J. Photochem. Photobiol. B: Biol., 14 (1992) 275-292
New Trends in Photobiology
Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy James
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
30, 1991; accepted
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 photosensitization 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, 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,
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 ALAinduced 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
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 . 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 . 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 . 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 , 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 . 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 . 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 . 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
Succinyl CoA (FEEDBACK CONTROL) *_______ ____
+ Iron -c_
Acid Protoporphyrinogen Oxidase
S-Aminolevulinic Acid Dehydrase
Deaminase + Vroporphyrinogen
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 ALAinduced 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 ALAinduced 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 . 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
benign and malignant abnormalities of the epidermis, and in carcinomas 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
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 . On the other hand, if the mechanism responsible for the synthesis of uroporphyrinogen 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 . If the conversion of PpIX into heme is slower than the rate at which PpIX is being synthesized, then PpIX will accumulate . 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 ALAinduced 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  or in the skin of human volunteers given ALA by either topical application or intradermal injection . The uroporphyrins are quite soluble in water, have a relatively short half-life in the body , 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 , are excreted by both the kidneys and the liver (via the bile to the bowel) but leave the body primarily with the feces . The uroporphyrins and coproporphyrins show no great affinity for the lipid of cell membranes . 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” . 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. Hemecontaining 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 photosensitization, and by an increase in the excretion of ALA and porphobilinogen without a corresponding increase in the excretion of uroporphyrin, coproporphyrin, or protoporphyrin. The metabolic abnormality is thought to involve defective utilization of porphobilinogen, with consequent accumulation of both porphobilinogen and ALA, the immediate precursor of porphobilinogen . Numerous investigators have examined
282 both ALA and porphobilinogen for possible neurotoxicity and found that high concentrations 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 . 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 . This produced useful information about ALA toxicity, and showed that the ALAinduced 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 . 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 . 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 . Others have reported that trace amounts of PpIX are extractable from certain tissues 24 h after the injection of ALA . 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 . 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 . 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 desquamation. There was no blister formation or skin necrosis . 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 adenocarcinoma 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 . 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 . 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 . 7.3. ALA-induced photosensitization of human tissues With a single exception, every human tissue that developed significant ALAinduced 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 When tissues that have been photosensitized to light, patients usually experience an irritation
of PpIX by ALA-induced PpIX are exposed of some sort, variously described as
“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.
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 fluorescence.
of ALA-induced PplX and hematoporphyrin
Hematoporphyrin derivative (HpD) and its various semipurified commercial preparations 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
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 . 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
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 photobleaching 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.
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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