Uptake Of Hematoporphyrin Derivative Research Articles

Selective accumulation of hematoporphyrin derivative in glioma through proton-coupled folate transporter SLC46A1

The mechanism of tumor-specific porphyrin accumulation is not clear. We investigated the expression of proton-coupled folate transporter SLC46A1 in glioma and aimed to clarify the relationship between tumor fluorescence and SLC46A1 expression.We confirmed the expression of SLC46A1 in surgical specimens from 24 glioma patients by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). We also investigated SLC46A1 expression in glioma cell lines by RT-PCR. The cellular uptake of hematoporphyrin derivative in vitro was measured with a microplate reader and fluorescence microscope. In these experiments, we used three human malignant glioma cell lines: U87, U251 and T98G. Immunohistochemistry showed SLC46A1 positivity in the malignant tumor lesion of each specimen. Strong positive SLC46A1 expression was observed in 33% of grade IV, 22% of grade III and 17% of grade II gliomas. All four randomly obtained malignant glioma frozen sections expressed SLC46A1 mRNA by RT-PCR. In vitro, U87 showed the least SLC46A1 expression, U251 was intermediate, and T98G showed the most expression. The amount of hematoporphyrin derivative (HpD) cellular uptake correlated with SLC46A1 expression. These results suggest that the accumulation of HpD in glioma cells is related to SLC46A1 function and SLC46A1 is involved in the mechanism of glioma fluorescence.

Photodynamic therapy of brain tumours: evaluation of porphyrin uptake versus clinical outcome

The objective of this study was to investigate whether the level of the photosensitizer haematoporphyrin derivative (HpD) uptake measured in tissue samples taken from brain tumour patients was associated with survival post-treatment with photodynamic therapy (PDT). The mean HpD uptake in tumour tissue was significantly higher in glioblastoma multiforme than anaplastic astrocytoma. Recurrent tumours had a higher mean uptake compared to primary tumours, which was evident in all grades of tumour. Among patients with GBM, there was a significant association between greater HpD uptake and survival ( HR = 0.26 [ 0.12 , 0.59 ] , p=0.001). There was also some evidence of a weak association between greater HpD uptake and survival among patients with AA, although the result was inconclusive ( HR = 0.73 [ 0.32 , 1.71 ] , p=0.472).

Photodynamic therapy: a 5-year study of its effectiveness in the treatment of posterior uveal melanoma, and evaluation of haematoporphyrin uptake and photocytotoxicity of melanoma cells in tissue culture

A human melanoma cell line RVH-421 which selectively demonstrates melanin synthesis when cultured in L15 Leibowitz medium but not in RPMI medium was used as a model to examine haematoporphyrin derivative (HPD) uptake and the photocytotoxicity of photodynamic therapy (PDT). Confocal scanning microscopy and extraction fluorometry showed HPD uptake in both non-pigmented and pigmented melanoma cells. Phototoxicity was determined by incubating pigmented and non-pigmented monolayer cells with HPD, exposing them to variable periods of white fluorescent light and calculating the number of viable cells in the samples relative to the controls. Both the non-pigmented and pigmented melanoma cells were affected by light-dependent cytotoxicity which was greater in the non-pigmented cells. Melanin or other substances may reduce the photo-oxidative effects of PDT. Posterior uveal melanomas in 36 patients were treated with PDT with the longest duration of tumour control being 6.5 years. Kaplan-Meier survival analysis showed that 76% of melanomas were not growing at the end of the first year, declining to 62% at the end of the second year, with 38% showing no signs of growth at the end of the fifth year. No eyes were lost as a result of PDT. Cox's hazards analysis showed that the degree of tumour pigmentation and patient age at therapy significantly influence the tumour response to PDT.

Photodynamic therapy of human ovarian epithelial carcinoma, OVCAR-3, heterotransplanted in the nude mouse

OBJECTIVE: Our objective was to develop an animal model for the study of photodynamic therapy in the treatment of human ovarian epithelial carcinoma.STUDY DESIGN: The human ovarian carcinoma OVCAR-3 was heterotransplanted into nude, athymic mice and treated with photodynamic therapy consisting of the hematoporphyrin derivative Photofrin II 10 ng/kg and argon-pumped dye laser light at 630 nm (200 J/cm2). Growth of tumors on one side of seven mice (treated tumors, n = 7) was compared with contralaterallurnors (control tumors, n = 8) not exposed to laser. Hematoporphyrin derivative uptake was determined in tumor and other tissues.RESULTS: Photodynamically treated tumors were completely ablated, and all remained absent. Hematoporphyrin derivative uptake was nonselective for tumor compared with other tissues.CONCLUSION: This model provides reproducible parameters for the study of photodynamic therapy In the treatment of gyneoologic malignancies and demonstrates the need for methods to increase selective uptake of hematoporphyrin derivatives. (AM J OBSTET GYNECOl 1992;167:1852-5.)

Selective Uptake of Hematoporphyrin Derivative into Human Cerebral Glioma

Abstract The uptake of hematoporphyrin derivative (HpD) into human cerebral glioma was measured using a porphyrin extraction technique. Patients with cerebral glioma were injected with HpD at a dose of 5 mg/kg body weight 24 hours before surgery and photoradiation therapy (PRT). Biopsies of tumor, and where possible, adjacent brain and normal brain were taken for analysis of HpD uptake. HpD was selectively localized into all grades of glioma, and there was a direct correlation between the grade of glioma and HpD level in the tumor. The levels were highest in glioblastoma multiforme (mean uptake of 5.9 mg of HpD/g of tumor wet weight) and lower in the intermediate-grade anaplastic astrocytoma (mean uptake of 2.4 mg/g of tumor) and the low-grade astrocytoma (1.6 mg/g of tumor), Uptake into normal brain tissue taken from HpD-sensitized patients was 0.2 mg/g. HpD was also localized into the “brain adjacent to tumor” region. The selective uptake into the low-grade glioma suggests that PRT may be of use as an adjuvant therapy in these tumors and the detection of HpD in this region indicates that PRT may control the spread of tumor infiltrating into the adjacent normal brain.

Selective uptake of hematoporphyrin derivative into human cerebral glioma

The uptake of hematoporphyrin derivative (HpD) into human cerebral glioma was measured using a porphyrin extraction technique. Patients with cerebral glioma were injected with HpD at a dose of 5 mg/kg body weight 24 hours before surgery and photoradiation therapy (PRT). Biopsies of tumor, and where possible, adjacent brain and normal brain were taken for analysis of HpD uptake. HpD was selectively localized into all grades of glioma, and there was a direct correlation between the grade of glioma and HpD level in the tumor. The levels were highest in glioblastoma multiforme (mean uptake of 5.9 micrograms of HpD/g of tumor wet weight) and lower in the intermediate-grade anaplastic astrocytoma (mean uptake of 2.4 micrograms/g of tumor) and the low-grade astrocytoma (1.6 micrograms/g of tumor). Uptake into normal brain tissue taken from HpD-sensitized patients was 0.2 microgram/g. HpD was also localized into the "brain adjacent to tumor" region. The selective uptake into the low-grade glioma suggests that PRT may be of use as an adjuvant therapy in these tumors and the detection of HpD in this region indicates that PRT may control the spread of tumor infiltrating into the adjacent normal brain.

Ablation of endometriotic implants in rabbits by hematoporphyrin derivative photoradiation therapy using the gold vapor laser

In a rabbit model, endometriotic lesions were utilized to study hematoporphyrin derivative (HPD) uptake and the effect of photoradiation using the gold vapor laser. Morphological studies confirmed successful induction of ectopic endometrial growth and the lesions displayed preferential HPD uptake compared to normal intraperitoneal structures. Gold vapor laser irradiation of the lesions in HPD pretreated animals produced necrosis of the endometriotic lesions leaving surrounding tissues healthy. Results from this study suggest that HPD-mediated photodynamic therapy may be a suitable means of treating endometriosis in the human.

Photoradiation therapy and its potential in the management of neurological tumors.

Photoradiation therapy is a form of local treatment that depends on the selective retention of a photosensitizer, such as hematoporphyrin derivative (HpD), by the tumor followed by treatment with light of an appropriate wavelength to activate the sensitizer in the tumor. The selective uptake of HpD by cerebral tumors has been demonstrated both in laboratory animal model studies and in clinical studies, and selective destruction of intracerebral tumors has been demonstrated in animal glioma models. The biological basis for photoradiation therapy and, in particular, the mechanisms for the selective uptake of the sensitizer into the tumor and the destruction of tumor with photoradiation therapy are discussed. Current evidence suggests that singlet oxygen is the major intermediary leading to cell damage, although other radicals such as hydrogen peroxide and hydroxyl radicals may be involved. Other studies suggest that the initial damage is to the blood vessels, and the tumor subsequently undergoes ischemic changes. Sixty-four patients treated with photoradiation therapy have been reported in the literature. The initial clinical studies were disappointing in their therapeutic effect but these studies often included treatment of recurrent gliomas and low doses of light were used. Technical advances, particularly in laser technology, have enabled more effective photoradiation therapy and the clinical trials are reviewed.

Temperature-dependence of haematoporphyrin derivative uptake in vitro

The uptake of haematoporphyrin derivative (HPD) by Ehrlich ascites tumour cells was investigated by measuring the fluorescence intensity of HPD bound to the cells at various temperatures. Analysis of the HPD fluorescence emission patterns suggests that there are two steps in the HPD binding process. First, a weak binding mode: the fluorescence peak appeared at 617 nm in the early incubation period below 25°C. Secondly, a stable binding mode: the fluorescence peak appeared at 630 nm in the late incubation period above 30°C, and it was considered that the HPD uptake was due mainly to facilitated diffusion. The Arrhenius plots of logarithmic values of the initial velocity and the reciprocal of the absolute temperatures in the HPD-uptake reaction showed a biphasic linear relationship having an inflexion point at 29±5°C. From the plots, the lower activation energy corresponded to the former mode, and the higher one to the latter mode. It was considered that the critical temperature might correlate with the phase change of the membrane lipid to which HPD molecules bound and by which they are transported. Furthermore, from the results of double vital staining with Hoechst 33258 and HPD it was shown that the uptake by late G1-, S- and early G2(+M)-phase cells increased somewhat in this order.

Hematoporphyrin Derivative Uptake in Uteri of Estrogen-Treated Ovariectomized Rats

ABSTRACT This study was devised to evaluate the influence of estrogen on the uptake of dihematoporphyrin ether (DHE) in the uterus of ovariectomized rats. The DHE uptake was measured by scintillation counting and the distribution by fluorescence microscopy. Estrogen treatment significantly (P < 0.001) increased the uterine uptake of DHE but had no effect on uptake by the spleen. Uptake levels in both tissues remained constant during the 72 hours after DHE injection. Changes in the anatomic distribution of DHE were found to be gradual and a function of time. At 3 hours after injection, the DHE was concentrated in the endometrial layer. After 6 hours, the DHE had disappeared from the endometrium and appeared in the myometrium and serosa; after 24 hours it was seen in the serosa only. It is concluded that although the quantity of DHE remains constant for 72 hours within the rat uterus, its distribution pattern within the organ layers is dynamic.

Hematoporphyrin Derivative Uptake and Tumor Cell Densitya

Hematoporphyrin Derivative Uptake and Tumor Cell Densitya

Uptake of hematoporphyrin derivative by atheromatous plaques: Studies in human in vitro and rabbit in vivo

Hematoporphyrin derivative, a photosensitive material used to identify and treat neoplastic tissue in humans, has been found to localize in atheromatous plaques in animals and has recently been found in postmortem human atherosclerotic plaques. It is not known whether human plaques take up hematoporphyrin derivative in vivo. In five patients undergoing surgical vascular procedures, specimens containing atheromatous plaques were removed and immediately incubated in autologous oxygenated blood at 37 degrees C with hematoporphyrin derivative at a clinically relevant concentration for 2 hours. On exposure to ultraviolet light, porphyrin fluorescence was noted throughout each plaque, whereas adjacent plaque-free tissue showed no fluorescence. To compare in vitro with in vivo hematoporphyrin derivative uptake by plaques, the fluorescence of three types of arterial lesions (induced by a high cholesterol diet, catheters or balloon injury) was studied in 16 New Zealand White rabbits. Each lesion fluoresced selectively with the same intensity whether hematoporphyrin derivative exposure was performed in vitro or in vivo. Fluorescence microscopy did not show a difference in the pattern of hematoporphyrin derivative fluorescence between in vitro and in vivo specimens. The results suggest that human atheromatous plaques should take up hematoporphyrin derivative in vivo and are, therefore, potentially suitable for photochemical treatment as a new therapeutic approach to atherosclerosis.

Porphyrin photosensitization and phototherapy.

Porphyrin photosensitization and phototherapy.

Photoradiation therapy: Current status and applications in the treatment of brain tumors

Photoradiation therapy is achieved when a photosensitizing drug is activated by light to form products that are lethal to tumor cells. The most commonly used drug is hematoporphyrin derivative, which is preferentially taken up and retained by malignant tissue. Photoactivation is usually produced by using a dye laser tuned at 630 nm (red light). The primary mechanism of neoplastic cell damage in photoradiation therapy involves the production of free radicals formed during illumination of hematoporphyrin derivative by light of this wavelength. The treatment would seem to damage first the tumor cell membrane, then the cytoplasmic inclusions, and finally the nucleus. Photoradiation therapy has been quite effective in the treatment of superficial malignancies, especially in skin, breast, eye, bladder, bronchus, and stomach. Experience with brain tumors is still limited. Important unresolved problems in the application of photoradiation therapy to gliomas include relative uptake of hematoporphyrin derivative into the tumor, limited light penetration of the tissue, local heating, and damage induced in normal brain by photoradiation therapy.

Effects of hematoporphyrin-derivative on mouse erythroleukemia cells in the absence of light irradiation

This paper concerns a general study on the effects of hematoporphyrin-derivative (HpD) on mouse erythroleukemia (MEL) cells, in the absence of light irradiation. In particular, HpD intrinsic cytotoxicity was evaluated at different doses and the results correlated with those referring to membrane functional and morphological changes. HpD uptake and release processes were also studied and compared with the above-mentioned results. In order to have an overall picture of HpD-cell interactions, time-resolved fluorescence measurements were performed on both undifferentiated and differentiated MEL cells. The results obtained indicate that, even at HpD doses exhibiting neither any cytotoxicity nor any morphological damage (1–10 μg/ml), membrane permeability alterations are observed. Thirty minutes of treatment are sufficient for HpD to develop its toxic effect: indeed, no differences in HpD influence on cell viability can be observed after 30 min, 60 min or 5 days of treatment. HpD cytotoxicity is reduced by high protein content in the incubation culture medium. The presence of both monomeric species and 580 nm emitting species was observed at cellular level. The latter is likelier in undifferentiated MEL cells, which also exhibit higher overall HpD uptake, as compared with differentiated MEL cells.

Uptake of hematoporphyrin derivative in bacteria and photosensitization of Propionibacterium acnes bacteria

Uptake of hematoporphyrin derivative in bacteria and photosensitization of Propionibacterium acnes bacteria

The nude mouse xenograft system: A model for photodetection and photodynamic therapy in head and neck squamous cell carcinoma

Mechanisms for hematoporphyrin derivative (HPD) localization in malignant tissue and in photodynamic therapy (PDT) have not been established. The authors' experience with human cancer xenografts in nude mice as a tumor system for the experimental study of these mechanisms is described. Human mucosal head and neck squamous cell carcinoma was successfully transplanted to nude mice in 29 per cent of trials and serially passed through multiple generations in three tumor lines. Time required for progressive growth averaged 13.9 days, and 300 mm3 tumors were obtained in four weeks. Take rates per passage varied, in part because of infection, and exponential growth rates varied among the three lines. However, within a single line exponential growth rates were similar. Average growth constants for the period of exponential growth were 0.14, 0.13, and 0.09 for the three lines. Fluorescent microscopy of excised xenografts revealed HPD fluorescence principally in connective tissue cells. Minimal fluorescence was seen in malignant epithelial cells. Spectrophotometric measures of HPD uptake in homogenized tissue showed highest values in liver. Tumor HPD levels were higher than those for either skin or muscle. The authors conclude that this tumor system can be used effectively to study HPD and PDT in head and neck cancer.

Uptake of hematoporphyrin derivative by valvular vegetations in experimental infective endocarditis.

Drugs that localize in valvular vegetations may be useful in the diagnosis and treatment of infective endocarditis. We therefore tested the hypothesis that parenterally injected hematoporphyrin derivative (HPD), which is concentrated in tumors and atherosclerotic plaques, localizes in the vegetations of experimental infective endocarditis. In 14 rabbits, various bacteria were given intra-arterially immediately after injury to the aortic valve. In 12 additional rabbits, sterile vegetations on the aortic valve were produced by the trauma caused by an indwelling catheter that had been in place over a long period. HPD, 2.5 mg/kg, was injected intravenously 1 to 2 days before the animals were killed in six rabbits with sterile vegetations and in seven rabbits with infected vegetations. In all rabbits, multiple vegetations on the aortic valve leaflets were identified. On exposure to ultraviolet light, strong porphyrin fluorescence of all vegetations, whether sterile or infected, was observed only in rabbits given HPD. In two rabbits given HPD 10 weeks after catheter implantation across the aortic valve, however, only mild fluorescence could be detected in healing endocardial vegetations. In frozen sections of HPD-laden lesions, a patchy distribution of fluorescence was observed that was similar to the pattern of HPD localization in atheromatous plaques. Since vegetations in experimental infective endocarditis selectively concentrate HPD, porphyrins could be useful in the diagnosis and treatment of infective endocarditis.

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.

Effect of hematoporphyrin derivative and photodynamic therapy on atherosclerotic rabbits

This study was performed to demonstrate selective uptake of hematoporphyrin derivative (HPD) within actively developing atheroma, to localize the site of uptake of HPD within the atheroma, and to determine the potential for photodynamic therapy (PDT) of atherosclerosis in the rabbit model. Fifteen rabbits were rendered atherosclerotic. Five rabbits received neither HPD nor PDT and 2 rabbits received HPD, 10 mg/kg intravenously, without subsequent irradiation. Eight other rabbits received 5 to 20 mg of HPD intravenously and subsequent intravascular 636-nm laser radiation to either the thoracic aorta or the aortic arch. A total of 32 to 288 J of laser energy was delivered through a 300-μ quartz fiber. All rabbits that received in vivo HPD had red fluorescence of their aortas when placed under ultraviolet light. The pattern of fluorescence corresponded precisely to the pattern of atheroma. In segments that received PDT, light microscopic examination revealed an accumulation of smooth muscle cells at the intimal surface. Fluorescence microscopy revealed a diminishing concentration gradient of HPD from intimal surface layers towards the media. Assessment of treated thoracic aortic segments revealed quantitative and qualitative differences compared with control segments. In the arch-treated segments, however, no changes were seen. It is concluded that HPD localizes within rabbit atheroma, can be detected by fluorescence and is deposited in a diminishing concentration gradient from lumen toward media. Irradiation with 636-nm light may induce qualitative and quantitative changes in atheroma.