Development and evaluation of diclofenac sodium thermorevesible subcutaneous drug delivery system

International Journal of Pharmaceutics 439 (2012) 120–126

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Development and evaluation of diclofenac sodium thermorevesible subcutaneous drug delivery system Fazli Nasir a , Zafar Iqbal a,∗ , Jamshaid A. Khan a , Abad Khan b , Fazli Khuda a , Lateef Ahmad a , Amirzada Khan a , Abbas Khan a , Abdullah Dayoo c , Roohullah a a

Department of Pharmacy, University of Peshawar, Peshawar-25120, Pakistan Department of Pharmacy, Abdul Wali Khan University Mardan, Ambar Campus, Pakistan c Faculty of Pharmacy, Sindh University Jamshooro, Sindh, Pakistan b

a r t i c l e

i n f o

Article history: Received 7 August 2012 Received in revised form 15 September 2012 Accepted 8 October 2012 Available online 17 October 2012 Keywords: Thermoreversible in situ gels Pluronics Methyl cellulose Diclofenac sodium Subcutaneous

a b s t r a c t The objective of current work was to develop and evaluate thermoreversible subcutaneous drug delivery system for diclofenac sodium. The poloxamer 407, methyl cellulose, hydroxypropyl methyl cellulose and polyethylene glycol were used alone and in combination in different ratios to design the delivery system. The physical properties like Tsol–gel, viscosity, clarity of solution and gel were evaluated. The in vitro release of the drug delivery system was evaluated using membrane less method and the drug release kinetics and mechanism was predicted by applying various mathematical models to the in vitro dissolution data. Rabbits were used as in vivo model following subcutaneous injection to predict various pharmacokinetics parameters by applying Pk-Summit software. The in vitro and in vivo data revealed that the system consisting of the poloxamer 407 in concentration of 20% (DP20) was the most capable formulation for extending the drug release and maintaining therapeutic blood level of DS for longer duration (144 h). The data obtained for drug content after autoclaving the solutions indicate that autoclaving results in 6% degradation of DS. The data also suggested that the studied polymers poloxamer, MC and PG are good candidate to extend the drug release possessing a unique thermoreversible property. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The application of the polymers in pharmaceutical sciences particularly in drug delivery systems and formulations is well known (Arnott et al., 1974). Many polymers show a decrease in solubility as their hydrophobicity increases upon temperature change. In such type of polymer solutions, three types of interactions are observed; (i) between the molecules of polymer, (ii) between water and polymer molecules and (iii) between the water molecules. This phenomenon is called hydrophobic effect (Gong et al., 2009a; Kang et al., 2006; Mulik et al., 2009; Tyagi et al., 2004). As a result of the increase in hydrophobicity the polymer chains are linked by physical reversible linkage, and gels can therefore return to solution after the temperature stimulus, causing gelation is removed (Kang et al., 2006). These formulations may sustain delivery of the drug for long period of time improving the patient’s compliance. Pluronic (poloxamer) is a group of nonionic surfactants consisting of polyethylene oxide (PEO) and polypropylene oxide (PPO) copolymers. The thermal gelation of pluronic is a result of

∗ Corresponding author. Tel.: +92 91 9239619; fax: +92 91 9218131. E-mail address: zafar [email protected] (Z. Iqbal). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.10.009

micellization which is due to the dehydration of hydrophobic poly propylene oxide blocks and hydration of poly ethylene oxide chains (Juhasz et al., 1989). Methyl cellulose is a synthetic methoxy derivative of cellulose. Methyl cellulose aqueous solution in concentration of 1–10% by weight forms a thermorevesible gel (Sarkar, 1979). HPMC is synthetic propylene glycol ether of methyl cellulose. Aqueous HPMC solution forms thermo-reversible gel on heating (Masae et al., 2001). Diclofenac sodium 2-[(2,6-dichlorophenyl)amino] benzeneacetic acid) a phenylacetic acid derivative is nonsteroidal anti-inflammatory agent used as analgesic and anti-rheumatic (Brogden et al., 1980; Catella-Lawson et al., 2001; Reynolds and Parfitt, 1989; Sallmann, 1986; Todd and Sorkin, 1988).

2. Materials and methods 2.1. Materials Diclofenac sodium (DS) purity 99.30% (manufactured by Suzhou Ausun Chemical Company Limited), methyl cellulose (MC) 15 cps, hydroxypropylylmethyl cellulose (HPMC, Methocel E5) manufactured by Dow Chemical and polyethylene glycol (PEG 6000) manufactured by Clariant GMBH were the kind gifts of Medicraft

F. Nasir et al. / International Journal of Pharmaceutics 439 (2012) 120–126

Pharmaceuticals Pvt. Ltd., Peshawar. Voren injection (DS) manufactured by Asian Continental Pharma, was purchased from local market. Methanol, acetonitrile, phosphoric acid, and triethylamine (HPLC grade), pluronic F-127 (poloxamer406), were purchased from Sigma–Aldrich (Oslo, Norway). Purified water was prepared using a Millipore ultra-pure water system (Milford, USA). White New Zealand rabbits were purchased from the Department of Pharmacy University of Peshawar. The procedure for use and care of animals for this study were approved by the Ethical Committee of Department of Pharmacy University of Peshawar.

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ranging from 5 to 400 rpm. All samples were analyzed in triplicate and for statistical significance Student’s t-test at p < 0.05 was performed. 2.6. Clarity of the formed gel of DS formulations The clarity of the DS solution and formed gels before and after autoclaving of the formulations was observed visually at 5 ◦ C, 25 ◦ C and 37 ◦ C. 2.7. Drug content of DS formulations

2.2. Instrumentation All of the samples were analyzed in triplicate for the drug content using HPLC method (Nasir et al., 2011) before and after autoclaving. Only samples with drug content within 100 ± 10% of labeled amount were accepted.

Perkin-Elmer HPLC system (Norwalk, USA), consisted of a pump (series 200), on-line vacuum degasser (series 200), auto-sampler (series 200), Peltier column oven (series 200), linked by a PE Nelson network chromatography interface (NCI) 900 with UV/VIS (series 200). The whole HPLC system was controlled by Perkin-Elmer Total chrom Workstation Software (version 6.3.1). Centrifuge (Centurion. Scientific Ltd.), Shaking water bath B.S.11 Lab Companion (Jelo Tech Korea), pH meter (Hanna instruments 8417, USA) and Autoclave HS-60 (Hansuin Medical Co. Ltd., Korea).

2.8. Determination of in vitro drug release from DS formulations The in vitro drug release was determined using membrane less dissolution model (Chandrashekhar, 1998). From each preparation, solution containing DS (2 ml) was transferred into tubes (ca. ≈ 10 ml). The solutions were equilibrated for 5 min to effect the gelation in digital shaking water bath maintained at 37 ± 1 ◦ C. Phosphate buffer pH 7.4 (2 ml), used as dissolution medium, was poured slowly on the surface of the gel not to disturb the surface layer. Whole of the dissolution medium was collected as a sample after predefined time intervals i.e.0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 24, 36, 48, 72, 96 and 120 h depending upon formulation that remain intact for the specified period of time. As soon as samples were collected fresh dissolution medium (2 ml) was added to the test tube. The samples were suitably diluted and analyzed for diclofenac content using HPLC method (Nasir et al., 2011).

2.3. Preparation of diclofenac sodium in situ gel formulations The drug and polymers were accurately weighed according to the composition for the formulations as shown in Table 1. Diclofenac sodium 5 mg/ml solutions with different polymers alone and in polymers combination were prepared using cold method (Schmolka, 1972; Wei et al., 2002). The polymer was dispersed in cold water with continuous stirring, the dispersion was then stored in refrigerator for 24 h to obtain clear polymer solution, DS was then added to this solution and dissolved with continuous stirring. The final solutions were sterilized by autoclaving at 121 ◦ C, 15 psi for 20 min and were evaluated for their physicochemical properties i.e. Tsol–gel, viscosity, clarity, drug content, in vitro drug release and in vivo pharmacokinetic parameters.

2.9. Drug release kinetics The data obtained from in vitro experiments was fitted to various mathematical models to assess the drug release kinetics.

2.4. Measurement of Tsol–gel DS formulations 2.9.1. Zero order kinetic model Qt = Q0 + K0 t

The Tsol–gel of the formulations was measured by tube inversion method (Gilbert et al., 1987; Vadnere et al., 1984). 2 ml of each solution was transferred to a 5 ml test tube in a digital Shaking water bath maintained at gelation temperature and sealed with a parafilm. The solutions were equilibrated for 5 min to effect the gelation. The gelation of the solutions was verified by inverting the test tube at 90◦ . Measurements were performed in triplicates and Student’s t-test at p < 0.05 was performed for statistical significance.

(1)

where Qt is the amount of drug dissolved in time t, Q0 is the initial amount of drug in the solution and K0 is zero order release constant. 2.9.2. First order kinetic model K1 t log Qt = log Q0 + 2.303

(2)

where Qt is the amount of drug dissolved in time t, Q0 is the initial amount of drug in the solution and Kt is first order release constant.

2.5. Measurement of steady shear viscosity of diclofenac sodium formulations

2.9.3. Higuchi model The model relates cumulative drug release versus square root of time as shown in Eq. (3). √ Q = KH t (3)

The steady shear viscosity before and after autoclaving was measured using cone and plate viscometer. A 0.5 ml sample of the solution was applied to the lower plate of the viscometer. The viscosity was taken using spindle 52 at 37 ± 0.1 ◦ C at a shear rate Table 1 Composition of diclofenac sodium thermoreversible gel. S. no.

Formulation

Diclofenac sodium (mg)

PL F127 (mg)

MC (mg)

PEG (mg)

Distilled water

1 2 3 4 5

DP18 DP20 DPM15/3 DMPG1.5/10 DMPG3/2

5.0 5.0 5.0 5.0 5.0

180 200 150 – –

– – 30.0 15.0 30

– – – 100.0 20.0

qs to 1 ml qs to 1 ml qs to 1 ml qs to 1 ml qs to 1 ml

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2.9.4. Hixson–Crowell model This model relates cubic root of the unreleased drug remaining in the dosage form versus time. As given by Eq. (4). 1/3

Q0

1/3

= Qt

+ KS t

(4)

2.9.5. Korsmeyer–Peppas model This model relates exponentially the drug release to the elapsed time. The equation is given as Eq. (5). Qt = Kk t n Q∞

(5)

2.10. In vivo evaluation of formulations 2.10.1. Animal handling Male New Zealand white rabbits weighing 2.5 ± 0.15 kg were used as animal model. Rabbits were kept individually in standard stainless steel cages, fed with a commercial laboratory rabbit diet and were freely allowed to water. 2.10.2. Drug administration and sampling Animals were divided into two groups of six rabbits. Each group was treated with either the representative DS in situ gel or the commercial diclofenac sodium injection by injecting (2 ml) subcutaneously. Blood sample (500 ␮l) from each animal was collected in ethylenediaminetetraacetic acid (EDTA) glass tubes from marginal ear vein 5 min before injecting the formulation and at predetermined time intervals i.e. 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96 and 120 h. Blood samples were then centrifuged at 1600 × g for 10 min at 4 ◦ C. The plasma was collected and stored at −20 ◦ C until analysis. DS content in plasma was determined using HPLC (Nasir et al., 2011). 3. Results and discussion 3.1. In vitro evaluation of in situ diclofenac sodium sol–gel formulations 3.1.1. Tsol–gel of DS formulations The transition temperature from sol–gel of the prepared in situ thermoreversible gels is depicted in Table 2. The data shows the effect of autoclaving at 121 ◦ C, 15 psi for 20 min on the Tsol–gel. The p-values (n = 3) obtained using Student’s t-test were 0.423, 0.225, 0.425, 0.478 and 1.00 for DPM18, DPM20, DPM15/3, DMPG3/2 and DMPG1.5/10, respectively, suggesting that autoclaving has no significant effect on the transition temperature of the in situ gels (Dumortier et al., 2006). 3.1.2. Viscosity of DS formulations Autoclaving has no significant effect on the viscosity, suggesting the results are consistent with previous studies (Dumortier et al., 2006), results are shown in Table 2.

Fig. 1. In vitro release profile of diclofenac sodium form various in situ sol–gel.

3.1.3. Clarity of the DS solutions and formed gels Solutions of all the formulations were checked for clarity at 5 ◦ C, 25 ◦ C and gelling temperature, all the solutions were clear indicating the solubility of the added ingredients at all the temperature excepts for the solutions containing MC (DMPG3/2 and DMPG1.5/10) that formed turbid gels at the gelling temperature. The turbidity may be due to the hydrophobic interaction of the methoxyl group of the polymer chain (Sarkar, 1979). The gel formed by the combination of pluronic and MC i.e. DMP15/3 were semitransparent. Autoclaving sterilization did not affect the clarity of these formulations. 3.1.4. Drug content of DS in situ gels The diclofenac sodium contents for the formulawere between 100.30 ± 0.40%–100.63 ± 0.49% tions and 94.00 ± 0.70%–94.23 ± 0.35% before and after autoclaving, respectively. Sterility is one of the prime requirements for parenteral delivery system. It is important to employ appropriate technique for sterilization. The data in Table 2 suggests that autoclaving significantly decreased the diclofenac content as indicated by the p-values. The decrease in % labeled amount of DS might be due to its degradation by exposure to high temperature during autoclaving. The results are consistent with the previous studies (Roy et al., 2001), to avoid the degradation of the DS, filtration at room temperature will be an ideal alternate technique for the sterilization. 3.1.5. In vitro drug release from DS in situ gels The release profiles of DS in situ gels (Fig. 1) show that all the formulation retarded the drug release above 12 h except for the formulation DMPG1.5/10. The t100% (time when 100% drug was released) was 4, 24, 72, 96 and 144 h for DMPG1.5/10, DMPG3/2, DPM15/3, DP18 and DP20, respectively. The slowest drug release was observed for the formulation DP20, that contains 20% pluronic F-127. The addition of 3% (w/v) MC to pluronic in DPM15/3 extended the drug release for about 72 h.

Table 2 Physicochemical evaluation of diclofenac sodium thermoreversible in situ gels. S. no.

Formulation

Drug content (%) before AC

1 2 3 4 5

DP18 DP20 DPM15/3 DMPG1.5/10 DMPG3/2

100.63 100.49 100.63 100.40 100.30

± ± ± ± ±

0.49 0.47 0.21 0.26 0.40

Clarity: +++++, good; +++, fair; –, poor.

Drug content (%) after AC 95.17 94.32 94.23 94.53 94.00

± ± ± ± ±

0.59 0.10 0.35 0.25 0.70

p value

Viscosity (cps) before AC

0.003 0.003 0.001 0.004 0.005

119.0 132.0 145.7 101.7 113.3

± ± ± ± ±

0.8 0.8 0.9 0.5 1.2

Viscosity (cps) after AC 117.67 130.7 145.0 102.7 112.7

± ± ± ± ±

1.5 0.5 0.8 1.2 0.9

p value

Tsol–gel (◦ C) before AC

0.057 0.270 0.529 0.478 0.270

33.33 31.0 34.85 36.2 35.3

± ± ± ± ±

0.24 0.41 0.24 0.24 0.24

Tsol–gel (◦ C) after AC 33.67 31.50 35.17 36.33 35.33

± ± ± ± ±

0.24 041 0.24 0.24 0.24

p value

Clarity before AC

Clarity after AC

0.423 0.225 0.425 0.478 1.00

+++++ +++++ +++ – –

+++++ +++++ +++ – –

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Table 3 Release parameters of diclofenac sodium from DP20, DP18, DPM15/3, DMPG3/2 and DMPG1.5/10 in situ sol–gel formulations. Formulation

DP20 DP18 DPM15/3 DMPG3/2 DMPG1.5/10

Zero order

Hixson–Crowell

Korsmeyer

R2

K0 h−1

First order R2

K1 h−1

Higuchi R2

KHh−0.5

R2

KH C(h−1/3)

n

R2

0.968 0.899 0.717 0.711 0.871

0.640 0.939 1.373 3.389 24.13

0.814 0.815 0.966 0.992 1.000

0.011 0.021 0.027 0.113 0.477

0.995 0.988 0.839 0.870 0.92

8.428 10.55 10.13 22.39 75.65

0.954 0.958 0.961 0.997 0.990

0.022 0.036 0.060 0.249 1.825

0.421 0.470 0.455 0.670 1.291

0.881 0.960 0.978 0.978 0.969

3.1.5.1. Drug release kinetics from DS in situ gels. The drug release from the thermoreversible hydrogels are governed by various parameters. Solubility of the drug in the polymer and water, water diffusion rate into the polymer gel, the drug diffusion rate from the polymer gel and dissolution of the polymer within the experimental conditions are important factors that affect the kinetics of drug release. The data obtained from the in vitro dissolution experiments were fitted to different mathematical model i.e. Zero order, First Order, Higuchi, Hixson–Crowell, Korsmeyer–Pappas, to predict the kinetics and release mechanism of the drug. The release constant and regression coefficient (r2 ) values obtained from the mathematical models are shown in Table 3. The data obtained shows that the formulations DP20 and DP18 follows zero order kinetics (Fig. 2) with r2 values of 0.968 and 0.899, respectively, indicating the drug release is independent of drug concentration within the system. While the formulations DPM15/3, DMPG3/2 and DMPG1.5/10 released the drug through first order kinetics (Fig. 3) with the r2 values of 0.996, 0.992 and 1.00, respectively. The data obtained for the formulations DP20 and DP18 best fits Higuchi model (Fig. 4) as indicated by the correlation coefficient i.e. 0.995 and 0.988, respectively, indicating the drug release from formulation followed Fickian diffusion. The DS release from Formulations DMPG3/2, DMPG1.5/10 and DPM15/3 is the result of polymer erosion as high linearity was obtained with the Hixson–Crowell model (Fig. 5), the r2 values were 0.997, 0.990 and 0.961, respectively. The value of release exponent “n” obtained by applying Korsmeyer–Pappas equation for the formulations DP20, DP18 and DPM15/3 was less than 0.5 (Fig. 6) indicating DS is released through Fickian diffusion from these formulations, while the drug release from DMPG3/2 followed anomalous release i.e. combination of

Fig. 2. % Cumulative diclofenac sodium release vs. time (zero order model) from diclofenac sodium in situ thermoreversible sol–gel.

Fig. 3. % Log cumulative diclofenac sodium remaining log(100)–log t vs. time (first order model) from diclofenac sodium in situ thermoreversible sol–gel.

diffusion and polymer surface erosion. The formulation DMPG1.5/10 released the drug through super case II mechanism indicating drug release as a result of rapid polymer erosion in the dissolution medium. It can be deduced from the data that with decrease in pluronic F-127 concentration results in weak gel, hence increase in the rate of water diffusion and rapid water uptake results in rapid dissolution of the drug and faster diffusion as the path length is reduced (Anderson et al., 2001). The decrease in concentration of the pluronic results in the increase in the number and size of the water channels, and mesh size of hydrogel resulting in high drug release (Alexandridis and Alan Hatton, 1995; Bhardwaj and Blanchard, 1996).

Fig. 4. % cumulative diclofenac Sodium release vs. Sq. Rt. time (Higuchi Model) from diclofenac sodium in situ thermoreversible sol–gel.

F. Nasir et al. / International Journal of Pharmaceutics 439 (2012) 120–126

± ± ± ± ± ± ± ± ± ±

Mean ± SD

4.038 0.671 2 4.608 5.493 38.19 6.953 5304.60 910.190 4.038 0.013 0.001 – 0.001 0.001 0.001 0.001 0.001 0.001 0.013 0.07 0.06 0.0 0.21 0.12 19.18 0.27 6.42 0.08 0.07 ± ± ± ± ± ± ± ± ± ± 6.785 2.724 48 85.757 88.058 3370.8 38.279 555.94 56.780 6.7851

p value p value

0.76 0.01 0.0 0.77 0.44 35.86 1.001 161.7 1.72 0.76 ± ± ± ± ± ± ± ± ± ± 9.071 2.640 1 32.50 35.78 815.65 22.793 1829.5 139.735 9.071

Mean ± SD p value

0.003 0.003 – 0.001 0.001 0.001 0.001 0.001 0.001 0.002 1.03 0.16 0.0 6.11 6.95 389.96 1.36 87.52 2.27 1.03 ± ± ± ± ± ± ± ± ± ± 18.363 2.3798 48 99.197 124.65 6329.5 50.775 1065.3 40.19 18.36

Mean ± SD p value

0.008 0.004 – 0.002 0.017 0.005 0.015 0.002 0.001 0.008 9.70 0.109 0.0 11.71 44.68 6097.78 13.19 152.59 5.19 9.70 ± ± ± ± ± ± ± ± ± ± 23.109 2.4017 48 156.61 202.22 14731 70.901 802.39 25.486 23.109

Mean ± SD

h ␮g/ml h ␮g h/ml ␮g h/ml ␮g h2 × h/ml h ml/kg ml/h/kg h E Half-life Cmax (obs) Tmax (obs) AUC (0–t) (obs area) AUC (0–∞) AUMC (0–∞) MRT (area) Vd (area)/kg CL (area)/kg Half-life from Vd and CL

DMPG3/2 DPL18 DPL20

Pk parameter

Fig. 6. Fraction (Mt /M) diclofenac sodium released vs. log time (Korsmeyer–Pappas model) from diclofenac sodium in situ thermoreversible sol–gel.

Unit

The prepared formulations and conventional commercial diclofenac sodium solutions were injected (5 mg/kg body weight) through subcutaneous route. The blood samples were collected and analyzed using HPLC. The plasma drug concentration as function of time of the formulations following the subcutaneous injection was plotted and shown in Fig. 7. The pharmacokinetic parameters of DS, calculated using non-compartmental model are shown in Table 4. The Cmax for DP18, DP20, DPM15/3 and DMPG3/2 was significantly high (Fig. 7) as compared to the conventional DS formulation. The Cmax obtained were in the order of DPM15/3 > DMPG3/2 > DP20 > DP18 > reference, the values were

Formulation

3.2. In vivo evaluation of DS in situ gels

Table 4 Pharmacokinetic parameters of diclofenac sodium commercial formulation and in situ gel after subcutaneous administration.

Formulation DPM15/3 contains 15% Pluronic P-F127 and 3% methyl cellulose. MC is a high molecular weight water soluble polymer, when dissolved in water the MC solutions are highly viscous which is concentration dependent, the high viscosity might be the reason for retarding the drug release from the formulation DPM15/3. In case of DPM15/3, high drug diffusion rate was observed that may be due to rapid water uptake that enhance the drug release rate (Gong et al., 2009b) from the formulation compared with the DPM20 and DPM18. The value of release exponent in case of DMPG1.5/10 suggests that the polymer degrades rapidly as a result of its dissolution and erosion. The formulation was not suitable for subcutaneous route as it could only retard the drug release for 4 h.

Mean ± SD

DPM15/3

Fig. 5. Cube root% diclofenac sodium remaining (W0 –Wt ) vs. time (Hixson–Crowell model) from diclofenac sodium in situ thermoreversible sol–gel.

0.003 0.001 – 0.001 0.001 0.001 0.001 0.000 0.001 0.003

Reference

0.19 0.061 0.0 0.15 0.09 12.43 0.31 4.56 0.10 0.19

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4. Conclusion

Fig. 7. Plasma concentration (␮g/ml) of diclofenac sodium at various time intervals after subcutaneous injection of commercial diclofenac sodium injection and DS in situ gels.

2.724 ± 0.06, 2.640 ± 0.01, 2.4017 ± 0.109, 2.3798 ± 0.16, and 0.671 ± 0.061, respectively. The higher Cmax may be due to the gel that remains intact and remains for the longer duration at the site of absorption releasing the drug continuously at constant rate. The Tmax obtained for the formulations DP18, DP20 and DPM15/3 was 48 h while for DMPG3/2 the Tmax was one hour for reference it was 2 h. The plasma drug concentration–time plot of formulations DP18, DP20 and DPM15/3 shows two peak concentrations, the first peak was observed at first hour indicating the burst release of drug (Hatefi and Amsden, 2002), which is due to transition lag time from solution to gel of the in situ gels. As the transition to gel phase is governed by micelle formation which is temperature and concentration dependent. The second high concentration peak is observed at 48 h, this second high concentration peak is due to the dissolution (Anderson et al., 2001) and degradation of the polymer with rapid drug release. The p-values were also significant for the pharmacokinetic parameters AUC0–t , AUC0–∞ , Vd and Cl and elimination half life. The values of AUC0–t , AUC0–∞ , MRT were significantly high compared to the reference, while the elimination half life, Vd and clearance were significantly low indicating the slow release of the drug from gel-formulations, The AUC0–t values indicate that bioavailability of diclofenac from DP18, DP20, DPM15/3 and DMPG3/2 was 21.5, 34, 18.6 and 6.5 fold, respectively, higher than the conventional solution. The relative bioavailability of DS from DP18, DP20, DPM15/3 and DMPG3/2 was 227%, 367%, 161% and 65%, respectively, higher compared with reference formulation. The elimination half-life obtained was in the order of DP20 > DP18 > DMPG3/2 > DPM15/3 > reference, the values were 23.109 ± 9.70, 18.363 ± 1.03, 9.071 ± 0.76, 6.785 ± 0.07 and 4.038 ± 0.19, respectively. Mean residence time (MRT) of all thermoreversible formulations was high compared to the reference formulation. The higher values of half life and MRT indicate that DS is slowly released from thermoreversible formulations maintaining the steady state plasma drug concentration. The plasma drug concentration–time plots indicates that the formulation DP20, DP18, DPM15/3 and DMPG3/2 maintained the plasma drug concentration well above the MEC of DS (Nishihata et al., 1988) for, 96, 72, 72 and 48 h, respectively. On the other hand the conventional commercial parenteral solution could only maintain the plasma drug concentration for 12 h.

In vitro evaluations of the formulations divulge that autoclaving has no significant effect on the Tsol–gel, viscosity and clarity of the solutions, while it significantly decreased the drug content of all the formulations. The in vitro drug release data indicates that the formulation DP20, DP18 follow the Higuchi model while DPM15/3, DMPG3/2 and DMPG1.5/10 followed Hixson–Crowell model. The drug release from the DP20 and DP18 was independent of drug concentration following zero order equation and drug release was concentration dependant in case of DPM15/3, DMPG3/2 and DMPG1.5/10. The release exponent values obtained from the Korsmeyer–Pappas equation suggests that the drug release from DP20 and DPM15/3 follow diffusion, formulations DP18 and DMPG3/2 followed anomalous transport, while drug release from DMPG1.5/10 followed super case II mechanism. The plasma drug–time profile values suggested that the in situ thermorevesible gels prepared with Pluronic, MC and their combination increased the absorption and prolong the elimination half-life of diclofenac sodium resulting in better bioavailability compared with the conventional parenteral solution. Among various in situ gels formulations DP20 was the most robust to extended the in vitro drug release for 120 h that was fully supported by the in vivo data obtained. The study also revealed that the combination of pluronic and methyl cellulose (DPM15/3) show comparable drug control and sustainability as that of the pluronic formulations. Consequently, the DS content from the administration of conventional parenteral preparation could not be detected in the plasma after 12 h indicating that it was eliminated from the body or below the limit of detection. The developed formulations are easy to manufacture, physically more stable and easy to administer as compared to suspensions, emulsions and ointments. These formulations will help in reducing the dosage frequency and also will maintain consistent plasma drug level and will reduce the systemic side effects. Acknowledgments The authors are highly grateful to the University of Peshawar for providing research financial support for the project under the Financial Support Research Program for the academic year 2010–11. References Alexandridis, P., Alan Hatton, T., 1995. Poly (ethylene oxide)–poly (propylene oxide)–poly(ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling. Colloids Surf. A 96, 1–46. Anderson, B.C., Pandit, N.K., Mallapragada, S.K., 2001. Understanding drug release from poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) gels. J. Control. Release 70, 157–167. Arnott, S., Fulmer, A., ScottI Cm, W.E., Moorhouse, R., Rees, D.A., 1974. The agarose double helix and its function in agarose gel structure. J. Mol. Biol. 90, 269–272. Bhardwaj, R., Blanchard, J., 1996. Controlled-release delivery system for the alpha MSH analog Melanotan-I using poloxamer 407. J. Pharm. Sci. 85, 915–919. Brogden, R.N., Heel, R.C., Pakes, G.E., Speight, T.M., Avery, G.S., 1980. Diclofenac sodium: a review of its pharmacological properties and therapeutic use in rheumatic diseases and pain of varying origin. Drugs 20, 24. Catella-Lawson, F., Reilly, M.P., Kapoor, S.C., Cucchiara, A.J., DeMarco, S., Tournier, B., Vyas, S.N., FitzGerald, G.A., 2001. Cyclooxygenase inhibitors and the antiplatelet effects of aspirin. N. Engl. J. Med. 345, 1809–1817. Chandrashekhar, G.U.N., 1998. Biodegradable injectable implant system for long term drug delivery using poly(lactic-co-glycolic) acid copolymers. J. Pharm. Pharmacol. l48, 669–674. Dumortier, G., Grossiord, J., Agnely, F., Chaumeil, J., 2006. A review of poloxamer 407 pharmaceutical and pharmacological characteristics. Pharm. Res. 23, 2709–2728. Gilbert, J.C., Richardson, J.L., Davies, M.C., Palin, K.J., Hadgraft, J., 1987. The effect of solutes and polymers on the gelation properties of pluronic F-127 solutions for controlled drug delivery. J. Control. Release 5, 113–118.

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