AYESHA YAQOOB, MAHMOOD AHMAD*, ASIF MAHMOOD and RAI MUHAMMAD SARFRAZ

Transcription

1 Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 6 pp. 1639ñ1648, 2016 ISSN Polish Pharmaceutical Society PREPARATION, IN VITRO AND IN VIVO CHARACTERIZATION OF HYDROPHOBIC PATCHES OF A HIGHLY WATER SOLUBLE DRUG FOR PROLONGED PLASMA HALF LIFE: EFFECT OF PERMEATION ENHANCERS AYESHA YAQOOB, MAHMOOD AHMAD*, ASIF MAHMOOD and RAI MUHAMMAD SARFRAZ Faculty of Pharmacy & Alternative Medicine, The Islamia University of Bahawalpur, Pakistan Abstract: Aim of present study was to develop metoprolol matrix patches using different enhancers. Combination of two hydrophobic polymers, ethyl cellulose and eudragit RL 100 (8 : 2) were used for preparation of unilaminated matrix patch. 10% w/w of isopropyl myristate (IPM), dimethyl sulfoxide (DMSO), span 20 (S20), Tween 20 (T20) and eucalyptus oil as enhancers and 40% of dibutyl phthalate as plasticizer were used. Prepared patches were evaluated for physical appearance, weight uniformity and thickness. FTIR studies were performed to assess compatibility among ingredients and developed formulation. Dissolution and permeation studies were performed to compare effects of enhancers. Surface morphology after release was examined by scanning electron microscopy. Selected formulation was subjected to in vivo studies by randomized crossover design in rabbits (n = 6) for pharmacokinetic comparison with oral solution administration. Physical evaluation revealed that translucent, flexible, non brittle patches of uniform weight and thickness were prepared. Release from patches followed Higuchi model. Mechanism of release was Fickian. Formulation containing IPM showed that release was by anomalous transport. Highest permeation flux was observed for formulation containing IPM with 2-fold enhancement in permeation. Permeation flux for patches was in order of formulation with no enhancer > IPM > T20 > S20 > DMSO = eucalyptus oil. Plasma concentration from in vivo studies exhibited sustained plasma levels of metoprolol after transdermal patch application in comparison to oral solution administration. Pharmacokinetic analysis of in vivo data elucidated that half life was increased 8 times when compared to oral administration, due to controlled release of drug for longer period of time. These findings suggested that hydrophobic transdermal patches of highly water soluble drug metoprolol were successfully prepared with 10% of IPM for sustained systemic delivery for prolonged half life. Keywords: isopropyl myristate, metoprolol, transdermal patch, hydrophobic, permeation enhancers, skin diffusion, in vivo Mortality due to cardiac pathologies increases with age and hypertension is one of the prevailing issues. Death rate related to hypertension has been increased in recent years. Treatment of hypertension requires long term therapy and more patient compliance. Metoprolol is widely prescribed for the treatment of hypertension. It undergoes extensive first pass metabolism and has short half-life. Frequent dosing reduces patient compliance that can be avoided by using transdermal route (1). For a drug to be delivered across skin it should have molecular weigh less than 500 Da and log P value from 1 to 3 (2). Thus, metoprolol is an ideal candidate for transdermal drug delivery system with log P value of 1.6 and molecular weight Da. A number of transdermal therapeutic systems (TTS) such as creams, patches, gels etc., are available for systemic delivery of drugs across skin. However, transdermal patches have an advantage over other systems because of ease of application and least cost of manufacturing. Transdermal patches are broadly classified as: drug in adhesive (DIA), polymer matrix and reservoir patches (3). Passive diffusion of drugs across skin is limited due to barrier properties of stratum corneum. Chemical and physical strategies are used to increase transport through skin by increasing permeability of skin and by providing driving force for drug molecule, respectively (4). IPM is an aliphatic acid ester. It is a promising permeation enhancer for topical formulations and is widely used as non volatile solvent in cosmetics and pharmaceutical industry. Aim of the present study was to develop hydrophobic transdermal patches of metoprolol by incorporating five different permeation enhancers * Corresponding author: ma786_786@yahoo.com; mobile: ; fax:

2 1640 AYESHA YAQOOB et al. and two hydrophobic polymers i.e., ethyl cellulose and eudragit RL 100 for transdermal delivery of metoprolol. EXPERIMENTAL Materials Metoprolol tartrate was received as a generous gift from Acto Laboratories, Karachi, Pakistan. Eudragit RL 100 (M/s Rohm Pharma GmbH, Darmstadt, Germany), ethyl cellulose (ethoxy contents % w/w; viscosity 10 cps, Sigma Aldrich, USA), ammonium dihydrogen phosphate (AppliChem, Germany), dichloromethane (Fisher Scientific, USA), polyvinyl alcohol (m.w , AppliChem, Germany) were purchased. Isopropyl myristate, eucalyptus oil, dimethyl sulfoxide were purchased from Aldrich Sigma (USA). Orthophosphoric acid, dibutyl phthalate, chloroform, span 20, Tween 20, micro filters of 0.45 micron, and HPLC grade acetonitrile and methanol were purchased from Merck (Germany). All other chemicals used were of analytical grade. Preparation of patches Patches were prepared by plate casting method. At first, PVA backing membrane was fabricated by preparing 4% w/v clear solution of PVA at 90 O C on hot plate magnetic stirrer. Five ml of PVA solution was poured in glass Petri dishes of 18.5 cm 2 area with the help of graduated pipette and evaporated in oven at 40 O C for 24 h. After drying, backing membrane was not removed from Petri dishes. In second step, 250 mg of ethyl cellulose (EC) and eudragit RL 100 (ERL) in ratio of 8 : 2 were dissolved in 10 ml of chloroform by sonication. Eighty mg of metoprolol, 40% w/w of dibutyl phthalate (100 mg) and 10% w/w of enhancer (25 mg) were added in clear polymeric solution. This clear matrix solution was poured on Petri dishes containing transparent backing membrane and allowed to evaporate under inverted funnel for 12 h at 40 O C. Dried films were taken out from Petri dishes and cut in circular pieces of 1.5 mm diameter for further analysis. Patches were evaluated for physical appearance, weight and thickness variation. Fourier transform infrared (FTIR) spectroscopy FTIR studies were conducted to assess compatibility between drug and excipients. FTIR spectra of drug, polymers and prepared transdermal films were recorded using attenuated total reflectance (ATR) Bruker FTIR (Tensor 27 series, Germany) apparatus with scanning range of cm -1. Raw material was grounded for uniformity of particle size before analysis and already prepared thin transparent films were directly scanned and analyzed by using Opus data collection software to identify any physical and chemical interaction. Dissolution studies Dissolution of patches was performed on Pharmatest apparatus attached with automated fraction collector (Pharma Test, Germany) by using paddle over disk method (U.S.P. apparatus no. 5). Disc assembly was prepared with stainless steel mesh of pore size 125 µm, paper clips and watch glass. Phosphate buffer ph 7.4 was used as dissolution medium (5). Disk assembly with releasing surface facing upward was placed in glass dissolution vessel containing 500 ml of phosphate buffer. Temperature was maintained at 32 ± 1 O C and media were stirred with paddles at speed of 50 rpm (6). Disk assembly was designed to reduce dead volume between vesselís bottom and patch holder and to kept patch release surface parallel to paddle blade bottom. Distance between disc and paddle blade was kept at almost 25 mm. Samples were withdrawn at time points 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 16, 20 and 24 h. Skin diffusion studies For diffusion studies, prepared rabbit skin was mounted between two compartments of Franz diffusion cell (PermeGear, USA). Receptor compartment was filled with phosphate buffer solution ph 7.4 and kept on stirring on multi plate magnetic stirrer (7). Temperature in receptor compartment was maintained at 32 ± 1 O C by automated thermostat. Patch of an area 1.77 cm 2 was placed on skin in donor compartment. Samples were taken from sampling port at predetermined time intervals. Absorbance of each sample was determined without any further dilution on UV-Vis spectrophotometer (IRMECO U2020, Germany) at λ max of 223 nm. Samples of skin penetration study were analyzed after appropriate dilution with phosphate buffer. Phosphate buffer ph 7.4 was used as blank. Concentration of drug at each sampling point was calculated from calibration curve. Elucidation of drug release mechanism Dissolution and skin diffusion data were subjected to various kinetic models by using DD Solver Æ Excel based ads in program (8) to elucidate release mechanism from patches. Zero order kinetics indicates that drug release rate is independent of the amount of drug in formulation. F t = K o t

3 Preparation, in vitro and in vivo characterization of hydrophobic patches of First order kinetic model indicates dependency of drug release on amount of drug in formulation at time t. K 1 t log Ft = log F o + ñññññññññ Higuchi plot deals with release of drug from uniform matrix formulation by diffusion. This model is used to describe release of water soluble drug from transdermal patches and modified released formulations (9). Ft = K H t 1/2 Korsmeyer Peppas kinetic model is expressed by following equation: F ñññ t = Kp t F n o where F t is amount of drug released at time t and F o indicates the amount of drug added in formulation. F t /F o is the fraction of metoprolol released. K o, K 1, K H and K p are release constants of zero order kinetics, first order model, Higuchi plot and Korsmeyer model, respectively. ìnî is release exponent of Korsmeyer Peppas plot and elucidate the behavior of diffusion release. As value of ìnî approaches 0.5, the release mechanism follows Fickian diffusion. When 0.5 < n > 1, the mechanism of diffusion release is anomalous. If n = 1, the release is Case-II transport (10). Patch surface morphology studies Patch surface was evaluated visually for smoothness and homogeneity before release study. The behavior of transdermal films after metoprolol release was observed under scanning electron microscope (SEM). Small pieces of patch after release were directly scanned under electron microscope (Quanta 250) using maker FEI Æ software. Surface morphology of patches further explained drug release mechanism. In vivo studies in rabbits Patch having the highest permeation flux was selected for in vivo studies in rabbits. It was a crossover single dose study. Six rabbits of weight kg were obtained from pharmacology animal house. Weight of each rabbit was noted and animals were properly labeled for identification. One day before experiment rabbits were kept fasting overnight but had free access to water. Twenty mg of metoprolol in form of solution was administered to rabbits with a plastic syringe (11). Blood samples were taken in EDTA tubes at time points of 0, 0.5, 1, 2, 3, 4 and 6 h. Plasma was separated by centrifugation at speed of 5000 rpm. Transdermal application of metoprolol patch was done after one week of washout period. One day before transdermal patch application, hairs were removed from abdominal region of rabbits with depilatory cream (Anne French, Pakistan). After hair removal, skin was carefully washed with distilled water. On the day of experiment, patch containing 80 mg of metoprolol was applied on hair free area with adhesive tape. Blood samples after transdermal application were taken at 0, 1, 3, 6, 9, 12, 24, 36 and 48 h. Plasma was separated and stored at -70 O C. On the day of analysis, plasma was allowed to come at room temperature. Plasma samples were prepared by liquid-liquid extraction with dichloromethane (1ñ2 ml) and concentrated on sample concentrator at 40 O C. Two hundred µl of mobile phase was used to reconstitute plasma samples. Two hundred µl of these prepared samples were analyzed by HPLC on Agilent Technologies series 1100 (USA) chromatograph with a pump and UV-Vis detector by using BDS Hypersil C 18 column ( mm, particle size 5.0 µm, Thermo Electron Co., USA). Mobile phase was a combination of ammonium dihydrogen phosphate (50 mm) solution and methanol (50 : 50, v/v) adjusted to ph Concentration of drug in plasma was determined by using calibration curve plotted by concentration versus peak area of drug in chromatograms of spiked plasma. In vivo data were subjected to pharmacokinetic analysis by using application package Kinetica Æ version 5.0 (Thermo Fisher Scientific, USA). RESULTS AND DISCUSSION Physical characterization Physical characterization of prepared hydrophobic patches of metoprolol was done visually. All patches showed reasonable tensile strength and were non-brittle. Surface was found smooth in appearance. Patches without enhancer as well as containing enhancers were translucent in their physical appearance. Smoothness, physical strength and transparency were increased due to the presence of enhancers. Backing membrane was firmly attached with matrix layer. However, improper storage for more than six months resulted in detachment of backing membrane and brittle patches. Weight/cm 2 and thickness were found uniform throughout prepared polymeric films. FTIR spectra analysis FTIR spectra of pure drug, polymers and prepared patches containing enhancers were recorded to assess physical and chemical interactions between drug and excipients. The most characteristic peaks

4 1642 AYESHA YAQOOB et al. of metoprolol spectra were at , and cm -1. All these characteristic peaks of pure drug were also present in the spectra of all prepared hydrophobic films containing drug (with or without enhancer). Selected FTIR spectra are shown in Figure 1. Thus, the FTIR spectra analysis indicated that no interaction occurred between metoprolol and film ingredients during formulation. The most characteristic peak of prepared films was at 1725 cm -1 which was observed in all formulations with or without enhancers. Dissolution studies Dissolution studies were performed to evaluate effects of enhancers (10% w/w) on release of drug. Amount of metoprolol released was plotted against time and released profile of patches with enhancer and without enhancer is presented in Figure 2. Initial burst release was observed for all formulations. After initial fast release of surface drug, release became slower. The initial burst release will be helpful to achieve therapeutic plasma levels in short time (12). Incorporation of enhancers had not affected amount of drug release up to 24 h from patches except in formulations containing DMSO. DMSO containing formulation showed more than 90% drug release within 12 h. All other formulations showed less than 90% of drug release (85.62 to 88.91%) up to 24 h. Release pattern was not altered for the formulations containing enhancers. However, presence of Figure 1. FTIR spectra of metoprolol (MET), EC, ERL and patch containing IPM (P-IPM)

5 Preparation, in vitro and in vivo characterization of hydrophobic patches of Figure 2. Metoprolol release from patches containing 10% of various enhancers. S 20 = span 20, T 20 = Tween 20 Table 1. Release kinetics of metoprolol from patches. Enhancer T 50% (h) T 90% (h) Zero order First order Higuchi's plot Korsmeyer Peppas R R R K H R n No enhancer Eucalyptus oil IPM S T DMSO S 20 = span 20, T 20 = Tween 20 enhancers affected the burst release properties of patches. The highest burst effect was observed for formulation containing DMSO with T 50% (time taken to release 50% of drug) of 2.18 h as shown in Table 1. T 50% is an indicator of fast release of drug (13). Less initial burst release was observed for formulation containing span 20 with T 50% of 8.14 h. The lowest value of T 90% (time to release 90% of drug) was observed for formulation containing DMSO. No significant statistical difference (p > 0.05) was observed between values of T 90% of other formulations. DMSO is commonly used as co-solvent in topical formulations. It is also called as ìuniversal solventî. It has polar nature and has ability to cross biological membranes. Due to solubilizing ability it is used as enhancer for topical and transdermal drug delivery. Smaller values of T 50% and T 90% can be related to co-solvent property of DMSO. To elucidate mechanism of release, data were subjected to release kinetic models. Release parameters are shown in Table 1. R value (correlation coefficient) was found in order of Korsmeyer Peppas > Higuchi > first order > zero order. As release followed Korsmeyer Peppas and Higuchi model, thus release was by diffusion of highly water soluble drug from matrix of hydrophobic patch. Value of release exponent ëní helps to understand behavior of diffusion release. Formulations, except that containing IPM, had ëní less than 0.5, thus release was by Fickian diffusion. For formulation containing IPM the value of ëní was 0.528, so release followed anomalous transport i.e., non-

6 1644 AYESHA YAQOOB et al. Fickian mechanism of diffusion. High values of Higuchi rate constant (K H ) was observed for patch containing DMSO. Thus, release from patches was sustained and diffusion controlled. Patch surface morphology studies Surface morphology of patches after drug release was examined under electron microscope. SEM images of patch containing no enhancer and that of containing IPM as enhancer are shown in Figure 3. The image of patch containing no enhancer showed unequal distribution of pores of different sizes. Patch containing IPM showed only large pores indicating that erosion mechanism of release was also involved in drug release. These results were also supported by release exponent ìnî of IPM containing patch, indicating non-fickian mechanism of diffusion. Smaller pores were less in patch containing IPM. Figure 3. SEM photographs of (A) patch without enhancer and (B) patch containing 10% IPM Skin diffusion studies Skin diffusion studies help to predict in vivo performance of transdermal patch. Aim of skin diffusion studies was to evaluate effects of 10% permeation enhancers on diffusion of metoprolol through excised rabbit skin. Amount of drug permeated (µg/cm 2 ) was plotted against time (h) as shown in Figure 4. Permeation parameters i.e., cumulative amount of drug permeated up to 30 h (Q 30, µg/cm 2 ), permeation flux at steady state (Jss, µg/cm 2 /h) and enhancement ratio (ER) are given in Table 2. Bar Figure 4. Metoprolol permeation through rabbit skin from patches containing 10% of various enhancers. S 20 = span 20, T 20 = Tween 20

7 Preparation, in vitro and in vivo characterization of hydrophobic patches of Figure 5. Bar chart of permeation flux of metoprolol through rabbit skin from patches containing various enhancers Figure 6. Plasma concentration-time profile of metoprolol after transdermal and oral administrations in rabbits (n = 6) chart of permeation flux of formulations containing different enhancers is shown in Figure 5. All enhancers containing formulations showed significantly higher (p < 0.05) values of permeation flux through rabbit skin as compared to formulations without enhancer. Highest permeation flux of ± µg/cm 2 /h was observed in formulation containing IPM with 2.12 folds enhancement in permeation as compared to control (no enhancer). IPM has also exhibited highest value of Q 30 ( ± ). Formulation containing Tween 20 showed 2.05 folds rise in permeation flux. Enhancement of

8 1646 AYESHA YAQOOB et al. permeation induced by various enhancers was in decreasing order of IPM > Tween 20 > Span 20 > DMSO = eucalyptus oil (p = 0.292). Results obtained in present study are supported by the findings of Yousuf et al., whereas IPM was evaluated as enhancer for combination of drugs: ketotifen fumarate and salbutamol sulfate (14). IPM has been declared best permeation enhancer in another work conducted by Das et al., where it was compared with another aliphatic ester isopropyl palmitate (IPP) for permeation enhancement of trazodone in a matrix patch (15). IPM is well tolerated permeation enhancer for topical formulations and is widely used as non volatile solvent in cosmetic and pharmaceutical industry (16). IPM disrupts highly order lipid structure of skin thus altering metoprolol permeability across skin. IPM is lipophilic in nature thus has innate ability to interact with lipid bilayers. IPM can also partition into polar phase (proteins) of skin because of its intermediate polar nature (15, 17). IPM belongs to class of aliphatic esters and according to Sato et al. this class of penetration enhancers act both by increasing partition coefficient of metoprolol for skin as well as by increasing its diffusivity to skin (18). In present study, Tween 20 was found to be more effective when compared to span 20. Span 20 and its ethoxylate derivative polysorbate 20 (Tween 20) belongs to class of non ionic surfactants. Both have same C12 fatty acid chain but differ in their polar head functional groups. Tween 20 is more hydrophilic than span 20. Being hydrophilic both non ionic surfactants exert their effects by an increase in skin hydration, resulting in increase in drug partitioning across skin and leading to higher penetration of metoprolol through rabbit skin. Results of permeation studies showed that DMSO and eucalyptus oil were equally effective for metoprolol with respect to penetration enhancement. Eucalyptus oil was found as effective permeation enhancer (30-fold increase in permeation) for another highly water soluble drug 5-florouracil in a study by William and Barry (19). Effects of DMSO as penetration enhancer can be related to its lipid extraction efficiency as well as partitioning increasing property (20). Table 2. Permeation parameters and kinetics of metoprolol through rabbit skin from patches. Enhancer Q 30 J ss ER Zero order First order Higuchi model No enhancer ± ± Eucalyptus oil ± ± IPM ± ± S ± ± T ± ± DMSO ± ± S 20 = span 20, T 20 = Tween 20 Table 3. Pharmacokinetic parameters (mean ± S.D.) of metoprolol in rabbits (n = 6) plasma after oral solution administration and transdermal patch application. Pharmacokinetic Oral Transdermal parameters solution patch C max (ng/ml) ± ± T max (h) 0.50 ± ± 1.77 AUC 0-t (ng h / ml) ± ± AUC 0- (ng h / ml) ± ± C ss ± ± 1.02 MRT (h) 4.71 ± ± 6.92 t 1/2 (h) 3.71 ± ± 5.85 % Relative bioavailability 96.22

9 Preparation, in vitro and in vivo characterization of hydrophobic patches of Results of kinetic analysis of permeation data are given in Table 2. Best fit equation for kinetic analysis of permeation data was in order of Higuchi > first order > zero order for formulation containing eucalyptus oil, DMSO, span 20 and for that containing no enhancer. Formulations containing Tween 20 and IPM, values of R 2 were in decreasing order of zero order > first order > Higuchi. These results were due to skin barrier which has slowed the permeation of drug leading to almost zero order permeation. In vivo studies Formulation containing 10% of IPM was selected for in vivo studies in rabbits (n = 6). Plasma concentration-time profile after metoprolol oral solution administration and transdermal patch application is shown in Figure 6. Metoprolol was declined faster after oral administration, whereas plasma concentration remained relatively stable for longer period of time after transdermal application. Pharmacokinetic parameters of both treatments are given in Table 3. Statistical analysis of in vivo data was performed and pharmacokinetic parameters were found significantly different (p < 0.05) after oral and transdermal administration. Maximum plasma concentration (C max ) after oral administration was ± ng/ml, in comparison to ± ng/ml for transdermal application. Time to reach maximum plasma concentration (T max ) was 0.5 h after oral administration whereas after transdermal application it was 9 h. This lower value of C max and higher value of T max after transdermal application of 80 mg dose showed that drug was released at sustained level from patch for longer period of time. Mean residence time (MRT) after transdermal administration (46.58 ± 6.92 h) was high in comparison to oral administration (4.71 ± 0.60 h). Plasma half life (t 1/2 ), an indicator of prolonged drug delivery, was extended from 3.71 ± 0.56 h after oral administration to ± 5.85 h after transdermal application. Relative bioavailability after transdermal patch application was 96.22%. Oral administration of 20 mg of metoprolol resulted in higher value of C max whereas C max in case of transdermal application of 80 mg patch was lower. However, steady state plasma concentration (C ss ) with no plasma fluctuation resulted in transdermal administration for prolonged period of time as indicated from t 1/2 and MRT and this will be helpful in avoiding side effects related to frequent oral administration. The higher value of MRT was due to continuous delivery of drug from patch present on skin in to systemic circulation. Rama et al. (21) performed comparative pharmacokinetic study of another β blocker - propranolol hydrochloride in rabbits (weight = kg) after application of patches of an area 18 cm 2 containing 30 and 45 mg drug (for 30 h). Oral solution was administered in dose of 10 mg/kg (6 h). In their study MRT was found 5 times higher than that of oral administration, whereas in the present study, MRT after transdermal patch application was found 18 times higher than that of oral administration (21). In vitro release and permeation studies revealed slow sustained delivery of drug from hydrophobic patches. When tested in rabbits, this slow and sustained drug delivery resulted in continuous and prolonged absorption of metoprolol through skin in to the systemic circulation. CONCLUSION Present study concludes that combination of two hydrophobic polymers EC and ERL can provide diffusion controlled release of metoprolol and highest permeation flux across excised rabbit skin in the presence of 10% w/w of different enhancers. Controlled and sustained drug delivery resulted in continuous and prolonged systemic absorption of highly water soluble drug metoprolol for prolonged plasma half life. Acknowledgment Authors are really thankful to higher education commission (HEC) of Pakistan for providing financial support for this study in form of indigenous scholarship. REFERENCES 1. Papp J., Horgos J., Szente V., Zelko R.: Int. J. Pharm. 392, 189 (2010). 2. Flynn G.L., Stewart B.: Drug Dev. Res. 13, 169 (1988). 3. Nishida N., Taniyama K., Sawabe T., Manome Y.: Int. J. Pharm. 402, 103 (2010). 4. Alexander A., Dwivedi S., Ajazuddin, 0TK%5BAuthor%5D&cauthor=true&cauthor_uid= Giri T.K., Saraf S. et al.: J. Control. Release 164, 26 (2012). 5. Jayaprakash S., Ramkanth S., Anitha P., Alagusundaram M.: Malay. J. Pharm. Sci. 8, 25 (2010). 6. Guyot M., Fawaz F.: Int. J. Pharm. 204, 171 (2000).

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