Studies on the influence of penetration enhancers on in vitro permeation of carvedilol across rat abdominal skin
By - 12/18/2008
(1 vote)
G. Ramesh, Vamshi Vishnu Y, Kishan V and Madhusudan Rao Y*
Centre for Biopharmaceutics and Pharmacokinetics, University College of Pharmaceutical Sciences, Kakatiya University, Warangal- 506 009 (A.P)
* For correspondence : ymrao123 @ yahoo.com
Current Trends in Biotechnology and Pharmacy, Vol.1 (1) 62-69 (2007) ISSN:0973 - 8916
Abstract
The aim of the investigation was to study the effect of penetration enhancers on the in vitro permeation of carvedilol across excised ratabdominal skin and to select suitable penetrationenhancer. Terpenes menthol, camphor, d-limonene and carvone; surfactants, Transcutoland Labrasol at 5 % w/v, were used as penetrationenhancers in the study. Skin permeation studieswere conducted in Franz diffusion cells usingexcised rat abdominal skin. Solutions containing5 % w/v camphor showed maximum permeation(451.20 µg?) in 24 hr with a flux of 5.23 µg/hr/cm2and was significantly different (p<0.05) toflux obtained with other permeation enhancers.Control (phosphate buffer saline, pH 7.4containing 40 % v/v polyethylene glycol) sampleshowed lowest permeation (59.18 µg?), with aflux of 0.67 ìg?/hr/cm2. The flux of carvedilolobtained from the solutions containing camphor,Transcutol, d-limonene, carvone, Labrasol andmenthol (5 % w/v) were 7.81, 7.26, 6.52, 5.91,4.21 and 2.28 times higher than that observedwith control, respectively. The flux obtained withcamphor was significantly higher (p<0.05) thanthe fluxes obtained with other penetrationenhancers. The present study suggests thatcamphor, Transcutol and d-limonene at 5 % w/vlevel may be used as penetration enhancers inthe development of transdermal drug deliverysystems.
Key words:
Carvedilol, Terpenes, Transcutol, Labrasol, Rat skin .
Introduction
Carvedilol is a non-selective â-adrenergicantagonist widely used in the treatment of mildto moderate essential hypertension and stableangina pectoris. It also possesses antioxidant andantiproliferative effects. That may enhance itsability to combat the deleterious effects ofsympathetic nervous system activities in heartfailure (1). It is well absorbed followed by oraladministration. The systemic availability isapproximately 25-35 % because of high first passmetabolism (2). To reduce its high first passmetabolism and enhance its bioavailability otherroutes of administration such as buccal (3), havebeen reported. The biological properties ofcarvedilol such as high first pass metabolism,low dose, need for long term treatment andrepetitive dosing make this drug an interestingcandidate for transdermal administration.
The transdermal route of administration has beenrecognized as one of the potential route for thelocal and systemic delivery of drugs. Theadvantages of transdermal delivery, includetherapeutic benefits such as sustained deliveryof drugs to provide a steady plasma profile, particularly for drugs with short half lives andhence reduced systemic side effects; reducing thetypical dosing schedule to once daily or evenonce weekly hence generating the potential forimproved patient compliance and avoidance ofthe first pass metabolism effect for drugs withpoor bioavailability (4). However, the highlyorganized structure of stratum corneum formsan effective barrier to the permeation of drugs,which must be modified if poorly penetratingdrugs are to be administered. The use of chemicalpenetration enhancers would significantlyincrease the number of drug molecules suitablefor transdermal delivery (5) . Terpenes present in naturally occurring volatile oils appear to beclinically acceptable enhancers (6). Moreover, awide variety of terpenes have been shown toincrease the percutaneous absorption of numberof drugs (7). In this investigation, the penetrationenhancers camphor, carvone, menthol, d-limonene, Transcutol and Labrasol at 5 % w/vconcentration were used. Excised rat abdominalskin was used for in vitro permeation studies.
Previous studies, reported carvedilol transdermal therapeutic systems based on polymethacrylates(8) and membrane controlled matrix type patchesusing non ionic surfactants as penetration enhancers (9). The objective of present study was to investigate the effect of penetration enhancers on the permeation of carvedilol across rat abdominal skin and to select suitable penetration enhancer(s).
Materials and methods
Materials
Carvedilol was provided by SunPharmaceuticals, India. D-limonene, carvonementhol and poly ethylene glycol 400 (PEG 400)were purchased from Merck, India. Camphorwas purchased from Sd fine chemicals India.Labrasol (PEG-8 caprylate/caprate), Transcutol(Diethyleneglycol monoethyl ether) were gifts from Gattefosse (Cedex, France). All otherchemicals and reagents used are of analyticalgrade.
Preparation of Rat Abdominal Skin
Albino rats weighing 150-200 gm were selectedfor permeation studies and the study wasconducted with the approval of institutionalethical committee. The animals were sacrificedusing anesthetic ether, hair of test animals wascarefully trimmed short (<2 mm) with a pair ofscissors and the full thickness skin was removedfrom the abdominal region. The epidermis wasprepared surgically by heat separation technique(10), which involved soaking the entireabdominal skin in water at 60° C for 45 sec,followed by careful removal of the epidermis.The epidermis was washed with water and usedfor ex vivo permeability studies. The thickness of the skin was measured with digital micrometer (Mitotoyo, Japan).
In vitro permeability studies
Franz diffusion cell with a surface area of 3.56cm2was used for in vitro permeation studies. Ratabdominal skin with a thickness of about 1.0 mmwas mounted between the compartments of thediffusion cell with stratum corneum facing thedonor compartment. The receiver phase is 12 mlof phosphate buffer saline (PBS) pH 7.4containing 40 % v/v of PEG 400, stirred at 500rpm on a magnetic stirrer; the whole assemblywas kept at 37 ± 0.5o C. PBS (pH 7.4), containing40 % v/v PEG 400 and 3 mg of carvedilol (3mL) was placed in the donor compartment.Carvedilol is practically insoluble in water hencea buffer PEG 400 system was used for solublizingcarvedilol. All individual solutions in donorcompartment were prepared separately with andwithout (5% w/v) penetration enhancers.Menthol, camphor, Transcutol, Labrasol, d-limonene and carvone as penetration enhancersand PBS pH 7.4 containing 40 % v/v PEG 400 as control were used in the study. The entiresetup was placed over magnetic stirrer andtemperature was maintained at about 37 ± 0.5oCby placing the diffusion cell in a water bath. Theamount of drug permeated was determined byremoving 1 mL of sample at appropriate timeintervals up to 24 hr, the volume was replenished with an equal volume of PBS pH 7.4 containing40 % v/v PEG 400. The drug content in thesamples was determined by high performanceliquid chromatography (HPLC) and theconcentration was corrected for sampling effectsaccording to the equation (11). C1 1 n = Cn (VT/VT - VS) (C n-1/ Cn-1) Where C1n is the corrected concentration of the is the measured concentration of nth sample, C n carvedilol in the n th sample, C is the measured n-1 concentration of the carvedilol in the (n -1)th sample, VT is the total volume of the receiverfluid and VS is the volume of the sample drawn.
Estimation of drug content in the sample by HPLC method
The HPLC system (Shimadzu, Japan) consistedof a LC–10AT solvent module, and a model UV-Visible Spectrophotometric detector (SPD–10A)with LC 10 soft ware. Carvedilol was quantifiedaccording to a reported method (12). The columnused was a Kromasil KR 100-5C8 (25 X 4.6 mmi.d, 5 µ). The mobile phase consisted ofacetonitrile, 15 mM orthophosphoric acid(37:63), and 0.25 v/v % triethylamine mixture,and was adjusted to pH 2.5 with orthophosphoricacid. The elute was monitored at 238 nm with aflow rate of 1 mL/min. The sensitivity was set to0.005 AUFS.
Data analysis
As described by Barry (13), the steady state flux(Js), lag time (TL), diffusion coefficient (D) andapparent permeation coefficient (Papp) are definedby Js = (dQ/dt)ss 1/A - (1) h2 D = 6T L dQ 1 1 - (2) P = Dt A C - (3) Where, A is the effective diffusion area; h, thethickness of skin; Cs, the concentration in thesaturated solution and (dQ/dt)ss is the steady stateslope.
Statistical Analysis
The Q24(cumulative amount permeated in 24 hr)and flux values obtained from the varioussystems were tested for significant differencesusing a one-way analysis of variance (ANOVA)or unpaired t test. If the significant differencesexist when ANOVA was used, the pair wisecomparison of different systems was done to findout statistical significant difference in parametersusing a Dunnet’s test. When the normality testfailed, Kruskal-Wallis one-way ANOVA wasused to find out if the significant differences existbetween different systems. The statisticalanalysis was conducted using SigmaStatsoftware version 1.0 (Jandel Corp., California).
Results and Discussion
Effect of Penetration Enhancers
The effect of enhancers on permeation of a drugusually depends upon physicochemicalcharacteristics of both permeant as well asenhancer molecule. Among enhancers, variousterpenes have been widely used for transdermaldelivery of compounds (13). The effects ofvarious penetration enhancers on thepercutaneous penetration profile of carvedilolwas shown in Table 1, Figs. 1 and 2. Thethickness of isolated skin was found to be inbetween from 844 to 1234 microns.Solutions containing 5 % w/v camphor showedmaximum permeation of 451.20 µg in 24 hr with a flux of 5.23 µg/hr/cm2, was significantly higher(p<0.05) than the amount of carvedilol permeatedby other permeation enhancers used in the study.Control sample showed lowest amount ofpermeation, 59.18 µg in 24 hr with a flux of 0.67µg/hr/cm2. Maximum flux (5.23 µg/hr/cm2)obtained with camphor at 5 % w/v, wassignificantly higher (p<0.05) than the fluxobtained with other penetration enhancers, butwas not significantly different (p<0.05) from theflux obtained with Transcutol. Transcutol (4.87µg/hr/cm2) and d-limonene (4.37 µg/hr/cm2)showed similar flux values with lag time of 0.30hr and 0.40 hr respectively.The comparison of carvedilol flux obtained fromdrug solutions containing 5 % w/v penetrationenhancer through excised rat skin was shown inFig 3. The effect of the various enhancers on theflux of carvedilol followed the order: Camphor> Transcutol > d-limonene > Carvone > Labrasol> Menthol > Control. The flux of carvedilolobtained from the solutions containing camphor,Transcutol, d-limonene, carvone, Labrasol andmenthol (5 % w/v) were 7.81, 7.26, 6.52, 5.91,4.21 and 2.28 times higher than that observedwith control, respectively. The main findings ofthe study are the accelerants enhance diffusionor partition and thus permeation of drugs. Thelipid partitioning theory was proposed by Barryto describe the mechanism of action ofpermeation enhancers (14), whether by (i)disruption of the highly ordered structure of SClipids, (ii) interactions with intracellular proteinsor (iii) improvement in partitioning of the drug,co enhancers or co solvent in to the stratumcorneum.In this study, we found that diffusion of carvedilolthrough the skin was increased by terpenes. It isreported that terpenes enhance diffusion of drugsby extracting lipids from stratum corneum (15,16), it results in reorganization of lipid domainand barrier disruption (17, 18). The mechanismof barrier disruption may be due to the competitive hydrogen bonding of oxygen-containing monoterpenes with ceramide headgroups, thereby breaking the interlamellarhydrogen bonding network of lipid bilayer ofstratum corneum and new polar pathways orchannels are formed (19, 20). DSC proved thebarrier disruptive action of terpenes, where therewas a shift in endothermic transition temperatureof stratum corneum lipids after treatment withterpenes (21). The other possible mechanism ofaction may be the lipid fluidizing activity ofterpenes containing essential oils. Fouriertransform infrared (FTIR) studies proved thelipid extractive action of terpenes from stratumcorneum, where there was a decrease in heightsand areas of both symmetric and asymmetric CH2stretching absorbance peaks of stratum corneumlipids (16, 22). Surfactants, Transcutol andLabrasol change lipid chain fluidity of the SCand improve drug partition (23).
Conclusion:
The present study suggests that camphor,Transcutol and d-limonene at 5 % w/vconcentration may be used as penetrationenhancers, for the transdermal delivery ofcarvedilol. The data obtained in the present studyusing rat abdominal skin cannot be translated toin vivo delivery in humans as other factors suchas cutaneous microvasculature, which preventsthe accumulation of the drug in the skin and thecutaneous metabolism of the drug whichsignificantly alter the permeation profile of thedrug. The results obtained from this study willbe helpful in the development of transdermaldrug delivery systems. Further work isrecommended to evaluate the optimumconcentration of camphor, Transcutol and dlimonene to meet the target flux.
Acknowledgements
The authors acknowledge the financial support received from AICTE, New Delhi, India. The authors acknowledge M/s Sun Pharma, Baroda,India and M/s Gattefosse, France for providinggift samples of carvedilol and Transcutol andLabrasol, respectively.
Referances
1. Packer, M., Coats, A.J., Fowler, M.B.,Kataus, H.A., Krum, H., Mohaosi, P.,Rouleau, J.L., Tenaera, M., Castaigme,A., Roecker, E.B., Schultz, M.K. and DeMets, D.L. (2001) Effect of carvedilol onsurvival in severe chronic heart failure. N.Engl. J. Med, 344: 1651-1658.
2. Trefor, M. (1994) Clinical pharmacokine-tics and pharmacodynamics of carvedilol.Clin Pharmacokiet, 26: 335-346.
3. Vamshi Vishnu, Y., Chandrasekhar, K.,Ramesh, G. and Madhusudan Rao, Y.(2007) Development of mucoadhesivepatches for buccal administration ofcarvedilol. Current Drug Del, 4: 27-39.
4. Guy, R.H. (1996) Current status and futureprospects of transdermal drug delivery.Pharm Res, 13 : 1765–1769.
5. Kanikannan, N., Andega, S., Burton, S.,Babu, R.J. and Singh, M. (2004). F, DrugDev. Ind. Pharm, 30: 205-212.
6. Williams, A.C. and Barry, B.W. (1991).Species differences in percutaneousabsorption of nicorandil, Pharm. Res, 8:17 -24.
7. Moghimi, H., Williams, A.C. and Barry,B.W. (1997). A lamellar matrix model forstratum corneum intercellular lipids. V.Effects of terpene penetration enhancers onthe structure and thermal behaviour of thematrix. Int. J.Pharm, 146: 41- 54.
8. Udhumansha Ubaidulla, Molugu, V.S.Reddy., Kumaresan Ruckmani., Farhan, J.Ahmad. and Roop, K. Khar (2007)Transdermal therapeutic system of carvedilol: effect of hydrophilic andhydrophobic matrix on in vitro and in vivocharacteristics, AAPS PharmSciTech, 8 (1)Article 2, E1-E8.
9. Yuveraj Singh, T., Chetan Singh, C andAnshu Sharma (2007) Development andevaluation of carvedilol transdermalpatches. Acta Pharm, 57: 151–159.
10. Kaidi Zhao and Jagdish Singh (1999) Invitro percutaneous absorption enhancementof propranolol hydrochloride throughporcine epidermis by terpenes/ethanol. J.Control Rel, 62: 359-366.
11. Hayton W.L. and Chen. T. (1982)Correction of perfusate concentration forsample removal. J Pharm Sci, 71: 820 -821.
12. Ramesh Gannu, Vamshi Vishnu Yamsani,and Yamsani Madhusudan Rao. (2007).New RP-HPLC method with UV-detectionfor the determination of carvedilol inhuman serum. J Liq. Chrom. Rel. Tech,30: 1677-1685.
13. Barry B.W. (1983). Penetration enhancersIn: Dermatological formulations. MarcelDekker, New York: 160-172.
14. Barry B.W (1991) Lipid proteinpartitioning theory of skin penetrationenhancement. J. Control. Rel, 15:237-248.
15. Ogiso, T. Lwaki, M, and Paky, T. (1995).Effect of various enhancers on transdermalpenetration of indomethacin and urea, andrelationship between penetrationparameters and enhancement factors. J.Pharm. Sci, 84: 482-428.
16. Kaidi Zhao. and Jagdish Singh. (2000).Mechanism(s) of in vitro percutaneousabsorption enhancement of tamoxifen byenhancers. J. Pharm. Sci, 89: 771-780.
17. Williams, A.C. and Barry, B.W. (1991). Theenhancement index concept applied toterpene penetration enhancers for humanskin and model lipophilic (oestrodiol) andhydrophilic (5-fluorouracil) drugs. Int JPharm, 74: 157-168.
18. Williams, A.C., Barry, B.W. (1991).Terpenes and the lipid protein partitioningtheory of skin permeation enhancement,Pharm. Res, 8: 17-24.
19. Cornwell, P.A., Barry, B.W. (1993). Theroutes of penetration of ions and 5-fluorouracil across human skin and themechanisms of action of terpene skinpenetration enhancers, Int. J. Pharm, 94:189-194.
20. Thomas, N.S., Panchagnula, R. (2003).Transdermal delivery of zidovudine: effectof vehicles on permeation across rat skinand their mechanism of action, Eur. J.Pharm. Sci, 18: 71-79.
21. Vaddi, H.K., Ho, P.C., Chan, S.Y. (2002).Terpenes in propylene glycol as skin-penetration enhancers: Permeation andpartition of haloperidol, fourier transforminfrared spectroscopy, and differentialscanning calorimetry, J. Pharm. Sci, 91:1639-1651.
22. Yamane, M.A., Williams, A.C., Barry, B.W.(1995). Terpene penetration enhancers inpropylene glycol/water co-solvent systems:effectiveness and mechanism of action, J.Pharm. Pharmacol, 47: 978 -989.
23. Takahashi, K., Sakano, H., Yoshida, M.,Namata, N. and Mizuno, N. (2001).Characterization of the influence of polyolfatty acid esters on the permeation ofdiclofenac through rat skin, J Control Rel,73: 351-358.
Table 1:Permeation parameters of Carvedilol through excised rat abdominal skin from PBS pH7.4 containing 40 % v/v of PEG 400 and 5 % w/v of Penetration enhancer.
a Cumulative amount (µg) of drug permeated per cm2, results are mean ± SD (n=3)
bJs Transdermal flux, values represent mean ± SD (n=3).
cpapp Permeability Coefficient, values represent mean ± SD (n=3).
dT Lag Time, values represent mean ± SD (n=3).
L e D Diffusion Coefficient, values represent mean ± SD (n=3).
Fig 1:Effect of terpenes as penetration enhancers on in vitro permeation of carvedilol throughrat abdominal skin, values represent mean ± S.D (n=3)
Fig 2:Effect of surfactants as penetration enhancers on in vitro permeation of carvedilolthrough rat abdominal skin, values represent mean ± S.D (n=3)
Fig 3:Comparison of carvedilol flux obtained from drug solutions containing 5 % w/vpenetration enhancer through excised rat skin, values represent mean ± S.D (n=3)