Preclinical Study of the Novel Vascular Occluding Agent, WST11, for Photodynamic Therapy of the Canine Prostate

Preclinical Study of the Novel Vascular Occluding Agent, WST11, for Photodynamic Therapy of the Canine Prostate Simone Chevalier,*,† Maurice Anidjar,† Eleonora Scarlata, Lucie Hamel, Avigdor Scherz,‡ Hervé Ficheux, Nicolas Borenstein, Laurence Fiette and Mostafa Elhilali From the Urologic Oncology Research Group, Research Institute, McGill University Health Center (SC, MA, ES, LH, ME), Montreal, Quebec, Canada, Weizmann Institute of Science (AS), Rehovot, Israel, and Steba Biotech (HF), Cedex and Institut Mutualiste Montsouris (NB, LF), Paris, France

Abbreviations and Acronyms Cmax ⫽ highest plasmatic WST11 value VTP ⫽ vascular targeted photodynamic therapy Submitted for publication August 9, 2010. Study received approval from the animal ethics committees of Research Institute, McGill University Health Center, Canada, and Institut Mutualiste Montsouris, France. Sponsored by STEBA Biotech, France. Supplementary material for this article can be obtained at http://muhc.ca/sites/default/files/ docs/WST11-VTP-Prostate.pdf. * Correspondence: Division of Urology, Research Institute of McGill University Health Centre, 1650 Cedar Ave., Montréal, Québec, Canada, H3G 1A4 (telephone: 514-934-1934, ext 44616; FAX: 514-934-8261; e-mail: simone.chevalier@ mcgill.ca). † Equal study contribution. ‡ Financial interest and/or other relationship with Steba.

Purpose: Vascular targeted photodynamic therapy with WST09 shows promise for recurrent prostate cancer after radiation but hydrophobicity in aqueous solutions limited application. We tested the safety and efficacy of WST11, a novel water soluble vascular occluding agent, for vascular targeted photodynamic therapy of the dog prostate and compared it to WST09 vascular targeted photodynamic therapy. Materials and Methods: Optical fibers were inserted in the prostate and connected to diode lasers. WST11 (Steba Biotech, Cedex, France) at varying doses, including a drug control with no light in 34 dogs, and WST09 (Steba Biotech) (2 mg/kg) in 3 dogs were infused during 10 minutes. Illumination was initiated at 5 or 10 minutes, and lasted up to 33.2 minutes based on laser fluence and delivered energy. Blood was collected for analysis and pharmacokinetics. The end point was at 1 week. Results: No vascular targeted photodynamic therapy associated change was observed in blood pressure or blood test values. Circulating WST11 increased with drug infusion and decreased rapidly during 1 hour to reach undetectable levels by 24 hours. All except 1 dog with bowel intussusception did well after vascular targeted photodynamic therapy with only mild urinary symptoms that resolved within 24 to 48 hours. Lung and liver were normal. Hemorrhage was present in all prostates except controls. This translated into necrosis at a WST11 threshold and within a window of doses at fixed illumination. Necrosis was associated with loss of the vessel endothelial layer. Fluence highly impacted necrosis. WST11 vascular targeted photodynamic therapy was advantageously comparable to WST09 vascular targeted photodynamic therapy, and optimally ablated about 5.0 cm3 of tissue per lobe and about 10 cm3 of the whole prostate. Conclusions: The safety and efficacy of WST11 vascular targeted photodynamic therapy in the dog prostate support clinical applications for prostate cancer and benign prostatic hyperplasia. Key Words: prostate, photochemotherapy, necrosis, blood vessels, dogs

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PHOTODYNAMIC therapy is used to treat nonmalignant and malignant diseases.1,2 The procedure involves the administration of light sensitive drugs, followed by illumination of target tissues at maximum sensitizer light ab-

sorption, resulting in the generation of cytotoxic, short-lived reactive oxygen species in the illuminated area.3 Photosensitizers are classified as tissue or vascular targeting according to respective activation sites, implying

0022-5347/11/1861-0302/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION

Vol. 186, 302-309, July 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.03.039

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RESEARCH, INC.

VASCULAR OCCLUDING AGENT WST11 FOR PHOTODYNAMIC THERAPY OF PROSTATE

long (days) vs short (minutes) drug-light intervals.4,5 To date vascular targeting has relied on the rapid uptake of sensitizers by endothelial cells.6 – 8 The bacteriochlorophyll derivatives WST09 and WST11, of which the latter was more recently discovered, target vessels as circulating sensitizers that generate superoxide and hydroxyl radicals in the lumen upon near infrared illumination.9,10 Instantaneous (about 1 minute) and nonreversible occlusion of tumor and peritumor vasculature,11 followed by tumor necrosis within 24 to 48 hours after illumination12–15 resulted in a greater than 75% cure rate in rodent models.16 –18 Rapid clearance from the circulation and minimal extravasation to surrounding tissues have decreased the risks of prolonged cutaneous photosensitivity and rendered WST09 and WST11 promising agents for VTP.12–15 Clinically WST09-VTP showed efficacy as salvage focal therapy for localized prostate cancer after radiation therapy failure in 28 patients.19 A complete response rate, defined as negative biopsies at 6 months, was observed in greater than 60% of patients who received maximal drug and light doses. The same regimen was applied in patients without previous treatment.20 However, the poor solubility of WST09 resulted in a few complications that limited clinical application and promoted the development of WST11.17,21 This novel vascular occluding agent has efficiently treated colon carcinoma in mice,18 and macular degeneration in rabbit and rat eyes.21 We preclinically assessed the safety of WST11 in dogs and its efficacy for VTP of the prostate compared to that of WST09-VTP. A threshold WST11 dose, light dose and drug-light interval to achieve maximal necrosis with minimal side effects were found and used to define parameters for WST11VTP human protocols.

MATERIALS AND METHODS Animals The study was approved by the animal ethics committees of Research Institute, McGill University Health Center, Canada, and Institut Mutualiste Montsouris, France. A total of 27 dogs (Laka, Saint-Basile-le-Grand, Quebec, Canada) a mean ⫾ SD of 8 ⫾ 4 years old and 10 (Marshall BioResources Europe, Lyon Cedex, France) a mean of 5 ⫾ 2 years old of different breeds and body weights were individually housed (see table). They received a regular canine diet with free access to water.

Photosensitizers, Lasers and Fibers We used WST11 (Tookad® Soluble, monolysotaurine, 2 K⫹, 400 mg powder), WST09 (Tookad, 2.5 mg/ml, kept in the dark at 4C) and diode lasers (Steba Biotech), including 753 nm for WST11 and 763 nm for WST09. WST11 was reconstituted before each dog treatment in mannitol (0.7%)/glucose (5%), sterilized using a 0.22 ␮m membrane

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(Millipore™) and kept wrapped on ice for less than 1 hour until infusion. Optical fibers with a 1 cm diffuser tip (Medlight, Ecublens, Switzerland) were calibrated before use. Lasers were used at 150 to 250 mW/cm fluence to deliver 100 to 400 J/cm energy per lobe per diffuser (see table).

Procedure and Followup Antibiotics (cefazolin) were administered 24 hours before (50 mg/kg orally), during (1 gm intravenously) and daily after (50 mg/kg orally) VTP. For WST09 premedication (10 mg diphenhydramine intravenously and 10 mg NovoPrednisone® orally) was given 24 hours before and immediately before surgery.14,22 Anesthesia (5 mg/kg ketamine and 0.4 mg/kg valium intravenously) was induced after sedation (0.2 mg/kg butorphanol, 0.125 mg/kg acepromazine and 0.04 mg/kg atropine subcutaneously) and controlled with isoflurane (2% to 3%). Vein lines were installed in the contralateral legs for infusion and blood sampling, respectively. Suprapubic vertical midline laparotomy was performed to expose the prostate. One or 2 transparent plastic needles were positioned in each lobe parallel to the urethra to insert fibers. Photosensitizers at various doses were infused for 10 minutes through a calibrated peristaltic pump using a syringe fitted to a 0.22 ␮m filter (see table). Illumination was arbitrarily initiated at 5 minutes after the start or end of infusion in 22 and 14 dogs, respectively, with duration based on laser fluence and delivered energy (see table). Blood was withdrawn preoperatively, during surgery for VTP from 2 to 60 minutes, and postoperatively at 24 and 48 hours, and 1 week to analyze total proteins, albumin, glucose, blood urea nitrogen/urea, creatinine, bilirubin, aminotransferase, aspartate aminotransferase, ␥-glutamyl transferase, alkaline phosphatase, creatinine kinase, cholesterol, ions (Na, K, Cl, Ca, P and Mg), hematocrit, hemoglobin, mean corpuscular hemoglobin volume and concentration, D-dimers, differential blood cell and platelet counts, and photosensitizer pharmacokinetics, as previously reported.21,22 Each dog was monitored daily. Analgesia (0.015 mg/kg buprenorphine intravenously and a 75 ␮g transdermal fentamyl patch) was provided for 2 to 3 days postoperatively. Except for dog 16, as described, sacrifice (90 mg/kg Euthanyl™ intravenously) was performed 1 week after VTP to allow assessment of safety during followup and efficacy by established necrosis.23

Tissue Processing The prostate was weighed, measured, inked, fixed in 10% buffered formaldehyde, cut from cranial to caudal into 3 to 5 mm slices with the central urethra, photographed to capture sequential images of gross hemorrhages in each slice, which appeared as brown-reddish areas, and paraffin embedded. The bladder, lower urethra, liver and lung/ respiratory tree (3 random pieces per lobe) were similarly submitted for histopathology. Macroscopic hemorrhage was recorded for each prostate and reported as the number of positive (colored) slices per total number of slices (5 to 7 per prostate). Hemorrhagic areas were expressed relative to urethral length (mean ⫾ SD 3.4 ⫾ 0.54 cm) for each lobe. Intensity was graded from 0 to 6⫹ based on color and covered surface.

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Experimental VTP conditions Photoproduct (dog No.) WST11: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16* 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 WST09: 35 36 37

Light (mins) Body Wt (kg)

Dose (mg/kg)

Fluence (mW/cm)

Energy (J/cm) (lobe 1/2)

Start

Duration (lobe 1/2)

11 9 44 8 13 13 37 32 28 9 39 32 33 30 23 31 13 32 9 6 12 36 31 35 27 37 11 13 10 11 11 9 11 11

2 2 2 2 4 4 7.5 7.5 10 10 15 15 20 20 30 30 5 5 5 5 2 2 5 5 5 5 5 5 5 5 5 5 2 2

150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 200 200 200 200 200 200 250 250 250 250 250 250 250 250 250 250 250 250

100/200 100/200 100/200 100/200 100/200 100/200 100/200 100/200 100/200 0/0 100/200 100/200 100/200 100/200 100/200 100/200 200/400 200/400 200/400 200/400 200/400 200/400† 200/200† 200‡/400 200/400† 400/400‡ 200/400† 200/400† 200/400† 200/400† 200/400† 200/400† 200/200‡ 200/200‡

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 10 10 10 10 10 10 10 10 10 10 10 10

11/22 11/22 11/22 11/22 11/22 11/22 11/22 11/22 11/22 — 11/22 11/22 11/22 11/22 11/22 11/22 16.6/33.2 16.6/33.2 16.6/33.2 16.6/33.2 16.6/33.2 16.6/33.2 13.3/26.7 13.3/26.7 13.3/26.7 13.3/26.7 13.3/26.7 13.3/26.7 13.3/26.7 13.3/26.7 13.3/26.7 13.3/26.7 10/13.2 10/13.2

10 9 10

2 2 2

150 150 150

100/200 200/200‡ 200/200‡

5 10 10

11/22.1 22.1/22.1 22.1/22.1

* Small bowel intussusception. † Two fibers per lobe placed about 1 cm apart in each lobe. ‡ Two fibers in 1 lobe placed about 1 cm apart.

Blocks corresponding to most hemorrhagic prostate slices in the dose escalating series were sectioned at 4 ␮m for hematoxylin and eosin staining. Blocks and sections were remeasured to calculate shrinkage of an average of 1.25-fold due to processing. Blood vessel integrity was assessed by immunohistochemistry using Factor VIII antibodies (Dako, Carpinteria, California) (1:800 dilution).24 Well established necrotic areas stained with hematoxylin and eosin were mapped, scanned, analyzed by morphometry using Image-Pro Plus, version 5.0 (MediaCybernetics®) and expressed in surfaces in mm2 in each lobe. Tissue destruction was also reported as a percent relative to each lobe surface, also measured by morphometry. Prostates with extensive necrosis, including those in dogs 17 to 20 and 24 to 34 in the WST11-VTP optimization series, and in dogs 36 and 37 in the WST09-VTP series,

were entirely submitted for histopathological analysis to reconstitute the prostate. In these prostates necrosis was expressed as total volume (Vnt) in cm3 by summing necrotic volumes in lobes 1 (VN1) and 2 (VN2) using the equation, VN1 ⫽ ⌺ (SN1 ⫻ 1.25) ⫻ L/n and VN2 ⫽ ⌺ (SN2 ⫻ 1.25) ⫻ L/n in 1 to n slices per lobe, where SN1 and SN2 were determined on hematoxylin and eosin stained sections from serial slices of each prostate, 1.25 represents the correction factor accounting for dehydration and shrinkage throughout processing, and L/n represents the thickness of each slice obtained from urethral length, measured in the fresh organ, on the number of slices.

Statistics Data were analyzed by linear regression with comparison of r2 values for trend line. Statistical significance was also

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Figure 1. Blood WST11 levels. A, Cmax as function of 2 to 30 mg/kg infused WST11 in dose escalating series (r2 ⫽ 0.90, see table). B, representative pharmacokinetics show variations in plasmatic WST11 with time in dog 7 (see table). Cmax was attained at about 10 to 11 minutes. VTP conditions were WST11 7.5 mg/kg, laser fluence 150 mW/cm, and energy 100 and 200 J with single 1 cm fiber per lobe.

assessed by the Wilcoxon matched pair or the unpaired t test, as indicated, using PRISM®. Results were considered statistically different at p ⬍0.05.

RESULTS Safety Each procedure was uneventful with stable blood pressure and no or slight to moderate changes in biochemical and hematological parameters, as described, which returned to initial values by 24 to 48 hours (data not shown). No link was noted between animal characteristics, photoproducts, WST11 doses and laser parameters. At followup the only VTP related observation was purple-reddish urine and occasional mild urethral bloody discharge on day 1. Appetite and behavior were normal except in dog 16, in which health deterioration led to sacrifice on day 4. Small bowel intussusception was identified. In each case the lower urinary tract appeared normal at necropsy except for moderate to severe epiploic adhesions to the prostate/bladder neck. Average prostate weight was 19.5 ⫾ 8.1 gm (range 7.5 to 49.0). The capsule showed light bluish coloration with WST09, which was only found occasionally for WST11. There was no macroscopic damage to the rectal wall or large bowel and macrothrombi or microthrombi in the liver, lung and bronchial tree (data not shown). VTP Efficacy and Dose Escalation In addition to drug control dog 10 with 10 mg/kg WST11 infusion without illumination, 15 dogs were enrolled in VTP to determine the effects of increasing the WST11 dose from 2 to 30 mg/kg at 150 mW/cm fluence emitting 100 and 200 J, respectively, through 1 central 1 cm diffuser fiber in each prostate lobe (see table). Circulating WST11 peaked at Cmax values that increased linearly with the dose at the

end of infusion at 10 to 11 minutes (r2 ⫽ 0.90, fig. 1, A). They decreased to about 10% of Cmax by 1 hour and were nondetectable at 24 hours (fig. 1, B). Macroscopically VTP resulted in hemorrhages centered at the fiber location (fig. 2, A), which were visible in all treated prostate lobes except control lobes. Hemorrhages extended from 2 to all slices over 1.2 to 3.5 cm in the craniocaudal axis. There was no correlation between the WST11 dose and hemorrhage in prostate slices, expressed in cm of spread (r2 ⬍0.21), color intensity (r2 ⬍0.31) and surfaces (r2 ⬍0.003).

Figure 2. Representative images show VTP induced hemorrhage and histological changes in nonnecrotic prostate lobes after WST11-VTP 4 mg/kg in dog 6 using fluence 150 mW/cm, and energy 100 and 200 J with single 1 cm fiber per lobe (see table). A, hemorrhagic spread centered around treatment fiber in each prostate lobe sliced from base to apex were more pronounced at 200 J (left) than at 100 J (right). B, section shows red blood cells (arrowhead) in affected parenchyma with disorganized and atrophic glands (arrows) and inflammation. H & E, reduced from ⫻20. C, intact microvessels with endothelium stained in hemorrhagic areas without necrosis. Factor VIII, reduced from ⫻64.

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Microscopically hemorrhages were characterized by red blood cells infiltrating the stroma, accompanied by atrophic, disorganized or dilated glands, intercellular edema and inflammation (fig. 2, B). Of the 30 treated lobes 15 showed no necrosis 1 week after VTP and blood vessels were intact (fig. 2, C). Necrosis was well established in the 15 other lobes, which also showed moderate (3 to 4⫹) to intense (6⫹) hemorrhages. Necrotic areas were smaller and did not correlate with hemorrhagic areas (r2 ⫽ 0.59, fig. 3, A to C). Tissue destruction was accompanied by loss of the vessel endothelial layer (fig. 3, D and E). In contrast, the endothelium was intact in vessels in immediately adjacent, nonnecrotic parts of hemorrhages (fig. 2, C). In this series necrosis was established in 6 of 15 lobes (40%) illuminated at 100 J and in 9 of 15 (60%) illuminated at 200 J. In lobes with necrosis mean ablated tissue area was 22 (range 7 to 57) and 57 mm2 (range 9 to 109), representing 12% and 22% of lobe surfaces at 100 and 200 J, respectively. The difference in necrotic areas was not statistically different by the Wilcoxon matched pair t test. With all lobes included the extent of necrosis at 200 J was superior to that of necrosis at 100 J (p ⬍0.008). The threshold WST11 dose for tissue destruction was about 4.0 mg/kg at 200 J with less consistent necrosis above 10 mg/kg. The urothelium, capsule and pericapsular nerves were preserved. Optimal Illumination We determined the extent of necrosis in the most hemorrhagic slice of each prostate resulting from WST11VTP done at 5.0 mg/kg and at higher light doses. In

Necrotic surface (mm2)

A 125

dogs 17 to 20 at 200 mW/cm and 200 J 3 of 4 lobes (75%) showed tissue destruction with a mean necrotic area of 109 mm2 (range 36 to 211) or 43% per affected lobe. All 4 lobes showed necrosis at 400 J and the mean affected surface was similar at 106 mm2 (range 73 to 226) or 43% per lobe. Necrotic areas at 200 and 400 J also did not differ when all values were compared using the Wilcoxon matched pair t test. VTP under the same light conditions but at 2 mg/kg WST11 in dogs 21 and 22 did not result in necrosis, although morphological changes were present over the entire lobes (data not shown). The best WST11-VTP mediated necrosis was achieved at 250 mW/cm laser fluence. For instance, 9 of 10 prostates showed necrosis at 5 mg/kg WST11 in dogs 23 to 32. The average ablated surface was 201 mm2 (range 25 to 227) or 60% per affected lobe at 400 J compared to 157 mm2 (range 48 to 275) or 50% at 200 J. The difference was not statistically significant by the Wilcoxon matched pair t test. However when all values were included, necrosis at 400 J was greater than at 200 J (Wilcoxon matched pair t test p ⬍0.02). The prostate in dog 23 showed only morphological changes. At this fluence decreasing WST11 to 2.0 mg/kg in dogs 33 and 34 compared advantageously to 5.0 mg/kg to generate necrosis (140 to 161 mm2 per lobe at 200 J and 231 to 247 at 400 J). Figure 4, A shows the impact of illumination on WST11-VTP mediated prostate necrosis, expressed as the mean surface per lobe. Comparison of the highest light fluence regimen at 250 mW/cm and 400 J to other fluences and energies was significant

B

C

D

E

100 75 50 25 0

100

200

Hemorrhagic area (mm2)

Figure 3. VTP mediated prostate necrosis. A, necrotic lesions as function of macroscopic hemorrhage extension. Data were obtained from prostate lobes illuminated at 200 J (r2 ⫽ 0.59). B, representative macroscopic hemorrhagic prostate slice. C, macroscopic view of corresponding section shows necrosis (circles) in dog 4 using WST11 2 mg/kg, fluence 150 mW/cm and energy 100 to 200 J in either lobe (see table). H & E. D, microscopic view of larger necrotic area of same prostate reveals severe hemorrhage (red blood cells), increased eosinophilia, and absent glands and cellular structures. H & E, reduced from ⫻20. E, microscopic view of larger necrotic area of same prostate shows disrupted endothelium lacking staining. Factor VIII stain, reduced from ⫻64.

VASCULAR OCCLUDING AGENT WST11 FOR PHOTODYNAMIC THERAPY OF PROSTATE

Necrotic surface (mm2/section)

A

307

Comparison With WST09-VTP Dogs 35 to 37 underwent WST09-VTP using established conditions (see table).22,25 Gross hemorrhage and tissue destruction of the glands and vascular endothelium were observed in all lobes, similar to WST11-VTP 1 week after VTP. The necrotic surface was 76 to 238 mm2 or 30% to 77% per lobe.

300 250 200 150

Fluence (mW/cm)

100 50

250 S3 S2 200

0 1

100

2 200

3 400

S1 150

Energy (J/cm)

B

Ablated Prostate Volume To determine overall VTP effects necrosis was quantified in serial blocks from the 17 most affected prostates and expressed in volume of ablated tissue per lobe. Figure 4, B shows that necrosis extended throughout several prostate slices beyond the 1 cm diffuser. At 250 mW/cm and 5 mg/kg WST11 in dogs 24 to 32 mean VTP ablated prostate volume was 4.3 (range 1.2 to 7.0) and 5.2 cm3 (range 0.6 to 7.2) per lobe at 200 and 400 J, respectively, totaling about 10 cm3 (range 1.8 to 14.2) in about 20 cm3 (19.5 gm) prostates. Also, VTP with WST11 at 2 mg/kg in dogs 33 and 34 yielded a similar necrotic volume of 3.5 to 3.6 and 6.8 to 7.8 cm3 per lobe at 200 and 400 J, respectively. WST11-VTP compared favorably to WST09-VTP with 3.1 to 5.1 cm3 of ablated tissue per lobe or 6.4 to 8.6 cm3 per prostate in dogs 36 and 37.

DISCUSSION

Figure 4. Optimal VTP induced prostate necrosis. A, influence of light parameters, including fluence 150 to 250 mW/cm and energy 100 to 400 J per fiber, on necrotic areas per lobe induced by WST11 in sections from most hemorrhagic prostate slices. B, slices from dog 18 prostate show WST11-VTP induced hemorrhage in specimens and mapped necrosis in macroscopic H & E stained sections of each lobe. R, right. L, left.

in inducing greater necrotic damage (200 mW/cm at 400 and 200 J unpaired t test p ⬍0.05 and p ⬍0.02, and 150 mW/cm at 100 and 200 J unpaired t test p ⬍0.0001 and ⬍0.0001, respectively). Further histopathological analysis revealed necrotic microfoci at the capsule in 10 prostates, at periprostatic nerves in 3 and at the urothelium in 3.

To our knowledge we report the first preclinical assessment of the novel vascular occluding agent WST11 for interstitial VTP of the prostate. The water solubility of WST11 represents a major advantage for clinical use since it eliminates the Cremophor® based formulation for solubilization and premedication to counteract cosolvent effects on blood pressure.14,17,22 In dogs blood pressure was remarkably constant during VTP and independent of WST11 dose, dog age, body weight and breed. Moreover, safety was noted at varying WST11 doses and light parameters using hematological and biochemical parameters together with good animal behavior and there were no side effects, such as urinary retention or gross hematuria. One dog did not attain the study end point but his condition (small bowel intussusception) was unlikely related to VTP. WST11-VTP appears to mostly respect the prostate anatomy, focally reaching the urothelium, capsule and in some instances the periprostatic nerves. In all dogs the colon/rectum and bladder were intact after VTP with WST11 and WST09, in line with reports of WST09 in normal and pre-irradiated dog prostates.14,22,25,26 Nevertheless, caution is required when translating safety data for adjacent organs to the human setting in regard to avoiding rectourethral fistula.19 Hence, the sensitivity of naïve vs previously irradiated prostates to WST11-VTP in humans needs further evaluation.

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When considering safety, a remarkable feature of WST11-VTP is its short half-life in the circulation. With practically a single rapid exponential clearance rate with a half-time of about 12 to 15 minutes in dogs, similar to that in rodents and rabbits,13,21 potential cutaneous phototoxicity shortly after treatment is further decreased compared to that of WST09, which clears in a bi-exponential manner with a half-time of about 6 to 7 hours for the slower phase.27 We first observed the efficacy of WST11-VTP by dose escalation in fixed light conditions. Infused WST11 doses correlated with circulating concentrations and illumination time overlapped with maximal WST11 blood levels. As such, 2 to 30 mg/kg WST11 were sufficient for light activation to trigger macroscopic hemorrhages centered on the fiber in each prostate lobe. As expected, no hemorrhages were observed in drug control dog 10 or in light controls.21 Gross hemorrhages did not translate into necrosis 1 week after VTP in 15 of the 30 lobes but rather into morphological changes associated with red blood cell leakage from intact vessels into the stroma. Nicotinamide adenine dinucleotide diaphorase staining, which rapidly distinguishes living vs injured cells, was not studied since prostates were harvested at a time when morphological changes characterizing necrosis were best observed by hematoxylin and eosin staining.23 Remarkably hemorrhage in the other 15 lobes comprised areas of well established necrosis surrounded by immediately adjacent nonnecrotic regions. Striking differences in blood vessel and glandular integrity were noted between these zones. Notably the endothelium was lost only in necrotic areas, indicating a vascular shutdown mechanism of WST11-VTP tissue destruction, as reported.17 Hence, VTP induced necrosis was

observed beyond a threshold level of activated WST11 and within a window of 4 to 7.5 mg/kg, given inconsistent or no necrosis at high drug doses. This observation likely reflects the need to increase light fluence with the increased light absorption at the lumen-endothelium interface. In this regard fractionation of drug or light doses was proposed to increase VTP effects.28,29 Illumination appeared the determinant to maximize necrosis. Increasing fluence to 250 mW/cm to generate 400 J per diffuser and activate 5 mg/kg WST11 enabled the ablation of large tissue surfaces, which translated into a volume of about 5 cm3 per lobe or about 10 cm3 in the prostate. Such light conditions allowed a lower WST11 dose (2 mg/kg) to destroy a comparable prostate volume with no influence of specific animal characteristics. WST11-VTP favorably compared to WST09-VTP,22 which in the current small cohort led to 3 to 5 cm3 necrosis per lobe. Together these findings and the absence of toxicity provide clear advantages for WST11 use in the clinical setting.

CONCLUSIONS WST11 is safe in dogs and efficient for VTP mediated ablation of large volumes of prostatic tissue by vascular occlusion. It compares advantageously to WST09-VTP. This study provides the basis for optimal treatment parameters to apply this promising new therapy to target the malignant and benign human prostate.

ACKNOWLEDGMENTS Dr. S. Moussa, Dr. K. Szymanski, F. Zouanat and M. E. Robitaille provided technical assistance.

REFERENCES 1. Dolmans DE, Fukumura D and Jain RK: Photodynamic therapy for cancer. Nat Rev Cancer 2003; 3: 380. 2. Huang Z: A review of progress in clinical photodynamic therapy. Technol Cancer Res Treat 2005; 4: 283. 3. Triesscheijn M, Baas P, Schellens JH et al: Photodynamic therapy in oncology. Oncologist 2006; 11: 1034. 4. Henderson BW, Waldow SM, Mang TS et al: Tumor destruction and kinetics of tumor cell death in two experimental mouse tumors following photodynamic therapy. Cancer Res 1985; 45: 572. 5. Moore CM, Pendse D and Emberton M: Photodynamic therapy for prostate cancer—a review of current status and future promise. Nat Clin Pract Urol 2009; 6: 18.

6. Solban N, Rizvi I and Hasan T: Targeted photodynamic therapy. Lasers Surg Med 2006; 38: 522. 7. Solban N, Selbo PK, Sinha AK et al: Mechanistic investigation and implications of photodynamic therapy induction of vascular endothelial growth factor in prostate cancer. Cancer Res 2006; 66: 5633. 8. O’Connor AE, Gallagher WM and Byrne. AT: Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy. Photochem Photobiol 2009; 85: 1053. 9. Vakrat-Haglili Y, Weiner L, Brumfeld V et al: The microenvironment effect on the generation of reactive oxygen species by Pd-bacteriopheophorbide. J Am Chem Soc 2005; 127: 6487.

10. Ashur I, Goldschmidt R, Pinkas I et al: Photocatalytic generation of oxygen radicals by the watersoluble bacteriochlorophyll derivative WST11, noncovalently bound to serum albumin. J Phys Chem A 2009; 113: 8027. 11. Madar-Balakirski N, Tempel-Brami C, Kalchenko V et al: Permanent occlusion of feeding arteries and draining veins in solid mouse tumors by vascular targeted photodynamic therapy (VTP) with Tookad. PLoS One 2010; 5: e10282. 12. Gross S, Gilead A, Scherz A et al: Monitoring photodynamic therapy of solid tumors online by BOLD-contrast MRI. Nat Med 2003; 9: 1327. 13. Mazor O, Brandis A, Plaks V et al: WST11, a novel water-soluble bacteriochlorophyll derivative; cellular uptake, pharmacokinetics, biodistribution and vascular-targeted photodynamic activity us-

VASCULAR OCCLUDING AGENT WST11 FOR PHOTODYNAMIC THERAPY OF PROSTATE

309

ing melanoma tumors as a model. Photochem Photobiol 2005; 81: 342.

ment and increase in overall therapeutic rate. Photochem Photobiol 2008; 84: 1231.

human lymph node metastases of prostate cancer. Can J Urol 2004; 11: 2146.

14. Chen Q, Huang Z, Luck D et al: Preclinical studies in normal canine prostate of a novel palladiumbacteriopheophorbide (WST09) photosensitizer for photodynamic therapy of prostate cancers. Photochem Photobiol 2002; 76: 438.

19. Trachtenberg J, Weersink RA, Davidson SR et al: Vascular-targeted photodynamic therapy (padoporfin, WST09) for recurrent prostate cancer after failure of external beam radiotherapy: a study of escalating light doses. BJU Int 2008; 102: 556.

15. Preise D, Oren R, Glinert I et al: Systemic antitumor protection by vascular-targeted photodynamic therapy involves cellular and humoral immunity. Cancer Immunol Immunother 2009; 58: 71.

20. Ahmed HU, Moore C and Emberton M: Minimallyinvasive technologies in uro-oncology: the role of cryotherapy, HIFU and photodynamic therapy in whole gland and focal therapy of localised prostate cancer. Surg Oncol 2009; 18: 219.

25. Huang Z, Haider MA, Kraft S et al: Magnetic resonance imaging correlated with the histopathological effect of Pd-bacteriopheophorbide (Tookad) photodynamic therapy on the normal canine prostate gland. Lasers Surg Med 2006; 38: 672.

16. Koudinova NV, Pinthus JH, Brandis A et al: Photodynamic therapy with Pd-Bacteriopheophorbide (TOOKAD): successful in vivo treatment of human prostatic small cell carcinoma xenografts. Int J Cancer 2003; 104: 782.

21. Berdugo M, Bejjani RA, Valamanesh F et al: Evaluation of the new photosensitizer Stakel (WST-11) for photodynamic choroidal vessel occlusion in rabbit and rat eyes. Invest Ophthalmol Vis Sci 2008; 49: 1633.

17. Brandis A, Mazor O, Neumark E et al: Novel water-soluble bacteriochlorophyll derivatives for vascular-targeted photodynamic therapy: synthesis, solubility, phototoxicity and the effect of serum proteins. Photochem Photobiol 2005; 81: 983. 18. Fleshker S, Preise D, Kalchenko V et al: Prompt assessment of WST11-VTP outcome using luciferase transfected tumors enables second treat-

22. Hetzel FW, Chen Q, Luck D et al: Preclinical studies of vascular acting photosensitizer bacteriopheophorbide for the treatment of prostate cancer. Proc SPIE 2004; 5315: 27. 23. Shaco-Levy R, Gordon JM, Feuermann D et al: On appropriate pathology for photothermal surgery. Lasers Surg Med 2004; 35: 28. 24. Ismail AH, Altaweel W, Chevalier S et al: Expression of vascular endothelial growth factor-A in

26. Huang Z, Chen Q, Trncic N et al: Effects of Pd-bacteriopheophorbide (TOOKAD)-mediated photodynamic therapy on canine prostate pretreated with ionizing radiation. Radiat Res 2004; 161: 723. 27. Weersink RA, Bogaards A, Gertner M et al: Techniques for delivery and monitoring of TOOKAD (WST09)-mediated photodynamic therapy of the prostate: clinical experience and practicalities. J Photochem Photobiol B 2005; 79: 211. 28. Dolmans DE, Kadambi A, Hill JS et al: Vascular accumulation of a novel photosensitizer, MV6401, causes selective thrombosis in tumor vessels after photodynamic therapy. Cancer Res 2002; 62: 2151. 29. Togashi H, Uehara M, Ikeda H et al: Fractionated photodynamic therapy for a human oral squamous cell carcinoma xenograft. Oral Oncol 2006; 42: 526.