Greta M. Garbo

Photodynamic Treatment of Transplantable Bladder Tumors in Rodents After Pretreatment with Chloroaluminum Tetrasulfophthalocyanine

Chloroaluminum tetrasulfophthalocyanine (AlPCS) was used as a photosensitizer for the photodynamic treatment of transplantable N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) induced urothelial tumors. Two groups of six rats each were injected with AlPCS (three micrograms./gm. body weight) and 24 hours after injection underwent photodynamic treatment with red light (greater than 590 nm., 360 joules/cm.2). Tumors examined four hours (Group I) and 24 hours (Group II) after the completion of phototreatment showed extensive hemorrhagic necrosis. Tumors treated with AlPCS alone showed no changes. In two other groups of six rats each, blood flow to tumors treated with AlPCS alone (Group III) and AlPCS plus light (Group IV) was measured using the radioactive microsphere technique. AlPCS plus light resulted in a significant decrease (p less than .05) in tumor blood flow within 10 minutes of completion of phototreatment while AlPCS alone had no effect on tumor blood flow. These findings are similar to those observed when higher doses (10 micrograms./gm. to 20 micrograms./gm. body weight) of hematophorphyrin derivative (HpD) and light were used for phototreatment of FANFT induced tumors. AlPCS is a stable sulfonated derivative of tetraazotetrabenzoporphyrin which absorbs maximally in the red portion of the visible spectrum, a region with good tissue penetration properties. These studies suggest the AlPCS may be a useful new agent for photodynamic therapy of cancer.

SITES and EFFICACY OF PHOTODAMAGE BY TIN ETIOPURPURIN in vitro USING DIFFERENT DELIVERY SYSTEMS

Photosensitization by tin etiopurpurin (SnET2) was determined in cell culture using sensitizer dissolved in ethanol or solubilized via three different delivery systems: Cremophor EL, γ‐cyclodextrin or Molecusol (a more water‐soluble cyclodextrin derivative). Sensitizer uptake was substantially more efficient when the delivery systems were employed, in terms of intracellular level needed to lethally photosensitize cells. We observed photodamage at both membrane and mitochondrial loci, but the former was better correlated with loss of viability.

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>SnNT2>ZnET2>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.

Use of liposomes, emulsions, or inclusion complexes may potentiate in-vivo effects of SnET2

The majority of second generation sensitizers being proposed as possible alternatives to hematoporphyrin derivative, in photodynamic therapy, are hydrophobic in nature. Consequently, specific carrier systems have to be developed for in vivo administration. As an attempt to understand the interactions of these delivery systems in vivo, plasma binding properties of the sensitizer SnET2 complexed with liposomes, emulsions or cyclodextrins have been studied. Additional studies have investigated the effect of the carrier system on the cytotoxicity of SnET2 on transplantable tumors. Preliminary data suggest that tumor response may be mediated by the choice of carrier system. Further studies appear to be necessary before the optimum thug/carrier system complex can be defined.

Purpurins and benzochlorins as sensitizers for photodynamic therapy

Purpurins and benzochlorins are hydrophobic second generation photosensitizers, which have sizable absorption in the 650-780 nm range and exhibit photodynamic activity against tumors after visible light treatment. This review summarizes the published data regarding these compounds: from the spectroscopic, photophysical and photochemical characteristics to the in vivo and in vitro photodynamic inactivation of tumors and normal tissue effects. Some mechanistic studies using both the Sn-etiopurpurin and/or the Cu-benzochlorin derivatives are also reviewed.

Binding of drugs to human plasma proteins, exemplified by Sn(IV)-etiopurpurin dichloride delivered in cremophor and DMSO.

1. The mode-delivery-effect upon the binding of Sn(IV)-etiopurpurin dichloride (SnET2) in human plasma has been studied by ultracentrifugation, combined with absorption and fluorescence spectroscopy. SnET2 was delivered to plasma either in Cremophore EL (CRM) or in dimethyl sulfoxide (DMSO). To facilitate interpretation, optical, conductivity and aggregation properties of SnET2 were obtained for various solutions. 2. The second order rate constant for the aggregation of SnET2 monomers seemed to be remarkably small, of the order of 10(3) M-1 min-1. 3. SnET2 was bound as monomeric entities. Such entities had environmental-sensitive fluorescent properties dependent on the type of protein or solvent (DMSO, CRM, H2O) with which they interacted. 4. SnET2 showed saturable binding with high density subfraction(s) of high density lipoproteins and with one or more high density proteins. Complete or substantial saturation was achieved at the SnET2 level of 3.5 micrograms/ml. Such binding might be mediated by apolipoprotein D and alpha 1-acid glycoprotein. 5. There was little effect of SnET2 concentrations (3.5-35 micrograms SnET2/ml) upon the plasma binding of SnET2, irrespective of the mode of delivery. 6. The percentages of SnET2 bound to low density lipoproteins (LDL), high density lipoproteins (HDL), and high density proteins (HDP) were 10, 70 and 20%, respectively, for delivery in DMSO. The value for LDL also includes binding with very low density lipoproteins (VLDL). For delivery in CRM the corresponding values were 20, 50 and 30%. Apparently, CRM interacted with HDL entities and reduced their affinity for SnET2. 7. The distribution pattern of SnET2 among lipoproteins reflects interactions with apoproteins and/or with surface phospholipids rather than with core lipid constituents of lipoproteins. 8. Conductivity measurements showed that SnET2 was partly an ionic entity in water. 9. The plasma binding of SnET2 is compared with the corresponding binding of other drugs, both tetrapyrroles and nontetrapyrroles.

Observations on the synthesis and in vivo photodynamic activity of some benzochlorins

Abstract— An improved synthesis of benzochlorins is reported. Demetallation of the meso‐hydroxymethylvinyl derivative of octaethylporphyrin, followed by treatment with sulfuric acid results in cyclization to generate the corresponding octaethylbenzochlorin in high yield. Prolonged treatment with acid generates the sulfonated derivative. These sensitizers were shown to be efficient photodynamic agents in vivo. Animals bearing a transplanted N‐{4‐(5‐nitro‐2‐furyl)‐2‐thiazolyl}formamide induced urothelial tumor were treated with either the benzochlorin or its sulfonated derivative. Irradiation of tumors 24 h later resulted in a significant tumoricidal effect in a short term assay. We conclude that benzochlorins warrant further examination as potential agents for use in photodynamic therapy.

Tin (IV) Etiopurpurin Dichloride: An Alternative To DHE?

Etiopurpurin is a photosensitizer developed from etioporphyrin I, which we have shown to be useful in PDT. Incorporation of tin into the macrocycle gives a derivative which appears to be more effective both in vitro and in vivo studies on tumor cytotoxicity, than the parent purpurin. In vitro studies have shown not only that tin etiopurpurin dichloride (SnET2C12) is an efficient photosensitizer, but also that its action spectrum appears to be consistent with both the absorption and emission spectra. In vivo studies include a histological examination of tumors treated with SnET2C12 and PDT which suggests that disruption of the tumor vasculature plays an active role in tumor necrosis; a twelve day dose response study to determine effective dose; a thirty day study to determine tumor regrowth; a study of normal tissue response to PDT and a study to determine the optimum time for phototherapy after injection of the drug. For these studies, the FANFT induced urothelial tumor line was used, although we have recently initiated studies on the R3327 AT rat anaplastic adenocarcinoma, a tumor which appears to be more resistant to PDT.

Purpurins: Improved Photosensitizers For Photodynamic Therapy

Two new groups of photosensitizers, purpurins and metallopurpurins were tested for photodynamic activity when combined with visible light (>590nm), in the treatment of transplantable urothelial tumors (FANFT induced) in Fischer 344 rats. In preliminary studies, animals were injected with 10 mg/kg body weight (b.w.) of the free base purpurins (NT1, NT2, NT2H2, GG1, ET2, ET2H2 and JP1) and treated with 360J/cm2 of red light. Tumors were excised, fixed in formalin and stained for light microscopic examination. Purpurin based photodynamic therapy (PDT) was found to cause extensive hemorrhagic tumor necrosis. Control tumors shielded from the light showed no histological changes. These experiments were repeated using the same experimental design for the metallopurpurins (ZnET2, SnET2, ZnNT2H2, SnNT2H2, AgNT2H2 and ZnNT1). These compounds were also found to cause extensive hemorrhagic necrosis. These studies were followed by a dose response analysis in which groups of animals were treated with decreasing doses of drug while the light dose was kept constant. Response was determined by tumor dry weight, 12 days after completion of PDT. For the free base purpurins, drug doses as low as 1.0 mg/kg b.w. caused significant tumor regression while seleted metallopurpurins caused tumor regression at doses as low as 0.5 mg/kg b.w. In a series of related experiments the effect of purpurins on tumor blood flow was investigated since it has previously been shown that tumor blood flow is disrupted during porphyrin and phthalocyanine phototherapy. By using the radioactive microsphere technique, it was shown that both the free base purpurins and metallopurpurins caused a rapid decrease in tumor blood flow.

Spectroscopic studies of tin ethyl etiopurpurin in homogeneous and heterogeneous systems

Tin ethyl etiopurpurin is a promising second generation photosensitizer for photodynamic treatment of cancer. This compound is only poorly soluble in aqueous media and, therefore, needs a delivery system for administration to animals. Successful tumor eradication has previously been reported, following light exposure of rats previously administered with the purpurin formulated as a Cremophor El emulsion, in dipalmitoyl-phosphatidyl-choline liposomes or with gamma cyclodextrins. In this paper, we describe some absorbance and fluorescence studies of tin ethyl etiopurpurin in homogeneous and heterogeneous systems. In general, absorption and emission maxima were found to be red shifted as the environment changed from polar to non-polar. The viscosity and dielectric constant of the medium affected the purpurin fluorescence intensity. The liposome preparations were characterized by particle size determination, differential scanning calorimetry and by sensitizer fluorescence quenching studies. Photobleaching studies also showed variation owing to changes in the environment in which the dye was located.