Photodiagnosis and Photodynamic Therapy (2004) 1, 145—155
REVIEW
Photodynamic therapy for pancreatic carcinoma: experimental and clinical studies Lakshmana Ayarua, Stephen G. Bownb, Stephen P. Pereira PhD, FRCPa,∗ a
Institute of Hepatology, Department of Medicine, Royal Free and University College London Medical School, 69-75 Chenies Mews, London WCIE6HX, United Kingdom b National Medical Laser Centre, Department of Surgery, Royal Free and University College London Medical School, London, United Kingdom Received 26 May 2004 ; received in revised form 28 July 2004; accepted 28 July 2004 Available online 18 September 2004 KEYWORDS Photodynamic therapy; Pancreatic carcinoma; Pancreas
Summary Pancreatic carcinoma is the sixth leading cause of cancer-related mortality in the United Kingdom, with an overall 5-year survival of less than 5%. Attempted curative surgery is possible in less than 20% of cases and is associated with a 5-year survival of just 10—20%. Palliative radio-chemotherapy improves symptoms of pancreatic cancer but rarely extends median survival beyond 12 months. There is a need to develop novel therapies that improve outcome. Photodynamic therapy, which is a way of producing localised non-thermal tissue necrosis with light, is currently under evaluation as a treatment for pancreatic cancer. This review will examine some of the mechanisms underlying photodynamic therapy, and the preclinical work, which has led to this treatment being piloted in human studies. © 2004 Elsevier B.V. All rights reserved.
Contents Introduction................................................................................................
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Photosensitising agents.....................................................................................
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Mechanisms of action....................................................................................... Direct tumour cell death ................................................................................. Vascular cell death ....................................................................................... Immune system activation................................................................................
147 147 147 148
Pancreatic adenocarcinoma ................................................................................
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Preclinical studies of PDT for pancreatic cancer .......................................................... Haematoporphyrin derivative PDT ......................................................................
149 149
* Corresponding author. Fax: +44 207 3800405.
E-mail address: [email protected] (S.P. Pereira).
1572-1000/$ — see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/S1572-1000(04)00038-9
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L. Ayaru et al. Aluminium sulphonated phthalocyanine PDT (ALSPc) ..................................................... Pheophorbide A PDT ..................................................................................... Aminolaevulinic acid (ALA) PDT .......................................................................... meso-Tetrahydoxyphenylchlorin (mTHPC) PDT ........................................................... PDT of human pancreatic carcinoma cells................................................................
149 150 150 151 151
PDT for pancreatic cancer: clinical studies .................................................................
152
References .................................................................................................
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Introduction Photodynamic therapy (PDT) is a way of producing localised non-thermal tissue necrosis with light. After administration of the photosensitiser, the tumour is irradiated with laser light at a wavelength compatible with the absorption spectrum of the drug, usually in the red or near-infrared region. This leads to excitation of the sensitiser from its ground state (singlet state) into a relatively longlived electronically-excited state (triplet state), via a short-lived excited singlet state [1]. The excited sensitiser can react directly via a Type I photooxygenation process with substrate, e.g. protein, lipid leading to free radical intermediates, that can react with oxygen to generate various reactive oxygen species. More commonly, the triplet can transfer its energy directly to oxygen to form singletoxygen (Type II reaction), which is assumed to be the key agent of cellular damage [2,3]. Ideally, PDT-induced tumour destruction should occur without damage to adjacent normal organs. In theory, selective retention of photosensitiser by tumour compared with normal tissue, and the resistance of collagen (which maintains mechanical integrity of organs) to PDT, could achieve this goal
Table 1
[4]. In practice, animal models of pancreatic cancer and clinical studies have demonstrated that normal tissue is commonly damaged if exposed to the same light dose as the tumour [5—7]. However, PDT treated tumour and normal tissue can heal largely by regeneration of normal tissue (with some scarring) and thus one can effectively achieve selective tumour necrosis [8].
Photosensitising agents A variety of photosensitisers have been investigated in pancreatic cancer models and clinical studies (see Table 1). Haematoporphyrin derivative (HpD), one of the earliest photosensitisers to be evaluated, is the product mixture formed upon solubilising haematoporphyrin in aqueous media. It consists of a mixture of mono-, di- and oligomers, all containing the porphyrin moiety. As the oligomeric fraction appeared to be largely responsible for phototoxicity, purification methods were developed to remove part of the mono- and dimers [9], resulting in the commercial product porfimer sodium (photofrin). However, it has several limitations which include its complexity (making it difficult to reproduce its
Photosensitisers investigated in studies of PDT for pancreatic cancer.
Photosensitiser Porphyrin like macrocycles Dihematoporphyrin ether Porfimer sodium Aluminium sulphonated phthalocyanine (ALSPc) Chlorins meso-Tetrahydroxyphenylchlorin (mTHPC)
Reference for pancreatic cancer study Mang and Wieman [13], Schroder et al. [6] Moesta et al. [14] Matthews and Chui [15], Nuutinen et al. [5], Chatlani et al. [16], al-Laith and Matthews [17] Mlkvy et al. [18,19] Bown et al. [20]
5-Aminolaevulinic acid (ALA) ALA esters
Regula et al. [21], Ratcliffe et al. [22] Whitaker et al. [23]
Hypericin Indocyanine green Pheophorbide A
Liu et al. [24] Tseng et al. [25] Evrard et al. [7], Hajri et al. [26]
Photodynamic therapy for pancreatic carcinoma composition), a small absorption peak (630 nm) in the red region of the visible spectrum, and a tendency to produce prolonged cutaneous photosensitivity [10]. These limitations have led to the development of a number of second generation photosensitisers. Aluminium sulphonated phthalocyanine (ALSPc) has a chemical structure that is related to the porphyrin configuration but with differences that enhance absorption of red light (long wavelength absorbance in the 650—700 nm region). It is a less complex mixture than porfimer sodium but has been shown to be similar in its biological effect [11]. meso-Tetrahydroxyphenylchlorin (mTHPC) is a chlorin that was first synthesized at Queen Mary College, London. It is chemically pure, has a strong absorption peak in the red part of the spectrum at 652 nm, and has a much higher singlet-oxygen yield than porfimer sodium so treatment times can be shortened [12]. Skin sensitivity usually lasts 2—3 weeks rather than the 2—3 months associated with porfimer sodium. ALA (5-aminolaevulinic acid) is a naturally occurring amino-acid and a precursor in the biosynthesis of haem. The enzymes involved in this biosynthesis convert ALA to the photocytotoxic compound protoporphyrin IX (PPIX). The last step in the formation of photochemically inactive heme is the incorporation of iron into PPIX under the action of ferrochelatase. By adding exogenous ALA, protoporphyrin IX may accumulate as this last step becomes rate-limiting. PPIX has an absorption peak at 635 nm and is metabolized to haem within a few hours so that skin sensitivity only lasts 1—2 days. Pheophorbide A has a wavelength of absorption of red light of 665 nm whilst indocyanine green (water soluble anionic dye) has a spectrum in the infrared region of light (800 nm), giving these photosensitisers the potential to cause greater depths of tumour necrosis than porfimer sodium. Hypericin has a dianthraquinone structure and is activated by ultraviolet and green light.
Mechanisms of action At least three main mechanisms for PDT-mediated tumour destruction have been proposed [3]. First, reactive oxygen species that are generated by PDT can kill tumour cells directly. Second, PDT damages the tumour-associated vasculature, which may lead to tumour infarction [27]. Finally, PDT can stimulate an immune response against tumour cells [28]. The relative importance of each mechanism in pancreatic cancer is unclear, but certainly varies with the photosensitiser used.
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Direct tumour cell death Reactive oxygen species can cause direct photodamage to many biological molecules, including proteins, lipids and nucleic acids [29—31], at sites where the photosensitiser accumulates, either by apoptosis or necrosis. The intracellular localisation of the photosensitiser determines in part the mechanism of cell death [32]. Haematoporphyrin derivative and porfimer sodium both localise in mitochondria due to their hydrophobicity and their affinity for a plasma binding site on the mitochondrial membrane [33—35]. More hydrophilic sensitisers such as the phthlocyanines and many chlorins, enter cells via endocytosis and hence accumulate mainly in lysosomes. Damage to mitochondria generally leads to apoptosis whereas plasma membrane and lysosomal damage can delay or even inhibit apoptosis and instead induces necrosis [36—38]. The potential mechanisms of PDT-induced apoptosis have generated much interest particularly as they provide the opportunity to refine treatment [39]. Several pathways have been identified, including: (i) permeabilisation of mitochondrial membranes with subsequent release of apoptosis-related proteins, e.g. cytochrome c and subsequent generation of activated caspases [40,41], (ii) activation of cell surface death receptors Fas and TNFR1, (iii) release of cathepsins from lysosomes with subsequent cleavage of Bid to produce a pro apoptotic protein tBid [42], and (i.v.) damage to the endoplasmic reticulum with release of calcium which can promote apoptosis [43]. Several in vitro studies have shown that apoptosis requires the presence of functional enzymes and is thus inhibited by high dose PDT [44—46]. Pancreatic carcinoma tissue and cells treated with PDT have been shown to undergo both necrosis and apoptosis. A pilot study treating human pancreatic tumours (median diameter 4.0 cm) with mTHPC PDT achieved a radius of necrosis of 9 mm (range: 7—11) around each treatment point [20]. A study by Hajri et al. [26] demonstrated that apoptosis was the primary mechanism of cell death, when human pancreatic tumour cell lines were exposed to low dose pheophorbide A PDT in vitro and after grafting into athymic mice. As gentle programmed cell death avoids PDT-induced tumour haemorrhage [7,16], they postulated that the absence of haemoglobin (which acts as a shield against tissue light penetration) would improve efficacy.
Vascular cell death The viability of tumour cells is dependent on an adequate blood supply [47]. PDT-related damage of
148 the endothelium of blood vessels leads to severe and persistent post-PDT tumour hypoxia/anoxia [48]. These vascular effects are caused by reversible contraction of endothelial cells resulting in the exposure of the basement membrane, vessel leakage, and thrombus formation [3,49,50]. Dolmans and colleagues studied the effect of a novel photosensitiser MV6401 in vivo on the microvasculature of a mammary tumour (MCaIV). The acute vascular effects were characterised by vasoconstriction rather than thrombus formation [51]. However, long-term vascular shutdown was mediated by thrombus formation, as evidenced by histological evaluation and inhibition with heparin. The release of thromboxane [52,53] and inhibition of nitric oxide [54] have been implicated as mediators of these pathophysiological events, which lead to ischaemic death of tumour cells.
Immune system activation Since PDT causes necrosis of tumour cells with subsequent generation of inflammatory mediators, e.g. lipid fragments and metabolites of arachidonic acid [1,43], stimulation of the immune system has been postulated as one of its mechanisms of action. Evidence for PDT-induced immune activation comes from initial studies more than 10 years ago, which reported infiltration of lymphocytes, leukocytes and macrophages into PDT-treated tissue [1,43,55]. In 1996, de Vree et al. reported that PDT in a rhabdomyosarcoma-bearing rat model activated neutrophil accumulation, which slowed tumour growth. Depletion of neutrophils decreased the PDT-mediated effect on tumour growth [56]. A related study by the same group investigated this relationship in more detail, and found that PDT in vitro induced contraction of the endothelial cells, permitting neutrophil adherence to the subendothelial matrix [57]. More recently, the inflammatory cytokines interleukin IL-6 and IL-1 (but not TNF alpha) have been shown to be upregulated in response to PDT [58]. The effects of PDT on surviving tumour and normal cells are important elements in determining the outcome, but in contrast to the photocytotoxic action of PDT have received relatively little attention. Wong et al. addressed this by studying the effects of the photosensitisers aminolaevulinic acid (ALA) and porfimer sodium on several epithelial cancer cell lines and normal epithelial and stromal cells [59]. They showed that both normal and malignant cells lost their responsiveness to IL-6 class cytokines (involved in cellular differentiation and inhibition of proliferation of various epithelial cells) and epi-
L. Ayaru et al. dermal growth factor (involved in cellular proliferation, motility, adhesion, invasion and angiogenesis), in a dose-dependent manner. The recovery of responsiveness occurred 48—72 h later and was accompanied by a resumption of cell proliferation. The role of the adaptive immune response was investigated by Korbelik et al. in a study of PDT of EMT6 mammary sarcoma in SCID and normal mice. A significantly lower therapeutic effect was seen in SCID mice, suggesting that the lack of an immune response was responsible for the difference in tumour cures [60]. This effect could be restored by adoptive transfer of T-lymphocytes of normal (Balb/c) mice into the SCID mice. In a later study by the same group, EMT6 sarcoma-bearing mice were selectively depleted of specific lymphoid cells (T cells). Whilst initial tumour ablation by PDT was not affected, long-term tumour cure rates decreased markedly after depletion. These results provide direct evidence that T-lymphocyte function is essential for the maintenance of long-term control of PDT-treated tumours [61]. Henderson’s group reported that a tumour-cell lysate isolated after PDT with porfimer sodium could be used to vaccinate mice against the development of further tumours, indicating the induction of tumour specific immunity [62]. These PDT vaccines seemed to induce a cytotoxic T-cell response that involved induction of IL-12 expression. PDT may also be directly harmful to immune cells. Lymphocytes can accumulate photosensitiser and be destroyed during subsequent illumination, leading to local suppression of the immune system [63—65]. Moreover, PDT of the skin has been shown to suppress the contact hypersensitivity response, a classical example of a cell-mediated delayed-type immune reaction. Jolles et al. [66] demonstrated, in an adoptive transfer study in mice, that the reduction of the contact hypersensitivity response also extended to areas other than the illuminated site, and was associated with the generation of suppressor cells. These disparate observations can be explained in part by the structure of the tumour microenvironment. In general, immune cells are found in the tumour stroma, separated from tumour cells by extracellular matrix and basal membrane-like structures [67,68]. By disrupting the tumour microenvironment, PDT may enable direct interaction between immune cells and tumour cells. After the initial PDT-induced damage to tumour cells and immune cells, a strong inflammatory reaction occurs locally which leads to influx and activation of undamaged immune cells from elsewhere [28].
Photodynamic therapy for pancreatic carcinoma
Pancreatic adenocarcinoma Worldwide, adenocarcinoma of the pancreas is one of the top 10 leading causes of cancer death, and ranks sixth as a cause of cancer death in the UK and USA [69,70]. Upto 15—20% of patients have resectable disease, but even with adjuvant therapy only around 20% of these survive to five years [71,72]. Patients with locally advanced inoperable disease can be treated with chemotherapy, radiotherapy, or some combination of the two but these therapies rarely increase median survival beyond 12 months. For those with metastatic disease at presentation, palliative chemotherapy can improve symptoms of advanced malignancy, but the median survival is only 3—6 months [73,74].
Preclinical studies of PDT for pancreatic cancer Haematoporphyrin derivative PDT Mang and Wieman [13] were the first to demonstrate the potential of PDT as a treatment for pancreatic cancer. In two animal models of pancreatic carcinoma, they studied the uptake, fluorescence kinetics and effect of dihematoporphyrin ether (DHE). The first model was that of a pancreatic carcinoma of acinar origin induced in rats by azaserine treatments and serially transplanted into the same strain subcutaneously and intraperitoneally. The second model was of a ductal pancreatic carcinoma induced by injection of N-nitrosobis (2-oxopropyl) amine (BOP) into hamsters. At 24 h after injection of the photosensitiser, the tumours in both models exhibited equal or higher concentrations of DHE compared with normal pancreas by both tissue fluorescence and chemical extraction procedures. These tumours were then exposed at laparotomy to 630 nm light from a dye laser (75 mW cm−2 , 30 min). The tumour tissue demonstrated a relatively normal fluorescence decay pattern, with haemorrhage and a resultant loss of measurable DHE concentration. In contrast, there was no necrosis of normal pancreas under the same conditions and photobleaching of the DHE as measured by fluorescence decay did not occur. The reasons for this relative resistance of normal pancreas are unclear but the authors postulated that a singlet-oxygen quenching agent may be present in normal pancreas which is absent in pancreatic cancer and other tissues. DHE was also investigated in a BOP induced pancreatic cancer model by Schroder and colleagues [6]. Three hours after the injection of photosensitiser, DHE was 2.4 times more concentrated in
149 pancreatic tumour than in the normal pancreas and PDT caused extensive tumour necrosis without any obvious effect on the non-tumour-bearing pancreas. However, there was considerable accumulation of DHE in the adjacent small bowel, resulting in perforation of the duodenum and jejunum and death of four (57%) of the animals after PDT. Similar studies have been carried out using a variety of second generation photosensitisers, including phthalocyanine, pheophorbide A, 5-aminolaevulinic acid and mTHPC, as discussed below. Aluminium sulphonated phthalocyanine PDT (ALSPc) In an early in vitro study of ALSPc-PDT on normal acinar pancreatic cells and azaserine-induced pancreatic carcinoma cells, photon activation of cellularly bound ALSPc with light >570 nm permeabilised the cells and caused an increase in amylase release from normal acinar cells but a dose dependent inhibition of amylase release from tumour cells [15]. Ultrastructural studies showed that this distinct effect was not due to differences in the microanatomical organization of normal or tumour derived cells. Under these experimental conditions, the cells were capable of releasing secretory products such as amylase as a result of cellular stimulation by PDT and not simply as a direct consequence of widespread membrane lysis. A subsequent study by the same group showed that this release of amylase by normal acinar cells was energy dependent and inhibited by the chelation of free cytoplasmic calcium but not by the removal of extracellular calcium [17]. The authors suggested that the initial PDT-induced release of amylase from the normal pancreas may activate membrane receptors or G-proteins, resulting in release of intracellular calcium rather than being secondary to the influx of external calcium as seems to occur in myeloma cells treated with PDT [75]. They noted however that this PDT action did not exactly reproduce agonist action (bethanecol induced amylase release), indicating that ALSPc may be acting at a more distal point in the signal transduction pathway. In contrast, removal of extracellular calcium reversed the inhibitory effect of PDT on tumour cells and produced a significant increase in amylase release, whereas chelation of free cytoplasmic calcium had no effect. Suggested explanations for these results were PDT targeting of a calcium dependent membrane site or that in the presence of external calcium PDT activation may cause excessive influx of calcium, which inhibits constitutive
150 secretion. A protein kinase C-mediated opening of the voltage-sensitive calcium channels found in the tumour cells, but not in normal acinar cells, was postulated as a putative signal transduction mechanism. The adverse effects of PDT on the small intestine prompted our group to focus on the normal pancreas and adjacent tissues in hamsters, using treatment parameters similar to those known to induce pancreatic necrosis [5]. In initial pharmacokinetic studies using chemical extraction and microscopic fluorescence, the highest levels of ALS2 Pc were seen in duodenal mucosa and bile duct walls 48 h after photosensitisation. The PDT effects of disulphonated aluminium phthalocyanine (ALS2 Pc) were examined after illumination with light at wavelength 675 nm. When a dose of 5 mol/kg of sensitiser was used, duodenal perforations, gastric ulcers and transudation of bile from the bile duct occurred. The lesions in the stomach and bile duct healed without complication though the duodenal perforations remained. Minor necrosis was seen in the normal pancreas at this dose but damage to major vessels did not occur. A dose reduction to 1 mol/kg still caused a concealed duodenal perforation but damage to other tissues was diminished. Possible explanations for these results were the high concentration of sensitiser in the duodenum at 48 h and the thinness of the hamster duodenal wall. Our group then studied the effect of AlS2 Pc in hamster pancreas and a BOP induced hamster pancreatic cancer model [16]. Peak concentrations of sensitiser in tumour, measured by distribution studies from chemical extraction, were noted at 24 h. The tumour: normal tissue ratios at 4, 24 and 48 h were 0.74:1, 3.0:1 and 2.3:1, respectively. At 48 h after injection (5 mg/kg) into the inferior vena cava, the sensitiser was activated at laparotomy by red light (675 nm) delivered using a 200 m fibre just touching the tumour. When 25 or 50 J light doses were used, damage appeared to extend to the edges of tumour nodules 5 mm in diameter and up to 8 mm in larger tumours. In this study, no damage was seen in the adjacent normal pancreas or stomach in any tumour-bearing animals until light doses of 200 J were reached.
Pheophorbide A PDT Evrard et al. [7] demonstrated the feasibility of using pheophorbide A to treat azaserine-induced pancreatic rat carcinoma. A dose ranging component to the study showed that a 9 mg/kg versus 3 mg/kg dosage increased sensitiser concentration by a factor of two in the pancreas and over five in the tumour and was thus chosen as the optimum dose.
L. Ayaru et al. Administration of 9 mg/kg gave a selectivity ratio of 13.5:1 between tumour and surrounding tissue. Laser light at 660 nm and 100 J/cm2 delivered at laparotomy produced selective tumour necrosis of about 10 mm diameter. Six of nine tumour-bearing rats treated with PDT remained disease free at 120 days compared with a 100% mortality of control rats within 35 days (P < 0.01). The main complication was duodenal injury.
Aminolaevulinic acid (ALA) PDT Our group investigated ALA-induced PPIX for PDT in Syrian golden hamsters with transplanted pancreatic cancers [21]. ALA was given either intravenously or orally (in single or fractionated bolus doses), with laser light delivered by means of a bare fibre touching the tissue surface or by external irradiation using a light-integrating cylindrical applicator. We anticipated that oral fractionated doses would result in better tissue uptake than a single oral bolus because ALA and its metabolite, porphobilinogen, are rapidly excreted in the urine after a bolus dose [76]. The peak plasma concentration of ALA in humans after a single dose is reached at 60 min and the half-life (t1/2 ) of its clearance from the circulation is only about 50 min [77], so frequently repeated doses should maintain a steady high blood level of ALA for a longer time. This hypothesis was confirmed by pharmacokinetic studies 1—24 h after light exposure which demonstrated that PPIX sensitisation in the tumour, as measured by quantitative fluorescence microscopy, was highest after intravenous administration (which avoids first pass metabolism) of 200 mg/kg ALA, followed in decreasing order by oral fractionated and oral single bolus doses (both 400 mg/kg). Laser light application at 630 nm to give 12—50 J from the bare fibre or 50 J/cm using surface illumination with the cylindrical applicator resulted in necrosis of up to 8 mm in diameter. Smaller tumours showed complete necrosis of the entire lesion, but larger ones usually had a rim of viable tumour on the side opposite the irradiated surface. In a randomised survival study (the only one performed to date in PDT for pancreatic cancer), animals treated with oral fractionated ALA (400 mg/kg) survived significantly longer than the untreated group according to Kaplan—Meier analysis of survival curves (logrank test, P < 0.02) (see Fig. 1), although all animals eventually died of recurrent tumour [21]. A major limitation of ALA-PDT is that ALA itself is hydrophilic, which restricts its penetration through cellular membranes and access to mitochondria. Esterification of ALA (generated by treatment of ALA
Photodynamic therapy for pancreatic carcinoma
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Figure 1 Kaplan—Meier survival curves for Syrian golden hamsters with transplanted pancreatic tumours comparing the PDT-treated group with the control, non-treated group. Regula et al. [21] reproduced with permission from Nature Publishing Group, www.nature.com.
hydrochloride with alcohol and thionyl chloride) makes it more lipophilic and improves transmembrane access. Once inside the cell, large amounts of free ALA can be generated by the action of nonspecific intracellular esterases in the majority of cells [78,79]. In azaserine-induced pancreatic carcinoma cell lines treated with a series of straight chain, branched and cyclo-␦ ALA esters, Whitaker et al. [23] examined both the generation of photoactive species and the resulting photocytotoxicity. They found that several ALA esters not only induced the formation of more PPIX, but they did so at a faster rate than ALA itself. This resulted in an enhanced photocytoxicity of some 270 times using the more potent ALA esters.
meso-Tetrahydoxyphenylchlorin (mTHPC) PDT Mlkvy et al. [18,19] from our unit carried out experiments to assess the effects of mTHPC on normal hamster pancreas (see Fig. 2) and in a hamster pancreatic cancer model created by injecting a BOP induced pancreatic cancer cell line into the gastric lobe of the pancreas. Fluorescence microscopy showed maximum levels of mTHPC in normal pancreas 2—4 days after sensitisation and in tumour at 4—5 days. After intravenous injection of 0.1 or 0.3 mg/kg mTHPC, the tumours were treated at laparotomy 2 or 4 days later with red light (50 J at 650 nm, continuous or fractionated) delivered via a single fibre touching the tumour surface. As ALS2 Pc PDT had been complicated by duodenal perforations, the duodenum was shielded from light using opaque paper. The maximum zone of necrosis
(seen 3 days after PDT) was 8.7 mm in diameter, increasing to 12.4 mm with light fractionation (see Fig. 3). Despite the use of duodenal shielding, the main complication was sealed duodenal perforation (in 3 of the 16 animals), which may have been due to slippage of the paper. Other adverse effects included transient bile duct obstruction and the development of multiple small duodenal diverticula. No effects were seen in the stomach or major blood vessels (aorta, vena cava and portal vein). There was no evidence of pancreatitis or cyst formation in the normal pancreas of animals that were kept alive for up to 3 months after PDT with any of the photosensitisers. PDT of human pancreatic carcinoma cells Recent preclinical studies have demonstrated the efficacy of PDT against human pancreatic carcinoma cells. Tseng et al. [25] exposed three separate human pancreatic ductal adenocarcinoma cells lines to indocyanine green in vitro, at a dose of 20 g/ml and 808 nm diode laser light (0.45 W for 5 min) and achieved over 90% cell killing. Liu et al. applied hypericin PDT to human pancreatic cell lines in vitro and to a pancreatic carcinoma model produced by injection of one of the cell lines into the shoulder and pancreatic bed of athymic nude mice. Pancreatic carcinoma growth was suppressed by 66.1 ± 0.2%, 91.2 ± 2.3% and 42.2 ± 8.1% in pancreatic cell lines, subcutaneous shoulder tumours and orthotopically transplanted pancreatic tumours, respectively [24]. It is apparent from these experimental studies that whilst PDT achieves impressive areas of necrosis, complete tumour eradication is usually not
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Figure 2 Normal pancreas 7 days after i.v. injection of 0.3 mg/kg mTHPC, and 3 days after light-delivery. Each treated area received 50 J light (50 MW for 1000 s). The lesion on the left had a single fraction of light, but for that on the right, the light was divided into four equal fractions with a break of 3 min between fractions. Scale 2.0 mm × 1.5 mm. Mlkvy et al. [19] reproduced with permission from Nature Publishing Group, www.nature.com.
possible. Reasons may include non-homogeneous distribution of photosensitiser within the tumour, decreased ability to kill tumour cells with increased distance from vascular supply [80], a reduction of tissue oxygen tension during and after illumination of photosensitised tissue [81,82] and inadequate light doses at all relevant sites.
PDT for pancreatic cancer: clinical studies As experimental work in the various studies outlined above had demonstrated efficient necrosis of pancreatic tumour tissue as well as an improvement in survival in one study [21], we undertook a phase 1 clinical study of PDT for pancreatic carcinoma [20]. A total of 16 patients with histologically confirmed inoperable pancreatic adenocarcinomas located in the head of the pancreas were recruited into the study. They had all presented with obstructive jaundice, which was relieved with plastic or metal biliary endoprostheses. In 14 cases, there was tumour involvement or proximity to the major vessels precluding attempted curative
resection and twelve patients had involvement of the duodenum detected at endoscopy or by computed tomography (CT). Patients were photosensitised with 0.15 mg/kg meso-tetrahydroxyphenylchlorin, which was selected as the photosensitiser because in the preclinical work it produced the largest zone of necrosis and required the lowest light dose at each treatment site. Photoactivation was performed three days later via means of percutaneously placed needles placed into the tumour under ultrasound or CT guidance. The light source was a diode laserdelivering red light at 652 nm, which was divided equally between the optical fibres using a beam splitter. The fibres were then inserted down to the tip of the needles to leave 3 mm of bare fibre in direct contact with the tumour during light delivery. The volume of necrosis produced by PDT treatment ranged from 9.0 to 60 cm3 . In most cases the necrosed area of tumour healed safely without changing in size. There were no free duodenal perforations but three patients developed duodenal obstruction, which may have been related to treatment. Two patients with tumour involving the gastroduodenal artery had significant gastrointestinal
Photodynamic therapy for pancreatic carcinoma
153 the distribution of laser effects to the extent of diseased tissue being treated, and ideally to extend the treated area beyond the tumour margins identified on pretreatment scans. The use of modified selection criteria, such as excluding patients with tumour encasement of a major artery or the duodenum, would also be expected to reduce the risk of major complications and allow treated areas to heal safely.
References
Figure 3 Photomicrographs of tumour nodules (see arrows) treated 3 days previously with a total light dose of 50 J (50 mW for 1000 s), delivered 4 days after 0.3 mg/kg mTHPC. (A) Continuous light (B) Fractionated light (four equal fractions with a 3-min break between fractions). Scale: 20 mm × 13 mm. Mlkvy et al. [18] reproduced with permission from Nature Publishing Group, www.nature.com, volume 76, pages 713—718.
bleeds, which were controlled with endoscopic or radiological intervention. In 14 cases the late stages of disease were dominated by local tumour invasion and re-growth of tumour from the edges of treated areas. The median survival time after PDT was 9.5 months (range: 4—36 months), which is comparable to the reported 5 (2.2—23) months median survival after chemotherapy or 9 (8—12.7) months with chemoradiotherapy [83]. This study demonstrated the feasibility of mTHPC PDT for pancreatic cancer using percutaneously placed light fibres. The morbidity was relatively low although problems did arise with tumours involving the duodenal wall or gastroduodenal artery. This therapy, however, was clearly palliative, as only 44% of patients were alive 1 year after PDT. Randomised controlled studies will be required to assess the true influence of PDT on survival, and its potential additional role to palliative chemotherapy in the management of this disease. Technical aspects of future studies will be to match
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REVIEW
Photodynamic therapy for pancreatic carcinoma: experimental and clinical studies Lakshmana Ayarua, Stephen G. Bownb, Stephen P. Pereira PhD, FRCPa,∗ a
Institute of Hepatology, Department of Medicine, Royal Free and University College London Medical School, 69-75 Chenies Mews, London WCIE6HX, United Kingdom b National Medical Laser Centre, Department of Surgery, Royal Free and University College London Medical School, London, United Kingdom Received 26 May 2004 ; received in revised form 28 July 2004; accepted 28 July 2004 Available online 18 September 2004 KEYWORDS Photodynamic therapy; Pancreatic carcinoma; Pancreas
Summary Pancreatic carcinoma is the sixth leading cause of cancer-related mortality in the United Kingdom, with an overall 5-year survival of less than 5%. Attempted curative surgery is possible in less than 20% of cases and is associated with a 5-year survival of just 10—20%. Palliative radio-chemotherapy improves symptoms of pancreatic cancer but rarely extends median survival beyond 12 months. There is a need to develop novel therapies that improve outcome. Photodynamic therapy, which is a way of producing localised non-thermal tissue necrosis with light, is currently under evaluation as a treatment for pancreatic cancer. This review will examine some of the mechanisms underlying photodynamic therapy, and the preclinical work, which has led to this treatment being piloted in human studies. © 2004 Elsevier B.V. All rights reserved.
Contents Introduction................................................................................................
146
Photosensitising agents.....................................................................................
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Mechanisms of action....................................................................................... Direct tumour cell death ................................................................................. Vascular cell death ....................................................................................... Immune system activation................................................................................
147 147 147 148
Pancreatic adenocarcinoma ................................................................................
149
Preclinical studies of PDT for pancreatic cancer .......................................................... Haematoporphyrin derivative PDT ......................................................................
149 149
* Corresponding author. Fax: +44 207 3800405.
E-mail address: [email protected] (S.P. Pereira).
1572-1000/$ — see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/S1572-1000(04)00038-9
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L. Ayaru et al. Aluminium sulphonated phthalocyanine PDT (ALSPc) ..................................................... Pheophorbide A PDT ..................................................................................... Aminolaevulinic acid (ALA) PDT .......................................................................... meso-Tetrahydoxyphenylchlorin (mTHPC) PDT ........................................................... PDT of human pancreatic carcinoma cells................................................................
149 150 150 151 151
PDT for pancreatic cancer: clinical studies .................................................................
152
References .................................................................................................
153
Introduction Photodynamic therapy (PDT) is a way of producing localised non-thermal tissue necrosis with light. After administration of the photosensitiser, the tumour is irradiated with laser light at a wavelength compatible with the absorption spectrum of the drug, usually in the red or near-infrared region. This leads to excitation of the sensitiser from its ground state (singlet state) into a relatively longlived electronically-excited state (triplet state), via a short-lived excited singlet state [1]. The excited sensitiser can react directly via a Type I photooxygenation process with substrate, e.g. protein, lipid leading to free radical intermediates, that can react with oxygen to generate various reactive oxygen species. More commonly, the triplet can transfer its energy directly to oxygen to form singletoxygen (Type II reaction), which is assumed to be the key agent of cellular damage [2,3]. Ideally, PDT-induced tumour destruction should occur without damage to adjacent normal organs. In theory, selective retention of photosensitiser by tumour compared with normal tissue, and the resistance of collagen (which maintains mechanical integrity of organs) to PDT, could achieve this goal
Table 1
[4]. In practice, animal models of pancreatic cancer and clinical studies have demonstrated that normal tissue is commonly damaged if exposed to the same light dose as the tumour [5—7]. However, PDT treated tumour and normal tissue can heal largely by regeneration of normal tissue (with some scarring) and thus one can effectively achieve selective tumour necrosis [8].
Photosensitising agents A variety of photosensitisers have been investigated in pancreatic cancer models and clinical studies (see Table 1). Haematoporphyrin derivative (HpD), one of the earliest photosensitisers to be evaluated, is the product mixture formed upon solubilising haematoporphyrin in aqueous media. It consists of a mixture of mono-, di- and oligomers, all containing the porphyrin moiety. As the oligomeric fraction appeared to be largely responsible for phototoxicity, purification methods were developed to remove part of the mono- and dimers [9], resulting in the commercial product porfimer sodium (photofrin). However, it has several limitations which include its complexity (making it difficult to reproduce its
Photosensitisers investigated in studies of PDT for pancreatic cancer.
Photosensitiser Porphyrin like macrocycles Dihematoporphyrin ether Porfimer sodium Aluminium sulphonated phthalocyanine (ALSPc) Chlorins meso-Tetrahydroxyphenylchlorin (mTHPC)
Reference for pancreatic cancer study Mang and Wieman [13], Schroder et al. [6] Moesta et al. [14] Matthews and Chui [15], Nuutinen et al. [5], Chatlani et al. [16], al-Laith and Matthews [17] Mlkvy et al. [18,19] Bown et al. [20]
5-Aminolaevulinic acid (ALA) ALA esters
Regula et al. [21], Ratcliffe et al. [22] Whitaker et al. [23]
Hypericin Indocyanine green Pheophorbide A
Liu et al. [24] Tseng et al. [25] Evrard et al. [7], Hajri et al. [26]
Photodynamic therapy for pancreatic carcinoma composition), a small absorption peak (630 nm) in the red region of the visible spectrum, and a tendency to produce prolonged cutaneous photosensitivity [10]. These limitations have led to the development of a number of second generation photosensitisers. Aluminium sulphonated phthalocyanine (ALSPc) has a chemical structure that is related to the porphyrin configuration but with differences that enhance absorption of red light (long wavelength absorbance in the 650—700 nm region). It is a less complex mixture than porfimer sodium but has been shown to be similar in its biological effect [11]. meso-Tetrahydroxyphenylchlorin (mTHPC) is a chlorin that was first synthesized at Queen Mary College, London. It is chemically pure, has a strong absorption peak in the red part of the spectrum at 652 nm, and has a much higher singlet-oxygen yield than porfimer sodium so treatment times can be shortened [12]. Skin sensitivity usually lasts 2—3 weeks rather than the 2—3 months associated with porfimer sodium. ALA (5-aminolaevulinic acid) is a naturally occurring amino-acid and a precursor in the biosynthesis of haem. The enzymes involved in this biosynthesis convert ALA to the photocytotoxic compound protoporphyrin IX (PPIX). The last step in the formation of photochemically inactive heme is the incorporation of iron into PPIX under the action of ferrochelatase. By adding exogenous ALA, protoporphyrin IX may accumulate as this last step becomes rate-limiting. PPIX has an absorption peak at 635 nm and is metabolized to haem within a few hours so that skin sensitivity only lasts 1—2 days. Pheophorbide A has a wavelength of absorption of red light of 665 nm whilst indocyanine green (water soluble anionic dye) has a spectrum in the infrared region of light (800 nm), giving these photosensitisers the potential to cause greater depths of tumour necrosis than porfimer sodium. Hypericin has a dianthraquinone structure and is activated by ultraviolet and green light.
Mechanisms of action At least three main mechanisms for PDT-mediated tumour destruction have been proposed [3]. First, reactive oxygen species that are generated by PDT can kill tumour cells directly. Second, PDT damages the tumour-associated vasculature, which may lead to tumour infarction [27]. Finally, PDT can stimulate an immune response against tumour cells [28]. The relative importance of each mechanism in pancreatic cancer is unclear, but certainly varies with the photosensitiser used.
147
Direct tumour cell death Reactive oxygen species can cause direct photodamage to many biological molecules, including proteins, lipids and nucleic acids [29—31], at sites where the photosensitiser accumulates, either by apoptosis or necrosis. The intracellular localisation of the photosensitiser determines in part the mechanism of cell death [32]. Haematoporphyrin derivative and porfimer sodium both localise in mitochondria due to their hydrophobicity and their affinity for a plasma binding site on the mitochondrial membrane [33—35]. More hydrophilic sensitisers such as the phthlocyanines and many chlorins, enter cells via endocytosis and hence accumulate mainly in lysosomes. Damage to mitochondria generally leads to apoptosis whereas plasma membrane and lysosomal damage can delay or even inhibit apoptosis and instead induces necrosis [36—38]. The potential mechanisms of PDT-induced apoptosis have generated much interest particularly as they provide the opportunity to refine treatment [39]. Several pathways have been identified, including: (i) permeabilisation of mitochondrial membranes with subsequent release of apoptosis-related proteins, e.g. cytochrome c and subsequent generation of activated caspases [40,41], (ii) activation of cell surface death receptors Fas and TNFR1, (iii) release of cathepsins from lysosomes with subsequent cleavage of Bid to produce a pro apoptotic protein tBid [42], and (i.v.) damage to the endoplasmic reticulum with release of calcium which can promote apoptosis [43]. Several in vitro studies have shown that apoptosis requires the presence of functional enzymes and is thus inhibited by high dose PDT [44—46]. Pancreatic carcinoma tissue and cells treated with PDT have been shown to undergo both necrosis and apoptosis. A pilot study treating human pancreatic tumours (median diameter 4.0 cm) with mTHPC PDT achieved a radius of necrosis of 9 mm (range: 7—11) around each treatment point [20]. A study by Hajri et al. [26] demonstrated that apoptosis was the primary mechanism of cell death, when human pancreatic tumour cell lines were exposed to low dose pheophorbide A PDT in vitro and after grafting into athymic mice. As gentle programmed cell death avoids PDT-induced tumour haemorrhage [7,16], they postulated that the absence of haemoglobin (which acts as a shield against tissue light penetration) would improve efficacy.
Vascular cell death The viability of tumour cells is dependent on an adequate blood supply [47]. PDT-related damage of
148 the endothelium of blood vessels leads to severe and persistent post-PDT tumour hypoxia/anoxia [48]. These vascular effects are caused by reversible contraction of endothelial cells resulting in the exposure of the basement membrane, vessel leakage, and thrombus formation [3,49,50]. Dolmans and colleagues studied the effect of a novel photosensitiser MV6401 in vivo on the microvasculature of a mammary tumour (MCaIV). The acute vascular effects were characterised by vasoconstriction rather than thrombus formation [51]. However, long-term vascular shutdown was mediated by thrombus formation, as evidenced by histological evaluation and inhibition with heparin. The release of thromboxane [52,53] and inhibition of nitric oxide [54] have been implicated as mediators of these pathophysiological events, which lead to ischaemic death of tumour cells.
Immune system activation Since PDT causes necrosis of tumour cells with subsequent generation of inflammatory mediators, e.g. lipid fragments and metabolites of arachidonic acid [1,43], stimulation of the immune system has been postulated as one of its mechanisms of action. Evidence for PDT-induced immune activation comes from initial studies more than 10 years ago, which reported infiltration of lymphocytes, leukocytes and macrophages into PDT-treated tissue [1,43,55]. In 1996, de Vree et al. reported that PDT in a rhabdomyosarcoma-bearing rat model activated neutrophil accumulation, which slowed tumour growth. Depletion of neutrophils decreased the PDT-mediated effect on tumour growth [56]. A related study by the same group investigated this relationship in more detail, and found that PDT in vitro induced contraction of the endothelial cells, permitting neutrophil adherence to the subendothelial matrix [57]. More recently, the inflammatory cytokines interleukin IL-6 and IL-1 (but not TNF alpha) have been shown to be upregulated in response to PDT [58]. The effects of PDT on surviving tumour and normal cells are important elements in determining the outcome, but in contrast to the photocytotoxic action of PDT have received relatively little attention. Wong et al. addressed this by studying the effects of the photosensitisers aminolaevulinic acid (ALA) and porfimer sodium on several epithelial cancer cell lines and normal epithelial and stromal cells [59]. They showed that both normal and malignant cells lost their responsiveness to IL-6 class cytokines (involved in cellular differentiation and inhibition of proliferation of various epithelial cells) and epi-
L. Ayaru et al. dermal growth factor (involved in cellular proliferation, motility, adhesion, invasion and angiogenesis), in a dose-dependent manner. The recovery of responsiveness occurred 48—72 h later and was accompanied by a resumption of cell proliferation. The role of the adaptive immune response was investigated by Korbelik et al. in a study of PDT of EMT6 mammary sarcoma in SCID and normal mice. A significantly lower therapeutic effect was seen in SCID mice, suggesting that the lack of an immune response was responsible for the difference in tumour cures [60]. This effect could be restored by adoptive transfer of T-lymphocytes of normal (Balb/c) mice into the SCID mice. In a later study by the same group, EMT6 sarcoma-bearing mice were selectively depleted of specific lymphoid cells (T cells). Whilst initial tumour ablation by PDT was not affected, long-term tumour cure rates decreased markedly after depletion. These results provide direct evidence that T-lymphocyte function is essential for the maintenance of long-term control of PDT-treated tumours [61]. Henderson’s group reported that a tumour-cell lysate isolated after PDT with porfimer sodium could be used to vaccinate mice against the development of further tumours, indicating the induction of tumour specific immunity [62]. These PDT vaccines seemed to induce a cytotoxic T-cell response that involved induction of IL-12 expression. PDT may also be directly harmful to immune cells. Lymphocytes can accumulate photosensitiser and be destroyed during subsequent illumination, leading to local suppression of the immune system [63—65]. Moreover, PDT of the skin has been shown to suppress the contact hypersensitivity response, a classical example of a cell-mediated delayed-type immune reaction. Jolles et al. [66] demonstrated, in an adoptive transfer study in mice, that the reduction of the contact hypersensitivity response also extended to areas other than the illuminated site, and was associated with the generation of suppressor cells. These disparate observations can be explained in part by the structure of the tumour microenvironment. In general, immune cells are found in the tumour stroma, separated from tumour cells by extracellular matrix and basal membrane-like structures [67,68]. By disrupting the tumour microenvironment, PDT may enable direct interaction between immune cells and tumour cells. After the initial PDT-induced damage to tumour cells and immune cells, a strong inflammatory reaction occurs locally which leads to influx and activation of undamaged immune cells from elsewhere [28].
Photodynamic therapy for pancreatic carcinoma
Pancreatic adenocarcinoma Worldwide, adenocarcinoma of the pancreas is one of the top 10 leading causes of cancer death, and ranks sixth as a cause of cancer death in the UK and USA [69,70]. Upto 15—20% of patients have resectable disease, but even with adjuvant therapy only around 20% of these survive to five years [71,72]. Patients with locally advanced inoperable disease can be treated with chemotherapy, radiotherapy, or some combination of the two but these therapies rarely increase median survival beyond 12 months. For those with metastatic disease at presentation, palliative chemotherapy can improve symptoms of advanced malignancy, but the median survival is only 3—6 months [73,74].
Preclinical studies of PDT for pancreatic cancer Haematoporphyrin derivative PDT Mang and Wieman [13] were the first to demonstrate the potential of PDT as a treatment for pancreatic cancer. In two animal models of pancreatic carcinoma, they studied the uptake, fluorescence kinetics and effect of dihematoporphyrin ether (DHE). The first model was that of a pancreatic carcinoma of acinar origin induced in rats by azaserine treatments and serially transplanted into the same strain subcutaneously and intraperitoneally. The second model was of a ductal pancreatic carcinoma induced by injection of N-nitrosobis (2-oxopropyl) amine (BOP) into hamsters. At 24 h after injection of the photosensitiser, the tumours in both models exhibited equal or higher concentrations of DHE compared with normal pancreas by both tissue fluorescence and chemical extraction procedures. These tumours were then exposed at laparotomy to 630 nm light from a dye laser (75 mW cm−2 , 30 min). The tumour tissue demonstrated a relatively normal fluorescence decay pattern, with haemorrhage and a resultant loss of measurable DHE concentration. In contrast, there was no necrosis of normal pancreas under the same conditions and photobleaching of the DHE as measured by fluorescence decay did not occur. The reasons for this relative resistance of normal pancreas are unclear but the authors postulated that a singlet-oxygen quenching agent may be present in normal pancreas which is absent in pancreatic cancer and other tissues. DHE was also investigated in a BOP induced pancreatic cancer model by Schroder and colleagues [6]. Three hours after the injection of photosensitiser, DHE was 2.4 times more concentrated in
149 pancreatic tumour than in the normal pancreas and PDT caused extensive tumour necrosis without any obvious effect on the non-tumour-bearing pancreas. However, there was considerable accumulation of DHE in the adjacent small bowel, resulting in perforation of the duodenum and jejunum and death of four (57%) of the animals after PDT. Similar studies have been carried out using a variety of second generation photosensitisers, including phthalocyanine, pheophorbide A, 5-aminolaevulinic acid and mTHPC, as discussed below. Aluminium sulphonated phthalocyanine PDT (ALSPc) In an early in vitro study of ALSPc-PDT on normal acinar pancreatic cells and azaserine-induced pancreatic carcinoma cells, photon activation of cellularly bound ALSPc with light >570 nm permeabilised the cells and caused an increase in amylase release from normal acinar cells but a dose dependent inhibition of amylase release from tumour cells [15]. Ultrastructural studies showed that this distinct effect was not due to differences in the microanatomical organization of normal or tumour derived cells. Under these experimental conditions, the cells were capable of releasing secretory products such as amylase as a result of cellular stimulation by PDT and not simply as a direct consequence of widespread membrane lysis. A subsequent study by the same group showed that this release of amylase by normal acinar cells was energy dependent and inhibited by the chelation of free cytoplasmic calcium but not by the removal of extracellular calcium [17]. The authors suggested that the initial PDT-induced release of amylase from the normal pancreas may activate membrane receptors or G-proteins, resulting in release of intracellular calcium rather than being secondary to the influx of external calcium as seems to occur in myeloma cells treated with PDT [75]. They noted however that this PDT action did not exactly reproduce agonist action (bethanecol induced amylase release), indicating that ALSPc may be acting at a more distal point in the signal transduction pathway. In contrast, removal of extracellular calcium reversed the inhibitory effect of PDT on tumour cells and produced a significant increase in amylase release, whereas chelation of free cytoplasmic calcium had no effect. Suggested explanations for these results were PDT targeting of a calcium dependent membrane site or that in the presence of external calcium PDT activation may cause excessive influx of calcium, which inhibits constitutive
150 secretion. A protein kinase C-mediated opening of the voltage-sensitive calcium channels found in the tumour cells, but not in normal acinar cells, was postulated as a putative signal transduction mechanism. The adverse effects of PDT on the small intestine prompted our group to focus on the normal pancreas and adjacent tissues in hamsters, using treatment parameters similar to those known to induce pancreatic necrosis [5]. In initial pharmacokinetic studies using chemical extraction and microscopic fluorescence, the highest levels of ALS2 Pc were seen in duodenal mucosa and bile duct walls 48 h after photosensitisation. The PDT effects of disulphonated aluminium phthalocyanine (ALS2 Pc) were examined after illumination with light at wavelength 675 nm. When a dose of 5 mol/kg of sensitiser was used, duodenal perforations, gastric ulcers and transudation of bile from the bile duct occurred. The lesions in the stomach and bile duct healed without complication though the duodenal perforations remained. Minor necrosis was seen in the normal pancreas at this dose but damage to major vessels did not occur. A dose reduction to 1 mol/kg still caused a concealed duodenal perforation but damage to other tissues was diminished. Possible explanations for these results were the high concentration of sensitiser in the duodenum at 48 h and the thinness of the hamster duodenal wall. Our group then studied the effect of AlS2 Pc in hamster pancreas and a BOP induced hamster pancreatic cancer model [16]. Peak concentrations of sensitiser in tumour, measured by distribution studies from chemical extraction, were noted at 24 h. The tumour: normal tissue ratios at 4, 24 and 48 h were 0.74:1, 3.0:1 and 2.3:1, respectively. At 48 h after injection (5 mg/kg) into the inferior vena cava, the sensitiser was activated at laparotomy by red light (675 nm) delivered using a 200 m fibre just touching the tumour. When 25 or 50 J light doses were used, damage appeared to extend to the edges of tumour nodules 5 mm in diameter and up to 8 mm in larger tumours. In this study, no damage was seen in the adjacent normal pancreas or stomach in any tumour-bearing animals until light doses of 200 J were reached.
Pheophorbide A PDT Evrard et al. [7] demonstrated the feasibility of using pheophorbide A to treat azaserine-induced pancreatic rat carcinoma. A dose ranging component to the study showed that a 9 mg/kg versus 3 mg/kg dosage increased sensitiser concentration by a factor of two in the pancreas and over five in the tumour and was thus chosen as the optimum dose.
L. Ayaru et al. Administration of 9 mg/kg gave a selectivity ratio of 13.5:1 between tumour and surrounding tissue. Laser light at 660 nm and 100 J/cm2 delivered at laparotomy produced selective tumour necrosis of about 10 mm diameter. Six of nine tumour-bearing rats treated with PDT remained disease free at 120 days compared with a 100% mortality of control rats within 35 days (P < 0.01). The main complication was duodenal injury.
Aminolaevulinic acid (ALA) PDT Our group investigated ALA-induced PPIX for PDT in Syrian golden hamsters with transplanted pancreatic cancers [21]. ALA was given either intravenously or orally (in single or fractionated bolus doses), with laser light delivered by means of a bare fibre touching the tissue surface or by external irradiation using a light-integrating cylindrical applicator. We anticipated that oral fractionated doses would result in better tissue uptake than a single oral bolus because ALA and its metabolite, porphobilinogen, are rapidly excreted in the urine after a bolus dose [76]. The peak plasma concentration of ALA in humans after a single dose is reached at 60 min and the half-life (t1/2 ) of its clearance from the circulation is only about 50 min [77], so frequently repeated doses should maintain a steady high blood level of ALA for a longer time. This hypothesis was confirmed by pharmacokinetic studies 1—24 h after light exposure which demonstrated that PPIX sensitisation in the tumour, as measured by quantitative fluorescence microscopy, was highest after intravenous administration (which avoids first pass metabolism) of 200 mg/kg ALA, followed in decreasing order by oral fractionated and oral single bolus doses (both 400 mg/kg). Laser light application at 630 nm to give 12—50 J from the bare fibre or 50 J/cm using surface illumination with the cylindrical applicator resulted in necrosis of up to 8 mm in diameter. Smaller tumours showed complete necrosis of the entire lesion, but larger ones usually had a rim of viable tumour on the side opposite the irradiated surface. In a randomised survival study (the only one performed to date in PDT for pancreatic cancer), animals treated with oral fractionated ALA (400 mg/kg) survived significantly longer than the untreated group according to Kaplan—Meier analysis of survival curves (logrank test, P < 0.02) (see Fig. 1), although all animals eventually died of recurrent tumour [21]. A major limitation of ALA-PDT is that ALA itself is hydrophilic, which restricts its penetration through cellular membranes and access to mitochondria. Esterification of ALA (generated by treatment of ALA
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Figure 1 Kaplan—Meier survival curves for Syrian golden hamsters with transplanted pancreatic tumours comparing the PDT-treated group with the control, non-treated group. Regula et al. [21] reproduced with permission from Nature Publishing Group, www.nature.com.
hydrochloride with alcohol and thionyl chloride) makes it more lipophilic and improves transmembrane access. Once inside the cell, large amounts of free ALA can be generated by the action of nonspecific intracellular esterases in the majority of cells [78,79]. In azaserine-induced pancreatic carcinoma cell lines treated with a series of straight chain, branched and cyclo-␦ ALA esters, Whitaker et al. [23] examined both the generation of photoactive species and the resulting photocytotoxicity. They found that several ALA esters not only induced the formation of more PPIX, but they did so at a faster rate than ALA itself. This resulted in an enhanced photocytoxicity of some 270 times using the more potent ALA esters.
meso-Tetrahydoxyphenylchlorin (mTHPC) PDT Mlkvy et al. [18,19] from our unit carried out experiments to assess the effects of mTHPC on normal hamster pancreas (see Fig. 2) and in a hamster pancreatic cancer model created by injecting a BOP induced pancreatic cancer cell line into the gastric lobe of the pancreas. Fluorescence microscopy showed maximum levels of mTHPC in normal pancreas 2—4 days after sensitisation and in tumour at 4—5 days. After intravenous injection of 0.1 or 0.3 mg/kg mTHPC, the tumours were treated at laparotomy 2 or 4 days later with red light (50 J at 650 nm, continuous or fractionated) delivered via a single fibre touching the tumour surface. As ALS2 Pc PDT had been complicated by duodenal perforations, the duodenum was shielded from light using opaque paper. The maximum zone of necrosis
(seen 3 days after PDT) was 8.7 mm in diameter, increasing to 12.4 mm with light fractionation (see Fig. 3). Despite the use of duodenal shielding, the main complication was sealed duodenal perforation (in 3 of the 16 animals), which may have been due to slippage of the paper. Other adverse effects included transient bile duct obstruction and the development of multiple small duodenal diverticula. No effects were seen in the stomach or major blood vessels (aorta, vena cava and portal vein). There was no evidence of pancreatitis or cyst formation in the normal pancreas of animals that were kept alive for up to 3 months after PDT with any of the photosensitisers. PDT of human pancreatic carcinoma cells Recent preclinical studies have demonstrated the efficacy of PDT against human pancreatic carcinoma cells. Tseng et al. [25] exposed three separate human pancreatic ductal adenocarcinoma cells lines to indocyanine green in vitro, at a dose of 20 g/ml and 808 nm diode laser light (0.45 W for 5 min) and achieved over 90% cell killing. Liu et al. applied hypericin PDT to human pancreatic cell lines in vitro and to a pancreatic carcinoma model produced by injection of one of the cell lines into the shoulder and pancreatic bed of athymic nude mice. Pancreatic carcinoma growth was suppressed by 66.1 ± 0.2%, 91.2 ± 2.3% and 42.2 ± 8.1% in pancreatic cell lines, subcutaneous shoulder tumours and orthotopically transplanted pancreatic tumours, respectively [24]. It is apparent from these experimental studies that whilst PDT achieves impressive areas of necrosis, complete tumour eradication is usually not
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Figure 2 Normal pancreas 7 days after i.v. injection of 0.3 mg/kg mTHPC, and 3 days after light-delivery. Each treated area received 50 J light (50 MW for 1000 s). The lesion on the left had a single fraction of light, but for that on the right, the light was divided into four equal fractions with a break of 3 min between fractions. Scale 2.0 mm × 1.5 mm. Mlkvy et al. [19] reproduced with permission from Nature Publishing Group, www.nature.com.
possible. Reasons may include non-homogeneous distribution of photosensitiser within the tumour, decreased ability to kill tumour cells with increased distance from vascular supply [80], a reduction of tissue oxygen tension during and after illumination of photosensitised tissue [81,82] and inadequate light doses at all relevant sites.
PDT for pancreatic cancer: clinical studies As experimental work in the various studies outlined above had demonstrated efficient necrosis of pancreatic tumour tissue as well as an improvement in survival in one study [21], we undertook a phase 1 clinical study of PDT for pancreatic carcinoma [20]. A total of 16 patients with histologically confirmed inoperable pancreatic adenocarcinomas located in the head of the pancreas were recruited into the study. They had all presented with obstructive jaundice, which was relieved with plastic or metal biliary endoprostheses. In 14 cases, there was tumour involvement or proximity to the major vessels precluding attempted curative
resection and twelve patients had involvement of the duodenum detected at endoscopy or by computed tomography (CT). Patients were photosensitised with 0.15 mg/kg meso-tetrahydroxyphenylchlorin, which was selected as the photosensitiser because in the preclinical work it produced the largest zone of necrosis and required the lowest light dose at each treatment site. Photoactivation was performed three days later via means of percutaneously placed needles placed into the tumour under ultrasound or CT guidance. The light source was a diode laserdelivering red light at 652 nm, which was divided equally between the optical fibres using a beam splitter. The fibres were then inserted down to the tip of the needles to leave 3 mm of bare fibre in direct contact with the tumour during light delivery. The volume of necrosis produced by PDT treatment ranged from 9.0 to 60 cm3 . In most cases the necrosed area of tumour healed safely without changing in size. There were no free duodenal perforations but three patients developed duodenal obstruction, which may have been related to treatment. Two patients with tumour involving the gastroduodenal artery had significant gastrointestinal
Photodynamic therapy for pancreatic carcinoma
153 the distribution of laser effects to the extent of diseased tissue being treated, and ideally to extend the treated area beyond the tumour margins identified on pretreatment scans. The use of modified selection criteria, such as excluding patients with tumour encasement of a major artery or the duodenum, would also be expected to reduce the risk of major complications and allow treated areas to heal safely.
References
Figure 3 Photomicrographs of tumour nodules (see arrows) treated 3 days previously with a total light dose of 50 J (50 mW for 1000 s), delivered 4 days after 0.3 mg/kg mTHPC. (A) Continuous light (B) Fractionated light (four equal fractions with a 3-min break between fractions). Scale: 20 mm × 13 mm. Mlkvy et al. [18] reproduced with permission from Nature Publishing Group, www.nature.com, volume 76, pages 713—718.
bleeds, which were controlled with endoscopic or radiological intervention. In 14 cases the late stages of disease were dominated by local tumour invasion and re-growth of tumour from the edges of treated areas. The median survival time after PDT was 9.5 months (range: 4—36 months), which is comparable to the reported 5 (2.2—23) months median survival after chemotherapy or 9 (8—12.7) months with chemoradiotherapy [83]. This study demonstrated the feasibility of mTHPC PDT for pancreatic cancer using percutaneously placed light fibres. The morbidity was relatively low although problems did arise with tumours involving the duodenal wall or gastroduodenal artery. This therapy, however, was clearly palliative, as only 44% of patients were alive 1 year after PDT. Randomised controlled studies will be required to assess the true influence of PDT on survival, and its potential additional role to palliative chemotherapy in the management of this disease. Technical aspects of future studies will be to match
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