solubility estimation in git

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Estimating drug solubility in the gastrointestinal tract

J.B. Dressmana, M. Vertzoni b, K. Goumas c , C. Reppasb ,

a Department of Pharmaceutical Technology, Johann Wolfgang Goe the University, Frankfurt, Germanyb Department of Pharmaceutical Technology, National & Kapodistrian University of Athens, Greece

c Department of Gastroenterology, Red Cross Hospital of Athens, Greece

Received 23 April 2007; accepted 10 May 2007

Available online 29 May 2007

Abstract

Solubilities measured in water are not always indicative of solubilities in the gastrointestinal tract. The use of aqueous solubility to predict oral

drug absorption can therefore lead to very pronounced underestimates of the oral bioavailability, particularly for drugs which are poorly soluble

and lipophilic. Mechanisms responsible for enhancing the luminal solubility of such drugs are discussed. Various methods for estimating intra-

lumenal solubilities are presented, with emphasis on the two most widely implemented methods: determining solubility in fluids aspirated from the

human gastrointestinal tract, and determining solubility in so-called biorelevant media, composed to simulate these fluids. The ability of the

biorelevant media to predict solubility in human aspirates and to predict plasma profiles is illustrated with case examples.

2007 Published by Elsevier B.V.

Keywords: Solubility; Gastrointestinal tract; Humans; Dogs; Simulated media; Oral absorption

Contents

1. Why estimate intra-lumenal solubility of drugs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591

2. Solubilization mechanisms in the gastrointestinal tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592

3. Procedures for aspirating and measuring solubility in gastrointestinal fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

4. Estimation of drug solubility in the stomach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

4.1. Fasted state, gastric . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

4.2. Fed state, gastric . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

5. Estimation of drug solubility in the small intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597

5.1. Fasted state, small intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597

5.2. Fed state, small intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598

6. Use of drug dissolution and solubility data in predicting plasma profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599

7. Conclusions future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601

1. Why estimate intra-lumenal solubility of drugs?

After oral administration, intra-lumenal drug concentrations

influence the rate of appearance in plasma and, in certain

situations they can determine the total amount reaching the

general circulation. In turn, the solubility under gastrointestinal

(GI) conditions sets the upper limit to the intra-lumenal

concentration that can be achieved.

Advanced Drug Delivery Reviews 59 (2007) 591602

www.elsevier.com/locate/addr

This review is part of the Advanced Drug Delivery Reviewstheme issue on

Drug solubility: How to measure it, how to improve it". Corresponding author. Department of Pharmaceutical Technology, National

& Kapodistrian University of Athens, Panepistimiopolis, 15771 Zografou

Athens, Greece. Tel.: +30 210 727 4678.

E-mail address: [email protected](C. Reppas).

0169-409X/$ - see front matter 2007 Published by Elsevier B.V.doi:10.1016/j.addr.2007.05.009
mailto:[email protected]://dx.doi.org/10.1016/j.addr.2007.05.009http://dx.doi.org/10.1016/j.addr.2007.05.009mailto:[email protected]


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The stomach, although not the primary site for drug

absorption, provides the first site at which an orally administered

formulation can quantitatively release its drug. For compounds

highly soluble at gastric pH, complete dissolution can occur in the

stomach. For such compounds, gastric emptying may well limit

the subsequent rate of absorption from the small intestine (e.g.

[1]). For poorly soluble weak acids like ibuprofen [2] littledissolution will occur in the stomach. By contrast, the small

intestine with its higher pH offers a more favorable environment

for dissolution of acids. For ibuprofen and similar weak acids,

emptying from the stomach becomes rate limiting to the onset of

dissolution and hence absorption. For poorly soluble neutral

compounds, dissolution will be slow in the gastric region and in

many cases will not be complete before the drug reaches the first

absorptive sites in the small intestine. Incomplete dissolution in

the GI tract of such compounds can severely restrict their oral

bioavailability. Finally, for poorly soluble weak bases, solubility

is likely to be higher in the (preprandial) stomach than elsewhere

in the GI tract. This can result in a supersaturation as the drugmoves out of the stomach into the higher pH small intestine.

Precipitation in the small intestine may result, though this process

appears to be hindered by the bile components[3].

In the fed state, the intra-gastric performance of immediate

release tablets has to date been studied primarily in dogs. In

these experiments, food components have been shown to delay

the dissolution of highly soluble compounds during gastric

residence [4,5]. Similar observations have been made very

recently in humans by Brouwers et al. [6]. In this study a food-

induced delay in the dissolution of fosamprenavir in the fed

stomach was reflected in changes in the plasma profile of

amprenavir[6].

In the small intestine, the drug concentration at the intestinalwall,Cw, is one of the two principal determinants of the rate of

drug uptake and transport across the cellular membrane of the

intestinal epithelium, the other being permeability. Depending

on the mechanism of transport, the drug flux through the

intestinal mucosa, J, can be described with the following

equations:

For passive transport : JCWPW 1

For carrier mediated transport : J JmaxCW

CWKM2

where Pw is the effective membrane permeability coefficient,

Jmaxis the maximum drug flux through the membrane and KMis the MichaelisMenten constant. Since the small intestine is

the primary site of absorption for the great majority of

compounds, the concentration of interest for the calculation of

flux is the one developed in this region. Intra-intestinal drug

concentrations after oral administration of drug powder were

first measured about ten years ago[7]. Recently, intra-intestinal

drug concentrations have been measured after oral drug

administration using procedures that do not perturb the natural

fluid balance and composition in the region substantially and,

thus, enable administration of marketed dosage forms and/or

typical meals[6,8,9,10]. These studies have proved very useful

in answering detailed questions about factors important to

absorption of several drugs. For example, it appears that in the

fed state concentrations of danazol in the aqueous phase of the

intestinal contents may not correlate with blood levels (despite

the limited aqueous solubility of this compound) [10], that

amprenavir's permeability from the marketed formulation is not

influenced by p-gP effects [9], and the delayed absorption offosamprenavir in fed state is not related to intra-intestinal but

rather to intra-gastric processes[6].

Drawbacks with direct measurements of intra-gastric or

intra-intestinal drug concentrations are the specialized proce-

dures used, the associated costs and ethical issues in terms of

exposing humans to the procedure and drugs without any direct

therapeutic benefit to the subject. As a result, data are usually

collected from just a limited number of subjects and studies

reported in the literature are few [6,8,9,10]. One way to

eliminate some of these drawbacks would be to use imaging

techniques. However, such techniques would also be expensive

to apply and, at best, they are still in their infancy with regard tothis application[11]. Another way to improve the cost:benefit

ratio of the experiments is to collect human aspirates without

prior administration of the drug and use them for measuring

the parameters of interest. Analogously, intestinal permeability

in humans is usually performed off-sitein cell cultures rather

than directly in human subjects. Interestingly, it has recently

been established that using human aspirates (containing the

drug and formulation excipients) as the medium for permeabil-

ity studies can lead to surprisingly different results than when

permeability is measured from solutions consisting of simple

buffers[9]. Similarly, it is expected that determining solubilities

in human aspirates will help us to estimate intra-lumenal

dissolution kinetics much better than studying solubility insimple buffer solutions (e.g.[12]).

Another reason that estimation of intestinal permeability and

intra-lumenal solubility has become of major interest during the

last decade is that both of these parameters are required for

application of the Biopharmaceutics Classification Scheme

principles[13] to drug development.

This article discusses the procedures necessary to obtain

reliable results for solubility in human aspirates and additionally

discusses whether the composition of the GI fluids can be

simulated with media that can be collected from animals or

manufactured in the laboratory.

2. Solubilization mechanisms in the gastrointestinal tract

Although the underlying driver for solubility in the GI fluids

is the aqueous solubility of the drug, the solubility in the GI tract

may additionally be influenced by the pH profile, by

solubilization via naturally occurring surfactants and food

components, as well as by complexation with food and native

components of the GI milieu. Since these additional influences

can result in orders of magnitude changes in solubility (see

Table 1for some examples), it is worthwhile addressing them in

some detail.

The pH profile in the GI tract is of primary importance for

drugs that can ionize in this range. Rearranging the Henderson

592 J.B. Dressman et al. / Advanced Drug Delivery Reviews 59 (2007) 591602


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Hasselbalch equation (e.g. [17]) we see that for a monobasic

compound the concentration of acid required for saturation of

the medium will be enhanced at pH values where ionizationoccurs:

Cs Cs;0 110pHpka

3

where Cs,0 is the solubility of the non-ionized acid form

(intrinsic solubility) and Cs is the total solubility (sum of

intrinsic solubility and the existing concentration of ionized

form) at the pH of interest. For a monoacidic compound the

equivalent equation is:

Cs Cs;0 110pkapH

4

Equations for more complex ionization reactions can be foundelsewhere in this issue[18].

The pH in the stomach in the fasted state has been the subject

of many studies over the years and the general consensus is that

in healthy adult humans the fasted pH usually lies in the range

pH 13. Elevated pH can be observed in a modest percentage of

elderly subjects due to waning ability to produce gastric acid.

The effect is particularly pronounced in the Japanese popula-

tion, although the incidence of achlorhydria there does appear to

be falling with time[19]. In North Americans, elevated pH in

the elderly is the exception rather than the rule [20]. On the

other hand, gastric pH can be elevated by pharmacological

interventions such as H2-receptor antagonists and proton pumpinhibitors, which are used widely in Western populations. By

contrast, hyper-secretion of acid is very rare, mostly associated

with specific diseases such as ZollingerEllison syndrome[21].

After meal intake, pH in the stomach usually rises due to

buffering effects of the meal contents, and may initially reach

values of up to 7, depending on meal composition. With the

continuous secretion of gastric acid, the pH value then trends

back down to baseline over a period of several hours[22,23].

In the small intestine the pH exhibits a profile, with lowest

pH proximally and somewhat higher pH values in the distal

regions. pH values in the duodenum in the fasted state tend to lie

slightly below neutral (pH 66.5) [22,23]. The pH in the

proximal small intestine is influenced more by meal intake than

the pH in the distal regions, as might logically be expected from

the huge swings in pH observed in the stomach between the

fasted and fed states. After meal intake, pH will be influenced

by the chyme coming into the small intestine from the stomach.

Thus, after meal intake the pH values may actually rise initially

in the proximal small intestine. With time, as the incoming

chyme becomes ever more acidic the pH will actually drop aslow as 55.5, even in the jejunum [22,23]. Meanwhile, pH

values in the distal ileum appear to remain stable at around pH

7.5 [24]. This is consistent with digestion and absorption

occurring primarily in the proximal part of the small intestine:

up to one-half of the small intestine can be removed without

disturbing the ability to sustain nutritional balance[25].

The pH in the proximal large intestine reverts to more acidic

values, typically between 5 and 6.5[26], due to fermentation of

undigested foodstuffs (cellulosics and the like) to short chain fatty

acids (e.g.butyrate, propionate, acetate) by the colonic bacteria.

The wide range of pH values encountered by ionizable drug

substances within the GI tract suggests that large swings insolubility may occur, with implications forCw(Eqs. (1) and (2))

and hence the efficiency of absorption.

The second major influence on solubility in the GI tract is

solubilization. Solubilization mechanisms include micellar

solubilization by either native or co-ingested surfactants,

binding to peptides or proteins and solubilization in lipid

components of the meal.

In the stomach, the source of surfactants is not so clear,

although it has been consistently observed that the surface

tension of gastric fluids is commensurate with a significant level

of surfactant (e.g. [23]). In some subjects, the reflux of bile

components into the stomach appears to be the source of

surfactant behavior, but in others no bile components can bedetected in gastric aspirates [27]. In addition to native

surfactants, meal intake offers the potential for solubilization

of drugs during gastric residence. Macheras et al. have

demonstrated that chlorothiazide and hydrochlorothiazide are

well solubilized by casein micelles in milk[28]whereas more

lipophilic compounds, such as indomethacin and diazepam, are

additionally solubilized into the milk fat [29]. On the other

hand, meal components can have an adverse effect on solubility

if an insoluble complex with the drug is formed. The classical

example here is complexation with calcium, which precipitates

bisphosphonates and tetracyclines, rendering them insoluble

and thus unavailable for absorption[30].In the small intestine the primary source of solubilization is

clear the bile components such as bile salt conjugates,

phospholipids and cholesterol team up (additionally with

lipolysis products in the fed state) to create mixed micelles

that can solubilize lipophilic molecules very well. Indeed,

correlations have been established for solubilization by mixed

micelles as a function of logPfor neutral compounds[31]. The

concentration of mixed micelles is much higher after meal

intake, as the gall bladder contracts in response to a meal and

empties its contents into the duodenum at the level of the

Sphincter of Oddi. In addition,in vitrostudies indicate that the

solubilization capacity of the micelles is enhanced by the

incorporation of lipolysis products [32]. It should be noted,

Table 1

Mean equilibrium solubility data in g/ml for three drugs, illustrating the large

differences between solubility in simple aqueous media and biorelevant media/

aspirates

Felodipine Ketoconazole Dipyridamole

(Intrinsic) aqueous solubility 1 a 6.9b 5.0b

Solubility in FaSSGF 1.4a

9054a

11,417a

Solubility in HGFfasted 0.4a 9025 a 8530 a

Solubility in FeSSIF 188 c 406540 d 181246 d

Solubility in HIFfed 412c 476989 d 160254 d

a From Ref.[15].b Intrinsic solubility (solubility of the non-ionized form) from Ref. [14].c Numbers were extracted from the graphs of Ref.[16].d Data vary with the aspirations times of HIFfed and with composition of

FeSSIF[14].

593J.B. Dressman et al. / Advanced Drug Delivery Reviews 59 (2007) 591602


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however, that absorption of lipolysis products is generally

completed in the jejunum and the bile salts are reabsorbed

actively in the ileum [33], thus solubilization effects are

restricted primarily to the upper small intestine.

As there is a paucity of information in the literature about

surface tension or surfactants in the colonic fluids, it is not

possible to comment at this time on potential solubilization inthis region.

Other factors that can influence the capacity of the GI tract to

dissolve drugs in a pharmacokinetically relevant way include

the volume of fluids available in the region(s) of interest and the

passage time of the drug/dosage from up to and through the

regions where the drug is most efficiently absorbed.

3. Procedures for aspirating and measuring solubility in

gastrointestinal fluids

Given the large number of factors affecting solubility,a priori

prediction of intra-lumenal solubility is practically impossible.Measurement of drug concentrations in situ requires highly

specialized expertise and is complicated and costly, with the result

that few studies of this kind have been performed (see above). A

third, physiologically relevant and generally more practical

approach to estimating drug solubility in the GI tract is to aspirate

fluids from the human GI tract and measure the solubility in these

fluids ex vivo. With this approach, issues include subject selection,

conditions under which the fluids are aspirated, maintaining the

quality of the aspirated sample ex vivo, the methodology for

measuring the solubility, and associated costs.

First, the composition of aspirated samples can vary

dramatically with the demographics and medical history of

the subjects from which the aspirates are collected. Early in thedevelopment process, the drug will typically be administered to

young, healthy subjects to assess safety. In bridging bioequi-

valence studies performed later in the clinical development,

formulations are also typically tested in healthy adults.

Therefore it seems reasonable to use a set of subjects with

similar characteristics to obtain aspirates of the GI fluids.

Second, it is necessary to write a well-defined protocol for

the aspiration studies. It is very important to stipulate fasting

requirements prior to the study day, to define water intake when

performing aspiration in the fasted state, and to exactly define

meal intake (composition, rate of ingestion) and timing of meal

intake in relation to timing of aspiration when performingaspiration in the fed state.

Aspirations can be performed after nasal or oral intubation,

with the nasal route considered more practical by gastroenter-

ologists since the tube arrives at the trachealesophageal

junction at an angle which is more favorable to entry into the

esophagus.

In the fasted state, aspirations after administration of 200

250 ml of water allow for the protocol to be closer to that of a

standard pharmacokinetic study. In addition, since water flux is

limited in the fasted stomach, such amounts of water will

dramatically affect the composition of gastric contents and,

therefore, the ability of the drug to dissolve during gastric

residence may be greatly modified. Additionally, for aspiration

from the fasted upper small intestine, administration of 200 or

250 ml water before the aspiration procedure will improve the

chances of aspirating adequate volumes.

In the fed state, aspirates will vary in composition with the

size and type of meal and with the time after the meal's

consumption (e.g.[23]). One way to resolve this problem would

be to administer the same meal typically administered inbioavailability/bioequivalence studies. However, solid meals

create problems with sample aspiration due to potential

clogging of the aspiration port. As a result, alternative liquid

meals have been suggested [34,35]. The duration of the

aspiration period after consumption of the meal should be as

long as the residence time in the region of the GI lumen from

which samples are aspirated.

The third issue is the handling of the samples after aspiration.

Upon collection, certain physicochemical parameters should be

measured immediately (e.g. pH and buffer capacity) and, to

maintain the composition of the aspirated sample until the

solubility experiment is performed, it is necessary to deactivateenzymes immediately using a method that itself only minimally

(or more preferably, not at all) affects the composition of the

sample, before storing the sample under deep-freeze conditions

(20 C or lower).

Individual or pooled aspirates can be used for solubility

estimations. Individual samples have the advantage of enabling

correlations with levels of specific components in each sample

to be developed and thus to determine the most important

factors affecting luminal solubility of the compound in question.

Pooled samples from a large number of volunteers offer greater

volumes, and results for solubility in the GI fluids can be

compared across a set of compounds. If samples are to be

pooled, the volume taken from each individual sample shouldbe held constant, to ensure that the pooled sample is equally

representative of all subjects.

The fourth issue is the methodology for measuring solubility.

To date, all available drug solubility data in human GI fluids

have been measured by equilibrium solubility methods which

are generally preferred to kinetic solubility measurements[17].

When measuring equilibrium solubility, an excess of pure drug

powder (typically two to three times higher than the expected

amount to needed to saturate the medium[36]) should be used.

On the one hand, the time allowed to reach equilibrium should

be as short as possible to minimize composition changes in the

aspirate. On the other hand, enough time should be allowed toenable the system to attain equilibrium. In some cases, a

supersaturation may be generated. This can be avoided in many

cases by using the most stable crystal form of the drug powder

at the highest possible level of purity. Another problem that can

arise during the solubility measurement is conversion of the

drug to another compound, especially if the conversion is

enzymatically catalyzed [37]. In such cases, kinetic solubility

measurements [38] may be more useful than the equilibrium

solubility approach.

Up till now, measurements of solubility in fed state human

aspirates have been made in the total luminal contents, with no

distinction between concentrations achieved in lipid, micellar and

aqueous phases. One problem with this approach is that it is



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difficult in some cases to separate the excess solid drug from the

rest of the sample prior to analysis without simultaneous phase

separation. The other is that the concentrations achieved in the

phase driving drug absorption may be over or underestimated if

only the total concentration is reported, leading to false estimates

of the expected impact on bioavailability. Postulating that it is the

micellar phase concentration which drives absorption, it would bepreferable to report solubilities determined in this phase of the

postprandial luminal contents to predict food effects.

The fifth issue relates to resources necessary to perform the

studies. Since collection of aspirates has to be carefully planned

and executed by highly trained and experienced staff, and since

the aspirates themselves require special handling to generate

reproducible and meaningful results, measurement of solubility

in human aspirates is a tedious and costly process. Hence, use of

human aspirates for screening the solubility of a large number of

compounds would not be cost-effective. Therefore, the

identification of alternative media for estimating luminal

solubility, e.g. intestinal aspirates from animals or simulatedGI fluids generated in the laboratory, would be highly desirable.

4. Estimation of drug solubility in the stomach

4.1. Fasted state, gastric

Typically, human gastric aspirates in the fasted state

(HGFfasted) are collected using simple naso- or orogastric

tubes from the antrum of healthy adult volunteers after a ten

hour (overnight) fast [27,39]. To better reflect the dosing

conditions in a standard pharmacokinetic study, it is preferable

to collect aspirates in suitably fasted subjects after they have

been administrated a glass of water (250 ml). Assuming thatin a bioavailability study the disintegrated particles of an

immediate release dosage form as well as any drug that has

already dissolved will empty from the stomach together with the

co-administered water, samples to be used for solubility studies

should be aspirated approximately 15 min to a half-hour after

water administration [15,23]. This timing will also facilitate

collection of enough volume (about 20 ml) to carry out a

solubility measurement. To avoid changes in aspirate compo-

sition, samples should be deep frozen immediately until

solubility studies are performed (typically to 80 C)[23,27].

Solubility data in HGFfasted samples to date have been

performed in the absence of enzyme inhibitors[15]. To preventmicrobial growth during the solubility experiments, 6 mM

NaN3 and 0.01 mM chloramphenicol can be added [39].

However, the effect of these agents on the solubility data has not

been elucidated. Immediately after equilibration, HGFfastedsamples can be either filtered through 0.45 m regenerated

cellulose filters (recommended)[15]or centrifuged (5000 rpm,

1020 min)[39]. Quantification of the dissolved compound is

typically performed with HPLC and standard curves are

constructed in the corresponding medium.

Theonly animal aspirates that have been studied as alternatives

toHGFfasted media for estimating intra-gastricsolubility are canine

gastric aspirates collected in the fasted state (CGFfasted)[15]. In

order to allow for the faster gastric emptying rates in dogs than in

humans in the fasted state, administration of a little more than

250 ml of water prior to aspirations with aspiration approximately

10 min after the water administration is recommended.

In contrast to the paucity of alternative animal models,

various simulated media have been proposed[40]. It should be

noted that media designed to simulate intra-gastric conditions

and frequently used in biorelevant dissolution studies tend tooverestimate intra-gastric solubility values if they contain

synthetic surfactants[15,39].

Fig. 1compares the solubility values of four compounds in

HGFfasted, CGFfasted and Fasted State Simulating Gastric Fluid

(FaSSGF), a biorelevant medium that contains only physiolog-

ically relevant substances[15,40]. CGFfasted, with its higher pH,

appears to be less useful than FaSSGF for predicting the intra-

gastric solubility of drugs. However, it is worth noting that

solubility data in FaSSGF are not always more predictive than

simple HCl solutions (Fig. 1, felodipine), so the search for a

ubiquitously applicable in vitro medium for the accurate

estimation of intra-gastric solubility continues[15].

4.2. Fed state, gastric

The problematic aspiration and the heterogenous composi-

tion of gastric contents after administration of typical solid

meals, the dramatically changing composition with time after

administration, and the presence of various phases (i.e. solid,

aqueous, micellar, and lipid) make drug solubility in the

stomach difficult to define and measure.

To facilitate the aspiration procedure and to reduce the

degree of heterogeneity of gastric contents, aspirates can be

collected after administration of a liquid meal (HGFfed).Table 2

shows the composition of two meals that have been recom-mended by the U.S. FDA, the composition of corresponding

liquid meals that have been used to facilitate aspiration of

samples from the fed stomach, and the composition of gastric

contents after administrations of these liquid meals to healthy

volunteers after a twelve hour fast.

When using aspirates as the solubility medium, there is an

important issue with respect to the changing composition of the

medium during the solubility experiment. Typically, termina-

tion of lipase activity can be achieved by transferring the

aspirate to glass vials containing a methanol solution of lipase

inhibitors (5% v/v: 100 mM diisopropyl fluorophosphate,

50 mM acetophenone, 250 mM phenylboronic acid) [42]whereas inhibition of pepsin's proteolytic activity can be

achieved by titrating the sample to pH 1[23]. Finally, to prevent

any bacterial growth, 5 l of an aqueous solution of 4% NaN3w/v and 5% w/v chloramphenicol per milliliter of gastric

aspirate can be added [42]. It goes without saying that all of

these additions can impact the solubility value measured, and,

therefore, deep-freezing of the aspirates immediately upon

collection at80 C may alternatively be considered.

What about the possibility of simulating the digestion

processin vitro? Indeed, the changing intra-gastric environment

with time after the meal's administration can be adequately

reproducedin vitrowith regard to pH and pepsin levels.Fig. 2

shows two relevant examples, with cow's milk (heat-treated)



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and Ensure Plus as the starting point for the media, in

comparison with the actual intra-gastric pH profile after

administration of 500 ml Ensure Plus or a normal solid/liquidmeal to healthy volunteers. With regard to sample work-up,

cow's milk is much more practical than using Ensure Plus as

the starting point for digestion. In addition, the lower nutrient

content of milk compared to Ensure Plus or to meals usually

administered in drug absorption studies [34] makes it more

representative of the intra-gastric conditions, where significant

amounts of secretions (i.e. significant dilutions of the meal)

occur[23]. At least partly for this reason, although cow's milk

deviates significantly from the caloric content of typical FDA

meals [34], the in vitro pH profile with time lies within theexpected intra-gastric values (Fig. 2).

Another issue when using intra-gastric fluids (either

simulated or aspirated) in the fed state is that of total

composition vs. aqueous phase composition. For example,

when using cow's milk, the aqueous phase may contain lipids,

casein micelles, and/or casein molecules and its physical

composition will depend on the pH [43]. Separation of the

Table 2

Composition and volumes of standard U.S. FDA meals and of liquid meals in comparison with the resulting intra-gastric composition after administration the liquid

meals to healthy fasted adults a

Meal suggested by

FDA until 2002[34]

Meal suggested by

FDA after 2002[41]

Glucose/Olive oil/

Egg meal[42]

HGFfedcomposition after

Glucose/Olive oil/Egg meal [42]

Ensure

Plus [23]

HGFfed composition after

Ensure Plus[23]

Proteins (g/l) 56.5 1516.6% of total

calories

33 N.M. 54.9 23.311.2

(30210 min)

Carbohydrates

(g/l)

142.3 2527.8% of total

calories

177 12010 200 152.149.1

(14 h) (30210 min)

Lipids (g/l) 52.6 6055.6% of total

calories

175 150 (13 h) 53.3 N.M.

50 (4 h)

pH 5.3 N.M. 7.0 4.02.5 6.7 6.42.7

(14 h) (30210 min)

Calories

(kcal)

648 9001000 960 N.A. 750 N.A.

Volume (ml) 513 N.M. 400 N.A. 500 N.A.

a N.A.: not applicable; N.M.: not measured.

Fig. 1. Solubility of ketoconazole, dipyridamole, miconazole and felodipine in HGFfasted, CGFfastedand media simulating intra-gastric conditions in the fasted state[15].

pHeq represents the pH value measured in the sample after solubility equilibrium had been attainted.



7/12

aqueous phase from the precipitated (digested) phase, the lipid

droplets, and the undissolved drug presents another challenge.

Usually, immediate centrifugation (up to 11,400 g, 10 C,

10 min) leads to separation of the aqueous phase from

precipitated/digested phase, most of lipid droplets, and the

undissolved drug [44]. If centrifugation does not work, i.e. if

drug solid particles cannot be separated, one must additionally

filter the supernatant. In this case the filter efficiency has to

balance the rate of filtration against the ability of the filter to

exclude undissolved material, and adsorption of the drug to the

filter has to be ruled out (Diakidou et al. unpublished data). Thepart of the sample to be analyzed (supernatant or filtrate) is then

treated analogously to a plasma sample, in that a further protein

precipitation step may be required, and can usually be analyzed

by standard HPLC techniques. Quantification of the drug in the

aqueous phase or in the whole (aspirated) medium is performed

with standard curves constructed in the corresponding medium.

This means that estimating solubility at various time points will

typically require the construction of several standard curves (to

take into account the changing composition with time after meal

administration).

As an alternative to measurement of solubility concomitant

with simulation of digestion, it is also possible to construct aseries of media to simulate the composition of the gastric

contents at various stages of the digestive process, so-called

snapshot media. The snapshot media reflect the composi-

tion of the gastric contents after administration of 500 ml Ensure

Plus, as reported by Kalantzi et al.[23]early, in the middle and

late in the digestive process. Parameters important to drug

solubility and dissolution such as pH, buffer capacity, surface

activity and osmolarity are all simulated in these media, which

use cow's milk as the starting point for preparation (Janen

et al. unpublished data). Such media have the advantage of

being completely defined, as opposed to starting with a

composition like cow's milk and simulating digestion, in

which case reproducibility may be an issue.

5. Estimation of drug solubility in the small intestine

5.1. Fasted state, small intestine

Human intestinal fluids in the fasted state (HIFfasted) are

usually collected at or about the beginning of the jejunum of

healthy fasted volunteers[6,8,9,16,23,27,37,39,45]. This site ispreferred, because it is possible to locate the aspiration tubes in

this region reproducibly (as opposed to the upper and middle

duodenum), the aspiration tubes can be placed in this region

reasonably quickly (as opposed to the lower small intestine), and

the volumes that can be aspirated are greater than is possible at

more distal locations in the small intestine. Some studies have

been conducted without prior administration of any water

[27,37,39,45]. It is also possible to use a procedure that involves

administration of 180250 ml water before the aspiration is

started[6,8,9,23]. This comes closer to simulating conditions in

a bioavailability study. In one version, the water is administered

via a tube to the antrum of the volunteer and samples areaspirated from the end of the duodenum as depicted in Fig. 3.

Alternatively, the water can be swallowed and aspirations can be

performed from a tube that allows access in the small intestine

[6,8,9]. In the latter case the tube has two lumens, both of which

end in the small intestine. One is used for aspirating samples and

the other for alleviation of any pressure reduction generated in

the intestine by the aspiration procedure. Regardless of the exact

methodology, aspirates are collected from the end of duodenum/

start of the jejunum and are typically stored at20 C (or lower),

until used [16,23,27,39,45]. Deactivation of trypsin can be

achieved by adding Phenylmethylsulfonyl fluoride (PMSF) to a

Fig. 3. Representative X-ray picture showing the position of a two-lumen tube in

the upper GI lumen of a volunteer who was administered a liquid meal into the

antrum of the stomach (administration ports) with sample aspiration from the

end of the duodenum (aspiration ports). Details of the specific tube can be foundelsewhere[23].

Fig. 2. pH values in the stomach after administration of 500 ml Ensure Plus to

healthy fasted adults [23] (box plots from 30 to 210 min), pH values in the

stomach after administration of a solid meal (1000 kcal, pH 5.7) to fasted healthy

subjects[22](box plots from 0 to 240 min), meanSD pH values in 500 ml

cow's milk after addition of acidic solutions of pepsin in physiologically

appropriate quantities [44] (), and meanSD pH values of 500 ml Ensure

Plus after addition of acidic solutions of pepsin in physiologically appropriate

quantities following the same procedure with that applied in milk ().



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final concentration of 1 mM[23]. Deactivation of lipase can be

achieved with one of the following methods: the cocktail

mentioned earlier for deactivation of lipase in the fed stomach

[42]at double the concentration[35,46]; using tetrahydrolistatin

(orlistat) at a final concentration of 1 mg/ml[16,47]; or usingp-

bromophenylboronic acid at a final concentration of 1 mM[48].

Regardless of the method used, the total volume of inhibitorsolution should be less than 2% (by volume) of the collected

luminal contents[35]. Sometimes, to prevent microbial growth

during the solubility tests, 6 mM NaN3 and 0.01 mM

chloramphenicol are added[39]. As with gastric aspirates, all

these treatments may affect the finally estimated solubility value

since they all impact the composition of the aspirated sample.

However, there are some data suggesting that orlistat does not

affect solubility data in HIFfasted [16]. To date, solubility data

in HIFfasted have been measured in aspirates that have been

treated several different ways: addition of orlistat[16], addition

of antimicrobial growth agents [39] or without addition of

chemicals (aspirates were simply deep frozen immediately uponcollection) [14]. After equilibrium, samples are centrifuged

(500010,000 rpm or at 10,000 g for about 1020 min)

[14,16,39], and then quantified with HPLC techniques. Per-

forming solubility studies in individual aspirates, Pedersen

et al. [39]were able to demonstrate that the total solubility of

danazol (logP= 4.53) correlated nicely with the bile salt content

of the aspirate, whereas for the less lipophilic hydrocortisone

(logP= 1.66) no correlation was seen. These results are

consistent with solubilization theory for bile salts[49].

Canine aspirates and simulated media have been proposed to

avoid the collection and use of human aspirates. One of the

disadvantages of using canine aspirates is that canine gall

bladder shows brief alternating excursions of filling andemptying with the number of emptying events exceeding the

filling events during phases II of the IMMCs[50]and this may

lead to highly variable estimations of solubility [14].

Simulated media contain phosphatidylcholine and bile salts

(usually trihydroxy bile salts of taurine having various levels of

purity), but usually contain a non-physiological buffer system

e.g. phosphates. Porter and Charman have reviewed this topic

[51]. One of the reasons for not using the physiological buffer

(bicarbonate) is that bicarbonates are unstable with time,

seeking to come to equilibrium with carbon dioxide in the

atmosphere. To maintain a constant pH, bicarbonate-containing

media must be continuously sparged with carbon dioxide andtitrated with sodium hydroxide[52,53]. This leads to changes in

buffer capacity, ionic strength and osmolality during the

experiment, changes which themselves are not physiological.

Due to these practical difficulties, bicarbonates are usually

replaced with stable buffer systems in media used for solubility

measurement. The anion in the buffer system may theoretically

affect the solubility product of a weakly basic compound with a

pkahigher than 5, the solubility of extremely highly lipophilic

compounds due to salting in/out properties (of the anion), and/

or the stability of the dissolving compound[54]. In addition, the

anion of the buffer system may be important for the conversion

of a prodrug to its active form [37]. For these reasons,

biorelevant media with alternative buffer species have been

suggested[54]. It should be pointed out, however, that to date

no data have been brought forward showing that the presence of

phosphates affects estimates of intra-intestinal solubility. In fact,

Kalantzi et al. were able to demonstrate that the fasted state

simulating intestinal fluids, originally proposed in 1998 [55],

are able to predict solubility in human aspirates well [14].The

use of a crude mixture of bile salts and maleates might slightlyimprove the prediction of intra-lumenal solubility in some cases

[14].

5.2. Fed state, small intestine

Human duodenal aspirates for solubility studies in the fed

state (HIFfed) have been collected and characterized after

administration of various liquid meals to the antrum or the

jejunum of healthy fasted volunteers (Table 2). Aspirated

samples are typically transferred quickly to glass vials

containing lipase and trypsin inhibitors as well as agents

for preventing microbial growth, as described above for theintestinal aspirates collected in the fasted state. However, since

these treatments may have an impact on the measured

solubility values, studies to date have been performed by

adding orlistat only [16] or no inhibitors [23] in the tested

aspirates.

Intestinal aspirates in the fed state are less heterogenous than

the corresponding gastric aspirates and, although their physi-

cochemical characteristics change with time, the changes are

not as pronounced as in gastric aspirates. In fact, the solubility

of ketoconazole and dipyridamole in pooled human aspirates

collected after administration of Ensure Plus was not

significantly affected by the time of aspiration, for times up to

120 min after administration of Ensure Plus[14].The solubility of compounds in HIFfedcan be several orders

of magnitude higher than the corresponding values in the fasted

state [14,16]. Although to date all relevant solubility experi-

ments have been performed using the entire aspirated sample, it

should be born in mind that a more detailed characterization of

solubility could be obtained if the phases are separated prior to

solubility determination. Complete phase separation is more

difficult in intestinal than in gastric aspirates because fine

emulsions may have been formed (seeFig. 4).

If solubility increases are phase-dependent (lipid, micellar,

aqueous), successful prediction of effects on absorption will be

contingent on correctly identifying the increase in solubility inthe absorption-relevant phase. For example, if the increase in

solubility is primarily due to incorporation of the drug in the

lipid phase, distribution into the aqueous phase may limit the

increase inin vivodissolution rate in the aqueous phase. On the

other hand, if the increase in solubility is due to micellar

solubilization in the aqueous phase, increases in dissolution and

subsequently absorption rates should follow NoyesWhitney's

considerations more directly [56]. Even so, the intra-lumenal

dissolution rate can be slower than that expected from solubility

data[16], most likely due to the slower diffusion of the micelle-

bound drug to the bulk solution[57].

Canine aspirates and simulated media have also been studied

as alternatives to HIFfedsamples. Persson et al.[16]found good



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agreement between canine and human results for drug solubility

in intestinal fluids when small (200 ml) meals were adminis-

tered to both species. However, when larger meals were

administered to both species, the solubilities in canine aspirates

overestimated solubilities in human aspirates. This is probably

due to the higher levels of bile salts in the postprandial canine

small intestine and makes extrapolation from canine to human

data rather precarious[14].An issue with simulated media is the identity of buffer

species used[54], although in this case (unlike in the fasted state

scenario) bicarbonate is only one of many buffers contributing

to the overall buffer capacity. In fact, data published to date

show that solubility in FeSSIF adequately predicts or only

slightly underestimates solubility in HIFfed [14]. Based on

Table 3however, FeSSIF is being redesigned to have a pH value

higher than 5 and contain less bile salt. Also, as demonstrated in

various in vitro setups[32,58], the simulated medium should

contain a biorelevant amount of lipolysis products in order to

adequately address the influence of meal digestion products on

solubility. This point is especially relevant for highly lipophilic

compounds, and a significant amount of work in this direction

has already been done to help optimize orally administered lipid

dosage forms[59].

6. Use of drug dissolution and solubility data in predicting

plasma profiles

Various examples in the literature have demonstrated the

utility of dissolution and solubility data measured under

biorelevant conditions in the prediction of drug plasma levelsand/or the fraction of drug absorbed[12,60,61,62].

For compounds with low dose:solubility ratios and which are

highly permeable, predictions are indeed quite successful, as is the

case with danazol in the fed state (using dissolution data collected

with the flow-through apparatus) (Fig. 5), glibenclamide in the

fasted state (using dissolution data collected with the rotating

Fig. 5. Mean observed plasma concentration profiles of danazol in the fasted ()

and in thefedstates() in comparisonwith predictedprofiles that were based on

dissolution data collected with the flow-through apparatus (32 ml/min) in a

mediumwith similar composition to HIFfasted composition (e.g. [39]) (),andat

8 ml/min in a medium with similar composition to HIFfed(e.g.[16,23]) (- - - - -).(Reproduced with permission from Ref.[60]).

Table 3

Average composition of contents in the upper small intestine after administration

of standard meals to healthy fasted adults a

Intestinal

composition after

400 ml glucose/

olive oil/egg mealb

[35,46]

Intestinal

composition

after 500 ml

Ensure Plusb

[23,14]

Intestinal composition

after NuTRIflex

administration

(180 ml over

90 min) c [16]

Proteins (g/l) N.M. 10 5.0 0.1

Carbohydrates

(g/l)

N.M. 5060 N.M.

Total neutral

lipids

55100 (g/l)

(14 h)

45.058.3 mM 221 mM

(0.53 h)

Phospholipids 3.05.8 mM 3 0.3 mM

(0.53 h)

Bile salts

(mM)

6.713.4 (14 h) 11.25.2 8.0 0.1

(0.53 h)

pH 67 6.65.2 6.1

(0.53.5 h)

a N.M.: not measured.b Please seeTable 2for the exact composition of this meal.c Unlike the other meals on this Table, NuTRIflex was administered directly

to the jejunum. NuTRIflex composition: pH: 5.4, proteins: 19 g/l, lipids:

12 mM, phospholipids: 3 mM [16]. 180 ml contains 138 kcal nitrogen 0.8 g,

amino acids 5.8 g, glucose 11.5 g, and lipids 7.2 g [16].

Fig. 4. Upper panel: Schematic representation of the various phases in intestinal

aspirates collected in the fed state (corresponding to the photograph shown in the

lower panel), after ultracentrifugation. Lower panel: Set of aspirated samples

from the end of the duodenum after administration of a glucose/olive oil/egg

meal [35,42,45] to a healthy volunteer. Numbers correspond to minutes after

administration of the meal. First row shows the samples immediately after

collection and second row shows the same samples after ultracentrifugation

(410,174 g, 37 C, 2 h).



10/12

paddle apparatus)[61] and troglitazone in the fed state (using

dissolution data collected with the rotating paddle apparatus) [12].

For compounds with high dose:solubility ratios and which

are highly permeable, predictions to date are successful in the

fed state, but in the fasted state the plasma profiles appear to be

affected significantly by the hydrodynamics.Fig. 6illustrates an

example with atovaquone tablets (Wellvone). The dose:solubility ratios of atovaquone in FaSSIF and FeSSIF have

been estimated to be 25 and 80 l, respectively. Dissolution data

collected with the rotating paddle apparatus and biorelevant

media in combination with the corresponding solubility data led

to successful prediction of plasma levels in the fed state. In the

fasted state, predictions, while much better than using

dissolution results in compendial media, were not as accurate

[12]. Another example is danazol in the fasted state. Its dose:

solubility ratio in HIFfasted is about 20 l [16]. By applying a

simulation methodology similar to that for atovaquone[12], the

predicted average plasma profiles for Danatrol capsules using

solubility data in HIFfasted[16]and dissolution data in FaSSIFwith the rotating paddle apparatus [55], provide much better

predictions than data collected in USP simulated intestinal fluid.

However, they fail to accurately predict the average maximum

concentration [60] (Fig. 7). Substantial improvements can be

achieved using solubilities close to those obtained in HIFfasted[16] and dissolution data obtained with the flow-through

apparatus[60](Fig. 5).

Figs. 57in combination with other relevant published data

suggest that although estimation of intra-lumenal dissolution

has greatly facilitated our ability to predict intra-lumenal

performance of solid dosage forms, hydrodynamics may in

some cases be crucial for accurate predictions of plasma

levels.

7. Conclusions

future directions

The ability to predict solubility in the upper gastrointestinal

tract would clearly be advantageous to discovery and

development programs in the pharmaceutical industry. Since

direct measurement of luminal concentrations is cumbersome,

recent efforts have been directed at determination of solubility

in human gastrointestinal fluids and in developing media which

can simulate these appropriately. As is evident from the

foregoing discussion, the collection of aspirates from the

human intestinal tract is fraught with technical challenges,

especially in the fed state. The reproducibility of solubility data

is highly dependent on the aspiration protocol and the way theaspirates are processed and stored. Careful attention also has to

be given to the experimental procedure of the solubility

determination itself. Although fluids from animals seem like a

reasonable way of addressing some of the challenges, results to

date with dogs have been disappointing. For all these reasons,

in vitro surrogate media for predicting drug solubility in the

upper gastrointestinal tract appear to be the way forward. With

media already available or being developed for the upper

gastrointestinal tract, the next logical step is to design media to

represent conditions in the lower regions. Work is currently

underway to characterize fluids collected from the proximal

colon and to use these as a basis for design of the corresponding

biorelevant media.

Fig. 7. Mean danazol plasma levels after single dose administration of one

Danatrol capsule (100 mg danazol per capsule) with 200 ml of water to nine

healthy fasted volunteers[60]() and predicted profiles that were constructed

using a simulation procedure identical with that used in Ref.[12]and dissolution

data collected with the rotating paddle apparatus (100 rpm) in USP simulated

intestinal fluid (.....) and in FaSSIF ()[55].

Fig. 6. (A) Median observed plasma data in the fasted state after single

administration of two Wellvone tablets to healthy subjects () and predicted

profiles that were based on dissolution data collected with the rotating paddle

apparatus (100 rpm) in USP simulated intestinal fluid (.....) and in FaSSIF ().

(B) Median observed plasma data in the fed state after single administration of

two Wellvone tablets to healthy subjects () and predicted profiles that were

based on dissolution data collected with the rotating paddle apparatus (100 rpm)

in USP simulated intestinal fluid (.....) and in FeSSIF (

). (Reproduced withpermission from Ref.[12]).



11/12

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