European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18

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European Journal of Pharmaceutics and Biopharmaceutics

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

Research paper

Development of a biphasic dissolution test for Deferasirox dispersible

tablets and its application in establishing an in vitro–in vivo correlation
Amal Al Durdunji 1, Hatim S. AlKhatib, Mutasim Al-Ghazawi ⇑
Faculty of Pharmacy, The University of Jordan, Amman, Jordan

a r t i c l e i n f o a b s t r a c t

Article history: In a biphasic dissolution medium, the integration of the in vitro dissolution of a drug in an aqueous phase
Received 23 July 2015 and its subsequent partitioning into an organic phase is hypothesized to simulate the in vivo drug absorp-
Revised 4 February 2016 tion. Such a methodology is expected to improve the probability of achieving a successful in vitro–in vivo
Accepted in revised form 10 February 2016
correlation. Dissolution of Dispersible tablets of Deferasirox, a biopharmaceutics classification system
Available online 16 February 2016
type II compound, was studied in a biphasic dissolution medium using a flow-through dissolution
apparatus coupled to a paddle apparatus. The experimental parameters associated with dissolution were
Keywords:
optimized to discriminate between Deferasirox dispersible tablets of different formulations. The dissolu-
Deferasirox
In vitro/in vivo correlations
tion profiles obtained from this system were subsequently used to construct a level A in vitro–in vivo
Flow-through apparatus correlation.
Paddle apparatus Ó 2016 Elsevier B.V. All rights reserved.
Biphasic dissolution medium

1. Introduction
Drug absorption is a composite process where the in vivo
dissolution, partitioning into and permeation through intestinal
mucosal cells as well as stability against the gastrointestinal envi-
ronment and first pass metabolism contributes to the rate and
extent of appearance of the drug in systemic circulation [1].
Dissolution testing has been extensively used by the pharma-
ceutical industry as a first line tool for evaluating the potential
ability of a dosage form to deliver a drug and make it bioavailable.
The ability of a dissolution testing procedure to predict the
performance of the dosage form in vivo is usually dependent on
the relevance of the in vitro dissolution test to the in vivo dissolu-
tion–permeation processes. The routinely used dissolution tests
are usually performed in aqueous buffered solutions. Such tests
are well suited for a quality control role; however, for understand-
ing the in vivo performance, more sophisticated media and condi-
tions which are more relevant to the physiological environment
found in the human gastrointestinal tract (GIT) are needed.
A biphasic dissolution system is expected to be particularly
suitable to study the in vitro dissolution and predict in vivo behav-
ior of formulations of BCS class II compounds. It involves the use of
two immiscible liquid layers, one is an aqueous phase and the
other is an organic one. In such a system, a drug is dissolved into
⇑ Corresponding author.
E-mail address: alghazam@ju.edu.jo (M. Al-Ghazawi).
1
Current address: Hikma Pharmaceuticals, Amman, Jordan.
the aqueous phase then partitions to the organic layer depending
on its distribution coefficient. The movement of the dissolved drug
into the organic layer allows dissolution in the aqueous phase to
continue by preventing the drug from accumulating in the aqueous
phase thus creating a sink condition.
Gibaldi and Feldman [2] were the first to use a biphasic dissolu-
tion medium to maintain a sink condition. They suggested that in a
dissolution rate – limited absorption, drug removal from the disso-
lution medium by partitioning is analogous to drug removal from
intestinal fluids by partitioning and permeation, in that both pro-
cesses do not allow the drug to accumulate in the release medium.
Besides overcoming the non-sink problems and mimicking a
larger portion of sequence of events involved in drug absorption,
a biphasic dissolution system can simplify the analytical task by
the action of the partitioning process as a filter that prevents
undissolved particles from moving into the organic phase, thus
removing interference/contribution of undissolved particles in
the analytical response when assaying the organic phase [3,4].
Different system configurations were reported when using
biphasic dissolution media. Hoa and Kinget [5] used a biphasic dis-
solution medium to obtain sink conditions for the water insoluble
drug, Artemisinin. They used USP apparatus II and added a second
two-blade stirrer to the paddle shaft to maximize the mixing effi-
ciency between the two phases which were water and chloroform.
In such an arrangement, a tablet was placed in a small glass cylin-
der; the bottom of the cylinder was closed by a paper filter and
attached to a disintegration apparatus motor in order to be moved
up and down in the aqueous phase. Another modification for USP

http://dx.doi.org/10.1016/j.ejpb.2016.02.006
0939-6411/Ó 2016 Elsevier B.V. All rights reserved.
10 A. Al Durdunji et al. / European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18
apparatus II was used by Grundy et al. [6,7] for the dissolution test
of nifedipine gastrointestinal therapeutic system in a biphasic
medium. They used a basket-paddle hybrid stirrer to prevent the
tablet or the released nifedipine suspension from depositing at
the bottom of the dissolution vessel (coning) and to provide
enough mixing at the interface between the organic and aqueous
layers.
Vangani et al. [3] and Shi et al. [4] combined the use of a USP IV
flow cell to study the dissolution phase with the use of a USP II ves-
sel to study the permeation phase using a bi-phasic dissolution
medium to achieve sink conditions.
In this report, we describe the application of a biphasic dissolu-
tion system in constructing an ‘‘In Vitro–In Vivo Correlation”
(IVIVC) for dispersible tablets containing 500 mg of Deferasirox
(DFX). DFX is an orally active iron chelating agent that it is highly
selective to iron (III) [8]. It is a tridentate ligand that binds iron with
high affinity in a 2:1 ratio [7]. DFX is mainly eliminated in the feces
due to hepatobiliary elimination of Deferasirox (including first-pass
elimination) and of its glucuronide. Renal excretion was only 8% of
the dose and included mainly the glucuronide conjugate [8].
DFX is classified as a BCS II type drug that is rapidly absorbed
following oral administration with a median time to maximum
plasma concentration of about 1.5–4 h [9], extensive protein bind-
ing (>98%) and a low plasma clearance of 3.53 (±0.87) L/h allowing
a single daily dosing [10].
The absolute bioavailability of DFX from ExjadeÒ, the originator
product, is about 70% [8,9].
2. Experimental
2.1. Materials
The following materials and supplies were purchased from their
stated suppliers and used as provided: 1-octanol (Fisher Scientific,
UK), sodium dihydrogen phosphate (VWR International, UK),
sodium hydroxide (Fisher Scientific, UK), sodium acetate anhy-
drous (Acros Organics, US), glacial acetic acid (Scharlau, Spain),
hydrochloric acid 37% (Merck, Germany), DFX (MSN Laboratories,
India), Titan 3 glass syringe filters with 0.7 lm pore size (Thermo
Scientific, Germany), glass beads of 1 mm diameter (Sotax,
Switzerland), and WhatmanÒ glass microfiber filters GF/F 25 mm
U (GE Healthcare, UK).
Three batches of different formulations of DFX 500 mg dis-
persible tablets (Test 1, Test 2 and Test 3) manufactured by Hikma
Pharmaceuticals, Jordan, were used in this study in addition to one
batch of ExjadeÒ500 mg dispersible tablets (Novartis, Batch Num-
ber S0063), the reference product of DFX 500 mg dispersible tablets
which was purchased from a local pharmacy in Amman, Jordan.
The three Hikma formulations were prepared using wet granu-
lation technology and contained the following excipients: crospovi-
done, povidone, sodium lauryl sulfate, microcrystalline cellulose,
silicon dioxide, Mg stearate. Test formulation 2 included also lac-
tose monohydrate in addition to the abovementioned materials.
The DFX material used in all formulations was crystalline in
nature and the main difference between the three formulations
was the particle size of DFX with d90 of 42 lm for Test 1 and
10 lm for both Test 2 and Test 3.
2.2. Methods
2.2.1. Determination of DFX solubility and distribution coefficient
The solubility of DFX at 37 °C in each of USP Phosphate Buffer
pH 6.8, USP Acetate buffer pH 4.5, 0.1 N HCl, 0.5% Tween 20 in
USP Phosphate buffer pH 6.8 and 1-octanol was determined in
duplicate. Excess of DFX was placed in glass vials containing
10 mLs of each of the above solvents. The vials were shaken over-
night while maintained at 37 °C in a shaking water bath (Grant
OLS200, Grant Instruments, UK). Thereafter, samples were with-
drawn and filtered through 0.7 lm glass syringe filters (Titan 3
glass syringe filters, Thermo Scientific, Germany) and the absor-
bance of DFX dissolved in the each sample was measured spec-
trophotometrically at a wavelength of 300 nm (UV-1700,
Shimadzu, Japan) and used to calculate DFX concentrations using
linear calibration curves.
The distribution coefficient of DFX between 1-octanol and phos-
phate buffer pH 6.8 was determined by dissolving 100 mg of DFX
in 50 mLs of 1-octanol in a 100 mL separatory funnel before adding
50 mL of the buffer to the separatory funnel and shaking for 30 min
(Gerhardt LS 500, Gerhardt, UK). After that, the two phases were
allowed to separate. A sample from the lower aqueous layer was
drained into a beaker for analysis and a sample from the upper
organic phase was withdrawn and analyzed spectrophotometri-
cally. The concentration of DFX in each phase was calculated using
linear calibration curves.
The distribution coefficient (log D) was calculated using the fol-
lowing equation:
Concentration in octanol
Log D ¼ log ð1Þ
Concentration in aqueous phase
2.2.2. In vitro dissolution
The dissolution test was performed on four tablets of each of
the three formulations of DFX dispersible tablets manufactured
by Hikma Pharmaceuticals (Test 1, Test 2 and Test 3) in addition
to the reference product, ExjadeÒ (Novartis, Batch Number S0063).
The bi-phasic dissolution medium, consisting of 500 mL of 1-
octanol as an organic phase and 300 mL of phosphate buffer pH
6.8 as an aqueous phase, was placed in a USP apparatus II vessel
(Varian VK7010, Vankel, United States) and stirred for 30 min to
allow equilibration between the two phases before the dissolution
run. An additional Teflon blade was fitted to the conventional USP
apparatus II shaft, and the two-blade configuration was intended
to enhance the efficiency of mixing of the two phases. A flow-
through apparatus (USP IV, CE 7 Smart, Sotax, Switzerland with
CP-7 Sotax piston pump) was coupled with the USP apparatus II.
A schematic presentation of the configuration of the dissolution
testing system used is shown in Fig. 1.
Each DFX tablet was dispersed in 10 mLs of deionized water for
about 1 min and the prepared suspension was transferred to the
USP apparatus IV cell (22.5 mm, 14 mL). The aqueous phase was
pumped from USP apparatus II to the USP apparatus IV cell at rate
of 30 mL/min. The return line from the USP apparatus IV cell was
kept immersed in the aqueous phase stirred in the USP apparatus
II vessel at rate of 120 rpm. Samples (5 mL) were withdrawn from
each phase at predetermined time intervals and were analyzed
spectrophotometrically at a wavelength of 300 nm (UV-1700, Shi-
madzu, Japan) and DFX concentrations were calculated using linear
calibration curves to determine the percentage released at each
time point using the following equation:
 
ðmg
mg
mLÞ
AbsðSn ÞStd: Conc: VðmLÞF
þ RSð1Þ C
Sðn1Þ
Abs ðStdÞ mL
 5 mL
% Released ¼  100%
500 mg
ð2Þ
where Abs(Sn) is the sample’s absorbance, Std. conc. is the standard
concentration in mg/mL, V is the volume of the medium in mL, F is
the sample’s dilution factor, Abs(std) is the standard’s absorbance, C
is the sample’s concentration, and 500 mg is the tablet labeled
claim.
 A. Al Durdunji et al. / European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18
Fig. 1. A schematic presentation of the configuration of the biphasic dissolution
system used.
Additionally, dissolution testing was performed in a single-
phase dissolution medium to compare the discriminatory power
of the two media types. The dissolution medium and testing condi-
tions recommended by the FDA dissolution database [11] for DFX
tablets were used for the single-phase dissolution method. The rec-
ommended method uses 900 mL of phosphate buffer pH 6.8 + 0.5%
tween 20 as a dissolution medium stirred in USP apparatus II at a
paddle speed of 50 rpm. In the single-phase dissolution experi-
ments, each of the six DFX tablets tested was dispersed in
100 mLs of the dissolution medium for about 1 min and the resul-
tant suspension was transferred to the dissolution vessel contain-
ing the rest of the medium. Samples (5 mL) were taken at
predetermined time intervals and were analyzed spectrophoto-
metrically at a wavelength of 300 nm (UV-1700, Shimadzu, Japan)
and concentrations were calculated using linear calibration curves
to determine the percentage released at each time point as
described above.
To compare the dissolution profiles of different formulas, the
model-independent similarity factor (f2) was used [12]:
8" #0:5 9
< Xn =
f 2 ¼ 50  log 1 þ ð1=nÞ ðRt  T t Þ2  100
: t¼1
;
where n is the number of data points and Rt and Tt are the % dis-
solved from each of the two formulations at a specific time point.
The dissolution profiles are considered similar if f2 is P50.
2.2.3. Pharmacokinetic studies on DFX dispersible tablets formulations
Human in vivo data were generated in three single-dose, fasted-
state, bioequivalence studies of crossover design for the three test
formulations against the originator ExjadeÒ. Data were generously
provided by Hikma Pharmaceuticals (Amman, Jordan).
The studies were all sponsored by Hikma Pharmaceuticals and
performed in the International Pharmaceutical Research Center
(IPRC, Amman, Jordan) and Pharma Medica (Toronto, Canada) in
compliance with the Declaration of Helsinki [13] after obtaining
the respective Institutional Review Board’s approval of the protocol
in each case. A written informed consent was read, understood and
signed by each volunteer before the start of the study.
11
The first two studies comparing ExjadeÒ to Test 1 and Test 2 for-
mulations enrolled Healthy male subjects, 18–50 years old, of Mid-
dle Eastern ethnicity with 8 subjects completing the first study and
6 completing the second study. The last study comparing ExjadeÒ
to Test 3 formulation enrolled Healthy male subjects, above
18 years old, of white, black, Asian or Latin ethnicity with 40 sub-
jects completing the study.
Total plasma concentrations of DFX were determined using a
validated LC-MS MS analytical method. The pharmacokinetic
parameters were calculated using non-compartmental analysis of
average plasma concentration–time profiles using WinNonlin (ver-
sion 5.3; Pharsight Corporation, Mountain View, CA), AUC was cal-
culated by trapezoidal rule, and maximum plasma concentration
(Cmax) and time to peak concentration (Tmax) were directly
obtained from the average plasma concentration–time profiles.
2.2.4. Data analysis and IVIVC
The fraction–absorbed of DFX from each of the tested formula-
tions was determined from the plasma concentration–time data
and calculated by numerical deconvolution using WinNonlin. The
plasma concentration–time profiles of the different formulations
were fitted to different compartmental models to determine the
best fit model based on Akaike information criterion (AIC) [14].
The obtained distribution and elimination rate constants (from
the best fit model) were then used to simulate an intravenous
(IV) plasma profile for each formula in order to fit a unit impulse
response (UIR). The plasma concentration–time profiles of the for-
mulations were then deconvoluted using the UIRs to determine the
fraction absorbed versus time or the input function in vivo for each
formulation.
The in vitro dissolution data representing the appearance of
DFX in the organic phase of the dissolution medium were fitted
to double Weibull model to obtain the in vitro input function. Lin-
ear regression analysis was applied to the in vivo and in vitro input
functions.
The predicted plasma concentrations were obtained from the
in vitro data by convolution using the IVIVC model and UIR; there-
after, the predicted and observed Cmax and AUC calculated by non-
compartmental analysis were compared to calculate the prediction
error by the following equation:
Predicted value  Observed value
%Prediction Error ¼  100%
Observed value
ð4Þ
ð3Þ 3. Results and discussion
3.1. Solubility and distribution coefficient of DFX
DFX solubility results are shown in Table 1. At lower pH values,
DFX is poorly soluble; the solubility was highest at pH 6.8 which
was expected considering the weakly acidic nature of DFX. How-
ever, even with the improvement of solubility at higher pH values,
DFX solubility classification remains ‘‘practically insoluble”. The
distribution coefficient values of DFX between phosphate buffer
pH 6.8 and 1-octanol were found to be 2.08, confirming the relative
lipophilic nature of DFX.
3.2. Pharmacokinetic data
The average plasma concentration–time profiles of DFX from
the four formulations are shown in Fig. 2.
The obtained AUC, Cmax and Tmax from the average plasma con-
centration–time profiles for the four formulations of DFX dis-
persible tablets are summarized in Table 2.
12 A. Al Durdunji et al. / European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18
Table 1
Solubility of DFX at 37 °C in different aqueous media.
Solvent Solubility (mg/mL) Standard
average, n = 2 deviation (mg/mL)
0.1 N HCl <0.004 – followed by the partitioning of DFX into the organic phase. The
Acetate buffer pH 4.5 <0.004 – apparent rate of appearance of DFX in the organic phase is a reflec-
Phosphate buffer pH 6.8 0.054 0.002
0.5% Tween 20 in USP 0.620 0.004
Phosphate buffer pH 6.8
1-Octanol 3.220 0.149
The analysis of the data showed that ExjadeÒ and Test 3 were
bioequivalent while ExjadeÒ and either of Test 1 or Test 2 were
not; however, the point estimates for Cmax and AUCinf in case of
Test 2 and ExjadeÒ were 90.2% and 106.6% respectively suggesting
that lack of bioequivalence is due to insufficient sample size of sub-
jects (n = 6) that yielded a wide confidence interval rather than a
formulation difference. The 90% confidence intervals of the ratios
of the means of the point estimates of the bioequivalence studies
are summarized in Table 3.
3.3. Dissolution results: biphasic medium
The aqueous phase used in the biphasic testing was composed
of phosphate buffer pH 6.8, and it was chosen because of the rela-
tively higher solubility of DFX at this pH. Similarly, 1-octanol was
chosen as the organic phase based on the solubility consideration
(3.2 mg/mL) in addition to the facts that it is immiscible with
water, does not evaporate at 37 °C, has relatively low viscosity
and has properties that mimic biological membranes [15].
Log D (1-octanol/phosphate buffer pH 6.8) of DFX was found to
be 2.08 and was judged enough to allow for effective partitioning
into the organic phase.
The volume of the organic phase we used was 500 mL. This
volume was chosen to allow for the establishment of a sink condi-
tion during dissolution testing of 500 mg DFX tablets based on a
calculation that the DFX concentration in the organic phase upon
complete release and partitioning of the drug into the organic
phase is less than one-third of DFX saturation solubility in
1-octanol. The volume of the aqueous phase needed to be as low
as possible to encourage partitioning of DFX into the organic phase
while still allowing for practical sampling and stirring in a USP
apparatus 2. An aqueous phase volume of 300 mLs was chosen as
a compromise.
The paddle speed showed a significant influence on DFX parti-
tioning. A speed of 100 rpm did not discriminate between DFX for-
mulations which showed different in vivo performance. On the
other hand, rotation speeds higher than 120 rpm resulted in vigor-
ous stirring and emulsification at the interface and were consid-
ered unsuitable. A paddle rotation speed of 120 rpm was able to
differentiate between formulations and was chosen for testing.
Considering the poor solubility of DFX in the aqueous phase, a
high rate of flow of the aqueous phase through the flow cell of
USP apparatus IV (30 mL/min) was used. Although this flow rate
is higher than the fluid flow rate through the intestine which has
been reported to vary from an average of 2.5 mL/min in fasting
subjects to as high as 20 mL/min after meals [16], it was chosen
to allow a complete dissolution within a reasonable time frame.
The presence or absence of glass beads inside the flow through
cell was also investigated. Glass beads in the cell are usually used
to provide laminar flow of the dissolution medium inside the cell;
otherwise, turbulent flow can develop [17]. Similar release profiles
were found regardless of the presence of glass beads and it was
decided to use glass beads in the flow through cell.
Fig. 3 shows the DFX cumulative release from the tested formu-
lations in the organic phase while Fig. 4 shows DFX cumulative
release in the aqueous phase using the biphasic dissolution condi-
tions described above.
The appearance of DFX in the organic phase is the result of two
consecutive processes, the dissolution of DFX in the aqueous phase
tion of the slower of these two processes which is expected to be
the dissolution into the aqueous phase with the organic phase act-
ing to generate a sink condition in this arrangement.
The f2 values calculated using the profiles generated from the
organic phase and with ExjadeÒ as a reference and either of Test
2 and Test 3 as test formulations were 73 and 80 respectively.
The f2 values suggest that the test formulations are similar to the
reference. However, in the case of Test 1, the f2 value calculated
using ExjadeÒ as a reference was 42.7 suggesting a lack of similar-
ity of the dissolution profiles.
These results correlate well with the observations from the
bioequivalence study where Test 3 was bioequivalent to ExjadeÒ.
While Test 2 and Test 1 were not bioequivalent, Test 2 had very
close point estimates of the ratios of Cmax and AUCinf (90.2% and
106.6% respectively) while those of Test 1 study were 60.08% and
76.84% respectively indicating a more obvious difference in perfor-
mance between Test 1 and ExjadeÒ.
The above results show the ability of the dissolution profiles in
the organic phase of a biphasic dissolution medium to rank order
formulations based on their in vivo performance.
The DFX appearance in the aqueous phase of the dissolution
medium is the net result of same processes described above; how-
ever, their arrangement in this case is not consecutive but rather
concomitant. The dissolution of DFX in the aqueous phase causes
DFX aqueous concentration to rise and the partitioning of DFX into
the organic phase causes it to decline. The rate of change of DFX
concentration in the aqueous phase is the net result of the two pro-
cesses. Reviewing the release profile of DFX in the aqueous phase
(Fig. 4) shows a rate of change of almost zero suggesting that a
steady state condition was established in the aqueous phase and
the rate of DFX appearance in the organic phase is equivalent to
DFX dissolution rate in the aqueous phase. This qualifies the suit-
ability of the chosen conditions in characterizing the dissolution
behavior of DFX tablets.
3.4. Dissolution results: single phase medium
Fig. 5 shows the dissolution results of DFX dispersible tablets
applying the single phase dissolution method. The % release
reached plateau within 10 min of the start of the run. More than
85% of DFX was released from ExjadeÒ, Test 2, Test 3 and as such,
an f2 value is not needed to indicate a similarity between Test 2,
Test 3 and ExjadeÒ. On the other hand, the f2 value comparing Test
1 and ExjadeÒ was 49 suggesting a borderline lack of similarity.
These findings resemble what was obtained in the biphasic dis-
solution system. However, the fact that the release profiles had
almost similar release rates and reached the plateau state within
10 min from the beginning of the dissolution runs reduces the
potential for correlation of these dissolution profiles with the
plasma concentration–time profiles which show a much longer
time frame.
3.5. Exploring IVIVC
The development of an IVIVC for an immediate release product
is a specific challenge that can be expected to be successful if the
dissolution is the rate limiting step for drug absorption, in such
case, biopharmaceutics classification system (BCS) type II com-
pounds which have high intestinal permeability and low solubility
are most suitable [18,19]. The low expectations for a successful
 A. Al Durdunji et al. / European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18 13

Fig. 2. Average total plasma concentration – time profiles after administration of a dose of 500 mg of Deferasirox in dispersible tablets formulations.

Table 2 so the model is capable of predicting the plasma drug concentra-

Observed pharmacokinetic parameters of 500 mg of Deferasirox administered in four tion from the in vitro dissolution profile while in a Multiple Level
dispersible tablets formulations.
C correlation, one or several pharmacokinetic parameters are cor-
Formula AUCinf (lg h/mL) Cmax (lg/mL) Tmax (h) related to the amount of drug dissolved at several time points [20].
Test 1 Average: 87.70 Average: 5.68 Average: 4.00 To explore a level ‘‘A” correlation, all formulations were used to
min: 30.94 min: 4.06 min: 3.00 build the model using IVIVC toolkit of the WinNonlin software. The
max: 331.39 max: 9.56 max: 6.00 biphasic dissolution profiles were fitted to double Weibull model
SD: 100.34 SD: 1.73 SD: 0.96
as shown in Fig. 6.
Test 2 Average: 153.53 Average: 10.68 Average: 4.50
min: 69.08 min: 8.41 min: 2.50 The plasma concentration–time profile of each formula was
max: 325.15 max: 15.70 max: 5.00 deconvoluted using its UIR (fitted from simulated IV data) to esti-
SD: 95.50 SD: 3.13 SD: 0.97 mate the fractions of DFX absorbed versus time for each formula.
Test 3 Average: 128.58 Average: 10.93 Average: 3.67
The simpler Wagner–Nelson deconvolution method could not be
min: 53.41 min: 3.52 min: 1.50
max: 202.04 max: 21.20 max: 4.50
used because DFX follows a multi-compartment model. The results
SD: 38.70 SD: 3.72 SD: 0.87 of the deconvolution process are shown in Fig. 7 as the fractions
ExjadeÒ Average: 128.39 Average: 10.29 Average: 3.33 absorbed versus time for the different formulations.
min: 54.02 min: 5.41 min: 1.50 A Levy plot which plots the time needed to have some percent-
max: 242.37 max: 25.60 max: 8.00
age absorbed in vivo on the Y-axis versus the time needed to have
SD: 41.76 SD: 4.12 SD: 1.25
the same percentage of drug dissolved in vitro on the X axis [31]
was used to estimate the time scaling (Fig. 8).
IVIVC for immediate release products show in the fact that com- Afterward, the fractions dissolved and absorbed were correlated
pendial and regulatory guidance have been provided for controlled (Fig. 9) to obtain the correlation model:
release products but not for immediate release ones [20,21].
A few examples of IVIVC for immediate release dosage forms
Fraction absorb: ¼ 2:322  Fraction diss:  ð0:161707
are available in the literature. They describe either Multiple Level
C correlations [22–26] or Level A correlations [27–30] since these  Time  0:000021Þ ð5Þ
are the correlations that are most useful in product development
and with regulatory acceptance. where 2.322 and 0.161707 are respectively the absorption and time
A Level A correlation is a point-to-point relationship between scaling factors, and 0.000021 is the time shift.
the in vitro dissolution and the in vivo input rate, and it utilizes To validate the model, fractions absorbed for all formulations
the entire dissolution profile and the entire in vivo plasma input were estimated using the correlation model, and then convolution

Table 3
Summary of the 90% confidence interval for the ratios of point estimates derived from the bioequivalence studies comparing the test formulations of 500 mg DFX dispersible
tablets to the reference product ExjadeÒ.

Study 90% confidence interval for the ratio (point estimate)

Cmax AUC0t AUCinf
Test 1 against ExjadeÒ (n = 8) 53.16–70.18% 61.81–76.69% 60.78–90.91%
(61.08%) (68.85%) (76.84%)
Test 2 against ExjadeÒ (n = 6) 61.74–131.78% 69.77–159.38% 75.38–150.89%
(90.20%) (105.45%) (106.65%)
Test 3 against ExjadeÒ (n = 40) 93.56–106.57% 95.11–104.40% 95.93–104.96%
(99.86%) (99.65%) (100.34%)
14 A. Al Durdunji et al. / European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18

Fig. 3. The release profiles of 500 mg of Deferasirox in dispersible tablet formulations from Reference formulation (ExjadeÒ), Test 1, Test 2 and Test 3 in the organic phase of
the biphasic dissolution medium (n = 4, results show average ± SD).

Fig. 4. The release profiles of 500 mg of Deferasirox in dispersible tablets formulations from Reference formulation (ExjadeÒ), Test 1, Test 2 and Test 3 in the aqueous phase of
the biphasic dissolution medium (n = 4, results show average ± SD).

Fig. 5. The release profiles of 500 mg of Deferasirox in dispersible tablet formulations from Reference formulation (ExjadeÒ), Test 1, Test 2 and Test 3 in the single phase
dissolution medium (n = 6, results show average ± SD).
 A. Al Durdunji et al. / European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18 15

Fig. 6. In vitro dissolved fractions of Reference formulation (ExjadeÒ), Test 1, Test 2 and Test 3 in the organic phase of the biphasic dissolution medium, fitted to double
Weibull model.

Fig. 7. Fractions absorbed over time of the tested 500 mg dispersible DFX tablet formulations: Reference formulation (ExjadeÒ), Test 1, Test 2 and Test 3.

was performed to obtain the predicted plasma concentration pro-
files as shown in Fig. 10.
Finally, the prediction errors for Cmax and AUCinf values were
calculated and are summarized in Table 4.
Employing the one-phase dissolution data to IVIVC modeling
produced the following correlation:
Fraction absorb: ¼ 1:179  Fraction diss:  ð0:0172  TimeÞ ð6Þ

Fig. 8. Levy plot for the tested 500 mg dispersible DFX tablet formulations: Reference formulation (ExjadeÒ), Test 1, Test 2 and Test 3.
16 A. Al Durdunji et al. / European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18

Fig. 9. Correlation between the Fraction Absorbed and the Fraction Dissolved (in the organic phase of a biphasic medium) for the tested 500 mg dispersible DFX tablet
formulations: Reference formulation (ExjadeÒ), Test 1, Test 2 and Test 3.

Fig. 10. Predicted plasma concentration–time profiles for the evaluated 500 mg dispersible DFX tablet formulations: Reference formulation (ExjadeÒ), Test 1, Test 2 and Test
3 calculated using the biphasic dissolution data.

where 1.179 and 0.0172 are respectively the absorption and time We have considered using actual intravenous bolus pharma-
scaling factors. cokinetic data instead of simulating it based on choosing the best
The predicted plasma concentration–time profiles and the pre- fitting multicompartment model. However, the only available data
diction errors for Cmax and AUCinf using single phase dissolution to us were that published by Séchaud et al. [10], which we tried to
data are illustrated in Fig. 11 and Table 5. use. However, the quality of the IVIVC developed using it was poor
Level A correlation, which is the most preferred level by the reg- and inferior to that generated using simulated IV data. This is prob-
ulatory authorities, was successfully achieved employing the two- ably the result of the nature of the abovementioned study which
phase dissolution. The internal validation results were conclusive, was conducted with a bioavailability objective in mind reflected
the prediction error values for Cmax and AUCinf for each formulation in the limited frequency of sampling in earlier time points and lim-
used in building the model were below 15% and the average abso- ited duration of sampling (8 h). The previous fact coupled with the
lute prediction error was below 10% for both pharmacokinetic infusion nature of the study resulted in the masking, or at least dis-
parameters, which is the acceptance criterion by the FDA for an torting the distribution characteristics of DFX.
IVIVC model [20]. None of the previous studies employing biphasic dissolution
system by coupling USP apparatus IV with USP apparatus II
achieved a level A correlation. Vangani et al. [3] combined USP IV
Table 4
with USP II to study the permeation phase using a bi-phasic disso-
Prediction errors for Cmax and AUCinf values based on the correlation model
constructed using the biphasic dissolution method. lution medium to achieve sink conditions. The dissolved drug
(AMG 517), a BCS class II compound, was continuously removed
Formulation Parameter Predicted Observed Prediction error %
from the aqueous phase (phosphate buffer, pH 6.8) into the organic
Test 1 Cmax (lg/mL) 5.3 5.7 6.9 phase (mixture of nonanol and cyclohexane 1:1) mimicking the
AUCinf (lg h/mL) 89.4 87.7 2.0
process of absorption. This dissolution system was able to provide
Test 2 Cmax (lg/mL) 9.6 10.7 10.4 a rank order correlation between in vitro and in vivo data (animal
AUCinf (lg h/mL) 154.5 153.5 0.6
data) for the different capsules and tablet formulations of AMG 517
Test 3 Cmax (lg/mL) 10.5 10.9 4.0 and discriminated bioequivalent and non-bioequivalent commer-
AUCinf (lg h/mL) 138.5 128.6 7.7
cial formulations of different drugs.
ExjadeÒ Cmax (lg/mL) 9.5 10.3 7.3 A similar biphasic in vitro test was used by Shi et al. [4] for
AUCinf (lg h/mL) 134.1 128.4 4.5
studying the release of Celecoxib, a BCS class II compound, from
 A. Al Durdunji et al. / European Journal of Pharmaceutics and Biopharmaceutics 102 (2016) 9–18 17

Fig. 11. Predicted plasma concentration–time profiles for the evaluated 500 mg dispersible DFX tablet formulations: Reference formulation (ExjadeÒ), Test 1, Test 2 and Test
3 calculated using the single phase dissolution method.

Table 5 4. Conclusion
Prediction errors for Cmax and AUCinf values based on the correlation model
constructed using the single phase dissolution method.
A biphasic dissolution method employing the flow-through
Formulation Parameter Predicted Observed Prediction error % apparatus and the USP paddle apparatus for discriminating the
Test 1 Cmax (lg/mL) 5.8 5.7 2.6 performance of different formulations of DFX dispersible tablets
AUCinf 78.8 87.7 10.1 was developed. It simulated two in vivo processes that contribute
(lg h/mL)
to drug absorption, dissolution and permeation, and it also pro-
Test 2 Cmax (lg/mL) 15.6 10.7 46.0 vided sink condition which is critical for the dissolution of drugs
AUCinf 158.2 153.5 3.1
of low solubility and high permeability. A level A IVIVC was suc-
(lg h/mL)
cessfully established. This system was shown to be superior to
Test 3 Cmax (lg/mL) 15.5 10.9 41.6
the one phase system in terms of discriminatory power between
AUCinf 123.2 128.6 4.2
(lg h/mL)
different formulations and ability to contribute to a successful
IVIVC.
ExjadeÒ Cmax (lg/mL) 14.7 10.3 43.0
AUCinf 124.8 128.4 2.8
(lg h/mL) Acknowledgments

The authors would like to acknowledge Hikma Pharmaceuticals

three different immediate release formulations. The organic phase
in this study was 1-octanol while the aqueous one was phosphate
buffer pH 6.8. The aqueous phase was pumped from a vessel con-
taining the biphasic medium into a flow-through setup that con-
tained the dosage unit. The human pharmacokinetics were
available for those three formulations and a rank order correlation
was obtained for the three formulations between their in vitro area
under the curve (AUC) of Celecoxib released in the octanol phase
and the in vivo AUC and Cmax. This relationship could not be estab-
lished when single phase dissolution tests under either sink or
nonsink conditions were used.
There is no literature available on the development of an IVIVC
for DFX. Establishing an IVIVC for this molecule is very useful for
the pharmaceutical industry while attempting to develop DFX dis-
persible tablets. The successful IVIVC model for this drug can be
used to minimize the number of bioequivalence studies needed
in the product development process and for bio-waiver requests.
Other than bio-waivers, a similar dissolution profile of a test
formulation against ExjadeÒ using the developed bi-phasic dissolu-
tion system provides a high guarantee for passing a well-designed
bioequivalence study because as proven here, formulation that was
not similar to ExjadeÒ failed the bioequivalence while similar ones
passed it.
Finally, the developed IVIVC can also be applied to establish a
specification for quality control dissolution testing for DFX dis-
persible tablets. A limit of not less than 80% release of the labeled
claim after 3 h is suggested to guarantee good in vivo performance.
for providing the results for the bioequivalence studies. Amal Al
Durdunji would like to acknowledge the support she received from
the Deanship of Academic Research at the University of Jordan to
support her MSc thesis under the supervision of Mutasim Al-
Ghazawi and Hatim S. AlKhatib.
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