Hypocrellin B‐loaded,folate‐conjugated polymeric micelle for intraperitoneal targeting of ovarian cancer in vitro and in vivo

Photodynamic therapy (PDT) is considered an innovative and attractive modality to treat ovarian cancer. In the present study, a biodegradable polymer poly (ethylene glycol) (PEG)‐poly (lactic acid)(PLA)‐folate (FA‐PEG‐PLA) was prepared in order to synthesize an active‐targeting, water‐soluble and pharmacomodulated photosensitizer nanocarrier. Drug‐loading content, encapsulation efficiency, in vitro and in vivo release were characterized, in which hypocrellin B (HB)/FA‐PEG‐PLA micelles had a high encapsulation efficiency and much slower control release for drugs compared to free drugs (P < .05). To evaluate the targeting ability of the HB/FA‐PEG‐PLA micelles, a cellular uptake study in vitro was carried out, which showed significantly enhanced uptake of HB/FA‐PEG‐PLA micelles in SKOV3 (FR+) compared to A2780 cancer cells (FR?). The enhanced uptake of HB/FA‐PEG‐PLA micelles to cancer cells resulted in a more effective post‐PDT killing of SKOV3 cells compared to plain micelles and free drugs. Binding and uptake of HB/FA‐PEG‐PLA micelles by SKOV3 cells were also observed in vivo after ip injection of folate‐targeted micelles in tumor‐bearing ascitic ovarian cancer animals. Drug levels in ascitic tumor tissues were increased 20‐fold (P < .001), which underscored the effect of a regional therapy approach with folate targeting. Furthermore, the HB‐loaded micelles were mainly distributed in kidney and liver (the main clearance organs) in biodistribution. These results showed that our newly developed PDT photosensitizer HB/FA‐PEG‐PLA micelles have a high drug‐loading capacity, good biocompatibility, controlled drug release, and enhanced targeting and antitumor effect, which is a potential approach to future targeting ovarian cancer therapy.     

Photodynamic therapy of locoregional breast cancer recurrences using a chlorin-type photosensitizer

Chest wall recurrences are a frequent problem in patients treated by mastectomy for breast cancer. Surgery and ionizing radiation are established treatment modalities in these cases. Photodynamic therapy (PDT) provides an alternative treatment modality using a photosensitizer and laser light to induce selective tumor necrosis. PDT was performed as compassionate use in 7 patients aged 57.6 years (+/-12.6 SD). A total of 89 metastatic skin nodes were treated in 11 PDT sessions. As photosensitizer meta-tetra(hydroxyphenyl)chlorin (m-THPC) was applied intravenously. Patients (n = 3) photosensitized with a drug dose of 0.10 mg/kg bodyweight were irradiated 48 hr after drug application at a lightdose of 5 J/cm(2). Patients (n = 4) were illuminated by an optical dose of 10 J/cm(2) 96 hr after photosensitization with 0.15 mg/kg. Laser light at a wavelength of 652 nm was generated by a diode laser and applied by a front lens light diffuser using a fluence rate of 20--25 mW/cm(2). PDT using m-THPC resulted in complete response in all patients. Response to treatment did not differ when using the 2 different drugdose protocols. Healing time depended mainly on the size of the illumination field but not on the lightdose. Pain score usually raised 1 day after PDT and lasted at higher levels for about 10 days. Healing time usually ranged between 8--10 weeks. Photodynamic technique offers a minimal-invasive, outpatient treatment modality for recurrent breast cancer on the chest wall with few side effects, high patient's satisfaction and with possible repetitive application.     

Optimizing photodynamic therapy: in vivo pharmacokinetics of liposomal meta-(tetrahydroxyphenyl)chlorin in feline squamous cell carcinoma.

PURPOSE: The aim of the present study was to optimize and simplify photodynamic therapy using a new liposomal formulation of the photosensitizer meta-(tetrahydroxyphenyl)chlorin [m-THPC (Foscan); liposomal m-THPC (Fospeg)] and to reduce systemic reactions to the photosensitizer. EXPERIMENTAL DESIGN: To examine the pharmacokinetics of liposomal m-THPC, we determined tissue and plasma variables in feline patients with spontaneous squamous cell carcinoma. In vivo fluorescence intensity measurements of tumor and skin were done with a fiber spectrophotometer after i.v. injection of m-THPC or liposomal m-THPC in 10 cats. Blood samples, drawn at several time points after photosensitizer administration, were analyzed by high-performance liquid chromatography. RESULTS: None of the liposomal m-THPC-treated cats showed side effects during or after drug injection. Fluorescence intensities, fluorescence ratios (tumor fluorescence divided by skin fluorescence), and bioavailability in the tumor were 2 to 4 times higher with liposomal m-THPC compared with m-THPC. Liposomal m-THPC concentration in the tumor increased constantly to reach a maximum at 4 hours after injection. Plasma concentration and bioavailability were approximately 3 times higher with liposomal m-THPC compared with m-THPC measured at the time points of highest plasma concentration. The distribution half-life was shorter with liposomal m-THPC, resulting in maximal tumor accumulation up to 5.5 times earlier. Maximal tumor accumulation and maximal fluorescence ratio with liposomal m-THPC occurred at the same time point, indicating maximal selectivity. In both groups, all cats responded to therapy. CONCLUSIONS: Liposomal m-THPC was well tolerated by all cats and seems to have superior pharmacokinetic properties compared with m-THPC. The efficacy of the drug warrants further study.     

Potent and specific antitumor effect of CEA‐targeted photoimmunotherapy

Conventional photodynamic therapy (PDT) for cancer is limited by the insufficient efficacy and specificity of photosensitizers. We herein describe a highly effective and selective tumor‐targeted PDT using a near‐infrared (NIR) photosensitizer, IRDye700DX, conjugated to a human monoclonal antibody (Ab) specific for carcinoembryonic antigen (CEA). The antitumor effects of this Ab‐assisted PDT, called photoimmunotherapy (PIT), were investigated in vitro and in vivo. The Ab‐IRDye conjugate induced potent cytotoxicity against CEA‐positive tumor cells after NIR‐irradiation, whereas CEA‐negative cells were not affected at all, even in the presence of excess photoimmunoconjugate. We found an equivalent phototoxicity and a predominant plasma membrane localization of Ab‐IRDye after both one and six hours of incubation. Either no or little caspase activation and membrane peroxidation were observed in PIT‐treated cells and a panel of scavengers for reactive oxygen species showed only partial inhibition of the phototoxic effect. Strikingly, Ab‐IRDye retained significant phototoxicity even under hypoxia. We established a xenograft model, which allowed us to sensitively investigate the therapeutic efficacy of PIT by non‐invasive bioluminescence imaging. Luciferase‐expressing MKN‐45‐luc human gastric carcinoma cells were subcutaneously implanted into both flanks of nude mice. NIR‐irradiation was performed for only the tumor on one side. In vivo imaging and measurement of the tumor size revealed that a single PIT treatment, with intraperitoneal administration of Ab‐IRDye and subsequent NIR‐irradiation, caused rapid cell death and significant inhibition of tumor growth, but only on the irradiated side. Together, these data suggest that Ab‐IRDye‐mediated PIT has great potential as an anticancer therapeutics targeting CEA‐positive tumors.     

Uptake and kinetics of 14C-labelled meta-tetrahydroxyphenylchlorin and 5-aminolaevulinic acid in the C6 rat glioma model.

Meta-tetrahydroxyphenylchlorin (m-THPC) and 5-aminolaevulinic acid (5-ALA) are two second-generation photosensitizers which are currently under investigation for photodynamic therapy (PDT) and photodynamic diagnosis (PDD). So far, the experience with these photosensitizers for use within brain tumours is limited. We examined the distribution and retention of 14C-labelled m-THPC and [14C]5-ALA in the rat C6 glioma brain tumour model. After intraperitoneal injection of m-THPC (71,909 d.p.m. microl(-1); 0.16 mg ml(-1) m-THPC; 0.3 mg kg(-1)), the following activities were found after 36 h: brain tumour 223,664 d.p.m. g(-1), brain contralateral to the tumour side 2567 d.p.m. g(-1), liver 369,959 d.p.m. g(-1) and skin 55,197 d.p.m. g(-1); 100,000 d.p.m. corresponding to 0.22 microg of m-THPC. After 7 days, the concentration of m-THPC decreased to 76,277 d.p.m. g(-1) in tumour and 635 d.p.m. g(-1) in brain. The radioactivity after intravenous administration of [14C]5-ALA (23,079 d.p.m. microl(-1); 40 mg ml(-1); 120 mg kg(-1)) increased within 15 min (59,634 d.p.m. g(-1) in tumour, 17,427 d.p.m. g(-1) in brain); after 8 h only a small amount (3653 d.p.m. g(-1) in tumour) remained. Brain adjacent to the tumour was also found to have a higher uptake of 5-ALA. This study provides basic information for the use of m-THPC and 5-ALA in brain tumours. Because of the different pharmacokinetic and toxicological profile, we recommend m-THPC for PDT and 5-ALA for PDD. Clinical trials now have to prove the superior phototoxic properties of these second-generation photosensitizers.     

Biodistribution and tumor localization of stealth liposomal tumor necrosis factor-α in soft tissue sarcoma bearing rats

The blood residence half-life and organ distribution of recombinant human tumor necrosis factor-α (TNF-α) encapsulated in sterically stabilized liposomes, were investigated in rats bearing a soft tissue sarcoma in the hind leg. We studied the decay in blood concentration of “empty” liposomes using the aqueous marker ⁶⁷gallium-desferal, as well as the blood concentration of soluble TNF-α and liposome encapsulated TNF-α using ¹²⁵I. Encapsulation efficacy of TNF-α was 24%. The pharmacokinetics of TNF-α were markedly altered after encapsulation in liposomes, with a 33-fold increase in mean residence time of TNF-α in the blood, and a concomitant 14-fold increase in the area under the plasma concentration vs. time curve for liposomal TNF-α. Although the liposomes exhibit Stealth characteristics, uptake by mononuclear phagocyte-rich organs (e.g., liver and spleen) was noticeable, especially at later time points. Encapsulation of TNF-α in sterically stabilized liposomes resulted in a marked increase in localization of the cytokine in tumor measured as total uptake over time. However, peak TNF-α concentration levels in tumor were not significantly enhanced compared with free TNF-α. Besides the augmented localization of TNF-α after encapsulation in sterically stabilized liposomes, a diminished toxicity was observed. Int. J. Cancer 77:901–906, 1998.© 1998 Wiley-Liss, Inc.     

Clinical pharmacokinetic studies of tetra(meta-hydroxyphenyl)chlorin in squamous cell carcinoma by fluorescence spectroscopy at 2 wavelengths

To optimize photodynamic therapy (PDT) and photodetection of cancer with second-generation photosensitizers, knowledge of important variables such as the uptake of the dye and the dye contrast between normal and tumoral tissue after injection is necessary. The pharmacokinetics of a second-generation photosensitizer, tetra(meta-hydroxyphenyl)chlorin (mTHPC), is presented. To study this in a clinical context, an apparatus based on fluorescence spectroscopy and a non-invasive optical fiber probe has been used. The mTHPC fluorescence is induced at 2 excitation wavelengths (420 and 520 nm) with different penetration depth. The pharmacokinetics of mTHPC in patients with a squamous-cell carcinoma in the oral cavity show a signal selectivity as high as 16 about 3 hr after i.v. injection for the more advanced carcinomas. The magnitude of this selectivity appears to correlate with the staging of the cancer, the more invasive tumors showing the highest selectivity. Results obtained at 420 and 520 nm show little difference. These pharmacokinetics can be used directly for optimizing photodetection with mTHPC. However, complementary information on the localization of the drug by fluorescence microscopy, and a correlation of this data with tumor necrosis efficacy, are needed to optimize PDT timing.     

Foscan-based photodynamic treatment in vivo: correlation between efficacy and Foscan accumulation in tumor, plasma and leukocytes

The tumoricidal effect of Foscan-mediated photodynamic therapy may involve both vessel and tumor cell destruction. The relevant importance of each mechanism seems to be defined by the time interval between photosensitizer administration and illumination (drug-light interval, DLI). Short drug-light intervals favor vascular damage due to the preferential photosensitizer accumulation in the tumor vasculature, whereas long drug-light intervals trigger direct tumor cell damage due to the dye localization in the tumor. The purpose of this study was to investigate the influence of tumor, plasma and leukocyte concentrations of Foscan at different times after photosensitizer delivery on PDT response. Both pharmacokinetic and tumor-response studies were carried out in nude mice bearing s.c. Colo26 tumors. One to 96 h after i.v. injection of 0.5 mg/kg Foscan, animals were exposed to 10 J/cm(2) 652-nm light delivered at 30 mW/cm(2). Mean tumor regrowth time was determined for each schedule of treatment and correlated to Foscan distribution in the compartments of interest at the time of illumination. PDT efficacy was greatest for irradiations performed at 6 and 12 h post Foscan injection and limited at 96 h. Unlike tumor and plasma Foscan concentrations, photosensitizer accumulation in leukocytes exhibited a good correlation with PDT efficacy. The results suggest that leukocytes could play an important role in the mechanism of PDT-induced vascular damage either by being one of the main effector compartments or by better reflecting Foscan accumulation in endothelial cells compared to plasma. The prevalence of indirect damage was highlighted by the fact that PDT efficacy was not modified by the use of a higher fluence rate of irradiation (160 mW/cm(2)), which depleted intratumor oxygen and did not restrain PDT-induced cell toxicity.     

Foscan-mediated photodynamic therapy for a peritoneal-cancer model: Drug distribution and efficacy studies

Distribution of the photosensitizer Foscan® (meta-tetrahydroxyphenylchlorin, mTHPC), after i.v. or i.p. injection, was investigated in Wag/Rij rats bearing i.p. tumours. These results were compared with the efficacy of mTHPC-mediated photodynamic therapy for illumination intervals of 4 hr to 3 days. For the distribution experiments a single tumour (CC531 colon carcinoma) was implanted intra-abdominally in a fat pad, or a cell suspension (1 × 10⁶ CC531 cells) was injected into the peritoneal cavity, which results in a dissemination of tumour nodules on the peritoneum. ¹⁴C-mTHPC was not selectively taken up in the single-tumour model after i.v. or i.p. injection, but higher concentrations were achieved for i.p. administration. For this tumour model the concentration ratios between tumour and normal tissue never exceeded a value of 3. In the disseminated-tumour model, an uptake of up to 40% of the injected dose was found per gram tumour at 4 hr after an i.p. injection and this resulted in very high (>14) concentration ratios of tumour to normal tissues. Low uptake was found after the i.v. injection route (1% of the injected dose per gram tumour) with lower tumour/normal tissue ratios (<8). The efficacy of i.p. photodynamic therapy (IPPDT) was evaluated using the single-tumour model only. The lower abdomen was illuminated at 4 hr to 3 days after mTHPC, and tumour size was repeatedly measured via a small laparoscopy. Significant delay in tumour regrowth was achieved for 6 J · cm⁻² at 1 day after i.v., or at 4 hr after i.p. mTHPC (p values 0.019 and 0.045 respectively). Response to PDT, of tumours implanted in the fat pad, was not greater for i.p. administration of the photosensitizer and there was a poor correlation between times of maximum drug uptake in tumours and optimal illumination times for PDT efficacy. Int. J. Cancer 73:230–235, 1997. © 1997 Wiley-Liss, Inc.     

Targeting tumor vasculature and cancer cells in orthotopic breast tumor by fractionated photosensitizer dosing photodynamic therapy

Photodynamic therapy (PDT) is a locally administered therapy currently being investigated in various clinical and preclinical settings. Tumor-host interaction is an important determinant of tumor biology and response to treatments. Here we report for the first time the effects of PDT on an orthotopic, murine mammary tumor model. PDT utilizes two individually nontoxic components: (a) the localization in the target site of a photosensitizing drug; and (b) the activation of the photosensitizer by light of an appropriate wavelength and energy. PDT after a single dose of the photosensitizer MV6401 induced drug dose-dependent, long-term blood flow shut down and tumor growth delay in the MCaIV tumor, grown in the mammary fat pad. The plasma half-life of MV6401 was approximately 20 min, and the drug was confined to the vascular compartment shortly after administration. However, it accumulated in the interstitial compartment at 2-6 h after the administration. Two equal MV6401 doses injected 4 h and 15 min before the light administration allowed the photosensitizer to localize in both vascular and tumor cell compartments. The fractionated drug dose PDT more effectively induced tumor growth delay than the same total dose given as a single dose either at 4 h or at 15 min before light administration. The long-term effect of the fractionated drug PDT on blood flow was also more extensive than single-dose PDT. Fractionated photosensitizer dosing PDT offers a new strategy to optimize PDT therapy.     

Differential up-regulation of metallothionein isoforms in well-differentiated nasopharyngeal cancer cells in vitro by photoactivated hypericin

Photoactivation of hypericin is known to generate reactive oxygen species and induce phototoxic effects. However, modulation of the cellular antioxidant defense would influence the extent and severity of the photodynamic effects. We have previously shown that hypericin-mediated photodynamic therapy (PDT) induced a significant reduction of Glutathione S-transferase activity. In this study, we investigated the phototoxic effects of hypericin-mediated PDT in nasopharyngeal cancer (NPC) in vitro and analyzed the expression of metallothionein (MT), a family of potential free radical scavengers. HK1 NPC cells were subjected to PDT treatment in vitro, and the effects on cell death were analyzed by flow cytometry (using propidium iodide and Annexin V staining) and transmission electron microscopy. The expression profile of MT-1E and MT-2A isoforms (the only functional MT isoforms expressed in HK1 NPC cells) were determined by quantitative real-time RT-PCR. The results showed that hypericin PDT induced necrotic cell death as evidenced by the absence of a subdiploid peak and decreased Annexin-V fluorescence. Ultrastructural examination verified the presence of cell necrosis. There was a significant up-regulation of MT-1E and MT-2A isoforms six hours following PDT, with an approximately 50-fold rise in the expression level of MT-1E and a 15-fold increase of MT-2A. Hence, despite the up-regulation of MT, cells still succumbed to PDT-induced necrosis. It appears that the oxidative stress induced by PDT overwhelmed the antioxidant defense mechanism such as the alteration of MT levels in tumor cells.     

Enhanced in vivo antitumor efficacy of poorly soluble PDT agent, meso-tetraphenylporphine, in PEG-PE-based tumor-targeted immunomicelles

Poorly soluble photosensitizer, meso-tetraphenylporphine (TPP), was solubilized using the polymeric micelles prepared from polyethylene glycol-phosphatidyl ethanolamine conjugate (PEG-PE). TPP-loaded PEG-PE micelles have been additionally modified with tumor-specific monoclonal 2C5 antibody (mAb 2C5), which resulted in significantly improved anticancer effect of the drug under the PDT conditions against murine Lewis lung carcinoma (LLC) In vivo in female C57BL/6 mice. Fourteen days after tumor inoculation, the mice with more than 2 mm diameter tumors were given an intravenous injection of 1 mg/kg of free TPP or TPP loaded into control PEG-PE micelles or into mAb 2C5-PEG-PE tumor-targeted immunomicelles. Twenty-four hours after the administration, the animals were anesthetized, and tumor sites were illuminated with light (630 nm) for 12 min. Microscopic evaluation of tumor response was conducted in some mice 24 h after light irradiation, and tumor size was followed in the remaining animals for another 35 days. The attachment of mAb 2C5 to TPP-loaded immunomicelles provided the maximum level of tumor growth inhibition. Enhanced tumor accumulation of TPP-loaded mAb 2C5-PEG-PE-immunomicelles was confirmed by gamma-imaging studies. The modification of the TPP-loaded polymeric micelles with tumor-specific antibodies could be used as a general approach to enhance the efficacy of PDT.     

Metabolic properties and photosensitizing responsiveness of mono-L-aspartyl chlorin e6 in a mouse tumor model.

A mouse mammary tumor model was used to evaluate metabolic properties of the photosensitizer mono-L-aspartyl chlorin e6 (NPe6) and to determine the optimal time interval between drug administration and light treatment for effective photodynamic therapy (PDT). Photosensitizer metabolism was evaluated by comparing tissue distribution patterns of NPe6 having 14C atoms positioned on either the tetrapyrrole ring or on the aspartyl residue. High performance liquid chromatographic analysis of photosensitizer extracted from tumor tissue was also obtained as a function of time after drug administration. NPe6 distribution in tissue samples and pharmacological calculations of area under the curve were similar for both forms of [14]NPe6. Likewise, metabolic contaminants of NPe6 were not detected by high performance liquid chromatographic analysis following extraction of the photosensitizer from tumor tissue. Maximal in vivo PDT effectiveness was achieved when light treatments were started within 2 h of drug injection. PDT effectiveness was decreased by 50% when light treatments were initiated 6 h after drug injection and was abolished with a 12-h interval between NPe6 injection and light exposure. Responsiveness to NPe6-mediated PDT was correlated with photosensitizer levels in the plasma but not in tumor tissue. These results show that NPe6 was not metabolized following in vivo administration and that the responsiveness of NPe6 mediated PDT was associated with vascular clearance of the photosensitizer.     

Enhancing the therapeutic responsiveness of photodynamic therapy with the antiangiogenic agents SU5416 and SU6668 in murine nasopharyngeal carcinoma models

Background: Photodynamic therapy (PDT) is a promising therapeutic modality using a tumor localizing photosensitizer and light to destroy tumor cells. A major limitation of PDT is tumor recurrence, which is partly due to neovascularization. Purpose: The objective of the present study was to determine whether combination therapy with PDT and antiangiogenic agents (i.e. SU5416 and SU6668) would be more effective in controlling tumor recurrence in a mouse model of human CNE2 poorly differentiated nasopharyngeal carcinoma compared with PDT or antiangiogenic agents administered alone. Methods: Athymic mice bearing CNE2 tumor xenografts received daily i.p. injections of 20 mg/kg SU5416 or 100 mg/kg SU6668 for 28 consecutive days either alone or following a single hypericin-PDT treatment. Results: Significant inhibition of CNE2 tumor growth was observed in all treatment groups. Differences in 4× tumor growth time, the number of mice with 4× tumor growth, tumor growth inhibition as well as the percent of mice surviving were not statistically significant among individual treatment groups. However, the number of mice with 4× tumor growth observed in SU6668 monotherapy and combined PDT and SU6668 treatment groups was significantly less than that in the control group (P<0.05 and 0.01, respectively). Moreover, compared with the control group, only the combined PDT and SU6668 treatment significantly extended survival of tumor-bearing host mice (P<0.05). The semiquantitative RT-PCR results showed that the expression of HIF-1, VEGF, COX-2 and bFGF were increased in PDT-treated tumor samples collected 24 h post-PDT, suggesting that PDT-induced damage to tumor microvasculature and the resultant hypoxia upregulate the expression of certain proangiogenic factors. Conclusions: The effectiveness of PDT can be enhanced by antiangiogenic treatment with the synthetic RTK inhibitors. Of the two synthetic RTK inhibitors tested, SU6668 was more effective than SU5416 in enhancing tumor responsiveness to PDT.     

Photodynamic eradication of amelanotic melanoma of the hamster with fast acting photosensitizers

Three porphycenes with fast pharmacokinetics were tested for their ability to photosensitize amelanotic hamster melanoma A-Mel-3 at short time intervals after injection. Laser light irradiation was performed at the time of maximal photosensitizer level in tumor tissue. Photodynamic therapy as short as 5 min after injection led to complete local tumor remission at a dosage of 1.4 μmol/kg for the porphycene CBPn. In comparison, Photofrin required 8.4 μmol/kg for local tumor remission in 5 of 6 animals with 24 hr accumulation time after injection. We propose a swift photodynamic protocol which can compete favorably with conventional techniques of tumor treatment. © 1996 Wiley-Liss, Inc.     

Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability

Marked differences in the tumor uptake of a ¹²⁵I-labeled monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA) were observed in 4 serially transplanted human colorectal carcinomas in nude mice. A comparative study showed that elevated values of measurable tumor vascular parameters, such as permeability, blood flow and blood volume, correlated better with high MAb tumor uptake than the concentration of target antigen in the tumor. In an attempt to modify the vascular parameters and to determine if this could increase antibody uptake by the tumor, rhTNFα (TNF) was injected i.t. or i.v. and antibody localization experiments were performed immediately thereafter. Results showed that the permeability of the tumor vessels increased 8 to 10 fold 1 hr after i.t. injection of TNF as compared to control tumors injected with saline. Tumor uptake of ¹²⁵I-labeled anti-CEA MAb, was 3 times higher 2 hr after i.v. injection and still 27% higher 22 hr later, as compared to results from controls. Intravenous injection of TNF simultaneously with the ^l25I- labeled anti-CEA MAb also resulted in a 2-fold increase in tumor uptake 4 hr after injection, but the increase was no longer significant 24 hr after injection. Interestingly after i.v. injection of TNF, the MAb concentration in the blood and other normal tissues, such as liver, kidneys, lungs and heart was decreased, resulting in significantly higher ratios of tumor to normal tissue. Taken together the results demonstrate that injection of TNF can increase tumor vascular permeability and improve radioantibody uptake. This raises the possibility of increasing the radiation dose delivered by antibody to the tumor in the course of radioimmunotherapy.     

Mechanisms for optimising photodynamic therapy: second-generation photosensitisers in combination with mitomycin C.

Mechanisms for improving photodynamic therapy (PDT) were investigated in the murine RIF1 tumour using meso-tetrahydroxyphenylchlorin (m-THPC) or bacteriochlorin a (BCA) as photosensitisers and comparing these results with Photofrin-mediated PDT. The 86Rb extraction technique was used to measure changes in perfusion at various times after interstitial PDT. Non-curative combinations of light doses with m-THPC and BCA PDT markedly decreased vascular perfusion. This decrease was more pronounced for both new photosensitisers than for Photofrin. Comparison of tumour perfusion after PDT with tumour response revealed an inverse correlation for all three photosensitisers, but the relationship was less clear for m-THPC and BCA. In vivo/in vitro experiments were performed after Photofrin or m-THPC PDT in order to assess direct tumour kill (immediate plating) vs indirect vascular effects (delayed plating). For both photosensitisers, there was little direct cell killing but clonogenic survival decreased as the interval between treatment and excision increased. When m-THPC PDT was combined with mitomycin C (MMC), light doses could be decreased by a factor of 2 for equal tumour effects. Lower light and m-THPC doses could be used compared with Photofrin PDT in combination with MMC. BCA PDT with MMC did not result in a greater tumour response compared with BCA PDT alone. Reduction in both light and photosensitiser does for effective PDT regimes in combination with MMC offers substantial clinical advantages, since both treatment time and skin photosensitisation will be reduced.     

Photofrin-mediated photodynamic therapy induces vascular occlusion and apoptosis in a human sarcoma xenograft model.

Photodynamic therapy (PDT) involves light activation of a photosensitizer, resulting in oxygen-dependent, free radical-mediated cell death. Little is known about the efficacy of PDT in treating human sarcomas, despite an ongoing clinical trial treating i.p. sarcomatosis. The present study evaluates PDT treatment of a human sarcoma xenograft in nude mice and explores the mechanism of PDT-mediated antitumor effect. Athymic nude mice, 6-8 weeks of age, were s.c. injected with 5 x 10(6) cells of the A673 human sarcoma cell line. Tumors were allowed to grow to a diameter of about 10 mm. Photofrin (PF), 10 mg/kg, was injected by tail vein, and 24 h later, 630 nm light was delivered to the tumor with fluences of 50, 100, 150, or 300 J/cm2 at a fluence rate of 250 mW/cm2. To assess the efficacy of PDT in the treatment of sarcomas, photosensitizer uptake/retention studies and dose-response studies were performed. Studies carried out to determine the mechanism of tumor response included tumor temperature measurements before, during, and after treatment; tumor vascular perfusion studies with laser Doppler; electron microscopic analysis of tumor sections for vascular occlusion; and analysis of tumor cryosections for endothelial cell damage, apoptosis, and necrosis. At all time points of analysis, photosensitizer levels were greater in tumor than in muscle. Dose-response studies showed that at 100 J/cm2, five of six mice had a complete response to treatment, one of six had a partial response, and no deaths occurred. Temperature measurements indicated that thermal injury did not contribute to tumor response. Vascular perfusion studies demonstrated a significant reduction in blood flow as early as 6 h after PDT. Electron micrographs revealed erythrostasis in tumor microvessels starting as early as 2 h after treatment and complete occlusion of blood vessels by 12 h. Starting as early as 4 h after PDT, apoptosis first appeared in endothelial cells lining the occluded blood vessels and became more widespread at later time points. PDT is an effective treatment for this human sarcoma xenograft in nude mice. The mechanism of tumor destruction in this model appears to be vascular damage with initial apoptosis in tumor endothelial cells and delayed tumor cell apoptosis. This therapy may be valuable in the treatment of patients with sarcomatosis.     

Tumor vascular microenvironment determines responsiveness to photodynamic therapy

The efficacy of photodynamic therapy (PDT) depends upon the delivery of both photosensitizing drug and oxygen. In this study, we hypothesized that local vascular microenvironment is a determinant of tumor response to PDT. Tumor vascularization and its basement membrane (collagen) were studied as a function of supplementation with basement membrane matrix (Matrigel) at the time of tumor cell inoculation. Effects on vascular composition with consequences to tumor hypoxia, photosensitizer uptake, and PDT response were measured. Matrigel-supplemented tumors developed more normalized vasculature, composed of smaller and more uniformly spaced blood vessels than their unsupplemented counterparts, but these changes did not affect tumor oxygenation or PDT-mediated direct cytotoxicity. However, PDT-induced vascular damage increased in Matrigel-supplemented tumors, following an affinity of the photosensitizer Photofrin for collagen-containing vascular basement membrane coupled with increased collagen content in these tumors. The more highly collagenated tumors showed more vascular congestion and ischemia after PDT, along with a higher probability of curative outcome that was collagen dependent. In the presence of photosensitizer-collagen localization, PDT effects on collagen were evidenced by a decrease in its association with vessels. Together, our findings show that photosensitizer localization to collagen increases vascular damage and improves treatment efficacy in tumors with greater collagen content. The vascular basement membrane is thus identified to be a determinant of therapeutic outcome in PDT of tumors.     

Light delivery over extended time periods enhances the effectiveness of photodynamic therapy.

PURPOSE: The rate of energy delivery is a principal factor determining the biological consequences of photodynamic therapy (PDT). In contrast to conventional high-irradiance treatments, recent preclinical and clinical studies have focused on low-irradiance schemes. The objective of this study was to investigate the relationship between irradiance, photosensitizer dose, and PDT dose with regard to treatment outcome and tumor oxygenation in a rat tumor model. EXPERIMENTAL DESIGN: Using the photosensitizer HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide), a wide range of PDT doses that included clinically relevant photosensitizer concentrations was evaluated. Magnetic resonance imaging and oxygen tension measurements were done along with the Evans blue exclusion assay to assess vascular response, oxygenation status, and tumor necrosis. RESULTS: In contrast to high-incident laser power (150 mW), low-power regimens (7 mW) yielded effective tumor destruction. This was largely independent of PDT dose (drug-light product), with up to 30-fold differences in photosensitizer dose and 15-fold differences in drug-light product. For all drug-light products, the duration of light treatment positively influenced tumor response. Regimens using treatment times of 120 to 240 min showed marked reduction in signal intensity in T2-weighted magnetic resonance images at both low (0.1 mg/kg) and high (3 mg/kg) drug doses compared with short-duration (6-11 min) regimens. Significantly greater reductions in pO(2) were observed with extended exposures, which persisted after completion of treatment. CONCLUSIONS: These results confirm the benefit of prolonged light exposure, identify vascular response as a major contributor, and suggest that duration of light treatment (time) may be an important new treatment variable.