An enteric polymer mitigates the effects of gastric pH on oral absorption of poorly soluble weak acid drugs from supersaturable formulations: A case study with dantrolene
Makoto Kataoka*, Ryo Nakanishi, Miyako Umesaki, Masaru Kobayashi, Keiko Minami, Haruki Higashino, Shoji Yamaguchi, and Shinji Yamashita
ABSTRACT
This study demonstrated that an enteric polymer can mitigate the effects of gastric pH on the oral absorption of a poorly water-soluble weak acid drug, dantrolene (DNT). An amorphous solid dispersion (ASD) of DNT with hydroxypropyl methylcellulose (HPMC) acetate succinate (ASD-HPMCAS) was prepared as the enteric released ASD (ER-SF). ASD with HPMC (ASD-HPMC) and DNT sodium salt were also used as immediate-release supersaturable formulations (IR-SFs) with and without water-soluble polymer, respectively. In vivo study with rats and in vitro study with a dissolution/permeation (D/P) system were performed to evaluate oral DNT absorption from each formulation under normal and high gastric pH conditions in rats and humans, respectively. The oral absorption of DNT from both IR-SFs in rats with a high gastric pH was significantly higher than that in rats with a normal gastric pH. In contrast, ASD-HPMCAS attenuated the difference in oral absorption between normal and high gastric pH conditions with significant improvement of DNT absorption. In vivo results implied that an enteric polymer delayed the onset of dissolution until after gastric emptying. ASD-HPMCAS generated supersaturation in the small intestine irrespective of gastric conditions, which was supported by the in vitro study using the D/P system. This study suggested that an enteric polymer is useful to mitigate the inter- and intra-individual differences in oral absorption of poorly water-soluble weak acid drugs.
Keywords; Dantrolene; Dissolution; Enteric polymer; Poor water solubility; Precipitation; Supersaturation; Weak acid drug
1. Introduction
Absorption of drugs administered orally as immediate-release (IR) formulations occurs as a result of a succession of the following processes: disintegration of formulations in the stomach, dissolution of drugs in the gastrointestinal (GI) fluid, and absorption of drugs across the intestinal epithelium. Therefore, a fraction of the absorbed dose (Fa) or absorption rate of drugs is often affected by GI states (1, 2). The difference in the gastric fluid pH can cause significant inter- and intra-individual differences in the oral absorption of administered drugs (3-8). These changes in oral absorption are common when poorly water-soluble drugs with a higher permeability classified in BCS (Biopharmaceutics Classification System (9)) class II are administered.
The dissolved concentration of drugs depends not only on their physicochemical properties and on the composition of intestinal fluid, but also on the dissolution process in the stomach. In the case of BCS class II weak base drugs, gastric dissolution is increased by the strong acidity of the gastric fluid (pH 1-2) based on the Henderson-Hasselbalch equation. This gastric fluid containing dissolved drugs moves down to the small intestine and the fluid pH increases to around 6.5 (10), which can induce supersaturation of the dissolved drug in the intestinal fluid. In patients with achlorhydria or hypogastric acidosis or those administered antacid agents, gastric acidity was significantly lower than that in healthy subjects (11-13). When such drugs were administered to patients with low gastric acidity, no or limited supersaturation in the small intestine was expected due to the high pH of the gastric fluid. These differences in the gastric pH can induce significant inter- and intra-individual variation in the oral absorption of BCS class II weak base drugs such as dipyridamole and ketoconazole (3, 4). Therefore, many formulations, such as salt forms, amorphous solid dispersions, lipid-based formulations, and use of a pH-modifier, may be applicable to overcome altered absorption caused by the difference in the gastric pH (14-17).
Regarding biowaiver based on BCS classification, an in vitro dissolution test for BCS class I drugs (more than 85% of drug dissolved within 30 min) and class III drugs (more than 85% of drug dissolved within 15 min) may substitute for in vivo bioequivalence studies (18, 19). Currently, extension of biowaivers for BCS class II weak acid drugs with an acid dissociation constant (pKa) less than 4.5 (20-22) is being considered. The solubility of such drugs in the stomach with a normal gastric pH may be significantly lower than that in the stomach with lower gastric acidity. However, poor solubility in the stomach may not affect the Fa because of the highly soluble conditions in intestinal fluid even if the precipitation of drugs in the stomach was due to IR-SFs such as salt forms. In contrast, when poorly water-soluble weak acid drugs with close to or higher pKa than the intestinal fluid pH were administered as IR-SFs, significant precipitation in the stomach was expected in the fasted subjects with normal gastric condition but not in those with a high gastric pH condition. These expected differences in the dissolution profiles of drugs in the stomach may lead to significant inter- and intra-individual differences in oral absorption of poorly water-soluble weak acid drugs administered as IR-SFs. Water-soluble polymers were employed as dispersion media and precipitation inhibitors for SFs. Hydroxypropyl methylcellulose (HPMC), polyvinyl pyrrolidone, their derivatives, and other water-soluble polymers were commonly used, and were able to significantly improve the oral absorption of poorly water-soluble drugs (23-26). Therefore, enteric-release supersaturable formulations with polymers that do not dissolve under strong acidic conditions can significantly reduce the inter-individual variations in oral absorption of poorly water-soluble weak acidic drugs with a relatively high pKa.
To demonstrate our theory, dantrolene (DNT) was used as a model poorly water-soluble weak acid drug with a higher pKa (7.5) than the intestinal fluid pH (27). As IR-SFs of DNT with or without water-soluble polymer, amorphous solid dispersion with HPMC (ASD-HPMC), and sodium salt (Na-salt) were used, respectively. An amorphous solid dispersion of DNT with enteric-release properties (ER-SF) was prepared with HPMC acetate succinate (ASD-HPMCAS). The oral administration study using rats with normal and high gastric pH conditions was performed for three SFs. Then, the in vitro evaluation was carried out using a dissolution/permeation (D/P) system (28), which was able to simultaneously evaluate drug dissolution and permeation with or without the gastric dissolution process to confirm our theory.
2. EXPERIMENTAL
2.1. Materials
Bovine serum albumin (BSA), egg-phosphatidylcholine (lecithin), Lactose, methylcellulose, Na-salt, omeprazole, and sodium taurocholate were obtained from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). HPMC and HPMCAS (HF grade, Shin-Etsu AQOAT®) were gifted from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Small gelatin capsules (PCcaps®) were supplied from Capsugel® Japan Inc. (Sagamihara, Kanagawa, Japan). The human colorectal adenocarcinoma cell line Caco-2 was purchased from the American Type Culture Collection (Rockville, MD) at passage 17. Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Sigma-Aldrich (St. Louis, MO). Non-essential amino acids (10 mM), fetal bovine serum (FBS), trypsin-EDTA (trypsin: 0.25%, EDTA: 1 mM), and an antibiotic-antimycotic mixture (penicillin: 10,000 U/mL, streptomycin: 10 mg/mL, amphotericin B: 25 g/mL; dissolved in 0.85% (w/v) sodium chloride aqueous solution) were purchased from Gibco Laboratories (Lenexa, KS). Cell culture inserts with polyethylene terephthalate filters (pore size: 3.0 m, growth area: 4.20 cm2) were obtained from Becton Dickinson Bioscience (Bedford, MA).
2.2. Preparation of a free form of DNT
A free form of DNT was obtained from Na-salt by the precipitation method. Na-salt was added to 0.1 M hydrochloric acid and mixed for 30 min. The precipitate was washed twice by purified water. Crystals of the obtained precipitate from Na-salt were characterized by powder X-ray diffraction (PXRD). PXRD patterns were obtained in reflectance mode using a Bruker D8 Discover (Bruker AXS K.K., Kanagawa, Japan) with a GADDS CS diffractometer equipped with a Cu Kα radiation and Hi-STAR area detector, operating at 40 kV and 40 mA. Data were collected from 5.0° to 45.0° 2θ, with an acquisition time of 120 s.
2.3. Preparation of amorphous solid dispersions of DNT
Amorphous solid dispersions of DNT with HPMC or HPMCAS were prepared by a solvent evaporation method. DNT and HPMC or HPMCAS (1:2 weight ratio) were dissolved into acetonitrile and dichloromethane solution (1:1). Each solution in sampling tubes was evaporated at 60°C with stirring at 400 rpm using a thermoshaker (MS-100, Hangzhou Allsheng Instruments Co., Ltd) in the fume hood. Obtained films were crushed with a mortar and pestle. Each formulation and pure polymers were analyzed by PXRD.
2.4. In vivo oral administration study using rats
All animal experiments were approved by the ethical review committee of Setsunan University and performed in accordance with the Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985). Male SD rats (weighing 250– 300 g) obtained from SHIMIDZU Laboratory Supplies Co., Ltd. (Kyoto, Japan) were deprived of food, but given free access to water for 18 h before the experiments.
Each capsule formulation was prepared by filling into a gelatin capsule (PCcaps®) with a designated filling kit (Capsugel® Inc., Morristown, NJ). Each formulation was diluted with lactose at a weight ratio of 7 on 3 of the formulation. A body of a capsule was placed to a stand of filling kit then 10 mg of each diluted formulation with lactose was filled into a body through a funnel. The capsule was tightly enclosed with a cap. Each capsule contained 1.0 mg of DNT as a respective formulation. For the oral administration study with a high gastric pH, rats were pretreated with 30 mg/kg of omeprazole in a 0.5 w/v% methylcellulose suspension (1 mL/kg by oral administration) one hour before the capsule administration. Omeprazole treatment was previously reported to increase the gastric pH of rats to around 6.0 within an hour and maintain the high pH for approximately 5 h thereafter (28). Five hundred microliters of water was immediately administered after capsule administration. Intravenous administration study was also performed using rats to calculate the oral bioavailability (BA) of DNT (data not shown). At pre-determined time points, blood samples (300 µL) were collected from the jugular vein. Blood samples were centrifuged, and plasma was maintained at -30°C before the quantification of DNT.
2.5. In vitro dissolution and permeation study
2.5.1. Preparation of Caco-2 cell monolayers
Caco-2 cell monolayers were obtained in accordance with a previously reported procedure (29). Briefly, Caco-2 cells were seeded on cell culture inserts at a density of 3 × 105 cells/insert with DMEM supplemented with 10% (v/v) FBS, 1% (v/v) non-essential amino acids, and 0.5% (v/v) antibiotic-antimycotic mixture (culture medium). Fresh culture medium (1.5 mL in the insert and 2.6 mL in the well) was replenished every 48 h during the initial 6 days and then every 24 h. After 18–21 days in culture, Caco-2 cell monolayers were utilized in subsequent experiments.
2.5.2. Preparation of different media for the D/P system
Hank’s balanced salt solution was used as a basic buffer solution in this study (transport medium, TM). Fasted-state simulated intestinal fluid for the D/P system contained 3 mM sodium taurocholate and 0.75 mM lecithin in TM (FaSSIFmod, pH 6.5). Simulated gastric fluid (SGF) and concentrated FaSSIFmod (FaSSIFmod8/6.5) were prepared by TM with an adjusted pH of 1.2 and TM with the addition of sodium taurocholate (4 mM) and lecithin (1 mM), respectively. The pH of FaSSIFmod8/6.5 was adjusted to 7.6. When SGF (1.5 mL) was added to FaSSIFmod8/6.5 (6.5 mL) in the apical compartment of the D/P system, the apical solution became FaSSIFmod with a pH of 6.5. As basal medium in the D/P system, TM containing BSA (4.5% w/v) with the pH adjusted to 7.4 was used.
2.5.3. In vitro dissolution and permeation study with the D/P system
The dissolution and permeation study with pH-shift (gastric dissolution process) was performed by a previously reported procedure (28). A Caco-2 cell monolayer with a support filter was mounted between the chambers of the D/P system after a 20-min pre-incubation with TMapical (1.5 mL) for the apical side and TMbasal (2.6 mL) for the basal side. For the study with pH-shift, the apical and basal sides of the D/P system with an attached Caco-2 cell monolayer were filled with 6.5 mL of FaSSIFmod8/6.5 and 4.0 mL of the basal medium, respectively. The small vessel for the gastric compartment was filled with 6 mL of SGF. Each formulation or DNT (2 mg as DNT) was added to the gastric vessel as a powder. After 10 min, 1.5 mL of the drug-containing medium in the gastric compartment and 1.5 mL of the basal solution were introduced into the apical and basal sides of the system, respectively. Aliquots of samples (0.1 mL) were routinely collected from the apical and basal solutions for 2 h. All apical samples were filtered through a polytetrafluoroethylene filter (Millex®-LH, pore size: 0.45 m, Millipore, Billerica, MA), and each filtrate (0.05 mL) was immediately mixed with 0.45 mL of the solution consisting of water and acetonitrile (50/50).
A dissolution and permeation study without the gastric dissolution process was performed in accordance with a previously reported procedure (29). Briefly, the apical and basal sides of the D/P system were filled with 8 mL of FaSSIFmod containing 10 mM HEPES (pH 6.5) and 5.5 mL of the basal medium. An appropriate amount of each formulation and DNT (0.5 mg as DNT) was applied to the apical solution as a powder. Sample collection and treatment were the same as described above.
The transepithelial electric resistance (TEER) of the Caco-2 cell monolayer was measured before and after the experiment by Millicell®-ERS (Millipore, Billerica, MA). All experiments were performed at 37°C under stirring at 200 rpm with a magnetic stirrer.
2.5.4. Prediction of oral absorption from in vitro experiments using the D/P system
The following equation (Eq. 1) was used to estimate the fraction of the dose absorbed (Fa%) of each drug in a fasted human (in vivo) from in vitro data; where Absmax is 100 (the maximum Fa), PA is the permeated amount during a 2-hr experiment (% of dose/2 h), PA50 is the predicted in vitro permeated amount giving a half-maximum Fa, and is Hill’s coefficient. Based on our previous findings (29), 0.334 and 0.883 were substituted for PA50 and , respectively.
2.6. Assay
All plasma samples (0.1 mL) were mixed with 0.9 mL of acetonitrile. The mixture was shaken and the supernatant (0.8 mL) was collected after centrifugation at 15,000 rpm for 20 min. After removal of the solvent under a vacuum at 40°C, the residue was dissolved in 0.1 mL of the solution consisting of 0.1% (v/v) formic acid and acetonitrile (50/50). For the in vitro experiments, all samples without BSA were analyzed without further treatment. For samples containing BSA from the experiments using the D/P system, the collected basal samples (0.1 mL) were mixed with 0.9 mL of acetonitrile. The mixture was shaken and the supernatant collected by centrifugation at 15,000 rpm (Himac CF15R, HITACHI, Tokyo, Japan) for 20 min.
The amount of DNT in the treated solution was measured using an ultra-performance liquid chromatography (UPLC) system (ACQUITY® UPLC, Waters, MA) equipped with a tandem mass spectrometer (ACQUITY® TQD, Waters, MA). A reversed-phase Waters Acquity® UPLC BEH C18 analytical column of 50 mm × 2.1 mm I.D. and 1.7-m particle size (Waters, MA) was used with a mobile phase consisting of 0.1% (v/v) formic acid in water (solvent A) and acetonitrile containing 0.1% (v/v) formic acid (solvent B) with a gradient time period. The initial mobile phase was 98% solvent A and 2% solvent B pumped at a flow rate of 0.3 mL/min. Between 0 and 1.0 min, the percentage of solvent B increased linearly to 95%, which was then maintained for 1.0 min. Between 2.01 and 2.5 min, the percentage of solvent B decreased linearly to 2%. This condition was maintained until 3 min, at which time the next sample was injected into the UPLC system. All treated samples were injected at a volume of 5 L into the UPLC system. The ionization conditions for the analysis of DNT were as follows: electrospray ionization, positive mode; source temperature, 150°C; desolvation temperature, 400°C; cone voltage, 30 V; and collision energy, 48 eV. Precursor and production ions (m/z) were 315.11 and 113.89, respectively.
2.7. Statistical analysis
All data are presented as means with the standard deviation (s.d.) for individual groups. Significance was assessed by the unpaired Student’s t-test and p-values of 0.05 or less were considered significant.
3. RESULTS
3.1. Preparation of crystal form and solid dispersions of DNT
3.1.1. Free form of crystalline DNT
When Na-salt was added to 0.1 M hydrochloric acid, the color of the solution immediately changed from orange (nature color of Na-salt) to clear, followed by precipitation of yellow particles (Fig. 1). The mass spectrometric and nuclear magnetic resonance results of the yellow precipitate suggested the precipitate to be pure DNT without degradation (data not shown). To characterize the crystallization of the free form of DNT, the crystal form of the precipitate and Na-salt were analyzed by PXRD. The diffraction pattern of yellow precipitate demonstrated a crystalline nature and a different form that of Na-salt (Fig. 1). These results suggested that the obtained yellow precipitate was a crystalline free form of DNT.
3.1.2. Preparation of amorphous solid dispersions of DNT
PXRD patterns of both solid dispersion formulations with HPMC and HPMCAS exhibited halo patterns with slight peaks at 19.98° and 26.00° for HPMCAS formulations, and 19.62° and 25.95° for HPMCAS formulations, respectively (Fig. 2) However, their degrees did not correspond to the strong peaks observed for the crystalline form of DNT (16.98°, 22.48°, and 28.54° shown in Fig. 1). This suggested that DNT in each formulation was dispersed as an amorphous form.
3.2. Oral absorption of DNT from different formulations in rats with normal and high gastric pH conditions
3.2.1. Effects of gastric pH on oral absorption of DNT from IR-SF without polymer
The plasma concentration-time profiles of DNT after oral administration of Na-salt without water-soluble polymer to rats under normal and high gastric pH conditions, together with that of the free form of DNT to normal rats are shown in Figure 3a. The oral absorption of DNT from Na-salt (AUC, BA) in rats with a normal gastric pH was comparable to that from the free form of DNT (Table 1). In the case of rats with a high gastric pH, DNT absorption (AUC) and the maximum concentration of DNT (Cmax) were 11- and 14-fold higher than that in normal rats, respectively (Table 1).
3.2.2. Effects of ASDs on oral absorption of DNT in rats with normal and high gastric pH conditions
Although ASD-HPMC significantly improved DNT absorption in normal rats compared with that from Na-salt, oral absorption of DNT from ASD-HPMC in rats with a high gastric pH was significantly higher than that in normal rats (Fig. 3b, Table 1). When DNT was administered to rats as ASD-HPMCAS, high absorption was observed compared with that from Na-salt and ASD-HPMC, irrespective of gastric pH conditions (Fig. 3c, Table 1). The initial plasma concentration-time profile of DNT in normal rats was lower than that in rats with a high gastric pH. However, no significant difference in AUC between rats with a normal and high gastric pH was observed (Fig. 3c, Table 1).
3.3. In vitro evaluation of SFs on oral absorption of DNT using the D/P system
3.3.1. Dissolution and permeation profiles of DNT from different forms in the D/P system with or without the gastric dissolution process
Each SF of DNT, Na-salt, ASD-HPMC, and ASD-HPMCAS, was applied to the D/P system as a solid form (without the gastric dissolution process) or solution of SGF obtained from the dissolution study with the gastric compartment (with the gastric dissolution process). The dissolved and permeated drugs in the D/P system were consecutively monitored for 2 h. No significant decreases were observed in the TEER value during the experiments (data not shown).
As negative control experiments, the free form of crystalline DNT was applied to the D/P system with and without the gastric dissolution phase (Fig. 4). The gastric dissolution condition did not affect dissolution or permeation of DNT, and both amounts were the lowest among all dosage forms examined in this study. When Na-salt was applied to the D/P system with the gastric dissolution phase, the dissolved amount plateaued without supersaturation (Fig. 4a). The time profiles of dissolved and permeated amounts were comparable to those observed with the free form of DNT (Fig. 4a, 4b). In contrast, the dissolved amount of DNT without the gastric dissolution phase exhibited a supersaturated dissolution profile as follows: DNT rapidly dissolved, and its dissolved amount then gradually decreased with time, resulting in higher permeation to the basal side (Fig. 4c, 4d). When ASD-HPMC was applied to the D/P system without the gastric dissolution phase, the dissolved amount of DNT (20.9±1.2% of dose at 2 h) was significantly higher than that in the D/P system with the gastric dissolution phase (9.58±0.76% of dose at 2 h) (Fig. 4a, 4c). Regarding dissolution profiles, the permeated amount (0.887±0.011% of dose at 2 h) without the gastric dissolution phase was significantly higher than that (0.641±0.069% of dose at 2 h) with the gastric dissolution phase (Fig. 4b, 4d). In the case of ASD-HPMCAS, the gastric dissolution phase did not influence the time profiles of the dissolved or permeated amount of DNT (Fig. 4).
3.3.2. Prediction of oral absorption in humans of DNT from in vitro data
Permeation data (% of dose/2 h) were substituted for PA in Eq. 1 for prediction of the human oral absorption (Fa%) of DNT under normal and high gastric pH conditions (refer to section 2.5.4.). Permeation data and prediction results are summarized in Table 1. The oral absorption of DNT from Na-salt, ASD-HPMC, ASD-HPMCAS, and the free form in fasted healthy subjects was predicted to be 35%, 64%, 65%, and 26%, respectively, based on their permeated amounts in the D/P system at 2 h. In fasted subjects with a high gastric pH, the predicted absorption of DNT was 63% from Na-salt, 70% from ASD-HPMC, 65% from ASD-HPMCAS, and 35% from the free form.
4. DISCUSSION
Abeele et al. investigated the GI behavior of diclofenac potassium in subjects under fasted (with or without a proton-pump inhibitor, PPI) and fed states by aspiration of the GI fluid (31). Diclofenac is a poorly water-soluble weak acid drug and has been used as a salt form with sodium or potassium for clinical treatment (32,33). Their study revealed that diclofenac significantly precipitated in the stomach with strong acidity even though most of the diclofenac dissolved in the gastric fluid after administration to PPI-treated subjects. However, dissolved concentrations of diclofenac in the duodenum were comparable to the total (dissolved and undissolved) concentration in aspirated samples irrespective of the difference in the gastric pH. As the pKa value of diclofenac (3.8) is significantly lower than the pH of the intestinal fluid, diclofenac is completely dissolved in the intestinal fluid. These dissolution behaviors strongly supported that pantoprazole, a PPI, did not affect the pharmacokinetics and pharmacodynamics of diclofenac after oral administration (34). Assuming that a similar phenomenon occurred in the dissolution process of DNT from each salt in the GI tract, the oral absorption is expected to be affected by the gastric dissolution process. In our previous study, the D/P system reflecting the gastric dissolution process was able to predict the effects of gastric pH on the oral absorption of BCS class II weak base drugs (28). Although pH differences in the stomach may influence the dissolution profiles of diclofenac from sodium salt in the stomach and small intestine, no significant effects on oral absorption were predicted. This correspondence between in vivo absorption and in vitro prediction of diclofenac absorption suggested that the D/P system is a useful tool to assess the effects of gastric pH on oral absorption of BCS class II weak acid drugs, including their salt forms, in addition to BCS class II weak base drugs.
The dissolved concentration profile of DNT in the intestine may be strongly regulated by not only the composition of the intestinal fluid and pH, but also the gastric dissolution process due to a higher pKa (7.5) when DNT was administered as SFs. Kambayashi and Dressman demonstrated that physiologically based pharmacokinetic (PBPK) modeling with dissolution and precipitation kinetics in the GI tract is useful for predicting the PK profile after the oral administration of Na-salt as clinical formulations (Dantrium® capsules) (35). Their PBPK modeling reflected the dissolution and precipitation of DNT in the stomach and gastric emptying, followed by the intestinal dissolution of an undissolved fraction of Na-salt and DNT precipitant. This suggested that precipitation of DNT from IR-SF, Na-salt, was one of the important factors to determine its absorption. As supersaturation and precipitation profiles of DNT from clinical formulations or intact Na-salt were significantly affected by dissolution media (28,35), the change in the gastric pH may influence the intestinal absorption of DNT. The effects of gastric pH on DNT absorption in humans have not been reported. The oral administration study was performed using rats with a normal or high gastric pH. In this study, the applied amount of DNT (0.5 mg) to the D/P system (1/100 of the clinical dose) was slightly more than that in a previous study (0.5 mg as Na-salt) because DNT (free form) was used as a model poorly water-soluble weak acid drug. Similar profiles to those in the previous study of the dissolution and permeation of DNT were observed (Fig. 4). In the D/P system without the gastric dissolution phase, supersaturation and precipitation were observed in the apical media, which induced higher permeation than that without supersaturation in the apical side. Hisada et al. reported that metastable precipitate from Na-salt produced a higher concentration than that of the free form of DNT. The supersaturated state was maintained until the metastable precipitate was converted to the free form of DNT (36). The oral absorption of DNT from Na-salt in rats with low gastric acidity was markedly higher than that in normal rats (Fig. 3a). Although this in vivo observation was consistent with the in vitro prediction, the tendency of change in the absorption in rats differed from the predicted absorption by the D/P system (Fig. 5). This discrepancy may be caused by the ratio between dose and body weight and/or fluid volume, which was 0.5 mg for the D/P system (50 mg for humans) and 1 mg for rat (corresponding to approximately 240 mg/60 kg). This higher oral dose of DNT in the animal study was set to enable detection of formulation effects. Thus, the effects of Na-salt on the oral absorption of DNT in the in vivo study demonstrated the pros and cons of this formulation technique more so than the in vitro study using the D/P system.
Water-soluble polymers, such as HPMC and polyvinyl pyrrolidone, were used for not only dispersion of drugs in an amorphous state, but also to prevent the supersaturated state from precipitating the drugs; therefore, these polymers were often contained in clinical formulations of SFs. As water-soluble polymers, HPMC and HPMCAS were employed in this study. ASD-HPMC significantly improved DNT absorption (BA: 39.6±13.9%) in rats with normal gastric conditions compared with that from Na-salt (5.50±4.08%), suggesting that HPMC inhibited the precipitation of DNT in the stomach. However, BA from ASD-HPMC (68.9±5.4%) in rats having a high gastric pH was still significantly higher than that in normal rats (Fig. 5a). This difference may have been caused by the dissolved concentration of DNT in the intestinal fluid. Although HPMC may prevent dissolved DNT in the stomach from precipitating under the normal acidic conditions in the stomach, the dissolved concentration in the gastric fluid was lower than that in the high gastric pH conditions. The dissolved and permeated amounts of DNT from ASD-HPMC observed in the D/P system (Fig. 4) supported these different absorption profiles in rats between normal and high gastric pH conditions.
When DNT was administered to normal rats as ER-SF with a precipitation inhibitor, ASD-HPMCAS, significant improvement of the oral absorption (63.4±15.6%) was observed compared with that from IR-SF. The initial absorption rate from ASD-HPMCAS in normal rats was slower than that in omeprazole treated rats (Fig. 3c), whereas this improved oral absorption was comparable to that in rats with a high gastric pH (83.5±20.4%, Fig. 5a and Table 1). Based on the D/P system, the dissolved amount of DNT from ASD-HPMCAS was approximately 20% in 2 h irrespective of pretreatment with strong acidic conditions, which led to a similar permeation profile of DNT on the basal side of the D/P system with or without the gastric dissolution phase (Fig. 4). These higher dissolved amounts of DNT from both ASD formulations may have been due to the prevention of precipitation by the supersaturated state of DNT. The oral absorption of DNT under normal and elevated gastric pH conditions was predicted to be 65% (Fig. 5b and Table 2). Therefore, ASD-HPMCAS can reduce the inter- and intra-individual differences in the oral absorption of DNT caused by the difference in the pH of gastric fluid, with significant improvement of oral absorption. Drug release from tables coated with enteric polymers such as HPMCAS and Eudragit® in bicarbonate buffer after pretreatment with a strongly acidic medium (0.1 M HCl) for 2 h significantly delayed compared to that in a phosphate buffer. This delayed disintegration of coated tablets caused by exposure with a strongly acidic condition corresponded well to that reported in the literature in the human small intestine (37). The dissolved amount of DNT from ASD-HPMCAS reached to be a plateau at a first sampling point, 15 min, irrespective of pretreatment with SGF (Fig. 4a,c). DNT release from ASD-HPMCAS could be unaffected by the pretreatment with SGF. This discrepancy between the dissolution of an enteric polymer in HEPES containing medium and that in intestinal bicarbonate might be a limitation to evaluate enteric formulations. Since powder formulations were used in this study, gastric emptying of such formulations in humans could be significantly more rapid compared to that of enteric-coated tablets (38-40), indicating that the gastric emptying could be shorter than that of enteric-coated tablets. It is thought that the influence of strong acidity in the stomach on drug release from ASD-HPMCAS can be small; however, this explanation should be investigated as future studies.
Whereas the D/P system without the gastric phase might overestimate the oral absorption of weak acid drugs under low gastric pH, the effect of gastric pH on oral absorption of DNT predicted by the D/P system corresponded to that observed in animal study. However, in order to perform precious prediction of oral drug absorption with high gastric pH using the D/P system, dissolution process of drugs in the stomach should be reflected. Since the fluid pH in the stomach with low acidity ranges from 4 to 7 (11-13), the gastric phase for the D/P system reflecting low gastric acidity should be carefully set up. Therefore, the establishment of gastric phase with low acidity to perform precious evaluation of oral drug absorption in humans with high gastric pH will be a subject for future study.
Based on our in vivo and in vitro findings, the time profiles of dissolved concentrations of DNT from each SF in the stomach and intestine are abstractly depicted in Figure 6. The difference in profiles after administration of IR-SF (A; Na-salt), IR-SF with water-soluble polymer (B; ASD-HPMC), and ER-SF with enteric polymer (C; ASD-HPMCAS) are discussed in detail as follows:
(A) In the normal gastric conditions, DNT immediately precipitated in the stomach and moved down to the small intestine. Poor solubility of the precipitant resulted in a similar oral absorption to that of the free form of DNT. When IR-SF was administered to rats with a high gastric pH, dissolved DNT in the stomach moved down to the intestine maintaining the supersaturated state. According to this supersaturated state in the intestine, DNT was well absorbed from the intestinal epithelium.
(B) The precipitation of DNT from the supersaturated state in the stomach was inhibited by the presence of HPMC. HPMC also prevented the precipitation of DNT in the intestinal tract from the supersaturated state (metastable precipitate), resulting in increased absorption. However, the inhibition effects in the stomach were more marked in rats with a high gastric pH than in normal rats. Therefore, the oral absorption under high gastric pH conditions was higher than that under normal gastric pH conditions.
(C) DNT was mainly released from ASD-HPMCAS at the absorption site, the intestinal tract, in both states, which maintained the supersaturated state (metastable precipitate). This higher dissolved concentration in the intestine led to the reduction of inter- and intra-individual differences in oral absorption and significantly improved oral absorption.
As these descriptions did not take the absorption process into account, the degree of supersaturation and dissolved concentration do not reflect the in vivo situation. An in vivo study with aspiration of the GI fluids will be performed on human subjects or large animals to confirm the above descriptions. However, the effects of gastric pH on oral absorption of poorly water-soluble weak acid drugs can be roughly predicted from the physicochemical properties, such as solubility and pKa, and dose strength when these drugs are administered as SFs. Furthermore, an in vitro system, such as the D/P system, provides valuable information about the effectiveness of various SFs.
5. CONCLUSION
In conclusion, we demonstrated the usefulness of supersaturable formulation with an enteric polymer (ER-SF) not only to improve oral absorption of DNT, but also to reduce differences in the oral absorption caused by the change in gastric pH. However, similar experiments are needed to confirm this application and will be a subject for future studies.
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