A+Transdermal+Microemulsion...

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

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A transdermal microemulsion-based hydrogel of nisoldipine: preparation, in vitro characterization

and in vivo pharmacokinetic evaluationYan 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

_____________________________________________________________________________________________________________

Abstract

The purpose of this study was to construct a microemulsion-based hydrogel (MEs-H) for the transdermal delivery of an anti-hypertensive 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 23.1±2.4 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 461.47±38.33 µg/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 first-pass 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

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

__________*Corresponding author. Address: China Pharmaceutical University, No.24, Tongjiaxiang, Nanjing 210009, China. Tel: +86-25-83271293; Fax: +86-25-83271293 E-mail: [email protected]

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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 time-consuming 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.

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 Gattefossé, (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

chromatography–mass spectrometry (LC/MS) grade. All other chemicals and reagents were of analytical grade.

Male Sprague-Dawley rats (180–200 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 μm,4.6 mm×250 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 40°C and the injection volume was 20 μl. The peak area correlated linearly with NS concentration in the range of 0.5–50.0 μg/ml with the lowest detection limit at 0.5 μg/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 80were chosen as surfactants. Cosurfactants selected wereTranscutol® and PEG400. An excess amount of NS was added to 2 ml of each excipient and turbine reciprocally at 32°C, then shook at 32°C for 72 h. Samples were centrifuged at 10,000 rpm for 10 minutes. The super-natant was separated and then diluted with ethanol. The diluted samples were filtered through 0.45 μm 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 32°C. After equilibration, the mixtures were assessed visually under light versus a dark background and determined as being MEs by their clarity and transparency.

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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 32°C 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 180–200 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 (0–4°C) 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 f rom 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 32±0.5°C 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 μm filter films and analyzed by HPLC. The cumulative amount of permeated drug per unit area of skin (Q, μg/cm2) at time point ‘n’ was calculated using Eq. (1).

(1)

Where Cn and Ci are the drug concentrations at sampling time point ‘n’ and ‘i’ respectively, V is the volume of receptor medium, S is the effective skin surface area. All the transdermal permeation experiments were repeated six times.

2.7. Drug solubility in the NS-MEs

An excess amount of NS was added to the mixture of oil, surfactant and cosurfactant, and then water was added drop by drop under gentle magnetic stirring at 32°C.

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].

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

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

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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 (220–250 g) were maintained at 25°C 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 NS-MEs-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. NS-MEs-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 –20°C 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 μl plasma, 10 μl (100 ng/ml) of nimodipine and 25 μl 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 40°C under a stream of air. The residue was reconstituted with 100 μl mobile phase and then NS was estimated using LC/MS.

Chromatographic separation was carried out at room temperature with an Kromasil C18 (5 μm, 250×4.6 mm) reversed-phase column on an LC/MS2020 system (Shimadzu Corporation，Japan). The column temperature was set at 40°C, and the injection volume was 5 μl. 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 350°C, DL temperature 250°C. 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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ng/ml with a coefficient of determination of 0.9999 and good calculated accuracy and precision. Peak area ratio (A1) of nisoldipine and internal standard was determined after extraction of the plasma samples containing the nisoldipine at different concentrations. Another peak area ratio (A2) was obtained from direct injection of the standard solution containing the nisoldipine and the internal standard at the same concentration with the plasma samples used above. The extraction recoveries of the drug from the rat plasma were more than 70% determined by comparing A1 and A2 described above. The RSD for intra and inter-day estimations were 2.43% and 1.93%, respectively, demonstrating good reproducibility.

2.11.3. Convolution method

Convolution and deconvolution method could be employed to predict the in vivo or in vitro data by a complicated calculation using the obtained information. To predict the drug plasma concentration of NS by the in vitro skin permeation studies, the convolution method were performed according to the previous research [26], with the in vitro permeation rate as input function and the i.v. plasma data as weight parameters.

In our study, plasma concentrations of NS against time after intravenous administration were fitted to a two compartment model of intravenous bolus using PKSolver program. The value of unit impulse parameters (A, B, α and β) could be estimated from the Eq. (2).

(2)In vitro skin permeation fluxes (J, μg/cm2/h) of each

time interval was calculated from the slope of the plot of drug amount permeated vs. time (h) according to the Eqs. (3),(4) and (5).

(3) (4) (5)

Where t1, t2 and t3 were the selected time points, Qt1, Qt2 and Qt3 were the permeated drug amount at each time point.

After the Laplace transforms [26-27], the transdermal drug concentration in rats can be calculated in Eq. 6.

(6)

(6)

The prediction error (%PE) was calculated by the Eq. (7) to quantitatively determine how well convolution method can accurately predict the in vivo plasma concentrations.

(7) Where C(obs) and C(pred) represent the observed and

predicted NS plasma concentration respectively.

2.12. Statistical analysis

Data were shown as mean±SD. Statistical data were analyzed by the two-tailed t-test and P values for significance were set at 0.05 or 0.01.

3. Results and discussion

3.1. Components for MEs

To maintain a larger solubility of NS in ME systems, suitable oils and surfactants were selected. The solubility of NS in various excipients was determined (Table 1). Among the various tested oils, the solubility of NS was highest in IPM, followed by Labrafil® M 1944, olive oil and oleic acid. It was also reported that IPM was a powerful enhancer for transdermal delivery since it could increase fluidity of lipid portion of the stratum corneum which resulted in a permeation enhancing effect [28-30]. The solubility of NS in surfactants showed that Cremophor EL was better than Tween 80. Cremophor EL is one of the nonionic surfactants which are widely used in topical and transdermal formulations as solublizing agents. Transcutol® also has a good dissolving property of the NS and it was often used as penetration enhancer in transdermal drug delivery systems [31-33]. IPM, Cremophor EL and Transcutol® were chosen respectively as the oil phase, surfactant and cosurfactant for the formulations of NS-MEs.

3.2. Construction of pseudo-ternary phase diagram

Pseudo-ternary phase diagrams were constructed to confirm the concentration ranges of the components. The phase diagrams with various weight ratios of Cremophor

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(P<0.005)

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312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

S

CVCQ

1-n

1i inn

5

t-t- BeAe=C(t)

1

t11 t

QJ

12

t1t22 t-t

Q-Q=J

23

t2t33 t-t

Q-Q=J

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tf

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tIf

)e-1()e-1()(

,t≤t≤0If

)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

BABA

tCBJAJtC

BJAJtC

100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

S

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1-n

1i inn

5

t-t- BeAe=C(t)

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t11 t

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)e-1()e-1()(

,t≤t≤0If

)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

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tCBJAJtC

BJAJtC

100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

S

CVCQ

1-n

1i inn

5

t-t- BeAe=C(t)

1

t11 t

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Q-Q=J

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Q-Q=J

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tf

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tIf

)e-1()e-1()(

,t≤t≤0If

)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

BABA

tCBJAJtC

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100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

S

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1-n

1i inn

5

t-t- BeAe=C(t)

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t11 t

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)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

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I

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100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

S

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1-n

1i inn

5

t-t- BeAe=C(t)

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Q-Q=J

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)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

BABA

tCBJAJtC

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100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

S

CVCQ

1-n

1i inn

5

t-t- BeAe=C(t)

1

t11 t

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Q-Q=J

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Q-Q=J

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)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

BABA

tCBJAJtC

BJAJtC

100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

S

CVCQ

1-n

1i inn

5

t-t- BeAe=C(t)

1

t11 t

QJ

12

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Q-Q=J

23

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Q-Q=J

]ee[)(

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,t≤t≤ tf

]ee[)(

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,t≤t≤ tIf

)e-1()e-1()(

,t≤t≤0If

)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

BABA

tCBJAJtC

BJAJtC

100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

J2

J3

S

CVCQ

1-n

1i inn

5

t-t- BeAe=C(t)

1

t11 t

QJ

12

t1t22 t-t

Q-Q=J

23

t2t33 t-t

Q-Q=J

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tf

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tIf

)e-1()e-1()(

,t≤t≤0If

)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

BABA

tCBJAJtC

BJAJtC

100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

1 11 13211


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

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 (X1), 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-

Components Excipients Solubility Mean ± SD (mg/ml)

Oil

IPM 4.77±0.23

Labrafil M1944 3.87±0.17

oleic acid 0.87±0.05

olive oil 2.39±0.15

SurfactantCremophor EL 5.64±0.35

Tween 80 4.67±0.42

Co-surfactantTransactol 23.18±1.48

PEG400 19.73±1.13

Table 1Solubility of NS in various excipients (n =3).

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

1 13221


1 1

MEs and the cumulative amount of NS permeated across rat skins per unit area after 72 h [Eq.(8)].

(8)where bi is the estimated coefficient for the factor

Xi. X1, X2, and X3 represent the average results of changing one factor at one time from its minimum to maximum value, and X1X2, X2X3, X1X3, and X1X2X3, the interactions show how the responses change when two or three factors change simultaneously.

Solubility and in vitro drug permeation amount during 72h (Q72h) of the seven selected formulations were presented in Table 2. With the help of Design expert 7.0 Software, Eqs. (9) and (10) were obtained to calculate and predict response values for other formulations.

As shown in Fig. 3, the in vitro permeation profiles of the tested ME formulations demonstrated that penetration through skin was a steady process during 72 h.Fig. 4A and 4B show the effect of the three factors on the two responses, the solubility and the in vitro drug cumulative permeation amount, respectively. The orange color represents the favorable area for the response, and the green and blue colors indicate the areas which have intermediate and least values of the response, respectively. From Fig. 4A, the largest cumulative permeation amount of nisoldipine was obtained when the content of water and Transcutol® were both moderate. It seemed that water and Transcutol® were contributed together to the increasing of the transdermal permeation amount. It was reported that the MEs can significantly increase the skin hydration [34], which may enhance the permeation ability of formulations [35]. Hence, the content of water in MEs was critical for drug permeation. The Fig. 4B shows that the solubility increased largely with the increase of Transcutol® probably due to its solubilization effect. The mechanism of skin permeation enhancement of Transcutol® was confirmed that it can increase the solubility of the penetrant in stratum corneum [36]. Therefore, the content of Transcutol® in

MEs was also important. As results shown, water and Transcutol® had to compromise to each other to obtained the largest transdermal permeation amount.

Optimization was performed through a multiple response method called desirability which reflects the desirable ranges for each response. The desirable ranges are from zero to one (least to most desirable respectively). Fig. 4C represents the 2D overlay contour plot showing the composition of the formulation which achieves the maximum desirability (1.0). The suggested formulation composed of IPM (3%, w/w), Cremophor EL (15%, w/w), Transcutol® (22%, w/w) and water (60%, w/w) was prepared and characterized.

3.4. Characterization of NS-MEs and NS-MEs-H

3.4.1. Droplet size

The size of NS-MEs and B-MEs were 22.4±0.3 and 23.1±0.5nm, respectively, which shown that the NS did not influence the droplet size of the MEs (p>0.05).

S

CVCQ

1-n

1i inn

5

t-t- BeAe=C(t)

1

t11 t

QJ

12

t1t22 t-t

Q-Q=J

23

t2t33 t-t

Q-Q=J

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tf

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tIf

)e-1()e-1()(

,t≤t≤0If

)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

BABA

tCBJAJtC

BJAJtC

100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

S

CVCQ

1-n

1i inn

5

t-t- BeAe=C(t)

1

t11 t

QJ

12

t1t22 t-t

Q-Q=J

23

t2t33 t-t

Q-Q=J

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tf

]ee[)(

]e-1[]e-1[)(

,t≤t≤ tIf

)e-1()e-1()(

,t≤t≤0If

)t-t(-)t-t(-2)t-t(-3)t-t(-3

32

)t-t(-)t-t(-1)t-t(-2)t-t(-2

21

t-1t-1

1

2222

1111

BABA

tCBJAJtC

I

BABA

tCBJAJtC

BJAJtC

100C(obs)

)pred(C-C(obs)%PE


32132

3121321XX66.57X-X6.14X+


(P<0.005)

32132

312132172hXX10487.40X-X1235.16X+

X1606.38X+X86.50X-243.06X+67.58X+181.41X=Q

(P<0.0001)

Formulation number Water (%) Surfactant (%) Cosurfactant (%) Solubility (mg/g) Q72h (μg/cm2)

1 70 15 12 7.24 ±0.56 181.41±16.21

2 50 35 12 7.86 ±0.52 67.58±6.32

3 50 15 32 10.31± 0.78 243.06±20.65

4 60 25 12 8.86 ±0.43 102.87±9.87

5 60 15 22 10.87 ±0.51 613.83±70.12

6 50 25 22 10.62± 0.73 464.11±42.65

7 56.7 21.7 18.7 8.20 ±0.62 81.71±8.21

Table 2Components, solubility and Q72h of seven different formulations (n = 6).

Fig. 3. Percutaneous permeation profiles of the tested ME formulations (mean±SD; n = 6): 1-7 represent the seven different formulations stated in Table 2.

0

100

200

300

400

500

600

700

0 12 24 36 48 60 72

Time(h)

Cum

ulat

ive

perm

eate

d am

ount

of

NS

(μg/

cm2 )

1234567

1 11 13231


Polydispersity index of NS-MEs and B-MEs were 0.147±0.013 and 0.204±0.021, 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

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.

1 13241


1 1

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 599.91±49.83 and 461.47±38.33 µg/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

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.

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

0

100

200

300

400

500

600

700

0 12 24 36 48 60 72Time(h)

Cum

ulat

ive

perm

eate

d am

ount

of

NS

(μg/

cm2 )

NS-MEs-H

NS-MEs

1 11 13251


administration plotted against time is shown in Fig. 7 (A).The PKSolver program was used to calculate the model parameters. A, B, α and β were 1098.626±133.490 ng/ml,256.779±34.883 ng/ml, 11.913±1.646 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, 6.96±0.58 (p<0.01 vs.J1), and 4.56±0.36 (p<0.01 vs.J2) µg/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 NS-MEs-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

calculate the pharmacokinetic parameters. Peak plasma concentration (Cmax) and time of its occurrence (Tmax) were read from the plasma concentration–time curve individually. Cmax and Tmax 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 day’streatment. 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.

Fig. 7. (A) Mean plasma concentration–time curve after bolus intravenous administration of NS (2 mg/kg) (means ± SD, n=6). (B) The in vitro permeation profile of the NS-MEs-H (2 mg/cm2) (means ± SD, n=6).

0

200

400

600

800

1000

1200

1400

1600

0.00 6.00 12.00 18.00 24.00 30.00Time (h)

Plas

ma

conc

entra

tion

of N

S(ng

/ml)

IV (A)

0

100

200

300

400

500

0 12 24 36 48 60 72Time(h)

(B)

Cum

ulat

ive

perm

eate

d am

ount

of

NS

(μg/

cm2 )

NS-MEs-H(B)

1 13261


1 1

.

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.

4. Conclusion

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

formulation was optimized using a simplex lattice

mixture design. The size of the NS-MEs was 23.1±2.4 nm and the spherical droplets existed in both NS-MEs and NS-MEs-H. The in vitro skin permeation and in vivo pharmacokinetic studies showed that NS-MEs-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

Table 3Pharmacokinetic parameters of NS after oral and transdermal administration of NS-MEs-H (n=6).

Parameters Transdermal NS-MEs-H Oral suspension

Tmax1 (h) 42.00±6.92 0.56±0.23

Cmax1 (ng/ml) 27.53±1.88 46.26±4.30

Tmax2 (h) － 24.47±0.07

Cmax2 (ng/ml) － 44.95±5.14

Tmax3 (h) － 48.44±0.09

Cmax3 (ng/ml) － 51.73±4.02

AUC0-72 (ng/ml*h) 1736.31±106.59 586.50±42.30

T1/2 (h) 7.61±0.70 4.62±0.47

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).

T (h) C(obs)a (ng/ml) C(pred)bng/ml) %PE

1 10.3±1.6 11.4 -10.8±17.4

2 12.1±1.8 14.4 -18.8±20.3

4 16.2±2.4 16.1 0.9±14.0

6 18.1±2.3 16.3 9.8±11.8

8 20.3±2.2 19.3 5.1±12.0

10 22.8±2.1 21.0 8.0±8.7

12 23.5±2.1 21.2 9.6±8.4

24 24.4±2.0 25.4 -4.2±8.5

36 25.0±1.7 25.4 -1.7±7.4

48 24.1±1.9 25.4 -5.5±9.0

60 23.3±2.8 25.4 -9.1±15.1

72 19.4±2.3 14.2 26.8±7.8

Table 4The prediction errors (%PE) of predicted plasma concentrations after transdermal administration of the NS-MEs-H (n=6).

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

0.00

10.00

20.00

30.00

40.00

50.00

60.00

0 12 24 36 48 60 72 84 96 108Time(h)

(A)

NS

plas

ma

conc

entra

tion

(ng/

ml)

Oral

Before the removal of the NS-MEs-HAfter the removal of the NS-MEs-HObserved

Predicted

Time (h)

1 11 13271


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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