Received: 2 February 2020 Revised: 15 April 2020 Accepted: 17 April 2020

DOI: 10.1002/ddr.21677

RESEARCH ARTICLE

In vitro–in vivo evaluation of tetrahydrozoline-loaded ocular in

situ gels on rabbits for allergic conjunctivitis management

Okur1
Neslihan Üstündag | Vildan Yozgatli2 | Mehmet Evren Okur3

1
Department of Pharmaceutical Technology,
Faculty of Pharmacy, University of Health
Sciences, Istanbul, Turkey
2
Department of Pharmaceutical Technology,
Faculty of Pharmacy, Ege University, _Izmir
Turkey
3
Department of Pharmacology, Faculty of
Pharmacy, University of Health Sciences,
_Istanbul, Turkey
Correspondence
Neslihan Üstünda
g Okur, Department of
Pharmaceutical Technology, Faculty of
Pharmacy, University of Health Sciences,
Istanbul, Turkey.
Email: neslihanustundag@yahoo.com
Abstract
Ocular allergy is one of the most common disorders of the eye surface. The conven-
tional eye drops lack of therapeutic efficacy due to low ocular bioavailability and
decreased drug residence time on eye surface. Hence, the present research work
aimed to formulate, optimize, and evaluate the in situ gel for ophthalmic drug deliv-
ery. The prepared in situ gel formulations were evaluated for clarity, pH, gelling
capacity, viscosity, osmolality, in vitro release study, and kinetic evaluation. ex vivo
corneal permeation/penetration study using goat and in vivo studies on rabbits were
also performed. Fourier-transformed infrared spectroscopy was also applied to study
possible interactions between drug and polymers. The formulations found to be sta-
ble, nonirritant, and showed sustained release of the drug for a period of up to 24 hr
with no ocular damage. The developed in situ gels loaded with tetrahydrozoline are
alternative and promising ocular candidates for the treatment of allergic
conjunctivitis.

KEYWORDS

eye irritation, in situ gel, rabbit, tetrahydrozoline, topical drug delivery

1 | I N T RO DU CT I O N 24 hr and most patients require more effective and longer-lasting

Last decades, allergies seem to dramatically increase. Ocular allergies
are of the most frequent ocular disorders which ophthalmologists have
to manage in clinical practice (Leonardi et al., 2019). Allergic conjuncti-
vitis involves the seasonal and perennial allergic conjunctivitis, vernal
and atopic keratoconjunctivitis, and contact blepharoconjunctivitis
(Leonardi et al., 2012). It has been reported that allergic conjunctivitis
is often under-diagnosed and consequently under-treated (La Rosa
et al., 2013; Rathi & Murthy, 2017). The symptoms of allergic conjunc-
tivitis can decrease the quality of life, interfere with productivity, and
lead to a considerable economic burden. The optimal management
includes the allergen avoidance, symptomatic relief, and pharmacologic
suppression of inflammatory responses using topical pharmacological
agents which decreased inflammation combined with non-
pharmacologic remedies (Bilkhu, Wolffsohn, Naroo, Robertson, &
Kennedy, 2014) which provide temporary symptomatic relief (Bielory,
O'Brien, & Bielory, 2012). Although this approach is effective for most
patients with mild symptoms, none of these medications last a full
treatment. Overall, topical antihistamines and mast cell stabilizers that
seem to be safe and well tolerated are prescribed. Additionally, topical
decongestants are frequently used as a first-line treatment due to their
availability over the counter and nonsteroidal anti-inflammatory drugs
are also chosen. Nonetheless, the current ocular medications in the
form of eye drops require frequent administration and exhibit limited
duration of action (Leonardi et al., 2019).
Ocular drug delivery is one of the most fascinating but challeng-
ing administration route (Siafaka et al., 2015). Various ocular drug
delivery routes have been recommended but the topical administra-
tion is the most common and safe route. Nonetheless, the ocular
physiological barriers as nasolacrimal drainage, tear dilution, tear turn-
over of the eye are challenging and should be overcome for the
drug to reach the tissue target (Arul Kumaran, Karthika, &
Padmapreetha, 2010). Thus, the conventional eye drops present low
ocular bioavailability minimizing their therapeutic efficacy (Üstünda

g-
Okur et al., 2015). Moreover, eye drops may induce severe side
effects such as irritation (Puranik & Tagalpallewar, 2015; Rewar,

Drug Dev Res. 2020;1–12. wileyonlinelibrary.com/journal/ddr © 2020 Wiley Periodicals, Inc. 1

2 OKUR ET AL.
Bansal, & Singh, 2014). In further, it has been stated that the repeated
ocular administration decreases patient compliance. Hence, the design
of novel drug delivery systems which are able to release the drug for
prolonged time maximizing the precorneal residence time is manda-
tory (Yang, Patel, Vadlapudi, & Mitra, 2013). Aside from drops, ocular
ointments have been proposed for their great ocular bioavailability
because of their great residence time of drug and low tear production.
On the other hand, such ointments lead to undesirable blurred vision
(Baranowski, Karolewicz, Gajda, & Pluta, 2014).
To avoid the aforementioned side effects and improve the thera-
peutic efficacy the paradigm of thermosensitive drug delivery systems
has been proposed (Ranch et al., 2019; Sapino et al., 2019; Üstünda

g ride (BZC) was supplied from Sigma-Aldrich, Germany. All the used
Okur, Yozgatlı, Okur, Yoltaş, & Siafaka, 2019). The thermosensitive
drug delivery systems as the in situ gels, change their form from liquid
to solid due to temperature changes (Khan, Aqil, Ameeduzzafar,
Imam, & Ali, 2015; Ranch et al., 2019). In situ activated gel-forming
systems can be described as viscous liquids which by changing physio-
logical conditions (pH, temperature, ionic strength, and UV) will shift
to a gel phase (Venkatesh, Liladhar, Kumar, & Shivakumar, 2011). Situ
gels, which can undergo gelation due to physiological conditions
exposure such as temperature or pH, could be of the most ideal for-
mulations for various diseases (Irimia et al., 2018). A variety of syn-
thetic or natural macromolecules are applied to form in situ gels

Okur et al., 2019).
(Üstündag
Tetrahydrozoline hydrochloride (THZ) or 2-[(1RS)-1, 2, 3,
4-tetrahydronaphthalen-1-yl]-4,5-dihydro-1H-imidazole is an
adrenoceptor agonist-decongestant, used for reducing the symptoms
of allergic conjunctivitis (Mohamed, Rizk, Yousry, & Frag, 2015). THZ
is a sympathomimetic agent with alpha adrenergic activity and its
main mechanism of action is the constriction of conjunctional blood
vessels and also it acts as a local vasoconstrictor so THZ serves to
reduce the redness of the eye caused by minor ocular irritants
(Gumustas et al., 2016; Shadoul, Kariem, Ibrahim, & Adam, 2016).
Tetrahydrolozine may cause ocular burning, stinging, dryness, head-
ache, insomnia, dizziness, tremors, palpitations, rebound erythema,
and congestion (Cordes, 2007). Thus, to decrease these adverse
effects, more current and innovative formulations need to be devel-
oped. Various formulations of THZ as solutions or suspensions are
used as a conjunctival decongestant for the treatment of allergic con-
junctivitis (Ali, Ghori, & Saeed, 2002; Al-rimawi, Zareer, Rabie, &
Quod, 2012). For example, mucoadhesive drops of THZ were pre-
pared using hyaluronic acid and poly(acrylic acid) based on a ternary
interaction drug–polymer–polymer. From in vivo studies, authors con-
firmed that the mucoadhesive drops were able to maintain drugs level
for 20 min after instillation whereas the drug was not detectable after
3 min when the marketed solution was applied (Sandri et al., 2006).
The current technology trends focus on the development of new,
topically applied ocular formulations that increase the retention time.
Hence, the aim of this study is the development of a thermosensitive
drug delivery system based on the dispersion of THZ-loaded in situ
gels, to increase the residence time of drug, sustained drug release,
and reducing the dosing frequency of formulation. The characteriza-
tion included various physicochemical parameters as clarity, gelation
capacity, pH values, rheological characteristics, and drug percentage.
Furthermore, the in vitro drug release, ex vivo permeation and pene-
tration, and in vivo studies were also evaluated.
2 | M A T E R I A L S A N D M ET H O D S
2.1 | Materials
THZ was gifted from Bilim _Ilaç, Turkey. Poloxamer 407 (P407) and
188 (P188) were kindly gifted from BASF, Turkey. Benzalkonium chlo-
solvents and chemicals were of HPLC or analytical grade. The dialysis
membranes were purchased from Spectrum and presented the follow-
ing characteristics: Spectra/por 4, d = 16 mm, and Mw = 12–14 kDa.
2.1.1 | Preparation of blank and drug-loaded in
situ gels
The preparation of in situ gels was performed by the dissolution of
specific amounts of 407 as well as 188 in aqueous medium via mag-

Okur et al., 2019; Üstündag
netically stirring (Üstündag -Okur, Yoltas, &
Yozgatli, 2016). Then, the optimal in situ gels were chosen based on
their pH values and gelation temperatures. The cold method was cho-
sen for the development of the in situ gels.
The drug-loaded formulation was developed by dissolving THZ,
BZC (as a preservative [0.02%w/w]) as well as sodium chloride (for
isotonicity [0.9%w/w]) in aqueous solutions of P407 and P188.

Okur et al., 2019). The formulation code and components
(Üstündag
of ocular in situ gels are shown in Table 1.
2.2 | Physicochemical characterization of
formulations
2.2.1 | Detection of Sol–Gel temperature (Tsol–gel)
To determine the Sol–Gel Temperature of the in situ gels, 20 g of cold
sample solution directed into a beaker and placed in a temperature-
controlled stirrer. Then, a thermometer was immersed in the sample
solution for constant monitoring. The solution was heated from 25 to
60  C with a rate at 2  C/min and it was continuously stirred at
200 rpm. As Sol–Gel Temperature was reported, the temperature that
the magnetic bar stopped due to gelation (Aksu et al., 2019).
2.2.2 | Gelling capacity
The gelling capacity was confirmed by placing a drop of the in situ gels
in a beaker at 34  C. The system was observed for the gelation time
(Mandal, Prabhushankar, Thimmasetty, & Geetha, 2012; S. Patel,
Koradia, & Parikh, 2015).
OKUR ET AL. 3

TABLE 1 Components of the obtained in situ gels

Formulation code P188 (%w/w) P407 (%w/w) Tetrahydrozoline hydrochloride (g) Benzalkonium chloride Physiological saline (q.s)
I-1 5 20 — 0.02 100
I-2 10 20 — 0.02 100
I-3 15 20 — 0.02 100
TI-1 5 20 0.05 0.02 100
TI-2 10 20 0.05 0.02 100
TI-3 15 20 0.05 0.02 100

2.2.3 | Determination of pH 2.3 | FT-IR spectroscopy

A pH meter (Mettler Toledo, Switzerland) was applied for the mea-
surement of the pH of the formulations. The measurements were
recorded three times and the average value was chosen.
2.2.4 | Drug content
The determination of drug content was carried out by dissolving 0.125 ml
of the in situ gels in the 25 ml mobile phase. HPLC analysis was followed
by the estimation of the drug percentage (Mandal et al., 2012).
ATR FT-IR Spectrometry was used to examine the spectrum of poly-
mers, drug, and ideal blank and drug-loaded formulations, to show
that substances are compatible with each other. The spectrums were
analyzed at 4 cm−1 spectral resolution in the frequency range of
4.000–400 cm−1 using ATR-FTIR Spectrometer (Perkin Elmer, Spec-
trum 100 FT-IR Spectrometer). The peak position was determined
using Perkin Elmer Spectrum Version 6.0.2 Software.
2.4 | Stability of THZ-loaded in situ gels

HLPC method
The HPLC analysis was performed using an HPLC system comprised
of a gradient pump and a UV detector (Agilent 1100). The used col-
umn was a C18 column (5 μm, 150 mm × 4.6 mm). The samples analy-
sis was done at 212 nm with a 1.2 ml/min flow rate at 25  C. A
mixture of acetonitrile and phosphate buffer (35:65) (v/v) was chosen
as the mobile phase. The pH of the phase was adjusted at 2.7 with
ortho-phosphoric acid (Ali et al., 2002).
The stability of a formulation is a very important parameter. Hence, the
drug-loaded in situ gels were stored at 4 ± 1  C in the refrigerator and
25 ± 2  C (relative humidity 60%) in the stability cabinet for 3 months.
After their storage, the investigation of drug content, pH, gelling time,
and visual appearance was done. The experiments were done triplicate

Okur et al., 2017).
(Üstüdag
2.5 | Osmolality measurement

2.2.5 | Spreadability of in situ gels
The evaluation of spreadability was recorded by transferring 0.1 g of
the drug-loaded formulations to the central position of a glass plate of

temperature equal to 32 C (10 cm × 10 cm). Then, the glass plate
was compressed under another glass plate of the same size. Thus, the
in situ gel was spread out in between the plates. After 60 s, the weight
was removed and the diameter of the spread area (cm) was detected.
-Okur
The experiment was carried out three times (Üstündag
et al., 2016).
2.2.6 | Rheological studies
A Brookfield viscometer LVDV-E model with a cylindrical spindle was
used for the determination of the viscosity of the in situ gels. First,
the samples placed in the sampler tube and analyzed with 200 rpm
using probe 07 at 4 ± 0.5  C by a circulating bath connected to the
viscometer adaptor before each experiment (Mandal et al., 2012).
The osmolarity of the developed formulations was measured by a
freeze point method using an Osmometer (Knauer, Germany) at room
temperature. The instrument was calibrated with standard sodium
chloride solutions. The experiments were measured triplicate.
2.6 | Sterility studies
The sterility of the formulations was performed at Laminar air flow
Cabinet (Haier HR40-IIA2) under aseptic conditions according to the
international pharmacopeia. The fluid thioglycollate medium and soya-
bean casein digest medium were used for anaerobic and for fungi and
aerobic bacteria, respectively. First, 1 ml of the in situ gel was trans-
ferred to each medium. Then, the plates were incubated at 35  C for
bacteria and 25  C for fungi for 2 weeks. The growth promotion test of
aerobes, anaerobes, and fungi was carried out at 35  C for 2 days, using
fluid thioglycollate media which was inoculated with 100 CFU of
Staphylococcus aureus ATCC 6538, Clostridium sporogenes ATCC

Okur et al., 2019).
19404, and Candida albicans ATCC 1023 (Üstündag
4 OKUR ET AL.
2.7 | In vitro drug release studies
The in vitro release experiment was performed using simulated tear fluid
as a dissolution medium at 50 rpm (Siafaka, Titopoulou, et al., 2015;
Üstünda
g Okur et al., 2019). The used temperature was 32–34 ± 0.5  C.
More specifically, an amount of in situ gels (5 g) was separated from the
release medium and inserted in the dialysis membrane. Then, 1 ml is ret-
racted from the release medium at specific time intervals from 30 min to
24 hr. Each time, 1 ml of the fresh medium was added. The analysis for
THZ drug content is performed using HPLC.
2.7.1 | Kinetic analysis of release data
To understand the possible release mechanism of THZ, the data from
in vitro drug release studies was exposed to various mathematical models.
The best-fit model was decided based on the highest regression values (r2)
for correlation coefficients for formulations. For the evaluation and the
determination of the release mechanism, kinetic models like zero order,
first order, Higuchi, and Hixson Crowell were applied. In addition, the
Korsemeyers–Peppas equation was also used for kinetic analysis. When
n = 1, the release rate is independent of time (zero-order) (case II transport).
In the case of n = 0.5, a fickian diffusion is declared but when 0.5 < n < 1.0,
both means diffusion and nonfickian transport are involved. Finally, in the
case of n > 1.0 super case II transport is apparent ‘n’ is the slope value of
log mt/m∞ versus log time curve. When the release mechanism is mainly
based on Fickian diffusion, a dimensionless expression of the Higuchi
model can be applied (Siafaka et al., 2019; Üstünda

g-Okur et al., 2015).
2.8 | Ex vivo permeation and penetration
experiments
The corneal permeation and penetration studies were carried out using
vertical Franz diffusion cells (Logan Instruments) and excised goat cor-
neas. Sink conditions were maintained in the receptor compartment.
The corneas were clamped between the donor and the receptor cham-
ber of cells. In further, a specific volume of the formulations (0.5 ml) and
simulated tear fluid (10 ml) was filled into the donor and receptor cham-
bers, respectively. The temperature of the cells was adjusted at 32
± 2  C with stirring. The samples were collected from 30 min to 24 hr.
After 24 hr, for the penetration experiment, each cornea was washed
and placed in 10 ml of methanol for 1 day in a mixer to determine the
penetration amount of THZ. The amount of penetrated and permeated
drug was analyzed via HPLC. The study was performed in triplicate.
2.9 | In vivo experimental studies
2.9.1 | Animals
In vivo experiments were performed according to the Association for
Research in Vision and Ophthalmology resolution for the use of
animals in research and the protocol was approved by the Local Ethics
Committee. For the in vivo experiments, New Zealand albino rabbits
of weight from 2.5 to 3.5 kg and no signs of ocular inflammatory were
chosen. Moreover, rabbits were housed in a room of 22 ± 1  C tem-
perature and humidity in air-conditioned chambers with an alternating
12 hr light–dark cycle. The rabbits had free access to a pellet diet and
water ad libitum. During the in vivo experiments, the animals were
placed in boxes, and their heads and eyes movements allowed.
2.9.2 | Detection of the THZ in rabbit tear
For the study, six rabbits were applied which during the experiment
could move their heads freely, and their eye movements were not
restricted. Schirmer Tear Test Strip (ERC®) was used for obtaining the
tears. For the study, 50 μL of sterilized THZ-loaded in situ gel (TI-3)
was administered for one time in the lower conjunctival sac of the
right eye. The exact volume of the marketed product (VISINE®) was
administered to the lower conjunctival sac of the left eye. After the
instillations, the rabbit's tears were collected at 0.5, 1, 2, 3, 4, 6, 8, and
24 hr using tear strip. The tear strip placed in the conjunctival sac and
the marked tear strip was reached to a certain level. Then, the strips
were immediately placed in Eppendorf tubes with 2 ml methanol and
stored at +4  C up to analyze. The HPLC analysis was chosen for the
determinations of THZ amount on tears (Byrro et al., 2012; Qi
et al., 2007).
2.9.3 | Ocular irritation test
The modified Draize test was performed to evaluate the possible ocu-
lar irritancy and/or damaging effects of THZ formulations. An amount
of 0.05 ml of in situ gels were administrated into the right eye of the
cornea. As a control, the left eye served, which was treated with
sodium chloride (0.9%). Afterward, the congestion, swelling, discharge,
and redness of the rabbits' eyes were examined for 6 hr after the
instillation and graded on a scale from 0 to 3, 0 to 4, 0 to 3, and 0 to
3, respectively. The irritation and corneal opacity were graded on a
scale from 0 to 4 (Chhonker et al., 2015).
2.10 | Statistical data analysis
The analysis of statistical data was carried out via the student's t-test
with p < .05 as the minimal level of significance.
3 | RESULTS AND DISCUSSION
3.1 | Preparation of ocular in situ gels
In this study, the cold method was applied for the preparation of the
thermosensitive in situ gels of THZ. Besides, there are also other
OKUR ET AL. 5

methods as the hot process which, however, leads to undesirable
physiochemical effects as the formation of polymeric lumps (Swain,
Patel, Gandhi, & Shah, 2019). There are various polymers which are
used for the preparation of the in situ gels, either natural or synthetic

Okur et al., 2019).
(Güven, Berkman, Şenel, & Yazan, 2019; Üstündag
Poloxamers are triblock thermoresponsive copolymers frequently
applied in pharmaceutical technology and ocular delivery. Herein, the
P407 and P188 were selected due to their great aqueous solubility,
clarity, safety, nonirritability, and concentration-dependent viscosity
(Soliman, Ullah, Shah, Jones, & Singh, 2019).
The prepared formulations were coded as TI-1-3. The drug was
loaded in 0.05% (w/w), since the currently marketed eye drops
entrapped the same drug amount. Moreover, BZC was added as a pre-
servative due to its amphipathic nature, high water solubility, and
superior antimicrobial effects (Üstünda

g Okur et al., 2019).
3.2 | Physicochemical characterization of ocular
formulations
the liquid formulations can be altered into gels with preferable viscos-
ity at 32–34 ± 2  C. Besides, the spreadability was in similar values
for the studied in situ gels.
One of the most significant parameters is the pH of the ocular
formulations. The pH of the eye should be maintained at normal levels
(4–8), since the alteration from acidic to alkali pH can lead to an eye
injury (Lim, Ah-Kee, & Collins, 2014). Thus, the newly prepared ocular
formulations should not change the neutral ocular pH (Baranowski
et al., 2014). The recorded pH of the in situ gels ranged between 6.87
and 7.010, which are among the acceptable limits. From this fact, it
can also be assumed that the in situ gels will not present irritancy and
-Okur et al., 2014). Simi-
immediate production of tear fluid (Üstündag
larly, Güven et al. demonstrated that ocular in situ gels based on
poloxamers and loaded with olopatadine showed that the pH of the
formulations was between 6.5 and 7 (Güven et al., 2019). In our previ-
ous study, which the in situ gels incorporated voriconazole, the pH

Okur et al., 2019).
results were also in the same limits (Üstündag
Finally, as it was expected the drug loading ranged between 94.76
and 96.45% which is desirable.

Characterization of ocular formulations is very significant since the

physicochemical properties can affect the stability and the perfor-
mance of the ocular formulations. Table 2 summarized the crucial fac-
tors of clarity, pH, the gelation time and temperature as well as
viscosity and spreadability. The ease of administration in case of a
highly viscous solution and gel forms retard its use and patient compli-
ance. The blurred vision and the lachrymation are associated with the
dosage form involving hydrogel (Destruel et al., 2020). The clarity of
the developed in situ gels followed by visual observation in the black
and white background was satisfactory and in the desirable limits.

Okur
Similar results were observed in our previous studies (Üstündag
et al., 2019; Üstündag-Okur et al., 2016). Their eye surface tempera-
ture has been measured at around 32  C which varies upon various
eye diseases (Velez-Montoya, Sniegowski, Erlanger, & Olson, 2015).
Thus, the gelation temperature of ocular in situ gels should be under

or near the 32 C. Herein, the in situ gels temperature was in the
range of 31.08 ± 0.34 to 33.53 ± 0.69, which is acceptable. In further,
the gelation capacity results demonstrated that the in situ gels could
immediately gelate in 2 s. A past study, which evaluated the use of in
situ gels against conjunctivitis showed that the gelation capacity is
affected by the polymer concentrations (Patil, Kadam, Bandgar, &
Patil, 2015). The viscosity experiment of the formulations showed that
3.3 | FTIR spectroscopy
In general, the possible interactions might occur between polymers
and drugs molecules can lead to undesirable effects as instability or
incompatibility of the formulations (Jayaraj et al., 2010; Siafaka

Okur et al., 2019).
et al., 2019; Siafaka, Okur, et al., 2019; Üstündag
The study of such interactions can be performed via FT-IR spectros-
copy which is a widely useful technique in pharmaceutical technology
(Siafaka et al., 2016; Siafaka, Barmbalexis, & Bikiaris, 2016a). First of
all, the polymeric components of the in situ gels P407 and P188 were
studied. The IR spectra of the poloxamers included the absorption
bands at 3400 cm−1 characteristic of the hydroxyl groups. In further,
at 2,889 and 2,891 cm−1, the C-H stretch of aliphatic groups is
recorded. At 1,349 and 1,340 cm−1, it is recorded the in-plane O-H
bend, while at 1108 cm−1, the C-O stretch is depicted (Figure 1a)

Okur
(Garala, Joshi, Shah, & Ramkishan, & Patel, 2013; Üstündag
et al., 2019).
The pure in situ gels (I1-3) demonstrate the presence of water
(Innocenzi, Malfatti, Carboni, & Takahashi, 2015) and BZC due to the
exhibition of the following bands at 2120 and 1642 (alkenes) cm−1,
respectively (Figure 1b). The remaining bands are similar to that of

TABLE 2 Physicochemical properties of ocular blank and tetrahydrozoline hydrochloride-loaded formulations

Formulation code Gelling temperature ( C) pH Clarity Gelation capacity (s) Spreadability (cm) Viscosity (cP) Drug content (%)
I-1 31.2 ± 0.1 7.375 ± 0.009 +++ 2.1 ± 0.1 1.575 ± 0.035 290.16 ± 1.3 —
I-2 33.867 ± 0.746 7.410 ± 0.003 +++ 2.0 ± 0.1 1.675 ± 0.035 360.98 ± 3.2 —
I-3 31.133 ± 1.002 7.630 ± 0.015 +++ 2.0 ± 0.1 1.525 ± 0.035 500.56 ± 3.5 —
TI-1 31.1 ± 0.53 6.873 ± 0.021 +++ 2.0 ± 0.1 3.100 ± 0.173 289.2 ± 1.8 94.76 ± 2.2
TI-2 33.53 ± 0.69 6.977 ± 0.045 +++ 1.8 ± 0.1 2.700 ± 0.265 351.7 ± 4.2 96.45 ± 3.397
TI-3 31.08 ± 0.34 7.010 ± 0.017 +++ 1.9 ± 0.1 2.167 ± 0.231 504.2 ± 3.5 95.78 ± 1.604
6 OKUR ET AL.
F I G U R E 1 FTIR spectroscopy results of poloxamers (a), blank in
situ gels (I-1, I-2, and I-3) (b), and THZ and THZ-loaded in situ gels
(TI-1, TI-2, and TI-3) (c). THZ, tetrahydrozoline hydrochloride
pure poloxamers, with negligible changes. Besides, it has been
reported that the blending of polymers can lead to miscible or immis-
cible blends (Siafaka et al., 2015); in this work, however, the FTIR
spectrum of the formulations did not show any strong shifting of the
peaks demonstrating the compatibility of the polymers and the possi-
ble stability of the formulations. THZ is an imidazole derivative; its
FTIR spectrum exhibits the characteristic bands of C-H stretching at
3000–2866 cm−1. At 1591 cm−1, it is recorded the C-C stretching,
whereas at the area between 1,400 and 1,300, the C-N stretching
−1
bands are found. The FTIR band appeared at 3360 cm can be
assigned to N-H stretching modes of vibrations. Furthermore, the
THZ-loaded in situ gels (Figure 1c) spectra did not reveal any
alterations, as it was expected. In fact, throughout literature in situ
gels prepared from these bands do not show any new peaks or strong
shiftings following the addition of the active ingredient (Makwana,

Okur et al., 2019). Accordingly, drugs
Patel, & Parmar, 2016; Üstündag
and excipients are compatible. In this work, the absence of newly


recorded peaks demonstrates the compatibility of the gels (Üstündag
Okur et al., 2018, 2019).
3.4 | Stability
From the stability results, it resulted that the ocular in situ gels were
stable for more than 3 months. It was not depicted any aggregation or
precipitation was and the THZ in situ gels present the same clarity
and viscosity as well as pH values with the initial values. Similar results
have been recorded for various in situ gels (Aksu et al., 2019;

Okur et al., 2019). Table 3
Gonzalez-Pizarro et al., 2019; Üstündag
summarizes the obtained data of the stability study.
3.5 | Osmolality measurements
The formulation should also be isotonic to avoid irritation upon
administration. Isotonicity is an important characteristic of the oph-
thalmic formulation. Isotonicity needs to be maintained to prevent tis-
sue damage or irritation of the eye (Destruel et al., 2020). The three
formulations exhibited similar osmolalities. The osmolarity of the
THZ-loaded in situ gels TI-1, TI-2, and TI-3 were found as 295.04
± 5.14, 296.45 ± 3.24, and 296.82 ± 6.47 mOsm, respectively. Serum
physiologic (0.9% NaCl solution) osmolarity was found as 297.02
± 2.63 mOsm. Formulations with osmolarity values between 150 and
320 mOsm/L do not produce ocular discomfort. Based on previous
findings, ocular solution osmolality lower than 100 mOsm/kg and
higher than 640 mOsm/kg may cause ocular irritation (Mahboobian,
Mohammadi, & Mansouri, 2020). Normal human eyes had an average
tear osmolarity values of approximately 302+/−8 mOsm/L (Potvin,
Makari, & Rapuano, 2015). Therefore, developed formulations are
appropriate for ocular applications.
3.6 | Sterility studies
Ophthalmic solutions except stable should also be sterile to avoid
new contamination of the eye. From the sterility studies, the in situ
gels did not show any turbidity in media after 14 days of incubation.
More specifically, it was not evidenced any microbial growth in fluid
thioglycollate medium and at 20–25  C in the case of soya-bean
casein digest medium. Furthermore, to control the used media for
the suitability of the sterility test, the growth promotion test was
done, and it was found that both microorganisms showed visible
growth in all media. And results show that all formulations are
found sterile. Patel et al. also derived similar sterility results
(N. Patel et al., 2016).
OKUR ET AL. 7

TABLE 3 Stability results of in situ gels after stored for 3 months

Formulation code Gelling temperature ( C) pH Clarity Gelling capacity (s) Spreadability (cm) Viscosity (cP) Drug content (%)
TI-1 31.5 ± 0.64 6.98 ± 0.5 +++ 2.0 ± 0.1 2.9 ± 0.2 309.605 ± 1.5 95.5 ± 0.4
TI-2 32.87 ± 0.23 6.76 ± 0.2 +++ 1.9 ± 0.1 2.8 ± 0.9 342.670 ± 1.5 96.23 ± 2.2
TI-3 31.4 ± 0.56 7.1 ± 0.7 +++ 1.9 ± 0.1 2.2 ± 0.3 490.85 ± 3.6 94.87 ± 0.9

3.7 | In vitro drug release studies 3.7.1 | Kinetic study for drug release

Figure 2 depicts the in vitro drug release studies of THZ, which were
carried out in simulated tear fluid. THZ is freely soluble in water,
which increases the risk of its clearance from the eye. In addition, this
clearance will lead to decreased therapeutic efficacy, since the drug
cannot obtain the maximum therapeutic levels. Hence, THZ release
should be sustained and prolonged. As it can be seen from Figure 2,
the prepared in situ gel TI-1-3 depicted prolonged release in simulated
tear fluid for almost 24 hr. Nonetheless, a small burst effect can be
seen since almost 20% of the drug is released within 30 min. More-
over, at the end of the day, the THZ was released from TI-1, TI-2, and
TI-3 formulations at high levels 97.017 ± 4.803%, 93.866 ± 6.855%,
and 88.745 ± 8.275%, respectively. Another study, which evaluated
the encapsulation of the water-soluble drug pilocarpine in ocular in
situ gels, exhibited that the water soluble drugs present a high burst
effect and fast release as solutions (Lee & Jeong, 2008). However,
when pilocarpine was formulated to in situ gels depicted a sustained
release rate (Miyazaki et al., 2001) Hence, in this study, it is remark-
able the prolonged release of the THZ.
Considering that the TI-3 showed a slower release rate and great
physicochemical properties, it was chosen for further examination
(ex vivo and in vivo performance).
The analysis and modeling of drug release is a rather complex phe-
nomenon, especially when polymers are involved for the develop-
ment of the formulations (Siafaka, Titopoulou, et al., 2015).
Normally, the drug release of gelling systems generated by swelling
polymers such as chitosan or poloxamers is driven by diffusion or
erosion controlled mechanism is noted, whereas the majority of the
systems exhibits a mechanism combination (Gu & Alexandridis,
2006; Siafaka, Zisi, Exindari, Karantas, & Bikiaris, 2016c). Herein, the
kinetics and mechanism of THZ release from the gels were evaluated
by using the zero-order, first-order, Higuchi, Hixson–Crowell, and
Korsmeyer–Peppas models. Generally, the zero-order model distin-
guishes drug delivery systems that do not corrode, and the active
molecule is released in a slow manner independent of the initial drug
concentration. On the other hand, the first-order model best charac-
terizes a system where the release is dependent on the initial drug
concentration. Furthermore, Higuchi's kinetic model is applied for
the characterization of the neat diffusion release mechanism of the
drug from the matrix, with no erosion or swelling. The Hixson–
Crowell cube root law characterizes the release from drug delivery
systems where surface area and diameter of particles or tablets are
altered (Gouda, Baishya, & Qing, 2017). The Korsmeyer–Peppas

FIGURE 2 In vitro release profile of THZ from the ocular formulations (TI-1, TI-2, and TI-3). THZ, tetrahydrozoline hydrochloride
8 OKUR ET AL.

TABLE 4 Release kinetics of tetrahydrozoline hydrochloride-loaded in situ gels by fitting to various mechanism models (n = 3)

Zero order First order Higuchi Hixson–Crowell Korsmeyer–Peppas

Models
Formulation r 2
n m r 2
n m r 2
n m r 2
n m r2 n
TI-1 0.959 12.216 21.728 0.990 4.842 −0.675 0.986 −24.397 60.002 0.993 −0.063 0.688 0.996 0.846
TI-2 0.979 6.96 21.48 0.987 4.822 −0.56 0.993 −28.622 58.869 0.997 −0.11 0.612 0.97 1.18
TI-3 0.985 8.056 16.082 0.986 4.73 −0.376 0.998 −22.825 47.809 0.995 −0.037 0.429 0.968 1.05

model can be used as a decision parameter between the Higuchi and Korsmeyer–Peppas power law equation states the type of diffusion,
zero-order models (Gouda et al., 2017). which was evaluated by value, n (release exponent) which is higher
The suitability of each model to describe the release kinetic of than 0.89 for TI-2 and TI-3, which implies that the drug release from
active ingredients from drug delivery systems was based on R2 calcula- the system follow Super case II transport.
tion for the formulations. The fitting of the in vitro release data into var-
ious kinetic models as listed in Table 4. The release data were fitted to
the Hixson–Crowell followed by first-order and Higuchi models for TI-1 3.8 | Ex vivo permeation and penetration studies
and TI-2 in situ gels, respectively. The release of TI-3 followed Higuchi using goat cornea
kinetics, which indicates the role of diffusion in the release from the in
situ gel-forming system. In this kinetic model, the release process is Ex vivo studies were used to predict further the permeation and pene-
dependent on formulation characteristics. Jabarian et al. prepared and tration of THZ in real time. In this work, goat cornea and simulated
kinetically evaluated octreotide-loaded in situ gels. They found that the
release of PEGylated octreotide fitted to the Higuchi kinetics (Erfani
Jabarian et al., 2013). Ricci et al. developed lidocaine gels by using
Poloxamer 407. It was revealed that Higuchi model was fitted to drug-
loaded gel. They found that the drug release from P407 gels is con-
trolled by gel dissolution in the acceptor solution and by drug diffusion
from the gel matrix (Ricci, Lunardi, Nanclares, & Marchetti, 2005).
In vitro release kinetics of THZ in situ gels were also evaluated
according to the Korsmeyer–Peppas model. It can be said that due to
the complexity of the gels, it is difficult to distinguish which model is
fitted the best since most of the values are quite similar. In addition, it
can be assumed that the release mechanism is driven by two steps:
(a) a slight burst effect due to polymer relaxation and (b) drug diffusion
phenomena. These results are similar to previously reported studies
involving gels (Cojocaru et al., 2015; Inal & Yapar, 2013). The model

F I G U R E 3 The obtained THZ concentrations from the THZ- F I G U R E 4 The obtained data of at 0, 1, 3, and 6 hr after the
loaded in situ gel (TI-3) and commercial product of THZ. THZ, instillation of TI-3 and commercial product of THZ. THZ,
tetrahydrozoline hydrochloride tetrahydrozoline hydrochloride
OKUR ET AL.
tear fluid of pH 7.4 were used. The optimal TI-3 in situ gel was
selected as the studied formulation. It can be revealed that the
amount of THZ permeated through the cornea was detected as
2.035 ± 0.062%. Also, the penetration of THZ to the corneal tissue
was detected as 0.582 ± 0.035%. The results are rational given that
the used drug is allergic, and the important factor is to increase the
residence time of the drug on the eye.
3.9 | In vivo studies
3.9.1 | Detection of the THZ in tear
The first studied parameter from in vivo studies was the observation
of THZ concentration in tear, which is observed in Figure 3. After the
administration of 50 μL TI-3 and the marketed product of THZ, the
tear samples were withdrawn using the Schirmer tear strip. The
amount of THZ in tear samples taken from the rabbit was successfully
measured using the validated HPLC method.
At the beginning of the experiment, the found concentration
of THZ was 9.47 and 3.4 μg/ml for TI-3 and product, respectively.
By the passage of time, the detected concentration was lowering
but the levels of drug found in TI-3 are higher that the detected
levels of the marketed product. At the end of the experiment
(360 min), no drug can be detected for the commercial product.
Nonetheless, for TI-3 almost 1.029 μg/ml can be recorded. This is
very significant since in most cases the drug from ocular formula-
tions cannot be detected after 6 hr (Üstünda

g Okur et al., 2019).
Consequently, the in situ gel can keep the drug amount stable for
hours.
3.9.2 | Ocular irritation test
Figure 4 demonstrates that the obtained data of ocular irritation
experiments follow 6 hr instillation. Herein, the Draize Rabbit Eye
Test was applied which is an easily handled and cost-effective
method (Wilson, Ahearne, & Hopkinson, 2015). The ocular irritation
test using New Zealand white rabbits is very important to predict
the possible irritation of newly formed ocular products on humans.
It can be concluded that it was not depicted any ocular damage or
clinically abnormal signs in the cornea, conjunctiva, or iris followed
by the eye instillation of TI-3 and THZ solution (commercial prod-
uct of THZ). Zero and occasionally one grade was demonstrated
while between control and treated eyes no difference was
depicted. In addition, redness must be controlled for in eyes that
receive formulations. From application to final tear sampling there
was no redness or irritation in the eyes of the rabbit used in this
experiment. These results were expected since the materials used
herein are not toxic as well as previous studies demonstrated that
in situ gels comprised from poloxamers did not show any sign of

Okur
irritation (N. Patel et al., 2016; Ranch et al., 2019; Üstündag
et al., 2019).
9
4 | CONC LU SION
Allergic conjunctivitis is an ocular superficial disorder where its effi-
cient management depends on the presence of the active substance
to the eye surface for an extended period. The conventional ocular
drops used for the allergic conjunctivitis present various disadvan-
tages. Consequently, the ocular application of gelling systems could
be advantageous for the disease management. Tetrahydrozoline is a
sympathomimetic agent with alpha adrenergic activity applied for
allergic conjunctivitis. The prepared thermosensitive in situ gels were
able to improve and prolong the release of tetrahydrozoline drug. This
fact will further lead to maximization of its efficacy. Furthermore, the
physicochemical characterization exhibits that the obtained in situ
gels were stable for 3 months with great gelation capacity and time.
The osmolality of formulations was close to that of the lacrimal fluid.
ex vivo permeation studies showed desirable permeation and penetra-
tion, whereas in vivo studies demonstrated that the drug can be
detected in the rabbits' tears even after 6 hr. In further, it was not
observed any ocular damage or irritation signs in the rabbits' eye after
the administration of the optimal carriers. To conclude, the developed
thermosensitive in situ gels can act as promising formulations for the
entrapment of tetrahydrozoline drug which can lead to a great thera-
peutic potentional on allergic conjunctivitis.
ACKNOWLEDG MENTS
The authors wish to thank the Bilim _Ilaç, Istanbul, Turkey. The authors
are grateful to Dr. Ayşegül Yoltaş for sterility studies.
CONFLIC T OF INT ER E ST
The authors declare no conflicts of interest.
OR CID
Neslihan Üstünda

g Okur https://orcid.org/0000-0002-3210-3747
RE FE RE NCE S
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