Drug Deliv. and Transl. Res.
DOI 10.1007/s13346-015-0271-x
RESEARCH ARTICLE
Dissolution enhancement of atorvastatin calcium
by co-grinding technique
Priyanka Prabhu 1 & Vandana Patravale 1
# Controlled Release Society 2015
Abstract Atorvastatin calcium (AC) is a BCS class II drug
which shows poor bioavailability due to inadequate dissolu-
tion. Solid dispersions present a promising option to enhance
the solubility of poorly soluble drugs. Co-grinding with hy-
drophilic excipients is an easy and economical technique to
improve the solubility of poorly soluble drugs and is free from
usage of organic solvents. The aim of the present study was to
explore novel carrier VBP-1 (organosulphur compound) for
formulating a solid dispersion by using a simple, commercial-
ly viable co-grinding technique to enhance the dissolution of
AC and to develop an oral formulation of the same. Compo-
sition of the solid dispersion was optimized based on the re-
lease profile in pH 1.2 buffer. The optimized solid dispersion
was further characterized for flow properties, DSC, FTIR
spectroscopy, XRD, contact angle, SEM studies and release
profile in phosphate buffer pH 6.8. The developed solid dis-
persion gave similar release profile as the innovator formula-
tion (Lipitor® tablets) in both pH 1.2 buffer and phosphate
buffer pH 6.8. The developed solid dispersion was formulated
into hard gelatin capsules (size 3). The developed capsules
were found to give similar release as the innovator formulation
in both pH 1.2 buffer and phosphate buffer pH 6.8. The de-
veloped capsules were found to be stable for a period of
6 months. Anti-hyperlipidemic efficacy studies in rats showed
higher reduction in cholesterol and triglyceride levels by the
developed capsules in comparison to pure AC. In conclusion,
novel carrier VBP-1 was successfully employed to enhance
the dissolution of AC using co-grinding technique.
* Vandana Patravale
vbp_muict@yahoo.co.in
1
Department of Pharmaceutical Sciences and Technology, Institute of
Chemical Technology, Matunga, Mumbai 400019, India
Keywords Atorvastatin calcium . Co-grinding . Solid
dispersion . Solubility . Wettability . Dissolution
Introduction
Recent advances in combinatorial chemistry and high
throughput screening have fuelled the speedy discovery of a
plethora of drugs having promising pharmacological profile
[1]. However, the major hitch is the poor water solubility of
nearly 40 % of these new chemical entities [2, 3]. Consequent-
ly, poor aqueous solubility leads to poor bioavailability, dos-
age escalation, huge inter- and intra-subject variation and
greater variability in plasma drug levels under fed versus
fasted states. Poor solubility of a drug also restricts the selec-
tion of delivery strategies in addition to difficult dissolution
testing methodology with low in vitro-in vivo correlation [4].
As a consequence, poorly soluble drugs end up being elimi-
nated in the preliminary stage itself before making it to the
clinic. There is a growing need for commercially viable solu-
bility enhancement strategies which can convert these poorly
soluble new chemical entities as well as existing Biopharma-
ceutical Classification System (BCS) class II and IV
drugs into formulations so as to overcome bioavailabil-
ity problems [5].
There are various strategies used to enhance the aqueous
solubility of poorly soluble drugs. These include cyclodextrin
complexation, use of surfactants, salt formation, particle size
reduction through nanosuspension formation and
micronization, pro-drug formation, use of solid dispersions,
etc. However, each of the aforementioned strategies has dis-
advantages: prodrug formation results in the generation of a
new chemical entity; salt formation is only possible for acidic
or basic compounds; use of surfactants and cyclodextrins
raises toxicity concerns; particle size reduction results in

agglomeration, poor wettability and flow properties and dust
generation [6]. Solid dispersions present a promising approach
to enhance release of poorly soluble drugs among all the
above-mentioned techniques [5, 7]. Solid dispersions can be
prepared by various methods such as melt fusion, hot melt
extrusion, solvent evaporation, kneading, spray drying, gel
entrapment, co-precipitation and co-grinding method [8].
Co-grinding with hydrophilic excipients is a very promis-
ing method to improve the solubility of poorly soluble drugs
owing to its simplicity and cost-effectiveness (as compared to
supercritical fluid and spray drying techniques) and complete
avoidance of organic solvents [4]. Numerous research groups
have successfully utilized the co-grinding method for improv-
ing solubility of various BCS class II and IV drugs such as
carbamazepine [9], fenofibrate [10], nifedipine [11], gliclazide
[12], aceclofenac [13] and piroxicam [14] to name a few.
Atorvastatin calcium (AC) belongs to the second gen-
eration of statins [15]. It is a synthetic reversible inhibitor
of the microsomal enzyme 3-hydroxy-3-methyl-glutaryl-
coenzyme A (HMG-CoA) reductase, an enzyme which
catalyses the conversion of HMG-CoA to mevalonate,
the rate-limiting step in de novo cholesterol biosynthesis.
It is presently used in the treatment of hypercholesterol-
emia [16]. It is insoluble in aqueous solutions of pH 4 and
lower and very slightly soluble in distilled water and
pH 7.4 phosphate buffer. The absolute bioavailability of
AC is reported to be 12 % and only 30 % of the drug is
absorbed on oral administration [17]. It is a BCS class II
drug and has poor bioavailability due to inadequate dis-
solution in the gastrointestinal tract [18].
Hence, the objective of the present investigation was to
explore novel carrier VBP-1 (organosulphur compound) by
using a simple, commercially viable co-grinding technique
for enhancing the dissolution of AC and to develop an oral
formulation of the same.
Materials and methods
Materials
Crystalline AC was obtained as a generous gift sample from
Cadila Pharmaceuticals, Ahmedabad, India, and used without
any further purification. VBP-1 (organosulphur compound)
was purchased from Healers Nutraceuticals Pvt. Limited,
Chennai, India. PVP K30 (Kollidon®30), PVP K25
(Kollidon®25), poloxamer 188 (Lutrol® F 68), poloxamer
407 (Lutrol® F 127) and vinylpyrrolidone-vinyl acetate co-
polymer (Kollidon®VA 64) were obtained as gift samples
from BASF India Limited, Mumbai, India. HPMC E5 LV
was obtained as gift sample from Colorcon Asia Pvt. Ltd,
Mumbai, India. Mannitol was obtained from S.D. Fine
Chemicals Limited, Mumbai, India. Triton WR 1339 was
Drug Deliv. and Transl. Res.
purchased from Sigma Chemical Co, St. Louis, MO. Aceto-
nitrile and methanol of HPLC grade were purchased from
S.D. Fine Chemicals Limited, Mumbai, India. Hard gelatin
capsules were obtained as gift samples from Associated Cap-
sules Pvt. Ltd, Mumbai, India.
Preparation of solid dispersion
AC along with the excipients was co-ground in a planetary
ball mill PM-100. Six zirconium oxide balls (30-mm
diameter) were used for 5 g of sample with processing time
of 8 min.
Analytical method development
Ultraviolet-visible spectroscopic method (UV-vis double
beam spectrophotometer Jasco Model 1503V, Jasco Inc. Ja-
pan) was developed in methanol to analyse the drug content.
UV spectrophotometric methods in pH 1.2 buffer and in phos-
phate buffer pH 6.8 (PB) were developed to study the in vitro
release profile of the developed formulation. A stability indi-
cating HPLC method was developed to evaluate the stability
of AC in the developed formulations subjected to stability
guidelines as per ICH guidelines. Reverse phase HPLC meth-
od was developed using HPLC system having following spec-
ifications: pump: Jasco PU-2080 Plus Intelligent HPLC pump
(Jasco, Japan); injector: a Rheodyne 7725 injector (Rheodyne,
USA); detector: Jasco UV-2075 Intelligent UV/vis detector
(Jasco, Japan) and integrator: a JascoBorwin Chromatography
Software (version 1.50) integrator software. Column used was
X-Terra® 250 × 4.6 mm C-18 column (5 μm) and a mobile
phase containing acetonitrile:methanol:0.02 M potassium
dihydrogen orthophosphate buffer pH 6.85 (40:40:20) was
employed at a flow rate of 0.5 ml/min. Injection volume was
20 μl and detection wavelength was 246 nm.
Optimization of solid dispersion
One of the most important tools to distinguish between
various batches of solid dispersions is dissolution testing.
Hence, for unbiased evaluation, the selection of proper
dissolution medium is very critical. AC is official in Indi-
an Pharmacopoeia, which prescribes PB as dissolution
medium [19]. Dissolution studies were also carried out
in pH 1.2 buffer. AC is insoluble in aqueous solutions
of pH 4 and lower and is very slightly soluble in pH 7.4
phosphate buffer [17]. Hence, initially, formulation devel-
opment was done using pH 1.2 buffer as a tool for screen-
ing solid dispersions. The optimized solid dispersion was
then tested for in vitro drug release in PB.
Drug Deliv. and Transl. Res.
Characterization of optimized solid dispersion
Differential scanning calorimetry (DSC)
The DSC measurements were performed on a DSC (Perkin
Elmer coupled with Pyris software) with a thermal analyser.
All accurately weighed samples (about 10–20 mg) were
placed in aluminium pans and sealed, before heating under
nitrogen flow (20 ml/min) at a scanning rate of 10 °C/min
from 35 to 200 °C. An empty aluminium pan was used as a
reference.
Fourier transform infrared spectroscopy (FTIR)
FTIR spectra were obtained by using Perkin Elmer Spectrum
100 FTIR Spectrometer. The samples (AC, VBP-1,
poloxamer 407, blank and solid dispersion) were previously
ground and mixed thoroughly with potassium bromide (KBr),
at 1:10 ratio (sample:KBr) approximately. The KBr pellets
were prepared by compressing the powders at a pressure of
5 tons for 1 min in hydraulic press.
X-ray diffraction (XRD)
Powder XRD patterns were measured using a wide-angle X-
ray diffractometer (Rigaku Miniflex Japan) with CuKα radi-
ation of intensity 1.54 A°. The scanning rate employed was
2.5°/min over the 2 to 60° diffraction angle (2θ) range.
Scanning electron microscopy (SEM)
Particle morphology was observed using Jeol JSM-6380 LA
analytical scanning electron microscope. The samples were
initially coated with platinum to conduct electricity using Jeol
JFC-1600 auto fine coater.
Contact angle studies
Contact angle (θ) is a quantitative measure of the wetting of a
solid by a liquid. It can be defined geometrically as the angle
created by a liquid at the three phase border where a liquid, gas
and solid meet. Low values of θ mean that the liquid spreads,
or wets well, while high values represent poor wetting. Con-
tact angle of less than 90° indicates that the liquid is able to
wet the solid. Contact angle of greater than 90° indicates that
the solid is non-wetting or hydrophobic. A zero contact angle
represents complete wetting [20]. Contact angles of pure AC,
AC with VBP-1 (1:1), AC with poloxamer 407 (1:0.5) and
solid dispersion were measured on Kruss G10 contact angle
measurement equipment using distilled water as medium. A
300-mg sample was compressed using 11-mm punch in tablet
press to get discs of constant hardness. A constant drop vol-
ume (5 μl) of double-distilled water was introduced on surface
of disc (Sessile drop) using syringe and contact angles were
measured by tangent method. Measurements were made in
triplicate.
Drug content estimation
Solid dispersion equivalent to 40-mg dose of the drug was
transferred to 50-ml volumetric flask. The volume was made
up with AR grade methanol. The solution was sonicated for
15 min. 0.1 ml of this solution was diluted to 10 ml with
methanol. Samples were prepared in triplicate. UVabsorbance
was recorded at 246 nm and drug content was determined.
In vitro release profile
The in vitro dissolution study was carried out in pH 1.2 buffer
and also in PB as the Indian Pharmacopoeia recommends PB
as the dissolution medium for AC [19]. The release profile of
the developed solid dispersion was compared with both pure
AC and innovator formulation (Lipitor® tablets) in both
pH 1.2 buffer and PB. The similarity factor (F2) was calculat-
ed for the optimized solid dispersion considering innovator
formulation as the reference formulation.
In vivo anti-hyperlipidemic efficacy studies
The in vivo efficacy of the developed solid dispersion was
tested using ‘Triton-induced hyperlipidemia’ in rats. The pro-
tocol for animal studies was approved by the Institutional
Animal Ethics Committee of the Institute of Chemical Tech-
nology (ICT/IAEC/2011/P10) and the work was carried out at
the Institute of Chemical Technology premises. In vivo study
was approved and performed in accordance with the guide-
lines of the animal ethics committee at Institute of Chemical
Technology. Triton is a non-ionic surfactant which causes hy-
perlipidemia via inhibition of peripheral lipoprotein lipase en-
zymes involved in elimination of lipid particles from the body.
The administration of Triton causes a temporary rise in lipid
levels, which reaches a peak at 18 to 24 h post administration
(phase I) and begins to reduce again the next day (phase II)
[21, 22].
Male Wistar rats were purchased from Haffkine Institute,
Mumbai, India. Rats (weighing between 150–250 g) were
divided into five groups (n = 6). Animals were grouped as
follows: group 1: control group receiving no formulation,
group 2: placebo group receiving placebo of solid dispersion,
group 3: pure drug group receiving only AC, group 4: solid
dispersion of AC and group 5: innovator formulation group.
The rats were fasted overnight and injected intraperitoneally
with 250 mg/kg Triton WR 1339 dissolved in 0.5 %
carboxymethyl cellulose (CMC) solution. The control group
of rats was given the vehicle (CMC solution 0.5 %), and
experimental groups were given pure AC (25 mg/kg body
 Drug Deliv. and Transl. Res.

Table 1 Composition and release profile of various solid dispersions dosing was performed by intubation using an 18-gauge feed-
prepared using co-grinding method
ing needle (the volume to be fed was 1.0 ml in all cases).
Batch Composition Ratio T50 % (min) % release Blood was withdrawn from retro-orbital sinus using heparin-
ized glass capillary and collected in Eppendorf tubes at 0, 24
A1 AC:VBP-1 1:1 – 42 and 48 h, and the serum was separated by centrifugation at 10,
A2 AC:VBP-1 1:2 38.1 53 000 g for 10 min. Serum cholesterol, triglycerides and high-
A3 AC:VBP-1 1:3 16.8 61 density lipoprotein (HDL) were estimated in all groups using
A4 AC:VBP-1 1:4 9.4 71 Merck diagnostic kits. Serum low-density lipoprotein (LDL)
A5 AC:VBP-1 1:5 6.3 72 and very low-density lipoprotein (VLDL) were estimated
A6 AC:Mannitol 1:1 20.1 64 using the following relation:
A7 AC:PVP K25 1:1 42.1 52
A8 AC:PVP K30 1:1 – 42 LDL ¼ cholesterol−HDL.
– VLDL
A9 AC:HPMC E5 1:1 – 40
VLDL ¼ triglyceride 5
A10 AC:Kollidon VA 64 1:1 – 41
A11 AC:P188 1:1 21.7 94
Statistical analysis of the collected data was performed
A12 AC:P407 1:1 3.9 100
using one-way analysis of variance (ANOVA) followed by
A13 AC:VBP-1:P188 1:4:1 <10 100
Tukey-Kramer multiple comparisons test with the help of
GraphPad Instat 3 software, and P < 0.05 was considered to
A14 AC:VBP-1:P188 1:3:1 100
be significant.
A15 AC:VBP-1:P188 1:2:2 100
A16 AC:VBP-1:P188 1:1:3 13 97
A17 AC:VBP-1:P188 1:2:1 98 Formulation development
A18 AC:VBP-1:P188 1:1.5:1.5 98
A19 AC:VBP-1:P188 1:1:2 18 95
The solid dispersion was filled in size 3 hard gelatin capsules
A20 AC:VBP-1:P188 1:2:0.5 99
(colour code: white-coloured body and pink-coloured cap).
A21 AC:VBP-1:P188 1:0.5:2 20 89
A22 AC:P188 1:0.5 24.5 91 Characterization of capsules
A23 AC:VBP-1:P407 1:1:1 100
A24 AC:VBP-1:P 407 1:0.5:1 100 The capsules were characterized for the following:
A25 AC:VBP-1:P407 1:1:0.5 100
A26 AC:VBP-1:P407 1:0.75:0.75 100 Uniformity of weight
A27 AC:VBP-1:P407 1:0.5:0.5 89
A28 AC:P407 1:0.5 9.0 87 The test was performed as stated in the Indian Pharmacopoeia
[23]. Each capsule was weighed. Each capsule was then
opened and without losing any part of the shell, the contents
weight) or the solid dispersion (equivalent to 25 mg/kg AC) or were completely removed. Each capsule shell was then
innovator formulation equivalent to 25 mg/kg AC. The oral weighed. The weight of the contents is the difference between

Fig. 1 In vitro release profiles of

AC with VBP-1 (n = 3)
Drug Deliv. and Transl. Res.

Fig. 2 In vitro release profiles of a AC with mannitol, PVP-K25 and PVP-K30; b AC with HPMC E5 and Kollidon VA 64 and c AC with poloxamer
188 and poloxamer 407 (n = 3)

the two weighings. The procedure was followed for a total of
20 capsules and the average weight was computed. The phar-
macopoeia specifies that not more than two of the individual
weights deviate from the average weight by 10 % and none
deviates by more than 20 %.
Disintegration time
The test was performed as stated in the Indian Pharmacopoeia
[24]. A basket and rack assembly was used to determine the
disintegration time. The pharmacopoeia states that the disin-
tegration time for hard gelatin capsules should not exceed
30 min.
Drug content
Contents from six capsules were emptied into a mortar,
amount of capsule fill equivalent to a single dose (40 mg)
was weighed and drug content of the capsules was determined
in accordance with the procedure mentioned in “Drug content
estimation” section.
In vitro release studies
The in vitro release performance of the developed capsules of
AC was compared with pure AC (40 mg) and innovator for-
mulation in both pH 1.2 buffer and PB. Consequently, F2
value was calculated for the developed AC capsules, consid-
ering innovator formulation as a reference in both pH 1.2
buffer and PB.
at time intervals of 1, 3 and 6 months, and their disintegration
time, drug content and in vitro release parameter (T90 %) in
pH 1.2 buffer and PB were determined.
Results and discussion
Analytical method development
The developed ultraviolet-visible spectroscopic methods were
found to be linear over the concentration range of 3–18, 4–24
and 3–21 μg/ml for methanol (λmax 246 nm), pH 1.2 buffer
(λmax 241 nm) and PB (λmax240 nm), respectively. The devel-
oped HPLC method was found to be linear over the concen-
tration range of 8–24 μg/ml. The retention time of AC was
found to be 6.075 min.
Optimization of solid dispersion
Table 1 gives the composition and release profile of various
solid dispersions prepared by co-grinding method. Dissolu-
tion testing revealed that the solid dispersions prepared using
VBP-1 exhibited better release than pure AC. The increase in
rate and extent of dissolution of AC may be attributed to the
improved wettability of AC by VBP-1. It was found that

Stability studies

Developed capsules were packed in AluAlu blisters. The de-

veloped capsules were then subjected to stability studies as per
ICH guidelines at 25 °C/60 % RH, 30 °C/65 % RH and 40 °C/
75 % RH. Stability indicating HPLC method described in
“Analytical method development” section was employed to
adjudge the stability of the drug. Also, capsules subjected to Fig. 3 In vitro release profiles of batches containing AC, VBP-1 and
various conditions of temperature and humidity were removed poloxamer 407 in various ratios (n = 3)

Table 2 Flow properties of solid dispersions
Parameters Results
A20 A25
Bulk density 0.7407 g/ml 0.4054 g/ml
Tapped density 0.9523 g/ml 0.4687 g/ml
Angle of repose 25.35° 19.74°
Carr’s index 22.22 13.50
Hausner ratio 1.29 1.16
increasing the ratio of VBP-1 to drug gave a proportionate
increase in release up to the ratio 4:1, after which a plateau
was reached, above which there was no further increase in the
release of AC (Fig. 1). However, VBP-1 alone could not give
the desired release, thus necessitating the addition of other
solubilizers to further improve the release. Other solubilizers,
namely mannitol, PVP K25, PVP K30, HPMC E5, Kollidon
VA 64, poloxamer 188 and poloxamer 407 were incorporated
in the ratio of drug: solubilizer (1:1) to select the solubilizer
which gave the greatest release at this ratio and use that
solubilizer in combination with VBP-1 in order to achieve
the desired release. Solid dispersion prepared using mannitol
exhibited a release of 64 % at the end of 1 h (Fig. 2a). Man-
nitol probably formed a hydrophilic boundary around the drug
particles resulting in better wetting of the drug in dissolution
medium [25]. Solid dispersion prepared using PVP K30 gave
no improvement in drug release, whereas PVP K25 gave a
slightly better release of about 52 % at the end of 1 h
(Fig. 2a). PVP K30 formed a sticky mass which was not easily
dispersed in the dissolution medium, giving rise to plug for-
mation and hence no dissolution improvement. Solid disper-
sions prepared using HPMC E5 and Kollidon VA 64 exhibited
no improvement in drug release probably because they formed
a mass which was not wetted easily and hence the drug release
was very poor (Fig. 2b). As shown in Fig. 2c, poloxamer 188
and poloxamer 407 were found to improve the release of AC
significantly. Poloxamer 188 gave a slower onset of release,
Fig. 4 DSC thermograms of a pure AC, b VBP-1, c poloxamer 407, d blank formulation and e solid dispersion
Drug Deliv. and Transl. Res.
but it increased the extent of dissolution of the drug, and 94 %
of the drug was released at the end of 1 h. Poloxamer 407, on
the other hand, was found to give a faster onset of release
(60 % release within 10 min) and 100 % release at the end
of 1 h. After looking at the promising results exhibited by
solid dispersions prepared using poloxamer 188 and
poloxamer 407, it was decided to explore different ratios of
VBP-1 and poloxamer 188 as well as VBP-1 and poloxamer
407 in order to study the influence of these excipients in com-
bination with VBP-1 on the release of AC.
Various trials were taken using VBP-1 and poloxamer 188
in different ratios. Poloxamer 188 in combination with VBP-
1 at 1:4 ratio (batch A13) gave a good release of 92 % within
10 min and complete release at the end of 1 h. Hence, further
trials were taken with the aim of reducing the amount of car-
rier required, as well as to study the effect of increasing VBP-1
relative to poloxamer 188 and vice versa for a given ratio of
drug:carrier. It was observed that increasing the ratio of VBP-
1 to poloxamer 188 (batches A14, A17 and A20) gave faster
onset of release, whereas the batches which had a lower
amount of VBP-1 as compared to poloxamer 188 (batches
A16 and A19) gave complete release at the end of 1 h, but
the onset of release was slower taking about 30 min to achieve
release comparable to that exhibited by batches having greater
amount of VBP-1. Batches containing equal amount of VBP-
1 and poloxamer 188 (batches A15 and A18) also gave similar
release as the batches containing a greater amount of VBP-1.
Thus, it was established that VBP-1 has the ability to rapidly
wet and solubilize the poorly soluble drug, thus resulting in a
rapid onset of release. All five batches, namely A14, A15,
A17, A18 and A20 gave almost same release, but A20 was
selected since it had the lowest carrier:drug ratio and a mini-
mum amount of poloxamer 188 required for achieving the
desired release. Poloxamer 188 possesses an amphiphilic
structure containing hydrophobic (propylene oxide block)
and hydrophilic regions (ethylene oxide block). The hydro-
phobic part provides a pool for drug and the hydrophilic por-
tion functions as an interface between the drug and dissolution
medium [26, 27]. Mixing of drug with poloxamer 188 leads to
Drug Deliv. and Transl. Res.
Fig. 5 FTIR spectra of a pure AC b VBP-1, c poloxamer 407, d blank formulation and e solid dispersion
enhanced wettability facilitating its easy dissolution. Mixing
of drug particles with poloxamer 188 results in close associa-
tion of the drug with polymer particles in the dry state. Expo-
sure to dissolution medium causes hydration of polymer sol-
ubilizing the drug particles in proximity and eventually releas-
ing the drug into the medium [28]. Trials were taken to prove
that both VBP-1 and poloxamer 188 in the ratio 2:0.5 were
required to achieve the desired release. As shown in Table 1,
neither poloxamer 188 nor VBP-1, when used alone, was
successful in achieving the desired release. Reversing the ratio
of VBP-1:poloxamer 188 (batch A21) showed a very slow
onset of release. Hence, it was proved that both VBP-1 and
poloxamer 188 were required in the ratio of 2:0.5 to achieve
the desired release.
Poloxamer 407 was found to give 100 % release at the end
of 1 h at 1:1 ratio of drug:poloxamer 407, so further trials were
taken to observe the effect of combination of VBP-1 and
poloxamer 407 on the release of AC. VBP-1 and poloxamer
407 in the ratio 1:1 gave 90 % release within 10 min and
100 % release at the end of 1 h. Hence, further trials were
taken with the aim of reducing the amount of carrier required
as well as to study the effect of reducing the amount of
poloxamer 407 relative to VBP-1 and vice versa. As shown
in Fig. 3, all three batches A23, A24, A25 and A26 gave
similar release. However, VBP-1 and poloxamer 407 in the
ratio 0.5:0.5 gave both a slower onset of release as well as
lower release at the end of 1 h. Hence, batch A25 was selected
Fig. 6 XRD spectra of a pure AC, b VBP-1, c poloxamer 407, d blank formulation and e solid dispersion
because it had the lowest carrier:drug ratio and a minimum
amount of poloxamer 407 required for achieving the desired
release. Two mechanisms may be responsible for the increase
in the release of AC in presence of poloxamer 407. The pres-
ence of carrier may avert aggregation of fine drug particles,
thus providing greater surface area for dissolution [29]. The
surface active character of the polymer aids in enhanced wet-
ting of the drug lowering interfacial tension between the me-
dium and the drug and, hence, results in higher dissolution
[30, 31].
Trials were taken to prove that both VBP-1 and poloxamer
407 in the ratio 1:0.5 were required to achieve the desired
release. As shown in Table 1, neither poloxamer 407 (batch
A28) nor VBP-1 (batch A1), when used alone, was successful
in achieving the desired release. Reversing the ratio of VBP-1
to poloxamer 407 (batch A24) also gave similar release, but
batch A25 was selected as it had the minimum concentration
of poloxamer 407 needed to achieve the desired release pro-
file. As both the batches A20 and A25 gave similar release
profiles (similarity factor), an attempt was made to select the
final formulation based on the flow properties: angle of re-
pose, Carr’s index and Hausner ratio, and moisture uptake.
Both the solid dispersions containing poloxamer 188 and
poloxamer 407 (batches A20 and A25) were kept in an open
petri dish for 7 days, and at the end of 7 days, the moisture
content was analysed using Shimadzu moisture analyser. On
the basis of Carr’s index and Hausner ratio, batch A20 showed

Fig. 7 SEM images of a pure AC
and b solid dispersion
poor flow properties (Table 2). Batch A25 showed excellent
flow properties. Also, batch A20 showed a moisture uptake of
0.522 while batch A25 showed uptake of 0.481. Thus, batch
A25 was finalized for further studies and dosage form
development.
Characterization of optimized solid dispersion
DSC
Figure 4 shows the DSC curves of AC, VBP-1, poloxamer
407, blank and solid dispersion. DSC curve of AC showed a
sharp endotherm at 156.38 °C corresponding to the melting
point of the drug. The broad endothermic peak from 75–
125 °C could be attributed to the loss of three molecules of
water. DSC curve of VBP-1 showed a sharp endothermic peak
at 113.71 °C corresponding to its melting point. DSC thermo-
gram of poloxamer 407 revealed an endothermic peak at
61.37 °C corresponding to its melting point. DSC thermogram
of blank formulation showed the characteristic peaks of VBP-
1 and poloxamer 407 at 114.24 and 57.33 °C, respectively.
DSC thermogram of solid dispersion showed endothermic
peaks at 55.72 and 108.01 °C corresponding to melting points
of poloxamer 407 and VBP-1, respectively. However, the en-
dothermic peak of AC at 156.38 °C disappeared and was
shifted to 203.51 °C. This indicated change in the crystallinity
of the drug through interaction with excipients.
Fig. 8 In vitro release profiles of
solid dispersion in a pH 1.2 buffer
and b phosphate buffer pH 6.8
(n = 3)
Drug Deliv. and Transl. Res.
FTIR
Figure 5 shows FTIR spectra of AC, VBP-1, poloxamer 407,
blank and solid dispersion. FTIR spectrum of AC (Fig. 5a)
showed characteristic peaks at 2967.03 cm −1 (C–H
stretching), 1655.11 cm−1 (asymmetric C=O stretching),
1577.52 cm−1 (symmetric C=O stretching), 1240 cm−1 and
1215.27 cm−1 (C–N stretching) and 3384.91 cm−1 (O–H
stretching). FTIR spectrum of blank (Fig. 5d) showed all the
characteristic peaks of VBP-1 and poloxamer 407. FTIR spec-
trum of solid dispersion (Fig. 5e) showed no major changes as
compared to pure AC indicating no change in molecular struc-
ture of the drug.
XRD
Figure 6 shows the XRD spectra of AC, VBP-1, poloxamer
407, blank and solid dispersion. XRD spectrum of pure AC
(Fig. 6a) showed characteristic diffraction peaks at 2θ values
of 9.16°, 11.84°, 12.18°, 16.98°, 19.44°, 21.58°, 22.58°,
23.26° and 23.68°. VBP-1 showed characteristic diffraction
peaks at 16.96°, 33.56° and 33.62° (Fig. 6b). XRD spectrum
of blank (Fig. 6d) showed no peak at 33.56° and 33.62°, but
showed a peak with reduced intensity at 33.26° indicating
change in crystal quality or orientation and reduced crystallin-
ity. Also the peak at 16.96° was shifted to 16.62°. XRD spec-
trum of solid dispersion (Fig. 6e) also showed peak of VBP-1
Drug Deliv. and Transl. Res.

Fig. 9 Effect of various

treatments on a cholesterol levels,
b triglyceride levels, c LDL levels
and d VLDL levels; data
expressed as mean ± standard
deviation (n = 6)

of reduced intensity at 33.16°. Poloxamer 407 showed major
diffraction peaks at 19.54° and 23.66° (Fig. 6c). XRD spectra
of both blank (Fig. 6d) and solid dispersion (Fig. 6e) showed a
reduction in the intensity of characteristic diffraction peaks of
poloxamer 407 suggesting reduced crystallinity of the latter.
XRD spectrum of solid dispersion (Fig. 6e) showed a reduc-
tion in the intensity of AC peak at 9.16° and shift in the 2θ
value to 9.22° indicating partial loss of crystallinity. Also,
characteristic diffraction peaks of AC at 11.84° and 12.18°
were absent in the diffractogram of solid dispersion. There
were no additional peaks in the diffractogram of solid disper-
sion, thus indicating absence of new compound formation.
Overall, the XRD analysis revealed change in the crystallinity
of AC, VBP-1 and poloxamer 407 on co-grinding together.

Table 3 Statistical significance

of anti-hyperlipidemic efficacy Groups Cholesterol Triglyceride LDL VLDL
data (n = 6) levels levels levels levels

24 h
Control v/s pure AC P < 0.001 P < 0.001 P < 0.001 P < 0.001
Control v/s placebo P < 0.05 P > 0.05 (ns) P > 0.05 (ns) P > 0.05 (ns)
Control v/s solid dispersion P < 0.001 P < 0.001 P < 0.001 P < 0.001
Control v/s innovator formulation P < 0.001 P < 0.001 P < 0.001 P < 0.001
Pure AC v/s solid dispersion P < 0.001 P < 0.001 P < 0.001 P < 0.001
Solid dispersion v/s innovator P < 0.001 P < 0.05 P > 0.05 (ns) P < 0.05
formulation
Pure AC v/s placebo P < 0.001 P < 0.001 P < 0.001 P < 0.001
Pure AC v/s innovator formulation P < 0.001 P < 0.05 P < 0.001 P < 0.05
Placebo v/s solid dispersion P < 0.001 P < 0.001 P < 0.001 P < 0.001
48 h
Control v/s pure AC P < 0.001 P < 0.001 P < 0.001 P < 0.001
Control v/s placebo P < 0.01 P > 0.05 (ns) P < 0.05 P > 0.05 (ns)
Control v/s solid dispersion P < 0.001 P < 0.001 P < 0.001 P < 0.001
Control v/s innovator formulation P < 0.001 P < 0.001 P < 0.001 P < 0.001
Pure AC v/s solid dispersion P < 0.001 P < 0.05 P < 0.01 P < 0.05
Solid dispersion v/s innovator P < 0.001 P > 0.05 (ns) P > 0.05 (ns) P > 0.05 (ns)
formulation
Pure AC v/s placebo P < 0.001 P < 0.001 P > 0.05 (ns) P < 0.001
Pure AC v/s innovator formulation P > 0.05 (ns) P > 0.05 (ns) P > 0.05 (ns) P > 0.05 (ns)
Placebo v/s solid dispersion P < 0.001 P < 0.001 P < 0.001 P < 0.001

ns not significant
 Drug Deliv. and Transl. Res.

Fig. 10 In vitro release profiles

of capsules in a pH 1.2 buffer and
b phosphate buffer pH 6.8 (n = 3)

SEM In vitro release profile

Figure 7 shows SEM photographs of AC and solid dispersion.
SEM images revealed that the crystallinity of the drug was
reduced in the solid dispersion.
Contact angle studies
Pure AC exhibited a high contact angle of 105° due its hydro-
phobic character. AC and VBP-1 together resulted in lowering
of contact angle to 40° attributed to the wetting property of
VBP-1. Also, AC and poloxamer 407 together showed a con-
tact angle of 35° due to the reduction in surface tension by
poloxamer 407. The extremely low contact angle of 25° ob-
tained for solid dispersion confirmed the significant improve-
ment in wettability of drug by both VBP-1 and poloxamer 407.
Drug content
Drug content of solid dispersion was found to be
99.69 ± 1.24 %.
As represented in Fig. 8, optimized solid dispersion gave
higher release as compared to pure AC in both pH 1.2 buffer
and PB. This could be due to improved wetting and solubili-
zation of AC by VBP-1 and poloxamer 407 in combination.
F2 values of 97.27 in pH 1.2 buffer and 98.50 in PB indicated
that the developed solid dispersion was similar to the innova-
tor formulation with respect to in vitro release in both the
media.
In vivo anti-hyperlipidemic efficacy studies
Figure 9 shows the serum cholesterol, triglyceride, LDL and
VLDL levels in different treatment groups at the end of 24 and
48 h. Table 3 shows the statistical significance of the data. The
developed solid dispersion exhibited statistically significant
reduction in cholesterol levels (P < 0.001), triglyceride levels
(P < 0.001), LDL levels (P < 0.001) and VLDL levels
(P < 0.001) as compared to pure AC at the end of 24 h. Also,
the developed solid dispersion showed significant reduction in
cholesterol levels (P < 0.001), triglyceride levels (P < 0.05),
LDL levels (P < 0.01) and VLDL levels (P < 0.05) as

Table 4 Results of capsules

subjected to stability studies Month Condition Drug content (%) Disintegration T90 % (min)
time (min)
pH 1.2 buffer PB

Zero day – 99.69 ± 1.24 2.5 12.7 6.5

1 month 25 °C/60 % RH 99.41 ± 0.96 2.5 13.3 7
30 °C/65 % RH 98.96 ± 1.45 2.6 13.3 7.5
40 °C/75 % RH 98.35 ± 0.76 2.7 13.3 7.9
3 months 25 °C/60 % RH 98.13 ± 2.13 2.7 11.9 6.7
30 °C/65 % RH 97.66 ± 1.45 2.9 12.9 7.5
40 °C/75 % RH 97.03 ± 0.85 3 13.9 9.1
6 months 25 °C/60 % RH 97.33 ± 1.67 2.9 11.9 7.9
30 °C/65 % RH 96.92 ± 1.87 3.2 14.7 9.3
40 °C/75 % RH 96.29 ± 2.32 3.2 16.4 12.2
Drug Deliv. and Transl. Res.
compared to pure AC at the end of 48 h. The innovator for-
mulation showed statistically significant reduction in choles-
terol levels (P < 0.001), triglyceride levels (P < 0.05), LDL
levels (P < 0.001) and VLDL levels (P < 0.05) as compared
to pure AC at the end of 24 h. However, at 48 h, there was
no statistically significant difference with respect to reduction
in cholesterol, triglyceride, LDL and VLDL levels between
innovator formulation and pure AC. The developed solid dis-
persion showed a significant reduction in cholesterol levels
(P < 0.001) as compared to innovator formulation at the end
of both 24 and 48 h. The developed solid dispersion also
showed a significant reduction in triglyceride levels
(P < 0.05) compared to innovator formulation at 24 h. How-
ever, at 48 h, there was no significant difference with respect
to triglyceride reduction between developed and innovator
formulation. There was no significant difference between de-
veloped and innovator formulation with respect to LDL reduc-
tion at the end of 24 and 48 h. The developed solid dispersion
showed significant reduction in VLDL levels (P < 0.05) com-
pared to innovator formulation at 24 h. However, at 48 h, there
was no significant difference with respect to VLDL reduction
between developed solid dispersion and innovator formula-
tion. There was no significant difference between the triglyc-
eride levels and VLDL levels of the control group and the
placebo group at the end of 24 and 48 h. There was a signif-
icant difference between the cholesterol levels of the control
group and the placebo group at the end of 24 h (P < 0.05) and
48 h (P < 0.01). There was no significant difference between
the LDL levels of the control group and the placebo group at
the end of 24 h; however, at the end of 48 h, there was a
significant difference (P < 0.05) between the LDL levels of
the control group and the placebo group. Pharmacodynamic
activity data suggests that the developed solid dispersion
showed better activity than pure AC and also the innovator
formulation. The enhanced activity of the solid dispersion
could probably be because of the increased dissolution of
AC which would have led to enhanced bioavailability and
hence increased activity.
Characterization of capsules
Uniformity of weight
The capsules showed an average weight of 100.105 ± 10 mg
and thus complied with the limits for uniformity of weight as
per Indian Pharmacopoeia.
Disintegration time
The capsules showed a disintegration time of 2.5 min and
thus complied with the limits for disintegration time as
per Indian Pharmacopoeia.
Drug content
Drug content of capsules was found to be 99.39 ± 1.13 %.
In vitro release studies
As shown in Fig. 10, the developed capsules showed higher
and faster drug release as compared to pure AC. The devel-
oped capsules were found to give 100 % release at the end of
1 h in pH 1.2 buffer and at the end of 30 min in PB. F2 values
of 99.60 in pH 1.2 buffer and 99.03 in PB indicated that the
developed capsules showed similar release as innovator for-
mulation in both the media.
Stability studies
Table 4 shows the drug content, disintegration time and
in vitro release parameter (T90 %) of capsules (subjected to
various conditions of temperature and humidity) in pH 1.2
buffer and PB. The developed capsules were found to be sta-
ble at the end of 6 months.
Conclusion
Co-grinding technique was successfully employed for formu-
lating solid dispersion of poorly water soluble drug AC. Com-
bination of VBP-1 and poloxamer 407 provided synergistic
effect on dissolution of AC. Novel carrier VBP-1 was found to
play a major role in the enhancement of solubility of AC as
solid dispersions prepared without VBP-1 failed to achieve
the desired release profile. The developed solid dispersion
was filled in hard gelatin capsules of size 3. The developed
capsules were found to give similar release as innovator for-
mulation (Lipitor® tablets) in both pH 1.2 buffer and PB. The
developed capsules were found to be stable at the end of
6 months. Anti-hyperlipidemic efficacy studies revealed the
superior lipid lowering potential of the developed solid dis-
persion in comparison to pure AC.
Acknowledgments The authors are thankful to University Grants
Commission for Junior Research Fellowship. The authors also wish to
thank Cadila Pharmaceuticals, Ahmedabad for the gift sample of AC;
BASF, Mumbai for excipients and Haffkine Institute, Mumbai for pro-
viding the animals. The authors also thank Dr. Dalapathi Gugulothu, Dr.
Harshad Shete, Dr. Soniya Jain, Jayesh Dhodi, Dr. S.S. Sathaye and Prof.
A.R. Juvekar for their guidance in animal studies and Rubicon Research
Private Limited, Mumbai for packaging of formulations.
Compliance with ethical standards All institutional and national
guidelines for the care and use of laboratory animals were followed.
Conflict of interest The authors declare that they do not have any
competing interests.

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