomerates. Meanwhile, the small transparent crystal particles from methyl tertbutyl ether have a prismatic structure. The solid form of LEV crystals obtained from different conditions should be identified as accurately as possible. The XRPD Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
patterns of the LEV solid forms were obtained and are illustrated in Figure 4. The diffraction peak positions of the crystals purchased from the supplier and those obtained from different solvents at different conditions were similar, as shown in the XRPD results. The result indicated that these LEV solid forms possess similar spatial arrangements. These LEV solid forms all have major peaks at 22values of 10.10°, 14.96°, 18.52°, 20.48°, 22.17°, 23.29°, 23.84°, 24.43°, 26.77°, 28.86°, 30.30°, and 31.97°. LEV Crystallization in Water The effects of LEV concentration and crystallization temperature in water on the crystallization of LEV are displayed in Figure 5. The crystals in 0.010, 0.025, 0.250, and 0.50 g/mL of LEV aqueous solution were not produced at 4°C because of the low concentration of LEV, and the XRPD patterns of the crystals obtained from 1.00, 1.50, 2.00, and 2.50 g/mL of LEV aqueous solution are shown in Figure 5a. The results show that no new diffraction peaks are observed in the XRPD patterns, which indicates that new solid forms were not produced in different DOI 10.1002/jps.24628
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Figure 4. XRPD patterns of the LEV crystals obtained from different solvents: (a) crystallization by slow solvent evaporation at 25°C (the curves from bottom to top: LEV APIs, water, methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, N,N-dimethyl formamide, chloroform, ethyl acetate, 1,4-dioxane, dimethylsulfoxide, and methyl tertbutyl ether), and (b) crystallization by cooling crystallization method from 25°C to 4°C (the curves from bottom to top: LEV APIs, water, methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, N,N-dimethyl formamide, chloroform, ethyl acetate, dimethylsulfoxide, and methyl tertbutyl ether).
Figure 5. Effect of LEV concentration in water solution on crystallization and solid form transformation at different temperatures (the LEV concentration of curves from bottom to top at 4 °C: 1.00, 1.50, 2.00, and 2.50 g/mL, and at 30°C, 50°C, 70°C, and 90°C: 0.010, 0.025, 0.250, 0.50, 1.00, 1.50, 2.00, and 2.50 g/mL). (a) 4°C, (b) 30°C, (c) 50°C, (d) 70°C, and (e) 90°C.
concentrations of LEV aqueous solution at 4°C. Therefore, the LEV concentration in water has no effect on the polymorphism of LEV at 4°C. The XRPD patterns of the crystals obtained from 0.010, 0.025, 0.250, 0.50, 1.00, 1.50, 2.00, and 2.50 g/mL of LEV aqueous solution at 25°C, 50°C, 75°C, and 90°C are shown in Figures 5b, 5c, 5d, and 5e, respectively. Similarly, any new diffraction peaks are not observed in all XRPD patterns. This phenomenon shows that new solid forms of LEV were not DOI 10.1002/jps.24628
produced at these aqueous solution conditions. Thus, the LEV watery solution concentration and crystallization temperature have no influence on the crystallization and polymorphism of LEV. Effect of Humidity on Solid-State LEV Further attempts with solution crystallization did not result in any new LEV solid forms. Hence, other techniques such as Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
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Figure 6. XRPD patterns of LEV solid forms: (a) effect of humidity on crystal transformation (the curves from bottom to top: 0−30 d, detected once every 5 d for 0–30 d). Condition: humidity, 75%; temperature, 50°C; (b) the crystal obtained from different concentrations of LEV aqueous solution via freeze drying (the LEV concentration of curves from bottom to top: 0.01, 0.05, 0.50, 1.00, 1.50, and 2.00 g/mL); (c) effect of milling on crystal transformation at various milling times (the detection time point of curves from bottom to top: 0, 0.5, 1.0, 1.5, and 2.0 h); (d) LEV solid forms obtained via melt cooling and quench cooling.
humidity, melt cooling, freeze-dryer, quench cooling, sublimation, as well as milling experiments were performed to investigate if LEV polymorphism occurs. The effect of humidity on LEV was investigated at conditions of 50°C and 75% relative humidity. The XRPD data were obtained for all the solid forms at 5 days intervals for up to 1 month. The LEV was stable for one month, as confirmed by XRPD results because no change in diffraction peak position was observed. The XRPD patterns of the effect of humidity on LEV form are shown in Figure 6a. Effect of Freeze-Drying on LEV Solid Form Amorphous and metastable forms could be generated by freezedrying. Thus, the freeze-drying technique was performed to investigate whether or not LEV exist the phenomenon of amorphous form and metastable form. Figure 7 shows the photomicrographs of the freezedried LEV product obtained from LEV aqueous solution with different concentrations. As shown in Figures 7b–7f
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(corresponding LEV concentrations: 0.05, 0.50, 1.00, 1.50, and 2.00 g/mL, respectively), the results illustrate that the particle size of LEV decreased with increasing LEV concentration. This phenomenon is probably due to the acceleration of LEV nucleation rate and the increase in crystallization rate caused by high LEV concentrations, resulting in the decrease of LEV particle size at high concentration. However, the particle size obtained from 0.01 g/mL of LEV aqueous solution was not the maximum (Fig. 7a). The LEV aqueous solution with low concentration does not contribute to the formation of large LEV particle sizes because the spacing of LEV molecules is high in the freeze-dried samples. As shown in Figure 6b, the XRPD patterns of the solid phase of LEV did not change with increasing LEV concentration during the freeze-drying experiments. The results indicate that no amorphous form and other solid forms were produced during the freeze-drying process of the LEV aqueous solution. Therefore, the LEV solid form is very stable during the freeze-drying process and is not influenced by the LEV concentration in solution.
DOI 10.1002/jps.24628
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Figure 7. Photomicrographs of the crystal obtained from different concentrations of LEV aqueous solution via freeze-drying (photomicrographs were magnified 4×). (a) 0.01, (b) 0.05, (c) 0.50, (d) 1.00, (e) 1.50, and (f) 2.00 g/mL.
Effect of Milling on LEV Solid Form
Stability Study of LEV Solid Phase
To understand further the mechanically activated polymorph conversion, milling experiments were performed on LEV at room temperature for different periods up to 3 h. Approximately 2 g of LEV was milled using a planetary mill, and the change in milling was monitored via XRPD. The XRPD results are shown in Figure 6c. The diffraction peak positions of the samples did not change with prolonged milling duration. This result indicates that the mechanical effect (milling) has no influence on the solid-state phase transformation of LEV, and the LEV solid phase is very stable in this process.
Solution crystallization, humidity, melt cooling, freeze-dryer, quench cooling, sublimation, and milling were applied to investigate the crystallization and polymorphism of LEV in detail. To the best of our knowledge, several techniques, including melt cooling, quench cooling, and milling, can convert solid drugs to amorphous and metastable forms.25,36,49,50 However, the results reveal that no new solid forms were formed when various methods of solid-state transformation were performed. Thus, only one solid form of LEV was obtained via various crystal
LEV Crystallization via Melt Cooling, Quench Cooling, and Sublimation The crystallization of LEV by melt cooling was performed at 4°C and 25°C. The XRPD patterns are displayed in Figure 6d, which indicates that these solid forms of LEV have similar diffraction peak positions with the LEV APIs. Therefore, these LEV solid forms possess the same spatial arrangements as the original solid form of LEV (APIs). Moreover, the transformation rate from molten LEV to its crystal state is very fast, and this procedure only took 2 min to complete without the formation of any meta-stable state at 25°C. The same result was obtained from the XRPD patterns of quench cooling. Thus, the solid form of LEV APIs is very stable at quench cooling condition. To investigate if LEV solid phase transformation will take place via vapor phase sublimation, experiments were carried out using a sublimation unit after the LEV was melted and the temperature was maintained at 125°C for 30 min. Spindly, needle-like, and transparent crystals were produced (shown in Fig. 8). The crystal morphology obtained via sublimation is different from those obtained via other methods. However, the XRPD pattern confirmed that the collected sublimed crystals were similar to the original LEV solid form. Thus, a new solid form of LEV was not produced via sublimation. DOI 10.1002/jps.24628
Figure 8. XRPD patterns and morphologies of the LEV crystals via sublimation (left: photo, and the scale bar in the bottom right corner of photo represents 0.5 cm; right: photomicrograph, and this needle-like crystals was magnified 4×). Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
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techniques and crystal transformation methods. This LEV solid form is the most stable form.
CONCLUSIONS The crystallization of LEV in different solvents was systematically investigated at different temperatures, and various crystal habits of LEV were observed. Meanwhile, the crystals produced in different solvents were characterized by XRPD and morphological analysis. Moreover, the effects of LEV concentration and crystallization temperature in aqueous solution on the crystallization of LEV were also investigated in detail, and no new solid form of LEV was observed in LEV watery solution with different concentrations at different temperatures. In addition, humidity, melt cooling, freeze-drying, quench cooling, sublimation, and milling did not induce a solid-state conversion of LEV. Therefore, the LEV solid form is very stable under various extreme conditions.
ACKNOWLEDGMENTS This work was supported by the Applied Basic Research Project of Sichuan Province (Grant No. 2014JY0042), the Testing Platform Construction of Technology Achievement Transform of Sichuan Province (Grant No. 13CGPT0049), and the National Development and Reform Commission and Education of China (Grant No. 2014BW011). We thank the College of Polymer Science and Engineering, Sichuan University, for providing instrumentation and infrastructure for the thermal analysis.
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An Investigation into the Polymorphism and Crystallization of Levetiracetam and the Stability of its Solid Form KAILIN XU, XINNUO XIONG, LIUQI GUO, LILI WANG, SHANSHAN LI, PEIXIAO TANG, JIN YAN, DI WU, HUI LI College of Chemical Engineering, Sichuan University, Chengdu 610065, China Received 9 June 2015; revised 21 July 2015; accepted 11 August 2015 Published online 1 September 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24628 ABSTRACT: Levetiracetam (LEV) crystals were prepared using different solvents at different temperatures. The LEV crystals were systematically characterized by X-ray powder diffraction (XRPD) and morphological analysis. The results indicated that many kinds of crystal habits exist in a solid form of LEV. To investigate the effects of LEV concentration, crystallization temperature, and crystallization type on crystallization and solid phase transformation of LEV, multiple methods were performed for LEV aqueous solution to determine if a new solid form exists in solid-state LEV. However, XRPD data demonstrate that the LEV solid forms possess same spatial arrangements that are similar to the original solid form. This result indicates that the LEV concentration, crystallization temperature, and crystallization type in aqueous solution have no influence on the crystallization and solid phase transformation of LEV. Moreover, crystallization by sublimation, melt cooling, and quench cooling, as well as mechanical effect, did not result in the formation of new LEV solid state. During melt cooling, the transformation of solid form LEV is a direct process from melting amorphous phase to the original LEV crystal phase, and the conversion C 2015 Wiley rate is very quick. In addition, stability investigation manifested that LEV solid state is very stable under various conditions. Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:4123–4131, 2015 Keywords: levetiracetam; crystals; morphology; X-ray powder diffractometry; solid state; stability
INTRODUCTION The solid phase diversity of active pharmaceutical ingredients (APIs), including pharmaceutical polymorphs,1–4 pseudo polymorphs (solvates and hydrates),5,6 salts,7,8 co-crystals,9,10 and amorphous solids,11–15 has become one of the most investigated areas in contemporary drug research because of the unique physicochemical properties, efficacy, and toxic side effects existing in different solid forms of drugs.16–18 Pharmaceutical solid form screening has rapidly developed into a general approach to improve the physicochemical properties of drugs, which can remarkably affect the solubility, bioavailability, hygroscopicity, melting point, stability, compressibility, and other performance characteristics of pharmaceutical products.2,17,19–21 A large number of methods can be applied to produce polymorphs of APIs, such as high-temperature-triggered transformation,16,22 cooling crystallization,23–25 pressure-induced transformation,22 milling,26,27 humidity,28 and solution crystallization by changing the crystallization temperature, solution concentration, cooling rate, solvent types, pH, and additive.19,29–34 Studies aimed at obtaining solid forms of relevant drug are greatly essential and meaningful to develop optimal drug crystals and to control the quality of the final state of the drug.35,36 In general, the stable polymorph should be used in the marketed formulation to prevent polymorphic transformation during manufacturing, delivery, or storage. Therefore, to examine the stabilities of crystal forms, investigating the polymorph transformation and understanding the process of solid-state transformation at different conditions are highly important. Levetiracetam (LEV), whose chemical name is (S)-2-(2oxopyrrolidin-1-yl) butanamide, is an ethyl analog of the Correspondence to: Hui Li (Telephone: +86-028-85405149; Fax: +86-02885401207; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 104, 4123–4131 (2015) C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
nootropic and dementia drug piracetam, and is used as an anticonvulsant in the treatment of epilepsy.37–39 Compared with other antiepileptic drugs, LEV has a unique mechanism for the treatment of epilepsy, and it has satisfactory pharmacokinetic characteristics and efficiency with minimal side effects.40 Moreover, LEV could prevent the progression to Alzheimer’s disease by reducing hippocampal hyperactivity in patients with cognitive impairment.41 Polymorphism and the solid state of drugs can have a significant impact on the clinical efficacy, toxic side effects, and quality of medicines. Thus, studies on polymorphism, polymorph transformation, and stability of solid state of LEV are very significant. The racemic compound of LEV, etiracetam, was reported to have three different solid-state phases, including two polymorphs and one hydrate phase.38 Moreover, the features of LEV molecular structure, including five freely rotatable bonds, four hydrogen acceptors, and two strong Hbond donor, make it very attractive in investigating whether chemically identical yet conformationally flexible LEV molecule exist in multiple crystal forms and conversion between poly˚ morphism. The crystal structure of LEV [a = 9.199 A, ˚ c = 6.272 A, ˚ $ = 108.65°, unit-cell volume V = b = 7.993 A, ˚ 3 , Z = 2, and space group P21 ] has been reported,42 436.962 A and systematic studies on LEV co-crystal were reported in several literatures.43–45 Two patents46,47 claim four polymorphs of LEV, as well as the corresponding preparation methods. But in our primary experiments, it was found that only one solid form of LEV was obtained after performing the same preparation methods multiple times. This work aims to search for various solid forms of LEV, and to investigate the stability of solid forms of LEV by changing the crystallization condition and mechanisms of solid-state transformation. The methods of potentially obtaining LEV polymorphs, including changing solvent type, crystallization temperature, LEV concentration in solution, humidity, physical effects, crystallization types, high temperature effects, and
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so on, were systematically investigated. The solid forms of LEV were prepared and characterized by X-ray powder diffraction (XRPD), and morphological analysis. Moreover, the effects of LEV concentration, crystallization temperature, and crystallization type in water on the formation of LEV polymorphs were also investigated in detail. The stability of LEV solid state was discussed under various conditions.
MATERIALS AND METHODS Materials The title compound, LEV (99% purity), in powder form was obtained from Zhejiang Jingxin Pharmaceutical Company, Ltd. (Shaoxing, China). Ultrapure water (18 M resistivity from a Millipore system; Massachusetts, USA) was used throughout the experiment. All analytical grade solvents were purchased from Kelong Company, Ltd. (Chengdu, China), and used without further purification. Methods Preparation of LEV Crystals In the solvent evaporation method, the LEV crystals were obtained by slow solvent evaporation and crystallization from different solvents of LEV at 25°C. Most of these crystals easily crystallized from a number of organic solvents, such as ethanol, methanol, 2-propanol, dimethylsulfoxide, acetone, acetonitrile, N, N-dimethyl formamide, chloroform, 1, 4-dioxane, methyl tertbutyl ether, ethyl acetate, and tetrahydrofuran. In the cooling crystallization method, the LEV crystals were crystallized and obtained from the LEV saturated solution of different solvents (25°C) by slowly decreasing the temperature from 25°C to 4°C. Crystallization Experiments in Water To investigate the effects of LEV concentration and crystallization temperature in water on the crystallization of LEV, the different concentrations of LEV aqueous solution from 0.01 to 2.5 g/mL was prepared. The samples were then placed in a thermotank to obtain crystals via slow solvent evaporation and crystallization at 4°C, 25°C, 50°C, 75°C, and 90°C. The obtained LEV crystals were then immediately analyzed by XRPD detection to avoid the effects caused by other factors.
periods (typically 2 min) were applied to limit the mechanical heating of the sample. The samples were milled for 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h to evaluate the effect of milling on the LEV. The milled samples were then immediately analyzed after the end of the milling process. Crystallization by Sublimation, Melt Cooling, and Quench Cooling Sublimation experiment was performed using a sublimation unit after the LEV was melted and the temperature was maintained at 125°C for 30 min. The crystals were then collected and detected as soon as possible. To investigate the effect of melt cooling on the solid-state transition of LEV, the LEV APIs samples were placed in an oven and subjected to melting at 125°C. The melted samples were cooled at 25°C and then to 4°C to obtain solid-state LEV. The samples were immediately monitored via XRPD to determine if the crystal phase was changed in this process. Quench cooling is a process in which the drug is first heated and melted at its melting point and then cooled at −20°C. In this study, 1 g of LEV was placed in a crucible and heated up to 125°C. The melted product was immediately covered with aluminum foil and kept at −20°C in a deep freezer for 24 h. The quench-cooled samples were analyzed as soon as possible. Analytical Techniques Single-Crystal X-Ray Diffraction Single-crystal data were collected using an Oxford Diffraction ˚ Xcalibur Nova system with Mo K" radiation (8 = 0.71073 A) at room temperature, and the 2 from 3.13° to 26.37°. The program package Olex2–1.1 (SHELX-97) was used for structure solution and refinement. Simulated XRPD patterns based on the single-crystal X-ray diffraction (SXRD) data were obtained using Mercury 2.4 software. Optical Microscopy The LEV crystals were placed in a transparent glass holder. The shape and surface morphology were recorded by an optical microscope (CX21; Olympus; Tokyo, Japan) equipped with a digital CCD camera and a thermostatic stage system (TS62, Instec; USA).
Freeze-Drying Experiments Different concentrations of LEV in aqueous solution (0.01, 0.05, 0.50, 1.00, 1.50, and 2.00 g/mL) were frozen and freeze-dried for 48 h (FD-1A-50; Biocool Instruments Company, Ltd., Beijing, China). The freeze-dried LEV was used for shape and surface morphology analysis and XRPD detection as soon as possible after drying. Milling Experiments Milling experiments were performed using a planetary mill (LNMN-QM 0.4L; Heishan Xinlitun Agate Handicrafts Company, Ltd.; Jinzhou, China) at room temperature. Four agate ball milling jars of 50 cm3 with 10 balls (ø = 10 mm) of the same material were used. Each milling was performed with 2 g of the powder to ensure homogeneous milling and reproducible results. The rotation speed of the solar disk was set to 400 rpm, and alternate milling periods (typically 10 min) with pause Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
Figure 1. XRPD pattern of LEV (red represents experimental pattern, and black belongs to the simulated pattern from LEV singlecrystal structure). DOI 10.1002/jps.24628
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Figure 2. Photos or photomicrograph of LEV crystals obtained from different solvents by slow solvent evaporation at 25°C (the scale bar in the bottom right corner of the photos represents 0.5 cm, and the photomicrograph was magnified 4×). (a) Water, (b) methanol, (c) ethanol, (d) isopropanol, (e) acetone, (f) acetonitrile, (g) tetrahydrofuran, (h) N,N-dimethyl formamide, (i) chloroform, (j) ethyl acetate, (k) 1,4-dioxane, (l) dimethylsulfoxide, (m) methyl tertbutyl ether, and (n) photomicrograph of the crystal obtained from methyl tertbutyl ether.
XRPD X-ray powder diffraction data were collected at room temperature using an X’Pert PRO diffractometer (PANalytical; Almelo, Netherlands) with a PIXcel 1D detector and Cu K" radiation ˚ generator setting: 40 kV and 40 mA). The (8 = 1.54056 A, diffraction data were collected in the 22 range from 5° to 50° with a step size of 0.01313° and a counting time of 30 ms/step.
RESULTS AND DISCUSSION SXRD Analysis The LEV crystal used for SXRD detection was obtained from methanol, and the SXRD analytical results are similar to the reported data.42 Namely, the LEV crystal produced in methanol has a monoclinic structure with space group P21 and unit-cell ˚ b = 8.006(3) A, ˚ c = 6.285(2) A, ˚ $= parameters: a = 9.211(3) A, ˚ 3 , and Z = 2. 108.371(5)°, unit-cell volume V = 439.8(3) A XRPD Analysis The experiment and simulated XRPD patterns of the LEV crystal were obtained and are illustrated in Figure 1. The DOI 10.1002/jps.24628
diffraction peak positions of the crystal obtained from the supplier and those simulated by the Mercury 2.4 software were similar, as shown in the XRPD results. The result indicated that the spatial arrangement of the LEV from methanol is similar to that of the LEV from the supplier. From the XRPD patterns, the LEV crystal has major peaks at 22 values of 10.10°, 14.96°, 18.52°, 20.48°, 22.17°, 23.29°, 23.84°, 24.43°, 26.77°, 28.86°, 30.30°, and 31.97°. Effect of Solvent Type and Crystallization Temperature on Solid-State LEV Crystallization can cause either changes in internal structure, leading to polymorphism and solvation, or, more frequently, changes in external properties, such as crystal habit, elongation ratio, mean particle size, and so on.48 Figure 2 shows the shape and surface morphology of LEV crystals obtained by slow solvent evaporation and crystallization from different solvents at 25°C. Most of the crystals have a prismatic and transparent crystal structure, including the crystals produced from methanol, isopropanol, acetonitrile, N, N-dimethyl formamide, chloroform, and ethyl acetate. However, particular crystals display isodimensional characteristics, and the crystals will grow Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
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Figure 3. Photos or photomicrograph of LEV crystals obtained from the LEV saturated solution of different solvents (25°C) by slowly decreasing the temperature from 25°C to 4°C. (The scale bar in the bottom right corner of the photos represents 0.5 cm, and the photomicrograph was magnified 4×). (a) Water, (b) methanol, (c) ethanol, (d) isopropanol, (e) acetone, (f) acetonitrile, (g) tetrahydrofuran, (h) N,N-dimethyl formamide, (i) chloroform, (j) ethyl acetate, (k) dimethylsulfoxide, and (l) photomicrograph of the crystal obtained from methyl tertbutyl ether.
around a central point to produce an agglomerate of tabular and prismatic crystals with a larger radius, such as crystals obtained from water, ethanol, 1,4-dioxane, and dimethylsulfoxide. The crystal produced from methyl tertbutyl ether is significantly smaller than the others probably because the solubility of LEV in methyl tertbutyl ether solution is obviously lower than in other solvents. The shape and surface morphology of LEV crystals obtained via cooling crystallization method is displayed in Figure 3. The result indicates that most of crystals also have a prismatic and transparent crystal structure, which is similar to the structure of the crystals obtained via slow solvent evaporation method at 25°C. However, the characteristic of the crystals produced from tetrahydrofuran and ethyl acetate is significantly different from the rest, where prismatic crystals usually agglomerate, as shown in Figures 3g and 3j. In addition, the crystals from dimethylsulfoxide are isodimensional with an irregular shape and have a tendency to form particle agglomerates. Meanwhile, the small transparent crystal particles from methyl tertbutyl ether have a prismatic structure. The solid form of LEV crystals obtained from different conditions should be identified as accurately as possible. The XRPD Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
patterns of the LEV solid forms were obtained and are illustrated in Figure 4. The diffraction peak positions of the crystals purchased from the supplier and those obtained from different solvents at different conditions were similar, as shown in the XRPD results. The result indicated that these LEV solid forms possess similar spatial arrangements. These LEV solid forms all have major peaks at 22values of 10.10°, 14.96°, 18.52°, 20.48°, 22.17°, 23.29°, 23.84°, 24.43°, 26.77°, 28.86°, 30.30°, and 31.97°. LEV Crystallization in Water The effects of LEV concentration and crystallization temperature in water on the crystallization of LEV are displayed in Figure 5. The crystals in 0.010, 0.025, 0.250, and 0.50 g/mL of LEV aqueous solution were not produced at 4°C because of the low concentration of LEV, and the XRPD patterns of the crystals obtained from 1.00, 1.50, 2.00, and 2.50 g/mL of LEV aqueous solution are shown in Figure 5a. The results show that no new diffraction peaks are observed in the XRPD patterns, which indicates that new solid forms were not produced in different DOI 10.1002/jps.24628
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Figure 4. XRPD patterns of the LEV crystals obtained from different solvents: (a) crystallization by slow solvent evaporation at 25°C (the curves from bottom to top: LEV APIs, water, methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, N,N-dimethyl formamide, chloroform, ethyl acetate, 1,4-dioxane, dimethylsulfoxide, and methyl tertbutyl ether), and (b) crystallization by cooling crystallization method from 25°C to 4°C (the curves from bottom to top: LEV APIs, water, methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, N,N-dimethyl formamide, chloroform, ethyl acetate, dimethylsulfoxide, and methyl tertbutyl ether).
Figure 5. Effect of LEV concentration in water solution on crystallization and solid form transformation at different temperatures (the LEV concentration of curves from bottom to top at 4 °C: 1.00, 1.50, 2.00, and 2.50 g/mL, and at 30°C, 50°C, 70°C, and 90°C: 0.010, 0.025, 0.250, 0.50, 1.00, 1.50, 2.00, and 2.50 g/mL). (a) 4°C, (b) 30°C, (c) 50°C, (d) 70°C, and (e) 90°C.
concentrations of LEV aqueous solution at 4°C. Therefore, the LEV concentration in water has no effect on the polymorphism of LEV at 4°C. The XRPD patterns of the crystals obtained from 0.010, 0.025, 0.250, 0.50, 1.00, 1.50, 2.00, and 2.50 g/mL of LEV aqueous solution at 25°C, 50°C, 75°C, and 90°C are shown in Figures 5b, 5c, 5d, and 5e, respectively. Similarly, any new diffraction peaks are not observed in all XRPD patterns. This phenomenon shows that new solid forms of LEV were not DOI 10.1002/jps.24628
produced at these aqueous solution conditions. Thus, the LEV watery solution concentration and crystallization temperature have no influence on the crystallization and polymorphism of LEV. Effect of Humidity on Solid-State LEV Further attempts with solution crystallization did not result in any new LEV solid forms. Hence, other techniques such as Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
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Figure 6. XRPD patterns of LEV solid forms: (a) effect of humidity on crystal transformation (the curves from bottom to top: 0−30 d, detected once every 5 d for 0–30 d). Condition: humidity, 75%; temperature, 50°C; (b) the crystal obtained from different concentrations of LEV aqueous solution via freeze drying (the LEV concentration of curves from bottom to top: 0.01, 0.05, 0.50, 1.00, 1.50, and 2.00 g/mL); (c) effect of milling on crystal transformation at various milling times (the detection time point of curves from bottom to top: 0, 0.5, 1.0, 1.5, and 2.0 h); (d) LEV solid forms obtained via melt cooling and quench cooling.
humidity, melt cooling, freeze-dryer, quench cooling, sublimation, as well as milling experiments were performed to investigate if LEV polymorphism occurs. The effect of humidity on LEV was investigated at conditions of 50°C and 75% relative humidity. The XRPD data were obtained for all the solid forms at 5 days intervals for up to 1 month. The LEV was stable for one month, as confirmed by XRPD results because no change in diffraction peak position was observed. The XRPD patterns of the effect of humidity on LEV form are shown in Figure 6a. Effect of Freeze-Drying on LEV Solid Form Amorphous and metastable forms could be generated by freezedrying. Thus, the freeze-drying technique was performed to investigate whether or not LEV exist the phenomenon of amorphous form and metastable form. Figure 7 shows the photomicrographs of the freezedried LEV product obtained from LEV aqueous solution with different concentrations. As shown in Figures 7b–7f
Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
(corresponding LEV concentrations: 0.05, 0.50, 1.00, 1.50, and 2.00 g/mL, respectively), the results illustrate that the particle size of LEV decreased with increasing LEV concentration. This phenomenon is probably due to the acceleration of LEV nucleation rate and the increase in crystallization rate caused by high LEV concentrations, resulting in the decrease of LEV particle size at high concentration. However, the particle size obtained from 0.01 g/mL of LEV aqueous solution was not the maximum (Fig. 7a). The LEV aqueous solution with low concentration does not contribute to the formation of large LEV particle sizes because the spacing of LEV molecules is high in the freeze-dried samples. As shown in Figure 6b, the XRPD patterns of the solid phase of LEV did not change with increasing LEV concentration during the freeze-drying experiments. The results indicate that no amorphous form and other solid forms were produced during the freeze-drying process of the LEV aqueous solution. Therefore, the LEV solid form is very stable during the freeze-drying process and is not influenced by the LEV concentration in solution.
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Figure 7. Photomicrographs of the crystal obtained from different concentrations of LEV aqueous solution via freeze-drying (photomicrographs were magnified 4×). (a) 0.01, (b) 0.05, (c) 0.50, (d) 1.00, (e) 1.50, and (f) 2.00 g/mL.
Effect of Milling on LEV Solid Form
Stability Study of LEV Solid Phase
To understand further the mechanically activated polymorph conversion, milling experiments were performed on LEV at room temperature for different periods up to 3 h. Approximately 2 g of LEV was milled using a planetary mill, and the change in milling was monitored via XRPD. The XRPD results are shown in Figure 6c. The diffraction peak positions of the samples did not change with prolonged milling duration. This result indicates that the mechanical effect (milling) has no influence on the solid-state phase transformation of LEV, and the LEV solid phase is very stable in this process.
Solution crystallization, humidity, melt cooling, freeze-dryer, quench cooling, sublimation, and milling were applied to investigate the crystallization and polymorphism of LEV in detail. To the best of our knowledge, several techniques, including melt cooling, quench cooling, and milling, can convert solid drugs to amorphous and metastable forms.25,36,49,50 However, the results reveal that no new solid forms were formed when various methods of solid-state transformation were performed. Thus, only one solid form of LEV was obtained via various crystal
LEV Crystallization via Melt Cooling, Quench Cooling, and Sublimation The crystallization of LEV by melt cooling was performed at 4°C and 25°C. The XRPD patterns are displayed in Figure 6d, which indicates that these solid forms of LEV have similar diffraction peak positions with the LEV APIs. Therefore, these LEV solid forms possess the same spatial arrangements as the original solid form of LEV (APIs). Moreover, the transformation rate from molten LEV to its crystal state is very fast, and this procedure only took 2 min to complete without the formation of any meta-stable state at 25°C. The same result was obtained from the XRPD patterns of quench cooling. Thus, the solid form of LEV APIs is very stable at quench cooling condition. To investigate if LEV solid phase transformation will take place via vapor phase sublimation, experiments were carried out using a sublimation unit after the LEV was melted and the temperature was maintained at 125°C for 30 min. Spindly, needle-like, and transparent crystals were produced (shown in Fig. 8). The crystal morphology obtained via sublimation is different from those obtained via other methods. However, the XRPD pattern confirmed that the collected sublimed crystals were similar to the original LEV solid form. Thus, a new solid form of LEV was not produced via sublimation. DOI 10.1002/jps.24628
Figure 8. XRPD patterns and morphologies of the LEV crystals via sublimation (left: photo, and the scale bar in the bottom right corner of photo represents 0.5 cm; right: photomicrograph, and this needle-like crystals was magnified 4×). Xu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:4123–4131, 2015
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techniques and crystal transformation methods. This LEV solid form is the most stable form.
CONCLUSIONS The crystallization of LEV in different solvents was systematically investigated at different temperatures, and various crystal habits of LEV were observed. Meanwhile, the crystals produced in different solvents were characterized by XRPD and morphological analysis. Moreover, the effects of LEV concentration and crystallization temperature in aqueous solution on the crystallization of LEV were also investigated in detail, and no new solid form of LEV was observed in LEV watery solution with different concentrations at different temperatures. In addition, humidity, melt cooling, freeze-drying, quench cooling, sublimation, and milling did not induce a solid-state conversion of LEV. Therefore, the LEV solid form is very stable under various extreme conditions.
ACKNOWLEDGMENTS This work was supported by the Applied Basic Research Project of Sichuan Province (Grant No. 2014JY0042), the Testing Platform Construction of Technology Achievement Transform of Sichuan Province (Grant No. 13CGPT0049), and the National Development and Reform Commission and Education of China (Grant No. 2014BW011). We thank the College of Polymer Science and Engineering, Sichuan University, for providing instrumentation and infrastructure for the thermal analysis.
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