European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

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European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier.com/locate/ejpb

Fasted and fed state human duodenal fluids: Characterization, drug solubility, and comparison to simulated fluids and with human bioavailability

D. Dahlgren a, M. Venczel b,c, J.-P. Ridoux b,c, C. Skjold¨ a, A. Müllertz d, R. Holm e, P. Augustijns f, P.M. Hellstrom¨ g, H. Lennernas¨ a,* a Department of Pharmaceutical Biosciences, Biopharmaceutics, Uppsala University, Sweden b Global CMC Development Sanofi, Frankfurt, Germany c Global CMC Development Sanofi, Vitry, France d Physiological Pharmaceutics, University of Copenhagen, Copenhagen, Denmark e Drug Product Development, Janssen R&D, Johnson & Johnson, Beerse, Belgium f Drug Delivery and Disposition, KU Leuven, Leuven, Belgium g Department of Medical Sciences, Gastroenterology/Hepatology, Uppsala University, Sweden

ARTICLE INFO ABSTRACT

Keywords: Accurate in vivo predictions of intestinal absorption of low solubility drugs require knowing their solubility in Bioavailability physiologically relevant dissolution media. Aspirated human intestinal fluids (HIF) are the gold standard, fol­ Food effects lowed by simulated intestinal HIF in the fasted and fed state (FaSSIF/FeSSIF). However, current HIF charac­ Drug solubility terization data vary, and there is also some controversy regarding the accuracy of FaSSIF and FeSSIF for Human intestinal fluids predicting drug solubility in HIF. This study aimed at characterizing fasted and fed state duodenal HIF from 16 Drug absorption Drug dissolution human volunteers with respect to pH, buffer capacity, osmolarity, surface tension, as well as protein, phos­ Drug delivery pholipid, and bile salt content. The fasted and fed state HIF samples were further used to investigate the equi­ librium solubility of 17 representative low-solubility small-molecule drugs, six of which were confidential industry compounds and 11 were known and characterized regarding chemical diversity. These solubility values were then compared to reported solubility values in fasted and fed state HIF, FaSSIF and FeSSIF, as well as with their human bioavailability for both states. The HIF compositions corresponded well to previously reported values and current FaSSIF and FeSSIF compositions. The drug solubility values in HIF (both fasted and fed states) were also well in line with reported solubility data for HIF, as well as simulated FaSSIF and FeSSIF. This indicates that the in vivo conditions in the proximal small intestine are well represented by simulated intestinal fluids in both composition and drug equilibrium solubility. However, increased drug solubility in the fed vs. fasted states in HIF did not correlate with the human bioavailability changes of the same drugs following oral administration in either state.

1. Introduction rate and solubility in the intestinal lumen, because only aqueous dis­ solved drug molecules cross the intestinal epithelial barrier. A systemically acting drug administered orally must be absorbed Thus, the rate-limiting step in intestinal drug absorption can be from the gastrointestinal (GI) tract and avoid first-passextraction in the either the solubility/dissolution rate of the drug in the GI luminal fluids gut wall and liver before it can exert its pharmacological effect. The or the intestinal permeability. In drug development, solubility is the fraction absorbed from the intestine is determined by the velocity more frequently observed limitation, because candidate drugs in drug (length/time) of drug transport across the apical intestinal cell mem­ discovery and early development tend to have limited aqueous solubility brane, i.e. the permeability. It is also determined by the drug dissolution and slow dissolution rate in the GI lumen [1]. This is related to the lead-

Abbreviations: HIF, human intestinal fluid; FaSSIF/FeSSIF, fasted and fed state simulated intestinal fluids; GI, gastrointestinal; SIF, simulated intestinal fluid. * Corresponding author at: Department of Pharmaceutical Biosciences, Uppsala University, Box 580, SE-751 23 Uppsala, Sweden. E-mail address: [email protected] (H. Lennernas).¨ https://doi.org/10.1016/j.ejpb.2021.04.005 Received 11 February 2021; Received in revised form 1 April 2021; Accepted 5 April 2021 Available online 16 April 2021 0939-6411/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251 findingtechniques applied, which often are based on molecular in silico Table 1 and in vitro screening of receptor-ligand interactions [2–4]. These Some physicochemical properties and BCS class of the 17 drug compounds in screening tools select for drugs that can interact with target receptors, this study. meaning they will typically be hydrophobic and/or lipophilic [5–7]. Compounds BCS class MM Class Log Log Tm ◦ Consequently, low aqueous solubility leading to low oral bioavail­ [46–48] (g/ (pKa) D7.4 D6.5 ( C) ability is a bottle neck in drug discovery and early development even mol) though this biopharmaceutical factor was recognized several decades A1150 II 440 Base 2.51 ago. Drug solubility and dissolution rate in water are therefore critical (13.67) biopharmaceutical parameters for any candidate drug or drug product A1260 IV 483 Acid (5) 7.75 83 A4356 II 410 Base 2.6 (pH and are investigated by a range of methods [8]. Early development re­ (2.8) 6.8) quires resource- and time-efficient methods to identify drugs with un­ Acid favorable biopharmaceutical and pharmacokinetic properties, such as (7.5) poor intestinal solubility. This typically happens when the solubility A7651 III 600 Base 2.6 (9.98) value in water is below a predetermined value, for example, a drug with A8942 II 400 Base > a dose number (Do) 5, as proposed by Lipinski [9]. However, high- (6.1) throughput water solubility values of a drug do not necessarily reflect A9530 II 600 Acid 3.4 (pH the compound solubility in the GI lumen. Especially for lipophilic drugs, (3.5) 6.8) the difference can be substantial. For instance, cinnarizine, fenofibrate, Base < µ (7.4) and danazol are three low-solubility drugs ( 5 g/mL in water), but aprepitant II 534 Base 4.3 4.5 252 they have 20-fold higher solubility in biorelevant fasted state intestinal (3.1) media than in plain aqueous buffer at pH 6.5 [10]. bromocriptine II 654 Base 3.8 3.5 215 Hence, it is important to investigate drug solubility in media that (6.7) carvedilol II 406 Base 0.96 1.0 114 give accurate in vivo absorption predictions [11]. The gold standard in (8.8) biopharmaceutical drug solubility assessment is human intestinal fluid felodipine II 384 Base 3.4 3.4 145 (HIF). By individual sampling of different parts of the GI tract at (5.4) different prandial states, HIF takes into account the large intra- and fenofibrate II 361 Neutral 5.28 5.28 80 inter-individual variabilities. This is important because food intake ibuprofen II 206 Acid 1.34 2.19 76 (4.8) triggers neural and hormonal signaling from the GI tract in response to ketoconazole II 531 Base 4.16 3.91 145 gastric distension and the chemical presence of nutrients. For instance, (6.8) digested dietary lipids in the lumen control GI motility, luminal pH, bile probucol II 516 Neutral 10.57 10.57 126 salts, and lipase secretion [12,13]. The fasted state HIF composition is tadalafil II 389 Neutral 1.64 1.64 301 valsartan II 435 Acid 1.08 2.46 116 also affected by the difference in gastric, intestinal bile and pancreatic (3.9/4.7) secretions during the human interdigestive motility cycle [14,15]. zafirlukast II 575 Acid 5.46 5.48 139 However, HIF is not practical for routine solubility investigations in (4.29) – preclinical drug development as it involves sampling from human sub­ Log D7.4/6.5 - n-octanol water partition coefficientat pH 7.4/6.5, MM molar mass, – – jects, a time- and resource-demanding approach. An alternative to HIF is pKa dissociation constant, Tm melting point values. The properties of the confidentialdrugs are from the literature [48] and the values of the known drugs are simulated intestinal fluid (SIF) in the fasted (FaSSIF) and fed (FeSSIF) calculated using chemicalaize.com for all values except Tm that was from https://p states. These in vitro compositions of bile salts and phospholipids at ubchem.ncbi.nlm.nih.gov/. appropriate pH, buffer capacity and osmolarity replicate the luminal GI conditions to better capture in vivo drug solubility and drug product performance. During the last two decades SIFs have been refined and diversity after a comprehensive analysis. Next, the solubility values from updated to identify which luminal components primarily affect the this study were compared to reported solubility values in fasted and fed solubility of different drugs [16,17]. Nonetheless, there is still contro­ state HIF, FaSSIF, and FeSSIF, as well as to their human bioavailability versy regarding their accuracy in predicting HIF drug solubility. This when administered in the fasted and fed states. This was to evaluate the applies especially to low-solubility compounds (<10 µM) in FaSSIF, and reproducibility of HIF solubility values, the accuracy of SIFs for pre­ for FeSSIF, where current data indicate an underestimation of HIF sol­ dicting solubility in HIF, and the potential of using HIF drug solubility ubility values [18]. In part, this is due to the limited number of drugs for predicting systemic exposure after oral dosing. that have had their solubility determined in HIF and to the version of SIF used in the solubility correlations. Further, data on HIF characteriza­ 2. Materials and methods tions are variable because of lack of consensus on the in vivo components and their concentrations, different food, drink, and sampling protocols, 2.1. Chemicals and the complexity of neural and endocrine responses in the GI tract [19]. 17 drugs were supplied from industrial partners. Six proprietary This investigation aimed at characterizing pooled fasted and fed state compounds (A1150, A1260, A4356, A7651, A8942, and A9530) along HIF from the duodenum of 16 volunteers with respect to pH, buffer with 11 model drugs (aprepitant, bromocriptine (Novartis Pharma AG, capacity, osmolarity, surface tension, total protein and phospholipid Basel, Switzerland), carvedilol, valsartan, and probucol (Tokyo chemi­ content, and bile salt compositions and concentrations. The fasted and cal industries, Japan), felodipine and zafirlukast (Astra Zeneca, ¨ fed state HIF samples were then used to investigate the equilibrium Goteborg, Sweden), fenofibrate (Chemagis LTD, Beer-Sheva, Israel), solubility of 17 representative low-solubility small-molecule drugs, ibuprofen (BASF, Ludwigshafen, Germany), ketoconazole (Janssen whereof six were confidential industry compounds and 11 were well- Pharmaceutical, Beerse, Belgium), tadalafil (AK scientific, Union City, known model drugs. These 11 were chosen for their structural USA)). The 17 drugs and some of their physicochemical properties are

241 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251 presented in Table 1. Ensure Plus (Abbott Nutrition, Chicago, IL). Orli­ water (t = 0). The samplings started with the fasted state, and the fed stat was obtained from Sigma-Aldrich, and Xylocaine (2% gel or 10% state sampling was performed on the same day directly after the fasted spray) from AstraZeneca AB. state sampling at Uppsala University, and at least one day after at the University of Leuven. The fasted state sampling started 10 min after intake of water, and 2.2. Molecular diversity analysis intestinal samples were collected by suction through the tube at 10-min intervals during 120 min using a syringe. The fed sampling started the A dataset of 691 pharmaceutical compounds used on the Swedish subject drinking 400 mL Ensure Plus nutritional supplement drink (600 market in the year 2001 was used as reference for the molecular di­ kcal, 29% lipids, 54% carbohydrates, 17% proteins) within 10 min. At versity analysis. Preparation and description of this dataset has been 20 min after the start of meal intake, fed subjects drank 240 mL of tap published elsewhere [20,21]. Permanently charged molecules (n = 17) water (t = 0). in the reference dataset were not included in the analysis herein. The fed state sampling procedure followed the same protocol as the Structures were imported in Maestro (Schrodinger¨ Release 2019–3: fasted one described above. After the last sampling time (t = 120 min) Maestro, Schrodinger,¨ LLC, New York, NY, 2019). The dataset of 11 the GI catheter was removed. All duodenal samples were immediately model compounds was compiled from the respective structures if they weighed, analyzed for pH, and stored on ice. To prevent lipolysis, the fed were present in the reference dataset. If not present, they were imported state duodenal fluids were immediately added the lipase inhibitor separately in Maestro as SMILES. The structures were prepared using ◦ (Orlistat 1 μM). Samples were centrifuged (4 C, 10 min, 4000 × g) to LigPrep (Schrodinger¨ Release 2019–3: LigPrep) in their neutral form. remove debris, whereupon all samples from each individual and pran­ Descriptors were calculated using QikProp (Schrodinger¨ Release dial state were pooled (2 pooled samples each per individual, fasted and 2019–3: QikProp) in default mode. Principal component analysis was ◦ fed state). The pooled individual samples were stored at 20 C and performed using default settings and visualized in SIMCA (version 15, shipped to SanofiR &D (Vitry, France) for detailed characterization and Umetrics, Umeå, Sweden). Included QikProp descriptors in the principal further pooling (2 population pools, fasted and fed state). Pooled sam­ component analysis were (descriptor abbreviation found in the PCA ples were characterized with respect to: collected volume, pH, osmo­ loading plot are given in parentheses): molecular weight (mol MW), larity, buffer capacity, surface tension, total protein, phospholipid and number of hydrogen bond acceptors (acceptHB), number of hydrogen bile salts content, and bile salts composition, as described in detail bond donors (donorHB), solvent accessible volume (volume), solvent below. accessible surface area (SASA), hydrophilic component of SASA (FISA), hydrophobic component of SASA (FOSA), weakly polar component of SASA (WPSA), carbon atom π-system component of SASA (PISA), Van 2.4. Human intestinal fluids physicochemical characterization der Waals surface of polar nitrogen and oxygen atoms (PSA), globularity (glob), and predicted octanol/water log P (QPlogPo/w). HIF were characterized in physicochemical terms of pH, buffer ca­ pacity, surface tension, and osmolarity. The first two of these were ◦ measured at 37 C and the latter two at ambient temperature. pH was 2.3. Subjects and intestinal sampling procedure measured using a pHM240 meter (Radiometer, Copenhagen, Denmark) and the buffer capacity was determined by titration on the same 2.3.1. Study subjects equipment. The surface tension was determined with the Wilhelmy plate The study was approved by the regional ethics committee for human method (Thermo Cahn 322 Dynamic Contact Angle Analyzer, Irvine, CA, research in Uppsala, Sweden (no: 2013/029) and Leuven, Belgium USA). The osmolarity was determined on a Vapro vapour pressure (S53791). Sixteen healthy male (n = 10) and non-pregnant female (n = osmometer (model 552O, Wescor Inc., Logan, UT, USA). 6) volunteers, 18–45 years of age who had not been exposed to frequent ionizing radiation in the previous year were eligible to participate. Before signing informed consent and enrolling in the study, volunteers 2.5. Drug solubility in human intestinal fluid aspirates were tested against inclusion and exclusion criteria. A clinical physician assessed volunteers to be healthy based on a physical examination (free The equilibrium drug solubility in intestinal aspirates was performed from illness or gastrointestinal disorders) and laboratory tests (hepatitis on 17 small-molecule compounds covering a broad chemical space. B, hepatitis C or HIV). This study was conducted at Uppsala University Eleven were well-known model drugs and six were confidentialindustry Hospital, Sweden, and Leuven University Hospitals, Belgium, in accor­ compounds. Solubility experiments were performed in duplicates (n = dance with the International Conference of Harmonization guidelines 2) in both fasted- and fed-state pooled HIF for each compound. for good clinical practice and the principles described in the World The solubility study and the analytical characterization of samples Medical Association Declaration of Helsinki. were carried out in Biosafety Level-2 laboratories. The pooled HIF ◦ samples were stored at minus 20 C. Before the solubility de­ 2.3.2. Study design and sampling procedure terminations, small volumes of intestinal fluidswere separately frozen to This intestinal sampling study was a crossover trial performed in the decrease the number of thawing processes of the main pooled intestinal fasted and fed states in 16 subjects: eight each at Uppsala University and fluidcontainers. After a 60 min thawing process, the fasted and fed HIF University of Leuven. After an overnight fasting period of at least 10 h, were weighted into vials, with 1 mL in each. To these vials an excess API all volunteers arrived at the hospital. Following topical anesthesia of the amount of 2 mg was added to obtain a saturated suspension with the nose-throat cavity with Xylocaine (2% gel or 10% spray), a double- target concentration of 2 mg/mL. All API containing vials were put on a ◦ lumen polyvinyl catheter fitted with a guide wire (Salem Sump Tube shaker for agitation for the duration of 24 h at 37.0 ± 0.5 C under light 14 Ch, external diameter 4.7 mm) was inserted through the nose and protection. At each of the seven sampling times (15, 30 and 60 min, and placed in the duodenum (third or fourth part). The GI position was 2, 3, 6 and 24 h) the shaking was stopped and 50 µL samples were taken checked by fluoroscopy and verified by pH and visual inspection of a and filtered using a microplate filtration process before the sample small volume of intraluminal fluid.The intubation was followed by a 20- analysis. After the sampling process the sampled volume was replaced min stabilization period, after which fasted subjects drank 240 mL of tap with HIF, and agitation was continued to reach the next sampling point.

242 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

Table 2 98% A, 0.5 min 98% B, 0.2 min 98% A). The standard calibration curve Composition of versions 1 [49] and 2 [50] Fasted and Fed State Simulated In­ had a linearity of R > 0.999 for all compounds. testinal Fluids (FaSSIF/FeSSIF). Compound FaSSIF FeSSIF 2.9. Statistical analysis and calculations Version Version Version Version 1 2 1 2 All results are presented as individual values or mean ± standard Bile salt (taurocholate) (mM) 3 3 15 10 deviation (SD) or as the ratio between the solubility in the fasted and fed Phospholipid (lecithin) (mM) 0.75 0.2 3.75 2 = – – – states (ratio average fed/average fasted) for both HIF and SIF. Cut-off Sodium dihydrogen phosphate 28.7 < < (mM) values for a difference in fed vs. fasted solubility was set to 3 ratio Acetic acid (mM) – – 144 – 0.33, based on the anticipated variability in solubility determinations Sodium chloride (mM) 105.9 – 173 125.5 between laboratories. This inter-laboratory variability depends, in turn, Maleic acid (mM) – – – 55.0 on the experimental protocol (e.g. amount of drug powder used) and Sodium hydroxide (mM) – – 101 81.7 Glyceryl monooleate (mM) – – – 5 lack of standardization of data analysis [23]. When there was more than Sodium oleate (mM) – – – 0.8 one reported solubility value in HIF or SIF, the lower value was used in pH 6.5 6.5 5 5.8 the correlations. Do was calculated using equation (1), by relating the Osmolality (mOsmol/kg) 270 180 635 390 maximum oral dose and gastric volume (250 mL) to the drug solubility Buffer capacity (mEq/pH/L) 10 10 76 25 in fasted and fed HIF. dose 1 Do = × The study was stopped after 24 h where also the pH of the samples were 250mL fastedHIF or fedHIF solubility (1) measured. A comparative study was performed to evaluate the solubility determinations after filtration (PTFE, 0.45 µm pore diameter) and This Do was theoretical and not related to the regulatory-definedDo – > centrifugation processes, but there were no significant differences. The based on aqueous solubility in the pH range of 1.0 6.8. A Do 1 predicts equilibrium solubility was defined as when the dissolution reached a that the intestinal volume is insufficientto dissolve the maximum dose, < steady-state plateau (±5% between time points) or at 24 h. whereas Do 1 predicts that all drug will be in solution in the intestines.

2.6. Solid state characterization 3. Results

The X-ray powder diffraction pattern of the solubility residues was 3.1. Molecular diversity of the drug data set compared with the pattern of the relevant solid phase API. This analysis showed no changes in solid state for any of the APIs, exemplified for Principal component analysis of the reference dataset with 674 Felodipine in Supplementary file 1. Analyses were carried out at room compounds and 12 physicochemical descriptors resulted in two prin­ temperature on a Brucker D4 Endeavor instrument using the Bragg- cipal components. In total, this explained 73% of the variance in the Brentano (vertical θ-θ configuration) parafocusing geometry. A sealed dataset. The score- and loading plots are presented in Fig. 1a and b, copper anode X-ray tube running at 40 kV and 35 mA was used (λ CuKα respectively, where visual analysis of the loading plot suggests that the average = 1.54178 Å). A counting time of 1 s per step in an angular general property directions in the plots are molecular size in principal ◦ ◦ ◦ range [2 40 ] with a 0.016 step size in 2θ was used for each short- component 1 (x-axis) and hydrophobicity in principal component 2 (y- term sample analysis. For each experiment, the powder was deposited axis). The predicted score plots of the 11 known compounds with HIF onto the surface of a sample holder. Then the sample was sealed with a solubility in the fasted and fed states are presented in Fig. 1c and d, Kapton® film for safety consideration. respectively.

2.7. Comparisons 3.2. Human intestinal fluids characterization and composition

Drug solubilities in the fasted and fed state HIF from this study were Characterizations of fasted- and fed-state HIF (described below) were compared to those from the literature, and to FaSSIF and FeSSIF. As the performed on pooled HIF samples from Uppsala University and Uni­ reported SIF solubility values were determined in different FaSSIF and versity of Leuven. The mean volumes (±SD) of HIF collected in each FeSSIF versions, their compositions are presented in Table 2. Solubilities individual during 120 min were 45.8 ± 14.3 and 59.2 ± 28.3 mL in the in fasted and fed state HIF were also compared to the drug bioavail­ fasted and fed states, respectively, and there were only minor differences ability or plasma exposure in the two states. between the two sites.

2.8. Bioanalyses 3.2.1. Duodenal pH, buffer capacity, osmolarity, surface tension, and total protein, phospholipid, and bile salt contents Protein concentration was measured using a Roche Cobas Mira Table 3 presents pH, buffer capacity, osmolarity, surface tension, and clinical chemistry analyzer (Basle, Switzerland) and a commercial total protein, phospholipid, and bile salt content of the pooled fasted- enzyme-based colorimetric assay (Bio-Rad Protein Assay; Bio-Rad Lab­ and fed-state HIF samples. The fed-state duodenal pH was slightly lower oratories, Hercules, CA, USA). The analytical method used for the than the fasted-state pH (5.96 vs. 6.83), and two parallel pH stability quantificationof bile salts and phospholipid content in HIF is described investigations one month apart on frozen samples showed no change in in detail by Riethorst et al, 2016 [22]. Drug HIF concentrations were pH over 8 h. There was only a minor difference in surface tension of the ◦ analyzed with a validated analytical method at Sanofi, France, on an fasted (34.3 mN/m) and fed (26.4 mN/m) samples at 37 C. The fed- Agilent 1290 Infinity 1 type HPLC equipment with a ZORBAX Eclipse state values were substantially higher than the fasted ones for buffer ◦ Plus C18 (2.1*50 mm 1.8 µL) column: H2O + HCOOH 0.05% (A) / ACN capacity at 37 C (3.3 fold), osmolarity (2.0 fold), and total protein (5.9 ◦ + HCOOH 0.035% (B), 1.1 mL min, 55 C, runtime 2.2 min (1.5 min fold), total phospholipid (19.5 fold), and total bile salt (2.5 fold).

243 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

Fig. 1. Principal component analysis results. The score plot ellipse is the confidenceregion based on Hotelling’s T2 (95%) implemented in SIMCA as outlier indicator. (a) Score plot of the reference dataset. (b) Loading plot of the reference dataset. Predicted score plot of the 11 known compounds investigated, colored according to human intestinal molar solubility in (c) fasted state and (d) fed state.

3.2.2. Duodenal bile salt composition Table 3 The bile salt concentrations and composition in the fasted and fed The mean (±SD) pH, buffer capacity, osmolarity, surface tension, and total states are presented in Fig. 2. There were only minor differences in the protein, phospholipid, and bile salt content of the pooled (n = 16 individuals) qualitative composition between the two prandial states. The most fasted and fed state human intestinal fluid (HIF) samples. pronounced difference (fraction vs. fraction) was for taurocholic acid Parameter Fasted HIF Fed HIF (5.2%), followed by glycochenodeoxycholic acid (2.5%), glycodeox­ pH, initial 6.83 5.96 ycholic acid (2.7%), and taurochenodeoxycholic acid, taurodeoxycholic Buffer capacity (mmol/L/pH unit) 5.4 ± 0.1 18.0 ± 0.8 acid, glycoursodeoxycholic acid (~1% each), while there were no dif­ Osmolarity (mmol/kg) 189 372 ferences (<0.5%) for glycocholic acid and tauroursodeoxycholic acid. Surface tension (mN/m) 34.3 26.4 Total protein content (g/L) 2.8 ± 0.4 16.5 ± 0.2 Total phospholipid content (mM) 0.19 ± 0.02 3.72 ± 0.11 Total bile salt content (mM) 3.52 8.91

244 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

TUDC TUDC 16 µM 29 µM 0.5% 0.3% TDC TDC 134 µM 274 µM TCDC 3.8% TCDC 3.1% 410 µM 1171 µM GUDC 11.6% 13.1% GUDC GC 62 µM GC 97 µM 1076 µM 1.8% 2723 µM 1.1% 30.6% 30.6%

GCDC GCDC 879 µM 2008 µM 25.0% 22.5% TC TC 507 µM 1742 µM 14.4% GDC 19.6% GDC 438 µM 862 µM 12.4% 9.7%

Fasted Total concentration 3522 µM Fed Total concentration 8907 µM

Fig. 2. Bile salt composition and their luminal concentrations in pooled (n = 16) fasted and fed state human intestinal fluids.Bile salt abbreviations as follows: TUDC (tauroursodeoxycholic acid); TC (taurocholic acid); TDC (taurodeoxycholic acid); TCDC (taurochenodeoxycholic acid); GCDC (glycochenodeoxycholic acid); GC (glycocholic acid); GDC (glycodeoxycholic acid); GUDC (glycoursodeoxycholic acid).

lower fasted-state HIF solubility tended to have increased solubility in Table 4 the fed state (Fig. 3B). This was especially apparent for drugs with a The two (A and B) individual equilibrium solubility measurements of 17 model solubility in fasted HIF below 30 µg/mL. drugs in pooled fasted and fed state human intestinal fluids(HIF). Presented are also previously reported mean ± SD (or range) solubility values of some of the drug compounds in fasted and fed state HIF [18,51]. 3.3.2. Comparison with reported drug solubility values in human intestinal fluids Compounds Fasted HIF solubility (µg/mL) Fed HIF solubility (µg/mL) Table 4 shows the reported equilibrium solubility in fasted- and fed- A B Reported A B Reported state HIF for 7 and 4 drugs, respectively, that were also included in the aprepitant 7 7 13 ± 2.9 119 113 present study. Reported values are compared to the ones obtained in this bromocriptine 0.19 0.37 17.7 26.1 study in Fig. 4A. There were only minor differences between the re­ ± carvedilol 15 17 36 0.5 150 197 ported solubility values and the study ones, for fasted and fed states. The felodipine 16 15 14 ± 0 181 183 413 ± 52.0 only drug for which there was a difference (>3 fold) was fenofibratein fenofibrate 1.57 1.14 19 ± 25.9 140 130 147 ± 59.5 ibuprofen 3112 3112 1990 2533 2456 the fasted state. ketoconazole 27 28 28–326 775 828 754–1087 probucol 3 2 0.9 ± 0.5 39 26 25 3.3.3. Comparison between human intestinal fluids solubility and reported tadalafil 7 8 16.4 16.4 bioavailability valsartan 4789 4692 4264 3943 zafirlukast 0.37 0.37 3 3 Fig. 4B shows the solubility ratio between fed vs. fasted HIF of 10 A1150 29.1 32.9 10.1 13.3 models drugs compared to reported changes in bioavailability or plasma A1260 1.1 1.2 113 91 exposure induced by food [24–33]. The doses (and fasted and fed state A4356 83.6 83.6 48.7 47.2 Do at this dose) for the 10 drugs were: ibuprofen 800 mg (1.0/1.3), A7651 812 799 518 536 valsartan 160 mg (0.1/0.2), tadalafil 20 mg (11/5), zafirlukast 50 mg A8942 21.5 23.1 35.3 27.5 A9530 17.1 22 87.4 72.3 (540/67), carvedilol 50 mg (13/1), felodipine (n/a), aprepitant 165 mg (94/6), ketoconazole 200 mg (29/1), bromocriptine 5 mg (71/1), pro­ bucol and fenofibrate145 mg (427/4). For all 10 drugs, there were only 3.3. Drug solubility in human intestinal fluids small changes (<50%) in food-induced bioavailability or exposure, which is in contrast to the substantial increase (>3 fold) in HIF solubility 3.3.1. Equilibrium solubility for 7 of these compounds. The two individual equilibrium solubility (n = 2) measurements of the model drugs in pooled fasted- and fed-state HIF are presented in 3.3.4. Comparison with reported drug solubility values in FaSSIF and Table 4. The variability (CV%) in the drug solubility values obtained FeSSIF from the two determinations were below 30%, with the exception of the Table 5 shows reported equilibrium solubility values in FaSSIF (11 least soluble drug, bromocriptine, in the fasted state (46%). For refer­ drugs) and FeSSIF (10 drugs, no value for probucol). For comparison, the ± ence, Table 4 also includes the reported mean ( SD), or range, solubility fasted- and fed-state mean (±SD) equilibrium solubility of the model values of the same drugs in fasted- and fed-state HIF. drugs in HIF, and the reported FaSSIF and FeSSIF solubility values, are Fig. 3A shows the ratio between drug solubility in fed- vs. fasted-state presented in Fig. 5A and B, respectively. HIF for the 17 drugs. For seven of them (A1150, A7651, ibuprofen, In the fasted state, only valsartan out of the 11 drugs had a higher valsartan, A4356, A8942, and tadalafil) there were no difference be­ solubility (>3 fold) in fasted HIF than FaSSIF, while 5 drugs (bromo­ tween their fed vs. fasted solubility ratios (definedas a fold difference in criptine, zafirlukast, fenofibrate, felodipine, carvedilol) had a lower the range of 0.33 to 3). The remaining ten drugs had a higher solubility solubility (<0.33 fold) in fasted HIF than in FaSSIF. In the fed state, 3 out > in the fed state (ratio 3), with the following rank order (low to high): of 10 drugs (fenofibrate,ketoconazole, valsartan) had a higher solubility A9530, zafirlukast, carvedilol, felodipine, probucol, aprepitant, keto­ (>3 fold) in fed HIF than FeSSIF, and only bromocriptine had a lower conazole, bromocriptine, A1260, fenofibrate. Of these ten drugs, eight solubility (<0.33 fold) in fed HIF than FeSSIF. In total, there were two had a substantially higher fed/fasted solubility ratio (>10). Drugs with a

245 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

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r 10 d e y t i l st i a b /f u l d o e f s

( 1 F I H

0.1 0 1 n n 6 2 il 0 t l e l t e e 0 e s lo o n l n t fe ta laf a i in c a o ti a o r a k d ip u it z p r 115 765 r a 435 894 d 953 lu e d b a ri 126 ib A A p ls A A A ir rv o o ep n c A f u a ta f a l r r o o o ib v a c fe p p c z a to m fen e ro k b B A1260 100 Fenofibrate Bromocriptine Ketoconazole Aprepitant Probucol Felodipine o

i 10

t Zafirlukast Carvedilol ) a d r e

y A9530 t st i l i a Tadalafil b /f u d l A8942

e Valsartan o f s ( 1 A4356 F

I Ibuprofen A7651 H A1150

0.1 0.1 1 10 100 1000 10000 Fasted HIF solubility (µg/mL)

Fig. 3. (A) Ratio between the mean drug solubility in the fasted compared to fed (fed/fasted) state human intestinal fluids(HIF) as reported in this study (Table 4). (B) Comparison between the increases in fed HIF vs. fasted HIF ratio compared to the solubilities of the drugs in fasted HIF. drugs (bromocriptine and valsartan) for which the values deviated>10 4. Discussion fold between HIF and SIF; regardless of prandial state, valsartan had higher solubility in HIF, and bromocriptine had a lower solubility in HIF This study collected and characterized duodenal HIF from 16 healthy than in SIF. volunteers in fasted and fed states with respect to pH, buffer capacity, Fed- vs. fasted-state solubility ratios in HIF and SIF are compared in osmolarity, surface tension, total protein and phospholipid content, and Fig. 6. The solubility ratio in HIF was higher (>3 fold) than in SIF for 3 bile salt compositions and total content. Pooled HIF samples were used out of 10 drugs (zafirlukast, bromocriptine, fenofibrate), i.e., SIF to determine equilibrium solubility of 17 low-solubility model drugs (11 underpredicted the food-induced increase in HIF solubility. This known and 6 confidential)in fasted and fed states. The 11 known drugs underprediction was primarily related to a higher solubility in FaSSIF were physicochemically characterized, and their solubility values in HIF than in fasted HIF (Fig. 5A, bromocriptine, zafirlukast, fenofibrate, were compared to reported values of: i) HIF solubility, ii) human felodipine, carvedilol), and to a lesser extent a higher solubility in fed bioavailability in the fasted and fed states, and iii) FaSSIF and FeSSIF HIF than in FeSSIF (Fig. 5B, only bromocriptine). solubility. Principal component analysis of the 11 known drugs predicted them 3.3.5. Dose number in fasted and fed state HIF into the chemical space of 674 marketed drugs [20,21]. The 11 drugs Table 6 shows the Do for 11 model drugs. Five of the drugs (apre­ were fairly well distributed in the hydrophobic chemical space without pitant, carvedilol, ketoconazole, bromocriptine and fenofibrate) had any extreme data points compared to the marketed drugs; see the score their Do reduced from values above 10 in fasted HIF to below 5 in fed plots in Fig. 1. Although there are directions in the score plot of the HIF. A high permeability drug with a Do below 5 is defined as a low- fasted-state HIF solubility that hold the relatively most soluble solubility compound, as proposed by Lipinski [9]. (ibuprofen and valsartan) and the least soluble compounds (bromo­ criptine and zafirlukast),no general trend for HIF solubility was evident

246 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

A 10000 This study Reported Fed state )

L 1000 m / g

µ - Fasted state ( 100 y t i l i 10 ub l o S

F 1 I H 0.1 e l t e l e n e e e l t o n n lo l e t n l o ra c ta i i zo f ra i zo c b u i ip d a ro b ip a u fi b p d e fi d b o e o rv n p o n o o r r l a o u o l o r n p p e c c ib n e c P fe a f to fe f to e e k k B 100 HIF fed/fasted solubility ratio Fed/fasted bioavailability ratio )

d 10 e t o i s t a a f R d/ e

(f 1

0.1 l t l t n n fi o e n le e te e a a il in o in a f rt l kas ta z t r ro a a d ip i a p b s d u e d p ri fi p l a irl rv o e n c u a t f a l r o o ib v a c e p c o n z f a to fe e rom k b

Fig. 4. (A) Comparison of the mean solubility in the fasted and fed state HIF in this study with reported values [18]. A (-) indicates a higher (3-fold) solubility in reported HIF compared to this study. (B) Comparison between the solubility ratios of fed vs. fasted state HIF in this study with reported bioavailability values for these two states. for the investigated physicochemical properties. Osmolarity, like pH, is a luminal parameter under strict physiological Luminal pH (and buffer capacity) has an impact on the solubility of regulation in vivo. The intestine rapidly reacts to variations in osmolarity weak acidic and basic drugs. The duodenal pH values in this study were to keep the intestinal fluidisotonic. Depending on luminal tonicity, this 6.83 and 5.96 in the fasted and fed states, respectively. This is well in is done by either secretion or absorption of osmolytes, coupled to passive line with human luminal pH values from three other aspiration studies water flux[38] . For instance, the luminal osmolarity increased from 50 in the proximal small intestine of healthy volunteers, which all report a to 170 mOsm in the first25 cm of the proximal small intestine following slightly higher pH in the fed than in the fasted state (e.g. 7.5 vs. 6.1 [34], intake of water, a process that occurs during approximately 3 min 6.78 vs. 6.22 [22], 6.3 vs. 6.1 [35]). An exact proximal small intestinal [39,40]. A comparison of the fasted and fed state osmolarity values in pH value is not readily defined,as the value is affected by the position of this study (189 and 372 mOsm) to those in FaSSIF and FeSSIF, showed the intestinal tube (pH increases aborally from the pyloric sphincter that our values closely resemble those in version 2 (180 and 390 mOsm). [35]); the interdigestive motility complex in the fasted state [14]; and Given the rapid luminal adjustment of osmolarity and its overall minor the dietary composition in the fed state [36]. Of these three factors, the impact on the solubility of small drug molecules [16], it is probably not luminal position is probably more important than the pH and buffer so important in drug solubility investigations. capacity of the food. This was supported by the higher pH observed in In this study, the surface tension of fasted- and fed-state HIF were the distal duodenum compared to the proximal jejunum (6.0 vs. 7.4, comparable (34.3 and 26.4 mN/m) and similar to reported fasted state although only a few centimeters apart) after administration of compa­ values (≈28 mN/m) [34]. All versions of FaSSIF (≥54 mN/m) and rable nutritional drinks[34,37] Comparison of fasted and fed state HIF to FeSSIF (≥40.5 mN/m) consequently overpredicted surface tension by at FaSSIF and FeSSIF, respectively, showed a high similarity between pH least 15 mN/m [41]. This overprediction may result in an increased drug and buffer capacity, except for FeSSIF-V1, for which the pH was lower dissolution rate of lipophilic compounds, as surface tension reflects the (5.0 vs. 5.96) and the buffer capacity higher (76 vs. 18 mmol/L/pH). wetting behavior, whereas the effect of surface tension on equilibrium

247 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

Table 5 reassessment of these parameters in SIF need not be prioritized. Reported mean (±SD) equilibrium solubility of the known model drugs in fasted Duodenal fluids in this study were collected and pooled during 120 and fed state simulated intestinal fluids (FaSSIF/FeSSIF). min following intake of 240 mL water (fasted state) or 400 mL of a Compounds Drug solubility (µg/ FaSSIF and FeSSIF versions References nutritional drink followed by 240 mL of water (fed state). It should be mL) (Table 2) mentioned that values can vary depending on individual variations in FaSSIF FeSSIF luminal composition and the time of sampling after intake [22]. How­ ever, there was an overall good correspondence between the pooled aprepitant 13.6 101.8 Fa/FeSSIF-V2 [52] bromocriptine 58 462 FaSSIF-V1, and FeSSIF-V1 [53] values in this study and reported pooled or mean/median HIF values in with pH 6.5 the literature. There was also a good correspondence of HIF in the fasted carvedilol 55.9 ± 305.0 Fa/FeSSIF-V1 [54] and fed states to FaSSIF and FeSSIF, especially for the later version of the ± 1.0 2.0 SIFs, which better captured the upper small intestinal pH, buffer ca­ felodipine 54.5 ± 237 ± Fa/FeSSIF-V1 [54] 3.7 1.0 pacity, osmolarity, and total bile salt content (Table 2). The luminal fenofibrate 9.6 ± 40 ± Revised Fa/FeSSIF-V1 with [10] composition data from this study supported the current compositions for 1.4 2.9 taurocholate and soybean FaSSIF and FeSSIF. However, their texture and viscosity could still be lecithin [55] improved, to better mimic intake of a solid meal rather than a liquid one. ± ± ibuprofen 1405 1905 Fa/FeSSIF-V1 [56,57] Luminal viscosity is a parameter that may impact the in vivo dissolution 17 9 ketoconazole 13.2 ± 248.9 Fa/FeSSIF-V2 [57] rate and/or drug product performance following oral intake [44]. The 0.1 ± 1.3 next level of in vitro dissolution models should also better reflect the probucol 1.6 ± FaSSIF-V2 [58] dynamic changes in the fasted state as well as during digestion. It would 0.7 also be clinically important to have improved GI models for certain GI tadalafil 5.9 ± 21.0 ± Fa/FeSSIF-V1 [59] 0.7 0.5 diseases [19]. valsartan 237 ± 76 ± 6 Fa/FeSSIF-V1 [60] For seven drugs in the fasted state and four in the fed state in this 20 study, there were previously reported equilibrium solubility values in zafirlukast 2.1 2.8 FaSSIF-V1, and FeSSIF-V1 [53] HIF (Fig. 4A). With one exception (fenofibrate in the fasted state), this with pH 6.5 comparison showed only minor differences, regardless of the different food and dosing regiments in the reported studies. This indicated that drug solubility should be minor [42]. Still, luminal amphiphilic com­ reproducibility was acceptable for drug equilibrium solubility de­ ponents (e.g., bile salts, fatty acids and phospholipids) in HIF may still terminations between laboratories using different aspiration techniques affect solubilization, and thereby increase the equilibrium solubility of and types of meals [18]. Further, the HIF solubility values in the fed state the same drugs (an effect unrelated to particle wetting). were for the same, or higher, than in the fasted state for all the drugs. Luminal protein content is low in the fasted state and in the fed state The drugs tended to have increased insolubility in the fed state if their primarily reflectsmeal composition. In this study, the duodenal protein solubility was low in the fasted state. This is logical given the inverse concentration increased from 2.8 ± 0.4 to 16.5 ± 0.2 g/L with 400 mL correlation between drug lipophilicity and water solubility, where the Ensure Plus (protein content 17 mg/mL). This can be compared to a rise addition of lipophilic constituents has a larger effect on the solubiliza­ in the jejunum from 1.0 ± 0.1 to 5.0 ± 0.1 g/L following 200 mL of tion of lipophilic (i.e. low solubility) compounds. Based on the limited Nutriflex (protein content 19 mg/mL) [34]. The difference in fed-state dataset in this study, the fed state had an insignificant impact on the protein content between the duodenum and jejunum after ingestion of equilibrium solubility of those drugs with a fasted state solubility above similar protein drinks illustrates the large capacity of the upper GI tract 30 µg/mL. to digest and rapidly absorb protein. The rapid luminal absorption of With the exception of the solubility values of valsartan and bromo­ digested proteins and its insignificant impact on drug solubility may criptine in fasted and fed state HIF, equilibrium solubility values in HIF explain why proteins are not included in any of the FaSSIF or FeSSIF and reported SIF were within a 10-fold difference. This was acceptable 〈 versions. considering the large interlaboratory variability (CV% 160) of the Bile salts and phospholipids (with or without fatty acids) form mixed water solubility values reported by Andersson et al., 2016, for the same micelles (6.5 nm in diameter) and vesicles (50 nm in diameter) in the compounds and identical experimental conditions [23]. The large HIF intestinal lumen, where the micelles predominate at high concentrations vs. SIF difference for valsartan and bromocriptine cannot be readily (i.e., fed state) [43]. Both micelles and vesicles facilitate the solubili­ explained by any compositional differences, or by any unique properties zation of lipophilic vitamins and drugs, and are thus important for in vivo of these two drugs. It is likely that the unusually large differences in the relevant drug solubility determinations. Reported bile salt concentra­ reported SIF values are associated with the experimental method of drug tions in the fasted state range from 1.4 to 8.1 mM, and from 3.6 to 24.0 solubility determination/quantification in those studies, as there were mM in the fed state [22]. The fasted- and fed-state values from this study no difference between the HIF solubility values in our study and re­ (3.52 and 8.91 mM) fell in the middle of the reported luminal concen­ ported HIF solubility values for either the fasted or fed states. It was also tration ranges. Total bile salts concentration was thus well captured in evident that the different ratio of fed vs. fasted solubility was higher in all versions of FaSSIF and FeSSIF, but with a better similarity in FeSSIF- HIF than in SIF, an effect generally related to a higher solubility in V2 than FeSSIF-V1. While the total bile salts content changed with FaSSIF than in fasted HIF. This cannot be attributed to any dissimilarities prandial state, the composition was essentially unaffected. These results in composition between these two fasted media, as they had only minor thus corroborate data from others who report similar bile salt compo­ differences in pH, buffer capacity, and phospholipid and bile acid con­ sitions in any one individual over time [22]. The corresponding reported centration. Based on the data in this study, it can be concluded that values for phospholipids in the fasted state range from 0.1 to 1.8 mM, current SIF media are generally successful in predicting HIF solubility. and from 1.2 to 6.0 mM in the fed state [22]. The fasted HIF phospho­ However, care must be taken when interpreting FaSSIF solubility data, lipid level in this study (0.19 mM) fell in the lower part of reported as these tended to slightly overpredict fasted-state HIF solubility, espe­ > values and the fed value (3.72 mM) in between. For phospholipids, the cially for low-solubility drugs (Do 5, which applies to all drugs except concentrations in FaSSIF-V2 capture the fasted HIF value better from ibuprofen and valsartan). These results are in opposite of previous ob­ this study, whereas FeSSIF was better than FeSSIF-V2. In summary, servations, which indicate that FaSSIF slightly underpredict HIF solu­ given the individual variability and the small differences in concentra­ bility for low-solubility drugs [18]. tions for both bile salts and phospholipids between HIF and SIF, Interestingly, the increase in fed- vs. fasted-HIF solubility in this study (accompanied by a decrease in Do) was not reflectedin increased

248 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

A Fasted state 10000 + HIF

) 1000 FaSSIF L m / - - - g 100 µ (

y - t i l i 10 - ub l o

S 1

0.1 t l t l l e te o n fi e o le n n in a c a in il o e a t kas r ta l z f rt p b u i a ip d a ro a ri u fi b p d d e s c irl o e a o rv n p l f o r r t l a o u a o a n p p fe c c ib v z fe a to rom e b k

B Fed state 10000 HIF + FeSSIF + )

L 1000 - m

/ + g µ ( 100 y t i l i ub l

o 10 S

1 t l t l fi e n te o e le n n as a in a il in o e a k l t ta r z f rt a p i b d ip a ro a u d ri p fi e d s irl a c e rv o n p l f t r o a l o u a a o p n c e c ib v z a fe f to rom e b k

Fig. 5. The mean solubility values in the fasted (A) and fed (B) state in human intestinal fluids (HIF) from this study (Table 4), as well as reported values in fasted (FaSSIF) and fed (FeSSIF) states of simulated intestinal fluids( Table 5). A (-) indicates a higher (3-fold) solubility in simulated fluidsthan in HIF, and (+) indicates a higher (3-fold) solubility in HIF than in simulated fluids. human bioavailability or exposure of the same drugs following dosing of disintegration of various solid dosage forms. Likewise, the effect of immediate release formulations in fasted and fed states. The results are prandial state on physiological parameters affecting drug absorption in contrast to the theory that an increase in solubility of a poorly soluble (such as gastric emptying rate and complexation with food and luminal compound should also increase its absorption [45]. The dissimilarity components) must be included. may be related to the free-dissolved drug concentration, or the molec­ ularly dissolved one, in the water phase of HIF/SIF; these may be un­ 5. Conclusion affected by addition of solubilizing constituents, such as bile acids, fatty acids and phospholipids. Further, the rapid absorption of fatty acids and This study generated a broad physicochemical and compositional phospholipids in the upper small intestine typically causes an immediate analysis of HIF in fasted and fed states and coupled these to equilibrium precipitation of liberated drug molecules thereby limiting the time- drug solubility measurements for a range of low-solubility drugs. The 11 window for supersaturation. Consequently, the data presented here known model drugs are well embedded in the lipophilic part of the suggest that an increased equilibrium solubility in fed HIF, compared to physicochemical space of marketed drugs. The HIF compositions cor­ fasted HIF or water, should not be interpreted to be predictive of ab­ responded well to previously reported values and to current FaSSIF and sorption of low solubility compounds. Other biopharmaceutical luminal FeSSIF compositions. The drug solubilities in HIF (both fasted and fed processes must also be taken into account, such as the size-related states) were also well in line with reported solubility values for HIF and behavior of drug crystals, precipitation/supersaturation, and the simulated FaSSIF and FeSSIF. This indicates that the in vivo conditions in

249 D. Dahlgren et al. European Journal of Pharmaceutics and Biopharmaceutics 163 (2021) 240–251

100 Appendix A. Supplementary data on o ti i ic t d re a rp Supplementary data to this article can be found online at https://doi. r e ov

y ld org/10.1016/j.ejpb.2021.04.005. fo

li t 3 i n tio b 10 c di u e bromocriptine References l r rp

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