Powder Technology 256 (2014) 336–344

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Powder Technology
journal homepage: www.elsevier.com/locate/powtec

Improved oral delivery of clonazepam through liquisolid powder

compact formulations: In-vitro and ex-vivo characterization
Krishna Sanka, Sravanthi Poienti, Abdul Bari Mohd, Prakash V. Diwan ⁎
Department of Pharmaceutics, School of Pharmacy, (Formerly Lalitha College of Pharmacy) Anurag Group of Institutions, Hyderabad, 500088 AP, India

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

Article history: The liquisolid powder compacts (LSPCs) proved to be the potential solubility improvement strategy for efficient
Received 30 August 2013 oral delivery of BCS class II and IV drugs. Henceforth, an attempt was made to improve the oral delivery of BCS
Received in revised form 3 February 2014 class II drug clonazepam (CLZ) by formulating into a novel LSPCs. Solubility studies were conducted in different
Accepted 9 February 2014
liquid vehicles, namely propylene glycol, span 20 and span 80. The LSPCs were formulated using propylene glycol
Available online 17 February 2014
as non volatile solvent. The effect of different formulation variables on LSPCs performance was evaluated using 32
Keywords:
factorial design. The selected independent variables were % of clonazepam in propylene glycol (X1) and % of
Clonazepam sodium starch glycolate (X2) and dependent variables were disintegration time (YDT) and % cumulative drug
Factorial design release at 15th minute (YQ15). LSPCs of CLZ formulated with propylene glycol at optimum drug concentration
Liquisolid powder compacts produced high dissolution profile with acceptable tablet properties. Fourier transform infra-red spectroscopy
Solubility (FTIR) studies revealed that there was no interaction between drug and polymers, differential scanning calorim-
Dissolution rate etry (DSC) and X-Ray Diffraction (XRD) indicated conversion of crystalline to amorphous form of the CLZ. Further
the permeation studies carried out in isolated rat intestine revealed that potential of LSPCs for enhanced perme-
ation of CLZ across rat intestinal barrier. The increase in permeation of clonazepam from LSPCs formulation across
rat intestine suggests the potential of LSPC formulation for improved oral delivery of CLZ.
© 2014 Elsevier B.V. All rigths reserved.

1. Introduction described by Spireas [34,35] are composed of a non-volatile, water mis-

Clonazepam (CLZ) is an anticonvulsant chloro-nitrobenzodiazepine
that enhances the gamma-amino butyric acid (GABA) receptor response.
CLZ exerts its action by binding to the benzodiazepine site of the GABA
receptors, causing an enhancement of the electric effect of GABA on the
neuron when it binds to the receptor. It is poorly soluble in water (at
25 °C b 0.1 mg/ml) [1]. According to the biopharmaceutical classification
(BCS), CLZ is in class II, which consists of drugs of low solubility and
high permeability. The rate of oral absorption of these class II drugs is
often controlled by the dissolution rate in the gastrointestinal (GI) tract
[2]. Therefore, together with permeability, the solubility and dissolution
behavior of a drug are key determinants of its oral bioavailability.
Different approaches have been attempted to increase water solubility
of poorly soluble drugs, such as conversion of a crystalline molecule to its
amorphous state [3,4] particle size reduction via micronization [5–10], co-
grinding [11–13], inclusion complexation [14–16], solid dispersions
[17–19], self emulsifying drug delivery system [20–22], nanosuspensions
[23–25] and adsorption of drugs to hydrophilic silica aerogels [26–28].
One of the most promising strategies for release enhancement is the
Liquisolid Powder Compacts (LSPCs) [29–35]. Liquisolid systems as
⁎ Corresponding author. Tel.: +91 9849005903 (Cell).
E-mail addresses: sreekrishnaspips@gmail.com (K. Sanka), diwanpv@gmail.com
(P.V. Diwan).
cible liquid vehicle, solid drug particles, and selected excipients (carrier
and coating materials). The liquid portion, which can be a liquid drug, a
drug suspension, or a drug solution in suitable non-volatile liquid vehi-
cles, is incorporated into the porous carrier material. Once the carrier is
saturated with liquid, a liquid layer is formed on the particle surface,
which is instantly adsorbed by the fine coating particles. Thus, an appar-
ently dry, free flowing, and compressible powder is obtained. Besides
drug release enhancement, the liquisolid approach is a promising tech-
nique because of the simple manufacturing process, low production
costs, and the possibility of industrial manufacture due to the good
flow and compaction properties of the liquisolid formulations. To
calculate the required amount of powder excipients (carrier and coating
materials), a mathematical approach for the formulation of liquisolid
systems has been developed by Spireas [36]. Depending on the excipient
ratio R of the powder substrate, an acceptably flowing and compressible
liquisolid powder can be obtained only if a maximum liquid load, named
“liquid load factor” (Lf), is not exceeded. The terms “acceptable flow”
and “acceptable compressibility” imply the desired and thus preselected
flow and compaction properties, which must be met by the final
liquisolid formulation. R represents the ratio between the mass of the
carrier (Q) and the coating (q) materials present in the formulation:
R ¼ Q =q: ð1Þ

http://dx.doi.org/10.1016/j.powtec.2014.02.026
0032-5910/© 2014 Elsevier B.V. All rigths reserved.
 K. Sanka et al. / Powder Technology 256 (2014) 336–344 337

Lf represents the ratio between the mass of the liquid portion W and Table 1
the carrier materials Q: Observed responses from 32 factorial design.

Formula Independent Dependent variables

code variables
L f ¼ W=Q : ð2Þ X1 X2 YQ15 (%) YDT (sec)

F1 −1 −1 55.59 ± 4.10 110 ± 5

F2 −1 0 77.49 ± 2.48 55.4 ± 4
With the desired amount of liquid, the amount of carrier and coating F3a −1 +1 90.27 ± 7.07 30.9 ± 2
material can be calculated if the liquid load factor Lf is known. F4 0 −1 52.63 ± 5.46 108.00 ± 6
The aim of this study was to increase the solubility and dissolution F5 0 0 62.84 ± 10.24 21.00 ± 2
F6 0 +1 63.11 ± 9.46 11.50 ± 3
properties of CLZ using liquisolid technique. In this study CLZ, a poorly F7 +1 −1 19.94 ± 0.67 95.00 ± 8
water-soluble, anticonvulsant drug was formulated into liquisolid com- F8 +1 0 48.45 ± 0.44 24.90 ± 6
pacts consisting of similar powder excipients and propylene glycol with F9 +1 +1 51.78 ± 1.10 11.00 ± 4
different drug concentrations in their liquid medications and super
Coded values Actual values
disintegrating agent in the final LSPCs. Further ex-vivo studies have
been carried out to assess the permeation of CLZ from LSPCs using iso- X1 (%) X2 (%)

lated rat intestine. −1 10 0

0 50 2
+1 90 4
2. Materials and methods
a
Indicates optimized formulation; X1% of CLZ in non-volatile solvent; X2% of sodium
2.1. Materials starch glycolate; YQ15% Cumulative drug release at 15th min; YDT disintegration time in
sec.

CLZ was a gift sample from Centaur Pharmaceuticals, India. Micro-

crystalline cellulose PH 102 (MCC PH 102) and sodium starch glycolate
were obtained from SD-Fine Chemicals Ltd., Mumbai, India. AEROSIL®
200 was obtained from Southern Chemicals, Hyderabad, India. Propylene statistical model incorporating interactive and polynomial terms was
glycol was provided by the Universal Laboratories Pvt., Ltd., Mumbai, used to evaluate the response (Eq. (3))
India. HPLC grade methanol and acetonitrile were obtained from Merck
Specialities Pvt., Ltd., Mumbai, India. HPLC grade orthophosphoric acid Y ¼ b0 þ b1 X1 þ b2 X2 þ b12 X1 X2 þ b11 X1 X1 þ b22 X2 X2 ð3Þ
and triethanolamine (TEA) are obtained from Thermo Fisher Scientific
India Pvt., Ltd., Mumbai. India. All other reagents and chemicals used
where Y is the dependent variable, b0 is the arithmetic mean of the 9
were of analytical grade.
trials, and bi is the estimated coefficient for the factor Xi. The X1 and X2
are the main effects; represent the average results of changing one factor
2.2. Methods
at a time from its low to high value. The interaction terms (X1X2) show
how the response changes when two factors are simultaneously varied.
2.2.1. HPLC analysis of clonazepam
The polynomial terms (X1X1 and X2X2) are included to investigate
The samples were assayed for clonazepam by using RP-HPLC method
nonlinearity.
reported earlier [37]. HPLC was equipped LC-20AD pumps, DGU-20A3
degasser, SPD-M20A Photo Diode Array Detector, LC Solutions 1.25 soft-
2.2.4. Preparation of liquisolid powder compacts
ware, Rheodyne injector fitted with 20 μL capacity (Shimadzu, Nakagyo-
LSPCs were formulated according to the 32 factorial design (Table 1).
Ku, Kyoto, Japan) and a Luna (Phenomenex®, USA) C18 column (5 μm,
The weighed amount of drug substance was dispersed in the calculated
4.6 mm × 250 mm). Isocratic elution was carried out at a flow rate
amount of non-volatile solvent (propylene glycol) termed as liquid
1 ml/min. The mobile phase consisted of 60:40 (%v/v) acetonitrile and
vehicle. The mixing procedure was conducted in three stages as
0.1%v/v aqueous TEA (pH 5.5), respectively and the detection wave-
described by Spireas et al. [42]. Firstly, the weighed quantity of carrier
length was set at 254 nm. Aliquots of 20 μL of each sample were injected
material (Microcrystalline cellulose PH 102) was blended with liquid
onto the column.
medication in order to evenly distribute the liquid medication into the
powder. Then, the calculated amount of coating material (AEROSIL®
2.2.2. Solubility studies
200) was added to the system under continuous triturating in a mortar.
Solubility studies were conducted by placing an excess amount of
Finally, to the above binary mixture super disintegrating agent
drug in each vehicle in a 2 ml Clicklock microcentrifuge tubes (Tarsons
i.e., sodium starch glycolate was added and mixed for a period of 10 to
Products Pvt. Ltd., Kolkata, India) containing 1.5 ml of the vehicle. The
20 min producing the final liquisolid powder which was compressed
mixture was vortexed and kept for 48 h at 25 °C in an orbital shaking
using table top pilot scale 10 station rotary tablet Rimek mini press-I
incubator (Remi Electrotechnik Ltd, CIS-2413L, Mumbai, India) to
(M/S Karnavati Engineering Ltd., Gujarat, India)
facilitate the solubilization [34,38]. The samples were centrifuged at
3000 rpm for 15 min to remove the undissolved drug. The supernatant
2.2.5. Micromeritic properties of prepared pre compressed liquisolid
was taken and the concentration of drug in each vehicle was quantified
powder systems
by RP-HPLC method.
A fixed funnel method was used to study the angle of repose (θ). A
weighed quantity of samples was transferred in to graduated cylinder
2.2.3. Experimental design for designing liquisolid powder compacts
from each batch to determine the bulk and tap density using USP-I
A 32 factorial design consists of two independent variables at three
tapped density tester (TD 1025, Labindia Instruments, Mumbai, India).
levels. According to this design, 9 runs were conducted in total [39–41].
The experiments were performed in triplicate. The parameters selected
The independent variables selected for this study were X1, % of CLZ
to study flow properties were determined using Eqs. (4), (5), (6), (7), (8)
in non-volatile solvent (propylene glycol); X2, % of superdisintegrant
(sodium starch glycolate). The dependent variables were Y1, amount of
drug release at 15th min (YQ15); and disintegration time (YDT). The levels h
Tan θ ¼ ð4Þ
of independent and dependent variables were listed in Table 1. A r
338 K. Sanka et al. / Powder Technology 256 (2014) 336–344
Mass
Bulk density ðσ bÞ ¼
Poured volume
Mass
Tap density ðσtÞ ¼
Tapped volume
ðσ t – σ bÞ
Carr’s Indexð%Þ ¼  100
σt
0 ðσ tÞ
Hausner s ratio ¼
ðσ bÞ
2.2.6. Post compression studies of the clonazepam liquisolid powder
compacts
The prepared LSPCs further evaluated for drug content (n = 6)
thickness (n = 20), hardness (n = 20), friability (n = 20) and weight
variation (n = 20). Drug content in each batch was determined by trit-
urating 20 tablets in a mortar with the help of pestle and the amount
equivalent to one average tablet was weighed and dissolved in 250 ml
volumetric flask containing 100 ml of solvent mixture of methanol
and water (1:1). The flask was placed in orbital shaking instrument
(Remi, Electrotechnik Ltd., Vasai, India). Temperature and rpm were
adjusted to 27 °C and 150, respectively. Shaking was continued for
72 h, and later was filtered through a 0.45 μm Millipore membrane filter
paper. The few ml of initial filtrate was discarded and sufficient volume
of filtrate was collected. The amount of drug was estimated by injecting
appropriated diluted samples in to HPLC. The thickness of CLZ liquisolid
powder compacts was determined by using digital Vernier calipers
(CD-6″CSX, Mitutoyo Corp., Kawasaki, Japan) which permits accurate
measurements and provides information on the variation between
tablets. The hardness of the LSPCs was evaluated using Monsanto hard-
ness tester (MHT-20, Kshitij International, Ambala, India), the mean
hardness of each formula was determined. The friability of prepared
formulations was determined using Roche friabilator (FT 1020, Labindia,
Mumbai, India). The disintegration time of the LSPCs was measured
using disintegration tester (DT 1000, Labindia, Mumbai, India). Weight
variation test was performed according to the official method (USP)
using an electronic balance (ATX224, Shimadzu, Japan).
2.2.7. In vitro dissolution studies
In vitro dissolution was carried out using USP II dissolution apparatus
(DS 8000, Labindia, Mumbai, India). LSPCs were placed in the vessel
containing 900 ml of HPLC water as suitable dissolution media at
37 ± 0.5 °C and paddle speed of 75 rpm. At predetermined time
intervals (5, 10, 15, 30, 45, 60, 90 and 120 min), an aliquot of 5 ml was
withdrawn and replaced with an equal volume of fresh dissolution
medium to maintain constant volume [43,44]. The samples were filtered
through the 0.45 μm Millipore membrane filter (NYLON 66, Axiva, Lab
filters, Delhi, India) and assessed for drug release using HPLC.
2.2.8. Fourier transforms infrared (FTIR) spectroscopy
Fourier transform infrared spectra of the Pure CLZ, MCC PH 102,
AEROSIL® 200, sodium starch glycolate and optimized CLZ loaded LSPC
were obtained using Fourier Transform Infrared Spectrophotometer
(Bruker, Alpha-T, Ettlingen, Germany). Samples were prepared using
KBr disks by means of hydraulic pellet press at a pressure of 10 t. The
samples were scanned from 4000 to 400 Cm−1. Spectra are analyzed
by OPUS-6.5 software.
2.2.9. Solid state characterization
ð5Þ
2.2.9.1. Differential scanning calorimetry (DSC). DSC thermograms of Pure
CLZ, MCC PH 102, AEROSIL® 200, sodium starch glycolate and opti-
mized CLZ loaded LSPC were analyzed by DSC (Model: SIIO-6300,
Japan). Samples (3–5 mg weighed to precision of 0.005 mg) were
ð6Þ
placed in aluminium pans and the lids were crimped using a crimper.
Thermal behavior of the samples was investigated at a scanning rate
of 10 °C/min, using nitrogen as blanket gas covering a temperature
range of 0 to 300 °C [45].
ð7Þ
2.2.9.2. X-ray diffraction (XRD). For further characterization of the crys-
talline state, the X-ray diffraction (XRD) patterns were determined
for Pure CLZ, MCC PH 102, AEROSIL® 200, sodium starch glycolate and
optimized CLZ loaded LSPC. The X-ray powder diffraction was obtained
ð8Þ using X-RD (Model: Stereoscans S120, Cambridge, UK). The samples
are loaded on to the diffractometer, then exposed to Cu-Kα radiation
(40 kV × 30 mA) and scanned over a range of 2θ, values from 10 to
80°at a scan rate of 0.05°/0.4 s [45].
2.2.10. Ex-vivo permeation studies
Male Wistar rats weighed between 180 and 200 g used in the study.
The animals were housed in separate cages and maintained under con-
trolled condition of temperature and the rats had free access to water
and food until they were sacrificed. The study was conducted with the
prior approval of the Institutional Animal Ethical Committee, School of
Pharmacy, Anurag Group of Institutions, Hyderabad, India.
The rats were sacrificed with excess ether inhalation. The abdomen
was opened and a segment of the ileum was removed and flushed
with Krebs–Ringer solution to remove the mucus and adhered intestinal
contents. One end of the intestine segment was tied and dose equivalent
crushed LSPCs of optimized formulation were introduced into the
lumen and were tightly closed. The tissue was placed in a 250 ml beaker
with continuous aeration and maintained at a temperature of 37 °C.
The beaker consists of 100 ml of phosphate buffer (pH 6.8). At predeter-
mined time intervals (5, 10, 15, 30, 45, 90 and 120 min) an aliquot of 1
ml was collected and replenished with equal volume of medium. The
samples were treated with an equal volume of methanol, centrifuged
and the supernatant was quantified for CLZ using HPLC. Control (pure
CLZ) and marketed formulation were also included in the study for
comparison [46,47].
The cumulative amount of CLZ permeated (Q) was plotted against
time (t). The steady state flux (Jss) was calculated from the slope of
linear portion of the cumulative amount permeated per unit area vs.
time plot. The permeability coefficient (Kp) of the CLZ through intestine
was calculated by dividing steady state flux with initial concentration of
CLZ donor compartment. The enhancement ratio (ER) was calculated by
using the following equation:
Jss of liquisolid formulation
ER ¼ : ð9Þ
Jss of control
2.2.11. Stability studies
Stability studies were carried out according to the ICH guidelines by
storing the CLZ loaded LSPC formulation at 40 °C/75% ± 5% RH for a
period of three months. The samples were withdrawn after 90 days
and analyzed for assay, disintegration time and in vitro release studies
[48–51].
3. Results and discussion
3.1. Solubility studies
The solubility of CLZ in propylene glycol, span 80, span 20 and water
is given in Table 2. The results show that the solubility of the CLZ is very
 K. Sanka et al. / Powder Technology 256 (2014) 336–344 339

Table 2 Table 4
Solubility of CLZ in various non-volatile solvents. Regression coefficients for the responses.

S.No. Solvent Solubility (mg/ml) S.No. Coefficients of regression parameters

1 Propylene glycol 6.48 ± 0.081 b0 b1 b2 b12 b11 b22 R2-value p-value

2 Span 80 3.46 ± 0.022
YQ15 58.01 −17.2 12.83 – – – 0.8958 0.0011
3 Span 20 0.44 ± 0.010
YDT 28.63 −10.98 −43.27 −1.22 7.7 27.3 0.9810 0.0087
4 Water 0.14 ± 0.070
YQ15% cumulative drug release at 15th min; YDT disintegration time in sec.

poor in water (0.14 mg/ml) whereas, propylene glycol enhanced the
solubility of CLZ to the level of 6.48 mg/ml. Solubility in span 80 and
span 20 was found to be 3.46 and 0.44, respectively. Thus, from the
above results of solubility studies among the non-volatile solvents
tested, propylene glycol could be a better choice as a solvent for the
formulation of LSPCs.
3.2. Precompression studies of the prepared liquisolid systems
The flowability of powder material plays a major role in the produc-
tion of pharmaceutical dosage forms in order to get a uniform feed as
well as reproducible filling of tablet dies, otherwise dose variations
will occur. Flow properties of the liquisolid powder systems were
studied by determining angle of repose (θ), Carr's index, Hausner's
ratio, bulk density (σ b) and tapped density (σ t) [52] and their results
are presented in Table 3.
As angle of repose (θ) is a characteristic of internal friction or
cohesion of the particles, the value of the angle of repose will be high
if the powder is cohesive and low if the powder is non-cohesive. As
presented in Table 3, angle of repose was in the range from 25.15 ±
0.14 to 28.07 ± 0.08 which indicates good flow ability of the powder
from all formulations. The values of bulk and tapped density were
found to be in the range from 0.511 ± 0.001 to 0.573 ± 0.003 g/cm3,
0.596 ± 0.02 to 0.657 ± 0.011 g/cm3, respectively. The Carr's index
(%) and Hausner's ratio was found to be in the range from 11.10 ±
0.10 to 17.16 ± 0.15, 1.11 ± 0.09 to 1.19 ± 0.0, respectively. These
values indicate that the micromeritic properties of all powder blends
from each batch are within the limits and they exhibit good flow prop-
erty and compressibility.
3.3. Application of experimental design for designing liquisolid tablets
polynomial equation can be used to draw a conclusion after considering
the magnitude of coefficient and the mathematical sign it carries (posi-
tive or negative). Results of ANOVA were depicted in Table 5 and the in-
vitro dissolution release profile of 9 trials vs pure CLZ was shown in
Fig. 1. To demonstrate the effect of the % of CLZ in propylene glycol
and % of sodium starch glycolate, the response surface plots were
generated for the dependent variables YQ15, YDT using Design-Expert®
Software (Stat-Ease Inc., Minneapolis).
3.3.1. Effect of formulation variables on release profile (YQ15)
The in vitro performance of CLZ loaded LSPCs showed release profile
depends upon the formulation variables i.e., % of CLZ in propylene glycol
(X1) and % of sodium starch glycolate (X2). The results showed that
optimized formulation of CLZ loaded LSPC demonstrated more than
85% drug release at Q15, when compared with that of pure drug which
demonstrated less than 10% drug release at Q15 at P b 0.05. The effect
of formulation variables on the amount of drug release at 15th minute
(YQ15), is given in Eq. (10).
YQ 15 ¼ 58:01−17:20X1 þ 12:83X2 : ð10Þ
In Eq. (10), b1 bears negative sign indicating an increase in the % of
CLZ in propylene glycol (X1) decreased the amount of drug release at
15th min, b2 bears positive sign in the same equations indicating an
increase in the % of sodium starch glycolate (X2) increase in amount of
drug release at 15th min.
3.3.2. Effect of formulation variables on disintegration time (YDT)
The effect of formulation variables i.e., CLZ % propylene glycol (X1)
and % of sodium starch glycolate (X2) on disintegration time (YDT), is
given in Eq. (11).

In the present investigation, the effect of % of CLZ in propylene glycol

(X1) and % of sodium starch glycolate (X2) on % cumulative drug release
at 15th min (YQ15) and disintegration time (YDT) is studied using 32
factorial design, revealed wide variation (Table 1). The data clearly
indicates that the dependent variables are strongly dependent on the
independent variables. The fitted equation relating the response YQ15,
YDT to the transformed factor are shown in Eqs. (10) and (11). The
value of correlation coefficient (Table 4) indicates good fit. The
YDT ¼ 28:63−10:98X1 −43:27X2 −1:22X1 X2 þ 7:7 X1 X1 þ 27:30 X2 X2 :
ð11Þ
In the above equation, b1 and b2 bear negative sign indicating an
increase in the % of CLZ in propylene glycol (X1) and % of sodium starch
glycolate (X2) decreased the disintegration time, b12 bears negative sign

Table 3
Micromeritic properties of prepared pre-compression liquisolid powders.

Formula code Angle of repose (θ) Bulk density (gm/cm3) Tapped density (gm/cm3) Carr's index (%) Hausner's ratio

F1 27.31 ± 0.3 0.573 ± 0.003 0.647 ± 0.006 11.10 ± 0.10 1.11 ± 0.09
F2 26.55 ± 0.49 0.541 ± 0.002 0.656 ± 0.003 17.16 ± 0.15 1.17 ± 0.06
F3 25.64 ± 0.56 0.563 ± 0.005 0.657 ± 0.011 13.91 ± 0.03 1.15 ± 0.005
F4 27.59 ± 0.09 0.524 ± 0.014 0.59 ± 0.0015 12.07 ± 0.11 1.126 ± 0.005
F5 26.51 ± 0.04 0.526 ± 0.005 0.596 ± 0.02 11.39 ± 0.01 1.123 ± 0.005
F6 28.07 ± 0.08 0.526 ± 0.015 0.630 ± 0.001 15.95 ± 0.066 1.19 ± 0.01
F7 26.61 ± 0.03 0.522 ± 0.001 0.627 ± 0.002 17.02 ± 0.06 1.17 ± 0.05
F8 27.36 ± 0.32 0.563 ± 0.029 0.655 ± 0.009 11.57 ± 0.15 1.13 ± 0.005
F9 25.15 ± 0.14 0.511 ± 0.001 0.601 ± 0.007 14.24 ± 0.22 1.15 ± 0.005
340 K. Sanka et al. / Powder Technology 256 (2014) 336–344

Table 5
Results of analysis of variance for measured response.

Parameters Degree of freedom Sum square Mean square f-Value p-Value

For YQ15
Regression 2 2762.52 1381.26 25.79 0.0011
Residual 6 321.33 53.56
Total 8 3083.85

For YDT
Regression 5 13560.05 2712.01 31.01 0.0087
Residual 3 262.37 87.46
Total 8 1322.42

YQ15% cumulative drug release at 15th min; YDT disintegration time in sec.

in the same equation indicating the interaction effect of X1X2 decreased

the disintegration time, b11 and b22 bear positive sign which indicates
that the interaction effect of X1X1 and X2X2 increased the disintegration
time.
The relationship between dependent and independent variables
was further elucidated using contour plots and 3D plots. The effects of
X1 and X2 and their interaction on YQ15 and YDT are given in Fig. 2. It
could be seen that increase in % of CLZ in propylene glycol and % of
sodium starch glycolate had a negative effect on YDT.

3.4. Post compression studies of the clonazepam liquisolid tablets

The CLZ loaded LSPCs were evaluated to determine the post com-
pression parameters, the results were shown in Table 6. The thickness
of the LSPCs was found to be ranged from 4.98 ± 0.1067 to 5.15 ±
0.125 mm. The hardness of the liquisolid compacts was measured by
Monsanto hardness tester and was found to be ranged from 4.76 ± Fig. 2. Response surface plot (A) and contour plot (B) showing effect of X1 and X2 on
response YDT.
0.28 to 5.16 ± 0.35 kg/cm2. The friability was found to be ranged from
0.498 ± 0.166 to 0.691 ± 0.193% which was below 1% for all the
formulations, which is an indication of good mechanical resistance of
the compacts. The weight variation for different formulations (F1–F9)
showed satisfactory results as per the United States Pharmacopeia CLZ loaded LSPC formulations, assay studies were performed which
(USP) limit (average weight ± 5%). For estimation of drug in different showed satisfactory results as per the USP i.e., CLZ tablets must
contain not less than 90% and not more than 110% of the labeled amount
of CLZ.

3.5. Fourier transform infrared spectroscopy

Drug-excipient interactions play a crucial role with respect to

the stability and potency of the drug. Fourier transform infrared
spectroscopy (FTIR) techniques have been used to study the physical
and chemical interaction between drug and excipients used. In the
spectra of optimized CLZ loaded formulation of LSPCs (Fig. 3, Table 7),
the peak characteristics to the excipients were present at almost
same positions, whereas CLZ peaks were also present, but at a reduced
intensity of absorption, indicating the trapping of CLZ inside the carrier
matrix. None of the spectra showed any peaks other than those
assigned to CLZ and excipients, which indicate that there is no differ-
ence between the IR patterns of the optimized formulation of CLZ and
pure drug.

3.6. Solid state characterization

3.6.1. Differential scanning calorimetry

Fig. 1. Dissolution release profiles of pure CLZ vs CLZ loaded LSPC formulations (F1–F9).
Differential scanning calorimetry (DSC) curve for CLZ showed a
sharp endothermic (melting) peak at 241 °C (Fig. 4, curve A). The CLZ
peak was absent in the DSC curve of Optimized formulation of CLZ
liquisolid powder compacts, indicating that the drug in final formula-
tion was less crystalline (more amorphous). Conversion of crystalline
 K. Sanka et al. / Powder Technology 256 (2014) 336–344 341

Table 6
Physical evaluation parameters of CLZ liquisolid powder compacts.

Formula code Thickness (mm) Hardness (kg/cm2) Friability (%) Weight variation (mg) Assay (%)

F1 5.11 ± 0.1067 5.03 ± 0.05 0.691 ± 0.193 448.2 ± 1.62 95 ± 3.12

F2 5.15 ± 0.125 5.06 ± 0.11 0.648 ± 0.165 447.5 ± 1.94 97 ± 2.84
F3 5.06 ± 0.124 5 ± 0.2 0.55 ± 0.157 450 ± 0.67 98.00 ± 2.02
F4 5.03 ± 0.047 5.16 ± 0.35 0.498 ± 0.166 448.2 ± 1.83 100 ± 1.03
F5 5.05 ± 0.095 4.93 ± 0.11 0.683 ± 0.186 448.7 ± 1.28 99.25 ± 3.41
F6 5.15 ± 0.047 4.83 ± 0.28 0.647 ± 0.194 447.9 ± 1.92 97.82 ± 2.03
F7 4.98 ± 0.1067 4.76 ± 0.25 0.594 ± 0.224 446.8 ± 1.75 98.33 ± 1.22
F8 5.11 ± 0.106 5 ± 0.2 0.645 ± 0.163 448.1 ± 1.55 99.88 ± 2.09
F9 5.08 ± 0.134 4.83 ± 0.28 0.610 ± 0.154 449 ± 1.38 97.26 ± 1.71

form of drug to amorphous form indicates enhanced solubility, which
led to improved dissolution release profile of the drug by formulation
of LSPCs.
3.6.2. X-Ray powder diffraction analysis
X-ray powder diffraction (XRD) analysis was used to assess the
degree of crystallinity of the LSPC constituents. CLZ showed major
peaks at 2θ values of 11.86, 14.79, 15.06, 18.27, 18.54, 20.07, 20.49,
22.84, 23.95, 24.34, 26.11, 27.16, 27.48, 27.81, 30.23° (Fig. 5, curve A).
Analysis of XRD patterns of the CLZ loaded optimized formulation
(Fig. 5, curve E) indicated that all the major peaks corresponding to
CLZ disappeared except peak at 22.84°, which show conversion of
crystalline form of drug to amorphous form due to addition of the
excipients to the formulation. A fall in degree of crystallinity means an
improvement in the amorphousness of a sample. Hence, from the
above discussion it was concluded that the LSPC technique resulted in
an amorphous form of CLZ with suitable excipients, which led to
improved dissolution release profile.

3.7. Ex-vivo intestinal permeation study

The rate and extent of absorption are not only dependent on the
dissolution rate rather than permeation across the gastrointestinal
tract but are also limiting factors for absorption. Therefore to have an
insight on the ability of liquisolid tablet formulations for improved
absorption, ex-vivo permeation study was carried out using the rat
intestinal segment. Since majority of the drugs get absorbed in vivo
from the small intestine it is more relevant to use the rat tissue [28].
Henceforth, we have used ileum portion to assess the potential of
LSPCs for improving the permeation of CLZ across the intestinal barrier.
Ex-vivo permeation data did not show significant (p N 0.05) differ-
ence between marketed formulation and free CLZ. The cumulative
amount permeated (CAP) across the intestine was calculated and repre-
sented in Fig. 6. The cumulative amount of CLZ permeated from control
to marketed formulation was 511 ± 28 and 405 ± 6 μg, respectively
and was significantly increased to 1492 ± 88 μg with optimized formu-
lation (P b 0.05) (Table 8). The flux was also increased significantly

Table 7
Characteristic peaks of free CLZ and CLZ in optimized LSPC.

S.No Compound Type of vibration Peaks (cm−1)

1 Pure drug CLZ N\H stretching 3106

Ar\H stretch 2971
C\H stretch
Ar\C\C stretch 1695
(in–ring)
Aromatics 1615
2 Optimized LSPC N\H stretching 3383
Ar\H stretch 2904
C\H stretch 1637
Aromatic
C\C stretch 1614
(in–ring)
Fig. 3. FTIR Spectra of A) Pure CLZ B) MCC PH 102 C) AEROSIL® 200 D) Sodium starch
Aromatics
glycolate E) Optimized CLZ loaded LSPCs.
342 K. Sanka et al. / Powder Technology 256 (2014) 336–344

Fig. 4. DSC thermograms of A) Pure CLZ B) MCC PH 102 C) AEROSIL® 200 D) Sodium
starch glycolate E) Optimized CLZ loaded LSPCs.

with optimized CLZ loaded LSPC formulation compared to control and

marketed formulation (P b 0.05). The ER was above 1 (3.80 ± 0.1) for
optimized CLZ loaded LSPCs (F3) and indicates enhanced permeation
compared to pure CLZ.

3.8. Stability study

Fig. 5. XRD patterns of A) pure CLZ B) MCC PH 102 C) AEROSIL® 200 D) Sodium starch
glycolate E) Optimized CLZ loaded LSPCs.
Stability study of optimized CLZ loaded LSPC formulations was
performed at 40 °C/75% ± 5% RH for three months. Samples were with-
drawn after 3 months and showed no significance (p N 0.05) change in
assay (%), and disintegration time (YDT). The similarity factor (f2) for the
optimized formulation was found to be 67.80 (Table 9), which indicates
good similarity of dissolution release profile, before and after stability
study.

4. Conclusion

In conclusion, the present study showed that LSPC technique could

be a promising strategy in improving dissolution of poorly water soluble
CLZ and wettability was improved by making a suspension in propylene
glycol, the water soluble, nonvolatile solvent. LSPCs could be prepared
using MCC PH 102 as a carrier, and AEROSIL® 200 as a coating material.
The FTIR studies revealed that excipients were compatible with the
drug. DSC and XRD studies showed that there is a decrease in crystallin-
ity of the CLZ in liquisolid compact formulation. A fall in crystallinity
means improved dissolution release profile. The optimized formulation
(F3) showed higher dissolution rate when compared with that of Fig. 6. Ex-vivo permeation of CLZ across rat intestine from Free CLZ, LSPCs and market
pure drug. formulation.
 K. Sanka et al. / Powder Technology 256 (2014) 336–344 343

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