Stabilization of a Supersaturated Solution of Mefenamic Acid from a Solid Dispersion with EUDRAGIT® EPO
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- Kojima, T., Higashi, K., Suzuki, T. et al. Pharm Res (2012) 29: 2777. doi:10.1007/s11095-011-0655-7
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The stabilization mechanism of a supersaturated solution of mefenamic acid (MFA) from a solid dispersion with EUDRAGIT® EPO (EPO) was investigated.
The solid dispersions were prepared by cryogenic grinding method. Powder X-ray diffractometry, in vitro dissolution test, in vivo oral absorption study, infrared spectroscopy, and solid- and solution-state NMR spectroscopies were used to characterize the solid dispersions.
Dissolution tests in acetate buffer (pH 5.5) revealed that solid dispersion showed > 200-fold higher concentration of MFA. Supersaturated solution was stable over 1 month and exhibited improved oral bioavailability of MFA in rats, with a 7.8-fold higher area under the plasma concentration-versus-time curve. Solid-state 1H spin–lattice relaxation time (T1) measurement showed that MFA was almost monomolecularly dispersed in the EPO polymer matrix. Intermolecular interaction between MFA and EPO was indicated by solid-state infrared and 13C-T1 measurements. Solution-state 1H-NMR measurement demonstrated that MFA existed in monomolecular state in supersaturated solution. 1H-T1 and difference nuclear Overhauser effect measurements indicated that cross relaxation occurred between MFA and EPO due to the small distance between them.
The formation and high stability of the supersaturated solution were attributable to the specifically formed intermolecular interactions between MFA and EPO.
Key wordssolid dispersionsupersaturationNMRoral bioavailabilityEUDRAGIT® EPO
In recent years, progress in combinatorial chemistry and high-throughput screening has enabled the pharmaceutical industry to synthesize and evaluate an enormous number of compounds in a short time. This has resulted in an increase in new drug candidates with intense pharmacological activity. However, this emphasis on pharmacological activity has resulted in the creation of complex-structured compounds with high molecular weight (1). These compounds exhibit much lower bioavailability than expected from their potential druggability because of poor water solubility and permeability. The United States Food and Drug Administration (FDA) has proposed a biopharmaceutical classification system (BCS) that categorizes drugs into 4 classes based on their permeability and solubility (2). Drugs showing high permeability and low water solubility have been classified into class II. Although these drugs have advantageous membrane permeability, they cannot act effectively because the dissolution process is a limiting factor for drug absorption. Therefore, improving the solubility of poorly water-soluble drugs is one of the most important tasks for enhancing the absorption of class II drugs, and various approaches have been investigated, such as cocrystal formation (3), inclusion complex formation with cyclodextrin (4,5), encapsulation of drugs into emulsion (6), and nanoparticle formation to increase surface area (7,8).
Amorphization of crystalline drugs is one of the most frequently used methods for improving water solubility (9). The arrangement of drug molecules in the amorphous state is disordered while those in the crystalline state are regularly arrayed. This results in molecules in the amorphous state that possess higher energy than molecules in the crystalline state (10). When amorphous drugs are dispersed into water, a supersaturated solution that exceeds the solubility of the crystalline drug at the same temperature can be obtained. However, it is thermodynamically difficult to prepare a stable amorphous drug by itself. Consequently, solid dispersions, where the drug molecules are dispersed monomolecularly into a carrier matrix, have been studied (11,12). Many water-soluble polymers can be used as carriers that accelerate amorphization of crystalline drugs and stabilize amorphous drugs in the solid state (13–15). These polymers can also inhibit recrystallization of drugs from supersaturated solution, resulting in stabilization of the supersaturated state in solution (16). For these reasons, solid dispersions are expected to improve in vitro dissolution and in vivo oral bioavailability of poorly water-soluble drugs (17). However, drug recrystallization may occur even in solid dispersions due to mechanical stress, heat, and humidity. There are few examples of solid dispersions of pharmaceutical products that offer long storage stability in the amorphous state over several years (18). Furthermore, supersaturated solutions obtained from solid dispersions are still thermodynamically unstable, and drug recrystallization in water must be induced gradually. Only a few papers have reported supersaturated solutions with long-term stability over the scale of several days.
EUDRAGIT®, which is a methacrylic acid copolymer, has various characteristics depending on its substituents. EUDRAGIT® has been widely used as a pharmaceutical excipient for enteric film-coating formation, sustained release, taste/smell masking, and wear/moisture prevention. Solid dispersions using these polymers as carriers have been documented (19–21).
Nuclear magnetic resonance (NMR) spectroscopy is an important technique that allows direct observation at the molecular level of both low-molecular-weight compounds and macromolecules. In the last 25 years, solid-state NMR has increasingly evolved thanks to the development of magic-angle spinning (MAS), cross polarization (CP), and high-power decoupling (21). In the pharmaceutical field, the solid-state NMR technique is used for studying structure/conformation, analyzing molecular motions, assigning resonances, and measuring internuclear distances (22). Solid-state 13C-NMR studies have revealed that the solid-state interactions of probucol/polyvinylpyrrolidone (PVP) and PVP/sodium dodecyl sulfate (SDS) on grinding play a key role in probucol nanoparticle formation (23). Multivariate analysis of solid-state 13C-NMR spectra of troglitazone/PVP dispersions have allowed prediction of the drug’s recrystallization behavior (24). In particular, NMR relaxation time can provide information on the molecular motions of specific atoms in a molecule. Therefore, it is a powerful tool for determining individual molecular motions in multicomponent systems such as pharmaceutical formulations (25). Schantz et al. evaluated the physical stability of amorphous binary mixtures of citric acid and paracetamol by relaxation time measurement (26), and Aso et al. reported that the miscibility of a drug and an excipient in solid dispersion can be evaluated by assessing its relaxation decay (27). Solid dispersions of ibuprofen or flurbiprofen with EUDRAGIT® RL100 have also been evaluated by 1H relaxation time measurement (20). These reports confirm the utility of solid-state NMR for the analysis of pharmaceutical formulations that include solid dispersion. In contrast, there have been few reports describing the molecular state of solid dispersions in aqueous media by solution-state NMR measurement because supersaturated solutions from solid dispersion are usually viscous, weak in concentration, and unstable for the time scale needed for NMR measurement. If these difficulties could be overcome by applying NMR techniques to long-stabilized supersaturated solutions, detailed structural information could be obtained. The combination of solution- and solid-state NMR spectroscopy could provide us with a deep understanding of solid dispersion because the molecular states both before and after dispersing in aqueous media could be evaluated.
In this study, a highly stable solid dispersion and highly concentrated supersaturated solution were prepared by utilizing cryogenic grinding of mefenamic acid (MFA) and EUDRAGIT® EPO (EPO) as a model of a poorly water-soluble drug and carrier polymer, respectively. An in vitro dissolution test and an in vivo oral absorption study in rats were performed to investigate the dissolution characteristics and the gastrointestinal absorption rate of MFA in supersaturated solution. The physicochemical properties of the solid dispersion were assessed by powder x-ray diffraction (PXRD), infrared (IR), and solid-state NMR including relaxation time measurement, to investigate the formation and stabilization mechanisms. Furthermore, solution-state NMR measurement was performed to investigate the molecular state of MFA and EPO in the supersaturated solution.
Materials and Methods
Preparation of Solid Dispersions
Solid dispersions were prepared by the cryogenic grinding method. Drug and polymer were mixed at a weight ratio of 24/76 to obtain a physical mixture (PM). The PM was ground in a TI-500ET vibration rod mill (CMT Co. Ltd., Fukushima, Japan) at −180°C for 90 min to prepare cryogenic-ground mixture (Cryo-GM).
Powder X-Ray Diffractometry
PXRD measurements were performed using a Miniflex II X-ray diffractometer (Rigaku, Tokyo, Japan) with the temperature at 25°C, voltage at 30 kV, current at 15 mA, scanning speed at 4°/min, and CuKα radiation source with a Ni filter.
In Vitro Dissolution Test
Dissolution tests were carried out at 37.0 ± 0.5°C for 180 min in 500 mL of 0.1 M acetate buffer (pH 5.5) using a USP type 2 apparatus NTR-VS6P (Toyama Sangyo, Osaka, Japan) with the paddle speed at 100 rpm. Samples containing 250 mg of drug were put into the dissolution vessel. Five milliliters of dissolution medium were withdrawn at the indicated periods (1, 3, 5, 7, 10, 15, 20, 25, 30, 60, 120, and 180 min) and immediately filtered by a 0.20 μm ADVANTEC® membrane filter (Tokyo Roshi Kaisha, Tokyo, Japan). The filtrate was diluted and analyzed with a V-650 UV–vis Spectrometer (JASCO, Tokyo, Japan). The determinations of MFA, IMC, and PXC concentrations were performed at λ = 345, 340, and 251 nm, respectively. In the meantime, an equal volume of the same medium was added to maintain constant volume. The dissolution data were obtained in triplicate.
All experiments were conducted according to guidelines approved by the Nihon University Animal Care and Use Committee (Nihon University, Japan). Adult male Wistar/ST rats (8 weeks old, 230–250 g body weight) were obtained from Japan SLC, Inc. (Shizuoka, Japan). The rats were housed in stainless steel cages with a 12 h light/dark cycle (light on from 8:00 am to 8:00 pm) under conditions of controlled temperature maintained at 25°C ± 1°C with a humidity of 55% ± 10% relative humidity (RH) for at least 1 week before use. Animals were fasted for 15 h prior to the experiments.
In Vivo Absorption Studies
Intact MFA was suspended in 0.5% carboxymethylcellulose (CMC) solution to prepare a 10 mg/mL dosing solution. PM or Cryo-GM (10 mg as MFA) was dispersed in 1 mL of 0.1 M acetate buffer solution (pH 5.5). The dosing solution at a dose of 2 mL/kg body weight was administered orally by gavage to rats. The dose was calculated to be 20 mg/kg as MFA. At the designated time points (0.5, 1, 2, 4, 6, 10, and 24 h after the administration of the drug, blood (0.5 mL) was collected from the jugular vein under pentobarbital anesthesia (40 mg/kg). The blood was centrifuged to separate the plasma. Each plasma sample was stored at −20°C until analysis.
Determination of MFA Concentrations in Plasma
Plasma concentrations of MFA were determined by a minor modification of the high-performance liquid chromatography (HPLC) method reported previously (28,29). Briefly, 50 μL of plasma sample in a 1.5 mL test tube was added 20 μL of methanol solution containing diclofenac (5 μg/mL) to serve as an internal standard. After acidification with 10 μL of 0.1 N HCl, the mixture was extracted with 1 mL of hexane/diethyl ether (50/50, v/v) by mechanical shaking for 5 min. After centrifugation at 11,400 × g (12,000 rpm) for 10 min at 4°C, the upper organic solvent layer was transferred to another tube. The residue mixture was extracted again with 1 mL of hexane/diethyl ether. The organic solvent was evaporated to dryness at 30°C. The evaporated residue was reconstituted with 200 μL of the HPLC mobile phase, and an aliquot of 20 μL was analyzed by using the HPLC apparatus (Shimadzu, Kyoto, Japan). The analytical column, C-18 CAPCELL PAK® (4.6 × 250 mm, 5 μm, Shiseido, Tokyo, Japan) was used at 40°C. The mobile phase of acetonitrile/0.05 M phosphate buffer (pH 7.5) (30/70, v/v) was pumped at a flow rate of 1.0 mL/min, and the column eluate was monitored at 280 nm. The retention times were 10.7 min for the internal standard and 12.8 min for MFA. The limit of detection was 10 ng/mL of MFA in plasma. Peakarea ratios for MFA relative to the internal standard were linearly related (r2 = 0.998) to the amount of MFA added to blank plasma in the range of 100–8,000 ng/mL. The intra- and inter-day assay coefficients of variation were calculated to be less than approximately 3% in concentrations ranging from 150 to 8,000 ng/mL. The mean analytical recovery was 95.3% in that range.
The highest plasma concentration of MFA was employed as Cmax, and the time to reach Cmax was defined as tmax. The area under the plasma concentration versus time curve (AUC) was calculated from 0 to 24 h using a linear trapezoidal rule.
Fourier Transform IR (FT-IR) Spectroscopy
IR spectroscopy measurement was performed with a FT-IR 300E spectrometer (JASCO, Tokyo Japan) using the KBr disk method. The IR spectra were obtained in the scan range of 650–4,000 cm−1 at a resolution of 4 cm−1 with 32 scans at a temperature of 25°C.
Solid-State NMR Spectroscopy
All solid-state NMR measurements were conducted using a JNM-ECA600 NMR spectrometer (JEOL, Tokyo, Japan) with a magnetic field of 14.09 T operating at the 1H Larmor frequency of 600.0 MHz and the 13C frequency of 150.0 MHz. Samples (ca. 100 mg) were placed as powders into 4 mm silicon nitride rotors. The 13C spectra were acquired using CP together with MAS at 15.0 kHz and high-power 1H decoupling at an inlet air temperature of 25.0°C. For each spectrum, the total number of accumulations (1,000–4,096) was acquired depending on the required signal-to-noise ratio. Pertinent acquisition parameters included relaxation delays of 2–16 s, a CP contact time of 5 ms, and a 1H 90° pulse of 2.7 μs. The total number of data points was 4,096 points per spectrum in each experiment, zero-filled to 16,384 points. All spectra were externally referenced to tetramethylsilane by setting the methine peak of hexamethylbenzene to 17.3 ppm. Spinning sidebands were confirmed by changing the rotational speed from 10.0 to 15.0 kHz. 1H spin–lattice relaxation time (T1) was measured using the standard [180°-τ-90°]n inversion recovery method with 1H 90° pulse of accurately determined for each sample; 13C-T1 was determined using a pulse sequence reported by Torchia (30).
Solution-State NMR Spectroscopy
All solution NMR measurements were performed using the JNM-ECA600 NMR spectrometer described in the previous section. Solution-state NMR samples, except unprocessed MFA, were dissolved at a concentration of 3 mg/mL as MFA in 0.1 M acetate buffer (pH 5.5), which was prepared by using D2O and put in glass sample tubes (5 mmϕ). Due to the low solubility of MFA at pH 5.5, unprocessed MFA was dissolved in CDCl3. 1H NMR spectra were recorded at 37.0°C using a relaxation delay of 5 s, 1H 45° pulse of 7.75 μs, spectral width of 15 ppm centered at 5.0 ppm, and spinning rate of 15 Hz. The signal of the solvent was used for 1H chemical shift referencing (CHCl3 for 7.25 and HDO for 4.67 ppm). For each spectrum, the total number of accumulations (32–128) was acquired depending on the required signal-to-noise ratio. 1H-T1 was measured using the standard [180°-τ-90°]n inversion recovery method. 1H difference nuclear Overhauser effect (NOE) experiments were applied to the same Cryo-GM solution sample. A difference NOE spectrum was obtained by subtracting a pair of spectra (ΔI = Ion-Ioff) acquired in an interleaved fashion. On- and off-resonance frequencies were at 8.00 ppm and −10.0 ppm, respectively; the measurements were conducted without sample spinning. Pertinent acquisition parameters included a relaxation delay of 5 s, 1H 90° pulse of 13.5 μs, total accumulation number of 512, NOE build-up time of 3 s, and irradiation attenuators varying from 65 to 77 dB.
Results and Discussion
Effect of EUDRAGIT® Species on Formation of MFA Solid Dispersion
In Vitro Dissolution of MFA from MFA/EPO Cryo-GM
In Vivo Oral Absorption
Further investigation was performed in order to mention that the changes in IR spectrum of MFA in MFA/EPO Cryo-GM were not the result of merely amorphization but formation of some intermolecular interaction with EPO. We tried to prepare amorphous MFA without EPO. Melt-quenching method was not appropriate for preparing amorphous MFA because MFA started to decompose immediately after the fusion around 230°C (33). Cryogenic grinding method and solvent evaporation method using methanol and dichloromethane were used. However, PXRD measurement showed that amorphous MFA was not formed according to those methods (data not shown). Hence, it was suggested that amorphous MFA could not form without adding additives at room temperature.
Since it was difficult to prepare amorphous MFA without additive, we prepared another solid dispersion of MFA with hydroxypropyl methylcellulose (HPMC, Fig. S1) by cryogenic grinding method and compared its IR spectrum with that of MFA/EPO Cryo-GM. PXRD pattern of MFA/HPMC Cryo-GM at a weight ratio of 24/76 showed hallo pattern (Fig. S2). IR measurement showed that the peak positions in MFA/HPMC PM were the superimposition of those in unprocessed MFA and HPMC (Fig. S3). In the IR spectrum of MFA/HPMC Cryo-GM, N-H stretching vibration peak of MFA did not change, while the C=O stretching vibration peak of MFA showed peak broadening and small shift to a higher wave number at 1,657 cm−1. The spectral changes were due to the overlap of two peaks: an unshifted one at 1,650 cm−1 and another shifted one around 1,680 cm−1. This result suggested the limited drug-polymer miscibility; non-interacted small crystalline MFA, which was not observed by PXRD measurement, coexisted with interacted MFA with HPMC (34). It was emphasized that the peak shift of C=O stretching vibration from PM to Cryo-GM was much larger in MFA/EPO system than in MFA/HPMC system, even when only the peak of interacted MFA with HPMC around 1,680 cm−1 was taken into consideration. The peak shift on the formation of molecularly-mixed solid dispersion should be derived from the breakage of drug crystal packing and subsequent arising new intermolecular interaction of drug with polymer (34). The remarkable difference in the peak position between MFA/EPO Cryo-GM and MFA/HPMC Cryo-GM could be derived from the strength of intermolecular interaction between drug and polymer. It was concluded that carboxyl group of MFA could strongly contribute to the intermolecular interactions with EPO in the Cryo-GM.
As shown in Fig. 2, aminoalkyl groups in EPO played an important role in the formation of intermolecular interactions with MFA. Since an aminoalkyl group is a proton-accepting functional group, it is reasonable that the proton-donating carboxyl group of MFA (where large peak shift was observed in IR spectroscopy) worked as the counterpart of the aminoalkyl groups of EPO. It was suggested that strong intermolecular interactions could occur between MFA and EPO in the Cryo-GM, such as electrostatic interaction or hydrogen bonding in addition to hydrophobic interaction.
Solid-State 1H-T1 Measurement
1H-T1 of MFA/EPO system in solid state
Solid-State 13C-CP/MAS NMR Spectroscopy
To clarify the molecular state of MFA, we focused on the region from 100 to 160 ppm. In the spectrum of MFA/EPO Cryo-GM, significant peak broadening was observed, compared with that of MFA/EPO PM (Fig. 6c and d). Peak broadening in the solid-state NMR spectrum could be ascribable either to a wider distribution of isotropic chemical shifts for the same carbons belonging to different molecules, or to an equable suppression of the molecular mobility of the carbons (20). Mobility of MFA molecules was significantly enhanced in the Cryo-GM, as discussed in the results of 1H-T1 measurement. Therefore, this line broadening was explained in terms of a wider distribution of chemical environments of the same carbons in different molecules due to the amorphous nature of the solid dispersions where drugs existed in various conformational situations (20).
The changes in chemical shift were discussed in order to observe the interaction of MFA and EPO. In the spectrum of MFA/EPO PM, all peaks were observed at the same position as in each individual constituent (Fig. 6c). On the other hand, the changes of chemical shift with peak broadening were observed in the spectrum of MFA/EPO Cryo-GM (Fig. 6d). The degree of peak shift in the aromatic ring that contained the carboxyl group (C1-C6) appeared to be greater than those of the other aromatic carbons (C1′-C6′). This implies that the electron density of the aromatic ring with a carboxyl group could be dramatically changed because of the strong intermolecular interaction via carboxyl groups. As for the spectrum of EPO, the peak derived from N-methyl and N-methylene carbons neighboring to nitrogen atoms shifted. These results in the CP/MAS spectra were consistent with the changes observed in the IR spectra, indicating the formation of intermolecular interaction between the carbonyl group of MFA and the aminoalkyl group of EPO. We therefore concluded that MFA molecules were monomolecularly dispersed in the EPO polymer matrix via intermolecular interactions.
Solid-State 13C-T1 Measurement
13C-T1 of MFA/EPO System in Solid State
13 C Assignment (ppm)
C1′, C3′ (139.1)
C2′, C3 (133.3)
C4′, C5′, C6′ (126.2)
In MFA/EPO Cryo-GM, 13C-T1 values of MFA within the slow motional region were significantly shortened compared with the unprocessed sample, indicating its amorphous nature with high molecular mobility. Meanwhile, EPO in the Cryo-GM within the extremely narrowing region showed longer 13C-T1 values than did the raw material. It has been reported that in the case of nifedipine-PVP and phenobarbital-PVP solid dispersions, the mobility of PVP carbonyl carbon was changed by hydrogen bond interactions, resulting in the change of 13C-T1 of PVP carbonyl carbon (42). Especially, 13C-T1 values at 58.0 and 45.9 ppm, which were assigned to the 13C next to nitrogen, were greatly changed. The formation of intermolecular interaction with MFA via the aminoalkyl group increased the localized motion of carbon atoms. These results correspond well with the chemical shift changes of 13C-CPMAS NMR spectra. We concluded that in MFA/EPO Cryo-GM, MFA had extremely high molecular mobility compared to its crystalline state, and EPO formed intermolecular interaction via the aminoalkyl group, resulting in the changes of localized molecular mobility.
Solution 1H-NMR Measurement
Solution 1H-T1 Measurement
1H-T1 of MFA/EPO system in solution state
1H Assignment (ppm)
Difference NOE Measurement
Effect of Drugs on Supersaturated Behavior of EPO Solid Dispersions
Solid dispersion of MFA prepared by cryogenic grinding with EPO showed long-term storage stability in the solid state. A highly supersaturated solution obtained by dispersing the solid dispersion into the buffer solution also exhibited high stability. When the supersaturated solution of MFA was orally administered to rats in vivo, an intense increase in bioavailability was observed, indicating the potential usability of EPO solid dispersions for improving the oral absorption of MFA. Solid- and solution-state NMR revealed that MFA and EPO formed interactions with each other at a molecular level. It was indicated that carboxyl group in MFA and aminoalkyl groups in EPO played an important role in the interactions. It was considered that specifically-formed intermolecular interactions between MFA and EPO resulted in the stabilization of the thermodynamically unfavorable monomolecular state in both the solid and solution state. Solubility enhancement by solid dispersion with EPO was also confirmed when IMC and PXC, 2 types of acidic NSAIDs, were used instead of MFA. Solid dispersions with EPO prepared by cryogenic grinding are extremely useful method that can be applied to various kinds of drugs. It should become one of the best answers for the problem concerning about improvement of water solubility and oral bioavailability of poorly water-soluble drugs.
Acknowledgments & DISCLOSURES
The authors gratefully acknowledge Associate Professor Dr. Hiroko Seki, Dr. Mamoru Imanari, and Dr. Jun Uzawa of the Chemical Analysis Center, Chiba University, for NMR measurement assistance. We would like to thank Evonik Degussa Japan Co. Ltd., for their generous gift of EUDRAGIT®. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho), Japan (21790032, 21590038) and by a Grant from the Japan Health Science Foundation.