Studies of tin ethyl etiopurpurin photodynamic therapy of the canine prostate.

PURPOSE: Previous studies have demonstrated the technical feasibility of destroying prostate tissue using photodynamic therapy for benign and malignant disease. A series of canine studies was performed to evaluate the systemic uptake and distribution of the photosensitizer tin ethyl etiopurpurin (SnET2) in the prostate and surrounding tissues, and determine the optimal combination of drug dose, light dose and time interval between drug and light administration using transurethral and transperineal interstitial light delivery. MATERIALS AND METHODS: Adult male mongrel source dogs received intravenous bolus injections of 0.5 or 1.0 mg./kg. SnET2 in 4 studies. In the first study the concentration of SnET2 in the prostate and surrounding tissue was measured at various time points after dosing. In the second study a tissue dose response relationship of SnET2-PDT was studied after transperineal interstitial light application. The third and fourth studies evaluated the tissue effects of combined transurethral and transperineal interstitial light application on SnET2 sensitized prostates. RESULTS: Substantial amounts of SnET2 were measured in the prostate between 24 and 168 hours after infusion. Drug and light dose dependent prostatic tissue necrosis and volume reduction were documented in the dose response relationship study. The combination of transurethral and transperineal light resulted in the extensive destruction of glandular epithelium with minimal damage to surrounding structures. Average prostate volume decreased 52%. Transperineal interstitial light delivery with multiple diffusers resulted in substantial glandular destruction of the prostate. An average volume reduction of more than 60% was achieved. CONCLUSIONS: SnET2-PDT is a viable minimally invasive treatment modality for prostate tissue destruction.

Recombinant PAI-1 inhibits angiogenesis and reduces size of LNCaP prostate cancer xenografts in SCID mice.

To understand the fundamental determinants of urokinase plasminogen activator (uPA) driven angiogenesis in cancer we studied how inhibition of uPA activity could reduce neovascularization and consequently reduce tumor size in experimental animals. Proteolytic enzymes are required to mediate tumor cell invasion to adjacent tissues and initiate the metastatic process. Many different human cancers commonly overexpress the urokinase plasminogen activator system, one of the proteolytic enzyme systems. Reduction of urokinase activity in cancer cells is evidently associated with diminished invasion and metastasis. However, it has been shown recently that inhibitors of uPA could reduce tumor size also. The mechanism of action leading to decline in tumor growth rate is not clear. Proteolysis is responsible for degradation of proteins, for invasion or metastasis, but not for the proliferate properties of the cancer cells. It is difficult to envision that diminishing the size of tumor is due to simply blocking of uPA activity of cancer cells. Instead, inhibitors of uPA may be interacting with the elements of the extracellular matrix, such the neovascular bed surrounding tumors that has been reported to contain high amounts of uPA and its receptor. Overall these data strongly suggest that inhibitors of urokinase limit cancer growth by inhibiting angiogenesis. However, it is possible also that uPA inhibitors could act on cancer cells directly or prevent angiogenesis by alternative mechanisms that are not related to uPA inhibition. Therefore, we examined if plasminogen activator inhibitor (PAI-1) could limit angiogenesis. If it does, it will provide definitive evidence of uPA/PAI-1 involvement in reduction of cancer growth. Indeed, our study demonstrates that exogenously applied 14-1b PAI-1 is a powerful inhibitor of angiogenesis in three different in vitro models and is a powerful anti-cancer agent in a SCID mice model inoculated with human LNCaP prostate cancer cells.

Computer model for cryosurgery of the prostate.

The objective of this study was to devise an interactive tool to assist in cryoablation therapy through computer modeling, simulation, and visualization. CryoSim, a software package, accepts a set of acquired and processed three-dimensional ultrasound images, then models heat diffusion (formation of the iceball) based on numerical approximation of the heat equation and knowledge of the thermal properties of the underlying tissues. Results of cryoexperiments were found to be significantly similar to those generated by CryoSim. Therefore, CryoSim provides a viable technique for predicting the outcome of cryosurgery, and establishes a platform for future automation of cryosurgery.

Inhibitors of urokinase reduce size of prostate cancer xenografts in severe combined immunodeficient mice.

Proteolytic enzymes are required to mediate tumor cell invasion and metastasis. The urokinase plasminogen activator (uPA) is commonly overexpressed by many human cancers. Therefore, uPA is a logical target to inhibit cancer invasion and metastasis. However, uPA inhibitors also reduce tumor growth. We used a mutated form of plasminogen activator inhibitor type 1 to conform a correlation between the inactivation of uPA and tumor size; we have compared these results with the action of p-aminobenzamidine and amiloride, known inhibitors of uPA. Our results show that blocking uPA by uPA inhibitors reduces tumor size in experimental animals. Our molecular simulation of docking inhibitors to the urokinase reveals that all tested small molecule inhibitors bind in proximity of uPA's specificity pocket, a critical site for future search of novel anticancer uPA inhibitors.

Transperineal photodynamic ablation of the canine prostate.

PURPOSE: Experiments were undertaken to determine the effects of transperineal interstitial photodynamic therapy on the canine prostate. MATERIALS AND METHODS: Mongrel dogs were injected intravenously with the photosensitizer, tin (II) ethyl etiopurpurin dichloride. Twenty-four hours later, 2 optical fibers were implanted in 1 hemisphere of the prostate, which was then treated with red light (660 nm.). RESULTS: Acutely, the treated areas showed extensive hemorrhagic necrosis. At 3 and 6 weeks, the treated lobes were largely replaced by fibrous connective tissue. CONCLUSION: Transperineal photodynamic therapy of the canine prostate is feasible. Further preclinical investigation is warranted to determine the applicability of this approach to the treatment of localized prostate cancer.

The effect of transurethral light on the canine prostate after sensitization with the photosensitizer tin (II) etiopurpurin dichloride: a pilot study.

A pilot study was undertaken to determine the effect of transurethral light on photosensitized periurethral prostatic tissue in the dog. Initial studies demonstrated that a sufficient level of the photosensitizer tin (II) etiopurpurin dichloride, SnET2, was present in the canine prostate 24 hours after intravenous administration to create a photodynamic effect. Gross and histologic examination of SnET2 photosensitized prostates treated transurethrally with 285 joules per cm. of red light (660 nm.) showed hemorrhagic necrosis as much as 1 cm. from the urethral wall. Three weeks after treatment, circumferential glandular atrophy was evident within the treatment area. The urethral mucosa, which was histologically absent at the 48-hour interval, regenerated by 3 weeks. Transurethral photodynamic treatment of the prostate is feasible, and its use for the treatment of benign prostatic hyperplasia warrants further investigation.

Photochemical ablation of intestinal mucosa for bladder augmentation.

Complications associated with enterocystoplasty include mucus production, electrolyte abnormalities, infections, stones and cancer at the vesicoenteric anastomosis. Removal of the intestinal mucosa with subsequent urothelialization may obviate these problems. We describe a unique approach whereby photodynamic therapy is used to de-epithelialize an ileal segment before augmentation. Enterocystoplasty was performed in 32 female Fischer 344 rats using a 1.5 cm. patch of terminal ileum. Of the 32 rats 24 survived at least 6 weeks before euthanasia. The experimental group (10 rats) received hematoporphyrin derivative intravenously 24 hours before surgery. The ileal patch was treated with red light for 20 minutes and then used for augmentation. There were 3 control groups, including 1 group of 5 rats that underwent augmentation alone, while the other 2 groups were augmented but received either light treatment (4 rats) or hematoporphyrin derivative (5 rats). Histological analysis revealed urothelialization of the augments treated with hematoporphyrin derivative and light, which did not occur in the controls. The preoperative and postoperative bladder capacities increased substantially in all groups. Mucus production and bacterial colonization were reduced while stone formation increased in the treated animals.

Photodynamic therapy of tumors: effects of hematoporphyrin derivative on normal rat intestine.

Hematoporphyrin derivative (HpD) is a complex mixture of dicarboxylic porphyrins in addition to dimers, oligomers and aggregates of variable sizes. The ability of this mixture (and enriched preparations thereof) to be retained by tumors and to sensitize them to destruction by light has led to worldwide studies of the treatment modality called photodynamic therapy (PDT). Understanding how PDT affects normal tissues surrounding the tumor is of crucial importance. Prior studies have documented that normal intestinal blood flow can be disrupted by HpD PDT. In addition, mucosal and submucosal damage to normal rat jejunum was observed following HpD PDT. The present study was designed to correlate the above observed changes with local levels of porphyrins. Three groups of Fischer 344 rats were injected with 0 (controls), 10 or 20 mg HpD/kg body weight. Twenty-four hours after the drug was injected, jejunal segments were excised and blood samples taken. Jejunal contents were obtained by saline perfusion and analyzed for porphyrins alongside the jejunal homogenates and the bloods. Jejunal segments of animals injected with 10 or 20 mg HpD/kg b.w. had porphyrin levels some 4- and 9-fold above controls (controls m +/- S.D. = 0.25 +/- 0.12 micrograms/g wet weight). For each injected dose the jejunal perfusates contained 2- and 3-fold higher levels of porphyrins (in transit) as the controls (controls, m +/- S.D. = 1.48 +/- 0.66 micrograms/g w.w.). In the general circulation, only 0.6% or 0.4% of the respective injected doses remained. After HpD i.v. injections, high levels of porphyrins were found in the small intestine and in transit.(ABSTRACT TRUNCATED AT 250 WORDS)

Iminium salt benzochlorins: structure-activity relationship studies.

An iminium salt of copper(II) octaethylbenzochlorin (CDS1) is an effective new photosensitizer despite the fact that it does not produce singlet oxygen, does not fluoresce and the triplet state lifetime can only be less than 20 ns. A number of octaethylbenzochlorin derivatives were synthesized in order to determine the structural component(s) that is(are) responsible for the photodynamic action of these new photosensitizers. Studies utilizing the N-(4-[5-nitro-2-furyl]-2-thiazolyl)formamide-induced urothelial tumor revealed that the coexistence of the copper inside the aromatic ring and the iminium group at the meso position are required for the photodynamic effect.

Enhanced skin allograft survival after photodynamic therapy. Association with lymphocyte inactivation and macrophage stimulation.

It has been found previously that peritoneal exposure to hematoporphyrin derivative (HpD) photodynamic therapy (PDT) can induce systemic immunosuppression of contact hypersensitivity. We have now found that HpD-PDT also significantly prolongs survival of murine skin allografts. Normal A/J mice transplanted with BALB/c skin rejected the grafts within 10 +/- 0.9 days. Recipient mice treated 24 hr previously with HpD-PDT rejected skin allografts at 16 +/- 1.2 days. HpD alone or irradiation alone had no effect on skin graft survival, nor did HpD-PDT administered shortly after grafting. Flow cytometric analyses showed a nearly complete depletion of peritoneal lymphocytes 3 days after HpD-PDT. Lymphocyte levels were normal in the spleen, an organ not directly targeted by the PDT treatment, but the cells were totally unresponsive to Con A and LPS mitogens. Conversely, peritoneal HpD-PDT caused a striking enhancement in macrophage function as measured by phagocytosis of antibody-coated sheep erythrocytes. Humoral immunity to hen egg-white lysozyme was not significantly changed by HpD-PDT. These results demonstrate that HpD-PDT causes systemic immunosuppression of cellular immunity which, in turn, allows prolonged survival of allografts. Humoral immunity appears to remain largely unaffected by HpD-PDT and macrophages become activated, suggesting that this therapy might be more effective in specifically targeting T cell-mediated immunity than current immunosuppressive treatments.

Copper benzochlorin, a novel photosensitizer for photodynamic therapy: effects on a transplantable urothelial tumor.

An iminium salt of octaethylbenzochlorin with copper in the aromatic ring, CDS1, was tested for its tumoricidal effects on the AY-27 N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide tumor line. CDS1 was found to be an effective photosensitizer in vivo when used in combination with either a xenon arc lamp or a pulsed alexandrite laser. Hemodynamically, CDS1 and light caused a rapid decrease in tumor blood flow. Skin photosensitization was found to be minimal when drug-injected mice were illuminated in a solar simulator.

Synthesis and in vivo photodynamic activity of some bacteriochlorin derivatives against bladder tumors in rodents.

Bacteriochlorins have been suggested as potential photosensitizers for use in photodynamic therapy. We have shown that bacteriochlorin-like macrocycles can be generated through cyclization of either 5,10- or 5,15-bis[(ethoxycarbonyl)vinyl]porphyrins; however, the resulting products are rapidly decomposed on exposure to air. More stable systems can be generated by Diels-Alder reactions between dienophiles such as dimethyl acetylenedicarboxylate or tetracyanoethylene, and vinylporphyrinones. Although spectroscopic properties of these latter products resemble those of porphyrinones rather than bacteriochlorins, in vivo studies using the N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide-induced rat bladder tumor (AY-27) transplanted into Fisher CDF (F344)/CrlBr rats demonstrated a powerful photodynamic response.

Treatment of the transplantable FANFT induced bladder tumors with the purpurin SnET2 and red light emitted by a pulsed frequency doubled Nd:YAG laser.

Photodynamic therapy of transplantable N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide-induced tumors engrafted onto Fischer CDF (F-344)/CrlBR rats that had been sensitized with the photosensitizer tin (ll) etiopurpurin dichloride was performed in combination with visible light (approximately equal to 660 nm) emitted by either a continuous wave argon-dye laser or a pulsed, frequency-doubled Nd:Yag laser. Tumor control was assessed either by tumor dry-weight 12 days after treatment or by the palpatory absence of tumor at 60 days after treatment. Both laser sources were effective in creating the desired photodynamic effect. This study demonstrates the potential for the use of a solid-state pulsed laser for photodynamic therapy when used in combination with the tumor sensitizer tin (ll) etiopurpurin dichloride.

Synthesis and in vivo activity of some porphyrindione derivatives with potential in photodynamic therapy.

A number of synthetic bacteriochlorins were prepared from porphyrindiones. The potential of these compounds as sensitizers for photodynamic therapy was examined using the FANFT-induced urothelial cell carcinoma (AY-27) transplanted into rats. In a number of cases, tumor regression was significant following treatment with both drug and light.

Metallopurpurins and light: effect on transplantable rat bladder tumors and murine skin.

Purpurins are modified chlorins with photodynamic properties. Their strong absorption in the red region of the visible spectrum makes them candidates for use in photodynamic cancer therapy. A series of metal derivatives of the free base purpurins have been synthesized and shown to cause tumor necrosis in transplantable tumors when exposed to visible light. In the following set of experiments, the effects of two metallo-derivatives (tin and zinc) of two purpurins, octaethylpurpurin (NT2) and etiopurpurin (ET2), and light on the N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide transplantable tumors in Fischer CDF(F344)/CrlBr rats were studied. The photodynamic activity was assessed by a short term assay using tumor dry weight 12 days after purpurin-PDT as a criterion of response. From these experiments it appears that SnET2 greater than SnNT2 greater than ZnET2 greater than ZnNT2 in photodynamic activity. SnET2 was further characterized by attempting to determine the time interval after systemic injection at which maximum therapeutic effect occurred. These studies shown that 24 h after metallopurpurin injection was the optimum time for treatment of tumors with visible light. In a final set of experiments, the effect of solar light on the skin of hairless mice injected with SnET2 was found to be much less injurious than with hematoporphyrin derivative.

Effects of hypothermia on testicular ischemia.

The ischemic effects of prolonged testicular torsion have been well documented; however, prevention or arrest of the damaging effects of prolonged ischemia has been incompletely studied. Two groups of Sprague-Dawley rats were subjected to varying lengths of bilateral testicular ischemia. Group I underwent normothermic ischemia for two, four, and six hours. Likewise, Group II underwent similar time periods of ischemia, however, after thirty minutes of normothermic ischemia the scrotum of each animal was placed into an ice bath maintained at 4C. Two weeks postoperatively, bilateral orchiectomy was performed. Histology of the testes of the two groups was compared. Neither group revealed significant destruction of the germinal epithelium after two hours of ischemia. Group I revealed only 25% preservation of the germinal epithelium at four hours and only 8% preservation at six hours of ischemia. In contrast, Group II which received ice showed 90% preservation of germinal epithelium at four hours and 85% preservation at six hours of ischemia. We conclude that external ice application significantly preserves seminiferous tubules at four and six hours of ischemic injury in the rat testicle.

Diels-Alder adducts of vinyl porphyrins: synthesis and in vivo photodynamic effect against a rat bladder tumor.

Benzoporphyrin derivatives have been proposed as potential photosensitizers for photodynamic therapy. We have prepared a number of benzoporphyrin derivatives and tested their effect, in combination with red light, on the N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) induced rat bladder tumor (AY-27) transplanted into Fischer CDF(F344)/CrlBr rats. Tetracyanoethylene (TCNE) adducts showed very little activity, which may be attributable to their poor tumor uptake or to chemical instability of the adduct under physiological conditions. However, dimethyl acetylenedicarboxylate (DMAD) adducts tested showed greater cytotoxic effect than hematoporphyrin derivative and similar activities to metallopurpurins when tested under the same protocol.

Hemodynamic effect of the metallopurpurin SnET2 and light on transplantable FANFT induced bladder tumor.

Changes in blood flow to transplantable N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide induced bladder tumors growing subcutaneously in the flanks of Fischer CDF (F344/CrlBR) rats were measured after photodynamic therapy with the photosensitizer tin (II) etiopurpurin dichloride using the radioactive microsphere technique. As with the other photosensitizers, hematoporphyrin derivative and chloroaluminum tetrasulfophtalocyanine, tin (II) etiopurpurin dichloride and light caused a rapid decrease in tumor blood flow in this tumor model. The decrease in blood flow occurred whether the vehicle for photosensitizer delivery was an emulsion or a liposome. Systemic heparinization of animals prior to light treatment did not alter changes in tumor blood flow.

New sensitizers for photodynamic therapy: controlled synthesis of purpurins and their effect on normal tissue.

Purpurins are a class of porphyrin derivative that have been shown to have good in vivo cytotoxicity to N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) induced rat bladder tumors (AY-27) implanted into Fisher 344 rats. The synthesis of purpurins from etioporphyrin I and coproporphyrin I proceeds in high yield and with a high degree of regioselectivity. Product formation can be rationalized in terms of relief of steric strain about the periphery of the purpurin macrocycle. The effect of therapeutic light doses using the rat footpad model suggests that, at therapeutic sensitizer doses, normal tissue damage is within acceptable limits, particularly for metalated purpurins.

New photosensitizers for photodynamic therapy: combined effect of metallopurpurin derivatives and light on transplantable bladder tumors.

A series of metallopurpurins was tested for their photodynamic activity against transplantable N-[4-(5-nitro-2-furyl)-2-thiazoyl]formamide-induced urothelial tumors growing in male Fischer CDF (F344/CrlBR) rats. Histological examination of tumors in animals treated with the metallopurpurins and red light (greater than 590 nm, 360 J/cm2) revealed tumor necrosis 24 h after completion of therapy. Control tumors showed no histological change. In 30-day tumor regrowth studies, 70% of animals treated with the metallopurpurin derivative SnET2 were free of tumors while 50% of the animals treated with the free-base purpurin ET2 were free of tumor. Metallopurpurins have intense absorptions in the red region of the visible spectrum, a region with good tissue penetration. The metallopurpurins are easily prepared from the corresponding purpurins with a high degree of purity. This study demonstrates the potential of these photosensitizers for photodynamic cancer therapy.