Influence of Impurities on the Solution-Mediated Phase Transformation of an Active Pharmaceutical Ingredient Takashi Mukuta,†,‡ Alfred Y. Lee,‡ Takeshi Kawakami,† and Allan S. Myerson*,‡ Process Chemistry Labs, Astellas Pharma Inc., 160-2, Akahama, Takahagi-shi, Ibaraki, 318-0001 Japan, and Department of Chemical and Environmental Engineering, Illinois Institute of Technology, Chicago, Illinois 60616
CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 4 1429-1436
Received October 14, 2004
ABSTRACT: The solution-mediated phase transformation of the metastable A form of an active pharmaceutical ingredient (1) to the stable B form is investigated in 2-propanol. The transformation behavior (or rate) is quantified using powder X-ray diffraction. The studies show that the rate of transformation is sensitive to the tailor-made impurities and that the presence of certain inhibitors reduces the rate of transformation. Concurrently molecular modeling studies are undertaken to investigate the incorporation of these structurally related impurities into the crystal lattice, and it is observed that the build-in approach used in morphology predictions for additive-host systems can be applied to evaluate the extent of impurity incorporation. The build-in approach employs the attachment energy method in which the host molecules are substituted by impurity molecules, and the relative incorporation energies are calculated for various crystal faces. The order of the relative incorporation energies of the structurally similar impurities is identical to the order of the percentages of the amount of impurities incorporated into the crystal lattice as determined by high performance liquid chromatography (HPLC). Introduction Polymorphism is the ability of a chemical entity to exist in more than one distinct crystalline form as a result of differences in the packing arrangement and/ or molecular conformation.1 This phenomenon is often observed in organic molecular crystals2 and is of paramount importance in the pharmaceutical industry where different solid forms of the same chemical compound can exhibit different physical and chemical properties as well as different solubility and dissolution, which in turn affects the bioavailability and stability of the drug substance. Pharmaceutical manufacturers are required by the Food and Drug Administration to consistently produce the desired polymorph of a drug.3-5 Discovery and characterization of polymorphs are crucial in the early stages of the development of the drug product, as unanticipated appearance or disappearance6 of a polymorph can impact the time to market for a drug, or in the case of ritonavir7,8 it can result in a withdrawal of a commercial pharmaceutical product. As a result, polymorph screening, in which a compound is crystallized in various process conditions under a variety of crystallization methods (e.g., sublimation, crystallization from the melt, vapor diffusion, thermal treatment, and crystallization from a single solvent or combinations of solvents), has been particularly important.9 More recently, high-throughput crystallization screens have been developed using a combinatorial approach to capture crystal form diversity.10-14 This approach enables a more comprehensive exploration of solid forms and has been applied to various highly polymorphic pharmaceutical compounds such as acetaminophen,15 MK-996,16 ritonavir,17 and sertraline HCl.16,18 It is important to identify the most stable polymorph as well as to fully understand and control the conditions * To whom correspondence should be addressed. Phone: 312-5673163. Fax: 312-567-7018. E-mail:
[email protected]. † Astellas Pharma Inc. ‡ Illinois Institute of Technology.
to obtain the desired solid form. Numerous methods and strategies have been used to control polymorphism, including capillary crystallization,19-21 laser-induced nucleation,22,23 solvent-drop grinding,24 spray drying,25 supercritical fluid crystallization,26 self-assembled monolayers,27,28 surfaces of metastable crystal forms,29 nanoporous polymer monoliths and glass matrixes,30 polymer heteronuclei,31 and organic single-crystal surfaces that direct the selectivity of polymorphs through epitaxial matching.32,33 In addition, designer additives have been shown to inhibit the formation of one polymorph, in turn promoting the crystallization of another polymorph.34,35 These additives have also been exploited to engineer crystal morphology36,37 and kinetically resolve chiral molecules.38,39 Similarly, impurities and synthesis byproducts can influence the nucleation and growth of polymorphs as can be seen in the case of terephthalic acid where an impurity induced twinning and inhibited a solid-state transformation, leading to the stabilization of the metastable form.40 Recent works have utilized additives or impurities in manipulating the polymorphic outcome.41-45 Structurally related additives or impurities may be incorporated into the host crystal lattice as crystal faces are sometimes unable to discriminate between the host and the additive/impurity molecule.46 This can lead to severe consequences as incorporated impurities can alter the physical and chemical properties of the crystals and quite possibly have toxicological effects. Thus, control and minimization of the impurity content in pharmaceutical products are of utmost importance. Molecular modeling techniques employing the attachment energy method have shown that impurity-modified crystal habit can be successfully predicted47-52 and that relative incorporation energies can be used as an indicator for the likelihood of impurity incorporation on crystal surfaces.51,52 In most cases, the crystallization of polymorphs often obeys Ostwald’s Law of Stages53 where the kinetically
10.1021/cg049646j CCC: $30.25 © 2005 American Chemical Society Published on Web 05/18/2005
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Figure 1. Molecular structures of compound 1 and the structurally related impurities.
metastable form initially appears followed by its transformation to the more stable polymorph. Understanding the different factors (e.g., solvent, temperature, and agitation rate) that affect the conversion between the forms is essential as these variables can facilitate or impede the transformation rate. In addition, impurities in the process can also impact the transformation behavior as evident in studies in which additives can stabilize the kinetic crystal form.40-45 In this work, the influence of four structurally related impurities on the solution-mediated phase transformation of an active pharmaceutical ingredient (1) is investigated. The polymorphic transformation is monitored using powder X-ray diffraction, and the extent of impurity incorporation is determined by high performance liquid chromotography (HPLC) and compared to relative incorporation energies derived for measuring the compatibility of impurity with the host crystal lattice. The four structurally related compounds were chemically synthesized and chosen as model inhibitors. Experimental Section Materials. Cyclohexane and 2-propanol were obtained from Pharmco Products (Brookfield, CT). Form B of compound 1 was supplied from Astellas Pharma Inc. and used without further purification. The A form was prepared from cyclohexane at reflux temperature. Form C was crystallized in a mixture of ethyl acetate and n-heptane. The structurally related compounds RS1, RS2, RS3, and RS4 as shown in Figure 1 were synthesized and have been identified by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Solubility and Dissolution. The solubility of each polymorph of compound 1 was measured in the temperature range of 0 to 40 °C and determined by high performance liquid chromatography (HPLC) using a Shimadzu HPLC system (YMC-GEL ODS column, injection volume was 5 µL, flow rate was 1.0 mL/min, UV detector was set at 210 nm and the mobile phase consisted of 40% acetonitrile/60% phosphoric acid buffer pH 5.0). The experimental setup consisted of a 50 mL glass vessel equipped with an agitator where 200 mg of compound 1 was suspended in 10 mL of 2-propanol at a desired temper-
Mukuta et al. ature controlled by a thermostat. After 30 min, the solution was filtered and the concentration of compound 1 in the supernatant was determined by HPLC. The residual crystal of compound 1 on the filter was dried, and powder X-ray diffraction was performed to ensure that the polymorph transformation did not occur during solubility measurement. Also, the stable polymorph concentration after the solutionmediated phase transformation was determined by HPLC in the absence and the presence of the impurities. To measure the amount of impurities incorporated in compound 1, the crystals were dissolved in the mobile phase. After dissolution, the amount of impurities in the effluent or the degree of impurity incorporation within the crystal lattice was determined by HPLC analysis. Crystallization Studies. The solution-mediated phase transformation of the metastable A form of compound 1 to the stable B form was carried out at 30 °C. Experiments were performed in a 100 mL three-neck flask equipped with a mechanical stirrer (stirring speed of 300 rpm). The temperature was chosen in to avoid the appearance of form C being mixed with form B since form C was more stable below 20 °C despite having a long transformation time from form B to form C. Different concentrations of impurities RS1, RS2, RS3, and RS4 were mixed with form A and added into 100 mL of 2-propanol. Samples were removed at desired time intervals; the solid phase was immediately filtered under reduced pressure, and the physically adsorbed solvent was removed by drying for 15 h at 30 °C. The phase composition in the solid was examined by powder X-ray diffraction, and the level of impurities incorporated in the crystal lattice was assessed by HPLC. Also the effects of seeding of the stable B form on the rate of transformation were studied. Two different seeding levels (0.1 and 0.5 wt %) were used in the presence of the most effective inhibitor. The form B seed crystals were added to the slurry of the A form at time 0 s and at 30 °C. Scanning Electron Microscopy. The morphology of each crystalline form was observed with a scanning electron microscope (SEM, Hitachi S-800). Samples were sputter coated with gold before examination to improve conductivity, and images were acquired at an operating voltage of 5 kV. Single-Crystal X-ray Structure Determination. Data were collected using a four axis single-crystal X-ray diffractometer (Rigaku AFC-7R) at ambient temperature. Measurements were carried out under the following conditions: graphite monochromated CuKR (λ ) 0.154178 nm) radiation; voltage, 40 kV; current, 40mA; and scanning speed, 8°/min. The crystal structures for all three polymorphs were solved by direct methods with the crystallographic software package, teXsan.54 Powder X-ray Diffraction. X-ray powder diffraction was obtained with a Rigaku Miniflex diffractometer with CuKR radiation (λ ) 0.15418 nm), and measurements were carried out at a power of 30 kv and 15 mA. Samples were manually ground into fine powder in a mortar and pestle and packed on glass slides for analysis. Data were collected from 3° to 30° with a scan rate and step size of 1°/min and 0.1°, respectively. Powder X-ray diffraction was utilized to quantify the relative amounts of forms A and B present in the mixture based on the differences between the two distinct powder patterns. For calculation purposes, the area of all the peaks in the scan range (3° to 30°) of both forms were utilized. The conversion to form B or the content of form A of the sample collected with time was determined on the basis of the area ratio of the X-ray peaks of the two crystalline forms. Binary mixtures of both polymorphic forms in various ratios were prepared in a mortar and pestle. Figure 2 shows typical powder patterns for a number of standard mixtures of different compositions (0, 30, 50, 70, and 100 wt % of form A). It can be seen that there are four unique peaks in the powder pattern for form A (2θ ) 6.8°, 13.4°, 21.6°, and 24.4°) that can be differentiated from peaks of pure form B. Thus, the ratio of the area of these four characteristic peaks for the two forms was chosen for use in the construction of a calibration curve for determining quantitatively the polymorphic composition. The calibration curve
Solution-Mediated Phase Transformation
Crystal Growth & Design, Vol. 5, No. 4, 2005 1431 Table 1. Crystallographic Data for Compound 1 Polymorphs crystal system space group a (Å) b (Å) c (Å) R (Å) β (Å) γ (Å) cell volume (Å3) Z Fcalc (g/cm3) temp (K) radiation wavelength R Rw
Figure 2. Powder X-ray diffraction patterns for a number of mixtures of form A and form B.
form A
form B
form C
triclinic P1 h 10.614(2) 13.419(4) 5.123(1) 90.84(2) 95.52(2) 88.98(3) 726.1(3) 2 1.41 293.2 CuKR 1.5418 0.1016 0.1788
monoclinic P21/c 10.488(7) 4.811(1) 28.263(1) 90 91.23(2) 90 1425(1) 4 1.436 293.2 CuKR 1.5418 0.0638 0.1063
triclinic P1 9.5060(8) 14.997(1) 5.276(1) 98.00(1) 101.76(1) 103.845(8) 700.8(2) 1 1.461 293.2 CuKR 1.5418 0.083 0.1312
energy is obtained to ensure that the conformation of the impurity molecule is adjusted in such a way that it is situated at its optimum position within the host crystal lattice. In this work, the DREIDING 2.2156 force field is used for all molecular simulations, van der Waals forces are modeled with the Lennard-Jones 12-6 expression, and hydrogen bonding energy is approximated using a Lennard-Jones-like 12-10 expression. Partial atomic charges are calculated with MOPAC using a modified neglect of diatomic overlap (MNDO) Hamiltonian approximation,57 and the Ewald summation technique is utilized for the summation of long-range van der Waals and electrostatic interactions under the periodic boundary conditions. In the final step, attachment energy58 calculations are performed with the impurity species in each symmetry position and the relative incorporation energy for each crystal face is given by b b′ sl att sl′ ∆b ) Ehkl - Ehkl,i ) (Ehkl + (1/2)Ehkl ) - Ki(Ehkl,i + att′ ) (1/2)Ehkl,i
Figure 3. Calibration curve for the degree of conversion of form A to form B using powder X-ray diffraction. for the extent of phase transformation of form A to form B is shown in Figure 3. Molecular Modeling. Relative incorporation or binding energy calculations, including molecular dynamics (MD) and mechanics simulations, were carried out with the software Cerius2. The molecular modeling methodology and details of the procedures for the build-in approach have been described elsewhere.47 Briefly, the build-in approach consists of four main steps. First molecular mechanics (MM) simulations are performed using a suitable potential function to predict the crystal morphology to identify the morphologically important faces. Next, in each symmetry position of the unit cell, the host molecule is replaced by an impurity molecule. Molecular mechanics simulations are then carried out again, using the conjugate gradient method55 to minimize the energy of the impurity molecule within the host crystal lattice, in combination with MD simulations, where external forces on the molecule are applied and Newton’s equations of motions are solved to compute the new atomic positions. This sequence of MM and MD simulations is repeated until a global minimum
att where Ebhkl, Esl hkl, and Ehkl are the incorporation energy of the host molecule on the {hkl} face, the slice energy, and the attachment energy of the {hkl} face, respectively. Ki is the ratio of the lattice energy of the pure crystal to the lattice energy of the crystal with the impurity in symmetry position i. The lattice energy is calculated by summing all the atom-atom interactions between a central molecule and all the surroundb′ ing molecules in the crystal. Ehkl,i is the incorporation energy sl′ of the impurity molecule on the {hkl} face, while Ehkl,i and att′ Ehkl,i are the slice and attachment energy of the {hkl} face with the impurity in symmetry position i, respectively. Minimum change in the relative incorporation energy (i.e., low ∆b) indicates where the impurities are most likely to incorporate.48,49 The energy is a useful measure on the compatibility of the impurity with the host crystal lattice and has been successful in predicting an impurity-modified crystal morphology47-52 and thus used herein to assess the impact of the impurities on the purity of the crystals.
Results and Discussion Crystal Structures. Compound 1 exists in three distinct crystalline forms. Table 1 summarizes the crystallographic data for each polymorph. All three crystal structures are rich in hydrogen bonds and are composed of sheets where it is observed that the crystal building block or growth unit for each modification is a centrosymmetric dimer interconnected by symmetrical NsH NtC interactions. In forms A and B, compound 1 molecules are packed to form the centrosymmetric aggregate between the hydroxyl hydrogen and the
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Figure 5. Solubility of the three polymorphs of compound 1 in 2-propanol.
Figure 4. Crystal packing of compound 1 polymorphs: (a) form A viewed along the c-axis; (b) form B viewed down the b-axis; and (c) form C viewed parallel to the c-axis. Hydrogen bonds are represented by the aqua dashed lines. The circled areas indicate the growth units of each form.
trifluoromethyl fluorine atoms (Figure 4a,b). In contrast, molecules in form C are organized in which an intermolecular hydrogen bond is formed between the hydroxyl hydrogen and the carbonyl oxygen atom of a neighboring molecule (Figure 4c). In form B, the fluorine atoms of the trifluoromethyl group in compound 1 are disordered and are assigned a site occupancy factor (SOF) of 0.7/0.3 as determined from refinement. In all three crystalline structures, there are two hydrogen bond donors, the amide hydrogen (N-H) and the hydroxyl hydrogen (O-H), within the molecule, and there are two hydrogen bond acceptors: for forms A and B, the cyano nitrogen (CtN) and the trifluoromethyl fluorine (CF3), and for form C the cyano nitrogen (Ct N) and the carbonyl oxygen (CdO). In addition, these donors and acceptors are involved in intramolecular hydrogen bonds with the exception of the trifluoromethyl group. The hydrogen bonding interaction between the amide hydrogen and cyano nitrogen links the growth
unit of each polymorph. Clearly, hydrogen bonding is an essential feature in the crystal structures of compound 1. The similar hydrogen bonding motifs in forms A and B suggest that disruptions in the hydrogen bonding sequence through the incorporation of an impurity might not only interfere with form A but also affect form B. On this basis, it might not be possible to stabilize the metastable A form by inhibiting the stable B form with an impurity that hinders the hydrogen bond formation; it would also likely disrupt the structure and the crystallization process of the metastable phase and possibly negate the suppression of the transformation to the more stable polymorph. Solubilities. The solubility curve of each form of compound 1 in 2-propanol in the temperature range of 0 to 40 °C is shown in Figure 5. The solubilities show that form B and form C, and form A and form C are enantiotropic with a transition temperature at 20.1 and 35.2 °C, respectively. Below these crossover temperatures, form C is the most stable (i.e., lowest solubility) with respect to the other form, whereas above these temperatures, the other polymorph is more stable. In contrast, the A and B form is a monotropic pair where form A is metastable relative to the B form. The solution-mediated transformation studies of form A to form B are carried out at 30 °C to avoid the appearance of form C being mixed with form B since it has the lowest free energy (most stable phase) below 20 °C despite the fact that the transformation rate from form B to form C is slow. Solution-Mediated Transformation. The solutionmediated phase transformation of compound 1 comprises three main steps: dissolution of the metastable phase, form A, nucleation of the stable phase, form B, and crystal growth of the stable form. The morphology of each form is shown in Figure 6. Both forms exhibit a platelike morphology, and thus it is difficult to identify the polymorph by the shape of the crystal or monitor the phase transformation by microscopy. Powder X-ray diffraction is employed to assess the rate of transformation; polymorphic fractions are measured based on the differences between the powder patterns of each form (Figure 2). Characteristic peaks for both polymorphs are used to construct the calibration curve to quantitatively measure the composition change of the two polymorphs in the slurry (Figure 3). At certain time intervals, samples of the crystal slurry are removed and the conversion of form A to form B is monitored. Figure 7
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Crystal Growth & Design, Vol. 5, No. 4, 2005 1433
Figure 6. SEM images (500×) of form A (left) and form B (right) of compound 1. Figure 9. Effect of impurity concentration on the transformation behavior of compound 1 at 30 °C.
Figure 7. Transformation behavior of form A to form B at 30 °C in 2-propanol. Figure 10. Effect of seeding on the transformation rate of compound 1 at 30 °C in 2-propanol.
Figure 8. Influence of impurities on the transformation behavior of compound 1 at 30 °C.
shows the transformation behavior of compound 1 in 2-propanol. In the first three experiments, the stirring speed is 300 rpm and initial transformation of form A occurs after the first hour. However, when the degree of agitation is reduced to 150 rpm, the time elapsed for the initial appearance of form B increases. Increasing agitation rate increases the crystallization kinetics (or the amount of secondary nucleation) of the stable phase, thus increasing the surface area of this phase and hence the transformation rate. The influence of structurally related impurities on the transformation rate is shown in Figure 8. Impurities RS1 and RS4 slightly retard the transformation rate, while with the addition of RS2 and RS3 transformation of the metastable A form to the stable form is hindered,
particularly RS2, where a trace amount of form B is observed after 30 h. Overall, the transformation behavior in the presence of RS1 and RS4 is very similar to the rate of the pure solution. To understand the effect of the doping level on the rate of transformation, two different impurity loadings (0.1 and 0.5 w/w%) for the two best inhibitors (RS2 and RS3) were added to the solution, and the results are shown in Figure 9. The stabilization of the metastable modification as reflected in a decrease in the transformation rate is sensitive to the doping level. High impurity concentrations (e.g., 0.5 w/w%) of RS2 and RS3 suppress the transformation to a greater extent than at low concentration consistent with the notion at low doping levels the nucleation rate and growth rate coefficients for the stable polymorph is similar to those in the absence of the impurities, whereas at high loadings the nucleation rate and growth rate coefficients decreases, resulting in an increase in transformation time.41 The influence of seeding with form B in combination with the addition of the inhibitor RS2 is shown in Figure 10. At low doping level and seeding, the transformation behavior resembles very closely to that in the absence of the impurity as conversion to the stable modification is completed after 2 h. For high impurity loading and seeding with form B, small fractions of the stable B form, as detected from the powder X-ray patterns, are observed after 6 h resulting in a 5-fold decrease in the initial appearance of the stable modification when compared to same doping level without seeding. In
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Table 2. Influence of Structurally Related Impurities on the Concentration (g/L) of Form B after the Solution-Mediated Phase Transformation at 30 °C in 2-Propanol 0 w/w% (pure) 0.1 w/w% 0.5 w/w% 1.0 w/w%
RS1
RS2
3.77 3.82 3.71
3.98 3.93 4.00
RS3
RS4
3.80 3.87 3.77
3.73 3.80 3.81
Mukuta et al. Table 3. Relative Incorporation Energies (kcal/mol) for Various Crystallographic Planes of Compound 1 in the Presence of Four Structurally Related Impuritiesa crystal faces {hkl}
Z
RS1
RS2
RS3
RS4
{100}
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
-2.883 -2.475 -2.419 -2.425 -3.530 -3.314 -3.304 -3.307 -2.874 -2.479 -2.323 -2.567 -3.005 -2.401 -2.373 -2.046 -2.818 -2.367 -2.340 -2.346 -2.137 -1.874 -1.864 -1.862
-7.405 -7.902 -7.608 -7.891 -6.868 -7.720 -7.515 -7.711 -5.564 -6.898 -6.128 -6.877 -5.898 -5.986 -5.791 -5.974 -5.270 -5.824 -5.528 -5.812 -5.664 -8.374 -5.736 -8.367
-4.345 -4.050 -3.985 -2.760 -4.621 -4.458 -4.214 -2.848 -4.154 -4.540 -3.941 -3.823 -4.012 -3.128 -4.021 -2.526 -4.141 -3.679 -4.521 -2.453 -3.948 -4.081 -3.454 -2.802
-14.475 -14.453 -15.987 -15.992 -15.288 -15.265 -16.797 -16.802 -14.068 -14.111 -14.882 -14.999 -12.685 -12.148 -11.986 -12.633 -12.34 -12.317 -13.878 -13.884 -11.824 -11.12 -11.548 -11.542
3.86
contrast, seeds that are ground together with a high level of RS2 impurities reveal that the stabilizing effect of the impurity is reduced as full conversion to the stable form is observed after 4 h. The addition of seeds in the process clearly decreases the transformation time as a result of secondary nucleation. As the size of the seeds decreases such as in the case of grinding, higher surface areas of the seeds are expected, in turn, increasing the mass transfer and the overall growth rate of the stable polymorph.59 Thus, the transformation rate is accelerated and the crystallization to form B is enhanced as the seeds act as a catalyst during the nucleation process. The initial appearance of form A and its subsequent disappearance and conversion to the stable B form follow Ostwald’s Law of Stages concerning the precipitation of the metastable modification followed by its transformation to the stable form. The driving force for the solution-mediated transformation is the difference in the free energy, specifically differences in the solubility between the stable and the metastable modifications. Transformation to the less soluble B form occurs at the expense of the more soluble (or metastable) A form and the process progresses faster as the solubility difference between the two forms becomes greater. Full conversion is obtained when the solution reaches saturation with respect to the stable polymorph and the metastable modification is completely dissolved. The significant retardation effect of the RS2 impurity is possibly due to its ability to inhibit the nucleation of form B, in turn, kinetically stabilizing the metastable phase, as reflected in the increase of the transformation time. The addition of the impurity most likely reduces the nucleation rate of form B, perhaps by disrupting and inhibiting the emerging nucleus. The crystal growth rate of form B might also be influenced by the impurity but not to a great extent considering that when small seed crystals were used in the presence of RS2, the transformation to form B proceeds much more rapidly compared with the addition of large form B seed crystals (Figure 10), suggesting that crystal growth of the stable form in solution ensues despite the impurity. The prolonged induction period of the stable phase might also be explained by examining the impact the impurities have on the solubility of compound 1. Impurities can influence or alter the solubility of a solute, in turn, affecting the crystallization process. The higher the doping level or impurity loading, the more pronounced the effect becomes. Table 2 shows the effect of the structurally related impurities on the concentration of the stable B form of compound 1 in the absence and presence of the impurities. Although the impurities are molecularly similar to compound 1, it is believed that the superior inhibitory impact of RS2 might also be a result of the increase in the solubility of form B. This sudden enhancement leads to a smaller driving force for the transformation process, thus lowering the rate
{102 h}
{011}
{110)
{102}
{211 h}
a Z is the symmetry position in the unit cell: (1) Z ) (x, y, z); (2) Z ) (-x, y + 1/2, -z + 1/2); (3) Z ) (-x, -y, -z); (4) Z ) (x, -y + 1/2, z + 1/2).
of transformation and stabilizing the metastable polymorph. Differences in the molecular structure of the impurities lead to a different outcome of the transformation behavior as each impurity molecule has unique modes of action and affects the crystallization process differently. The suppression of the stable B form with RS2 as determined from powder X-ray diffraction quantification also reveals that the doping level is another factor that affects the conversion process and that a sufficient level of impurities is needed to hinder the formation of the stable modification. Impurity Incorporation. Structurally related impurities may enter the host crystal lattice and replace the host molecule at lattice sites as a result of its molecular compatibility. Relative incorporation energies are calculated and used to evaluate the extent of the impurity incorporation. The molecular modeling approach is a modification of Hartman and Perdok’s classical theory for predicting crystal morphology.58 It requires the substitution of the host molecule with the impurity molecule and the calculation of attachment energies for both the pure and the impurity-modified crystal surfaces. Table 3 shows the relative incorporation energies for compound 1 doped with the four structurally related impurities in each crystallographic position. The position of each impurity within the host lattice is optimized through a sequence of molecular mechanic and molecular dynamics simulations. The likelihood of an impurity incorporating into the crystal structure of form B of compound 1 can be indicated by low values of the relative incorporation energies. Crystal surfaces that have a minimum change in the energy are where the impurity will most likely to incorporate. Thus relative incorporation energies can be used to measure how easily an impurity can replace the host molecule on a given crystal plane. It can be seen that the impurity RS1 has the lowest relative incorporation energies
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Crystal Growth & Design, Vol. 5, No. 4, 2005 1435
Table 4. Summary of Impurity Incorporation as Determined by HPLC in Compound 1 for Different Concentrations of Impurities 0.1 w/w% RS1 RS2 RS3 RS4
0.5 w/w%
crystal
filtrate
crystal
filtrate
3.89 mg (95.8%) 1.14 mg (40.0%) 3.27 mg (72.3%) 0.404 mg (9.8%)
0.17 mg (4.2%) 1.71 mg (60.0%) 1.25 mg (27.7%) 3.73 mg (90.2%)
15.47 mg (93.7%) 6.97 mg (48.9 mg) 12.41 mg (68.1%) 0.369 mg (2.1%)
1.04 mg (6.3%) 7.29 mg (51.1%) 5.81 mg (31.9%) 17.2 mg (97.9%)
signifying that it can easily enter the crystal lattice and substitute for the host molecules in various crystal faces of form B of compound 1, in particular, the {211h } surface where there is a minimal change in the energy for each symmetry position. In contrast, large energy losses for the RS4 impurity are observed suggesting that segregation into the compound 1 crystal is least favorable, while for the impurity RS3, low incorporation energies are observed in symmetry position 4 for various crystallographic lattice planes. The impurity molecule, RS2, yields moderate energetic values suggesting that the uptake may not be extensive as compared with RS1 and RS3 impurities. The extent of impurity incorporation in compound 1 crystals for two different concentrations of impurities as determined by HPLC is shown in Table 4. In excellent agreement with the relative incorporation energies, RS1 appears to easily replace the host molecule in the crystal lattice of the stable phase, form B, and incorporates more extensively when compared with the other structurally similar impurities. The order of the impurities uptake is RS1 > RS3 > RS2 > RS4, which mirrors the order of the relative incorporation energies. Although studies have shown that calculations from the modified attachment energies have been successful in the prediction of impurity-modified crystal habit47-52 as impurities can affect the individual growth rate of crystal faces, the effects of impurities on the morphology of compound 1 are not examined as the intent of the simulations is to assess the impact of impurities on the purity of the crystals and possibly understand how the incorporation of impurities can be exploited for the stabilization of a metastable polymorph. Crystallographic lattice planes are unable to discriminate between the host and the impurity molecules as a result of its molecular similarity and compatibility. Thus, the impurities easily incorporate onto the crystal surface. Once incorporated, the impurity can disrupt the lattice and the normal hydrogen bonding sequence. In the case of the RS2 and RS3 impurities, the hydrogen bonding interactions are maintained intact as the substitution of either impurity in the crystal lattice is still involved in the hydrogen bond network through its cyano nitrogen, amide hydrogen, hydroxyl hydrogen, and trifluoromethyl fluorine atoms. In contrast, the hydrogen bond sequence is broken upon substitution of the impurities RS1 and RS4, specifically the hydrogen bond donor-acceptor pairing, the hydroxyl hydrogen atom, and the trifluoromethyl fluorine atom. It would be expected that since the growth unit for form B is disrupted, the crystallization of the stable form will be inhibited; however, this is not observed as the trans-
formation rate for the metastable A form to the more stable phase is only slightly hindered. With the impurity RS2, the level of incorporation may have not been extensive as for RS1 and RS3, but the inhibitory effect is considerably greater when compared with the other structurally related impurities. This can be attributed to its ability to hinder the nucleation and crystal growth of form B and possibly its influence on the solubility of the stable modification, form B. The observed increase of the concentration of form B in the presence of the RS2 impurity suggests that the impurity can increase the solubility of form B, which would lead to a reduction in the driving force for the phase transformation, namely, the free energy difference between the two crystalline forms. Relative incorporation energies derived from the modified attachment energy calculations enable us to assess the compatibility of an impurity to incorporate into the crystal structure. In addition to accurately simulating crystal habits in the presence of additives or impurities as reported in other studies,47-52 the energetic calculations can be employed to design or screen additive/impurity molecules to hinder the transformation of a metastable modification to a stable phase by examining the differences in the level of incorporation between crystalline forms. An example where this might be advantageous is in the case of 4-methyl-2nitroacetanilide where it has been observed that as the incorporation efficiency of isomorphic additives increases in the high-energy form, the rate of transformation decreases, suggesting that a necessary amount of impurities incorporated is required to stabilize the metastable phase.43 Conclusions The solution-mediated phase transformation of an active pharmaceutical ingredient is influenced by the presence of tailor-made impurities as certain structurally related inhibitors, namely RS2, suppressed the transformation of the metastable A form to the stable B form, while other impurities have a slight or moderate effect on the transformation rate. Powder X-ray diffraction is employed for quantitative measurements of polymorph composition in the slurry. The driving force for the transformation is differences in the solubility between the two forms. The kinetic stabilization of the metastable phase in the presence of the RS2 impurity is a result of its ability to disrupt the nucleation and crystal growth of the stable polymorph and its enhancement in the solubility of form B, which in turn leads to a reduction in the driving force. The inhibitors are molecularly similar to compound 1; consequently, the impurities may be incorporated into the host crystal lattice. The compatibility of an impurity substituting for the host molecule in the lattice can be measured by calculation of the relative incorporation energies. It is demonstrated that the modified attachment energy calculations can be an effective indicator on how likely the impurity molecule will incorporate as the order of the impurities uptake, as determined by HPLC, mirrors the relative incorporation energies. Although the level of incorporation did not correspond to the inhibitory effect of the impurities, incorporation energies may be used to stabilize the metastable phase in cases in which
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the transformation rate is retarded by an increase in the incorporation level of an additive or impurity. This might be extremely beneficial in cases where the metastable modification is desired due to its higher solubility and improved bioavailability. The energetic calculations can enable molecules to be rationally designed to inhibit the crystallization of the stable form and hence stabilize the metastable modification. Also the modeling approach might reduce the development time and the labor-intensive batch experiments that are performed to determine which additives could be effective inhibitors. Furthermore, relative incorporation energies can be employed to screen the impact of impurities on the purity of the crystals and be utilized to simulate its effects on the crystal morphology. References (1) Brittain, H. G. Polymorphism in Pharmaceutical Solids; Marcel Dekker: New York, 1999. (2) Bernstein, J. Polymorphism in Molecular Crystals; Oxford University Press: New York, 2002. (3) Byrn, S.; Pfeiffer, R.; Ganey, M.; Hoiberg, C.; Poochikian, G. Pharm. Res. 1995, 12, 945. (4) Byrn, S. R.; Pfeiffer, R. R.; Stowell, J. G. Am. Pharm. Rev. 2002, 5, 92. (5) Raw, A. S.; Furness, M. S.; Gill, D. S.; Adams, R. C.; Holcombe, F. O., Jr; Yu, L. X. Adv. Drug. Delivery Rev. 2004, 56, 397. (6) Dunitz, J. D.; Bernstein, J. Acc. Chem. Res. 1995, 28, 193. (7) Chemburkar, S. R.; Bauer, J.; Deming, K.; Spiwek, H.; Patel, K.; Morris, J.; Henry, R.; Spanton, S.; Dziki, W.; Porter, W.; Quick, J.; Bauer, P.; Donaubauer, J.; Narayanan, B. A.; Soldani, M.; Riley, D.; McFarland, K. Org. Process Res. Dev. 2000, 4, 413. (8) Bauer, J.; Spanton, S.; Henry, R.; Quick, J.; Dziki, W.; Porter, W.; Morris, J. Pharm. Res. 2001, 18, 859. (9) Guillory, J. Generation of Polymorphs, Hydrates, Solvates and Amorphous Solids. In Polymorphism in Pharmaceutical Solids; Brittain, H. G., Ed.; Marcel Dekker: New York, 1999; pp 183-226. (10) Storey, R. A.; Docherty, R.; Higginson, P. D. Am. Pharm. Rev. 2003, 6, 100. (11) Hilfiker, R.; Berghausen, J.; Blatter, F.; Burkhard, A.; De Paul, S. M.; Freiermuth, B.; Geoffroy, A.; Hofmeier, U.; Marcolli, C.; Siebenhaar, B.; Szelagiewicz, M.; Vit, A.; von Raumer, M. J. Therm. Anal. Cal. 2003, 73, 429. (12) Morissette, S. L.; Almarsson, O.; Peterson, M. L.; Remenar, J. F.; Read, M. J.; Lemmo, A.; Ellis, S.; Cima, M. J.; Gardner, C. R. Adv. Drug Del. Rev. 2004, 56, 275. (13) Gardner, C. R.; Almarsson, O.; Chen, H.; Morissette, S.; Peterson, M.; Zhang, Z.; Wang, S.; Lemmo, A.; GonzalezZugasti, J.; Monagle, J.; Marchionna, J.; Ellis, S.; McNulty, C.; Johnson, A.; Levinson, D.; Cima, M. Comput. Chem. Eng. 2004, 28, 943. (14) Storey, R.; Docherty, R.; Higginson, P.; Dallman, C.; Gilmore, C.; Barr, G.; Dong, W. Cryst. Rev. 2004. 10, 45. (15) Peterson, M. L.; Morissette, S. L.; McNulty, C.; Goldsweig, A.; Shaw, P.; LeQuesne, M.; Monagle, J.; Encina, N.; Marchionna, J.; Johnson, A.; Gonzalez-Zugasti, J.; Lemma, A. V.; Ellis, S. J.; Cima, M. J.; Almarsson, O. J. Am. Chem. Soc. 2002, 124, 10958. (16) Almarsson, O.; Hickey, M. B.; Peterson, M. L.; Morissette, S. L.; Soukasene, S.; McNulty, C.; Tawa, M.; MacPhee, J. M.; Remenar, J. F. Cryst. Growth Des. 2003, 3, 927. (17) Morissette, S. L.; Soukasene, S.; Levinson, D.; Cima, M. J.; Almarsson, O. Proc. Nat. Acad. Sci. U.S.A. 2003, 100, 2180. (18) Remenar, J. F.; MacPhee, J. M.; Larson, B. K.; Tyagi, V. A.; Ho, J. H.; McIlroy, D. A.; Hickey, M. B.; Shaw, P. B.; Almarsson, O. Org. Process Res. Dev. 2003, 7, 990. (19) Chyall, L. J.; Tower, J. M.; Coates, D. A.; Houston, T. L.; Childs, S. L. Cryst. Growth Des. 2002, 2, 505. (20) Hilden, J. L.; Reyes, C. E.; Kelm, M. J.; Tan, J. S.; Stowell, J. G.; Morris, K. R. Cryst. Growth Des. 2003, 3, 921. (21) Childs, S. L.; Chyall, L. J.; Dunlap, J. T.; Coates, D. A.; Stahly, B. C.; Stahly, G. P. Cryst. Growth Des. 2004, 4, 441.
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