Low Buffer Capacity and Alternating Motility Along The Human Gastrointestinal Tract Implications For in Vivo Dissolution and Absorption of Ionizable Drugs

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Article
Low Buffer Capacity and Alternating Motility Along
The Human Gastrointestinal Tract: Implications for In
Vivo Dissolution and Absorption of Ionizable Drugs
Bart Hens, Yasuhiro Tsume, Marival Bermejo, Paulo Paixao, Mark Koenigsknecht, Jason R.
Baker, William L. Hasler, R. Lionberger, Jianghong Fan, Joseph Dickens, Kerby A. Shedden,
Bo Wen, Jeffrey Wysocki, Raimar Löbenberg, Allen Lee, Ann F. Fioritto, Gregory E. Amidon,
Alex Yu, Gail Benninghoff, Niloufar Salehi, Arjang Talattof, Duxin Sun, and Gordon L. Amidon
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00426 • Publication Date (Web): 24 Jul 2017
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Page 1 of 37 Molecular Pharmaceutics
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3 Submitted to Molecular Pharmaceutics ((featured topic issue, Industry-Academic Collaboration in Oral
4 Biopharmaceutics: The European IMI OrBiTo Project)
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6 Low Buffer Capacity and Alternating Motility Along the Human
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Gastrointestinal Tract: Implications for in vivo Dissolution and
9 Absorption of Ionizable Drugs
10
‡
11 Bart Hens1, ‡Yasuhiro Tsume1, ‡Marival Bermejo2, ‡Paulo Paixao3, ‡Mark J. Koenigsknecht1, ‡Jason
12 R. Baker4, ‡William L. Hasler4, ‡Robert Lionberger5, ‡Jianghong Fan5, Joseph Dickens6, Kerby
13 Shedden6, Bo Wen1, Jeffrey Wysocki1, Raimar Loebenberg7, Allen Lee2, Ann Frances1, Greg
14 Amidon1, Alex Yu1, Gail Benninghoff1, Niloufar Salehi8, Arjang Talattof1, Duxin Sun1, ‡Gordon L.
15 Amidon1*
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18
‡
19 Bart Hens, Yasuhiro Tsume, Marival Bermejo, Paulo Paixao, Mark J. Koenigsknecht, Jason R.
20 Baker, William L. Hasler, Ann Frances Robert Lionberger, Jianghong Fan, Duxin Sun and Gordon L.
21 Amidon are the primary authors.
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24 1
Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor,
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26 Michigan 48109, USA
27 2
Department Engineering Pharmacy Section, Miguel Hernandez University, San Juan de Alicante,
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03550 Alicante, Spain
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30 3
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av.
31 Professor Gama Pinto, 1649-003 Lisboa, Portugal
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33 Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor,
34 Michigan 48109, USA
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36 Office of Generic Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug
37 Administration, Silver Spring, MD, USA
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39 Department of Statistics, University of Michigan 48109, Ann Arbor, USA
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Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Canada
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42 8
Center for the Study of Complex Systems and Department of Chemical Engineering, University of
43 Michigan, Ann Arbor, Michigan 48109-2136, USA
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45 Corresponding Author
46 * Prof. Dr. Gordon L. Amidon
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48 428 Church St.
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50 College of Pharmacy
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University of Michigan
53 Ann Arbor, MI 48109-1065
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55 Phone: +(1) 734-764-2226
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Fax: +(1) 734-764-6282
58 Email: glamidon@med.umich.edu
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ACS Paragon Plus Environment
Molecular Pharmaceutics Page 2 of 37
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3 Graphical Abstract
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24 Keywords
25 clinical study; in vivo dissolution; local drug concentration in the GI tract; ibuprofen; immediate
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27 release; bioequivalence; bioavailability; oral absorption; buffer capacity
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30 Abstract
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In this study, we determined the pH and buffer capacity of human gastrointestinal (GI) fluids
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(aspirated from the stomach, duodenum, proximal jejunum and mid/distal jejunum) as a function of
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time, from 37 healthy subjects after oral administration of an 800 mg immediate-release tablet of
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38 ibuprofen (reference listed drug; RLD) under typical prescribed bioequivalence (BE) study protocol
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40 conditions in both fasted and fed state (simulated by ingestion of a liquid meal). Simultaneously,
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42 motility was continuously monitored using water-perfused manometry. The time to appearance of
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44 phase III contractions (i.e. housekeeper wave) was monitored following administration of the
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46 ibuprofen tablet. Our results clearly demonstrated the dynamic change in pH as a function of time and,
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48 most significantly, the extremely low buffer capacity along the GI tract. The buffer capacity on
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50 average was 2.26 µmol/mL/∆pH in fasted state (range: 0.26 and 6.32 µmol/mL/∆pH) and 2.66
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52 µmol/mL/∆pH in fed state (range: 0.78 and 5.98 µmol/mL/∆pH) throughout the entire upper GI tract
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54 (stomach, duodenum, proximal and mid/distal jejunum). The implication of this very low buffer
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56 capacity of the human GI tract is profound for the oral delivery of both acidic and basic active
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58 pharmaceutical ingredients (APIs). An in vivo predictive dissolution method would not only require a
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3 bicarbonate buffer, but more significantly, a low buffer capacity of dissolution media to reflect in vivo
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5 dissolution conditions.
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3 Introduction
4 Predicting in vivo outcomes with computational software today, including variation in oral
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6 absorption, requires accurate and biorelevant input functions reflecting the dynamic environment of
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8 the gastrointestinal (GI) tract. The in vitro USP 1 and 2 dissolution tests are very useful in a quality
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10 control (QC) setting and, in some cases, these tests can be used for a biowaiver of in vivo
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12 bioequivalence (BE) studies for immediate release (IR) dosage forms (BCS class I (high solubility,
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14 high permeability) and III (high solubility, low permeability) compounds) since gastric emptying is on
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16 average slower than the in vitro dissolution of the API. Thus, gastric emptying would control plasma
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18 variation of two oral products (i.e. marketed and generic) with the same API. Nevertheless, these QC
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20 methods do not capture the inter- and intra-individual differences of GI variables as well as in
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22 systemic outcomes (plasma AUC, Cmax and Tmax) after oral administration of oral IR drug products.1,2
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24 In the case of slower dissolution and/or particulary lower solubility, various physiological GI variables
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26 will impact the disintegration and dissolution of the formulation and drug, and thus the time-dependent
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28 presentation of the drug to the intestinal absorbing membrane along the intestinal tract.3
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31 As recently demonstrated, the impact of gastric motility and the variation in the fasted state on
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33 oral drug absorption may be one of the major determinants of variability in bioavailability, particularly
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35 Cmax among normal subjects in the fasted state.4 The time of intake of the oral dosage form relative to
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37 the fasted state cyclic motility pattern (i.e. migrating motor complex (MMC)5) will cause significant
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39 variation in gastric emptying and presentation of the drug to the absorbing membrane and thus,
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41 ultimately, systemic availability of the drug. In fasting conditions, the GI muscular system contracts
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43 with a cyclical periodic interdigestive MMC, in which the phases of quiescence (phase I) are followed
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45 up by a phase of intermittent contractions (phase II) and a final phase of very strong contractions
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47 (phase III) resulting in a motility varying gastric emptying rate.5,6
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When no digestible food is administered, this cyclical fasted state motility pattern will be
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repeated periodically. In 1973, Heading and colleagues found a strong correlation between plasma
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Cmax, Tmax and the rate of gastric emptying of acetaminophen (expressed as gastric half-life; T1/2,G), an
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early demonstration that gastric emptying is the rate-limiting step for absorption of acetaminophen
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58 (which is a BCS class I compound).7 More recently, similar results were noted for BCS class III
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3 compounds, i.e. fluorouracil and diethylcarbamazine, when applying periodic gastric emptying rates
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5 with computational modeling software developed to reflect the GI motility variation based on
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7 available published data.4 These results suggest that strong phase III contractions stimulate gastric
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9 emptying of drug content in such way that a large amount of drug is directly available for absorption
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11 in the small intestine, being a main determinant of the plasma Cmax and Tmax of the drug. Most
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13 significantly, in BE studies, we usually dose randomly relative to the this motility cycle and, therefore,
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15 introduce this random variable, independent of dosage form, into normal BE studies.
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17 Further, the dynamic nature of the pH along the GI tract, and most significantly the very low
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19 buffer capacity of the GI fluids, will substantially alter drug dissolution for ionizable drug
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21 substances.8,9 In the case of carboxylic acids (e.g. ibuprofen, diclofenac, ketoprofen, flurbiprofen,
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23 naproxen, etc.), the dissolved fraction of drug substance along the GI tract depends on the existing
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25 luminal pH and buffer capacity.10–12 The pKa values reported for this group of medicines range from
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27 3.9 to 5.8, where solubility and dissolution rate are highly dependent on the duodenal pH and buffer
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29 capacity at the time of gastric emptying.13 At low physiological pH values (e.g. stomach), these drug
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31 substances are poorly soluble in gastric fluids (pH 1-2) and become much more soluble in the dynamic
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pH range of the intestinal fluids. However, while a static intestinal pH value of 6.5 (fasted state
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simulated intestinal fluid (FaSSIF)) and 6.8 (USP simulated intestinal fluid (SIF)) are frequently used
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in in vitro dissolution experiments, the GI environment is known to be much more dynamic with a pH
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ranging from 2.4 to 7.5 in the duodenum.14,15 Duodenal pH and buffer capacity were described as
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42 highly variable depending on the active pancreatic and mucosal cell secretion of bicarbonate to
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44 neutralize gastric content variably emptying from the stomach, depending on the fasted state motility
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46 cycle.9,16–19 As stated by Dooley and colleagues, the contribution of each phase to the cycle length in
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48 the antrum is 55% for phase I, 41% for phase II and 4% for phase III.20 The pancreatic bicarbonate
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50 secreted into the small intestine varies in the fasted state with the motility cycle (4-21 mM bicarbonate
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52 concentration)16,21. In addition to pancreatic secretions, duodenal epithelium cells secrete bicarbonate
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54 too and protect the duodenal epithelium from the gastric fluids discharged from the stomach.9
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3 The reported differences in buffer capacity/pH values of human intestinal fluids are highly
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5 variable.22 This dynamic, intestinal environment is, however, not reflected in conventional dissolution
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7 media (e.g. FaSSIF and USP SIF). FaSSIF (pH 6.5) and USP SIF (pH 6.8) retaining a strong buffer
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9 capacity of 12 and 18.4 µmol/mL/∆pH, respectively, equal to a 5 and 7.7 times higher buffer capacity
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11 compared to human intestinal fluid (ranging from 2.4-5.6 µmol/mL/∆pH).12,23 Further, much larger
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13 dissolution volumes of simulated GI fluids are used in current in vitro dissolution methodologies than
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15 are present in the human GI tract based on magnetic resonance imaging (MRI) studies.24 This may
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17 certainly contribute as one of the major factors for poor in vivo dissolution predictions. The applied
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19 phosphate buffer is not a biorelevant reflection of the more complex bicarbonate/CO2 buffer25, the
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21 predominant buffer in the GI tract.9 In vitro dissolution studies demonstrated the added value of using
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23 bicarbonate buffers instead of phosphate buffers.9,16,26 For example, physiological bicarbonate buffers
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25 proved to be more discriminative of the drug release behavior of enteric-coated formulations for
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27 ileocolonic delivery, resulting in better reflections of in vivo disintegration-dissolution times than
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29 observed for conventional phosphate buffers.26,27 As stated by Tsume and colleagues, the adoption of
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31 inappropriate buffer media for in vitro BE studies would likely cause a failure in achieving an in vitro-
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in vivo correlation (IVIV-C) and BE/bioavailability (BA) for oral drug products.12
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36 The broad aim of this project was to map the link between the drug product disintegration and
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38 drug dissolution in vivo, the presentation of the drug to the intestinal mucosal absorbing surface, and
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40 systemic availability of an orally administered ibuprofen tablet (800 mg; RLD)28. Ibuprofen is highly
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42 permeable and shows no limits in dissolving at the neutral pH of the small intestine (BCS class 2a
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44 drug), as demonstrated by Tsume et al.12 However, we hypothesize that inter-individual variability in
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46 systemic outcome for ibuprofen may be caused by differences in drug release and dissolution of the
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48 drug along the entire GI tract. Therefore, this research proposal wants to explore how (i) alternating
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50 motility patterns and (i) alternating buffer capacity and pH changes along the GI tract contribute to
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inter-individual variability in systemic response of ibuprofen (in terms of Cmax, Tmax and AUC).
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3 Materials and Methods
4 Chemicals
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6 Acetonitrile and pH buffer solutions (pH 4.00, 7.00 and 10.00) were purchased from VWR
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8 International (West Chester, PA). Methanol and HCl were obtained from Fisher Scientific (Pittsburgh,
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10 PA). Ibuprofen, ibuprofen-D3 and NaOH were received from Sigma-Aldrich (St. Louis, MO). Mineral
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12 oil was purchased from Acros Organics (Morris Plains, NJ). Immediate-release tablets of ibuprofen
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14 administered in the clinical study were obtained from Dr. Reddy’s Laboratories Limited (Shreveport,
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16 LA; IBU™ – Ibuprofen Tablets, USP, 800 mg, Lot number L400603). Pulmocare® was obtained from
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18 Abbott Nutrition (Lake Forest, IL). Purified water (i.e. filtrated and deionized) was used for the
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20 analysis methods (Millipore, Billerica, Massachusetts).
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23 Intraluminal and Systemic Profiling of Ibuprofen in Healthy Volunteers
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25 Samples collected in this study were part of clinical trial NCT02806869. The institutional review
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27 boards at the University of Michigan (IRBMED, protocol number HUM00085066) and the
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29 Department of Health and Human Services, Food and Drug Administration (Research Involving
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31 Human Subjects Committee/RIHSC, protocol number 14-029D) both approved the study protocol. All
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33 subjects provided written informed consent in order to participate. The study was carried out
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35 accordance with the protocol, International Conference on Harmonization Good Clinical Practice (ICH
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37 GCP) guidelines, and applicable local regulatory requirements. The protocol of the clinical study has
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39 recently been described by Koenigsknecht et al.15 Briefly, two experimental treatment arms were
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41 tested in 25 subjects: intake of one IR tablet of ibuprofen (IBU™ – Ibuprofen Tablets, USP, 800 mg,
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43 Dr. Reddy’s Laboratories Limited) in fasted state with water or in fed state conditions simulated by
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45 intake of a liquid meal (Pulmocare®) prior to drug administration with approximately 250 mL of
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47 water. Of all 25 subjects, 12 individuals performed a second study visit in order to generate intra-
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49 subject variability data (Table 1).
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5 Table 1: Overview of the study course in terms of (i) the ID of all subjects, (ii) test condition that was performed (fasted or fed state) and
6 (iii) the number of study visits. (*) Subject B003 withdrew from study 2.5 h after dose administration. (*1) Only plasma samples and motility
7 data were collected for subject B044; no GI fluid collection. Data included in the table are derived from Koenigsknecht et al.15
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9 Subject ID Treatment Arm Study Visit #1 Study Visit #2
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11 B002 Fed X X
12 *B003 Fasted X
13 B004 Fasted X X
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15 B005 Fasted X X
16 B006 Fasted X
17 B008 Fed X
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B017 Fasted X X
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20 B020 Fed X X
21 B022 Fed X X
22 B026 Fed X
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24 B031 Fed X
25 B034 Fed X
26 B041 Fed X
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B042 Fasted X X
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29 B043 Fed X X
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* B044 Fasted X
31 B046 Fed X
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33 B049 Fasted X X
34 B052 Fasted X
35 B053 Fasted X
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B055 Fasted X X
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38 B060 Fed X X
39 B063 Fasted X
40 B065 Fasted X X
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42 B066 Fed X
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45 In total, 20 fasted and 17 fed state test conditions were performed. Upon arrival in the hospital,
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47 a customized aspiration multi-channel catheter (body length 292 cm; MUI Scientific, Mississauga,
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49 ON) was intubated via the mouth and positioned in the mid-jejunum, proximal jejunum, duodenum
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51 and stomach. Each segment contains aspiration and motility channels to aspirate GI fluids and to
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53 monitor motility patterns along the GI tract, respectively. Positioning and guiding of the catheter were
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55 verified by fluoroscopy (Figure 1).
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Stomach
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Figure 1: Fluoroscopic image of the position of a 23-channel catheter in the GI tract of subject ID B022, study visit #2. The arrows indicate
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31 After checking the positioning of the catheter, volunteers were asked to remain in a hospital bed in an
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33 upright sitting position. After performing a baseline motility test of 3-5 h (see next paragraph), the
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35 ibuprofen tablet was administered together with 250 mL of water containing 25 mg of USP grade
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37 phenol red as a non-absorbable marker for monitoring GI fluid changes related to dilution, secretion,
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39 and absorption. To induce the fed state condition, volunteers were asked to drink two cans of
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41 Pulmocare® (total volume of 474 mL, containing 29.6 g of proteins, 44.2 g of fat, 25 g of
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43 carbohydrates and a total amount of 710 calories) prior to dose administration. Volunteers were not
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45 obliged to drink the total amount of administered water and/or liquid meal to avoid any feeling of
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nausea at the start of the study. The study drug, water, and/or Pulmocare was swallowed by the
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50 subject and was not administered via the GI catheter. After study drug administration, GI fluids were
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52 aspirated at specific predetermined time points for 7 h. Immediately after aspiration of GI fluids, pH
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54 was measured ex vivo using a pH electrode (Mettler InLab® Micro Pro, Mettler-Toledo LLC,
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56 Columbus, OH), suitable for measuring pH in small or large volumes. After oral administration of the
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58 drug, GI fluids were aspirated at specific predetermined time points for 7 h. The GI fluid samples were
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3 centrifuged at 21,000 x g for 5 minutes and the supernatant was collected and stored at -80°C until
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5 analysis. Blood samples were collected for up to 24 h to monitor ibuprofen systemically. Blood
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7 samples were added to venous blood collections tubes (K2EDTA (spray-dried), 7.2 mg) and plasma
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9 was separated from blood samples by centrifugation and stored at -80°C until analysis. Blood samples
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11 were collected for 24 h to monitor ibuprofen systemically. LC-MS/MS analyses were performed using
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13 a Shimadzu HPLC system interfaced to an AB SCIEX QTRAP 5500 mass spectrometer by a TurboV
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15 electrospray ionization (ESI) source (Applied Biosystems/MDS Sciex, Toronto, Canada). Ibuprofen-
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17 D3 was used as an internal standard (IS) to normalize variation during sample preparation and LC-
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19 MS/MS analyses. Chromatographic separation was achieved using a 2.1 x 50 mm, 3.5 µm Agilent
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21 ZORBAX Extend-C18 column. The injection volume was 5 µL and the flow rate was kept constantly
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23 at 0.4 mL/min. Mobile phase A and B were water and acetonitrile, respectively. Both water and
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25 acetonitrile contained 0.1% acetic acid (v/v). The flow gradient was initially 98:2 v/v of A:B for 0.5
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27 min, linearly ramped to 5:95 A:B over 1 min. To completely wash the column, the gradient was held
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at 5:95 A:B for 2 min and then returned to 98:2 over 0.5 min. This condition was held for 3 min prior
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to the injection of the sample and ibuprofen was detected at 2.48 min. The mass spectrometer was
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operated at ESI negative ion mode ([M-H]-) and multiple reaction monitoring (MRM) was used for
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monitoring the transitions of m/z 205.1→159.1 and m/z 208.2→161.2 for ibuprofen and ibuprofen-D3
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38 (IS), respectively. Protein precipitation with methanol was used to extract ibuprofen from the human
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40 plasma. 60 µL of each plasma sample was mixed with 180 µL of methanol containing 500 ng/mL
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42 ibuprofen-D3 in a 96-well plate. The mixture was vortexed for 60 seconds at a high speed. The 96-
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44 well plate was centrifuged at 3000 x g for 20 min to precipitate proteins at 4 °C. The clear supernatants
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46 were collected and 5 µL of the supernatant was injected for LC-MS/MS analysis. Sample preparation
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48 of GI fluid was similar to the plasma samples. All of the GI fluid samples were diluted 10-fold with
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50 the blank human plasma before protein precipitation in methanol containing 500 ng/mL ibuprofen-D3
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52 in order to reduce matrix effect on GI fluid samples and ibuprofen in GI fluid was quantified with the
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54 plasma calibration curve. Stock solutions of ibuprofen (2 mg/mL) and ibuprofen-D3 (IS) (5 mg/mL)
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56 were prepared in methanol and dimethyl sulfoxide, respectively. To prepare the calibration standards,
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58 the 2 mg/mL ibuprofen stock solution was diluted to 500 µg/mL using methanol, which was spiked
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3 with blank human plasma to provide a final ibuprofen plasma concentration of 10 µg/mL. Then serial
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5 dilution with the blank human plasma was carried out to provide a series of calibration standards from
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7 2.5-10,000 ng/mL. Quality control (QC) plasma solutions at 5, 25, 250, 5000, and 10000 ng/mL were
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9 prepared similarly using separately weighed methanol stock solutions of ibuprofen (2 mg/mL).
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11 Methanol was used to further dilute the stock ibuprofen-D3 solution to 500 ng/mL for protein
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13 precipitation during sample preparation, also to minimize the variation. According to the FDA
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15 guidelines, validation procedures were performed in human plasma and GI fluid diluted with human
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17 plasma.15,29 These procedures included: (A) specificity and selectivity; (B) recovery and matrix effects
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19 at low (0.015 µg/mL), medium (0.1 and 10 µg/mL), and high (35 µg/mL) concentrations; (C)
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21 calibration curve with a correlation coefficient (r) of more than 0.98; (D) precision and accuracy, with
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23 the intra-day and inter-day assay precision and accuracy estimated by analyzing six replicates at three
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25 QC levels; (E) stability, with all stability studies conducted at three concentration levels in the
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27 biomatrix at room temperature, 4 °C, −20 °C, and −80 °C; and (F) dilution integrity, with experiments
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29 carried out with blank biomatrix. Accuracy and precision errors (< 15%) met the FDA requirements
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31 for bioanalytical method validation. All samples were analyzed by the PK Core at the University of
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Michigan.
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Intraluminal Profiling of Periodic Motility Patterns along the GI Tract in Healthy Volunteers
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After positioning, the catheter was connected to a computer console that generated real-time
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42 manometry recordings in the different segments of the GI tract (Medical Measurement Systems,
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44 Dover, NH). The manometric channels attached to the catheter were perfused with sterilized water at a
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46 rate up to 2 mL/min and served as intestinal pressure recording ports to assess intestinal motility. Each
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48 segment contained four motility channels to monitor pressure events. Baseline intestinal motility was
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50 evaluated for 3-5 h prior to study drug administration of the tablet. Subsequently, GI motility was
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52 measured continuously for 7 h. In fasting conditions, periods of active GI motility (phase II-III)
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54 alternated between periods of quiescence (phase I). After food intake, the proximal stomach (fundus)
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56 serves as a reservoir and will initiate tonic contractions/relaxations similar in strength as phase II
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3 contractions (approximately 3 contractions per min).30,31 The strength and number of pressure events
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38 Figure 2: Water-perfused manometry recording of the MMC in subject B049, study visit #2. The indicated area demonstrates the initiation
39 of antral phase III activity, propagating more distally towards the intestinal segments as a function of time.
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42 We identify MMC phase III motility periods using spectral density estimation and penalized
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44 logistic regression. The spectral density estimate of a manometry sample decomposes the sample into
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46 a series of periodic components of decreasing frequencies and associated energies for each periodic
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48 component.32 Figure 2 demonstrates the start of a powerful antral phase III contractions defined as the
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50 occurrence of regular 2-3 contractions per minute for at least 2 minutes with an average amplitude of
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52 75 mmHg.5,33 Duodenal phase III contractions are characterized by 11-12 contractions per minute with
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54 an average amplitude of 33 mmHg which can last for at least 3 minutes.5 As the contractile activity
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56 propagates, it becomes less spatiotemporally organized resulting in slower propulsion rates in the
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58 distal small bowel.34,35 The corresponding spectral density estimate of a phase III period will have high
59
60 12
1
2
3 energy levels in the 10-12 cycles/min components, leading to a concentrated spectrum. During non-
4
5 phase III motility, the spectral density will have a more diffuse spectrum. Using penalized logistic
6
7 regression, we find that the proportion of energy in the 9-12 cycle/min frequencies are an important
8
9 predictor of phase III motility.
10
11
12 Measurement of Buffer Capacity in Aspirated Human Intestinal Fluids Ex Vivo
13 Briefly, buffer capacity was measured by adding accurate amounts of HCl to 100 µL of fasted
14
15
or fed duodenal/jejunal fluid and measuring the pH change.36 In case of gastric samples, fasted state
16
17
gastric fluids were titrated with accurate amounts of NaOH, whereas fed state gastric fluids were
18
19
titrated with accurate amounts of HCl due to the elevation of pH by liquid meal intake. For each
20
21
22
sample, 50 µL of pure mineral oil was added in order to prevent the loss of carbon dioxide (CO2)
23
24 during buffer capacity measurements.9 Accurate and precise pH measurements were assured by
25
26 calibration of the pH electrode (Mettler InLab® Micro Pro, Mettler-Toledo LLC, Columbus, OH)
27
28 using three different pH buffer solutions (pH 4.00, pH 7.00, and pH 10.00) prior to the start of each
29
30 experimental day, resulting in a Pearson Coefficient of Determination (R2) of 95 ± 0.9% (mean ± SD).
31
32 The sample has a buffer capacity value of 1 when one equivalent of strong acid or alkali is required to
33
34 change the pH value of 1 L by one pH unit.37
35
36
37
38 Neutralizing Effect of Bicarbonate on Gastric Acid and the Effect of Ibuprofen Dissolution on pH
39 The available bicarbonate (HCO3-) present in the intestinal tract reacts with hydrogen ions (H+)
40
41 to neutralize the gastric acid (HCl) emptied from the stomach. The formation of carbonic acid (H2CO3)
42
43
produces, in turn, CO2 and H2O through a dehydration reaction:
44
45
46 - + ⇌
⇌
+
(Reaction 1)
47
48 This mentioned pathway is a reversible process and concentrations of each of the component are
49
50 depending on the corresponding equilibrium constants. As gastric fluids (pH 2=0.01 M HCl) would
51
52 empty the stomach as a first order kinetic process, a mass transport analysis was performed that
53
54 describes the appearing duodenal concentrations of H+:38
55
56

= −
57
(Equation 1)
58

59
60 13
1
2
3 where is the concentration of HCl that appears in the duodenum as a function of time t , Q stands
4
5 for the amount of HCl present in the stomach (0.01 M HCl in 35 mL of fluids = 350 µmol)39, V# is the
6
7 constant duodenal volume (40 mL)40, k % is the first order gastric emptying rate constant (0.046 min-1)
8
9 and F is the constant flow rate (1.6 mL/min) from duodenal compartment to the more distal segments
10
11 of the intestinal tract.38 The maximum concentration of HCl that was observed in the duodenum was
12
13 used to react with HCO3- (Reaction 1) present in the duodenal compartment (minimum concentration
14
15 of 4 mM and maximum concentration of 21 mM, as reported in literature).16,21 Based on the simulated
16
17 concentrations of HCl in the duodenum, the reaction with HCO3- and corresponding duodenal pH can
18
19 be carried out by the following calculation:
20
21
() * +(),-. / +0 23 ),-. / ()1 ,-. +0
22 &' -) = ()1 ,-. +0
⇌ ( + = (),-. / +0
⇌ −456 ( + = 7 (Equation 2)
23
24
25
26 where Ka (HCO3-) is equal to 4.47x10-7. [HCO3-]f and [H2CO3]f are the final concentrations of HCO3-
27
28 and H2CO3 after reaction of H+ (maximal simulated H+ concentration) and HCO3- (0.2 M).
29
30 Besides the fluctuations that may occur due to differences in H+ stomach secretions and
31
32 duodenal HCO3- secretions, pH along the intestinal tract can be altered by the present drug. Dissolution
33
34
of a weak acid or base may decrease or increase intestinal pH, depending on the strength of the acid
35
36
(Ka) or base (Kb). Dissolution of ibuprofen (weak acid (HA); Ka = 1.23 x 10 -5) in the intestinal fluids
37
38
can be described by following reaction:
39
40
41 8 +
⇌ 8- + (Reaction 2)
42
43
44 Based on the amount (X) of ibuprofen that dissociates as A- and H+, the drop in pH can be perceived
45
46 by the following calculation:
47
48
() * +(F/ + G1 G1
49 &' 9:;7<5=> = 1,23 × 10E = = = ⇌ ( + = H = I&' × (8+ ⇌ −456 ( + = 7
()F+ ()F+ ()F+
50
51 (Equation 3)
52
53
54 The value that will be applied for [HA] is equal to the average solution concentrations as observed in
55
56 the fasted duodenum as a function of time in this study.
57
58
59
60 14
1
2
3 Results and Discussion
4 Oral Drug Behavior of Ibuprofen Along the GI tract: Monitoring of Intraluminal Concentrations,
5 Post-dose Phase III Contractions, pH and Buffer Capacity
6
7 Several studies have determined the ibuprofen plasma levels after oral administration of
8
9 different dose strengths of the drug in fasting and fed state conditions.41–44 Generally, all studies
10
11 demonstrated a decrease in systemic Cmax/AUC (ratio Cmax fed/fasted: 0.81; ratio AUCfed/fasted: 0.90) and a
12
13 delay in Tmax (ratio Tmax fed/fasted: 1.33) after oral administration of ibuprofen in postprandial conditions.
14
15 Similar ratios fed/fasted state were observed in this study (0.69, 0.77 and 1.67 for Cmax, AUC, and Tmax
16
17 respectively). A more controlled and delayed gastric emptying of ibuprofen in the fed state conditions
18
19 may explain the lower and delayed exposure of ibuprofen in plasma as compared to the fasting state
20
21 conditions as it has been demonstrated that the intake of a liquid meal delays or slows gastric
22
23 emptying significantly.38 The results of this study with simultaneous intestinal sampling and
24
25 manometry recording of GI motility are in line with earlier published systemic data on ibuprofen. In
26
27 this study, we investigated the causes of both inter- and intra-subject variability. For example, in fasted
28
29 state conditions, plasma Cmax ranged from 28100 to 89700 ng/mL, while plasma Tmax varied from 1 to
30
31 8 hour. In fed state conditions, plasma Cmax and Tmax varied from 5700 to 64572 ng/mL and 1 to 8
32
33 hour, respectively. Thus we hypothesized that the underlying cause of this variability in plasma levels
34
35 is due to (i) the time of drug intake relative to the present interdigestive cyclic motility in the stomach
36
37 (i.e. phase I, II or III), both subject to subject and within the subject and (ii) the dynamic change in pH
38
39 as well as the base pH difference along the GI tract.
40
41
42 The time to appearance of the maximal concentration of ibuprofen in plasma (plasma Tmax) is
43
44 plotted as a function of the time to appearance of the maximal solution concentration of ibuprofen in
45
46 the duodenum (duodenal Tmax; Figure 3A) and in the jejunum (jejunal Tmax; Figure 3B).
47
48
49
50
51
52
53
54
55
56
57
58
59
60 15
1
2
3
4
5 A B
9 9
6
7 8 8
8 7 7
Plasma Tmax (h)

Plasma Tmax (h)
9 6 6
10
5 5
11
12 4 4
13 3 y = 0.6932x + 1.506 3 y = 1.008x + 0.6247
14 (0.158 - 0.612)
2 2 (0.139 - 0.497)
R² = 0.4062
15 R² = 0.6534
1 1
16
0 0
17
0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
18
Duodenal Tmax (h) Jejunal Tmax (h)
19
20 Fasted State Fed State Fasted State Fed State
21
22
23 Figure 3: Plot of plasma Tmax (h) as a function of (A) duodenal Tmax (h) and as a function of (B) jejunal Tmax. Fasted and fed state results are depicted by the blue and orange dots, respectively. Trendlines for both graphs
24 are given by the black line and expressed by the slope and intercept. The Pearson Coefficient of Determination is expressed as R2. Standard errors of slope and intercept, respectively, of the linear regression are
25 indicated in parenthesis. Regression for both plots was significant with p < 0.05 (Analysis of Variance, ANOVA).
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42 16
43
44
45
46 ACS Paragon Plus Environment
47
48
1
2
3 Figure 3A and Figure 3B clearly illustrate the link between the time of maximal concentrations of
4
5 ibuprofen in solution (dissolved) appearing in the intestine and in blood. Ibuprofen is a weak acid
6
7 (pKa ~4.85) and dissolution of ibuprofen is favored at pH > 4.85, while very low intrinsic solubility
8
9 concentrations are expected in the stomach in fasting state conditions (pH 1-2). The pH is highly
10
11 variable and fluctuating along the GI tract3,8,22,45,46, and this will have a major influence on the
12
13 dissolution of the drug.12,47 Figure 4A and Figure 4B shows the pH values as measured in this study at
14
15 the time of the duodenal/jejunal Tmax appeared in both fasted and fed state conditions, respectively,
16
17 with the red horizontal line approximating the pKa of ibuprofen.
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 17
1
2
3
4
Fasted State Fed State
5A B
6
7 8 8
8 7 7
9
6 6
10
11 5 5
12
pH
pH
4 4
13
14 3 3
15 2 2
16
1 1
17
18 0 0
19
20
21
22 pH at Duodenal Tmax pH at Jejunal Tmax pH at Duodenal Tmax pH at Jejunal Tmax
23
24
25 Figure 4: (A) pH values as measured at the time Tmax of ibuprofen appeared in duodenum (blue bars) and jejunum (yellow bars) in fasted state conditions. (B) pH values as measured at the time Tmax of ibuprofen
26 appeared in duodenum (blue bars) and jejunum (yellow bars) in fed state conditions. The red line indicates the pKa value of ibuprofen (pKa ~4.85). F1 stands for ‘Fasted State – Visit #1’; F2 stands for ‘Fasted State –
Visit #2’; P1 stands for ‘Fed State – Visit #1’; P2 stands for ‘Fed State – Visit #2’.
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42 18
43
44
45
47
48
1
2
3 The pH values measured at the time of appearance of maximal solution concentrations of
4
5 ibuprofen in the duodenum/jejunum were higher than the pKa of the drug (~4.85) and favorable for
6
7 dissolution of ibuprofen at the duodenal/jejunal Tmax. This was observed for the majority of the
8
9 subjects (30 out of 36 subjects). Furthermore, based on the individual plots (data not shown), it was
10
11 indeed shown that at the time when the plasma Cmax appeared, solution concentrations in the intestinal
12
13 tract were associated with pH values higher than the pKa of ibuprofen, sufficient to ensure complete
14
15 dissolution of ibuprofen (i.e. solution and total concentrations are equal) for almost all subjects (10 out
16
17 of 11 for the fasted state; 7 out of 11 for the fed state). Remarkably, whenever the first wave of phase
18
19 III contractions was observed after administration of the tablet, the appearance of the plasma Cmax was
20
21 on average 1 h later in fasting state conditions: the strong burst of phase III contractions, often referred
22
23 to as ‘the housekeeper wave’48,49, would likely stimulate gastric emptying of ibuprofen such that large
24
25 quantities of ibuprofen enter the small intestine and dissolve. If this arrival in the small intestine is
26
27 accompanied with a sufficient pH to promote ibuprofen’s dissolution (as observed in Figure 4), we
28
29 suggest that motility and pH are the major determinants for the appearance of plasma Cmax and Tmax of
30
31 the drug.
32
33
Based on research reports, there is a clear link observed between the rate of duodenal
34
35
secretions (i.e. bile, pancreatic enzymes and bicarbonate) and the appearance of the interdigestive
36
37
cyclic motility phases of the MMC: pancreatic enzyme and bile secretion peak in the late phase II
38
39
contractions (emptying of approximately 25% of gallbladder contents), whereas gastric acid and
40
41
42 bicarbonate secretion into the duodenum peak during the initiation of phase III contractions.19,50–52
43
44 This link between GI motility and secretions has been defined as the ‘secretomotor complex’, which
45
46 appears to be regulated by hormonal (e.g. motilin and pancreatic peptide) and neural (e.g. vagus nerve
47
48 stimulation mediated by acetylcholine) stimuli.53,54 As confirmed by Vantrappen and colleagues, a rise
49
50 in duodenal pH in humans was observed following the start of duodenal phase III contractions, which
51
52 would be expected to be reflected in the pH values we observed (> 4.85) at the time of appearance of
53
54 the duodenal/jejunal Tmax (Figure 4).17
55
56 In this study, the pH along the GI tract was highly fluctuating as a function of time and
57
58 ibuprofen was present in the GI tract up to 7 hours based on aspiration of luminal samples. Figure 5
59
60 19
1
2
3 shows the average pH profiles as a function of time for the different GI segments in fasted and fed
4
5 state conditions.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 20
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34 Figure 5: Mean pH versus time profiles in fasting (n=20) and fed state (n=17) conditions as measured in the stomach, the duodenum and the jejunum (mean + SD). Data obtained from Koenigsknecht et al.15
35
36
37
38
39
40
41
42 21
43
44
45
47
48
1
2
3 Gastric pH was observed to be highly variable in fasted (range 1.1-7.47) and fed (range 1.1-
4
5 7.39) states. The average and median pH observed in fasted state were 2.50 and 2.25, respectively. The
6
7 average and median pH in fed state were 4.04 and 3.95, respectively. Ingestion of the liquid meal (pH
8
9 6.66) resulted in an initial increase in gastric pH, which slowly decreased as a function of time
10
11 returning to fasted state conditions.15 Both the average and pH range were in line as observed in other
12
13 studies where gastric pH was measured after aspiration of gastric fluids in healthy volunteers.14,55,56
14
15 Duodenogastric reflux and/or saliva swallowed by the volunteer may contribute to higher gastric pH
16
17 values in fasted state conditions, as discussed in the literature and as observed in this study.14
18
19
20 Duodenal pH values ranged from 1.71 to 7.57 in fasted state, with a mean value of 4.93 and a
21
22 median value of 4.91. Jejunal pH values ranged from 2.2 to 6.75 in fasted state, with a mean value of
23
24 5.55 and a median value of 5.62. Less variability in pH was observed in the jejunal segment compared
25
26 to the duodenum. As the main function of the duodenum is alkalizing and mixing the gastric content
27
28 with pancreatic secretions (e.g. bicarbonate, amylase, lipase, trypsinogen), variability in pH would be
29
30 more expected in this part of the intestine compared to the more distal segments (i.e. jejunum and
31
32 ileum).57 Recently published data demonstrated that pH values for aspirated duodenal fluids as a
33
34 function of time in a wide range from 3.4 to 8.3 and 4.7 to 7.1 in fasted and fed state, respectively.45
35
36 Regarding fed state conditions, duodenal pH values ranged from 1.27 to 7.21 (mean: 5.26; median:
37
38 5.35), whereas jejunal pH values ranged from 4.46 to 7.77 (mean: 6.04; median: 6.10).
39
40
41 The pH along the GI tract is a dynamic physiological variable. The wide range of pH that has
42
43 been measured in all volunteers can significantly affect drug dissolution of ibuprofen along the
44
45 intestinal tract and thus the amount of drug in solution and available for absorption. Figure 6 depicts
46
47 the average profiles of solution concentrations of ibuprofen, total concentrations and pH as a function
48
49 of time in fasted state duodenum (A) and jejunum (B), as well as in fed state duodenum (C) and
50
51 jejunum (D). Moreover, the average time of appearance of post-dose phase III contractions (2.02 and
52
53 4.64 h in fasted and fed state, respectively) is indicated by the green line, while the average plasma
54
55 Tmax (2.98 and 4.88 h in fasted and fed state, respectively) is indicated by the dark blue line.
56
57
58
59
60 22
1 Fasted State Duodenum Fasted State Jejunum

2
3 3000000 8 3000000 8
4 2000000 2000000
5 1000000 6 1000000 6
Conc IBU (ng/mL)
Conc IBU (ng/mL)

6
7 600000 600000
pH
pH
4 4
8
400000 400000
9
2 2
10 200000 200000
11
12 0 0 0 0
13 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time (h) Time (h)
14
15 Total Conc Duodenum Post-dose Phase III Contr Total Conc Jejunum Post-dose Phase III Contr
16 pH Duodenum pH Jejunum
17 Solution Conc Duodenum
Plasma Tmax
Solution Conc Jejunum
Plasma Tmax
18
19
20 Fed State Duodenum Fed State Jejunum
21 8 8
1200000 1200000
22
23 800000 6 800000 6
Conc IBU (ng/mL)
Conc IBU (ng/mL)

24 300000
25 600000
pH
26
4 pH 4
200000
27 400000
28 2 2
100000
200000
29
30 0 0 0 0
31 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
32 Time (h) Time (h)
33 Total Conc Duodenum Total Conc Jejunum

Post-dose Phase III Contr Post-dose Phase III Contr
34 pH Jejunum
pH Duodenum
35 Plasma Tmax Solution Conc Jejunum Plasma Tmax
Solution Conc Duodenum
36
37
Figure 6: Average solution concentrations of ibuprofen (IBU) (blue line), total concentrations of IBU (gray area) and pH profiles (red line) of all subjects as measured in fasted state duodenum (A), fasted state jejunum
38 (B), fed state duodenum (C) and fed state jejunum (D). The green line indicates the average time when phase III contractions occurred after intake of the tablet. The dark blue line represents the average plasma Tmax.
39 Data presented as mean + SD.
40
41
42 23
43
44
45
47
48
1
2
3 Based on the average and individual profiles, the present pH determined the fraction dissolved
4
5 of ibuprofen along the intestinal tract, indicated by the same profile pattern between solution
6
7 concentrations and pH in fasted state (Figure 6A and 6B). In postprandial conditions, duodenal pH
8
9 (mean pH 5.26) was decreasing as a function of time, resulting in more solid ibuprofen in the collected
10
11 samples in the last three hours of aspiration (Figure 6C), whereas jejunal pH (mean pH 6.04) remained
12
13 relatively constant as a function of time, resulting in dissolution of ibuprofen along the jejunal tract
14
15 (Figure 6D). Gastric emptying of ibuprofen will likely be slowed down in postprandial conditions,
16
17 which is observed in the delayed plasma Tmax in fed state conditions compared to fasted state
18
19 conditions (2.97 h versus 4.88 h, respectively). This is due to the later maximal concentrations of
20
21 ibuprofen in the intestinal tract (Figure 3; orange dots). Further, as depicted in Figure 7C and 7D, the
22
23 onset of phase III contractions was delayed in fed state conditions relative to the fasted state,
24
25 indicating a slow release of ibuprofen from the stomach to the small intestine. The plasma Cmax values
26
27 of all volunteers are depicted as a function of the post-dose appearance of phase III contractions in
28
29 Figure 7.
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 24
1
2
3
4
5 100000
6
7 90000
8 y = - 6713.467 + 69802.07
80000
9
Plasma Cmax (ng/mL)

(1702.21 – 6298.96)
10 70000
11 R² = 0.3204
60000
12
50000
13
14 40000
15 30000
16
17 20000
18 10000
19
0
20 0 1 2 3 4 5 6 7 8
21
Time to Phase III contractions post-dose (h)
22
23
24
25
26 Figure 7: Plot of plasma Cmax (h) as a function of time of appearance of phase III contractions after oral intake of ibuprofen. Fasted and fed state results are depicted by the blue and orange dots, respectively. The
trendline is given by the black line and the Pearson Coefficient of Determination is expressed as R2. Standard errors of slope and intercept, respectively, of the linear regression are indicated in parenthesis. Regression
27 was significant with p < 0.05 (Analysis of Variance, ANOVA).
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42 25
43
44
45
47
48
1
2
3 The delayed appearance of phase III contractions in fed state conditions was positively linked
4
5 to a decreased systemic exposure of ibuprofen (in terms of Cmax), which clearly illustrates the effect of
6
7 motility on systemic drug exposure. It should be noted that subject B041 - visit #1 in fed state
8
9 conditions, ingested only 7 mL of Pulmocare® and, therefore, demonstrated a fast onset of phase III
10
11 contractions, occurring in one hour after intake of the tablet. The time to onset of phase III
12
13 contractions is associated with the rate of gastric emptying of the liquid meal which, in turn, will
14
15 depend on the caloric intake of the meal. Moreover, the amplitude and origin (i.e. antral versus
16
17 duodenal) of these contractions observed in fasted and fed state may reveal how they affect
18
19 formulation disintegration and drug release along the GI tract and is also subject of interest for future
20
21 research.
22
23
24 Where most in vitro predictive dissolution tests focus on a constant pH and high buffer
25
26 capacity during dissolution testing, it should be noted that this is not an adequate reflection of the in
27
28 vivo situation. In the case of ionized drugs, depending on pKa, the fluctuating pH along the intestinal
29
30 tract is one of the major causes in explaining inter- and intra-individual variability in drug exposure
31
32 since it affects the dissolved fraction of a drug. The changing intestinal pH is determined by the buffer
33
34 capacity of these fluids. Figure 8 depicts buffer capacity of the aspirated GI fluids as a function of time
35
36 in fasted (A) and fed (B) state.
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 26
1
2
3
4
5
6
7
8 A 12 B 12
Buffer Capacity (µmol/mL/∆pH)

10 12 10
10

11 10
8 8
12
13 6 8
6
14
15 4 6
4
16
2 4
17 2
18 2
0 0
19 0 1 2 3 4 5 6 7 8
20 0 0 1 2 3 4 5 6 7 8
Time (h) Time (h)
21 0 1 2 3 4 5 6 7 8
22 Time (h)
23
24
Buffer Capacity Stomach Buffer Capacity Duodenum Buffer Capacity Jejunum
25
26
27
28
29 Figure 8: Buffer capacity values of the aspirated fluids in (A) fasting state conditions and (B) fed state conditions as a function of time (h). The orange line represents buffer capacity data of the aspirated gastric fluids;
30 the yellow line represents buffer capacity data of the aspirated duodenal fluids; the blue line represents buffer capacity data of the aspirated jejunal fluids. Data presented as mean + SD.
31
32
33
34
35
36
37
38
39
40
41
42 27
43
44
45
47
48
1
2
3 Surprisingly, the buffer capacity of the luminal fluid along the entire GI tract is extremely low.
4
5 This is likely the reason why ibuprofen was still present in the luminal samples throughout the entire 7
6
7 hours of aspiration, as a dynamic pH was observed that will determine the fraction dissolved (Figure
8
9 6). Only a few publications in the literature report buffer capacity of GI fluids. To the extent of our
10
11 knowledge, buffer capacity has never been measured for GI fluids at several sites in the upper GI tract
12
13 based on aspirated samples as a function of time; usually this was only measured for pooled samples.
14
15 The measured buffer capacity in the intestinal fluids derived from this study is in line with reported
16
17 results in literature: Fadda et al. reported an average of 3.23 µmol/mL/∆pH for aspirated fasted state
18
19 jejunal fluids, whereas Persson and colleagues measured a buffer capacity of 2.8 µmol/mL/∆pH for
20
21 fasted human intestinal fluids (HIF).36,58 In fed HIF, Persson et al. reported a buffer capacity of 14.6
22
23 µmol/mL/∆pH (single value), remarkably higher than the average buffer capacity measured in this
24
25 study for all fed state aspirates in the duodenum and jejunum (2.16 µmol/mL/∆pH and 1.81
26
27 µmol/mL/∆pH, respectively).58 Based on the two plots (Figure 8A and 8B), values in fed state were on
28
29 average slightly higher than fasted state values as the intake of the liquid meal will stimulate
30
31 pancreatic secretions (e.g. amylase, lipase, and bicarbonate secretion) to facilitate food digestion.57
32
33
The buffer capacity of gastric fluids is reported to range between 7-18 µmol/mL/∆pH after intake of
34
35
250 mL of water14 and 4.7-27.6 µmol/mL/∆pH after intake of 240 mL of water.59 When a liquid meal
36
37
(Ensure Plus®) was administered, a rise in gastric buffer capacity was observed: reported values
38
39
between 14-28 µmol/mL/∆pH14 and 22.5-30.0 µmol/mL/∆pH60, which are higher than observed in our
40
41
42 clinical study (ranging between 2.33 and 5.98 µmol/mL/∆pH on average for all subjects). This
43
44 observation is likely due to differences in administered volumes/types of liquid meals between studies.
45
46 In general, the measured buffer capacity values are extremely low on average and five times
47
48 lower compared to the buffer capacity of simulated intestinal fluids, frequently applied in dissolution
49
50 testing. For example, FaSSIF (pH 6.5) and USP SIF (pH 6.8) both demonstrate retaining a strong
51
52
buffer capacity of 12 and 18.4 µmol/mL/∆pH, which is not suitable for in vitro predictive BE
53
54
dissolution testing, maintaining a constant pH value during the entire dissolution experiment.
55
56
Furthermore, besides the low buffer capacity of human intestinal fluids, differences in secretion rates
57
58
59
60 28
1
2
3 between H+ (approximately 17 mmol/h)61 from the parietal cells in the stomach and HCO3- (8
4
5 mmol/h)62 from the pancreas and mucosal cells in the duodenum, will likely contribute to the observed
6
7 pH fluctuation in the upper small intestine. Moreover, the low luminal concentrations of bicarbonate in
8
9 combination with the limited available duodenal fluid volume (i.e. less than 50 mL free fluid available
10
11 for drug dissolution), is likely to be an additional reason for this dynamic change in pH.24 It should be
12
13 noted that the length and intensity of motor contractions, e.g. phase III, and secretory rates are variable
14
15 between and within humans due to the time of the day, time of dosing, and the length of time after
16
17 food ingestion.50
18
19
20 Therefore, the important role of buffer capacity needs to be reflected in preclinical in vitro/in
21
22 silico models in order to make a good prediction of the systemic appearance of an oral drug product.
23
24 This in vivo predictive dissolution method would likely be more complex than the current QC
25
26 methods. The iPD method would be useful for developing optimized formulations and it may be more
27
28 appropriately to refer to it as formulation predictive dissolution (fPD) testing. This method would be
29
30 able to discriminate between inter- and intra-individual differences in systemic exposure of a drug
31
32 product and would be extremely useful for oral drug product development and optimization. The use
33
34 of more complex, multicompartmental models (e.g. gastrointestinal simulator (GIS)63, artificial
35
36 stomach-duodenum (ASD) model64 and the two-phase dissolution model65,66) attempt to integrate the
37
38 variables of the dynamic GI environment, capturing the critical formulation and product variables,
39
40 such as gastric emptying, intestinal secretions and the potential for drug absorption, resulting in a
41
42 better understanding of formulation and/or drug behavior for certain classes of drugs and/or
43
44 formulations. While much public policy debate is required, these fPD models may eventually be useful
45
46 in BE determinations (e.g. BCS biowaiver extensions) and/or may lead to a reduction in the number of
47
48 human subjects required for in vivo BE trials and minimize the number of failed BE studies.47
49
50
51 Neutralizing Effect of Bicarbonate on Gastric Acid and the Effect of Ibuprofen Dissolution on pH
52
53 Measured bicarbonate concentrations in the upper small intestine have been reported to be 4-
54
55 21 mM with an average of 15 mM.16,21 If we assume that gastric fluids (pH 2=0.01 M HCl) would
56
57 empty the stomach as first kinetic order process (Equation 1), the maximum concentration of protons
58
59
60 29
1
2
3 (H+) that would enter the small intestine is 3.4 mM. As this concentration would interact with the
4
5 present bicarbonate (a minimum of 4 mM and a maximum of 21 mM), a measured intestinal pH of
6
7 5.46 and 6.21 would be observed for both situations (Equation 2). Regarding the dynamic environment
8
9 of the GI tract, concentrations of HCl entering the duodenum and concentrations of bicarbonate
10
11 present in the duodenum may vary, resulting in fluctuations of pH levels up to 5 units which would not
12
13 be uncommon in the upper part of the intestinal tract.61
14
15
16 In addition, the dissolution of an acidic or basic drug in these fluids would be expected to alter
17
18 the pH of the intestinal fluids. Based on the measured solution concentrations of ibuprofen in the
19
20 duodenum, a drop in pH of approximately two units can be estimated due to the drug (Equation 3) in
21
22 an aqueous environment with a negligible buffer capacity (Figure 9).
23
24
25 12
26 10
27
28 8
29
pH
6
30
31 4
32 2
33
34 0
0 1 2 3 4 5 6 7 8
35
36 Time (h)
37
38 Figure 9: Impact of drug dissolution of ibuprofen on pH in an aqueous environment with a negligible buffer capacity. Data presented as
39 mean + SD.
40
41
42
43 A similar drop in pH was observed when a dissolution experiment was performed for one
44
45 tablet of ibuprofen (400 mg) in 500 mL of 10 mM phosphate buffer dissolution media at a pH of 6.0
46
47 and 6.8 (buffer capacity 1.6-2.8 µmol/mL/∆pH).12 Dissolution of ibuprofen resulted in a drop in pH
48
49 from 6.0 to 5.1 and from 6.8 to 6.1, respectively, not as outspoken as observed in our study. Based on
50
51 the administered dose, the drop in pH along the intestinal tract may have a significant effect on drug’s
52
53 dissolution. Performing standard QC dissolution methods didn’t seem to affect the in silico systemic
54
55 outcome of lesinurad (weak acid, pKa 3.2) for different batches of the drug, differing in particle size.67
56
57 However, selection of the right buffer and applying biorelevant buffer concentrations is of utmost
58
59
60 30
1
2
3 importance in developing a predictive dissolution test to link GI variability with the systemic exposure
4
5 of the drug, which could explain the possible failures in BE studies. It should be noted that the
6
7 frequently applied phosphate (pKa 7.2) buffer (e.g. FaSSIF and USP SIF) is not a biorelevant
8
9 reflection of the bicarbonate (pKa 6.35) buffer as observed in vivo. Differences in drug release
10
11 behavior have been reported for both buffers during in vitro dissolution experiments.27 Further, the
12
13 dynamic change in buffer capacity along the intestinal tract is due to the CO2 concentrations in the GI
14
15 fluids, both in solution (CO2(aq)) and the luminal gas phase (CO2(g)) as these concentrations directly
16
17 determine the formation of bicarbonate. The concentration of CO2 dissolved in water follows Henry’s
18
19 Law constant (KH) and the partial pressure of gaseous CO2 (i.e. J,-1 :
20
21
22
6 ⇌
KL (Reaction 3)
23
24
(,-1 'N +
25 &)M OPQ1
(Reaction 4)
26
27
28 The formation of bicarbonate can be produced by sparging gaseous CO2 equilibrated with
29
30 water. At different pH values, the formation of bicarbonate will differ and so will the buffer capacity,
31
32 as experimentally demonstrated by Krieg and colleagues (Table 2).25
33
34
35 Table 2: Effect of sparged CO2 on the formation of bicarbonate in water at different pH values. Data derived from Krieg et al.25
36
37 Bicarbonate Buffer Concentration (mM)
38
39 Buffer Capacity in Parentheses (µmol/mL/∆pH)
40
41 %CO2
42
43 pH 5% 10% 15% 20% 40% 60%
44
45
5.5 0.19 0.38 0.57 0.76 1.52 2.29 (0.61)
46
47
6 0.6 1.20 1.81 2.41 4.82 7.23
48
49
50 6.5 1.9 3.81 (2.10) 5.71 (3.10) 7.62 (4.20) 15.23 22.85
51
52 7 6.02 12.04 18.07 24.10 48.17 72.26
53
54 7.5 19.04 (2.50) 38.08 57.13 76.17 152.34 228.51
55
56
57
58
59
60 31
1
2
3 At lower pH values (pH 5.5), the equilibrium solubility of CO2 is too low, even at high partial
4
5 pressures, to form meaningful bicarbonate buffer concentrations. Under atmospheric pressure
6
7 conditions, the percentage of CO2 is equal to 400 ppm (0.04% ~ 0.304 mmHg). In the stomach of
8
9 healthy volunteers, measured luminal percentages of CO2 ranged between 4-10%. In the proximal
10
11 duodenum, this value was significantly higher: 66%.68,69 This means that the partial pressure of CO2 in
12
13 the upper small intestine can be 1650 times higher than the partial pressure measured in the
14
15 atmosphere likely explained by the formed CO2 after the neutralizing reaction of HCl and bicarbonate
16
17 (Reaction 1). As derived from Table 2, a maximal concentration of bicarbonate (and thus a maximal
18
19 buffer capacity) can be obtained at a neutral pH (pH 7-7.5) along the intestinal tract. As CO2 (g) may
20
21 evaporate after intestinal aspiration, it should be noted that values mentioned in literature for aspirates
22
23 may underestimate the effective buffer capacity. However, as reported by Fadda et al., buffer capacity
24
25 measurements immediately performed on fluid collection and after storage did not significantly
26
27 differ.36 It can be stated that the formation of bicarbonate along the intestinal tract is a dynamic and
28
29 complex phenomenon that depends on the rate of bicarbonate secretion (secretomotor complex), the
30
31 circulating CO2 (aq)/ CO2 (g) along the GI tract and the dehydration reaction of H2CO3 which is
32
33
formed by the reaction of H+ and HCO3- itself.
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 32
1
2
3 Conclusion and Future Directions
4
5
6 The fluctuating pH and buffer capacity along the GI tract have been shown to be a major
7
8 determinant of inter- and intra-subject variability in systemic exposure of ibuprofen. The pH presented
9
10 in the different segments of the intestinal tract determine the solution concentrations, total
11
12 concentrations and absorption of ibuprofen from the intestinal lumen. This study clearly demonstrated
13
14 the link between the plasma Tmax and duodenal/jejunal Tmax of ibuprofen after the administration of the
15
16 800 mg ibuprofen tablet to healthy volunteers. At the time these maximal duodenal/jejunal
17
18 concentrations of ibuprofen were reached, pH values in these aspirated samples were higher than the
19
20 pKa value of ibuprofen, supporting the dissolution of the drug. The dynamic change in pH along the
21
22 GI tract was explained by the limited buffer capacity of the aspirated fluids. The plasma Cmax was, in
23
24 turn, positively linked with the time to appearance of the first phase III contractions after oral
25
26 administration of ibuprofen. These results demonstrate how the dynamic pH, explained by the limited
27
28 buffer capacity, and the time of drug intake relative to the cyclic motility pattern will result in not only
29
30 inter-subject variability but also intra-subject variability in systemic availability (Cmax and Tmax) for a
31
32 BCS class 2a drug.70
33
34
35 Based on the results of this study, we expect that this very low buffer capacity and dynamic
36
37 pH are also essential and determining for the absorption of basic drugs, where the levels of
38
39 supersaturation and rates of precipitation will be affected by the dynamic change of pH and buffer
40
41 capacity along the intestinal tract.71 Future in vivo studies should focus on how (i) the amplitude of
42
43 contractions, (ii) the origin of contractions (i.e. antral versus duodenal), (iii) the gastric emptying rate
44
45 (linked to the ingested amount of calories) and (iv) intestinal transit times (based on concentrations of
46
47 a non-absorbable marker), may account for variability in systemic outcome of the administered drug
48
49 product. Finally, based on the results of this study, adjustments to the GIS model (e.g. use of
50
51 bicarbonate buffer) will be performed in order to adequately predict the in vivo outcome of oral drug
52
53 absorption and to capture the GI and plasma variability as observed in the clinical study.
54
55
56
57
58
59
60 33
1
2
3 Acknowledgments
4 This work was supported by grant # HHSF223201510157C and grant # HHSF223201310144C by the
5
6 U.S. Food and Drug Administration (FDA). This report represents the scientific views of the authors
7
8 and not necessarily that of the FDA.
9
10
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Low Buffer Capacity and Alternating Motility Along The Human Gastrointestinal Tract Implications For in Vivo Dissolution and Absorption of Ionizable Drugs

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Molecular Pharmaceutics is published by the American Chemical Society. 1155

Plasma Tmax (h)

1 Fasted State Duodenum Fasted State Jejunum

Conc IBU (ng/mL)

Conc IBU (ng/mL)

Conc IBU (ng/mL)

32 Time (h) Time (h)

33 Total Conc Duodenum Total Conc Jejunum

Plasma Cmax (ng/mL)

Buffer Capacity (µmol/mL/∆pH)

Buffer Capacity (µmol/mL/∆pH)

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