Optimization Of Ibuprofen Delivery Through Rat Skins Biology Essay

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The topical delivery of non-steroidal anti-inflammatory drugs such as Ibuprofen as an effective analysis and anti-inflammatory compounds has been explored as a potential method of avoiding the first pass effects and the gastric irritation that may occur when it is used orally. Ibuprofen was formulated into many topical preparations to reduce the adverse effects simultaneously avoid the hepatic first-pass metabolism; however it is difficult to obtain effective concentration by topical delivery of Ibuprofen due to its low skin permeability. The aim of this study was to develop two kind of microemulsions formulations and this experiment focused on the screening of Ibuprofen loaded microemulsions and evaluation the influence of two types of microemulsions on Ibuprofen skin permeability. In both microemulsions, oil was similar but they varied in surfactant and co-surfactant. The effect of independent variables on skin permeability parameters was evaluated with full factorial design. Results demonstrate that novel formulations were more effective than traditional formulation as skin enhancer. In novel formulation, any increase in percentage of sum of surfactant and co-surfactant had increasing effect on Jss. On the other hand, the proportion of surfactant/ co-surfactant had reverse correlation with Jss. In traditional formulations, increase of both variables: sum of percentage of surfactant and co-surfactant and proportion of surfactant/co-surfactant results in Jss promotion. Comparison between two kinds of microemulsions illustrated that, novel formulations were more effective as Ibuprofen topical carrier in contrast to traditional microemulsions due to lower amounts of surfactant and co-surfactant and less irritating effect.

Keywords: Ibuprofen; NSAID; Microemulsion; Permeability; Topical preparations.

1. Introduction

The advantages of transdermal drug delivery rout include good patient compliance, low systemic side effect, and avoidance of the hepatic first pass effect and so better therapeutic efficacy [1]. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID) can be applied in systemic treatment of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. The topical administration of Ibuprofen may be useful for the patients ever since it reduces the adverse effects, on the other hand, it avoids the hepatic first-pass metabolism and applied relatively consistent drug levels in the site of action. However, it is difficult to maintain effective concentrations in topical drug delivery due to Ibuprofen poor skin permeability [2]. In order to enhance the permeation of Ibuprofen through skin, different formulations have been tested such as supersaturated solutions, mucoadhesive patches and vehicle containing non-ionic surfactants or fatty acid [3]. Transdermal drug delivery of ibuprofen has been reported in various papers. Polyoxyethylene (5) cetyl/oleyl a non-ionic surfactant showed enhancement effect on skin permeation of ibuprofen [4]. Supersaturated system has been used to enhance the penetration of ibuprofen through human skin [5]. Results showed significant flux enhancement was obtained from supersaturated solution compare to the saturated solution.

Microemulsions are clear, thermodynamically stable, isotropic liquid mixtures of oil, water, surfactant and co-surfactant. An approximate droplet diameter of 100 nm was estimated for microemulsions [6]. Nobody can deny the widespread application of microemulsions in different fields such as pharmaceutics, food, and so many industries [7]. They offer a promising vehicle for increasing the aqueous solubility of poorly water-soluble drugs considerably which is usually necessary for parenteral application [8]. Microemulsions have many advantage, for instance, enhance drug solubility, perfect thermodynamic stability, ease of manufacturing and permeation over conventional formulations that convert them to important drug delivery systems. Microemulsions are suitable pharmaceutical formulation for drug delivery to and through the skin [9]. Microemulsion can improve transdermal delivery of lipophilic and hydrophilic compound with different mechanisms. The correlation between microemulsion structure/ composition with its drug delivery effects have been reported and a few studies have shown that internal structure of microemulsion should allow free diffusion of the drug to optimized cutaneous delivery from them [6]. Microemulsion- based hydrogel formulation was applied for ibuprofen topical delivery. Microemulsions could increase the topical delivery of ibuprofen 5.72- 30 times compare with the control [7].

The objective of this project is to evaluate the potential of traditional ( was made with common surfactant and cosurfactant ) and novel (was made with new surfactant and cosurfactant agents) microemulsions to effectively ibuprofen transdermal delivery through rat skin and to identify those factors that influencing the transport with full- factorial experimental design and optimize its topical delivery.

2. Materials and methods

2.1. Materials

Ibuprofen was received from Caspian Tamin, Rashat, Iran. Isopropyl myristate was obtained from Panreac, Spain. Span 20, Tween 80, Acetonitryl, Acetic acid glacial, Choloroform, Methanol, Liquid paraffin, Ethoxylated Caster oil all were purchased of Merck, Germany. PEG 400 was bought from Fluka, England. PEG-8 Caprylic/ Capric Glycerides (L.A.S.), Ethoxydiglycol (Transcutol CG), and Caprylocaproyl macrogolglycerides (Labrasol) were gift from GATTEFOSSE, France. All other reagents were highly commercially available.

2.2. Animals

Female adult Wistar rats (weighing 100-150 g) with the age of 10-12 weeks were purchased from Animals Laboratory, Jundishapur University of Medical Sciences, Ahvaz, Iran. The animals were treated according to the principles for the care and use of laboratory animals and approval for the studies was given by the Ethical Committee of the Ahvaz Jundishapur University of Medical Sciences

2.3. Determination of solubility of Ibuprofen in different oil mixtures

In order to find out perfect oily phase, drug solubility was measured in isopropyl myristate, liquid paraffin and Ethoxylated Caster oil. A mixture of surfactant: co-surfactant: oily phase (ratio 1:1:1) was prepared containing drug in excess, mixed for 24 hours at room temperature, then centrifuged at 3000 rpm for 10 min. In the following the undissolved drug was filtered, after dilution with methanol it was measured by HPLC (Waters, USA). Similar experiments were performed regarding to oily phase solely simultaneously [7]. Finally ibuprofen solubility was determined in various oils.

2.4. Microemulsions preparation

Ternary phase diagrams for determination of the components and their concentration ranges were constructed on the basis of large existence of microemulsion without drug or in the presence of 5% ibuprofen [10]. Two main formulations were prepared: Novel and traditional preparations. Table 1 summarizes all the components present in both formulations. Major variables take part in determination of microemulsion concentration range are including mass ratio of surfactant (S) to co-surfactant (C), mixture weight percentage (S+C), and oily and aqueous phase weight percentage (Tab. 1). At initial step, in a 10 ml tube, the minimum volume of S/C ratio was hold in order to investigate the surfactant weight. In the next step the minimum volume of oily phase was added to latter mixture, and the amounts of oily phase was determined. The appearance of microemulsion was examined visually. In the following, we obtain a turbid mixture by application of tube shaker (Kavosh Mega, Iran). Water titration method was used as well. A volume of 20 µl water was added and by repeating this procedure we come to a clear solution. Clear solutions with low viscosities are the sign of microemulsions formations. The addition of water continued until we observed turbidity in the sample, this is where the titration stopped, and evaluation of other formulation begins. Such procedure was repeated for different oily phase at constant volume of S/C ratio [11].

Formulation

Surfactant

Co-surfactant

Oily phase

S/C ratio

S+C (µl)

Oily phase volume(µl)

Span20, Tween 80

(1:1 mass ratio)

PEG400

Isopropyl myristate

1:1

100

100

Traditional

1:2

150

150

1:3

200

200

250

250

Novel

L.A.S

Labrasol

Isopropyl myristate

1:1

100

50

1:3

200

150

250

200

300

250

350

400

Table1. Different components of novel and traditional formulations.

2.5. Mean droplet size and distribution

According to full-factorial design, both novel and traditional formulations were selected for mean droplet size determination by application of Particle Size Analyzer (Malvern, England). Polydispersity index was measured by Dynamic Light Scattering Spectrophotometer at 25 and 632 nm with 90 angle [12].

2.6. Microemulsion characterization

2.6.1. Viscosity determination

The viscosity of each formulation was measured by application of DV-Ó€Ó€Ó€ viscometer (Brookfield, U.S.A) at 25 in triplicate. The measurement was performed with a number 40 spindle and the shear rate was adjusted at different rpm [12].

2.6.2. Stability

The stability of microemulsion formulations was evaluated through clarity and phase separation observation, on the other hand, particle size analysis was used for droplet size determination formulations at 40 for up to 3 months. In addition, the centrifuge test was performed to determine the physical stability of microemulsion at 5000 rpm for 10 min [12].

2.7. HPLC analysis

HPLC analysis was used for ibuprofen measurement. The column was a Novapac C18 column (4µ, 4.6 mm internal diameter Ã- 25 cm). The mobile phase was acetate buffer (pH 4.1)-acetonitryl (pH 5.1) 50: 50 ratio and 0.7 ml/min flow rate. Wavelength detection was considered at 264 nm. No interference of other components was detected, however, all samples filtered through an aqueous 0.45µm pore size membrane filter before the injection carry out.

2.8. In vitro permeation studies

Full thickness of abdomen skin was separated from newly sacrificed Wistar rat with ether. The subcutaneous fat was completely removed and stored at freezer at -20. For carrying out the permeability studies we keep freeze skin at room temperature previously. The skin was mounted on the diffusion cell (Malek Teb, Iran). It must be considered that the epidermal side (skin surface area of 4.9 0.12, receiver and donor volume of 30 and 5 ml, respectively) should cover the diffusion cell completely with the face up. Both receiver and donor chambers were filled with water and remained for 16 hours at room temperature, then the skins were removed, dried and their thickness was measured [13]. The equilibrated diffusion cell was maintained at 37 for 1 hour. Volume of Ibuprofen microemulsion 5%w/v formulation (up to 5 ml) was applied as donor phase. During the experiments cells were exposed to a magnet stirring in water bath. acetate buffer 70% (pH 4.1) and acetonitryl 10% as received phase stirred permanently at 37 and 300 rpm, whereas skin temperature was 32. At each interval (0.5, 1, 2, 3, 4, 5, 6, 7, 8 hours) a volume of 1 ml was removed from receiver phase, at the same time a new volume of 1 ml was replaced in receiver chamber. The permeability test was performed triplicate. The microemulsion formulations were prepared according to full-factorial design

2.9. Study design for preparation of microemulsions and permeation

Several parameters influence on final properties of microemulsions and permeation through rat skin. Full-factorial design was used concerning with 3 variables at 2 levels (Tab.2). According to the table S/C ratio and percentage of sum of surfactant and co-surfactant in both novel and traditional formulations were the same whereas the oily phase in novel formulation and aqueous phase in traditional formulation were different. For obtaining more documents we evaluated ternary-phase diagrams. In recent study, the influence of independent variables on particle size and Ibuprofen permeation parameters through rat skin considering as response was investigated (P, Tlag, Jss). The interactions intensity of variables on each response were estimated through simultaneous multiple regression. The optimized formulation was selected and its permeation through rat skin on the basis of surface response technique was evaluated.

Novel formulations

Traditional formulations

Variables

3:1

1:1

3:1

1:1

High level

S/C

Low level

50

70

40

65

High level

S+C

Low level

-

-

18

8

High level

W%

Low level

20

10

20

10

High level

Oil%

Low level

Table2. Presentation of independent variables and levels in novel and traditional formulations.

2.12. Statistical analysis

The data was demonstrated as mean S.D. The statistical analysis was according to two-way t-test or variance analysis, following by full-factorial design using Minitab Ó€Ó€ software. In order to figure out the relation between dependent and independent variables, we applied simultaneously multi regression test.

3. Results and discussion

3.1. Ibuprofen aqueous solubility

Ibuprofen aqueous solubility was measured triplicate estimated as 0.880.04 mg/ml, accordingly Ibuprofen is very slightly soluble which is consistent with USP report. On the other hand, to confident about conformation of sink condition in Ibuprofen permeation study through rat skin, drug solubility in receiver phase containing acetate buffer 70% (pH 4.1) and acetonitryl 10% was evaluated. The results demonstrate that Ibuprofen solubility is 32524 mg/ml (n=3).

3.2. Ibuprofen solubility at different oily phases

Table 3 demonstrates drug solubility at various oily phase and mixture of oil/ Tween 80/ PEG 400 with 1: 1: 1 ratio. As it is illustrated Ibuprofen has the highest solubility in Isopropyl Myristate and secondly shows appropriate solubility in liquid paraffin and Ethoxylated Caster oil, respectively. In addition, drug solubility in a mixture of Isopropyl Myristate accompanied by surfactant and co-surfactant was much more higher in contrast to other oily mixtures, however, addition of surfactant and co-surfactant leads to a decrease of drug solubility in the presence of Isopropyl Myristate whereas for other oils showed an increasing trend regarding drug solubility. Chen et.al obtained similar results in 2006 [15]. In recent study, drug solubility was evaluated in Isopropyl Myristate, Isopropyl Palmitate, Oleic acid, Ethyl Oleate, and a mixture of mentioned oils in combination with Tween 80 and propylene glycol as surfactant and co-surfactant, respectively. Results indicate that the highest drug solubility belongs to oleic acid primarily then Isopropyl Myristate, while addition of surfactant and co-surfactant to isopropyl Myristate results in decrease of drug solubility. Ibuprofen solubility in Isopropyl Myristate equals to 0.160.015 g/ml.

Carrier

Solubility

Isopropyl Myristate

0.1960.029

Liquid paraffin

0.1380.019

Ethoxylated Caster Oil

0.1120.014

Isopropyl myristate:Tween80:Peg400

0.1760.025

Liquid paraffin:Tween80:PEG400

0.1550.017

Ethoxylated Caster Oil:Tween80:PEG400

0.1240.010

Table3. Demonstration of Ibuprofen solubility at different oil: surfactant: co-surfactant mixtures.

3.3. Phase studies

The systems were composed of Tween 80: Span 20 as surfactant and PEG 400 as co-surfactant. The phase diagrams were used to investigate the microemulsion regions. The pseudo-ternary phase diagrams were constructed in various weight ratios of S/C (Fig. 1)

Fig.1. The pseudo-ternary phase diagrams of S/C system at the 1: 1, 1: 2 and 1: 3 weight ratios for traditional formulations at 25 .

The translucent microemulsion region can be observed in phase diagrams. The diagrams indicate that a rise in S/C ratio leads to a more extensive microemulsion regions and presence of much more water in the structures. All the microemulsions contain 4-20% water, about 30-70% mixture of surfactant and co-surfactant and 25-64% oil. On the other aspect, traditional formulations contain low amounts of water and are W/O type, Two types of non-ionic surfactant were used for preparation of traditional formulations, however, a combination of non-ionic and ionic surfactant is essential to develop the microemulsion region [14].

For preparation of novel formulations LAS and Labrasol were used as surfactant and co-surfactant, respectively. Figure 2 gives information about phase studies for novel microemulsions. The results indicate that the microemulsion regions are wider in novel preparation in contrast to traditional. In addition, in both novel and traditional formulations the ranges of microemulsion concentrations developed while we prepare 1:3 ratio of S/C in comparison to 1:1. In novel formulations 5-88% water, 1-30% oil and 10-85% combination of surfactant and co-surfactant exist consequently, high amounts of water can be held in microemulsion construction. From this aspect novel preprations are more economic and lower irritation effect observes due to the less content of surfactant. In such formulations microemulsions are of O/W type since they contain great amounts of water and observation of micellar constructions is predictable [14].

Fig.2. The pseudo-ternary phase diagrams of S/C system at the 1:1, 1:2 and 1:3 weight ratios for novel formulations at 25 .

According to full-factorial design 8 novel and traditional formulations selected, respectively and characterization of each formulation examined separately before permeation study through rat skin (Tab.4, 5).

Formulation No.

Full-factorial state

S/C ratio

(S+C) %

Aqueous phase volume%

1

+++

3:1

65

18

2

++-

3:1

65

8

3

+--

3:1

40

8

4

+-+

3:1

40

18

5

-++

1:1

65

18

6

--+

1:1

40

18

7

-+-

1:1

65

8

8

---

1:1

40

8

Tab.4. illustration of traditional formulations used in permeation studies

Formulation No.

Full-factorial state

S/C ratio

(S+C) %

Aqueous phase volume%

1

+++

3:1

70

20

2

++-

3:1

70

10

3

+--

3:1

50

10

4

+-+

3:1

50

20

5

-++

1:1

70

20

6

--+

1:1

50

20

7

-+-

1:1

70

10

8

---

1:1

50

10

Tab.5. illustration of novel formulations used in permeation studies

3.4. Characterization studies

Viscosity:

Among traditional formulations 1, 2, 5, 7 have the highest viscosity but 3, 4, 6, 8 formulations showed low degree of viscosity. The following results illustrate that the major factor affecting viscosity is percentage of surfactant and co-surfactant.

The same experiments were performed concerning novel preparations. Formulations 1, 2, 5 containing a great amount of surfactant and co-surfactant showed a high value of viscosity, although formulation 7 did not follow the similar trend. Comparison between novel and traditional preparations indicates that traditional formulations have much more attitude to be dispersed rather than novels. Apart from that the percentage of surfactant and co-surfactant in traditional formulations has more effect on viscosity in contrast to novel ones. We expect that apart from mentioned parameters, other factors such as oil percentage and S/C ratio influence on viscosity changes in novel formulations. However, the viscosity was not significantly higher in traditional formulations than novel ones (p= 0.085), (Tab.7, 8).

Mean particle size:

For traditional preparations we put all formulations in two categories: first, the ones with mean particle size below 25 nm that prepared by a ratio of 1:1 S/C including formulations number 5, 6, 7, 8. The second group has the mean particle size above 30 nm consist of a great ratio of S/C, including formulations number 1, 2, 3, 4. In addition, the difference between mean particle size in two categories is significant (p=0.005). In conclusion, it seems that the amounts of co-surfactant plays an important role in mean particle size variations, on the other hand, PEG 400 in traditional preparations as a co-surfactant forms a film around dispersed was resulting in a decrease of particle size. As it was observed the polydispersity index demonstrates uniformity of particle size in all formulations.

Similar tests were carried out regarding novel preparations. All the novel formulations showed mean particle size below 15 nm except formulations 2, 5, 7, 8, thus great consumption of co-surfactant in S/C ratio and high percentage of surfactant and co-surfactant leads to decrease in particle size. In conclusion, particle size in novel formulations significantly is less than traditional (p=0.0016), thus LAS and Labrasol components forms a film round the particles which causes decrease in interface forces and more small particle size. Such investigations are consistent to phase studies in which a greater microemulsion region belongs to novel formulations. It should be considered that both traditional and novel formulations showed appropriate uniformity of particles (Tab.7, 8).

Different regression analysis was use for evaluation of independent variables on microemulsion particle size.

For traditional formulation results indicate that S/C (p=0.001) and S+C % significantly affect mean particle size while S/C ratio has the highest effect among all. However, surfactant and co-surfactant percentage have diverse relationship with particle size changes (Fig. 3)

Fig.3. Presentation of independent variables on mean particle size for traditional formulations.

Similar experiments were performed related to novel formulations. The results illustrate that in novel preparations, both S/C and percentage of surfactant and co-surfactant parameters affect mean particle size, although this relation is not completely significant (p value of 0.135 and 0.085, respectively).

It should be considered that for novel preparations the percentage of surfactant and co-surfactant critically influence on mean particle size (Fig. 4) whereas for traditional formulations the most important factor is S/C ratio. The results show that for novel preparations the interaction of three variables was magnificently affect mean particle size variations.

Fig.4. the effect of independent variables on mean particle size for novel formulations.

Formulation No.

Viscosity (cps)

Mean particle size(nm)

Polydispersity index

1

603.1

301.4

0.30.01

2

562.3

301.3

0.250.009

3

451.6

371.0

0.310.013

4

411.1

351.4

0.280.012

5

572.2

220.7

0.190.014

6

402.3

231.1

0.20.008

7

532.8

190.4

0.220.007

8

441.9

250.9

0.220.011

Table7. Characterization of traditional formulations (meanS.D, n=3).

Formulation No.

Viscosity (cps)

Mean particle size(nm)

Polydispersity index

1

501.7

190.5

0.240.011

2

482.3

130.7

0.190.008

3

421.5

250.9

0.150.005

4

431.2

200.8

0.210.007

5

472.0

100.4

0.280.009

6

401.8

180.6

0.250.006

7

411.6

150.3

0.120.004

8

381.4

160.2

0.150.006

Table8. Properties of novel formulations (meanS.D, n=3)

3.5. Stability

Both novel and traditional formulations did not showed any change of phase separation during 6 months. Amounts of Ibuprofen in different formulations at 40 for 3 months were 943 % (n=8) and 972% (n=8) for traditional and novel formulations, respectively. However, traditional formulations showed a little instability at 40 which is due to thermal sensitivity.

3.6. Ibuprofen effect on microemulsion structure

In order to evaluate the effect of Ibuprofen on microemulsion formulation Ibuprofen was added to the formulation in two steps: first, the addition of drug was carried out to oily phase containing surfactant and co-surfactant in the following the microemulsion was formed by water titration. In the second procedure, Ibuprofen was added after formation of microemulsion. The results illustrate that traditional formulations were transparent in first procedure although they became initially turbid in the second procedure. On the other hand, novel preparations were transparent in both procedures.

Traditional formulations are mostly W/O microemulsions because after addition of Ibuprofen, turbidity of formulation observes as a result of particle size growth, however, Ibuprofen causes reformation of microemulsion as it establishes a surfactant film [15]. Since novel formulation contains bicontineous structure, surfactant film has the capacity to avoid particle size growth and it maintains the formulation in microemulsion region.

3.7. Ibuprofen microemulsion permeation studies from rat skin

Different parameters were investigated through permeation studies, including flux (Jss), permeability coefficient (p), incubation time (Tlag) and diffusion coefficient (D). The linear slope of accumulative drug amount against time curve is considered as Jss. We obtained P by Jss=PC in which C represents the drug concentration in donor phase and has a value of 0.88 mg/ml for control group and 50 mg/ml for microemulsions. On the other hand, by crossing the steady state section of permeation profile on the horizontal axis, D parameter can be easily found.

(Equation-1)

Since h demonstrates skin thickness and practically does not show the real pathway for drug permeability, the diffusion coefficient is defined as apparent D. on the other hand, confirmation of sink condition was necessary for calculation of Jss and p parameters therefore the maximum concentration at receiver phase was less than 3% of drug solubility. Laplace transformation technique was used according to finite and infinite dose also science software to obtain less error through calculations. In this technique because of estimation of momentary velocity the error comes to its lowest level [16]. For simulation of skin into normal condition, skin samples were hydrated from approximately 10 to 20%. Samples thickness were 34045 µ (n=35).

Traditional formulation:

In this experiment, the permeability parameters were calculated according to cumulative amounts of drug by application of full-factorial design. (Tab.9)

Formulation

Jss (µg/.h)

P (

D (appearance)

()

Tlag (h)

Full-factorial state

Control

83.56.3

128

0.00690.0008

2.60.17

-

1

1377.9

2.750.29

0.0120.005

1.860.12

+++

2

1339.4

2.660.31

0.010.007

1.970.14

++-

3

1204.5

2.40.18

0.00660.0005

2.390.25

+--

4

1236.2

2.460.21

0.00970.0008

2.270.18

+-+

5

1187.7

2.360.17

0.00760.0004

2.320.15

-++

6

1125.1

2.240.12

0.00970.0006

2.440.1

--+

7

1153.9

2.30.21

0.0110.0003

2.350.19

-+-

8

1074.3

2.140.16

0.00640.0006

2.40.19

---

Table9. Presentation of different parameters interfere with Ibuprofen permeation through rat skin for traditional formulations (meanS.D, n=3).

The following regression equation demonstrates the relation between independent variables and Jss:

(Equation-2)

S/C ratio and (S+C) % are significantly affect Jss as an increase in two mentioned factor leads to elevation of Jss (p=0.001 and 0.004). The percentage of aqueous phase was not significantly affect Jss (p=0.085). Surfactant deforms the skin structure simultaneously causes an increase in Jss, on the other hand, through an increase in surfactant and co-surfactant a decreasing trend in solubility of ibuprofen occurs and promotion of drug thermodynamic activity leads to Jss elevation. Comparison between Jss parameter in control group and microemulsions indicate that formulation 1 and 2 are significantly (p<0.05) different.

The same regression analysis was performed concerning with Tlag parameter. For more information take a glance at following equation:

(Equation-3)

The relation between S/C and (S+C) % with Tlag was significant (p=0.05), an increase in named factors leads to decrease in Tlag which represents the effect of surfactant on skin structure. Comparison between control group and microemulsions illustrates that formulation 1, 2, 4 could result in a significant decrease of Tlag.

On the other hand, formulations number 1, 2, 4, 6, 7 show higher value of diffusion coefficient in contrast to control group (p<0.05). Consequently the Equation 7 demonstrates the relation between independent variables and D parameter.

(Equation-4)

Any of the independent variables significantly influence on D parameter. While we compare the results obtained from D studies and simultaneously P parameter we investigate that the major effect of variables on P parameter is due to the influence on distribution coefficient between skin and formulation. Eventually, surfactant and co-surfactant facilitate the distribution of drug through skin. Considering the fact that the relation between the effect of surfactant and co-surfactant was insignificant with D parameter thus the influence of surfactant mixture is mainly focused on skin ability for solubility of Ibuprofen.

-Novel formulation

Similar experiments were carried out for novel preparations (table 9). The following equation indicates the effect of independent variables on Jss for novel formulations:

(Equation-5)

The results illustrate that three variables are significantly affect Jss (p<0.05). It should be noted that the relation between S/C ratio and oil phase percentage with Jss is diverse although (S+C) % is directly influence on Jss, subsequently higher consumption of surfactant in the formulation results in the promotion of Jss and this is diversely true for traditional formulations.

Formulation

Jss (µg/.h)

P (

D (appearance)

()

Tlag (h)

Full-factorial state

Control

78.36.54

135.67.5

0.00750.0007

2.760.185

-

1

15112.8

3.020.27

0.01230.001

1.350.14

+++

2

16910.3

3.380.2

0.0150.0009

1.270.09

++-

3

14714.5

2.950.28

0.0120.0013

1.330.12

+--

4

1447.6

2.850.17

0.01010.0008

1.660.11

+-+

5

17515.8

3.50.33

0.0250.0017

0.970.066

-++

6

17018.11

3.40.36

0.0170.001

1.180.006

--+

7

19516.6

3.90.33

0.01860.0015

1.050.008

-+-

8

18016.1

3.650.35

0.020.0016

1.20.075

---

Table10. Presentation of different parameters interfere with Ibuprofen permeation through rat skin for novel formulations (meanS.D, n=3).

It could be pointed that the above results are closely connected to phase behaviors, thus the higher amounts of Labrasol in novel formulations may form bicontineous structures and actually development of skin permeability [17]. In traditional formulations, PEG 400 plays a key role as co-surfactant although it was not magnificently affect permeability. The amounts of oily phase in the formulation make the Jss parameter to be decreased, in other words, Jss has a higher value in microemulsions in contrast to control group and we conclude that it is an indicator of absorption enhancer in novel microemulsions (p<0.05).

Several investigations were carried out to figure out the influences of independent variables on Tlag and D parameters for novel formulations, as well.

(Equation 6)

According to regression analysis we found out that S/C ratio was directly and significantly affect Tlag (p=0.018), in other words, the more this ratio, the higher Tlag (fig 5). An increase in surfactant amount leads to decrease of Tlag by forming bicontineous structures. Studies demonstrate that this fact accelerate the permeation of drug in microemulsion which is mainly affect Tlag parameter [17]. As a matter of fact, all novel formulations significantly decrease Tlag and are capable of accelerating drug onset of action.

Fig.5. the relation between independent variables and Tlag for novel formulations.

A similar pattern was observed in D parameter studies as it is obvious in the following equation:

(Equation 7)

S/C ratio is the only factor which is in diverse and significant relation to D parameter (p=0.017), from another aspect the higher amounts of co-surfactant in formulation results in D parameter elevation (fig 6). In conclusion, co-surfactant in novel formulation can promote drug diffusion as well as changing diffusion coefficient. On the basis of equation, P parameter will change by K and D parameters, mention the fact that S/C ratio diversely affect P and D, it seems that the enhancing effect of novel formulation on P was due to D and K parameter had less importance. The incredible role of Labrasol in novel formulations results in promotion of D parameter in contrast to control group (p<0.05).

Fig. 6. The relation between independent variables and D parameter for novel formulations.

Comparison between the effects of two formulations on permeability parameters indicated that maximum enhancement of Jss for novel and traditional formulations were 2.5 and 1.65 respectively. It seams that he kind of oil phase and cosurfactant and s/c ratio and (s+c)% are very important variables that influence ibuprofen permeation through rat skin. The effect of microemulsion on ibuprofen permeation through porcine skin was reported [7]. In this study various formulations were made by ethyl oleate as oil phase, Tween 80 as surfactant and propylene glycol as cosurfactant. Results showed microemulsions increased ibuprofen permeation rate 5.72-30 times. The internal structure of microemulsion plays critical role on cutaneous delivery and it seams that ibuprofen release from microemulsion in present study is lower than microemulsion made by ethyl oleate, Tween 80 and propylene glycol.

3.8. Skin permeation optimization

In order to achieve a formulation with optimized permeation through rat skin active variables that significantly influence the response were used and skin permeation were performed with this active variables using a central composite design (CCD). For this purpose additional formulations were provided with 23 factorial design and central points (four formulations) and the effect of active variables on skin permeation parameters were evaluated.

In traditional preparations, D and P parameters were eliminated from field of study since there were not significantly related to independent variables. Thus both Jss and Tlag parameters considered appropriate for the goal of formulation optimization. Other studies indicate that water percentage showed no significant relation with both Tlag and Jss factors which makes the role of S/C ratio and (S+C) % more apparent in microemulsion optimization (table 11, fig 7&8). Since aqueous phase content and Jss are not significantly related the following equation obtains:

Jss= 2.1 + 0.29 (S+C) + 5.28 (S/C)

(Equation-8)

Therefore (S+C) and (S/C) demonstrate direct and significant correlation with Jss. The modified equation for Tlag follows the below pattern:

(Equation-9)

In contrast to Jss, any increasing in amount of (S+C) and (S/C) decrease Tlag.

ANOVA

P - value

Factor coefficient

Factor

Response

92.08 = F

98% =

0.01

120.450

intercept

0.001

7.7

S/C

0.004

5.125

(S+C)%

0.085

1.87

W%

0.105

1.542

(S/C) Ã- (S+C)%

0.719

-0.208

(S/C) Ã- W%

0.942

0.042

(S+C)% Ã- W%

0.432

0.046

(S/C) Ã- (S+C)% Ã- W%

5.53 = F

93%=

0. 02

2.250

intercept

0.045

-0.127

S/C

0.048

-0.125

(S+C)%

0.569

-0.027

W%

0.235

-0.082

(S/C) Ã- (S+C)%

0.517

-0.03

(S/C) Ã- W%

0.922

-0.007

(S+C)% Ã- W%

0.61

0.001

(S/C) Ã- (S+C)% Ã- W%

Table11. Regression coefficient and statistical analysis concerning with independent variables and permeation parameters through rat skin for traditional formulations in central composite design.

Fig.7. Response surface plot regarding the relationship between Jss, W% and (S+C)% (a).

The relationship between Jss, W% and S/C (b). demonstration of Jss, (S+C) % and S/C (c) for traditional formulations.

Fig.8. Response surface plot concerning with independent variables (W%, (S+C) %, S/C) and Tlag for traditional microemulsion formulations.

In order to verifying the above equation, two different formulations were prepared, thus Tlag and Jss were estimated and compared with each parameter calculated with equation. The results illustrate that the optimized formulations concerning with traditional preparation were ones containing 65% of (S+C) with S/C 1:3 ratio at minimum and maximum values of Jss and Tlag, respectively (table 12).

Calculative parameters

Experimental parameters

Independent variables

Formulation No.

(S+C)%

S/C

2.34

117

2.4

138.8

55

2

1

2.24

120.9

2.3

122

65

2

2

Table12. Properties of central composite formulations accompanied by Jss and Tlag in traditional preparation.

In recent study the above investigations were analyzed regarding novel preparations as well. The results indicate that both Jss and P parameters were significantly related to independent variables, whereas T lag with S/C and (S+C) % as D parameter with S/C followed such a relationship (table 13, fig 9&10). The modified equations for novel microemulsions are as follow:

Jss= 21.11+ 0.99 (oil%) + 0.212 (S+C)% + 0.25 (S/C)

(Equation-10)

Tlag= 0.78- 0.011 (S+C)% + 0.19 (S/C)%

(Equation-11)

D= 0.012 - 0.0093 (S/C)

(Equation-12)

ANOVA

P - value

Factor coefficient

Factors

response

27.72= F

97.6 %=

0.001

166.37

intercept

0.002

-13.62

S/C

0.026

6.13

(S+C)%

0.023

-6.37

Oil%

0.15

1.12

(S/C) Ã- (S+C)%

0.17

1.13

(S/C) Ã- Oil%

0.09

-3.12

(S+C)% Ã- Oil%

0.25

-0.62

(S/C) Ã- (S+C)% Ã- Oil%

7.21= F

91.6%=

0.008

1.25

intercept

0.018

0.105

S/C

0.056

0.009

(S+C)%

0.378

0.038

Oil%

0.218

-0.001

(S/C) Ã- (S+C)%

0.196

-0.064

(S/C) Ã- Oil%

0.394

-0.038

(S+C)% Ã- Oil%

0.269

-0.024

(S/C) Ã- (S+C)% Ã- Oil%

5.8= F

89.6%=

0.08

0.016

intercept

D

0.017

-0.0038

S/C

0.231

0.0013

(S+C)%

0.971

-0.00004

Oil%

0.654

-0.00029

(S/C) Ã- (S+C)%

0.514

-0.00089

(S/C) Ã- Oil%

0.471

0.00096

(S+C)% Ã- Oil%

0.642

-0.00014

(S/C) Ã- (S+C)% Ã- Oil%

Table 13. Regression coefficient and statistical analysis concerning with independent variables and permeation parameters through rat skin.

Response surface plots for novel microemulsion preparations were brought underneath:

Fig.9. Response surface plot regarding independent variables (oil%, (S+C) %, S/C) and Jss in novel formulations.

Fig.10. Response surface plot concerning with independent variables (S+C )%, S/C and Tlag for novel formulations.

According to above results the optimized novel formulation contains minimum S/C ratio apart from maximum of (S+C) % with 1 and 70% values, respectively. In order to verifying the above equations, two different formulations were prepared, thus Tlag and Jss were estimated and compared with each parameter calculated with equation. (table 14). It should be pointed that such a formulation possesses minimum Tlag in contrast to a maximum values of Jss with optimized permeability through rat skin.

Calculative parameters

Experimental parameters

Independent variables

Formulation No.

Oil%

(S+C)%

S/C

1.14

116

1.22

119

15

60

2

1

1.05

193

1.12

190

15

70

2

2

Table14. Central composite characterizations for novel formulations.

Conclusion

Phase diagrams indicated more extensive microemulsion region with a rise in S/C. wider microemulsion region was provided with novel formulsion. Particle size in novel formulsions was significantly less than traditional ones. It seams that LAS and Labrasol produced elastic film round the particles.

In permeation experiments, the correlation with Jss and S/C ratio was directly and diversely for traditional and novel formulations respectively. It suggests that the effect of cosurfactant on Jss was more effective than traditional cosurfactant.

In conclusion novel formulation demonstrates better formulation for ibuprofen transdermal delivery. Novel formulations produced smaller particles size and higher Jss. Jss of ibuprofen in novel formulations controlled with s/c ratio, oil % and (s+c) % in this manner that any increasing in co surfactant increased Jss.

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