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Gayatri et al: Chemical Permeation Enhancers for Transdermal Delivery of Thiocolchicoside 3827

Research Paper

Chemical Permeation Enhancers for Transdermal Delivery of Thiocolchicoside: Assessment of Ex-vivo Skin Flux and In-vivo Pharmacokinetics

P. Gayatri1, M. Ajitha2, Kiran Kumar3, P. Pavan Kumar4, and Y. Madhusudan Rao5* 1,3,5Vaagdevi Institute of Pharmaceutical Sciences, Bollikunta, 506005, Warangal, India; 2Centre for Pharmaceutical Sciences, IST, JNTUH, 500072, Hyderabad, India; and 4Dr. Reddy’s Laboratories, Bachupally, 500090, Hyderabad, India.

Received June 5, 2017; accepted July 13, 2017

ABSTRACT

Present investigation comprises the study of ex-vivo skin flux and in-vivo pharmacokinetics of Thiocolchicoside (THC) from transdermal films. The films were fabricated by solvent casting technique employing combination of hydrophilic and hydrophobic polymers. A flux of 18.08 μg/cm2h and 13.37μg/cm2h was achieved for optimized formulations containing 1, 8-cineole and oleic acid respectively as permeation enhancers. The observed flux values were higher when compared to passive control (8.66 μg/cm2h). Highest skin permeation was observed when 1,8-cineole was used as chemical

permeation enhancer and it considerably (2-2.5 fold) improved the THC transport across the rat skin. In vivo studies were performed in rabbits and samples were analysed by LC-MS-MS. The mean area under the curve (AUC) values of transdermal film showed about 2.35 times statistically significant (p<0.05) improvement in bioavailability when compared with the oral administration of THC solution. The developed transdermal therapeutic systems using chemical permeation enhancers were suitable for drugs like THC in effective management of muscular pain.

KEYWORDS: Thiocolchicoside; 1,8-cineole; Oleic acid; Skin flux; Passive control; Bioavailability.

Introduction

Thiocolchicoside is a semisynthetic sulfur derivative of colchicoside, a glucoside contained in Colchicum autumnale plant (Perucca et al., 1995). Thiocolchicoside has a selective affinity for -amino butyric acid (GABA) receptors and acts on the muscular contracture by activating the GABA – nergic inhibitory pathways thereby acting as a potent muscle relaxant (Sutherland et al., 2002). Clinically the compound is used in the treatment of muscular, rheumatic, traumatic and neurological disorders. Anti-inflammatory and analgesic effects are also reported in animal models. Oral bioavailability of drug is about 25% due to incomplete gastrointestinal absorption (Perucca et al., 1995) and hepatic first pass effect (Artusi et al., 2003). The low oral bioavailability restricts its use, thus an alternate route of administration is desirable to deliver high concentrations in blood to treat management in muscle pain.

Authors have reported the enhancement of thiocolchicoside skin flux by Iontophoresis (Artusi et al., 2004), but very few reports are available for the

transdermal delivery of THC using essential oils and fatty acids as chemical permeation enhancers. Transdermal drug delivery (TDD) offers an advantageous mode of drug administration by eliminating first pass hepatic metabolism and providing sustained drug release for a prolonged period of time, it is painless when compared to needles and therefore offers superior patient compatibility (Pankaj and Samir, 2009). Skin allows the movement of very few foreign particles across it. A unique hierarchical structure of lipid-rich matrix with embedded keratinocytes in the upper strata (15 μm) of skin, stratum corneum (SC), is responsible for this barrier (Bouwstra, 1997).

Transdermal delivery allows the passage of low molecular weight drug molecules across the skin. They are typically characterized by high Log P (>1.5) and low MW 500 Da (Bos and Meinardi, 2000), the biggest challenge today in transdermal drug delivery is to permeate high molecular weight and hydrophilic drug molecules safely and effectively through the skin. To overcome this challenge advances in chemical permeation enhancement have been made in the past

International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 10Issue 5 September – October 2017

MS ID: IJPSN-06-06-17-GAYATRI

3827

ABBREVIATIONS: THC, Thiocolchicoside; ACE, Aceclofenac; IS, Internal standard; TS, Tensile strength; EB, Elongation at break;Jss, Study state flux; kP,Permeability coefficient; ER, Enhancement ratio; DS, Drug solution; PE, Permeation enhancer; ng, nanogram; mcg,microgram.

3828 Int J Pharm Sci Nanotech Vol 10; Issue 5 September October 2017

couple of decades using various classes of chemical permeation enhancers and reporting various novel permeation enhancers.

Physicochemical properties of THC are not favourable for transdermal permeation across the skin because of its high molecular weight and low Octonal/water partition coefficient. Thus an attempt was made to enhance the transdermal permeation of THC using chemical enhancer like, 1, 8-cineole and oleic acid. Chemical penetration enhancer may act by disrupting the highly ordered structure of stratum corneum lipid, interacting with intercellular protein or by increasing partition of the drug, co-enhancer or solvent into the stratum corneum (Pankaj and Samir, 2009). Oleic acid is a fatty acid shown to be effective as permeation enhancer for many drugs, for example increasing the flux of salicylic acid and 5-flourouracil flux through human skin membrane in vitro (Williams and Barry, 2012). 1,8-cineole is the principal terpene element within eucalyptus oil, polar group present in this terpenes provide better enhancement for hydrophilic permeants and this molecule was one of a series of 17 monoterpenes and terpenoids evaluated as enhancers for the model hydrophilic drug 5-flourouracil in human skin in vitro (Williams and Barry 2012).

In the present study, we attempted to demonstrate chemical permeation enhancement techniques to improve ex-vivo skin flux and in-vivo bioavailability with respect to passive control and oral administration.

Materials and Methods

Materials

Thiocolchicoside was obtained as gift samples from Sun Pharmaceutical Industries ltd (Ahmedabad, Gujarat, India). Eudragit RL 100, Eudragit RS 100 and Hydroxy propyl methyl cellulose (HPMC) were gift samples obtained from Dr. Reddy’s Laboratories Ltd, Hyderabad, India. 1, 8-cineole and Oleic acid were purchased from Sigma Aldrich Bangaluru, India and SD fine chemicals, Mumbai, Maharashtra, India. Other chemicals used in the study were of analytical grade, purchased from Merck Ltd, Mumbai. India.

Preparation of Rat Skin Membrane for Ex-vivo Permeation Studies

Albino rats were used for the ex vivo studies. The animal study was conducted with the permission of Institutional Animal Ethical Committee, Vaagdevi Institute of Pharmaceutical Sciences, Warangal, India. The rats were sacrificed using anaesthetic ether. The hair present in the abdominal region was trimmed carefully and full thickness skin was removed. The epidermis was prepared surgically by heat separation technique, which involved soaking the entire abdominal skin in water at 60 °C for 45 minutes followed by careful removal of epidermis (Gannu et al., 2011; Levang et al., 1999). The skin was taken out washed with water and was kept at room temperature before use.

Preparation of THC Transdermal Films

Matrix type THC transdermal films were fabricated by film casting technique employing different ratios of Eudragit RS 100, Eudragit RL 100 and HPMC E-15. The combinations of polymeric mixtures were dissolved in 20 mL of solvent (consisting of 1:1 ratio of dichloromethane and Methanol). Small amount of polymer was added to solvent to prevent the formation of lumps. Required quantity of THC was dissolved in 5 mL of solvent mixture. The drug solution was added slowly to the polymer solution and mixed thoroughly to get a uniform solution. Propylene glycol (12% w/w) was added to the polymeric solution as a plasticizer and was vortexed for 5 minutes. The mixture was kept aside for 10 minutes to remove the entrapped air bubbles and was transferred into petri plates. The rate of evaporation of the solvent was controlled by inverting a funnel over the petri plate. Drying of these films was carried out at room temperature for overnight. The films were removed carefully and were cut to the size of 3.56cm2 and stored in desiccators (Gannu et al., 2011; Gayatri et al., 2016). Films containing permeation enhancers 1,8-cineole and oleic acid were also prepared in the same fashion explained above by adding different concentrations of permeation enhancers (2.5, 5.0 & 7.5% w/w) to propylene glycol and then mixed with polymeric solution. Fabrications design and compositional details of films are represented in below Table 1.

TABLE 1

Fabrications design of Thiocolchicoside Transdermal films.

Formulation code

THC HPMC

E15 Eudragit RS 100

Eudragit RL 100

% 1,8-cineole

% Oleic acid

TRS1 1 …. 20 ….. ….. …..TRS2 1 5 15 ….. ….. …..TRS3 1 7.5 12.5 ….. ….. …..TRS4 1 10 10 ….. ….. …..TRS5 1 12.5 7.5 ….. ….. …..TRS6 1 15 5 ….. ….. …..TRL1 1 …. …. 20 ….. …..TRL2 1 5 …. 15 ….. …..TRL3 1 7.5 …. 12.5 ….. …..TRL4 1 10 …. 10 ….. …..TRL5 1 12.5 …. 7.5 ….. …..TRL6 1 15 …. 5 ….. …..TCN1 1 12.5 7.5 ….. 2.5 …..TCN2 1 12.5 7.5 ….. 5.0 …..TCN3 1 12.5 7.5 ….. 7.5 …..TOA1 1 12.5 7.5 ….. ….. 2.5TOA2 1 12.5 7.5 ….. ….. 5.0TOA3 1 12.5 7.5 ….. ….. 7.5

THC- Thiocolchicoside TRS- Thiocolchicoside Transdermal films fabricated with HPMC E 15 + Eudragit RS 100 TRL- Thiocolchicoside Transdermal films fabricated with HPMC E 15 + Eudragit RL 100 TCN- Thiocolchicoside Transdermal films fabricated with HPMC E 15 + Eudragit RS 100+ 1,8-cineole TOA- Thiocolchicoside Transdermal films fabricated with HPMC E 15 + Eudragit RS 100+ Oleic acid Films were fabricated using a solvent system of 1:1 ratio of Methanol : DCM, using 12% of Propylene Glycol Each 3.56 cm2 film contains 4 mg of Thiocolchicoside


Evaluation of Physicochemical Properties Weight and thickness variation: Each formulation

was prepared in triplicate. Ten films with area 3.56 cm2

were cut and selected randomly; weight and thickness variation of films was measured using digital balance and digital screw gauge respectively. The mean and standard deviation values were calculated.

Estimation of drug content: Three films from each formulation series were taken, cut into small pieces and were dissolved in 10 mL of solvent mixture (1:1 ratio of DCM: methanol). The resulting mixture was suitably diluted and was filtered through membrane filter (0.45 μ). The drug content was estimated by HPLC.

Moisture absorption studies: Circular films having an area 3.56 cm2 were weighed accurately and placed in desiccators containing 100 mL of standard aluminium chloride solution to maintain 79.5 % RH. The films were weighed after 72 h and the procedure was repeated until they showed a constant weight. The percentage of moisture uptake was calculated as difference between the final and initial weight with respect to the initial weight. (Devi et al., 2003; Gayatri et al., 2016).

Moisture loss studies: The fabricated films were weighed individually and kept in a vacuum desiccators containing fused calcium chloride at room temperature for 72 h. The films were individually weighed after 72 h until they showed a constant weight. The percentage of moisture content was calculated as a difference between initial weight and final weight with respect to final weight (Devi et al., 2003; Gayatri et al., 2016).

Mechanical properties: Microprocessor based advanced force gauze equipped with a motorized test stand (Ultra Test, Mecmesin, West Sussex, and UK), equipped with 25 kg load cell was used to study the mechanical properties of the films. The Tensile strength (T.S) and elongation of break (E.B) were measured using the film strips. The film strip (60 × 10mm) was held between two clamps positioned at a distance of 3 cm. During measurement, the top clamp at a rate of 2 mm s-1pulled the strips to a distance till the film broke. The force and elongation were measured when the films were broken (Gannu et al., 2011; Gayatri et al., 2016). The mechanical properties were calculated using the equation 1 and 2.

T.S (Kg mm–2) = [Force at break (Kg)/ Initial cross sectional area of the film (mm–2)] …..(1) E.B (%mm–2) = [Increase in length (mm)/ Original length × 100/Cross sectional area (mm–2) …..(2) Ex-vivo permeation studies of THC drug solution

through rat skin (Passive Control): Ex-vivo permeation studies were conducted using Vertical Franz diffusion Cell. This study was conducted to check the transdermal flux of THC through albino rat abdominal skin in absence of permeation enhancers to serve as a passive control for comparison.

Permeation of THC from transdermal films using chemical enhancer (1, 8-cineole and Oleic Acid) and without chemical enhancer: Ex-vivo permeation studies of THC transdermal films (3.56 cm2) containing

chemical permeation enhancer 1,8-cineole, oleic acid and films without chemical enhancer were conducted using Vertical Franz diffusion Cell. The purpose was to study the effect of different concentration of permeation enhancers 1,8-cineole and oleic acid (2.5, 5.0 and 7.5 % w/w) on ex-vivo skin flux of THC from transdermal films. Excised albino rat abdominal skin was placed between the donor and receptor compartments of the diffusion cell with stratum corneum facing the donor compartment. The fabricated film was placed over the skin. Phosphate buffer saline pH 7.4 was used as the receptor fluid. The entire assembly was kept on the magnetic stirrer. The temperature of the receptor fluid was maintained at 37 ± 2 °C with the help of thermostat. 1.5 mL of receptor fluid was collected as samples at pre-determined time points and was replaced with same volume of phosphate buffer saline pH 7.4 to maintain the sink conditions. Samples were collected at 0.5, 1, 2, 4, 6, 8, 12, and 24 h and were stored under refrigerated conditions until the analysis was carried out. All the experiments were conducted in triplicate. Cumulative amount of THC permeated was determined by HPLC.

High-performance liquid chromatography (HPLC) method for analysis of ex vivo samples: The HPLC method used to quantity the THC from ex-vivo skin diffusive samples was developed on Phenomenix Luna® C18 column (150 x 4.6mm.i.d.5μm) using a mobile phase of ammonium di hydrogen phosphate buffer: Acetonitrile (60:40), adjusted to pH 4.5 with glacial acetic acid. The eluent was monitored by UV detector at 264 nm at a flow rate of 1.0 mL/min. The injection volume was 50 μL. The in vitro calibration curves was linear (R2 > 0.997) over the concentration range of 100 to 4000 ng/mL (7 points, n = 6).

Determination of flux and permeability coefficient: A cumulative amount of THC permeated in μg/cm2 was calculated and plotted against time. THC flux (Jss, μg/cm2 h) at steady state was calculated by dividing the slope of the linear portion of the curve by area of the exposed skin surface (3.56 cm2). Permeability coefficient was calculated by dividing the flux by amount of drug loaded initially. Enhancement ratio was calculated by dividing flux achieved with permeation enhancer and with that of without of permeation enhancer. The observations are noted in Table 4.

Stability studies: The stability studies were conducted for the optimized formulations. The films from different formulations were wrapped in primary pack aluminium foil and placed in a secondary pack of carton to charge in stability Chambers at temperature of 40 ± 2 °C, 75 ± 5 % RH for 6 months. The samples were withdrawn at regular interval of 1, 3 and 6 months and analysed for drug content using the HPLC.

Skin irritation studies: Skin irritation studies were performed on rabbits with approved study protocol (File. No. IAEC/13/VCPS/2016) by Institutional ethical committee, Vaagdevi Institute of pharmaceutical sciences, Warangal, Telangana, India. The dorsal surface


of the skin was shaved and was cleaned. THC transdermal film was applied and development of erythema was monitored for a week.

In vivo bioavailability studies in rabbits: The bioavailability study protocol (File.No.IAEC/13/VCPS/ 2016) was reviewed and approved by Institutional ethical committee, Vaagdevi Institute of pharmaceutical sciences, Warangal, Telangana, India. Rabbits weighing 2.3 ± 0.5 kg were selected for the study. Before commencing the studies animals were allowed for fee access to food and water. They were fasted for 12 hours before dosing. Bioavailability study was carried out by Latin square cross over design dividing the rabbits in two groups each containing six rabbits. The bioavailability of THC from the transdermal films containing 1, 8- cineole as permeation enhancer was compared with an oral solution (control). The oral solution was prepared by dissolving THC in water (0.5mg/mL) and was administered to first group of rabbits through feeding tube followed by rinsing with 12mL of water. The dorsal surface of the second group of rabbits was shaved with electrical shaver and was cleaned properly. Optimized transdermal film (TCN2) containing 4mg of THC & 5.0% 1, 8- cineole was applied to the second group of rabbits with an impermeable backing membrane and was fixed with the help of adhesive tape. After first phase the second phase study was followed vice versa and was conducted after two weeks of wash period. After administration of oral solution and application of TCN2 film, blood samples (3mL) were collected from marginal ear vein at specific time intervals for 36h. Blood samples were collected and were centrifuged for 20 min at 4000 rpm. The separated serum samples were stored in micro - centrifuge tubes at -20 0C until analysis (Gannu et al., 2010). The samples were subjected for suitable extraction procedure and estimated by LC-MS-MS method.

Estimation of THC from rabbit serum using high performance liquid chromatographic separation with mass spectrometric detection (LC-MS-MS) method: LC-MS-MS method was used for the estimation of THC in rabbit plasma samples using Aceclofenac (ACE) as internal standard (IS). Liquid-liquid extraction method was optimized for quantification of THC. To 50 μL of plasma sample 50 μL of ACE was added and was vortexed for 10 sec. 1 mL of ethyl acetate was added to the test tube and vortexed for 10 minutes. The above mixture was vortexed for 20 minutes, then centrifuged at 4000 rpm for 10 minutes. The supernatant was transferred in a test tube and was evaporated to dryness. 0.5mL of reconstitution solution was added to the dried residue vortexed for 1 minute and was transferred to auto injector vial for further analysis.

Chromatographic separation was carried out on a Gemini 3 μm, NX- C18 110 A 50 × 4.6mm column. The gradient mobile phase consist of diluted ammonia solution as solvent A and 100% acetonitrile solution as solvent B, pumped at 0.7mL/min. Column elute was analysed by Applied bio system API 2000 LC-MS-MS with multiple reaction monitoring mode, equipped with

electron spray probe interfaced to a liquid chromatograph, operating in electron spray, positive –single-ion mode to monitor 401.90 m/z for THC and 353.90 m/z for IS. The detector response was linear in the concentration range of 05 to 200 ng/mL for plasma samples. Inter day and intraday precession and accuracy was less than 15%. The method was found to be good for the determination of plasma concentrations range to be studied.

Pharmacokinetic analysis: Pharmacokinetic parameters Cmax (ng mL-1), Tmax (h) and area under the cure (AUC) of THC were estimated from each rabbit using the computer programme, KINETICA software 2000 (Version 5, Innaphase corporation, Philadelphia, USA) after the administration of oral solution and a transdermal film. The relative bioavailability FT for chemical method of permeation technique using transdermal film was calculated using the equation 3

Relative bioavailability FT = [AUC] Patch/ [AUC] Oral suspension .....(3) Statistical analysis: Minitab® 17.2.1 was used for

statistical comparisons. Results were expressed as mean ± SD, study was considered significant at 95% confidences interval.

Results and Discussions

Evaluation of Prepared Transdermal Films

THC transdermal films were fabricated employing various concentrations of Eudragit RL 100, Eudragit RS 100 and HPMC E-15 with permeation enhancer 1,8-cineole and oleic acid. The fabricated films were transparent indicating the drug is uniformly distributed in the polymeric matrix. The values of weight variation, thickness and drug content uniformity are shown in (Table 2) showing the uniformity of weight of films according to the %RSD values, which is less than 6. Thickness of the films in the respective series varied from 195 ± 12.8μm to 207 ± 14.3 μm (TRS) and 197 ± 11.8μm to 203 ± 12.2 μm (TRL) indicating no significant change in the thickness of film with the change of polymer concentration. The drug content in fabricated films was ranged from 96.2 ± 3.5 to 101.4 ± 3.5 for TRS series whereas films of TRL series were ranged from 98.4 ± 2.6 to 101.7 ± 4.12.

Percentage Moisture uptake and Moisture Loss

The mean % moisture absorption ranged from 3.21 to 7.48 % in TRS series and 3.08 to 7.22 % in TRL series. The mean % moisture content in the films ranged from 0.88 to 1.97 % in TRS series and 0.90 to 1.65 % in TRL series whereas mean % moisture absorption and loss for films containing permeation enhancers were found lesser than that of without permeation enhancer films, it may be due to the hydrophobic nature of incorporated permeation enhancers and is represented in (Figure 1). Low moisture uptake and presence of small moisture content protects the transdermal film from microbial contamination and becoming brittle during storage.


TABLE 2

Physicochemical and mechanical properties of THC transdermal films.

Formulation code Weight (mg) Thickness (μm) Assay (%) T.S (kg/mm2) EB (%mm2)

TRS1 158.3 ± 5.2 201 ± 15.2 96.2 ± 3.5 2.13 ± 0.20 9.7 ± 1.28 TRS2 161.6 ± 4.5 197 ± 11.6 100.2 ± 2.8 2.26 ± 0.21 9.3 ± 1.51 TRS3 159.7 ± 4.8 196 ± 13.2 101.4 ± 3.5 2.44 ± 0.25 9.9 ± 2.2 TRS4 160.6 ± 6.1 203 ± 13.3 98.8 ± 1.8 2.96 ± 0.38 11.2 ± 4.9 TRS5 162.3 ± 3.8 207 ± 14.3 99.7 ± 3.1 3.78 ± 0.44 12.4 ± 3.8 TRS6 157.3 ± 6.3 195 ± 12.8 99.2 ± 5.1 3.64 ± 0.12 11.9 ± 3.27 TRL1 161.5 ± 4.3 202 ± 13.2 100.3 ± 4.2 1.81 ± 0.07 6.78. ± 2.1 TRL2 156.9 ± 5.6 197 ± 11.8 101.7 ± 4.1 1.84 ± 0.15 7.89 ± 4.2 TRL3 160.3 ± 5.3 203 ± 12.2 98.4 ± 2.6 1.90 ± 0.22 8.32 ± 2.9 TRL4 163.0 ± 3.9 199 ± 15.5 99.6 ± 3.2 1.93 ± 0.31 8.74 ± 4.6 TRL5 158.2 ± 4.7 198 ± 10.4 99.3 ± 4.3 1.95 ± 0.13 9.06 ± 3.3 TRL6 162.5 ± 5.4 205 ± 12.2 100.1 ± 3.1 2.01 ± 0.22 9.2 ± 3.7 TCN1 163.3 ± 3.5 201 ± 13.9 100.1 ± 3.3 3.72 ± 0.6 12.6 ±1.48 TCN2 165.8 ± 4.1 205 ± 11.7 99.5 ± 2.8 3.91 ± 0.52 13.0 ± 3.30 TCN3 166.7 ± 4.3 199 ± 10.5 100.2 ± 1.9 4.0 ± 0.48 14.7 ± 4.12 TOA1 166.3 ± 3.5 208 ± 12.6 98.4 ± 2.8 4.1 ± 0.39 15.3 ± 3.77 TOA2 167.2 ± 6.0 210 ± 14.6 99.8 ± 2.0 4.3 ± 0.22 16.7 ± 4.92 TOA3 164.9 ± 5.2 206 ± 11.3 101.3 ± 3.7 4.5 ± 0.62 17.2 ± 3.83

Values, Mean ± SD (n=3)


Fig. 1. Moisture absorption and loss studies results of THC transdermal films.

Mechanical Properties of Films

A soft and weak polymer is characterized by low TS and E/B; a hard and brittle polymer is defined by a moderate TS and low E/B; a soft and tough polymer is characterized by a moderate TS and high E/B; whereas a hard and tough polymer is characterized by a high TS and E/B (Gannu et al., 2011; Aulton et al., 1981). Thus a suitable transdermal film should possess a high TS and E/B. The results of tensile strength and elongation at break are indicated in the (Table 2). The formulation TRS5 and TRL6 exhibited greater values of TS (3.78 ± 0.12 kg/mm2 and 2.01 ± 0.22 kg/mm2 for TRS6 and TRL6 respectively). Optimized formulations TRS5 (12.4 ± 3.8mm2) and TRL6 (9.2 ± 3.7 mm2) showed greater values of elongation at break in their respective series.

Thus, as the concentration of hydrophilic polymer HPMC E-15 increases in TRS series the TS decreases and E/B increases. Both TS and E/B found to be increased with increase in concentration of HPMC E-15 in TRL series (Gannu et al., 2011). The above observations reveal that the formulation TRS5 and TRL6 films were found to be strong and were not brittle.

Assessment of THC Skin Flux through Rat Skin (Passive control)

The flux achieved by transdermal permeation of THC drug solution across the skin was found to be 8.66μg/cm2h. This assessment was done to understand the ability of THC permeation across the rat skin. The results clearly indicate that the drug has a capability of permeation though the skin by passive diffusion, but the achieved flux was less. Hence there was a need of superior enhancement techniques to enhance the permeability of drug though skin. The amount permeated through rat skin profile shown in (Figure 2).


Fig. 2. Ex-Vivo permeation of THC drug solution through rat abdominal skin.

Assessment of THC Skin Flux from Transdermal Films (without chemical permeation enhancer)

Results of the ex-vivo permeation studies from the THC films without chemical permeation enhancer are presented in (Figure 3 & 4). The maximum skin flux was observed in formulation TRS5 (11.31μg/cm2h) and TRL6 (10.21μg/cm2h) in respective series and were considered to be the optimized formulations from each series. Drug THC in films was available as more solubilised form in presence of hydrophilic and hydrophobic polymers and present in higher concentrations at permeation site, due


to this high concentration gradient across the membrane drug has passively permeated though the rat abdominal skin. The presence of propylene glycol in the formation may also contribute for higher transdermal flux compared to the drug solution.


Fig. 3. Ex-vivo permeation profiles of thiocolchicoside transdermal films – TRS & TRL series.


Fig. 4. Ex-vivo permeation profiles of thiocolchicoside transdermal films – TRS & TRL series.

Assessment of THC Skin Flux from Transdermal films using Chemical Permeation Enhancers

The outcomes of the ex-vivo permeation studies from the THC films containing 1, 8-cineole and oleic acid are presented in (Table 3 & Figure 5). Skin fluidity was observed in formulation TCN2 containing 5.0 % w/w of 1, 8-cineole (18.08 μg/cm2h) and TOA2 containing 5.0 %w/w oleic acid (13.37μg/cm2h). There was an improvement in permeation rates of the films with 1, 8-cineole which was about 1.5–2.0 fold higher, and permeation rates of films with oleic acid was about 1.0–1.5 fold higher, than in those without enhancer. The effectiveness of permeation enhancer was determined by comparing drug flux in the presence and absence of each enhancer.

TABLE 3

Ex vivo skin permeation study results for THC drug solution and transdermal films.

System Flux

(μg/cm2h) (%) kP (%) ER

Drug solution 8.66 0.22 - Film without Enhancer (TRL6) 10.21 0.25 1.17 Film without Enhancer (TRS5) 11.31 0.28 1.30 Film with oleic acid (TOA2) 13.37 0.33 1.51 Film with 1,8 cineole (TCN2) 18.08 0.45 2.05


Fig. 5. Ex-vivo Permeation Profiles of thiocolchicoside transdermal films with (Oleic acid & 1,8 cineole).

According to lipid-protein-partitioning theory permeation enhancers act by three important mechanisms: disruption of highly ordered lipid structure between the corneocytes, so increasing intercellular diffusivity, interaction with intracellular protein to promote permeation through the corneocyte, and increased partitioning of the drug or co enhancer into the tissue (Barry 1988; Barry, 1987; Williams and Barry, 1991). The enhancement ratio of THC using terpene was tabulated as 2.1 folds, suggesting the permeation of THC was enhanced by 1-8 cineole by disrupting the highly ordered structure of stratum corneum there by increasing the diffusivity. THC is hydrophilic in nature and its solubility in 1-8 cineole is less than water stating there is no increase in partitioning of the drug. The ex vivo study suggests enhancement in permeation of THC by using permeation enhancer is due to disruption of lipid layers of stratum corneum but not due to partitioning effect. The results of the study also hold good with the published report where the permeability coefficient of 5-flurouracil was increased to 90 folds using 1-8 cineole as permeation enhancer (Barry, 1987). The activity of 1-8 cineole as permeation enhancer towards several drugs including procaine, indomethacin, bupranolol, dibucaine and bezocaine across the hairless mouse skin has been reported (Barry, 1987). Good enhancement effects on the permeation of 5-Fluorouracil across human epidermis was observed using terpenes in propylene glycol/water (Yamane et al., 1995(a)).

In previous studies oleic acid has been used to enhance the transdermal delivery of dihydroergotamine through rabbit skin (Niazy, 1991), 5-Fluorouracil (Yamane et al., 1995(b)), and Naphazoline and sodium salicylate (Green et al., 1988) through human skin. Oleic acid acts as penetration enhancers by stratum corneum lipid fluidization and lipid phase separation (Aarti et al., 1995). Propylene glycol (12%) is used in the formulation of transdermal film as plasticizer, it is also reported its use as permeation enhancer when used with other chemical penetration enhancers. Propylene glycol probably works by solvating alpha keratin and occupying hydrogen-bonding site, thus reducing the drug/tissue binding and may be excessive amount of water present itself function as permeation enhancer (Barry, 1987).


From our study it is observed that 1- 8 cineole and oleic acid can be used as penetration enhancers for transdermal delivery of THC. The presence of propylene glycol in the formulation also enhanced the permeation of drug through skin to some extent. The highest skin flux was observed in the films containing 1, 8-cineole, followed by the films containing oleic acid. 1, 8-cineole showed the best enhancement for THC across the rat skin and was considered to be the formulation for testing bioavailability in rabbits. The presence of oleic acid in the films also enhanced the permeation rate but to a lesser extent.

Stability Studies

The optimized formulations (TRS5 and TCN2) were subjected to stability studies. On storing the transdermal films at 40 ± 2°C/75 ± 5% RH for 6 months 0.98% (TRS5) and 1.07% (TCN2) drop in assay was observed. The observed degradation is less than 5 % from initial value in the formulation. Hence the fabricated films are stable at accelerated stability conditions. Results represented in (Figure 6).


Fig. 6. Stability study results of THC transdermal films (% Drug content estimation at initial & 40°C/75% RH 6M).

Skin Irritation Studies

The absence of redness and irritation indicates that fabricated transdermal films were non-irritant. The photographs showing absence of redness and erythema after one week upon application of optimized formulation is represented in (Figure 7).

Fig. 7. Skin irritation studies on Healthy rabbits.

In vivo Bioavailability Studies

Pharmacokinetic studies were carried out in rabbits and serum samples obtained were suitably extracted and analysed by LC-MS-MS method. (Figure 8, 9a & 9b) shows typical mass spectra of thiocolchicoside parent ion, chromatograms obtained after spiking rabbit serum samples at 2h and 8h upon application of TCN2 film. Comparative serum profiles of THC oral solution and from transdermal films containing 1, 8 cineole as chemical permeation were shown in (Figure 10). Relative bioavailability study shows that THC is successfully released from the fabricated transdermal films and is transported well through different layers of stratum corneum and improved skin permeation was observed when 1, 8 cineole was used as chemical permeation enhancer. The pharmacokinetic parameters Cmax (ng mL–1), Tmax (h) and area under the cure (AUC) from the studied therapeutic systems are represented in (Table 4).

Fig. 8. Mass spectra of Thiocolchicoside (full Scan of THC for parent ion).


Fig. 9a. Chromatogram after spiking rabbit serum sample at 2h upon application of TCN2 film.

Fig. 9b. Chromatogram after spiking rabbit serum sample at 8h upon application of TCN2 film.


Fig. 10. Comparative mean serum concentration profile of THC drug solution and transdermal film with permeation enhancer (PE).

TABLE 4

Pharmacokinetic parameters of THC (Oral vs transdermal).

Pharmacokinetic Parameters THC Drug Solution Film TCN2

Cmax (ng/mL) 23.36 31.33 Tmax (h) 1.5 8.0 AUC 0-t (ng.h/mL) 173.15 406.26


The Cmax was found to be 23.56 ± 18.26 and 33.85 ± 25.3ng mL–1 after administration of THC as oral solution and transdermal film with chemical permeation enhancer 1, 8 cineole respectively. The absorption kinetic values were higher when THC is administered transdermally when compared to oral route. When THC is given orally it is incompletely absorbed but when given via transdermal route the permeation of drug was increased to greater extent. Tmax values in all rabbits


were prolonged for transdermal administration when compared with oral route and the difference was found to statistically significant (p<0.05).

The total mean area under the curve (AUC0-t ) from transdermal films containing with 1, 8 cineole was found to be higher than the oral solution and the improvement was statistically significant (p<0.05). In the present study the enhancement of bioavailability in rabbits was demonstrated when 1, 8- cineole is used as chemical permeation enhancer. Thus transdermal therapeutic systems of THC can enhance the bioavailability by 2.35 times than that of oral administration and can be effectively used in the management of muscle pain.

Conclusions

Thiocolchicoside matrix type transdermal films were successfully fabricated using combination of hydrophilic and hydrophobic polymers. The films showed good mechanical properties. Drug remains stable in the films on storage. Improvement in transdermal permeation across rat skin was achieved using 1,8 cineole. The pharmacokinetic study in rabbits showed enhancement in the bioavailability of THC films by 2.35 folds. The present study confirms the feasibility of developing transdermal filmusing 1,8 cineole as chemical permeation enhancer for drugs like THC in effective management of muscle pain.

Acknowledgements

One of the authors (P. Gayatri) thank Sun pharmaceutical industries Ltd for providing the thiocolchicoside drug, Dr. Reddy’s laboratories ltd for providing different grades of HPMC and Eudragit polymers.

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Address correspondence to: Y. Madhusudan Rao, Director, Vaagdevi Group of Pharmacy Colleges, Warangal, Telangana, India. Mob: 9849466620 E-mail: [email protected]; [email protected]

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