Digestive processes in the GI tract and their impact on the fate of oral drugs and formulations

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1 UMR7281 Bioenergetics and Engineering of Proteins Enzymology of Supramolecular Systems group Digestive processes in the GI tract and their impact on the fate of oral drugs and formulations Frédéric ric Carrière re UNGAP F2F meeting, Leuven, 09 March carriere@imm.cnrs.fr

Direct food-drug drug interaction ex: solubilisation of poorly water soluble drugs in dietary lipids Stimulation of digestive processes, secretions by food ex: changes in ph, release of

gelatin capsules by pepsin Digestion of LBF by lipases Impact of food on the drug target and efficacy ex: target in the GI tract lumen Ex: Orlistat effects depending on the type of meal

2 Direct food-drug drug interaction ex: solubilisation of poorly water soluble drugs in dietary lipids Stimulation of digestive processes, secretions by food ex: changes in ph, release of digestive enzymes, bile secretion, gastric emptying Impact on drug release from capsules, formulations Ex: ph-dependent release of gastro-protected pancreatic enzymes in PERT Digestion of gelatin capsules by pepsin Digestion of LBF by lipases Impact of food on the drug target and efficacy ex: target in the GI tract lumen Ex: Orlistat effects depending on the type of meal Food-drug drug interaction upon intestinal absorption and beyond ex: digestion of dietary lipids leads to the production and release of chylomicrons that can be vehicules for poorly water soluble molecules

3 Digestive enzymes

4 An overview of lipid digestion

Digestion of dietary lipids in humans Gastric lipase (HGL) Gastrointestinal lipolysis Pancreatic Lipase (HPL) + colipase Pancreatic lipase-related protein 2 (PLRP2)

5 Digestion of dietary lipids in humans Gastric lipase (HGL) Gastrointestinal lipolysis Pancreatic Lipase (HPL) + colipase Pancreatic lipase-related protein 2 (PLRP2) Carboxylester hydrolase (CEH, BSSL) Pancreatic phospholipase A2 (spla2-gib) Micellar solubilization of lipolysis products Intestinal absorption of lipolysis products

6 Other pancreatic enzymes involved in lipid digestion Carboxyl Ester Hydrolase (CEH) Other names: Cholesterol Esterase Carboxyl Ester Lipase Bile Salt-Stimulated Lipase (also present in mother s milk) Substrates: cholesterol esters monoglycerides vitamin A and E esters Phospholipids Galactolipids Pancreatic Lipase -Related Protein 2 (PLRP2) Substrates: Phospholipids (PLA1) Galactolipids Monoglycerides Triglycerides vitamin A and E esters Pancreatic Phospholipase A2 (spla2-ib) Substrates: Phospholipids

Pancreatic lipase Monoglycerides Phospholipids

7 Many (most) components of lipid-based formulations can be digested by GI lipolytic enzymes Enzymes Substrates Vegetable oils Diglycerides MCT Gastric lipase colipase Pancreatic lipase Monoglycerides Phospholipids Polyethoxylated Castor oil CEH PLRP2 Tween Polysorbate PEG esters CITREM spla2-ib

8 Substrate preferences of lipolytic enzymes in the GI tract Emulsions Triglycerides Diglycerides Classical PL Gastric Lipase Mixed micelles Phospholipids Galactolipids Monoglycerides PLRP2 CEH Phospholipase A2

9 Lipase activities on medium chain tri-,, di- and monoglycerides Fernandez et al. BBA Molecular and Cell Biology of Lipids (2007) 1771:

10 Lipase activities on PEG mono- and diesters Fernandez et al. BBA Molecular and Cell Biology of Lipids (2007) 1771:

11 Quantitative studies of lipid digestion in humans Carrière et al. Gastroenterology (1993) 105: Carrière et al. Am. J. Physiol. (2001) 281:G16-G28 Carrière et al. Clin. Gastroenterol. Hepatol. (2005) 3:28-38

12 What specifically happens in the stomach Drastic variations in ph What is the relevant ph to be selected for static in vitro models of gastric digestion?

13 Gastric ph levels and variations Homogenized Liquid test meal N = 10 Gastric ph during a meal % Carrière et al. Digestion 2001, 64:46 53 Time (min) Meal gastric emptying %

14 Homogenized Liquid test meal N = 30 Gastric ph during meal digestion A B % 95% Time (min) Meal gastric emptying (%) Sams et al. Food & Function 2016, 7:30-45

15 Homogenized Liquid test meal N = 30 Gastric ph A All data points Mean ph values vs Time Data curve-fitting using Polynomial model B Solid- liquid test meal N = 53 Gastric ph C D Time (min) Meal gastric emptying (%) Sams et al. Food & Function 2016, 7:30-45

16 What specifically happens in the stomach Drastic variations in ph Lipid and protein digestion starts in the stomach

17 Lipase outputs and lipolysis during a meal Stomach Pylorus HGL secretion: : mg /3 hours Intragastrique lipolysis : 10-25% HPL secretion : mg /3 hours (depending on the amounts of TG ingested) Duodenum Treitz HGL is stable in the small intestine contents during a meal HGL acts in synergy with HPL Lipolysis level at the Angle of Treitz : 30-60%

18 What specifically happens in the stomach Drastic variations in ph Lipid and protein digestion starts in the stomach The digestion of lipid-based formulation also starts in the stomach

Gastric lipolysis of lipid-based formulations (LBF) Oils Emulsion Fine emulsion Microemulsion Micellar solution Limited dispersion SEDDS SEDDS SMEDDS 1500 IIIA-MC

19 Gastric lipolysis of lipid-based formulations (LBF) Oils Emulsion Fine emulsion Microemulsion Micellar solution Limited dispersion SEDDS SEDDS SMEDDS 1500 IIIA-MC Gastric lipase specific activity (U/mg) I-LC II-LC IIIA-LC I-MC LBFs II-MC IIIB-MC IV Bakala N Goma et al. Pharm. Res. (2015) 32:

20 In vitro lipolysis of Labrasol under conditions simulating the gastric (1) and duodenal (2) phases of digestion % of the initial amounts of each compound (1) Gastric lipase (2) + Human pancreatic juice Time (min) C8-C10 triacylglycerols C8-C10 diacylglycerols C8-C10 monoacylglycerols PEG-8 diesters Free PEG-8 released Fernandez et al. Pharmaceutical Research (2009) 26:

21 What specifically happens in the stomach Drastic variations in ph Lipid and protein digestion starts in the stomach The digestion of lipid-based formulation also starts in the stomach Short and medium chain free fatty acids ( C12) are absorbed in the stomach

22 What specifically happens in the stomach Drastic variations in ph Lipid and protein digestion starts in the stomach The digestion of lipid-based formulation also starts in the stomach Short and medium chain free fatty acids ( C12) are absorbed in the stomach Does the gastric step of digestion favors the emulsification of lipids or coalescence of lipid droplets?

milk In vitro / in vivo digestion by gastric lipase de Oliveira et al.

23 Gastric lipolysis can promote lipid droplet coalescence RHM: raw human milk PHM: pasteurized human milk PHHM: pasteurized and homogenized human milk In vitro / in vivo digestion by gastric lipase de Oliveira et al. Food Chemistry (2016) 211: de Oliveira et al. Clin Nutr ESPEN (2017) 20:1-11.

24 MRI imaging Gastric digestion can promote lipid droplet coalescence / creaming Hussein et al. Eur J Lipid Sci Technol (2015) 117:31 36 Steingötter et al. Am J Clin Nutr (2015) 101:

as well as late re-emulsification emulsification In vitro gastric digestion of milk fat globules (newborn conditions) ph changes only (from ph 6 to 4 in 180 min) + gastric lipase + gastric

25 as well as late re-emulsification emulsification In vitro gastric digestion of milk fat globules (newborn conditions) ph changes only (from ph 6 to 4 in 180 min) + gastric lipase + gastric lipase + pepsin min min 0-30 min min Bourlieu et al. Food Chemistry (2015) 182: Also seen in vivo by MRI imaging, see: Steingötter et al. Am J Clin Nutr (2015) 101:

26 What specifically happens in the stomach Drastic variations in ph Lipid and protein digestion starts in the stomach The digestion of lipid-based formulation also starts in the stomach Short and medium chain free fatty acids ( C12) are absorbed in the stomach The gastric step of digestion may favor both emulsification and coalescence of lipid droplets Lipid emulsion reshuffling is possible in the stomach Synergistic effects of digestive processes (lipolysis and proteolysis) have to be considered

27 What happens in the small intestine Rather stable ph, but may change with disease (EPI) and drug treatment (PPI)

28 Duodenal ph Bile acid levels Duodenal ph Liquid meal Solid-liquid meal Liquid meal + PPI Solid-liquid meal in CP BA duodenal concentration (mm) Solid-liquid meal Solid-liquid meal in CP CMC 4 mm Time (min) Time (min) PPI: proton pump inhibitor CP: chronic pancreatitis Bakala N Goma et al. Therapeutic Delivery (2012) 3: Humbert et al.. submitted

29 8 6 Borgström et al. J. Clin. Invest. (1957)

30 What happens in the small intestine Rather stable ph, but may change with disease (EPI) and drug treatment (PPI) Lipid and protein digestion is completed Bile secretion favors both the emulsification of lipids and the micellar solubilization of lipolysis products The micellar solubilization of lipolysis products allows their dispersion and diffusion towards the intestinal brush border and enterocytes where intestinal absorption occurs.

31 The lipolysis reaction Class I insoluble, non-swelling, amphiphilic lipids separated lipid phase oil-in-water emulsion weak interaction with water (including non-ionized FFA) Class II insoluble, swelling, amphiphilic lipids lamellar structures and micelles, higher interaction with water (including FFA soaps) FFA FFA FFA FFA TAG DAG MAG Glycerol Mainly gastric and pancreatic lipases Mainly gastric and pancreatic lipases CEH PLRP2 Gastric Lipase

32 E Lipase Bile salt Free fatty acids (FFA) Monoglycerides (MAG) Fat TAG DAG E* MAG E 1) Lipolysis 2) Micellar solubilization Enterocyte 3) Intestinal absorption

33 What happens in the small intestine Rather stable ph, but may change with disease (EPI) and drug treatment (PPI) Lipid and protein digestion is completed Bile secretion favors both the emulsification of lipids and the micellar solubilization of lipolysis products The micellar solubilization of lipolysis products allows their dispersion and diffusion towards the intestinal brush border and enterocytes where intestinal absorption occurs. Lipolysis products are not just vehicules for calories or essential fatty acids: they are also signaling molecules that tune digestive physiology through the release of GI hormones

34 Free fatty acids (FFA) Bile salt Intestinal lumen CD36 Enterocyte I-cell Circulation CCK chylomicrons

35 FFA and CCK mediate the adaptation of pancreatic lipase secretion to the amounts of fat in the diet TAG Lipase 600 FFA (oleic acid) CCK (cholecystokinin) Exocrine pancreatic secretion Pancreatic lipase outputs (mg) Fat amounts in test meal (g)

36 Studies with the lipase inhibitor Orlistat confirmed the role of FFA in CCK release and pancreatic exocrine secretion TAG + Orlistat Lipase 350 FFA Postprandial plasma CCK levels Pancreatic lipase secretion (mg, anti-hpl ELISA) Controls Xenical (120 mg Orlistat) Exocrine pancreatic secretion 0 Liquid meal Shak Iso Solid meal hamburger, fries, butter, string beans Schwizer et al. Am J Physiol (1997); Hildebrand et al. Gastroenterology (1998); Carrière et al. Am J Physiol (2001)

CCK secretion is stimulated by the fatty acids released in the upper part of the duodenum, triggers pancreatic secretion and thus food digestion, CCK known to be reduced in presence of lipase

37 CCK secretion is stimulated by the fatty acids released in the upper part of the duodenum, triggers pancreatic secretion and thus food digestion, CCK known to be reduced in presence of lipase inhibitors Relationships between fatty acids and the gut hormones involved in the control of GI tract physiology, food uptake and digestion CCK Ghrelin GLP-1 PYY Acylated by C8 to be functional, C8 partly comes from the diet (milk, MCT), is released by preduodenal lipase in the stomach where it can be directly absorbed Triggered by fatty acids arriving in the distal part of the intestine, thus presumably depends on the rate of fat digestion and absorption, important for ileal brake and satiety mechanisms, can be bypassed if digestion and absorption are too fast

A satiety mechanism know as the «ileal brake» Fat ingestion Reduced gastric emptying Reduced intestinal motility Decrease in pancreatic secretion Decrease in bile secretion Decrease in gastric

38 A satiety mechanism know as the «ileal brake» Fat ingestion Reduced gastric emptying Reduced intestinal motility Decrease in pancreatic secretion Decrease in bile secretion Decrease in gastric secretion Reduced food intake PYY GLP-1 secreted by L-cells in the ileum and colon Maljaars et al. Physiology & Behavior (2008) Undigested fat and non-absorbed FFA reaching the ileum

39 What happens in the small intestine Rather stable ph, but may change with disease (EPI) and drug treatment (PPI) Lipid and protein digestion is completed Bile secretion favors both the emulsification of lipids and the micellar solubilization of lipolysis products The micellar solubilization of lipolysis products allows their dispersion and diffusion towards the intestinal brush border and enterocytes where intestinal absorption occurs. Lipolysis products are not just vehicules for calories or essential fatty acids: they are also signaling molecules that tune digestive physiology through the release of GI hormones Lipolysis products can contribute to the solubilization of lipophilic molecules

40 In vitro lipolysis of Labrasol under conditions simulating the gastric (1) and duodenal (2) phases of digestion % of the initial amounts of each compound (1) Gastric lipase (2) + Human pancreatic juice Time (min) C8-C10 triacylglycerols C8-C10 diacylglycerols C8-C10 monoacylglycerols PEG-8 diesters Free PEG-8 released Fernandez et al. Pharmaceutical Research (2009) 26:

products of Labrasol digestion are therefore as important as initial components

41 Effects of Labrasol lipolysis on the solubility of Cinnarizine (an anti-histaminic drug) Gastric phase Duodenal phase No enzyme Active enzymes The products of Labrasol digestion are therefore as important as initial components for the solubilization of Cinnarizine Fernandez et al. Pharmaceutical Research (2009) 26:

42 Lipolysis of various LBF and solubilization of Danazol Danazol in the aqueous phase (%) IV II-LC IIIA LC IIIB MC A I-MC II-MC IIIA MC IV II-LC IIIB MC IIIA LC II-MC IIIA MC I-MC B Lipolysis level (% total FA) Fatty acid released (mmol) Danazol solubilization correlates with lipolysis. The products of LBF digestion are therefore as important as initial LBF components Williams et al. J Pharm Sci (2012) 101: Carrière. Biochimie (2016) 125:

43 Impact of the physical state of lipids on digestion and absorption rates

44 Effects of milk treatment on the size of fat droplets and lipolysis rate Droplet size In vitro gastric lipolysis The smaler the size, the higher the specific surface and lipolysis rate Bourlieu et al. Food Chemistry (2015) 182: Benzonana and Desnuelle. BBA (1965) 105:

45 Effects of various test meals on fat digestion Liquid test meal (Shak( Iso ): complete meal containing proteins, carbohydrates and a fine lipid emulsifion stabilized by lecithins and monoglycerides (= good lipase substrate) Mixed liquid/solid test meal: Hamburger, french fries+cooking oil, string beans, butter, containing fat from various origins and different physicochemical states (= not a very good substrate for lipases)

Fat digestion along the human GI tract (lipolysis levels, %) Stomach Solid-liquid liquid meal Pylorus 9.3 ± 6.3 Duodenum Treitz 27.7 ± 6.8 Emulsified liquid meal 24.4 ± 5.

46 Fat digestion along the human GI tract (lipolysis levels, %) Stomach Solid-liquid liquid meal Pylorus 9.3 ± 6.3 Duodenum Treitz 27.7 ± 6.8 Emulsified liquid meal 24.4 ± ± 5.6 Jejunum Ileum Pre-emulsified emulsified fat is digested more rapidly Colon Feces >96 >96 % of ingested fat: <4 <4 Carrière et al. Am. J. Physiol. (2001) 281:G16-G28

Fat from pre-emulsions emulsions is absorbed more rapidly than non-emulsified fat Kinetics of chylomicron levels in plasma (TAG, mm) Healthy volunteers n=9 BMI=22 [TAG 0 ]=0.

47 Fat from pre-emulsions emulsions is absorbed more rapidly than non-emulsified fat Kinetics of chylomicron levels in plasma (TAG, mm) Healthy volunteers n=9 BMI=22 [TAG 0 ]=0.8 Obese subjects n=9 BMI=32 [TAG 0 ]=1.4 Chylomicron TAG (mm) 40g spread on bred 40g in emulsion Dairy fat in the meal Time (min) Vors et al. Am J Clin Nutr (2013); Etude LIPINFLOX, INRA, CNIEL, CRNH Rhône-Alpes

48 Correlations between lipolysis and adsorption rates In vitro digestion Fatty acid absorption in rats Lipolysis level (%) Gastric lipolysis Casein-Flaxseed oil emulsion Soja Lecithin-Flaxseed oil emulsion * * * * A Duodenal lipolysis * α-linolenic acid (mg/ml lymph) AUC (mg/ml*h) C * Casein Soja lecithin * * B * Time (min) Time (h) Couëdelo et al. Food and Function (2015) 6:

49 Surfactants in digestive processes Surfactants naturally present in food or produced along the GI tract - Proteins - Phospholipids - Bile salts - Lipolysis products (fatty acids, lysophospholipids, monoglycerides) - Mucins Surfactants from pharmaceutical or dietary formulations (many) - Tweens / Polysorbates - PEG esters - Phospholipids - Monoglycerides - Cremophor Surfactants usually favour emulsification and lipolysis rates, but they can also limit enzyme access to its substrate and reduce lipolysis (e.g., enzyme desorption from the oil-water interface or creation of an interfacial barrier)

In vitro digestion of various flaxseed oil emulsions 70 Gastric lipolysis Duodenal lipolysis 60 50 Lecithin-Flaxseed oil emulsion Casein-Flaxseed oil emulsion Tween 80-Flaxseed oil emulsion 50.3 ±2.

50 In vitro digestion of various flaxseed oil emulsions 70 Gastric lipolysis Duodenal lipolysis Lecithin-Flaxseed oil emulsion Casein-Flaxseed oil emulsion Tween 80-Flaxseed oil emulsion 50.3 ±2.0 % Tween 80 4µm Sodium caseinate 5µm Lipolysis level (%) ±4.3 % µm Lecithin ± 0.8 % ±0 % 15µm ± 2.9 % 2.1 ± 2.7 % Time (min) Couëdelo et al. Food and Function (2015) 6:

51 In vitro digestion modeling initiatives (2012) 101(9): , cited 93 times Pharma Ex: LFCS consortium (2014) 5: , cited 387 times Food Ex: EU COST Infogest

The INFOGEST network still exists with 6 active working groups WG4: Lipases and lipid digestion A round-robin robin study on assays of lipase activities in pancreatins and gastric lipase sources is

52 The INFOGEST network still exists with 6 active working groups WG4: Lipases and lipid digestion A round-robin robin study on assays of lipase activities in pancreatins and gastric lipase sources is in progress, with the aim of identifying sources of enzymes for in vitro digestion studies and to establish the standard methods for lipase assays Establish bridges between UNGAP and INFOGEST networks: joint workshops? New gastric lipase supplier: See

53 Thank you for your attention! Food-drug interactions

L1, 2 : Biochemical Aspects of Digestion of Lipids, Proteins, and Carbohydrates

L1, 2 : Biochemical Aspects of Digestion of Lipids, Proteins, and Carbohydrates OBJECTIVES: Understand the process of digestion of dietary lipids, protein and carbohydrates including, the organs involved,