Mechanism of In Vitro Percutaneous Absorption Enhancement of Carvedilol by Penetration Enhancers
Percutaneous Absorption Enhancement of CarvedilolSaima Amin, Kanchan Kohli, and Roop K. KharDepartment of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
Showkat R. MirDepartment of Pharmacognosy, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
Krishna K. PillaiDepartment of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
The effect of penetration enhancers like tulsi (basil) oil,eucalyptus oil, clove oil, black cumin oil, oleic acid and Tween80 on the percutaneous absorption of model lipophilicdrug-carvedilol was investigated using excised rat abdominalskin. Transdermal flux, permeability coefficient and enhance-ment factor were calculated for each penetration enhancer. Blackcumin oil (5% v/v) was selected on the basis of its highestenhancement in permeation and was evaluated further for itsmode of action using DSC, FTIR and histological studies. Theresults indicated that the oil shows its action by extraction of lipidsfrom stratum corneum as well as by loosening the hydrogenbonds between ceramides subsequently leading to fluidization ofthe lipid bilayer.
Topical administration of drugs through the skin is aviable route for delivery of potent, low molecular weightdrugs that undergo extensive hepatic metabolism leadingto their low bioavailability.[1] But to deliver drugs through
skin is difficult as the outermost layer of the skin, thestratum corneum (SC) forms an excellent barrier againstpermeation of drugs because of its rigid lipid lamellarlayer. Therefore, such drugs must possess certain charac-teristics like sufficient lipophilicity, partition coefficientand low molecular weight.[2] The transdermal permeationof such drugs can also be improved by increasing thermo-dynamic activity of the drugs in transdermal formulation(push effect) or by the use of permeation enhancer (pulleffect).[3,4] The physical enhancement by iontophoresis orsonophoresis is also a mode of increasing the transdermalpermeation of drugs especially the high molecular weightdrugs like peptides and proteins.[5,6] To increase the per-meation of drugs through the skin, inclusion of penetrationenhancers (PEs) have been reported to reversibly reducethe barrier resistance of skin and to allow the drug topenetrate to the viable tissues and enter the systemiccirculation.[7] The objective of the present research workwas to gain background information on various types ofpenetration enhancers and their mechanism of penetrationenhancement. PEs are known to follow either of the threepermeation routes, namely, lipid rich route, water richroute and a pore (shunt route).[8] In the present researchvarious PEs, broadly belonging to terpenes, fatty acids andsurfactants, have been studied for their percutaneousabsorption enhancing effects. All these differ in theirmechanism of alteration in skin structures like, terpenestend to partition into the SC and increase the diffusionwhile fatty acids cause drug solubilization, increase parti-tioning and cause disruption of the SC components.[9,10]
Tulsi (basil) oil is a volatile oil obtained from leavesof Ocimum basilicum by steam distillation and mainly
Received 7 March 2008, Accepted 23 May 2008.Address correspondence to Saima Amin, Department of
Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, India; E-mail: [email protected]
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contains linalool, methylchavicol, eugenol, camphor andmethyl cinnamate. Eucalyptus oil is the volatile oilobtained from leaves of Eucalyptus globulus and containscineole, terpineol, sesquiterpene alcohols and aliphaticalcohols. Clove oil is the volatile oil obtained by steamdistillation of flower buds of Eugenia caryophyllata andmainly contains eugenol and eugenyl acetate. Black cuminoil is a fixed oil obtained from seeds of Nigella sativa andis known to contain linoleic and oleic acid along withother free fatty acids.[11] These oils are classified asgenerally regarded as safe (GRAS) by Food and DrugAdministration. It was further postulated that since the oilcontains different types of fatty acids, a synergistic effectcan be expected.
The general techniques used to study the biophysicalmeasurements of skin components are Fourier TransformInfrared Spectroscopy (FTIR), Differential ScanningCalorimetry (DSC) and histological studies. The mechanismof alterations by penetrants are indicated by change in theFTIR specifically in the frequency of C-H2 stretchingabsorbance (which indicates the change in the lipid organi-zation in the SC) or change in the band width of C-H2stretching absorbance (which indicates the motion of thelipid chains). However, a decrease in intensity of absor-bance, if observed, is due to C-H stretching indicatinglipid extraction.[12] Another mode of analyzing the lipidphase transition caused by penetrants is measuring thephase transition temperature or enthalpy of skin samples inDSC thermograms.[13,14] The changes in transition temper-ature occur due to change in the organization of lipidbilayer. The four major transitions T1, T2, T3 and T4 areindicative of the lipids and proteins organized in theSC.[15] Histological changes are also useful to understandthe mode of action of penetrants. The penetrant-treated SCshows loosening of the layered SC and widening ofinterspaces.[16]
The effect of PEs like tulsi (basil) oil, eucalyptus oil,clove oil, black cumin oil, oleic acid and Tween 80 on thepercutaneous absorption of model lipophilic drug-carvedilolwas undertaken. Carvedilol has low oral bioavailability(nearly 25% po) as it undergoes extensive hepatic metabo-lism and thus the therapy requires frequent dose adminis-tration per day resulting in lack of patient compliance. Thedrug is a large molecular weight moiety (406.5 Da) havinglog P value 0.62.[17] Although the drug is lipophilic innature, it shows low skin permeation. In-vitro skinpermeation study was carried out using excised abdominalskin of albino Wistar rats (200–250 g/Female). The per-meability coefficient, transdermal flux and enhancementfactor were calculated and interpreted. FTIR, DSC andhistological studies of the control and black cumin oiltreated skin samples were carried out to investigate itsmode of alteration of skin permeability.
MATERIALS AND METHODS
Materials
Carvedilol was generously gifted by WockhardtPharmaceuticals Ltd. India, for the research work. Tulsi(basil) oil, eucalyptus oil, clove oil, black cumin oil, oleicacid and Tween 80 were purchased from CDH chemicals.Isopropyl alcohol was purchased from Merck India Ltd.All other chemicals used were of analytical grade.
Methods
In Vitro Percutaneous Absorption Studies
To carry out the in vitro percutaneous absorptionstudies, a Franz diffusion cell of 24 ml volume and 2 cminternal diameter was fabricated. The cell consisted of twohalf cells, the donor and the receiver cell, which were heldtogether with springs. The area of diffusion was calculatedto be 3.142 cm2. The skin was excised from the abdominalregion after sacrificing the animals. The skin was washedwith distilled water and hair was removed by a clipper.The excised skin was mounted between the two half cellsof the Franz diffusion cell in such a way that SC side facedthe donor compartment where as dermis faced the receivercompartment and the apparatus was assembled in positionusing the springs. The donor compartment was kept emptywhile the receiver compartment was filled with a mixtureof phosphate buffer saline pH 7.4 and acetonitrile (ACN)in 4:6 ratio and stirred at 500 rpm. The temperature wasmaintained at 37 ± 0.5°C by placing the diffusion cell inthe organ bath. The ACN-buffer solution was replacedevery half an hour to stabilize the skin which was evidentby recording the UV absorption of the ACN-buffersolution. The zero absorption indicated the completestabilization.[18]
Following the stabilization of the skin, the donorchamber was filled with 1% w/v drug solution with orwithout PE (5% v/v). The sample of 1 ml volume waswithdrawn at different time intervals till 48h and analyzedby UV spectrophotometry at λmax 242 nm after appropriatedilution.[19] The permeation rate (flux) of the drug wascalculated from the slope of the amount of drug permeated(μg/cm2) vs. time (h) plot. The permeability coefficient(cm/h) was calculated as the quotient of flux and drug con-centration in the donor compartment (mg/cm3). The resultsof this preliminary skin permeation study are given inTable 1. The effectiveness of a PE was determinedcomparing the permeation rate of carvedilol in the pres-ence and absence of PE. This was defined as enhancementfactor.
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Differential Scanning Calorimetry (DSC)
The rat abdominal skin was freshly excised and wastreated with 0.0001% (w/v) trypsin overnight at 37°C.The skin was then washed with distilled water and storedat 37°C after evaporation of water.[20] The dried skin wascut into pieces of small dimension and then placed in thevials containing the IPA and IPA-black cumin oil to actas IPA-treated control and PE-treated skin samples,respectively. The samples were kept in the solution for24 h at 37°C. The untreated and treated skin sampleswere dried, cut to the weight of 5 ± 2 mg and then sealedin aluminum hermetic pans and equilibrated for 1 hbefore the DSC (Pyris 6, Perkin Elmer, Germany) wasrun. Scanning rate was 10°C/min over the temperaturerange of 10–300°C.
Fourier Transform Infrared Spectroscopy (FTIR)
Dried skin samples prepared as per method discussedabove were cut into pieces and incubated in the vials for24 h at 37°C with IPA and IPA-black cumin oil. The skinsamples after removal of moisture were investigated forstructural changes by recording its FTIR on Bio-Rad WinIR equipped with a plate with a hole in the center assample device. The sample was scanned from wavenumber 800 cm−1 to 3000 cm−1.[14]
Histology of Rat Skin
The abdominal rat skin samples treated with IPA andIPA-black cumin oil were removed immediately afterscarifying the animals and biopsies were fixed on cardboard to stretch the skin samples, the same were then dippedin 10% v/v buffer formalin. The tissue samples wereembedded in paraffin blocks and vertically sectioned at 5 μmthickness. Sections were stained with hematoxyline-eosinand examined under microscopy.[21,22]
RESULTS AND DISCUSSION
Permeation Profile of Carvedilol Using PEs
The PEs – clove oil, eucalyptus oil, tulsi (basil) oil,oleic acid, Tween 80 and black cumin oil – were studied as5% v/v solution. All the PEs showed increase in theamount of drug permeated. The results are shown inFigure 1. Amongst all PEs explored for carvedilol solu-tion, black cumin oil showed highest enhancement factor(6.40). Transdermal flux and permeability coefficient werealso found to be high (0.0102 ± 0.00005 mg/cm2/h and0.00192 ± 1×10−5 cm/h, respectively) thus indicating itspotential to help the drug to traverse the skin. The presentresearch work also tried to establish the mechanism of blackcumin oil as PE using DSC, FTIR and histological studies.
Thermal Analysis for Study of Lipid Fluidity in SC
The skin barrier function is known to reside in the SC.One of the techniques to study the physicochemicalcharacteristics of the SC barrier is thermoanalysis.[13] The
Table 1 Permeability parameters and enhancement factor for different penetration enhancers
DSC thermograms of IPA-treated control and IPA-blackcumin oil treated skin samples are shown in Figure 2. TheDSC of an untreated SC (Thermogram not shown) showedfour major transitions. The first one at 39.45°C (T1) and isassociated with the melting point of sebaceous lipids. Thesecond and the third transitions appearing at 72.24°C and84.81°C (T2 and T3) are associated with phase transitions ofthe lipid bilayers from lamellar state to the liquid-crystallinestate. The fourth transition occurred at 95.02°C (T4) and isassociated with denaturation of α-keratin.[23,24] Changes ofthe phase-transition enthalpies and temperatures of theintercellular lipids and alterations of α-keratin denaturationpeak indicate an interaction of respective compounds withSC.[25] These changes indicate increase in the fluidity of thelipid bilayer, i.e. an interaction of skin lipids with the PEcausing the alteration in the ordered state of the skin.[26] TheDSC of IPA-treated control sample showed a single peak at97.70°C with an area value of 479.180 mJ and the corre-sponding enthalpy of 95.83 J/g. However, the treatment ofdried SC with black cumin oil revealed that the phase transi-tion temperature shifted to 121.524°C along with a reduc-tion in the peak area, the treated skin showed the peak areavalue of 253.499 mJ. There was also a reduction in theenthalpy value and was found to be 50.700 J/g which isquite a significant change. Moreover, the first three transi-tion peaks are lipid-based and disappeared after extractionwith IPA in both IPA-treated control and IPA- black cuminoil-treated samples. All these considerations show that 5%of black cumin oil has the potential of causing not only thealteration in the skin protein (α-keratin denaturation) but italso caused fluidization of the skin (extraction of lipids)thus creating the passage for the drug to traverse the dermis.
Infrared Spectroscopic Studies of Black Cumin Oil Treated and Untreated SC
The molecular vibrations of the lipids and proteins arerelated to various peaks in the FTIR spectrum of SC. Theabsorption bands at 2923 and 1744 cm−1 are due to pres-ence of esters (lipids) and amides (proteins), respectively.The height and the area of these bands are proportional tothe order of the skin samples. Any extraction of lipidsfrom SC results in decrease in their peak height andarea.[27,28] Ceramides present in SC form hydrogen bondsbetween each other in lipid bilayer, thus causing the splitin peak at 1650 cm−1 (amide I) which was found in modelmembrane prepared from SC lipids by Moore andRerek.[29] A similar split in the peak at 1650 cm−1 regionwas observed in our control sample (Figure 3). FTIR of ratskin after 24 h treatment with a 5% black cumin oilshowed non-significant shifts in wave numbers but signifi-cant changes in the peak heights and area. FTIR revealedthe disappearance of split in peak near 1650 cm−1 regionand appearance of a single peak that indicates breaking ofhydrogen bonds network existing in α-keratin and thus theexpansion of corneocytes of skin by black cumin oilcannot be overlooked.[30] Since the main barrier in thepermeation is the lipid bilayer, this effect may notbe significant, unless the lipid bilayer fluidity increases.The FTIR of the black cumin oil treated skin samplesdisplayed characteristics peaks at 2925 cm−1 (for esters)and 1725 cm−1 (for amides) but the height of the peakwas lesser than the control. It indicated that black cuminoil alters the permeability of skin by loosening the hydro-gen bonds between ceramides because of competitive
Figure 2. Differential Scanning Calorimetry (DSC) of the rat stratum corneum; (b) black cumin oil and (c) control.
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hydrogen bonding. The hydrogen bonds at head ceram-ides break as fatty acids enter into lipid bilayer of SC.[28]
A disruption of SC integrity is also brought about by thelipid extraction by black cumin oil solution. Thus, it isconcluded that 5% black cumin oil increased the perme-ation potential of SC significantly in comparison tocontrol via mechanism of breaking of H-bonds and lipidextraction.
Histological Examination of SC
Sections of control group revealed well defined epi-dermal and dermal layers as shown in photomicrograph.The keratin layer was also well formed and was present justadjacent to the top most layer of the epidermis. Dermis wasdevoid of any inflammatory cells as clearly seen in Figure4a. Skin appendages were also within normal limits. How-ever, sections of the black cumin oil-treated skin biopsiesshowed thinning of the keratin layer in the epidermis. Thefollicular lumen in the epidermis still showed the kerati-nized cells. The other layers of the epidermis were normal.Dermis showed a mild separation of the collagen fibres dueto dermal edema. Interestingly, no inflammatory infiltratewas seen in the dermis (Figure 4b). Black cumin oilbrought about protein modification of the SC. Furthermore,the black cumin oil chemically contains high concentrationof fatty acids which could have opened the dense proteinstructure of the SC making it permeable and increasing thedrug diffusion across the membrane. The black cumin oilmight have also increased the partitioning of the drug intothe horny layer by altering the solubility characteristics ofthe SC. Thus, an increased diffusion is expected due toalterations in the proteins, partitioning of drug and subse-quent fluidization of bilipid layers of the SC. Our findingssuggest that greater extraction of the SC lipids led to
greater permeability of carvedilol. The increase in the per-meability of the drug may be due to increased drugdiffusivity through the partially delipidised SC.[31,32]
CONCLUSIONS
The black cumin oil (5%) showed the greatestenhancement of carvedilol permeability amongst all thetested PEs. The present study identifies the mechanism(s)of in-vitro percutaneous absorption enhancement ofcarvedilol through rat abdominal skin by 5% v/v of blackcumin oil. Enhancement in the permeability of carvedilolby black cumin oil is attributed to increase in lipid extrac-
Figure 3. FTIR of the rat stratum corneum; (b) black cumin oiland (c) control.
Figure 4. (a) High power photomicrograph of section of ratskin from control group showing details of epidermal structure.The keratin layer is well formed and lies just adjacent to thetopmost cellular layer of the epidermis (HE, 400×). (b) Highpower photomicrograph of section of black cumin oil treated ratskin showing details of epidermal structure. The keratin layer isreduced in thickness and separation of collagen fibres by spacesis clearly seen (arrow) (HE, 400×).
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tion and increased drug diffusion through partially delipi-dised SC. Chemical evaluation is underway to documentthe constituents of the black cumin oil using GC-MS andfurther correlate the penetration enhancement effect tospecific constituent(s).
ACKNOWLEDGMENTS
The author (SA) is grateful for financial assistanceprovided by Council for Scientific and Industrial Research(CSIR), India.
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