metodo dinamica de disolucion

Designing a Dynamic Dissolution Method: A Review ofInstrumental Options and Corresponding Physiology of Stomachand Small Intestine

MARTIN CULEN, ANNA REZACOVA, JOSEF JAMPILEK, JIRI DOHNAL

Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno 612 42, Czech Republic

Received 4 November 2012; revised 10 February 2013; accepted 13 February 2013

Published online 12 March 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23494

ABSTRACT: Development of new pharmaceutical compounds and dosage forms often requiresin vitro dissolution testing with the closest similarity to the human gastrointestinal (GI) tract.To create such conditions, one needs a suitable dissolution apparatus and the appropriatedata on the human GI physiology. This review discusses technological approaches applicable inbiorelevant dissolutions as well as the physiology of stomach and small intestine in both fastedand fed state, that is, volumes of contents, transit times for water/food and various solid oraldosage forms, pH, osmolality, surface tension, buffer capacity, and concentrations of bile salts,phospholipids, enzymes, and Ca2+ ions. The information is aimed to provide clear suggestionson how these conditions should be set in a dynamic biorelevant dissolution test. © 2013 WileyPeriodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2995–3017, 2013Keywords: dynamic dissolution; gastrointestinal physiology; biorelevant dissolution; gas-trointestinal transit; dissolution; in vitro models; bioequivalence; bioavailability

INTRODUCTION

Dissolution testing of oral dosage forms can serve twopurposes: quality control (QC) and research and de-velopment (R&D). For QC purposes, such as lot-to-lotconsistency testing, simpler dissolution methods aresuccessfully used. On the contrary, dissolution testingof new compounds or dosage forms for R&D purposesmay benefit from additional methods, which are morecomplex and more biorelevant. These should be ableto successfully predict in vivo dissolution behavior,and thus minimize the number of bioavailability (BA)or bioequivalence (BE) studies needed to make a suc-cessful formulation. To introduce the current instru-mental options in this field, some model apparatusesdescribed so far in literature are discussed. The com-plexity of these methods is compared as well as theirdifferent demands on dissolution a media.

The issue of dissolution media, in itself, has beengiven considerable attention in the recent years, andthe knowledge in this field has significantly pro-gressed. A proof may be the development of biorel-evant dissolution media, for example, fasted-state-

Correspondence to: Martin Culen (Telephone: +420-608904147;E-mail: [email protected])Journal of Pharmaceutical Sciences, Vol. 102, 2995–3017 (2013)© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association

simulated gastric fluid (FaSSGF), fed-state-simulatedgastric fluid (FeSSGF), fasted-state-simulated smallintestinal fluid—updated version (FaSSIF V2), andfed-state-simulated small intestinal fluid–updatedversion (FeSSIF V2).1,2

These media, however, do not allow full simulationof real-time dynamic processes going on in vivo. It isa logical assumption that the most precise in vitroprediction of in vivo conditions would be achieved bythe closest possible mimicking of the in vivo environ-ment. In dissolutions, this would be represented bya dynamic dissolution method simulating continuouschanges occurring in the gastrointestinal tract (GIT).Such method basically needs only one medium that isprogressively changed as it travels through the simu-lated digestive tract. The dosage form, in this case, isstill a part of this environment. It is not transferredto another separate medium during the course of theexperiment as it is in classical dissolution methods.So far, this dynamic approach is best represented bythe artificial digestive tract TIM-1.3–6

This type of testing, however, entails some difficul-ties not encountered with classical dissolution meth-ods. The best examples are the transfer of the dissolu-tion medium between compartments and the dynamicchange of the medium in the process of the exper-iment (changing pH, enzymes concentrations, etc.).

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To simulate these, a proper and accurate knowledgeof the related physiology is in place. This is, neverthe-less, complicated primarily by great intra- and inter-subject variability, possible gender differences, andfurther variables that occur in pathological states.These problems have been discussed in some criti-cal reviews, which quoted the extreme values for par-ticular GI parameters and pointed out the gaps andcontradictory facts present in the current knowledgeof human GIT.7–11 The aim of this review, on the con-trary, is to summarize GI parameters that immedi-ately concern dynamic dissolution testing and to pro-vide concrete values that could be used for in vitrosimulation of the GIT, while at the same time keep-ing in mind the difficulties involved.

DISSOLUTION APPARATUSES

The following text discusses some of the dissolutionapparatuses used for testing of oral dosage forms. Be-sides the compendial apparatuses, the attention ispaid to instruments that can be used for partial or fullsimulation of dynamic GI processes. The applicabilityof the technical solutions for this type of research isdiscussed.

Compendial Dissolution Apparatuses

At present, there are four types of compendial dis-solution apparatuses used for testing of oral dosageforms. The first two are the United States Pharma-copeia (USP) 1 basket and the USP 2 paddle appara-tus. They have a simple design and can be success-fully used for QC purposes, such as lot-to-lot qualitytesting. However, because of their simplicity, they lackany stronger resemblance to the GIT, which is a draw-back when trying to simulate in vivo GI conditions.They usually use unphysiologically large volumes ofdissolution media (usually 500–1000 mL), their hy-drodynamics is not physiologically relevant, and theyenable the use of one only dissolution medium at atime.12 Some of the discussed aspects were improvedin the other two compendial apparatuses: USP 3 (re-ciprocating cylinder) and USP 4 (flow thorough cell).A dosage form in these apparatuses can be exposed tovarious dissolution media during one experiment tosimulate the changing conditions during its passagethrough GIT. Also hydrodynamics in USP 4 apparatuscan be set closer to natural conditions.13

Modified Vessel-Based Apparatuses

The following apparatuses comprise important mod-ifications that enable creation of dynamic conditionswhile still using simple vessels.

For example, an interesting upgrade of a USP 2 ap-paratus was achieved by equipping it with a buretteand a pH sensor.14 This way, the pH of a phosphatebuffer medium could be changed during the exper-

iment, from 2 to 5.5 and then to 6.8. This enabledcontinuous dissolution testing of the dosage form atvarious pH values while still keeping it in the samemedium. Although only simple buffer solutions wereused in this study, a creation of some simple dynamicbiorelevant conditions would be imaginable in thisconfiguration even despite the apparatus having onlyone compartment. This could be performed by start-ing with a biorelevant gastric medium and addingpancreatin and bile salts (BS) at the time when thepH is raised to the duodenal value. Such change wouldof course affect the whole medium at once, whereasin a mutlicompartmental setup, the change is pro-gressive and depends on the speed of the transfer ofthe medium into the successive compartment. Suchsimulation could be performed in the following ap-paratuses that consist of two vessels interconnectedby a peristaltic pump.15,16 One of these apparatuses,the artificial stomach–duodenum (ASD) model, wasemployed by Carino et al.,15 who used this appara-tus to simulate dissolution of carbamazepine in a dogmodel. As the authors used only simple buffer solu-tions in this study, it would be interesting to knowwhether their autosampling device with UV/VIS de-tector would be able to deal with addition of enzymesand BS at least in simulation of fasted state, andalso what would be the required modifications, if any(e.g., addition of another filter). In fed state, a mediumcontaining a nutrition drink or a homogenized mealwould probably completely restrict the use of an au-tosampler connected to a detector because of inten-sive clogging. Besides the ASD model, another typeof a two-compartmental apparatus using a peristalticpump was used by Kostewicz et al.16 Here, the au-thors used fasted- and fed-state-simulated media, andtheir sampling was manual with a syringe filtrationstep and an high-performance liquid chromatography(HPLC) analysis. Dilution of the duodenal medium bythe inflowing gastric medium was left unaddressed.pH was also not raised back to the original value; itwas maintained only by the action of the containedbuffer.

The both discussed apparatuses are interesting be-cause of their use of peristaltic pumps to transferthe medium between the compartments. This tech-nical solution can allow creation of fully dynamic con-ditions, where the gastric medium would be trans-formed into a duodenal medium when it arrives intothe duodenal compartment. The change in the duo-denum would comprise active rising of the pH of thediluted duodenal content back to its original value,and in case of using a biorelevant medium, also BSand enzymes would be added to make up for thedecrease in their concentrations after dilution bythe gastric juice. Generally put, there would be in-tentional changes applied to the medium to mimicthe changing chyme in GIT. In the first mentioned

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DESIGNING A DYNAMIC DISSOLUTION METHOD 2997

two-compartmental apparatus, there was not much tochange regarding the composition, as the dissolutionmedium was only a buffer solution.15 Nevertheless,the pH in the duodenal compartment was actively al-tered by a constant inflow of a fresh medium, andexcess duodenal medium was removed to maintain aconstant volume of 30 mL. In the second apparatus, byKostewicz et al.,16 pH, volume, and the composition ofthe duodenal fluid were all left without intervention.An improved method was presented by Psachouliaset al.,17 who also used a gastric and a duodenal com-partment, but here, the dilution of the duodenal fluid(FaSSIF V2plus) was compensated by an inflow of aconcentrated medium from a third vessel.

Artificial Digestive Systems

Simulated digestion is naturally a dynamic process.Therefore, there is no possibility of using preparedmedia that will each simulate only one part of the GIT.In artificial digestive systems, gastric juice is intro-duced into the artificial stomach, and in the presenceof food its properties are changed as the result of thedigestion process. If the apparatus contains also fur-ther compartments, the medium continues throughthe system and is gradually transformed into smallintestinal (SI) medium.

Dynamic Gastric Model

This apparatus, developed by IFR, Norwich, UK,represents a computer controlled artificial model ofstomach. The dynamic gastric model (DGM) is de-signed to simulate physical forces applied in stom-ach, along with gastric secretions, while working withphysiological volumes and transit times. The appa-ratus is divided into two sections, simulating fun-dus and antrum, and is also able to process nonho-mogenized meals.18–20 A duodenal compartment mayalso be added. Because of the precise simulation ofgastric processes, the apparatus is a so far unsur-passed artificial model of stomach. For this reason,it might be of great value for dissolution testing ofdosage forms with performance dependent on physi-cal stresses. Nevertheless, for formulations that areonly little affected by the gastric physical forces, otherand in this regard simpler methods are sufficient.

TIM-1

The TNO Nutrition and Food Research center inZeist, The Netherlands has developed a mutlicom-partmental artificial GI system TIM-1. Its designwas thoroughly described by Minekus et al.21 Theapparatus consists of four interconnected compart-ments: stomach, duodenum, jejunum, and ileum (seeFig. 1). The number of compartments and the simu-lation of stomach and the whole SI is one of the maindifferences compared with other apparatuses. Theapparatus maintains successive transport of chyme

Figure 1. Scheme of TIM-1 system: (1) stomach, (2) duo-denum, (3) jejunum, (4) ileum, and (5) dialysis unit. (Reed-ited according to Ref. 22.)

through these compartments and ensures peristalticmovement. This allows simulation of the physicalforces applied in GIT. pH of the dissolution mediumis controlled using pH electrodes and altered by in-jection of water or acid solution into the stomach,and electrolytes or basic solution into the SI com-partments. This allows mimicking the changes inchyme pH as they naturally occur in vivo duringthe passage of chyme through the GIT. Gastric, bil-iary, and pancreatic secretions are maintained bycomputer-controlled pumps. Sink conditions for wa-ter, dissolved drugs, and small molecules, such asproducts of digestion, are created by pumping the con-tents of jejunum and ileum through hollow fiber dial-ysis units.6 The dissolved active pharmaceutical in-gredients (APIs)content is determined in the dialysisfluid, which eliminates the need for laborious filtra-tion and centrifugation performed to exclude undis-solved API particles and other content that may laterclog the HPLC instrument. This advantage will beeven more pronounced in simulation of fed state,where the medium requires more preparation stepsbefore the analysis.

The TIM-1 apparatus provides full simulation ofthe GIT and surpasses both the compendial and othernoncompendial methods in this respect. It also fullyrepresents the dynamic dissolution approach, as thedissolution medium is continually changed through-out the experiment as it passes from one compartmentto another. As such, it may be regarded as a modelinstrument in this field of research. Nevertheless,

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because of the complexity of this instrument, its dis-advantages include laborious preparation and manip-ulation during an experiment, which, of course, re-quires higher number of personnel and also higherdemands on maintenance than in the case of simplerinstruments. Another disadvantage is the long timeneeded for one experiment (approximately a wholeday), but this is also because of the long net timeneeded for simulation of GIT transit. A significantlimiting factor is also the high cost of these experi-ments.

The use of TIM-1 was originally intended for studyof nutrition effects. As such, it was utilized for ex-ample to assess bioaccessibility of folates, iron andphosphorous, heterocyclic amines, and "-tocopheroland lycopene.22–26 In pharmaceutical research, TIM-1 was used by Blanquet et al.6 to study behavior ofacetaminophen powder and its SR formulation de-pending on the presence of food. The tmax values forin vitro and in vivo results were determined by theauthors as similar. Nevertheless, no quantitative linkcould be found, which was attributed to the follow-ing possible in vivo factors: metabolization by epithe-lial cells, first pass effect, larger distribution volumein vivo, and renal clearance. These influences couldbe, of course, calculated by special simulation soft-ware if the in vitro and in vivo results were to becorrelated. But in this case, the software can thencalculate also the effect of absorption, which meansthat the main asset of the membranes can be seen inmaintaining of sink conditions for the studied APIsand the digestion products. The disadvantages of thedialysis units are their cost and the need to createand additional circuit for their use. Other studiesthat described the use of TIM-1 in pharmaceuticalresearch provided comparison of its results with re-sults from compendial dissolution methods. In one ofthese, Souliman et al.5 used TIM-1 to establish level Ain vitro–in vivo correlation (IVIVC) for immediate re-lease (IR) acetaminophen (paracetamol) dosage form,with correlation coefficient of 0.9128 and 0.9984 forfasted and fed state, respectively. The absorption pro-file from TIM-1 in the fed state was almost identicalto the in vivo absorption profile, and TIM-1 providedbetter results than the standard USP 2 apparatus.5

Souliman et al. also compared dissolution of theo-phylline hydrophilic matrix tablets in TIM-1, USP2, and USP 4 apparatuses.3 In this case, the authorsstated that TIM-1 provided better prediction of in vivoresults than the other methods involved as the meanratio between the in vitro results from TIM-1 andthe in vivo data was 1.02, whereas the best resultof the compendial methods was ratio of 0.88. Nayloret al.27 processed the TIM-1 absorption data of an IRformulation of paroxetine hydrochloride hemihydrateon GastroPlusTM software.27 Data from TIM-1, in this

study, allowed more accurate prediction of in vivo dis-solution than the data from USP 2 apparatus.

Despite the studies mentioned, it would be benefi-cial if more IVIVC studies employing this instrumentwere published, as this would help to better evidenceits capabilities in the field of dissolutions.

PHYSIOLOGICAL DATA REQUIRED FORSIMULATION OF STOMACH AND SMALLINTESTINE

Biorelevant dissolution media have been thoroughlystudied during the last years. The developed simu-lated media may serve as a reference for choosing theright concentration of various components and the ef-fect of these components on physicochemical parame-ters of the dissolution medium. The latest versions ofthese media are FaSSGF, FeSSGF, FaSSIF V2, FeS-SIF V2, and a modified FaSSIF V2plus (with addedlipolysis products).2,17 Furthermore, three “snaphot”versions of FeSSIF (early, middle, and late FeSSIF)have been developed to address changes in the SI af-ter food intake.2 The media have been fine tuned, andthey carry important information on the compositionof simulated GI fluids in certain parts of GIT at cer-tain separate time points after administration of adosage form. However, the situation in a dynamic sim-ulation is more complicated. To create dynamic condi-tions the medium from one separate time point mustbe transformed into the medium from the followingseparate time point. This review specifically discussesthese dynamic aspects, the parameters that are crit-ical to simulate the changing of the medium and itstransit through an artificial digestive tract. The aim ofthe following section is to provide information neededfor designing a method, where the biorelevant disso-lution medium goes from the stomach compartmentinto the compartments of SI, merging with the fluidsalready contained there.

Volumes of GI Contents

Fasted State Volumes

Table 1 comprises volumes reported for gastric andSI liquid contents, measured in various studies forfasted state, using magnetic resonance imaging ortube aspiration.28–33 For fasted stomach, a startingvolume of 30 mL of gastric fluid can be suggested asan average value acceptable for biorelevant dissolu-tions. This volume would be increased by additional200–250 mL of water used in in vivo studies to swal-low a dosage form. It must be kept in mind that thisaddition of water dilutes the gastric resting contentand lowers the concentrations of all the contents. Theresulting volume of around 230–280 mL is, however,lower than the volume needed in normal USP I and II



Table 1. Volumes of Gastric and SI Liquid Contents in FastedState (Mean ± SD)

Stomach (mL) Small Intestine (mL)

45 ± 18 (Ref. 28) 105 ± 72 (Ref. 28)27 (Ref. 29) 86 (Ref. 30)28 (Ref. 31) 109 ± 36 (Ref. 34)26.7 ± 18.9 (Ref. 32) 112 ± 27 (Ref. 34)23.9 ± 24.4 (Ref. 33) 165 ± 22 (Ref. 35)24 (Ref. 36)

apparatuses to completely immerse the basket or thepaddle. Moreover, it must be considered that in dy-namic dissolution this volume will be further quicklydecreased as the medium is transferred into duode-num. Therefore, in a dynamic dissolution test, thestomach compartment must be able to work with vol-umes near 50 mL. When rotating mixing is to be em-ployed, smaller vessels can be applied as in the caseof the two two-compartmental apparatuses discussedabove.15,16

The generally accepted value for the liquid contentin fasted SI from literature is 100 mL, which alsocorresponds with the values reviewed in Table 1.9

Fed-State Volumes

Postprandial volume of gastric contents is determinedby the amount of ingested food and is therefore notfurther discussed. The manner of gastric food emp-tying is then thoroughly analyzed in the followingsection on transit times.

In case of SI fluids, several studies reported de-crease in their volumes after meal intake. Such de-crease was for example observed in a case where thestarting SI volume before meal was 86 mL, but only34 mL was measured 90 min after breakfast.30 More-over, additional 150 mL of stomach food content en-tered the SI during this interval. This means the dis-appearance of approximately 200 mL of liquid, only50 mL of which was found to enter the colon. In thesame experiment, at later time points, the volumerose between 90 and 180 min, and it maintained asteady state between 180 and 405 min. After 405 min,the volume sharply fell, and in 420 min another meal(4184 kJ) was ingested.30 The initial intestinal liq-uid resorption was explained to be caused by low-molecular-weight saccharides, which can be readilyabsorbed along with water, and which were widelypresent in the administered meal (pudding, orangejuice). The theory is also supported by decrease ofSI liquid content after ingestion of a glucose solutionand the opposite sharp volume increase after inges-tion of a solution of unabsorbable mannitol.30 The re-ported decrease was also observed by Schiller et al.,28

who measured a volume of 105 mL of SI liquids (dis-tributed in four pockets) in fasted subjects, whereas

in other subjects 60 min after meal intake (63.2% car-bohydrates) they measured a volume of only 54 mL(distributed in six pockets in the SI). In this work,the decrease could also be added to the absorption oflow-molecular saccharides from food.

These findings indicate that the starting SI volumeof 100 mL should be decreased when simulating fedstate. Also, at the beginning of a fed-state experimentthere should be simulated a heavy resorption of theSI fluid, along with some metabolites, as it was ob-served in the studies discussed above. Furthermore,there is the complication associated with the fluidsbeing not present as one bolus in the SI. This meansthat the administered dosage form, once it reachesthe SI, does not have the whole volume of chyme to itsdisposal, but instead, it dissolves only in one and sig-nificantly smaller part of the volume at a time. Thiscan be solved by dividing the SI into several com-partments. This is partly addressed in TIM-1 withits three SI compartments. Whether it would be de-sirable to include more than three SI compartmentsto simulate the reported four to six pockets is ques-tionable and may prove more laborious than effective.In a two compartmental vessel-type apparatus, suchas the stomach duodenum models mentioned above,the medium from the second compartment should bepumped out, to reduce its volume during the inflowof the gastric juice. Here, the dosage form would betechnically still in one “pocket” but this single pocketwill not contain the volume normally present in thewhole SI.

Migratory Motor Complex and Peristaltic Contractions

To describe the residence times for dosage forms, it isnecessary to understand the basic functioning of GImotility cycle, which is controlled by migratory motorcomplex (MMC).37 This cycle is active only in fastedstate and comprises three phases. Phase I is a quies-cent phase with very low contractile activity. PhaseII has irregular activity, which gradually increases inboth amplitude and frequency, and ends in phase IIIwith maximum contractile activity, where the expul-sion of large objects such as undigested food contentor tablets is possible.37 Coleman et al.38 measuredthe phases I, II, and III to last 60 ± 34, 58 ± 44,and 6.0 ± 2.1 min, respectively. Oberle et al.39 mea-sured quite similar durations of 46 ± 24, 107 ± 68,and 8.1 ± 4.3 min, respectively. Mojaverian et al.40

determined that Heidelberg capsule had no effect onthe duration of the phases in duodenum. Their mea-sured times (mean ± SE), for the phases I, II, andIII, were 54 ± 9.3, 22 ± 8.1, 5.3 ± 0.7 min, respec-tively, with the capsule present. Without the capsule,the durations were 31.6 ± 10.1, 58.9 ± 32, 4.2 ±0.7 min, respectively. VanTrappen et al.41 determined


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phases I and II to last 112.5 ± 11.4 min (mean ± SE)together. Kellow et al.42 measured the whole cycle inproximal jejunum to last 108 ± 7 min (mean ± SE),with the most cycle lengths falling between 60 and90 min, and relative contributions of phases I, II, andIII being 21 ± 2, 67 ± 3, and 12 ± 1 (mean percent-age ± SE), respectively. According to the authors, theMMC was mostly occurring in the proximal jejunumregion, and it weakened in both proximal and distaldirection away from this point, that is, only 39% ofthe proximal jejunal MMCs progressed to proximalileum, and only 74% occurred in stomach.

It is clear that the interdigestive motility cycleshows great variability, but if its average durationwould have to be defined for simulation purposes, itwould be 1–2 h.

In fed state, the MMC is replaced by regular toniccontractions which mix food and move it toward theantrum.38 Only after the completion of digestion,when all the digested food content is transferred fromstomach to duodenum, the MMC should start anew.Because the fed-state motility patterns are directlylinked with gastric emptying of dosage forms, theyare discussed in the section on gastric residence times(GRT).

The MMC is also linked with frequency of con-tractions. In the case of the interdigestive motilitycycle, Stotzer et al.43 defined phase III by havingthree pressure waves per minute, phase II by hav-ing at least two contractions per 10 min, and phaseI by having less than two contractions per 10 min.43

Kellow et al.42 associated phase II and III with con-tractions of frequency of 1–3/min. The regular antralcontractions after a meal of 323 kJ were measuredto occur 3.0 ± 0.3 times per minute.38 This frequencywas also observed by Weitschies et al.44 in a digest-ing distal part of stomach, but not in its proximalpart. This lower physical stress in the proximal stom-ach was in this case assumed to cause delay in ab-sorption of felodipine contained in hydrogel-basedextended release (ER) tablets in cases where thetablets were retained in proximal stomach. In duo-denum, the frequency of contractions during phaseIII, which lasted 8.7 min, was 11.7/min.42 In ter-minal jejunum, the contraction frequency of 8.5/minwas measured, and the phase III lasted for 13.8 min.In general, the movement of dosage forms in SIand in fasted-state stomach was described as phasesof relative peace interrupted by rapid movements(unfortunately a detailed description of these pat-terns was not given).45,46 Moreover, the regular re-peating of these “velocity spikes,” throughout thewhole measurement, indicated that they occurredoutside phase III. In fed stomach, in five of sixsubjects, this behavior with velocity spikes was notobserved.46

The contractions create specific hydrodynamic con-ditions, which were evaluated in relation to the clas-sical USP 1 and 2 apparatuses.12,47–49 The hydrody-namics created in an apparatus simulating peristalticmixing would be, however, completely different. Here,studies reporting the frequency of contractions alongwith manometric measurements offer more helpfulinformation. Such manometric recording was for ex-ample measured by Cassilly et al.50 using manomet-ric probes and SmartPill. These results have shownthat the strong contractions, just before the expul-sion of the pill from stomach, reached 300 mmHg,whereas the ordinary contractions were usually be-low 25 mmHg.50 The manometric measurements likethese could also be performed inside of a biorelevantdissolution apparatus to create physiology matchingconditions. So far, the only simulation to mimic thepresence of the stronger contractions of 300 mmHgwas performed by Garbacz et al.51 with their stress-test apparatus, where the dosage form was held inan enclosure with inflating and deflating balloon andthe whole enclosure rotated above the surface of themedium, thus being alternately immersed. The au-thors here assessed a role of two stress phases; affect-ing a tablet upon its expulsion from stomach and intocaecum. These two stress phases markedly increasedthe dissolved concentration of diclofenac from its ERtablet and resulted in two distinct increases in itsdissolution profiles.

At the end of this section, it is worth mentioningthat all the data stated here indicate that a constantrotation of a paddle is far from biorelevant. A biorele-vant simulation should mimic a peristaltic movementwith separate pattern for digestive phase, separatecontraction pattern for the following interdigestivemotility with its three different phases, the simu-lation should mimic the observed repeating velocityspikes and also the stronger contraction force dur-ing phase III. From technical point of view, it is clearthat a normal vessel apparatus with a paddle cannotsimulate these conditions. An improvement in thisregard is offered by the USP 3 apparatus, where it ispossible to program a sequence with changing num-ber of dips per minute.52 In an apparatus with peri-staltic mixing the simulation of an irregular pattern isonly a matter of appropriate setting of operation soft-ware. The challenging part may be the simulation ofalternating contraction force (simulating strong con-tractions during phase III). Of course, the benefit ofsuch accurate simulation of peristalsis, over a classi-cal monotonous one, would have to be thoroughly eval-uated. In the end, it could be found that its main useis only evaluation of robustness of ER dosage forms. Adifferent complication, here, is the contemporary ab-sence of complex data on the whole issue. Studies withthe main aim of providing clear quantification of the



periodicity and intensity of contractions are currentlyrequired.

GI Transit Times

The GI transit times are the most debatable param-eter discussed in this review. They seem to be themost affected by biological variability, and moreover,their determination is not exact. The measurementsof GI transit times suffer from a disadvantage thatthey require to determine the position of the studiedmatter (water, food, dosage form) in the GIT, which isnot always exact and can be burdened by both instru-mental and human inaccuracies and errors. This ispronounced by the fact that this issue has been stud-ied in many smaller studies in 30 year long periodof time. This, of course, means that the studies dif-fered in both hardware and software tools, availableat the time of their undertaking, for positioning of thedosage forms studied. The studies were performed byvarious scientific teams, which allows for interlabo-ratory variability in evaluation of the location of theobjects in GIT. Also, the positioning in itself is noteasy. Even if the patient takes up the same positionin each measuring made during a 9 h long period, itsbowels may have slightly rearranged. Furthermore,there is the factor of sampling times; the results nat-urally vary if the measurements are taken twice anhour or each 5 min. All these variables together mayhave caused serious discrepancies between the stud-ies. Moreover, there is still the issue of intra- and

intersubject variability. Most of the studies were car-ried out on around eight subjects, and therefore theysometimes ended with eight different results prevent-ing any reasonable averaging. Also, if each study fol-lows a slightly different protocol, they may yield com-pletely different or contradictory results, which is, infact, true for some of the studies discussed further.

Thus, the determination of GI transit times is dis-advantaged compared with determination of physic-ochemical parameters and composition of GI fluids,which are more or less influenced only by biologicalvariability.

Gastric Emptying Times for Water and Food

Ingestion of water or saline does usually not alter themotility cycle pattern.53 Vice versa, it can be said thatthe effect of MMC on gastric emptying of water haslow significance, although the emptying of 200 mL ofwater was actually shown to be faster in phase III,than in phases I and II.53 But because of the shortduration of phase III, and the resulting low odds ofthe liquid being ingested directly before this phase,the average values reported for emptying of wateradministered nonspecifically during the MMC corre-spond well to those determined exclusively for phaseI and II. The emptying times for water and variousmeals are shown in Table 2. The reported gastric emp-tying half-times for nonnutrition water solutions, ofvolumes around 250 mL, are usually no longer than15 min with the emptying plot being described as log

Table 2. Gastric Emptying Times for Water and Meals

Ingested Water or MealT50% (Mean ± SD, min),If Not Stated Otherwise

Other DescriptionCharacteristics

Lag Times(min)

200 mL of water39 11.8 ± 8.2300 mL of water31 15.8200 mL of saline (administered

with a solid meal 1230 kJ)54Amount retained: 44%

at 10 min,1

10% at 60 min (medianvalues)

200 mL of 25% dextrose solution(administered with a solid meal1230 kJ)54

Amount retained: 91%at 10 min,

6

38% at 60 min (medianvalues)

Solid meal (1230 kJ) (administeredwith 200 mL of saline)54

Amount retained: 36%at 120 min (median)

37

Solid meal (1230 kJ) (administeredwith 200 mL of 25% dextrosesolution)54

Amount retained: 91%at 120 min (median)

95

122 mL of water + meal (1067 kJ,2% fat)50

95 ± 6 T90% (min) = 194 ± 11

38 ± 5% retained at120 min

Solid meal (1200 kJ)55 64 ± 32 51 (median)Liquid meal (1351 kJ)38 40 ± 13Liquid meal (2050 kJ)38 44 ± 15Solid meal (2050 kJ)38 105 ± 21Solid meal (2242 kJ)56 72 ± 18


3002 CULEN ET AL.

linear.31,39 Many of the studies also reported a lagphase preceding the onset of emptying. In a studywith 200 mL of water ingested, the gastric emptyingwas preceded by a lag time of 5.4 ± 6.7 min.39 But onthe contrary, in another study with 300 mL of wateringested, no lag was observed.31

The emptying times for ingested meals are gener-ally longer than for liquids. Also, solid meals werereported to have longer emptying than liquid mealsof the same energetic value.38 Hunt and Stubbs57

summarized results of several studies and came toa conclusion that the emptying of food is not depen-dent on its volume but on its caloric content, with theemptying being slower for more caloric food, to pro-vide a more or less constant energy inflow into theduodenum (kJ/min). However, they also stated thatthe mechanism could not fully compensate for high-energy meals, where the energy content transferredto duodenum was still higher than for light meals.The authors also reported that stomach emptied thesame caloric amount irrespective of the nutrient con-tent—whether carbohydrates, fats, or proteins.

The emptying curve for solid meal was describedby Coupe et al.55 to be linear with a slow final phase.This was also reported by Collins et al.54 for a solidmeal and a simultaneously administered dextrose so-lution but with a preceding lag phase (see Table 2).On the contrary, the same solid meal administeredwith saline instead of the glucose solution emptied inan exponential fashion and also faster with a shorterlag phase. What is interesting on these findings isthat the solid meal was retained in proximal stomachwhile the liquids were being emptied. The simulationof this effect would be currently probably only possi-ble on the DGM. Still, it is questionable whether suchcomplex simulation and its results would be worththe trouble compared with simpler dissolution witha homogenized or liquid meal contained in only onegastric compartment.

The results in Table 2 clearly show that the gastricemptying of liquid meals is faster than of solid meals.An interesting fact is that two liquid meals includedin Table 2, determined by different methods but com-ing from the same study, had almost identical gastricemptying half times, even despite they differed in en-ergetic value by 700 kJ. Also, the emptying times ofthe solid meals from Table 2 do not increase withtheir energetic content, as would be expected. Hence,it is clear that more measurements would have tobe obtained, to at least determine a border betweenemptying half times for light and heavy meals.

GRTs for Dosage Forms

The GRTs for nondisintegrating dosage forms showgreat variability, and there are still many questionsabout how and in what manner do the individual pa-rameters affect the process of gastric emptying. The

following text summarizes the current findings onGRTs, and shows which results are and which arenot in agreement, thus allowing to confirm or disputethe influence of a particular effect.

MMC. The importance of MMC phase III on gas-tric emptying of dosage forms was demonstrated forexample by Cassilly et al.50 using SmartPill (26.8 ×11.7 mm). In fasted state, this device was emptiedfrom stomach with the first upcoming MMC phase IIIin four of five subjects, and in the fifth subject, it emp-tied only with the second following phase III. In fedstate, the situation was more complicated. Accordingto the principle discussed in the section on the MMC,the digestion phase must finish before the interdiges-tive phase can start. Therefore, if a dosage form isto be emptied during phase III, occurring at the endof the interdigestive phase, its emptying time shouldequal the sum of the time needed to digest and emptythe food from stomach plus the length of the wholenew interdigestive MMC cycle that starts after theend of the digestion. Using SmartPill, this scenariowas observed only in four of 14 subjects where thedevice was found to empty near the first phase IIIoccurring at 239 ± 23 min.50 Also, at this time therewas still 10 ± 4.3% of the food left in the stomach,which shows that the motility cycle started soonerthan all the food was emptied. In other five subjects,the SmartPill was emptied only in the second phaseIII, and in yet other five it emptied with isolated distalantral contractions independently of phase III. In onefurther subject, the SmartPill resided in stomach formore than 7 h.50 The study of emptying times using adevice such as SmartPill can be regarded as very ac-curate because the arrival into duodenum is signaledby increased pH measured by the capsule. Thus, thelocalization does not depend only on external monitor-ing techniques, such as gamma scintigraphy. The factthat the emptying of solids from stomach must notalways solely depend on phase III was also observedby Coupe et al.55 on emptying of a radiotelemetry cap-sule (25 × 8 mm), as in one subject the capsule leftstomach outside of the phase III assumingly duringstrong contractions at the end of the gastric prandialphase. This type of emptying, occurring during thedigestive state, was also observed for 1.5 and 3.0 mmcubic radiopaque markers, which all emptied beforethe onset of phase III.43 The emptying in this case oc-curred during irregular postprandial motility, whichwas similar in its motility index to late phase II. Thesefacts show that in most cases solid objects empty fromstomach during: digestion phase, first phase III, orsecond phase III; and at the same time none of theseoptions seems to be clearly prevalent. This arousesthe question whether it is necessary to perform dis-solution tests simulating each of the three emptyingscenarios, or whether it is generally sufficient to work



with a model where the emptying occurs in the firstphase III (being the middle option). Given that a sin-gle biorelevant dissolution test may take a whole day(especially in fed state), a testing of one pill under allthree emptying conditions will take 6 days if each ex-periment is repeated only twice. Therefore, the cost/benefit ratio will assumingly not be very favorable inthis case, and such thorough testing may be neededonly for some modified release dosage forms.

Food Effect. Food induced prolongation of GRT iscurrently probably the only noncontroversial effectrelated to gastric emptying. This effect was very welldescribed in a complex work by Davis et al.,58 whichsummed up results of several studies. The authors re-ported the fasted-state GRTs for various dosage formsto range up to approximately 1.5 h. GRTs after inges-tion of light breakfast (1500 kJ) were shown to bedelayed to 2–4 h, and after ingestion of heavy break-fast (3600 kJ) the prolongation was so marked that anadministered osmotic pump (24 × 7 mm) remained inthe stomach in all six subjects for more than 9 h.58

A similarly pronounced prolongation of GRT (for ametal tablet of 11 × 6 mm) was achieved in anotherwork by subsequent ingestion of meals and snacks in2.5 h intervals.59 This regime was suggested to main-tain continuous digestion and a resulting almost 10 hlong GRT. This prolongation caused by ingestion of asecond meal was also tested in relation to energeticcontent of the first administered meal.60 In this study,a lunch (second meal) was ingested at 4 h after admin-istration of 12 mm tablets and the first meal. The GRTwas 7.6 h long for the subjects that consumed heavybreakfast (3000 kJ) and 5.1 h for subjects that wereon lighter breakfast (1500 kJ). Very good possible ex-planations for this effect were given by Abrahamssonet al.61 in a study with a 9.6 h long gastric residencemeasured for a 9 mm tablet. The authors of the studystated that the effect could have been accounted forthe meals which followed at 4, 6, and 10 h after thetablet administration, but they also came with twoother interesting options: absence of strong waves, ortheir inability to expel the tablet. The evidence forthe latter one has been described in the already men-tioned study by Cassilly et al.,50 where the SmartPillemptied during the second MMC phase III. The for-mer option regarding the absence of stronger contrac-tions, that is, the phase III, is in good agreement withthe aforementioned findings of Kellow et al.,42 whomeasured the incidence of MMC cycles in esophagusand stomach to be only 56% and 74%, respectivelyof the cycles occurring in proximal jejunum. Still, in-dependently on the cause of these reported 9 h longGRTs, the advantage of simulation of such long exper-iments over a shorter one would have to be evaluated.

Here, it must be mentioned that the dosage formcan also be retained independently of these effects.

This was very well documented by Davis et al.60 onemptying of two simultaneously administered 12 mmtablets, which did not always empty at once. There-fore, if one tablet empties and the other does not, itmeans that the contractions ought to be present andsufficiently strong (except for cases of the reported“fortuitous emptying” where the tablet is coinciden-tally expelled by a peristaltic wave), and the retentionof the second tablet is possibly caused by its entrap-ment in antrum or generally its inability to reach thesphincter. Concurrent emptying of the both 12 mmtablets, in the aforementioned study, was observedonly in four of eight subjects, fed a 1500 kJ meal, andonly in five of eight subjects, who received a 3000 kJbreakfast.60 In subjects where the two tablets emp-tied separately, the differences between the emptyingtimes ranged from 40 to 105 min in the light fed stateand from 50 to 490 min in the subjects after heavybreakfast. It is obvious that if emptying of two iden-tical tablets can be so variable in one subject, thecomparison of results between studies will be verydifficult, and only average values can be taken asrepresentative.

Simply put, all the discussed results clearly indi-cate that presence of food delays gastric emptyingof dosage forms. This is also well supported by thecomparison of results from different prandial statesshown in Table 4. Here, the data have been dividedinto three categories: those measured in fasted state,after light meal, and after heavy meal. The table or-derly shows the effect of food on prolongation of GRT.The ranges for fasted, light fed, and heavy fed satesoverlap only minimally, and the mean and medianvalues are distinctly higher for each successive mealenergy category. The only exception is the range forpellets after heavy meal, but this is due to a single de-viating value of 78 min (the other values were above181 min).

Size. A widely discussed factor to influence GRT isthe size of the objects/dosage forms to be emptied.The most interesting results, concerning this issue,are discussed in more detail. For example, opposingresults obtained in the course of one study were givenby Ibekwe et al.,62 who measured almost identicalGRTs for an 8 mm tablet and a large Bravo R© capsule(25 × 5 mm) in fasted state, but not in prefeed regime(tablets administered 30 min before breakfast). Here,the larger capsule had 1 h longer gastric residencethan the smaller tablet. Opposed to this, Podczecket al.63 measured faster gastric empting for larger12 mm tablets than for 6.6 mm tablets. The fact thatthe GRT tend to occur in one common broad interval,irrespective of the size of the dosage form involved,is also visible in the summaries in Table 4. Here, thereviewed results for single unit dosage forms were di-vided into three groups according to their size: dosage


3004 CULEN ET AL.

forms measuring ≤12, ≤19, and ≥20 mm. The range,mean, and median parameters for the first two groupsare practically identical. This means that the evalu-ated objects measuring 16, 17, and 19 mm had com-parable GRTs to the smaller objects, below 12 mm.Hence, it can be assumed that the size effect doesnot influence this size range. However, in case of thethird group, with size of 20 mm and more, the meanand median values are higher than for the smallerobjects, in both fasted and light fed state, (unfortu-nately, in heavy meal group only one measurement isavailable). The weight of this finding is reduced by thefact that only three results were available for fastedand light fed state alike, and also by the finding ofIbekwe et al.62 where the 8 and 25 mm dosage formshad the same GRTs in one study. Still, the option thatthese extralarge objects are emptied slower shouldnot be completely dismissed. At this point, it is alsoworth mentioning the two included GRTs for two 19mm tablets. Their residence times were measured af-ter administration of a 420 kJ liquid meal and wereamong the shortest in the light meal category, last-ing 91 and 97 min. But it can be expected that if asolid light meal with higher energy content (i.e., onesimilar to the other meals in this category) would beadministered, the GRTs would acquire higher values,possibly similar to the objects above 20 mm.

It is evident that the current data on this subjectdo not allow drawing any conclusions, and that weneed more studies or rather a sufficiently large one tounambiguously determine the influence of object sizeon gastric empting. Until then, the only correct optionfor determination of GRT for any type of formulationis to average the available data.

Emptying of Pellets. Although the emptying of pel-lets, that is, multiple unit dosage forms, technicallycomes under the subject of size, it is discussed sep-arately because of the specific behavior. In an ear-lier study, Davis et al.58 proposed that pellets emptyfaster from stomach than larger single unit dosageforms. Later, however, also opposite findings were ob-tained. For example, in a study performed in patientsafter light breakfast, an 11.5 mm tablet had fasteremptying (90 ± 35 min) than was the T50% emptyingtime of 0.8–1.1 mm pellets (105 ± 45 min).64 Earlier, itwas also believed that pellets can leave stomach alongwith food, as long as they fall under a certain cutoffsize, for example, 1.4 or 2 mm.37,58,65 However, overthe years of research the situation has proven moredifficult as there have been published works that statethe direct opposite. For example, Coupe et al.64 foundsmall pellets (Ø 0.8–1.1 mm) to empty slower thanfood (T50% for pellets = 151 ± 81 min; T50% for food= 64 ± 32 min) with only two subjects emptying thefood and the pellets simultaneously. The emptyingdid not show any unified trend. Clarke et al.66 stated

that pellets of 0.5 and 4.75 mm did not empty withconcurrently administered orange juice. The authorsalso measured no statistically significant differencebetween the GRTs of the two sizes, which disprovesthe “cutoff size” theory. The existence of cutoff sizecan also be excluded upon reviewing the data fromTable 3, as these show that the GRTs of pellets areclearly not linked to their size. Another thing worthmentioning, in connection with pellets, is the man-ner of their emptying. Unlike nondisintegrating SUdosage forms, pellets have the ability to leave stomachgradually. Abrahamsson et al.61 determined the meanemptying curve as “approximately linear”. Postpran-dially, Kenyon et al.67 also observed a linear trend insome subjects, but in fasted state the curve was ex-ponential. On top of this, Clarke et al.68 found threedifferent emptying profiles for pellets of three differ-ent densities in one subject. This nicely demonstratesthe variability of gastric emptying of pellets. A GIsimulation could therefore maintain either linear orexponential trend, with none of these being prefer-entially supported by in vivo data. The only commonfinding among the studies on emptying of pellets waspresence of a lag phase.61,64,66–69 The reported val-ues for duration of the lag phase in fed state were2–3 h and 106 min.61,69 Without food, the reportedlags lasted 46 and 101 min (after ingestion of orangejuice).69,68 The reported lag times are yet another ev-idence against the simultaneous emptying of pelletswith food, as such long lags were not reported for theemptying of food, or in majority of the studies no lagswere reported at all. For simulations, this means thatthe pellets should be somehow retained in the stom-ach compartment and released only later on. On onehand, this may seem more difficult to simulate thanto leave the pellets freely swim and exit the artifi-cial stomach along with food. But on the other hand,if the pellets are left to freely disperse, there is nocertainty that their distribution would be identicaleach time, and that each time they would exit in thesame manner. These problems could be solved, for ex-ample, by closing the pellets into several baskets orperforated bags and then transferring these one byone into the duodenal compartment. Such transportwould also be in agreement with work of Yuen et al.,69

who found that pellets emptied from stomach in bo-luses (the boluses were more frequent and smaller infed state). The pellets enclosed in these bags couldalso be transferred in this manner between the com-partments of the SI (if they still do not disintegrate).This would be supported by a finding that little orno further spreading was observed in SI after the re-lease of pellets from stomach.64,69,70 Such technicalsolution would enable to transfer the pellets at de-sired times, and it would also solve the problem ofpellet transport through peristaltic pumps employedfor example in the above mentioned ASD model, as



Table 3. GRTs for Nondisintegrating Dosage Forms and Solid Objects

Dosage Form (Density)/Size (mm) Ingested Water or MealGRT (Mean ± SD, min), If Not Stated

Otherwise

Pellets67 150 mL of water T50% = 30 ± 24Breakfast (3000 kJ) T50% = 78 ± 18

Pellets/0.8–1.1 (Ref. 55) 100 mL of water plus meal (1200 kJ) T50% = 151 ± 81T50% = 129 (median)

Pellets/1.18–1.14 (Ref. 69) 150 mL of water T50% = 70 ± 27 (mean ± SE)T90% = 110 ± 38 (mean ± SE)Lag = 46 ± 20 (mean ± SE)

150 mL of water plus breakfast(2240 kJ)

T50% = 181 ± 17 (mean ± SE)

T90% = 257 ± 32 (mean ± SE)Lag = 106 ± 18 (mean ± SE)

Pellets (overall mean for 1.0, 1.5, and2.4 g/cm3 pellets)/1.2–1.4 (Ref. 68)

200 mL of orange juice T50% = 179

Lag = 101Pellets (1.5 and 2.4 g/cm3 pooled)/4.75

(Ref. 66)200 mL of orange juice T50% = 117

Pellets (1.5 g/cm3)/0.5 (Ref. 66) T50% = 125Pellets (2.6 g/cm3)/0.5 (Ref. 66) T50% = 141Tablet/5 (Ref. 71) 150 mL of water 37 (median)

150 mL of water plus standardbreakfast (2146 kJ)

149 (median)

150 mL of water; standard breakfast(2146 kJ) ingested 45 min afterthe tablet

39 (median)

5 Tablets/5 (Ref. 72) 200 mL of water Day 1: T50% = 25 ± 4 (mean ± SE)Day 2: T50% = 30 ± 6 (mean ± SE)Day 3: T50% = 113 ± 78 (mean ± SE)

Matrix tablet/8.2 (Ref. 73) 150 mL of water 89 ± 21150 mL of water plus high-fat

breakfast; other mealsadministered after 4 and 10 h

298 ± 104

Tablet/9 (Ref. 61) 200 mL of water plus standardbreakfast (2800 kJ), other mealsadministered after 4, 6, and 10 h

576 ± 372

Pellets (1.2–1.3 g/cm3)/0.4–0.6(Ref. 61)

T50% = 216 ± 72

Metal tablet/11 × 6 (Ref. 59) No water/meal 38 ± 11250 mL of water 38 ± 8Milk (652 kJ) 128 ± 14250 mL of liquid meal Fresubin

(1000 kJ, 3.4 g/100 mL)152 ± 6

Continental breakfast (2200 kJ) 249 ± 24Matrix tablet/11 (Ref. 44) 200 mL of water 53 ± 61

23 (median)Standard breakfast 210 ± 35

Tablet/11.5 (Ref. 64) 100 mL of water plus light breakfast 90 ± 35Pellets/0.8–1.1 (Ref. 64) T50% = 105 ± 45Tablet (1.5 g/cm3)/12 (Ref. 63) 50 mL of water 14.8 (median)Tablet (3.7 g/cm3)/12 (Ref. 63) 24.6 (median)Tablet (1.41 g/cm3)/6.6 (Ref. 63) 58 (median)Tablet (2.85 g/cm3)/6.6 (Ref. 63) 82 (median)2 Tablet/12 (Ref. 60) Light breakfast (1500 kJ) 306 ± 49 (mean ± SE)

Heavy breakfast (3000 kJ) 459 ± 44 (mean ± SE)Tablet/16.4 × 7.3 × 5.1 (Ref. 45) 200 mL of water 72 ± 25Tablet/17 × 4 (Ref. 74) 200 mL of water (20–23 years old subjects): 38.4 ± 7.2

(mean ± SE), 34.2 (median)(63–76 years old subjects): 51.6 ± 13.2

(mean ± SE), 39 (median)200 mL of water plus breakfast

(1600 kJ)(20–23 years old subjects): 180.0 ± 72.0

(mean ± SE), 126 (median)(63–76 years old subjects) 198 ± 49.8

(mean ± SE), 192 (median)

contin-ued


3006 CULEN ET AL.

Table 3. Continued.

Dosage Form (Density)/Size (mm) Ingested Water or MealGRT (Mean ± SD, min), If Not Stated

Otherwise

Tablet/19 × 6 × 9 (Ref. 46) 240 mL of water plus 100 mL ofClinutren R© ISO (420 kJ)

97.4 ± 44.6 (enteric coated)

91.3 ± 26.0 (nondisintegrating)Heidelberg capsule/20 × 7 (Ref. 40) 60 mL of water 114 ± 66Heidelberg capsule/20 × 4 (Ref. 75) Food: 16.7 kJ per kg of body weight 210 ± 48Osmotic pump Osmet/24 × 7 (Ref. 76) Light breakfast (1500 kJ) 183 ± 77 (mean ± SE)

Heavy breakfast (3600 kJ) Approximately 600Pellets/0.7–1.2 (Ref. 76) Light breakfast (1500 kJ) T50% = 119 ± 15 (mean ± SE)

Heavy breakfast (3600 kJ) T50% = 285 ± 45 (mean ± SE)Bravo R© pH capsule/25 × 5 × 6

(Ref. 62)150 mL of water 62 ± 21

150 mL of water; standard breakfastadministered 30 min post dose

160 ± 127

Tablet/8 (Ref. 62) 150 mL of water 63 ± 46150 mL of water; standard breakfast

administered 30 min postdose100 ± 109

SmartPill/26.8 × 11.7 (Ref. 50) 150 mL of water 92 ± 44 return to MMC phase III in 87 ±30 min

122 mL of water + meal (1067 kJ,2% fat)

261 ± 22 return to MMC phase III in239 ± 22 min

there is always the risk of the pellets being damagedby the pumps.15 In an apparatus with vessels similarto those in USP 1 and 2 apparatuses, the transfer ofsuch pellet filled bags would not constitute a problem.But of course, in a more sophisticated model such asTIM or DGM the implementation of these extra pelletenclosures may be technically more challenging.

Density. Density of solid objects is regarded as one ofthe parameters that may influence gastric emptying.There have been conducted many studies to clarifythis effect, but they have ended with opposite results.The positive effect of density on emptying of pellets

has been assessed by Devereux et al.,77 who founda significant difference between gastric emptying of1 mm pellets with densities of 1.5 and 2.8 g/cm3, andthat both in fasted and in fed states. Also, Clarkeet al.66 measured a statistically significant prolon-gation of GRT in case of higher density small pel-lets (2.6 g/cm3, Ø 0.5 mm, GRT50% = 141 min) com-pared with lighter pellets of the same size (1.5 g/cm3,GRT50% = 125 min), administered with orange juice.But here, the difference was not large, and the ef-fect of density was statistically insignificant in caseof large pellets (Ø 4.75 mm). Negative proof for the ef-fect of density was also confirmed in another study by

Table 4. Overall Summary of Reviewed GRTs for Nondisintegrating Dosage Forms and Solid Objects

Studied Objects Prandial State Range Mean Median

Single unit dosage formsor objects of all sizes

Fasted 38–114 65 62

Light meal ≤ 1600 kJ 90–306 172 180Heavy meal > 1600 kJ 249–600 436 459

Single unit dosage formsor objects ≤ 12 mm

Fasted 38–89 56 53


Single unit dosage formsor objects ≤ 19 mm

Fasted 39–89 55 52


Single unit dosage formsor objects ≥ 20 mm

Fasted 62–114 89 92

Light meal ≤ 1600 kJ 183–261 218 210Heavy meal > 1600 kJ 600 600 600

Pellets Fasted state 30–70 50 50Orange juice 117–179 140 133Light meal ≤ 1600 kJ 105–151 125 119Heavy meal > 1600 kJ 78–285 190 199



Clarke et al.,68 where they studied gastric emptyingof 1.2–1.4 mm pellets with three different densities(1.5, 2.0, and 2.4 g/cm3, all administered with orangejuice), and measured only very small and statisticallyinsignificant differences. Apart from pellets, Podczecket al.63 evaluated the effect of density on emptying of6.6 and 12 mm sized tablets in fasted state, but withpositive results. The smaller tablets had densities of1.41 and 2.85 g/cm3. The larger tablets were in twoversions, with densities of 1.5 and 3.7 g/cm3. The au-thors found that the GRTs were longer for tabletswith higher density, but at the same time, the sizeeffect has overridden the effect of density, that is, the12 mm tablets of 3.7 g/cm3 emptied faster than 6.6 mmtablets of 1.41 g/cm3. From these contradictory find-ings, it is obvious that the link between the densityof objects and their gastric emptying has yet to beproperly investigated.

In general, the gastric emptying is a very complexprocess with many variables, not all of which are clearso far. The following conclusions may be drawn. Theexpulsion of nondigestible solid objects was shown notto be solely dependent on phase III of the MMC cy-cle. There seems to be a universal agreement thatthe GRT prolongs with increasing energy content offood. No clear link between size, and density of objectsand their GRT was proven so far. The evidence tendsto show that pellets are not emptied simultaneouslywith ingested food or liquids.

To offer a simple summary of the results on GRT ofvarious nondisintegrating dosage forms and solid ob-jects reviewed in Table 3, we have divided these intoseveral groups according to the nature of the studiedobjects and the experimental conditions applied, andcharacterized these using basic parameters: range,mean, median. Only the mean values from Table 3were selected for this purpose. The fasted-state datainclude experiments performed with or without anywater. Because the energy contents of meals from allreviewed studies were either ≤1600 or ≥2146 kJ, allmeals below 1600 kJ were conveniently put into onecategory, labeled as light meals, and the rest was la-beled as heavy meals. Data without specified mealenergy content and without stating whether light orheavy meal were omitted. The results of this compari-son are shown below, in Table 4. The calculated meanand median values may also serve as an approximatereference for simulation aims.

SI Transit Times for Water and Food

The reviewed small intestinal transit times (SITTs)for food and water, in Table 5, range from 2.7 to 4.0 h.The information is important as any dosage formcontent disintegrated/dissolved in stomach is furthertransferred with the digested food or water.

Table 5. SITTs for Water and Meals

Ingested Water or MealTransit Time (Mean ± SD, h),

If Not Stated Otherwise

Water78 4.0 ± 0.8Solution79 3.0 ± 0.3Radiolabeled solution70 4.1 ± 0.5 (mean ± SE)Non-digestible solid fiberparticles79

2.7 ± 0.3

Solid food80 3.6 ± 0.3Solid food (2242 kJ)56 4.0 ± 1.4

SITTs for Dosage Forms

The transit of dosage forms through the whole SIwas stated in literature to last approximately 3–4 h,regardless of the dosage form type or the energeticvalue of the meal ingested.58,81 The dosage formspends only few seconds or minutes of this time induodenum, where a possible retropulsion to stom-ach may occur.82 Given the biological variability, itis natural that also other SITT values were reportedin literature. From the reviewed results (not includ-ing the prefeed regimes), only four measured valuesfall below the 3 h, with the lowest of them being 2.32h, and only three are higher than 4.2 h.83 In caseof two higher values of 5.7 and 8 h (median), mea-sured by a radiotelemetry method, the longer SITTswere suggested to be caused by inaccurate location ofthe radiotelemetry device or long intervals betweenthe measurements.84,85 Another higher SITT value of5.3 ± 1 h was determined using gamma-scintigraphyby Goodman et al.46 The authors accounted the longertransit for a combination of size of the tablet 19 × 6 ×9 mm and its higher density of 2.2 g/cm3. This is,however, disputable as an even larger Bravo R© cap-sule (25 × 5 × 6 mm) with a similar density (havingweight of 1.5 g) had SITT below 4 h.62 Another factorthat may have caused or contributed to these pro-longations of SITT, is stagnation at ileo-caecal junc-tion that was reported for both pellets and SU dosageforms.62,64,67,69,75 This period of stagnation in thesestudies could have been longer than in the majorityof others. The following text discusses other formula-tion and physiological parameters that according tothe reviewed works may affect the SITT of dosageforms.

Size. A positive effect of dosage form size on SITTwas reported for example by Clarke et al.,66 who foundthat pellets sized 0.5 and 4.75 mm had significantlydifferent SITTs (see Table 6). Another example of sizediscrimination was reported for a large Bravo R© cap-sule, which had prolonged residence compared withan 8 mm tablet.62 On the contrary, the size effectwas excluded in other studies which compared SITTof pellets and single-unit dosage forms.61,64,76 Here,


3008 CULEN ET AL.

Table 6. SITTs for Nondisintegrating Dosage Forms and Solid Objects

Dosage form (Density)/Size (mm) Prandial StateSITT (Mean ± SD, h), If Not Stated

Otherwise

Pellets67 Fasted 3.7 ± 1.6Breakfast (3000 kJ) 3.2 ± 1.1

Pellets/0.7–1.4 (Ref. 70) Light breakfast 3.4 ± 0.52 (mean ± SE)Pellets/1.18–1.14 (Ref. 69) Fasted 4.17 ± 0.48 (mean ± SE)

Breakfast (2240 kJ) 3.9 ± 0.4 (mean ± SE)Pellets/1.2–1.4 (Ref. 68) 200 mL of orange juice 4.1Pellets (1.5 and 2.4 g/cm3 pooled)/4.75

(Ref. 66)200 mL of orange juice 3.55

Pellets (1.5 g/cm3 pellets)/0.5 (Ref. 66) 2.73Pellets (2.6 g/cm3)/0.5 (Ref. 66) 3.1Tablet/5 (Ref. 71) Fasted 3.4 (median)

Standard breakfast (2146 kJ) 3.5 (median)Standard breakfast (2146 kJ)

ingested 0.75 h after the tablet2.35 (median)

5 Tablets/5 (Ref. 72) Fasted Day 1: 3.08 ± 0.48 (mean ± SE)Day 2: 3.45 ± 0.6 (mean ± SE)Day 3: 2.96 ± 0.85 (mean ± SE)

Tablet/8.2 (Ref. 73) Fasted 3.1 ± 0.7High-fat breakfast; other meals

administered after 4 and 10 h3.2 ± 0.4

Tablet/11.5 (Ref. 64) Light breakfast 3.03 ± 1.15Pellets/0.8–1.1 (Ref. 64) 3.48 ± 1.00

2 Tablets/12 (Ref. 60) Light breakfast (1500 kJ) 2.83 ± 0.50 (mean ± SE)Heavy breakfast (3000 kJ) 3.15 ± 0.85 (mean ± SE)

Tablet/16.4 × 7.3 × 5.1 (Ref. 45) Fasted 4.1 ± 0.25Capsule/16.8 × 4.6 (Ref. 28) Fasted 7 h after the capsule administration,

three of 12 capsules emptied tocolon

3 h after the capsule administrationa meal was administered (3381 kJ)

4 h after the capsule administration,five of 11 capsules emptied to colon

6 h after the capsule administrationa meal was ingested (3381 kJ)

7 h after the capsule administration,12 of 12 capsules emptied to colon

Tablet/17 × 4 (Ref. 74) Fasted (20–23 years old subjects): 3.0 ± 0.5(mean ± SE) 3.0 (median)

(63–76 years old subjects): 3.3 ± 0.74(mean ± SE) 3.1 (median)

Breakfast (1600 kJ) (20–23 years old subjects): 3.2 ± 0.56(mean ± SE) 3.0 (median)

(63–76 years old subjects): 3.4 ± 0.56(mean ± SE) 3.1 (median)

Tablet/19 × 6 × 9 (Ref. 46) Clinutren R© ISO (420 kJ) 5.33 ± 1Osmotic pump Osmet/24 × 7 (Ref. 76) Light breakfast (1500 kJ) 3.18 ± 0.40 (mean ± SE)Pellets/0.7–1.2 (Ref. 76) Light breakfast (1500 kJ) 3.13 ± 0.46 (mean ± SE)

Heavy breakfast (3600 kJ) 3.36 ± 0.46 (mean ± SE)Bravo R© pH capsule/25 × 5 × 6 (Ref. 62) Fasted 3.75 ± 1.67

0.5 h after the capsuleadministration a standardbreakfast was administered

2.83 ± 1.37

Tablet/8 (Ref. 62) Fasted 2.32 ± 1.020.5 h after the capsule

administration a standardbreakfast was administered

1.77 ± 0.72

Radiotelemetry capsule (Ref. 83) Fasted 5.7

the differences were either minimal or statistically in-significant. The option that the SITT is not influencedby the size of the objects involved also correlates withthe results from various studies shown in Table 6. Thereviewed transit times tend to be very similar fallingin the aforementioned range of 3–4 h irrespective ofthe dimensions of the objects.

Density. The effect of density on prolongation ofSITT was reported by Clarke et al.,66 but only in thecase of smaller pellets (0.5 mm). The SITT of largerstudied pellets (4.75 mm) was unaffected. The effectof density on SITT of 1 mm pellets (1.5 and 2.8 g/cm3)was not confirmed in a study by Devereux et al.77

Thus, unless a sound evidence for the effect of density



is provided, it is not possible to admit that density isa factor of influence here.

Food. As already mentioned, the SI transit time doesnot seem to be affected by prandial state (whetherfasted or fed) nor by the energetic value of food. How-ever, based upon the evidence from some studies,it may be affected by meals ingested only after theadministration of dosage form. For example, Faddaet al.71 measured the SITT for a 5 mm tablet to last204 and 210 min in fasted and fed state, respectively.However, in a prefeed regime (tablet administered 45min before the breakfast) the median SITT lasted only141 min. Moreover, those tablets that emptied beforethe meal administration (i.e., in less than 45 min)had a median SITT of only 100 min. The rest of thetablets that left stomach after the arrival of the foodhad a “normal” SITT of 185 min. These results showthat after ingestion of food, the SI tries to quicklyempty itself and make space for the next meal, thusthe tablets that were already in the SI when the foodwas ingested experienced a faster SI transit.28 Quitesimilar results were also obtained by Ibekwe et al.,62

who measured SITT of an 8 mm tablet to be 139 ± 61min in fasted state, 144 ± 78 min in fed state, and 106± 43 under prefeed conditions (tablet administration30 min before meal). Confusing is the fact that a largeBravo R© capsule had also shortened SITT in this study,in the prefeed regime (SITT in fasted state 225 ± 100min; 170 ± 82 min in prefeed regime), but it emp-tied from stomach only 2 h later after the ingestionof the “prefeed meal.” It is clear that the mechanismproposed in the previously mentioned study was notthe cause here, and the reason for the accelerated SItransit is unknown. Otherwise, the shortening of SItransit after ingestion of food was also described in astudy by Schiller et al.28 Taking the aforementionedinto consideration, there is one thing worth giving athought. In most of the studies on GI transit the sub-jects are offered a lunch, usually at 4 h after the startof the experiment, and also other snacks are takenat later time points. Therefore, given that the small

bowel transit is accelerated by following meals, thereemerges a question, whether all the measurementswere not influenced by those following lunches andsnacks. To the authors’ knowledge, the answer wasso far provided only by Khosla et al.,72 who found norelationship between ingestion of a lunch at 4 h afteradministration of five 5 mm tablets and the SITT ofthese tablets.

It is obvious that there is currently no clear evi-dence supporting the effect of any of the discussedparameters on the SI transit, with the exception ofthe influence of prefeed regime. Also, despite the re-ported shorter and longer transit times, the vast ma-jority of the reviewed SITTs (see Table 6) still fallwithin the earlier proposed range of 3–4 h. Thus, forsimulation purposes this is the applicable value. Theminority of deviating transit times may be useful forthe study of processes occurring in the small bowel,but from a simulation view they are not significantunless observed in many more studies.

pH

pH in Fasted State

The pH values for stomach and individual parts of SIin fasted state are reviewed in Table 7. The valuescorrespond well to the work by Dressman et al.,86

which stated that the pH value for in vitro dissolutionin fasted stomach should be set between 1.5 and 2.0.This also agrees with statements of other authors.87

A gender difference in gastric pH was reported byFeldman et al.,97 who measured a mean pH of 2.16in 252 men, and an average pH value of 2.79 in 113women. On the contrary, no such difference was ob-served by Russell et al.,98 on a smaller group of 79elderly patients. An existing gender difference in gas-tric pH could be important for dissolution tests ofdrugs intended for patients of only one sex (e.g., hor-monal contraceptives, drugs for treatment of benignprostatic hyperplasia, etc.).

When simulating the gastric conditions, it must bealso considered that in a BA/BE study the dosage

Table 7. pH Values for GIT in Fasted State (Mean ± SD)

Stomach Duodenum Jejunum Ileum

1.4 ± 0.4 (Ref. 62) 5.7 (Ref. 88) 6.6 ± 0.5 (Ref. 83) 6.5 (Ref. 86)1.4 ± 0.5 (Ref. 33) 5.8 ± 0.8 (Ref. 75) 6.6 (Ref. 86) 7.2 ± 0.4 (Ref. 62)1.7 (median)89 5.9 (median)90 6.7 (Ref. 91) 7.3 ± 0.7 (at ileocaecal junction)75

1.8 (median)92 6.1 (median)89 6.8 ± 0.3 (Ref. 62) 7.4 (Ref. 86)2.8 ± 1.1 (Ref. 91) 6.2 (median)87 6.8 ± 0.4 (Ref. 93) 7.5 ± 0.5 (Ref. 83)1.0–2.5 (Ref. 83) 6.5 ± 0.3 (Ref. 62) 6.8 (median)90 7.5 (median)90

6.5 (Ref. 94) 4.4–6.5 (Ref. 86) 6.8–8.0 (Ref. 86)6.7 ± 0.9 (Ref. 91)6.7 (Ref. 95)6.84 (median)96

7.0 ± 0.4 (Ref. 93)7.2 (Ref. 92)


3010 CULEN ET AL.

forms are administered with water, in most cases withvolume around 200–250 mL. This amount of water di-lutes the gastric juices and results in an increase ingastric pH. Kalantzi et al.87 measured pH value of 2.4at 20 min after administration of 250 mL of water. ThepH decreased to 1.7 in around 60 min. The simulationof this pH decrease would be probably unnecessarywhen considering that the gastric emptying half-timefor water is around 15 min and the whole additionalvolume (e.g., 250 mL) should be out of stomach inaround 30 min. Therefore, it would be sufficient if thepH would be kept at a value around 2.5 the wholetime.

On the basis of the data from Table 7, the pHin a simulated duodenum could be set to a valuearound 6.5, and for jejunum the approximately rangeof 6.6–6.8 can be considered representative. HigherpH was then measured in ileum, where the averagevalue would be slightly above 7.

pH in Fed State

After a meal, the original gastric content is diluted byfood. As the volume of food which is usually given inBE studies is much greater than the initial volume ofthe fluids in fasted stomach, it is logical that the gas-tric content then acquires similar pH value as the foodalone. Youngberg et al.88 found that after a standardmeal (4184 kJ) the pH increased to a value around 5.In 60 min, the pH was around 3, and it further de-creased to 2, and lower after almost 2 h. The samestandard meal was administered in a study by Dress-man et al. where the initial postprandial pH was also5, decreased to 3 in 56 min, and reached value of 2 af-ter 107 min.89 Evan higher pH values were observedafter administration of 500 mL of Ensure Plus R© (3138kJ).87 In 30 min after administration, the pH was 6.4(pH of Ensure Plus R© is 6.6). In 210 min, it decreasedto 2.7 (see Fig. 2).

Figure 2. Gastric pH after ingestion of 500 mL of EnsurePlus R©. (Reedited according to Ref. 84.)

In SI, the pH postprandially slowly decreases be-cause of the influx of the gastric contents.99 The val-ues measured in duodenum after administration ofEnsure Plus R© are shown in Table 8. The pH after in-gestion of Ensure Plus R© and Scandishake Mix R© wasreported to return to the initial fasted-state valuesafter 300 min.99

Osmolality

In fasted state, both the gastric and the duodenalcontents are hypotonic, whereas the jejunal contentreaches isotonic range (see Table 9). Twenty minutesafter ingestion of 250 mL of water, osmolality in anaspirated gastric content reached 98 mOsm/kg andafter 40 min it stabilized at around 140 mOsm/kg.87

In fed state, 30 min after ingestion of 500 mL ofEnsure Plus R©, the osmolality in the stomach wasmeasured to reach 559 mOsm/kg, and it graduallydecreased to 217 mOsm/kg at around 210 min.87

The osmolality in pooled duodenal samples, 30 minafter intake of 500 mL of Ensure Plus R©, was 408mOsm/kg.95 It further rose to 416 mOsm/kg at 60 minand reached the value of 453 mOsm/kg at 120 min.At 180 min, there was a decrease to 368 mOsm/kg.95

Similarly, Kalantzi et al.87 measured the duodenalcontent to be hyperosmotic after ingestion of 500 mLof Ensure Plus R© with the decrease to isosmotic val-ues in 210 min. Similar trend was also observed byClarysse et al.,99 when at 60 min after ingestion of 400mL of Ensure Plus R© the osmolality showed maximumof 516 mOsm/kg, after which it gradually decreased.In the same study, the osmolality maximum of 619mOsm/kg was observed at the very beginning of thesampling period after ingestion of a fat enriched Scan-dishake Mix R©. The decline in osmolality was steeperin this case and ended at around 120 min.99

Data on postprandial osmolality in jejunum andileum, as well as data on postprandial osmolality afterstandard meals are lacking. The starting osmolalityof a physiologically relevant medium should automat-ically fall in the physiological range. In fasted state,the osmolality at later time points will also reflect thecomposition of the medium and should not be hardto maintain at a desired level. In fed state, the laterosmolality values will be largely dependent on thesimulated digestion process and the present sink con-ditions. Thus, here, it will be much harder to simulatethe physiological time profile.

Surface Tension

Lowering of surface tension markedly increases thesolubility of compounds. In biorelevant dissolutionmethods the specific physiological levels should bemaintained through the already contained enzymes,BS, and phospholipids (all these are discussed in sep-arate sections below). The use of unphysiological sur-factants is discouraged. The reported surface tension



Table 8. pH Values Measured Postprandially in Small Intestine at Various Time Intervals

Ingested Liquid Meal Fasted State 30 min 60 min 120 min 180 min 210 min

Ensure Plus R© 500 mL95 6.7 6.6 6.5 5.8 4.9Ensure Plus R© 500 mL87 6.2 (median) 6.6 (median) 5.2 (median)

values for stomach and duodenum, in both fasted andfed states, are listed in Table 10. The postprandialsurface tension was reported to show some fluctua-tion but without any specific trend.87

Buffer Capacity

Physiological buffer capacity values in fasted stom-ach, 20 min after ingestion of 250 mL of water, weremeasured to be 7 mmol/(L �pH), and they stabilizedat around 18 mmol/(L �pH) at 40 min and later.87

After ingestion of 500 mL of Ensure Plus R©, the buffercapacity was raised to approximately 14–28 mmol/(L�pH).

In fasted duodenum, the buffer capacity was re-ported to be 5.6 mmol/(L �pH).95,87 After ingestionof 500 mL of Ensure Plus R©, the duodenal buffer ca-pacity acquired values in the ranges of 18–30 mmol/(L �pH) and 22–30 mmol/(L �pH), without any cleartime-dependent trend.95,87

In a biorelevant medium, the buffer capacity willnot only be determined by concentration of a spe-cific buffer (preferably bicarbonate), but it will be alsoheavily influenced by the components such as BS andenzymes, and of course by food in fed state. Moreover,it is arguable whether it is necessary to maintain anyspecific level of buffer capacity in an apparatus withautomatically controlled and constantly adjusted pH,for example, TIM-1.

Pepsin

The main importance of pepsin in biorelevant dissolu-tions is the postprandial proteolysis of proteins fromfood. In fasted-state pepsin is inactive, as it requirespH of around 3–6 to function, but it represents a phys-iological agent for lowering the surface tension.100

This helps in avoiding the use of non-physiologicalsurfactants, such sodium lauryl sulfate, TWEEN, orothers.

Pepsin levels in resting fasted gastric fluid were re-ported to fall in the range of 0.83–1.27 mg/mL.87 How-ever, 20 min after ingestion of 250 mL of water thepepsin concentration was measured to be only 0.11

mg/mL, and it rose to 0.22 mg/mL at 60 min.87 In fedstomach, the pepsin concentration ranged between0.26 and 0.58 mg/mL, during the whole experiment87

The recommended pepsin level for dissolution ofgelatin capsules in a two-tier test (with and with-out enzymes) was assessed as ≤750,000 units (3.2g)/1000 mL, and this method was also included intoUSP.101 The concentration of 3.2 mg/mL is, however,one order higher than the above reported values of0.11–0.58 mg/mL. Also, Digenis et al.102 later reportedthat this value could successfully predict BE of mod-erately cross-linked gelatin capsules but it may failfor highly cross-linked capsules, where lower concen-trations may prove more discriminatory.

Bile Salts

BS in Stomach

Bile salts play an important role as a physiological sol-ubilizing agent. Their presence in stomach originatesfrom duodenal reflux. As can be seen in Table 11, theirlevels may reach almost 1 mM. However, Kalantziet al.87 assumed that after administration of water,the reflux should be suppressed by the flow of the liq-uid out of stomach, and that may be the reason whythe higher levels around 1 mM were measured dur-ing fasted resting conditions in older studies. Also, the250 mL of water administered with the dosage formwas found to dilute the BS concentration to around0.1 mM. In this concentration, the BS have proba-bly only small effect on reduction of surface tension,and therefore the need for their presence in simulatedfasted gastric environment is questionable.

BS in Small Intestine

The measured BS levels in fasted SI ranged from 2.6to 6.4 mM (see Table 11). This range was assumed torepresent the fasted state also by Soderlind et al.105

The authors also found that in this concentration thestructure of BS had small effect on solubility of neu-tral compounds, and that one type of BS is sufficientto represent the in vivo conditions; with taurocholic

Table 9. Osmolality Values Measured in Fasted State for Stomach, Duodenum, andJejunum (Mean ± SD, If Not Stated Otherwise)

Stomach (mOsm/kg) Duodenum (mOsm/kg) Jejunum (mOsm/kg)

196 (median)92 137 ± 54 (Ref. 93) 200 ± 68 (Ref. 93)221 ± 15 (Ref. 91) 178 (Ref. 87) 273 (median)92

197 (Ref. 95) 278 ± 16 (Ref. 91)224 (Ref. 5)


3012 CULEN ET AL.

Table 10. Surface Tension Values for Stomach and Duodenum

Fasted Stomach (mN/m) Fed Stomach (mN/m) Fasted Duodenum (mN/m) Fed Duodenum (mN/m)

41.9–45.7 (after 250 mLof water)87

30–31 (after 500 mLof Ensure Plus R©)87

32.3 (Ref. 87) 28.1–28.8 (after 500 mLof Ensure Plus R©)87

33.6 (Ref. 91) 33.6 (Ref. 95) 27.8–28.6 (after 500 mLof Ensure Plus R©)95

(TC) acid and glycocholic (GC) acid being the bestcandidates, as they are the most present in the hu-man intestine.105 Perez de la Cruz Moreno et al.93

also measured these two bile acids to be the mostabundant. They assessed the actual distribution of in-dividual BS/acids in duodenal aspirates to be: TC 42± 17%, GC 16 ± 4%, sodium glycochenodeoxycholate(GCDC) 16 ± 10%, sodium glycodeoxycholate (GDC)12 ± 8%, sodium taurochenodeoxycholate (TCDC) 8 ±2%, sodium taurodeoxycholate (TDC) 5 ± 3%, cholicacid (C) 0.5 ± 0.4%, sodium tauroursodeoxycholate(TUDC) 0.3 ± 0.2%, chenodeoxycholic acid (CDC) 0.3± 0.4%, and deoxycholic acid (DOC) 0.3 ± 0.2%. In je-junal aspirates, the percentage distribution was: TC48 ± 18%, GC 19 ± 7%, GCDC 12 ± 8%, GDC 8 ± 5%,TCDC 8 ± 2%, TDC 4 ± 2%, C 0.3 ± 0.2%, TUDC 0.3± 0.2%, CDC 0.1 ± 0.06%, and DOC 0.08 ± 0.03%.

On the basis of these findings, one may assumethat 3–6 mM of TC would be appropriate for simu-lation of fasted state in duodenum and jejunum. Thesame BS concentration could be possibly set also forileum, which is the active reabsorption site for BS,unlike jejunum, where the BS transport is passive. Ingeneral, the reabsorption saves approx. 95% of all BSfrom chyme.106

After a meal, the BS concentration in SI increases.Table 12 comprises duodenal values measured afteringestion of commonly used liquid meals. It can beseen that at 30–60 min after the introduction of aliquid meal, the BS concentration reaches approxi-mately 11 mM, and it slowly starts to fall at the latertime points.

The determination of a suitable BS concentrationfor a dynamic in vitro dissolution system, however,is not that simple, as the BS also play an importantrole in digestion of lipids. As Mac Gregor et al. as-sumed, there is a sink condition for lipolytic prod-ucts in the SI; mainly for free fatty acids (FA) and

2-monoglycerides (MGC).107 FA and MGC bind BSthrough formation of mixed micelles, and thus de-crease the amount of free BS. Lipolytic products are,however, continuously absorbed by the intestinal walland are removed from the micelles, thus liberating BSback to the intestinal juice. This way, a small amountof BS is enough to complete the whole lipolysis.107

Nevertheless, the simulation of such sink conditionsmay prove challenging. In TIM-1, the removal of BSwas maintained by dialysis of chyme against a solu-tion with lower BS concentration.21 Otherwise, cre-ation of a selective removal only for BS may provevery difficult, and therefore the dialysis seems to bethe only option so far. If the sink conditions are notincluded, the optional way to complete the whole lipol-ysis is to use unphysiologically high amounts of BS.Mac Gregor et al.107 used concentration of 260 mM,which raised the lipolysis of medium chain triglyc-erides to 63% in half an hour compared with 8% and24% achieved with 8 mM and 32 mM BS concentra-tions, respectively. Still, after this period the lipolysisrate represented a plateau even despite of the 260 mMconcentration. Another problem to be expected withsuch unphysiologically high BS concentrations is adramatically increased solubility of mainly lipophiliccompounds. As Mithani et al.108 have found, the sol-ubility of such compounds linearly increases with theBS concentration, once it is above the critical micelleconcentration. In this respect, it must be mentionedthat 1 mol of BS was found to solubilize 1 mol of lipoly-tic products.109 The type of BS used was found not tohave effect on the lipolysis.107

To propose the correct BS concentrations for dy-namic simulations, it is clear that their effect on disso-lution results must be evaluated simultaneously withthe effect of lipolytic products and the general extentof lipolysis. Currently, studies dealing with this wholeissue would be very helpful.

Table 11. BS Concentrations for Stomach, Duodenum, and Jejunum in Fasted State (Mean ±SD, If Not Stated Otherwise)

Stomach (mM) Duodenum (mM) Jejunum (mM)

0.08 (Ref. 103) 2.6 (Ref. 93) 1.52 ± 1.77 (Ref. 91)0.08 (60 min after 250 mL of water)104 2.6 (Ref. 87) 2.1 (median)92

0.1 (median)92 2.6 (median)99 3.5 (Ref. 93)<0.5 (Ref. 87) 2.82 (Ref. 95) 3–6 (Ref. 86)0.82 ± 0.57 (Ref. 91) 4.6–6.4 (Ref. 86)



Table 12. BS Concentrations for Duodenum in Fed State Measured at Various TimeIntervals

Fasted State 30 min 60 min 90 min 120 min 180 min

Ingested Liquid Meal (mM)

Ensure Plus R© 500 mL95 2.82 10.7 11.8 6.62 3.72Ensure Plus R© 500 mL87 11.2 5.2Ensure Plus R©104 9.211Ensure Plus R© 400 mL99 2.6 8.3Scandishake Mix R© 300 mL99 2.6 11.9

Phospholipids

Phospholipids play an important role in dissolutionmedia, as they are used as physiological surfactants,and can greatly influence solubility. For fasted duode-num, the phospholipid concentration was reported tobe in range of 0.03–0.15 mM, but a higher value of 0.6mM was also reported.99,110 As a more useful informa-tion may serve the findings of Soderlind et al.,105 whoperformed solubility studies of 24 model compounds,and determined that 0.2 mM phospholipid concentra-tion used in the newer FaSSIF V2 better reflected thedissolution in aspirated human intestinal fluid thanthe higher 0.75 mM concentration used in the olderFaSSIF V1. Therefore, the concentration of 0.2 mMrepresented solely by commonly used lecithin wouldbe recommended as optimal.

In fed state, after intake of 500 mL of Ensure Plus R©,the pooled phospholipid concentrations were higherthan in the fasted state and were found to decreasein time.95 At 30 min after the meal, the phospholipidlevel was 5.77 mM. In 60 min, it was 4.31 and de-creased further to 2.99 mM in 120 min. In 180 min,the concentration was below the level of quantifica-tion (0.01 mM). Somewhat lower fed state phospho-lipid concentrations were chosen for early, middle, andlate FeSSIF; these were 3, 2, and 1 mM, respectively.2

Although the values from the simulated media may betaken as more relevant in this case, a more thoroughinvestigation of fed state phospholipid concentrationsand they role on solubility of compounds would be inplace.

Pancreatic Enzymes

Lipase

Jantratid et al.2 found that the postprandial lipaselevels reported in literature ranged in the interval of500–1000 U/mL. However, in aspirated fed-state in-testinal fluid, the lipid concentration in the micellarphase form was not more than 70 mM. Thus, it wassuggested that only 70 U/mL would be sufficient tocomplete the lipolysis, and the suggested optional ad-dition of pancreatin to the FeSSIF V2 medium was100 lipase units per milliliter plus CaCl2 in concen-tration of 5 mM.2 Lipase activities ranging in the in-

terval of 163–300 U/mL were reported by Zangen-berg et al.,109 who also stated that these levels exceedthe physiological requirements for the lipolysis rate.Other than that, Mac Gregor et al.107 found that thelipase activity had only minor effects on the lipoly-sis rate compared with Ca2+ and BS concentrations.Therefore, the previously suggested lipase activity of100 U/mL would be recommended.

Besides the evaluation of enzymatic activity permilliliter, also the enzymatic output was reported.Keller et al.111 stated the average secretion of lipasein fasted state to be around 1000 U/min. This meansthat the coadministered 250 mL of water, if emptiedfrom stomach in 30 min, will acquire 30 kU of li-pase activity in duodenum, which is more than 1000U/mL. In fed state, the heavier secretion will resultin even higher activity per milliliter. Postprandially,the maximal secretion of approximately 3000–6000U/min was measured between 20 and 60 min afteringestion of a meal with at least around 500 kcal. Se-cretion of around 2000–4000 U/min was determinedfor later postprandial time points with a return to thebasal secretion at the end of the digestion process.111

Amylase

The enzymatic activity of amylase in duodenum wasfound to be around 100–400 U/mL in fasted state and150–300 U/mL during the whole fed state.111 Amylaseoutput in fasted state was summarized to be rang-ing approximately from 50 to 250 U/min.111 In fedstate, the maximum secretion was put to be around500–1000 U/min, with possible highest values being2000 U/min. The later secretion was stated to be ap-proximately 500 U/min.

Trypsin

Duodenal enzymatic activity of trypsin betweenmeals was stated to range around 20–50 U/mL, ris-ing to 80–180 U/mL early after a meal. The later lev-els were around 60–100 or even 150 U/mL.111 Thevalues for trypsin secretion gathered from variousstudies were stated to range from less than 20 to1000 U/min in fasted state, with the most commonfindings to be around 50–100 U/min. The maximalpostprandial secretion was evaluated to range from


3014 CULEN ET AL.

200 to 1000 U/min, and the later plateau output wasfound to range from 150 to 500 U/min.

Ca2+

The Ca2+ in chyme is required for pancreatic lipaseactivity. It is also reported to form soaps with productsof lipolysis, thus removing FA from oil–water inter-face and enabling the completion of the in vitro lipol-ysis. The median Ca2+ concentration in fasted stom-ach was found to be 0.6 mM. In fasted duodenum, thevalues ranged between 0.5 and 3 mM, and in fasted je-junum the median concentration was only 0.3 mM.92

In fed stomach and duodenum, the Ca2+ concentra-tion naturally depends on the ingested food.109 There-fore, the Ca2+ concentration for fed state has beenmainly evaluated in regards to the actual lipolyticactivity. For example, a 20 mM Ca2+concentration,along with 6.6 mM of BS, was used to simulatelipolysis of soybean oil, and the process was com-plete after 40 min.109 On the contrary, Mac Gregoret al.107 argued against the Ca2+ driven lipolysis,and stated that FA-Ca2+ soap formation was slow,and preferred high BS concentration for the suffi-cient lipolysis drive. Still, both the unphysiologicallyhigh BS or Ca2+ concentrations and the resulting ex-cess amounts of unabsorbed formed soaps may haveunpredictable effects on the whole dissolution pro-cess and solubility of the tested compounds. In thislight, the better options seem to be either to proceedwith lower physiological concentrations of both com-ponents and sacrifice the completion of lipolysis, or toemploy an absorption step and create sink conditionsfor the lipolysis products. All these options currentlyrequire further research and comparison with deter-mine which provides the best prediction of in vivoresults.

CONCLUSIONS

It seems that the main problem of biorelevant dy-namic dissolutions today are not the missing piecesof knowledge of GIT, but the current availability ofappropriate instruments and the general reluctanceto use more complex media and methods. Many disso-lution tests are still performed on classical USP appa-ratuses using simple buffer solutions, while trying nu-merous conditions to gain the full picture, which couldbe otherwise obtained using only few or just one fullybiorelevant test. The solution to this problem could bean introduction of a middle category apparatus, onethat will similarly as TIM-1 provide an accurate sim-ulation of GIT, while being at the same time simpler,easier to maintain, and much more affordable thanTIM-1. This could increase the popularity of biorele-vant dissolutions and speed up their progress.

The second and main part of this article, discussingthe functioning of the human stomach and SI, showed

that there are still certain areas that would requirefurther study. For example, the description of jejunaland ileal contents is poor compared with the knowl-edge available on duodenum, which is understand-able as the duodenum is better accessible. Also, thereis missing a complex description of postprandial GIfluids after ingestion of standard meals, as most ofthe currently available data originate from studiesusing liquid nutrition drinks. These are of course alsovery valuable as the dissolutions are easier to performusing nutrition drinks, but it would still be beneficialto compare them with in vivo fed state data for stan-dard meals. Other than that, the dynamic simulationswould benefit from more extensive study of enzymaticdigestion processes and their connection with eitherincluded or absent sink conditions, and of course theeffect of these on prediction abilities of correspondingdissolution methods.

ACKNOWLEDGMENTS

This study was supported by IGA VFU Brno, ProjectNo. 84/2012/FaF.

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