Article pubs.acs.org/molecularpharmaceutics
Effect of Surfactants, Gastric Emptying, and Dosage Form on Supersaturation of Dipyridamole in an in Vitro Model Simulating the Stomach and Duodenum A. Mitra and H. M. Fadda* Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Butler University, Indianapolis, Indiana 46208, United States ABSTRACT: The purpose of this study was to investigate the influence of gastric emptying patterns, surfactants, and dosage form on the supersaturation of a poorly soluble weakly basic drug, dipyridamole, using an in vitro model mimicking the dynamic environment of the upper gastrointestinal tract, and, furthermore, to evaluate the usefulness of this model in establishing correlations to in vivo bioavailability for drugs with solubility/dissolution limited absorption. A simulated stomach duodenum model comprising four compartments was used to assess supersaturation and precipitation kinetics as a function of time. It integrates physiologically relevant fluid volumes, fluid transfer rates, and pH changes of the upper GI tract. Monoexponential gastric emptying patterns simulating the fasted state were compared to linear gastric emptying patterns simulating the fed state. The effect of different surfactants commonly used in oral preparations, specifically, sodium lauryl sulfate (SLS), poloxamer-188, and polysorbate-80, on dipyridamole supersaturation was investigated while maintaining surface tension of the simulated gastric fluids at physiological levels and without obtaining artificial micellar solubilization of the drug. The supersaturation behavior of different dose strengths of dipyridamole was explored. Significant levels of dipyridamole supersaturation were observed in the duodenal compartment under all the different in vivo relevant conditions explored. Dipyridamole supersaturation ratios of up to 11-fold have been observed, and supersaturation has been maintained for up to 120 min. Lower duodenal concentrations of dipyridamole were observed under linear gastric emptying patterns compared to mononexponential gastric emptying. The mean duodenal area under concentration−time curves (AUC60min) for the dipyridamole concentration profile in the duodenal compartment is significantly different for all the surfactants explored (P < 0.05). Our investigations with the different surfactants and comparison of dosage form (solution versus suspension) on the precipitation of dipyridamole revealed that crystal growth, rather than nucleation, is the rate-limiting step for the precipitation of dipyridamole. A linear dose−response relationship was found for the mean in vitro duodenal area under concentration−time curves (AUC∞) in the dose range of 25 mg to 100 mg (R2 = 0.886). This is in agreement with the pharmacokinetic data of dipyridamole reported in the literature. The simulated stomach duodenum model can provide a reliable and discriminative screening tool for exploring the effect of different physiological variables or formulations on the supersaturation/precipitation kinetics of weakly basic drugs with solubility limited absorption. The amount of drug in solution in the duodenal compartment of the SSD correlates to bioavailability for the weakly basic drug, dipyridamole, which has solubility limited absorption and undergoes supersaturation/precipitation. KEYWORDS: crystallization, biorelevant, in vitro−in vivo correlation (IVIVC), Biopharmaceutics Classification System, simulated gastric fluid (SGF)
1. INTRODUCTION Precipitation of drug particles in the lumen of the gastrointestinal (GI) tract can contribute to explaining the large intraand interindividual variability in drug exposure as well as reduced bioavailability observed for some low solubility drugs. For drug precipitation to occur, a supersaturated drug solution needs to exist. Supersaturation and precipitation of some weakly basic drugs is a physiologically triggered phenomenon arising from the pH gradient from the stomach to the small intestine. Weak bases are ionizable and display much higher solubility under the acidic conditions of the stomach compared to the small intestine. For weakly basic drugs, two species exist © 2014 American Chemical Society
in solution, their proportions depending on pH. At low pH such as in the gastric environment of a young adult in the fasted state, the ionized form of the drug (BH+) predominates. At high pH, the free base (B) which has a lower solubility compared to its salt counterpart predominates. After the drug dissolves in the stomach and reaches the small intestine, a basic drug may exist in the supersaturated state, that is, in solution at Received: Revised: Accepted: Published: 2835
March 11, 2014 May 29, 2014 June 30, 2014 July 15, 2014 dx.doi.org/10.1021/mp500196f | Mol. Pharmaceutics 2014, 11, 2835−2844
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Figure 1. Schematic diagram of the simulated stomach duodenum model with flow rates mimicking those of the fasted upper gastrointestinal tract.
kinetics and rate-limiting steps of precipitation. The SSD model is a four-compartment model integrating physiologically relevant fluid volumes, fluid transfer rates, and pH of the upper GI tract. The removal of drug from the duodenum is also taken into account. With a dynamic model, it is less likely to see an overprediction of precipitation in the GI lumen as has been observed with other in vitro methods.4,7 The SSD has been modeled after the system described by Vatier et al.8 and Carino et al.,9 who have respectively used the model to study antacidinduced resistance to gastric acidification and in selection of the solid form of carbamazepine that gives the highest bioavailability. The objective of this study is to utilize the physiologically relevant conditions of the in vitro SSD model to probe the effect of gastric emptying conditions simulating the fasted and fed states on the supersaturation of dipyridamole. The dosage form of dipyridamole (solution vs suspension) is also explored. The effect of different surfactants commonly used in oral preparations, specifically sodium lauryl sulfate, poloxamer-188, and polysorbate-80, on dipyridamole supersaturation is investigated while maintaining surface tension of the simulated gastric fluids at physiological levels and without obtaining artificial micellar solubilization of the drug. Despite there being an abundance of evidence exploring the effect of different polymeric excipients on drug supersaturation, there is a paucity of work on the influence of surfactants. The supersaturation behavior of different dose strengths of dipyridamole available in the market is studied and compared to published in vivo drug exposure data. We test the hypothesis that the amount of drug in solution in the duodenal compartment of the SSD correlates to bioavailability for drugs with solubility/dissolution limited absorption undergoing supersaturation/precipitation.
a concentration above its solubility. Once supersaturation is reached, the drug can precipitate almost immediately or remain in a supersaturated state for some time before precipitating out. It is important to gain insight into the kinetics of precipitation as it will determine the amount of drug available for absorption. The extent and duration of supersaturation of drugs in the small intestine depend on a multitude of factors including the solid-state properties of the drug as well as GI luminal conditions including pH, motility patterns, and food components, to name but a few.1,2 The supersaturation behavior of the model weakly basic drug, dipyridamole, is to be explored here. Dipyridamole is poorly soluble in water, lipophilic, and categorized as a BCS class II (low solubility, high permeability) compound. It is available as a tablet formulation in different dose strengths (25, 50, 75, and 100 mg). Weakly basic drugs are of great interest due to their physiologically triggered supersaturation in the small intestine; however, they also comprise the majority of active pharmaceutical ingredients (APIs) in the market for oral use. The supersaturation behavior of dipyridamole is to be explored here using an in vitro model simulating the dynamic conditions of the upper GI tract. Several in vitro models of varying complexity have been used for studying drug supersaturation. Single-step, pH-shift methods have been adopted to explore formulation-induced supersaturation.3 A limitation with single-step models is that they do not address the dynamic environment of the stomach and small intestine and therefore do not correlate to in vivo. While Kostewicz et al.,4 Psachoulias et al.,5 and Gu et al.6 have used dynamic systems with continuous transfer of gastric contents into the small intestine, these systems are still rather simple as they do not simulate the monoexponential fasted gastric emptying patterns and the continuous gastric and duodenal secretions, all of which can impact the duodenal luminal environment. It is important to use simulative in vitro models to elucidate and explore the significance of the effect of different physiological parameters and additives on drug supersaturation. Here we probe the influence of gastric emptying patterns, surfactants, and dosage form on the supersaturation of a model weakly basic drug, dipyridamole, in a simulated stomach duodenum (SSD) model that mimics the dynamic environment of the GI tract. We explore the utility of the SSD model in studying supersaturation of dipyridamole under different conditions and its capability of predicting in vivo trends in the bioavailability of different doses of dipyridamole. Concentration−time profiles are compared to gain an insight into the
2. MATERIALS AND METHODS 2.1. Materials. Dipyridamole (pKa 6.4), ≥98% purity powder was purchased from Sigma-Aldrich (St. Louis, MO). Poloxamer-188 was kindly donated by BASF (Florham Park, New Jersey). All salts used to make up the gastric and duodenal simulated fluids as well as the HPLC buffer were of analytical grade and purchased from VWR (Radnor, PA). All other materials were purchased from Sigma-Aldrich (St. Louis, MO). 2.2. Conditions and Defined Parameters of the Simulated Stomach Duodenum Model. Figure 1 illustrates a schematic of the simulated stomach duodenum (SSD) model. Physiologically relevant conditions that mimic the dynamic conditions of the upper gastrointestinal (GI) tract of humans 2836
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fluids from the cells of the gastric mucosa and emptying of fluids into the duodenum. Gastric juice output in adults has been reported to be 2−3 L/day.19 Ottenjann et al.20 has also measured the basal gastric juice output in 17 adults and found it to be at a mean of 96.6 mL/h. Hence the gastric acid secretion rate from a reservoir compartment into the gastric compartment was set at a constant rate of 1.7 mL/min in the SSD. The fluid in the gastric reservoir compartment was set to be of similar composition to the basal gastric fluid explored in the different experiments. 2.2.4. Duodenal Secretions. Physiological duodenal secretions include bile, pancreatic, and mucosal secretions. The daily production of bile has been amounted to approximately 600 mL per day (0.4 mL/min).21,22 Under basal conditions, pancreatic secretion is very limited and has been assessed to be less than 6 mL/h (0.1 mL/min) in adults.23 The submucosa of the duodenum consists of duodenal (Brunner’s) glands which secrete alkaline mucus that helps to neutralize acidic chyme. Total volume secreted by crypts has been approximated to be 1800 mL/day (1.25 mL/min).24 Therefore, a duodenal secretion rate of 1.75 mL was utilized in the SSD. The composition of the duodenal reservoir compartment was 0.05 M, pH 6.5 phosphate buffer. 2.2.5. Emptying from the Duodenal Compartment. Volume of duodenal content was maintained constant at 30 mL. This was achieved through the use of a vacuum pump which allowed the removal of any fluid exceeding 30 mL. It is important to try and mimic drug emptying patterns from the duodenum as the buildup of drug in the compartment will overpredict precipitation. The concentration of drug in the supersaturated solution and particle concentration act as the driving force for nucleation and crystallization.25 In the SSD model under fasting conditions, the median transfer rate from the duodenal compartment is 3.65 mL/min (corresponding to the sum of the median gastric emptying rate (1.9 mL/min) and secretions into the duodenum (1.75 mL/min)). This happens to be not too far off the rate at which dipyridamole leaves the duodenum through either absorption or transit into the lower GI compartments. Dipyridamole has high permeability with an absorption rate constant of 0.07 min−1,26 corresponding to 2.1 mL/min when the duodenal volume is 30 mL. Kerlin et al.27 reported the flow of small intestinal contents over the average MMC to be 0.73 mL/min. Therefore, the theoretical rate at which dipyridamole leaves the duodenum through either absorption or transit is 2.83 mL/min. For drugs with poor permeability, this duodenal emptying pattern may not be applicable and different emptying rates will need to be employed. Another limitation of the SSD model is that there is no drug absorption compartment which would be useful for poor permeability drugs or for drugs where significant absorption occurs beyond the duodenum. 2.2.6. Composition of the Gastric and Duodenal Compartments. Modified fasted state simulated gastric fluid (mFaSSGFSLS) was chosen to simulate the gastric environment. m-FaSSGFSLS as has been proposed by Aburub et al.28 was used throughout except when exploring surfactant effect. This comprises 0.2% NaCl, 1.75 mM sodium lauryl sulfate (SLS), pH 2.0, and a surface tension of ∼34 mN/m. This was judged to be the most suitable as it has been shown to achieve a surface tension similar to that of gastric fluids (34 to 45 mN/m) without artificial micellar solubilization. This absence of artificial micellar solubilization was confirmed with solubility measurements of dipyridamole.
were adopted. Water jacketed glass vessels were used for the stomach and duodenum compartments, and the temperature of the vessel contents was maintained at 37 °C through the use of a water circulator. The basal gastric volume was set as 50 mL based on the mean fluid volume reported in fasted adults by Schiller et al.10 and Gentilcore et al.11 Duodenal volume was set at 30 mL as suggested by Vatier et al.8 Agitation in the compartments was set at a constant rate (200 rpm) using digital magnetic stirrers. While this does not simulate the hydrodynamics of the GI tract, agitation was set at a constant rate to systematically investigate the influence of other variables. Transfer of contents from the gastric to the duodenal compartments was set at different patterns and rates to simulate the fasted and fed states. Details of these patterns are provided below. The gastric and duodenal in vivo environments are not stagnant; there are constant secretions into these compartments that have also been incorporated into the SSD model and are further discussed. Drug transfer from the duodenum is also taken into account. 2.2.1. Fasted State Gastric Emptying Patterns into the Duodenum. In the fasted state, gastric emptying patterns of non-nutrient liquids is nonlinear, following a monoexponential pattern.12,13 The emptying of non-nutrient liquids has been measured by several groups through various means, and while there is considerable variation, a median emptying half-life (T1/2) close to 15 min has been frequently reported.14−16 Therefore, to simulate the fasted state in the SSD model, gastric emptying from the stomach to the duodenal compartment was programmed to follow a monoexponential pattern with a T1/2 of 15 min until basal gastric volume has been restored. Once this basal volume (50 mL) has been restored, gastric emptying was set to be maintained at a constant rate of 1.7 mL/min to equal the rate of basal gastric acid secretions. In one set of experiments explored in this study, the gastric emptying T1/2 was set to 30 min as this value has also been observed.11,17 Since gastric emptying times are likely to undergo intraday and interindividual variations, it is therefore of interest to explore the impact of these variations on drug supersaturation as it may contribute to explaining the variability in drug exposure of low solubility drugs. 2.2.2. Fed Gastric Emptying Patterns into the Duodenum. Fasted and fed gastric emptying patterns are very different. Gastric emptying in the fed state includes an initial lag period (reduction of food to small particles) followed by a linear pattern which is proportional to caloric content (1−3 kcal min−1).11,18 There are receptors in the duodenum which regulate gastric emptying so that the rate of delivery of energy is constant. To simulate gastric emptying patterns in the fed state, an average emptying rate of 2 kcal min−1 was explored in the SSD. A standard, low fat, FDA breakfast is approximately 500 kcal. Assuming that this calorie content has a fluid volume of 250 mL, emptying rate of the nutrients will be 1 mL min−1 (500 kcal/250 mL = 2 kcal mL−1; 2 kcal min−1/2 kcal mL−1 = 1 mL min−1). This, summed to the basal gastric emptying rate of 1.7 mL min−1 (explained later), gastric emptying mimicking fed state of the SSD was set at a constant rate of 2.7 mL min−1. This set of experiments is of interest as it explores the influence of the different fasted and fed gastric emptying patterns on drug supersaturation while maintaining all the other variables constant. 2.2.3. Gastric Secretions. A further important simulation in the SSD model is gastric acid secretions. The contents of the stomach are not stagnant; there is a continuous secretion of 2837
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2.6. Exploring the Effects of Different Surfactants on Dipyridamole Supersaturation. The effects of poloxamer188 and polysorbate-80 on dipyridamole supersaturation were compared to that of SLS. These surfactants were added to simulated gastric media containing 0.01 M HCl and 0.2% NaCl. Poloxamer surfactants are triblock copolymers having a polyethylene oxide (PEO)−propylene oxide (PPO)−polyethylene oxide (PEO) structure. The three different surfactants were used at levels below their critical micelle concentration (CMC), and the surface tension was kept constant at ∼35 to 45 mN/m to match the surface tension values of the fasted stomach contents.31,32 The surface tension of gastric fluids with different surfactant concentrations was measured using the du Noüy ring method. For poloxamer-188, a concentration of 0.05% was used as this is below the CMC of the surfactant while providing a surface tension value similar to that of the fasted stomach. This was in agreement with the surface tension values reported by Kabanov et al.33 For polysorbate-80, a concentration of 0.0015% was found to be below the CMC of the surfactant while providing a surface tension value similar to that of the fasted stomach. This is in agreement with findings by Wan and Lee.34 The solubility of dipyridamole was explored at a range of surfactant concentrations to confirm that the concentration of surfactant used was below the CMC. 2.7. Exploring the Effects of Dosage Form on Dipyridamole Supersaturation. 50 mg of dipyridamole was allowed to completely dissolve in the gastric medium, after which the solution was transferred into the duodenal compartment. In another experiment, dipyridamole was not completely dissolved in the gastric medium and a few particles were allowed to remain to act as seeds for crystal growth. The only difference between these two experiments is that in one of them dipyridamole is in complete solution, while in the other it exists as a suspension (predominantly in solution however with a few undissolved particles remaining). 2.8. Data Analysis and Statistics. Area under the curve (AUC) for the concentration−time profiles in the duodenal compartment was integrated from 0 to a specific time or 0 to ∞ and expressed as AUCtmin and AUC∞ respectively. AUC was calculated using the trapezoidal rule. Supersaturation ratio, S, of dipyridamole in the duodenal compartment was determined from the concentration of drug in solution, C, and the equilibrium solubility of the drug, Ceq, in the medium at the measured pH, as determined by the log solubility−pH profile.
pH 6.5, 0.05 M phosphate buffer was chosen to simulate the duodenal compartment. pH 6.5 corresponds to the luminal pH of the upper small intestine,29 and phosphate buffer is a compendial buffer that can be reproducibly prepared. Bile salts and phospholipids were not introduced into the duodenal media so that we are able to systematically explore the influence of surfactants in gastric fluid without any confounding variables. 2.3. Exploring Supersaturation of Dipyridamole with the Simulated Stomach Duodenum Model. Dipyridamole powder (100 mg, unless stated otherwise) was added to the gastric compartment of the SSD comprising 50 mL of mFaSSGF. This was followed by the addition of a typical dosing volume of 240 mL of water to simulate how patients take their medication.30 Drug solution and any undissolved powder were transferred from the gastric into the duodenal compartment under various emptying patterns and rates through the use of a programed ChemTec pump (SciLog, Madison, WI, USA). This sequence of events is more realistic compared to previous supersaturation studies whereby the drug is dissolved in a solution of simulated gastric fluid and the dosing fluid (typically water) is not included in the gastric compartment.4−6 The pH was continuously monitored in the gastric and duodenal compartments. Sample volume of 1 mL was taken from the gastric and duodenal compartment every 10 min. Samples were filtered to remove any undissolved or precipitated drug. The first 0.5 mL of filtrate was discarded and the remainder diluted by 20-fold with mobile phase. PVDF, 0.45 μm filters were used throughout, and validation experiments showed no drug adsorption onto the filter. The concentration of drug was quantified using HPLC−UV. All experiments were conducted at least in triplicate. 2.4. Dipyridamole HPLC Assay. The HPLC system consisted of an Agilent 1200 series with a PDA detector (Agilent Technologies, Santa Clara, CA). The HPLC method was adapted from that described by USP (2012) for dipyridamole tablets. The mobile phase comprised 75% methanol and 25% dibasic sodium phosphate buffer adjusted to pH 4.6. A reverse phase C18 column (5 μm, 150 × 4.6 mm) (Zorbax ODS, Agilent, Santa Clara, CA, USA) was used. The flow rate was set to 1.3 mL/min and PDA detector wavelength at 288 nm. 50 μL injection volumes were used, and dipyridamole retention time of 4.6 min was observed. The method was validated, and standard curves were prepared for every run. The lower limit of quantification for dipyridamole was determined to be 0.2 μg/mL. 2.5. Dipyridamole Solubility Assay. Solubility of dipyridamole in different media was measured using the shake-flask solubility method at 37 °C. Excess drug was added to glass vials containing the appropriate media. These were shaken for 24 h on a thermal rocker and samples taken at 6, 12, and 24 h. Saturation solubility of dipyridamole was achieved within 6 h. The suspensions were filtered using PVDF, 0.45 μm filters, and the filtrate was diluted with mobile phase and drug content quantified by HPLC−UV. A pH−log solubility profile was obtained for dipyridamole by measuring its solubility under different pH conditions: pH 2 (0.01 M HCl) and pH 4, 5.5, 6, 6.5, and 8 in 0.05 M phosphate buffer. The final pH once drug saturation solubility was achieved was noted and used for the dipyridamole pH solubility curve fitting. Solubility measurements were also conducted in the different gastric media with different surfactants and at different surfactant concentrations.
S=
C Ceq
Statistical analysis was performed using Student’s t test or oneway analysis of variance followed by Tukey post hoc comparison (P < 0.05 defining significance).
3. RESULTS AND DISCUSSION 3.1. Dipyridamole Solubility. The solubility of dipyridamole in simulated gastric fluid comprising 0.01 M HCl, 0.2% NaCl was measured to be 4.97 ± 0.26 mg/mL. The solubility was not significantly different when the specified concentrations of the different surfactants were included (P > 0.05). The solubility of dipyridamole in pH 6.5 phosphate buffer is poor and was measured to be 5.75 ± 0.6 μg/mL. The pH−solubility profile of dipyridamole is illustrated in Figure 2. 2838
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Figure 2. pH−solubility profile of dipyridamole.
3.2. In Vitro Dipyridamole Supersaturation in Modified Fasted Simulated Gastric Fluid (mFaSSGF). The concentration−time profiles of dipyridamole in the stomach and duodenal compartments were accurately measured over time in the simulated stomach duodenum (SSD) model mimicking the dynamic nature of the upper GI tract. Significant levels of dipyridamole supersaturation were observed in the duodenal compartment under all the different in vivo relevant conditions explored. At supersaturation, the concentration in solution, C, is higher than the equilibrium solubility of the drug, Ce. The control experiment was set up to simulate the fasted upper gastrointestinal tract; it comprised mFaSSGF in the stomach compartment, 0.05 M pH 6.5 phosphate buffer in the duodenal compartment, and a monoexponential emptying of gastric contents from the stomach into the duodenum with an emptying half-life (T1/2) of 15 min. 100 mg of dipyridamole was added to mFaSSGF followed by the addition of 240 mL of water. Significant supersaturation of dipyridamole was achieved under these physiologically relevant, control conditions (Figure 3). A 10-fold degree of supersaturation was achieved, and precipitation was slow, with dipyridamole remaining in the supersaturated state for up to 120 min in the dynamic duodenal compartment. 3.3. Influence of Gastric Emptying Patterns and Rates on in Vitro Supersaturation of Dipyridamole. Slower gastric emptying (GE) patterns, monoexponential emptying with a T1/230min, give rise to initial dipyridamole concentrations in the duodenum similar to that observed with a GE T1/215min (Figure 4). The precipitation kinetics are however different, with a faster onset of precipitation observed at a GE T1/230min. A greater degree of supersaturation stability and, therefore, higher dipyridamole concentrations are observed with the faster rate of GE for the first hour. The supersaturation ratio is overall higher during the first hour for the GE T1/215min compared to the GE T1/230min (Figure 5). At 75 min, however, duodenal concentrations at the slower rate of GE (T1/230min) start to exceed those at the faster rate (T1/215min). Interestingly, this change in the trend of drug concentration is also observed with the supersaturation ratios at the different GE rates.
Figure 3. Measured dipyridamole concentrations (solid line) and predicted solubility (dashed line) in the stomach (a) and duodenal compartment (b) of the SSD (mean ± SD). mFaSSGFSLS in the gastric compartment with a gastric emptying T1/215min. Dipyridamole dose 100 mg.
The higher degree of supersaturation stability with a T1/215min observed during the first hour may be explained by the lower pH in the duodenum (measured to be ∼0.2 pH unit lower), which can be attributed to the faster gastric fluid entry. Rate of gastric fluid emptying into the duodenum under a T1/215min exceeds that of T1/230min during the first 40 min as can be observed from the slopes of the monoexponential emptying curves (Figure 6). Thereafter, rate of gastric fluid entry into the stomach under a T1/230min exceeds that under a T1/215min. Consequently, the pH in the duodenal compartment becomes lower at the slower rate of gastric emptying. It is close to this time point that a shift in the concentrations as well as supersaturation ratios of dipyridamole is observed at the two transfer rates. Precipitation kinetics at T1/230min slow down with this fall in pH, and the drop in dipyridamole concentration to the final equilibrium concentration becomes slower. These results illustrate the importance of luminal pH in the supersaturation stability of weakly basic drugs. It is therefore critical when studying drug supersaturation using in vitro models to accurately simulate the pH shift of the upper GI using realistic transfer rates. Kostewicz et al.4 have investigated the effect of different gastric fluid transfer rates on precipitation kinetics. While they do not see a trend in maximum concentrations at different transfer rates, they observe faster precipitation kinetics at higher transfer rates. They attribute this difference to the greater number of seed crystals generated at higher transfer rates. A limitation of the model used by Kostewick et al. is the use of a 2839
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Figure 6. Illustration of the gastric emptying patterns at the different monoexponential emptying rates.
adopted different constant gastric emptying rates and observed negligible effects on the drug concentrations available for absorption. While the multicompartment model utilized by Gu et al. allows for fluid emptying from the duodenal compartment, the volume of fluid in the duodenal compartment is allowed to reach as high as 500 mL thus accommodating higher amounts of drug in solution. A comparison of the constant fed state gastric emptying transfer rate (2.7 mL/min) to the fasted emptying pattern of T1/230min reveals lower dipyridamole concentrations with the constant flow rate (2.7 mL/min) (Figure 7). This can again be explained by the higher pH in duodenal compartment which is attributable to the slower rate of gastric fluid entry into the duodenum. At 80 min, however, the gastric emptying rates into the duodenum become similar under both fasted and fed conditions, and interestingly the duodenal dipyridamole concentrations also become similar. The above results illustrate that different gastric emptying patterns and transfer rates impact drug supersaturation and precipitation kinetics. Drug concentrations in the duodenum will therefore be different, which will ultimately affect bioavailability of poorly soluble, highly permeable drugs. Large variations in dipyridamole plasma concentration profiles exist. The bioavailability of an oral dose of 100 mg of dipyridamole was found to vary from 37 to 66%.26 In another study exploring a 50 mg oral dose of dipyridamole, the bioavailability was found to be 52 ± 23%.35 These variations cannot be explained by first pass effects alone, however, and have been mainly attributed to the variable absorption of dipyridamole due to its poor solubility and dissolution.26,35 Intra- and interindividual variations in gastric emptying patterns impact dipyridamole concentrations in the duodenum and along with other factors may thus contribute to its variable absorption. Moreover, the findings of our study lead us to infer that small changes in intestinal pH can have a high impact on dipyridamole supersaturation behavior. Several in vivo studies have illustrated the impact of gastric pH on dipyridamole bioavailability. Russell et al. showed that absorption and mean AUC of dipyridamole were lower in elderly patients with achlorhydria compared to those with a normal gastric pH.36 Similar results were also seen in rats where a 1 h delay of Tmax and ∼62% reduction of AUC for dipyridamole was observed in omeprazole-treated rats compared to normal rats.37 Raised gastric pH will also raise duodenal pH since duodenal fluids have poor buffer capacity;38 thus impacting dipyridamole bioavailability.
Figure 4. Dipyridamole concentrations in the stomach (a) and duodenal compartments (b) of the SSD (mean ± SD). Comparison of the different rates of monoexponential gastric emptying, T1/215min versus T1/230min. mFaSSGFSLS in the gastric compartment and dipyridamole dose 100 mg.
Figure 5. Supersaturation ratio−time profiles of dipyridamole in the duodenal compartment of the SSD. Comparison of the different rates of monoexponential gastric emptying, T1/215min versus T1/230min. mFaSSGFSLS in the gastric compartment and dipyridamole dose 100 mg.
constant transfer rate from the stomach to the duodenum; physiological gastric emptying in the fasted state, however, follows a monoexponential emptying pattern until the basal volume (50 mL) of gastric fluid is reached. Furthermore, in the aforementioned model, fluid is not removed from duodenal compartment and therefore drug is allowed to accumulate, which acts a driving force for drug precipitation. Gu et al.6 2840
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Figure 7. Dipyridamole concentrations in the gastric (a) and duodenal (b) compartments of the SSD (mean ± SD). Comparison of the different gastric emptying patterns, monoexponential gastric emptying T1/230min, versus constant flow rate 2.7 mL/min mFaSSGFSLS in the gastric compartment and dipyridamole dose 100 mg.
Figure 8. Comparison of the effect of different surfactants on dipyridamole concentrations in the stomach (a) and duodenal compartments (b) of the SSD (mean ± SD). mFaSSGF in the gastric compartment, dipyridamole dose 100 mg, and monoexponential gastric emptying T1/215min.
3.4. Influence of Surfactants on in Vitro Supersaturation of Dipyridamole. The effect of pharmaceutical polymers on drug supersaturation/precipitation inhibition has been thoroughly explored and reviewed,39−41 while work with surfactants has been limited. Sodium lauryl sulfate (SLS) at concentrations below its CMC has been shown to act as a good precipitation inhibitor for tacrolimus formulations prepared by ultrarapid freezing.42 Poloxamers have been shown to inhibit precipitation of celecoxib at concentrations above their CMC through drug partitioning into micelles.43 Studies with nifedipine have shown precipitation inhibition with poloxamer 407 through increased aqueous solubility or hydrogen bonding.44 Here we directly compare the precipitation inhibition potency of surfactants commonly used in oral pharmaceutical dosage forms at concentrations below their CMC, while maintaining a surface tension similar to that of the fasted stomach. The surfactants explored, sodium lauryl sulfate (SLS), poloxamer-188, and polysorbate-80, were added to simulated gastric fluid (0.01 M HCl, 0.2% NaCl). The absence of micellar solubilization of dipyridamole at the concentrations of surfactants explored was confirmed. The solubility of dipyridamole in the simulated media with different surfactants was similar to that in 0.01 M HCl with 0.2% NaCl, 4.97 ± 0.26 mg/mL (P > 0.05). The surfactants explored here have all successfully stabilized supersaturated dipyridamole solutions, however to different extents (Figure 8). Dipyridamole supersaturation ratios of up to
11-fold have been observed and supersaturation has been maintained for up to 120 min. The AUC60min for the dipyridamole concentration profile in the duodenal compartment is significantly different for all the surfactants explored (P < 0.05) (Table 1). The AUC60min and AUC120min in the Table 1. Comparison of the Influence of Surfactants on Dipyridamole Concentrations and Supersaturation in the Duodenal Compartment of the SSD surfactant sodium lauryl sulfate (SLS) poloxamer-188 polysorbate-80
AUC60min (μg min/mL) (mean ± SD)
Cmax (μg/mL)
max obsd supersatn ratio
5170.5 ± 506
117.0
10.1
2603.8 ± 282 1975.4 ± 118
115.3 75.9
11.2 8.2
simulated gastric fluid with SLS (mFaSSGF) is over 2-fold that observed in the other surfactant systems despite that the predicted solubilities, as extrapolated from the pH−solubility profiles are similar (i.e., the differences seen are not attributable to pH). Similar maximum supersaturation ratios are achieved for simulated gastric fluids containing SLS and poloxamer-188; however, higher dipyridamole concentrations are maintained for a longer period of time in the duodenal compartment in the presence of SLS. In the case of poloxamer-188, dipyridamole 2841
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AUC60min profile were also found to be similar (P > 0.05). AUC60min for 50 mg solution and suspension were 4608 ± 546 and 4447 ± 99 μg min/mL, respectively. This further supports the hypothesis that crystal growth, not nucleation, is the ratelimiting step in the precipitation of dipyridamole. 3.5. Comparison of Duodenal Concentration Profiles of Different Dose Strengths of Dipyridamole. The stomach and duodenal concentration profiles of different dose strengths of dipyridamole available in the market (25, 50, and 100 mg of powder) were explored. The concentration profiles for the dipyridamole 25 mg dose are illustrated in Figure 9. At this lower dose, the maximum degree of
concentrations in the duodenal compartment decline much faster and the degree of supersaturation from 15 min onward is much lower than for the SLS system. Supersaturation is maintained for 75 and 115 min in the poloxamer-188 and SLS systems, respectively. As for the polysorbate-80 system, the maximum supersaturation ratio is 20% lower and supersaturation was maintained for a similar duration to the poloxamer-188 system. This greater precipitation inhibition observed in the case of SLS compared to the other surfactants is counterintuitive. Unlike in the simulated gastric fluids with other surfactants, dipyridamole did not immediately dissolve in mFaSSGF and therefore drug particles were transferred into the duodenal compartment. Drug precipitation/crystallization is normally separated into nucleation and crystal growth. It is therefore thought that, if some particles of the weak base escape dissolution in the stomach and end up in the duodenum, they will act as nuclei for precipitation in the small intestine, thus making precipitation more pronounced.45 In turn, if nucleation is slow, a supersaturated drug solution will be maintained for longer.46 The results of this study, however, show that the presence of nuclei does not accelerate precipitation in the case of dipyridamole. It therefore appears that the rate-limiting step in the precipitation of dipyridamole in the systems studied is crystal growth rather than nucleation. Evidence from optical microscopy supports this; we observed crystal growth over time, and the rate of growth was shown to be faster in the polysorbate-80 and poloxamer-188 systems in which drug particles were initially absent. Evidence suggests that surfactants contribute to precipitation inhibition by alteration of the bulk properties such as surface tension or saturation solubility.47−49 Both these parameters, however, have been kept constant in the three simulated gastric fluids we have explored and therefore cannot explain the differences in the supersaturation kinetics we are observing. Surfactants have also been shown to act as precipitation inhibitors by allowing drug partitioning into micelles.41 Again, this process is not plausible in our systems as all the surfactants were used at concentrations below their CMC. One of the mechanisms by which the surfactants explored can inhibit precipitation in the systems studied here is through improved solvation of dipyridamole. Hydrophobic interactions can improve solvation, which therefore increases the activation energy necessary for desolvation during nucleation and crystal growth.50 Another mechanism for precipitation inhibition is excipient adsorption onto the surface of small embryo drug particles which retards crystal growth through blocking of the active surface and steric stabilization.42 In the case of SLS an additional process would be steric stabilization through electrostatic repulsion. While the SLS tails will be orientated onto the surface of the dipyridamole crystals, the sulfate groups will be ionized, causing electrostatic repulsion and thus inhibiton of crystal growth. To probe the proposed hypothesis that crystal growth is the rate-limiting step for the precipitation of dipyridamole, we investigated the effect of dosage form (solution versus suspension) on the supersaturation/precipitation kinetics of a 50 mg dose of this drug in the SSD model. In the case where dipyridamole is in a suspension (few drug particles undissolved and transferring into duodenal compartment), the precipitation kinetics were observed to be similar to when dipyridamole is completely in solution in the gastric compartment. The drug concentration profiles in the duodenal compartment and the
Figure 9. Measured dipyridamole concentrations (solid line) and predicted solubility (dashed line) in the stomach (a) and duodenal compartment (b) of the SSD (mean ± SD). mFaSSGFSLS in the gastric compartment with a gastric emptying T1/215min. Dipyridamole dose 25 mg.
supersaturation is 3.6. It is noteworthy that a lower pH was observed in the duodenal compartment with a low dose of dipyridamole, thus giving rise to a higher equilibrium solubility of the drug for the first 60 min. This can contribute to explaining the lower degree of supersaturation compared to the high dipyridamole dose. These findings are in agreement with those by Psachoulias et al.45 where the supersaturation of dipyridamole was studied in the small intestinal luminal contents of fasted adults. The authors reported a generally lower degree of supersaturation when the low dose strength of dipyridamole was administered compared to the high dose strength. The AUC∞ calculated in the duodenal compartment for the 25 mg dipyridamole dose strength is 3350 ± 140 μg min/mL. A 2842
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linear dose−response relationship was found for the mean duodenal area under concentration−time curves (AUC∞) in the dose range of 25 mg to 100 mg (R2 = 0.886) (Figure 10).
Article
AUTHOR INFORMATION
Corresponding Author
*4600 Sunset Ave., Indianapolis, IN 46208. Tel: +1 317 940 8574. Fax: +1 317 940 3046. E-mail:
[email protected] Notes
The authors declare no competing financial interest.
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REFERENCES
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Figure 10. Correlation between dipyridamole dose and duodenal area under concentration−time profile (AUC∞).
This is in agreement with the medical information for Persantine (Boehringer Ingelheim, Germany)51 whereby a linear dose−response relationship was also observed in adults over this dose range. This proves the hypothesis that the amount of drug in solution in the duodenal compartment of the SSD correlates to drug bioavailability for drugs like dipyridamole with solubility/dissolution limited absorption undergoing supersaturation/precipitation. However, for drugs with poor permeability or where liver and intestinal first pass extraction limit bioavailability, in vitro−in vivo correlations with the SSD model alone cannot be assumed. Those drugs would also benefit from in silico physiologically based pharmacokinetic modeling such as with GastroPlus or Simcyp which allow simulation of drug absorption from different intestinal compartments and predictions of metabolism, distribution and clearance. In silico modeling would compliment the drug concentration profiles provided by the SSD model thus improving in vitro−in vivo correlations. This is the scope of our future research.
4. CONCLUSIONS The in vitro simulated stomach duodenum (SSD) model provides a reliable and discriminative screening tool for exploring the effect of different physiological variables or formulations on drug supersaturation/precipitation. The model is physiologically relevant and provides concentration−time profiles that can be used to predict the relative in vivo bioavailability of weakly basic drugs with solubility limited absorption. The SSD model also provides a good tool for assessing the influence of the dosage form, whether solid or liquid, on precipitation kinetics and for gaining insight into the rate-limiting steps of drug precipitation. Furthermore, the results suggest that variations in gastric emptying patterns can have a significant effect on duodenal drug supersaturation profiles. Surfactants commonly used in oral formulations have been shown to successfully stabilize supersaturated dipyridamole solutions to different extents and without obtaining artificial micellar solubilization of the drug. 2843
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