GYAN VIHAR UNIVERSITY

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1 7. In Vitro Skin Permeation Studies In vitro skin permeation studies were performed on a Franz diffusion cell with an effective diffusional area of cm2 and 4 ml of receiver chamber capacity using rat abdominal skin. The automated transdermal diffusion cell sampling system (SFDC6, Logan Inst, Avalon, NJ) was used for these studies. The fullthickness rat skin was excised from the abdominal region, and hair was removed with an electric clipper.the subcutaneous tissue was removed surgically, and the dermis side was wiped with isopropyl alcohol to remove adhering fat. The cleaned skin was washed with distilled water and stored in the deep freezer at 21 C until further use. The skin was brought to room temperature and mounted between the donor and receiver compartment of the Franz diffusion cell, where the stratum corneum side faced the donor compartment and the dermal side faced the receiver compartment. Initially the donor compartment was empty and the receiver chamber was filled with ethanolic phosphate buffered saline (PBS) ph 7.4 (20:80% vol/vol). The receiver fluid was stirred with a magnetic rotor at a speed of 600 rpm, and the assembled apparatus was placed in the Logan transdermal permeation apparatus and the temperature maintained at 32 C ± 1 C. All the ethanolic PBS was replaced every 30 minutes to stabilize the skin. It was found that the receiver fluid showed negligible absorbance after 4.5 hours and beyond, indicating complete stabilization of the skin. After complete stabilization of the skin, 1 ml of nanoemulsion formulation (20 mg/ml AT calicum) or 1 g of CG (20 mg/g) was placed into each donor compartment and sealed with paraffin film to provide occlusive conditions. Samples were withdrawn at regular intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,20, 22, and 24 hours), filtered through a membrane filter, and analyzed for drug content by UV spectrophotometer at λ max of 274 nm. The formulation GM1 provided the highest 174

2 release as compared with the other nanoemulsion formulations. The formulation F1 was also converted into nanoemulsion formulations by adding 1% wt/wt Carbopol 940 and was coded as GM2. The skin permeation profile of the optimized nanoemulsion formulation was compared with nanoemulsion (GM1) and CG using the Dunnett test of 1-way analysis of variance (ANOVA). Permeation Data Analysis The cumulative amount of drug permeated through the skin (mg/cm2) was plotted as a function of time (t) for each formulation. Drug flux (permeation rate) at steady state (Jss) was calculated by dividing the slope of the linear portion of the graph by the area of the diffusion cell. The permeability coefficient (Kp) was calculated by dividing Jss by the initial concentration of drug in the donor cell (C0): K p = J s s C 0 (1) Enhancement ratio (Er) was calculated by dividing the Jss of the respective formulation by the Jss of the control formulation: E r = J s s of formulation J s s of control (2) Table 7: Permeability Parameter of Different Formulations (n= 3)* Formulations Jss SD(mg/cm 2 /h) K p SD(cm/h) E r GM Tablet Suspension API drug Suspension GM Tablet Suspension API drug Suspension

3 Skin Irritation Test The skin irritation test was carried out on male Swiss albino mice weighing 20 to 25 g. The animals were kept under standard laboratory conditions, with temperature of 25 C ± 1 C and relative humidity of 55% ± 5%. The animals were housed in polypropylene cages, 6 per cage, with free access to a standard laboratory diet (Lipton feed, Mumbai, India) and water ad libitum. A single dose of 10 μl of the nanoemulsion was applied to the left ear of the mouse, with the right ear as a control. The development of erythema was monitored for 6 days using the method of Van-Abbe et al. Bioavailability Study Of Atorvastatin Nanoemulsion In Rats The ultimate test of success of a formulation depends on its in vivo performance and for the assertion of the same the in vivo studies were conducted in a suitable animal model. Approval to carry out in vivo study was obtained from Jamia Hamdard, Institutional Animal Ethics Committee and their guidelines were followed for the studies (173/CPCSEA, 28 Jan 2000; Form no 337). The animals used for in vivo experiments were adult wistar female albino rats obtained from Central Animal House of Hamdard University, New Delhi, India. The nanoemulsion formulation GM1 that showed the highest release profile of drug in in vitro studies was subjected to in vivo studies. Following is the description of the procedure followed during the in-vivo studies Specification for the animals used Animals used for the study: Rats a) Species: Albino Wistar 176

4 b) Age/ weight/size gm c) Gender: Female d) Total no. of formulations: Three GM1 Nanoemulsion Tablet, 10 mg (Atorlip 5) Drug Suspension of API e) No. of animals in each group: Six per group f) Total time of study and no. of blood samples for each animal: The blood sampling was carried out for around 48 hours and 10 to 12 samples of blood were taken from each animal in the group. g) Actual time interval for sampling: As per the Table Dose Calculation Dose for the rats was calculated on the basis of body weight of the animal model (Balck et al., 1999, Shen and Zhong., 2006; Freireich, E.J., 1996). For the present study 6 mg/kg (Shen and Zhong., 2006) body weight of the Atorvastatin Ca was used. As the bioavailability study was conducted with an average animal weight of about gm, accordingly the calculation was done. Hence, 1.2 mg of the drug containing formulation was fed to each animal in the designated group. The blood samples were withdrawn as per the plan given in the Table

5 Preparation of doses from different formulations a)tablet (Atorlip -Label claim 10 mg) Six tablets with label claim of 10 mg (Atorlip ) were taken, crushed in mortar, and mixed with 50 ml of double distilled water (1.2 mg/ml). Each rat in this group was given 1.0 ml using oral feeding needle. b) API drug suspension 12 mg of Atorvastatin powder was exactly weighed and mixed with 10 ml of double distilled water (1.2 mg/ml). Thereafter, each rat was given 1.0 ml of this suspension using oral feeding needle. c) Nanoemulsion (GM1) Based on the average weight (200 g) of each rat in this group a dose of Atorvastatin (1.2 mg) was constituted into 1 ml of working formula (Table 7.1). The formulation was then given to the rats orally with the help of feeding needle. 178

6 Table 7.1: Working formula for nanoemulsion (GM1 Oil : Sefsol 218+ Oleic acid, Surfactant : Tween 20, Cosurfactant : Carbitol Volume of components for Nanoemulsion Formulation Code Oil (ml) Surfactant Co surfactant Aqueous Atorvastatin (ml) (ml) phase (ml) (mg) GM Procedure The animals were kept under standard laboratory conditions at a temperature of 25 ± 2 C and relative humidity (55 ± 5%). The animals were housed in animal cages, six per cage, with free access to standard laboratory feed (Lipton feed, Mumbai, India) and water ad libitum. The formulations were given orally using oral feeding needle. Dose for the rats was calculated based on the weight of the rats (6 mg/ kg) (Balck et al., 1999; Shen et al., 2005; Shen and Zhong., 2006). The rats were anesthetized using diethyl ether and blood samples (0.5 ml) were withdrawn from the tail vein of rat at 0 (pre-dose), 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 6, 12, 24, 36 and 48 h in micro centrifuge tubes. Tubes were stored at room temperature, 25 ± 2 C and relative humidity (55 ± 5%) for 30 minutes. The clotted blood was then centrifuged at 5000 rpm for 30 min. The serum was separated and stored at -21 C until drug analysis was carried out using RP- HPLC method. The collected serum was extracted with ethyl-acetate for analysis and centrifuged at 5000 rpm for 5 min and dried at room temperature. After evaporating the ethyl acetate solid residue was reconstituted using 100 µl mobile phase and then analyzed by RP-HPLC method (Chapter 5, Page no ). The drug from each collected serum was extracted with ethyl-acetate (5 ml) by using Sodium phosphate buffer (ph 7, 1 ml) as a protein-precipitating agent. The samples were 179

7 injected and the area was noted down in triplicate. The mean areas obtained by HPLC are given in the Table Pharmacokinetic and statistical analysis Pharmacokinetic parameters were calculated by using non-compartmental analysis also called as Model independent analysis using WinNonLin version 4.0 (Pharsight Corp., Mountain View, CA). All pharmacokinetic (PK) parameters (t max, C max, AUC 0 t, AUMC 0 t and MRT 0 t) were calculated individually for each subject in the group and the values were expressed as mean ±SD. The comparative in-vivo bioavailability profiles of nanoemulsion formulation GM1, API suspension and Tablet suspension were shown in the Table 7.4. Table 7.2: Comparative observation table for in-vivo bioavailability studies in rat animal model (n=6). Sampling time SNEDDS Tablet suspension API suspension (Hours) (Mean Concn SD) (Mean Conc SD) (Mean Conc SD)

8 Concentration (ng/ml) CHAPTER 7 IN-VITRO AND IN-VIVO STUDIES Table 7.3: Relative pharmacokinetic parameters of different formulations containing AT Calcium (n = 6). Formulation t max a C max b (ng/ml) AUC 0 t c Rel. BA f (hrs) (ng.h/ml) % GM Tablet Suspension API drug Suspension a time of peak concentration; b peak of maximum concentration; e area under the concentration time profile curve until last observation; f Relative bioavailability of formulations Comparative bioavailability profile in Rat Time (Hrs) SNEDDS Formulations Tablets Suspension API Suspension 181

9 Fig 7.1: Comparative In-vivo absorption profile of atorvastatin using different formulations in rat model 7.4. Result and discussion In Vitro Skin Permeation Studies In vitro skin permeation studies were performed to compare the release of drug from 6 different nanoemulsion formulations (GM1-GM6), NG1, and CG, all having the same quantity (2% wt/wt) of AT calicum. In vitro skin permeation was highest in formulation F1 and lowest for CG (Figures 3 and 4). The formulation GM1 showed an intermediate skin permeation profile. The skin permeation profile of GM1 was significantly different when compared with that of CG and GM1 (P <.05). The significant difference in AT calicum permeation between nanoemulsion formulations, GM1, and CG was probably due to the mean size of internal phase droplets, which were significantly smaller in nanoemulsions. The maximum release in GM1 could be due to having the lowest droplet size and lowest viscosity of all the nanoemulsions. Permeation Data Analysis Permeability parameters like steady-state flux (Jss), permeability coefficient (Kp), and enhancement ratio (Er) were significantly increased in nanoemulsions and the GM1 formulation as compared with CG (P <.05). This is because nanoemulsions and GM1 excipients contain permeation enhancers like Labrafil, Triacetin, Tween 80, and Transcutol P. The permeability parameters of different formulations are given in Table 7. Skin Irritation Test The skin irritation test was performed to confirm the safety of the optimized nanoemulsion formulation. Van-Abbe et al 27 mentioned that a value between 0 and 9 indicates that the applied formulation is generally not an irritant to human skin. The mean skin irritation score for formulation GM1 was 2.12 ± From this it was concluded that the optimized 182

10 nanoemulsion formulation was safe to be used for transdermal drug delivery. AT Calcium plasma concentration values as assessed by the HPLC studies are shown in Table 7.2 while the graphical representation of the drug in rat serum is depicted in Fig The pharmacokinetic profiles are shown in (Table 7.3), C max of AT Calcium in GM1, tablet suspension and API suspension was ± , ± , and ± ng/ml respectively whereas AUC0 t was ± , ± and ± ng.hr/ml respectively. The percentage relative bioavailability of the GM1 was found to be % and % as compared to the Tablet suspension and the API drug suspension of AT Calcium respectively, indicating the influence of the nanosizing of the oil droplets on the bioavailability. The quick onset of the drug action in the body is usually depicted by the presence of a lesser Tmax value. There was a marked difference in the Tmax of GM1 formulations in comparison to the Tablet suspension and API drug suspension. It was found to be 0.96±0.102 hrs. It could be deduced from the above results that the formulation GM1 acted successfully in vivo and was able to reach a maximum concentration in minimum possible time while also having an increased extent of bioavailability. As can be seen in Table 7.2, and 7.3, tmax and C max of GM1 were 0.96±0.102 h and ± ng/ml respectively as compared to tablet suspension which were ±1.95 h and ± ng/ml, and API drug suspension 2.04-±1.94 h and ± ng/ml respectively. Statistically, the difference in t max of GM1 was significant (p<0.05) when compared to t max of tablet suspension and API drug suspension where as the difference was insignificant (p>0.05) when compared to tablet (Table 7.2). The difference in C max of GM1 formulation was highly significant (p<0.01) when compared with the tablet suspension formulation and API drug suspension. It was also observed that AUC 0 t GM1 formulation was 183

11 ± ng.h/ml and thus the difference was highly significant (p<0.01) as compared to AUC 0 t ( ± ) and ( ± ) of tablet suspension and API suspension formulations respectively (Table 7.3). 184