Distinguishing between Two Polymorphs of the Drug Cimetidine

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Polymorphic Discrimination Using Atomic Force Microscopy: Distinguishing between Two Polymorphs of the Drug Cimetidine A. Danesh, X. Chen, M. C. Davies, C. J. Roberts,* G. H. W. Sanders, S. J. B. Tendler, and P. M. Williams School of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham, U.K. NG7 2RD M. J. Wilkins SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Harlow, Essex, U.K. Received April 20, 1999. In Final Form: July 27, 1999

Introduction Polymorphism describes the ability of an element or a compound to crystallize into more than one form.1 Identification of polymorphism is important in the pharmaceutical industry, since each crystal form may exhibit different physicochemical properties. Examples of problems caused as a consequence of polymorphism include the antibiotic ampicillin (bioavailability differences due to changes in solubility and absorption profile),1 the steroid prednisolone (dissolution rate fluctuations),1 and most recently the anti-AIDS drug protease inhibitor “Ritanovir” (different dissolution and possibly different absorption profile).2 Hence, a thorough understanding of, and a systematic characterization of polymorphic forms expressed by an active drug is necessary to maintain both high product quality and reproducibility.3 The regulatory expectations for the characterization of new drugs have also been expanded to include the polymorph types and their purity.4 The pharmaceutical industry utilizes a variety of strategies for the characterization of polymorphism in drugs. Although a universal approach is desirable, different compounds may require different strategies and techniques.3 Some of the techniques employed either singly or in combination are optical microscopy (crystal shape, optical properties), X-ray crystallography (lattice parameters), infrared spectroscopy (IR) (molecular vibration), differential scanning calorimetery (DSC) (thermodynamic behavior), and electron microscopy (high-resolution morphology).1,3,5 In this paper we describe how tapping mode atomic force microscopy (TM-AFM) may be used to distinguish and characterize polymorphs and demonstrate this potential for the drug cimetidine. Cimetidine, N′′-cyano-N-methyl-N′-{2-[[(5-methyl-1Himidazol-4-yl)methyl]thio]ethyl}guanidine,6 is a specific competitive histamine H2-receptor antagonist, which inhibits the release of histamine-stimulated gastric acid.7 There are seven known forms of cimetidine, the anhydrous polymorphic forms A, B, C, and D, and the hydrated forms M1, M2, and M3. These can be produced using a variety of well-established crystallization conditions.8 (1) Halebian, J.; McCrone, W. J. Pharm. Sci. 1969, 58, 911. (2) Simpson, D. Pharm. J. 1998, 261, Aug. 1, 150. (3) Lian, Yu; Susan, M.; Reutzel, A.; Gregory, A. Pharm. Sci. Technol. Today 1998, 1, 118. (4) Byrn, S. R. Pharm. Res. 1995, 12, 945. (5) Wall, M. G. Pharm. Manuf. 1986, 3, 33. (6) Shibata, M.; Kokubo, H.; Morimoto, K.; Morisaka, K.; Ishida, T.; Inoune, M. J. Pharm. Sci. 1983, 72, 1436. (7) Hall, N. Chem. Br. 1997, 25.

The only polymorphic forms used in the manufacture of pharmaceuticals are A (tablets) and B (suspension). Form A is preferred to other polymorphs since it is easier to obtain in a crystallographically pure state9 and easier to handle, particularly in large-scale operations due to good flow properties and lack of adherence to machinery. Form A may be crystallized at room temperature from nonaqueous solvents such as acetone, acetonitrile, and 2-propanol (IPA).9 Form B can be formed by slowly cooling a hot aqueous solution 15% (w/w), from 343 K.8 It has been reported that the crystalline form B may also contain a small degree of amorphous, noncrystalline material,10 inhibiting the acquisition of adequate crystallographic data. Previous work on identifying different phases of cimetidine polymorphs has illustrated that the forms could not be differentiated by thermal techniques because of the similarity in their melting points. However, light microscopy, X-ray crystallography, and IR spectroscopy were found suitable for polymorphic differentiation.10,11 The crystal structure of form A has been determined by X-ray single-crystal diffraction which showed it to possess prismatic and monoclinic crystals12 of space group P21/C. Prodic-Kojic et al.13 have reported the cell constants to be a ) 6.82 Å, b ) 18.813 Å, c ) 10.374 Å, β ) 111.306°. However, similar structural determination has not been possible for form B, because the crystals are too thin.10 Since the inception of the AFM by Binnig et al. (1986),14 it has become an important tool for imaging the topography of various types of substrates at a range of resolutions, from the micrometers to the molecular scale.15,16 AFM has also been extended to the direct measurement of discrete intermolecular forces.17-24 The relatively recent development of TM-AFM27 has generally overcome the problems of sample damage which can occur when imaging (8) Hegedus, B.; Gorog, S. J. Pharm., Bio. Anal. 1985, 3, 303. (9) British Patent Specification 1 543 238, 1976. (10) Bauer-Brandl, A. Int. J. Pharm. 1996, 140, 195. (11) Tudor, A. M.; Davies, M. C.; Melia, C. D.; Lee, D. C.; Mitchell, R. C.; Hendera, P. J.; Church, S. J. Spectrochim. Acta 1991, 47A, 1389. (12) Bavin, P. M. G. Anal. Profiles Drug Subst. 1984, 13, 127. (13) Prodic-Kojiz, B.; Kajfes, F.; Belin, B.; Toso, R.; Sujic, V. Gazz. Chim. Ital. 1979, 109, 535. (14) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930. (15) Butt, H. J.; Jaschke, M.; Ducker, W.; Bioelectrochem. Bioener. 1995, 38, 191. (16) Hansma, H. G.; Hoh, J. H. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 115. (17) Hoh, J. H.; Cleveland, J. P.; Pratter, C. B.; Revel, J. P.; Hansma, P. K. J. Am. Chem. Soc. 1993, 114, 4917. (18) Boland, T.; Ratner, B. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5297. (19) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Guntherodt, H. J.; Misevic, G. Science 1995, 267, 1173. (20) Dammer, U.; Hegner, M.; Anselmetti, D.; Wagner, P.; Dreier, M.; Huber, W.; Guntherodt, H. J. Biophys. J. 1996, 70, 2437. (21) Florin, E. L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (22) Allen, S.; Chen, X.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Edwards, J. C.; Roberts, C. J.; Sefton, J.; Tendler, S. J. B.; Williams, P. M. Biochemistry 1997, 36, 7457. (23) Cappella, B.; Baschieri, P.; Frediani, C.; Miccoli, P.; Ascoli, C. IEEE Eng. Med. Biol. 1997, 58. (24) Allen, S.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. TIBTECH 1997, 15, 101. (25) Zhong, Q.; Inniss, D.; Kjoller, K.; Elings, V. B. Surf. Sci. 1993, 290, L688. (26) Magonov, S. N.; Elings, V.; Whangbo, M. H. Surf. Sci. 1997, 375, L385. (27) Akari, S. O.; Van der vegte, E. W.; Grim, P. C. M.; Belder, G. F.; Koutsos, V.; Ten Brinke, G.; Hadziioannous, G. Appl. Phys. Lett. 1994, 65, 1915.

10.1021/la990470a CCC: $19.00 © 2000 American Chemical Society Published on Web 10/14/1999

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soft or weakly adsorbed samples in contact mode AFM. In addition, the availability in TM-AFM of simultaneously acquired phase image26-30 provides further surface information. By mapping the phase shift of cantilever oscillation compared to the driving signal during a tapping mode scan, phase imaging facilitates detection of variations in composition, adhesion, friction, and viscoelasticity.31 Further surface-related information might also be obtained by measuring the amplitude and/or phase lag of cantilever oscillation against tip-sample distance.32 We refer to such data as amplitude phase distance (a-p,d) measurements. Previously AFM has been used to distinguish between polymorphs by high-resolution imaging.33,34 In this report, we propose an alternative method of utilizing AFM to distinguish between two polymorphic forms based upon phase imaging and a-p,d measurements and discuss the origin of the results in relation to surface physicochemical properties. Experimental Section Sample Preparation. Disks of mixtures of cimetidine polymorphs A and B (100/0, 50/50, 0/100) were prepared by placing approximately 200 mg of the material (SmithKline Beecham, Harlow, U.K.) in a press dye under vacuum pump at a pressure of 10 ton for 5 min. This produced disks of approximately 13 mm in diameter and 1 mm depth. FT-Raman spectroscopy was performed on crystals before and after compression revealing identical spectra (results not shown) suggesting no effect of compaction on polymorphic forms. Contact Angle Measurement. Static contact angles35 were measured in air using the sessile drop method by placing a 2 µL drop of distilled water using a microsyringe onto the surface of the sample. Contact angle measurements were made immediately using a Vickers Ealing goniometer (Ealing Electrooptics plc, U.K.). Measurements were made on both sides of the drop and repeated on at least four drops. AFM Imaging. A Nanoscope IIIa MultiMode AFM (Digital Instruments, Santa Barbara, CA) was employed. All images were acquired in tapping mode under ambient conditions, using the E-type scanner (10 µm × 10 µm × 2 µm) and silicon TESP tips (Digital Instruments). AFM images were acquired with cantilevers oscillating just below their resonant frequencies (ca. 300 kHz). All images were taken at a scan rate between 1 and 2 Hz, with a 512 × 512 pixel resolution. Tapping Mode Amplitude-Phase Distance Measurements (a-p,d). Tapping mode (TM) a-p,d measurements were conducted with the same AFM tips used for TM-AFM imaging. During the TM a-p,d measurements, the cantilevers were driven at a constant driving amplitude just below the cantilever’s free status resonance. Following the establishment of stable tapping, the constant-amplitude feedback control was temporarily cut off and a low-frequency oscillation (typically 1 Hz) voltage was applied on the z-piezo. This results in a periodic displacement (typically 100 nm) of the scanner in the z-direction (vertical to the sample surface), during which time the cantilever’s oscillation amplitude and phase angle with respect to the driving signal were recorded against the scanner’s relative displacement. Comparisons were made between a-p,d curves obtained from the surface of pure cimetidine disks in ambient air conditions (28) Magonov, S. N.; Cleveland, J.; Elings, V.; Denley, D.; Whangbo, M. H. Surf. Sci. 1997, 389, 201. (29) Schmitz, I.; Schreiner, M.; Friedbacher, G.; Grasser-bauer, M. Appl. Surf. Sci. 1997, 115, 190. (30) Spatz, J. P.; Sheiko, S.; Moller, M.; Winkler, R. G.; Reineker, P.; Marti, O. Langmuir 1997, 13, 4699. (31) Babcock, K. L.; Prater, C. B. Digital Instruments 1995. (32) Chen, X.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Davies, J.; Dawkes, A. C.; Edwards, J. C. Ultramicroscopy 1998, 75, 171. (33) Lotz, B. Macromol. Symp. 1995, 94, 97. (34) Snetivy, D.; Vancso, G. J.; Rutledge, G. C. Macromolecules 1992, 25, 7037. (35) Good, R. T. J. Adhes. Sci. Technol. 1992, 6, 1269.

Table 1. A List of Contact Angle Values (deg) for Cimetidine Polymorphs A and B no. of measurements

cimetidine A

cimetidine B

1 2 3 4 average

31.8 34.8 28.8 38.0 33.4

58.9 59.0 54.6 52.0 56.1

and in low humidity conditions. Humidity was reduced by enclosing the apparatus in an airtight enclosure containing dried silica gels for 2 h. A humidity and temperature indicator HMI 31 (Vaisala, Finland) was used to monitor humidity.

Results and Discussion Contact Angles. The contact angles measured for disks of cimetidine forms A and B are presented in Table 1. The values obtained indicate that the contact angles on the surface of cimetidine A disks (an average of 36°) are lower than those from the surface of cimetidine B disks (an average of 56°), indicating the increased hydrophilic nature of form A over B. AFM Images. Images of pure disks of cimetidine A and B are shown in parts a and c of Figure 1, respectively. The corresponding phase images (Figure 1b and Figure 1d) illustrate a one-component homogeneous surface with no significant phase contrast. This is due to a constant tip-sample interaction as the same species is present on the surface of each sample. The small phase contrast observed in these images results from topography-induced phase shifts. Panel e of Figure 1 shows a TM-AFM image of cimetidine disk composed of a 50:50 mixture of A and B. The phase image (Figure 1f) illustrates a two-component surface, with the presence of dark and light regions. Since, the phase lag of cantilever is sensitive to the tip-sample interaction, the contrast observed is related to differences in surface properties of cimetidine A and B. The darker regions, indicating a larger phase shift, are attributed to stronger attractive forces.32 A-p,d Measurements. To investigate the observed contrast in the phase data, an analysis of the a-p,d measurements was taken. The curves can reveal further detail of the tip-sample interaction by relating the amplitude or phase signal against tip-sample distance over a single point on the surface.32 Parts a and b of Figure 2 show a-p,d curves measured on the surface of pure cimetidine A and B disks, respectively. At point a in Figure 2a, the cantilever is vibrating at its resonant frequency (long distance away from the sample surface) and is moving toward the surface at a steady rate (solid line) without any changes in its amplitude or phase. At point b the cantilever is close enough to the surface that it starts to experience attractive dominated forces; thus the amplitude dampens and the phase lag increases (positive phase shift) as the tip moves toward the surface. At point c the tip enters a repulsive force dominated regime where the amplitude continues to decrease, but the phase starts to decrease (negative phase shift) as the tip continues to move forward until point d, a predetermined point of maximum load. The tip is then retracted from the sample surface, and the amplitude and phase shift of the tip are depicted by the thinner line. The phase contrast observed from the phase images corresponds to the difference in the phase-shift values for the A and B forms that occurs at the chosen amplitude set-point of a particular image.32 Parts a and b of Figure 2 illustrate that form A has a larger attractive region compared to form B, which indicates the presence of stronger attractive force between

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Figure 1. 10 µm × 10 µm images of (a) polymorph A disk, height image, (b) polymorph A disk phase image, (c) polymorph B disk height image, (d) polymorph B disk phase image, (e) 50:50 mixed disk height image, and (f) 50:50 mixed disk phase image. All images have a height scale of 300 nm and a phase scale of 180°.

the tip and the surface of form A disks.32 This stronger force of adhesion is also indicated by the hysteresis present between the extension and retraction curves and could be due to either surface properties of cimetidine A or the

presence of a contamination/ water layer. Such differences in the a-p,d curves for A and B can be employed to unequivocally identify the different forms in a mixed sample. An example of this for a 50:50 mixed disk can be

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Figure 2. Amplitude and phase to distance (a-p,d) curves from (a) polymorph A disk, (b) polymorph B disk, (c) dark region of a 50:50 mixed disk, and (d) light region of a 50:50 mixed disk.

seen in parts c and d of Figure 2 (note different AFM tip than that used for the data in Figure 2a,b). The a-p,d measurements from the dark regions of the 50:50 mixed disks (Figure 2c) correlate well with those obtained on the surface of pure cimetidine A disks (Figure 2a), and those obtained from the light regions of the same image (Figure 2d) correlate well with those from pure cimetidine B disks (Figure 2b). Contact angle measurements indicated a difference in hydrophilicity and hence wettability between the polymorphs, with form A being the more hydrophilic. To investigate the possible role of adsorbed water in inducing phase contrast, a-p,d curves were taken on the surface of pure cimetidine A and B disks at different relative humidity (RH) conditions (38% and 6.5%). For form B no changes in a-p,d data were observed (data not shown); however, for form A significant changes resulted (Figure 3a,b). It is clear that the length of the attractive region is reduced under low humidity conditions, and consequently, a significant part of the larger attractive force experienced between the tip and form A surface is attributed to capillary forces between the tip and a surface moisture layer. Conclusion

Figure 3. Amplitude and phase to distance (a-p,d) curve from the surface of polymorph A disk, in (a) ambient air and (b) low humidity.

We can conclude that TM-AFM can be used to distinguish between crystalline polymorphs A and B of cimetidine. Phase imaging may be used to demonstrate the presence of more than one polymorph in a mixed blend, and a-p,d curves can be used as a fingerprint to identify the different components of the blend, by providing information on repulsive/attractive property of the dominated tip-sample interactions. In the case of cimetidine,

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a-p,d curves illustrated that form A has a larger attractive region compared to form B, a significant part of which is shown to be due to attractive capillary forces. However, it is important to note that other polymorphic systems need to be investigated to evaluate the usefulness of the proposed technique as a generic means of polymorphic characterization. This application of AFM could be of particular use to the pharmaceutical development sector, since it combines polymorphic discrimination and surface property differentiation. The method proposed requires relatively

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small quantities of the sample with little preparation. We believe that TM-AFM could play an important role in identifying polymorphs and characterizing their surface properties. Acknowledgment. A.D. thanks SmithKline Beecham and the EPSRC for the provision of a studentship. G.W.H.S. thanks the BBSRC for funding. X.C. thanks Orthoclinical Diagnostics and the BBSRC for funding. LA990470A