Synthesis, Characterization and Biocompatibility of N-palmitoyl L-alanine-based Organogels as Sustained Implants of Granisetron and Evaluation of thier Antiemetic Effect

Article abstract of DOI:10.1007/s11095-018-2433-2

Purpose: To assess the gelation power of N-palmitoyl L-alanine derivatives in injectable oils and to use the best chosen organogel as parenteral implant of granisetron for the treatment of emesis. Methods: Twelve N-palmitoyl L-alanine derived organogels were developed and evaluated in terms of morphology, thermal properties and in vivo performance. The ability of the selected formula to form in situ gel upon subcutaneous injection in rats and its biocompatibility were monitored over 2?weeks by histopathological examination of the injection site. Results: The acid derivative (N-palmitoyl L-alanine; PA) was superior to ester derivatives. The chosen formula (PA/safflower oil 10% w/v) was successful in forming an in situ gel of granisetron when subcutaneously injected in rats, lasting for 2?weeks and proved to be biocompatible by histopathological examination. Moreover, it exerted an extended antiemetic activity by decreasing the cisplatin-induced pica for a duration of 96?h and reduced preprotachykinin A mRNA expression and Substance P level for up to 4?days (gastric tissue) or 5?days (medulla oblongata) in rats. Conclusion: Granisetron organogel could be considered as a safe, sustained-release and supportive anticancer treatment in both acute and chronic emesis as well as an accompanying treatment with chemotherapeutics in cancer cases.

Full text of DOI:10.1007/s11095-018-2433-2

Pharm Res (2018) 35:149
https://doi.org/10.1007/s11095-018-2433-2
RESEARCH PAPER
Synthesis, Characterization and Biocompatibility of N-palmitoyl
L-alanine-based Organogels as Sustained Implants of Granisetron
and Evaluation of thier Antiemetic Effect
1
2
3
3
Hala Bakr El-Nassan & Aliaa Nabil ElMeshad & Walaa Wadie & Rabab H. Sayed
Received: 18 March 2018 /Accepted: 18 May 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
.
.
.
ABSTRACT
KEY WORDS antiemesis cisplatin granisetron
.
Purpose To assess the gelation power of N-palmitoyl L-
alanine derivatives in injectable oils and to use the best chosen
organogel as parenteral implant of granisetron for the treat-
ment of emesis.
N-palmitoyl L-alanine organogel
ABBREVIATIONS
Methods Twelve N-palmitoyl L-alanine derived organogels
were developed and evaluated in terms of morphology, ther-
mal properties and in vivo performance. The ability of the se-
lected formula to form in situ gel upon subcutaneous injection
in rats and its biocompatibility were monitored over 2 weeks by
histopathological examination of the injection site.
Results The acid derivative (N-palmitoyl L-alanine; PA) was
superior to ester derivatives. The chosen formula (PA/safflow-
er oil 10% w/v) was successful in forming an in situ gel of
granisetron when subcutaneously injected in rats, lasting for
CGC
DSC
GRAS
H-bond
LA
Critical gelling concentration
Differential scanning calorimetry
Generally Regarded As Safe
Hydrogen bond
N-lauroyl L-alanine
N-lauroyl L-alanine methyl ester
LAM
LMWOGs Low molecular weight organogelators
NMP
PA
N-methyl pyrrolidone
N-palmitoyl L-alanine
PAB
PAE
PAM
PBS
SAM
Sc
TEM
Tg–s
Ts–g
Tt
N-palmitoyl L-alanine butyl ester
N-palmitoyl L-alanine ethyl ester
N-palmitoyl L-alanine methyl ester
Phosphate buffer saline
N-stearoyl L-alanine methyl ester
Subcutaneous
Transmission electron microscopy
The gel-sol temperatures
The sol-gel temperatures
The transition temperature
2
weeks and proved to be biocompatible by histopathological
examination. Moreover, it exerted an extended antiemetic ac-
tivity by decreasing the cisplatin-induced pica for a duration of
9
6 h and reduced preprotachykinin A mRNA expression and
Substance P level for up to 4 days (gastric tissue) or 5 days
medulla oblongata) in rats.
(
Conclusion Granisetron organogel could be considered as a
safe, sustained-release and supportive anticancer treatment in
both acute and chronic emesis as well as an accompanying
treatment with chemotherapeutics in cancer cases.
INTRODUCTION
*
Hala Bakr El-Nassan
hala_bakr@hotmail.com
Gels are semi-solid systems composed of two components; a
liquid solvent phase (either polar or nonpolar) and a gelling
agent (also referred to as a gelator) which is often present in
low concentration (<15%) (1–6). This gelator self-assembles,
via physical or chemical interactions, in the presence of an
appropriate solvent into an extensive three-dimensional mesh
network. The latter prevents solvent flow as a result of capil-
lary forces and surface tension (1–6). Gels can be classified on
the basis of the polarity of solvents into hydrogels (polar
1
2
3
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy
Cairo University, Cairo 11562, Egypt
Pharmaceutics and Industrial Pharmacy Department, Faculty of Pharmacy
Cairo University, Cairo 11562, Egypt
Pharmacology and Toxicology Department, Faculty of Pharmacy, Cairo
University, Cairo 11562, Egypt

149 Page 2 of 17
El-Nassan, ElMeshad, Wadie and Sayed (2018) 35:149
solvent) and organogels (organic solvent). Organogels can be
further classified based on the nature of the gelators into low
molecular weight organogelators (LMWOG) and polymeric
gelators (1–6). LMWOG are typically ≤3000 Da (7) and they
display higher organogelation abilities than polymeric gelators
mixture (19). Besides, they are biodegradable and biocompat-
ible with the biological milieu which makes them successful
drug carriers (1–6,19,20). Recently, there has been significant
work on the development of biocompatible organogels based
on generally regarded as safe (GRAS) materials (4) and fatty
N-acylamino esters represented a good example of GRAS
materials. For example, GRAS formulations containing
7.5% N-stearoyl L-alanine methyl ester derivative (SAM) in
safflower oil showed good biocompatibility over 8 weeks eval-
uation period when injected as implants in rats (21). During
designing fatty N-acylamino acid as organogelators, many
amino acids have been examined including glycine (13,22),
L-serine (20,22), L-valine (13,23,24), L-phenylalanine
(13,20,22), glutamic acid (20,25), isoleucine (23), and L-
alanine (12,13,20,22) with various saturated and unsaturated
fatty acids including stearic, lauric, myristic, oleic and palmitic
acids. However, most of the studies done focused mainly on
the physical characteristics and the mechanisms of gel forma-
tion of hydrocarbons. There is little information in the litera-
ture on their pharmaceutical applications and probably L-
alanine based gelators have received considerable attention
and represent promising platforms for long-term sustained
drug delivery (21,26).
Recently, there has been a growing interest in the develop-
ment of parenteral sustained delivery systems administered via
intravenous or subcutaneous routes. This type of delivery sys-
tem can avoid first-pass metabolism and the harsh environ-
ment within the gastrointestinal tract while simplifying the
dosing schedule (1–6). The self-assembling, biodegradable,
and hence biocompatible properties of fatty N-acylamino acid
organogels meet the criteria necessary for their use as in situ
forming drug delivery systems. The latter are in sol form be-
fore administration, but undergo in-situ gelation to form a gel
within the body (6). The in situ forming gels can be prepared
by mixing hydrophilic organic solvents [such as ethanol or N-
methyl pyrrolidone (NMP)] with the gel components to pre-
vent them from gelling and keeps them in the sol state at room
temperature. Once injected into the body, the solvent diffuses
quickly to the surrounding tissues and the sol converts to gel in
a short period of time (27,28). Indeed, subcutaneously-
injected in situ-forming organogels prepared from L-alanine
derivatives in safflower oil were able to release leuprolide over
a period of 3–4 weeks compared to the control group, which
showed activity for only 2 days (28). The same system was also
used in the sustained delivery of rivastigmine where the
organogel provided a 5-fold lower burst effect than the control
oil formulations, followed by sustained release of the drug for
up to 11 days (29).
(8,9). Gels can also be categorized either as physical or chem-
ical gels depending on the type of interactions involved in the
gelation process (1–6). Chemical gels involve alteration in
chemical bonds usually through formation of covalent bonds
between the gelator molecules, whilst physical gels involve
physical interactions (weak interactions such as H-bonding,
van derWaals forces and π-π stacking) among the gelator mol-
ecules. These weak interactions result in the formation of ag-
gregates long enough to overlap and induce solvent gelation.
Physical gels are thermo-reversible, appearing as a gel below a
critical temperature and sol above this temperature (1–6).
Many factors such as steric effects, rigidity and polarity
affect the molecule’s aggregating tendency (1–6). In general,
it is believed that the presence of long chain alkyl units or
steroidal groups is essential for the formation of a stable
LMWOG gel (4,6,7,10). Although not essential for the gela-
tion process, many organogelators are characterized by their
chirality. Many of the existing organogelators possess at least
one stereogenic center. The presence of chiral centers within
the gelators aids in the development of a compact molecular
packing, which provides a thermodynamic and kinetic stabil-
ity to the organogels (6,11). For example, the gelator N-
stearoyl alanine methyl ester displayed higher transition tem-
peratures when used in the enatiomerically pure L-form as
opposed to the racemic form (4,12,13). Besides, β-alanine de-
rived organogelators with no chiral center failed to gel aliphat-
ic hydrocarbons and its gelation ability was inferior to L-
alanine gelators (11).
LMWOG claim advantages of viscoelasticity and thermo-
reversibility (2,5). Other benefits include ease of preparation
and thermodynamic stability (their structure integrity is main-
tained over long periods) (5). They are also characterized by
their chemical and biological stability and they resist microbial
contamination (5). Besides, both hydrophobic and hydrophilic
drugs can be incorporated in organogels (5). Finally, the use of
biocompatible, biodegradable and non-immunogenic gelators
makes them safe for long term applications (5). Many
LMWOGs have been discovered in the last two decades
(1–6). The most important gelator families include fatty and
amino acids, organometallic compounds, steroids, amide- or
urea compounds, nucleotides and dendrimers (1–6,14–16).
However, only few of them have current applications in drug
delivery, probably due to the lack of information on their
biocompatibility and toxicity (1–6,14,17,18). Among the var-
ious chemical classes of LMWOGs, fatty N-acylamino acids
and esters have received considerable attention during the last
two decades. Fatty N-acylamino esters gelators are character-
ized by their ability to selectively form organogels in water/oil
In 2005, Motulsky et al. (21) reported the gelation power of
N-lauroyl L-alanine methyl ester (LAM) and N-stearoyl L-
alanine methyl ester (SAM) on safflower oil. The authors esti-
mated the gelation power of lauroyl (C ) and stearoyl (C ) L-
1
2
18
alanine derivative and the stearoyl derivatives proved to be

N-palmitoyl L-alanine Organogels (2018) 35:149
Page 3 of 17 149
more effective gelators since they have higher transition tem-
presence of long chain hydrophobic group (palmitoyl chain),
and the presence of amide group to ensure H-bonding of the
gelator]. However, the role of the ester group was not thor-
oughly investigated. Therefore, three ester derivatives were
investigated, namely, methyl, ethyl and butyl esters in order
to determine the effect of increasing the alkyl chain of the ester
group on the gelation power. Besides, the acid derivative was
also investigated due to its previously reported high gelation
power on organic solvent (13,19,26). In addition, three inject-
able oils were examined, namely: safflower, sesame and soy-
bean oils. Thereby, twelve gels were developed and charac-
terized and the formula with suitable characteristics was cho-
sen for further examination as drug delivery system for
granisetron. Moreover, the selected organogel was loaded
with granisetron and its ability to form in situ gel upon subcu-
taneous (sc) injection in rats was examined. The biocompati-
bility of the selected formula was monitored over 2 weeks by
histopathological examination of the injection site and tissues
around it. Furthermore, the antiemetic activity of the selected
granisetron organogel was assessed in cisplatin-induced
model of emesis in rats by monitoring the occurrence
and duration of pica, mRNA expression of preprotachykinin
A (PPT-A) and substance P level in both gastric and medulla
oblongata tissues.
perature (T ). Also, the methyl ester was superior to ethyl ester
t
and both were superior to their acid counterparts which ex-
hibited very high T . However, the gelation power of
t
palmitoyl (C ) derivatives was not investigated. Their char-
1
6
acters especially T was predicted (in our postulation) to be
t
superior to lauroyl (C ) and inferior to stearoyl (C )
1
2
18
and thus the use of the acid derivative might be possible
The acid derivative tends to give stronger H-bonding
[
than the ester and thus its gels will be far stronger than that
formed by the ester derivative].
N-Palmitoyl L-alanine and its methyl ester were reported
by Pal and coworkers (13) as organogelators of hydrocarbons.
However, up to our knowledge, the gelation power of N-
palmitoyl L-alanine derivatives on injectable oils and the sub-
sequent use of the resulting organogels as parenteral drug
delivery implants have not been examined before.
Granisetron is a serotonin or 5-hydroxytryptamine 3 (5-
HT3) antagonist which is used to overcome nausea and
vomiting symptoms usually associated with cancer chemother-
apy, radiation therapy, postoperative, and for the treatment of
hyperemesis gravidarum (severe nausea and vomiting occur-
ring during the early pregnancy stage). Chemotherapeutic
agents destroy the lining cells of the stomach releasing seroto-
nin in the bloodstream, which in turn activates 5HT3 recep-
tors in the brain causing nausea and vomiting. Granisetron
blocks the serotonin 5-HT3 neuroreceptors in the brain, so
prevents the serotonin released in the bloodstream from bind-
ing to their receptors and causing emesis (30). Based on the
hypothesis that granisetron acts as an antiemetic solely on the
acute level (31), various immediate acting dosage forms of
MATERIALS AND METHODS
Materials
Granisetron hydrochloride was a kind gift of Amoun
Pharmaceutical Company, Cairo, Egypt. L-alanine methyl
ester hydrochloride, palmitoyl chloride, triethylamine, N-
methyl pyrrolidone (NMP), super refined safflower, sesame
and soybean oils were purchased from Sigma-Aldrich,
Canada Ltd., and used as received. Cisplatin was obtained
from Sigma-Aldrich, St. Louis, MO, USA. All other ingredi-
ents were of pure analytical grade.
granisetron are present in the market as oral solution
®
(
Kytril , Roche, UK) (32,33); but these dosage forms are ad-
ministered orally to patients already suffering from emesis and
this rules them out from being considered as the optimum
dosage forms for granisetron. In addition, immediate release
granisetron tablets were formulated by Nagendrakumar et al.
releasing the drug in the mouth cavity (33). However, intro-
®
ducing granisetron transdermal patches (Sancuso ) in the
USA in 2008 by 3 M Drug Delivery systems (St. Paul,
Minnesota, USA) (34) for controlling nausea and vomiting in
cancer patients treated with chemotherapy for a duration up
to five consecutive days proved the chronic antiemetic activity
of granisetron. This opened the way for formulating sustained
release dosage forms of granisetron especially that transder-
mal patches suffer from the difficulty to be retained on the
patient’s skin for a long time due to either sweating or while
taking a shower.
The aim of the present study was to investigate the gelation
power of various N-palmitoyl L-alanine derivatives on inject-
able vegetable oils. Upon designing the organogelators, the
basic requirements for effective gelators were taken into ac-
count [the presence of stereogenic center (L-alanine), the
Animals
Adult male Wistar rats (200–250 g) were obtained from the
animal house of Faculty of Pharmacy, Cairo University,
Egypt. Rats were housed at constant temperature (25
± 2°C), humidity (60 ± 10%), and a 12/12-h light/dark
cycle and were kept on a standard pellet diet and given
water ad libitum. The study was performed in accordance
with ethical procedures and policies approved by the Animal
Care and Use Committee of the Faculty of Pharmacy, Cairo
University, Cairo, Egypt (PI 816) and complies with the Guide
for the Care and Use of Laboratory Animals published by the
US National Institutes of Health (NIH Publication No. 85–23,
revised 2011).

149 Page 4 of 17
El-Nassan, ElMeshad, Wadie and Sayed (2018) 35:149
Chemical Synthesis
J = 7.2 Hz), 0.95 (t, 3H, J = 7.2 Hz), 1.27 (m, 24H), 1.41 (d,
3H, J = 7.2 Hz), 1.61–1.67 (p, 2H, J = 7.2 Hz), 2.20 (t, 2H,
Melting points (mp) were determined using Stuart SMP20
apparatus and were uncorrected. The FTIR spectra were re-
corded on Shimadzu IR 435 spectrophotometer and values
J = 7.6 Hz), 2.36 (t, 2H, J = 7.2 Hz), 4.23 (m, 4H), 4.58–4.65
(p, 1H, J = 7.2 Hz), 6.07 (br d, 1H, J = 7.2 Hz, D O ex-
2
1
3
changeable); C NMR (100 MHz, CDCl ) 13.6, 14.1, 18.7,
3
−
1
1
13
were represented in cm . The H NMR and C NMR
spectra were carried out on Bruker 400 MHz and 100MHz
spectrophotometer, respectively, Faculty of Pharmacy, Cairo
University, Cairo, Egypt. Tetramethylsilane (TMS) was used
as an internal standard and chemical shifts were recorded in
ppm on δ scale and coupling constants (J) were given in Hz.
Elemental analyses were carried out at the regional center for
mycology and biotechnology, Al-Azhar University, Cairo,
Egypt, using PerkinElmer PE 2400 CHN Elemental
Analyzer. Analytical thin layer chromatography (TLC) on sil-
ica gel plates containing UV indicator was employed routinely
to follow the course of the reactions and to check the purity of
products. All reagents and solvents were purified and dried by
standard techniques. Methyl N-palmitoyl L-alaninate (PAM)
and N-palmitoyl L-alanine (PA) were synthesized according to
the method reported by Pal et al. (13).
19.0, 22.6, 24.7, 25.6,29.0, 29.2, 29.3, 29.4, 29.5, 29.6, 29.6,
29.6, 30.5, 31.9, 33.8,36.6, 47.9, 65.3, 172.7, 173.4;Anal.
Calcd for C H NO : C, 72.01; H, 11.82; N, 3.65. Found:
2
3
45
3
C, 72.26; H, 11.69; N, 3.57.
N-n-Hexadecanoyl (L)-alanine; N-palmitoyl L-alanine (PA)
This compound was prepared by alkaline hydrolysis of methyl
N-palmitoyl L-alaninate (PAM) according to the method re-
ported by Pal et al. (13) Yield: 94%; mp: 92–93°C (reported
−1
91–93°C (13)); IR (cm ): 3321 (NH), 2954, 2848 (CH-ali-
1
phatic), 1701 (acid C=O), 1647 (amide C=O); H NMR
(400 MHz, CDCl ) 0.90 (t, 3H, J = 6.6 Hz), 1.27 (m, 24H),
3
1.41 (d, 3H, J = 6.6 Hz), 1.61–1.67 (p, 2H, J = 7.2 Hz), 2.35
(t, 2H, J = 7.5 Hz), 4.60 (br s, 1H), 6.12 (br s, 1H, D O ex-
2
1
3
changeable); C NMR (100 MHz, CDCl ) 14.1, 17.9, 22.6,
3
24.6, 25.5, 29.0, 29.2, 29.3, 29.4, 29.5, 29.5, 29.6, 29.6, 29.6,
General Procedure for the Synthesis of Alkyl N-palmitoyl
31.9, 33.9, 36.4, 179.0, 179.6.
L-alaninates:
Formation of Organogels
A mixture of methyl, ethyl or butyl L-alaninate HCl salts
(
10 mmol), triethylamine (2.02 g, 20 mmol) in methylene chlo-
The ability of the prepared organogelators (PAM, PAE, PAB
or PA) to gel in three injectable vegetable oils: safflower oil,
sesame oil and soybean oil was examined at four different
concentrations (1, 5, 7.5 and 10% w/v) and was investigated
using the inverted tube method. Briefly, an amount of each
gelator (10, 50, 75 and 100 mg) was weighed and placed into a
4 mL screw-capped vial, and then 1 mL of each oil was added.
The mixture was heated in a hot water bath to 50 °C to
dissolve the solid completely and then allowed to cool over-
night at room temperature. Gelation was confirmed when the
mixture did not flow upon inversion of the vial. The best
chosen gel formulations were loaded with granisetron. The
drug (10 mg) was dispersed in 1 mL oil using bath sonicator
(S30H Elmasonic, Elma Schmidbauer GmbH, Singen,
Germany) for 5 min. The gelator was added and the temper-
ature of the oil was raised to 50 °C till complete dissolution.
To boost the syringeability of granisetron organogel in case of
in vivo study, 10% v/v NMP was added and mixed till the gel
structure was completely lost.
ride (25 mL) was stirred at room temperature for 30 min.
Palmitoyl chloride (2.75 g, 10 mmol) was added dropwise
and the reaction mixture was stirred at room temperature
for 5 h then kept overnight. The solvent was concentrated
under reduced pressure, and the solid formed was filtered,
washed with water (50 mL) and saturated sodium carbonate
solution (50 mL), then dried and crystallized from ethanol.
N-n-Hexadecanoyl Ethyl (L)-alaninate; Ethyl N-palmitoyl L-
alaninate (PAE). Yield: 91%; mp: 61–62°C (reported 62°C
(
−
1
35)); IR (cm ): 3323 (NH), 2920, 2850 (CH-aliphatic),
1
1
741 (ester C=O), 1647 (amide C=O); H NMR
(400 MHz, CDCl ) 0.90 (t, 3H, J = 7.0 Hz), 1.27 (m, 24H),
3
1
.41 (d, 3H, J = 7.1 Hz), 1.61–1.68 (p, 2H, J = 7.2 Hz), 2.20
(t, 2H, J = 7.7 Hz), 2.36(t, 3H, J = 7.2 Hz),4.23 (q, 2H, J =
7
6
1
2
1
3
.2 Hz), 4.58–4.65 (p, 1H, J = 7.2 Hz), 6.09 (br d, 1H, J =
1
3
.4 Hz, D O exchangeable); C NMR (100MHz, CDCl )
2
3
4.1, 18.6, 22.6, 24.7, 25.6, 28.8,29.0, 29.2, 29.3, 29.4,
9.6, 29.6, 31.9, 33.9, 35.3, 36.6, 45.1, 47.9, 61.5, 172.8,
73.4;Anal. Calcd for C H NO : C, 70.94; H, 11.62; N,
Characterization of the Organogels
2
1
41
3
.94. Found: C, 70.86; H, 11.79; N, 4.05.
Critical Gelling Concentration
N-n-Hexadecanoyl Butyl (L)-alaninate; Butyl N-palmitoyl L-
−
1
alaninate (PAB). Yield: 93%; mp: 54–55°C; IR (cm ): 3325
The critical gelation concentration (CGC) was studied and
confirmed by the inverted tube method. Briefly, after the
weight of the organogelator needed to gel in the oil was
(
(
NH), 2956, 2848 (CH-aliphatic), 1739 (ester C=O), 1647
amide C=O); H NMR (400 MHz, CDCl ) 0.90 (t, 3H,
1
3

N-palmitoyl L-alanine Organogels (2018) 35:149
Page 5 of 17 149
determined, the weight was decreased to the minimum weight
of the organogelator needed to gel 1 mL oil. This is noted as
the critical gelling concentration (CGC). The gelation behav-
ior of the L-alanine derivatives together with their correspond-
ing CGC was summarized in Table I.
with molecular weight cut-off of 12,000–14,000 which was
soaked in the receptor medium overnight. The components
in the donor compartment were monitored and the time taken
to change to the gel state was recorded. Besides, 1 mL samples
of the receptor medium were collected at predetermined in-
tervals for 3 days. The receptor medium was compensated
with fresh medium kept at the same temperature and the
concentration of granisetron in the samples was determined
using a UV- spectophotometer at 302 nm with reference to a
Differential Scanning Calorimetry (DSC)
Phase transition diagrams were established by DSC.
Thermograms of gels formed by PAM at concentrations of
2
pre-constructed calibration curve (R = 0.999, n = 3).
7
.5 and 10% w/v in different oils and PA at concentrations
of 5, 7.5 and 10% w/v in different oils were recorded using a
Shimadzu DSC-60 (Kyoto, Japan). The organogels (5 mg)
were weighed in standard aluminum pans that were then
sealed and the gels were allowed to set overnight at ambient
conditions. All the samples were heated from 30 to 100°C at a
heating rate of 20°C/min. At least two heating and cooling
scans per sample were recorded. The parameters estimated
were the gel-sol transition temperature (Tg–s) and the sol-gel
transition temperature (Ts–g) which were determined by en-
dothermic and exothermic peak maxima during the heating
and cooling phases, respectively.
Release Kinetics. The release kinetics was computed by fitting
the release data to the following four mathematical
models (36):
&
&
&
&
Zero order: M / M = k t
t ∞ 0
(
−k t)
First order: M / M = 1 – exp.
t
∞
1
Higuchi model: M / M = k t
H 1/2
t
∞
Power law model: M / M = k n
t
∞
t
where M / M is the fraction of drug released at time t and k ,
t
∞
0
k , k , and k represent zero-order release constant, first-order
1
H
release constant, Higuchi constant, and Korsemeyer-Peppas
constant, respectively. In the power law model, n is the release
Gel Imaging
The chosen gel was diluted with safflower oil in the ratio 1: 10,
spread as a thin layer over a glass slide and examined using a
light microscope (Leica Microsystems, Wezlar, Germany).
Photomicrographs were taken using a digital camera (Victor,
Yokohama, Japan). Moreover, a drop of the diluted chosen
gel was placed on a copper grid (200-mesh, Science Services,
Munich, Germany), left to dry in the air, and examined under
a transmission electron microscope (TEM 1230; JEOL,
Tokyo, Japan) at 80 kV.
Tab le I Gelation Ability of N-palmitoyl L-alanine Derivatives in Various Oils
at 20°C
a
Compound Concentration (% w/v) Physical characteristics
Safflower oil Sesame oil Soybean oil
PAM
PAE
PAB
PA
1%
S
S
S
5%
V
V
V
7.5%
G
G
6
G
G
7
G
G
7
10%
In Vitro Release Study
CGC
1%
S
S
S
A preliminary experiment was done to study the ability of the
chosen organogel (PA/safflower oil 10% w/v) to lose its gel
state and change to liquid form upon adding NMP during
preparation; and also to study its ability to revert to its original
gel state when NMP diffused through body fluids. Phosphate
buffer saline (PBS) was prepared as a medium simulating
5%
S
S
S
7
.5%
0%
S
S
S
1
G
8
G
9
G
9
CGC
1%
S
S
S
5
7
1
%
S
S
S
®
blood (pH 7.4). A vertical Franz diffusion cell, with an effec-
.5%
0%
V
V
V
2
tive area of 3.14 cm , was used to carry out the experiment.
G
10
S
V
V
One gram of the chosen organogel (PA/safflower oil 10% w/
v) prepared using 10% v/v NMP was placed in the donor
compartment and 25 mL of PBS (pH 7.4) was placed in the
receptor compartment. The temperature of the cell was main-
tained at 37 °C and the receptor compartment was stirred on
a magnetic stirrer (WiseStir MSH-20D, Daigger, USA) at
CGC
1%
>10
S
>10
S
5
7
1
%
G
G
G
2.5
G
G
G
4
G
G
G
4
.5%
0%
CGC
1
00 rpm. The donor and receptor compartments were sepa-
a
rated by a cellulose membrane (Spectrum Medical Inc., USA)
S soluble; V viscous liquid; G gel

149 Page 6 of 17
El-Nassan, ElMeshad, Wadie and Sayed (2018) 35:149
exponent, indicative of the mechanism of drug release. The
values of k , k , k , and n were determined by fitting the
Antiemetic Activity
0
1
H
release data into the respective equations.
Kaolin Preparation. Kaolin was prepared according to a previ-
ously described method (38). Briefly, analytical grade kaolin
and acacia were mixed at a ratio of 99:1 followed by addition
of distilled water to form a thick paste. The paste was
further rolled and cut into pieces resembling regular rat
food pellets. The pellets were dried completely at room tem-
perature for 72 h.
Stability of Gels
The best chosen organogel (PA/safflower 10% w/v) was
stored for 6 months in a stability cabinet (Climacell,
Medcenter, Einrichtungen GmbH, MMM group, Germany)
at 25 °C and 75% relative humidity. The gel appearance and
CGC were re-evaluated after 1, 2, 4 and 6 months and com-
pared to the freshly prepared organogel.
Experimental Design. In preliminary experiment, rats received
intraperitoneal injection (ip) injection of cisplatin in a
dose of 3, 6, or 10 mg/kg. Accordingly, cisplatin at a
dose of 10 mg/kg was chosen to induce pica in the subsequent
experiment. This dose was also reported in previous study by
Han et al. (38).
In Vivo Study
Biocompatibility Assessment
Rats (n = 150) were randomly allocated into six
groups (n = 25 per group); the normal control group
that received normal saline ip; granisetron solution group that
received granisetron solution (0.1 mg/kg, ip) (39); granisetron
organogel group that received granisetron organogel (0.5 mg/
kg, sc); cisplatin group that received normal saline and cisplat-
in (10 mg/kg, ip) 30 min subsequent to the administration of
normal saline; cisplatin + granisetron solution group that re-
ceived granisetron solution and cisplatin 30 min subsequent to
the administration of granisetron solution; and cisplatin +
granisetron organogel group that received granisetron
organogel and cisplatin 30 min subsequent to the administra-
tion of granisetron organogel. Granisetron solution was then
administrated every 24 h for the observation period.
At 24, 48, 72, 96 and 120 h after administration of cisplat-
in, 5 rats from each group were sacrificed by cervical
dislocation under light anesthesia. Gastric tissues and
brains were then removed, and medulla oblongata was
dissected. The tissues were stored at −80°C and used for fur-
ther analysis.
The ability of the chosen organogel (PA/safflower oil
1
0% w/v) to form in situ gel upon injection in rats and the
possible irritation that could occur in the rats’ tissues upon
injection were evaluated. The organogel was prepared under
aseptic conditions: the safflower oil and the NMP were initially
filtered each through nylon membrane filter (0.22 μm, Sigma-
Aldrich, USA). A homogenous solution of PA in safflower oil
(10% w/v) was first prepared by heating, then NMP (10% v/v)
was added while hot to inhibit gel formation. The solution was
collected in a sterile syringe (1 mL) while hot and allowed to
cool to room temperature. For medicated organogel, 10 mg of
granisetron were first dispersed in safflower oil 10% w/v and
the same procedure was followed.
Eight male Wistar rats were used in the experiment which
lasted for 14 days. The rats were randomly assigned into 2
groups. Rats in group 1 were sc injected in the dorsal area with
0
.01 mL of safflower oil and served as control, while rats in
group 2 were sc injected with 0.01 mL of the chosen
organogel. The animals were sacrificed after 14 days. The
formation of gels was checked, the semisolid gels were excised
from the injection site and their sizes were determined. The
tissues adjacent to the injection site were examined for histo-
pathological changes.
Monitoring of Pica Behavior. The kaolin pellets were introduced
into the cages 3 days prior to drug administration to allow the
rats to adapt to its presence. The animals’ kaolin and food
intakes as well as body weight were measured daily at 9 am.
In order to accurately measure kaolin and food intakes, re-
maining kaolin and food were collected, including that spilled
outside the containers. The collected kaolin and food were
dried for 24 h and weighed to obtain dry weight values.
Histopathological Study
The tissues surrounding the injection sites were fixed in 10%
formol saline for 24 h. Washing was done by water followed
by serial dilutions of (methyl, ethyl and absolute ethyl) alcohols
which were used for dehydration. Specimens were cleared in
xylene and embedded in paraffin at 56°C in hot air oven for
Quantitative Real-Time Polymerase Chain Reaction Analysis of
mRNA Expression of Preprotachykinin A (PPT-A) in Gastric
Tissue and Medulla Oblongata. Total RNA was extracted from
the frozen tissue samples using the RNeasy Mini Kit (Qiagen,
Valencia, CA) according to the manufacturer’s protocol. The
RNA concentration and purity were spectrophotometrically
determined at the optical density OD 260/280 nm. The RNA
2
4 h. Paraffin beeswax tissue blocks were prepared for section-
ing at 4 μm thickness by slidge microtome. The obtained
tissue sections were collected on glass slides, deparaffinized,
stained with hematoxylin and eosin (H & E) stain for exami-
nation by the light electric microscope (37).

N-palmitoyl L-alanine Organogels (2018) 35:149
Page 7 of 17 149
1
integrity was assessed using agarose gel electrophoresis and
ethidium bromide staining. Equal amounts of RNA (2 mg)
were reverse transcribed into complementary DNA (cDNA)
using Superscript Choice systems (Life Technologies, Breda,
Netherlands) according to the manufacturer’s guidelines. To
assess the expression of PPT-A gene, quantitative real-time
PCR was performed using SYBR Green PCR Master Mix
1647 cm− corresponding to ester and amide C=O bonds,
respectively. Besides, the NH band appeared at 3317–
−
1
1
3325 cm . H NMR spectra of the ester derivatives
(PAM, PAE and PAB) showed an exchangeable broad
doublet signal at δ 6.07–6.09 ppm corresponding to the
NH proton together with the appearance of the ester
1
3
alkyl protons. The C NMR spectra of (PAM, PAE and
PAB) displayed two signals at δ 172–174 ppm corresponding
to the two C=O carbons.
Alkaline hydrolysis of methyl N-palmitoyl L-alaninate
(PAM) afforded N-palmitoyl L-alanine (PA) as reported earli-
er by Pal et al. (13). The disappearance of the ester C=O band
in the IR spectrum of the acid derivative PA, together with the
(Applied Biosystems, Foster City, CA, USA) as described by
the manufacturer. Briefly, in a 25-μL reaction volume, 5 μL of
cDNA was added to 12.5 μL of 2× SYBR Green Master Mix
and 200 ng of each primer. The sequence of the primer was
5’-ACCAGATCAAGGAGGCAATG-3′ as a forward primer
and 5′- GCCCATTAGTCCAACAAAGG-3′ as a reverse
primer. PCR reactions included 10 min at 95°C for activation
of AmpliTaq Gold DNA polymerase, followed by 40 cycles at
−
1
appearance of the acid C=O band at 1701 cm pro-
vided a proof for the formation of PA. The H NMR
1
9
5°C for 15 s (denaturing) and 60°C for 1 min (annealing/
spectrum of PA revealed the disappearance of the character-
1
3
extension). The relative expression of target genes was obtain-
ed using the 2
istic signals due to COOCH protons. Besides, its C NMR
3
−ΔΔCT
formula as described previously by Pfaffl
spectrum showed two signals at δ 179 ppm corresponding to
(
(
40) using Glyceraldehyde-3-phosphate dehydrogenase
GAPDH) as a housekeeping gene.
the two C=O carbons.
Characterization of Gels
Measurement of Levels of Substance P in Gastric Tissue and
Medulla Oblongata. The levels of Substance P in gastric tissue
and medulla oblongata were determined using an Enzyme-
Linked Immunosorbent Assay (ELISA) kit according to
the manufacturer’s instructions (R&D Systems, Minneapolis,
MN, USA). Protein content was measured according to the
method of Bradford (41). Values were expressed as ng/mg
protein.
Formation of Organogel and the Critical Gelling Concentration
(CGC)
N-Palmitoyl L-alanine derivatives (PAM, PAE, PAB and PA)
were tested for their ability to gelatinize injectable vegetable
oils at four different concentrations (1, 5, 7.5 and 10%
w/v) using the inverted tube method. The reason for
using the specific concentrations of the organogelator
was to cover the low (1% w/v), intermediate (5 and
Statistical Analysis
7.5% w/v) and high concentration (10% w/v) in order to
Data were expressed as means ± standard error (SEM).
Results were analyzed using one-way-analysis of variance test
obtain a gelator with the minimum CGC for practical use as
a drug delivery system.
(
ANOVA) followed by Tukey Kramer multiple comparison’s
The results of the gelation ability of each compound in the
tested oils together with their CGC were displayed in Table I.
In this work, all the tested compounds were soluble in the
tested oils and all the gels formed were optically opaque gels.
The results from Table I indicated that none of the com-
pounds formed gel at 1% w/v. Only the acid derivative PA
was able to gelatinize all the oils tested (safflower, sesame and
soybean oils) at concentration level of 5% w/v. The methyl
ester derivative PAM formed gels in all oils at concentration of
7.5% w/v. On the other hand, the ethyl and butyl ester de-
rivatives (PAE and PAB, respectively) formed gels only at
10% w/v. It should be noted that the butyl ester PAB even
did not gelate at concentration level of 10% w/v in sesame and
soybean oils.
test. For all statistical tests, the level of significance was set at
P < 0.05. GraphPad Prism® software package, version 5
(
GraphPad Software, Inc., USA) was used to carry out all
statistical tests.
RESULTS AND DISCUSSION
Synthesis of N-palmitoyl L-alanine Derivatives
Figure 1a outlines the synthesis of the target N-palmitoyl L-
alanine derivatives (PAM, PAE, PAB or PA).
In this work, L-alanine esters were acylated with palmitoyl
chloride in methylene chloride to afford N-palmitoyl L-
alanine methyl (PAM), ethyl (PAE) and butyl esters (PAB) in
excellent yields (91–93%). The formation of the ester deriva-
tives (PAM, PAE and PAB) was confirmed by FTIR spectra
The CGC values (Table I) of all the organogelators lied
between 2.5% and > 10% w/v. The lowest CGC was ob-
served with PA in safflower oil (2.5% w/v). The CGC ob-
served for PA in both sesame and soybean oils was 4% w/v.
The highest CGC was observed by PAB in sesame and
−
1
that showed two carbonyl bands at 1739–1741 cm and

149 Page 8 of 17
El-Nassan, ElMeshad, Wadie and Sayed (2018) 35:149
Fig. 1 (a) Scheme of preparation
of N-palmitoyl L-alanine derivatives;
photographs showing gelation of:
(b) PAM and (c) PA in different oils
by invert tube method.
soybean oils (>10% w/v). PAM attained CGC of 6% w/v in
safflower oil, whereas it was 7% w/v in both sesame and
soybean oils. As for PAE, the CGC was 8% w/v in safflower
oil and was 9% w/v in sesame and soybean oils. Finally, PAB
was able to gel in safflower oil at 10% w/v. Figure 1b, c depict
the photographs of PAM and PA gels at different concentra-
tions and in various oils respectively.
strong H-bonding was represented schematically in Fig. 2.
From the CGC results obtained, it was noticed that safflower
oil was the optimum oil to continue work with; as all the
organogelators attained lower CGC in this oil compared to
the other oils used in the study. It was concluded that the gels
formed by PAE and PAB were excluded at this stage and no
further testing was done on them.
Structurally, the compounds tested belonged to the same
series and differed only in the nature of the functional group
on L-alanine, being ester (methyl, ethyl and butyl) or acid
moiety. The gelation abilities of the organogelators were com-
pared using CGC values. From the CGC results obtained it
can be concluded that increasing the hydrophobicity and alkyl
chain length of the ester moiety from methyl to ethyl to butyl
reduced the gelation power. As expected, the CGC of the acid
derivative PA were superior to that of the methyl ester and this
result was consistent with the previously published data (21)
that suggested that the acid formed strong H-bond due to
dimer formation and thus its ability to form gel was higher
than that of ester derivatives. A proposed explanation of this
Thermotropic Behavior of the Gels (DSC)
The thermotropic behavior of the gels formed by PAM and
PA was investigated using DSC. The gels were heated from
30 to 100°C at a heating rate of 20°C/min. The transition
temperatures (Tg–s) and (Ts–g) and the enthalpies of
PAM and PA organogels were summarized in
Table II and Fig. 3a, b. Figure 3c) illustrated the
DSC thermogram for the gel formed by the acid derivative
PA in soybean oil at 10% w/v showing several (Tg–s) transi-
tions. From the results in Table II, the following general re-
marks were observed:

N-palmitoyl L-alanine Organogels (2018) 35:149
Page 9 of 17 149
&
For all the samples tested, the gel-sol (Tg–s) temper-
atures were higher than the sol-gel temperatures
derivatives (13). Herein, obtaining SEM micrographs of the
prepared gels was not possible due to failure in obtaining the
xerogels. However, images taken by transmission electron mi-
croscopy (TEM) for PA 2% w/v in safflower oil confirmed the
presence of fibrous networks (Fig. 5a, b).
(
(
Ts–g). (Tg–s) ranged between 38 and 52°C, while,
Ts–g) ranged between 27 and 35°C. Thus, all (Tg–s)
exceeded (Ts–g) by more than 10°C (hysteresis behavior).
Examining the DSC thermograms for the gels (for exam-
ple Fig. 3c) revealed the presence of more than one (Tg–s)
transition which may be attributed to the polymorphic
character of the aggregate.
It was reported earlier that increasing the concentration of
the gelator is known to increase the thermal stability of the
gel which means an increase in the (Tg–s) value. However,
the DSC results of the organogels formed by PAM in
different oils indicated that increasing the concentration
from 7.5 to 10% w/v was not accompanied by significant
increase in (Tg–s) values, thus the optimum concentration
for PAM is 7.5% w/v. Whilst, for the acid derivative PA in
sesame and soya bean oils, increasing the concentration
from 5 to 7.5 to 10% w/v was accompanied by increase in
&
&
In Vitro Release Study
The addition of NMP (10% v/v) to PA (10% w/v) in safflower
oil prevented the gel formation. The solution changed to gel
when mixed with PBS at 37°C within 5 min. without emulsi-
fication. Figure 5c illustrated the release pattern of granisetron
from the two selected gels: 10% w/v in safflower oil and in
sesame oil. The release of granisetron was significantly de-
layed by formulation of organogels compared to drug solution
of the same concentration and showed a biphasic pattern. The
rate of granisetron released from sesame organogel during the
first 6 h was 1.79%/h, whereas that released from safflower
organogel was 3.14%/h. The rates of drug release after 6 h
from the respective gels were 0.1 and 0.27%/h. By fitting the
release data to different mathematical models in order to pre-
dict the order of drug release from the organogels, results
showed that the release of granisetron followed a diffusion
model (showing the highest coefficient of determination
(Tg–s) values (Fig. 3a, b).
&
The (Tg–s) values of the gels formed by acid derivative PA
were higher than those of the methyl ester PAM at the
same concentration (Fig. 3a, b). This might be attributed
to the increase in H-bonding and van der Waals interac-
tion in the acid derived organogels especially at higher
concentrations. These results were consistent with the pre-
viously published results of N-stearoyl and N-lauroyl-L-al-
anine derivatives (21).
2
[R ]) (Table III). This meant that the drug was entrapped
within the gel network structure and for the drug to be re-
leased it had to first diffuse from the organogel matrix.
&
The (Tg–s) and (Ts–g) values obtained by palmitoyl deriv-
atives (C ) were higher than those reported for N-lauroyl
Stability of the Gels
1
6
derivatives (C ) and lower than those reported for N-
The appearance of the chosen gel formula (PA/safflower 10%
w/v) remained unchanged for 6 months when stored at ambi-
ent conditions. The CGC value was the same as obtained for
the freshly prepared gels.
1
2
stearoyl derivatives (C ) (21).
1
8
&
&
Regarding the type of oil used, no significant DSC differ-
ence can be determined between the various oils used and
comparable (Tg–s) results were obtained in various oils.
The stored gels were not only thermoreversible, but also
they were susceptible to external stimuli such as application of
mechanical shaking and ultrasound treatment. Thus, they
converted into sol state upon shaking or sonication and
returned to gel state upon resting. Here, the organogels
formed by acid derivative (PA) tolerated the shaking stress
(especially at 10% w/v) more than the ester derivatives at the
same concentration. This observation might be explained in
view of the fact that acid derivatives formed more stable gels
due to dimer formation (their intermolecular H-bonding were
stronger than those formed by ester derivatives).
g-s
s-g
The organogelator enthalpies (ΔH ) and (ΔH ) are re-
ported in Table II. Here also, both enthalpies increased
with increasing organogelator concentration of the acid
from 7.5 to 10% w/v.
&
The highest enthalpies were obtained with the PA/sesame
g–s
oil organogels at 10% w/v (ΔH = −61.40 mJ) and this
result was consistent with the rheology study where this gel
exhibited the highest yield stress (30 Pa) (data not shown).
Based on the DSC results, the acid derived gels at 10% w/v
concentration were chosen for further testing.
In Vivo Study
Gel Imaging
Biocompatibility Studies
The optical micrographs of the formed gels (Fig. 4) showed the
rod-like aggregates formed by the organogelators as expected
from previously published data on N-palmitoyl L-alanine
Previously published reports of N-lauroyl L-alanine (LA) and
N-lauroyl L-alanine methyl ester (LAM) (21,26) ruled out the
use of acid derivative and preferred the use of ester derivatives

149 Page 10 of 17
El-Nassan, ElMeshad, Wadie and Sayed (2018) 35:149
O
O
H
N
H
O
H
N
O
O
O
H
O
H
N
H
N
O
O
O
OH
H
O
H
O
N
O
H
N
O
O
O
H
O
H
O
O
O
H
N
H
O
O
O
OH
OH
H
N
O
H
O
N
O
H
N
O
H
O
H
O
H
H
N
O
N
H
O
O
H
O
O
N
O
H
N
O
O
O
H
O
O
H
O
H
H
N
O
N
H
O
O
OH
OH
O
H
O
N
O
H
N
O
H
O
H
O
H
O
H
O
N
O
O
N
H
O
H
O
N
O
H
N
O
H
O
H
O
O
Fig. 2 Schematic representation of the possible interactions between N-palmitoyl L-alanine (PA) molecules; dotted lines represent H-bonds.
for the preparation of a depot formulation in spite of their
inferior properties and these reports justified that by two rea-
sons: 1) LA did not exhibit hysteresis behavior and 2) LA
emulsified the soybean oil in PBS, resulting in the loss of the
gel integrity. However, in this work, it was found that the acid
derived gels at 10% w/v concentration exhibited high values
of (Tg-s) (50–52°C) and low values of (Ts-g) (30–32°C) and
thus exhibited pronounced hysteresis behavior.
An attempt was done to enhance the syringability of the
chosen gel formula [PA in safflower oil (10% w/v)] by adding
10% v/v NMP during the preparation of the gel formula. This
inhibited the formation of the expected gel matrix, thus easing
Table II Thermotropic Behavior
of the Organogels as Measured by
Differential Scanning Calorimetry
g–s
c
s–g
d
Organogelator/oil
Concentration
% w/w)
Heating a
Freezing
Ts–g (°C)
ΔH (mJ)
ΔH (mJ)
b
(
Tg–s (°C)
PAM/safflower oil
PAM/sesame oil
PAM/soybean oil
PA/safflower oil
7.5
43.88
49.13
43.48
38.70
47.65
46.03
50.26
49.11
46.59
40.69
43.76
50.74
45.04
48.55
52.06
27.63
32.47
ND
−30.45
−14.93
−45.37
−40.74
−48.24
ND
1.33
2.85
ND
1
0
7.5
1
0
ND
ND
7.5
28.01
35.39
31.88
28.56
32.82
ND
1.10
1.46
0.67
1.09
1.38
ND
1
5
7
1
5
7
1
5
7
1
0
−10.78
−16.62
−29.09
−35.45
−58.02
−61.40
−14.52
−34.04
−40.35
.5
0
PA/sesame oil
PA/soybean oil
.5
0
27.84
30.13
28.97
28.15
32.61
1.59
2.72
0.77
1.13
2.11
.5
0
a
Tg–s: gel-to-sol temperature
Ts–g: sol-to-gel temperature
b
c
g–s
ΔH : gel-to-sol enthalpy variation
d
s–g
ΔH : sol-to-gel enthalpy variation
ND not determined

N-palmitoyl L-alanine Organogels (2018) 35:149
Page 11 of 17 149
Fig. 3 Graphical representation of
DSC results of: (a) PAM in different
oils at two different concentrations
(
7.5 and 10% w/v); (b) PA in
different oils at three different
concentrations (5, 7.5 and
1
0% w/v) and (c) Thermogram for
the gel formed by the acid derivative
PA/soybean oil 10% w/v.
the injection of the formula. The ability of the gel formula to
revert to gel upon administration to rats was possible due to
the fact that NMP, upon injection, could diffuse
through body fluids and leaving the formula in an in
situ gel matrix or network (27,28). Moreover, PA (10%
w/v) in safflower oil in the presence of 10% v/v NMP
was found to form a gel when mixed with PBS at 37°C within
this case might be suitable for the preparation of a depot
formulation.
The chosen gel formula (PA/safflower 10% w/v) was eval-
uated in Wistar rats to assess its ability to form in situ gel upon
sc injection and to evaluate the possible irritation that could
occur in the rats’ tissues upon injection. The rats were divided
into 2 groups: group 1 rats were sc injected with safflower oil
and served as control and group 2 rats were sc injected with
the chosen gel formula. The experiment duration was 14 days.
5
min. without emulsification. Thus, the acid derived gels in
Fig. 4 Optical micrograph of: (a)
PAM/ safflower oil 7.5% w/v and
(b) PAM/ sesame oil 7.5% w/v.

149 Page 12 of 17
El-Nassan, ElMeshad, Wadie and Sayed (2018) 35:149
Fig. 5 (a) TEM micrographs of
PA/safflower oil 2% w/v, bar =
1
0 μm; (b) TEM micrographs of
PA/safflower oil 2% w/v, bar =
μm and (c) average release profile
5
of granisetron from different
organogels and granisetron solution
in saline phosphate buffer (pH 7.4)
(n = 3).
No death among rats of the two groups occurred. The sc
injection, revealed no histopathological alteration in the epi-
dermal and dermal layer as well as the subcutaneous tissue
and underlying musculature (Fig. 6c–f). These results proved
the safety, non-irritancy and biocompatibility of the prepared
gels as in situ gel forming drug delivery systems.
injection of PA gels resulted in bean-like gels which remained
quite morphologically intact upon excision (Fig. 6a, b). A mac-
roscopic observation of the injection site after 14 days after
administration of PA in safflower oil (10% w/v) revealed no
great difference in the gel’s integrity. The average shape of the
formed in situ gel was elliptical with an average size of 2 ±
Antiemetic Activity
0
.2 cm length and 0.8 ± 0.1 cm in width (n = 4). Accordingly,
the gels formed by PA/safflower oil (10% w/v) are considered
as promising sustained release drug delivery systems.
Rats react to emetic/nausea-producing stimuli, such as cis-
platin, with altered feeding habits, manifested by pica or in-
creased consumption of kaolin. The profiles of cisplatin-
induced pica in rats are similar to clinical findings for
cisplatin-induced emesis in humans and can be used as a
model of nausea in humans (39,42). As shown in Fig. 7a,
cisplatin ip injection promoted kaolin intake by rats at 24 h
post administration, compared to the normal control group,
and this effect continued for 120 h. This enhanced kaolin
intake was attenuated upon pretreatment with granisetron
solution, compared with cisplatin group, at 24 h and 48 h
only. The ability of granisetron solution to only abolish the
acute phase of cisplatin-induced pica was previously reported
by other authors (39,43). Interestingly, pretreatment with
granisetron organogel led to a marked decrease in kaolin in-
take at 24 h, 48 h, 72 h and 96 h (Fig. 7a).
Histopathological Study
Histopathological examination of the injection site and its sur-
rounding tissues in rats of the two groups, after 14 days of sc
Tab le I II Kinetic Analysis of the Release Data According to Different Models
2
Order of release
Coefficient of determination (R )
Zero
First
Higuchi diffusion
Organogel in sesame oil
Organogel in safflower oil
0.638
0.725
0.416
0.502
0.819
0.889

N-palmitoyl L-alanine Organogels (2018) 35:149
Page 13 of 17 149
Fig. 6 Photographs of: in situ gels
formed in the dorsal area of rats
after 14 days of sc injection of
PA/safflower oil/NMP: (a) intact
under rat skin; (b) excised sc tissue
of rat from the dorsal area showing
the gel formation; histopathological
examination of the injection site and
its surrounding tissues in rats after
1
1
4 days of sc injection: (c) in group
showing normal histological
structure of epidermis and dermis;
d) in group 1 showing normal
(
histological structure of
subcutaneous tissue and
musculature; (e) in group 2 showing
normal histological structure of
epidermis and dermis and (f) in
group 2 showing normal histological
structure of subcutaneous tissue and
musculature (H & E, 16X).
Cisplatin injection induced a significant reduction in nor-
mal food intake from 24 h to 120 h post administration, com-
pared to the normal control group (Fig. 7b). This was consis-
tent with the previous studies which showed that cisplat-
in induced significant pica accompanied by reduced
food intake within 8 h of its administration that contin-
ued for 5 days (39,42). Food intake was considerably
improved upon pretreatment with granisetron solution,
compared with cisplatin group, at 24 h only. However,
pretreatment with granisetron organogel led to a marked im-
provement in normal food intake at 24 h, and extended for
emetic circuitry in the medulla oblongata and then induces an
emetic reflex (44). These observations are the rationale that 5-
HT3 receptor antagonists, such as granisetron, are
regarded as the first choice for the prevention and treatment
of emesis (45,46).
Previous studies have shown that Substance P is involved in
the patho-aetiology of emesis, nausea, anorexia and inhibition
of gastric emptying (47,48). In fact, chemotherapeutic agents
can increase the release of not only 5-HT, but also Substance
P from the EC cells of gastric mucosa and brainstem (49,50).
Tatsushima et al. (51) reported that the level of Substance P in
the cerebrospinal fluid of rats was increased 72 h after an ip
injection of cisplatin. Moreover, the expression of PPT-A
mRNA, which encodes mainly for Substance P, was reported
to be increased in the medulla oblongata by motion stimuli
that induced pica behavior in rats (52). Yamamoto et al. (39)
found that PPT-A mRNA in the medulla oblongata was up-
regulated between 24 and 120 h after the injection of cisplatin
and the authors concluded that Substance P production via 5-
HT3 receptors in the medulla oblongata is involved in the
development of both the acute and delayed phases of
cisplatin-induced pica. Consistently, the present finding
showed that cisplatin injection significantly increased PPT-A
mRNA expression (Fig. 8a, b) and Substance P level (Fig. 8c,
9
6 h (Fig. 7b).
Cisplatin injection led to a marked body weight re-
duction in duration of 24 to 120 h post administration,
as compared to normal control group. Granisetron so-
lution produced a significantly lower body weight loss at
2
4 h only. In contrast, granisetron organogel pretreat-
ment afforded protection against body weight reduction
at 24 h and a significant trend of less reduction at 48 h
and 72 h (Fig. 7c).
The aetiology of cisplatin-induced emesis has been postu-
lated to be that 5-HT released from the enterochromaffin
(EC) cells in the gut mucosa stimulates the 5-HT3 receptor
on adjacent vagal afferent nerves, which activate the central

149 Page 14 of 17
El-Nassan, ElMeshad, Wadie and Sayed (2018) 35:149
Fig. 7 Effects of granisetron solution and organogel on cisplatin-induced (a) pica, anorexia (b) and body weight changes (c). Values were
expressed as mean of 5 experiments ± SEM. One-way ANOVA followed by Tukey–Kramer post-hoc test were used for statistical analysis of data. *: significantly
different from the control group at P < 0.05. #: significantly different from the cisplatin group at P < 0.05.
d) in gastric tissue and medulla oblongata at 24 h post admin-
istration, as compared to normal control group and these
findings lasted during the observation period.
Pretreatment with granisetron solution significantly
inhibited the increased PPT-A mRNA expression in gastric
tissue at 24 h only, and in medulla oblongata at 24 h and 48 h,

N-palmitoyl L-alanine Organogels (2018) 35:149
Page 15 of 17 149
Fig. 8 Effects of granisetron solution and organogel on the mRNA expression of preprotachykinin A (PPT-A) in gastric tissue (a) and
medulla oblongata (b), and the levels of Substance P in gastric tissue (c) and medulla oblongata (d). Values were expressed as mean of 5
experiments ± SEM. One-way ANOVA followed by Tukey–Kramer post-hoc test were used for statistical analysis of data. *: significantly different from the control
group at P < 0.05. #: significantly different from the cisplatin group at P < 0.05.
as compared with cisplatin group (Fig. 8a, b). The cisplatin-
induced increase in Substance P levels in gastric tissue and
medulla oblongata was inhibited by granisetron solution until
acid derivative showing superiority to the esters derivatives in
terms of critical gelling concentration, differential scanning cal-
orimetry and thermotropic behavior. N-palmitoyl L-alanine
acid derivative in safflower oil (10% w/v) was successful in
forming an in situ gel, upon injection subcutaneously in rats,
maintaining its gel matrix for 14 days and the histopathological
studies proved its biocompatibility. Organogels-loaded
granisteron significantly decreased pica for extended duration
(96 h), mRNA expression of preprotachykinin A (PPT-A) and
substance P level in both gastric (96 h) and medulla oblongata
(120 h) tissues in cisplatin-induced model in rats. Granisetron
organogels alleviated acute and chronic emesis in rats and thus
could be considered a safe, sustained and supportive treatment
in cancer cases.
4
8 h only (Fig. 8c, d). This observed effect was in agreement
with other authors (38,39). The granisetron organogel was,
however, effective in reducing cisplatin-induced rise in PPT-
A mRNA expression for up to 4 (in gastric tissue) or 5 days (in
medulla oblongata) depending on the tissue (Fig. 8a, b).
Pretreatment with granisetron organogel also attenuated the
increment in Substance P level in gastric tissue and medulla
oblongata until 120 h, except at 96 h in medulla oblongata no
significant difference was observed (Fig. 8c, d).
CONCLUSION
Acknowledgments and Disclosures. The authors are grateful to
Prof. Dr. Adel Bakeer Kholoussy, Professor of Pathology,
Faculty of Veterinary Medicine, Cairo University for carrying
N-palmitoyl L-alanine derivatives were successful
organogelators in safflower, sesame and soybean oils with the

149 Page 16 of 17
El-Nassan, ElMeshad, Wadie and Sayed (2018) 35:149
2
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amino acid derivatives: potent biomimetic hydro/organogelators as
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out the histopathological study. The authors declare that there
are no conflicts of interest to declare.
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