INTRODUCTION:

Atorvastatin calcium is a synthetic lipid-lowering agent. It is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, an early and rate-limiting step in cholesterol biosynthesis. The molecular weight of Atorvastatin calcium is 1155.36 D which is more than 500D so it is suitable technique for this drug. It is rapidly absorbed after oral administration, the maximum plasma concentrations occur within 1 to 2 hours. The absolute bioavailability of atorvastatin calcium is approximately 14%. The food also decreases the rate and extent of drug absorption by approximately 25%. ^{(1, 2)}

Iontophoresis is a process by which the transport of ions into or through the skin is increased by application of an external electrical field across the skin. Iontophoresis is only now being studied systemically for its application to the fields of the drug therapy, diagnosis and monitoring. Iontophoresis includes use of low-level electric current (0.5mA/cm²) to drive both charged and even uncharged compound across the skin at rate very much greater than passive permeability.

The highly polar, high molecular weight and frequently charged nature of these compounds has provoked considerable new research into the mechanism and application of electrically controlled drug delivery through the skin. In particular the potential of Iontophoresis to truly control transdermal transport rates is a singular advantage and there is good evidence that the current profile can be manipulated to vary the kinetics and extend of drug absorption⁽³⁾.

The aim of the present study was to formulate and evaluate the iontophoretic transdermal patches of atorvastatin calcium and investigates the influence of electrical factors like current density, current profile and current application duration and device related factors like electrode material on the iontophoretic transport of atorvastatin calcium through the rat skin.

MATERIALS AND METHODS:

Materials:

Atorvastatin calcium was obtained as gift sample from Micro lab- Bangalore, India. Sodium alginate, HPMC K4M, Sodium CMC, potassium dihydrogen ortho phosphate, sodium chloride were purchased from S.D fine- chem. Ltd., Mumbai, India. Iontophoretic instrument was custom designed.

Preparation of monolithic matrix film:

Solvent casting method: ⁽⁴⁾

Transdermal films were prepared by solvent casting method. The different polymers were accurately weighed and transferred to a 20 ml beaker; the beaker was kept on magnetic stirrer to dissolve the polymer. The mixture was stirred continuously to prevent the formulation of lumps of polymer .Stirring was continued till a clear solution was obtained. Then the accurately weighed quantity of Atorvastatin calcium was added and stirring was continued. The resulting solution was then transferred into the Petri dish. It was covered with inverted funnel to control the rate of evaporation of the solvent system. Finally it was kept in the hot air oven at 40⁰c for drying and kept undisturbed for overnight and packed with aluminum foil.

Table no 1: Formulation Design

Ingredients	F1	F2	F3
Atorvastatin calcium	80 mg	80 mg	80 mg
Sodium alginate	400 mg	-	-
HPMC K4M	-	400 mg	-
Sodium Carboxy methyl cellulose	-	-	400 mg
Glycerin	0.4 ml	0.4 ml	0.4 ml

EVALUATION OF PREPARED TRANSDERMAL PATCHES: ^{(5, 6,
7,8)}

Physical Appearance: The prepared patches were evaluated for their physical appearance.

Uniformity of weight:

The film was cut into 10 patches of 1cm²each and their average weight was calculated. Percentage deviation from average weight for each patch was also determined.

Film thickness uniformity:

The thickness of each patch was measured at the different sites using screw gauge and the average thickness was calculated.

Folding endurance:

This was determined by repeatedly folding the patches until it shows any crack or break. The number of times the film could be folded without breaking/cracking gave the value of folding endurance. Five randomly selected patches for each formulation were tested.

Percentage moisture loss:

The film was weighed and kept in a desiccators containing calcium chloride at 40^oC in a drier for at least 24 h or more until it showed a constant weight. The moisture content was the difference between the constant weight taken and the initial weight and was reported in terms of percentage (by weight) moisture content.

Percentage moisture loss = Initial weight - Final weight x 100

Initial weight

Percentage moisture absorption:

The film were weighed accurately and placed in desiccators containing 100 ml of saturated solution of aluminum chloride (79.50 % RH). After 3 days, the films were taken out and weighed, the percentage of moisture uptake was calculated as the difference between final and initial weight with respect to initial weight.

Percentage moisture absorption = Final weight - Initial weight x 100

Initial weight

Drug content uniformity⁽⁹⁾

The prepared patch was cut into 1cm² and put into 10 ml diffusion medium used respectively and stirred continuously using a mechanical stirrer and sample was withdrawn at the end of three hours and the drug content was determined by UV spectrophotometer at 245nm.

Preparation of skin: ⁽¹⁰⁾

Full thickness skin excised from abdominal portion of male Wistar rats (200-250g) was used in all the experiments. Before excision, the abdominal part of the skin was shaved with electrical shaver and uniform circle was made on the abdomen making the precise section to be positioned between the half cells. This was done to avoid the differences of stretching the skin during removal. Using phosphate buffer Ph 7.4 as a receptor solution.

Fig:- Physical appearance of monolithic transdermal film

Electrodes:

All transport studies were carried out using a pair of platinum wire (99.99% purity, 0.5 mm diameter) as working electrodes with negatively charged electrode (cathode) in the donor compartment and positively charged electrode (anode) in the receptor compartment.

Design of Modified Franz Diffusion Cell for passive method: ^(11,12,13)

Franz diffusion cell consists of an upper donor compartment and the lower receptor compartment, surrounded by water jacket for circulation of water to maintain the temperature inside at 37 ± 1⁰C.The uniformity of solution in the receptor phase was maintained by stirring at high speed of 50 rpm (approx) using a tiny magnetic bead the volume of receptor compartment was maintained at 60 ml. The receptor compartment was provided with the sampling port on one side, to withdraw 3 ml of sample at the predetermined time intervals for estimation of drug content by UV spectrophotometer.

Diffusion Cell for iontophoresis^{(3,11,14,15,16)}:

In vitro permeation studies were carried out by using a diffusion cell with a diffusion surface area of 2.3cm². Diffusion cell placed on the receptor compartment acted as donor compartment. The receptor compartment was filled with phosphate buffer PH 7.4 and receptor phase was stirred with small magnetic beads to mix contents uniformly. Receptor phase was maintained at 37±1°C. Rat skin along with loaded polymeric film was tied to the donor compartment placed in position with the receptor compartment. Surface of the membrane dipped in the receptor fluid. The anode was placed in the donor compartment and the cathode was placed in the receptor compartment. Current 0.5 mA/cm² was applied for period of 2 hours by using platinum electrode. 5ml sample drawn out at regular intervals up to 24 hrs. The drug content of collected sample was determined by UV visible spectrophotometer at 245nm. After each interval the same quantity of fresh medium was replaced immediately.

Effect of current density and duration:

The experiment was conducted with constant direct current of 0.3, 0.5 and 0.7 mA/cm²applied to optimized formulation F1 and period 2 hrs. The constant electric current 0.5 mA/cm²was applied to remaining formulations F2 and F3 for 2 hrs.

Accelerated Stability Studies: ⁽¹⁷⁾

The optimized patches were subjected to stability studies to evaluate any change in the performance when exposed to accelerated conditions of environment during storage, handling transport and use. The patches were packed in the aluminium foil and kept at 40± 2^oC and 75 ±5% RH as per ICH guidelines. (ICH Q1A [R2] Stability testing of new drug substances and products).

RESULTS:

Table no 2: Physicochemical evaluation of Prepared Transdermal Patches

S. No.	Formulation code	Weight variation in mg	Thickness in mm	Folding endurance	Percentage Moisture loss	Percentage Moisture absorbed
1	F₁	7.670.40	0.410.05	1582.51	1.87  0.03	6.17 0.17
2	F₂	8.530.58	0.520.11	1722.51	2.58  0.09	4.12  0.22
3	F₃	6.7 0.20	0.38  0.01	1672.51	1.20  0.16	2.14  0.14

* All values are expressed as mean ± S.D, n= 3.

Table no: 3 Drug Content Uniformity

Formulation code	Percentage of drug in 1 sq cm
Formulation code	1	2	3	4	5	*Mean
F1	98.76	96.90	98.31	98.56	98.73	98.25
F2	94.65	94.60	94.35	94.60	94.62	94.56
F3	89.50	89.25	89.50	89.57	89.42	89.45

Table no 4: Comparative in - vitro % Drug Permeation Data for all Formulation

Time in hours	F1 passive	F2 passive	F3 passive	F1 0.3 mA/cm²	F1 0.5 mA/cm²	F1 0.7 mA/cm²	F2 0.5 mA/cm²	F3 (0.5 mA/cm²)
0.5	4.23	5.70	3.98	6.28	9.31	11.13	4.71	3.43
1	10.24	10.44	7.67	11.41	11.71	12.79	8.70	6.85
2	14.97	16.60	15.35	16.57	14.90	15.88	12.37	11.96
4	23.44	21.11	21.35	21.11	22.50	23.96	15.19	13.78
6	35.63	29.10	28.77	29.57	31.10	32.55	21.21	19.24
10	44.44	41.74	39.28	43.16	44.20	45.71	35.36	32.68
14	51.60	51.02	46.27	55.71	58.47	59.97	44.87	43.09
18	62.54	61.81	58.11	69.44	71.53	73.53	60.73	58.04
22	68.42	71.10	63.63	76.77	78.58	80.65	73.85	70.61
24	76.27	73.28	69.58	80.40	82.78	86.43	79.75	77.51

Graph no 1: Comparative Plot of In Vitro % Drug Permeation of all Formulation

Graph no 2: Comparative Plot Of In Vitro % Drug Permeation of Formulation F1 by Using Different Current Density

Table no: 5 Kinetics values obtained from different plots Formulations (F1-F3)

Formulation	Zero order plot	First order plot	Higuchi plot	Korsmeyer Peppa’s Plot
Formulation	R²	R²	R²	R²
F1 (passive)	0.9357	0.9468	0.9357	0.9304
F2 (passive)	0.925	0.9317	0.925	0.9561
F3 (passive)	0.9278	0.9486	0.9275	0.9924
F1 (0.3 mA/cm²)	0.9523	0.9909	0.963	0.9867
F1 (0.5 mA/cm²)	0.9484	0.9899	0.9647	0.9891
F1 (0.7 mA/cm²)	0.9422	0.9808	0.9674	0.9879
F2 (0.5 mA/cm²)	0.9899	0.9571	0.9061	0.9664
F3 (0.5 mA/cm²)	0.9933	0.9579	0.8944	0.971

SEM Image of optimized formulation F1

SEM Image of F1 After 90 days

DISCUSSION:

In the present study three formulations (F1-F3) with different polymer (sodium alginate, HPMC K4M and sodium CMC) were prepared and evaluated for various physico-chemical parameter, In-vitro drug permeation study by passive method and in-vitro Permeation study by Iontophoresis. On the basis of drug content and In-vitro permeation studies, the best formulation was selected.

The cumulative amount of Atorvastatin calcium permeated through excised skin under various conditions of current density, and duration of current application shows a substantial increase in the amount of drug permeated compared to passive diffusion across the skin. Table no 4 enumerate the details of drug release under various time intervals.

Total 3 formulations were prepared with different polymers (table no 1). The prepared transdermal patches were then evaluated for various physico-chemical tests like thickness, folding endurance, weight variation, percentage moisture loss, and percentage moisture absorption and drug content uniformity and viscosity of matrix mixtures. The thickness of the transdermal patches was uniform in all formulations and they were found to be flexible and smooth. The weight variation of the all film value ranged from 6.72 ±0.20

to 8.53±0.40 (Table No.2). The thickness of the all film value ranged from 0.38±0.0.005

to 0.52±0.115 (Table No.2).

The results were found to be uniform with low SD value. The folding endurance of the all film value ranged from 158±2.510

to 172±2.516 (Table No.2). The percentage moisture loss of the all film value ranged from 1.20±0.03

To 2.58±0.16 %( Table No.2).

The percentage moisture absorption of the all film value ranged from 2.14±0.14 t

o 6.17±0.0.22% (Table No.2). The viscosity of all formulations ranged from 8.85

±0.38 to 8140 ± 181.4 c^p.

The results of all physico-chemical tests were found to be satisfactory. Drug content was also found to be uniform among the all formulations and ranged from 89.50 to 98.76 % (Table No. 3).

Passive diffusion experiment:

In vitro permeation studies were carried out using rat skin in a diffusion cell. The cumulative percentage drug permeation was found to be 76.27%, 73.28%, and 69.58% for formulation F1, F2, and F3 respectively. The studies indicated that this film were permeable to drug Atorvastatin calcium showed lesser permeable.

Iontophoresis of Atorvastatin calcium:

In vitro permeation studies were carried out on rat skin in a diffusion cell by using Iontophoresis (0.5mA/cm²) for 2hour with direct current. Iontophoresis of Aorvastatin calcium Increase pore size of the skin and help in an easy permeation of drug through skin. The cumulative percentage drug permeation was found to be 82.78%, 79.75%, and 77.51 % for formulation F1, F2, and F3 respectively.

In this respect formulation F1 showed best result among all formulation. Formulation F1 showed better rate controlling membrane as after application of current of 0.5mA/cm²on the transdermal patch. The formulation F1 was subjected to different current density Viz 0.3mA/cm², 0.5mA/cm²and 0.7mA/cm²Permeability gradually increased and cumulative percentage drug permeation was found to be 80.40 %, 82.78% and 86.43 % respectively. The rate of drug delivery was found to be proportional to the current flowing through circuit.

Mechanism of drug permeation:

In order to understand the complex mechanism of drug permeation from the passive and Iontophoresis transdermal patches, the in vitro Atorvastatin calcium permeation data were fitted to Korsmeyer Peppa’s permeation model and interpretation of permeation exponent values (n) enlightens in understanding the permeation mechanism from the dosage form^(18,19). The permeation exponent values thus obtained were from 0.61 to 0.76. Based on these values we can say that the formulations F1 to F3 (passive and Iontophoresis) exhibited (non-fickian transport) diffusion mechanism. The drug permeation was diffusion controlled as the plot of Higuchi’s model was found to be linear (r>0.9357). While the formulations F1 to F3 showed higher R² values for zero order plot indicating that drug permeation followed zero order kinetics. This finding (table no 5) reveals that above a particular concentration, Polymers are capable of providing almost zero order drug release.

After 90 days accelerated stability studies was performed, all various physico-chemical properties were not changed relatively normal evaluation parameters. The physical appearance only slightly changed (SEM image).

CONCLUSION:

Atorvastatin calcium being high molecular weight can be more effectively transported iontophoretically, by optimizing current intensity transdermal penetration of the drug can be improved compared to passive diffusion. Linear regression analysis of the drug dissolution profile and the drug diffusion profile showed that the mechanism of drug release was following diffusion pattern. The prepared patches could provide the delivery of drug at a controlled rate across intact skin and might be used for transdermal purposes. The formulations really improve the bioavailability, patient compliance and avoiding the first pass metabolism. Further work has to be carried out to establish the therapeutic utility of these systems in humans.

ACKNOWLEDGEMENTS:

The authors are thankful to the management, the Erode College of pharmacy and research institute, for providing necessary facilities to carry out this work.

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Received on 03.09.2010 Modified on 11.10.2010

Research J. Pharm. and Tech. 4(5): May 2011; Page 730-734

Table no 2: Physicochemical evaluation of Prepared Transdermal Patches

Percentage of drug in 1 sq cm

1

2

3

4

5

*Mean

SEM Image of optimized formulation F1

SEM Image of F1 After 90 days

Mechanism of drug permeation: