Toxicology in Vitro 26 (2012) 645–648

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Toxicology in Vitro

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Toxicological implications of the delivery of fentanyl from gel extractedfrom a commercial transdermal reservoir patch

Gabriela Oliveira, Jonathan Hadgraft, Majella E. Lane ⇑Department of Pharmaceutics, School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 January 2012Accepted 26 February 2012Available online 3 March 2012

Keywords:FentanylTransdermal patchReservoirIn vitroHuman skin

0887-2333/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.tiv.2012.02.007

⇑ Corresponding author. Tel.: +44 207 753 5821; faE-mail address: majella.lane@btinternet.com (M.E

Fentanyl in a rate controlling membrane (RCM) transdermal patch form has been available since the early1990s for outpatient management of chronic pain. Fatalities associated with misuse or overuse of fenta-nyl patches have been reported. Concerns have also been raised about the possibility that defects in suchpatches may result in leaking of the reservoir of the patch onto patients’ skin and consequent overdose. Inorder to investigate the possibility of fentanyl toxicity arising from leaking of patches, the permeation offentanyl from the reservoir gel of a commercially available fentanyl transdermal patch was examinedin vitro. Finite doses of the formulation were applied to human skin and permeation was monitored, at32 �C under non-occluded conditions, for 48 h. Similar levels of skin permeation of fentanyl from the1% gel formulation were obtained for the two skin donor samples tested. After 48 h, the dose of fentanylthat had permeated was 7.4 (±3.6)% and 7.7 (±1.9)% of the respective total amounts applied. At the end ofthe experiment, most of the drug was found in the residual formulation at the skin surface (i.e. 63–66%).For both the skin samples, a relatively small amount of the fentanyl applied (2–3%) was present in theskin at 48 h after application. The maximum flux from the data generated was between 6 and 24 h overwhich time frame it was 0.3 lg/cm2/h. Assuming spreading of leaked gel over an area of 100 cm2, thiswould result in a plasma level of 0.6 ng/mL. The anticipated plasma levels from a 100 lg/h patch areknown to be approximately 2.5 ng/ml. Thus, the maximum increase in the plasma levels from a patchwhich leaks gel is calculated to be, at most, about 25%.

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1. Introduction

Fentanyl is a potent opioid narcotic which was first synthesisedby Janssen in 1959 (Andrews and Prys-Roberts, 1983). The drug is al-opioid receptor agonist and is estimated to be 80 times more po-tent than morphine as an analgesic (Mather, 1983). The first pub-lished report of fentanyl skin permeation appeared in the 1970s(Michaels et al., 1975) and by the early late 1980s transdermalpatches were available for clinical evaluation with subsequentcommercial availability in the 1990s. The early patch design con-tained the drug in a reservoir, with a rate-controlling membrane(RCM) moderating the delivery of the drug to the skin surfaceand a separate adhesive to ensure skin contact. Advances in adhe-sive technology subsequently led to the development of drug-in-adhesive matrix patches where the adhesive serves as the carrierfor the drug but also as the skin adhesive.

Toxicity and death from fentanyl intoxication with RCM fentanyltransdermal patches have been reported and have been associated

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x: +44 870 165 9275.. Lane).

with inappropriate use of the patches (Edinboro et al., 1997) oroveruse (Reeves and Ginifer, 2002; Lilleng et al., 2004). Specific con-cerns relating to defects in the RCM patch device and leakage of thegel from the reservoir onto intact skin have prompted product re-calls in 2004 and in 2008. Although leaking patches may result ina spreading of the gel on the skin, the question of how this processmay affect transdermal delivery has not been explored to date.

Recently we have designed and evaluated the in vitro skin per-meation of a range of liquid fentanyl formulations (Santos et al.,2011). After 24 h, the maximum cumulative flux we observed forfinite doses of supersaturated systems was of the order of0.05 lmol cm�2 (16.9 lg cm�2), representing �20% of the applieddose. The aim of the present study was to determine the in vitroskin flux of fentanyl from the reservoir constituents of a RCMfentanyl patch and to compare it with our earlier formulations.In addition, knowledge of the permeation characteristics of fenta-nyl from the reservoir material should facilitate our understandingof how the gel itself influences fentanyl permeation as distinctfrom the intact patch. Hence, possible toxic events may bepredicted.

646 G. Oliveira et al. / Toxicology in Vitro 26 (2012) 645–648

2. Materials and methods

2.1. Materials

Fentanyl Transdermal System 100 mcg/h patches were obtainedfrom Watson Pharma, USA. Phosphate buffered saline tablets(Dulbecco A, pH 7.3 ± 0.2 at 25 �C) tablets for the receptor phasewere obtained from Oxoid Ltd. (UK) For the HPLC analysis, 1-hep-tanesulfonate (sodium salt) monohydrate (for ion pairing chroma-tography, P99.0%, Aldrich) was supplied by Sigma Aldrich, UK andperchloric acid (70% v/v, AnalaR�, BDH) was supplied by VWR, UK.Polyoxyethylene 20 oleyl ether (Brij98, Aldrich) was supplied bySigma Aldrich, UK. All other materials were of analytical gradeand obtained from Fisher Scientific UK, unless otherwise stated.

2.2. Methods

2.2.1. Skin preparationFull thickness abdominal skin samples were obtained from two

Caucasian female donors following cosmetic surgery, in accordancewith NHS Research Ethics Committee approval and informed pa-tient consent (REC 11/LO/0389). The tissue was stored in a freezerat �20 �C until required and the preparation of human epidermalskin membranes for permeation studies was carried out by theheat separation method (Kligman and Christophers, 1963) whichhas been reported previously (Dias et al., 2007; Watkinson et al.,2009).

2.2.2. In vitro permeation studies using human skinSkin permeation studies were conducted using static glass

Franz diffusion cells, placed in a temperature controlled water bathat 34 (±1) �C. The temperature of the skin surface was 32 (±1) �C.The diffusional area (approx. 1 cm2) was accurately measured foreach Franz cell. Non-occluded, finite dose conditions were selectedto closely mimic the clinical situation. The heat separated epider-mal membranes were thawed and cut to appropriate size usingscissors. The skin was placed in the Franz cells on the filter papersupport to confer extra mechanical strength and to help maintainskin integrity during the experiment. Phosphate buffered salinewith 0.002% sodium azide (PBS) was used as the receptor solution(pH = 7.4). The receptor solution was degassed by high speed stir-ring under vaccum in a Nuova II Stirrer connected to a vacuumpump for �30 min prior to use in the permeation studies. TheFranz cells were assembled using vacuum grease (Dow Corning�,supplied by VWR, UK) and a metallic clamp to create a leak proofseal between donor and receptor compartments. A known volumeof the receptor solution was added to each Franz cell (�4 mL) andthe skin was allowed to equilibrate with the receptor solution forat least 1 h before starting the experiment. Skin barrier integritywas assessed prior to the beginning of the experiment by measur-ing skin impedance. Sink conditions were maintained throughoutthe duration of the experiment.

Fig. 1. Cumulative amount of fentanyl permeated from the gel formulation (finitedose, non-occluded donor) through the skin of donor 1 (n = 6) and donor 2 (n = 7) at32 �C. Mean ± SD.

2.2.3. Application of the gel formulationThe gel containing fentanyl was squeezed out into hermetically

sealed amber flasks to facilitate application of finite doses at theskin surface. Finite doses of each of the gel formulations weregently, rapidly and evenly spread at the surface of the skin usinga custom made glass spreader. The target dose was 10 mg/cm2

and the actual dose of the gel applied was determined gravimetri-cally for each individual Franz cell (Sartorius balance, ±0.0001 gaccuracy). The actual weights of the applied formulations were12.1 ± 2.9 mg/cm2 for skin donor 1 (n = 6) and 9.4 ± 1.5 mg/cm2

(n = 7) for skin donor 2.

2.2.4. Analysis of permeation samplesThe contents of the receptor phase were continuously stirred

throughout the experiment with a small Teflon coated magneticflea. Receptor phase samples (1 mL) were taken at 2, 4, 24, 33and 48 h, following application of the gel formulation. Receptorphase samples were also taken before starting the experiment(t = 0) to check for drug contamination in the receptor phase andanalytical interference from material leaching from the skin. Sam-pling occurred at the designated time points with volume replace-ment using fresh receptor solution. The permeation samples wereanalysed for fentanyl content using HPLC with UV detection.

2.2.5. Mass balance studiesAt the end of the permeation studies, the surface of the skin was

washed with 1 mL of a 6% (w/v) solution of polyoxyethylene 20oleyl ether and gently rubbed with a soft cotton bud to ensurecomplete removal of the residual gel formulation from the foldsand furrows of the stratum corneum surface. This procedure was re-peated consecutively six times, after which the cotton buds wereextracted with 1 mL of methanol. After washing the skin surface,the amount of fentanyl remaining in the skin was extracted fourtimes using 1 mL of methanol each time. All samples were centri-fuged for 5 min at 13.2(�1000) rpm/16.1(�1000) rcf (Eppendorfcentrifuge model 5425R) prior analysis. The mass balance protocolwas validated for both skin surface wash and skin surface washand skin extraction procedures.

2.2.6. HPLC–UV quantification method details and validationSkin permeation samples were analysed using a 1100 Series

Hewlett Packard HPLC system, equipped with a diode array detec-tor, and data were acquired and analysed using ChemStation for LC3D software by Agilent Technologies, UK. The HPLC column, mobilephase and flow rate as well as detection wavelength details were asreported previously (Santos et al., 2011). The HPLC quantificationmethod was assessed for selectivity of sample analysis and wasalso validated for accuracy, precision and linearity over the quan-tification range of 0.05–10 lg/mL The method also showed goodinjection reproducibility (CV < 5%; n = 5) and accuracy. The esti-mated limit of detection (LOD) for fentanyl using this quantifica-tion method was 0.02 lg/mL.

2.3. Data analysis

The skin permeation of fentanyl was evaluated by plotting thecumulative amount permeated per unit surface area of the

Table 1Cumulative amount of fentanyl permeated through skin donors 1 (n = 6) and 2 (n = 7),including mass balance recovery values.

Time(h)

Skin donor 1 Skin donor 2

Mean SD RSD% Mean SD RSD%

Dose applied (lg/cm2) 121.2 28.5 23.5 93.9 14.8 15.7Cumulative amount permeated

(lg/cm2)0 0.0 0.0 0.0 0.02 0.2 0.3 182.2 0.0 0.1 264.66 0.9 0.8 94.1 0.4 0.3 72.1

24 5.8 1.9 32.2 5.5 1.6 28.833 7.1 2.1 30.0 6.5 1.9 28.748 8.4 2.5 29.7 7.3 2.2 29.9

Amount recovered at skin surface(lg/cm2)

80.0 22.5 28.2 58.8 12.1 20.6

Amount recovered from the skin(lg/cm2)

2.7 0.7 24.5 2.5 1.4 56.9

Total amount recovered (lg/cm2) 91.1 22.4 24.6 68.6 13.8 20.1

Table 2Amount of fentanyl permeated through skin donors 1 (n = 6) and 2 (n = 7), includingthe mass balance recovery values. The data are presented in terms of percentage ofthe dose applied.

Time(h)

Skin donor 1 Skin donor 2

Mean SD RSD% Mean SD RSD%

% Dose applied whichpermeated

0 0.0 0.0 0.0 0.02 0.1 0.2 160.0 0.0 0.1 264.66 0.8 0.8 105.3 0.5 0.3 73.8

24 5.1 2.6 50.0 5.9 1.5 25.833 6.3 3.1 48.8 6.9 1.7 24.248 7.4 3.6 48.8 7.7 1.9 24.1

Recovery at skinsurface (%)

66.1 10.4 15.7 62.7 9.0 14.3

Recovery from theskin (%)

2.3 0.7 30.3 2.6 1.4 55.5

Total Recovery (%) 75.8 11.8 15.5 73.0 9.6 13.2

G. Oliveira et al. / Toxicology in Vitro 26 (2012) 645–648 647

membrane (in lg/cm2), as well as the percent of the dose applied(% dose applied), against the collection time in hours.

3. Results

The exact dose of fentanyl applied was 121.2 ± 28.5 lg/cm2 forskin donor 1 (n = 6) and 93.9 ± 14.8 lg/cm2 for skin donor 2 (n = 7).The skin permeation of fentanyl was evaluated by plotting thecumulative amount of fentanyl permeated per unit surface area

Fig. 2. Amount of fentanyl permeated from the gel formulation (finite dose, non-occluded donor) through the skin of donor 1 (n = 6) and donor 2 (n = 7) at 32 �C,represented in terms of the percentage of the dose of fentanyl applied permeatedover time. Mean ± SD.

of the skin (lg/cm2) against collection time in hours, and is shownin Fig. 1.

Table 1 shows the amount of fentanyl permeated at each timepoint from the 1% gel formulation for skin donors 1 and 2, and alsoshows the recoveries obtained following the mass balance study.

The same data, expressed as a percentage of the original doseapplied to the skin, are shown in Table 2.

4. Conclusions

The skin permeation of fentanyl obtained from the 1% gel for-mulation was very similar for the two skin donors, with 7.4(±3.6)% and 7.7 (±1.9)% of the dose applied permeating after 48 hfor skin donors 1 and 2, respectively. Figs. 1 and 2 show a charac-teristic sigmoidal profile as expected for a finite dose. However theprojected plateau is significantly lower than 100%, this is in accor-dance with the large amount of fentanyl being found on the skinsurface. For both skin donors, most of the applied drug remainedin the residual phase of the formulation (i.e. skin surface) at theend of the experiment (i.e. 63–66%). A comparatively smalleramount of fentanyl (2–3% of the dose applied) was extracted fromthe skin at the end of the experiment. Interestingly, the cumulativepercentage permeated is approximately half the percentage ob-served for the supersaturated systems we have previously evalu-ated (Santos et al., 2011).

Using the in vitro data it is possible to predict what will happenif a patch is cut and deliberately spread onto the skin surface. If it isnot deliberately spread the area over which the gel will spread willnot be large as the gel is viscous. To estimate the largest possibleeffect, gel from a 100 lg/h patch will be considered. Also it willbe assumed that a fresh patch is damaged to give the maximumpossible body burden

Before these considerations it is also important to note thatwhen the patch is damaged ethanol will evaporate and fentanylremaining in the patch will crystallise and become unavailablefor delivery. The patch on the skin will stop delivering at the samerate; plasma levels will start to drop. Under normal conditionsRCM patches do not stop delivering fentanyl because the active de-pletes, it is because ethanol in the patch is exhausted and the fen-tanyl can no longer be held in solution.

Published information (Medical Officer Review, 1990) showthat the amount of gel that can be squeezed out of the patch is24%. Since the patch contains 1 mL of gel, this equates to 240 llgel. The maximum area over which 1 ml can be spread is400 cm2 (Medical Officer Review, 1990). Therefore, the maximumarea over which the gel that is squeezed from the patch is approx-imately 100 cm2. The maximum flux from the data generatedabove is between 6 and 24 h over which time frame it is 0.3 lg/cm2/h. The Medical Officer Review also provides the pharmacoki-netics of fentanyl (Cl = 46.3 l/h and Vss = 398 l). It is therefore pos-sible to calculate the steady state plasma levels of fentanyl thatwould result from gel delivering fentanyl at 100 � 0.3 lg/cm2/h(30 lg/h). It is 30/46.3 ng/ml (0.6 ng/ml).

Immediately after the patch is cut, the plasma levels will dropas the patch fails to deliver. There will be a lag phase over whichfentanyl from the spread gel will diffuse across the skin. Afterabout 6 h the fentanyl plasma levels will rise and there will be agradual increase in the plasma levels by 0.6 ng/ml over the base-line (the dropping levels from the fentanyl extant in the plasma).After a 24 h period these too will begin to drop.

It is also possible to interpolate the data in Fig. 1 to estimate theapparent dose applied (i.e. the total amount that will permeate).This can be achieved by fitting the data to an appropriate finitedose solution to Fick’s second law of diffusion (Anissimov andRoberts, 2001). This may be done in Scientist� (Micromath Ltd.)

648 G. Oliveira et al. / Toxicology in Vitro 26 (2012) 645–648

which uses a Laplace Transform procedure. The apparent dose forthe two donors (i.e. the interpolated plateau levels) are 9.2 ± 1.9and 9.7 ± 3.4 lg/cm2. The data analysis also provides values ofD/l2 from which the conventional lag time (l2/6D) can be estimated,where D represents the drug diffusion coefficient and l representsthe pathlength for diffusion. The lag times for the two donors are10.7 and 9.5 h. These values are consistent with the known perme-ation of fentanyl.

The impact on the plasma levels can be estimated. The antici-pated levels from a 100 lg/h patch are 2.5 ng/ml (Duragesic�

Patient information leaflet, 2003). Therefore if these levels do notdrop over the first 6 h, and if the gel starts to deliver at its maxi-mum rate immediately on spreading, the increase in the plasmalevels can, at most, be about 25%. This is the worst-case scenario.However whilst undesirable, it is therefore highly unlikely thatadverse effects will be seen as a result of fentanyl gel contacting in-tact skin after cutting a fentanyl patch.

5. Conflict of interest statement

JH acts as a consultant for Watson Pharma.

References

Andrews, C.J.H., Prys-Roberts, C., 1983. Fentanyl – A review. Clin. Anaesthesiol. 1,97–122.

Anissimov, Y.G., Roberts, M.S., 2001. Diffusion modeling of percutaneous absorptionkinetics: 2. Finite vehicle volume and solvent deposited solids. J. Pharm. Sci. 90,504–520.

Dias, M., Hadgraft, J., Lane, M.E., 2007. Influence of membrane-solvent-soluteinteractions on solute permeation in skin. Int. J. Pharm. 340 (1-2), 65–70.

Duragesic� patient information leaflet. Janssen, May 2003.Edinboro, L.E., Poklis, A., Trautman, D., Lowry, S., Backer, R., Harvey, C.M., 1997. Fatal

fentanyl intoxication following excessive transdermal application. J. ForensicSci. 42, 741–743.

Kligman, A.M., Christophers, E., 1963. Preparation of isolated sheets of humanstratum corneum. Arch. Dermatol. 88 (1963), 702–705.

Lilleng, P.K., Mehlum, L.I., Bachs, L., Morild, I., 2004. Deaths after intravenous misuseof transdermal fentanyl. J. Forensic Sci. 49, 1364–1366.

Mather, L.E., 1983. Clinical pharmacokinetics of fentanyl and its newer derivatives8, 422–446.

Medical Officer Review, 1990. Volume 2, Pharmacokinetics & Pharmacodynamics.Alza Corporation, pp. 1–92.

Michaels, A.S., Chandrasekaran, S.K., Shaw, J.E., 1975. Drug permeation throughhuman skin: theory and in vitro experimental measurement. AIChE J. 21 (1975),985–996.

Reeves, M.D., Ginifer, C.J., 2002. Fatal intravenous misuse of transdermal fentanyl.Med. J. Aust. 177, 552–553.

Santos, P., Watkinson, A.C., Hadgraft, J., Lane, M.E., 2011. Formulation issuesassociated with transdermal fentanyl delivery. Int. J. Pharm. 416 (2011), 155–159.

Watkinson, R.M., Herkenne, C., Guy, R.H., Hadgraft, J., Oliveira, G., Lane, M.E., 2009.Influence of ethanol on the solubility, ionization and permeation characteristicsof ibuprofen in silicone and human skin. Skin Pharmacol Physiol. 22 (1), 15–21.