Inhibitory Effect of Hydroxypropyl Methylcellulose Acetate Succinate

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Inhibitory effect of hydroxypropyl methylcellulose acetate succinate on drug recrystallization from a supersaturated solution assessed using nuclear magnetic resonance measurements Keisuke Ueda, Kenjirou Higashi, Keiji Yamamoto, and Kunikazu Moribe Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp400278j • Publication Date (Web): 11 Sep 2013 Downloaded from http://pubs.acs.org on September 16, 2013

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Molecular Pharmaceutics

Inhibitory effect of hydroxypropyl methylcellulose acetate succinate on drug recrystallization from a supersaturated solution assessed using nuclear magnetic resonance measurements Keisuke Ueda†, Kenjirou Higashi†, Keiji Yamamoto, and Kunikazu Moribe∗ †

These authors contributed equally to this work Graduate School of Pharmaceutical Sciences, Chiba University 1-8-1 Inohana, Chuo-ku, Chiba 2608675, Japan

∗

Corresponding Author: Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan; Tel.: +81-43-226-2866; Fax: +81-43-226-2867; E-mail: [email protected] ACS Paragon Plus Environment

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Table of contents graphic

Acetyl

― STD spectrum ― 1D spectrum

CBZ

Hydroxypropyl Succinoyl

2.8

2.0 δ (ppm)

1.0

HPMC-AS

ACS Paragon Plus Environment

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ABSTRACT

We examined the inhibitory effect of hydroxypropyl methylcellulose acetate succinate (HPMC-AS) on drug recrystallization from a supersaturated solution using carbamazepine (CBZ) and phenytoin (PHT) as model drugs. HPMC-AS HF grade (HF) inhibited the recrystallization of CBZ more strongly than that by HPMC-AS LF grade (LF). 1D-1H-NMR measurements showed that the molecular mobility of CBZ was clearly suppressed in the HF solution compared to that in the LF solution. Interaction between CBZ and HF in a supersaturated solution was directly detected using nuclear Overhauser effect spectroscopy (NOESY). The cross-peak intensity obtained using NOESY of HF protons with CBZ aromatic protons was greater than that with the amide proton, which indicated that CBZ had hydrophobic interactions with HF in a supersaturated solution. In contrast, no interaction was observed between CBZ and LF in the LF solution. Saturation transfer difference NMR measurement was used to determine the interaction sites between CBZ and HF. Strong interaction with CBZ was observed with the acetyl substituent of HPMC-AS although the interaction with the succinoyl substituent was quite small. The acetyl groups played an important role in the hydrophobic interaction between HF and CBZ. In addition, HF appeared to be more hydrophobic than LF because of the smaller ratio of the succinoyl substituent. This might be responsible for the strong hydrophobic interaction between HF and CBZ. The intermolecular interactions between CBZ and HPMC-AS shown by using NMR spectroscopy clearly explained the strength of inhibition of HPMC-AS on drug recrystallization.

KEYWORDS HPMC-AS, supersaturated solution, recrystallization inhibition, 1H-NMR, NOESY, STD-NMR


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INTRODUCTION More than 40% of drug candidates are poorly water soluble, and the poor solubility sometimes hampers drug development.1,2 Many methods are available to improve drug dissolution characteristics, such as nanoparticle formation,3,4 cyclodextrin inclusion complex formation,5,6 and encapsulation into drug carriers.7 In addition, amorphization is an effective method.8 Compared to the crystalline form, the amorphous form of a compound has higher energy because of its random array of molecules. Once the amorphous drugs are dissolved in an aqueous solution, a supersaturated solution, in which the drug is apparently dissolved more than their solubility, can be formed. However, the supersaturated solution cannot maintain a high concentration of the drug for a long time because of drug recrystallization. Therefore, stabilization of the supersaturated solution by adding inhibitors of recrystallization has been investigated.9 Solid dispersion, where drug molecules are dispersed and stabilized in polymer matrices,10 is practically useful as a solid dosage form because it can achieve both amorphization of drug and stabilization of the supersaturated solution. Solid dispersions with different kinds of polymers such as polyvinylpyrrolidone (PVP),11,12 methacrylate copolymers (Eudragit®),11,13 hydroxypropyl methylcellulose (HPMC),12 and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) have been studied.12,14,15 Among these polymers that form solid dispersions, HPMC-AS has a strong inhibitory effect on drug recrystallization in solution and forms a quite stable supersaturated solution with high apparent solubility.12,14 Parallel artificial membrane permeability assay (PAMPA), rat intestinal perfusion method, and Caco-2 monolayer assay showed that the supersaturated solution with HPMC-AS showed significant drug permeation.16,17 Supersaturated solution of a drug with HPMC-AS showed no decrease in apparent permeability with an increase in apparent solubility. On the contrary, an increase in apparent solubility via cyclodextrins, surfactants, and cosolvents decreased the apparent permeability.16-18 These results clearly indicated that the supersaturated solution with HPMC-AS was more advantageous than


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other approaches to improve drug solubility to increase the oral absorption of poorly water-soluble drugs. Few studies have examined the intermolecular interaction between the drug and polymer in a supersaturated solution although a large number of studies have been performed to determine the interactions in a solid dispersion. This could be because of the low drug concentration and low stability of the supersaturated solution. Recently, we evaluated the molecular state of a highly stable supersaturated solution of a poorly water soluble drug, carbamazepine (CBZ), and HPMC-AS by using 1D-1H-NMR measurement and measurement of NMR relaxation time.17 A previous study showed that the mobility of CBZ in the HPMC-AS solution decreases with the self-association of CBZ and an increase in the microviscosity around CBZ.17 In addition, the molecular state of CBZ in the HPMC-AS solution indicated its permeation behavior.17 However, the specific intermolecular interaction between CBZ and HPMC-AS has not been clarified thus far. NMR spectroscopy is a powerful tool to detect the intermolecular interactions between different components. Nuclear Overhauser effect spectroscopy (NOESY) measurements have been generally used to directly observe the intermolecular interactions.19,20 The intermolecular interactions between a drug and cyclodextrin or surfactants have been investigated by using NOESY.21-23 The cross peaks in the NOESY spectrum strongly indicate that the spatial location of protons in the different compounds is within 5 Å.24 Saturation transfer difference (STD) NMR measurement is another method that can be used along with conventional NOE experiments to obtain detailed information about molecular interactions. The STD-NMR experiment is highly sensitive and effective in determining intermolecular interactions in a solution containing large molecules. This method has been often used for examining intermolecular interactions between a ligand and protein.25-27 This study aimed to investigate the intermolecular interactions between the drug and HPMC-AS, which acts an inhibitor of recrystallization of the drug from a supersaturated solution. We used 2 types of HPMC-AS with different ratio of substituents, AS-LF (LF) and AS-HF (HF) to compare the


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inhibitory effect on recrystallization of the drug molecules, CBZ and phenytoin (PHT). Intermolecular interactions between CBZ and HPMC-AS were investigated by using NMR techniques such as 1D-1HNMR, NOESY, and STD-NMR. We compared the 1D-1H-NMR spectra of different concentrations of CBZ and HPMC-AS solutions. NOESY and STD-NMR measurements were used to directly detect the intermolecular interaction between the drug and HPMC-AS. Finally, we discussed the inhibitory effect of HPMC-AS on drug recrystallization based on the strength of intermolecular interaction between CBZ and HPMC-AS.

EXPERIMENTAL SECTION MATERIALS CBZ and PHT were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Wako chemicals Co. (Tokyo, Japan), respectively. Two types of HPMC-AS, Shin-Etsu AQOAT® type AS-LF (LF) and AS-HF (HF), were kindly gifted by the Shin-Etsu Chemical Co. (Tokyo, Japan). All other materials were of reagent grade. The chemical structures of CBZ, PHT, and HPMC-AS are shown in Figure 1.

METHODS Evaluation of Drug Recrystallization in HPMC-AS Solution CBZ (100 mg) or PHT (50 mg) was dissolved in 1 mL of dimethyl sulfoxide (DMSO). The DMSO solution was added to the HPMC-AS solution (0.05 M phosphate buffer, pH 6.8) at a DMSO concentration of 2% (v/v) for CBZ and 1% (v/v) for PHT solution. The mixed solutions were shaken in a water bath at 150 rpm and 37°C. The solution was sampled at definite time intervals and then filtered through a cellulose ester membrane filter (0.45 µm). The concentration of the drug in the filtrate was determined using high-performance liquid chromatography (HPLC).

HPLC Conditions


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The sample solutions after filtration were diluted with acetonitrile before quantification. The samples were applied to a Shodex® ODS column (5 µm, 150 mm × 4.6 mm) at 37°C. The mobile phase consisted of 50% (v/v) acetonitrile and 50% (v/v) phosphate buffer (pH 7.4). The injection volume was 5 µL, and the flow rate was 1 mL/min. The concentration of CBZ and PHT was determined by measuring UV absorption at 285 and 225 nm, respectively.

Sample Preparation for NMR Measurements HPMC-AS solutions at different concentrations were prepared with H2O/D2O (9/1, v/v). CBZ was dissolved in DMSO-d6 at the concentration of 10, 20, and 40 mg/mL, and PHT was 10 mg/mL. The CBZ and PHT solutions were added to the HPMC-AS solution (0.05 M phosphate buffer, pH 6.8) at a DMSO-d6 concentration of 2% (v/v) and 1% (v/v), respectively. The temperature was controlled at 37°C during sample preparation process.

1D-1H-NMR Measurements All NMR measurements were performed using the ECX-400 NMR system (9.39T, JEOL Resonance Inc., Tokyo, Japan). The sample solution was transferred into a 5-mm NMR sample tube. The 1H-NMR spectrum was obtained at 37°C. Trimethylsilyl propionate (TSP) was used as an internal reference. The water signal was suppressed using WATERGATE W5 solvent suppression method.

NOESY Measurements NOESY experiments were performed using the standard three-pulse sequence, including WATERGATE W5 solvent suppression. The spectrum was recorded using 1024 data points in the t2 time domain, 256 t1 increments, and 32 scans. The mixing times were 0.1, 0.5, 1, 1.5, and 2 s. A relaxation delay of 3 s was used between the scans. A sine apodization function was used in both dimensions before Fourier transformation.


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STD-NMR Measurements The STD-NMR spectra were obtained using the STD sequence, including WATERGATE W5 solvent suppression. The on-resonance irradiation of proton was 2.12 and 3.57 ppm for HPMC-AS, 7.56 ppm for CBZ, and 7.49 ppm for PHT. The off-resonance irradiation of proton were adjusted to -0.4 ppm. The length of selective Gaussian pulse was set to 64 ms, and the number of selective pulse was changed from 5 to 80.

RESULTS AND DISCUSSION Evaluation of Drug Recrystallization in the HPMC-AS Solution The concentration profiles of CBZ as a function of incubation time are shown in Figure 2. Concentration of CBZ in the phosphate buffer without HPMC-AS markedly decreased to 238 µg/mL and reached to the solution equilibrium within 1 day. The equilibrium concentration of CBZ was similar to that of the solubility concentration of CBZ at 37°C (ca. 220 µg/mL).28 On the contrary, the HF solution at 0.4 mg and 3.2 mg/mL showed a significant inhibitory effect on recrystallization of CBZ from the supersaturated state. The CBZ concentration in the HF solution despite reaching a plateau continued to be higher than the equilibrium solubility concentration of CBZ. Previous studies have shown that the supersaturated solution from the solid dispersion with HF showed a stable high concentration of CBZ.17 The long-term supersaturated state of CBZ in the HF solution shown in this study could be attributed to the strong inhibitory effect of drug recrystallization by HF. Compared to the low concentration of HF (0.4 mg/mL), the high concentration of HF (3.2 mg/mL) showed a greater inhibition of CBZ recrystallization in the solutions. This indicated that inhibitory effect on drug recrystallization depended on the HF concentration. Comparison between HF and LF indicated that the concentrations of CBZ after 7 days incubation in the LF solutions were lesser than those in the HF


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solutions although the concentrations of CBZ in the LF solution were greater than the equilibrium solubility concentration of CBZ. Thus, the HF solution was more effective than the LF solution for inhibiting CBZ recrystallization. Our results were consistent with those reported previously by Tanno et al., where the HF solution had a stronger inhibitory effect on nifedipine recrystallization than the LF solution.14 The difference in the chemical structure between LF and HF is mainly because of the difference in the ratio of the acetyl and succinoyl groups. The acetyl and/or succinoyl groups could play an important role in inhibiting drug recrystallization by HPMC-AS. The effect of HF concentration on inhibition of CBZ recrystallization was further evaluated. The supersaturation level of CBZ at 7 days after addition of DMSO was plotted against the HF concentration (Figure 3a). We observed a sharp increase in the CBZ concentration with an increase in the HF concentration from 0 to 0.4 mg/mL. However, the rate of increase in the CBZ concentration decreased when the HF concentration reached around 0.4 mg/mL. The supersaturation level of CBZ was about 3 times of the solubility at higher HF concentration up to 3.2 mg/mL. The concentration profile shown in our study was clearly different from that for typical solubilizing agents such as surfactants.29-31 Concentration profiles of drug against solubilizing agent shows a linear increase in drug concentration when the concentration of a solubilizing agent exceeds the critical micelle concentration. The CBZ concentration reached a ceiling at 0.4 mg/mL of HF solution because of limitation of increasing the CBZ concentration at a supersaturated state in HF solution or structural change of HF in the presence of CBZ. The supersaturation level of PHT as a function of HF concentration is shown in Figure 3b. The increase in the tendency of supersaturation level of PHT was similar to that of CBZ. PHT concentration sharply increased at HF concentration from 0 to 0.025 mg/mL and then the rate of increase in the PHT concentration decreased. The critical HF concentration around 0.025 mg/mL in the concentration profile of PHT was different from that of CBZ around 0.4 mg/mL, which showed that the critical HF concentration where the drug concentration reached the ceiling was dependent on the property of drug. These results indicated that nonlinear increase in the supersaturation level with HF concentration was


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not because of structural changes in HF but because of the limitation of supersaturated state of drug in the HF solution.

1D-1H-NMR Measurements The 1D-1H-NMR spectra of CBZ in HF and LF solutions at different concentrations of CBZ are shown in Figure 4. CBZ peaks were observed at 5.5–8.0 ppm and HPMC-AS peaks at 1.0–4.0 ppm. H1 peaks of CBZ around 5.7 ppm were broader than H2–H6 peaks (7.0–8.0 ppm) because H1 was exchangeable.32 The CBZ peak in the HF solutions (Figures 4d, e, and f) were broader than the peaks of CBZ alone (Figure 4a); however, the CBZ peaks in LF solutions (Figures 4b and c) were almost same as those of CBZ alone. HF in aqueous solution could suppress the molecular mobility of CBZ, while LF had a little effect on the molecular state of CBZ. The mobility of CBZ in the HF solution was highly suppressed at high concentrations of CBZ. On the contrary, peak widths of CBZ in the LF solutions did not change even at concentrations of CBZ as high as 400 µg/mL. The mobility changes of CBZ in the LF solution were too small to be observed by using NMR measurements. Because the peak width of TSP used as internal standard was constant in all spectra, the mobility suppression of CBZ in HF solution could not be because of increase in the viscosity of the aqueous solution. Different molecular states of CBZ in the HF and LF solutions could lead to different recrystallization behavior of CBZ as shown in the previous section. Inhibition of CBZ recrystallization in the HF solution may be because of suppression of mobility of the CBZ molecules. Chemical shift of CBZ changed depending on CBZ concentrations in HF and LF solutions (Figure 4). CBZ peaks shifted upfield depending on CBZ concentration. Chemical shifts of H1 and H6 were 5.723 and 7.116 ppm, respectively, at the CBZ concentration of 200 µg/mL (Figures 4a, b, and d) and were 5.718 and 7.111 ppm, respectively, at 400 µg/mL (Figures 4c and e). Chemical environment of CBZ molecule was not affected by coexistence of HPMC-AS but completely depended on CBZ concentration although the shifted values were very small. Previous studies have reported similar concentration-


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dependent changes in the chemical shift of aromatic compounds as those reported in our study.33,34 Selfassociation of aromatic compounds at the higher concentration induced the upfield shift of the peaks.33,34 The upfield shift of CBZ peaks with an increase in the CBZ concentration could be because of self-association of CBZ. In contrast, the difference of chemical shift and peak shape was not observed for HPMC-AS. The molecular state changes of HPMC-AS were not markedly affected by the coexistence of CBZ. 1D-1H-NMR measurements of CBZ/HF solution were performed by changing the HF concentrations to evaluate the effect on the molecular state of CBZ (Figure 5). Chemical shifts of all CBZ peaks did not change among the HF solutions. On the contrary, peak width of CBZ broadened with an increase in the HF concentration. Suppression in the mobility of CBZ depended on the HF concentration; a higher HF concentration had a stronger effect on suppression of mobility of CBZ. CBZ recrystallization was inhibited more effectively at higher concentrations of HF (Figures 2 and 3). These results confirmed that the mobility suppression of CBZ in the HF solution contributed to the inhibition of CBZ recrystallization. 1D-1H-NMR experiments could not show specific changes in CBZ spectra by the co-existence of LF. On the other hand, peak broadening could be observed in the HF solution, which was dependent on the HF concentration. The peak of CBZ broadened at high concentrations of CBZ in the HF solution but not in the LF solution. Inhibition mechanism of drug recrystallization by polymer has been discussed based on nucleation inhibition and crystal growth inhibition. It is reported that crystal growth inhibition is achieved by the adsorption of polymer on drug crystal surface, preventing extension of the crystal.35-37 In contrast, nucleation inhibiting mechanism by polymer has not been clearly understood, because nucleation process occurs on the molecular level and in a quite short time. Anwar et al. simulated the effect of additive on crystal nucleation.38 The authors mentioned that the additive with high affinity to solute molecules could coexist within solute aggregation, leading to the disruption of solute molecules packing. In this study, the nucleation of CBZ was strongly suppressed by HF in supersaturated solution,


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because any precipitations of CBZ from CBZ/HF solution (800/3,200 µg/mL) could not be observed at least for 24 hours (data not shown). It was speculated that HF could penetrate into the interstices between CBZ molecules and prevent the emerging CBZ crystallization. The interaction between CBZ and HF in prenuclear aggregations achieved crystal nucleation inhibition of CBZ and efficiently stabilized CBZ supersaturated solution. The mobility suppression of CBZ molecules in supersaturated solution could be derived from the prenuclear aggregations of CBZ/HF.

NOESY measurements The intermolecular interaction between CBZ and HPMC-AS was directly investigated by using NOESY techniques. The NOESY spectrum of CBZ/HF solution (800/3200 µg/mL) with mixing time of 1.5 s is shown in Figure 6. Cross peaks were observed between the peaks of CBZ around 7.0–8.0 ppm and those of HPMC-AS peaks at 1.0–4.0 ppm. This showed that intermolecular interactions could exist between CBZ and HF. We compared the intensity of the cross peaks to discuss the interaction between CBZ and HF in detail. Figure 7a shows the 1D-spectrum obtained by slicing the NOESY spectrum (Figure 6) at 7.56 ppm corresponding to H2–H5 peaks of CBZ. The positive NOESY peaks of HF protons as well as H1 and H6 of CBZ were clearly observed in this sliced spectrum. The relative NOESY peak intensity, which was calculated from the cross peak intensity divided by diagonal peak intensity at 7.56 ppm, was evaluated by changing the mixing time (Figures 7b and c). The relative NOESY peak intensity of H1 and H6 increased with an increase in the mixing time and reached plateau at mixing time of 1.5 s. The cross peaks observed in NOESY experiments with short mixing time was because of transit NOE. A sharp increase in the relative NOESY peak intensities of H1 and H6 with the mixing time from 0.1 to 0.5 s were mainly because of the transient NOE. In contrast, the relative NOESY peak intensity of all HF protons continued to increase in a linear manner. Spin diffusion occurs especially in macromolecules such as protein and polymer, which sometime causes additional increases in NOE peaks.39-41 The spin diffusion could occur within the HF after build-up of NOE from CBZ.


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Figure S1 shows the 1D-spectra sliced at HF proton peaks in NOESY spectrum (Figure 6). The CBZ peaks around 7.0-8.0 ppm were clearly observed in these sliced spectra, which confirmed the intermolecular interaction between CBZ and HF. However, the identification of a specific interaction site between CBZ and HF was difficult because the signal/noise ratio of cross peaks between CBZ and HF was not sufficient to be analyzed. Further, NOESY experiments were performed on the CBZ/LF and CBZ/HF solutions with the same concentration of CBZ/HPMC-AS at 400/3200 µg/mL to clarify the difference in the molecular state between these solutions (Figures S2 and S3). The spectrum of CBZ/HF (400/3200 µg/mL) solution (Figure S3) was similar to that of CBZ/HF (800/3200 µg/mL) solution (Figure 6), which showed the cross peaks between CBZ and HF. Meanwhile, in the spectrum of CBZ/LF (400/3200 µg/mL) solution, no cross peaks that showed the intermolecular interaction between CBZ and LF could be detected (Figure S2). This clearly represented that the interaction between CBZ and HF was stronger than that between CBZ and LF. We compared the 1D-spectra sliced at CBZ peak at 7.56 ppm from the NOESY spectra of CBZ/LF and CBZ/HF solutions (Figure 8). HPMC-AS peaks around 1.0–4.0 ppm could not be observed in the spectrum of CBZ/LF solution although those peaks were clearly observed in the spectrum of CBZ/HF solution. The NOESY peak of H6 in the spectrum of CBZ/LF solution was negative in contrast to the positive peak in the spectrum of CBZ/HF solution. The negative cross peak in the spectrum of CBZ/LF solution could be typical because it was derived from the intramolecular interaction within the CBZ molecule, which had high mobility in the extreme-narrowing limit (ωτc ≪ 1).42 CBZ mobility in the LF solution was less restricted even by the coexistence of LF. In contrast, H6 in CBZ/HF solution showed positive NOESY peaks (ωτc ≫ 1),42 which indicated that the mobility of CBZ was markedly suppressed. These results were consistent with the results that the suppression in the mobility of CBZ was not observed in the LF solution but in the HF solution by using 1D-1H-NMR measurements (Figure 4). The 1D-spectra sliced at HPMC-AS peaks from the NOESY spectra of CBZ/LF and CBZ/HF solutions are shown in Figure S4. The NOESY peaks of HPMC-AS around 1.0– ACS Paragon Plus Environment

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4.0 ppm were observed in both these spectra, and the intensities of these peaks were almost same. However, the NOESY peaks of CBZ around 7.0–8.0 ppm could be observed only in the spectrum of CBZ/HF solution. This result supported the discussion that HF had stronger intermolecular interactions with CBZ than those with LF.

STD-NMR The STD-NMR experiments were performed to determine interaction sites between CBZ and HF. The STD spectrum of CBZ/HF solution (800/3200 µg/mL) at the on-resonance of HF peaks at 3.57 is shown in Figure 9. The H1-H6 peaks of CBZ were observed in the STD spectrum, which showed that the saturation transfer of magnetization occurred from HF to CBZ. Relative STD intensity (%) of CBZ peak; ratio of the peak intensity in the STD spectrum against that in 1D-1H-NMR spectrum was plotted against the number of saturation pulse (Figures 9b). The relative STD intensity of CBZ peak increased with the number of saturation pulses, which showed that relative STD intensity of CBZ peak was dependent on the saturation of HF. The saturation transfer of magnetization occurred from HF to CBZ. In addition, the relative STD intensity was apparently greater in H2-H6 than in H1. Interaction between CBZ and HF was strongly formed through the aromatic part of CBZ rather than its amide one. The inhibitory effect of HPMC-AS on drug recrystallization has been observed in many kinds of poorly water-soluble drugs, independent of their different functional groups.15 In this study, unlike broadening of the NMR peaks of CBZ, the NMR peak of a hydrophilic compound, TSP, showed no change in the HF solution. STD-NMR experiments were also performed for PHT/HF solution (100/3200 µg/mL) with on-resonance of HF peaks at 3.57 (Figure S5). The saturation transfer of magnetization from HF to PHT was clearly observed. STD peaks of aromatic protons of PHT indicated the hydrophobic interaction of PHT with HF. These results strongly supported the assumption that hydrophobic interactions between a drug and HPMC-AS played an important role in the inhibitory effect on drug recrystallization. We also performed STD-NMR experiments with on-resonance of CBZ peaks at 7.56 ppm. Figure 10a


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shows the STD spectrum with the number of saturation pulse at 80, overlapped with the 1D-1H-NMR spectrum normalized with the peak intensity at 2.12 ppm. All the HF peaks were detected in the STD spectrum although the relative STD intensity was different between the HF peaks. Relative STD intensity of the acetyl peaks around 2.2 ppm (2.03%) was the largest in the HF peaks. The relative intensity of methyl proton of hydroxypropyl substituent around 1.2 ppm was 1.50% and that of the peaks around 3.0–4.0 ppm, which contained cellulose chain, methyl substituent, and hydroxypropyl substituent, was 1.47–1.72%. On the contrary, the relative STD intensity of succinoyl peaks around 2.5 ppm (0.54%) was quite small and thus the peaks could not be clearly observed. The relative STD intensity of succinoyl peaks was distinctly smaller than that of other peaks. Relative STD intensity of HF peaks was also evaluated in PHT/HF solution (100/3200 µg/mL) with on-resonance of PHT peak at 7.49 ppm (Figure S6). The largest value of relative STD intensity of acetyl substituent peaks (2.12 ppm) among HF peaks was observed. In contrast, STD peaks of succinoyl substituent could not be clearly observed. The hydrophilic succinoyl substituent, which can ionize, may not participate in the intermolecular interaction with these drugs. Hence, the hydrophobic interaction between HF and poorly water-soluble drugs rather than ionic or hydrogen bonds may affect the inhibitory effect on drug recrystallization. This hydrophobic interaction could be between aromatic parts of drugs and HF substituents except the succinoyl substituent. To evaluate the intermolecular site of interaction of HF and CBZ, the relative STD intensity of acetyl, hydroxypropyl, and succinoyl peaks was plotted against the number of saturation pulses (Figure 10b). The relative STD intensity of acetyl peaks continued to increase according to the number of saturation pulses and was the highest among the HF peaks at any number of saturation pulses. In addition, the hydroxypropyl peaks showed an increase in the relative STD intensity with the number of saturation pulses. However, the relative STD intensity of succinoyl peaks remained small despite an increase in the number of saturation pulses. These results confirmed that the interaction strength increased in the order of acetyl, hydroxypropyl, and succinoyl substituents.


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Here, the difference of inhibitory effect on drug recrystallization between HF and LF solution was discussed based on the interaction site shown by NMR experiments. Higher percentage of acetyl group in HF could partly contribute to stronger inhibitory effect on drug recrystallization in HF solution than in LF solution because the acetyl group of HPMC-AS could have the strongest interaction with CBZ. However, the higher percentage of acetyl group in HF was not sufficient to completely explain the large difference between HF and LF solutions. Another contribution to the stronger inhibitory effect on drug recrystallization could be the smaller percentage of succinoyl substituent in HF. Ilevbare et al. reported that the cellulose derivative which contained carboxylic acid showed pH-dependent inhibiting effect on drug recrystallization.43 Ionization of carboxylic acid reduced the affinity of the polymer with hydrophobic drug surface and weakened the inhibiting effects. In order to evaluate the ionization effect of HPMC-AS on the drug recrystallization inhibition, CBZ concentration was monitored in LF solution of pH 5.5 (Figure 11), which is the solubility threshold of LF.14 Higher concentration of CBZ in LF solution could be observed at pH 5.5 than that at pH 6.8, although CBZ solubility was almost same at both pH (CBZ solubility: 238 ± 14 µg/mL at pH 5.5 and 242 ± 4 µg/mL at pH 6.8). This result confirmed that the hydration of HPMC-AS network could also inhibit the interaction of HPMC-AS with hydrophobic drugs such as CBZ and PHT. Here, we need to consider the ionization state of succinoyl substituent in LF and HF at pH 6.8 to discuss the difference of inhibiting strength of drug recrystallization. The succinoyl substituent of LF is fully hydrated at pH 6.8, disturbing the significant interactions with hydrophobic drugs. In contrast, HF with the solubility threshold at pH 6.5 has less ionized succinoyl substituent, effectively allowing such interactions.14 Meanwhile, it should be mentioned that the inhibiting effect of CBZ recrystallization by LF at 5.5 was still weaker than that by HF at pH 6.8. Higher percentage of acetyl group in HF surely contributed to stronger inhibition effect of drug recrystallization. It was concluded that strong inhibition effect of drug recrystallization by HF could be achieved by the efficient hydrophobic interaction between acetyl substituent and drugs under less disturbance by ionized succinoyl substituent.


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NOESY and STD-NMR experiments directly proved that intermolecular interactions existed between CBZ and HF in the supersaturated solution. Several studies have been performed about the interaction between a drug and polymer in the drug-solubilized solution.44,45 However, no studies about the intermolecular interactions in a supersaturated solution have been performed thus far. Thus, to our knowledge, this is the first study in which the intermolecular interaction between a drug and polymer in a supersaturated solution was directly detected. Thus, our study showed the intermolecular interactions in a supersaturated solution that are useful to understand the mechanism of inhibition of drug recrystallization.

CONCLUSIONS Recrystallization of CBZ and PHT from supersaturated solutions was highly inhibited in HF solution than in LF solution. The supersaturation level of a drug increased in a non-linear manner with increase in the concentration of HF. This is unlike that observed in a typical solution in which solubilization by using surfactants showed a linear increase in the drug concentration. The critical HF concentration, where the rate of increase in the supersaturation level was significantly reduced, was different between CBZ and PHT. Inhibition of drug recrystallization by HF was mainly influenced by the stability of the drug in the supersaturated state and not by structural changes of HF. Suppression in the molecular mobility of CBZ and intermolecular interaction between CBZ and HPMC-AS were clearly observed in the CBZ/HF solution by NMR measurements. NMR spectroscopy showed that stronger intermolecular interactions in HF than in LF had a good relationship with the strength of the inhibitory effect on drug recrystallization. NOESY and STD-NMR experiments indicated that hydrophobic interactions were observed between CBZ and HF. In addition, STD-NMR measurements revealed strength of interaction between each substituent of HF and CBZ. Acetyl substituent showed the strongest interaction with CBZ, while the interaction of the succinoyl substituent was quite small. The order of interaction strength indicated that the interaction of CBZ with HF was stronger than that with LF.


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The inhibitory effect of HPMC-AS on drug recrystallization from a supersaturated solution could be clearly understood based on molecular level information obtained by NMR measurements. Understanding the mechanism underlying the interaction between a drug and polymer in a supersaturated solution will provide insights in designing the formulation that efficiently maintains a high concentration of poorly water-soluble drugs.

ACKNOWLEDGMENTS This study was partly supported by the Japan Health Sciences Foundation for Public-private sector joint research on Publicly Essential Drugs and by Grants-in-Aid for Scientific Research (C) (JSPS, 24590045, 25460032), for Young Scientist (B) (JSPS, 24790041) from the Japan Society for the Promotion of Sciences. We also thank the Shin-Etsu Chemical Co. (Tokyo, Japan) for gifting HPMCAS.


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FIGURE CAPTIONS Figure 1. Chemical structures of (a) CBZ, (b) PHT, and (c) HPMC-AS. Proton numbering of CBZ represents peak assignment in 1H-NMR spectra. Figure 2. Concentration profile of CBZ in ( ) phosphate buffer, (●) LF solution of 0.4 mg/mL, (×) LF solution of 3.2 mg/mL, (■) HF solution of 0.4 mg/mL, and (▲) HF solution of 3.2 mg/mL (pH 6.8, n = 3, mean ± S.D.). Initial CBZ concentration in each solution was 2000 µg/mL. Figure 3. Supersaturation level of (a) CBZ and (b) PHT as a function of HF concentration at 7 days after addition of DMSO solution (n = 3, mean ± S.D.). The inserts shows the enlarged profiles at the HF concentration from 0 to 0.4 mg/mL. Figure 4. 1D-1H-NMR spectra of CBZ in (a) CBZ (200 µg/mL), (b) CBZ/LF (200/3200 µg/mL), (c) CBZ/LF (400/3200 µg/mL), (d) CBZ/HF (200/3200 µg/mL), (e) CBZ/HF (400/3200 µg/mL), and (f) CBZ/HF (800/3200 µg/mL) solutions. Figure 5. 1D-1H-NMR spectra of CBZ (200 µg/mL) in HF solution. Concentration of HF: (a) 0, (b) 0.0125, (c) 0.05, (d) 0.2, (e) 0.8, and (f) 3.2 mg/mL. Half widths (ν1/2) of H6 were shown in figure. Figure 6. NOESY spectrum of CBZ/HF (800/3200 µg/mL) solution at mixing time of 1.5 s. Figure 7. (a)1D-spectrum sliced at 7.56 ppm from 2D-NOESY spectrum of CBZ/HF (800/3200 µg/mL) solution. (b, c) Relative NOESY peak intensity (cross peak intensity/diagonal peak intensity at 7.56 ppm) plotted against mixing time. Figure 8. 1D-spectrum sliced at 7.57 ppm from NOESY spectrum of (a) CBZ/LF (400/3200 µg/mL)


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and (b) CBZ/HF (400/3200 µg/mL) solutions. Figure 9. (a) STD-NMR spectra of CBZ/HF (800/3200 µg/mL) at on-resonance of 3.57 ppm with number of saturation pulse at 80. (b) Relative STD intensity was plotted against number of saturation pulse at on-resonance of 3.57 ppm. Relative STD intensity (%) was calculated by dividing the peak intensity in the STD spectrum with that in 1D-1H-NMR spectrum. Figure 10. (a) 1D-1H-NMR spectrum (black) and STD-NMR spectrum (red) at on-resonance of 7.56 ppm of CBZ/HF (800/3200 µg/mL) solution. The spectra were normalized with peak intensity at 2.12 ppm. (b) Relative STD intensity plotted against number of saturation pulse at on-resonance of 7.56 ppm.

Figure 11. Concentration profile of CBZ in (●) LF solution of 3.2 mg/mL at pH 6.8, (○) LF solution of 3.2 mg/mL at pH 5.5, and (▲) HF solution of 3.2 mg/mL at pH 6.8 (n = 3, mean ± S.D.). Initial CBZ concentration in each solution was 2000 µg/mL.


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Figure 1.

H1 (a)

(c) H2

H3

H2

H4

H3

H4 H5

H5 H6

H6 Ratio of substituent (moles/ring unit)

(b)

Substituent

LF

HF

-CH3

1.87

1.89

-CH2CH(CH3)OH

0.25

0.25

-COCH3

0.48

0.67

-COCH2CH2COOH

0.37

0.18


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Figure 2.

2000

CBZ concentration (µ µg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1600

1200

800

400

0 0

1

2

3

4

5

6

7

Time (days)


25


(a)

(b) PHT concentration (µ µg/mL)

Figure 3.


900 800 700 600 500 400 300 200

1000 800 600 400 200 0 0

100

0.1

0.2

0.3

0.4

HF Concentration (mg/mL)

160 140 120

PHT concentration (µg/mL)

1000

CBZ concentration (µg/mL)

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100 80 60 40 20

160 120 80 40 0 0

0.1

0.2

0.3

0.4


0

0 0

0.8

1.6

2.4


3.2

0

0.8

1.6

2.4

3.2



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Figure 4.


27


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Figure 5.


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Figure 6.


29


(a)

H2~H5

(b) 0.8 H6 H1

0.4

0 0.0

0.5

1.0

1.5

Mixing time (s)

H6

2.0

Relative NOESY peak intensity

Figure 7.

Relative NOESY peak intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(c) 0.2

3.57ppm 3.43ppm 2.12ppm

0.1

0 0.0

0.5

1.0

1.5

2.0

Mixing time (s)

Cellulose chain, methyl, hydroxypropyl

H1

Acetyl 3.57 ppm 3.43 ppm 2.12 ppm


Hydroxypropyl

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Figure 8.


31


Figure 9.

Relative STD intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(b)

7 6 5 4 3 2 1 0


7.56ppm 7.10ppm 0

20

40

7.48ppm 5.71ppm 60

80

Number of saturation pulse

Acetyl (-CH3) H2~H5 H6

(a)

H1

Succinoyl (-CH2-)


Hydroxypropyl (-CH3)

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Figure 10.

(a)


Relative STD intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

(b)

Acetyl (-CH3) Hydroxypropyl (-CH3) Succinoyl (-CH2-)

2

1

0 0

20

40

60

Number of saturation pulse Acetyl (-CH3)

Succinoyl (-CH2-)


80

Hydroxypropyl (-CH3)

33


Figure 11.


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2000

1600

1200

800

400

0 0

1

2

3

4

5

6

7

Time (days)


34

Inhibitory Effect of Hydroxypropyl Methylcellulose Acetate Succinate

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