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Photodynamic Therapy Using Verteporfin Photosensitization in the Pancreas and Surrounding Tissues in the Syrian Golden Hamster
Original Paper Received: February 9, 2006 Accepted: July 20, 2006 Published online: April 18, 2007
Pancreatology 2007;7:20–27 DOI: 10.1159/000101874
Photodynamic Therapy Using Verteporfin Photosensitization in the Pancreas and Surrounding Tissues in the Syrian Golden Hamster Lakshmana Ayaru a Johannes Wittmann b A.J. MacRobert b Marco Novelli c Stephen G. Bown b Stephen P. Pereira a a The UCL Institute of Hepatology, Department of Medicine; b National Medical Laser Centre, and c Department of Pathology, Royal Free and University College London Medical School, University College London, London, UK
Key Words Meso-tetrahydroxyphenylchlorin Photodynamic therapy Syrian golden hamster Verteporfin photodynamic therapy Verteporfin photosensitization
Abstract Background/Aim: Photodynamic therapy (PDT) is a potential treatment for locally advanced pancreatic cancer. We aimed to assess the safety of interstitial PDT using verteporfin (benzoporphyrin derivative monoacid A – a novel photosensitizer with a short drug-light interval and limited cutaneous photosensitivity) in the Syrian golden hamster, and compare it to meso-tetrahydroxyphenylchlorin (mTHPC) which we have previously evaluated in preclinical and clinical studies. Methods: Verteporfin (2 mg/kg) was administered at laparotomy by inferior vena caval injection (n = 57), with plasma levels quantified at 5, 15, 30, 60 and 240 min, and 24 h. 15 min after photosensitization, tissues (liver, pancreas, duodenum, colon, aorta) were illuminated with 690 nm red laser light (150 mW), at a range of light doses (1–100 J/cm2). The PDT effects on the targeted organ and adjacent structures were assessed at post-mortem on days 3 and 21, or at the time of death. Results: The elimination half-life of verteporfin was 4–5 h. Light doses of 10, 25 and 50 J/cm2 were safe in the hamster pancreas, liver and colon, respectively, and
© 2007 S. Karger AG, Basel and IAP 1424–3903/07/0071–0020$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
produced coagulative necrotic lesions of 3 (range 3–4), 10 (9–10) and 7 (7–8) mm diameter. Collagen was resistant to damage and lesions healed mainly by regeneration of normal tissue. At higher light doses, necrosis extended to the edge of organs, sometimes causing sealed duodenal perforations as seen with mTHPC. Conclusion: The safety profile of verteporfin is very similar to mTHPC, with the advantages of a shorter drug-light interval and drug elimination time. Phase I clinical studies using this photosensitizer in pancreatic cancer should be feasible. Copyright © 2007 S. Karger AG, Basel and IAP
Introduction
Photodynamic therapy (PDT) is a way of producing localized non-thermal tissue necrosis with light, and is currently under evaluation as a treatment for pancreatic cancer [1]. This malignancy has a poor prognosis and novel therapies are urgently needed. We and others have shown in a series of experimental and clinical studies of PDT that safe and efficient necrosis of pancreatic tumour tissue is possible [2–6]. Previous photosensitizers that have been evaluated for this indication include haematoporphyrin derivative (HpD) and meso-tetrahydroxyphenylchlorin (mTHPC) Dr. Steve Pereira The UCL Institute of Hepatology Royal Free and University College London Medical School 69–75 Chenies Mews, London WCIE 6HX (UK) Tel. +44 20 7679 6510, Fax +44 20 7380 0405, E-Mail [email protected]
[4, 6–8]. However, HpD and its derivative porfimer sodium have several limitations which include their complexity (making it difficult to reproduce their composition), a small absorption peak (630 nm) in the red region of the visible spectrum, and prolonged cutaneous photosensitivity of 1–3 months [9]. mTHPC is chemically pure and has a strong absorption peak in the red part of the spectrum at 652 nm, but is inconvenient to use in clinical practice as it has a drug-light interval of 48–72 h and skin sensitivity lasting 2–3 weeks. Verteporfin (benzoporphyrin derivative monoacid ring A) is a chlorin-like molecule that exists as an equal mixture of two regioisomers, BPD-MAC and BPD-MAD. It is a potent second-generation photosensitizer which is licensed for the treatment of choroidal neovascularization secondary to age-related macular degeneration [10]. Verteporfin has several biological properties which could potentially improve the effectiveness and tolerability of pancreatic PDT. First, it has a short drug light interval (DLI) of 15–60 min. Second, it has an absorption peak at 690 nm and a higher quantum yield for singlet oxygen than mTHPC, which may allow for greater depths of necrosis to be achieved. Finally, the compound is cleared rapidly from the body, with skin photosensitivity times of only 24–48 h [11, 12]. In this study, we examined the effects of verteporfin PDT on the normal pancreas and surrounding tissues, with the aim of optimizing treatment parameters for tissue necrosis and avoiding unacceptable damage to surrounding tissues. Whilst in preclinical studies it has been relatively easy to destroy small volumes of solid tumours with verteporfin PDT [13–16], there has been little work on its effect on surrounding tissues. In theory, selective retention of photosensitizer by tumour over that of normal tissue could result in selective destruction of tumours. In practice, normal tissue is commonly damaged if exposed to the same light dose as the tumour, although selective tumour necrosis can still be achieved if PDT parameters are optimized since both tumour and normal tissue can heal by regeneration of normal tissue [2, 17, 18]. The first aim of this study was to determine the effect of verteporfin PDT on the normal pancreas and surrounding normal tissues under conditions known to produce necrosis in transplanted cancers. We also aimed to determine if the effects of verteporfin PDT on normal tissues were similar to those found with mTHPC, which we have previously used in experimental and clinical studies of pancreatic PDT [4, 8].
Verteporfin Photodynamic Therapy
Methods Photosensitizer Verteporfin (lipid-formulated benzoporphyrin monoacid A BPD-MA) was a gift from QLT, Inc. (Vancouver, Canada). A stock saline solution of verteporfin was reconstituted according to the manufacturer’s instructions and stored at 4 ° C in the dark. Animal Model All experiments were undertaken on normal female Syrian golden hamsters (100–120 g) under general anaesthesia using inhaled halothane (ICI Pharmaceuticals, Cheshire, UK) and intramuscular Hypnorm (fentanyl and fluanisone, Janssen Pharmaceuticals, High Wycombe, UK). Following surgery, analgesia was administered subcutaneously using buprenorphine hydrochloride (Reckitt & Colman Products Ltd, Hull, UK). Verteporfin Plasma and Tissue Fluorescence Pharmacokinetics To determine the pharmacokinetic profile of verteporfin, blood and tissue (liver, pancreas and colon) samples were taken from culled animals at 5, 15, 30, 60 min, 3 and 24 h after administration of 2 mg/kg verteporfin via bolus inferior vena caval injection at laparotomy. Two animals were studied at each time point. Potassium oxalate/sodium fluoride tubes were used for blood collection. Plasma was collected after centrifugation and frozen immediately. High-performance liquid chromatography and capillary electrophoresis with laser-induced fluorescence were used to quantify BPD-MAC and BPD-MAD. Three sections from each tissue sample were taken for fluorescence microscopy. Control sections were taken from unsensitized animals. Tissues removed were immediately frozen in a bath of isopentane (BDH, UK) cooled in liquid nitrogen. Frozen sections of 5 m thickness were cut (cryostat E microtome, Reichert) and stored at –70 ° C. Tissue sections were prepared and imaged with a minimum of light exposure to avoid bleaching of photosensitizer. Phase contrast microscopy with a slow-scan cooled chargecoupled device (CCD) camera (Wright Instruments Ltd, Enfield, London, UK) was used to image and quantify fluorescence in the cryosections. The fluorescence was excited using an 8-mW helium-neon laser (632.8 nm) and detected between 660 and 710 nm using band pass and long pass (Schott) filters. A false colour-coded image of the fluorescence signal in counts per pixel was produced in order to quantify the fluorescence intensity over specified areas. Three measurements were made and averaged per section. Photodynamic Therapy In preliminary experiments carried out on the liver of unsensitized hamsters using 150 mW at the fibre tip (100 J), no thermal effects were seen. Therefore, 150 mW and a range of energies from 1 to 100 J were studied, as these parameters are similar to those shown to produce necrosis of solid tumours in other experimental models using verteporfin PDT [15, 19]. In anaesthetized hamsters, 2 mg/kg of verteporfin was administered via inferior vena caval injection at laparotomy with laser light delivered after an interval of 15 min. This drug-light interval was chosen as previous work had shown that the induced vascular photodynamic effect produced a better cure rate of sol-
Pancreatology 2007;7:20–27
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Fig. 1. Linear plot of mean verteporfin concentrations in normal hamsters.
id tumours than DLIs of 3 h, which resulted in a direct cytotoxic effect [20, 21]. At laparotomy, light from a semiconductor laser was delivered at a wavelength of 690 nm (peak absorption for verteporfin), via a 400-m cleaved fibre just touching the surface of the tissue to be irradiated. For colonic PDT, the fibre was passed through the anti-mesenteric colon wall approximately 1 cm distal to the caecum so that it touched the mucosa of the opposite side. Only one site was treated in each animal. Two animals were studied for each light dose. The experiments were divided into two groups: (1) Duodenal lobe of the pancreas, upper duodenum (adjacent to the pancreas and bile duct, but clear of the ampullary region), caecum and liver were treated with light energies of 1, 5, 10, 25, 50 and 100 J. The aorta and inferior vena cava were treated with 10 J. Animals were culled 3 days after treatment (the time of maximal necrosis based on previous studies by our group of PDT for pancreatic cancer [8]). At post-mortem, the treated areas were excised and the maximum diameter of pancreatic lesions quantified with a micrometer. The minimum (a) and maximum (b) perpendicular diameters of the nearly circular liver lesions were measured and the surface area calculated using the formula ab/4. After treatment, representative specimens were fixed in formalin, sectioned and stained with haematoxylin and eosin for conventional light microscopy. (2) Based on the results from section one, the maximum likely tolerable light energies were delivered to the pancreas (10 J), liver (25 J) and colon (25 J) of a second group of hamsters 15 min after intravenous injection of 2 mg/kg of verteporfin. The animals were weighed every 3 days and followed up for 21 days to assess long-term survival after PDT, and on day 21 to assess healing of tissues. All studies were performed under the authority of experiment licences issued by the Home Office (UK).
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Fig. 2. a Fluorescence intensity (in arbitrary counts of units/pixel) 8SD as a function of time after administration of 2 mg/kg verteporfin in normal hamster pancreas, liver and colon. Each point is based on three measurements taken from each of 2 animals. b Fluorescence microscopy images of pancreas, liver and colon 15 min after injection of 2 mg/kg of verteporfin and no injection control tissues.
Results
Verteporfin Is Cleared Rapidly from Plasma and Normal Tissues The maximum plasma concentration of verteporfin was observed 15 min after intravenous injection, at the time when the first sample was taken. Thereafter, there was a clear biexponential decline of plasma verteporfin concentrations, with a rapid distribution phase followed by a moderately rapid elimination phase. The estimated half-life of verteporfin was 4–5 h (fig. 1). Ayaru /Wittmann /MacRobert /Novelli / Bown /Pereira
15 min after drug injection
Control (no drug)
Pancreas
Liver
Colon
b
2
Tissue fluorescence was measured in arbitrary units of counts per pixel corrected for background levels using sections from control animals. The relative levels of verteporfin in the pancreas, liver and colon over time and fluorescent images 15 min after drug injection are shown in figures 2a and b, respectively. In all the tissues studied, the peak levels of verteporfin fluorescence were seen 5 min to 3 h after sensitization, with the highest values recorded in liver tissue. Consistent with the rapid plasma clearance, verteporfin concentration fell in all tissues as evidenced by fluorescence levels at or close to background 24 h after injection.
Verteporfin PDT Produces Efficient Predictable Necrosis of Normal Tissues In control animals (no verteporfin) treated with 100 J of light there were no macroscopic or microscopic effects on the liver at the power level of 150 mW, thus excluding a thermal effect being produced using these parameters.
Verteporfin Photodynamic Therapy
Pancreatology 2007;7:20–27
Liver and Colon The effects of verteporfin PDT were initially tested on the liver and colon (fig. 4), as lesions in these tissues tended to be well defined and easy to measure, thus providing a guide as to which energies would be tolerated by the 23
normal pancreas. In these tissues, PDT produced wellcircumscribed areas of coagulative necrosis confirmed histologically. Increasing the light energy proportionately increased the volume of necrosis in both liver and colon, thus allowing for some predictive planning of PDT (fig. 3). Light doses to the liver of 50 J and above caused necrosis to extend beyond the edge of the liver and into the adjacent stomach. Similarly, light doses to the colon of 50 J or above resulted in a large inflammatory mass and adhesions between the bowel serosa and the abdominal wall causing bowel obstruction.
Fig. 3. Volume of liver necrosis (8SD) with increasing light
doses.
Pancreas, Duodenum and Large Vessels A light dose of 10 J to the pancreas resulted in a median pancreatic necrosis diameter of 3 mm (range
Fig. 4. Normal liver and colon 3 days after an intravenous injection of 2 mg/kg of verteporfin and light delivery (25 J, 690 nm, 150 mW). a Macroscopic appearance of liver lesion (arrow). b Macroscopic view of colon showing serosal blanching and inflammation (arrow). c HE stain showing coagulative necrosis (CN) and adjacent normal liver (NL).
Fig. 5. Normal pancreas 3 days after an intravenous injection of 2 mg/kg verteporfin and light delivery (10 J, 690 nm, 150 mW). a, b HE stains of (a) coagulative pancreatic necrosis (CN) and adjacent normal pancreas (NP) and (b) adjacent duodenal ulceration (arrow). c Masson’s trichrome stain showing underlying submucosal collagen (arrow) which is resistant to damage.
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Ayaru /Wittmann /MacRobert /Novelli / Bown /Pereira
3–4 mm) (fig. 5a) and minor adjacent duodenal mucosal damage (fig. 5b). Macroscopically, necrosis in the pancreas had a yellowish red appearance with surrounding oedema. Microscopically there were well-demarcated areas of necrosis. No necrosis was seen in the stomach of animals whose pancreas had been treated even if the tip of the fibre was placed in the duodenal lobe of the pancreas only about 3 mm from the greater curve of the stomach. Duodenal collagen was largely unaffected by PDT at this light energy (fig. 5c). A light dose of 25 J and above to the normal pancreas produced adjacent duodenal obstruction together with sealed perforations and adhesions. 10 J directly to the duodenum produced 6–7 mm of necrosis on the serosal surface but no associated obstruction or perforation. Three days after 10 J of energy was applied there were no macroscopic changes to large vessels (aorta and inferior vena cava). Verteporfin PDT Is Safe when Applied to Normal Tissues Light doses of 25 J applied to either the colon or liver and 10 J to the pancreas were associated with 100% survival at 21 days. Due to the high metabolism of hamsters, a sick animal that is not eating will rapidly lose weight and this was used as a surrogate marker for general wellbeing after treatment (fig. 6). Pancreatic and liver PDT at laparotomy was responsible for a weight loss of about 20% of body weight over the first 6 days. After this time, hamsters gained weight appropriately. Colonic PDT was much better tolerated than at the other two sites, with minimal weight loss after treatment. Lesions of the pancreas, liver and colon healed without any obvious macroscopic signs of necrosis detectable at 21 days. In particular, there were no signs of bowel obstruction or perforation.
Fig. 6. Weight change after verteporfin PDT.
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 [22, 23]. Approximately 15–20% of patients have resectable disease, but even with adjuvant therapy only around 20% of these survive to 5 years [24, 25]. Patients with locally advanced irresectable disease can be treated with chemotherapy, radiotherapy, or some combination of the two but these therapies rarely increase median survival beyond 12 months. PDT is a potential treatment for locally advanced pancreatic cancer. As experimental and clinical studies have
shown it can be tolerated by the pancreas and surrounding normal tissue. Photosensitizers evaluated to date for pancreatic cancer, which include sodium porfimer and mTHPC, have a number of limitations including prolonged cutaneous photosensitivity and long DLIs, limiting their tolerability and convenience for clinical use [1]. Verteporfin, a second-generation photosensitizer, has a number of biological properties which make it suitable for pancreatic PDT. The rapid systemic clearance of verteporfin is responsible for its short period of cutaneous photosensitivity of !48 h [12]. In the present study, verteporfin had an estimated half-life of 4–5 h in the hamster, which is similar to the reported half life of 5–6 h in humans [11], and it was rapidly cleared from the plasma and normal tissues by 24 h. We delivered verteporfin PDT after a short drug-light interval of 15 min and achieved efficient necrosis in all tissues studied. This short DLI has been shown to produce a vascular effect and better tumour cures than longer DLIs which produce predominantly cytotoxic damage [20, 21]. PDT has the potential for selective destruction of locally advanced tumours. There is indeed some selectivity in the uptake of verteporfin, mTHPC and sodium porfimer in pancreatic and other tumours, in the region of 2–3 times that of adjacent normal tissue [6, 18, 26]. In practice, this level of selectivity is rarely enough to achieve completely selective necrosis when tumour and normal tissue are exposed to the same light dose. In the present study, the maximal tolerated light dose by the pancreas
Verteporfin Photodynamic Therapy
Pancreatology 2007;7:20–27
Discussion
25
was 10 J, which produced median diameters of pancreatic necrosis of 3 mm – similar to the 4-mm lesions we saw with mTHPC PDT in the normal pancreas [8]. We observed the highest levels of tissue fluorescence and largest areas of necrosis for a given light energy in the liver, probably due its vascularity and role in drug uptake and metabolism. In the pancreas, the safe production of large necrotic lesions was limited by the small size of the gland and short distance from the fibre tip to adjacent organs. Achieving larger volumes of pancreatic necrosis should be feasible in the human pancreas, as we have previously shown in a pilot phase I/II study of mTHPC for pancreatic cancer [4]. In the present study, we identified parameters for verteporfin PDT capable of achieving safe necrosis in the normal pancreas, colon and liver. The most serious complications of adjacent duodenum and colon obstruction and sealed perforation predictably occurred after increasing the energy delivered. Previous studies by our group using mTHPC [8] and AlS2Pc [17] PDT on normal pancreas identified the duodenum as the most vulnerable organ – in contrast to the relatively thick-walled stomach which also lies in close proximity. In our earlier experimental studies, shielding of the duodenum reduced the risk of injury to the bowel wall, but in the present study we identified treatment parameters which made this unnecessary. In our clinical study of mTHPC for pancreatic
cancer [4], duodenal perforation did not occur, due in part to the thicker human duodenum and the greater distance from the tip of the fibre in the pancreas to the duodenum. We also found that the biology of verteporfin PDT was similar to that of other photosensitizers used for gastrointestinal and pancreatic PDT, in that collagen was largely resistant to damage [27] and tissues healed mainly by regeneration of normal tissues with minimal scarring [28]. These data suggest that it should be safe to treat human pancreatic cancers with verteporfin PDT, since some selectivity of tumour necrosis is achievable by limiting the light dose reaching organs outside the pancreas and maintaining the mechanical integrity of adjacent bowel [28]. Whilst we did not examine the effects of verteporfin PDT in an experimental model of pancreatic cancer, there is good evidence for the efficacy of this photosensitizer in necrosing other solid tumours [13–16]. Moreover, the results of our previous preclinical and clinical studies using mTHPC PDT indicate that at least as much necrosis can be achieved in pancreatic cancers as in the normal pancreas [3, 5, 6, 8, 18]. In conclusion, the safety profile of verteporfin PDT in the Syrian golden hamster is very similar to mTHPC, with the advantages of a shorter drug-light interval and drug elimination time. Phase I/II clinical studies are planned.
References 1 Ayaru L, Bown SG, Pereira SP: Photodynamic therapy for pancreatic carcinoma: experimental and clinical studies. Photodiagn Photodyn Ther 2004;1:145–155. 2 Evrard S, Keller P, Hajri A, Balboni G, Mendoza-Burgos L, Damge C, Marescaux J, Aprahamian M: Experimental pancreatic cancer in the rat treated by photodynamic therapy. Br J Surg 1994; 81:1185–1189. 3 Chatlani PT, Nuutinen PJ, Toda N, Barr H, MacRobert AJ, Bedwell J, Bown SG: Selective necrosis in hamster pancreatic tumours using photodynamic therapy with phthalocyanine photosensitization. Br J Surg 1992; 79: 786–790. 4 Bown SG, Rogowska AZ, Whitelaw DE, Lees WR, Lovat LB, Ripley P, Jones L, Wyld P, Gillams A, Hatfield AW: Photodynamic therapy for cancer of the pancreas. Gut 2002;50:549– 557. 5 Regula J, Ravi B, Bedwell J, MacRobert AJ, Bown SG: Photodynamic therapy using 5aminolaevulinic acid for experimental pancreatic cancer – prolonged animal survival. Br J Cancer 1994;70:248–254.
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6 Mikvy P, Messman H, MacRobert AJ, Pauer M, Sams VR, Davies CL, Stewart JC, Bown SG: Photodynamic therapy of a transplanted pancreatic cancer model using meta-tetrahydroxyphenylchlorin. Br J Cancer 1997;76: 713–718. 7 Mang TS, Wieman TJ: Photodynamic therapy in the treatment of pancreatic carcinoma: dihematoporphyrin ether uptake and photobleaching kinetics. Photochem Photobiol 1987;46:853–858. 8 Mlkvy P, Messmann H, Pauer M, Stewart JC, Millson CE, MacRobert AJ, Bown SG: Distribution and photodynamic effects of mesotetrahydroxyphenylchlorin in the pancreas and adjacent tissues in the Syrian golden hamster. Br J Cancer 1996; 73:1473–1479. 9 Ayaru L, Bown SG, Pereira SP: Photodynamic therapy for pancreatic and biliary tract carcinoma. Int J Gastrointest Cancer 2005; 35:1–13.
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10 Wu L, Murphy RP: Photodynamic therapy: a new approach to the treatment of choroidal neovascularization secondary to age-related macular degeneration. Curr Opin Ophthalmol 1999;10:217–220. 11 Houle JM, Strong A: Clinical pharmacokinetics of verteporfin. J Clin Pharmacol 2002; 42:547–557. 12 Houle JM, Strong HA: Duration of skin photosensitivity and incidence of photosensitivity reactions after administration of verteporfin. Retina 2002;22:691–697. 13 Chen B, Pogue BW, Zhou X, O’Hara JA, Solban N, Demidenko E, Hoopes PJ, Hasan T: Effect of tumor host microenvironment on photodynamic therapy in a rat prostate tumor model. Clin Cancer Res 2005; 11: 720– 727. 14 Richter AM, Waterfield E, Jain AK, Allison B, Sternberg ED, Dolphin D, Levy JG: Photosensitising potency of structural analogues of benzoporphyrin derivative in a mouse tumour model. Br J Cancer 1991; 63:87–93.
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15 Richter AM, Waterfield E, Jain AK, Canaan AJ, Allison BA, Levy JG: Liposomal delivery of a photosensitizer, benzoporphyrin derivative monoacid ring A, to tumor tissue in a mouse tumor model. Photochem Photobiol 1993;57:1000–1006. 16 Pogue BW, O’Hara JA, Demidenko E, Wilmot CM, Goodwin IA, Chen B, Swartz HM, Hasan T: Photodynamic therapy with verteporfin in the radiation-induced fibrosarcoma-1 tumor causes enhanced radiation sensitivity. Cancer Res 2003; 63:1025–1033. 17 Nuutinen PJ, Chatlani PT, Bedwell J, MacRobert AJ, Phillips D, Bown SG: Distribution and photodynamic effect of disulphonated aluminium phthalocyanine in the pancreas and adjacent tissues in the Syrian golden hamster. Br J Cancer 1991; 64:1108–1115. 18 Schroder T, Chen IW, Sperling M, Bell RH Jr, Brackett K, Joffe SN: Hematoporphyrin derivative uptake and photodynamic therapy in pancreatic carcinoma. J Surg Oncol 1988; 38:4–9.
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19 Allison BA, Waterfield E, Richter AM, Levy JG: The effects of plasma lipoproteins on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative. Photochem Photobiol 1991;54:709– 715. 20 Fingar VH, Kik PK, Haydon PS, Cerrito PB, Tseng M, Abang E, Wieman TJ: Analysis of acute vascular damage after photodynamic therapy using benzoporphyrin derivative. Br J Cancer 1999;79:1702–1708. 21 Kurohane K, Tominaga A, Sato K, North JR, Namba Y, Oku N: Photodynamic therapy targeted to tumor-induced angiogenic vessels. Cancer Lett 2001;167:49–56. 22 Jemal A, Thomas A, Murray T, Thun M: Cancer statistics, 2002. CA Cancer J Clin 2002;52:23–47. 23 Parkin DM, Pisani P, Ferlay J: Global cancer statistics. CA Cancer J Clin 1999;49:33–64.
24 Neoptolemos JP, Stocken DD, Friess H, Bassi C, Dunn JA, Hickey H, Beger H, FernandezCruz L, Dervenis C, Lacaine F, Falconi M, Pederzoli P, Pap A, Spooner D, Kerr DJ, Büchler MW: A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. N Engl J Med 2004; 350:1200–1210. 25 Li D, Xie K, Wolff R, Abbruzzese JL: Pancreatic cancer. Lancet 2004;363:1049–1057. 26 Richter AM, Cerruti-Sola S, Sternberg ED, Dolphin D, Levy JG: Biodistribution of tritiated benzoporphyrin derivative (3H-BPDMA), a new potent photosensitizer, in normal and tumor-bearing mice. J Photochem Photobiol B 1990;5:231–244. 27 Barr H, Tralau CJ, Boulos PB, MacRobert AJ, Tilly R, Bown SG: The contrasting mechanisms of colonic collagen damage between photodynamic therapy and thermal injury. Photochem Photobiol 1987;46:795–800. 28 Bown SG, Lovat LB: The biology of photodynamic therapy in the gastrointestinal tract. Gastrointest Endosc Clin N Am 2000; 10: 533–550.
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Pancreatology 2007;7:20–27 DOI: 10.1159/000101874
Photodynamic Therapy Using Verteporfin Photosensitization in the Pancreas and Surrounding Tissues in the Syrian Golden Hamster Lakshmana Ayaru a Johannes Wittmann b A.J. MacRobert b Marco Novelli c Stephen G. Bown b Stephen P. Pereira a a The UCL Institute of Hepatology, Department of Medicine; b National Medical Laser Centre, and c Department of Pathology, Royal Free and University College London Medical School, University College London, London, UK
Key Words Meso-tetrahydroxyphenylchlorin Photodynamic therapy Syrian golden hamster Verteporfin photodynamic therapy Verteporfin photosensitization
Abstract Background/Aim: Photodynamic therapy (PDT) is a potential treatment for locally advanced pancreatic cancer. We aimed to assess the safety of interstitial PDT using verteporfin (benzoporphyrin derivative monoacid A – a novel photosensitizer with a short drug-light interval and limited cutaneous photosensitivity) in the Syrian golden hamster, and compare it to meso-tetrahydroxyphenylchlorin (mTHPC) which we have previously evaluated in preclinical and clinical studies. Methods: Verteporfin (2 mg/kg) was administered at laparotomy by inferior vena caval injection (n = 57), with plasma levels quantified at 5, 15, 30, 60 and 240 min, and 24 h. 15 min after photosensitization, tissues (liver, pancreas, duodenum, colon, aorta) were illuminated with 690 nm red laser light (150 mW), at a range of light doses (1–100 J/cm2). The PDT effects on the targeted organ and adjacent structures were assessed at post-mortem on days 3 and 21, or at the time of death. Results: The elimination half-life of verteporfin was 4–5 h. Light doses of 10, 25 and 50 J/cm2 were safe in the hamster pancreas, liver and colon, respectively, and
© 2007 S. Karger AG, Basel and IAP 1424–3903/07/0071–0020$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
produced coagulative necrotic lesions of 3 (range 3–4), 10 (9–10) and 7 (7–8) mm diameter. Collagen was resistant to damage and lesions healed mainly by regeneration of normal tissue. At higher light doses, necrosis extended to the edge of organs, sometimes causing sealed duodenal perforations as seen with mTHPC. Conclusion: The safety profile of verteporfin is very similar to mTHPC, with the advantages of a shorter drug-light interval and drug elimination time. Phase I clinical studies using this photosensitizer in pancreatic cancer should be feasible. Copyright © 2007 S. Karger AG, Basel and IAP
Introduction
Photodynamic therapy (PDT) is a way of producing localized non-thermal tissue necrosis with light, and is currently under evaluation as a treatment for pancreatic cancer [1]. This malignancy has a poor prognosis and novel therapies are urgently needed. We and others have shown in a series of experimental and clinical studies of PDT that safe and efficient necrosis of pancreatic tumour tissue is possible [2–6]. Previous photosensitizers that have been evaluated for this indication include haematoporphyrin derivative (HpD) and meso-tetrahydroxyphenylchlorin (mTHPC) Dr. Steve Pereira The UCL Institute of Hepatology Royal Free and University College London Medical School 69–75 Chenies Mews, London WCIE 6HX (UK) Tel. +44 20 7679 6510, Fax +44 20 7380 0405, E-Mail [email protected]
[4, 6–8]. However, HpD and its derivative porfimer sodium have several limitations which include their complexity (making it difficult to reproduce their composition), a small absorption peak (630 nm) in the red region of the visible spectrum, and prolonged cutaneous photosensitivity of 1–3 months [9]. mTHPC is chemically pure and has a strong absorption peak in the red part of the spectrum at 652 nm, but is inconvenient to use in clinical practice as it has a drug-light interval of 48–72 h and skin sensitivity lasting 2–3 weeks. Verteporfin (benzoporphyrin derivative monoacid ring A) is a chlorin-like molecule that exists as an equal mixture of two regioisomers, BPD-MAC and BPD-MAD. It is a potent second-generation photosensitizer which is licensed for the treatment of choroidal neovascularization secondary to age-related macular degeneration [10]. Verteporfin has several biological properties which could potentially improve the effectiveness and tolerability of pancreatic PDT. First, it has a short drug light interval (DLI) of 15–60 min. Second, it has an absorption peak at 690 nm and a higher quantum yield for singlet oxygen than mTHPC, which may allow for greater depths of necrosis to be achieved. Finally, the compound is cleared rapidly from the body, with skin photosensitivity times of only 24–48 h [11, 12]. In this study, we examined the effects of verteporfin PDT on the normal pancreas and surrounding tissues, with the aim of optimizing treatment parameters for tissue necrosis and avoiding unacceptable damage to surrounding tissues. Whilst in preclinical studies it has been relatively easy to destroy small volumes of solid tumours with verteporfin PDT [13–16], there has been little work on its effect on surrounding tissues. In theory, selective retention of photosensitizer by tumour over that of normal tissue could result in selective destruction of tumours. In practice, normal tissue is commonly damaged if exposed to the same light dose as the tumour, although selective tumour necrosis can still be achieved if PDT parameters are optimized since both tumour and normal tissue can heal by regeneration of normal tissue [2, 17, 18]. The first aim of this study was to determine the effect of verteporfin PDT on the normal pancreas and surrounding normal tissues under conditions known to produce necrosis in transplanted cancers. We also aimed to determine if the effects of verteporfin PDT on normal tissues were similar to those found with mTHPC, which we have previously used in experimental and clinical studies of pancreatic PDT [4, 8].
Verteporfin Photodynamic Therapy
Methods Photosensitizer Verteporfin (lipid-formulated benzoporphyrin monoacid A BPD-MA) was a gift from QLT, Inc. (Vancouver, Canada). A stock saline solution of verteporfin was reconstituted according to the manufacturer’s instructions and stored at 4 ° C in the dark. Animal Model All experiments were undertaken on normal female Syrian golden hamsters (100–120 g) under general anaesthesia using inhaled halothane (ICI Pharmaceuticals, Cheshire, UK) and intramuscular Hypnorm (fentanyl and fluanisone, Janssen Pharmaceuticals, High Wycombe, UK). Following surgery, analgesia was administered subcutaneously using buprenorphine hydrochloride (Reckitt & Colman Products Ltd, Hull, UK). Verteporfin Plasma and Tissue Fluorescence Pharmacokinetics To determine the pharmacokinetic profile of verteporfin, blood and tissue (liver, pancreas and colon) samples were taken from culled animals at 5, 15, 30, 60 min, 3 and 24 h after administration of 2 mg/kg verteporfin via bolus inferior vena caval injection at laparotomy. Two animals were studied at each time point. Potassium oxalate/sodium fluoride tubes were used for blood collection. Plasma was collected after centrifugation and frozen immediately. High-performance liquid chromatography and capillary electrophoresis with laser-induced fluorescence were used to quantify BPD-MAC and BPD-MAD. Three sections from each tissue sample were taken for fluorescence microscopy. Control sections were taken from unsensitized animals. Tissues removed were immediately frozen in a bath of isopentane (BDH, UK) cooled in liquid nitrogen. Frozen sections of 5 m thickness were cut (cryostat E microtome, Reichert) and stored at –70 ° C. Tissue sections were prepared and imaged with a minimum of light exposure to avoid bleaching of photosensitizer. Phase contrast microscopy with a slow-scan cooled chargecoupled device (CCD) camera (Wright Instruments Ltd, Enfield, London, UK) was used to image and quantify fluorescence in the cryosections. The fluorescence was excited using an 8-mW helium-neon laser (632.8 nm) and detected between 660 and 710 nm using band pass and long pass (Schott) filters. A false colour-coded image of the fluorescence signal in counts per pixel was produced in order to quantify the fluorescence intensity over specified areas. Three measurements were made and averaged per section. Photodynamic Therapy In preliminary experiments carried out on the liver of unsensitized hamsters using 150 mW at the fibre tip (100 J), no thermal effects were seen. Therefore, 150 mW and a range of energies from 1 to 100 J were studied, as these parameters are similar to those shown to produce necrosis of solid tumours in other experimental models using verteporfin PDT [15, 19]. In anaesthetized hamsters, 2 mg/kg of verteporfin was administered via inferior vena caval injection at laparotomy with laser light delivered after an interval of 15 min. This drug-light interval was chosen as previous work had shown that the induced vascular photodynamic effect produced a better cure rate of sol-
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Fig. 1. Linear plot of mean verteporfin concentrations in normal hamsters.
id tumours than DLIs of 3 h, which resulted in a direct cytotoxic effect [20, 21]. At laparotomy, light from a semiconductor laser was delivered at a wavelength of 690 nm (peak absorption for verteporfin), via a 400-m cleaved fibre just touching the surface of the tissue to be irradiated. For colonic PDT, the fibre was passed through the anti-mesenteric colon wall approximately 1 cm distal to the caecum so that it touched the mucosa of the opposite side. Only one site was treated in each animal. Two animals were studied for each light dose. The experiments were divided into two groups: (1) Duodenal lobe of the pancreas, upper duodenum (adjacent to the pancreas and bile duct, but clear of the ampullary region), caecum and liver were treated with light energies of 1, 5, 10, 25, 50 and 100 J. The aorta and inferior vena cava were treated with 10 J. Animals were culled 3 days after treatment (the time of maximal necrosis based on previous studies by our group of PDT for pancreatic cancer [8]). At post-mortem, the treated areas were excised and the maximum diameter of pancreatic lesions quantified with a micrometer. The minimum (a) and maximum (b) perpendicular diameters of the nearly circular liver lesions were measured and the surface area calculated using the formula ab/4. After treatment, representative specimens were fixed in formalin, sectioned and stained with haematoxylin and eosin for conventional light microscopy. (2) Based on the results from section one, the maximum likely tolerable light energies were delivered to the pancreas (10 J), liver (25 J) and colon (25 J) of a second group of hamsters 15 min after intravenous injection of 2 mg/kg of verteporfin. The animals were weighed every 3 days and followed up for 21 days to assess long-term survival after PDT, and on day 21 to assess healing of tissues. All studies were performed under the authority of experiment licences issued by the Home Office (UK).
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Fig. 2. a Fluorescence intensity (in arbitrary counts of units/pixel) 8SD as a function of time after administration of 2 mg/kg verteporfin in normal hamster pancreas, liver and colon. Each point is based on three measurements taken from each of 2 animals. b Fluorescence microscopy images of pancreas, liver and colon 15 min after injection of 2 mg/kg of verteporfin and no injection control tissues.
Results
Verteporfin Is Cleared Rapidly from Plasma and Normal Tissues The maximum plasma concentration of verteporfin was observed 15 min after intravenous injection, at the time when the first sample was taken. Thereafter, there was a clear biexponential decline of plasma verteporfin concentrations, with a rapid distribution phase followed by a moderately rapid elimination phase. The estimated half-life of verteporfin was 4–5 h (fig. 1). Ayaru /Wittmann /MacRobert /Novelli / Bown /Pereira
15 min after drug injection
Control (no drug)
Pancreas
Liver
Colon
b
2
Tissue fluorescence was measured in arbitrary units of counts per pixel corrected for background levels using sections from control animals. The relative levels of verteporfin in the pancreas, liver and colon over time and fluorescent images 15 min after drug injection are shown in figures 2a and b, respectively. In all the tissues studied, the peak levels of verteporfin fluorescence were seen 5 min to 3 h after sensitization, with the highest values recorded in liver tissue. Consistent with the rapid plasma clearance, verteporfin concentration fell in all tissues as evidenced by fluorescence levels at or close to background 24 h after injection.
Verteporfin PDT Produces Efficient Predictable Necrosis of Normal Tissues In control animals (no verteporfin) treated with 100 J of light there were no macroscopic or microscopic effects on the liver at the power level of 150 mW, thus excluding a thermal effect being produced using these parameters.
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Liver and Colon The effects of verteporfin PDT were initially tested on the liver and colon (fig. 4), as lesions in these tissues tended to be well defined and easy to measure, thus providing a guide as to which energies would be tolerated by the 23
normal pancreas. In these tissues, PDT produced wellcircumscribed areas of coagulative necrosis confirmed histologically. Increasing the light energy proportionately increased the volume of necrosis in both liver and colon, thus allowing for some predictive planning of PDT (fig. 3). Light doses to the liver of 50 J and above caused necrosis to extend beyond the edge of the liver and into the adjacent stomach. Similarly, light doses to the colon of 50 J or above resulted in a large inflammatory mass and adhesions between the bowel serosa and the abdominal wall causing bowel obstruction.
Fig. 3. Volume of liver necrosis (8SD) with increasing light
doses.
Pancreas, Duodenum and Large Vessels A light dose of 10 J to the pancreas resulted in a median pancreatic necrosis diameter of 3 mm (range
Fig. 4. Normal liver and colon 3 days after an intravenous injection of 2 mg/kg of verteporfin and light delivery (25 J, 690 nm, 150 mW). a Macroscopic appearance of liver lesion (arrow). b Macroscopic view of colon showing serosal blanching and inflammation (arrow). c HE stain showing coagulative necrosis (CN) and adjacent normal liver (NL).
Fig. 5. Normal pancreas 3 days after an intravenous injection of 2 mg/kg verteporfin and light delivery (10 J, 690 nm, 150 mW). a, b HE stains of (a) coagulative pancreatic necrosis (CN) and adjacent normal pancreas (NP) and (b) adjacent duodenal ulceration (arrow). c Masson’s trichrome stain showing underlying submucosal collagen (arrow) which is resistant to damage.
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Ayaru /Wittmann /MacRobert /Novelli / Bown /Pereira
3–4 mm) (fig. 5a) and minor adjacent duodenal mucosal damage (fig. 5b). Macroscopically, necrosis in the pancreas had a yellowish red appearance with surrounding oedema. Microscopically there were well-demarcated areas of necrosis. No necrosis was seen in the stomach of animals whose pancreas had been treated even if the tip of the fibre was placed in the duodenal lobe of the pancreas only about 3 mm from the greater curve of the stomach. Duodenal collagen was largely unaffected by PDT at this light energy (fig. 5c). A light dose of 25 J and above to the normal pancreas produced adjacent duodenal obstruction together with sealed perforations and adhesions. 10 J directly to the duodenum produced 6–7 mm of necrosis on the serosal surface but no associated obstruction or perforation. Three days after 10 J of energy was applied there were no macroscopic changes to large vessels (aorta and inferior vena cava). Verteporfin PDT Is Safe when Applied to Normal Tissues Light doses of 25 J applied to either the colon or liver and 10 J to the pancreas were associated with 100% survival at 21 days. Due to the high metabolism of hamsters, a sick animal that is not eating will rapidly lose weight and this was used as a surrogate marker for general wellbeing after treatment (fig. 6). Pancreatic and liver PDT at laparotomy was responsible for a weight loss of about 20% of body weight over the first 6 days. After this time, hamsters gained weight appropriately. Colonic PDT was much better tolerated than at the other two sites, with minimal weight loss after treatment. Lesions of the pancreas, liver and colon healed without any obvious macroscopic signs of necrosis detectable at 21 days. In particular, there were no signs of bowel obstruction or perforation.
Fig. 6. Weight change after verteporfin PDT.
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 [22, 23]. Approximately 15–20% of patients have resectable disease, but even with adjuvant therapy only around 20% of these survive to 5 years [24, 25]. Patients with locally advanced irresectable disease can be treated with chemotherapy, radiotherapy, or some combination of the two but these therapies rarely increase median survival beyond 12 months. PDT is a potential treatment for locally advanced pancreatic cancer. As experimental and clinical studies have
shown it can be tolerated by the pancreas and surrounding normal tissue. Photosensitizers evaluated to date for pancreatic cancer, which include sodium porfimer and mTHPC, have a number of limitations including prolonged cutaneous photosensitivity and long DLIs, limiting their tolerability and convenience for clinical use [1]. Verteporfin, a second-generation photosensitizer, has a number of biological properties which make it suitable for pancreatic PDT. The rapid systemic clearance of verteporfin is responsible for its short period of cutaneous photosensitivity of !48 h [12]. In the present study, verteporfin had an estimated half-life of 4–5 h in the hamster, which is similar to the reported half life of 5–6 h in humans [11], and it was rapidly cleared from the plasma and normal tissues by 24 h. We delivered verteporfin PDT after a short drug-light interval of 15 min and achieved efficient necrosis in all tissues studied. This short DLI has been shown to produce a vascular effect and better tumour cures than longer DLIs which produce predominantly cytotoxic damage [20, 21]. PDT has the potential for selective destruction of locally advanced tumours. There is indeed some selectivity in the uptake of verteporfin, mTHPC and sodium porfimer in pancreatic and other tumours, in the region of 2–3 times that of adjacent normal tissue [6, 18, 26]. In practice, this level of selectivity is rarely enough to achieve completely selective necrosis when tumour and normal tissue are exposed to the same light dose. In the present study, the maximal tolerated light dose by the pancreas
Verteporfin Photodynamic Therapy
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Discussion
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was 10 J, which produced median diameters of pancreatic necrosis of 3 mm – similar to the 4-mm lesions we saw with mTHPC PDT in the normal pancreas [8]. We observed the highest levels of tissue fluorescence and largest areas of necrosis for a given light energy in the liver, probably due its vascularity and role in drug uptake and metabolism. In the pancreas, the safe production of large necrotic lesions was limited by the small size of the gland and short distance from the fibre tip to adjacent organs. Achieving larger volumes of pancreatic necrosis should be feasible in the human pancreas, as we have previously shown in a pilot phase I/II study of mTHPC for pancreatic cancer [4]. In the present study, we identified parameters for verteporfin PDT capable of achieving safe necrosis in the normal pancreas, colon and liver. The most serious complications of adjacent duodenum and colon obstruction and sealed perforation predictably occurred after increasing the energy delivered. Previous studies by our group using mTHPC [8] and AlS2Pc [17] PDT on normal pancreas identified the duodenum as the most vulnerable organ – in contrast to the relatively thick-walled stomach which also lies in close proximity. In our earlier experimental studies, shielding of the duodenum reduced the risk of injury to the bowel wall, but in the present study we identified treatment parameters which made this unnecessary. In our clinical study of mTHPC for pancreatic
cancer [4], duodenal perforation did not occur, due in part to the thicker human duodenum and the greater distance from the tip of the fibre in the pancreas to the duodenum. We also found that the biology of verteporfin PDT was similar to that of other photosensitizers used for gastrointestinal and pancreatic PDT, in that collagen was largely resistant to damage [27] and tissues healed mainly by regeneration of normal tissues with minimal scarring [28]. These data suggest that it should be safe to treat human pancreatic cancers with verteporfin PDT, since some selectivity of tumour necrosis is achievable by limiting the light dose reaching organs outside the pancreas and maintaining the mechanical integrity of adjacent bowel [28]. Whilst we did not examine the effects of verteporfin PDT in an experimental model of pancreatic cancer, there is good evidence for the efficacy of this photosensitizer in necrosing other solid tumours [13–16]. Moreover, the results of our previous preclinical and clinical studies using mTHPC PDT indicate that at least as much necrosis can be achieved in pancreatic cancers as in the normal pancreas [3, 5, 6, 8, 18]. In conclusion, the safety profile of verteporfin PDT in the Syrian golden hamster is very similar to mTHPC, with the advantages of a shorter drug-light interval and drug elimination time. Phase I/II clinical studies are planned.
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