Solid-Phase Mediated Methodology To Incorporate Drug into

Article pubs.acs.org/crystal

Solid-Phase Mediated Methodology To Incorporate Drug into Intermolecular Spaces of Cyclodextrin Columns in Polyethylene Glycol/Cyclodextrin-Polypseudorotaxanes by Cogrinding and Subsequent Heating Marina Ogawa,†,# Kenjirou Higashi,†,# Sachie Namiki,† Nan Liu,† Keisuke Ueda,† Waree Limwikrant,†,‡ Keiji Yamamoto,† and Kunikazu Moribe*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8675, Japan Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, 447 Sri Ayudhya Road, Ratchatewi, Bangkok 10400, Thailand

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S Supporting Information *

ABSTRACT: In this study, a new preparation method was developed to obtain drug/(polyethylene glycol/cyclodextrinpolypseudorotaxane (PEG/CD-PPRX)) complexes in which drugs were incorporated into the intermolecular spaces of CD columns in PEG/CD-PPRXs. This method was solid-phase mediated and used cogrinding and subsequent heating. Guest drug and CD in PEG/CD-PPRX were amorphized by cogrinding, and then crystallization of CD was promoted by subsequent heating. A previously reported sealed-heating method using the gas phase was not applicable for poorly sublimated and thermally unstable drugs such as piroxicam (PXC) and hydrocortisone, whereas this new method allowed these drugs to be incorporated into the intermolecular spaces of γ-CD columns. Furthermore, salicylic acid (SA) and salicylamide were successfully incorporated into the intermolecular spaces of CD columns using α-CD instead of γ-CD. Powder X-ray diffraction and solution-state 1H nuclear magnetic resonance measurements revealed that complexation followed the stoichiometric rule and that the size of the guest drug determined whether complexation occurred. Accurate control of preparation conditions (temperature and water content) was required to obtain complexes with high CD crystallinity. Changes in the molecular state and mobility of each component during the formation process of the PXC/(PEG/γ-CD-PPRX) and SA/(PEG/α-CD-PPRX) complexes were evaluated using solid-state NMR measurements. Finally, dissolution enhancement and sublimation suppression of SA in the SA/ (PEG/α-CD-PPRX) complex were demonstrated.

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INTRODUCTION Cyclodextrin (CD), a cyclic oligosaccharide, is a widely known host compound with a cavity into which various guest compounds can be incorporated. α-CD, β-CD, and γ-CD are composed of 6, 7, and 8 glucopyranose units, respectively, that are bound by α(1 → 4) glycosidic linkages. The internal diameters of α-CD, β-CD, and γ-CD depend on the number of glucopyranose units and are 4.7−5.3 Å, 6.0−6.5 Å, and 7.5−8.3 Å, respectively. The depth is the same for each: 7.9 Å.1 CD inclusion complexes have been used for products in various fields, including in food, cosmetic, toiletry, and pharmaceutical products.2 In particular, CD is used in the pharmaceutical field to improve the taste, stability, and solubility of drugs and to mask their smell.3,4 Polymers are also included in the inner cavity of a CD series and form CD-polypseudorotaxane (PPRX) with a CD columnar structure.5 The formation of © 2017 American Chemical Society

CD-PPRX depended on the size of the guest polymer and the host CD cavity. α-CD incorporated a single polyethylene glycol (PEG) chain to form PEG/α-CD-PPRX, whereas γ-CD, which has a larger cavity size than α-CD, incorporated doublestranded PEG chains.6,7 CD-PPRX has attracted attention as a new supramolecular material and is being developed as a molecular machine and shuttle in electrical and material fields owing to its specific characteristics.8 Application of CD-PPRX as a new drug delivery carrier has already begun in the pharmaceutical field. Hydrogels of CD-PPRX that include various CDs, and polymers have been developed as sustained release carriers for low molecular weight drugs, antibodies, and Received: September 24, 2016 Revised: January 3, 2017 Published: January 18, 2017 1055

DOI: 10.1021/acs.cgd.6b01410 Cryst. Growth Des. 2017, 17, 1055−1068

Crystal Growth & Design

Article

DNA.9−11 Furthermore, CD-polyrotaxane, in which both ends of CD-PPRX are capped by bulky molecules, has been investigated for targeted drug delivery.12,13 “CD inclusion complex”, which contains the guest drug inside the CD cavity, has been widely studied. “CD complex”, in which drug is incorporated into the intermolecular spaces of CD columns, has also been investigated.14,15 In our previous study, drugs with high sublimation character were incorporated into the intermolecular spaces of γ-CD columns using a sealedheating technique.16,17 Ternary γ-CD inclusion complexes, which incorporated different kinds of drugs in the inner CD cavity and intermolecular spaces of γ-CD columns, were formed using this technique.18 Furthermore, drug/(PEG/γ-CD-PPRX) complexes, in which drugs were incorporated into the intermolecular spaces of γ-CD columns in PEG/γ-CD-PPRX, were prepared.16,19 These complexes had crystallographically unique structures because a polymer and a low molecular weight compound were incorporated in the same crystal structure. In particular, application in the pharmaceutical field is expected because the dissolution of poorly water-soluble drugs was clearly improved by the complex formation. However, the sealed-heating technique uses the gas phase for the complex formation and requires a long heating process, generally over 1 h. This makes it difficult to apply the sealed-heating technique to guest drugs with poor sublimation that are thermally unstable. In addition, the sealed-heating technique did not achieve complex formation of guest drug with PEG/α-CDPPRX using α-CD instead of γ-CD. Therefore, for their wider application, it is essential to establish an alternative technique to prepare various drug/(polymer/CD-PPRX) complexes. Drug/CD inclusion complexes have been prepared using several methods, including coprecipitation, kneading, sealedheating, and cogrinding.20 Cogrinding is a versatile method of complex formation using the solid phase and is utilized to prepare crystalline complexes such as inclusion complexes and cocrystals.21,22 Mechanical force and thermal energy are important factors in complex formation by cogrinding. Mechanical force promotes amorphization of the starting materials along with intermolecular interaction between each component, and thermal energy allows the simultaneous crystallization of the complex.23,24 By applying this knowledge to the present study, a cogrinding and subsequent heating technique was developed as a new method to incorporate guest drug into the intermolecular spaces of CD columns. Piroxicam (PXC) and hydrocortisone (HCT), which display poor sublimation character and low thermal stability, were used as model drugs to form complexes with PEG/γ-CD-PPRX. Salicylic acid (SA) and salicylamide (SAM) were also utilized as model drugs for complex formation with PEG/α-CD-PPRX, instead of PEG/γ-CD-PPRX. The effect of preparation conditions and composition on complex formation was investigated in order to elucidate the complexation mechanism. A variety of analytical techniques, including powder X-ray diffraction (PXRD) measurement, thermal analysis, and nuclear magnetic resonance (NMR) spectroscopy, have been employed to characterize drug/CD inclusion complexes in the solid state.25 Solid-state NMR measurement is a powerful tool that is useful for evaluating the local chemical environment of each component because each NMR peak reflects the state of the component at a nuclear level.26 Moreover, molecular mobility can be evaluated by comparing spectra obtained from different NMR pulse sequences. In our previous study, the molecular state of the guest drug in drug/(PEG/γ-CD-PPRX) complexes

was evaluated using the generally used solid-state 13C crosspolarization (CP)/magic-angle spinning (MAS) NMR measurement, although a detailed evaluation has not yet been carried out.16,19 In this study, we investigated the change in molecular state and mobility of all the components (drug, CD, and PEG) during the complex formation process using multiple solid-state NMR techniques, including 1H MAS, 13C CP/MAS, and 13C pulse saturation transfer (PST)/MAS NMR measurements. Finally, dissolution tests and thermogravimetry (TG) measurements were conducted in order to assess the pharmaceutical properties of the obtained drug/(PEG/CDPPRX) complex.

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EXPERIMENTAL SECTION

Materials. α- and γ-CD were kindly provided by Cyclochem Co., Ltd. (Japan). PEG 2000, PXC (melting point at 198−200 °C), HCT (melting point at 217−220 °C), and SA (melting point at 157−159 °C) were purchased from Wako Pure Chemical Industries, Ltd. (Japan). SAM (melting point at 138−142 °C) was obtained from Iwaki Seiyaku Co., Ltd. (Japan). All of the materials were of reagent grade. Methods. Preparation of Drug/(PEG/γ-CD-PPRX) Complexes by Cogrinding and Subsequent Heating. PEG/γ-CD-PPRX was prepared using a coprecipitation method as described in a previous report.27 γ-CD aqueous solution (100 mg/mL) and PEG aqueous solution (100 mg/mL) were mixed at a volume ratio of 1:1. The obtained suspension was stirred at 25 °C for 2 days, and then stored at 25 °C for 1 day. The precipitate was filtrated and dried at 25 °C for 1 day to prepare PEG/γ-CD-PPRX with a tetragonal-columnar (TC) form of γ-CD. The sample was dried at 100 °C for 12 h in vacuo, and the structure of γ-CD changed from the TC form to the hexagonalcolumnar (HC) form. The PEG/γ-CD-PPRX was mixed with the guest drugs (PXC and HCT) at various molar ratios of drug to γ-CD using a vortex mixer for 3 min to prepare the physical mixture (PM). The PM (1.0 g) was coground using an MM400 ball mill (Verder Scientific, Germany) at 4 °C for 60 min to prepare the ground mixture (GM). The GM was dried at 25 °C and 0% relative humidity (RH) in a desiccator containing diphosphorus pentoxide for 2 days and humidified at 25 °C and 11% RH in a desiccator containing a saturated solution of lithium chloride for 1 day. Thereafter, 100 mg of GM was sealed in a 2.0 mL glass ampule and heated in a gas chromatograph oven (GC-12A; Shimadzu, Japan) to obtain heated-GM. The heating conditions were at 170 °C for 5 min and at 160 °C for 5 min to prepare the complexes with PXC and HCT, respectively. Preparation of Drug/(PEG/α-CD-PPRX) Complexes by Cogrinding and Subsequent Heating. PEG/α-CD-PPRX was prepared using the coprecipitation method reported previously by Harada et al.6 α-CD aqueous solution (145 mg/mL) and PEG aqueous solution (150 mg/ mL) were mixed at the volume ratio of 10:1. The obtained suspension was stirred at 25 °C for 1 h, and then stored at 25 °C for 1 day. The filtrated precipitate was dried at 25 °C for 1 day, and then at 100 °C for 12 h in vacuo to obtain PEG/α-CD-PPRX with the HC form of αCD. The PEG/α-CD-PPRX was mixed with the guest drugs (SA, SAM, PXC, and HCT) at various molar ratios of drug to α-CD using a vortex mixer for 3 min to prepare the PM. The PM (1.0 g) was coground using the vibration ball mill at 4 °C for 60 min to prepare the GM. The GM was dried at 25 °C and 0% RH for 2 days and humidified at 25 °C and 11% RH for 1 day. Thereafter, 100 mg of GM was sealed in a 2.0 mL glass ampule. The GMs of SA and SAM were heated at 140 °C for 30 min and at 130 °C for 30 min to prepare heated-GM, respectively. The GMs of PXC and HCT were heated at 200 °C for 5 min. Preparation of Drug/(PEG/CD-PPRX) Complexes by SealedHeating. The sealed-heated sample (SH) was prepared using a method described in our previous report.16,19 PEG/γ-CD-PPRX was mixed with PXC and HCT at the molar ratio of PXC/γ-CD = 1:1 and HCT/γ-CD = 2:3 using a vortex mixer for 3 min to prepare the PM. PEG/α-CD-PPRX was also mixed with SA and SAM at the molar ratio 1056



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Figure 1. Powder X-ray diffraction (PXRD) patterns of (a) piroxicam (PXC) crystal, (b) polyethylene glycol (PEG)/γ-cyclodextrin (γ-CD)polypseudorotaxane (PPRX), (c) PXC/(PEG/γ-CD-PPRX) physical mixture (PM) (PXC/γ-CD = 1:1(mol/mol)), (d) PXC/(PEG/γ-CD-PPRX) ground mixture (GM) (PXC/γ-CD = 1:1), (e) PXC/(PEG/γ-CD-PPRX) GM (PXC/γ-CD = 2:1), (f) PXC/(PEG/γ-CD-PPRX) heated-GM (PXC/γ-CD = 1:1), and (g) PXC/(PEG/γ-CD-PPRX) heated-GM (PXC/γ-CD = 2:1). Characteristic peaks of (●) PXC crystal, (⧫) hexagonalcolumnar (HC) form of γ-CD, and (▲) monoclinic-columnar (MC) form of γ-CD are represented in the figure. of drug/α-CD = 1:1. The PM (200 mg) was sealed in a 2.0 mL glass ampule and heated for 3 h to obtain the SH. The heating temperature of PXC, HCT, SA, and SAM was 170 °C, 160 °C, 150 °C, and 130 °C, respectively. Quantitative Determination of the Molar Ratio of Guest Drug to CD in the Complex. The molar ratio of drug to CD in the complex was determined using the established method described in a previous study by Kurozumi et al.28 The heated-GM (200 mg) at a molar ratio of PXC/γ-CD = 2:1, HCT/γ-CD = 1:1, SA and SAM/α-CD = 2:1, which contained an excess amount of each drug, was suspended in diethyl ether (15 mL) and washed three times. Filtrated samples were dried at 40 °C for 1 h in vacuo to prepare pure drug/(PEG/CDPPRX) complexes. The dried sample was completely dissolved in dimethyl sulfoxide-d6 (DMSO-d6) and quantitative determination was conducted using solution-state 1H NMR measurements. The molar ratio of drug to CD was calculated by integrating the respective characteristic 1H peaks observed for each complex. Analytical Techniques. PXRD. PXRD patterns were obtained using a Miniflex II diffractometer (Rigaku, Japan). Measurement conditions were as follows: target, Cu; filter, Ni; voltage, 30 kV; current, 15 mA; scanning speed, 4°/min; and scanning angle, 3−35°. Solution-State 1H NMR Spectroscopy. 1H NMR experiments were performed using a JNM-ECA500 NMR spectrometer (JEOL Resonance, Japan) operating at a magnetic field of 11.7 T. Measurement conditions were as follows: temperature, 25 °C; spinning rate, 15 Hz; 1H 90° pulse width, 12.75 μs; relaxation delay,

5 s; number of scans, 8; data points, 16384; and internal standard, tetramethylsilane (0.0 ppm). Water Content Determination. Water content was measured by the Karl Fischer method using an MKC-500 moisture titrator (Kyoto Electronics Manufacturing Co., Ltd., Japan). Solid-State NMR Spectroscopy. Solid-state NMR spectra were acquired using a JNM-ECX400 NMR spectrometer (JEOL Resonance, Japan) operating at a magnetic field of 9.39 T. 1H MAS, 13C CP/MAS, and 13C PST/MAS NMR measurements were performed at 25 °C. For each spectrum, the total number of accumulations acquired was dependent on the required signal-to-noise ratio. 1H MAS NMR measurement conditions were as follows: spinning rate, 15 kHz; 1H 90° pulse width, 2.95 μs; relaxation delay, 2.5−60 s; data points, 4096; and internal standard, silicone rubber (0.12 ppm). 13C CP/MAS NMR measurement conditions were as follows: spinning rate, 15 kHz; decoupling method, two-pulse phase-modulation; 1H 90° pulse width, 2.95 μs; contact time, 2 ms in the system of PXC/(PEG/γ-CD-PPRX) complex formation and 1 ms in the system of SA/(PEG/α-CD-PPRX) complex formation; relaxation delay, 2.5−60 s; data points, 4096; and internal standard, hexamethylbenzene (17.3 ppm). 13C PST/MAS NMR measurement conditions were as follows: spinning rate, 5 and 15 kHz; decoupling method, two-pulse phase-modulation; 13C 90° pulse width, 3.25 μs; relaxation delay, 10−30 s; data points, 4096; and internal standard, hexamethylbenzene (17.3 ppm). Solubility and Dissolution Tests. Solubility studies of SA using Japanese Pharmacopoeia (JP) XVII, the first dissolution media (JP first, pH 1.2) at 37 °C were performed before dissolution experiments. 1057



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Figure 2. PXRD patterns of (a) salicylic acid (SA) crystal, (b) PEG/α-CD-PPRX, (c) SA/(PEG/α-CD-PPRX) PM (SA/α-CD = 1:1), (d) SA/ (PEG/α-CD-PPRX) sealed-heated sample (SH) (SA/α-CD = 1:1), (e) SA/(PEG/α-CD-PPRX) GM (SA/α-CD = 1:1), (f) SA/(PEG/α-CDPPRX) GM (SA/α-CD = 2:1), (g) SA/(PEG/α-CD-PPRX) heated-GM (SA/α-CD = 1:1), and (h) SA/(PEG/α-CD-PPRX) heated-GM (SA/αCD = 2:1). Characteristic peaks of (●) SA crystal and (⧫) HC form of α-CD are represented in the figure. An excess of SA powder was added to the JP first fluid. The covered container with the suspension was rotated by rotation type stirrer in a thermostatic bath at 37 °C with a 50 rpm for 24 h. The sample was filtered through a 0.45 μm membrane, and the concentration of SA was determined by HPLC measurement (column; 150 × 4.6 mm, 3 μm particle size Cadenza CD-C18 column (Imtacket Corporation, Japan), mobile phase; acetonitrile/0.01 M phosphate buffer (pH 2.6) = 1:1 (v/v), and λ; 300 nm). The dissolution test was carried out with a dissolution test apparatus (Toyama Sangyo, Japan) following the JP apparatus II (paddle) method. The dissolution profile of SA was studied in 500 mL of JP first dissolution media at a paddle rotation speed of 50 rpm and a temperature of 37.0 ± 0.5 °C. The dissolution experiment was initiated by putting the powder samples containing 100 mg of SA into a dissolution vessel. This experiment at the SA concentration of 0.2 mg/mL was under a sink condition since the solubility of SA in JP first fluid was determined to be 2.66 ± 0.07 mg/ mL (n = 3, mean ± S.D.) at 37 °C. A 5 mL aliquot of the sample was withdrawn at specific intervals with dissolution medium replacement, and these samples were filtered through a 0.45 μm membrane. The concentration of SA was also determined by HPLC measurement. TG. TG curves were obtained by Rigaku TG8120 (Japan) using the conditions: sample weight 5−10 mg, heating rate 5 °C/min, temperature range 30−200 °C.

prepare PXC/(PEG/γ-CD-PPRX) complex using the sealedheating method established in our previous study.16,19 The PM (PXC/γ-CD = 1:1) of PXC and PEG/γ-CD-PPRX was sealedheated at 170 °C for 3 h, but the color of the sample changed from white to brown (data not shown). This indicated that PXC was decomposed by the long heating process. We also performed sealed-heating the PM at 170 °C for 5 min (data not shown). The SH shows a similar PXRD pattern as the PM. Thus, it was difficult to obtain a complex with PXC and PEG/γCD-PPRX using the sealed-heating technique with a short time. Figure 1 shows the change in PXRD patterns during the formation of PXC and PEG/γ-CD-PPRX complexes by cogrinding and subsequent heating. The PXRD pattern of the PM (PXC/γ-CD = 1:1, Figure 1c) was the superimposition of those of the PXC crystal (Figure 1a) and the HC form of γ-CD in the PPRX (Figure 1b).29 In the PXRD pattern of GM (PXC/ γ-CD = 1:1, Figure 1d), all crystalline peaks disappeared and a halo pattern was observed. This indicated that all the components in the GM existed in an amorphous state. Heated-GM (PXC/γ-CD = 1:1, Figure 1f) displayed a monoclinic-columnar (MC) form of γ-CD.27 Our previous study revealed that the crystal structure of γ-CD changed to the MC form with large intermolecular spaces, rather than the HC form, following the incorporation of guest drug into the intermolecular spaces of γ-CD columns.16,19 Therefore, PXC

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RESULTS AND DISCUSSION Complex Formation of Drug and PEG/CD-PPRX by Cogrinding and Subsequent Heating. Complex Formation of Drug and PEG/γ-CD-PPRX. First, we attempted to 1058



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was observed. However, it was difficult to apply this technique for complexation with PEG/α-CD-PPRX. Figure 2d shows the PXRD pattern of the SH (SA/α-CD = 1:1) prepared by sealedheating at 150 °C for 3 h. Diffraction peaks of both crystalline SA (Figure 2a) and the HC form of α-CD (Figure 2b) were observed, and the SH displayed a diffraction pattern similar to that of the PM (Figure 2c).30,31 This confirmed that SA/(PEG/ α-CD-PPRX) complex was not obtained using the sealedheating method. In the present study, the developed cogrinding and subsequent heating method was applied to form complexes between SA and PEG/α-CD-PPRX. All crystalline peaks disappeared and a halo pattern was observed in GM (SA/αCD = 1:1, Figure 2e). All the components (SA, PEG, and αCD) existed in amorphous states in the GM. In the PXRD pattern of heated-GM (SA/α-CD = 1:1, Figure 2g), the diffraction peaks of the HC form of α-CD appeared, whereas crystalline SA peaks did not. In contrast, GM (SA/α-CD = 2:1, Figure 2f) displayed crystalline SA peaks in addition to the characteristic halo pattern of the GM. Diffraction peaks of both crystalline SA and the HC form of α-CD were observed in heated-GM (SA/α-CD = 2:1, Figure 2h). In order to determine the complex formation of SA and PEG/α-CD-PPRX in the heated-GM, the excess amount of SA crystal in heated-GM (SA/α-CD = 2:1) was washed out using diethyl ether, and the molar ratio of SA to α-CD in the complex was determined by solution-state 1H NMR measurement (Figure S3a). The integration ratio of SA peak (Hg) to α-CD peak (H1) was determined to be 0.90:6.00. The molar ratio of SA to α-CD was calculated to be 0.90:1.00 by taking into account the six glucopyranose units of α-CD. Therefore, SA/(PEG/α-CDPPRX) complex was formed with the integer molar ratio of SA/ α-CD = 1:1 by cogrinding and subsequent heating. Furthermore, another drug/(PEG/α-CD-PPRX) complex, using SAM instead of SA as a guest drug, was prepared using the same procedure in order to evaluate the applicability of this preparation method. The PXRD (Figure S4) and solution-state 1 H NMR (Figure S3b) studies confirmed the complex formation of SAM and PEG/α-CD-PPRX at a molar ratio of SAM/α-CD = 1:1 in a manner similar to SA. The reason that complexes of guest drugs (SA and SAM) with PEG/α-CD-PPRX were not formed using the sealedheating method, but were formed using the cogrinding and subsequent heating method, was related to the structure of αCD. In the sealed-heating method, vaporized guest drugs were incorporated into the intermolecular spaces of the CD columns using the gas phase. Vaporized guest drugs cannot get into the intermolecular spaces of α-CD columns because α-CD is composed of six glucopyranose units, has a rigid structure, and forms a firm columnar crystal structure.32 However, during the cogrinding and subsequent heating method, the rigid crystal structure of α-CD was broken into an amorphous state during the first step of the cogrinding process. Guest drugs were dispersed in monomolecular and/or cluster states in amorphous PEG/α-CD-PPRX matrices. Thereafter, the drugs were incorporated into the intermolecular spaces during recrystallization of α-CD through subsequent heating. Drug/ (PEG/α-CD-PPRX) complex was only obtained using the cogrinding and subsequent heating method, even though drug/ (PEG/γ-CD-PPRX) complex was formed using the sealedheating method and the cogrinding and subsequent heating method. γ-CD, which is composed of eight glucopyranose units, has a larger cavity size and more flexible structure than αCD.33 Therefore, vaporized guest drugs are more easily

was incorporated into the intermolecular spaces, and PXC/ (PEG/γ-CD-PPRX) complex was formed in the heated-GM. The molar ratio of PXC to γ-CD in the complex was determined by varying the initial molar ratio of PXC to γ-CD. GM (PXC/γ-CD = 2:1, Figure 1e) showed a halo pattern similar to that of GM (PXC/γ-CD = 1:1). On the other hand, heated-GM (PXC/γ-CD = 2:1, Figure 1g) displayed crystalline PXC peaks in addition to the characteristic peaks of the MC form of γ-CD. PXC in the GM existed in monomolecular and/ or cluster states in disordered PEG/γ-CD-PPRX matrices because PXC was amorphized, even when a large amount of PXC was present. Some of the PXC was not incorporated into intermolecular spaces of γ-CD columns during complex formation by heating and was individually crystallized. The molar ratio of PXC to γ-CD in the PXC/(PEG/γ-CD-PPRX) complex was quantitatively determined by solution-state 1H NMR measurement (Figure S1a). Heated-GM (PXC/γ-CD = 2:1) containing an excess amount of PXC was suspended in diethyl ether. PXC crystals that did not interact with PEG/γCD-PPRX were washed out and pure PXC/(PEG/γ-CDPPRX) complex was obtained. The integration ratio of PXC peak (Hn) to γ-CD peak (H1) was calculated to be 0.98:8.00 in the 1H NMR spectrum of the complex dissolved in DMSO-d6. The molar ratio of PXC to γ-CD was calculated to be 0.98:1.00 because γ-CD is composed of eight glucopyranose units. The molar ratio of PXC/γ-CD in PXC/(PEG/γ-CD-PPRX) complex formed by cogrinding and subsequent heating was determined to be an integer ratio at 1:1. Stoichiometric complex formation of PXC and PEG/γ-CD-PPRX indicated that PXC was regularly positioned and monomolecularly dispersed in specific intermolecular spaces of γ-CD columns.14,15,19 In order to evaluate the applicability of the cogrinding and subsequent heating method, preparation of HCT/(PEG/γ-CDPPRX) complex was attempted using HCT as a guest drug. HCT is poorly sublimated and thermally unstable. The color of the sample containing HCT changed from white to brown during the sealed-heating process, indicating that HCT was pyrolytically decomposed and the sealed-heating method did not result in formation of a complex composed of HCT and PEG/γ-CD-PPRX. Figure S2 shows the changes in PXRD patterns during complex formation. GM (HCT/γ-CD = 2:3, Figure S2d) showed a halo pattern, whereas only the characteristic diffraction peaks of the MC form of γ-CD were observed in heated-GM (HCT/γ-CD = 2:3, Figure S2f). This result indicated that HCT was incorporated into the intermolecular spaces of γ-CD columns. GM (HCT/γ-CD = 1:1, Figure S2e), which had a larger proportion of HCT, displayed a halo pattern, whereas heated-GM (HCT/γ-CD = 1:1, Figure S2g) exhibited MC form peaks of γ-CD in addition to crystalline HCT peaks. This indicated that the excess HCT that was not incorporated into the intermolecular spaces during the complex formation crystallized individually in a manner similar to that of PXC. The molar ratio of HCT to γ-CD in the complex was determined to be 2.19:3.00 by solution-state 1H NMR measurement (Figure S1b). The PXRD and solutionstate 1H NMR studies clearly indicated that a HCT/(PEG/γCD-PPRX) complex was formed with the molar ratio of HCT/ γ-CD = 2:3 and confirmed the applicability of the cogrinding and subsequent heating method. Complex Formation of Drug and PEG/α-CD-PPRX. In our previous study, the formation of complexes composed of various guest drugs and PEG/γ-CD-PPRX by sealed-heating 1059



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Figure 3. PXRD patterns of (a) PXC crystal, (b) PEG/α-CD-PPRX, (c) PXC/(PEG/α-CD-PPRX) PM (PXC/α-CD = 1:2), (d) PXC/(PEG/αCD-PPRX) GM (PXC/α-CD = 1:2), and (e) PXC/(PEG/α-CD-PPRX) heated-GM (PXC/α-CD = 1:2). Characteristic peaks of (●) PXC crystal and (⧫) HC form of α-CD are represented in the figure.

Table 1. Molar Ratio of Drug to CD in Various Drug/(PEG/CD-PPRX) Complexesa

a

(*1) Prepared by cogrinding and subsequent heating method. (*2) Prepared by sealed-heating method. N.C., not crystallized.

incorporated into the intermolecular spaces of the γ-CD columns than those of the α-CD columns using the sealedheating method, even when the crystal columnar structure of γCD is retained. The ability of the host to form complexes with guest drugs was compared between PEG/CD-PPRX complexes using α-CD and γ-CD. The formation of complexes with PEG/α-CD-PPRX was attempted using PXC and HCT as guest drugs, both of which formed a complex with PEG/γ-CD-PPRX. The size of the intermolecular spaces in the α-CD columns was expected to be smaller than those of the γ-CD columns because the cavity size of α-CD is smaller than that of γ-CD. Harata et al. reported the crystal structure of m-nitrophenol/α-CD = 2:1 complex where m-nitrophenol is included in the cavity of α-CD and also incorporated into the intermolecular spaces between the α-CD columns. 14 The molar ratio of m-nitrophenol in the intermolecular spaces against α-CD is 1:1. Therefore, the size of the intermolecular spaces in the α-CD columns could fit to the guest drug of single aromatic ring. Even if it was possible, only a small amount of drug would be incorporated into the intermolecular spaces of α-CD columns. Therefore, the initial molar ratio of drug/α-CD was set at 1:2 so that the ratio of drug to α-CD was small. The PXRD patterns of both GMs

(PXC/α-CD and HCT/α-CD = 1:2) displayed a halo pattern (Figures 3d and S5d), indicating that both drug and α-CD were amorphized by cogrinding. The characteristic halo pattern of heated-GM sharpened slightly compared with that of GM, probably due to the transformation of a disordered α-CD columnar structure into a more highly ordered one (Figures 3e and S5e), although the diffraction peaks of the crystalline drug and the HC form of α-CD were not observed. Therefore, complexes of PXC and HCT with PEG/α-CD-PPRX could not be formed by cogrinding and subsequent heating. The reason the complexation of guest drugs (PXC and HCT) proceeded with PEG/γ-CD-PPRX, but not with PEG/α-CD-PPRX, was also related to the size of the intermolecular space in the CD column. It was reported that inclusion complexes of PXC and HCT with γ-CD were formed at the molar ratios of PXC/γ-CD = 1:1 and HCT/γ-CD = 2:3 in the solid state.34,35 In our previous study, the intermolecular space of γ-CD columns in the MC form was assumed to be as large as that inside the γCD cavity.19 The crystallization of γ-CD occurred by heating because the intermolecular space had sufficient molecular size to incorporate PXC and HCT. On the other hand, there were no reports that these guest drugs were included in α-CD, presumably because of the small cavity size. Assuming that the 1060



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size of intermolecular spaces of α-CD columns is similar to that of α-CD cavities, it would be difficult to incorporate PXC and HCT into the intermolecular spaces because of the bulky size of the drug molecules. These drugs were amorphized with PEG/α-CD-PPRX during the cogrinding process and formed intermolecular interactions. However, crystallization of α-CD did not occur due to the steric hindrance of the bulky guest drugs. Table 1 summarizes the results of drug/(PEG/CD-PPRX) complex formation. Our previous study revealed that SA and SAM were incorporated into the intermolecular spaces of γ-CD columns at the molar ratio of drug/γ-CD = 2:1 using the sealed-heating method.16,19 In contrast, the molar ratio of drug/ α-CD was 1:1 in the complexes of these drugs with PEG/αCD-PPRX using cogrinding and subsequent heating. The difference in the incorporated amount of drug was explained by the different size of the intermolecular spaces in α-CD and γCD columns. Therefore, a smaller amount of drug was incorporated into α-CD columns than into γ-CD columns because the size of the spaces in the former was limited compared with the size of the spaces in the latter. Effect of Preparation Conditions on Drug/(PEG/CDPPRX) Complex Formation. The effect of mechanical force and thermal energy during the cogrinding process and water content during the heating process on complex formation was investigated. Cogrinding is a preparation method for molecular complexes, such as inclusion complexes and cocrystals, using the solid phase.21,22 It was reported that the formation of an amorphous intermediate is the most likely mechanism of crystalline complex formation during the cogrinding method.24,36 Mechanical force and thermal energy are critical factors involved in the cogrinding process. Amorphization of two or more components occurs along with formation of intermolecular interaction by mechanical force, and then crystallization of the complex is promoted by thermal energy. The cogrinding process broke the crystal structure of both drug and CD in the PPRX and induced amorphization accompanied by the formation of intermolecular interactions between the drug and CD. The temperature during cogrinding in this study was controlled under cold conditions at 4 °C, and the crystallization of CD by thermal energy was suppressed. The effect of thermal energy during heating on the crystallization of CD was evaluated. Figure 4a shows the change in PXRD patterns of SA/(PEG/α-CD-PPRX) GM (SA/α-CD = 1:1) after heating at various temperatures. Crystallization of α-CD in the HC form began at 100 °C, proceeded with the rise in temperature, and was completed at 140 °C. Ground PEG/α-CD-PPRX, which was used for comparison, displayed a halo pattern, indicating that α-CD was amorphized by cogrinding (Figure 4b). Even when the ground PEG/α-CD-PPRX was heated to 200 °C, the PXRD pattern was constant and no crystallization occurred. Therefore, SA dispersed in amorphous PEG/α-CD-PPRX matrices promoted the crystallization of α-CD. The crystallization was facilitated at temperatures that were close to or higher than the glass transition temperature (Tg) of the amorphous mixture, which was approximated from the Tg of the individual components.24 Ground PEG/α-CD-PPRX by itself remained in the amorphous state until the temperature reached 200 °C, indicating that the Tg was immensely high. In addition, SA is a highly crystallizing compound according to the Pajula’s classification.37 The Tg of SA amorphous was not determined because of the instability of SA amorphous, but would be

Figure 4. PXRD patterns of (a) SA/(PEG/α-CD-PPRX) GM (SA/αCD = 1:1) and (b) ground PEG/α-CD-PPRX after heating at variable temperature. Heating temperature is described in the upper left of each PXRD pattern. Characteristic peaks of (⧫) HC form of α-CD are represented in the figure.

expected to be quite low. Therefore, complex formation occurred at a lower temperature in the GM in which SA with a low Tg was dispersed than in the crystallization of PEG/αCD-PPRX alone. The influence of water content in the GM on the crystallization of CD during the heating process was investigated. Figure 5 shows the PXRD patterns of both SA/ (PEG/α-CD-PPRX) GM (SA/α-CD = 1:1) and the corresponding heated-GM after humidifying at 25 °C and variable RH for 1 day. The PXRD patterns of GM humidified at 0 and 11% RH maintained a halo pattern, whereas those humidified at more than 33% RH exhibited crystalline SA peaks. SA in GM maintained its amorphous state under humidified conditions less than 11% RH. Heated-GM prepared at 11% RH displayed the diffraction peaks of the HC form of α-CD more clearly than that prepared at 0% RH, indicating a higher crystallinity of αCD. The crystallization occurred at a lower temperature when water with a Tg of −138 °C was contained in the sample.38,39 The water content of GM humidified at 11% RH was determined to be approximately 3% using the Karl Fischer method. This small amount of water promoted the crystallization of α-CD and enabled the formation of complexes with high α-CD crystallinity. Evaluation of the Molecular State and Mobility of Each Component in the Complexes. PXC/(PEG/γ-CDPPRX) Complex. The change in molecular state and mobility of each component during the PXC/(PEG/γ-CD-PPRX) complex formation process was evaluated using solid-state NMR spectroscopy. Figure S6 represents the full 13C CP/MAS and 13C PST/MAS NMR spectra, which emphasize the signals 1061



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PEG peaks in the PST spectrum of GM (Figure 6e) did not increase as much, indicating that the molecular mobility of PEG in GM was lower than that in heated-GM. The intensity of PEG peaks in the PST spectrum of ground PEG/γ-CD-PPRX (Figure 6g) was compared with that of PEG/γ-CD-PPRX (Figure 6h) in order to evaluate the effect of γ-CD crystallinity on the molecular mobility of PEG. The PEG peak intensities in PEG/γ-CD-PPRX with crystalline γ-CD were stronger than those in ground PEG/γ-CD-PPRX with amorphous γ-CD. The molecular mobility of PEG in ground PEG/γ-CD-PPRX was suppressed compared with that of PEG/ γ-CD-PPRX, regardless of the presence of drug. When γ-CD was arranged regularly with a columnar crystal structure, the PEG included in the cavity was freely mobile within a specific space.47,48 In contrast, when the columnar crystal structure was broken in amorphous γ-CD, free mobile space was not formed. Therefore, the molecular mobility of PEG was suppressed in the ground PEG/γ-CD-PPRX. The PST spectra of heated-GM and PEG/γ-CD-PPRX were compared to evaluate the effect of PXC incorporation on the mobility of PEG. The intensities of the PEG peaks from the heated-GM were stronger than those from PEG/γ-CD-PPRX, indicating that the molecular mobility of PEG increased in heated-GM owing to the incorporation of PXC. 1H MAS NMR measurement was performed in order to confirm the difference in molecular mobility between PEG in heated-GM and PEG/γCD-PPRX (Figure 6i−l). Heated-GM (Figure 6j) and PEG/γCD-PPRX (Figure 6l) displayed sharp PEG peaks, whereas the PEG peaks broadened in GM (Figure 6i) and ground PEG/γCD-PPRX (Figure 6k). In addition, the PEG peak in heatedGM significantly sharpened (half line width: 76.3 Hz) compared with that in PEG/γ-CD-PPRX (half line width: 142.6 Hz). In general, in the rigid solid sample, all 1H peaks broadened because of a strong homonuclear 1H−1H dipolar interaction, and it was difficult to obtain resolved 1H NMR spectra. In contrast, guest molecules included in host molecules displayed fast mobility and sharp peaks in the 1H NMR spectra owing to the averaging of homonuclear 1H−1H dipolar interactions.49−51 Therefore, it was confirmed that PEG in heated-GM had higher molecular mobility than PEG in PEG/γCD-PPRX. In our previous study, the molecular state of the drug in SA/(flurbiprofen/γ-CD) ternary complex was evaluated using solid-state NMR measurement.18 New hydrogen bonds between SA and γ-CD were formed by the incorporation of SA into the intermolecular spaces, and the interaction between flurbiprofen and γ-CD inside the cavity changed. In PXC/ (PEG/γ-CD-PPRX) complex formation, new intermolecular interactions between PXC and γ-CD were formed by the incorporation of PXC into the intermolecular spaces. Thereafter, γ-CD−γ-CD intermolecular interactions, which formed the columnar stacking structure, were partially broken. The intermolecular interaction between PEG and γ-CD inside the cavity was weakened, leading to an increase in PEG mobility. PXC peaks from GM (Figure 7b,d) and heated-GM (Figure 7c,e) were broadened compared with those from PXC crystal (Figure 7a). This peak broadening was explained by the wider distribution of the isotropic chemical shift, indicating that PXC in both GM and heated-GM existed in an amorphous state.42,52 There was no difference between the CP and PST spectra of GM and heated-GM, which suggested that PXC did not have high mobility in the complexes. The 13C peaks of PXC, indicated as peak “i” in the GM (Figure 7f), had different chemical shift values and peak shapes than those in the heated-

Figure 5. PXRD patterns of (a) SA/(PEG/α-CD-PPRX) GM (SA/αCD = 1:1) after storing at 40 °C, 0% relative humidity (RH) for 2 days and then humidifying at 25 °C under variable RH for 1 day and (b) the corresponding SA/(PEG/α-CD-PPRX) heated-GM (SA/α-CD = 1:1). The RHs are described in the upper left of each PXRD pattern. Characteristic peaks of (●) SA crystal and (⧫) HC form of α-CD are represented in the figure. The water content of GM humidified at 25 °C, 11% RH was determined as about 3% by the Karl Fischer method.

of components with high molecular mobility and low molecular mobility, respectively.40 Each carbon peak of PXC, γ-CD, and PEG was assigned by referring to previous reports.41−44 Figure 6 represents the expanded 13C CP/MAS and 13C PST/MAS NMR spectra of GM and heated-GM and those of ground PEG/γ-CD-PPRX and PEG/γ-CD-PPRX. The γ-CD peaks in the CP spectrum of GM (Figure 6a) broadened compared with those of heated-GM (Figure 6b). This peak broadening could have reflected the crystallinity change of γCD because γ-CD in heated-GM existed in the crystalline state, whereas γ-CD in GM existed in the amorphous state. The γ-CD peak shape of heated-GM was different from that of PEG/γCD-PPRX (Figure 6d). The peaks of C-1 and C-4, belonging to the backbone of the γ-CD structure, were split for PEG/γ-CDPPRX. γ-CD in both heated-GM and PEG/γ-CD-PPRX existed in a crystalline state, although with different crystal structures (MC vs HC form). In addition, the crystallinity of γ-CD in heated-GM was lower than that in PEG/γ-CD-PPRX obtained using the coprecipitation method. This was because heated-GM was prepared using a cogrinding process, which reduced the crystallinity of the obtained compound. The difference in the spectra of heated-GM and PEG/γ-CD-PPRX could have been due to the difference in the crystal structure and crystallinity of γ-CD. The intensity of PEG peaks relative to CD peaks was significantly increased in the PST spectrum of heated-GM (Figure 6f) compared with its CP spectrum. It has been reported for a variety of crystalline polymer/CD-PPRX complexes that the relative intensities of PEG peaks to CD peaks in the PST spectrum are stronger than those in the CP spectrum.43,45,46 These studies revealed that the polymer in polymer/CD-PPRX with crystalline CD exhibited high molecular mobility. Therefore, PEG in the heated-GM also underwent extremely fast motion. In contrast, the intensities of 1062



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Figure 6. 13C cross-polarization (CP)/magic-angle spinning (MAS) (a−d), 13C pulse saturation transfer (PST)/MAS (e−h), and 1H MAS (i−l) NMR spectra of (a, e, i) PXC/(PEG/γ-CD-PPRX) GM (PXC/γ-CD = 1:1), (b, f, j) PXC/(PEG/γ-CD-PPRX) heated-GM (PXC/γ-CD = 1:1), (c, g, k) ground PEG/γ-CD-PPRX, and (d, h, l) PEG/γ-CD-PPRX.

different crystallinity of α-CD. These results suggested that heated-GM prepared using the cogrinding process led to relatively low crystallinity of α-CD compared to that in PEG/αCD-PPRX prepared using the coprecipitation method. However, the reason the difference in crystallinity appeared to be rather large is not only explained by the difference in preparation method. In addition, it was reported that CD crystallinity depended on the strength of the intermolecular hydrogen bonding among CDs.54 Therefore, we proposed that the α-CD−α-CD intermolecular interaction forming the columnar crystal structure was weakened by the new intermolecular interaction between SA and CD, resulting in the lower crystallinity of α-CD in heated-GM. The intensity of the PEG peak was increased in the PST spectrum of heated-GM (Figure 8f) compared with that in the CP spectrum, whereas the increase in the intensity of the PEG peak was small for the GM (Figure 8e). The PEG peak significantly sharpened in the 1H MAS NMR spectrum of heated-GM (half line width: 75.2 Hz, Figure 10j). The difference between GM and heated-GM was similar to the result of the system of PXC/(PEG/γ-CD-PPRX) complex formation. In other words, PEG in the SA/(PEG/α-CD-PPRX) complex had high molecular mobility, and the mobility of PEG was suppressed in the GM. Furthermore, the intensity of the

GM (Figure 7g). The chemical shift variation reflected the difference in the chemical environment surrounding PXC; that is, PXC in the GM was dispersed in a cluster state in disordered PEG/γ-CD-PPRX matrices, whereas PXC in the heated-GM existed in a monomolecular state in the intermolecular spaces. Furthermore, PXC peaks in heated-GM sharpened slightly, compared with those in GM, indicating that monomolecular PXC in the heated-GM had higher molecular mobility or orientation. SA/(PEG/α-CD-PPRX) Complex. The full 13C CP/MAS and PST/MAS NMR spectra from the formation of complexes made of SA and PEG/α-CD-PPRX are shown in Figure S7. Each carbon peak was assigned by referring to previous reports.6,16,53 The expanded CP spectra of GM and heated-GM are shown in Figure 8a,b, and those of ground PEG/α-CDPPRX and PEG/α-CD-PPRX are shown in Figure 8c,d. The broadened peaks of α-CD in GM (Figure 8a) reflected the amorphous state of α-CD, whereas the sharpened peaks of αCD in heated-GM (Figure 8b) reflected the crystalline state of α-CD. Chemical shift values of α-CD in heated-GM were similar to those in PEG/α-CD-PPRX (Figure 8d), indicating the same crystal structure of α-CD was present with the HC form. On the other hand, the α-CD peaks from heated-GM were broader than those from PEG/α-CD-PPRX, reflecting the 1063



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Figure 7. 13C CP/MAS (a−c) and 13C PST/MAS NMR (d, e) spectra of (a) PXC crystal, (b, d) PXC/(PEG/γ-CD-PPRX) GM (PXC/γ-CD = 1:1), and (c, e) PXC/(PEG/γ-CD-PPRX) heated-GM (PXC/γ-CD = 1:1). The enlarged C-i peaks in (b, c) are represented in (f, g), respectively.

PEG peak was stronger in the PST and 1H spectra of heatedGM than in those of PEG/α-CD-PPRX (Figure 8h,l). This demonstrated that the molecular mobility of PEG increased because of the incorporation of SA into the intermolecular spaces of α-CD columns. This result was consistent with the fact that the incorporation of PXC caused an increase in the molecular mobility of PEG in the PXC/(PEG/γ-CD-PPRX) complex. The carbonyl carbon peak of SA, represented as “a”, was upfield shifted in the CP and PST spectra of GM (172.5 ppm, Figure 9b,d) compared with that of SA crystal (176.2 ppm, Figure 9a). Because SA exists in a dimer structure in its crystalline state, this upfield shift could have resulted from the breakage of the dimeric structure of SA and subsequent formation of intermolecular interactions between monomeric SA and α-CD following cogrinding.16,55 The chemical shift value of the carbonyl carbon in heated-GM (Figure 9c,e) was similar to that in GM, indicating that SA was still dispersed as a monomer in the intermolecular spaces. The SA peaks

broadened in both the CP and PST spectra of GM, whereas sharp SA peaks were detected in the PST spectrum, but not the CP spectrum, of heated-GM. This suggested that SA molecules in the heated-GM exhibited a high mobility, similar to liquid.50,56,57 13C PST/MAS NMR measurement with a reduced MAS rate was conducted to confirm the liquid-like mobility of the SA molecules (Figure 9f). The SA peaks became broader when the MAS rate decreased from 15 to 5 kHz. Components with high molecular mobility displayed broad peaks similar to those with low molecular mobility under slow MAS conditions when the averaging of dipolar interactions did not work efficiently.58 In addition, the peak of the phenyl ring proton in SA was observed in the 1H MAS NMR spectrum of the heated-GM (Figure 8j), but not in that of the GM (Figure 8i). These results clearly indicated that SA molecules in heatedGM displayed liquid-like motion, whereas the mobility of SA molecules was suppressed in GM. Scheme 1 shows a schematic representation of the molecular state of each component during the formation of the PXC/ 1064



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Figure 8. 13C CP/MAS (a−d), 13C PST/MAS (e−h), and 1H MAS NMR (i−l) spectra of (a, e, i) SA/(PEG/α-CD-PPRX) GM (SA/α-CD = 1:1), (b, f, j) SA/(PEG/α-CD-PPRX) heated-GM (SA/α-CD = 1:1), (c, g, k) ground PEG/α-CD-PPRX, and (d, h, l) PEG/α- CD-PPRX.

to the improvement of wettability by the coexistence of PEG and α-CD. The heated-GM demonstrated further enhancement of SA dissolution where almost SA dissolved within 15 min. This could be ascribed to the monomolecular dispersion of SA in the intermolecular spaces between α-CD columns. The enhancement of drug dissolution by the drug/(PEG/α-CDPPRX) complex formation agreed with the previous results that drug dissolution was improved by the complexation with PEG/ γ-CD-PPRX.16,19 The TG measurements was conducted in order to evaluate the sublimation character of SA from the SA/ (PEG/α-CD-PPRX) complex (Figure 10, bottom). The SA in the PM showed a rapid weight loss upon heating, and almost all of SA (11.5 wt %) was sublimated until 160 °C. On the other hand, the sublimation of SA from the heated-GM was significantly suppressed. It has been well-known that the sublimation of drug included in the CD cavity is suppressed owing to the interactions between the drug and inner surface of CD. Therefore, the intermolecular interactions which was detected in the solid state NMR measurement could stabilize the SA at the intermolecular spaces between the PEG/α-CDPPRX columns.

(PEG/γ-CD-PPRX) and SA/(PEG/α-CD-PPRX) complexes. Drug, CD, and PEG in GM existed in an amorphous state, and drugs were dispersed in a cluster state in disordered PEG/CDPPRX matrices. The molecular mobility of drug and PEG was suppressed in GM. As for heated-GM, CD existed in a crystalline state with the MC form of γ-CD in the PXC/(PEG/ γ-CD-PPRX) complex and the HC form of α-CD in the SA/ (PEG/α-CD-PPRX) complex. The molecular mobility of PEG inside the CD cavities was higher than its mobility in the bulk state. On the other hand, the molecular mobility of PXC and SA was different in each complex. PXC molecules were monomolecularly dispersed in the intermolecular spaces of γCD columns, and their molecular mobility was suppressed, similar to that in GM. In contrast, SA molecules displayed liquid-like motion in the intermolecular spaces of α-CD columns.50,56 CD crystallized to an ordered columnar structure, and a specific molecular space was formed both inside and outside the CD during the complexation. PEG and SA, which have less steric hindrance, are freely mobile in each space; however, when the bulky guest drug, PXC, was incorporated into the intermolecular spaces of γ-CD columns, it did not display liquid-like motion because of the limited space. Pharmaceutical Properties of Drug/(PEG/CD-PPRX) Complex. The dissolution of SA from the SA/(PEG/α-CDPPRX) complex in JP first medium (pH = 1.2) was assessed (Figure 10, top). The SA crystal showed a slow dissolution because of its poor wettability and solubility. The SA dissolution from the PM was rather improved probably due

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CONCLUSIONS A new methodology utilizing a cogrinding and subsequent heating technique successfully incorporated PXC and HCT into the intermolecular spaces formed by γ-CD columns in the PEG/γ-CD-PPRX complex and SA and SAM into those formed by α-CD columns in the PEG/α-CD-PPRX complex. These 1065



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Scheme 1. Molecular States of Drug, CD, and PEG in GM and Heated-GM of the (a) PXC/(PEG/γ-CD-PPRX) System and (b) SA/(PEG/α-CD-PPRX) System

Figure 9. 13C CP/MAS (a−c) and 13C PST/MAS NMR (d−f) spectra of (a) SA crystal, (b, d) SA/(PEG/α-CD-PPRX) GM (SA/α-CD = 1:1), and (c, e, f) SA/(PEG/α-CD-PPRX) heated-GM (SA/α-CD = 1:1). The MAS rate was (a−e) 15 kHz and (f) 5 kHz.

complexes were not formed using the previously reported sealed-heating technique. Stoichiometric complexation occurred depending on the size of the guest drug and host CD. Thermal energy and water content were crucial factors in the process of promoting the crystallization of CD. Changes in the molecular state and mobility of the drug, CD, and PEG during complex formation were clarified using multiple solid-state NMR techniques. Guest drug mobility in the intermolecular spaces varied significantly. The molecular mobility of bulky PXC in the space of γ-CD columns was suppressed, and the molecular mobility of small SA in the space of α-CD columns was high, similar to liquid. The SA/(PEG/α-CD-PPRX) complex showed the dissolution enhancement and also sublimation suppression of SA, demonstrating the utility of the complexes in pharmaceutical sciences. The possibility of forming various drug/(PEG/CD-PPRX) complexes was demonstrated by the developed cogrinding and subsequent heating method. This method is applicable for guest drugs with poor sublimation and thermally unstable character. The intermolecular spaces of α-CD columns also became a new host space for small guest drugs using this method. It is essential to clarify the host−guest interaction between each component for the precise design of the complex because the

Figure 10. Dissolution profiles in pH 1.2 dissolution medium (n = 3 mean ± S.D.) (top) and thermogravimetric (TG) curves (bottom) of (a) SA crystal, (b) SA/(PEG/α-CD-PPRX) PM, and (c) SA/(PEG/αCD-PPRX) heated-GM.

complex is composed of multiple components, including drug, CD, and PEG. The knowledge at a nuclear level obtained by 1066



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(15) Kamitori, S.; Toyama, Y.; Matsuzaka, O. Carbohydr. Res. 2001, 332, 235−240. (16) Higashi, K.; Ideura, S.; Waraya, H.; Moribe, K.; Yamamoto, K. Cryst. Growth Des. 2009, 9, 4243−4246. (17) Higashi, K.; Tozuka, Y.; Moribe, K.; Yamamoto, K. J. Pharm. Sci. 2010, 99, 4192−4200. (18) Higashi, K.; Ideura, S.; Waraya, H.; Moribe, K.; Yamamoto, K. J. Pharm. Sci. 2011, 100, 325−333. (19) Higashi, K.; Waraya, H.; Lin, L. K.; Namiki, S.; Ogawa, M.; Limwikrant, W.; Yamamoto, K.; Moribe, K. Cryst. Growth Des. 2014, 14, 2773−2781. (20) Mura, P. J. Pharm. Biomed. Anal. 2015, 113, 226−238. (21) Ramos, A. I.; Braga, T. M.; Silva, P.; Fernandes, J. A.; RibeiroClaro, P.; Lopes, M.d.F.S.; Paz, F. A. A.; Braga, S. S. CrystEngComm 2013, 15, 2822−2834. (22) Sarceviča, I.; Orola, L.; Nartowski, K. P.; Khimyak, Y. Z.; Round, A. N.; Fábián, L. Mol. Pharmaceutics 2015, 12, 2981−2992. (23) Moribe, K.; Masaki, M.; Kinoshita, R.; Zhang, J.; Limwikrant, W.; Higashi, K.; Tozuka, Y.; Oguchi, T.; Yamamoto, K. Int. J. Pharm. 2011, 420, 191−197. (24) Frišcǐ ć, T.; Jones, W. Cryst. Growth Des. 2009, 9, 1621−1637. (25) Vogt, F. G.; Strohmeier, M. Mol. Pharmaceutics 2012, 9, 3357− 3374. (26) Skorupska, E.; Jeziorna, A.; Kazmierski, S.; Potrzebowski, M. J. Solid State Nucl. Magn. Reson. 2014, 57−58, 2−16. (27) Kawasaki, J.; Satou, D.; Takagaki, T.; Nemoto, T.; Kawaguchi, A. Polymer 2007, 48, 1127−1138. (28) Kurozumi, M.; Nambu, N.; Nagai, T. Chem. Pharm. Bull. 1975, 23, 3062−3068. (29) Uyar, T.; Hunt, M. A.; Gracz, H. S.; Tonelli, A. E. Cryst. Growth Des. 2006, 6, 1113−1119. (30) Hwang, M. J.; Bae, H. S.; Kim, S. J.; Jeong, B. Macromolecules 2004, 37, 8820−8822. (31) Topchieva, I. N.; Tonelli, A. E.; Panova, I. G.; Matuchina, E. V.; Kalashnikov, F. A.; Gerasimov, V. I.; Rusa, C. C.; Rusa, M.; Hunt, M. A. Langmuir 2004, 20, 9036−9043. (32) Harangi, J.; Béke, G.; Harangi, M.; Mótyán, J. A. J. Inclusion Phenom. Mol. Recognit. Chem. 2012, 73, 335−339. (33) Raffaini, G.; Ganazzoli, F. Chem. Phys. 2007, 333, 128−134. (34) Vikmon, M.; Kolbe, I.; Szejtli, J.; Redenti, E.; Ventura, P. Preparation and characterization of piroxicam alkali-salt γ-cyclodextrin complexes. In Proceedings of the Ninth International Symposium on Cyclodextrins: Santiago de Compostela, Spain; May 31−June 3, 1998, Labandeira, J. J. T.; Vila-Jato, J. L., Eds.; Springer: Netherlands: Dordrecht, 1999; pp 281−284. (35) Uekama, K.; Fujinaga, T.; Hirayama, F.; Otagiri, M.; Yamasaki, M. Int. J. Pharm. 1982, 10, 1−15. (36) Seefeldt, K.; Miller, J.; Alvarez-Núñez, F.; Rodríguez-Hornedo, N. J. Pharm. Sci. 2007, 96, 1147−1158. (37) Pajula, K.; Taskinen, M.; Lehto, V.; Ketolainen, J.; Korhonen, O. Mol. Pharmaceutics 2010, 7, 795−804. (38) Andronis, V.; Yoshioka, M.; Zografi, G. J. Pharm. Sci. 1997, 86, 346−351. (39) Jayasankar, A.; Somwangthanaroj, A.; Shao, Z. J.; RodríguezHornedo, N. Pharm. Res. 2006, 23, 2381−2392. (40) Liu, N.; Higashi, K.; Kikuchi, J.; Ando, S.; Kameta, N.; Ding, W.; Masuda, M.; Shimizu, T.; Ueda, K.; Yamamoto, K.; Moribe, K. J. Phys. Chem. B 2016, 120, 4496−4507. (41) Hasegawa, Y.; Higashi, K.; Yamamoto, K.; Moribe, K. Mol. Pharmaceutics 2015, 12, 1564−1572. (42) Redenti, E.; Zanol, M.; Ventura, P.; Fronza, G.; Comotti, A.; Taddei, P.; Bertoluzza, A. Biospectroscopy 1999, 5, 243−251. (43) Harada, A.; Suzuki, S.; Okada, M.; Kamachi, M. Macromolecules 1996, 29, 5611−5614. (44) Harada, A.; Li, J.; Kamachi, M. Macromolecules 1994, 27, 4538− 4543. (45) Harada, A.; Okada, M.; Li, J.; Kamachi, M. Macromolecules 1995, 28, 8406−8411.

solid-state NMR spectroscopies will be helpful for designing new and useful drug/(PEG/CD-PPRX) complexes in the future. We expect that the application of this method will contribute to the development of new formulations by improving pharmaceutical properties such as dissolution character and stability.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.6b01410. 1 H NMR spectrum of drug/(PEG/CD-PPRX) complex in DMSO-d6 to determine the molar ratio (drug to CD) (Figures S1 and S3). Changes in PXRD patterns of HC/ (PEG/γ-CD-PPRX) and SAM/(PEG/α-CD-PPRX) complex formation (Figures S2 and S4). Changes in PXRD patterns of HC and PEG/α-CD-PPRX system during cogrinding and subsequent heating (Figure S5). Full 13C CP/MAS and PST/MAS NMR spectra of the PXC/(PEG/γ-CD-PPRX) and SA/(PEG/α-CD-PPRX) system (Figures S6 and S7) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-43-226-2865. Fax: +81-43-226-2867. E-mail: [email protected]. ORCID

Kunikazu Moribe: 0000-0003-0162-5152 Author Contributions #

M.O. and K.H. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Cyclochem Co. for the kind provision of α-CD and γ-CD. This research was supported by a JSPS KAKENHI Grant-in-Aid for Scientific Research (C) 15K07885.

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