5-Aminolevulinic Acid (ALA)-InducedProtoporphyrin IX Fluorescence and

Photodynamic Effects in the Rat Bladder:An In Vivo Study Comparing Oral and

Intravesical ALA AdministrationShi-Chung Chang, MD,1,2* Gio Buonaccorsi, PhD,1

Alexander J. MacRobert, PhD,1 and S.G. Bown, MD1

1National Medical Laser Centre, University College London Medical School, London,United Kingdom

2Department of Urology, Tz’u-Chi General Hospital, Taiwan

Background and Objective: Photodynamic therapy (PDT) using5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX)for sensitization is a promising treatment for carcinoma in situand diffuse premalignant changes of the bladder. We studied thebiodistribution of PpIX in a range of tissues with oral and intra-vesical routes of administration of ALA and compared the pho-todynamic effects on bladder and skin.Study Design/Materials and Methods: Normal Wistar rats weregiven oral or intravesical ALA and PpIX levels in the liver, kid-ney, skin, and bladder measured by fluorescence microscopy ontissue sections. At the time of maximum PpIX levels, the bladderand skin on the back were illuminated with light at 630 nm andthe PDT effects compared.Results: PpIX fluorescence in the urothelium after 200 mg/kggiven intravesically was comparable to that found after 100mg/kg orally. The ratio of PpIX levels between the urotheliumand the underlying muscle was the same for both routes of ad-ministration, although there appeared to be more selectivity ofurothelial PDT necrosis after intravesical administration. Skinphotosensitization was greater after oral ALA, the epidermalPpIX level being three times higher than after intravesical ad-ministration for comparable urothelial levels and the PDT effectbeing more marked.Conclusions: Intravesical instillation is preferable to oral admin-istration of ALA for PDT ablation of the urothelium of the ratbladder without damage to the underlying tissue layers and forminimizing skin photosensitivity. The technique is now ready forclinical trials. Lasers Surg. Medicine 20:254–264, 1997.© 1997 Wiley-Liss, Inc.

Key words: 5-Aminolevulinic acid (ALA); bladder cancer; fluorescencemicroscopy; photodynamic therapy; skin photosensitization;intravesical therapy

INTRODUCTION

Photodynamic therapy (PDT) is a modalityreceiving increasing attention in many medicalspecialties for the treatment of early malignan-cies [1,2]. By the combined action of a photosen-sitizer and light illumination in the presence of

Contract grant sponsors: Compassion Relief of Tzu-Chi Foun-dation and the Imperial Cancer Research Fund, London,U.K.*Correspondence to: Dr. Shi-Chung Chang, M.D., PhD, De-partment of Medicine, Tzu-Chi College of Medicine 16, Hsin-Sheng South Road, Hua-Lien, Taiwan.Accepted for publication 19 May 1996.

Lasers in Surgery and Medicine 20:254–264 (1997)

© 1997 Wiley-Liss, Inc.

tissue oxygen, photochemical cellular destructioncan be achieved if a sufficient amount of photo-sensitizer is present in the tissue and light of ap-propriate wavelength and energy is delivered[3]. Hematoporphyrin derivative (HpD) or itspurified ether/ester (Photofrin) is the most fre-quently used photosensitizer in current PDT stud-ies, but the prolonged cutaneous photosensitiza-tion, which lasts for 4–6 weeks, is an obviousdisadvantage. Research on developing new photo-sensitizers is being widely pursued, but the prob-lem of cutaneous photosensitivity remains. An al-ternative approach is to use local administrationsuch as intratumoral injection or intravesical ad-ministration, which has met with varying degreesof success [4–6].

The use of 5-aminolevulinic acid (ALA), aprecursor of protoporphyrin IX (PpIX), has beenextensively studied in vitro [7], in vivo [8,9], andin clinical trials [10–12] in recent years and is apromising new approach for PDT. ALA is anaturally occurring photoinactive intermediaryin the cellular biosynthetic pathway for heme. Inthis chain reaction, ALA is converted in smallamounts to coproporphyrin and uroporphyrin,but mainly to PpIX, the predominant porphyrinspecies responsible for photosensitization [10].Since the conversion of PpIX to heme is therate-limiting step and is controlled by ferroche-latase and tissue iron, bypassing the normalregulatory feedback mechanism with excessexogenous ALA results in temporary accumula-tion of PpIX [10]. By this means, photodynamictherapy has been successfully undertaken for thetreatment of skin cancer [10,11] and other humanmalignancies [12,13]. The use of ALA/PpIX forPDT of dysplastic lesions in hollow organs suchas the bladder has attracted much interest, sincethe risk of significant systemic side effects shouldbe minimal [12,13]. As previous urological expe-rience with HpD-based PDT for bladder cancertreatment showed a high incidence of complica-tions (particularly scarring of detrusor muscleand contracted bladders) [14], we studied thefeasibility of instilling ALA directly into thebladder as a means of achieving selectiveurothelial sensitization compared with the under-lying muscle [6] and assessed the photodynamicresponses after light illumination [15]. Further toour previous studies, in the present study, wecompared the intensity of PpIX fluorescence invarious organs at different times after givingeither oral or intravesical ALA and evaluatedthe difference of photodynamic effects on uri-

nary bladder and skin using the two routes ofsensitization.

MATERIALS AND METHODS

Animals and Anesthesia

Female Wistar rats, weighing 100–160 g,were used in this study. The ALA solution wasadministered by gastric gavage to rats anesthe-tized with a mixture of Halothane and O2 (volumeratio 1:2). However, for longer procedures such astransurethral instillation of ALA and laser treat-ment, general anesthesia was used with intra-muscular injection of 0.1 ml/kg Hypnorm (fenta-nyl and fluanisone, Jansen Pharmaceuticals,Grove, UK) and 1 mg/kg diazepam.

Photosensitizer

ALA (ALA.HCl) was obtained as a 98% purepowder from DUSA Pharmaceuticals, (New York,NY). As in our previous study [15], we used 10%ALA solution (100 mg/ml) at pH 5.5 for bladderinstillation. This was prepared immediately priorto administration by buffering the ALA solutionwith saturated sodium bicarbonate.

ALA Administration

Bladder instillation (BI) of ALA was accom-plished by infusing a 10% solution at the dose of200 mg/kg (0.2 ml/100 gm body weight) throughan 18-gauge Teflon catheter, which had been in-serted transurethrally and was kept in the blad-der for 2 hours while the rats remained undergeneral anesthesia. Throughout the text, instilla-tion time refers to the time ALA was kept in thebladder with the animal under general anesthe-sia, whereas retention time refers to the sum ofinstillation and postinstillation times until theanimals were killed (for the biodistributionstudy), or treated (to look at the photodynamiceffect).

Oral administration (Oral) of ALA was un-dertaken by giving ALA solutions at doses of 100,200, and 400 mg/kg body weight through a bulb-tip gavage needle into the stomach under inhala-tion anesthesia. As rats are incapable of vomitingor regurgitation, all animals ingested the full de-livered dose. All the rats resumed normal activitywithin 5 minutes of terminating the anesthetic.The oral doses used were those found in prelimi-nary studies to give urothelial levels of PpIX com-parable to those found in our previous study usingintravesical ALA.

PDT with Intravesical and Oral ALA 255

Fluorescence MicroscopyFor each ALA dose and route of administra-

tion, 2–3 rats were studied. The control groupconsisted of two rats receiving nothing and twoothers with bladder instillation of 0.9% normalsaline (0.24–0.3 ml, depending on body weight).The rats were killed and specimens from the uri-nary bladder, liver, kidney, and back skin werecollected 1, 2, 3, 4, 5, 7, 9, and 24 hours afteradministration of ALA. The tissues harvestedwere initially placed in precooled isopentane(BDH Chemicals, Poole UK) and then transferredto liquid nitrogen before being processed to thinfrozen sections (10 mm) for fluorescence imaging.

Microscopic imaging and quantification ofALA-induced PpIX fluorescence was done with aninverted phase contrast microscope (OlympusIMT-2) attached to a slow-scan, charge coupleddevice (CCD) camera (Wright Instruments, Lon-don, UK, model 1, 385 × 578 pixels). The setup ofthis highly sensitive photometric system has beendescribed in previous studies on fluorescence im-aging after administration of photosensitisingagents [9,16]. The slow-scan CCD system permit-ted highly reproducible signal calibration, whichwas checked using a thin film on a glass slideunder a coverslip of a mounting medium (Fluor-omount, BDH Poole, UK) containing aluminiumphthalocyanine at a concentration of 10 mg/ml. Inbrief, an 8 mW helium neon laser (632.8 nm) wasused to excite the PpIX and the emitted fluores-cence was detected between 660 and 710 nm, usinga combination of bandpass and longpass filters.This excitation wavelength is ideal for PpIX butless so for uro- or coproporphyrins, which showpeak absorption at slightly shorter wavelengthsnearer 620nm [17]. The fluorescence signals wereprocessed by an IBM personal computer into afalsely color-coded image depicting the mean sig-nal per pixel. The software also allowed fluores-cence data to be quantified digitally over definedareas from which the mean counts per pixel couldbe calculated. After fluorescence imaging, the sec-tions were fixed in formaldehyde and stained withhematoxylin and eosin for histologic matching.

The fluorescence intensity of specific tissuelayers measured in the bladder and skin (urothe-lium, lamina propria, muscularis propria, skinepidermis, dermis, and hair follicle) was the av-erage measurement from > 20 representativeblocks (minimum 10×10 pixels) with correctionfor background autofluorescence. However, forsections of the solid organs (liver, kidney, and ab-dominal muscle), the fluorescence quantification

profile was the average of 10–12 representativeblocks (minimum 350×500 pixels) taken fromvarious areas. No correction for background auto-fluorescence was applied for these organs.

Photodynamic Therapy

The control group consisted of three ratsreceiving laser illumination without ALA sensi-tization. The experimental groups were strati-fied into Group A: Intravesical ALA (200 mg/ kg), and 50J light; Group B: Oral ALA (100 mg/kg) and 50J light; Group C: Oral ALA (200 mg/kg)and 25J light; Group D: Oral ALA (200 mg/kg)and 50J light, and Group E: Oral ALA (400 mg/kg) with 50J light dose. The light source was acopper vapor-pumped dye laser (Oxford Lasers)tuned to 630 nm, the beam being deliveredthrough a 200-mm silicon-coated quartz fibre. Atthe optimal time after sensitization (5 hours withintravesical, 4 hours with 200 and 400 mg/kgoral, and 3 hours with 100 mg/kg oral ALA), asmall abdominal incision was made under gen-eral anesthesia to expose the urinary bladder. Thebladder was then eased upward and placed onthe abdomen, emptied of its urinary contents witha transurethral cannula, and filled with 0.3 ml of10% intralipid solution to ensure homogenouslight scattering. At this volume the bladder wasapproximately spherical in shape, which furtherensured isotropic light distribution. The tip of abare laser fiber (without cladding) was placed ∼ 3mm from the top of the bladder after insertionthrough the dome. No intralipid leakage was seenon puncturing the bladder as the fenestrationsealed tightly around the fiber. The power fromthe fibre tip was calibrated before and after eachtreatment and was set at 100 mW for 250 or 500seconds giving a total light dose of 25 or 50J,respectively, per treatment. Following PDT, ani-mals were killed at 2, 3, and 7 days and the blad-der sectioned and stained for histological assess-ment. Four rats, two with intravesical (200 mg/kg) and two with oral ALA (400 mg/kg), weretreated at a light dose of 50J and were kept alivefor 6 months after PDT for long-term compari-sons. These specimens were stained with Van Gie-son for assessment of collagen fibrils in the blad-der wall.

Eight rats (2 without drug, 2 with intraves-ical ALA, 2 with 200 mg/kg oral, and 2 with 100mg/kg oral ALA) were investigated for the skinphotosensitivity study. All were treated 3 hoursafter receiving ALA. The fur overlying the lowerabdomen was shaved and the exposed skin was

256 Chang et al.

illuminated with red light (630 nm) through a200-mm laser fiber placed 1 cm above the surfaceat a power of 100 mW (power density 100 mW/cm2) for 500 seconds (50J/cm2). The animals werekilled 3 days after light illumination and thetreated areas examined macroscopically and sec-tioned and stained with hematoxylin and eosin (H& E) for histologic evaluation.

RESULTS

Fluorescence Microscopy

The highest levels of PpIX fluorescence werepresent in the urothelium (Fig. 1A,B). Levels rosefaster following oral administration of ALA (Fig.2). At the higher oral doses (200 and 400 mg/kg),fluorescence in the urothelium peaked at 6–7hours, but at 100 mg/kg, it was at 3 hours. Withbladder instillation of 200 mg/kg, the peak uro-thelial level occurred between 4 and 7 hours, atwhich time it was similar to that found 3 hoursafter 100 mg/kg orally. It was notable that blad-der instillation gave a more sustained level ofPpIX in the urothelium, whereas the level fluc-tuated more after oral administration. Althoughthe peak urothelial intensity was generallyhigher in the oral groups, the ratio between thelevel in the urothelium and in the underlying lay-ers remained similar as the fluorescence signalsin the lamina propria and muscle were alsohigher with oral administration (Fig. 3). The re-sults in the liver are shown in Figure 4. At theearly times following oral ALA, fluorescence inthe liver was much higher than after bladder in-stillation. The signal observed probably arises

from protoporphyrin, uroporphyrin, and copropor-phyrin (see Discussion). However, at longertimes, the differential declined and was virtuallyabolished after 7 hours. The fluorescence levelsin the renal cortex resembled those of liver, butwide fluctuations in the readings prevented anyclear pattern being identified (Fig. 5). It was no-table that readings of fluorescence levels at timezero, which indicated the baseline autofluores-cence in the liver and kidneys without ALA ad-ministration, were much higher than in the otherorgans measured, presumably due to the presenceof endogenous porphyrins. Despite the lack of aclear pattern in terms of porphyrin pharmacoki-netics, hepatic fluorescence signals returned tobaseline values by 24 hours after ALA adminis-tration.

PpIX fluorescence levels in the epidermaland dermal layers of the skin on the back areshown in Figures 6 and 7. In each layer, signalsfrom the oral groups were generally higher thanthose from the BI group. At an oral dose of 100mg/kg, which gave a peak urothelial PpIX levelsimilar to that from 200 mg/kg given by BI, PpIXin the epidermis was twice as high as in the BIgroup. Intravesical instillation also resulted inless PpIX buildup in the hair follicles.

Photodynamic Therapy

Macroscopic findings. Control bladdersthat underwent light illumination only showed noevidence of tissue injury. Under the most severeconditions (Group E, 400 mg/kg oral ALA and 50Jlight), 3 of the 12 rats died within 48 hours. Oth-ers in the group that survived the early stages

Fig. 1. A. Fluorescence microscopy of rat urinary bladder 5hours after instillation of ALA showing higher PpIX(White) intensity in urothelium than in the underlying

layers (Red). The fluorescence scale is shown in the color barat the top. B. Corresponding histological section H&E stain(×150).

PDT with Intravesical and Oral ALA 257

were physically unwell until 7 days after PDT.The perioperative findings of rats in Group D (200mg/kg oral ALA and 50J light) were similar toGroup E, and two died within 48 hours of PDT.However, with a reduced light dose: Group C (200mg/kg oral ALA and 25J light), or reduced ALAdose: Group B (100 mg/kg oral ALA and 50Jlight), no perioperative mortality was seen. Sim-ilarly, for intravesically sensitized rats in GroupA, no PDT-associated death or perioperative dis-tress was observed. On necropsy of the rats thatdied within 48 hours of PDT (Group D and E), themost probable cause of death was extravasation ofurine with possible peritonitis as accumulation ofturbid fluid in the abdomen was a consistent find-ing. The cause of urinary extravasation remainsuncertain, although there is the possibility ofleakage through the site of fibre puncture, whichcould have become an established perforation af-ter PDT.

Whatever the dose of oral ALA, 48 hours af-ter treatment the bladder looked inflamed, puffy,and edematous. Only the five animals describedabove showed evidence of perforation, although aperivesical fatty tissue reaction was prominent inmost. The bladders receiving 50J of light con-tained some tissue debris and were particularlythin and flaccid on filling with formalin. Thelower light dose of 25J provoked less tissue reac-tion. By the seventh day, perivesical fatty inflam-mation was subsiding, although was not com-pletely resolved. Animals with intravesical ALA(Group A), however, demonstrated much milderchanges macroscopically at these times afterPDT. Six months following PDT, the appearanceof bladders and the surrounding tissue in animals

that had been sensitized with oral or intravesicalALA were macroscopically indistinguishablefrom those of control rats, even though the onesgiven oral ALA had received the highest drug andlight doses.

Microscopic findings. Histologically, noevidence of urothelial damage was discovered inrats receiving light but no ALA. However, mildinflammatory cell infiltration in the serosa andouter layer of muscularis propria was seen, prob-ably due to handling. The bladder lesions 2–3days after PDT were more severe in the oral ALAgroup than in the instillation group. Thosetreated with 50J showed full thickness tissue de-struction with fibrinoid necrosis of arterioles inmost parts of the bladder (Fig. 8). At 25J, theurothelium was ablated with preservation of partof the underlying structures. However, muscledamage of some degree seemed inevitable. Inter-estingly, little urothelial destruction was ob-served in Group B, although the fluorescence in-tensity at 3 hours was as high as that at 5 hoursin Group A. Despite nearly complete regenerationof damaged urothelium by 7 days after PDT,subepithelial fibroblast infiltration was seen, es-pecially in the lamina propria. In contrast, thehistological changes found with intravesical ALAshowed less prominent but more selective urothe-lial ablation without lamina propria or muscledamage (Fig. 9), even though they were treatedwith 50J. By the 7th day, bladder epithelium hadregenerated and fibroblast infiltration was hardlyseen in either lamina propria or muscle. The find-ings 6 months after PDT revealed significantlymore collagen fibrils in both the lamina propriaand the muscularis propria of the oral group com-pared with the intravesical group (Figure 10).The microscopic findings are summarized in Ta-ble 1.

Skin. Macroscopically, no visible lesion wasseen in the control and intravesically sensitizedrats. Those having oral ALA showed brownishdiscoloration of the skin especially in the centrewhere the light was greater. Microscopically, con-trol rats treated with light alone showed normalskin architecture (Fig. 11A). In the ALA instilla-tion group, focal epidermolysis with very mildkeratolysis was observed. The epidermis and der-mis were normal, whereas the loose connectivetissue layer showed mild infiltration of inflamma-tory cells (Fig. 11B). In contrast, the skin his-tology of those receiving oral ALA demonstratedextensive destruction of the epidermis and der-mis. There was intensive inflammatory cell infil-

Fig. 2. Plot of PpIX fluorescence intensity in the urotheliumafter oral or intravesical ALA.

258 Chang et al.

tration where the dermis meets the underlyingconnective tissue layer (Fig. 11C). The micro-scopic findings were more marked in thosetreated with 200 mg/kg compared with thosegiven 100 mg/kg orally.

DISCUSSION

Although PDT has the potential for treatingbladder tumors and some of the preliminary re-sults using HpD as a sensitizer seemed encourag-ing [18–20], its universal acceptance is unlikelyunless the concerns of detrusor muscle damage[14] and prolonged skin photosensitization [21]can be satisfactorily resolved. In our previousstudy using 10% ALA solution at pH 5.5 admin-istered intravesically and treating the bladder

with a light dose of 50J (630 nm), we were able toinduce homogenous urothelial ablation with onlynegligible damage to the underlying layers [15].The selective necrosis of the transitional celllayer produced with ALA induced PpIX is there-fore relatively mild and well tolerated. Althoughthe duration of skin photosensitization with ALAis only 1–2 days [12], measures that could reduceits severity would be welcome. From this study,intravesical administration is the preferred routefor treating the bladder as for comparable levelsof urothelial PpIX, there was less full thicknessbladder damage and less skin photosensitivity.

For any substance to be efficiently trans-ported into the urothelium after intravesical ad-ministration, it should have a low molecular

Fig. 3. A. Plot of PpIX fluorescence intensity in the lamina propria. B. Muscularispropria after giving ALA via the oral or intravesical route.

Fig. 4. Plot of PpIX fluorescence intensity in the liver aftergiving ALA via the oral or intravesical route. Fig. 5. Plot of PpIX fluorescence intensity in the kidney

showing a fluctuating pattern of PpIX after giving ALA viathe oral or intravesical route.

PDT with Intravesical and Oral ALA 259

weight (<200), be watersoluble, and yet be suffi-ciently lipophilic for binding with plasma mem-branes [22]. The biochemical properties of ALA(amphiphilic with molecular weight of 131) fulfilthese criteria. Amino acids of similar molecularweight to ALA also have been shown to diffusethrough the urothelium over a concentration gra-dient [23]. The buildup of PpIX fluorescence in theurothelium after instilling the ALA solution isgood evidence to support the permeability of theurothelium to ALA [6]. After penetrating the su-perficial umbrella cells, it may take some time todiffuse into deeper transitional cell layers and tobe converted to PpIX. The duration of bladder in-stillation is important as there is continuousPpIX generation in the cells, so the tissue level ofPpIX can build up through the constant contact ofthe urothelium with ALA. The relatively con-stant level of urothelial PpIX 4–7 hours after

bladder instillation is another advantage overoral administration as it makes photodynamic ef-fects in the bladder more uniform and predictable.Systemic administration, however, provides ALAfor PpIX biosynthesis through the vasculature, somost ALA would be expected in the best vascu-larised layers, the urothelium, and lamina pro-pria. Nevertheless, conversion to PpIX may stillbe more efficient in the urothelium due to thegreater metabolic activity of these cells, as wasshown by Loh et al. [24]. From our previous report[24], peak PpIX levels in the epithelium herereached earlier at lower ALA doses (3 h at 100mg/kg, 7–8h at 200 and 400 mg/kg). It is alsonoteworthy that 400 mg/kg orally produced nohigher peak PpIX in the urothelium than 200 mg/

Fig. 6. PpIX fluorescence intensity in the epidermis of backskin after oral or intravesical ALA.

Fig. 7. PpIX fluorescence intensity in the dermis of back skinafter oral or intravesical ALA.

Fig. 8. Microscopic picture 48 hours after PDT showing fullthickness bladder damage with fibrinoid necrosis of vesselsand prominent inflammatory cell infiltration. No evidence ofselectivity between layers is seen (50J, 4 h after 400 mg/kgorally) (H&E stain, ×60).

Fig. 9. Microscopic picture of bladder 48 hours after PDTshowing selective destruction of urothelium and relativepreservation of the muscularis propria (50J, 5 h after bladderinstillation of 200 mg/kg) (H&E stain, ×60).

260 Chang et al.

kg. It is probable that above 200 mg/kg, the avail-able ALA saturates the synthetic capacity of theurothelium so further increases of PpIX levels areimpossible.

This report has shown better selectivity ofPDT necrosis of the urothelium compared with the

underlying layers using intravesical ALA. Thiswas unexpected as the fluorescence microscopystudies showed that the selectivity of PpIX distri-bution between layers was the same for bothroutes of administration. This may be becausewith bladder instillation, the PpIX is located in

Fig. 10. Microscopic finding of bladder 6 months after PDT showing (A) slightlyincreased collagen fibrils in the lamina propria (200 mg/kg ALA, intravesical, 50J).B marked deposit of collagen in the lamina propria (400 mg/kg ALA orally) (HVGstain × 100).

TABLE 1. Histological Changes of Bladder Wall (2–3, 7, and 180 days) after PDT in DifferentExperimental Groups*

GroupUrothelium

damageOedema

Necrosis ofurotheliumand L.P.a

Musclenecrosis

Inflamm. cell infiltration FibroblastL.P.a M.P.b L.P.a M.P.b Serosa L.P.a M.P.b Serosa

Control (light illumination only)− − − − − ± − ± − − −

A (BIc 200 mg/kg, 50J)2–3d diffuse ++ + ++ + ++ ++ + − − −7d − − − − − + ± − + ± −180d − − − − −- − − − ++ + +

B (Oral 100 mg/kg, 50J)2–3d patchy − − ± ++ ++ + + ++ + ±7d − − − − + ++ + + + + +

C (Oral 200 mg/kg, 25J)2–3d extensive ++ + +++ ++ + + + − + −7d − − − − ± + ± − + ± ±

D (Oral 200 mg/kg, 50J)2–3d extensive ++ + +++ +++ ++ + + ++ ++ +7d patchy − − + ± + + ± ++ + +

E (Oral 400 mg/kg, 50J)2–3d extensive ++ ++ +++ +++ + + + ++ ++ +7d patchy ± ± + ± + + ± ++ + +180d − − − − − − − − +++ ++ ++

*Treatment parameters: ALA concentration, light dose. Grade of histological changes: −: none, ±: minimal, +: mild; + +:moderate; + + +: severe.aLamina propria.bMuscularis propria.cBladder instillation.

PDT with Intravesical and Oral ALA 261

particularly sensitive regions of the urothelialcells, whereas with oral administration it is not.Some of the PpIX seen after oral administrationmay have been synthesized in the liver andreached the bladder via the bloodstream. Never-theless, this does suggest that intravesical ALA ispreferable for management of superficial bladdercancer with PDT, particularly for field change dis-ease.

The substantially higher porphyrin levels(mostly PpIX, but possibly with smaller amountsof uroporphyrins and coproporphyrins [5]) de-tected in the liver soon after oral ALA are likelyto be due to rapid absorption and first pass con-version in the liver. The levels after oral and in-travesical sensitization do not become comparableuntil 7 hours after delivery, although they arestill relatively high at this time. Intravesical ad-ministration would appear to slow down ratherthan eliminate systemic absorption as there arestill substantial amounts of PpIX detected in theliver, but if the delay in absorption is comparableto the time for PpIX to be metabolized to heme,then the levels in tissues other than the bladderare likely to stay low at all times, reducing the

risks of systemic phototoxicity. From our clinicalstudies, transient elevation of serum aspartateaminotransferases was found in one-third (6/18)of patients receiving 60 mg/kg oral ALA [12]. Wedid not carry out any enzyme assays in this study,but it seems unlikely that rises would be seenafter intravesical administration, although thiswarrants further studies.

With the kidney, the situation is different.With bladder instillation, in addition to systemicabsorption, there is also the possibility of uretericreflux, which might explain our rather variableresults as seen in Figure 5. Clinically, preventionof vesico-ureteral reflux can be achieved by re-duction of the intravesical pressure during instil-lation by reducing the volume of fluid used. Wehave no evidence at present that increased levelsof PpIX in the kidney, both at cellular and sub-cellular levels, do any harm in the absence of light,but this does need further investigation. Defini-tive answers can be provided only by examiningchanges in subcellular structure or the presence oftissue specific substances (such as Tamm Horsfallprotein) in the renal tubule or the glomerulus afterprolonged contact with ALA and PpIX.

Fig. 11. Microscopic picture of abdominal skin. A. Skin sec-tion with light only (50J/cm2) but without ALA shows nodetectable lesion (H&E stain, ×60). B. Mild sloughing of epi-dermis with keratolysis and with inflammatory cell infiltra-tion in the connective tissue after intravesical sensitizationwith 200 mg/kg ALA and 50J/cm2 (H&E stain, ×60). C. Ex-tensive destruction of epidermis and dermis after oral sensi-tization with 100 mg/kg ALA and 50J/cm2 (H&E stain, ×150).

262 Chang et al.

Using fluorescence microscopy for quantita-tive studies of the tissue distribution of photosen-sitizers, there is always concern that the resultsmight not be as accurate as using a chemical ex-traction method. However, the advantage is thatfluorescence levels in the various tissue layerscan be resolved, whereas extraction techniquesonly provide an averaged, although absolute,measurement. It could be argued that fluores-cence quenching may differ between layers, butprevious studies with ALA on rat colon and stom-ach do not support this contention as microscopicfluorimetry showed good correlation with abso-lute levels measured separately in the mucosaand muscle [25]. A similar study on the bladderwould not be feasible because of the thinness ofthe urothelium. Further, it may not be reliable tocompare the absolute porphyrin levels in differentorgans solely on the basis of fluorescence micros-copy, especially if several different porphyrin spe-cies are present. However, using a chemical ex-traction method, Schoenecker et al. [26] foundthat the concentration of liver and kidney porphy-rins 4 hours after intraperitoneal injection of 200mg/kg ALA were 35.8±10.1 and 20.1±11.3 mg/mg, respectively. The ratio of liver/kidney concen-tration of porphyrins was 1.8 (range 1–5.2) intheir study, which is roughly comparable to ourfigure of 2.6 using fluorescence microscopy. Thusit sees likely that the fluorescence measurementsare giving reasonably accurate values for the rel-ative levels of porphyrin between organs and be-tween different layers of each organ.

In this study we demonstrated that lightalone was not able to induce any detectable skinlesion on macro or microscopic examination. How-ever, the degree of epidermolysis and keratolysiswas considerably more prominent in rats receiv-ing oral ALA (100 or 200 mg/kg) than with blad-der instillation. Our study on the skin was verylimited, but it does suggest that there is less cu-taneous photosensitivity giving the ALA intra-vesically. This is not a major clinical problem, asphotosensitivity clears within 1–2 days anyway,but it is another factor in favor of bladder instil-lation.

Another possible advantage of intravesicalALA for photodynamic management of urinarybladder cancer with field change disease is theseemingly better selectivity achievable betweenthe urothelium and underlying layers over thatwith oral ALA. The resultant subepithelial fibro-blast infiltration 7 days after PDT is apparentlymore with oral ALA-based PDT. Further evalua-

tion of the bladder at 6 months with VanGiesonstain showed more collagen fibrils in the laminapropria after oral ALA sensitization than instilla-tion, although the animals in the oral groupwere given 400 mg/kg rather than the 100 mg/kgthat would have given comparable urothelial lev-els of PpIX. The actual role of collagen in thebladder is poorly defined, but less collagen depos-ited in the lamina propria is likely to mean lesschance for the bladder to become functionally im-paired as a result of altered elasticity.

Management of bladder cancer, unlike mostother malignancies, is focused primarily on theprevention of recurrence, which is estimated ashigh as 40–60% at 12 months after initial treat-ment of papillary tumors [27]. Measures such asintravesical chemotherapy with antineoplasticagents [28] and Bacillus Calmette-Guerin (BCG)[29] have been tried with some success. However,for effective prevention of recurrence, the best op-tion is to replace the diseased urothelium, whichmay be in patches or be generalised, with healthytransitional cell lining. PDT of the bladder offersthe possibility of achieving this by destroyingurothelium without damaging the underlyingmuscle and repairing the necrosed area with re-generation of normal tissue. Our experimentssuggest that PDT with ALA has potential fortreating dysplasia of the urothelium, even if it isgeneralised, and for treating superficial bladdercancers (provided the PpIX reaches the deeperparts of the tumor), or for prevention of recur-rence. The preferred route of administration is in-travesical as urothelial levels of PpIX are moresustained, systemic effects are less and cutaneousside effects are milder than after oral administra-tion.

ACKNOWLEDGMENTS

Dr. Shi-Chung Chang is funded by the Com-passion Relief of Tzu-Chi Foundation in Taiwan,and Professor Bown by the Imperial Cancer Re-search Fund, London, UK.

REFERENCES

1. Pass HI. Photodynamic therapy in oncology: mechanismsand clinical use. J Natl Cancer Inst 1993; 85:443–456.

2. Marcus SL. Clinical photodynamic therapy: the continu-ing evolution. In: Henderson BW, Dougherty TJ, eds.Photodynamic Therapy: Basic Principles and ClinicalApplications. New York: Marcel Dekker, 1992, pp 219–268.

3. Foote CS. Photosensitized oxidation and singlet oxygen:

PDT with Intravesical and Oral ALA 263

consequences in biological systems. In: Pryor WA, ed.Free Radicals in Biology, Vol. II. New York: AcademicPress, 1976, pp 85–124.

4. Amano T, Prout GR, Lin CW. Intratumor injection as amore effective means of porphyrin administration forphotodynamic therapy. J Urol 1988; 139:392–395.

5. Kennedy JC, Pottier RH. Endogenous protoporphyrin IX,a clinically useful photosensitizer for photodynamic ther-apy. J Photochem Photobiol B:Biol 1992; 14:275–292.

6. Chang SC, MacRobert AJ, Bown SG. Biodistribution ofprotoporphyrin IX in rat urinary bladder after intraves-ical instillation of 5-aminolevulinic acid. J Urol 1996;155:1744–1748.

7. Malik Z, Lugaci H. Destruction of erythroleukaemic cellsby photoactivation of endogenous porphyrins. Br J Can-cer 1987; 56:589–595.

8. Peng Q, Moan J, Warloe T, Nesland JM, Rimington C.Distribution and photosensitizing efficiency of porphy-rins induced by application of exogenous 5-aminole-vulinic acid in mice bearing mammary carcinoma. Int JCancer 1992; 52:433–443.

9. Bedwell J, MacRobert AJ, Phillips D, Bown SG. Fluores-cence distribution and photodynamic effect of ALA-in-duced PpIX in the DMH rat colonic tumour model. Br JCancer 1992; 65:818–824.

10. Kennedy JC, Pottier RH. Endogenous protoporphyrin IX,a clinically useful photosensitizer for photodynamic ther-apy. J Photochem Photobiol B:Biol 1992; 14:275–292.

11. Cairnduff F, Stringer MR, Hudson EJ, Ash DV, BrownSB. Superficial photodynamic therapy with topical5-aminolaevulinic acid for superficial primary and sec-ondary skin cancer. Br J Cancer 1994; 69:605–608.

12. Regula J, MacRobert AJ, Gorchein A, Buonaccorsi G,Thorpe SM, Spencer GM, Hatfield ARW, Bown SG. Pho-tosensitisation and photodynamic therapy of oesoph-ageal, duodenal, and colorectal tumours using 5 amino-laevulinic acid induced protoporphyrin IX—a pilot study.Gut 1995; 36:67–75.

13. Mlkvy P, Messmann H, Regula J, Conio M, Pauer M,Millson CE, MacRobert AJ, Bown SG. Sensitization andphotodynamic therapy of esophageal, duedenal, and co-lonic tumors with 5-aminolaevulinic acid (ALA) inducedprotoporphyrin IX (PpIX). In: Cortese DA, ed. Proceed-ings of 5th International Photodynamic Association Bi-ennial Meeting. Washington: SPIE, 2371, 1994, pp 370–374.

14. Harty JI, Amin M, Wieman TJ, Tseng MT, Ackerman D,Broghamer W. Complications of whole bladder dihemato-porphyrin ether photodynamic therapy. J Urol 1989; 141:1341–1346.

15. Chang SC, MacRobert AJ, Bown SG. Photodynamic ther-apy on rat urinary bladder with intravesical instillationof 5-aminolevulinic acid. Light diffusion and histologicalchanges. J Urol 1996; 155:1749–1753.

16. Chan WS, MacRobert AJ, Phillips D, Hart IR. Use ofcharge coupled device for imaging of intracellularphthalocyanines. Photochem Photobiol 1989; 50:617–624.

17. Pottier RH, Chow YFA, LaPiante JP, Truscott TG,Kennedy JC, Beiner LA. Noninvasive technique for ob-taining fluorescence excitation and emission spectra invivo. Photochem Photobiol 1986; 44:679–687.

18. Hisazumi H, Misaki T, Miyoshi N. Photoradiation ther-apy of bladder tumors. J Urol 1983; 130:685–687.

19. Benson RC Jr, Kinsey JH, Cortese DA, Farrow GM, UtzDC. Treatment of transitional cell carcinoma of the blad-der with hematopoprhyrin derivative phototherapy. JUrol 1983; 130:1090–1095.

20. D’Hallewin MA, Baert L, Marijnissen JPA, and StarWM. Whole bladder wall photodynamic therapy with insitu light dosimetry for carcinoma in situ of the bladder.J Urol 1992; 148:1152–1155.

21. Lowdell CP, Gilson D, Ash DV, Holroyd JA, Vernon D,Brown SB. An attempt to reduce skin photosensitivity inclinical photodynamic therapy using oral activated char-coal. Lasers Med Sci 1992; 7:351–356.

22. Bridges JW, Sargent NS, Upshall DG. Rapid absorptionfrom the urinary bladder of a series of n-alkyl carbamate:a route for the recirculation of drug. Br J Pharmac 1979;66:283–289.

23. Morris CR, Bryan GT. Absorption by the mouse and raturinary bladder of glycin, tryptophan and glucose. Na-ture 1966; 210:857–858.

24. Loh CS, MacRobert AJ, Bedwell J, Regula J, Krasner N,Bown SG. Oral versus intravenous administration of5-aminolaevulinic acid for photodynamic therapy. Br JCancer 1993; 68:41–51.

25. Loh CS, Vernon D, MacRobert AJ, Bedwell J, Bown SG,Brown SB. Endogenous porphyrin distribution inducedby 5-aminolaevulinic acid in the tissue layers of the gas-trointestinal tract. J Photochem Photobiol B: Biol 1993;20:47–54.

26. Schoenecker JA Jr, Egger NG, Anderson KE, GourleyWK, Weinman SA. Photosensitizer accumulation kinet-ics and photoxicity in aminolevulinic acid (ALA) treatedhepatocytes and hepatoma cells in vivo and in vitro. Gas-troenterology 1994; 106(suppl): A978(abstract).

27. Hall RR, Parmar MKB, Richards AB, Smith PH. Pro-posal for changes in cystoscopic follow up of patients withbladder cancer and adjuvant intravesical chemotherapy.BMJ 1994; 308:257–260.

28. Nseyo UO, Lamm DL. Therapy of superficial bladder can-cer. Current Opinion Urol 1994; 4:275–280.

29. Herr HW, Laudone VP, Badalament RA, Oettgen HF,Sogani PC, Freedman BD, Melamed MR, Whitmore WFJr. Bacillus Calmette-Guerin therapy alters the progres-sion of superficial cancer. J Clin Oncol 1988; 6:1450–1455.

264 Chang et al.