Bevernage, IJP, 2013, Evaluation of Gastric Drug Supersaturation and Precipitation

8/10/2019 Bevernage, IJP, 2013, Evaluation of Gastric Drug Supersaturation and Precipitation

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J. Bevernage et al. / International Journal of Pharmaceutics453 (2013) 2535 27

Fig.1. Typical degree ofsupersaturation (DS)timeprofiles fora saturatedsolution (DS= 1)anda supersaturatedsolutionwithorwithoutprecipitationinhibitor; summarizing

metrics forsupersaturation assessment based on thearea under these DStime profiles, are illustrated.

fraction, followed by dilution to limit any (further) precipitation.

Since they enable faster phase separation, filtration techniques

are preferred. However, the use of filtration may be hindered by

adsorption issues, especially if only small sample volumes are

available; in that case, centrifugation might be superior for phase

separation (Psachoulias et al., 2011). Both techniques have their

limitations in the separation of small particles so that thepresence

of nanoparticles cannot be excluded. It may therefore be valuable

to confirm the absorption-enhancing capacity of supersaturating

formulations by fluxmeasurements across an epithelial cell layer

(Brouwers andAugustijns, 2012;Mellaerts et al., 2010).

2.2. Detecting precipitation

As an alternative to the quantitative approach of evaluat-

ing supersaturation, detection of precipitation has also been

applied to assess supersaturation stability. Different methodolo-

gies to detect precipitation have been described, including visual

inspection and light microscopy (Raghavan et al., 2001), spectro-

photometric UV/VIS absorbance-time measurements (Ozaki et al.,

2012; Chandran et al., 2011), and nephelometric turbidity mea-

surements(Warrenet al., 2010). Precipitationdetection techniques

avoid phase separation and subsequent drug quantification, lend-

ing them to medium- to high-throughput screening assays and

makingthemsuitablefor continuous in-processprecipitationmon-

itoring. Typically, the lag time before appearance of any crystals

(induction or nucleation time) is detected as, for instance, a sud-

denincrease inabsorbanceandused to rank differentformulationsor precipitation inhibitors. Arnoldet al. proposed theuseof in-line

Raman spectrophotometrymonitoringduringprecipitationassays.

Changes inRaman spectra were notonlyused todefine thenuclea-

tion time but also to predict the amount of precipitated drug over

time; a good correlation with the actual precipitated amount was

observed (R2: 0.995) suggesting that a quantitative approach for

precipitation evaluation is feasiblewithout the need forphasesep-

aration (Arnold et al., 2011).

2.3. Solid state analysis of the precipitate

Solid state analysis of precipitated drug may be a useful tool

to investigate the mechanism of precipitation and excipient-

mediatedprecipitation inhibition.Usui etal.used scanningelectron

microscopy, X-ray diffraction and excipient content analysis to

revealthe influenceofpolymer (HPMC,HPC and PVP) onthecrystal

shapeofprecipitatedRS-8359 (Usui et al., 1997). Sassene et al.ana-

lyzed cinnarizineprecipitate formedduringthe in vitro lipolysisof

a SMEDDSformulation(Sassene et al., 2010). X-ray powder diffrac-

tion and polarized light microscopy illustrated that cinnarizine

precipitated in an amorphous rather than a crystalline form. Since

the amorphous form dissolved significantly faster than crystalline

cinnarizine, redissolutionmay limit the impact of precipitation on

the bioperformance of the cinnarizine SMEDDS. Finally, potential

polymorphic transitions during precipitationmay further compli-

cate the supersaturation behavior andwarrant solid state analysis

(Llins andGoodman, 2008; Singhal and Curatolo, 2004).

3. An overview of in vitro supersaturation assays

Multiple in vitro assays to evaluate supersaturation, precipi-

tation or precipitation inhibition are reported in literature. They

differ in (1)approaches togeneratesupersaturation, (2) techniques

to measure supersaturation/precipitation, and (3) experimental

conditions (Fig. 2). This section provides an overview of the

main experimental setups for supersaturation evaluation of both

non-formulated drugs and drugs formulated in SDDS. Critical

experimental variablesandconcernswillbe discussed in Section4.

3.1. Supersaturation evaluation of non-formulated drugs

To evaluate the supersaturation/precipitation potential ofnative (non-formulated) drugs and/or the precipitation inhibition

capacity of excipients, induction of supersaturation is the starting

point. Various methods to generate supersaturated drug concen-

trations can be found in the field of crystallization chemistry,

including solventevaporationor freezing,additionof ionsthatpar-

ticipate in the precipitation process, dissolution of unstable high

energy forms, change of temperature, pH-shift and solvent shift

(Rodrguez-Hornedo and Murphy, 1999). In the context of test-

ing the supersaturation potential of non-formulated drugs, only

solvent- or pH-shift methods have been commonly applied.

3.1.1. Solvent shift methods

The solvent shift or co-solvent quenchmethod is a popular and

simpleapproach to createsupersaturation (Yamashitaet al., 2011;


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28 J. Bevernage et al./ International Journal of Pharmaceutics 453 (2013) 2535

Fig. 2. Differentmedia andapproaches to generate supersaturationthat arecommonlyused in in vitro supersaturationassays.

Warren et al., 2010; Brewster et al., 2008). In practice, the poorly

water soluble drug is first dissolved in a water miscible solvent

with a significantly higher solubilizing capacity for the drug than

the aqueous environment in which supersaturation is being eval-

uated. A variety of solvents have been applied for this purpose,

including DMSO (Bevernage et al., 2011; Yamashita et al., 2010),

DMF (Vandecruys et al., 2007), DMA (Vandecruys et al., 2007), PEG

(Carlert et al., 2010) and propylene glycol (Warren et al., 2010).

Next, a fraction of this co-solvent solution is added to the aqueous

medium under investigation such that supersaturation is gener-

ated as a result of thesolubility difference. Finally, supersaturation

and/or precipitation canbe monitored bymeans of the aforemen-tioned techniques (see Section 2). Knowing the solubility of the

drug in the aqueousmedium, anydesired initial degreeof supersa-

turation can easily be set by tuning the drug concentration in the

co-solvent solution and/or the transferred amount. Possible alter-

ations in drug solubility in the aqueous medium upon addition of

the solvent should be taken into account.

The solvent shift technique is broadly applicable for anypoorly

water solubledrugthat canbedissolvedat significantlyhighercon-

centration ina watermiscible co-solvent.Because of theuseofdrug

solutions, the process of inducing supersaturation can be readily

automated and requires only limited amounts of drug compound.

As a result, the implementation of high-throughput solvent shift

setups is popular in early drug discovery. Obviously, the biorele-

vance of the solvent shift technique is often questionable.

3.1.2. pH-shift methods

As an alternative to the solvent shift method, a pH-shift can

be performed to assess the supersaturation potential of ionizable

drugs. Due to ionization, the solubility of molecules can increase

dramatically in polar aqueous solvents. In such cases, a shift in pH

that results in reduced ionization will rapidly decrease the drug

solubility (as expressed in the HendersonHasselbalch equation)

and inducea supersaturatedstate.This implies thatpH-shiftmeth-

odscanonlybeapplied to investigate thesupersaturation behavior

of neutral drug species. Compared to a solvent shift, a pH-shift

may be considered more biorelevant forweakly basic compounds

as the natural pH shift occurring upon transfer from the fasted

state stomach to the small intestine might induce supersaturated

concentrations in vivo. pH-shifts can be performed in one com-

partment where for example an acidic solution of a basic drug is

supplementedwith a buffering agent to increase thepH above the

materialspKa(Carlert et al., 2010;Yamashitaet al., 2010;Mellaerts

et al., 2008; Overhoff et al., 2008). Alternatively, a pH shift can

be achieved upon bolus or continuous transfer between two com-

partments: for instance, Kostewicz et al. monitored precipitation

of weakly basic drugs in an intestinal compartment (neutral pH)

resulting from continuous infusion from an acidic compartment

with dissolved compound (Kostewicz et al., 2004).

3.1.3. Potentiometric methodsAn alternative pH shift approach to investigate supersaturation

phenomena of ionizable drugs is the method of chasing equilib-

rium solubility or CheqSol systemintroduced by Sirius (Box et al.,

2006). This potentiometric techniquewas originally developed to

measure the intrinsic solubility (i.e. the solubility of the unionized

form) of weak acids and bases but also provides a way to quan-

tify theextentanddurationof supersaturation(Box et al., 2009). In

brief, a solution of the ionizable drug is titrated, during which pH

and UV absorbance of the solution are carefully monitored. At the

start, the pH is adjusted to completely dissolve the drug in its ion-

ized form. The solution of ionized solute is back titrated by adding

measured quantities of a titrant (KOH for basic compounds and

HCl foracidiccompounds) to form the less soluble unionizedform,

resulting in supersaturationandsubsequentprecipitationwhich isdetected by an increase in apparent absorbance using a fiber optic

dip probe. As soon as a sufficient quantity of precipitate has been

formed, the process of chasing equilibrium starts by repeated pH-

induced alterations from a supersaturated to a subsaturated state

andvice versa.Duringthis process, the small pH changes resulting

from gradual precipitation or dissolution can be used to calculate

theconcentrationofneutralspeciesasa functionof timeusingmass

andchargebalanceequations,provided thataccurate knowledgeof

(1) the pKa(s) and concentrationof the ionizable drug and (2) total

volumeandconcentration of added titrant, is available (Stuart and

Box, 2005).

For many ionizable drugs (the so-called chasers), supersatu-

rated concentrations of neutral species well above their intrinsic

equilibrium solubility have been recorded prior to precipitation.


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Drugs that precipitate immediately to the intrinsic equilibrium

solubility (non-chasers) have no or limited potential for super-

saturation. For chasers, the concentrationtime profile of neutral

species recorded during titration provides an idea of the extent

and duration of the formed supersaturated solution. As such, the

Cheqsol method may also be applied to investigate excipient

effectsonthesupersaturation stabilityof ionizabledrugs (Boxetal.,

2009). Using the Cheqsol approach, Hsieh et al. examined super-

saturation and precipitation of 10 weak bases and correlated the

results with the solid state properties of the formed precipitate

(crystalline versus amorphous) (Hsieh et al., 2012). While short-

lived supersaturationwas associatedwith crystallineprecipitation,

prolonged supersaturation occurred in caseof amorphous precipi-

tation.In thiscontext, theyalso foundthepH-titrationmethodtobe

a practicalwaytodeterminethesolubilityof amorphousmaterials.

3.1.4. High throughput supersaturation assays

The lack of a theoretical basis for the selection of supersatu-

ration strategies and, hence, the need for experimental input in

early stages of drug development, has driven the development of

medium to high throughput supersaturation assays for the rapid

generation of supersaturation data while consuming only limited

amounts of compound and media. In this context, Vandecruys

et al. developed a screening method based on a solvent shift toidentify excipients that affect supersaturation (Vandecruys et al.,

2007). Drug candidates dissolved in DMA or DMF were gradu-

ally added to the dissolution medium in presence or absence of

excipient (10ml, 0.01N HCl, USP buffer pH 4.5 or 6.8 or water,

stirred at 600 rpm using a magnetic stirrer bar) until visual detec-

tion of precipitation; subsequently, the dissolution medium was

sampled, filtered and analyzed (UV absorbance) over a period of

120min. Similarly, Yamashitaet al.developed a 96-wellbasedhigh

throughput format toevaluatethe capacityof excipientsto stabilize

itraconazole supersaturation (Yamashita et al., 2011). Itraconazole

was dissolved in DMSO fromwhich 4l was added to FaSSIF con-

taining 0.015% excipient. Shaking of the plate was provided and

at appropriate times, samples were taken and filtered using a filter

plate. Interestingly, theresults obtainedusingthis highthroughputsolvent shift methodwere confirmed in the dissolution profiles of

soliddispersions containingidenticalexcipients usingthe Japanese

Pharmacopeia paddle dissolution method (300ml FaSSIF, 50rpm).

In addition, similar results were obtained using a pH-shift to gen-

erate the supersaturated state (Yamashita et al., 2010).

Thehigh throughput induction of supersaturation is often com-

binedwithonlinedetectionof precipitationtogenerate immediate

results. Forinstance,Warrenet al.employeda solvent shiftmethod

accompanied with turbidity measurements to monitor danazol

precipitation kinetics in presence of various polymers (Warren

et al., 2010).

3.2. Supersaturation evaluation of formulated drugs

Toevaluate the behaviorofSDDS, supersaturationshouldnotbe

induced aspart of the assay,but as an inherent characteristicof the

formulation. Predictive performance evaluation requires an assay

that provides an adequate aqueous environment, relevant for the

gastrointestinal tract.While critical variables(e.g. sinkversus non-

sink conditions,mediumselection, etc.) arediscussed in Section 4,

this paragraphprovides an overview of the different experimental

setups that have been reported for SDDS evaluation.

3.2.1. Dissolutionmethods for the evaluation of SDDS

To evaluate thedissolution propertiesof formulations thathave

the potential to inducedrug supersaturation, traditional one com-

partment/one phase setups, often based on USP I or II apparatus,

are commonly applied. To facilitate rapid sample treatmentwhen

handling supersaturated samples (phase separation and dilution),

alternative dissolution setups, such as rotating syringes (Curatolo

et al., 2009; Dong et al., 2007) or microcentrifuge tubes directly in

thecentrifuge (Curatolo et al., 2009; Friesenet al., 2008) have been

proposed.

To test formulations that rely on the gastrointestinal pH gradi-

entto generatesupersaturation, thispHshiftneeds tobesimulated

in the dissolution method. In general, it is advisable to include

an acidic phase in the evaluation of any SDDS that may release

drug in the stomach, in view of the different dissolution and/or

precipitationkinetics in gastric versus intestinalmedia (Bevernage

et al., 2012b). Bothone compartment (Carlert et al.,2010;DiNunzio

et al., 2008,2010;Mellaerts et al., 2008;Miller et al., 2007,2008a,b)

and two compartment (Carlert et al., 2010; Van Speybroeck et al.,

2010b; Mellaerts et al., 2008) pH shift approaches have been

applied for this purpose. Finally, a multi-compartment dissolution

setupcomprising a gastric, intestinal andabsorptioncompartment

have been used to predict precipitation from formulationsofweak

bases such as dipyridamoleandcinnarizine (Guet al., 2005). Com-

pared to conventional dissolution tests, this multi-compartment

dissolution setup appeared to be more predictive for the in vivo

exposure.

3.2.2. Higher throughput evaluation of SDDS

The time pressure and limited availability of API early in the

pharmaceutical development cycle associated with modern drug

formulation configuration requires fast and economic methods to

evaluate SDDS. However, experience with higher throughput dis-

solution methods, especially in an SDDS context, is fairly limited.

Daietal.developeda 96-wellbasedprecipitationmethodtorapidly

evaluate drugprecipitation fromliquid formulationsupondilution

in the gastrointestinal tract (Dai et al., 2007). Drug and excipients

dissolved in n-propanol were dispensed to and mixed in a 96-well

plate. Upon evaporation of the solvent using a centrifugal vacuum

evaporator, the liquidformulationwas formedin situ afterwhichit

was diluted using biorelevantmedia. After thedesired incubation,

samples were transferred to a filter plate and the resulting filtrate

was diluted and analyzed. The obtained results correlated withtraditional USP dissolution testing and allowed high throughput

discrimination between fast, medium and slow precipitating for-

mulations. Similarly, Chandran et al. were able to identify various

stages of precipitation using an online UV-absorbance technique

after dilution of solubilized formulations (Chandran et al., 2011).

Higher throughput dissolution studies involving solid SDDS are

limited to solvent cast dissolutions. Although solvent casts can-

not be considered as true formulations, their dissolution behavior

can provide useful information for solid dispersion development,

as illustrated by Shanbhag et al. (2008). Drug and excipient solu-

tions in volatile solvents were used to prepare solvent casts in

a 96-well plate. Dissolution was performed through addition of

300l simulated intestinal fluid (SIF) to each well. Samples were

filtered using a filter plate and the resulting filtrate was dilutedprior to quantification with UV spectroscopy. Although this dis-

solution screening method does not provide information on the

physicalstateof thesolidformulations, it appearedquitesuccessful

in identifying formulations that significantlyenhanceddissolution

and bioavailability (Shanbhag et al., 2008).

3.2.3. Evaluation of lipid based formulations as SDDS

Inherent to their lipophilic nature, poorly water soluble drugs

can be more soluble in oils and other non-polar solvents. From

this perspective, lipid-based formulations have been developed

to deliver drug in solubilized form in the gastrointestinal tract

(Kuentz,2012;Kohli etal., 2010). Typically, theabsorptionenabling

properties of lipid based systems have been attributed to the

enhanced solubilizing capacity of gastrointestinal fluids upon


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dispersionanddigestionof the formulation components. Recently,

however, it has become clear that supersaturation/precipitation

phenomena also contribute to the bioperformance of lipid-based

delivery systems (Anby et al., 2012; Porter et al., 2011) In addition

to dispersion (Mohsin et al., 2009), digestion has been recognized

asa trigger forsupersaturation andsubsequent precipitation(Anby

et al., 2012; Sassene et al., 2010; Porter et al., 2004). In vitro

performance evaluation of lipid-based formulations thus requires

supersaturation/precipitationassaysthatincludea digestion(lipol-

ysis) step. Recently, different in vitro lipolysis models for the

evaluation of lipid-based delivery systems have been reviewed by

Larsen et al. (2011). Typically, lipid formulations are dispersed in

digestion medium (containing bile salts and phospholipids) that

reflects fasted state conditions; digestion is initiated by addition

of pancreatic enzymes. During lipolysis, the pH is maintained by

titrationof liberated fatty acidsusing a pH-stat titrationunit. Sam-

ples are taken throughout the experiment andsupplementedwith

an inhibitor of the lipolysis process to stop any further digestion.

(Ultra)centrifugation is applied to separate precipitated material

from the lipid and aqueous phases. Quantifying drug in any of the

separated phases allows insight in the extent of precipitation.

One of the major challenges in predicting the absorption

enhancing capacity of lipid-based formulations, is to capture the

complex interplay between solubilization, supersaturation andprecipitation in an environment with continuously evolving col-

loidal characteristics dueto lipolysis (Porter et al., 2011). Although

optimization and standardization is still ongoing (Williams et al.,

2012), a number of interesting in vitroin vivo correlations have

already been established (Anby et al., 2012; Fatouros et al., 2008;

Porter et al., 2004).

4. Critical variables in in vitro supersaturation evaluation

When designing in vitro assays for supersaturation evaluation,

variouschoiceshavetobemadeconcerningtheappliedexperimen-

tal conditions, including the mode of supersaturation induction

(discussed in Section 3.1), sink versusnon-sink conditions, the test

medium, hydrodynamics and the inclusion of an absorptive com-partment (Figs. 24). As will be illustrated in this section, these

experimental conditionsmayall significantlyaffect theoutcomeof

a supersaturation assay and require careful consideration.

4.1. Sink versus non-sink conditions

Currently, SDDS are often evaluated using slightly adapted one

compartment compendial dissolution methods. As these compen-

dial tests are usually run under sink conditions (large dissolutionvolumes or high surfactant concentration), they are only suited

to test release kinetics, e.g. in a quality control context. However,

in vivo predictive tools for evaluation of solubility-limited absorp-

tionandSDDSas absorption-enabling strategy, require application

ofmorebiorelevantnon-sinkconditions inthedissolutionmedium.

Only these circumstances allow adequate investigation of super-

saturation and precipitation. The need for non-sink conditions

during SDDS testing has recently been discussed in a commen-

tary by Augustijns and Brewster where meaningful in vitroin

vivocorrelationsfor silica basedformulationsandsoliddispersions

were only achieved using non-sink in vitro dissolution approaches

(Augustijns andBrewster,2012). VanSpeybroecket al. investigated

the absorption of fenofibrate from silica formulations with differ-

ent pore size in vivo (Van Speybroeck et al., 2010a). The achieved

bioavailability appeared inversely correlated with the pore size

(Fig. 5A), which could be predicted by in vitro dissolution studies

under non-sink (supersaturating) conditions (Fig. 5C). In contrast,

in vitro dissolution studies undersinkconditions, suggesting faster

release with larger pore size, did not correlate with the in vivo

outcome (Fig.5B). Also for entericmicroparticles containing carba-

mazepine,Donget al. illustrated thatin vivoplasmaconcentrations

were better predicted from in vitro dissolution kinetics obtained

under non-sink versus sink conditions (Dong et al., 2007).

4.2. Hydrodynamics

It is known form crystallization chemistry that hydrodyna-

mics will influence the nucleation and crystal growth process of

drug molecules (Baldyga and Orciuch, 2001; Manth et al., 1996).For instance, Dalvi et al. showed that a higher diffusivity of drug

Fig. 3. Different stirring and shaking patterns used in supersaturationassays.


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Fig. 4. Inclusionof an acceptor compartment in in vitro supersaturation assessment.

molecules in solution, dependent of the applied hydrodynamics,

will cause an increase in nucleation rate (Dalvi and Dave, 2010).

In general, the increased kinetic energy resulting from extensive

mixing may assist in overcoming the activation hurdle for nuclei

formation (Brouwers et al., 2009; Lindfors et al., 2008). However,

when evaluating the intraluminal supersaturation/precipitation

potential of drugs, very few studies have addressed the aspect of

hydrodynamics (Fig. 3). Carlert et al. compared the in vitro precip-

itation of a basic BCS class II drug (AZD0865) in a stirring model

(USP 2 mini-vessel setup, paddle speed of 150rpm) versus a shak-

ingmodel (85cycles/min,amplitude2 cm)(Carlert et al., 2010). The

observed precipitation rate was remarkably slower in the shakingmodel compared to the stirringmodel illustrating the importance

of the applied hydrodynamics in supersaturation/precipitation

evaluation. Since the in vivo gastrointestinal motility in the fasted

state is expected to be relatively low (Dressman et al., 1998) com-

pared to the thorough mixing that is usually applied in in vitro

dissolution/precipitation assays (McAllister, 2010), it has been

hypothesized that these in vitroassays oftenoverestimate precipi-

tation (Psachoulias et al.,2012). It is clear thatmore researchon the

nature of in vivo hydrodynamics and its implementation in disso-

lution/precipitation models (DArcy et al., 2009), may significantly

improve thebiorelevance of in vitro precipitation assessment.

4.3. Medium selection

From dissolution testing, it is well known that simple aque-

ous buffer solutions, designed for quality control purposes, are

not sufficient to accurately predict the in vivo performance of

drug formulations. Therefore,more biorelevant dissolution media

that simulate fasted and fed state conditions in the stomach

and the small intestine, have been developed (Jantratid et al.,

2008). Although not yet optimal, the use of these biorelevant

media has significantly improved the accuracy of in vitroin vivo

correlations (Dressman et al., 1998). Also precipitation kinetics

may be affected by components present in gastrointestinal flu-

ids, including bile salts and phospholipids. To this point, Lehto

et al. investigatedthecomplexdissolution andprecipitationbehav-

ior of carbamazepine in fasted state simulated intestinal fluid

(FaSSIF) (Lehto et al., 2009); intermolecular interactions between

sodiumtaurocholateandcarbamazepinewereshown to inhibitthe

dihydrate crystal formation, thereby preventing carbamazepine

precipitation.

Bycomparingtheprecipitationbehaviorofpoorlysolublemodel

compounds inhumanversus simulatedgastrointestinalfluids,Bev-

ernage et al. illustrated the importance of a careful selection of

media for supersaturation studies (Bevernage et al., 2010, 2011,

2012a). To predict precipitation kinetics in the intestinal envi-

ronment, simple aqueous buffer solutions at pH 6.5 should be

avoided as they significantly overestimate the stability of super-

saturation. ThecommonlyusedFaSSIF performsreasonablywell in

predicting the precipitation behavior in fasted state human fluids.For the fed state, in contrast, fed state simulated intestinal fluid

(FeSSIF) may significantly underestimate precipitation. Regarding

precipitation simulation in the gastric environment, fasted state

simulated gastric fluid (FaSSGF), containing small amounts of tau-

rocholate and lecithin (Vertzoni et al., 2007), should be used in

place ofUSP-simulatedgastricfluid, since theformerunderpredicts

precipitation. The capacity of buffer solutions and simulated gas-

trointestinal fluids to predict excipient-mediated supersaturation

stabilization in human fluids, appears to be inconsistent, limiting

the usefulness of screening assays for precipitation inhibitors. A

better understanding of themechanisms underlying precipitation

inhibition is clearly needed (Brouwers andAugustijns, 2012).

4.4. Temperature

For practical reasons, the majority of in vitro supersaturation

assays are currently performed at room temperature instead of a

more relevant temperature of 37 C. Literature on the importance

of this temperature difference in supersaturation evaluation is

veryscarce. Using Ramanspectrophotometry, Alonzoet al. demon-

strated an altered dissolution behavior of amorphous felodipine

at 37 C versus 25 C, resulting from temperature-dependent

felodipine crystallization kinetics upon contact with the dissolu-

tionmedium.As such, dissolutionofamorphous felodipine induced

supersaturation at 25C but not at 37 C (Alonzo et al., 2010). This

example illustrates thatsupersaturation/precipitationdataat 25 C

canonlybeextrapolatedto 37 Cwith caution; forpredictionof the

in vivo performance of SDDS, it is advisable towork at 37C.


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Fig. 5. In vitro behavior andin vivoperformanceof fenofibrate loadedinto orderedmesoporous silicamaterialswith varying pore sizes (2.7nm,4.4nm,7.3 nm). Thein vivo

performance of the differentformulations in rats after gastric dosing (A). In vitro dissolution tests under sink (B)and non-sink (C) conditions.

4.5. Implementing an acceptor/absorptive compartment

Implementingan acceptorcompartmentwith sinkconditionsto

simulate absorption has proven to increase the predictive power

of dissolution tests for drugs suffering from dissolution-limited

absorption or administered as modified release formulations(McAllister,2010). Since thepossibilityof permeationmaynotonly

affect dissolution butalso precipitation kinetics, the addition of an

acceptorcompartmentmaybe crucial for thepredictive evaluation

of SDDS. In a thermodynamically unstable supersaturated system,

permeation into a sink compartmentmay act as an alternative for

precipitation to lower the systems Gibbs free energy. Recently,

Bevernage et al. demonstrated that precipitation of loviride was

significantly reduced in the presence of an absorption compart-

ment simulated in the Transwell Caco-2 model. As a result, the

stabilityof supersaturation observed in a classic one-compartment

setup without an absorption compartment did not correlate with

transport across theCaco-2monolayer (Bevernageet al., 2012a).

In practice, thesimulation of absorption hasbeenaccomplished

by different experimental approaches, including the addition ofan immiscible organic layer as absorptive sink (biphasic model),

or the integration of an actual absorption compartment sepa-

rated from the dissolution medium by a filter/pump combination

or a Caco-2 monolayer (Kataoka et al., 2012; McAllister, 2010).

The added value of combined dissolution-absorption systems for

the evaluation of SDDS was demonstrated by Shi et al. (2010).

The authors used a biphasic system to assess release profiles of

the poorly soluble drug celecoxib from three formulations (the

commercial Celebrex capsule, a solution containing co-solvent

and surfactant and a supersaturable self-emulsifying drug deliv-

ery system (S-SEDDS)). The setup consisted of a USP IV apparatus

for aqueous dissolution under non-sink conditions connected to a

USPII apparatuscontainingan additionalwater immiscibleoctanol

layer creating an acceptor compartment with sink conditions.

For comparison purposes, release profiles were also assessed in

a monophasic system under both sink and non-sink conditions.

None of thereleaseprofiles obtained inaqueousmediausingeither

monophasic (Fig.6A) orbiphasicsetups (Fig.6B) coulddiscriminate

among the three formulationsandpredict the in vivo bioavailabil-

ity of celecoxib. Only the concentration profiles obtained in theoctanol phase of the biphasic system (Fig. 6C) could be correlated

to the invivooutcome (Fig. 6D), illustratingthe necessityof a com-

bined dissolutionabsorption model to allow reliable in vitroin

vivo correlations.

5. In vivo evaluation of supersaturation

Considering the large number of experimental variables that

mayaffect the in vivo predictive power of in vitro supersaturation

assays, the bioperformance of SDDS can often only be established

using in vivo studies in animals or humans. These in vivo studies

are also necessary as a reference for the optimization of existing

in vitro assays. Importantly,classic pharmacokinetic studies donot

allowfor thedemonstrationof supersaturation/precipitationat thesite of absorption, making it extremely difficult to judge the pre-

cise contribution of intraluminal supersaturation to the observed

plasma concentrationtime profile. Bothdirect and indirectmeth-

ods have been described to provide evidence of the intraluminal

supersaturation behavior of drugs.

5.1. Indirect evaluation of intraluminal supersaturation

The indirect approach combines in vitro dissolution data with

modeling techniques to simulate intraluminal drug behavior and

explain the observed pharmacokinetic profiles in vivo. In a study

by Shono et al., physiologically based pharmacokinetic model-

ing (PBPK modeling) using STELLA software was used to predict

plasmaprofilesof theweaklybasicdrugnelfinavir,basedon invitro


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Fig. 6. Dissolutionprofiles of celecoxib(CEB)from3 types of formulationsobtainedin a singlephasedissolutiontest (A), theaqueous phase of a biphasic dissolution test (B)

and theoctanol phase of a biphasic dissolution test (C). Therelative in vivo AUCandCmax of the respective formulationsare also included (D).

dissolution data from nelfinavir mesylate tablets in biorelevant

media, standard gastrointestinal parameters and the disposition

pharmacokineticsof nelfinavir (Shonoetal.,2011). Inthefasted,but

not the fed state, accurate simulation of the in vivo performance of

nelfinavir required the implementationof drugprecipitationbased

on the crystal growth theory, in the PBPK model. This suggests

that intestinal precipitation limits the fasted state absorption of

the weakly basic nelfinavir. Similarly, Takano et al. used deconvo-lution of plasma concentrationtime profiles and PBPK modeling

to simulate the in vivo intraluminal drug concentration of a novel

farnesyltransferase inhibitor (FTI-2600) (Takano et al., 2010). After

administrationofthecrystallineHCl saltto dogs, themeasuredCmaxand AUC0-infwere 4-fold higher compared to the free base. Based

on the PK data, intraluminal concentrations in the small intestine

were modeled. For the free base, simulated intestinal concentra-

tions agreedwith the equilibrium solubility; for the salt, however,

a 4-fold higher intestinal concentration was predicted, indicating

that intestinal supersaturation was responsible for the improve-

ment in bioavailability.

5.2. Direct evaluation of intestinal concentrations

In an attempt to directly evaluate in vivo supersaturation and

precipitation, Psachoulias et al. applied a duodenalaspirationtech-

nique after intragastric administration of an acidic solution of 2

weakly basic drugs (dipyridamole and ketoconazole) in healthy

adults(Psachouliaset al.,2011). Both the totaldrug content andthe

soluble drug fraction in duodenal aspirateswere determined upon

sampling. To allow assessment of supersaturation, equilibrium

solubilitieswerealsodetermined ineachaspirate,aftertheaddition

of an excess of crystalline drug. Based on these data, the fraction

precipitatedandtherelativesupersaturation indexwerecalculated

as a function of time. Although the duodenal fluids appeared to be

supersaturatedat several earlytimepoints (540min), onlylimited

precipitation (maximum 7% and 16% for dipyridamole and keto-

conazole, respectively) was observed, indicating that absorption

of these weakly basic drugs is only minimally affected by duode-

nalprecipitation.Interestingly,previouslyassessedprecipitationof

dipyridamole in simulated intestinal fluids using an in vitro trans-

fermodel overestimatedduodenal precipitationwhencomparedto

the in vivo observations (Kostewicz et al., 2004). This emphasizes

the need for in vivo reference data on intraluminal supersatura-

tion andprecipitationto optimize invitroassays(Psachouliaset al.,

2012).

6. Concluding remarks

The increasing awareness of the potential of supersaturation

as an enabling formulation approach for drugs suffering from

solubility-limited absorption, stimulates the need for in vivo

predictive supersaturation/precipitation assays. Since contempo-

rary evaluation methods are mostly adapted dissolution tests,

they cannot be simply considered biorelevant in a supersa-

turation/precipitation context. Although several experimental

variables have been identified as being essential for the reliable

simulationofSDDS(e.g.medium,dissolution rate, transit,hydrody-

namics, absorption), their integration in preclinical assays remains

immature. This is mainly due to (1) our erratic understandingof the multifactorial process of precipitation and precipitation

inhibition, especially in a complex and varying environment as

the gastrointestinal tract, and (2) the lack of in vivo reference

data on intraluminal supersaturation behavior to guide model

optimization. Facing these challenges will be key to the successful

adoption of the supersaturation concept as a rational formulation

strategy.

References

Alonzo, D.E., Zhang, G.G.Z., Zhou, D., Gao, Y., Taylor, L.S., 2010. Understanding thebehaviorof amorphouspharmaceuticalsystemsduringdissolution. Pharm. Res.27, 608618.

Anby,M.U.,Williams,H.D., McIntosh,M., Benameur,H., Edwards,G.A.,Pouton,C.W.,

Porter, C.J.H., 2012. Lipid digestion as a trigger for supersaturation: evaluation


10/11


11/11


Rodrguez-Hornedo,N.,Murphy, D., 1999. Significance of controlling crystallizationmechanisms andkinetics in pharmaceutical systems.J. Pharm. Sci.88, 651660.

Sassene,P.J., Knopp,M.M.,Hesselkilde, J.Z., Koradia,V.,Larsen,A., Rades, T.,Mllertz,A., 2010. Precipitation of a poorly soluble model drug during in vitro lipolysis:characterizationand dissolution of theprecipitate. J. Pharm. Sci. 99, 49824991.

Shanbhag, A., Rabel, S., Nauka, E., Casadevall, G., Shivanand, P., Eichenbaum, G.,Mansky, P.,2008.Methodfor screeningof solid dispersion formulationsof low-solubility compoundsminiaturization and automation of solvent casting anddissolution testing. Int. J. Pharm. 351, 209218.

Shi, Y., Gao, P., Gong, Y., Ping, H., 2010. Application of a biphasic test for character-ization of in vitro drug release of immediate release formulations of celecoxib

and its relevanceto in vivoabsorption.Mol. Pharm.7, 14581465.Shono, Y., Jantratid,E., Dressman,J.B., 2011.Precipitation in the smallintestinemay

play a more important role in the in vivo performance of poorly soluble weakbases in the fasted state: case example nelfinavir. Eur. J. Pharm. Biopharm. 79,349356.

Singhal, D., Curatolo,W., 2004.Drug polymorphismanddosageformdesign:a prac-tical perspective. Adv.Drug Deliv. Rev. 56, 335347.

Stegemann, S., Leveiller, F., Franchi, D., de Jong, H., Lindn, H., 2007. When poorsolubility becomes an issue: from early stage to proof of concept. Eur. J. Pharm.Sci. 31, 249261.

Stuart, M., Box, K., 2005. Chasing equilibrium:measuring the intrinsic solubility ofweak acids andbases.Anal. Chem. 77, 983990.

Takano, R.,Takata, N., Saito,R., Furumoto, K.,Higo, S.,Hayashi, Y.,Machida, M., Aso,Y., Yamashita, S., 2010. Quantitative analysis of theeffect of supersaturationonin vivodrug absorption. Mol. Pharm.7, 14311440.

Usui, F.,Maeda, K.,Kusai, A.,Nishimura, K.,Yamamoto, K.,1997.Inhibitoryeffects ofwater-soluble polymers on precipitation of RS-8359. Int. J. Pharm.154, 5966.

Van Speybroeck, M., Mellaerts, R., Mols, R., Thi, T.D., Martens, J.A., Van Humbeeck,J., Annaert, P., Van denMooter, G., Augustijns, P., 2010a. Enhanced absorption

of the poorly soluble drug fenofibrate by tuning its release rate from orderedmesoporous silica. Eur. J. Pharm.Sci. 41, 623630.

Van Speybroeck, M., Mols, R., Mellaerts, R., Thi, T.D., Martens, J.A., Humbeeck, J.V.,Annaert, P., Mooter, G.V., den Augustijns, P., 2010b. Combined use of orderedmesoporous silica and precipitation inhibitors for improved oral absorption ofthepoorly solubleweakbase itraconazole.Eur. J.Pharm.Biopharm.75, 354365.

Vandecruys, R., Peeters, J., Verreck, G., Brewster, M.E., 2007. Use of a screeningmethod to determine excipients which optimize the extent and stability ofsupersaturated drug solutions and application of this system to solid formu-lationdesign. Int. J. Pharm.342, 168175.

Vertzoni,M., Pastelli, E., Psachoulias, D., Kalantzi, L., Reppas, C., 2007. Estimation of

intragastric solubility of drugs: in what medium? Pharm.Res. 24, 909917.Warren, D.B., Benameur,H.,Porter, C.J.H., Pouton, C.W., 2010. Using polymericpre-

cipitation inhibitorsto improve theabsorptionof poorly water-soluble drugs: amechanistic basis for utility. J. Drug Target. 18, 704731.

Williams,H.D., Sassene,P., Kleberg,K., Bakala-Ngoma,J.-C., Calderone,M., Jannin,V.,Igonin,A., Partheil,A., Marchaud,D., Jule,E.,Vertommen, J.,Maio,M.,Blundell,R.,Benameur,H., Carrire, F.,Mllertz,A., Porter, C.J.H., Pouton, C.W.,2012. Towardthe establishment of standardized in vitro tests for lipid-based formulations,part 1: method parameterization and comparison of in vitro digestion profilesacross a range of representative formulations. J. Pharm. Sci. 101,33603380.

Wu, H., Khan, M.A., 2011. Quality-by-design: an integratedprocess analytical tech-nology approach to determinethe nucleationand growthmechanisms during adynamic pharmaceutical coprecipitationprocess.J. Pharm. Sci.100, 19691986.

Yamashita, T., Kokubo, T., Zhao, C., Ohki, Y., 2010. Antiprecipitantscreening systemfor basicmodel compoundsusing bio-relevantmedia. J. Assoc. Lab.Automat.15,306312.

Yamashita, T., Ozaki, S., Kushida, I., 2011. Solvent shift method for anti-precipitantscreeningof poorlysoluble drugs using biorelevantmediumand dimethyl sulf-oxide. Int. J. Pharm.419, 170174.

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