Transdermal Delivery of Lercanidipine Hydrochloride

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Transdermal Delivery of Lercanidipine Hydrochloride: Effect of Chemical Enhancers and Ultrasound Pallavi K. Shetty1, Neelam A. Suthar1, Jyothsna Menon1, Praful B. Deshpande1, Kiran Avadhani1, Raghavendra V. Kulkarni2 and Srinivas Mutalik1,* 1

Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576104, Karnataka, India; 2Department of Pharmaceutical Technology, BLDE Pharmacy College, BLDE University Campus, Bijapur, Karnataka, India Abstract: The effects of permeation enhancers and sonophoresis on the transdermal permeation of lercanidipine hydrochloride (LRDP) across mouse skin were investigated. Parameters including drug solubility, partition coefficient, drug degradation and drug permeation in skin were determined. Tween-20, dimethyl formamide, propylene glycol, poly ethylene glycol (5% v/v) and different concentration of ethanol were used for permeation enhancement. Low frequency ultrasound was also applied in the presence and absence of permeation enhancers to assess its effect on augmenting the permeation of drug. All the permeation enhancers, except propylene glycol, increased the transdermal permeation of LRDP. Sonophoresis significantly increased the cumulative amount of LRDP permeating through the skin in comparison to passive diffusion. A synergistic effect was noted when sonophoresis was applied in presence of permeation enhancers. The results suggest that the formulation of LRDP with an appropriate penetration enhancer may be useful in the development of a therapeutic system to deliver LRDP across the skin for a prolonged period (i.e., 24 h). The application of ultrasound in association with permeation enhancers could further serve as non-oral and non-invasive drug delivery modality for the immediate therapeutic effect.

Keywords: Lercanidipine hydrochloride, Transdermal, Ultrasound, Chemical enhancer, Skin delivery. INTRODUCTION Delivery of drug across the skin to systemic circulation to achieve a therapeutic effect is commonly known as transdermal drug delivery and it contrasts from simple topical delivery of drugs [1]. Transdermal drug delivery systems (TDDS) are the dosage forms that comprise the transport of drugs to viable skinlayers for local therapeutic effect, and a major portion of drug is carried to the blood circulation [25]. Transdermal drug delivery is one of the ideal routes of drug administration as it bypasses first-pass metabolism, eliminates gastro intestinal (GI) side effects and maintains the constant plasma concentration of drug for an extended period of time [6-8]. Lercanidipine hydrochloride (LRDP), a dihydropyridinecalcium channel blocker, is one of the widely used drugs in the treatment of hypertension due to its selectivity and specificity on the smooth vascular cells. It is being administered in the form of tablets via oral route, which is associated with several disadvantages, viz. low oral bioavailability (44%) due to extensive first pass metabolism and increased frequency of administration due to lower biological half-life (2-5 h). In addition to these disadvantages, low daily dose (2.5-20 mg) *Address correspondence to this author at the Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576104, Karnataka, India; Tel: +91-820-2922482; Fax: +91-820-2571998; E-mail: [email protected] 1567-2018/13 $58.00+.00

and low molecular weight makes this drug as an ideal candidate for transdermal drugs delivery [9-14]. However skin is a strong barrier that inhibits the permeation of to reach the systemic circulation. Hence, although LRDP is an ideal candidate to deliver it across the skin, it is difficult to achieve therapeutic concentrations in blood without aid of permeation enhancement approaches. Transdermal permeation of drugs can be increased by physical (sonophoresis, iontophoresis, electroporation and microneedles) and chemical (chemical permeation enhancers like solvents, surfactants, fatty acids and terpenes) enhancement approaches [3,15]. Among various physical enhancement techniques, sonophoresis (application of ultrasound) is an effective and widely used technique to successfully enhance the permeation of drugs across the skin [16]. Generally this form of ultrasound uses low frequency ( 20 KHz) to increase transdermal permeation of drugs by cavitation, micro-streaming and heating [17,18]. Ultrasound 20 KHz has increased permeation enhancement up to 1000 times higher than that induced by therapeutic ultrasound [19]. Proteins like insulin, ginterferon and erythropoietin were successfully transported across cadaver skin in vitro by application of low-frequency ultrasound [20]. In a previous study, 3-5 min of ultrasound application increased transdermal permeation of mannitol and physostigmine across hairless rat skin in vivo by up to 15-fold [21]. Synergism in skin permeability was observed when ultrasound and sodium lauryl sulphate were combined [22]. We previously reported the © 2013 Bentham Science Publishers

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drastic enhancement in the transdermal permeation of tizanidine hydrochloride using the combination of ultrasound and chemical enhancers [18]. Apart from increasing the transdermal permeation, sonophoresis along with enhancers reduces the severity of chemical enhancers required to achieve target permeation rate [23-26]. Moreover, if the desired permeation rate is achieved only with chemical enhancers, then sonophoresis alone or in combination with chemical enhancers can be used to deliver the drug in “bolus” (such as immediately acting tablets) in short time period (say 30 min). The effect of few permeation enhancers (dimethyl sulfoxide, eugenol, citral, sodium lauryl sulphate and isopropyl myristate) on the transdermal permeation of LRDP has been reported [9]. However many important parameters such as effect of popularly and widely used enhancers (propylene glycol, polyethylene glycol, Tween-20, dimethyl formamide and different concentrations of ethanol), stability of LRDP in skin, target flux calculation, etc have not been studied. Moreover, there is no literature available on the effect of sonophoresis alone or the combination of sonophoresis and chemical enhancers on the permeation of LRDP across the skin. Hence the present study has been undertaken to assess i) the effect of some chemical permeation enhancers, ii) sonophoresis and iii) the combination of chemical enhancers and sonophoresis on the permeation of LRDP across mouse skin. MATERIALS Lercanidipine hydrochloride (LRDP) was a gift sample from Cipla Laboratories, Mumbai, India. n-Octanol, isopropyl myristate (IPM), poly ethylene glycol (PEG), propylene glycol (PG), dimethyl formamide (DMF) and Tween-20 were purchased from Sigma, MN, USA. All the other chemicals used were of analytical/ reagent/HPLC grade. METHODS Solubility Studies [27,28] Saturation solubility of LRDP was determined in aqueous buffer solutions of different pH (pH 4.5, 7.4 and 9.2). The solubility of LRDP was also determined in the presence of tested permeation enhancers (5% w/v of PG, PEG, Tween-20 and DMF and different concentrations (10 - 70% v/v) of ethanol. An excess amount of drug was added to different solutions (as shown above) and the mixture was placed on rotary shaker for 24 h at room temperature. The concentration of LRDP in these saturated solutions was determined spectrophotometrically at 242 nm after passing through 0.45 m membrane filter. Determination of Partition Coefficient The partition coefficient values of LRDP in different systems like n-octanol/water, IPM/water and mineral oil/water were determined. Ten ml of oil phase (n-octanol/ IPM/ mineral oil) was added to an equal volume of drug solution (10 g/ml) in water in a separating funnel. The system was kept at room temperature for 24 h with intermittent shaking. Finally aqueous layer was separated, clarified by

Shetty et al.

centrifugation at 1000 rpm for 5 min, and assayed by RP-HPLC after passing through 0.45 m membrane filter [29,30]. Degradation of LRDP in Skin The in vitro skin degradation of LRDP was assessed in different extracts such as epidermis, dermis and skin extracts [18,27]. Freshly excised mouse skin was fixed in the diffusion cell, with stratum corneum facing the donor compartment and the dermis facing the receptor compartment. Both compartments were filled with water. After 24 h, the donor (epidermal extract) and receptor (dermal extract)solutions were collected separately. Skin extract was prepared by homogenizing freshly excised skin (1 cm2 area) in 10 ml distilled water for 10 min in an ice bath. The mixture was centrifuges at 3000 rpm for 20 min and supernatant was collected. Drug solution in water was mixed with 5 ml of the epidermal, dermal and skin extracts separately. The samples were shaken in a water bath at 32 °C and at 50 rpm. At different time intervals up to 6 h, the concentration of drug in each solution was determined by RP-HPLC. Drug solution in water, without any extracts, was used as a control [28]. In Vitro Skin Permeation Studies In vitro skin permeation studies were conducted using vertical type diffusion cells with 1 cm2 surface area. The receptor compartment capacity was 3.5 ml. Membrane for permeability studies used was full thickness skin from the dorsal section of Swiss albino mice of 6-8 weeks old. The hair from abdominal region of mice was removed on the previous day of the permeation experiment using an electrical hair clipper [31]. The freshly excised mouse skin was fixed on the diffusion cell with epidermis facing the donor chamber. The receptor compartment was filled with water. Two ml of drug suspension in water, with or without penetration enhancer (10-70% of ethanol or 5% of other enhancers), was placed in the donor compartment and sealed with parafilm. The receptor solution was continuously stirred by magnetic bead. At regular time intervals up to 24 h, 500 l of receptor solution was withdrawn and assayed for LRDP using RP-HPLC. The samples from receptor compartment were replaced with fresh receptor solution at each sampling to maintain the appropriate volume [28,30]. Application of Ultrasound Low-frequency ultrasound was applied to the donor solution using a probe sonicator (VibraCell, VC 130, Sonics and Materials, CT, USA). The distance between the probe of the sonicator and the skin was about 0.5 cm. Following parameters were set for ultrasound application: Time: 30 min; Amplitude: 30; Output of 7–8W/cm2; On–off cycle (pulser): 1 s (‘on’ for 1 s, followed by ‘off’ and then ‘on’ for 1 s). In a previous study, the temperature of the donor solution was gradually getting increased to 45°C following ultrasound application at an on–off cycle of 5 s [17]. Hence, in order to control the increases in temperature during sonication, the donor solution was replaced every 2 min [27]. During this changing time, ultrasound was not applied. The example of sampling at 10th min is given below:

Transdermal Delivery of Lercanidipine Hydrochloride

Current Drug Delivery, 2013, Vol. 10, No. 0

Ultrasound

Donor Solution Replacement

Ultrasound

Donor Solution Replacement

Ultrasound

Donor Solution Replacement

Ultrasound

2 min

1 min

2 min

1 min

2 min

1 min

1 min

Even with above schedule, the temperature of donor solution was found to increase up to 38 °C. To evaluate the effect of this elevated temperature on the skin permeation of LRDP, ‘temperature control’ experiments (without sonication) were also conducted at 38-39 °C [18,23].

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by Dunnet’s posthoc-test using Graph Pad Prism software. The data obtained in skin permeation studies upon application of ultrasound (Table 2) was analysed by two-way ANOVA followed by Bonferroni’ sposthoc-test for multiple group comparison. In both sets of statistical analyses, ‘p’ value less than 0.05 was considered statistically significant.

HPLC Analysis of LRDP The samples were analyzed for the concentration of LRDP using an integrated HPLC system (LC-2012CHT, Shimadzu, Kyoto, Japan) equipped with low pressure quaternary gradient pump along with dual wavelength UV detector and auto sampler. The chromatographic data was processed using LC solution 1.24 SP1 software. The column used was a RP C18 column (GraceVydac; particle size 5 m; 250 x 4.6 mm), maintained at 25 °C. The mobile phase consisted of acetonitrile: phosphate buffer (10mM, pH 3.5±0.2 adjusted with dilute orthophosphoric acid) in a ratio of 70:30 v/v and flow rate was 1 ml/ min. Total run time for each sample was 7 min. The detector wavelength was set at 242 nm and the volume of injection was 50 l/min [32-34]. The method was validated with respect to calibration curve (R2: >0.9990; over the concentration range of 1 to 100 g/ml), precision of the area (RSD values ranging between 0.05 and 2%) and accuracy (between 98 and 102% at different concentrations). The retention time of LRDP was found to be 7.16 min and the peak was devoid of any interfering peaks from skin. Statistical Analysis The data of solubility, flux and drug content of skin were analysed by One-way analysis of variance (ANOVA) followed Table 1.

RESULTS & DISCUSSION Solubility Studies The solubility in different buffer solutions was conducted to select a suitable receptor medium for LRDP. The solubility was found to be highest in distilled water in comparison with other pH buffer solutions tested. The results of solubility studies are as follows: Distilled water: 1006 g/ml; pH 4.5:654.1 g/ml; pH 7.4:470.5 g/ml; pH 9.2: 231.2 g/ml. As pH increased the solubility of LRDP decreased. As LRDP showed a good solubility in water compared to other solvents, it was selected as a medium for permeation studies. Solubility studies of LRDP in different vehicles containing different amounts of ethanol and different penetration enhancers were also conducted (Table 1). In the presence of tested chemical enhancers (5% v/v in distilled water), LRDP showed increased solubility when compared to distilled water alone. Tween-20 and DMF significantly (p