Effects of the Various Solvents on the In vitro Permeability of Indomethacin through Whole Abdominal Rat Skin

Purpose : The aim of the present study was to evaluate the effect of some solvents on in vitro ARRB, showing the greatest enhancement ratio (ER flux ) based on flux followed by Tween 80, plurololeique, polyethylene glycol 400 and labrasol. The ER flux for all the solvents was higher than ER D . Plurololeique, isopropyl myristate and Tween 80 significantly increased diffusion coefficient (p<0.05) compared to hydrated rat skin. Lipid fluidization, lipid disruption structure and the irreversible denaturation of proteins in the SC layer of skin by isopropyl myristate, Tween 80 and plurololeique, as indicated by FT-IR and DSC, is the main factor for higher ER flux and ER D ratios compared to control.


INTRODUCTION
The skin, our body ' s largest organ, is generally considered as an impermeable protective barrier against mechanical, chemical, microbial and physical hazards. The success of a topical drug to be used for systemic drug delivery depends on the ability of the drug to penetrate via skin in sufficient quantities to achieve the desired effect [1]. Nowadays, transdermal route has become one of the most successful strategies for systemic drug delivery.
Transdermal delivery has many advantages among them, avoidance of first pass metabolism of drug, controlled and continuous drug delivery, reduction of dosing frequency, patient compliance enhancement, facilitation of drug localization at target site, avoidance of the risks of injections and reduction in toxic level of drugs [2][3][4][5][6].
Skin permeation of drugs is slightly section of percutaneous absorption. Permeation is the penetration from one layer into another layer of skin. The lipid matrix of stratum corneum layer plays a meaningful role in determining the permeability of substances through the skin. The factors that influence transdermal absorption include biological properties of skin, physicochemical properties of drug, vehicle and dosing concentrations. The biological factors consist of thickness of the skin, regional site, age, blood flow rate and skin conditioning can influence the skin permeation of drugs. Moreover, the physiological characteristics of the drug and vehicle play a significant role in determining percutaneous absorption. These factors include the molecular properties of the drug and vehicle. Skin permeation can be improved by several strategies: increasing the diffusivity in the skin by disturbance of SC lipid matrix, strengthening drug solubility in the skin, enhancing the degree of saturation of the drug in formulation [7,8].
Permeation of drugs through skin is the principle of transdermal delivery. Passive permeation of drug through skin often depends on two major physicochemical properties such as, drug solubility and partition coefficient in SC. Two possible strategies for improving the drug permeation are reversible enhancement of solvent and modification of the drug thermodynamic activity [7]. Hereby, drug transdermal permeation may be affected by solvent type and drug molecule thermodynamic activity. So, selection of a suitable solvent for preparation of topical formulations greatly improves drug delivery.
Enhancer solvents such as water, propylene glycol and oleic acid are known as permeation enhancer by different mechanisms, including disruption of the organized intercellular lipid structure of the stratum corneum, fluidizing the membrane lipids, altering cellular proteins, and extracting intercellular lipids by mostly non-polar solvents. Increase in the diffusivity of stratum corneum has been reported for polar solvent materials, e.g., dimethyl sulfoxide and dimethyl formamide [2,[6][7][8].
Recently, the lipid organization and microstructure of the skin has been examined using various techniques including DSC and FTIR [6,[9][10].
Indomethacin is a non-steroidal anti-inflammatory drug widely used as analgesic, antipyretic and for relief of dysmenorrheal pain symptoms. The most widely reported side effect of indomethacin as a member of NSAID drugs is gastrointestinal ulceration, accompanied by anemia due to bleeding. Administration of drug via dermal route may eliminate gastric irritation, minimizes the systemic toxicity and achieve a better therapeutic response [11]. The use of a topical drug for dermal and transdermal drug delivery depends on the ability of the drug to penetrate via skin in sufficient quantities to achieve the desired effect [1].
The aim of this research was to evaluate the effect of several hydrophilic and hydrophobic solvents on in vitro skin permeability of indomethacin with a view to designing and developing a suitable transdermal drug delivery system for indomethacin.

MATERIALS AND METHODS
Indomethacin was purchase from Darou pakhsh Company (Tehran, Iran). Plurololeique and Labrasol were obtained as gift samples (Gattefosse, France). Isopropyl myristate, Tween 80 and PEG 400 were obtained from Merck (Germany).

Solubility Tests
The solubility of indomethacin was determined in Plurololeique, Labrasol, Tween 80, Isopropyl myristate and PEG 400 (polyethylene glycol 400). An excess amount of indomethacin was added to 5 ml of the solvent. The mixture was submerged in a water bath for 24 h at 37°C and allowed to equilibrate. Then, suspension mixture was centrifuged for 10 min at 3000 rpm, filtered, diluted and the dissolved drug was measured by a validated UV spectrophotometric method at 321 nm [12].

Animal Studies
Male adult Wistar rats (weighing 200 -250 g) and aged 10 -12 weeks were purchased from Animals Laboratory, Jundishapur University of Medical Sciences, Ahvaz, Iran. The hair on the abdominal skin was removed with an electric clipper, taking care not to damage the skin. Prior to sacrificing the rats, They were anaesthetized with ether and abdominal full-thickness skin was removed and any extraneous subcutaneous fat was cleaned from the dorsal side using cooled pure acetone solution. Whole skin thickness was measured using a digital micrometer. The animals were treated according to the principles for the care and use of laboratory animals. The procedures followed comply with standard international guidelines [13].

Differential Scanning Calorimetry (DSC)
The solvents, which induced changes in structure of whole skin, were determined using a DSC instrument (Mettler Toledo DSC 1 system). Approximately 5 -10 mg of skin samples were placed in hermetically sealed aluminum pans. Simultaneously, an empty hermetically sealed pan was used as a reference. Skin samples were exposed to heat ranging from 20 to 200°C (scan rate: 5°C/min). All experiments were carried out in triplicate. Enthalpy (∆H) values were measured from endothermic and exothermic transitions of the thermo grams [2,10].

In-vitro Permeation Studies
In-vitro permeation studies were carried out using vertical glass Franz diffusion cells with an approximated effective diffusion area of 5.73 cm 2 . The receptor compartment volume contained 30ml. Whole skin sample was hydrated and then mounted between the donor and receptor compartments of the cell with the stratum corneum facing the donor medium. Indomethacin (1% w/v), dissolved in the test solvent, was in the donor compartment and the receptor cell was filled with methanol: phosphate buffer (pH 7) (2:1). The Franz diffusion cell was placed and clamped in a 37±0.5°C water bath with a magnetic stirrer. The receptor medium was stirred with a small magnetic bead at 200 rpm. At predetermined time intervals (0.5, 1, 2, 3,……, 56 h), a 2ml sample was withdrawn from the receptor medium and immediately exchanged for an equivalent volume of fresh methanol : phosphate buffer (2:1) to maintain sink condition. The samples were filtered and the permeated amount of indomethacin was analyzed by UV spectroscopy method at 321nm. Each of the test solvents was used as blank [2,14].

Data Analysis of Permeations
Indomethacin concentration was corrected for sampling effects according to Eq-1 [7,8].
where C 1 n is the corrected concentration of the n th sample, Cn is the observed concentration of indomethacin in the n th sample, C n-1 is the obtained concentration of indomethacin in the (n-1) th sample, V T is the total volume of the receptor fluid medium and V S is the volume of the withdrawn sample. The corrected indomethacin concentration divided by the area of the skin exposed to donor solution to calculate the cumulative amount of indomethacin permeated per unit area which was plotted against time. Steady-state flux (mg/cm 2 /h) was calculated from the linear portion of the slope of the permeation curve. Permeability coefficient (Kp, cm/h) of indomethacin through the skin was calculated as in Eq-2 [7,8]: Where J ss is steady-state flux and C v is the initial concentration of indomethacin in the cell diffusion donor compartment.

Statistical Methods
Statistical comparisons were made using oneway ANOVA and p < 0.05 was considered statistically significant. Correlation analysis was performed by least square linear regression method. All statistical analyses were conducted using Minitab software (version 16.0).

Solubility Study
The results of solubility study are shown in Table 1. The results indicate the highest solubility in Tween 80 followed by PEG 400, labrasol, isopropyl myristate and plurololeique in that order.

FT-IR
Spectral analysis involved examination of change in peak wave number position and their intensities from 4000cm -1 -500cm -1 ( Fig. 1 and Table 2, Table 3, Table 4

Differential Scanning Calorimetry (DSC)
Thermotropic states of skin treated with different solvents evaluated by comparing for mean transition temperature (T m ) and their enthalpies (∆H). Table 5 shows the temperature transition and enthalpy amounts after solvent treatment and the thermograms are displayed in Fig. 2. Two endothermic transitions as obtained from the thermogram of hydrated whole rat skin were observed around 67.5 (Tm 1 ) and 112°C (Tm 2 ) were obtained in thermogram of hydrated whole rat skin. Tm 1 and Tm 2 transitions appeared to be due to melting of lipids and irreversible denaturation of intracellular keratin, respectively.  [20]. It has been shown that Tm 1 corresponded to lipid transformation from a lamellar to disordered state, Tm 2 is due to the melting of lipid -protein (keratin) complex [22], or disruption of polar head groups of lipids [20][21][22][23], Tm 3 is known to occur during the irreversible denaturation of proteins in the SC [20]. It was observed that Tm 1 was shifted by isopropyl myristate, labrasol and plurololeique to lower melting points and ∆H 1 decreased when compared to control. In addition, Tm 1 was removed by Tween 80, while Tm 1 was shifted byPEG400 to higher melting point and ∆H 1 increased in comparison with control. The results indicated that all of the solvents caused Tm 2 shift to higher melting points (with the exception of labrasol) and lower enthalpy that suggest possible denaturation of protein [2].

In vitro Skin Permeation of Indomethacin
The effect of solvents on indomethacin permeability with hydrated skin as control is expressed in Table 6 in which ER flux is ratio of drug flux after and before skin pretreatment with solvent and ER D is drug diffusion coefficient after and before skin pretreatment with solvents. All solvents increased permeability across rat skin compared to control. Isopropyl myristate provided the best effect on indomethacin flux, followed by Tween 80, plurololeique, PEG 400, and labrasol in that order.   ERflux = ratio of flux after and before treatment with solvent ERD = ratio of diffusion coefficient after and before treatmentwith solvent ERP= ratio of permeability coefficient after and before treatment with solvent Significant increase in diffusion coefficient (p < 0.05) resulted with plurololeique with the highest ER D followed by Isopropyl myristate, Tween 80, labrasol and PEG 400. ER flux of all solvents were higher than their ER D , indicating that the solvents increased flux more than they did diffusion coefficient.
The solvents showed significant decrease in permeability coefficient (p < 0.05) compared to water solution of indomethacin.

DISCUSSION
The results show that all solvents increased drug flux through skin more than they did diffusion. Indomethacin falls in Biopharmaceutical Classification System (BCS) class II, so that the low solubility of indomethacin in water suggests that partitioning from stratum corneum to vital epidermis the rate-limiting step. All solvents with different lipophilic properties increased partitioning and flux from stratum corneum to vital periderm. The results of this study indicate significant correlation between drug solubility in various solvents and ER D (ER D = 9.7-1.07 drug solubility amount in solvents, p = 0.036) so that with decrease of drug solubility in solvent, ER D value is increased. The low solubility of drug in solvents might have led to higher thermodynamic activity and hence less resistance of skin, thus resulting increase in diffusion coefficient. ER D data after skin treatment with PEG400 correlate with FT-IR and DSC results as they indicate that PEG400 interacts mostly with SC keratins (∆H 2 of skin treated with PEG400 less than control) , and does not alter significantly SC lipid organization. The shift of 2821.65cm -1 and 1572.27cm -1 bands to a lower wave numbers (2754.62cm -1 and1539.97cm -1 ) provided red shift which indicates partial orientation of lipid groups leading to strengthening of SC barrier properties. Treatment with penetration modifiers may have led to extraction of lipids from SC membrane as usually occurs with enhancers. Increasing another probable mechanism is the intensity at the particular band representing retardation in the case of retardants [2,15]. Lipid fluidization, lipid disruption and the irreversible denaturation of proteins in the SC layer of skin by isopropyl myristate, Tween 80 and plurololeique, as indicated by FT-IR and DSC, are the main factors for higher ER flux and ER D ratios in comparing to control. Group Increase in enthalpy and transition Tm 1 by PEG 400 may be due to bilayer cohesion, which is in contrast to lipid extraction and fluidization. The shift in Tm to lower temperatures can be interpreted as disruption of the lipid bilayer and the irreversible denaturation of proteins in the SC layer of skin, while decrease in ∆H is related to fluidization of lipid in lipid bilayer and lipid -protein complex [2,[19][20][21][22][23][24][25][26].
The spectra for whole skin rat treated with labrasol obtained the relative red shift in 1716.94cm -1 band that indicates formation of strong hydrogen bonds within the lipid molecules leading to strengthening of SC barrier properties, thus resulting in decrease in diffusion coefficient. Isopropyl myristate is hydrophobic and hence miscible with lipid, thus resulting in decrease in the melting point of the stratum corneum lipids. Plurololeique is a water-in-oil surfactant with HLB equal to 6 and form water-in -oil emulsion and can act as oily phase and hence miscible with lipid. Therefore, it may cause lipid disruption, lipid fluidization in the SC layer of skin.

CONCLUSION
The results obtained indicate that all solvents tested increased flux and diffusion coefficient across abdominal rat skin. Lipid fluidization, disruption lipid structure, lipid extraction and the irreversible denaturation of proteins in the SC membrane by isopropyl myristate, Tween 80 and plurololeique are the main factors for higher ER D in comparison to polyethylene glycol 400 and labrasol.