Preparation and in vitro percutaneous penetration of simvastatin ethosome gel

Abstract

To prepare ethosome gel containing simvastatin ethosome and investigate the permeation behavior of simvastatin from ethosome gel. Cumulative permeation quantity in unit area and intradermal retention were the indicators to evaluate the effects of simvastatin in vitro percutaneous permeation behavior. Cumulative permeation quantity in unit area of simvastatin ethosome gel was significantly higher than other agents (P < 0.05), the intradermal retention of simvastatin ethosome gel, simvastatin gel containing 1%, and 3% menthol were significantly higher than simvastatin gel (P < 0.05). Ethosome gel could enhance the skin permeation and accumulation in a depot of simvastatin.

Keywords::

• ethosome
• gel
• percutaneous penetration
• simvastatin

Introduction

Simvastatin is a HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitor. It has been shown to be especially effective in reducing low-density lipoprotein cholesterol (LDL) and improve other conditions that are not influenced by lipid levels, including coronary artery diseases (Lagrost et al. 1999). Simvastatin has a high degree of selectivity in liver after oral administration, almost 95% simvastatin is metabolized by liver and excreted through bile. Only less than 5% of the active ingredient is found in the blood circulation after oral administration (Krysiak and Okopien 2010). Existing simvastatin formulations include tablet, capsule, dry suspension, orally disintegrating tablet, and particles without lubricant, and they have low bioavailability after oral administration but cause a huge burden on liver.

Ethosome system contains a large amount of ethanol which brings a good transdermal property (Barry 2001). We prepared a transdermal formation, simvastatin ethosome gel, which simvastatin can penetrate through the skin and into the blood circulation. Therefore the transdermal administration of simvastatin ethosome could be expected to relieve the burden of liver and much lesser dosage of simvastatin ethosome could be bioequivalent to the oral administration which has 5% bioavailability.

Materials

Reagents

Simvastatin ethosomes (An et al. 2011) (self-made, 20110309); Carbomer, Qingdao Tianliyuan biotechnology corporation. Triethanolamine, Tianjin FuYu fine chemicals. Menthol, Tianjin Kemiou chemicals. Simvastatin standard reference substance (mass fraction of 100%), National institutes for food and drug control. Simvastatin API (mass fraction of 98.7%), Zhejiang Hisun pharmaceutical Co., Ltd.

Instruments

Agilent 1200 (G1314B UV detector, G1314A pump, Rev.B.04.02[98] chromatography workstation). ZTY intelligent transdermal diffusion instrument TP3A (temperature range: ‐40°C~room temperature, temperature precision: ± 0.1°C, transdermal instrument diffusion area: 3.4 cm², receiving cup capacity: 10 ml, transdermal Cup: 6 cups), Gongyi Yuhua instrument Co., Ltd.

Animals

Kunming Mice, male, 20 ± 5 g, experimental animal center, Tongji medical college, Huazhong University of science and technology. Animals certificate: SCXK (E) 2004˜2007.

Methods

Preparation of simvastatin gel

0.5 g carbomer was sprinkled in 8 ml water and placed in the refrigerator overnight for swelling, adjusted pH to 7 with triethanolamine (about 0.67 g), then added the other components according to the prescriptions and distilled water to 50 g, kept stirring until the uniform swelling. The concentration of simvastatin in each preparation was 1.2 mg/g, menthol in prescription Ⅱ and prescription Ⅲ were 1% and 3%, respectively (Table I).

Table I. Prescriptions of simvastatin gel.

Prescription	I	II	III	IV
Simvastatin ethosome	0	0	0	40 ml
Simvastatin	0.06 g	0.06 g	0.06 g	0
Carbomer	0.5 g	0.5 g	0.5 g	0.5 g
Glycerol	0.5 g	0.5 g	0.5 g	0.5 g
Menthol	0	0.5 g	1.5 g	0
Ethanol （75%）	30 ml	30 ml	30 ml	0
Distilled water	To 50 g	To 50 g	To 50 g	To 50 g

Process of mouse skin

Executed the mice by cervical dislocation, shaved off the hair on the abdominal skin by electric shaving machine, pinned the stripped abdominal skin on the foam board and wetted with NS, removed the subcutaneous fat and adhesion complexes carefully, then flushed it with purified water and NS (Chen et al. 2010, Dubey et al. 2010, de la Presa et al. 2009).

In vitro percutaneous permeation experiments

Percutaneous permeation device is shown as Figure 1. The skin was mounted onto the cups of transdermal diffusion apparatus and trimmed to suitable size. Each diffusion cell contained ethanol–saline (3:7), the diffusion cells were placed in 37°C water bath and stirred at 150 rpm (Madsen et al. 2011). Covered 0.25g gel of each preparation (Preparation I, II, III and Ⅳ, simvastatin concentration 1.2 mg/g) on the horny layer uniformly. Drew off 0.5 ml receptor medium at the time of 2, 4, 6, 8, 12, and 24 h, then refilled the same amount of fresh medium in the receptor compartments. The content of simvastatin in the 0.5 ml receptor medium was filtered and determined by HPLC.

Figure 1. Percutaneous permeation device.

Removed the mouse skin after the above-mentioned experiment, washed the skin surface with NS and cut it into pieces, homogenized the pieces with 5.0 ml ethanol for 10 min and ultrasonic extracted for 30 min, filtered with 0.22 μm microporous membrane (Chen et al. 2010, Dubey et al. 2010, de la Presa et al. 2009). The content of simvastatin in extraction was determined by HPLC.

The formula and statistical methods

Qn is cumulative penetration amount in unit area; V is volume of receptor medium; Cn and Ci are simvastatin concentrations of sample at the n time and the i time; Vi is each sample volume; A is the diffusion area. This formula is used to calculate the simvastatin cumulative permeation amount in per unit area of rat skin.

SPSS 17.0 statistical software was used to analysis cumulative permeation amount differences and the intradermal retention differences of four agents group.

Results

Cumulative permeation amount in unit area

Cumulative permeation amount in unit area of four agents was calculated according to formula 3.4, the results were shown in Table II and Figure 2.

Figure 2. Cumulative permeation amount per unit area.

It can be seen from Table II that compared with Preparation I, the cumulative permeation amount in unit area (Qn) of Preparation Ⅱ was significantly higher at 8 h after the penetration (P < 0.05), Preparation Ⅲ and Ⅳ showed differences from 4 h after the penetration. Compared with Preparation Ⅱ, the cumulative permeation amount in unit area (Qn) of Preparation III was significantly higher at 4 h after the penetration (P < 0.05). Compared with Preparation Ⅲ, the cumulative permeation amount in unit area (Qn) of Preparation Ⅳ was significantly higher at 4 h after the penetration (P < 0.05).

Table II. Cumulative amounts of different preparations at different times (Character± s, n = 3).

Time (h)	Preparation I (μg ml−^1)	Preparation II (μg ml−^1)	preparation III (μg ml−^1)	Preparation IV (μg ml−^1)
2	1.70 ± 0.34	2.44 ± 0.48	2.46 ± 0.57	2.55 ± 0.43
4	3.35 ± 1.04	4.78 ± 0.66	5.47 ± 0.75*	7.74 ± 1.01*^＃▲
6	5.14 ± 0.81	6.84 ± 0.80	8.42 ± 1.17*	11.13 ± 1.12*^＃▲
8	6.57 ± 0.98	10.65 ± 1.07*	12.78 ± 0.68*^＃	17.25 ± 1.10*^＃▲
12	7.62 ± 0.90	12.81 ± 1.21*	16.04 ± 1.27*^＃	20.70 ± 0.89*^＃▲
24	9.40 ± 0.84	15.87 ± 0.72*	18.87 ± 1.13*	23.15 ± 0.96*^＃▲

*Compared with preparation I, P < 0.05; ＃ Compared with Preparation II, P < 0.05; ▲ Compared with Preparation Ⅲ, P< 0.05;

Penetration kinetics equation

Fitting kinetic equation of penetration behavior according to data in Table II. The penetration behavior of various gels was fitted by Zero order kinetics equation, first-order kinetic equation, Higuchi equation and the equation WeibuU.

During 2˜8 h, Qt to t fitted the first -order kinetic equation.

Preparation Ⅰ: Qt = 0.0825 + 0.8212 t R² = 0.9982

Preparation ⅡI: Qt = ‐0.4996 + 1.3353 t R² = 0.9795

Preparation Ⅲ: Qt = ‐1.1973 + 1.6959 t R² = 0.9905

Preparation Ⅳ：: Qt = ‐2.2074 + 1.3748 t R² = 0.9892

During 8˜24 h, Qt to t^1/2 fitted the higuchi equation.

Preparation Ⅰ：Qt = 2.8569 + 1.3424 t^1/2 R² = 0.9947

Preparation ⅡI: Qt = 0.3842 + 3.6104 t^1/2 R² = 0.9996

Preparation ⅢI：Qt = 5.5307 + 2.7779 t^1/2 R² = 0.9341

Preparation ⅣV: Qt = 11.6980 + 2.2308 t^1/2 R² = 0.8557

Intradermal retention of simvastatin

Table III and Figure 3 were the results of simvastatin retentions in mouse skin, the simvastatin retentions of Preparations II, Preparation III and Preparation IV were significantly higher than PreparationI (P < 0.05).

Table III. Simvastatin retention in mouse skin.

	Retention amount (μg ml−^1)	x-± s (μg·ml−^1)	RSD (%)
Preparation I	15.19
	15.54	16.07 ± 1.24	0.08
	17.48
Preparation II	30.01
	28.81	29.32 ± 0.62*	0.02
	29.13
Preparation III	25.39
	34.85	30.29 ± 4.74*	0.15
Preparation IV	30.61
	27.26
	43.67	34.57 ± 8.35*	0.24
	32.77

Compared with preparation I, P < 0.05.

Figure 3. Simvastatin retention in mouse skin.

Discussions

Carbomer was selected as matrix to prepare gels in order to keep gels semi-solid, uniformed and refined. Gel has many advantages, such as easy to spread and form transparent film after coating on the skin, strong adhesion, fast release speed, long retention time, easy to clean and no irritation to the skin, which is suitable for the skin application.

Not like most water white gels, the simvastatin ethosome gel was milky white and opaque, similar to cream. The surfactant in the simvastatin ethosome resulted in the different characters.

The human skin temperature is 33.5°C ± 0.5°C. The temperature of receiving fluid was controlled at 37°C in this experiment, which is higher than normal skin temperature. In view of the lower activity of the isolated skin, a higher temperature can improve the activity to make the experimental results more accurate.

During 2˜8 h, Qt to t fitted the linear equations, which indicated the fast penetration rate in initial stage. During 8˜24 h, Qt to t_1/2 fitted the Higuchi equation, which indicated that the penetration rate was slowed down to maintain a steady state.

The correlation coefficients of the equations fluctuated at 0.99, some correlation coefficients are smaller or even less than 0.9. The differences of the mouse weight and the thickness of mouse skins may influence the penetration capacity, considering that the correlation coefficient can be appropriately narrowed when the ripple factor is larger, the lower correlation coefficients were acceptable.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by the key scientific and technological projects of Shandong Province in 2008.