J. Photochem. Photobiol. B: Biol.

, 14 (1992) 275-292 275

New Trends in Photobiology (Invited Review)

Endogenous protoporphyrin IX, a clinically useful

photosensitizer for photodynamic therapy

James C. Kennedy” and Roy H. Pottierb

Departments of “Oncology and Pathology, bDepatiment of Urology, Queen’s University, Kingston,
Ont. K7L 3N6 (Canada); and “pbDepartment of Chemishy and Chemical Engineering, The Royal
Military College of Canada, Kingston, Ont. K7K 5LO (Canada)

(Received December 30, 1991; accepted January 31, 1992)

Abstract

The tissue photosensitizer protoporphyrin IX (PpIX) is an immediate precursor of heme

in the biosynthetic pathway for heme. In certain types of cells and tissues, the rate of
synthesis of PpIX is determined by the rate of synthesis of 5-aminolevulinic acid (ALA),
which in turn is regulated via a feedback control mechanism governed by the concentration
of free heme. The presence of exogenous ALA bypasses the feedback control, and thus
may induce the intracellular accumulation of photosensitizing concentrations of PpIX.
However, this occurs only in certain types of cells and tissues. The resulting tissue-specific
photosensitization provides a basis for using ALA-induced PpIX for photodynamic therapy.
The topical application of ALA to certain malignant and non-malignant lesions of the
skin can induce a clinically useful degree of lesion-specific photosensitization. Superficial
basal cell carcinomas showed a complete response rate of approximately 79% following
a single exposure to light. Recent preclinical studies in experimental animals and human
volunteers indicate that ALA can induce a localized tissue-specific photosensitization if
administered by intradermal injection. A generalized but still quite tissue-specific photo-
sensitization may be induced if ALA is administered by either subcutaneous or intraperitoneal
injection or by mouth. This opens the possibility of using ALA-induced PpIX to treat
tumors that are too thick or that lie too deep to be accessible to either topical or locally
injected ALA.

Keywords: Photodynamic therapy, photochemotherapy, cancer, protoporphyrin IX, 5

aminolevulinic acid, porphyria.

1. Introduction

When certain types of mammalian cells are exposed to adequate concentrations

of 5aminolevulinic acid (ALA) under appropriate conditions, they synthesize and
accumulate photosensitizing concentrations of protoporphyrin IX (PpIX). However,

Elsevier Sequoia
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certain other types of cells do not become photosensitized when exposed to ALA
under similar conditions. Such cellular specificity provides a basis for the clinical use
of ALA-induced PpIX in photodynamic therapy (PDT).
There are several very practical questions that must be answered before ALA-
induced PpIX can be used routinely for PDT. Which types of cells and tissues become
photosensitized, and which do not? By what routes can ALA be administered? What
is the minimum effective dose by each route, and what toxicities or other hazards are
associated with the use of each route? What is the time course for the development
of photosensitization in specified tissues, and how long does it take the PpIX to be
cleared from those tissues? In addition, there is the very basic question of why ALA
induces only certain types of cells to synthesize and accumulate photosensitizing
concentrations of PpIX. The answer to this last question may have substantial relevance
for the development of new clinical applications.

2. Tissue specificity of ALA-induced PpIX

The systemic administration of an adequate dose of ALA to experimental animals

leads to the development of strong PpIX fluorescence in certain tissues but not in
others. In mice, strong fluorescence develops in the skin, in the mucosa of the oral,
vaginal, and anal cavities, in the salivary glands, in the bile ducts and gall bladder,
and in the seminal vesicles. Certain other organs show weaker PpIX fluorescence, but
there is no detectable fluorescence in skeletal or cardiac muscles [l-3]. Fluorescence
microscopy of the skin, bladder, and uterus showed strong fluorescence in the epidermis,
endometrium, and urothelium without significant fluorescence in the dermis, the elastic
cartilage of the ear, the myometrium, or the muscles of the bladder [4]. Flow cytometry
of the cells of the peripheral blood and bone marrow revealed that there was very
little increase in PpIX fluorescence following the systemic administration of ALA [3].
In human cancer patients the topical application of an adequate dose of ALA can
induce strong PpIX fluorescence in tumors that originate in the epidermis (basal cell
carcinoma, squamous cell carcinoma, adenocarcinoma of the sebaceous gland), in the
bronchi (squamous cell carcinoma of the lung), in mammary tissue (adenocarcinoma
of the breast), and in the salivary gland (squamous cell carcinoma of the parotid) [5,
61. However, no PpIX fluorescence was induced in human renal cell carcinoma. On
the basis of such observations it appears that ALA-induced PpIX accumulates primarily
in tissues that line surfaces (epidermis, conjunctiva, oral mucosa, respiratory mucosa,
vaginal mucosa, rectal mucosa, serosal surfaces, endometrium, urothelium) or in glands
with ducts that empty onto such surfaces (liver, sebaceous glands, mammary glands,
salivary glands, seminal vesicles). In contrast, major tissues of mesodermal origin
(muscle, connective tissue, cartilage, bone marrow and blood) do not develop significant
PpIX fluorescence after a single dose of ALA in viva. However, cells in culture may
behave quite differently. The addition of ALA to malignant cells of hemopoietic origin
[7-lo] or to Schwann cells, fibroblasts, and macrophages in mature chick dorsal root
ganglion cultures [ll] may lead to a substantial accumulation of porphyrins.
It is of interest that ALA-induced PpIX can be used as a selective herbicide [12,
131 or as an insecticide [14, 151. When sprayed with a solution of ALA under specified
conditions, only certain species of plants accumulate enough PpIX to be destroyed
by subsequent exposure to sunlight.
 277

3. Is ALA-induced PplX synthesized in situ?

A few minutes after ALA is injected into mice or rats, the bile ducts begin to
fill with a micellar solution of PpIX in bile. Since this quickly passes into the bowel,
it seems possible that some of that PpIX may be absorbed and thus enter the general
circulation. Alternatively, some of the PpIX that is synthesized in the liver may enter
the blood directly. Consequently, we must give serious consideration to the possibility
that the ALA-induced PpIX that accumulates in the skin, oral mucosa, salivary glands
etc. was not synthesized in situ, but instead was synthesized by the liver and reached
the tissues in question via the blood. The tissue specificity described above would then
be a consequence of the differential uptake and/or retention of the PpIX from the
blood, and the use of ALA to induce PpIX would be merely a complicated way of
introducing PpIX into the body. If so, we might expect that exogenous PpIX would
show the same tissue specificity if injected directly into the circulation. However,
injected PpIX and ALA-induced PpIX have such different tissue specificities that the
above hypothesis appears to be highly improbable.

3.1. Most nucleated cells can synthesize Pprx

The failure of ALA to induce PpIX fluorescence in certain types of cells and
tissues is probably not because of a complete absence of the necessary metabolic
machinery. We must assume that every cell in the body is able to convert nutrients
into a biologically useful form of chemical energy. In the presence of adequate supplies
of molecular oxygen the vast majority of nucleated cells use “oxidative phosphorylation”
for this purpose [16]. The process involves an electron transport chain that produces
a biologically useful form of chemical energy by the controlled oxidation of partially
processed fragments of nutrients. Since heme-containing enzymes (cytochromes) are
directly involved in transport of the electrons (by reversible alterations in the valence
of the heme iron), it follows that most cells must be capable of synthesizing both
heme and its immediate precursor (Fig. 1). Also, since heme is synthesized within
the mitochondria [17], any cell that contains functional mitochondria should have at
least some capacity to synthesize PpIX. Why then does exogenous ALA induce the
accumulation of detectable amounts of PpIX in certain types of cells but not in others?
Possibly the different types of cells may have very different capacities to synthesize
heme. Alternatively, different feedback control mechanisms may be operating in the
different types of cells [ll].

3.2. AL.A can induce PpU: synthesis in vitro

The systemic administration of ALA to normal mice induces PpIX fluorescence
in the parotid glands [4]. We cannot rule out a possible role for the liver in any such
in vivo model. However, if fresh slices of parotid gland from a normal mouse are
incubated with ALA in vitro under appropriate conditions, strong PpIX fluorescence
develops [18]. The liver certainly cannot have synthesized the PpIX that accumulates
in the parotid gland under these in vitro conditions. The same applies to the porphyrins
that can be induced by ALA in cultures of malignant hemopoietic cells [7-lo] or in
mature cultures of chick dorsal root ganglions [13]. All such porphyrins must have
been synthesized in situ.

3.3. Localbation of ALA leads to localization of PpIX

Evidence that PpIX is synthesized in situ in mouse skin (rather than synthesized
in the liver and then transported via the blood to the skin) was obtained by injecting
278

Glycine + Succinyl CoA

Ferrochelatase
S-Aminolevuliaic (FEEDBACK
+ Iron
Acid Synthase CONTROL)
*_______ ____ Heme -c_

5Aminolevulinic Acid
Protoporphyrinogen
S-Aminolevulinic Oxidase
Acid Dehydrase
a II
Porphobilinogen Protoporphyrinogen IX

Copro-
Deaminase Deaminase + porphyrinogen
Vroporphyrinogen III Oxidase

J
Uroporpbyrinogen I Uroporphyrinogen III I> Coproporphyrinogen III

Uroporphyrinogen
Decarboxylase

\
Coproporphyrinogen

Fig. 1. Simplified biosynthetic pathway for heme. Fluorescing and photosensitizing compounds
are enclosed in rectangles, with protoporphyrin IX highlighted. The S-aminolevulinic acid/heme
feedback control is indicated by a dotted arrow. The principal biosynthetic route for ALA-
induced protoporphyrin IX is indicated by the large arrows.

a small volume of ALA solution into the subcutaneous space near the middle of the
tail. Diffusion of ALA from this site was so slow that the epidermal cells overlying
the injection site were exposed to the ALA for a significant period of time. The ALA-
induced PpIX fluorescence that followed was limited to the immediate area about
the injection site [l]. Analogous experiments using intradermal injections of ALA in
human volunteers resulted in strong PpIX fluorescence and photosensitization that
remained localized to the site of injection [19]. The topical application of ALA to
superficial basal cell and squamous cell carcinomas induced PpIX fluorescence and
photosensitization that remained localized to the site of ALA application 151. Similar
localization of PpIX fluorescence and photosensitization was observed following the
topical application of ALA to adenocarcinoma of the sebaceous gland, actinic keratoses,
psoriasis, skin abrasions, and skin nodules of carcinoma of the breast, carcinoma of
the lung, and squamous cell carcinoma of the parotid [5,6]. In none of these experimental
models was there any sign of generalized fluorescence or photosensitization, as would
be expected if the ALA that was injected or applied at a localized site had been
absorbed into the general circulation, metabolized in the liver, and then returned to
the general circulation in the form of PpIX. Consequently, we now have direct evidence
for the ALA-induced biosynthesis of PpIX in situ in normal epidermis, in various
 279

benign and malignant abnormalities of the epidermis, and in carcinomas derived from
the mammary gland, the parotid gland, and the bronchial mucosa.

4. How is the biosynthetic pathway for heme regulated?

Cells of the erythroid series in the bone marrow routinely synthesize large amounts
of heme (to make hemoglobin), but the systemic administration of a single dose of
ALA to mice causes only a very slight increase in the fluorescence of cells in the
marrow and in the peripheral blood. Muscle cells contain large amounts of heme in
the form of myoglobin, but the systemic administration of a single large dose of ALA
to mice induces no detectable PpIX fluorescence in either skeletal muscle, cardiac
muscle, myometrium, or the muscles of the urinary bladder. Liver contains a large
amount of heme in the form of catalase and other heme-containing enzymes, but in
this case the systemic administration of a single dose of ALA rapidly induces the
synthesis of a large amount of PpIX. It appears that PpIX biosynthesis is regulated
differently in different tissues, and that such differences may be responsible for at
least some of the tissue specificity that is characteristic of ALA-induced PpIX.

4.1. Regulation of heme biosynthesis in erythroid cells

Although there has been much research in this area during the past 20 years,
the mechanism responsible for regulating heme biosynthesis in erythroid cells has not
yet been clearly identified. It is apparent that large amounts of heme are synthesized
only when erythroid cells are at a certain stage in their maturation, and that the
process of differentiation is linked in some manner to the regulation of heme biosynthesis
[9, 10, 20-221. There is some evidence that regulators of iron metabolism influence
heme biosynthesis [23-271. Exogenous ALA can stimulate the biosynthesis of heme
in certain types of erythroleukemic cells, but it is apparent that factors other than
the concentration of ALA are limiting. However, under suitable conditions, exogenous
ALA can induce the synthesis of photosensitizing concentrations of PpIX in malignant
cells of the hemopoietic series [7, 81. There would appear to be a potential clinical
application if such photosensitization could be induced in human leukemia cells in
vivo, without inducing a similar degree of photosensitization in the normal cells of
the blood or marrow. Since the enzyme profiles of normal cells and of cells derived
from them by malignant transformation are not identical, it appears possible that the
biosynthetic pathways for heme in normal and malignant cells of the hemopoietic
system may differ in a manner that might permit the induction of a useful degree of
differential photosensitization [28, 291.

4.2. Regulation of heme biogwthesis in the liver

Under normal conditions the rate of synthesis of heme is regulated so that it
matches the rate at which free heme is removed from the system. In the liver (and
in certain other tissues) the synthesis of heme is controlled by a feedback mechanism
in which the presence of free heme inhibits the synthesis of ALA, a distant precursor
of heme [17, 3&33]. Thus, if heme is used up as quickly as it is synthesized, there
will be little free heme and therefore little feedback inhibition of the synthesis of
ALA. Under such conditions the concentration of ALA will increase, and eventually
(since ALA is a precursor of heme) there will be a corresponding increase in the
synthesis of heme. However, if the heme now is produced faster than it can be removed
from the system, free heme will accumulate and inhibit the synthesis of ALA, which
280

in turn will lead to a decrease in the synthesis of heme. Thus, under normal conditions
the demand for heme controls the rate of synthesis of heme, and therefore the rate
of synthesis of PpIX also.

5. How does exogenous ALA induce the accumulation of PpIX?

Although there are multiple enzyme-catalysed steps between ALA and heme (Fig.
l), in the liver and certain other tissues the whole process appears to be regulated
by the single feedback control system described in Section 4.2 above. Under normal
conditions the maximum rate at which ALA is synthesized and enters the biosynthetic
pathway for heme is always less than the maximum rate of the slowest of the subsequent
steps in that pathway. Consequently, each step always has ample reserve capacity and
intermediates do not accumulate. However, this is not so if the ALAlheme feedback
control is bypassed by the presence of a large excess of exogenous ALA. With the
concentration of ALA no longer limiting, the rate of synthesis of the first intermediate
(porphobilinogen) is determined primarily by the maximum capacity of the enzyme
system that is responsible for that specific step. What happens at each subsequent
step may vary from tissue to tissue, since this will be determined by the relative
capacities of the various processes that are involved. For example, if porphobilinogen
is being synthesized at a rate that exceeds the maximum capacity of the next step in
the pathway (the synthesis of uroporphyrinogen), then porphobilinogen will accumulate
[34]. On the other hand, if the mechanism responsible for the synthesis of uropor-
phyrinogen still has some reserve capacity even though porphobilinogen is being
produced at the maximum possible rate, then the rate of synthesis of uroporphyrinogen
will be limited by the rate of synthesis of porphobilinogen. The same principle applies
at each subsequent step in the biosynthetic pathway. If the first step in the decarboxylation
of uroporphyrinogen is slower than the synthesis of uroporphyrinogen, then there will
be an accumulation of uroporphyrinogen, and subsequently of uroporphyrin also [35].
If the conversion of PpIX into heme is slower than the rate at which PpIX is being
synthesized, then PpIX will accumulate [36]. However, it is important to note that a
rate-limiting step upstream in the pathway may greatly influence subsequent events
downstream. For example, even though the maximum capacity for the synthesis of
PpIX from protoporphyrinogen may greatly exceed the maximum capacity of the
subsequent iron-dependent step by which PpIX is converted into heme, PpIX will not
accumulate if an even slower process is located anywhere upstream of PpIX in the
biosynthetic pathway for heme. The presence or absence of such a rate-limiting step
may explain why only certain types of cells accumulate PpIX when exposed to high
concentrations of exogenous ALA.

5. I. ALA-induced uropoq?hyrin and/or coproporphytin

Uroporphyrins I and III and coproporphyrins I and III are potent tissue sensitizers
[371. Since the uroporphyrins are derived from uroporphyrinogen and the coproporphyrins
from coproporphyrinogen, under certain circumstances we might expect that ALA-
induced deregulation of the biosynthetic pathway for heme would lead to the accumulation
of one or more of these porphyrins [35, 38-401. For example, uroporphyrin III might
accumulate if uroporphyrinogen III was being synthesized faster than it could be
decarboxylated to form heptacarboxy porphyrin III. Some of the metabolic diseases
known as porphyrias provide examples of disease processes in which a partial defect
 281

in the enzymatic machinery at one point in the heme biosynthesis pathway leads to
the accumulation of uroporphyrin and/or coproporphyrin [17, 411.
The fluorescence emission and excitation spectra of PpIX in skin are readily
distinguished from the corresponding spectra of uroporphyrins and coproporphyrins.
However, neither uroporphyrins nor coproporphyrins could be detected in the skin
of mice given large doses of ALA by intraperitoneal injection [2] or in the skin of
human volunteers given ALA by either topical application or intradermal injection
[19]. The uroporphyrins are quite soluble in water, have a relatively short half-life in
the body [42], and are excreted primarily via the kidneys [17J The coproporphyrins,
which are somewhat less soluble in water and have a longer half-life in the body [42],
are excreted by both the kidneys and the liver (via the bile to the bowel) but leave
the body primarily with the feces [17]. The uroporphyrins and coproporphyrins show
no great affinity for the lipid of cell membranes [43]. With such characteristics they
are not likely to accumulate anywhere except in the kidneys or liver during the process
of excretion, or in necrotic areas of tumors where a transient tissue concentration
differential may develop as the porphyrin is more rapidly cleared from the tissues
with better circulation. In contrast, PpIX is only slightly soluble in water at physiological
pH and shows a strong affinity for membrane lipids. Consequently, we might expect
PpIX to be retained by those tissues (other than liver) within whose mitochondria it
has been synthesized.

5.2. Other techniques for increasing the accumulation of &IX

Even though the addition of exogenous ALA might bypass the ALAtheme feedback
control, and thus free the synthesis of PpIX from its normal regulation, PpIX will
not accumulate if any one of its precursors is being produced so slowly that the
mechanism responsible for the conversion of PpIX into heme always has some excess
capacity. Given our present level of knowledge, it probably would not be feasible to
increase the rate of synthesis of the precursor in question. However, it might be
possible to induce PpIX to accumulate in such cells by slowing the conversion of
PpIX into heme, in effect creating an artificial “hepatic protoporphyria” [17]. Several
techniques for inhibiting the addition of iron to PpIX are in routine use in various
experimental systems [36,44]. Unfortunately, toxicity could be a major problem. Heme-
containing enzymes are essential for energy metabolism, and the potential danger of
even a transient interruption in their synthesis is probably unacceptable. Alternatively,
it might be possible to bypass the rate-limiting step that is preventing the accumulation
of PpIX by adding some precursor of PpIX that lies downstream from that particular
step. However, there are serious problems of cost, solubility, and ease of access to
the interior of cells associated with the in viva use of any precursor of PpIX other
than ALA.

6. Prechical studies of ALA-induced PpIX

6.1. Potential neurotoxicity of ALA and/or PpIX
The acute intermittent (Swedish) form of hepatic porphyria is a metabolic disease
characterized by severe neurological abnormalities in the absence of skin photosen-
sitization, and by an increase in the excretion of ALA and porphobilinogen without
a corresponding increase in the excretion of uroporphyrin, coproporphyrin, or pro-
toporphyrin. The metabolic abnormality is thought to involve defective utilization of
porphobilinogen, with consequent accumulation of both porphobilinogen and ALA,
the immediate precursor of porphobilinogen [17]. Numerous investigators have examined
282

both ALA and porphobilinogen for possible neurotoxicity and found that high con-
centrations of ALA may lead to changes in behaviour, in cell membrane function,
and in neuromuscular and spinal cord transmission [ll, 45-741. Consequently, since
we had been giving large doses of ALA systematically to various experimental animals
in order to induce the synthesis of PpIX, we too looked for evidence of ALA neurotoxicity.
A toxic and near lethal intraperitoneal injection of ALA (1000 mg ALA per kilogram
of body weight) caused a transient depression of motor nerve conduction velocity in
mice that was indistinguishable from the depression caused by a toxic and near lethal
intravenous injection of hematoporphyrin derivative (80 mg HpD per kilogram of body
weight). Complete recovery of neurological function occurred in both groups of mice
7-10 days following the injection [l]. However, since the systemic administration of
ALA into normal mice induces the synthesis of large amounts of PpIX, this type of
experiment could not distinguish the possible neurotoxicity of ALA as such from the
possible toxicity of the PpIX that it induced.
In vitro studies helped provide such a distinction. Neurotoxicity was assayed by
quantitating the inhibition of neurite outgrowths of chick embryo neuroblasts during
stimulation by nerve growth factor. Uroporphyrin, coproporphyrin, protoporphyrin, and
hematoporphyrin derivative caused dose-dependent toxicity, with protoporphyrin being
the most toxic since it produced 50% inhibition of neurite outgrowth (in the dark)
at a concentration of only 50 nM. In contrast, ALA produced no detectable toxicity
even at a concentration of 1.5 mM [75]. A study by Whetsell et al. in which ALA
was added to mature (three-week old) organotypic cultures of chick dorsal root ganglion
showed no evidence of toxicity after 48 h at ALA concentrations up to 10 mM [ll].

6.2. Phannacokinetics of ALA-induced PpLX

The intraperitoneal or subcutaneous injection of ALA into mice induces the
biosynthesis of PpIX in numerous organs and tissues. The use of a non-invasive
technique permitted detailed pharmacokinetic studies of PpIX fluorescence in the skin
of mice that had been given various doses of ALA by intraperitoneal injection [2].
This produced useful information about ALA toxicity, and showed that the ALA-
induced PpIX was cleared from the skin within 24 h of injection. A cat given ALA
by subcutaneous injection developed generalized photosensitization of the skin, with
complete recovery within 24 h [3]. Human volunteers given ALA by either topical
application to various types of skin lesions or intradermal injection of normal skin
developed localized photosensitization which vanished within 24 h [3]. ALA-injected
mice were killed at regular intervals following injection and the PpIX fluorescence in
various organs and tissues measured. No tissue showed more than background levels
of PpIX fluorescence at 24 h post-injection [3]. Others have reported that trace amounts
of PpIX are extractable from certain tissues 24 h after the injection of ALA [76]. It
appears then that ALA-induced PpIX is almost completely cleared from the body
within 24 h of its induction, no matter what the route used for administration.

6.3. Photosensitization by ALA-induced PpLX

The exposure of ALA-injected albino mice to white light led to transient loss of
hair and microscopic evidence of damage to the pilosebaceous units and basal cell
layer of the skin. There was no skin necrosis or any other evidence of gross phototoxic
damage. However, the number of pilosebaceous units per unit area of skin was reduced,
apparently permanently [4]. The exposure of ALA-injected tumor-bearing mice to red
light caused gross tumor necrosis. There was necrosis of some of the overlying skin
also, but only minimal phototoxic damage to adjacent skin within the treatment field
 283

[3]. Human volunteers given localized injections of ALA by intradermal injection and
then exposed to sunlight developed localized areas of relatively mild phototoxic damage,
with localized erythema and edema followed by hyperpigmentation and slight des-
quamation. There was no blister formation or skin necrosis [19]. Certain types of
malignant hemopoietic cells can be photosensitized if cultured under appropriate
conditions in the presence of ALA [7, 81. Human volunteers who took ALA by mouth
developed dose-dependent photosensitization of the skin [77, 781. It is apparent then
that exogenous ALA may induce the synthesis and accumulation of a high enough
concentration of PpIX to cause clinically significant photosensitization of certain organs
and tissues.

7. Clinical studies of topical ALA-induced PpIX

To date, most such studies have involved the topical application of ALA to lesions
of the skin [6, 791. In a few cases we injected the ALA, either directly into a tumor
or intradermally into the skin overlying a tumor. The lesions treated have been primarily
superficial basal cell carcinomas, with some superficial squamous cell carcinomas and
actinic keratoses. During the past 3 years we have used topical ALA-induced PpIX
to treat more than 300 superficial basal cell carcinomas, with a complete response
rate at 3 months of approximately 79% following a single treatment. We have used
the same technique to provide palliation for a few patients with ulcerated skin nodules
of breast carcinoma: one patient each with ulcerated skin nodules of squamous cell
carcinoma of the lung, squamous cell carcinoma of the parotid, or renal cell carcinoma,
and one patient who had widespread dermal and epidermal involvement by adeno-
carcinoma of the sebaceous gland. In addition, we have used topical ALA plus light
to treat a few patients with psoriasis, with variable results. Other clinical studies of
ALA-induced PpIX plus light to treat superficial basal cell carcinomas, superficial
squamous cell carcinomas, and/or psoriasis are in progress in Vienna (Austria),
Rotterdam (Netherlands), Leeds (UK), Boston (USA), and Buffalo (USA). Treatment
of superficial squamous cell carcinoma in a patient with xeroderma pigmentosum has
been reported [79].

7.1. Penetration of the stratum comeum by ALA

The development of PpIX fluorescence following the topical application of ALA
to skin indicates that a significant amount of the ALA has penetrated the stratum
corneum. Normal skin varies in its ability to resist the penetration of ALA. Since thin
skin presents a less effective barrier than thick skin, there is some degree of variation
from site to site on the same patient. Oriental or native American skin blocks the
penetration of ALA better than do most occidental skins, and (as first reported by
W. M. Star of Rotterdam) the very fair and unusually thin skin of certain northern
Europeans may allow quite significant penetration of ALA. The heavily freckled skin
that characterizes some of the Celtic race (especially those with red hair) appears to
be penetrated in a punctate manner, since the PpIX fluorescence that follows the
topical application of ALA to what appears to be normal skin usually takes the form
of a scattering of tiny irregular spots. Various types of benign abnormalities of the
skin are associated with increased permeability to ALA. These include open wounds
and abrasions, inflammation fromvarious causes (but not inflammatory breast carcinoma),
psoriasis, and weeping lesions of any origin. Skin that shows evidence of chronic sun
damage usually permits increased penetration of ALA, as do areas of actinic keratosis.
284

On the other hand, the hyperkeratosis associated with verruca vulgaris greatly inhibits
the penetration of ALA.
The abnormal layer of keratin that is produced by superficial basal cell or squamous
cell carcinomas is rapidly penetrated by ALA. However, since the adjacent normal
skin is less permeable, it is not necessary to restrict the topical application of ALA
to the lesion itself (although an attempt to do so might be considered if the patient
has very fair or sun-damaged skin). The observed specificity of the fluorescence for
such skin lesions is a result of the relative impermeability of the normal skin to ALA.
The ALA that penetrates the stratum corneum diffuses through the epidermis
and into the dermis. However, even though there may be sufficient ALA in the dermis
to induce strong PpIX fluorescence in the epidermal appendages (such as pilosebaceous
units) that lie within the dermis, the dermal cells as such do not develop significant
PpIX fluorescence or become photosensitized [4]. Consequently, it is possible to destroy
cancers of epidermal origin without causing serious injury to the dermis. This minimizes
scarring.

Z2. ALA-induced fiuorescence of human tissues

The topical application of ALA can induce PpIX fluorescence in non-malignant
cells of the epidermis and in various cancers derived from the epidermis: in 100% of
more than 300 superficial basal cell carcinomas treated, in most (but not all) superficial
squamous cell carcinomas treated, and in adenocarcinoma of the sebaceous gland (one
case only). The topical application of ALA to cutaneous secondaries that had penetrated
through the epidermis induced PpIX fluorescence in all nodules of carcinoma of the
breast treated, in squamous cell carcinoma of the parotid (one case only), and in
squamous cell carcinoma of the lung (one case only). In contrast, skin nodules of
renal cell carcinoma (one case only) did not develop PpIX fluorescence. Since there
was no barrier to the penetration of ALA into these renal cell carcinoma nodules,
the observed lack of response presumably had a biochemical basis. It should be noted
that the normal hemopoietic system (which is of mesodermal origin) has a very large
capacity to synthesize heme, but it could not be induced to do so in vivo by the
administration of large amounts of exogenous ALA [3].

7.3. ALA-induced photosensitization of human tissues

With a single exception, every human tissue that developed significant ALA-
induced PpIX fluorescence showed some degree of phototoxic damage after being
exposed to light. The exception was a nodule of squamous cell carcinoma that developed
intense PpIX fluorescence but showed no visible evidence of phototoxic damage
following our standard dose of light. We did not attempt to measure the concentration
of glutathione or other possible singlet oxygen traps in this tumor. Other squamous
cell carcinomas in the same patient developed phototoxic damage as expected when
treated with topical AL4 plus light.
A few superficial squamous cell carcinomas that showed little or no ALA-induced
PpIX fluorescence developed substantial phototoxic damage following exposure to light.
Since all of these tumors contained brown pigment, it seems likely that PpIX was
induced but its red fluorescence absorbed by the pigment.

Z 4. H&amine-like reaction during photoactivation of PpIX

When tissues that have been photosensitized by ALA-induced PpIX are exposed
to light, patients usually experience an irritation of some sort, variously described as
 285

“itching”, “tingling”, “stinging”, “pricking”, “burning”, “throbbing”, “ants biting the

skin”, or “a worm crawing under the skin”. The sensation is usually noticed after less
than 1 min of exposure, rises to a peak within the next few minutes, and then gradually
decreases to reach a background level that patients often describe as “a mild sunburn”.
As with such sunburn, there is usually little or no discomfort 24 h later, although the
treated area may be somewhat tender to the touch for several days.
Immediately following completion of the treatment, the lesion is normally quite
edematous. There may be a serous exudate. In many patients the skin immediately
adjacent to the lesion is slightly edematous also, and there may be a small zone of
erythema. The reaction of the surrounding tissue tends to be more spectacular in
patients who have the “Celtic” type of skin. Such patients often develop a typical
“wheal and flare” (bee-sting) reaction in the skin immediately adjacent to the treated
lesion, the “flare” of erythema sometimes extending for more than 10 cm. Both the
wheal and the flare fade over a period of several hours.
This histamine-like reaction is more of a nuisance than a hazard. It complicates
the treatment of senile patients since they may require sedation to keep them from
reacting to the localized irritation during treatment, and a few patients who have a
very low pain threshold find it difficult to tolerate. The topical application of 2%
lidocaine gel reduces but does not eliminate the discomfort. We are in the process
of evaluating the use of oral antihistamines for this purpose.

7.5. Photobleaching of ALA-induced @IX

ALA-induced PpIX is photobleached very readily in vivo. If an adequate dose
of light has been given, at completion of treatment there should be no detectable
PpIX fluorescence within the treatment field. However, a relatively weak renewal of
PpIX fluorescence and photosensitization may occur during the first few minutes or
hours following treatment, as ALA that was in transit in the tissue is converted into
PpIX. Patients therefore should be warned that they may experience a mild histamine
reaction (itching, etc.) at the treatment site if they do not protect it from exposure
to sunlight during the first 24 h following treatment.
The readiness with which ALA-induced PpIX is photobleached has one very
important clinical consequence. Since no photosensitizer is completely specific for
malignant tissue, there will always be a small amount of PpIX in the normal tissues
within the treatment field. However, this is photobleached to an inactive form so early
in the course of the treatment that the non-malignant tissues experience no more
phototoxic damage from a very large dose of light than they do from the standard
dose. In practice, this means that in most clinical situations it is possible to stop
worrying about giving the adjacent normal tissues an overdose of light. In order to
avoid giving a dose of light too small to eradicate the malignant target tissue, we
normally give at least double what we consider to be the minimum effective dose.
There is one exception. If the skin immediately adjacent to a malignant lesion develops
significant ALA-induced PpIX fluorescence, we reduce the dose of light, and if
technically possible we shield some of the adjacent skin with aluminum foil. Although
ALA-induced PpIX has not caused any second or third degree bums of normal skin
to date, caution is advised if it is necessary to treat an unusually large area of skin
with severe actinic damage, inflammation, or atrophy. Under such conditions, the
treatment field should be examined under W immediately prior to treatment, and
the dose of light reduced if the non-malignant skin shows significant PpIX fluo-
rescence.
8. Comparison of ALA-induced PplX and hematoporphyrin derivative

Hematoporphyrin derivative (HpD) and its various semipurified commercial prep-

arations are complex and somewhat variable mixtures of various porphyrin monomers,
dimers, polymers, and aggregates. HpD and other preformed photosensitizers are
usually administered by intravenous injection, although they have been used topically
for the experimental treatment of various superficial skin lesions [80-901. Some of the
photosensitizing material in the circulation tends to accumulate preferentially in certain
types of normal and malignant tissues, and as the HpD is gradually cleared from the
rest of the body, a clinically useful concentration differential may develop in adjacent
tissues. HpD does not necessarily accumulate within the malignant cells of a tumor,
but is often found in association with the tumor vasculature or interstitial structures
[91-931. There is some evidence that the malignant cells in such tumors may die
primarily because of phototoxic damage to the tumor blood supply. The mechanism
responsible for the preferential accumulation of HpD by malignant tissues may be a
function of the increased solubility of HpD in plasma membrane lipids at the relatively
low pH that characterizes many tumors [94-951. If so, it might be expected that
intravenously injected HpD would tend to accumulate preferentially in the endothelial
cells of tumor capillaries and venules, since these are the first cells that the circulating
HpD encounters at the relatively low pH that characterizes the extracellular fluid in
many tumors.
ALA is not itself a photosensitizer. If administered systemically, it readily passes
through the endothelial cells of the capillaries, enters the extracellular fluid, and then
diffuses into adjacent cells. In certain types of cells (but not others) it may induce
the biosynthesis of photosensitizing concentrations of PpIX. This is the primary
mechanism responsible for the observed tissue specificity. If administered topically, its
tissue specificity is determined (as before) by differences in the biochemical profiles
of the various cells into which it diffuses, but also by differences in its rate of diffusion
through the stratum comeum and other barriers to diffusion. Since PpIX is synthesized
within the mitochondria of living cells, it accumulates inside those cells rather than
in the extracellular space. Little or no PpIX is present in the general circulation. To
date, ALA-induced PpIX has been used to treat patients primarily by topical application
[5, 61.

8.1. Problems
Although HpD has been and continues to be a useful tissue photosensitizer for
the treatment of cancer, it has several serious defects. First, the relatively slow rate
of clearance of HpD from the skin and certain other normal tissues means that a
patient who has received a standard dose of HpD by intravenous injection must avoid
exposure to sunlight for at least 2 weeks following that injection. A relatively small
proportion of patients will retain clinically significant concentrations of HpD in the
skin for up to 3 months. Exposure of the skin to sunlight during this period of
photosensitization may result in severe phototoxic damage. Since it often is quite
inconvenient for patients to avoid exposure to sunlight for an extended period of time,
many clinicians have been reluctant to use HpD to treat lesions for which there is
any reasonable alternative form of therapy. Thus, systemic HpD is rarely used to treat
the non-malignant lesions of psoriasis or the malignant but relatively benign basal cell
carcinomas of the skin.
The slow rate of clearance of HpD from the skin can cause major problems if
the tumor being treated is both large and growing rapidly. For reasons that are both
 287

technical and biological, it is difficult to eradicate a large tumor with a single course
of treatment. If the tumor in question has a short doubling time, then the cells that
survived the PDT may proliferate so rapidly that the tumor is back to its original
volume within 2-3 weeks following treatment. Unfortunately, it often takes longer
than 2-3 weeks to clear the skin of HpD. If subsequent injections of HpD must be
given before the skin is completely free of residual HpD (in order to catch the partially
destroyed tumor while it is still relatively small), the concentration of HpD in the
skin may build up to dangerously phototoxic levels. This also reduces the HpD
concentration differential between normal and malignant tissues. In one patient with
a rapidly growing carcinoma of the breast, we caused full thickness skin necrosis by
giving her five standard doses of HpD and light at intervals of 1 month. A small
amount of the tumor survived the fifth treatment, but the skin did not.
ALA-induced PpIX is cleared from the skin within 24 h of systemic, topical, or
intradermal administration. Inconvenience for the patient is minimized, but of equal
importance, we have found it possible to repeat a course of PDT as often as every
other day without accumulating a photosensitizing concentration of PpIX in the skin.
Although HpD shows a useful degree of specificity for malignant tissues, under
certain conditions this is not enough to permit the giving of a lethal dose of light to
every part of the tumor without giving an overdose to at least some parts of the
normal tissues within the treatment field. The choice then is between allowing some
of the tumor to survive, or causing an undesirable amount of damage to some of the
non-malignant tissue. For example, if a superficial tumor of the skin involves the
bridge of the nose, the tip, and both sides, the treatment field will consist of a complex
set of curved surfaces that are very difficult to illuminate evenly because of the variation
in the cosine correction factor [96]. Under such conditions, it is very difficult to avoid
underexposing some parts of the treatment field while overexposing (and thus damaging)
others. Another common problem caused by insufficient tissue specificity occurs when
chest wall nodules of carcinoma of the breast are treated by an external beam. These
nodules typically arise in the dermis, and then erode into both the epidermis and the
subcutaneous space. The concentration of HpD in the nodules is substantially higher
than in the overlying skin. However, there is unavoidable attenuation of the external
beam as it passes into the deeper tissues. Consequently, in order to ensure that the
deep surface of each nodule receives a lethal dose of light, it would be necessary to
give the skin surface a dose of light so large that even the relatively low concentration
of HpD in the skin would be sufIicient to produce serious damage. In actual clinical
practice, whenever we use HpD with an external beam to treat multiple chest wall
secondaries of breast carcinoma, the dose of light is adjusted to produce a moderate
sunburn reaction of the skin within the treatment field. This is close to a tissue
tolerance dose for normal skin. Such a dose can eradicate very superficial breast cancer
nodules, but because of beam attenuation with increasing tissue penetration it generally
causes only partial destruction of the deeper nodules.
Since ALA-induced PpIX photobleaches very rapidly, the effect of PDT on a
given cell is determined primarily by the concentration of PpIX in that cell rather
than by the dose of light that it receives. Cells that contain only a small amount of
PpIX at the onset of PDT will very shortly contain none, and consequently will
experience no additional damage during the remainder of the exposure. It is possible
then to “overdose” part of the treatment field in order to make sure that some other
part receives the “normal” dose, yet to do so without causing serious damage to
normal tissues in the overdosed part of the field. Also, it is possible to safely “overdose”
normal tissues at the surface of the field in order to ensure that an adequate dose
288

of light reaches the deep surface of a tumor. However, that tumor will be killed only
if its cells have accumulated sufficient PpIX to cause cell death before rapid pho-
tobleaching reduces the PpIX concentration to a harmless level.

9. Summary

Topical ALA-induced PpIX has been shown to have clinical value for the treatment
of superficial basal cell carcinomas and certain other malignant and non-malignant
lesions of the skin. The rapid photobleaching of ALA-induced PpIX in normal skin
normally permits the use of very large doses of photoactivating light without danger
of serious phototoxic damage. Studies in experimental animals and in human volunteers
indicate that both the localized injection and the systemic administration of ALA may
have value in the treatment of other types of cancer. Although numerous toxicity and
neurotoxicity studies of ALA and its metabolic products have been carried out [ll,
34-661, more detailed studies are urgently required.

Acknowledgments

This review was supported by the Ontario Cancer Treatment and Research
Foundation, the National Cancer Institute of Canada, and the Department of National
Defence (Canada).

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