CRYSTAL GROWTH & DESIGN
Part of the Special Issue: Facets of Polymorphism in Crystals.
2008 VOL. 8, NO. 1 114–118
Concomitant Hydrate Polymorphism in the Precipitation of Sparfloxacin from Aqueous Solution Antonio Llinàs,*,‡ Jonathan C. Burley,§,⊥ Timothy J. Prior,4 Robert C. Glen,‡ and Jonathan M. Goodman*,‡ Pfizer Institute for Pharmaceutical Materials Science and UnileVer Centre for Molecular Informatics, Department of Chemistry, UniVersity of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom, Leicester School of Pharmacy, Faculty of Health and Life Sciences, De Montfort UniVersity, The Gateway, Leicester LE1 9BH, United Kingdom, and Department of Chemistry, UniVersity of Hull, Cottingham Road, Kingston-upon-Hull HU6 7RX, United Kingdom ReceiVed September 20, 2007; ReVised Manuscript ReceiVed NoVember 28, 2007
ABSTRACT: The Potentiometric Cycling for Polymorph Creation [PC]2 has been applied to sparfloxacin, a third-generation fluoroquinolone antibiotic. Two different trihydrate phases precipitate from aqueous solution. X-ray data indicate that one of these is a previously unknown polymorph of sparfloxacin trihydrate. Because both forms crystallize from solution at the same time, the two crystalline forms are concomitant polymorphs that precipitate in a thermodynamically controlled ratio. Introduction One of the key properties of a drug that can determine bioavailablity is the solubility of the neutral form of the drug. The solubility of formulated drugs can be affected by several parameters, including the solid state form in which the drug is delivered. Amorphous forms generally have higher solubility than crystalline forms, but do not find favor in oral dosage regimes as they exhibit a strong tendency to crystallize, which can lead to an abrupt decrease in solubility on storage. Crystalline forms are usually more stable and, therefore, find greater favor in oral dosage routes. However, it is well-known that stoichiometrically and chemically identical crystalline forms can potentially exist in a variety of solid forms, a phenomenon known as polymorphism.1 Because the different polymorphs possess different crystal structures, they can exhibit different physical properties, including color, crystallite morphology, flow properties, melting points, enthalpies of fusion, solubility, and dissolution rates. Therefore, the determination of solubility, and the understanding and control of polymorphism, are strongly related issues that are extremely relevant to the design of new treatments. Measurements of solubility are frequently plagued by difficulties in achieving true thermodynamic equilibrium. Measurements of solubility by different researchers, under different conditions, can differ by several orders of magnitude because of these difficulties.2 This is a major issue in drug development and understanding of the different efficacies of different formulations. The recently developed method of “chasing equilibrium” allows for a reliable and self-consistent determination of both intrinsic and kinetic solubilities3 and has been * Corresponding author. Tel: (44)(0)1223-336434. Fax: (44) (0)1223-763076. E-mail:
[email protected] (A.L.);
[email protected] (J.M.G.). ‡ University of Cambridge. § De Montfort University. 4 University of Hull. ⊥ Current address: Boots Science Building, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom.
fully validated.4 The intrinsic solubility is defined as the solubility of a compound in its free acid or free base form. We have recently described use of the “chasing equilibrium” method to determine the intrinsic solubility of diclofenac starting from three different solid forms and were able to show that the measured intrinsic solubility was not a function of the solid form of the starting material. This is to be expected if the method probes intrinsic solubility, and this study acted as a further validation of the reliability of this approach.5 In a subsequent paper, we further extended this to the reproducible generation of two crystalline anhydrous polymorphs of sulindac from aqueous solution by using a new technique [PC]2 (potentiometric cycling for polymorph creation).6 The two polymorphs exhibited intrinsic solubilities that differed by a factor of 7. This may have significant implications for bioavailability if transformations between the two forms can occur on storage or in vivo. In this paper, we report the results of our studies of the solubility of sparfloxacin (5-amino-1-cyclopropyl-7-[(3R,5S)3,5-dimethylpiperazin-1-yl]-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Scheme 1), a third-generation fluoroquinolone antibiotic used to treat a variety of diseases, including pneumonia and chronic bronchitis, often under the brand name Zagam.2 It has been withdrawn from human use in the United States because of severe adverse reactions including cardiotoxicity and phototoxicity, but it is still used to treat animals. Sparfloxacin was selected as part of an ongoing project for the accurately determination of solubilities for a range of drug molecules and investigate possible polymorphic behavior. In the solid state, it is available both as an anhydrous form and a trihydrate. Crystal structures, determined from single-crystal data, have been reported for both an anhydrous form7 (triclinic, centric space group P1¯) and a trihydrate8 (monoclinic, centric space group P21/n). Results and Discussion We investigated the solubility of sparfloxacin in water using the “chasing equilibrium” method, which is documented
10.1021/cg700908m CCC: $40.75 2008 American Chemical Society Published on Web 01/02/2008
Hydrate Polymorphism in Precipitation of Sparfloxacin
Crystal Growth & Design, Vol. 8, No. 1, 2008 115
Scheme 1. Precipitation of Sparfloxacin
elsewhere.3,5,6 Experiments were performed at 25 ( 0.1 °C and 0.150 M ionic strength adjusted with KCl. The CheqSol method relies on a potentiometric titration and, therefore, the accuracy of the intrinsic solubilities values obtained by this method is proportional to the precision of the measurement of the pKa of the compound to be studied (an error of 1 pKa unit will produce an error of 1 log unit on the solubility values). The pKa values for sparfloxacin were determined potentiometrically at exactly the same conditions (temperature and ionic strength) that were later used to perform the solubility experiments. Sparfloxacin is a zwitterion at neutral pH, and has two pKa values: 8.904 ( 0.055 and 6.403 ( 0.020. The higher pKa is assigned to the secondary amine on the piperazine ring and the lower to the carboxylic acid (Scheme 1). The solubility experiments were started by dissolving an accurately weighed amount of sparfloxacin (0.117-0.230 mg) in 10.00 mL of 0.150 M KCl solution. This solution was then brought to pH 12 by adding a basic titrant (KOH) so that all of the sparfloxacin dissolved as the zwitterion (2, Scheme 1) changed to the more soluble anion (3, Scheme 1). Acid (HCl) was now used as the titrant to move the solution toward the acidic region (Figure 1). It is convenient to follow this process using a Bjerrum plot, which plots the average number of bound protons (hydrogen-ion binding capacity) against pH. Figure 1 shows the Bjerrum plot for sparfloxacin and the data for one of the solubility experiments. Two different curves can be derived from samples at equilibrium: the nonprecipitation curve (Figure 1, dashed line), which represents the hydrogen binding capacity of the solute during the aqueous titration with no precipitate present, and the precipitation curve (Figure 1, dash-dot line), which represents the hydrogen binding capacity of the solute at equilibrium with some solid precipitated. The experimental data follow the nonprecipitation Bjerrum curve (Figure 1, dashed line) up to the point, at around pH 9.1, where precipitation is detected by the use of a spectroscopic dip probe placed inside the titration vessel. The kinetic solubility is derived at this point (Figure 1). The thermodynamic measurement is obtained by forcing the system to cycle between a supersaturated state and a subsaturated one several times (usually eight cycles is enough) and the intrinsic solubility value is derived from the mass and charge balance equations when the pH gradient
(dpH/dt) is zero (Figure 2), All the crossing points between supersaturated and subsaturated conditions lie close together on the precipitation theoretical curve. The pH changes very little during these cycles (Figure 3). Sparfloxacin was analyzed by X-ray powder diffraction analysis of the material received from Sigma-Aldrich (Figure 4) and was consistent with the previously reported triclinic crystal structure of the anhydrous form7 and with the stated composition from the manufacturer. During the solubility measurements, we found no evidence of salt precipitation or phase transformations. The kinetic solubility was measured as
Figure 1. Bjerrum curve for sparfloxacin (1 of 10). The experimental data follows the theoretical curve (dashed line) up to the precipitation point (full circle), when it jumps onto the precipitation curve. The direction of titration is towards the acidic region (right to left). nH represents the average number of bound protons (hydrogen-ion binding capacity). The pKa values of sparfloxacin have been superimposed on the Bjerrum curves. At a pH (see Scheme 1) below the first pKa (pKa1 ) 6.41), the piperazine and the acid are both protonated; at a pH between pKa1 ) 6.41 and pKa2 ) 8.89, the main species is the zwitterion (piperazine protonated and acid deprotonated); and at a pH above pKa2 ) 8.89, the main species is the anionic form, piperazine deprotonated (neutral), and acid deprotonated (negative).
116 Crystal Growth & Design, Vol. 8, No. 1, 2008
Figure 2. Chasing equilibrium of sparfloxacin (1 of 10 runs) of pH gradient plotted against concentration. The pH gradient (dpH/dt) changes from a positive value (neutral form is precipitating) in the supersaturated region to a negative one (neutral form is redissolving) in the subsaturated one: the rate of pH change is zero at the crossing points (dpH/dt ) 0). In this run, eight cycles were performed. The intrinsic solubility value shown (S0 ) 424.1 µM or 167 µg mL-1) is the mean of eight individual values from these eight cycles.
Figure 3. Plot of pH vs. time for sparfloxacin. Once the oscillation between the supersaturated and subsaturated system has begun (after around 50 min), the pH gradients caused by precipitating or dissolving solid cause the pH to remain stable around a value of 10.6.
5600 ( 670 µg mL-1. The intrinsic solubility was found to be 167 ( 14 µg mL-1 (mean of 10 experiments). The equilibrium precipitate was isolated from this experiment and subjected to X-ray powder diffraction analysis. The resultant powder pattern resembled that reported for the trihydrate8 (and the pattern could be partially fitted using this single-phase model using the Rietveld method9), but contained several extra diffraction peaks (Figure 4 asterisks). This led us to consider the possibility of concomitant crystallization of two solid forms in our experiments. Although we have previously had some success in solving crystal structures from powder diffraction data,10–15 we were unable to index the extra Bragg peaks, with matters complicated by the likely presence of two phases (the previously reported trihydrate plus the new phase). We were able to isolate crystals of sufficient size to undertake a full structural characterization using a laboratory diffractometer and to show that we had isolated a new polymorph of sparfloxacin trihydrate distinct from that reported previously. A full structural study was performed and details of the refined structure have been deposited with the Cambridge Crystallographic Data Centre and are available on request from the Centre. We were also fortunate in having access to the microcrystal diffraction facility on Station 9.8 at the Synchrotron Radiation Source at Daresbury
Llinàs et al.
Figure 4. X-ray powder diffraction data for the solid forms considered in these experiments. The data for the “as-received” and the “precipitate” samples are real and were collected as described; the data for the Sivalakshmidevi structure were calculated on the basis of the reported crystal structure. All data are normalized for intensity (Imax ) 10 000). Asterisks show some extra diffraction peaks corresponding to the new phase.
Laboratories, U.K. The synchrotron experiments were performed contemporaneously with the laboratory experiments and yielded information not available from the laboratory experiments. A crystal that appeared to be single was selected from the precipitate. This could not be indexed using the reported unit cells of either the anhydrous or the known trihydrate, and a full data set suitable for crystal structure determination was therefore collected. The crystal structure determined from this data set was that of a stoichiometric trihydrate identical to that from our laboratory experiments and therefore different to that previously reported by Sivalakshmidevi.8 A more detailed analysis of these data, with emphasis on the polymorphism, is reported below. In both the new and the known structures, the molecular conformations are nearly identical and in both polymorphs the hydrogen bonding of the sparfloxacin molecules is very similar. A comparison of the two crystal structures is presented in Figure 5, It can be seen that despite the similarity of the two structures at the level of individual molecules, the two structures are distinguished by strong differences in relative positioning and orientation of the various molecules in space, i.e., crystal packing. The clear and unambiguous difference between the two crystal structures indicates that we have isolated a new, previously unknown, polymorph of sparfloxacin trihydrate. Because both crystallize from aqueous solution at the same time, the two crystalline forms of the trihydrate are concomitant polymorphs16 that have the same thermodynamic stability (∆G ) RT ln K, where K is the solubility product and all other terms have their standard meaning). For full validation of the concomitant nature of the polymorphism exhibited by sparfloxacin trihydrate, we reanalyzed the X-ray powder diffraction data of the bulk precipitate using the Rietveld method, employing a structural model comprising both trihydrates. As shown in Figure 6, this model is fully consistent with the observed data, with the model giving excellent goodness of fit indicators (χ2 ) 2.540, Rwp ) 7.28, Rp ) 5.68) and a visually satisfactory fit. The refined ratio of the two hydrates was found to be 88.8(4):11.2(1) (%) (the majority phase corresponding to the new hydrate characterized in this study). Standard deviations are taken from the GSAS software employed17 and include contributions from the covariance matrix. As with all quoted standard deviations, they are dependent on
Hydrate Polymorphism in Precipitation of Sparfloxacin
Figure 5. Crystal structures for both polymorphs of sparfloxacin. (a) Molecular conformation of the new polymorph isolated in this study; (b) that of the previously reported structure isolated by Sivalakshmidevi; (c) crystal packing of the new polymorph; (d) that reported by Sivalakshmidevi.
Figure 6. Rietveld refinement utilizing a structural model that comprises both polymorphs of sparfloxacin trihydrate. The reflections for the new polymorph, phase 1, are the uppermost of the markers. Those of the Sivalakshmidevi phase are the lower.
the model employed and represent only statistical uncertainties; the true uncertainties will include systematic uncertainties in addition and are therefore expected to be significantly larger. To investigate whether these two phases were at equilibrium, we collected different precipitates after 8, 16, and 60 cycles at pH 10.6. The powder diffraction patterns of these samples were identical, indicating that the phase distribution was not changing as the experiment proceeded. Different polymorphs usually have different solubilities, and so the solubility measurements should vary with the number of cycles, if the ratio of the polymorphs changed with time. The solubility measurements did not change with the number of cycles performed, indicating either that the polymorphs have the same solubility, or else are present in a constant ratio. As a final check that the ratio was thermodynamically controlled, a run was stopped after the equilibrium was reached (16 cycles at pH 10.6), part of the solid suspension was filtered and analyzed by powder X-ray diffraction. The rest of the precipitate and the solution were stored together in a sealed vial at room temperature. After six months, the solid was filtered and submitted to powder X-ray. The data yielded results extremely similar to the pattern collected for the initial
Crystal Growth & Design, Vol. 8, No. 1, 2008 117
precipitate and again indicated the presence of two polymorphs of the trihydrate in approximate ratio 9:1. Given the lack of change in the ratios of the two hydrates, coupled with the fact that we observe only a single value for the intrinsic solubility of the solution, we conclude that the observed concomitant polymorphism appears to be an equilibrium property of the sparfloxacin:water system. A further confirmation of the concomitant crystallization of both trihydrate polymorphs was unexpectedly uncovered in a final re-examination of the original image frames collected in the single-crystal synchrotron experiment. The presence of extremely weak extra reflections was noted. These could be separately indexed using the same centric monoclinic unit cell (P21/n) as reported by Sivalakshmidevi for the original trihydrate.8 This indicates that the “single” crystal selected in this experiment actually comprised mainly the new phase, with a very small crystal of the original hydrate attached. The original images were reprocessed to yield two intensity files, one for each of the trihydrates. Both could be solved and refined, although given the very small amount of the Sivalakshmidevi phase, the reliability of the refinement is not as high as that previously reported. As with the laboratory data, refinement of the new P21 polymorph proceeded readily to yield excellent goodness of fit indicators. To summarize the single-crystal data collection: from the laboratory data we have demonstrated the existence of a new, acentric polymorph of sparfloxacin trihydrate; from the synchrotron data, we have clear and unequivocal evidence of the concomitant crystallization of two polymorphs of the trihydrate through the serendipitous event of inadvertently selecting a “non-single” crystal to characterize. Both laboratory and synchrotron data are in full agreement regarding the structure of the new polymorph. The results reported in this paper act as further confirmation of the validity of the “chasing equilibrium” method for determining accurate and reproducible solubilities, as well as extending the utility of the [PC]2 method to include the interconversion of polymorphic hydrates. They also indicate that the [PC]2 method is capable of differentiating between kinetic and thermodynamic control of crystallization in concomitant polymorphic systems. Experimental Section Solubility Measurements. The apparatus used to perform the solubility determinations was a GLpKa titrator and a D-PAS spectrometer controlled from a computer running Refinement Pro and CheqSol software (Sirius Analytical Instruments Ltd.). All experiments were performed in 0.15 M KCl solution under nitrogen atmosphere, at 25 ( 0.1 °C, using standardized 0.5 M HCl and 0.5 M KOH solutions. Powder X-ray Diffraction. X-ray powder diffraction was performed using Co KR1 radiation (λ ) 1.79 Å) on a Stoe Stadi-P diffractometer operating in Debye–Scherrer geometry. The sample was contained in a 0.7 mm diameter borosilicate capillary (Lindemann glass). Data sets with relatively high signal-noise ratios suitable for structure solution were collected at 290 K (approximate counting time: 24 h per data set). Laboratory Single-Crystal X-Ray Diffraction. Single crystals of (4) were immersed in perfluoropolyether oil, which protects them from atmospheric oxygen and moisture,18 mounted on thin glass fibers, and placed in a low-temperature N2 stream. Crystals were examined on a Nonius Kappa CCD diffractometer using thin slice κ and ω-scans at 150 (2) K utilizing Mo KR radiation. Low temperatures were achieved using an Oxford Instruments Cryostream cooler. Data for this sample were corrected for absorption anomalies using the SORTAV utility. Structures were solved by direct methods using SHELXTL software. Refinements were made on F2 with all non-H atoms refined anisotropically.19
118 Crystal Growth & Design, Vol. 8, No. 1, 2008 Synchrotron Single-Crystal Diffraction. Single-crystal X-ray diffraction data were collected at the microcrystal diffraction facility on station 9.8 of the Synchrotron Radiation Source, STFC Daresbury Laboratory, U.K. Crystals were covered in perfluoropolyether oil and mounted at the end of a two-stage glass fiber and cooled to 150 K in a nitrogen gas cryostream. Data were collected using a Bruker D8 diffractometer fitted with an APEXII CCD area detector. Data collection nominally comprised a sphere of reciprocal space by three series of ω-scans with different crystal Φ orientation angles. Each frame employed a 0.3° rotation in ω and a 2 s exposure time. Reflection intensities were integrated using standard procedures, allowing for the plane polarized nature of the primary synchrotron beam. Semiempirical corrections were applied to model absorption and incident beam decay. Unit-cell parameters were refined using all observed reflections. The structures were solved by routine automatic direct methods. The structures were completed by least-squares refinement based on all unique measured F2 values and difference Fourier methods.
Acknowledgment. Financial support by Pfizer is gratefully acknowledged, and the assistance of Dr. John Davies in collection of and structure solution from the laboratory X-ray data is likewise acknowledged. Supporting Information Available: Crystallographic information files of the new polymorph of sparfloxacin trihydrate (synchrotron and laboratory single-crystal X-ray diffraction) (CIF). This information is available free of charge via the Internet at http://pubs.acs.org.
References (1) Bernstein, J. Polymorphism in Molecular Crystals; Oxford University Press: Oxford, U.K., 2002. (2) Avdeef, A. Physicochemical profiling (solubility, permeability and charge state). Curr. Top. Med. Chem. 2001, 1, 277–351. (3) Stuart, M.; Box, K. Chasing equilibrium: measuring the intrinsic solubility of weak acids and bases. Anal. Chem. 2005, 77, 983–90. (4) Box, K. J.; Volgyi, G.; Baka, E.; Stuart, M.; Takacs-Novak, K.; Comer, J. E. Equilibrium versus kinetic measurements of aqueous solubility, and the ability of compounds to supersaturate in solution--a validation study. J. Pharm. Sci. 2006, 95, 1298–307. (5) Llinàs, A.; Burley, J. C.; Box, K. J.; Glen, R. C.; Goodman, J. M. Diclofenac solubility: independent determination of the intrinsic solubility of three crystal forms. J. Med. Chem. 2007, 50, 979–83.
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CG700908M
