Improved Human Bioavailability of Vemurafenib, a Practically

Insoluble Drug, Using an Amorphous Polymer-Stabilized Solid

Dispersion Prepared by a Solvent-Controlled Coprecipitation
Process
NAVNIT SHAH,1 RAMAN M. IYER,1 HANS-JUERGEN MAIR,2 DUK SOON CHOI,1 HUNG TIAN,1 RALPH DIODONE,2
2

KARSTEN FAHNRICH,
ANNI PABST-RAVOT,2 KIN TANG,1 EMMANUEL SCHEUBEL,2 JOSEPH F. GRIPPO,3 SEBASTIAN
3
A. MOREIRA, ZENAIDA GO,1 JAMES MOUSKOUNTAKIS,1 THERESA LOUIE,1 PRABHA N. IBRAHIM,4 HARPREET SANDHU,1
LINDA RUBIA,2 HITESH CHOKSHI,1 DHARMENDRA SINGHAL,1 WASEEM MALICK1
1

Pharmaceutical and Analytical R&D, Hoffmann-La Roche Inc., Nutley, New Jersey, 07110

Pharma Global Technical Development, F. Hoffmann-La Roche Ltd., Basel, Switzerland

Clinical Pharmacology, Hoffmann-La Roche Inc., Nutley, New Jersey, 07110

Non-clinical Development, Plexxikon Inc., Berkeley, California, 94710

Received 10 October 2012; revised 26 November 2012; accepted 30 November 2012

Published online 29 December 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23425
ABSTRACT: The present work deals with improving the solubility of vemurafenib, a practically insoluble drug, by converting it into an amorphous-solid dispersion using a solventcontrolled precipitation process. The dispersion containing vemurafenib and hypromellose acetate succinate (HPMCAS), an enteric polymer, is termed microprecipitated bulk powder (MBP),
in which the drug is uniformly dispersed within the polymeric substrate. HPMCAS was found
to be the most suitable polymer for vemurafenib MBP, among a series of enteric polymers
based on superior physical stability and drug-release characteristics of the MBP. The MBP
provided a greater rate and extent of dissolution than crystalline drug, reaching an apparent
drug concentration of 2835 :g/mL, almost 30-fold higher than solubility of crystalline drug
at 1 :g/mL. The supersaturation was also maintained for more than 4 h. Upon exposure to
high temperature and humidity, the MBP was destabilized, resulting in crystallization and
lower dissolution rate. The control of moisture and temperature is essential to maintain the
stability of the MBP. In a relative human bioavailability study, vemurafenib MBP provided
a four- to fivefold increase in exposure compared with crystalline drug. Improving solubility
with an amorphous-solid dispersion is a viable strategy for the development of practically insoluble compounds. 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
J Pharm Sci 102:967981, 2013
Keywords: glass transition; amorphous; solid dispersion; precipitation; dissolution;
solubility; X-ray diffractometry; thermal analysis; absorption; bioavailability

INTRODUCTION AND BACKGROUND

Because 85% of drugs sold around the world are orally
administered, the properties of a drug molecule that
govern oral absorption are critical to its development.
The Biopharmaceutics Classification System (BCS) is
a guide for predicting intestinal absorption based on
Correspondence to: Raman M. Iyer (Telephone: +973-235-5862;
Fax: +973-235-3769; E-mail: raman.iyer@roche.com)
Journal of Pharmaceutical Sciences, Vol. 102, 967981 (2013)
2012 Wiley Periodicals, Inc. and the American Pharmacists Association

the two parameters, aqueous solubility and intestinal permeability.1,2 Although permeation enhancers
could be used in a limited manner to improve permeability of poorly permeable drugs, from a formulation
perspective, solubility enhancement using formulation intervention is the key driver for greater bioavailability. An increasing number of drug molecules being
discovered belong to BCS class II or IV, with poor
solubility being the primary concern. Based on the
NoyesWhitney theory of dissolution,3 the dissolution
rate is a function of the concentration gradient, which

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967

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SHAH ET AL.

increases with the maximum solubility or saturated

concentration achievable. Some of the more conventional approaches to enhance this solubility include
the use of excipients such as solubilizers and lipidbased surfactants for micellar solubilization,46 an increase in the surface area available for dissolution by
micronization or nanoparticles using sizing-down (top
down) or building-up (bottom up) technologies,7,8 and
complexation using cyclodextrins.9
Over the past two decades, solid dispersions in polymeric carriers have been investigated as a means
of improving the bioavailability of poorly soluble
drugs.1016 The polymeric carriers reportedly increase
the rate and extent of drug dissolution, in turn leading to greater bioavailability. The polymers seem to
maintain the drug in a dissolved state at levels higher
than their thermodynamic solubility limits for several hours, albeit as an unstable system. Because
drug dissolution precedes absorption, the dissolved
drug could be absorbed before the unstable system
reaches its thermodynamic equilibrium state via precipitation of the drug and loss of bioavailability. Seen
in this manner, the carrier excipient becomes a critical component that governs the drugs solubility and
bioavailability. Several techniques are available to
create a stabilized drug-carrier solid dispersion where
the drug exists in varying states of crystallinity or
in an amorphous state.17 Non-polymer-based amorphous conversion such as comilling/cogrinding with
inorganic silicates18 has been used for select drugs.
Polymer-based techniques of solid dispersion could be
simple, moderately difficult, or complex. Comelting
and melt quenching19 are simple approaches, whereas
examples of moderate ones are solvent evaporation
under vacuum,20 spray drying,21 and lyophilization.22
Solventantisolvent precipitation and hotmelt extrusion are examples of complex techniques where
solubility differences, thermal stability, and so forth,
play a major role in the type of solid dispersion
obtained.23,24 The most challenging of these are
the ones where the drug is converted from a crystalline to an amorphous state and maintained with
a stabilizing polymer. The present work deals with
the conversion of crystalline vemurafenib into an
amorphous-solid dispersion stabilized with an enteric
polymer using a solvent-controlled precipitation process. The amorphous-solid dispersion is termed MBP
and stands for microprecipitated bulk powder.

MATERIALS AND METHODS

Vemurafenib is a practically water insoluble compound with a melting point around 272 C and the
structure shown below. Its physicochemical properties and solubility are summarized in Table 1. The
solubility of vemurafenib in various conventional organic solvents was 5 mg/mL at 25 C, with the exJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013

Table 1.

Physicochemical Properties of Vemurafenib

Molecular formula
Molecular weight
Melting point (by DSC)
Glass transition (Tg )
Partition coefficient
Aqueous vehicles (:g/mL)
Aqueous buffers (pH 3 and 7)
Fasted simulated intestinal fluid
Organic solvents (mg/mL)
Dimethyl sulfoxide
Methanol
Acetonitrile
Dichloromethane
Isopropanol
Acetone

C23 H18 ClF2 N3 O3 S

489.93 Da
272.1 C
105 C107 C
3.0
<0.1
<2
>50
4.57
1.40
1.95
3.56
<6

ception of dimethylacetamide (DMA), in which the

solubility was >500 mg/mL.

Preparation of MBP by Coprecipitation

Vemurafenib was prepared by Chemical Synthesis at
Roche (Nutley, New Jersey) in a crystalline form and
used for preparation of amorphous MBP. Hypromellose acetate succinate (HPMCAS) NF (Aqoat AS-LF)
and hypromellose phthalate (HPMCP) NF (HP-55)
were obtained from Shinetsu Corporation, Japan.
R
L 100-55 was obtained from Evonik CorEudragit
poration, New Jersey. All the excipients were used
as received. The measured Tg value (rounded off
to the nearest whole number) of HPMCAS was
R
L 100-55 and
120 C, whereas that of Eudragit

HPMCP are 110 C and 133 C, respectively, from the

literature.24,25 All the polymers evaluated are enteric
in nature and soluble at pH of 5.5 and above.
Amorphous-solid dispersions were prepared by a
solvent-controlled coprecipitation process as shown in
Figure 1. In this process, drug and polymer in a ratio
of 40:60 (w/w) were dissolved in DMA to a solids content of 15% (w/w). The DMA solution was introduced
at ambient temperature into 0.01 N HCl maintained
at 2 C5 C. The DMAacid ratio was maintained at
1:10 (w/w). The resulting precipitate was washed with
cold, dilute 0.01 N HCl followed by cold water to result in a DMA content of less than 0.2% (w/w). The wet
precipitate was dried under vacuum at a temperature
of 35 C to get a moisture content of 2% (w/w) or less.
The resulting dry solid dispersion sample, designated
as MBP, was analyzed as outlined below.
The amorphous MBP of vemurafenib prepared
R
L 100-55
using HPMCP, HPMCAS, and Eudragit
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IMPROVED HUMAN BIOAVAILABILITY OF VEMURAFENIB

Figure 1. Coprecipitation process for preparation of MBP.

was analyzed in the solid state, using powder X-ray

diffraction (pXRD) immediately after preparation and
after storing under accelerated stress-stability conditions of 40 C/75% relative humidity (RH) for up to
4 weeks. Amorphous MBP of vemurafenib and HPMCAS was prepared in a drugpolymer ratio of 30:70
(w/w) using the process outlined in Figure 1. The MBP
was further analyzed using additional analytical techniques as outlined in following sections to demonstrate its superior dissolution rate and bioavailability.
Powder X-Ray Diffraction (pXRD)
A Bruker D8 Advance powder X-ray diffractometer
(Bruker AXS Inc., Germany) was used to obtain the
pXRD pattern. The X-ray was applied at a voltage of
40 kV and a current intensity of 40 mA. The samples were analyzed over a 2 range of 4 40 with
a scanning rate (step size) of 0.9 /min. The X-ray
patterns of the samples were calculated and analyzed by a commercial software package (MDI Jade 9,
Materials Data Inc., California).
The X-ray powder diffraction pattern of MBP was
recorded and compared with the patterns of the
amorphous and crystalline vemurafenib reference
materials and a physical mixture of vemurafenib and
HPMCAS in the same ratio as MBP. The fraction of
crystalline vemurafenib was calculated using the patterns of amorphous reference material and crystalline
reference material. Using this technique, the limit of
detection of crystalline material was achieved at 1.6%
and the limit of quantitation at 4.8%.
Determination of Tg by Differential Scanning
Calorimetry
The thermal profile of amorphous MBP of vemurafenib with HPMCAS was evaluated using a TA
Q2000 modulated differential scanning calorimeter
(DSC, TA Instruments, New Castle, Delaware). The
DOI 10.1002/jps

969

instrument was calibrated with indium reference

(melting point, 156.6 0.2 C) and maintained under
an inert atmosphere with nitrogen purging. Sample
amounts of 58 mg were loaded in T-zero pans and
scanned at a rate of 3 C/min, modulated at a rate of
0.3 C every 50 s. Experiments were carried out in
triplicate.
In addition, a thermal profile of amorphous MBP of
vemurafenib was generated using a multifrequency
R
) measuretemperature modulated DSC (TOPEM
ment, where a conventional linear temperature ramp
is overlaid with stochastic temperature pulses of varyR
studies were carried out on a
ing duration. TOPEM
DSC-1 scanning calorimeter with a nitrogen cooling
accessory (Mettler, Greifensee, Switzerland). The instrument was calibrated for melting onset and heat
of fusion with reference materials and maintained
under inert atmosphere with nitrogen. All samples
were prepared around 1.02.5 mg in hermetic 40-:L
aluminum pans and scanned at a rate of 1 C/min,
stochastically modulated in pulses between 15 and
30 s and a pulse height of 1 C. Glass transitions were
evaluated from the reversing heat flow. The temperature values reported for a given parameter such as
Tg may slightly differ for the same material because
of different sources or measurement techniques.

Scanning Electron Microscopy

The morphology of the amorphous MBP was characterized using scanning electron microscopy (SEM).
The SEM analysis was carried out using a highresolution SEM (FEI Nova Nano SEM 230) at an
accelerating voltage of 5 kV. Prior to analysis, the
samples were sputtered with 20-nm of gold using a
sputter coater (Bal-tec Med 020, Leica Microsystems,
Buffalo Grove, Illinois). As a comparison with MBP,
excipient HPMCAS and vemurafenib were precipitated separately using the same procedure as that
of the MBP and analyzed by SEM.

Solid Dosage Formulation

The amorphous MBP was formulated into 40-mg
strength capsules of vemurafenib using a dryblending and a wet-mixing process and designated
MBP-1 and MBP-2, respectively. The particle size of
the MBP was 60 :m at d50% and 220 :m at d90% .
For comparative purposes, capsules of crystalline vemurafenib with poloxamer F127 as a solubilizer were
also developed for dissolution and pharmacokinetic
(PK) studies. The particle size of crystalline vemurafenib employed in these studies was d50% 3 :m
and d100% <13 :m. In addition, the amorphous MBP
was also formulated into tablets of vemurafenib
240-mg strength using a dry granulation process.
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SHAH ET AL.

Determination of pH solubility of MBP Polymers

Polymer sample (100 mg) was dispersed in 3 mL of
50 mM ammonium acetate buffer solvent at pH ranging from 2.1 to 8.0. The dispersions were tumbled at
25 C for 24 h. The supernatant was filtered through
0.2-:m polyvinylidene fluoride (PVDF) membrane filter and mixed with acetonitrile in ratio of 1:1.5 and
assayed for dissolved polymer using a validated highperformance liquid chromatography (HPLC) method
with gradient elution and detection with charged
aerosol detector (CAD).
Dissolution Studies
The dissolution of MBP was evaluated using USP
flow-through cell (USP Apparatus 4) and USP paddle (Apparatus 2). The flow-through cell consists of
a reservoir with the dissolution medium, a 22.6-mm
test cell, and a pump that forces the medium, maintained at 37 C, through the cell. A sample of 20-mg
MBP (6 mg of vemurafenib) was loaded into phosphate buffer at pH 6.8 containing 0.05% hexadecyltrimethylammonium bromide (HTAB), a cationic
surfactant, with medium flow rate of 10 mL/min.
Samples were obtained at various time intervals from
2.5 to 180 min and analyzed using a validated HPLC
method with detection by UV at 254 nm.
The dissolution of MBP was also evaluated using
USP Apparatus 2 at 75 rpm with two different media
maintained at 37 C. In the discriminating studies between crystalline and amorphous forms, fasted simulated intestinal fluid (FaSSIF), a biorelevant medium
was used. FaSSIF is a simulated physiological fluid
of the small intestine in the fasted state with a composition described in the literature.26,27 The 40-mg
strength capsule was placed in 500 mL of FaSSIF
medium for dissolution. Fluid samples were withdrawn at specified intervals and the amount dissolved
was analyzed using a validated HPLC method with
detection by UV at wavelength of 305 nm. In the stability and quality-control studies evaluating the impact of stress factors on dissolution of vemurafenib
amorphous MBP, a medium consisting of 50 mM phosphate buffer at pH 6.8 with either 0.09% or 1% HTAB
was used. The dissolution of 240-mg strength tablet
was conducted in 900 mL of this medium and analyzed similar to FaSSIF studies. Henceforth, the
FaSSIF and phosphate buffer studies will be referred
to as Apparatus 2(a) and 2(b), respectively, for clarity.
Where applicable, crystalline vemurafenib was evaluated as a micronized powder with a particle size of
d50% 3 :m and d100% <13 :m.
Storage Stability of Amorphous MBPTemperature and
Moisture
Microprecipitated bulk powder sample (SACR29701)
prepared using the process outlined above was stored
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under open and closed conditions at 25 C/60% RH,

30 C/75% RH, and 40 C/75% RH and tested for crystallinity and moisture content by pXRD and Karl
Fischer titration, respectively, at various intervals for
up to 24 months. The drug content of the sample was
assayed using a validated HPLC method and UV detection at a wavelength of 254 nm. The purity of vemurafenib and percentages of the substances were
determined using a validated HPLC method.
Relative Bioavailability Study in Healthy Volunteers
The primary objective of this randomized, open-label,
three-period crossover study was to assess the relative bioavailability and PK of vemurafenib with two
different amorphous-MBP capsule formulations of
40-mg strength, MBP-1 and MBP-2, against a formulation containing crystalline drug (reference at the
time of study) with 10% poloxamer F127. The study
was conducted in 18 healthy, male subjects of 18
65 years of age (inclusive) with body mass index (BMI)
between 18 and 32 kg/m2 (inclusive). This design
was the most-efficient approach to determine relative bioavailability using a relatively small number
of subjects with a total duration of 84 days, including screening of subjects. The clinical study was conducted in accordance with the protocol and all applicable laws and regulations including, but not limited
to, the International Conference on Harmonization
Guideline for Good Clinical Practice, the Code of
Federal Regulations, and the ethical principles that
have their origins in the Declaration of Helsinki. Prior
to study initiation, the protocol, amendments, and
informed consent form (ICF) were reviewed and approved by the institutional review board (Independent Investigational Review Board Inc., Plantation,
Florida). Following a screening phase (21 days), subjects were randomized to receive the following study
treatments:
Treatment A

Treatment B

Treatment C

Reference crystalline formulation, 900 mg of

vemurafenib dosed as 3 300-mg powder-filled
hard gelatin capsules. Note: In Period 3, the
dose was decreased to 300 mg
(3 100-mg capsules).
Test MBP-1 (dry blend), 160 mg of vemurafenib
dosed as 4 40-mg powder-filled hard gelatin
capsules.
Test MBP-2 (wet granulated), 160 mg of
vemurafenib dosed as 4 40-mg powder-filled
hard gelatin capsules.

In each treatment, vemurafenib was administered

following an overnight fast of at least 8 h. Subjects
continued to fast for 4 h after dose, at which time
they were given a standardized meal and water ad
libidum. Grapefruit juice and grapefruits were prohibited from 48 h prior to first dosing until after the
last PK sample was collected. No caffeine or alcohol
DOI 10.1002/jps

IMPROVED HUMAN BIOAVAILABILITY OF VEMURAFENIB

was consumed from 48 h prior to PK sampling until the last PK sample was taken on Day 8. Subjects
were discharged after collection of the 48-h (Day 3)
PK sample. A minimum washout of 14 days was given
between doses.
Blood samples were collected at time zero and at
30-min and 1-, 2-, 4-, 6-, 8-, 10-, 12-, 18-, 24-, 30-, 36-,
48-, 72-, 96-, 120-, 144-, and 168-h time points after
dosing and assayed using a validated HPLC method.
PK parameters were calculated using standard
R
noncompartmental methods via WinNonlin V5.2
(Pharsight, St. Louis, Missouri). All calculations used
actual times recorded, with the deviation at 5 min at
1-h through 48-h periods and 1 h at 72-h through
168-h periods. The following parameters were calculated to determine the relative bioavailability:
AUC0last
AUC0inf
Cmax

Area under the plasma-concentration and time curve

up to the last quantifiable concentration.
Area under the plasma-concentration and time curve
extrapolated to infinity.
Maximum observed plasma concentration.

RESULTS AND DISCUSSION

Effect of Enteric Polymers on Stability of MBP Solid
Dispersions of Vemurafenib
Solvent-controlled precipitation processes have been
reported in the literature to form crystalline nanostructured particles with rapid dissolution rates The
precipitation processes have used various combinations of aqueous and organic solutions or suspensions of drug with or without suitable crystallization inhibitors that are sprayed into cryogenic
liquids (e.g., liquid nitrogen), resulting in frozen
nanoparticles.28,29
However, for drugs that are practically insoluble in
aqueous media, crystalline nanoparticles, upon storage, can act as seeds that induce crystallization resulting in an increase in the structural order over
time and decrease in solubility. Therefore, for such
drugs, a conversion to stabilized amorphous form
seems the only viable approach to improve kinetic
solubility and bioavailability. In the present study,
crystalline vemurafenib was converted to MBP, a
polymer-stabilized amorphous-solid dispersion, using
a solvent-controlled coprecipitation process.
A solution of vemurafenib and stabilizing polymeric
excipient in a ratio of 30:70 (w/w) in DMA is introduced under ambient conditions into dilute 0.01 N
HCl maintained at 2 C5 C. Because of the insolubility of drug and polymer in acidic aqueous media,
the components of the mixture are precipitated simultaneously with the vemurafenib being dispersed
in the polymeric matrix.
DOI 10.1002/jps

971

Because the coprecipitation process relies on low

solubility of vemurafenib and polymer in the precipitation medium (antisolvent), the choice of polymers
with pH-dependent solubility is desirable as the pH
could be modulated as needed to ensure polymer is
insoluble in the antisolvent. The acidic nature of the
antisolvent is thus highly amenable for use of enteric
polymers as stabilizing matrices for amorphous drug
with this process.
The vemurafenib MBP prepared with the three
different polymers was amorphous upon preparation
(Fig. 2a), but upon storage at 40 C/75% RH in open
containers for up to 4 weeks, the MBP prepared with
HPMCP exhibited a crystalline peak at the characteristic 2 of 25 after 2 weeks of storage (Fig. 2b);
R
L 100-55 provided
whereas HPMCAS and Eudragit
MBP that remained amorphous for up to 4 weeks
(Fig. 2c).
In the most-stable MBP-containing vemurafenib
R
L 100-55, the veand either HPMCAS or Eudragit
murafenib seems to be uniformly embedded in the
ionic polymer within the amorphous-solid dispersion,
the embedding enabled by coprecipitation or quenching of drugpolymer from solution phase in the antisolvent at a low temperature. The vemurafenib is
thus immobilized within the polymer to form a homogeneous amorphous-solid dispersion, the MBP. The Tg
R
of MBP containing HPMCAS and Eudragit
L 100-55

was measured to be 102 C and 111 C, respectively, as

seen from Figure 3, indicating that both polymers provide MBP with close Tg values and stabilize the MBP
by immobilizing the drug because of their high Tg values. Such immobilizing effect of high-glassy polymers
on small molecules has been reported for spray dried
dispersions (SDDs) prepared with HPMCAS.30
Vemurafenib MBP prepared with HPMCAS and
R
L 100-55 retained its amorphicity as meaEudragit
sured by pXRD and Tg when stored up to 4 weeks
at 40 C/75% RH. The polymer matrix is believed to
prevent nucleation and crystallization.
The solubility of the drug and polymer in the solvent of choice (e.g., DMA), the interaction of drug with
the polymer via hydrogen bonding and other weak
forces, Tg of drug and polymer, and process conditions
such as temperature and homogenization are some of
the critical factors that could be used to explain the
varying stability profile seen between vemurafenib
MBPs prepared with different types of polymers prepared using the process outlined in Figure 1.
Solubility parameters based on group contribution
from various moieties in the drug molecule may be utilized in silico to determine drugpolymer interactions.
The solubility of a majority of drugs in polymer is estimated in the range of 2%8%, whereas drugpolymer
miscibility is estimated to be greater when the difference in solubility parameter between the two components is less than 7 units.3133 In addition, molecular
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SHAH ET AL.

Figure 2. pXRD profile of vemurafenib MBP with drugpolymer ratio of 40:60 (w/w).
R
L 100-55, HPMCP, HPMCAS, crystalline vemurafenib),
(A) Time zero (from top: Eudragit

R
L 100-55,
(B) after 2-week storage at 40 C/75% RH, open (from top: HPMCP, Eudragit

HPMCAS, crystalline vemurafenib), (C) after 4-week storage at 40 C/75% RH, open (from top:
R
L 100-55, HPMCAS, crystalline vemurafenib).
Eudragit

motion of drug in an amorphous dispersion at temperatures even below its Tg has been reported and
may additionally be the cause of instability in MBP
prepared with HPMCP polymers.34
The drugpolymer interaction in MBP is postulated
to occur via hydrogen bonding because the structure
of HPMCAS (Aqoat AS-LF) suggests several hydrogen bonding sites due to the OH and COOH functional groups provided by 8% acetyl and 15% succinoyl
groups bound to the cellulose backbone.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013

The ability of HPMCAS to form hydrogen

bonds with griseofulvin in a spray-dried amorphous dispersion was shown using Fourier transform
infrared (FT-IR) scan. The carbonyl group in griseofulvin was shown to form hydrogen bonds with
the hydroxyl group in the HPMCAS, resulting in
an extended stability of the drug for more than
18 months. This hydrogen-bonding interaction was
further enhanced by addition of [N-(2-hydroxypropyl)
methacrylate] (PHPMA).35 In solid dispersions of indomethacin with povidone up to 30% (w/w) drug
loading similar to that in vemurafenib MBP, the
drugpolymer interaction in solid state was inferred
as hydrogen bonding that inhibits the intrahydrogen bonding that forms carboxylic acid dimers of indomethacin, which is fully eliminated at 30% (w/w)
polymer.36
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IMPROVED HUMAN BIOAVAILABILITY OF VEMURAFENIB

Figure 3. mDSC profile of vemurafenib MBP indicating

Tg at the midpoint of transition region (solid line: MBP with
R
L 100-55).
HPMCAS; dashed line: MBP with Eudragit

In addition to solid-state stability, upon dispersion

in aqueous medium, the polymeric matrix of the MBP
can maintain supersaturation of the drug in the aqueous medium for significant time period because the
amorphous form has a faster dissolution rate than
crystalline form. The polymer can potentially interact
with functional sites on drugs via hydrogen bonding
and other weak interactions, thus inhibiting a fast
precipitation of the amorphous form following supersaturation. Such a prolonged supersaturation phase
can result in an increased bioavailability. The crystalgrowth inhibitory effect of polymers containing carboxyl groups such as carboxymethylcellulose, alginic
acid, polymethacrylic acid, and polyacrylic acid has
been demonstrated in the study of crystal growth of
inorganic hydrates such as calcium sulfate.37
R
Although both HPMCAS and Eudragit
L 100-55
stabilized amorphous vemurafenib MBP in solid
state, HPMCAS was able to maintain a higher level
of supersaturation. In dissolution studies shown in
Figure 4, the percentages of drug dissolved over a
R
L 100-55
5-h period from HPMCAS and Eudragit
MBP dispersions were 90% and 55%, respectively.
These represent supersaturated concentration levels
of 40 and 22 :g/mL for the two MBPs, respectively,
an almost 100-fold increase from solubility of the
crystalline vemurafenib. In a comparative study of
HPMCAS, HPMCP, povidone, and acrylic polymers on
their ability to enhance drug dissolution from nifedipine solid dispersions, HPMCAS showed the highest
level of drug dissolved from its solid dispersion in a
buffer of pH 6.8.38 Based on the pH-solubility profile of
R
L 100-55 shown in Figure 5,
HPMCAS and Eudragit
HPMCAS has a two- to threefold greater solubility
R
L
in the enteric pH range of 6.88.0 than Eudragit
100-55, which may explain the lower level of supersaturation observed with the latter during dissolution. It
DOI 10.1002/jps

973

Figure 4. Dissolution profiles of vemurafenib MBP with

drugpolymer ratio of 40:60 (w/w) in USP Apparatus 2(b)
with 0.09% HTAB (filled square: MBP with HPMCAS; open
R
L 100-55).
square: MBP with Eudragit

Figure 5. pH-solubility profile of MBP polymers (filled

square: MBP with HPMCAS; open square: MBP with
R
L 100-55).
Eudragit

may be theorized that higher the solubility of a polymer, greater the interaction with the drug, leading to
greater solubility and degree of supersaturation.
In addition to the type of polymer, the drugpolymer ratio is probably one of the critical factors that
can impact physical stability of the MBP. The stability of solid dispersions of ritonavir with PEG 8000
was found to decrease with increasing drug loading
from 10% to 20% to 30%, leading to phase separation and crystallization.39 In dispersions of itraconaR
E 100, phase separation was obzole with Eudragit
served above 13% drug loading, indicating that drug
load is a critical factor that impacts stability of the
dispersion.40 In studies with drug-HPMCP systems,
the amount of drug incorporated has been found to
be one of the significant factors, with a solid solution
obtained at a drug loading no greater than 20%.41
The superior nature of HPMCAS in forming stable
solid dispersions at high drug loading compared with
HPMCP has been observed by others.42 In most cases
of amorphous dispersion, 20% (w/w) drug in polymer
seems nominal. In addition, the rate of dispersion
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SHAH ET AL.

(quenching) of solid solution in the antisolvent is also

a factor. Faster precipitation at higher speed is favorable for quenching, but can also lead to greater degree
of plasticization due to increasing mass transfer and
loss of drug as part of the dispersion formation.41
In addition to the choice of polymers, the final state
of the MBP is controlled by the levels of moisture and
residual solvents in the system. The residual moisture and solvents can lower the glass-transition temperature (Tg ) of the MBP, enhance the mobility of the
drug molecule in the MBP, and facilitate nucleation
and finally crystallization of the drug. Therefore, it
is critical that the process conditions such as precipitation rate (quenching rate), temperature, filtration, and drying rates and solventantisolvent ratios
employed in manufacturing the MBP are highly controlled and reproducible from batch to batch.
Thermal and Morphological Evaluation of Vemurafenib
MBP Prepared with HPMCAS
The polymer screening studies indicated HPMCAS to
be the most stabilizing polymer. MBP was prepared
with vemurafenib and HPMCAS in a ratio of 30:70
(w/w) and evaluated with the objective of determining
the phase homogeneity by pXRD and glass-transition
temperature, Tg , as the two main characteristics. Although pXRD is a measure of the long-range order
(or lack thereof), the Tg is the temperature at which
the change in free energy, G, and its first- and secondorder functions of volume and heat capacity, respectively, with temperature is gradual (unlike melting
point that exhibits a sharp discontinuity) as the sample transitions from a liquid or highly viscous state

to a supercooled glassy state.43 The glass transition

of two amorphous substances that have two separate
glass-transition temperatures when measured separately can be used to investigate the quality of the
mixture of these substances. A single Tg is a good indicator that they exist in a single homogenous phase,
whereas more than one Tg is indicative of incomplete mixing or phase separation of drug and polymer
phases.
The Tg values of amorphous vemurafenib and
HPMCAS were 107 C and 119 C, respectively, as seen
from Figure 6. A physical mixture of these two components in a ratio of 30:70 (w/w) exhibited two glasstransition temperatures because of the two separate
phases produced by a simple mixing of the components, whereas the amorphous MBP of vemurafenib
exhibited a single Tg in the range of 97 C102 C from
R
both mDSC and TOPEM
measurements.
The presence of two Tg values for amorphous physical mixtures of drug and HPMCAS has been reported earlier.30 In addition, upon phase separation
of drug and polymer, a solid dispersion exhibiting a
single Tg can begin to show two Tg values as seen for
griseofulvinpovidone mixtures containing 30% (w/w)
drug in povidone.44
The GordonTaylor (GT) equation (Eq. 1), based
on the additivity of free volumes of individual
components,45 could be used to predict the Tg of a
binary mixture, given their individual Tg and weight
fraction in the mixture.

Tg mix =

w1 Tg1 + Kw2 Tg2

w1 kw2

(1)

R
Figure 6. Modulated DSC profile (TOPEM
) indicating Tg (Acrystalline vemurafenib,
BHPMCAS, Cphysical amorphous mixture of vemurafenib and HPMCAS, Dvemurafenib
MBP).

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013

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IMPROVED HUMAN BIOAVAILABILITY OF VEMURAFENIB

where w is the weight fraction of drug, Tg is the glasstransition temperature and suffixes 1 and 2 represent
drug and polymer, respectively, and k is the so-called
GT constant.
The Tg of HPMCAS-based MBP containing 30%
and 40% (w/w) vemurafenib were 370.0 and 375.1 K,
R
DSC and
respectively, as measured by TOPEM
mDSC, respectively; whereas the predicted values using GT equation were 385.1 and 383.9 K, respectively.
A deviation (decrease) of Tg mix from the GT equation
is indicative of an interaction between vemurafenib
and HPMCAS in the MBP. Depending on the nature
of the interactions between drug and polymer, both
negative and positive deviations from ideality have
been observed.46,47 It is also noted that amorphous
materials absorb moisture and plasticize when exposed to high humidity, resulting in greater mobility
and lower Tg . However, their hydrophobic nature can
minimize this effect. At 60%75% RH, HPMCAS absorbs about 4% water, much less than polymers like
povidone and HPMC because of its greater relative
hydrophobicity.30
The SEM images of precipitated vemurafenib, precipitated HPMCAS and MBP are shown in Figure 7.
In general, the MBP particles [panels (e) and (f)] have
a comparable smooth and round surface. On the edges
of broken particles, the inner structure of the particles
can be seen, where under a surface film (<500 nm), a
spongy network structure can be observed. The pores
in this spongy structure are in range of 50200 nm
and are accompanied by bigger bubbles (with a range
of 310 :m). The surface seemed to be built up from
compressed spongy structures. SEM images of high
shear precipitated HPMCAS [panels (c) and (d)] show
smooth particles built up from several layers. Only
on some edges, the inner structure of the HPMCAS
particles can be seen, which seem to be porous than
in the MBP. The precipitated vemurafenib particles
[panels (a) and (b)] are 530 :m and are partially
hollow. These particles consist of agglomerated, small
crystals with an approximate size between 200 and
1000 nm. The precipitation condition, similar to that
for MBP, results in crystalline vemurafenib.
In the SEM images of MBP [panels (e) and (f)],
traces of some characteristic structural elements of
the HPMCAS can be seen with similar surface texture related to folding of the particle and a porous,
spongy inner structure. However, no evidence of crystalline vemurafenib can be observed in MBP, neither as crystalline structures within the HPMCAS
nor as separate crystals. In the MBP, vemurafenib
seemed to be uniformly spread in an amorphous form
within the polymer, supporting the formation of a
highly spongy inner structure. At less than 100 nm
[panel (f)], no differentiation in domains of the ingredients in the inner and outer structure can be
observed. Therefore, it is inferred that MBP conDOI 10.1002/jps

975

sists of a homogeneous dispersion of vemurafenib and

HPMCAS.
Drug Release StudiesRelease of Vemurafenib from
MBP Formulations
The dissolution profiles from the flow-through cell
studies are shown in Figure 8. At 120 min, 100%
of drug was released and dissolved from the MBP,
whereas amorphous vemurafenib and physical mixture of crystalline vemurafenib and HPMCAS exhibited about 10% drug release, indicating the need for
an amorphous dispersion of vemurafenib and HPMCAS that significantly improves the dissolution rate.
Furthermore, MBP samples with similar BrunauerEmmett-Teller (BET) area values were differentiated
based on their particle size with faster drug release
from MBP with particle size of 48 :m compared with
125 :m. With decrease in BET area and further increase in particle size, the percentage dissolved was
significantly lower at a given time point (not shown
here). This clearly indicates the role of HPMCAS as
a stabilizing polymer for the MBP and the need for
vemurafenib and HPMCAS to be present as an amorphous MBP instead of as an amorphous physical mixture for greater rate and extent of dissolution.
The results from dissolution of stressed and unstressed vemurafenib MBP against micronized crystalline drug using USP Apparatus 2(a) are shown in
Figure 9. The FaSSIF medium was also able to discriminate between crystalline, partially crystalline,
and amorphous vemurafenib. Unstressed amorphous
MBP provided faster rate and extent of dissolution,
whereas amorphous MBP stressed for 18 h at 40 C/
100% RH exhibited a slower dissolution rate and
lower extent of dissolution. Capsule containing crystalline drug had the lowest dissolution as expected on
the basis of its solubility in FaSSIF. The lower dissolution of stressed MBP was presumably due to a

Figure 8. Dissolution profiles of vemurafenib MBP samples in USP Apparatus 4. (a) MBP with d50% 48 :m,
BET area23.1 m2 /g; (b) MBP with d50% 125 :m, BET
area24.2 m2 /g; (c) amorphous vemurafenib; (d) physical mixture of micronized crystalline vemurafenib and
HPMCAS.
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976

SHAH ET AL.

Figure 7. Scanning electron micrographs: (a) and (b) Precipitated vemurafenib; (c) and (d)
precipitated HPMCAS; (e) and (f) vemurafenib MBP.

partial conversion of the amorphous to a crystalline

form as observed by pXRD in Figure 10. Subjecting
amorphous material to such high levels of temperature and humidity stress conditions can also result in
sintering phenomenon48 that could make the mateJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013

rial harder and slow down disintegration of capsule

fill material. This, in turn, could lower the rate and
extent of dissolution.
Although incomplete dissolution of amorphous
MBP was observed because of nonsink condition, the
DOI 10.1002/jps

IMPROVED HUMAN BIOAVAILABILITY OF VEMURAFENIB

Figure 9. Dissolution profiles of MBP and crystalline vemurafenib in USP Apparatus 2(a). (a) Unstressed vemurafenib MBP; (b) stressed vemurafenib MBP; (c) metastable
crystalline vemurafenib; (d) stable crystalline vemurafenib.

Figure 10. pXRD profiles of stressed capsules of

vemurafenib MBP. (Topstressed vemurafenib MBP;
bottomvemurafenib MBP control).

ability of FaSSIF to differentiate between levels of

crystallinity seems useful in early screening of amorphous forms. In addition, the FaSSIF was able to
differentiate between crystalline polymorphs of vemurafenib as shown in Figure 9. The metastable crystalline form exhibited an initial enhanced solubility
but precipitated into the stable crystalline form whose
dissolution reached levels similar to that of the stable
crystalline drug in micronized state.
An apparent dissolution concentration of 35 :g/mL
of vemurafenib with a supersaturation concentration
of 28 :g/mL was obtained with the HPMCAS-based
MBP, almost 20- to 30-fold higher than crystalline
drug solubility of 12 :g/mL. Furthermore, the supersaturation was maintained in the medium for more
than 4 h. The superior ability of HPMCAS to provide stable dispersions and supersaturated concentrations upon dissolution has been reported.49,50 In
studies with felodipine dispersions (similar solubility
profile as vemurafenib), an apparent solution concentration of up to 14 :g/mL followed by supersaturaDOI 10.1002/jps

977

tion of up to 11 :g/mL within 4 h was obtained with

75% HPMCAS.50 A similar effect of HPMCAS increasing drug dissolution and maintaining supersaturation has been observed with about fivefold increase
in concentration of free drug and drug in micelles
from spray-dried drug-HPMCAS dispersions.51 The
high level of supersaturation from HPMCAS-based
spray-dried dispersions has been attributed to the formation of stable, amorphous drug/polymer nanostructures and nanoaggregates that can rapidly dissolve
because of their small size (20300 nm) and a large
surface area. The impact of surface area of solid dispersions on drug dissolution has been reported.51 Particles with an average diameter of 200300 nm were
observed for felodipine dispersion during early dissolution phase52 by dynamic light scattering. The vemurafenib MBP samples evaluated in the flow-through
USP Apparatus 4 had surface area in the 2324 m2 /g
range. The high surface areas lead to more rapid levels of supersaturation, higher than those seen for lowsurface-area solid dispersions, which undergo crystallization during slow dissolution.
As mentioned earlier, the pXRD method was able
to detect crystalline vemurafenib at levels as low
as 1.6%. X-ray peak height ratios have been used
to determine percentage crystallinity.53 A correlation
seems to exist between the percentage crystallinity of
vemurafenib MBP and dissolution using USP Apparatus 2(b) with 1% HTAB in the medium, as shown in
Figure 11. It was observed that the percentage of drug
dissolved from the MBP at 30 min was >90% up to a
level of 4% crystallinity but decreased significantly
as the crystalline content became higher.
Effect of Temperature and Humidity (Storage
Conditions) on Stability of MBP
Crystallization in amorphous systems occurs primarily as a function of temperature, water content, and
storage time. The stability of amorphous materials
can be improved by storage well below the Tg and
by protection from plasticizers such as residual solvents and moisture that can lower Tg , thereby inducing mobility of the drug and eventual crystallization.
The water content, in turn, is impacted by the initial moisture content of the drug product, its storage
humidity and packaging conditions.
The amorphous nature of the MBP under different processing conditions and storage conditions of
temperature and relative humidity was investigated
using pXRD as a primary indicator. The physical stability of vemurafenib MBP was influenced by its moisture content, with faster crystalline conversion at
higher temperature and relative humidity of storage.
The moisture content of MBP stored under different
conditions of stress is shown in Table 2.
The moisture content of samples stored up to
6 months under closed conditions was dependent on
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013

978

SHAH ET AL.

Figure 11. Dissolution @ 30 min as a function of percentage of crystalline form (determined

by pXRD analysis) in vemurafenib MBP tablets (coefficient of correlation = 0.9753).

the initial moisture content of the MBP, whereas both

samples stored under open conditions at 30 C/75%
RH and 40 C/75% RH reached an equilibrium moisture level of 4% within 2 months under open storage. This is further supported by the moisture vapor sorption study, the results of which showed the
MBP achieving a moisture content of 4%5% (w/w) at
60%75% RH.
The impact of temperature and humidity on moisture content and percentage of crystalline content
of vemurafenib 240-mg tablets upon storage for
15 months under open and closed conditions in glass
bottles is shown in Table 3. The greater ability of
HPMCAS to stabilize amorphous dispersions upon exposure to high levels of relative humidity compared
with hygroscopic polymers like povidone is known.54
Under open conditions of storage, no significant traces
(2%) of crystalline vemurafenib were detected after
15 months at 25 C/60% RH, but crystalline traces up
to 6% were observed at 30 C/75% RH and as high
Table 2.

as 24% at 40 C/75% RH. The moisture content under

these open conditions was 3.5%4.5%. However, under closed conditions in glass bottles, the moisture
content remained in the range of 1.3%1.6% with
the result that even after 15 months, the crystalline
traces were 2% under the severe stress conditions
of 40 C/75% RH. Thus, a combination of high humidity and temperature can result in destabilization of
the amorphous MBP via a change in the moisture
content.
With a Tg of 137 C, water has a very high free
volume and sorbed moisture can penetrate into the
bulk phase of the MBP and significantly increase the
total free volume of the mixture. This typically leads
to a lower Tg and increased molecular mobility due to
breakage of the intermolecular hydrogen bonds.5557
This mobility is further enhanced by temperature
that increases the rate of relaxation of the amorphous
glass resulting in nucleation and crystal growth. Such
effects have been observed with amorphous sucrose

Moisture Content of Vemurafenib MBP upon Storage

Closed Storage

Months of
Storage

5C

30 C/75% RH

0
1
2
3
6

0.8
1.40
ns
1.0
1.0

0.8
1.3
ns
1.1
1.3

0
1
2
3
6

1.8
2.1
ns
2.0
1.8

1.8
2.1
ns
2.1
2.1

40 C/75% RH

Open Storage
30 C/75% RH

40 C/75% RH

MBP initial moisture <1%a

0.8
1.4
ns
1.2
1.3

0.8
3.4
3.6
4.0
3.9

0.8
3.4
3.6
4.1
ns

MBP initial moisture 2%b

1.8
2.3
ns
2.5
2.2

1.8
3.5
3.6
4.0
3.8

1.8
3.3
3.5
4.1
ns

a Mean

of duplicate samples.
of triplicate samples.
ns, No sample.
b Mean

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IMPROVED HUMAN BIOAVAILABILITY OF VEMURAFENIB

979

Table 3. Effect of Storage Temperature and Humidity on Moisture and Crystalline Content of Vemurafenib MBP Tablets Stored for
15 MonthsOpen and Closed Storage
Open Storage
Storage Conditions
25 C/60% RH
30 C/75% RH
40 C/75% RH

Closed Storage

Moisture Content (%)

Crystalline Content (%)

Moisture Content (%)

Crystalline Content (%)

3.5
4.5
4.5

2
6
24

1.31.6

<LOD
<LOD
2

and lactose where a critical amount of moisture was

deemed necessary for crystals to form.58,59 Therefore,
the control of moisture and temperature via protective packaging is essential to maintain amorphous
nature of MBP upon storage.

talline Phase 1 formulation were 3.6 and 3.1, respectively. These data show that the MBP formulation was
superior to the crystalline formulation and support
the development of amorphous MBP for improving
the bioavailability of practically insoluble drugs.

Assessment of the Relative Bioavailability of

Vemurafenib: Amorphous MBP Versus Crystalline Form
The primary study variables for assessment of the
relative bioavailability of the two MBP formulations versus crystalline formulation were AUC0inf ,
AUC0last , and Cmax . The relative exposure based on
log-transformed values of AUC and Cmax of the MBP
formulations relative to crystalline formulation was
estimated with 90% confidence intervals. Relative
bioavailability was estimated using the exposure ratios based on AUC and Cmax of Treatments B and C
relative to Treatment A as follows: Relative bioavailability = [AUC or Cmax MBP/AUC or Cmax Phase 1]
[Dose Phase 1/Dose MBP).
Although data from periods 1, 2, and 3 were used in
the analysis for both the MBP-1 and MBP-2 formulations, only the Period 3 data was used for the Phase 1
formulation generated with 100-mg capsule containing crystalline vemurafenib. The Period 1 and 2 data
for Phase 1 formulation were generated with 300-mg
capsules that exhibited low polymorphic stability and
resulted in lower than expected exposures.
The mean dose-normalized vemurafenib plasma
concentration versus time profiles shown in Figure 12
indicates that both the MBP formulations have nearly
identical exposures. The exposure (AUC0inf ) following a single 160-mg dose was similar 86.2 52.1 :Mh
and 79.8 42.8 :Mh for MBP-1 and MBP-2, respectively. The MBP formulations exhibited higher exposures after a single dose of 160 mg than micronized
crystalline formulations exhibited after a single dose
of 300 mg (AUC0inf 34.2 23.8 :Mh).
Based on the single-dose PK data of vemurafenib,
the mean values of AUC0inf from both MBP formulations were comparable with each other and both
were higher than the reference Phase 1 formulation.
The relative BA of the MBP-1 and MBP-2 formulations was 4.4 to 4.7 times compared with the Phase 1
formulation. Similarly, the Cmax was comparable for
both MBP formulations and greater than that for the
crystalline Phase 1 formulation. The Cmax ratios for
MBP-1 and MBP-2 formulations compared with crysDOI 10.1002/jps

SUMMARY AND CONCLUSIONS

Vemurafenib is a practically insoluble drug in its stable and unstable crystalline forms, the poor solubility leading to low bioavailability. Conventional technologies such as hotmelt extrusion and spray-drying
technologies were not suitable to form solid dispersion because of very high melting point and very low
solubility in low-boiling solvents. A solvent-controlled
precipitation process was developed that produced an
amorphous-solid dispersion stabilized by HPMCAS,
an enteric polymer. The dispersion was termed MBP.
The faster and higher dissolution rate of vemurafenib
from MBP dispersion compared with crystalline formulation was demonstrated in drug-release studies.
HPMCAS was found to be the best polymer to prepare stable MBP among a series of enteric polymers
based on superior physical (amorphous) stability of
the MBP and greater dissolution of drug from MBP
over time, resulting in a prolonged period of supersaturation in the dissolution medium. The homogeneity of amorphous MBP dispersion was confirmed by
pXRD and thermal analysis, where a single Tg was
obtained and by image analysis using SEM. Furthermore, upon exposure to high levels of temperature
and humidity in an unprotected (open) condition, the
MBP was found to absorb moisture and induce phase
separation between drug and polymer, resulting in
significant crystalline conversion and subsequent
lowering of dissolution rate. Therefore, the control of
moisture and temperature via protective packaging
is essential to maintain amorphous nature of MBP
upon storage. Vemurafenib amorphous MBP was also
incorporated into tablet-dosage form, which exhibited
excellent clinical efficacy and long-term storage stability. The MBP provided an almost fivefold increase
in exposure compared with crystalline form in a relative bioavailability study in man, demonstrating the
superior utility of amorphous form in improving absorption via drug dissolution and hence the bioavailability.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013

980

SHAH ET AL.

Figure 12. Comparison of dose-normalized exposure data among three capsule formulations:
Phase 1 crystalline vemurafenib and two MBP amorphous vemurafenib formulations.

Based on this study, the preparation of MBP via

a solvent-controlled coprecipitation process is considered a viable strategy for the development of stable
amorphous-solid dispersion of practically insoluble
compounds when more conventional technologies of
improving solubility are not feasible.

ACKNOWLEDGMENTS
The authors thank Dr. Keith Nollop of Plexxikon Inc.
for assistance in the design and conduct of the relative human bioavailability study; Dr. Roumen Radinov, Dr. Peter Luetolf, and Muriel Cordon Federspiel
for preparing some of the MBP samples; and Bharat
Patel for providing support in MBP testing studies.

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013