Michael T. Tseng

Complications of Whole Bladder Dihematoporphyrin Ether Photodynamic Therapy

Photodynamic therapy with dihematoporphyrin ether was used to treat superficial bladder tumors in 7 patients with a followup of at least 1 year. Each patient received treatment to the whole bladder and those with papillary lesions received additional focal treatment. At 3 months 4 of the 5 patients with papillary tumors (stages Ta and T1) and 1 of the 2 with diffuse carcinoma in situ (Tis) were free of disease. However, at 1 year only 3 patients remained free of disease. Of 5 patients with an increase in irritative bladder symptoms 4 had a contracted bladder, hydroureteronephrosis and vesicoureteral reflux. Deep bladder biopsies showed replacement of smooth muscle by fibrous tissue. Six patients had mild to moderate skin phototoxicity. We conclude that although photodynamic therapy is an attractive and exciting method to treat cancer, its use with dihematoporphyrin ether in cases of bladder carcinoma can be associated with significant complications. The correct treatment parameters for safe, effective therapy are not known to date.

The effect of photodynamic therapy on the microcirculation

Photodynamic therapy (PDT) is a new form of cancer therapy involving tumor localization by photosensitizing drugs such as dihematoporphyrin ether (DHE). Light irradiation of drug-sensitized tissue results in photoactivation of DHE and tumor necrosis. The mechanism of action is incompletely understood but involves the generation of singlet oxygen which produces cytotoxic effects on tissues containing the compound. In addition, microcirculatory aberrations have been described during PDT. We have studied the acute effects of PDT on the microcirculation using in vivo television microscopy of the rat cremaster. This has enabled us to observe the effects of PDT on both paired and unpaired arterioles and venules using two different wavelengths of activating light (blue, 450-490 nm, and green, 530-560 nm). For both wavelengths of activating light, significant reduction in blood flow of all vessels was seen during PDT. This, in combination with the formation and embolization of platelet thrombi, resulted in stasis of blood flow in 80% of arterioles and 90% of venules. Observation for 2 hr after the completion of photoactivation revealed reperfusion in 20% of the arterioles but none of the venules. Blood flow was reduced by a combination of vasoconstriction and platelet thrombus formation. Reducing the total activating energy from 120J/cm2 to 24J/cm2 significantly reduced the response in venules and the incidence of stasis in both arterioles and venules. We conclude that the photoactivation of DHE results in significant vasoconstriction and thrombosis of normal vessels and that if these effects are seen at later times after DHE administration and in tumor neovasculature they may contribute to the mechanism by which PDT results in tumor necrosis.

Photodynamic therapy induced ultrastructural alterations in microvasculature of the rat cremaster muscle.

Abstract— Photodynamic therapy disrupts blood flow to tumors and produces tumor necrosis. These effects may be due to a localized generation of singlet oxygen. The current studies used direct observations of the rat cremaster microvasculature to examine the vascular effects of PDT. The objective of the morphological examination was to delineate the structural basis for the altered blood flow in photodynamic therapy. Dihematoporphyrin ether given 30 min or 48 h prior to the experiment was activated with green light (wavelength530–560 nm, 120 J/cm2). After the in vivo activation the tissues were prepared for electron microscopy. Light alone induced little or no change in the luminal content or vessel wall. On exposure to activating light both acute (30 min) and long term (48 h) dihematoporphyrin ether pretreated samples displayed formation of luminal aggregates, granulocyte margination and migration, and endothelial cell and smooth muscle cell damage. The latter was more pronounced in the arterioles than the venules. Perivascular changes included interstitial edema and damage to striated myocytes. Some of the alterations such as interstitial edema may be transient; however, smooth and skeletal muscle cell injury are important in normal and tumor tissue necrosis after photodynamic therapy.

Verapamil Enhancement of Chemotherapeutic Efficacy in Human Bladder Cancer Cells

The calcium influx blocker verapamil has been used to overcome drug resistance in several tumor systems. The possible in vitro enhancement of drug efficacy was assessed in bladder cancer cell line T24. Combination of thiotepa and doxorubicin hydrochloride with verapamil significantly reduced the survival and growth of T24 cells after as little as 1 hour of drug exposure. An increase in doxorubicin hydrochloride-induced inhibition of [3H]thymidine uptake resulted when verapamil was administered. However, this trend was not demonstrated when combined with thiotepa. It appears that verapamil enhances thiotepa-induced cytotoxicity while it potentiates the antimitotic nature of doxorubicin hydrochloride. The data presented is consistent with the postulate that verapamil alters active efflux of drug from malignant cells and suggests that verapamil has a role in the clinical management of bladder cancer.

ANALYSIS OF PULMONARY MICROVASCULATURE CHANGES AFTER PHOTODYNAMIC THERAPY DELIVERED TO DISTANT SITES

Abstract— Photodynamic therapy (PDT) can exert local damage by direct tumor cytotoxicity, by disruption of the microvas‐culature or by a combination of these effects. Although systemic effects after PDT of small tissue areas (< 1% total body surface area) are unlikely, treatment of larger areas may result in an accumulated effect leading to toxicity. Several investigators have described animal death after high dose PDT to tumors on the hind limb of animals and hypothesized that a toxic shock syndrome caused by vasoactive agents released after PDT is responsible. Because one of the most vulnerable organs to toxic shock injury is the lung, we studied the systemic effects of local PDT to this organ by intravital microscopy using a pulmonary window chamber. The PDT treatment conditions (25 mg/kg Photofrin®, 24 h, 150 J/cm2 630 nm, maximum area 6.28 cm2) were chosen that produce systemic toxicity and lethality in rats. Adhesion of leukocytes in the lung was monitored in vivo using anti‐CD‐13‐labeled microspheres. The progression of pulmonary edema was assessed by monitoring the leakage of rhodamine‐labeled albumin and by wet‐to‐dry lung weight ratios. Although an increased leukocyte adherence was observed and a significant number of animals died after the extensive PDT treatment, no biologically significant lung edema could be demonstrated. These data indicate that lung edema and acute respiratory distress syndrome is not the cause of death in these animals and that the toxicity is related to other mechanisms including circulatory shock after extensive muscle damage.

THYMIDINE UPTAKE, INCORPORATION AND DNA POLYMERASE ACTIVITY IN MURINE BLADDER TUMOR CELL,MBT–2, EXPOSED TO UV ACTIVATED DIHEMATOPORPHYRIN ETHER

Abstract— Photodynamic therapy (PDT) is a new modality for treatment of malignancy. In this paper, we reported the effect of UV activated dihematoporphyrin ether (DHE) on [3H] thymidine uptake and DNA synthesis in murine bladder tumor cells,MBT–2. Exponentially growing cells were pretreated with 0.05–5μg/ml of DHE for 30 min in complete darkness prior to irradiation with 0.15‐0.90 J/cm2 of UV light (265 nm). The rates of thymidine uptake and DNA synthesis were suppressed in a DHE concentration and photic energy dependent manner. Double reciprocal analysis on the kinetics of the thymidine uptake and DNA synthesis indicated that the inhibition was non‐competitive, i.e. decrease in both the apparent Km value and maximum velocity in DHE plus UV light treated cells. The activities of DNA polymerase a and (3 were determined by [*H] dATP incorporation into DNA of permeabilizedMBT–2 cells. DNA polymerase a activity was approximately 60% of the control after 0.45 J/cm2 of UV light exposure; a further inhibition of DNA polymerase a was observed when 0.5–5ng/W of DHE and UV photoradiation were combined. In contrast, a slight stimulation of DNA polymerase fJ was noted after a similar treatment. This study demonstrates that photodynamic therapy‐induced suppression of DNA synthesis inMBT–2 cells is a complex process involving in reduction of thymidine transport as well as the perturbation of the enzymes involved in DNA synthesis.

Systemic influence of intravesical chemotherapy with verapamil

SummaryThe influence of the calcium blocker verapamil (VR) on systemic toxicity resulting from the intravesical instillation of Adriamycin (ADM) and thiotepa (THT) was assessed in mice. Eighty per cent of the animals receiving THT+VR developed a generalized alopecia. Data gathered at necroscopy failed to reveal any trauma to the major organs or the presence of a drug-induced myelosuppression. Combination of ADM and VR did not produce and enhancement of systemic toxicity, manifest as myelosuppression. The drug combination did not produce a cardiomyopathy as assessed by histologic examination. The use of VR in combination with antineoplastic agents posed no more of a threat to the animals than did the use of cytotoxin alone.

Ultrastructural Evaluation of Murine Bladder Epithelium Exposed to Verapamil

SummaryThe effect of single or multiple instillations of high verapamil concentrations on the cytoarchitecture of the bladder epithelium was assessed by electron microscopy. Ruthenium red was used to evaluate the surface mucopolysaccharide coats and the integrity of junctional complexes between luminal or nonluminal cells was found in any experimental animals, nor was there a breakdown of the junctional complexes between luminal cells. These data suggest that verapamil may be safely used intravesically as adjunct to standard chemotherapy.

The influence of photodynamic therapy on the ultrastructure of the normal rat bladder

Reduced bladder capacity is a major side effect for patients receiving photodynamic therapy (PDT) for bladder cancer. A rat bladder model has been developed to address both the vascular and tissue effects of the photodynamic treatment of the urinary bladder. Bladders were exteriorized and positioned in a plexiglass tissue bath. Effects on microvasculature were assessed during PDT of the bladder by recording luminal diameter changes in arterioles and venules. Animals receiving Photofrin II (10 mg kg-1) 30 min prior to PDT scored a statistically significant reduction in the diameter of the red blood cell column in the vessels, whereas administration of Photofrin II 48 h prior to PDT was ineffective. Morphological changes included significant endothelial and vascular myocyte damage in the 30 min PDT group alone. Among the other tissue components, the mucosal lining was minimally affected and the response of the muscularis was highly variable. Smooth muscle cell changes ranged from mild contraction to frank necrosis with many of the affected cells located near the altered vascular beds. These data suggest that the clinical symptoms of reduced bladder capacity can be accounted for by vascular damage and myocyte sensitivity. Further refinements in the Photofrin II and light doses used in therapy may reduce bladder complications and allow for better management of bladder cancer.

In vitro and in vivo responses of a murine transitional cell carcinoma to doxorubicin, mitoxantrone and aclacinomycin-A

SummaryIn vitro and in vivo effects of mitoxantrone, aclacinomycin-A and doxorubicin were examined in a transplantable murine transitional bladder carcinoma, FCB. The in vitro parameters used included monolayer growth kinetics, tumor stem-cell colony formation and autoradiographic analysis of thymidine labeling. Monolayer growth kinetics revealed that both mitoxantrone and aclacinomycin-A resulted in reductions in FCB cell growth, which were significantly higher (41% and 65%, respectively) than those seen with doxorubicin treatment (22%). Similarly, by the stem-cell assay, an increased reduction in colony formation was seen in aclacinomycin-A (98%) and mitoxantrone (91%) treated cultures when compared with doxorubicin (51%) treated cultures. Autoradiographic data revealed that 24-h exposure with both aclacinomycin-A and mitoxantrone significantly inhibited thymidine incorporation (98% and 80% respectively), which was an increase over doxorubicin (19%). In vivo studies revealed that aclacinomycin-A treatment increased the mean life span of C57BL mice by 60.6% when compared with a 33.6% increase in doxorubicin-treated animals and a 19.7% increase in mitoxantrone-treated animals. Both the in vitro and in vivo data suggest that aclacinomycin-A is a superior drug when used against this specific murine bladder tumor cell and that further testing of this agent for its efficacy in other urologic tumors is justified.