International Journal of Pharmaceutics 453 (2013) 2535

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

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

Review

Evaluation of gastrointestinal drug supersaturation and precipitation: Strategies

and issues
Jan Bevernage a,1 , Joachim Brouwers a,1 , Marcus E. Brewster b , Patrick Augustijns a,
a
b

Laboratory for Pharmacotechnology and Biopharmacy, KU Leuven, O&N 2, Herestraat 49 Box 921, 3000 Leuven, Belgium
Drug Product Development, Pharmaceutical Development and Manufacturing Sciences Janssen Research and Development, Johnson & Johnson, Beerse, Belgium

a r t i c l e

i n f o

Article history:
Received 23 September 2012
Received in revised form
13 November 2012
Accepted 15 November 2012
Available online 27 November 2012
Keywords:
Supersaturation
Intestinal absorption
Precipitation

a b s t r a c t
Supersaturating drug delivery systems (SDDS) hold the promise of enabling intestinal absorption for
difcult-to-formulate, poorly soluble drug candidates based on a design approach that includes (1) converting the drug into a high energy or rapidly dissolving system which presents a supersaturated solution
to the gastrointestinal environment and (2) dosage form components that act to stabilize the formed
metastable drug solution through nucleation and/or crystal growth inhibition. The appropriate development and study of SDDS require that useful and biorelevant supersaturation and precipitation assays are
available. This review summarizes different methodological aspects of currently available in vitro assays,
including the generation of supersaturation (solvent shift, pH shift or formulation-induced), the quantication of supersaturation and the detection of precipitation. Also down-scaled approaches, including
96-well plate setups, are described and situated in the pharmaceutical development cycle based on their
consumption of API as well as time requirements. Subsequently, the ability to extrapolate in vitro supersaturation assessment to the in vivo situation is discussed as are direct and indirect clinical tools that can
shed light on SDDS. By emphasizing multiple variables that affect the predictive power of in vitro assays
(e.g. the nature of the test media, hydrodynamics, temperature and sink versus non-sink conditions), this
review nally highlights the need for further harmonization and biorelevance improvement of currently
available in vitro procedures for supersaturation and precipitation evaluation.
2012 Elsevier B.V. All rights reserved.

Contents
1.
2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In vitro supersaturation evaluation: fundamental aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Quantitating supersaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1.
Distinguishing supersaturation from solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
Solid phase separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Detecting precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Solid state analysis of the precipitate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
An overview of in vitro supersaturation assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Supersaturation evaluation of non-formulated drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Solvent shift methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
pH-shift methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.
Potentiometric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.4.
High throughput supersaturation assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Supersaturation evaluation of formulated drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Dissolution methods for the evaluation of SDDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2.
Higher throughput evaluation of SDDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3.
Evaluation of lipid based formulations as SDDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +32 16 33 03 01; fax: +32 16 33 03 05.

E-mail address: Patrick.Augustijns@pharm.kuleuven.be (P. Augustijns).
1
These authors equally contributed to the manuscript and should both be considered as rst author.
0378-5173/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2012.11.026

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4.

5.

6.

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

Critical variables in in vitro supersaturation evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.1.
Sink versus non-sink conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Hydrodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Medium selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.
Implementing an acceptor/absorptive compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In vivo evaluation of supersaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Indirect evaluation of intraluminal supersaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Direct evaluation of intestinal concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

t = DSt 1 =

The impediments associated with obtaining sufcient oral

bioavailability for poorly soluble drugs (Stegemann et al., 2007;
Lipinski, 2000) has compelled pharmaceutical scientists to develop
formulations that enable absorption by providing for intestinal
supersaturation, i.e. drug concentrations that exceed the equilibrium solubility in the intestinal environment. In view of their
potential, the use of these supersaturating drug delivery systems
(SDDS) has received increased attention (Brouwers et al., 2009).
Supersaturation is a thermodynamically metastable state that constitutes the driving force for precipitation. Therefore, the clinical
benet of SDDS in enhancing drug bioavailability will greatly
depend on the stability of the induced supersaturated state and,
hence, the kinetics of precipitation. Appropriate evaluation of
supersaturation, precipitation and possibly precipitation inhibition
is therefore key for the efcient development and optimization of
SDDS. As the in vivo evaluation of intraluminal SDDS behavior is
burdened with practical difculties, efforts have been directed to
the development of predictive in vitro precipitation assays. A multitude of approaches designed from different perspectives have been
reported in literature (Gao and Shi, 2012; Wu and Khan, 2011; Li
et al., 1998), illustrating the need for harmonization and rationalization. This review provides an overview of the reported in vitro
and in vivo approaches to evaluate the supersaturation potential of
drug molecules, the precipitation inhibition capacity of excipients,
and the performance of oral SDDS. In particular, this review highlights specic requirements and issues that need to be addressed
to achieve more predictive supersaturation evaluation as well as to
translate these assays to practical bioavailability concerns.

2. In vitro supersaturation evaluation: fundamental aspects

2.1. Quantitating supersaturation
The in vitro evaluation of supersaturation essentially involves
(1) inducing supersaturation in the medium of interest (see
below) and (2) assessing drug concentrations in solution as a
function of time. The latter step resembles classic concentration
assessment during traditional dissolution testing. However, useful
supersaturation quantitation requires that the measured drug
concentrations are combined with the equilibrium solubility of
the drug in the test medium under consideration, with inclusion of
solubility altering components that are added or which comprise
the formulation. In this way, the measured drug concentration can
be expressed relative to the equilibrium solubility as the degree
of supersaturation (DS) (Eq. (1)) or the supersaturation index ()
(Eq. (2)).
DSt =

Ct
Ceq

(1)

Ct Ceq
Ceq

30
30
30
31
31
32
32
32
33
33
33

(2)

where Ct represents the drug concentration at the time of measurement, and Ceq is the equilibrium solubility of the drug in the
test medium. The quantication of supersaturation allows identifying the saturation extent (DS < 1,  < 0: subsaturated, DS = 1,
 = 0: saturated, DS > 1,  > 0: supersaturated) as a measure of the
thermodynamic tendency for precipitation (Rodrguez-Hornedo
and Murphy, 1999). Instead of concentrationtime proles, results
from supersaturation assays can be expressed as DStime proles
as depicted in Fig. 1, allowing for a better appreciation of the
supersaturation behavior. Based on the area under the curve (AUC)
of the DStime proles, summarizing metrics have been applied
(Fig. 1). The supersaturation factor represents the fold increase in
AUC of the DStime curve compared to the saturated condition.
Similarly, the potential precipitation inhibition capacity of an
excipient can be expressed as the excipient gain factor, representing the increase in AUC of the DStime curve upon inclusion of the
excipient (Bevernage et al., 2011, 2012b).
2.1.1. Distinguishing supersaturation from solubilization
The determination of the equilibrium solubility in conditions
equivalent to the supersaturation assay enables one to distinguish
between supersaturation induction and thermodynamic solubilization as absorption-enhancing strategy for poorly water soluble
drugs. This distinction is essential to fully understand and appreciate the behavior of an SDDS and its potential usefulness. In
contrast to solubilization, a supersaturated state is thermodynamically unstable and one should be aware of precipitation as a
potential limitation on the biopharmaceutical potential of SDDS. If
the supersaturated state remains metastable for a sufcient period
of time, however, it may be more efcient than solubilization
in promoting absorption. In contrast to solubilization, supersaturation typically increases the free drug concentration, without
altering the tendency of the drug to permeate into and across
the epithelial monolayer. As such, the increased drug concentration is readily available for absorption. This is conrmed in studies
that assessed the transepithelial permeation from supersaturated
solutions (Bevernage et al., 2012a; Miller et al., 2012; Mellaerts
et al., 2008). For instance, in absence of precipitation, loviride
transport across Caco-2 monolayers linearly increased with the
degree of supersaturation that was induced apically (Bevernage
et al., 2012a). In case of solubilization based on micellization (surfactants), complexation (cyclodextrins) or even cosolvency, the
solubility enhancement may be compromised by a reduction in
permeability (Beig et al., 2012; Miller et al., 2012; Dahan et al.,
2010).
2.1.2. Solid phase separation
The quantitative evaluation of supersaturation/precipitation
requires immediate ltration or (ultra)centrifugation to accurately
separate non-dissolved or precipitated drug from the dissolved

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

27

Fig. 1. Typical degree of supersaturation (DS)time proles for a saturated solution (DS = 1) and a supersaturated solution with or without precipitation inhibitor; summarizing
metrics for supersaturation assessment based on the area under these DStime proles, are illustrated.

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

Since they enable faster phase separation, ltration techniques
are preferred. However, the use of ltration 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 the presence
of nanoparticles cannot be excluded. It may therefore be valuable
to conrm the absorption-enhancing capacity of supersaturating
formulations by ux measurements across an epithelial cell layer
(Brouwers and Augustijns, 2012; Mellaerts et al., 2010).
2.2. Detecting precipitation
As an alternative to the quantitative approach of evaluating supersaturation, detection of precipitation has also been
applied to assess supersaturation stability. Different methodologies to detect precipitation have been described, including visual
inspection and light microscopy (Raghavan et al., 2001), spectrophotometric UV/VIS absorbance-time measurements (Ozaki et al.,
2012; Chandran et al., 2011), and nephelometric turbidity measurements (Warren et al., 2010). Precipitation detection techniques
avoid phase separation and subsequent drug quantication, lending them to medium- to high-throughput screening assays and
making them suitable for continuous in-process precipitation monitoring. Typically, the lag time before appearance of any crystals
(induction or nucleation time) is detected as, for instance, a sudden increase in absorbance and used to rank different formulations
or precipitation inhibitors. Arnold et al. proposed the use of in-line
Raman spectrophotometry monitoring during precipitation assays.
Changes in Raman spectra were not only used to dene the nucleation 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 feasible without the need for phase separation (Arnold et al., 2011).

microscopy, X-ray diffraction and excipient content analysis to

reveal the inuence of polymer (HPMC, HPC and PVP) on the crystal
shape of precipitated RS-8359 (Usui et al., 1997). Sassene et al. analyzed cinnarizine precipitate formed during the in vitro lipolysis of
a SMEDDS formulation (Sassene et al., 2010). X-ray powder diffraction and polarized light microscopy illustrated that cinnarizine
precipitated in an amorphous rather than a crystalline form. Since
the amorphous form dissolved signicantly faster than crystalline
cinnarizine, redissolution may limit the impact of precipitation on
the bioperformance of the cinnarizine SMEDDS. Finally, potential
polymorphic transitions during precipitation may further complicate the supersaturation behavior and warrant solid state analysis
(Llins and Goodman, 2008; Singhal and Curatolo, 2004).
3. An overview of in vitro supersaturation assays
Multiple in vitro assays to evaluate supersaturation, precipitation or precipitation inhibition are reported in literature. They
differ in (1) approaches to generate supersaturation, (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 variables and concerns will be discussed in Section 4.
3.1. Supersaturation evaluation of non-formulated drugs

2.3. Solid state analysis of the precipitate

To evaluate the supersaturation/precipitation potential of

native (non-formulated) drugs and/or the precipitation inhibition
capacity of excipients, induction of supersaturation is the starting
point. Various methods to generate supersaturated drug concentrations can be found in the eld of crystallization chemistry,
including solvent evaporation or freezing, addition of ions that participate 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 testing the supersaturation potential of non-formulated drugs, only
solvent- or pH-shift methods have been commonly applied.

Solid state analysis of precipitated drug may be a useful tool

to investigate the mechanism of precipitation and excipientmediated precipitation inhibition. Usui et al. used scanning electron

3.1.1. Solvent shift methods

The solvent shift or co-solvent quench method is a popular and
simple approach to create supersaturation (Yamashita et al., 2011;

28

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

Fig. 2. Different media and approaches to generate supersaturation that are commonly used in in vitro supersaturation assays.

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

water soluble drug is rst dissolved in a water miscible solvent
with a signicantly higher solubilizing capacity for the drug than
the aqueous environment in which supersaturation is being evaluated. 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 generated as a result of the solubility difference. Finally, supersaturation
and/or precipitation can be monitored by means of the aforementioned techniques (see Section 2). Knowing the solubility of the
drug in the aqueous medium, any desired initial degree of supersaturation can easily be set by tuning the drug concentration in the
co-solvent solution and/or the transferred amount. Possible alterations 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 any poorly
water soluble drug that can be dissolved at signicantly higher concentration in a water miscible co-solvent. Because of the use of drug
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 biorelevance 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 induce a supersaturated state. This implies that pH-shift methods can only be applied to investigate the supersaturation behavior
of neutral drug species. Compared to a solvent shift, a pH-shift
may be considered more biorelevant for weakly 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 compartment where for example an acidic solution of a basic drug is
supplemented with a buffering agent to increase the pH above the
materials pKa (Carlert et al., 2010; Yamashita et al., 2010; Mellaerts
et al., 2008; Overhoff et al., 2008). Alternatively, a pH shift can
be achieved upon bolus or continuous transfer between two compartments: 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 methods
An alternative pH shift approach to investigate supersaturation
phenomena of ionizable drugs is the method of chasing equilibrium solubility or CheqSol system introduced by Sirius (Box et al.,
2006). This potentiometric technique was 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 quantify the extent and duration of 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 ionized form. The solution of ionized solute is back titrated by adding
measured quantities of a titrant (KOH for basic compounds and
HCl for acidic compounds) to form the less soluble unionized form,
resulting in supersaturation and subsequent precipitation which is
detected by an increase in apparent absorbance using a ber optic
dip probe. As soon as a sufcient quantity of precipitate has been
formed, the process of chasing equilibrium starts by repeated pHinduced alterations from a supersaturated to a subsaturated state
and vice versa. During this process, the small pH changes resulting
from gradual precipitation or dissolution can be used to calculate
the concentration of neutral species as a function of time using mass
and charge balance equations, provided that accurate knowledge of
(1) the pKa (s) and concentration of the ionizable drug and (2) total
volume and concentration of added titrant, is available (Stuart and
Box, 2005).
For many ionizable drugs (the so-called chasers), supersaturated concentrations of neutral species well above their intrinsic
equilibrium solubility have been recorded prior to precipitation.

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

Drugs that precipitate immediately to the intrinsic equilibrium

solubility (non-chasers) have no or limited potential for supersaturation. For chasers, the concentrationtime prole 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
effects on the supersaturation stability of ionizable drugs (Box et al.,
2009). Using the Cheqsol approach, Hsieh et al. examined supersaturation 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 shortlived supersaturation was associated with crystalline precipitation,
prolonged supersaturation occurred in case of amorphous precipitation. In this context, they also found the pH-titration method to be
a practical way to determine the solubility of amorphous materials.
3.1.4. High throughput supersaturation assays
The lack of a theoretical basis for the selection of supersaturation 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 to
identify excipients that affect supersaturation (Vandecruys et al.,
2007). Drug candidates dissolved in DMA or DMF were gradually added to the dissolution medium in presence or absence of
excipient (10 ml, 0.01 N HCl, USP buffer pH 4.5 or 6.8 or water,
stirred at 600 rpm using a magnetic stirrer bar) until visual detection of precipitation; subsequently, the dissolution medium was
sampled, ltered and analyzed (UV absorbance) over a period of
120 min. Similarly, Yamashita et al. developed a 96-well based high
throughput format to evaluate the capacity of excipients to stabilize
itraconazole supersaturation (Yamashita et al., 2011). Itraconazole
was dissolved in DMSO from which 4 l was added to FaSSIF containing 0.015% excipient. Shaking of the plate was provided and
at appropriate times, samples were taken and ltered using a lter
plate. Interestingly, the results obtained using this high throughput
solvent shift method were conrmed in the dissolution proles of
solid dispersions containing identical excipients using the Japanese
Pharmacopeia paddle dissolution method (300 ml FaSSIF, 50 rpm).
In addition, similar results were obtained using a pH-shift to generate the supersaturated state (Yamashita et al., 2010).
The high throughput induction of supersaturation is often combined with online detection of precipitation to generate immediate
results. For instance, Warren et al. employed a solvent shift method
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
To evaluate the behavior of SDDS, supersaturation should not be
induced as part of the assay, but as an inherent characteristic of the
formulation. Predictive performance evaluation requires an assay
that provides an adequate aqueous environment, relevant for the
gastrointestinal tract. While critical variables (e.g. sink versus nonsink conditions, medium selection, etc.) are discussed in Section 4,
this paragraph provides an overview of the different experimental
setups that have been reported for SDDS evaluation.
3.2.1. Dissolution methods for the evaluation of SDDS
To evaluate the dissolution properties of formulations that have
the potential to induce drug supersaturation, traditional one compartment/one phase setups, often based on USP I or II apparatus,
are commonly applied. To facilitate rapid sample treatment when

29

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
the centrifuge (Curatolo et al., 2009; Friesen et al., 2008) have been
proposed.
To test formulations that rely on the gastrointestinal pH gradient to generate supersaturation, this pH shift needs to be simulated
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
precipitation kinetics in gastric versus intestinal media (Bevernage
et al., 2012b). Both one 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
setup comprising a gastric, intestinal and absorption compartment
have been used to predict precipitation from formulations of weak
bases such as dipyridamole and cinnarizine (Gu et al., 2005). Compared 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 conguration requires fast and economic methods to
evaluate SDDS. However, experience with higher throughput dissolution methods, especially in an SDDS context, is fairly limited.
Dai et al. developed a 96-well based precipitation method to rapidly
evaluate drug precipitation from liquid formulations upon dilution
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 liquid formulation was formed in situ after which it
was diluted using biorelevant media. After the desired incubation,
samples were transferred to a lter plate and the resulting ltrate
was diluted and analyzed. The obtained results correlated with
traditional USP dissolution testing and allowed high throughput
discrimination between fast, medium and slow precipitating formulations. 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 cannot 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 solutions in volatile solvents were used to prepare solvent casts in
a 96-well plate. Dissolution was performed through addition of
300 l simulated intestinal uid (SIF) to each well. Samples were
ltered using a lter plate and the resulting ltrate was diluted
prior to quantication with UV spectroscopy. Although this dissolution screening method does not provide information on the
physical state of the solid formulations, it appeared quite successful
in identifying formulations that signicantly enhanced dissolution
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 et al., 2010). Typically, the absorption enabling
properties of lipid based systems have been attributed to the
enhanced solubilizing capacity of gastrointestinal uids upon

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

dispersion and digestion of 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
as a trigger for supersaturation and subsequent precipitation (Anby
et al., 2012; Sassene et al., 2010; Porter et al., 2004). In vitro
performance evaluation of lipid-based formulations thus requires
supersaturation/precipitation assays that include a digestion (lipolysis) 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
reects fasted state conditions; digestion is initiated by addition
of pancreatic enzymes. During lipolysis, the pH is maintained by
titration of liberated fatty acids using a pH-stat titration unit. Samples are taken throughout the experiment and supplemented with
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 and
precipitation in an environment with continuously evolving colloidal characteristics due to 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,
various choices have to be made concerning the applied experimental conditions, including the mode of supersaturation induction
(discussed in Section 3.1), sink versus non-sink conditions, the test
medium, hydrodynamics and the inclusion of an absorptive compartment (Figs. 24). As will be illustrated in this section, these

experimental conditions may all signicantly affect the outcome of

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 compendial tests are usually run under sink conditions (large dissolution
volumes 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 absorption and SDDS as absorption-enabling strategy, require application
of more biorelevant non-sink conditions in the dissolution medium.
Only these circumstances allow adequate investigation of supersaturation and precipitation. The need for non-sink conditions
during SDDS testing has recently been discussed in a commentary by Augustijns and Brewster where meaningful in vitroin
vivo correlations for silica based formulations and solid dispersions
were only achieved using non-sink in vitro dissolution approaches
(Augustijns and Brewster, 2012). Van Speybroeck et al. investigated
the absorption of fenobrate from silica formulations with different 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 under sink conditions, suggesting faster
release with larger pore size, did not correlate with the in vivo
outcome (Fig. 5B). Also for enteric microparticles containing carbamazepine, Dong et al. illustrated that in vivo plasma concentrations
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 hydrodynamics will inuence 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 supersaturation assays.

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

31

Fig. 4. Inclusion of 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 precipitation of a basic BCS class II drug (AZD0865) in a stirring model
(USP 2 mini-vessel setup, paddle speed of 150 rpm) versus a shaking model (85 cycles/min, amplitude 2 cm) (Carlert et al., 2010). The
observed precipitation rate was remarkably slower in the shaking
model compared to the stirring model 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) compared to the thorough mixing that is usually applied in in vitro
dissolution/precipitation assays (McAllister, 2010), it has been
hypothesized that these in vitro assays often overestimate precipitation (Psachoulias et al., 2012). It is clear that more research on the
nature of in vivo hydrodynamics and its implementation in dissolution/precipitation models (DArcy et al., 2009), may signicantly
improve the biorelevance of in vitro precipitation assessment.

sodium taurocholate and carbamazepine were shown to inhibit the

dihydrate crystal formation, thereby preventing carbamazepine
precipitation.
By comparing the precipitation behavior of poorly soluble model
compounds in human versus simulated gastrointestinal uids, Bevernage 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 environment, simple aqueous buffer solutions at pH 6.5 should be
avoided as they signicantly overestimate the stability of supersaturation. The commonly used FaSSIF performs reasonably well in
predicting the precipitation behavior in fasted state human uids.
For the fed state, in contrast, fed state simulated intestinal uid
(FeSSIF) may signicantly underestimate precipitation. Regarding
precipitation simulation in the gastric environment, fasted state
simulated gastric uid (FaSSGF), containing small amounts of taurocholate and lecithin (Vertzoni et al., 2007), should be used in
place of USP-simulated gastric uid, since the former underpredicts
precipitation. The capacity of buffer solutions and simulated gastrointestinal uids to predict excipient-mediated supersaturation
stabilization in human uids, appears to be inconsistent, limiting
the usefulness of screening assays for precipitation inhibitors. A
better understanding of the mechanisms underlying precipitation
inhibition is clearly needed (Brouwers and Augustijns, 2012).

4.3. Medium selection

4.4. Temperature
From dissolution testing, it is well known that simple aqueous buffer solutions, designed for quality control purposes, are
not sufcient 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 signicantly improved the accuracy of in vitroin vivo
correlations (Dressman et al., 1998). Also precipitation kinetics
may be affected by components present in gastrointestinal uids, including bile salts and phospholipids. To this point, Lehto
et al. investigated the complex dissolution and precipitation behavior of carbamazepine in fasted state simulated intestinal uid
(FaSSIF) (Lehto et al., 2009); intermolecular interactions between

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
very scarce. Using Raman spectrophotometry, Alonzo et al. demonstrated an altered dissolution behavior of amorphous felodipine
at 37 C versus 25 C, resulting from temperature-dependent
felodipine crystallization kinetics upon contact with the dissolution medium. As such, dissolution of amorphous felodipine induced
supersaturation at 25 C but not at 37 C (Alonzo et al., 2010). This
example illustrates that supersaturation/precipitation data at 25 C
can only be extrapolated to 37 C with caution; for prediction of the
in vivo performance of SDDS, it is advisable to work at 37 C.

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

Fig. 5. In vitro behavior and in vivo performance of fenobrate loaded into ordered mesoporous silica materials with varying pore sizes (2.7 nm, 4.4 nm, 7.3 nm). The in vivo
performance of the different formulations 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

Implementing an acceptor compartment with sink conditions to
simulate absorption has proven to increase the predictive power
of dissolution tests for drugs suffering from dissolution-limited
absorption or administered as modied release formulations
(McAllister, 2010). Since the possibility of permeation may not only
affect dissolution but also precipitation kinetics, the addition of an
acceptor compartment may be crucial for the predictive evaluation
of SDDS. In a thermodynamically unstable supersaturated system,
permeation into a sink compartment may act as an alternative for
precipitation to lower the systems Gibbs free energy. Recently,
Bevernage et al. demonstrated that precipitation of loviride was
signicantly reduced in the presence of an absorption compartment simulated in the Transwell Caco-2 model. As a result, the
stability of supersaturation observed in a classic one-compartment
setup without an absorption compartment did not correlate with
transport across the Caco-2 monolayer (Bevernage et al., 2012a).
In practice, the simulation of absorption has been accomplished
by different experimental approaches, including the addition of
an immiscible organic layer as absorptive sink (biphasic model),
or the integration of an actual absorption compartment separated from the dissolution medium by a lter/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 proles 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 delivery system (S-SEDDS)). The setup consisted of a USP IV apparatus
for aqueous dissolution under non-sink conditions connected to a
USP II apparatus containing an additional water immiscible octanol
layer creating an acceptor compartment with sink conditions.

For comparison purposes, release proles were also assessed in

a monophasic system under both sink and non-sink conditions.
None of the release proles obtained in aqueous media using either
monophasic (Fig. 6A) or biphasic setups (Fig. 6B) could discriminate
among the three formulations and predict the in vivo bioavailability of celecoxib. Only the concentration proles obtained in the
octanol phase of the biphasic system (Fig. 6C) could be correlated
to the in vivo outcome (Fig. 6D), illustrating the necessity of a combined dissolutionabsorption model to allow reliable in vitroin
vivo correlations.
5. In vivo evaluation of supersaturation
Considering the large number of experimental variables that
may affect 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 do not
allow for the demonstration of supersaturation/precipitation at the
site of absorption, making it extremely difcult to judge the precise contribution of intraluminal supersaturation to the observed
plasma concentrationtime prole. Both direct and indirect methods 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 proles in vivo. In a study
by Shono et al., physiologically based pharmacokinetic modeling (PBPK modeling) using STELLA software was used to predict
plasma proles of the weakly basic drug nelnavir, based on in vitro

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

33

Fig. 6. Dissolution proles of celecoxib (CEB) from 3 types of formulations obtained in a single phase dissolution test (A), the aqueous phase of a biphasic dissolution test (B)
and the octanol phase of a biphasic dissolution test (C). The relative in vivo AUC and Cmax of the respective formulations are also included (D).

dissolution data from nelnavir mesylate tablets in biorelevant

media, standard gastrointestinal parameters and the disposition
pharmacokinetics of nelnavir (Shono et al., 2011). In the fasted, but
not the fed state, accurate simulation of the in vivo performance of
nelnavir required the implementation of drug precipitation based
on the crystal growth theory, in the PBPK model. This suggests
that intestinal precipitation limits the fasted state absorption of
the weakly basic nelnavir. Similarly, Takano et al. used deconvolution of plasma concentrationtime proles and PBPK modeling
to simulate the in vivo intraluminal drug concentration of a novel
farnesyltransferase inhibitor (FTI-2600) (Takano et al., 2010). After
administration of the crystalline HCl salt to dogs, the measured Cmax
and AUC0-inf were 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 concentrations agreed with the equilibrium solubility; for the salt, however,
a 4-fold higher intestinal concentration was predicted, indicating
that intestinal supersaturation was responsible for the improvement 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 duodenal aspiration technique after intragastric administration of an acidic solution of 2
weakly basic drugs (dipyridamole and ketoconazole) in healthy
adults (Psachoulias et al., 2011). Both the total drug content and the
soluble drug fraction in duodenal aspirates were determined upon
sampling. To allow assessment of supersaturation, equilibrium
solubilities were also determined in each aspirate, after the addition
of an excess of crystalline drug. Based on these data, the fraction
precipitated and the relative supersaturation index were calculated
as a function of time. Although the duodenal uids appeared to be
supersaturated at several early time points (540 min), only limited
precipitation (maximum 7% and 16% for dipyridamole and ketoconazole, respectively) was observed, indicating that absorption

of these weakly basic drugs is only minimally affected by duodenal precipitation. Interestingly, previously assessed precipitation of
dipyridamole in simulated intestinal uids using an in vitro transfer model overestimated duodenal precipitation when compared to
the in vivo observations (Kostewicz et al., 2004). This emphasizes
the need for in vivo reference data on intraluminal supersaturation and precipitation to optimize in vitro assays (Psachoulias et 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 contemporary evaluation methods are mostly adapted dissolution tests,
they cannot be simply considered biorelevant in a supersaturation/precipitation context. Although several experimental
variables have been identied as being essential for the reliable
simulation of SDDS (e.g. medium, dissolution rate, transit, hydrodynamics, absorption), their integration in preclinical assays remains
immature. This is mainly due to (1) our erratic understanding
of 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.
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