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Mechanistic Differences in Permeation Behavior of Supersaturated and Solubilized Solutions of Carbamazepine Revealed by Nuclear Magnetic Resonance Measurements Keisuke Ueda,† Kenjirou Higashi,† Waree Limwikrant, Shuichi Sekine, Toshiharu Horie, Keiji Yamamoto, and Kunikazu Moribe* Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan S Supporting Information *

ABSTRACT: A solid dispersion (SPD) of carbamazepine (CBZ) with hydroxypropyl methylcellulose acetate succinate (HPMC-AS) was prepared by the spray drying method. The apparent solubility (37 °C, pH 7.4) of CBZ observed with the SPD was over 3 times higher than the solubility of unprocessed CBZ. The supersaturated solution was stable for 7 days. A higher concentration of CBZ in aqueous medium was also achieved by mixing with Poloxamer 407 (P407), a solubilizing agent. From permeation studies of CBZ using Caco-2 monolayers and dialysis membranes, we observed improved CBZ permeation across the membrane in the supersaturated solution of CBZ/HPMC-AS SPD. On the contrary, the CBZsolubilized P407 solution exhibited poor permeation by CBZ. The chemical shifts of CBZ on the 1H NMR spectrum from CBZ/ HPMC-AS SPD solution were not altered significantly by coexistence with HPMC-AS. In contrast, an upfield shift of CBZ was observed in the CBZ/P407 solution. The spin−lattice relaxation time (T1) over spin−spin relaxation time (T2) indicated that the mobility of CBZ in the HPMC-AS solution was much lower than that in water. Meanwhile, the mobility of CBZ in P407 solution was significantly higher than that in water. NMR data indicate that CBZ does not strongly interact with HPMC-AS. CBZ mobility was suppressed due to self-association and microviscosity around CBZ, which do not affect permeation behavior. Most of the CBZ molecules in the CBZ/P407 solution were solubilized in the hydrophobic core of P407, and a few were free to permeate the membrane. The molecular state of CBZ, as evaluated by NMR measurements, directly correlated with permeation behavior. KEYWORDS: solid dispersion, HPMC-AS, supersaturated solution, Caco-2 permeation, 1H NMR spectroscopy, NMR relaxation time

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methylcellulose (HPMC), 9−11 poly(vinylpyrrolidone) (PVP),9−13 methacrylate copolymers (Eudragit),10,13 and hydroxypropyl methylcellulose acetate succinate (HPMCAS)11,14 can achieve longer times in the supersaturated state. HPMC-AS is used as an additive for coating and as a stabilizing agent for tablets. It inhibits drug crystallization and functions as a good stabilizer for SPDs with amorphous drugs.11,14,15 Friesen et al. have reported that stable supersaturated solutions of various drugs can be formed by SPD with HPMC-AS.14 Caco-2 cell monolayers are generally accepted as a good in vitro model of absorption in the intestine since results obtained from permeability studies using this model correlate well with in vivo data.16,17 Caco-2 permeation studies have been performed using supersaturated solutions induced by solvents and salt.18,19 Both supersaturated solutions showed improved permeation by poorly water-soluble drugs. Permeation of

INTRODUCTION Many drugs developed in recent years are poorly water-soluble. Among such drugs, the biopharmaceutical classification system class II drugs show good permeability.1 Their bioavailability can be improved to desirable levels by altering their dissolution properties. Many methods, such as nanoparticle formation,2,3 cyclodextrin inclusion complex formation,4,5 and encapsulation into drug carriers6 are used to enhance the dissolution rate and solubility of drugs. Amorphization has also been focused on as an effective method.7 Compared to its crystalline form, the amorphous form of a compound has higher energy because the random array in the amorphous form leads to a higher dissolution rate and solubility. However, amorphous drugs can easily crystallize, with the result that supersaturated states in water are maintained for only very short times. Solid dispersion (SPD), where drug molecules are dispersed and stabilized in polymer matrices, has been investigated as a method for improving the stability of amorphous drugs.8 When SPDs are dispersed in water, the drugs can recrystallize after a short period in the supersaturated state. However, it has been reported that SPDs along with polymers, such as hydroxypropyl © 2012 American Chemical Society

Received: Revised: Accepted: Published: 3023

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the Shin-Etsu Chemical Co. (Tokyo, Japan) and BASF (Ludwigshafen, Germany), respectively. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotic−antimycotic solution were purchased from SigmaAldrich Japan (Tokyo, Japan), Thermo Scientific (Yokohama, Japan), and Life Technologies (Maryland, USA), respectively. All materials were of reagent grade. The chemical structures of CBZ, HPMC-AS, and P407 are schematically represented in Figure 1.

supersaturated solutions in Caco-2 cells using SPDs has also been assessed.14,20,21 However, drug permeation from such supersaturated solutions has not been well-characterized, due to the poor stability of supersaturated solutions and occasional polymer effects on Caco-2 monolayers. The formulation of a stable supersaturated solution would allow us to investigate drug permeation through Caco-2 monolayers. Furthermore, permeation studies using dialysis membranes, where polymer effects on the membrane are negligible, are an even more simplified permeation model for quantitative analysis.22 The combination of the Caco-2 monolayer with the dialysis membrane model is likely to provide a more detailed understanding of mechanisms of drug permeation through membranes. Understanding the relationship between the molecular state of a drug within a supersaturated solution and its permeability is necessary for designing new formulations with improved bioavailability.23 Although many reports on permeation studies are available, there are few reports on the molecular state of drugs in supersaturated solutions.13,24 Hence, the mechanism underlying stabilization of supersaturated drug solutions is not yet clearly understood. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for investigating the molecular state of drugs. Chemical shifts in NMR spectra reflect differences in the chemical environment of molecules. The encapsulation of drugs in polymeric micelles or the interaction of drugs with polymers in supersaturated solutions can be evaluated by NMR on the basis of associated changes in chemical shift.13,25 Information about the mobility of molecules has previously been obtained using relaxation times, such as spin−lattice relaxation time (T1) and spin−spin relation time (T2). 1H NMR peak widths reflect molecular mobility, and the broadening of 1H NMR peaks demonstrates suppression of mobility.26,27 T2 values were calculated from the peak width of 1H NMR spectra according to the equation (half peak width = 1/πT2). However, this equation is applicable only when the field inhomogeneity effect and modulation of J-coupling are negligible. Molecular mobility can be assessed more quantitatively by comparison of experimentally determined T1 and T2 values using pulse techniques, such as inversion recovery and spin echo.28−30 T1/T2 correlates well with the correlation time (τc), which can be used as a direct index of molecular mobility.31,32 The final goal of this study was to clarify the effect of the molecular state of drugs in a supersaturated solution on drug permeation properties. To this end, we studied the stable carbamazepine (CBZ)/HPMC-AS supersaturated solution from an SPD prepared by the spray-drying method. Permeation studies of CBZ from supersaturated solutions were conducted using both Caco-2 monolayers and dialysis membranes. The molecular state of the drug in supersaturated solution was assessed using NMR techniques, including analysis of the relaxation time. The relationship between molecular states of the drug and its dissolution and permeation properties are discussed. In these studies, P407, which is a well-known solubilizing agent, was used to evaluate differences between supersaturated and solubilized drug solutions.

Figure 1. Chemical structures of (a) CBZ, (b) HPMC-AS, and (c) P407. Proton numbering of CBZ and P407 represents the peak assignment in 1H NMR spectra.

Methods. Preparation of Physical Mixtures. Physical mixtures (PMs) of CBZ with HPMC-AS and P407 were prepared by vortex mixing in a vial for 3 min. Preparation of CBZ/HPMC-AS SPD by the Spray-Drying Method. The PMs of CBZ and HPMC-AS were dissolved in dichloromethane to a total solid concentration of 3% (w/v). The weight percent of CBZ in PMs was 6.3%, 11.8%, 16.7%, 21.1%, and 25.0%. The solution was 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, 70 °C; outlet temperature, 45 °C; atomizing pressure, 0.05 MPa; nozzle diameter, 0.7 mm (liquid), and 1.7 mm (gas). Powder X-ray Diffraction Measurement. Powder X-ray diffraction (PXRD) measurements were performed to evaluate the formation of CBZ/HPMC-AS SPD by the spray-drying process. 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, 3−40°. Measurements of Dissolution. Measurements of dissolution were performed using the dissolution method II (Paddle method), as specified in the Japanese Pharmacopoeia XVI. Hanks’ balanced salt solution (HBSS, containing 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.4) was used as the dissolution medium to evaluate dissolution behavior in the same buffer used in Caco-2 permeation studies. Each sample, with 400 mg CBZ, was dispersed in 500 mL of

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EXPERIMENTAL SECTION Materials. CBZ (pKa = 13.4)33 was purchased from Tokyo Chemical Industry (Tokyo, Japan). HPMC-AS (Shin-Etsu AQOAT type AS-HF) and ethylene oxide−propylene oxide block copolymer (P407, poloxamer 407) were kindly gifted by 3024

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HBSS at 37 °C. The solution was stirred with a rotation paddle at a speed of 250 rpm because the wettability of CBZ is very low. The solution (3 mL) was sampled at defined intervals and then filtered through a cellulose ester membrane (0.45 μm). An equal volume of fresh buffer was replaced after each sampling. The concentration of CBZ in the filtrate was determined by high-performance liquid chromatography (HPLC). High-Performance Liquid Chromatography. Sample solutions were diluted with acetonitrile before quantification. CBZ concentrations were determined by measuring UV absorption at 285 nm.34 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 retention time of CBZ was 2.9 min. Preparation of Sample Solution for Permeation Studies. To prepare unprocessed CBZ and CBZ/HPMC-AS PM solution, CBZ or CBZ/HPMC-AS PM, with HPMC-AS at 3 mg/mL, were dispersed in HBSS and stirred vigorously for more than 12 h at 37 °C to reach equilibrium. The CBZ/ HPMC-AS SPD sample was dispersed in HBSS (HPMC-AS concentration: 3 mg/mL) and stirred vigorously for 2 h at 37 °C to obtain CBZ/HPMC-AS SPD solution. For the CBZ/ P407 solution, CBZ was added to a P407 solution (60 mg/ mL), while stirring at 37 °C until the solution became clear. CBZ concentrations in CBZ/HPMC-AS SPD and CBZ/P407 solutions were changed from 200 to 800 μg/mL, where all CBZ was apparently dissolved. We confirmed that the CBZ concentration in each solution remained stable for 24 h at 37 °C. Cell Culture. Caco-2 cells were grown in DMEM, supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/ mL streptomycin, and 0.25 mg/mL amphotericin B. Caco-2 cells at passage numbers 19−27 were seeded at a density of 100 000 cells/cm2 on Transwell (Massachusetts, USA) plates with 0.4 μm polyester membranes coated with collagen. Spent medium was replaced 3 times a week. Caco-2 cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 for at least 25 days. The transepithelial electrical resistance (TEER) of Caco-2 monolayers was confirmed to be >500 Ω cm2 before use in permeation studies. Assessment of Permeation through Caco-2 Monolayers. Culture medium in the apical and basal compartments of Caco2 monolayers was replaced with HBSS 30 min prior to conducting permeation studies. The cells were preincubated in a humidified atmosphere with 5% CO2 at 37 °C. The buffer in the basal compartment was then replaced with 1.5 mL of fresh HBSS, and the buffer in the apical compartment was replaced with 0.5 mL of sample solution. At time intervals of 30, 60, 90, 120, and 180 min, the solution in the basal compartment was sampled (0.1 mL) and replaced with fresh HBSS. Amounts of CBZ in sampled solutions were determined by HPLC. The TEER of the Caco-2 monolayers was routinely measured after the permeation study to confirm cell viability. Assessment of Permeation through the Dialysis Membrane. The pore size of the dialysis membrane used in these studies excludes molecules with molecular weight (MW) > 3500−5000 Da. Thus, CBZ, which has a MW of 263, can cross the dialysis membrane, while the polymers, HPMC-AS (MW 18 000) and P407 (MW 12 500), cannot. Hence, the dialysis membrane can separate drug associated with polymeric micelles from that in the aqueous phase. Three milliliters of sample solution was poured into a dialysis tube (lot number: 3251975,

tube diameter: 10 mm, tube length: 100 mm) of cellulose ester with a MW cutoff of 3500−5000 Da (Spectrum Laboratories, Texas, USA), which was closed at one end. After filling, both sides of the tube were closed, while being careful to exclude air bubbles. The tube was then placed into the dissolution test vessel, containing 900 mL of HBSS at 37 °C. To study the selfdiffusion kinetics of CBZ, the osmotic pressures of the solutions inside and outside the tube were equalized by adding sucrose (MW 342) into the outer solution. The solution in the vessel was stirred with a paddle at 50 rpm. The solution outside the tube was sampled every 5 min for 1 h, and the amount of CBZ was determined by HPLC. Determination of Permeation Constant. The apparent permeation constant through a dialysis membrane is calculated on the basis of drug partition and diffusion kinetics. The method reported by Melik-Nubarov et al. was used to determine drug concentration in micelles.35 In the micellar solution, the drug exists either in the aqueous phase or in micelles. The molar ratio of the drug in the aqueous phase (α) is represented by eq 1. α=

Cw Cw + Cm

(1)

where Cw and Cm represent the drug concentrations in the aqueous phase and in micelles, respectively. The rate of drug diffusion from the dialysis membrane to the outer solution is represented by eq 2. dCout = kCw dt

(2)

where Cout represents the drug concentration in the outer compartment of the dialysis tube at sink conditions and k indicates the rate constant of drug diffusion across the dialysis membrane. By combining eqs 1 and 2, Cout can be calculated by eq 3. Cout = C0(1 − e−α kt)

(3)

where C0 represents the initial concentration of drug in the dialysis membrane. This equation can be transferred to eq 4 α kt = ln C0 − ln(C0 − Cout)

(4)

The apparent permeation constant k′ (k′ = αk) is determined by plotting the ln(Co − Cout) against t. The value of α for each polymer solution was theoretically calculated by applying k obtained from unprocessed drug solution. Sample Preparation for 1H NMR Measurements. Phosphate buffer with deuterium oxide (D2O−phosphate buffer) and deuterated chloroform (CDCl3) were used as NMR solvents. D2O−phosphate buffer contained the same compounds as phosphate buffered saline (pH 7.4). To prepare unprocessed CBZ, CBZ the drug was dispersed in D2O− phosphate buffer (CBZ concentration: 200 μg/mL) and stirred vigorously for more than 12 h at 37 °C. The CBZ/HPMC-AS SPD sample was dispersed in D2O−phosphate buffer (HPMCAS concentration: 3 mg/mL) and stirred vigorously for 2 h at 37 °C to obtain the CBZ/HPMC-AS SPD solution. For the CBZ/P407 solution, CBZ was added to a P407 solution (60 mg/mL) while stirring at 37 °C until the solution became clear. In addition, CBZ was dissolved in CDCl3 at a concentration of 1 mg/mL. 1 H NMR Measurements. All 1H NMR measurements were performed using a JEOL ECX-400 NMR system (9.39T, 3025

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Tokyo, Japan). The sample solution was transferred into a 5 mm NMR sample tube. 1H NMR spectra were obtained at 37 °C with 15 Hz sample spinning. Tetramethylsilane (TMS) and trimethylsilyl propionate (TSP) were used as internal references for samples in CDCl3 and D2O−phosphate buffer, respectively. T1 and T2 Measurements. Relaxation measurements were performed at 37 °C without spinning. T1 was analyzed by performing an inversion−recovery experiment. The delay between pulse sequences was more than 5T1 to allow the spin system to relax to equilibrium. τ intervals of each sample were started from less than T1/10 to more than 5T1. Twenty points of τ interval were used to evaluate T1 by nonlinear curve fitting. T1 was calculated using JEOL Delta ver. 5.01 software (Tokyo, Japan). T2 was measured by using the Carr Purcell Meiboom Gill (CPMG) sequence. More than 5T1 was set between consecutive experiments to allow the spin system to relax to thermal equilibrium. Measurements were conducted at 20 points to evaluate T2 by nonlinear curve fitting. The dipolar intramolecular T1 and T2 are defined by the following equations.31 2 ⎞ τc 4τc 1 3 ⎛ μ ⎞ γ 4ℏ2 ⎛ ⎜ ⎟ ⎜ ⎟ = + 2 2 2 2 6 T1 10 ⎝ 4π ⎠ r ⎝ 1 + ω τc 1 + 4ω τc ⎠

Figure 2. Powder X-ray diffraction patterns of (a) unprocessed CBZ; (b) HPMC-AS; (c) CBZ/HPMC-AS PM, containing CBZ at 21.1% (w/w); and CBZ/HPMC-AS SPD, containing CBZ at (d) 6.3%; (e) 11.8%; (f) 16.7%; (g) 21.1%; and (h) 25.0% (w/w).

(5)

2 ⎞ 5τc 2τc 1 3 ⎛ μ ⎞ γ 4ℏ2 ⎛ ⎜ ⎟ ⎟ ⎜3τc + = + 2 2 2 2 6 T2 20 ⎝ 4π ⎠ r ⎝ 1 + ω τc 1 + 4ω τc ⎠

(6)

where μ is the magnetic dipole moment, ℏ is the Planck constant, γ is the gyromagnetic ratio, r is the distance between the 2 nuclei carrying the magnetic dipole moment, ω is the Larmor frequency (400 MHz of 1H resonance frequency in this study), and τc is the correlation time of the molecular tumbling motion. τc could be described from the Stokes−Einstein eq 7.31 τc =

4πηa3 3kT

(7)

where η is the viscosity, a is the molecular hydrodynamic radius, k is the Boltzman’s constant, and T is the absolute temperature. T1/T2 is correlated with τc and could be used to evaluate relative molecular motion, although T1/T2 is constant in the extreme narrowing regime and T1/T2 grows as the square of the τc in the slow motional regime, where the τc is much longer than the inverse of the Lamor frequency.

Figure 3. Dissolution profiles of CBZ (800 μg/mL) from (⧫) unprocessed CBZ, (■) CBZ/HPMC-AS PM, (▲) CBZ/HPMC-AS SPD, and (×) CBZ/P407 p.m. (n = 3, mean ± SD). Concentrations of HPMC-AS and P407 in PM and SPD solutions were 3 mg/mL and 60 mg/mL, respectively.

RESULTS AND DISCUSSION PXRD Measurements. Characteristic peaks of CBZ were observed in the PM sample (Figure 2). Halo patterns were observed in the CBZ/HPMC-AS SPD samples, with the weight ratio of CBZ from 6.3% to 25.0% (Figure 2). These results confirmed the amorphization of CBZ in the SPD samples and the formation of the CBZ/HPMC-AS solid dispersion. Dissolution Test. Figure 3 shows the dissolution profiles of each sample (CBZ: 800 μg/mL). For unprocessed CBZ, the concentration of CBZ reached approximately 350 μg/mL within 1 h, after which it decreased and was maintained at approximately 250 μg/mL, indicating its equilibrium state. The solubility of CBZ in aqueous solutions at pH 7.4 has been reported to be approximately 250 μg/mL.36 The observed supersaturated state of CBZ in the early stage of the dissolution test should be due to the transformation of CBZ anhydrate to

CBZ hydrate in the dissolution medium. For the CBZ/HPMCAS PM sample, the equilibrium concentration of CBZ was approximately 250 μg/mL, and was maintained for 7 days (Figure S1 of the Supporting Information (SI)). This result indicates that the coexistence of HPMC-AS in the solution did not affect the solubility of CBZ. On the other hand, SPD with HPMC-AS led to rapid CBZ dissolution. The equilibrium concentration of CBZ in the SPD sample was 3 times higher than that of the PM sample. This may be attributable to amorphization of CBZ in the HPMC-AS matrix in the solid state. Furthermore, the supersaturated state of CBZ was maintained for 7 days (Figure S1 of the SI), demonstrating high stability. The strong inhibition of drug crystallization by HPMC-AS solution has been previously reported.11,15 Once CBZ is dissolved in HPMC-AS solution, crystallization of CBZ is greatly suppressed. In addition, we evaluated the stability of supersaturated solutions after filtration to confirm that

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Figure 4. Cumulative amount of CBZ in the basal compartment of transwell plates after addition of sample solutions to the apical compartment (n = 3−4, mean ± SD). (a) Sample solution with 200 μg/mL CBZ (*p < 0.01 and **p < 0.001 vs unprocessed CBZ sample), (b) sample solution with 800 μg/mL CBZ (*p < 0.01 and **p < 0.001 vs unprocessed CBZ sample), (c) different sample solutions of the CBZ/HPMC-AS SPD system, and (d) different sample solutions of the CBZ/P407 system. CBZ concentration is shown in the figure, and concentrations of HPMC-AS and P407 were 3 mg/mL and 60 mg/mL, respectively.

Caco-2 Permeation Study. Figure 4a shows the permeation profiles of samples containing 200 μg/mL CBZ. When CBZ concentrations were lower than its intrinsic solubility, there was no apparent difference in the profiles among unprocessed CBZ, PM, and SPD samples. It appeared that HPMC-AS had no apparent enhancing effects on CBZ permeation through the Caco-2 monolayer. In contrast, the cumulative amount of CBZ decreased when using the CBZ/ P407 system. CBZ could be incorporated into the polymeric micelles of P407, and since the size of the polymer itself is quite large,42,43 this would account for the lower CBZ permeation across Caco-2 monolayers. The amount of CBZ that was not incorporated into P407 micelles was low, and consequently, permeation of CBZ into the basal compartment decreased. It has been reported that poloxamer functions as a permeability enhancer due to its inhibitory effect on transport by Pglycoprotein (P-gp).44 However, CBZ is not a substrate of Pgp,45 and poloxamer effectively enhances permeability when used at concentrations under the cmc.46 Hence, the influence of P407 on Caco-2 cell membranes could be negligible. The results of permeation studies when using CBZ concentrations of 800 μg/mL are shown in Figure 4b. For unprocessed CBZ and PM samples, the solution was turbid white and saturated with CBZ because of solubility limitations.

stabilization was not due to the remaining solid phase of solid dispersion. After filtration, the transparent supersaturated solution also retained a high concentration (data not shown). The dissolution profile of CBZ/P407 p.m. revealed a smooth increase in CBZ concentration up to 800 μg/mL, from the early stages of the dissolution test (Figure 3). This phenomenon can be explained by the solubilizing effect of P407 and is supported by the preliminary observation that solubilized CBZ concentrations increased to 903 μg/mL when P407 concentrations were increased from 40−60 mg/mL (Table S1 of the SI). Kadam et al. reported that enhancement of CBZ solubility by P407 depends on P407 concentration.37 CBZ could be solubilized in P407 micelles at a P407 concentration of 60 mg/mL, because this concentration is much greater than its critical micelle concentration (cmc) of approximately 0.02% wt at 37 °C38,39 and is less than 16% wt. at which P407 gelation can occur.40,41 In contrast, improvement of the dissolution behavior of CBZ/HPMC-AS PM was not observed in this study. These different dissolution profiles between PM solutions using HPMC-AS and P407 could be due to mechanistic differences in the enhancement of CBZ concentrations by the polymer. These results enabled us to distinguish between CBZ-solubilized in P407 solutions and CBZ-supersaturated HPMC-AS solutions. 3027

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The profile of CBZ/P407 samples was similar to that of unprocessed CBZ and CBZ/HPMC-AS PM samples. The data suggest that the amount of free CBZ in the CBZ/P407 solution was similar to that in the unprocessed CBZ and PM suspensions, even though the apparent concentration of CBZ in the P407 solution was much higher, as shown by the dissolution test. In contrast, the amount of CBZ in the basal compartment when using the CBZ/HPMC-AS SPD solution was approximately 3 times more than that for the saturated CBZ solution. These results indicate that CBZ was not incorporated into polymeric HPMC-AS but existed as small molecules that can permeate the Caco-2 monolayer. Membrane permeation by CBZ when using CBZ/HPMC-AS SPD and CBZ/P407 solutions at different CBZ concentrations was investigated (Figures 4c and d). Permeation by CBZ was directly proportional to CBZ concentration in the CBZ/ HPMC-AS SPD solution (Figure 4c). Increments in the cumulative amount of permeated CBZ in the basal compartment were observed, even at concentrations greater than CBZ solubility. On the other hand, permeation by CBZ when using the CBZ/P407 solution was not greater than that when using saturated solutions, that is, unprocessed CBZ and PM solutions, although CBZ permeation increased with increasing CBZ concentrations (Figure 4b and d). These results are in agreement with previous permeation studies on solubilized systems, such as cyclodextrin47 and surfactants.48 The results of these permeation studies clearly show that permeation of Caco2 cells by CBZ is not explained by CBZ solubility, as determined by the dissolution test. Therefore, we propose that the molecular state of CBZ in HPMC-AS solutions is different to that in polymeric micelles of P407. Dialysis Permeation Study. Figure 5 shows the profiles for permeation of CBZ through a dialysis membrane. The

Table 1. Apparent Permeation Rate Constant (k′) through the Dialysis Membrane (n = 3, mean ± SD), Portion of Drug Molecule in Aqueous Phase (α), and Theoretical Concentration of Drug Molecules in the Aqueous Phase. The Data Were Calculated from Results of Studies of Permeation through Dialysis Membranes concentration (μg/mL) k′ (× 10−2) α theoretical drug concentration in aqueous phase (μg/mL)

unprocessed CBZ 200

CBZ/HPMCAS SPD 800/3000

CBZ/P407 solution 800/60000

2.24 ± 0.34 -

2.21 ± 0.01 0.987 790

0.95 ± 0.08 0.425 340

mL for the CBZ/HPMC-AS SPD solution. This value was 3 times greater than CBZ solubility and almost equal to the total concentration of CBZ in the CBZ/HPMC-AS SPD solution. On the other hand, the concentration in the CBZ/P407 system was limited to 340 μg/mL, which was less than half of the total concentration. 1H NMR measurements were conducted for the solutions inside and outside the dialysis membranes to confirm the lack of membrane permeation by HPMC-AS (Figure S2 of the SI). NMR spectra of the inner solution revealed peaks for both CBZ (7−8 ppm) and HPMC-AS (0−6 ppm), while NMR spectra for the outer solution showed only CBZ peaks and those of HPMC-AS. This result confirmed that HPMC-AS does not permeate through the dialysis membrane under the conditions of our study, and that interactions of CBZ with HPMC-AS are not strong, since almost all of the CBZ in the CBZ/HPMC-AS SPD solution was not associated with HPMC-AS. 1 H NMR Measurement. Figure 6 shows the 1H NMR spectra of CBZ, CBZ/HPMC-AS, and P407 in D2O− phosphate buffer. The 1H peaks of CBZ were detected in the low field range around 7−8 ppm, while the chemical shift of

Figure 5. Cumulative amount of CBZ that permeated the dialysis membrane at 37 °C (n = 3, mean ± SD). CBZ concentration is shown in the figure, and concentrations of HPMC-AS and P407 were 3 mg/ mL and 60 mg/mL, respectively.

cumulative amount of permeated CBZ from the CBZ/HPMCAS SPD solution was significantly greater than that from the CBZ/P407 system. This result was consistent with the Caco-2 cell permeation studies, confirming that differences in permeability between the CBZ/HPMC-AS and the CBZ/ P407 solutions are not derived from polymer effects on Caco-2 cells, but from different molecular states of CBZ in each solution. Table 1 shows the numerical values from the profiles shown in Figure 5. The theoretical concentration of CBZ in the aqueous phase, which is the concentration of CBZ without incorporation into the polymer, was calculated to be 790 μg/

Figure 6. 1H NMR spectra of (a) CBZ, (b) HPMC-AS, and (c) P407 in D2O−phosphate buffer at 37 °C. Peaks were identified as proton numberings or substituents. 3028

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Figure 7. 1H NMR spectra of the CBZ/HPMC-AS SPD system. (a) 200 μg/mL CBZ in D2O−phosphate buffer, CBZ/HPMC-AS SPD in D2O− phosphate buffer at CBZ/HPMC-AS concentrations of (b) 200/3000 μg/mL, (c) 400/3000 μg/mL, and (d) 800/3000 μg/mL and (e) 1000 μg/mL CBZ in CDCl3 at 37 °C.

both polymers was in the high field range between 0−6 ppm. Figure 7 shows the expanded 1H NMR spectra of CBZ/ HPMC-AS SPD system between 6.7 and 8.2 ppm. The change in the CBZ chemical shift between unprocessed CBZ and CBZ/HPMC-AS SPD solutions with the same CBZ concentration (200 μg/mL) was rather small. This result suggested that the chemical environment of CBZ in each solution was not significantly different. In contrast, the peak for CBZ in the SPD solution was much broader than that for unprocessed CBZ in D2O−phosphate buffer. The CBZ peaks in the CBZ/HPMCAS SPD solution broadened with increasing concentrations of CBZ, although little change in chemical shift was observed (Figures 7b−d). This suggests that the molecular mobility of unprocessed CBZ was significantly different from that of CBZ in the CBZ/HPMC-AS SPD solution, although the chemical environment surrounding the CBZ molecules was not remarkably different. The chemical shift of the drug peak changed when polymers were present in the solution due to differences in the chemical environment surrounding the drug, for example, hydrophobic or hydrophilic environments.49,50 However, the chemical shift of CBZ was not strongly affected by HPMC-AS, implying that the interaction between CBZ and HPMC-AS is not strong. The broadening of NMR peaks can be attributable to changes in molecular mobility,26,27 and we propose that suppression of the molecular mobility of CBZ in HPMC-AS solution resulted in the peak broadening observed in these studies. Figure 8 shows expanded 1H NMR spectra of the CBZ/P407 system, which clearly show that the CBZ chemical shift and peak shape were different in the presence of P407. Compared to the chemical shifts of unprocessed CBZ in

D2O−phosphate buffer, those of CBZ in the CBZ/P407 system shifted upfield. Similar upfield shifts of drug peaks in poloxamer solutions have been observed in a previous study.25 The encapsulation of the drug into P407 a polymeric micelle core, a hydrophobic environment, likely resulted in this upfield shift of the drug peak, as has been previously reported for P407 micelles.25 Here, a single H6 peak of CBZ in P407 solution was observed. This confirmed that the exchange between free CBZ and encapsulated CBZ was faster than the NMR time scale under these conditions. The shape of the CBZ peak obtained for the CBZ/P407 solution was sharper than that obtained for unprocessed CBZ in D2O−phosphate buffer and was different from that obtained for the CBZ/HPMC-AS solution. This meant that the mobility of CBZ in the P407 solution was higher than that in the unprocessed CBZ solution. The chemical shift and peak width suggested that CBZ mobility in the CBZ/P407 system was similar to that of CBZ in CDCl3, rather than CBZ in D2O−phosphate buffer. Here, the peak shapes of CBZ shown in Figures 7 and 8 were compared to evaluate the state of CBZ in each solution. The rank order of peak width was as follows: CBZ/HPMC-AS solution in D2O−phosphate buffer > unprocessed CBZ in D2O−phosphate buffer > CBZ/P407 solution in D2O−phosphate buffer > unprocessed CBZ in CDCl3. This revealed that the molecular mobility of CBZ, a poorly water-soluble drug, in CDCl3, a hydrophobic environment, was higher than that in D2O−phosphate buffer, a hydrophilic environment. Similar changes in 1H NMR spectra, where the peak of an aromatic compound was broadened in different solvents with chemical shift changes, have been previously reported.51 Sodium chloride content in the solution 3029

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Figure 8. 1H NMR spectra of the CBZ/P407 system. (a) 200 μg/mL CBZ in D2O−phosphate buffer, CBZ/P407 solution in D2O−phosphate buffer at CBZ/P407 concentrations of (b) 200/60 000 μg/mL, (c) 400/60 000 μg/mL, and (d) 800/60 000 μg/mL and (e) 1000 μg/mL CBZ in CDCl3 at 37 °C.

has also been shown to influence peak broadening.52 Changes in NMR spectra were due to self-association of the aromatic compound.51,52 Self-association of CBZ may also be related to the broadening of the 1H NMR peak of CBZ in D2O− phosphate buffer containing unprocessed CBZ and CBZ/ HPMC-AS SPD. NMR Relaxation Time Measurements. Differences in CBZ mobility in each solution were quantitatively analyzed by measuring NMR relaxation times. NMR relaxation times, T1 and T2, were experimentally determined for CBZ and polymer in each solution (Table 2). In addition, T1/T2, which correlates with τc, was also calculated (Table 2). T1/T2 represents differences in the molecular mobility of CBZ in different solutions; the larger the T1/T2 ratio, the lower the mobility. The T1/T2 values of the H2−H6 peaks of unprocessed CBZ in CDCl3 were the lowest, approximately 1.5, compared to those in other solutions. This low T1/T2 value can be explained by the fact that CBZ freely dissolves in CDCl3. For unprocessed CBZ in D2O−phosphate buffer, the T1/T2 value of the H6 peak remained low at 1.2, but that of the H2−H5 peaks were larger, from 9.8−13. This difference in T1/T2 values between CBZ protons in D2O−phosphate buffer and CDCl3 could be due to self-association of CBZ. Thus, an additional experiment was performed to determine CBZ self-association. 1H NMR spectra of unprocessed CBZ in D2O−phosphate buffer were compared in different magnetic fields at 9.39 and 14.10 T (Figure S3 of the SI). The peak width of H6 in 14.10 T was almost the same as that in 9.39 T. On the other hand, the peak width of H2−H5 in 14.10 T was clearly broader than that in 9.39 T. The chemical interchange between free and self-associated states of

CBZ may lead to such chemical shift modulation. The difference between chemical shifts in two states, that is, free and self-associated states, was large for H2−H5 but small for H6. Thus, H2−H5 of CBZ may predominantly participate in self-association, and thisindicates that CBZ was not freely mobile in D2O−phosphate buffer, a poor solvent for CBZ, due to self-association. The T1/T2 values of CBZ in the CBZ/HPMC-AS SPD solution were 160 for H2−H5 and 250 for H6; these values were dramatically larger than those of CBZ in the other solutions that were analyzed. Such large T1/T2 values indicate that the mobility of CBZ in HPMC-AS solution is very low. The T1/T2 value of HPMC-AS, in the range of 7.4−18, was considerably lower than that of CBZ in the HPMC-AS solution, regardless of the MW of CBZ and HPMC-AS. These results suggest that the suppression of mobility of CBZ in HPMC-AS solution was not predominantly due to encapsulation of CBZ in polymer micelles, since the mobility of CBZ should be similar to that of the polymeric micelle core if encapsulation had occurred. We also conducted 1H NMR experiments in different magnetic fields to evaluate the chemical exchange modulation effect (Figure S4 of the SI). Although the peak of CBZ was sharper in higher magnetic fields, the CBZ in CBZ/HPMC-AS SPD solution was still broad compared to unprocessed CBZ in D2O−phosphate buffer. These data confirmed that the modulation of chemical exchange in CBZ/HPMC-AS SPD solution was not the main reason for peak broadening. Since HPMC-AS inhibits crystallization,11,15 HPMC-AS and CBZ must interact by some mechanism. However, the large difference in the T1/T2 values of CBZ and HPMC-AS in 3030

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CBZ. In contrast, the diffusion coefficient of CBZ decreased in CBZ/HPMC-AS SPD solution compared to that in unprocessed CBZ solution, although it was much larger than that of HPMC-AS in CBZ/HPMC-AS SPD solution. The smaller diffusion coefficient of CBZ in CBZ/HPMC-AS SPD solution could be due to changes in microviscosity and the molecular radius of CBZ, according to the Stokes−Einstein equation,53 which correlates with our conclusions from analyses of NMR relaxation time. In contrast to the T1/T2 values of CBZ in CBZ/HPMC-AS solution, the T1/T2 values of CBZ in the CBZ/P407 system, approximately 2.5−3.9, were smaller than those of unprocessed CBZ in D2O−phosphate buffer. CBZ in P407 polymer micelles could be relatively mobile compared to unprocessed CBZ in D2O−phosphate buffer, but still less mobile than unprocessed CBZ in CDCl3, according to the ranking of T1/T2 values. The hydrophobic unit (Ha and Hb) of P407 had larger T1/T2 values, between 3.5 and 4.9, than the hydrophilic unit (Hc), which were in the range 1.8−2.2. 1H motion in the hydrophobic core of P407 polymer micelles was more restricted than that in the hydrophilic shell. The comparable T1/T2 values between the 1H of CBZ and the hydrophobic unit of P407 suggest that CBZ was encapsulated in the hydrophobic core of P407 polymeric micelles. NMR relaxation of CBZ in P407 micelles was mainly dominated by intermolecular relaxation by P407. The reason for T1/T2 values of CBZ in P407 solution being lower than those of unprocessed CBZ in D2O− phosphate buffer was probably due to self-association of CBZ in D2O−phosphate buffer. Structure and Permeation Mechanism of CBZ from CBZ/HPMC-AS Supersaturated Solution and CBZ/P407 Solubilized Solution. Figure 9 shows the structures of CBZ/

Table 2. Spin−Lattice Relaxation Times (T1), Spin−Spin Relaxation Times (T2), and T1/T2 for 1H-NMR Peaks of CBZ and Polymer at 37 °C δ (ppm)

T1 (s)

T2 (s)

Unprocessed CBZ in CDCl3 H6 6.94 3.1 2.3 H4 7.33 3.8 2.4 H3 7.37 3.3 2.5 H5 7.43 3.2 2.6 H2 7.49 4.0 2.5 Unprocessed CBZ in D2O−Phosphate Buffer CBZ H6 7.12 3.0 2.5 H4 7.49 4.0 0.39 H3 7.55 4.9 0.39 H5 7.57 4.1 0.42 H2 7.58 5.1 0.39 CBZ/HPMC-AS SPD in D2O−Phosphate Buffer CBZ H6 7.10 2.4 0.0097 H4 H3 7.56a 2.0a 0.012a H5 H2 HPMC-AS 1.17−3.60 1.2−2.2 0.077−0.18 CBZ/P407 in D2O−Phosphate Buffer CBZ H6 7.07 1.2 0.32 H4 7.45 1.4 0.41 H3 7.51 1.3 0.52 H5 7.54 1.5 0.47 H2 7.55 1.5 0.43 P407 Ha 1.13−3.54 0.41−0.49 0.083−0.13 Hb Hc 3.64−3.87 0.56−1.2 0.28−0.54

CBZ

T1/T2 1.3 1.6 1.4 1.2 1.6 1.2 10 12 9.8 13 250 160a 7.4−18 3.9 3.4 2.5 3.2 3.4 3.5−4.9 1.8−2.2

a

The peaks of H2, H3, H4, and H5 overlapped due to peak broadening.

CBZ/HPMC-AS solution may indicate that interaction with HPMC-AS is not the only reason for suppression of CBZ mobility in HPMC-AS solution. According to eq 7, viscosity and molecular hydrodynamic radius can also affect the correlation time. The macroviscosity of solutions with and without HPMC-AS showed no significant difference (Table S2 of the SI). This confirmed that macroscopic changes, such as gelation of HPMC-AS, did not occur under these conditions. However, microviscosity can be different from macroviscosity in the case of polymeric solutions, and CBZ mobility could be suppressed due to a microviscosity increment when HPMC-AS is present in the solution. In addition, an increase in molecular radius due to self-association of CBZ, which was also observed with unprocessed CBZ in D2O−phosphate buffer, may also contribute to suppression of CBZ mobility. Changes in both microviscosity and CBZ self-association could result in a much larger correlation time for CBZ in CBZ/HPMC-AS SPD solution. Diffusion coefficients were determined by 1H NMR measurements to confirm the macroscopic state of CBZ and HPMC-AS in CBZ/HPMC-AS SPD solutions (Table S3 of the SI). The diffusion coefficient of HPMC-AS did not dramatically change despite the presence of CBZ. Particle size distribution, as assessed by the dynamic light scattering method, was quite similar between HPMC-AS solution and CBZ/HPMC-AS SPD solution (Figure S5 of the SI), supporting the hypothesis that the structure of HPMC-AS was not altered by the presence of

Figure 9. Hypothesized molecular states of CBZ in CBZ/HPMC-AS SPD and CBZ/P407 solutions.

HPMC-AS and CBZ/P407 solutions as postulated from permeation studies and NMR measurements. As for the P407 solution, the high concentrations of CBZ can be explained by CBZ encapsulation in poloxamer micelles.37 NMR measurements indicate that CBZ in the CBZ/P407 solution can be encapsulated in the polymer micelle core. The molecular mobility of CBZ in the hydrophobic core increased. However, due to the size of the polymer micelles, CBZ in the polymer micelles could not permeate Caco-2 monolayers or dialysis membranes. Permeation studies using Caco-2 monolayers and dialysis membranes demonstrated that almost all of the CBZ in the HPMC-AS solution could permeate efficiently. Indeed, interactions between CBZ and HPMC-AS should not be strong as those with P407 micelles. It has been suggested that the 3031

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CBZ/HPMC-AS supersaturated solution exists as a homogeneous phase with CBZ dissolved in the polymer solution. NMR measurements suggested that CBZ is self-associated in the HPMC-AS solution. CBZ was not completely incorporated in HPMC-AS but was slightly displaced from the HPMC-AS structure. We propose that HPMC-AS suppresses the CBZ aggregation process leading to crystallization and stabilizes selfassociated CBZ in solution.

CONCLUSIONS Dissolution characteristics of CBZ were greatly improved in CBZ/HPMC-AS SPD. HPMC-AS had a strong inhibitory effect on drug recrystallization leading to prolonged stabilization of the supersaturated solution but did not have a solubilizing effect similar to P407. In contrast to CBZ solubilization by P407, stable supersaturation by HPMC-AS was effective in enhancing CBZ permeation through Caco-2 monolayers and dialysis membranes. The physicochemical environment of CBZ in P407 solution is hydrophobic, while that of the HPMC-AS solution is similar to aqueous solution without polymer. Te mobility of CBZ in HPMC-AS solution was significantly suppressed, without strict incorporation into the polymer. Suppression of molecular mobility may result from CBZ self-association and interaction with HPMC-AS. Any of these differences in CBZ molecular state when in different polymer solutions could underlie its different permeation behaviors. A formulation of supersaturated solutions using polymers that prevent recrystallization is likely to be more effective in improving the permeation behavior of poorly watersoluble drugs, compared to solubilized solutions. For example, such formulations could be useful in the setting of oral drug delivery, where the fluid within the intestinal lumen changes in both composition and volume over time. ASSOCIATED CONTENT

S Supporting Information *

Dissolution profiles, solubilizing effect of P407, 1H NMR data and spectra, sample viscosities, diffusion coefficient measurements, and particle size distributions. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

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: moribe@p. chiba-u.ac.jp. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by Research on Publicity Essential Drugs and Medical Devices of Japan Health Sciences Foundation and by the OTC Self-Medication Promotion Foundation (Sato Pharmaceutical Co., Ltd.). We would like to thank Dr. Mamoru Imanari for helpful discussions relating to NMR experiments. We also thank the Shin-Etsu Chemical Co. (Tokyo, Japan) for gifting HPMC-AS and BASF (Ludwigshafen, Germany) for gifting P407. 3032

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