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Supersaturation in human gastric fluids
European Journal of Pharmaceutics and Biopharmaceutics 81 (2012) 184–189
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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
Supersaturation in human gastric fluids Jan Bevernage a, Bart Hens a, Joachim Brouwers a, Jan Tack b, Pieter Annaert a, Patrick Augustijns a,⇑ a b
Laboratory for Pharmacotechnology and Biopharmacy, KU Leuven Leuven, Belgium Department of Gastroenterology, University Hospitals Leuven, Leuven, Belgium
a r t i c l e
i n f o
Article history: Received 21 November 2011 Accepted in revised form 31 January 2012 Available online 11 February 2012 Keywords: Supersaturation Biorelevant Precipitation inhibition Solubility Excipients
a b s t r a c t Purpose: The current study reports on supersaturation, precipitation and excipient mediated precipitation inhibition of five poorly soluble drugs (loviride, glibenclamide, itraconazole, danazol, and etravirine) in human and simulated gastric fluids. Method: Upon induction of supersaturation in human gastric fluids (HGFs), simulated gastric fluid (SGF), and fasted state simulated gastric fluid (FaSSGF) using a solvent shift method, supersaturation and precipitation were assessed as a function of time. In addition, the precipitation inhibitory capacity of three polymers (EudragitÒ E PO, HPMC-E5, and PVP K25) was investigated. Results: Supersaturation in human gastric fluids was observed for all model compounds, but proved to be relatively unstable (fast precipitation), except for itraconazole. Only modest excipient-mediated stabilizing effects on supersaturation were observed using HPMC-E5 and EudragitÒ E PO whereas PVP K25 exerted no effect. In contrast to SGF, the observed precipitation behavior in FaSSGF was similar to the behavior in human gastric fluids. Conclusion: The present study demonstrates that supersaturation stability of drugs in human gastric fluids is in general inferior to supersaturation stability in intestinal fluids. As the potential for excipient mediated precipitation inhibition in gastric fluids was only limited, our data suggest that supersaturation should preferably be targeted to the intestine. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Due to high throughput drug discovery methodologies and the selection of targets requiring more lipophilic compounds, an increasing number of drug candidates suffer from limited aqueous solubility [1,2]. As dissolution is a prerequisite for intestinal absorption, oral bioavailability for these drug candidates is often solubility or dissolution limited and therefore insufficient. Faced with this challenge, formulation scientists have developed different strategies to compensate for poor solubility. These strategies are often based on circumventing the dissolution step through drug solubilized formulations (e.g., co-solvent solutions, lipid formulations) or enhancing the dissolution rate (e.g., salt formation, solid dispersion, nanoparticles) [3]. Using these types of formulations, intestinal drug concentrations are not necessarily limited by solubility as drug supersaturation (i.e., the concentration of dissolved drug exceeds the thermodynamic solubility of the drug in that specific medium) may be generated upon dilution or contact of these formulations with gastrointestinal fluids. As passive
⇑ Corresponding author. Laboratory for Pharmacotechnology and Biopharmacy, O&N 2, Herestraat 49, Box 921, KU Leuven 3000 Leuven, Belgium. Tel.: +32 16 33 03 01; fax: +32 16 33 03 05. E-mail address: [email protected] (P. Augustijns). 0939-6411/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2012.01.017
intestinal absorption is driven by intraluminal concentrations, enhanced free drug concentrations fueled by supersaturation may enable oral administration of drugs suffering from solubility limited absorption. Generation of supersaturation in vitro accompanied with enhanced absorption in vivo has been described for a variety of formulations, categorized as supersaturating drug delivery systems (SDDSs) [3]. For drug supersaturation to be beneficial, the supersaturated state has to be maintained long enough to allow absorption. Indeed, as supersaturation is thermodynamically unstable and the driving force for precipitation itself, the generated supersaturated state is transient by definition; consequently, the free drug concentration will drop as precipitation occurs. A swift decrease in free drug concentration (fast precipitation) may therefore limit the impact of supersaturation on intestinal absorption. At that point, deceleration of drug precipitation through inclusion of so-called ‘‘precipitation inhibitors’’ could be beneficial. A variety of pharmaceutical excipients have been proven to be effective for precipitation inhibition including polymers [3–9], surfactants [4,10,11], and cyclodextrines [5,12]. To gain insight in the potential clinical benefit of SDDSs for intestinal absorption, a thorough understanding of the precipitation kinetics and the added value of potential precipitation inhibitors is crucial, especially under biorelevant conditions. As drug absorption takes place in the intestine, most studies focus on the characterization of drug supersaturation and excipient-mediated
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precipitation inhibition in media representative of the intestinal environment. As such, supersaturation along with positive effects of precipitation inhibitors has been demonstrated in human and simulated intestinal fluids [13,14]. However, upon ingestion of an oral formulation, passage through the stomach is inevitable. Induction of supersaturation upon contact with gastric simulation fluids has been described for different types of formulations including self emulsifying drug delivery systems [9,15,16], silica based formulations [17], and solid dispersions [18]. Since no absorption from the stomach is expected to occur, gastric supersaturation has to be maintained until transit to the intestine in order to be beneficial for absorption. Premature precipitation before entering the duodenum may significantly reduce the subsequent intestinal drug absorption. Therefore, knowledge of the precipitation kinetics in the stomach environment is essential to predict the in vivo performance of many SDDSs. However, a systematic investigation of drug supersaturation and possible excipient-mediated precipitation inhibition in gastric fluids is still lacking. Moreover, frequently used simulated gastric fluids have never been benchmarked as relevant media for supersaturation/precipitation studies that are supposed to mimic the in vivo situation. This study aimed to fill this gap as it explored, for the first time, supersaturation for five poorly soluble drugs (itraconazole, glibenclamide, loviride, etravirine, and danazol) (Table 1) in human gastric aspirates, simulated gastric fluid (SGF), and fasted state simulated gastric fluid (FaSSGF). Additionally, the precipitation inhibitory capacity of three commonly used polymers (EudragitÒ E PO, HPMC-E5, and PVP K25) was evaluated in the respective simulated and human gastric fluids.
3. Methods 3.1. Sampling of human gastric fluids (HGFs) Gastric fluids were collected from four healthy volunteers (two female, two male, between 19 and 35 years old) in the fasted state and used for solubility measurements and supersaturation assays. The procedure for collecting HGF followed the tenets of the Declaration of Helsinki and was approved by the Committee of Medical Ethics of the University Hospitals Leuven, Belgium (ML3242). All volunteers provided written informed consent to participate in this study. The HGFs were collected every 15 min for up to 120 min after the intake of water (200 ml). We refer to a previous study for a more detailed description of the aspiration protocol [19]. One pooled sample was made by combining the aspirates from the four volunteers. The pooling was performed in order to obtain media with average characteristics. The pH of the HGF pool was measured immediately after pooling, before and after experiments (Hamilton Slimtrode) (pH 1.55); the HGF pool was stored at 30 °C until usage in solubility or supersaturation assays. 3.2. Preparation of fasted state simulated gastric fluid (FaSSGF) and simulated gastric fluid (SGF) For validation purposes, solubility and supersaturation were also evaluated in simulated gastric fluid without pepsin (SGF) (pH 1.2) and fasted state simulated gastric fluid (FaSSGF) (pH 1.6). SGF was prepared according to the USP protocol. FaSSGF, including lecithin and sodium taurocholate as surface tension lowering agents, was prepared as described by Vertzoni et al. [20].
2. Materials 3.3. Solubility measurement Loviride and itraconazole were kindly donated by Johnson & Johnson Pharmaceutical Research and Development (Beerse, Belgium); etravirine was provided by Tibotec-Virco Virology bvba (Beerse, Belgium); danazol and fenofibrate were obtained from Indis (Aartselaar, Belgium). Hydroxypropylmethylcellulose, grade E5 (HPMC-E5) (Colorcon Ltd, Kent, UK), polyvinylpyrrolidone K25 (PVP K25) (BASF, Ludwigshafen, Germany), and EudragitÒ E PO (Evonik industries, Darmstadt, Germany) were received as free samples. Sodium taurocholate (practical grade) [ICN Biomedicals, Inc (Eschwege, Germany)] and Phospholipon 90G (lecithin) [Nattermann Phospholipid Gmbh (Köln, Germany)] were used as received. Acros Organics (Geel, Belgium) supplied methanol (MeOH), dimethylsulfoxide (DMSO), sodium acetate trihydrate, and NaH2PO4H2O, while NaCl, acetonitrile, and hydrochloric acid were provided by Fisher Scientific (Leicestershire, UK). Chloroform and acetic acid were from Chemlab NV (Zedelgem, Belgium); NaOH pellets were obtained from BDH Laboratory Supplies (Poole, UK). Water was purified with a Maxima system (Elga Ltd., High Wycombe Bucks, UK). Double-lumen polyvinyl catheters [(Salem Sump Tube 14 Ch (external diameter 4.7 mm), Sherwood Medical, Petit Rechain, Belgium] were used for the aspiration of human gastric fluids.
The thermodynamic solubility of the model compounds was determined using the standard shake flask method in SGF, FaSSGF, and the fasted HGF pool, in presence or absence of pharmaceutical polymers at a concentration of 0.05% (w/w) (EudragitÒ E PO, HPMC-E5 or PVP K25). All solubility experiments were performed in triplicate; solid phase separation was achieved using filtration (0.2 lm regenerated cellulose, Macherey Nagel, Düren, Germany) for SGF and FaSSGF samples or centrifugation (15 min, 20,817 g at 37 °C) for HGF samples. When centrifugation was used, approximately 2 mg of drug compound was added to microcentrifuge tubes containing 500 ll of HGF. Samples for filtration were prepared by adding approximately 20 mg of drug to 10 ml of SGF or FaSSGF in a test tube. Prior to centrifugation or filtration, the samples were allowed to equilibrate in a prewarmed shaking incubator [37 °C at 130 rpm (Incubator shaker series 25D, New Brunswick Scientific Co., Inc., Edison, NJ)]. Since previously reported equilibrium times in biorelevant media for poorly soluble drugs showed that equilibrium was always reached within 24 h [21,22], the equilibration time was set at 24 h. Filters were saturated with 9 ml of sample. The filtrate/supernatant was diluted twice in methanol. The diluted samples of SGF and FaSSGF were used as such for
Table 1 Physicochemical properties of the five model drugs used in the present study. Compound
Ionization behavior
Molecular weight (g/mol)
# H-donors
# H-acceptors
cLogP
Loviride Itraconazole Glibenclamide Etravirine Danazol
Neutral Weakly basic (pKa 2.2/3.9) Weak acid (pKa 5.5) Weakly basic (pKa 4.5) Neutral
351 706 494 435 337
2 0 3 2 1
6 11 6 6 3
3.98 7.14 3.79 5.54 3.38
pKa values were calculated using Marvinsketch software (Chemaxon Ltd.).
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further analysis. In the case of HGF, precipitation and separation of proteins present in HGF was achieved by performing a second centrifugation (5 min, 20,817g at 37 °C) after dilution. The resulting supernatant was used for quantification. 3.4. Supersaturation assay Supersaturation was investigated using the solvent-shift method. The experimental setup consisted of a water bath at 37 °C with 40 ml dissolution vessels. The vessels contained 20 ml SGF or FaSSGF, or, due to limited availability, 2 ml HGF with or without 0.05% (w/w) predissolved polymer, and were equilibrated at 37 °C. Preliminary experiments demonstrated no significant influence of the test volume on supersaturation behavior for the five model compounds used (data not shown). Magnetic stirrer bars provided mixing of the medium (400 rpm). Stock solutions in DMSO of various drug concentrations were prepared to induce an initial degree of supersaturation (DS) equal to 20 based on the determined solubility in the respective media. After adding the DMSO stock solutions to the test medium, samples (3.5 ml SGF/FaSSGF, 200 lL HGF) were taken at 5, 15, 30, 45, and 60 min and filtered (0.2 lm regenerated cellulose, Macherey Nagel, Düren, Germany) in the case of SGF and FaSSGF samples (first 3 ml was discarded to saturate the filter material) or centrifuged (10 min, 20,817 g, 37 °C) in the case of HGF samples. The resulting filtrate/supernatant was directly diluted twice in MeOH before analysis. Diluted HGF samples were centrifuged again (5 min, 20,817g) to separate precipitated proteins; the obtained supernatant was used for analysis. All supersaturation experiments were performed in triplicate. 3.5. Analysis All drug concentrations were determined by reversed phase HPLC analysis with UV detection. An aliquot (50 lL) of the diluted supernatants, obtained as described above, was injected into a Waters HPLC system consisting of an alliance 2695 separations module and a Novapak C-18 column under radial compression (Waters, Milford, MA). UV signals were detected by a Waters UV detector (W2487). All chromatographic methods were run in the isocratic mode. A detailed description of the chromatographic methods for the different model compounds can be found in Table 2. The observed peaks were integrated using Empower Pro (Empower 2) software. Calibration curves were made in mobile phase. Samples were diluted to fit in the range of the linear calibration curve. Precision and accuracy were assessed by analyzing standard samples (n = 5) at a high and a low level. Relative standard deviations and bias below 5.0% were obtained at all concentrations and for all model drugs. 3.6. Data analysis and presentation Degree of supersaturation (DS) – time profiles, supersaturation factors, and excipient gain factors were calculated as described
before [14]. In short, DS-time profiles were constructed by dividing the concentration measured at a particular time point by the equilibrium solubility of the corresponding drug in exactly the same medium (Fig. 1). The area under the DS-time curve was calculated using Graphpad Prism software (Graphpad software Inc.) to generate the supersaturation factor (SF) by dividing the areas under the DS-time profiles up to 60 min (AUC60min) with the AUC60min for a saturated solution (DS: 1) (Eq. (1)).
SF HGF ¼
Area A þ Area B Area A
ð1Þ
Finally, to assess the extent to which excipients stabilize supersaturation, the excipient gain factor (EGF) was calculated by dividing the AUC60min of the DS-time profile in the presence of excipient by the AUC60min of the DS-time profile in the absence of excipient (Eq. (2)).
EGF ¼
Area A þ Area B þ Area C Area A þ Area B
ð2Þ
3.7. Statistical analysis To determine the significance (p < 0.05) of the excipient gain factor, one sided ANOVA tests were performed using the Dunnet’s multiple comparison test as a post test (Graphpad software Inc.). 4. Results and discussion 4.1. Solubility To allow a clear interpretation of supersaturation experiments, distinction between solubilizing and supersaturation effects is a prerequisite. Therefore, thermodynamic solubilities were determined for the five model compounds in SGF, FaSSGF, and HGF. Mean solubility values in the respective media are depicted in Fig. 2 and illustrate that the five selected model compounds offer a relatively broad low-solubility range. The solubilities of the model compounds are significantly higher in HGF than those obtained in the simulated fluids (except for itraconazole, for which the solubility in SGF was not significantly different from that obtained in HGF. This could be attributed to the lower pH of SGF compared to FaSSGF and HGF). The inaccurate prediction of the solubility in HGF using FaSSGF or SGF is in line with a previously published report, which states that accurate intragastric solubility prediction using simulated fluids is problematic [22]. 4.2. Supersaturation in simulated and human gastric fluids Based on the determined solubility values, supersaturation experiments were performed for all model compounds in SGF, FaSSGF, and HGF. To level the thermodynamic tendency for precipitation, the initial degree of supersaturation was always adjusted to 20. Supersaturation factors were calculated from the DS-time
Table 2 Parameters for the HPLC analysis of the five model compounds used in the present study.
Mobile phase Flow rate (ml/min) Retention time (min) Detection UV (nm) Quantification limit (lM) a b c
Etravirine
Itraconazole
Loviride
Danazol
Glibenclamide
20/80 Buffera/MeOHb 1.0 6.2 312 0.004
20/80 Buffera/MeOHb 1.5 6.8 265 0.02
50/50 Buffera/AcNc 1.0 7.7 366 0.2
85/15 Buffera/MeOHb 1.0 6.0 288 0.04
20/80 Buffera/MeOHb 1.0 4.5 300 0.15
25 mM sodium acetate buffer, pH 3.5. Methanol. Acetonitrile.
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Fig. 1. Degree of supersaturation-time profiles for loviride in human gastric fluids (HGF) (j) and in HGF + 0.05% polymeric precipitation inhibitor ðNÞ. Mean ± SD (n = 3), starting from an initial degree of supersaturation of 20 after solvent shift. In addition, the saturation profile (DS = 1) of loviride in HGF is indicated (d).
profiles as explained in Fig. 1. An overview of the supersaturation factors obtained for the different model compounds in the respective media can be found in Fig. 3. As was the case in intestinal fluids [13,14], supersaturation was observed in gastric fluids without inclusion of precipitation inhibitors. Supersaturation in HGF, however, appeared to be limited for 4 out of 5 model compounds (loviride, glibenclamide, etravirine and danazol), with mean supersaturation factors around 2, indicating relatively fast precipitation. For itraconazole, supersaturation in HGF was remarkably more stable (supersaturation factor around 10). As itraconazole is a weakly basic drug and therefore ionized at the pH applied in the test conditions (pH 1.2, 1.6 and 1.55 for SGF, FaSSGF and HGF respectively), one could argue that charge repulsion might aid in the stabilization of the supersaturated state. However, in view of the limited supersaturation stability observed for the also charged weak base etravirine, it seems that charge repulsion cannot be invoked as the main determinant for supersaturation stability of weakly basic compounds. When comparing precipitation behavior in human versus simulated fluids, it is clear that FaSSGF provides a good surrogate medium to predict the supersaturation behavior in HGF. The use of SGF in supersaturation experiments appears to be cumbersome as it significantly overestimated the supersaturation factor for 3 out of 5 model compounds (glibenclamide, itraconazole, and
Fig. 2. Solubility (mean ± SD, n = 3) of the different model compounds in fasted state human gastric fluid (HGF) (gray bars), fasted state simulated gastric fluid (FaSSGF) (open bars), and simulated gastric fluid (SGF) (black bars). Indicates a significant (p < 0.05) difference in the solubility obtained in SGF/FaSSGF compared with HGF.
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Fig. 3. Supersaturation factors for the five model compounds in fasted state human gastric fluids (HGFs) (gray bars), fasted state simulated gastric fluid (FaSSGF) (open bars), and simulated gastric fluid (SGF) (black bars) in the absence of any pharmaceutical excipient. Mean ± SD (n = 3); the dotted line (- - - -) represents the saturated situation (supersaturation factor = 1).
danazol). These data demonstrate the added value of FaSSGF compared with SGF for precipitation prediction in gastric fluids. 4.3. Excipient mediated precipitation inhibition in human and simulated gastric fluids As significant precipitation was observed, three pharmaceutical polymers, EudragitÒ E PO, HPMC-E5, and PVP K25, were selected to test their precipitation inhibitory capacity in FaSSGF and HGF. The selection of polymers was motivated by their presence as precipitation inhibitors in many previous supersaturation studies [5,23]. SGF as simulation medium was not included for further precipitation inhibition studies in view of the aforementioned underestimation of precipitation. As a clear judgment on the precipitation inhibitory capacity of excipients requires assessment of possible solubilizing effects, thermodynamic solubilities were determined in simulated and human gastric fluids including 0.05% (w/w) excipient. At the applied concentration, only EudragitÒ E PO had a significant influence on the solubility, inducing an up to 178-fold increase in solubility at the applied concentration depending on the model compound (Fig. 4). Taking into account the solubilizing effect, supersaturation experiments were conducted as before in FaSSGF and HGF including the respective polymers. The excipient
Fig. 4. Mean solubility (mean ± SD, n = 3) of the different model compounds in human gastric fluids (HGFs) in the absence (open bars) and the presence of 0.05% EudragitÒ E PO (gray bars).
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any tested model compound in FaSSGF or HGF. EudragitÒ E PO significantly reduced precipitation of loviride in HGF and of loviride and danazol in FaSSGF. HPMC-E5 provided only significant precipitation inhibition in FaSSGF for loviride and itraconazole. Overall, excipient mediated precipitation inhibition proved compound and excipient dependent and was rather limited, resulting in remarkably low mean excipient gain factors, not exceeding 1.7 in HGF. Previous precipitation inhibition studies performed in intestinal human aspirates using identical drug/polymer combinations confirmed the inability of PVP K25 to inhibit precipitation. The performance of HPMC-E5 in human intestinal aspirates was similar (loviride, etravirine) or better (danazol) than that obtained in HGF [14]. EudragitÒ E PO has previously been identified as effective precipitation inhibitor for danazol [23]; however, this could not be confirmed in the current study despite the use of similar polymer concentrations. An explanation for this discrepancy might be the fact that the considerable capacity of EudragitÒ E PO to solubilize danazol at the applied concentration (Fig. 4) was not taken into account in the previous study. To predict excipients effects, FaSSGF seemed to slightly overestimate the precipitation inhibitory capacity of some excipients. However, in view of the small excipient gain factors, it is difficult to judge the true predictive power of FaSSGF. 4.4. Gastric versus intestinal supersaturation The supersaturation behavior and excipient mediated precipitation inhibition (HPMC-E5 and PVP K25) of danazol, etravirine, and loviride have previously been investigated in human intestinal fluids [14]. Apart from the use of intestinal fluids instead of gastric fluids, the experimental design was identical to the one applied in the current study (including the same initial tendency for precipitation, i.e., DS = 20), allowing comparison of gastric versus intestinal supersaturation behavior. The supersaturation factors for danazol, etravirine, and loviride obtained in fasted state HGF and human intestinal fluid (HIF) are depicted in Fig. 6. It is clear that supersaturation appeared much more stable in intestinal versus gastric fluids (minimum 2.3-fold difference in supersaturation factor). Previously, Van Speybroeck et al. also found that co-solvent induced supersaturation involving indomethacin and glibenclamide was more stable in simulated intestinal fluid than in simulated gastric fluid [24]. If a drug is released to generate supersaturated concentrations in the stomach, premature precipitation prior to transfer to the small intestine is therefore very likely to occur, impairing the potential positive effect of gastric supersaturation on intestinal absorption. Taking into account the limited supersaturation stability and the previously discussed minimal precipitation inhibitory capacity of excipients in HGF, one may
Fig. 5. Excipient gain factors of HPMC-E5 (A), EudragitÒ E PO (B), and PVP (C) for the model compounds in HGF (gray bars) and FaSSGF (open bars). Mean ± SD (n = 3). Indicates a significant (p < 0.05) increase in the AUC60min of the DS-time profile compared with the no excipient condition.
gain factor (EGF), i.e., the excipient-induced fold increase in AUC60min of the DS-time profile. Mean EGFs of the respective polymers for the model compounds in FaSSGF and HGF are depicted in Fig. 5. PVP K25 was unable to significantly inhibit precipitation of
Fig. 6. Supersaturation factors for loviride, etravirine, and danazol in fasted state human gastric fluid (HGF) (gray bars) versus human intestinal fluid (HIF) (open bars) in the absence of any pharmaceutical excipient. Mean ± SD (n = 3); the dotted line (- - - -) represents the saturated situation (supersaturation factor = 1).
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conclude that the targeted generation of supersaturation in the intestinal environment is more likely to result in enhanced absorption. The possible advantage of targeted intestinal supersaturation has been reported before. Kondo et al. found absorption of the anticancer drug HO-221 to be doubled when a pH-dependent carrier (hypromellose phthalate, HP55) was used in its supersaturable formulation [25]. Miller et al. demonstrated that supersaturated concentrations of itraconazole are preferably targeted to the small intestine to attain adequate absorption [8]. Significant improvements in the in vivo oral absorption of itraconazole (better than results reported for SporanoxÒ in the literature) were achieved, using EudragitÒ L100-55 as a pH-dependent carrier matrix including 20% CarbopolÒ 974P as a stabilizer [26].
5. Conclusion Despite the fact that supersaturation research usually focuses on the intestinal environment, dilution/disintegration of supersaturating drug delivery systems may already induce supersaturation in the stomach. In order to be beneficial for absorption, gastric supersaturation should be maintained until transfer to the intestine. Therefore, the current study assessed supersaturation and excipient-mediated precipitation inhibition in human gastric aspirates. Supersaturation was observed in HGF for model compounds with different physicochemical properties. As previously seen in human intestinal aspirates [13], the supersaturation stability appeared compound and medium dependent. While commonly used simulated gastric media (SGF and FaSSGF) were unsuccessful in accurate prediction of the solubility in HGF, FaSSGF was able to predict the precipitation behavior in HGF. In contrast, SGF overestimated supersaturation stability in 3 out of 5 cases suggesting that the use of FaSSGF is to be preferred for the biorelevant evaluation of drug precipitation. In general, precipitation occurred relatively fast in HGF indicating limited supersaturation stability. Comparison of the supersaturation stability in HGF with FaHIF illustrated the inferior supersaturation stability in HGF. Moreover, achieving substantial excipient-mediated precipitation inhibition in HGF or FaSSGF proved to be difficult with only modest effects of EudragitÒ E PO and HPMC-E5 and no effect of PVP K25. Taking into account the limited supersaturation stability in HGF and the modest capacity of excipients to slow down gastric precipitation, one could argue that supersaturation should be targeted to the small intestine. Acknowledgements This research was funded by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), as well as from grants from the Research Foundation Flanders (FWO) and from the ‘‘Onderzoeksfonds’’ of the KU Leuven, Belgium. We also wish to thank Rita Vos (Gastroenterology, University Hospitals Leuven, Belgium) for her assistance during the in vivo studies. Joachim Brouwers is a postdoctoral fellow of the FWO. References [1] C.A. Lipinski, Drug-like properties and the causes of poor solubility and poor permeability, J. Pharmacol. Toxicol. Methods 44 (2000) 235–249. [2] R.D. Connors, E.J. Elder, Using a portfolio of particle growth technologies to enable delivery of drugs with poor water solubility, Drug Del. Technol. 4 (2004) 78–83.
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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
Supersaturation in human gastric fluids Jan Bevernage a, Bart Hens a, Joachim Brouwers a, Jan Tack b, Pieter Annaert a, Patrick Augustijns a,⇑ a b
Laboratory for Pharmacotechnology and Biopharmacy, KU Leuven Leuven, Belgium Department of Gastroenterology, University Hospitals Leuven, Leuven, Belgium
a r t i c l e
i n f o
Article history: Received 21 November 2011 Accepted in revised form 31 January 2012 Available online 11 February 2012 Keywords: Supersaturation Biorelevant Precipitation inhibition Solubility Excipients
a b s t r a c t Purpose: The current study reports on supersaturation, precipitation and excipient mediated precipitation inhibition of five poorly soluble drugs (loviride, glibenclamide, itraconazole, danazol, and etravirine) in human and simulated gastric fluids. Method: Upon induction of supersaturation in human gastric fluids (HGFs), simulated gastric fluid (SGF), and fasted state simulated gastric fluid (FaSSGF) using a solvent shift method, supersaturation and precipitation were assessed as a function of time. In addition, the precipitation inhibitory capacity of three polymers (EudragitÒ E PO, HPMC-E5, and PVP K25) was investigated. Results: Supersaturation in human gastric fluids was observed for all model compounds, but proved to be relatively unstable (fast precipitation), except for itraconazole. Only modest excipient-mediated stabilizing effects on supersaturation were observed using HPMC-E5 and EudragitÒ E PO whereas PVP K25 exerted no effect. In contrast to SGF, the observed precipitation behavior in FaSSGF was similar to the behavior in human gastric fluids. Conclusion: The present study demonstrates that supersaturation stability of drugs in human gastric fluids is in general inferior to supersaturation stability in intestinal fluids. As the potential for excipient mediated precipitation inhibition in gastric fluids was only limited, our data suggest that supersaturation should preferably be targeted to the intestine. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Due to high throughput drug discovery methodologies and the selection of targets requiring more lipophilic compounds, an increasing number of drug candidates suffer from limited aqueous solubility [1,2]. As dissolution is a prerequisite for intestinal absorption, oral bioavailability for these drug candidates is often solubility or dissolution limited and therefore insufficient. Faced with this challenge, formulation scientists have developed different strategies to compensate for poor solubility. These strategies are often based on circumventing the dissolution step through drug solubilized formulations (e.g., co-solvent solutions, lipid formulations) or enhancing the dissolution rate (e.g., salt formation, solid dispersion, nanoparticles) [3]. Using these types of formulations, intestinal drug concentrations are not necessarily limited by solubility as drug supersaturation (i.e., the concentration of dissolved drug exceeds the thermodynamic solubility of the drug in that specific medium) may be generated upon dilution or contact of these formulations with gastrointestinal fluids. As passive
⇑ Corresponding author. Laboratory for Pharmacotechnology and Biopharmacy, O&N 2, Herestraat 49, Box 921, KU Leuven 3000 Leuven, Belgium. Tel.: +32 16 33 03 01; fax: +32 16 33 03 05. E-mail address: [email protected] (P. Augustijns). 0939-6411/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2012.01.017
intestinal absorption is driven by intraluminal concentrations, enhanced free drug concentrations fueled by supersaturation may enable oral administration of drugs suffering from solubility limited absorption. Generation of supersaturation in vitro accompanied with enhanced absorption in vivo has been described for a variety of formulations, categorized as supersaturating drug delivery systems (SDDSs) [3]. For drug supersaturation to be beneficial, the supersaturated state has to be maintained long enough to allow absorption. Indeed, as supersaturation is thermodynamically unstable and the driving force for precipitation itself, the generated supersaturated state is transient by definition; consequently, the free drug concentration will drop as precipitation occurs. A swift decrease in free drug concentration (fast precipitation) may therefore limit the impact of supersaturation on intestinal absorption. At that point, deceleration of drug precipitation through inclusion of so-called ‘‘precipitation inhibitors’’ could be beneficial. A variety of pharmaceutical excipients have been proven to be effective for precipitation inhibition including polymers [3–9], surfactants [4,10,11], and cyclodextrines [5,12]. To gain insight in the potential clinical benefit of SDDSs for intestinal absorption, a thorough understanding of the precipitation kinetics and the added value of potential precipitation inhibitors is crucial, especially under biorelevant conditions. As drug absorption takes place in the intestine, most studies focus on the characterization of drug supersaturation and excipient-mediated
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precipitation inhibition in media representative of the intestinal environment. As such, supersaturation along with positive effects of precipitation inhibitors has been demonstrated in human and simulated intestinal fluids [13,14]. However, upon ingestion of an oral formulation, passage through the stomach is inevitable. Induction of supersaturation upon contact with gastric simulation fluids has been described for different types of formulations including self emulsifying drug delivery systems [9,15,16], silica based formulations [17], and solid dispersions [18]. Since no absorption from the stomach is expected to occur, gastric supersaturation has to be maintained until transit to the intestine in order to be beneficial for absorption. Premature precipitation before entering the duodenum may significantly reduce the subsequent intestinal drug absorption. Therefore, knowledge of the precipitation kinetics in the stomach environment is essential to predict the in vivo performance of many SDDSs. However, a systematic investigation of drug supersaturation and possible excipient-mediated precipitation inhibition in gastric fluids is still lacking. Moreover, frequently used simulated gastric fluids have never been benchmarked as relevant media for supersaturation/precipitation studies that are supposed to mimic the in vivo situation. This study aimed to fill this gap as it explored, for the first time, supersaturation for five poorly soluble drugs (itraconazole, glibenclamide, loviride, etravirine, and danazol) (Table 1) in human gastric aspirates, simulated gastric fluid (SGF), and fasted state simulated gastric fluid (FaSSGF). Additionally, the precipitation inhibitory capacity of three commonly used polymers (EudragitÒ E PO, HPMC-E5, and PVP K25) was evaluated in the respective simulated and human gastric fluids.
3. Methods 3.1. Sampling of human gastric fluids (HGFs) Gastric fluids were collected from four healthy volunteers (two female, two male, between 19 and 35 years old) in the fasted state and used for solubility measurements and supersaturation assays. The procedure for collecting HGF followed the tenets of the Declaration of Helsinki and was approved by the Committee of Medical Ethics of the University Hospitals Leuven, Belgium (ML3242). All volunteers provided written informed consent to participate in this study. The HGFs were collected every 15 min for up to 120 min after the intake of water (200 ml). We refer to a previous study for a more detailed description of the aspiration protocol [19]. One pooled sample was made by combining the aspirates from the four volunteers. The pooling was performed in order to obtain media with average characteristics. The pH of the HGF pool was measured immediately after pooling, before and after experiments (Hamilton Slimtrode) (pH 1.55); the HGF pool was stored at 30 °C until usage in solubility or supersaturation assays. 3.2. Preparation of fasted state simulated gastric fluid (FaSSGF) and simulated gastric fluid (SGF) For validation purposes, solubility and supersaturation were also evaluated in simulated gastric fluid without pepsin (SGF) (pH 1.2) and fasted state simulated gastric fluid (FaSSGF) (pH 1.6). SGF was prepared according to the USP protocol. FaSSGF, including lecithin and sodium taurocholate as surface tension lowering agents, was prepared as described by Vertzoni et al. [20].
2. Materials 3.3. Solubility measurement Loviride and itraconazole were kindly donated by Johnson & Johnson Pharmaceutical Research and Development (Beerse, Belgium); etravirine was provided by Tibotec-Virco Virology bvba (Beerse, Belgium); danazol and fenofibrate were obtained from Indis (Aartselaar, Belgium). Hydroxypropylmethylcellulose, grade E5 (HPMC-E5) (Colorcon Ltd, Kent, UK), polyvinylpyrrolidone K25 (PVP K25) (BASF, Ludwigshafen, Germany), and EudragitÒ E PO (Evonik industries, Darmstadt, Germany) were received as free samples. Sodium taurocholate (practical grade) [ICN Biomedicals, Inc (Eschwege, Germany)] and Phospholipon 90G (lecithin) [Nattermann Phospholipid Gmbh (Köln, Germany)] were used as received. Acros Organics (Geel, Belgium) supplied methanol (MeOH), dimethylsulfoxide (DMSO), sodium acetate trihydrate, and NaH2PO4H2O, while NaCl, acetonitrile, and hydrochloric acid were provided by Fisher Scientific (Leicestershire, UK). Chloroform and acetic acid were from Chemlab NV (Zedelgem, Belgium); NaOH pellets were obtained from BDH Laboratory Supplies (Poole, UK). Water was purified with a Maxima system (Elga Ltd., High Wycombe Bucks, UK). Double-lumen polyvinyl catheters [(Salem Sump Tube 14 Ch (external diameter 4.7 mm), Sherwood Medical, Petit Rechain, Belgium] were used for the aspiration of human gastric fluids.
The thermodynamic solubility of the model compounds was determined using the standard shake flask method in SGF, FaSSGF, and the fasted HGF pool, in presence or absence of pharmaceutical polymers at a concentration of 0.05% (w/w) (EudragitÒ E PO, HPMC-E5 or PVP K25). All solubility experiments were performed in triplicate; solid phase separation was achieved using filtration (0.2 lm regenerated cellulose, Macherey Nagel, Düren, Germany) for SGF and FaSSGF samples or centrifugation (15 min, 20,817 g at 37 °C) for HGF samples. When centrifugation was used, approximately 2 mg of drug compound was added to microcentrifuge tubes containing 500 ll of HGF. Samples for filtration were prepared by adding approximately 20 mg of drug to 10 ml of SGF or FaSSGF in a test tube. Prior to centrifugation or filtration, the samples were allowed to equilibrate in a prewarmed shaking incubator [37 °C at 130 rpm (Incubator shaker series 25D, New Brunswick Scientific Co., Inc., Edison, NJ)]. Since previously reported equilibrium times in biorelevant media for poorly soluble drugs showed that equilibrium was always reached within 24 h [21,22], the equilibration time was set at 24 h. Filters were saturated with 9 ml of sample. The filtrate/supernatant was diluted twice in methanol. The diluted samples of SGF and FaSSGF were used as such for
Table 1 Physicochemical properties of the five model drugs used in the present study. Compound
Ionization behavior
Molecular weight (g/mol)
# H-donors
# H-acceptors
cLogP
Loviride Itraconazole Glibenclamide Etravirine Danazol
Neutral Weakly basic (pKa 2.2/3.9) Weak acid (pKa 5.5) Weakly basic (pKa 4.5) Neutral
351 706 494 435 337
2 0 3 2 1
6 11 6 6 3
3.98 7.14 3.79 5.54 3.38
pKa values were calculated using Marvinsketch software (Chemaxon Ltd.).
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further analysis. In the case of HGF, precipitation and separation of proteins present in HGF was achieved by performing a second centrifugation (5 min, 20,817g at 37 °C) after dilution. The resulting supernatant was used for quantification. 3.4. Supersaturation assay Supersaturation was investigated using the solvent-shift method. The experimental setup consisted of a water bath at 37 °C with 40 ml dissolution vessels. The vessels contained 20 ml SGF or FaSSGF, or, due to limited availability, 2 ml HGF with or without 0.05% (w/w) predissolved polymer, and were equilibrated at 37 °C. Preliminary experiments demonstrated no significant influence of the test volume on supersaturation behavior for the five model compounds used (data not shown). Magnetic stirrer bars provided mixing of the medium (400 rpm). Stock solutions in DMSO of various drug concentrations were prepared to induce an initial degree of supersaturation (DS) equal to 20 based on the determined solubility in the respective media. After adding the DMSO stock solutions to the test medium, samples (3.5 ml SGF/FaSSGF, 200 lL HGF) were taken at 5, 15, 30, 45, and 60 min and filtered (0.2 lm regenerated cellulose, Macherey Nagel, Düren, Germany) in the case of SGF and FaSSGF samples (first 3 ml was discarded to saturate the filter material) or centrifuged (10 min, 20,817 g, 37 °C) in the case of HGF samples. The resulting filtrate/supernatant was directly diluted twice in MeOH before analysis. Diluted HGF samples were centrifuged again (5 min, 20,817g) to separate precipitated proteins; the obtained supernatant was used for analysis. All supersaturation experiments were performed in triplicate. 3.5. Analysis All drug concentrations were determined by reversed phase HPLC analysis with UV detection. An aliquot (50 lL) of the diluted supernatants, obtained as described above, was injected into a Waters HPLC system consisting of an alliance 2695 separations module and a Novapak C-18 column under radial compression (Waters, Milford, MA). UV signals were detected by a Waters UV detector (W2487). All chromatographic methods were run in the isocratic mode. A detailed description of the chromatographic methods for the different model compounds can be found in Table 2. The observed peaks were integrated using Empower Pro (Empower 2) software. Calibration curves were made in mobile phase. Samples were diluted to fit in the range of the linear calibration curve. Precision and accuracy were assessed by analyzing standard samples (n = 5) at a high and a low level. Relative standard deviations and bias below 5.0% were obtained at all concentrations and for all model drugs. 3.6. Data analysis and presentation Degree of supersaturation (DS) – time profiles, supersaturation factors, and excipient gain factors were calculated as described
before [14]. In short, DS-time profiles were constructed by dividing the concentration measured at a particular time point by the equilibrium solubility of the corresponding drug in exactly the same medium (Fig. 1). The area under the DS-time curve was calculated using Graphpad Prism software (Graphpad software Inc.) to generate the supersaturation factor (SF) by dividing the areas under the DS-time profiles up to 60 min (AUC60min) with the AUC60min for a saturated solution (DS: 1) (Eq. (1)).
SF HGF ¼
Area A þ Area B Area A
ð1Þ
Finally, to assess the extent to which excipients stabilize supersaturation, the excipient gain factor (EGF) was calculated by dividing the AUC60min of the DS-time profile in the presence of excipient by the AUC60min of the DS-time profile in the absence of excipient (Eq. (2)).
EGF ¼
Area A þ Area B þ Area C Area A þ Area B
ð2Þ
3.7. Statistical analysis To determine the significance (p < 0.05) of the excipient gain factor, one sided ANOVA tests were performed using the Dunnet’s multiple comparison test as a post test (Graphpad software Inc.). 4. Results and discussion 4.1. Solubility To allow a clear interpretation of supersaturation experiments, distinction between solubilizing and supersaturation effects is a prerequisite. Therefore, thermodynamic solubilities were determined for the five model compounds in SGF, FaSSGF, and HGF. Mean solubility values in the respective media are depicted in Fig. 2 and illustrate that the five selected model compounds offer a relatively broad low-solubility range. The solubilities of the model compounds are significantly higher in HGF than those obtained in the simulated fluids (except for itraconazole, for which the solubility in SGF was not significantly different from that obtained in HGF. This could be attributed to the lower pH of SGF compared to FaSSGF and HGF). The inaccurate prediction of the solubility in HGF using FaSSGF or SGF is in line with a previously published report, which states that accurate intragastric solubility prediction using simulated fluids is problematic [22]. 4.2. Supersaturation in simulated and human gastric fluids Based on the determined solubility values, supersaturation experiments were performed for all model compounds in SGF, FaSSGF, and HGF. To level the thermodynamic tendency for precipitation, the initial degree of supersaturation was always adjusted to 20. Supersaturation factors were calculated from the DS-time
Table 2 Parameters for the HPLC analysis of the five model compounds used in the present study.
Mobile phase Flow rate (ml/min) Retention time (min) Detection UV (nm) Quantification limit (lM) a b c
Etravirine
Itraconazole
Loviride
Danazol
Glibenclamide
20/80 Buffera/MeOHb 1.0 6.2 312 0.004
20/80 Buffera/MeOHb 1.5 6.8 265 0.02
50/50 Buffera/AcNc 1.0 7.7 366 0.2
85/15 Buffera/MeOHb 1.0 6.0 288 0.04
20/80 Buffera/MeOHb 1.0 4.5 300 0.15
25 mM sodium acetate buffer, pH 3.5. Methanol. Acetonitrile.
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Fig. 1. Degree of supersaturation-time profiles for loviride in human gastric fluids (HGF) (j) and in HGF + 0.05% polymeric precipitation inhibitor ðNÞ. Mean ± SD (n = 3), starting from an initial degree of supersaturation of 20 after solvent shift. In addition, the saturation profile (DS = 1) of loviride in HGF is indicated (d).
profiles as explained in Fig. 1. An overview of the supersaturation factors obtained for the different model compounds in the respective media can be found in Fig. 3. As was the case in intestinal fluids [13,14], supersaturation was observed in gastric fluids without inclusion of precipitation inhibitors. Supersaturation in HGF, however, appeared to be limited for 4 out of 5 model compounds (loviride, glibenclamide, etravirine and danazol), with mean supersaturation factors around 2, indicating relatively fast precipitation. For itraconazole, supersaturation in HGF was remarkably more stable (supersaturation factor around 10). As itraconazole is a weakly basic drug and therefore ionized at the pH applied in the test conditions (pH 1.2, 1.6 and 1.55 for SGF, FaSSGF and HGF respectively), one could argue that charge repulsion might aid in the stabilization of the supersaturated state. However, in view of the limited supersaturation stability observed for the also charged weak base etravirine, it seems that charge repulsion cannot be invoked as the main determinant for supersaturation stability of weakly basic compounds. When comparing precipitation behavior in human versus simulated fluids, it is clear that FaSSGF provides a good surrogate medium to predict the supersaturation behavior in HGF. The use of SGF in supersaturation experiments appears to be cumbersome as it significantly overestimated the supersaturation factor for 3 out of 5 model compounds (glibenclamide, itraconazole, and
Fig. 2. Solubility (mean ± SD, n = 3) of the different model compounds in fasted state human gastric fluid (HGF) (gray bars), fasted state simulated gastric fluid (FaSSGF) (open bars), and simulated gastric fluid (SGF) (black bars). Indicates a significant (p < 0.05) difference in the solubility obtained in SGF/FaSSGF compared with HGF.
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Fig. 3. Supersaturation factors for the five model compounds in fasted state human gastric fluids (HGFs) (gray bars), fasted state simulated gastric fluid (FaSSGF) (open bars), and simulated gastric fluid (SGF) (black bars) in the absence of any pharmaceutical excipient. Mean ± SD (n = 3); the dotted line (- - - -) represents the saturated situation (supersaturation factor = 1).
danazol). These data demonstrate the added value of FaSSGF compared with SGF for precipitation prediction in gastric fluids. 4.3. Excipient mediated precipitation inhibition in human and simulated gastric fluids As significant precipitation was observed, three pharmaceutical polymers, EudragitÒ E PO, HPMC-E5, and PVP K25, were selected to test their precipitation inhibitory capacity in FaSSGF and HGF. The selection of polymers was motivated by their presence as precipitation inhibitors in many previous supersaturation studies [5,23]. SGF as simulation medium was not included for further precipitation inhibition studies in view of the aforementioned underestimation of precipitation. As a clear judgment on the precipitation inhibitory capacity of excipients requires assessment of possible solubilizing effects, thermodynamic solubilities were determined in simulated and human gastric fluids including 0.05% (w/w) excipient. At the applied concentration, only EudragitÒ E PO had a significant influence on the solubility, inducing an up to 178-fold increase in solubility at the applied concentration depending on the model compound (Fig. 4). Taking into account the solubilizing effect, supersaturation experiments were conducted as before in FaSSGF and HGF including the respective polymers. The excipient
Fig. 4. Mean solubility (mean ± SD, n = 3) of the different model compounds in human gastric fluids (HGFs) in the absence (open bars) and the presence of 0.05% EudragitÒ E PO (gray bars).
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any tested model compound in FaSSGF or HGF. EudragitÒ E PO significantly reduced precipitation of loviride in HGF and of loviride and danazol in FaSSGF. HPMC-E5 provided only significant precipitation inhibition in FaSSGF for loviride and itraconazole. Overall, excipient mediated precipitation inhibition proved compound and excipient dependent and was rather limited, resulting in remarkably low mean excipient gain factors, not exceeding 1.7 in HGF. Previous precipitation inhibition studies performed in intestinal human aspirates using identical drug/polymer combinations confirmed the inability of PVP K25 to inhibit precipitation. The performance of HPMC-E5 in human intestinal aspirates was similar (loviride, etravirine) or better (danazol) than that obtained in HGF [14]. EudragitÒ E PO has previously been identified as effective precipitation inhibitor for danazol [23]; however, this could not be confirmed in the current study despite the use of similar polymer concentrations. An explanation for this discrepancy might be the fact that the considerable capacity of EudragitÒ E PO to solubilize danazol at the applied concentration (Fig. 4) was not taken into account in the previous study. To predict excipients effects, FaSSGF seemed to slightly overestimate the precipitation inhibitory capacity of some excipients. However, in view of the small excipient gain factors, it is difficult to judge the true predictive power of FaSSGF. 4.4. Gastric versus intestinal supersaturation The supersaturation behavior and excipient mediated precipitation inhibition (HPMC-E5 and PVP K25) of danazol, etravirine, and loviride have previously been investigated in human intestinal fluids [14]. Apart from the use of intestinal fluids instead of gastric fluids, the experimental design was identical to the one applied in the current study (including the same initial tendency for precipitation, i.e., DS = 20), allowing comparison of gastric versus intestinal supersaturation behavior. The supersaturation factors for danazol, etravirine, and loviride obtained in fasted state HGF and human intestinal fluid (HIF) are depicted in Fig. 6. It is clear that supersaturation appeared much more stable in intestinal versus gastric fluids (minimum 2.3-fold difference in supersaturation factor). Previously, Van Speybroeck et al. also found that co-solvent induced supersaturation involving indomethacin and glibenclamide was more stable in simulated intestinal fluid than in simulated gastric fluid [24]. If a drug is released to generate supersaturated concentrations in the stomach, premature precipitation prior to transfer to the small intestine is therefore very likely to occur, impairing the potential positive effect of gastric supersaturation on intestinal absorption. Taking into account the limited supersaturation stability and the previously discussed minimal precipitation inhibitory capacity of excipients in HGF, one may
Fig. 5. Excipient gain factors of HPMC-E5 (A), EudragitÒ E PO (B), and PVP (C) for the model compounds in HGF (gray bars) and FaSSGF (open bars). Mean ± SD (n = 3). Indicates a significant (p < 0.05) increase in the AUC60min of the DS-time profile compared with the no excipient condition.
gain factor (EGF), i.e., the excipient-induced fold increase in AUC60min of the DS-time profile. Mean EGFs of the respective polymers for the model compounds in FaSSGF and HGF are depicted in Fig. 5. PVP K25 was unable to significantly inhibit precipitation of
Fig. 6. Supersaturation factors for loviride, etravirine, and danazol in fasted state human gastric fluid (HGF) (gray bars) versus human intestinal fluid (HIF) (open bars) in the absence of any pharmaceutical excipient. Mean ± SD (n = 3); the dotted line (- - - -) represents the saturated situation (supersaturation factor = 1).
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conclude that the targeted generation of supersaturation in the intestinal environment is more likely to result in enhanced absorption. The possible advantage of targeted intestinal supersaturation has been reported before. Kondo et al. found absorption of the anticancer drug HO-221 to be doubled when a pH-dependent carrier (hypromellose phthalate, HP55) was used in its supersaturable formulation [25]. Miller et al. demonstrated that supersaturated concentrations of itraconazole are preferably targeted to the small intestine to attain adequate absorption [8]. Significant improvements in the in vivo oral absorption of itraconazole (better than results reported for SporanoxÒ in the literature) were achieved, using EudragitÒ L100-55 as a pH-dependent carrier matrix including 20% CarbopolÒ 974P as a stabilizer [26].
5. Conclusion Despite the fact that supersaturation research usually focuses on the intestinal environment, dilution/disintegration of supersaturating drug delivery systems may already induce supersaturation in the stomach. In order to be beneficial for absorption, gastric supersaturation should be maintained until transfer to the intestine. Therefore, the current study assessed supersaturation and excipient-mediated precipitation inhibition in human gastric aspirates. Supersaturation was observed in HGF for model compounds with different physicochemical properties. As previously seen in human intestinal aspirates [13], the supersaturation stability appeared compound and medium dependent. While commonly used simulated gastric media (SGF and FaSSGF) were unsuccessful in accurate prediction of the solubility in HGF, FaSSGF was able to predict the precipitation behavior in HGF. In contrast, SGF overestimated supersaturation stability in 3 out of 5 cases suggesting that the use of FaSSGF is to be preferred for the biorelevant evaluation of drug precipitation. In general, precipitation occurred relatively fast in HGF indicating limited supersaturation stability. Comparison of the supersaturation stability in HGF with FaHIF illustrated the inferior supersaturation stability in HGF. Moreover, achieving substantial excipient-mediated precipitation inhibition in HGF or FaSSGF proved to be difficult with only modest effects of EudragitÒ E PO and HPMC-E5 and no effect of PVP K25. Taking into account the limited supersaturation stability in HGF and the modest capacity of excipients to slow down gastric precipitation, one could argue that supersaturation should be targeted to the small intestine. Acknowledgements This research was funded by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), as well as from grants from the Research Foundation Flanders (FWO) and from the ‘‘Onderzoeksfonds’’ of the KU Leuven, Belgium. We also wish to thank Rita Vos (Gastroenterology, University Hospitals Leuven, Belgium) for her assistance during the in vivo studies. Joachim Brouwers is a postdoctoral fellow of the FWO. References [1] C.A. Lipinski, Drug-like properties and the causes of poor solubility and poor permeability, J. Pharmacol. Toxicol. Methods 44 (2000) 235–249. [2] R.D. Connors, E.J. Elder, Using a portfolio of particle growth technologies to enable delivery of drugs with poor water solubility, Drug Del. Technol. 4 (2004) 78–83.
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