Direct NMR Monitoring of Phase Separation Behavior of Highly

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Direct NMR monitoring of phase separation behavior of highly supersaturated nifedipine solution stabilized with hypromellose derivatives Keisuke Ueda, Kenjirou Higashi, and Kunikazu Moribe Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on June 5, 2017

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Molecular Pharmaceutics

Direct NMR monitoring of phase separation behavior of highly supersaturated nifedipine solution stabilized with hypromellose derivatives Keisuke Ueda, Kenjirou Higashi, and Kunikazu Moribe∗,

†

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

ACS Paragon Plus Environment

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Table of Contents/Abstract Graphic


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ABSTRACT

We investigated the phase separation behavior and maintenance mechanism of supersaturated state of poorly water-soluble nifedipine (NIF) in hypromellose (HPMC) derivative solutions. Highly supersaturated NIF formed NIF-rich nano-droplet through phase separation from aqueous solution containing HPMC derivative. Dissolvable NIF concentration in the bulk water phase was limited by the phase separation of NIF from the aqueous solution. HPMC derivatives stabilized the NIF-rich nanodroplets and maintained the NIF supersaturation with phase-separated NIF for several hours. The size of the NIF-rich phase was different depending on the HPMC derivatives dissolved in aqueous solution, although the droplet size had no correlation with the time for which NIF supersaturation was maintained without NIF crystallization. HPMC acetate and HPMC acetate succinate (HPMC-AS) effectively maintained the NIF supersaturation containing phase-separated NIF compared with HPMC. Furthermore, HPMC-AS stabilized NIF supersaturation more effectively in acidic conditions. Solution 1

H NMR measurements of NIF-supersaturated solution revealed that HPMC derivatives distributed into

the NIF-rich phase during the phase separation of NIF from the aqueous solution. The hydrophobicity of HPMC derivative strongly affected its distribution into the NIF-rich phase. Moreover, the distribution of HPMC-AS into the NIF-rich phase was promoted at lower pH due to the lower aqueous solubility of HPMC-AS. The distribution of a large amount of HPMC derivatives into NIF-rich phase induced the strong inhibition of NIF crystallization from the NIF-rich phase. Polymer distribution into the drug-rich phase directly monitored by solution NMR technique can be a useful index for the stabilization efficiency of drug-supersaturated solution containing a drug-rich phase.

KEYWORDS Supersaturated solution, 1H NMR measurement, phase separation, hypromellose, nifedipine


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INTRODUCTION The improvement in dissolution and absorption of a poorly water-soluble drug is an important task in formulation studies as most new drug candidates have poor water solubility.1,2 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 However, apparent improvement of drug concentration using such techniques does not always improve the absorption in the small intestine. Strongly encapsulated drugs in solubilizing agents, such as micelle, cyclodextrin, and emulsion, usually exhibit low permeability,9-11 resulting in a failure in improvement of drug absorption. Therefore, increasing drug concentration in the bulk water phase can directly improve drug permeation and enhance intestinal absorption of poorly water-soluble drugs.10,12 Amorphous drugs possess higher energy compared with crystal forms.13,14 The drug in its amorphous form temporarily reaches a concentration higher than that of its crystalline form, forming the drugsupersaturated solution. However, the supersaturated drug would be in a metastable state and easily recrystallize to its stable crystalline form.15 Therefore, water-soluble polymers such

as

polyvinylpyrrolidone,16-18 methacrylate copolymers,19,20 hypromellose (HPMC),18,21 and hypromellose acetate succinate (HPMC-AS)22-24 have been used as crystallization inhibitors of the supersaturated drug. Previous studies indicated that these polymers slowed drug nucleation and crystal growth25,26 and achieved the long-term maintenance of the drug supersaturation.27,28 Recent advances in the crystallization inhibitor have enabled the poorly water-soluble drugs to achieve a high level of supersaturation. The increase in drug supersaturation results in the phase separation of drug from aqueous solution, known as liquid-liquid phase separation or glass-liquid phase separation,29,30 where drug molecules cannot dissolve in the bulk water phase as homogeneous phase and form the drug-rich phase. Drug concentration dissolved in the bulk water phase is limited by the ACS Paragon Plus Environment

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phase-separated concentration, which is similar to the predicted solubility of the amorphous drug.31,32 Drug permeation becomes almost constant at the drug concentration above the phase-separated concentration.31,32 The drug-rich phase can also work as a reservoir to maintain the phase-separated concentration,33 although the drug-rich phase is unstable. Therefore, the drug concentration is seldom maintained at phase-separated concentration due to the drug crystallization. Some polymers stabilize the drug-rich phase in aqueous solution and maintain the drug supersaturation at the phase-separated concentration.34,35 Previous study confirmed that polymers pre-dissolving in aqueous solution are included in the drug-rich phase.34 However, the correlation between the polymer inclusion and the stabilization of the drug-rich phase was not clearly understood, as the polymer inclusion behavior could not be directly monitored. The phase separation behavior of the supersaturated drug has been evaluated using various techniques including ultraviolet (UV) spectroscopy, fluorescence spectroscopy, and light scattering.30,31,36 However, the molecular states of the supersaturated drug and crystallization inhibitors are still unclear. Solution NMR techniques allow the evaluation of the molecular states of drugs and additives even in ever-changing solutions.37,38 Chemical shifts in the NMR spectra reflect differences in the chemical environments of molecules. NMR peak width reflects molecular mobility, and NMR peak broadening demonstrates mobility suppression.39,40 Furthermore, the amount of the dissolving component in a solution can be quantified by the integration of the NMR peak area.41 This technique can provide detailed information on changes in molecular states of drugs and crystallization inhibitors in the supersaturated solutions. Thus, stabilization and maintenance mechanism of drug-supersaturated solution can be investigated by solution NMR techniques. The molecular interaction between drugs and crystallization inhibitors and the mobility suppression of supersaturated drugs, which contribute toward stabilization of drug supersaturation, are demonstrated by solution NMR techniques.23,27 In this study, solution 1H NMR techniques were applied to reveal maintenance mechanism of the drugsupersaturated solution containing an NIF-rich phase. A poorly water-soluble nifedipine (NIF) was used as the model drug. The solution behavior of crystallization inhibitor, HPMC derivative, in NIFACS Paragon Plus Environment

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supersaturated solution containing NIF-rich phase was directly monitored by NMR measurement. Correlation between the maintenance ability of NIF-supersaturated solution and the solution behavior of HPMC derivatives was discussed based on the revealed molecular state.

EXPERIMENTAL SECTION MATERIALS NIF was purchased from Wako Chemicals Co., (Tokyo, Japan). HPMC (type TC-5E) and HPMC-AS (type AS-MF) were donated by Shin-Etsu Chemical Co., (Tokyo, Japan). We synthesized HPMC acetate (HPMC-Ac) according to a method described by Miller et al.42 In brief, 60 g of HPMC was dissolved in 400 mL of acetic acid at 90ºC. After dissolving HPMC, 90 mL of acetic anhydrate was added and stirred at 90ºC for 1.5 h. Distilled water was added to the solution to stop the reaction. The solution was dialyzed to remove acetic acid, and finally, the HPMC-Ac powder was obtained by spray drying. All other materials were of reagent grade, and the chemical structures of NIF and HPMC derivatives are shown in Figure 1. The substituent ratio of the HPMC derivatives was determined using solution 1H NMR (Table 1).


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Figure 1. Chemical structures of (a) nifedipine (NIF) and (b) hypromellose derivative. Carbon numbering of NIF represents peak assignment in NMR spectra.

Table 1. Substituent ratios of HPMC, HPMC-Ac, and HPMC-AS (average number/glucose ring unit).

-CH3

-CH2CH(CH3)OH

-COCH3

HPMC

1.89

0.25

HPMC-Ac

1.89

0.25

0.23

HPMC-AS

1.88

0.24

0.52

-COCH2CH2COOH

0.26

METHODS Preparation of NIF-supersaturated Solution HPMC derivative solutions were prepared by dissolving 1 mg of HPMC derivative in 1 mL of 0.05 M phosphate buffer (pH 7.4 and pH 6.0). NIF stock solution was prepared by dissolving 10 mg of NIF in 1 mL of dimethyl sulfoxide (DMSO). This stock solution was added to the HPMC derivative solutions at a DMSO concentration of 4 % (v/v) at 25°C to prepare NIF-supersaturated solutions. The NIF-supersaturated solutions were shaken at 200 rpm in a water bath at 25°C. Solutions were sampled at 5 min, 30 min, 1 h, 2 h, 4 h, and 8 h and ultracentrifuged at 150,000 ×g for 15 min. NIF concentrations in the supernatant were determined using HPLC. All experiments were performed in the dark to prevent photodegradation of NIF.

HPLC Conditions After ultracentrifugation, the supernatants were diluted with acetonitrile and applied to Shodex® ODS columns (5 µm, 150 mm × 4.6 mm) at 40°C. The mobile phase comprised 50% (v/v) acetonitrile and ACS Paragon Plus Environment

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50% (v/v) phosphate buffer (pH 7.4). The injection volume was 5 µL, and the flow rate was set at 1 mL/min. NIF concentrations were determined by measuring UV absorbance at 235 nm.

Cryogenic-Transmission Electron Microscopy Measurements The nano-droplet formed in the NIF-supersaturated solution was evaluated using cryogenictransmission electron microscopy (cryo-TEM) measurements. A drop of sample solution was placed on the 200-mesh copper grids with a perforated polymer film (Nisshin EM Co., Tokyo, Japan) following hydrophilic treatment and vitrified in liquid ethane controlled at −178ºC after the excess sample solution was removed using the filter paper. The sample was stored in liquid nitrogen. The imaging was performed in a Gatan 626 cryo-holder (Gatan Inc., Pleasonton, California, USA) at 120 kV using a JEM-2100F (JEOL Co., Tokyo, Japan).

Sample Preparation for NMR Measurements The 0.05 M phosphate buffer (pH 7.4 and pH 6.0; pH meter reading) with HPMC derivative was prepared in D2O containing trimethylsilyl propionate (TSP). DMSO-d6 solutions containing 10 mg/mL NIF were added to the HPMC derivative solutions at a final DMSO-d6 concentration of 0.5% and 4% (v/v). The temperature was maintained at 25°C during sample preparation. All the preparation processes were performed in the dark to prevent photodegradation of NIF.

Solution 1H NMR Measurements All NMR measurements were performed using the ECA-500 NMR system (11.75 T, JEOL Resonance Inc., Tokyo, Japan). The sample solution was transferred into a 5-mm NMR sample tube. The solution 1

H NMR spectrum was obtained at 25°C. TSP was used as the internal reference, and NIF and HPMC

derivative concentrations in the solutions were determined using the calibration curves.


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RESULTS AND DISCUSSION De-supersaturation Test of NIF in HPMC Derivative Solutions Figure 2 represents the NIF concentration profile in HPMC derivative solutions (1 mg/mL) after the addition of the NIF stock solution (10 mg/mL, in DMSO) to the HPMC derivative solutions at 4% (v/v). The represented NIF concentration in the Y-axis of Figure 2 is the concentration of the supernatant after ultracentrifugation at 150,000 ×g. In the phosphate buffer without HPMC derivative, NIF rapidly recrystallized and reached the equilibrium concentration, which was similar to the crystalline solubility of NIF in the phosphate buffer containing 4% DMSO (Table S1). In contrast, the NIF concentration reduction stopped at once in all HPMC derivative solutions at NIF concentrations of 200–250 µg/mL. The NIF crystalline solubility was not improved in HPMC derivative solution compared with the intrinsic solubility of NIF crystal (Table S1). Hence, HPMC derivatives maintained the high NIF concentration not by the solubilization effect but by the crystallization inhibition of NIF in the NIFsupersaturated solution.22 Drug crystallization from the supersaturated solution is inhibited by slowing drug nucleation and crystal growth using the crystallization inhibitors, which maintains the drug supersaturated state.25,26,43 Figure S1 represents the appearance of HPMC derivative solutions immediately following the addition of the NIF stock solution. NIF precipitation in the HPMC derivative solutions was not observed, while the solutions were slightly turbid. At a high supersaturated level of the drug, phase separation between aqueous phase and drug-rich phase can be observed. Subsequently, the drug-rich droplet is formed.29,44 The slight turbidity of the NIF-supersaturated solution could be due to the formation of NIF-rich droplet in HPMC derivative solutions. The appearance of the NIFsupersaturated solution was different among the various HPMC derivative solutions; the HPMC-AS solution was much clearer than HPMC and HPMC-Ac solutions. Figure 3 presents the cryo-TEM images of the NIF-supersaturated solutions. In all NIF-supersaturated solutions, a 50–150 nm sphere can be observed, which confirms the presence of the NIF-rich nano-droplet. The particle sizes of the nanodroplet were different among the various HPMC derivative solutions. In HPMC-AS solutions, the


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particle size of the NIF-rich nano-droplet was smaller than that in HPMC and HPMC-Ac solutions, which coincides with the differences in solution appearance (Figure S1). In the de-supersaturation test of NIF in HPMC-AS solutions, the NIF concentration reached a plateau at approximately 200 µg/mL (Figure 2). The NIF concentration was slightly different among the HPMC derivative solutions; in the initial metastable stage, the NIF concentration was around 230 µg/mL for HPMC solution at pH 7.4 and 6.0 and HPMC-AS solution at pH 7.4 and around 200 µg/mL for HPMCAc solution at pH 7.4 and HPMC-AS solution at pH 6.0. The phase separation of NIF from the aqueous solution should limit the NIF concentration in the bulk water phase. The measured NIF concentration in the initial metastable stage might reflect the dissolved NIF concentration in the bulk water phase. However, it is unclear whether the NIF-rich nano-droplets were completely removed by ultracentrifugation. Hence, the NIF concentration dissolved in the bulk water phase was further discussed based on the NMR measurements.

400

NIF concentration (µg/mL)

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Dose concentration

without HPMC derivative (pH 7.4) HPMC (pH 7.4) HPMC-Ac (pH 7.4) HPMC-AS (pH 7.4) HPMC (pH 6.0) HPMC-AS (pH 6.0)

350 300 250 200 150 100 50 0 0

60

120

180

240

300

360

420

480

Time (min)


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Figure 2. NIF concentration profiles in HPMC derivative solutions (n = 3, mean ± S.D.). The concentration of HPMC derivative solutions was 1 mg/mL, and the dose concentration of NIF was 400 µg/mL.

Figure 3. Cryogenic-transmission electron microscopy (cryo-TEM) images of the NIF-supersaturated solution in (a) HPMC solution (pH 7.4), (b) HPMC-Ac solution (pH 7.4), (c) HPMC-AS solution (pH 7.4), and (d) HPMC-AS solution (pH 6.0) immediately following preparation.

Further concentration reduction of NIF from the initial metastable stage was inhibited in each HPMC derivative solution for several hours. The maintenance time of NIF supersaturation at the initial metastable stage was different among the HPMC derivative solutions. HPMC maintained NIF supersaturation at the initial metastable stage only for 0.5 h at pH 7.4 and 6.0, while HPMC-Ac and HPMC-AS maintained NIF supersaturation for 4 h and 1 h at pH 7.4, respectively. It is noteworthy that the maintenance time was 2 h in HPMC-AS solution at pH 6.0. HPMC-AS more effectively maintained NIF supersaturation at a lower pH, whereas HPMC solution showed the same maintenance time at various pH. A previous report demonstrated that crystallization inhibition by HPMC-AS containing the ACS Paragon Plus Environment

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succinoyl substituent is stronger at a lower pH. This was speculated to be due to the increase in hydrophobicity of HPMC-AS in acidic condition.22 Maintenance time of NIF supersaturation at the initial metastable stage varied depending on the HPMC derivative species dissolved in aqueous solution. The variation cannot be explained by the size of the NIF-rich nano-droplet. Hence, the solution behavior of HPMC derivative in the aqueous solution containing NIF-rich nano-droplet was investigated by solution 1H NMR measurement for understanding of the mechanism underlying supersaturation maintenance.

Solution 1H NMR Measurements Figure 4 represents solution 1H NMR spectra of HPMC-AS solution (pH 7.4, prepared using D2O) with and without NIF. The stock solution of NIF (10 mg/mL) was added to the HPMC-AS solution at 0.5% and 4% (v/v) to prepare HPMC-AS solution containing NIF at 50 and 400 µg/mL, respectively. HPMC-AS peaks were detected in the higher magnetic field, whereas the aromatic protons of NIF were detected in the lower magnetic field. On comparing the spectra of different NIF concentrations, NIF at a high concentration of 400 µg/mL showed a broad peak. Solution viscosity reduces the molecular mobility of the dissolved components, resulting in peak broadening.45 However, as the peak width of TSP did not change with the NIF concentration (data not shown), peak broadening at the high concentration was not due to increase in solution viscosity. The existence of the NIF-rich nano-droplets can broaden the NMR peak of NIF. To clarify the detected component of NIF by solution 1H NMR, NIF concentrations were calculated using the NIF peaks from 5.4 to 8.0 ppm on the basis of TSP peak. Solution 1H NMR detected the NIF concentration to be 49 µg/mL in the HPMC-AS solution containing NIF at 50 µg/mL. In contrast, the NIF concentration was determined to be 215 µg/mL in the HPMC-AS solution containing NIF at 400 µg/mL. Therefore, the detected NIF concentration was much smaller than the dose concentration of NIF. Figure 5 represents the solution 1H NMR spectra of NIF in various HPMC derivative solutions. The peaks for NIF in the HPMC derivative solutions were also found to be


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broad. In particular, in HPMC-Ac and HPMC-AS solutions, NIF mobility was strongly suppressed. The NIF concentration detected by solution 1H NMR was calculated from the NIF peak area (Table 2). Similar to the HPMC-AS solution (pH 7.4), NIF at approximately 200 µg/mL was not detected in all the HPMC derivative solutions using 1H NMR spectroscopy. The detectable NIF concentration was higher in HPMC solution (pH 7.4 and 6.0) and HPMC-AS solution (pH 7.4) than in HPMC-Ac solution (pH 7.4) and HPMC-AS solution (pH 6.0). The re-orientational correlation time of NIF molecules in the NIF-rich nano-droplet of around 100 nm should be much longer than that of the molecules dissolved in the bulk water phase due to slow diffusion and high viscosity of NIF-rich nano-droplet. This resulted in significant broadening and finally led to disappearance of NIF peak in the solution 1H NMR spectrum.46 Hence, the NIF detected in solution 1H NMR spectrum should reflect the NIF dissolved in bulk water phase.

Figure 4. Solution 1H NMR spectra of HPMC-AS solution (1 mg/mL, pH 7.4) containing NIF at the dose concentrations of 0 (black line), 50 (red line), and 400 µg/mL (blue line).


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Figure 5. Solution 1H NMR spectra of NIF-supersaturated solution in (a) HPMC solution at pH 7.4, (b) HPMC-Ac solution at pH 7.4, (c) HPMC-AS solution at pH 7.4, (d) HPMC solution at pH 6.0, and (e) HPMC-AS solution at pH 6.0 immediately following the preparation. The concentration of HPMC derivative solutions was 1 mg/mL, and the dose concentration of NIF was 400 µg/mL.

Table 2. NIF and HPMC derivative concentrations detected on solution 1H NMR spectra of NIFsupersaturated solutions (dose concentration of NIF: 400 µg/mL) in HPMC derivative solutions (1 mg/mL).

pH

7.4

6.0

Polymer

HPMC

HPMC-Ac

HPMC-AS

HPMC

HPMC-AS

Detected NIF concentration (µg/mL)

225

180

215

224

181

Detected HPMC derivative concentration (mg/mL)

0.975

0.677

0.843

0.966

0.600

To confirm the detectable components using 1H NMR techniques, the spectra were obtained for the NIF supersaturated solution after ultracentrifugation at 150,000 ×g for 15 min. The solution 1H NMR spectra of the supersaturated NIF in various HPMC derivative solutions after ultracentrifugation are


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shown as the red line in Figure 6, which overlapped with that obtained before ultracentrifugation (black line). The ultracentrifugation sharpened the peak of NIF in all HPMC derivative solutions. Especially for the HPMC and HPMC-Ac solution, the peak width of NIF in the ultracentrifuged solution at the dose concentration of NIF at 400 µg/mL was similar to that in the HPMC-AS solution containing NIF of 50 µg/mL (Figure 4), which did not form the NIF-rich nano-droplets. The existence of NIF-rich nanodroplets could broaden the NIF peak. Specifically, ultracentrifugation removed almost all the NIF-rich nano-droplets in the HPMC and HPMC-Ac solutions. In contrast, the NIF peaks were still broadened in the HPMC-AS solution after ultracentrifugation (Figure 6c and d). The cryo-TEM images show that the NIF-rich nano-droplets were relatively smaller in the HPMC-AS solutions than they were in the HPMC and HPMC-Ac solutions (Figure 3). Ultracentrifugation at 150,000 ×g for 15 min did not completely remove the NIF-rich nano-droplets from the HPMC-AS solutions. The exchange of NIF molecules between the NIF-rich nano-droplets and bulk water phase should broaden the peak of NIF in the solution 1H NMR spectrum. The comparison of the NIF detected using solution 1H NMR measurements showed there were no clear differences in the NIF peak area before and after ultracentrifugation in all the HPMC-derivative solutions. It is noteworthy that even for the HPMC and HPMC-Ac solution containing relatively large particles of the NIF-rich phase, the NIF peak area in the solution 1H NMR spectra was not changed after the removal of NIF-rich nano-droplets. Hence, the components of NIF detected using the solution 1H NMR spectrum could represent the NIF components dissolved in the bulk water phase. However, it remains unclear whether the NIF molecules in the NIF-rich nano-droplets that were several tens of nanometers could be detected using solution 1H NMR measurements. Thus, the NIF concentration detected using the solution 1H NMR spectrum might have been slightly overestimated compared to that in the bulk water phase for the HPMC-AS solutions.


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Figure 6. Solution 1H NMR spectra of NIF-supersaturated solution (black line) before and (red line) after ultracentrifugation of (a) HPMC solution at pH 7.4, (b) HPMC-Ac solution at pH 7.4, (c) HPMCAS solution at pH 7.4, and (d) HPMC-AS solution at pH 6.0 immediately following the preparation. The concentration of HPMC derivative solutions was 1 mg/mL and the dose concentration of NIF was 400 µg/mL.

HPMC-AS peak intensity was marginally reduced in the HPMC-AS solution containing NIF at 400 µg/mL compared with HPMC-AS solution without NIF; however, the intensity did not change with the ACS Paragon Plus Environment

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addition of 50 µg/mL NIF (Figure 4). To clarify the detected component of HPMC-AS by solution 1H NMR, HPMC-AS concentrations were calculated using the methyl proton of hydroxypropyl substituent at around 1.2 ppm. The detectable concentration of HPMC-AS on solution 1H NMR spectrum was 0.84 mg/mL for the HPMC-AS solution (pH 7.4, 1.00 mg/mL) containing 400 µg/mL NIF. A part of HPMCAS (0.16 mg/mL) disappeared from the solution 1H NMR spectrum by the addition of NIF. As mentioned above, NIF within the NIF-rich nano-droplet was not reflected on the solution 1H NMR spectrum. Thus, the undetectable HPMC-AS incorporated into the NIF-rich phase. The inclusion of HPMC-AS into the highly viscous NIF-rich nano-droplet led to the disappearance of HPMC-AS peaks from the solution 1H NMR spectrum. In contrast, the chemical shift and shape of HPMC-AS peaks did not change due to the presence of the NIF-rich nano-droplet. The NIF-rich nano-droplet had no effect on the molecular state of dissolved components of HPMC-AS in aqueous solution. Then, solution 1H NMR measurements were conducted using the different concentration of HPMC-AS. Figure 7 presents the detectable NIF and HPMC-AS concentrations after the addition of NIF stock solution at the dose concentration of 400 µg/mL to HPMC-AS solutions (pH 7.4) at 0.5, 1, and 2 mg/mL. The detectable concentration of NIF was similar among the HPMC-AS solutions. In every concentration of HPMC-AS, the detectable concentration of HPMC-AS was reduced compared with the initial concentration. Interestingly, the reduction rate of HPMC-AS concentration was similar among the HPMC-AS solutions (around 85%). The amount of HPMC-AS incorporated into the NIF-rich phase increased with the elevation of the initial HPMC-AS concentration.


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Detected concentration by 1H NMR (µg/mL)


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2000

1600

NIF HPMC-AS

87.0%

1200 84.3 %

800 85.2%

400

0 0.5 mg/mL 1 mg/mL 2 mg/mL Initial concentration of HPMC-AS

Figure 7. NIF and HPMC-AS concentrations detected using solution 1H NMR spectra immediately after the addition of NIF stock solution at 400 µg/mL to HPMC-AS solution (pH 7.4). The percentage of detectable component against the initial concentration of HPMC-AS is shown above the bars. NIF and HPMC-AS concentrations are mean value of duplicate results.

The solution 1H NMR spectra of methyl proton of hydroxypropyl substituent in each HPMCderivative were also compared before and after the addition of the supersaturated NIF (Figure S2). Similar to the HPMC-AS solution (pH 7.4), the peak intensity of each HPMC derivative was reduced by the addition of supersaturated NIF. The concentrations of HPMC derivatives determined by the detectable peaks on the solution 1H NMR spectra have been presented in Table 2. The HPMC derivative distribution into the NIF-rich phase was promoted in the HPMC-Ac solution (pH 7.4) and HPMC-AS solution (pH 6.0). HPMC-Ac showed stronger hydrophobic properties compared with HPMC due to the presence of the acetyl substituent. High hydrophobicity of HPMC-Ac led to its large distribution into the NIF-rich phase from the bulk water phase during the aggregation process of the NIF-rich phase. Moreover, the amount of HPMC-AS incorporated into the NIF-rich phase varied with the pH; HPMC showed pH-independent inclusion. HPMC-AS showed pH-dependent solubility in aqueous solution and could not dissolve in acidic condition. The solution pH strongly affected the dissolving state of the HPMC-AS containing carboxylic acid; the 1H peaks for the succinoyl substituent were different in


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aqueous solutions at pH 7.4 and 6.0 (Figure S3). The lower solubility of HPMC-AS in aqueous solution at pH 6.0 compared with that at pH 7.4 resulted in the higher distribution of HPMC-AS into the NIFrich phase. The hydrophobicity of HPMC derivative strongly affected its solubility in the aqueous phase as well as the NIF-rich phase. This should play an important role in the HPMC derivative distribution into the NIF-rich phase during aggregation. For understanding NIF supersaturation in detail, HPMC-AS solution (pH 7.4, 1 mg/mL) at the NIF dose concentration of 400 µg/mL (Figure 8a) was monitored in real time by solution 1H NMR. The NIF and HPMC-AS concentrations detected by solution 1H NMR were plotted against the time interval (Figure 8b). The spectral changes in NIF were not observed during the 2-h storage period, and the NIF concentration was maintained at around 210 µg/mL. The detected concentration of HPMC-AS was also constant for 2 h. The molecular state and the concentration of dissolved components in the bulk water phase did not change during the maintenance of the NIF concentration at approximately 210 µg/mL. However, the NIF concentration began to decrease after 3 h of storage and reached 126 µg/mL after 4 h of storage, indicating NIF crystallization. Concurrently, HPMC-AS concentration began to increase after 3 h of storage. After distribution into the NIF-rich phase, HPMC-AS could re-dissolve into the aqueous phase. As HPMC-AS cannot be retained in the NIF crystalline lattice, it was released into the aqueous phase by NIF crystallization within the NIF-rich phase.


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Figure 8. (a) Real-time monitored solution 1H NMR spectra of NIF-supersaturated solution (dose concentration of NIF: 400 µg/mL) in HPMC-AS solution (1 mg/mL, pH 7.4) and (b) NIF and HPMCAS concentrations detected on the spectra. Storage times in the NMR sample tubes after the preparation of NIF-supersaturated solution are presented for each spectrum.


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Figure 9 presents the schematic illustration of the phase separation behavior of NIF in HPMC derivative solution. When excess NIF above the phase-seperated concentration is dissolved in the aqueous solution, the phase separation of NIF from bulk water phase occurs. In the absence of HPMC derivative, the NIF -rich phase rapidly aggregates in the aqueous solution, and NIF crystallizes to form NIF precipitation. HPMC derivatives are included into the NIF-rich phase during aggregation. The amount of HPMC derivative included into the NIF-rich phase depends on the initial amount of the dissolved HPMC derivative in the aqueous solution. The HPMC derivative inclusion into NIF-rich phase hinders the rapid aggregation of NIF-rich phase and maintains the nano-droplets of the NIF-rich phase. The NIF concentration in the bulk water phase is maintained at the initial metastable stage with the coexistence of the NIF-rich nano-droplet. Furthermore, the HPMC derivative incorporated into the NIF-rich phase suppresses the re-orientation of NIF and subsequent NIF nucleation. Until the NIF crystallization occurs, the included HPMC derivative is retained in the NIF-rich phase. However, once the NIF nucleation in the NIF-rich phase begins, the HPMC derivative re-dissolves into the aqueous solution. Then, the distribution of HPMC derivative into the NIF-rich phase is cancelled, followed by further NIF crystallization.

Figure 9. A schematic illustration of HPMC derivative inclusion into NIF-rich phase in NIFsupersaturated solution.


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The concentration of NIF dissolved in the bulk water phase and maintenance time of NIF supersaturation at the initial metastable stage (Figure 2) were discussed on the basis of the revealed solution behavior of HPMC derivatives. The concentration of NIF dissolved in the bulk water phase with the coexisting NIF-rich nano-droplets was approximately 200 µg/mL and similar among the HPMC derivative solutions (Table 2). A previous study reported that the NIF concentration at which phase separation commences in phosphate buffer at 25ºC was 10 times higher than its crystalline solubility.47 The phase separation concentration changes depending on the crystalline solubility in aqueous solution. For instance, adding an organic solvent to an aqueous medium increases the drug concentration at which phase separation commences in accordance with the increase in drug crystalline solubility.48 Furthermore, the maintained drug concentration at the initial metastable state after phase separation depends on the crystallization inhibitor in the aqueous solution, especially for fast crystallizing drugs such as NIF.34 The limitation of NIF concentration in bulk water phase, indicating the NIF concentration at which phase separation begins to occur, might not be accurately determined using the present method because of the initial rapid reduction of the NIF concentration from 400 µg/mL. However, since maintained NIF concentration dissolved in the bulk water phase were approximately 200 µg/mL at initial metastable stage for all HPMC-derivative solutions, the dissolvable NIF concentration in the bulk water phase should be approximately 200 µg/mL in the phosphate buffer containing 4% DMSO at 25ºC. The maintenance time of NIF supersaturation with the coexistence of NIF-rich phase was longer in HPMC-Ac solution (pH 7.4) and HPMC-AS (pH 6.0) solution, where the HPMC derivative inclusion into the NIF-rich phase was higher. The NIF-supersaturated solution containing the NIF-rich nano-droplet should be stabilized by hindering re-orientation of NIF and subsequent NIF nucleation within the NIF-rich phase owing to the inclusion of HPMC derivative. The distribution of a large amount of HPMC derivative into NIF-rich phase led to further stabilization of NIF supersaturation at the initial metastable stage. Herein, we suggested that the polymer inclusion into


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the drug-rich phase can be a good index for prediction of stabilization of the drug supersaturation containing the drug-rich phase.

CONCLUSIONS Solution 1H NMR experiments revealed that HPMC derivatives rapidly distributed into the NIF-rich phase during the formation of the NIF-rich nano-droplet. The increase in hydrophobicity of HPMC derivative promoted its inclusion into the NIF-rich phase. The limitation of the NIF concentration dissolved in the bulk water phase was similar among the HPMC derivatives solutions while the larger distribution of the HPMC derivatives into the NIF-rich phase more strongly stabilized the NIFsupersaturated solution containing NIF-rich nano-droplet. Therefore, polymer inclusion into the drugrich phase monitored by solution NMR techniques can be a good index for the maintenance ability of drug-supersaturated solutions.

ACKNOWLEDGMENTS This study was partly supported by the Research on Development of New Drugs from Japan Agency of Medical Research and Development (AMED) and the Grant-in-Aid for Young Scientists (B) (JSPS, 16K18859) from the Japan Society for the Promotion of Sciences. We thank Shin-Etsu Chemical Co., (Tokyo, Japan) for donating HPMC and HPMC-AS.


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Direct NMR Monitoring of Phase Separation Behavior of Highly

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