Selective Crystallization of the Metastable Form IV Polymorph of Tolbutamide in the Presence of 2,6-Di-O-methyl-β-cyclodextrin in Aqueous Solution Yoh Sonoda,† Fumitoshi Hirayama,† Hidetoshi Arima,† Yoshihiro Yamaguchi,† Wolfram Saenger,‡ and Kaneto Uekama*,†
CRYSTAL GROWTH & DESIGN 2006 VOL. 6, NO. 5 1181-1185
Graduate School of Pharmaceutical Sciences, Kumamoto UniVersity, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan, and Insitute fu¨r Kristallographie, Freie UniVersita¨t Berlin, Takustrasse 6, D-14195 Berlin, Germany ReceiVed January 2, 2006; ReVised Manuscript ReceiVed February 28, 2006
ABSTRACT: It is of great importance in the pharmaceutical fields to discover, produce, and isolate crystalline polymorphs of a given solid drug and to control their polymorphic transformations. In this paper, we report that a cyclic oligosaccharide derivative, 2,6-di-O-methyl-β-cyclodextrin, is useful for detection and isolation of Ostwald’s intermediate metastable polymorphs occurring during an early stage of crystallization. The metastable Form IV of a hyperglycemic agent, tolbutamide, exclusively crystallized from an aqueous solution of 2,6-di-O-methyl-β-cyclodextrin, whereas the stable Form I crystallized in the absence of the cyclodextrin. The selective crystallization of the metastable Form IV was attributable to an inhibition of the solution-mediated transformation of the metastable form to the stable form by the complexation with 2,6-di-O-methyl-β-cyclodextrin. Introduction Polymorphism is defined as the ability of a substance to exist in two or more crystalline phases that have different arrangements or conformations of the molecules in the crystal lattice.1,2 Different polymorphs exhibit different physicochemical properties such as solubility, dissolution rate, bioavailability, and chemical and physical stabilities. For the development of highquality drugs, it is important to select a proper polymorph and to be able to control its polymorphic transformation(s),3,4 and diverse crystal forms provide valuable intellectual property protection in drug development.5,6 Traditional methods to obtain stable polymorphs of a compound are recrystallization under different conditions by varying solvent composition, temperature, or concentration.7,8 Recent advanced approaches control crystal surfaces at the molecular level on the basis of crystal structures, which include crystallizations with tailormade additives using structurally related molecules9 or polymers,10 utilization of epitaxial crystal growth11 or polymer heteronuclei,12 and seeding with appropriate polymorphs.13 In the pharmaceutical fields, metastable polymorphs are purposefully employed to ensure higher aqueous solubility of poorly water soluble drugs, leading to improved bioavailability.14-16 Polymorphic transformations proceed generally via the solid-solid or the solution-mediated mechanism.17,18 The solid-solid transformation is dependent on internal rearrangements or conformational changes of the molecules in crystals. On the other hand, the solution-mediated transformation is controlled by differences in solubility of stable and metastable forms, where a metastable form with higher solubility appears first from solution, and it then dissolves, nucleates, and transforms into a stable form with the lowest solubility, according to “Ostwald’s Rule of Stages”.19 Therefore, we usually obtain only the final form that is most stable under the experimental conditions. On the other hand, it is difficult to isolate the intermediate metastable forms that occur during crystallization and transform rapidly to the stable form. * To whom correspondence should be addressed. E-mail: uekama@ gpo.kumamoto-u.ac.jp. † Kumamoto University. ‡ Freie Universita ¨ t Berlin.
Cyclodextrins (CDs), cyclic oligosaccharides consisting of usually six to eight D-glucose units, form inclusion complexes with various molecules in aqueous solution and in solid states, and are successfully utilized for improvement of pharmaceutical properties of drugs.20-22 We expected a priori that the solutionmediated polymorphic transformation of compounds will be suppressed by inclusion complex formation with CDs because CDs generally enhance the solubility of drugs in water, slow the diffusion rate of drugs due to an increase in molecular volume, and inhibit aggregation of drugs due to the masking of intermolecular interaction sites by inclusion formation. On the basis of these premises, we studied effects of CDs on the crystallization and polymorphic transformation of an oral hypoglycemic agent, tolbutamide,23 in aqueous solution.24 In this paper, we report that 2,6-di-O-methyl-β-CD (DM-β-CD) markedly suppresses the solution-mediated polymorphic transformation of a Ostwald’s metastable form of tolbutamide to its stable form by inclusion complex formation, yielding exclusively the metastable form. Experimental Section Materials. R-CD, β-CD, γ-CD, 2-hydroxypropyl-R-CD (HP-R-CD, degree of substitution (DS) of 2-hydroxypropyl groups is 4.8), HP-βCD (DS 4.8), 2,6-di-O-methyl-R-CD (DM-R-CD), and 2,6-di-O-methylβ-CD (DM-β-CD) were supplied by Japan Maize Co. DM-β-CD was contained as a major component and the over-methylated homologue, 2,6-per-O-methyl-3A-O-methyl-β-CD, was a minor component in a ratio of about 3:1, as determined by mass spectrometry. It was difficult to separate the minor over-methylated component by means of recrystallization and column chromatography. Tolbutamide, p-hydroxybenzoic acid, and its ethyl, n-propyl and n-butyl esters were purchased from Wako Pure Chemical Co. Other chemicals and solvents were of analytical reagent grade, and deionized double-distilled water was used throughout the study. Crystallization. The crystallization of the hypoglycemic drug in the absence and presence of various CDs in aqueous solution was conducted as follows: for example, tolbutamide was dissolved at 5.0 mM concentration in the absence and in the presence of CDs at various concentrations in pH 8.0 sodium phosphate buffer (20 mL, prepared with 0.1 M H3PO4/0.1 M NaOH) in a 50 mL beaker at room temperature. The solution was slowly titrated with aqueous 0.5 M HCl solution (about 2 mL) to pH 6.8 where tolbutamide did not precipitate
10.1021/cg060001o CCC: $33.50 © 2006 American Chemical Society Published on Web 03/29/2006
1182 Crystal Growth & Design, Vol. 6, No. 5, 2006 yet. The solution was paper-filtered, and the filtrate was put in a refrigerator (4 °C) for 1 day unless otherwise stated. The precipitated tolbutamide crystals were collected by filtration. In experiments of the competitive inclusion complexation, the competitors (benzoic acid derivatives) were dissolved at different concentrations in solutions with 5 mM tolbutamide and 5 mM DM-β-CD in pH 8.0 sodium phosphate buffer solutions, and the pH was changed to 6.8 with 0.5 M HCl solution. The crystallization was conducted at the same conditions as described above. The contents of different polymorphs were determined by powder X-ray diffractometry (Rigaku RINT 2500) under the following conditions: Ni-filtered Cu-KR radiation (1.542 Å), 40 kV, 40 mA, divergent slit of 1.74 mm (1°), scanning slit of 0.94 mm (1°), receiving slit of 0.15 mm, and goniometer angular increment of 1°/ min (Figure S1 in the Supporting Information for the calibration curve of Forms I and IV). Concentrations of tolbutamide in the filtrates were determined by a UV spectroscopic method at 230 nm. Standard Form I crystals of tolbutamide were prepared by dissolving the drug (5 g) in benzene (10 mL) at 70 °C, by slowly adding hexane (5 mL) and keeping the resulting solution at room temperature. Standard Form IV crystals of tolbutamide were prepared by the spray-drying method: a mixed solvent of ethanol/dichloromethane (1.2:1 v/v), an air flow rate of 0.4 m3/min, an air pressure of 1.0 kgf/cm2, and the inlet and outlet temperatures of 85 and 55 °C, respectively.23 Inclusion Complex Formation. The interaction of tolbutamide with CDs was studied by the solubility method and 1H NMR spectroscopy. The solubility method was conducted according to the method of Higuchi and Connors25: an excess amount of tolbutamide (about 5 mg) was added in a test tube containing CD solutions at various concentrations in water (1 mL), and the mixture was shaken at 4 °C or 25 °C for about 5 days. After equilibrium was attained, an aliquot (0.5 mL) was taken by a cotton-plugged pipet, diluted appropriately with water, and analyzed for tolbutamide by UV spectroscopy at 230 nm. The interaction of DM-β-CD with the competitors was also studied by the solubility method under the same conditions except for the added amounts (5-25 mg) and the analytical wavelengths (λmax for each competitor). The stability constant (Kc) of CD complexes was calculated by the equation of Kc ) slope/[intercept (1 - slope)]25 using slopes and intercepts of the initial straight line portion of the phase solubility diagrams. 1H NMR spectra were taken at 25 °C on a JEOL JNM-R 500 spectrometer operating at 500 MHz, using a 5-mm sample tube. Tolbutamide (5.0 mM) and CDs (2.5-10 mM) were dissolved in 0.1 M borate buffer/D2O solution (pH meter reading of 9.0). The continuous variation plots26 were made under a constant concentration (5 mM) of host and guest molecules. The 2D-ROESY spectra were taken at concentrations of guest (10 mM) and host (10 mM) compounds. 1H NMR signals of DM-CDs were assigned according to the report of Onda.27
Sonoda et al.
Figure 1. Powder X-ray diffraction diagrams of crystals obtained after 24 h from 5 mM tolbutamide in pH 6.8 sodium phosphate buffer (made with 0.1 M H3PO4/0.1 M NaOH) in the absence and presence of 5 mM R-CD, β-CD, γ-CD, HP-R-CD, HP-β-CD, DM-R-CD, DM-β-CD. or 35 mM glucose at 4 °C.
Results and Discussion Figure 1 shows powder X-ray diffraction patterns of crystals precipitated from an aqueous, buffered (pH 6.8) 5 mM tolbutamide solution in the absence and presence of various CDs (5 mM) after 24 h. It is apparent that in the absence of CDs, tolbutamide crystallized into stable Form I, giving the diffraction peaks typical for tolbutamide Form I crystal,23 e.g., 2θ ) 8.7, 12.1, 17.5, and 19.9°. Stable Form I crystals of tolbutamide were also obtained from the solutions containing 5 mM parent R-, β-, and γ-CDs, HP-R-CD, HP-β-CD, DM-R-CD, and 35 mM glucose. By sharp contrast, the solution containing 5 mM DMβ-CD yielded exclusively metastable Form IV crystals,23 which showed a different X-ray diffraction pattern compared to Form I, e.g., diffraction peaks typical for tolbutamide Form IV crystal at 2θ ) 10.6, 18.0, 18.9, and 27.1°. The crystalline polymorphs differed in habits, Form I being platelike and Form I being needlelike, as shown in Figure 2. In all cases, tolbutamide crystals were recovered in 60-70% yield of the initially added amounts, and CDs had not cocrystallized with tolbutamide at the concentrations used (
