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                                Sirish Vodithala et al. / International Journal of Biopharmaceutics. 2010; 1(1): 39-45.

                                                                                                                          ISSN 0976 – 1047

                               International Journal of Biopharmaceutics

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                    Sirish Vodithala*, Sadhna Khatry1, Nalini Shastri1, M. Sadanandam1
                 Department of Pharmaceutics, Sri Venkateshwara College of Pharmacy, Madhapur, Hyderabad -500081.

         In-situ gelling systems are viscous polymer-based liquids that exhibit sol-to-gel phase transition on the ocular surface
due to change in a specific physico-chemical parameter like temperature, ionic strength, or pH. A major problem in ocular
therapeutics is the attainment of optimal drug concentration at the site of action, which is compromised mainly due to pre-corneal
loss resulting in only a small fraction of the drug being ocularly absorbed. The effective dose administered can be altered by
increasing the retention time of medication into the eye by using in situ gel forming systems, thereby preventing the tear drainage.
The aim of the present investigation is to prepare and evaluate novel in situ ocular gelling system (Thermo-reversible gelling
systems) of Ketorolac tromethamine. These gelling systems involve the use of Poloxamer (Pluronic F 127) as thermo reversible
polymer and Methyl cellulose as release retardant. The formulations were evaluated for clarity, pH measurement, gelling
capacity, drug content estimation, rheological study, in vitro drug release, ocular irritation studies (as per draize test) and ex-vivo
corneal permeation studies using isolated goats cornea. The developed formulations showed sustained release of drug for upto 5
hrs. The formulations were found to be non-irritating with no ocular damage.

Keywords: Ketorolac tromethamine, in situ gels, Draize test, ex-vivo studies, Pluronic F 127.

Ophthalmic drug delivery is one of the interesting and                           of actions. Approaches to improve the ocular bioavailability
challenging endeavors facing the pharmaceutical scientist                        aim at increasing the corneal permeability by using
today. The anatomy, physiology, and biochemistry of the                          penetration enhancers or prodrugs, and prolonging the
eye render this organ highly impervious to foreign                               contact time with the ocular surface by using viscosity-
substances.                                                                      enhancing or in-situ gelling polymers. The in-situ gelling
A significant challenge to the formulator is to circumvent                       polymers undergo sol-to-gel phase transition on exposure to
the protective barriers of the eye without causing permanent                     the physiological conditions present in the eye. In-situ
tissue damage. The major problem encountered with topical                        gelling systems are viscous polymer-based liquids that
administration is the rapid pre-corneal loss caused by                           exhibit sol-to-gel phase transition on the ocular surface due
nasolacrimal drainage and high tear fluid turnover which                         to change in a specific physico-chemical parameter
leads to only 10% drug concentrations available at the site                      (temperature, ionic strength, or pH) (Ashim K. Mitra, 2003;
                                                                                 Gibaldi M et al 1982). In situ gel forming systems can be
* Corresponding Author                                                           classified as temperature dependent systems (e.g Pluronics
                                                                                 [El-Kamel, 2002; Cho et al., 2003; Cho et al., 2005; Qi et
                                                                                 al., 2007]), Tetronics (Spancake et al., 1991) and
Sirish Vodithala                                                                 polymethacrylates), pH triggered systems (e.g Carbopol
Email:                                                 (Aggarwal et al., 2005; Sultana et al., 2006; Wu et al.,
                                                                                 2007) and cellulose acetate phthalate (Gurny et al., 1985)
                                                                                 and ion activated systems (e.g Gelrite [Balasubramaniam et
                              Sirish Vodithala et al. / International Journal of Biopharmaceutics. 2010; 1(1): 39-45.

al., 2003, 2005]) and sodium alginate (Liu et al., 2006). The                  simulated tear fluid (STF) is sodium chloride 0.670 g,
principal advantage of in-situ gelling systems is the easy,                    sodium bicarbonate 0.200 g, calcium chloride dihydrate
accurate and reproducible administration of a dose                             0.008 g, purified water upto 100.0 g. The optimized
compared to the application of preformed gels. They have                       formulation for thermo-reversible ocular gels is shown in
an advantage over preformed gels that they can be easily                       Table 3.
instilled in liquid form, and are capable of prolonging the                    Characterization of Thermo reversible ocular gels
residence time of the formulation on the surface of the eye                    a. Clarity:
due to gelling.                                                                The clarity of the formulations before and after gelling was
Ketorolac tromethamine is a nonsteroidal anti-inflammatory                     determined by visual examination of the formulations under
drug, used to treat seasonal allergic conjunctivitis.                          light alternatively against white and black backgrounds.
Temperature induced insitu ocular gels of Ketorolac                            b. pH:
tromethamine were prepared by using polymers like                              Formulation was taken in a beaker and 0.1M NaOH was
Pluronic F 127 and methyl cellulose. Pluronic F 127 (Wen-                      added dropwise with continuous stirring. pH was checked
Di Ma et al., 2008) was used as a thermo-reversible                            using pH meter ( µ pH Systronics digital pH meter)
polymer and methyl cellulose was used to sustain the drug                      c. Assay:
release. The present investigation deals with development                      Accurately weighed amount KT insitu gel equivalent to 5mg
and evaluation of temperature induced insitu ocular gels of                    of drug was taken in a 100ml volumetric flask. Simulated
ketorolac tromethamine. The dosage regimens prepared                           Tear Fluid (STF pH 7.4) was added to it and kept on
provide ease in application and provides sustained drug                        magnetic stirrer to dissolve the drug. The volume was made
release with reduced frequency of administration.                              to 100ml with STF (pH 7.4).and filtered using Whatmann
Materials and Methods:                                                         filter paper (No 42). 10ml aliquot of the above solution was
Ketorolac tromethamine was obtained as a gift sample from                      taken and diluted to 100ml with STF (pH 7.4). The
Symed labs; Hyderabad. Pluronic F 127 was obtained as gift                     absorbance of sample solution was determined at 322nm
sample from Dr. Reddy’s Laboratories, Hyderabad. Methyl                        against STF (pH 7.4) as blank after suitable dilution with
cellulose was obtained as gift sample from Hychem                              STF.
laboratories, Hyderabad. Glass cylinders were fabricated by                    d. In-vitro Dissolution studies:
Murthy labs works, Hyderabad. A.P. Rabbits used for the                        In-vitro drug release studies of samples were carried out by
Draize eye irritancy test are from the college animal house                    using modified USP apparatus II paddle method with STF
and the Goat’s cornea for the ex-vivo studies was obtained                     (pH 7.4) as dissolution medium. A glass cylinder of 2.5 cm
from the local slaughter house.                                                in diameter open at both ends was designed for the purpose
Preliminary studies for optimum amount of Pluronic F                           of the study. Dialysis membrane previously soaked in STF
127 and Methyl cellulose for insitu gellation                                  (pH 7.4) was taken, dried, and tied on to one end of the
Preliminary studies were conducted using different                             glass cylinder and to this one ml of the formulation was
concentrations of Pluronic F127 (Eve Ruel-Garie PY et al.,                     accurately pipetted (Satish kumar P et al., 2008). The glass
2004 ) and Methyl cellulose. Based on the gellation studies,                   cylinder was attached to the shaft of USP apparatus II, in
Pluronic F127 and Methyl cellulose were selected as                            place of basket as shown in Figure 1. The cylinder was then
polymers for thermo-reversible gels. Placebo systems using                     suspended in 50 mL of dissolution medium maintained at 34
different amounts of Pluronic F 127 and methyl cellulose                       ± 0.5°C such that the membrane just touched the dissolution
were taken as shown in Table 1. For optimizing the amount                      medium. The speed of the metallic device shaft was set at
of Pluronic F127 and Methyl cellulose, gellation studies                       50 rpm. Aliquots were withdrawn at intervals of 1, 2, 3, 4,
were carried at 34 º ±. 0.5º C. Preliminary studies revealed                   and 5 hours and replaced by equal volumes of dissolution
optimum results with 15 % Pluronic F127 and 2% Methyl                          medium. Aliquots were suitably diluted with STF (pH 7.4)
cellulose.                                                                     and analyzed by UV Spectrophotometer at 322 nm. The
Preparation of the formulations                                                percent release of the drug was computed as shown on
Preparation of in-situ gelling systems:                                        Table 4 and the graph of percent drug release versus time
Aqueous solutions of various concentrations of pluronic F                      were plotted as shown in Fig 2.
127 and methyl cellulose (formulation codes KT-T1, KT-                         Data treatment of dissolution studies
T2, KT-T3, KT-T4, KT-T5 and KT-T6) were prepared,in                            Various models like zero order, first order, Higuchi models,
order to optimize the compositions for suitable for use as                     and Korsemeyer & Peppas were tested for explaining the
insitu gelling systems for ocular use as shown in Table 2.                     kinetics of drug release based on the release rate data as as
The gelling capacity was determined by placing a drop of                       shown in Table 7.
the gel in a vial containing 2 ml of simulated tear fluid                      e. FTIR spectroscopy
freshly prepared and equilibrated at 34º ± 0.5 º C and                         FTIR spectra of drug, and formulation were obtained.
visually assessing the gel formation. The composition of                       Sample is suspended between KBr plates, and examined in
                              Sirish Vodithala et al. / International Journal of Biopharmaceutics. 2010; 1(1): 39-45.

0.1mm KBr sealed cell, and scan for 16 times. The                              SHIMADZU and the FTIR spectrum was recorded from
instrument model used for FTIR was Prestige 21,                                3800 cm-1 to 650 cm-1.
f. Rheological Evaluation:                                                     h. Ex vivo corneal permeation studies using goat’s
Viscosity of formulation was determined before and after                       cornea
gellation by using Brookfield’s viscometer (DV II model) in                    Goat corneas were used to study the permeation across the
the small volume adaptor and the angular velocity was                          corneal membrane. Whole eyeballs of goat were procured
increased gradually from 10, 20, 50 and 100 rpm. The                           from a slaughter house and transported to laboratory in cold
hierarchy of the angular velocity was reversed. The average                    condition in normal saline maintained at 4ºC. The cornea
of two readings was taken to calculate the viscosity of the                    were carefully removed along with a 5–6 mm of
gels. Gellation was induced in formulation by raising the                      surrounding scleral tissue and washed with cold saline. The
temperature to 34 ºC ± 0.5 ºC (Katarina Edsman et                              washed corneas were kept in cold freshly prepared solution
al.,1998).                                                                     of tear buffer of pH 7.4. (Himanshu Gupta et al., 2007).The
g. Ocular Irritancy test                                                       study was carried out by using Franz-diffusion cell in such a
The optimized formulation was evaluated for in vivo                            way that corneum side is continuously remained in an
performance in animal model (Rabbits). The protocol is                         intimate contact with formulation in the donor compartment.
approved by college ethical committee (Ethical committee                       The receptor compartment was filled with STF pH 7.4 at 34
Registration number is CPCSEA/IAEC/Reg. No.                                    º C ± 0.5 º C. The receptor medium was stirred on a
518/2009).                                                                     magnetic stirred. The samples were withdrawn at different
Three rabbits (Albino rabbits) were used for this study.                       time intervals and analyzed for drug content. Receptor phase
They were housed and maintained in the animal house at                         were replenished with an equal volume of STF (pH 7.4) at
room temperature (27ºC) during the period of the study.                        each time interval. The percent drug released was plotted
They were fed with standard diet and water. The animals                        against time to get dissolution rate curves.
were placed in cages and the eyes were marked as test and                      Results and Discussions
control. The control group received no sample and the test                     Pluronic F 127 and Methyl cellulose in the concentrations of
eye received the formulation (0.5ml), and the eyes were                        15% and 2% were found to be better carrier system because
observed for the ocular irritancy (includes the macroscopic                    it shows optimum gellation. With the increase in the
observation of cornea, iris, and conjunctiva) (Satish kumar P                  concentration of pluronic F 127 and methyl cellulose the
et al., 2008; Himanshu Gupta et al., 2007)                                     gellation capacity increases.

Table 1: Gellation studies with Pluronic F 127 and Methyl cellulose
     S.No         Pluronic F 127 (% wt/vol)           Methyl Cellulose (% wt/vol)                    Gellation          Gellation Capacity

      1                       13                                        1                               YES                     +
      2                       13                                        2                               YES                     +
      3                       15                                        1                               YES                     +
      4                       15                                        2                               YES                    ++
      5                       20                                        -                               YES                   +++
      6                       25                                        -                               YES                   +++

-     No gellation
+     Gels after a few minutes and dissolves rapidly.
++    Gelation immediate and remains for few hours.
+++ Gelation immediate and remains for extended period of time
                              Sirish Vodithala et al. / International Journal of Biopharmaceutics. 2010; 1(1): 39-45.

Table 2: Formulation of Thermo-reversible ocular gels containing Pluronic F 127 and Methyl Cellulose

                                Concentration of Methyl cellulose                     Concentration of
Concentration of Pluronic F                (%wt/vol)                                   KT(%wt/vol)                         Formulation code
     127 (%wt/vol)

            13 & 1                                  1                                                                           KT-T1
            13 & 2                                  2                                                                           KT-T2
            15 & 1                                  1                                0.5%                                       KT-T3
            15 & 2                                  2                                                                           KT-T4
              20                                    --                                                                          KT-T5
             25                                     --                                                                          KT-T6

Table 3: Optimized Formulation of Thermoreversible ocular Gels of KT (KT-T4)

                         Ingredients                                                                 Amount (% wt/vol)
                        Pluronic F 127                                                                          15
                       Methyl Cellulose                                                                          2
                             Drug                                                                               0.5
                     Benzalkonium chloride                                                                     0.02
                            Water                                                                             20mL
                         0.1 N NaOH                                                                  q.s for pH adjustment

Table 4: Comparative Dissolution profiles of Thermo-reversible ocular gels

     Time                                                               Formulation code
                         KT-T1                      KT-T2                 KT-T3                 KT-T4                   KT-T5        KT-T6
       1                 44.86                      31.38                  53.83                40.01                   32.89        25.47
       2                 54.56                      48.40                  66.28                60.14                   50.35        41.46
       3                 67.13                      59.83                  73.33                75.60                   59.83        46.73
       4                 82.80                      78.04                  89.84                87.38                   80.33        56.88
       5                 90.77                      88.74                    --                 96.30                   90.77        65.37

Table 5: Comparative dissolution profiles of Formulation (KT-T4) and Marketed product (Acular 0.5%)

       Time (Hrs)                               Formulation (KT-T4)                                              Marketed product
                                                                                                                  (Acular 0.5%)
             0                                              0                                                           0
             1                                            40.02                                                       48.27
             2                                            60.14                                                       68.09
             3                                            75.6                                                        87.09
             4                                            87.39                                                      100.32
             5                                            96.3                                                        91.08
             6                                            78.72                                                       83.83
                               Sirish Vodithala et al. / International Journal of Biopharmaceutics. 2010; 1(1): 39-45.

Table 6: Shows the pH, viscosity (before and after gellation), gellation capacity, and the drug content of different thermo-
reversible gels.

     Formulation code                pH measurement                Gelling capacity              Drug content (%)
         KT-T1                            7.4                              +                            79.53
         KT-T2                            7.4                              +                            85.23
         KT-T3                            7.4                              +                            88.61
         KT-T4                            7.4                              ++                           99.69
         KT-T5                            7.4                             +++                           98.76
         KT-T6                            7.4                             +++                          100.15
                                   VISCOSITY FOR FORMULATION CODE KT-T4
                 RPM                      Viscosity in cps (spindle no. S     Viscosity in cps (spindle no. S 34) After
                                               34) Before gellation                           gellation
                  10                                   1236                                     3012
                  20                                    954                                     2481
                  50                                    721                                     1842
                  100                                   594                                     1089

Table 7: Different Release Rate Constants of Thermo-reversible ocular gels of Ketorolac tromethamine (Formulation code KT-

 Formulation            Parameters              Zero Order                First order              Higuchi               Korsemeyer & Peppas
                            K                      0.072                     0.010                  1.168                       1.828
                            r2                    0.9921                    0.9790                 0.9902                      0.9971

Table 8: Diffusion profile of formulation (KT-T4) on goat cornea

             Time (Hrs)                                                              % Drug Release
                 0                                                                         0
                 1                                                                       30.62
                 2                                                                        47
                 3                                                                       63.55
                 4                                                                       76.10
                 5                                                                       84.80

Figure 1: Dissolution apparatus
                             Sirish Vodithala et al. / International Journal of Biopharmaceutics. 2010; 1(1): 39-45.

Figure 2: Effect of polymers ratio on dissolution of                          Figure 4: FT-IR studies of Thermoreversible ocular gels of
Ketorolac tromethamine Thermoreversible ocular gels                           Ketorolac tromethamine

Figure 3 .Comparative Dissolution Profiles of formulation                     Figure 5: Viscosity before and after gellation (KT-T4)
(KT-T4) and Marketed product (Acular 0.5%)

Figure 6: Diffusion profile of Thermoreversible ocular gel of KT (on Goat cornea)

Characterization of thermo-reversible insitu ocular gels                      official medium was selected for all dissolution studies.
of ketorolac tromethamine                                                     Optimized formulation of Thermo-reversible insitu ocular
Assay: The thermo-reversible insitu ocular gels of KT                         gels was subjected to in vitro dissolution testing in STF (pH
prepared complied with the requirements of assay. The                         7.4). Thermo-reversible insitu ocular gels KT-T4 showed
results for assay were 99.69 %.                                               highest dissolution rate as shown in Table 4 and Fig 2.
In vitro dissolution: Simulated tear fluid (pH 7.4) the
                              Sirish Vodithala et al. / International Journal of Biopharmaceutics. 2010; 1(1): 39-45.

FTIR spectroscopy                                                              curves. Table 8 shows the percent drug release and Fig. 6
It was performed by KBR pellet method. The principal                           shows the percent drug release.
peaks of Ketorolac tromethamine were observed at 3353,                         CONCLUSIONS
3082, 2922, 2953cm-1, 1613.The characteristic peak for                         Rationale of the present study was to improve the pre-
formulation were found at 2969 cm-1 as shown in Fig 4.                         corneal residence time, and sustain the drug release by
Ocular Irritancy studies:                                                      utilizing the approach of in situ gelling systems using
Ocular irritation studies indicate that KT-T4 was a                            various polymers. It was envisaged that this techniques
nonirritant. The formulation was very well tolerated by the                    would prove successful in case of formulations prepared
eye. No ocular damage or abnormal clinical signs to the                        with the drug (KT).
cornea, iris, or conjunctivae were visible                                     In situ gelling systems were prepared using Pluronic F 127
Ex vivo corneal permeation studies:                                            and Methyl cellulose (15% pluronic F 127 and 2% methyl
Corneal permeation studies were performed using isolated                       cellulose) as polymers for thermo reversible ocular gels.
goat’s cornea on Franz-diffusion cell using STF (pH 7.4) at                    Acknowledgements:
34 ± 0.5 º C. The samples were withdrawn at regular                            The authors are thankful to Sri Venkateshwara college of
intervals and analyzed for drug content. The percent drug                      Pharmacy, Madhapur, Hyderabad, for proving the facilities
released was plotted against time to get dissolution rate                      for the present work.


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