Photochemistry and Photobiology Vol. 55, No. 1, pp, 145-157, 1992

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REVIEW ARTICLE

HOW DOES PHOTODYNAMIC THERAPY WORK? BARBARA W. HENDERSON* and THOMAS J. DOUGHERTY Division of Radiation Biology, Roswell Park Cancer Institute, Buffalo, NY, USA

(Received 16 April 1991; accepted 10 June 1991)

INTRODUCTION

Those readers already familiar with the field of photodynamic therapy (PDT)t will consider this title somewhat presumptuous since it implies that the answer to the posed question is known. Indeed, answers to many questions regarding PDT have been found over the past decade, but a comprehensive understanding of all mechanisms involved in PDT tumor destruction has not yet emerged. This paper will attempt to deal with this complex subject by giving a sequential account of the effects occurring during PDT tissue treatment on a cellular and tissue level. Photodynamic therapy is based on the dye-sensitized photooxidation of biological matter in the target tissue (Foote, 1990). This requires the presence of a dye (sensitizer) in the tissue to be treated. Although such sensitizers can be naturally occurring constituents of cells and tissues, in the case of PDT they are introduced into the organism as the first step of treatment. In the second step, the tissuelocalized sensitizer is exposed to light of wavelength appropriate for absorption by the sensitizer. Through various photophysical pathways, also involving molecular oxygen, oxygenated products harmful to cell function arise and eventual tissue destruction results. In keeping with the chronological nature of this review, the subject matter will be divided into the

following sections: sensitizer administration and distribution in cells and tissues, light delivery and distribution in tissues, the photodynamic effect, acute effects of PDT on cells and tissues, and finally, late effects of PDT. The ever-increasing number of novel sensitizers makes it necessary that we confine our discussions to those with at least proven preclinical usefulness for PDT. We will also try to focus as much as possible on mechanisms known to be relevant to the in vivo PDT response, although this will force us to pass over many intriguing aspects of PDT on the molecular level (Gomer et al., 1991). SENSITIZER DELIVERY AND DISTRIBUTION IN CELLS AND TISSUES

The first step towards PDT tumor treatment is the delivery of the photosensitizer to the target tissue. The radiolabeling of sensitizers while maintaining their photophysical, photochemical and biological characteristics, has allowed detailed preclinical studies of pharmacokinetics and tissue distribution for Photofrin I1 (PII) (Bellnier et al., 1989c), the sensitizer of current clinical use, as well as certain sulfonated phthalocyanines (PcS) (Rousseau et al., 1990), mono-L-aspartyl chlorin e6 (NPe6) (Gomer and Ferrario, 1990) and benzoprophyrin derivative (BPD-MA) (Richter et al., 1990). In mice, [14C]PII is cleared from the blood with kinetics fitting a triexponential equation with elimination half-lives of 4 h, 9 days and 36 days. At 24 h after injection, about 1% of the total injected ‘To whom correspondence should be addressed. remains in the circulation, with some tAbbreviationsF:ALA, aminolevulinic acid; A P C S , ,,,~ , ,,, , ~ ,material ~ chloroaluminum phthalocyanine, mono, di, tri, tetra or material (approx. 0.0lY0) still detectable at 75 days. mixed sulfonation; bChla, bacteriochlorophyll a ; BPD- More than 65% of the administered sensitizer is MA, benzoporphyrin derivative-monoacid; EDKC, N , N’-bis(2-ethyl-l,3-dioxolane)kryptocyanine;EMT6, ex- excreted in the feces over 192 h. Similar clearance perimental mouse mammary tumor; ESR, ear swell- kinetics have been determined for I4C-labeled diing response; FaDu, human squamous carcinoma; and trisulfonated gallium PcS (GaPcS2 and GaPcS3) Ga phthalocyanine, di or trisulfonated; iso- with circulating material still being detected 21 days BoSiNc, bis(di-i-butyloctadecylsiloxy)silicon 2,3-naph- after injection into mice. [3H]BPD-MA in mice is thalocyanine; LDH, lactate dehydrogenase; LDL, low density lipoproteins; MPH, methylpheophorbide hexyl still detectable in the blood at 168 h post injection, ether; NPe6, MACE, mono-L-aspartyl chlorin e6; PII, while [I4C]NPe6 drops close to the limits of detecPhotofrin 11; PcS, sulfonated phthalocyanines; PDT, tion by 96 h. photodynamic therapy; RIF, radiation induced fibroMost sensitizers studied to date are distributed to sarcoma; SMTF, spontaneous mammary tumor, fast and are retained by normal as well as neoplastic growing; SnETZ, Sn etiopurpurin;TNF, tumor necrosis factor; TPPS, o, ., tetraphenylporphine, mono or tetra tissues. In mice, [14C]PII was found, in order of decreasing sensitizer levels, in the following normal sulfonated; ZnPC, Zn phthalocyanine. 145

BARBARA W. HENDERSON and THOMAS J. DOUGHERTY

146

tissues: liver, adrenal gland, urinary bladder > pancreas, kidney, spleen > stomach, bone, lung, heart > skin > muscle brain. Very similar distributions were found in mice for the other above-mentioned sensitizers, as well as for sulfonated chloroaluminum phthalocyanine (AlPcS,) by Chan et al. (1988), who used extraction and fluorescence spectroscopy of unlabeled material. As a rule it can be stated that these, and practically all other so far studied sensitizers have a pronounced affinity for all tissues high in reticulo-endothelial components. What varies considerably between sensitizers are the time intervals between administration and peak sensitizer tissue levels, and sensitizer retention in tissues. While peak NPe6 levels are reached between 2 and 60 min and peak BPD-MA levels at 3 h after injection, it takes 5-10 h and 24-48 h respectively for PI1 and GaPcS. Photofrin I1 and GaPcSs have cleared only insignificantly from normal tissues by 48 h post injection, with, for example, 61% of peak PI1 levels in the spleen and about 30% in lung still being present 75 days after drug administration. In contrast, BPD-MA levels have diminished by 50-60% and Npe6 concentrations by 8S-9OYo of peak levels in spleen at 48 h. It is of interest to note that while sensitizers such as PII, PcS and Npe6 are cleared with little or no detectable drug metabolism, BPDMA seems to be metabolized in vivo to an inactive form. Bacteriochlorophyll a (bChla), also an effective sensitizer, is metabolized to various, also photodynamically active, materials (Henderson et al., 1991a). These differences in sensitizer uptake and clearance in normal tissue have important implications for PDT treatment, in particular where PDT late effects (especially skin photosensitivity) are concerned. From the above it is clear that sensitizers do not accumulate selectively in neoplastic tissues. Nevertheless, some treatment selectivity is observed in rodent tumor models, and seems to be much more pronounced in certain human neoplastic conditions, such as malignant skin lesions. Although the reasons for the at least partially selective tumor response are not totally understood, they are likely to be due to some degree of differential sensitizer concentration between tumor and surrounding normal tissues. Ratios of tumorlskin for subcutaneously or intradermally implanted rodent systems differ between sensitizers as well as tumor types. Chan et al. (1988), determining AIPcS, levels 24 h after injection, found ratios of about 2:l for the mouse UV-2237 fibrosarcoma, but of about 1O:l for the Colo 26 carcinoma. Photofrin I1 tumorkkin ratios characteristically are less than 2: 1 in transplantable rodent tumors (Bellnier et al., 1989~).We have recently found that PI1 uptake in subcutaneous, mixed sarcomas, arising spontaneously in transgenic mice, was identical to that in subcutaneous, implanted tumor systems (unpublished). It is possible that very different ratios might be found in

*

tumors arising from tissues other than skin or muscle-such preclinical systems have not been extensively studied, with the exception of glioma models, which show large differentials between tumor and normal brain due to the breakdown of the blood-brain barrier (Kaye and Hill, 1990). The pancreas represents the unusual situation of little or no sensitizer differential between normal and neoplastic pancreas-both for PI1 and AlPcS,-but a large differential in treatment response (Mang and Wieman, 1987; Chatlani er al., 1990). The reasons for this phenomenon are not yet clear, but may have to d o with distribution of sensitizer among different tumor compartments and/or different sensitivities of normal and tumor-derived cells (Mathews and Cui, 1990). An entirely different class of sensitizers, the cationic dyes, some of which show high specificity for carcinoma cells in virro (Oseroff ef al., 1986), have so far shown disappointing tumor accumulation in vivo-an iodinated rhodamine-123 showed no differential between skin and several different implanted tumor types (Kinsey et al., 1989), but some novel cationic compounds may hold promise for the future (Cincotta et al., 1990). A highly innovative idea of sensitizer “delivery” to tumors has recently been advanced by Kennedy et al. (1990), where the topical administration of 5aminolevulinic acid (ALA), a precursor of protoporphyrin IX, is used to stimulate the local biosynthesis of this endogenous photosensitizer in skin cancer lesions. While overall tissue uptake of sensitizers has been fairly well analyzed, the sensitizer distribution within the tumor tissue has not yet been well defined. In fact, this aspect of PDT is hazy and confusing, as the following examples will illustrate. In the stroma-rich SMTF tumor in mice the sensitizer ratio of hematoporphyrin derivative, the forerunner of PII, between stroma and tumor parenchyma has been found to be about 5:l (Bugelski et al., 1981). In the stroma-poor RIF mouse tumor 90% of total tumor PI1 can be recovered in the isolated tumor cell portion (Henderson and Fingar, 1989). Yet, overall PI1 accumulation and response to PDT in these tumor models are nearly identical. Similarly, the more hydrophilic sulfonated derivative of tetraphenylporphines (TPPS4) was found to distribute preferentially to tumor stroma in a subcutaneous pancreatic mouse tumor model, while the more lipophilic derivative (TPPS,) localized in the parenchyma-yet, again, overall tumor content with these compounds was the same (Kessel et a / . , 1987). In vitro photodestruction of isolated tumor cells after in vivo drug exposure indicates some degree of tight binding to tumor cells for a wide range of sensitizers tested (Fig. 1). In addition to tumor cells, binding of sensitizers to endothelial cells has long been assumed, and Weintraub et al. (1988) detected AlPcS, localized in sinusoidal endothelial cells of mouse spleen and lymph nodes. As already indi-

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147

degree of drug lipophilicity tends to favor binding to LDL, while hydrophilic sensitizers bind more avidly to albumin, other factors such as aggregation properties, charge distribution and polarity etc. also play important roles (Kongshaug et ol., 1989). The lo-' limited applicability of in vitro drug uptake studies to the in vivo situation is best illustrated by work with PcS with different degrees of sulfonatiow while the tetrasulfonated compounds proved com10-2 pletely inactive in vitro due to their hydrophilicity and resultant exclusion from cells, they produced considerable tumor and tumor cell photosensitization after in vivo drug exposure (Fig. 1) (Brasseur et al., 1988). Prebinding of sensitizer to lo-' 0 10 20 30 40 50 60 7 0 80 various lipoprotein fractions has shown a significant, 2 . but very transient, improvement of overall tumor/ J/cm in vitro skin ratios for [14C]BPD-MA, but whether tumor Figure 1. Photosensitization of RIF tumor cells after cell uptake was increased was not determined accumulation of sensitizer in vivo for 24 h and illumination (Allison et ol., 1990). Sensitizer delivery to LDL is in virro after tumor cell isolation. Conditions: isoBoSiNc also being pursued for Zn-phthalocyanine (ZnPc) (0.13 pmol/kg in cremophore, 775 nm), bChla (16.2 pmoVkg in Tween 80, 780nm), MPH (0.4 pmoUkg in by incorporating the material into liposomes of Tween 80, 665 nm), PI1 (10 mg/kg in HBSS, 630 nm), specific lipid composition, which favors transfer AIPcS,.,.,,,, (6.8 pmoVkg in HBSS, 680 nm). These drug of the sensitizer to LDL (Ginevra et al., 1990). doses lead to complete tumor response (complete disap- We have recently delivered the Si-naphthalocyanine pearance of tumor bulk within 24-48 h; this does not necessarily imply tumor cure) when combined in vivo with isoBoSiNc in mice in vivo via cremophore oil emulsion or small unilamellar liposomes of varying lipid 135 J/cm2 of external light. composition, assuring that drug was present in unaggregated form, and found no difference in tumor cated by organ distribution studies, photosensitizers cell sensitization and overall response (Mayhew et accumulate to the highest degree in components of al., 1990). the reticulo-endothelial system, which include free The subcellular distribution of sensitizers, studied and fixed macrophages in the connective tissue, mainly in in vitro systems, will only be briefly dealt Kupffer cells of the liver, and red pulp macrophages with here, since there are no indications yet that it of the spleen (Chan et al., 1988; Bugelski et al., matters greatly with regard to the in vivo effective1981). Mast cells have been found to also retain ness. A case in point may be found in in vitro studies high levels of sensitizer (Bugelski, unpublished). with PI1 and NPe6, where PII, after initial binding Surprisingly, the formed blood elements seem to to the plasma membrane, eventually redistributes stay largely sensitizer-free, despite their direct to other lipophilic membrane sites, and where NPe6 access to the highest sensitizer concentrations preferentially localizes in lysosomes (Roberts et al., (Bellnier et al., 1989c; Chan et al., 1988). In addition 1988)-yet both can cause efficient tumor tissue to the cellular tumor components, sensitizers also destruction (Nelson et al., 1988). However, more may bind to the fibrous tissue matrix (collagen, subtle consequences of intracellular sensitizer distrielastic and reticular fibers) (Musser et al., 1982). bution may yet be detected. As pointed out in an There has been much speculation on the mechan- excellent overview by Moan et al. (1989), lipophilic, ism of increased tumor accumulation and/or reten- anionic dyes generally localize in membrane struction of photosensitizers, not the least since under- tures (including plasma, mitochondrial, endoplasstanding of these mechanisms might allow us to mic reticulum and nuclear membranes), while further improve tumor targeting of sensitizers, and hydrophilic materials seem to accumulate in lysowith it treatment selectivity. A most likely under- somes. Certain cationic sensitizers accumulate preflying factor is simple pooling of sensitizer in tumors erentially in mitochondria due to electrical potential due to leaky tumor vasculature and poor lymphatic gradients across the mitochondrial membrane drainage. A more specific mechanism of uptake (Oseroff et al., 1986). via a lipoprotein pathway has also been suggested, based on the heightened low-density lipoprotein LIGHT DELIVERY AND DISTRIBUTION IN TISSUES (LDL) receptor mediated endocytosis activity of neoplastic cells. This has stimulated extensive As the second component for PDT tumor treatresearch concerning the binding and distribution ment it is necessary to bring photosensitizer-activaproperties of sensitizers among the various plasma ting light to the target tissue. The scientific basis protein classes, and their imphcations for sensitizer involved and technology to achieve this have been delivery (Kessel, 1986; Jori, 1989). Although a high recently reviewed by Wilson (1989), and will only 1 oo

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BARBARA W. HENDERSON and THOMAS J. DOUGHERTY

be dealt with briefly here. Light thence in tissue decreases exponentially with the distance; the effective penetration depth is inversely proportional to the effective attenuation coefficient (a = 1 / depth). ~ The latter is influenced by the optical absorption (due to endogenous tissue chromophores, mainly hemoglobin) and by optical scattering within the tissue. Both of these parameters differ from tissue to tissue, with liver, for example, affording especially poor light penetration due to its high hemoglobin content, and brain tissue being particularly light scattering. On the average, however, a is about 1-3 mm at 630 nm, the wavelength used for clinical treatment with PII, while penetration is approximately twice that at 700-850 nm (Svaasand, 1984; Wilson er al., 1985). The increased penetration depth of longer wavelength light is the major incentive for the deveiopment of sensitizers absorbing at such wavelengths, and isoBoSiNc (776 nm) and bChla (780 nm) fall into this category (Firey and Rodgers, 1987; Borland er al., 1987). The absorption of light by the photosensitizer itself can limit tissue light penetration-this phenomenon has been termed “self-shielding” and is particularly pronounced with sensitizers which absorb very strongly at the treatment wavelength (Wilson er al., 1986; Dougherty and Potter, 1991). Many sensitizers are prone to photo-destruction during light exposure, a process called “photobleaching” (Potter er a[., 1987; Svaasand et a [ . , 1990). This not only counteracts its self-shielding properties, but may have beneficial effects regarding treatment differential. These are based on the following considerations: there exists a threshold PDT dose to produce tissue necrosis (Fingar ef al., 1987; Fingar and Henderson, 1987; Patterson er al., 1990a). If photobleaching occurs (which does not have such a threshold) before this threshold is achieved, no tissue damage is incurred. This is clearly a desirable situation for normal tissue exposed to therapeutic light during therapy-it is clearly an undesirable situation for the tumor tissue to be treated. However, it is possible to take advantage of the differential sensitizer uptake such as between tumors involving the skin and normal skin. While for PI1 in mice this ratio is near unity, in humans it is estimated to be 5-10 (Potter, unpublished). Figure 2 is a plot of E, photodynamic dose, vs depth, 2 (expressed in terms of the attenuation coefficient a) for different combinations of drug and light doses. At the highest drug dose (2.0 mg/kg) there is an exponential fall-off of E with depth since at this PI1 dose photobleaching is minimal at the highest acceptable normal tissue dose (36 J/cm2). However, at a drug dose of 1.0 mg/kg, where photobleaching becomes significant, a threshold is found which extends from a2 = 1 (Jo = 200 J/cm2) to a 2 = 2.5 (Jo = lo00 J/cmZ).If a = (U3) mm-’, then 2 = 3 and 7.5 mm respec-

I

I

/3=0.036cm2/joul

30

-0

n

1

2

3

4

5

2 6

aZ Figure 2. Graphs of the photodynamic dose, E , as a function of depth from the tissue surface, Z . The horizontal axis is in units of aZ,a dimensionless number. Knowledge of a, the total attenuation coefficient of the tissue allows a scale to be assigned to the horizontal axis. If a = (113) mm-I, then the aZ axis is marked in units of 3 mm and aZ = 2 would correspond to Z = 6 mm (from Potter er al., Photochem. Photobiol. 46,97, 1987).

tively. Thus, the net result is that one can achieve greater depth of tumor necrosis while sparing the normal skin. The realization of the complex interplay between all these parameters, sensitizer distribution and oxygen during PDT has given rise to the concept of “total photodynamic dose”, and efforts are under way to design and build instruments which will allow the monitoring of all these aspects during PDT treatment (Profio and Doiron, 1987; Wilson, 1989). THE PHOTODYNAMIC EFFECT

Similar to ionizing radiation, the damage-initiating interactions of PDT occur within a very small time frame. It must be kept in mind that photodynamic interactions will take place wherever sensitizer, light of appropriate wavelength and oxygen are present simultaneously. Briefly, following the absorption of light, the sensitizer is transformed from its ground state (singlet state) into an electronically excited state (triplet state, lifetime 10-3-10 s) via a short-lived excited singlet state. The excited triplet can undergo two kinds of reactioncit can react directly either with substrate or solvent by hydrogen atom or electron transfer to form radicals and radical ions, which after interaction with oxygen can produce oxygenated products (Type I reaction); or it can transfer its energy to oxygen directly to form singlet oxygen (lo2,lifetimes 4 x s in water, 50-100 x s in lipid, 0.6 x s in cellular environment), a highly reactive, oxidative species (Type I1 reaction) (Foote, 1990; Moan, 1990). Type I and Type I1 reactions may occur simultaneously, and the ratio between

Review Article

the two processes is highly influenced by sensitizer, substrate and oxygen concentration, as well as the binding of sensitizer to substrate. There is much indirect evidence to suggest that singlet oxygen is the major damaging species in PDT, but direct measurement of singlet oxygen production in complex biological systems appears to be extremely difficult (Patterson et al., 1990a), and most indirect methods such as the use of chemical quenchers of reactive intermediates or D 2 0 (which prolongs the singlet oxygen lifetime and so can increase photosensitization) are not entirely specific for singlet oxygen (Weishaupt et al., 1976; Henderson and Miller, 1986; Truscott et al., 1988; Foote, 1990). In particular, indications are that superoxide ion (0I ) may be involved in some aspects of PDT damage (Athar et a f . , 1989). From the above it is evident that PDT effects should be oxygen-dependent. This is indeed the case for most sensitizers (Moan and Sommer, 1985; Henderson and Fingar, 1987; Brasseur et al., 1985), with the exception of some cationic sensitizers such as the cyanine dye EDKC (Oseroff et al., 1986), which seems to act by oxygen-independent mechanisms. For PI1 photosensitization of cells in vitro, full effects are observed at about 5% O2 levels, with a half-value at about 1% 02.No photosensitization can be observed in the absence of measurable oxygen. Induction of tissue hypoxia in vivo by clamping also abolishes any PDT effects (Gomer and Razum, 1984). Thus, similar to ionizing radiation effects, where hypoxic cells are less sensitive than well oxygenated ones, one might expect tissue oxygenation levels to play a significant role in influencing PDT treatment effectiveness. ACUTE EFFECTS OF PHOTODYNAMIC THERAPY ON CELLS AND TISSUES

Cellular effects

Singlet oxygen lifetime and diffusion distance, in a cellular environment, are limited by its avid reactivity with and quenching by cell constituents such as histidine, tryptophan, cholesterol, etc. Moan (1990) has estimated the diffusion distance in cells to be about 0.1 pm. Therefore, cell damage mediated by '02will occur close to its site of generation, and can-due to the wide and varied sensitizer distribution within cells-affect virtually all cellular components. For lipophilic, anionic dyes, this means damage of membranes in general, including the plasma-, mitochondrial-, lysosomal-, endoplasmic reticulum- and nuclear membranes (Moan et al., 1989). Photoperoxidation of membrane cholesterol and other unsaturated phospholipids leads to changes in membrane permeability, loss of fluidity, cross-linking of aminolipids and polypeptides, and inactivation of membrane associated enzyme systems and receptors (Girotti, 1990). While the inhi-

149

bition of mitochondrial enzymes has frequently been considered a key event in PDT cell lethality (Gibson et al., 1989; Salet and Moreno, 1990), there is evidence that the inactivation of membrane transport systems, depolarization of the plasma membrane and inhibition of DNA repair enzymes may precede inactivation of mitochondrial, cytosolic and lysosoma1 enzymes (Moan et al., 1989; Boegheim et a f . , 1987; Specht and Rodgers, 1990). Since hydrophilic sensitizers localize somewhat preferentially in lysosomes, disruption of these structures and resulting release of hydrolytic enzymes into the cytoplasm seem to be the major cause of cell lethality with such compounds as NPe6 or tetrasulfonated tetraphenylporphine (TPPS4) (Roberts et al., 1989a; Moan et al., 1989). The preferential mitochondrial accumulation of cationic sensitizers accounts for the predominance of mitochondrial damage induced by these dyes (Salet and Moreno, 1990). In view of the almost non-detectable uptake of, at least, the more lipophilic porphyrin and phthalocyanine sensitizers inside the cell nucleus, the development of documented DNA damage (DNA strand breaks, alkalilabile lesions, DNA-DNA and DNA-protein crosslinking, sister chromatid exchanges) poses an interesting question (Gomer, 1980; Ramakrishnan et al., 1989). Moan has recently suggested that the damage may originate at the nuclear membrane (Moan et al., 1989). Whatever the primary lethal insult(s) may be, with PI1 the consequence is a rapid loss of cell integrity. When monitored by "Cr-release assay, cell disintegration after lethal PII-PDT treatment in vitro is complete within 4 h (Bellnier and Dougherty, 1982). However, leakage of lactate dehydrogenase (LDH) from PDT treated cells commences practically immediately following light exposure, and is complete within 30 min (Henderson and Donovan, 1989). Associated with this cell membrane damage is the release of inflammatory and immune mediators. A wide range of eicosanoids are released in parallel with LDH from all cell types studied in vitro following PII-photosensitization (Table l), and this process can be inhibited by blockage of phospholipase A2 as well as prostaglandin endoperoxide synthetase pathways (Lim et al., 1986; Henderson and Donovan, 1989). Histamine, another inflammatory mediator, is liberated by PDT through mast cell degranulation (Kerdel et al., 1987). All of these substances are potent, fast acting and vasoactive-either constrictive or dilatory-and evidence exists to implicate them in the development of PDT induced vascular damage to be discussed below. Release of the procoagulant Factor VIII from AlPcS,-sensitized endothelial cells in vitro has also been observed following within 10 min of light treatment (Ben Hur et af., 1988). Finally, the release of tumor necrosis factor (TNF), which by itself can cause vascular damage, from PII-sensitized macrophages has been found to peak within 3-6 h

BARBARA W. HENDERSON and THOMAS J. DOUCHERW

150 Table 1

Cell type

Sensitizer Eicosanoids released

EMT6 (mouse mammary carcinoma)

PI1

0

PGEZ

> Q)

.c PGEZ, lZHETE, HHT. LTBI, AA

stimulated

PI1 PI I

PGEZ, l2HETE IZHETE, HHT. AA

Endothelium: bovine

PI1

6Keto-PGF,,' I2HETE PGF,, &Keto-PGF,,,

0 -

0 [L

24

human

PI I

human?

UroJ Pp8 PGF2,

AA

Rat mast cellst

v) Q)

3 -

PI1 RIF (mouse fibrosarcoma) PI I FaDu (human squamous carcinoma) Macrophages: resident

PGE,

PP

PGDZ, PGE2, &Keto-PGF,,

*6-Keto-PGF,, is a stable metabolite of PGII.

tH.W. Lim (1989). SUroporphyrin. IProtoporphyrin.

after light exposure in vitro, making it somewhat unlikely that it would be involved in the most immediate PDT response (Evans et af., 1990). The acute, direct, lethal effects on tumor cells, as on all cells, will depend on the localization of the sensitizer on or in the cells, the photodynamic efficiency of the sensitizer in that environment, the light dose reaching the cells, and the kinetics of vascular occlusion and oxygen supply. The direct tumor cell photosensitizing potential under optimal light and oxygenation conditions can be analyzed by in vivolin vitro techniques for given therapeutic in vivo doses of different sensitizers (Fig. 1). Among these, phthalocyanines show the greatest promise for direct tumor cell attack, while bChla and Si-naphthalocyanine (isoBoSiNc) show the least. Light and oxygen limitations will determine how much of that potential can be realized during in vivo treatment, as discussed below. Vascular effects

The most easily and rapidly discernable acute effects of PDT in vivo are of a vascular nature. The development of microscopic and macroscopic PDT tissue damage has been well described for a number of sensitizers, including PII, purpurins, phthalocyanines and Npe6 (Reed et al., 1988,1989a; Nelson et al., 1988; Morgan et al., 1990). Here, only several key points will be made, which pertain to the timeframe and succession of events-PI1 will be taken as an example. In experimental tumor systems, clumps of aggregated platelets may be observed in the microvasculature within seconds of light

48

72

96

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T i m e a f t e r s e n s i t i z e r i n j e c t i o n (h)

Figure 3. Changes in sensitizer plasma levels (-) and vascular photosensitivity (---) as a function of time after sensitizer administration. (B) TPPS, (1 FmoUkg), (0) MPH (0.4 FmoVkg), (V) PI1 (10 mgkg). Sensitizer plasma levels were determined spectroscopically. Vascular photosensitivity was assessed through fluorescein angiography of the mouse back skin (Fingar and Henderson, 1987) by determining the minimum light dose necessary to induce vascular shut-down in the treatment field. Fluorescein was injected by tail vein within 5 min of completion of light treatment and its distribution within the treatment field was assessed under UV light immediately thereafter. Light (648,665 and 630 nm respectively, 75 mW/cm2). exposure, followed by transient vasoconstriction, vasodilation and eventual complete blood stasis and hemorrhage. Gross edema and erythema are always the first signs of a PDT response, particularly where skin is involved in the treatment field. These changes usually precede any detectable tumor cell or endothelial cell damage. In the clinical situation, these vascular events are marked by blanching or hemorrhaging of the treated tissue. The time interval between initiation of damage and vascular occlusion may vary from tumor to tumor and with different sensitizers, but eventual vessel occlusion seems to be a general phenomenon accompanying PDT. Collapse of the treated tissue and scab formation ensues as early as 24 h post treatment. The major determinant for vascular photosensitivity appears to be the level of circulating photosensitizer. Vascular occlusion can be induced rapidly upon light exposure of tissue shortly after drug injection with most sensitizers tested, with the exception of cationic compounds. With time allowed between administration of sensitizer and illumination, however, the light doses necessary for vascular occlusion begin to vary greatly. If one follows the relative changes in plasma sensitizer levels and vascular photosensitivity from 1 h after dye injection onward, one can observe that these go hand in hand for the lipophilic compounds PI1 and MPH (Fig. 3). A very different pattern can be found for the very hydrophilic TPPS4, where vascular photosensitivity increases over 24 h post injection despite

Review Article

isoBoSiNc ‘

0

2

4

6

8

‘

‘

‘

‘

I

“

10 12 1 4 16 18 2 0 2 2 24

T i m e a f t e r s e n s i t i z e r i n j e c t i o n (h)

Figure 4. Changes in vascular photosensitivity as a function of time after sensitizer administration. TPPS, (1 pnolikg), MPH (0.4 FmoVkg), PI1 (10 mgkg), isoBoSiNc (0.13 pmoVkg). Vascular photosensitivity was assessed as in Fig. 3.

rapid initial plasma clearance. With further time the decline in vascular photosensitivity is consistent with decreased drug plasma levels. If one analyzes the early changes in vascular photosensitivity more closely, a peak in sensitivity at about 1 h post injection can also be detected for lipophilic compounds (degree of lipophilicity isoBoSiNc > MPH %-% TPPS4, PI1 is a mixture of differently lipophilic materials) (Fig. 4). These relationships seem to indicate the strong dependence on sensitizer solubility characteristics of sensitizer delivery and/or binding to a sensitive vascular target. The relationship between circulating sensitizer levels and vascular damage has been observed earlier by Bellnier and Dougherty (1989b) with PII, when the mouse ear swelling response was used as endpoint. Likewise, Reed et al. (1989a) have described massive mural thrombus formation and blood stasis in normal rat urinary bladder vasculature, if PII-PDT was carried out when blood drug levels were high, but only minor and short-lived effects, if tissue levels were high but blood levels were low. Once complete vascular occlusion has occurred, it seems to be irreversible, at least when assayed by fluorescein angiography , and tissue necrosis usually follows (Fingar and Henderson, 1987). It should be pointed out here that one can also distinguish between acute and delayed vascular occlusion; for example, while more than 250 J/cm2 are required for acute vessel shutdown (detectable at completion of light delivery) with bChla and isoBoSiNc (light 24 h post injection of a therapeutic drug dose in mice), vessels remain open during delivery of 135 J/cm*, but will undergo complete occlusion in the treatment field within 3-4 h post illumination. To detect the latter phenomenon, fluorescein angiography is carried out at hourly intervals after comPA? 5511-K

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pletion of light treatment, and the observed delayed vascular shut-down patterns appear not significantly different from the acute effects. These time relationships may profoundly influence PDT mechanisms, since they determine how much oxygen is available during light treatment for direct photodynamic killing of tumor cells. The consequences of vascular damage for the tumor microenvironment are severe. Light treatment in vivo of mouse and rat tumors, as well as normal tissues, after PI1 administration at therapeutic doses leads to reduction of blood flow velocity and induction of detectable hypoxic tumor cell fractions within minutes of illumination (Star et al., 1986; Reed et al., 1988; Henderson and Fingar, 1987,1989). Both of these effects increase with time during and after light treatment. This rapid shift of cells into hypoxia, where they are protected from further PDT damage due to the oxygen limitation of the photodynamic processes, is potentially limiting to direct tumor cell photodestruction. Induction of tumor hypoxia is, in keeping with vascular photosensitization, highly dependent on injected drug dose and less so on light dos-in the RIF mouse model, 24 h post drug injection, a PI1 dose of 10 mglkg and 45 J/cmZ will cause a hypoxic tumor cell fraction of about 9% within the light delivery time (10 min), while a PI1 dose of 100 mg/kg and 4.5 J/cmZ (1 min, same drugnight product) will induce 26% hypoxic cells. This deviation from the usual drug/light reciprocity is probably due to circulating sensitizer remaining after the high dose administration. The analysis of changes in tumor oxygenation during PDT has recently been carried further by Tromberg et al. (1990) through noninvasive, real-time monitoring of tissue oxygenation, utilizing transcutaneous oxygen electrodes on transplanted tumors in rabbits. These measurements distinguished three consecutive processesinitial consumption of oxygen through the photodynamic process, i.e. presumably production of ‘O2 from ground state molecular oxygen, followed by pathophysiologic alterations in regional blood supply (hypoxia) and finally total vascular occlusion (ischemia). Hypoxia may be reversible, depending on treatment dose. The limited reversibility of hypoxia may be exploitable for maintenance of tumor oxygenation during treatment by light fractionation schemes, which allow for short light interruptions and re-establishment of blood flow during the interval (Gibson et al., 1990). It must be emphasized, however, that all these cases must be taken as the models they are-the time frame of oxygenation changes in response to PDT will depend highly on the tumor type, probably the individual tumor, as well as the dose just discussed and time parameters. Thus, the blood flow in a rat ocular retinoblastoma-like tumor was found to initially increase in response to a therapeutic dose of PII-PDT (Horsman and Winther, 1989), and pre-

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liminary blood flow studies in human tumors have shown initial increases as well as decreases (Fingar, personal communication). That vascular destruction by itself, without any contribution from direct tumor cell effects, can lead to tumor cures in mice can be shown by exposing tumors to light immediately after sensitizer injecI tion, when blood drug levels are high and tumor cell localization of sensitizer is absent (Henderson and Bellnier, 1989). Fast clearing sensitizers, such AIPcS, as protoporphyrin and NPe6, can be made to work through this principle, and are only effective when treatment is delivered while circulating sensitizer is lo-’ high. This is consistent with the observation by 0 1 2 3 Denekamp et al. (1983), that nutritional deprivation T i m e post light t r e a t m e n t (h) of tumors through simple clamping of the blood supply for at least 15 h will lead to tumor cures. Delay or prevention of vascular occlusion and its Figure 5. Reduction in clonogenic RIF tumor cells as a physiologic consequences through pharmacological function of time after PDT tumor treatment in vivo. bChla means was found to also delay or prevent tumor (16.2 pmolkg), PI1 (10 mg/kg), AIPcS, (6.8 pmolkg); light (780, 630 and 680 nm respectively; 135 J/cm*, cell death in mouse and rat tumors, at least for 75 mW/cm2). Anoxia was induced by asphyxiation. PII-PDT (Fingar et al., 1988; Fingar and Wieman, 1990). The impact of tumor bed destruction on mouse tumor curability can be shown through ing anti-inflammatory drugs such as aspirin or indoexperiments where normal tissues surrounding the methacin before and during PDT exhibit diminished tumor are shielded from light exposure (Fingar and tissue response. Again, the difficulty in extrapolaHenderson, 1987). These show that “sterilization” ting from in vitro experiments to the in vivo situation of a margin of normal tissue vasculature underlying should be pointed out here-while in vivo PDT the tumor is necessary for cure-probably to pre- causes platelet aggregation as one of its first effects, vent nutritional resupply to still viable tumor cells exposure of isolated platelets to PI1 doses closely through diffusion or angiogenesis. equivalent to clinical exposure and light in v i m Are tumor vessels more sensitive to PDT than leads to very rapid, drug dose dependent, inhibition normal vessels, as is the case with hyperthermia of platelet aggregation (Zieve et af., 1966; Hender(Song, 1980)? No significant differential in sensi- son, unpublished data), therefore implying that the tivity has been found by Reed et al. (1989b), but in in vivo effects require additional participants such rodent tumors vascular shut-down slightly preceding as, for example, elements from the vascular wall. shut-down of the surrounding tissue can be achieved Similarly, in vitro PDT of endothelial cells leads to with all sensitizers tested if one adjusts the PDT rapid, PGIl mediated inhibition of platelet aggredoses carefully-these conditions, however, are gation, when small amounts of endothelial cell culalways subcurative. ture supernatants are transferred to anticoagulated No significant induction of vascular photo- platelet suspensions (Henderson et af., 1991b). No sensitivity has been observed with cationic benzo- aggregation inducing effects have been detected in phenothiazinium sensitizers (Cincotta el al., 1990) any of these experiments, and the question of the or Victoria Blue-BO (Henderson and Oseroff, initiation of vascular damage must remain open for the present. unpublished data). It is difficult to pinpoint the initiating event(s) in PDT-induced vascular damage, since any disturTumor response bance at the vascular wall may cause a cascade of succeeding events, involving the vessel lining, vessel The time course of tumor andlor tissue response support structures and formed blood elements, and to PDT depends on the relative contributions from leading eventually to vascular obstruction. The ra- tumor cell and vascular photosensitization, whereby pidity of events implicates fast-acting, potent particularly the latter can be manipulated by varying mediators, such as those released from cells after the time interval between drug injection and light PDT as described earlier. The fact that in rats delivery andlor drug dose. Direct lethal in vivo administration of the eicosanoid synthesis inhibitor tumor cell damage is expressed in the reduction of indomethacin could inhibit vascular occlusion clonogenic tumor cells at the completion of light induced by in vivo PDT, as well as appearance of treatment, while further reductions (delayed tumor circulating thromboxane, supports this hypothesis cell kill) reflect influences of the tumor environment (Fingar and Wieman, 1990). However, no docu- and follow the kinetics of vascular disturbance. mentation exists to date to show that patients receiv- These relationships are demonstrated in Fig. 5

u

Review Article under therapeutic conditions in vivo (i.e. treatment reduces clonogenic tumor cells to limits of detection (3-4 logs] within 2 4 4 8 h) for three sensitizers with very different direct tumor celUvascular photosensitivity patterns. For comparison the cell death kinetics following simple tumor anoxia have been added. Bacteriochlorophyll a, which has a maximum direct cell kill potential of about 50% (Fig. l ) , limited by rapid photobleaching (Henderson et al., 1991a), and demonstrates no vascular shutdown until 3-4 h after light delivery, shows this reduction of clonogenic cells already by the end of light treatment. Further reduction does not occur until vascular shutdown commences. Photofrin 11, which has a direct cell kill potential of about 2 logs, but where vascular shutdown begins within minutes of light delivery, realizes a direct cell kill of only 20-30%, but further cell death proceeds immediately, to be equal to that induced by anoxia within 3 h and again to reach limits of detection by 24 h. Phthalocyanine (AlPcS,), which has the highest direct cell kill potential (about six times that of PII) and relatively less vascular photosensitivity, kills at least 10 times more tumor cells outright, and further cell kill proceeds immediately due to vascular shutdown setting in by the end of light delivery. All three sensitizers have a high 7-day complete response rate, but no tumor cures can be achieved under these conditions in the mouse model with bChla. What, if any, impact these mechanistic differences might have in clinical treatments remains to be seen. It has recently been argued (Patterson er al., 1990b) that the importance of direct cell kill effects may be underestimated in the kind of analyses presented above, since histologic evaluation of tumor necrosis following PDT shows extremely clear demarcation lines between ‘‘live’’ and “dead” tissue, which correspond with depth of light penetration and seem inconsistent with the concept of cell kill due to vascular occlusion reaching beyond the tumor boundaries. It must be realized, however, that histologically apparent necrotic tumor tissue can contain as many as 20% clonogenic tumor cells (Fingar et al., 1987)-more than enough to rapidly repopulate a tumor under good nutritional conditions. Thus, while direct cell kill is reflected in histologic evaluations, it does not determine tumor cure. LATE EFFECTS OF PHOTODYNAMIC THERAPY

Prolonged cutaneous photosensitivity is the major PI1 late effect of concern. By anecdotal accounts, all patients undergoing PII-PDT will remain photosensitive to some degree for up to 6 weeks after drug injection (Dougherty ef al., 1990; Mullooly er al., 1990). No well controlled, prospective clinical studies of cutaneous photosensitivity have yet been carried out, however. In preclinical studies the choice of the experimental endpoint may be critical to the interpretation of

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results. As pointed out by Bellnier and Dougherty (1989b), the frequently employed mouse ear-swelling response (ESR) model reflects levels of circulating photosensitizer (at least with PII), while the murine foot response model seems to be influenced by both circulating and tissue localized sensitizer. In our experience the vascular shutdown model of the mouse back skin also reflects circulating drug levels. The response of all of these models is characterized by acute erythema and edema, blanching, and in severe cases, followed by necrosis. Complete vascular shutdown within the light field almost always leads to eventual skin necrosis. Prolonged skin photosensitivity can conceivably be abrogated by rapid photosensitizer clearance due to excretion and/or sensitizer breakdown, photobleaching of sensitizer or pharmacologic intervention, either to speed sensitizer clearance or interfere with the mechanisms of sensitization. A number of newly developed sensitizers promise to significantly improve the problem of prolonged cutaneous sensitivity. In mice, AIPcS, induced skin sensitivity was found to decline within 2 weeks after injection, while PI1 sensitivity lasted for 2 monthsthis did not correlate well with decline of drug extractable from the skin, which was not significantly different for the two compounds (Tralau et al., 1989). The metallopurpurin SnET2 produces no murine skin response by 9 days after injection (Morgan ef al., 1990), while the fast clearing NPe6 (MACE) causes no skin photosensitivity after 24 h post injection in mice or guinea pigs (Roberts el al., 1989b; Gomer, 1990). With bChla, which is chemically unstable in vivo, skin photosensitivity totally subsides within 5 days (Henderson et al., 1991a). Among the above sensitizers, NPe6 and bChla are particularly photodegradable. The gradual photobleaching of sensitizer (PII) remaining in the skin following PDT has been advocated by Mang et al. (1987) and Boyle and Potter (1987) as a means of diminishing skin photosensitivity, and some anecdotal clinical evidence seems to support this point. Attempts to accelerate the clearance of PI1 in mice through the use of absorbing agents, diuretics and modifiers of porphyrin metabolism, as well as to interfere with the inflammatory effects of photosensitization have met with no or mild success (Manyak et al., 1988). Earlier attempts have tried to suppress skin photosensitivity in porphyric mice through the use of singlet oxygen and free radical quenching carotenoids, and some benefit was observed when skin-fold thickness was measured, none when ESR was taken as endpoint (MathewsRoth, 1984). Photoprotection through sulfhydrylcontaining compounds (WR-2721 and related compounds), commonly considered protectors against ionizing radiation, was suggested by Dillon et al. (1988). A recent study showed no therapeutic gain through this approach when protectors were

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administered before P D T treatment, since tumor and skin were equally protected (Bellnier and Dougherty, 1989a). Another aspect of P D T which should be considered under “late effects” is the nature of tissue healing after P D T induced damage. According to Barr et al. (1990), preservation of the structural and functional integrity of collagen after PII- and AlPcS,-PDT is superior to that after hyperthermia treatment. This leads to maintenance of the mechanical strength of organs such as the colon o r bladder, and may contribute to the excellent cosmetic results after clinical PDT of skin lesions. The observed infiltration of PDT-treated tissue with lymphocytes, plasma cells and histiocytes suggests an immune response (Shumaker and Hetzel, 1987). High levels of interleukin 1-beta, interleukin 2 and tumor necrosis factor-alpha have been found in patients’ urine u p to 50 days after high dose bladder PDT treatment (Nseyo et al.. 1990). This was always concurrent with severe inflammatory symptoms, and it remains to be seen whether cytokine release in vivo represents a specific PDT response, or whether it is simply related to the degree of inflammation present. Finally, in discussing late effects of tumor treatments, the troublesome possibility of mutagenic and carcinogenic events must b e considered. The only information concerning these aspects comes from in vitro studies with porphyrins and phthalocyanines. Using the production of 6-thioguanine a n d o r ouabain resistant mutants in Chinese hamster cells as endpoint, no statistically significant mutagenic potential was found with H p D or AIPcS, (Gomer et al., 1983; Ben-Hur er al., 1987). More recently, mutagenicity similar to that induced by equitoxic doses of UV-C irradiation, but lower than ionizing radiation, has been demonstrated for AIPcS, at the heterozygous thymidine kinase locus in a strain of mouse lymphoma cells deficient in the repair of D N A double strand breaks (Evans et al., 1989). CONCLUDING REMARKS

The complex nature of the tissue response to PDT treatment has yet to be fully elucidated. Ongoing, intensified studies of structure/activity relationships for a wide variety of different sensitizers should clarify many of the remaining open questions as well as stimulate new avenues of research. Finally, the currently proceeding Phase 111 clinical trials with PI1 and the approaching clinical testing of second generation sensitizers will determine whether PDT will earn a lasting place in the armament of cancer treatment. Acknowledgemenu-The authors wish to thank Drs. J. E. van Lier, M. Kenney and M. A. J. Rodgers for supplying various sensitizers and Drs. D. A. Bellnier, T. Gessner, E. Mayhew and J. Sweeney for valuable collaboration. Work performed in this laboratory was supported by NIH

grants CA 42278, CA 42683 and CA 16716 from the National Cancer Institute. REFERENCES

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