Equilibrium State at Supersaturated Drug Concentration Achieved by

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Equilibrium State at Supersaturated Drug Concentration Achieved by Hydroxypropyl Methylcellulose Acetate Succinate: Molecular Characterization Using H NMR Technique 1

Keisuke Ueda, Kenjirou Higashi, Keiji Yamamoto, and Kunikazu Moribe Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500588x • Publication Date (Web): 27 Feb 2015 Downloaded from http://pubs.acs.org on March 3, 2015

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Molecular Pharmaceutics

Equilibrium State at Supersaturated Drug Concentration Achieved by Hydroxypropyl Methylcellulose Acetate Succinate: Molecular Characterization Using 1H NMR Technique Keisuke Ueda†, ‡, Kenjirou Higashi†, Keiji Yamamoto†, and Kunikazu Moribe∗, †

†

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 2608675, Japan ‡

Research Fellow of Japan Society for the Promotion of Science, Japan

∗

Corresponding Author: Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan; Tel.: +81-43-226-2865; Fax: +81-43-226-2867; E-mail: [email protected]

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Table of contents graphic


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ABSTRACT

The maintenance mechanism of the supersaturated state of poorly water-soluble drugs, glibenclamide (GLB) and chlorthalidone (CLT), in hydroxypropyl methylcellulose acetate succinate (HPMC-AS) solution was investigated at a molecular level. HPMC-AS suppressed drug crystallization from supersaturated drug solution and maintained high supersaturated level of drugs with small amount of HPMC-AS for 24 h. However, the dissolution of crystalline GLB into HPMC-AS solution failed to produce supersaturated concentrations, although supersaturated concentrations were achieved by adding amorphous GLB to HPMC-AS solution. HPMC-AS did not improve drug dissolution and/or solubility but efficiently inhibited drug crystallization from supersaturated drug solutions. Such an inhibiting effect led to the long-term maintenance of the amorphous state of GLB in HPMC-AS solution. NMR measurements showed that HPMC-AS suppressed the molecular mobility of CLT depending on their supersaturation level. Highly supersaturated CLT in HPMC-AS solution formed a gel-like structure with HPMC-AS, in which the molecular mobility of the CLT was strongly suppressed. The gel-like structure of HPMC-AS could inhibit the reorganization from drug prenuclear aggregates to the crystal nuclei and delay the formation of drug crystals. The prolongation subsequently led to the redissolution of the aggregated drugs in aqueous solution and formed the equilibrium state at the supersaturated drug concentration in HPMC-AS solution. The equilibrium state formation of supersaturated drugs by HPMC-AS should be an essential mechanism underlying the marked drug concentration improvement.

KEYWORDS hydroxypropyl

methylcellulose

acetate

succinate,

amorphous

drug,

supersaturated

solution,

crystallization inhibition, 1H NMR measurement


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INTRODUCTION Most new drug candidates have poor water-solubility,1,2 which results in insufficient blood concentration of the drugs. Several methods have been developed to overcome the poor solubility, such as nanoparticle formation,3,4 cyclodextrin inclusion complex formation,5,6 and encapsulation into drug carriers.7,8 Furthermore, solid dispersion, in which poorly water-soluble drugs are dispersed into the polymer matrix in an amorphous state, is a useful method for improving drug dissolution properties.9 The amorphization leads to quite high drug dissolution rates and solubility as a result of higher energy of amorphous forms compared with that of crystal forms.10 Water-soluble polymers such as polyvinylpyrrolidone,11,12 methacrylate copolymers,11,13 hydroxypropyl methylcellulose (HPMC),12 and hydroxypropyl methylcellulose acetate succinate (HPMC-AS)12,14,15 have been used as solid dispersion carriers that facilitate amorphization of drugs in solid dispersions. Such polymers inhibit drug crystallization from supersaturated drug solutions formed by the dissolution of amorphous drugs. The supersaturation level achieved by solid dispersions depends on the inhibition of drug crystallization by each polymer. Inhibition of drug crystallization by polymers in supersaturated drug solution has been phenomenologically confirmed.11,16,17 We previously showed that HPMC-AS strongly inhibited drug crystallization and facilitated long-term maintenance of supersaturated drug solutions.18 Previous studies indicate that drug crystallization from supersaturated solution is inhibited by slowing drug nucleation and crystal growth.19,20 However, inhibition of drug crystallization has only been demonstrated at the macroscopic level. Molecular investigations of drug crystallization are required to elucidate mechanisms that allow maintenance of supersaturated drug solutions. Till date, we have revealed hydrophobic interactions between drugs and HPMC-AS in supersaturated drug solutions using NMR measurements.18 However, how these hydrophobic interactions inhibit crystallization remains unclear.


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When drug precipitates are formed from supersaturated drug solution, some aggregation of solute molecules and formation of crystal nuclei should occur.21,22 Rapid changes of the drug molecular state during crystallization hamper the elucidation of the crystallization mechanism. To clarify the role of polymers in inhibiting drug crystallization, detailed understanding of the drug molecular state in supersaturated solution is essential. NMR techniques allow the evaluation of molecular states even in ever-changing solutions.23,24 Chemical shifts in NMR spectra reflect differences in chemical environments of molecules. Furthermore, 1H NMR peak widths reflect molecular mobility, and the broadening of 1H NMR peaks demonstrates suppression of mobility.25,26 This technique can provide detailed information of changes in molecular states of drugs and crystallization inhibitors in the supersaturated solutions. In the present study, we evaluated the effects of HPMC-AS coexistence on drug precipitation and dissolution in aqueous solution using glibenclamide (GLB) and chlorthalidone (CLT) as poorly watersoluble drugs. The relationship of the formation of supersaturated solution with the molecular state of a solid drug component was investigated using the dissolution test of crystalline and amorphous drugs in HPMC-AS solution. In addition, recrystallization of amorphous drugs in HPMC-AS solution was investigated to clarify the effect of dissolved HPMC-AS on the reorganization of drug molecules. The molecular state of supersaturated drugs in HPMC-AS solution was evaluated by 1H NMR measurements. Finally, we have discussed the inhibition of drug crystallization in HPMC-AS solution to clarify the maintenance mechanism of supersaturated solution by HPMC-AS.

EXPERIMENTAL SECTION MATERIALS GLB and CLT were purchased from Wako Chemicals Co. (Tokyo, Japan). HPMC-AS (Shin-Etsu AQOAT® type AS-HF) was a kind gift from the Shin-Etsu Chemical Co. (Tokyo, Japan). All other


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materials were of reagent grade, and chemical structures of GLB, CLT, and HPMC-AS are shown in Figure 1.

Figure 1. Chemical structures of (a) glibenclamide (GLB), chlorthalidone (CLT), and (c) hydroxypropyl methylcellulose acetate succinate (HPMC-AS). Proton numbering of CLT represents peak assignment in 1H NMR spectra.

METHODS Evaluation of Drug Crystallization in HPMC-AS Solution HPMC-AS solution was prepared by the dissolving of HPMC-AS into 0.05 M phosphate buffer (pH 6.8), including sodium dihydrogenphosphate and disodium hydrogenphosphate. GLB (10 mg) or CLT (150 mg) was dissolved in 1 mL of dimethyl sulfoxide (DMSO). The DMSO solution was added to HPMC-AS solution (0.05 M phosphate buffer, pH 6.8) at a DMSO concentration of 2% (v/v). Mixed solutions were shaken at 150 rpm in a water bath at 37°C. Solutions were sampled at 0.5, 1, 2, 4, 8, 16,


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and 24 h and filtered through a 0.45-µm cellulose ester membrane filter. Drug concentrations were determined in filtrates using HPLC.

HPLC Conditions After filtration, sample solutions were diluted with acetonitrile and were applied to Shodex® ODS columns (5 µm, 150 mm × 4.6 mm) at 40°C. The mobile phase comprised 50% (v/v) acetonitrile and 50% (v/v) phosphate buffer (pH 7.4) containing potassium dihydrogenphosphate and disodium hydrogenphosphate. The injection volume was 5 µL, and the flow rate was 1 mL/min. Concentrations of GLB and CLT were determined by measuring UV absorbance at 275 and 229 nm, respectively.

Preparation of Amorphous GLB Amorphous GLB was prepared using a spray-drying technique. GLB was dissolved in dichloromethane/methanol (1/1, v/v) at a concentration of 3% w/v. The solution was then fed into a spray-dryer (ADL311S, Yamato Scientific, Tokyo, Japan) at a rate of 4 g/min. Spray-drying was performed under the following conditions: inlet temperature, 90°C; outlet temperature, 56°C; atomizing pressure, 0.05 MPa; and nozzle diameter, 0.7 mm (liquid) and 1.7 mm (gas).

Dissolution Test Dissolution tests were performed using the paddle method, as specified in the United States Pharmacopeia. Each sample was dispersed in a glass vessel containing 500 mL dissolution medium at 37°C. Phosphate buffer (0.05 M, pH 6.8) with and without 1000 µg/mL HPMC-AS were used as dissolution medium. The solution was stirred with a rotation paddle at a speed of 150 rpm. The solution (1 mL) was sampled at defined intervals and filtered through 0.45-µm cellulose ester membranes. The drug concentrations were determined in the filtrates using HPLC.


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Evaluation of Recrystallization of Amorphous GLB in HPMC-AS Solution An excess amount of amorphous GLB (500 mg) was dispersed in 50 mL of HPMC-AS solution (0.05 M phosphate buffer, pH 6.8). The suspension was then vigorously stirred at 40°C and sampled at 30 min, 1, 3, 6, and 18 h. Samples were transferred to centrifuge tubes and centrifuged at 4000 rpm for 20 min, and the precipitated powder was dried under reduced pressure. The dried powder was evaluated using powder X-ray diffraction (PXRD) measurement.

PXRD Measurement The powder sample was filled into a glass plate for PXRD measurements. PXRD measurements were conducted using MiniFlex II (Rigaku, Tokyo, Japan) under the following conditions: target, Cu; filter, Ni; voltage, 30 kV; current, 15 mA; scanning speed, 4°/min; scanning angle, 5°–40°.

Sample Preparation for NMR Measurements For NMR measurements, 0.05M phosphate buffer with and without HPMC-AS were prepared in D2O containing trimethylsilyl propionate (TSP). DMSO-d6 solutions containing CLT were added to the phosphate buffer with and without HPMC-AS at a final DMSO-d6 concentration of 2% (v/v). The temperature was controlled at 37°C during the sample preparation process.

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H 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. TSP was used as an internal reference, and CLT and HPMC-AS concentrations were determined in solutions with reference to calibration curves using TSP as internal standard.


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RESULTS AND DISCUSSION Inhibitory Effect of HPMC-AS on Drug Crystallization Figure 2 shows concentration profiles of GLB and CLT in HPMC-AS solution. The concentrations of GLB and CLT in HPMC-AS solutions at 100 and 1000 µg/mL were kept constant without crystallization, whereas those of GLB and CLT decreased in the absence of HPMC-AS. Long-term maintenance of supersaturated GLB and CLT solutions in HPMC-AS solutions should be a result of the inhibitory effect of drug crystallization by HPMC-AS. The term drug supersaturation is defined as drug concentrations above the intrinsic solubility.16 GLB and CLT concentrations in 100 µg/mL HPMC-AS solution were kept at 200 and 3000 µg/mL for 24 h, respectively. The molar concentrations of GLB, CLT and HPMC-AS were calculated taking the molecular weight into account. The molar concentrations of GLB and CLT were approximately 0.4 and 8.8 µmol/mL, respectively, while that of the glucose unit of HPMC-AS was approximately 0.4 µmol/mL. The molar concentration of CLT was around 20-times higher than that of the glucose unit of HPMC-AS. A small amount of HPMC-AS compared with CLT achieved efficient inhibition of crystallization of CLT. Drug solubilization by micelles using solubilizer such as surfactants or polymers has been reported to improve drug concentrations.27,28 However, the concentration improvement requires a much higher amount of solubilizer than the drug.27,28 The solubilization stoichiometrically occurred by drug encapsulation into micelles; the drug concentration was linearly increased with an increase in the solubilizer concentration. In contrast, drug concentration improvements in the presence of HPMC-AS showed a nonlinear relationship with HPMC-AS concentrations,18 suggesting that the inhibition of GLB and CLT crystallization by HPMC-AS is not derived from stoichiometric interactions of dissolved drugs with HPMC-AS.


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3000 2500 2000 1500 1000 500 0 0

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Time (h)

Time (h)

Figure 2. Concentration profile of (a) GLB and (b) CLT in (▲) phosphate buffer, (■) 100 µg/mL HPMC-AS solution, and (●) 1000 µg/mL HPMC-AS solution (n = 3, mean ± S.D.). Initial GLB and CLT concentrations were 200 and 3000 µg/mL, respectively.

GLB Dissolution Test in HPMC-AS Solutions Dissolution properties of crystalline and amorphous GLB were evaluated in aqueous solution with and without HPMC-AS to evaluate the necessary conditions for achieving the supersaturated concentration of drugs in HPMC-AS solution (Figure 3). Amorphous GLB was prepared by spray-drying of GLB in dichloromethane/methanol (1/1, v/v) (Figure S1). Dissolution rates of crystalline GLB in HPMC-AS solution were slightly slower than those in the absence of HPMC-AS. Cellulose polymers such as HPMC adsorb drug crystal surfaces in aqueous solution and limit direct contact of water molecules and drug crystal surfaces.29,30 It is suggested that the dissolution of GLB from GLB crystal surface could be decreased by such an adsorption effect of HPMC-AS. In terms of GLB concentration, the crystal GLB did not exceed its intrinsic solubility in the presence of HPMC-AS in aqueous solution. When the drug crystal reaches the solubility equilibrium at the drug intrinsic solubility, the adsorption rates of dissolved drugs onto crystal surface accord with desorption rates of drug molecules from crystal surface. Thus, HPMC-AS did not improve the equilibrium concentration through mechanisms affecting drug adsorption and/or desorption. A solubilizing system such a micellar solution improves drug ACS Paragon Plus Environment

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concentration by drug encapsulation into the micellar phase. The distribution of drugs into the micellar phase reduces the dissolved drug in bulk water phase and leads to a higher drug concentration than the intrinsic solubility. However, dissolution tests of crystalline drugs in HPMC-AS solution revealed that HPMC-AS did not form alternative phase distributions when the crystalline drug was dispersed into HPMC-AS solution. 180 160 140

GLB concentration (µg/mL)

GLB concentration (µ µg/mL)

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120 100 80 60 40

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Time (h)

Figure 3. Dissolution profiles of GLB from (▲) crystal GLB and (△) amorphous GLB in phosphate buffer and from (●) crystal GLB and (○) amorphous GLB in 1000 µg/mL HPMC-AS solution (n = 3, mean ± S.D.). GLB powder (100 mg) was dispersed into 500 mL of medium.

Unlike crystalline GLB, amorphous GLB had a high dissolution rate and reached a concentration of approximately 20 µg/mL within 20 min (Figure 3), exceeding crystalline GLB solubility (Table S1). The significant improvement of dissolution rates and solubility of GLB should be according to the amorphization and/or morphological change of GLB by spray-drying. However, in the absence of HPMC-AS, the GLB concentration rapidly decreased from a transient supersaturated state to the intrinsic solubility of crystalline GLB. Amorphous GLB should be transformed to crystalline GLB through dissolution in aqueous solution, resulting in solution equilibrium at crystalline GLB solubility. In contrast, the GLB concentration in HPMC-AS solution continued to increase and reached around 170 µg/mL in 12 h, and dissolution tests showed no reductions in GLB concentrations for 12 h. The no ACS Paragon Plus Environment

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reduction of GLB concentration should be due to GLB crystallization inhibition by HPMC-AS as shown in Figure 2b. Although HPMC-AS did not improve GLB concentrations of solubility equilibrium between dissolved GLB and crystalline GLB, HPMC-AS efficiently sustained supersaturated concentrations of GLB, following dissolution of amorphous GLB. Higher concentrations of amorphous GLB in HPMC-AS solution should be due to higher theoretical solubility of the amorphous form of GLB than that of the crystalline form.31 The achievement of supersaturated concentration of GLB should require the prolongation of the appearance of crystalline GLB transformed from amorphous GLB because crystalline GLB did not exceed its intrinsic solubility in HPMC-AS solution. In addition to the inhibitory effect of drug crystallization from dissolved drug, the long-term maintenance of amorphous GLB allowed high supersaturation level of GLB in HPMC-AS solution.

Recrystallization of Amorphous GLB in HPMC-AS Solution The effects of dissolved HPMC-AS on recrystallization of amorphous GLB was evaluated. PXRD measurements were conducted for precipitated powder after aqueous dispersion of amorphous GLB (Figure 4). Dispersion of amorphous GLB into aqueous solution resulted in GLB recrystallization in 30 min in the absence of HPMC-AS, as indicated by characteristic peaks of GLB crystals in the PXRD pattern at 30 min (Figure 4a). Amorphous GLB rapidly recrystallized in aqueous solution. The drug recrystallization is derived from crystallization of dissolved drug in aqueous solution and/or solid–solid transformation from the amorphous to crystal form.31-33 To determine the degree of solid–solid transformation of GLB, amorphous GLB was stored at 40°C under 96% relative humidity (RH) for several days. Under these conditions, recrystallization of amorphous GLB did not occur for 3 days (Figure S2), indicating that solid–solid transformation of amorphous GLB to the crystalline form was slow. The immediate recrystallization of amorphous GLB in aqueous solution was mainly due to the crystallization of dissolved GLB in aqueous solution from amorphous GLB powder.


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Figure 4. Changes in powder X-ray diffraction (PXRD) patterns of amorphous GLB powder after dispersion into (a) phosphate buffer, (b) 100 µg/mL HPMC-AS solution, and (c) 1000 µg/mL HPMCAS solution. Times after dispersion of spray-dried GLB powder into solutions are presented for each PXRD pattern.

In contrast to aqueous solution without HPMC-AS, HPMC-AS in aqueous solution strongly suppressed recrystallization of amorphous GLB (Figure 4b-c). A halo pattern of amorphous GLB was observed for 3 and 6 h in 100 mg/mL HPMC-AS and 1000 mg/mL HPMC-AS solutions, respectively. ACS Paragon Plus Environment

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This strong inhibitory effect of HPMC-AS on GLB recrystallization mainly occurred in the crystallization process of dissolved GLB. HPMC-AS could suppress the reorganization of GLB molecules into crystal nuclei, leading to delay of appearance of crystalline GLB. The supersaturated concentrations of GLB following continuous dissolution of amorphous GLB into HPMC-AS solution (Figure 3) was derived from such a long-term stabilization of amorphous GLB in aqueous solution.

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H NMR Measurements Molecular states of drug and HPMC-AS in supersaturated solutions were evaluated using NMR

techniques. Figure 5a shows the real-time monitored 1H NMR spectra of supersaturated CLT starting at 3000 µg/mL in the absence of HPMC-AS. The peak intensity of CLT decreased with crystallization of CLT from the supersaturated solution. The CLT concentrations were calculated from the peak areas of H1–H7 and plotted against time (Figure 5b). In agreement with the data shown in Figure 2a, CLT concentrations decreased with time. Moreover, the chemical shift of CLT from H1 to H6 of CLT was down-field shifted with the decrease of CLT concentrations (Figure 5a and 6). The chemical shifts depending on solute concentration have been observed in previous studies.34,35 Self-association of solute molecules induces changes in molecular states and corresponding chemical shifts of solute molecules. During crystallization of supersaturated solutes, molecules initially aggregate, and crystal nuclei are formed through reorganization of aggregates.21,22 The observed chemical shift changes of CLT likely reflected changes in numbers of self-associated CLT molecules. CLT crystallization led to decreased CLT concentrations and reduced its self-association.


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Figure 5. (a) Real-time monitored 1H NMR spectra of CLT in phosphate buffer. Times after addition of DMSO solution containing CLT are presented for each spectrum. The initial CLT concentration was 3000 µg/mL; (b) CLT concentrations were calculated from peak areas of H1–H7 real-time monitored 1H NMR spectra after addition of DMSO solution containing CLT.


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Figure 6. 1H NMR spectra of CLT solution at the CLT concentrations of (black line) 250 and (red line) 3000 µg/mL in phosphate buffer. Data were normalized to the peak height of H7.

Figure 7 shows the 1H NMR spectra of CLT/HPMC-AS solutions with varying CLT concentrations. HPMC-AS peaks were observed at higher magnetic fields (0.8–4.2 ppm) than CLT peaks (7.0–8.8 ppm). The chemical shifts of CLT except H7 were up-field shifted with the increase of CLT concentrations and were similar to those of solutions containing CLT alone (Figure 5a). Self-association of CLT mainly induced chemical environmental changes around CLT. When the line width of CLT peaks in HPMC-AS solution was compared with that in the absence of HPMC-AS, the CLT peaks were dramatically broadened in the presence of HPMC-AS. Overlapped spectra at the same CLT concentrations of 250 and 3000 µg/mL are shown in Figure 8. The black and red lines represent CLT spectra in aqueous solution without HPMC-AS and 1000 µg/mL HPMC-AS solution, respectively. The chemical shifts of CLT peaks at the same concentration were not changed in the presence of HPMC-AS, confirming that chemical environmental changes around CLT was mainly induced by self-association of CLT. In contrast, broadening of CLT peaks in HPMC-AS solution was clearly observed at both concentrations of CLT. Line widths of 1H NMR peaks increased with decreasing mobility.25,26 The slower mobile components of CLT were increased in the presence of HPMC-AS in aqueous solution. Suppression of


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mobility by HPMC-AS was more pronounced at higher CLT concentrations, with half widths of H7 peaks being 6.63 and 23.50 Hz at 250 and 3000 mg/mL, respectively. Similar mobility suppression of poorly water-soluble drug, carbamazepine, was previously observed in the supersaturated solution stabilized by HPMC-AS.18 Suppression of molecular mobility by HPMC-AS was promoted by the formation of supersaturation, although line widths of CLT peaks did not differ between different CLT concentrations in the absence of HPMC-AS (Figure 6).

Figure 7. 1H NMR spectra of CLT in 1000 µg/mL HPMC-AS solutions. CLT concentrations varied between 0 and 3000 µg/mL.


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Figure 8. 1H NMR spectra of (black line) CLT and (red line) CLT/HPMC-AS solutions at CLT concentrations of (a) 250 and (b) 3000 µg/mL, respectively. HPMC-AS concentration was 1000 µg/mL.

Although line widths of HPMC-AS peaks in 1H NMR spectra did not change with CLT concentrations (Figure 7), HPMC-AS peak heights decreased at higher CLT concentrations. The molecular state of HPMC-AS could be changed by the addition of CLT stock solutions in DMSO. CLT and HPMC-AS concentrations were calculated from peak areas of 1H NMR spectra using calibration curves of intact solution and were expressed as a percentage of the theoretical concentration (Figure 9). Almost all CLT and HPMC-AS were detected in 1H NMR spectrum at the CLT/HPMC-AS dose concentrations of 250/1000 µg/mL. In contrast, detectable HPMC-AS concentrations decreased with increasing CLT ACS Paragon Plus Environment

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concentrations, and were reduced to 55% in the presence of 3000 µg/mL CLT. The mobility of a part of HPMC-AS should become slow enough not to be detected by solution NMR measurement. Cellulose polymers such as HPMC can form gel depending on the concentration and temperature in aqueous solution.36 However, HPMC-AS did not form the gel by itself at the present conditions. HPMC-AS peaks were not decreased at the unsaturated CLT concentration of 250 µg/mL, suggesting that 1H NMRdetectable HPMC-AS decreases with supersaturation of CLT. In a previous study, molecular dynamics of the poorly water-soluble drug phenytoin and HPMC and HPMC-AS in aqueous solution were simulated.37 The simulation indicated that such polymers forms a gel-like phase with drug molecules entrapped inside, which considerably slows drug diffusion and leads to maintenance of highly supersaturated concentrations of drug in aqueous solution.37 HPMC-AS could form gel-like structure with the supersaturated CLT in aqueous solution, leading to strong suppression of HPMC-AS molecular mobility and diminished 1H NMR peaks of HPMC-AS. During aggregation of CLT molecules in supersaturated CLT solution, HPMC-AS interacted with CLT and formed a gel-like structure. Detectable HPMC-AS in 1H NMR spectra were reduced with increasing CLT concentrations, suggesting that the formation of gel-like structures depended on the degree of supersaturation. Stronger CLT aggregation induced higher gelation rates of HPMC-AS and diminished detectable HPMC-AS concentrations in 1H NMR spectra.


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CLT

100 Detectable components in 1H NMR spectrum (%)

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Figure 9. Percent detection of added CLT and HPMC-AS concentrations in 1H NMR spectra. CLT and HPMC-AS concentrations are presented in the table as the mean of duplicates; these were calculated from peak areas of H1–H7 and acetyl protons (approximately 2.0 ppm) in 1H NMR spectra, respectively.

To determine the effects of HPMC-AS concentrations on gel-like structure formation, 1H NMR experiments were performed in 100, 500, and 1000 µg/mL HPMC-AS solutions containing 3000 µg/mL CLT (Figure 10). Calculated concentrations of CLT and HPMC-AS from 1H NMR peaks are presented in Figure 9; detectable percentage of HPMC-AS were similar around 55% for all HPMC-AS concentrations, although undetectable HPMC-AS amount in 1H NMR spectra increased with the increase of initial HPMC-AS concentrations. These data indicate that formation of gel-like structures depended on both CLT and HPMC-AS concentrations. Stronger suppression of CLT mobility at higher CLT concentrations in HPMC-AS solution (Figure 7) indicates higher rates of gel-like structure formation with higher initial CLT concentrations. Similar dependency of mobility suppression of CLT on the amount of gel-like structure can be observed in Figure 10; the CLT peaks were more broadened at higher HPMC-AS concentrations.


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Figure 10. 1H NMR spectra of CLT/HPMC-AS solutions at concentrations of (black line) 3000/100, (orange line) 3000/500, and (red line) 3000/1000 µg/mL.

In contrast with 1H NMR-determined concentrations of HPMC-AS, calculated CLT concentrations from 1H NMR peaks were similar to initial CLT concentrations in case of all CLT/HPMC-AS solutions (Figure 9). Most CLT could remain dissolved and enough high mobility to be detected by solution NMR techniques in HPMC-AS solutions, although gel-like structures of HPMC-AS were not detected. It was speculated that the CLT supersaturation in HPMC-AS solution induced the transient gel-like structure formation, and then CLT immediately redissolved into aqueous solution from the gel-like structures (Figure 9). At the equilibrium state, almost all the gel-like structure should be composed of HPMC-AS due to the redissolution of CLT. To investigate the effects of gel-like structures on the molecular state of CLT, NMR measurements were conducted before and after ultracentrifugation of CLT/HPMC-AS solution at 100,000 ×g for 20 min. Ultracentrifugation of 3000/1000 µg/mL CLT/HPMC-AS solution produced a semitransparent gel at the bottom of the tube. The NMR spectrum of the supernatant after ultracentrifugation was compared with those of the original CLT/HPMC-AS solution (Figure 11). ACS Paragon Plus Environment

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Although ultracentrifugation induced very little changes in the peak area or concentration of CLT, CLT peaks were sharpened following ultracentrifugation. Molecular mobility of CLT increased with the ultracentrifugation. In contrast, ultracentrifugation had little effect on HPMC-AS peaks, indicating that suppression of CLT mobility in HPMC-AS solution was mainly derived from the interaction of CLT with the NMR-undetectable component of HPMC-AS, which should be a gel-like structure. However, CLT peaks still broadened in comparison with CLT alone solution, indicating that a part of CLT interacted with HPMC-AS in the ultracentrifuged solution. Notably, the mobility of internal reference TSP was not affected by removal of gel-like HPMC-AS structures by ultracentrifugation (insert spectrum of Figure 11). Thus, gel-like structures of CLT and HPMC-AS may locally occur around CLT molecules, without affecting solution viscosity. HPMC-AS interacted with aggregated CLT and formed a gel-like structure following the addition of CLT solution to HPMC-AS solution. The gel-like structures of CLT and HPMC-AS delayed the reorganization of CLT into crystal lattices. The complex formation caused the redissolution of CLT into aqueous solution and facilitated an equilibrium state at the supersaturated CLT concentrations, while a part of HPMC-AS remained as gel form in CLT/HPMCAS solution.


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Figure 11. 1H NMR spectra of CLT/HPMC-AS solutions at 3000/1000 µg/mL (black line) before and (red line) after ultracentrifugation at 100,000 ×g for 20 min. Insert spectra represent TSP peaks of each solution.

Maintenance of Drug Supersaturation in HPMC-AS Solution The effect of HPMC-AS on drug supersaturation formation and molecular state of solid component of drug in supersaturated solution were evaluated using GLB. On the other hand, in order to clarify the character of dissolved component of drug in the supersaturated solution, NMR experiments was conducted using CLT which could achieve the high concentration of drug enough to be detected in solution 1H NMR spectrum. Then, the mechanism of drug crystallization inhibition by HPMC-AS was comprehensively considered, based on these experimental results using GLB and CLT. The scheme in Figure 12 shows dissolution and precipitation behaviors of poorly water-soluble drugs in HPMC-AS solution. In the presence of excess drug molecules in aqueous solution, dissolved drug molecules rapidly aggregate into crystal nuclei and precipitate with crystal growth. Upon solution equilibrium between adsorption and desorption of drug molecules onto crystal surfaces, drug concentrations in the saturated solution correspond with intrinsic drug solubility in aqueous solution. Similarly, dispersions of crystalline drug in aqueous solution without HPMC-AS reach saturated equilibrium. Although HPMCAS did not improve the intrinsic solubility of crystalline drugs, it maintained supersaturated drug concentrations once excess amount of drug molecules were dissolved in HPMC-AS solution. In addition, the transformation of crystalline drug from amorphous one was strongly delayed by HPMCAS in aqueous solution. In the process of prenuclear aggregation formation from the supersaturated drug, HPMC-AS interacted with the prenuclear aggregates and form gel-like structures that prevent reorganization into crystal nuclei. Hence, the formation of gel-like structures delays the crystallization of drug from the supersaturated solution. These inhibitory effects enable redissolution of prenuclear ACS Paragon Plus Environment

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drug aggregates into aqueous solution and formation of the equilibrium state in HPMC-AS solution even at the supersaturated drug concentration. Although HPMC-AS prolonged increased drug concentrations, the initial dissolution of excess drug in aqueous solution have to be achieved by improvement of intrinsic drug dissolution properties such as those by drug amorphization in solid dispersion formulations.

Figure 12. Hypothesized mechanism of prolonged drug supersaturation in HPMC-AS solution.

CONCLUSIONS HPMC-AS facilitated the formation of equilibrium state at the supersaturated concentration of poorly water-soluble drugs with a quite low amount of HPMC-AS compared with dissolved drugs. Moreover, ACS Paragon Plus Environment

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the appearance of drug crystals in aqueous solution was efficiently delayed in the presence of HPMCAS, leading to long-term maintenance of amorphous drug in aqueous solution. The addition of amorphous drug to HPMC-AS solution produces highly supersaturated drug solutions. 1H NMR measurements revealed that HPMC-AS slowed the mobility of drug components in supersaturated solutions with diminishing HPMC-AS in solution NMR spectra. HPMC-AS–drug gel-like structures were formed during drug aggregation and prevented the formation of drug crystal nuclei. HPMC-AS– drug gel-like structures can be formed depending on the supersaturation level of the drug. The formation of gel-like structures allows the redissolution of drug prenuclear aggregations into aqueous solution and maintenance of supersaturated drug concentrations in HPMC-AS solutions. The equilibrium state at supersaturated drug concentration in HPMC-AS solution proposed in this study will help to find necessary conditions for solid dispersion formulations intending efficient improvement of drug concentration.

ACKNOWLEDGMENTS This study was partly supported by Health and Labor Sciences Research Grants for Research on Development of New 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 providing HPMC-AS.


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Equilibrium State at Supersaturated Drug Concentration Achieved by

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