pH INDUCED IN-SITU GEL FORMULATION OF
LOMEFLOXACIN HCL
S. J. SHANKAR*, ANUDEEP KALIKONDA
Department of Pharmaceutical Technology, PES College of Pharmacy
*PES College of Pharmacy,
Department of Pharmaceutical Technology,
50 feet road, Hanumantha nagar, BSK Ist stage,
Bangalore-560050, karnataka, India.
e-mail: shankarsj@rediffmail.com
ABSTRACT
The effective dose administered ophthalmically may be altered by increasing the
retention time of medication into the eye by using an in situ gel forming system. The
aim of this work was to prepare and evaluate an ophthalmic delivery system of an
antibacterial drug Lomefloxacin HCl based on the concept of pH induced in situ
gellation. Carbopol 940 was used as the gelling agent in combination with HPMC
E50LV and HPMC E4M as viscosity enhancing agent and formulations were
evaluated for in vitro gelling capacity, rheological study and percentage drug release.
The in vitro gelling capacity was carried out using simulated tear fluid showing a
higher gelling capacity for Carbopol 940. All the formulations were found to exhibit
pseudo plastic behaviour for both the solution and gel. The developed formulations
provide sustained release of drug from formulation at the site of action over 8 hrs.
Drug release was followed by zero-order kinetic with increased time of residence of
the formulation in precornea. The optimized formulations were tested in albino rabbits
(male) using the draize test protocol with cross over studies and found to be nonirritant to the rabbit eye. The selected formulations also subjected to stability studies,
which show that no changes observed in pH, drug content and clarity during 6 weeks
of studies. Thus, the developed system may be valuable alternative to the
conventional system.
KEY WORDS
pH induced insitu gel, Lomefloxacin HCl, Carbopol 940, HPMC E50LV,
HPMC E4M
INTRODUCTION
Ophthalmic drug delivery is one of the most interesting and challenging endeavors
facing the pharmaceutical scientist. The anatomy, physiology, and biochemistry of the
eye render this organ exquisitely impervious to foreign substances. The challenge to
the formulator is to circumvent the protective barriers of the eye without causing
permanent tissue damage. The development of newer, more sensitive diagnostic
techniques and therapeutic agents render urgency to the development of more
successful ocular delivery system.1Drugs may be delivered to treat the precorneal
region for such infections as conjuctivitis, and blepharitis, or to provide intra ocular
treatment via the cornea for diseases such as glaucoma and uveitis.2 The eye drops is
easy to instil but suffers from the inherent drawback that the majority of the
medication is immediately diluted in the tear as soon as the eye drop is instilled into
the cul-de-sac and is rapidly drained away from the precorneal cavity by constant tear
flow. The higher the drug concentration in the eye drop solution, the greater the
amount of drug lost through lacrimal-nasal drainage system. Subsequent absorption of
this drained drug, if it is high enough, may result in undesirable systemic side effects.3
It is widely accepted that increasing the viscosity of a drug formulation in the
precorneal region leads to an increased bioavailability, due to slower drainage from
the cornea, several concepts for the in-situ gelling systems have been investigated.
These systems can be triggered by pH, temperature and ion activation.6
In the present work, an attempt has been made to formulate pH-induced in-situ gel as
an ophthalmic drug delivery system. In situ gels are made from polymers that exhibit
phase transition due to physicochemical change in the environment. They can be
conveniently dropped as a solution into the conjuctival sac in the eye. Upon contact
with the lacrimal fluid, the polymer changes its conformation to form a gel. This
delivery system has the ease of administration similar to an opthalmic solution and
has a long retention time because of the gel formation. Different polymers for
preparing pH induced in-situ gels have been evaluated.
MATERIALS AND METHODS
Lomefloxacin HCl (IPCA Limited, Ratlam, India), HPMC E50LV &HPMC E4M
(Color cone Asia Ltd., Verna, India), Carbopol 940 and all other excipients used are
from S.D. Fine Chemicals Ltd., Mumbai and were HPLC grade.
Preparation of pH Induced In-Situ Gelling System:
The pH sensitive polymers like Carbomer derivatives, Carbopol, forms aqueous
solution in the unionized form. These polymers undergo reversible ionization at pH
7.4, physiological condition, to form a stiff gel network, which swells and forms large
aqueous pores. Hence, Carbopol 940 was selected for preparation of pH induced insitu gelling system.
Preparation of Gelling System:
The buffer salts were dissolved in 50 ml of purified water; HPMC (E50LV / E4M)
was added to hydrate. Carbopol 940 was sprinkled over this solution and allowed to
hydrate overnight. The solution was stirred with an overhead stirrer. Lomefloxacin
Hydrochloride was dissolved in small quantity of water, Benzalkonium Chloride
(Preservative) and Sodium chloride (Isotonocity adjusting agent) was added to this
solution; the drug solution was added to the polymer solution under constant stirring
until a uniform solution was obtained. Purified water was then added to make up the
volume to 100ml. Formulation pH was adjusted to pH 5 with the help of 0.5 M
Sodium Hydroxide. This solution was filtered through 0.2m filter paper. The
optimized formulations were sterilized in an autoclave at 121º C and 15 psi for 15
minutes.
Evaluation of Prepared Gelling System:
Drug Content Analysis:
Drug content analysis of prepared in-situ gelling systems was carried out using
Spectrophotometric method. The assay of these formulations was carried out by
pipetting 0.1 ml of all four optimized formulations, and it was diluted up to 100 ml of
Simulated Tear Fluid (pH 7.4). The absorbance was measured at 281.5 nm using UVVisible spectrophotometer.
In-Vitro Gellation:
The Gelling capacity of the formulations containing different ratio of Carbopol 940
and HPMC (E50LV / E4M) was evaluated. It was performed by placing a drop of
polymeric solution in vials containing 1 ml of Simulated Tear Fluid, freshly prepared
and equilibrated at 34C, and visually assessed the gel formed and time for gellation
as well as time taken for the gel formed to dissolve.
Sterility Testing:
Sterility testing were intended for detecting the presence of viable form of
microorganisms and were performed for aerobic and anaerobic bacteria and fungi by
using Fluid Thioglycolate Medium and Soyabean Casein Digest medium, respectively
as per the Indian Pharmacopoeia.
Rheological Studies:
Rheological properties of the prepared gelling systems under the different Shear rates
(2, 4, 6, 10, 20, and 30 rpm) were measured at non-physiological (pH 5 and 25C) and
physiological condition (pH 7.4 and 34C), respectively. The hierarchy of the shear
rate was reversed, and the average of two reading was used to calculate the viscosity.
In-Vitro Release Studies:
In-vitro drug release from the formulations was studied by the diffusion process. Here
the pH of the Lacrimal fluid and the blinking rate of the eye were taken into
consideration and were simulated. The in-vitro release of Lomefloxacin HCl was
studied through cellophane membrane using diffusion cell. The cellophane membrane
was soaked overnight in the receptor medium (Simulated Tear Fluid, pH 7.4). It was
tied to one end of a glass diffusion cell. The diffusion cell was filled with 2 ml of the
formulation and suspended in 100 ml of receptor containing beaker by assuring that
the membrane was just touched the receptor medium surface.The whole assembly was
transferred on magnetic stirrer and was maintained at 34C  1C and 22 rpm. The
drug samples (1ml) were withdrawn at the interval of 30 minutes from receptor
medium and replaced by equal volumes of the receptor medium. The samples were
diluted with appropriate receptor medium and analyzed by a UV-Visible
spectrophotometer at 281.5 nm using receptor medium as a blank. Comparative
evaluations of prepared formulations were compared with that of marketed
formulations.
Pharmacokinetic Release Studies:
All the optimized formulations were subjected to study the release kinetics of it. So
the best fit kinetic model was determined for the optimized formulations using
analysis software.
Antimicrobial Efficacy Studies:
The antimicrobial efficacy studies were carried out to ascertain the biological activity
of the optimized formulations. Staphylococcus aureus, Pseudomonas aeruginosa and
E.coli were used as the test organisms. These were determined by Agar diffusion test
employing Cup-Plate method.
Ocular Irritancy Studies:
It is measured according to Draize test.According to this test, the amount of the test
substance applied to the eye is normally 100 l placed into the lower cul-de-sac with
observation of the various criteria made at a designated required time interval of 1hr,
24 hrs, 48 hrs, 72 hrs, and 1 week after administration.A total four albino rabbits
(male) weighing 1.5-2 kg was used for the present study. The sterile formulations
were instilled twice a day for a period of 7 days. Rabbits were observed periodically
for redness, swelling, watering of the eye. The evaluation was made according to the
Draize test protocol.
Accelerated Stabilities Studies:
Stability studies were carried out on optimized formulations according to International
Conference on Harmonization (ICH) guidelines.A sufficient quantity of formulations
in previously sterilized vials was stored in desiccators containing a saturated solution
of sodium chloride, which gives a relative humidity of 75±5 %. The desiccators were
placed in a hot air oven maintained at a temperature 40C0.5C and at room
temperature. Samples were withdrawn at 7, 14, 21 and 30days followed by 30 days
till 90 days. The logarithms of percent drug remaining were calculated and plotted
against time in days.
RESULTS AND DISCUSSION
Preparation of pH Induced In-Situ Gelling System:
The prepared formulations were evaluated for their viscosity using a Rheometer.
3.2. Evaluation of Prepared In-Situ Gelling Systems:
Evaluation of Visual appearance, Clarity, pH, and Drug Content:
All the prepared in-situ gelling systems were evaluated for preliminary steps such as
Visual appearance, Clarity, pH, and Drug content. These formulations were
transparent and clear. The pH of the formulations were found to be 50.5, and drug
content was in between 99.38 % to 99.71% shown in table 2.
Interaction Studies:
The result of these studies reveals that there were no definite changes obtained in the
bands of drug with respect to pure drug. Shown in Figures 1 & 2
Sterility Testing:
All the prepared in-situ gelling systems were evaluated for the sterility. After 7 days
of incubation the results showed that the no microbial growth was found in all
formulations.
Rheological Studies:
Results obtained reveals that the viscosity of all formulations decreased as the shear
rate increased, which showed the character of pseudoplastic fluid. The results were
shown in table 3.
In-Vitro Release Studies:
Results (shown in table 4) reveal that all formulations exhibited sustained release of
the drug (above 88 %) from the Carbopol 940 and HPMC E50LV /E4M network over
8-hours. Cellulose derivatives like, HPMC E50LV / E4M dissolve in water and yield
much more viscous solution compared to Carbopol 940 solution. Thus, the increase in
viscosity might have contributed to the decrease in rate of drug release from these
formulations.
Pharmacokinetic Release Studies:
The best fit kinetic model for the optimized formulation were the zero order and
peppas model which suggest that the drug release was independent of the
concentration and occurred by diffusion mechanism. The polymer can absorb a
significant amount of water to form to elastic gel and at the same time, release the
dissolved entrapped drug by diffusion through swollen regions of the gel. With a
hydrophilic Carbopol-HPMC gel, tear fluid would except to diffuse into the gel
interior and leach out water soluble drug such as Lomefloxacin HCl.
Antimicrobial Efficacy Studies:
Clear zones of inhibition were obtained in the case of all formulations. The diameter
of zone of inhibition produced by formulations against all test microorganisms are
calculated. The antimicrobial effect of the Lomefloxacin in prepared in-situ gelling
systems is probably due to a fairly rapid initial release of the drug into the viscous
solution formed by dissolution of gel, followed by formation of a drug reservoir that
permits the drug to be released to the target site relatively slowly.
Ocular Irritancy Studies:
All the formulations were found to be non-irritating with no ocular damage or
abnormal clinical signs to the cornea, iris, and conjuctiva observed.
Accelerated Stabilities Studies:
All the Formulations were analyzed for Visual appearance, Clarity, pH and Drug
Remaining. 90 days studies reveal that there were no changes observed in Visual
changes and Clarity. All the formulations showed slight changes in pH, but it were in
acceptable limits ( 0.5). Study of drug content remaining in all formulations reveals
that there were no definite changes observed for drug
CONCLUSION
The developed formulations are a viable alternative to conventional Lomefloxacin
HCl eye drop by virtue of its abilities that it cannot only be readily administered and
decreases the frequency of administration, thus resulting in better patient acceptance,
but also prolong the precorneal residence time to get higher bioavailability and reduce
the systemic side effects caused by the drainage from the nasolacrimal duct.
Hence, “pH Induced In-Situ Gelling System of an Anti-infective drug (Lomefloxacin
HCl) for Sustained Ocular Delivery” was successful experiment.
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Table 1: Formulation of in-situ gels:
S. No.
Ingredients
1
2
3
4
5
6
7
Lomefloxacin HCl
Carbopol 940
HPMC E50LV
HPMC E4M
Sodium Chloride
Benzalkonium Chloride
Acetate Buffer (pH 5) up
to
Concentrations (% w/v)
CHL 2
CHL 3
0.3
0.3
0.4
0.2
1.5

0.4

0.9
0.9
0.01
0.01
100
100
CHL 1
0.3
0.3
1.5

0.9
0.01
100
CHL 4
0.3
0.3

0.4
0.9
0.01
100
Table 2: Preliminary evaluation of Visual appearance, Clarity, pH, and Drug Content.
Evaluation steps
Visual Appearance
Clarity
pH
Drug Content
CHL 1
Transparent
Clear
4.99
99.71 %
CHL 2
Transparent
Clear
4.97
99.38 %
CHL 3
Transparent
Clear
5.01
99.43 %
CHL 4
Transparent
Clear
4.98
99.67 %
Table 3: Rheological studies of Formulations
Shear
Rate
(rpm)
2
4
6
10
20
30
Viscosity (cps) of Formulations at Viscosity (cps) of Formulationsat
Non physiological Condition (pH 5) Physiological Condition (pH 7.4)
CHL 1
CHL 2
CHL 3
CHL 4
CHL 1
CHL 2
CHL 3
CHL 4
102.3
90.1
78.2
60.0
35.2
26.9
109.4
92.3
80.4
56.1
40.3
22.9
98.4
86.7
71.5
53.9
45.3
27.1
101.7
89.2
74.3
58.1
50.7
22.8
221.0
132.5
113.5
105.8
87.9
72.6
234
125.1
103
92.3
83.9
63.8
242.1
133.9
101.3
88.1
70.9
51.3
254.5
156
109.4
85.6
68.2
57.5
Table 4: In-Vitro Release Profile:
S. No.
Time
(min.)
Pure drug % release
of CHL 1
% release % release
of CHL 2 of CHL 3
% release
of CHL 4
Marketed eye
drops
1
2
3
4
5
6
7
8
9
10
1
12
13
14
15
16
30
60
90
120
150
180
210
240
270
300
330
360
390
420
450
480
24.83
52.23
72.10
85.96
97.15
11.33
14.93
19.08
23.61
27.50
33.11
37.76
41.96
48.03
52.28
58.65
64.71
69.65
76.65
81.70
88.96
10.83
15.26
19.58
22.45
27.00
32.10
36.90
42.43
47.83
51.30
57.78
63.16
68.60
78.25
84.15
90.00
22.50
49.88
62.55
79.16
90.28
96.88
12.33
16.45
20.31
25.70
30.08
35.03
40.06
44.90
50.35
55.33
61.36
67.28
72.25
78.26
86.50
94.63
Figure 1: IR spectra of pure drug
11.66
15.45
21.60
25.65
31.18
35.76
41.08
44.31
50.9
54.56
60.91
68.65
72.96
79.81
87.56
95.55
Figure 2: IR spectra of Formulation CHL 4
Figure 3: Comparative In-Vitro Release of Marketed Eye Drop and Prepared In-Situ
Gels
In-vitro Drug Release
100
Pure Drug
90
CHL 1
70
CHL 2
60
50
CHL 3
40
CHL 4
30
Lomebact (Marketed
Product)
20
10
Time in Minites
480
450
420
390
360
330
300
270
240
210
180
150
120
90
60
30
0
0
% Release of Drug
80