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Enantiotropically Related Albendazole Polymorphs
Enantiotropically Related Albendazole Polymorphs MARCO B. PRANZO,1 DYANNE CRUICKSHANK,2 MASSIMO CORUZZI,1 MINO R. CAIRA,2 RUGGERO BETTINI1 1
Department of Pharmacy, University of Parma, Viale G.P. Usberti 27/A, 43124 Parma, Italy
2
Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
Received 9 October 2009; revised 23 November 2009; accepted 27 November 2009 Published online 28 January 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22072 ABSTRACT: In the present study we report the solid-state properties of albendazole (ABZ) re-crystallized from different solvents for comparison with the commercially available form. Crystalline phases were characterized as to thermal behavior, X-ray diffractometry, both on powder and single crystal, and solubility in methanol or 0.1 N HCl. The relevant thermodynamic parameters were calculated from solubility measurements at different temperatures. The recrystallization of ABZ both from methanol and N,N-dimethylformamide afforded a new stable polymorph form (Form II) enantiotropically related to the commercially available ABZ (Form I), the latter being the metastable form at ambient temperature. Both forms proved to be physically quite stable, likely due to a high-energy barrier for the activation of the interconversion. ABZ in the solid state represents a rather complex system in which the molecular structural differences that could be associated with the polymorphism are of at least four possible types, or combinations of these: (a) tautomeric; (b) different conformations of either or both of the side-chains attached to the bicyclic ring system; (c) the occurrence of molecular disorder or its absence; (d) no essential difference in molecular structure but different hydrogen bonding arrangements in the two polymorphs. ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3731–3742, 2010
Keywords:
albendazole; polymorphism; enantiotropy; crystal structure; solid state
INTRODUCTION Albendazole (ABZ), methyl [5-(propylthio)-1-H-benzimidazol-2-yl] carbamic acid methyl ester, is among the most effective broad-spectrum anthelmintic agents.1,2 The molecule was patented in 19753 and the drug is described as occurring as colorless crystals with melting point between 208 and 2108C, and being practically insoluble in water.4 ABZ therapy is very important in systemic cestode infections especially in inoperable or disseminated cases of hydatidosis1 and neurocystercosis1,5,6 both in human and veterinary medicine.7 ABZ is undetected7–10 or present at extremely low concentrations2,7,9,11–13 in blood plasma after oral administration in various animal species and in man, due to an extensive first-pass metabolism occurring in the enterocytes and in liver cells as well as the very low intestinal absorption stemming from the unfavorable aqueous solubility.5 Both these effects result in a low and erratic bioavailability. The assignment Correspondence to: Ruggero Bettini (Telephone: 390521905089; Fax: 390521905006; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 3731–3742 (2010) ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association
of ABZ to class II or IV of the Biopharmaceutics Classification System is still a matter of debate.14 Irrespective of this, the insufficient aqueous solubility is an issue that has to be addressed by pharmaceutical formulators. In this respect, solid dispersions with polyvinylpyrrolidone,5 liquid formulations with Transcutol115 and ABZ b-cyclodextrin complexes16 were prepared to improve the aqueous solubility and dissolution characteristics of ABZ. On the other hand, it is known that solid-state properties may play a crucial role in dissolution rate and solubility, especially when polymorphs with different thermodynamic stability are involved. These properties deeply influence all steps of drug product development from the drug candidate discovery, through its processability and production of the dosage form, up to the in vivo performance.17–19 In fact, differences in crystal packing as well as in lattice energy and entropy often result in significant changes in physical properties, such as density, hardness, tablettability, refractive index, melting point, enthalpy of fusion, vapor pressure, solubility, dissolution rate, as well as other thermodynamic and kinetic properties and even color between different polymorphs of the same drug substance.18
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Despite the fact that ABZ was discovered more than 35 years ago and its use in therapy is well established, no reports are presently available on its solid-state characterization nor on its possible polymorphism. The aim of the present work was to study the solidstate properties of ABZ re-crystallized from different solvents for comparison with the commercially available form. Crystalline phases were characterized as to thermal behavior, X-ray diffractometry, both on powder and single crystal, and solubility in methanol or 0.1 N HCl. The relevant thermodynamic parameters were calculated from solubility measurements at different temperatures.
MATERIALS AND METHODS ABZ Crystallization An amount corresponding to about 100 mg of ABZ (Sigma, Steinheim, Germany) was dissolved in 100 mL of methanol, (HPLC grade, 99.9% minimum assay, Carlo Erba Reagents, Milan, Italy) or N,Ndimethylformamide, DMF, (99.8% assay, Fluka Chemie GmBH, Buchs, Switzerland) and stirred at 408C until a clear solution was obtained. The filtered solution was allowed to spontaneously evaporate under ambient conditions until ABZ crystallization was complete. Colorless crystals formed after a few days from methanol, whereas light brown crystals were collected after about 1 month from DMF. Thermal Analysis Differential scanning calorimetry (DSC) was performed on an indium calibrated Mettler DSC 821e (Mettler Toledo, Columbus, OH) driven by STARe software (Mettler Toledo). DSC traces were recorded by placing precisely weighed quantities (1–5 mg) in a sealed and pierced 40 mL aluminium pan. Scans were performed between 30 and 2258C at 5 K min1 or between 30 and 2708C at 40 K min1 under a flux of dry nitrogen (100 mL min1). Each powder sample was analyzed at least in triplicate. Thermogravimetric analysis (TG 50, Mettler Toledo) was carried out on ABZ samples placed in 70 mL alumina pans with a pierced cover. Samples were heated under a flux of dry nitrogen (100 mL min1) at 5 K min1 in the 30–2258C temperature range.
Single Crystal X-Ray Analysis of Form II of ABZ Microscopic examination of many batches of ABZ obtained by re-crystallization from methanol and DMF revealed a constant crystal morphology, namely poorly formed laminae with typical thickness only 10 mm. A specimen viewed normal to the thin section is shown in Figure 1. Eventually, a specimen with thickness 0.06 mm was identified and cut to a more equant shape (0.18 mm 0.18 mm 0.06 mm) to minimize X-ray absorption error and ensure sufficient volume for adequate X-ray diffraction intensity. This was mounted on a cryoloop with Paratone oil (Exxon Chemical Co., Houston, TX), placed on a Nonius Kappa CCD four-circle diffractometer and cooled in a stream of nitrogen vapor to 213(2) K using an Oxford Cryostream cooler (Oxford Cryosystems, Oxford, UK) to ensure a rigid mount. No phase change accompanied the cooling process. Data collection (COLLECT software20) with Mo ˚ ) involved a combination Ka-radiation (l ¼ 0.71073 A of f- and v-scans of 1.008 and 0.508, respectively. All data were corrected for Lorentz-polarization effects. Absorption effects were negligible for the specimen used (transmission range 0.9555–0.9858). Unit cell refinement and data-reduction were performed using DENZO-SMN and SCALEPACK.21 The monoclinic crystal system was established from the Laue symmetry (2/m) and the space group C2/c from systematic absences, centrosymmetry being indicated by the hE2 1i value of 0.922. The structure was solved using direct methods (program SHELXS-9722) and refined by full-matrix least-squares against F2 (program SHELXL-9723). After location of the nonhydrogen atoms of the asymmetric unit (a single molecule of ABZ), it was noted that the propylthio-chain was disordered over two chemically equivalent positions. Each component was modeled with a fixed site occupancy of 0.5, based on very similar electron densities for the atoms comprising the two side-chains as well as intermolecular distance constraints. Following treatment of the non-H atoms, special care was exercised in the location of hydrogen atoms, given the possibility of
X-Ray Diffraction Studies X-ray diffraction patterns on powder were recorded on a bench-scale diffractometer (Rigaku, Tokyo, Japan) using a Cu Ka radiation source at 30 kV voltage over the scanning range (2u) 5–358 (scanning speed of 0.058/min). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
Figure 1. Typical morphology of a crystal of albendazole obtained by re-crystallization from methanol or DMF. DOI 10.1002/jps
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tautomerism for this molecule. All H atoms were located unequivocally in difference Fourier electron density maps. In particular, both N atoms of the imidazole ring were found to bear H atoms, while no electron density attributable to a hydrogen atom was found on the exocyclic N atom. Figure 2a shows the conventional structural diagram for ABZ and Figure 2b the structure of the tautomer found in the crystal of Form II. The disordered model was completed with half-atoms of hydrogen added at positions C7, C8 of the phenyl ring. Except for one of the atoms of a disordered chain component (C18A), all non-H atoms refined anisotropically. H atoms were generally added in a riding model at idealized positions based on their stereochemistries, clearly established from electron density maps, and were assigned isotropic thermal parameters 1.2–1.3 times those of their parent atoms. Crystal data and refinement parameters are reported in Table 1. Molecular parameters were calculated with program PLATON.24 Solubility Determination The solubility of both commercially available and re-crystallized ABZ was determined at 258C in CH3OH or HCl 0.1 N. The latter solubility determination was also performed in the 25–1008C interval. An excess of drug was suspended in the selected solvent and stirred with a Vortex three times within 1 h. Thereafter, the suspension was allowed to settle for 48 h, then it was filtered (0.45 mm) and the filtrate was diluted and analyzed by HPLC to determine the ABZ
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Table 1. Crystal Data and Refinement Parameters for Form II of Albendazole Parameter Chemical formula Formula weight Dcalc (g cm3) Crystal system Space group ˚) a (A ˚ b (A) ˚) c (A a (8) b (8) g (8) ˚ 3) V (A Formula units (Z) m (Mo Ka) (mm1) T (K) Reflections Parameters Completeness (%) R1 (on F > 4s ( F)) wR2 (on F2, F > 4s ( F)) Goodness-of fit (S) Final D/s ˚ 3) Dr min.; max. (e A
Form II C12H15N3O2S 265.33 1.401 Monoclinic C2/c 23.934(5) 5.440(1) 19.586(6) 90.0 99.29(1) 90.0 2516.6(10) 8 0.255 213(2) 2157 204 97.8 0.0723 0.1547 1.033 <0.001 0.257; 0.259
concentration. Each determination was performed in triplicate. High-Performance Liquid Chromatography (HPLC) ABZ in solution was quantified according to the method described in the European Pharmacopoeia25 using an isocratic HPLC system (LC-10 ATvp, Shimadzu, Tokyo, Japan) equipped with a Spherisorb1 column (5 mm, 4.6 mm 250 mm, ODS2, Waters, Milford, MA) and a DAD detector (SPDM10Avp, Shimadzu) set between 250 and 260 nm. The volume of the injected samples was 20 mL (Rheodyne injector 7010). As mobile phase a mixture consisting of 30% (v/v) of ammonium dihydrogen phosphate solution (1.67 g L1) and 70% (v/v) of CH3OH at a flow rate of 0.7 mL min1 was used. ABZ retention time was 6 min. The method was validated for linearity (R2 ¼ 0.999), limit of detection (1.1 mg mL1), limit of quantification (3.6 mg mL1) and relative standard deviation (<1.5%).
RESULTS AND DISCUSSION Figure 2. The conventional structural diagram for albendazole (a) and the structure of the tautomer occurring in the stable crystal Form II (b). The latter also shows the disorder of the propylthio-chain established in this study, each component being present at 50% site occupancy. DOI 10.1002/jps
For ABZ re-crystallization, two polar solvents were selected, one protic and one aprotic, differing mainly in their volatility (boiling points 64.8 and 1538C for methanol and DMF, respectively). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
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As stated above the microscopic examination of ABZ crystals obtained from both methanol and DMF revealed a laminar morphology. The different solvent volatility resulted in a larger average dimension for crystals obtained from DMF than from methanol likely due to the slower crystal growth: as the supersaturation was attained slowly, as in the case of DMF, crossing of the metastable region was slow; then the process of particle growth overtook the process of nucleation, eventually leading to large crystals. The commercial product consisted of very small crystals showing a strong tendency to aggregation (Fig. 3). TGA analysis carried out at the scanning rate of 5 K min1 on the different ABZ specimens indicated a weight loss of about 1.5% and about 13% in the 30– 1508C and 175–2258C intervals, respectively. No significant differences were observed among the samples tested. This indicates that the re-crystallization process did not result in the formation of solvate forms. In fact the theoretical content of solvent for a ABZ monosolvate form would be 21.6% and 10.8% (w/w) for DMF and methanol, respectively, whereas the water content by weight in a hypothetical monohydrate form would be 6.3%. DSC traces recorded from the commercial ABZ in the 30–2258C range at 5 K min1 (Fig. 4A) showed a single broad endothermic peak at 202.3 2.28C (onset 189.1 2.98C) likely ascribable to final melting with decomposition. This interpretation is supported by the literature data4 and by the above mentioned weight loss recorded in the melting interval by TGA. The traces relevant to the ABZ re-crystallized both from methanol and from DMF were not different from that of the commercial form, except for a small endothermic phenomenon at 1508C (onset 1428C) immediately followed by an exotherm. The DSC traces recorded upon scanning at higher rate (40 K min1) (Fig. 4B) put in better evidence some interesting features: the curve for the commercial material, again, presented just one endothermic peak around 2208C (onset 212.8 2.28C), whereas the re-crystallized materials, in addition to the peak around 2208C, presented also a clear endothermic peak at about 1608C, closely followed by an exothermic one. Although the two phenomena could not be separated, the enthalpy change associated with the first one was measured. The relevant data are reported in Table 2. It should be emphasized that the differences between the enthalpies associated with both the first and the second peak of the studied materials were not statistically significant (Student’s t-test: p > 0.05). Despite the large number of papers available in literature presenting the thermal behavior of ABZ JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
Figure 3. Pictures taken at the polarized light microscope of (a) commercial albendazole crystals and re-crystallized albendazole (b) from methanol, (c) from DMF. Original magnification 20.
alone or in various carriers26–29 this is the first time that a thermal event other than final melting is presented for ABZ. We hypothesized that the endo–exo thermal events observed between 140 and 1808C in the re-crystallized ABZ could be ascribed to the melting of a crystal form not yet described in literature, followed by a recrystallization leading to a crystal phase corresponding to the commercial one that eventually melted with decomposition. This new crystal phase stems from the re-crystallization process. DOI 10.1002/jps
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Figure 4. DSC traces of commercial albendazole (curves a), and albendazole re-crystallized from DMF (curves b) and from methanol (curves c). Scan carried out at 5 K min1 (panel A) and at 40 K min1 (panel B).
The hypothesis was verified by X-ray diffraction on powder. Figure 5 reports the diffraction patterns obtained from the commercial and re-crystallized ABZ crystals. The comparison between the pattern of the commercially available material and that of the ABZ re-crystallized from DMF (Fig. 5a) evidenced some differences related to the shape of the peaks at 78 and around 258 2u and the presence of three peaks (10.58, 158, and 308 2u) in the pattern of the recrystallized product that are not observed in that of the commercial one. Moreover, the latter shows a peak around 11.58 2u not present in the re-crystallized ABZ. On the other hand, the diffraction patterns of the re-crystallized materials were practically superimpo-
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sable (Fig. 5b). The only noticeable difference was represented by the higher intensity of the diffraction peaks of ABZ re-crystallized from DMF, most likely ascribable to different extents of crystallinity and preferred orientation. On the basis of these findings it can be affirmed that the re-crystallization of ABZ at ambient temperature both from methanol and DMF leads to a new polymorph (Form II) having a melting temperature at 1608C which re-crystallizes during the DSC scan to give rise to the polymorph melting at 2208C (Form I). The fusion and re-crystallization of the low melting polymorph can be better evidenced when a high scanning rate is used in the thermal analysis. The remarkable similarity between the DSC curves of the two re-crystallized products indicates that the polymorphs obtained upon solvent evaporation may depend on the crystallization conditions, in particular the temperature, rather than on the type of solvent used in the re-crystallization process. These observations allow us to assume that the two polymorphs likely constitute an enantiotropic pair with a transition temperature lower than 1408C; in this respect, the lower-melting crystal would represent the stable form at ambient temperature. The solubility in methanol and 0.1 N HCl aqueous solution was determined at 258C, both for investigating possible differences between commercial and recrystallized ABZ and to serve as a coarse indicator of the relative thermodynamic stability of the two crystal forms. The values obtained are reported in Table 3. ABZ Form I (commercial) proved to be more soluble than Form II (re-crystallized from DMF). The difference was low but statistically significant in both solvents ( p < 0.01 by Student’s t-test). It is worth stressing that the accurate determination of equilibrium solubility of two polymorphs is often a difficult task as solution-mediated conversion may occur in the timeframe of the measurements. Furthermore, we are aware that the approach is conceptually incorrect since, by definition, only the stable form is ‘‘thermodynamically allowed’’ to achieve equilibrium solubility. With respect to this concern DSC analysis was carried out on the solid phases recovered at the end of the equilibrium solubility experiments. For both materials no change of phase was noticed, thus it was concluded that ABZ Form I, although
Table 2. Peak Temperatures and Relevant Enthalpies of ABZ Samples
Commercial product Re-crystallized from DMF Re-crystallized from methanol
1st Peak, 8C
DH 1st Peak, J g1
2nd Peak, 8C
DH 2nd Peak, J g1
— 159.0 (0.9) 160.3 (0.5)
— 10.6 (2.6) 8.4 (0.2)
220.7 (0.1) 220.4 (0.6) 218.7 (1.78)
133.5 (2.1) 119.7 (18.9) 137.2 (5.6)
Standard deviation in parenthesis (n ¼ 3). DOI 10.1002/jps
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Figure 5. X-ray diffraction patterns on powder of albendazole samples: (panel a) commercial and re-crystallized from DMF; (panel b) re-crystallized from DMF and re-crystallized from methanol.
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Table 3. Solubility of Commercial or DMF Re-Crystallized ABZ in Methanol and in HCl 0.1 N Aqueous Solution at 258C
Commercial product (Form I) Re-crystallized from DMF (Form II)
Solubility in Methanol
Solubility in HCl 0.1 N
1.45 (0.47) 0.72 (0.05)
0.54 (0.17) 0.30 (0.07)
Standard deviation in parenthesis (n ¼ 3).
thermodynamically unfavorable at 258C, was kinetically stable enough for allow the determination of a reliable solubility value. The differences in solubility observed are useful to gain some insights into the thermodynamic relationship between the two polymorphs. Form II displays the lower solubility in both solvents; therefore, it should be considered the more stable form at 258C. Furthermore, the possibility to determine the temperature range of thermodynamic stability by measurements of equilibrium solubility of different polymorphs is well established.30–32 Therefore, in order to verify the correctness of our assumption about the enantiotropic relationship, the ABZ II ! I transition temperature was estimated by measuring the ABZ solubility in aqueous HCl solution at different temperatures in the 25–1008C interval. This solvent was selected because of the ease of analysis. The obtained data are reported in Figure 6 both as concentration (mass per unit volume) versus temperature (panel A) and as classical van’t Hoff plot (panel B). For both plots in Figure 6 the regression lines (second order polynomial and linear regression for panel A and B, respectively) relevant to the solubility data of the two forms intersect at around 808C. This point represents the solid–solid transition temperature and this observation confirms that ABZ Form I represents the metastable polymorphs at ambient temperature. It can be noted from Figure 6 panel B that in the temperature range studied a linear relationship exists between the logarithm of the solubility and the inverse of the absolute temperature (R2 ¼ 0.9 and 0.94 for Form I and Form II, respectively). Therefore, the apparent enthalpies of solution of the two polymorphs can be calculated from the relevant slope according to the equation lnCs ¼
DHs 1000 þb T R
(1)
where Cs is the molar solubility, DHs is the heat of solution at saturation, R is the gas constant, T is the absolute temperature, and b is a constant. DOI 10.1002/jps
Figure 6. Solubility of albendazole polymorphs in the 25–1008C range in 0.1 N aqueous HCl solution represented in mass per unit volume (panel A) and as van’t Hoff type plot (panel B). Form I square, Form II circle. The bars represent the standard deviation.
The values obtained were 21.2 and 10.4 kJ mol1 for Form II and Form I, respectively. From these figures the enthalpy of transition from Form I to Form II, DHI ! II, was calculated as 10.8 kJ mol1. At constant temperature and pressure the free energy difference between the two Forms, DGT, can be calculated from the logarithm of the ratio between the relevant solubility values DGT ¼ RT ln
Csmetastable Csstable
(2)
At 258C the free energy change computed from the solubility data obtained in HCl aqueous solution was 1.5 kJ mol1. This value is not significantly different from that computed from solubility data obtained in methanol at the same temperature (1.7 kJ mol1), thus justifying the calculation of the thermodynamic relationships on the basis of the assumption that Henry’s law applies and the obtained values are independent of the solvent used. The values of DGT are reported in Figure 7 as a function of the absolute temperature in the 25–1008C temperature interval. In this plot the transition JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
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Figure 7. Gibbs free energy variation as a function of the absolute temperature for the albendazole Form I and Form II system.
temperature can be graphically evaluated by looking at the point where DGT becomes zero. Finally, the entropy change for the transition of Form I to Form II at a particular temperature T, was calculated from the following equation DST ¼
DHI!II DGT T
(3)
At 258C DST is 31.5 J K1 mol1. At the transition point (350 K) DGT is 0 and Eq. (3) affords the entropy change as 30.9 J K1 mol1. Thermodynamic relationships are summarized in Figure 8 that provides a semi-quantitative energy versus temperature representation and highlights the relevant transition points as well as the relative stability of the two ABZ Forms.
These data put into evidence that the newly isolated ABZ crystal phase and the commercially available one constitute an enantiotropic pair, the commercially available Form being metastable at ambient temperature and thermodynamically stable above 350 K. It is worth emphasizing that for pharmaceutical applications attention to these new findings is advised. In fact, the use of a metastable form may be advantageous (e.g., for exploiting higher solubility in the GI tract) only when the kinetics of conversion would be slow, namely when the energetic barrier Form I $ Form II cannot be overcome during specific storage conditions. In this respect, the metastable phase should be kept cool and dry and should not be too finely subdivided.31 On the other hand, one should be aware that many pharmaceutical operations may cause an undesired change from the metastable to the stable Form, especially those in which the conversion could be mediated by solubilization.33 As a general consideration, ABZ Form I is evidently physically (kinetically) stable, being a commercialized product (although with the warning: keep under refrigeration). Form II proved to be physically quite stable as well, as indicated by DSC experiments in which a scanning rate of 5 K min1 was high enough to allow it to ‘‘pass through’’ the transition temperature and to put into evidence the melting and recrystallization of Form II (see, Fig. 4A). We also heated Form II at 1108C in a dry nitrogen atmosphere under static conditions for 48 h without detecting any phase change. A progressive Form II ! I transition was instead observed upon heating the ABZ Form II at 1308C. This condition afforded complete conversion in <20 h. X-Ray Structure of Form II The molecular and crystal structure reported here is the first for any ABZ crystal modification, despite the longevity of this compound. This is likely due to the practical difficulty of obtaining suitable single crystals. Figure 9 shows the molecular structure occurring in the crystal of Form II. Despite the fairly severe limitations of crystal quality and size mentioned above, as well as significant molecular disorder, the
Figure 8. Semi-quantitative energy versus temperature diagram for the albendazole Form I and Form II pair. G, Gibbs free energy; H, enthalpy; Ttrans, transition temperature; TmI and TmII, melting temperature for Form I and Form II, respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
Figure 9. Structure and atomic numbering of the albendazole molecule in Form II. Thermal ellipsoids are drawn at the 50% probability level. DOI 10.1002/jps
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Table 4. Selected Molecular Parameters for Form II of Albendazole
Table 5. Hydrogen Bond Data for Form II of Albendazole D–H A
˚) Bond Distances (A N1–C2 N10–C2 N1–C5 C11–O12 Bond angles (8) C5–N1–C2 N1–C2–N3 N3–C2–N10
a
1.348(5) 1.330(5) 1.394(5) 1.221(5)
N3–C2 N3–C4 N10–C11 C11–O13
1.344(5) 1.396(5) 1.345(5) 1.364(5)
109.0(3) 108.2(4) 130.8(4)
C4–N3–C2 N1–C2–N10 C2–N10–C11
109.2(3) 120.9(4) 118.7(3)
structural refinement proceeded reasonably well, all parameters converging satisfactorily and no abnormal residual electron density being evident. The most significant molecular features are the locations of the H atoms on N1 and N3, and the twofold disorder of the propylthio-chain. As expected, the atoms of the side-chains display significantly higher thermal vibration than the rest of the molecule. The five-membered ring is symmetrical, the bond distances N1–C2 and N3–C2 being equal and longer than N10–C2 (Tab. 4), in accord with the formal bond orders indicated in Figure 2b. The corresponding N–C and N – – C distances in N-(2benzimidazolyl)-O-methyl carbamate (refcode SEDZUW34), which has the same formal bonding arrangement, are respectively 1.352(4), 1.364(6), and ˚ . These values do not differ significantly 1.328(5) A from their counterparts listed in Table 4. Further ring symmetry is indicated in the present study by the equality of the endocyclic angles at N1 and N3. The bond angle N3–C2–N10 exceeds N1–C2–N10 by 108
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N1–H1 N10 N3–H3 O12b N3–H3 O12
˚) D–H (A
˚) H A (A
˚) D A (A
D–H A (8)
0.87 0.87 0.87
1.96 2.20 2.25
2.816(5) 2.928(4) 2.735(5)
168 140 115
a b
1/2 x, 5/2 y, 2 z. 1/2 x, 1/2 þ y, 3/2 z.
due to formation of a six-membered ring as a result of intramolecular hydrogen bonding (N3–H3 O12, Tab. 5). Atom H3 is in fact involved in an intermolecular H-bond as well, as described below. The formal bonding pattern C2 – N10–C11 occurring in this tautomer is also easily distinguished from the alternative tautomeric possibility C2–N10(H)–C11 by the value of the bond angle subtended at N10 (118.7(3)8 in the present case), which is the same as the reported value of 118.5(4)8 in N-(2-benzimidazolyl)-O-methyl carbamate. Instead, in benzimidazole analogues in which N10 bears a hydrogen atom, and either one or both endocyclic N atoms bear a hydrogen atom, the angle subtended at N10 is significantly larger (range 121.8–123.78 in the closely related molecules BEWLOF,35 BMCBIB,36 QESBOG37). Thus, there is ample evidence from the present Xray structure that the ABZ tautomer in Form II corresponds to that shown in Figure 2b. Molecular association via intermolecular hydrogen bonding is extensive, with two well-defined motifs occurring, one finite and the other infinite. The former is a centrosymmetric dimer with graph set R22 (8),38 shown at the top of Figure 10, in which the
Figure 10. Stereoview showing details of the primary hydrogen bonding motifs and the generation of nonparallel molecular stacks in the crystal structure of albendazole, Form II. DOI 10.1002/jps
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unique H-bond is N1–H1 N10a (Tab. 5). The second is a continuous spiral array of molecules parallel to the crystallographic twofold screw axis (unique Hbond N3–H3 O12b). The net effect of these interactions is to produce infinite stacks of dimers, alternate stacks along the c-axis being rotated by 808 relative to each other. This is evident in the packing diagram projected down the c-axis while the full hydrogen bonding scheme is evident in the b-axis projection (Fig. 11). Nearly isostructural hydrogen bonding networks occur in the crystal structure of N-(2benzimidazolyl)-O-methyl carbamate6. Other interactions such as p-stacking and C–H O interactions do not play a significant role in the present crystal structure. The experimental and computed PXRD patterns for Form II of ABZ are shown in Figure 12, together with the experimental PXRD pattern of the commercial material, Form I. The three most intense reflections in the computed pattern39 are those occurring at 2u values 7.498, 24.968, and 25.958. The primary contributors to these three characteristic peaks (with their relative intensity contributions in parentheses) are reflections from the crystal planes 200 (1.00), 510 (0.55), and 511 (0.56), respectively. The high intensities of the first two peaks may be rationalized with
Figure 11. Crystal packing diagrams projected down the c-axis (top), the b-axis (bottom). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
Figure 12. PXRD patterns for albendazole: experimental (a) and computed (b) of Form II, and the experimental pattern of the metastable Form I (c).
reference to the packing diagrams (Fig. 11), as follows. In addition to having a high inherent ˚ ) are multiplicity, the 200 planes (d ¼ 11.810 A straddled by the ‘‘heavy’’ sulfur atoms (Fig. 11, bottom). The 510 planes are populated by the closely ˚ ) running diagonally (/) in spaced sheets (d ¼ 3.567 A Figure 11 (top) while the geometrically equivalent planes, with indices 510 coincide with sheets inclined along the other diagonal (\). These striking packing features can thus easily be reconciled with the X-ray data. (In the case of the third strong peak, rotation of Fig. 11 (top) around the b-axis is necessary to view the relevant planes 511; these coincide with a ˚] set of closely stacked molecular sheets [d ¼ 3.434 A crystallographically distinct from those described above.) The 2u values 7.498, 24.968, and 25.958 for the three most intense reflections in the computed pattern correspond with those at 2u ¼ 7.308, 24.658, and 25.558, respectively, in the experimental PXRD pattern. Peak locations at slightly higher angles in the former case are due to unit cell shrinkage at the lower temperature of the single crystal X-ray analysis compared with ambient temperature for the experimental PXRD record. Relatively poor agreement between corresponding peak intensities in the computed and experimental PXRD patterns indicates significant preferred orientation in the sample. This is not surprising given the typical morphology shown in Figure 1. As regards Form I, the commercial material whose structure is unknown, the presence of strong peaks in the experimental PXRD pattern around 2u 7.08 and 24–258 (Fig. 12) suggests that similar crystal DOI 10.1002/jps
ENANTIOTROPICALLY RELATED ALBENDAZOLE POLYMORPHS
packing features to those described above for Form II might obtain.
CONCLUSIONS From the presented data it can be concluded that the re-crystallization of ABZ both from methanol and DMF affords a new stable polymorphic form enantiotropically related to the commercially available ABZ, the latter being the metastable form at ambient temperature. Both forms proved to be physically quite stable, likely due to a high-energy barrier for the activation of the interconversion. ABZ represents a rather complex system in which the molecular structural differences that could be associated with the polymorphism are of at least four possible types, or combinations of these: (a) tautomeric (with e.g., both N atoms of the 5-ring protonated [as in the crystal structure determined in the present work], or with only one of these two N atoms protonated [as in the conventional structural depiction of the ABZ molecule]); (b) different conformations of either or both of the side-chains attached to the bicyclic ring system; (c) the occurrence of molecular disorder (as we have for the propylthioside chain) or its absence; (d) no essential difference in molecular structure but different hydrogen bonding arrangements in the two polymorphs. The findings reported in the present paper clearly put into evidence the need both for ABZ manufacturers and pharmaceutical formulators to carefully consider the crystallization procedure and to perform specific test to characterize the obtained crystal phase as well as to pay particular attention to possible undesired phase change (from the metastable to the stable) stemming from pharmaceutical operations implying heating, mechanical stress, or partial solubilization.
SUPPORTING INFORMATION The CIF file for the single crystal X-ray structure described has been deposited at the Cambridge Crystallographic Data Centre (CCDC 736654).
ACKNOWLEDGMENTS This work was supported by a grant of the Italian Ministry of Education University and Research (MIUR) through the PRIN 2006 program. MRC and RB also wish to acknowledge financial support of the Executive Programme of Scientific and Technological Co-operation between the Italian Republic and Republic of South Africa 2008–2010. DOI 10.1002/jps
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REFERENCES 1. Cook GC. 1990. Use of benzimidazole chemotherapy in human helminthiases: Indications and efficacy. Parasitol Today 6:133– 136. 2. Marriner S. 1986. Anthelmintic drugs. Vet Rec 118:181–184. 3. Gyurik RJ, Theodorides VJ. 1975. Methyl 5-propylthio2-benzimidazolcarbamate. US patent, ed.: SmithKline Corporation. 4. O’Neil MJ, editor. 2006. The Merck Index. 40th edition. Whitehouse Station, NJ: Merch Research Laboratoires. p 39. 5. Torrado S, Torrado S, Torrado JJ, Caerdoniga R. 1996. Preparation, dissolution and characterisation of albendazole solid dispersions. Int J Pharm 140:247–250. 6. Del Brutto OH, Sotelo J, Roman GC. 1993. Therapy for neurocysticercosis: A reappraisal. Clin Infect Dis 17:730–735. 7. Batzias GC, Theodosiadou E, Delis GA. 2004. Quantitative determination of albendazole metabolites in sheep spermatozoa and seminal plasma by liquid chromatographic analysis with fluorescence detection. J Pharm Biomed Anal 35:1191–1202. 8. Swarnkar CP, Sanyal PK, Singh D, Khan FA, Bhagwan PS. 1998. Comparative disposition kinetics of albendazole in sheep following oral and intraruminal administration. Vet Res Commun 22:545–551. 9. Mirfazaelian A, Dadashzadeh S, Rouini MR. 2002. Effect of gender in the disposition of albendazole metabolites in humans. Eur J Clin Pharmacol 58:403–408. 10. Mirfazaelian A, Rouini MR, Dadashzadeh S. 2002. Dose dependent pharmacokinetics of albendazole in human. Biopharm Drug Dispos 23:379–383. 11. Alvarez LI, Sanchez SF, Lanusse CE. 1997. Modified plasma and abomasal disposition of albendazole in nematode-infected sheep. Vet Parasitol 69:241–253. 12. Prichard RK, Hennessy DR, Steel JW, Lacey E. 1985. Metabolite concentrations in plasma following treatment of cattle with five anthelmintics. Res Vet Sci 39:173–178. 13. Garcia JJ, Bolas-Fernandez F, Torrado JJ. 1999. Quantitative determination of albendazole and its main metabolites in plasma. J Chromatogr B 723:265–271. 14. Lindemberg M, Kopp S, Dressman JB. 2004. Classification of orally administered drugs on the World Health Organization Model list of Essential Medicines according to the biopharmaceutics classification system. Eur J Pharm Biopharm 58:265– 278. 15. Torrado S, Torrado S, Caerdoniga R, Torrado JJ. 1996. Formulation parameters of albendazole solution. Int J Pharm 140:45–50. 16. Castillo JA, Palomo-Canales J, Garcia JJ, Lastres JL, Bolas F, Torrado JJ. 1999. Preparation and characterization of albendazole beta-cyclodextrin complexes. Drug Dev Ind Pharm 25: 1241–1248. 17. Haleblian J, McCrone W. 1969. Pharmaceutical applications of polymorphism. J Pharm Sci 58:911–929. 18. Grant DJW. 1999. Theory and origin of polymorphism. In: Brittain GH, editor. Polymorphism in pharmaceutical solids, ed. New York: Marcel Dekker. pp. 1–33. 19. Vippagunta SR, Brittain HG, Grant DJW. 2001. Crystalline solids. Adv Drug Del Rev 48:3–26. 20. Nonius BV. 2002. COLLECT Nonius. Delft, the Netherlands: Nonius BV. 21. Otwinowski Z, Minor W. 1997. Processing of X-ray diffraction data collected in oscillation mode. In: Carter CW, Sweet RM, editors. Methods in enzymology, ed. New York: Academic Press. pp. 307–326. 22. Sheldrick G. 1990. Phase annealing in SHELX-90: Direct methods for larger structure. Acta Crystallogr A46:467–473. 23. Sheldrick GM. 1997. SHELX-97: Program for the refinement of crystal structures. University of Go¨ ttingen, Germany. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
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24. Spek AL. 1990. PLATON: A multipurpose crystallographic tool. Acta Crystallogr A46:C34. 25. 2008. Monograph 01/2008:1386. European Pharmacopoeia 6.0, ed. Strasbourg, France: EDQM. pp. 1122–1123. 26. Alanazi FK, El-Badry M, Ahmed MO, Alsarra IA. 2007. Improvement of albendazole dissolution by preparing microparticles using spray-drying technique. Sci Pharm 75:63–79. 27. Moriwaki C, Costa GL, Ferracini CN, de Moraes FF, Zanin GM, Pineda EAG, Matioli G. 2008. Enhancement of solubility of albendazole by complexation with b-cyclodextrin. Brazilan J Che Eng 25:255–267. 28. Kalaiselvan R, Mohanta GP, Manna PK, Manavalan R. 2006. Studies on mechanism of enhanced dissolution of albendazole solid dispersions with crystalline carriers. Indian J Pharm Sci 68:599–607. 29. Kalaiselvan R, Mohantha GP, Manna PK, Manavalan R. 2006. Multicoponent system of albendazole with cyclodextrins and hydroxyacids. Acta Pharm Sci 48:19–33. 30. Aguiar AJ, Zelmer JE. 1969. Dissolution behavior of polymorphs of chloramphenicol palmitate andmefenamic acid. J Pharm Sci 58:983–987. 31. Carstensen JT. 2001. Polymorphism. In: Carstensen JT, editor. Advanced Pharmaceutical Solids, ed. New York: Marcel Dekker. pp. 117–131. 32. Brittain HG, Grant DJW. 1999. Effect of polymorphism and solid-state solvation on solubility and dissolution rate. In: Brittain HG, editor. Polymorphism in pharmaceutical solids, ed. New York: Marcel Dekker. pp. 291–329.
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33. Bettini R, Bonassi L, Castoro V, Rossi A, Zema L, Gazzaniga A, Giordano F. 2001. Solubility and conversion of carbamazepine polymorphs in supercritical carbon dioxide. Eur J Pharm Sci 13:281–286. 34. Sokol VI, Davydov VV, Porai-Koshits MA, Zaitsev BE, Palishkin MV, Sheban GV, Pakhomov VI, Kukalenko SS. 1988. Crystal and molecular structure of N-(2-benzimidazolyl)-Oalkyl carbamates. Izv Akad Nauk SSSR Ser Khim (Russ) (Russ Chem Bull) 37:1306–1311. 35. Iriepa I, Villasante FJ, Galvez E, Bellanato J, Martin A, Gomez-Sal P. 2004. Synthesis, spectroscopic and crystallographic study of some carbamates from an azabicyclic chloroformate and primary heterocyclic amines. New J Chem 28:618–624. 36. Blaton NM, Peeters OM, De Ranter CJ. 1980. (5-Benzoyl-1Hbenzimidazol-2-yl)carbamic acid methyl ester hydrobromide (Mebendazole.HBr), C16H14BrN3O3. Cryst Struct Commun 9:181–186. 37. Sepassi K, Nichol GS, Yalkowsky SH. 2006. The napsylate salt of carbendazim. 2-(Methoxycarbonylamino)benzimidazolium naphthalene-1-sulfonate. Acta Crystallogr Sect E: Struct Rep Online E62:o5172–o5173. 38. Etter MC. 1990. Encoding and decoding hydrogen bond patterns of organic compounds. Acc Chem Res 12:120– 126. 39. Yvon K, Jeitschko W, Parthe E. 1977. LAZY PULVERIX: A computer program for calculating X-ray and neutron diffraction powder patterns. J Appl Crystallogr 10:73–74.
DOI 10.1002/jps
Department of Pharmacy, University of Parma, Viale G.P. Usberti 27/A, 43124 Parma, Italy
2
Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
Received 9 October 2009; revised 23 November 2009; accepted 27 November 2009 Published online 28 January 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22072 ABSTRACT: In the present study we report the solid-state properties of albendazole (ABZ) re-crystallized from different solvents for comparison with the commercially available form. Crystalline phases were characterized as to thermal behavior, X-ray diffractometry, both on powder and single crystal, and solubility in methanol or 0.1 N HCl. The relevant thermodynamic parameters were calculated from solubility measurements at different temperatures. The recrystallization of ABZ both from methanol and N,N-dimethylformamide afforded a new stable polymorph form (Form II) enantiotropically related to the commercially available ABZ (Form I), the latter being the metastable form at ambient temperature. Both forms proved to be physically quite stable, likely due to a high-energy barrier for the activation of the interconversion. ABZ in the solid state represents a rather complex system in which the molecular structural differences that could be associated with the polymorphism are of at least four possible types, or combinations of these: (a) tautomeric; (b) different conformations of either or both of the side-chains attached to the bicyclic ring system; (c) the occurrence of molecular disorder or its absence; (d) no essential difference in molecular structure but different hydrogen bonding arrangements in the two polymorphs. ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3731–3742, 2010
Keywords:
albendazole; polymorphism; enantiotropy; crystal structure; solid state
INTRODUCTION Albendazole (ABZ), methyl [5-(propylthio)-1-H-benzimidazol-2-yl] carbamic acid methyl ester, is among the most effective broad-spectrum anthelmintic agents.1,2 The molecule was patented in 19753 and the drug is described as occurring as colorless crystals with melting point between 208 and 2108C, and being practically insoluble in water.4 ABZ therapy is very important in systemic cestode infections especially in inoperable or disseminated cases of hydatidosis1 and neurocystercosis1,5,6 both in human and veterinary medicine.7 ABZ is undetected7–10 or present at extremely low concentrations2,7,9,11–13 in blood plasma after oral administration in various animal species and in man, due to an extensive first-pass metabolism occurring in the enterocytes and in liver cells as well as the very low intestinal absorption stemming from the unfavorable aqueous solubility.5 Both these effects result in a low and erratic bioavailability. The assignment Correspondence to: Ruggero Bettini (Telephone: 390521905089; Fax: 390521905006; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 3731–3742 (2010) ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association
of ABZ to class II or IV of the Biopharmaceutics Classification System is still a matter of debate.14 Irrespective of this, the insufficient aqueous solubility is an issue that has to be addressed by pharmaceutical formulators. In this respect, solid dispersions with polyvinylpyrrolidone,5 liquid formulations with Transcutol115 and ABZ b-cyclodextrin complexes16 were prepared to improve the aqueous solubility and dissolution characteristics of ABZ. On the other hand, it is known that solid-state properties may play a crucial role in dissolution rate and solubility, especially when polymorphs with different thermodynamic stability are involved. These properties deeply influence all steps of drug product development from the drug candidate discovery, through its processability and production of the dosage form, up to the in vivo performance.17–19 In fact, differences in crystal packing as well as in lattice energy and entropy often result in significant changes in physical properties, such as density, hardness, tablettability, refractive index, melting point, enthalpy of fusion, vapor pressure, solubility, dissolution rate, as well as other thermodynamic and kinetic properties and even color between different polymorphs of the same drug substance.18
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Despite the fact that ABZ was discovered more than 35 years ago and its use in therapy is well established, no reports are presently available on its solid-state characterization nor on its possible polymorphism. The aim of the present work was to study the solidstate properties of ABZ re-crystallized from different solvents for comparison with the commercially available form. Crystalline phases were characterized as to thermal behavior, X-ray diffractometry, both on powder and single crystal, and solubility in methanol or 0.1 N HCl. The relevant thermodynamic parameters were calculated from solubility measurements at different temperatures.
MATERIALS AND METHODS ABZ Crystallization An amount corresponding to about 100 mg of ABZ (Sigma, Steinheim, Germany) was dissolved in 100 mL of methanol, (HPLC grade, 99.9% minimum assay, Carlo Erba Reagents, Milan, Italy) or N,Ndimethylformamide, DMF, (99.8% assay, Fluka Chemie GmBH, Buchs, Switzerland) and stirred at 408C until a clear solution was obtained. The filtered solution was allowed to spontaneously evaporate under ambient conditions until ABZ crystallization was complete. Colorless crystals formed after a few days from methanol, whereas light brown crystals were collected after about 1 month from DMF. Thermal Analysis Differential scanning calorimetry (DSC) was performed on an indium calibrated Mettler DSC 821e (Mettler Toledo, Columbus, OH) driven by STARe software (Mettler Toledo). DSC traces were recorded by placing precisely weighed quantities (1–5 mg) in a sealed and pierced 40 mL aluminium pan. Scans were performed between 30 and 2258C at 5 K min1 or between 30 and 2708C at 40 K min1 under a flux of dry nitrogen (100 mL min1). Each powder sample was analyzed at least in triplicate. Thermogravimetric analysis (TG 50, Mettler Toledo) was carried out on ABZ samples placed in 70 mL alumina pans with a pierced cover. Samples were heated under a flux of dry nitrogen (100 mL min1) at 5 K min1 in the 30–2258C temperature range.
Single Crystal X-Ray Analysis of Form II of ABZ Microscopic examination of many batches of ABZ obtained by re-crystallization from methanol and DMF revealed a constant crystal morphology, namely poorly formed laminae with typical thickness only 10 mm. A specimen viewed normal to the thin section is shown in Figure 1. Eventually, a specimen with thickness 0.06 mm was identified and cut to a more equant shape (0.18 mm 0.18 mm 0.06 mm) to minimize X-ray absorption error and ensure sufficient volume for adequate X-ray diffraction intensity. This was mounted on a cryoloop with Paratone oil (Exxon Chemical Co., Houston, TX), placed on a Nonius Kappa CCD four-circle diffractometer and cooled in a stream of nitrogen vapor to 213(2) K using an Oxford Cryostream cooler (Oxford Cryosystems, Oxford, UK) to ensure a rigid mount. No phase change accompanied the cooling process. Data collection (COLLECT software20) with Mo ˚ ) involved a combination Ka-radiation (l ¼ 0.71073 A of f- and v-scans of 1.008 and 0.508, respectively. All data were corrected for Lorentz-polarization effects. Absorption effects were negligible for the specimen used (transmission range 0.9555–0.9858). Unit cell refinement and data-reduction were performed using DENZO-SMN and SCALEPACK.21 The monoclinic crystal system was established from the Laue symmetry (2/m) and the space group C2/c from systematic absences, centrosymmetry being indicated by the hE2 1i value of 0.922. The structure was solved using direct methods (program SHELXS-9722) and refined by full-matrix least-squares against F2 (program SHELXL-9723). After location of the nonhydrogen atoms of the asymmetric unit (a single molecule of ABZ), it was noted that the propylthio-chain was disordered over two chemically equivalent positions. Each component was modeled with a fixed site occupancy of 0.5, based on very similar electron densities for the atoms comprising the two side-chains as well as intermolecular distance constraints. Following treatment of the non-H atoms, special care was exercised in the location of hydrogen atoms, given the possibility of
X-Ray Diffraction Studies X-ray diffraction patterns on powder were recorded on a bench-scale diffractometer (Rigaku, Tokyo, Japan) using a Cu Ka radiation source at 30 kV voltage over the scanning range (2u) 5–358 (scanning speed of 0.058/min). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
Figure 1. Typical morphology of a crystal of albendazole obtained by re-crystallization from methanol or DMF. DOI 10.1002/jps
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tautomerism for this molecule. All H atoms were located unequivocally in difference Fourier electron density maps. In particular, both N atoms of the imidazole ring were found to bear H atoms, while no electron density attributable to a hydrogen atom was found on the exocyclic N atom. Figure 2a shows the conventional structural diagram for ABZ and Figure 2b the structure of the tautomer found in the crystal of Form II. The disordered model was completed with half-atoms of hydrogen added at positions C7, C8 of the phenyl ring. Except for one of the atoms of a disordered chain component (C18A), all non-H atoms refined anisotropically. H atoms were generally added in a riding model at idealized positions based on their stereochemistries, clearly established from electron density maps, and were assigned isotropic thermal parameters 1.2–1.3 times those of their parent atoms. Crystal data and refinement parameters are reported in Table 1. Molecular parameters were calculated with program PLATON.24 Solubility Determination The solubility of both commercially available and re-crystallized ABZ was determined at 258C in CH3OH or HCl 0.1 N. The latter solubility determination was also performed in the 25–1008C interval. An excess of drug was suspended in the selected solvent and stirred with a Vortex three times within 1 h. Thereafter, the suspension was allowed to settle for 48 h, then it was filtered (0.45 mm) and the filtrate was diluted and analyzed by HPLC to determine the ABZ
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Table 1. Crystal Data and Refinement Parameters for Form II of Albendazole Parameter Chemical formula Formula weight Dcalc (g cm3) Crystal system Space group ˚) a (A ˚ b (A) ˚) c (A a (8) b (8) g (8) ˚ 3) V (A Formula units (Z) m (Mo Ka) (mm1) T (K) Reflections Parameters Completeness (%) R1 (on F > 4s ( F)) wR2 (on F2, F > 4s ( F)) Goodness-of fit (S) Final D/s ˚ 3) Dr min.; max. (e A
Form II C12H15N3O2S 265.33 1.401 Monoclinic C2/c 23.934(5) 5.440(1) 19.586(6) 90.0 99.29(1) 90.0 2516.6(10) 8 0.255 213(2) 2157 204 97.8 0.0723 0.1547 1.033 <0.001 0.257; 0.259
concentration. Each determination was performed in triplicate. High-Performance Liquid Chromatography (HPLC) ABZ in solution was quantified according to the method described in the European Pharmacopoeia25 using an isocratic HPLC system (LC-10 ATvp, Shimadzu, Tokyo, Japan) equipped with a Spherisorb1 column (5 mm, 4.6 mm 250 mm, ODS2, Waters, Milford, MA) and a DAD detector (SPDM10Avp, Shimadzu) set between 250 and 260 nm. The volume of the injected samples was 20 mL (Rheodyne injector 7010). As mobile phase a mixture consisting of 30% (v/v) of ammonium dihydrogen phosphate solution (1.67 g L1) and 70% (v/v) of CH3OH at a flow rate of 0.7 mL min1 was used. ABZ retention time was 6 min. The method was validated for linearity (R2 ¼ 0.999), limit of detection (1.1 mg mL1), limit of quantification (3.6 mg mL1) and relative standard deviation (<1.5%).
RESULTS AND DISCUSSION Figure 2. The conventional structural diagram for albendazole (a) and the structure of the tautomer occurring in the stable crystal Form II (b). The latter also shows the disorder of the propylthio-chain established in this study, each component being present at 50% site occupancy. DOI 10.1002/jps
For ABZ re-crystallization, two polar solvents were selected, one protic and one aprotic, differing mainly in their volatility (boiling points 64.8 and 1538C for methanol and DMF, respectively). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
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As stated above the microscopic examination of ABZ crystals obtained from both methanol and DMF revealed a laminar morphology. The different solvent volatility resulted in a larger average dimension for crystals obtained from DMF than from methanol likely due to the slower crystal growth: as the supersaturation was attained slowly, as in the case of DMF, crossing of the metastable region was slow; then the process of particle growth overtook the process of nucleation, eventually leading to large crystals. The commercial product consisted of very small crystals showing a strong tendency to aggregation (Fig. 3). TGA analysis carried out at the scanning rate of 5 K min1 on the different ABZ specimens indicated a weight loss of about 1.5% and about 13% in the 30– 1508C and 175–2258C intervals, respectively. No significant differences were observed among the samples tested. This indicates that the re-crystallization process did not result in the formation of solvate forms. In fact the theoretical content of solvent for a ABZ monosolvate form would be 21.6% and 10.8% (w/w) for DMF and methanol, respectively, whereas the water content by weight in a hypothetical monohydrate form would be 6.3%. DSC traces recorded from the commercial ABZ in the 30–2258C range at 5 K min1 (Fig. 4A) showed a single broad endothermic peak at 202.3 2.28C (onset 189.1 2.98C) likely ascribable to final melting with decomposition. This interpretation is supported by the literature data4 and by the above mentioned weight loss recorded in the melting interval by TGA. The traces relevant to the ABZ re-crystallized both from methanol and from DMF were not different from that of the commercial form, except for a small endothermic phenomenon at 1508C (onset 1428C) immediately followed by an exotherm. The DSC traces recorded upon scanning at higher rate (40 K min1) (Fig. 4B) put in better evidence some interesting features: the curve for the commercial material, again, presented just one endothermic peak around 2208C (onset 212.8 2.28C), whereas the re-crystallized materials, in addition to the peak around 2208C, presented also a clear endothermic peak at about 1608C, closely followed by an exothermic one. Although the two phenomena could not be separated, the enthalpy change associated with the first one was measured. The relevant data are reported in Table 2. It should be emphasized that the differences between the enthalpies associated with both the first and the second peak of the studied materials were not statistically significant (Student’s t-test: p > 0.05). Despite the large number of papers available in literature presenting the thermal behavior of ABZ JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
Figure 3. Pictures taken at the polarized light microscope of (a) commercial albendazole crystals and re-crystallized albendazole (b) from methanol, (c) from DMF. Original magnification 20.
alone or in various carriers26–29 this is the first time that a thermal event other than final melting is presented for ABZ. We hypothesized that the endo–exo thermal events observed between 140 and 1808C in the re-crystallized ABZ could be ascribed to the melting of a crystal form not yet described in literature, followed by a recrystallization leading to a crystal phase corresponding to the commercial one that eventually melted with decomposition. This new crystal phase stems from the re-crystallization process. DOI 10.1002/jps
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Figure 4. DSC traces of commercial albendazole (curves a), and albendazole re-crystallized from DMF (curves b) and from methanol (curves c). Scan carried out at 5 K min1 (panel A) and at 40 K min1 (panel B).
The hypothesis was verified by X-ray diffraction on powder. Figure 5 reports the diffraction patterns obtained from the commercial and re-crystallized ABZ crystals. The comparison between the pattern of the commercially available material and that of the ABZ re-crystallized from DMF (Fig. 5a) evidenced some differences related to the shape of the peaks at 78 and around 258 2u and the presence of three peaks (10.58, 158, and 308 2u) in the pattern of the recrystallized product that are not observed in that of the commercial one. Moreover, the latter shows a peak around 11.58 2u not present in the re-crystallized ABZ. On the other hand, the diffraction patterns of the re-crystallized materials were practically superimpo-
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sable (Fig. 5b). The only noticeable difference was represented by the higher intensity of the diffraction peaks of ABZ re-crystallized from DMF, most likely ascribable to different extents of crystallinity and preferred orientation. On the basis of these findings it can be affirmed that the re-crystallization of ABZ at ambient temperature both from methanol and DMF leads to a new polymorph (Form II) having a melting temperature at 1608C which re-crystallizes during the DSC scan to give rise to the polymorph melting at 2208C (Form I). The fusion and re-crystallization of the low melting polymorph can be better evidenced when a high scanning rate is used in the thermal analysis. The remarkable similarity between the DSC curves of the two re-crystallized products indicates that the polymorphs obtained upon solvent evaporation may depend on the crystallization conditions, in particular the temperature, rather than on the type of solvent used in the re-crystallization process. These observations allow us to assume that the two polymorphs likely constitute an enantiotropic pair with a transition temperature lower than 1408C; in this respect, the lower-melting crystal would represent the stable form at ambient temperature. The solubility in methanol and 0.1 N HCl aqueous solution was determined at 258C, both for investigating possible differences between commercial and recrystallized ABZ and to serve as a coarse indicator of the relative thermodynamic stability of the two crystal forms. The values obtained are reported in Table 3. ABZ Form I (commercial) proved to be more soluble than Form II (re-crystallized from DMF). The difference was low but statistically significant in both solvents ( p < 0.01 by Student’s t-test). It is worth stressing that the accurate determination of equilibrium solubility of two polymorphs is often a difficult task as solution-mediated conversion may occur in the timeframe of the measurements. Furthermore, we are aware that the approach is conceptually incorrect since, by definition, only the stable form is ‘‘thermodynamically allowed’’ to achieve equilibrium solubility. With respect to this concern DSC analysis was carried out on the solid phases recovered at the end of the equilibrium solubility experiments. For both materials no change of phase was noticed, thus it was concluded that ABZ Form I, although
Table 2. Peak Temperatures and Relevant Enthalpies of ABZ Samples
Commercial product Re-crystallized from DMF Re-crystallized from methanol
1st Peak, 8C
DH 1st Peak, J g1
2nd Peak, 8C
DH 2nd Peak, J g1
— 159.0 (0.9) 160.3 (0.5)
— 10.6 (2.6) 8.4 (0.2)
220.7 (0.1) 220.4 (0.6) 218.7 (1.78)
133.5 (2.1) 119.7 (18.9) 137.2 (5.6)
Standard deviation in parenthesis (n ¼ 3). DOI 10.1002/jps
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Figure 5. X-ray diffraction patterns on powder of albendazole samples: (panel a) commercial and re-crystallized from DMF; (panel b) re-crystallized from DMF and re-crystallized from methanol.
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Table 3. Solubility of Commercial or DMF Re-Crystallized ABZ in Methanol and in HCl 0.1 N Aqueous Solution at 258C
Commercial product (Form I) Re-crystallized from DMF (Form II)
Solubility in Methanol
Solubility in HCl 0.1 N
1.45 (0.47) 0.72 (0.05)
0.54 (0.17) 0.30 (0.07)
Standard deviation in parenthesis (n ¼ 3).
thermodynamically unfavorable at 258C, was kinetically stable enough for allow the determination of a reliable solubility value. The differences in solubility observed are useful to gain some insights into the thermodynamic relationship between the two polymorphs. Form II displays the lower solubility in both solvents; therefore, it should be considered the more stable form at 258C. Furthermore, the possibility to determine the temperature range of thermodynamic stability by measurements of equilibrium solubility of different polymorphs is well established.30–32 Therefore, in order to verify the correctness of our assumption about the enantiotropic relationship, the ABZ II ! I transition temperature was estimated by measuring the ABZ solubility in aqueous HCl solution at different temperatures in the 25–1008C interval. This solvent was selected because of the ease of analysis. The obtained data are reported in Figure 6 both as concentration (mass per unit volume) versus temperature (panel A) and as classical van’t Hoff plot (panel B). For both plots in Figure 6 the regression lines (second order polynomial and linear regression for panel A and B, respectively) relevant to the solubility data of the two forms intersect at around 808C. This point represents the solid–solid transition temperature and this observation confirms that ABZ Form I represents the metastable polymorphs at ambient temperature. It can be noted from Figure 6 panel B that in the temperature range studied a linear relationship exists between the logarithm of the solubility and the inverse of the absolute temperature (R2 ¼ 0.9 and 0.94 for Form I and Form II, respectively). Therefore, the apparent enthalpies of solution of the two polymorphs can be calculated from the relevant slope according to the equation lnCs ¼
DHs 1000 þb T R
(1)
where Cs is the molar solubility, DHs is the heat of solution at saturation, R is the gas constant, T is the absolute temperature, and b is a constant. DOI 10.1002/jps
Figure 6. Solubility of albendazole polymorphs in the 25–1008C range in 0.1 N aqueous HCl solution represented in mass per unit volume (panel A) and as van’t Hoff type plot (panel B). Form I square, Form II circle. The bars represent the standard deviation.
The values obtained were 21.2 and 10.4 kJ mol1 for Form II and Form I, respectively. From these figures the enthalpy of transition from Form I to Form II, DHI ! II, was calculated as 10.8 kJ mol1. At constant temperature and pressure the free energy difference between the two Forms, DGT, can be calculated from the logarithm of the ratio between the relevant solubility values DGT ¼ RT ln
Csmetastable Csstable
(2)
At 258C the free energy change computed from the solubility data obtained in HCl aqueous solution was 1.5 kJ mol1. This value is not significantly different from that computed from solubility data obtained in methanol at the same temperature (1.7 kJ mol1), thus justifying the calculation of the thermodynamic relationships on the basis of the assumption that Henry’s law applies and the obtained values are independent of the solvent used. The values of DGT are reported in Figure 7 as a function of the absolute temperature in the 25–1008C temperature interval. In this plot the transition JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
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Figure 7. Gibbs free energy variation as a function of the absolute temperature for the albendazole Form I and Form II system.
temperature can be graphically evaluated by looking at the point where DGT becomes zero. Finally, the entropy change for the transition of Form I to Form II at a particular temperature T, was calculated from the following equation DST ¼
DHI!II DGT T
(3)
At 258C DST is 31.5 J K1 mol1. At the transition point (350 K) DGT is 0 and Eq. (3) affords the entropy change as 30.9 J K1 mol1. Thermodynamic relationships are summarized in Figure 8 that provides a semi-quantitative energy versus temperature representation and highlights the relevant transition points as well as the relative stability of the two ABZ Forms.
These data put into evidence that the newly isolated ABZ crystal phase and the commercially available one constitute an enantiotropic pair, the commercially available Form being metastable at ambient temperature and thermodynamically stable above 350 K. It is worth emphasizing that for pharmaceutical applications attention to these new findings is advised. In fact, the use of a metastable form may be advantageous (e.g., for exploiting higher solubility in the GI tract) only when the kinetics of conversion would be slow, namely when the energetic barrier Form I $ Form II cannot be overcome during specific storage conditions. In this respect, the metastable phase should be kept cool and dry and should not be too finely subdivided.31 On the other hand, one should be aware that many pharmaceutical operations may cause an undesired change from the metastable to the stable Form, especially those in which the conversion could be mediated by solubilization.33 As a general consideration, ABZ Form I is evidently physically (kinetically) stable, being a commercialized product (although with the warning: keep under refrigeration). Form II proved to be physically quite stable as well, as indicated by DSC experiments in which a scanning rate of 5 K min1 was high enough to allow it to ‘‘pass through’’ the transition temperature and to put into evidence the melting and recrystallization of Form II (see, Fig. 4A). We also heated Form II at 1108C in a dry nitrogen atmosphere under static conditions for 48 h without detecting any phase change. A progressive Form II ! I transition was instead observed upon heating the ABZ Form II at 1308C. This condition afforded complete conversion in <20 h. X-Ray Structure of Form II The molecular and crystal structure reported here is the first for any ABZ crystal modification, despite the longevity of this compound. This is likely due to the practical difficulty of obtaining suitable single crystals. Figure 9 shows the molecular structure occurring in the crystal of Form II. Despite the fairly severe limitations of crystal quality and size mentioned above, as well as significant molecular disorder, the
Figure 8. Semi-quantitative energy versus temperature diagram for the albendazole Form I and Form II pair. G, Gibbs free energy; H, enthalpy; Ttrans, transition temperature; TmI and TmII, melting temperature for Form I and Form II, respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
Figure 9. Structure and atomic numbering of the albendazole molecule in Form II. Thermal ellipsoids are drawn at the 50% probability level. DOI 10.1002/jps
ENANTIOTROPICALLY RELATED ALBENDAZOLE POLYMORPHS
Table 4. Selected Molecular Parameters for Form II of Albendazole
Table 5. Hydrogen Bond Data for Form II of Albendazole D–H A
˚) Bond Distances (A N1–C2 N10–C2 N1–C5 C11–O12 Bond angles (8) C5–N1–C2 N1–C2–N3 N3–C2–N10
a
1.348(5) 1.330(5) 1.394(5) 1.221(5)
N3–C2 N3–C4 N10–C11 C11–O13
1.344(5) 1.396(5) 1.345(5) 1.364(5)
109.0(3) 108.2(4) 130.8(4)
C4–N3–C2 N1–C2–N10 C2–N10–C11
109.2(3) 120.9(4) 118.7(3)
structural refinement proceeded reasonably well, all parameters converging satisfactorily and no abnormal residual electron density being evident. The most significant molecular features are the locations of the H atoms on N1 and N3, and the twofold disorder of the propylthio-chain. As expected, the atoms of the side-chains display significantly higher thermal vibration than the rest of the molecule. The five-membered ring is symmetrical, the bond distances N1–C2 and N3–C2 being equal and longer than N10–C2 (Tab. 4), in accord with the formal bond orders indicated in Figure 2b. The corresponding N–C and N – – C distances in N-(2benzimidazolyl)-O-methyl carbamate (refcode SEDZUW34), which has the same formal bonding arrangement, are respectively 1.352(4), 1.364(6), and ˚ . These values do not differ significantly 1.328(5) A from their counterparts listed in Table 4. Further ring symmetry is indicated in the present study by the equality of the endocyclic angles at N1 and N3. The bond angle N3–C2–N10 exceeds N1–C2–N10 by 108
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N1–H1 N10 N3–H3 O12b N3–H3 O12
˚) D–H (A
˚) H A (A
˚) D A (A
D–H A (8)
0.87 0.87 0.87
1.96 2.20 2.25
2.816(5) 2.928(4) 2.735(5)
168 140 115
a b
1/2 x, 5/2 y, 2 z. 1/2 x, 1/2 þ y, 3/2 z.
due to formation of a six-membered ring as a result of intramolecular hydrogen bonding (N3–H3 O12, Tab. 5). Atom H3 is in fact involved in an intermolecular H-bond as well, as described below. The formal bonding pattern C2 – N10–C11 occurring in this tautomer is also easily distinguished from the alternative tautomeric possibility C2–N10(H)–C11 by the value of the bond angle subtended at N10 (118.7(3)8 in the present case), which is the same as the reported value of 118.5(4)8 in N-(2-benzimidazolyl)-O-methyl carbamate. Instead, in benzimidazole analogues in which N10 bears a hydrogen atom, and either one or both endocyclic N atoms bear a hydrogen atom, the angle subtended at N10 is significantly larger (range 121.8–123.78 in the closely related molecules BEWLOF,35 BMCBIB,36 QESBOG37). Thus, there is ample evidence from the present Xray structure that the ABZ tautomer in Form II corresponds to that shown in Figure 2b. Molecular association via intermolecular hydrogen bonding is extensive, with two well-defined motifs occurring, one finite and the other infinite. The former is a centrosymmetric dimer with graph set R22 (8),38 shown at the top of Figure 10, in which the
Figure 10. Stereoview showing details of the primary hydrogen bonding motifs and the generation of nonparallel molecular stacks in the crystal structure of albendazole, Form II. DOI 10.1002/jps
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unique H-bond is N1–H1 N10a (Tab. 5). The second is a continuous spiral array of molecules parallel to the crystallographic twofold screw axis (unique Hbond N3–H3 O12b). The net effect of these interactions is to produce infinite stacks of dimers, alternate stacks along the c-axis being rotated by 808 relative to each other. This is evident in the packing diagram projected down the c-axis while the full hydrogen bonding scheme is evident in the b-axis projection (Fig. 11). Nearly isostructural hydrogen bonding networks occur in the crystal structure of N-(2benzimidazolyl)-O-methyl carbamate6. Other interactions such as p-stacking and C–H O interactions do not play a significant role in the present crystal structure. The experimental and computed PXRD patterns for Form II of ABZ are shown in Figure 12, together with the experimental PXRD pattern of the commercial material, Form I. The three most intense reflections in the computed pattern39 are those occurring at 2u values 7.498, 24.968, and 25.958. The primary contributors to these three characteristic peaks (with their relative intensity contributions in parentheses) are reflections from the crystal planes 200 (1.00), 510 (0.55), and 511 (0.56), respectively. The high intensities of the first two peaks may be rationalized with
Figure 11. Crystal packing diagrams projected down the c-axis (top), the b-axis (bottom). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 9, SEPTEMBER 2010
Figure 12. PXRD patterns for albendazole: experimental (a) and computed (b) of Form II, and the experimental pattern of the metastable Form I (c).
reference to the packing diagrams (Fig. 11), as follows. In addition to having a high inherent ˚ ) are multiplicity, the 200 planes (d ¼ 11.810 A straddled by the ‘‘heavy’’ sulfur atoms (Fig. 11, bottom). The 510 planes are populated by the closely ˚ ) running diagonally (/) in spaced sheets (d ¼ 3.567 A Figure 11 (top) while the geometrically equivalent planes, with indices 510 coincide with sheets inclined along the other diagonal (\). These striking packing features can thus easily be reconciled with the X-ray data. (In the case of the third strong peak, rotation of Fig. 11 (top) around the b-axis is necessary to view the relevant planes 511; these coincide with a ˚] set of closely stacked molecular sheets [d ¼ 3.434 A crystallographically distinct from those described above.) The 2u values 7.498, 24.968, and 25.958 for the three most intense reflections in the computed pattern correspond with those at 2u ¼ 7.308, 24.658, and 25.558, respectively, in the experimental PXRD pattern. Peak locations at slightly higher angles in the former case are due to unit cell shrinkage at the lower temperature of the single crystal X-ray analysis compared with ambient temperature for the experimental PXRD record. Relatively poor agreement between corresponding peak intensities in the computed and experimental PXRD patterns indicates significant preferred orientation in the sample. This is not surprising given the typical morphology shown in Figure 1. As regards Form I, the commercial material whose structure is unknown, the presence of strong peaks in the experimental PXRD pattern around 2u 7.08 and 24–258 (Fig. 12) suggests that similar crystal DOI 10.1002/jps
ENANTIOTROPICALLY RELATED ALBENDAZOLE POLYMORPHS
packing features to those described above for Form II might obtain.
CONCLUSIONS From the presented data it can be concluded that the re-crystallization of ABZ both from methanol and DMF affords a new stable polymorphic form enantiotropically related to the commercially available ABZ, the latter being the metastable form at ambient temperature. Both forms proved to be physically quite stable, likely due to a high-energy barrier for the activation of the interconversion. ABZ represents a rather complex system in which the molecular structural differences that could be associated with the polymorphism are of at least four possible types, or combinations of these: (a) tautomeric (with e.g., both N atoms of the 5-ring protonated [as in the crystal structure determined in the present work], or with only one of these two N atoms protonated [as in the conventional structural depiction of the ABZ molecule]); (b) different conformations of either or both of the side-chains attached to the bicyclic ring system; (c) the occurrence of molecular disorder (as we have for the propylthioside chain) or its absence; (d) no essential difference in molecular structure but different hydrogen bonding arrangements in the two polymorphs. The findings reported in the present paper clearly put into evidence the need both for ABZ manufacturers and pharmaceutical formulators to carefully consider the crystallization procedure and to perform specific test to characterize the obtained crystal phase as well as to pay particular attention to possible undesired phase change (from the metastable to the stable) stemming from pharmaceutical operations implying heating, mechanical stress, or partial solubilization.
SUPPORTING INFORMATION The CIF file for the single crystal X-ray structure described has been deposited at the Cambridge Crystallographic Data Centre (CCDC 736654).
ACKNOWLEDGMENTS This work was supported by a grant of the Italian Ministry of Education University and Research (MIUR) through the PRIN 2006 program. MRC and RB also wish to acknowledge financial support of the Executive Programme of Scientific and Technological Co-operation between the Italian Republic and Republic of South Africa 2008–2010. DOI 10.1002/jps
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DOI 10.1002/jps