Iontophoretic Delivery of ALA Provides a Quantitative Model for ALA Phartnacokinetics and PpIX Phototoxicity in Hutnan Skin

Lesley E. Rhodes, Maria M. Tsoukas, R. Rox Anderson, and Nikiforos Kollias Wellman Laborato ri es of PhotolTl cdicine, Massachuscrts General Hospital. Harvard Uni versity, Bos ton. Mass:1Chllsetts. U.S.A.

Photodynamic therapy (PDT) with topical 5-aminole­vulinic acid (ALA) is increasingly employed for skin cancer, yet ALA dosing is crude. Using iontophoresis, we developed a rapid and quantifiable system for topical ALA delivery, with measurement of subse­quent PpIX fluorescence and phototoxicity. ALA was iontophoresed fron1 a 2°1c, solution into upper inner arm skin of 13 healthy volunteers. Six doses of ALA were delivered with a series of charges varying frOl1"1 3-120 milliCoulombs (mC); four additional doses were given with a charge of 60 mC. Five hours post-iontophoresis, sites were irradiated with broad­band yellow-red light, the series of six ALA doses r eceiving 100 ]lcm2

, while the four identical doses received 6.25, 12.5, 25, and 50 ]lcm2

, respectively. Resultant erythel11a was measured by reflectance spectroscopy. The tinle course of PpIX fluorescence w as ALA-dose-dependent. With charge $; 24 mC,

To pi cal appli cation of 5-amino levulini c ac id (ALA) is al.1 in cre asin gly popu lar m ethod o f ph otosensitizatio n in the photodynaJ11i c th e rapy (PDT ) o f skin tumo rs (Kenned y ci ai, 199 0: Kennedy and Po ttier. '1992: W o lf ('I ai , 1993; C airndutF ci al . 1994: Svanberg ('I al .

1994 ; Lan g el (/1. 1995) . ALA is a prec urso r of th e endogen o us flu o rescent photosensitizer protopo rphyrin IX (PpIX) . pro du ced as th e p e nultimate step in he m e biosynthesis. Applicati o n o f excessive am o un ts of ALA enables bypass o f a rate-limi tin g step. i,e., fee dba ck inhibi t ion b y he m e o n ALA syn thase . resulting in acc u­mulatio n of PpIX . Activati o n o f th e sensitizer by v isibl e lig ht gen e rates single t o xygen. whi ch cau ses se lecti ve damage of cell s containin g th e ph o tosensitizer (Weishaupt ('I ai, 197(, ; M o an and Somme r, '1985) . ALA-induced PplX a 'cumula tes pred o minantl y ill th e epidermi s and skin appenda ges fo ll o wing bo th topi cal and sys te mic appli catio n (D ivaris el al. 1990: G ofF ('I al. · 1992 : Szeimi es el ai, 1994). The mu ch hi gher leve ls fo und in no n-me lan o ma skin can cers th an in normal skin arc attributed to i.nGre:lsed ALA uptake thro ug h abno rmally permeable stratum corne um . al tho ugh tum o r

M anuscript rece ived Ma y 13. 'I 096 ; rc vi ~cd Aug u st" 15. 1996: acce p ted fo r publica tion August 29, 199(, .

R.cprin t requests to : Dr. Les ley E. R.hodes. Dennato lo!;y Uni t. Uni versity of Liverpool. 1'. 0 . Uo~ 147. Live rpool L69 3UX. U K.

Abbreviations: ALA. 5-amin olcvulinic acid: PpI X. prowporph yrin IX: PDT, photodynamic therapy.

PpIX fluorescence peaked a t 3 h and returne d t o zero at 9-10 h, whereas charges> 24 I11C had a su st a ine d peak at 5-10 h, falling to zero by 24 h. Pre-ir r a dia tion , PpIX fluorescence correlated with ALA dose (r = 1.0). PpIX fluorescence fe ll imm ediat ely post-irra dia tion (p < 0.0001); recovery l evels at 3 h correl a ted with ALA dose (p < 0.0001). Delayed eryth e ma corre lated with ALA dose and irradiation dose (p < 0.0001, p < 0.01, respective ly). Both PpIX fluoresce nce in t e nsity pre-irradiation and fall in PpIX fluorescence pos t­irradiation correlated with e rythe ma (r = 0 . 98). Hence, PpIX synthe sis is A LA-close-dependent, and phototoxicity can be predicted frol11 ALA dose , irra­diation dose, and photo bleaching o f PpIX . This r e ­producible system allows a ccurate dosiI11etry in top­ical PDT and fa c ilitates st udy of ALA m etabolism. KCJI I/Jovds: iOlltoplroJ'csislpIJotolJlcac/rillg 1 plrotodyrrarrric arcmpJ', ] [livest D emrat(li 108:87-91 J 1997

ce ll s ma y a lso show higher prod uction o f ALA-induced po rp hyrins

(Iinum<l 1'1 a/. 1994). T opi ca l A LA-induced PplX is m o re con venient th an other

sensitizers and rouces o f applica ti o n in PDT beca use th e p hotosen­sitivity pro du ced is ne ither sys tem ic n or p ro lo n ged. Prev io us stu dies o f P DT with topi ca l ALA in no rm,ll and abn o rmal skin have usua lly in volved applica ti o n of e x.cess ALA in crea m bases. T h erefore . th e precise re latio nship between ALA dose and the resul tan t Au o rescel1t and ph o totoxic efFects has not been esta bli shed . M o reover . because ALA is a hig hly polar m o lecule. ALA cn!ams m uSf be applied fo r ho urs bdol'e su fti cie nt amo llnts arc absorbed. Hence . it has been diffi cult to study the kin etics of Ppl X indu ctio n du e to th e de lay in curred in th e pe ne tration of ALA in to epidermis.

T h e w ate r-soluble and io nic nature of ALA ma kes it am en able to io n to pho res is. T hi s technique in vo lves the active d e li very of sm all charged m o lecules in to th e skin b y rhe app lica tio n o f :m electri ca l current. The positi ve o r negative d ru g ions ac t as charge ca rrie rs ac ross the hig h- impedance stratum corneum, such that the (iru g is rapidl y de livered to th e epidermis. T h e m e th od is pa ill less an d lI o llill vasive . and co nsistent qu antities of drug arc de livered . All elec tri cal po tential by itself docs not in crease skin pen neab ili ty (Ga ngarosa 1'1 Il l. 1980). Successful de li vcry of wa ter- so lubl e and io nized dru gs has been described (Ty le 1986) . and atte m prs have been m ade to trea t a w ide ran ge of derm ato logic con ditio ns (Sloa n and So ltani . 198(,). It has been shown tha r ALA ca n he iontopho re­sed ac ross 111 0 ll se skin ;11 ,,;/ re> (G reen ('I al. 1,)<) I) . b u t th ere arc n o repo rted srudi es us in g th is m ethod to r de li very of ALA in PDT.

(1022-202X/97 / S I(l.50 • COI'Y "i g l ll' '' ~ 1997 b y T he Society fi.)r In vestigative Dermatology. Illc.

87

88 RH ODES ET / IL

A linear re lationsh ip cx i.sts between levels of PplX Auorescen ce and its con centrations in so lu tion (Le vecki s el ai, 1994) a nd in tissue (Loh ci ai, 1993). Spectroscopic measure m e nt of PplX fluorescen ce in th e sk in was th e re fo re use d in this study as ~ simple, nonin vasive m eth o d of d emon stratin g the ph o(Qsen sit izer (Pottie r cI ai, 1986; Svanherg cI ai, 1994). H e re we report ALA appli ca tion by ionto­ph o resis in human skin, whi ch d efI n es a quantita tive system for ALA delive ry. A series of increasing ALA doses were app li ed to n o rmal skin w ithi n seconds to minutes, follo wed by measurement of PplX Auorescence. Photosensitization b y PplX was assessed quantitative ly b y measuring the e rythem :1 1 respon se and con 'e1ated w ith ALA dose, irr;ldi ation dose, and photoblea c hing of PpIX.

MATER.I ALS AN I M ETHODS

Subjects and Study Design Approva l was granced by the Etllics ConH11ittcc of Massachusetts Ge neral H ospita l. and illfo rnlCd con sen t was obtained. Thirtee n he<11th y vo lunteers were stud ied (median age 37, ran ge 24 -4 5 y, 1() male).

5-A lllinolcvlllinic acid hydrochlO1'ide (5-ALA-HCL. DUSA I'hanllaccu­tical s. Inc .. T"rryroWll , NY) WH, made into a 2'1., so lu t.ion with steri!c de io lli zed water. This waS iontophorescd into healthy skin of the upper inJ1 e r ann fronl a dru g deli very charnbc[ (Moor JIl st rUI11 CJlts , AX.I11 ins tcr.

Devon, UK) using a battery-powered cOlltrollahle direct curre llt source (Phorese r II system, lO ME!) , Salt Lake C ity, Uca h). The chamber had a circLll ar cenrral delivery aperture of 1 elll diameter where ALA solution was htold in 'direct contact with skjn, and e lectri cal current was uni formly di$t.r ibu tcd over the arc a. The chamber "vas cO llll ected t.o the anode, and all indilTerent clectrode was placed 6 inches fi'om the chamber . A currenI' ofU.2 rn A was used tll1'oughout the study. The dose o r ALA administered was va ri ed hy the charge d ·livered; the time of iontopho resis was adj usted as fo llo ws: charge (milli oulombs. mC) = current (mA) X time (seconds) . A series of six ALA doses WilS applied hy illcreil sing the time of iontophoresis fi'om 15 s to 10 min, i.e., with charge 3-12lJ me. Sices were washed innll cd i:ltd y after iontopho res is in order to remO ve rf.!s idual A LA soluti o n . All cxperi,nents elllployed the follow ing comrois: iOlltopho resis of diluent (water) alone; reversa l of iOll top ho res is polarity ; app li cation of A LA and c hanlbcr to th e sk in w ith o ut iOllto ph o res is.

T he time course o f PplX flu o resce nce fo llowillg ALA iOllto phoresis was first dctcnnined in four subj c<.:ts usin g; flu o rescence spectroscopy (sec below). A sel;"s of f1 v" charges fi'om 6-120 mC were ndllli ni stered . Flu orescence was m C:lSlIr"d at 1- 1.5 ho url y interval s in the first 12 h, and at 2 4 h. In th e second part of the study, '10 subj ects received light cx posures to sices of A LA iontoph o res is. A se ries of six charges in creasin g fronl 3-'120 mC was iOll topho rescd, :lIld four additiollal sites received 60 mC each . Contro ls were illcll.ldcd as abo ve , together with control sites of iontophnre­sed ALA alone alld light exposure alone. T he light SOllrCl! wilS a high­press llre 5U-w sodiulll lalll p (peak wave le ll gth 5H9 11111 . range 550-800 1I1l1). This was p os iti o ned 6 in c hes fro m the skin . g ivin f{ :l lInifornl irradian cc at' the sk ill surl';lCe of 85 tIl W/C I1I 2 , as lII easured with a spcctroradio l11ete r (llItcrnatiol\a l Light , New buryport, MA). All , ites Wl'I'e irradiated simulta­neollsly at S h following ionto phoresis. T he sites of the sc ri es of six doses of ALA received all irradiation dose of 100 jlC 111

2, whi le the four sites of

identical ALA doses (60 ll1e) rccei""d the geoll1etric sc ries 0[6.25 .1 2.5 . 25, and 50 ) / cn,> . re spectively. Flnoresccncc was measured pre-irradiatio ll , im11lcdi;)tcly post-in·a tliation. and ;lI: :> and 24 h post-irradiation . Eryt.hema was assc:,;scd vi~ LJ:lll y (grildc 0 (0 ++ + ) inunc.diate1 y post- exposure, and <"n 3 ;r"d 24 h, alld qU <l lltilied by rcflect'"lee spectroscopy.

Fluorescence Spectroscopy A Au o rcscc ll cc spectrolll e ter :lI1d associ­ated softw:rre were p rovided by Spex Industries, Inc. (Edison, NJ) . 11..:rdia­tioll frol11 a Xe ll OIl arC lam p W;lS foclised into an exc ita ri o n Hloll0chrol11ator co upled to n bifurcated f1b croptic cable. enablillg conduct;o ll of c"citnrion radiati on to the skill surface and co llection of fluorcscence radiation . which w as directed into th e c.!nrnlll CC sli t: of :III Cilli ssio ll lll o n OChnJ IlI<ltor. PplX fluorescen ce s.p ectra ,"vC rc d e tected with all exc itatio n wave le n gth of 4 I II 1'1111 and ellli ssion sc"n of 450 - 80(1 rim. revea ling the ch:U-;l cte ri sti c 636- and 704-11)11 pe:lk5. Comparison with Ppl X standard (Si~lI1a C hemica ls, Sr Louis, M O) is shown (Fig 1). PplX fluorescellce was quall tiflCd in arbitrary 1I11if;:o; frOJl1 th e ;lrc01 undcr thc CI11iss io l1 c urve._

Reflectance Spectroscopy Erythema was quantified wit.h a rcliectance instrulll en t (Dcl'l1 latospectrol1'lete r, COrtex Technology, H"dsllnd, Dcn­I11 nrk) . which mca sLlrcs the red and gre,," light rel1 ectcd by th" skin. A t'eadollt, the "crythcllla index," rc];ltcs to the :1I110 1lJlt of blood ill the upper derl1li s (D ifl'cy l'/ "I . I ()H4). The erythema index of adjncent 1IIlirradi:lI:ed COlltrol sk in wa. subtr:lc tc d fro)"n that of the tes t s ites to CO lllPCIl S:ltc for

T H E JOURN /\L OF INVESTIGATIVE \)EJl. .. M /\TO LOGY

100

Figure 1. F1uore~cencc ~

elnission spectra of porphy- · 80 .£

rin obt"ined from skin (--) · u .0 cotnpared with PpIX stan- ~ · u dard (- - -). The fluorescen ce · 40 a spcctrUlll III sk in was ohtained ~ 6 h tollowill~ ionto pho resis of · 20

ALA with a charge of 24 rnC. '~ .. Excira riol1 wavclen gth was 41 II 0: 0

5S0 .00 650 700 nnl.

750 800

Wavelength loml

lntcr-st1l~)ect difFe rc IH':c s in baseline ~ kit\ co lo r. T ripli cn tc readin gs \Vcn.~

tnken and mean difference in erythema index calculated.

Statistical AnaJysis Diflc re ll ccs bc twce ll the IIl ca fi S W<.! I"C' assessed by ana1 ysis o f vari~ncc .

R ESU LTS

B oth PplX Auorescen ce and photo toxi c ity following irradiatioll were 1 0c~l i zed to the 1-cm diameter applj catio ll s ite~. fontophore sis ca u sed e ither n o sen sation or a mild pric king sen sation. N o erythema occurred followin g ion tophoresis of ALA or dilue nr. Followin g irradiati o n , ,lI1ill1l1ledi,lte whea l and Aare reac tion was seen in som e cases . A ll subj ects sho,"" cd an initial re action of erythe ma with e dema. T he edema sett led within hours, leaving an eryth ema that persisted for several days . Th is was fo ll owed by a variable d egree of pigm entation, w ith a mOre intense resp onse at th e higher ALA d oses. No blister fo rlll 'ltion o r skin necrosis was o bserved.

Time Course of PpIX Fluorescence Is Dependent on the ALA Dose No bac kg ro und PplX Auo rescence wa s d etectable. In two individual s aV il il a ble for frequ ent read in gs, PplX Auorescen ce was d etected as ea rl y as 15 min fo llowing io ntopho res is. The maximum PplX Auorescen ce was d epende n t o n ALA dose, with hjgher and later peak in te n sity at highe r ALA dme.~ (p < 0.00 1). PrIX fluores ce n ce after charges of 5 24 m C pe;d<ed ca rly ,m d brie fl y , at 3 h, and (ell to zero by 9 -10 h (Fig 2). T h e hig her d oses

o f > 24 mC sh owed a la ter and m o re su stained pea k be tween 5-1 0 h and fe ll to zero b y 2 4 h. The initial rate o f increase of PplX fluorescen ce wa s proportional to the c harge of ALA, suggesting thac even th e highest ALA doses in th is study did nor full y sn turate the e n zym e steps leading to Ppl X synthesis. No Auorescen ce was d e te cted at th e cOlltl'01 s ites of ALA applic ation with o ut ionto­phoresis, but a sm a ll am o unt of fluorescen ce «1 U'!!" of that of the test sites) occurred w h ere ALA was applied by reve rse c l/l'rent.

Both PpIX Fluorescence and the Erythen1<ll Response to PDT Are Dependent on the Dose of ALA

PplX FI JJore.m:IICI' Is Rl'lalerilo ALA Dos/' PplX flu o rescen ce at 5 h post-ionto phores is, i. e .. before irr;ldiation in the 10 subjects recei v­ing PDT, i.ncrease d lincarl y with log'lri rhm of ALA dose (r == J .D. j) < 0'(1001; Fig 3) .

R ccOIN:ry o[ PpfX FJIIV},(,SCl'IICC n.lin J>I/OI(J"/('nclrill<~ by Lt<:11I £.'-1105///'(, Is

Related 10 ALII D osc Immediate ly following exposure to 100 J lig ht p e r C111

2, Ppl X Huoresccll ce fe ll to n c ar zero. demonstra tin g

tha t thi s exposu re dose wa s suffi c ient to complete ly ble ac h the photose n sitizer (p < 0.0001; Fig 4). Thre e hours later, recovery of PplX Hu o rescen ce occurred du e to continued synthesis of PplX at

doses of ALA delive red w ith > 3 m C, and recovery levels co rrelated with ALA dose (I' < (UlO()l; Fig 4). T h e rate of re-synthesis of

PplX post-expoHlrc was an average of 54'!!" of the initial rate.

Er)' i// cl//ni R cspoll sc 10 PDT Drpl'IIds 011 t/,(' Dos/' (~r A LA Io n to­phores is of ALA o r dilucnt res ulted in no in crea se ill the eryth ema index. Fo ll owing J 00 ) irradi ation pe r c m ", rhe mean iOlHophoretic c harge o f ALA that resulted in ;ll11i/1 il11 al e ry the mal response wa s 17 (SD 17) 1l1C. The diffe re n ce ill e ry the ma index betwee n that of each tes t site :ll1d contro l sk in agreed we ll with the g rade of

VO L. IIiH. NO. I .I /\ NUAR Y 1')')7

2.5

() ~ 2.0 < II () 1.5 r:: II () .. II .... 1.0 0

~ X 0 .5 a. a..

0.0 ~ 0 3 6 9 12 24

Hours

2.5

()

// ~ 2.0 < II () 1.5 r:: II / () .. II

0 1.0 ::J :;:

X 0 .5

~ Q.. a..

0 .0 0 3 6 9 12 24

Hours

Figure 2. The early rate of PplX synthesis and the hei ght and t iming of peak leve ls reached depend 011 ALA dose. Five doses of ALA wc re gi ve n b y in cre asil1 g the c harge of iOlltopho res is ( •. 6 !l1e: • . 12 mC: *. 24 mC: •. 60 mC: • . 12 1) mC). and ALA-induced " p i X syllthcsis was quantified by Auorescence spectroscopy (arbitrary uni ts). Daw fi'oll1 two indiyiduais arc showll .

e r ythem a determilled visually and in creased w ith inc reasill g A LA d ose ( I' = 0.99 , I' < (J . n()!) I : Fig 5 ). A c lose correlatio ll wa s o bse rved between inten sity of Ppl X flu o rescell ce pre-irrad iatioll and the difference ill erythema in dex pos t- irrad iati o ll (I' = 0.97).

Both Photoblcaching ofPpIX and thc Erythcmal Responsc to PDT Are Dcpendent on Light Exposure Dose

Fn ll ill Ppl X r:I1I(lr('sl'I' IICC Dl'jJl'llds Oil 1111' ['-,."dinli"l1 Dose No signi f­icant diffe rence was o bserved in the pre-irradiati o n Ppl X fluOl'es­cen ce at th e fo ur sites g iven 60 m C ALA. Immediately post­irradiation, Ppl X flu o rescence fe ll ill rel ;l t ion to lig h t dose (I' < 0.000 1: Fig 6) , but th e hi g hest dose was in slI fl'ic ie ll t to e illse co mplete photobleaching. Pp lX flll o rescen ce showed e viden ce of recovery at 3 h post-exposure.

2.0

U 3 •• S

u c . u • 0

0

x O.S

.f 0.0

10 .00

ALA dose (mel

1000

Figure 3. PplX fluorescence is strongly correlated with ALA dose. Ppl X l"luorcscc ll cC \vas ql1amified by spectroscopy 5 It t{) I­

lowi Jlg iolltopiloresis of ALA usiJlg six charge., (3- 120 mC) . n = 111 . lIIeaJl ::t SEM are pl oned; I' = I .n .

ALA I ()NTL) I'H O I (E~ I S ANI) I'IIOTOT OX IC EFFECTS 89

() ::::> < 1.5

CII 0 r:: CII 0 1.0 II> CII L.

0 :::l

;;::

X 0.5

a. a..

3mC 6mC 12mC 24mC 60mC 120mC

ALA dose

Figure 4. PplX fluorescence falls to minimal levels inllnediatcly post- irradiation but shows recovery at 3 h. Six doses of ALA \\'" re iOJl top ilorescd wirlt charges of3- 120 mC. aJld after 3 It all sires rccci\'cd '100 J irrad iatio n per cn1 1

. Levels ofPpl X Auorcscc ll cc a n : sh oW Tl p rc-irr;ld ia ri o n (cI/JI' 11 (11 11111111"') , inlll ll!diardy post-irradi ation (solid [ (1/f/11 1I1S). a11d :\ h posr­irradiatioJl (rl'flss-ha/rhcd rollllllll s). JI = 1 n. means :!: SEM arc ploned.

Eryllll' lIInl /?. l'SI"l/' .'" 10 PDT DC[I/'llds 011 Ihl' [ rradilll i,," D"SI' T he l1I e:l n ex posure dose tha t produ cecl a m in im al e rythema fo ll o,wing a GO-m C charge de li ve rin g ALA wa s 13.6 (Sf) 13) J / cm - . T he erythema l m easuremen ts ag reed we ll w ith visu all y assessed e ry­thema. The differen ce in erythem a ind ex fi'om con tro l skin co rre­lated with exposure dose (I' = 0.98, I' < n.n I).

Photobleaching of PpIX Predicts the Phototoxic Effect T he absolu te dro p in PplX (iu o rescence d ue to p horobl eachin g dll rin g li gh t exposure was p lotted against the m easured e rythemal respo nse "for th e co mbill ed dat;1 of th e series of six ALA closes given l Oll J li ght per cm 2 and the to ur iden tica l ALA doses g i\' en 6.25-50

J li ght per C III ~ (Fig 7) . A high corre latio n was fO lllld (I' = 0.98).

D ISCUSS ION

We h ave developecl a quan tifi ab le and reproducibl e syste m for topica l ALA de li ve ry to the sk in . Prec ise dosin g o f ALA res ulted in predic tab le levcls of skin PplX fluorescence and ph otosensiti,·ity. Applicatio n o f ALA by the rapId m ethod of Ion tophoreSIS, there­fore, has the potential to a ll o w detailed study of ALA / PpIX pharmacokin etics , and to prod uce a desi red degree of phototox ic ity in the PDT of sk in d isord ers . Foll ow ing activation of Ppl X by irr;ldiatio n , cle layed e rythem a co rre latecl w ith both the delivered charge of ALA and the ligh t dose. Furthermore, the absolute fall in Ppl X flu o rescence fo llowi ng light exposure predi 'ted phototo :>;ic­ity, suggesting that i/l "i"" Au o rescence spectroscopy may be lI sed to

accura te ly d irect the dosi m etry of topi ca l A LA Pf)T. T he rate- limi tin g step for ALA uptake into skin lies at the level

o f the stra tum co rn e llm (GofF ('I nl, 1992). Ion tOph ores is rap idl y clriv es ALA throu gh thi s ba rri e r w ithin seconds o r minlltes. In pre vio us studi es an in te rva l o r hOllrs occurred betwee n A LA

g ) ~ 2

;; \ c

/ 10 IDa

ALA dose (me)

1000

Fig ure 5. Phototoxic e£rect de­pends 011 ALA dose , T he crythe­mal response was quanti flcd by rc­fle c ta n ce spcc rro ll1 crry: resu lrs are expressed ;lS diflcrcll cc ill cryth cl113 in d ex fror11 llll ir rad i:Hcd contro l ski ll . n = -10. tl1 C;1 n s ~ SEM arc

plotted; r = 0.99 .

90 IU~ODES ET I lL

() ::J c( 1.5

Q) 0 c: Q) 0 1.0 III Q) ... 0 .= u.. X 0 .5

c.. 0..

6.25 12.5 25 50

Irradiation dose (J/cm2 )

Figurc 6. Fall in Pp IX flu orcsccn cc dcpcnds on irradiation dosc, an d P pIX fl uor csccn ce shows rccovery at 3 h. I' our identica l doses of ALA were iomopho,.escd with a charge of 60 mC, and site, were irrad iated with 6.25-50 J /crn 2

• respectively. at 5 h. Pp lX fl uorescence was quantified by spec troscopy p re-irradiatio n (OPl'1I ro lfllllll s) , iI111lH.:dia tt:! ly )Jost-irrad ia tion (black ( 0 /1111/11 .1') , and 3 h post-i rrad i:ltion (cross·hatehl'd Cli /lllllll .l' ) . n = 10, mcallS ± SEM arc pl otted.

app li cation and detectio n of Pp lX flu orescen ce (Ken ned y ci ai, 1990; Sva n berg c/ (/1. 1994; Szeimies 1'1 (/1, 1994). It has bee n im possi b le to d istin guish the proportion of thi s de la y d ue to absorption of ALA cream fi'o m that d ue to co n ve rsion of ALA to PpIX. In con trast, we detected PpIX fl uorescence within 15 min of ion top ho resis of ALA, de monstratin g earl y satura ti on of fe rroche­latase.

T he fl uorescence kin etics of pplX show that the peak accu mu­lation in th e ski n is hi gher , m o re susta in ed, and occurs late r w ith th e highe r ALA doses. consisten t w ith m urin e studies us ing systemi c ALA (Pottier 1'/ (/1, 1986, van de l' Veen cl " I, 1994). Foll owin g app li cation of 20'% ALA crea m , max imum PpIX flu o resce nce o ccurs at 4-6 h (M ali k ('I (/1, '1995). O llr data show th at the nlte o f PpIX synthesis is slower at the lower ALA doses, i.e., limi ted b y ava ih, bili ty of substrate, bu t the de layed pea ks of Pp lX flu o rescence at ch arges > 24 m C suggest the li miting factor at hig her ALA doses is the hem e biosyn thetic path way. T he flu o rescence k.inetics cou ld a lso reflect the ce ll u lar d istl'ibutio n of Pp lX (Gau llie ,' ct nl, 1995; Ste in bach ('{ nl, 19(5), the 3-h pea k signi fy in g acc umul atio n o f

U 2.0

=> .5

~ 1 5

~ j 1.0

x 0. a.. 0 ,5 4

C

;;; . u. 0.0 ,.1----,-=---,---- -,-----,

Difference in ery them a index

Figure 7. Photo toxicity correl ates with photoblcaching o f PpIX . T he erythema l response was quantified by reflectance spectrometry as the di fference in crythc rna index between irrad iated sites a l1 d contro l unirradi­ated ski n. Data poin ts comprise six doses of ALA (charges of 3-'120 mC) irradia ted with 100 J light per cm2

, and fo ur identical doses of ALA (charge 60 me ) irrad iated with ( .. 25-50 J li l': I1 t per cm' . respectively. 11 = 10. mean ± SEM arc plotted. r = O.9R .

T H E J OU I~AL 0 1' INVESTIGATIV E DEI~ATOLOGY

PplX in an ini ti al compart111en t, w hereas hi gh er ALA d oses ca use PpIX accu111ulation in a seco nd compartm ent wi th la rger capacity and lo nger re ten tio n . T he lower doses of ALA-induced Pp l X were cleared compl etely by 9 -1 0 h fo ll owin g io ntopho res is, w hi ch could prove con venien t if these d oses are su fi:lc ien t to trea t skin lesion s. H ighe r do ses were cleared from the skin withjn 24 h , consis tent w ith repor ts of ALA admiJl istered b y othe r rou tes (Kenn edy an d Pottie r 1992; Gran t cl nl, 1993).

O ur da ta support the suppositio n tlw t photobleachin g of Ppi X and p hotodynamic injury to ce ll s correspo nd quanti tative ly, w hich is in 'lg reem en t with the find in gs in coloni c tumo rs (O rth cl nl, 1994). T his is an ticipated beca use the rap id destructio n of PpIX is cau sed primarily b y sin glet oxygen , w hich is also responsibl e f()r th e p hotodyn amic injury of cell s (M oan and Sommer, 1985; H end erson and Finga l', 1987), predo minantly via m em brane lipid peroxidation . In anim al studies, reciproca l re la tio nships fo r phototox icity ex.ist, over som e lim its, fo r d rug and ligh t d ose, altho ug h these are confo unded by th e loss of photosens itiz in g abili ty through photo­bleachin g (Fing'lr and H end erson, 1987; M ang cl nl, 1987). O ur data n o w suggest tha t the m agni tude of lo cal Pp lX photobl eachin g itself accurately pred ic ts the degree of ti ssue dam age, indepe ndent of the cond itions used to cause th e photobl eachin g, i.e . , the total li ght d ose o r the Ppl X dose. T issue Ppl X and lipid ox ida tio n assays are needed to confirm that red uctio n in PplX corre lates w ith the detected photobl ea ching and the res ul tin g p.hototoxic dam age . Good corre latio n is known to exist, however , between levels of flu orescen ce an d extracta bl e sensitizer (M ang el nl, 1987 , Lo h ('I nl. '19(3) an d between microscopic lo cali zatio n of Ppl X flu o rescen ce and tissue d am age (D ivaris ('I nl, 19(0) .

Io n tophoresis could provid e a rapid m eth od fo r de li ve ry of ALA into skin les io llS, mak.in g pro lo n ged c,'ea m appli cation un necessa ry. Penetration of ALA is a sc ve re limi tatio n in the treatm en t of no dul ar basal cell c<Hcino m <ls (Ca irn d u fF et al. 1994; Svan berg el nl. 1994; Pen g el nl. 1995). perhaps du e to the ir 110 rmal ep ide rm is. Fu ture stud ies shou ld there fo re exa min e 'w hether io ntoph o res is can d e li ve r suffi c ien t am o unts of ALA fo r the ir tr ea tm en t. We suspect tIlH t su ffic ien tl y hig h doses fo r tu m o r n ecrosis w ere reached in o ur ex pe rimen ts , beca use no n-m elano ma skin tu mors are 1I10 re sensi­tive than norm al skin to PDT (D iva ris ('I (/1, 1990). Furth er, because PpJX flu o rescence showed a simple expo n en tial re lation ship to ALA dose within the limits of our ex perimen ts, la rge r q uan tit ies of Ppl X co ul d be gene rated by in creasin g the charge o f ALA. Pre limina ry da ta (not shown) suggest the ALA dose de li vered is not augm en ted b y in creasin g th e concen tra tion of ALA solutio n to 20% bu t that rec iprocity ex ists between c urren t and applicatio n tim e .

M easured d e li very of ALA to the epidermis b y ion topho res is h as en abl ed the c lea r dem onstrati on of a co rre latio n betweell photo­bleach ed photosensitizcr and photobio logic response and illu strates the poten tial fo r precise dosimetry in P DT. T hi s novel q uan titative system f.'1C ili tates fur ther study of skin photobi o logy and ALA pharm acok inetics. in cl uding the e ffects of recurren t ALA appli ca­tio n on th e heme biosyn th e tic pathway, w hi ch becom es importan t if repeated treatm ent is used fo r skin tumo rs (Ca lzavara- Pin to n 1995) o r in flam matory disorde rs slI ch as pso rias is (Boehn cke ('/ nl. 1994).

REFERE NCES

Bnchl1 ckc W- H. ~tcrr)' W. 1(:llIfin:lI1 l1 ll: T reatm en t o fpsod asis hy topica l p h otociy_

1I :J llli e therapy w ith po lych rum atic ligh r (letter) . Lllllc('1 343:81) I , 199'1 C ai m ci ufr F. Srrin gcr MR. H udson I~ J . A ... h DV. Brow li SB: Supcri'icia I ph olodYll:I lllic

rh crapy w ith t:op ic:d S- <lminll lacv ulini c a ..: id (t H' su pc r l'i ci al pri m ary :lI1d sCc.:O l1 d~I·Y

skill call Cc r. Bl'j e m/('(' (,9:(,OS-008. 1994 C:!lz,lvnrOl- Pi lito n PG: Rcpc.-itivc photodynami c the rapy with topi cal 1~-:lI l1 ill o l ac v ll ­

lini e acid as an approp ria tc ;,lppn ) :Jch W the ro u ti nc trea tm ent or su per ficial lIo ll - me lanom a sk ill t:lIrnOll rs . J Phol(lcit t'llJ PhOfOb i() / B 29:S3-S7 . 1995

D iney IJL. O li ver ItJ. Farr PM : A po rta hi e instrullI eli t fo r q uantifyi ll g eryth ema ill d uccd b y ul trav io le t rad ialio ll . /Jr.l Dcnll tllol III :()()3-67 2. 1984

Diva ris DXG. Kenll cdy Je t Pottie r IU-I : Ph OLOlOXic dalll age to schan:o llS glands and h:li r fo lli cles of mice fo ll owi l1~ systcm ic ad1l1illi srra rio rl o f 5-a m ill o lcv ulin ic acid corn.:l:llcs with loca li sed pro to po rph yr in IX A\l orcscclln :. Alii) 1)(/(110 1 (I: 143-14 8. 199q

VOL. l OR. NO. I JANUAR Y I')n

Fi n gar VH. I-knel l-'rson B\XI: Dru g and light dnsc dCPClldclH;c of p ltorodYliamic th e rapy: a stud y or l-lIlIlor .lI1d Il orllwl tissue respoll se. PltIU"r/H'/11 Plwl{)/J j() / 4 6:~ :I7-H4 I . I ')H7

G al1 g:lro ~ iI LIl . Pa rk NH. \Viggins CA . HiIlJ M: Increased ~C I1 C lr;lIiO Il of" n u n clct' ITu­

iy[Cs ini"t ) m o use ski ll d uring iOll fo ph nrctil- wa leI' tran spo rl . J PI/am, Ex}' Thfl 2 12:377-]H I . 19HII

Gau lli er J - M . Gczc M . Sa lllll s R. Mel o TSE. Ma zic rc .I -C . 13azill M. Modicrc 1'. Dubcrtrct L: Suhcellu la r loc:lI izatio ll o r and p llOtOSC Il Siriz;ltio ll hy p rotoporph yri ll I X ill hUl1 1:l n kc ra tin ocyrcs and ~ihrohbs l s Luiti v ill-c d wid l 5- :l11li ll OlcV lIlini c acid. P/iOIOI'i' L'/11 I'I,()/ ohiol 62: 114 - 122, 1995

Goff l3A . l3ac hor It. I{nlli;\ !" N , HaS;11l T : EHccts uf pliufndyna lllic tlll~ ra py wi l'li top ica l appli c,\t'io ll ur S-;lI11iIl Olcvu li nic acid n n I1 nrllla l :oi kin of IliIi rl t..·ss g uinea pigs. -' P/IIJ(od, t'1I1 1)/wll./Ji,,/ B 15:239 -25 1, 11)')2

Cra nt WE. Ho pper C, Madto hert AJ. Speight PM . llown SC: Pho lOd Yll amic th e rapy o r ora l c:lm:e r: ph otose nsitizatio n w ith :oiy!H"emic :l millo lacv ulinic ac id. U I//(( "

3·12 : 147- 148. 1993 Green PC. I-l inz It.S . Kim A. Szok:1 FeJr. G Il Y R II : In ll w pil o reric deliver), of:l series

o f tripe-prides anoss the skill ill " ifm. P//(Inu /(I'S H: I I :! I - I 127. 199 I H e nderso n U\'(/ . Fin ga r V 1-1 : R cl;llio llsh ip of' HlI ll ot lr h ypox ia and resp o llse W

p h o t'odynalilic IrC;ltlll cn t in an cxpcrim e ll tal l1lo \1 :oic t lllllOr. C ,mf tT /{( ','i 47::\1 10-3 11 4 . 19 H7

linullli.l S, Farshi SS . O rt e l B. 1-I :lsan T : A m echanisti c study or cellul:J r ph o todeSITlH':­t iOi I w ith 5-a l1lino laevlIlin ic ac id- ind uccd po rph yrin. IJrj C (lI/(CI' 70:2 1- 28 . I Y94

Ken n ed y Je . Po tTi er RJ-I : Endogeno ll s protoporph yrin IX . il clinica ll y lI ~e rll l p holD­sen sitize r ro r ph otodynami c th erap y. ) P/lOlor/U' /1I fJlliltob iCl/lJ 14:275-21)2 . 1992

Ke nn ed y Je, POlti c r rU-l . Pro ss D C : Ph otod YIl:Ullic t hc rap y w ith c lldngeno lls l'roro­p o rphyrin IX: b:lsi c principles and prese nt dinical t!xpl:ricn ce. J Plwr"r/Il'1I1 Plw,c>/ii,,1 H 0: 1 ~ 3- 14 H. 1 9~U

La n g S. Baumg artner R . SO'lIck R . Le llllig A. G Ulillallll R. Fc)'h J: Ph orodyn ;nll k diag no sis and thl.'rap), of n euplasm s of the f:lt.: ial skin alier topic al admini stration

of 5-:lIllin o lev ulinic acid . l.AfI)' /lorhiIl Od fl'/O,~if 74:t)5-8? 1995 Levcc.:kis J . UUrlIJL. Brow n NJ . It.CCl l M\V lt: I< ille ti cs ofcndog-c llOllS protopo rpllyrin

IX inductio ll hy :lmin o lcvulin ic ;lci d :prc.1ill1ina ry sru d ics in thl~ hl:ldder. J Unl/ I 52:55()-SS3. 1994

Loh es. Verno n D. Mm.- Rohert AJ . Bed we ll J . 130w II SG. Bruwn S U: Endoge no lls porphyrin di srr ihllti o ll in d uced h y 5-a lllill nlcvlIlin ic acid ill rhe ti ssue la yers o f til e g :lstroi ll t'es tinal tract. j P"lli(Jr/H'''' PIIt'l e"J i,l/ lJ 20 :-17-54. 1')93

Ma li k Z . I<osn.:n ich G. Iloinllan L. Ehrenbcrg U. O rc ll st:cin A: 'Topi ca l :lppiicat"io ll o( 5-il lllill o le vu linic ac id. DMSO and EDT!\ : prll tllporphyrin IX act.: l1ll1ul:i rion ill ski ll and 1'1I11HIIII'S of mi ce . J PI'l lfllChcl/l J)llOilJhiol H 2H :2 1.1-2 1 X, I I)() 5

A LA IONTOPI-I OH..ES IS AND I'HOTOTOXIC EFFECTS 91

Mang TS. Donghcrr)" TJ . Pottc, W it. 13(»'1c DC. So me, S. MoanJ : I'h o!ohloa"hin !,: of po rp hyri ll s lIsed ill photodyn amic therapy ;llld illlplic:lrio ns fo r rhcr:IPY. Plulwrlu'/1/ P/l(lrob;(,/ -15:50 I-SO(I. 1987

/'\1\0;111 J. SOlllnl er S: Oxn;cll dependen ce of the p hoto.~e l1 s iti z illg cl1cc t of h acl1lato­

porph yrin derivati vc in N I-II K 3025 ce lls. Callfer n"s -IS: 160H-Ih l tl. I 'JHS anh K. J{o lli~ K. Gcnzc F. Ru ck 1\ : Pho todyn amic thcrapy uf cspe rimcnrai colo ni c

tlI lllOUI'S with S-;llll ino lcvu lilli c-a d d- itl llu t.:cd c lldngc llotls pnrpilrrins. J e mlCeT

I<<,s IiI/ 0 1/( ,,1 120:057-66 1. 19')4

Pe nt; Q. \Va rloe T. Moan J. I-I eyenb hl 1-1 . Stecn 1-113. Ncsland JM . C ic rcksky K-E:

I istrihutio ll or 5-amin o levu lil1i c acid- indu ced porphyrin s ill n o dul o ll lce ra tive basa l ce ll carc illOilla. Phor"t/Il'1II l 'II t1r, lhi,,1 62:906-913. I ')t) 5

Po tTie r IU-I, C how YFA , LaPI:1I1te J - P. Truscott T G. J{ c IIIH.·d y .I C . Be in cr LA :

NO II - ill v:ls i\'c I'cclmiquc for obrai l lill ~ Uuorcsccll ce ~XCi l :H"i oll and l! lllis"iOlI SpcCt!':1 ill lIill(l . P/IOrtltiH'lIl Photl,/)i,,1 -1-1 :(,79-6t-17. 198(,

Sloa n .113. So lrani KS: Io n topho resis in nc rmato logy . .1 r l", ,-lend DCT",flItll I 5:(,7 I-(,H-I.

I ') S6 Sh: inh;tch P. \Vcing;1I1dr H . U:1\11l1 garrn e l' R . KI'icgllla ir M. I-Iof. .. t:idt(·r F. Kn iichd It:

C e llu b r ntl OrCSCCll ce of the e ll dogcnotls photose nsiti zer jJrol"Ol'o rl' ilyrin I X

fo ll owing cxpo sure ro 5-a min o lc"11Iinic acid . Pltoll'(/1I'1/1 P/lClfilj, i,,/ (,2:8H7-895 .

1995 Svanberg K. An dersso n T. Ki ll allncr D. \Xf .11lg I. SrCnr;l1 l1 U. Andersson-Engels S. L3crg

R. J o han sson J. Svanh(,.· rg S: Ph o todynam ic rhc rapy of llo ll -mclalloll1:l m alignant 1'lll1l0llrS of the skin u s in~ topical I)-amin o levulini c add scnsiriz ;1 ri o ll and laser

irradiatio ll . /3,. ) Oel"/II n'o/ 130:743-70 1. IY94 Szc.illlics It - IV\. Sassy T. La ndtha lc r M : Pcn c trarion POICIH:Y o f ropical app lied

O-:lIll ino lcVlllin ic ;Ic in lo r p ho todynam ic Ihe rap ), o f hasa l cc ll ca rc in o m a. P/WfcJ ­

dU'11/ P/wtl) IJj()/ 51):73-7(,. 191)4 Ty lc P: IOll topho rc ric de\' icl!S I() r d rug delive ry. Plitt,.", Rt'J 3:3 1 S-J26. 1 9~('.

V:III der Vcen N , van Lccngoed HLLM . Slar \V M : III pil'<' fluorescen cc killl:li c s ~lIld

I'hol odyn:lI11ic rhe rapy usin g 5- alllill o lacv lIlini c add-induccd porphYTi n : in ­c rcased dam age after Illultiple irradiatio ns. Hrj . 11 1/((" 70:R67- S72 . 199-1

\Vcish :llIpl I<.R . G Ull"lc r eJ. Doughl.' rt)' TJ : Idcntilical io n of si ngle l oxr~ell a:o. the

cytoloxic :Igelll ill pi1ot:oillaClivari o ll of llIurin e rUlll or. C llIln.,. R('s 36:2326-2321},

197(, \Vo lrp. It.i e~c.:r E. I<erl H : Topic;!1 phorodyn:lI1li c Ih er:.p), with cndogell o us po rph yrins

aftc r a l~,pli c atiol1 o f 5 :lll1 ill Olcv ulinic ac ici: a ll :1 lterllad vl' treatm cnt lllo dalily 1'01'

so lar kcraroses . superficia l s qU :UIl O U !) ce ll carci nomas and hastd ,-ell carcin o ma s?.I

.-1111 .·karl 1)/.,.1I1f1l111 2H: 17-2 1. 1993