High-Throughput Surveys of Crystal Form Diversity of Highly Polymorphic Pharmaceutical Compounds O ¨ rn Almarsson,* Magali B. Hickey, Matthew L. Peterson, Sherry L. Morissette, Stephen Soukasene, Chris McNulty, Mark Tawa, J. Michael MacPhee, and Julius F. Remenar
CRYSTAL GROWTH & DESIGN 2003 VOL. 3, NO. 6 927-933
TransForm Pharmaceuticals, Inc., 29 Hartwell Avenue, Lexington, Massachusetts 02421 Received April 10, 2003;
Revised Manuscript Received August 4, 2003
ABSTRACT: Surveys of crystal form diversity of two test compounds, 1 (an experimental angiotensin II antagonist) and 2 (sertraline HCl, the active drug in Zoloft), have been performed with high-throughput (HT) crystallization. Compound 1 was found to have at least 18 crystal forms based on a HT recrystallization experiment using diverse solvents, compared with nine forms originally reported from a traditional screening effort. The efficiency of screening in HT mode is estimated to be about 2 orders of magnitude greater than traditional bench-scale approaches. The multiple patented forms of 2 have been summarized and evaluated based on published information, which is found to include several transient species and at least one mixture of known phases. A comparison between results of HT experiments and data on the disclosed forms shows that the HT effort generates the viable crystal forms; highly unstable hydrates and one metastable polymorph IV were not observed. In attempting to recover form IV, a novel acetic acid solvate was discovered and characterized by single crystal X-ray diffraction. Additionally, a previously undisclosed ethyl acetate hemisolvate of 2 was identified as an intermediate en route to form T1. The study demonstrates that highly polymorphic pharmaceutical compounds can be surveyed by HT form experimentation, and that an HT strategy coupled with critical analysis of reported form diversity can be used to rank the utility of crystal forms. Introduction Polymorphism is relatively common in pharmaceutical solids.1 The late Dr. Walter McCrone stated somewhat provocatively that “the number of forms of a given compound is proportional to the time and money spent on research on the compound.”2 Indeed, screening for and characterization of polymorphs are considered important activities in drug development, due to the impact that the crystal form can have on drug properties and performance.3 For example, almost two years after FDA approval, the HIV protease inhibitor ritonavir4 appeared as a previously unknown, thermodynamically more stable polymorph.5 The new polymorph caused the existing capsule product to fail its regulatory specifications, thus forcing removal of the capsule from the market until the product could be reformulated to meet the necessary performance criteria. A recent study demonstrates the existence of even more crystal forms of ritonavir.6 The unpredictable nature of polymorphism requires early and thorough examination of form space to minimize the risk of surprises in drug development. In general, when polymorphs of a development compound are observed, the decision to pursue a particular form is critically dependent on resolution of issues of stability, process control, drug product performance, and patent protection. Traditional methods to generate polymorphs (as well as hydrates and solvates) include manual techniques of slurry conversion, recrystallization from a selection of process-relevant solvents, and thermal microscopy.7 * Corresponding author: O ¨ rn Almarsson, Ph.D., TransForm Pharmaceuticals, Inc., 29 Hartwell Avenue, Lexington, MA 02421; (781) 674-7894 [tel]; (781) 863-6519 [fax]; e-mail: almarsson@ transformpharma.com.
Recently, high-throughput (HT) polymorphism screening has been developed with the aim of proactively and comprehensively addressing form diversity, and hence accelerating the identification of a potential polymorph issue.6,8 Occasionally, highly polymorphic compounds (materials having many more forms than other polymorphic systems, such as acetaminophen and ritonavir) are encountered that present special challenges for drug development. One of the most striking published examples of polymorphism in a development compound is the angiotensin II receptor antagonist 1 (Scheme 1), which is a potassium salt that has nine reported crystal forms.9,10 A question that merits consideration is whether HT crystallization experiments could reproduce and even enhance the form diversity of 1 on a shorter time scale with a comparatively small amount of material. We report herein results from HT crystallization, carried out in a 4-week period using a novel system applied to solid forms of 1. The efficiency of form surveys between HT mode and traditional screening is compared. We also report results of a 6-week HT crystallization study of the selective serotonin reuptake inhibitor (SSRI) sertraline HCl (2, Scheme 1)4 as another example of a highly polymorphic compound. The results are contrasted with a comprehensive analysis of the form diversity reported in numerous patents relating to this drug. For both test compounds 1 and 2, extensive and rapid form screening on the order of thousands of crystallization trials was demonstrated to elicit the numerous crystal forms of these highly polymorphic materials. Experimental Section Materials. The crystalline hydrate of 1 was donated by Merck & Co., Inc. Compound 2 was obtained from Interchem
10.1021/cg034058b CCC: $25.00 © 2003 American Chemical Society Published on Web 09/10/2003
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Crystal Growth & Design, Vol. 3, No. 6, 2003 Scheme 1.
Test Compounds Used in HT Polymorphism Study
Corporation (Paramus, NJ) as crystalline form II.11a Both materials were of high chemical purity (>99% and 99.8% by HPLC area for 1 and 2, respectively), and no further purification was performed prior to use in solubility measurements and crystallization experiments. Solvents were purchased from Sigma-Aldrich, Spectrum or Fisher, generally as HPLC grade, and were used without further purification. HPLC-grade distilled water (at pH just over 5) was obtained from Fisher. Only borosilicate glass or polypropylene components were allowed to contact the solvents during storage. Single-use glass tubes packaged in shrink-wrapped deep-well plates were purchased from Spike International (Wilmington, NC) and loaded into aluminum 96-well arrays prior to dispensing of compound and solvents into the tubes. Methods. HT crystallization trials were performed using CrystalMax technology.12 Multiple parallel crystallizations were carried out for each compound according to methods previously described.6,8 Crystallization of 1 was carried out in fifteen 96-well aluminum blocks holding borosilicate tubes containing the crystallization mixtures, which were rendered supersaturated by heating to 70 °C for 2 h followed by a 1 °C/min cooling ramp to 5 °C. The cooling profiles of the aluminum racks that comprise the heat source for the blocks were roughly linear. The individual mixtures of drug and solvent, devised by use of proprietary design software, were created by first dispensing the compound into the glass tubes from an organic solvent using a Tecan miniprep (TECAN, Durham, NC), followed by drying under a stream of nitrogen (TurboVap 96, Zymark Corp. Hopkinton MA). Next, water and/ or organic solvents were dispensed combinatorially into the tubes using a Cartesian SynQuad 32-channel dispenser (Cartesian Technologies, Irvine, CA). Duplicates of each composition were prepared; identical samples were located in different arrays in each case. Each tube in a 96-tube array was sealed within 15 s of combinatorial dispensing with a Teflon-coated crimp top to avoid evaporation of organic solvents. A selection of 21 diverse solvents was used as single solvents or as various binary and ternary solvent combinations. Three nominal concentrations of 1 (corresponding to 1-3 mg of material in each tube) were employed to vary supersaturation levels. The concentrations of 1 were based on room temperature solubility measurements in pure solvents, which were performed by blank solvent-corrected UV-visible spectroscopic absorption at 250 nm using a Cary spectrometer (Varian Instruments, Palo Alto, CA). In addition to the 1440 solvent-mediated crystallizations, a separate hydrate screen was conducted at room temperature as a slurry conversion in one 96-well block. Hydrate screening of 1 was performed using methanol-water and acetonitrile-water mixtures of selected water activity.13 Crystallization of 2 was carried out in thirty-two 96-well blocks according to a method similar to that employed for 1. Tubes contained from 1 to 2.5 mg of 2. A selection of 24 diverse solvents was used combinatorially in a total of 3100 trials. Two nominal concentration levels and two crystallization temperatures of 5 and 25 °C were employed for two identical sets of blocks with duplicates of each composition at each temperature. Raman spectra of the solids obtained in the crystallization process were collected on a Nicolet Almega dispersive system fitted with a 30 mW NIR laser at 785 nm. Powder X-ray diffraction (PXRD) on samples in boron rich glass or quartz capillaries (Charles Supper Co., Natick, MA) in transmission mode was performed on a Rigaku D/MAX Rapid image plate
Almarsson et al. diffractometer (Rigaku/MSC, Woodlands, TX) employing CuKR radiation with a 0.3 mm collimator and a 2.0 kW source operating at 46 kV/40 mA. Preferred orientation effects were minimized by collecting PXRD data in transmission mode, while oscillating around the omega-axis from 0 to 5 deg and spinning 360 deg about the phi-axis at 2 deg/s. In addition, the image plate output data (full Debye rings) were inspected for evidence of preferred orientation (manifested as spots along a given reflection plane). In each case, powder samples did not exhibit significant orientation effects. Cluster classification of PXRD patterns was performed using proprietary software (Inquire). The software first filters the PXRD patterns to remove background and to accentuate diffraction peaks and shoulders. Then, peaks are picked using standard derivative methods, and amplitudes are calculated. Finally, a hierarchical clustering algorithm is used in conjunction with a distance measure related to the Tanimoto coefficient8,12 to bin spectra. This method uses peak positions, rather than amplitudes, to discriminate between different patterns. The software allows a user to select a window over which two peaks are considered to be at the same position (a tolerance value, which is generally up to 0.2), as well as a minimum height for a filtered peak to be considered for clustering. Additionally, regions of 2-theta can be selected to focus on ranges of d spacings of interest. With appropriate settings, which can be interactively adjusted by the user, the software is able to identify and bin a PXRD pattern that has only one peak feature in a slightly different location than in other patterns. Thermal analyses were performed on a Q1000 mDSC and Q500 TGA (TA Instruments, Wilmington DE). Heating rates of 10 °C/min were employed. IR off-gas analysis was conducted using a Nicolet FT-IR system (Nicolet, Madison, WI). Thermal microscopy was performed on a Zeiss Axioplan microscope (Zeiss, Thornwood, NY) with a Mettler Toledo FP90 central processor equipped with an FP82HT hot stage (Mettler, Hightstown, NJ). Heating rate of 5 °C/min was employed and photomicrographs were taken by a Zeiss Axiocam Type 1 SNO641 (Zeiss, Thornwood, NY) at one degree intervals.
Results and Discussion The structure of test compound 1 contains eight torsion angles that give rise to multiple possible conformations. In the original polymorphism study of 1, 10 discrete forms were reported, based on powder X-ray diffraction (PXRD).9 One of the forms was described as being an amorphous phase. Of the nine crystalline forms, one is only obtained by heating to >190 °C.14 Some 1500 crystallization trials were conducted in HT mode using thermally driven supersaturation of 1 from diverse solvents. Over a period of 7 days, 186 solids, corresponding to a 13% hit rate, were harvested and analyzed. Following Raman clustering, over 80 of those solids were analyzed by capillary PXRD. In Table 1 are summarized the powder patterns for several different forms of 1 from the HT screen. Cluster analysis of the PXRD patterns15 shown in Figure 1 suggests the presence of at least 18 distinct forms resulting from the solvent-mediated recrystallization experiment. To the extent that comparison was feasible, at least five of the previously known forms were also generated in the HT experiments.14,16 The hydrate (originally named form I), which was the desirable form for development, was obtained under conditions of slurry conversion with aqueous solvent mixtures in the HT screen. Importantly, form D was also found in the HT screen. This form was an apparent example of a “disappearing polymorph”.17 In the original report, the authors stated that D became elusive once form I
High-Throughput Surveys of Crystal Form Diversity
Crystal Growth & Design, Vol. 3, No. 6, 2003 929 Table 2. Comparison of Traditional and HT Mode Crystal Form Screening for 1
Figure 1. Cluster diagram of PXRD pattern similarity of 1. Table 1. Powder X-ray Diffraction Data for Crystal Forms of 1 Found in HT Screen form Aa Da Ea Ga Ia L M N O P H Q R S T U V W
peaks from powder pattern 3.83, 7.21, 8.17, 10.53, 13.27, 13.81, 14.79, 15.23 4.07, 8.15, 8.59, 9.59, 12.33, 13.45, 13.77, 15.73, 16.43, 18.61, 19.07, 24.21, 25.57 3.75, 7.27, 8.19, 8.53, 10.15, 11.23, 13.39, 14.71, 15.55, 16.31, 17.91, 20.61, 23.51 4.91, 5.79, 6.69, 9.03, 10.47, 13.43, 14.21, 15.45, 18.31, 20.13, 25.65 3.81, 7.21, 8.10, 10.50, 11.41, 13.65, 14.71, 15.23, 18.23, 19.23, 19.59, 20.65, 21.55, 22.43, 24.03, 26.25 4.15, 7.49, 8.25, 9.23, 11.07, 12.33, 13.19, 15.01, 15.67, 17.79, 19.09, 20.83, 22.45, 23.85, 24.99, 25.49 3.67, 7.49, 9.97, 11.85, 13.87, 14.67, 20.45, 23.17, 26.23, 27.11, 28.49, 28.77 4.15, 4.45, 7.39, 7.91, 8.31, 8.95, 11.15, 12.49, 15.03, 15.51, 17.63, 20.85, 22.37, 23.93 4.21, 8.77, 13.99, 15.99, 16.61 4.25, 7.09, 7.53, 8.83, 9.65, 13.49, 14.83, 15.73 16.71, 17.51, 24.31, 25.39 3.81, 5.71, 7.91, 10.845 13.49, 15.23, 15.95, 18.25, 20.39, 24.05, 25.27, 26.41 3.83, 4.71, 7.43, 8.17, 9.37, 10.35, 15.13, 17.93, 19.19, 24.07 4.21, 8.79, 10.13, 13.99, 15.95, 16.57, 19.63, 21.93, 24.23, 25.79 4.05, 4.88, 8.05, 13.85, 15.61, 24.09 3.77, 7.23, 8.51, 10.19, 10.57, 13.39, 14.69 6.95, 8.25, 9.95, 11.21, 11.83, 13.23, 14.55, 15.81, 16.55, 17.05, 17.83, 18.87, 20.39, 21.41, 23.15, 25.23, 26.27, 27.07, 28.87 3.67, 7.17, 7.51, 10.69, 15.05, 23.89 3.58, 4.35, 5.05, 8.91, 10.25, 14.04, 16.11, 16.67, 19.64, 24.47
a We have retained the alphabetical naming convention used in ref 9; the symbol denotes a form described in that paper.
appeared.9 Clearly, sufficient experimentation with diverse conditions, such as is enabled by HT methods, affords the possibility to regenerate an elusive form. On the basis of the combined results, HT crystallization demonstrates that 1 has more crystal forms than was originally suspected. Some efficiency metrics of form screening for 1 in traditional and HT mode are presented in Table 2. It is evident that form screening for the development compound can be achieved more efficiently in HT mode: More experiments can be attempted in a shorter time period, leading to an estimated efficiency increase of 120-fold over a manual setup. (The number is calculated as the product of 8-fold time acceleration and a 15-fold increase in the number of trials conducted in HT mode vs traditional screening, respectively.) The personnel requirement is similar for the two modes of screening:
metric
traditional
HT mode
number of experiments time frame (weeks) people requirement amount of material used number of crystalline forms found
ca. 100 ca. 32 ca. 2 >10 g 9
1500 4 1-2
