Lipids in the stomach – Implications for the evaluation of food effects on oral drug absorption
Mirko Koziolek1,2, Frédéric Carrière3, Christopher J. H. Porter2,4
* Corresponding author:
Dr. Mirko Koziolek
1 Center of Drug Absorption and Transport, Department of Biopharmacy and Pharmaceutical Technology, University of Greifswald, Germany
Felix-Hausdorff-Str. 3, D-17487 Greifswald, Germany [email protected]
2 Drug Delivery Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Victoria, 3052, Australia
3 Aix-Marseille Université, CNRS, UMR 7282 Enzymologie Interfaciale et de Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
4 ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Victoria, 3052, Australia
1 Contents Contents ...... 2
Abstract ...... 3
Key words ...... 3
Abbreviations ...... 4
1 Introduction ...... 5
2 Lipid processing in the stomach...... 6
2.1 Human Gastric Lipase ...... 7
2.2 Parameters affecting gastric lipolysis...... 12
2.2.1 Emulsification ...... 12
2.2.2 Luminal pH value...... 14
2.2.3 Gastric motility ...... 15
2.2.4 Presence of amphiphiles ...... 17
3 The impact of lipids on gastric function ...... 18
3.1 Effects on gastric emptying ...... 19
3.2 Effects on gastric motility ...... 22
3.3 Effects on gastric luminal conditions ...... 22
3.4 Downstream effects on intestinal conditions ...... 23
4 Lipids in oral drug delivery ...... 24
5 In vitro techniques to assess lipid digestion and its effects on drug release from oral dosage forms ...... 26
5.1 In vitro test methods ...... 26
5.2 Biorelevant media ...... 29
5.3 Lipase selection ...... 33
5.4 In vitro studies investigating the effect of lipids and lipid digestion on drug solubility and drug release from solid oral dosage forms in the stomach...... 36
6 Conclusion ...... 40
References ...... 41
2 Abstract Food effects on oral drug bioavailability can have significant impact on the provision of safe and reliable oral pharmacotherapy. A mechanistic understanding of the events that contribute to the occurrence of food effects is therefore critical. An increased oral bioavailability is often seen for poorly water-soluble drugs after co-administration with lipids, including lipids in food, and is commonly explained by the ability of lipids to enhance drug solubility in intestinal luminal fluids. In contrast, the impact of lipids on drug solubilisation in the stomach has received less attention. This is in spite of the fact that lipid digestion is initiated in the stomach by human gastric lipase and that gastric events also initiate emulsification of lipids in the gastrointestinal tract. The stomach therefore acts to ‘pre-process’ lipids for subsequent events in the intestine and may significantly affect downstream events at intestinal drug absorption sites. In this article, the mechanisms by which lipids are processed in the stomach are reviewed and the potential impact of these processes on drug absorption discussed. Attention is also focused on in vitro methods that are used to assess gastric processing of lipids and their application to better understand food effects on drug release and absorption.
Key words Gastric lipolysis; food effect; human gastric lipase; in vitro digestion; lipids; biorelevant dissolution; milk
3 Abbreviations AUC Area under the curve
CCK Cholecystokinin
DAG Diacylglycerol
DGL Dog Gastric Lipase
EMA European Medicines Agency
FDA United States Food and Drug Administration
FFA Free Fatty acid
HGF Human Gastric Fluid
HGL Human Gastric Lipase
LBF Lipid Based Formulations
LCFA Long chain fatty acid
LCT Long chain triacylglycerol
LFCS Lipid Formulation Classification System
MAG Monoacylglycerol
MCFA Medium chain fatty acid
MCT Medium chain triacylglycerol
MRI Magnetic resonance imaging
PEI Pancreatic exocrine insufficiency
PWSD Poorly water-soluble drugs rDGL Recombinant Dog Gastric Lipase
RGE Rabbit Gastric Extract
SGF Simulated Gastric Fluid
TAG Triacylglycerol
SCFA Short chain fatty acid
4 1 Introduction Food-drug interactions can affect both the safety and efficacy of oral pharmacotherapy and thus, are still regarded as a significant challenge in oral drug delivery. Food effects on oral drug bioavailability are not highly predictable as they are the result of the complex interplay between the physiological conditions within the human GI tract and the properties of the drug substance and the dosage form (1). Food may enhance or reduce drug absorption after oral administration. One area of growing interest is the potential for food to enhance the bioavailability (i.e. a positive food effect) of poorly water-soluble drugs (PWSD). This is an increasingly important issue in the pharmaceutical sciences as a large proportion of drugs in development suffer from low water solubility and large variations in absorption are apparent with food ingestion (2). In this context, it is important to identify and comprehend the physiological processes that contribute to food effects on oral drug bioavailability. The ingestion of dietary lipids is regarded as one of the main contributors to positive food effects of PWSD such as cyclosporine or amiodarone (3). Such effects are often explained by the high solubility of lipophilic drugs in dietary lipids or in micelles formed by lipolysis products, bile salts and phospholipids, resulting in increased drug dissolution. However, these effects are strongly affected by the dynamic changes occurring upon gastrointestinal lipid digestion.
When considering the impact of lipids on drug dissolution and absorption, the gastric phase of digestion has been largely neglected, but recent studies and experiences suggest that the role of gastric digestion should not be underestimated. For example, the decrease in gastric emptying rate that has long been known to occur in the fed state, dictates that the time period for gastric dissolution is lengthened and therefore the gastric content may be an important dissolution medium, especially for PWSD administered in fed state (4). It was also shown recently by magnetic resonance imaging (MRI) that lipids and lipolysis products account for up to 10 % of the gastric medium over a time frame of several hours. (5). During this period, ingested lipids experience significant chemical and structural changes that may have direct or indirect consequences for oral drug delivery (e.g. changed drug solubility in luminal fluids, delayed gastric emptying).
This review therefore aims to describe the effects of lipids and lipolysis in the human stomach on oral drug bioavailability. We first provide a brief overview of gastric lipolysis and the effect of lipids and lipolysis products on gastric physiology. Subsequently, we present in vitro methods that may be applied to study the effect of lipids and lipolysis products on drug release from a dosage form, dissolution and solubility. The review focusses primarily on the role of lipids in the stomach and the downstream effects that this may have on intestinal processes. Nonetheless, processes in the small intestine can also play an important role in the occurrence of food effects. In particular, the alkaline secretion of bicarbonate, bile salts and pancreatic lipases leads to significant structural changes and thus, affects intestinal drug concentration as well as drug absorption from the small intestine. These
5 aspects were recently summarised in reviews published by Porter, Trevaskis and Charman (6) and Bakala-N’Goma, Amara, Dridi, Jannin and Carrière. (7).
2 Lipid processing in the stomach In the US, the average intake of lipids amounts to 120-150 g per day (8). The majority of lipids present in our daily diet are triacylglycerols (TAG), whereas diacylglycerols (DAG), monoacylglycerols (MAG) and free fatty acids (FFA) are present at lower levels. Since lipids are mainly absorbed in the form of MAG and FFA, the TAG must be first hydrolysed via lipolysis, a process mediated by enzymes (lipases) present in the lumen of the stomach and the small intestine (7,9). Since lipid absorption, as well as the major part of lipolysis, takes place in the small intestine, the role of the stomach has often been overlooked. Nonetheless, gastric lipolysis contributes to 10 – 30 % of overall lipolysis and in addition, has an important role in promoting the action of pancreatic lipolytic enzymes (10–12). The stomach also mediates the direct absorption of short and medium chain ( After mastication and oesophageal transit, lipids enter the stomach either in form of solid fat, larger fat droplets or an emulsion. Within the stomach, the action of intense peristaltic waves in the antrum supports the formation of a crude emulsion. At the lipid-water interface formed by lipid dispersion, the water-soluble gastric lipase hydrolyses TAG, forming primarily DAG and FFA (14). Gastric lipolysis of TAG comprising medium-chain fatty acids (MCFA) is more efficient than that of TAG comprising LCFA since the latter do not dissociate from the lipid-water interface as readily as the more polar medium-chain digestion products. In the absence of fatty acid acceptors (e.g. bile salt micelles), LCFA rapidly inhibit the activity of gastric lipase in the stomach (15,16), which limits the extent of intragastric lipolysis when compared to intestinal lipolysis. The transfer of this crude emulsion to the duodenum is controlled by the process of gastric emptying. In the small intestine, TAG and DAG are further processed into absorbable 2-MAG and FFA mainly by the action of pancreatic lipase and colipase. Whereas FFA with chain lengths of 12 C- atoms or less are absorbed directly via the enterocytes into the portal vein, FFA with longer chain lengths follow a different pattern. After uptake into enterocytes, they are re-esterified into TAG, incorporated into chylomicrons and, subsequently, enter the lymphatic transport pathway (11). The two main processes that are usually considered when examining gastric lipid processing are: 1) lipid emulsification resulting from mechanical mixing via peristalsis and 2) lipid digestion under the control of human gastric lipase (HGL). These processes typically combine to reduce the particle 6 size of lipid emulsion droplets in the stomach, but gastric lipolysis can also lead to the partial coalescence of lipid droplets initially present in food emulsions and thus, to an increase in particle size. This has been recently shown by particle size analysis of milk and infant formulas upon in vitro (17,18) and in vivo (19) gastric digestion. The formation and presence of emulsions in the stomach can provide lipophilic environments into which poorly water-soluble drugs may partition, preventing drug precipitation and potentially enhancing drug absorption. However, enzymatic digestion processes in the stomach cause dynamic changes to the physicochemical properties of the gastric content and thus, also affect drug release and drug solubility. A detailed description of these processes is provided in the following section to aid the reader in comprehending possible interactions between gastric lipid processing and drug release in the fed stomach. 2.1 Human Gastric Lipase Human gastric lipase (HGL), a globular protein with a molecular weight of 50 kDA, is co- localised with pepsinogen in chief cells that are present mainly in the proximal stomach (9,20,21). Food intake triggers the secretion of HGL via the release of neuro-hormonal stimuli such as gastrin or acetylcholine. Borovicka and co-workers demonstrated maximum HGL secretion rates of up to 1 mg/min after intragastric infusion of a liquid test meal (22). However, owing to dilution into gastric content, HGL concentrations are typically at their lowest directly after food intake. During digestion, gastric lipase accumulates and concentrations increase towards a maximum equivalent to that of basal gastric juice (23) (Fig. 1). It can be further seen in Fig. 1 that the inter-individual variability of the HGL concentration is high when plotted against time. Interestingly, if the HGL concentration is plotted as a function of meal gastric emptying, the inter-individual variability of HGL concentration is clearly reduced indicating that the HGL concentration in the stomach is the result of the interplay between secretion and gastric emptying. 7 Fig. 1 HGL concentration as a function of time (left) and as a function of meal gastric emptying (right) after intake of a mixed solid-liquid test meal at t= 0 min by 53 individuals. The dots represent the experimental HGL concentration measured in aspirated gastric juice, whereas the solid line represents the best-fitting curve. The activity of HGL in gastric juice was measured with the pH-stat method using tributyrin as a substrate at pH 6.0. Reproduced from Sams et al. (23) with permission from The Royal Society of Chemistry. The gastric lipase outputs seen in response to different meals are summarised in Table I. It should be noted that lipase activity also depends on the diet of the subjects prior to in vivo investigation. For example, after a high-fat diet for two weeks, lipase activity was found to be higher when compared to a low-fat diet (24). 8 Table I Human gastric lipase (HGL) output in response to different test meals. Share of Number of Total HGL output Test meal total Ref subjects mean lipolysis Liquid meal (375 kcal) n=15 23 ± 8 mg 10 ± 1 % (14) Liquid meal (375 kcal) 22 ± 14 mg n=20 (25) Minced solid meal 15 ± 5 mg Liquid meal (375 kcal) 22 ± 15 mg 24 ± 6 % n=9 (26) Minced solid meal 17 ± 5 mg 9 ± 6 % Minced solid meal n=6 14.7 ± 8.7 mg 7.3 ± 5,9 % (27) Emulsified test meal n=5 9 – 12% (28) (960 kcal) Liquid meal (375 kcal) n=6 15 ± 8 mg 24 ± 8 % (29) Liquid meal (400 kcal) n=6 25 ± 7 mg (22) The products generated by gastric lipolysis can be analysed by chromatographic methods such as thin layer chromatography (TLC) coupled with densitometry (26), TLC coupled with flame ionisation detection (21,30), high performance liquid chromatography coupled with light scattering detection (31), mass spectrometry (32) or high-resolution gas chromatography (33). Beside this characterisation of the activity of gastric lipase under “physiological” conditions in vivo, various assays have been developed to estimate lipase activity under optimised in vitro conditions and have been employed to estimate the concentration of active enzyme in biological samples. A comprehensive review of the analytical procedures used for the characterisation of lipases has been published by Beisson and colleagues (34). To this point, the pH-stat technique, where lipid digestion is quantified via titration of liberated FA, has been the most commonly employed method to estimate gastric lipase activity using various TAG substrates. Tributyrin is often used for a sensitive detection at low pH values (14,15). These assays have significantly contributed to the current knowledge of HGL secretion levels and their action on lipids in the stomach. HGL hydrolyses TAG ester bonds on the glycerol backbone (35) with a stereo-preference for the sn-3-position, leading to preferential formation of sn-1,2-DAG (36). HGL is less active on DAG, and MAG are usually poorly hydrolysed (7). Nevertheless, gastric lipase has the intrinsic ability to hydrolyse all three ester bonds present in TAG (35), whereas pancreatic lipase does not cleave the ester bond at the sn-2 position. Gargouri and co-workers demonstrated that the hydrolysis rates of TAG formed from short and long chain fatty acids were comparable and thus, there is no significant intrinsic chemo-specificity towards short-, medium- or long-chain fatty acids (9). But as described above, the activity of gastric lipase is inhibited by the accumulation of LCFA at the lipid-water interface (16) and so in practice gastric lipolysis of long chain TAG is lower than that of medium and 9 short chain TAG. Notably, the composition and fatty acid distribution of different dietary lipids can vary (37). For instance, in the case of milk and butter, short- and medium-chain fatty acids are predominantly conjugated to the sn-3-position of the TAG backbone and thus, released preferentially during gastric lipolysis (38,39). HGL is a water-soluble enzyme acting on water-insoluble substrates. Therefore, enzyme adsorption at the lipid-water interface is a prerequisite for enzyme activity. The binding mechanism of gastric lipase to emulsified TAG is not completely elucidated, but involves conformational changes in a lid domain that controls access to the active site upon lid opening (40,41). The N-terminal tetrapeptide has also been shown to be important for binding to long chain TAG emulsions (42). This probably involves the N-terminal hydrophobic residues (Leu-Phe) that are part of the interfacial recognition site, and also the presence of a charged residue (Lys4), that is involved in the stabilisation of the lid domain via a salt bridge with Glu225 present in the lid (41). Site-directed mutagenesis of Lys4, Glu225 and R229 in recombinant HGL has recently elaborated the key role of these residues in locking the open conformation of the lid and substrate selectivity for short-, medium- and long-chain TAG (43). The active site of HGL consists of a catalytic triad of serine, histidine and aspartate residues, as found in serine proteases and other lipases. For similar catalytic mechanisms, optimal enzyme activity is usually observed above neutrality due to the pKa of histidine. However, in contrast, gastric lipase displays its optimum activity at slightly acidic pH values (pH 4 – 5.4). This is due to the fact that the enzyme adsorption step that precedes lipolysis is pH-dependent with an optimum at acidic pH values. This is the rate limiting step for gastric lipase activity (44). The activity of gastric lipase is generally expressed in international enzyme units (U / 1 U = 1 µmol of FFA released per minute) and can be determined using a pH-stat titration method. For most in vitro tests, pure triacylglycerols such as triolein or tributyrin are used to study the activity of gastric lipase. Tributyrin is often the preferred substrate to study gastric lipase kinetics, because direct titration of free fatty acids can be performed at low pH values owing to the low pKa of short- chain fatty acids. The activities of purified HGL, obtained by using tributyrin as the substrate are summarised in Table II. 10 Table II Specific activities of native human gastric lipase (HGL) determined using the pH-stat method under optimized assay conditions. Gastric lipase activity Substrate Ref. 1200 U/mg tributyrin, pH 6.0 (14) 1300 U/mg tributyrin, pH 4.5 (25) 318 U/mg tributyrin, pH 5.6 (22) 1160 U/mg tributyrin, pH 6.0 (15) 600 U/mg Intralipid®, pH 4.5 – 5.5 In contrast, the use of long-chain TAG (e.g. Intralipid®, triolein), rather than short chain TAG such as tributyrin, requires back titration due to the higher pKa value of long chain fatty acid (resulting in incomplete ionisation at typical experimental pH). The application of long-chain TAG is therefore less convenient for studying enzyme kinetics in routine assays. Although standardised conditions are sometimes applied, data from different studies are often not comparable as can be seen in Table II (11). It is therefore important to note that the standard pH-stat assay employed for gastric lipase activity has some limitation as it is usually carried out under non-physiologically relevant conditions established for measuring the highest enzyme turnover and thus, providing the most sensitive detection of the enzyme through its activity range. It has been shown, for example, that HGL specific activity under these conditions is 40 times higher than the specific activity of HGL when measured against TAG in a test meal (25). Nevertheless, even based on the low levels obtained using TAG as a substrate, the complete lipolysis of a solid meal should be possible within 120 min. These low rates of gastric lipase activity are supported by in vivo data obtained with the same test meals (25) and they reflect the average physiological turnover of gastric lipase. In contrast, using a specific activity of 1,300 U/mg as recorded for HGL using TBU as substrate under optimised conditions, the complete lipolysis of dietary TAG by HGL would be expected to be complete within 1 to 3 min, which is clearly unrealistic (25). Thus, the different activity values that can be found in the literature have to be interpreted and used with care as they only apply to the test conditions employed in that particular study. Where the pH-stat technique is employed to perform in vitro digestion studies that inform understanding of lipid digestion in vivo, it is important to use physiologically relevant assay conditions. These considerations are valid for both gastric and pancreatic lipases and were taken into account, for instance, when developing the method employed by the LFCS consortium to examine the in vitro lipolysis of lipid-based formulations under intestinal conditions (45). Since the pH-stat technique is not available in all laboratories, alternative assays of gastric lipase activity have been 11 tested but they are often impaired by the need to work at low pH values. Instead of using purified HGL, some authors also propose the use of human gastric fluids. The difficulty here is the high variability of enzyme activity as well as the instability of gastric fluids. Storage of gastric juice or fluid samples is critical, because gastric lipase is destroyed by pepsin after denaturation at highly acidic pH values (pH 0.5-1.0) (46). This process is amplified upon freezing/thawing and therefore, the pH of samples should be increased up to pH 6 before freezing. Recently, a spectrophotometric pH indicator-based lipase assay (PHLIBA) was developed for measuring lipase activities on tributyrin and tricaprylin in microtiter platers (47). It allowed for the continuous measurement of the activity of both rabbit and dog gastric lipases across a broad pH range. The maximum specific activities measured at pH 5 – 5.5 were only 2 to 4-fold lower than those measured with the pH-stat technique, suggesting that this new assay has good sensitivity. 2.2 Parameters affecting gastric lipolysis The activity of HGL is affected by various factors that are summarised in Fig. 2 and discussed in the following paragraphs. Fig. 2 Parameters affecting HGL activity in the human stomach. 2.2.1 Emulsification As for all lipases, the degree of emulsification of the water-insoluble lipid substrate (e.g. dietary lipid), and thus the specific surface available for enzyme adsorption is critical for the action of gastric lipase, since the enzyme hydrolyses lipids preferentially at the lipid-water interface (17). In the absence of amphiphiles, HGL shows no activity, because it is denatured at the high energy TAG- water interface (9). However, amphiphiles including phospholipids and proteins in foods are present in the stomach and facilitate the activity of gastric lipase by lowering the interfacial tension (48). Taking the example of the standard high-fat and high-calorie breakfast recommended by FDA and EMA for studies aiming at the investigation of food effects on oral bioavailability (FDA breakfast), 12 the main sources of lipids are butter and milk (49). In both cases, the lipids are already present in the form of natural emulsions stabilised by phospholipids and milk proteins such as casein (50). In case of other lipids such as in bacon, mastication and gastric peristalsis have to support the release and emulsification of lipids. Overall, gastric mucins and emulsifying food components such as proteins or phospholipids can stabilise emulsions pre-ingested or formed in the stomach. Digestion products generated during gastric lipolysis such as FFA may also contribute to the stabilisation of food emulsions. Armand and colleagues have shown that the gastric processing of a coarse (10 – 100 µm droplets), emulsified test meal composed of olive oil, egg yolk and sucrose leads to the formation of a fine emulsion with median droplet sizes in the range of 20 – 40 µm (28). Here, the most significant changes in emulsion droplet sizes are observed in the first hour after meal intake. Later, the released LCFA inhibit gastric lipase activity and thus, further reductions in emulsion droplet sizes are not observed. This demonstrates that the degree of lipid emulsification in the stomach can be increased by digestive processes, but does not elaborate the behaviour of non-emulsified dietary fat. Gastric lipolysis can also lead to coalescence of lipid droplets, as has been observed during in vitro digestion of milk and infant formulas (17). Further complications arise from the realisation that the lipid phase from a solid-liquid test meal is emptied from the stomach at a slower rate compared to the aqueous phase (51), whilst the lipids in a homogenised and emulsified liquid test meal are emptied at the same rate (52). In case of typical solid-liquid test meals, gastric samples show the presence of very large lipid droplets in gastric sampling tubes suggesting phase separation in the later stages of gastric digestion. The formation of a lipid layer on top of the gastric contents have been also observed for the FDA standard breakfast by MRI (5). It is clear therefore, that lipid coalescence can occur under gastric digestion conditions. One has therefore to be careful when discussing the physical state of lipids in the stomach, because both emulsification and coalescence processes can occur and these processes are tightly linked to the initial physical state of the dietary lipids ingested. Unfortunately, most in vivo studies have been performed with emulsified liquid test meals or lipid emulsions as it is almost impossible to collect gastric and duodenal samples after administration of solid meals. However, the conclusions drawn from these experiments might not reflect entirely what occurs with mixed solid-liquid meals such as the FDA standard meal. The size of the intragastric emulsion and the nature of emulsifiers also affect the activity of gastric lipase. As seen for other lipases, the larger the lipid-water interface, the more lipase molecules can adsorb. Armand and co-workers have shown, for example, that the gastric lipolysis rate is higher after administration of a fine emulsion (< 1 µm) when compared to a coarse emulsion (10 µm) (53). However, protonated LCFA that are generated during digestion of long chain glycerides accumulate at the oil-water-interface. Together with phospholipids, LCFA create novel particles at the surface of the lipid droplet that bind and inactivate HGL. Therefore, without PL, the external addition of oleic 13 acid alone does not affect HGL activity (16). In the study of Armand and co-workers, the droplet size of the fine emulsion increased within the first hour, whereas the median droplet size of the coarse emulsion was relatively constant (53). The inhibitory LCFA concentration for emulsions with different droplet sizes was 107-122 µmol/m2 (16). Despite higher lipolysis rates, the larger surface area generated by the fine emulsions is able to delay the inhibitory effect on HGL. Hence, after 4 h, gastric contributions to overall lipolysis was 37 % for the fine emulsion as compared to 16 % for the coarse emulsion (53). 2.2.2 Luminal pH value The luminal pH value is another highly important parameter for the stability and activity of HGL. Human gastric lipase is stable at pH values from pH 2 – 7 and its maximum lipolytic activity is at pH 5 – 5.4 (Fig. 3) (7,23). 1200 Intralipid (LCFA) tributyrin (SCFA) 1000 800 600 400 200 Specific HGL activity (U/mg) 0 3 4 5 6 7 8 pH Fig. 3 Specific activity of HGL as a function of pH on 30 % Intralipid® and tributyrin as substrates. Reproduced from Gargouri et al. (15) with permission from Elsevier. During gastric digestion, the pH increases up to values of pH 5 to 6 after meal ingestion depending on the type of meal, and then drops back to baseline pH values of pH 1 – 2 over several hours (54). HGL is acid-stable and remains active down to pH 2, which ensures sufficient lipolysis over the time course of gastric digestion. Nonetheless, at strongly acidic pH values below pH 1, HGL is inactivated irreversibly with a half inactivation time of 43 ± 9 min (46). As such, the buffering effect of the ingested meal is needed for optimum HGL activity. The pH decrease observed during the time course of gastric digestion reduces HGL activity and therefore, it might be expected that the highest lipolysis rates are observed within the first hour after meal intake. In contrast, Carrière and colleagues have shown in dogs that the pyloric output of free fatty acids reaches its maximum ~ 1 h after meal intake. This can be explained by the higher gastric lipase levels at this time, which compensate for the loss in activity caused by decreasing pH values (14). Interestingly, the mean pH value of around 6 within the duodenal lumen remains favourable for the action of HGL and thus, HGL can also contribute to lipolysis outside the stomach (14). This has been confirmed in patients with severe chronic pancreatitis and exocrine pancreatic insufficiency, who had almost no residual 14 pancreatic lipase (27). The high stability of HGL in human duodenal contents at around pH 6 and in the presence of food has been shown using simultaneous ELISA and activity measurements (14). Nonetheless, it is difficult to estimate exactly, the quantitative contribution of HGL to intestinal lipolysis under normal conditions, since the levels of pancreatic lipase in the intestine are much higher than that of HGL in healthy subjects. The intragastric pH value also influences the stability of lipid emulsions, since the surface charge of many emulsions entering the stomach depends on pH (55). For instance, the surface charge of emulsions stabilised by proteins can become positive at acidic pH values, due to protonation, which may cause instability of the emulsion and thus, phenomena like flocculation or coalescence (56). Depending on the type of emulsifier, these processes can be further promoted by the action of gastric enzymes such as pepsin or lipase. Golding and co-workers, for example, showed that an emulsion stabilised by whey protein was unstable after the addition of pepsin (57). In all cases, any increase in emulsion droplet size will reduce the surface area available for HGL and thus, further decrease the rate of gastric lipolysis. 2.2.3 Gastric motility Gastric motility can also have an impact on intragastric emulsion stability, however, this is often overestimated. Although the application of high stirring rates in different in vitro tests enables the formation of a homogeneous sample, it does not reflect human GI physiology. The proximal parts of the stomach represent a region, where incoming food is basically stored and where mixing is poor. In contrast, in the distal stomach (i.e. the antrum), antral contraction waves enable homogenisation of the gastric contents and mixing with gastric secretions. However, consideration of the frequency and propagation speed of antral contraction waves, suggests that the stomach provides only gentle mixing of its contents (4,58). The generation of a retropulsive jet stream is probably the most important driver of the emulsification of lipids (59). However, 3D computer simulations based on computational fluid dynamics and a numerical model suggest that this phenomenon is limited to a small region in the antrum and is highly dependent on the viscosity of the gastric content (Fig. 4) (60). 15 Fig. 4 3D computer simulations describing the viscosity dependent generation of a retropulsive jet stream in the distal stomach by antral contraction waves. The two figures describe the hydrodynamics in the central plane of the stomach. Thereby, the streamlines indicate the fluid flow within the stomach at the time point of maximum occlusion in the antrum for two Newtonian fluids of different viscosity (1 mPas and 1000 mPas, respectively). The velocity of fluid flow is given by the colour code at the left of each example. Reprinted from Ferrua et al. (60) with permission from Elsevier. It was shown recently by MRI that in the case of acid-unstable emulsions, the shear conditions within the stomach are not sufficient to prevent phase separation phenomena like flocculation or coalescence (61,62). As such a floating fat layer was evidenced on top of the gastric content after administration of acid-unstable emulsions (Fig. 5). Fig. 5 MR images showing the phase separation of a coarse sunflower oil-in-water emulsion in the human stomach. Reprinted from Hussein et al. (61) with permission from The American Society for Nutrition. Nonetheless, the intense shear and significant forces that can be created during gastric emptying may provide a high degree of homogenisation during gastric emptying into the small intestine (63,64). The subsequent secretion of alkaline bicarbonate, bile and pancreatic lipase is also expected to lead to further rapid changes in the emulsion structure, with bile being the most important mediator of 16 spontaneous lipid emulsification. When considering biorelevant media that reflect the conditions within the human gastrointestinal tract, the physical state in which lipids are present, differs significantly between stomach and small intestine. These differences must be taken into account when evaluating drug release and solubility under simulated physiological conditions. By using (stable) emulsions, for example, rather than coarse emulsions or non-emulsified fat it is likely that the solubility of poorly water-soluble drugs in gastric contents will be overestimated. 2.2.4 Presence of amphiphiles As described above, the presence of surface active agents is also important for HGL activity. HGL shows no activity on pure triglycerides in the absence of amphiphiles, mainly because it is irreversibly denatured at high interfacial tension (15,48). HGL activity on tributyrin is thus lost above 15 mN/m but also below 6 mN/m. In the latter case, which is observed in the presence of synthetic detergents (e.g. Tween® 80, Triton® X-100, benzalkonium chloride) or highly tensioactive proteins (e.g. melitin, myoglobin), HGL inhibition may result from the impairment of its adsorption at the lipid-water interface. Maximum HGL activity is observed at interfacial tensions ranging from 8 – 13 mN/m, which can be obtained in the presence of bile salts like taurodeoxycholate (0.5-2 mM) or less tensioactive proteins like -lactoglobulin, ovalbumin and serum albumin (48). The type of emulsifier/surfactant is therefore crucial for gastric lipase activity. The highest in vitro activities of HGL are typically observed in the presence of bile salts, BSA and/or phospholipids like egg phosphatidylcholine (48) and a combination of these surfactants has been used to establish the standard assay conditions for the sensitive measurement of gastric lipase activity on various triglyceride substrates (15). Bile salts are the preferred amphiphile for use in HGL standard assays even though they are not physiologically relevant for simulating gastric conditions. In vivo, these endogenous surfactants are secreted via the bile duct into the duodenum and are not present in fed gastric contents at significant levels, except in rare cases of duodenogastric reflux (65,66). In most studies investigating the concentration of bile salts in fed gastric aspirates, the concentrations were found to be very low (< 0.5 mM) or even below the limit of detection (67–69). Therefore, under normal in vivo conditions or physiologically-relevant in vitro conditions, dietary proteins and phospholipids will be the main amphiphiles influencing HGL activity in the gastric environment. Considering emulsions with similar droplet size, lecithin is usually the emulsifier leading to the highest rates of gastric lipolysis, while emulsions made with casein are hydrolysed at a lower rate and those made with Tween® 80 are not hydrolysed at all under gastric conditions (70,71). There are several possible mechanisms by which amphiphiles can impact gastric lipase activity (72). The first is via the regulation of interfacial tension, independently from a direct effect of the amphiphile on the enzyme. The dependence of gastric lipase activity on interfacial tension has been 17 shown previously (albeit not via the effects of amphiphiles) using monomolecular lipid films and the barostat technique (73,74). Second, some highly tensioactive amphiphiles may impair gastric lipase adsorption and activity at the oil-water interface by either displacing the adsorbed lipase molecules or by forming a compact viscoelastic adsorption layer (75). Third, amphiphile molecules may interact directly with the enzyme and impact its structure and kinetic properties. Local conformation changes that affect “lid” opening and monomer binding in the active site have been observed for instance with -octyl glucoside (41) while SDS can lead to complete enzyme denaturation. Certain physicochemical properties of the gastric contents such as viscosity or ionic strength can also affect emulsification and emulsion stability (55). The activity of HGL is typically not directly influenced by bulk viscosity (76), but changes in the viscoelastic properties of interfacial layers may impact its adsorption and activity (75). The emulsification process may also be hindered as a higher viscosity of the gastric content decreases the shear movements within the stomach (59). Pasquier and colleagues showed in an in vitro study that gastric emulsification is decreased significantly if the viscosity of the medium is increased by the addition of dietary fibre (76). However, in case of emulsions already present in the stomach, nutrients such carbohydrates (e.g. polysaccharides from toast or potato products) can stabilise emulsions by increasing the viscosity of the intragastric content and forming so-called quasi-emulsions. The increased stability of a coarse O/W-emulsion after addition of a food thickener (locust bean gum) was well illustrated in an MRI study by Hussein and colleagues (61). However, due to technical difficulties, the viscosity of the intragastric content after administration of the FDA breakfast has not been determined to this point and it can only be speculated as to the possible effects. In addition, owing to secretions coming from the gastric wall and low gastric mixing it is very likely that luminal viscosity is highly inhomogeneous. 3 The impact of lipids on gastric function Lipids are known to affect the in vivo performance of solid oral dosage forms and to contribute to the occurrence of food effects. Apart from direct effects on drug release and solubility, lipids and lipolysis products cause specific physiological responses in the human gastrointestinal tract, which may affect oral drug delivery. Various studies have shown that the sensing of long chain fatty acids in the small intestine causes the release of various gastrointestinal hormones such as cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1) and peptide YY (77). Medium chain fatty acids are also involved in the activation by acylation of ghrelin, another gastrointestinal hormone released in the stomach that triggers hunger (13). An overview of the effects of these hormones on gastrointestinal function is given elsewhere (77,78). The following section provides an overview of the specific effects of lipids and lipolysis products on gastric emptying, secretion and motor function. For a broader overview of the effects of food on human GI physiology, the interested reader is directed to reviews such as those by Camilleri or Schulze (58,79). 18 3.1 Effects on gastric emptying The process of gastric emptying is, in large part, controlled by the caloric density, the nature of calories (e.g. carbohydrates, proteins or lipids) and the volume of the gastric content (80–83). In order to guarantee maximum nutrient absorption, chemo-receptors (e.g. sensors for pH or nutrients such as glucose or fatty acids) within the duodenum monitor the properties of the chyme and adapt the gastric emptying rate via neural and humoral pathways (79). This enables a constant flow of nutrients to the duodenum. The gastric emptying rate is typically 2 – 4 kcal/min (4,58,84). Owing to the high caloric density of lipids (9 kcal/g), their effect on gastric emptying is comparatively high. In the case of stable emulsions, the intragastric lipid fraction is typically constant over time and drops only immediately prior to complete meal emptying from the stomach (5,28,85). After intake of the FDA standard breakfast, the intragastric fat share remains relatively constant at a level of 6 – 10 % (V/V) over a period of more than 6 h. This dictates that lipid effects on drug solubility can be present even hours after food intake. In line with this, several studies have described increased bioavailability of poorly water-soluble drugs such as quazepam or blonanserin even after administration 4 h after food intake (86,87). MRI data show that lipids are emptied constantly into the duodenum during gastric digestion of a meal, but that the fat fraction in the fundus and antrum may differ significantly (5). Thus, over a post-prandial period of more than 4 h, the fat fraction in the fundus was reportedly higher than in the antrum, indicating lipid retention in the proximal stomach (Fig. 6) (5). From a physiological point of view, such behaviour is beneficial as HGL is secreted by chief cells in the fundus (88). Fig. 6 Intragastric fat fraction (%) in fundus, antrum and total stomach over time (means of n = 6 ± SD) after intake of the FDA standard breakfast measured by MRI. Differentiation into fundus and antrum was not possible at t = 0 min and t = 375 min due to low volumes in antrum. Reprinted with permission from Koziolek et al. (5). Copyright (2017) American Chemical Society. The formation of a fat layer on top of the gastric content is common and can influence gastric emptying (61,89). For example, the gastric emptying of two different olive oil emulsions was recently studied, where one was acid labile leading to lipid phase separation, whereas the other was acid stable. 19 In this case the acid labile emulsion, where lipid phase separation was evidenced resulted in rapid emptying of the aqueous phase of the gastric content, whereas in case of the acid-stable emulsion the gastric volume decreased constantly (62,90). Kunz and colleagues further showed that ingestion order affects gastric emptying of a mixture containing a fat (mayonnaise on toast) and non-fat (pasta with tomato sauce) component (Fig. 7). When the fat was ingested first, gastric emptying was faster, reflecting rapid dilution of the mayonnaise with gastric fluid. The extent of layering was also stronger if fat was ingested first, since the acidic pH value of gastric fluid is likely to result in cracking of the emulsion and lipid phase separation. Where the non-fat component was administered first, the meal buffering capacity and the higher pH in fundus may have prevented initial emulsion breakage and reduced gastric emptying (91). Fig. 7 Effect of ingestion order on gastric emptying of a solid meal. The subjects in this study received the solid meal, which was composed of a fat (F) and a non-fat (NF) component in two different study conditions (F-NF vs. NF-F). The data are presented as means (of the fitted curves) + SEM. *Significant difference (p < 0.05). Reprinted from Kunz et al. (91) with permission from WILEY. The specific effects of lipids on gastric function are largely mediated by the gastrointestinal hormone cholecystokinin (CCK). The release of CCK slows gastric emptying and inhibits antral motility and gastric acid secretion. CCK release is stimulated by long chain fatty acids (LCFA) that are sensed by G-protein coupled receptors in the intestinal wall (79,92). Since CCK is produced by I- cells in the proximal small intestine, it is likely that secretion is stimulated by LCFA released in the upper part of the duodenum (93). LFCA released in the stomach and then emptied through the pylorus into the upper intestine might therefore be important for CCK release and the stimulation of pancreatic and bile secretions. The link between LCFA production and CCK release is mediated by LCFA receptors. Of these, CD36 is considered to be the main contributor (94). CD36 also mediates fat perception in taste buds and triggers the initiation of the cephalic phase (i.e. prior to food ingestion and driven by anticipation) of digestion (95). The potential for an anticipatory reaction to lipid based formulations has not been considered to this point, but may also have an impact on their digestion in the upper GI tract. 20 Owing to the sn-3-stereospecifity of HGL, the molecular structure of the ingested lipids (e.g. milk) is highly relevant to effects on gastrointestinal function. For instance, the chain length of the fatty acids released has a major influence on the release of CCK (96,97). Whereas fatty acids with a chain length of C12 or higher stimulate CCK release, fatty acids of shorter chain length do not lead to such effects (98). Instead, medium-chain fatty acids (MCFA) increase the plasma concentrations of the hunger-stimulating hormone ghrelin (99). Ghrelin, produced by X-cells in the gastric corpus, initiates food uptake (100), and also stimulates growth hormone release (101). Ghrelin needs to be acylated by octanoic acid (C8) to be functional and octanoylation is mediated by ghrelin-O- acyltransferase (GOAT) (102). Since MCFA with less than 12 C-atoms can be absorbed in the stomach (103) and C8 lipids are used for the acylation of ghrelin (104,105), it might be assumed that gastric lipolysis plays an important role in the release of MCFA from dietary lipids (e.g. from milk or coconut oil) for ghrelin acylation in the stomach. However, the mechanism by which MCFA are absorbed and then used for ghrelin octanoylation has not been studied in detail. In a recent study in rats, in which a specific inhibitor of gastric lipolysis was administered, gastric absorption of MCFA was reduced, but there was no effect on ghrelin acylation (13), suggesting that C8 fatty acids could be obtained from alternate sources. Since MCFA are often present in lipid based formulations, their impact on gastrointestinal physiology might usefully be investigated further. The influence of FFA on gastric emptying has been demonstrated by various groups (97,98,106). In a recent study by Little and co-workers, the oral administration of an emulsion containing 40 g of oleic acid resulted in a decrease in GE rate from the 2-4 kcal/min typically seen for food to only 0.1 - 0.4 kcal/min for the long chain lipid emulsion (97). This study well illustrates the importance of gastric lipolysis in the regulation of gastric function, even though the contribution of HGL to overall lipolysis is only moderate (10 – 30 %). Dietary lipids and lipolysis products affect gastric emptying not only by their presence in the proximal small intestine, but also by their presence in more distal parts of the small intestine. The “ileal brake” for example, describes a phenomena whereby sensing of TAG, SCFA and LCFA in the distal small intestine results in a decrease in gastric emptying (107– 109). This intestinal feedback mechanism involves gastrointestinal hormones such as CCK, glucagon- like-peptide-1 and peptide YY (107,109,110). However, Reppas and colleagues showed that TAG are typically not present in distal ileum, whereas DAG and FFA can be found in fed ileal contents (111). A summary of the properties and gastrointestinal effects of short and long-chain fatty acids is given in Table III. 21 Table III Properties and gastrointestinal effects of short/medium-chain and long-chain fatty acids. Short/medium-chain Long-chain fatty acids fatty acids Physical state at body water- insoluble solids when saturated; temperature Partly water-soluble liquids water-insoluble liquids when unsaturated (e.g. oleic acid) Inhibition of lipase no yes for intragastric lipolysis1 activity a) direct absorption into 1. uptake into enterocytes portal vein 2. re-esterification into TAG Absorption b) absorption in the stomach 3. assembly into chylomicrons (up to C12) 4. transport via lymph Effects on CCK no yes Effects on ghrelin yes (by octanoylation) no Contribution to yes yes “ileal brake” 1 The inhibition of gastric lipase by LCFA is not caused by a direct inhibition, but by the entrapment of HGL in particles consisting of LCFA and phospholipids (16), as well as by the mass action law when LCFA remain at the lipid-water interface. This type of inhibition is usually abolished in the presence of fatty acid acceptors like bile salt micelles in the small intestine. 3.2 Effects on gastric motility The release of CCK also affects gastric motility by reducing antral motility and favouring fundic relaxation. This allows the stomach to store larger amounts of food. The increase in storage capacity is known as gastric accommodation (79). McLaughlin and colleagues have shown that the occlusive diameter of the antral contractions in the fed stomach (soup) decreases as CCK release is stimulated by free fatty acids with C > 12 (98). In line with these results, Feltrin and colleagues have also demonstrated that the number and amplitude of antral contraction waves are significantly reduced after intraduodenal infusion of lauric acid (C12:0) when compared to decanoic acid (C10:0) (96). Thus, the products of gastric lipolysis decrease gastric mixing, and this in turn is expected to affect intragastric emulsion stability and potentially, drug release from solid oral dosage forms. 3.3 Effects on gastric luminal conditions Lipids in food also affect the intragastric pH profile after meal ingestion as the intraluminal pH of the fed stomach is the result of the buffer capacity of the meal, the pH value and the volume of the fasted state content as well as the rate of gastric acid secretion and gastric emptying. Typically, the gastric pH reaches a maximum in the stomach directly after meal intake and decreases over time towards a highly acidic baseline value (54,112). The pH profile also strongly depends on the amount and composition of the ingested food. For instance, components such as proteins or fatty acids have a strong effect on the buffer capacity of the gastric content (68,113). The secretion of gastric acid from 22 parietal cells is stimulated directly or indirectly by histamine, gastrin and acetylcholine. These pathways are activated either centrally or by stimuli in the stomach such as distension, pH or protein (114). Acidic gastric pH values are needed for optimum protein digestion (115), but decrease the activity of HGL. In contrast, the products of gastric lipolysis, free fatty acids, have an inhibitory effect on gastric acid secretion via stimulation of CCK release in the small intestine (116). Thus, lipids are expected to increase the buffer capacity of the gastric content and inhibit gastric acid secretions. 3.4 Downstream effects on intestinal conditions In addition to effects on gastric function, gastric lipolysis can also have an impact on intestinal conditions via alterations to the release of CCK (Fig. 8). LCFA formed in the stomach and sensed in the small intestine can trigger effects such as gallbladder emptying and exocrine pancreatic secretions including, amongst others, pancreatic lipase (117). In studies performed with the lipase inhibitor Orlistat, for example, even small amounts of free fatty acids were sufficient to induce CCK release (117). These effects may have a significant impact on intestinal drug solubility, since mixed micelles formed by bile salts, phospholipids and lipid digestion products are able to substantially enhance the solubilisation of poorly water-soluble drugs. Furthermore, the pH shift from acidic to almost neutral pH brought about by secretion of bicarbonate can also affect intestinal drug solubility (118). A thorough description of these processes is given in reviews published by Dressman, Vertzoni, Goumas & Reppas (118), Hörter & Dressman (119) and Porter, Trevaskis and Charman (6). Fig. 8 The release of CCK is stimulated by various triggers such as the presence of LCFA in the small intestine, which leads to various changes of GI function in stomach and small intestine. Finally, the physiological response of the human GI tract after meal ingestion is not only the consequence of the ingested lipids, but also of other nutrients such as carbohydrates and proteins as 23 well as being affected by the structural and physicochemical properties of the food. With respect to the FDA breakfast, this provides a significant challenge to the human GI tract and is likely to lead to the maximum effects that food is likely to have on gastric emptying and secretion. It was shown in recent studies that the gastric emptying of this meal takes more than 6 h and pH values drop from pH 4 – 5 directly after meal intake to highly acidic pH values over several hours (pH ≤ 1) (5,54). In this case, the specific contribution of lipids to such a large effect is difficult to determine. For instance, the release of CCK is stimulated by a variety of a substances including lipids, carbohydrates and proteins (77). Nonetheless, the products of gastric lipolysis can have great impact on gastric function including gastric emptying, gastric pH and gastric motility - all processes that are expected to impact oral drug delivery. Each of those parameters may alter drug release and solubility in the fed stomach and must therefore be taken into account when evaluating food effects on oral drug bioavailability. For instance, the positive food effects commonly seen for poorly water-soluble drugs may not just reflect the increases in drug solubilisation that are likely in the intestine, but, for weak bases for example, may also reflect the impact of lipids on gastric emptying, increasing transit time in the acidic environment of the fed stomach. The broader implications of the impact of lipids in the stomach on drug absorption are discussed below. 4 Lipids in oral drug delivery The development and use of lipid based formulations (LBF) to enhance the oral bioavailability of poorly water-soluble drugs was based largely on the realisation that co-administration of PWSD with lipids in food commonly enhances drug absorption, and that the major driver of this effect was an increase in apparent drug solubility in gastric and intestinal fluids. Since the introduction of this concept some 50 years ago, many research groups have employed LBF to promote the oral bioavailability of different poorly water-soluble drugs (3,6,120). The experiences gained in the design and testing of these LBF have contributed to a much deeper understanding of drug-lipid interactions as well as the development of valuable in vitro tools to assay both lipolysis and drug solubilisation. In the following section we summarize the most relevant aspects of these studies where they relate to an understanding of lipid effects in the stomach. For further information, the interested reader is referred to the following reviews (6,120). By way of introduction, it is appropriate to point out that not all poorly water-soluble drugs are similarly lipophilic (and therefore have similar affinities for lipophilic environments). Typically, PWSD are viewed across two extremes. Firstly, drugs that are poorly water soluble and also truly lipophilic. These so called “grease ball” drugs, have high lipid solubilities, typically show significant positive food effects and their high lipid solubility allows ready formulation into LBF. In contrast, “brick dust” molecules are similarly poorly soluble in water, but have poor lipid solubility, often reflecting very strong crystal lattices (and high melting point). These types of molecules may also 24 show significant positive food effects, but their low intrinsic solubility in lipids generally precludes formulation in LBF (although recent studies suggest that pairing ionisable drugs with lipophilic counterions to form low melting ionic liquids may address some of those concerns) (121). From a formulation perspective the inclusion of more hydrophilic surfactants and co-solvents into LBF may also help to promote drug solubility in the formulation. (122). The range of possible LBF has been summarised by Pouton and colleagues in the Lipid Formulation Classification System (LFCS), (123,124) (Table IV). Table IV The Lipid Formulation Classification System. composition properties I lipids not self-emulsifying, digestion required II lipids + water-insoluble surfactants self-emulsifying, high lipid III lipids + surfactants + co-solvents self-emulsifying, lower lipid IV water-soluble surfactants + co-solvents formation of micelles The behaviour of these different types of formulation on dispersion into biorelevant media and on digestion by pancreatic enzymes has subsequently been examined in detail by a consortium of academic and industrial scientists (the LFCS consortium). Whilst these studies focussed largely on lipid digestion by pancreatic enzymes, the general behaviours are relevant to events in the stomach during the digestion of lipids in food. A more limited number of studies were also performed to specifically explore the impact of gastric lipolysis on formulation (and drug) behaviour (125). Examination of the components of the FDA standard breakfast - two eggs fried in butter, two strips of bacon, two slices of toast with butter, four ounces of hash brown potatoes, and eight ounces of whole milk - reveals the presence of lipids that are mainly saturated triacylglycerols (TAG) of animal origin such as butter or bacon (49,126). Surface-active components are also present and are derived either from exogenous (egg lecithin, milk proteins) or endogenous sources (bile salts, bile phospholipids, mucins). It is apparent therefore that there is likely to be much to learn about the digestion of lipids and amphiphilic components in food from recent studies of the digestion of similar lipids and synthetic amphiphiles in LBF. The methods used to assess drug behaviour during in vitro lipid digestion, and the common themes that have evolved from these studies are addressed below. The experiences with LBFs contributed much to the current understanding of the physicochemical processes underlying the formation and processing of emulsions in the human GI tract. In case of poorly water-soluble drugs, it is likely that the process of micellar solubilisation in the gut is highly relevant for the occurrence of food effects (6). The solubilisation via incorporation into micelles is most beneficial for drugs with log P > 3. However, the solubilisation capacity of a drug is limited and depends on the properties of the surfactant (type, critical micellar concentration), the medium (e.g. 25 pH), and the drug (e.g. pka) (2). However, without external addition of surface-active substances the formation of mixed micelles is limited in the stomach due to low gastric bile salt concentrations. Based on the information presented above, it can be summarised that several parameters affect the luminal drug solubility in the presence of lipids. These are 1) the nature and characteristics of the ingested lipid, 2) the presence of surfactants, 3) the rate and extent of lipid digestion and 4) the characteristics of the digestion products. Together these processes lead to the formation of colloidal structures comprising endogenous and exogenous components that promote drug solubilisation. 5 In vitro techniques to assess lipid digestion and its effects on drug release from oral dosage forms In vitro methods have been widely used to profile the behaviour of LBF during formulation dispersion and digestion (127), but have been used much less frequently to better understand food effects. In this section, the in vitro methods that have been used to study either gastric lipolysis or lipid effects on drug solubility or dissolution, including the evaluation of food effects, are discussed. Since the source of lipase or the choice of medium employed for these types of studies is critical, these aspects are discussed in more detail. 5.1 In vitro test methods The pH-stat titration method, which is a reference method for the determination of lipase activity, has become an attractive in vitro approach to study gastrointestinal lipolysis. As was described above, this method allows the investigation of lipid digestion under standardised conditions. The system is typically composed of a reaction vessel, in which the temperature is kept constant (37 °C), containing a digestion medium that is stirred at a certain rate (often 100 – 200 rpm), a pH-meter and a titration control unit. The pH value of the medium inside the vessel is set to a certain endpoint value. During lipolysis, the released fatty acids cause a decrease of the pH value of the medium, which is monitored by the pH-meter. Consequently, the titration control unit counterbalances the drop in pH by adding NaOH to the medium in order to maintain the pre-set pH endpoint value. In this manner, the exact amount of FFA released during digestion can be determined either directly, if the pH endpoint is much higher than the pKa of the FFA, or after back titration at alkaline pH values (pH 9) if the pH endpoint is below the pKa of the FFA generated during the test. Alternatively, apparent values can be corrected using pre-established correction factors based on fatty acid ionisation as a function of pH (25,128). The higher apparent pKa values of long-chain fatty acids (e.g. pKa of 9.85 for oleic acid) are a limitation to the use of the pH-stat technique as a continuous titration method at physiologically relevant acidic pH values (125,129,130). The shift in the pKa of the carboxylic acid function of LCFA above that of a classical carboxylic acid is caused by the environment at the lipid-water interface 26 (surface potential, presence of phospholipid headgroups, etc.) (131). It can also result from the self- assembly of fatty acids into pre-micelles, micelles (132), lamellar or crystalline aggregates (133). As such, the structures that are formed strongly depend on the pH value of the medium. For instance, in case of 80 mM oleic acid/ sodium oleate, micelles are formed at pH values of pH 10 and higher, vesicles are present at pH 8 – 10 and oil phases eventuate at pH values below pH 8 (134). Due to this limitation, the pH-stat technique is most appropriate to study intestinal lipolysis rather than gastric lipolysis, but can be used for both if the appropriate constraints are recognised. For example, the pH- stat technique was recently employed to continuously measure the lipolysis of medium-chain emulsions under consecutive gastric and duodenal lipolysis conditions (129). Owing to increased interest in LBFs during the last decades, the pH-stat method has been increasingly used to study the dispersion and digestion of these formulations under simulated gastrointestinal conditions. LBFs are usually designed to be administered in fasted state (indeed often they are utilised to try to match fed state conditions, without the need for food co-administration or to minimise the clinical variability that comes with different food use). Fasted administration results in relatively short gastric transit times and exposure to relatively low gastric pH. As such residence times in the stomach are short and the activity of HGL is low. In vitro investigations aimed at characterising drug release have therefore mainly focussed on intestinal lipolysis and the test protocols are often designed to be fit for this purpose. By comparing the test conditions described in the literature, it can be seen that the methods also differ in lipase selection, the addition of further excipients, the presence of phospholipids, bile salt choice and/or stirring speed (135). This realisation led, in large part, to the generation of the LFCS consortium that developed a standardised protocol for the assessment of drug solubilisation during intestinal processing of LBF and also published detailed evaluations of the effect of changes to bile salt concentration, drug load, lipid concentration and formulation characteristics on formulation performance (45,136,137). Outside of the drug formulation literature, Li and co-workers also demonstrated that test conditions (e.g. bile concentration, calcium concentration, lipid concentration, droplet size) are critical determinants of the degree of lipolysis observed in in vitro experiments used for simulation of intestinal lipolysis of food components (138). Of more relevance to the current discussion, an increasing appreciation of the role of gastric dispersion and digestion on LBF performance led the LFCS consortium to also establish an in vitro test method for gastric lipolysis. The test protocol was designed to allow study of the influence of gastric dispersion and lipolysis on the drug release behaviour of eight different LBF under fasted state conditions (125). In this study that was published by Bakala N’Goma and colleagues, recombinant dog gastric lipase (rDGL) was chosen to simulate HGL due to its comparable lipolytic activity towards triacylglycerols of different chain length (see full protocol in Table V). It could be shown that all LBFs were digested by rDGL over a wide pH range of pH 2 – 7. Maximum lipolytic activity was obtained at pH 4 for class II and IIIa formulation with medium-chain TAG, which further contained 27 Tween® 85 or Cremophor® EL. The higher specific activity towards medium-chain TAGs was explained by the different dispersion properties of MC and LC formulations, which led to larger lipid- water interfaces for the MC formulations as well as improved emulsification of the TAGs and their digestion products. Moreover, the products of lipolysis of long-chain TAGs also inhibit enzyme activity as was mentioned earlier. The authors concluded that even after fasted administration where gastric lipolysis is relatively low, the consideration of gastric lipase is important, because HGL retains its activity in the small intestine. In a recent publication, Christophersen and co-workers further suggested a test protocol for fed state studies (139). In this work, drug release from two self-nano-emulsifying drug delivery systems (SNEDDS) with cinnarizine was compared to drug release from a conventional IR tablet with the same drug in a two-step lipolysis model. As can be seen in Table V, the medium for gastric lipolysis was based on milk and lipase A from Candida Antarctica was used to simulate HGL activity. Interestingly, the fed state test protocol was not able to reproduce the results for the two SNEDDS formulations from an in vivo dog study, which indicates that further optimisation is necessary. However, the authors were able to confirm the positive food effect observed in vivo with their in vitro data. Table V Gastric lipolysis protocol for fasted and fed state (125,139). Fasted state Fed state (LFCS protocol) (Copenhagen protocol) Tested pH range 1.5 – 7.0 pH 6 Medium volume 40 mL 200 mL Digestion medium 2 mM Tris-maleate milk 1.4 mM CaCl2 150 mM NaCl 3 mM NaTDC 0.75 mM phosphatidylcholine Stirring speed 450 rpm not specified (propeller with d = 25 mm) Lipase recombinant dog gastric lipase Candida Antarctica lipase A Lipolysis time 5 min 60 min Titrant 0.1 M NaOH 5.0 M NaOH (back titration to pH 9) The major advantage of the pH-stat method is its simplicity, which allows for highly reproducible test conditions. Nonetheless, analogous to standard dissolution methods, the simplicity of such a conventional method also limits physiological relevance. For example, the static conditions in terms of pH and lipase concentrations maintained during the in vitro experiments do not accurately reproduce the highly changeable conditions observed in vivo. Furthermore, the high stirring speeds employed do not well simulate the shear conditions in the human stomach. 28 To overcome the limitations of these simple in vitro methods, highly complex models such as the Dynamic Gastric Model (DGM) and the TNO TIM-1 system, have been applied in order to investigate in more detail the effect of digestion processes on drug release from oral dosage forms. A thorough description of these models is given elsewhere (140–143). An overview of the different test approaches is given in Fig. 9. The major advantage of these systems is the potential to investigate digestion and/or drug release under highly dynamic conditions that better take into account gastric emptying, motility and secretion. These systems can provide helpful information on drug release in the fed stomach. However, their complexity precludes widespread application. Fig. 9. Figure to show the range of physiologically relevant dissolution test devices, of increasing complexity, that can potentially be used to study the effects of lipids and lipolysis on drug release from solid oral dosage forms. 5.2 Biorelevant media The potential effect of lipids and lipid digestion products on drug solubility and dissolution is typically studied using biorelevant test media in either traditional dissolution tests or lipolysis/digestion tests. However, although standardised media (FaSSIF, FeSSIF) are broadly applied for the simulation of intestinal conditions, even among different laboratories worldwide, there is no convention regarding a standard medium to mimic the gastric environment. The current state of the art was summarised in a comprehensive review by Baxevanis, Kuiper and Fotaki (144). The application of the FDA standard breakfast for in vitro dissolution testing is not common, since the use of a solid, heterogeneous meal results in complex analytical challenges. The shredding of the meal and dilution with the aid of artificial gastrointestinal juice, as proposed by several authors, is one way to allow application of this medium for disintegration and dissolution studies (145–147), but does not accurately reflect human physiology. For example, it was shown by MRI that the gastric 29 content after food ingestion is highly heterogeneous and thus, the situation generated by shredding represents a different scenario in terms of viscosity and particle size (5). This may have dramatic consequences for processes like drug disintegration and drug diffusion within the medium. It is therefore likely that certain effects will be over- or underestimated in vitro. Moreover, gastric secretion and emptying rates are highly variable and depend on numerous interrelated factors. The generation of accurate in vitro models therefore requires rich physiological data sets to inform their development, but the collection of this data has been hampered in the case of solid meals by problems associated with aspiration of samples of gastric contents. Large particles cause clogging of collection tubes and the process of aspiration may also lead to significant structural changes of the medium. Data on in vivo digestion of the FDA breakfast, in particular with respect to lipid processing, are therefore not currently available. Owing to the technical problems associated with the use of the FDA standard breakfast, full-fat milk and the nutrient drink Ensure® Plus are frequently used to simulate the gastric conditions present after the ingestion of the FDA breakfast (144,145). Ensure® Plus is an emulsion containing 1.5 kcal/mL and where approximately 26% of the calories are derived from fat. Klein showed that different physicochemical parameters of Ensure Plus® are comparable to the homogenised FDA breakfast, which is assumed to describe the initial situation in the stomach (145). Therefore, Ensure® Plus has been used in several different studies, as a biorelevant dissolution medium, to simulate the conditions in the fed stomach. It has also been used to simulate the fed state in in vivo studies, since the clogging of the aspiration tubes is less likely (148). Kalantzi et al. characterised human gastric fluids (HGF) sampled 30, 60 and 120 min after intake of 500 mL Ensure® Plus to provide data on pH, buffer capacity, osmolality and protein content (68). The authors also used the aspirates to determine the solubility of ketoconazole and dipyridamole in these samples (149). Further nutrient drinks or parenteral emulsions that are frequently used to simulate fed gastric conditions are Scandishake® Mix, Nutrison® and Intralipid® (144). It should be noted that all these systems represent perfect emulsions, which does not reflect the physiological situation in the human stomach. Therefore, their application in biorelevant dissolution testing may lead to misleading results. Full-fat milk is also commonly employed as a potential biorelevant medium for in vitro testing and is also used as the basis for Fed State Simulated Gastric Fluid (FeSSGF). Milk was proposed as a biorelevant dissolution medium in different studies published in the late 1980s by the group of Panos Macheras (150–152). The use of cow’s milk has the advantage that it represents the main liquid component of the FDA breakfast and simulates, at least partly, the initial luminal conditions in the stomach. Bovine milk is an oil-in-water emulsion with up to 4 % fat content. TAGs are the main fraction (> 95 %) of milk lipids and are located within the core of the milk fat globules (39,50,153). The TAGs of milk are composed of more than 400 different saturated, monounsaturated and polyunsaturated fatty acids. Blasi and colleagues (Table VI) report that the most common fatty acids 30 of bovine milk TAG are palmitic acid (32 %), oleic acid (19 %), myristic acid (13 %), stearic acid (7 %) and butyric acid (6 %) (33). Interestingly, the location of the fatty acids on the glycerol backbone follows a consistent pattern: SCFA at the sn-3-position, MCFA at the sn-2 and sn-3-position and LCFA mainly at the sn-1-position and sn-2-position (with the exception of oleic acid) (33,153– 156). In general, the sn-3-position, which is preferred by gastric lipase, is occupied by butyric, lauric and oleic acid. Notably, it has been reported that up to 36 % of milk TAGs contain an SCFA at the sn- 3-position together with two LCFA at the sn-1 and sn-2-position (33,39). Table VI Fatty acid composition of TAG from native cow’s milk (n=5, from Blasi et al. (33)).* Total FA Intrapositional composition Interpositional composition FA composition (% (% mol, mean ± SD) (% mol, mean ± SD) mol, mean ± SD) sn-1 sn-2 sn-3 sn-1 sn-2 sn-3 C4:0 6.0±0.8 1.3±1.2 0.4±0.2 17.9±1.7 6.0±5.0 2.2±1.2 91.8±3.8 C6:0 2.9±0.6 0.3±0.1 0.9±0.2 8.2±2.0 2.8±1.1 10.1±4.5 87.1±5.3 C8:0 1.7±0.2 0.3±0.1 0.2±0.1 4.7±0.6 5.9±2.0 4.7±2.0 89.4±2.0 C10:0 3.4±0.6 1.1±0.3 1.4±0.5 8.7±1.8 9.7±2.7 12.4±5.6 77.9±3.8 C12:0 3.9±0.9 2.5±0.6 4.8±1.2 5.1±1.4 20.0±0.5 39.1±7.2 40.9±7.2 C14:0 13.1±1.2 11.7±1.4 22.8±1.3 8.2±1.8 27.3±1.2 53.5±2.8 19.2±2.5 C16:0 31.6±2.6 46.8±2.4 44.1±3.9 12.0±2.2 45.5±1.5 42.8±1.6 11.6±1.3 C16:1 1.8±0.3 1.5±0.3 2.5±0.4 1.9±0.6 26.5±5.4 42.2±4.7 31.3±5.7 C18:0 6.6±1.4 11.1±1.1 5.3±1.4 5.2±2.0 52.6±7.5 24.2±2.3 23.2±5.5 C18:1 19.2±4.3 21.5±3.1 15.7±4.4 25.1±5.5 34.9±2.5 24.8±2.6 40.3±1.1 C18:2 1.9±0.2 1.7±0.2 1.9±0.3 2.7±0.4 27.8±1.6 29.8±3.8 42.4±3.4 C18:3 0.2±0.1 0.2±0.1 0.2±0.1 0.3±0.1 36.3±6.5 24.2±5.6 39.5±8.8 * The intrapositional composition describes the distribution of the fatty acids for a defined position of the triacylglycerol molecule. On the other hand, the interpositional composition describes the distribution of each fatty on the three positions of the triacylglycerol molecule. Since control of the physicochemical properties of the medium is one of the main prerequisites for dissolution testing, Jantratid and co-workers proposed a semi-dynamic approach that employed a step-wise change in the dissolution medium over time – resulting in media representative of “early”, “middle” and “late” gastric conditions (157). These media are made from mixtures of full-fat milk and acetate buffer and consider the changes in pH value, osmolality, buffer capacity and fat content over time. They are based on the physicochemical characterisation of pooled human gastric fluid (HGF) sampled 30 min, 60 min and 120 min after administration of 500 mL of Ensure® Plus by Kalantzi et al. (68). This also led to the development of three sets of milk-based biorelevant media with increasing complexity. These media provide different “snap-shots” of GI conditions at different times and also aim to represent the different phases of gastric digestion. The composition of the three media were based on (1) the physicochemical properties of HGF, (2) the physicochemical properties of HGF plus the presence of pepsin or (3) the physicochemical properties of HGF plus the presence of pepsin and lipase (Lipase RN from Rhizopus niveus) (149). 31 A similar approach was suggested recently by Markopoulos and colleagues, based on the idea of using media of differing levels of complexity that can be used for the biorelevant dissolution testing of solid oral dosage forms (Fig. 10). Whereas level I media are solely based on pH and buffer capacity, the presence of lipids is taken into account for level II media by the addition of Lipofundin®, an oil-in- water emulsion based on soybean oil. The use of this emulsion is in contrast to the snap-shot media proposed by Jantratid, who used milk. However, the media proposed by Markopoulos and colleagues also consider three different stages of gastric transit (early, middle, late) and simulate ongoing lipid digestion, gastric secretion and gastric emptying by different ratios of Lipofundin® and the aqueous buffer and decreasing osmolality. For level III media, the authors proposed that additional properties of the fed stomach could also be simulated including for instance, viscosity, dietary proteins or enzymatic digestion processes, but specific recommendations on the composition of such media were not published (158). Fig. 10 Snap-shot media of different complexity proposed by Markopoulos et al. (158). Although full-fat milk and Ensure® Plus provide important insights into dissolution behaviour in the fed stomach, their fat content of 3.5 % (V/V) and ~ 5 % (V/V), respectively, is clearly below the value of around 9.5 % measured directly after intake of the FDA meal (5). However, the question of how much fat is needed to stimulate a ‘food effect’ in vivo is not simple to answer and is likely to be different from drug to drug. For example, in a study by Kossena and co-workers, it was shown that the administration of 2 g of lipid had at least partial effects and led to various physiological effects such as gallbladder contraction and elevated intestinal bile salt levels, whereas 10 g of lipid appeared to 32 result in almost a complete ‘food effect’ (159). Similar effects have been reported after co- administration of the poorly water-soluble drug danazol with 10 g of lipid where increases in absorption analogous to the increases seen on co-administration with food were apparent (160). Thus, even relatively small amounts of lipids can cause changes to drug absorption. One additional factor that is not taken into account when employing biorelevant media in static experiments is that the decrease in gastric pH and changes to enzyme activity seen in vivo over time are likely to affect the stability and the properties of any lipid emulsion present, which in turn could influence drug dissolution (112,161). 5.3 Lipase selection The addition of a lipase to experiments investigating drug release under simulated gastric conditions can increase the physiological relevance of the in vitro test method. Unfortunately, HGL is not commercially available in purified or recombinant form. Therefore, alternative approaches have been employed to simulate gastric lipolysis (Table VII). These include the use of human gastric fluids or purified human gastric lipase, as well as the use of animal and microbial alternatives to HGL. The use of purified HGL is ideal, but is also the most laborious way to investigate gastric lipolysis and is not compatible with high throughput experimentation (162,163). As can be seen from Table VII, the activity of purified HGL is significantly higher than that of human gastric fluid. Although this approach is not suitable for routine experiments due to ethical, economical and technical reasons, it allows the physiologically relevant characterisation of an in vitro test method and may serve as a suitable reference method to alternative lipases. 33 Table VII Activity of gastric lipases of different origins. Lipase Substrate Lipase activity Ref. tributyrin 1160 U/mg (pH 6) Human Gastric Lipase medium-chain TAG 760 U/mg (pH 6) (15) Intralipid® 30% 620 U/mg (pH 5.4) tributyrin 570 U/mg (pH 5.5) Dog Gastric Lipase trioctanoin 760 U/mg (pH 6) (35) Intralipid® 30% 950 U/mg (pH 4) tributyrin 1200 U/mg (pH 5.5) medium-chain TAG Rabbit Gastric Lipase 850 U/mg (pH 5-6) (164) (C8-C10) 250 U/mg (pH 4) Intralipid® 30% tributyrin 562 U/mg (pH 5.7) recombinant Human Gastric Lipase trioctanoin 730 U/mg (pH 5-6) (165) (expressed in insect cells) Intralipid® 30% 434 U/mg (pH 3.5) recombinant Dog Gastric Lipase tributyrin 250 U/mg (pH 5.25) (166) (expressed in tobacco plants) Intralipid® 30% 950 U/mg (pH 3.5) tributyrin 310 U/mg (pH 5.4) recombinant Human Gastric Lipase 172 U/g tissue (167) (expressed in tobacco plants) Intralipid® 30% 116 U/g tissue recombinant Human Gastric Lipase tributyrin 1,192 U/mg (pH 5.5) (expressed in the yeast Pichia trioctanoin 1,480 U/mg (pH 5.5) (43) pastoris) Intralipid® 20% 489 U/mg (pH 4) The small-scale expression of recombinant human gastric lipase has been successfully performed in different organisms including insect cells (165,168) and yeasts (43,169–171). Vardakou and co- workers have also described the production of recombinant HGL (rHGL) in tobacco plants that yielded relatively high quantities of enzyme (50 – 100 mg) even at laboratory scale (100 – 200 g of leaf) (167). The advantage of using recombinant Human Gastric Lipase over Rabbit Gastric Lipase (RGL) or fungal lipases for the in vitro digestion of infant formula that are mainly based either on MCT or LCT was demonstrated in a recent publication by Sassene and colleagues (172). It was shown in this work that rHGL preferably releases MCFA, but also releases some LCFA. On the other hand, the fungal lipase from Rhizopus oryzae did not display any preference for a certain chain length, whereas RGL was only active on MCT. This latter result is not in agreement, however, with previous studies showing that RGL and rabbit gastric extracts (RGE) are active on LCT present in Intralipid® (164), test meals (30), various emulsions of milk fat, olein and rapeseed oil (70), cow´s milk and infant formulas (17) and human milk (18). Studies on acid-stable lipases for pancreatic enzyme replacement therapy in patients with pancreatic exocrine insufficiency (PEI) have led to the identification of dog gastric lipase (DGL) as a 34 prospective acid-stable lipase and a promising alternative to human gastric lipase (173). Indeed, DGL shows high activity on long-chain TAG at low pH values (35) and is well adapted to the acidic intestinal conditions observed in PEI patients (chronic pancreatitis and cystic fibrosis) who have a reduced bicarbonate secretion. Recombinant dog gastric lipase (rDGL) has been produced in both transgenic tobacco (166) and corn (174) as a therapeutic enzyme but it can also be regarded as one possibility for a suitable lipase for simulation of gastric lipolysis. rDGL is active from pH 2 – 7, shows a comparable lipolytic activity on triacylglycerols to HGL, shares 85.7 % of the amino acid sequence of HGL and its 3D structure is almost superimposable with HGL (41,125). However, for acid-stable lipases to be applicable in mainstream pharmaceutical in vitro testing, they must be available in large quantities at high (and reproducible) quality at relatively reasonable cost. Since these criteria cannot, at this time, be fulfilled by animal or recombinant human gastric lipases, microbial lipases (e.g. from Rhizopus sp. or Aspergillus sp.) are being increasingly employed for pharmaceutical in vitro studies (139,175,176). The activity of microbial lipases is typically lower than that of HGL and thus, the amount of enzyme applied has to be adjusted. Christophersen and co- workers suggest the use of Candida Antarctica lipase A, which has a pH activity profile comparable to human gastric lipase. For example, in a recent study aimed at the investigating drug release from a self-nanoemulsifying drug delivery system in the fed state, the enzyme activity employed in in vitro lipolysis testing was modelled on HGL activity in the fed state (40 U/mL) (139). Whilst microbial lipases provide a ready source of acid-stable lipase, their use is complicated by the realisation that different lipase sources show different pH optima, activities and specifies, when compared to HGL. To this point we are not aware of studies that have specifically compared the effect of different lipases on drug release or solubility. Interestingly, the in vitro digestion protocol described in the recently published consensus paper of the European COST Infogest initiative, excludes the use of an alternative lipase for gastric lipolysis due to the unavailability of relevant alternatives at the time of publication and instead recommends performing in vitro experiments without the use of an artificial gastric lipase (177). Rabbit gastric extract (RGE) might be one possible alternative (30) and has several advantages: (1) it is easily accessible in crude form, (2) it contains further enzymes such as pepsin and (3) the specific activity of its lipase towards certain TAG is comparable with HGL (30,125). RGE may also become commercially available soon as a new company called Lipolytech (Marseille, France) was recently launched in order to develop this product. In the near future, RGE from Lipolytech will be tested in a round-robin study by several labs within the INFOGEST research network. An excellent review summarising the importance of lipase selection and progress in developing alternative lipases was published recently by Sams and co-workers (23). 35 5.4 In vitro studies investigating the effect of lipids and lipid digestion on drug solubility and drug release from solid oral dosage forms in the stomach Despite the limitations mentioned above, several groups have successfully applied in vitro methods to study the effects of lipids and lipid digestion products on drug release from solid oral dosage forms. The relevance of gastric digestion processes on the solubility of poorly water-soluble drugs and drug release has been examined in vitro by Diakidou and co-workers (149,176). In the first of two studies, drug release from felodipine extended release tablets was investigated during milk digestion, the latter stimulated by addition of acidic solutions of either pepsin alone or pepsin plus lipase (Lipase RN from Rhizopus niveus). The highest drug solubility and highest drug release was obtained in media where milk was digested with pepsin and lipase. Thus, the presence of the lipase favoured the dissolution of felodipine under simulated gastric conditions by increasing gastric drug solubility (176). In a second study, the solubility of two poorly water-soluble drugs (i.e. ketoconazole and dipyridamole) was evaluated in human gastric fluid sampled after ingestion of Ensure® Plus. Both drugs are weak bases with reported pKa values of 5.7-6.4 in case of dipyridamol and 2.9 (pKa1) and 6.5 (pKa2) in case of ketoconazole (149). 36 Fig. 11 Solubility of dipyridamole (A) and of ketoconazole (B) in biorelevant media of different complexity. Reprinted from Diakidou et al. (149) with permission from WILEY. This data set was then compared with data gathered in various aqueous buffers of different pH, and in milk digested with pepsin and/or lipase (Fig. 11). As expected the solubility of the basic drugs was higher in acidic simulated gastric fluids, but for both drugs, solubility in HGF was clearly higher than in aqueous buffers at the same pH, suggesting that an increase in ionisation caused by a decrease in pH was only partly responsible for the increased solubility. When studied in milk-based media that were comparable to the snap-shot (“early” and “middle”) media described earlier, the solubilities of both drugs were low in undigested samples. However, after addition of HCl and enzymes, drug solubility increased dramatically. For ketoconazole, drug solubility in digested milk was comparable with that in HGF. In the case of the dipyridamole , however, solubility in milk digested with pepsin and lipase was up to 18.8 times higher than in HGF sampled 120 min after Ensure® Plus administration (149). The data suggest that gastric digestion may be an important determinant of the gastric solubilisation of PWSD, but also show the need for optimisation of the methods employed to assess solubility enhancement. 37 The gastric solubility of ketoconazole was also examined by Ghazal and colleagues, where solubility and intrinsic dissolution rate (IDR) were investigated in a variety of media containing additives such as milk, proteins, amino acids, and carbohydrates (178). Despite positive effects (i.e. increasing solubility and faster dissolution) for most of the additives, the highest solubility and highest IDR were revealed under acidic conditions in Simulated Gastric Fluid (SGF) pH 1.2. The solubilities measured in media containing 1:1 mixtures of SGF pH 3 and different types of milk were also increased when compared to SGF pH 3, but were still lower than in SGF pH 1.2. Interestingly, in this case solubility did not appear to be dependent on fat content. However, it should be noted that digestion was not initiated in this study, which may lead to differences among the different types of milk. Nonetheless, this work illustrated that multiple factors can enhance gastric drug solubility and that fat content alone does not always maximally contribute to improved solubility in the fed state. Since the products of gastric lipolysis are surface-active, it is likely that the wetting of a drug is another parameter that contributes to enhanced drug dissolution under gastric conditions (179). The impact of lipids and the potential digestion of lipids used in the formulation of PWSD on aqueous solubility in vitro has been examined in detail by Fernandez and co-workers using a two-step digestion model (180). This model involved a gastric phase of 30 min, during which formulations are digested at slightly acidic conditions (pH 5.5) using recombinant Dog Gastric Lipase (rDGL), and a subsequent intestinal phase (pH 6.25) that is initiated by the addition of Porcine Pancreatic Extract. The in vitro digestion of two different macrogolglycerides that are often used for formulating microemulsions (Labrasol® and Gelucire® 44/14) resulted in significant changes of the aqueous solubility of the two model compounds, piroxicam and cinnarizine. In case of piroxicam, simulation of gastric lipolysis of the two solubilising excipients caused precipitation of the drug in the intestinal phase and thus, led to a decreased solubility in the aqueous phase. In case of cinnarizine, gastric lipolysis of the excipients had no negative effect on the aqueous solubility. Notably, the hydrolysis of Labrasol® did even prevent the precipitation of this drug in the intestinal phase. Lipids can also influence drug release via the formation of a film around the dosage form that prevents disintegration and drug release. This may be a particular problem for modified release dosage forms, although the origin of the film and its in vivo relevance is still not clear. Abrahamsson and co-workers investigated disintegration times and drug release profiles of different hydrogel matrix tablets containing metoprolol tartrate in vitro and in vivo (dogs) under fasted and fed state conditions (181). They reported that a film, which was formed mainly by proteins that precipitate on the surface of the tablets, caused delayed disintegration and drug release in the fed state. However, in studies testing the same tablets in vivo in Labrador dogs, co-administration with a nutrient drink delayed in vivo disintegration, whereas co-administration with a high-fat meal did not cause this effect (181). Franek and colleagues published further data on HPMC tablets containing caffeine, which were tested in vitro in the presence of Ensure® Plus. It was shown again in this study that drug release was 38 delayed under simulated fed gastric conditions. This effect was also attributed to the formation of a hydrophobic film around the tablets(182). In contrast to the findings by Abrahamsson et al. (181), Williams and colleagues demonstrated with the aid of MRI and confocal fluorescence imaging that a fat (rather than protein) layer was formed around HPMC matrix tablets in the presence of different fat emulsions, and that this may also affect in vitro drug release. In this case, it was not protein, which was responsible for the film formation as this layer was not formed in low-fat milk, but was formed in protein-free Intralipid® (Fig. 12) (183). Whilst these in vitro studies show consistent film formation, it is unknown whether the formation of a hydrophobic film around solid oral dosage forms has in vivo relevance as the intense shear forces arising in the stomach may quickly destroy the film. This assumption is supported by experiments in dogs, which were performed by Kalantzi and co-workers (184). In this study, in vitro drug release in USP apparatus II and IV from two immediate release tablets with paracetamol was delayed regardless of the shear conditions, but this effect could not be confirmed in vivo. In another study performed by Jain and co-workers, it was shown by Magnetic Marker Monitoring that erosion rates of HPMC matrix tablets were much faster (and not slower) under postprandial conditions when compared to fasted conditions (185). Fig. 12 Deposition of fat on the surface of HPMC matrix tablets in milk (left) and Intralipid® 30% (right) over time investigated with confocal fluorescence imaging. Reprinted from Williams et al. (183) with permission from Elsevier. 39 Conclusion Lipids in the stomach, and in the intestine, have manifold effects on human gastrointestinal function and can thus affect drug release from oral dosage forms in different ways. For highly lipophilic drugs (e.g. cyclosporine), lipids typically enhance drug absorption (positive food effect) and this is usually attributed to increases in intestinal drug solubilisation in mixed micelles comprising bile salts, phospholipids and lipid digestion products. However, events within the stomach may also impact drug release and solubility, either directly by changes in gastric pH, solution conditions and emptying rates, or indirectly via downstream changes to intestinal conditions. Recent progress in the in vitro assessment of lipid-based formulations has helped to better understand the interactions between gastric lipid processing and oral drug delivery and has also promoted the development of more physiologically relevant in vitro test methods. As such several biorelevant test media are now available to assess possible effects of food (and digested food) in the stomach, including those caused by lipids and their lipolysis products. While in vitro simulation of intestinal lipid digestion is possible using pancreatic extracts as enzyme sources, simulation of gastric lipolysis remains challenging because of limited access to a reliable and reproducible source of gastric lipase. This has limited development of standard methods for the in vitro testing of gastric drug solubility and dissolution under physiologically relevant conditions. Recent efforts to progress commercial sources of gastric lipase will hopefully address this limitation and provide the means to better understand food effects on oral drug bioavailability. Acknowledgement This work was supported by a fellowship within the Postdoc-Program of the German Academic Exchange Service (DAAD). 40 References 1. Fleisher D, Li C, Zhou Y, Pao L-H, Karim A. Drug, Meal and Formulation Interactions Influencing Drug Absorption After Oral Administration: Clinical Implications. Clin Pharmacokinet. 1999;36(3):233–54. 2. Williams HD, Trevaskis NL, Charman SA, Shanker RM, Charman WN, Pouton CW, et al. Strategies to address low drug solubility in discovery and development. Pharmacol Rev. 2013;65(1):315–499. 3. Charman WN, Porter CJH, Mithani S, Dressman JB. Physiochemical and physiological mechanisms for the effects of food on drug absorption: the role of lipids and pH. J Pharm Sci. 1997;86(3):269–82. 4. Koziolek M, Garbacz G, Neumann M, Weitschies W. Simulating the postprandial stomach: Physiological considerations for dissolution and release testing. Mol Pharm. 2013;10(5):1610– 22. 5. Koziolek M, Grimm M, Garbacz G, Kühn JP, Weitschies W. Intragastric volume changes after intake of a high-caloric, high-fat standard breakfast in healthy human subjects investigated by MRI. Mol Pharm. 2014/04/05. 2014;11(5):1632–9. 6. Porter CJH, Trevaskis NL, Charman WN. Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nat Rev Drug Discov. 2007;6(3):231–48. 7. Bakala-N’Goma JC, Amara S, Dridi K, Jannin V, Carrière F. Understanding the lipid- digestion processes in the GI tract before designing lipid-based drug-delivery systems. Ther Deliv. 2012;3(1):105–24. 8. Feinle-Bisset C, Azpiroz F. Dietary lipids and functional gastrointestinal disorders. Am J Gastroenterol. 2013;108(5):737–47. 9. Gargouri Y, Moreau H, Verger R. Gastric lipases: biochemical and physiological studies. Biochim Biophys Acta. 1989;1006(3):255–71. 10. Bernbäck S, Bläckberg L, Hernell O. Fatty acids generated by gastric lipase promote human milk triacylglycerol digestion by pancreatic colipase-dependent lipase. Biochim Biophys Acta. 1989;1001(3):286–93. 11. Armand M. Lipases and lipolysis in the human digestive tract: where do we stand? Curr Opin Clin Nutr Metab Care. 2007;10(2):156–64. 12. Gargouri Y, Pieroni G, Rivière C, Lowe PA, Saunière JF, Sarda L, et al. Importance of human gastric lipase for intestinal lipolysis: an in vitro study. Biochim Biophys Acta. 1986;879(3):419–23. 13. Lemarié F, Cavalier J-F, Garcia C, Boissel F, Point V, Catheline D, et al. Effect of preduodenal lipase inhibition in suckling rats on dietary octanoic acid (C8:0) gastric absorption and plasma octanoylated ghrelin concentration. Biochim Biophys Acta. 2016;1861(9):1111–20. 41 14. Carrière F, Barrowman JA, Verger R, Laugier R. Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans. Gastroenterology. 1993/09/01. 1993;105(3):876–88. 15. Gargouri Y, Sarda L, Ferrato F, Verger R. Kinetic assay of human gastric lipase on short and long chain triacylglycerol emulsions. Gastroenterology. 1986;19–22. 16. Pafumi Y, Lairon D, De La Porte PL, Juhel C, Storch J, Hamosh M, et al. Mechanisms of inhibition of triacylglycerol hydrolysis by human gastric lipase. J Biol Chem. 2002;277(31):28070–9. 17. Bourlieu C, Ménard O, De La Chevasnerie A, Sams L, Rousseau F, Madec MN, et al. The structure of infant formulas impacts their lipolysis, proteolysis and disintegration during in vitro gastric digestion. Food Chem. 2015;182:224–35. 18. De Oliveira SC, Bourlieu C, Ménard O, Bellanger A, Henry G, Rousseau F, et al. Impact of pasteurization of human milk on preterm newborn in vitro digestion: Gastrointestinal disintegration, lipolysis and proteolysis. Food Chem. 2016;211:171–9. 19. de Oliveira SC, Bellanger A, Olivia M, Pladys P, Le Gouar Y, Dirson E, et al. Impact of homogenization of pasteurized human milk on gastric digestion in the preterm infant: A randomized controlled trial. Clin Nutr ESPEN. 2017;20:1–11. 20. Moreau H, Bernadac A, Gargouri Y, Benkouka F, Laugier R, Verger R. Immunocytolocalization of human gastric lipase in chief cells of the fundic mucosa. Histochemistry. 1989;91(5):419–23. 21. Moreau H, Laugier R, Gargouri Y, Ferrato F, Verger R. Human preduodenal lipase is entirely of gastric fundic origin. Gastroenterology. 1988;95(5):1221–6. 22. Borovicka J, Schwizer W, Mettraux C, Kreiss C, Remy B, Asal K, et al. Regulation of gastric and pancreatic lipase secretion by CCK and cholinergic mechanisms in humans. Am J Physiol. 1997;273(2 Pt 1):G374–80. 23. Sams L, Paume J, Giallo J, Carrière F. Relevant pH and lipase for in vitro models of gastric digestion. Food Funct. 2015; 24. Armand M, Hamosh M, DiPalma JS, Gallagher J, Benjamin SB, Philpott JR, et al. Dietary fat modulates gastric lipase activity in healthy humans. Am J Clin Nutr. 1995/07/01. 1995;62(1):74–80. 25. Carrière F, Renou C, Lopez V, De Caro J, Ferrato F, Lengsfeld H, et al. The specific activities of human digestive lipases measured from the in vivo and in vitro lipolysis of test meals. Gastroenterology. 2000;119(4):949–60. 26. Carrière F, Renou C, Ransac S, Lopez V, De Caro J, Ferrato F, et al. Inhibition of gastrointestinal lipolysis by Orlistat during digestion of test meals in healthy volunteers. Am J Physiol Gastrointest Liver Physiol. 2001;281(1):G16–28. 27. Carrière F, Grandval P, Renou C, Palomba A, Priéri F, Giallo J, et al. Quantitative study of 42 digestive enzyme secretion and gastrointestinal lipolysis in chronic pancreatitis. Clin Gastroenterol Hepatol. 2005;3(1):28–38. 28. Armand M, Borel P, Dubois C, Senft M, Peyrot J, Salducci J, et al. Characterization of emulsions and lipolysis of dietary lipids in the human stomach. Am J Physiol. 1994;266(3 Pt 1):G372-81. 29. Renou C, Carrière F, Ville E, Grandval P, Joubert-Collin M, Laugier R. Effects of Lansoprazole on Human Gastric Lipase Secretion and Intragastric Lipolysis in Healthy Human Volunteers. Digestion. 2001;63(4):207–13. 30. Capolino P, Guérin C, Paume J, Giallo J, Ballester J-M, Cavalier J-F, et al. In Vitro Gastrointestinal Lipolysis: Replacement of Human Digestive Lipases by a Combination of Rabbit Gastric and Porcine Pancreatic Extracts. Food Dig. 2011;2(1–3):43–51. 31. Carrière F, Rogalska E, Cudrey C, Ferrato F, Laugier R, Verger R. In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases. Bioorg Med Chem. 1997;5(2):429–35. 32. Mottram HR, Evershed RP. Elucidation of the composition of bovine milk fat triacylglycerols using high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry. J Chromatogr A. 2001;926(2):239–53. 33. Blasi F, Montesano D, De Angelis M, Maurizi A, Ventura F, Cossignani L, et al. Results of stereospecific analysis of triacylglycerol fraction from donkey, cow, ewe, goat and buffalo milk. J Food Compost Anal. 2008;21(1):1–7. 34. Beisson F, Tiss A, Rivière C, Verger R. Methods for lipase detection and assay: a critical review. Eur J Lipid Sci Tech. 2000;102(2):133–53. 35. Carrière F, Moreau H, Raphel V, Laugier R, Benicourt C, Junien JL, et al. Purification and biochemical characterization of dog gastric lipase. Eur J Biochem. 1991;202(1):75–83. 36. Rogalska E, Ransac S, Verger R. Lipases, Stereoselectivity of Lipases II. Stereoselective Hydrolysis of Triglycerides by Gastric and Pancreatic Lipases. J Biol Chem. 1990;265(33):20271–6. 37. Hunter JE. Studies on Effects of Dietary Fatty Acids as Related to Their Position on Triglycerides. Lipids. 2001;36(7):655–68. 38. Lindmark-Månsson H. Fatty acids in bovine milk fat. Food Nutr Res. 2008;52:1–3. 39. Jensen RG. The composition of bovine milk lipids: January 1995 to December 2000. J Dairy Sci. 2002;85(2):295–350. 40. Roussel A, Canaan S, Egloff M-P, Rivière M, Dupuis L, Verger R, et al. Crystal structure of human gastric bipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest. J Biol Chem. 1999;274(24):16995–7002. 41. Roussel A, Miled N, Berti-Dupuis L, Rivière M, Spinelli S, Berna P, et al. Crystal structure of the open form of dog gastric lipase in complex with a phosphonate inhibitor. J Biol Chem. 43 2002;277(3):2266–74. 42. Bernbäck S, Bläckberg L. Human gastric lipase. The N-terminal tetrapeptide is essential for lipid binding and lipase activity. Eur J Biochem. 1989;182(3):495–9. 43. Sams L, Amara S, Chakroun A, Coudre S, Paume J, Giallo J, et al. Constitutive expression of human gastric lipase in Pichia pastoris and site-directed mutagenesis of key lid-stabilizing residues. Biochim Biophys Acta. 2017;1862(10):1025–34. 44. Chahinian H, Snabe T, Attias C, Fojan P, Petersen SB, Carrière F. How gastric lipase, an interfacial enzyme with a Ser-His-Asp catalytic triad, acts optimally at acidic pH. Biochemistry. 2006;45(3):993–1001. 45. Williams HD, Sassene P, Kleberg K, Bakala-N’Goma JC, Calderone M, Jannin V, et al. Establishment of standardized in vitro tests for lipid-based formulations, part 1: Method parameterization and comparison of in vitro digestion profiles across a range. J Pharm Sci. 2012;101(9):3360–80. 46. Ville E, Carrière F, Renou C, Laugier R. Physiological study of pH stability and sensitivity to pepsin of human gastric lipase. Digestion. 2002;5:73–81. 47. Camacho-Ruiz MA, Mateos-Díaz JC, Carrière F, Rodriguez JA. A broad pH range indicator- based spectrophotometric assay for true lipases using tributyrin and tricaprylin. J Lipid Res. 2015;56(5):1057–67. 48. Gargouri Y, Pieroni G, Lowe PA, Sarda L, Verger R. Human gastric lipase. The effect of amphiphiles. Eur J Biochem. 1986;156:305–10. 49. FDA. Guidance for Industry: Food-Effect Bioavailability and Fed Bioequivalence Studies. 2002. 50. Singh H, Gallier S. Nature’s complex emulsion: The fat globules of milk. Food Hydrocoll. 2017;68:81–9. 51. Cortot A, Phillips SF, Malagelada JR. Gastric emptying of lipids after ingestion of a solid- liquid meal in humans. Gastroenterology. 1981;80(5 pt 1):922–7. 52. Cortot A, Phillips SF, Malagelada JR. Gastric emptying of lipids after ingestion of an homogenized meal. Gastroenterology. 1979;76(5):939–44. 53. Armand M, Pasquier B, André M, Borel P, Senft M, Peyrot J, et al. Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract. Am J Clin Nutr. 1999;70(6):1096–106. 54. Koziolek M, Schneider F, Grimm M, Modeβ C, Seekamp A, Roustom T, et al. Intragastric pH and pressure profiles after intake of the high-caloric, high-fat meal as used for food effect studies. J Control Release. 2015;220(Part A):71–8. 55. Golding M, Wooster TJ. The influence of emulsion structure and stability on lipid digestion. Curr Opin Colloid Interface Sci. 2010;15(1–2):90–101. 56. Singh H, Ye A, Horne D. Structuring food emulsions in the gastrointestinal tract to modify 44 lipid digestion. Prog Lipid Res. 2009;48(2):92–100. 57. Golding M, Wooster TJ, Day L, Xu M, Lundin L, Keogh J, et al. Impact of gastric structuring on the lipolysis of emulsified lipids. Soft Matter. 2011;7(7):3513. 58. Schulze K. Imaging and modelling of digestion in the stomach and the duodenum. Neurogastroenterol Motil. 2006/02/21. 2006;18(3):172–83. 59. Ferrua MJ, Kong F, Singh RP. Computational modeling of gastric digestion and the role of food material properties. Trends Food Sci Technol. 2011;22(9):480–91. 60. Ferrua MJ, Singh RP. Computational modelling of gastric digestion: Current challenges and future directions. Curr Opin Food Sci. 2015;4:116–23. 61. Hussein MO, Hoad CL, Wright J, Singh G, Stephenson MC, Cox EF, et al. Fat Emulsion Intragastric Stability and Droplet Size Modulate Gastrointestinal Responses and Subsequent Food Intake in Young Adults 1 – 4. J Nutr. 2015;1–8. 62. Marciani L, Wickham MSJ, Bush D, Faulks RM, Wright J, Fillery-Travis AJ, et al. Magnetic resonance imaging of the behaviour of oil-in-water emulsions in the gastric lumen of man. Br J Nutr. 2007;95(2):331. 63. Weitschies W, Wilson CG. In vivo imaging of drug delivery systems in the gastrointestinal tract. Int J Pharm. 2011/08/09. 2011;417(1–2):216–26. 64. Dillard S, Krishnan S, Udaykumar HS. Mechanics of flow and mixing at antroduodenal junction. World J Gastroenterol. 2007/04/26. 2007;13(9):1365–71. 65. Boulby P, Moore R, Gowland PA, Spiller RC. Fat delays emptying but increases forward and backward antral flow as assessed by flow-sensitive magnetic resonance imaging. Neurogastroenterol Motil. 1999;11(1):27–36. 66. Hausken T, Li XN, Goldman B, Leotta D, Odegaard S, Martin RW. Quantification of gastric emptying and duodenogastric reflux stroke volumes using three-dimensional guided digital color Doppler imaging. Eur J Ultrasound. 2001/08/23. 2001;13(3):205–13. 67. Efentakis M, Dressman JB. Gastric juice as a dissolution medium: Surface tension and pH. Eur J Drug Metab Pharmacokinet. 1998;23(2):97–102. 68. Kalantzi L, Goumas K, Kalioras V, Abrahamsson B, Dressman JB, Reppas C. Characterization of the human upper gastrointestinal contents under conditions simulating bioavailability/bioequivalence studies. Pharm Res. 2006;23(1):165–76. 69. Bergström CAS, Holm R, Jørgensen SA, Andersson SBE, Artursson P, Beato S, et al. Early pharmaceutical profiling to predict oral drug absorption: Current status and unmet needs. Eur J Pharm Sci. 2013/11/13. 2014;57:173–99. 70. Vors C, Capolino P, Guérin C, Meugnier E, Pesenti S, Chauvin M-A, et al. Coupling in vitro gastrointestinal lipolysis and Caco-2 cell cultures for testing the absorption of different food emulsions. Food Funct. 2012;3(5):537. 71. Couëdelo L, Amara S, Lecomte M, Meugnier E, Monteil J, Fonseca L, et al. Impact of various 45 emulsifiers on ALA bioavailability and chylomicron synthesis through changes in gastrointestinal lipolysis. Food Funct. 2015;6(5):1726–35. 72. Delorme V, Dhouib R, Canaan S, Fotiadu F, Carrière F, Cavalier JF. Effects of surfactants on lipase structure, activity, and inhibition. Pharm Res. 2011;28(8):1831–42. 73. Gargouri Y, Pieroni G, Ferrato F, Verger R. Human gastric lipase: A kinetic study with dicaprin monolayers. Eur J Biochem. 1987;169(1):125–9. 74. de La Fournière L, Ivanova MG, Blond JP, Carrière F, Verger R. Surface behaviour of human pancreatic and gastric lipases. Colloids Surf B Biointerfaces. 1994;2(6):585–93. 75. Scheuble N, Lussi M, Geue T, Carrière F, Fischer P. Blocking Gastric Lipase Adsorption and Displacement Processes with Viscoelastic Biopolymer Adsorption Layers. Biomacromolecules. 2016;17(10):3328–37. 76. Pasquier B, Armand M, Castelain C, Guillon F, Borel P, Lafont H, et al. Evaluation and lipolysis of triacylglycerols are altered by viscous soluble dietary fibres in acidic gastric medium in vitro. Biochem J. 1996;314:269–75. 77. Karhunen LJ, Juvonen KR, Huotari A, Purhonen AK, Herzig KH. Effect of protein, fat, carbohydrate and fibre on gastrointestinal peptide release in humans. Regul Pept. 2008;149(1– 3):70–8. 78. Delzenne N, Blundell J, Brouns F, Cunningham K, De Graaf K, Erkner A, et al. Gastrointestinal targets of appetite regulation in humans: ILSI Supplement. Obes Rev. 2010;11(3):234–50. 79. Camilleri M. Integrated Upper Gastrointestinal Response to Food Intake. Gastroenterology. 2006;131(2):640–58. 80. Kwiatek MA, Menne D, Steingoetter A, Goetze O, Forras-Kaufman Z, Kaufman E, et al. Effect of meal volume and calorie load on postprandial gastric function and emptying: studies under physiological conditions by combined fiber-optic pressure measurement and MRI. Am J Physiol Gastrointest Liver Physiol. 2009/09/26. 2009;297(5):G894-901. 81. Goetze O, Steingoetter A, Menne D, van der Voort IR, Kwiatek MA, Boesiger P, et al. The effect of macronutrients on gastric volume responses and gastric emptying in humans: A magnetic resonance imaging study. Am J Physiol Gastrointest Liver Physiol. 2006/08/29. 2007;292(1):G11-7. 82. Doran S, Jones KL, Andrews JM, Horowitz M. Effects of meal volume and posture on gastric emptying of solids and appetite. Am J Physiol Regul Integr Comp Physiol. 1998;275(5):R1712–8. 83. Hunt JN, Smith JL, Jiang CL. Effect of meal volume and energy density on the gastric emptying of carbohydrates. Gastroenterology. 1985;89(6):1326–30. 84. Calbet JA, MacLean DA. Role of caloric content on gastric emptying in humans. J Physiol. 1997;498(Pt 2):553–9. 46 85. Armand M, Borel P, Pasquier B, Dubois C, Senft M, André M, et al. Physicochemical characteristics of emulsions during fat digestion in human stomach and duodenum. Am J Physiol. 1996;271(1 Pt 1):G172-83. 86. Yasui-Furukori N, Takahata T, Kondo T, Mihara K, Kaneko S, Tateishi T. Time effects of food intake on the pharmacokinetics and pharmacodynamics of quazepam. Br J Clin Pharmacol. 2003;55(4):382–8. 87. Saruwatari J, Yasui-Furukori N, Inoue Y, Kaneko S. Effect of dose timing in relation to food intake on systemic exposure to blonanserin. Eur J Clin Pharmacol. 2010;66(9):899–902. 88. Edelbroek M, Horowitz M, Maddox A, Bellen J. Gastric emptying and intragastric distribution of oil in the presence of a liquid or a solid meal. J Nucl Med. 1992;33(7):1283–90. 89. Boulby P, Gowland P, Adams V, Spiller RC. Use of echo planar imaging to demonstrate the effect of posture on the intragastric distribution and emptying of an oil/water meal. Neurogastroenterol Motil. 1997;9(1):41–7. 90. Marciani L, Faulks RM, Wickham MSJ, Bush D, Pick B, Wright J, et al. Effect of intragastric acid stability of fat emulsions on gastric emptying, plasma lipid profile and postprandial satiety. Br J Nutr. 2008/08/06. 2009;101(6):919–28. 91. Kunz P, Feinle-Bisset C, Faas H, Boesiger P, Fried M, Steingötter A, et al. Effect of ingestion order of the fat component of a solid meal on intragastric fat distribution and gastric emptying assessed by MRI. J Magn Reson Imaging. 2005;21(4):383–90. 92. Little TJ, Feinle-Bisset C. Effects of dietary fat on appetite and energy intake in health and obesity - Oral and gastrointestinal sensory contributions. Physiol Behav. 2011;104(4):613–20. 93. Hildebrand P, Petrig C, Burckhardt B, Ketterer S, Lengsfeld H, Fleury A, et al. Hydrolysis of dietary fat by pancreatic lipase stimulates cholecystokinin release. Gastroenterology. 1998;114(1):123–9. 94. Sundaresan S, Shahid R, Riehl TE, Chandra R, Nassir F, Stenson WF, et al. CD36-dependent signaling mediates fatty acid-induced gut release of secretin and cholecystokinin. FASEB J. 2013;27(3):1191–202. 95. Laugerette F, Passilly-Degrace P, Patris B, Niot I, Febbraio M, Montmayeur JP, et al. CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions. J Clin Invest. 2005;115(11):3177–84. 96. Feltrin KL, Little TJ, Meyer JH, Horowitz M, Smout AJPM, Wishart J, et al. Effects of intraduodenal fatty acids on appetite, antropyloroduodenal motility, and plasma CCK and GLP-1 in humans vary with their chain length. Am J Physiol Gastrointest Liver Physiol. 2004;287(3):R524–33. 97. Little TJ, Russo A, Meyer JH, Horowitz M, Smyth DR, Bellon M, et al. Free Fatty Acids Have More Potent Effects on Gastric Emptying, Gut Hormones, and Appetite Than Triacylglycerides. Gastroenterology. 2007;133(4):1124–31. 47 98. McLaughlin J, Grazia Lucà M, Jones MN, D’Amato M, Dockray GJ, Thompson DG. Fatty acid chain length determines cholecystokinin secretion and effect on human gastric motility. Gastroenterology. 1999;116(1):46–53. 99. Fukumori R, Sugino T, Shingu H, Moriya N, Kobayashi H, Hasegawa Y, et al. Ingestion of medium chain fatty acids by lactating dairy cows increases concentrations of plasma ghrelin. Domest Anim Endocrinol. 2013;45(4):216–23. 100. Cummings DE, Shannon MH. Roles for ghrelin in the regulation of appetite and body weight. Arch Surg. 2003;138(4):389–96. 101. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth- hormone-releasing acylated peptide from stomach. Nature. 1999;402(6762):656–60. 102. Al Massadi O, Tschöp MH, Tong J. Ghrelin acylation and metabolic control. Peptides. 2011;32(11):2301–8. 103. Egelrud T, Ollvecrona T, Helander H. Studies on Gastric Absorption of Lipids in the Suckling Rat. Scand J Gastroenterol. 1971;6(4):329–33. 104. Nishi Y, Hiejima H, Hosoda H, Kaiya H, Mori K, Fukue Y, et al. Ingested medium-chain fatty acids are directly utilized for the acyl modification of ghrelin. Endocrinology. 2005;146(5):2255–64. 105. Nishi Y, Mifune H. Chapter Nineteen – Ghrelin Acylation by Ingestion of Medium-Chain Fatty Acids. In: Methods in Enzymology. 2012. p. 303–15. 106. Hunt JN, Knox MT. A relation between the chain length of fatty acids and the slowing of gastric emptying. J Physiol. 1968;194(2):327–36. 107. van Avesaat M, Troost FJ, Ripken D, Hendriks HF, Masclee a a M. Ileal brake activation: macronutrient specific effects on eating behavior? Int J Obes. 2014;(April):1–37. 108. Van Citters GW, Lin HC. The ileal brake: a fifteen-year progress report. Curr Rep Gastroenterol. 1999;1(5):404–9. 109. Cuche G, Cuber JC, Malbert CH. Ileal short-chain fatty acids inhibit gastric motility by a humoral pathway. Am J Physiol Gastrointest Liver Physiol. 2000;279(5):G925–30. 110. Hellström PM, Grybäck P, Jacobsson H. The physiology of gastric emptying. Best Pr Res Clin Anaesthesiol. 2006;20(3):397–407. 111. Reppas C, Karatza E, Goumas C, Markopoulos C, Vertzoni M. Characterization of Contents of Distal Ileum and Cecum to Which Drugs/Drug Products are Exposed during Bioavailability/Bioequivalence Studies in Healthy Adults. Pharm Res. 2015;32(10):3338–49. 112. Dressman JB, Berardi RR, Dermentzoglou LC, Russell TL, Schmaltz SP, Barnett JL, et al. Upper gastrointestinal (GI) pH in young, healthy men and women. Pharm Res. 1990/07/01. 1990;7(7):756–61. 113. Simonian HP, Vo L, Doma S, Fisher RS, Parkman HP. Regional postprandial differences in pH within the stomach and gastroesophageal junction. Dig Dis Sci. 2006/01/18. 48 2005;50(12):2276–85. 114. Schubert ML, Peura DA. Control of gastric acid secretion in health and disease. Gastroenterology. 2008/05/14. 2008;134(7):1842–60. 115. Piper DW, Fenton BH. pH stability and activity curves of pepsin with special reference to their clinical importance. Gut. 1965;6(5):506–8. 116. Borovicka J, Schwizer W, Guttmann G, Hartmann D, Kosinski M, Wastiel C, et al. Role of lipase in the regulation of postprandial gastric acid secretion and emptying of fat in humans: a study with orlistat, a highly specific lipase inhibitor. Gut. 2000;46(6):774–81. 117. Degen L, Matzinger D, Drewe J, Nisslé S, Maecke H, Lengsfeld H, et al. Role of free fatty acids in regulating gastric emptying and gallbladder contraction. Digestion. 2007;74(3– 4):131–9. 118. Dressman JB, Vertzoni M, Goumas K, Reppas C. Estimating drug solubility in the gastrointestinal tract. Adv Drug Deliv Rev. 2007/06/30. 2007;59(7):591–602. 119. Hörter D, Dressman JB. Influence of physicochemical properties on dissolution of drugs in the gastrointestinal tract. Adv Drug Deliv Rev. 2001/03/22. 2001;46(1–3):75–87. 120. Feeney OM, Crum MF, McEvoy CL, Trevaskis NL, Williams HD, Pouton CW, et al. 50 years of oral lipid-based formulations: Provenance, progress and future perspectives. Adv Drug Deliv Rev. 2016; 121. Sahbaz Y, Williams HD, Nguyen TH, Saunders J, Ford L, Charman SA, et al. Transformation of poorly water-soluble drugs into lipophilic ionic liquids enhances oral drug exposure from lipid based formulations. Mol Pharm. 2015;12(6):1980–91. 122. Mu H, Holm R, Müllertz A. Lipid-based formulations for oral administration of poorly water- soluble drugs. Int J Pharm. 2013;453(1):215–24. 123. Pouton CW. Lipid formulations for oral administration of drugs: Non-emulsifying, self- emulsifying and “self-microemulsifying” drug delivery systems. Eur J Pharm Sci. 2000;11(SUPPL. 2):93–8. 124. Pouton CW, Porter CJH. Formulation of lipid-based delivery systems for oral administration: Materials, methods and strategies. Adv Drug Deliv Rev. 2008;60(6):625–37. 125. Bakala-N’Goma JC, Williams HD, Sassene P, Kleberg K, Calderone M, Jannin V, et al. Toward the establishment of standardized in vitro tests for lipid-based formulations. 5. Lipolysis of representative formulations by gastric lipase. Pharm Res. 2015;32(4):1279–87. 126. EMA. Guideline on the Investigation of Drug Interactions [Internet]. CPMP/EWP/560/95/Rev. 1 Corr.* Committee for Human Medicinal Products (CHMP); 2012. 127. Carrière F. Impact of gastrointestinal lipolysis on oral lipid-based formulations and bioavailability of lipophilic drugs. Biochimie. 2016;125:297–305. 128. Fernandez S, Jannin V, Rodier JD, Ritter N, Mahler B, Carrière F. Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides 49 and PEG esters. Biochim Biophys Acta. 2007;1771(5):633–40. 129. Chatzidaki MD, Mateos-Diaz E, Leal-Calderon F, Xenakis A, Carrière F. Water-in-Oil microemulsions versus emulsions as carriers of hydroxytyrosol: an in vitro gastrointestinal lipolysis study using the pHstat technique. Food Funct. 2016;7:2258–69. 130. Kanicky JR, Shah DO. Effect of Degree, Type, and Position of Unsaturation on the pKa of Long-Chain Fatty Acids. J Colloid Interface Sci. 2002;256(1):201–7. 131. Egret-Charlier M, Sanson A, Ptak M, Bouloussa O. Ionization of Fatty Acids At the Lipid - Water Inter Face. FEBS Lett. 1978;89(2):313–6. 132. Kanicky JR, Shah DO. Effect of premicellar aggregation on the pKa of fatty acid soap solutions. Langmuir. 2003;19(6):2034–8. 133. Cistola DP, Hamilton JA, Jackson D, Small DM. Ionization and phase behavior of fatty acids in water: application of the Gibbs phase rule. Biochem. 1988;27(6):1881–8. 134. Morigaki K, Walde P. Fatty acid vesicles. Curr Opin Colloid Interface Sci. 2007;12(2):75–80. 135. Larsen AT, Sassene P, Müllertz A. In vitro lipolysis models as a tool for the characterization of oral lipid and surfactant based drug delivery systems. Int J Pharm. 2011;417(1–2):245–55. 136. Williams HD, Anby MU, Sassene P, Kleberg K, Bakala-N’Goma JC, Calderone M, et al. Toward the establishment of standardized in vitro tests for lipid-based formulations. 2. The effect of bile salt concentration and drug loading on the performance of type. Mol Pharm. 2012;9(11):3286–300. 137. Devraj R, Williams HD, Warren DB, Müllertz A, Porter CJH, Pouton CW. In vitro digestion testing of lipid-based delivery systems: calcium ions combine with fatty acids liberated from triglyceride rich lipid solutions to form soaps and reduce the solubilization capacity of colloidal digestion products. Int J Pharm. 2013;441(1–2):323–33. 138. Li Y, Hu M, McClements DJ. Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pH-stat method. Food Chem. 2011;126(2):498–505. 139. Christophersen PC, Christiansen ML, Holm R, Kristensen J, Jacobsen J, Abrahamsson B, et al. Fed and fasted state gastro-intestinal in vitro lipolysis: In vitro in vivo relations of a conventional tablet, a SNEDDS and a solidified SNEDDS. Eur J Pharm Sci. 2014;57:232–9. 140. McAllister M. Dynamic Dissolution: A Step Closer to Predictive Dissolution Testing? Mol Pharm. 2010;7(5):1374–87. 141. Koziolek M, Garbacz G, Neumann M, Weitschies W. Simulating the postprandial stomach: biorelevant test methods for the estimation of intragastric drug dissolution. Mol Pharm. 2013/05/10. 2013;10(6):2211–21. 142. Guerra A, Etienne-Mesmin L, Livrelli V, Denis S, Blanquet-Diot S, Alric M. Relevance and challenges in modeling human gastric and small intestinal digestion. Trends Biotechnol. 2012;30(11):591–600. 50 143. McClements DJ, Li Y. Review of in vitro digestion models for rapid screening of emulsion- based systems. Food Funct. 2010;1(1):32–59. 144. Baxevanis F, Kuiper J, Fotaki N. Fed-state gastric media and drug analysis techniques: Current status and points to consider. Eur J Pharm Biopharm. 2016;107:234–48. 145. Klein S, Butler J, Hempenstall JM, Reppas C, Dressman JB. Media to simulate the postprandial stomach I. Matching the physicochemical characteristics of standard breakfasts. J Pharm Pharmacol. 2004;56(5):605–10. 146. Radwan A, Amidon GL, Langguth P. Mechanistic investigation of food effect on disintegration and dissolution of BCS class III compound solid formulations: the importance of viscosity. Biopharm Drug Dispos. 2012;33:403–16. 147. Radwan A, Ebert S, Amar A, Münnemann K, Wagner M, Amidon GL, et al. Mechanistic Understanding of Food Effects: Water Diffusivity in Gastrointestinal Tract Is an Important Parameter for the Prediction of Disintegration of Solid Oral Dosage Forms. Mol Pharm. 2013;10(6):2283–90. 148. van den Abeele J, Brouwers J, Mattheus R, Tack J, Augustijns P. Gastrointestinal Behavior of Weakly Acidic BCS Class II Drugs in Man - Case Study Diclofenac Potassium. J Pharm Sci. 2015;n/a-n/a. 149. Diakidou A, Vertzoni M, Dressman JB, Reppas C. Estimation of intragastric drug solubility in the fed state: comparison of various media with data in aspirates. Biopharm Drug Dispos. 2009/08/01. 2009;30(6):318–25. 150. Macheras P, Koupparis M, Tsaprounis C. Drug dissolution studies in milk using the automated flow injection serial dynamic dialysis technique. Int J Pharm. 1986;33(1–3):125–36. 151. Macheras PE, Koupparis MA, Antimisiaris SG. Effect of temperature and fat content on the solubility of hydrochlorothiazide and chlorothiazide in milk. J Pharm Sci. 1989/11/01. 1989;78(11):933–6. 152. Macheras PE, Koupparis MA, Antimisiaris SG. Drug binding and solubility in milk. Pharm Res. 1990/05/01. 1990;7(5):537–41. 153. Lopez C. Milk fat globules enveloped by their biological membrane: Unique colloidal assemblies with a specific composition and structure. Curr Opin Colloid Interface Sci. 2011;16(5):391–404. 154. Michalski MC. Specific molecular and colloidal structures of milk fat affecting lipolysis, absorption and postprandial lipemia. Eur J Lipid Sci Tech. 2009;111(5):413–31. 155. Parodi PW. Positional distribution of fatty acids in triglycerides from milk of several species of mammals. Lipids. 1982;17(6):437–42. 156. Breckenridge WC, Kuksis A. Specific distribution of short-chain fatty acids in molecular distillates of bovine milk fat. J Lipid Res. 1968;9(3):388–93. 157. Jantratid E, Janssen N, Reppas C, Dressman JB. Dissolution media simulating conditions in 51 the proximal human gastrointestinal tract: an update. Pharm Res. 2008/04/12. 2008;25(7):1663–76. 158. Markopoulos C, Andreas CJ, Vertzoni M, Dressman JB, Reppas C. In-vitro simulation of luminal conditions for evaluation of performance of oral drug products: Choosing the appropriate test media. Eur J Pharm Biopharm. 2015;93:173–82. 159. Kossena GA, Charman WN, Wilson CG, O’Mahony B, Lindsay B, Hempenstall JM, et al. Low dose lipid formulations: effects on gastric emptying and biliary secretion. Pharm Res. 2007;24(11):2084–96. 160. Charman WN, Rogge MC, Boddy AW, Berger BM. Effect of Food and a Monoglyceride Emulsion Formulation on Danazol Bioavailability. J Clin Pharmacol. 1993;33(4):381–6. 161. van Aken GA, Bomhof E, Zoet FD, Verbeek M, Oosterveld A. Differences in in vitro gastric behaviour between homogenized milk and emulsions stabilised by Tween 80, whey protein, or whey protein and caseinate. Food Hydrocoll. 2011;25(4):781–8. 162. Moreau H, Abergel C, Carrière F, Ferrato F, Fontecilla-Camps JC, Cambillau C, et al. Isoform purification of gastric lipases. Towards crystallization. J Mol Biol. 1992;225(1):147–53. 163. Aoubala M, Douchet I, Laugier R, Hirn M, Verger R, De Caro A. Purification of human gastric lipase by immunoaffinity and quantification of this enzyme in the duodenal contents using a new ELISA procedure. Biochemistry. 1993;1169(2):183–8. 164. Moreau H, Gargouri Y, Lecat D, Junien JL, Verger R. Purification, characterization and kinetic properties of the rabbit gastric lipase. Biochim Biophys Acta. 1988;960:286–93. 165. Wicker-Planquart C, Canaan S, Rivière M, Dupuis L, Verger R. Expression in insect cells and purification of a catalytically active recombinant human gastric lipase. Protein Eng. 1996;9(12):1225–32. 166. Gruber V, Berna PP, Arnaud T, Bournat P, Clément C, Mison D, et al. Large-scale production of a therapeutic protein in transgenic tobacco plants: Effect of subcellular targeting on quality of a recombinant dog gastric lipase. Mol Breed. 2001;7(4):329–40. 167. Vardakou M, Sainsbury F, Rigby N, Mulholland F, Lomonossoff GP. Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system. Protein Expr Purif. 2012;81(1):69–74. 168. Canaan S, Dupuis L, Rivière M, Faessel K, Romette JL, Verger R, et al. Purification and interfacial behavior of recombinant human gastric lipase produced from insect cells in a bioreactor. Protein Expr Purif. 1998;14(1):23–30. 169. Bodmer MW, Angal S, Yarranton GT, Harris TJ, Lyons A, King DJ, et al. Molecular cloning of a human gastric lipase and expression of the enzyme in yeast. Biochim Biophys Acta. 1987;909(3):237–44. 170. Crabbe T, Weir AN, Walton EF, Brown ME, Sutton CW, Tretout N, et al. The Secretion of Active Recombinant Human Gastric Lipase by Saccharomyces cerevisiae. Protein Expr Purif. 52 1996;7(3):229–36. 171. Smerdon GR, Aves SJ, Walton EF. Production of human gastric lipase in the fission yeast Schizosaccharomyces pombe. Gene. 1995;165:313–8. 172. Sassene PJ, Fanø M, Mu H, Rades T, Aquistapace S, Schmitt B, et al. Comparison of lipases for in vitro models of gastric digestion: lipolysis using two infant formulas as model substrates. Food Funct. 2016;7(9):3989–98. 173. Aloulou A, Carrière F. Gastric lipase: an extremophilic interfacial enzyme with medical applications. Cell Mol Life Sci. 2008/01/24. 2008;65(6):851–4. 174. Zhong Q, Gu Z, Glatz CE. Extraction of recombinant dog gastric lipase from transgenic corn seed. J Agric Food Chem. 2006;54(21):8086–92. 175. Mercuri A, Passalacqua A, Wickham MSJ, Faulks RM, Craig DQ, Barker SA. The effect of composition and gastric conditions on the self-emulsification process of ibuprofen-loaded self- emulsifying drug delivery systems: a microscopic and dynamic gastric model study. Pharm Res. 2011/02/25. 2011;28(7):1540–51. 176. Diakidou A, Vertzoni M, Abrahamsson B, Dressman JB, Reppas C. Simulation of gastric lipolysis and prediction of felodipine release from a matrix tablet in the fed stomach. Eur J Pharm Sci. 2009/05/12. 2009;37(2):133–40. 177. Minekus M, Alminger M, Alvito P, Ballance S, Bohn T, Bourlieu C, et al. A standardised static in vitro digestion method suitable for food - an international consensus. Food Funct. 2014/05/08. 2014;5(6):1113–24. 178. Ghazal HS, Dyas AM, Ford JL, Hutcheon GA. The impact of food components on the intrinsic dissolution rate of ketoconazole. Drug Dev Ind Pharm. 2014;1–8. 179. Luner PE, Van Der Kamp D. Wetting characteristics of media emulating gastric fluids. Int J Pharm. 2001/02/13. 2001;212(1):81–91. 180. Fernandez S, Chevrier S, Ritter N, Mahler B, Demarne F, Carrière F, et al. In vitro gastrointestinal lipolysis of four formulations of piroxicam and cinnarizine with the self emulsifying excipients Labrasol® and Gelucire® 44/14. Pharm Res. 2009;26(8):1901–10. 181. Abrahamsson B, Albery T, Eriksson A, Gustafsson I, Sjöberg M. Food effects on tablet disintegration. Eur J Pharm Sci. 2004/05/26. 2004;22(2–3):165–72. 182. Franek F, Holm P, Larsen F, Steffansen B. Interaction between fed gastric media (Ensure Plus®) and different hypromellose based caffeine controlled release tablets: Comparison and mechanistic study of caffeine release in fed and fasted media versus water using the USP dissolution apparatus 3. Int J Pharm. 2014;461(1–2):419–26. 183. Williams HD, Nott KP, Barrett DA, Ward R, Hardy IJ, Melia CD. Drug release from HPMC matrices in milk and fat-rich emulsions. J Pharm Sci. 2011/07/19. 2011;100(11):4823–35. 184. Kalantzi L, Polentarutti B, Albery T, Laitmer D, Abrahamsson B, Dressman J, et al. The delayed dissolution of paracetamol products in the canine fed stomach can be predicted in vitro 53 but it does not affect the onset of plasma levels. Int J Pharm. 2005;296(1–2):87–93. 185. Jain AK, Söderlind E, Viridén A, Schug B, Abrahamsson B, Knopke C, et al. The influence of hydroxypropyl methylcellulose (HPMC) molecular weight, concentration and effect of food on in vivo erosion behavior of HPMC matrix tablets. J Control Release. 2014;187:50–8. 54 Legend to Figures Fig. 1 HGL concentration as a function of time (left) and as a function of meal gastric emptying (right) after intake of a mixed solid-liquid test meal at t= 0 min by 53 individuals. The dots represent the experimental HGL concentration measured in aspirated gastric juice, whereas the solid line represents the best-fitting curve. The activity of HGL in gastric juice was measured with the pH-stat method using tributyrin as a substrate at pH 6.0. Reproduced from (23) with permission from The Royal Society of Chemistry. Fig. 2 Parameters affecting HGL activity in the human stomach. Fig. 3 Specific activity of HGL as a function of pH on 30% Intralipid and tributyrin as substrates. Reproduced from Gargouri et al. (15) with permission from Elsevier. Fig. 4 3D computer simulations describing the viscosity dependent generation of a retropulsive jet stream in the distal stomach by antral contraction waves. The two figure reflect the hydrodynamics in the central plane of the stomach. The streamlines indicate the fluid flow within the stomach at the time point of maximum occlusion in the antrum for two Newtonian fluids of different viscosity (1 mPas and 1000 mPas, respectively). The velocity of fluid flow is given by the colour code at the left of each example. Reprinted from Ferrua et al. (60) with permission from Elsevier. Fig. 5 MR images showing the phase separation of a coarse sunflower oil-in-water emulsion in the human stomach. Reprinted from Hussein et al. (61) with permission from The American Society for Nutrition. Fig. 6 Intragastric fat fraction (%) in fundus, antrum and total stomach over time (means of n = 6 ± SD) after intake of the FDA standard breakfast measured by MRI. Differentiation into fundus and antrum was not possible at t = 0 min and t = 375 min due to low volumes in antrum. Reprinted with permission from Koziolek et al. (5). Copyright (2017) American Chemical Society. Fig. 7 Effect of ingestion order on gastric emptying of a solid meal. The subjects in this study received the solid meal, which was composed of a fat (F) and a non-fat (NF) component in two different study conditions (F-NF vs. NF-F). The data are presented as means (of the fitted curves) + SEM. *Significant difference (p < 0.05). Reprinted from Kunz et al. (91) with permission from WILEY. Fig. 8. The release of CCK by triggers such as the presence of LCFA in the small intestine leads to various changes of GI function in stomach and small intestine. Fig.9 Figure to show the range of physiologically relevant dissolution test devices, of increasing complexity, that can potentially be used to study the effects of lipids and lipolysis on drug release from solid oral dosage forms. Fig. 10. Snap-shot media of different complexity proposed by Markopoulos et al. (158). Fig. 11 Solubility of dipyridamole (A) and of ketoconazole (B) in biorelevant media of different complexity. Reprinted from Diakidou et al. (149) with permission from WILEY. Fig. 12 Deposition of fat on the surface of HPMC matrix tablets in milk (left) and Intralipid® 30% (rigt) over time investigated with confocal fluorescence imaging. Reprinted from Williams et al. (183) with permission from Elsevier. 55