Drug Delivery

ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: https://www.tandfonline.com/loi/idrd20

Novel tablet formulation of amorphous

candesartan cilexetil solid dispersions involving P-
gp inhibition for optimal drug delivery: in vitro and
in vivo evaluation

Gurunath Surampalli, Basavaraj K. Nanjwade, P. A. Patil & Rakesh Chilla

To cite this article: Gurunath Surampalli, Basavaraj K. Nanjwade, P. A. Patil & Rakesh Chilla
(2016) Novel tablet formulation of amorphous candesartan cilexetil solid dispersions involving P-gp
inhibition for optimal drug delivery: in�vitro and in�vivo evaluation, Drug Delivery, 23:7, 2124-2138,
DOI: 10.3109/10717544.2014.945017

To link to this article: https://doi.org/10.3109/10717544.2014.945017

Published online: 31 Jul 2014.

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ISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, 2016; 23(7): 2124–2138

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.945017

RESEARCH ARTICLE

Novel tablet formulation of amorphous candesartan cilexetil solid

dispersions involving P-gp inhibition for optimal drug delivery:
in vitro and in vivo evaluation
Gurunath Surampalli1, Basavaraj K. Nanjwade2, P. A. Patil3, and Rakesh Chilla4
1
Department of Pharmacology, Vaagdevi Institute of Pharma Sciences, Warangal, Andhra Pradesh, India, 2Faculty of Pharmacy, Department of
Pharmaceutics, Omer Al-Mukhtar University, Tobruk, Libya, 3Department of Pharmacology, International Medical Programme, USM-KLE, Belgaum,
Karnataka, India, and 4Department of Pharmaceutics, Vaagdevi Institute of Pharma Sciences, Warangal, Andhra Pradesh, India

Abstract Keywords
Objective: The aim of this study was to develop a novel tablet formulation of amorphous Candesartan, direct compression, naringin,
candesartan cilexetil (CAN) solid dispersion involving effective P-gp inhibition for optimal drug oral bioavailability
delivery by direct compression (DC) method.
Methods: To accomplish DC, formulation blends were evaluated for micromeritic properties. History
The Carr index, Hausner ratio, flow rate and cotangent of the angle a were determined. The
tablets with and without naringin prepared by DC technique were evaluated for average Received 26 May 2014
weight, hardness, disintegration time and friability assessments. The drug release profiles were Revised 11 July 2014
determined to study the dissolution kinetics. In vivo pharmacokinetic studies were conducted in Accepted 11 July 2014
rabbits. Accelerated stability studies were performed for tablets at 40 ± 2  C/75% RH ± 5% for
6 months.
Results: FTIR studies confirmed no discoloration, liquefaction and physical interaction between
naringin and drug. The results indicated that tablets prepared from naringin presented a
dramatic release (82%) in 30 min with a similarity factor (76.18), which is most likely due to the
amorphous nature of drug and the higher micromeritic properties of blends. Our findings
noticed 1.7-fold increase in oral bioavailability of tablet prepared from naringin with mean Cmax
and AUC0–12 h values as 35.81 ± 0.13 mg/mL and 0.14 ± 0.09 mg h/mL, respectively. The tablets
with and without naringin prepared by DC technique were physically and chemically stable
under accelerated stability conditions upon storage for 6 months.
Conclusion: These results are attractive for further development of an oral tablet formulation of
CAN through P-gp inhibition using naringin, a natural flavonoid as a pharmaceutical excipient.

Introduction Solid dispersions have been the focus of growing interest

during the last decades to develop poorly soluble compounds
Candesartan cilexetil (CAN) is an esterified prodrug of
like CAN to increase dissolution rate and impact saturation
candesartan and an angiotensin II receptor antagonist. Ester
solubility. In these dispersions, the drug is often present as
hydrolysis during absorption from gastrointestinal tract rapidly
partially amorphous or amorphous substance (Yonemochi
and completely bio-activates CAN to its active parent drug,
et al., 1997, 1999; Mura et al., 2002; Dai et al., 2007).
candesartan. A very low aqueous solubility (0.0003 mg/mL)
Previously, we reported that freeze-drying is the most suitable
is the major drawback of CAN to develop an oral dosage form
preparative technique among various pharmaceutical inter-
for its therapeutic application and efficacy. Incomplete
ventions for preparing amorphous solid dispersions of poorly
absorption from the gastrointestinal (GI) tract is reported due
water-soluble drug, CAN (Gurunath et al., 2014).
to the low solubility of CAN across the physiological pH range.
Together, we demonstrated that, improved aqueous
CAN is classified as a class II drug in the Biopharmaceutics
solubility followed by effective P-gp efflux pumps inhibition
Classification System (BCS) based on its solubility across
in the GI tract, the CAN oral bioavailability can significantly
physiologically relevant pH conditions and absorption char-
be enhanced through freeze-dried solid dispersions using
acteristics (Vijaykumar et al., 2009).
naringin, as a novel pharmaceutical excipient (Gurunath et al.,
2013, 2014).
Recently, Li et al. (2013, 2014) reported the acute and
chronic oral toxicity studies of naringin at a single dose of
Address for correspondence: Mr. S. Gurunath, H.No 2-10-910, Bank
Colony, Subedari, Wadepalli, Warangal 506370, Andhra Pradesh, India. 16 g/kg that did not produce any mortality, abnormal changes
Tel: +91 9966555091. Email: s.gurunath1979@gmail.com in body weights and food consumption, undesirable clinical
DOI: 10.3109/10717544.2014.945017
signs, toxicologically related alterations in hematology,
clinical biochemistry and macroscopic findings upon expos-
ure of rats. It is rational to establish that LD50 of rats is greater
than 16 g/kg when treated with single oral dose of naringin.
Therefore, naringin could be considered as reasonably safe
with Hodge and Sterner scale but non-toxic with Gosselin,
Smith and Hodge scale. According to the study, the no
observed adverse effect level (NOAEL) of naringin is
anticipated to be more than 1250 mg/kg/day to Sprague
dawley rats following daily oral administrations for 13 weeks
or 6 months.
P-gp-mediated drug efflux significantly intervenes the
delivery of the drugs that requires a very small dose for their
pharmacological actions or the drugs that show extremely
slow diffusion and dissolution rates. Those small amounts of
drugs cannot reach the blood circulation in adequate quantity
as it decreases drug absorption and sometimes, that can be life
threatening (Amin, 2013).
This impact on the bioavailability has been recognized in
research using mdr1a/1b knockout mice, where the absorption
of paclitaxel, a substrate of P-gp, was greater than that in
wild-type mice (Schinkel et al., 1997; Sparreboom et al.,
1997; Woo et al., 2003). These findings established that about
half of paclitaxel dose administered is expelled out into the
gut lumen by P-gp and simply a small amount of drug is lost
by gut wall and liver metabolism. Thus, the effective
inhibition P-gp-mediated efflux can significantly improve
the oral bioavailability of paclitaxel.
Traditionally, the oral route is the most frequently accepted
route of drug administration. Tablets are considered the most
attractive dosage form by patients and industry for drug
delivery (Allen et al., 2005; Rudnic, 2005; Staniforth, 2007;
Emery et al., 2009).
Manufacturing tablets, a chiefly directly compressed tablet
is clear-cut manufacturing process that involves low cost,
hence attractive to pharmaceutical laboratories (Strydom
et al., 2011).
The term ‘‘direct compression’’ (DC) is employed to
describe the process where tablets are compressed directly
from the powder blends of active ingredients and suitable
excipients. No pre-treatment by wet or dry granulation is
involved for the powder blends. The DC is more economical,
decreases the cycle time of the products and is simple in terms
of the requirements of good manufacturing practices. The DC
is environment-friendly with less number of steps, without
involving water and temperature, and finally the stability of
the final product can be improved (Gohel & Jogani et al.,
2005; McCormick, 2005; Strydom et al., 2011).
To the best of our understanding, there are no available
published reports or works demonstrating the effects of
naringin as P-gp inhibitor as a novel concept of employing
naturally occurring bio-flavonoid as a pharmaceutical excipi-
ent with absorption-improving potential formulated as com-
pressed tablets with significant improvement in the
dissolution rate of CAN through amorphous forms.
Consequently, our study involves CAN; an ester prodrug
that rapidly de-esterifies in vivo during absorption from the
GI tract to produce its active metabolite, candesartan
(McClellan & Goa, 1998) was used as a model drug. CAN
is not only an extremely poor lipophilic compound
An in vitro and in vivo evaluation of novel tablet formulation 2125
(Vijaykumar et al., 2009) but also its active metabolite,
candesartan is a substrate of P-gp (also known as MDR1,
ABCB1; Zhou et al., 2009). The bioflavonoid, called
naringin, was identified as an extremely safe; naturally
occurring novel pharmaceutical excipient to show effective
P-gp-mediated cellular efflux inhibition of P-gp substrate,
candesartan (Lassoued et al., 2012; Gurunath et al., 2013,
2014).
Hence, our present investigation aims:
(a) To prepare novel tablet formulation by DC method with
and without naringin to combine the two systems
(improved poor water-solubility through amorphization
and effective P-gp efflux pump inhibition) to one system.
(b) To evaluate the formulation blends for micromeritic
properties.
(c) To evaluate the physical characteristics of directly
compressed tablets with and without naringin.
(d) Pharmacokinetic evaluation of finished tablets with and
without naringin in rabbits.
(e) To perform accelerated stability studies for tablets with
and without naringin at 40 ± 2  C/75% RH ± 5% upon
storage for 6 months.
Materials and methods
Candesartan cilexetil (of purity more than 99%, melting
temperature ¼ 163 ± 0.5  C) was kindly obtained as a gift
sample from the Dr. Reddy’s Labs (Hyderabad, India).
Polyvinyl pyrrolidone K30 (PVP-K30) was obtained from
SD Fine Chemicals Ltd., Mumbai, India. Candesartan,
paracetamol, naringin, spray-dried lactose, Avicel PH 102,
mannitol, magnesium stearate, talc, Na2HPO4, KH2PO4,
NaOH, KCl, citric acid NaCl, CaCl2, MgCl2 and NaHCO3
were obtained from Sigma Aldrich (Hyderabad, India).
Acetonitrile, methanol of HPLC grade and orthophosphoric
acid were supplied by E-Merck India Ltd., Mumbai. All other
chemicals were of analytical grade. All drug solutions were
freshly prepared before use.
Drug–excipient interaction studies
It was imperative to understand the compatibility between the
drug under study and the novel excipient used within the
system while designing a tablet formulation. As a result, it is
necessary to conform that drug is not interacting with
excipient for a period of 4 weeks under experimental
conditions (40 ± 5  C and 75 ± 5% RH). The infrared absorp-
tion spectra were run between 4000 and 400 cm1 for the pure
drug and the physical mixture of drug and excipient. The IR
spectra of pure drug and a mixture of excipient and drug are
shown in Figure 1(a–c).
Formulation of blends
Freeze-dried CAN-loaded (1:2 w/w, CAN: PVP-K30) solid
dispersions (Gurunath et al., 2014) and excipients, like
naringin, Avicel PH 102, spray-dried lactose, mannitol,
were co-grounded in pestle mortar (without talc and magne-
sium stearate) and were passed through mesh no. 60. Finally,
talc and magnesium stearate were added and mixed for 5 min
to obtain formulation blends (Table 1).
2126 G. Surampalli et al. Drug Deliv, 2016; 23(7): 2124–2138

Figure 1. FT-IR spectrum of (a) Candesartan

cilexetil (CAN), (b) Naringin (NGN) and (c)
a physical mixture of pure candesartan
cilexetil and Naringin.

Micromeritic studies of the formulation blends with
novel excipient (naringin)
Bulk and tapped densities determination of the blends
Apparent density (b) and tapped density (t) were measured
through the apparent volume indirectly (Qiu et al., 2009).
The tests were performed with an automatic compactor
(Tapped Density Volumeter, Sisco, India). The bulk density
was determined by gradually pouring the samples into a
100 mL graduated glass cylinder up to 60% of the container
volume. The mass associated with the filled volume (60 mL)
was evaluated on an analytical balance. The bulk density was
DOI: 10.3109/10717544.2014.945017 An in vitro and in vivo evaluation of novel tablet formulation 2127
Table 1. Composition of 100 mg tablets (FDS-NT and FDS-T). Tablets preparation by direct compression method
with FSD-NT and without FSD-T naringin
FDS-NT Quantity FDS-T Quantity
(with naringin) (mg) (without naringin) (mg) A preliminary assessment was made to test the ability of the
Freeze-dried solid 24 Freeze dried solid 24 blends to form tablets by DC with and without naringin.
dispersions dispersions An optimized tablet formulations were prepared as listed in
(1:2 w/w, drug to (1:2 w/w, drug to Table 1. Fifty tablets for each formulation were prepared.
carrier ratio) carrier ratio)
Naringin 15 b-Cyclodextrin 15
Table 1 shows the compositions and sample designations of
Spray-dried lactose 18 Spray-dried lactose 18 the tablets. The entire constituents as shown in Table 1 were
Mannitol 18 Mannitol 18 co-grounded for 5–10 min in a glass mortar pestle for each
Avicel PH 102 22 Avicel PH 102 22 formulation separately and were passed through sieve no. 60.
Magnesium stearate 01 Magnesium stearate 01
Talc 02 Talc 02 The mixed blends of excipients were compressed to produce
convex faced tablets weighing 100 mg each, respectively, with
5 mm diameter by means of a single punch tablet machine
(Cadmach, Ahmedabad). The compression force and time
calculated using the relation between the weight (g) and the
were 15 kN and varied from 30 to 34 s for both tablet
apparent volume (mL). The following formula was used to
formulations, respectively.
calculate the bulk density.
The tapped density was obtained by b ¼ VMb tapping a Physical evaluation of tablets
graduated glass cylinder containing a known amount of
samples. The volume after 100 taps (V100) and 500 taps (V500) According to official methods, all tablets (FSD-NT and FSD-
was recorded. In cases where the difference between V100 and T) were evaluated for weight uniformity, friability, thickness
V500 was more than 2%, an additional cycle of 500 taps was and hardness. Twenty tablets were used for weight variation
carried out until variations were less than 2%. determination using an electronic balance (Sartorius, India).
The tapped density (g/mL) was obtained from the mass (g) Twenty tablets (Jadhav et al., 2011) were weighed separately
and the final volume of solids (mL). The following formula and collectively using digital balance.
was used to calculate the tapped density. The average weight of one tablet was obtained by dividing
the total sum of 20 tablets. The weight variation test is an
M acceptable method for determining the drug content uniform-
t ¼
Vt ity of tablets. For an average weight equal to 130 mg or less,
the maximum of 10% weight variation is considered satis-
Hausner ratio and Carr’s index determination factory. Ten tablets were used to determine the tablet hardness
using a Monsanto tablet hardness tester (Campbell
The Hausner ratio (HR) is the association between t and b Electronics, Mumbai, India). About 40 N (1 kg ¼ 10 N),
and is linked to inter-particle resistance. It can be used to tablet breaking force is considered adequate for mechanical
evaluate the powder flow properties. The Carr index (CI%) is stability. Tablet friability was calculated as the percentage
expressed as a percentage which is the measure between the weight loss of 10 tablets at 25 rpm for 4 min using a friabilator
apparent and compact densities. The compressibility percent- (Sisco, India). The weight loss should not be greater than 1%
age indirectly provides a concept of the cohesion, size w/w. The disintegration time was determined at 37 ± 0.5  C
uniformity and surface area of powders or their mixtures in purified water with a disintegration tester (Sisco, India)
(Staniforth, 2007). The compressibility index (I) is calculated fitted with sintered disks. The disintegration time is recorded
as follows: for an average of six measurements. Results are shown in
t  b Tables 2 and 3.
I¼  100
t
Dissolution study
where t is the tapped density and b is the bulk density.
Hausner ratio (HR) is an indirect measure of ease of The USP II Paddle dissolution apparatus (TDT-08 L,
powder flow that is calculated as follows. Electrolab, India) was used to perform the dissolution studies.
The tablets were placed in 900 mL phosphate buffer (pH 6.8)
t maintained at 37 ± 0.5  C, stirring at 50 rpm. At periodical
Hr ¼
b intervals of 0, 2, 4, 6, 10, 15, 20, 25 and 30 min, samples were
where t is tapped density and b is bulk density. Smaller collected, filtered through 0.45 mm and replaced with a fresh
Hausner ratio (51.25) signifies improved flow properties than dissolution medium.
higher ones (41.25). Samples withdrawn at predetermined time intervals,
filtered in-line and assayed using an HPLC method.
Chromatographic separation was accomplished using a
Flow rate and the cotangent of angle a analysis
Shimadzu HPLC system (LC-20 A series) equipped with a
The blends flow rate was evaluated with a flow tester from UV detector, micro-volume double plunger pump (10 mL/
the flow time of the sample. The blends (50 g) were poured stroke) and manual injector type model 7725-port sample
through the funnel (12 mm) attached to the stiff equipment. injection valve was employed. The system was controlled
The funnel was opened and the time taken to release the through SCL-10Avp or SCL-10 A software (Conquer
samples was noted (Schüssele & Bauer-Brandl, 2003). Scientific Lab Equipment, San Diego, CA).
2128 G. Surampalli et al.
Table 2. Micromeritic properties of formulation blends (Mean ± SD, n ¼ 3).
Parameters
Formulation Bulk Density (g/mL) Tapped Density (g/ml)
FDS-NT 0.567 ± 0.034 0.640 ± 0.023
FDS-T 0.618 ± 0.029a 0.696 ± 0.039a
a
p40.05 when compared to FDS-NT.
Table 3. Physical properties of FSD-NT and FSD-T tablets (Mean ± SD, n ¼ 3/6).
Parameters
Formulation Thicknessa (mm) Weighta (mg)
FDS-NT 3.028 ± 0.045 100.4 ± 4.24
FDS-T 3.049 ± 0.027c 100.9 ± 3.17c
a
Average of three determinations.
b
Average of six determinations.
c
p40.05 statistically insignificant when compared to FDS-NT.
HPLC analysis was performed under the following condi-
tions: a Shim-pack VP-ODS or equivalent ODS column
(Particle size 5 mm, column dimension: I.D 4.6 mm  length
150 mm) maintained at room temperature. An isocratic
mobile phase composed of acetonitrile:methanol (60:40%
v/v) was adjusted for pH 6.0 with orthophosphoric acid at
ambient temperature and the injection volume was 20 mL at a
flow rate of 1 mL/min. The detective wavelength was 255 nm.
The mobile phase was filtered through a 0.45 mm filter in a
Millipore solvent-filtration apparatus before use.
HPLC method was validated prior to assay as previously
reported (Gurunath et al., 2013), which was carried out for
linearity, precision, accuracy, limit of detection and quanti-
fication according to US Pharmacopoeia (The United States
Pharmacopoeia, 2007) and International Conference on
Harmonization (ICH) guidelines [ICH (Q2R1), 2007]
The similarity factor (f2) was calculated to compare the
release profiles of two formulations under similar conditions
of dissolution media, agitation rate, pH and temperature
employed, to rule out or understand the influence of naringin
on drug release as novel excipient. The similarity between
two dissolution profiles was considered significant when its
value falls between 50 and 100. Drug release studies were
carried out in triplicate.
Pharmacokinetic study
Drug administration
Six male (2.8–3.1 kg) healthy adult albino rabbits were
divided into two groups. Each group consisting of three
rabbits each. Each rabbit was housed individually in separate
cages under environmentally controlled conditions (25 ± 2  C,
12 h light/dark cycle). Prior to each experiment, the rabbits
were starved for 24 h and were allowed free access to tap
water. Tablets (FSD-NT and FSD-T, each of 100 mg with
5 mm diameter) were orally administered into the stomach of
rabbits using a gastric intubation tube (made of silicone
rubber) with one tablet placed on the tip of tube. Care was
taken to prevent any airway obstruction. The study was
conducted as two-periods, two treatments crossover design for
2 weeks as follows.
Drug Deliv, 2016; 23(7): 2124–2138
Hausner’s ratio Carr’s index Time of flow (s) Cotangent of angle a
1.13 ± 0.031 11.40 ± 2.442 2.1 ± 0.442 0.042 ± 0.072
1.12 ± 0.023a 11.20 ± 1.837a 2.3 ± 0.837a 0.041 ± 0.057a
Friabilitya (%) Hardnessa (kg/cm2) Disintegration time (min)b
0.44 ± 0.047 3.8 ± 0.12 5.86 ± 1.4
0.46 ± 0.044c 3.8 ± 0.13c 6.03 ± 4.6c
Group I: Received tablet (FSD-T) in a randomized
sequence (100 mg each; p.o.; n ¼ 3) in two occasions with
wash out period.
Group II: Received tablet (FSD-NT) in a randomized
sequence (100 mg each; p.o.; n ¼ 3) in two occasions with
wash out period.
The prior approval for conducting the experiments in
rabbits was obtained from our Institutional Animal
Committee with registration number 1533/PO/a/11/CPCSEA.
Sampling procedures
Blood samples (2 mL) were collected at 0.0, 0.25, 0.5, 0.75, 1,
1.5, 2, 4, 6, 8 and 12 h of drug administration from a marginal
ear vein into K3 EDTA tubes (J. K. Diagnostics, Rajkot, India)
and placed on ice, protected from light. Samples were stored
at 20  C until assayed.
Preparation of plasma samples
Liquid–liquid extraction technique was employed for all the
plasma samples. An aliquot of 500 mL plasma mixed with
25 mL of internal standard (paracetamol, 10 mg/mL) was
spiked with 3 mL of HPLC grade acetonitrile by vortex
mixing for 3 min. The mixture was centrifuged at 10 500 rpm
for 10 min using a Remi-Model RM 12 centrifugal device, the
supernatant was filtered through 0.45 mm filter (Millipore)
and the organic supernatant was evaporated to dryness at
room temperature. The residue was reconstituted with 200 mL
of mobile phase, then the solution was centrifuged at
15 000 rpm for 10 min; finally, an aliquot of 20 mL of the
solution was injected into the HPLC (Shimadzu, LC- 20 A
series) equipped with a UV detector at a flow rate of 1 mL/
min and the run time was 5 min. The mobile phase comprised
60% acetonitrile: 40% methanol at pH 6.0 was determined at
UV wavelength of 255 nm.
HPLC analysis
The chromatograms were acquired using LC solutions
software (Shimadzu, Japan) from a Shimadzu HPLC system
(LC-20 A series) equipped with a UV detector. The retention
times for candesartan and internal standard (IS, paracetamol)
DOI: 10.3109/10717544.2014.945017
were approximately 3.93 and 2.85 min, respectively. Linear
regression analysis was employed for standard curves
obtained from drug/internal standard peak area ratios as a
function of plasma concentration versus time data was
analyzed and the oral pharmacokinetic data were developed.
Accelerated stability studies
In this study, the accelerated stability studies were performed
for tablets prepared with and without naringin because these
studies are more convenient and less time-consuming over
long-term stability studies, which will take longer time to
evaluate the physical and chemical stability of the product
under normal conditions of temperature and humidity.
Moreover, tablet formulations containing active pharma-
ceutical ingredient (API) in any high-energy amorphous form
(including solid dispersions) are expected to enhance solu-
bility, dissolution rate and consequently, oral bioavailability
of poor water-soluble drugs. However, the amorphous regions
of the dispersed drug in the carrier are thermodynamically
unstable and are therefore susceptible to recrystallization
upon storage (Hecq, 2005).
Hence, in this study, the prepared tablets (FDT-NT and
FDT-T) were subjected to accelerated stability studies where
the product is stored under extreme condition of temperature
and humidity of 40 ± 2  C/75% RH ± 5% RH for 6 months,
respectively.
Tablets (FDT-NT and FDT-T; 100 mg each) were wrapped
in aluminum foil to prevent the formulation from exposure to
light to simulate the aluminum packaging, i.e. Alu–Alu
packing, of drug products and stored in airtight containers
which is impermeable to solid, liquid and gases. These packed
samples of tablets in airtight containers were subjected to
accelerated stability studies for a period of 6 months as per
ICH guidelines [ICH-Q1A (R2) 2003] at the extreme
temperature and relative humidity conditions of 40 ± 2  C/
75% RH ± 5% RH in a humidity-controlled oven (Thermolab,
India). The samples from airtight containers were withdrawn
at 1, 3 and 6 months and evaluated for physical appearance,
drug content, hardness, disintegration time and in vitro drug
release at 30 min against initial (0 month) values.
The physical stability was analyzed to monitor for
potential re-crystallization of the solid dispersions in prepared
tablets with and without naringin using XRPD studies, while
the chemical stability was studied with HPLC to evaluate for
the formation of any degradation product/s under storage
conditions for 1, 3 and 6 months, respectively. Any physical
and chemical changes observed during storage for 6 months
were compared against initial (0-month) diffractrograms and
the peak areas, respectively.
Drug content determination
The content uniformity was assessed according to USP
requirements. The test is used to ensure that every tablet
contains the amount of drug substance intended with a
negligible variation among tablets within a batch upon
storage. Three tablets from each formulation (FDT-NT and
FDT-T; 100 mg each) at initial (0), 1, 3 and 6 months were
tested randomly. Each tablet was weighed individually and
crushed to a powder in a glass mortar. The powder blend
An in vitro and in vivo evaluation of novel tablet formulation 2129
equivalent to 10 mg of drug (CAN) was weighed and
dissolved in 10 mL of methanol, filtered using 0.45 mm filter
paper and an aliquot of 20 mL of the resulting filtrate was
injected into HPLC to determine the concentration of CAN
from tablet samples. All assays were carried out in triplicate.
Hardness testing
The hardness or crushing strength of the tablets was measured
using a Monsanto Hardness Tester (Campbell Electronics,
Mumbai, India). Three tablets from each formulation
(FDT-NT and FDT-T; 100 mg each) were tested randomly at
initial (0), 1, 3 and 6 months, respectively, and the average
reading was noted. The hardness is measured in kg/cm2.
X-ray powder diffraction studies
X-ray powder diffraction (XRPD) analyses were performed
for crushed powder samples of two tablets (FDT-NT and
FDT-T; 100 mg each) at initial (0), 1, 3 and 6 months,
respectively, using an X-ray diffractometer (PW 1729, Philips,
Netherlands). The samples were irradiated with monochro-
matized Cu Ka radiation (1.5406 Å) and analyzed between 5
and 50 2y. The voltage and current used were 40 kV and
40 mA, respectively. The range and the chart speed were
2  103 CPS and 10 mm/degree 2y, respectively.
Degradation studies using HPLC
Three tablets from each formulation (FDT-NT and FDT-T;
100 mg each) were tested for degradation studies at initial (0),
3 and 6 months, respectively. Each tablet was weighed
individually and crushed to a powder in a glass mortar. The
powder blend equivalent to 10 mg of pure drug (CAN) was
weighed and dissolved in 10 ml of methanol, filtered using
0.45 mm filter paper and an aliquot of 20 mL of the resulting
solution was injected into the HPLC to observe the peaks
corresponding to the pure drug (CAN) and degradation
products, respectively. All assays were carried out in
triplicate.
In vitro dissolution studies
Drug release studies were carried out in triplicate for two
tablets (FDT-NT and FDT-T; 100 mg each) at initial (0), 3 and
6 months, respectively, as described above (refer ‘‘Dissolution
study’’ section). The similarity factor (f2) was calculated to
compare the release profiles of two tablets under similar
conditions of dissolution media, agitation rate, pH and
temperature employed, to understand the influence of storage
conditions on drug release profiles for a period of 6 months.
The similarity between two dissolution profiles at initial
(0), 3 or 6 months was considered significant when its value
falls between 50 and 100.
Data analysis
Pharmacokinetic analysis
Plasma concentrations of candesartan in rabbits were plotted
against time and non-compartmental analysis was used to
determine the pharmacokinetic parameters using Kinetica 5.0
Software (Adept Scientific Ltd., Amor Way, Letchworth,
2130 G. Surampalli et al.
Garden City, Herts, United Kingdom). The area under the
plasma concentration versus time curve up to the last
quantifiable time point, AUC0–t was obtained by the linear
trapezoidal method. The AUC0–t was extrapolated to infinity
(AUC0–1) by adding the quotient Clast/Kel, where Clast
represents the last measured concentration and Kel represents
the apparent terminal rate constant. The half-life of the
terminal elimination phase was obtained using the relation-
ship t1/2 ¼ 0.693/Kel. The Cmax and Tmax were obtained
directly from the data. Oral clearance (Cl) was calculated as
dose divided by AUC0–1. The apparent volume of distribu-
tion was obtained from the equation Vd ¼ D/(AUC0–1)  Kel.
Mean residence time (MRT) was determined by division of
area under the first moment curve (AUMC) by AUC0–1.
Statistical analysis
Data was expressed as mean ± standard deviation (±SD).
The statistical significance was determined using
One-way ANOVA followed by Dunnett’s test or by a
Student’s t-test. Results were considered statistically signifi-
cant from the control when p50.05 and very significant when
p50.01.
Results
FTIR spectroscopy
Figure 1(a–c) shows the FT-IR spectra of pure CAN, naringin
and their physical mixture under study. The spectrum of CAN
shows prominent peaks at 3410, 2941, 2860,1753, 1716,
1614, 1548 and 1282 cm1 corresponding to N–H stretching,
C–H aromatic stretching, C–H aliphatic stretching, C ¼ O
stretching in ester, C ¼ O stretching in acid, C ¼ N stretching,
C ¼ C stretching and –C–O–ester.
The IR spectrum of naringin exhibited characteristic IR
bands at around 3408 (broad), 3387 (broad), 2920, 1691 and
1616 cm1 due to more than one O–H aromatic stretching,
C–H aliphatic stretching, C ¼ O and C ¼ C bonds,
respectively.
The physical mixture showed the superimposed spectra of
CAN crystals and naringin. The crystalline form of CAN and
naringin has sharper and significant peaks at all above-
mentioned prominent regions. No shifting of peaks was
observed with either pure drug or naringin in the physical
mixture spectra. It might be speculated that the no intermo-
lecular bonding was formed between the molecules of two
systems indicating absence of any chemical interaction
between CAN and excipient (naringin).
Evaluation of micromeritic properties
Results of flowability, bulk and tapped density, Hauser’s ratio
and Carr’s index
According to the results presented in Table 2, it is possible to
conclude that the blended powder has flow properties and
compaction performance appropriate for pharmaceutical
applications. The results show that naringin as an excipient
displayed a good flow, in view of the fact that the values of
flow time and cotangent of angle a were less than 10 s and
0.1, respectively (Table 2).
Drug Deliv, 2016; 23(7): 2124–2138
Physical characterization of tablets
Results of average weight, hardness and friability
Direct compression (DC) method was used due to its ease of
manufacture and lower cost. We optimized (Table 1) the
tablet formulation for 100 mg prepared from naringin for
hardness value, 3.8 kg/cm2, which was significantly (p50.05)
greater than the hardness value, 1.5 kg/cm2 for 85 mg tablet
without naringin. This signifies that naringin formulates the
tablet harder than the moderate value (1.5 kg/cm2) without
naringin. The significant (p50.05) change in the hardness
formulated with naringin may be attributed to the glycosidic
linkages present in the flavonoid glycoside. However, the
mechanism is unclear and needs further elucidation.
Since, there is no such official specification specified in
any pharmacopoeia with reference to hardness of material and
hardness test for uncoated tablets. Formulator is supposed to
decide and maintain the range of hardness during preparation
based upon requirements in addition to type of formulation.
However, minimum acceptable hardness of uncoated tablets is
40 N (4.08 kg/cm2) approximately (Banker, 1987).
Therefore, to equilibrate the hardness (3.8 kg/cm2) of
tablets prepared from naringin, hardness of the tablets without
naringin was increased with increased concentration of
b-cyclodextrin at constant compression force (15 kN) for
both tablets (FSD-NT and FSD-T) in our preliminary trials.
Consequently, the tablets were assessed for their weight
variation, thickness, disintegration time, hardness and friabil-
ity. The results in Table 3 reveal that the tablets at hardness
3.8 kg/cm2, the disintegration time, friability and thickness
remained invariable for both tablets (FSD-NT and FSD-T)
with no significant change in their weight variation.
Dissolution study
Figure 2 features the dissolution release profiles from tablet
formulations (FSD-NT and FSD-T) in the dissolution
medium. In vitro dissolution results showed that tablets
(FSD-NT and FSD-T) provided an immediate release of the
drug with no significant differences in their release profiles
under similar conditions of pH medium, agitation rate,
temperature and dissolution media, respectively.
Figure 2. Dissolution profiles of FSD-NT and FST-T tablets showing
cumulative amount of drug release in 900 mL of phosphate buffer
(pH 6.8). Mean ± SD (n ¼ 3).
DOI: 10.3109/10717544.2014.945017 An in vitro and in vivo evaluation of novel tablet formulation 2131

To describe the release behavior of CAN from the
formulations, the data was fitted into a first-order kinetic
model. The tablet formulations (FSD-NT and FSD-T) showed
a good correlation to first-order kinetics equation (r2 ¼ 0.992)
suggesting that the existence of linear correlation between
concentration and ratios of peak areas.
In reality, the release profiles were superimposable under
analogous conditions tested. The similarity factor (f2) was
used to compare the dissolution profiles that reached to a
value of 76.18 in phosphate buffer (pH 6.8) at 50 rpm.
Pharmacokinetic study
In vivo studies in rabbits were conducted in order to evaluate
whether an immediate plasma concentration profile of
candesartan from the newly compressed tablet formulations
(FSD-NT and FSD-T) as a solid dosage form could be
achieved.
It has been reported that CAN must be transformed to
candesartan to achieve antihypertensive effects (Vijaykumar
et al., 2009). The plasma concentrations of candesartan were
determined by HPLC method to evaluate the pharmacokinetic
behavior of FSD-NT against FSD-T. The plasma concentra-
tion–time profiles of candesartan after single-dose oral
administration of each tablet with and without naringin is
presented in Figure 3 and the corresponding pharmacokinetic
parameters are summarized in Table 4. The experimental
results showed a significant (p50.001) difference occurred
between the pharmacokinetic profiles of FSD-NT in com-
parison to that of FSD-T.
Taken together, at each time point, the plasma concentra-
tion of candesartan from FSD-NT was higher than those
measured for the plain FSD-T. These are evidenced by our
previous reports (Gurunath et al., 2013, 2014), which showed
high apparent permeability coefficient in vitro and also higher
Cmax in vivo with freeze-dried CAN solid dispersions in the
presence of naringin.
The peak concentrations (Cmax) of FSD-NT and FSD-T
were found to be 88.44 ± 13.8 and 122.23 ± 34.5 mg/mL,
respectively, which was significantly improved (p50.01) over
1.38 when compared to the peak concentration of FSD-T.
However, there was no change in the time to reach peak
concentration (tmax) for both formulations (FSD-NT and
FSD-T, 2 h), which was consistently similar to our previously
demonstrated pharmacokinetic studies, indicating that cande-
sartan could be absorbed more rapidly after its prodrug, CAN
was formulated into freeze-dried solid dispersion.
In particular, areas under the concentration–time curves
(AUC0–12 h) for FSD-NT (644.03 ± 78.6 mg h/mL, respect-
ively) were enhanced by 1.5-fold over the area under the
concentration–time profiles of FSD-T (428.51 ± 67.8 mg
h/mL; p50.01) paralleled with a reduction in candesartan
clearance from 3235.64 ± 231.7 to 1878.62 ± 115.7 mL/min.
In addition, a significant change (p50.01) in the elimin-
ation half life was observed between two tablet formulations
(FSD-NT and FSD-T) from 7.15 ± 3.9 to 4.86 ± 2.5 h,
respectively. This revealed that the oral bioavailability of
candesartan could be dramatically increased in the presence
of naringin. The significantly enhanced oral absorption of
candesartan from FSD-NT was consistent after its direct
delivery to the intestine, which indicated that the intestinal
absorption was predominant in the improved oral absorption
of CAN.
The therapeutic effect of CAN occurred at 4–6 h after the
oral administration of free-CAN tablets (Israili, 2000; Gleiter
& Mörike, 2002; Husain et al., 2011). Hence, the more rapid
absorption of candesartan from FSD-NT tablet formulation
could be helpful in treatment of hypertension or heart failure
in clinical therapeutics.
Accelerated stability studies
The results of the accelerated stability studies showed, no
significant differences in drug content uniformity, hardness,
disintegration time and in vitro dissolution profiles before
(0-month) and after the storage periods (1, 3 or 6 months) for
tablets with and without naringin at extreme condition of
temperature and relative humidity of 40 ± 2  C/75% RH ± 5%.
The stability data of tablets (FDT-NT and FDT-T) are
shown in Tables 5–7 for percentage of drug content, hardness
and disintegration time to evaluate the effect of storage for
Table 4. Pharmacokinetic parameters (Mean ± SD, n ¼ 6) of candesartan
following oral administration of FSD-T and FSD-NT tablets.

Parameters FSD-T (100 mg) FSD-NT (100 mg)

AUC(0–t) mg/mL h 428.51 ± 67.8 644.03 ± 78.6a
AUC(0–in) mg/mL h 515.09 ± 83.4 887.17 ± 93.4a
AUC(extra) mg/mL h 85.58 ± 13.4 243.14 ± 64.8a
AUMC (0–t) mg/mL h  h 1792.04 ± 180.4 2816.16 ± 217.8a
AUMC (0–in) mg/mL h  h 3438.48 ± 234.3 8243.05 ± 545.7a
AUMC (extra) mg/mL h  h 1646.43 ± 169.2 5426.89 ± 287.8a
Cmax (mg/mL) 88.44 ± 13.8 122.23 ± 34.5a
Tmax (h) 2.0 ± 1.4 2.0 ± 1.2
t1/2 (h) 4.86 ± 2.5 7.15 ± 3.9a
Vd (L) 1.36 ± 0.8 1.16 ± 0.9a
Clearance (mL/min) 3235.64 ± 231.7 1878.62 ± 115.7a
MRT (h) 6.67 ± 2.7 9.29 ± 4.8a
K (h  1) 0.142 ± 0.31 0.09 ± 0.42a
RB (%) – 66.5

Data are presented as mean ± SD (n ¼ 6). Cmax, maximum plasma

Figure 3. Plasma concentration–time profiles (mean ± SD, n ¼ 6) of concentration; Tmax, time to reach peak concentration; AUC, area
candesartan following oral administration of FSD-NT and FSD-T tablet under the plasma concentration–time curve; t1/2, elimination half-life.
a
(100 mg) formulations. Significant at p50.01 compared to FSD-T.
2132 G. Surampalli et al. Drug Deliv, 2016; 23(7): 2124–2138

Table 5. Percentage of drug content for FDT-T and FDT-NT tablets from accelerated stability studies (Mean ± SD, n ¼ 3).

Drug content (%)

Parameter
Initial 1st-month at 3rd-month at 6th-month at
Formulation (0-month) 40 ± 2  C/75% RH ± 5% RH 40 ± 2  C/75% RH ± 5% RH 40 ± 2  C/75% RH ± 5% RH
FDT-T 98.4 ± 0.44 99.2 ± 0.28a 97.4 ± 0.96a 97.1 ± 1.75a
FDT-NT 98.7 ± 1.83 99.7 ± 0.60a 98.3 ± 0.61a 97.8 ± 1.67a
a
Average of three determinations with p40.05 statistically insignificant when compared to initial (0-month) values.

Table 6. Hardness of FSD-T and FSD-NT tablets from accelerated stability studies (Mean ± SD, n ¼ 3).

Hardness (kg/cm2)
Parameter
Initial 1st-month at 3rd-month at 6th-month at
Formulation (0-month) 40 ± 2  C/75% RH ± 5% RH 40 ± 2  C/75% RH ± 5% RH 40 ± 2  C/75% RH ± 5% RH
FDT-T 3.80 ± 0.12 4.01 ± 0.62a,b 4.08 ± 0.32a,b 4.05 ± 0.52a,b
FDT-NT 3.80 ± 0.13 3.81 ± 0.43a 3.83 ± 0.73a 3.85 ± 0.23a
a
Average of three determinations with p40.05 statistically insignificant when compared to initial (0-month) values.
b
p50.01 when compared to initial (0-month) values.

Table 7. Disintegration time of FSD-T and FSD-NT tablets from accelerated stability studies (Mean ± SD, n ¼ 3).

Disintegration time (min)

Parameter
Initial 1st-month at 3rd-month at 6th-month at
Formulation (0-month) 40 ± 2  C/75% RH ± 5% RH 40 ± 2  C/75% RH ± 5% RH 40 ± 2  C/75% RH ± 5% RH
FDT- T 5.86 ± 1.4 6.95 ± 1.12a,b 7.12 ± 1.36a,b 7.65 ± 1.67a,b
FDT-NT 6.03 ± 1.6 6.17 ± 1.17a 6.26 ± 1.42a 6.12 ± 1.63a
a
Average of three determinations with p40.05 statistically insignificant when compared to initial (0- month) values.
b
p50.01 when compared to initial (0-month) values.

6 months. Percentage drug content of two tablets with and
without naringin were found within the range of 97.1–99.7%
which meets the USP guidelines as shown in Table 5. The
results in Table 6 reveal no significant (p40.05) difference in
hardness values (3.8 kg/cm2) of tablets prepared from
naringin (FSD-NT) as compared to initial 0-month.
However, for tablets without naringin (FDT-T), there was
significant difference in hardness (p50.01) values at tem-
perature and relative humidity of 40 ± 2  C/75% RH ± 5%,
respectively, wherein hardness was increased with time,
prolonging the disintegration time of the tablets (Table 7);
but, in all cases, disintegration time was within the specified
limit (within 15 min) of official monographs for uncoated
tablets (Indian Pharmacopoeia, 2007).
Figure 4 features the dissolution release profiles from
tablets (FDT-NT and FDT-T) in the dissolution medium at
initial (0), 3 and 6 months, respectively. In vitro dissolution
results showed that tablets (FDT-NT and FDT-T) provided an
immediate release of the drug with no significant differences
in their release profiles upon storage for 6 months as
compared to initial (0-month) release under the similar
conditions of pH medium, agitation rate, temperature and
dissolution media, respectively.
In fact, the release profiles of initial (0), 3 or 6 months
were superimposable under analogous conditions tested. The
similarity factor (f2) was used to compare the dissolution
profiles for 3 and 6 months, respectively, that reached to a
value of 65.23 (3-month; FDT-T); 71.43 (6-month; FDT-T)
and 75.78 (3-month; FDT-NT); 76.86 (6-month; FDT-NT) as
compared to initial (0-month) profiles in phosphate buffer
(pH 6.8) at 50 rpm.
No significant effect on the dissolution profiles of tablets
without naringin (FDT-T) was observed due to the changes in
hardness and disintegration time upon storage for 6 months.
This was clearly evidenced from the dissolution profiles of
FDT-T tablets (Figure 4a) with no significant (p40.05)
changes as compared to initial (0-month) release profile with
the similarity factors (f2) as 65.23 and 71.43 for 3 and 6
months, respectively.
In addition, the Figure 5(a,b) depicts the XRPD patterns of
pure drug (CAN), hydrophilic polymer (PVP-K30), FDT-NT
and FDT-T tablets, respectively, to evaluate the effect of
storage at 40 ± 2  C/75% RH ± 5% for different time periods.
The diffraction pattern of pure drug (CAN) exhibited intensity
peaks at 2y values of 9.8 , 9.9 , 10 , 10.1 , 11.6 , 17.2 ,
20.3 , 23.3 , 24.9 , 27.7 and 29.2 (Figure 5). The spectrum
of PVP-K30 exhibited complete absence of any diffraction
peaks, which is characteristic of an amorphous compound
showing the XRPD halo (Figure 5).
Neither FDT-NT nor FDT-T showed any signs of
re-crystallization during the storage period as depicted in
Figure 5(a,b). The diffractrograms of tablets (FDT-NT and
FDT-T) at initial (0-month), 1, 3 and 6 months showed
peaks similar to PVP-K30 and absence of any major
diffraction peaks corresponding to pure drug (CAN) indicat-
ing that the drug is present in an amorphous state, which is in
agreement with our previously published report (Gurunath
et al., 2014).
DOI: 10.3109/10717544.2014.945017
Figure 4. Dissolution profiles of (a) FSD-T and (b) FSD-NT tablets showing cumulative amount of drug release in 900 mL of phosphate buffer (pH 6.8)
over storage for 3 and 6 months, respectively (Mean ± SD, n ¼ 3).
The HPLC results in Figure 6 showed no peaks other than
those corresponding to pure drug (CAN) at retention time
(3.95 ± 0.04 min) for tablets (FDT-NT and FDT-T) upon
storage for 6 months. The observed peak areas of both tablets
corresponding to pure drug (CAN) upon storage for 6 months
were similar to those of their initial (0-month) peak areas with
similar retention time values.
Discussion
Candesartan cilexetil (CAN), an anti-hypertensive agent, is
widely indicated for the treatment of hypertension or heart
failure in clinical therapeutics. Data available on its poor
solubility, limited intestinal absorption and disposition
showed encouraging results with variable absorption, poor
efficacy and bioavailability profiles. Numerous factors such
as low aqueous solubility, poor dissolution properties and
poor apparent permeability due to intrinsically low absorptive
membrane permeability contribute to the low and erratic oral
bioavailability of drugs. More recently, secretory membrane
transporters (e.g. P-gp and BCRP) have also been implicated
in controlling the oral bioavailability and variability of drug
absorption. As a result, novel formulations of CAN exhibiting
better oral absorption and bioavailability need to be
developed.
Our FT-IR drug-excipient interaction studies showed
no discoloration, liquefaction and physical interaction
between the drug and the novel excipient used. No
significant shift in the peak was observed which revealed
that both the drug and excipient are compatible with each
other. The FTIR spectra of drug, excipient and mixture of
drug and excipient are shown in the Figure 3(a–c),
respectively.
The formulation blends illustrated good packing and
flowability as indicated by Hausner’s ratio value and Carr’s
index. The Carr’s compressibility index can be used as an
indication to produce a uniform blend that predicts the flow
properties. Compressibility index values that fall between
5 and 15% indicate excellent flow whereas those fall between
12 and 16% represent good flow. Conversely, values greater
An in vitro and in vivo evaluation of novel tablet formulation 2133
than 23% and up to 35% are acknowledged as poor flow
materials.
The Hauser’s ratio (HR) obtained as a result of the ratio
between the bulk density and compacted density of powders is
correlated with the cohesion and adhesion forces of particles.
Hausner demonstrated that powders such as coarse spheres,
had ratios of approximately 1.25 with low inter-particle
friction, whereas Hausner ratios greater than 1.5 showed more
cohesive, less free-flowing powders such as flakes.
Therefore, it is established that values lower than 1.25 are
related to a good flow, whereas values higher than 1.25 are
related to a poor flow, in view of the fact that flow and
cohesive properties are inversely proportional (Staniforth,
2007; Ofori-Kwakye et al., 2010).
The Carr’s index and Hausner ratio based on the
cohesiveness between the particles only reflect the potential
for consolidation of powders. To determine the ease and speed
of flow, it is imperative to determine the flow rate by means
of the time and cotangent of a angle.Since the values of flow
time and cotangent of angle a were less than 10 s and 0.1,
respectively, the formulation blends exhibited good flow
properties.
Hardness is an element of interest to the compression
process that can be used for equipment adjustment and
calibration to obtain the quality of the finished product. In
general, higher the compression force, greater will be the
consolidation and deformation of the powder in the diet. This
will results in the greater the contact points in the material
being compacted with the low porosity of the tablet. There
will be an inclination to increase the hardness and disinte-
gration time.
Alternatively, low hardness will be an indirect measure of
the inefficiency of the process of consolidation and compac-
tion of the powders in tablets. Harder tablets will have inferior
friability with a difficulty to be ejected from the press (Kuentz
& Leuenberger, 2000; Armstrong, 2007; Staniforth, 2007;
Niazi, 2009).
In our preliminary trials, the ability of b-cyclodextrin as
effective diluent (Late & Banga, 2010) in place of naringin to
2134 G. Surampalli et al. Drug Deliv, 2016; 23(7): 2124–2138

Figure 5. (a) XRPD diffractograms of single components of CAN and PVP-K30 together with tablets prepared (a) without naringin–FSD-T at initial
(0 month), FSD-T at 1st month, FSD-T at 3rd month and FSD-T at 6th month, respectively, upon storage for 6 months at 40 ± 2  C/75% RH ± 5% and
(b) with naringin–FSD-NT at initial (0 month), FSD-NT at 1st month, FSD-NT at 3rd month and FSD-NT at 6th month, respectively, upon storage for
6 months at 40 ± 2  C/75% RH ± 5%.
DOI: 10.3109/10717544.2014.945017 An in vitro and in vivo evaluation of novel tablet formulation 2135

Figure 6. Typical HPLC chromatograms of FSD-T and FSD-NT upon storage for 6 months at 40 ± 2  C/75% RH ± 5%.

formulate tablets containing freeze-dried CAN-loaded out or understand the influential role of naringin on drug
solid dispersions prepared by the DC was demonstrated release.
in order to equilibrate the hardness of FSD-NT to that of Besides, the increase in the oral bioavailability of hydro-
FSD-T tablets at constant compression force and time to rule phobic drugs of low water solubility using water-soluble
2136 G. Surampalli et al.
excipients, these diluents (naringin or b-cyclodextrin) can also
act as fillers for such tablets.
The analogous disintegration time values for FSD-NT and
FSD-T formulations may also attribute to the superior water
solubility nature of naringin (i.e. 1 mg of naringin dissolved in
mL of water) due to the hydroxyl groups and glycosidic
linkages present in the flavonoid and hydrophilic nature of
b-cyclodextrin, respectively.
Although hardness is not an attribute that can be directly
correlated with drug release, a greater hardness value may be
beneficial for maintaining tablet integrity and controlling
drug delivery. From a comparison of the release profiles,
increasing or equilibrating the hardness of the FSD-T to that
of FSD-NT using b-cyclodextrin facilitated the release of
CAN, most likely because the b-cyclodextrin is a hydrophilic
polymer, allowing the penetration of the dissolution medium
and thus showing similarity in the rate of drug release to that
of tablet formulation with naringin (FSD-NT).
Both FSD-NT and FSD-T tablets offered dramatic
improvements in the rate as well as the extent of drug release
(Figure 5) and presented the highest (80%) drug release in
30 min. In the early 10-min period, there were about 54 and
57% increase of drug release from both tablets, respectively.
This improved drug release attributed to the presence of
amorphous form of CAN, as confirmed by our earlier
reported DSC and PXRD studies (Gurunath et al., 2014).
Other factors such as increased wettability, solubilization of
the drug by the carrier at the diffusion layer, the reduction or
absence of aggregation, agglomeration increased the rate
of dissolution and higher micromeritic properties exerted by
excipients might have been contributed to improved drug
release from these tablet formulations. This can clearly be
evidenced by superimposable drug release profiles with
similarity factor value between 50 and 100 (76.18).
Based on all of the above findings, the possible mechan-
isms for an increased release rate from both tablets have been
postulated and are summarized as follows: (i) reduction of
crystallinity, (ii) increased water absorption by the hydro-
philic carrier and excipients which leads to increased
wettability, (iii) solubilization effect by the carrier and
excipients, (iv) phase transition from crystalline to amorphous
nature and (v) higher micromeritic properties exerted by
naringin or b-cyclodextrin.
The results of the in vivo pharmacokinetic study demon-
strated that rabbits exhibited the moderate inter-individual
variation in the plasma concentrations of candesartan, which
may be reflective of absorption, metabolism and elimination
differences between individuals. As shown in Figure 3,
FSD-NT was absorbed significantly from the intestinal
regions and reached a maximum peak plasma concentration
at about 2 h after the drug treatment as compared to FSD-T
indicating that the presence of naringin contributed signifi-
cantly for the enhanced intestinal absorption of candesartan
from its tablet formulation as a P-gp inhibitor.
All the Cmax and AUC0t values of FSD-NT were much
greater than the Cmax and AUC0t values of FSD-T at the
same dose of 100 mg, which may explain that the dissolution
of CAN along with effective P-gp inhibition significantly
contributed to improved intestinal absorption. This is also
attributed to the presence of solid state (amorphous) of drug
Drug Deliv, 2016; 23(7): 2124–2138
improved through wetting of drug particles and localized
solubilization by hydrophilic carriers.
In addition, there was also concomitant decrease in plasma
clearance and volume of distribution with increased the
plasma concentration and AUC as shown in Figure 3 and
Table 1, with FSD-NT tablet formulation. The small volume
of distribution, low oral clearance and increased mean
residence time obtained in this study indicated that the drug
distributes primarily in the extracellular spaces.
The above-mentioned facts may attribute to high protein
binding at physiological pH, as previously reported (Gleiter &
Mörike, 2002; Husain et al., 2011). The significant change in
MRT and Kel suggests that the increased time for which the
drug remains in the body.
The relative absorption (RA) is defined as the AUC0–t
values of FSD-NT compared to that of FSD-T, which was
relatively higher for FSD-NT as depicted in Table 2. The
increased rate of absorption could be attributed to the
concomitant and synergistic inhibition of P-gp present in
the intestine as a plausible explanation for prominent increase
in oral bioavailability of CAN.
Accelerated stability studies for the tablet formulations
(FDT-NT and FDT-T) did not show any significant changes in
their physical appearance, drug contents, disintegration time
and in vitro drug release profiles at 30 min (p40.01). Upon
6 months of storage at 40 ± 2  C/75% RH ± 5% RH (Figure 4),
the tablets (FSD-NT and FSD-T) demonstrated similar
dissolution profiles as compared to that of initial 0-month
release profiles indicating that CAN was stable in the
presence of PVP-K30 and other excipients like naringin.
Tablets prepared from naringin showed no significant
changes in the hardness and disintegration time as observed
for tablets without naringin. Although significant changes
were noticed in the hardness and disintegration time of FDT-T
tablets probably due to the loss of moisture, but no significant
change was observed in their dissolution profiles upon storage
for 6 months indicating that both formulations (FSD-NT and
FSD-T) were fairly stable at storage time and conditions.
Figure 5 represents XRPD diffractrograms of tablet
formulations (1, 3 and 6 months over storage at 40 ± 2  C/
75% RH ± 5% RH) showing no signs of re-crystallization in
the samples. Hence, there was no influence of storage time as
well as storage conditions on the XRPD patterns of the drug
in the samples of tablets, exhibiting similar XRPD patterns
that overlay the initial (0-month) patterns, thus retaining the
amorphous nature of the drug.
Furthermore, the results of the chemical stability by HPLC
analyses showed no peaks other than those corresponding to
pure drug (CAN) at retention time (2.85 ± 0.04 min) for
tablets (FDT-NT and FDT-T) upon storage for 6 months
suggesting that the tablets did not undergo decomposition or
degradation upon storage over 6 months’ study, indicating that
the assay procedure is selective for the analysis of CAN
without interference of the excipients. This could be attributed
to the evolved interactions between CAN and PVP-K30
(Barmpalexis et al., 2013) or glycosidic linkages of naringin,
which requires further elucidation.
Oral formulations of CAN having better efficacy and less
toxicity could be developed using appropriate P-gp inhibitor.
However, further studies are required to compare the
DOI: 10.3109/10717544.2014.945017 An in vitro and in vivo evaluation of novel tablet formulation
effectiveness of naringin with other novel P-gp inhibitors to
enable pharmacokinetic modulation of candesartan or its
prodrug, CAN. Genetic differences mediated by P-gp in both
hepatic metabolism and intestinal transport may be an
important contributing factor in the wide inter-individual
variability in the absorption and disposition of candesartan.
These results suggest that the administration of CAN in the
form of tablet dosage form formulated with safe and naturally
occurring P-gp inhibitor may improve the therapeutic index of
CAN for the treatment of hypertension and other associated
complications of hypertension.
Conclusion
The success of DC method in the design of oral dosage forms
requires the study of the physical properties of the formula-
tion blends. The blends formed more suitable tablets with and
without naringin, as a result of improved flow and compress-
ibility of the novel excipients. The compression time, force
and amount of excipients used did not influence friability,
hardness and drug release. The dissolution profiles demon-
strated that the proposed formulations produced more than
80% of drug release within 30 min. No change in dissolution
profiles and release kinetics were noticed between two
formulations, most likely due to the similarity in nature of
the excipients. Another improvement that can be associated
with the developed tablets was that no other additional
ingredients in the formulations were required since naringin
acted as filler in the DC process. Consequently, the current
results demonstrate that the intestine plays an important role
in the net absorption and disposition of CAN, which showed
that naringin increased significantly the oral bioavailability of
candesartan, suggesting the involvement of P-glycoprotein in
CAN disposition. It also revealed that naringin, a
P-glycoprotein inhibitor can be used as pharmaceutical
excipient for developing oral dosage forms to increase
intestinal permeability and in vivo pharmacokinetic perform-
ance of CAN to enhance oral bioavailability. Also, under the
accelerated stability conditions evaluated in this study, tablets
prepared with and without naringin involving CAN solid
dispersions prepared by DC process were physically and
chemically stable over 6 months as compared to initial
(0-month) physical and chemical parameters.
However, due to the complexity of the in vivo model, the
role of P-gp and other putative secretory efflux transporters is
not yet clear and needs to be further elucidated. Hence,
further studies are required for comprehensive and multi-
disciplinary evaluation of various claims to make effective
use of these products.
Declaration of interest
Authors declare no conflicts of interest in the publication of
this research work.
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