Rathapon Asasutjarit, Ph.D. Development of Thermoresponsive Diclofenac Ophthalmic In Situ Gels Formulaitons By Rathapon Asasutjarit, Ph.D. Department of Pharmaceutical Technology, Faculty of Pharmacy, Rangsit University Asira Fuongfuchat, Ph.D. National Metal and Materials Technology center, National Science and Technology Development Agency (NSTDA)

Outline Introduction Objectives Methods Results and discussion Conclusions Acknowledgements

Introduction Eye drop solution: convenient to use by instilling into conjunctival sac of the eye with accurate dose. Bioavailability is very poor: blinking reflex and lacrimal secretion. - Inserts, ointments and suspensions. Eye drop solution Insert Ointment

- In situ gels: pH, specific ion and temperature - The thermoresponsive in situ gel is an in situ gel system responding to a shift in temperature. - It is liquid at low temperature and becomes gel when in contact with higher temperature.

Diclofenac is one of non-steroidal anti-inflammatory drugs approved for ocular use. Diclofenac is available in the drug market as an eye drop solution. It has been used intensively in the case of post-op inflammatory with 3-4 times of administration. Thus, minimized frequency of administration, once or twice daily, is necessary for increasing patient compliance.

- My previous work (Asasutjarit, 2008): - Formulation of thermoresponsive diclofenac ophthalmic in situ gels contained either : Pluronic F127 (Poloxamer 407) or : Combination of Pluronic F127 and hydroxypropylmethylcellulose - Phase transition temperatures were lower than room temperature.

Objectives The purposes of this study were to develop and to evaluate the formulation of thermoresponsive diclofenac ophthalmic in situ gels possessing optimum phase transition temperature.

Methods, Results and discussion Preparation of thermoresponsive diclofenac ophthalmic gels and products evaluation : 0.1% w/w diclofenac sodium ophthalmic gels containing various gelling agent contents : prepared on a weight basis by the cold method Formulation (Rx) Gelling agent (%w/w) F127 F68 Carbopol 940 1. F127-20 20 2. F127-20+F68-8 8 3. F127-20+F68-11 11 4. F127-20+F68-11+C.1 0.1 5. F127-20+F68-11+C.3 0.3 6. F127-20+F68-11+C.5 0.5

: Phosphate buffer pH 7.4 was used in the formulations as vehicle except for the formulations containing carbopol 940, phosphate buffer pH 5.5 was used. : All samples were analyzed spectrophotometrically for the content uniformity at a wavelength of 276 nm. : Only samples with diclofenac sodium content within 100± 10% of labeled amount were accepted. : They were measured their pH in triplicate by using a pH meter.

2. Determination of phase behavior and sol-gel transition temperature of thermoresponsive gels 2.1 Test tube inverting method : 2±1°C (storage temperature) : 27±1°C (room temperature) : 35±1°C (ophthalmic temperature).

- storage modulus (G´) and loss modulus (G´´) at varied temperatures 2.2 Temperature sweep test : Rheometer - storage modulus (G´) and loss modulus (G´´) at varied temperatures Gel phase Phase transition Sol phase

Preparation and evaluation of thermoresponsive diclofenac ophthalmic gels (continued) Formulation (Rx) %Labeled amount *Flow ability at various temperatures Sol-gel transition temperature (°C) pH 2±1 (°C) 27±1 (°C) 35±1 (°C) 1. F127-20 103.2±1.0 +++ - 20.7±0.1 7.4±0.1 2. F127-20+F68-8 103.3±1.2 + 27.2±0.3 7.3±0.1 3. F127-20+F68-11 102.6±0.6 31.6±1.8 4. F127-20+F68-11+C.1 103.2±0.8 32.7±1.1 5.4±0.1 5. F127-20+F68-11+C.3 103.3±0.9 31.7±0.5 4.8±0.1 6. F127-20+F68-11+C.5 36.3±1.4 4.7±0.1 (n=3, mean± SD), *Flow ability: +++ = very good; ++ = good; + = flow; - = not flow

3. Gelling capacity test Drop 20-μl of samples A test tube containing 2-ml of simulated tear fluid (STF) (pH 7.4) equilibrated at 35 °C. The visual assessment of gel formation and dissolution was performed in triplicate (Wu et al., 2007).

Time for remaining gel in STF (min) Formulation (Rx) Time for remaining gel in STF (min) 3. F127-20+F68-11 20.2±0.8 4. F127-20+F68-11+C.1* 40.4±0.7 5. F127-20+F68-11+C.2 45.3±0.6 6. F127-20+F68-11+C.3 49.0±0.4 The formulations containing carbopol 940 were eroded by STF more slowly than the formulation without carbopol 940 markedly. - This is probably that carbopol 940 consisting in the formulations led to more stiff gel when pH of the environment was increased. - Since formulation Rx 4. (F127-20+F68-11+C.1) had an optimum sol-gel transition temperature, pH and gelling capacity, it was chosen as a representative for the further experiments.

4. Steady shear sweep test : The experiment was carried out at 27±1 °C and 35±1 °C by a rheometer for determination of flow behavior of Rx 4. (F127-20+F68- 11+C.1) : The initial and final shear rates were 0.05 and 100 s-1, respectively.

Pseudoplastic flow Rheogram of Rx 4. (F127-20+F68-11+C.1)

Viscosity profile of Rx 4. (F127-20+F68-11+C.1) - Ocular shear rate is about 0.03 s-1 during interblinking and 4,250- 28,000 s-1 during blinking.

5. Eye irritation test : 4-New Zealand white rabbit 20-µl of the sample was instilled into the conjunctival sac of rabbit’s right eye The left was kept as a control without manipulation. 0, 5, 10, 30 min, 1, 6, 12, 24, 48 and 72 hours

(DiPasquale and Hayes, 2001) Lesions Grades Cornea No ulceration or opacity Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible 1 Easily discernible translucent area, details of iris slightly obscured 2 Nacreous area, no details or iris visible, size of pupil barely discernible 3 Complete corneal opacity, iris not discernible 4 Iris Normal Markedly deepened folds, congestion, swelling, moderate circumcorneal injection,(any o these separately or combined) iris still reacting to light (sluggish reaction is positive) No reaction to light, hemorrhage, gross destruction(any or all of these) (DiPasquale and Hayes, 2001)

(DiPasquale and Hayes, 2001) Lesions Grades Conjunctive Redness (refers to palpebral and bulbar conjunctivae, excluding cornea and iris) Vessels normal Some blood vessels definitely hyperemic (injected) 1 Diffuse, crimson color, individual vessels not easily discernible 2 Diffuse beefy red 3 Chemosis No swelling Any swelling above normal (includes nictitating membranes) Obvious swelling with partial eversion of lids Swelling with lids about half closed Swelling with lids more than half-closed 4 (DiPasquale and Hayes, 2001)

Results of eye irritation test of Rx 4. (F127-20+F68-11+C.1) Lesions Grades Cornea No ulceration or opacity Iris Normal Conjunctive Vessels normal Chemosis No swelling b a: the test eye b: the controlled eye

6. Diclofenac absorption in the eye - 8-New Zealand white rabbit Group 1 : Rx 4. (F127-20-+F68-11+C.1) Group 2 : Diclofenac eye drop solution (Original) - Sampling time: 5, 10, 30 min, 1, 6, and 12 hours, - Sampling volume: 200 µl - Zoletil 100® im - High-performance liquid chromatrography (HPLC)

Diclofenac concentrations in aqueous humor Cmax gel = 3,900 ng/ml Rx 4. (F127-20+F68-11+C.1) Cmax solution = 1,517 ng/ml Diclofenac eye drop solution (Original) Diclofenac concentrations in aqueous humor

Conclusions - 20% w/w pluronic F127, 11% w/w pluronic F68 and 0.1% w/w carbopol 940. - Exhibited solution character predominantly at the storage temperature and room temperature and became gel at the ophthalmic temperature. - Sol-gel transition temperature was 32.7±1.1 °C. - It was convenient to use without keeping in a refrigerator. - It did not cause eye irritation in rabbits. - It increased concentration of diclofenac in aqueous humor and prolonged the contact time of diclofenac in the eye. - This formulation had a potential for the further study in patients suffering from inflamed eye.

Acknowledgements The authors gratefully acknowledge - Mr. Chanwit Kammat, - Mr. Thumrongrat Pukpinyo, - Ms. Watunya Witisil, - Ms. Suthira Thansanchokpibull - Ms. Susiri Preedasuttichit - Ass. Prof. Aree Thayananuphat, DVM, Ph.D. - Research Institute, Rangsit University for the research grant No. 33/50.

Thank you