`
`ty of Topically Administered Steroids: A
`Bioavailabili
`‘Mass Balance” Technique
`
`Daniel A.W. Bucks, M.A., James R. McMaster, M.A., Howard I. Maibach, M.D., and Richard H. Guy, Ph.D.
`Departments of Pharmacy, Pharmaceutical Chemistry, and Dermatology, University of California, San Francisco, San Francisco,
`California, U.S.A.
`
`The percutaneous absorption of four steroids (hydrocorti-
`sone, estradiol,
`testosterone, and progesterone) has been
`measured in vivo in man under occluded and “protected”
`(Le, covered, but non-occlusive) conditions. The experi-
`mental approach, involving simple modificationsof standard
`radiochemical methodology, has enabled excellent “mass
`balance” and dose accountability to be achieved. Conse-
`quently,theutility of the procedurefor the measurement of
`in vivo topical bioavailability can be inferred. In addition,
`because of
`the precision and accountability ofthe results, the
`technique offers a potential means to establish quantitative
`structure-penetration relationships for skin absorption in
`man. It was found that steroid absorption increased with
`increasinglipophilicity up to a point, but that penetration of
`progesterone(the most hydophobic analog studied) did not
`continue the trend and wasatleast partly rate-limited by slow
`
`interfacial transport at the stratum corneum-viable epidermis
`boundary. Comparison of data obtained from the occluded
`and “protected” experiments permitted the effect of occlu-
`sion (defined as the complete impairmentofpassive transepi-
`dermal waterloss at the application site) to be assessed. Oc-
`clusion significantly increased percutaneous absorption of
`estradiol, testosterone, and progesterone but did noteffect
`the penetration of hydrocortisone. A mechanism is proposed
`to explain whythe absorption of the morelipophilic steroids
`is enhanced by occlusion but that of the most water-soluble
`(i.e., hydrocortisone)is not. It is suggested that the rate-de-
`terminingrole of the sequential steps involved in percutane-
`ous absorption can be revealed by experiments of the type
`described using related series of homologous or analogous
`chemicals. J Invest Dermatol 90:29-33, 1988
`
`n the development of a dosage form intended for topical
`administration on the skin,an essential step is to determine
`the percutaneous absorption of the drug. Of the various al-
`ternatives available for the assessment of skin penetration,
`there is little doubt that an in vivo measurement in manis
`most appropriate and desirable [1]. However, in vivo percutaneous
`absorption experiments in man are much more difficult to perform
`than either animal modelorin vitro penetration studies. Further-
`more, mostofthe in vivo investigations which have beencarried out
`have notallowed accountability of the applied dose and, hence, have
`not
`producedresults which can be interpreted unequivocally.
`The majority of humanin vivo percutaneousabsorption measure-
`ments have used indirect radiochemical methods [2-6]. Typically, a
`144C labeled chemical is applied topically from a volatile solvent
`vehicle and penetration is evaluated
`from the excretion of the C
`radiolabel over the next 5-10 d. Correction for incomplete elimi-
`nation is madebyperforminganidentical protocolafter intravenous
`or intramuscular administration of the same '“C labeled material.
`Theapproachhas someclearlimitations: any conclusionsare based
`on euliclahel data, not specific information about the parent com-
`pound andits metabolites; the elimination profile after topical and
`parenteral dosing must be assumedidentical;thefate of that fraction
`of the topical dose which is not absorbed immediately into the skin
`post-application is not controlled so that the meaning of“dose” in
`this situation is usually poorly defined.
`
`
`Manuscript received September10, 1987; accepted for publication Febru-
`ary 5, 1988.
`Reprint requests to: Dr, Richard H. Guy, School of Pharmacy, Box 0446,
`UCSF,San Francisco, CA 94143
`
`In this paper, simple modifications of the conventional in vivo
`experimentare described and the improvementin resulting data
`quality is illustrated for four steriods: progesterone, testosterone,
`estradiol, and hydrocortisone. The procedures involve i) covering
`the applicationsite for the entire duration of the study, ii) washing
`the dosed skin surface at the end ofthe dosing period,andiii) on
`occasion, when monitoring ofurinary excretionis terminated, tape-
`stripping
`the upperlayer of stratum corneum. The key improve-
`ment afforded by these changesis that the radiolabeled dose can be
`totally accountedfor,i.e., mass balanceis possible. The approach has
`beenapplied to both single and multiple-dosing regimens and mea-
`surements have been made under both occlusive and non-occlusive
`(“protected”) covering conditions. The results obtained demon-
`strate that the technique may havesignificant potential for estab-
`lishing quantitative structure-penetration relationships for skin ab-
`sorption in man, and revealing quantitatively the effects of
`occlusion on the transport of compoundsacross the cutaneous bar-
`rier.
`
`MATERIALS AND METHODS
`
`The penetrants considered were four steroids: progesterone, testos-
`terone, estradiol, and hydrocortisone. The C-labeled chemicals
`(RPI Corp., Mount Prospect, IL) were applied in acetone to the
`ventral forearm of healthy male volunteers (n > 5), from whom
`informed consent, approved by the UCSF Committee on Human
`Research, had beenpreviously obtained. Chemicaland radioactivity
`doses were 4 g/cm? and1 4Ci/cm?,respectively; the area ofappli-
`cation was 2.5 cm? and the dose was administered in 20 pl of ace-
`tone.
`After evaporation of the vehicle (<0.5 min), the applicationsite
`was coveredwith a semirigid,
`polypropylene Hilltop* (Hilltop Re-
`search,Inc., Cincinatti, OH) chamber (HTC), which was affixed to
`
`0022-202X/88/$03.50 Copyright © 1988 by The Society for Investigative Dermatology, Inc.
`
`29
`
`  
`
`

 
`
`MYLAN- EXHIBIT 1022
`
`

`

`30
`
`BUCKS ET AL
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`the skin with hypoallergenic adhesive tape. The cotton pads, with
`whichthe chambers are supplied, were removedpriorto application
`on the subjects’ forearms. In the occluded studies, intact chambers
`were employed; for the penetration experiments under “‘protected”
`conditions, the chambers were “ventilated” by boring several small
`holes through the plastic (such that about 50% of the surface area
`was exposed). To preventloss of surface material (squames, undis-
`solved penetrant,etc.), the roof of the chamber was covered with a
`piece of Gore-Tex® (W.L. Gore & Associates, Inc., Elkton, MD)
`membrane(0.2 um poresize). It was found that Gore-Tex®did not
`impede transepidermal water loss to any significant extent and
`hence the objective of dosing site protection without occlusivity
`wasachieved (Bucksetal, in press).
`The subjects collected their urine for 7 d, post-steroid applica-
`tion, according to the schedule: 0-4, 4-8, 8-12, and 12-24-h;
`day 2, 3, 4, 5, 6, and 7. Urine volumeswere determined gravimetri-
`cally for each time period, and duplicate 3-ml samples were ana-
`lyzed for radioactivity. '*C-Toluene wasadded,as an internalstan-
`dard, to a third 3-ml sample to determine quenching. The percent
`“dose”? (as total radioactivity) excreted was determined for each
`time interval. At 24 hafter dosing, the chamber (or chamber +
`Gore-Tex") was removed,placed in scintillation fluid, and seques-
`tered '*C was counted. An appropriate quench correction was again
`made. The application site was washed with a standardized proce-
`dure [7] using 5 cotton balls consecutively soaked in soap solution
`(Ivory Liquid Soap, Proctor and Gamble Co., Cincinatti, OH; di-
`luted 1:1 with water), water, soap solution, water, and water. All
`washings were collected and were processed forliquid scintillation
`counting to assay for residual surface chemical. For the remaining
`6 d of the urine collection period, the administration site was again
`covered with a (new) chamber. Finally, this chamber wasalso as-
`sayed for '*C-chemical;also,at this time, in the “protected” experi-
`ments, stratum corneum at the site of application was stripped 10
`times with adhesive tape (Scotch Cellophane Tape*, 3M,St. Paul,
`MN)andthe skin strips were analyzed for residual radioactivity
`(once more, corrected accordingly forscintillation quenching).
`
`A parallel protocol was also performed following a multiple-dos-
`ing regimen [8] for testosterone, estradiol, and hydrocortisone
`under occluded conditions. The compounds were applied every
`24 h for 14 d at a dose of 4 zg/cm? to the sameskin site. Thefirst
`and eighth applicationsutilized '*C-labeled drug and urinary excre-
`tion for 7 d (using the collection schedule described above) after
`eachof these doses was followed. In these studies, the 24-h washing
`procedure was performed daily (prior to that day’s dosing) and a
`new chamberwas provided on each occasion.
`Partition coefhcients of the penetrants between isopropyl myris-
`tate and water, and tetradecane and water, were determined using 4
`standard technique [9]. Octanol-waterpartition coefficients were
`obtained fromthe literature [10].
`RESULTS
`
`Data fromthesingle dose experiments performed underocclusive
`conditions are presented in Table I and should be compared to the
`correspondingresults from the “protected” studies given in Table
`Il. Total recoveries are, in general, high and weregreater for the
`“protected” measurements. These experiments were performed
`after the occludedinvestigation and incorporatedobligatory evalua-
`tions of i)
`'C-radiolabeled sequestered on the second HTC,ii)
`chemical in the second set of washings, and iii) material remaining
`in the upperlayers of the stratum corneum at the end of 7 d. This
`more thorough determination of penetrantdisposition probably ac-
`counts for the improved mass balance in the “protected” studies.
`Thepercentage dose absorbed columns in Tables I and II show the
`effect of occlusion on the topicalbioavailability of the four steroids.
`With the exception of hydrocortisone, unpaired t-tests show that
`occlusion significantly increases the
`percutaneous absorption (p
`<0.01) of these compoundsin man. Thisfindingis further empha-
`sized in Fig 1, which shows,for each ofthe foursteroids, the rate of
`excretion of radiolabel following their topical application under
`both occluded and “protected”conditions. To optimizeclarity, data
`for estradiol, testosterone, and progesteroneare plotted semi-loga-
`rithmically because of the itheree in absorption between oc-
`
`Table I. Disposition of Topically Applied *C-labeled Steroids Following a Single Dose under Occluded Conditions
`Percentage of Applied Dose*
`
`
`
` Steroid Absorbed? 1st HTC* Ist Wash4 2nd HTC" 2nd Wash! Total
`
`
`
`
`
`
`
`
`
`68 + 3.9
`n.d.t
`nde
`36 + 3.0
`28 + 5.6
`4.0 + 2.4
`Hydrocortisone
`87+ 13
`n.d.é
`0.5403
`1847.2
`41+10
`27+ 6.4
`Estradiol
`90 + 8.4
`n.dt
`0.3+0.2
`3.0 + 4.1
`41+8.4
`46+ 15
`Testosterone
`80 + 5.5
`
`46+ 10
`1.2+0.8
`07 + .02
`01 + 0.0
`Progesterone
`33+ 8.9
`* Mean + standard deviation (n = 5, except for progesterone, for which n = 6).
`* Values corrected for incomplete renal elimination [3].
`© Material sequestered on Hilltop chamber (HTC) removed at 24 h post-dosing.
`“ Chemical found in combined washings performed 24 h post-dosing.
`* Material sequestered on HTC removed at end of measurement period.
`§ Chemical found in combined washings performedat end of experiment.
`© n.d.: not determined.
`
`
`Table II. Disposition of Topically Applied '*C-labeled Steroids Following a Single Dose Under“Protected” Conditions
`Percentage of Applied Dose*
`
`
`
`
` 7 Steroid Absorbed? ist HTC* Ist Wash? 2nd HTC* 2nd Wash SC “strips"® Total
`
`
`
`Hydrocortisone
`4441.7
`271i
`51+ 18
`Se 17
`2.72 1.3
`25 1
`89 + 5.6
`Estradiol
`3.44 1.2
`38+ 13
`58 + 12
`0.7+0.4
`0.3+ 0.4
`O1+01
`100 + 0.9
`Testosterone
`18 + 8.6
`46+7.5
`30+ 15
`1.4+0.4
`0.1 + .08
`n.d.
`96 + 2.0
`
`Progesterone
`13+6.3
`5447.7
`27+ 8.7
`1.2 + 0.6
`0.3 + 0.4
`n.d.)
`96+3.4
`
`* Mean + standard deviation (n = 6).
`* Values corrected for incomplete renal elimination [3].
`* Material sequestered on HTC + Gore-Tex® removed at 24 h post-dosing.
`4 Chemical found in combined washings performed 24 h post-dosing.
`* Material sequestered on HTC + Gore-Tex® removed at end of measurementperiod.
`Chemical found in combined washings performed at end of experiment.
`* 4C-radiolabel present in 10 tape strippings of stratum corneum (SC) removedafter final washing procedure.
`* n.d: not determined.
`
`

`

`VOL. 91, NO.
`
`1
`
`JULY 1988
`
`STEROID BIOAVAILABILITY 31
`
`HYDROCORTISONE ABSORPTION
`
`ESTRADIOL ABSORPTION
`
`Table IV. Oil-Water Partition Coefhcients of Steroids Studied
`
`
`
` Steroid log Kopw" log Kip lon Kyypw*
`
`
`
`
`
`@DOSE/HOUR o
`a SARaDaneani
`
`20
`
`40
`
`60
`
`80
`
`100 120 140 160
`
`0
`
`20
`
`40
`
`60
`
`#80
`
`100 120 140 160
`
`TIME (HAS.)
`TESTOSTERONE ABSORPTION
`
`TIME (HRS.)
`PROGESTERONE ABSORPTION
`
`"DOSE/HOUR
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100 120 140 160
`
`0
`
`20
`
`40
`
`60
`
`«80
`
`100
`
`120 140 160
`
`TIME (HRS }
`
`TIME (HRS.)
`
`Figure 1. Urinary excretion rates (mean % dose per hour) as a function of
`time following topical application of four steroids under occluded (open
`square) and “protected” (filled square) conditions. A, hydrocortisone; B, es-
`tradiol; C, testosterone; D, progesterone.
`
`cluded and “protected” measurements; for hydrocortisone, on the
`other hand, a linear graphis presented and the occluded and “‘pro-
`tected” results essentially superimpose.
`The multiple-dose measurements, which were performed under
`occlusion, are summarized in Table III. Again, total recoveries of
`applied radioactivity were good. An analysis of variance showed
`that for each ofthe steroids there was no significant difference (p
`>0.05): a) in the percentage dose absorbed dermally between the
`first and eighth doses under occlusion and b) between the multidose
`absorption figures and the percentage dose absorbed following a
`single dose under occluded conditions (Tables I and II) [with the
`ssible exception ofestradiol for which marginally significant dif-
`eer = 0.04) in percutaneous absorption between thefirst
`and eighth doses of the multidose regimen and between thefirst
`dose of the multiple application study and the single acute dose
`study were found].
`Finally, in Table IV,partition coefficientsof the steroids between
`each ofthreeoil phases (octanol, isopropyl myristate, tetradecane)
`and waterare reported.
`
`Hydrocortisone
`Estradiol
`Testosterone
`Progesterone
`
`1.61
`2.49
`3.32
`3.87
`
`—0.19 + 0.02
`2.33 + 0.04
`1.98 + 0.002
`2.62 + 0.005
`
`—2.17 + 0.03
`—0.027 + 0.003
`0.68 + 0.02
`2.27 £0.11
`
`* Kopw = Octanol-waterpartition coefficient [9,10].
`* Kiyw = Isopropyl myristate-water partition coefficient (mean + standard devia-
`tion; n = 6).
`© Kypw = Tetradecane-water partition coefficient
`n= 6),
`
`(mean + standard deviation;
`
`DISCUSSION
`
`The experiments reported in this paper highlight threeissues: 1) the
`accountability of the applied chemical dose and the potentialutility
`of the technique for measurement oftopical bioavailability; 2) the
`effect of occlusion onthe in vivo skin permeation ofsteroids; and 3)
`the relationship between percutaneousabsorptionandtherelative
`lipophilicity afche penetrant.
`The massbalances achieved in this work are generally high and
`often approach 100%. A conventional
`in vivo approach [2-6]
`would
`have only revealed the percent dose absorbed columns in
`Tables I-III. Disposition ofthe remainderof the applied radioactiv-
`ity would remain unknown. The importance ofrepeating the wash-
`ing procedures and chamberanalysis at the end of the 7-d experi-
`mentalperiodis indicated in the improved accountabilities observed
`in the “protected” (Table II) and multiple-dosing (Table III) stud-
`ies. Further support for this contention has recently been observed
`in our laboratory for a series of para-substituted phenols [11], for
`which, again,essentially complete mass balance has been recorded.
`It
`is
`pertinent to note that in Table II hydrocortisone, the least
`lipophilic steroid, is significantly measurable in the stratumcor-
`neumat 7-d post-dosing. The amountrecoveredis clearly relevant
`whenconsidered in relation to the level of percutaneous absorption.
`Thepersistence of hydrocortisone in the stratum corneumfor this
`prolonged period suggests chemical-tissue interaction of appreci-
`able strength. Althoughthe nature ofthis “binding” phenomenon
`is not revealed by these experiments, the effect clearly goes beyond
`simple depotbehavior. This hypothesis is reinforced by thefact that
`the more lipophilic estradiol is barely detectable in the stratum
`corneum at the end of the experiment (Table Il). In addition, the
`recentinvestigation [12] using para-substituted phenolic penetrants
`has revealed the samepattern: phenols with more polar para-substit-
`uents (¢.g., ~NH,, ~NHCOCH;, —NHCOC;Hs) show pro-
`longed stratum corneum residence, whereas more lipophilic ana-
`logs (p-CN, p-I) do not.
`InFig 2, the percentage dose absorbed for each steroidis plotted as
`a function of penetrant octanol/waterpartition coefficient; results
`obtained under occluded and “renacredl* conditions are compared.
`
`Table II. Disposition of Topically Applied C-labeled Steroids Following Multiple Dose Under Occluded Conditions
`Percentage of Applied Dose*
`Steroid
`Dose
`Absorbed*
`Ist HTC!
`1st Wash*
`2nd HTC’
`2nd Washes
`
`Hydrocortisone
`1st
`3.5:c& 1.3
`2347.7
`53411
`3.5+1.4
`2.6+0.8
`8th
`3.1 + 1.0
`32+ 5.4
`33+ 7.5
`7T4A+OS
`4.84 1.7
`Ist
`387.9
`47+ 12
`14+ 6.8
`0.6 + 0.8
`0.5+ 0.6
`8th
`22+7.1
`37 £ 9.9
`2125.2
`0.4 + 0,2
`0.5 + 0.2
`Ist
`51+ 10
`4649.1
`1.71.0
`0.2+0.1
`.06 + .06
`8th
`50+ 9.5
`379.7
`43+5.4
`0.2+0.2
`.06 + .04
`
`
`Total
`
`85+4.3
`81+2.5
`100 + 3.9
`812+ 6.0
`99+4.3
`92+17
`
`Estradiol
`
`‘Testosterone
`
`
`* Mean + standard deviation (n = 5, except for hydrocortisone 8th dose, for which n = 4),
`+ The Ist and 8th doses of a daily dosing regimen, lasting 14 d, were “C-radiolabeled,
`© Values corrected for incomplete renal elimination [3].
`4 Material sequestered on HTC removed at 24 h post-dosing.
`* Chemical found in combined washings performed 24 h post-dosing.
`£'4C-labeled material sequestered on HTC removed at 48 h post-dosing.
`® “C-labeled chemical found in combined washings performed at 48 h post-dosing.
`
`

`

`32
`
`BUCKS ET AL
`
`THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
`
`Absorbed
`
`%Dose
`
`log Ko/w
`
`Figure 2. Percutaneous absorption offour steroids (mean % dose absorbed)
`as a function of octanol/water
`partition coefficient (K,,/) under occluded
`(open square) and “protected”
`(filled square) conditions.
`
`With the exception of hydrocortisone, unpaired t-tests reveal that
`thereis significantly (p <0.01) more penetrant absorbed under oc-
`clusion than under protected conditions. Althoughit is generally
`accepted dogmathatocclusion increases percutaneous absorption,
`quantificationoftheeffectin vivois scant
`(13,14].It is also believed,
`on the whole, that occlusion increases transdermal penetration for
`all compounds,butthat,in particular, more water-soluble materials
`will exhibit greatest enhancement. However,ourresults show that
`the least lipophilic steroid, hydrocortisone, appears unaffected by
`occlusion. This observation also contradicts an earlier study [15]
`which showeda clear promotion of absorption for hydrocortisone
`whentheapplication site was occluded with thin plastic film (Saran
`Wrap). However,in this previous experiment, the skin site was not
`washeduntil 4 d post-dosing, during which time the occlusive pro-
`tection remained continuouslyin place. Thereis, in addition, some
`evidenceto suggest that continuedfrictional contact combined with
`skin flexing produces a “rubbing”effect which maycause an eleva-
`tion in percutaneous absorption [16,17]. While the plastic film re-
`mains in direct contact with the skin surface, the HTC does not.
`The occlusion-induced enhancementin absorption seenfortheli-
`pophilic steroids may be understoodby a consideration of the steps
`involved in percutaneous penetration. Following application, the
`chemical musti) diffuse from the skin surface through the stratum
`corneum,1i) partition from the stratum corneum into the much
`more aqueous in nature viable epidermis, iii) diffuse through the
`epidermis and upper dermis, and iv) encounter the cutaneous mi-
`crovasculature and gain access to the systemic pool. Occlusion leads
`to hydration of the stratum corneum and must, therefore, exertits
`effect(s) on one or both of the first two steps. If hydration simply
`decreased the viscosity of the stratum corneum transport pathway
`(now believed to involve the intercellular lipid-filled channels
`[18,19]), then the penetration ofall chemicals should be equally
`enhancedbyocclusion. An alternative possibility is that the stratum
`corneum-viable epidermis partitioning stepis altered. Hydration of
`the stratum corneum will reduce the effective partition coefficient
`ofthe penetrant between the stratum corneum andviable epidermis
`(because the twotissue phases now appear moresimilar). Theeffect
`ofthis decrease will be to increase the kinetics of transfer of pene-
`trant from stratum corneum to viable epidermis, a change that
`should becomeprogressively more apparentas the lipophilicity of
`the absorbing molecule increases (20).
`The importanceofthe partitioning step discussed aboveis further
`implied by the dependence of percutaneous absorption on steroid
`lipophilicity (Fig 2, Table IV). Penetration does not continue to
`increase with increasing lipophilicity. This attenuation in absorp-
`tion implies a shift in the rate-determining step from stratum cor-
`neumdiffusion to transfer across the stratum corneum-viable epi-
`dermis interface,
`a process which should become slower
`as
`penetrantlipophilicity increases. Once more, results with a series of
`
`para-substituted phenols are comparable [11]. The possibility that
`the parabolic form ofpercutaneous absorption versuslog K is caused
`by decreased surface availability as a result of increased association
`between the penetrant and the HTC has been considered. We be-
`lieve that this explanation is not valid for two reasons: First, the
`dependency of HTC-recovered dose on penetrantlipophilicity 's
`weak.Second,literature data for the absorption ofthe four steroids
`under openapplication, i.e., non-protected, conditions [3] show 4
`similar trend: frydmenetacie 1.9 + 1.6%;estradiol, 10.6 + 4.9%:
`testosterone, 13.2 + 3.0%; progesterone, 10.8 + 5.8%, In this case,
`no consistently available alsorcare surface was accessible to the
`applied compounds. Interestingly, only the result for estradiol in
`chk earlier study is significantly different from the corresponding
`absorption values in Table II (“‘protected” conditions).
`In summary, this paper presents evolving improvements in iM
`vivo percutaneous absorption methodology. The approach is com-
`lementary to the recently described experiments of Rougieret al
`21-26]. The results demonstrate mass alanis and dose account
`ability, a meansto studytheeffects ofocclusion on skin penetration,
`and, in the long term, the potential to define chemical structure-
`percutaneous absorption relationships in man.
`
`This research was supported by grants from the National Institutes of Health
`(GM-33395 and HD-23010) to RHG, whois the recipient ofa Special Emphasis
`Research Career Award (KO1-0H00017) from CDC/NIOSH. Wethank the
`Dermatopharmacy group at UCSF for helpful discussions and input, Allen R.
`Guizzetti for supplying the Gore-Tex® membrane, and Andrea Mazel for manw-
`script preparation.
`
`REFERENCES
`
`2.
`
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`4. Feldmann RJ, Maibach HI: Absorption of some organic compounds
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`STEROID BIOAVAILABILITY 33
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