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In vitro dissolution of fluconazole and dipyridamole in Gastrointestinal Simulator (GIS), predicting in vivo dissolution and drug-drug interaction caused by acid-reducing agents Kazuki Matsui, Yasuhiro Tsume, Gregory E Amidon, and Gordon L. Amidon Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00135 • Publication Date (Web): 18 May 2015 Downloaded from http://pubs.acs.org on May 21, 2015

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Molecular Pharmaceutics

In vitro dissolution of fluconazole and dipyridamole in Gastrointestinal Simulator (GIS), predicting in vivo dissolution and drug-drug interaction caused by acid-reducing agents

Kazuki Matsui1,2, Yasuhiro Tsume1, Gregory E. Amidon1, Gordon L. Amidon1,*

1

College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-

1065, United states 2

Pharmacokinetics & Safety Laboratory, Discovery Research, Pharmaceutical Research Center,

Mochida Pharmaceutical Company Limited, 722 Uenohara, Jimba, Gotemba, Shizuoka 4128524, Japan

KEYWORDS: dissolution, GIS, BCS, drug-drug interactions, IVIVC, in vivo predictive dissolution methodology, supersaturation, precipitation, dipyridamole, fluconazole

ACS Paragon Plus Environment

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ABSTRACT

Weakly basic drugs typically exhibit pH-dependent solubility in the physiological pH range, displaying supersaturation and/or precipitation along the gastrointestinal tract. Additionally, their oral bioavailabilities may be affected by co-administration of acid-reducing agents that elevate gastric pH. The purpose of this study was to assess the feasibility of a multicompartmental in vitro dissolution apparatus, Gastrointestinal Simulator (GIS), in predicting in vivo dissolution of certain oral medications. In vitro dissolution studies of fluconazole, a BCS class I, and dipyridamole, a BCS class II weak bases (class IIb), were performed in the GIS as well as USP apparatus II and compared with the results of clinical drug-drug interaction (DDI) studies. In both USP apparatus II and GIS, fluconazole completely dissolved within 60 min regardless of pH, reflecting no DDI between fluconazole and acid-reducing agents in a clinical study. On the other hand, 7-fold and 15-fold higher concentrations of dipyridamole than saturation solubility were observed in the intestinal compartments in GIS with gastric pH 2.0. Precipitation of dipyridamole was also observed in the GIS and the percentage of dipyridamole in solution was 45.2 ± 7.0%. In GIS with gastric pH 6.0, mimicking the co-administration of acid-reducing agents, the concentration of dipyridamole was equal to its saturation solubility and the percentage of drug in solution was 9.3 ± 2.7%. These results are consistent with the clinical DDI study of dipyridamole with famotidine, which significantly reduced the Cmax and AUC. An In situ mouse infusion study combined with GIS revealed that high concentration of dipyridamole in the GIS enhanced oral drug absorption, confirming the supersaturation of dipyridamole. In conclusion, GIS was shown to be a useful apparatus to predict in vivo dissolution for BCS class IIb drugs.


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INTRODUCTION In 1995, Amidon et al. devised a Biopharmaceutics Classification System (BCS) which classified oral active pharmaceutical ingredients (APIs) into 4 categories by their physicochemical properties of solubility and membrane permeability.1 The BCS classification permits the rate-determining process in oral drug absorption for certain BCS classes to be estimated.2 For instance, BCS class III drugs have high aqueous solubility but low permeability, thus their oral absorption is limited by membrane permeability. In other cases, dissolution rate or solubility may be the rate-limiting step in oral absorption of BCS class II drugs which are categorized as low solubility and high permeability drugs. BCS classification thus provides us important information for oral medications. Combinatorial chemistry, high-throughput screening and structure based drug design have been introduced to improve the efficiency of the drug discovery process, but simultaneously an increase in molecular complexity (eg: increased molecular weight and lipophilicity) has resulted in reducing aqueous solubility of many drug candidates.3 These situations in drug discovery and development emphasize the importance of evaluating in vivo oral drug dissolution profile in the gastrointestinal (GI) tract. To achieve a comprehensive assessment of drug dissolution, absorption, and bioavailability, the development of appropriate in vitro dissolution methodologies which incorporate gastrointestinal physiology may be one of great value.4 Conventional and compendial dissolution tests using USP apparatus I and II have been widely used for the purpose of quality control and batch-to-batch comparison of pharmaceutical products. Additionally, dissolution data obtained in these apparatuses can provide biowaiver eligibility for immediate release oral products of BCS class I.5, 6 The USP-type dissolution


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studies are appropriate tools to evaluate the dissolution of BCS class I drugs. However, these apparatuses may be unsuitable for the assessment of poorly soluble drugs because the dissolution profile of these drugs is largely dependent on physiological factors in the GI tract like intestinal fluid volume, media components, buffer capacity, hydrodynamics, drug absorption, and pH condition.7-9 Since the USP dissolution tests often do not adopt these GI physiologies, they may not be considered as in vivo predictive dissolution methodologies especially for poorly soluble drugs.10 To overcome the limitation of USP-type dissolution tests, several in vivo predictive dissolution methodologies like dissolution/permeation (D/P) system,11, 12 biphasic dissolution test,13 the artificial stomach-duodenum (ASD) apparatus,14-16 TNO gastrointestinal tract model (TIM)17, 18 and the gastrointestinal simulator (GIS)19, 20 have been developed and investigated.4 Dissolution tests using biorelevant media,21, 22 and physiological buffer23, 24 reportedly predict better in vivo dissolution of test medications. To improve the prediction of in vivo dissolution for certain drugs, the ideal dissolution methodologies should be selected based on physicochemical properties of oral drug products. BCS sub-classification approach proposed by Tsume et al. may help the selection of dissolution methodologies.25 According to the proposed sub-classification approach, the dissolution profile of BCS class II weak acid and base drugs should be assessed with methodologies incorporating pH changes in the GI tract because these drugs have pHdependent solubility. Since dissolution will be the rate limiting step for oral drug absorption of BCS class II drugs, the accurate prediction of in vivo dissolution is important. For weakly basic drugs, the drastic pH change from the stomach to the duodenum and the proximal jejunum can have a significant impact on dissolution profiles. When weak base drugs exit an acidic condition in the stomach and enter a neutral pH condition in the small intestine,


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they may undergo supersaturation and subsequent precipitation.26 As the extent and duration of supersaturation have great influence on the oral bioavailability of BCS class II basic drugs (BCS class IIb), there have been many attempts to capture those phenomena with in vitro dissolution methodologies.16, 27, 28 Since the supersaturation state is a metastable condition in the GI tract, an in vitro apparatus which incorporates the physiological changes in the GI tract would be necessary to assess in vivo dissolution. Moreover, it has been reported that drug-drug interactions (DDI) caused by acid-reducing agents lower systemic exposure of oral weak base drugs and often limits their therapeutic applications.29 Thus, several experiments have been attempted to predict the extent of DDI using animals,30, 31 in vitro devices,32, 33 in silico simulations,34 and combinations of these methodologies.35 A GIS has been developed and investigated to achieve better in vitro-in vivo correlations (IVIVC) for oral medications.20 The GIS consists of three chambers which represent stomach, duodenum, and jejunum. Each chamber has different fluid volumes, pH, and buffer species that approximate in vivo conditions. Previous work with this apparatus using BCS class I drugs in combination with in silico technology, GastroPlusTM demonstrated the utility of the GIS system and better reflects the physiological and environmental changes in the human GI tract in the fasted state.20 Therefore, GIS may provide reliable information to predict the in vivo dissolution of BCS class IIb drug products, but detailed investigation has not been carried out. In this study, fluconazole, a BCS class I drug, and dipyridamole, a BCS class IIb drug, were selected as test drugs having high membrane permeability. Dissolution studies with USP apparatus II and GIS were conducted to assess the suitability of these in vitro dissolution methodologies to predict in vivo dissolution. Additionally, elevated gastric pH was used in in vitro dissolution experiments to assess the influence of acid-reducing agents in drug dissolution


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studies with USP apparatus II and GIS. These experiments were conducted to determine if the in vitro dissolution results could be related to in vivo performance. To assess the in vivo relevance of high drug concentrations of dipyridamole in the GIS, an in situ mouse infusion study was also performed in combination with the GIS. EXPERIMENTAL SECTION Materials Fluconazole immediate release tablets (200 mg) (Teva Pharmaceuticals USA, North Wales, PA) and dipyridamole immediate release tablets (25 mg) (Zydus Pharmaceuticals USA, Pennington, NJ) were obtained through University of Michigan Hospital. Fluconazole, dipyridamole, ketoconazole, potassium phosphate monobasic, and sodium chloride (NaCl) were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO) and used as received. Acetonitrile, trifluoroacetic acid (TFA), Formic acid (FA) and methanol were purchased from Fisher Scientific Inc. (Pittsburgh, PA). All chemicals were either analytical or HPLC grade. Dissolution study with USP apparatus II The dissolution studies of fluconazole and dipyridamole in USP apparatus II were conducted using a Hanson SR6 Dissolution Test Station (Chatsworth, CA). Dissolution was performed at a rotational speed of 50 rpm at 37°C in 300 mL of simulated gastric fluid (SGF) at pH 2.0 (SGFpH2.0, 10-2 N HCl with 34.2 mM NaCl), SGF at pH 6.0 (SGFpH6.0, 10-6 N HCl with 34.2 mM NaCl), or simulated intestinal fluid (SIF) (SIFpH6.5, 50 mM potassium phosphate buffer with 15.4 mM NaCl, pH 6.5). The fluid compositions were based on US Pharmacopoeia 24 with modified pH adjusted by hydrochloric acid. These fluids were prepared without enzymes. Three


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hundred mL of buffer was chosen for this set of experiments to directly compare dissolution results with other in vitro dissolution systems used in this study and to more closely reflect in vivo fluid volumes. SGFpH6.0 was chosen to represent the elevated pH of gastric fluid after the intake of acid-reducing agents. One tablet of 200 mg fluconazole or two tablets of 25 mg dipyridamole were placed into each buffer media to start the dissolution study. Samples were manually collected at 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 45, 60, 90, 120, 150, and 180 min. All samples were immediately centrifuged (2,000×g, 30 sec) and their supernatants were diluted with the equal volume of methanol. Dissolved drug concentration was measured by HPLC analysis. Dissolution study with Gastrointestinal Simulator (GIS) In the previous study, the GIS system was designed to represent the physiological conditions of the human GI tract in the fasted state. The dissolution studies of fluconazole and dipyridamole with GIS were performed following the previous method with slight modifications.20 The diagram of the GIS is shown in Figure 1. Briefly, the GIS had three chambers, which represent stomach (GISstomach), duodenum (GISduodenum), and jejunum chambers (GISjejunum). Four peristaltic pumps were used for fluid transfer (Ismatec REGLO pump, IDEX Health and Science, Glattbrugg, Switzerland). Initially, the GISstomach had either 50 mL of SGFpH2.0 or SGFpH6.0 with 250 mL of distilled water to represent the dose volume. The GISduodenum was filled with 50 mL of SIFpH 6.5. The GISjejunum was empty at first. Dissolution studies were started by dosing one tablet of 200 mg fluconazole or two tablets of 25 mg dipyridamole into the GISstomach. During the test, simulated gastric fluid (SGFpH2.0 or SGFpH6.0) and simulated duodenal fluid (100 mM sodium phosphate buffer with 30.8 mM NaCl, pH 6.5) were introduced into the GISstomach and GISduodenum at the constant rate of


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1 mL/min respectively. All buffers were warmed to 37°C. Gastric components were introduced into the duodenal chamber at the first-order rate, which was set at 8 min as a gastric halfemptying time. In a previous study, the gastric half-emptying time between 5 and 10 min for the GIS provided a good prediction of in vivo PK profiles of BCS class I drugs.20 Thus, the gastric half-emptying time in this study was set at 8 min as an average value. The fluid transfer rate from the GISstomach to the GISduodenum was controlled by computer to decrease the gastric fluid volume at first-order rate, thus the fluid volume in the GISstomach at time t (Vstomach) can be represented as: Vstomach=Vstomach,initial*exp(-ln(2)*t/8). Where Vstomach,initial is the initial volume of the GISstomach which is in total 300 mL (50 mL of SGF with 250 mL of distilled water as the dose volume). To maintain the fluid volume in the GISduodenum equal to 50 mL, duodenal contents were transferred into the GISjejunum at an appropriate rate. The overhead paddle speeds in the GISstomach and GISduodenum were 20 rpm, and they do a quick high speed burst every 25 seconds to mimic the contractions in the stomach and the duodenum.15 The GISjejunum was stirred at a constant speed using a stir bar, representing weak contractions in the distal site.36 The duration of the experiments was set at 32 min (0.53 hr) considering the rapid absorption of dipyridamole in human healthy subjects (Tmax=0.58 hr).37 Thus, this experimental condition was designed to assess the effect of the acid-reducing agents on the drug absorption only in the upper intestine. Samples (200 µL) were manually collected at 0, 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, and 32 min from each chamber. All samples were immediately centrifuged (2,000×g, 30 sec) and 100 µL of supernatant were diluted with the equal volume of methanol. Drug concentration was measured by HPLC analysis. Data analysis of GIS


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In the GIS, once dosed drug was partially disintegrated in the GISstomach, both undissolved and dissolved drug will be transferred into the GISduodenum and GISjejunum through the tubing and peristaltic pump. Therefore, the drug amount in solution in each chamber can be described by the following equations. ()

()

()

= ( ) ( ) − ( ) ( ) − ( ) ( )

(1)

= () () + ( ) ( ) − () () − () ()

(2)

= () () + () () − () ()

(3)

Where Xd(s), Xd(d), and Xd(j) are the drug amount in solution in the GISstomach, GISduodenum, and GISjejunum, respectively. Xud(s), Xud(d), and Xud(j) are the amount of drug in solid (undissolved) state in the GISstomach, GISduodenum, and GISjejunum. kd(s), kd(d), and kd(j) represent the drug dissolution rate constants in each chamber. kp(s), kp(d), and kp(j) are the drug precipitation rate constants in each chamber. The transfer rate constants of dissolved drug from the GISstomach to GISduodenum and from the GISduodenum to GISjejunum are kd(s-d) and kd(d-j), respectively and are determined by experimental setup. The drug dissolution rate constants as well as the precipitation rate constants are time-dependent rate constants. Xd(s) was experimentally determined from drug concentration-time profile in the GISstomach and Vstomach. If there is no precipitation in the GISduodenum and GISjejunum and also if no undissolved drug is transferred into these two chambers from the GISstomach, then kp(s), kp(j), Xud(d), and Xud(j) may be assumed to be zero. In these cases, equations (2) and (3) are simplified to: ()

= ( ) ( ) − () ()

(4)


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()

= () ()

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(5)

Theoretical drug concentration curves were calculated following equations (4) and (5) based on the assumption that neither the precipitation nor the transfer of undissolved particles from the previous chamber occurs in the GISduodenum and GISjejunum. These assumptions, in effect, assume that drug is only dissolving in the GISstomach and only drug in solution is transferred into the GISduodenum and GISjejunum. Thus, if the observed drug concentration is lower than the theoretical concentration, drug precipitation may be assumed to be happening in the chamber. If the observed drug concentration is greater than the theoretical concentration, additional particle transfer and dissolution in the GISduodenum and GISjejunum is likely occurring. The comparison of in vitro dissolution with in vivo clinical outcomes In vivo pharmacokinetic parameters (AUC and Cmax) of fluconazole and dipyridamole in human were compared with the results of in vitro dissolution studies with USP apparatus II and GIS. To assess the suitability of these in vitro dissolution methodologies to predict the impaired drug absorption caused by acid-reducing agents, the ratios of drug amount in solution in two different gastric pH conditions (pH 2.0 and 6.0) in USP apparatus II and GIS were expressed as reduction ratios caused by the raised gastric pH. In USP apparatus II, drug amount in solution in 300 mL of SGFpH2.0 at the end of the study was compared with that in 300 mL of SGFpH6.0 using a following equation.

(Reduction ratio in USP) = 1 −

(#$%& '()%*+ ,* -).%+,)* ,* /01234.6 ) (#$%& '()%*+ ,* -).%+,)* ,* /01237.6 )

(6)


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The reduction ratio in drug amount in solution in GIS at the end of the study (32 min) was also calculated using a following equation.

(Reduction ratio in GIS) = 1 −

('()%*+ ,* -).%+,)* ,* +:; 0?=@A>B '*# 0A>B D,+: /01234.6 ) ('()%*+ ,* -).%+,)* ,* +:; 0?=@A>B '*# 0A>B D,+: /01237.6 )

(7) Since fluconazole and dipyridamole are highly permeable drugs, the drug in solution in the sum of the GISduodenum and GISjejunum was regarded as the bioavailable drug. Drug absorption from the stomach was assumed to be zero or negligible. In situ mouse infusion study of dipyridamole All animal experimental protocols were approved by the University of Michigan Committee of Use and Care of Animals (UCUCA). All animals were housed following the University of Michigan Unit for Laboratory Animal Medicine guidelines. C57BL/c mice ranged from 20-30 g were used for this study. Animals were randomly assigned to the individual experimental groups. After overnight fasting (10-12 hr), mice were anesthetized with an intramuscular injection of 5 mg/kg xylazine and 80 mg/kg ketamine and then placed on a heated pad (Harvard Apparatus Inc., Holliston, MA) at 37 °C. The abdomen was opened by a midline incision of 1.5 cm and carefully exposed proximal jejunum was cannulated with flexible PVC tubing (2.29 mm i.d., Fisher Scientific Inc., Pittsburgh, PA). A GIS dissolution study with two tablets of 25 mg dipyridamole (50 mg) was conducted as described in the EXPERIMENTAL SECTION. Two minutes after starting the GIS


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dissolution, the fluid in the GISjejunum was rapidly drawn into tubing and pumped into mouse proximal jejunum via cannulated PVC tubing at the rate of 0.1 mL/min for 30 min using a peristaltic pump. In these studies, either SGFpH2.0 or SGFpH6.0 was employed as a gastric fluid in the GISstomach to compare each condition. To create a saturated solution of dipyridamole for comparison purposes, two tablets of 25 mg dipyridamole were introduced into 300 mL of SIFpH6.5 and stirred overnight at room temperature. After filtration, saturated solution was pumped into mouse small intestine at the rate of 0.1 mL/min for 30 min using a peristaltic pump. Mouse blood was collected from the abdominal portion of the vena cava 60 min after infusion of dipyridamole solution. Blood samples were centrifuged at 9,700×g for 10 min at 4°C and supernatant was kept at -20°C until analysis. HPLC analysis The concentration of fluconazole and dipyridamole was determined by a gradient HPLC method using a Waters HPLC system (Waters, Inc., Milford, MA). The HPLC system consisted of two Waters pumps (model 515), a Waters autosampler (WISP model 712), and a Waters UV detector (996 photodiode arraydetector) controlled by Waters Millennium 32 software (Version 3.0.1). Analytical column was a ZORBAX Eclipse XDB-C18 column (3.5 µm, 4.6 × 150 mm) equipped with a guard column. The mobile phases are 0.1% TFA containing water (Solvent A) and 0.1% TFA containing acetonitrile (Solvent B). The flow rate was set at 1.0 mL/min and Solvent B gradient was changing from 20%-50% at a rate of 7.7%/min during a 10 min run. The wavelength of the UV detector was 260 nm for fluconazole and 290 nm for dipyridamole.


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Bioanalytical method Mouse plasma was mixed with 4-times volume of methanol (50 µg/mL ketoconazole as internal standard) and vortexed for 3 min. After centrifugation (9,700×g for 10 min at 4°C), the supernatant was transferred to LC-MS vial for analysis. Dipyridamole concentration in supernatant was analyzed by LCMS-2010EV (Shimadzu Scientific Instruments, Kyoto, Japan) equipped with an ESI (electrospray ionization) source. The Shimadzu LC–MS system consisting of Shimadzu LC-20AD pumps with DGU-20A in-line vacuum degasser units, and SIL-20A HT autosampler with a XTerra MS-C18 column (5.0 µm, 2.0 × 50 mm) was used for the separation and the effluent from the column was introduced directly to the ionization source. The system was controlled by Shimadzu LCMS solution software (version 3) to collect and process data. All samples were run with 0.1% FA containing water (Solvent A) and 0.1% FA containing acetonitrile (Solvent B) with Solvent B gradient changing from 20% to 95% at a rate of 16.7%/min over a 14 min run. The ESI probe was operated with a detector voltage of 1.5 kV, CDL temperature of 200°C, heat block of 200°C, and nebulizing gas flow of 1.2 mL/min in positive mode. The drying gas was N2 delivered at 0.1 MPa. Statistics All results were expressed as mean ± standard deviation (SD). One-way ANOVA (analysis of variance) was used to assess differences for multiple comparisons. Differences were considered statistically significant at p < 0.05. RESULTS Dissolution studies of fluconazole and dipyridamole with USP apparatus II


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The results of fluconazole dissolution with USP apparatus II is shown in Figure 2a. Regardless of buffer pH and buffer species, a 200 mg fluconazole tablet dissolved rapidly (>50% dissolved in 10 minutes) and completely dissolved in 300 mL of SGFpH2.0, SGFpH6.0, or SIFpH6.5 within 60 minutes. In contrast, dipyridamole exhibited a pH-dependent aqueous dissolution rate (Figure 2b). Although two 25 mg dipyridamole tablets dissolved rapidly (>50% dissolved in 10 minutes) and completely in SGFpH2.0, less than 10% of dipyridamole was dissolved in both SGFpH6.0 and SIFpH6.5 after 60 minutes. Due to the low buffer capacity of SGFpH6.0, the pH level in SGFpH6.0 was increased to 6.5 after dipyridamole dosing. Therefore, dissolved drug amount in SGFpH6.0 was in the same level as in SIFpH6.5. Dissolution studies of fluconazole and dipyridamole with GIS The fluconazole dissolution study was conducted in GIS and the drug concentration in each chamber is shown in Figure 3. Regardless of the gastric pH, either SGFpH2.0 or SGFpH6.0, fluconazole exhibited similar dissolution profiles in each of the GIS chambers (Figures 3a-c). The observed concentration of fluconazole in the GISduodenum and GISjejunum was compared with the theoretical curve, assuming dissolution only in the GISstomach. Fluconazole dissolution profiles in the GISduodenum and GISjejunum mostly matched the theoretical concentration curves in both GIS with SGFpH2.0 and with SGFpH6.0 conditions, suggesting neither supersaturation nor precipitation. Figure 4 shows the percentage of fluconazole in solution in each chamber. Fluconazole was quickly dissolved in the GISstomach regardless of the pH (Figure 4a) and the gastric pH affected neither the dissolution rate nor the amount of fluconazole in solution in the GISduodenum


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and GISjejunum (Figures 4b and 4c). More than 80% of fluconazole was observed as solution in the sum of the duodenal and jejunal chambers (Figure 4d). The dissolution profile of dipyridamole was also assessed with the GIS. The observed drug concentration is presented in Figure 5. In contrast to fluconazole, raised gastric pH reduced dipyridamole dissolution in the GISstomach (Figure 5a), which led to lower concentrations in the GISduodenum and GISjejunum (Figures 5b and 5c). When the pH of the gastric fluid was 2.0, drug concentration in the GISstomach increased to 234 ± 27 µg/mL. The maximum concentrations in the GISduodenum and GISjejunum were 125 ± 13 µg/mL at 11 min and 56 ± 4 µg/mL at 17 min, respectively. Since the saturated concentration of dipyridamole in 50 mM phosphate buffer at pH 6.5 was 8.1 ± 0.4 µg/mL, the observed drug concentrations in these chambers were 15-fold and 7-fold higher than the saturated solubility, suggesting supersaturation. Meanwhile, when the pH of the gastric fluid was raised to pH 6.0 to simulate DDI condition in the case of coadministration of acid-reducing agents, the concentration of dipyridamole in each chamber only reached its saturated level (~10 µg/mL). The theoretical and saturated concentrations of dipyridamole were plotted along with the observed dipyridamole concentration in the GISduodenum and GISjejunum (Figures 5b and 5c). When the gastric pH was 2.0, the observed drug concentration in both chambers stayed above the saturated concentration, indicating supersaturation. However, the measured drug concentration in both chambers fell below the theoretical lines, suggesting possible precipitation of some dipyridamole. In contrast, neither supersaturation nor the precipitation was observed when the gastric pH was 6.0 in GIS.


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Figure 6 shows the percentage of dipyridamole in solution in each chamber. Under lower gastric pH conditions, approximately 95% of dosed dipyridamole was in solution in the total of the three chambers within 8 minutes. Due to precipitation, the percentage of total drug amount in solution was reduced to 45.2 ± 7.0% at 32 minutes after starting the dissolution test (Figure 6e). In contrast, when the pH of gastric fluid was raised, the percentage of total drug amount in solution was 9.3 ± 2.7%. In the sum of the duodenal and jejunal chambers, the percentage of drug amount in solution at 32 minutes after starting the dissolution test was 40.4 ± 5.7% in the GIS with SGFpH2.0 and 8.9 ± 2.5% in the GIS with SGFpH6.0 (Figure 6d). At 32 minutes, 4.8 ± 1.3% and 0.4 ± 0.2% of drug in solution remained in the gastric chamber with gastric pH 2.0 and pH 6.0, respectively (Figure 6a). The comparison of dissolution results in USP apparatus II and GIS with in vivo clinical outcomes The reduction ratios in drug amount in solution in USP apparatus II and in GIS were calculated following equations (6) and (7). In vivo AUC ratio and Cmax ratio for the low gastric pH condition to achlorhydric condition were also calculated from clinical DDI studies data.37, 38 The results are summarized in Table 1. The dissolution study of fluconazole with USP apparatus II and GIS revealed that the pH of the gastric fluid didn’t affect the percentage of drug amount in solution very much. In contrast, the increased gastric pH resulted in a large reduction in the amount of dipyridamole in solution by 93% in USP apparatus II and by 78% in GIS, respectively. In situ mouse infusion study of dipyridamole


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Figure 7 indicates the plasma concentration of dipyridamole in the mouse infusion study. When the jejunal fluid from the GIS with SGFpH6.0 and saturated solution in SIFpH6.5 were infused into mouse jejunum, dipyridamole concentration in plasma was 2 ± 1 ng/mL and 6 ± 2 ng/mL, respectively. Meanwhile, plasma concentration in mice infused with the fluid in the GISjejunum with SGFpH2.0 was 67 ± 28 ng/mL, which is 11- to 34-fold higher than the high gastric pH condition. Discussion Poorly soluble drugs with ionizable characteristics at physiological pH often show distinctive dissolution patterns in the GI tract. Their dissolution is influenced not only by physicochemical drug properties such as intrinsic solubility, pKa, particle size, and dosage form, but also by gastrointestinal physiology like pH, fluid volume, fluid components, gastric emptying time, and motility.7-9 BCS class IIb drugs tend to dissolve to reach a high concentration in the acidic stomach. When this high concentration of weak base drugs enters into the duodenum and proximal jejunum, supersaturation and/or precipitation may occur. However, elevated gastric pH will result in lower concentrations of dissolved drug in the stomach as, for example, with the concomitant use of acid-reducing agents such as proton pump inhibitors, histamine 2A receptor blockers, and antacids. Concomitant administration of these acid-reducing agents is known to impair dissolution and oral bioavailability.29, 39, 40 The multi-compartmental in vitro dissolution apparatus such as the ASD, TIM, and GIS may be used to capture the dissolution of weakly basic drugs because they incorporate physiological factors such as pH, fluid volume, and fluid components into in vitro dissolution methodologies.14-19 Here, we argue that the GIS more accurately simulates physiological gastric emptying because the gastric transfer rate is adjusted to fit in vivo conditions as demonstrated in previous work characterizing the PK profiles of


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metoprolol and propranolol.20 Since the gastric transfer rate may have a significant impact on the process of supersaturation and precipitation, the GIS may be a more relevant in vitro apparatus to predict in vivo dissolution. In this study, fluconazole and dipyridamole were selected as test drugs. Fluconazole is a triazole antifungal drug (log P ; 0.5, pKa; 2.56, 2.94, and 11.01).41 Due to its high aqueous solubility and membrane permeability, fluconazole is categorized as a BCS class I41 drug and its oral bioavailability is greater than 90%.42 In addition, oral bioavailability of fluconazole is not affected by the concomitant use of acid-reducing agents which raise the gastric pH up to 6.0.38 Dipyridamole is a phosphodiesterase enzyme inhibitor and therapeutically used for antithrombosis. Because of its physicochemical characteristics (Log P ; 2.74, pKa; 6.24),43 dipyridamole is classified as a BCS IIb drug. As dipyridamole exhibits pH-dependent solubility in the physiological pH range44, the intraluminal concentration of dipyridamole was observed to be higher than its saturated solubility in humans.45 Also, the co-administration of acid-reducing agents significantly reduced the oral bioavailability of dipyridamole.37 Thus, dipyridamole is a typical BCS class IIb drug with ideal characteristics to assess the utility of GIS. In USP apparatus II, all tested buffers did not significantly alter the dissolution profile of fluconazole regardless of pH and buffer species (Figure 2a), suggesting that the pH change caused by gastrointestinal transit from the stomach to the duodenum or co-administration of acid-reducing agents should not affect the dissolution profile of fluconazole. These observations were consistent with the results in the GIS dissolution study (Figure 3). Neither supersaturation nor precipitation was observed and the gastric pH didn’t change the dissolution profile of fluconazole (Figures 3 and 4). The drug concentration in the GISduodenum was immediately increased toward the same level as in the GISstomach (Figure 3b). It resulted from the rapid


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disintegration and dissolution of a fluconazole tablet in the GISstomach as well as the rapid fluid transfer from GISstomach to the GISduodenum at early time points. Dosed fluconazole tablet was disintegrated within a minute and small particles were visibly transferred to the GISduodenum. This observation was ascertained by the comparison of the drug concentration-time profile in the GISduodenum with theoretical curve. As shown in Figure 3b, the drug concentration-time profile in the GISduodenum was slightly higher than the theoretical curve at early time points, suggesting the dissolution of fluconazole particles in the GISduodenum. In vitro dissolution results of fluconazole in USP apparatus II and GIS were confirmed by the clinical DDI study that cimetidine pretreatment raised the gastric pH up to > 6.0, but didn’t change the oral bioavailability of fluconazole in healthy subjects (Table 1).38 These findings demonstrate that USP apparatus II, as well as the GIS, is sufficient to assess the dissolution of BCS class I drugs which are highly soluble. On the other hand, dipyridamole has a pH-dependent dissolution profile which was demonstrated by USP apparatus II dissolution study, showing that dipyridamole dissolved well in acidic pH but not in neutral pH (Figure 2b). It suggested that the pH change in the GI tract might induce supersaturation or precipitation. It also suggested that elevated gastric pH might reduce the oral bioavailability of dipyridamole due to lower dissolution of dipyridamole in the pH-elevated stomach. However, the extent and duration of supersaturation and precipitation cannot be predicted from conventional USP dissolution studies because USP dissolution apparatus I and II typically use a single vessel with a fixed pH. Also, the impact of elevated gastric pH on the systemic exposure of dipyridamole cannot be predicted. Even in a single vessel whose pH level was changed according to predefined time course by titration with sodium


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hydroxide, it does not consider gastric empting which should have significant impact on the precipitation, thus is still insufficient to predict in vivo dissolution of dipyridamole. In the GIS, when the pH of the gastric fluid was set at 2.0, the maximum concentrations of dipyridamole in the GISduodenum and GISjejunum were 15-fold and 7-fold higher than saturated drug solution concentration, respectively (Figure 5). Times to maximum concentration were 11 min and 17 min in the GISduodenum and GISjejunum , indicating rapid absorption of dipyridamole in subjects with low gastric pH. The higher than saturated drug concentrations in the GISduodenum and GISjejunum were maintained over 32 min. The observed drug concentration was compared with human intraluminal concentration of dipyridamole. Psachoulias et al. have measured dipyridamole concentration in aspirated intestinal contents in human after the intake of dipyridamole 30 mg or 90 mg in HCl solution (pH 2.7) and revealed that the concentration up to 10 min post dosing was approximately 80 µg/mL and 250 µg/mL, respectively.45 In our GIS study, dipyridamole concentration in the duodenal chamber was 125 ± 13 µg/mL at 11 min when 50 mg dose was used, consistent with their results. The precipitation of diyridamole was also observed in the GIS by comparing the measured drug concentration with the theoretical drug concentration curve (Figure 5). The percentage of precipitated drug was approximately 50% in the GIS dissolution study after 32 minutes (Figure 6). However, Psachoulias et al. observed the fraction precipitation in aspirated intestinal fluids exhibiting less than 7% of dosed dipyridamole (90 mg). This discrepancy may come from differences in dosage form. When solid drug is dosed as an immediate release dosage form, the formulation disintegrates in the stomach and undissolved particles may be expected to be present. As some of those particles exit the stomach into the duodenum, precipitation might be accelerated due to the presence of undissolved drug particles as potential nuclei. In contrast,


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Psachoulias’s group used completely dissolved drug solution to eliminate undissolved particles, which might reduce precipitation and cause this difference. Another possible reason might be the absence of absorption compartment in the dissolution apparatus. Similar findings were previously pointed out.32, 46 Drug absorption from the GI tract will apparently reduce the drug amount from the GI lumen, hence, slow drug precipitation. In line with those studies, the results obtained in the GIS clearly suggest the importance of absorption process in assessing in vitro dissolution of drugs with low aqueous solubility. The raised gastric pH condition, simulating the concomitant use of acid-reducing agents, critically reduced dipyridamole concentration in the GISstomach, which triggered low concentrations in the GISduodenum and GISjejunum (Figure 5). The reduction ratios in drug amount in solution caused by elevated gastric pH were calculated in USP apparatus II and GIS. The elevated gastric pH reduced the percentage of drug amount in solution by 93% and 78% in USP apparatus II and GIS, respectively (Table 1). These ratios were compared with the results of the clinical DDI study. With concomitant use of famotidine, a 79% reduction in dipyridamole Cmax and 37% reduction in AUC were observed in the clinical DDI study.37 Since neither metabolic nor transporter-mediated DDI was not reported between dipyridamole and famotidine, this reduced Cmax and AUC should derived from pH-dependent solubility of dipyridamole.44 Considering this information, USP apparatus II overestimated the extent of DDI because no precipitation was observed in a single vessel with a fixed pH. On the other hand, the reduction ratio of dipyridamole in GIS was quite similar to the reduction of dipyridamole in Cmax, but less so with the reduction in AUC. In the clinical study, dipyridamole exhibits relatively late Tmax (2.25 hr) in famotidine-treated subjects compared with the Tmax in control subjects (0.58 hr).37 This result suggests that the duodenum and the proximal jejunum may be the main absorptive


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site of dipyridamole in subjects with low gastric pH condition. Although the distal jejunum and the ileum are still absorptive sites for dipyridamole, the total amount absorbed from these sites would be minimal because most of drug would be absorbed from the upper intestine. However, no observation of the supersaturation in the duodenum and the proximal jejunum of famotidinetreated subjects may cause that the distal jejunum as well as the ileum may make a relatively large contribution to the absorption of dipyridamole in famotidine-treated subjects, resulting in delayed Tmax. Therefore, in vitro dissolution results in the GIS could not fully capture in vivo dissolution profile of dipyridamole in famotidine-treated subjects because of the relatively short dissolution study (0.53 hr). The presented GIS study was focused on drug dissolution in the duodenum and the proximal jejunum. It means that the current experimental condition is designed to assess the effect of acid-reducing agents on the drug absorption from the upper intestine, but not from the whole GI tract. Thus, it is possible that the onset of drug absorption, which is represented by Cmax, was able to be well captured, but the entire drug absorption profile, which is signified by AUC, was not simulated in GIS. A prolongation of experimental time course or an addition of extra compartments as distal intestine might be helpful to address this hypothesis. In order to assess the observation of high drug concentration of dipyridamole in GIS for oral absorption, an in situ mouse infusion study was performed in combination with GIS dissolution study. Figure 7 reveals that the supersaturation of dipyridamole observed in the GIS with SGFpH2.0 enhanced oral drug absorption in mice. Moreover, the infusion study using the duodenal fluid, in which dipyridamole concentration was higher than in the jejunal fluid, resulted in 3- to 4-fold higher plasma drug concentration (data not shown). Considering these results, drug concentration in the GIS system has the potential to be an excellent predictor of its systemic


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exposure in the case of dipyridamole. Hence, GIS appears to be an improved methodology to assess the pH-effect on BCS class II drugs with weak base properties like dipyridamole. Besides the well designed gastric half-emptying time, GIS might have advantages over other multi-compartmental dissolution methodologies. The artificial stomach-duodenum (ASD) model, which has two chambers representing the stomach and duodenum, is one of the most validated apparatuses to capture in vivo drug dissolution.14, 15 Compared to the ASD model, the GIS has an additional compartment as a proximal jejunal chamber, adding some extra values to the GIS. Since the supersaturation as well as the precipitation of BCS class IIb drugs may occur not only in the duodenum but also in the proximal jejunum, the dissolution and precipitation behavior in the proximal jejunal chamber should not be negligible. Thus, the existence of the jejunal chamber may improve the predictability of in vivo dissolution of BCS class IIb drugs. In the present study, smaller extent of supersaturation of dipyridamole was observed in the GISjejunum than in the GISduodenum (Figures 5b and c), which suggested that the effect of supersaturation on drug absoption may be overestimated in the absence of the proximal jejunal chamber. Moreover, the pH level in the GISduodenum was ranging from 5.9 to 6.5, whereas the pH in the GISjejunum was more stable and between 6.3 and 6.5 (data not shown). The reported pH value in the jejunum is in narrow range compared with in the duodenum.9 Therefore, drug dissolution and precipitation profile in the upper intestine may be well captured in the GIS. TNO intestinal model (TIM) is widely recognized as an excellent in vivo predictive dissolution apparatus as well as the ASD.17 TIM has several compartments, mimicking the whole GI tract, and incorporates much more physiological factors than the ASD and the GIS. Therefore, TIM might be an advanced dissolution apparatus in terms of physiological relevance in the GI tract. However, the complexity of TIM system may require a lot of time and instruments in preparation


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and operation, which results in limiting its practical application as a screening tool. The GIS can be used in drug candidate screening processes and has a capability of higher throughput performance. Though the GIS system was able to capture the DDI of fluconazole and dipyridamole in the case of elevated pH by acid-reducing agents, there might be adjustable experimental conditions to further improve the prediction of in vivo dissolution for certain oral drug products. The use of biorelevant dissolution media is known to improve the accuracy of IVIVC of lipophilic drugs in the fasted state.8, 21 Also, the utilization of biorelevant dissolution media may alter precipitation kinetics. Moreover, absorption process should be taken into account. To mimic the continuous drug removal from the GI tract, the incorporation of absorptive site might advance in vivo predictability of GIS.13 These modifications for certain drugs may improve the prediction of in vivo dissolution and might be necessary to validate the theory and the experimental condition of the GIS. Also, combining the experimental results of the GIS with in silico simulation may be a prominent approach to verify the predictability of the GIS. In conclusion, USP apparatus II was able to forecast the gastric pH effect on the dissolution of fluconazole, but not dipyridamole because of dipyridamole’s more complex dissolution profile. Meanwhile, the GIS captured the supersaturation and precipitation of dipyridamole as well as the potential reduction of drug absorption caused by acid-reducing agents. These data suggest that the GIS is a suitable methodology to assess in vivo dissolution for low soluble drugs, especially for BCS class IIb drugs.


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FIGURE CAPTIONS Figure 1. Diagram of the Gastrointestinal Simulator (GIS). Figure 2. Drug % dissolved-time profiles of a tablet of 200 mg fluconazole (a) and two tablets of 25 mg dipyridamole (b) in USP apparatus II at different fluids and pH. Each drug was dosed into 300 mL of 10-2 N HCl (pH 2.0), 10-6 N HCl (pH 6.0), or 50 mM phosphate buffer (pH 6.5). Closed, open, and gray circles are representing the dissolution profiles in 10-2 N HCl, 10-6 N HCl, and 50 mM phosphate buffer (pH 6.5), respectively. Each data point represents mean ± SD (n = 3). Figure 3. Drug concentration-time profiles of fluconazole in the gastric (a), duodenal (b), and jejunal chambers (c) at different gastric conditions in GIS. Closed and open circles represent a condition with gastric pH 2.0 and 6.0, respectively. Black and gray lines indicate the theoretical concentration curves at a condition with gastric pH 2.0 and 6.0, respectively. Each data point represents mean ± SD (n = 3). Figure 4. Drug amount in solution-time profiles of 200 mg fluconazole with GIS in the gastric (a), duodenal (b), and jeunal chambers (c) and drug amount in solution-time profiles in the sum of duodenal and jejunal chambers (d) and in the sum of the gastric, duodenal, and jejunal chambers (e). Closed and open circles represent a condition with gastric pH 2.0 and 6.0, respectively. Each data point represents mean ± SD (n = 3). Figure 5. Drug concentration-time profiles of dipyridamole in the gastric (a), duodenal (b), and jejunal chambers (c) at different gastric conditions in GIS. Closed and open circles represent a condition with gastric pH 2.0 and 6.0, respectively. Black and gray lines indicate the theoretical


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concentration curves at a condition with gastric pH 2.0 and 6.0, respectively. Dotted lines represent the saturated concentration at pH 6.5. Each data point represents mean ± SD (n = 3). Figure 6. Drug amount in solution-time profiles of 50 mg dipyridamole with GIS in the gastric (a), duodenal (b), and jeunal chambers (c) and drug amount in solution-time profiles in the sum of the duodenal and jejunal chambers (d) and in the sum of the gastric, duodenal, and jejunal chambers (e). Closed and open circles represent a condition with gastric pH 2.0 and 6.0, respectively. Each data point represents mean ± SD (n = 3). Figure 7. Mouse plasma concentration of dipyridamole in infusion study. Black and gray bars represent the infusion combined with GIS in case of gastric pH 2.0 and pH 6.0, respectively. White bar represents the infusion with saturated solution. Each data point represents mean ± SD; n = 4-7 (*, p < 0.05; **, p < 0.01).


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FIGURES Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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Figure 6.


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Figure 7.


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TABLE Table 1. The comparison of in vitro dissolution results with clinical data Clinical DDI study In vitro dissolution results with acid-reducing agent

Compound

gastr ic pH

dissolved amount in USP apparatus

dissolved amount in GIS

mg

Reduct ion ratio (%)

mg


Cmax gastri c pH µg/m L

AUC

Reduct ion µg*hr ratio /mL (%)


Fluconazole

2.0

189.8

-

175.3

-

-

3.43

-

161.5

-

200 mg

6.0

189.6

0

156.3

11

>6.0

3.43

0

161.7

0

Dipyridamole

2.0

52.3

-

20.2

-

1

1.58

-

4.26

-

50 mg

6.0

3.4

93

4.4

78

5.9

0.33

79

2.69

37


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AUTHOR INFORMATION Corresponding Author: Gordon L. Amidon Phone: 734-764-2464. FAX: 734-764-6282. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT The authors would like to thank Gail Benninghoff for her excellent secretarial work, and Susumu Takeuchi for his valuable comments. This work was supported by NIH Grant HHSF223201310144C. ABBREVIATIONS API, active pharmaceutical ingredient; AUC, area under the curve; BCS, biopharmaceutics classification system; DDI, drug-drug interaction; GIS, Gastrointestinal Simulator; GI tract, gastrointestinal tract; IVIVC, in vitro-in vivo correlations; NaCl, Sodium chloride; SGF, simulated gastric fluid; SIF, simulated intestinal fluid; USP, United States Pharmacopeia; REFERENCES 1. Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm Res 1995, 12, (3), 413-20. 2. Lennernäs, H.; Abrahamsson, B. The use of biopharmaceutic classification of drugs in drug discovery and development: current status and future extension. J Pharm Pharmacol 2005, 57, (3), 273-85. 3. Lipinski, C. A. Drug-like properties and the causes of poor solubility and poor


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