Gastric and duodenal diclofenac concentrations in healthy volunteers

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Gastric and duodenal diclofenac concentrations in healthy volunteers after intake of the FDA standard meal: in vivo observations and in vitro explorations Jari Rubbens, Joachim Brouwers, Jan Tack, and Patrick Augustijns Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00865 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Molecular Pharmaceutics

Gastric and duodenal diclofenac concentrations in healthy volunteers after intake of the FDA standard meal: in vivo observations and in vitro explorations

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Jari Rubbens1; Joachim Brouwers1; Jan Tack2; Patrick Augustijns1*

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1

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[email protected]; [email protected]; [email protected]

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2

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Herestraat 49 Box 701, 3000 Leuven, Belgium; [email protected]

KU Leuven Drug Delivery & Disposition, Gasthuisberg O&N2, Herestraat 49 Box 921, 3000 Leuven, Belgium,

KU Leuven Translational Research Center for Gastrointestinal Disorders (TARGID), Gasthuisberg O&N1,

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*Corresponding author at: KU Leuven Drug Delivery & Disposition, Campus Gasthuisberg O&N 2, Box 921,

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Herestraat 49, 3000 Leuven, Belgium.

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E-mail address: [email protected] (P. Augustijns).

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KEYWORDS

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Gastrointestinal drug disposition

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Solid meal

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Oral drug delivery

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FDA standard breakfast

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Gastrointestinal

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Pharmacokinetics

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Food effects

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Clinical pharmacokinetics

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ACS Paragon Plus Environment

Molecular Pharmaceutics 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 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

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ABSTRACT

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This study investigated gastrointestinal drug concentrations of the weakly acidic drug diclofenac when

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dosed to healthy volunteers after intake of the FDA standard meal. In gastrointestinal aspiration

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studies, postprandial conditions are usually achieved using liquid or homogenized meals. However,

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these liquid meals may have a substantially different impact on the gastrointestinal physiology

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compared to a solid meal. To evaluate the effect on the gastrointestinal behavior of diclofenac, five

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healthy volunteers were recruited into a clinical study. Twenty minutes prior to diclofenac ingestion

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(Cataflam®, 50 mg potassium diclofenac), the volunteers were asked to eat a solid meal with the

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following composition corresponding to the FDA standard meal: 2 eggs, 2 bacon strips, 2 toasts, 4

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ounces of hash brown potatoes and 8 ounces of milk. Gastric and duodenal fluids were collected as a

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function of time and blood samples were collected to link the gastrointestinal behavior to systemic

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exposure. In vivo observations were complemented with in vitro research to obtain a mechanistic

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understanding of diclofenac’s intraluminal behavior. Ingestion of the solid meal resulted in

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intraluminal pH-profiles similar to earlier studies with a liquid meal. However, intraluminal drug

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disposition differed. In the stomach, a substantial fraction of diclofenac appeared dissolved, despite

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an unfavorable acidic pH. Successive in vitro tests suggested that the dissolution of diclofenac is higher

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in the complex gastric media resulting from FDA standard meal ingestion compared to liquid meal

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ingestion. Despite the favorable pH and in contrast to a previous study with a liquid meal, significant

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amounts of solid foods were observed in the intestine. Further in vitro tests revealed adsorption of

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dissolved diclofenac molecules to bacon fragments present in the FDA standard meal. This adsorption

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negatively affected the permeation of diclofenac across a physical barrier, suggesting that in vivo

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absorption is affected as well. Being the first time a gastrointestinal aspiration study is combined with

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the administration of a solid meal, the present study demonstrates that the intraluminal behavior of

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diclofenac (and possibly other drugs) heavily depends on the consistency and composition of the

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accompanied meal. 2


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1. Introduction

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Traditional pharmacokinetic studies rely on drug plasma concentrations to evaluate a drug’s in vivo

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performance. These studies do not provide any information on intraluminal drug and formulation

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behavior, though the biopharmaceutical performance of a drug can be influenced by a variety of

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physiological and physicochemical factors at the level of the gastrointestinal (GI) tract. 1 The exact

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mechanisms of how a specific systemic exposure is achieved are often speculated or insufficiently

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understood. 2 GI aspiration studies have been proven to be a useful tool to characterize gastrointestinal

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drug disposition.

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duodenal fluids are collected as a function of time by means of aspiration. These aspirates can be

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analyzed for drug concentration, pH, solubilizing capacity, osmolality and bile salt concentration.

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These results help in obtaining a more mechanistic insight into how oral formulation ingestion leads

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to a specific systemic exposure. 4 3

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Clinical pharmacokinetic trials always include a trial arm in which the formulation is administered in

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fasted volunteers. This fasted state is defined as abstinence from food for at least 10 hours according

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to the Food and Drug Administration (FDA) Guidance for Industry on Food-Effect Bioavailability (BA)

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and Fed Bioequivalence (BE) Studies. 6 This means that in real-life, people can be considered as being

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fasted only in the morning. Volunteers are usually also tested in a fed state to assess postprandial

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effects on the rate and extent of drug absorption. A fed state can induce beneficial or detrimental

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effects on intestinal absorption. 7 Food can alter a drug’s bioavailability by delaying gastric emptying,

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stimulating bile flow, altering gastrointestinal pH, increasing splanchnic blood flow, changing luminal

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metabolism, and physically or chemically interacting with the drug. 6 The FDA Guidance for Industry on

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Food-Effect BA and Fed BE Studies recommends using a meal with a high caloric and high fat content

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in order to provide the largest effect on gastrointestinal physiology and systemic exposure. As an

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example, the following composition is given, often referred to as the ‘FDA standard meal’: two eggs

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fried in butter, two strips of bacon, two slices of toast with butter, four ounces of hash brown potatoes

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Healthy volunteers are dosed with a formulation, after which gastric and/or

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and eight ounces of whole milk. The FDA standard meal should contain approximately 800 to 1000 kCal

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of which roughly 150 kCal from protein, 250 kCal from carbohydrate and 500-600 kCal from fat. It

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should be noted that this meal is not meant to represent a standard meal used in daily practice, but

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rather to provoke the highest possible food effect. 6

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To simulate fed state conditions in gastrointestinal aspiration studies, liquid or homogenized meals are

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usually administered (e.g. Scandishake®, Ensure® or mixed meals).

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bought, ready-to-use, feasibility when combined with intubation through nose or mouth), these liquid

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meals may have a substantially different impact on the gastrointestinal physiology compared to the

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solid FDA standard meal. Not all of these liquid meals deliver approximately half of their energy in the

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form of fat as suggested by the FDA. 6 This discrepancy may be of considerable relevance as fat is

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emptied slower from the stomach than carbohydrates and proteins because of its higher caloric

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density.

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compared to a solid meal, which can influence intragastric formulation disintegration.

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ingestion, liquid meals spread throughout the entire stomach and are emptied by pressure of the

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fundus. Emptying starts rapidly after ingestion and follows a first order kinetic proportional to the

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volume present in the stomach. 13 Solid meals, however, are stocked in the fundus and proceed to the

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antrum for trituration. 12 Solid foods are demolished until particles less than 1 or 2 mm can pass the

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pylorus. The gastric emptying of solids has a lag phase after which a constant emptying occurs (zero

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order kinetic). 13

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The present study investigated gastrointestinal drug concentrations of the weakly acidic diclofenac

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when dosed to volunteers after intake of the FDA standard meal. Diclofenac is a weakly acidic BCS Class

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II drug, pKa’s are reported ranging from 3.8 to 4.21.

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aspiration study is combined with the administration of a solid meal. Blood samples were collected to

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link the gastrointestinal behavior to systemic exposure. In vivo observations were complemented with

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in vitro research to obtain a mechanistic understanding of diclofenac’s intraluminal behavior when

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dosed with a solid meal.

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3 8 9

Though convenient (store-

Moreover, the stomach processes and empties liquid meals in a different manner

14151617

1 12 13

After

This is the first time a gastrointestinal

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2. Materials and Methods

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2.1.Chemicals

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Sodium diclofenac was purchased from Fagron (Nazareth, Belgium). 13C6-diclofenac was

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purchased from Alsachim (Illkirch Graffenstaden, France). Cataflam® (50 mg potassium diclofenac,

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Novartis, Basel, Switzerland) was obtained via the hospital pharmacy of the University Hospitals

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Leuven (Belgium). Methanol was purchased from Biosolve (Valkenswaard, the Netherlands). Dimethyl

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sulfoxide (DMSO) was purchased from Acros Organics (Geel, Belgium). Acetonitrile was supplied by

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Fisher Scientific (HPLC grade; Leicestershire, UK). Water was purified with a Maxima system (Elga Ltd.,

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High Wycombe Bucks, UK). Formic acid (HCOOH) was ordered from Biosolve (99%; Valkenswaard, the

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Netherlands). Sodium acetate trihydrate (NaOAc·3H2O) was purchased from Chem-Lab (Zedelgem,

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Belgium). Acetic acid was acquired from VWR International (99%–100% p.a.; Dublin, Ireland). Disodium

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monohydrogenphosphate dihydrate (Na2HPO4·2H2O) and sodium dihydrogenphosphate monohydrate

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(NaH2PO4·H2O) were acquired from Sigma–Aldrich (St. Louis, Missouri). Fasted state simulated

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intestinal fluid (FaSSIF), fed state simulated intestinal fluid (FeSSIF) and fasted state gastric fluid

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(FaSSGF) powder was purchased from Biorelevant (London, U.K.) Pancreatin from procine pancreas (8

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x USP specifications) was purchased from Sigma-Aldrich (St. Louis, Missouri).

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2.2.Clinical study

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Five healthy volunteers (HVs) (three males, two females) were recruited into a clinical study. HVs were

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fasted for at least 12 hours prior to testing. One tablet of Cataflam® (50 mg diclofenac potassium) was

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administered with 240 mL of tap water in fed state conditions. The study obeyed the tenets of the

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Declaration of Helsinki and Tokyo. The study was registered in the European Clinical Trials Database

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(EudraCT 2013-004636-29), approved by the Federal Agency of Health and Medicines (FAHMP,

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646493) and by the Committee of Medical Ethics of the University Hospitals Leuven (ML10131).

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Exclusion criteria were (a history of) gastrointestinal disorders, use of medication, pregnancy and

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infection with hepatitis B, C or HIV. All volunteers provided written informed consent. Prior to 6


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participation, female volunteers were checked for pregnancy. Volunteers were intubated through the

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mouth or nose with two double-lumen polyvinyl catheters [Argyle Salem Sump Tube, 14 Ch (4.7 mm x

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108 cm); Covidien, Dublin, Ireland]. One catheter was positioned in the antrum of the stomach, the

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other one in the duodenum. The position of the catheters was checked using X-ray fluoroscopy.

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Subsequently, volunteers were seated in a hospital bed for the duration of the trial. Twenty minutes

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prior to Cataflam® ingestion, the volunteers were asked to eat a solid meal with a composition in

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accordance with the FDA’s guide for food-effect BA and fed BE studies. 6 An overview of the meal

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ingredients, brands and a caloric breakdown can be found in Table 1. All meal ingredients were

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purchased from Aldi market (Leuven, Belgium). This meal was prepared before the study and briefly

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reheated in a microwave before consumption. The Cataflam® tablet was administered with 240 mL of

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tap water. Gastric and duodenal fluids (maximum 3 mL) were aspirated through the catheters with the

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help of 50 mL catheter tip syringes (Terumo Europe, Leuven, Belgium). Gastrointestinal aspirates were

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taken every 15 minutes for 5 hours. Immediately after aspiration, samples were prepared for

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diclofenac analysis. Venous blood samples were collected in heparinized tubes (BD Vacutainer

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systems, Plymouth, U.K.) every 15 minutes during the first 5 hours and at 6, 7 and 8 hours after

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Cataflam® administration.

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2.2.1. Sample preparation

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The pH of the gastrointestinal fluids was measured immediately after aspiration (Hamilton Knick

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Portamess®, Bonaduz, Switzerland). Aliquots of the aspirated gastric and duodenal samples were

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diluted 50-fold in methanol:water (50:50, v/v) to assess total diclofenac content (solid + solute) (see

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section 2.4). The remaining aliquot was centrifuged (20,817g, 5 min; microcentrifuge 5424; VWR

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International), upon which the supernatant was diluted 50-fold in methanol:0.1 M phosphate buffer

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pH 7 (50:50, v/v) to assess the dissolved diclofenac concentration (see section 2.4).

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Venous blood samples were stored on ice during the clinical trial after which they were centrifuged

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(1,699g, 15 min, 4 °C; Centrifuge 5804R, Eppendorf, Hamburg, Germany) to obtain plasma. A protein 7



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precipitation step was performed in which 100 µL of plasma was added to 400 μL of methanol

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containing the internal standard (10 nM of 13C6-diclofenac). This mixture was vortexed and centrifuged

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at 20,238g (5 min, 4 °C; Centrifuge 5804R, Eppendorf, Hamburg, Germany); the supernatant was used

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for analysis (see section 2.4).

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2.3.In vitro studies

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2.3.1. Assessment of diclofenac dissolution and solubility in simulated fed state gastric

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medium

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The dissolution and solubility of diclofenac from a Cataflam® formulation was investigated in simulated

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fed state gastric media. All in vitro tests were performed in triplicate, FDA standard meal was

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homogenized [mixed with a Tomado Stick mixer (Amsterdam, the Netherlands)] to enable the use of

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a representative meal sample with a correct ratio of all meal components. Mixtures of FaSSGF:FDA

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standard meal (20mL:20g) and FaSSGF:Ensure® Plus (20mL:20mL) were equilibrated for 30 minutes at

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37°C and continuous magnetic stirring (900 rpm). The pH was adjusted by adding 3.2 mL 1M HCl,

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resulting in a pH of 2.08 for the FaSSGF:FDA standard meal mixture and a pH of 2.04 for the

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FaSSGF:Ensure® Plus mixture. The dissolution of diclofenac was monitored by adding one tablet of

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Cataflam® to these mixtures. Samples of 0.5 mL were collected at 5, 10, 20, 30, 45, 60, 90 and 120 min.

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The pH was measured again after 120 min. Immediately after sampling, centrifugation (20,817g, 5 min;

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microcentrifuge 5424, VWR International, Belgium) was performed, followed by a 100-fold dilution of

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the supernatant in methanol:0.1 M phosphate buffer pH 7 (50:50, v/v) to assess the dissolved

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diclofenac concentration (see section 2.4).

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The dissolution of diclofenac was further evaluated after removal of solid food particles from the

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simulated fed state gastric medium created with the mixed FDA standard meal. To this end, the

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medium of FaSSGF:FDA standard meal mixture (50:50, w/v) was equilibrated for 30 minutes at 37°C

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and continuous magnetic stirring (900 rpm) and subsequently shortly centrifuged at low speed (106 g,

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1 min; Centrifuge 5804R, Eppendorf, Hamburg, Germany). The pellet containing solid food particles 8


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was discarded. Microcentrifuge tubes containing 1 mL of the resulting medium or a 50:50

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FaSSGF:Ensure® Plus medium (v/v) were spiked with diclofenac originating from three sources: 1.25

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mg of a crushed Cataflam® tablet (potassium diclofenac), 1 mg sodium diclofenac powder or 4 µL of a

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100 mM sodium diclofenac stock solution in DMSO (corresponding to 0,13 mg sodium diclofenac).

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These microcentrifuge tubes were subsequently placed in a shaking incubator (KS4000i incubator; Ika,

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Staufen, Germany) at 200 rpm and 37°C. Samples were collected at 10, 30, 60, 120 and 180 min. The

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pH was measured again after 180 min. Following centrifugation (20,817g, 5 min; microcentrifuge 5424,

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VWR International, Belgium), a 50-fold dilution of the supernatant in methanol:0.1 M phosphate buffer

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pH 7 (50:50, v/v) was used to assess the dissolved diclofenac concentration.

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2.3.2. Assessment of diclofenac adsorption to food components

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All adsorption studies were performed in media simulating the fed intestinal environment, created by

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adding mixed meals or mixed meal components to a FeSSIF-V2 solution. The influence of several food

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components on the recovery of diclofenac from a solution of 100 µM in FeSSIF-V2 pH 5.8 was tested,

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unless stated otherwise. First, the recovery of diclofenac was tested in the presence of mixed FDA

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standard meal, liquid meal and all FDA standard meal components separately. The impact of bacon

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fragments was further studied in more detail. An overview of all test conditions can be found in Table

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2. All adsorption studies were performed in triplicate.

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One milliliter of a 100 µM diclofenac solution in FeSSIF-V2 was transferred to a microcentrifuge tube

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containing 65, 125 or 250 mg of either mixed FDA standard meal or one of its mixed components. In

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the case of Ensure® Plus (liquid meal, 600 kcal; Sorgente, Houten, The Netherlands) and whole milk,

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65, 125 or 250 µL was added to the diclofenac solution. The microcentrifuge tubes were placed in a

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shaking incubator (KS4000i incubator; Ika, Staufen, Germany) at 200 rpm and 37°C for 30 min. After

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30 min, the tubes were briefly vortexed, upon which a sample was taken and diluted 50-fold in

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methanol:0.1 M phosphate buffer pH 7 (50:50, v/v) to assess the total amount of diclofenac present.

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Subsequently, samples were centrifuged (20,817g, 5 min; microcentrifuge 5424; VWR International), 9



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followed by a 50-fold dilution of the supernatant in methanol:0.1 M phosphate buffer pH 7 (50:50, v/v)

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to assess the dissolved diclofenac concentration. The recovery of diclofenac was expressed as a

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percentage of the measured initial concentration (Ranged from 95 µM to 105 µM).

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Using the same methodology, the influence of the quantity of bacon fragments on the recovery of

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diclofenac was tested by adding either 65, 125 or 250 mg of mixed bacon fragments to 1 mL of test

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solution. The impact of surface area available for adsorption was investigated by adding 125 mg of

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bacon to 1 mL of test solution, either as a single weighted piece of bacon (low surface area) or as mixed

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bacon fragments (high surface area); diclofenac adsorption as a function of time was tested by adding

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125 mg of mixed bacon fragments to 1 mL of test solution and monitoring the recovery at 5, 10, 30,

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60, 120 and 180 min.

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In the experiments described so far, adsorption of diclofenac was investigated directly in FeSSIF-V2. To

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assess the potential of bacon to adsorb diclofenac in more biorelevant conditions, an initial acidic

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incubation step and enzymes were included to simulate both passage of bacon fragments through the

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stomach as well as digestion. In detail: 125 mg of mixed bacon fragments were added to 450 µL of

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FaSSGF (pH 1.6) containing 1 mg/mL of pancreatin. This mixture was placed in a shaking incubator

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(KS4000i incubator; Ika, Staufen, Germany) at 200 rpm and 37°C for 30 min. Hereafter, 450 µL of

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FeSSIF-V2 (pH 5.8) containing 10 mg/mL pancreatin was added, followed by incubation for another 30

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min. Subsequently, 100 µL of a 1 mM diclofenac solution in FeSSIF-V2 (pH 5.8) was added upon which

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diclofenac recovery was assessed (final concentration 100 µM), as described previously after a final 30

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min incubation step.

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To exclude the possibility of biased adsorption results due to a potential direct effect of the complex

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medium resulting from incubating bacon fragments with FeSSIF-V2, the recovery of diclofenac was

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determined in this medium after removal of the bacon fragments. Two mixtures of mixed bacon

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fragments and FeSSIF-V2 (1:8 w/v) were incubated for 30 min at 200 rpm and 37°C (KS4000i incubator;

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Ika, Staufen, Germany). One mixture was shortly centrifuged at low speed (106 g, 1 min; Centrifuge

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5804R, Eppendorf, Hamburg, Germany) to separate the bacon pellet from the medium. As such, this 10


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medium did not contain any bacon (MediumNB) The second mixture was centrifuged for a longer time

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at higher speed (1 699 g, 15 min, 4 °C; Centrifuge 5804R, Eppendorf, Hamburg, Germany) to separate

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both the bacon pellet and the upper lipid layer from the remaining supernatant. As such, this medium

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did not contain bacon or lipids (MediumNB, NL). Upon spiking 100 µM diclofenac from a DMSO stock

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solution, the recovery of diclofenac from both mediumNB and mediumNB, NL was assessed as described

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previously.

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Finally, the possibility of biased adsorption results due to potential diclofenac degradation was

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excluded by determining the recovery of diclofenac by means of a protein precipitation approach using

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acetonitrile. One mL of a 100 µM diclofenac solution in FeSSIF-V2 was incubated with 125 mg of mixed

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bacon fragments for 30 minutes. Two mL of acetonitrile was added, upon which the mixture was

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vortexed for 1 min and centrifuged (1 699g, 5 min, 4 °C; Centrifuge 5804R, Eppendorf, Hamburg,

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Germany). The supernatant was diluted 50-fold in methanol:0.1 M phosphate buffer pH 7 (50:50, v/v)

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to assess the diclofenac concentration.

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2.3.3. Influence of diclofenac adsorption on permeation

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The influence of diclofenac adsorption to bacon on its permeation behavior was investigated using the

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artificial membrane insert-system (AMI-system).

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weight cutoff, 2 kDa; thickness, 20 mm; flat width, 44 mm; dry diameter, 28 mm) were wetted in

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purified water 30 min before use. These membranes were then mounted between 2 plastic rings with

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a surface area of 4.91 cm2. These insert systems were placed in a 6-well plate in a shaking incubator

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(Thermostar; BMG Labtech, Offenburg, Germany) at 37°C and 300 rpm. The donor compartment (665

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µl) consisted of either a 100 µM diclofenac solution in FeSSIF-V2 (reference condition) or a 100 µM

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diclofenac solution in FeSSIF-V2 pre-incubated with 85 mg of mixed bacon fragments in a shaking

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incubator (30 min, 200 rpm, 37°C KS4000i incubator; Ika, Staufen, Germany) (test condition). The

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acceptor compartment consisted of 2 mL of a D-ɑ-tocopheryl polyethylene glycol 1000 succinate

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(TPGS) solution in purified water (0.2% w/v). Sampling of the acceptor compartment was performed

18

Regenerated cellulose membranes (molecular

11



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at 5, 10, 20, 30 and 60 min. Samples of 100 µL were taken and diluted 1:1 in MeOH/H20 (50:50 v/v).

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The withdrawn volumes were replaced with fresh TPGS solution. The absolute cumulative amount of

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diclofenac appearing in the acceptor compartment was expressed as function of time. All experiments

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were performed in triplicate.

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2.4. Analysis

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All samples were analyzed using analytical methods described by Van Den Abeele et al.

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samples originating from gastrointestinal fluids and in vitro tests were analyzed by RP-HPLC with UV

259

detection (279 nm; Chromaster 5410 UV detector, VWR International). Separations were performed

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on a Novapak C18 column under radial compression (4 μm, 8 ×100 mm, Waters, Milford, MA, USA).

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Diclofenac was isocratically eluted with a flow rate of 1mL/min using methanol:25 mM sodium acetate

262

buffer pH 3.5 (82:18 v/v). The method was proven to be lineair, accurate and precise.

263

Plasma samples were analyzed by RP-HPLC with MS-MS detection (Acquity H-class UPLC, Waters,

264

Milford, MA, USA and Xevo TQ-S micro Waters, Milford, MA, USA). In brief, separation was performed

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using a Kinetex XB - C18 column (2.6 μm, 2.1 × 50 mm; Phenomenex, Utrecht, the Netherlands) held

266

at 35 °C. Methanol (solvent A) and 0.05% formic acid in water (solvent B) were used as eluens at 500

267

µL/min. Gradient elution was performed as follows: 65% of solvent A during 0.9 min, followed by 95%

268

A for 1.6 min. After 2.5 min, solvent A decreased from 95% to 65%. An MS/MS positive ionization mode

269

was carried out with an HESI source on a Xevo TQ-S micro mass detector (Waters, Milford, MA, USA).

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The mass transitions were m/z 296.11 → 214.00 (collision energy: 30 V) for diclofenac and m/z 302.11

271

→ 220.00 (collision energy: 30 V) for 13C6-diclofenac. Calibration curves were made on the day of

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analysis by serial dilution in plasma. The method was proven to be linear, accurate and precise. For

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further details on these analytical methods we refer to Van Den Abeele et al. 19

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3. Results and discussion

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3.1.Gastric drug disposition

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3.1.1. In vivo observations

19

In brief,

12


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Ingestion of the FDA standard meal (934 kCal) resulted in an elevation of the stomach pH up to 4.6

278

(±1.6 SD, n=5), followed by a gradual decline back to basal conditions over the course of five hours

279

(insert Fig. 1). The course of this pH-profile is similar to observations from gastrointestinal aspiration

280

studies using a liquid meal. 3 8 The disintegration of the Cataflam® tablet in the stomach did not start

281

immediately as no diclofenac was detected in gastric aspirates during the first hour after ingestion.

282

(Fig. 1) In one volunteer, no intragastric diclofenac was detected until 3 hours after ingestion. Van Den

283

Abeele et al. observed a similar average delay in intragastric Cataflam® disintegration when

284

administered with a liquid meal (i.e. Ensure® Plus). 19 Brouwers et al. accounted a delay in intragastric

285

drug release from Telzir® tablets (fosamprenavir) upon intake with a liquid meal (i.e. Scandishake Mix®)

286

to the formation of a food-dependent precipitation layer on the tablet surface impairing water

287

ingression. 3 20 The observation of delayed tablet disintegration in the present study suggests that such

288

a layer may also be formed in the presence of (partially digested) solid food particles.

289

Five hours after intake of the tablet, diclofenac was still present in the stomach of all volunteers. (Fig.

290

1) Van Den Abeele et al. observed complete clearance of diclofenac from the stomach 4 h after

291

administration of Cataflam® with Ensure® Plus.

292

attributed to differences in caloric content, meal composition and meal viscosity between the FDA

293

standard meal and the liquid meal. The FDA standard meal used in this study contains 934 kCal, while

294

Van Den Abeele et al. used a liquid meal containing 600 kCal. 19 Gastric emptying supplies the intestine

295

with a constant rate of caloric content (1 to 4 kCal/min) through a negative feedback mechanism by

296

duodenal receptors. 21 22 23 Thus, a 900 kCal meal will take longer to empty from the stomach compared

297

to the 600 kCal meal. Furthermore, among the major food components, fat is emptied slower than

298

proteins and carbohydrates. 11 10 As 47% of the calories in the FDA standard meal originates from fat

299

(compared to 30% for Ensure® Plus), this will further affect gastric emptying. Finally, the gastric

300

emptying of solids only begins after a lag phase for solid trituration, which is absent after intake of a

301

liquid meal. 13

19

This discrepancy in gastric emptying may be

13



Page 14 of 32

302

By the time tablet disintegration in the stomach was initiated, the gastric pH had dropped to values

303

below the pKa of diclofenac (insert Fig. 1). In literature, pKa values ranging from 3.8 to 4.21 are

304

reported.

305

solely on pH, this would result in an environment with a low solubilizing capacity for the weakly acidic

306

diclofenac (local pH < pKa diclofenac). Indeed, following intake of a liquid meal, Van Den Abeele et al.

307

did observe mainly solid drug material in the stomach after administration of Cataflam®.

308

present study, however, a substantial fraction of intragastric diclofenac appeared to be in solution.

309

(Fig. 1)

310

151617

In this manuscript we will refer to a pKa of 4.1 as reported by Rhfols et al.

14

19

Based

In the

3.1.2. In vitro explorations

311

The dissolution of diclofenac in a fed gastric environment was further investigated in vitro. Note that

312

these in vitro studies are primarily qualitative studies to compare the behavior of diclofenac in

313

simulated gastric media containing a liquid meal versus a homogenized solid meal. Mixtures of FaSSGF

314

and either FDA standard meal or Ensure® Plus were made and the pH was adjusted to 2 as observed

315

between 2 and 4 hours in the in vivo fed stomach pH-profile (insert Fig. 1). Tablet disintegration and

316

subsequent drug dissolution was monitored by adding one Cataflam® tablet to these media. (Fig. 2)

317

Following disintegration of the Cataflam tablet, the pH raised to 3.40 and 3.37 after 120 min for the

318

FaSSGF:FDA standard meal and the FaSSGF:Ensure® Plus mixtures, respectively. Higher diclofenac

319

concentrations were obtained in the medium with FDA standard meal compared to the medium with

320

Ensure® Plus. (Fig. 2) The peak in diclofenac concentration at 20 min followed by a decrease suggests

321

a supersaturation and precipitation event in the presence of FDA standard meal components.

322

Furthermore, the higher concentrations indicate higher solubility and/or extensive precipitation

323

inhibition in the presence of solid food components. These results suggest that the FDA standard meal

324

affects intragastric diclofenac dissolution and solubility in a different way than the liquid meal and

325

through factors other than pH. The solubility of lipophilic drugs like diclofenac (log P = 4,4) will indeed

326

increase when (food-derived) surface active compounds are present.

24

These effects are most 14


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327

pronounced in an environment of low solubility, which is the acidic gastric environment for diclofenac.

328

Both the FDA standard breakfast and Ensure® Plus include components with solubilizing capabilities.

329

(egg lecithin, milk proteins, triglycerides,…) 625 The results in Fig. 2. suggest an improved dissolution

330

and/or a higher solubility of diclofenac in an acidic environment in the presence of the FDA standard

331

breakfast compared to the liquid meal. This can be the result of the FDA standard breakfast containing

332

a relatively high amount of fat compared to Ensure® plus (47% of the calories from fat versus 30 %,

333

respectively).

334

This apparent beneficial effect on diclofenac dissolution was further evaluated in the absence of solid

335

particles and using various sources of diclofenac. Solid food particles were removed from the simulated

336

fed state gastric medium created with FDA standard meal to exclude any direct influence of food

337

particles on diclofenac dissolution or solubility. Furthermore, removal of solid particles allowed using

338

identical volumes of fed state gastric medium created with either FDA standard meal or Ensure® Plus

339

can be used. Three sources of diclofenac were used: a crushed Cataflam® tablet (potassium diclofenac),

340

sodium diclofenac powder or sodium diclofenac stock solution. This allowed us to evaluate whether

341

the apparent increase in diclofenac dissolution is an intrinsic characteristic of the medium or is

342

coherent with the source of diclofenac (formulation, pure salt form and solution). Regardless of the

343

diclofenac source, the observed concentrations of diclofenac were consistently higher in the medium

344

created with the FDA standard meal, once more indicating that the FDA standard meal affects

345

intragastric diclofenac dissolution and/or precipitation in a different way compared to the liquid meal.

346

(Fig. 3.) The discrepancy in dissolution is most pronounced when a crushed Cataflam® tablet is used as

347

diclofenac source indicating that the formulation containing the potassium salt of diclofenac is most

348

prone to this effect. Though no equilibrium concentrations were reached within the time frame of the

349

experiment, these observations also suggest a difference in diclofenac solubility due to media

350

composition. After 180 min, the pH of the FaSSGF:FDA standard meal media spiked with a crushed

351

Cataflam® tablet increased from 2.08 to 2.40. The pH in all other test conditions increased less than 15



352

0.1 units after 180 min. Further tests are necessary to identify the factors contributing to the observed

353

increase in diclofenac concentrations/solubility (e.g. pH, osmolality, ionic strength, temperature, bile

354

acid activity…). For now, our data suggests that the complex intragastric medium resulting from FDA

355

standard meal ingestion benefits local intraluminal diclofenac solubility.

356

3.2.Intestinal drug disposition

357

3.2.1. In vivo observations

358

The delayed tablet disintegration and gastric emptying clearly affected the duodenal concentration-

359

time profile as diclofenac arrived relatively late in the duodenum. On average, diclofenac

360

(concentrations ≥ 5% of the Cmax) could not be detected until 105 min after drug administration. (Fig.

361

4) Even though diclofenac is known to (re)dissolve quickly in the neutral intestinal environment of the

362

small intestine 8 19, significant amounts of solid diclofenac were observed in the duodenum when co-

363

administrated with the FDA standard meal. (Fig. 4) Upon administration of Cataflam® with a liquid

364

meal, Van den Abeele et al. noticed the presence of some solid diclofenac in the intestine at a few

365

initial time points only, possibly due to occasional rapid emptying of not-yet-disintegrated tablet

366

fragments. 19 In the present study, however, solid diclofenac was present at multiple sampling times

367

up to 5 h after intake. (Fig. 4)

368

different impact on diclofenac’s gastrointestinal behavior compared to a liquid meal, despite similar

369

local pH profiles.

370

19

Again, this observation suggests that the FDA standard meal has a

3.2.2. In vitro explorations

371

A possible explanation for the significant amounts of solid diclofenac observed in the intestine, might

372

involve direct adsorption of intraluminal diclofenac to meal components present in the FDA standard

373

meal. This hypothesis was investigated by a series of in vitro tests. First, fed intestinal media were

374

simulated by mixing a solution of diclofenac in FeSSIF-V2 with a homogenized FDA standard meal or

375

Ensure® Plus. Total and dissolved diclofenac were measured after 30 minutes of equilibration. Fig. 5

376

depicts diclofenac recovery (total and dissolved) as a function of the amount of FDA standard meal or 16


Page 16 of 32

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377

Ensure® plus added. The recovery of diclofenac was expressed of the initial concentration (100 µM

378

diclofenac in FeSSIF-V2, pH 5.8). Due to dilution with liquid food components, recoveries less than

379

100% were observed for total diclofenac. However, an even lower recovery of dissolved diclofenac was

380

observed in the medium mixed with 250 mg FDA standard meal. No effect was seen when Ensure®

381

Plus or lower quantities of FDA standard meal were added. The pH of the FeSSIF-V2 test solution was

382

not substantially affected by meal addition (pH increase ≤ 0.2).

383

The decrease in diclofenac recovery upon addition of 250 mg FDA standard meal was further explored

384

by testing the components of the FDA standard meal separately: milk, potatoes, eggs, toast and bacon.

385

(Fig. 6 a) While potatoes, eggs and toast all reduced the recovery of dissolved diclofenac, mixed bacon

386

fragments affected diclofenac recovery most. This effect increased with higher amounts of bacon

387

present (Fig. 6b). Also a higher surface area of the bacon reduced the recovery of diclofenac, as

388

observed when comparing the effect of adding either a single bacon piece (low surface area) versus

389

mixed bacon fragments of equal weight (high surface area) to a diclofenac solution (Fig. 6b). These

390

data seem to confirm the hypothesis that diclofenac adsorbs to components of the FDA standard meal,

391

in particular to bacon. As illustrated in Fig. 7, the decrease in concentrations due to adsorption

392

appeared to be relatively fast: the recovery of diclofenac already dropped to 59% (SD ±2) within the

393

first 5 min and slowly decreased further as a function of time.

394

To exclude an effect of components present in the complex medium which had been in contact with

395

bacon, diclofenac recovery was tested in the absence of bacon fragments. Following centrifugation of

396

the bacon-FeSSIF-V2 mixture (see Materials and Methods), mediumNB (excluding bacon fragments) and

397

mediumNB, NL (excluding lipids and bacon fragments) were created and tested. The excellent recovery

398

of diclofenac from these two media (Fig. 6b) suggests that interaction with solid bacon fragments is

399

responsible for the observed reduction in diclofenac recovery following centrifugation.

400

To exclude any influence of diclofenac degradation, a protein precipitation was performed using

401

acetonitrile. The observed recovery of 93% (SD ±0.04) indicates that very little diclofenac is degraded;

402

most likely, other factors (e.g. extraction efficiency and components of the bacon fragments dissolving 17



Page 18 of 32

403

in acetonitrile) contributed to the recovery not being 100%. Based on this test, it is unlikely that

404

diclofenac degradation is responsible for the disappearance of diclofenac from a solution containing

405

bacon fragments.

406

Overall, these in vitro results suggest that diclofenac adsorbs to the surface of bacon (and possibly

407

other meal components), effectively lowering the concentration of diclofenac in solution. In vivo, meal

408

components are partly processed before reaching the intestine. To simulate this, an acidic incubation

409

step and enzymatic degradation of the bacon fragments were included in the in vitro adsorption

410

studies. Following digestion in (i) acidic medium (FaSSGF, pH 1.6) including pancreatin, and (ii)

411

simulated intestinal medium (FeSSIF-V2, pH 5.8) including pancreatin, bacon fragments reduced

412

diclofenac recovery to 22.4 % (SD ±6.3) after 30 min at a final pH of 5.51. Since non-processed bacon

413

fragments (no incubation with acid or enzymes) reduced diclofenac recovery to 43.9 % (SD ±8.9) (Fig.

414

6), these data may suggest that diclofenac adsorption to bacon (or bacon degradation products) might

415

be more pronounced in vivo compared to our findings in vitro.

416

In this study, an adsorption interaction is described between a pharmaceutical compound

417

and a common meal component. This interaction may occur in vivo when Cataflam® is administered

418

with a meal including bacon and possibly other common meal components (potatoes, eggs and toast).

419

Bacon fragments masticated and/or grinded by the stomach to sizes smaller than 1 or 2 mm can pass

420

the pylorus and may adsorb diclofenac in the intestine. 13 This interaction could clarify why a significant

421

fraction of diclofenac was not in solution in the intestine when Cataflam® was administered with the

422

FDA standard meal. The observed adsorption interaction is not specific to bacon. Less diclofenac was

423

recovered in the presence of potatoes, eggs and toast as well. (Fig. 6) Diclofenac as a lipophilic

424

molecule could have a high affinity for the fat content of food compounds. The drug could also bind to

425

proteins present in these food components, similar to drug binding to plasma proteins in the systemic

426

circulation. To the best of our knowledge no literature is available in which the adsorption of drugs to

427

food components has been thoroughly studied. It would be interesting to further explore the

18


Page 19 of 32 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 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


428

underlying mechanism of this adsorption interaction and whether it is also relevant for other drugs

429

and meal components.

430 431

3.2.3. Influence of diclofenac adsorption on permeation

432

Since the intraluminal diclofenac concentration is the driving force for permeation across the intestinal

433

mucosa, the presence of intraluminal bacon fragments may hamper intestinal absorption. The

434

influence of bacon fragments on permeation of diclofenac across a physical barrier was investigated in

435

vitro using the AMI-system. This cell-free absorption model has been proven to be efficient at

436

evaluating the passive intestinal permeation of poorly water-soluble drugs, including BCS class II

437

weakly acidic compounds. 18 26 The permeation of diclofenac from a solution in FeSSIF-V2 was tested

438

in the absence or presence of mixed bacon fragments. The cumulative amount of diclofenac

439

permeating across the membrane as function of time is given in Fig. 8. Adsorption of diclofenac to

440

bacon fragments clearly affected permeation in the AMI-system. In the presence of bacon, only 9.4

441

nmol (±1.0 SD, n=3) permeated the membrane after 60 min, versus 25.7 nmol (±3.5 SD, n=3) in the

442

absence of bacon. This observation suggests that the adsorption of diclofenac to small bacon

443

fragments passing the pylorus may negatively affect absorption in vivo.

444

3.3.Systemic concentrations

445

The average systemic concentration-time profile when Cataflam® is administered with a solid meal is

446

given in Fig. 9. The average Tmax was 237 min (±109 SD, n=5). One volunteer had a strikingly high Tmax

447

at 420 min, accompanied with a high intestinal Tmax at 285 min (intestinal sampling stopped at 300

448

min). It is possible that the ingested Cataflam® tablet settled inside the food bolus in the gastric fundus.

449

Weitschies et al. observed a direct correlation between the residence time of a tablet in the proximal

450

stomach and the appearance of the drug in plasma. 27 Thakker et al. observed an average Tmax of 375

451

min (± 138 min SD,n=12) after administration of a 150 mg diclofenac hydrogel bead capsule to healthy

452

volunteers fed with the FDA standard breakfast.

28

19



453

The average AUC0-8h in this study amounted to 2.35 µM h (±1.08 SD, n=5). In 4 out of 5 volunteers,

454

diclofenac was still present in the systemic circulation at t = 8 h (concentrations ≥ 5% of Cmax). In

455

comparison, Chen et al. reported an average AUC0-12h of 3,63 µM (±0,87 SD, n=35) and complete

456

clearance of diclofenac from the systemic circulation at 8 h after intake of a 50 mg diclofenac tablet

457

with an FDA standard meal. Unfortunately, the publication by Chen et al. does not mention the time

458

between feeding and drug administration, making it hard to interpret the observed differences.

459

Scallion et al. reported an AUC0-8h of 4.92 µM h ± 1.29 (SD, n = 23) and elimination of diclofenac from

460

the systemic circulation within 8 h when a 50 mg soft gelatin capsule was administered to volunteers

461

fed with the FDA standard breakfast. 29 In a study by Van Den Abeele et al., Cataflam® was administered

462

to healthy volunteers fed with a liquid meal. In the intestine, solid diclofenac was only observed at a

463

few initial time points, possibly due to occasional rapid emptying of not-yet-dissolved tablet fragments.

464

The AUC0-5h for intestinal dissolved diclofenac concentrations was 166.4 µM h (SD ± 112.1, n=6). After

465

5 h of sampling, no diclofenac was observed in the duodenum. In the present study, the AUC0-5h for

466

intestinal dissolved diclofenac concentrations was observed to be 42.7 µM h (SD ± 20.8, n=5). Solid

467

diclofenac was present consistently at multiple intestinal sampling times up to 5 h after formulation

468

ingestion due to diclofenac adsorption to food components. In section 3.2.3 we have demonstrated

469

that adsorbed diclofenac is not readily available for permeation (See section 3.2.3. of the manuscript).

470

Further digestion may result in the release of adsorbed diclofenac from these food components and

471

eventually lead to absorption. In accordance with this delayed absorption, systemic diclofenac

472

concentrations are still observed at t=8h, contrary to the study by Van Den Abeele et al. Furthermore,

473

a lower systemic Cmax was observed compared to the study by Van Den Abeele et al. [0.98 ± (SD ± 0.46,

474

n=5), versus 3.5 µM (SD ± 1.0, n=6), respectively]. Finally, it should be noted that our study combined

475

systemic blood sampling with gastrointestinal aspiration which involves placement of an aspiration

476

catheter through the pylorus. Though Longstreth et al. indicated no effect of one transpyloric tube

477

(diameter 4 mm) on gastric emptying of low viscous and grinded solid foods, no research has been

478

performed on the potential impact on the gastric emptying of unprocessed (i.e. neither grinded nor 20


Page 20 of 32

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479

mixed) solid meals. 30 The presence of a transpyloric catheter may hamper efficient antral milling of

480

solid particles and as such slow down gastric emptying. The effect of the gastrointestinal sampling

481

method on gastric emptying of solid meals is currently under investigation.

482

4. Conclusions

483

For the first time, a gastrointestinal aspiration study was combined with the administration of a solid

484

meal. Healthy volunteers were asked to eat the FDA standard meal, upon which a Cataflam® tablet

485

was ingested with water and gastric and duodenal diclofenac concentrations were monitored.

486

Ingestion of the solid meal resulted in intraluminal pH-profiles similar to various earlier studies with a

487

liquid meal. However, intraluminal drug disposition differed. In the stomach, a substantial fraction of

488

diclofenac appeared dissolved, despite the unfavorable pH. Successive in vitro tests suggested that the

489

dissolution of diclofenac is higher in the complex gastric media resulting from FDA standard meal

490

ingestion compared to liquid meal ingestion. Further research on the intragastric solubility of

491

diclofenac in the presence of the FDA standard meal is needed to identify the exact mechanism(s) for

492

the higher intragastric diclofenac concentrations. In the intestine, significant amounts of non-dissolved

493

diclofenac were observed. In vitro tests revealed adsorption of dissolved diclofenac molecules to bacon

494

fragments (and potentially other meal components) present in the FDA standard meal. This adsorption

495

interaction negatively affected the permeation of diclofenac across a physical barrier. Consequently,

496

the importance of a direct drug-food interaction such as adsorption cannot be underestimated and

497

further research regarding this topic is warranted. Overall, the present study demonstrates that the

498

intraluminal behavior of diclofenac (and possibly other drugs) heavily depends on the consistency and

499

composition of the accompanied meal.

500

4. Acknowledgments

501

This study was supported by the Research Foundation – Flanders (FWO) (PhD fellowship 11Z2615N

502

and Research grant No. G.0769.14N). This work has received support from the Innovative Medicines

503

Initiative Joint Undertaking (http://www.imi.europa.eu) under Grant Agreement No. 115369, 21



Page 22 of 32

504

resources of which are composed of financial contribution from the European Union's Seventh

505

Framework Program and EFPIA companies' in kind contribution.

506

22


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507

Tables

508

Table 1. FDA standard meal: composition, brands used, and caloric value.

509

Composition of the FDA standard meal Food component Brand used 2 eggs Mamie Poule 2 bacon strips Délifin 2 toasts Délipain 4 ounces of hash brown potatoes Nicola 8 ounces of milk Milsa Total calories: 934 kCal (fat 47%, carbohydrates 35%, proteins 18%)

510

Table 2.

511

Overview of the conditions used to assess diclofenac adsorption to food componentsa. Meals

512

Meal components

Bacon – influence of quantity and surface area 65 µL Ensure ® Plus 125 mg homogenized potatoes 65 mg homogenized bacon 125 µL Ensure ® Plus 125 mg homogenized bacon 250 mg homogenized bacon 250 µL Ensure ® Plus 125 mg homogenized eggs ± 125 mg solid bacon fragment 65 mg homogenized FDA standard meal 125 mg homogenized toast 125 mg homogenized FDA standard meal 125 µL milk 250 mg homogenized FDA standard meal aAdsorption was assessed by determining diclofenac recovery from 1 mL of a 100 µM diclofenac solution in FeSSIF-V2, pH 5.8

23



513

Figures

514 515

Figure 1. Gastric concentration-time profiles for diclofenac following administration of a Cataflam®

516

tablet (50 mg diclofenac potassium) with 240 mL of water in healthy volunteers fed with the FDA

517

standard meal. Black lines and gray areas represent the dissolved and total diclofenac content (solid +

518

solute expressed as concentration), respectively (mean + S.E.M., n = 5). The insert depicts the pH of

519

the gastric fluids as a function of time (mean + S.E.M., n = 5). The dashed line represents the pKa of

520

diclofenac (4.1).

300

Concentration (µM)

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 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

Page 24 of 32

200

100

0 0

30

60

90

120

Time (min)

521 522

Figure 2. Dissolution of diclofenac from a Cataflam® tablet (50 mg diclofenac potassium) in (●)

523

FaSSGF:Ensure® Plus, pH 2.04 (20mL:20mL) and (o) FaSSGF:FDA standard meal, pH 2.08 (20mL:20g).

524

(mean ± S.D., n = 3)

24


Page 25 of 32

FaSSGF:FDA standard meal FaSSGF:Ensure® plus

80 60 40 20 0 0

50

100

Time (min)

150

b

c

100

Concentration (µM)

100

Concentration (µM)

a Concentration (µM)

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 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


80 60 40 20

100 80 60 40 20 0

0 0

50

100

150

0

Time (min)

50

100

150

Time (min)

525 526

Figure 3. Diclofenac dissolution in (●) FaSSGF:Ensure® Plus, pH 2.04 (50:50 v/v) and (o) FaSSGF:FDA

527

standard meal, pH 2.08 (50:50 m/v) after removal of solid particles. (a) Concentration-time of

528

diclofenac from a crushed Cataflam® tablet (diclofenac potassium). (b) Concentration-time profile of

529

diclofenac from sodium diclofenac powder (c) Concentration-time profile of diclofenac from a DMSO

530

stock solution made with sodium diclofenac powder. (mean ± S.D., n = 3)

531 532

Figure 4. Intestinal concentration-time profiles for diclofenac following administration of a Cataflam®

533

tablet (50 mg diclofenac potassium) with 240 mL of water in healthy volunteers fed with the FDA

534

standard meal. Black lines and gray areas represent the dissolved and total diclofenac content (solid

535

+ solute expressed as concentration), respectively (mean ± S.E.M., n = 5). Inserts depict the pH of the

536

intestinal fluids as a function of time (mean ± S.E.M., n = 5). The dashed line represents the pKa of

537

diclofenac (4.1).

25



80 80 60 60 40 40 20 20 0 0

80 80 60 60 40 40 20 20 0

Recovery (%) Recovery (%)

bb100 100

Recovery (%) Recovery (%)

100 aa100

65 µL

125 µL

250 µL

65 mg

0

®

Plus 65 µL Ensure125 µL added 250 µL

125 mg

250 mg

65 mg standard 125meal mg added250 mg FDA

Ensure® Plus added

FDA standard meal added

538 539

Figure 5. Recovery of diclofenac in the presence of ascending quantities of liquid or solid meal. One

540

milliliter of a 100 µM diclofenac solution in FeSSIF-V2, pH 5.8 was added to microcentrifuge tubes

541

containing quantities of liquid or solid meal. After 30 min of equilibration, total (black bars) and

542

dissolved (gray bars) diclofenac amounts were determined. The recovery of diclofenac was expressed

543

as a percentage of the concentration measured from the initial 100 µM solution in FeSSIF-V2. (a)

544

Recovery in the presence of a liquid meal (Ensure® Plus). (b) Recovery in the presence of a mixed

545

solid meal (FDA standard meal). (mean ± S.D., n = 3).

Meal components added 100

Bacon added

b 100

80

Recovery (%)

a Recovery (%)

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 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

Page 26 of 32

60 40

50

20 0

Milk Potatoes Eggs

Toast

Bacon

0 65 mg 125 mg 250 mg 65 mg

125 mg

250 mg

Low High Surface Surface Aqueous supernatant Emulsified supernatant High surface Low area surface area Medium NB, NL MediumNB Area Area

546 547

Figure 6. (a) Recovery of diclofenac in the presence of 125 mg (potatoes, eggs, toast, bacon) or 125 µL

548

(milk) of FDA standard meal components. These components were transferred into a microcentrifuge

549

tube containing a solution of 100 µM of diclofenac in FeSSIF-V2. Recovery was expressed as a 26


Page 27 of 32 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 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


550

percentage of the concentration measured from the initial 100 µM solution. (b) Recovery of diclofenac

551

in the presence of ascending quantities of mixed bacon, recovery in mediumNB and mediumNB, NL(see

552

materials and methods, recovery expressed as percentage of theoretical concentration) and recovery

553

in the presence of a single bacon piece (low surface area) versus mixed bacon pieces (high surface

554

area). All recoveries were assessed after 30 min. (mean ± S.D., n = 3).

555

27



Diclofenac recovery in Fessif-V2

Recovery (%)

100 80 60 40 20 0 0

30

60

90

120

150

180

Time (min) 556 557

Figure 7. Diclofenac recovery as a function of time in a mixture of 125 mg of mixed bacon pieces,

558

added to 1 mL of a 100 µM diclofenac solution in FeSSIF-V2. Recovery was expressed as a percentage

559

of the concentration measured in the initial solution. (mean ± S.D., n = 3).

560 561 Cumulative permeation (nmol)

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 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

Page 28 of 32

562

30

Diclofenac solution Diclofenac solution + 125 mg bacon

20

10

0 0

20

40

60

Time (min)

563

Figure 8. Permeation of diclofenac in the AMI-system. Donor solution: 100 µM diclofenac in FeSSIF-V2

564

with or without 125 mg of mixed bacon. Acceptor solution: 0.2% TPGS (w/v). (mean ± S.D., n = 3)

28


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565 566

Figure 9. (o) Average systemic diclofenac concentration-time profile after administration of a

567

Cataflam® tablet (50 mg diclofenac potassium) with 240 mL of water in healthy volunteers fed with

568

the FDA standard meal. (mean ± S.E.M., n = 5). (Δ) Average systemic diclofenac concentration-time

569

profile after administration of a Cataflam® tablet (50 mg diclofenac potassium) with 240 mL of water

570

in healthy volunteers fed with Ensure® plus. (mean ± S.E.M., n = 6). Data adapted from Van Den Abeele

571

et al. 19

572 573 574

29



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Gastric and duodenal diclofenac concentrations in healthy volunteers

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