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



                                                                                                                       ISSN 0976 – 1047




                                                                                                                        IJB
                               International Journal of Biopharmaceutics


                                          Journal homepage: www.ijbonline.com


    DEVELOPMENT AND EVALUATION OF THERMOREVERSIBLE OCULAR
              GELS OF KETOROLAC TROMETHAMINE

             Sirish Vodithala *, Sadhna Khatry 1, Nalini Shastri 1, Dr. M. Sadanandam 1
           *,1
                 Department of Pharmaceutics, Sri Venkateshwara College of Pharmacy, Madhapur, Hyderabad -500081

Abstract:
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

Introduction
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., M. Gibaldi et al 1982). In situ gel forming systems
* Corresponding Author                                                         can be 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])
Sirish Vodithala                                                               and polymethacrylates), pH triggered systems (e.g Carbopol
Email: vodithalasirish@gmail.com                                               (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
                                                                                                                                       40
                               Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 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
                                                                                                                                           41
                               Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 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 ,.
g. Ocular Irritancy test                                                      2007).The study was carried out by using Franz-diffusion
The optimized formulation was evaluated for in vivo                           cell in such a way that corneum side is continuously
performance in animal model (Rabbits). The protocol is                        remained in an intimate contact with formulation in the
approved by college ethical committee (Ethical committee                      donor compartment. The receptor compartment was filled
Registration number is CPCSEA/IAEC/Reg. No.                                   with STF pH 7.4 at 34 º C ± 0.5 º C. The receptor medium
518/2009).                                                                    was stirred on a magnetic stirred. The samples were
Three rabbits (Albino rabbits) were used for this study.                      withdrawn at different time intervals and analyzed for drug
They were housed and maintained in the animal house at                        content. Receptor phase were replenished with an equal
room temperature (27ºC) during the period of the study.                       volume of STF (pH 7.4) at each time interval. The percent
They were fed with standard diet and water. The animals                       drug released was plotted against time to get dissolution rate
were placed in cages and the eyes were marked as test and                     curves.
control. The control group received no sample and the test                    Results and Discussions
eye received the formulation (0.5ml), and the eyes were                       Pluronic F 127 and Methyl cellulose in the concentrations of
observed for the ocular irritancy (includes the macroscopic                   15% and 2% were found to be better carrier system because
observation of cornea, iris, and conjunctiva) (Satish kumar P                 it shows optimum gellation. With the increase in the
et al., 2008, Himanshu Gupta et al., 2007)                                    concentration of pluronic F 127 and methyl cellulose the
                                                                              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
                                                                                                                                           42
                              Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 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
                                                                                                                                             43
                                Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 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-
T4).

 Formulation            Parameters              Zero Order               First order              Higuchi              Korsemeyer & Peppas
                            K                      0.072                    0.010                  1.168                      1.828
    KT-T4
                            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
                                                                                                                                      44
                              Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 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
                                                                                                                                     45
                               Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 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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