Journal of Liposome Research

ISSN: 0898-2104 (Print) 1532-2394 (Online) Journal homepage: http://www.tandfonline.com/loi/ilpr20

Niosomes for oral delivery of nateglinide: in situ–in

vivo correlation

Amal A. Sultan, Sanaa A. El-Gizawy, Mohamed A. Osman & Gamal M. El

Maghraby

To cite this article: Amal A. Sultan, Sanaa A. El-Gizawy, Mohamed A. Osman & Gamal M. El
Maghraby (2018) Niosomes for oral delivery of nateglinide: in�situ–in�vivo correlation, Journal of
Liposome Research, 28:3, 209-217, DOI: 10.1080/08982104.2017.1343835

To link to this article: https://doi.org/10.1080/08982104.2017.1343835

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Published online: 02 Jul 2017.

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ISSN: 0898-2104 (print), 1532-2394 (electronic)

J Liposome Res, 2018; 28(3): 209–217

! 2017 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/08982104.2017.1343835

RESEARCH ARTICLE

Niosomes for oral delivery of nateglinide: in situ–in vivo correlation

Amal A. Sultan, Sanaa A. El-Gizawy, Mohamed A. Osman, and Gamal M. El Maghraby

Department of Pharmaceutical Technology, College of Pharmacy, University of Tanta, Tanta, Egypt

Abstract Keywords
Niosomes have been claimed to enhance intestinal absorption and to widen the absorption Drug release, in situ intestinal absorption,
window of acidic drugs. This was reported after monitoring the intestinal absorption in situ. in vivo, niosomes, nateglinide
Accordingly, the aim of this work was to investigate the effect of niosomal encapsulation on
intestinal absorption and oral bioavailability of nateglinide. This was conducted with the goal of History
correlation between in situ intestinal absorption and in vivo availability. The drug was
encapsulated into proniosomes. The niosomes resulting after hydration of proniosomes were Received 24 January 2017
characterized with respect to vesicle size and drug entrapment efficiency. The in situ rabbit Revised 22 May 2017
intestinal absorption of nateglinide was monitored from its aqueous solution and niosomes. Accepted 13 June 2017
Streptozotocin was used to induce diabetes in albino rats which were then used to assess the
hypoglycemic effect of nateglinide after oral administration of aqueous dispersion and
niosomal systems. The prepared vesicles were in the nanoscale with the recorded size being
283 nm. The entrapment efficiency depended on the pH of the formulation. The in situ
intestinal absorption reflected non-significant alteration in the membrane transport parameters
of the drug after niosomal encapsulation compared with the free drug solution. In contrast,
niosomes showed significant improvement in the rate and extent of the hypoglycemic effect
compared with the unprocessed drug. This discrepancy can be attributed to different transport
pathway for the drug after niosomal inclusion with the vesicles undergoing translymphatic
transport which can minimize presystemic metabolism. However, this requires confirmatory
investigations. In conclusion niosomes can enhance oral bioavailability of nateglinide with the
absorption being through nontraditional pathway.

Introduction (Avdeef, 2007). Taking these specifications into consider-

ation, the variability in nateglinide oral bioavailability can be
Nateglinide is a potent oral hypoglycemic agent which
explained. Increasing the passive diffusion characteristics of
stimulates insulin release. The drug is characterized by a
the drug irrespective to the pH of the absorption site can at
relatively rapid onset and shorter duration of action compared
least reduce the potential variability in the oral bioavailability
with earlier class, sulphonylureas (McLeod, 2004; Maggi
of nateglinide. Colloidal drug delivery systems, including self
et al., 2013). The drug is weakly acidic with a pKa of 3.1
microemulsifying systems and peceol containing niosomes
resulting in a pH dependent solubility and ionization (Bruni
showed some success in this respect (Sultan et al., 2016).
et al., 2011). The dependence of the degree of ionization of
Niosomes are vesicular nanostructures which have been
nateglinide on the pH at the absorption site makes it candidate
successfully utilized for enhancing oral bioavailability of
for a narrow absorption window with greater chance for
lipophilic drugs (Bansal et al., 2013; Jadon et al., 2009).
passive absorption in the upper part of the gastrointestinal
These vesicular structures employ nonionic surfactant and
tract (GIT). In addition to possible passive absorption from
cholesterol as the principle components exhibiting better
the upper GIT, nateglinide is thought to be absorbed from the
stability profile compared with liposomes (Abdelaziz et al.,
small intestine via a carrier mediated transport (H+ driven
2015). The efficiency and characteristics of niosomes are
transporter) (Itagaki et al., 2005; Okamura et al., 2002). This
continuously improved by incorporation of secondary addi-
carrier mediated transport suffers from being site specific
tives to the membrane structures. These additives include
which provide additional possibility for the localized absorp-
membrane fluidizers such as oleic acid, lecithin and peceol.
tion window. In addition to site specific absorption,
The later showed a promising capacity to enhance intestinal
nateglinide is poorly water soluble and hydrophobic.
absorption of drugs after incorporation in niosomes
These characteristics resulted in slow dissolution rate
(Abdelaziz et al., 2015; Sultan et al., 2016). The benefit
will become even greater if the vesicular system was prepared
Address for correspondence: Gamal M. El Maghraby, Department of
as pro-concentrate (e.g. proniosomes). The proniosomes have
Pharmaceutical Technology, College of Pharmacy, University of Tanta,
Tanta, Egypt. Tel: +2 0403336007 (ext: 359). Fax: +2 0403335466. the advantage of greater physical stability with a higher
E-mail: gmmelmag@yahoo.com potential for advancing into a dosage form compared with the
210 A. A. Sultan et al.
corresponding niosomes (Akhilesh et al., 2012; Venkatesh
et al., 2014).
The mechanisms of enhanced oral absorption from peceol
containing niosomes were monitored using the in situ rabbit
intestinal technique. Such strategy can discriminate between
diffusive and convective transport but cannot probe the
possibility of intact vesicular absorption (Sultan et al., 2016).
Accordingly, the main objective of this study was to perform
in situ–in vivo correlation of nateglinide absorption after
loading into peceol containing niosomes. This correlation was
conducted with reference to the absorption from the unpro-
cessed drug. The goal was to search the existence of different
absorption pathway for the vesicular system compared with
the free drug solution.
Materials and methods
Materials
Nateglinide was procured from All Pro Corporation, Qingdao,
Shandong, China. Glyceryl monooleate (Peceol) was a gift
from Gattefosse, Saint-Priest Cedex, Lyon, France.
Streptozotocin was purchased from MP Biomedicals, LLC,
Illkirch, France. Acetonitrile (HPLC specifications) was from
SDFCL (S D Fine-Chem Limited), Mumbai, Maharashtra,
India. Sorbitan monostearate (Span 60) was obtained from
Sigma Chemical Co. (St. Louis, MO). Cholesterol, ethanol,
potassium dihydrogen phosphate, potassium chloride, sodium
chloride and disodium hydrogen phosphate were ordered from
El-Nasr Pharmaceutical Chemicals Company, Cairo, Egypt.
Chromatography
Quantitative determination of nateglinide was achieved using
high pressure liquid chromatography (HPLC) employing an
Agilent system (Agilent technologies 1260 infinity, DE,
Germany). This computer controlled equipment was sup-
ported with UV detector (VWD 1260) and an auto-sampling
facility (TCC 1260). A mixture containing 65% acetonitrile
and 35% of filtered potassium dihydrogen phosphate aqueous
solution (10 mM, pH 3) was pumped at 1.3 ml/minute through
ODS reversed phase column (15  0.46 cm; GL Sciences Inc.,
Tokyo, Japan). The average particle size of the stationary
phase was 5 mm. The drug concentration in the effluent was
quantified at 210 nm. The method was validated according to
the ICH guidelines (ICH, 1996).
Solubility determination of nateglinide
An excess amount of nateglinide was added to each of
distilled water, 0.01 N HCl and phosphate buffered saline
(PBS) adjusted to pH 6.6 and 7.4. The mixtures were
incubated at ambient temperature (25 ± 1 C) for 48 h with
continuous magnetic stirring. The resulting suspensions were
filtered through microporous membrane filters (0.45 mm). The
filtrate was suitably diluted and neutralized (if required)
before HPLC analysis.
Preparation of proniosomes and niosomes
Niosomes comprising Span 60, cholesterol, peceol and the
drug (1.2: 0.3: 0.3: 0.009 g) were prepared according to the
previously established method (El Maghraby et al., 2014).
J Liposome Res, 2017; 28(3): 209–217
Briefly, the above components were heated with 1.5 g of
ethanol on a water bath (65 ± 1  C) to form clear dispersion.
Water (1.5 ml) was added and the system was maintained on
the water bath with gentle mixing until clarity. This was
removed from the water bath and mixing continued to
produce proniosomal gel on cooling. The proniosomes were
hydrated by gradual addition of the aqueous phase under
continuous mixing to form 50 ml of crude niosomes. The
crude niosomes were left to undergo complete hydration at
ambient temperature for an overnight. This was suitably
diluted and bath sonicated for 30 min when needed.
Particle size analysis
The niosomal size was recorded using a Zetasizer from
Malvern Instruments Ltd (Malvern, Worcestershire, UK)
which employs dynamic light scattering in particle size
measurement. This was achieved after suitable dilution of
niosomal dispersion with pre-filtered distilled water. The
equipment was maintained at 25  C with a diffraction angle of
90 . The recorded data were expressed as Z-average values
with the particle size distribution being revealed as poly-
dispersity index (PDI).
Product recovery and entrapment efficiency
determination
The product recovery was calculated from the weight of
proniosomes recovered relative to the weight of components
used in preparation. This was expressed as percentage (Yuksel
et al., 2016). The entrapment efficiency (EE) was evaluated
after removal of the free drug from niosomal dispersion by
dialysis. Niosomes (5 ml) were thus loaded into dialysis bags
(Cellulose tubing, Serva, Germany) before incubation into
100 ml of PBS for 4 h. Nateglinide concentration in the
dialysate was measured by the validated HPLC method. The
concentration of the free drug was corrected for volume to
calculate the amount of unentrapped drug which was used to
calculate the EE based on the following equation (El
Maghraby et al., 2014):
Entrapment efficiency ð%Þ ¼ ½ðCt  Cf Þ=Ct   100:
where Ct is the total amount of the drug added to 5 ml
niosomes and Cf is the amount of free drug.
Drug release study
Nateglinide release was monitored after loading known
volume 5 ml) of unprocessed drug dispersion in PBS or
niosomes (each containing 45 mg/ml of drug) into dialysis
bags (Cellulose dialysis tubing, Serva, Germany) which were
incubated at 37  C in 50 ml of 0.01 N HCl containing sodium
lauryl sulfate (0.5% w/v). The study was extended to
investigate the effect of pH on niosomal drug release
employing PBS adjusted to pH 6.6 or 7.4 as release media.
The release medium was intermittently mixed and sampled
periodically and was replenished after each sample with
equivalent volume of the corresponding fresh medium
(preheated to 37  C). The drug content of each sample was
determined using the HPLC method. The cumulative amounts
of drug released were plotted as a function of time to produce
DOI: 10.1080/08982104.2017.1343835
the release profile. The effect of nateglinide niosomal
encapsulation and pH of the release medium were assessed
by comparing the release efficiency which was calculated
from the area under the release profile at time t relative to
that calculated assuming 100% release during the same time
(Khan, 1975). The release studies were conducted in
triplicates.
Preparation of perfusion fluid
The perfusion fluid comprised nateglinide as true aqueous
solution in PBS (control) or encapsulated into niosomes with
the drug concentration being 45 mg/ml in both cases. The
niosomes were obtained by dilution of the crude niosomal
dispersion with PBS (1 in 4) followed by bath sonication
for 30 min. The pH of the perfusion fluid was adjusted to 6.6
and 7.4 for duodenum and jejuno-ileum, respectively. The
prepared perfusion fluids were equilibrated to physiologic
body temperature which was maintained throughout the study.
In situ intestinal absorption studies
Figure 1 presents a schematic illustration of the in situ
intestinal perfusion technique. The study protocol and animal
manipulations were certified by the ethical committee,
College of Pharmacy, University of Tanta (approval number,
206014). Male albino rabbits (six rabbits, 2–2.5 kg in weight)
were used in monitoring the absorption of the drug after
in situ perfusion through the duodenum (15 cm) and jejuno-
ileum (30 cm). The intestinal test segment was exposed after
well-established surgical procedures (Osman et al., 2006;
Sultan et al., 2017). The surgical manipulation was conducted
on the fasting animals which were anesthetized by intramus-
cular injection of two consecutive doses of ketamine HCl
(45 mg/kg each). An extra dose (25 mg/kg) can be injected for
complete anesthesia if required. To prevent any possible
convulsion, a muscle relaxant (Chlorpromazine HCl, 2 mg/kg)
was injected 10 min before ketamine. The required abdominal
area was shaved before midline abdominal longitudinal
incision. The test segment was exposed, ligated and
cannulated from both sides before cleaning by perfusion of
warm PBS. Uniform flow rate was maintained by arranging
the target segment in a uniform multi-S-pattern. Cotton wool
moistened with warm PBS (37  C) was used as a cover for the
intestinal segment to maintain physiologic temperature.
The test formulation was constantly delivered through the
Perfusion solution
Inlet
Outlet
0.27
Syringe pump Exposed
intestinal
(Qin) segment
Figure 1. A schematic illustration of in situ intestinal perfusion technique. Qin is the inlet flow rate (ml/min), Cin is the amount of drug perfused, Cout is
the amount of drug remaining and Fa is the fraction of the drug absorbed.
Niosomes for oral delivery of nateglinide 211
intestinal segments at a rate of 0.27 ml/minute using a syringe
pump (Harvard-22; Harvard Apparatus, Millis, MA). The
perfusate was collected periodically. The volume of each
sample was measured before centrifugation and determination
of the remaining concentration of nateglinide. The actual
length of the target segment was measured at the end of the
experiment after sacrificing the rabbit.
Data analysis
Absorptive clearance
The flow rate and the sampling intervals were used to
calculate the theoretical volume of each sample. This was
subtracted from the actual sample volume to estimate the net
water flux. This was used to normalize the measured drug
concentration to calculate the actual amount remaining per
unit time {C(out)}. The initial drug concentration in the
perfusion fluid was multiplied by the actual flow rate to
produce the amount of drug perfused per unit time {C(in)}.
The ratio between the amount of the drug entering the
segment and that remaining after perfusion was computed.
The samples collected during the time intervals of
70–120 min were assumed to represent the steady state
absorption. The mean of the ratios of the amount of drug
entering the segment and that remaining after perfusion
during these intervals was considered as the steady state
fraction remaining after perfusion, {C(out)/C(in)}ss. This
together with the average flow rate within the intestine (Q,
ml/min) were used to compute the permeability surface area
product (PeA, cm3/min) in which the apparent permeability
coefficient (Pe) is normalized to the effective surface area (A).
This was achieved using the following equation (Gan et al.,
2002):

CðoutÞ =CðinÞ ss ¼ expðPeA=QÞ ð1Þ
Rearrangement of Equation (1) will produce Equation (2).


PeA ¼  Q  ln CðoutÞ =CðinÞ ss ð2Þ
The fraction absorbed (Fa) was computed using Equation
(3).

Fa ¼ 1  CðoutÞ =CðinÞ ss ¼ 1  expðPeA=QÞ ð3Þ
The length of the intestine remaining after complete drug
absorption was taken as the anatomical reserve length (ARL)
(Cin) (Cout)
(Fa)
212 A. A. Sultan et al.
and was computed from Equation (4) in which L* represents
the original length of the intestinal segment (cm) and l* is the
intestinal length along which 95% absorption was achieved
(L95%, cm). l* was calculated thus from Equation (4) and
ARL was computed using Equation (5). ARL can be used as a
marker for the degree of drug absorption with positive values
reflecting complete drug absorption from a given intestinal
segment (Sultan et al., 2017).
0:05 ¼ expðPeAl =QÞ ð4Þ
ARL ¼ L  l ð5Þ
Effect of water flux on intestinal absorption
Intestinal absorption of drugs can result from the contribution
of transcellular and/or paracellular pathways. The later is
dependent on the water flux effect. Taking this into consid-
eration, the rate of drug absorption (Js) can be expressed using
Equation (6) (Lifson et al., 1968). This equation considers
both the transcellular diffusive process ‘Ks(C Cp)’ and the
paracellular convective transport ‘sJwC’.


Js ¼ Ks C  Cp þ s Jw C ð6Þ
Where ‘‘Ks’’ is the diffusive permeability coefficient, ‘‘C’’
and ‘‘Cp’’ are the drug concentrations in the intestinal lumen
and plasma, respectively. ‘‘s’’ is the sieving coefficient of
the drug and ‘‘Jw’’ is the water flux which is an absorption
secretion mechanism. The above equation can be simplified to
Equation (7) considering the existence of sink conditions at
steady state in which the steady state flux is abbreviated as
‘‘Jss’’ (mg/min) with the ‘‘Css’’ (mg/ml) describing the
concentration of the drug remaining for absorption at steady
state.
Jss ¼ Ks Css þ s Jw Css ð7Þ
Rearrangement of Equation (7) gives Equation (8) which
describes the overall absorptive clearance through different
pathways (Jss/Css) that is experimentally estimated as PeA
Equation (2).
Jss =Css ¼ Ks þ s Jw ð8Þ
A plot of the overall absorptive clearance as a function of
the water flux is linear. The slope of this linear plot provides
an estimate for the sieving coefficient. The intercept of this
line with the Y axis reflects the diffusive part of the overall
absorptive clearance (Sultan et al., 2017).
Investigation of the hypoglycemic effect of
nateglinide
This investigation was conducted using 12 male albino rats
with an average weight of 200 g. The study protocol including
the manipulation of rats and the study period was designed
and approved by the Ethical Committee of the College of
Pharmacy, University of Tanta (Approval number, 108016).
The study involved induction of diabetes which was by single
intra peritoneal injection of freshly prepared streptozotocin
solution in 0.1 M citrate buffer (pH 4.5). The dose of
J Liposome Res, 2017; 28(3): 209–217
streptozotocin was 50 mg/kg body weight (Hemalatha et al.,
2004). The injected animals were given a free access to 5%
glucose solution and food to overcome the possibility of
sudden streptozotocin induced hypoglycemia
(Balasubramaian et al., 2004). Two days after injection,
food was restricted for 2 h at the end of which the rats were
anesthetized by ether inhalation before blood sampling from
the tail vein. The blood glucose level was determined using
Gluco DR super sensor (Allmedicus, Gyeonggi, Korea).
Diabetes was confirmed in those rats recording a fasting
blood glucose level above 250 mg/dl.
On the day of experiment the rats were fed pellet diet for
30 min at the end of which the food was restricted and free
access to water was allowed. Two hours after food restriction
the blood glucose level of each animal was measured. This
stable blood glucose level was recorded as the zero time
reading. The test formulation (2.5 mg/ml of unprocessed drug
dispersion in water or the proniosomal formulation) was
administered orally using a feeding syringe. The amount of
drug in proniosomal formulation was the sum of the entrapped
drug and free drug. The administered volume was adjusted to
deliver a dose equivalent to 5 mg/kg. Blood samples were
taken at predetermined time intervals ‘‘0.25, 0.5, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 and 24 h’’ for determination of blood glucose
level. The blood glucose profile was constructed by plotting
the recorded blood glucose concentration as a function of
time. The area above blood glucose profile (AAC) was
calculated by non-linear trapezoidal rule. The reduction in
blood glucose level was calculated with reference to the zero
time level. The time of maximum glucose reduction (Tmax)
was determined by monitoring the rate of change in blood
glucose. The calculated parameters were used for comparison.
Results and discussion
Chromatography
Nateglinide was separated as a baseline to baseline peak after
a retention time of 3.7 min. The standard curve was linear in
the tested concentration range (2–20 mg/ml). The linearity was
indicated from the correlation coefficient which was calcu-
lated to be 0.9999. The straight line equation of the standard
curve was Y ¼ 41.707X  3.6117. The accuracy of the assay
was ensured from the percent of the drug recovered in each
sample relative to nominal concentration (98–102%). The
method was shown to be precise with the repeatability being
indicated from the relative standard deviation values which
ranged from 0.1 to 1.4% for inter and intra-day parameters.
The sensitivity of the method was reflected from the LOD and
LOQ values which were 0.02 and 0.05 mg/ml, respectively.
Solubility determination of nateglinide
This study was conducted with the goal of investigating the
effect of pH on the solubility of nateglinide. The recorded
solubility values were 48.2, 12.5, 183.6 and 727.7 mg/ml in
water, 0.01 N HCl, PBS adjusted at pH 6.6 and 7.4,
respectively. These results highlight the dependence of drug
solubility on the pH of the medium with higher pH values
enhancing the solubility of the drug. This correlates with the
acidic nature of nateglinide. Similar findings have been
DOI: 10.1080/08982104.2017.1343835
Figure 2. Particle size distribution of niosomes.
recorded by other investigators for another acidic drug with
the results being similarly explained (Mokhtar et al., 2008).
Particle size analysis
The particle size of niosomes was expressed as the Z-average
which was recorded to be 283 ± 63 nm. The polydispersity
index (PDI) was taken as a measure for the homogeneity of
the vesicular dispersion with respect to their size. The vesicles
were characterized as heterogeneous population as indicated
from the value of PDI which was 0.49 ± 0.04. This is further
reflected from the particle size distribution which is shown as
multimodal distribution (Figure 2). The heterogeneity is
expected with vesicles prepared by bath sonication after
hydration of proniosomes. Similar pattern was recorded for
vesicular systems prepared using the same technique
(Alomrani et al., 2011; Sultan et al., 2016). It is important
to highlight that the same size range was shown for niosomes
prepared using the same technique after monitoring their size
and morphology by scanning electron microscope (Abdelaziz
et al., 2015).
Product recovery and drug entrapment efficiency
The proniosomal formulation was prepared with a yield of
95.3%. This high recovery reflected both the appropriateness
of proniosomes components and suitability of the technique
employed for preparation (Yuksel et al., 2016). Determination
of nateglinide entrapment efficiency in niosomes involved
separation of unentrapped drug. The estimated entrapment
efficiency depended on the pH of the dispersion medium with
the recorded levels being 54.3% and 39.6% at pH 6.6 and pH
7.4, respectively. These values are expected based on the
acidic nature of the drug which allows for higher fraction of
unionized form in the acidic pH with the degree of ionization
increasing at higher pH value. Taking into consideration the
fact that the unionized species can be entrapped in the lipid
bilayer with the ionic form being located in the aqueous
compartment, the recorded lower entrapment efficiency at pH
7.4 can be explained. The same hypothesis was used to
explain the dependence of the encapsulation efficiency of
drugs into vesicular systems on the physicochemical proper-
ties of the drug (El Maghraby et al., 2005). Similar
encapsulation pattern was published after entrapping flurbi-
profen (acidic drug) into a vesicular system hydrated with
aqueous phase at different pH values (Mokhtar et al., 2008).
More recently, furosemide, another acidic drug behaved
Niosomes for oral delivery of nateglinide 213
similarly with respect to encapsulation in niosomes at
different pH values (Sultan et al., 2016).
Drug release study
The rate of drug release from vesicular systems is sensitive to
temperature change with the release rate increasing at higher
temperature values (Papahadjopoulos et al., 1973).
Accordingly, nateglinide release was monitored at 37  C to
mimic the experimental condition of both in situ intestinal
perfusion and in vivo studies. To further mimic the in vivo
situation, the niosomal drug release was conducted in
simulated gastric and intestinal pH. The study design allows
explanation of the intestinal perfusion as well as the in vivo
data where proniosomes disperses after administration form-
ing niosomes. Figure 3 shows the release profiles of
nateglinide from aqueous drug dispersion in PBS and
niosomes at different pH values. The rate of drug release
from niosomes was slower than that from the unprocessed
drug solution with the release efficiency being 11.8% and
36.4% for niosomes and plain drug, respectively (Figure 3).
Similar results have been previously recorded by other
investigators for niosomal drug release relative to the
unprocessed drug (Jadon et al., 2009). Considering niosomal
drug release, the release profiles reflected the dependence of
nateglinide release on the pH of the release medium with the
release efficiency increasing at the higher pH value. The
calculated niosomal release efficiency values were 11.8%,
38.9% and 42.7% at pH 1.2, 6.6 and 7.4, respectively. This
indicates that niosomes will retain most of the entrapped drug
in the stomach. The recorded release data can be explained on
base of the acidic nature of the drug which will preferentially
partition out to release media having pH values greater than
its pKa where the drug will exist in the ionized status.
In situ intestinal absorption studies
The membrane transport parameters and the transport path-
ways of nateglinide were researched using the in situ rabbit
intestinal absorption technique. These determinations were
conducted after perfusion of the drug in the form of simple
aqueous solution and niosomal dispersion. The niosomal
dispersion was prepared in such a way to mimic the expected
in vivo dispersion of proniosomes after oral administration.
In situ intestinal perfusion was employed in this study to take
the advantages of absence of possible drug food interaction
and elimination of the possible fluctuation in gastric emptying
214 A. A. Sultan et al.
80
Niosomes
Cumulative amount released (%)
60
40
20
0
0 50
Time (min)
80
pH 7.4
Cumulative amount released (%)
60
40
20
0
0 50
Time (min)
Figure 3. Release profiles of nateglinide from aqueous drug dispersion (control) and niosomes at pH 1.2 (top) and the effect of pH on niosomal drug
release (bottom). Error bars are within the symbol size.
rate with strict control on the viability of the tissue and
circulatory functions. This technique can also provide good
evidence on the absorption mechanisms (Osman et al., 2006;
Sultan et al., 2016). The test animal offers similar gastro-
intestinal physiology to human which can further add to the
advantages of this technique (Maeda et al., 1977; Pade &
Stavchansky, 1997). The membrane transport of the drug was
monitored from the duodenum and jejuno-ileum. The rec-
orded parameters are tabulated in Table 1. The dependence of
nateglinide absorptive clearance on the water flux is graph-
ically determined as shown in Figure 4.
The recorded intestinal membrane transport parameters of
nateglinide aqueous solution revealed incomplete absorption
from the duodenal segment. This was indicated from the
negative value of the ARL. The same parameter indicated
complete absorption from the jejuno-ileal segment (Table 1).
This result is against the expectations which are based on the
high degree of ionization of this drug in the jejuno-ileal
segment (at pH 7.4). This may be explained on the base of the
J Liposome Res, 2017; 28(3): 209–217
Control
100 150
pH 6.6 pH 1.2
100 150
specialized absorption mechanism (H+ driven transporter)
which has been reported for nateglinide (Itagaki et al., 2005;
Okamura et al., 2002). Figure 4 shows the dependence of the
absorptive clearance of nateglinide on the water flux which is
reflected from the value of the slope of the plotted line that
was significantly different from zero (P50.01) in both
segments. This finding suggests a role for the paracellular
pathway in nateglinide absorption from both segments.
Considering the absorptive clearance value at zero water
flux, the transcellular pathway is responsible for 25.8% and
25.5% of the absorptive clearance of nateglinide from the
duodenum and jejuno-ileum, respectively (Table 2). Taking
this estimation into consideration, paracellular pathway plays
a major role in nateglinide absorption from both segments.
Perfusion of nateglinide in niosomal dispersion did not
affect the membrane transport parameters compared with the
data recorded after delivery of the drug in the form of simple
aqueous solution (Table 1). With respect to the transport
pathway, the paracellular pathway was dominant with the
DOI: 10.1080/08982104.2017.1343835 Niosomes for oral delivery of nateglinide 215
Table 1. The membrane transport parameters of nateglinide in the perfused intestinal segments.

Control Niosomes
Parameter Duodenum Jejuno-ileum Duodenum Jejuno-ileum
PeA/L (ml/min cm) 0.007* (0.0003) 0.004* (0.0007) 0.008** (0.0007) 0.004** (0.0004)
%Fa/L 2.3* (0.2) 1.5* (0.3) 2.6* (0.4) 1.4* (0.1)
L 95% (cm) 113.3 (16.2) 166.0 (28.4) 106.2 (11.1) 189.5 (24.4)
ARL (cm) 93.3 (16.2) 14.0 (28.4) 86.2 (11.1) 9.5 (24.4)
Jw/L (ml/min cm) 0.005** (0.0003) 0.003** (0.0001) 0.006** (0.0004) 0.003** (0.0002)

PeA/L is absorptive clearance per unit length, %Fa/L is the percentage fraction absorbed normalized to the segment length, L95% is the length
corresponding to 95% absorption, ARL is the anatomical reserve length and Jw/L is the water flux per unit length. Values between brackets are SD
(n ¼ 3).
*Significant difference between duodenum and Jejuno-ileum (P50.05).
**Significant difference between duodenum and Jejuno-ileum (P50.01).

(a) 0.015 (b) 0.009

PeA/L (ml/min.cm)

PeA/L (ml/min.cm)
0.01 0.006

0.005 y = 0.9685x + 0.0017 0.003

y = 1.0228x + 0.0011

0
0
0 0.002 0.004 0.006 0.008 0.01
0 0.002 0.004 0.006

JW/L(ml/min.cm) JW/L(ml/min.cm)

(c) 0.02 (d) 0.009

0.015
PeA/L (ml/min.cm)

PeA/L (ml/min.cm)

0.006

0.01

0.003
0.005 y = 1.1152x + 0.0011 y = 1.1532x + 0.0006

0 0
0 0.005 0.01 0.015 0 0.002 0.004 0.006
JW/L(ml/min.cm) JW/L(ml/min.cm)

Figure 4. Absorptive clearance versus water flux plots of nateglinide from duodenum (left) and jejuno-ileum (right). The data were recorded after
perfusion of nateglinide aqueous solution (a, b) and niosomal dispersion (c, d).

Table 2. The intestinal absorption pathways of nateglinide after in situ

perfusion of simple drug solution (control) and niosomes.
fraction permeating via this route increasing compared with
that calculated after perfusion of drug solution (Table 2). This
Control Niosomes
finding can be attributed to the potential of digestion products Absorption
of niosomes components to widen the tight junctions with pathway Duodenum Jejuno-ileum Duodenum Jejuno-ileum
subsequent increase in the fraction permeating via this route Transcellular (%) 25.8 25.5 14.4 14.8
(Subramanian & Ghosal, 2004). The recorded results are Paracellular (%) 74.2 74.5 85.6 85.2
contrary to the published data on the same carrier with other
drugs (Sultan et al., 2016). This may be explained on the base hypothesis is supported by the recorded data which indicate
of the specialized absorption mechanism of nateglinide (H+ a trend of reduced membrane transport parameters after
driven transporter) which is capable of transporting the free niosomal encapsulation in case of the jejuno-ileum which is
ionized form of nateglinide. The fraction of the free form is the major site for the transporters. However, question
expected to decrease after niosomal encapsulation. This remains about the presence or absence of translymphatic
216 A. A. Sultan et al. J Liposome Res, 2017; 28(3): 209–217

niosomal permeation. If exists, it can affect the bioavailability
and subsequently the pharmacodynamic effect of
nateglinide. This possibility cannot be tested using the in
situ intestinal absorption technique. Accordingly, the study
was extended to conduct in situ–in vivo correlation (see
below).
Evaluation of the hypoglycemic effect of nateglinide
proniosomes
The pharmacodynamic effect can be taken as a measure for the
oral bioavailability. This reflects the overall drug absorption
irrespective to the absorption mechanism. This strategy is
widely adopted in literature (El Maghraby et al., 2015; Sahoo
et al., 2014). Thus diabetic rats were employed to monitor the
hypoglycemic effect of nateglinide after oral administration in
the form of aqueous dispersion or proniosomes encapsulated
drug. The recorded blood glucose levels were plotted as a
function of time. These levels are shown in Figure 5 with the
calculated area above the blood glucose versus time curves
(AAC) being tabulated in Table 3. The unprocessed nateglinide
produced gradual reduction in the concentration of blood
glucose. The maximum effect was recorded 1 h after admin-
istration (Tmax). This pharmacodynamic parameter correlates
1000
proniosomes
800
Blood glucose (mg/dl)
well with the recorded corresponding pharmacokinetic param-
eter which was estimated after monitoring the plasma concen-
tration of orally administered nateglinide (Keilson et al., 2000;
McLeod, 2004). It is important to emphasize that the blood
glucose continued to reduce gradually after this Tmax, probably
due to the additive effect of drug administration and fasting
status of the rats (Figure 5). This hypothesis is supported by the
findings of other investigators who detected lower blood
glucose level after oral administration of nateglinide to fasting
subject compared with that recorded in case of untreated
fasting subjects (Gribble et al., 2001). Similar hypoglycemic
response was noticed for the same drug in other studies (El
Maghraby et al., 2015).
Administration of nateglinide niosomes proconcentrate
resulted in rapid reduction in blood glucose level. This effect
was characterized with rapid onset with the maximum
reduction being noticed after 0.25 h. This rapid hypoglycemic
effect can be partially attributed to the presence of residual
ethanol in the formulation which could enhance initial rapid
absorption of the free nateglinide (Fagerberg et al., 2015).
The membrane fluidizing effect of peceol may also contribute
to the recorded efficacy of niosomes (Risovic et al., 2004;
Sultan et al., 2016). This effect was maintained by subsequent
niosomal absorption. This rapid hypoglycemia was associated
with increase in the extent of drug absorption as reflected
from the significant increase in the AAC after niosomal
Unprocessed drug encapsulation compared with the unprocessed drug dispersion

(P50.005) (Table 3). The overall data indicate enhanced

600
overall oral bioavailability. Enhanced bioavailability can be
achieved from enhanced dissolution, absorption and/or
400 modulated presystemic metabolism. Considering the lipid
nature of niosomes, they can enhance bioavailability of drugs
200
after oral administration via a mechanism similar to that
0 postulated for the traditional lipid based carriers. The
0 4 8 12 16 20 24 membrane fluidization with subsequent augmentation of
Time (h) transcellular pathway may be considered as the first possible
approach. Other tactics can depend on the effect of lipid
Figure 5. The blood glucose level recorded after oral administration of
unprocessed nateglinide aqueous dispersion or nateglinide proniosomes digestion products which improve the paracellular pathway by
to diabetic rats as a function of time. widening the tight junctions (Subramanian & Ghosal, 2004).
The last hypothesis utilizes possible translymphatic absorp-
tion of intact niosomes. The later mechanism can provide
Table 3. Effect of niosomal encapsulation of nateglinide on its additional advantage as it bypasses the presystemic dispos-
hypoglycemic effect after oral administration to diabetic rats. ition. Considering the recorded similar in situ transport
parameters of nateglinide from the vesicles and free drug
Reduction of blood glucose (mg/dl) solution together with recorded in vivo superiority of the
Time (h) Unprocessed drug Proniosomes vesicles, the lymphatic absorption can be taken as the most
probable factor in enhanced bioavailability from vesicular
0.25 13.4 (4.8) 263.6 (23.2)
0.5 21.2 (10.3) 367.7 (44.4) systems. Enhanced oral delivery was recorded after admin-
1 129.8 (27.7) 423.6 (41.5) istration of griseofulvin niosomes. This was explained on the
2 142.8 (25.5) 414.4 (41) base of translymphatic transport of niosomes (Jadon et al.,
3 187.6 (42.1) 479 (41.9)
4 227.5 (41.7) 468.9 (46.7)
2009). Similar explanation was hypothesized after recording
5 269.3 (41.3) 479.4 (47.6) improved intestinal absorption and widened absorption
6 260.5 (42.3) 480.9 (37.2) window of furosemide after niosomal encapsulation (Sultan
7 258.9 (58) 508.5 (42.8) et al., 2016).
8 266.2 (69.6) 484.5 (42.4)
9 248.7 (84.3) 478.6 (49.5)
10 372 (90.6) 510.4 (49.2)
24 24.8 (44.5) 264.4 (73.2)
Conclusions
AACa (mg h/dl) 4959 (468) 14,170 (1555) In situ rabbit intestinal absorption studies revealed a similarity
Values between brackets are SEM (n ¼ 6). between the intestinal absorption of nateglinide from simple
a
AAC is the area above the blood glucose versus time profile. solution and niosomal dispersion with the later increasing
DOI: 10.1080/08982104.2017.1343835
the fraction absorbed via paracellular pathway. In vivo
investigations reflected the superiority of niosomes encapsu-
lated nateglinide compared with the corresponding nategli-
nide aqueous dispersion. This can suggest a possibly different
transport pathway for niosomes encapsulated drug with
subsequent bypass of the presystemic metabolism. However,
this hypothesis merits further future investigations.
Disclosure statement
No potential conflict of interest was reported by the authors.
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