Asian Journal of Pharmaceutical Sciences 2012, 7(5):316-328

A transdermal microemulsion-based hydrogel of nisoldipine: preparation, in vitro characterization and in vivo pharmacokinetic evaluation
Yan Xiao, Zhiyi Lin, Jianping Liu*, Wenli Zhang, Ling Wang, Panpan Yu
Department of Pharmaceutics, China Pharmaceutical University, No.24, Tongjiaxiang, Nanjing, China
Received 4 August 2012; Revised 17 October 2012; Accepted 3 December 2012

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Abstract
The purpose of this study was to construct a microemulsion-based hydrogel (MEs-H) for the transdermal delivery of an antihypertensive drug nisoldipine (NS) and to evaluate, predict its in vivo pharmacokinetic behaviors in rats using convolution method. Compositions of the nisoldipine microemulsions (NS-MEs) were determined by simplex lattice mixture design. The nisoldipine microemulsion-based hydrogel (NS-MEs-H) was prepared by adding carbomer. The NS-MEs and NS-MEs-H were characterized by size, morphology and in vitro skin permeation behaviors. In vivo drug plasma concentrations in rats were determined by LC/MS. The drug bioavailability of NS-MEs-H was compared with that of oral suspension. Pharmacokinetic behaviors of NS-MEs-H were predicted by convolution method. The optimized NS-MEs were composed of isopropyl myristate (IPM, 3%, w/w), Cremophor EL (15%, w/w), Transcutol (22%, w/w) and water (60%, w/w). The size of the NS-MEs was 23.12.4 nm and the spherical droplets existed in both NS-MEs and NS-MEs-H. The cumulative amount of NS permeated from NS-MEs and NS-MEs-H were 599.91 49.83 and 461.4738.33 g/cm2 during 72 h, respectively. The pharmacokinetic results showed that transdermal administration of NS-MEs-H could maintain a steady drug plasma concentration in three days. The NS bioavailability of NS-MEs-H was 1.55-fold increased than that of the oral administration. Percent prediction errors of most drug plasma concentrations were below 10%. The NS-MEs-H has the potential to provide a long effective treatment of hypertension, and the convolution method could be utilized to predict in vivo pharmacokinetic behaviors of transdermal drug delivery systems in the preliminary study. Keywords: Transdermal drug delivery; Microemulsion; Pharmacokinetics; Nisoldipine; LC-MS; Convolution ____________________________________________________________________________________________________________

1. Introduction
NS, a calcium antagonist of the 1, 4-dihydropyridine class, can reduce vascular resistance and blood pressure by inhibiting calcium uptake of myocardial and smooth muscle cells. Immediate-release (5 and 10 mg) and controlled-release (10, 20, 30 mg and 40 mg) oral preparations for NS have been approved in a number of countries for the treatment of hypertension and angina pectoris [1-3]. However, absolute bioavailability of the oral tablet is only 5.5%, as a result of significant firstpass metabolism in the gastrointestinal tract and liver [4]. Now many anti-hypertension drugs have been incorporated into the transdermal delivery system [5-6]. Transdermal drug delivery system can avoid the drug degradation in the gastrointestinal tract and the metabolism in the liver, also sustain a steady plasma
__________ *Corresponding author. Address: China Pharmaceutical University, No.24, Tongjiaxiang, Nanjing 210009, China. Tel: +86-25-83271293; Fax: +86-25-83271293 E-mail:jianpingliu1293@163.com

level to decrease side effects particularly those associated with plasma levels fluctuation [7]. However, the outermost layer of skin which mainly consist of stratum corneum forms a main barrier against permeation of drugs, because of its rigid lipid lamellar structure. Thus, methods which can improve skin penetration of active ingredients to achieve therapeutically relevant levels are critical for transdermal formulations. MEs are a dispersion with droplet size from 10 to 100 nm and do not have the tendency to coalesce [8-10]. MEs form spontaneously with appropriate amounts of a lipophilic and a hydrophilic ingredient, as well as a surfactant and a cosurfactant [11]. MEs have several specific physicochemical properties such as transparency, optical isotropy, low viscosity and thermodynamic stability[11-12]. As efficient drug carriers, MEs have been widely employed in both transdermal and dermal delivery of drugs [13-14]. The main mechanisms to explain the advantages of MEs for the transdermal delivery of drugs include the high solubility potential for hydrophilic drugs of ME systems, permeation enhancing effect of the ingredients of MEs, and the increased

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Asian Journal of Pharmaceutical Sciences 2012, 7(5):316-328

thermodynamic activity of the drug in the carriers [9,11-12]. Most of the MEs have very low viscosity, which may restrict their application to the transdermal delivery field due to inconvenient use [15]. The addition of hydrogels, e.g. carrageenan and carbomer, into ME can lead to the formation of hydrogel-thickened ME with a weak gel behavior and the increase of viscosity [16-18]. Several of anti-hypertension drugs have been incorporated in MEs for transdermal delivery [19-20]. In this investigation, NS-MEs-H has been employed to deliver NS transdermally. Usually, it would be cost-consuming and timeconsuming to evaluate in vivo pharmacokinetic behaviors of various formulations, moreover, the in vivo drug plasma concentrations after transdermal administration were hard to detect due to their low concentration. Thus the estimation of in vivo drug concentration by in vitro permeation results was necessary. Convolution method, out of many mathematical approaches, serves as an efficient tool to predict drug plasma concentration based on in vitro data. Through this approach, the in vivo data of various formulations could be calculated and estimated without a lot of experiments in the formulation development stage. In this study, O/W NS-MEs have been developed and optimized using a simplex lattice design. The NS-MEs was incorporated into Carbopol 980 to form a NS-MEs-H. The NS-MEs and NS-MEs-H were characterized by size, morphology and in vitro skin permeation studies. In vivo pharmacokinetic behaviors after transdermal administration of NS-MEs-H were estimated. The drug plasma concentration in rats was predicted using a convolution method and the prediction ability was also evaluated.

chromatographymass spectrometry (LC/MS) grade. All other chemicals and reagents were of analytical grade. Male Sprague-Dawley rats (180200 g and 220 250 g) were obtained from the animal centre of China Pharmaceutical University (Nanjing, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. 2.2. HPLC analysis of NS in vitro The concentrations of NS in the samples were analyzed by HPLC (Shimadzu LC-10A, Kyoto, Japan). NS was separated by a reversed phase C18 column (5 m, 4.6 mm250 mm, Kromasil) and detected using UV detection at a wave length of 237nm. A 80:20 (v/v) mixture of methanol and water was used as mobile phase with a flow rate of 1 ml/min. The column temperature was 40C and the injection volume was 20 l. The peak area correlated linearly with NS concentration in the range of 0.550.0 g/ml with the lowest detection limit at 0.5 g/ml, and the mean correlation coefficient was 0.9998. 2.3. Screening of components for MEs To find out appropriate components that have good solubilizing capacity of NS, the solubility of NS in different oils, surfactants and cosurfactants were determined. Oils employed were Labrafil M 1944, olive oil, oleic acid and IPM. Cremophor EL and Tween 80 were chosen as surfactants. Cosurfactants selected were Transcutol and PEG400. An excess amount of NS was added to 2 ml of each excipient and turbine reciprocally at 32C, then shook at 32C for 72 h. Samples were centrifuged at 10,000 rpm for 10 minutes. The supernatant was separated and then diluted with ethanol. The diluted samples were filtered through 0.45 m membrane filter and solubility of NS was determined by HPLC. 2.4. Pseudo-ternary phase diagrams To determine the concentration of components for the existing range of the ME, the pseudo-ternary phase diagrams were constructed using water titration method. Different phase diagrams were prepared with the 1:4, 1:2, 1:1 and 2:1 weight ratio of surfactant to cosurfactant. For each phase diagram, the ratios of oil to the mixture of surfactant and cosurfactant were varied as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9 (w/w). Water was added drop by drop under gentle magnetic stirring at 32C. After equilibration, the mixtures were assessed visually under light versus a dark background and determined as being MEs by their clarity and transparency.

2. Materials and methods

2.1. Materials Nisoldipine and nimodipine were purchased from Shandong Boyuan Pharmaceutical Co., Ltd. (Shandong, China). Isopropyl myristate (IPM), olive oil, oleic acid and Tween 80, were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Labrafil M 1944 (Oleoyl macrogolglyceride) and Transcutol (Diethylene glycol monoethyl ether) were donated by Gattefoss, (Saint Priest, France). Polyoxyl 35 castor oil (Cremophor EL) was donated by BASF, (Ludwigshafen, Germany). Carbopol 980 was obtained by Noveon, Inc. (Cleveland, USA). Polyethylene Glycol400 (PEG400) was purchased from XiLong Chemical Plant (Wuxi, China). Water was deionized and filtered in our lab. Methyl alcohol was high pressure liquid chromatography (HPLC) or liquid

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Asian Journal of Pharmaceutical Sciences 2012, 7(5):316-328

2.5. Preparation of NS-MEs NS-MEs were prepared by dissolving 0.4% (w/w) NS in the mixture of oil, surfactant and cosurfactant, then water was added drop by drop with the help of gentle magnetic stirring at 32C and the final preparation was made up to 100% (w/w). The MEs were sealed tightly and stored at ambient temperature. 2.6. In vitro permeation studies Male Sprague-Dawley rats weighing 180200 g were sacrificed using an anesthetic ether. After hair was removed carefully with a razor, the full thickness abdominal skin was cautiously excised and the subcutaneous tissues were trimmed. Before use, the skins were washed with water and examined for their integrity. If not used immediately, the skin was always kept in refrigerator (04C) and limited to be used within 3 days. The rat abdominal skin was mounted onto a modified Franz diffusion cell with the stratum corneum facing the donor cell and the in vitro transdermal permeation behaviors of the drug from the NS-MEs were investigated. The receptor volume was 17 ml and the area of diffusion was 2.92 cm2. Before the permeation experiment, the receptor cell was filled with normal saline containing 20% ethanol as the receiver medium. The receptor media was maintained at 320.5C and magnetically stirred at 250 rpm. The ME formulation was applied on the stratum corneum (0.5 g/cm2, the NS concentration in ME was 4 mg/g) by gently pipetting the solution into the donor cell. At the time points 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 24.0, 36.0, 48.0, 60.0, and 72.0 h, 5 ml of the receptor media was taken and then replaced with the same amount of the fresh receptor medium. The permeation samples were filtered through 0.45 m filter films and analyzed by HPLC. The cumulative amount of permeated drug per unit area of skin (Q, g/cm2) at time point n was calculated using Eq. (1).
Qn Cn V

Drug solubility in different ME formulations was measured according to the method described in the section of Screening of components for MEs. 2.8. Formulation optimization The component proportion of MEs was optimized by using the simplex lattice experiment design [21-22]. In this research, the concentration of surfactant, cosurfactant and water, were selected as independent variables. Two critical values for evaluation of the transdermal MEs-H, the cumulative amount of NS permeated through excised rat skins per unit area and the drug solubility in the MEs, were taken as responses. As shown in Fig. 1, the simplex lattice design for a three factor system can be represented as an equilateral triangle with seven points on it. A, B and C represent three formulations containing the maximum amount of one component; with the other two components has the minimum concentration. Other three points located at the halfway points between vertices (AB, BC, and AC) represent three formulations containing the average of the minimum and maximum amounts of the two components. The last one is at the center point (ABC) represents a formulation containing one third of each component. The concentrations of those three factors change simultaneously but always keep their total concentration constant. Seven ME formulations were prepared based on the rules. The responses for them were used to fit an equation to predict each response values of all possible MEs [23].

S

n -1

i 1

Ci 5

(1)

Where C n and C i are the drug concentrations at -t point sampling time C(t) = Ae Be - tn and i respectively, V is the volume of receptor medium, S is the effective skin Q surface area. All the transdermal permeation experiments J t1 were1 repeated six times. t1
Q t2 - Q t1 2.7.J Drug 2 = solubility in the NS-MEs t 2 - t1

Fig. 1. The schematic diagram of simplex lattice design for a three-component system.

2.9. Construction of NS-MEs-H Carbomer was selected as hydrogel matrix based on previous reports [21,24]. NS-MEs-H was prepared according to the established method [18]. Carbopol 980 was swelled in a little water for 24 h and a high viscous gel was obtained, and then the NS-MEs were slowly added to the viscous gel of Carbopol 980 under magnetic stirring. The pH values were subsequently regulated to 6 9, and NS-MEs-H was obtained. The concentration of

An excess amount of NS was added to the mixture Q -Q t2 J 3 = t3 of oil, surfactant t 3 - t 2 and cosurfactant, and then water was added drop by drop under gentle magnetic stirring at 32C. If 0 t t 1 ,

C (t )

J1 A

(1 - e -t )

J1B

(1 - e - t )
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If t 1 t t 2 , C (t ) J2A [1 - e - ( t - t1 ) ] J2B [1 - e - ( t - t1 ) ] C (t1 )

[ Ae - ( t - t1 ) Be - ( t - t1 ) ]

Asian Journal of Pharmaceutical Sciences 2012, 7(5):316-328

Carbopol 980 in NS-MEs-H was 1% (w/w). The final concentration of NS in NS-MEs-H was 0.4% (w/w). 2.10. Characterization of the NS-MEs and the NS-MEs-H 2.10.1. Droplet size Size of NS-MEs and the blank microemulsions without NS (B-MEs) were determined using dynamic light scattering (Zetasizer 3000 HSA, Malvern, UK). Triplicate measurements were made and the samples were diluted appropriately with distilled water. 2.10.2. Visualization by transmission electron microscopy (TEM) The nisoldipine hydrogel (NS-H) was prepared by removing IPM from NS-MEs-H. The preparation steps of NS-H were the same as that of NS-MEs-H. The four formulations including NS-MEs, B-MEs, NS-MEs-H and NS-H were diluted appropriately with aqueous phase. A drop of each sample was added to a copper grid coated with carbon film and air-dried; 2% (w/v) phosphotungstic acid solution was then dropped and covered onto the grids. After air-dried at room temperature, the samples were observed by TEM (H-7650, Hitachi, Japan). 2.10.3. In vitro permeation behaviors In vitro permeation behaviors of NS-MEs (0.4%, w/w) and NS-MEs-H (0.4%, w/w) were studied according to the method described previous. 2.11. In vivo studies 2.11.1. Animal experiment Male Sprague-Dawley rats (220250 g) were maintained at 25C for the study. The intravenous administration study was used to estimate the NS pharmacokinetic parameters employed in the convolution method. The NS bioavailability from transdermal NSMEs-H was compared with that from oral suspension. The intravenous injection was prepared by dissolving the NS in the normal saline containing Cremophor EL (20%, v/v). The concentration of NS was 0.5 mg/ml. The oral suspension was prepared by suspending NS in the normal saline containing 0.5% (w/v) of sodium carboxy methyl cellulose (4 mg/ml). The animals were randomly divided into three groups (n=6) for transdermal administration, oral administration and intravenous administration respectively. For the rats transdermal administrated with NS-MEs-H, the

abdominal hair was carefully clipped by a razor without any skin damage and washed with distilled water. NSMEs-H (5 g, the concentration of NS was 4 mg/g) was attached closely on the abdominal skin (10 cm2) of each rat with the help of a device made in our lab. After the administration period, NS-MEs-H was removed at 72 h. Rats in oral group were given intragastric NS suspension 14 mg/kg three times (the second and third time was 24h and 48h respectively after blood sampling). The rats employed in the intravenous study were administrated by NS injection via the tail vein at a dose of 2 mg/kg. Blood samples (0.2 ml) were collected from the plexus venous in the eye ground at 1, 2, 4, 6, 8, 12, 18, 24, 36, 48, 60, 72, 84, 96 and 108 h after transdermal administration of NS-MEs-H, 0.08, 0.17, 0.33, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 24.17, 24.33, 24.5, 26, 30, 32, 36, 48, 48.17, 48.33, 48.5, 50, 54, 56, 60, 72 h after oral administration and 0.083, 0.167, 0.333, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after intravenous administration. The plasma was obtained by centrifugation at 4000 rpm for 15 min and stored at 20C before analysis. 2.11.2. LC/MS analysis of NS Plasma levels of NS were estimated by LC/MS [25]. Nimodipine was used as internal standard. To 50 l plasma, 10 l (100 ng/ml) of nimodipine and 25 l of 1.0 M NaOH solution was added and then extracted with 2 ml of 2-Methoxy-2-methylpropane (MTBE), for 5 min. Organic layer (1.8 ml) was separated and evaporated to dryness at 40C under a stream of air. The residue was reconstituted with 100 l mobile phase and then NS was estimated using LC/MS. Chromatographic separation was carried out at room temperature with an Kromasil C18 (5 m, 2504.6 mm) reversed-phase column on an LC/MS2020 system (Shimadzu CorporationJapan). The column temperature was set at 40C, and the injection volume was 5 l. The mobile phase consisted of methanol-water (70:30, v/v) at a flow rate of 1 ml/min, but only 0.2 ml/min eluent volume was introduced into the spectrometer because it was split at the ratio of 2:8 before entering the ESI interface. The single-quadruple mass spectrometer fitted with an electro spray ionisation (ESI) source was used for analysis. It was operated in the positive ion mode with following parameters: probe voltage +4.50 kV (+ESI), nebulizer gas flow 1.5 l/min, drying gas flow 15.0 l/min, block heater 350C, DL temperature 250C. The [(M+Na)+, m/z 411] for nisoldipine and [(M+Na) +, m/z 441] for nimodipine were selected as detecting ions, respectively. The quantification was performed using the ratio of the peak areas of the NS and of nimodipine. Linearity was established for the concentration range from 0.5 to 2000

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J 1 = Ae -t Be - t C(t) t 1 t3 Q -Q Q t2 t2 J3 = = Q t3 J 3 t t Q Q 3 2 Q t t 3 2 t1 J12 = t1t2 J - tt 1, t1 t 2 Q If 0 t t 1 , t3 - Q t2 If 0 t 1, If 0 t t 1 J3 = t 3 - t-2 Q Q J1 A J B J A J 1B B t3 t2 t2 t 1 Pharmaceutical tt tt 1A t1 Asian Journal Sciences 7(5):316-328 JC J C (t ) (1 - e ) of (1 - e - t ) 3( J1 J1 t2012, (= ) ( 1--e e-- ) 1--e e-- 2 C t ) ( 1 ) (( 1 )) t t 3 2 2 1 If 0 t t 1 , If t 1 t t 2 J , A Q Q If 0 If tt3 t ,2,, tt 1 tt ttt2 J B 1 t (t ) 1 (1 - of e -0.9999 ) 1 and (1 - e - t ) J 3 =1 t - t 2 ng/ml with a coefficient of C determination J A - ( t - t1 ) J2B C (t1 ) J12A A B - t -- C((tt1)) 3 2 - ( t -J B tJ )t ((tt--tt1)J - -2 t2 ) )1 ( t - t1 ) [1 - e - ( t - t1 ) ] A 1 1B 2 1 [ Ae- 2 C (t ) and [1 - e Peak ] A e([ B ][[ 1e [ (J C(6) B C(( [ 1 et- 1--) e ((tt--tt11))]] Be C tt)) 1 ---e ) (1 -e 1 e ]] 1 e [ Ae ( t - t1 ) good calculated accuracy precision. area ratio A B A B If t t t , If 0 t t , A B 2 was determined 1 (A1) of nisoldipine and internal1 standard If plasma t 2 t t 3J , A containing the f t If ttt tt ,,, -t- ( t - tJ) 2 3 II tt J2B C ( t1 ) J A B --( 1 23 after extraction of the samples - ( t - t1 ) - ( tf - t1 ) 2 t t1 ) 1 2 (1 -[e (1e - e t) ] C (t ) [1 - e ] [1 - e C (t ) ] Ae ) 1 1 B J 3 A - ( t -Another J3B ( ) C t J A J B C t2 nisoldipine at different concentrations. peak J A J B ((( tt1 ) ) - t2 ) ( t t ) ( t t ) ( t t ) e A 2 ( ) 3 B 32 C )))e ttt ttt ))) ] C (( t(t -tt-1 ) 2 2 ] 2( 2 2 2) - ((t tt --t tt1 (( -tt2 1 3 3 2 )[ [ C (t ) [1 - e ] [1 [ Ae[[ B ] 2 2 C(( 1--e e] J2 [ 1--e e--- A e-- B C tt)) 1 ] [ 1 ]] e B e ] [A A e B area ratio (A 2) was obtained from direct injection of B A B A B If t 2 t t 3 , If tA t t , A B 1 2 the standard solution containing the nisoldipine and If t ) Jt C (t1 ) J-3C A J3B (tt23),--C C C(obs) (pred)- ( t - twith C(obs) C (pred pred )t 2 ) J 2 B- ( t - t 2-) -( ( t -( t1 ) ( t - t 22 - t) 2A the internal standard at the same %PE %PE 100 (t ) [1 -[e ] [ Ae - ( t - t1 ) Be C(obs) Be [1 - e ]( t - t1 ) ] C (t ) concentration [1 - e 1002 ) ] the [1 - e -C ] A e %PE 100 C(obs) J J (tB C C(obs) A A C(obs) B - ( t - t 2 ) - ( t -t 2 ) 3A 3B 2) plasma samples used above. The extraction recoveries C (t ) [1 - e ] [1 - e ] [ Ae - ( t - t 2 ) B A B of the drug from theY rat plasma were more than 70% The prediction error (%PE) was calculated by the Eq. = b1X1 + b 2 X 2 + b-3C X + b) + X X + X X Xb IY f t1 tb ,X Y = b X + b Xb +b b X + b X1X X2 + +b b13X X1X X3 + + b 23X X 2X X3 + +b b123X X1X X 12 X1X 2 + b13 X1X 3t 23 2 1 3 1X 1 2 2 123 3X 32 12X 2b 32 = b + + + C(obs) (3 pred 1 2 3 3 12 1 2 13 1 3 b 23 2 3 123 1 100 determined by comparing A%PE and A2 described above. The (7) to quantitatively determine how well convolution 1 J A J B ( ) C t C(obs) C(obs) - C-( pred ( t - t 2)) ( t -t 2 ) ( t -t 2 ) 3 3 Solubility = 7.24X1 + 7.86X + 8.38X X Solubility = 7.24X +7.86X 7.86X +10.31X 10.31X +5.24X 5.24X X22 +8.38X 8.38X X 3 Be C (X t ) 2 [ 11e3 ] 2 [1 - e -3 ] 1 [ Ae - %PE 100 RSD for intra and inter-day estimations were 2.43% and 3 + 5.24X method can accurately predict the in vivo plasma 2 + 10.31X 1 1+ 2+ 3+ 1X 1X Solubility = 7.24X + 1 2 1 3 C(obs) A B Y = b X + b X + b X + b X X + b X X + b X X + b X X X 1 1 2 2 3 3 12 1 2 13 1 3 23 2 3 123 1 2 3 1.93%, respectively, demonstrating good reproducibility. concentrations. + 6.14X + 6.14X 6.14X 2X X 3 --66.57X 66.57X1X X 2X X3 2X 3 - 66.57X1X 2 X 3 + 3 1 2 3 (P< (P< Y = b1X X +2b(P<0.005) 1 + b 22( pred 3X 1X C(obs) C ) 3 + b12 X1X 2 + b13 X1X 3 + b 23 X 2 X 3 + b123 X 1X 2 + 8.38X1X 3 100 Solubility = 7.24X1 + 7.86X 2 + 10.31X 3 %PE + 5.24X (7) 2.11.3. Convolution method C(obs) Solubility = 7.24X + 7.86X 10.31X 3 + 5.24X X +1606.38X 8.38X1X 31X Q72h = 181.41X1 + 6.14X 67.58X - 86.50X X 1606.38X X Q =C(obs) 181.41X + 67.58X ++ 243.06X -86.50X 86.50X X2 1606.38X X3 - 66.57X1 167.58X 1 2+ 3X 172h 2+ 3 72h 11 22 31 2+ 2X 3 243.06X 2 X 3 Where Q = 181.41X + + 243.06X X and C(pred) represent the observed and 2 2 1 3 (P<0.005) Y = b1X1 + b 2 X1 + b12 X1X 2 +3b13 X1X 3 1+ b 2 +b 3 X-3 23 X 2 X 3 + b123 X1X + 6.14X X 66.57X X + 1235.16X X 10487.40X X X + 1235.16X 10487.40X X X Convolution and deconvolution method could be plasma 2 concentration 2 1 respectively. 2 3 2 3 1 2 3 predicted NS 2X3 3 - 10487.40X 1 2 3 + 1235.16X X2 X3 1 (P< (P<0.0001) (P< (P< employed to predict the in vivo or in vitro data by a 7.24X + 7.86X + 10.31X + 5.24X X + 8.38X X Q72h = 181.41X1 + 67.58X2 + 243.06X3 -Solubility 86.50X1X= + 1606.38X X 1 2 3 1 2 1 3 2 complicated calculation using the obtained information. 2.12. Statistical analysis 1 3 + 6.14X X 3 - 66.57X Q72h = 181.41X 67.58X + 1235.16X2X3 - 10487.40X1X 2X3 1X 2 X 3 1 +2 2 + 243.06X 3 - 86.50X1X 2 + 1606.38X1X3(P<0 To predict the drug plasma concentration of NS by the (P<0.0001) + 1235.16X X3 2 Xas 3 - 10487.40X 1X 2Statistical in vitro skin permeation studies, the convolution method Data were shown meanSD. data (P< were performed according to the previous research [26], were analyzed by1the two-tailed t -test and P values for Q72h = 181.41X + 67.58X + 243.06X 86.50X X 2 3 1 2 + 1606.38X1X3 with the in vitro permeation rate as input function and the significance were set at 0.05 or 0.01. + 1235.16X 2 X3 - 10487.40X1X 2 X3 (P<0 i.v. plasma data as weight parameters. In our study, plasma concentrations of NS against 3. Results and discussion n administration -1 time after C intravenous were fitted to a two Ci 5 n V i 1 compartment model of intravenous bolus using PKSolver 3.1. Components for MEs Qn S of unit impulse parameters (A, B, program. The value n -1 and ) could be from To maintain a larger solubility of NS in ME systems, n -1Ci Cn V estimated 5 the Eq. (2). i 1 C V C 5 t t n i Q (2) suitable oils and surfactants were selected. The solubility n = Ae i 1 C(t) S Be Q n S In vitro skin permeation fluxes (J, g/cm2/h) of each of NS in various excipients was determined (Table 1). Q time interval was calculated from the slope of the plot Among the various tested oils, the solubility of NS was J 1 t1 -t - t C(t) = Ae Ben -t permeated -- t of drug amount vs. time (h) according to the highest in IPM, followed by Labrafil M 1944, olive 1 t n 1 1 C(t) = Ae Be n n-1 1C C V 5 C V C 5 n i n i Cn (5). Cii 5 Eqs. (3),(4) and oil and oleic acid. It was also reported that IPM was ii 1 1 n V Q Qn QQ - Q t1 ii 1 1 n Q t1 t2 n S n2 a powerful enhancer for transdermal delivery since it = S JJ Q S t1 J1 1 t 1 t 2 - t1 (3) could increase fluidity of lipid portion of the stratum t1 t t corneum which resulted in a permeation enhancing effect Q Q t t Q Q t3 t2 C(t) = Ae Be -t - t t2 t1 C(t) =Q Ae Be = Q C(t) Ae Be 3= = JJ t2 t1 2 (4) [28-30]. The solubility of NS in surfactants showed that 2 t t J2 = t2 3 - t1 2 t t Cremophor EL was better than Tween 80. Cremophor EL 2 1 Q Qt1 t1 J 0Q t 1t2 , t1 Q -Q JIf t1t 1 t3 1 (5) is one of the nonionic surfactants which are widely used J J 1 Q J 1 = t 1 1 t3 - Q t2 tt1 J33 t3 t in topical and transdermal formulations as solublizing 3 = 1 J A J B 2 t 1t 1 t3 C (t )Q (1 -te - ) the (1 - e - t )time points, Q , 2t1 Q Q -Q Q t2 Where t , t and were selected agents. Transcutol also has a good dissolving property t2 t1 Q J = 1 2 3 t1 t2 t1 J =t 2 If 0 t , 2= J 1 2 -t If 0Q Qt2 and of the NS and it was often used as penetration enhancer 2 1 1,the permeated drug amount at each time 1 tttwere ttt 22 1 If t 1 t3 t - t 2 , -t J A J1B t 1 point. in transdermal drug delivery systems [31-33]. IPM, JA Q Q C (t )Q ( 1 - e -t ) J 1 B (1 - e - t ) Qt3 -Q Q 1 t2 t3 t2 J A J B C (t1 ) J = C t ( ) ( 1 e ) ( 1 e ) t3 t2 J = ( t t ) ( t t ) ( t - t1 ) ( t - tand 2 2 3 After the Laplace transforms [26-27], the transdermal EL 1 1 1 ) ] Transcutol were chosen respectively 3 J3 Be - C= (t ) [1 - e ] [1 - e ] [ Ae Cremophor t t t t 3 2 3 2 t 3 - t 2 in rats can be calculated A B drugIf concentration in Eq. 6. as the oil phase, surfactant and cosurfactant for the t1 t t2, If t t t , 1 2 If 0 t t , formulations of NS-MEs. If 0t t tA ,3, If t t 1 1 If 0 ,t 2 t J J2B C (t1 ) - ( t - t1 ) - ( t - t1 ) - ( t - t1 ) - ( t - t1 ) 21 J A J B C ( t ) C (t ) J [ 1 e ] [ 1 e ] [ A e - )J1 B - ( t - t1 ) Be - ( t - t1 ) ] 2 2 B- t - ( t - t1 ) 1 ) A J JJ A BJ - tt ((tt--tt1J 1 A C ( t C ( t ) [ 1 e ] [ 1 e ] [ A e B e ] t 1 1 J A B ) ( t t ) ( t t ) 3 3 2 A B C ( ) ( 1 e ( 1 e ) 2 ] t ) t 2 1] ( C t) ( ( 1 -e e( 1 -e e- )e Construction C ( t)) 1 ( [1 e [1 Be - ( t - t 2 )of ] pseudo-ternary phase diagram C tt ( 1 )) 1 ) A B [ Ae 3.2. 2 A B If t 2 t t 3 , If ttt 2 t t3 , (6) If If 1 2 Pseudo-ternary phase diagrams were constructed to 1 2,,, - C(pred) If t1 ttt ttt2 C(obs) J A J3B C (t 2 ) ( t t ) ( t t ) - ( t - t 2 ) - ( t -t 2 ) 3 2 2 %PE J 100 J A J B ( ) C t C (t ) [ 1 e ] [ 1 e ] [ A e B e ] ( t t ) ( t t ) ( t t ) ( t t ) 3 3 2 A J B C ( t ) confirm the concentration ranges of the components. The 2 2 2 ] A[ J2 B [1 - e-- C(( t1 ) [ Ae t --tt11 )) ] J ( t --tt112 )) ] C - t --tt11) -- t --tt11)) 2 ((( (( ( ) (( ( C( (tt t) ) JJ2 1-e e-- B 2A 2B C(obs) t B tt - t1 - t1 tt - t1 tt - t1 C 1 ] 1 e ] [ A e B e ] 11) 2 A diagrams C ) 1 e ] [ 1-e e-((t t ] [A A e- B e-e ] various weight ratios of Cremophor 1) ] 1) ] 1) 1) ] C (( t) 1 --e [ e B e [[[ [[1 A B phase with A B A B bB A+ Y = b1X X + b 3) X3 + b 1 + b 22 12 X1X 2 + b13 X1X 3 23 X 2 X 3 + b123 X1X 2 X 3 C(obs) C (pred %PE 100 I f t t t , C(obs) C ( pred ) I f t t t , 2 3 3, I%PE f t 22 t t 3 100 C(obs) Solubility = C(obs) 7.24X1 + 7.86XJ + 10.31X 3 + 5.24X1 X 2 2 2+ 1X J A B C ( ) J A J3 B C ( )8.38X ( t t ) - t --tt22)) - t --3 t 22)) -- tt--tt22)) 3 -(( t t22)) ] J ((( ((( ((( 2) J 33A ( C ttt2 - t -t - tt - tX )1] tt -b tt t C ( t ) [ 1 e [ 1 e [ A e B e ] 33B Y = b X + b X + b X + b X X + b X + b X X + X X X B C ( t ) [ 1 e ] [ 1 e ] [ A e B e] 2 2 2 2 2 2) 2) ] 1 1 2 2 3 3 12 1 2 13 3 23 2 3 123 1 2 3- t 2 + 6.14X XX - 66.57X X X 1 C (t= ) [ 1 e ] [ 1 e ] [ A e e 320 2 3 1 2 3 Y b1X + b X + b + b X X + b X X + b X X + b X X X A B 1 2 2 3 3 12 1 2 13 1 3 23 2 3 123 1 2 3 A B (P<0.005) A B 1 Solubility = 7.24X + 7.86X 1 2 + 10.31X 3 + 5.24X1X 2 + 8.38X1X 3 C(obs) C pred C(obs) C pred Solubility = 7.24X C(obs) ---C ((( pred ))) 1 + 7.86X 2 + 10.31X 3 + 5.24X1X 2 + 8.38X1X 3 %PE 100 %PE 100 Q72h 67.58X %PE = 181.41X 100 +C(obs) 6.14X X3 - 66.57X 1 +2 2 + 243.06X 1X 2 X 33 - 86.50X1X 2 + 1606.38X1X3 C(obs) + 6.14X X 66.57X X X (P<0.005) C(obs)

Q t3 - Q Q t2 - Q t1 J3 = J 2 = t2 t 3 - t 2 t 2 - t1

t1 2

Asian Journal of Pharmaceutical Sciences 2012, 7(5):316-328

Table 1 Solubility of NS in various excipients (n =3). Components Excipients IPM Oil Labrafil M1944 oleic acid olive oil Surfactant Co-surfactant Cremophor EL Tween 80 Transactol PEG400 Solubility Mean SD (mg/ml) 4.770.23 3.870.17 0.870.05 2.390.15 5.640.35 4.670.42 23.181.48 19.731.13

sample was cloudy or showed the phase separation, it represented the turbid and conventional emulsions. The area of existence in ME was enlarged when the ratio of surfactant to cosurfactant increased. 3.3. Formulation optimization of NS-MEs The goal for the formulation optimization was to obtain a stable ME formulation with a large drug solubility (above 8 mg/g) and maximum transdermal permeation amount. In our design, the three independent variables were the concentrations of water (X 1 ), Cremophor EL (X2) and Transcutol (X3). Based on the results of pseudo-ternary diagrams, the concentration of oil was fixed on 3%. The water content was determined from 50% to 70% and the ranges of Cremorpor EL and Transcutol were 15%-35% and 12%-32%, respectively. The two responses (Y) were the drug solubility in NS-

EL to Transcutol were depicted in Fig. 2. As shown in Fig. 2, if the sample was an isotropic and clear solution, it was defined as a ME (black area); if the

Fig. 2. Pseudo-ternary phase diagrams of MEs. (Km: the ratio of surfactant to co surfactant. The black area represent the existence of microemulsions.)

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J2B

If t 1 t t 2 ,

J A J B C (t1 ) C (t ) 2 [1 - e - ( t - t1 ) ] 2 [1 - e - ( t - t1 ) ] [ Ae - ( t - t1 ) Be - ( t - t1 ) ] C ( t ) [ Ae - ( t - t1 ) Be - A B ) ( t - t1 ) 1 ] [1 - e - ( t - t1 ) ] ] tB If t 2A t 3 , J3B

J A J Asian B Journal of Pharmaceutical C (t 2 ) Sciences 2012, 7(5):316-328 C (t ) 3 [1 - e - ( t - t 2 ) ] 3 [1 - e - ( t - t 2 ) ] [ Ae - ( t - t 2 ) Be - ( t - t 2 ) ] ( ) C t A B [ Ae - ( t - t 2 ) Be - ( t - t 2 ) ] ) 2 ] [1 - e - ( t - t 2 ) ] A B C(obs) - C(pred) MEs and MEs was also important. As results shown, water and %PE the cumulative amount 100 of NS permeated across C(obs) rat skins per unit area after 72 h [Eq.(8)]. Transcutol had to compromise to each other to obtained 100 Y = b1X1 + b 2 X 2 + b 3 X 3 + b12 X1X 2 + b13 X1X 3 + b 23 X 2 X 3 +the b123 X1X 2 X largest transdermal permeation amount. 3 X 3 + b12 X1X 2 + b13 X1X 3 + b 23 X 2 X 3 + b123 X1X 2 X 3 (8) Optimization was performed through a multiple Solubility + 10.31X 3 + for 5.24X 1 + 7.86X 2 coefficient 1X 2 + 8.38X1X 3 where bi = is 7.24X the estimated the factor response method called desirability which reflects 86X 2 + 10.31X 3 + 5.24X X + 8.38X X2 + 6.14X X 66.57X X X X . X , X , and X represent the average results of the desirable ranges for each response. The desirable 1 2 1 3 3 1 2 3 i 1 2 3 (P<0.005) - 66.57X1X 2 X 3 changing one factor at one time from its minimum to ranges are from zero to one (least to most desirable (P<0.005) maximum value, and X1X2, X2X3, X1X3, and X1X2X3, the respectively). Fig. 4C represents the 2D overlay contour Q72h = 181.41X1 + 67.58X2 + 243.06X3 - 86.50X1X 2 + 1606.38X1X3 interactions show how the responses change when two or plot showing the composition of the formulation which 8X2 + 243.06X3 - 86.50X + 1606.38X X3 1235.16X X 1X 2+ 1 3 - 10487.40X1X 2 X3 2 three factors change simultaneously. achieves the maximum desirability (1.0). The suggested (P<0.0001) 10487.40X1X 2X3 Solubility and in vitro drug permeation amount during formulation composed of IPM (3%, w/w), Cremophor (P<0.0001) 72h (Q 72h) of the seven selected formulations were EL (15%, w/w), Transcutol (22%, w/w) and water presented in Table 2. With the help of Design expert 7.0 (60%, w/w) was prepared and characterized. Software, Eqs. (9) and (10) were obtained to calculate 700 and predict response values for other formulations. As shown in Fig. 3, the in vitro permeation profiles of 1 600 the tested ME formulations demonstrated that penetration 2 3 through skin was a steady process during 72 h. 500 4 Fig. 4A and 4B show the effect of the three factors on 5 the two responses, the solubility and the in vitro drug 6 400 cumulative permeation amount, respectively. The orange 7 color represents the favorable area for the response, 300 and the green and blue colors indicate the areas which have intermediate and least values of the response, 200 respectively. From Fig. 4A, the largest cumulative permeation amount of nisoldipine was obtained when the 100 content of water and Transcutol were both moderate. It seemed that water and Transcutol were contributed 0 together to the increasing of the transdermal permeation 0 12 24 36 48 60 72 amount. It was reported that the MEs can significantly 3.4. Characterization of NS-MEs and NS-MEs-H Time(h) increase the skin hydration [34], which may enhance Fig. 3. Percutaneous permeation profiles of the tested ME the permeation ability of formulations [35]. Hence, the formulations (meanSD; n = 6): 1-7 represent the seven content of water in MEs was critical for drug permeation. different formulations stated in Table 2. The Fig. 4B shows that the solubility increased largely with the increase of Transcutol probably due to its 3.4.1. Droplet size solubilization effect. The mechanism of skin permeation enhancement of Transcutol was confirmed that it The size of NS-MEs and B-MEs were 22.40.3 and can increase the solubility of the penetrant in stratum 23.10.5nm, respectively, which shown that the NS corneum [36]. Therefore, the content of Transcutol in did not influence the droplet size of the MEs (p>0.05).
Table 2 Components, solubility and Q72h of seven different formulations (n = 6). Formulation number 1 2 3 4 5 6 7 Water (%) 70 50 50 60 60 50 56.7 Surfactant (%) 15 35 15 25 15 25 21.7 Cosurfactant (%) 12 12 32 12 22 22 18.7 Solubility (mg/g) 7.24 0.56 7.86 0.52 10.31 0.78 8.86 0.43 10.87 0.51 10.62 0.73 8.20 0.62 Q72h (g/cm2) 181.4116.21 67.586.32 243.0620.65 102.879.87 613.8370.12 464.1142.65 81.718.21
Cumulative permeated amount of NS (g/cm)
2

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Fig. 4. 2D-contour plots: (A) the effect of the formulation variables on the solubility; (B) the effect on the in vitro drug cumulative permeation amount; (C) the overall desirability.

Polydispersity index of NS-MEs and B-MEs were 0.147 0.013 and 0.2040.021, respectively. 3.4.2. Visualization by transmission electron microscopy Fig. 5 shows the TEM images of the NS-MEs, B-MEs, NS-MEs-H and NS-H. According to the images, the spherical droplets were existed in the NS-MEs and B-MEs, which represented the structure of MEs. The drop size of most droplets was below 50 nm. The gel network in NS-MEs-H and NS-H were observed. Several droplets of which size was around 50 nm were found in the NS-MEs-H. The finding confirmed that NS-MEs-H still maintained the microstructure of MEs. However, some researchers thought that the addition of

carbomer resulted in the transform from MEs to lamellar structure or a highly ordered microstructure or to a highly structured cubic microgel by adding carrageenan [15,37]. No droplet was observed in the NS-H and only the gel network was observed, this finding was consistent with the previous research [18]. The hydrogel showed a ordered structure of gel network. The spherical droplets in NS-MEs-H were distributed in the three-dimensional gel network of continuous phase. 3.4.3. In vitro permeation behaviors of the NS-MEs and NS-MEs-H The in vitro skin permeation behaviors of NS from the NS-MEs and NS-MEs-H are shown in Fig. 6. The in

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Fig. 5. The TEM images of the optimized MEs and the hydrogel: (A) B-MEs; (B) NS-MEs; (C) NS-H; (D) NS-MEs-H.
Cumulative permeated amount of NS (g/cm )

vitro permeation profiles of the NS-MEs and NS-MEs-H suggest that the formulation may maintain a steady in vivo plasma concentration in three days. The cumulative permeation amount of NS-MEs and NS-MEs-H are 599.9149.83 and 461.4738.33 g/cm2 respectively. The NS-MEs-H has slower permeation rate and lower cumulative permeation amount in 72 h compared with the NS-MEs. The reason was that the viscosity of the NS-MEs-H was much higher and the hydrogel of the NS-MEs-H constrained the release and permeation of the NS. 3.5. Prediction and evaluation of in vivo pharmacokinetic behaviors 3.5.1. Model parameters from a bolus intravenous injection The plasma concentration of NS after intravenous

700 600 500 400 300 200 100 0 0

NS-M Es-H NS-M Es

12

24

36 Time(h)

48

60

72

Fig. 6. The in vitro permeation profile (means SD, n=6): ()NS-MEs and ()NS-MEs-H.

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administration plotted against time is shown in Fig. 7 (A). The PKSolver program was used to calculate the model parameters. A, B, and were 1098.626133.490 ng/ml, 256.77934.883 ng/ml, 11.9131.646 h -1 and 0.907 0.113 h-1, respectively. 3.5.2. Input function from in vitro skin penetration data From Fig. 7 (B), the in vitro skin permeation of NS from the NS-MEs-H was divided into three stages, including a slower release stage during the first 6 h, a rapid and steady release phase from 6 to 60 h, and a slower release phase from 60 to 72 h at last. When t1, t2, t3 is 6, 60, and 72 h, the corresponding J1, J2, J3 is 4.38 0.55, 6.960.58 (p<0.01 vs.J1), and 4.560.36 (p<0.01 vs.J2) g/cm2/h calculated by the Eqs. (3) (4) and (5), respectively. The first slow release from the formulation was due to the reason that the components of the MEs as penetration enhancers was not rapidly diffused from the gel network to skin, therefore delayed penetration enhancing effect. Then the steady release phase was obtained due to the controlled released effect of the polymer. At last 12 h, the slower permeation rate was recorded for the reason that the drug concentration in the NS-MEs-H was not sufficient to obtain a driving force for the high steady permeation rate. 3.5.3. In vivo pharmacokinetic behaviors Pharmacokinetic parameters of transdermal NSMEs-H and oral NS suspension were estimated for each rat by using a computer program, PKSolver (Version 2.0), and the average of six values was calculated. Noncompartmental analysis was employed to
Plasma concentration of NS(ng/ml)

calculate the pharmacokinetic parameters. Peak plasma concentration (Cmax) and time of its occurrence (Tmax) were read from the plasma concentrationtime curve individually. C max and T max after oral administration were recorded three times after each dose. The pharmacokinetic parameters are shown in Table 3. After dose normalization, the bioavailability of transdermal administration was 1.55-fold higher than that of oral suspension. The higher bioavailability of the transdermal NS-MEs-H was due to the avoidance of the first pass effect. In addition, the transdermal NS-MEs-H can avoid the fluctuation of drug plasma concentration. From Fig. 8, we can obviously concluded that transdermal NS-MEs-H can provide a much steadier plasma concentration-time curve compared with the oral suspension in three days treatment. Thus, a more stable anti-hypertensive effect could be obtained. The predicted plasma concentrations were calculated using convolution method by the Eq. 6 presented previous. Fig. 8 illustrates the observed and predicted plasma concentrations of NS after transdermal administration of the optimized NS-MEs-H. From Fig.8, almost every observed data was distributed around the predicted curve. Further more, we calculated the %PE. As shown in Table 4, most of the absolute %PE values during the application period were below 10%. The low values of %PE suggested that the predictions of in vivo drug level from the in vitro permeation data were in good agreement with the actual observed concentration data, this is in accordance with the previous research [26]. However, a few in vivo plasma concentrations were not predicted well as the %PE values may exceed 10%. This may due to the batch to batch variation of the formulation and the individual variation of the animals.
Cumulative permeated amount of NS (g/cm )
500 NS-M Es-H 400 300 200 100 0 0 12 24

1600 1400 1200 1000 800 600 400 200 0 0.00 6.00 12.00 18.00 24.00 IV

(A)

(B)

36 48 60 72 Time(h) (B) (means SD, n=6). (B) Fig. 7. (A) Mean plasma concentrationtime curve after bolus intravenous administration of NS (2 mg/kg)

30.00

Time (h)

The in vitro permeation profile of the NS-MEs-H (2 mg/cm2) (means SD, n=6).

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60.00 Oral
NS plasma concentration (ng/ml)

50.00 40.00 30.00 20.00 10.00 0.00

Before the removal of the NSM Es-H After the removal of the NSM Es-H Observed Predicted

12

24

36

48 (A) Time (h)

Time(h)

60

72

84

96

108

Fig. 8. NS plasma concentration vs. time profile in rats: after three oral administration of NS suspensions and transdermal administration of the NS-MEs-H (20 mg/10 cm2). Table 3 Pharmacokinetic parameters of NS after oral and transdermal administration of NS-MEs-H (n=6). Parameters Tmax1 (h) Cmax1 (ng/ml) Tmax2 (h) Cmax2 (ng/ml) Tmax3 (h) Cmax3 (ng/ml) AUC0-72 (ng/ml*h) T1/2 (h) Transdermal NS-MEs-H Oral suspension 42.006.92 27.531.88 1736.31106.59 7.610.70 0.560.23 46.264.30 24.470.07 44.955.14 48.440.09 51.734.02 586.5042.30 4.620.47 Table 4 formulation was optimized using a simplex lattice

The prediction errors (%PE) of predicted plasma concentrations after transdermal administration of the NS-MEs-H (n=6). T (h) 1 2 4 6 8 10 12 24 36 48 60 72 C(obs)a (ng/ml) C(pred)bng/ml) 10.31.6 12.11.8 16.22.4 18.12.3 20.32.2 22.82.1 23.52.1 24.42.0 25.01.7 24.11.9 23.32.8 19.42.3 11.4 14.4 16.1 16.3 19.3 21.0 21.2 25.4 25.4 25.4 25.4 14.2 %PE -10.817.4 -18.820.3 0.914.0 9.811.8 5.112.0 8.08.7 9.68.4 -4.28.5 -1.77.4 -5.59.0 -9.115.1 26.87.8

. The good prediction ability suggested that the convolution method could be employed to estimate the preliminary in vivo pharmacokinetic behaviors of the long-term transdermal preparations. In the further studies, an ideal in vivo plasma concentration curve could be estimated by the pharmacodynamic studies. Than the best in vitro release behaviors can be calculated by deconvolution method. Thus the formulation that has a larger drug permeation rate or a longer period of sustained drug release can be obtained.

C(obs): Observed NS plasma concentration; b C(pred): Predicted NS plasma concentration

a

4. Conclusion
In conclusion, the MEs systems for transdermal delivery of nisoldipine were investigated and its

mixture design. The size of the NS-MEs was 23.1 2.4 nm and the spherical droplets existed in both NSMEs and NS-MEs-H. The in vitro skin permeation and in vivo pharmacokinetic studies showed that NSMEs-H could maintain a steady skin permeation rate and drug plasma concentration in three days. The in vivo plasma concentration was predicted by convolution method. A good prediction ability suggests that the

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convolution method could be used to estimate the in vivo pharmacokinetic behaviors of long-term transdermal drug delivery systems in the further research.

Acknowledgments
This study is financially supported by the major project of National Science and Technology of China for new drugs development (No.2009ZX09310-004), the Fundamental Research Funds for the Central Universities (No.JKY2011017) and National Found for Fostering Talents of Basic Science(No.J0630858).

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