A Pilot Trial of Vascular-Targeted Photodynamic Therapy in Renal Tissue

Abstract

Purpose

Vascular-targeted photodynamic therapy (VTP) represents the newest generation of photodynamic therapy and a new paradigm for minimally-invasive ablative therapy. We report on a pilot trial of VTP to evaluate the effect on porcine renal tissue.

Materials and Methods

Pigs underwent continuous infusion of WST-09 and concurrent illumination with interstitial laser at a wavelength of 763 nm to the lower pole of the kidney. Drug doses were 0.5–1.0mg/kg and light doses 100–200 Joules. Nuclear renography was performed on POD 5. On POD 7 arteriography, pyelography, computed tomography of the abdomen, and necropsy was performed.

Results

Four of seven animals completed therapy and all evaluations. Three animals died, 1 from surgical complications, and 2 due to an anaphylactoid reaction to the cremophor solvent in the compound. All kidneys in the surviving animals functioned on nuclear renography. Renal function remained unchanged. No lesions or urine leaks were visible on imaging. On necropsy lesion sizes ranged from 5×4×3mm to 7×7×14mm, depending on drug/light dose. Histology showed a distinct demarcation between the treated zone and surrounding parenchyma at the higher doses. The lesions were well-demarcated, with necrotic tubules, glomerular fibrinoid necrosis, thrombosis of capillary loops, interstitial hemorrhage, and lymphocytic infiltrates.

Conclusions

Significant tissue effect with some necrosis was seen at these low drug/light combinations. This study provides the initial proof of principle that justifies further preclinical investigation of VTP for treatment of renal tumors. A newer water-based formulation should reduce the incidence of reactions in swine. This newer formulation will allow for further safe investigation of this novel treatment paradigm.

INTRODUCTION

Photodynamic therapy (PDT) is a unique form of therapy by which a photochemical reaction generates free radicals with the capability of causing tissue injury and necrosis. PDT is much different from other modalities using laser energy, such as interstitial laser coagulation, laser thermal ablation, or laser interstitial thermal therapy, which generate thermal energy. In these latter cases, heat is the primary cause of tissue ablation, whereas with PDT the generation of reactive oxygen species (ROS) causes tissue injury. Currently approved indications for PDT include age-related macular degeneration, skin cancer and other cutaneous disorders, brain, lung, esophageal, gastric and bladder cancer, cervical dysplasia and cervical cancer ¹. New areas of investigation include its use for endovascular applications ².

The clinical experience with PDT in urology is limited. Until recently the major role for PDT in urology has been for the diagnosis and treatment of bladder cancer, but it is currently undergoing Phase II/III clinical trials in patients with primary prostate cancer who failed radiation therapy ^{3,4, 5}. The shortcomings of current popular photosensitizers have held back development significantly. These include the use of shorter wavelengths (which have less tissue penetration), phototoxicity requiring prolonged avoidance of sun exposure, and slow clearance.

A new generation of PDT is now available with unique properties that are advantageous in comparison to traditional PDT. These properties include rapid clearance resulting in minimal phototoxicity and sequestration within the vascular compartment, which may have some advantages from a selectivity and toxicity perspective ^6–8. The unique targeting by the new generation of PDT agents has motivated this application to be termed vascular-targeted photodynamic therapy (VTP). On the basis of this recent availability of VTP, we performed a pilot trial evaluating its safety and efficacy for treatment of renal tissue.

MATERIALS AND METHODS

Animal care and surgical procedures

This study was approved by the institutional animal safety committee. Animals were maintained in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care, International and in accordance with current U.S. Department of Agriculture, Department of Health and Human Services, and National Institutes of Health regulations and standards. Domestic female juvenile pigs (30 kg) were obtained from a local breeder and were held for a minimum of 7 days for acclimation and observation prior to enrollment on the protocol. Any pigs with clinical signs of disease (anorexia, diarrhea, nasal discharge, coughing, etc.) were rejected and removed from the facility. Food was withheld for at least 12 hours prior to surgery, while water was available at all times.

Pigs were premedicated with intramuscular (IM) atropine 0.04mg/kg and sedated with ketamine 30mg/kg plus acepromazine 0.3mg/kg IM, then intubated and maintained on isoflurane 1–3% in oxygen. The pigs were mechanically ventilated at 10–12 breaths per minutes and tidal volume 10–15 ml/kg. An intravenous catheter was placed in the auricular vein and the external jugular vein. Intravenous fluids (0.9% NaCl) were given throughout surgery. Pigs were under stable anesthesia for an average of 30 minutes before beginning the procedure. After induction of general anesthesia, animals were placed in left lateral decubitus position and laparoscopic access was obtained with a Veress needle. Pneumoperitoneum was established at a pressure of 15 mm Hg. One 12-mm port and two 5-mm ports were placed. The lower pole of the right kidney was exposed and a laser fiber was inserted 1 cm into the anterior lower pole of the kidney. Treatment was initiated 5 minutes after WST-09 injection was started. After treatment was completed, the laser fiber was withdrawn, all laparoscopic ports were removed, and the skin was closed using wire staples. Animals received standard post-operative care including analgesia, antibiotics, and routine postoperative evaluations.

Photosensitizer preparation, laser methodology, and delivery of PDT

WST-09 (Tookad®, palladium-bacteriopheophorbide, Negma-Lerads, France) was supplied as a dark-violet liquid in sterile glass vials. The compound was stored at 4-degrees Celsius in fully darkened conditions and all vials and delivery devices (intravenous tubing, connectors, syringes, etc.) were wrapped in aluminum foil to prevent exposure to ambient light. All manipulation of the drug occurred at very low light conditions, including darkening of the operating room and lowering the laparoscopic light source to the lowest possible intensity. Intravenous injection of WST-09 was performed by continuous infusion using a digital microinjector programmed to deliver the entire dose steadily over 20 minutes. Drug doses used were 0.5 and 1.0 mg/kg.

Five minutes after initiation of drug infusion, a 600 micron silica laser fiber fitted with a diffuser tip producing a homogeneous radial light pattern was introduced transcutaneously and inserted 1 cm into the lower pole of the kidney. Interstitial illumination was initiated at a wavelength of 763 nm using a Ceralas® PDT 763 medical laser system (Ceramoptec GmbH, Bonn, Germany). Light doses used were 100 and 200 Joules (J). Light fluence was kept low so as to avoid any confounding effects from thermal injury ⁹. It is possible however that when light excitation is performed on circulating sensitizers such as with WST-09 the thermal effect is minimized, allowing for higher light regimens, but we have not had the opportunity to evaluate this ¹⁰. A schematic of the treatment design is given in Figure 1.

Figure 1.

Timeline of drug infusion and illumination parameters.

Evaluation during and after treatment

Laboratory studies consisting of a complete blood count, serum electrolytes, blood urea nitrogen, and creatinine tests were performed on the day of surgery just prior to treatment and on postoperative days (POD) 5 and 7. Nuclear renography was performed on POD 5, using ethylene dicysteine technetium-99 to evaluate differential renal function. On POD 7 arteriography, pyelography, and renal-protocol computed tomography of the abdomen were performed just prior to necropsy. Animals were euthanized by exsanguination under deep anesthesia at the completion of the final imaging study.

Necropsy and histology evaluation was performed by a single veterinary pathologist (LCS) unaware of treatment parameters and uninvolved with the treatment itself. After exploratory laparotomy the treated kidney was harvested. The zone of treatment was readily identified visually. The kidney was bisected in 2 dimensions perpendicular to the surface where the treatment occurred, and the lesion was measured in 3 dimensions. Photographs were taken, and tissue samples were harvested in formalin for histologic evaluation. Histology was performed by standard hematoxylin and eosin (H&E) staining.

RESULTS

A total of 7 animals underwent experiments. In the first 5 animals, 2 died within minutes of starting the drug infusion. Two other animals required medical management of hypotension during drug infusion. Before proceeding further, all possibilities for these events were evaluated, and it was believed that the animals were having an anaphylactoid reaction to the cremophor solvent in the compound. Subsequent animals were treated proactively with generous intravenous hydration and medical management. These steps prevented any further mortality. One animal had a bowel injury during initial laparoscopic access and was sacrificed prior to drug administration. In all, 4 animals completed treatment and all evaluations prior to sacrifice. All kidneys in the 4 animals completing therapy functioned on nuclear renography on POD 5 (Figure 2A). Laboratory studies remained stable and within normal limits. Imaging prior to sacrifice on POD 7 showed no visible lesions on arteriography or CT scan, and no urine leaks were seen on retrograde pyelography in any of the surviving animals (Table 1).

Figure 2.

All figures are from the same animal (#421). A) Nuclear renogram on POD 5 showing symmetrical renal function. B) Photograph of kidney one week after receiving 0.5 mg/k of WST-09 and a laser light dose of 200 J. The site of treatment corresponds to the cortical depression. C) Bisection of the kidney through the treatment zone shows a pale central column of necrosis surrounded by a hyperemic zone. D) Photomicrograph of a section at the site of depression of the capsule that shows the margin of the lesion with viable kidney to the right, a zone basophilia comprised of inflammation and mineralization, and necrotic renal parenchyma in the left corner. Original magnification, 100x. E) This region of the lesion has loss of tubules, necrosis of glomeruli, and diffuse lymphocytic inflammation. Original magnification, 250x.

Table 1.

Animal	Drug dose (mg/kg)	Light dose (J)	Status	CT scan results day 7	Arteriography results day 7	Pyelography results day 7	Serum creatinine (mg/dl) Day 0, 5, 7	Lesion size day 7, mm (length × width × depth)
379	0.5	100	Died *	--	--	--	--	--
381	0.5	100	Died **	--	--	--	--	--
378	0.5	200	Survived	Negative	Negative	Negative	1.6, 1.6, 1.4	5 × 4 × 3
421	0.5	200	Survived	Negative	Negative	Negative	1.4, 1.4, 1.2	7 × 7 × 7
380	1.0	100	Died **	--	--	--	--	--
422	1.0	100	Survived	Negative	Negative	Negative	1.3, 1.2, 1.3	7 × 10 × 7
423	1.0	200	Survived	Negative	Negative	Negative	1.4, 1.3, 1.6	7 × 7 × 14

^*Surgical complication

^**Hypotension

On necropsy, the zone of treatment was clearly visible as a depression of the capsular surface (Figure 2B) without any surrounding organ damage. Histologic evaluation of the lesion at the cortical level showed widespread loss and necrosis of tubules (Figure 2D and 2E). Outright coagulative necrosis was seen in the central portion of the outer cortex, surrounded by an accumulation of granular basophilic material compatible with mineral deposition. The most significant and dramatic changes occurred to glomeruli, which were either lost (outright necrosis), or had significant hemorrhage and thrombosis of capillary loops. However, there was not complete necrosis of all the central tissue. At the edge of the treatment zone the glomerular lesions were segmental and affecting only portions of the glomerular tuft. In the cortex and medulla the remaining tubules were filled with blood or hyaline casts. Some were seen to show enlargement and basophilia of the epithelium along with poor polarity indicating attempts at repair. The interstitium of the lesion was infiltrated diffusely by lymphocytes, with the most intense lymphocytic infiltrates seen in the medulla. Arterioles in the interstitium exhibited hypertrophy of the smooth muscle of the tunica media as well as enlargement of endothelial cells.

DISCUSSION

This study demonstrates the following findings. One, the cremophor required as a solvent for the photosensitizer can cause an anaphylactoid reaction in swine. Once discovered, this was readily managed by appropriate intraoperative maneuvers. Two, VTP can otherwise be delivered safely, in a targeted fashion to a specific location in the kidney, without surrounding damage to nearby structures, and without discernible effects on renal function. Three, tissue effects were seen with every treatment and seemed to follow a dose-response (Figure 3), although this is based on limited data. And four, these relatively low drug-light dose combinations were insufficient to create reproducible homogeneous necrosis in normal kidney tissue. The use of higher light/drug doses and multiple fibers should amplify the necrosis, and as well the effect on malignant tissue is potentially more dramatic, but we are not yet able to test these hypotheses.

Figure 3.

Bubble graph showing relationship of drug dose, light fluence, and volume of therapy lesion. Volume is represented by the relative size of the circles (numbers in circles are mm³), and was calculated using the formula: 4/3π*radius1*radius2*radius3 (Source: Spiegel, Murray R. Mathematical Handbook of Formulas and Tables. Schaum’s Outline series in Mathematics. McGraw-Hill Book Co., 1968).

PDT relies on the 3 interacting components of light, drug, and molecular oxygen. The drug is a photosensitizer, usually delivered intravenously. Light of a specific wavelength generates ROS from the photosensitizer. ROS cause organelle (mitochondria), cellular, and microvascular damage via oxidation of lipids, lipoproteins and proteins ¹¹. Membrane injury induces apoptosis if the mitochondrial membrane is injured by means of protease release, whereas if the cell membrane is injured, necrosis ensues due to cell lysis ¹². Vascular damage and collapse is also an important mechanism of tissue destruction in addition to cytotoxicity and apoptosis ^12,13. These initial experiments were designed to allow these physiological processes to take place over the course of a week prior to tissue evaluation.

The history of PDT started with initial studies at the Mayo clinic nearly 50 years ago using derivatives of hematoporphyrin ¹⁴. The subsequent clinical development and experience with porphyrin-based PDT was performed by researchers at Roswell Park Cancer Institute, leading to the development of the first-generation photosensitizer. This compound (Porfimer sodium, Axcan Scandipharm Inc., Birmingham, Alabama) absorbed light at less than 640 nm ¹⁵. At this wavelength light does not penetrate tissues deeply, partly because of tissue pigmentation and absorption by endogenous porphyrins ⁹. Besides poor penetration, other limitations of the first generation photosensitizers include a long half-life and significant, prolonged skin phototoxicity. Aminolevulinic acid (ALA) is an intermediary in the heme pathway, forming the primary backbone of porphyrin synthesis and is another compound used for PDT ¹⁶. Exogenously delivered ALA appears to have selectivity for tumor tissues, possibly due to increased vascular permeability and decreased turnover by tumor cells ⁴. Since these initial developments, a variety of other porphyrin, non-porphyrin, and ALA-based photosensitizers have become available for a variety of specific diagnostic and therapeutic indications ^16,17. PDT allows for double targeting by mechanical and cellular mechanisms. Direct mechanical targeting is accomplished by placing the laser fiber directly into the tissue of interest, while the cellular targeting is accomplished by the higher affinity of photosensitizers for accumulation into tumor cells, their organelles, or their microvasculature. This multiple targeting confines the treatment to a precise area.

One alternative to porphyrin-based derivatives is the use of bacteriochlorophyll. WST-09 is an agent from this novel family in which the central magnesium ion has been replaced by a palladium ion. Studies have shown that WST-09 has not only differential effects on tumor and non-tumor tissues in pre-clinical studies, but in addition, the drug remains sequestered in the vascular compartment, resulting in significant improvements in drug toxicity, including rapid clearance, and possibly better selectivity ^18,19. Regarding the toxicity, in a Phase 1–2 trial of WST-09 in patients with locally recurrent prostate cancer after radiation therapy, no skin phototoxicity was seen, even when patients were challenged with skin light application ²⁰. Regarding the selectivity, one of the most unique properties of this compound is the “vascular-targeted” application, whereby the most significant damage occurs to the microvasculature, owing to the fact that the compound is restricted to the vascular space. This is evident in the current study given the findings occurring in the renal vasculature and particularly the glomeruli. Also, light penetrates deeper into tissues due to the longer wavelength. On a practical level, since illumination is performed within minutes of injection, no separate clinic visit or prolonged waiting time would be necessary as is the case with most other compounds presently available.

This pilot study shows that VTP can be applied to the kidney and results in localized tissue damage without affecting the non-targeted renal tissue or overall renal function, which opens up the possibility to explore this novel application further. Future efforts should use a water-based version, WST-11, which will avoid the reactions due to the cremophor solvent ⁸. The drug-light doses used in this pilot study were insufficient to cause consistent areas of necrosis, and thus higher drug doses and light fluence should be tested in order to explore how the dynamic kinetics of drug and light interaction manifest in renal tissue. More than likely, multiple simultaneous interstitial laser fibers placed as an array will be necessary for treatment over larger areas, similar to brachytherapy for prostate cancer. Even though the effect on tumor is expected to be more significant due to prior studies showing a preferential accumulation of photosensitizer in tumor microvasculature, this will be difficult to judge in pre-clinical trials owing to the absence of a large animal model of RCC.

CONCLUSION

A significant, localized tissue effect was seen in all animals surviving, including limited necrosis at these relatively low drug/light combinations. VTP therapy of the kidney does not impair renal function. Aside from the acute anaphylactoid reaction seen in the initial animals, the treatment is well-tolerated with no post-therapy side effects noted during the study period, and a new formulation will make this treatment even safer. This study provides the initial proof of principle that VTP can function as a novel minimally invasive nephron-sparing modality for the treatment of renal tumors. Further pre-clinical development is needed before any clinical trials.

Key of Definitions for Abbreviations

ALA

aminolevulinic acid

Hg

mercury

IM

intramuscular

J

Joules

Kg

kilograms

Mg

milligrams

Ml

milliliters

NaCl

sodium chloride

Nm

nanometers

PDT

photodynamic therapy

POD

postoperative day

RCC

renal cell carcinoma

ROS

reactive oxygen species

VTP

vascular-targeted photodynamic therapy