Effect of albumin on the fluorescence quantum yield of porphyrin -based agents for fluorescent diagnostics.

BACKGROUND: Among modern methods of tumor diagnosis, fluorescent methods are considered one of the most prospective. Diagnostic agents (DAs) spread throughout the body by the bloodstream, so, the DA molecules are often transported by albumins and can be affected by these proteins. In our study we evaluate the effect of complex formation between bovine serum albumin (BSA) and three fluorescence DA's (Photolon, Photoditazin and Dimegin) on their fluorescent quantum yields. METHODS: Electron absorption spectroscopy and fluorescence spectroscopy were carried out to calculate fluorescence quantum yields of the DAs using Rhodamine 6G as a standard fluorescent dye. RESULTS: For all three DA's dissolved in phosphate buffer with pH 7.5 (close to that of blood) the addition of albumin resulted in bathochromic shift of the Soret band as well as change of amplitudes of absorption bands. Similar changes were observed for fluorescence spectra of all DAs that are connected with complex formation between DA and albumin. The presence of isobestic point suggests that DA can present in the solution only in two states, free and BSA-bound. Chlorine-based DA's demonstrate about 1.5-times higher fluorescence quantum yield in PBS than Dimegin. Nevertheless, the addition of BSA to the solutions of all DA's decreases sharply their fluorescence quantum yield to approximately equal values. CONCLUSION: The complex formation between DA and albumin equalize fluorescence efficacies of all studied DAs, so the results of photodymanic diagnostics using the specific DA will depend on other factors.

Pharmacokinetics, tissue distribution and excretion of a new photodynamic drug deuxemether.

Deuxemether was a new photodynamic drug effective for many kinds of solid tumor therapy, which was mainly composed of 3-(or 8-)-(1-methoxyethyl)-8-(or 3-)-(1-hydroxyethyl)-deutero-porphyrin IX (MHD) and 3,8-di(1-methoxyethyl)-deuteroporphyrin IX (DMD). The aims of this study were to elucidate its pharmacokinetic characteristics, tissue distribution, plasma protein binding and excretion properties and underlying mechanisms of deuxemether in rats based on the simultaneous determination of MHD and DMD. The pharmacokinetic profiles of both MHD and DMD in rats after intravenous doses were linear and best fitted to a two compartment model, characterized with a rapid distribution phase (MHD: t(1/2)alpha, 0.09-0.14 h; DMD: t 1/2 alpha, 0.07-0.11h) and a relatively slow elimination phase (MHD: t 1/2 beta, 2.03-3.20 h; DMD: t 1/2 beta, 2.51-3.20 h). The tissue distributions of MHD and DMD in rats were rather limited as evidenced from their low distribution volume (0.75-1.70 L/kg) and the results of tissue distribution study. Protein binding of MHD and DMD were moderate (65.36-89.99% for MHD; 45.43-76.23% for DMD), independent of drug concentrations and similar between human and rat plasma over a concentration range of 0.50-50.0 microg/mL. Both MHD and DMD were predominantly (>74.1%) eliminated from rats as the parent drugs through the hepatobiliary systems and finally excreted into the feces. The multidrug resistance-associated proteins 2 (MRP2) inhibitors, bromosulfophthalein and probenecid, substantially inhibited the hepatobiliary elimination of MHD and DMD while the P-gp inhibitor digoxin had little effect, suggesting that MRP2 may contribute to the rapid and extensive hepatobiliary excretion of deuxemether. There were no significant differences between MHD and DMD for all pharmacokinetic characteristics studied. In conclusion, this study provided firstly the full pharmacokinetic characteristics and mechanisms of deuxemether, which would be helpful for its clinical regiment design.

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.

Boron neutron capture therapy for undifferentiated thyroid carcinoma: preliminary results with the combined use of BPA and BOPP.

We have shown the selective uptake of borophenylalanine (BPA) by undifferentiated human thyroid cancer (UTC) ARO cells both in vitro and in vivo. Moreover, a 50% histologic cure of mice bearing the tumor was observed when the complete boron neutron capture therapy was applied. More recently we have analyzed the biodistribution of BOPP (tetrakis-carborane carboxylate ester of 2,4-bis-(alpha,beta-dihydroxyethyl)-deutero-porphyrin IX) and showed that when BOPP was injected 5 days before BPA, and the animals were sacrificed 60 min after the i.p. injection of BPA, a significant increase in boron uptake by the tumor was found (38-45 ppm with both compounds vs. 20 ppm with BPA alone). Five days post the i.p BOPP injection and 1h after BPA the ratios were: tumor/blood 3.75; tumor/distal skin 2. Other important ratios were tumor/thyroid 6.65 and tumor/lung 3.8. The present studies were performed in mice transplanted with ARO cells and injected with BOPP and BPA. Only in mice treated with the neutron beam and injected with the boronated compounds we observed a 100% control of tumor growth. Two groups of mice received different total absorbed doses: 3.00 and 6.01 Gy, but no further improvement in the outcome was found compared to the previous results using BPA alone (4.3 Gy).

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.

Photoproduct formation from a zinc benzochlorin iminium salt detected by fluorescence microscopy.

When examined by fluorescence microscopy, tumor cells loaded with a zinc benzochlorin iminium salt showed a very faint deep-red fluorescence that was rapidly transformed to a substantially brighter red-orange fluorescence. Fourier transfer spectroscopy analysis with a red-sensitive detection system revealed that an initial fluorescence at 770 nm was gradually converted to 640 nm fluorescence during excitation. Image analysis showed that photoproduct formation was accompanied by a change in the site of drug localization from the cytoplasm to the nucleus. These studies illustrate the power of interferometry for the characterization of photoproducts and changes in sensitizer localization during photoproduct formation.

Can cellular phototoxicity be accurately predicted on the basis of sensitizer photophysics?

The phototoxicity of three structurally related photosensitizers (PS), deuteroporphyrin IX (DP) and monobromo (Br-DP) and dibromo (Br2-DP) derivatives, was studied in murine L1210 leukemia cells. These compounds were chosen on the basis of heavy-atom-induced differences in triplet yield, phi T, and lifetime, tau T, and used as tools to test a model for phototoxicity based on photophysical parameters. All three porphyrins were found to localize preferentially in the plasma membrane of L1210 cells by confocal fluorescence microscopy. A poor correlation was observed between the measured photodynamic efficacies of these PS and a model using photophysical parameters determined by laser flash photolysis in homogeneous solution. However, an excellent correlation was obtained when the same parameters measured directly in the cells were used. The biological microenvironment of the porphyrins in cells induces significant changes in the photophysics of the PS. Reduction in fluorescence yield, phi F, and phi T observed for Br2-DP in cell suspensions arises from self association of the molecule due to increased hydrophobicity and high local concentrations. The photophysical model was also tested for its ability to handle variations in the oxygen dependence of cellular phototoxicity of these PS. The good correlation achieved between laser flash photolysis data determined in cells and the measured phototoxicity under air, 1.5% and 0.5% O2-saturated conditions, proves the intermediacy of singlet oxygen. This study gives further credence to the direct use of photophysical techniques to elucidate photochemical mechanisms in biological media while highlighting the potential pitfalls of using solution data to predict photosensitizing potential.

Tumor versus vascular photodamage in a rat tumor model.

The [4-(5-nitro-2-furyl)-2-thiazoyl] formamide (FANFT)-induced urothelial tumor in the rat is found to express the mdr gene. The resulting multidrug resistance (MDR) phenotype results in the expression of an outward transport system that prevents cellular accumulation of certain weakly cationic agents. Among the latter is a photosensitizer with known efficacy for the FANFT tumor, the copper benzochlorin iminium salt. FANFT cells are protected from direct cell kill mediated by this drug, suggesting that the substantial delay in tumor regrowth from this tumor/sensitizer combination can be attributed to vascular effects.

Photodynamic therapy of the ciliary body with tin ethyl etiopurpurin and tin octaethyl benzochlorin in pigmented rabbits.

BACKGROUND AND OBJECTIVES: The authors used a pigmented rabbit model to investigate two photosensitizers, tin ethyl etiopurpurin (SnET2) and tin octaethyl benzochlorin (BNZ 203), to determine their potential for creating ciliary body injuries during photodynamic therapy (PDT). MATERIALS AND METHODS: The biodistribution of SnET2 (n = 10) and BNZ 203 (n = 9) was studied by fluorescence microscopy using a low light detection system, based on charged-coupled device photography, with digital image processing at 1 and 24 hours after injection. PDT with SnET2 (n = 8; 664 +/- 7-nm light; 75 mW/cm2; 50 or 100 J/cm2; 1-mm spot size) and BNZ 203 (n = 6; 689 nm; 75 mW/cm2; 50 or 100 J/cm2; 1-mm spot size) was performed at 24 hours post-injection. The control subjects for SnET2 (n = 5) and BNZ 203 (n = 3) were given a maximal light dose (100 J/cm2). RESULTS: Both photosensitizers demonstrated an intravascular distribution at 1 hour that shifted to a ciliary body distribution at 24 hours (SnET2 much greater than BNZ 203). In addition, the SnET2 demonstrated suborgan localization to the nonpigmented ciliary body epithelium. Both photosensitizing agents were able to produce selective injury to the rabbit ciliary body (SnET2 much greater than BNZ 203), with evidence of a small component of thermal damage (SnET2 greater than BNZ 203). CONCLUSIONS: PDT with SnET2 or BNZ 203 can produce selective injury to the pigmented rabbit ciliary body. The nonpigmented ciliary body epithelium exhibits selective retention of SnET2. This finding warrants further investigation.

Photophysics and intracellular distribution of a boronated porphyrin phototherapeutic agent.

The absorption and emission spectra, fluorescence quantum yields and lifetimes and triplet state properties of a boronated porphyrin, the tetrakiscarborane carboxylate ester of 2,4-(alpha, beta-dihydroxyethyl) deuteroporphyrin IX (BOPP), have been determined. This compound is an alternative photodynamic therapy (PDT) agent that exhibits highly selective tumor localization, with the potential to be used in conjunction with boron neutron capture therapy. The photophysical characteristics of BOPP are similar to other porphyrins and it exhibits marked aggregation and acid-base speciation under typical physiological conditions. In particular, protonation of the porphyrin imino (-N=) nitrogens occurs in the pH 5-7 region and influences the photophysical properties. Time-resolved confocal fluorescence imaging of the intracellular distribution of BOPP in C6 glioma cells indicates distinct subcellular localization and heterogeneity of emission. The results are interpreted and discussed in terms of the possible mechanisms for cellular uptake and localization.

A comparative study on the pharmacokinetics and biodistribution of boronated porphyrin (BOPP) and sulfhydryl boron hydride (BSH) in the RG2 rat glioma model.

Boron neutron capture therapy is a treatment modality for cancer that depends on the specific uptake of boron by the tumor cells. The infiltrative growth of malignant gliomas requires that boron reach and accumulate in migrating cells outside the margin of the tumor; thus, it is important that the biodistribution of new boron compounds is also studied in the surrounding healthy brain tissue. This study is undertaken in the present work, in which the biodistribution and pharmacokinetics of sulfhydryl boron hydride (BSH) and boronated porphyrin (BOPP) in the RG2 rat glioma model are investigated. This model mimics the characteristics of human glioma with cells migrating into the surrounding brain. The animals were infused intravenously with either BSH (25 micrograms or 175 micrograms of boron per gram of body weight) or BOPP (12 micrograms of boron per gram body weight). For the low dose of BSH, the maximum tumor-boron content was 8 ppm at approximately 9 hours after the infusion with a tumor-to-blood ratio of 0.6. At the higher dose, the corresponding figures were 15 ppm after 12 hours with a tumor-to-blood ratio of 0.5. For BOPP, a tumor-boron concentration of 81 ppm was achieved 24 hours after the infusion and sustained in that range for at least 72 hours. The tumor-to-blood ratio at 24 hours was slightly above 6, but continued to increase as the blood was cleared. These results indicate that both compounds are spread into the normal brain tissue following the same pathways as the migrating tumor cells and in this way can be taken up even in distant tumor cell foci.

Enhanced boron uptake in RG 2 rat gliomas by electropermeabilization in vivo--a new possibility in boron neutron capture therapy.

Accumulation of boron in tumor tissue is an indispensable requirement for boron neutron capture therapy and it is important that the uptake is as high as possible. In this work we have studied the influence of electropermeabilization in vivo on the uptake of boron in normal and RG 2 glioma bearing Fischer 344 rats. Two different boron compounds, a sulfhydryl boron hydride (BSH) and a boronated porphyrin (BOPP), have been investigated. The rats were infused intravenously during 5 min with 175 micrograms BSH/g body weight or 12 micrograms BOPP/g body weight. Two electrodes were placed 5 mm apart in the brain and electropermeabilization was performed with eight square 400 V pulses at 4 and 7 min after the end of the infusion. After 6 h the animals were killed, and the boron content in the tumors and the surrounding brain was measured with neutron-activated autoradiography. In electropermeabilized healthy animals the BOPP uptake was low and limited to the electrode lesions, whereas BSH was spread extensively throughout the hemisphere. Rats with gliomas showed doubled (BOPP) to 10-fold (BSH) uptake of boron in the tumor when electropermeabilization was performed as compared with untreated animals. We conclude that electropermeabilization in the future may provide an interesting possibility to increase the uptake of certain boron compounds before neutron capture therapy.

Impaired accumulation of a cationic photosensitizing agent by a cell line exhibiting multidrug resistance.

Transport and accumulation of copper benzochlorin iminium salt (CDS1), a cationic photosensitizing agent, were examined using the P388/ADR murine leukemia, which exhibits the MDR (multidrug resistance) phenotype, and the wild-type parent cell line, P388. The recent availability of radioactive CDS1 permitted kinetic studies at drug levels in the submicromolar range. Exclusion of CDS1 by P388/ADR cells could be demonstrated, indicating that this agent is a substrate for the outward transport system associated with MDR. These results have implications with regard to the efficacy of cationic photosensitizers against this common neoplastic phenotype. The CDS1 was readily accumulated by P388 cells and by P388/ADR cells when the outward transport system was inhibited. Under these conditions, CDS1 was tightly bound and could not be washed out even when the outward transport system was reactivated.

111In-labeled Mn-metalloporphyrin for tumor imaging.

We synthesized and developed a new tumor imaging agent, 111In-labeled metalloporphyrin (111In-ATN-10) which consists of a carrier (ATN-10) of the tumor imaging possessing both a non-radioactive manganese complex in the porphyrin ring and a bifunctional chelating group (DTPA) attached to its side chain. The images of the three kinds of tumors were delineated more clearly by 111In-ATN-10 than 67Ga-citrate. Moreover, there was no photosensitivity in ATN-10. 111In-ATN-10 is studied as a new tumor positive scintigraphic agent instead of 67Ga-citrate.

Absorption of zinc deuteroporphyrin IX 2,4-bis-glycol by the neonatal rat small intestine in vivo.

Zinc deuteroporphyrin IX 2,4-bis-glycol (ZnBG) is a potent inhibitor of heme oxygenase and may be useful in the prevention of neonatal jaundice. Enteral administration could be advantageous clinically, but it has been only minimally effective with other metalloporphyrins in rats and humans. Thus, the absorption of ZnBG by the small intestine in vivo was examined. ZnBG was administered enterally at 40 mumol/kg to 2-week-old suckling rats via in situ catheterization of the small intestine. Within 15 min ZnBG was absorbed by the small intestine, as it was measured in portal and systemic venous plasma, intestine, kidney, liver, and spleen. Concentrations exceeding 5.0 microM were found in plasma within 30 min, and 9.4 microM was found in the liver after 30 min. A total of 4.6% of the administered ZnBG dose was measured in plasma and tissues.

Selective in vivo tumor localization of heptacarboxylic porphyrin isomer I in a bladder tumor model: a novel technique to modulate porphyrin localization.

We were the first to report that uroporphyrin isomer I is a superior tumor localizer when compared with hematoporphyrin derivative. In the present study, we have examined the tumor localization of heptacarboxylic porphyrin isomer I (hepta-P) using a bladder tumor model. We have also compared it to that found with uroporphyrin isomer I (Uro-P). We now show, for the first time, that (hepta-P) isomer I can be selectively retained in bladder malignant cells, a novel observation which has not yet been described by other investigators. Furthermore, we have provided a novel technique to modulate and manipulate blood protein binding to porphyrin in a controlled manner, such that the tumor localization properties can be effectively utilized without prolonged retention in the skin and to produce high uptake in the tumor, i.e., a higher therapeutic ratio. The biodistribution of hepta-P in different organs is presented.