Systemic effects of orally-administered zinc and tin (IV) metalloporphyrins on heme oxygenase expression in mice.

Some metalloporphyrins (Mps) inhibit heme oxygenase (HO), the rate-limiting enzyme in the production of bilirubin, and are potential compounds for the treatment of neonatal jaundice. We studied the safety and efficacy of Mps following oral administration. Adult HO-1-luc reporter mice were administered 30 micromol/kg body weight of tin mesoporphyrin (SnMP), zinc bis glycol deuteroporphyrin (ZnBG), or zinc protoporphyrin (ZnPP), or vehicle by oral gavage. Bilirubin production was measured as total body carbon monoxide (CO) excretion (VeCO). HO activity was quantitated via CO measurements by gas chromatography. HO-1 protein was determined by Western blot. HO-1 transcription levels were assessed by in vivo bioluminescence imaging. A significant 28% decrease in bilirubin production occurred within 3 h of SnMP treatment and persisted beyond 48 h. Bilirubin production decreased 15% and 9% by 3 h after administration of ZnBG and ZnPP, respectively, but returned to baseline within 48 h. Maximal inhibition of liver, spleen, and intestine HO activity was seen at 3 h with inhibitory effects decreasing in the order: SnMP > or = ZnBG > or = ZnPP. After SnMP treatment, HO-1 transcription increased 5.7-fold after 24 h. Furthermore, liver and spleen HO-1 protein significantly increased 3.7- and 2.0-fold, respectively, after 24 h. HO-1 transcription and protein were not affected in ZnBG- or ZnPP-treated mice. We conclude that the three Mps are absorbed at different rates in the mouse and affect bilirubin production and HO-1 expression in a tissue- and time-dependent manner.

Toxicity, biodistribution, and convection-enhanced delivery of the boronated porphyrin BOPP in the 9L intracerebral rat glioma model.

PURPOSE: To investigate the toxicity, biodistribution, and convection-enhanced delivery (CED) of a boronated porphyrin (BOPP) that was designed for boron neutron capture therapy and photodynamic therapy. METHODS AND MATERIALS: For the toxicity study, Fischer 344 rats were injected with graded concentrations of BOPP (35-100 mg/kg) into the tail vein. For boron biodistribution studies, 9L tumor-bearing rats received BOPP either systematically (intravenously) or locally. RESULTS: All rats that received 70 mg/kg BOPP and 70% of rats that received < or = 60 mg/kg BOPP i.v. either had to be euthanized or died within 4 days of injection. In the biodistribution study, boron levels were relatively high in liver, kidney, spleen, and adrenal gland tissue, and moderate levels were found in all other organs. The maximum tumor boron concentration was 21.4 mug/g at 48 h after i.v. injection; this concentration of boron in brain tumors is at the low end of the range considered optimal for therapy. In addition, the tumor/blood ratio (approximately 1.2) was not optimal. When BOPP was delivered directly into intracerebral 9L tumors with CED, we obtained tumor boron concentrations much greater than those obtained by i.v. injection. Convection-enhanced delivery of 1.5 mg BOPP produced an average tumor boron level of 519 mug/g and a tumor/blood ratio of approximately 1850:1. CONCLUSIONS: Our study demonstrates that changing the method of BOPP delivery from i.v. to CED significantly enhances the boron concentration in tumors and produces very favorable tumor/brain and tumor/blood ratios.

Biopharmaceutics of boronated radiosensitizers: liposomal formulation of MnBOPP (manganese chelate of 2,4-(alpha, beta-dihydroxyethyl) deuterioporphyrin IX) and comparative toxicity in mice.

Binary treatment modalities such as photodynamic therapy (PDT) and neutron capture therapy (NCT) combine low-toxicity electromagnetic irradiation with an appropriate radiation sensitizer to enhance selectivity for tumor targets. The porphyrin derivative tetrakiscarborane carboxylate ester of 2,4-(alpha, beta-dihydroxyethyl) deuterioporphyrin IX (BOPP) shows tumor-selective uptake and is active in both treatment modalities. BOPP also chelates paramagnetic ions such as Mn(2+), and therefore its tissue accumulation and selectivity can be detected noninvasively by using magnetic resonance imaging. However, local and systemic toxicity appears elevated for the Mn(2+) chelate (MnBOPP), but is poorly characterized. Here we have developed a liposomal formulation of MnBOPP and compared its toxicity with that of MnBOPP administered to mice in saline. The optimal liposome composition and maximal capacity to accommodate MnBOPP were investigated by differential scanning calorimetry and by encapsulation efficiency. MnBOPP was encapsulated quantitatively at up to 12 mol % (drug:lipid) in liposomes of varying composition, and remained incorporated during extended dialysis. Phase separation of drug- and lipid-rich domains was observed above 12% drug. MnBOPP in buffered saline was lethal to animals at 90 micromol/kg, and caused severe necrosis at the injection site at dose levels of 60 micromol/kg or greater. In contrast, MnBOPP formulated in liposomes was well tolerated at the highest tested dose of 135 micromol/kg, with the elimination of local toxicity.

In vivo and in vitro photodynamic studies with benzochlorin iminium salts delivered by a lipid emulsion.

Benzochlorin iminium salts (BIs) are hydrophobic photosensitizers based on an octaethylbenzochlorin nucleus that absorb in the near-IR region of the visible spectrum. In these studies the photodynamic activities of the zinc, copper and metal-free BI derivatives were compared in vivo in C3H-HeJ mice bearing a mammary adenocarcinoma tumor line. In vitro studies were also performed with the radiation-induced fibrosarcoma tumor line. An argon-pumped Ti-sapphire laser tuned to deliver light between 710 and 800 nm or an Oriel arc-lamp filtered to deliver broadband light above 590 nm were used as light source. A lipid emulsion was used as the delivery system for sensitizers in all studies. A pronounced solvent dependence was observed for the Q band for each of all iminium salts examined. As an example, the metal-free (BI) derivative had an absorption maximum at 798 nm in dichloromethane and at 727 nm in serum. The action spectra showed a greater PDT response at blue-shifted wavelengths for each of the three iminium salts both in vivo and in vitro. Among the three derivatives, the zinc analog (ZnBI) produced the greatest tumor regression at the low drug/light dose of 0.7 (mumole/kg and 200 J/cm2. These results indicate that iminium salts have characteristics that may make them promising third-generation photosensitizers.

Mitochondria are the functional intracellular target for a photosensitizing boronated porphyrin.

A photosensitizing boron-containing porphyrin derivative denoted BOPP, which is selectively localised into mitochondria, has been tested on Namalwa cells, in each of two genetic configurations: rho+ cells containing normal mtDNA and mitochondrial respiratory functions, or rho0 cells lacking mtDNA and devoid of mitochondrial oxidative phosphorylation. After short-term cellular uptake for 18 h, BOPP (30 micrograms/ml) was not cytotoxic, but did show marked phototoxicity in Namalwa rho+ cells, concomitant with substantial reduction of mitochondrial respiratory activity. After long-term (3 days or more) exposure to BOPP without light, growth of Namalwa rho+ cells was inhibited at concentrations significantly above 30 micrograms/ml. At such concentrations BOPP was shown to have direct inhibitory effects on mitochondrial azide-sensitive respiration of p+ cells. By contrast, BOPP showed neither cytotoxic nor phototoxic effects in rho0 cells. These results indicate functional mitochondria to be a major cellular target in vivo after BOPP uptake and photoactivation.

Selective tumor kill of cerebral glioma by photodynamic therapy using a boronated porphyrin photosensitizer.

The prognosis for patients with the high-grade cerebral glioma glioblastoma multiforme is poor. The median survival for primary tumors is < 12 months, with most recurring at the site of the original tumor, indicating that a more aggressive local therapy is required to eradicate the unresectable "nests" of tumor cells invading into adjacent brain. Two adjuvant therapies with the potential to destroy these cells are porphyrin-sensitized photodynamic therapy (PDT) and boron-sensitized boron neutron capture therapy (BNCT). The ability of a boronated porphyrin, 2,4-(alpha, beta-dihydroxyethyl) deuteroporphyrin IX tetrakiscarborane carboxylate ester (BOPP), to act as a photosensitizing agent was investigated in vitro with the C6 rat glioma cell line and in vivo with C6 cells grown as an intracerebral tumor after implantation into Wistar rats. These studies determined the doses of BOPP and light required to achieve maximal cell kill in vitro and selective tumor kill in vivo. The data show that BOPP is more dose effective in vivo by a factor of 10 than the current clinically used photosensitizer hematoporphyrin derivative and suggest that BOPP may have potential as a dual PDT/BNCT sensitizer.

Photosensitization with etiobenzochlorins and octaethylbenzochlorins.

The photophysical and photobiological properties of a series of etiobenzochlorins were evaluated in cell culture using murine leukemia L1210 cells. In the series of agents tested, the chlorin-(mono)sulfonate was the most efficacious, the tin chlorin somewhat less so and the tin chlorin-sulfonate much less active. The parent chlorin was essentially inactive at the limit of solubility. Photodamage was assessed by measuring alterations in surface hydrophobicity (via a two-phase partitioning procedure), amino acid transport and membrane potential. Additional information was provided from fluorescence microscopy, which was used to identify sites of sensitizer binding and effects of photodamage on the binding patterns of fluorescent probes specific for mitochondria, lysosomes and plasma membranes. Effects of photodamage on fluorescence lifetime distribution of the membrane probe trimethylaminodiphenyl hexatriene were examined. The data obtained were consistent with localization of the parnet etiobenzochlorin and tin derivative at lysosomal loci, the chlorin-sulfonate at plasma and mitochondrial membranes and tin-sulfonate at the cell surface.

Sites of photodamage by the iminium salt of a copper octaethylbenzochlorin.

Because the benzochlorin derivative copper (II) alpha-meso-N,N'-dimethyloctaethylbenzochlorin iminium chloride (CDS1) is not fluorescent, sites of drug localization in L1210 cells were detected by indirect methods involving using a series of fluorescent probes. The CDS1-mediated cytotoxicity was associated with mitochondrial damage, a decreased membrane potential and an increase in the heterogeneity of membrane sites of binding of a polar analog of diphenylhexatriene. Although CDS1 is a cationic compound, its accumulation was not impaired in a cell line exhibiting the multidrug resistance phenotype.

Photocytotoxicity of water-soluble metalloporphyrin derivatives.

A new water-soluble porphyrin derivative, 2,4-bis(1-decyloxyethyl)-deuteroporphyrinyl-6,7-bisaspart ic acid (C10-DP), and its metal complexes (Ga, I(n), Zn, Mn, Cu, Ni and Fe) were examined for their physicochemical properties (absorption, fluorescence, triplet lifetime and partition coefficient) and photocytotoxicity on HeLa cells. The five derivatives with longer (> 1 ms) triplet lifetimes (free base, Zn, Ga, I(n) and Sn complexes) exhibited remarkable photocytotoxicity, and the other derivatives (Mn, Cu, Ni and Fe), which had or were deduced to have fairly short (< 0.01 ms) triplet lifetimes, manifested no photocytotoxicity, indicating that the triplet lifetime of these derivatives played a significant role in their photocytotoxicity. Cellular fluorescence due to C10-DP and its gallium complex was observed mainly on the plasma membrane at the concentrations showing significant photocytotoxicity with low (< 32.6%) cytotoxicity in the dark (2-10 microM).

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.

In vivo effects of porphyrins on bacterial DNA.

The DNA damage in intact Staphylococcus aureus and E. coli cells induced by photosensitized deuteroporphyrin or hemin is described. Treatment of S. aureus cultures with hemin or photosensitized deuteroporphyrin (Dp) caused time-dependent changes in the plasmidial DNA profiles. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected by hemin and by photosensitized Dp, since its degradation products were detected after exposing the bacterial cells to the porphyrin drugs. Photosensitization of E. coli cells, pretreated with Dp and polymyxin B nonapeptide (PMBNP), also resulted in plasmidial damage. No such damage occurred when E. coli cultures were treated with hemin and PMBNP. The above results can be tightly correlated with the antimicrobial action of porphyrins. Their damage to the bacterial DNA seems to reflect one of the in vivo effects of these porphyrins.