SHARAN 2ND JOURNALCLUB

8/9/2019 SHARAN 2ND JOURNALCLUB

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JOURNAL CLUBTRANSDERMAL IONTOPHORETIC DELIVERY OF

TERBINAFINE HYDROCHLORIDE:QUANTITATION

OF DRUG LEVELS IN STRATUM CORNEUM ANDUNDERLYING SKIN

Vishal Sachdeva, Srujana Siddoju, Yi-Ying Yu, Hyun D. Kim, Phillip M.Friden, Ajay K. Banga

BYSHARAN KUMAR REDDY B

M.Pharm I.P (IIsem)SPIPS

VIDHYANAGAR, HANAMKONDA.

SOURCE:INTERNATIONAL JOURNAL OFPHARMACEUTICS 388(2010 )


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IntroductionTerbinane (TBF) is a synthetic allylamineantifungal agent used for the treatment of supercialfungal infections of the skin and nail.The stratum corneum is the primary site of action for terbinane in supercial cutaneous fungal infections.In order to treat fungal infections effectively, thedrug must be present at the site of action at aconcentration above the minimum inhibitoryconcentrations (M IC) during the entire treatment

period.


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CONTD.Oral administration has been shown to be associatedwith drug-drug interactions (inhibition of CYP2D6,an important phase I metabolizing enzyme),hepatotoxicity, gastrointestinal and systemic sideeffects, lactose intolerance and other adverse effects.An improved topical drug delivery approach could

overcome these limitations as it provides immediateaccess to the site of infection and reduces unwantedsystemic drug exposure.However, a major limitation of topical delivery for

skin infections is poor bioavailability .


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CONTD.Results have shown a bioavailability of less than 5%in humans even after a weeks treatment period.One of the more powerful enhancement techniques isiontophoresis, as it is the only one that provides a

physical driving force to move drugs through biological membranes.This technique involves the use of small amounts of

physiologically acceptable electric current (in the Arange) to drive charged or neutral drug molecules

across the skin or nail.


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CONTD..Currently,oral and topical terbinane formulations areindicated only for supercial cutaneous fungal infections(dermatomycosis).Its use in the treatment of deep seated skin fungalinfections (subcutaneous / cutaneous mycoses) isconsidered ineffective, most likely due to its inability to

deliver adequate drug levels into the deeper epidermis or dermis layers of the skin where the infection exists.Hence, it would be benecial to investigate whether

higher drug levels in deeper skin layers could be attained

with the use of iontophoresis.


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CONTD.In this study,iontophoresis was used to deliver terbinanehydrochloride into and across hairless rat skin in vitro .

Subsequently, drug levels were determined in the stratum corneum(using tape stripping method), the underlying skin (using skinextraction method) and the receptor compartment.Studies to identify the rate limiting barrier for the penetration of terbinane into the skin and predominant process driving the drugduring iontophoresis were performed.The effect of current density, duration of current application and

the presence or absence of formulation during the post-iontophoretic period were also investigated.


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MaterialsTerbinane hydrochlorideSodium BorateHexaneFormic acidAcetonitrile (HPLC grade)Ammonium acetateSilver wire (0.5mm diameter) and silver chlorideMale hairless rats, 810 weeks old weighing 350 400 g


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Skin isolation and preparationAbdominal skin was freshly excised and prepared for use in each in vitro study.

Rats were euthanized by CO 2 asphyxiation and gentlylaid in the area prepared for surgery.Following skin isolation, subcutaneous fat (if present)was carefully removed.

The skin was then cleaned using de-ionized water andcut into appropriate size.These skin pieces were then mounted on the receptor compartment of the vertical Franz diffusion cells.


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Preparation of electrodesA planar coil of silver wire was prepared manuallyand used as anode.

The cathode was custom made by coating a melt of silver chloride on a ne silver wire.The coating procedure was continued until a uniformand sufcient coat of silver chloride was obtained.The electrodes were freshly prepared on the day of the experiment.


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Permeation experiments

Vertical Franz diffusion cells.Receptor compartment : receptor buffer (5ml, consisting of 10% ethanol,30% propylene glycol, and 10mM sodiumchloride in de-ionized water, pH 5.8).Temperature: 37CTerbinane hydrochloride (4%, w/w) formulation (0.5ml, pH3.48) was then added to the donor chamber as donor solution.

The anode (silver wire coil) was placed in the donor chamber and the cathode (custom made silver chloride electrode) wasinserted into the receptor compartment through the samplingarm to perform anodal iontophoresis.Samples obtained were protected from light and air untilanalyzed using HPLC.


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Tape stripped skin studies

In order to determine the primary barrier to the penetration of terbinane into the skin, both passive permeation and anodal iontophoresis

(0.4mA/cm2)were performed for 1h across intact(with stratum corneum) and tape stripped (withoutstratum corneum) hairless rat skin.Trans-epidermal water loss (TEWL) measurementswere recorded using a closed chamber evaporimeter asan indirect measure to ensure complete removal of stratum corneum by the tape stripping process.Approximately 15 tape strips were able to remove theentire stratum corneum, as evidenced by an increase in

TEWL values to 810 times the base value (obtained before tape stripping).


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CONTDFollowing 1h permeation study, skin sampleswere cleaned of excess formulation, removed

from the Franz cells and the drug wasextracted.The amount of drug delivered into theunderlying skin in the presence and absenceof the stratum corneum was compared for

both passive and iontophoretic groups.


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Effect of current density and duration

of current applicationThe inuence of current density on the amount of drugdelivered into the stratumcorneum and the underlying skinwas investigated by conducting anodal iontophoresis for 1h atdifferent current density values of 0.2, 0.3 and 0.4mA/cm 2.For the duration of current application study, anodaliontophoresis was performed for 15, 30, 45 and 60 min at acurrent density of 0.3mA/cm 2.

Tape stripping and skin extraction was performedimmediately after the study to determine the amount of drugdelivered into the stratum corneum and underlying skin,respectively.


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Post-iontophoretic delivery studies

To study drug permeation following iontophoretic delivery, 1 h

anodal iontophoresis at 0.4 mA/cm2

was followed by 23 h of passive permeation (post-iontophoretic period).In order to investigate whether the presence of the drugformulation has an impact on permeation during the post-treatment period, studies were performed with (formulation not

removed, FNR or formulation partially removed; FPR) or without (formulation completely removed; FCR) theformulation in the donor compartment.At the end of 24 h ( 1 h treatment + 23 h post-treatment period),tape stripping and skin extraction studies were performed .


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Tape strippingThe treated area on each skin sample (0.64 cm 2) wastape stripped 15 times using 3 M Transpore tape .The rst two tape strips were discarded to avoidoverestimation of the amount of terbinane in thestratum corneum due to excess supercial formulation

at the skin surface.Extraction of drug from the strips was performed atroom temperature for 34 h while shaking on a roller shaker. Samples were directly analyzed using HPLC.


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Skin extractionThe area of the tape stripped skin exposed toformulation was punch biopsied and cut into small

pieces using a scalpel blade. These skin pieces were

transferred to tubes containing 1 ml of 5 M NaOHand incubated for 2 h at 60C.Following incubation, the mixture was cooled toroom temperature and neutralized using 100 l HCl(5N).Borate buffer (pH 10, 1.5 ml) was added and thetubes were vortexed. Hexane (6 ml) was added toeach tube and the drug was extracted by shaking on aroller mixer for 60min.

The contents were then centrifuged for 10 min at8000 g .


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CONTDThe upper organic layer containing extracted drug was separated and freshhexane (6 ml) was added to the original tubes to extract the remaining drugfrom the aqueous layer using the above procedure.The organic layer isolated previously was completely evaporated usingnitrogen ushing to concentrate the drug.Following the second extraction, the tubes were centrifuged and placed in a-80C freezer to freeze the aqueous layer and obtain the entire organiclayer.This organic layer was reconstituted into the tubes containing drugconcentrate obtained earlier and vortexed. A solution (1.5 ml) of sulfuric

acid (0.5 M) and isopropyl alcohol (85:15) was added to these tubes to back extract the drug from the organic layer by shaking for 30 min.The tubes were centrifuged at 8000 g for 10 min and the upper organiclayer was completely evaporated. 50l of the bottom layer was injected intothe HPLC column for analysis, following ltration through a 0.22m lter.


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Recovery studiesIn order to determine the extraction efciency, 50l of standardsolutions having a known amount of drug were injected

supercially into hairless rat skin samples (mean weight 250mg) and allowed to equilibrate for 3 h.Four different amounts (5, 25, 125 and 250g) were injectedinto the skin pieces with three replicates for each amount.Following equilibration, the drug was extracted from the skin.

The actual amount extracted was determined using standardcurve (0.5500 g).The extraction efciency was found to be 75.0%. This valuewas taken into consideration while calculating the actualamount of drug present in the unknown skin samples.


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Quantitative analysisTerbinane hydrochloride was quantied by high-

performance liquid chromatography using an Alliancesystem (Waters Corp., MA, USA) with a photodiode

array detector at 233 nm.Isocratic elution was performed using RP-18Phenomenex column .The mobile phase was acetonitrile, water and 100 mMammonium formate, adjusted to pH 3.75 using formicacid (60:30:10, v/v).At a ow rate of 1.5 ml/min and 30C columntemperature, the retention time was ~6 min.The run time employed was 13 min and the injection

volume was 50l.


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Amount of drug delivered to different skin layers andreceptor compartment during permeation studies


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ConclusionAnodal iontophoresis enhanced the delivery of terbinane hydrochloride, a water soluble form of ahydrophobic drug, into the skin. Rapid delivery to thetarget site was achieved with the use of this technique.Furthermore, iontophoretic delivery of terbinane intothe soft skin tissues, such as those surrounding thenails, could be advantageous in ensuring continuousdrug levels at the site of infection, which couldtranslate into less frequent administrations and/or reduced chance for recurrence.


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ReferencesAlba, N., Naik, A., Guy, R.H., Kalia, Y.N., 2006. Iontophoresis: clinical applications andfuture challenges. In: Smith, E.W., Maibach, H. I. (Eds.), Percutaneous Penetration Enhancers,2nd ed. Taylor & Francis Group, pp. 177219.Banga, A.K., 1998. Electrically Assisted Transdermal and Topical Drug Delivery. Taylor &Francis, Bristol, PA.Denouel, J., Keller, H.P., Schaub, P., Delaborde, C., Humbert, H., 1995. Determination of terbinane and its desmethyl metabolite in human plasma b y high-performance liquidchromatography. J. Chromatogr. B: Biomed. Appl. 663, 353359.Morimoto, Y., Hatanaka, T., Sugibayashi, K., Omiya, H., 1992. Prediction of skin permeabilityof drugs: comparison of human and hairless rat skin. J. Pharm. Pharmacol. 44, 634639.Paturi, J., Kim, H.D., Chakraborty, B., Friden, P.M., Banga, A.K., 2009. Transdermal andintradermal iontophoretic delivery of dexamethasone sodium phosphate: quantication of thedrug localized in skin. J. Drug Target..

Schafer-Korting, M., Schoellmann, C., Korting, H.C., 2008. Fungicidal activity plus reservoir effect allow short treatment courses with terbinane in tinea pedis. Skin Pharmacol. Physiol.21, 203210.Volpato, N.M., Nicoli, S., Laureri, C., Colombo, P., Santi, P., 1998. In vitro acyclovir distribution in human skin layers after transdermal iontophoresis. J. Control. Release 50, 291 296.


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SHARAN 2ND JOURNALCLUB